Early IL-2 production by mouse dendritic cells is the result of microbial-induced priming

Dendritic cells (DCs) are professional APCs able to initiate innate and adaptive immune responses against invading pathogens. Different properties such as the efficient Ag processing machinery, the high levels of expression of costimulatory molecules and peptide-MHC complexes, and the production of cytokines contribute in making DCs potent stimulators of naive T cell responses. Recently we have observed that DCs are able to produce IL-2 following bacterial stimulation, and we have demonstrated that this particular cytokine is a key molecule conferring to early bacterial activated DCs unique T cell priming capacity. In the present study we show that many different microbial stimuli, but not inflammatory cytokines, are able to stimulate DCs to produce IL-2, indicating that DCs can distinguish a cytokine-mediated inflammatory process from the actual presence of an infection. The capacity to produce IL-2 following a microbial stimuli encounter is a feature shared by diverse DC subtypes in vivo, such as CD8 alpha(+) and CD8 alpha(-) splenic DCs and epidermal Langerhans cells. When early activated DCs interact with T cells, IL-2 produced by DCs is enriched at the site of cell-cell contact, confirming the importance of DCs-derived IL-2 in T cell activation.     

Phagosomal Acidification Prevents Macrophage Inflammatory Cytokine Production to Malaria,and Dendritic Cells Are the Major Source at the Early Stages of Infection: IMPLICATION FOR MALARIA PROTECTIVE IMMUNITY DEVELOPMENT*

Inflammatory cytokines produced at the early stages of malaria infection contribute to shaping protective immunity and pathophysiology. To gain mechanistic insight into these processes, it is important to understand the cellular origin of cytokines because both cytokine input and cytokine-producing cells play key roles. Here, we determined cytokine responses by monocytes, macrophages, and dendritic cells (DCs) to purified Plasmodium falciparum and Plasmodium berghei ANKA, and by spleen macrophages and DCs from Plasmodium yoelii 17NXL-infected and P. berghei ANKA-infected mice. The results demonstrate that monocytes and macrophages do not produce inflammatory cytokines to malaria parasites and that DCs are the primary source early in infection, and DC subsets differentially produce cytokines. Importantly, blocking of phagosomal acidification by inhibiting vacuolar-type H⁺-ATPase enabled macrophages to elicit cytokine responses. Because cytokine responses to malaria parasites are mediated primarily through endosomal Toll-like receptors, our data indicate that the inability of macrophages to produce cytokines is due to the phagosomal acidification that disrupts endosomal ligand-receptor engagement. Macrophages efficiently produced cytokines to LPS upon simultaneously internalizing parasites and to heat-killed Escherichia coli, demonstrating that phagosomal acidification affects endosomal receptor-mediated, but not cell surface receptor-mediated, recognition of Toll-like receptor agonists. Enabling monocytes/macrophages to elicit immune responses to parasites by blocking endosomal acidification can be a novel strategy for the effective development of protective immunity to malaria. The results have important implications for enhancing the efficacy of a whole parasite-based malaria vaccine and for designing strategies for the development of protective immunity to pathogens that induce immune responses primarily through endosomal receptors.     

Oncostatin M induces dendritic cell maturation and Th1 polarization

Oncostatin M (OSM) is a pleiotropic cytokine and a member of the gp130/IL-6 cytokine family that has been found to be involved in both pro- and anti-inflammatory responses in cell-mediated immunity. Maturation of dendritic cells (DCs) is crucial for initiation of primary immune responses and is regulated by several stimuli. In this study, the role of OSM in the phenotypic and functional maturation of DCs was evaluated in vitro. Stimulation with OSM upregulated the expression of CD80, CD86, MHC class I and MHC class II and reduced the endocytic capacity of immature DCs. Moreover, OSM induced the allogeneic immunostimulatory capacity of DCs by stimulating the production of the Th1-promoting cytokine IL-12. OSM also increased the production of IFN-γ by T cells in mixed-lymphocyte reactions, which would be expected to contribute to the Th1 polarization of the immune response. The expression of surface markers and cytokine production in DCs was mediated by both the MAPK and NF-κB pathways. Taken together, these results indicate that OSM may play a role in innate immunity and in acquired immunity by enhancing DCs maturation and promoting Th1 immune responses.     

Activation of invariant NKT cells by the helminth parasite schistosoma mansoni

Mouse CD1d-restricted NKT cells, including invariant (i)NKT cells, are innate cells activated by glycolipid Ags and play important roles in the initiation and regulation of immune responses. Through their ability to promptly produce large amounts of Th1 and/or Th2 cytokines upon TCR engagement, iNKT cells exert crucial functions in the immune/inflammatory system during bacterial, protozoan, fungal, and viral infections. However, their roles during metazoan parasite infection, which are generally associated with strong Th2 responses, still remain elusive. In this study, we show that during the course of murine schistosomiasis, iNKT cells exhibit an activated phenotype and that following schistosome egg encounter in the liver, hepatic iNKT cells produce both IFN-gamma and IL-4 in vivo. We also report that schistosome egg-sensitized dendritic cells (DCs) activate, in a CD1d-dependent manner, iNKT cells to secrete IFN-gamma and IL-4 in vitro. Interestingly, transfer of egg-sensitized DCs promotes a strong Th2 response in recipient wild-type mice, but not in mice that lack iNKT cells. Engagement of TLRs in DCs is not necessary for iNKT cell stimulation in response to egg-sensitized DCs, suggesting an alternative pathway of activation. Finally, we propose that self, rather than parasite-derived, CD1d-restricted ligands are implicated in iNKT cell stimulation. Taken together, our data show for the first time that helminths can activate iNKT cells to produce immunoregulatory cytokines in vivo, enabling them to influence the adaptive immune response.     

Successful induction of clinically competent dendritic cells from granulocyte colony-stimulating factor-mobilized monocytes for cancer vaccine therapy

Recent studies have suggested that dendritic cell (DC)-based immunotherapy is one promising approach for the treatment of cancer. We previously studied the clinical toxicity, feasibility, and efficacy of cancer vaccine therapy with peptide-pulsed DCs. In that study, we used granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood monocytes as a cell source of DCs. However, previous investigations have suggested that G-CSF-mobilized peripheral blood monocytes produce reduced levels of proinflammatory cytokines such as interleukin (IL)-12 and tumor necrosis factor (TNF)-α. These T helper (Th)-1-type cytokines are thought to promote antitumor immune response. In this study, we assessed the functional abilities of DCs generated from G-CSF-mobilized monocytes obtained from 13 patients with CEA-positive advanced solid cancers. Peripheral blood mononuclear cells were obtained from leukapheresis products collected before and after systemic administration of G-CSF (subcutaneous administration of high-dose [5–10 μg/kg] human recombinant G-CSF for five consecutive days). In vitro cytokine production profiles after stimulation with lipopolysaccharide (LPS) were compared between monocytes with and without G-CSF mobilization. DCs generated from monocytes were also examined with respect to cytokine production and the capacity to induce peptide-specific T cell responses. Administration of G-CSF was found to efficiently mobilize peripheral blood monocytes. Although G-CSF-mobilized monocytes (G/Mo) less effectively produced Th-1-type cytokines than control monocytes (C/Mo), DCs generated from G/Mo restored the same level of IL-12 production as that seen in DCs generated from C/Mo. T cell induction assay using recall antigen peptide and phenotypic analyses also demonstrated that DCs generated from G/Mo retained characteristics identical to those generated from C/Mo. Our results suggest that G-CSF mobilization can be used to collect monocytes as a cell source for the generation of DCs for cancer immunotherapy. DCs generated in this fashion were pulsed with HLA-A24-restricted CEA epitope peptide and administered to patients safely; immunological responses were induced in some patients.     

Hepatitis C virus core and nonstructural protein 3 proteins induce pro- and anti-inflammatory cytokines and inhibit dendritic cell differentiation

Antiviral immunity requires recognition of viral pathogens and activation of cytotoxic and Th cells by innate immune cells. In this study, we demonstrate that hepatitis C virus (HCV) core and nonstructural protein 3 (NS3), but not envelope 2 proteins (E2), activate monocytes and myeloid dendritic cells (DCs) and partially reproduce abnormalities found in chronic HCV infection. HCV core or NS3 (not E2) triggered inflammatory cytokine mRNA and TNF-alpha production in monocytes. Degradation of I-kappa B alpha suggested involvement of NF-kappa B activation. HCV core and NS3 induced production of the anti-inflammatory cytokine, IL-10. Both monocyte TNF-alpha and IL-10 levels were higher upon HCV core and NS3 protein stimulation in HCV-infected patients than in normals. HCV core and NS3 (not E2) inhibited differentiation and allostimulatory capacity of immature DCs similar to defects in HCV infection. This was associated with elevated IL-10 and decreased IL-2 levels during T cell proliferation. Increased IL-10 was produced by HCV patients' DCs and by core- or NS3-treated normal DCs, while IL-12 was decreased only in HCV DCs. Addition of anti-IL-10 Ab, not IL-12, ameliorated T cell proliferation with HCV core- or NS3-treated DCs. Reduced allostimulatory capacity in HCV core- and NS3-treated immature DCs, but not in DCs of HCV patients, was reversed by LPS maturation, suggesting more complex DC defects in vivo than those mediated by core or NS3 proteins. Our results reveal that HCV core and NS3 proteins activate monocytes and inhibit DC differentiation in the absence of the intact virus and mediate some of the immunoinhibitory effects of HCV via IL-10 induction.     

TNF-alpha drives human CD14+ monocytes to differentiate into CD70+ dendritic cells evoking Th1 and Th17 responses

Many mechanisms involving TNF-alpha, Th1 responses, and Th17 responses are implicated in chronic inflammatory autoimmune disease. Recently, the clinical impact of anti-TNF therapy on disease progression has resulted in re-evaluation of the central role of this cytokine and engendered novel concept of TNF-dependent immunity. However, the overall relationship of TNF-alpha to pathogenesis is unclear. Here, we demonstrate a TNF-dependent differentiation pathway of dendritic cells (DC) evoking Th1 and Th17 responses. CD14(+) monocytes cultured in the presence of TNF-alpha and GM-CSF converted to CD14(+) CD1a(low) adherent cells with little capacity to stimulate T cells. On stimulation by LPS, however, they produced high levels of TNF-alpha, matrix metalloproteinase (MMP)-9, and IL-23 and differentiated either into mature DC or activated macrophages (M phi). The mature DC (CD83(+) CD70(+) HLA-DR (high) CD14(low)) expressed high levels of mRNA for IL-6, IL-15, and IL-23, induced naive CD4 T cells to produce IFN-gamma and TNF-alpha, and stimulated resting CD4 T cells to secret IL-17. Intriguingly, TNF-alpha added to the monocyte culture medium determined the magnitude of LPS-induced maturation and the functions of the derived DC. In contrast, the M phi (CD14(high)CD70(+)CD83(-)HLA-DR(-)) produced large amounts of MMP-9 and TNF-alpha without exogenous TNF stimulation. These results suggest that the TNF priming of monocytes controls Th1 and Th17 responses induced by mature DC, but not inflammation induced by activated M phi. Therefore, additional stimulation of monocytes with TNF-alpha may facilitate TNF-dependent adaptive immunity together with GM-CSF-stimulated M phi-mediated innate immunity.     

Atrial natriuretic peptide polarizes human dendritic cells toward a Th2-promoting phenotype through its receptor guanylyl cyclase-coupled receptor A

Atrial natriuretic peptide (ANP) is a cardiovascular hormone secreted mainly by the cardiac atria and regulates the volume-pressure homeostasis. The action of ANP is mediated by its receptor, guanylyl cyclase-coupled receptor A (GC-A). In this study, we explored the possibility that ANP and GC-A may play a role in the dendritic cell (DC)-mediated immune regulation. We first examined the expression of GC-A in human monocyte-derived DCs in comparison with monocytes and found that DCs but not monocytes express GC-A at both the mRNA and protein levels. DCs responded to ANP with an increase in intracellular cGMP in a dose-dependent manner, indicating that GC-A expressed on DCs is functional. Furthermore, treatment of DCs with ANP decreased production of IL-12 and TNF-alpha and conversely increased that of IL-10 upon stimulation with LPS. In accordance with this change of cytokine production, DCs treated with ANP plus LPS promoted differentiation of naive CD4(+) T cells into a Th2 phenotype. Finally, we presented evidence that ANP affected cytokine production of fresh whole blood stimulated with LPS in line with the above-mentioned results. These results indicate that ANP polarizes human DCs toward a Th2-promoting phenotype through GC-A and thus can regulate immune responses.     

IFN-alpha skews monocyte differentiation into Toll-like receptor 7-expressing dendritic cells with potent functional activities

IFN-alpha is an important cytokine for the generation of a protective T cell-mediated immune response to viruses. In this study, we asked whether IFN-alpha can regulate the functional properties of dendritic cells (DCs). We show that monocytes cultured in the presence of GM-CSF and IFN-alpha can differentiate into DCs (IFN-alpha-derived DCs (IFN-DCs)). When compared with DCs generated in the presence of GM-CSF and IL-4 (IL-4-derived DCs), IFN-DCs exhibited a typical DC morphology and expressed, in addition to DC markers CD1a and blood DC Ag 4, a similar level of costimulatory and class II MHC molecules, but a significantly higher level of MHC class I molecules. After maturation with CD40 ligand, IFN-DCs up-regulated costimulatory, class I and II MHC molecules and expressed mature DC markers such as CD83 and DC-lysosome-associated membrane protein. IFN-DCs were endowed with potent functional activities. IFN-DCs secreted large amounts of the inflammatory cytokines IL-6, IL-10, TNF-alpha, IL-1beta, and IL-18, and promoted a Th1 response that was independent of IL-12p70 and IL-18, but substantially inhibited by IFN-alpha neutralization. Furthermore, immature IFN-DCs induced a potent autologous Ag-specific immune response, as evaluated by IFN-gamma secretion and expansion of CD8(+) T cells specific for CMV. Also, IFN-DCs expressed a large number of Toll-like receptors (TLRs), including acquisition of TLR7, which is classically found on the natural type I IFN-producing plasmacytoid DCs. Like plasmacytoid DCs, IFN-DCs could secrete IFN-alpha following viral stimulation or TLR7-specific stimulation. Taken together, these results illustrate the critical role of IFN-alpha at the early steps of immune response to pathogens or in autoimmune diseases.     

Macrophage colony-stimulating factor drives cord blood monocyte differentiation into IL-10(high)IL-12absent dendritic cells with tolerogenic potential

Immature dendritic cells (DCs) induce tolerance and mature DCs induce inflammatory immune responses. However, the likelihood of maturation of immature DCs in vivo limits its potential application for suppression of unwanted immune reactions in vivo. The aim of this study was to generate DCs with anti-inflammatory properties in both the immature and mature states. GM-CSF combined with IL-4 drives monocyte differentiation into DCs. As M-CSF is a critical cytokine in development of the monocytic lineage and its level is dramatically elevated in immunosuppressive conditions, we investigated whether M-CSF could replace GM-CSF and generate DCs with distinct functions from umbilical cord blood monocytes. Highly purified umbilical cord blood monocytes cultured with M-CSF and IL-4, in a GM-CSF-independent fashion, differentiated into IL-10(high)IL-12absent cells with a DC phenotype (termed M-DC). Single time stimulation with immature DCs (both M-DCs and DCs) derived from cord blood induced hyporesponsive and regulatory CD4+ T cells. In contrast to mature DCs, mature M-DCs induced decreased Th1 differentiation and proliferation of naive CD4+ T cells in both primary and secondary allogeneic MLR and showed tolerogenic potential. These results demonstrate an unrecognized role for M-CSF in alternative differentiation of monocytes into anti-inflammatory M-DCs and suggest that M-CSF-induced DCs may be of use for suppressing unwanted immune responses.     

Il-10 is a central regulator of cyclooxygenase-2 expression and prostaglandin production

IL-10 is a potent anti-inflammatory and immune regulatory cytokine. IL-10(-/-) mice produce exaggerated amounts of inflammatory cytokines when stimulated with LPS, indicating that endogenous IL-10 is a central regulator of inflammatory cytokine production in vivo. PGs are lipid mediators that are also produced in large amounts during the inflammatory response. To study the role of IL-10 in the regulation of PG production during the acute inflammatory response, we evaluated LPS-induced cyclooxygenase (COX) expression and PG production in wild-type (wt) and IL-10(-/-) mice. LPS-induced PGE(2) production from IL-10(-/-) spleen cells was 5.6-fold greater than that from wt spleen cells. LPS stimulation resulted in the induction of COX-2 mRNA and protein in both wt and IL-10(-/-) spleen cells; however, the magnitude of increase in COX-2 mRNA was 5.5-fold greater in IL-10(-/-) mice as compared with wt mice. COX-1 protein levels were not affected by LPS stimulation in either wt or IL-10(-/-) mice. Neutralization of IFN-gamma, TNF-alpha, or IL-12 markedly decreased the induction of COX-2 in IL-10(-/-) spleen cells, suggesting that increased inflammatory cytokine production mediates much of the COX-2 induction in IL-10(-/-) mice. Treatment of IL-10(-/-) mice with low doses of LPS resulted in a marked induction of COX-2 mRNA in the spleen, whereas wt mice had minimal expression of COX-2 mRNA. These findings indicate that, in addition to IL-10's central role in the regulation of inflammatory cytokines, endogenous IL-10 is an important regulator of PG production in the response to LPS.     

Interplay between CD8α+ dendritic cells and monocytes in response to Listeria monocytogenes infection attenuates T cell responses

During the course of a microbial infection, different antigen presenting cells (APCs) are exposed and contribute to the ensuing immune response. CD8α(+) dendritic cells (DCs) are an important coordinator of early immune responses to the intracellular bacteria Listeria monocytogenes (Lm) and are crucial for CD8(+) T cell immunity. In this study, we examine the contribution of different primary APCs to inducing immune responses against Lm. We find that CD8α(+) DCs are the most susceptible to infection while plasmacytoid DCs are not infected. Moreover, CD8α(+) DCs are the only DC subset capable of priming an immune response to Lm in vitro and are also the only APC studied that do so when transferred into β2 microglobulin deficient mice which lack endogenous cross-presentation. Upon infection, CD11b(+) DCs primarily secrete low levels of TNFα while CD8α(+) DCs secrete IL-12 p70. Infected monocytes secrete high levels of TNFα and IL-12p70, cytokines associated with activated inflammatory macrophages. Furthermore, co-culture of infected CD8α(+) DCs and CD11b+ DCs with monocytes enhances production of IL-12 p70 and TNFα. However, the presence of monocytes in DC/T cell co-cultures attenuates T cell priming against Lm-derived antigens in vitro and in vivo. This suppressive activity of spleen-derived monocytes is mediated in part by both TNFα and inducible nitric oxide synthase (iNOS). Thus these monocytes enhance IL-12 production to Lm infection, but concurrently abrogate DC-mediated T cell priming.     

Thymic stromal lymphopoietin is produced by dendritic cells

Thymic stromal lymphopoietin (TSLP) is a type 1 cytokine that contributes to lymphopoiesis and the development of asthma and atopic dermatitis. TSLP acts on multiple lineages, including dendritic cells (DCs), T cells, NKT cells, eosinophils, and mast cells, mediating proliferation and survival and linking innate and adaptive immune responses. TSLP is produced by a range of cells, including epithelial cells, fibroblasts, stromal cells, and keratinocytes. DCs are important primary targets of TSLP, and we unexpectedly demonstrated that DCs also produce TSLP in response to TLR stimulation and that this is augmented by IL-4. Moreover, we demonstrated that when mice were challenged with house dust mite extract, lung CD11c(+) DCs expressed TSLP mRNA at an even higher level than did epithelial cells. These data suggested that DCs not only respond to TSLP but also are a source of TSLP during pathogen and/or allergen encounter.     

Induction of dendritic cell maturation by IL-18

IL-18 is a pluripotent proinflammatory cytokine produced primarily by antigen presenting cells involved in numerous aspects of immune regulation most notably on lymphoid cells. The effect of IL-18 stimulation on cells in the myeloid compartment, however, has been poorly studied. Human monocytes did not respond to IL-18. However, the human myelomonocytic cell line KG-1 and monocyte-derived dendritic cells (generated by GM-CSF+IL-4) showed a marked increase in CD83, HLA-DR, and several costimulatory molecules upon stimulation with IL-18. Furthermore, IL-18 decreased pinocytosis of these cells and increased their ability to stimulate alloreactive T cell proliferation, all characteristics of mature dendritic cells. These results suggest that IL-18 is involved in the maturation of myeloid DCs, but not differentiation of monocytes into DCs. The finding that IL-18 is involved in the maturation of dendritic cells is both novel and unexpected and indicates another important role for IL-18 as a key regulator of immune responses.     

Inflammatory blood monocytes contribute to tumor development and represent a privileged target to improve host immunosurveillance

Progressing tumors in humans and mice are frequently infiltrated by a highly heterogeneous population of inflammatory myeloid cells that contribute to tumor growth. Among these cells, inflammatory Gr-1(+) monocytes display a high developmental plasticity in response to specific microenvironmental signals, leading to diverse immune functions. These observations raise the question of the immune mechanisms by which inflammatory monocytes may contribute to tumor development. In this study, we found that adoptive transfer of normal inflammatory Gr-1(+) monocytes in tumor-bearing mice promotes tumor growth. In this tumoral environment, these monocytes can differentiate into tolerogenic dendritic cells (DCs) that produce IL-10 and potently induce regulatory T cell responses in vivo. Moreover, diverting the differentiation of Gr-1(+) monocytes into tolerogenic DCs by forced expression of IL-10 soluble receptor and IL-3 in tumor cells improves host immunosurveillance by reducing the regulatory T cell frequency and by inducing immunogenic DCs in the tumor. As a consequence, tumor growth is strongly reduced. Our findings indicate that Gr-1(+) monocytes represent a valuable target for innovative immunotherapeutic strategies against cancer.     

High expression of antioxidant proteins in dendritic cells: possible implications in atherosclerosis

Dendritic cells (DCs) display the unique ability to activate naive T cells and to initiate primary T cell responses revealed in DC-T cell alloreactions. DCs frequently operate under stress conditions. Oxidative stress enhances the production of inflammatory cytokines by DCs. We performed a proteomic analysis to see which major changes occur, at the protein expression level, during DC differentiation and maturation. Comparative two-dimensional gel analysis of the monocyte, immature DC, and mature DC stages was performed. Manganese superoxide dismutase (Mn-SOD) reached 0.7% of the gel-displayed proteins at the mature DC stage. This important amount of Mn-SOD is a primary antioxidant defense system against superoxide radicals, but its product, H(2)O(2), is also deleterious for cells. Peroxiredoxin (Prx) enzymes play an important role in eliminating such peroxide. Prx1 expression level continuously increased during DC differentiation and maturation, whereas Prx6 continuously decreased, and Prx2 peaked at the immature DC stage. As a consequence, DCs were more resistant than monocytes to apoptosis induced by high amounts of oxidized low density lipoproteins containing toxic organic peroxides and hydrogen peroxide. Furthermore DC-stimulated T cells produced high levels of receptor activator of nuclear factor kappaB ligand, a chemotactic and survival factor for monocytes and DCs. This study provides insights into the original ability of DCs to express very high levels of antioxidant enzymes such as Mn-SOD and Prx1, to detoxify oxidized low density lipoproteins, and to induce high levels of receptor activator of nuclear factor kappaB ligand by the T cells they activate and further emphasizes the role that DCs might play in atherosclerosis, a pathology recognized as a chronic inflammatory disorder.     

Oncostatin M induces tissue-type plasminogen activator and plasminogen activator inhibitor-1 in Calu-1 lung carcinoma cells

Oncostatin M (OSM) is a glycoprotein cytokine that is produced by activated T-lymphocytes, monocytes, and macrophages. In a DNA synthesis assay, OSM reduced tritiated thymidine incorporation by 53% in Calu-1 lung carcinoma cells. Radiolabeled cDNAs from untreated Calu-1 cells and 30-h OSM-treated cells were used to probe duplicate nylon membrane cDNA expression arrays. This study revealed OSM-mediated expression of mRNAs encoding tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1). Northern blot analysis showed that the steady-state level of tPA mRNA is nearly undetectable in Calu-1 cells. Exposure of these cells to OSM for 30 h increased tPA mRNA expression by 20-fold and PAI-1 mRNA expression by 5-fold. Exposure of these cells to other gp130 receptor family cytokines, including leukemia inhibitory factor (LIF), interleukin-6 (IL-6), and IL-11, do not significantly affect DNA synthesis or induction of tPA/PAI-1. Western blot studies demonstrated that OSM mediates a marked increase in secretion of the tPA protein. Secreted tPA was present in the conditioned medium almost exclusively as tPA/PAI-1 complexes. Inhibitor studies demonstrated that OSM-mediated induction of tPA and PAI-1 mRNAs is largely dependent upon activation of the MEK1/2 pathway. The JAK3/STAT3 pathway potentially serves a secondary role in these regulatory events.     

Suppression of IL-12 production by soluble CD40 ligand: evidence for involvement of the p44/42 mitogen-activated protein kinase pathway

IL-12 is a key cytokine in skewing immune responses toward Th1-like reactions. Human monocytes/macrophages produce high amounts of bioactive IL-12 when a priming signal (IFN-gamma or GM-CSF) precedes a second signal (e.g., LPS). We and others have previously shown that preincubation with LPS before this stimulation procedure can efficiently and selectively suppress the production of IL-12 by human monocytes. In this study, we show that an almost complete suppression of IL-12 production can also be observed after preincubation of monocytes with costimulatory cell surface molecules that bind to members of the TNFR superfamily (CD40 ligand, TNF-related activation-induced cytokine (TRANCE)). The suppression of IL-12 was observable on the mRNA and protein levels and was not due to endogenous production of known IL-12 antagonists (i.e., IL-10, IL-4, and PGE(2)), to an increased number of cells undergoing apoptosis, nor to down-regulation of the IFN-gamma or CD40 receptor. Cell surface expression of the costimulatory molecules CD80 and CD86 was not reduced by the preincubation procedure, and only a moderate reduction of IL-6 production was observed. Several studies have identified signal transduction pathways that are activated by CD40 signaling, including activation of mitogen-activated protein kinases. The presence of the extracellular signal-related kinase-specific mitogen-activated protein kinase kinase 1/2-specific inhibitors PD98059 and U0126 abrogated suppression induced by sCD40 ligand or other second signals. This indicates that activation of extracellular signal-regulated kinase 1/2 contributes to the underlying mechanism of IL-12 suppression. This mechanism may be relevant in other inflammatory responses and may help to develop therapeutic strategies in Th1-mediated diseases.