Dendritic cells (DCs) orchestrate immune responses according to their state of maturation. In response to infection, DCs differentiate into mature cells that initiate immune responses, while in the absence of infection, most of them remain in an immature form that induces tolerance to self Ags. Understanding what controls these opposing effects is an important goal for vaccine development and prevention of unwanted immune responses. A crucial question is what cytokine(s) regulates DC maturation in the absence of infection. In this study, we show that IL-6 plays a major role in maintaining immature DCs. IL-6 knockout (KO) mice had increased numbers of mature DCs, indicating that IL-6 blocks DC maturation in vivo. We examined this effect further in knockin mice expressing mutant versions of the IL-6 signal transducer gp130, with defective signaling through either Src homology region 2 domain-containing phosphatase 2/Gab/MAPK (gp130F759/F759) or STAT3 (gp130FxxQ/FxxQ), and combined gp130 and IL-6 defects (gp130F759/F759/IL-6 KO mice). Importantly, we found STAT3 activation by IL-6 was required for the suppression of LPS-induced DC maturation. In addition, STAT3 phosphorylation in DCs was regulated by IL-6 in vivo, and STAT3 was necessary for the IL-6 suppression of bone marrow-derived DC activation/maturation. DC-mediated T cell activation was enhanced in IL-6 KO mice and suppressed in gp130F759/F759 mice. IL-6 is thus a potent regulator of DC differentiation in vivo, and IL-6-gp130-STAT3 signaling in DCs may represent a critical target for controlling T cell-mediated immune responses in vivo.

We found IL-6-STAT3 pathway suppresses MHC class II (MHCII) expression on dendritic cells (DCs) and attenuates T cell activation. Here, we showed that IL-6-STAT3 signaling reduced intracellular MHCII alphabeta dimmer, Ii, and H2-DM levels in DCs. IL-6-mediated STAT3 activation decreased cystatin C level, an endogenous inhibitor of cathepsins, and enhanced cathepsin activities. Importantly, cathepsin S inhibitors blocked reduction of MHCII alphabeta dimer, Ii, and H2-DM in the IL-6-treated DCs. Overexpression of cystatin C suppressed IL-6-STAT3-mediated increase of cathepsin S activity and reduction of MHCII alphabeta dimer, Ii, and H2-DM levels in DCs. Cathepsin S overexpression in DCs decreased intracellular MHCII alphabeta dimer, Ii, and H2-DM levels, LPS-mediated surface expression of MHCII and suppressed CD4(+) T cell activation. IL-6-gp130-STAT3 signaling in vivo decreased cystatin C expression and MHCII alphabeta dimer level in DCs. Thus, IL-6-STAT3-mediated increase of cathepsin S activity reduces the MHCII alphabeta dimer, Ii, and H2-DM levels in DCs, and suppresses CD4(+) T cell-mediated immune responses.

Mast cells are thought to participate in a variety of immune responses, such as parasite resistance and the allergic reaction. Mast cell development depends on stem cell factor (Kit ligand) and its receptor, c-Kit. Gab2 is an adaptor molecule containing a pleckstrin homology domain and potential binding sites for SH2 and SH3 domains. Gab2 is phosphorylated on tyrosine after stimulation with cytokines and growth factors, including KitL. Gab2-deficient mice were created to define the physiological requirement for Gab2 in KitL/c-Kit signaling and mast cell development. In Gab2-deficient mice, the number of mast cells was reduced markedly in the stomach and less severely in the skin. Bone marrow-derived mast cells (BM-MCs IntroductionMast cells are hematopoietic-lineage cells that participate in immunoglobulin (Ig)E-associated immune responses, including allergic reactions and parasite resistance (see Galli 1 for a review). It was recently shown that mast cells also participate in the innate immunity to bacterial infection, in which IgE may not be involved. 2 Genetic evidence indicates that Kit ligand (KitL) and its receptor, c-Kit, play essential roles in mast cell development. Mutations in the mouse Kit ligand and c-Kit genes (Steel and White spotting) lead to defects in the development of melanocytes, germ cells, erythroid cells, basophils, and mast cells. 3-5 c-Kit is a receptor-type tyrosine kinase that displays some homology with platelet-derived growth factor receptors. The binding of KitL to c-Kit induces the dimerization and transphosphorylation of c-Kit. Tyrosyl-phosphorylated c-Kit recruits signaling molecules containing the Src homology 2 (SH2) domain, such as phosphatidyl inositol (PI)-3 kinase, 6 phospholipase C␥1, 7,8 Grb2, and the Src kinase, 9 to c-Kit and initiates cytoplasmic signaling. In addition to KitL/c-Kit signaling, interleukin (IL)-3 is also involved in mast cell development. IL-3-deficient mice maintain a basal level of mast cells, whereas mast cells fail to expand in response to infection by the nematode Stronglyoides venezuelensis. 10 Double-mutant Kit W /Kit W-v , IL-3 Ϫ/Ϫ mice display a more severe reduction in mast cell and basophil expansion elicited by the nematode infection than do single-mutant mice. 10 The result suggests that IL-3 is not essential for the generation of mast cells in a resting state but that it is required for the increase in mast cells in the immune response elicited by parasites. On the other hand, KitL-mediated signals are required for the development of the basal level of mast cells.Gab2 is a member of the Gab/DOS family of adapter molecules, which contain a pleckstrin homology (PH) domain and potential binding sites for the SH2 and SH3 domains. [11][12][13][14] Gab2 is tyrosine phosphorylated on stimulation by growth factors, cytokines, and Tand B-cell antigen receptors, including KitL and IL-3, and phosphorylated Gab2 binds SHP-2 and p85 PI-3 kinase. 11,15,16 Overexpression of Gab2 enhances the activation of cytokine-dependent ERK mitogen-activated protei...

Rheumatoid arthritis (RA) is a polygenic autoimmune disease. The autoimmunity develops from synergistic actions of genetic and environmental factors. We generated a double-mutant mouse by crossing two murine models of RA, a gp130 mutant knock-in mouse (gp130F759/F759) and an HTLV-1 pX transgenic mouse (pX-Tg), in a C57BL/6 background, which is resistant to arthritis. The mice spontaneously developed severe arthritis with a much earlier onset than the gp130F759/F759 mice and with a much higher incidence than did the pX-Tg mice. The symptoms of gp130F759/F759 mice, including lymphoadenopathy, splenomegaly, hyper-gamma-globulinemia, autoantibody production, increases in memory/activated T cells and granulocytes in the peripheral lymphoid organs, and a decrease in the class II MHC(bright) CD11c+ population, were augmented in the double mutants. Marked reductions in incidence, severity and immunological abnormalities were seen in the triple mutant, IL-6-/-/gp130F759/F759/pX-Tg, indicating that the arthritis in the double mutant is IL-6 dependent. gp130F759/F759/pX-Tg is a unique mouse model for RA.

Grb2-associated binder 1 (Gab1) is a member of the Gab/daughter of sevenless family of adapter molecules involved in the signal transduction pathways of a variety of growth factors, cytokines, and Ag receptors. To know the role for Gab1 in hematopoiesis and immune responses in vivo, we analyzed radiation chimeras reconstituted with fetal liver (FL) cells of Gab1−/− mice, because Gab1−/− mice are lethal to embryos. Transfer of Gab1−/− FL cells of 14.5 days post-coitum rescued lethally irradiated mice, indicating that Gab1 is not essential for hematopoiesis. Although mature T and B cell subsets developed normally in the peripheral lymphoid organs, reduction of pre-B cells and increase of myeloid cells in the Gab1−/− FL chimeras suggested the regulatory roles for Gab1 in hematopoiesis. The chimera showed augmented IgM and IgG1 production to thymus-independent (TI)-2 Ag, although they showed normal responses for thymus-dependent and TI-1 Ags, indicating its negative role specific to TI-2 response. Gab1−/− splenic B cells stimulated with anti-δ-dextran plus IL-4 plus IL-5 showed augmented IgM and IgG1 production in vitro that was corrected by the retrovirus-mediated transfection of the wild-type Gab1 gene, clearly demonstrating the cell-autonomous, negative role of Gab1. Furthermore, we showed that the negative role of Gab1 required its Src homology 2-containing tyrosine phosphatase-2 binding sites. Cell fractionation analysis revealed that nonfollicular B cells were responsible for the augmented Ab production in vitro. Consistent with these results, the Gab1 gene was expressed in marginal zone B cells but not follicular B cells. These results indicated that Gab1 is a unique negative regulator specific for TI-2 responses.