1: J Biol Chem. 2005 Sep 2;280(35):30838-44. Epub 2005 Jul 5. Vaccinia virus protein A52R activates p38 mitogen-activated protein kinase and potentiates lipopolysaccharide-induced interleukin-10. Maloney G, Schroder M, Bowie AG. Viral Immune Evasion Group, School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland. Vaccinia virus (VV) has many mechanisms to suppress and modulate the host immune response. The VV protein A52R was previously shown to act as an intracellular inhibitor of nuclear factor kappaB (NFkappaB) signaling by Toll-like receptors (TLRs). Co-immunoprecipitation studies revealed that A52R interacted with both tumor necrosis factor receptor-associated factor 6 (TRAF6) and interleukin-1 receptor-associated kinase 2 (IRAK2). The effect of A52R on signals other than NFkappaB was not determined. Here, we show that A52R does not inhibit TLR-induced p38 or c-Jun amino N-terminal kinase (JNK) mitogen activating protein (MAP) kinase activation. Rather, A52R could drive activation of these kinases. Two lines of evidence suggested that the A52R/TRAF6 interaction was critical for these effects. First, A52R-induced p38 MAP kinase activation was inhibited by overexpression of the TRAF domain of TRAF6, which sequestered A52R and inhibited its interaction with endogenous TRAF6. Second, a truncated version of A52R, which interacted with IRAK2 and not TRAF6, was unable to activate p38. Because interleukin 10 (IL-10) production is strongly p38-dependent, we examined the effect of A52R on IL-10 gene induction. A52R was found to be capable of inducing the IL-10 promoter through a TRAF6-dependent mechanism. Furthermore, A52R enhanced lipopolysaccharide/TLR4-induced IL-10 production, while inhibiting the TLR-induced NFkappaB-dependent genes IL-8 and RANTES. These results show that although A52R inhibits NFkappaB activation by multiple TLRs it can simultaneously activate MAP kinases. A52R-mediated enhancement of TLR-induced IL-10 may be important to virulence, given the role of IL-10 in immunoregulation. PMID: 15998638 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: Infect Immun. 2005 Jul;73(7):3990-8. Role of mitogen-activated protein kinases and NF-kappaB in the regulation of proinflammatory and anti-inflammatory cytokines by Porphyromonas gingivalis hemagglutinin B. Zhang P, Martin M, Michalek SM, Katz J. Department of Pediatric Dentistry, University of Alabama at Birmingham, 845 19th Street South, BBRB 258/5, Birmingham, Alabama 35294-2170, USA. Hemagglutinin B (HagB) is a nonfimbrial adhesin expressed on the surface of Porphyromonas gingivalis and has been implicated as a potential virulence factor involved in mediating the attachment of the bacteria to host cells. However, the molecular mechanisms underlying host responses to HagB and their roles in pathogenesis have yet to be elucidated. Mitogen-activated protein kinases (MAPKs) are activated following engagement of a variety of cell surface receptors via dual tyrosine and threonine phosphorylation and are thought to be involved in various cellular responses. The purpose of this study was to determine the role of intracellular signaling pathways including the MAPKs and NF-kappaB in regulating the production of proinflammatory and anti-inflammatory cytokines following stimulation of murine macrophages with recombinant HagB (rHagB). Stimulation of peritoneal macrophages with rHagB resulted in the production of the proinflammatory cytokines interleukin-12p40 (IL-12p40), gamma interferon (IFN-gamma), and tumor necrosis factor alpha, as well as the anti-inflammatory cytokine IL-10. We also demonstrated the activation of extracellular signal-related kinase (ERK), c-Jun NH2-terminal protein kinase (JNK), and p38 MAPKs by rHagB-stimulated macrophages. Furthermore, blocking of the ERK and p38 signaling pathways by using specific inhibitors revealed differential regulatory roles in the rHagB-mediated production of proinflammatory and anti-inflammatory cytokines. ERK and p38 were important in down-regulation of IL-12p40 and IFN-gamma production and up-regulation of IL-10 production. The enhanced levels of IL-12p40 in rHagB-stimulated macrophages by inhibition of ERK or p38 activity were partially attributable to the inhibition of IL-10 production. Moreover, NF-kappaB was found to be critical for up-regulation of IL-12p40 and down-regulation of IL-10 production in rHagB-stimulated macrophages. Taken together, our results demonstrate a role for the p38 and ERK pathways and the transcription factor NF-kappaB in modulating key immunoregulatory cytokines involved in the development of immune responses to P. gingivalis HagB. PMID: 15972486 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Neuroimmunomodulation. 2005;12(2):113-6. Evidence of an anti-inflammatory role for Vasogen's immune modulation therapy. Nolan Y, Campbell VA, Bolton AE, Lynch MA. Department of Physiology, Trinity College, Dublin, Ireland. We have reported that Vasogen's immune modulation therapy (IMT), a procedure involving intramuscular administration of autologous/syngeneic blood, which has been exposed ex vivo to increased temperature, UVC light and oxidation, prevents several LPS-induced inflammatory changes in the hippocampus. Here, we investigated neuroprotective effects of IMT in cortical tissue, and report that the treatment acts as an anti-inflammatory and antioxidative agent, reducing the concentration of TNFalpha and the accumulation of reactive oxygen species. The data couple these changes with an increase in the concentration of the anti-inflammatory cytokine IL-10, and a decrease in activation of the stress-activated protein kinase, c-jun N-terminal kinase. Consistent with these putative protective effects of IMT, we report that the LPS-induced increase in TUNEL staining, which is indicative of cell death, is prevented by IMT. PMID: 15785113 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: J Immunol. 2005 Feb 15;174(4):2098-105. Regulation of IL-10 gene expression in Th2 cells by Jun proteins. Wang ZY, Sato H, Kusam S, Sehra S, Toney LM, Dent AL. Department of Microbiology and Immunology and Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN 46202, USA. IL-10 is a key regulatory cytokine produced by T lymphocytes. Although Th2 cells are a major source of IL-10, little is known about IL-10 gene regulation in Th2 cells. High levels of IL-10 mRNA transcription are induced in the Th2 clone D10 after PMA plus ionomycin (P/I) stimulation; however we found that the IL-10 promoter was not inducible by P/I in D10 cells. We therefore sought regulatory regions in the IL-10 gene that could promote P/I-activated transcription in Th2 cells. Two strong DNase I-hypersensitive sites (DHSSs) were identified in the IL-10 gene in mouse T cells, and conserved noncoding sequences (CNSs) between the mouse and human IL-10 genes were also identified. One IL-10 DHSS maps within or next to a highly conserved CNS region, CNS-3. The CNS-3 region contains an AP-1 site that binds JunB and c-Jun proteins specifically in Th2 cells and not in Th1 cells. The CNS-3 element activates transcription from the IL-10 promoter after P/I stimulation and is responsive to c-Jun and JunB. Retroviral mediated-expression of either c-Jun or JunB in primary T cells led to a large increase in IL-10 expression, and inhibition of AP-1 activity by a dominant negative form of c-Jun in primary T cells strongly repressed IL-10 expression. IFN-gamma was relatively unaffected by modulations in AP-1 activity. These data indicate that we have identified a novel regulatory element that can specifically activate transcription of the IL-10 gene in Th2 cells via the AP-1/Jun pathway. PMID: 15699140 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Gastroenterology. 2005 Feb;128(2):376-92. Infliximab induces potent anti-inflammatory responses by outside-to-inside signals through transmembrane TNF-alpha. Mitoma H, Horiuchi T, Hatta N, Tsukamoto H, Harashima S, Kikuchi Y, Otsuka J, Okamura S, Fujita S, Harada M. Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan. BACKGROUND AND AIMS: Both infliximab (chimeric anti-tumor necrosis factor [TNF]-alpha antibody) and etanercept (p75 TNF-alpha receptor/immunoglobulin G fusion protein) are effective against rheumatoid arthritis, but only infliximab induces clinical remission in Crohn's disease. To clarify this difference in clinical efficacy, we investigated reverse signaling through transmembrane TNF-alpha (mTNF) by these 2 anti-TNF agents. METHODS: We stably transfected wild-type and cytoplasmic serine-replaced mutant forms of mTNF in human Jurkat T cells. Cells were stimulated with infliximab and etanercept and then analyzed for E-selectin expression, reactive oxygen species accumulation, apoptosis, and cell cycle distribution by flow cytometry. Interleukin-10 and interferon-gamma were measured by enzyme-linked immunosorbent assay. Phospho-c-Jun NH2-terminal kinase, Bax, Bak, p21(WAF1/CIP1), caspase-8, and caspase-3 were examined by immunoblotting. RESULTS: Both anti-TNF agents induced E-selectin expression, but only infliximab induced interleukin-10 production, apoptosis, and G0/G1 cell cycle arrest. Apoptosis and cell cycle arrest were abolished by substitution of all 3 cytoplasmic serine residues of mTNF by alanine residues. Infliximab induced accumulation of reactive oxygen species and up-regulation of Bax, Bak, and p21(WAF1/CIP1) proteins, suggesting the involvement of p53 activation. Moreover, phosphorylation of c-Jun NH2-terminal kinase was necessary for infliximab-induced apoptosis and cell cycle arrest. CONCLUSIONS: We revealed the mTNF motifs and the downstream intracellular molecular events essential for reverse signaling through mTNF. The biologic effects of mTNF elicited by infliximab should be important action mechanisms of this potent anti-inflammatory agent in addition to the neutralization of soluble TNF-alpha. These observations will provide insight into the novel role of mTNF in inflammation. PMID: 15685549 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Eur J Immunol. 2004 Jun;34(6):1695-704. AgC10, a mucin from Trypanosoma cruzi, destabilizes TNF and cyclooxygenase-2 mRNA by inhibiting mitogen-activated protein kinase p38. Alcaide P, Fresno M. Centro de Biologia Molecular, CSIC-UAM, Universidad Autonoma de Madrid, Madrid, Spain. Secretion of proinflammatory mediators by activated macrophages plays an important role in the immune response to Trypanosoma cruzi. We have previously reported that AgC10, a glycosylphosphatidylinositol-anchored mucin from T. cruzi, inhibits TNF secretion by activated macrophages (de Diego, J., Punzon, C., Duarte, M. and Fresno, M., Alteration of macrophage function bya Trypanosoma cruzi membrane mucin. J. Immunol. 1997. 159: 4983-4989). In this report we have further investigated the molecular mechanisms underlying this inhibition. AgC10 inhibited TNF, IL-10 and cyclooxygenase-2 (COX-2) synthesis by macrophages activated with LPS or LPS plus IFN-gamma in a dose-dependent manner. AgC10 did not affect other aspects of macrophage activation induced by LPS, such as inducible nitric oxide synthase (iNOS) expression. AgC10 also had no effect on TNF or COX-2 transcription or the induction of their promoters but inhibited the stability of TNF and COX-2 mRNA, which are regulated post-transcriptionally by the mitogen-activated protein kinase (MAPK) p38 pathway. AgC10 was found to inhibit both the activation and the activity of p38 MAPK, since MAPK activated protein kinase-2 (MAPKAP-K2 or MK-2) phosphorylation was also strongly inhibited. This led to TNF and COX-2 mRNA destabilization. In contrast, AgC10 did not affect p38 activation induced by TNF. Furthermore, AgC10 inhibition must lie upstream in the MAPK activation pathway by LPS, since this mucin also inhibited extracellularly regulated kinase (ERK) and Jun kinase (JNK)activation. PMID: 15162440 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: J Neuroimmunol. 2004 Jun;151(1-2):12-23. Evidence of a protective effect of phosphatidylserine-containing liposomes on lipopolysaccharide-induced impairment of long-term potentiation in the rat hippocampus. Nolan Y, Martin D, Campbell VA, Lynch MA. Department of Anatomy/Neuroscience, University College, Cork, Ireland. Lipopolysaccharide (LPS) has a negative impact on long-term potentiation (LTP) in the rat hippocampus, which has been correlated with increased concentration of interleukin-1 beta (IL-1 beta) and activation of p38 and c-Jun N-terminal kinase (JNK). It has been documented that phosphatidylserine (PS)-containing liposomes induce anti-inflammatory signals and we report that pre-treatment of rats with PS liposomes prevented these LPS-induced effects while also inhibiting microglial activation. We also observed increased concentration of the anti-inflammatory cytokine interleukin-10 (IL-10), whose intracerebroventricular injection administration mimicked the effects of PS liposomes on LTP. This suggests that administration of PS liposomes protects against the deleterious effects of LPS possibly through generation of the anti-inflammatory cytokine IL-10. PMID: 15145599 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: 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] --------------------------------------------------------------- 9: J Immunol. 2003 Dec 15;171(12):6827-37. Peroxisome proliferator activator receptor-gamma ligands, 15-deoxy-Delta(12,14)-prostaglandin J2 and ciglitazone, reduce systemic inflammation in polymicrobial sepsis by modulation of signal transduction pathways. Zingarelli B, Sheehan M, Hake PW, O'Connor M, Denenberg A, Cook JA. Division of Critical Care, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA. basilia.zingarelli@cchmc.org Peroxisome proliferator activator receptor-gamma (PPARgamma) is a nuclear receptor that controls the expression of several genes involved in metabolic homeostasis. We investigated the role of PPARgamma during the inflammatory response in sepsis by the use of the PPARgamma ligands, 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) and ciglitazone. Polymicrobial sepsis was induced by cecal ligation and puncture in rats and was associated with hypotension, multiple organ failure, and 50% mortality. PPARgamma expression was markedly reduced in lung and thoracic aorta after sepsis. Immunohistochemistry showed positive staining for nitrotyrosine and poly(ADP-ribose) synthetase in thoracic aortas. Plasma levels of TNF-alpha, IL-6, and IL-10 were increased. Elevated activity of myeloperoxidase was found in lung, colon, and liver, indicating a massive infiltration of neutrophils. These events were preceded by degradation of inhibitor kappaBalpha (IkappaBalpha), activation of IkappaB kinase complex, and c-Jun NH(2)-terminal kinase and, subsequently, activation of NF-kappaB and AP-1 in the lung. In vivo treatment with ciglitazone or 15d-PGJ(2) ameliorated hypotension and survival, blunted cytokine production, and reduced neutrophil infiltration in lung, colon, and liver. These beneficial effects of the PPARgamma ligands were associated with the reduction of IkappaB kinase complex and c-Jun NH(2)-terminal kinase activation and the reduction of NF-kappaB and AP-1 DNA binding in the lung. Furthermore, treatment with ciglitazone or 15d-PGJ(2) up-regulated the expression of PPARgamma in lung and thoracic aorta and abolished nitrotyrosine formation and poly(ADP-ribose) expression in aorta. Our data suggest that PPARgamma ligands attenuate the inflammatory response in sepsis through regulation of the NF-kappaB and AP-1 pathways. PMID: 14662889 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: Cell Mol Life Sci. 2003 Nov;60(11):2516-25. Suppression of TGF-beta signaling by conophylline via upregulation of c-Jun expression. Atsumi S, Nagasawa A, Koyano T, Kowithayakornd T, Umezawa K. Microbial Chemistry Research Center, 3-14-23 Kamioosaki, Shinagawa-ku, Tokyo 141-0021, Japan. atsumi@bikaken.or.jp In the course of screening for inhibitors of transforming-growth factor-beta (TGF-beta) functions we found that conophylline, a vinca alkaloid, inhibited TGF-beta-induced apoptosis in rat hepatoma cells. Because conophylline also inhibited TGF-b-induced promoter activity in mink lung cells, we studied the mechanism of the inhibition in this cell line. Conophylline did not inhibit nuclear translocation of Smad2. Instead, we found that conophylline increased the expression of c-Jun, which had been earlier shown to interact with the corepressor TGIF to suppress the transcriptional activity dependent on Smad2. Conophylline attenuated the interaction between the Smad2 complex and p300 but enhanced that between the Smad2 complex and TGIF. In cells overexpressing c-Jun, suppression of promoter activity induced by TGF-beta and the enhancement of the association of the Smad2 complex with TGIF were also observed. Thus, our data suggest that inhibition of TGF-beta-induced promoter activity by conophylline can be attributed to its potency in modulating the interaction of downstream transcriptional factors via upregulation of c-Jun expression. PMID: 14625694 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: J Biol Chem. 2003 Dec 19;278(51):51075-84. Epub 2003 Oct 2. Analysis of interleukin-1 beta-induced cell signaling activation in rat hippocampus following exposure to gamma irradiation. Protective effect of eicosapentaenoic acid. Lynch AM, Moore M, Craig S, Lonergan PE, Martin DS, Lynch MA. Department of Physiology, Trinity College Institute of Neuroscience, Trinity College, Dublin 2, Ireland. amlynch@tcd.ie Among the many reported effects of irradiation in cells is activation of the stress-activated protein kinase, c-Jun N-terminal kinase (JNK), which has been shown to result in apoptotic cell death. The trigger that leads to JNK activation has not been identified, although, in rat hippocampus at least, irradiation-induced apoptosis has been coupled with increased accumulation of reactive oxygen species (ROS). Significantly, irradiation-induced changes in hippocampus are abrogated by treatment of rats with the polyunsaturated fatty acid, eicosapentaenoic acid (EPA). A close coupling between ROS accumulation and concentration of the pro-inflammatory cytokine, interleukin-1 beta (IL-1 beta) in hippocampus has been reported, and the evidence suggests that IL-1 beta may be responsible for the enhanced ROS production. Here we set out to assess the possibility that whole body gamma-irradiation increases IL-1 beta concentration in hippocampus and to investigate the consequences of such a change. We present evidence that reveals that the irradiation-induced increase in IL-1 beta concentration in hippocampus is accompanied by increased expression of IL-1 type I receptor and IL-1 accessory protein and increased activation of IL-1 receptor-activated kinase. These changes, which were coupled with increased activation of JNK and evidence of apoptotic cell death, were absent in hippocampus of rats that received EPA treatment. Significantly, EPA treatment enhanced hippocampal IL-10 concentration that was inversely correlated with IL-1 beta concentration. The data are consistent with the idea that EPA exerts anti-inflammatory and neuroprotective effects in the central nervous system. PMID: 14525971 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: J Immunol. 2003 Jul 15;171(2):717-25. Role of the phosphatidylinositol 3 kinase-Akt pathway in the regulation of IL-10 and IL-12 by Porphyromonas gingivalis lipopolysaccharide. Martin M, Schifferle RE, Cuesta N, Vogel SN, Katz J, Michalek SM. Department of Microbiology, University of Alabama, Birmingham, AL 35294, USA. Stimulation of the APC by Porphyromonas gingivalis LPS has been shown to result in the production of certain pro- and anti-inflammatory cytokines. However, the signaling pathways that regulate these processes are currently unknown. In the present study, the role of the phosphatidylinositol 3 kinase (PI3K)-Akt pathway in regulating P. gingivalis LPS-induced production of IL-10, IL-12 p40, and IL-12 p70 by human monocytes was investigated. P. gingivalis LPS selectively activates the PI3K-Akt pathway via Toll-like receptor 2, and inhibition of this pathway results in an abrogation of extracellular signal-regulated kinase 1/2 phosphorylation, whereas the activation of p38 and c-Jun N-terminal kinase 1/2 kinases were unaffected. Analysis of cytokine production following stimulation of monocytes with P. gingivalis LPS revealed that inhibition of the PI3K pathway differentially regulated IL-10 and IL-12 synthesis. IL-10 production was suppressed, whereas IL-12 levels were enhanced. Inhibition of P. gingivalis LPS-mediated activation of the PI3K-Akt pathway resulted in a pronounced augmentation of NF-kappaB p65 that was independent of IkappaB-alpha degradation. Furthermore, the ability of the PI3K-Akt pathway to modulate IL-10 and IL-12 production appears to be mediated by the selective suppression of extracellular signal-regulated kinase 1/2 activity, as the MEK1 inhibitor PD98059 closely mimicked the effects of wortmannin and LY294002 to differentially regulate IL-10 and IL-12 production by P. gingivalis LPS-stimulated monocytes. These studies provide new insight into how engagement of the PI3K-Akt pathway by P. gingivalis LPS affects the induction of key immunoregulatory cytokines that control both qualitative and quantitative aspects of innate and adaptive immunity. PMID: 12847238 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: Psychiatry Clin Neurosci. 2003 Apr;57(2):153-9. Interleukin-10 gene promoter polymorphism is not associated with schizophrenia in the Korean population. Jun TY, Pae CU, Kim KS, Han H, Serretti A. Department of Psychiatry, College of Medicine, Catholic University of Korea, St Mary's Hospital, Seoul, Korea. The present study was aimed at examining the interleukin (IL)-10 gene promoter region polymorphic variants in patients with schizophrenia in the Korean population. Two hundred and thirty-three Korean patients diagnosed to have schizophrenia on the basis of Diagnostic and Statistical Manual of Mental Disorders (4th edn; DSM-IV) and 181 normal healthy controls participated in the present study. The DNA was extracted from whole blood using proteinase K and the IL-10 gene promoter region was amplified by polymerase chain reaction. Gene typing was performed by restriction fragment length polymorphism and single-strand conformation polymorphism. Distribution of the alleles and haplotypes in patients with schizophrenia was not significantly different from those of controls. The present study suggests that IL-10 gene promoter polymorphism is not associated with the development of schizophrenia in the Korean population. PMID: 12667161 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: Shock. 2003 Feb;19(2):150-6. Differential regulation of cytokines and transcription factors in liver by curcumin following hemorrhage/resuscitation. Gaddipati JP, Sundar SV, Calemine J, Seth P, Sidhu GS, Maheshwari RK. Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, USA. Inflammatory cytokines interleukin 1 (IL-1), IL-2, IL-6, and tumor necrosis factor-alpha (TNF-alpha) have been recognized as important mediators of pathophysiological and immunological events associated with shock. These inflammatory events after hemorrhage and resuscitation are characterized by the activation of transcription regulators such as nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1). Curcumin, an anti-inflammatory remedy used in Indian medicine, is known to suppress NF-kappaB and AP-1 activation and also to reduce ischemia-reperfusion injuries in animal models. Therefore, the aim of this study was to determine whether administration of curcumin before hemorrhagic shock has any salutary effects on cytokines and the redox-sensitive transcription factors NF-kappaB and AP-1. mRNA levels of IL-1alpha, IL-1beta, IL-2, IL-6, IL-10, and TNF-alpha were determined by reverse transcriptase-polymerase chain reaction in rat livers collected at 2 and 24 h after hemorrhage/resuscitation. The effect of curcumin on the activation of NF-kappaB and AP-1 was determined by electrophoretic mobility shift assays. Significant increases in the levels of liver cytokines IL-1alpha, IL-1beta, IL-2, IL-6, and IL-10 were observed in the 2-h posthemorrhage/resuscitation group compared with sham animals. In contrast, oral administration of curcumin for 7 days followed by hemorrhage/resuscitation regimen resulted in significant restoration of these cytokines to depleted levels, and, in fact, IL-1beta levels were lower than sham levels. Also, the 24-h postresuscitation group showed similar patterns with some exceptions. NF-kappaB and AP-1 were differentially activated at 2 and 24 h posthemorrhage and were inhibited by curcumin pretreatment. Serum aspartate transaminase estimates indicate decreased liver injury in curcumin-pretreated hemorrhage animals. These results suggest that protection against hemorrhage/resuscitation injury by curcumin pretreatment may result from the inactivation of transcription factors involved and regulation of cytokines to beneficial levels. PMID: 12578124 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 15: J Immunol. 2002 Nov 15;169(10):5660-72. Differential regulation of CD44 expression by lipopolysaccharide (LPS) and TNF-alpha in human monocytic cells: distinct involvement of c-Jun N-terminal kinase in LPS-induced CD44 expression. Gee K, Lim W, Ma W, Nandan D, Diaz-Mitoma F, Kozlowski M, Kumar A. Department of Pediatrics, Research Institute, Children's Hospital of Eastern Ontario, University of Ottawa, 401 Smyth Road, Ottawa, Ontario, Canada, K1H 8L1. Alterations in the regulation of CD44 expression play a critical role in modulating cell adhesion, migration, and inflammation. LPS, a bacterial cell wall component, regulates CD44 expression and may modulate CD44-mediated biological effects in monocytic cells during inflammation and immune responses. In this study, we show that in normal human monocytes, LPS and LPS-induced cytokines IL-10 and TNF-alpha enhance CD44 expression. To delineate the mechanism underlying LPS-induced CD44 expression, we investigated the role of the mitogen-activated protein kinases (MAPKs), p38, p42/44 extracellular signal-regulated kinase, and c-Jun N-terminal kinase (JNK) by using their specific inhibitors. We demonstrate the involvement, at least in part, of p38 MAPK in TNF-alpha-induced CD44 expression in both monocytes and promonocytic THP-1 cells. However, neither p38 nor p42/44 MAPKs were involved in IL-10-induced CD44 expression in monocytes. To further dissect the TNF-alpha and LPS-induced signaling pathways regulating CD44 expression independent of IL-10-mediated effects, we used IL-10 refractory THP-1 cells as a model system. Herein, we show that CD44 expression induced by the LPS-mediated pathway predominantly involved JNK activation. This conclusion was based on results derived by transfection of THP-1 cells with a dominant-negative mutant of stress-activated protein/extracellular signal-regulated kinase kinase 1, and by exposure of cells to JNK inhibitors dexamethasone and SP600125. All these treatments prevented CD44 induction in LPS-stimulated, but not in TNF-alpha-stimulated, THP-1 cells. Furthermore, we show that CD44 induction may involve JNK-dependent early growth response gene activation in LPS-stimulated monocytic cells. Taken together, these results suggest a predominant role of JNK in LPS-induced CD44 expression in monocytic cells. PMID: 12421945 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 16: Shock. 2002 Oct;18(4):336-41. Kinetics of mitogen-activated protein kinase family in lipopolysaccharide-stimulated mouse Kupffer cells and their role in cytokine production. Jiang JX, Zhang Y, Ji SH, Zhu P, Wang ZG. Research Institute of Surgery, Daping Hospital, The Third Military Medical University, Chongqing, China. This study was designed to systemically investigate the kinetics of extracellular signal-regulated kinase (ERK) 1/2, p54 c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinases (MAPKs) in lipopolysaccharide (LPS)-stimulated Kupffer cells (KC) simultaneously at the levels of protein expression, phosphorylation, and kinase activity, respectively, and their role in mediating pro- and anti-inflammatory cytokines. The protein expression, phosphorylation, and activities of these MAPKs in LPS-stimulated primary mouse KCs were determined with SDS-PAGE and western blotting using nonphosphorylated or phosphospecific antibodies or their corresponding substrates. The levels of TNF-alpha and IL-10 in culture supernatants were measured with ELISA kits. The results revealed that LPS stimulation, although not up- or downregulating the protein expression of ERK1/2, p54JNK, and p38 MAPKs in KCs, could induce rapid and significant activation of these kinases, with parallel profiles of changes in both phosphorylation and kinase activities. Although ERK1/2, p54JNK, and p38 kinases in LPS-stimulated KCs have similar kinetics of activation, the activation of ERK1/2 and p38 kinases was the most prominent. Inhibition of p38 MAPK with SB203580 inhibited the production of TNF-alpha and IL-10 by LPS-stimulated KCs, whereas blockade of ERK1/2 with PD98059 could reduce TNF-alpha production, but did not affect IL-10 production. Furthermore, PD98059 and SB203580 had an additive effect on TNF-alpha production, but PD98059 did not augment the SB203580-induced inhibition of IL-10 production. These data indicate that LPS stimulation, although not inducing any change in protein expression, results in rapid activation of ERK1/2, p54JNK, and p38 kinases in KCs, and that they may have different importance in the regulation of pro- and anti-inflammatory responses by LPS-stimulated KCs. PMID: 12392277 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 17: J Immunol. 2002 Oct 15;169(8):4522-30. Pseudomonas aeruginosa activates human mast cells to induce neutrophil transendothelial migration via mast cell-derived IL-1 alpha and beta. Lin TJ, Garduno R, Boudreau RT, Issekutz AC. Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada, B3J 3G9. tong-jun.lin@dal.ca The mechanisms of neutrophil (PMN) recruitment to Pseudomonas aeruginosa infection remain incompletely defined. Mast cells (MC) involvement in this process has not been studied previously. In this study, we demonstrate that human cord blood-derived MC phagocytose P. aeruginosa and release mediators that activate HUVEC monolayers for supporting PMN transmigration. Pretreatment of supernatants from P. aeruginosa-MC cocultures with neutralizing anti-IL-1alpha plus anti-IL-1beta Abs, or IL-1R antagonist before addition to HUVEC for stimulation completely abrogated MC-induced PMN transmigration, while anti-TNF-alpha treatment had no effect. The expression of E-selectin and ICAM-1 on HUVEC, the latter a ligand for PMN CD11/CD18, was significantly up-regulated by P. aeruginosa-induced MC mediators. Pretreatment of human PMN with anti-CD18 mAb or pretreatment of HUVEC with a combination of three mAbs (against ICAM-1, ICAM-2, and E-selectin) inhibited by 85% the MC-dependent PMN transmigration. Moreover, P. aeruginosa-induced production of IL-1alpha and IL-1beta was down-regulated by IL-10 and dexamethasone. This study demonstrates for the first time that MC may mediate P. aeruginosa-induced PMN recruitment via production of IL-1alpha and beta. These findings have important implications for diseases involving P. aeruginosa infection and suggest novel targets for modulating P. aeruginosa-induced inflammation. PMID: 12370389 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 18: J Immunol. 2002 Oct 1;169(7):3801-10. Th2 cytokine production from mast cells is directly induced by lipopolysaccharide and distinctly regulated by c-Jun N-terminal kinase and p38 pathways. Masuda A, Yoshikai Y, Aiba K, Matsuguchi T. Laboratory of Host Defense and Germfree Life, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya, Japan. amasuda@med.nagoya-u.ac.jp Mast cells secrete multiple cytokines and play an important role in allergic inflammation. Although it is widely accepted that bacteria infection occasionally worsens allergic airway inflammation, the mechanism has not been defined. In this study, we show that LPS induced Th2-associated cytokine production such as IL-5, IL-10, and IL-13 from mast cells and also synergistically enhanced production of these cytokines induced by IgE cross-linking. LPS-mediated Th2-type cytokine production was abolished in mouse bone marrow-derived mast cells derived from C3H/HeJ mice, suggesting that Toll-like receptor 4 is essential for the cytokine production. Furthermore, we found that mitogen-activated protein kinases including extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase, and p38 kinase were activated by LPS stimulation in bone marrow-derived mast cells. Inhibition of extracellular signal-regulated kinase activation has little effect on LPS-mediated cytokine production. In contrast, inhibition of c-Jun N-terminal kinase activation significantly suppressed both IL-10 and IL-13 expression at both mRNA and protein levels. Interestingly, although inhibition of p38 did not down-regulate the mRNA induction, it moderately decreased all three cytokine productions by LPS. These results indicate that LPS-mediated production of IL-5, IL-10, and IL-13 was distinctly regulated by mitogen-activated protein kinases. Our findings may indicate a clue to understanding the mechanisms of how bacteria infection worsens the clinical features of asthma. PMID: 12244175 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 19: Neuroimmunomodulation. 2002;10(1):40-6. Attenuation of LPS-induced changes in synaptic activity in rat hippocampus by Vasogen's Immune Modulation Therapy. Nolan Y, Minogue A, Vereker E, Bolton AE, Campbell VA, Lynch MA. Department of Physiology, Trinity College, Dublin, Ireland. Systemic injection of lipopolysaccharide (LPS) blocks the expression of long-term potentiation in the hippocampus of the rat. This is coupled with increased IL-1beta concentration and c-Jun NH(2)-terminal kinase activity, as well as an increase in the number of cells displaying apoptotic characteristics in the hippocampus. Vasogen's Immune Modulation Therapy (IMT) is a procedure involving intramuscular administration of syngeneic blood which has been exposed ex vivo to elevated temperature, oxidation and ultraviolet light. We report that Vasogen's IMT significantly abrogates these LPS-induced effects with a concomitant increase in the concentration of the anti-inflammatory cytokine IL-10. These data suggest that Vasogen's IMT may play a protective role against the deleterious effects of immune insults in the brain. Copyright 2002 S. Karger AG, Basel PMID: 12207162 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 20: Surgery. 2002 Aug;132(2):360-4. Impaired activation of mitogen-activated protein kinases after hemorrhagic shock. Khadaroo RG, Lu Z, Powers KA, Papia G, Kapus A, Rotstein OD. Department of Surgery, University Health Network, and University of Toronto, Ontario, Canada. BACKGROUND: Patients sustaining major trauma are at risk of developing organ dysfunction. We have previously shown that resuscitated hemorrhagic shock primes for increased lung injury in response to lippolysaccharide (LPS), in part by preventing upregulation of the counterinflammatory cytokine IL-10. Because the mitogen-activated protein kinase (MAPK) family is known to participate in LPS signaling, we hypothesized that altered upstream signaling through these kinases might contribute to impaired LPS-simulated IL-10 release after shock and resuscitation. METHODS: Rats were bled to a mean arterial pressure of 40 mm Hg and maintained for 1 hour, then resuscitated. Alveolar macrophages were retrieved at the end of resuscitation and exposed to LPS (0.5 microg/mL). Western blotting for p38, extracellular-regulated protein kinase, and c-Jun NH2-terminal kinase was performed on whole cell lysates. In some studies, the alveolar macrophages were preincubated with the p38 inhibitor or the extracellular-regulated protein kinase inhibitor before LPS stimulation. IL-10 levels were measured by enzyme-linked immunosorbent assay. RESULTS: LPS caused an early activation in all members of the MAPK family, whereas antecedent shock both delayed and attenuated the LPS induction. To discern whether this reduction in LPS-stimulated MAPK activation after shock might contribute to reduced IL-10, specific inhibitors were used. Inhibition of p38 MAPK completely inhibited LPS-induced IL-10 production, whereas blockade of extracellular-regulated protein kinase pathway had no effect. CONCLUSIONS: Shock resuscitation impairs LPS-induced activation of the members of the MAPK family. For the critical counterinflammatory cytokine IL-10, inhibition of p38 activation appears to contribute to the reduced levels of this cytokine in response to LPS. This study provides in vitro evidence for altered signaling through p38 MAPK, as a mechanism leading to failed upregulation of a counterinflammatory cytokine, and thus the propagation of an unrestrained proinflammatory state. Restoration of normal signaling may represent an effective strategy to reverse this effect. PMID: 12219035 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 21: J Cell Biochem. 2002;86(1):1-11. Inhibition of the p38 pathway upregulates macrophage JNK and ERK activities, and the ERK, JNK, and p38 MAP kinase pathways are reprogrammed during differentiation of the murine myeloid M1 cell line. Hall JP, Davis RJ. Howard Hughes Medical Institute and Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA. Mitogen-activated protein (MAP) kinases have been implicated as important mediators of the inflammatory response. Here we report that c-Jun NH(2)-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 MAP kinase activities are reprogrammed during the IL-6 induced macrophage-like differentiation of the murine myeloid M1 cell line. Moreover, p38 inhibition upregulates JNK and ERK activity in M1 cells and in thioglycollate-elicited peritoneal exudate macrophages. IL-6-induced M1 differentiation also induces expression of the anti-inflammatory cytokine IL-10, and p38 inhibition potentiates this increase in IL-10 expression in an ERK-dependent manner. Thus, we speculate that during inflammatory conditions in vivo macrophage p38 may regulate JNK and ERK activity and inhibit IL-10 expression. These data highlight the importance of p38 in the molecular mechanisms of macrophage function. Copyright 2002 Wiley-Liss, Inc. PMID: 12112010 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 22: Eur J Pharmacol. 2002 Jun 20;446(1-3):177-85. A novel anti-fibrotic agent pirfenidone suppresses tumor necrosis factor-alpha at the translational level. Nakazato H, Oku H, Yamane S, Tsuruta Y, Suzuki R. Department of Immunology, Shionogi Discovery Research Laboratories, Shionogi & Co. Ltd., 2-5-1, Mishima, Settsu, Osaka 566-0022, Japan. A new experimental drug pirfenidone (5-methyl-1-phenyl-2-1H-pyridine-2-one) has been reported to have beneficial effects for the treatment of certain fibrotic diseases. Here, we studied the anti-inflammatory activities of pirfenidone by investigating the mechanism of its inhibitory effect on cytokine production. In RAW264.7 cells, a murine macrophage-like cell line, pirfenidone suppressed the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) by a translational mechanism, which was independent of activation of the mitogen-activated protain kinase (MAPK) 2, p38 MAP kinase, and c-Jun N-terminal kinase (JNK). In the murine endotoxin shock model, pirfenidone potently inhibited the production of the proinflammatory cytokines, TNF-alpha, interferon-gamma, and interleukin-6, but enhanced the production of the anti-inflammatory cytokine, interleukin-10. The in vivo model also showed that pirfenidone suppressed the cytokine production by a translational mechanism, though interleukin-10 transcription was activated by pirfenidone. These findings show that pirfenidone inhibits the production of the proinflammatory cytokine selectively at the translational level. Therefore, cytokine inhibitory activities play an important role in the anti-inflammatory activities of pirfenidone. Coupled with the fact that this inhibitory effect is selective, translational, and not for total protein synthesis, this drug may have a clinical effect on inflammation and fibrosis with very low toxicity. PMID: 12098600 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 23: Crit Rev Oral Biol Med. 2002;13(3):229-37. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) as modulators of both innate and adaptive immunity. Ganea D, Delgado M. Department of Biological Sciences, Rutgers University, Newark, NJ 07102, USA. dganea@andromeda.rutgers.edu The structurally related neuropeptides VIP and PACAP are released within the lymphoid organs following antigenic stimulation, and modulate the function of inflammatory cells through specific receptors. In activated macrophages, VIP and PACAP inhibit the production of pro-inflammatory agents (cytokines, chemokines, and nitric oxide), and stimulate the production of the anti-inflammatory cytokine IL-10. These events are mediated through the VIP/PACAP effects on de novo expression or nuclear translocation of several transcription factors, i.e., NFkappaB, CREB, c-Jun, JunB, and IRF-1. The in vivo administration of VIP/PACAP results in a similar pattern of cytokine and chemokine modulation, which presumably mediates the protective effect of VIP/PACAP in septic shock. In addition, VIP/PACAP reduce the expression of the co-stimulatory molecules B7.1/B7.2, and the subsequent stimulatory activity of macrophages for T-helper cells. In T-cells expressing specific VIP/PACAP receptors, VIP and PACAP inhibit the expression of FasL through effects on NFkappaB, NFAT, and Egr2/3. The reduction of FasL expression has several biological consequences: inhibition of antigen-induced cell death in CD4 T-cells, inhibition of the FasL-mediated cytotoxicity of CD8 and CD4 effectors against direct and bystander targets, and promotion of long-term memory Th2 cells, through a positive effect on the survival of Th2, but not Th1, effectors. The various biological effects of VIP and PACAP are discussed within the range of a general anti-inflammatory model. Publication Types: Review Review, Tutorial PMID: 12090463 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 24: Int Immunol. 2002 Jun;14(6):555-66. A co-stimulatory molecule on activated T cells, H4/ICOS, delivers specific signals in T(h) cells and regulates their responses. Arimura Y, Kato H, Dianzani U, Okamoto T, Kamekura S, Buonfiglio D, Miyoshi-Akiyama T, Uchiyama T, Yagi J. Departments of Microbiology and Immunology, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. We examined the co-stimulatory activity of H4/ICOS on murine activated CD4(+) T cells and found that the cross-linking of H4/ICOS enhanced their proliferation, in addition to raising IFN-gamma, IL-4 and IL-10 production to levels comparable to those induced by CD28. However, IL-2 production was only marginally co-stimulated by H4/ICOS. This distinct pattern of lymphokine production appears to be induced by a specific intracellular signaling event. Compared with CD28, H4/ICOS dominantly elicited the Akt pathway via phosphatidylinositol 3-kinase. In addition, mitogen-activated protein kinase family kinases were activated in different ways by CD28 and H4/ICOS. The strong phosphorylation of p46 c-Jun N-terminal kinase was observed upon CD28 co-stimulation, but was less potently induced by H4/ICOS. The strain diversity in the induction of H4/ICOS was recognized. The expression of H4/ICOS on BALB/c activated CD4(+) T cells was >6-fold higher compared with C57BL/6 activated CD4(+) T cells. Furthermore, BALB/c activated CD4(+) T cells exhibited more T(h)2-deviated lymphokine production as compared with C57BL/6 activated CD4(+) T cells and signaling through H4/ICOS during the primary stimulation of naive CD4(+) T cells promoted the generation of T(h)2 cells. Thus, the difference in H4/ICOS expression on activated CD4(+) T cells, which is regulated among the mouse strains, may also regulate the polarization of T(h) cells. PMID: 12039907 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 25: J Biol Chem. 2002 Aug 9;277(32):29094-100. Epub 2002 May 28. c-Jun associates with the oncoprotein Ski and suppresses Smad2 transcriptional activity. Pessah M, Marais J, Prunier C, Ferrand N, Lallemand F, Mauviel A, Atfi A. INSERM U 482, Hopital Saint-Antoine, 184 Rue du Faubourg Saint-Antoine, 75571, Paris Cedex 12, France. The Smad proteins are key intracellular effectors of transforming growth factor-beta (TGF-beta) cytokines. The ability of Smads to modulate transcription results from a functional cooperativity with the coactivators p300/cAMP-response element-binding protein-binding protein (CBP), or the corepressors TGIF and Ski. The c-Jun N-terminal kinase (JNK) pathway, another downstream target activated by TGF-beta receptors, has also been suggested to inhibit TGF-beta signaling through interaction of c-Jun with Smad2 and Smad3. Here we show that c-Jun directly interacts with Ski to enhance the association of Ski with Smad2 in the basal state. Interestingly, TGF-beta signaling induces dissociation of c-Jun from Ski, thereby relieving active repression by c-Jun. Moreover, activation of JNK pathway suppressed the ability of TGF-beta to induce dissociation of c-Jun from ski. Thus, the formation of a c-Jun/Ski complex maintains the repressed state of Smad2-responsive genes in the absence of ligand and participates in negative feedback regulation of TGF-beta signaling by the JNK cascade. PMID: 12034730 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 26: Psychiatry Clin Neurosci. 2002 Apr;56(2):177-80. Report on IL-10 gene polymorphism at position -819 for major depression and schizophrenia in Korean population. Jun TY, Pae CU, Chae JH, Bahk WM, Kim KS, Han H. Department of Psychiatry, St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea. The present study was carried out to examine the relationship of interleukin (IL)-10 gene polymorphism at position -819 for major depression and schizophrenia in the Korean population. DNA was extracted from 92 Korean patients with major depression, 141 Korean patients with schizophrenia, and 146 ethnically matched controls. DNA was amplified by a polymerase chain reaction-based method and digested by MaeIII, and the restriction fragment length polymorphism of two alleles, IL-10*C and IL-10*T, were assessed. There were no significant differences in genotype frequencies of IL-10*T/T, IL-10*T/C, and IL-10*C/C as well as allelic frequencies of IL-10*T and IL-10*C between patients with major depression and controls in the Korean population. Comparison of genotype and allelic frequencies of IL-10 gene between patients with schizophrenia and controls were also not significant. The present study suggests that IL-10 gene polymorphism at position -819 does not confer susceptibility to major depression and schizophrenia, at least in the Korean population. Further systematic studies including various clinical variables would be required. PMID: 11952921 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 27: J Immunol. 2002 Feb 15;168(4):1759-69. Distinct role of p38 and c-Jun N-terminal kinases in IL-10-dependent and IL-10-independent regulation of the costimulatory molecule B7.2 in lipopolysaccharide-stimulated human monocytic cells. Lim W, Ma W, Gee K, Aucoin S, Nandan D, Diaz-Mitoma F, Kozlowski M, Kumar A. Department of Pediatrics, University of Ottawa, Ottawa, Ontario, Canada. The costimulatory molecule B7.2 (CD86) plays a vital role in immune activation and development of Th responses. The molecular mechanisms by which B7.2 expression is regulated are not understood. We investigated the role of mitogen-activated protein kinases (MAPK) in the regulation of B7.2 expression in LPS-stimulated human monocytic cells. LPS stimulation of human monocytes resulted in the down-regulation of B7.2 expression that could be abrogated by anti-IL-10 Abs. Furthermore, SB202190, a specific inhibitor of p38 MAPK, inhibited LPS-induced IL-10 production and reversed B7.2 down-regulation, suggesting that LPS-induced B7.2 down-regulation may be mediated, at least in part, via regulation of IL-10 production by p38 MAPK. In contrast to human promonocytic THP-1 cells that are refractory to the inhibitory effects of IL-10, LPS stimulation enhanced B7.2 expression. This IL-10-independent B7.2 induction was not influenced by specific inhibitors of either p38 or p42/44 MAPK. To ascertain the role of the c-Jun N-terminal kinase (JNK) MAPK, dexamethasone, an inhibitor of JNK activation, was used, which inhibited LPS-induced B7.2 expression. Transfection of THP-1 cells with a plasmid expressing a dominant-negative stress-activated protein/extracellular signal-regulated kinase kinase 1 significantly reduced LPS-induced B7.2 expression, thus confirming the involvement of JNK. To study the signaling events downstream of JNK activation, we show that dexamethasone did not inhibit LPS-induced NF-kappaB activation in THP-1 cells, suggesting that JNK may not be involved in NF-kappaB activation leading to B7.2 expression. Taken together, our results reveal the distinct involvement of p38 in IL-10-dependent, and JNK in IL-10-independent regulation of B7.2 expression in LPS-stimulated monocytic cells. PMID: 11823508 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 28: 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] --------------------------------------------------------------- 29: Br J Pharmacol. 2001 Nov;134(5):1029-36. A novel transcription factor inhibitor, SP100030, inhibits cytokine gene expression, but not airway eosinophilia or hyperresponsiveness in sensitized and allergen-exposed rat. Huang TJ, Adcock IM, Chung KF. Thoracic Medicine, Chang Gung Memorial Hospital, Keelung Branch, Taiwan, Republic of China. 1. We examined the effect of SP100030, a novel inhibitor of activator protein-1 (AP-1) and nuclear factor (NF)-kappa B transcription factors, in a rat model of asthma. 2. Sensitized Brown-Norway rats were treated with SP100030 (20 mg kg(-1) day(-1) for 3 days) intraperitoneally prior to allergen challenge. Allergen exposure of sensitized rats induced bronchial hyperresponsiveness (BHR), accumulation of inflammatory cells in bronchoalveolar lavage (BAL) fluid, and also an increase in eosinophils and CD2(+), CD4(+) and CD8(+) T-cells in the airways together with mRNA expression for IL-2, IL-4, IL-5, IL-10, and IFN-gamma. 3. Pre-treatment with SP100030 inhibited BAL lymphocyte influx (P<0.03), specifically reduced CD8(+) T-cell infiltration in the airway submucosa (P<0.03), and mRNA expression for IL-2, IL-5, and IL-10 (P<0.05). Neutrophil, eosinophil, and CD4(+) T-cells accumulation in the airways and BHR were not affected by SP100030. 4. Our results indicate that suppression of IL-2 and IL-5 mRNA expression may not necessarily lead to suppression of BHR. The expression of IL-5 mRNA may contribute to the airway accumulation of eosinophils, but does not correlate with the extent of eosinophilia. 5. The joint AP-1 and NF-kappa B inhibitor, SP100030, selectively inhibits CD8(+) T-cells, and mRNA expression of both Th1 and Th2 cytokines in vivo, but does not inhibit allergen-induced airway eosinophilia and BHR. PMID: 11682451 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 30: J Biol Chem. 2001 Dec 7;276(49):45564-72. The anti-inflammatory cytokine, interleukin (IL)-10, blocks the inhibitory effect of IL-1 beta on long term potentiation. A role for JNK. Kelly A, Lynch A, Vereker E, Nolan Y, Queenan P, Whittaker E, O'Neill LA, Lynch MA. Trinity College Institute of Neuroscience, Department of Physiology and Department of Biochemistry, Trinity College, Dublin 2, Ireland. Several effects of the proinflammatory cytokine, interleukin-1 beta (IL-1 beta), have been described in the central nervous system, and one area of the brain where marked changes have been reported is the hippocampus. Among these changes are an IL-1 beta-induced inhibition of long term potentiation (LTP) in perforant path-granule cell synapses and an attenuation of glutamate release in synaptosomes prepared from the hippocampus. Evidence suggests that, at least in circulating cells, the anti-inflammatory cytokine, IL-10, antagonizes certain effects of IL-1. We investigated the effect of IL-10 on IL-1 beta-induced inhibition of LTP and glutamate release. The evidence presented indicates that IL-1 beta stimulates the stress-activated protein kinase, c-Jun-activated protein kinase (JNK), and IL-1 receptor-associated kinase, which may explain its inhibitory effect on release and LTP, and that IL-10 reversed the IL-1 beta-induced stimulation of JNK activity and inhibition of release and LTP. We observed that IL-10 abrogated the stimulatory effect of IL-1 beta on superoxide dismutase activity and reactive oxygen species production, whereas the H(2)O(2)-induced inhibition of LTP was also blocked by IL-10. We present evidence that suggests that the action of IL-10 may be mediated by its ability to induce shedding of the IL-1 type I receptor. PMID: 11581275 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 31: J Immunol. 2001 Sep 15;167(6):3339-45. Mycobacteria-induced TNF-alpha and IL-10 formation by human macrophages is differentially regulated at the level of mitogen-activated protein kinase activity. Reiling N, Blumenthal A, Flad HD, Ernst M, Ehlers S. Department of Immunochemistry and Biochemical Microbiology and Immunology and Cell Biology, Research Center Borstel, Borstel, Germany. nreiling@fz-borstel.de The clinical course of mycobacterial infections is linked to the capacity of pathogenic strains to modulate the initial antimycobacterial response of the macrophage. To elucidate some of the mechanisms involved, we studied early signal transduction events leading to cytokine formation by human monocyte-derived macrophages (MDM) in response to clinical isolates of Mycobacterium avium. TNF-alpha production induced by M. avium was inhibited by anti-CD14 mAbs, but not by Abs against the macrophage mannose receptor. Analysis of mitogen-activated protein (MAP) kinase activation (extracellular signal-regulated kinase 1/2, p38, and c-Jun NH(2)-terminal kinase) showed a rapid phosphorylation of all three subfamilies in response to M. avium, which was inhibited by anti-CD14 Abs. Using highly specific inhibitors of p38 (SB203580) and MAP kinase kinase-1 (PD98059), we found that activation of the extracellular signal-regulated kinase pathway, but not of p38, was essential for the M. avium-induced TNF-alpha formation. In contrast, IL-10 production was abrogated by the p38 inhibitor, but not by the MAP kinase kinase-1 inhibitor. In conclusion, M. avium-induced secretion of TNF-alpha and IL-10 by human macrophages is differentially regulated at the level of MAP kinase activity. PMID: 11544323 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 32: Int Immunopharmacol. 2001 Jun;1(6):1173-82. Decreased serum IgE level, decreased IFN-gamma and IL-5 but increased IL-10 production, and suppressed cyclooxygenase 2 mRNA expression in patients with perennial allergic rhinitis after treatment with a new mixed formula of Chinese herbs. Yang SH, Hong CY, Yu CL. Institute of Clinical Medicine, National Yang-Ming University School of Medicine, Taipei, Taiwan. A new mixed formula of Chinese herbs containing Shin-yi-san + Xiao-qing-long-tang + Xiang-sha-liu-jun-zi-tang by the weight of 9 + 3 + 3 g/day was prescribed for the treatment of patients with perennial allergic rhinitis for 3 months (the composition of each herb is shown in the tables of the article). We classified the patients into high (H-IgE) and low IgE (L-IgE) groups according to the titer of serum total IgE (> 200 KIU/l in H-IgE vs. < 200 KIU/l in L-IgE) and the presence of house dust mite-specific IgE. The nasal symptomatic score in the high IgE group was significantly improved from 7.19 +/- 0.18 before treatment to 2.67 +/- 0.37 after treatment. In addition, the serum total and house dust mite-specific IgE level were also decreased after treatment. For elucidating the working mechanism of the mixed formula, the Th1 (IFN-gamma) and Th2 (IL-4, IL-5, IL-10 and IL-13) cytokine production by phytohemagglutinin (PHA)-stimulated mononuclear cells (2 x 10(6) cells/ml) and cyclooxygenase type 2 (COX-2) mRNA expression in LPS or IL-13-stimulated PMN were compared before and after 3 months of treatment. We found that the mixed formula treatment significantly enhanced IL-10 but decreased IFN-gamma and IL-5 production by PHA-stimulated mononuclear cells. The IL-5 production was also decreased by PHA-stimulated lymphocyte. In addition, the COX-2 mRNA expression in stimulated PMN was significantly suppressed after treatment. These results suggest that the new mixed formula treatment is beneficial to the patients with perennial allergic rhinitis via modulating the function of lymphocytes and neutrophils. Publication Types: Clinical Trial PMID: 11407311 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 33: J Clin Invest. 2001 Jun;107(11):1459-68. Modulation of T-cell responses to alloantigens by TR6/DcR3. Zhang J, Salcedo TW, Wan X, Ullrich S, Hu B, Gregorio T, Feng P, Qi S, Chen H, Cho YH, Li Y, Moore PA, Wu J. Human Genome Sciences Inc., Rockville, Maryland, USA. jun_zhang@hgsi.com TR6 (DcR3) is a new member of the TNF receptor (TNFR) family that lacks a transmembrane domain in its sequence, indicating that it is a secreted molecule. TR6 can bind to FasL and prevent FasL-induced apoptosis; it can also associate with LIGHT, another TNF family member. The role of TR6 in immune responses was investigated in this study. According to flow cytometry, recombinant human TR6-Fc binds to human LIGHT expressed on 293 cells or on activated human T cells and competes with the LIGHT receptor TR2 for the binding to LIGHT on these cells. Human TR6 could cross-react with mouse LIGHT in immunoprecipitation. TR6-Fc also downregulates cytotoxic T lymphocyte activity in vitro and graft-versus-host responses in mice. Moreover, TR6-Fc modulates lymphokine production by alloantigen-stimulated mouse T cells. TR6-Fc ameliorated rejection response to mouse heart allograft. These results indicate that TR6 can dampen T-cell responses to alloantigens. Such regulatory effects of TR6 probably occur via interference with interaction between pairs of related TNF and TNFR family members, LIGHT/TR2 being one of the possible candidate pairs. PMID: 11390428 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 34: Proc Natl Acad Sci U S A. 2001 May 22;98(11):6198-203. c-Jun interacts with the corepressor TG-interacting factor (TGIF) to suppress Smad2 transcriptional activity. Pessah M, Prunier C, Marais J, Ferrand N, Mazars A, Lallemand F, Gauthier JM, Atfi A. Institut National de la Sante et de la Recherche Medicale U 482, Hopital Saint-Antoine, 184 Rue du Faubourg Saint-Antoine, 75571, Paris Cedex 12, France. The Sma and Mad related (Smad) family proteins are critical mediators of the transforming growth factor-beta (TGF-beta) superfamily signaling. After TGF-beta-mediated phosphorylation and association with Smad4, Smad2 moves to the nucleus and activates expression of specific genes through cooperative interactions with DNA-binding proteins, including members of the winged-helix family of transcription factors, forkhead activin signal transducer (FAST)-1 and FAST2. TGF-beta has also been described to activate other signaling pathways, such as the c-Jun N-terminal Kinase (JNK) pathway. Here, we show that activation of JNK cascade blocked the ability of Smad2 to mediate TGF-beta-dependent activation of the FAST proteins. This inhibitory activity is mediated through the transcriptional factor c-Jun, which enhances the association of Smad2 with the nuclear transcriptional corepressor TG-interacting factor (TGIF), thereby interfering with the assembly of Smad2 and the coactivator p300 in response to TGF-beta signaling. Interestingly, c-Jun directly binds to the nuclear transcriptional corepressor TGIF and is required for TGIF-mediated repression of Smad2 transcriptional activity. These studies thus reveal a mechanism for suppression of Smad2 signaling pathway by JNK cascade through transcriptional repression. PMID: 11371641 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 35: Gut. 2001 May;48(5):610-22. Absence of endogenous interleukin 10 enhances early stress response during post-ischaemic injury in mice intestine. Zingarelli B, Yang Z, Hake PW, Denenberg A, Wong HR. Children's Hospital Medical Center, Division of Critical Care, 3333 Burnet Avenue, Cincinnati, Ohio 45229, USA. bzingarelli@chmcc.org BACKGROUND: Interleukin 10 (IL-10) exerts a wide spectrum of regulatory activities in immune and inflammatory responses. AIMS: The aim of this study was to investigate the role of endogenous IL-10 on modulation of the early inflammatory response after splanchnic ischaemia and reperfusion. METHODS: Intestinal damage was induced by clamping the superior mesenteric artery and the coeliac trunk for 45 minutes followed by reperfusion in IL-10 deficient mice (IL-10(-/-)) and wild-type controls. RESULTS: IL-10(-/-) mice experienced a higher rate of mortality and more severe tissue injury compared with wild-type mice subjected to ischaemia and reperfusion. Splanchnic injury was characterised by massive epithelial haemorrhagic necrosis, upregulation of P-selectin and intercellular adhesion molecule 1, and neutrophil infiltration. The degree of oxidative and nitrosative damage was significantly higher in IL-10(-/-) mice than in wild-type littermates, as indicated by elevated malondialdehyde levels and formation of nitrotyrosine. Plasma levels of the proinflammatory cytokines tumour necrosis factor alpha and interleukin 6 were also greatly enhanced in comparison with wild-type mice. These events were preceded by increased immunostaining and activity of the stress regulated c-Jun NH(2) terminal kinase and activation of the transcription factor activator protein 1 in the cellular nuclei of damaged tissue. CONCLUSIONS: These data demonstrate that endogenous IL-10 exerts an anti-inflammatory role during reperfusion injury, possibly by regulating early stress related genetic response, adhesion molecule expression, neutrophil recruitment, and subsequent cytokine and oxidant generation. PMID: 11302957 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 36: Cryobiology. 2000 Dec;41(4):301-14. Marked difference in tumor necrosis factor-alpha expression in warm ischemia- and cold ischemia-reperfusion of the rat liver. Lutterova M, Szatmary Z, Kukan M, Kuba D, Vajdova K. Laboratory of Perfused Organs, Slovak Centre for Organ Transplantation, Institute of Preventive and Clinical Medicine, Limbova 14, 83301 Bratislava, Slovakia. Although tumor necrosis factor-alpha has been implicated in liver injury after both warm ischemia- and cold ischemia-reperfusion, it is unclear whether reactivity of the liver to these stimuli is similar with regard to cytokine expression. Here we compare the effects of warm and cold ischemia on tumor necrosis factor-alpha expression and test the hypothesis that cold ischemia preceding warm ischemia causes overexpression of this cytokine. Rat livers were flushed out with University of Wisconsin solution and subjected to varying periods of warm ischemia, cold ischemia, or cold ischemia plus warm ischemia followed by reperfusion using a blood-free perfusion model. Tumor necrosis factor-alpha and interleukin-10 release into the perfusate and bile were measured by ELISA, and expression of these cytokines and that of c-fos, c-jun, and c-myc were studied by reverse-transcriptase polymerase chain reaction. We found high levels of tumor necrosis factor-alpha in the perfusates of livers subjected to warm ischemia-reperfusion, whereas minimal or no tumor necrosis factor-alpha was detected in livers subjected to cold ischemia-reperfusion or to cold ischemia plus warm ischemia-reperfusion. Reverse-transcriptase polymerase chain reaction confirmed the above findings and showed that immediate early genes were expressed in reperfused groups of livers. Measurements of cytokine release into bile showed that neither tumor necrosis factor-alpha nor interleukin-10 were upregulated by cold ischemia-reperfusion. The results suggest that (1) warm ischemia- and cold ischemia-reperfusion of rat liver lead to very different outcomes with regard to tumor necrosis factor-alpha expression and (2) cold ischemia preceding warm ischemia prevents upregulation of tumor necrosis factor-alpha. Copyright 2000 Academic Press. PMID: 11222027 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 37: J Immunol. 2000 Dec 15;165(12):7096-101. Synergy and cross-tolerance between toll-like receptor (TLR) 2- and TLR4-mediated signaling pathways. Sato S, Nomura F, Kawai T, Takeuchi O, Muhlradt PF, Takeda K, Akira S. Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan. A family of Toll-like receptor (TLR) mediates the cellular response to bacterial cell wall components; murine TLR2 and TLR4 recognize mycoplasmal lipopeptides (macrophage-activating lipopeptides, 2 kDa (MALP-2)) and LPS, respectively. Costimulation of mouse peritoneal macrophages with MALP-2 and LPS results in a marked increase in TNF-alpha production, showing the synergy between TLR2- and TLR4-mediated signaling pathways. Macrophages pretreated with LPS show hyporesponsiveness to the second LPS stimulation, termed LPS tolerance. The LPS tolerance has recently been shown to be primarily due to the down-regulation of surface expression of the TLR4-MD2 complex. When macrophages were treated with MALP-2, the cells showed hyporesponsiveness to the second MALP-2 stimulation, like LPS tolerance. Furthermore, macrophages pretreated with MALP-2 showed reduced production of TNF-alpha in response to LPS. LPS-induced activation of both NF-kappaB and c-Jun NH(2)-terminal kinase was severely impaired in MALP-2-pretreated cells. However, MALP-2-pretreated macrophages did not show any reduction in surface expression of the TLR4-MD2 complex. These findings indicate that LPS-induced LPS tolerance mainly occurs through the down-regulation of surface expression of the TLR4-MD2 complex; in contrast, MALP-2-induced LPS tolerance is due to modulation of the downstream cytoplasmic signaling pathways. PMID: 11120839 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 38: J Immunol. 2000 Oct 1;165(7):3951-8. Exaggerated human monocyte IL-10 concomitant to minimal TNF-alpha induction by heat-shock protein 27 (Hsp27) suggests Hsp27 is primarily an antiinflammatory stimulus. De AK, Kodys KM, Yeh BS, Miller-Graziano C. Department of Surgery, University of Massachusetts Medical School, Worcester 01655, USA. Unlike more well-studied large heat shock proteins (hsp) that induce both T cell antiinflammatory (IL-10, IL-4) and macrophage proinflammatory (TNF-alpha, IL-15, IL-12) cytokines, hsp27, a small hsp, has been primarily identified as a substrate of mitogen-activated protein kinase-activated protein kinase-2 involved in the p38 signaling pathway and activated during monocyte IL-10 production. Hsp27 can also act as an endogenous protein circulating in the serum of breast cancer patients and a protein whose induction correlates to protection from LPS shock. However, the cytokine-stimulating properties of hsp27 have been unexplored. In this study, exogenous hsp27 is demonstrated for the first time as a potent activator of human monocyte IL-10 production, but only a modest inducer of TNF-alpha. Although exogenous hsp27 stimulation activated all three monocyte mitogen-activated protein kinase pathways (extracellular signal-related kinase (ERK) 1/2, c-Jun N-terminal kinase, and p38), only p38 activation was sustained and required for hsp27 induction of monocyte IL-10, while both ERK 1/2 and p38 activation were required for induction of TNF-alpha when using the p38 inhibitor SB203580 or the ERK inhibitor PD98059. Hsp27's transient activation of the c-Jun N-terminal kinase pathway, which can down-regulate IL-10, may contribute to its potent IL-10 induction. Hsp27's ERK 1/2 activation was also less sustained than activation by stimuli like LPS, possibly contributing to its modest TNF-alpha induction. The failure of either PD98059 or anti-TNF-alpha Ab to substantially inhibit IL-10 induction implied that hsp27 induces IL-10 via activation of p38 signaling independently of TNF-alpha activation and may be predominantly an antiinflammatory monokine stimulus. PMID: 11034403 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 39: FASEB J. 2000 Oct;14(13):2065-74. Adenosine inhibits IL-12 and TNF-[alpha] production via adenosine A2a receptor-dependent and independent mechanisms. Hasko G, Kuhel DG, Chen JF, Schwarzschild MA, Deitch EA, Mabley JG, Marton A, Szabo C. Inotek Corp., Beverly, Massachusetts 01915, USA. haskoge@umdnj.edu Interleukin 12 (IL-12) is a crucial cytokine in the regulation of T helper 1 vs. T helper 2 immune responses. In the present study, we investigated the effect of the endogenous purine nucleoside adenosine on the production of IL-12. In mouse macrophages, adenosine suppressed IL-12 production. Although the order of potency of adenosine receptor agonists suggested the involvement of A2a receptors, data obtained with A2a receptor-deficient mice showed that the adenosine suppression of IL-12 and even TNF-alpha production is only partly mediated by A2a receptor ligation. Studies with adenosine receptor antagonists or the adenosine uptake blocker dipyridamole showed that adenosine released endogenously also decreases IL-12. Although adenosine increases IL-10 production, the inhibition of IL-12 production is independent of the increased IL-10. The mechanism of action of adenosine was not associated with alterations of the activation of the p38 and p42/p44 mitogen-activated protein kinases or the phosphorylation of the c-Jun terminal kinase. Adenosine failed to affect steady-state levels of either IL-12 p35 or p40 mRNA, but augmented IL-10 mRNA levels. In summary, adenosine inhibits IL-12 production via various adenosine receptors. These results support the notion that adenosine-based therapies might be useful in certain autoimmune and/or inflammatory diseases. PMID: 11023991 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 40: Shock. 2000 Aug;14(2):144-9. Spermine differentially regulates the production of interleukin-12 p40 and interleukin-10 and suppresses the release of the T helper 1 cytokine interferon-gamma. Hasko G, Kuhel DG, Marton A, Nemeth ZH, Deitch EA, Szabo C. Inotek Corporation, Beverly, Massachusetts 01915, USA. Polyamines are endogenous immunomodulatory molecules. Recent studies revealed that polyamines suppress the production of proinflammatory cytokines and nitric oxide. In the present study, we investigated the effect of the polyamines spermine, spermidine, and putrescine on the production of interleukin (IL)-12 p40, IL-10, and interferon (IFN-gamma) in mouse peritoneal macrophages and spleen cell suspensions. Spermine, but not spermidine or putrescine, suppressed, in a concentration-dependent manner, the production of IL-12 p40 by lipopolysaccharide (LPS)-stimulated macrophages. The effect of spermine was post-transcriptional, because steady-state levels of messenger ribonucleic acid (mRNAs) for IL-12 (p35 and p40) were not affected. In contrast to its inhibitory effect on IL-12 p40, spermine (0.3-3 microM) augmented IL-10 production. The down-regulation of IL-12 p40 by spermine was independent of enhancement of IL-10 by this agent, for spermine retained its ability to suppress IL-12 production in peritoneal macrophages obtained from IL-10-deficient mice. The alterations in cytokine production by spermine did not involve an effect on early intracellular pathways of LPS signal transduction, including the p38 or p42/44 mitogen-activated protein kinases, or the c-jun terminal kinase. In spleen cell suspensions, spermine suppressed the release of IFN-gamma induced either by LPS or anti-CD3 antibody. In summary, spermine exerts anti-inflammatory effects by suppressing IL-12 and IFN-gamma and by augmenting the production of IL-10. PMID: 10947158 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 41: Liver Transpl Surg. 1999 Jul;5(4):282-93. Activation of nuclear factor-kappaB during orthotopic liver transplantation in rats is protective and does not require Kupffer cells. Bradham CA, Schemmer P, Stachlewitz RF, Thurman RG, Brenner DA. Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Reperfusion after liver transplantation results in the induction of tumor necrosis factor-alpha (TNFalpha) as well as activation of the stress-associated signaling proteins, c-Jun N-terminal kinase (JNK), activating protein-1 (AP-1), and nuclear factor-kappaB (NF-kappaB). To test the hypothesis that Kupffer cells are involved in the activation of signal transduction cascades during rat liver transplantation, Kupffer cells were depleted from donor liver using gadolinium chloride (GdCl3), and then the activation of JNK, AP-1, and NF-kappaB were assessed after transplantation. The results showed that GdCl3 treatment did not inhibit the activation of these stress signals, although transplanted livers were depleted of Kupffer cells and partially protected from reperfusion injury. Interleukin-6 (IL-6) and IL-10 messenger RNAs (mRNAs) were induced by transplantation, and the induction was suppressed by Kupffer cell depletion. The induction of TNFalpha mRNA and serum protein during liver transplantation was unaffected by GdCl3. These results show that Kupffer cells are not a major source of TNFalpha production after liver transplantation and that stress-signaling protein activation occurs independently of Kupffer cells. Transplantation strongly activates the transcription factor NF-kappaB, which blocks TNFalpha-mediated apoptosis in hepatocytes in vitro. To assess the role of NF-kappaB activation during liver transplantation, the IkappaBalpha superrepressor was expressed in donor livers using adenoviral-mediated gene transfer. Inhibition of NF-kappaB resulted in increased serum alanine aminotransferase levels after 3 hours of transplantation. In addition, the blockade of NF-kappaB resulted in increased histological tissue injury and increased hepatic terminal deoxyribonucleotide transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining, indicating apoptosis. These results show that NF-kappaB activation has a protective role in the transplanted liver. PMID: 10388501 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 42: J Immunol. 1999 Apr 1;162(7):3865-72. Extracellular signal-regulated kinase, stress-activated protein kinase/c-Jun N-terminal kinase, and p38mapk are involved in IL-10-mediated selective repression of TNF-alpha-induced activation and maturation of human peripheral blood monocyte-derived dendritic cells. Sato K, Nagayama H, Tadokoro K, Juji T, Takahashi TA. Department of Cell Processing, Institute of Medical Science, University of Tokyo, Japan. TNF-alpha or IL-10 has been implicated to reversibly regulate physiological states of dendritic cells (DCs). However, little is known about dual stimulations of these cytokines on DC properties and the intracellular signaling events that are responsible for the regulation of these states. Here, we show that a family of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase 2 (ERK2), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and p38mapk, are potentially involved in IL-10-mediated selective suppression of TNF-alpha-induced changes of the monocyte-derived DC properties. TNF-alpha induced the cluster formation of the cells and the enhancement of cell surface expression levels of CD83, CD86, and HLA-DR, and T cell stimulatory capacity, whereas the capacities for the endocytosis and the chemotactic migration were suppressed in these cells. Treatment of monocyte-derived DCs with IL-10 resulted in the reduction of the cell surface expression levels of CD86, HLA-DR, and T cell stimulatory capacity, whereas both endocytic and chemotactic migratory capacities were increased by IL-10. Dual stimulations of monocyte-derived DCs with TNF-alpha and IL-10 selectively antagonized their respective effects on these DC properties. TNF-alpha induced tyrosine phosphorylation and enzymatic activation of ERK2, SAPK/JNK, and p38mapk, whereas IL-10 did not induce these events. Dual stimulations of TNF-alpha plus IL-10 abolished TNF-alpha-induced changes of these MAPKs in DCs. These results suggest that the blockage in the MAPKs cascades contributes to IL-10-mediated repression of TNF-alpha-induced changes of DC properties. PMID: 10201904 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 43: J Biol Chem. 1999 Feb 26;274(9):5835-42. CD40 signaling of monocyte inflammatory cytokine synthesis through an ERK1/2-dependent pathway. A target of interleukin (il)-4 and il-10 anti-inflammatory action. Suttles J, Milhorn DM, Miller RW, Poe JC, Wahl LM, Stout RD. Department of Biochemistry and Molecular Biology, Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614, USA. suttles@etsu.edu Ligation of CD40 on monocytes through its interaction with CD40 ligand (CD154) present on activated T helper cells, results in activation of monocyte inflammatory cytokine synthesis and rescue of monocytes from apoptosis induced through serum deprivation. Both of these consequences of CD40 stimulation have been shown to be dependent on the induction of protein tyrosine kinase activity. CD40-mediated activation of protein tyrosine kinase activity and subsequent inflammatory cytokine production are abrogated by treatment of monocytes with the T helper type 2 cytokines interleukin 4 (IL-4) and interleukin 10 (IL-10). In the current study we demonstrate that stimulation of monocytes through CD40 resulted in the phosphorylation and activation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) mitogen-activated protein kinases, whereas phosphorylation of mitogen-activated protein kinases family members p38 and c-Jun N-terminal kinase was not observed in response to this stimuli over the time course examined. PD98059, an inhibitor of the upstream activator of ERK1/2, the MAP/ERK kinase MEK1/2, suppressed IL-1beta and tumor necrosis factor-alpha production in a dose-dependent fashion. Pretreatment of monocytes with IL-4 and IL-10 inhibited CD40-mediated activation of ERK1/2 kinase activity when used individually, and are enhanced in effectiveness when used in combination. Together, the data demonstrate that CD40-mediated induction of IL-1beta and tumor necrosis factor-alpha synthesis is dependent on a MEK/ERK pathway which is obstructed by signals generated through the action of IL-4 and IL-10. PMID: 10026206 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 44: Blood. 1999 Jan 1;93(1):208-16. Mechanism of interleukin-10 inhibition of T-helper cell activation by superantigen at the level of the cell cycle. Perrin GQ, Johnson HM, Subramaniam PS. Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA. gperrin@micro.ifas.ufl.edu We have analyzed the effects of interleukin-10 (IL-10) on the entry of quiescent CD4(+) T cells into the cell cycle upon stimulation with the superantigen staphylococcal enterotoxin B (SEB). IL-10 arrested cells at G0/G1. IL-10 treatment prevented the downregulation of p27(Kip1), an inhibitory protein that controls progression out of the G0 phase of the cell cycle. IL-10 also prevented the upregulation of the G1 cyclins D2 and D3, proteins necessary for entry and progression through the G1 phase of the cell cycle. Associated with the inhibition of the cell cycle, IL-10 suppressed SEB induction of interleukin-2 (IL-2). Addition of exogenous IL-2 to IL-10-treated cells significantly reversed the antiproliferative effects of IL-10. Moreover, IL-10 effects on the early G1 proteins p27(Kip1) and cyclin D2 were similarly reversed by exogenous IL-2. Although this reversal by IL-2 was pronounced, it was not complete, suggesting that IL-10 may have some effects not directly related to the suppression of IL-2 production. Cell separation experiments suggest that IL-10 can effect purified CD4(+) T cells directly, providing functional evidence for the presence of IL-10 receptors on CD4(+) T cells. IL-10 also inhibited expression of IL-2 transcriptional regulators c-fos and c-jun, which also inhibit other cell functions. Our studies show that the mechanism of IL-10 regulation of quiescent CD4(+) T-cell activation is mainly by blocking induction of IL-2 that is critical to downregulation of p27(Kip1) and upregulation of D cyclins in T-cell activation and entry into the cell cycle. PMID: 9864163 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 45: Immunology. 1998 Mar;93(3):350-7. Kinetics of cytokine and NFAT gene expression in human interleukin-2-dependent T lymphoblasts stimulated via T-cell receptor. Sareneva T, Matikainen S, Vanhatalo J, Melen K, Pelkonen J, Julkunen I. Department of Virology, National Public Health Institute, Helsinki, Finland. T cells respond to mitogenic or antigenic stimulation by proliferation and by turning on cytokine gene expression. Here we have analysed the kinetics and nature of cytokine production in human peripheral blood-derived T lymphoblasts stimulated with anti-CD3 antibodies or Lens culinaris lectin (LCL). T cells were purified from peripheral blood mononuclear cells (PBMC) and primarily activated with anti-CD3 antibodies and cultured in the presence of interleukin-2 (IL-2). Anti-CD3-restimulated T cells (mainly CD8+) produced IL-2, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) and low levels of IL-4 and IL-10 transcripts and proteins. No IL-6 gene expression was observed. In LCL-stimulated cells the cytokine production pattern was very similar. Steady-state mRNA levels of IL-2, IL-10 and IFN-gamma peaked at 3 hr after anti-CD3 stimulation and declined rapidly thereafter. The kinetics of TNF-alpha mRNA expression was faster, being at its peak level 1 hr after stimulation. Anti-CD3-stimulated IL-2 gene expression was down-regulated by protein synthesis inhibitor, whereas IL-10, IFN-gamma and TNF-alpha genes were readily induced independent of ongoing protein synthesis. T-cell receptor stimulation also induced a very rapid expression of c-jun, c-fos and NFATc1 (NFATc) genes, the gene products of which are involved in cytokine gene expression. In conclusion, the cytokines synthesized by IL-2-dependent T cells were predominantly IL-2, IFN-gamma and TNF-alpha. PMID: 9640245 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 46: Biol Neonate. 1997;72(6):329-36. Constitutive expression c-fos, c-jun, and NF kappa B mRNA is in nucleated fetal blood cells and up-regulation of c-fos and c-jun with anti-CD3 stimulation. Jarvis JN, Zhao L, Xu CS, Moore HT, Berry S. Division of Immunology/Rheumatology, Wayne State University School of Medicine, Detroit, Mich., USA. james-jarvis@ouhsc.edu Fetal and neonatal lymphocytes are relatively resistant to activation and cytokine production when stimulated either via their T-cell antigen receptors or lectins. The molecular mechanism(s) responsible for this phenomenon have not been clearly elucidated. We have hypothesized that such defects in fetal/neonatal T-cell activation may be due to lack of expression of the transcriptional regulatory elements required for T-cell activation. We used reverse transcriptase-polymerase chain reaction to examine both fetal and term neonatal cord bloods for mRNA expression of three transcription factors implicated in T-cell activation: c-jun, c-fos, and NF kappa B (p50 subunit). We demonstrate that mRNAs for all three of these regulatory factors are expressed in fetal blood cells by the 27th week of gestation and in term cord bloods. Activation of term infant cord blood mononuclear cells with anti-CD3 monoclonal antibodies resulted in up-regulation of both c-jun and c-fos mRNAs within 15 min of stimulation. However, secretion of IL-2 by anti-CD3-stimulated cord blood mononuclear cells was still blunted compared with control cells from adults. We conclude that fetal nucleated blood cells constitutively express important genes for cytokine regulation and are able to increase intracellular accumulation of the mRNAs for these factors in response to anti-CD3 stimulation. Thus, qualitative differences in the capacity to regulate these factors could not be shown in fetal blood cells. Quantitative experiments comparing binding of these transcription factors to the IL-2 promoter are currently under investigation. PMID: 9428992 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 47: Leukemia. 1996 Aug;10(8):1308-16. Effects of IL-10 and IL-4 on LPS-induced transcription factors (AP-1, NF-IL6 and NF-kappa B) which are involved in IL-6 regulation. Dokter WH, Koopmans SB, Vellenga E. Department of Medicine, University of Groningen, The Netherlands. Interleukin-10 (IL-10), like IL-4, is known to inhibit cytokine expression in activated human monocytes. We showed that both IL-10 and IL-4 inhibit LPS-induced IL-6 mRNA and protein expression by inhibiting the transcription rate of the IL-6 gene. The strong inhibition of the IL-6 transcription rate prompted us to study the effect of IL-10 and IL-4 on the expression of transcription factors. We questioned whether or not IL-10 and IL-4 affected the expression of transcription factors that are known to be involved in the control of the IL-6 transcription rate, namely activator protein-1 (AP-1), nuclear factor IL-6 (NF-IL6), and nuclear factor kappa B (NF-kappaB). In electrophoretic mobility shift assays (EMSAs) we showed that IL-10 and IL-4 inhibited LPS-induced AP-1 binding activity. The inhibiting effect of IL-4 was slightly more pronounced than that of IL-10. Downregulation of LPS-induced AP-1 was accompanied, and thus possibly explained, by a reduced expression at mRNA level of the two major components of the AP-1 complex, namely c-fos and c-jun as determined by Northern experiments. Binding activity of NF-IL6 was also strongly inhibited by IL-4 whereas IL-10 showed no effect. NF-IL6 mRNA levels were not affected by IL-10 or IL-4, suggesting that IL-4 affects binding activity of preexisting NF-IL6. Neither IL-10 nor IL-4 inhibited LPS-induced NF-kappa B binding activity. In agreement with this finding, Northern experiments where p65 and p105 mRNA levels were determined, demonstrated that expression of these components of the NF-kappa B transcription factor were not affected by IL-10 or IL-4. Furthermore, neither IL-10 nor IL-4 showed any effect on I-kappa B mRNA expression as determined by Northern experiments. Thus, IL-10 and IL-4 similarly affect IL-6 expression. However, for IL-4 this was accompanied with a reduction of AP-1 and NF-IL6 binding activity whereas IL-10 only inhibited AP-1 binding activity. PMID: 8709636 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 48: J Autoimmun. 1996 Jun;9(3):331-9. A new type of CD4+ suppressor T cell completely prevents spontaneous autoimmune diabetes and recurrent diabetes in syngeneic islet-transplanted NOD mice. Han HS, Jun HS, Utsugi T, Yoon JW. Department of Microbiology and Infectious Diseases, Faculty of Medicine, University of Calgary, Alberta, Canada. Insulin-dependent diabetes mellitus (IDDM) in the non-obese diabetic (NOD) mouse results from effector T cell-mediated autoimmune processes directed against pancreatic beta cells. These effector T cell-mediated beta cell-specific autoimmune processes can be blocked by transfusion with supressor T cells. The CD4+ autoreactive T cell clone, NY4.2, isolated from lymphocytes infiltrating the pancreatic islets of NOD mice was transfused into young NOD mice and none of the animals became diabetic. Similarly, when NY4.2 cells were transfused into acutely diabetic NOD mice prior to syngeneic islet transplantation, the grafted islets were not destroyed and the animals maintained normoglycaemia. This investigation was initiated to determine how our cloned CD4+ autoreactive suppressor T cells are able to inhibit effector T cell-mediated beta cell destruction in NOD mice. We found that NY4.2 cells, which responded to self MHC class II determinants, had a significant immunosuppressive effect on proliferative responses of splenic effector T cells from NOD mice. This suppressive activity of the NY4.2 cells was a result of soluble factors secreted by them. The clone was found to produce substantial amounts of transforming growth factor beta (TGF-beta), IL-10, and IFN-gamma, but not IL-2 or IL-4, indicating that this T cell clone is not a member of either the classic Th1 or Th2 cell type. The suppressive activity of NY4.2 cells was abrogated by treatment with anti-TGF-beta antibodies, but not by treatment with anti-IL-10 or anti-IFN-gamma antibodies. On the basis of these observations, we suggest that a new type of CD4+ suppressor T cell, NY4.2, by secreting TGF-beta, can prevent effector T cell-mediated beta cell destruction. PMID: 8816968 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 49: Proc Natl Acad Sci U S A. 1994 Aug 30;91(18):8602-6. Monocyte deactivation by interleukin 10 via inhibition of tyrosine kinase activity and the Ras signaling pathway. Geng Y, Gulbins E, Altman A, Lotz M. Department of Medicine, University of California at San Diego, La Jolla 92093. Activation of monocytes by bacterial lipopolysaccharides (LPSs) is a central component in the pathogenesis of septic shock syndrome. Interleukin 10 (IL-10) is a potent monocyte-deactivating factor and transcriptionally inhibits LPS-induced expression of proinflammatory mediators. The intracellular signaling pathways of LPS have been only partially characterized and mechanisms of IL-10 signaling remain unknown. We show that LPS activates the protein tyrosine kinase (PTK) p56lyn and that this is associated with tyrosine phosphorylation of the protooncogene product Vav. These events are completely blocked by the tyrosine kinase inhibitor herbimycin A. LPS also increases Ras activation in monocytes. LPS-triggered phosphorylation of mitogen-activated protein kinase is a downstream activation event that is also reduced by herbimycin A. Analysis of the IL-10 effects shows that it completely inhibits the p56lyn tyrosine kinase activation and all other subsequent events in this pathway including Ras activation. The IL-10 effects are selective since it reduced PTK-dependent cytokine mRNA expression but not the PTK independent induction of c-jun and c-fos mRNA in LPS-activated monocytes. These results identify the Ras signaling pathway as a component of intracellular signaling in LPS-stimulated monocytes and define early events in this response as targets of monocyte deactivation by IL-10. PMID: 8078929 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------