E J Enyedy

Walter Reed National Military Medical Center, Washington, Washington, D.C., United States

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Publications (13)66.89 Total impact

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    ABSTRACT: 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.
    Cellular Immunology 03/2002; 215(2):151-61. · 1.74 Impact Factor
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    ABSTRACT: B cells from patients with systemic lupus erythematosus (SLE) display increased responses following cross-linking of the surface antigen receptor. We explored the possibility that the increased responses are at least partially due to simultaneous cross-linking of the complement receptor 2 (CR2). To this end, we stimulated fresh B cells from SLE patients with an anti-IgD antibody conjugated to the Epstein-Barr virus gp350 protein, which binds to CR2, and recorded the free intracytoplasmic calcium response during the first 10 min. Despite the fact that SLE B cells were found to express half as many surface CR2 as normal B cells, both peak responses and the percentage of responding cells were significantly increased in the former. These observations suggest that regulatory molecules such as CR2 are involved in the increased B cell responses in SLE patients. We propose that certain immune complexes that circulate in the sera of SLE patients that have anti-surface immunoglobulin specificities and are decorated with natural ligands of CR2, such as C3d, elicit and promote B cell overactivity.
    Lupus 02/2002; 11(5):299-303. · 2.78 Impact Factor
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    ABSTRACT: 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. 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. 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. 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.
    Arthritis & Rheumatology 02/2002; 46(1):163-74. · 7.48 Impact Factor
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    ABSTRACT: B lymphocytes from patients with systemic lupus erythematosus (SLE) display enhanced B cell antigen receptor (BCR)-mediated early signal transduction events, including increased fluxes of intracytoplasmic calcium ([Ca(2+)](i)). Because crosslinking of FcgammaRIIb1 (CD32) in normal B cells suppresses the BCR-initiated signal transduction process, we investigated whether the increased BCR-initiated [Ca(2+)](i) response in SLE B cells is the consequence of decreased FcgammaRIIb1-mediated suppression. To this end, we used flow cytometry to study the [Ca(2+)](i) responses of indo-1-loaded negatively gated B cells stimulated with F(ab')(2) fragments or whole IgG anti-human micro Ab. We found that the ratio of F(ab')(2) to whole anti-micro Ab [Ca(2+)](i) response was significantly lower in SLE B cells compared to B cells from patients with other systemic rheumatic diseases or normal individuals (P < 0.01). Because the surface expressions of FcgammaRIIb1 and surface IgM were similar in B cells from SLE patients and disease and normal controls, these data indicate a decrease in FcgammaRIIb-mediated suppression in SLE B cells. In addition, the whole IgG anti-micro Ab but not its F(ab')(2) fragment caused increased redistribution of SH2 domain-containing inositol 5'phosphatase in SLE compared to normal and disease control B cells. In conclusion, deficient FcgammaRIIb1-mediated suppression contributes to the augmented [Ca(2+)](i) responses of human SLE B cells.
    Clinical Immunology 12/2001; 101(2):130-5. · 3.77 Impact Factor
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    ABSTRACT: T cells from the majority of patients with systemic lupus erythematosus (SLE) express significantly lower levels of T cell receptor zeta chain, a critical signaling molecule. However, TCR/CD3 triggering of SLE T cells shows increased phosphorylation of downstream signaling intermediates and increased [Ca2+]i response, suggesting the presence of alternative signaling mechanisms. We investigated whether Fcepsilon receptor type I gamma chain (FcepsilonRIgamma) could substitute for TCR zeta chain and contribute to T cell signaling in SLE. T cells were purified from the peripheral blood of 21 patients with SLE and 5 healthy volunteers. The expression of FcepsilonRIgamma was investigated using immunoblotting, reverse transcriptase-polymerase chain reaction, and flow cytometry methods. Involvement of the FcepsilonRIgamma in T cell signaling was studied by immunoprecipitation and/or immunoblotting after TCR/CD3 stimulation. Western blotting and densitometric analysis showed that the expression of FcepsilonRIgamma in SLE T cells was 4.3-fold higher than in normal T cells (P < 0.001). Flow cytometric analyses of T lymphocyte subsets revealed that the proportions of FcepsilonRIgamma+,CD3+, FcepsilonRIgamma+,CD4+, and FcepsilonRIgamma+, CD8+ cells were significantly greater in SLE patients than in healthy controls (P < 0.001). Immunoprecipitation of SLE T cell lysates with an anti-FcepsilonRIgamma antibody showed that FcepsilonRIgamma associates with the tyrosine kinase Syk and the CD3epsilon chain, suggesting that FcepsilonRIgamma is functionally involved in TCR signaling. These results demonstrate that the FcepsilonRIgamma chain is expressed at high levels in a large proportion of SLE T cells. The increased expression of FcepsilonRIgamma chain in SLE T cells may account in part for the aberrant antigen receptor-initiated signaling and contribute to the diverse cellular abnormalities found in this disease.
    Arthritis & Rheumatology 06/2001; 44(5):1114-21. · 7.48 Impact Factor
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    ABSTRACT: 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. 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. 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. 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.
    Arthritis & Rheumatology 06/2001; 44(6):1336-50. · 7.48 Impact Factor
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    ABSTRACT: ObjectiveT cells from patients with systemic lupus erythematosus (SLE) display antigen receptor–mediated signaling aberrations associated with defective T cell receptor (TCR) ζ chain protein and messenger RNA (mRNA) expression. This study was undertaken to explore the possibility that coding-region mutations/polymorphisms of the TCR ζ chain could account for its decreased expression and altered signaling in SLE T cells.MethodsTCR ζ 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.ResultsDNA sequence analysis revealed several widely distributed missense mutations and silent polymorphisms in the coding region of the TCR ζ 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 ζ chain. A splice variant of the TCR ζ 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 ζ 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 ζ 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 ζ 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.
    Arthritis & Rheumatology 05/2001; 44(6):1336 - 1350. · 7.48 Impact Factor
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    ABSTRACT: ObjectiveT cells from the majority of patients with systemic lupus erythematosus (SLE) express significantly lower levels of T cell receptor ζ chain, a critical signaling molecule. However, TCR/CD3 triggering of SLE T cells shows increased phosphorylation of downstream signaling intermediates and increased [Ca2+]i response, suggesting the presence of alternative signaling mechanisms. We investigated whether Fcϵ receptor type I γ chain (FcϵRIγ) could substitute for TCR ζ chain and contribute to T cell signaling in SLE.MethodsT cells were purified from the peripheral blood of 21 patients with SLE and 5 healthy volunteers. The expression of FcϵRIγ was investigated using immunoblotting, reverse transcriptase–polymerase chain reaction, and flow cytometry methods. Involvement of the FcϵRIγ in T cell signaling was studied by immunoprecipitation and/or immunoblotting after TCR/CD3 stimulation.ResultsWestern blotting and densitometric analysis showed that the expression of FcϵRIγ in SLE T cells was 4.3-fold higher than in normal T cells (P < 0.001). Flow cytometric analyses of T lymphocyte subsets revealed that the proportions of FcϵRIγ+,CD3+, FcϵRIγ+,CD4+, and FcϵRIγ+, CD8+ cells were significantly greater in SLE patients than in healthy controls (P < 0.001). Immunoprecipitation of SLE T cell lysates with an anti-FcϵRIγ antibody showed that FcϵRIγ associates with the tyrosine kinase Syk and the CD3ϵ chain, suggesting that FcϵRIγ is functionally involved in TCR signaling.Conclusion These results demonstrate that the FcϵRI γ chain is expressed at high levels in a large proportion of SLE T cells. The increased expression of FcϵRI γ chain in SLE T cells may account in part for the aberrant antigen receptor–initiated signaling and contribute to the diverse cellular abnormalities found in this disease.
    Arthritis & Rheumatology 05/2001; 44(5):1114 - 1121. · 7.48 Impact Factor
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    ABSTRACT: A vast majority of systemic lupus erythematosus (SLE) patients display decreased expression of TCR zeta-chain mRNA, a critical signaling molecule implicated in the selection of the TCR repertoire and in the prevention of autoimmunity. To identify the molecular mechanisms involved in the downregulation of TCR zeta-chain transcripts in SLE T cells, we investigated the possibility of polymorphisms/mutations in the promoter and the 3' untranslated region. PCR, cloning and sequence analysis of the promoter region from the genomic DNA showed significantly higher number of polymorphisms in SLE T cells compared to non-SLE control subjects (P = 0.044). Promoter sequence was also analysed from granulocytes to delineate the possibility of somatic mutations in activated SLE T cells. Promoter polymorphisms were significantly higher in granulocytes of SLE patients compared to non-SLE controls (P = 0.048), suggesting that these polymorphisms were of genomic origin. Nucleotide analysis of the promoter sequence revealed a -76T insertion compared to the published sequence, in all of the SLE samples and controls. RT-PCR analysis of the TCR zeta-chain 3' untranslated region showed a 344 bp product in addition to the expected 906 bp product. Cloning and sequence analysis of the 344 bp product indicated that it is an alternatively spliced form with both splicing donor and acceptor sites, resulting in deletion of nucleotides 672-1233 of TCR zeta-chain mRNA. Unlike the nomal TCR zeta-chain, the expression of TCR zeta-chain with the alternatively spliced 344 bp 3' untranslated region was higher in SLE T cells compared to non-SLE controls. The number of mutations/polymorphisms in the 906 bp TCR zeta-chain 3' untranslated region were significantly higher in SLE T cells compared to non-SLE subjects (P = 0.032). Frequent mutations/polymorphisms and aberrant splicing of the downstream 3' untranslated region may affect the stability and/or transport of TCR zeta-chain mRNA, leading to its downregulation in SLE T cells.
    Journal of Autoimmunity 04/2001; 16(2):133-42. · 8.15 Impact Factor
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    ABSTRACT: Dexamethasone is a potent anti-inflammatory and immunosupressive agent that has complex, yet incompletely defined, effects on the immune response. Here, we explored the effect of dexamethasone on the expression of TCR zeta chain and TCR/CD3-induced early signaling events in human T lymphocytes. Immunoblotting studies using TCR zeta chain specific mAb showed a dose-dependent biphasic effect of dexamethasone on TCR zeta chain expression, that is, it was increased when cells were incubated with 10 nM, whereas the expression was decreased when incubated with 100 nM dexamethasone. The dose-dependent biphasic effect of dexamethsone on the TCR zeta chain expression was also revealed by FACS analysis of permeabilized cells. Time course studies showed that upregulation of the TCR zeta chain at 10 nM dexamethasone reached maximum levels at 24 h and remained elevated up to 48 h. Other subunits of the TCR/CD3 complex were minimally affected under these conditions. The increased expression of the TCR zeta chain following treatment with 10 nM dexamethasone correlated with increased anti-CD3 antibody-induced tyrosine phosphorylation of the TCR zeta chain and downstream signaling intermediate ZAP-70 and PLC gamma with faster kinetics. Similarly, the induction of TCR zeta chain expression at 10 nM dexamethasone correlated with increased and more sustained TCR/CD3-mediated [Ca(2+)](i) response. Reporter gene assays using TCR zeta chain promoter-driven luciferase gene constructs in Jurkat cells showed that treatment with 10 nM dexamethasone increased TCR zeta chain promoter activity and that the region between -160 and +58 was responsible for the observed effect. These results suggest that dexamethasone primarily acts at the transcriptional level and differentially modulates TCR zeta chain expression and antigen receptor-mediated early signaling events in human peripheral T lymphocytes.
    Cellular Immunology 03/2001; 208(1):62-71. · 1.74 Impact Factor
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    ABSTRACT: 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) zeta chain in human T cells. At 10 nM, it induced the expression of TCR zeta chain whereas at 100 nM, it inhibited its expression. In parallel to the upregulation of TCR zeta chain, the TCR/CD3-mediated [Ca(2+)](i) response was enhanced in 10 nM Dex-treated cells. However, at 100 nM, Dex treatment enhanced TCR/CD3-mediated [Ca(2+)](i) response without the induction of TCR zeta 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 [Ca(2+)](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 [Ca(2+)](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 [Ca(2+)](i) response suggesting that the effect of Dex is mediated through the glucocorticoid receptor. Although the lipid-raft association of the TCR zeta chain was not significantly increased, high-dose of Dex increased the amount of ubiquitinated form of the TCR zeta 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 zeta chain and form more distinct clusters upon TCR/CD3 stimulation. These results suggest that high dose of Dex perturbs the membrane distribution of TCR zeta chain leading to more functional signaling clusters that result in increased TCR/CD3-mediated [Ca(2+)](i) response independent of TCR zeta chain expression.
    Journal of Cellular Biochemistry 01/2001; 83(3):401-13. · 3.06 Impact Factor
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    ABSTRACT: After heat treatment, human T lymphocytes downregulate the T-cell receptor (TCR)/CD3-mediated [Ca(2+)](i) response and production of inositol triphosphate. Here we demonstrate that heat treatment of T lymphocytes at sublethal temperature decreases the expression of TCR zeta chain, which plays a critical role in the regulation of TCR/CD3-mediated signal transduction. Downregulation of TCR zeta 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 epsilon or TCR alphabeta chains was minimally affected. Consistent with the decrease in TCR zeta 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 zeta chain content in the detergent-insoluble membrane fraction showed that heat treatment induces translocation of soluble TCR zeta chain to the cell membranes. In addition, the mRNA level of TCR zeta chain was reduced in heat-treated T cells. Correlative with the downregulation of TCR zeta chain mRNA, the level of the TCR zeta 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 zeta chain by increasing its association with the membranes and decreasing the transcription of the TCR zeta gene. Decreased expression of the TCR zeta chain is apparently responsible for the decreased TCR/CD3 responses of T cells.
    Journal of Cellular Biochemistry 10/2000; 79(3):416-26. · 3.06 Impact Factor
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    ABSTRACT: The fate of the lymphocyte is determined by integration of signals delivered after the binding of antigen to the surface antigen receptor, signals delivered by cytokines that bind to their surface receptors, and signals initiated after the engagement of other surface receptors, known as costimulatory molecules. The summation of this input determines whether the immune cell will become stimulated, ignore the signal (anergy), or die (apoptosis). Antigen-receptor signaling events are abnormal in lupus lymphocytes, manifested by increased calcium responses and hyperphosphorylation of several cytosolic protein substrates. Further down, at the gene transcription level, the activity of the nuclear factor kappaB is decreased. These events are underwritten by defective T cell receptor zeta chain expression, overexpression of the gamma chain of the Fc(epsilon)RI that functions as an alternate of zeta chain, and decreased p65 -Rel A protein that is responsible for the inducible NFkappaB activity. Accumulated research data have enabled us to begin deciphering the molecular basis of the abnormal lupus lymphocyte and may lead to the development of new medicinal treatments for lupus.
    Current Opinion in Rheumatology 10/2000; 12(5):355-63. · 5.19 Impact Factor

Publication Stats

360 Citations
66.89 Total Impact Points

Institutions

  • 2002
    • Walter Reed National Military Medical Center
      Washington, Washington, D.C., United States
  • 2000–2002
    • Walter Reed Army Institute of Research
      • Center for Military Psychiatry and Neuroscience Research
      Silver Spring, Maryland, United States
  • 2000–2001
    • Uniformed Services University of the Health Sciences
      • Department of Medicine
      Maryland, United States