Summary of Research Achievements

The immune system plays a pivotal role in the defense reaction against infectious microorganisms. A variety of cytokines, such as interleukin 6(IL-6) are critically involved in the regulation of the growth and differentiation of lymphocytes in the immune system. Furthermore, cytokines are involved in hematopoiesis, nervous system, endocrine system and bone metabolism, which are important to maintain the homeostasis.

We found that pleural effusion cells of the patients with tuberculous pleurisy produce a large amount of soluble factors capable of inducing immunoglobulin production in B lymphocytes in Osaka Prefectural Habikino Hospital in the late 1970 (J. Immunol. 126:517-522, 1981). Furthermore, we found that the same culture supernatant of the pleural effusion cells contains an active factor having the same biological activity and physicochemical properties of a cytokine, which is now called IL-6 (J. Immunol. 128:1903-1908, 1982). We continued purification and characterization of IL-6 in Kumamoto University (J. Immunol. 132:229-234, 1984, J. Immunol. 133:798-802, 1984.) and finally succeeded on the molecular cloning of human IL-6 in Osaka University in 1986 (Nature 324:73-76, 1986). Since then, it has been shown that IL-6 is a multifunctional cytokine acting in the immune response, acute phase response, nervous system, endocrine system and bone metabolisms. Furthermore, we found that a large amount of IL-6 is present in the synovial fluids of patients with rheumatoid arthritis (RA), first suggesting the involvement of IL-6 in RA (Eur. J. Immunol. 18:1797-1801, 1988) and IL-6 is a growth factor of myeloma (Nature 332:83-85, 1988.) (see reviews Immunology Today, 11, 443-449, 1990, Intern. Rev. Immunol., 16:249-284, 1998 ). To understand the molecular mechanisms of IL-6 action, we cloned IL-6 receptor alpha chain and gp130 (Science 241:825-828, 1988., Cell 58:573-581, 1989, Cell 63:1149-1157, 1990.). Furthermore, we established IL-6 induces two major signal transduction pathways, the Stat3 signal and the SHP2/Gab/MAPK signal in a manner dependent on the YxxQ motif and the Y759 of gp130, respectively (EMBO J. 15:1557, 1996, EMBO J. 15, 3651-3658, 1996, Immunity, 5:449, 1996, EMBO J, 16, 6670-6677, 1998, Mol. Cell Biol18, 4109-4117, 1998, Immunity, 11, 709-719, 1999, Blood, 93, 1806-1816, 1999, see reviews, Cytokine Growth Factor Reviews, 8, 241-252, 1997 and Oncogene, 19: 2548, 2000). In order to clarify the in vivo roles of each of the two signals of gp130, we generated a series of knock-in mouse lines in which the gp130-mediated SHP2 or Stat3 signal is selectively disrupted (Immunity, 12, 95, 2000). To make the SHP2 signal-deficient mice (gp130F759/F759), we mutated Y759 of gp130 to phenylalanine (F759) . The results indicate that the Y759-dependent signals negatively regulate the biological responses elicited by the Stat3-mediated signals in vivo. The most intriguing finding is that the gp130F759/F759 mice develop RA-like joint disease accompanied by autoantibody production and accumulated memory/activated T cells (J. Exp Med. 196: 979, 2002). This is the first definitive evidence showing that IL-6 is critically involved in spontaneous autoimmune disease and showed that abnormal cytokine signal can induce autoimmune disease like RA. Hirano is actually one of discoverers of IL-6 and during the last 30 years, he and his colleagues have very much contributed to clarification of the molecular mechanisms of IL-6 action in vitro and in vivo. Based on a large amount of a series of studies, we finally showed the disturbance of IL-6 signal is actually involved in the process of RA-like disease. Then, we have tried to clarify molecular basis of the RA-like joint disease in F759 mice. Studies using bone-marrow transplantation and various knock-out strains, we demonstrated that the arthritis is CD4 T cell-dependent, and interestingly, the gp130 F759 mutation is necessary in cells of non-hematopoietic origin. In response to IL-6 stimulation, these non-hematopoietic cells from F759 mice produce a large amount of T-cell survival factor, IL-7, leading to the activation of CD4 T cells by homeostatic proliferation. This homeostatic proliferation of CD4 T cells is important for the development of the RA-like disease (J Exp Med. 203:1459, 2006). Thus we proposed a model where the interaction between non-hematopoietic tissues and immune system plays critical role in causing autoimmune disease. It is recently found that IL-6 together with TGFbeta induces Th17, which has been considered to play a pivotal role in causing autoimmune diseases and inflammation, indicating that IL-6 is located upstream of IL-17. In fact, we showed gp130 and STAT3 in T cells is essential for Th17 development (Int Immunol. 19: 695, 2007). We found that IL-6 is not only an IL-17 inducing factor, but also a target gene of IL-17 in non-hematopoietic tissues including fibroblast cells. Importantly, Il-6 is a critical downstream target gene of IL-17 for autoimmune arthritis in F759 mice. Intriguing finding we made is that IL-17-induced IL-6 gene expression through NF-kB activation is augmented in the presence of IL-6. This synergistic induction of IL-6 is mediated through an interaction between NF-kB and STAT3. More importantly, this IL-6 positive feedback loop in type1 collagen positive tissues is required for autoimmune arthritis in F759 mice. IL-6 can induce Th17 cells and therefore once IL-6 positive feedback loop is initiated in type1 collagen positive non-hematopoietic tissues, enhanced IL-6 production is resulted in the enhanced Th17 development, giving rise to further enhanced production of IL-6. We named this positive loop as an IL-6 amplifier, This IL-6 amplifier system is activated and enhanced in F759 mice where IL-6-mediated negative feedback through SOCS3 is defective. Therefore, we concluded that any event, including virus infection capable of activating IL-6 amplifier through either STAT3 activation or NF-kB activation or both plays critical role in causing autoimmune diseases. This scenario could be applied in general autoimmune model such as experimental autoimmune encephalomyelitis (EAE), because we showed that MOG-specific Th17-induced EAE is dependent on STAT3, which is a central molecule of IL-6 amplifier in type 1 collagen expressing tissue.(Immunity. 29: 628, 2008).

The regulation of cell movement plays critical roles in early embryonic development and the immune responses. It is suggested that gp130/JAK/STAT3 signal regulates cell movement. Spemann's organizer can specify the fate of the surrounding tissue and initiate highly integrated cell movements during gastrulation. Although the cell movement that occurs during gastrulation is well understood, little is known of the molecular mechanisms that initiate and coordinate this process. We showed that mouse and zebrafish STAT3 is required for the correct morphogenetic movements during gastrulation (Dev. Cell. 2: 363-375, 2002). To clarify the molecular mechanisms by which gp130/JAK/STAT3 signal transduction pathway regulates cell movement, we have recently identified Liv1/Zip6, a zinc transporter, as one of target genes of Stat3 and showed that Liv1/Zip6 is critically involved in cell movement of zebrafish gastrulation (Nature. 429: 298, 2004). The finding of the link between gp130/STAT3 signaling and zinc transporter led us to focus on a role for zinc in immunity. Consequently and also serendipitously, we opened a door for a new field of research, the zinc signaling (Adv Immunol. 97: 149, 2008). Zinc is important for the immune system, including dendritic cell maturation (Nat Immunol. 7: 971, 2006) and mast cell degranulation (J Immunol. 177: 1296, 2006 and J Cell Biol. 177: 637, 2007). In addition, the zinc system is involved in more variety of biological processes than we initially thought. By generating mice strains lacking zinc transporters, Zip13 we found that Zip13 is required for connective tissue development. Furthermore, Zip13 is required for TGFbeta/BMP signaling (PLoS ONE. 3: e3642, 2008). These results showed that extracellular stimuli could dynamically modulate intracellular status of Zinc and its concentration and its distribution through the change of expression profile of zinc transporters, affecting intracellular signaling molecules, such as Snail, Smad and tyrosine phosphatase so on (Nature. 429: 298, 2004; Nat Immunol. 7:971, 2006; J. Cell Biol., 177:637, 2007 and PLoS ONE 3:e3642, 2008). Based on these facts, we proposed that zinc acts as an intracellular signaling molecule. In addition, we observed that intracellular zinc concentration is rapidly changed in mast cells after FceR stimulation independently on the change of zinc transporter expression in several minutes after the stimulation. We named this phenomenon as “Zinc wave”(J. Cell Biol., 177:637, 2007). Since the change of intracellular zinc concentration or zinc distribution through the change of expression profile of zinc transporter is occurred several hours after the extracellular stimulation, we named this type zinc signaling as “Late zinc signaling” and the former one, Zinc wave type as “Early zinc signaling” (Adv. Immunol. 97: 149, 2008).

These studies on IL-6 and its signal transduction pathways have provided the molecular basis for the cytokine system in general. We also showed that zinc is not only an essential nutrient as a component of our body, but also it plays a role as an intracellular signaling molecule. This should open the new research area, “Zinc signaling”. Thus our studies have greatly contributed to the understanding of the molecular mechanisms of not only an immune system but also a variety of biological reactions and their disorders in general. In particular, our pioneering work to isolate interleukin 6, determine it properties and explore its role in the onset in inflammatory diseases, such as arthritis paved the way for the development of a new drug for rheumatoid arthritis and related inflammatory diseases.

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Summary of Research Achievements