1: J Immunol. 2005 Sep 1;175(5):3354-9. Stat3 in resident macrophages as a repressor protein of inflammatory response. Matsukawa A, Kudo S, Maeda T, Numata K, Watanabe H, Takeda K, Akira S, Ito T. Department of Pathology and Experimental Medicine, Graduate School of Medical Sciences, School of Medicine, Kumamoto University, Honjo, Kumamoto, Japan. matsu@kaiju.medic.kumamoto-u.ac.jp Inflammation is counterbalanced by anti-inflammatory cytokines such as IL-10, in which Stat3 mediates the signaling pathway. In this study, we demonstrate that resident macrophages, but not other cell types, are important targets of IL-10 in a murine model of acute peritonitis. Injection of thioglycollate i.p. induced a considerable number of neutrophils and macrophages in the peritoneum, which was significantly augmented in mice with a cell-type specific disruption of the Stat3 gene in macrophages and neutrophils (LysMcre/Stat3flox/- mice). The augmented leukocyte infiltration was accompanied by increased peritoneal levels of TNF-alpha, MIP-2, KC chemokine (KC), and MCP-1/CCL2. Stat3 was tyrosine phosphorylated in peritoneal resident macrophages as well as infiltrating leukocytes in the littermate controls, suggesting that Stat3 in either or both of these cells might play a regulatory role in inflammation. The peritoneal levels of TNF-alpha, MIP-2, KC, and MCP-1 were similarly elevated in LysMcre/Stat3flox/- mice rendered leukopenic by cyclophosphamide treatment as compared with the controls. Adoptive transfer of resident macrophages from LysMcre/Stat3flox/- mice into the control littermates resulted in increases in the peritoneal level of TNF-alpha, MIP-2, KC, and MCP-1 after i.p. injection of thioglycollate. Under these conditions, control littermates harboring LysMcre/Stat3flox/- macrophages exhibited an augmented leukocyte infiltration relative to those received control macrophages. Taken together, these data provide evidence that resident macrophages, but not other cell types, play a regulatory role in inflammation through a Stat3 signaling pathway. Stat3 in resident macrophages appears to function as a repressor protein in this model of acute inflammation. PMID: 16116228 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: Endocrinology. 2005 Nov;146(11):5003-11. Epub 2005 Aug 4. Urocortin 2 suppresses host resistance to Listeria monocytogenes infection via up-regulation of interleukin-10. Sashinami H, Kageyama K, Suda T, Nakane A. Department of Bacteriology, Hirosaki University School of Medicine, Zaifu-cho 5, Hirosaki, Aomori 036-8562, Japan. Previous studies have showed that corticotropin-releasing factor (CRF) modulates immune response during inflammation. We investigated the effect of CRF family peptides on host resistance to Listeria monocytogenes infection in mice. When mice were administered ip with CRF, urocortin (Ucn), or Ucn2 30 min prior a sublethal infection with L. monocytogenes, the numbers of bacteria in the organs of Ucn2-treated mice were dramatically increased, and most of these mice succumbed. However, host resistance to the infection was retained in CRF- or Ucn-treated mice. The suppressive effect of Ucn2 was dependent on CRF receptor type 2 because an antagonist to the receptor canceled the effect of Ucn2. IL-10 production was significantly increased, and interferon-gamma and TNFalpha production was decreased in the spleens of Ucn2-treated mice, compared with those in Ucn2-untreated control mice. The effect of Ucn2 was canceled by treatment with anti-IL-10 monoclonal antibody and in IL-10-deficient mice. The expression and activation of signal transducers and activators of transcription (STAT) 3 were up-regulated, and the expression and activation of STAT1 were down-regulated in the spleens from Ucn2-treated mice, compared with vehicle-treated mice. Moreover, suppression of TNFalpha production and augmentation of IL-10 production and expression and activation of STAT3 by Ucn2 treatment were observed in heat-killed L. monocytogenes-stimulated macrophages. These results suggested that Ucn2 suppresses host resistance to L. monocytogenes infection via up-regulation of IL-10 production. PMID: 16081642 [PubMed - in process] --------------------------------------------------------------- 3: Nat Immunol. 2005 Aug;6(8):844-51. Epub 2005 Jul 17. Erratum in: Nat Immunol. 2005 Sep;6(9):954. Comment in: Nat Immunol. 2005 Aug;6(8):756-7. Stimulation of the vagus nerve attenuates macrophage activation by activating the Jak2-STAT3 signaling pathway. de Jonge WJ, van der Zanden EP, The FO, Bijlsma MF, van Westerloo DJ, Bennink RJ, Berthoud HR, Uematsu S, Akira S, van den Wijngaard RM, Boeckxstaens GE. Division of Gastroenterology and Hepatology, Academic Medical Center, Amsterdam, The Netherlands. Acetylcholine released by efferent vagus nerves inhibits macrophage activation. Here we show that the anti-inflammatory action of nicotinic receptor activation in peritoneal macrophages was associated with activation of the transcription factor STAT3. STAT3 was phosphorylated by the tyrosine kinase Jak2 that was recruited to the alpha7 subunit of the nicotinic acetylcholine receptor. The anti-inflammatory effect of nicotine required the ability of phosphorylated STAT3 to bind and transactivate its DNA response elements. In a mouse model of intestinal manipulation, stimulation of the vagus nerve ameliorated surgery-induced inflammation and postoperative ileus by activating STAT3 in intestinal macrophages. We conclude that the vagal anti-inflammatory pathway acts by alpha7 subunit-mediated Jak2-STAT3 activation. PMID: 16025117 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Am J Respir Cell Mol Biol. 2005 Oct;33(4):406-11. Epub 2005 Jul 13. Interleukin-10 induces inhibitory C/EBPbeta through STAT-3 and represses HIV-1 transcription in macrophages. Tanaka N, Hoshino Y, Gold J, Hoshino S, Martiniuk F, Kurata T, Pine R, Levy D, Rom WN, Weiden M. Division of Pulmonary & Critical Care Medicine, Department of Medicine, N.Y.U. School of Medicine, New York, NY 10016, USA. Pulmonary tuberculosis (TB) has been characterized by inflammation with increased pro- or anti-inflammatory cytokines produced by macrophages. We have reported that IFN produces inhibitory C/EBPbeta and represses transcription of the HIV-1 LTR in macrophages. STAT-1 and type I IFN receptor knockout mice have macrophages that are defective in IFN signaling, yet LPS stimulation induces inhibitory C/EBPbeta, demonstrating that other cytokines can induce this repressor. LPS or Mycobacterium tuberculosis-derived lipoarabinomannan induce the anti-inflammatory cytokine interleukin (IL)-10, which represses the HIV-1 LTR in differentiated THP-1 macrophages by inducing inhibitory C/EBPbeta. In contrast, in undifferentiated THP-1 monocytes, IL-10 did not inhibit HIV-1 replication or induce C/EBPbeta. IL-10 signal transduction uses STAT-3, and macrophages from STAT-3-/- mice fail to produce inhibitory C/EBPbeta after LPS or IL-10 stimulation. Transfection of STAT-3 into THP-1 cells enhances C/EBPbeta promoter activity. THP-1 differentiation also increases STAT-3 protein, but not STAT-3 gene transcription, and induces a translational regulator, CUG-binding protein, that was essential for production of C/EBPbeta. Differentiation induced post-transcriptional regulation is required to produce inhibitory C/EBPbeta in response to IL-10. Only macrophages are able to repress HIV-1 LTR promoter activity and inhibit viral replication in response to IL-10 or type I IFN. PMID: 16014896 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Gastroenterology. 2005 Jul;129(1):185-203. Growth hormone inhibits signal transducer and activator of transcription 3 activation and reduces disease activity in murine colitis. Han X, Sosnowska D, Bonkowski EL, Denson LA. Cincinnati Children's Hospital Medical cetner and the University of Cincinnati College of Medicine, Ohio, USA. BACKGROUND & AIMS: Constitutive signal transducer and activator of transcription (STAT) 3 activation promotes chronic inflammation and epithelial proliferation in murine colitis and human inflammatory bowel disease. SHP-2, through binding to the glycoprotein 130 signaling receptor, negatively regulates STAT3 activation. Growth hormone reduces disease activity and promotes mucosal healing in colitis and can activate SHP-2. METHODS: We hypothesized that growth hormone administration would reduce disease activity in experimental colitis and that this would involve modulation of SHP-2/glycoprotein 130 association and STAT3 activation. RESULTS: Growth hormone administration improved weight gain and colon histology in interleukin 10-null mice with colitis. Growth hormone reduced apoptosis and increased proliferation of crypt epithelial cells while increasing apoptosis of lamina propria mononuclear cells. Growth hormone increased SHP-2/glycoprotein 130 association and reduced colonic STAT3 activation in interleukin 10-null mice and in biopsy samples from patients with Crohn's colitis. Expression of the antiapoptotic protein bcl-2 was increased in crypt epithelial cells after growth hormone treatment. Growth hormone increased SHP-2/glycoprotein 130 binding and reduced interleukin 6-dependent STAT3 activation in the T84 human colon carcinoma and Jurkat human T-cell leukemia lines. CONCLUSIONS: Growth hormone administration improves weight gain and reduces disease activity in interleukin 10-null mice with colitis. The improvement in disease activity is associated with increased SHP-2/glycoprotein 130 binding and reduced STAT3 activation in both murine and Crohn's colitis. Growth hormone may be a useful therapy in inflammatory bowel disease, in terms of both improving anabolic metabolism and enhancing mucosal healing. PMID: 16012947 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: J Immunol. 2005 Jul 1;175(1):469-77. ERK activation following macrophage FcgammaR ligation leads to chromatin modifications at the IL-10 locus. Lucas M, Zhang X, Prasanna V, Mosser DM. Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA. We have previously demonstrated that macrophages stimulated in the presence of immune complexes produce high levels of IL-10. We now examine the mechanism of IL-10 superinduction. We report that the enhanced production of IL-10 correlates with a rapid and enhanced activation of two MAPKs, ERK and p38. The inhibition of either ERK or p38 prevented IL-10 induction, indicating that both MAPKs were required for IL-10 synthesis. By chromatin immunoprecipitation assay, we demonstrate that activation of ERK leads to the phosphorylation of serine 10 on histone H3 at the il-10 gene, making the promoter more accessible to transcription factors generated in response to p38 activation. Inhibition of ERK activation prevented histone modifications, and decreased the binding of Sp1 and STAT3 to the IL-10 promoter. We conclude that the activation of ERK following FcgammaR ligation leads to a remodeling of the chromatin at the il-10 locus, making it more accessible to transcription factors. The rapid and transient regulation of transcription factor accessibility to the IL-10 promoter by MAPK activation represents a novel way that the production of this cytokine is regulated. PMID: 15972681 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: J Immunol. 2005 Jun 15;174(12):7823-32. STAT-1 mediates the stimulatory effect of IL-10 on CD14 expression in human monocytic cells. Rahimi AA, Gee K, Mishra S, Lim W, Kumar A. Department of Pathology and Laboratory Medicine, University of Ottawa, Ottawa, Ontario, Canada. IL-10, an anti-inflammatory cytokine, has been shown to exhibit stimulatory functions including CD14 up-regulation on human monocytic cells. CD14-mediated signaling following LPS stimulation of monocytic cells results in the synthesis of proinflammatory cytokines. Our results show that LPS-induced CD14 expression on monocytic cells may be mediated by endogenously produced IL-10. To investigate the molecular mechanism by which IL-10 enhances CD14 expression, both human monocytes and the promyelocytic HL-60 cells were used as model systems. IL-10 induced the phosphorylation of PI3K and p42/44 ERK MAPK. By using specific inhibitors for PI3K (LY294002) and ERK MAPKs (PD98059), we demonstrate that LY294002 either alone or in conjunction with PD98059 inhibited IL-10-induced phosphorylation of STAT-1 and consequently CD14 expression. However, IL-10-induced STAT-3 phosphorylation remained unaffected under these conditions. Finally, STAT-1 interfering RNA inhibited IL-10-induced CD14 expression. Taken together, these results suggest that IL-10-induced CD14 up-regulation in human monocytic cells may be mediated by STAT-1 activation through the activation of PI3K either alone or in concert with the ERK MAPK. PMID: 15944287 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: Proc Natl Acad Sci U S A. 2005 Jun 14;102(24):8686-91. Epub 2005 Jun 3. The primary mechanism of the IL-10-regulated antiinflammatory response is to selectively inhibit transcription. Murray PJ. Department of Infectious Diseases, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38105, USA. peter.murray@stjude.org The antiinflammatory cytokine IL-10 inhibits the production of multiple, diverse inflammatory mediators from activated macrophages and dendritic cells, a process requiring STAT3 activation. However, the mechanisms involved in the broad inhibitory effects of IL-10 are controversial. I eliminated the contribution of the major confounding variable to understanding the antiinflammatory response, the 3' UTR region of inflammatory mediator genes, through knock-in mutation and analysis of the effects of IL-10 on transcription rate of inflammatory genes. IL-10 activates STAT3 to act indirectly by selectively inhibiting gene transcription independent of general effects on NF-kappaB or posttranscriptional mRNA processing through a process that reduces the overall transcriptional rate of specific genes. PMID: 15937121 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: Eur J Immunol. 2005 Jun;35(6):1877-85. Lipopolysaccharide primes neutrophils for a rapid response to IL-10. Cassatella MA, Tamassia N, Crepaldi L, McDonald PP, Ear T, Calzetti F, Gasperini S, Zanderigo F, Bazzoni F. Department of Pathology, Section of General Pathology, University of Verona, Verona, Italy. marco.cassatella@univr.it Responsiveness of human neutrophils to IL-10 was recently shown to be strictly dependent on the levels of IL-10R1 expression. Activation of signal transducer and activator of transcription 3 (STAT3) phosphorylation and induction of suppressor of cytokine signaling (SOCS)-3 protein by IL-10 are in fact negligible in circulating or freshly isolated ("time 0") neutrophils, but become readily measurable in neutrophils cultured for 4 h in the presence or absence of LPS. In this study, we show that modulation by IL-10 of LPS-induced TNF-alpha, CXCL8/IL-8 and IL-1 receptor antagonist (IL-1ra) mRNA accumulation in neutrophils already expressing a functional IL-10R and antigenic SOCS-3 (i.e. in "4-h-cultured" neutrophils) occurs with kinetics that are similar to those observed in "time 0" neutrophils, depends on de novo protein synthesis, but does not require SOCS-1, SOCS-3, heme oxygenase and Bcl-3 induction. By contrast, we show that IL-10 alone rapidly modulates the expression of TNF-alpha, CXCL8/IL-8 and IL-1ra mRNA, without any new protein synthesis requirement, if neutrophils have been previously exposed to LPS for at least 4 h. These findings suggest that LPS prepares neutrophils to optimally respond to IL-10 in terms of rapid gene modulation via mechanisms that, presumably, depend on specific LPS-induced protein(s). PMID: 15864776 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: Rheumatology (Oxford). 2005 Aug;44(8):983-8. Epub 2005 Apr 19. Inhibition of IL-10-induced STAT3 activation by 15-deoxy-Delta12,14-prostaglandin J2. Ji JD, Kim HJ, Rho YH, Choi SJ, Lee YH, Cheon HJ, Sohn J, Song GG. Division of Rheumatology, Department of Internal Medicine, College of Medicine, Korea University, 126-1, Anam-dong 5-Ga, Sungbuk-Gu, Seoul 136-705, South Korea. jjdjmesy@korae.ac.kr OBJECTIVES: 15-Deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) is a natural ligand that activates the peroxisome proliferator-activated receptor (PPAR)-gamma, a member of the nuclear receptor family implicated in the regulation of lipid metabolism and adipocyte differentiation. Recent data have shown that 15d-PGJ2 exerts anti-inflammatory action via inhibition of the interferon gamma (IFN-gamma)-induced Jak-STAT signalling pathway. The anti-inflammatory effect of IL-10 is mediated via activated STAT3 (signal transducer and activator of transcription 3). In this study, we investigated whether 15d-PGJ2 inhibit IL-10-induced STAT activation. METHODS: We used western blotting, flow cytometric analysis and a real-time polymerase chain reaction. RESULTS: 15d-PGJ2 blocked IL-10-induced STAT1 and STAT3 activation in primary human monocytes, macrophages and THP-1 cells. Inhibition was not specific for IL-10, as induction of STAT activation by IFN-gamma and IL-6 was also inhibited by 15d-PGJ2. Inhibition of IL-10 signalling was induced within 1 h after pretreatment of 15d-PGJ2. Other PPARgamma agonists, such as troglitazone, did not inhibit IL-10 signalling. Treatment with GW9662, a specific PPARgamma antagonist, had no effect on 15d-PGJ2-mediated inhibition of IL-10 signalling even at higher concentrations (50 microM), indicating that 15d-PGJ2 affects the IL-10-induced Jak-STAT signalling pathway via an PPARgamma-independent mechanism. Actinomycin D had no effect on 15d-PGJ2-mediated inhibition of IL-10 signalling, indicating that inhibition of IL-10 signalling occurs independently of de novo gene expression. Also, inhibitors of extracellular signal-regulated kinase (ERKs) (PD98059), p38 MAPK (mitogen-activated protein kinase) (SB203580) and protein kinase C (PKC) (GF109203X, calphostin C) had no effect on 15d-PGJ2-mediated inhibition of IL-10 signalling. These results show that MAPKs and PKC are not involved in the inhibition of IL-10 signalling. CONCLUSIONS: We showed that 15d-PGJ2 non-specifically inhibits STAT signalling of the anti-inflammatory cytokine IL-10 as well as the proinflammatory cytokine IFN-gamma. These findings indicate the possibility that 15d-PGJ2 can have adverse effects in the management of diseases in which IL-10 plays a critical role in the suppression of inflammation. PMID: 15840591 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: Zhonghua Wai Ke Za Zhi. 2005 Jan 1;43(1):6-9. [Influence on the cytokines expression on hepatic tissue by inhibition the signal pathway of inflammatory mediators following extensive hepatectomy in rats] [Article in Chinese] Mao YL, Yu Z, Sang XT, Lu X, Yang ZY, Zhong SX. Liver Surgery Center, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100730, China. maoy@public3.bta.net.cn OBJECTIVE: To investigate the impact of AG490, a cytokine signaling inhibitor, on cytokine signaling pathway with phosphorylation levels of Janus kinase 2 (Jak2) and singal transducers and activators of transcription 3 (Stat3), and liver pro-/anti-inflammatory cytokine expressions. METHODS: Rats were divided into two groups after surgery: control group, without treatment; AG490 group, with AG490 (1 mg.kg(-1).12 h(-1)) administration intraperitoneally, immediately and through 36 hs after the operation. Western blotting was used to detect the levels of phosphorylated Jak2 and Stat3. Semi-quantitative RT-PCR was employed to examine Interleukin-6 (IL-6) and Interleukin-10 (IL-10) expression. RESULTS: At 8 h and 12 h post-operatively, the phosphorylation levels of Jak2 and Stat3 were significantly inhibited in the AG490 group when compared with the control group. The DNA levels of IL-6 in the liver of the AG490 group rat at the same time points were also decreased, whereas IL-10 levels markedly increased. These changes made the ratio of IL-6/IL-10 dropped significantly. CONCLUSIONS: AG490 ameliorates the overwhelming inflammatory response via a mechanism of blocking cytokine signaling transduction and consequently suppresses the ratio of pro-/anti-inflammatory cytokine expression, which exerts potential clinical implications of use of anti-inflammatory agents in hepatic surgery. PMID: 15774164 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: J Immunol. 2005 Mar 15;174(6):3695-702. IL-22 inhibits epidermal differentiation and induces proinflammatory gene expression and migration of human keratinocytes. Boniface K, Bernard FX, Garcia M, Gurney AL, Lecron JC, Morel F. Laboratoire Cytokines et Inflammation, UPRES EA 3806, Pole Biologie Sante, Universite de Poitiers, Poitiers, France. IL-22 belongs to a family of cytokines structurally related to IL-10, including IL-19, IL-20, IL-24, and IL-26. In contrast to IL-10, IL-22 has proinflammatory activities. IL-22 signals through a class II cytokine receptor composed of an IL-22-binding chain, IL-22RA1, and the IL-10RB subunit, which is shared with the IL-10R. In the present study, we show that short-term cultured human epidermal keratinocytes express a functional IL-22R but no IL-10R. Accordingly, IL-22 but not IL-10 induces STAT3 activation in keratinocytes. Using a cDNA array screening approach, real-time RT-PCR, and Western blot analysis, we demonstrate that IL-22 up-regulates, in a dose-dependent manner, the expression of S100A7, S100A8, S100A9, a group of proinflammatory molecules belonging to the S100 family of calcium-binding proteins, as well as the matrix metalloproteinase 3, the platelet-derived growth factor A, and the CXCL5 chemokine. In addition, IL-22 induces keratinocyte migration in an in vitro injury model and down-regulates the expression of at least seven genes associated with keratinocyte differentiation. Finally, we show that IL-22 strongly induces hyperplasia of reconstituted human epidermis. Taken together, these results suggest that IL-22 plays an important role in skin inflammatory processes and wound healing. PMID: 15749908 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: J Immunol. 2005 Mar 15;174(6):3148-52. IL-10-independent STAT3 activation by Toxoplasma gondii mediates suppression of IL-12 and TNF-alpha in host macrophages. Butcher BA, Kim L, Panopoulos AD, Watowich SS, Murray PJ, Denkers EY. Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA. bab26@cornell.edu Infection of mouse macrophages by Toxoplasma gondii renders the cells resistant to proinflammatory effects of LPS triggering. In this study, we show that cell invasion is accompanied by rapid and sustained activation of host STAT3. Activation of STAT3 did not occur with soluble T. gondii extracts or heat-killed tachyzoites, demonstrating a requirement for live parasites. Parasite-induced STAT3 phosphorylation and suppression of LPS-triggered TNF-alpha and IL-12 was intact in IL-10-deficient macrophages, ruling out a role for this anti-inflammatory cytokine in the suppressive effects of T. gondii. Most importantly, Toxoplasma could not effectively suppress LPS-triggered TNF-alpha and IL-12 synthesis in STAT3-deficient macrophages. These results demonstrate that T. gondii exploits host STAT3 to prevent LPS-triggered IL-12 and TNF-alpha production, revealing for the first time a molecular mechanism underlying the parasite's suppressive effect on macrophage proinflammatory cytokine production. PMID: 15749841 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: J Neurochem. 2005 Feb;92(3):505-18. AG490 prevents cell death after exposure of rat astrocytes to hydrogen peroxide or proinflammatory cytokines: involvement of the Jak2/STAT pathway. Gorina R, Petegnief V, Chamorro A, Planas AM. Departament de Farmacologia i Toxicologia, IIBB-CSIC, IDIBAPS, Rossello 161, Planta 6, 08036 Barcelona, Spain. Janus kinases/STAT pathway mediates cellular responses to certain oxidative stress stimuli and cytokines. Here we examine the activation of Stat1 and Stat3 in rat astrocyte cultures and its involvement in cell death. H(2)O(2), interferon (INF)-gamma and interleukin (IL)-6 but not IL-10 caused cell death. Stat1 was phosphorylated on tyrosine (Tyr)-701 after exposure to H(2)O(2), INF-gamma or IL-6 but not IL-10. Tyr-705 pStat3 was observed after H(2)O(2), IL-6 and IL-10. Also, H(2)O(2) induced serine (Ser)-727 phosphorylation of Stat1 but not Stat3. The degree of Tyr-701 pStat1 by the different treatments positively correlated with the corresponding reduction of cell viability. AG490, a Jak2 inhibitor, prevented Tyr-701 but not Ser-727, Stat1 phosphorylation. Also, AG490 inhibited Tyr-705 Stat3 phosphorylation induced by H(2)O(2) and IL-6 but did not prevent that induced by IL-10. Furthermore, AG490 conferred strong protection against cell death induced by INF-gamma, IL-6 and H(2)O(2). These results suggest that Jak2/Stat1 activation mediates cell death induced by proinflammatory cytokines and peroxides. However, we found evidence suggesting that AG490 reduces oxidative stress induced by H(2)O(2), which further shows that H(2)O(2) and/or derived reactive oxygen species directly activate Jak2/Stat1, but masks the actual involvement of this pathway in H(2)O(2)-induced cell death. PMID: 15659221 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 15: J Immunol. 2005 Jan 1;174(1):310-9. Erratum in: J Immunol. 2005Apr 15;174(8):5133. O'Neill, Emma J [added]. IL-2 overcomes the unresponsiveness but fails to reverse the regulatory function of antigen-induced T regulatory cells. Anderson PO, Sundstedt A, Yazici Z, Minaee S, O'Neill EJ, Woolf R, Nicolson K, Whitley N, Li L, Li S, Wraith DC, Wang P. Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, London EC1A 7ED, United Kingdom. Intranasal administration of peptide Ac1-9[4Y], based on the N-terminal epitope of myelin basic protein, can induce CD4(+) T cell tolerance, and suppress experimental autoimmune encephalomyelitis induction. The peptide-induced regulatory T (PI-T(Reg)) cells failed to produce IL-2, but expressed IL-10 in response to Ag and could suppress naive T cell responses in vitro. Analysis of Jak-STAT signaling pathways revealed that the activation of Jak1, STAT3, and STAT5 were induced in tolerant T cells after Ag stimulation in vivo. In addition, the expression of suppressor of cytokine signaling 3 was induced in tolerant T cells, suggesting that cytokines regulate the tolerant state of the PI-T(Reg) cells. Stimulation of PI-T(Reg) cells in vitro with IL-10 induced Jak1 and STAT3 activation, but not STAT5, suggesting that IL-10 is important, but not the only cytokine involved in the development of T cell tolerance. Although IL-2 expression was deficient, stimulation with IL-2 in vitro induced Jak1 and STAT5 activation in PI-T(Reg) cells, restored their proliferative response to antigenic stimulation, and abrogated PI-T(Reg)-mediated suppression in vitro. However, the addition of IL-2 could not suppress IL-10 expression, and the IL-2 gene remained inactive. After withdrawal of IL-2, the PI-T(Reg) cells regained their nonproliferative state and suppressive ability. These results underline the ability of the immune system to maintain tolerance to autoantigens, but at the same time having the ability to overcome the suppressive phenotype of tolerant T cells by cytokines, such as IL-2, during the protective immune response to infection. PMID: 15611254 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 16: Mol Ther. 2004 Dec;10(6):1085-95. Bystander activity of Ad-mda7: human MDA-7 protein kills melanoma cells via an IL-20 receptor-dependent but STAT3-independent mechanism. Chada S, Mhashilkar AM, Ramesh R, Mumm JB, Sutton RB, Bocangel D, Zheng M, Grimm EA, Ekmekcioglu S. Introgen Therapeutics, Inc., Houston, TX 77030, USA. s.chada@introgen.com The melanoma differentiation-associated gene-7 (mda-7/IL24) is a unique member of the IL-10 family of cytokines, with ubiquitous tumor cell proapoptotic activity. Transduction of tumor or normal cells with the mda-7 gene results in secretion of glycosylated MDA-7 protein. Recent data indicate that secreted MDA-7 protein functions as a pro-Th1 cytokine and as a potent antiangiogenic molecule. MDA-7 protein binds two distinct type II cytokine heterodimeric receptor complexes, IL-20R1/IL-20R2 (type 1 IL-20R) and IL-22R1/IL-20R2 (type 2 IL-20R). In this study we analyzed the activity of glycosylated secreted MDA-7 against human melanoma cells. MDA-7 protein induces phosphorylation and nuclear translocation of STAT3 in melanoma cells via both type 1 and type 2 IL-20R. MDA-7 induces dose-dependent cell death in melanoma tumor cells. MDA-7 receptor engagement results in up-regulation of BAX and subsequent apoptosis induction; this effect is mediated by STAT3-independent signaling. Additional IL-10 family members (IL-10, -19, -20, and -22) also activate STAT3; however, these ligands do not activate death pathways in melanoma. In normal cells, MDA-7 can bind to its cognate receptors and induce phosphorylation of STAT3, without cytotoxic sequelae. This study defines a tumor-selective cytotoxic bystander role for secreted MDA-7 protein and identifies a novel receptor-mediated, STAT3-independent, and PKR-independent death pathway. PMID: 15564140 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 17: Immunology. 2004 Nov;113(3):281-92. Interleukin-10 suppression of myeloid cell activation--a continuing puzzle. Williams LM, Ricchetti G, Sarma U, Smallie T, Foxwell BM. The Kennedy Institute of Rheumatology Division, Imperial College, London, UK. b.foxwell@ic.ac.uk Efforts to identify the signal transduction pathways used by interleukin-10 (IL-10) have resulted in limited success. The anti-inflammatory effects elicited by IL-10, and the mechanisms by which these are mediated, are still relatively unknown. Understanding the signalling mechanisms behind the suppression of cytokine expression by IL-10 could be of potential therapeutic interest. Although the consensus is that the Janus kinase, Jak1, as well as the signal transducer and activator of transcription STAT3 are central, much controversy exists about the participation and roles of many other signalling pathways targeted by IL-10. The mechanisms of cytokine suppression proposed by various groups have included transcriptional, post-transcriptional and post-translational regulation of IL-10 target genes; nevertheless no unifying model has emerged thus far. Here we would like to highlight novel findings and discuss their implications in the context of current understanding of IL-10 signalling. Publication Types: Review Review, Tutorial PMID: 15500614 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 18: J Dermatol Sci. 2004 Oct;36(1):51-6. Hapten-induced contact hypersensitivity is enhanced in Tyk2-deficient mice. Hosogi M, Tonogaito H, Aioi A, Hamada K, Shimoda K, Muromoto R, Matsuda T, Miyachi Y. Biochemistry Laboratory, Pias Corporation, 1-3-1 Murotani, Nishi-ku, Kobe 651-2241, Japan. miwahoso@kuhp.kyoto-u.ac.jp BACKGROUND: Previous studies have shown that Tyk2, a member of the Janus family of protein tyrosine kinases, which are activated by a variety of cytokines, plays a crucial role in interleukin (IL)-12-mediated T-cell functions such as IFN-gamma production. On the other hand, hapten-induced contact hypersensitivity (CHS) is mediated by IFN-gamma producing CD8+ T cells and regulated by CD4+ T cells. OBJECTIVE: This study hypothesized that the CHS response might be reduced in Tyk2-deficient mice because of a lack of IFN-gamma production from CD4+ and CD8+ T cells. METHODS: The CHS reaction was evoked in wild-type and Tyk2-deficient mice and the ears of the mice were examined to measure for several cytokines. RESULTS: Ear swelling during CHS was significantly enhanced in Tyk2-deficient mice compared with the controls. IL-12 and IFN-gamma levels at the reaction sites in Tyk2-deficient mice were significantly lower than in the controls, whereas IL-2 and IL-4 levels were elevated. Furthermore, STAT3- and STAT4-phosphorylation in the draining lymph node cells of Tyk2-deficient mice decreased. CONCLUSION: These results suggest that the lack of Tyk2-mediated signal transduction enhances a compensative pathway during CHS. PMID: 15488705 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 19: J Biol Chem. 2004 Dec 24;279(52):54358-68. Epub 2004 Oct 13. Interleukin-10 induces uteroglobin-related protein (UGRP) 1 gene expression in lung epithelial cells through homeodomain transcription factor T/EBP/NKX2.1. Srisodsai A, Kurotani R, Chiba Y, Sheikh F, Young HA, Donnelly RP, Kimura S. Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. UGRP1 is a downstream target gene for homeodomain transcription factor T/EBP/NKX2.1, which is predominantly expressed in lung epithelial cells, and may play an anti-inflammatory role in lung inflammation. To understand the role of UGRP1 in inflammation, its expression was investigated in relation to cytokine signaling. In vivo experiments using mouse embryonic lung organ culture and intranasal administration of interleukin (IL) 10 revealed that constitutive expression of Ugrp1 mRNA is enhanced by IL-10. Increase of protein levels was also demonstrated by immunohistochemistry using embryonic lungs. This IL-10 induction of Ugrp1 gene expression occurs at the transcriptional level when examined using mouse embryonic lung primary cultures. In human lung NCI-H441 cells that in contrast to mouse lung cells, do not exhibit constitutive expression of the gene, expression of the UGRP1 gene was induced in a rapid and stable fashion. Two T/EBP, but not STAT3, binding sites located in the human UGRP1 gene promoter are responsible for IL-10 induction of the UGRP1 gene as judged by transfection, gel shift, and chromatin immunoprecipitation analyses. The IL-10 receptor chains, IL-10R1 and IL-10R2, are expressed in H441 cells, however, STAT3 was only weakly activated upon IL-10 treatment. In contrast, STAT3 was strongly activated when the cells were treated with other cytokines such as IL-22 and interferon-beta but UGRP1 expression was not increased. Together these results demonstrate that IL-10 induces UGRP1 gene expression in lung epithelial cells through a T/EBP/NKX2.1-dependent pathway. The results further suggest that UGRP1 might be a target for IL-10 anti-inflammatory activities in the lung. PMID: 15485815 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 20: J Biol Chem. 2004 Dec 3;279(49):51697-703. Epub 2004 Oct 1. IRAK1 serves as a novel regulator essential for lipopolysaccharide-induced interleukin-10 gene expression. Huang Y, Li T, Sane DC, Li L. Department of Medicine, Wake Forest University School of Medicine, Winston Salem, North Carolina 27157, USA. Being one of the key kinases downstream of Toll-like receptors, IRAK1 has initially thought to be responsible for NFkappaB activation. Yet IRAK1 knock-out mice still exhibit NFkappaB activation upon lipopolysaccharide (LPS) challenge, suggesting that IRAK1 may play other un-characterized function. In this report, we show that IRAK1 is mainly involved in Stat3 activation and subsequent interleukin-10 (IL-10) gene expression. Splenocytes from IRAK1-deficient mice fail to exhibit LPS-induced Stat3 serine phosphorylation and IL-10 gene expression yet still maintain normal IL-1beta gene expression upon LPS challenge. Mechanistically, we observe that IRAK1 modification upon LPS challenge leads to its modification, nuclear distribution, and interaction with Stat3. IRAK1 can directly use Stat3 as a substrate and cause Stat3 serine 727 phosphorylation. In addition, nuclear IRAK1 binds directly with IL-10 promoter in vivo upon LPS treatment. Atherosclerosis patients usually have elevated serum IL-10 levels. We document here that IRAK1 is constitutively modified and localized in the nucleus in the peripheral blood mononuclear cells from atherosclerosis patients. These observations reveal the mechanism for the novel role of IRAK1 in the complex Toll-like receptor signaling network and indicate that IRAK1 regulation may be intimately linked with the pathogenesis and/or resolution of atherosclerosis. PMID: 15465816 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 21: Shock. 2004 Oct;22(4):333-9. Impaired induction of IL-10 expression in the lung following hemorrhagic shock. Khadaroo RG, Fan J, Powers KA, Fann B, Kapus A, Rotstein OD. Departments of Surgery, University Health Network and University of Toronto, Toronto, Ontario, Canada. The balance between pro- and anti-inflammatory cytokines is considered to be an important determinant of the magnitude of inflammation in a number of disease states. We previously showed that resuscitated hemorrhagic shock augmented LPS-induced release of proinflammatory molecules by alveolar macrophages (AM). In the present studies, we evaluated the expression and regulation of the counter inflammatory cytokine IL-10 in the lung using this model. We hypothesized that impaired up-regulation of IL-10 in shock/resuscitated animals might serve as a mechanism contributing to accentuated lung inflammation. In a rodent model, animals exposed to LPS alone exhibited enhanced IL-10 mRNA levels in lung tissue as well as in AM, but antecedent shock/resuscitation delayed and attenuated the LPS-induced IL-10 mRNA levels. The ability of shock to attenuate LPS-stimulated IL-10 was also seen in the protein levels. This effect correlated with an augmented expression of cytokine-induced neutrophil chemoattractant (CINC) mRNA. Shock/resuscitated animals given exogenous IL-10 had reduced proinflammatory response, as shown by decreased expression of CINC mRNA and decreased neutrophil sequestration in the lung. Shock/resuscitation plus LPS markedly reduced the transcription rate of IL-10 mRNA compared to LPS alone but did not affect IL-10 mRNA stability. Reduced IL-10 transcription was not caused solely by impaired nuclear translocation of STAT3 and Sp1/Sp3 transcription factors because LPS-induced nuclear translocation of these factors was augmented by antecedent shock. Considered together, these findings show that shock/resuscitation suppresses LPS-induced IL-10 expression by AM in the lung by inhibiting IL-10 gene transcription. Failed up-regulation of counter inflammatory cytokines may contribute to augmented organ dysfunction in trauma patients. PMID: 15377888 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 22: Biochem Biophys Res Commun. 2004 Sep 3;321(4):828-34. STAT3 activation in macrophages following infection with Salmonella. Lin T, Bost KL. Department of Biology, University of North Carolina at Charlotte, Charlotte, NC 28223, USA. The induction of signal transducer and activators of transcription (STATs) in macrophages is necessary for cellular activation, and we investigated the activation of STAT3 in these cells following infection with Salmonella. Increased activation of STAT3 was observed at 6 and 24 h post-infection in the mesenteric lymph nodes and spleens when compared to control mice. CD11b+ cells isolated from the mesenteric lymph nodes of infected mice demonstrated increased STAT3 activation as early as 6 h following infection. Culturing bone marrow-derived macrophages with Salmonella resulted in translocation of STAT3 to the nucleus and STAT3 phosphorylation as early as 30 min post-exposure. Increased STAT3 activation was also observed in the lymphoid organs or in macrophages from mice deficient for IL-6 or IL-10 production following infection. Taken together, these studies clearly demonstrate an early increase in the activation of STAT3 in vivo and in vitro following infection with wild type Salmonella. PMID: 15358102 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 23: J Immunol. 2004 Sep 1;173(5):3383-91. Shaping phenotype, function, and survival of dendritic cells by cytomegalovirus-encoded IL-10. Raftery MJ, Wieland D, Gronewald S, Kraus AA, Giese T, Schonrich G. Institute of Virology, Charite Medical School, Berlin, Germany. Human dendritic cells (DCs) are essential for the antiviral immune response and represent a strategically important target for immune evasion of viruses, including human CMV (HCMV). Recently, HCMV has been discovered to encode a unique IL-10 homologue (cmvIL-10). In this study we investigated the capacity of cmvIL-10 to shape phenotype, function, and survival of DCs. For comparison we included human IL-10 and another IL-10 homologue encoded by EBV, which does not directly target DCs. Interestingly, cmvIL-10 strongly activated STAT3 in immature DCs despite its low sequence identity with human IL-10. For most molecules cmvIL-10 blocked LPS-induced surface up-regulation, confirming its role as an inhibitor of maturation. However, a small number of molecules on LPS-treated DCs including IDO, a proposed tolerogenic molecule, showed a different behavior and were up-regulated in response to cmvIL-10. Intriguingly, the expression of C-type lectin DC-specific ICAM-grabbing nonintegrin, a receptor for HCMV infection found exclusively on DCs, was also enhanced by cmvIL-10. This phenotypic change was mirrored by the efficiency of HCMV infection. Moreover, DCs stimulated with LPS and simultaneously treated with cmvIL-10 retained the function of immature DCs. Finally, cmvIL-10 increased apoptosis associated with DC maturation by blocking up-regulation of the antiapoptotic long form cellular FLIP. Taken together, these findings show potential mechanisms by which cmvIL-10 could assist HCMV to infect DCs and to impair DC function and survival. PMID: 15322202 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 24: Blood. 2005 Jan 15;105(2):689-96. Epub 2004 Jul 13. STAT3 regulates NF-kappaB recruitment to the IL-12p40 promoter in dendritic cells. Hoentjen F, Sartor RB, Ozaki M, Jobin C. Center for Gastrointestinal Biology and Diseases, University of North Carolina at Chapel Hill, USA. Interleukin-10-deficient (IL-10(-/-)) mice develop an IL-12-mediated intestinal inflammation in the absence of endogenous IL-10. The molecular mechanisms of the dysregulated IL-12 responses in IL-10(-/-) mice are poorly understood. In this study, we investigated the role of nuclear factor-kappa B (NF-kappaB) and signal transducers and activators of transcription 3 (STAT3) in lipopolysaccharide (LPS)-induced IL-12p40 gene expression in bone marrow derived-dendritic cells (BMDCs) isolated from wild-type (WT) and IL-10(-/-) mice. We report higher IL-12p40 mRNA accumulation and protein secretion in LPS-stimulated BMDCs isolated from IL-10(-/-) compared with WT mice. LPS-induced NF-kappaB signaling is similar in IL-10(-/-) and WT BMDCs as measured by IkappaBalpha phosphorylation and degradation, RelA phosphorylation and nuclear translocation, and NF-kappaB transcriptional activity, with no down-regulatory effects of exogenous IL-10. Chromatin immunoprecipitation demonstrated enhanced NF-kappaB (cRel, RelA) binding to the IL-12p40 promoter in IL-10(-/-) but not WT BMDCs. Interestingly, LPS induced STAT3 phosphorylation in WT but not IL-10(-/-) BMDCs, a process blocked by IL-10 receptor blocking antibody. Adenoviral gene delivery of a constitutively active STAT3 but not control green fluorescence protein (GFP) virus blocked LPS-induced IL-12p40 gene expression and cRel recruitment to the IL-12p40 promoter. In conclusion, dysregulated LPS-induced IL-12p40 gene expression in IL-10(-/-) mice is due to enhanced NF-kappaB recruitment to the IL-12p40 promoter in the absence of activated STAT3. PMID: 15251981 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 25: J Leukoc Biol. 2004 Sep;76(3):719-26. Epub 2004 Jul 7. Heme oxygenase 1 expression induced by IL-10 requires STAT-3 and phosphoinositol-3 kinase and is inhibited by lipopolysaccharide. Ricchetti GA, Williams LM, Foxwell BM. Kennedy Institute of Rheumatology Division, Imperial College London, Hammersmith, UK. Heme-oxygenase 1 (HO-1) is a stress-response protein with anti-inflammatory activity. This study has examined the regulation of HO-1 expression by the anti-inflammatory factor, interleukin (IL)-10 and whether HO-1 could account for the function of the cytokine. IL-10-induced expression of HO-1 required the activation of signal transducer and activator of transcription (STAT)-3 but not p38 mitogen-activated protein kinase. However, expression of HO-1 also required the activation of the phosphatidylinositol-3 kinase pathway, a signaling mechanism not required for the anti-inflammatory activity of IL-10. Moreover, induction of HO-1 expression was not restricted to IL-10, as IL-6, a cytokine known to activate STAT-3, could also induce the protein. In human macrophages, lipopolysaccharide inhibited HO-1 expression induced by IL-10. Also, inhibition of HO-1 activity by the specific inhibitor zinc-II-protoporphyrin-IX had no effect on the anti-inflammatory function of IL-10. In summary, although IL-10 does regulate HO-1 expression, it does not appear to play a significant role in the anti-inflammatory activity of the cytokine. PMID: 15240748 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 26: J Leukoc Biol. 2004 Sep;76(3):735-42. Epub 2004 Jun 24. Role of endogenous IL-10 in LPS-induced STAT3 activation and IL-1 receptor antagonist gene expression. Carl VS, Gautam JK, Comeau LD, Smith MF Jr. University of Virginia Health System, Department of Medicine, Digestive Health Center of Excellence and Microbiology, Charlottesville, VA 22908-0708, USA. The regulation of secretory interleukin (IL)-1 receptor antagonist (sIL-1Ra) in response to IL-10 is unique. In contrast to most cytokines, the lipopolysaccharide (LPS)-induced expression of the sIL-1Ra gene is enhanced by concomitant treatment with IL-10. Cotreatment of RAW 264.7 cells with IL-10 + LPS resulted in at least a twofold increase in sIL-1Ra promoter activity and mRNA expression compared with LPS alone; IL-10 alone had no effect on promoter activity or mRNA expression. Examination of sIL-1Ra mRNA expression in bone marrow-derived macrophages (BMDM) resulted in identical results. Transfection of RAW 264.7 cells with the sIL-1Ra/luc reporter and a dominant-negative signal transducer and activator of transcription (STAT)3 (Y705A) expression plasmid inhibited the enhanced response induced by exogenous IL-10 in the presence of LPS. The presence of a functional STAT3-binding site within the proximal sIL-1Ra promoter was demonstrated. As IL-10 is produced by LPS-stimulated macrophages, a role for endogenously produced IL-10 in the response of the sIL-1Ra gene to LPS was suggested. This was confirmed in IL-10-deficient BMDM, which when compared with normal BMDM, had significantly decreased LPS-induced sIL-1Ra mRNA levels that could be restored by exogenously provided IL-10, which induced a fivefold increase of LPS-induced IL-1Ra mRNA in cells from IL-10-/- BMDM. Western blot analysis of phosphorylated STAT3 from wild-type and IL-10-/- BMDM and IL-10 neutralization experiments demonstrated a role for endogenously produced IL-10 in the LPS-induced STAT3 activity. Together, these results demonstrate that endogenously produced IL-10 plays a significant role in LPS-induced sIL-1Ra gene expression via the activation of STAT3. PMID: 15218058 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 27: J Gen Virol. 2004 Jun;85(Pt 6):1401-12. A new member of the interleukin 10-related cytokine family encoded by a poxvirus. Bartlett NW, Dumoutier L, Renauld JC, Kotenko SV, McVey CE, Lee HJ, Smith GL. Department of Virology, Faculty of Medicine, Imperial College London, St Mary's Campus, Norfolk Place, London W2 1PG, UK. Poxviruses express numerous proteins involved in manipulating the host immune response. Analysis of the primary sequence and predicted structure of the 134R protein of Yaba-like disease virus (Y134R) indicated that it is similar to cellular proteins of the IL-10 family, specifically IL-19, IL-20 and IL-24. A flag-tagged Y134R was expressed from mammalian cells and identified as a secreted, monomeric glycoprotein that stimulated signal transduction from class II cytokine receptors IL-20Ralpha/IL-20Rbeta (IL-20R type1) and IL-22R/IL-20Rbeta (IL-20R type 2). Y134R induced phosphorylation of signal transducers and activators of transcription, their translocation to the nucleus and the induction of reporter gene expression. In contrast, Y134R was unable to induce similar responses from either the IL-22 or IFN-lambda (IL-28A, IL-28B, IL-29) class II cytokine receptors. To examine the role Y134R plays during a poxvirus infection, a vaccinia virus recombinant expressing Y134R was constructed and tested in a murine intranasal infection model. Compared with control viruses, the virus expressing Y134R had a reduced virulence, manifested by reduced weight loss, signs of illness and virus titres in infected organs. These results demonstrate that Y134R is a new viral member of the IL-10-related cytokine family and that its activity in vivo affects virus virulence. PMID: 15166422 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 28: Biochim Biophys Acta. 2004 May 24;1689(1):22-32. The role of interleukin-10 in the regulation of the systemic inflammatory response following trauma-hemorrhage. Schneider CP, Schwacha MG, Chaudry IH. Department of Surgery, University of Alabama at Birmingham, G094 Volker Hall, 1670 University Blvd., Birmingham, AL 35294-0019, USA. Pro-inflammatory cytokine release after shock is central in the development of subsequent multiple organ dysfunction syndrome. Some studies suggest that interleukin-10 (IL-10) is an immunosuppressive mediator after injury or sepsis, while others suggest that IL-10 is an important regulator of the pro-inflammatory response. We hypothesized that in a model of trauma and hemorrhagic shock (TH), IL-10 regulates pro-inflammatory cytokine activity via an autocrine effect on cytokine mRNA transcription in Kupffer cells early after TH. To study this, male C3H/HeN mice were sham-operated or subjected to TH. Plasma levels of TNF-alpha, IL-6 and PGE(2) were elevated following TH. A sharp peak in IL-10 levels was observed at 2 h after the insult. Kupffer cell (KC) depletion prior to TH reduced plasma IL-6, IL-10 and TNF-alpha levels, whereas treatment with anti-IL-10 after TH increased IL-6 and TNF-alpha levels. Kupffer cell mRNA expression for IL-6, IL-10 and TNF-alpha was elevated in the TH group and further increased by anti-IL-10 treatment. These findings indicate that KC-dependent IL-10 regulates the early systemic inflammatory response after TH. Thus, while IL-10 is an important mediator of immunosuppression following traumatic injury, it also is beneficial with regard to its ability to counter-regulate the early inflammatory response under such conditions. PMID: 15158910 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 29: Int Immunopharmacol. 2004 May;4(5):679-91. Interleukin-22 activates STAT3 and induces IL-10 by colon epithelial cells. Nagalakshmi ML, Rascle A, Zurawski S, Menon S, de Waal Malefyt R. Department of Experimental Pathology and Pharmacology, DNAX Research Inc., 901 California Avenue, Palo Alto, CA 94304-1104, USA. Human interleukin-22 (IL-22), a cytokine with structural homology to IL-10, is produced by activated T cells. The IL-22 receptor complex consists of a ligand-binding chain, the IL-22R1 and a signal-transducing chain, the IL-10R2. The aim of this study is to identify potential target cells and associated biological activity of IL-22 by identifying cell types that specifically express high levels of IL-22R1 as the expression of IL-10R2 is ubiquitous. Expression of IL-22R1 mRNA, as analyzed by real time quantitative polymerase chain reaction (PCR), was observed in human tumor cell lines of stromal or epithelial origin derived from liver, pancreas, colon and lung tissue. Furthermore, we examined the ability of IL-22 to activate the JAK-Signal Transducer and Activator of Transcription (STAT) pathway in epithelial cells of the colon. IL-22 induced the phosphorylation of STAT1 and STAT3 in Colo205, a colon epithelial cell line. Consequently, IL-22 upregulated mRNA for Suppressor of Cytokine Signaling 3 (SOCS3), a STAT3-responsive gene. Further analyses, by real time quantitative PCR, on a panel of chemokines and immune function related genes revealed that IL-22 induced expression of the acute phase proteins alpha-Antichymotrypsin and Serum Amyloid A, as well as IL-10 mRNA and protein production by Colo205. Induction of IL-10 by IL-22, in Colo205 cells, could be inhibited in the presence of a neutralizing antibody against IL-10R2. IL-22-mediated effects on the Colo205 cells were also inhibited in the presence of IL-22 binding protein (IL-22BP), a soluble receptor with structural similarity to IL-22R1. The high levels of expression of IL-22R1 observed in epithelial cells of the colon and the ability of IL-22 to upregulate production of acute phase proteins and IL-10 in Colo205 cells, suggest a functional role for IL-22 in intestinal inflammation. PMID: 15120652 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 30: Int Immunopharmacol. 2004 May;4(5):649-67. MDA-7/IL-24 is a unique cytokine--tumor suppressor in the IL-10 family. Chada S, Sutton RB, Ekmekcioglu S, Ellerhorst J, Mumm JB, Leitner WW, Yang HY, Sahin AA, Hunt KK, Fuson KL, Poindexter N, Roth JA, Ramesh R, Grimm EA, Mhashilkar AM. Introgen Therapeutics, Inc., 2250 Holcombe Blvd., Houston, TX 77030, USA. s.chada@introgen.com The melanoma differentiation associated gene-7 (mda-7) cDNA was isolated by virtue of being induced during melanoma differentiation. Initial gene transfer studies convincingly demonstrated potent antitumor effects of mda-7. Further studies showed that the mechanism of antitumor activity was due to induction of apoptosis. Most striking was the tumor-selective killing by mda-7 gene transfer--normal cells were unaffected by Adenoviral delivery of mda-7 (Ad-mda7). A variety of molecules implicated in apoptosis and intracellular signaling are regulated by Ad-mda7 transduction. Different apoptosis effector proteins are regulated in different tumor types, suggesting that Ad-mda7 may regulate various signaling pathways. mda-7 encodes a secreted protein, MDA-7, which has now been designated as IL-24, and is a novel member of the IL-10 cytokine family. MDA-7/IL-24 protein is actively secreted from cells after mda-7 gene transfer. In human peripheral blood mononuclear cells (PBMC), STAT3 activation by MDA-7/IL-24 is followed by elaboration of secondary Th1 cytokines, demonstrating that MDA-7/IL-24 is a pro-Th1 cytokine. Furthermore, MDA-7/IL-24 is antagonized by the prototypic Th2 cytokine IL-10. MDA-7/IL-24 protein is endogenously expressed in cultured NK and B-cells and is also expressed in dendritic cells in tissues. MDA-7/IL-24 protein is expressed in nevi and melanoma primary tumors, to varying degrees, but is rarely expressed in malignant melanoma or other human tumors evaluated. Indeed, loss of MDA-7/IL-24 protein expression correlates strongly with melanoma tumor invasion and disease progression. The "bystander" effects proposed for MDA-7/IL-24 protein include immune stimulation, antiangiogenesis and receptor-mediated cytotoxicity. Thus, mda-7 is a unique multifunctional cytokine in the IL-10 family and may have potent antitumor utility in a clinical setting. Publication Types: Review Review, Tutorial PMID: 15120650 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 31: Allergy. 2004 May;59(5):505-14. Differential expression of IL-10 receptor by epithelial cells and alveolar macrophages. Lim S, Caramori G, Tomita K, Jazrawi E, Oates T, Chung KF, Barnes PJ, Adcock IM. Department of Thoracic Medicine, Imperial College School of Medicine, National Heart and Lung Institute, Dovehouse Street, London, UK. BACKGROUND: Interleukin (IL)-10 is a pleiotropic cytokine with a broad spectrum of immunosuppressive and anti-inflammatory effects. IL-10 secretion from alveolar macrophages is defective in patients with asthma and lower concentrations of IL-10 are found in bronchoalveolar lavage (BAL) from asthmatic patients than in normal control subjects. Reduced IL-10 may result in exaggerated and more prolonged inflammatory responses in asthmatic airways. IL-10 acting through the IL-10 receptor (IL-10R) stimulates the transcription factors STAT1 and STAT3. METHODS: We investigated IL-10 and IL-10R expression in normal and asthmatic bronchial epithelium and BAL macrophages using reverse transcription-polymerase chain reaction, immunohistochemistry and Western blotting. The functional effect of IL-10 was examined using granulocyte-macrophage-colony stimulating factor, enzyme-linked immunosorbent assay and Western blotting for phosphorylated STAT1 and STAT3. RESULTS: IL-10 was not expressed in epithelial cells; furthermore these cells did not express the IL-10R and had no functional response to exogenous IL-10. Bronchial epithelial cells expressed variable levels of phosphorylated STAT1 and STAT3 with no change in expression between normal subjects and asthmatics. IL-10 protein and IL-10R expression was detected in alveolar macrophages from all subjects. CONCLUSION: Our study suggests that the bronchial epithelium is not a source of IL-10 and cannot respond to exogenous IL-10 because of a lack of IL-10R expression. PMID: 15080831 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 32: Oncogene. 2004 Apr 29;23(20):3530-40. Rituximab inhibits p38 MAPK activity in 2F7 B NHL and decreases IL-10 transcription: pivotal role of p38 MAPK in drug resistance. Vega MI, Huerta-Yepaz S, Garban H, Jazirehi A, Emmanouilides C, Bonavida B. Department of Microbiology, Immunology, and Molecular Genetics, Jonsson Comprehensive Cancer Center, University of California, 10833 Le Conte Ave. A2-060 CHS, Los Angeles, CA 90095, USA. We have recently reported that Rituximab (anti-CD20) sensitizes drug-resistant 2F7 and 10C9 B Non-Hodgkin's lymphoma (NHL) cell lines to the apoptotic effects of various chemotherapeutic drugs by downregulation of IL-10 and Bcl-2 expression. The mechanism by which Rituximab induces downregulation of IL-10 was examined. We hypothesized that Rituximab may inhibit p38 MAPK activity that regulates IL-10 expression via Sp1. Treatment of 2F7 cells with Rituximab or the p38 inhibitor SB203580 inhibited the constitutive p38 MAPK activity and resulted in the inhibition of Sp1, IL-10, STAT3, and Bcl-2. Inhibition of the Src-family PTKs, Lyn, and Src-family PTKs upstream signaling molecules of the p38MAPK pathway, by PP2, a specific Src-family kinase inhibitor, resulted in the inhibition of p38MAPK and IL-10 expression. In addition to p38 MAPK, Rituximab also inhibited NF-kappaB activity. Inhibition of the Src PTKs, MAPK, and NF-kappaB activities by Rituximab or by specific chemical inhibitors sensitized the cells to CDDP-mediated apoptosis. The above signaling-mediated effects by Rituximab were observed with similar kinetics beginning at 1 h following treatment. Thus, altogether, these results demonstrate that signaling by Rituximab results in the inhibition of the p38MAPK pathway, which in turn inhibits the transcription of IL-10 via Sp1. Inhibition of the IL-10 autocrine/paracrine loop results in the inhibition of STAT3 activity and, consequently, inhibition of Bcl-2 expression and sensitization to drugs-apoptosis. Further, Rituximab-mediated signaling identifies several new intracellular targets in NHL that may be of potential therapeutic interest for the development of new drugs in the treatment of drug-refractory NHL tumor cells. PMID: 15077178 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 33: J Immunol. 2004 Apr 1;172(7):4630-6. Recruitment of STAT3 for production of IL-10 by colon carcinoma cells induced by macrophage-derived IL-6. Herbeuval JP, Lelievre E, Lambert C, Dy M, Genin C. Groupe Immunite des Muqueuses et Agents Pathogenes, University of Saint Etienne, Saint Etienne, France. herbeuvj@mail.nih.gov The immunosuppressive cytokine IL-10 is associated with poor prognosis in colon cancer. Although macrophages are involved in antitumor defenses, production of IL-10 by tumor cells may permit malignant cells escape to cell-mediated immune defenses. To investigate interactions between macrophages and tumor cells in humans, we cultured macrophages isolated from patients and tested the effect of these macrophages on the production of IL-10 by several tumor cell lines. Macrophages were isolated from pleural effusions of patients with malignancy and from noncancer control patients. We demonstrated that culture supernatants of macrophages from both sources strongly stimulated IL-10 production by the three different human colon adenocarcinoma cell lines, Colo 205, Colo 320, and HT29. Recombinant IL-6, but not IL-10, TNF-alpha, and IFN-alpha, stimulated the secretion of IL-10 by colon tumor cells. mAbs against IL-6 and IL-6R prevented the effect of macrophage culture supernatants and of rIL-6, respectively, on the production of IL-10 by the three cell lines. Cocultures of macrophages and colon cancer cells showed that these tumor cells first stimulated macrophages to produce IL-6, which was then followed by IL-6-induced IL-10 production by colon cancer cells. Finally, we showed that IL-10 gene regulation was mediated by STAT3, which was phosphorylated after the binding of IL-6 to IL-6R. This is the first demonstration that IL-6, secreted by macrophages, can induce a STAT3-mediated IL-10 production by colon tumor cells. PMID: 15034082 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 34: J Biol Chem. 2004 Jun 4;279(23):24724-32. Epub 2004 Mar 4. Inhibition of interleukin-10 by the immunomodulator AS101 reduces mesangial cell proliferation in experimental mesangioproliferative glomerulonephritis: association with dephosphorylation of STAT3. Kalechman Y, Gafter U, Weinstein T, Chagnac A, Freidkin I, Tobar A, Albeck M, Sredni B. Cancer, AIDS, and Immunology Research Institute, Faculty of Life Sciences, Bar Ilan University, Ramat Gan 52900, Israel. Mesangial cell (MC) proliferation is essential for the pathogenesis and progression of glomerular disease. Using an acute model of mesangial proliferative glomerulonephritis (Thy1 GN), we show that neutralization of interleukin (IL)-10 greatly ameliorated the disease as expressed by both decreased MC expansion and proteinuria. Treatment with the tellurium compound AS101 (ammonium trichloro(dioxoethylene-o,o')tellurate) resulted in favorable effects provided that the compound was administered 24 h before insult, whereas partial effects were obtained when administered after insult. We identified STAT3 as playing a pivotal role in IL-10-induced MC proliferation in vitro and in vivo. IL-10 activates MC STAT3 in vitro as expressed by its phosphorylation and nuclear translocation. The role of STAT3 in MC proliferation induced by IL-10 was deduced from results showing that IL-10-induced proliferation was abrogated if MC transfected with STAT3 antisense oligonucleotides were used or if cells were incubated with inhibitors of STAT3. AS101 deactivates STAT3 in control but not in MC transfected with IL-10 antisense oligonucleotides. Inactivation of STAT3 prevents reduction of MC proliferation by AS101. We further demonstrate the role of STAT3 in the regulation of cell cycle and survival regulatory proteins by AS101 in MC via inhibition of IL-10. IL-10 increased MC expression of Bcl-2 and Bcl-X1 and simultaneously decreased the levels of p27kip1. These survival factors were decreased by AS101 in a STAT3- and IL-10-dependent manner, whereas p27kip1 was similarly increased. In Thy1 GN, phosphorylated STAT3 in glomerular MC peaked at day 6 and correlated with MC expansion. Neutralization of IL-10 or its inhibition by AS101 abolished phosphorylation of STAT3. This effect positively correlated with amelioration of the disease. These in vitro and in vivo studies indicate that the autocrine MC growth factor IL-10 induces MC proliferation via STAT3. We suggest that IL-10 or its downstream target STAT3 might be therapeutic targets for kidney diseases induced by mesangial proliferation. PMID: 15001575 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 35: Cancer Cell. 2004 Feb;5(2):111-2. Inactivation of Stat3 in tumor cells: releasing a brake on immune responses against cancer? Gamero AM, Young HA, Wiltrout RH. Laboratory of Experimental Immunology, National Cancer Institute, Center for Cancer Research, Frederick, MD 21702, USA. A model of immune evasion mediated by tumors expressing constitutively activated Stat3 was recently proposed in Nature Medicine by Wang et al., suggesting opportunities for a new therapeutic approach for cancer. Publication Types: Review Review, Tutorial PMID: 14998485 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 36: Cancer Res. 2004 Mar 1;64(5):1843-52. Ammonium trichloro(dioxoethylene-o,o')tellurate (AS101) sensitizes tumors to chemotherapy by inhibiting the tumor interleukin 10 autocrine loop. Sredni B, Weil M, Khomenok G, Lebenthal I, Teitz S, Mardor Y, Ram Z, Orenstein A, Kershenovich A, Michowiz S, Cohen YI, Rappaport ZH, Freidkin I, Albeck M, Longo DL, Kalechman Y. C.A.I.R. Institute, Faculty of Life Sciences, Bar Ilan University, Ramat Gan, Israel. srednib@mail.biu.ac.il Cancer cells of different solid and hematopoietic tumors express growth factors in respective stages of tumor progression, which by autocrine and paracrine effects enable them to grow autonomously. Here we show that the murine B16 melanoma cell line and two human primary cultures of stomach adenocarcinoma and glioblastoma multiforme (GBM) constitutively secrete interleukin (IL)-10 in an autocrine/paracrine manner. This cytokine is essential for tumor cell proliferation because its neutralization decreases clonogenicity of malignant cells, whereas addition of recombinant IL-10 increases cell proliferation. The immunomodulator ammonium trichloro(dioxoethylene-o,o')tellurate (AS101) decreased cell proliferation by inhibiting IL-10. This activity was abrogated by exogenous addition of recombinant IL-10. IL-10 inhibition by AS101 results in dephosphorylation of Stat3, followed by reduced expression of Bcl-2. Moreover, these activities of AS101 are associated with sensitization of tumor cells to chemotherapeutic drugs, resulting in their increased apoptosis. More importantly, AS101 sensitizes the human aggressive GBM tumor to paclitaxel both in vitro and in vivo by virtue of IL-10 inhibition. AS101 sensitizes GBM cells to paclitaxel at concentrations that do not affect tumor cells. This sensitization can also be obtained by transfection of GBM cells with IL-10 antisense oligonucleotides. Sensitization of GBM tumors to paclitaxel (Taxol) in vivo was obtained by either AS101 or by implantation of antisense IL-10-transfected cells. The results indicate that the IL-10 autocrine/paracrine loop plays an important role in the resistance of certain tumors to chemotherapeutic drugs. Therefore, anti-IL-10 treatment modalities with compounds such as AS101, combined with chemotherapy, may be effective in the treatment of certain malignancies. PMID: 14996748 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 37: J Immunol. 2004 Feb 15;172(4):2613-20. Inhibition of IL-10 receptor function in alveolar macrophages by Toll-like receptor agonists. Fernandez S, Jose P, Avdiushko MG, Kaplan AM, Cohen DA. Department of Microbiology, University of Kentucky Medical Center, Lexington, KY 40536, USA. Despite an immunosuppressive lung environment, alveolar macrophages (AM) retain the capacity to respond to microorganisms. This report demonstrates that IL-10, constitutively produced by normal alveolar epithelium, stimulates signal transduction through the IL-10R on AM and that IL-10R function can be inhibited by stimulation of Toll-like receptor (TLR) on AM. IL-10 mRNA and protein were constitutively expressed in normal alveolar epithelium of mice, and IL-10R were constitutively expressed on normal murine AM. Stimulation of AM through TLR2, TLR4, or TLR9 was sufficient to inhibit IL-10R signal transduction, including phosphorylation and nuclear translocation of STAT3 transcription factor. Inhibition of IL-10R function by TLRs was not associated with a decrease in IL-10R expression, but did require expression of the myeloid differentiation factor 88 adaptor protein. Continuous exposure of macrophages to IL-10 caused sustained expression of the chemokine receptors CCR1 and CCR5. However, the addition of TLR ligands inhibited IL-10-induced expression of CCR1 and CCR5. Finally, exposure of macrophages to TLR ligands blocked the ability of IL-10 to inhibit the induction of TNF-alpha by C2-ceramide. These findings demonstrate a novel regulatory mechanism that may allow AM to overcome inhibitory effects of constitutive IL-10 in the lungs that may permit a more effective response to pulmonary infections. PMID: 14764735 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 38: J Immunol. 2004 Feb 15;172(4):2006-10. Cutting edge: IL-26 signals through a novel receptor complex composed of IL-20 receptor 1 and IL-10 receptor 2. Sheikh F, Baurin VV, Lewis-Antes A, Shah NK, Smirnov SV, Anantha S, Dickensheets H, Dumoutier L, Renauld JC, Zdanov A, Donnelly RP, Kotenko SV. Division of Therapeutic Proteins, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA. The receptor for IL-26 (AK155), a cytokine of the IL-10 family, has not previously been defined. We demonstrate that the active receptor complex for IL-26 is a heterodimer composed of two receptor proteins: IL-20R1 and IL-10R2. Signaling through the IL-26R results in activation of STAT1 and STAT3 which can be blocked by neutralizing Abs against IL-20R1 or IL-10R2. IL-10R2 is broadly expressed on a wide variety of tissues, whereas only a limited number of tissues express IL-20R1. Therefore, the ability to respond to IL-26 is restricted by the expression of IL-20R1. IL-10, IL-19, IL-20, IL-22, and IL-24 fail to signal through the combination of IL-10R2 and IL-20R1 proteins, demonstrating that this receptor combination is unique and specific for IL-26. PMID: 14764663 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 39: Life Sci. 2004 Feb 20;74(14):1739-49. Ovarian carcinoma cells inhibit T cell proliferation: suppression of IL-2 receptor beta and gamma expression and their JAK-STAT signaling pathway. Wang H, Xie X, Lu WG, Ye DF, Chen HZ, Li X, Cheng Q. Womens Hospital, School of Medicine, Zhejiang University, Hangzhou, 310006, China. Deficient T cell immune function and intracellular signaling in cancer patients may result from effects of tumors or their products on lymphocytes. Recently, it was demonstrated that several ovarian carcinoma cell lines could produce soluble factors that inhibited T cell proliferation. The aim of this study is to assess the effect of supernatants from 3 ovarian carcinoma cell lines (OVCAR3, CAOV3, SKOV3) on signal transduction elements that are linked to the IL-2R and its JAK-STAT pathway. A profound inhibition of proliferation, lower level of IFN-gamma and higher level of IL-10 gene expression were observed when CD8+ T cells were co-cultured with supernatants from 3 ovarian carcinoma cell lines. Cell cycle studies on inhibited CD8+ T cells showed most of them were growth arrested in G0/G1 phase. Western blot analysis showed that tumor supernatants suppressed expression of JAK3 and tyrosine phosphorylation of STAT5. JAK1 was not altered and the inhibition of STAT3 only appeared in OVCAR3 cells. Tumor supernatants also partially blocked induction of IL-2R beta and gamma chains expression. These findings suggest that ovarian carcinoma cells may suppress T cell proliferation through inhibition IL-2 dependent signaling pathways, which may be a mechanism of ovarian carcinoma induced immunosuppression. PMID: 14741732 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 40: Am J Physiol Cell Physiol. 2004 Jun;286(6):C1302-11. Epub 2004 Jan 21. Selective activation of STAT3 in human monocytes stimulated by G-CSF: implication in inhibition of LPS-induced TNF-alpha production. Nishiki S, Hato F, Kamata N, Sakamoto E, Hasegawa T, Kimura-Eto A, Hino M, Kitagawa S. Department of Physiology, Osaka City University Medical School, Asahi-machi, Abeno-ku, Osaka 545-8585, Japan. Lipopolysaccharide (LPS) induced tumor necrosis factor (TNF)-alpha production in human monocytes, which was dependent on activation of extracellular signal-regulated kinase (ERK), p38, c-Jun NH(2)-terminal kinase (JNK), and nuclear factor (NF)-kappa B. LPS-induced TNF-alpha production was inhibited by granulocyte colony-stimulating factor (G-CSF) and interleukin (IL)-10. G-CSF, like IL-10, exerted the inhibitory effect even when simultaneously added with LPS. Among the signaling pathways, signal transducer and activator of transcription 3 (STAT3) was selectively activated in monocytes stimulated by G-CSF or IL-10. G-CSF-mediated inhibition of LPS-induced TNF-alpha production as well as G-CSF-induced STAT3 phosphorylation and suppressor of cytokine signaling 3 mRNA expression were prevented by pretreatment of monocytes with AG-490, an inhibitor of Janus kinase 2. G-CSF did not affect LPS-induced activation of ERK, p38, JNK, and NF-kappa B, indicating that G-CSF affects the pathway downstream or independently of these signaling molecules. G-CSF-induced, but not IL-10-induced, STAT3 phosphorylation was attenuated in the presence of LPS. These findings suggest that G-CSF, like IL-10, inhibits LPS-induced TNF-alpha production in human monocytes through selective activation of STAT3, and the immunomodulation observed in vivo by G-CSF administration may be partly ascribed to the direct effect of G-CSF on monocyte functions. PMID: 14736711 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 41: J Immunol. 2004 Jan 1;172(1):567-76. Signal transducer and activator of transcription 3 is the dominant mediator of the anti-inflammatory effects of IL-10 in human macrophages. Williams L, Bradley L, Smith A, Foxwell B. Kennedy Institute of Rheumatology Division, Imperial College London, ARC Building, 1 Aspenlea Road, Hammersmith, London W6 8LH, United Kingdom. The signaling mechanism by which the anti-inflammatory cytokine IL-10 mediates suppression of proinflammatory cytokine synthesis remains largely unknown. Macrophage-specific STAT3-null mice have demonstrated that STAT3 plays a critical role in the suppression of LPS-induced TNF-alpha release, although the mechanism by which STAT3 mediates this inhibition is still not clear. Using an adenoviral system, we have expressed a dominant negative (DN) STAT3 in human macrophages to broaden the investigation to determine the role of STAT3 in IL-10-mediated anti-inflammatory signaling and gene expression. Overexpression of STAT3 DN completely inhibited IL-10-induced suppressor of cytokine signaling 3, tissue inhibitor of MMP-1, TNF receptor expression, and the recently identified IL-10-inducible genes, T cell protein tyrosine phosphatase and signaling lymphocyte activation molecule. STAT3 DN also blocked IL-10-mediated inhibition of MHC class II and COX2 expression. In agreement with the studies in STAT3-null mice, overexpression of the STAT3 DN completely reversed the ability of IL-10 to inhibit LPS-mediated TNF-alpha and IL-6 production. However, real-time PCR analysis showed that STAT3 DN expression did not affect immediate suppression of TNF-alpha mRNA, but did reverse the suppression observed at later time points, suggesting a biphasic regulation of TNF-alpha mRNA levels by IL-10. In conclusion, although STAT3 does appear to be the dominant mediator of the majority of IL-10 functions, there are elements of its anti-inflammatory activity that are STAT3 independent. PMID: 14688368 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 42: Invest Ophthalmol Vis Sci. 2003 Dec;44(12):5206-11. Interleukin-10 receptor signaling through STAT-3 regulates the apoptosis of retinal ganglion cells in response to stress. Boyd ZS, Kriatchko A, Yang J, Agarwal N, Wax MB, Patil RV. Department of Ophthalmology, University of Nebraska Medical Center, 985145 Nebraska Medical Center, Omaha, NE 68198-5145, USA. PURPOSE: Interleukin (IL)-10 has recently been shown to promote survival of neurons and glia. The purpose of this report is to investigate whether IL-10 has any role in protecting retinal ganglion cells (RGCs) from death under conditions in which growth factors are removed, or in which oxidative stress is present. Signal transduction pathways that activate IL-10 signaling in RGCs were studied in both stress conditions. METHODS: Effects of various interleukins on the viability of the RGC cell line was determined, and apoptotic cells were quantified. Immunoblot analysis was preformed to identify the IL-10 receptor (IL-10R) and phosphorylated or nonphosphorylated Akt and STAT-3 proteins in RGC extracts. Immunohistochemistry was performed on the rat retinal sections to identify native IL-10R. RESULTS: Apoptosis of RGCs in the absence of growth factors with or without dexamethasone (1 microM) occurred in 68.5% +/- 3.4% and 53.4% +/- 2.6% of cells, respectively, after 96 hours. Addition of IL-10 at a concentration of 50 ng/mL significantly reduced the apoptotic population of RGCs to 28.2% +/- 2.3% in the absence of growth factors with dexamethasone and to 31% +/- 2.7% in the absence of growth factors alone. RGCs as well as native retina expressed functional IL-10R as determined by immunoblot analysis and by the ability of IL-10 to phosphorylate Stat-3. However, IL-10 failed to phosphorylate Akt in these cells. CONCLUSIONS: IL-10 caused a 59% and 42% reduction in the apoptotic population of serum-deprived cells with and without dexamethasone treatment, respectively. These observations establish that activation of IL-10R promotes survival of RGCs and this survival-promoting activity is due to IL-10 signaling through the Stat-3 pathway, which inhibits the cell death and not through the Akt cell survival pathway. PMID: 14638718 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 43: J Immunol. 2003 Nov 15;171(10):5034-41. Reprogramming of IL-10 activity and signaling by IFN-gamma. Herrero C, Hu X, Li WP, Samuels S, Sharif MN, Kotenko S, Ivashkiv LB. Department of Medicine, Hospital for Special Surgery, Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10021, USA. One important mechanism of cross-regulation by opposing cytokines is inhibition of signal transduction, including inhibition of Janus kinase-STAT signaling by suppressors of cytokine signaling. We investigated whether IFN-gamma, a major activator of macrophages, inhibited the activity of IL-10, an important deactivator. Preactivation of macrophages with IFN-gamma inhibited two key anti-inflammatory functions of IL-10, the suppression of cytokine production and of MHC class II expression. Gene expression profiling showed that IFN-gamma broadly suppressed the ability of IL-10 to induce or repress gene expression. Although IFN-gamma induced expression of suppressor of cytokine signaling proteins, IL-10 signal transduction was not suppressed and IL-10 activation of Janus kinases and Stat3 was preserved. Instead, IFN-gamma switched the balance of IL-10 STAT activation from Stat3 to Stat1, with concomitant activation of inflammatory gene expression. IL-10 activation of Stat1 required the simultaneous presence of IFN-gamma. These results demonstrate that IFN-gamma operates a switch that rapidly regulates STAT activation by IL-10 and alters macrophage responses to IL-10. Dynamic regulation of the activation of different STATs by the same cytokine provides a mechanism by which cells can integrate and balance signals delivered by opposing cytokines, and extends our understanding of cross-regulation by opposing cytokines to include reprogramming of signaling and alteration of function. PMID: 14607900 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 44: J Immunol. 2003 Nov 1;171(9):4792-800. Novel and detrimental effects of lipopolysaccharide on in vitro generation of immature dendritic cells: involvement of mitogen-activated protein kinase p38. Xie J, Qian J, Wang S, Freeman ME 3rd, Epstein J, Yi Q. Myeloma Institute for Research and Therapy and Arkansas Cancer Research Center, Little Rock 72205, USA. Dendritic cells (DCs) are recognized as major players in the regulation of immune responses to a variety of Ags, including bacterial agents. LPS, a Gram-negative bacterial cell wall component, has been shown to fully activate DCs both in vitro and in vivo. LPS-induced DC maturation involves activation of p38, extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinases, and NF-kappaB. Blocking p38 inhibits LPS-induced maturation of DCs. In this study we investigated the role of LPS in the in vitro generation of immature DCs. We report here that in contrast to the observed beneficial effects on DCs, the presence of LPS in monocyte culture retarded the generation of immature DCs. LPS not only impaired the morphology and reduced the yields of the cultured cells, but also inhibited the up-regulation of surface expression of CD1a, costimulatory and adhesion molecules. Furthermore, LPS up-regulated the secretion of IL-1beta, IL-6, IL-8, IL-10, and TNF-alpha; reduced Ag presentation capacity; and inhibited phosphorylation of ERK, but activated p38, leading to a reduced NF-kappaB activity in treated cells. Neutralizing Ab against IL-10, but not other cytokines, partially blocked the effects of LPS. Inhibiting p38 (by inhibitor SB203580) restored the morphology, phenotype, and Ag presentation capacity of LPS-treated cells. SB203580 also inhibited LPS-induced production of IL-1beta, IL-10, and TNF-alpha; enhanced IL-12 production; and recovered the activity of ERK and NF-kappaB. Thus, our study reveals that LPS has dual effects on DCs that are biologically important: activating existing DCs to initiate an immune response, and inhibiting the generation of new DCs to limit such a response. PMID: 14568957 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 45: Chem Immunol Allergy. 2003;83:1-23. Transcription factor activation in human neutrophils. Cloutier A, McDonald PP. Pulmonary Division, Faculty of Medicine, Universite de Sherbrooke, Sherbrooke, Quebec, Canada. Publication Types: Review Review, Tutorial PMID: 12947976 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 46: Biol Pharm Bull. 2003 Aug;26(8):1076-81. Interleukin-10-induced CCR5 expression in macrophage like HL-60 cells: involvement of Erk1/2 and STAT-3. Makuta Y, Sonoda Y, Yamamoto D, Funakoshi-Tago M, Aizu-Yokota E, Takebe Y, Kasahara T. Department of Biochemistry, Kyoritsu College of Pharmacy, Tokyo, Japan. As an immunosuppressive and anti-inflammatory cytokine, IL-10 was recently reported to play roles in CCR5 expression in human monocytes. CCR5 promoter regions contain Oct-2, TCF-1alpha, GATA, and STAT binding sites. Here, we studied the signals involved in the CCR5 expression in IL-10-stimulated cells using the HL-60 cell line. HL-60 cells were stimulated with PMA and differentiated to macrophage-like cells, then stimulated with IL-10. IL-10 induced significant expression of CCR5 protein and CCR5 mRNA in these cells. The induction of CCR5 by IL-10 was inhibited by a MEK-1 inhibitor, PD98059. In addition, IL-10 induced tyrosine (Tyr) phosphorylation of Erk, as well as serine (Ser) and Tyr phosphorylation of STAT-3. Tyr phosphorylation of Erk and Ser phosphorylation of STAT-3 were inhibited by PD98059, while Tyr phosphorylation of STAT-3 was not inhibited by PD98059. DNA binding activity of STAT-3 was observed by the stimulation with IL-10, which was inhibited by PD98059. These results first indicate that Erk1/2 and STAT-3 regulate CCR5 expression, and that Erk-mediated phosphorylation of Ser is required for full stimulation of STAT-3 in CCR5 expression. PMID: 12913253 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 47: Cancer Res. 2003 Aug 1;63(15):4472-80. Rapamycin inhibits the interleukin 10 signal transduction pathway and the growth of Epstein Barr virus B-cell lymphomas. Nepomuceno RR, Balatoni CE, Natkunam Y, Snow AL, Krams SM, Martinez OM. Department of Surgery, Stanford University School of Medicine, 1201 Welch Road, Stanford, CA 94305, USA. EBV-infected B-cell lymphomas are a potentially life-threatening complication in bone marrow and solid organ transplant recipients. Immunosuppressive drugs required to prevent allograft rejection also impair anti-EBV T-cell immunity, thereby increasing the risk of EBV-associated disease. Here we demonstrate that the immunosuppressant rapamycin (RAPA) has a strong antiproliferative effect in vitro on B-cell lines derived from organ transplant recipients with EBV-associated posttransplant lymphoproliferative disorder (PTLD). Furthermore, RAPA significantly inhibits or delays the growth of solid tumors established from EBV-infected B-cell lines in a xenogeneic mouse model of PTLD. RAPA acts via cell cycle arrest, induction of apoptosis, and, most importantly, inhibition of interleukin 10 secretion, a necessary autocrine growth factor. The reduced interleukin 10 production is accompanied by corresponding decreases in the constitutive activation of the growth-promoting transcription factors signal transducer and activator of transcription 1 and 3. Thus, RAPA can limit B-cell lymphoma growth while simultaneously providing immunosuppression to prevent graft rejection in patients who are otherwise at risk for EBV-associated PTLD. Moreover, these findings may have application to other EBV-associated malignancies. PMID: 12907620 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 48: J Biol Chem. 2003 Sep 26;278(39):37874-80. Epub 2003 Jul 11. Interleukin-10 induction of nitric-oxide synthase expression attenuates CD40-mediated interleukin-12 synthesis in human endothelial cells. Cattaruzza M, Slodowski W, Stojakovic M, Krzesz R, Hecker M. Department of Cardiovascular Physiology, University of Gottingen, 37073 Gottingen, Germany. Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine in Th1 cell-mediated chronic inflammatory diseases such as, e.g. Crohn's disease. Moreover, IL-10 has been shown to limit the progression of atherosclerosis, presumably by influencing endothelial cell function. Here we demonstrate that under pro-inflammatory conditions expression of the human IL-10 receptor gene is enhanced in endothelial cells in vitro and in vivo. Subsequent exposure to IL-10 results in an up-regulation of both endothelial nitric-oxide synthase (NOS-3) expression and activity. Gel mobility shift analyses and decoy oligonucleotide experiments suggest that this effect of IL-10 is mediated through activation of the transcription factor STAT-3 (signal transducer and activator of transcription-3). One functional consequence of IL-10 up-regulation of NOS-3 abundance in cultured endothelial cells is the attenuation of CD154-induced IL-12 p40 expression. Moreover, CD154-induced IL-12 p40 expression is enhanced after blockade of NOS-3 activity but attenuated in the presence of exogenous nitric oxide. Increased NOS-3 expression may, thus, be one mechanism by which IL-10 exerts its anti-inflammatory effects in Th1 cell-mediated chronic inflammatory diseases. PMID: 12857749 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 49: J Immunol. 2003 Jul 1;171(1):285-90. IFN-alpha induces the human IL-10 gene by recruiting both IFN regulatory factor 1 and Stat3. Ziegler-Heitbrock L, Lotzerich M, Schaefer A, Werner T, Frankenberger M, Benkhart E. Institute for Immunology, University of Munich, Munich, Germany. lzh1@le.ac.uk The anti-inflammatory cytokine IL-10 can be induced by type I IFNs, but the molecular mechanisms involved have remained elusive. With in silico analysis of the human IL-10 promoter we identified a module consisting of an IFN regulatory factor 1 (IRF-1) site and a Stat3 site. We demonstrate that IFN-alpha will induce the binding of IRF-1 and Stat3 to the respective motifs. Mutational analysis revealed that inactivation of the IRF-1 motif substantially reduces trans-activation from 5- to 2-fold and that inactivation of the Stat3 motif completely ablates trans-activation by IFN-alpha. The dominant role of Stat3 in this module was confirmed with the blockade of trans-activation by a dominant negative Stat3. By contrast, Stat1 contributes a minor proportion to the DNA binding to the Stat site, and overexpression will counteract Stat3-mediated trans-activation. The data show that IFN-alpha induces the IL-10 gene via a module consisting of interdependent IRF-1 and Stat3 motifs. Of note, LPS-induced trans-activation does not target this module, since it is independent of the IRF-1 motif but completely depends on Stat3. PMID: 12817009 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 50: Nat Immunol. 2003 Jun;4(6):551-6. Epub 2003 May 18. Comment in: Nat Immunol. 2003 Jun;4(6):507-9. IL-6 induces an anti-inflammatory response in the absence of SOCS3 in macrophages. Yasukawa H, Ohishi M, Mori H, Murakami M, Chinen T, Aki D, Hanada T, Takeda K, Akira S, Hoshijima M, Hirano T, Chien KR, Yoshimura A. Institute of Molecular Medicine and Department of Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093-0641, USA. Whereas interleukin-6 (IL-6) is a proinflammatory cytokine, IL-10 is an anti-inflammatory cytokine. Although signal transducer and activator of transcription 3 (STAT3) is essential for the function of both IL-6 and IL-10, it is unclear how these two cytokines have such opposing functions. Here we show that suppressor of cytokine signaling 3 (SOCS3) is a key regulator of the divergent action of these two cytokines. In macrophages lacking the Socs3 gene or carrying a mutation of the SOCS3-binding site in gp130, the lipopolysaccharide-induced production of tumor necrosis factor (TNF) and IL-12 is suppressed by both IL-10 and IL-6. SOCS3 specifically prevents activation of STAT3 by IL-6 but not IL-10. Taken together, these data indicate that SOCS3 selectively blocks signaling by IL-6, thereby preventing its ability to inhibit LPS signaling. PMID: 12754507 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 51: Nat Immunol. 2003 Jun;4(6):546-50. Epub 2003 May 18. Comment in: Nat Immunol. 2003 Jun;4(6):507-9. SOCS3 regulates the plasticity of gp130 signaling. Lang R, Pauleau AL, Parganas E, Takahashi Y, Mages J, Ihle JN, Rutschman R, Murray PJ. Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA. Suppressor of cytokine signaling (SOCS) proteins are feedback inhibitors of the Janus kinase (JAK) and signal transducer and activator of transcription (STAT) signaling pathway. SOCS3 is upregulated by several signals in macrophages and has been implicated as a regulator of various signaling pathways. Here we show that phosphorylation of STAT3 is prolonged in mouse Socs3-deficient macrophages after stimulation with interleukin-6 (IL-6) but not IL-10, indicating that SOCS3 specifically affects signaling mediated by IL-6 and gp130. IL-6 induces a wider transcriptional response in Socs3-deficient macrophages than in wild-type cells; this response is dominated by interferon (IFN)-regulated genes owing to an excess of STAT1 phosphorylation. Thus, SOCS3 functions to control the quality of the response to IL-6 and prevents the activation of an IFN-induced program of gene expression. PMID: 12754506 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 52: Nat Immunol. 2003 Jun;4(6):540-5. Epub 2003 May 18. Comment in: Nat Immunol. 2003 Jun;4(6):507-9. SOCS3 negatively regulates IL-6 signaling in vivo. Croker BA, Krebs DL, Zhang JG, Wormald S, Willson TA, Stanley EG, Robb L, Greenhalgh CJ, Forster I, Clausen BE, Nicola NA, Metcalf D, Hilton DJ, Roberts AW, Alexander WS. Cancer and Haematology Division, The Walter and Eliza Hall Institute of Medical Research and the Cooperative Research Centre for Cellular Growth Factors, 1G Royal Parade, Parkville, Victoria 3050, Australia. Members of the suppressor of cytokine signaling (SOCS) family are potentially key physiological negative regulators of interleukin-6 (IL-6) signaling. To examine whether SOCS3 is involved in regulating this signaling, we have used conditional gene targeting to generate mice lacking Socs3 in the liver or in macrophages. We show that Socs3 deficiency results in prolonged activation of signal transducer and activator of transcription 1 (STAT1) and STAT3 after IL-6 stimulation but normal activation of STAT1 after stimulation with interferon-gamma (IFN-gamma). Conversely, IL-6-induced STAT activation is normal in Socs1-deficient cells, whereas STAT1 activation induced by IFN-gamma is prolonged. Microarray analysis shows that the pattern of gene expression induced by IL-6 in Socs3-deficient livers mimics that induced by IFN-gamma. Our data indicate that SOCS3 and SOCS1 have reciprocal functions in IL-6 and IFN-gamma regulation and imply that SOCS3 has a role in preventing IFN-gamma-like responses in cells stimulated by IL-6. PMID: 12754505 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 53: J Clin Invest. 2003 May;111(9):1297-308. Comment in: J Clin Invest. 2003 May;111(9):1284-6. Toll-like receptor-dependent production of IL-12p40 causes chronic enterocolitis in myeloid cell-specific Stat3-deficient mice. Kobayashi M, Kweon MN, Kuwata H, Schreiber RD, Kiyono H, Takeda K, Akira S. Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan. Stat3 plays an essential role in IL-10 signaling pathways. A myeloid cell-specific deletion of Stat3 resulted in inflammatory cytokine production and development of chronic enterocolitis with enhanced Th1 responses in mice. In this study, we analyzed the mechanism by which a Stat3 deficiency in myeloid cells led to the induction of chronic enterocolitis in vivo. Even in the absence of Stat1, which is essential for IFN-gamma signaling pathways, Stat3 mutant mice developed chronic enterocolitis. TNF-alpha/Stat3 double-mutant mice developed severe chronic enterocolitis with enhanced Th1 cell development. IL-12p40/Stat3 double-mutant mice, however, showed normal Th1 responses and no inflammatory change in the colon. RAG2/Stat3 double-mutant mice did not develop enterocolitis, either. These findings indicate that overproduction of IL-12p40, which induces potent Th1 responses, is essential for the development of chronic enterocolitis in Stat3 mutant mice. Furthermore, enterocolitis was significantly improved and IFN-gamma production by T cells was reduced in TLR4/Stat3 double-mutant mice, indicating that TLR4-mediated recognition of microbial components triggers aberrant IL-12p40 production by myeloid cells, leading to the development of enterocolitis. Thus, this study clearly established a sequential innate and acquired immune mechanism for the development of Th1-dependent enterocolitis. PMID: 12727921 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 54: J Immunol. 2003 Mar 15;170(6):3263-72. Activation of STAT3 by IL-6 and IL-10 in primary human macrophages is differentially modulated by suppressor of cytokine signaling 3. Niemand C, Nimmesgern A, Haan S, Fischer P, Schaper F, Rossaint R, Heinrich PC, Muller-Newen G. Institut fur Biochemie and Klinik und Lehrstuhl fur Anasthesiologie, Universitatsklinikum RWTH Aachen, Aachen, Germany. On human macrophages IL-10 acts as a more potent anti-inflammatory cytokine than IL-6, although both cytokines signal mainly via activation of the transcription factor STAT3. In this study we compare IL-10 and IL-6 signaling in primary human macrophages derived from blood monocytes. Pretreatment of macrophages with PMA or the proinflammatory mediators LPS and TNF-alpha blocks IL-6-induced STAT3 activation, whereas IL-10-induced activation of STAT3 remains largely unaffected. Although LPS induces the feedback inhibitor suppressor of cytokine signaling 3 (SOCS3) in macrophages, inhibition of IL-6 signal transduction by LPS occurs rapidly and does not depend on gene transcription. We also found that pretreatment of macrophages with IL-10 inhibits subsequent STAT3 activation by IL-6, whereas IL-10-induced STAT3 activation is not affected by preincubation with IL-6. This cross-inhibition is dependent on active transcription and might therefore be explained by different sensitivities of IL-10 and IL-6 signaling toward the feedback inhibitor SOCS3, which is induced by both cytokines. In contrast to the IL-6 signal transducer gp130, which has been previously shown to recruit SOCS3 to one of its phosphotyrosine residues (Y759), peptide precipitation experiments suggest that SOCS3 does not interact with phosphorylated tyrosine motifs of the IL-10R. Taken together, different sensitivities of IL-10 and IL-6 signaling toward mechanisms that inhibit the Janus kinase/STAT pathway define an important mechanism that contributes to the different anti-inflammatory potencies of these two cytokines. PMID: 12626585 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 55: J Immunol. 2003 Feb 1;170(3):1383-91. Suppressor of cytokine signaling 1 inhibits IL-10-mediated immune responses. Ding Y, Chen D, Tarcsafalvi A, Su R, Qin L, Bromberg JS. Carl C. Icahn Institute for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA. IL-10 has proved to be a key cytokine in regulating inflammatory responses by controlling the production and function of various other cytokines. The suppressor of cytokine signaling (SOCS) gene products are a family of cytoplasmic molecules that are essential mediators for negatively regulating cytokine signaling. It has been previously shown that IL-10 induced SOCS3 expression and that forced constitutive expression of SOCS3 inhibits IL-10/STAT3 activation and LPS-induced macrophage activation. In this report, we show that, in addition to SOCS3 expression, IL-10 induces SOCS1 up-regulation in all cell lines tested, including Ba/F3 pro-B cells, MC/9 mast cells, M1 leukemia cells, U3A human fibroblasts, and primary mouse CD4(+) T cells. Induction of SOCS molecules is dependent on STAT3 activation by IL-10R1. Cell lines constitutively overexpressing SOCS proteins demonstrated that SOCS1 and SOCS3, but not SOCS2, are able to partially inhibit IL-10-mediated STAT3 activation and proliferative responses. Pretreatment of M1 cells with IFN-gamma resulted in SOCS1 induction and a reduction of IL-10-mediated STAT3 activation and cell growth inhibition. IL-10-induced SOCS is associated with the inhibition of IFN-gamma signaling in various cell types, and this inhibition is independent of C-terminal serine residues of the IL-10R, previously shown to be required for other anti-inflammatory responses. Thus, the present results show that both SOCS1 and SOCS3 are induced by IL-10 and may be important inhibitors of both IL-10 and IFN-gamma signaling. IL-10-induced SOCS1 may directly inhibit IL-10 IFN-gamma signaling, while inhibition of other proinflammatory cytokine responses may use additional IL-10R1-mediated mechanisms. PMID: 12538698 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 56: Clin Cancer Res. 2003 Jan;9(1):316-26. Inhibition of constitutive STAT3 activity sensitizes resistant non-Hodgkin's lymphoma and multiple myeloma to chemotherapeutic drug-mediated apoptosis. Alas S, Bonavida B. Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles School of Medicine, University of California, Los Angeles, California 90095, USA. Hematopoietic malignancies have been shown to depend on cytokine growth factor autocrine/paracrine loops for growth and differentiation. This results in the constitutive activation of cytokine-mediated transcription factors like signal transducer and activators of transcription (STAT) 3 in non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM). Recent evidence demonstrates that cytokines also contribute to a drug-resistant phenotype in many tumor cell types. We hypothesized that inhibitors of the STAT3 pathway would sensitize drug-resistant and endogenous cytokine-dependent NHL and MM tumor cells to the cytotoxic effects of chemotherapeutic drugs. We examined an AIDS-related NHL cell line, 2F7, known to be dependent on interleukin (IL)-10 for survival and an MM cell line, U266, known to be dependent on IL-6 for survival. IL-10 and IL-6 signal the cells through the activation of Janus kinase (JAK)1 and JAK2, respectively. Thus, we investigated the effect of two chemical STAT3 pathway inhibitors, namely, piceatannol (JAK1/STAT3 inhibitor) and tyrphostin AG490 (JAK2/STAT3 inhibitor), on the tumor cells for sensitization to therapeutic drugs. We demonstrate by phosphoprotein immunoblotting analysis and electrophoretic mobility shift analysis that piceatannol and AG490 inhibit the constitutive activity of STAT3 in 2F7 and U266, respectively. Furthermore, piceatannol and AG490 sensitize 2F7 and U266 cells, respectively, to apoptosis by a range of therapeutic drugs including cisplatin, fludarabine, Adriamycin, and vinblastine. The specificity of the inhibitors was corroborated in experiments showing that piceatannol had no effect on U266 and, likewise, AG490 has no effect on 2F7. The sensitization observed by these inhibitors correlated with the inhibition of Bcl-2 expression in 2F7 and Bcl-xL expression in U266. Altogether, these results demonstrate that STAT3 pathway inhibitors are a novel class of chemotherapeutic sensitizing agents capable of reversing the drug-resistant phenotype of cytokine-dependent tumor cells. PMID: 12538484 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 57: J Leukoc Biol. 2003 Jan;73(1):145-54. Adherence influences monocyte responsiveness to interleukin-10. Petit-Bertron AF, Fitting C, Cavaillon JM, Adib-Conquy M. UP Cytokines & Inflammation, Institut Pasteur, 28 rue Dr Roux, 75015 Paris, France. We studied the effects of adherence on the properties of interleukin (IL)-10 on monocyte-enriched peripheral blood mononuclear cells. We found that the decrease of CD11b expression induced by IL-10 was enhanced by adherence. Toll-like receptor (TLR)2 and TLR4 mRNA, as well as TLR4 surface expression, were significantly up-regulated by IL-10 in adherent cells. The absence of adherence prevented the inhibitory effects of IL-10 on lipopolysaccharide-induced tumor necrosis factor (TNF) and granulocyte-colony stimulating factor production and increased IL-1beta production and soluble TNF receptor II release in IL-10-pretreated cells. Similarly, the absence of adherence amplified the enhancement of phagocytosis induced by IL-10. Tyk2 and signal transducer and activator of transcription 3 (STAT3) phosphorylation and suppressor of cytokine signaling 3 (SOCS3) expression were induced by IL-10 in both conditions, but a longer activation and/or expression were observed in adherent monocytes. Finally, heme oxygenase-1, an anti-inflammatory molecule, was induced by IL-10 in adherent monocytes, whereas its expression remained low in nonadherent cells. Altogether, these data illustrate that adherence modulates the properties and the anti-inflammatory effects of IL-10. PMID: 12525572 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 58: Biochem J. 2003 Mar 1;370(Pt 2):391-6. Cloning of a new type II cytokine receptor activating signal transducer and activator of transcription (STAT)1, STAT2 and STAT3. Dumoutier L, Lejeune D, Hor S, Fickenscher H, Renauld JC. The Ludwig Institute for Cancer Research, Brussels Branch, and the Experimental Medicine Unit, Christian de Duve Institute of Cellular Pathology, Universite Catholique de Louvain, avenue Hippocrate 74, B-1200-Brussels, Belgium. In the present paper, we report the identification of a new gene encoding a transmembrane protein of 520 amino acids, showing 22% amino acid identity with the extracellular domain of the interleukin (IL)-20 receptor. This gene, termed likely interleukin or cytokine receptor-2 ( LICR2 ), is located on chromosome 1, at 25 kb from the IL22R (IL-22 receptor) gene, and is constitutively expressed in most tissues. A chimaeric receptor, consisting of the extracellular domain of the IL-10 receptor alpha chain and the intracellular domain of LICR2, activated signal transducer and activator of transcription (STAT)1, STAT2, STAT3 and STAT5 upon IL-10 stimulation, in a Janus kinase 1-dependent manner. In contrast, none of the IL-10-related cytokines described so far could activate LICR2-transfected cells, suggesting that LICR2 is a signalling receptor for a new cytokine of the IL-10 family. PMID: 12521379 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 59: J Leukoc Biol. 2002 Dec;72(6):1198-205. Interleukin-10 differently regulates monocyte chemoattractant protein-1 gene expression depending on the environment in a human monoblastic cell line, UG3. Ikeda T, Sato K, Kuwada N, Matsumura T, Yamashita T, Kimura F, Hatake K, Ikeda K, Motoyoshi K. Third Department of Internal Medicine, National Defense Medical College, Saitama, Japan. Interleukin (IL)-4, IL-10, and IL-13 affect monocyte/macrophage functions including regulation of cytokine production. We analyzed the regulatory effects of these cytokines on cytokine production using a human monoblastic cell line, UG3. It is interesting that IL-10 up-regulated, whereas IL-4 and IL-13 down-regulated monocyte chemoattractant protein-1 (MCP-1) production by unstimulated UG3 cells. IL-10-induced expression of MCP-1 mRNA occurred without de novo protein synthesis at transcriptional and post-transcriptional levels. The enhancement of binding activity of nuclear factor Sp1 (Sp-1) and signal transducer and activators of transcription (STAT)1 and 3 but not nuclear factor kappaB (NF-kappaB) was associated with this IL-10-induced MCP-1 expression. Furthermore, IL-10 suppressed lipopolysaccharide (LPS)-induced NF-kappaB binding but not Sp-1. The present results suggest IL-10 has two contrasting actions on the MCP-1 production of monocytes/macrophages, between the resting and activated conditions. The combination of activated Sp-1 and STATs is important for IL-10-induced MCP-1 expression in resting monocytes/macrophages, and the inhibition of LPS-induced NF-kappaB binding is crucial for down-regulation of MCP-1 by IL-10 in stimulated monocytes/macrophages. PMID: 12488502 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 60: J Pathol. 2003 Jan;199(1):84-9. Expression of STAT3 and its phosphorylated forms in mantle cell lymphoma cell lines and tumours. Lai R, Rassidakis GZ, Medeiros LJ, Leventaki V, Keating M, McDonnell TJ. Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. The pathogenesis of mantle cell lymphoma (MCL) is incompletely understood, although cyclin D1 overexpression leading to deregulated cell proliferation is probably important. Recent data suggest that interleukin (IL)-10 can increase the proliferative activity of MCL cells. STAT3 (signal transducer and activator of transcription 3) is the signal transducer of IL-10, and STAT3 is activated by phosphorylation. The hypothesis of this study is that STAT3 is activated in MCL. The expression of the two phosphorylated (i.e. active) forms of STAT3, pSTAT3-tyr (phosphorylated at the tyrosine(705) residue) and pSTAT3-ser (phosphorylated at the serine(727) residue), was assessed in four MCL cell lines and 12 MCL tumours using western blots and/or immunofluorescence staining techniques. All MCL cell lines expressed STAT3, but only one had detectable pSTAT3-tyr and none had pSTAT3-ser. Addition of IL-10 rapidly resulted in expression of pSTAT3-tyr but not pSTAT3-ser. All eight cases of frozen MCL tumours examined had detectable pSTAT3-tyr and pSTAT3-ser. Immunofluorescence studies using four formalin-fixed, paraffin wax-embedded MCL tumours demonstrated cytoplasmic localization of STAT3, as opposed to the nuclear localization of the pSTAT3 species. In conclusion, these findings provide evidence that STAT3 is constitutively activated in MCL, supporting the concept that STAT3 signalling may be important in the pathogenesis of these tumours. Copyright 2002 John Wiley & Sons, Ltd. PMID: 12474230 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 61: Br J Pharmacol. 2002 Dec;137(7):1011-20. Inhibition of cytokine-induced JAK-STAT signalling pathways by an endonuclease inhibitor aurintricarboxylic acid. Chen CW, Chao Y, Chang YH, Hsu MJ, Lin WW. Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan. 1. Inducible nitric oxide (iNOS) is thought to involve in host defence and tissue damage in inflammatory loci. In previous study, we have found that the endonuclease inhibitor aurintricarboxylic acid (ATA) can protect macrophages from cell death induced by bacterial lipopolysaccharide. This action is through the interruption with signalling pathways for NF-kappa B and AP-1 activation, and thus iNOS expression. In this study we have addressed the effects of ATA on JAK-STAT signalling pathways. 2. In murine RAW 264.7 macrophages, IFN-gamma-mediated NO production and iNOS expression were concentration-dependently reduced by the presence of 3-100 micro M ATA. 3. IFN-gamma-induced STAT1 activation, as assessed from its tyrosine phosphorylation, nuclear translocation, binding to specific DNA response element and evoked IRF-1 reporter gene assay, were concomitantly inhibited by ATA. However, ATA did not alter IFN-gamma binding to RAW 264.7 cells. 4. The activities of JAK1 and JAK2, the upstream kinases essential for STAT1 signalling in response to IFN-gamma, were also reduced by ATA. 5. Moreover, IL-4, IL-10, GM-CSF and M-CSF elicited tyrosine phosphorylation of STAT3, STAT5 and/or STAT6 in macrophages were diminished by the presence of ATA. 6. Taken together, we conclude that ATA can interfere JAK-STAT signalling pathways in response to cytokines. This action contributes to the inhibition of IFN-gamma-induced iNOS expression. PMID: 12429573 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 62: Int Immunol. 2002 Oct;14(10):1145-53. Molecular basis of the synergistic production of IL-1 receptor antagonist by human neutrophils stimulated with IL-4 and IL-10. Crepaldi L, Silveri L, Calzetti F, Pinardi C, Cassatella MA. Department of Pathology, General Pathology Unit, University of Verona, Strada Le Grazie 4, 37134 Verona, Italy. In this study, we report that the release of IL-1 receptor antagonist (IL-1ra) from IL-4-stimulated neutrophils is markedly enhanced in the presence of IL-10. We also show that up-regulation of IL-1ra release by IL-10 in IL-4-stimulated neutrophils takes place through IL-1ra mRNA stabilization and enhancement of IL-1ra de novo synthesis. Furthermore, we report that the ability of IL-10 to up-regulate IL-1ra mRNA expression in IL-4-treated neutrophils requires 5-6 h and it is preceded by the acquisition of the capacity to activate Stat3 tyrosine phosphorylation. This latter response to IL-10 was strictly dependent on the levels of expression of IL-10R1, which were in fact significantly increased by IL-4 in cultured neutrophils via a signaling pathway sensitive to the serine/threonine kinase inhibitor H-7. Collectively, our data emphasize the central role of IL-10R1 expression in regulating cell responsiveness to IL-10. In addition, the fact that IL-10 strongly up-regulates IL-1ra production in IL-4-activated neutrophils uncovers a novel mechanism whereby IL-10 and IL-4 cooperate to negatively modulate the inflammatory responses. PMID: 12356680 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 63: J Immunol. 2002 Sep 1;169(5):2253-63. Shaping gene expression in activated and resting primary macrophages by IL-10. Lang R, Patel D, Morris JJ, Rutschman RL, Murray PJ. Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, TN 38015, USA. IL-10 regulates inflammation by reducing cytokine and chemokine production from activated macrophages. We performed microarray experiments to identify possible effector molecules of IL-10 and to investigate the global effect of IL-10 on the transcriptional response induced in LPS-activated macrophages. To exclude background effects of endogenous IL-10, macrophages from IL-10-deficient mice were used. IL-10 up-regulated expression of a small number of genes (26 and 37 after 45 min and 3 h, respectively), including newly identified and previously documented targets such as suppressor of cytokine signaling-3 and IL-1 receptor antagonist. However, the activation program triggered by LPS was profoundly affected by IL-10. IL-10 repressed 62 and further increased 15 of 259 LPS-induced genes. For all genes examined, the effects of IL-10 were determined to be STAT3-dependent. These results suggest that IL-10 regulates STAT3-dependent pathways that selectively target a broad component of LPS-induced genes at the mRNA level. Publication Types: Validation Studies PMID: 12193690 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 64: Antiviral Res. 2002 Aug;55(2):331-9. Interleukin-10 induces transcription of the early promoter of human papillomavirus type 16 (HPV16) through the 5'-segment of the upstream regulatory region (URR). Arany I, Grattendick KG, Tyring SK. Department of Microbiology/Immunology, The University of Texas Medical Branch, Galveston, TX 77555-1070, USA. aranyistvan@uams.edu The effects of various proinflammatory cytokines on the transcription of human papillomaviruses (HPVs) have been demonstrated. On the other hand, the role of anti-inflammatory cytokines has not been elaborated, despite the fact that levels of interleukin-10 (IL-10) have been found significantly elevated in cervical dysplasias or carcinomas as well as in the cervix of HIV-positive individuals. These conditions are also associated with elevated viral transcription. Thus, the impact of IL-10 on HPV transcription might be important in pathogenesis of cervical lesions in both immunocompetent or immunosuppressed individuals. In this paper we describe the effects of IL-10 on the transcription of HPV type 16. We found that treatment of HPV 16-positive cervical carcinoma cells with IL-10 increased mRNA levels of the E7 early gene at the level of transcription. Similarly, IL-10 significantly and dose-dependently induced the transcription from the HPV early promoter in a reporter system. Employing deletion mutants we determined that this induction is mapped to the 5' segment of the URR. Transient transfection of an antisense-STAT3-expression vector abolished IL-10-induced reporter activity as well as HPV 16 E7 expression. This suggests that STAT3 either directly binds to the URR and stimulates transcription or affects expression and/or binding of transcription factors that bind to the 5'-region. Our findings suggest a mechanism by which--in addition to its immunosuppressive effects--IL-10 might enhance persistence and progression of HPV-related lesions under conditions (e.g. dysplastic progression, HIV infection) when the cytokine expression in the cervical microenvironment changes. Copyright 2002 Elsevier Science BV. PMID: 12103433 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 65: J Biol Chem. 2002 Sep 13;277(37):33676-82. Epub 2002 Jun 26. Interleukin-22 (IL-22) activates the JAK/STAT, ERK, JNK, and p38 MAP kinase pathways in a rat hepatoma cell line. Pathways that are shared with and distinct from IL-10. Lejeune D, Dumoutier L, Constantinescu S, Kruijer W, Schuringa JJ, Renauld JC. Ludwig Institute for Cancer Research, Brussels Branch, Experimental Medicine Unit, Universite de Louvain, avenue Hippocrate 74, B-1200 Brussels, Belgium. IL (interleukin)-22 is an IL-10-related cytokine; its main biological activity known thus far is the induction of acute phase reactants in liver and pancreas. IL-22 signals through a receptor that is composed of two chains from the class II cytokine receptor family: IL-22R (also called ZcytoR11/CRF2-9) and IL-10Rbeta (CRF2-4), which is also involved in IL-10 signaling. In this report, we analyzed the signal transduction pathways activated in response to IL-22 in a rat hepatoma cell line, H4IIE. We found that IL-22 induces activation of JAK1 and Tyk2 but not JAK2, as well as phosphorylation of STAT1, STAT3, and STAT5 on tyrosine residues, extending the similarities between IL-22 and IL-10. However our results unraveled some differences between IL-22 and IL-10 signaling. Using antibodies specific for the phosphorylated form of MEK1/2, ERK1/2, p90RSK, JNK, and p38 kinase, we showed that IL-22 activates the three major MAPK pathways. IL-22 also induced serine phosphorylation of STAT3 on Ser(727). This effect, which is not shared with IL-10, was only marginally affected by MEK1/2 inhibitors, indicating that other pathways might be involved. Finally, by overexpressing a STAT3 S727A mutant, we showed that serine phosphorylation is required to achieve maximum transactivation of a STAT responsive promoter upon IL-22 stimulation. PMID: 12087100 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 66: J Immunol. 2002 Jun 15;168(12):6404-11. Involvement of suppressor of cytokine signaling-3 as a mediator of the inhibitory effects of IL-10 on lipopolysaccharide-induced macrophage activation. Berlato C, Cassatella MA, Kinjyo I, Gatto L, Yoshimura A, Bazzoni F. Department of Pathology, Section of General Pathology, University of Verona, Verona, Italy. Previous studies have shown that IL-10 can induce the expression of the suppressor of cytokine signaling 3 (SOCS-3) mRNA in human monocytes and neutrophils, suggesting that the capacity of IL-10 to inhibit the expression of LPS-inducible proinflammatory genes may depend on SOCS-3 induction. However, no direct experimental evidence has been provided to support such hypothesis. Herein, we show that stable transfection of SOCS-3 into the mouse macrophage cell line J774 resulted in an inhibition of NO, TNF-alpha, IL-6, and GM-CSF secretion in response to LPS at levels similar to those exerted by IL-10 in LPS-stimulated wild-type J774. Constitutive SOCS-3 expression also down-regulated the mRNA expression of inducible NO synthase and IL-6 and impaired the production of TNF-alpha, mainly at a post-transcriptional level. In addition, SOCS-3-transfected cells displayed a constitutive expression of the IL-1R antagonist gene, consistent with the observation that IL-10 enhances IL-1R antagonist mRNA in LPS-stimulated wild-type cells. Furthermore, in peritoneal macrophages harvested from mice carrying heterozygous disruption of the SOCS-3 gene, IL-10 was less effective in repressing LPS-stimulated TNF-alpha and NO production. Taken together, our data show that SOCS-3 inhibits LPS-induced macrophage activation, strongly supporting the idea that it plays a role in the molecular mechanism by which IL-10 down-modulates the effector functions of LPS-activated macrophages. Finally, we show that forced expression of SOCS-3 significantly suppresses the ability of IL-10 to trigger tyrosine phosphorylation of STAT3. Therefore, SOCS-3 functions both as an LPS signal inhibitor and as a negative feedback regulator of IL-10/STAT3 signaling. PMID: 12055259 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 67: Crit Rev Immunol. 2001;21(5):427-49. Interleukin-10 in the brain. Strle K, Zhou JH, Shen WH, Broussard SR, Johnson RW, Freund GG, Dantzer R, Kelley KW. Department of Animal Sciences, College of Medicine, University of Illinois, Urbana 61801, USA. Interleukin (IL)-10 is synthesized in the central nervous system (CNS) and acts to limit clinical symptoms of stroke, multiple sclerosis, Alzheimer's disease, meningitis, and the behavioral changes that occur during bacterial infections. Expression of IL-10 is elevated during the course of most major diseases in the CNS and promotes survival of neurons and all glial cells in the brain by blocking the effects of proapoptotic cytokines and by promoting expression of cell survival signals. Stimulation of IL-10 receptors regulates numerous life- or death-signaling pathways--including Jak1/Stat3, PI 3-kinase, MAPK, SOCS, and NF-kappaB--ultimately promoting cell survival by inhibiting both ligand- and mitochondrial-induced apoptotic pathways. IL-10 also limits inflammation in the brain; it does so by three major pathways: (1) reducing synthesis of proinflammatory cytokines, (2) suppressing cytokine receptor expression, and (3) inhibiting receptor activation. Finally, IL-10 induces anergy in brain-infiltrating T cells by inhibiting cell signaling through the costimulatory CD28-CD80/86 pathway. The multiple functions of IL-10 in the brain will create new and intriguing vistas that will promote a better understanding of neurodegenerative diseases. These discoveries could lead to development of innovative approaches for the use of antiinflammatory cytokines in major debilitating diseases of the CNS. Publication Types: Review PMID: 11942558 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 68: Virology. 2001 Nov 10;290(1):91-8. HTLV-1 cell lines differ in constitutively activated signaling pathways that can be altered by cytokine exposure. Liang W, Hague B, Zhao T, Kindt TJ. Molecular and Cellular Immunogenetics Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA. wliang@niaid.nih.gov Examination of signaling pathways used by HTLV-1-infected rabbit cell lines revealed differences between one, RH/K30, that mediates asymptomatic infection and another, RH/K34, that causes lethal experimental leukemia. Both lines are IL-2 independent; RH/K30 produces IL-4 while RH/K34 produces IL-10. Examination of the Jak/STAT (Janus kinase/signal transducer and activator of transcription) activation of the lines revealed constitutive phosphorylation of Jak1 in both STAT6 phosphorylation, not previously reported for HTLV-1 cells, was observed in RH/K30; STAT1 and STAT3 were phosphorylated in RH/K34. Treatment with cytokines altered the activation of the STAT proteins: IL-2 induced STAT5 phosphorylation in both lines. Supernatant from RH/K34 or IL-10 induced STAT3 phosphorylation in RH/K30 cells. Supernatant from RH/K30 or IL-4 induced STAT6 phosphorylation in RH/K34 cells, which could be reversed with a Jak kinase inhibitor--AG-490. PMID: 11883009 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 69: Immunity. 2002 Feb;16(2):219-30. Chronic intestinal inflammatory condition generates IL-10-producing regulatory B cell subset characterized by CD1d upregulation. Mizoguchi A, Mizoguchi E, Takedatsu H, Blumberg RS, Bhan AK. Immunopathology Unit, Department of Pathology, Massachusetts General Hospital, Boston, MA 02114, USA. amizoguchi@partners.org B cells possess a variety of immune functions that are involved in normal and abnormal immune responses, including autoimmune disorders. Through murine models of intestinal inflammation, we here demonstrate a B cell subset that is induced in gut-associated lymphoid tissues and is characterized by CD1d upregulation. This B cell subset appears under a chronic inflammatory environment, produces IL-10, and suppresses progression of intestinal inflammation by downregulating inflammatory cascades associated with IL-1 upregulation and STAT3 activation rather than by altering polarized T helper responses. This study indicates that B cells, by producing cytokines such as IL-10, can act as regulatory cells in immunologically mediated inflammatory reactions. PMID: 11869683 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 70: J Neuroimmunol. 2002 Jan;122(1-2):9-19. IL-10 promotes survival of microglia without activating Akt. Strle K, Zhou JH, Broussard SR, Venters HD, Johnson RW, Freund GG, Dantzer R, Kelley KW. Laboratory of Immunophysiology, Department of Animal Sciences, University of Illinois, 207 ERML, 1201 W. Gregory Dr., Urbana, IL 61801, USA. IL-10 is an anti-inflammatory cytokine that has recently been shown to promote survival of neurons and glia. Here we establish that IL-10 induces phosphorylation of Stat3 on Tyr(705) and serves as a survival factor for N13 microglial cells. Recombinant IL-10 (10 ng/ml) decreases growth factor withdrawal-induced apoptosis by 50%, as assessed by TUNEL. In contrast to IL-10, IGF-I increases enzymatic activity of PI 3-kinase and causes phosphorylation on serine(473) of Akt but does not prevent microglial apoptosis. These data establish that IL-10 activates Stat3 and inhibits the mitochondrial pathway of cell death without activating the Akt cell survival pathway. PMID: 11777539 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 71: Cell Immunol. 2001 Oct 10;213(1):62-71. Splenic and peritoneal B-1 cells differ in terms of transcriptional and proliferative features that separate peritoneal B-1 from splenic B-2 cells. Fischer GM, Solt LA, Hastings WD, Yang K, Gerstein RM, Nikolajczyk BS, Clarke SH, Rothstein TL. Department of Microbiology, Boston University Medical Center, Boston, Massachusetts 02118, USA. B-1 cells constitute a distinct B cell subset with characteristic phenotypic and functional features. B-1 cells are highly represented among peritoneal lymphocytes; substantial numbers of B-1 cells are also located within splenic tissue. Here a number of differences in transcription factor and gene expression were identified that separate peritoneal B-1 and splenic B-2 cells, and then splenic B-1 cells obtained from immunoglobulin transgenic mice were tested for these parameters. Splenic B-1 cells resembled splenic B-2 cells rather than peritoneal B-1 cells in terms of nuclear expression of DNA-binding STAT3, CREB, and PU.1, with respect to transcriptional activation of IL-10, and in the failure to enter cell cycle in response to PMA. Splenic B-1 cells (B-1S) appear to constitute a unique population of B-1 cells, which, while sharing with peritoneal B-1 cells (B-1P) certain phenotypic features, differ from them in transcription factor and gene expression and in signaling for cell cycle progression. (c)2001 Elsevier Science. PMID: 11747357 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 72: J Immunol. 2001 Dec 15;167(12):6884-92. Differential IL-10R1 expression plays a critical role in IL-10-mediated immune regulation. Ding Y, Qin L, Zamarin D, Kotenko SV, Pestka S, Moore KW, Bromberg JS. Institute for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA. yaozhong.ding@mssm.edu In this study, we characterized the differential receptor-binding specificity, affinity, and Janus kinase-STAT activation of cellular IL-10 (cIL-10) compared with viral IL-10 (vIL-10). Only cells expressing IL-10R1 bind human IL-10 or vIL-10. IL-10R2 does not bind to cIL-10 or vIL-10 alone and its presence does not enhance the receptor-binding affinity of cIL-10 or vIL-10, but it is essential for both cIL-10- and vIL-10-mediated signal transduction and immune regulation. Responses initiated by cIL-10 and vIL-10 were compared in B cell and mast cell lines, and demonstrated that the inability of vIL-10 to stimulate immune responses, as compared with human IL-10, is due to failure to initiate signaling. Absent signal transduction is due to low level expression of cell surface IL-10R1, since overexpressing IL-10R1 allows vIL-10 to initiate cIL-10-like signals and subsequent biological responses. These results are similar in primary cells, since splenocytes respond to both cIL-10 and vIL-10, while thymocytes respond only to cIL-10 and have very low mouse IL-10R1 but not mouse IL-10R2 expression. These data demonstrate that IL-10R1 expression plays a critical role in determining whether cells respond to IL-10. Modulation of cell surface IL-10R1 density might be an important mechanism for determining whether IL-10 leads to immunostimulation or immunosuppression in vivo. PMID: 11739506 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 73: Mol Cell Biol. 2001 Dec;21(24):8301-17. Influenza virus infection induces metallothionein gene expression in the mouse liver and lung by overlapping but distinct molecular mechanisms. Ghoshal K, Majumder S, Zhu Q, Hunzeker J, Datta J, Shah M, Sheridan JF, Jacob ST. Department of Molecular and Cellular Biochemistry, College of Medicine, The Ohio State University, 333 Hamilton Hall, 1645 Neil Ave., Columbus, OH 43210, USA. Metallothionein I (MT-I) and MT-II have been implicated in the protection of cells against reactive oxygen species (ROS), heavy metals, and a variety of pathological and environmental stressors. Here, we show a robust increase in MT-I/MT-II mRNA level and MT proteins in the livers and lungs of C57BL/6 mice exposed to the influenza A/PR8 virus that infects the upper respiratory tract and lungs. Interleukin-6 (IL-6) had a pronounced effect on the induction of these genes in the liver but not the lung. Treatment of the animals with RU-486, a glucocorticoid receptor antagonist, inhibited induction of MT-I/MT-II in both liver and lung, revealing a direct role of glucocorticoid that is increased upon infection in this induction process. In vivo genomic footprinting (IVGF) analysis demonstrated involvement of almost all metal response elements, major late transcription factor/antioxidant response element (MLTF/ARE), the STAT3 binding site on the MT-I upstream promoter, and the glucocorticoid responsive element (GRE1), located upstream of the MT-II gene, in the induction process in the liver and lung. In the lung, inducible footprinting was also identified at a unique gamma interferon (IFN-gamma) response element (gamma-IRE) and at Sp1 sites. The mobility shift analysis showed activation of STAT3 and the glucocorticoid receptor in the liver and lung nuclear extracts, which was consistent with the IVGF data. Analysis of the newly synthesized mRNA for cytokines in the infected lung by real-time PCR showed a robust increase in the levels of IL-10 and IFN-gamma mRNA that can activate STAT3 and STAT1, respectively. A STAT1-containing complex that binds to the gamma-IRE in vitro was activated in the infected lung. No major change in MLTF/ARE DNA binding activity in the liver and lung occurred after infection. These results have demonstrated that MT-I and MT-II can be induced robustly in the liver and lung following experimental influenza virus infection by overlapping but distinct molecular mechanisms. PMID: 11713267 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 74: Blood. 2001 Nov 15;98(10):3030-4. Protein tyrosine phosphatase epsilonC selectively inhibits interleukin-6- and interleukin- 10-induced JAK-STAT signaling. Tanuma N, Shima H, Nakamura K, Kikuchi K. Division of Biochemical Oncology and Immunology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan. Protein tyrosine phosphatase (PTP) epsilon (PTPepsilon) exists as 2 forms generated by alternative promoter usage. It has recently been reported that a cytosolic isoform of PTPepsilon (PTPepsilonC) when over-expressed in murine M1 myeloid cells inhibits interleukin-6 (IL-6)- and leukemia inhibitory factor-induced activation of Janus kinases (JAKs), thereby suppressing STAT3 tyrosine phosphorylation and STAT3 signaling. This study characterizes an inhibitory action of PTPepsilonC on IL-6 signaling and also reveals that PTPepsilonC inhibitory activity is independent of other potential negative regulators, such as SHP-2 and SOCS family proteins. Furthermore, it analyzes the selectivity of PTPepsilonC action toward several cytokines. On IL-6 stimulation, expression of PTPepsilonC-DA, a catalytically inactive mutant of PTPepsilonC, results in an earlier onset of STAT3 tyrosine phosphorylation, suggesting different modes of action between PTPepsilonC and other negative regulators. In addition, the study shows PTPepsilonC-DA enhances activation of STAT1 by IL-6 as well. In terms of specificity to cytokines, over-expressed PTPepsilonC also inhibits IL-10-induced tyrosine phosphorylation of STAT3 in M1 cells, whereas PTPepsilonC does not affect either interferon-beta- and interferon-gamma-induced tyrosine phosphorylation of STATs or expression of STAT transcriptional targets. Among cytokines tested, the inhibitory effect of PTPepsilonC is selective to IL-6- and IL-10-induced JAK-STAT signaling. PMID: 11698287 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 75: Transplantation. 2001 Oct 27;72(8):1416-22. Alterations in transcription factor binding at the IL-2 promoter region in anergized human CD4+ T lymphocytes. Heisel O, Keown P. Department of Medicine, Vancouver General Hospital, 910 West 10th Avenue, Vancouver, B.C., Canada. BACKGROUND: The mechanisms responsible for the induction of clonal anergy are not well understood. We have utilized an in vitro model of human T cell anergy to explore the perturbations in cell signaling at the level of interleukin (IL)-2 gene transcription and to define the contribution of other cytokines to this effect. METHODS: An in vitro model of clonal anergy was established by using CD4+ T lymphocytes from healthy human donors. Cells were anergized by prestimulation with an anti-CD3 monoclonal antibody (mAb) followed by restimulation 72 hr later with anti-CD3 mAb with or without anti-CD28. RESULTS: CD4+ T cells, anergized with anti-CD3 monoclonal antibody (OKT3) prestimulation, displayed a marked reduction in proliferation (P=0.0036) and IL-2 production (P<0.0001). Co-incubation with IL-10 reduced cellular proliferation in OKT3/CD28 pretreated cells by 19% (P=NS) and reduced IL-2 production by 40% (P=0.0024). Anergized T cells demonstrated a reduced binding activity of the AP-1 complex to the IL-2 promoter. Supershift experiments and Western blots confirmed that the binding of c-Fos, JunB, and JunD, but not of FosB, was reduced in anergized cells. At the sis-inducible element (SIE)-binding region of the c-Fos promoter, Stat3 binding was reduced. CONCLUSIONS: T cell anergy, induced by prestimulation with OKT3, is characterized by reduced proliferation and a profound decrease in IL-2 production. Anergy can be prevented by co-incubation with anti-CD28 and partially re-established by IL-10. Anergy is accompanied by a reduction in AP-1 binding to the IL-2 promoter, with selective reduction in binding of c-Fos, JunB, and JunD. Defective binding for Stat3 at the c-Fos promoter suggests an involvement of the Jak-Stat pathway. PMID: 11685114 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 76: J Immunol. 2001 Oct 15;167(8):4436-42. IL-10 inhibits apoptosis of promyeloid cells by activating insulin receptor substrate-2 and phosphatidylinositol 3'-kinase. Zhou JH, Broussard SR, Strle K, Freund GG, Johnson RW, Dantzer R, Kelley KW. Department of Animal Sciences, University of Illinois, Urbana, IL 61801, USA. IL-10 is well known to be a potent inhibitor of the synthesis of proinflammatory cytokines, but noninflammatory hemopoietic cells also express IL-10Rs. Here we show that IL-10 directly affects progenitor myeloid cells by protecting them from death following the removal of growth factors. Murine factor-dependent cell progenitors cultured in the absence of growth factors were 43 +/- 1% apoptotic after 12 h. Addition of IL-10 at a concentration as low as 100 pg/ml significantly reduced the apoptotic population to 32 +/- 3%. At 10 ng/ml, IL-10 caused a 4-fold reduction in the apoptotic population (11 +/- 1%). The anti-apoptotic activity of IL-10 was significantly inhibited with a neutralizing IL-10R Ab. Factor-dependent cell progenitor promyeloid cells expressed functional IL-10Rs, as assessed by precipitation of a 110-kDa protein with an Ab to the IL-10R and by the ability of IL-10 to activate Jak1 and Tyk2 and to phosphorylate tyrosine 705 on Stat-3. IL-10 increased tyrosyl phosphorylation of insulin receptor substrate-2 and stimulated the enzymatic activity of both phosphatidylinositol 3'-kinase and Akt. The anti-apoptotic activity of IL-10 was blocked by inhibition of phosphatidylinositol 3'-kinase. Wortmannin and LY294002 also totally inhibited activation of extracellular signal-related kinase (ERK)1/2 by IL-10. Direct inhibition of ERK1/2 with the mitogen-activated protein kinase/ERK kinase inhibitor PD98059 partially, but significantly, impaired the anti-apoptotic activity of IL-10. These data establish that activation of the IL-10R promotes survival of progenitor myeloid cells. This survival-promoting activity is totally due to IL-10 stimulating the insulin receptor substrate-2/PI 3-kinase/Akt pathway, which increases the anti-apoptotic activity of ERK1/2. PMID: 11591769 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 77: J Immunol. 2001 Oct 1;167(7):3545-9. Cutting edge: STAT activation by IL-19, IL-20 and mda-7 through IL-20 receptor complexes of two types. Dumoutier L, Leemans C, Lejeune D, Kotenko SV, Renauld JC. Ludwig Institute for Cancer Research, Brussels Branch, Avenue Hippocrate 74, B-1200 Brussels, Belgium. IL-10-related cytokines include IL-20 and IL-22, which induce, respectively, keratinocyte proliferation and acute phase production by hepatocytes, as well as IL-19, melanoma differentiation-associated gene 7, and AK155, three cytokines for which no activity nor receptor complex has been described thus far. Here, we show that mda-7 and IL-19 bind to the previously described IL-20R complex, composed by cytokine receptor family 2-8/IL-20Ralpha and DIRS1/IL-20Rbeta (type I IL-20R). In addition, mda-7 and IL-20, but not IL-19, bind to another receptor complex, composed by IL-22R and DIRS1/IL20Rbeta (type II IL-20R). In both cases, binding of the ligands results in STAT3 phosphorylation and activation of a minimal promoter including STAT-binding sites. Taken together, these results demonstrate that: 1) IL-20 induces STAT activation through IL-20R complexes of two types; 2) mda-7 and IL-20 redundantly signal through both complexes; and 3) IL-19 signals only through the type I IL-20R complex. PMID: 11564763 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 78: J Immunol. 2001 Aug 15;167(4):2312-22. Up-regulation of IL-10R1 expression is required to render human neutrophils fully responsive to IL-10. Crepaldi L, Gasperini S, Lapinet JA, Calzetti F, Pinardi C, Liu Y, Zurawski S, de Waal Malefyt R, Moore KW, Cassatella MA. Department of Pathology, General Pathology Unit, University of Verona, Verona, Italy. We have recently shown that IL-10 fails to trigger Stat3 and Stat1 tyrosine phosphorylation in freshly isolated human neutrophils. In this study, we report that IL-10 can nonetheless induce Stat3 tyrosine phosphorylation and the binding of Stat1 and Stat3 to the IFN-gamma response region or the high-affinity synthetic derivative of the c-sis-inducible element in neutrophils that have been cultured for at least 3 h with LPS. Similarly, the ability of IL-10 to up-regulate suppressor of cytokine signaling (SOCS)-3 mRNA was dramatically enhanced in cultured neutrophils and, as a result, translated into the SOCS-3 protein. Since neutrophils' acquisition of responsiveness to IL-10 required de novo protein synthesis, we assessed whether expression of IL-10R1 or IL-10R2 was modulated in cultured neutrophils. We detected constitutive IL-10R1 mRNA and protein expression in circulating neutrophils, at levels which were much lower than those observed in autologous monocytes or lymphocytes. In contrast, IL-10R2 expression was comparable in both cell types. However, IL-10R1 (but not IL-10R2) mRNA and protein expression was substantially increased in neutrophils stimulated by LPS. The ability of IL-10 to activate Stat3 tyrosine phosphorylation and SOCS-3 synthesis and to regulate IL-1 receptor antagonist and macrophage-inflammatory protein 1beta release in LPS-treated neutrophils correlated with this increased IL-10R1 expression, and was abolished by neutralizing anti-IL-10R1 and anti-IL-10R2 Abs. Our results demonstrate that the capacity of neutrophils to respond to IL-10, as assessed by Stat3 tyrosine phosphorylation, SOCS-3 expression, and modulation of cytokine production, is very dependent on the level of expression of IL-10R1. PMID: 11490020 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 79: Cancer Res. 2001 Jul 1;61(13):5137-44. Rituximab inactivates signal transducer and activation of transcription 3 (STAT3) activity in B-non-Hodgkin's lymphoma through inhibition of the interleukin 10 autocrine/paracrine loop and results in down-regulation of Bcl-2 and sensitization to cytotoxic drugs. Alas S, Bonavida B. Department of Microbiology, Immunology and Molecular Genetics, Jonsson Comprehensive Cancer Center, University of California Los Angeles School of Medicine, University of California, Los Angeles, California 90095, USA. Development of the chimeric mouse antihuman CD20 antibody, Rituximab, presented a notable advance in the treatment of patients with non-Hodgkin's lymphoma (NHL). Its use allowed the specific targeting of tumor B cells without the systemic toxicity of traditional therapies. The mechanisms by which Rituximab induces its antitumor activity are not fully understood. We have shown previously that Rituximab down-regulates Bcl-2 expression in some B-NHL cell lymphoma lines through an interleukin 10 (IL-10)-dependent autocrine loop, an effect that renders the resistant cells susceptible to chemotherapeutic drugs. The objective of this study was to delineate the signaling pathway by which Bcl-2 is controlled by Rituximab and IL-10. We hypothesized that the down-regulation of IL-10 by Rituximab decreases activation of the signal transducer and activator of transcription 3 (STAT3) protein, which in turn, is responsible for decreased levels of Bcl-2. We demonstrate by phosphoprotein immunoblotting and gel shift analyses that endogenous IL-10 induces activation of STAT3 in the 2F7 cell line. Furthermore, we show that Rituximab and anti-IL-10 antibody treatment decreases the ability of STAT3 to bind to its DNA binding site. The decrease in STAT3 activation by these treatments correlates with a decrease in Bcl-2 expression. Additionally, piceatannol, an inhibitor of STAT3 activation, down-regulates the expression of Bcl-2. Altogether, these results demonstrate that Bcl-2 expression is under the regulation of the STAT3 signaling pathway, which is regulated by endogenously secreted IL-10. Hence, Rituximab-induced down-regulation of IL-10 expression is responsible for the down-regulation of Bcl-2 and sensitization of NHL cells by therapeutic drugs. Furthermore, these findings support the notion that circulating IL-10 in vivo may control the resistance of NHL to drug-mediated cytotoxicity. PMID: 11431352 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 80: J Immunol. 2001 Apr 1;166(7):4312-8. Regulatory activity of autocrine IL-10 on dendritic cell functions. Corinti S, Albanesi C, la Sala A, Pastore S, Girolomoni G. Laboratory of Immunology, Istituto Dermopatico dell'Immacolata, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy. s.cortini@idi.it IL-10 is a critical cytokine that blocks the maturation of dendritic cells (DCs), but the relevance of autocrine IL-10 on DC functions has not been investigated. In this study, we found that immature monocyte-derived DCs released low but sizeable amounts of IL-10. After stimulation with bacteria, LPS, lipoteichoic acid, or soluble CD40 ligand, DCs secreted high levels of IL-10. Addition of an anti-IL-10-neutralizing Ab to immature DCs as well as to soluble CD40 ligand- or LPS-maturing DCs led to enhanced expression of surface CD83, CD80, CD86, and MHC molecules and markedly augmented release of TNF-alpha and IL-12, but diminished IL-10 mRNA expression. Moreover, DCs treated with anti-IL-10 Ab showed an increased capacity to activate allogeneic T cells and primed naive T cells to a more prominent Th1 polarization. DC maturation and IL-10 neutralization were associated with enhanced accumulation of the IL-10 receptor binding chain (IL-10R1) mRNA and intracellular IL-10R1 protein. In contrast, surface IL-10R1 and IL-10 binding activity diminished in mature DCs. These results indicate that autocrine IL-10 prevents spontaneous maturation of DCs in vitro, limits LPS- and CD40-mediated maturation, and increases IL-10 production by DCs. Moreover, IL-10R expression appears to be regulated by both transcriptional and posttranscriptional mechanisms. Endogenous IL-10 and IL-10R can be relevant targets for the manipulation of DC functions. PMID: 11254683 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 81: J Exp Med. 2001 Feb 19;193(4):471-81. CIS3/SOCS3/SSI3 plays a negative regulatory role in STAT3 activation and intestinal inflammation. Suzuki A, Hanada T, Mitsuyama K, Yoshida T, Kamizono S, Hoshino T, Kubo M, Yamashita A, Okabe M, Takeda K, Akira S, Matsumoto S, Toyonaga A, Sata M, Yoshimura A. Institute of Life Science, Kurume University, Kurume 839-0861, Japan. Immune and inflammatory systems are controlled by multiple cytokines, including interleukins (ILs) and interferons. These cytokines exert their biological functions through Janus tyrosine kinases and signal transducer and activator of transcription (STAT) transcription factors. We recently identified two intrinsic Janus kinase (JAK) inhibitors, JAK binding protein (JAB; also referred to as suppressor of cytokine signaling [SOCS1]/STAT-induced STAT inhibitor [SSI1]) and cytokine-inducible SH2 protein (CIS)3 (or SOCS3/SSI3), which play an essential role in the negative regulation of cytokine signaling. We have investigated the role of STATs and these JAK inhibitors in intestinal inflammation. Among STAT family members, STAT3 was most strongly tyrosine phosphorylated in human ulcerative colitis and Crohn's disease patients as well as in dextran sulfate sodium (DSS)-induced colitis in mice. Development of colitis as well as STAT3 activation was significantly reduced in IL-6-deficient mice treated with DSS, suggesting that STAT3 plays an important role in the perpetuation of colitis. CIS3, but not JAB, was highly expressed in the colon of DSS-treated mice as well as several T cell-dependent colitis models. To define the physiological role of CIS3 induction in colitis, we developed a JAB mutant (F59D-JAB) that overcame the inhibitory effect of both JAB and CIS3 and created transgenic mice. DSS induced stronger STAT3 activation and more severe colitis in F59D-JAB transgenic mice than in their wild-type littermates. These data suggest that hyperactivation of STAT3 results in severe colitis and that CIS3 plays a negative regulatory role in intestinal inflammation by downregulating STAT3 activity. PMID: 11181699 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 82: Eur J Immunol. 2001 Feb;31(2):665-71. Lymphokine dependence of STAT3 activation produced by surface immunoglobulin cross-linking and by phorbol ester plus calcium ionophore treatment in B cells. Fan H, Rothstein TL. Department of Microbiology, Boston University School of Medicine, Boston, MA, USA. Stimulation of B cells by surface immunoglobulin (sIg) triggering, or through the mitogenic combination of phorbol ester and calcium ionophore, is accompanied by activation of STAT transcription factors. The mechanism responsible for the delayed nuclear accumulation of phosphorylated STAT3 was examined in detail, focusing on the role of B cell-derived lymphokines. sIg-induced activation of STAT3 was partially inhibited in B cells obtained from IL-6- or IL-10-deficient mice, and was partially blocked by neutralizing antibodies directed against either of these lymphokines. sIg-induced STAT3 activation was completely inhibited by combining IL-6- and IL-10-specific neutralizing antibodies, or by adding individual neutralizing antibodies to B cells obtained from lymphokine-deficient animals. In contrast, IL-10 alone appeared to account for STAT3 activation resulting from B cell stimulation with phorbol ester and calcium ionophore. In keeping with these results, soluble IL-6 and IL-10 were found in supernatant fluid obtained from stimulated B cells. This work indicates that a lymphokine pathway is responsible for STAT3 activation that occurs late after B cell stimulation, and points out differences in B cell activation that result from stimulation through the antigen receptor and through pharmacological mimicry of signaling mediators. PMID: 11180132 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 83: Cell. 2001 Jan 12;104(1):9-19. Interleukin 20: discovery, receptor identification, and role in epidermal function. Blumberg H, Conklin D, Xu WF, Grossmann A, Brender T, Carollo S, Eagan M, Foster D, Haldeman BA, Hammond A, Haugen H, Jelinek L, Kelly JD, Madden K, Maurer MF, Parrish-Novak J, Prunkard D, Sexson S, Sprecher C, Waggie K, West J, Whitmore TE, Yao L, Kuechle MK, Dale BA, Chandrasekher YA. Department of Genetics, ZymoGenetics, Inc., 1201 Eastlake Avenue E, Seattle, WA 98102, USA. A structural, profile-based algorithm was used to identify interleukin 20 (IL-20), a novel IL-10 homolog. Chromosomal localization of IL-20 led to the discovery of an IL-10 family cytokine cluster. Overexpression of IL-20 in transgenic (TG) mice causes neonatal lethality with skin abnormalities including aberrant epidermal differentiation. Recombinant IL-20 protein stimulates a signal transduction pathway through STAT3 in a keratinocyte cell line, demonstrating a direct action of this ligand. An IL-20 receptor was identified as a heterodimer of two orphan class II cytokine receptor subunits. Both receptor subunits are expressed in skin and are dramatically upregulated in psoriatic skin. Taken together, these results demonstrate a role in epidermal function and psoriasis for IL-20, a novel cytokine identified solely by bioinformatics analysis. PMID: 11163236 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 84: Immunity. 2000 Oct;13(4):549-60. Partial impairment of cytokine responses in Tyk2-deficient mice. Karaghiosoff M, Neubauer H, Lassnig C, Kovarik P, Schindler H, Pircher H, McCoy B, Bogdan C, Decker T, Brem G, Pfeffer K, Muller M. Institute of Animal Breeding and Genetics, Veterinary University of Vienna, Austria. To assess the role of the Janus kinase (Jak) family member Tyk2, we have generated Tyk2-/- mice. In contrast to other Jaks, where inactivation leads to a complete loss of the respective cytokine receptor signal, Tyk2-/- mice display reduced responses to IFNalpha/beta and IL-12 and a selective deficiency in Stat3 activation in these pathways. Unexpectedly, IFNgamma signaling is also impaired in Tyk2-/- mice. Tyk2-/- macrophages fail to produce nitric oxide upon lipopolysaccharide induction. Tyk2-/- mice are unable to clear vaccinia virus and show a reduced T cell response after LCMV challenge. These data imply a selective contribution of Tyk2 to the signals triggered by various biological stimuli and cytokine receptors. PMID: 11070173 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 85: J Immunol. 2000 Nov 1;165(9):5227-37. Inhibition of IL-6 and IL-10 signaling and Stat activation by inflammatory and stress pathways. Ahmed ST, Ivashkiv LB. Graduate Program in Immunology, Weill Graduate School of Medical Sciences, and Department of Medicine, Hospital for Special Surgery, Weill Medical College of Cornell University, New York, NY 10021, USA. The development and resolution of an inflammatory process are regulated by a complex interplay among cytokines that have pro- and anti-inflammatory effects. Effective and sustained action of a proinflammatory cytokine depends on synergy with other inflammatory cytokines and antagonism of opposing cytokines that are often highly expressed at inflammatory sites. We analyzed the effects of the inflammatory and stress agents, IL-1, TNF-alpha, LPS, sorbitol, and H(2)O(2), on signaling by IL-6 and IL-10, pleiotropic cytokines that activate the Jak-Stat signaling pathway and have both pro- and anti-inflammatory actions. IL-1, TNF-alpha, and LPS blocked the activation of Stat DNA binding and tyrosine phosphorylation by IL-6 and IL-10, but not by IFN-gamma, in primary macrophages. Inhibition of Stat activation correlated with inhibition of expression of IL-6-inducible genes. The inhibition was rapid and independent of de novo gene induction and occurred when the expression of suppressor of cytokine synthesis-3 was blocked. Inhibition of IL-6 signaling was mediated by the p38 subfamily of stress-activated protein kinases. Jak1 was inhibited at the level of tyrosine phosphorylation, indicating that inhibition occurred at least in part upstream of Stats in the Jak-Stat pathway. Experiments using Stat3 mutated at serine 727 and using truncated IL-6Rs suggested that the target of inhibition is contained within the membrane-proximal region of the cytoplasmic domain of the gp130 subunit of the IL-6 receptor and is different from the SH2 domain-containing protein-tyrosine phosphatase/suppressor of cytokine synthesis-3 docking site. These results identify a new level at which IL-1 and TNF-alpha modulate signaling by pleiotropic cytokines such as IL-6 and IL-10 and provide a molecular basis for the previously described antagonism of certain IL-6 actions by IL-1. PMID: 11046056 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 86: FEBS Lett. 2000 Sep 1;480(2-3):132-6. IL-10 attenuates IFN-alpha-activated STAT1 in the liver: involvement of SOCS2 and SOCS3. Shen X, Hong F, Nguyen VA, Gao B. Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298, USA. Interleukin-10 (IL-10) has been used in the treatment of viral hepatitis in interferon-alpha (IFN-alpha) non-responders while patients who have high levels of IL-10 are poorly responsive to IFN-alpha. The mechanism underlying such controversial functions of IL-10 remains unknown. Here we demonstrated that injection of IL-10 into mice attenuated IFN-alpha-induced signal transducer and activator transcription factor (STAT)1 tyrosine phosphorylation in the liver. Reverse transcriptase-polymerase chain reaction assay demonstrated that mouse liver expressed high levels of IL-10 receptor 2 (IL-10R2) but low levels of IL-10R1. Injection of IL-10 into mice activated STAT3 but not STAT1 tyrosine phosphorylation and induced suppressor of cytokine signal 2 (SOCS2), SOCS3, and cytokine-inducible SH2 protein (CIS) mRNA expression in the liver. Furthermore, overexpression of SOCS2 or SOCS3 inhibited IFN-alpha-induced reporter activity in hepatic cells. These findings suggest that IL-10 inhibits IFN-alpha-activated STAT1 in the liver, at least in part, by inducing SOCS2, SOCS3, and CIS expression, which may be responsible for the resistance of IFN-alpha therapy in patients who have high levels of IL-10 and recommends that IL-10 treatment for viral hepatitis should be cautious. PMID: 11034314 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 87: Am J Pathol. 2000 Oct;157(4):1177-86. Novel protective effects of stem cell factor in a murine model of acute septic peritonitis. Dependence on MCP-1. Bone-Larson CL, Hogaboam CM, Steinhauser ML, Oliveira SH, Lukacs NW, Strieter RM, Kunkel SL. Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan. University of California, Los Angeles, California, USA. Mast cells participate in the host response during sepsis and have been shown to have a protective effect in a murine model of acute septic peritonitis and multi-organ failure initiated by cecal ligation and puncture (CLP). Stem cell factor (SCF) is a hematopoietic cytokine important in mast cell proliferation and activation. In the present study, we examined the protective effects of a single intraperitoneal injection of SCF given 2 hours before CLP surgery in mice. Four days after the CLP surgery, SCF pretreatment significantly improved mouse survival from 29 to 56% and mast cells were absolutely required for this effect. Immunoneutralization studies revealed that the SCF-stimulated release of monocyte chemoattractant protein-1 (MCP-1) into the septic peritoneal cavity contributed to the protective effect of SCF in this model. One potential cellular source of MCP-1 was the SCF-activated mast cell. In addition, SCF pretreatment significantly augmented circulating levels of SCF and the immunomodulatory cytokine interleukin-10 in septic mice, in part because the SCF pretreatment seemed to promote the release of both mediators from the liver. Additional hepatic effects of SCF treatment included an accelerated expression of hepatic levels of signal transducer and activator of transcription-3 (STAT-3) in CLP mice pretreated with SCF. Taken together, the findings from the present study demonstrate that the intraperitoneal delivery of SCF has a major protective effect in a murine model of CLP. PMID: 11021822 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 88: Oncogene. 2000 Jul 27;19(32):3675-83. Oncostatin M and interleukin 6 inhibit cell cycle progression by prevention of p27kip1 degradation in HepG2 cells. Klausen P, Pedersen L, Jurlander J, Baumann H. Department of Haematology, The Finsen Centre, Rigshospitalet, Copenhagen, Denmark. We analysed the regulation of G1-phase progression in relation to cytokine receptor signalling in HepG2 hepatoma cells, stably transduced with the IL-10 receptor after stimulation with Oncostatin M (OSM), IL-6, Leukaemia Inhibitory Factor (LIF) and IL-10. All cytokines induced STAT3 phosphorylation to approximately the same level, but only OSM, and to a lesser extent IL-6, induced STAT5 phosphorylation. The cytokines also stimulated phosphorylation of ERK in the order of decreasing effectiveness: OSM > IL-6 > LIF > IL-10. The same order of activity of the cytokines was observed on inhibition of DNA synthesis and accumulation of cells in the G1-phase of the cell cycle. These processes were accompanied by a decrease in cyclin A expression and CDK2 activity, and enhanced accumulation of p27kip1. The level of p27kip1 mRNA expression was unaffected by the cytokines, and maintenance of the elevated level of p27kip1 occurred independently of de novo protein synthesis. Furthermore, inhibition of proteasomal activity increased the level of p27kip1 in the unstimulated cells to the same level as in OSM-treated cells. Inhibition of MEK activation completely abrogated OSM and IL-6 induced p27kip1 accumulation, while expression of dominant negative STAT5 decreased the OSM and IL-6 mediated inhibition of DNA-synthesis and partially inhibited p27kip1 accumulation. PMID: 10951574 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 89: J Immunol. 2000 Aug 1;165(3):1612-7. Role of Stat3 in lipopolysaccharide-induced IL-10 gene expression. Benkhart EM, Siedlar M, Wedel A, Werner T, Ziegler-Heitbrock HW. Institute for Immunology, University of Munich, Munich, Germany; Institute of Mammalian Genetics, Neuherberg, Germany; and Genomatix Software GmbH, Munich, Germany. IL-10 is a unique cytokine because it is anti-inflammatory and immunosuppressive. IL-10 is regulated at the level of transcription, but the critical motifs and the relevant transcription factors controlling this gene have remained elusive to date. We now report that a sequence at -120 bp in the human IL-10 promoter binds Stat3 but no other Stat proteins. Mutation of this motif abrogates LPS-induced trans-activation. Overexpression of dominant negative Stat3 suppresses promoter activity, while wild-type Stat3 leads to an enhancement of this activity. Our results show that Stat3, by binding to a single motif in the IL-10 promoter, is controlling expression of the human IL-10 gene. PMID: 10903771 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 90: J Immunol. 2000 May 1;164(9):4607-15. Stat3-dependent induction of p19INK4D by IL-10 contributes to inhibition of macrophage proliferation. O'Farrell AM, Parry DA, Zindy F, Roussel MF, Lees E, Moore KW, Mui AL. Department of Molecular Biology, DNAX Research Institute, Palo Alto, CA 94304, USA. anne_marie.ofarrell@pharma.novartis.com We have previously reported that IL-10 inhibits proliferation of normal bone marrow-derived macrophages and of the monocyte/macrophage cell line J774. Activation of Stat3 was shown to be necessary and sufficient to mediate inhibition of proliferation. To investigate further the mechanism of growth arrest, we examined the effect of IL-10 on expression of cell cycle inhibitors. We found that IL-10 treatment increases expression of the cyclin-dependent kinase inhibitors p19INK4D and p21CIP1 in macrophages. IL-10 cannot induce p19INK4D expression or block proliferation when Stat3 signaling is blocked by a dominant negative Stat3 or a mutant IL-10Ralpha which does not recruit Stat3 in J774 cells, whereas p21CIP1 induction is not affected. An inducibly active Stat3 (coumermycin-dimerizable Stat3-Gyrase B), which suppresses J774 cell proliferation, also induced p19INK4D expression. Sequencing of the murine p19INK4D promoter revealed two candidate Stat3 binding sites, and IL-10 treatment activated a reporter gene controlled by this promoter. These data suggest that Stat3-dependent induction of p19INK4D mediates inhibition of proliferation. Enforced expression of murine p19INK4D cDNA J774 cells significantly reduced their proliferation. Use of antisense p19INK4D and analysis of p19INK4D-deficient macrophages confirmed that p19INK4D is required for optimal inhibition of proliferation by IL-10, and indicated that additional IL-10 signaling events contribute to this response. These data indicate that Stat3-dependent induction of p19INK4D and Stat3-independent induction of p21CIP1 are important components of the mechanism by which IL-10 blocks proliferation in macrophages. PMID: 10779764 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 91: Proc Natl Acad Sci U S A. 2000 Feb 15;97(4):1695-700. Human cytomegalovirus harbors its own unique IL-10 homolog (cmvIL-10). Kotenko SV, Saccani S, Izotova LS, Mirochnitchenko OV, Pestka S. Department of Molecular Genetics, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ 08854-5635, USA. kotenkse@umdnj.edu We identified a viral IL-10 homolog encoded by an ORF (UL111a) within the human cytomegalovirus (CMV) genome, which we designated cmvIL-10. cmvIL-10 can bind to the human IL-10 receptor and can compete with human IL-10 for binding sites, despite the fact that these two proteins are only 27% identical. cmvIL-10 requires both subunits of the IL-10 receptor complex to induce signal transduction events and biological activities. The structure of the cmvIL-10 gene is unique by itself. The gene retained two of four introns of the IL-10 gene, but the length of the introns was reduced. We demonstrated that cmvIL-10 is expressed in CMV-infected cells. Thus, expression of cmvIL-10 extends the range of counter measures developed by CMV to circumvent detection and destruction by the host immune system. PMID: 10677520 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 92: J Exp Med. 2000 Jan 17;191(2):213-24. A single amino acid determines the immunostimulatory activity of interleukin 10. Ding Y, Qin L, Kotenko SV, Pestka S, Bromberg JS. Department of Microbiology and Immunology, The University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA. Cellular interleukin 10s (cIL-10s) of human and murine origin have extensive sequence and structural homology to the Epstein-Barr virus BCRF-I gene product, known as viral IL-10 (vIL-10). Although these cytokines share many immunosuppressive properties, vIL-10 lacks several of the immunostimulatory activities of cIL-10 on certain cell types. The molecular and cellular bases for this dichotomy are not currently defined. Here, we show that the single amino acid isoleucine at position 87 of cIL-10 is required for its immunostimulatory function. Substitution of isoleucine in cIL-10 with alanine, which corresponds to the vIL-10 residue, abrogates immunostimulatory activity for thymocytes, mast cells, and alloantigenic responses while preserving immunosuppressive activity for inhibition of interferon gamma production and prolongation of cardiac allograft survival. Conversely, substitution of alanine with isoleucine in vIL-10 converts it to a cIL-10-like molecule with immunostimulatory activity. This single conservative residue alteration significantly affects ligand affinity for receptor; however, affinity changes do not necessarily alter specific activities for biologic responses in a predictable fashion. These results suggest complex regulation of IL-10 receptor-ligand interactions and subsequent biological responses. These results demonstrate that vIL-10 may represent a captured and selectively mutated cIL-10 gene that benefits viral pathogenesis by leading to ineffective host immune responses. The ability to manipulate the activity of IL-10 in either a stimulatory or suppressive direction may be of practical value for regulating immune responses for disease therapy, and of theoretical value for determining what aspects of IL-10 activity are important for normal T cell responses. PMID: 10637267 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 93: Blood. 1999 Oct 15;94(8):2880-9. Interleukin-10 (IL-10) selectively enhances CIS3/SOCS3 mRNA expression in human neutrophils: evidence for an IL-10-induced pathway that is independent of STAT protein activation. Cassatella MA, Gasperini S, Bovolenta C, Calzetti F, Vollebregt M, Scapini P, Marchi M, Suzuki R, Suzuki A, Yoshimura A. Department of Pathology, University of Verona, Verona, Italy. MCNCSS@borgoroma.univr.it We have recently shown that, in human neutrophils, interleukin-10 (IL-10) fails to induce specific DNA-binding activities to the gamma-interferon response region (GRR), a regulatory element located in the FcgammaRI gene promoter, which is required for transcriptional activation by IL-10 and interferon gamma (IFNgamma) in monocytic cells. In this study, we report that IL-10 is also unable to induce the binding of STAT1 or STAT3 to the serum-inducible element (hSIE/m67), despite the fact that both proteins are expressed in neutrophils. Whereas IFNgamma and granulocyte colony-stimulating factor (G-CSF) are efficient inducers of STAT1 and STAT3 tyrosine phosphorylation in polymorphonuclear neutrophils (PMN), IL-10 fails to trigger STAT1 and STAT3 tyrosine and serine phosphorylation, therefore explaining its inability to induce the FcgammaRI expression in these cells. By contrast, we demonstrate that IL-10 alone represents an efficient stimulus of CIS3/SOCS3 mRNA expression in neutrophils. CIS3/SOCS3 belongs to the recently cloned cytokine-inducible SH2-containing protein (CIS) gene family (which also includes CIS1, CIS2, CIS4, CIS5, and JAB) that is believed to be, at least in part, under the control of STAT transcription factors and whose products are potential modulators of cytokine signaling. Moreover, IL-10 synergizes with lipopolysaccharide (LPS) in upregulating CIS3/SOCS3 mRNA expression in PMN through a mechanism that involves mRNA stabilization. In contrast to CIS3/SOCS3, mRNA transcripts encoding other family members are unaffected by IL-10 in neutrophils. Finally, transfection of CIS3/SOCS3 in murine M1 myeloid cells suppresses LPS-induced growth arrest, macrophage-like differentiation, and nitric oxide synthesis, but not IL-6 mRNA expression. Collectively, our data suggest that, in neutrophils, the activation of STAT1 and STAT3 phosphorylation is neither required for CIS3/SOCS3 induction by IL-10 nor involved in the regulatory effects of IL-10 on cytokine production. PMID: 10515892 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 94: Immunology. 1999 Jun;97(2):226-31. Interleukin-10 activates heat-shock protein 90beta gene expression. Ripley BJ, Stephanou A, Isenberg DA, Latchman DS. Department of Molecular Pathology, The Windeyer Institute of Medical Sciences, University College London, London, UK. Elevated levels of the cytokine interleukin-10 (IL-10) have been reported in patients with active systemic lupus erythematosus (SLE). Any role for IL-10 in the pathogenesis of SLE is likely to involve the activation of expression of specific genes within its target cells. We have previously reported elevated levels of the 90 000 MW heat-shock protein (hsp 90) and autoantibodies to hsp 90 in patients with SLE. Recent studies have shown that the cytokine IL-6 activates hsp 90 gene expression via specific transcription factors that include STAT-3 (signal transducer and activator of transcription 3). In view of the known role of STAT proteins in IL-10 signalling pathways, we have investigated the effect of IL-10 on hsp 90 gene expression. Here we report that IL-10 enhances the expression of hsp 90 in both a human hepatoma cell line (HepG2) stably expressing the human IL-10 receptor and peripheral blood mononuclear cells (PBMC). In reporter gene assays IL-10 is able to activate both the hsp 90alpha and hsp 90beta promoters directly. Furthermore, a short region of the hsp 90beta promoter which is activated in response to IL-10, contains a STAT-3 binding site. This element but not a mutant derivative unable to bind STAT-3, is able to confer a response to IL-10 on a heterologous promoter. These results may be understood in terms of the shared signalling mechanisms of IL-10 and IL-6 and provide evidence of a role for IL-10 in the overexpression of hsp 90 in SLE, with possible pathological consequences. PMID: 10447736 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 95: J Interferon Cytokine Res. 1999 Jun;19(6):563-73. The interleukin-10 signal transduction pathway and regulation of gene expression in mononuclear phagocytes. Donnelly RP, Dickensheets H, Finbloom DS. Division of Cytokine Biology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA. donnelly@cber.fda.gov Interleukin-10 (IL-10) activates a diverse array of functional responses in mononuclear phagocytes. Functional IL-10 receptor (IL-10R) complexes are tetramers consisting of two IL-10R1 polypeptide chains and two IL-10R2 chains. Binding of IL-10 to the extracellular domain of IL-10R1 activates phosphorylation of the receptor-associated Janus tyrosine kinases, JAK1 and Tyk2. These kinases then phosphorylate specific tyrosine residues (Y446 and Y496) on the intracellular domain of the IL-10R1 chain. Once phosphorylated, these tyrosine residues (and their flanking peptide sequences) serve as temporary docking sites for the latent transcription factor, STAT3 (signal transducer and activator of transcription-3). STAT3 binds to these sites via its SH2 (Src homology 2) domain, and is, in turn, tyrosine-phosphorylated by the receptor-associated JAKs. It then homodimerizes and translocates to the nucleus where it binds with high affinity to STAT-binding elements (SBE) in the promoters of various IL-10-responsive genes. One of these genes, SOCS-3 (Suppressor of Cytokine Signaling-3) is a member of a newly identified family of genes that inhibit JAK/STAT-dependent signaling. Moreover, the ability of IL-10 to induce de novo synthesis of SOCS-3 in monocytes correlates with its ability to inhibit expression of many genes in these cells, including endotoxin-inducible cytokines such as tumor necrosis factor-alpha (TNF-alpha) and IL-1. Thus, the ability of IL-10 to inhibit gene expression in monocytes is associated with its ability to rapidly induce synthesis of SOCS-3. Publication Types: Review Review, Tutorial PMID: 10433356 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 96: J Immunol. 1999 Mar 1;162(5):2457-61. Cutting edge: clustered AU-rich elements are the target of IL-10-mediated mRNA destabilization in mouse macrophages. Kishore R, Tebo JM, Kolosov M, Hamilton TA. Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44122, USA. In the present study we show that IL-10-mediated inhibition of inflammatory gene expression can be mediated by an AU-rich element (ARE) cluster present in the 3' untranslated region (3'UTR) of sensitive genes. A series of chloramphenicol acetyl transferase (CAT) reporter gene constructs were prepared in which different fragments from the IL-10-sensitive KC mRNA 3'UTR were placed downstream of the coding region of the reporter gene CAT. CAT mRNA containing the KC 3'UTR was markedly destabilized as compared with the control CAT mRNA, and the decay rate was further increased in cells stimulated with IL-10. The KC 3'UTR contains an ARE cluster and three isolated ARE motifs. The ARE cluster spanning nucleotides 378-399 appeared to be both necessary and sufficient to mediate sensitivity to IL-10 because a 116-nucleotide fragment that contains the cluster conferred sensitivity, while mutation of the sequence between positions 378 and 399 eliminated sensitivity. The destabilizing effect of IL-10 was relatively selective, as the stability of chimeric CAT mRNAs was not modulated in cells treated with IFN-gamma or IL-4. PMID: 10072482 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 97: Immunity. 1999 Jan;10(1):39-49. Enhanced Th1 activity and development of chronic enterocolitis in mice devoid of Stat3 in macrophages and neutrophils. Takeda K, Clausen BE, Kaisho T, Tsujimura T, Terada N, Forster I, Akira S. Department of Biochemistry, Hyogo College of Medicine, Nishinomiya, Japan. We have generated mice with a cell type-specific disruption of the Stat3 gene in macrophages and neutrophils. The mutant mice are highly susceptible to endotoxin shock with increased production of inflammatory cytokines such as TNF alpha, IL-1, IFN gamma, and IL-6. Endotoxin-induced production of inflammatory cytokines is augmented because the suppressive effects of IL-10 on inflammatory cytokine production from macrophages and neutrophils are completely abolished. The mice show a polarized immune response toward the Th1 type and develop chronic enterocolitis with age. Taken together, Stat3 plays a critical role in deactivation of macrophages and neutrophils mainly exerted by IL-10. PMID: 10023769 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 98: Shock. 1998 Nov;10(5):309-18. Therapeutic implications of interleukin-10 in surgical disease. Selzman CH, Shames BD, Miller SA, Pulido EJ, Meng X, McIntyre RC Jr, Harken AH. Department of Surgery, University of Colorado Health Sciences Center and The Veterans Affairs Hospital, Denver 80262, USA. craig.selzman@UCHSC.edu Pharmacological therapy of surgical disease often involves manipulating the physiologic balance between pro- and anti-inflammatory responses. Many agents target only one aspect of the inflammatory cascade. Originally identified as a protein elaborated by T-lymphocytes, IL-10 appears to globally inhibit cytokine production. The purpose of this manuscript is to examine the immunomodulatory and anti-inflammatory effects of interleukin-10 (IL-10) in an attempt to define the clinical utility of IL-10, both as a marker of and as a therapeutic strategy for intervention in inflammatory and immune-mediated diseases. IL-10 is elaborated from multiple sources and has diverse cellular effects to regulate immune and inflammatory responses. Accumulating evidence suggests that the anti-inflammatory influence of IL-10 observed at the cellular level may be manipulated to impact the immune and inflammatory-mediated responses associated with injury and sepsis, gastrointestinal and cardiovascular disease, and transplantation. In conclusion, IL-10 is an important mediator of immune and anti-inflammatory responses in surgical disease and, as such, has therapeutic promise as an immunomodulator and as an anti-inflammatory agent. Publication Types: Review Review, Tutorial PMID: 9840644 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 99: J Immunol. 1998 Jan 15;160(2):911-9. High affinity receptor for IgG (Fc gamma RI/CD64) gene and STAT protein binding to the IFN-gamma response region (GRR) are regulated differentially in human neutrophils and monocytes by IL-10. Bovolenta C, Gasperini S, McDonald PP, Cassatella MA. Department of General Pathology, University of Verona, Italy. Since IL-10 has been shown to up-regulate the expression of the high affinity receptor for IgG (FcgammaRI/CD64) in human monocytes, we examined whether the cytokine exerts a similar action toward polymorphonuclear neutrophils (PMN). Unexpectedly, we found that in neutrophils, IL-10 failed to induce either the mRNA accumulation or the surface expression of FcgammaRI. Consistent with these findings, stimulation of PMN with IFN-gamma, but not with IL-10, resulted in the induction of specific DNA-binding activities to the IFN-gamma response region (GRR), a regulatory element located in the FcgammaRI gene promoter, required for transcriptional activation. In electrophoretic mobility shift assays (EMSAs), we confirmed that in PBMC, IL-10 induces the binding to the GRR of both STAT1 and STAT3, two members of the STAT family. In neutrophils, however, these activators did not bind to the GRR in response to IL-10, despite the fact that both STAT1 and STAT3 are expressed in these cells. On the other hand, IFN-gamma was an efficient inducer of STAT1 binding to the GRR in both PMN and PBMC. The lack of inducible GRR-binding activity in IL-10-treated PMN could not be ascribed to a lack of IL-10R, and did not appear to reflect an inhibitory effect of the cytokine. Taken together, our data suggest that IL-10 is unable to induce FcgammaRI gene expression in neutrophils because the intracellular signaling pathway triggered by the cytokine is impaired at the level of, or upstream of, STAT1 and/or STAT3 activation. PMID: 9551929 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 100: EMBO J. 1998 Feb 16;17(4):1006-18. IL-10 inhibits macrophage activation and proliferation by distinct signaling mechanisms: evidence for Stat3-dependent and -independent pathways. O'Farrell AM, Liu Y, Moore KW, Mui AL. Department of Molecular Biology, DNAX Research Institute, Palo Alto, CA 94304-1104, USA. Interleukin-10 (IL-10) limits inflammatory responses by inhibiting macrophage activation. In macrophages, IL-10 activates Stat1 and Stat3. We characterized IL-10 responses of the J774 mouse macrophage cell line, and of J774 cells expressing wild-type hIL-10R, mutant hIL-10R lacking two membrane-distal tyrosines involved in recruitment of Stat3 (hIL-10R-TyrFF), a truncated Stat3 (DeltaStat3) which acts as a dominant negative, or an inducibly active Stat3-gyraseB chimera (Stat3-GyrB). A neutralizing anti-mIL-10R monoclonal antibody was generated to block the function of endogenous mIL-10R. IL-10 inhibited proliferation of J774 cells and of normal bone marrow-derived macrophages, but not J774 cells expressing hIL-10RTyrFF. Dimerization of Stat3-GyrB by coumermycin mimicked the effect of IL-10, and expression of DeltaStat3 blocked the anti-proliferative activity of IL-10. For macrophage de-activation responses, hIL10R-TyrFF could not mediate inhibition of lipopolysaccharide-induced TNFalpha, IL-1beta or CD86 expression, while DeltaStat3 did not interfere detectably with these IL-10 responses. Thus signals mediating both anti-proliferative and macrophage de-activation responses to IL-10 require the two membrane-distal tyrosines of IL-10R, but Stat3 appears to function only in the anti-proliferative response. PMID: 9463379 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 101: Blood. 1997 Jun 1;89(11):4146-52. Characterization of interleukin-10 receptor expression on B-cell chronic lymphocytic leukemia cells. Jurlander J, Lai CF, Tan J, Chou CC, Geisler CH, Schriber J, Blumenson LE, Narula SK, Baumann H, Caligiuri MA. Department of Molecular and Cell Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA. B-cell chronic lymphocytic leukemia (B-CLL) cells accumulate in vivo in the G0/G1 phase of the cell cycle, suggesting that their malignant expansion is due, at least in part, to a delay in cell death. However, the cellular or molecular factors responsible for a delay in B-CLL cell death are unknown. B-CLL cells do express receptors for interferon-alpha (IFN-alpha) and IFN-gamma, and activation of both has been shown to promote B-CLL survival in vitro by preventing apoptosis. The interleukin-10 (IL-10) receptor is another member of the IFN receptor family, but its ligand, IL-10, has been reported to induce apoptosis in B-CLL cells. In the current study, we undertook a biochemical analysis of IL-10 receptor expression on freshly isolated B-CLL cells and characterized the functional responsiveness of IL-10 binding to its constitutively expressed receptor. We show that B-CLL cells bind IL-10 with significant specificity and express between 47 and 127 IL-10 receptor sites per cell, with a dissociation constant in the range of 168 to 426 x 10(-12) mol/L. Ligand binding and activation of the IL-10 receptor expressed on B-CLL cells results in the phosphorylation of signal transducer and activator of transcription 1 (STAT1) and STAT3 proteins. This pattern of STAT protein phosphorylation is identical to IL-10 receptor activation on normal cells and similar to IFN-alpha (STAT1 and STAT3) and IFN-gamma (STAT1) receptor activation in CLL. Further, in consecutive samples of fresh blood obtained from patients with B-CLL cells, the addition of IL-10 inhibited B-CLL proliferation, enhanced B-CLL differentiation, but did not induce apoptosis. Indeed, IL-10, like IFN-gamma, was able to significantly reduce the amount of B-CLL cell death caused by hydrocortisone-induced apoptosis. We conclude that cytokines, which signal through the interferon family of receptors, have comparable functional effects on B-CLL cells. PMID: 9166857 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 102: Neurochem Int. 1997 Apr-May;30(4-5):433-9. Biological activity of interleukin-10 in the central nervous system. Zocchia C, Spiga G, Rabin SJ, Grekova M, Richert J, Chernyshev O, Colton C, Mocchetti I. Department of Cell Biology, Georgetown University, School of Medicine, Washington, DC 20007, USA. Cytokines play a crucial role as mediators of inflammation. Astrocytes and microglia are the two major glial cells involved in the central nervous system immune responses. In this study we examined the effects of interleukin-10 (IL-10), one of the naturally occurring inhibitory cytokines, on different types of glial cells in culture such as rat astrocytes, hamster microglia and C6-2B glioma cells. Phosphorylation of signal transducers and activators of transcription (STAT) proteins was used as a marker for IL-10 activity. Within minutes, IL-10 elicited a strong and weak increase in STAT3 and STAT1 phosphorylation, respectively, in human T lymphocytes, suggesting that STAT3 is a main IL-10 signaling event in these cells. In contrast, IL-10 failed to induce STAT3 in glial cells, but elicited a weak increase in STAT1 phosphorylation in microglia and C6-2B glioma cells only, suggesting that in some glial cell population(s) IL-10 may produce cellular responses via activation of the STAT1 pathway. Moreover, in C6-2B cells, IL-10 elicited a decrease in the level of basic fibroblast growth factor mRNA. A similar decrease was observed in adult rat hypothalamus, indicating that this cytokine may regulate glial production of trophic factors. Our data suggest that IL-10 may play a role in glial cell differentiation and proliferation. PMID: 9106258 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 103: J Biol Chem. 1996 Nov 1;271(44):27954-61. Stat3 recruitment by two distinct ligand-induced, tyrosine-phosphorylated docking sites in the interleukin-10 receptor intracellular domain. Weber-Nordt RM, Riley JK, Greenlund AC, Moore KW, Darnell JE, Schreiber RD. Center for Immunology, Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110, USA. schreiber@immunology.wustl.edu Recent work has shown that IL-10 induces activation of the JAK-STAT signaling pathway. To define the mechanism underlying signal transducer and activator of transcription (STAT) protein recruitment to the interleukin 10 (IL-10) receptor, the STAT proteins activated by IL-10 in different cell populations were first defined using electrophoretic mobility shift assays. In all cells tested, IL-10 activated Stat1 and Stat3 and induced the formation of three distinct DNA binding complexes that contained different combinations of these two transcription factors. IL-10 also activated Stat5 in Ba/F3 cells that stably expressed the murine IL-10 receptor. Using a structure-function mutagenesis approach, two tyrosine residues (Tyr427 and Tyr477) in the intracellular domain of the murine IL-10 receptor were found to be redundantly required for receptor function and for activation of Stat3 but not for Stat1 or Stat5. Twelve amino acid peptides encompassing either of these two tyrosine residues in phosphorylated form coprecipitated Stat3 but not Stat1 and blocked IL-10-induced Stat3 phosphorylation in a cell-free system. In contrast, tyrosine-phosphorylated peptides containing Tyr374 or Tyr396 did not interact with Stat3 or block Stat3 activation. These data demonstrate that Stat3 but not Stat1 or Stat5 is directly recruited to the ligand-activated IL-10 receptor by binding to specific but redundant receptor intracellular domain sequences containing phosphotyrosine. This study thus supports the concept that utilization of distinct STAT proteins by different cytokine receptors is dependent on the expression of particular ligand-activatable, tyrosine-containing STAT docking sites in receptor intracellular domains. PMID: 8910398 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 104: FEBS Lett. 1996 Oct 7;394(3):365-70. IL-10 induces DNA binding activity of three STAT proteins (Stat1, Stat3, and Stat5) and their distinct combinatorial assembly in the promoters of selected genes. Wehinger J, Gouilleux F, Groner B, Finke J, Mertelsmann R, Weber-Nordt RM. University of Freiburg Medical Center, Department of Hematology and Oncology, Freiburg, Germany. Interaction of IL-10 with its receptor leads to the activation of STAT transcription factors. Herein we report the IL-10 dependent simultaneous activation of three STAT transcription factors: Stat1, Stat3, and Stat5. Upon IL-10 treatment multiple Stat proteins become simultaneously activated, and bind to different promoters with equal kinetics but form distinct homo- and heterodimeric transcriptionally active complexes depending on the STAT-consensus elements of a selected gene promoter. Upon IL-10 treatment Stat1, 3, and 5 bind to the GRR of the FcgammaRI gene, activated Stat1 and 3 bind to the SIE sequence of the c-fos promoter and transcriptionally active Stat5 assembles at the PRL-STAT consensus sequence of the beta-casein gene. Thus, functionally relevant STAT dimerization is influenced by the activated cytokine receptor as well as the specific STAT consensus sequence present in a specific gene promoter. PMID: 8830676 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 105: Blood. 1996 Aug 1;88(3):809-16. Constitutive activation of STAT proteins in primary lymphoid and myeloid leukemia cells and in Epstein-Barr virus (EBV)-related lymphoma cell lines. Weber-Nordt RM, Egen C, Wehinger J, Ludwig W, Gouilleux-Gruart V, Mertelsmann R, Finke J. Department of Hematology & Oncology, Albert-Ludwigs-University Medical Center, Freiburg, Germany. Although various molecular mechanisms of STAT protein (signal transducers and activators of transcription) activation have been identified, little is known about the functional role of STAT-dependent transcriptional activation. Herein we report the constitutive nuclear localization, phosphorylation, and DNA-binding activity of STAT proteins in leukemia cells and lymphoma cell lines. With the use of oligonucleotide probes derived from the Fc gamma RI promoter, the beta-casein promoter and a STAT-binding element in the promoter of the Bci-2 gene constitutive activation of STAT proteins was detected in untreated acute T- and C/B-leukemia cells (3 of 5 and 12 of 19 patients, respectively). Supershift analyses using Stats 1-6 specific antisera showed the constitutive DNA binding activity of Stat5 in these cells. Confocal microscopy revealed the nuclear localization of Stat5 and Western blot analyses showed tyrosine phosphorylation of Stat5 in nuclear extracts of acute leukemia cells. In contrast, peripheral blood mononuclear cells did not display constitutive STAT-DNA interaction. Further studies were performed on freshly isolated acute myeloid leukemia cells as well as on cell line derived K562, lymphoblastoid cells (LCL), and Burkitt's lymphoma cells (BL). Fluorescence microscopy, gelshift, and supershift experiments showed the nuclear localization and constitutive DNA-binding activity of Stat5 in K562 cells. Stat1 and Stat3 were constitutively activated in freshly isolated AML cells (10 of 14 patients) and in Epstein Barr virus-positive or interleukin-10 expressing permanent LCL and BL cells. Thus, these data indicate a differential pattern of STAT protein activation in lymphoid or myeloid leukemia and in lymphoma cells. PMID: 8704235 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 106: J Biol Chem. 1996 Jun 14;271(24):13968-75. Receptors for interleukin (IL)-10 and IL-6-type cytokines use similar signaling mechanisms for inducing transcription through IL-6 response elements. Lai CF, Ripperger J, Morella KK, Jurlander J, Hawley TS, Carson WE, Kordula T, Caligiuri MA, Hawley RG, Fey GH, Baumann H. Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York 14263, USA. The cytoplasmic domain of the receptor for interleukin 10 (IL-10R) contains two box 3 sequence motifs that have been identified in the signal-transducing receptor subunits for IL-6-type cytokines and noted to be required for activating STAT3 and inducing transcription through IL-6-responsive elements. To determine whether the IL-10R has signaling functions similar to IL-6R in cells normally expressing these receptors, leukocytes of the B-, T-, and NK-cell lineages were treated with either cytokine. Both cytokines activated factors that bound to the sis-inducible element and included STAT1 and STAT3. The cell response to IL-10 characteristically differed from that to IL-2/IL-15, IL-4, and interferon gamma. The signaling capabilities of the IL-10R for activating specific STAT proteins and inducing gene transcription were defined by reconstitution of receptor functions in transfected tissue culture cells. COS-1 cells, co-expressing the human IL-10R and individual STAT proteins, confirmed a preference of the IL-10R for STAT3 and STAT1. Unlike many hematopoietin receptors, the IL-10R did not detectably activate STAT5. The IL-10R, together with reporter gene constructs containing different IL-6-responsive gene elements, reconstituted in hepatoma cells an induction of transcription by IL-10 that was comparable to that by IL-6. This regulation could not be appreciably modified by enhanced expression of STAT proteins. The similar actions of IL-10R and IL-6R on the induction of endogenous IL-6-responsive genes were demonstrated in hepatoma cells stably expressing the IL-10R. These receptor functions required the presence of the box 3 motifs, as shown by the analysis of the mouse IL-10R constructs containing progressively truncated cytoplasmic domains. The data demonstrate that the IL-10R, unlike other members of the interferon receptor family, is highly effective in recruiting the signaling pathways of IL-6-type cytokine receptors. PMID: 8662928 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 107: J Immunol. 1995 Aug 1;155(3):1079-90. IL-10 induces the tyrosine phosphorylation of tyk2 and Jak1 and the differential assembly of STAT1 alpha and STAT3 complexes in human T cells and monocytes. Finbloom DS, Winestock KD. Food and Drug Administration, Center for Biologics Evaluation and Research, Bethesda, MD 20892-4555, USA. IL-10 affects monocytes and T cells by driving the progression of immune responsiveness such that Th2 lymphocyte-mediated effects predominate. In this report, we show that in monocytes and T cells IL-10 stimulates tyrosine phosphorylation of the signal transducers and activators of transcription, STAT1 alpha and STAT3, in a differential manner such that the relative formation of homo- and heterodimers varies between the two cell types. Moreover, monocytes express a novel IL-10-stimulated STAT protein with an M(r) of 70 kDa that is recognized by the anti-STAT3 Ab but is not observed in T cells. IL-10 treatment of both T cells and monocytes results in the ligand-induced tyrosine phosphorylation of tyk2 and Jak1, but not Jak2 or Jak3. Selective modulation of immune responsiveness by IL-10 in cells such as monocytes and T cells may result in part from the differential activation of STAT protein pairs. PMID: 7543512 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------