IL-6/IL-6 receptor system and its role in physiological and pathological conditions.

IL (interleukin)-6, which was originally identified as a B-cell differentiation factor, is a multifunctional cytokine that regulates the immune response, haemopoiesis, the acute phase response and inflammation. IL-6 is produced by various types of cell and influences various cell types, and has multiple biological activities through its unique receptor system. IL-6 exerts its biological activities through two molecules: IL-6R (IL-6 receptor) and gp130. When IL-6 binds to mIL-6R (membrane-bound form of IL-6R), homodimerization of gp130 is induced and a high-affinity functional receptor complex of IL-6, IL-6R and gp130 is formed. Interestingly, sIL-6R (soluble form of IL-6R) also binds with IL-6, and the IL-6-sIL-6R complex can then form a complex with gp130. The homodimerization of receptor complex activates JAKs (Janus kinases) that then phosphorylate tyrosine residues in the cytoplasmic domain of gp130. The gp130-mediated JAK activation by IL-6 triggers two main signalling pathways: the gp130 Tyr759-derived SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase-2)/ERK (extracellular-signal-regulated kinase) MAPK (mitogen-activated protein kinase) pathway and the gp130 YXXQ-mediated JAK/STAT (signal transducer and activator of transcription) pathway. Increased IL-6 levels are observed in several human inflammatory diseases, such as rheumatoid arthritis, Castleman's disease and systemic juvenile idiopathic arthritis. IL-6 is also critically involved in experimentally induced autoimmune diseases. All clinical findings and animal models suggest that IL-6 plays a number of critical roles in the pathogenesis of autoimmune diseases. In the present review, we first summarize the IL-6/IL-6R system and IL-6 signal transduction, and then go on to discuss the physiological and pathological roles of IL-6.

IL-6 and IL-1 synergistically enhanced the production of MMPs from synovial cells by up-regulating IL-6 production and IL-1 receptor I expression.

In the present study, we investigated potential synergism between IL-6 and IL-1 for the production of matrix metalloproteinases (MMPs) by the synovial cell line SW982. Cells were cultured with different combinations of IL-6, soluble IL-6 receptor (sIL-6R) and IL-1beta for 24h and production of MMPs was then measured. IL-6+sIL-6R, but not IL-6 alone, induced MMP-13 and MMP-3 production. IL-1beta also induced production of MMPs. Of interest, addition of IL-6+sIL-6R together with IL-1beta synergistically increased MMP production. Next, we analyzed the mechanism responsible for the synergistic effects of IL-6+sIL-6R and IL-1beta in combination. IL-1beta-induced MMP production was significantly augmented in the presence of sIL-6R. IL-1beta as well as IL-6+sIL-6R induced IL-6 production. Moreover, IL-6+sIL-6R significantly augmented expression of IL-1RI, but not IL-1RII, in SW982 cells. Responsiveness to IL-1beta was much higher in IL-6+sIL-6R-pretreated cells than non-treated cells in terms of MMP production. Finally, IL-6+sIL-6R-induced IL-1RI expression was inhibited by a STAT pathway inhibitor, but not a MAPK pathway inhibitor. These results suggest that increased expression of IL-1RI stimulated by IL-6+sIL-6R and the increased production of IL-6 on exposure to IL-1beta and IL-6+sIL-6R are involved in the observed synergistic effect on the production of MMPs by SW982 cells.

Development of human-human hybridoma from anti-Her-2 peptide-producing B cells in immunized NOG mouse.

OBJECTIVE: Numerous monoclonal antibodies have been developed for the purpose of medical treatments, including cancer treatment. For clinical application, the most useful are human-derived antibodies. In this study, we tried to prepare designed antigen-specific antibodies of completely human origin using immunodeficient mouse. METHODS: Nonobese diabetic/severe combined immunodeficient/IL-2 receptor gamma null mouse (NOG) mouse was used to reconstitute the human immune system with umbilical cord blood hematopoietic stem cells (CB-NOG mouse) and to prepare human-derived Her-2-epitope-specific antibodies. Hybridoma lines were prepared by fusing the human myeloma cell line Karpas707H. RESULTS: Serum of immunized NOG mouse contained human-derived immunoglobulin M (IgM) antibodies specific for a short peptide sequence of 20 amino acids, including the epitope peptide of apoptotic Her-2 antibody CH401. Hybridoma lines were successfully prepared with spleen B cells obtained from the immunized CB-NOG mouse. One of these cell lines produced human IgM against the epitope peptide that can recognize surface Her-2 molecule. CONCLUSION: We could produce human-derived IgM antibody against Her-2 epitope peptide in CB-NOG mouse, succeeding in generation of human hybridoma-secreting IgM against a given peptide.

Anti-interleukin 6 receptor antibody inhibits murine AA-amyloidosis.

OBJECTIVE: AA-amyloidosis is a severe complication in chronic inflammatory diseases. AA-amyloidosis is caused by the deposition of insoluble fibrils containing AA amyloid protein derived from serum amyloid A (SAA), which is synthesized by inflammatory cytokine stimulation. We examined whether anti-interleukin 6 receptor (IL-6R) antibody prevented the development of AA-amyloidosis in mouse models. METHODS: A transient model was induced by the injection of amyloid enhancing factor (AEF) and adjuvant treatment in C57BL/6 mice. Monoclonal IgG1 antibody, MR16-1, was injected intraperitoneally just once before the injection of AEF and adjuvant. After 2 and 5 weeks, mice were sacrificed and histologically examined. In contrast, a chronic model was induced by AEF injection into IL-6 transgenic mice. One week later, in order to avoid neutralizing antibody production, MR16-1 (200 mg/kg) was injected intravenously. MR16-1 (5 mg/kg) was injected subcutaneously twice a week from the next week. Three and 6 weeks after AEF injection, mice were sacrificed and histologically examined. RESULTS: In the transient model, amyloid deposition was observed in the spleen, liver, and kidney as early as 2 weeks after treatment. MR16-1 completely prevented amyloid deposition. Although IL-6 production was not suppressed, SAA production was significantly suppressed. In the chronic model, substantial amyloid deposition was seen in multiple organs and tissues as well as the spleen, liver, and kidney. MR16-1 suppressed amyloid deposition in many organs, even when injected one week after AEF injection; it showed a tendency to decrease SAA and IL-6 levels were decreased. CONCLUSION: IL-6 is a key cytokine for the induction of AA-amyloidosis, and anti-IL-6R therapy appears promising for the treatment of AA-amyloidosis.

Functional CD5+ B cells develop predominantly in the spleen of NOD/SCID/gammac(null) (NOG) mice transplanted either with human umbilical cord blood, bone marrow, or mobilized peripheral blood CD34+ cells.

OBJECTIVE: Human CD5+ B cells are the major B cell subset in fetal spleen and umbilical cord blood (CB), and their number gradually diminishes in both spleen and peripheral blood from infancy through childhood while conventional B cells increase. In this study, we investigated whether CD5+ cells differentiate from adult hematopoietic stem cells (HSCs) as well as fetal ones in immunodeficient mice. METHODS: In our system, NOD/SCID/gammac(null) (NOG) mice were transplanted with CD34+ cells from CB (hCB model), adult bone marrow (hBM model), and mobilized peripheral blood (hMPB model). RESULTS: In these model mice, a high proportion of CD19+IgM+CD5+ mature B cells appeared in the spleen, regardless of the CD34+ cell origin, 4 to 8 weeks after transplantation, while the majority were CD19+IgM-CD5- immature B cells in BM. The CD19+CD5- BM cells showed to express CD5 after the coculture with NOG spleen cells. In the sera of immunized hCB model mice with DNP-KLH, antigen-specific IgM but not IgG was enhanced. CONCLUSION: Our results show that adult CD34+ cells develop into functional CD5+ B cells in NOG spleen as much as fetal CD34+ cells do.

A resorcylic acid lactone, 5Z-7-oxozeaenol, prevents inflammation by inhibiting the catalytic activity of TAK1 MAPK kinase kinase.

TAK1, a member of the mitogen-activated kinase kinase kinase (MAPKKK) family, participates in proinflammatory cellular signaling pathways by activating JNK/p38 MAPKs and NF-kappaB. To identify drugs that prevent inflammation, we screened inhibitors of TAK1 catalytic activity. We identified a natural resorcylic lactone of fungal origin, 5Z-7-oxozeaenol, as a highly potent inhibitor of TAK1. This compound did not effectively inhibit the catalytic activities of the MEKK1 or ASK1 MAPKKKs, suggesting that 5Z-7-oxozeaenol is a selective inhibitor of TAK1. In cell culture, 5Z-7-oxozeaenol blocked interleukin-1-induced activation of TAK1, JNK/p38 MAPK, IkappaB kinases, and NF-kappaB, resulting in inhibition of cyclooxgenase-2 production. Furthermore, in vivo 5Z-7-oxozeaenol was able to inhibit picryl chloride-induced ear swelling. Thus, 5Z-7-oxozeaenol blocks proinflammatory signaling by selectively inhibiting TAK1 MAPKKK.

The mycotoxin penicillic acid inhibits Fas ligand-induced apoptosis by blocking self-processing of caspase-8 in death-inducing signaling complex.

Upon engagement with Fas ligand (FasL), Fas rapidly induces recruitment and self-processing of caspase-8 via the adaptor protein Fas-associated death domain (FADD), and activated caspase-8 cleaves downstream substrates such as caspase-3. We have found that penicillic acid (PCA) inhibits FasL-induced apoptosis and concomitant loss of cell viability in Burkitt's lymphoma Raji cells. PCA prevented activation of caspase-8 and caspase-3 upon treatment with FasL. However, PCA did not affect active caspase-3 in FasL-treated cells, suggesting that PCA primarily blocks early signaling events upstream of caspase-8 activation. FasL-induced processing of caspase-8 was severely impaired in the death-inducing signaling complex, although FasL-induced recruitment of FADD and caspase-8 occurred normally in PCA-treated cells. Although PCA inhibited the enzymatic activities of active recombinant caspase-3, caspase-8, and caspase-9 at similar concentrations, PCA exerted weak inhibitory effects on activation of caspase-9 and caspase-3 in staurosporine-treated cells but strongly inhibited caspase-8 activation in FasL-treated cells. Glutathione and cysteine neutralized an inhibitory effect of PCA on caspase-8, and PCA bound directly to the active center cysteine in the large subunit of caspase-8. Thus, our present results demonstrate that PCA inhibits FasL-induced apoptosis by targeting self-processing of caspase-8.

Actin cytoskeleton is required for early apoptosis signaling induced by anti-Fas antibody but not Fas ligand in murine B lymphoma A20 cells.

Murine B lymphoma A20 cells are highly sensitive to Fas-mediated death signals induced by anti-Fas antibody Jo2 or cross-linked Fas ligand (FasL). We have found that the microfilament poison cytochalasin D blocks Fas-mediated apoptosis induced by Jo2 but not FasL in A20 cells. The induction of Fas-mediated apoptosis by Jo2 was antagonized by anti-Fcgamma RII/RIII receptor (FcgammaR) antibody, and defective in FcgammaR-negative A20 cells. Since the induction of Jo2-mediated apoptosis in FcgammaR-negative A20 cells was reversed by the addition of wild type A20 cells or the cross-linking agent protein A, Fas-expressing bystander A20 cells seem to be killed by other A20 cells that capture and cross-link monomeric Jo2 via FcgammaR. Although cytochalasin D affected FcgammaR-mediated cross-linking of Jo2 molecules, the drug markedly inhibited the intracellular signaling pathway induced by Jo2. The blockade of Jo2-induced apoptosis by cytochalasin D occurred upstream of caspase-8 activation. Thus, these observations suggest that actin cytoskeleton is required for early apoptosis signaling induced by Jo2, but not physiological FasL.