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Mesenchymal stromal cells (MSC) are involved in tissue repair and in the regulation of immune responses. MSC express Toll-like receptors (TLR) known to link innate and adaptive immunity. We hypothesized that TLR signaling could influence human MSC (hMSC) function. Here, we show that hMSC express TLR1, TLR2, TLR3, TLR4, TLR5, and TLR6 but not TLR7, TLR8, TLR9, and TLR10. In inflammatory conditions mimicked by culturing hMSC in an inflammatory environment, TLR2, TLR3, and TLR4 are upregulated, whereas TLR6 is downregulated. Interleukin (IL)-1 beta, IL-6, IL-12p35 and transforming growth factor-beta mRNAs are constitutively expressed by hMSC. Inflammation leads to an increase in IL-1 beta, IL-6, IL-12p35, and transforming growth factor-beta transcription and is characterized by IL-23p19 and IL-27p28 transcription. In this setting, poly(I:C) further augments IL-6, IL-12p35, IL-23p19, and IL-27p28 transcription, whereas lipopolysaccharide (LPS) increases IL-23p19 and IL-27p28 transcription. By upregulating TLR3 and TLR4 transcription, inflammation increases the hMSC responsiveness to LPS and poly(I:C), leading to a proinflammatory shift in their cytokine profile. The hMSC osteogenic potential does not change after TLR triggering but stimulation with LPS and poly(I:C) results in a decrease in their immunosuppressive capabilities. In conclusion, TLR activation in hMSC may affect their function and could modify their in vivo fate, especially in an inflammatory context., ((c) 2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Conventional PKC (cPKC)-alpha regulates TRIF-dependent IFN response factor 3 (IRF3)-mediated gene transcription, but its role in MyD88-dependent TLR signaling remains unknown. Herein, we demonstrate that PKC-alpha is induced by several MyD88-dependent TLR/IL-1R ligands and regulates cytokine expression in human and murine DC. First, inhibition of cPKC activity in human DC by cPKC-specific inhibitors, Gö6976 or HBDDe, downregulated the production of classical inflammatory/immunomodulatory cytokines induced by TLR2, TLR5 or IL-1R but not by TLR3 stimulation. Similarly, dominant negative PKC-alpha repressed Pam(3)CSK(4) induced NF-kappaB- and AP-1-driven promoter activities in TLR2-expressing human embryonic kidney 293 T cells. Dominant negative PKC-alpha inhibited NF-kappaB reporter activity mediated by overexpression of MyD88 but not TRIF. Unexpectedly, BM-derived DC from PKC-alpha(-/-) mice exhibited decreased TNF-alpha and IL-12p40 production induced by both MyD88- and TRIF-dependent ligands. Furthermore, PKC-alpha is coupled to TLR2 signaling proximal to MyD88 since MAPK and IkappaB kinase-alpha/beta phosphorylations and IkappaBalpha degradation were inhibited in PKC-alpha(-/-) BM-derived DC. Finally, co-immunoprecipitation assays revealed that PKC-alpha physically interacts with Pam(3)CSK(4) activated TLR2 in WT but not in MyD88(-/-) DC. Collectively this study identifies a species-specific role of PKC-alpha as a key component that controls MyD88-dependent cytokine gene expression in human and mouse but differentially regulates production of TRIF-dependent cytokines.

CCR7(+ )central memory (T(CM)) CD4(+) T cells play a central role in long-term immunological memory. Recent reports indicate that a proportion of CD4(+) T(CM) is able to produce effector cytokines. The phenotype and the role of this subset remain unknown. We characterized cytokine-producing human CD4(+) T(CM) specific for cleared protein and persistent viral Ag. Our results demonstrate that the type of Ag stimulation is a major determinant of CD4(+) T(CM) differentiation. CMV-specific T(CM) were significantly more differentiated than protein Ag-specific T(CM) and included higher proportions of IFN-gamma-producing cells. The expression of killer cell lectin-like receptor G1 (KLRG1) by protein Ag- and CMV-specific T(CM) was associated with increased production of effector cytokines. KLRG1(+) T(CM) expressed high levels of CD127, suggesting that they can survive long term under the influence of IL-7. The induction of KLRG1(+) T(CM) may therefore represent an important target of vaccination against pathogens controlled by cellular immune responses.

A large body of evidence establishing the existence of an underlying T-cell disorder in a subset of patients fulfilling hypereosinophilic syndrome (HES) diagnostic criteria has accumulated over the past decade, resulting in the definition of a novel HES variant termed "lymphocytic" HES. Although end-organ complications of hypereosinophilia are generally benign, with predominant cutaneous manifestations, long-term prognosis is overshadowed by an increased risk of developing T-cell lymphoma, as a result of malignant transformation of aberrant T cells years after HES diagnosis. Therapeutic strategies should target pathogenic T cells in addition to eosinophils, but the practical implications remain largely unexplored.

The function of Ag-specific central (T(CM)) and effector (T(EM)) memory CD4+ T lymphocytes remains poorly characterized in vivo in humans. Using CD154 as a marker of Ag-specific CD4+T cells, we studied the differentiation of memory subsets following anti-hepatitis B immunization. Hepatitis B surface Ag (HBs)-specific memory CD4+T cells were heterogeneous and included T(CM) (CCR7+CD27+) and T(EM) (CCR7(-)CD27(+/-)). HBs-specific T(CM) and T(EM) shared the capacity to produce multiple cytokines, including IL-2 and IFN-gamma. Several years postimmunization, approximately 10% of HBs-specific memory CD4+ T cells were in cycle (Ki67+) and the proliferating cells were CCR7+. These results suggest that the model of functional specialization of T(CM) and T(EM) cannot be applied to protein vaccine Ags and support the concept that T(CM) are capable of self-renewal and contribute to maintain the pool of memory cells.

Rapid, simple, and reliable assays to monitor allogeneic responses are essential for the safe development of novel protocols of tailored immunosuppression. Herein, we describe a real-time polymerase chain reaction method based on interleukin-2 and interferon-gamma mRNA quantification upon stimulation of whole blood with allogeneic T cell-depleted peripheral blood mononuclear cells. The technique requires only small blood volumes and results can be obtained within 48 hours. Data obtained in a liver transplant patient receiving a tolerance induction protocol based on the infusion of donor-type hematopoietic stem cells suggest that this rapid whole blood mixed lymphocyte reaction assay could be valuable for the monitoring of patients undergoing solid organ or hematopoietic stem cell transplantation.

Aims/hypothesis: Human pancreatic duct cells are closely associated with islet beta cells, and contaminate islet suspensions transplanted in Type 1 diabetes mellitus patients. Activated duct cells produce cytotoxic mediators and possibly contribute to the pathogenesis of Type 1 diabetes mellitus or islet graft rejection. As CD40 transduces activation signals involved in inflammatory and immune disorders, we investigated CD40 expression on duct cells and their response to CD40 engagement., Methods: CD40 expression on human pancreatic duct cells was analysed by flow cytometry and immunohistochemical analyses. To assess the function of CD40 expression on duct cells, activation of the transcription factor nuclear factor-kappa B was determined using electrophoretic mobility shift assay and ELISA. Cytokine mRNA levels were quantified by real-time RT-PCR, and protein levels by Luminex technology., Results: Isolated human pancreatic duct cells and Capan-2 cell lines were found to express constitutively CD40. The expression of CD40 on duct cells was confirmed in vivo on human normal and pathological pancreatic specimens. CD40 ligation on Capan-2 cells induced rapid nuclear factor-kappa B activation, and supershift assays demonstrated that p50/p65 heterodimers and p50/p50 homodimers were present in the activated complexes in the nucleus. This activation was accompanied by tumour necrosis factor-a and interleukin-1beta mRNA accumulation. Tumour necrosis factor-alpha protein secretion was confirmed in CD40-activated Capan-2 cells and in isolated human pancreatic duct cells., Conclusions/interpretation: Interaction between activated T lymphocytes expressing CD40 ligand and duct cells expressing CD40 may contribute to the immune responses involved in Type 1 diabetes mellitus and islet graft rejection. Interfering with CD40-mediated duct cell activation could alleviate beta cell damage of immune origin.

There is a need for simple and sensitive assays to assess innate and adaptive immune responses to microbial agents and vaccines. Herein, we describe a whole blood method allowing to measure the induction of cytokine synthesis at the mRNA level. The originality of this method consists in the combination of PAXgene tubes containing an mRNA stabilizer for blood collection, the MagNA Pure instrument as an automated system for mRNA extraction and RT-PCR reagent mix preparation, and the real-time PCR methodology on the Lightcycler for accurate and reproducible quantification of transcript levels. We first demonstrated that this method is adequate to measure the induction of interleukin (IL)-1beta and IL-1 receptor antagonist (IL-1 RA) mRNA upon the addition of bacterial lipopolysaccharide (LPS) to whole blood. We then showed that this approach is also suitable to detect the production of mRNA encoding T cell-derived cytokines in whole blood incubated with tetanus toxoid as a model of in vitro immune response to a recall antigen. Finally, we demonstrated that this methodology can be used successfully to assess inflammatory as well as T cell responses in vivo, as it allowed to detect the induction of IL-1beta and IL-1 RA after injection of LPS in healthy volunteers, and also the induction of IL-2 upon recall immunisation with tetanus vaccine.

Bordetella pertussis toxin (PTX), a key component of acellular pertussis vaccines, is known to be endowed with adjuvant properties. In experiments designed to get insights into the interactions between PTX and circulating immune cells, we first observed that addition of PTX to adult whole blood induced the release of IL-12 and TNF-alpha as well as maturation of myeloid dendritic cells (DC). These effects were still present with a toxin mutant devoid of ADP-ribosyltransferase activity but not with a formaldehyde-inactivated toxin. These findings indicate that cytokine production and DC maturation require the intact structure of PTX but not its enzymatic activity. Secondly, studies on DC generated in vitro by culturing monocytes with IL-4 and GM-CSF showed that PTX directly stimulates MHC class II and costimulatory molecules up-regulation, cytokine synthesis and NF-kappa B activation. Finally, comparison of data obtained in adult vs. cord blood revealed deficient responses of neonatal DC to PTX. These data suggest new applications of PTX and PTX mutants as vaccine adjuvants.

The quantification of the cytokine mRNAs synthesised by peripheral blood cells should make it possible to estimate a "peripheral immune statute". However, an accurate quantification can only be performed from a fresh whole blood sample in which mRNA is protected against nuclease digestion, and where gene transcription is inhibited. As discussed in this note, this has been made possible by the use of surfactant reagents such as tetradecyltrimethylammonium oxalate. We performed RT-PCR for the quantification of IL-10 and IFN-gamma mRNAs spontaneously produced in peripheral blood. The results showed pronounced higher IFN-gamma transcript levels in whole blood compared to peripheral blood mononuclear cells (PBMC) from the same individuals, while no significant difference was observed for IL-10 mRNA. The higher amounts of IFN-gamma mRNA observed in blood can be attributed at least to mRNA degradation. Using a real time PCR technique, we indeed demonstrated that blood IFN-gamma mRNA is rapidly degraded in vitro, the t 1/2 being worth approximately one hour at room temperature.

Real-time PCR represents a new methodology that accurately quantifies nucleic acids. This has been made possible by the use of fluorogenic probes, which are presented in two forms, namely hydrolysis probes (also called TaqMan probes) and hybridisation probes. We decided to apply this methodology to cytokine mRNA quantification and this led us to the development of a protocol that provides an easy way to develop and perform rapidly real-time PCR on a Lightcycler instrument. It was made possible by the use of freely available software that permits a choice of both the hydrolysis probe and the primers. We firstly demonstrated that the reproducibility of the method using hydrolysis probes compares favourably with that obtained with hybridisation probes. We then applied this technique to determine the kinetics of IL-1ra, IL-1beta, IL-5, IL-13, TNF-alpha and IFN-gamma induction upon stimulation of human peripheral blood mononuclear cells (PBMC) by phytohaemagglutinin (PHA). Finally, the method was also used successfully to demonstrate that IFN-alpha induces IL-10 mRNA accumulation in human monocytes.

We recently observed a clonal expansion of CD3(-)CD4(+) T cells secreting Th2-type cytokines in patients presenting chronic hypereosinophilia. As clonal T cells isolated from such patients did not spontaneously secrete cytokines in vitro, we reasoned that costimulatory signals delivered by antigen-presenting cells might be required to induce their full activation. To address this question, we investigated in two such patients the responses of CD3(-)CD4(+) T cells to dendritic cells (DC). DC elicited proliferation and production of interleukin-5 (IL-5) and IL-13 by clonal cells from patient 1 and upregulated their expression of CD25 (IL-2R-alpha). These effects were abolished when blocking monoclonal antibodies (MoAbs) against IL-2R-alpha and IL-2 were added to cocultures, indicating critical involvement of an autocrine IL-2/IL-2R pathway. Cells from patient 2 were stimulated by DC to produce Th2 cytokines only when rIL-2 or rIL-15 was added to cocultures. In both patients, addition of inhibitory MoAbs against B7-1/B7-2 or CD2 to cocultures resulted in dramatic reduction of cytokine production and inhibited CD25 upregulation. Thus, TCR/CD3-independent activation of clonal Th2 cells by DC is an IL-2-dependent process, which requires signaling through CD2 and CD28.

Trypanosoma cruzi, a parasitic protozoan, is the etiological agent of Chagas' disease. Despite the many immune system disorders recognized in this infection and the crucial role played by dendritic cells (DC) in acquired immune responses, it was not known whether these cells could be infected by T. cruzi trypomastigotes and the consequences of such an infection on their immune functions. We now provide evidence that human monocyte-derived DC can be infected by T. cruzi and can support its intracellular multiplication. Interestingly, this infection has functional consequences on immature DC and on their maturation induced by lipopolysaccharide (LPS). First, after T. cruzi infection, the basal synthesis of interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-alpha) was impaired. Furthermore, the process of maturation of DC induced by LPS was drastically affected by T. cruzi infection. Indeed, secretion of cytokines such as IL-12, TNF-alpha, and IL-6, which are released normally at high levels by LPS-activated DC, as well as the up-regulation of HLA-DR and CD40 molecules, was significantly reduced after this infection. The same effects could be induced by T. cruzi-conditioned medium, indicating that at least these inhibitory effects were mediated by soluble factors released by T. cruzi. Taken together, these results provide new insights into a novel efficient mechanism, directly involving the alteration of DC function, which might be used by T. cruzi to escape the host immune responses in Chagas' disease and thus might favor persistent infection.

Described is the identification of a novel series of compounds that blocks the activation of two key transcription factors, AP-1 and NF-kappa B. These transcription factors regulate the expression of several critical proinflammatory proteins and cytokines and represent attractive targets for drug discovery. Through the use of high throughput screening and solution-phase parallel synthesis, inhibitors of both NF-kappa B and AP-1 were identified. In subsequent testing, these compounds were also shown to block both IL-2 and IL-8 levels in the same cells. One of the most potent compounds in this series, 28, was active in several animal models of inflammation and immunosuppression, thus validating the importance of AP-1 and NF-kappa B as potential therapeutic targets. The synthesis and preliminary structure-activity relationships of these compounds is addressed.

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.

To investigate the responses of dendritic cells (DC) during Gram-negative infections, we analyzed the effects of graded doses of LPS on the cytokine profile, phenotype, and allostimulatory potential of human DC generated by culturing plastic-adherent PBMC in presence of IL-4 and granulocyte-macrophage-CSF. First, we found that LPS stimulates the production of high levels of TNF-alpha, IL-6, IL-8, IL-12 by DC and up-regulates their expression of HLA-DR, B7-1, B7-2, and CD40. The effects of LPS were dose dependent, with a significant stimulatory effect already observed at a concentration of 0.1 ng/ml and a plateau being reached at 10 ng/ml. These phenotypic changes correlated with increased allostimulatory properties of LPS-activated DC because DC treated with LPS were significantly more efficient than untreated DC in eliciting IL-2 and IFN-gamma synthesis by alloreactive T cells and stimulating their proliferation. Experiments using neutralizing anti-IL-12 mAb indicated that LPS-induced IL-12 is responsible for the increased production of IFN-gamma but not for the increased proliferation during MLR. Finally, we observed that the DC responses to low levels of LPS (1 ng/ml) were dramatically inhibited by a blocking anti-CD14 mAb, although DC do not express CD14 molecules on their membrane. Experiments using serum depleted of soluble CD14 (sCD14) and sCD14 either purified from human serum or in recombinant form further established that DC respond to LPS via a soluble CD14-dependent pathway.

In order to study the immunopathological consequences of repeated exposure to bacterial superantigens, we evaluated the production of cytokines, the profile of serum immunoglobulins and the tissue damage in BALB/c mice injected twice a week for 3 weeks with 50 micrograms of staphylococcal enterotoxin B (SEB). First, we found that neither interleukin 2 (IL-2) nor interferon gamma (IFN-gamma) were detectable in serum after the 6th injection of SEB whereas both cytokines were released in the circulation after the first injection. In contrast, interleukin 4 (IL-4) and interleukin 10 (IL-10) serum levels were similar after the first and the sixth injection of SEB. Likewise, spleen cells from mice injected for 3 weeks with SEB produced much lower levels of IL-2 and IFN-gamma than spleen cells from control mice in response to SEB in vitro whereas their production of IL-10 was not significantly altered. Both IL-4 and IL-10 were found to be secreted by purified T cells from SEB-treated mice when rechallenged in vitro with SEB in presence of human accessory cells. This TH2-type cytokine profile was associated with a marked hyperimmunoglobulinemia and the appearance of anti-mouse immunoglobulin autoantibodies. By light microscopy, no tissue lesions were observed in mice chronically injected with SEB but mesangial deposits of IgG were found in their kidneys by immunofluorescence. We conclude that T cell anergy induced by repeated injections of SEB in BALB/c mice is associated with a persistent production of TH2-type cytokines and a polyclonal B cell activation.

Background: The release of cytokines after cardiopulmonary bypass may play an important role in postoperative morbidity. The release of proinflammatory cytokines, such as tumor necrosis factor alpha, interleukin (IL)-6 and IL-8, is even greater in patients undergoing heart transplantation (HTx) than coronary artery grafting. We tested the hypothesis that in HTx patients the earlier administration of steroids, before rather than after cardiopulmonary bypass as usual, can reduce the inflammatory response., Methods: In 20 consecutive patients who underwent HTx or heart-lung transplantation (HLTx), plasma levels of tumor necrosis factor alpha, IL-6, IL-8, and anti-inflammatory cytokine IL-10 were measured before heparin administration, at aortic cross-clamping and declamping, and 0.5, 1, 1.5, 2, 4, 12, and 24 hours after aortic declamping. In 10 patients (group I, 6 HTx and 4 HLTx), 500 mg of methylprednisolone was first given as usual at 1.5 hours after aortic declamping (at the end of cardiopulmonary bypass). In the next 10 patients (group II, 6 HTx and 4 HLTx), the first doses of methylprednisolone were given 1 hour before operation. In both groups, 125 mg of methylprednisolone were given every 8 hours thereafter during the first postoperative day., Results: The ischemic time and cardiopulmonary bypass time were similar in the two groups (166 +/- 16 minutes versus 157 +/- 13 minutes, and 192 +/- 21 minutes versus 186 +/- 20 minutes, respectively, mean +/- standard error of the mean). At 30 minutes after aortic declamping and throughout the next 4 hours, tumor necrosis factor alpha levels were significantly lower in group II than in group I (all p < 0.03). Interleukin-8 values 1 hour after declamping were also lower in group II than in group I (49 +/- 15 pg/mL versus 130 +/- 38 pg/mL, p < 0.02). Interleukin-10 levels were significantly higher in group II than in group I from 30 minutes after declamping through 2 hours after (all p < 0.03). Interleukin-6 levels were similar in the two groups., Conclusions: Earlier steroid administration in the immunosuppressive protocol for HTx or HLTx may be preferable to reduce the inflammatory response to cardiopulmonary bypass, as reflected by a lower production of tumor necrosis factor alpha and IL-8, and a greater release of IL-10.

Cardiac surgery with cardiopulmonary bypass triggers an inflammatory response involving proinflammatory cytokines such as tumor necrosis factor-alpha, interleukin-6, and interleukin-8. To elucidate the pathophysiology of this cytokine response, we explored the possible differences in cytokine responses between patients undergoing heart transplantation and those undergoing coronary artery bypass grafting. Plasma levels of tumor necrosis factor-alpha, interleukin-6, interleukin-8, and interleukin-10 were measured in eight patients undergoing heart transplantation (mean age 44 years) and eight patients undergoing coronary artery bypass grafting (mean age 61 years). Duration of cardiopulmonary bypass and ischemic time were both longer in the heart transplantation group than in the coronary artery bypass grafting group (133 +/- 26 min vs 100 +/- 31 min, p < 0.05, and 130 +/- 47 min vs 58 +/- 21 min, p < 0.005, respectively). Samples were collected before heparin administration, at aortic crossclamping and declamping, and at 0.5, 1, 1.5, 2, 4, 12, and 24 hours after declamping. Tumor necrosis factor-alpha levels were significantly higher 30 minutes after aortic declamping in the heart transplantation group than in the coronary artery bypass grafting group (68 +/- 30 vs 18 +/- 5 pg/ml, p < 0.05). Interleukin-6 and interleukin-8 levels were also significantly higher 90 minutes after declamping in patients undergoing heart transplantation than in those undergoing coronary artery bypass grafting (310 +/- 63 vs 169 +/- 24 pg/ml, p < 0.05, and 73 +/- 17 vs 24 +/- 5 pg/ml, p < 0.01, respectively). Furthermore, interleukin-6 and interleukin-8 values 90 minutes after declamping were significantly correlated with the ischemic time (r = 0.72 and r = 0.82, respectively, both p < 0.05). Interleukin-10 levels in both groups rose to reach a peak value of around 115 pg/ml 1 hour after declamping. Patients undergoing heart transplantation exhibited a second peak of tumor necrosis factor-alpha, interleukin-8, and interleukin-10 levels 12 hours after declamping, probably related to the administration of rabbit antihuman thymocyte immunoglobulin (Thymoglobuline) 3 hours after declamping. Interleukin-6 levels decreased more significantly 12 and 24 hours after declamping in patients undergoing heart transplantation, probably related to methylprednisolone therapy. In conclusion, cardiopulmonary bypass is associated with the production of both proinflammatory and antiinflammatory cytokines. The production of proinflammatory cytokines in patients undergoing heart transplantation is higher than that in patients undergoing coronary artery bypass grafting, and this increase could be related to the longer duration of ischemia in the former group. The later course of cytokine levels after heart transplantation may be further influenced by immunosuppressive therapy.

In addition to cytotoxic and proinflammatory mediators, eosinophils can produce a variety of cytokines and growth factors. Besides interleukin (IL)-5, we show in the present work that human eosinophils can synthesize interferon (IFN)-gamma and IL-10, by RT-PCR, in situ hybridization and immunostaining. Double-labelling procedures revealed the coexpression of IL-5 and IL-10 but not IL-5 and IFN-gamma, indicating the existence of subpopulations of eosinophils expressing type 1 or type 2 cytokines. IFN-alpha efficiently used for the treatment of hypereosinophilic syndromes can significantly decrease eosinophil degranulation and IL-5 release by eosinophils, through binding to a receptor for IFN-alpha. Thus, eosinophils can represent major sources of cytokines with regulatory functions, especially in allergic diseases and parasitic infections.

Interest for cytokines involvement in experimental and human liver injury has considerably increased over recent years. The present paper reviews the current knowledge in the field of alcoholic cirrhosis. The role of interleukin-1, tumor necrosis factor, interleukin-6, interleukin-8 and interleukin-10 are discussed together with their possible diagnostic and prognostic significance. Prospects for future investigations such as immunotherapy are underlined.

Interleukin-10 (IL-10) is a potent inhibitor of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) production and has been shown to protect mice from endotoxin shock. As IFN-gamma is another important mediator of LPS toxicity, we studied the effects of IL-10 on LPS-induced IFN-gamma synthesis in vitro and in vivo. First, we found that the addition of recombinant human IL-10 (rhIL-10) (10 U/ml) to human whole blood markedly suppressed LPS-induced IFN-gamma release while neutralization of endogenously synthesized IL-10 resulted in increased IFN-gamma levels. The ability of rIL-10 to inhibit LPS-induced IFN-gamma synthesis was also observed in vivo in mice. Indeed, administration of 1000 U recombinant mouse IL-10 (rmIL-10) 30 min before and 3 h after challenge of BALB/c mice with 100 micrograms LPS resulted in a threefold decrease in peak IFN-gamma serum levels. We then examined the production and the role of IL-10 during murine endotoxemia. We found that LPS injection causes the rapid release of IL-10, peak IL-10 serum levels being observed 90 min after LPS challenge. Neutralization of endogenously produced IL-10 by administration of 2 mg JES5-2A5 anti-IL-10 monoclonal antibody (mAb) 2 h before LPS challenge resulted in a marked increase in both TNF and IFN-gamma serum levels while irrelevant isotype-matched mAb had no effect. The enhanced production of inflammatory cytokines in anti-IL-10 mAb-treated mice was associated with a 60% lethality after injection of 500 micrograms LPS, while all mice pretreated with control mAb survived. We conclude that the rapid release of IL-10 during endotoxemia is a natural antiinflammatory response controlling cytokine production and LPS toxicity.

Three susceptible mouse strains, i.e., BALB/c (H-2d), C57BL/6 (H-2b), and major histocompatibility complex-congenic BALB.B10 (H-2b), were infected intravenously with 4 x 10(6) CFU of live Mycobacterium bovis BCG and analyzed 4 weeks later for in vitro spleen cell cytokine secretion in response to purified protein derivative (PPD), BCG culture filtrate (CF), BCG cellular extract, total BCG, the purified extracellular 30-32-kDa antigen (the fibronectin-binding antigen 85), or the intracellular 65-kDa heat shock protein. C57BL/6 and BALB.B10 mice produced 5- to 10-fold more gamma interferon and interleukin-2 (IL-2) when stimulated with CF, PPD, and antigen 85 than BALB/c mice did. When stimulated with BCG extract and whole BCG, gamma interferon and IL-2 levels were generally lower and comparable in the three strains. IL-4 was detected in spleen cell culture supernatants from infected BALB/c mice but not from C57BL/6 or BALB.B10 mice. IL-5 could not be detected. C57BL/6 and BALB.B10 spleen cells also produced more tumor necrosis factor alpha and IL-6 after stimulation with PPD and CF than BALB/c cells did. Finally, BCG vaccination generated efficient protective immunity in C57BL/6 and BALB.B10 mice but not in BALB/c mice. These data suggest that secreted mycobacterial CF antigens selectively induce a strong TH1 response in BCG-infected C57BL/6 and BALB.B10 mice, whereas in BALB/c mice this response is partly counterbalanced by TH2 cells.

Cytokines produced by antigen-presenting cells govern the fate of helper T-cell responses. Herein, we review the impact of interleukin (IL)-23 and IL-27 on the outcome of T-helper (Th) 17 cell responses and discuss their impact in the pathogenesis of T-cell-mediated inflammatory disorders of autoimmune or allergic origin. We then discuss how type I interferons might influence the course of autoimmune diseases by tipping the balance between IL-12 family members. [ABSTRACT FROM AUTHOR]

Characterization of the Toll-like receptor (TLR) family and associated signalling pathways provides a key molecular basis for our understanding of the relationship between exposure to microbial products and susceptibility to immune-mediated disorders. Indeed, ligation of TLR controls innate and adaptive immune responses by inducing synthesis of pro- as well as anti-inflammatory cytokines and activation of effector as well as regulatory lymphocytes. TLRs are therefore considered as major targets for the development of vaccine adjuvants, but also of new immunotherapies. Herein, we review the potential of TLR ligands as a novel class of pharmaceuticals for the prevention or treatment of allergic disorders. [ABSTRACT FROM AUTHOR]

We report an adolescent girl with a history of angiolymphoid hyperplasia with eosinophilia (ALHE) diagnosed at the age of 10 years. The patient also suffered from chronic persistent multiresistant herpes simplex virus infection. Atherosclerotic occlusive disease of the abdominal aorta and its major branches was observed at the age of 17 years, necessitating vascular surgical intervention 1 year later because of disease progression. Histological examination of the aorta disclosed widespread atherosclerosis and high levels of gene expression of both T-helper cell type (Th) 1- and Th2-derived cytokines. This suggests that a highly stimulated systemic immune response including increased production of both Th1- and Th2-derived cytokines such as interferon-γ and interleukin-4 may result in severe atherosclerotic lesions at a very young age. In addition, the patient developed a peripheral T-cell lymphoma at the age of 18 years. Neither systemic atherosclerosis nor T-cell lymphoma has been reported in association with ALHE. It is suggested that a highly stimulated dysfunctional immune response may play a key role in persistent inflammatory disease and premature development of atherosclerosis as well as malignant transformation of T cells. [ABSTRACT FROM AUTHOR]

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 μg/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. [ABSTRACT FROM AUTHOR]

The injection of the 145-2C11 anti-CD3 MoAb in mice induces a polyclonal T cell activation resulting in the release of several cytokines, including interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α). As these cytokines are known to be involved in the host defence against Trypanosoma cruzi, we measured serum levels of IFN-γ and TNF-α after injection of the 145-2C11 MoAb in the course of experimental murine Chagas' disease. Compared with control mice, T. cruzi-infected BALB/c mice were found to be primed to secrete very high levels of IFN-γ and TNF-α from the second and the first week of infection, respectively, up to the chronic phase. In vivo cell depletion experiments indicated that CD8+ T cells were responsible for these dramatic hyperproductions of IFN-γ and TNF-α. While all control mice survived anti-CD3 MoAb injection, a high lethality rate was observed in T. cruzi-infected mice within 24 h after anti-CD3 MoAb challenge. Pretreatment with neutralizing anti-IFN-γ MoAb or depletion of CD8+ T cell population dramatically decreased the mortality induced by anti-CD3 MoAb in T. cruzi-infected mice. Finally, we showed that anti-CD3 MoAb injection in T. cruzi-infected mice was followed by a massive release of nitric oxide (NO) metabolites, which was partially reduced by IFN-γ or TNF-α neutralization. The administration of the NO synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) before anti-CD 3 MoAb challenge did not prevent and even enhanced lethality in T. cruzi-infected mice, suggesting that NO overproduction and lethal shock are not causally related. We conclude that injection of anti-CD3 MoAb in the course of experimental Chagas' disease induces a CD8+ cell-dependent shock mediated by IFN-γ and TNF-α. [ABSTRACT FROM AUTHOR]