Differential expression and molecular associations of Syk in systemic lupus erythematosus T cells.

Diminished expression of TCR zeta and reciprocal up-regulation and association of FcRgamma with the TCR/CD3 complex is a hallmark of systemic lupus erythematosus (SLE) T cells. In this study we explored whether differential molecular associations of the spleen tyrosine kinase Syk that preferentially binds to FcRgamma contribute to pathological amplification of signals downstream of this "rewired TCR" in SLE. We detected higher amounts of Syk expression and activity in SLE compared with normal T cells. Selective inhibition of the activity of Syk reduced the strength of TCR-induced calcium responses and slowed the rapid kinetics of actin polymerization exclusively in SLE T cells. Syk and ZAP-70 also associated differently with key molecules involved in cytoskeletal and calcium signaling in SLE T cells. Thus, while Vav-1 and LAT preferentially bound to Syk, phospholipase C-gamma1 bound to both Syk and ZAP-70. Our results show that differential associations of Syk family kinases contribute to the enhanced TCR-induced signaling responses in SLE T cells. Thus, we propose molecular targeting of Syk as a measure to control abnormal T cell responses in SLE.

FcRgamma chain does not replace CD3zeta chain in CD3zeta-deficient T lymphocytes of patients with gastric adenocarcinoma.

Defective CD3zeta chain expression has been reported in T lymphocytes of patients with inflammatory diseases, such as systemic lupus erythematosus or osteoarthritis, and with cancer. In lupus, the absent CD3zeta chain is replaced by the FcRgamma chain, rendering the T cells hyper responsive. However, there are no data on T lymphocytes from patients with cancer. In this study, the presence of the FcRgamma chain and its associated kinase, Syk, was analysed in patients with gastric adenocarcinoma and healthy subjects. Western blot and immunoprecipitation experiments were carried out with total cell or lipid raft extracts from fresh peripheral blood mononuclear cells or T lymphocytes, and Herpesvirus saimiri-derived T-cell lines (of blood or tissue origin). Our results revealed that the absent CD3zeta chain in cancer T lymphocytes was not replaced by FcRgamma either in fresh T cells or T-cell lines, in contrast to lupus T cells. This altered expression of signalling molecules in T lymphocytes of cancer patients, would explain their low proliferative capacity. Our T-cell lines represent tools to unveil the signalling abnormalities of cancer T lymphocytes.

Stability and translation of TCR zeta mRNA are regulated by the adenosine-uridine-rich elements in splice-deleted 3' untranslated region of zeta-chain.

Systemic lupus erythematosus (SLE) T cells display reduced expression of TCR zeta protein. Recently, we reported that in SLE T cells, the residual TCR zeta protein is predominantly derived from an alternatively spliced form that undergoes splice deletion of 562 nt (from 672 to 1233 bases) within the 3' untranslated region (UTR) of TCR zeta mRNA. The stability and translation of the alternatively spliced form of TCR zeta mRNA are low compared with that of the wild-type TCR zeta mRNA. We report that two adenosine-uridine-rich sequence elements (AREs), defined by the splice-deleted 3' UTR region, but not an ARE located upstream are responsible for securing TCR zeta mRNA stability and translation. The stabilizing effect of the splice-deleted region-defined AREs extended to the luciferase mRNA and was not cell type-specific. The findings demonstrate distinct sequences within the splice-deleted region 672 to 1233 of the 3' UTR, which regulate the transcription, mRNA stability, and translation of TCR zeta mRNA. The absence of these sequences represents a molecular mechanism that contributes to altered TCR zeta-chain expression in lupus.

Increased caspase-3 expression and activity contribute to reduced CD3zeta expression in systemic lupus erythematosus T cells.

T cells isolated from patients with systemic lupus erythematosus (SLE) express low levels of CD3zeta-chain, a critical molecule involved in TCR-mediated signaling, but the involved mechanisms are not fully understood. In this study we examined caspase-3 as a candidate for cleaving CD3zeta in SLE T cells. We demonstrate that SLE T cells display increased expression and activity of caspase-3. Treatment of SLE T cells with the caspase-3 inhibitor Z-Asp-Glu-Val-Asp-FMK reduced proteolysis of CD3zeta and enhanced its expression. In addition, Z-Asp-Glu-Val-Asp-FMK treatment increased the association of CD3zeta with lipid rafts and simultaneously reversed the abnormal lipid raft preclustering, heightened TCR-induced calcium responses, and reduced the expression of FcRgamma-chain exclusively in SLE T cells. We conclude that caspase-3 inhibitors can normalize SLE T cell function by limiting the excessive digestion of CD3zeta-chain and suggest that such molecules can be considered in the treatment of this disease.

Decreased stability and translation of T cell receptor zeta mRNA with an alternatively spliced 3'-untranslated region contribute to zeta chain down-regulation in patients with systemic lupus erythematosus.

The molecular mechanisms involved in the aberrant expression of T cell receptor (TCR) zeta chain of patients with systemic lupus erythematosus are not known. Previously we demonstrated that although normal T cells express high levels of TCR zeta mRNA with wild-type (WT) 3' untranslated region (3' UTR), systemic lupus erythematosus T cells display significantly high levels of TCR zeta mRNA with the alternatively spliced (AS) 3' UTR form, which is derived by splice deletion of nucleotides 672-1233 of the TCR zeta transcript. Here we report that the stability of TCR zeta mRNA with an AS 3' UTR is low compared with TCR zeta mRNA with WT 3' UTR. AS 3' UTR, but not WT 3' UTR, conferred similar instability to the luciferase gene. Immunoblotting of cell lysates derived from transfected COS-7 cells demonstrated that TCR zeta with AS 3' UTR produced low amounts of 16-kDa protein. In vitro transcription and translation also produced low amounts of protein from TCR zeta with AS 3' UTR. Taken together our findings suggest that nucleotides 672-1233 bp of TCR zeta 3' UTR play a critical role in its stability and also have elements required for the translational regulation of TCR zeta chain expression in human T cells.

Defective CD3zeta chain expression in Herpesvirus saimiri (HVS)-derived T-cell lines in gastric adenocarcinoma.

Low expression of the CD3zeta chain has been reported in patients with cancer and it has been suggested that tumor-derived factors are involved in its downregulation. The expression of CD3zeta chain was measured in T-cell lines from patients with gastric adenocarcinoma and healthy volunteers and grown in vitro for several months and, hence, in the absence of any tumor-derived factors. T-cell lines of mucosal origin were obtained by Herpesvirus saimiri transformation from gastric cancer patients. The expression of CD3zeta and CD3epsilon was measured by flow cytometry and Western-blot analysis. Calcium mobilization and apoptosis rate were also measured. The levels of CD3zeta, but not CD3epsilon, chain on the cell surface were significantly reduced in T-cell lines derived from patients with gastric cancer when cultured in the absence of IL-2. Western-blot analysis of total cell extracts or lipid raft fractions confirmed this finding. Calcium mobilization, a measure of signal transduction, was reduced in T cell lines from patients with gastric cancer. We conclude that T cells from patients with cancer express lower levels of CD3zeta. This downregulation is not caused by a direct effect of tumor-derived factors but, rather, it appears to be inherent to the patient cells. The low CD3zeta expression would render T lymphocytes unable to control the growth of tumor cells.

Alterations in lipid raft composition and dynamics contribute to abnormal T cell responses in systemic lupus erythematosus.

In response to appropriate stimulation, T lymphocytes from systemic lupus erythematosus (SLE) patients exhibit increased and faster intracellular tyrosine phosphorylation and free calcium responses. We have explored whether the composition and dynamics of lipid rafts are responsible for the abnormal T cell responses in SLE. SLE T cells generate and possess higher amounts of ganglioside-containing lipid rafts and, unlike normal T cells, SLE T cell lipid rafts include FcRgamma and activated Syk kinase. IgM anti-CD3 Ab-mediated capping of TCR complexes occurs more rapidly in SLE T cells and concomitant with dramatic acceleration of actin polymerization kinetics. The significance of these findings is evident from the observation that cross-linking of lipid rafts evokes earlier and higher calcium responses in SLE T cells. Thus, we propose that alterations in the lipid raft signaling machinery represent an important mechanism that is responsible for the heightened and accelerated T cell responses in SLE.

Reconstitution of deficient T cell receptor zeta chain restores T cell signaling and augments T cell receptor/CD3-induced interleukin-2 production in patients with systemic lupus erythematosus.

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) zeta chain, a subunit of the TCR/CD3 complex. This study was undertaken to explore the possibility that forced expression of TCR zeta 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 zeta 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 zeta chain-transfected cells. RESULTS: In SLE T cells transfected with TCR zeta 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 gamma chain was diminished in SLE T cells transfected with the zeta chain expression vector, and the surface membrane clusters of cell signaling molecules were redistributed to a more continuous pattern. TCR zeta 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 kappaB in the nuclear fractions of these T cells. CONCLUSION: These results suggest that reconstitution of deficient TCR zeta chain can reverse the TCR/CD3-mediated signaling abnormalities as well as the defective IL-2 production in T cells of patients with SLE.

Forced expression of the Fc receptor gamma-chain renders human T cells hyperresponsive to TCR/CD3 stimulation.

High level expression of Fc epsilon RI gamma chain replaces the deficient TCR zeta-chain and contributes to altered TCR/CD3-mediated signaling abnormalities in T cells of patients with systemic lupus erythematosus. Increased responsiveness to Ag has been considered to lead to autoimmunity. To test this concept, we studied early signaling events and IL-2 production in fresh cells transfected with a eukaryotic expression vector encoding the Fc epsilon RI gamma gene. We found that the overexpressed Fc epsilon RI gamma chain colocalizes with the CD3 epsilon chain on the surface membrane of T cells and that cross-linking of the new TCR/CD3 complex leads to a dramatic increase of intracytoplasmic calcium concentration, protein tyrosine phosphorylation, and IL-2 production. We observed that overexpression of Fc epsilon RI gamma is associated with increased phosphorylation of Syk kinase, while the endogenous TCR zeta-chain is down-regulated. We propose that altered composition of the CD3 complex leads to increased T cell responsiveness to TCR/CD3 stimulation and sets the biochemical grounds for the development of autoimmunity.

Oxidative stress is involved in the heat stress-induced downregulation of TCR zeta chain expression and TCR/CD3-mediated [Ca(2+)](i) response in human T-lymphocytes.

Exposure of human T-lymphocytes to heat downregulates TCR zeta chain expression and inhibits (TCR)/CD3-mediated production of inositol triphosphate and [Ca(2+)](i) signaling. Here we investigated whether oxidative stress is involved in the heat-induced downregulation of TCR/CD3-mediated signaling. To this end, we have studied the effect of a thiol antioxidant, N-acetyl-L-cysteine (NAC), and a non-thiol antioxidant, allopurinol, on heat-induced downregulation of TCR/CD3-mediated signaling. We found that preincubation of cells with 10mM NAC significantly reversed the downregulation of TCR/CD3-mediated [Ca(2+)](i) response and restored the suppression of TCR zeta chain protein expression as well as prevented its increased membrane distribution in heat-treated cells. NAC also reversed the downregulation of TCR zeta chain mRNA expression and the active 94kDa TCR zeta chain transcription factor, Elf-1, in heat-treated cells. Consistent with the increase in the TCR zeta chain, preincubation with NAC increased the levels of antigen receptor-induced tyrosine phosphorylation of several cytosolic proteins. Finally, treatment with NAC was able to reverse the suppression of IL-2 production in heat-treated cells. Inactive analog, N-acetylserine, failed to reverse the heat-induced downregulation of TCR/CD3-mediated signaling. Allopurinol, another potent non-thiol antioxidant, also restored the TCR/CD3-mediated [Ca(2+)](i) response in heat-treated cells. These results demonstrate that antioxidants restore the expression of TCR zeta chain and reverse the TCR/CD3-mediated signaling abnormalities associated with heat stress and suggest that heat shock-induced oxidative stress is a mediator of the heat-induced biochemical damage that leads to downregulation of signaling in human T-lymphocytes.

Effect of trichostatin A on human T cells resembles signaling abnormalities in T cells of patients with systemic lupus erythematosus: a new mechanism for TCR zeta chain deficiency and abnormal signaling.

Trichostatin A (TSA) is a potent reversible inhibitor of histone deacetylase, and it has been reported to have variable effects on the expression of a number of genes. In this report, we show that TSA suppresses the expression of the T cell receptor zeta chain gene, whereas, it upregulates the expression if its homologous gene Fc(epsilon) receptor I gamma chain. These effects are associated with decreased intracytoplasmic-free calcium responses and altered tyrosine phosphorylation pattern of cytosolic proteins. Along with these effects, we report that TSA suppresses the expression of the interleukin-2 gene. The effects of TSA on human T cells are predominantly immunosuppressive and reminiscent of the signaling aberrations that have been described in patients with systemic lupus erythematosus.

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

OBJECTIVE: T cells from the majority of patients with systemic lupus erythematosus (SLE) display antigen receptor-mediated signaling aberrations associated with defective T cell receptor (TCR) zeta chain expression. The TCR zeta chain, a critical signaling molecule, exists in multiple molecular forms and membrane fractions with distinct functions in antigen-mediated signaling processes. This study was undertaken to investigate the complete spectrum of expression of the different forms and distribution of the TCR zeta chain in SLE T cells. METHODS: T cells were isolated from 48 SLE patients and 21 healthy subjects. The expression of various forms of the TCR zeta chain was investigated by immunoblotting with specific antibodies. The lipid raft-associated form of the zeta chain was determined by quantitating the solubilized zeta chain after disruption of the lipid rafts by cholesterol depletion using methyl-betacyclodextrin. The distribution of the zeta chain was investigated by fluorescence microscopy. RESULTS: The phosphorylated 21- and 23-kd forms and the detergent-insoluble membrane-associated form of the TCR zeta chain and alternatively spliced zeta chain were significantly decreased in SLE T cells. In contrast, major ubiquitinated forms of the zeta chain were increased in these cells. We also identified up-regulation of a novel 14-kd form of the zeta chain in SLE T cells. Resting SLE T cell membranes had an increased percentage of the residual membrane-bound zeta chain in the lipid rafts. Fluorescence microscopy findings indicated that the residual zeta chain is more clustered on the cell membranes of SLE T cells. CONCLUSION: These results suggest that, in addition to the 16-kd form, expression of other molecular forms and fractions of the TCR zeta chain as well as its membrane distribution are abnormal in SLE T cells. Increased lipid raft association and surface clustering of the zeta chain may explain the molecular mechanisms underlying the signaling abnormalities in these cells.