The presence of GM-CSF and IL-4 interferes with effect of TGF-beta1 on antigen presenting cells in patients with multiple sclerosis and in rats with experimental autoimmune encephalomyelitis

The possibility to generate and expand tolerogenic dendritic cells (DC) with TGF-β1 in vitro opens new therapeutic perspectives for the treatment of autoimmune diseases. In the present study, GM-CSF+IL-4 induced the differentiation of DC from adherent peripheral blood mononuclear cells, which had a higher expression of HLA-DR, CD86 and CD1a and the capacity to stimulate T cells. TGF-β1 alone slightly promoted the generation of antigen presenting cells (APC) with higher expression of CD14, but did not differentiate them into E-cadherin + Langerhans cell (LC)-like DC. TGF-β1-driven APC exhibited the morphology, phenotypes and functions of tolerogenic immature DC, and had lower capacity to stimulate T cells. In vivo experiment demonstrates that TGF-β1-treated APC exhibited the therapeutic potential in Lewis rats with experimental autoimmune encephalomyelitis (EAE), followed by increase of IL-10 production in lymph nodes and decrease of inflammatory cells in spinal cords. Most importantly, GM-CSF/IL-4 used in DC preparation abolished the effect of TGF-β1 to induce tolerogenic APC in vitro and in vivo. The results reveal that the usage of GM-CSF for the generation of tolerogenic DC should not be copied from DC preparation for anti-tumor therapy.     

IL-10 induces CCR6 expression during Langerhans cell development while IL-4 and IFN-gamma suppress it.

Immune responses are initiated by dendritic cells (DC) that form a network comprising different populations. In particular, Langerhans cells (LC) appear as a unique population of cells colonizing epithelial surfaces. We have recently shown that macrophage-inflammatory protein-3alpha/CCL20, a chemokine secreted by epithelial cells, induces the selective migration of LC among DC populations. In this study, we investigated the effects of cytokines on the expression of the CCL20 receptor, CCR6, during differentiation of LC. We found that both IL-4 and IFN-gamma blocked the expression of CCR6 and CCL20 responsiveness at different stages of LC development. The effect of IL-4 was reversible and most likely due to the transient blockade of LC differentiation. In contrast, IFN-gamma-induced CCR6 loss was irreversible and was concomitant to the induction of DC maturation. When other cytokines involved in DC and T cell differentiation were tested, we found that IL-10, unlike IL-4 and IFN-gamma, maintained CCR6 expression. The effect of IL-10 was reversible and upon IL-10 withdrawn, CCR6 was lost concomitantly to final LC differentiation. In addition, IL-10 induced the expression of CCR6 and responsiveness to CCL20 in differentiated monocytes that preserve their ability to differentiate into mature DC. Finally, TGF-beta, which induces LC differentiation, did not alter early CCR6 expression, but triggered its irreversible down-regulation, in parallel to terminal LC differentiation. Taken together, these results suggest that the recruitment of LC at epithelial surface might be suppressed during Th1 and Th2 immune responses, and amplified during regulatory immune responses involving IL-10 and TGF-beta.     

Bone cells from patients with quiescent Crohn's disease show a reduced growth potential and an impeded maturation

Patients with Crohn's disease (CD) are at increased risk of developing osteoporosis. The mechanism underlying bone loss in CD patients is only partly understood. Inflammation is thought to contribute by causing a disturbed bone remodeling. In this study, we aimed to compare functional characteristics of osteoblasts from CD patients and controls, as osteoblasts are one of the effector cells in bone remodeling. The study included 18 patients with quiescent CD and 18 healthy controls. Bone cells obtained from iliac crest biopsies were cultured in the absence and presence of the inflammatory cytokines IL-1α, IL-1β, IL-6, TNF-α, IL-10, and TGF-β. At various time points, cell proliferation and differentiation were analyzed. Bone cells from CD patients showed a prolonged culture period to reach confluence and a decreased cell number at confluence. CD patient-derived bone cell cultures produced higher alkaline phosphatase levels, whereas osteocalcin levels were considerably reduced compared to control cultures. At the proliferation level, the responsiveness to inflammatory cytokines was similar in bone cells from CD patients and controls. At the differentiation level, CD cultures showed an increased responsiveness to IL-6 and a decreased responsiveness to TGF-β. Responsiveness to the other cytokines tested was unaffected. In summary, we show a reduced growth potential and impeded maturation of bone cells from quiescent CD patients in vitro. These disease-related alterations combined with an unchanged sensitivity of CD patient-derived bone cells to inflammatory cytokines, provide a new insight in the understanding of CD-associated bone loss.     

Altered dendritic cells (DC) might be responsible for regulatory T cell imbalance and autoimmunity in nonobese diabetic (NOD) mice

Nonobese diabetic (NOD) mice spontaneously develop diabetes, an auto-immune disease characterized by the destruction of insulin-secreting beta-cells by autoreactive T cells. Defects in development and/or functions of dendritic cells (DC) might be critical in eliciting the auto-immune reaction to beta cells in this model. In this paper, DC differentiation in NOD mice was investigated in vitro using bone marrow-derived progenitors (BM-DC) in the presence of GM-CSF and IL-4 or spleen-derived progenitors in the presence of GM-CSF and early acting cytokines such as Flt-3L and IL-6 (SPL-DC). In both culture systems, the absolute number of NOD DC generated was strongly reduced as compared to control strains. In addition, both BM-DC and SPL-DC from NOD mice show defective differentiation into mature DC in conventional culture conditions as indicated by low expression of MHC class II and CD80 molecules among CD11c positive cells and low capacity to stimulate allogeneic T cells. However, DC achieved full maturation when exposed to LPS, except for MHC class II expression that remained decreased. Ex vivo analysis confirmed an unusual phenotype of NOD DC. Both sets of results are thus consistent with a specific defect of DC maturation in these mice.     

IL-10 Conditioning of Human Skin Affects the Distribution of Migratory Dendritic Cell Subsets and Functional T Cell Differentiation

In cancer patients pervasive systemic suppression of Dendritic Cell (DC) differentiation and maturation can hinder vaccination efficacy. In this study we have extensively characterized migratory DC subsets from human skin and studied how their migration and T cell-stimulatory abilities were affected by conditioning of the dermal microenvironment through cancer-related suppressive cytokines. To assess effects in the context of a complex tissue structure, we made use of a near-physiological skin explant model. By 4-color flow cytometry, we identified migrated Langerhans Cells (LC) and five dermis-derived DC populations in differential states of maturation. From a panel of known tumor-associated suppressive cytokines, IL-10 showed a unique ability to induce predominant migration of an immature CD14⁺CD141⁺DC-SIGN⁺ DC subset with low levels of co-stimulatory molecules, up-regulated expression of the co-inhibitory molecule PD-L1 and the M2-associated macrophage marker CD163. A similarly immature subset composition was observed for DC migrating from explants taken from skin overlying breast tumors. Whereas predominant migration of mature CD1a⁺ subsets was associated with release of IL-12p70, efficient Th cell expansion with a Th1 profile, and expansion of functional MART-1-specific CD8⁺ T cells, migration of immature CD14⁺ DDC was accompanied by increased release of IL-10, poor expansion of CD4⁺ and CD8⁺ T cells, and skewing of Th responses to favor coordinated FoxP3 and IL-10 expression and regulatory T cell differentiation and outgrowth. Thus, high levels of IL-10 impact the composition of skin-emigrated DC subsets and appear to favor migration of M2-like immature DC with functional qualities conducive to T cell tolerance.     

The effect of pro-inflammatory cytokines on immunophenotype,differentiation capacity and immunomodulatory functions of human mesenchymal stem cells

Mesenchymal stem cells (MSCs), as cells with potential clinical utilities, have demonstrated preferential incorporation into inflammation sites. Immunophenotype and immunomodulatory functions of MSCs could alter by inflamed-microenvironments due to the local pro-inflammatory cytokine milieu. A major cellular mediator with specific function in promoting inflammation and pathogenicity of autoimmunity are IL-17-producing T helper 17 (Th17) cells that polarize in inflamed sites in the presence of pro-inflammatory cytokines such as Interleukin-1β (IL-1β), IL-6 and IL-23. Since MSCs are promising candidate for cell-based therapeutic strategies in inflammatory and autoimmune diseases, Th17 cell polarizing factors may alter MSCs phenotype and function. In this study, human bone-marrow-derived MSCs (BM-MSC) and adipose tissue-derived MSCs (AD-MSC) were cultured with or without IL-1β, IL-6 and IL-23 as pro-inflammatory cytokines. The surface markers and their differentiation capacity were measured in cytokine-untreated and cytokine-treated MSCs. MSCs-mediated immunomodulation was analyzed by their regulatory effects on mixed lymphocyte reaction (MLR) and the level of IL-10, TGF-β, IL-4, IFN-γ and TNF-α production as immunomodulatory cytokines. Pro-inflammatory cytokines showed no effect on MSCs morphology, immunophenotype and co-stimulatory molecules except up-regulation of CD45. Adipogenic and osteogenic differentiation capacity increased in CD45+ MSCs. Moreover, cytokine-treated MSCs preserved the suppressive ability of allogeneic T cell proliferation and produced higher level of TGF-β and lower level of IL-4. We concluded pro-inflammatory cytokines up-regulate the efficacy of MSCs in cell-based therapy of degenerative, inflammatory and autoimmune disorders.     

Optimization of human Th17 cell differentiation in vitro<Emphasis Type="Italic">:</Emphasis> evaluating different polarizing factors

Regarding discrepancies that exist among different studies which have tried to clarify critical factors in human Th17 cell differentiation, the aim of this study was to identify the best condition for human Th17 differentiation and to clarify the possible role of TGF-β in differentiation of these cells. Naïve CD4⁺ T cells were isolated from cord blood samples and cultured either in X-VIVO 15 serum-free medium or RPMI 1640 containing 10% FBS. Purified cells were treated with different combinations of polarizing cytokines (TGF-β, IL-1β, IL-6, IL-23 and IL-21) followed by analysis of the expression of characteristic genes and their relevant cytokines by real-time quantitative RT-PCR and ELISA method, respectively. Our data indicate that a combination of TGF-β plus IL-6 and IL-23 cytokines in X-VIVO 15 serum-free medium could be applied as the best condition for developing human Th17 cells in compare with other studied cytokine treatments. It is shown that TGF-β could be considered as a positive regulator for human Th17 cell differentiation only if applied in average concentrations. Interestingly, polarizing treatments in absence of TGF-β, induced double-secreting Th17 cells which co-express IL-17 and IFN-γ whereas polarization in presence of TGF-β-induced single-secreting (only IL-17 expressing) Th17 cells.     

Granulocyte macrophage colony-stimulating factor enhances the modulatory effect of cytokines on monocyte-derived multinucleated giant cell formation and fungicidal activity against Paracoccidioides brasiliensis

Multinucleated giant cells (MGC) are cells present in characteristic granulomatous inflammation induced by intracellular infectious agents or foreign materials. The present study evaluated the modulatory effect of granulocyte macrophage colony-stimulating factor (GM-CSF) in association with other cytokines such as interferon-gamma (IFN-γ), tumour necrosis factor-alpha, interleukin (IL)-10 or transforming growth factor beta (TGF-β1) on the formation of MGC from human peripheral blood monocytes stimulated with Paracoccidioides brasiliensis antigen (PbAg). The generation of MGC was determined by fusion index (FI) and the fungicidal activity of these cells was evaluated after 4 h of MGC co-cultured with viable yeast cells of P. brasiliensis strain 18 (Pb18). The results showed that monocytes incubated with PbAg and GM-CSF plus IFN-γ had a significantly higher FI than in all the other cultures, while the addition of IL-10 or TGF-β1 had a suppressive effect on MGC generation. Monocytes incubated with both pro and anti-inflammatory cytokines had a higher induction of foreign body-type MGC rather than Langhans-type MGC. MGC stimulated with PbAg and GM-CSF in association with the other cytokines had increased fungicidal activity and the presence of GM-CSF also partially inhibited the suppressive effects of IL-10 and TGF-β1. Together, these results suggest that GM-CSF is a positive modulator of PbAg-stimulated MGC generation and on the fungicidal activity against Pb18.     

Regulation of GM-CSF-Induced Dendritic Cell Development by TGF-β1 and Co-Developing Macrophages

Using a culture system of bone marrow progenitor cells with GM-CSF and TGF-β1, a study was performed to analyze the effect of TGF-β1 on the development of dendritic cells (DC) and to elucidate the regulatory role of macrophages co-developing with dendritic cells. The results demonstrate that DC generated in the presence of TGF-β1 were immature with respect to the expression of CD86, nonspecific esterase activity and cell shape. Such inhibitory effects of TGF-β1 were dependent on FcR⁺ macrophages, which were depleted by panning. TGF-β1 did not appear to inhibit the commitment of progenitor cells to the DC lineage. In addition, TGF-β1 also acted directly on the intermediate stage of DC to prevent their over-maturation, which results in a preferential decrease in MHC class II, but not in CD86, in the presence of TNF-α. FcR⁺ suppressive macrophages were also shown to facilitate DC maturation when stimulated via FcR-mediated signals even in the presence of TGF-β1. These results indicate that TGF-β1 indirectly and directly regulate the development of DC and that co-developing macrophages have a regulatory role in DC maturation.     

Role of TGF-β and IL-6 in dendritic cells,Treg and Th17 mediated immune response during experimental cerebral malaria

The role of cytokines in Plasmodium infection have been extensively investigated, but pro and anti inflammatory cytokines mediated imbalance during malaria immune-pathogenesis is still unrevealed. Malaria is associated with the circulating levels of Interleukin-6 (IL-6) and transforming growth factor β (TGF-β), but association between these two cytokines in immune response remains largely obscured. Using mouse model, we proposed that IL-6 and TGF-β are involved in immune regulation of dendritic cells (DC), regulatory T cells (Treg), T-helper cells (Th17) during P. berghei ANKA (PbA) infection. Association between the cytokines and the severity of malaria was established with anti-TGF-β treatment resulting in increased parasitemia and increased immunopathology, whereas; anti-IL-6 treatment delays immunopathology during PbA infection. Further, splenocytes revealed differential alteration of myeloid DC (mDC), plasmocytoid DC (pDC), Treg, Th17 cells following TGF-β and IL-6 neutralization. Interestingly anti-TGF-β reduces CD11c⁺CD8⁺ DC expression, whereas anti-IL-6 administration causes a profound increase during PbA infection in Swiss mice. We observed down regulation of TGF-β, IL-10, NFAT, Foxp3, STAT-5 SMAD-3 and upregulation of IL-6, IL-23, IL-17 and STAT-3 in splenocytes during PbA infection. The STAT activity probably plays differential role in induction of Th17 and Treg cells. Interestingly we found increase in STAT-3 and decrease in STAT-5 expression during PbA infection. This pattern of STAT indicates that possibly TGF-β and IL-6 play a crucial role in differentiation of DCs subsets and Treg/Th17 imbalance during experimental cerebral malaria (ECM).     

Recombinant human bone morphogenetic protein-2 enhances expression of interleukin-6 and transforming growth factor-β1 genes in normal human osteoblast-like cells

The process of recombinant human bone morphogenetic protein-2 (rhBMP-2)-induced endochondral ossification involves (1) the proliferation and differentiation of mesenchymal cells into chondroblasts and osteoblasts; (2) the production and maturation of cartilage and bone matrix; and (3) the differentiation of circulating osteoclast precursor cells into osteoclasts. Currently the molecular mechanisms of these complex sequential events are unknown. It seemed reasonable to us to assume that communication between cells through soluble mediators during bone induction by rhBMP-2 may play an important role in the sequential differentiation of chondroblasts, osteoblasts, and osteoclasts. We have therefore used a human osteoblast-like initial transfectant cell line (HOBIT) to study the effect of rhBMP-2 on gene expression of interleukin-6 (IL-6) and transforming growth factor-β1 (TGF-β1), both of which affect osteogenesis and ostoeclastogenesis. Our results have demonstrated that rhBMP-2 acts on HOBIT cells to stimulate expression of IL-6 and TGF-β1 genes and the production of IL-6. Enhancement of gene expression of IL-6 and TGF-β1 by rhBMP-2 was both sensitive (half maximal effect at approximately 10 ng/ml) and potent (maximum induction was approximately four and threefold greater than controls, respectively). Time course studies showed that the induction of TGF-β1 and IL-6 mRNA occurs within short periods—4 and 8 hours after exposure to rhBMP-2, respectively. Interestingly, these effects, however, were not accompanied by the mitogenic action of rhBMP-2. It suggests that rhBMP-2 enhances IL-6 and TGF-β1 production during osteogenesis and at least in part mediates the complex sequential differentiation of chondroblasts, osteoblasts, and osteoclasts during rhBMP-2-induced endochondral ossification. © 1994 wiley-Liss, Inc.     

Inhibition of the Differentiation of Monocyte-Derived Dendritic Cells by Human Gingival Fibroblasts

We investigated whether gingival fibroblasts (GFs) can modulate the differentiation and/or maturation of monocyte-derived dendritic cells (DCs) and analyzed soluble factors that may be involved in this immune modulation. Experiments were performed using human monocytes in co-culture with human GFs in Transwell® chambers or using monocyte cultures treated with conditioned media (CM) from GFs of four donors. The four CM and supernatants from cell culture were assayed by ELISA for cytokines involved in the differentiation of dendritic cells, such as IL-6, VEGF, TGFβ1, IL-13 and IL-10. The maturation of monocyte-derived DCs induced by LPS in presence of CM was also studied. Cell surface phenotype markers were analyzed by flow cytometry. In co-cultures, GFs inhibited the differentiation of monocyte-derived DCs and the strength of this blockade correlated with the GF/monocyte ratio. Conditioned media from GFs showed similar effects, suggesting the involvement of soluble factors produced by GFs. This inhibition was associated with a lower stimulatory activity in MLR of DCs generated with GFs or its CM. Neutralizing antibodies against IL-6 and VEGF significantly (P<0.05) inhibited the inhibitory effect of CM on the differentiation of monocytes-derived DCs and in a dose dependent manner. Our data suggest that IL-6 is the main factor responsible for the inhibition of DCs differentiation mediated by GFs but that VEGF is also involved and constitutes an additional mechanism.     

Short communication: Concentration of TGF-β1, IL-10 and IL-6 in boar seminal plasma and TGF-β1 level in different fractions of ejaculates

Levels of the cytokines transforming growth factor (TGF)-β1, interleukin (IL)-10 and IL-6 in the boar seminal plasma (SP) as well as TGF-β1 level in different fractions of ejaculate were studied. These cytokines was chosen because of their expected effect on tissue immune response, i.e. suppressive (TGF-β1 and IL-10) and pro-inflammatory (IL-6). Three whole ejaculates from five boars A-E, (n=15) were sampled weekly to evaluate the levels of seminal plasma TGF-β1, IL-10 and IL-6 as well as their fluctuations over time. The effect of different storage temperatures, -20°C or -80°C, on the level of seminal plasma TGF β1 was also tested (three boars, two fractions in one ejaculate). In addition, in 4 different fractions of ejaculates: the pre-sperm-rich (Pre-SRF), first 10 ml of sperm-rich (10SRF), the rest of the sperm-rich fraction (Rest-SRF) and the rest of the ejaculate (RE) fraction, were collected from three boars (A-C) on four different occasions for TGF-β1 evaluation. In the whole ejaculates (n=15), a wide range in the concentration of the cytokines TGF-β1 (20.4 - 766.5 pg/mL) and IL-10, (73.7 - 837.3 pg/mL), was found. For IL-6, the concentration was low (range 11.5 - 30.9 pg/ml) and only detected in four out of 15 collections (from two boars). The mean levels of TGF-β1 and IL-10 between individual boars varied but were not statistical different. The level of TGF-β1 in Pre-SRF, Rest-SRF and RE fractions was significantly lower in boar A than the other boars. A significantly higher concentration of TGF-β1 was found in the 10SRF than in the other fractions. Different storage temperatures (-20°C or -80°C) did not affect the seminal plasma TGF-β1 level after one year of storage. To conclude: Boar seminal plasma contained TGF- β1 and IL-10 but with high individual variation. IL-6 was low or undetectable. The TGF- β1 level was highest in the first 10 mL of the sperm-rich fraction of the ejaculate. Further studies are needed on the role of different levels of cytokine in boar semen on porcine female reproductive tissue, especially for TGF- β1.     

Maturation and Antigen Loading Protocols Influence Activity of Anticancer Dendritic Cells

The practical use of dendritic cell-based vaccines in anticancer therapy is limited by a lack of standards for dendritic cell (DC) generation, as well as standard procedures for controlling their activation and the technique of DC loading with nucleic acids encoding tumor antigens. Analyzing the currently available data, the most promising cocktails for DC maturation were selected and a comparative study of the cocktails and time of maturation on the capacity of DC to activate T-cell immune response has been performed. A study of the expression of surface markers and the production of IL-12, IL-6, and IL-10 cytokines, as well as the efficacy of T-cell activation showed that the use of the standard 7-day maturation protocol is preferable to the 4-day maturation protocol. Cocktails composed of TNF-α, IL-1β, IFN-α, IFN-γ, and poly(I:C), as well as TNF-α, IL-1β, IFN-γ, R848, and PGE2 were shown to be the most efficient activators of DCs. A comparison of the efficacy of different methods of DNA transfection into DCs and RNA delivery using alphavirus vectors demonstrated the superiority of magnet-assisted transfection (MATra) to other protocols.     

TRAF6 mediates IL-1β/LPS-induced suppression of TGF-β signaling through its interaction with the type III TGF-β receptor

Transforming growth factor-β1 (TGF-β1) is an important anti-inflammatory cytokine that modulates and resolves inflammatory responses. Recent studies have demonstrated that inflammation enhances neoplastic risk and potentiates tumor progression. In the evolution of cancer, pro-inflammatory cytokines such as IL-1β must overcome the anti-inflammatory effects of TGF-β to boost pro-inflammatory responses in epithelial cells. Here we show that IL-1β or Lipopolysaccharide (LPS) suppresses TGF-β-induced anti-inflammatory signaling in a NF-κB-independent manner. TRAF6, a key molecule in IL-1β signaling, mediates this suppressive effect through interaction with the type III TGF-β receptor (TβRIII), which is TGF-β-dependent and requires type I TGF-β receptor (TβRI) kinase activity. TβRI phosphorylates TβRIII at residue S829, which promotes the TRAF6/TβRIII interaction and consequent sequestration of TβRIII from the TβRII/TβRI complex. Our data indicate that IL-1β enhances the pro-inflammatory response by suppressing TGF-β signaling through TRAF6-mediated sequestration of TβRIII, which may be an important contributor to the early stages of tumor progression.     

Cyclooxygenase-2-issued prostaglandin e(2) enhances the production of endogenous IL-10, which down-regulates dendritic cell functions

PGE(2) is a well-known immunomodulator produced in the immune response by APCs, such as dendritic cells (DCs), the most potent APC of the immune system. We investigated the PGE(2) biosynthetic capacity of bone marrow-derived DC (BM-DC) and the effects of PG on the APC. We observed that BM-DC produce PGE(2) and other proinflammatory mediators, such as leukotriene B(4) and NO, after LPS exposure. Constitutively present in BM-DC, cyclooxygenase (COX)-1 did not contribute significantly to the total pool of PGE(2) compared with the LPS-induced COX-2-produced PGE(2). Treatment of BM-DC with exogenous PGE(2) induced the production of large amounts of IL-10 and less IL-12p70. In addition, selective inhibition of COX-2, but not COX-1, was followed by significant decrements in PGE(2) and IL-10, a concomitant restoration of IL-12 production, and an enhancement of DC stimulatory potential. In contrast, we found no demonstrable role for leukotriene B(4) or NO. In view of the potential of PGE(2) to stimulate IL-10, we examined the possibility that the suppressive effect of PGE(2) is mediated via IL-10. We found that exogenous IL-10 inhibits IL-12p70 production in the presence of NS-398, a COX-2 selective inhibitor, while the inhibitory effects of PGE(2) were totally reversed by anti-IL-10. We conclude that COX-2-mediated PGE(2) up-regulates IL-10, which down-regulates IL-12 production and the APC function of BM-DC.     

Inhibition of IL-6, TNF-alpha, and cyclooxygenase-2 protein expression by prostaglandin E2-induced IL-10 in bone marrow-derived dendritic cells

Several endogenously produced mediators, including cytokines such as IL-6, IL-10, and TNF-alpha and prostanoids such as prostaglandin E(2) (PGE(2)), regulate dendritic cell (DC) function and contribute to immune homeostasis. In this study, we report that exogenous PGE(2) enhances the production of IL-10 from bone marrow-derived DC (BM-DC). IL-6, but not TNF-alpha, release is enhanced by PGE(2) in the presence of anti-IL-10, suggesting that endogenous IL-10 masks PGE(2)-induced IL-6. Furthermore, both exogenous IL-10 and PGE(2) inhibit LPS-induced IL-6 and TNF-alpha, whereas selective inhibition of cyclooxygenase-2 (COX-2) or addition of anti-IL-10 causes the reverse effects. Exogenous IL-10, but not IL-6, dose-dependently suppresses COX-2 protein expression and PGE(2) production, and TNF-alpha does not reverse this effect. In contrast, anti-IL-10 up-regulates prostanoid production by LPS-stimulated BM-DC. Taken together, our results show that in response to PGE(2), BM-DC produce IL-10, which in turn down-regulates their own production of IL-6-, TNF-alpha-, and COX-2-derived prostanoids, and plays crucial roles in determining the BM-DC pro-inflammatory phenotype.     

The Src-like adaptor protein regulates GM-CSFR signaling and monocytic dendritic cell maturation

GM-CSF is an important cytokine involved in myeloid differentiation and inflammatory processes. Signaling through the GM-CSFR also plays a critical role in the generation of monocyte-derived dendritic cells (DC). In this article, we report that the Src-like adaptor protein (SLAP) functions as a negative regulator of the GM-CSFR. In bone marrow-derived DC (BM-DC) lacking SLAP and the closely related SLAP2, downregulation of GM-CSFRβ is impaired, leading to enhanced phosphorylation of Jak2 and prolonged activation of Akt and Erk1/2 in response to GM-CSF stimulation. Compared with wild-type bone marrow, SLAP/SLAP2(-/-) bone marrow gave rise to similar numbers of CD11c(+) and CD11b(+) DC, but SLAP/SLAP2(-/-) BM-DC failed to acquire high levels of MHC class II, CD80, and CD86, indicating an impairment in maturation. Furthermore, MHC class II expression in SLAP/SLAP2(-/-) BM-DC was rescued by decreasing GM-CSF concentration, suggesting that enhanced GM-CSF signaling mediates the block in maturation. In addition, SLAP/SLAP2(-/-) BM-DC produced less IL-12 and TNF-α in response to LPS compared with controls and failed to stimulate T cells in an MLR. Ag-specific T cell activation assays showed that SLAP/SLAP2(-/-) BM-DC were less robust at inducing IFN-γ secretion by DO11.10 T cells. These results indicated that SLAP-mediated GM-CSFR regulation is important for the generation of functionally mature monocytic DC.     

TGF-β is necessary for induction of IL-23R and Th17 differentiation by IL-6 and IL-23

TGF-β and IL-6 induce Th17 differentiation, and IL-23 is required for expansion and maintenance of Th17 cells. Recently, it was shown that IL-6 up-regulates IL-23R mRNA in naive CD4⁺ T cells and therefore IL-6 and IL-23 synergistically promote Th17 differentiation. However, the molecular mechanism whereby IL-6 and IL-23 induce Th17 differentiation and the relevance to TGF-β remain unknown. Here, we found that IL-6 up-regulated IL-23R mRNA expression, and IL-6 and IL-23 synergistically augmented its protein expression. The combination induced Th17 differentiation, and TGF-β1 further enhanced it. IL-6 augmented endogenous TGF-β1 mRNA expression, whereas the amount of TGF-β produced was not enough to induce Th17 differentiation by IL-6 alone. However, unexpectedly, the up-regulation of IL-23R and induction of Th17 differentiation by IL-6 and IL-23 were almost completely inhibited by anti-TGF-β. These results suggest that the induction of IL-23R and Th17 differentiation by IL-6 and IL-23 is mediated through endogenously produced TGF-β.     

IL-1β and TGF-β act antagonistically in induction and differentially in propagation of human proinflammatory precursor CD4+ T cells

Cytokines are critical messengers that control the differentiation of Th cells. To evaluate their impact on the fate of human naive CD4(+) T cells from cord and adult blood, early T cell differentiation was monitored after T cell activation in the presence of pro- and anti-inflammatory cytokines. Interestingly, the analysis of Th cell lineage-specific molecules revealed that IL-1β on its own mediates differentiation of Th cells that secrete a wide range of proinflammatory cytokines and stably express CD69, STAT1, IFN-γ, and IL-17. Notably, our data suggest that IL-1β induces Th17 cells independent of RORC upregulation. In contrast, TGF-β that triggers RORC prevents Th17 cell development. This suppressive function of TGF-β is characterized by inhibition of STAT1, STAT3, and CD69. However, after repeated anti-CD3 and anti-CD28 stimulation, we observe that TGF-β provokes an increase in Th17 cells that presumably relies on reactivation of a default pathway by preferential inhibition of IFN-γ. Hence, our data extend the view that the principal cytokines for determining Th cell fate are IL-12 for the Th1 lineage, IL-4 for the Th2 lineage, and TGF-β in conjunction with IL-6 for the Th17 lineage. We propose that IL-1β induces a general proinflammatory Th cell precursor that, in the presence of the lineage-specifying cytokines, further differentiates into one of the specific Th cell subpopulations.