1: J Immunol. 2005 Jul 15;175(2):1041-6. Identification of a macrophage-specific chromatin signature in the IL-10 locus. Saraiva M, Christensen JR, Tsytsykova AV, Goldfeld AE, Ley SC, Kioussis D, O'Garra A. Divisions of Immunoregulation, Immune Cell Biology and Molecular Immunology, National Institute for Medical Research, The Ridgeway, London NW7 1AA, United Kingdom. msaraiv@nimr.mrc.ac.uk The molecular mechanisms that regulate expression of the immunosuppressive cytokine IL-10 remain poorly understood. In this study, by measuring sensitivity to DNase I digestion, we show that production of IL-10 by primary mouse bone marrow-derived macrophages stimulated through pattern recognition receptors was associated with chromatin remodeling of the IL-10 locus. We also demonstrate that the IL-10 locus is remodeled in primary Th2 cells and IL-10-producing regulatory T cells that have been differentiated in vitro. Strikingly, a novel DNase I-hypersensitive site (HSS-4.5) was identified in stimulated macrophages, but not in T cells. We show that hyperacetylated histones were recruited to this site in stimulated macrophages. Furthermore, HSS-4.5 is highly conserved and contains a putative NF-kappaB binding site. In support of a function for this site, NF-kappaB p65/RelA was recruited to HSS-4.5 in vivo and its activation was required for optimal IL-10 gene expression in LPS-stimulated macrophages. PMID: 16002704 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: J Immunol. 2005 Jun 15;174(12):8116-24. Curcumin inhibits immunostimulatory function of dendritic cells: MAPKs and translocation of NF-kappa B as potential targets. Kim GY, Kim KH, Lee SH, Yoon MS, Lee HJ, Moon DO, Lee CM, Ahn SC, Park YC, Park YM. Department of Microbiology and Immunology, and National Research Lab of Dendritic Cell Differentiation & Regulation and Medical Research Institute, Pusan National University College of Medicine, Pusan, South Korea. Curcumin has been shown to exhibit anti-inflammatory, antimutagenic, and anticarcinogenic activities. However, the effect of curcumin on the maturation and immunostimulatory function of dendritic cells (DC) largely remains unknown. In this study, we examined whether curcumin can influence surface molecule expression, cytokine production, and their underlying signaling pathways in murine bone marrow-derived DC. DC were derived from murine bone marrow cells and used as immature or LPS-stimulated mature cells. The DC were tested for surface molecule expression, cytokine production, dextran uptake, the capacity to induce T cell differentiation, and their underlying signaling pathways. Curcumin significantly suppressed CD80, CD86, and MHC class II expression, but not MHC class I expression, in the DC. The DC also exhibited impaired IL-12 expression and proinflammatory cytokine production (IL-1beta, IL-6, and TNF-alpha). The curcumin-treated DC were highly efficient at Ag capture, via mannose receptor-mediated endocytosis. Curcumin inhibited LPS-induced MAPK activation and the translocation of NF-kappaB p65. In addition, the curcumin-treated DC showed an impaired induction of Th1 responses and a normal cell-mediated immune response. These novel findings provide new insight into the immunopharmacological role of curcumin in impacting on the DC. These novel findings open perspectives for the understanding of the immunopharmacological role of curcumin and therapeutic adjuvants for DC-related acute and chronic diseases. PMID: 15944320 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Scand J Gastroenterol. 2005 Jan;40(1):103-8. Role of nuclear factor-kappaB, reactive oxygen species and cellular signaling in the early phase of acute pancreatitis. Shi C, Zhao X, Wang X, Andersson R. Department of Surgery, Lund University Hospital, Lund, Sweden. OBJECTIVE: Increased knowledge of regulation of signaling proteins in acute pancreatitis (AP) could potentially contribute to the development of novel agents targeted at regulation of cellular signaling. MATERIAL AND METHODS: Severe AP was induced by administration of 5% sodium taurodeoxycholate in rats. Thirty minutes prior to induction of AP, the animals had an intraperitoneal injection of the antioxidant, N-acetylcysteine (NAC; 10 mg/kg), the NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC; 100 mg/kg), the ERK inhibitor, PD-98059 (1 mg/kg), or the tyrosine kinase inhibitor, Genistein (1 mg/kg). Plasma levels of IL-6 and IL-10 were determined by ELISA and myeloperoxidase (MPO) levels were measured in the pancreas and lungs 3 and 6 h after sham operation or induction of AP. RESULTS: AP results in significant increases in plasma levels of IL-6 at 6 h and IL-10 at 3 and 6 h. Plasma levels of IL-6 were significantly decreased after administration of NAC. NAC pretreatment also increased the ratio of IL-10/IL-6. MPO levels in the pancreas (at 3 and 6 h) and lungs (3 h) were significantly increased in animals with pancreatitis. Pretreatment with NAC, PDTC, PD98059 or Genistein significantly decreased MPO levels in the pancreas at 3 and 6 h and following the administration of PD-98059 or NAC at 6 h. Pretreatment with NAC significantly decreased MPO levels in the lungs at 3 h. CONCLUSIONS: Pretreatment with NAC could regulate the pro-and anti-inflammatory cytokine balance, probably through NF-kappaB and ROS signaling pathways. The regulation of signaling pathways during the sequestration of neutrophils in acute pancreatitis seems to vary between organs. PMID: 15841722 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: J Immunol. 2005 Mar 1;174(5):2990-9. IL-10 gene-deficient mice lack TGF-beta/Smad signaling and fail to inhibit proinflammatory gene expression in intestinal epithelial cells after the colonization with colitogenic Enterococcus faecalis. Ruiz PA, Shkoda A, Kim SC, Sartor RB, Haller D. Centre for Nutrition and Food Research, Immunobiology of Nutrition, Technical University of Munich, Freising-Weihenstephan, Germany. Nonpathogenic enteric bacterial species initiate and perpetuate experimental colitis in IL-10 gene-deficient mice (IL-10(-/-)). Bacteria-specific effects on the epithelium are difficult to dissect due to the complex nature of the gut microflora. We showed that IL-10(-/-) mice compared with wild-type mice fail to inhibit proinflammatory gene expression in native intestinal epithelial cells (IEC) after the colonization with colitogenic Gram-positive Enterococcus faecalis. Interestingly, proinflammatory gene expression was transient after 1 wk of E. faecalis monoassociation in IEC from wild-type mice, but persisted after 14 wk of bacterial colonization in IL-10(-/-) mice. Accordingly, wild-type IEC expressed phosphorylated NF-kappaB subunit RelA (p65) and phosphorylated Smad2 only at day 7 after bacterial colonization, whereas E. faecalis-monoassociated IL-10(-/-) mice triggered persistent RelA, but no Smad2 phosphorylation in IEC at days 3, 7, 14, and 28. Consistent with the induction of TLR2-mediated RelA phosphorylation and proinflammatory gene expression in E. faecalis-stimulated cell lines, TLR2 protein expression was absent after day 7 from E. faecalis-monoassociated wild-type mice, but persisted in IL-10(-/-) IEC. Of note, TGF-beta1-activated Smad signaling was associated with the loss of TLR2 protein expression and the inhibition of NF-kappaB-dependent gene expression in IEC lines. In conclusion, E. faecalis-monoassociated IL-10(-/-), but not wild-type mice lack protective TGF-beta/Smad signaling and fail to inhibit TLR2-mediated proinflammatory gene expression in the intestinal epithelium, suggesting a critical role for IL-10 and TGF-beta in maintaining normal epithelial cell homeostasis in the interplay with commensal enteric bacteria. PMID: 15728512 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Immunology. 2005 Mar;114(3):313-21. Interleukin-10 (IL-10) mediated suppression of IL-12 production in RAW 264.7 cells also involves c-rel transcription factor. Rahim SS, Khan N, Boddupalli CS, Hasnain SE, Mukhopadhyay S. Centre for DNA Fingerprinting and Diagnostics (CDFD), Nacharam, Hyderabad, India. Interleukin-10 (IL-10) is known to inhibit IL-12 production in macrophages primarily at the transcriptional level with the involvement of p50 and p65 nuclear factor-kappaB (NF-kappaB). We demonstrate that the c-rel transcription factor also plays a major role in IL-10-mediated IL-12 suppression. Treatment of macrophages with recombinant IL-10 inhibited nuclear c-rel levels, whereas addition of neutralizing anti-IL-10 antibody up-regulated both nuclear c-rel levels and IL-12 production by macrophages. Decreased nuclear c-rel was associated with a reduction in phosphorylation of inhibitory kappa B alpha (IkappaBalpha) in the cytoplasm, indicating that IL-10 prevents degradation of IkappaBalpha and the subsequent translocation of c-rel into the nucleus. Treatment with leptomycin B, a known inhibitor of c-rel at a concentration of 10 nm, when used with anti-IL-10 antibody, resulted in reduced expression of IL-12. In a complementary experiment, in vitro transient expression of p65 NF-kappaB could not rescue the inhibitory effect of IL-10 on IL-12 production, suggesting that NF-kappaB alone was not sufficient to restore IL-12 production during IL-10 treatment. However, over-expression of c-rel resulted in IL-12 restoration upon stimulation with lipopolysaccharide plus interferon-gamma during IL-10 treatment. Our studies highlight the involvement of c-rel in IL-10-mediated IL-12 regulation. PMID: 15720433 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Int J Cancer. 2004 Oct 20;112(1):155-60. Nucleosomes activate NF-kappaB in endothelial cells for induction of the proangiogenic cytokine IL-8. Tanner JE. Laboratory of Anti-Nucleosome Antibody Therapeutics, Procyon Biopharma, Inc., Montreal, Quebec, Canada. jetanner@tantecbiosystems.com Solid tumors often exhibit regions undergoing apoptosis and necrosis, also referred to as "aponecrosis", juxtaposed to sites of active angiogenesis. We explored whether nucleosomes resulting from aponecrosis induced the angiogenic factor IL-8 in vascular endothelial cells. Results indicate that nucleosomes induced IL-8. Nucleosomes increased IL-6 and IL-8 mRNA but not IL-10, TNF-alpha, VEGF or FGF-2 mRNA. Induction of IL-8 by nucleosomes in endothelial cells appeared to be the result of NF-kappaB/RelA transcription factor activation. The increased expression of IL-8 in vascular endothelial cells following nucleosome stimulation suggests that aponecrosis could play an important role in the promotion of tumor angiogenesis. Copyright 2004 Wiley-Liss, Inc. PMID: 15305388 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: Blood. 2005 Jan 15;105(2):689-96. Epub 2004 Jul 13. STAT3 regulates NF-kappaB recruitment to the IL-12p40 promoter in dendritic cells. Hoentjen F, Sartor RB, Ozaki M, Jobin C. Center for Gastrointestinal Biology and Diseases, University of North Carolina at Chapel Hill, USA. Interleukin-10-deficient (IL-10(-/-)) mice develop an IL-12-mediated intestinal inflammation in the absence of endogenous IL-10. The molecular mechanisms of the dysregulated IL-12 responses in IL-10(-/-) mice are poorly understood. In this study, we investigated the role of nuclear factor-kappa B (NF-kappaB) and signal transducers and activators of transcription 3 (STAT3) in lipopolysaccharide (LPS)-induced IL-12p40 gene expression in bone marrow derived-dendritic cells (BMDCs) isolated from wild-type (WT) and IL-10(-/-) mice. We report higher IL-12p40 mRNA accumulation and protein secretion in LPS-stimulated BMDCs isolated from IL-10(-/-) compared with WT mice. LPS-induced NF-kappaB signaling is similar in IL-10(-/-) and WT BMDCs as measured by IkappaBalpha phosphorylation and degradation, RelA phosphorylation and nuclear translocation, and NF-kappaB transcriptional activity, with no down-regulatory effects of exogenous IL-10. Chromatin immunoprecipitation demonstrated enhanced NF-kappaB (cRel, RelA) binding to the IL-12p40 promoter in IL-10(-/-) but not WT BMDCs. Interestingly, LPS induced STAT3 phosphorylation in WT but not IL-10(-/-) BMDCs, a process blocked by IL-10 receptor blocking antibody. Adenoviral gene delivery of a constitutively active STAT3 but not control green fluorescence protein (GFP) virus blocked LPS-induced IL-12p40 gene expression and cRel recruitment to the IL-12p40 promoter. In conclusion, dysregulated LPS-induced IL-12p40 gene expression in IL-10(-/-) mice is due to enhanced NF-kappaB recruitment to the IL-12p40 promoter in the absence of activated STAT3. PMID: 15251981 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: Clin Exp Immunol. 2004 Jan;135(1):64-73. Molecular mechanisms of interleukin-10-mediated inhibition of NF-kappaB activity: a role for p50. Driessler F, Venstrom K, Sabat R, Asadullah K, Schottelius AJ. Research Business Area Dermatology, Research Laboratories of Schering AG, Berlin, Germany. Nuclear factor kappa B (NF-kappaB) is a transcription factor pivotal for the development of inflammation. A dysregulation of NF-kappaB has been shown to play an important role in many chronic inflammatory diseases including rheumatoid arthritis, inflammatory bowel disease and psoriasis. Although classical NF-kappaB, a heterodimer composed of the p50 and p65 subunits, has been well studied, little is known about gene regulation by other hetero- and homodimeric forms of NF-kappaB. While p65 possesses a transactivation domain, p50 does not. Indeed, p50/p50 homodimers have been shown to inhibit transcriptional activity. We have recently shown that Interleukin-10 exerts its anti-inflammatory activity in part through the inhibition of NF-kappaB by blocking IkappaB kinase activity and by inhibiting NF-kappaB already found in the nucleus. Since the inhibition of nuclear NF-kappaB could not be explained by an increase of nuclear IkappaB, we sought to further investigate the mechanisms involved in the inhibition of NF-kappaB by IL-10. We show here that IL-10 selectively induced nuclear translocation and DNA-binding of p50/p50 homodimers in human monocytic cells. TNF-alpha treatment led to a strong translocation of p65 and p50, whereas pretreatment with IL-10 followed by TNF-alpha blocked p65 translocation but did not alter the strong translocation of p50. Furthermore, macrophages of p105/p50-deficient mice exhibited a significantly decreased constitutive production of MIP-2alpha and IL-6 in comparison to wild type controls. Surprisingly, IL-10 inhibited high constitutive levels of these cytokines in wt macrophages but not in p105/p50 deficient cells. Our findings suggest that the selective induction of nuclear translocation and DNA-binding of the repressive p50/p50 homodimer is an important anti-inflammatory mechanism utilized by IL-10 to repress inflammatory gene transcription. PMID: 14678266 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: Infect Immun. 2003 May;71(5):2498-507. Role of innate immune factors in the adjuvant activity of monophosphoryl lipid A. Martin M, Michalek SM, Katz J. Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA. Monophosphoryl lipid A (MPL) is a nontoxic derivative of lipopolysaccharide (LPS) that exhibits adjuvant properties similar to those of the parent LPS molecule. However, the mechanism by which MPL initiates its immunostimulatory properties remains unclear. Due to the involvement of Toll-like receptors in recognizing and transducing intracellular signals in response to LPS, the aim of the present study was to determine the ability of MPL to utilize the Toll-like receptor 2 (TLR2) and TLR4. We provide evidence that MPL differentially utilizes TLR2 and TLR4 for the induction of tumor necrosis factor alpha, interleukin 10 (IL-10), and IL-12 by purified human monocytes as well as by human peripheral blood mononuclear cells. Assessment of NF-kappa B activity demonstrated that MPL utilized TLR2 and especially TLR4 for the activation of NF-kappa B p65 by human monocytes. In addition, stimulation of human monocytes by MPL led to an up-regulation of the costimulatory molecules CD80 and CD86, an effect that could be reduced by pretreatment of cells with a monoclonal antibody to TLR2 or TLR4. Analysis of MPL-induced activation of the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinases revealed that MPL utilized both TLR2 and TLR4 for the phosphorylation of ERK1/2, while TLR4 was the predominant receptor involved in the ability of MPL to phosphorylate p38. Moreover, using selective inhibitors for MAP kinase kinase (PD98059) and p38 (SB203580), we show that ERK1/2 exhibited differential effects on production of TNF-alpha and IL-12 p40 by human monocytes, whereas MPL-induced activation of p38 appeared to be predominantly involved in production of IL-10 and IL-12 p40 by MPL-stimulated monocytes. Taken together, these findings aid in understanding the cellular mechanisms by which MPL induces host cell activation and subsequent adjuvant properties. PMID: 12704121 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: J Immunol. 2003 Apr 15;170(8):3963-70. Cross-linking of CD32 induces maturation of human monocyte-derived dendritic cells via NF-kappa B signaling pathway. Banki Z, Kacani L, Mullauer B, Wilflingseder D, Obermoser G, Niederegger H, Schennach H, Sprinzl GM, Sepp N, Erdei A, Dierich MP, Stoiber H. Institute of Hygiene and Social Medicine, Leopold-Franzens University and Ludwig Boltzmann Institute for AIDS Research, Innsbruck, Austria. Dendritic cells (DC) represent a unique set of APCs that initiate immune responses through priming of naive T cells. Maturation of DC is a crucial step during Ag presentation and can be induced by triggering a broad spectrum of DC surface receptors. Although human DC express several receptors for the Fc portion of IgG which were described to play an important role in Ag internalization, little is known about the effects of IgG or immune complexes on DC maturation. In this study, we show that cross-linking of FcgammaR-type II (CD32) with immobilized IgG (imIgG) can induce maturation of human monocyte-derived DC via the NF-kappaB signaling pathway. IgG-mediated maturation was accompanied by a moderate increase of IL-10 secretion, whereas no IL-12 production was observed. Involvement of CD32 was further supported by experiments with the anti-CD32 mAb, which blocked IgG-triggered DC maturation and cytokine secretion significantly. Furthermore, DC cultivated in the presence of imIgG induced allogeneic T cell proliferation. Because this imIgG-induced maturation was considerably impaired in monocyte-derived DC from systemic lupus erythematosus patients, we suggest that DC, which matured in the presence of immune complexes, may contribute to prevention of pathological immune responses. PMID: 12682223 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: Proc Natl Acad Sci U S A. 2002 Nov 26;99(24):15770-5. Epub 2002 Nov 11. NCX-1015, a nitric-oxide derivative of prednisolone, enhances regulatory T cells in the lamina propria and protects against 2,4,6-trinitrobenzene sulfonic acid-induced colitis in mice. Fiorucci S, Antonelli E, Distrutti E, Del Soldato P, Flower RJ, Clark MJ, Morelli A, Perretti M, Ignarro LJ. Clinica di Gastroenterologia ed Endoscopia Digestiva, Dipartimento di Medicina Clinica, Patologia Universita di Perugia, Italy. fiorucci@unipg.it NCX-1015 is a nitric oxide (NO)-releasing derivative of prednisolone. In this study we show NCX-1015 protects mice against the S. A. development and induces healing of T helper cell type 1-mediated experimental colitis induced by intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS). The beneficial effect of NCX-1015 was reflected in increased survival rates, improvement of macroscopic and histologic scores, a decrease in the mucosal content of T helper cell type 1 cytokines (protein and mRNA), and diminished myeloperoxidase activity in the colon. In contrast to its NO derivative, only very high doses of prednisolone were effective in reproducing these beneficial effects. NCX-1015 was 10- to 20-fold more potent than the parent compound in inhibiting IFN-gamma secretion by lamina propria mononuclear cells. Protection against developing colitis correlated with inhibition of nuclear translocation of p65Rel A in these cells. In vivo treatment with NCX-1015 potently stimulated IL-10 production, suggesting that the NO steroid induces a regulatory subset of T cells that negatively modulates intestinal inflammation. PMID: 12427966 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: J Dermatol Sci. 2002 Feb;28(2):159-70. Nuclear factor kappaB (NF-kappaB) activation by hydrogen peroxide in human epidermal keratinocytes and the restorative effect of interleukin-10. Ikeda M, Hirose Y, Miyoshi K, Kodama H. Department of Dermatology, Kochi Medical School, Nankoku, 783-8505, Kochi, Japan. ikedamit@kochi-ms.ac.jp The heterodimeric form of nuclear factor kappaB (NF-kappaB), NF-kappaB1/RelA, is one of the pluripotential transcription factors that activates various genes encoding cytokines and cell adhesion molecules. To clarify the involvement of radical oxygen species in the NF-kappaB activation pathway in keratinocytes, we examined the effect of hydrogen peroxide (H(2)O(2)) on the activation of NF-kappaB in cultured normal human epidermal keratinocytes. After the treatment of keratinocytes with 300 microM H(2)O(2), a translocation of NF-kappaB from the cytoplasm to nucleus was observed in an immunofluorescence study using anti-human NF-kappaB1 and anti-human RelA antibodies. Specific DNA binding was observed with the nuclear extract prepared from the H(2)O(2)-treated keratinocytes by the electrophoretic mobility shift assay. The presence of N-acetyl-L-cysteine or pyrrolidine dithiocarbamate during H(2)O(2) treatment prevented the nuclear localization of NF-kappaB. The involvement of radical oxygen species in the NF-kappaB activation pathway was suggested. Pretreatment of keratinocytes with 10 ng/ml of recombinant human interleukin-10 (IL-10) for 24 h suppressed the nuclear translocation of NF-kappaB induced by H(2)O(2). IL-10, which increases in ultraviolet-irradiated skin and suppresses delayed type hypersensitivity in vivo, may play an inhibitory role in cutaneous inflammation by inhibiting the NF-kappaB activation pathway in keratinocytes. PMID: 11858955 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: J Leukoc Biol. 2001 Jul;70(1):30-8. Long-term-impaired expression of nuclear factor-kappa B and I kappa B alpha in peripheral blood mononuclear cells of trauma patients. Adib-Conquy M, Asehnoune K, Moine P, Cavaillon JM. Departement de Physiopathologie, Institut Pasteur, 75724 Paris Cedex 15, France. Nuclear factor (NF)-kappa B expression and dimer characteristics were studied in peripheral blood mononuclear cells (PBMCs) of major-trauma patients and healthy controls. Analysis of PBMCs on days 1, 3, 5, and 10 after trauma revealed that expression of both p65p50 heterodimers and p50p50 homodimers was significantly reduced compared with that in controls. In vitro lipopolysaccharide (LPS) stimulation of PBMCs induced NF-kappa B translocation. However, throughout the survey, p65p50 activation remained significantly lower in trauma patients than in controls. After LPS stimulation in vitro, the p65p50/p50p50 ratio was significantly lower in PBMCs from trauma patients than from healthy controls. The ex vivo expression of I kappa B alpha was higher in PBMCs of controls than of trauma patients. LPS did not induce I kappa B expression in PBMCs from trauma patients, but strong induction was obtained with staphylococci, suggesting that this defect is not universal and depends on the nature of the activating signal. Although no direct correlation was found between levels of interleukin-10 or transforming growth factor-beta and NF-kappa B, these immunosuppressive cytokines were significantly elevated in trauma patients by 10 days after admission. The long-term low-basal and LPS-induced nuclear translocation of NF-kappa B recalled long-term immunoparalysis observed in patients with severe inflammatory stress such as trauma. PMID: 11435482 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: Br J Pharmacol. 2001 May;133(1):49-60. The biphasic immunoregulation of pyrimidylpiperazine (Y-40138) is IL-10 sensitive and requires NF-kappa B targeting in the alveolar epithelium. Haddad JJ, Safieh-Garabedian B, Saade NE, Land SC. Oxygen Signalling Group, Centre for Research into Human Development, Tayside Institute of Child Health, Faculty of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY. j.j.haddad@dundee.ac.uk 1. Pyrimidylpiperazine (Y-40138), a synthetic derivative of N-[1-(4-([4-(pyrimidin-2-yl)piperazin-1-yl]methyl)phenyl)cyclopropyl] acetamide, is a novel dual regulator of pro- and anti-inflammatory cytokines in vivo. The aim of the present study was to determine the signal transduction mechanisms implicated in vitro. 2. In alveolar epithelial cells, pre-treatment (30 min) with Y-40138 reduced LPS-induced biosynthesis of IL-1 beta, IL-6 and TNF-alpha, an effect paralleled by up-regulating an anti-inflammatory counter-loop mediated through IL-10. 3. This differential regulation of pro- and anti-inflammatory signals was accompanied by an inhibition of the nuclear localization of selective NF-kappa B subunits, particularly NF-kappa B(1) (p50), RelA (p65), the major transactivating member of the Rel family, RelB (p68) and c-Rel (p75). In addition, Y-40138 blockaded, in a dose-dependent manner, the LPS-induced nuclear activation of NF-kappa B. 4. Analysis of the upstream pathway involved in Y-40138-dependent retardation of LPS-induced NF-kappa B translocation/activation revealed the involvement of an I kappa B-alpha sensitive pathway. Pre-treatment with Y-40138 ameliorated LPS-induced degradation of I kappa B-alpha in the cytosolic compartment and retarded its phosphorylation, suggesting the involvement of an upstream kinase. 5. Recombinant IL-10 (0 -- 10 ng ml(-1)) blockaded, in a dose-dependent manner, LPS-induced biosynthesis of IL-1 beta, IL-6 and TNF-alpha. Furthermore, rhIL-10 reduced the DNA binding activity of NF-kappa B. Immunoneutralization of endogenous IL-10 by a polyclonal alpha IL-10 (5 microg ml(-1)) reversed the inhibitory effect of Y-40138 on pro-inflammatory cytokines and partially restored the DNA binding activity of NF-kappa B. 6. These results indicate that Y-40138 mediated dual immunoregulation of pro- and anti-inflammatory cytokines is IL-10 sensitive and mediated through the I kappa B-alpha/NF-kappa B signal transduction pathway. PMID: 11325794 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 15: Am J Respir Crit Care Med. 2000 Nov;162(5):1877-83. NF-kappaB expression in mononuclear cells of patients with sepsis resembles that observed in lipopolysaccharide tolerance. Adib-Conquy M, Adrie C, Moine P, Asehnoune K, Fitting C, Pinsky MR, Dhainaut JF, Cavaillon JM. Unite d'Immuno-Allergie, Institut Pasteur, Paris, France; Service de Reanimation Medicale, Hopital Cochin Port-Royal, Paris, France. The expression of NF-kappaB was studied in freshly isolated peripheral blood mononuclear cells (PBMC) of patients with severe sepsis and major trauma. The expression of p65p50 heterodimer, the active form of NF-kappaB, was significantly reduced for all patients as compared with control subjects. The p50p50 homodimer, an inhibitory form of NF-kappaB, was reduced in the survivors of sepsis and in patients with trauma. Subsequent in vitro stimulation of PBMC with lipopolysaccharide (LPS) did not induce further NF-kappaB nuclear translocation: the survivors of sepsis and trauma patients showed low expression of both p65p50 and p50p50, whereas nonsurvivors of sepsis showed a predominance of the inactive homodimer and a low p65p50/p50p50 ratio when compared with control subjects. In the later group of patients there was a reverse correlation between plasma IL-10 levels and the p65p50/p50p50 ratio after in vitro LPS stimulation (r = -0.8, p = 0.04). The reduced expression of nuclear NF-kappaB was not due to its inhibition by IkappaBalpha, as very low expression of IkappaBalpha, as well as low levels of p65 and p50 were found in the cytoplasm of PBMC from patients with sepsis and trauma when compared with control subjects. These results demonstrate that upon LPS activation, PBMC of patients with systemic inflammatory response syndrome show patterns of NF-kappaB expression that resemble those reported during LPS tolerance: global down-regulation of NF-kappaB in survivors of sepsis and trauma patients and the presence of large amounts of the inactive homodimer in the nonsurvivors of sepsis. PMID: 11069829 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 16: Cytokine. 2000 Sep;12(9):1348-55. Interleukin 10 (IL-10)-mediated inhibition of inflammatory cytokine production by human alveolar macrophages. Raychaudhuri B, Fisher CJ, Farver CF, Malur A, Drazba J, Kavuru MS, Thomassen MJ. Department of Pulmonary and Critical Care Medicine, The Cleveland Clinic Foundation, 9500 Euclid Avenue Cleveland, Ohio 44195-5038, USA. Alveolar macrophages are an important source of inflammatory cytokines in the lung. IL-10 has been shown to inhibit inflammatory cytokine production by human alveolar macrophages, but mechanisms are unclear. The purpose of the present study was to investigate whether IL-10 modified cytokine production by interference with transcriptional pathways. Alveolar macrophages were obtained from healthy controls by fiberoptic bronchoscopy and incubated with LPS+/-IL-10. Results indicated that steady state mRNA levels of tumour necrosis factor-alpha (TNF) and interleukin 1-beta (IL-1) decreased in the presence of IL-10. Consequently, electrophoretic mobility shift assays were performed using end-labelled nuclear factor-kappa B (NF-kappa B) or activator protein-1 (AP-1) probe. NF-kappa B binding was decreased in extracts from macrophages incubated for 4 h with LPS+IL-10 in comparison to those incubated with LPS alone. IL-10 also inhibited TNF secretion and NF-kappa B activation induced by another stimulus, staphylococcal toxin. Supershift assays revealed the presence of both p50 and p65 subunits of NF-kappa B. AP-1 was not affected by IL-10. Further examination of mechanisms indicated that IL-10 delayed the LPS-mediated degradation of the inhibitor protein I kappa B, thus delaying the nuclear translocation of the p65 subunit. These observations provide the first evidence that IL-10 antagonizes cytokine transcription in human alveolar macrophages by impeding the nuclear translocation of NF-kappa B by delaying the degradation of I kappa B. Copyright 2000 Academic Press. PMID: 10975994 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 17: Arthritis Rheum. 2000 Jun;43(6):1244-56. Macrophage-derived cytokine and nuclear factor kappaB p65 expression in synovial membrane and skin of patients with psoriatic arthritis. Danning CL, Illei GG, Hitchon C, Greer MR, Boumpas DT, McInnes IB. University of Glasgow, UK. OBJECTIVE: Monocyte-derived cytokines are important mediators in synovitis and represent novel therapeutic targets. This study was undertaken to analyze their expression in synovial membrane (SM) of patients with psoriatic arthritis (PsA) compared with that in skin of patients with PsA and SM of patients with rheumatoid arthritis (RA). METHODS: Multiple synovial biopsy samples (24 from patients with PsA, 20 from patients with RA, 5 from patients with osteoarthritis [OA]) and skin biopsy samples (lesional and perilesional skin from 25 PsA patients) were obtained. Standard leukocyte antigens, cytokines (tumor necrosis factor alpha [TNFalpha], interleukin-1apha [IL-1alpha], IL-1beta, IL-15, and IL-10) and the transcription factor nuclear factor KB (NF-kappaB; active p65 subunit) were localized and quantified immunohistochemically by light microscopy and digital image analysis. RESULTS: Sublining cellular infiltration, lymphoid aggregation, and vascularity were similar in PsA and RA SM. Lining layer thickness was greater in RA SM, associated with more CD68+ macrophages. In PsA SM, TNFalpha, IL-1alpha, IL-1beta, IL-15, and IL-10 were primarily localized to lining layer and perivascular macrophages, as were cells expressing the active subunit of NF-kappaB (p65). TNFalpha, IL-1p, and IL-15 expression in PsA lining layer was less than that in RA lining layer, likely reflecting lower macrophage numbers. In sublining areas, levels of TNFalpha and IL-15 were lower in PsA patients than in RA patients, whereas IL-lalpha and IL-1beta expression was equivalent. IL-10 was identified at similar levels in RA and PsA SM lining layer and sublining. Expression of NF-kappaB (p65) was equal in lining layer from both patient groups, but lower in PsA than RA sublining. Histologic findings did not correlate with clinical parameters of disease. Cytokine expression in skin did not correlate directly with that in SM. Cytokine expression was greater in PsA and RA SM than in OA SM. CONCLUSION: This study shows, for the first time, that monocyte-derived cytokines are found in PsA SM and demonstrates the relative paucity of the antiinflammatory cytokine IL-10 in PsA skin and SM. Significant divergence from RA SM expression was observed, despite similar clinical and demographic features in the 2 patient groups. PMID: 10857783 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 18: J Immunol. 1999 Jun 1;162(11):6442-50. In vivo inhibition of NF-kappa B in T-lineage cells leads to a dramatic decrease in cell proliferation and cytokine production and to increased cell apoptosis in response to mitogenic stimuli, but not to abnormal thymopoiesis. Ferreira V, Sidenius N, Tarantino N, Hubert P, Chatenoud L, Blasi F, Korner M. Laboratoire d'Immunologie Cellulaire et Tissulaire, Centre National de la Recherche Scientifique UMR 7627, Hopital de la Pitie-Salpetriere, Paris, France. To understand the role of NF-kappa B complexes in T cell development and activation, we have generated transgenic mice in which RelA and c-Rel complexes were selectively inhibited in the T-lineage cells by specific expression of a trans-dominant form of I kappa B alpha. Transgene expression did not affect the thymic development, but led to lowered numbers of splenic T cells and to a dramatic decrease in the ex vivo proliferative response of splenic T lymphocytes. Analysis of IL-2 and IL-2R alpha expression demonstrated that the perturbation of the proliferation response was not attributable to an abnormal expression of these genes. In contrast, expression of IL-4, IL-10, and IFN-gamma was strongly inhibited in the transgenic T cells. The proliferative deficiency of the transgenic T cells was associated with an increased apoptosis. These results point out the involvement of NF-kappa B/Rel family proteins in growth signaling pathways by either regulating proteins involved in the IL-2 signaling or by functionally interfering with the cell cycle progression. PMID: 10352258 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 19: Leuk Lymphoma. 1998 Apr;29(3-4):239-48. Interleukin-10 gene expression and adult T-cell leukemia. Mori N, Prager D. Department of Medicine, UCLA School of Medicine, Los Angeles, USA. Numerous studies have shown spontaneous IL-10 gene expression and synthesis in a variety of peripheral blood or bone marrow-derived leukemic cells. These include B-cells derived from various lymphoproliferative disorders. Since little is known regarding IL-10 expression in leukemic T-cells, we examined clinical specimens of patients with adult T-cell leukemia (ATL) for IL-10 expression. Sera from ATL patients show increased levels of IL-10 when compared with sera from healthy donors. IL-10 is constitutively produced by ATL cells and also by human T-cell leukemia virus type I (HTLV-I)-infected cell lines. It is thought that HTLV-I infection induces gene expression for IL-10. In this review, a transcriptional regulation of IL-10 gene expression by HTLV-I Tax and the possible role of the NF-kappaB pathway are described. PMID: 9684922 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 20: Eur J Immunol. 1998 May;28(5):1719-26. IL-10-mediated suppression of TNF-alpha production is independent of its ability to inhibit NF kappa B activity. Clarke CJ, Hales A, Hunt A, Foxwell BM. Kennedy Institute of Rheumatology, Hammersmith, London, GB. IL-10 has a well-characterized anti-inflammatory role that includes the suppression of inflammatory cytokine (e.g. TNF-alpha) production by monocytic/macrophage cells. Both transcriptional and post-transcriptional/translational mechanisms have been proposed to explain this process. In this study we observed that IL-10 inhibited nuclear NF kappa B DNA binding activity without affecting I kappa B degradation or translocation of NF kappa B subunits to the nucleus. While the suppression of NF kappa B in 70Z/3 pre-B cells correlated with suppression of NF kappa B transcriptional activity and expression of surface IgM, it did not correlate with the production of TNF-alpha mRNA or protein in RAW 264.7 macrophages. Similar observations in the macrophages were made with a second anti-inflammatory cytokine, IL-4. Therefore we conclude that although IL-10 or IL-4 can suppress NF kappa B activity, this appears to have little effect on the expression of the TNF-alpha gene and is unlikely to be the basis of the anti-inflammatory effects of these cytokines. PMID: 9603479 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 21: Nat Med. 1996 Sep;2(9):998-1004. Local administration of antisense phosphorothioate oligonucleotides to the p65 subunit of NF-kappa B abrogates established experimental colitis in mice. Neurath MF, Pettersson S, Meyer zum Buschenfelde KH, Strober W. Laboratory of Immunology, University of Mainz, Germany. Chronic intestinal inflammation induced by 2,4,6,-trinitrobenzene sulfonic acid (TNBS) is characterized by a transmural granulomatous colitis that mimics some characteristics of human Crohn's disease. Here, we show that the transcription factor NF-kappa B p65 was strongly activated in TNBS-induced colitis and in colitis of interleukin-10-deficient mice. Local administration of p65 antisense phosphorothioate oligonucleotides abrogated clinical and histological signs of colitis and was more effective in treating TNBS-induced colitis than single or daily administration of glucocorticoids. The data provide direct evidence for the central importance of p65 in chronic intestinal inflammation and suggest a potential therapeutic utility of p65 antisense oligonucleotides as a novel molecular approach for the treatment of patients with Crohn's disease. PMID: 8782457 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------