CD3 ζ defects in systemic lupus erythematosus.

The prototype autoimmune disease, systemic lupus erythematosus (SLE), has been known to be associated with deficiency of ζ chain, a component of the T-cell receptor-CD3 complex. Comprehensive analysis has shown that expression of the CD3 ζ chain is attenuated or absent in over half of SLE patients. Furthermore, aberrant transcripts of the CD3 ζ chain, including spliced variants lacking exon 7 or having a short 3'-untranslated region, have been detected in SLE T cells. Although attenuated expression of the CD3 ζ chain is also observed in cancer patients, infections and other autoimmune diseases, sustained attenuation of the CD3 ζ expression accompanied with aberrant transcripts are only observed in SLE. In this study, the authors review the unique features of CD3 ζ defects observed in SLE and discuss the molecular basis of the defects by recent findings in animal models, single-nucleotide polymorphisms and genome-wide association studies.

Regulatory mechanisms for the production of BAFF and IL-6 are impaired in monocytes of patients of primary Sjögren's syndrome.

INTRODUCTION: In this study, we investigated possible aberrations of monocytes from patients with primary Sjögren's syndrome (pSS). We focused on B-cell-activating factor of the TNF family (BAFF) and IL-6 because they are both produced by monocytes and are known to be involved in the pathogenesis of pSS. METHODS: Peripheral monocytes were prepared from both pSS patients and normal individuals. The cells were stimulated in vitro with IFN-γ, and the amounts of IL-6 and soluble BAFF (sBAFF) produced by the cells were quantitated. The effect of sBAFF itself on the production of IL-6 was also studied. To investigate the response of pSS monocytes to these stimuli, the expression levels of the genes encoding BAFF receptors and IL-6-regulating transcription factors were quantitated. RESULTS: Peripheral pSS monocytes produced significantly higher amounts of sBAFF and IL-6 than normal monocytes did, even in the absence of stimulation. The production of these cytokines was significantly increased upon stimulation with IFN-γ. The elevated production of IL-6 was significantly suppressed by an anti-BAFF antibody. In addition, stimulation of pSS monocytes with sBAFF induced a significant increase in IL-6 production. Moreover, the expression levels of a BAFF receptor and transcription factors regulating IL-6 were significantly elevated in pSS monocytes compared to normal monocytes. CONCLUSIONS: The results of the present study suggest that the mechanisms underlying the production of sBAFF and IL-6 are impaired in pSS monocytes. Our research implies that this impairment is due to abnormally overexpressed IL-6-regulating transcription factors and a BAFF receptor. These abnormalities may cause the development of pSS.

Effect of interleukin-2 on synthesis of B cell activating factor belonging to the tumor necrosis factor family (BAFF) in human peripheral blood mononuclear cells.

B cell activating factor belonging to the tumor necrosis factor family (BAFF) is a cytokine, indispensable for B cell survival, maturation, and activation. Over-expression of BAFF leads to lupus like disease in mice and the serum level of BAFF is elevated in human lupus. However, little is known about BAFF synthesis and its regulation. In this study, we examined the effects of a series of inflammatory cytokines on BAFF production in human peripheral blood mononuclear cells (PBMCs) in vitro. We found interleukin-2 (IL-2) strongly and dose-dependently stimulated BAFF synthesis in PBMCs, and an anti-IL-2 antibody neutralized the effect. Furthermore, T and NK cells produced BAFF with IL-2 stimulation. From these observations, IL-2 is one of the regulatory cytokines having a positive effect on BAFF synthesis in human peripheral T and NK cells. Persistent over-production of IL-2 might lead to up-regulation of BAFF synthesis in PBMCs in pathological conditions such as lupus.

Down-regulation of Fas-ligand mRNA in Sjögren's syndrome patients with enlarged exocrine glands.

We have reported that Sjögren's syndrome (SS) patients with enlarged exocrine glands (EEG) formerly referred to as Mikulicz's disease were defective with Fas-ligand (FasL) expression in PBL and lacrimal glands (LGs). To investigate the mechanisms of reduced FasL expression in SS patients with EEG, FasL mRNA expression level was determined using real-time PCR. The FasL gene promoter region (from - 1197 to - 3) was also amplified using PCR and specific primers. Expression of the FasL mRNA in the LGs and PBLs of three SS patients with EEG was significantly decreased. Direct sequencing revealed a heterozygous point mutation ( - 259T/C) in the FasL gene promoter region in one SS patient with EEG. A luminescent beta-galactosidase (beta-gal) reporter assay using a pbetagal Enhancer Vector demonstrated that beta-gal activity from the vector including the mutant ( - 259C) FasL (pbetagal/mFasL) gene promoter region (735 +/- 42) was similar (p = 0.13) to that from a pbetagal Enhancer Vector without the gene promoter region (603 +/- 66). On the other hand, the beta-gal activity was significantly lower (p < 0.0001) than that from a vector including the wild-type ( - 259T) FasL (pbetagal/wFasL) (3226 +/- 148). In conclusion, the down-regulation of FasL in SS patients with EEG may be due to transcriptional regulation, and the point mutation at - 259T/C in the FasL gene promoter region may lead to the down-regulation of FasL mRNA expression and the lymphoproliferative process observed in SS patients with EEG.

Aberrant expression of BAFF in T cells of systemic lupus erythematosus, which is recapitulated by a human T cell line, Loucy.

B cell-activating factor of the tumor necrosis factor (TNF) family, or BAFF, is mainly produced in monocytes and dendritic cells, and indispensable for proliferation, differentiation and survival of B cells. BAFF is a type II membrane-bound protein and the extracellular C-terminal fragment is released from the cells as soluble BAFF (sBAFF), which binds to specific receptors on B cells. Accumulating evidence suggests that BAFF plays an important role in the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus (SLE). In this study, we developed a sensitive sandwich ELISA system to quantify the amount of sBAFF using our own mAb. Treatment of peripheral T cells of SLE patients with an anti-CD3 antibody triggered robust expression of BAFF and subsequent release of sBAFF from the cells. On the other hand, the stimulus induced only marginal elevation of sBAFF from normal T cells. These data indicate that BAFF is expressed in T cells upon stimulation at least under pathological conditions. Expression of BAFF was also largely induced in a human T cell line, Loucy (American Type Tissue Collection CRL-2629), in response to several stimuli, while other T cell lines so far examined produced the cytokine almost constitutively. These data suggest that Loucy recapitulates some of the characteristics of SLE T cells. Investigation of molecular and cellular mechanisms of production of BAFF in Loucy demonstrated that expression of BAFF was regulated through a signal transduction pathway which involves c-jun NH2-terminal kinase and p38, and that shedding of BAFF was catalyzed by a membrane-bound protease, furin.

T cell abnormalities in systemic lupus erythematosus.

Because of the consensus that T cells play a central role in the pathogenesis of systemic lupus erythematosus (SLE), we explored the molecular basis of the defective function of SLE T cells for expression of signal transduction molecules, as well as surface structures such as adhesion molecules, by extensively testing peripheral blood T cells from SLE patients. Upregulated expression and function of adhesion molecules was observed in T cells from patients with active SLE who had specific clinical manifestations such as vasculitis, epithelitis and arthritis, but proximal signal transduction was defective. Comprehensive analysis to identify the molecules responsible for the defects showed the expression of the TCR zeta chain was attenuated, or absent in more than half of SLE patients. Moreover, the aberrant transcripts of the TCR zeta chain, including spliced variants lacking exon 7 and with a short 3' UTR, were detected in SLE T cells. Although attenuated expression of the TCR zeta chain is also observed in patients with cancers, infections and other autoimmune diseases, sustained attenuation of TCR zeta expression and aberrant transcripts are only observed in SLE. In this review we discuss the unique features of the TCR zeta defects in SLE.

Therapeutic targets of misguided T cells in systemic lupus erythematosus.

It is widely accepted that T cells with defective function play a central role in the pathogenesis of systemic lupus erythematosus (SLE). The detailed molecular mechanism underlying the aberrant function of SLE T cells is now being revealed. The TCR zeta chain, transcription factor, elf-1, inflammation signal transducer NF-kB, and PKC theta have been identified as the responsible molecules. In contrast to the defective signal transduction molecules, surface structures such as adhesion molecules, and co-stimulators have been reported to increase in their expression and function. Glucocorticoids and immunosuppressive agents have greatly improved the outcome of acute diseases and 5-year survival rate. However, it is suggested that long-term survival and quality of life appears to be unsatisfactory. Although the medical management of SLE is not sufficient to warrant long-term survival of young patients, recent progress in anti-cytokine biologics therapy against rheumatoid arthritis (RA) has facilitated searching for the molecular targets of SLE. In this report, we briefly review the molecular basis of SLE pathogenesis, and discuss possible therapeutic targets in this disease, focusing particularly on signal transduction and adhesion molecules in T cells.

TCR zeta mRNA with an alternatively spliced 3'-untranslated region detected in systemic lupus erythematosus patients leads to the down-regulation of TCR zeta and TCR/CD3 complex.

The reduction or absence of TCR zeta-chain (zeta) expression in systemic lupus erythematosus (SLE) patients is thought to be related to the pathogenesis of SLE. Recently, we reported the predominant expression of zeta mRNA containing an alternatively spliced 3'-untranslated region (3'UTR; zetamRNA/as-3'UTR) and a reduction in the expression of zeta mRNA containing the wild-type 3'UTR (zetamRNA/w-3'UTR) in T cells from SLE patients. Here we show that AS3'UTR mutants (MA5.8 cells deficient in zeta protein that have been transfected with zetamRNA/as-3'UTR) exhibit a reduction in the expression of TCR/CD3 complex and zeta protein on their cell surface as well as a reduction in the production of IL-2 after stimulation with anti-CD3 Ab compared with that in wild-type 3'UTR mutants (MA5.8 cells transfected with zetamRNA/w-3'UTR). Furthermore, the real-time PCR analyses demonstrated that the half-life of zetamRNA/as-3'UTR in AS3'UTR mutants (3 h) was much shorter than that of zetamRNA/w-3'UTR in wild-type 3'UTR mutants (15 h). Thus, the lower stability of zetamRNA/as-3'UTR, which is predominant in SLE T cells, may be responsible for the reduced expression of the TCR/CD3 complex, including zeta protein, in SLE T cells.

Quantitative analysis of lacrimal gland function, apoptotic figures, Fas and Fas ligand expression of lacrimal glands in dry eye patients.

The purpose of our study was to examine the correlation between the lacrimal gland function and apoptotic figure, Fas and Fas ligand (FasL) expression in the lacrimal gland. A total of 15 dry eye patients (nine Sjögren's syndrome and six non-Sjögren's syndrome-type dry eye) were recruited for the study. Lacrimal function was evaluated by Schirmer tests 1 and 2. Lacrimal gland biopsies were performed and sections were analyzed by immunohistochemistry using APO2.7, an antibody to Fas and FasL. Quantitative analysis of fluorescein staining was performed by a scanning laser microscopy. Schirmer test 2 results were lower in Sjögren's syndrome-type dry eye and were associated with positive staining of acinar cells with APO2.7 and of infiltrating lymphocytes with FasL. There was a good correlation between the results of Schirmer test 2 and APO2.7 and FasL staining. Lacrimal gland dysfunction is related to the apoptotic figure of acinar cells possibly induced by FasL on the infiltrating lymphocytes.