Vishal G. Warke

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 FcRγ 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.

The FcRγ Subunit and Syk Kinase Replace the CD3ζ-Chain and ZAP-70 Kinase in the TCR Signaling Complex of Human Effector CD4 T Cells

The TCR-mediated signals required to activate resting T cells have been well characterized; however, it is not known how TCR-coupled signals are transduced in differentiated effector T cells that coordinate ongoing immune responses. Here we demonstrate that human effector CD4 T cells up-regulate the expression of the CD3ζ-related FcRγ signaling subunit that becomes part of an altered TCR/CD3 signaling complex containing CD3ε, but not CD3ζ. The TCR/CD3/FcRγ complex in effector cells recruits and activates the Syk, but not the ZAP-70, tyrosine kinase. This physiologic switch in TCR signaling occurs exclusively in effector, and not naive or memory T cells, suggesting a potential target for manipulation of effector responses in autoimmune, malignant, and infectious diseases.

T cell signaling abnormalities in systemic lupus erythematosus are associated with increased mutations/polymorphisms and splice variants of T cell receptor ζ chain messenger RNA

OBJECTIVE T cells from patients with systemic lupus erythematosus (SLE) display antigen receptor-mediated signaling aberrations associated with defective T cell receptor (TCR) zeta chain protein and messenger RNA (mRNA) expression. This study was undertaken to explore the possibility that coding-region mutations/polymorphisms of the TCR zeta chain could account for its decreased expression and altered signaling in SLE T cells. METHODS TCR zeta chain mRNA from 48 SLE patients, 18 disease controls, and 21 healthy volunteers was reverse transcribed, amplified by polymerase chain reaction, and cloned, and complementary DNA (cDNA) was sequenced. DNA sequences from multiple clones were analyzed for silent single-nucleotide polymorphisms, mutations, and splice variations, to promote the identification of heterozygosity. RESULTS DNA sequence analysis revealed several widely distributed missense mutations and silent polymorphisms in the coding region of the TCR zeta chain, which were more frequent in SLE patients than in patients with other rheumatic diseases or healthy controls (P < 0.0001). Several of the missense mutations were located in the 3 immunoreceptor tyrosine activation motifs or the GTP binding domain, and this could lead to functional alterations in the TCR zeta chain. A splice variant of the TCR zeta chain with a codon CAG (glutamine) insertion between exons IV and V was found in half of the SLE and control samples. Two larger spliced isoforms of the TCR zeta chain, with an insertion of 145 bases and 93 bases between exons I and II, were found only in SLE T cells. We also identified various alternatively spliced forms of the TCR zeta chain resulting from the deletion of individual exons II, VI, or VII, or a combined deletion of exons V and VI; VI and VII; II, III, and IV; or V, VI, and VII in SLE T cells. The frequency of the deletion splice variants was significantly higher in SLE than in control samples (P = 0.004). These variations were observed in cDNA and may not reflect the status of the genomic DNA. CONCLUSION These findings demonstrate that heterogeneous mutations/polymorphisms and alternative splicing of TCR zeta chain cDNA are more frequent in SLE T cells than in T cells from non-SLE subjects and may underlie the molecular basis of known T cell signaling abnormalities in this disease.

Decreased Stability and Translation of T Cell Receptor ζ mRNA with an Alternatively Spliced 3′-Untranslated Region Contribute to ζ Chain Down-regulation in Patients with Systemic Lupus Erythematosus

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

Forced Expression of the Fc Receptor γ-Chain Renders Human T Cells Hyperresponsive to TCR/CD3 Stimulation

High level expression of FcεRIγ chain replaces the deficient TCR ζ-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 FcεRIγ gene. We found that the overexpressed FcεRIγ chain colocalizes with the CD3ε 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 FcεRIγ is associated with increased phosphorylation of Syk kinase, while the endogenous TCR ζ-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.

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 ζ 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 ζ chain gene, whereas, it upregulates the expression if its homologous gene Fcε receptor I γ 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. J. Cell. Biochem. 85: 459–469, 2002. Published 2002 Wiley‐Liss, Inc.

NaCN-induced chemical hypoxia is associated with altered gene expression.

Sodium cyanide (NaCN)-induced chemical hypoxia is known to increase intracellular free calcium concentration and reduce cell survival, but its effect on gene expression has not been studied. In this study, we designed primers to conduct a rapid and reliable assay for the expression of mRNA of inducible nitric oxide synthase (iNOs), tumor suppressor protein p53, Bcl-2, heat shock protein 70 (HSP-70), and β-actin in human intestinal epithelial T84 cells and Jurkat T cells. NaCN-induced chemical hypoxia increased iNOs and HSP-70 mRNA in both types of cells, whereas p53 and Bcl-2 mRNA were singularly induced in T84 cells and Jurkat T cells, respectively. In both cell types, treatment of hypoxic cells with a reversible selective iNOs inhibitor, Nω-nitro-L-arginine (LNNA), blocked iNOs, Bcl-2, and HSP-70 mRNA, but increased p53. The NaCN-induced hypoxia was also found to increase caspase-3 cellular activity in both cell types. Treatment with LNNA alone decreased the basal caspase-3 cellular activity. A prior treatment of LNNA significantly inhibited the NaCN-induced increase in the cellular activity of this apoptotic enzyme. This is the first report to show that NaCN-induced chemical hypoxia alters both stress-related gene expression and caspase-3 cellular activity and can be regulated by the iNOs inhibitor LNNA. Since NaCN has been included in the ‘National chemical terrorism threat’ list, by the US Department of Defense, our studies provide useful insight in the development of molecular sensors to detect early exposure to this chemical terrorism threat.

Constitutive NO synthase regulates the Na+/Ca2+ exchanger in human T cells: role of [Ca2+]i and tyrosine phosphorylation.

For many types of cells, heat stress leads to an increase in intracellular free calcium concentration ([Ca2+]i) that has been shown to trigger a wide variety of cellular responses. In T lymphocytes, for example, heat stress stimulates pathways that make them more susceptible to Fas/CD95‐mediated apoptosis. Because of our interest in understanding more about the response of lymphocytes to various stressors, we used human peripheral and Jurkat T lymphocytes to investigate the effect of heat stress on calcium homeostasis. We found that peripheral and Jurkat T cells both exhibit cNOs activity but not iNOs activity. Heat stress increased NO production, which was inhibited by LNNA (a cNOs inhibitor) but not L‐NIL (an iNOs inhibitor). Heat stress increased [Ca2+]i in Jurkat T cells by decreasing the Km of the cell surface membrane Na+/Ca2+ exchanger for extracellular Ca2+. Heating also increased cNOs phosphorylation at tyrosine residues. In cells incubated with LNNA, heat stress promoted an increase in [Ca2+]i and a decrease in [Na+]i greater than in cells heated without LNNA, a larger decrease in Km of the Na+/Ca2+ exchanger for extracellular Ca2+, and decreased phosphorylation of cNOs. Our results suggest that cNOs plays an important regulatory role after heat stress. Heating appears to increase the phosphorylation of cNOs that is complexed with the Na+/Ca2+ exchanger to decrease its activity. This process is related to increased expression of Fas/CD95 on the cell surface, which might explain the apoptotic diathesis of lymphocytes after heat stress. J. Cell. Biochem. 89: 1030–1043, 2003. Published 2003 Wiley‐Liss, Inc.

Oxidative stress is involved in the heat stress-induced downregulation of TCR ζ chain expression and TCR/CD3-mediated [Ca2+]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.

Heat stress downregulates TCR ? chain expression in human T lymphocytes

After heat treatment, human T lymphocytes downregulate the T‐cell receptor (TCR)/CD3‐mediated [Ca2+]i response and production of inositol triphosphate. Here we demonstrate that heat treatment of T lymphocytes at sublethal temperature decreases the expression of TCR ζ chain, which plays a critical role in the regulation of TCR/CD3‐mediated signal transduction. Downregulation of TCR ζ chain in heat‐treated T cells was observed at 8 h and reached a maximum at 16 h. Under these conditions, the expression of CD3 ϵ or TCR αβ chains was minimally affected. Consistent with the decrease in TCR ζ chain, a reduction in the level of TCR/CD3 induced tyrosine phosphorylation of several cellular protein substrates, and a delay in the kinetics of peak tyrosine phosphorylation was observed in heat‐treated T cells. Interestingly, analysis of the TCR ζ chain content in the detergent‐insoluble membrane fraction showed that heat treatment induces translocation of soluble TCR ζ chain to the cell membranes. In addition, the mRNA level of TCR ζ chain was reduced in heat‐treated T cells. Correlative with the downregulation of TCR ζ chain mRNA, the level of the TCR ζ chain transcription factor Elf‐1 was also reduced in heat‐treated cells. We conclude that heat stress causes a decrease in the level of TCR ζ chain by increasing its association with the membranes and decreasing the transcription of the TCR ζ gene. Decreased expression of the TCR ζ chain is apparently responsible for the decreased TCR/CD3 responses of T cells. J. Cell. Biochem. 79:416–426, 2000.