Carolyn U. Fisher

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.

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

Diminished expression of TCR ζ and reciprocal up-regulation and association of FcRγ 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 FcRγ 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-γ1 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.

Increased caspase-3 expression and activity contribute to reduced CD3ζ expression in systemic lupus erythematosus T cells

T cells isolated from patients with systemic lupus erythematosus (SLE) express low levels of CD3ζ-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 CD3ζ 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 CD3ζ and enhanced its expression. In addition, Z-Asp-Glu-Val-Asp-FMK treatment increased the association of CD3ζ with lipid rafts and simultaneously reversed the abnormal lipid raft preclustering, heightened TCR-induced calcium responses, and reduced the expression of FcRγ-chain exclusively in SLE T cells. We conclude that caspase-3 inhibitors can normalize SLE T cell function by limiting the excessive digestion of CD3ζ-chain and suggest that such molecules can be considered in the treatment of this disease.

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.

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.

High dose of dexamethasone upregulates TCR/CD3-induced calcium response independent of TCR ζ chain expression in human T lymphocytes*

Glucocorticoids are very potent anti‐inflammatory and immunosuppressive agents that modulate cellular immune responses, although, the molecular mechanisms that impart their complex effects have not been completely defined. We have previously demonstrated that dexamethasone (Dex), a synthetic glucocorticoid, biphasically modulates the expression of TCR (T cell receptor) ζ chain in human T cells. At 10 nM, it induced the expression of TCR ζ chain whereas at 100 nM, it inhibited its expression. In parallel to the upregulation of TCR ζ chain, the TCR/CD3‐mediated [Ca2+]i response was enhanced in 10 nM Dex‐treated cells. However, at 100 nM, Dex treatment enhanced TCR/CD3‐mediated [Ca2+]i response without the induction of TCR ζ chain expression. Because the classical transcriptional model of glucocorticoid action cannot account for the effects of high dose of Dex, here we studied alternative mechanisms of action. We show that, increased and more sustained TCR/CD3‐mediated [Ca2+]i response was also observed in 100 nM Dex‐treated cells in the presence of actinomycin D or cycloheximide suggesting that cellular transcription and/or de novo protein synthesis are not required for the induction. The TCR/CD3‐mediated hyper [Ca2+]i response in 100 nM Dex‐treated cells was readily reversible by short‐term culture in steroid‐free medium. RU‐486, a competitive antagonist of Dex, inhibited the increase in [Ca2+]i response suggesting that the effect of Dex is mediated through the glucocorticoid receptor. Although the lipid‐raft association of the TCR ζ chain was not significantly increased, high‐dose of Dex increased the amount of ubiquitinated form of the TCR ζ chain in the cell membrane along with increased levels of actin. Fluorescence microscopy showed that high‐dose of Dex alters the distribution of the TCR ζ chain and form more distinct clusters upon TCR/CD3 stimulation. These results suggest that high dose of Dex perturbs the membrane distribution of TCR ζ chain leading to more functional signaling clusters that result in increased TCR/CD3‐mediated [Ca2+]i response independent of TCR ζ chain expression. J. Cell. Biochem. 83: 401–413, 2001.