Dysregulated cytokine expression and signaling are major contributors to a number of autoimmune diseases. Interleukin-17A (IL-17A) and IL-6 are important in many disorders characterized by immune self-recognition, and IL-6 is known to induce the differentiation of T helper 17 (Th17) cells. Here we described an IL-17A-triggered positive-feedback loop of IL-6 signaling, which involved the activation of the transcription factors nuclear factor (NF)-kappaB and signal transducer and activator of transcription 3 (STAT3) in fibroblasts. Importantly, enhancement of this loop caused by disruption of suppressor of cytokine signaling 3 (SOCS3)-dependent negative regulation of the IL-6 signal transducer gp130 contributed to the development of arthritis. Because this mechanism also enhanced experimental autoimmune encephalomyelitis (EAE) in wild-type mice, it may be a general etiologic process underlying other Th17 cell-mediated autoimmune diseases.

IL-17-producing Th (Th17) comprise a distinct lineage of pro-inflammatory Th that are major contributors to autoimmune diseases. Treatment with IL-6 and transforming growth factor beta (TGFbeta) induces naive CD4+ T cells to generate Th17, which also requires expression of the IL-6/TGFbeta target RORgammat. We reported that IL-6 transduces two signaling pathways via tyrosine redidues of the signal transducer gp130: one depends on signal transducers and activators of transcription (STAT)-3 activation and the other on Src homology region 2 domain-containing phosphatase 2 (SHP2)/Grb2 associated binder (Gab)/mitogen-activated protein kinase (MAPK) activation. Here, we showed that CD4+ T cells carrying a mutant gp130 that transduces the SHP2/Gab/MAPK pathway but not the STAT3-mediated one failed to develop into Th17, while CD4+ T cells whose mutant gp130 transduces the STAT3 signal only generated Th17, indicating that IL-6 acts directly on T cells through the tyrosine residues of gp130 required for STAT3 activation to promote the development of Th17. Moreover, we found that gp130-STAT3 pathway is essential for Th17 development and for the expression of RORgammat by using T cells specifically lacking gp130 and STAT3. Noteworthy is that the regulatory T cell (Treg) percentages and numbers were comparable between all mutant mice we tested in vivo, although we showed that IL-6-gp130-STAT3 pathway suppressed Treg development in vitro. Thus, we conclude that IL-6 acts directly to promote the development of Th17 by activating the T cell gp130-STAT3 pathway but has a minimum effect on Treg development at least in the steady state in vivo. Therefore, blockade of IL-6-gp130-STAT3 pathway in CD4+ T cells could be a good target for controlling unwanted Th17-mediated immune responses including autoimmune diseases.

Mice homozygous for the F759 mutation in the gp130 interleukin (IL)-6 receptor subunit have enhanced gp130-mediated signal transducer and activator of transcription (STAT)3 activation and spontaneously developed a lymphocyte-mediated rheumatoid arthritis-like joint disease. Here, we show that the development of the disease is dependent on both major histocompatibility complex (MHC) II–restricted CD4+ T cells and IL-6 family cytokines. In spite of the necessity for CD4+ T cells, the gp130 mutation was only required in nonhemtopoietic cells for the disease. The gp130 mutation resulted in enhanced production of IL-7. Conditional knockout of STAT3 in nonlymphoid cells showed that the enhancement of IL-7 production was dependent on STAT3 activation by IL-6 family cytokines. Homeostatic proliferation of CD4+ T cells was enhanced in gp130 mutant mice and acceleration of homeostatic proliferation enhanced the disease, whereas the inhibition of homeostatic proliferation suppressed the disease. Anti–IL-7 antibody treatment inhibited not only the enhanced homeostatic proliferation, but also the disease in gp130 mutant mice. Thus, our results show that autoimmune disease in gp130 mutant mice is caused by increased homeostatic proliferation of CD4+ T cells, which is due to elevated production of IL-7 by nonhematopoietic cells as a result of IL-6 family cytokine-gp130-STAT3 signaling.

Crystal structures of L-2-haloacid dehalogenase from Pseudomonas sp. YL complexed with monochloroacetate, L-2-chlorobutyrate, L-2-chloro-3-methylbutyrate, or L-2-chloro-4-methylvalerate were determined at 1.83-, 2.0-, 2.2-, and 2.2-Å resolutions, respectively, using the complex crystals prepared with the S175A mutant, which are isomorphous with those of the wild-type enzyme. These structures exhibit unique structural features that correspond to those of the reaction intermediates. In each case, the nucleophile Asp-10 is esterified with the dechlorinated moiety of the substrate. The substrate moieties in all but the monochloroacetate intermediate have a D-configuration at the C 2 atom. The overall polypeptide fold of each of the intermediates is similar to that of the wild-type enzyme. However, it is clear that the Asp-10 -Ser-20 region moves to the active site in all of the intermediates, and the Tyr-91-Asp-102 and Leu-117-Arg-135 regions make conformational changes in all but the monochloroacetate intermediates. Ser-118 is located near the carboxyl group of the substrate moiety; this residue probably serves as a binding residue for the substrate carboxyl group. The hydrophobic pocket, which is primarily composed of the Tyr-12, Gln-42, Leu-45, Phe-60, Lys-151, Asn-177, and Trp-179 side chains, exists around the alkyl group of the substrate moiety. This pocket may play an important role in stabilizing the alkyl group of the substrate moiety through hydrophobic interactions, and may also play a role in determining the stereospecificity of the enzyme. Moreover, a water molecule, which is absent in the substrate-free enzyme, is present in the vicinities of the carboxyl carbon of Asp-10 and the side chains of Asp-180, Asn-177, and Ala-175 in each intermediate. This water molecule may hydrolyze the ester intermediate and its substrate. These findings crystallographically demonstrate that the enzyme reaction proceeds through the formation of an ester intermediate with the enzyme's nucleophile Asp-10.1 is a unique enzyme that catalyzes the hydrolytic dehalogenation of L-2-haloacids to produce the corresponding D-2-hydroxyacids with an inversion of the C 2 -configuration. Various L-DEXs exhibiting very similar sequences have been isolated from different bacterial sources. Of all the enzymes, L-DEX YL, isolated from a 2-chloroacrylate-utilizable bacterium, Pseudomonas sp. YL, is an unusual L-DEX, in that it is relatively thermostable even though it is derived from a mesophilic bacterium (1). It is a dimeric enzyme formed by two identical subunits of 232 amino acid residues. We have already reported the crystallization of L-DEX YL (2) and its crystal structure determined by a 2.5-Å resolution x-ray analysis (3). The enzyme has a core domain of ␣/␤-structure, which differs topologically from those of the ␣/␤ hydrolase fold family proteins (4), along with a subdomain having a four-helix-bundle structure. Our ion spray mass spectrometric study showed that the dehalogenation of the L-2-haloacid catalyzed by L-DEX YL proceeds i...