Kazuhisa Nozawa

Levels of IL-12 in the sera of patients with systemic lupus erythematosus (SLE)— relation to Th1- and Th2-derived cytokines

IL‐12 is a cytokine that induces Th1‐derived cytokines (interferon‐gamma (IFN‐γ) and IL‐2). The significance of IL‐12 in human autoimmunity is no clear, and the serum levels of IL‐12 in SLE are not clearly established. Therefore, we examined the levels of IL‐12 in 39 patients with active SLE, with sandwich ELISA. The levels of IL‐12 in patients were significantly higher than in normal subjects. Patients with high levels of IL‐12 also had high levels of IFN‐γ, while their levels of IL‐13 were significantly lower than in patients with normal levels of IL‐12. Patients with pulmonary involvement had high levels of IL‐12, and steroid therapy decreased the IL‐12 level in three patients. In a retrospective study of seven patients, various changes of IL‐12 and IL‐13 were recognized before disease flare. Thus, in SLE patients, the level of IL‐12 was increased and this increase was related to the change of Th1‐ or Th2‐derived cytokines with some organ involvement.

Soluble Fas molecule in the serum of patients with systemic lupus erythematosus

The serum level of soluble Fas (sFas) molecules in 35 patients with SLE was determined by enzyme-linked immunosorbent assay (ELISA) and its relation to other lymphocyte activation markers and clinical parameters was examined. The level of sFas increased significantly compared to that in normal subjects, consistent with previous reports. There was a significant correlation between the level of sFas and that of sCD4, suggesting some relation between sFas and activation of CD4+ T cells. Patients with lymphopenia tended to have low levels of sFas, making it possible to hypothesize that sFas protects against apoptosis. Although the change in the level of sFas with steroid therapy was variable, some relation to the differential activation of T cell subsets was suggested.

Connective tissue growth factor promotes articular damage by increased osteoclastogenesis in patients with rheumatoid arthritis

IntroductionA protein analysis using a mass spectrometry indicated that there are serum proteins showing significant quantitative changes after the administration of infliximab. Among them, connective tissue growth factor (CTGF) seems to be related to the pathogenesis of rheumatoid arthritis (RA). Therefore, this study was conducted to investigate how CTGF is associated with the disease progression of RA.MethodsSerum samples were collected from RA patients in active or inactive disease stages, and before or after treatments with infliximab. CTGF production was evaluated by ELISA, RT-PCR, indirect immunofluorescence microscopy, and immunoblotting. Osteoclastogenesis was evaluated using tartrate-resistant acid phosphatase (TRAP) staining, a bone resorption assay and osteoclasts specific catalytic enzymes productions.ResultsThe serum concentrations of CTGF in RA were greater than in normal healthy controls and disease controls. Interestingly, those were significantly higher in active RA patients compared to inactive RA patients. Furthermore, the CTGF levels significantly were decreased by infliximab concomitant with the disease amelioration. In addition, tumour necrosis factor (TNF)α can induce the CTGF production from synovial fibroblasts even though TNFα can oppositely inhibit the production of CTGF from chondrocytes. CTGF promoted the induction of the quantitative and qualitative activities of osteoclasts in combination with M-CSF and receptor activator of NF-κB ligand (RANKL). In addition, we newly found integrin αVβ3 on the osteoclasts as a CTGF receptor.ConclusionsThese results indicate that aberrant CTGF production induced by TNFα plays a central role for the abnormal osteoclastic activation in RA patients. Restoration of aberrant CTGF production may contribute to the inhibition of articular destruction in infliximab treatment.

Giantin is the major Golgi autoantigen in human anti-Golgi complex sera

Anti-Golgi complex antibodies (AGAs) are primarily associated with systemic lupus erythematosus and Sjögrens syndrome. Here we report on the immunoreactivity of AGAs against five Golgi autoantigens (giantin, golgin-245, golgin-160, golgin-95/GM130, and golgin-97) and provide data from epitope mapping on the most common Golgi autoantigen, namely giantin. A total of 80 human sera containing AGAs, as defined by indirect immunofluorescence on HEp-2 cells, were analyzed by ELISA using recombinant autoantigens and immunoprecipitation. The proportion of AGA sera that reacted with the five Golgi autoantigens was correlated with the molecular mass of the Golgi antigens. Autoantibodies to giantin, the largest Golgi autoantigen, were the predominant AGAs, being found in 50% of the AGA sera. Epitope mapping of giantin was performed using six recombinant fragments spanning the entire protein. Antigiantin-positive sera with low titer autoantibodies recognized epitopes in the carboxyl-terminal fragments that are proximal to the Golgi membrane, whereas higher titer sera exhibited strong reactivity to amino-terminal and central domains that are likely to extend from the Golgi membrane into the cytoplasm. Our working hypothesis is that aberrantly expressed Golgi complex autoantigens may be released into the immune system when cells undergo lysis. By virtue of a carboxyl-terminal transmembrane domain, giantin is likely to be more stably associated with the cytoplasmic face of the Golgi complex than are other golgins, which are peripheral proteins. The stable association of giantin with the putative released Golgi complex may contribute to its preferential autoantigenicity.

Preferential Blockade of CD8+ T Cell Responses by Administration of Anti-CD137 Ligand Monoclonal Antibody Results in Differential Effect on Development of Murine Acute and Chronic Graft-Versus-Host Diseases

We investigated the effect of CD137 costimulatory blockade in the development of murine acute and chronic graft-vs-host diseases (GVHD). The administration of anti-CD137 ligand (anti-CD137L) mAb at the time of GVHD induction ameliorated the lethality of acute GVHD, but enhanced IgE and anti-dsDNA IgG autoantibody production in chronic GVHD. The anti-CD137L mAb treatment efficiently inhibited donor CD8+ T cell expansion and IFN-γ expression by CD8+ T cells in both GVHD models and CD8+ T cell-mediated cytotoxicity against host-alloantigen in acute GVHD. However, a clear inhibition of donor CD4+ T cell expansion and activation has not been observed. On the contrary, in chronic GVHD, the number of CD4+ T cells producing IL-4 was enhanced by anti-CD137L mAb treatment. This suggests that the reduction of CD8+ T cells producing IFN-γ promotes Th2 cell differentiation and may result in exacerbation of chronic GVHD. Our results highlight the effective inactivation of CD8+ T cells and the lesser effect on CD4+ T cell inactivation by CD137 blockade. Intervention of the CD137 costimulatory pathway may be beneficial for some selected diseases in which CD8+ T cells are major effector or pathogenic cells. Otherwise, a combinatorial approach will be required for intervention of CD4+ T cell function.

The Increased Interleukin-13 in Patients with Systemic Lupus Erythematosus: Relations to Other Thl-, Th2-Related Cytokines and Clinical Findings

The levels of interleukin-13 (IL-13) in patients with systemic lupus erythematosus (SLE) were examined and related to other Thl-/Th2- related cytokines, clinical manifestations and other markers. Serum levels of IL-13 and other cytokines, soluble markers were measured by enzyme-linked immunosorbent assay (ELISA). Patients with active SLE had a significantly increased level of EL-13. Most patients with high levels of IL-13 had higher levels of IL-6, and some patients had high levels of ylFN. These patients were divided into two groups according to the patterns of these increased cytokines; one with a high level of only Th2 related cytokines (IL-13 or IL-6) and another with high levels of both Th2 related and Thl related cytokines (γIFN or IL-2). The latter patients had high levels of soluble CD8 and CD23, and some of them had hemolytic anemia or pulmonary involvement, while most of the former patients had nephropathy. Thus, in SLE, the levels of IL-13 were increased, and the heterogeneity of increased Th2- and Thl-related cytokines was related to that of activation markers and clinical manifestations

Possible role of the JAK/STAT pathways in the regulation of T cell-interferon related genes in systemic lupus erythematosus.

Changes in gene expression in CD3+ T cells associated with disease progression in systemic lupus erythematosus (SLE) patients were determined. The genes related to SLE disease-related activities were identified and their gene regulatory networks were investigated. Analyses of gene expression were performed by both DNA microarray and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). The expression of certain genes including interferon (IFN) regulatory factor (IRF)-related genes, such as IFN-regulated, -related, and -signature genes was increased in the active phase of SLE. Pathway network analyses suggested that these IRF-related genes are regulated through the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. JAK/STAT pathway-mediated regulation of IRF-related genes may have an important role in the disease activity of SLE. Inhibitors of JAK/STAT cascade may be useful as therapeutic agents.

Inhibition of Connective Tissue Growth Factor Ameliorates Disease in a Murine Model of Rheumatoid Arthritis

OBJECTIVE We have shown that connective tissue growth factor (CTGF) plays an important role in the pathogenesis of rheumatoid arthritis (RA). This study was undertaken to evaluate the effects of blockade of the CTGF pathway on the development of collagen-induced arthritis (CIA) in mice. METHODS Arthritis was induced in DBA/1J mice by immunization with a combination of type II collagen (CII) and Freunds complete adjuvant. We evaluated the development of arthritis in mice with CIA left untreated versus treated with neutralizing anti-CTGF monoclonal antibody (mAb). RESULTS Inhibition of CTGF in mice treated with neutralizing anti-CTGF mAb significantly ameliorated arthritis compared to the untreated mice with CIA. Serum levels of matrix metalloproteinase 3 were reduced by anti-CTGF mAb treatment. Moreover, blockade of CTGF decreased interleukin-17 expression on purified CD4+ T lymphocytes. Although the expression of the retinoic acid receptor-related orphan receptor γt gene was not suppressed by anti-CTGF mAb treatment, that of interferon regulatory factor 4 (IRF-4) and IκBζ (Nfkbiz), which are other important molecules for the differentiation of Th17 cells, was suppressed. In addition, blockade of CTGF inhibited pathologic proliferation of T lymphocytes in response to CII restimulation in vitro. Moreover, aberrant osteoclastogenesis in mice with CIA was restored by anti-CTGF mAb treatment. CONCLUSION Our findings indicate that blockade of CTGF prevents the progression of arthritis in mice with CIA. Anti-CTGF mAb treatment suppresses pathologic T cell function and restores aberrant osteoclastogenesis in mice with CIA. CTGF may become a new target for the treatment of RA.

Decreased IL-4 producing CD4+ T cells in patients with active systemic lupus erythematosus-relation to IL-12R expression

The balance of interferon- n (IFN- n ) and/or interleukin-4 (IL-4) producing T cells and interleukin-12 receptor (IL-12R) expression on T cells were evaluated in patients with active systemic lupus erythematosus (SLE). Assessment of intracellular IFN- n and/or IL-4 were conducted with cytoplasmic staining. IL-12R presenting T cells were also assessed by flowcytometry without in vitro stimulation. In SLE, the number of IFN- n producing CD4 + T cells was increased, and the absolute number of IL-4 producing CD4 + T cells was significantly decreased. Although the ratio of IL-12R presenting CD4 + T cells was significantly greater, the absolute number did not increase. The ratio of IFN- n /IL-4-producing CD4 + T cells correlated with the SLE disease activity index (SLEDAI) and was significantly higher among patients with lupus nephritis. Therefore, the imbalance of IFN- n /IL-4 producing CD4 + T cells was due to the decrease in IL-4 producing CD4 + T cells and may play an important pathogenic role in active SLE.

Down-regulation of CD72 and increased surface IgG on B cells in patients with lupus nephritis.

The significance of both the acceleratory and inhibitory functions of the CD72 molecule was investigated among patients with systemic lupus erythematosus (SLE) during modification of B cell differentiation. Expression of the CD72 molecule and mRNA on B cells was decreased in SLE with lupus nephritis, while CD100 expression on both CD4+ T cells and CD8+ T cells was not significant in comparison with the controls. When the relationship between CD72 expression and other B cell markers was examined, decreased expression of CD72 was associated with differences in the stage of differentiation. In patients with decreased expression of CD72, switching to IgG was evident, and the disease stage was started to severe. In patients with lupus nephritis, the decreased expression of CD72 was related to class switching on B cells, suggesting that CD72 is a useful marker for determining class switching of B cells in lupus nephritis.