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Interleukin (IL)-23 is a heterodimeric cytokine of the IL-12 family. Human IL-23 is known to induce interferon (IFN)-gamma production and proliferation in T cells, preferentially in the CD45RO+ memory subset. Yet, its role in the differentiation of human naive T cells remains largely unknown. We investigated the effect of recombinant human (rh)IL-23 on cord blood CD4+ and CD8+ T cells during polyclonal activation. The IL-23 receptor complex was not detectable in resting naive T cells. Nevertheless, both IL-23 receptor subunits, IL-12Rbeta1 and IL-23R, were rapidly induced after activation in both naive CD4+ and CD8+ T cells. In both cell types, rhIL-23 enhanced IFN-gamma production. This effect was demonstrable as early as 2 days after activation, illustrating that a functional IL-23 receptor is rapidly induced in naive T cells upon activation. In naive CD8+ T cells, rhIL-23 specifically induced the secretion of IL-17, a pro-inflammatory cytokine. Moreover, rhIL-23 significantly increased the production of IL-10 in both naive CD4+ and CD8+ T cells. IL-17 and IL-10 levels were not affected by the addition of rhIL-12. We conclude that IL-23 induces a specific cytokine profile, remarkably distinct from IL-12, in activated human naive T cells.

Viral interleukin (IL)-10 (vIL-10) has been widely described as an immunoregulatory cytokine that does not possess the T-cell costimulatory activities of cellular IL-10; it was therefore believed to be a more potent tolerogenic mediator. The immunosuppressive properties of this cytokine are partly attributed to its capacity to render dendritic cells (DCs) unable to undergo full maturation and to activate T cells. We reported here that myeloid DCs retrovirally transduced with vIL-10 had an impaired production of IL-12 and a decreased expression of MHC class II molecules but had minor defects in costimulatory molecule expression and no alteration on CCR5 and CCR7 expression. In mixed leukocyte reaction, vIL-10-transduced C57BL/6 bm12 (MHC class II mismatch) DCs had a reduced capacity to stimulate C57BL/6 wild-type CD4+ T-cell proliferation. We show that bm12 vIL-10-transduced DC administration in CD8-/- C57BL/6 mice promoted IFN-gamma production, down-regulated TH2-type cytokine production, and did not induce skin graft tolerance. These findings suggest that vIL-10-transduced DC may surprisingly facilitate Th1-type inflammatory responses in vivo.

Secondary pneumococcal pneumonia is a serious complication during and shortly after influenza infection. We established a mouse model to study postinfluenza pneumococcal pneumonia and evaluated the role of IL-10 in host defense against Streptococcus pneumoniae after recovery from influenza infection. C57BL/6 mice were intranasally inoculated with 10 median tissue culture infective doses of influenza A (A/PR/8/34) or PBS (control) on day 0. By day 14 mice had regained their normal body weight and had cleared influenza virus from the lungs, as determined by real-time quantitative PCR. On day 14 after viral infection, mice received 10(4) CFU of S. pneumoniae (serotype 3) intranasally. Mice recovered from influenza infection were highly susceptible to subsequent pneumococcal pneumonia, as reflected by a 100% lethality on day 3 after bacterial infection, whereas control mice showed 17% lethality on day 3 and 83% lethality on day 6 after pneumococcal infection. Furthermore, 1000-fold higher bacterial counts at 48 h after infection with S. pneumoniae and, particularly, 50-fold higher pulmonary levels of IL-10 were observed in influenza-recovered mice than in control mice. Treatment with an anti-IL-10 mAb 1 h before bacterial inoculation resulted in reduced bacterial outgrowth and markedly reduced lethality during secondary bacterial pneumonia compared with those in IgG1 control mice. In conclusion, mild self-limiting influenza A infection renders normal immunocompetent mice highly susceptible to pneumococcal pneumonia. This increased susceptibility to secondary bacterial pneumonia is at least in part caused by excessive IL-10 production and reduced neutrophil function in the lungs.

Background: Graft ischemia-reperfusion injury (IRI) resulting from postreperfusion inflammatory reaction remains a major cause of complications after liver transplantation. In this article, the authors investigated the effect of anti-inflammatory cytokine interleukin (IL)-10 on IRI, in a preclinical model of liver transplantation in pigs., Methods: Donor pigs received IL-10 or saline at the start of liver graft harvesting. After 5 hr of cold ischemia, liver grafts were transplanted into untreated recipient pigs. IRI severity was measured in recipients by transaminase release and by cellular infiltration and necrosis on liver biopsy specimens., Results: Donor IL-10 administration attenuated IRI, as indicated by significant reduction of mean peak of transaminase in recipients of grafts from IL-10-treated donors. In contrast, no significant differences in cell infiltration or amount of necrosis were observed on liver biopsy specimens between groups., Conclusions: Donor preconditioning with IL-10 may constitute an interesting pharmacologic approach to reduce IRI severity after liver transplantation.

Inflammation is commonly observed in liver diseases and is frequently complicated by fibrosis and cirrhosis in end-stage disease. The only curative treatment for cirrhotic patients is liver transplantation. However, organ shortage as well as an increasing organ demand call for early treatment of liver disease and prevention of fibrosis. Experimental data have shown the critical role of pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in the development of liver injury. Here, we review our work on the role of endogenously produced interleukin-10 (IL-10), a potent anti-inflammatory cytokine, in several experimental models of acute and chronic liver injury. First, in acute macrophage-mediated hepatitis induced by galactosamine/lipopolysaccharide administration, IL-10 neutralisation led to a more severe liver damage, whereas IL-10 injection, even delayed, was able to limit liver necrosis. A similar protective effect of IL-10 was observed in acute T cell-mediated hepatitis induced by concanavalin A (Con A) injection. The immunoregulatory role of IL-10 was then established after repeated exposition to Con A. In carbon tetrachloride liver injury, two other properties of IL-10 have been suggested: modulation of hepatocyte proliferation and modulation of liver fibrosis. Finally, the potential therapeutic applications in human liver disease as well as the potential side effects are discussed.

Background: Mechanisms of action of extracorporeal photochemotherapy (ECP) in graft-versus-host disease are incompletely understood. It has been proposed that phagocytosis of apoptotic bodies by monocytes and macrophages induces their activation, a process that increases production of immunosuppressive cytokines. We analyzed apoptosis and cytokine secretion in an autologous coculture system combining peripheral blood lymphocytes (PBL) obtained after ex vivo ECP treatment and nonirradiated peripheral blood mononuclear cells (PBMC)., Methods: We studied 11 leukapheresis products treated by ECP from six patients with resistant limited or extensive chronic graft-versus-host disease. Purified PBL obtained by monocyte depletion of the buffy coat from leukapheresis products were mixed with nonirradiated PBMC. Nonirradiated PBL were used as control. Cytokine production was tested at the mRNA level by real-time reverse transcriptase-polymerase chain reaction for interleukin (IL)-10, IL-1 receptor antagonist (IL-1Ra), IL-1beta, tumor necrosis factor-alpha, and IL-12p40., Results: Morphologic analysis and flow cytometry displayed important lymphocyte apoptotic features culminating at 24 to 48 hr. Coculture of patient's PBMC with ultraviolet-irradiated PBL as compared with nonirradiated PBL resulted in significant increase of IL-10 mRNA (3418+/-1015 versus 10596+/-3402 mRNA copy numbers; P=0.001) and IL-1Ra mRNA (23890+/-6166 versus 41767+/-10181 mRNA copy numbers; P=0.001). Incubation with a neutralizing anti-IL-10 monoclonal antibody resulted in a marked decrease of IL-1Ra mRNA levels., Conclusion: Our findings are consistent with the fact that ECP modifies patient's autologous lymphocytes by inducing a process of apoptosis that activates monocytes and macrophages, leading to increased synthesis of IL-10 and IL-1Ra mRNAs. The induction of this latter mediator is dependent on IL-10.

To clarify the functional consequences of adenine nucleotides action on human monocyte-derived dendritic cells (DC), we have systematically compared the effects of adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), an ATP analog active on the P2Y(11) receptor, on the responses to three DC stimuli, TNF-alpha, LPS, sCD40L, tested at various concentrations, using two different IL-12 assays. We observed that ATPgammaS potentiated the IL-12p40 release induced by TNF-alpha, but also by lipopolysaccharides (LPS) and soluble CD40 ligand (sCD40L). This potentiation was observed as long as the IL-12p40 concentration under agonist stimulation remained below a threshold value close to 10 ng/ml; inhibition was observed above this value. The combinations ATPgammaS-TNF-alpha and ATPgammaS-sCD40L were unable to induce detectable bioactive IL-12p70 production and at concentrations of LPS that induced a significant stimulation of IL-12p70, the effect of ATPgammaS was purely inhibitory. Our results also show that ATPgammaS synergized with LPS and sCD40L, but not TNF-alpha, to stimulate IL-10 production. In conclusion, we have clarified the discrepancies in the literature concerning the action of adenine nucleotides on DC and our study supports the concept that, like prostaglandin E(2) and other agents increasing cyclic AMP, they favor either a Th2 response or tolerance.

Background: Interleukin-10 (IL-10) is currently being extensively studied in clinical trials for the treatment of Crohn's disease (CD). Only marginal effects have, however, been reported, and the dose-response curve was bell-shaped contrasting with the reported data from in vitro experiments., Aim: To use another in vitro model to analyze the effect of rhIL-10 and rhIL-4 on the spontaneous mucosal TNF-alpha secretion in patients with CD, and to characterize the phenotype of the cells targeted by rhIL-10., Methods: Non-inflamed colon biopsies from CD patients were cultured for 16 hours in presence of different concentrations of rhIL-10 or rhIL-4. The numbers of TNF-alpha-secreting cells among isolated lamina propria mononuclear cells (LPMNC) were estimated by Elispot., Results: Both rhIL-10 and rhIL-4 down-regulate TNF-alpha secretion by LPMNC from CD patients, with a more pronounced effect with rhIL-10. These effects were closely linked to the cytokine concentrations used, with a bell-shaped dose-response curve. Residual TNF-alpha secretion, in the presence of optimal rhIL-10 concentration was mainly attributable to CD3+ T cells. In contrast, at higher rhIL-10 concentrations, CD3- cells contributed significantly to the TNF-alpha secretion., Conclusions: The in vitro model we used, demonstrates that IL-4, but mostly IL-10, efficiently suppresses TNF-alpha secretion in LPMNC from CD patients, with a dose-response curve similar to results obtained in vivo. Resistance at high rhIL-10 concentrations was associated with a change in the phenotype of TNF-alpha-secreting cells.

Dendritic cells (DC) are the most potent presenters of alloantigens and therefore are responsible for the induction of allograft rejection. Genetic modifications of DC allowing the expression of a tolerogenic molecule may render them immunosuppressive. We transduced bone marrow-derived DC with recombinant MFG retrovirus encoding either viral interleukin (vIL)-10 or Fas ligand (FasL) to induce transplantation tolerance. Up to 10 ng/ml of bioactive vIL-10 was produced by DC after transfer of the corresponding gene. Although the inhibitory properties of vIL-10-transduced DC were revealed in vitro in a mixed lymphocyte culture, no clear down-regulation of the allogeneic response was observed in vivo after single or multiple injections of those DC overexpressing vIL-10. When we transduced wild-type bone marrow-derived DC with recombinant MFG retrovirus encoding murine FasL, cells quickly died, probably because of suicidal or fratricidal Fas-dependent death. Indeed, only DC from Fas-deficient lpr mice survived to FasL gene transfer. Those FasL-transduced lpr DC exhibited a strong cytotoxic activity against Fas-positive targets in vitro. DC overexpressing FasL did not behave as immunosuppressive DC in vivo. The subcutaneous injection of FasL+ lpr DC in MHC class II-disparate mice hyperactivated the allospecific proliferation of T cells in the draining lymph nodes compared with mice treated with control-transduced DC. These results argue against the development of FasL+ DC or vIL-10-secreting DC as immunosuppressive tools in vivo. The alternative pathways of T-cell activation triggered by these genetically modified DC need to be investigated.

The repeated injection of bacterial superantigens (SAg), such as staphylococcus enterotoxin (SE) A or B, has been shown in mice to induce a state of unresponsiveness characterized by the lack of secretion of Th1 lymphokines, such as IL-2 and IFN-gamma, following subsequent SAg challenge. We made the observation, in vivo as well as in vitro, that unresponsiveness to SAg could be transferred from SEA- to SEB-reactive T cells (and reversibly from SEB- to SEA-specific T cells) in C57BL/6 mice but not in BALB/c mice. Since C57BL/6 mice, unlike BALB/c mice, possess TCR V(beta)3+ and V(beta)11+ T cells able to react with both SEA and SEB, we hypothesized that SAg-unresponsive V(beta)3(+) and V(beta)11+ T cells could mediate linked suppression of other SAg-reactive T cells. To analyze further this possibility, spleen cells from BALB/c mice made unresponsive to SEB were tested for their capacity to suppress the response of normal BALB/c cells to SEB. The production of both IFN-gamma and IL-2 following SEB stimulation was greatly impaired in co-cultures containing CD4(+) T cells, but not CD8(+) T cells, isolated from unresponsive animals. In vivo, the production of both IFN-gamma and IL-2 responses to SEB was dramatically reduced in animals adoptively transferred with unresponsive spleen cells. This suppression was abrogated in recipients injected with neutralizing anti-IL-10 antibodies. Moreover, in animals made unresponsive to SEB, SAg-reactive CD4(+) T cells were found to express high levels of CTLA-4, a molecule recently described to play an essential role in the suppressive function of regulatory T cells. Taken together these results demonstrate that the repetitive injection of SAg induces the differentiation of regulatory CD4(+) T cells capable of suppressing SAg-reactive naive T cells.

Weekly injections of Concanavalin A (Con A) were performed in BALB/c mice to evaluate the pattern of cytokine production and liver injury. High serum levels of tumor necrosis factor alpha (TNF-alpha), interleukin 2 (IL-2), IL-4, and interferon gamma (IFN-gamma) were found in the serum after the first 2 injections of Con A but rapidly decreased from the third injection. Conversely, IL-10 serum levels after repeated Con A challenge increased by 7 times from week 1 to 20. In vivo depletion studies indicated that CD4(+) T cells are essential in IL-10 production. Hepatocyte necrosis was only observed after the first injections of Con A whereas centrilobular inflammatory infiltrates persisted up to 20 weeks. Perisinusoidal liver fibrosis was also increasingly detected in BALB/c mice, whereas no fibrous change was observed in nude mice after 6 weeks of Con A challenge. The number of stellate cells, detected by immunostaining, increased after 20 weeks of Con A injections. Liver cytokine messenger RNA (mRNA) expression after 20 weeks showed expression of transforming growth factor beta1 (TGF-beta1), IL-10, and IL-4 whereas IL-2 was no more expressed. The present study shows that mice repeatedly injected with Con A develop liver fibrosis. The cytokine-release pattern observed after 1 injection of Con A is rapidly shifted towards an immunomodulatory phenotype characterized by the systemic production of large amounts of IL-10.

Background: Activating anti-CD3 monoclonal antibodies (mAbs), such as OKT3, are potent immunosuppressive agents that are widely used in clinical transplantation. We investigated whether the in vivo induction of T cell unresponsiveness contributes to the immunosuppressive properties of the anti-mouse-CD3 mAb 145-2C11., Methods: After a single in vivo administration of 145-2C11 residual T cells were restimulated in vivo and in vitro to assess cytokine production. Mice were also transplanted with allogeneic skin 9 days after 145-2C11 administration to investigate whether the immunosuppressive properties of the antibody persist after the reexpression of the T cell receptor., Results: Pretreatment with anti-CD3 mAbs caused a profound deficit in both interleukin- (IL) 2 and interferon- (IFN) y secretion upon restimulation in vivo, whereas IL-4 was only partially inhibited and IL-10 production was significantly increased. Purified T cells obtained from mice injected with anti-CD3 mAb also displayed deficient IL-2 and IFN-gamma production together with persisting IL-4 and IL-10 secretion. 145-2C11 had immunosuppressive properties that per sisted after the reexpression of the T cell receptor because mice transplanted with allogeneic skin 9 days after a single anti-CD3 mAb injection still had significantly prolonged graft survival (14.1+/-0.6 days vs. 10.7+/-0.4 days in controls, P<0.02). Blocking IL-4 and IL-10 by neutralizing mAbs further prolonged skin graft survival in mice injected with 145-2C11 (18.3+/-0.7 vs. 14.8+/-0.6 days, P<0.02)., Conclusion: The in vivo administration of the 145-2C11 anti-CD3 mAb results in the selective inhibition of Thl-type cytokine secretion upon restimulation, which correlates with a state of immunosuppression. The persistent production of Th2-type cytokines does not contribute to the anti-CD3 mAb-mediated prolonged survival of skin allografts in our experimental model.

Background: The first administration of CD3 monoclonal antibodies, such as anti-human CD3 (OKT3), induces a massive release of several cytokines, including tumor necrosis factor alpha (TNF-alpha), interferon (IFN)-gamma, interleukin (IL)-2, IL-3, IL-6, and granulocyte-macrophage colony-stimulating factor., Methods: Cytokine levels in patient's sera were measured by specific ELISA. In vitro cultures were performed using OKT3-stimulated peripheral blood mononuclear cells and/or whole blood from patients and normal controls., Results: Here we describe that OKT3 administration to human renal allograft recipients also leads to a significant release of IL-10. Contrasting with most OKT3-induced cytokines, such as TNF-alpha whose release is transient, IL-10 levels show a more progressive increase, they peak only by 4-8 hr after the first OKT3 injection and persist longer. Thus, significant IL-10 levels are still detectable at the time of the second and the third OKT3 injection. Administration of corticosteroids, 1 hr before the first OKT3 injection, significantly reduced both TNF-alpha and IL-10 release. Experiments were performed to evaluate the source(s) of IL-10 and its (their) influence on the initial T-cell activation. When stimulated in culture with soluble OKT3, the production of IL-10 was dependent on the cooperation between T lymphocytes and monocytes. It is important that, as assessed through the use of a specific neutralizing antibody, the endogenous IL-10 produced in the co-culture system exerted a negative feed-back on the release of the other pro-inflammatory CD3-induced cytokines, which was reproducible., Conclusion: These results are supportive of a major role of IL-10 in the down-modulation of the OKT3-triggered T-cell activation cascade.

Mycophenolate mofetil (MMF) is a new immunosuppressive agent currently used in organ transplantation and under evaluation in immune-mediated inflammatory disorders such as rheumatoid arthritis. Although MMF was shown to inhibit purine nucleotide synthesis in lymphocytes, it is still unclear whether it might also exert direct antiinflammatory actions in vivo. To address this question, we evaluated the effects of MMF administration on the responses of mice to a single challenge with bacterial lipopolysaccharide (LPS). We observed that MMF treatment inhibits the release of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) upon LPS injection whereas it promotes IL-10 production. In parallel, MMF was found to protect mice from LPS-induced lethality. Inhibition of TNF-alpha release was also observed in IL-10-deficient mice indicating that it does not exclusively depend on the upregulation of IL-10 endogenous synthesis. In view of the differential effects of MMF on the LPS-induced production of TNF-alpha and NO on one hand and that of IL-10 on the other hand, we conclude that beside its immunosuppressive action at the lymphocyte level, MMF is also endowed with antiinflammatory properties.

The role of the anti-inflammatory cytokine interleukin-10 (IL-10) was investigated in the mouse model of liver injury induced by carbon tetrachloride (CCl4). To address the role of endogenous IL-10 production, acute hepatitis was induced by CCl4 in C57Bl/6 IL-10 gene knock out (KO) and wild-type (WT) mice. After CCl4 challenge, serum and liver levels of tumor necrosis factor-alpha (TNF-) and serum levels of transforming growth factor-beta 1 (TGF-beta1) increased and were significantly higher in IL-10 KO mice, whereas IL-6 serum levels were only slightly increased compared with WT mice. At histological examination, the livers disclosed a significantly more prominent neutrophilic infiltration in IL-10 KO mice 12 and 24 hours after CCl4 injection. In contrast, hepatocyte necrosis, evaluated by histological examination and serum alanine aminotransferase levels, was only marginally affected. The proliferative response of hepatocytes, assessed by the proliferating cell nuclear-antigen labeling index, was significantly increased in IL-10 KO mice, compared with WT mice 48 hours after CCl4 injection. Finally, repeated CCl4 injections led to more liver fibrosis in IL-10 KO mice after 7 weeks. In conclusion, endogenous IL-10 marginally affects the hepatocyte necrosis although it controls the acute inflammatory burst induced by CCl4. During liver repair, it limits the proliferative response of hepatocytes and the development of fibrosis.

We examined the effects of IL-10 on tumour necrosis factor-alpha (TNF-alpha) and NO production by LPS-activated macrophages and on the ability of these cells to control Trypanosoma cruzi infection. We first observed that the addition of rIL-10 to macrophages of the J774 cell line decreased their synthesis of TNF-alpha but increased their release of NO in a dose-dependent manner. In parallel, treatment of J774 cells with rIL-10 resulted in a better control of T. cruzi infection involving up-regulation of NO synthesis, as it was not observed in presence of N-nitro-L-arginine methyl ester (L-NAME), a competitive inhibitor of NO synthase. The enhancing effect of rIL-10 on NO production was not observed on peritoneal macrophages from wild-type C57Bl/6 mice, but well on macrophages from IL-10 knock-out mice. The control of NO production by endogenous IL-10 was confirmed by the demonstration that neutralization of IL-10 secreted by LPS-activated macrophages from wild-type mice inhibited their production of NO and, in parallel, their ability to control T. cruzi infection. Taken together, these data demonstrate that both exogenous and endogenous IL-10 up-regulate the production of NO by LPS-activated macrophages and improve thereby their ability to clear T. cruzi infection.

Interleukin-10 is an ubiquitous cytokine which plays a major regulatory role in the course of inflammatory responses by downregulating the synthesis of cytokines. In this paper, we summarize the major biological properties of IL-10 and the current knowledge of the molecular mechanisms by which IL-10 inhibits the expression of genes encoding proinflammatory cytokines. We then review the factors upregulating IL-10 synthesis and we present the concept that IL-10 is a stress cytokine produced not only in response to microbial pathogens but also to cellular injuries of diverse origins.

Purpose: The aim of this study was to investigate the relation between interleukin (IL) 10, tumor necrosis factor alpha (TNF alpha), IL-1, and IL-6 levels in patients with septic shock and relate these cytokine levels to the development of organ failure., Patients and Methods: In 11 patients with septic shock of recent onset, blood was sampled for determinations of TNF, IL-1, IL-6, and IL-10. The degree of organ failure was scored for four organ systems (respiratory, hepatic, renal, hematologic) in the first 48 hours of the study., Results: The APACHE II score was 21 +/- 4. Three patients died. IL-10 levels were directly correlated with TNF levels (r = 0.73, P < .05) and IL-6 levels (r = 0.67, P < .05); and inversely correlated with total C3 (r = -0.73, P < .05) and CH50 (r = -0.68, P < .05). Both IL-10 and TNF levels were correlated to the organ failure score (r = 0.75 and r = 0.68, both P < .01). Six patients with high IL-10 levels (> 60 pg/mL) had lower C3 (37 +/- 11 v 62 +/- 10 mg/dL) and CH50 (32 +/- 7 v 68 +/- 19%), and higher organ failure scores (5.7 +/- 0.8 v 3.8 +/- 1.3) than those with low IL-10 levels (all P < .05)., Conclusion: Although IL-10 has an inhibitory effect on the production of cytokines, it is released together with TNF and IL-6 in patients with septic shock. IL-10 blood levels are directly related to the severity of inflammation and the development of organ failure in septic shock.

The mechanisms of interleukin-12 (IL-12) toxicity were studied in mice using a schedule (murine rIL-12, 400 ng/mouse, intraperitoneally [IP] once daily for 5 days) that markedly reduced body weight and food intake. On day 5, IL-12-treated mice had elevated serum and spleen IFN-gamma and tumor necrosis factor (TNF). Serum sTNFR-P75 and corticosterone (CS) were also elevated. IL-12 toxicity was partially prevented by anti-IFN-gamma antibodies or dexamethasone (DEX). A pre-dose of IL-12 (200 ng/mouse on day -14) completely prevented the toxicity of subsequent IL-12. The IL-12 predose also inhibited IL-12-induced IFN-gamma levels, but did not modify IL-12-induced CS, TNF or sTNFR-P75. A protective effect was observed with a predose of lipopolysaccharide (LPS) or murine recombinant (r)IL-10. The protective effect of the IL-12 predose was reduced by coadministration of anti-IFN-gamma, but a predose of murine rIFN-gamma was not protective, suggesting that IFN-gamma is necessary but not sufficient for the protective effect of IL-12. The IL-12 predose specifically protected against IL-12 toxicity and did not modify LPS toxicity. These data indicate that IL-12 can induce tolerance to its own toxicity, probably through a downregulation of IL-12-induced IFN-gamma but independently of endogenous glucocorticoids. IFN-gamma, and possibly IL-10, might be important in this tolerance.

Background & Aims: Reactive oxygen intermediates and cytokines are key effectors in reperfusion injury after liver ischemia. We hypothesized that reactive oxygen intermediates act as a signal for the release of tumor necrosis factor (TNF) and interleukin 10 (IL-10) after reperfusion of cold-preserved livers., Methods: An endotoxin-free isolated perfused mouse liver system was designed. Harvested mouse livers were stored at 4 degrees C for 0-28 hours and reperfused for 90 minutes with a warm oxygenated Hank's balanced salt solution (alone or with additives). Cytokine messenger RNA (mRNA) from whole liver was measured by reverse-transcription polymerase chain reaction. Cytokine protein levels and liver injury assessed by alanine aminotransferase levels were evaluated in liver effluent during reperfusion., Results: TNF and IL-10 mRNA and protein concentrations were increased after reperfusion of ischemic livers. N-Acetylcysteine and allopurinol dramatically decreased TNF (-64% and -62%) and IL-10 (-49% and -57%) levels in the effluents, as did an inhibitor of the transcription factor NF-kappaB mobilization (-73% and -76% for TNF and IL-10, respectively). Liver injury was decreased by -40%, -43%, and -54% for the three inhibitors, respectively., Conclusions: Reactive oxygen intermediates are involved in TNF and IL-10 release after reperfusion of cold-preserved livers.

Background: We conducted a randomized, double-blind, placebo-controlled, rising single-dose study to investigate the effects of recombinant human (rh) interleukin (IL) 10 in renal transplant patients who received OKT3 as induction therapy., Methods: Patients received 0.1 (n=6), 1 (n=6), or 10 microg/kg (n=3) rhIL-10 or placebo (n=6) intravenously 30 min before the first injection of 5 mg of OKT3. We monitored IL-10 serum levels, the effect of rhIL-10 on OKT3-induced cytokine production, clinical toxicity, and the incidence of immunization against OKT3., Results: Serum IL-10 levels in the three experimental groups reached 0.8+/-0.2, 7.9+/-1.3, and 118.6+/-7.3 ng/ml (mean+/-SEM), respectively, 30 min after rhIL-10 injection. Peak plasma levels of tumor necrosis factor-alpha (TNF-alpha) were reduced from 2953+/-1599 pg/ml in patients injected with OKT3 and placebo to 447+/-155, 703+/-246, and 459+/-246 pg/ml in patients injected with 0.1, 1, and 10 microg/kg rhIL-10, respectively. Values for 24-hr TNF-alpha area under the curve decreased from 8988+/-3551 pg x hr/ml in control patients to 2284+/-494, 3950+/-955, and 2420+/-931 pg x hr/ml for the 0.1, 1, and 10 microg/kg rhIL-10 dose groups, respectively (P=0.045). There was also a trend toward reduced plasma levels of IL-2, IL-8, and interferon-gamma in rhIL-10-pretreated patients. Although none of the patients who received placebo or 0.1 or 1 microg/kg rhIL-10 developed an IgM antibody response directed against OKT3 during the first 10 days, this occurred in all three patients who received the highest rhIL-10 dose. In two of these patients, neutralization of OKT3 was associated with a reversible acute rejection episode., Conclusions: Pretreatment with doses of up to 1 microg/kg rhIL-10 is safe and reduces the release of TNF-alpha induced by OKT3. However, higher doses might promote early sensitization to OKT3.

We evaluated the effects of interleukin (IL)-10 on the maturation of human dendritic cells (DC) induced either by lipopolysaccharide (LPS) or CD40 engagement. For this purpose, DC generated by culturing plastic-adherent peripheral blood mononuclear cells for 7 days with granulocyte/macrophage-colony-stimulating factor and IL-4 were incubated for 3 days with either LPS (10 ng/ml) or 3T6 fibroblasts transfected with the gene encoding CD40 ligand, in absence or presence of IL-10. First we found that the membrane expression of CD83, a marker of mature DC, was inhibited by IL-10 when induced by LPS but not by CD40 engagement. Likewise, IL-10 inhibited LPS-induced but not CD40-dependent CD86 (B7.2) up-regulation on DC. Furthermore, IL-10 inhibited the production of IL-8 and tumor necrosis factor-alpha by DC when activated by LPS but not by CD40. In contrast, IL-10 inhibited IL-12 production in both activation systems. We conclude that IL-10 differentially influences LPS-dependent and CD40-dependent pathways of DC maturation.

In this study, we examined the effects of interleukin-10 (IL-10) on the outcome of experimental cerebral malaria (CM), a lethal neurological syndrome that occurs in susceptible strains of mice after infection with Plasmodium berghei ANKA (PbA). Constitutive IL-10 mRNA levels were significantly higher in the spleen and brain of resistant animals. In vivo neutralization of endogenous IL-10 in CM-resistant mice induced the neurological syndrome in 35.7% of these mice, as opposed to 7.7% in controls. IL-10 inhibited PbA antigen-specific interferon-gamma (IFN-gamma) production in vitro but not tumour necrosis factor (TNF) serum levels in vivo. Susceptible mice, on the other hand, were significantly protected against CM when injected with recombinant IL-10. Overall, our findings suggest that IL-10 plays a protective role against experimental cerebral malaria.

Experimental T-cell-mediated hepatitis induced by concanavalin A (Con A) involves the production of proinflammatory cytokines. Because interleukin (IL)-10 is a potent anti-inflammatory cytokine derived from macrophages and T cells and is produced within the liver, we investigated the role of IL-10 in modulating the hepatotoxicity and the secretion of cytokines following in vivo injection of Con A. IL-10 is produced early in the serum after Con A challenge. Neutralization of endogenous IL-10 by monoclonal antibodies (mAbs) increases the secretion of tumor necrosis factor alpha (TNF-alpha) (+111%), interferon gamma (IFN-gamma) (+92%), and IL-12 (+730%) 8 hours after Con A injection, and increases the hepatotoxicity, assessed by serum alanine transaminase (ALT) (+174%) measurement and by histology, 24 hours after induction of hepatitis. Conversely, preadministration of recombinant IL-10 reduces the production of these proinflammatory cytokines (-47%, -80%, and -47% for TNF-alpha, IL-12, and IFN-gamma, respectively), and decreases neutrophil infiltration and ALT serum concentration (-74%) 8 hours after Con A challenge. We conclude that IL-10, either endogenously produced or exogenously added, has a hepatoprotective role in Con A-induced hepatitis, through its suppressive property on proinflammatory cytokine production, and that it might be of therapeutic relevance in human liver diseases involving activated T cells.

Background & Aims: Interleukin (IL)-10 is a potent anti-inflammatory cytokine. Its role in modulating liver injury induced by galactosamine and lipopolysaccharide (Gal/LPS) was investigated., Methods: The effects of recombinant IL-10 (rIL-10), anti-IL-10 monoclonal antibodies, or gadolinium chloride (GdCl3) pretreatment were studied in mice challenged with Gal/LPS. Tumor necrosis factor (TNF) alpha and IL-10 serum concentrations were measured and liver injury was assessed by alanine aminotransferase (ALT) serum concentrations and by histology., Results: (1) IL-10 is produced early and together with TNF-alpha after Gal/LPS challenge. (2) Anti-IL-10 pretreatment increases TNF-alpha (+443%, P = 0.04), ALT (+160%, P = 0.04) serum levels, and the percentage of severe necrosis compared with control monoclonal antibodies. (3) Administration of rIL-10 30 minutes before Gal/LPS decreases TNF-alpha (-67%, P = 0.02), ALT (-94%, P = 0.01) serum concentrations, and the proportion of severe necrosis. The hepatoprotective effect is still observed when rIL-10 is injected 30 or 120 minutes after Gal/LPS. (4) GdCl3 pretreatment protects against hepatotoxicity, decreases TNF-alpha, but increases IL-10 serum concentrations., Conclusions: These results indicate that IL-10 protects the liver in the Gal/LPS mouse model. Increased IL-10 and decreased TNF-alpha secretion are potentially involved in the hepatoprotection observed after GdCl3 pretreatment.

We evaluated the effects of interleukin (IL)-10 on the differentiation of dendritic cells (DC) obtained by culturing plastic-adherent peripheral blood mononuclear cells for 7 days in presence of granulocyte/macrophage-colony-stimulating factor (GM-CSF) + IL-4. The addition of IL-10 at the initiation of culture resulted in the generation of macrophage-like cells with expressing high levels of CD14 and decreased levels of CD1a and CD1c. Furthermore, cells generated in presence of IL-10 secreted lower levels of IL-12, but higher levels of IL-8 compared with DC generated in absence of IL-10, both spontaneously and after CD40 engagement. Finally, cells generated in presence of IL-10 were less efficient than DC in stimulating the production of IL-2, interferon-gamma, and IL-4 by allogeneic T cells. We conclude that IL-10 prevents the generation of DC induced by GM-CSF + IL-4 and favors the development of macrophages with a lower T cell stimulatory potential, but secreting higher levels of IL-8 than DC.

Objective: To examine the role of endogenous interleukin-4 (IL-4) and interleukin-10 (IL-10) and the therapeutic effect of the addition of IL-4 and IL-10 in early and established murine collagen-induced arthritis (CIA)., Methods: Murine recombinant IL-4, IL-10, or the combination was given intraperitoneally twice daily from the day of arthritis onset up to 7-10 days of CIA in DBA/1 mice. Anti-IL-4, anti-IL-10, or both antibodies were given intraperitoneally before or after the onset of CIA. The effect of cytokine or anticytokine treatment was monitored visually by macroscopic scoring. Histology and reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed at the end of the treatment period., Results: IL-4 alone did not provoke any effect, IL-10 slightly suppressed the arthritis, but a more pronounced amelioration was found with the combination. This cooperative effect was noted after early treatment but also occurred when the start of treatment was delayed until 1 week after onset. Apart from suppression of macroscopic signs of inflammation, combined treatment with IL-4/IL-10 also reduced cellular infiltrates in the synovial tissue and caused pronounced protection against cartilage destruction. Moreover, levels of mRNA for tumor necrosis factor alpha (TNF alpha) and IL-1 were highly suppressed both in the synovial tissue and in the articular cartilage. In contrast, levels of IL-1 receptor antagonist (IL-1Ra) mRNA remained elevated, which suggests that the mechanism of protection may be related to suppressed production of TNF alpha and IL-1, with concomitant up-regulation of the IL-1Ra/IL-1 balance. However, accelerated onset of CIA and increased severity could be achieved with neutralizing anti-IL-10 antibodies. This expression could be further optimized with a combination of anti-IL-4 and anti-IL-10 antibodies, although anti-IL-4 alone was without effect., Conclusion: Our data are consistent with a dominant role of IL-10 in the natural suppression of arthritis expression, whereas combined treatment with IL-4 and IL-10 appears of potential therapeutic value, not only at the onset, but also in established arthritis.

Interactions between membrane-bound molecules were previously shown to be involved in the induction of tissue factor-dependent monocyte procoagulant activity (PCA) by activated T cells. To investigate the potential role of the CD40/CD40 ligand (CD40L) pathway in this process, we first determined the effects of blocking anti-CD40 or anti-CD40L monoclonal antibodies (mAb) on the development of monocyte PCA during mixed lymphocyte reaction (MLR) between allogeneic peripheral blood mononuclear cells (PBMC). The strong inhibitory effect exerted by both mAb (mean percentages of inhibition: 88 and 91% for anti-CD40 and anti-CD40L mAb, respectively) indicates that CD40/CD40L interactions are required for the induction of PCA in MLR. These data led us to measure monocyte PCA after incubation of PBMC or purified monocytes with a stimulating anti-CD40 mAb (BL-C4) or with 3T6 fibroblasts transfected with the gene encoding CD40L. In both systems, we found that CD40 engagement strongly induced monocyte PCA which was related to tissue factor expression as shown by flow cytometric analysis. Finally, we observed that recombinant interleukin (IL)-10, which inhibits lipopolysaccharide-induced PCA, did not significantly influence CD40-dependent PCA. We conclude that CD40 engagement on monocytes induces tissue factor-dependent PCA through an IL-10-resistant pathway. These findings have implications for the control of coagulation events triggered by interactions between T cells and monocytes.

The effects of recombinant interleukin (IL)-10 and the role of endogenous IL-10 were determined in C57B1/6 mice with pneumonia induced by intranasal inoculation with 10(6) cfu of Streptococcus pneumoniae. Pneumonia induced sustained expression of IL-10 mRNA and protein in lungs, but IL-10 remained undetectable in plasma. Intranasal inoculation of S. pneumoniae in combination with IL-10 (1500 U/mouse) resulted in decreased lung concentrations of tumor necrosis factor-alpha (TNF) and interferon (IFN)-gamma, increased bacterial counts in lungs and blood, and early lethality. Conversely, pretreatment (-2 h) of mice with an anti-IL-10 monoclonal antibody (2 mg/mouse) was associated with increased lung levels of TNF and IFN-gamma, reduced bacterial counts in lungs and plasma 40 h after the inoculation, and prolonged survival. These results indicate that during pneumococcal pneumonia, IL-10 attenuates the proinflammatory cytokine response within the lungs, hampers effective clearance of the infection, and shortens survival.

IL-10 is an endogenous antiinflammatory cytokine that inhibits TNF biosynthesis and protects mice from lipopolysaccharide (LPS)-induced lethality. As synthetic glucocorticoids are widely used as antiinflammatory agents, we analysed the effects of methylprednisolone administration on IL-10 biosynthesis during murine endotoxaemia. We found that low doses of methylprednisolone (2-10 mg/kg) markedly inhibited TNF production but did not affect serum levels of IL-10, while a high methylprednisolone dose (50 mg/kg) increased LPS-induced IL-10 levels. In parallel, we observed that LPS-induced IL-10 production is TNF-independent in this experimental setting. Experiments conducted in vitro indicated that methylprednisolone (from 0.01 to 100 micrograms/ml) also increased the biosynthesis of IL-10 by LPS-activated mouse peritoneal macrophages. We conclude that methylprednisolone differentially regulates IL-10 and TNF production induced by LPS both in vivo and in vitro at the macrophage level.

Adenosine is a potent endogenous antiinflammatory agent released by cells under metabolically unfavorable conditions. Its effects on the production of IL-10 by human monocytes were presently investigated. Pre-incubation with adenosine dose-dependently enhanced IL-10 release by TNF stimulated human monocytes (+29, +58, and +116% at 1, 10, and 100 muM, respectively.) Adenosine also significantly enhanced IL-10 production after hydrogen peroxide and LPS stimulation and dose-dependently inhibited TNF secretion. Pre-incubation was not mandatory to achieve these effects, since addition of adenosine at the time of or 30 min after the stimulus led to the same results. Blocking IL-10 with anti-IL-10 mAbs partially restored adenosine-induced TNF inhibition. The enhanced IL-10 production was not observed when cells were preincubated with adenosine A1 or A2 receptor agonists (R-phenylisopropyladenosine, 5'-N-ethylcarboxamido-adenosine, and 2-chloroadenosine) and was not affected by pretreatment with theophyllin, an antagonist of both A1 and A2 receptors, or with dipyridamole, an inhibitor of adenosine cellular uptake. In conclusion, adenosine, in the submillimolar concentration range, increases IL-10 secretion by stimulated monocytes. This phenomenon participates in TNF inhibition, a known property of adenosine, but is not mediated through the occupancy of A1 or A2 receptors. This may represent a novel antiinflammatory property of adenosine by which it could modulate inflammation and limit ischemia-reperfusion injury.

Because interleukin-10 (IL-10) has potent immunosuppressive and anti-inflammatory properties and is produced by some cancers, we hypothesized that its production might play a role in carcinogenesis by inhibiting adequate antitumoral immune responses. To test this hypothesis, retroviral vectors containing the IL-10 cDNA were generated and used to infect B16F1 melanoma cells that were injected subcutaneously in syngeneic mice. Surprisingly, IL-10 gene transfer resulted in a loss of tumorigenicity that was proportional to the amount of IL-10 secreted. Histological analysis showed massive area of necrosis of these tumor cells, with infiltration of polymorphic inflammatory cells. Parental cells simultaneously implanted had decreased tumorigenicity only when mixed with IL10-producing cells, but not when injected contralaterally, suggesting that their eradication is mediated mostly by a local phenomenon. Host T lymphocytes and natural killer (NK) cells were involved in this eradication because IL-10-producing cells grew in nude mice and in CD8+ or NK-depleted mice. Finally, mice injected with IL-10-secreting cells developed an antitumoral systemic immune response able to protect them against a subsequent challenge with parental cells. These results demonstrate that, in some settings, IL10 may have in vivo immunostimulating and proinflammatory properties that need to be considered in its therapeutic development.

IL-10 production during endotoxic shock is part of a protective mechanism that involves IL-10-induced inhibition of TNF synthesis. We sought to determine the role of IL-10 in septic peritonitis induced by cecal ligation and puncture (CLP). CLP led to a rapid induction of IL-10 mRNA in various organs of C57BI/6 mice. In liver, IL-10 mRNA was detectable within 1 h following CLP, while in spleen and lungs, IL-10 mRNA was detected from 2 to 4 h and onward. IL-10 protein became detectable in plasma 2 h after CLP, reaching peak concentrations after 12 h (12.7 +/- 5.7 ng/ml). Pretreatment (-2 h) with anti-IL-10 mAb resulted in higher plasma TNF levels following CLP when compared with mice treated with control mAb. Plasma IL-1 activity and IFN-gamma remained undetectable in virtually all mice. Anti-IL-10 enhanced mortality after CLP (p < 0.05 by log-rank test). Addition of anti-TNF mAb did not influence the increased mortality associated with anti-IL-10 treatment. Septic peritonitis is associated with sustained production of IL-10 in various organs, which serves to protect the host against lethality.

Monocytes of patients with alcoholic cirrhosis produce higher amounts of tumor necrosis factor-alpha (TNF-alpha) after lipopolysaccharide (LPS) stimulation. The mechanisms of the overproduction remain undefined. IL-10 (IL-10) is an antiinflammatory cytokine known to downregulate TNF-alpha secretion by monocytes. The present study analyzes IL-10 production by monocytes and its control on TNF-alpha secretion in alcoholic cirrhosis. LPS-stimulated monocytes from alcoholic cirrhotics (n = 13) showed decreased IL-10 (median, 240 pg/mL [40 to 500] upsilon 513 pg/mL [152 to 1,335]; P = .01) compared with controls (n = 13). Cells from cirrhotic patients were normally responsive to recombinant IL-10, which induced a dose dependent decrease of TNF-alpha secretion. On the other hand, preincubation with anti-IL-10 monoclonal antibodies led to significant increase in TNF-alpha secretion in controls (median, 7,325 to 16,800 pg/mL; P = .002) but not in cells from cirrhotic patients (16,535 to 20,450 pg/mL; P = .14), abolishing the difference in TNF-alpha production between cirrhotic patients and controls. It is concluded that defective IL-10 secretion by monocytes from alcoholic cirrhotic patients could be involved in the characteristics TNF-alpha overproduction observed in this disease.

Interleukin-10 is a potent macrophage-deactivating cytokine that inhibits lipopolysaccharide-induced tumor necrosis factor production. We determined the plasma levels of immunoreactive interleukin-10 in 16 patients with septic shock and in 11 patients with circulatory shock of nonseptic origin. In septic shock, interleukin-10 levels peaked during the first 24 h (median: 48 pg/ml) and decreased progressively till Day 5. In nonseptic shock, interleukin-10 plasma levels also increased during the first 24 h but to a lesser extent (median: 17 pg/ml). In septic shock patients, interleukin-10 plasma levels were positively correlated with tumor necrosis factor (r = 0.8, p = 0.01) and with parameters of shock severity including lactate levels (r = 0.56, p < 0.05) and correlated negatively with blood platelet counts (r = -0.65, p < 0.05). The decreased production of tumor necrosis factor-alpha and interleukin-6 after in vitro incubation of whole blood from septic shock patients with lipopolysaccharide was not influenced by in vitro neutralization of interleukin-10. We conclude that interleukin-10 is produced in patients with circulatory shock of septic and nonseptic origin and that the production of this anti-inflammatory cytokine during septic shock correlates positively with the intensity of the inflammatory response.

Most of the immunosuppressive effects of interleukin-10 (IL-10) are related to functional inhibition of antigen-presenting cells (APC). Herein, we investigate the influence of recombinant (r)IL-10 on human dendritic cells (DC) purified from peripheral blood of healthy volunteers. First, we found that rIL-10 inhibited in a dose-dependent manner the proliferative responses as well as the production of IL-2 and interferon-gamma (IFN-gamma) in mixed lymphocyte reaction (MLR) between purified T cells and DC. This rIL-10 effect could be attributed to a direct effect on DC, as DC preincubated with rIL-10 were found to be deficient in the induction of alloreactive T cells even when anti-IL-10 neutralizing mAb was added at the time of MLR. Flow cytometric analysis indicated that rIL-10 did not modify the expression of ICAM-1 (CD54) and B7-1 (CD80), but decreased HLA-DR and B7-2 (CD86) expression at the DC surface. We conclude that the inhibitory effect of rIL-10 on primary alloreactive T cell responses involves down-regulation of class II MHC and B7-2 expression at the DC surface.

Interleukin (IL)-10 is a novel cytokine produced by a variety of cells, including monocytes/macrophages, upon exposure to lipopolysaccharide (LPS). Recent observations indicate that, in turn, IL-10 exerts suppressive effects on macrophage response to LPS. Because mesangial cells are also a target for LPS, we have examined the potential role of IL-10 in the regulation of mesangial cell response to LPS. To this aim, we have studied the synthesis and the autocrine/paracrine function of IL-10 in cultured mouse mesangial cells. IL-10 mRNA expression and IL-10 protein secretion were determined by a reverse transcription polymerase chain reaction technique and a specific enzyme-linked immunosorbent assay, respectively. No IL-10 mRNA expression was detectable in unactivated cells. LPS induced IL-10 mRNA expression in a dose-dependent fashion (1 to 100 micrograms/ml). In addition, LPS induced IL-10 protein release that was both dose dependent (1 to 100 micrograms/ml) and time dependent (24 to 72 hours). We have also studied the effect of IL-10 on the production of inflammatory mediators by LPS-activated mouse mesangial cells. Whereas recombinant IL-10 inhibited the generation of tumor necrosis factor-alpha (TNF-alpha) and IL-1 beta by 90 and 60%, respectively, it did not affect the formation of nitric oxide-derived nitrite (NO2-) and nitrate (NO3-). As shown by the use of anti-IL-10 monoclonal antibody, endogenously produced IL-10 affected the generation of TNF-alpha but neither that of IL-1 beta nor that of NO2- and NO3-. Finally, we have examined whether conditions known to also reduce the generation of TNF-alpha modified the expression of IL-10. Of all the conditions tested, only the addition of desferrioxamine and transforming growth factor-beta were found to increase IL-10 release. Together, these data demonstrate that mesangial cell-derived IL-10 has important regulatory effects on the inflammatory response of these cells to LPS because of its capacity to blunt TNF-alpha generation.

Background/aims: Inflammatory events are believed to play an important role in the pathogenesis of acute pancreatitis. Interleukin 10 (IL-10) recently emerged as a major anti-inflammatory cytokine, inhibiting the secretion of proinflammatory cytokines by monocytes and/or macrophages. The potential protective role of IL-10 in a model of acute necrotizing pancreatitis in mice was tested., Methods: Animals received two intraperitoneal injections of either 1000 U recombinant IL-10 or control supernatant before and during induction of acute pancreatitis with repeated cerulein injections (seven intraperitoneal injections of 50 micrograms/kg at hourly intervals)., Results: Systemic amylase and lipase release peaked 9 hours after the first cerulein injection. This peak was significantly reduced by IL-10 treatment. Histologically, edema and inflammation of the pancreas were observed in both groups, whereas necrosis was dramatically reduced in IL-10-treated animals. Serum tumor necrosis factor levels were undetectable in this model; reverse-transcriptase polymerase chain reaction analysis of resected pancreatic tissues performed at the time of maximal morphological alterations showed a dramatically decreased expression of tumor necrosis factor alpha messenger RNA after IL-10 treatment compared with control pancreatitis., Conclusions: IL-10 is able to decrease the severity of experimental acute pancreatitis, mainly by inhibiting the development of acinar necrosis. Inhibition of local tumor necrosis factor alpha might explain, at least in part, the protective effect of IL-10.

The superantigen staphylococcal enterotoxin B (SEB) induces a defect in interleukin (IL)-2 production by T cells expressing specific T cell receptor V beta domains. The present study was undertaken to determine the capacity of T cells, made deficient in IL-2 production by exposure to SEB in vivo, to secrete interferon (IFN)-gamma and IL-10 and to induce pathology upon SEB rechallenge. For this purpose, BALB/c mice received two intraperitoneal injections of 100 micrograms SEB with a 48-h interval. First, we compared peak serum levels of IL-2, IFN-gamma and IL-10 after SEB rechallenge with those measured after a single SEB injection in control mice. The expected defect in IL-2 production in SEB-pretreated mice was associated with a major increase in IL-10 and IFN-gamma levels which were about fivefold higher than in controls. Experiments in mice depleted of CD4+ or CD8+ cells as well as studies in which purified T cell populations were rechallenged with SEB in vitro indicated that both CD4+ and CD8+ cells from SEB-pretreated mice were primed for IL-10 and IFN-gamma production. Furthermore, SEB-pretreated mice were sensitized to the toxic effects of the superantigen as indicated by a 30-70% lethality rate (vs. 0% in naive mice) within 48 h after SEB rechallenge. IFN-gamma was involved in the lethal syndrome as it could be prevented by injection of neutralizing anti-IFN-gamma monoclonal antibody. We conclude that SEB-reactive T cells made deficient for the production of IL-2 by exposure to SEB in vivo are primed for IFN-gamma and IL-10 production, and that IFN-gamma up-regulation is involved in the shock syndrome occurring upon SEB rechallenge.

Interleukin (IL) 10 inhibits endotoxin (lipopolysaccharide; LPS) induced tumor necrosis factor (TNF) production in vivo and in vitro. In turn, IL-10 is induced by LPS and acts as a negative feedback to limit TNF production. We investigated the effects of IL-10 on brain TNF and IL-1 beta production induced by a central LPS administration in mice. Because central LPS also induces peripheral TNF, we also measured the serum TNF levels. A single intracerebroventricular injection of murine recombinant IL-10 (75 ng/mouse) simultaneously with LPS (2.5 micrograms/mouse) almost completely inhibited brain TNF production. The brain IL-1 beta production was also inhibited, as was the serum concentration of the acute-phase protein serum amyloid A. On the other hand, intracerebroventricular administration of an anti-IL-10 monoclonal antibody (JES5-2A5; 60 micrograms/mouse) potentiated brain TNF and IL-1 beta production. Identical results were obtained when the serum TNF levels were measured. IL-10 did not affect the LPS-induced increase of serum corticosterone, the main endogenous inhibitor of TNF production, or the induction of IL-6. These results indicate that LPS-induced IL-10 can act as an important endogenous inhibitor of brain TNF production and suggest an anti-inflammatory role for IL-10 in the central nervous system.

Interleukin-10 (IL-10) is known to be spontaneously produced by human peripheral blood mononuclear cells. In order to define the cell type in which IL-10 gene is spontaneously expressed we used the reverse polymerase chain reaction for IL-10 mRNA expression, which was also used to study the effects of cycloheximide (CHX). First, we found that IL-10 mRNA is spontaneously expressed in monocytes and B cells but not T cells from healthy donors, and second, we demonstrated that CHX superinduces IL-10 mRNA in monocytes and B cells. Experiments including nuclear run-on analyses established that the effects of CHX on IL-10 gene expression involve both gene transcription and mRNA stabilization.

Interleukin (IL)-10 has an important antiinflammatory effect by inhibiting endotoxin-induced production of proinflammatory cytokines, such as tumor necrosis factor-alpha and IL-1. Since both cytokines are produced in massive amounts during fulminant meningococcal septic shock and are associated with severity of disease, IL-10 was measured in plasma samples of 25 consecutive children with fulminant meningococcal septic shock shortly after admittance to a pediatric intensive care unit. High levels of IL-10 (median, 6021 pg/mL; range, 137-24,600) were found in surviving patients (median, 1268 pg/mL; range, 137-24,600) and in those who died (median, 9915 pg/mL; range, 3996-14,100). IL-10 levels correlated weakly (r = .38; P = .055) with severity of disease as measured by the Glasgow meningococcal septicemia prognostic score. The findings indicate that IL-10 is produced in massive amounts in the initial phase of fulminant meningococcal septic shock.