Kensei Tsuzaka

TCRζ mRNA with an Alternatively Spliced 3′-Untranslated Region Detected in Systemic Lupus Erythematosus Patients Leads to the Down-Regulation of TCRζ and TCR/CD3 Complex

The reduction or absence of TCR ζ-chain (ζ) expression in systemic lupus erythematosus (SLE) patients is thought to be related to the pathogenesis of SLE. Recently, we reported the predominant expression of ζ mRNA containing an alternatively spliced 3′-untranslated region (3′UTR; ζmRNA/as-3′UTR) and a reduction in the expression of ζ mRNA containing the wild-type 3′UTR (ζmRNA/w-3′UTR) in T cells from SLE patients. Here we show that AS3′UTR mutants (MA5.8 cells deficient in ζ protein that have been transfected with ζmRNA/as-3′UTR) exhibit a reduction in the expression of TCR/CD3 complex and ζ 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 ζmRNA/w-3′UTR). Furthermore, the real-time PCR analyses demonstrated that the half-life of ζmRNA/as-3′UTR in AS3′UTR mutants (3 h) was much shorter than that of ζmRNA/w-3′UTR in wild-type 3′UTR mutants (15 h). Thus, the lower stability of ζmRNA/as-3′UTR, which is predominant in SLE T cells, may be responsible for the reduced expression of the TCR/CD3 complex, including ζ protein, in SLE T cells.

Defective expression and tyrosine phosphorylation of the T cell receptor zeta chain in peripheral blood T cells from systemic lupus erythematosus patients

We have reported that tyrosine phosphorylation and expression of the T cell receptor zeta chain (TCR ζ) was decreased in two systemic lupus erythematosus (SLE) patients with an abnormal TCR ζ lacking exon‐7. To examine further the TCR ζ defect and any possible relationship with specific clinical features, we studied the expression of TCR ζ in peripheral blood T cells from 44 patients with SLE, 53 with other rheumatic diseases (30 rheumatoid arthritis (RA), 11 systemic sclerosis (SSc) and 12 primary Sjögrens syndrome(SjS)) and 39 healthy individuals. Flow cytometric analysis demonstrated a significant decrease in the expression of TCR ζ in SLE (P < 0·001), but not in the other rheumatic diseases. Immunoprecipitation experiments confirmed that the expression of TCR ζ in SLE T cells was decreased dramatically (normal: 111·4 ± 22·6%, SLE: 51·6 ± 37·4%, P < 0·0001). The decrease in TCR ζ did not correlate with disease activity, or with the dose of prednisolone (PSL). There were, however, three SLE patients in whom the level of TCR ζ expression normalized after treatment, suggesting that mechanisms responsible for the TCR ζ defect appear to be heterogeneous. These results confirm the defective expression and altered tyrosine phosphorylation of TCR ζ in a large proportion of SLE patients, suggesting that it may play an important role in T cell dysfunction in SLE.

A Splice Variant of the TCR ζ mRNA Lacking Exon 7 Leads to the Down-Regulation of TCR ζ, the TCR/CD3 Complex, and IL-2 Production in Systemic Lupus Erythematosus T Cells

The reduction or absence of TCR ζ-chain (ζ) expression in patients with systemic lupus erythematosus (SLE) is thought to be a factor in the pathogenesis of SLE. We previously reported a splice variant of ζ mRNA that lacks the 36-bp exon 7 (ζ mRNA/exon 7(−)) and is accompanied by the down-regulation of ζ protein in T cells from SLE patients. In this study, we show that EX7− mutants (MA5.8 cells deficient in ζ protein that have been transfected with ζ mRNA/exon 7(−)) exhibit a reduction in the expression of TCR/CD3 complex and ζ 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 (WT) mutants (MA5.8 cells transfected with the WT ζ mRNA). Furthermore, real-time PCR analyses demonstrated that ζ mRNA/exon 7(−) in EX7− mutants was easily degraded compared with ζ mRNA by the WT mutants. Pulse-chase experiment showed ζ protein produced by this EX7− mutants was more rapidly decreased compared with the WT mutants. Thus, the lower stability of ζ mRNA/exon 7(−) might also be responsible for the reduced expression of the TCR/CD3 complex, including ζ protein, in SLE T cells.

Up-regulation of αEβ7, a novel integrin adhesion molecule, on T cells from systemic lupus erythematosus patients with specific epithelial involvement

OBJECTIVE To determine the possible role of a novel integrin, alphaEbeta7, in the pathogenesis of systemic lupus erythematosus (SLE). METHODS Expression of alphaEbeta7 was examined on peripheral blood lymphocytes (PBL) from normal subjects (n = 25) and patients with SLE (n = 31), primary Sjogrens syndrome (n = 7), or polymyositis/dermatomyositis (n = 8) by cytofluorometry and/or immunoprecipitation. Adhesion of alphaEbeta7+ T cells to HSG epithelial cells was investigated using a confocal image analyzer. RESULTS After phytohemagglutinin stimulation, expression of alphaEbeta7 on PBL, especially on CD8+ T cells, was significantly higher in SLE patients than in normal subjects (P<0.01). Elevated alphaEbeta7 expression was associated with the presence of oral ulcers or serositis (P<0.05). Activated SLE T cells with enhanced alphaEbeta7 expression strongly adhered to HSG; this adhesion was partially blocked by anti-alphaEbeta7. CONCLUSION Expression and adhesion of alphaEbeta7 on activated PBL was significantly increased in patients with SLE with epithelial involvement. This suggests a role of this novel integrin in tissue-specific retention of activated PBL, due to increased alphaEbeta7-E-cadherin interaction, which may contribute to epithelial inflammation.

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

IntroductionIn this study, we investigated possible aberrations of monocytes from patients with primary Sjögrens 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.MethodsPeripheral 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.ResultsPeripheral 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.ConclusionsThe 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.

Abnormal expression and function of Fas ligand of lachrymal glands and peripheral blood in Sjögren's syndrome patients with enlarged exocrine glands

The objective of our study was to investigate the possibility of Fas ligand protein abnormalities in certain types of Sjögrens syndrome patients with enlarged exocrine glands. Fas ligand expression by lymphocytes infiltrating the lachrymal glands and by peripheral blood monocytes in Sjögrens syndrome patients with enlarged exocrine glands was assessed immunohistologically and by immunoblotting. Cytotoxicity of peripheral blood monocytes and sensitivity to steroids in Sjögrens syndrome patients with enlarged exocrine glands were studied by functional assay. Minimal Fas ligand expression was detected in the lymphocytes of the lachrymal glands and a decreased level of Fas ligand was found in peripheral blood monocytes as assessed by immunoblotting. Functional assay confirmed the decreased cytotoxicity of lymphocytes in Sjögrens syndrome patients with enlarged exocrine glands, and that it is not affected by anti‐Fas ligand antibody. By contrast, the sensitivity of lymphocytes in Sjögrens syndrome patients with enlarged exocrine glands to steroids was increased. These observations suggest that abnormal expression and function of Fas ligand occurs in Sjögrens syndrome patients with enlarged exocrine glands.

Critical Role of the Fifth Domain of E-Cadherin for Heterophilic Adhesion with αEβ7, But Not for Homophilic Adhesion

The integrin αEβ7 is expressed on intestinal intraepithelial T lymphocytes and CD8+ T lymphocytes in inflammatory lesions near epithelial cells. Adhesion between αEβ7+ T and epithelial cells is mediated by the adhesive interaction of αEβ7 and E-cadherin; this interaction plays a key role in the damage of target epithelia. To explore the structure-function relationship of the heterophilic adhesive interaction between E-cadherin and αEβ7, we performed cell aggregation assays using L cells transfected with an extracellular domain-deletion mutant of E-cadherin. In homophilic adhesion assays, L cells transfected with wild-type or a domain 5-deficient mutant formed aggregates, whereas transfectants with domain 1-, 2-, 3-, or 4-deficient mutants did not. These results indicate that not only domain 1, but domains 2, 3, and 4 are involved in homophilic adhesion. When αEβ7+ K562 cells were incubated with L cells expressing the wild type, 23% of the resulting cell aggregates consisted of αEβ7+ K562 cells. In contrast, the binding of αEβ7+ K562 cells to L cells expressing a domain 5-deficient mutant was significantly decreased, with αEβ7+ K562 cells accounting for only 4% of the cell aggregates, while homophilic adhesion was completely preserved. These results suggest that domain 5 is involved in heterophilic adhesion with αEβ7, but not in homophilic adhesion, leading to the hypothesis that the fifth domain of E-cadherin may play a critical role in the regulation of heterophilic adhesion to αEβ7 and may be a potential target for treatments altering the adhesion of αEβ7+ T cells to epithelial cells in inflammatory epithelial diseases.

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 Sjögrens syndrome and six non-Sjögrens 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 Sjögrens 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.

Altered Distribution of Aquaporin 5 and its C-Terminal Binding Protein in the Lacrimal Glands of a Mouse Model for Sjögren's Syndrome

Purpose: To investigate the distribution and expression of aquaporin 5 (AQP5) and its C-terminal binding protein in the apical membrane of the lacrimal glands (LGs) in a mouse model for Sjögrens syndrome (SS). Methods: The LGs of NOD mice (mouse model for SS) and ICR mice (normal control) were homogenized and delivered into the affinity columns bound to synthetic AQP5 C-terminal peptide. The eluates were analyzed by electrophoresis and liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS) techniques.Results: AQP5 from the NOD mice exhibited the capacity to bind a 21-kDa protein that was lacking in the ICR mice. Instead, ICR mouse expressed a 17-kDa AQP5 binding protein that was absent in LGs of SS. LC-MS/MS analysis revealed these respective proteins to be major urinary protein 4 (MUP4) and prolactin-inducible protein (PIP). The treatment of ICR mice with antisense PIP oligonucleotides decreased immunostaining of AQP5 in the apical membrane. Conclusions: These observations suggest that the binding of PIP to the C-terminal portion of AQP5 may cause AQP5 to be transported to the apical membrane of LGs. Correction of the aberrant binding of PIP to the AQP5 C-terminus could normalize AQP5 trafficking to the apical membrane, leading to a treatment for patients with SS.

DNA microarray gene expression profile of T cells with the splice variants of TCRζ mRNA observed in systemic lupus erythematosus

We have reported that the TCRζ mRNA with alternatively spliced 3′ UTR (ζ mRNA/as-3′-untranslated region (UTR)) and ζ mRNA lacking exon 7 (ζ mRNA/exon 7−) observed in systemic lupus erythematosus patient T cells can lead to down-regulation of both ζ and TCR/CD3 complexes. To determine whether these T cells expressing decreased ζ exhibit differential transcription patterns, we transfected retrovirus vectors containing wild-type ζ cDNA, ζ cDNA/as-3′ UTR, and ζ cDNA/exon 7− into murine T cell hybridoma MA5.8 cells which lack ζ expression to construct the MA5.8 mutants WT, AS3′ UTR, and EX7−, respectively. FACS analyses demonstrated reduced cell surface expression of ζ and TCR/CD3 complexes on the AS3′ UTR mutant and the EX7− mutant in comparison to that on the WT mutant. Total RNA was collected after stimulating the MA5.8 mutants with anti-CD3 Ab. Reverse-transcribed cDNA was applied to the mouse cDNA microarray containing 8691 genes, and the results were confirmed by real-time PCR. The results showed that 36 genes encoding cytokines and chemokines, including IL-2, IL-15, IL-18, and TGF-β2, were down-regulated in both the AS3′ UTR mutant and the EX7− mutant. Another 16 genes were up-regulated in both, and included genes associated with membranous proteins and cell damage granules, including the genes encoding poliovirus receptor-related 2, syndecan-1, and granzyme A. Increased protein expression of these genes was confirmed by Western blot and FACS analyses. Identification of these responsive genes in T cells in which the ζ and TCR/CD3 complexes were down-regulated may help to better understand the pathogenesis of systemic lupus erythematosus.