Keiko Yoshimoto

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.

Interaction of KLRG1 with E‐cadherin: New functional and structural insights

The killer cell lectin‐like receptor G1 (KLRG1) is an inhibitory receptor expressed by memory T cells and NK cells in man and mice. It is frequently used as a cell differentiation marker and members of the cadherin family are ligands for KLRG1. The present study provides new insights into the interaction of mouse KLRG1 with E‐cadherin. Firstly, we demonstrate that co‐engagement of KLRG1 and CD3/TCR in a spatially linked manner was required for inhibition arguing against the notion that KLRG1‐ligation per se transmits inhibitory signals. Secondly, experiments with T cells carrying Y7F‐mutant KLRG1 molecules with a replacement of the tyrosine residue to phenylalanine in the single ITIM indicated that the inhibitory activity of KLRG1 is counteracted to some degree by increased interaction of KLRG1+ T cells with E‐cadherin expressing target cells. Thirdly, we demonstrate that deletion of the first or the second external domain of E‐cadherin abolished reactivity in KLRG1‐reporter cell assays. Finally, we made the intriguing observation that KLRG1 formed multimeric protein complexes in T cells in addition to the previously described mono‐ and dimeric molecules.

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.

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.

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.

Baseline levels of soluble interleukin-6 receptor predict clinical remission in patients with rheumatoid arthritis treated with tocilizumab: implications for molecular targeted therapy

Interleukin-6 (IL-6) is a monomeric protein that binds to either soluble or membrane-bound IL-6 receptors (IL-6R)1 ,2 Tocilizumab, a humanised anti-IL-6R monoclonal antibody, binds to soluble IL-6R (sIL-6R) and membrane-bound IL-6R, blocking signal transduction pathways through competitive inhibition of IL-6 binding.3 We have previously demonstrated that baseline plasma tumour necrosis factor (TNF) levels are associated with the clinical response to infliximab, anti-TNF monoclonal antibody binding to soluble and membrane-bound TNF.4 Therefore, it is tempting to speculate that baseline serum levels of sIL-6R, rather than those of IL-6, are associated with clinical response to tocilizumab in patients with rheumatoid arthritis (RA). To test this hypothesis, we analysed serum levels of IL-6 and sIL-6R before tocilizumab treatment in our institution and evaluated their association with clinical remission. Consecutive patients with RA in our institution who commenced 8 mg/kg tocilizumab treatment every 4 weeks as the first biologic agent between March 2010 and April 2012 were included. At baseline, serum levels of IL-6 and sIL-6R were measured by electrochemiluminescence assay with the Ultra-Sensitive Kit (Meso Scale …

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.

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.

Decreased mrna expression of two FOXP3 isoforms in peripheral blood mononuclear cells from patients with rheumatoid arthritis and systemic lupus erythematosus

Both the number and functional capacity of T-regulatory (Treg) cells are known to be decreased in various autoimmune diseases. FOXP3, an essential transcription factor for Treg cells, has three isoforms in humans, wild, and exon 2- and exon 2-exon 7-lacking, although their role in autoimmunity is not clearly understood. Here, we investigated the messenger RNA (mRNA) expression of the major wild and exon-2 isoforms in peripheral mononuclear cells by quantitative PCR methods in 56 subjects, consisting of 23 rheumatoid arthritis (RA) and 25 systemic lupus erythematosus (SLE) patients, and 8 healthy controls (HCs). Although mRNA expression of the two isoforms did not directly correlate with clinical disease activity, relative expression of both was significantly lower in SLE and RA patients than in HCs. Furthermore, we found a significant statistical correlation between the two isoforms, suggesting that they are similarly regulated. Decreased expression of these isoforms in RA and SLE may reflect Treg cell abnormalities in these autoimmune diseases.