Abnormal expression of various molecular forms and distribution of T cell receptor ? chain in patients with systemic lupus erythematosus

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101

Objective. T cells from a majority of patients with systemic lupus erythematosus (SLE) display antigen receptor-mediated signaling aberrations associated with defective T cell receptor (TCR) chain, a subunit of the TCR/CD3 complex. This study was undertaken to explore the possibility that forced expression of TCR chain may reverse the known signaling abnormalities and defective interleukin-2 (IL-2) production in SLE T cells.Methods. Freshly isolated SLE T cells were transfected with TCR chain construct in a eukaryotic expression vector at high efficiency, by a recently developed nucleoporation technique. Restoration of TCR/ CD3-mediated signaling was studied in the chaintransfected cells.Results. In SLE T cells transfected with TCR chain, surface expression of TCR chain was increased and the TCR/CD3-induced increased free intracytoplasmic calcium concentration response was normalized, as was hyperphosphorylation of cellular substrates. Simultaneously, the previously noted increased expression of the Fc receptor ␥ chain was diminished in SLE T cells transfected with the chain expression vector, and the surface membrane clusters of cell signaling molecules were redistributed to a more continuous pattern. TCR chain replacement also augmented the expression of diminished TCR/CD3-mediated IL-2 production in SLE T cells, associated with increased expression of the p65 subunit of nuclear factor B in the nuclear fractions of these T cells. Conclusion. These results suggest that reconstitution of deficient TCR chain can reverse the TCR/ CD3-mediated signaling abnormalities as well as the defective IL-2 production in T cells of patients with SLE.It is well recognized that T cells from patients with systemic lupus erythematosus (SLE) display a number of signaling abnormalities (1). Many of the identified molecular aberrations explain certain established cell and cytokine defects, whereas the mechanisms of other defects have not yet been elucidated. Our group and others have demonstrated that the expression of the subunit of the T cell receptor (TCR) is decreased in a majority of SLE patients (2-4) and that this defect persists over time and is independent of disease activity (5).Despite the decreased expression of the TCR chain in SLE T cells, crosslinking of the TCR/CD3 complex leads to increased free intracytoplasmic calcium concentration ([Ca 2ϩ ] i ) response (6) and protein tyrosine phosphorylation (2,4). These events appear to occur because the Fc receptor (FcR) ␥ chain becomes a functional part of the TCR/CD3 complex (7). In support

Section: Discussionmentioning

confidence: 91%

Section: Tcr Chain Transfection Down-regulates Fcr␥ Expression In Slementioning

confidence: 96%

Section: Patients and Controlsmentioning

confidence: 99%

Section: Patients and Controlsmentioning

confidence: 99%

Section: Transfection and Expression Of Tcr Chain In Sle T Lymphocytesmentioning

confidence: 99%

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Reconstitution of deficient T cell receptor ζ chain restores T cell signaling and augments T cell receptor/CD3–induced interleukin‐2 production in patients with systemic lupus erythematosus

Nambiar

Fisher

Warke

et al. 2003

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101

Increased ubiquitination and reduced expression of LCK in T lymphocytes from patients with systemic lupus erythematosus

Jury¹,

Kabouridis²,

Abba³

et al. 2003

Objective. To explore regulation of proximal signaling and composition of lipid rafts in T lymphocytes from patients with systemic lupus erythematosus (SLE).Methods. The expression, phosphorylation, and degradation of lipid raft-associated signaling molecules in T lymphocytes from 50 patients with SLE compared with 28 healthy controls and 22 rheumatoid arthritis patients were investigated. Lipid raft and nonraft fractions from T cells were isolated by ultracentrifugation. Proteins in the lipid raft and nonraft fractions were analyzed by Western blotting and probed for phosphotyrosine activity and for LCK, LAT, and CD3. Immunoprecipitation experiments were performed to assess protein ubiquitination in T cell lysates. T cell phenotype and levels of intracellular LCK were determined by flow cytometry.Results. LCK, an essential signaling molecule for T cell activation, was significantly reduced in both lipid raft and nonraft fractions of T lymphocytes from patients with active SLE compared with controls, and the reduction was independent of treatment. To identify the likely causes of reduced LCK, we explored the possibility that chronic activation of T lymphocytes underlies LCK degradation. The results revealed an increase in protein ubiquitination, and specifically LCK ubiquitination, in T cells from SLE patients. However, our findings suggest that the increase in ubiquitination is independent of T cell activation.Conclusion. LCK is reduced in T cell lipid rafts from patients with SLE. This reduction appears to be independent of activation and may be associated with abnormal ubiquitin-mediated regulation mechanisms. Systemic lupus erythematosus (SLE) is an autoimmune rheumatic disease characterized by abnormalities in T and B lymphocyte function and receptormediated signaling pathways (for review, see refs. 1-3).T lymphocytes from lupus patients exhibit numerous functional abnormalities, such as increased apoptosis, skewed cytokine production, and decreased proliferative responses to soluble antigens (4). Recently, several defects in proximal T cell signaling events have been identified. These include reduced expression and function of the protein tyrosine phosphatase (PTP) molecule CD45 (5), aberrant regulation of Src kinase molecules LCK and FYN (6-8), diminished expression of T cell receptor (TCR) chain (9,10), abnormal protein tyrosine phosphorylation of proximal signaling molecules (7,11), and increased CD3-mediated intracellular calcium mobilization (12).Ligation of the TCR leads to a rapid increase in protein tyrosine phosphorylation by recruitment of proximal protein tyrosine kinases (PTKs), the Src-family and Syk/ZAP-70 kinases, which are responsible for the initiation and amplification of the activation signal (for review, see ref. 13). Membrane-bound Src kinases (LCK and FYN) phosphorylate paired tyrosine residues in the immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR chain and CD3 complex. This leads to the recruitment and activation of the cytoplasmic PTK ZAP-70 by interaction of the...

Decreased Lyn expression and translocation to lipid raft signaling domains in B lymphocytes from patients with systemic lupus erythematosus

Flores-Borja¹,

Kabouridis²,

Jury³

et al. 2005

112

Objective. B lymphocytes from patients with systemic lupus erythematosus (SLE) are hyperactive and produce anti-double-stranded DNA (anti-dsDNA) autoantibodies. The cause or causes of B cell defects in SLE are unknown. In this study, we determined the level and subcellular distribution of Lyn protein, a key negative regulator of B cell receptor signaling, and assessed whether altered Lyn expression is characteristic of B cells in the setting of SLE.Methods. Negative selection was used to isolate B lymphocytes from blood. Lipid raft signaling domains were purified from B cells obtained from 62 patients with SLE, 15 patients with rheumatoid arthritis, and 31 healthy controls, by gradient ultracentrifugation. The total Lyn protein level was determined by Western blotting, confocal microscopy, and fluorescein-activated cell sorting (FACS). The distribution of Lyn into lipid raft and nonlipid raft domains was determined by Western blotting and confocal microscopy. Lyn content in B cell subpopulations was determined by FACS. In order to assess B lymphocyte activity, we used 3 Hthymidine incorporation and enzyme-linked immunosorbent assay to measure spontaneous proliferation and IgG and cytokine production by B cells.Results. This study revealed that B lymphocytes Patients with systemic lupus erythematosus (SLE) manifest immunologic abnormalities that include spontaneous B lymphocyte proliferation, hyperresponsiveness to physiologic stimuli, and altered pattern of production of and responses to cytokines (1-3). One consequence of these abnormalities is the production of pathogenic autoantibodies to nuclear antigens, including double-stranded DNA (dsDNA). Although anti-dsDNA autoantibodies have features indicating T lymphocytedriven responses, several studies suggest that the production of anti-dsDNA autoantibodies could also be attributable to intrinsic B cell defects (4). Furthermore, studies in gene-deficient mice have revealed that defects in negative regulators of B cell receptor (BCR) signaling, CD22, Fc␥ receptor II (Fc␥RII), or CD72, result in production of anti-dsDNA autoantibodies (5-7). In patients with SLE, there is evidence that a reduction in expression of Fc␥RIIA and CD22 relates to anti-dsDNA production (8,9). However, the molecular basis for the relationship between BCR signaling defects and SLE immunopathology remains unclear.