Proc. Nati. Acad. Sci. USAVol. 89, pp. 300-304, January 1992Biochemistry

Sulfhydryl oxidation down-regulates T-cell signaling and inhibitstyrosine phosphorylation of phospholipase Cyl

(T-celi antigen receptor/N-ethylmaleimide/neutrophils/glutathione/phosphotyrosine)

STEVEN B. KANNER*t, TERRANCE J. KAVANAGHL, ANGELIKA GROSSMANN§, SHIu-LOK Hu*,JOSEPH B. BOLEN¶, PETER S. RABINOVITCH§, AND JEFFREY A. LEDBETTER**Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA 98121; Departments of *Medicine and §Pathology, University of Washington, Seattle,WA 98195; and $Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543

Communicated by Leonard A. Herzenberg, October 8, 1991 (received for review July 15, 1991)

ABSTRACT Early events in both T-cell receptor (CD3)-and CD4-induced signal transduction pathways include tyro-sine phosphorylation of protein substrates, the generation ofphosphatidylinositol-phosphate breakdown products, and themobilization of intraceflular Ca2'. Oxidative stress in T cellsmediated by sulfhydryl-reactive nonpolar maleimides wasshown previously to down-regulate both receptor-mediatedCa2+ mobilization and interleukin 2 production. Here we showthat N-ethylmaleimide suppresses both CD3- and CD4-inducedCa2' responses in human T cells correlating with a reductionin the level of phospholipase Cy1 (PLCyl) tyrosine phosphor-ylation. The inhibition of tyrosine phosphorylation of PLCy1and additional protein substrates was observed at concentra-tions of N-ethylmaleimide above 20 ,uM, whereas lower con-centrations of oxidant appeared to increase tyrosine kinaseactivity following cell stimulation. Sulfhydryl oxidation did notdirectly affect the catalytic activity of PLCy1, since immu-nopurified enzyme from N-ethylnaleimide-treated T cells wasfully active. Although N-ethylmaleimide treatment of T cellsdid not cause a direct effect on total pp561k kinase activitymeasured in vitro, the interaction between CD4 and pp56Ik wasoxidation-sensitive in vivo. However, CD3-induced signalingwas inhibited at N-ethylmaleimide concentrations lower thanthat required for CD4/pp56Ick dissociation, suggesting thatCD3-associated tyrosine kinase activity involves acutely sensi-tive regulatory thiols. In addition to chemically induced sulf-hydryl oxidation, naturally regulated cellular redox statesappear to dictate the potential for T-cell responsiveness, sincedegranulating human peripheral blood neutrophils inhibitedCD3-induced Ca2+ mobilization in T lymphocytes. These dataindicate that signal transduction in T cells involves the activa-tion of PLCy1 by tyrosine phosphorylation through an oxida-tion-sensitive intermediate between surface receptors and ty-rosine kinases, perhaps including the interaction between CD4and pp56Ick.

Stimulation ofT cells through specific antigen presentation orby crosslinking surface receptors with antibody results inactivation of the phosphatidylinositol-specific phospholipaseC (PI-PLC) pathway. Early events in the signal transductioncascade include tyrosine phosphorylation of multiple cellularsubstrates (1, 2), generation of the PI-PLC second messen-gers diacylglycerol and inositol trisphosphate (3-5), and themobilization of intracellular Ca2' (1, 5, 6). These signalsfacilitate activation of protein-serine kinases, expression ofthe interleukin 2 (IL-2) receptor a chain (CD25), IL-2 pro-duction, and ultimately T-cell clonal expansion and effectorfunctions (5).

Studies of the redox state ofT lymphocytes have suggestedthat T-cell responses are affected by oxidative stress. Spe-cifically, receptor-induced mobilization of intracellular Ca2"is inhibited by the permeant sulfhydryl-reactive nonpolarmaleimide, N-ethylmaleimide (NEM) (7, 8). In addition, theimmunosuppressive effects of normal neutrophils on T lym-phocytes involve oxidative metabolic products of the neu-trophil myeloperoxidase system (9, 10). Further, T cells withlow levels ofglutathione, a metabolic regulator of thiol levels,have decreased capacity to proliferate (11, 12). Clearly, thesestudies indicate that the redox status of intracellular thiolsplays a role in the responsiveness of T cells to externalstimuli.The T-cell receptor (CD3) signals through an associated

protein-tyrosine kinase activity (5). Further, CD4 receptorsassociate with the tyrosine kinase pp56lck (13-15), which isactivated by CD4 receptor crosslinking, thereby stimulatingtyrosine phosphorylation and downstream PI-PLC signaling(13-18). CD4 interaction with CD3 increases PI-PLC cou-pling (19) and is critical for antigen-specific T-cell respon-siveness (16-18). In this report, we show that thiol-specificoxidation in T cells results in down-modulation of CD3- andCD4-induced protein-tyrosine kinase activity, PLC'yl tyro-sine phosphorylation, and PLCyl activity measured as afunction of Ca2' mobilization. This may represent a mech-anism whereby neutrophils regulate T-lymphocyte stimula-tory responses.

MATERIALS AND METHODSCell Culture. Fresh human peripheral blood lymphocytes

(PBLs) were isolated by density-gradient centrifugation onlymphocyte separation medium (Organon). Neutrophils wereisolated from human PBLs on Ficoll/Hypaque gradients(ICN). The CEM.6 T-cell line (20) was grown in RPMI 1640supplemented with 10o fetal bovine serum.Monoclonal Antibodies (mAbs) and Receptor Crosslinking.

CD3, CD4, and PLCyl mAbs were described previously (19,21). Rabbit antiserum to PLCyl was generated with ovalbu-min-conjugated PLCyl peptide (residues 1268-1290) andrabbit antiserum to pp56lck was generated as described (22).mAbs were conjugated to biotin with biotin-succinimide(Sigma) as described (19). Avidin (Sigma) was used tocrosslink biotin-conjugated mAbs at a 5:1 (wt/wt) ratio. CD3and CD4 receptors were ligated by incubating 107 cells withbiotinylated mAbs (5 ,ug/ml) each for 5 min at 37°C, followedby the addition of S ,tg of avidin per microgram of mAb.

Abbreviations: NEM, N-ethylmaleimide; PBL, peripheral bloodlymphocyte; PLC, phospholipase C; PI-PLC, phosphatidylinositol-specific PLC; mAb, monoclonal antibody; [Ca2l]i, intracellular Ca2+concentration; IL-2, interleukin 2; PIP, phosphatidylinositol 4,5-bisphosphate.tTo whom reprint requests should be addressed.

300

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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NEM Oxidation of Cells. Cells (107 per ml) were incubatedin growth medium with various concentrations of NEM(Sigma) for 15 min at 370C.

Immunoprecipitation, Immune-Complex Kinase Assays,and Western Immunoblotting. Immunoprecipitation reac-tions, immune-complex kinase assays, and Western immu-noblotting with affinity-purified rabbit antibody to phospho-tyrosine or mAbs were performed as described (23-25).

Cytoplasmic Ca2+ Measurements. Intracellular Ca2+ con-centration ([Ca2+]) was measured using indo-1 (MolecularProbes) and a model 50 HH/2150 flow cytometer (OrthoInstruments, Westwood, MA) as described (26). For neutro-phil assays, neutrophils stimulated with opsonized zymosanwere incubated with PBLs at a ratio of 1:1 for 3 hr prior to[Ca2+], measurements.PLCyl Assay. Immunoprecipitates of PLCyl were pre-

pared using rabbit antiserum to PLCy1 on protein A-Seph-arose beads. The washed beads were incubated with 0.2 ,Ci(7.4 kBq) of [3H]phosphatidylinositol 4,5-bisphosphate([3H]PIP2; New England Nuclear) in 2.5% octyl 8-D-glucoside/1 mM CaCl2/0.3 mM PIP2/0.6 mM EGTA/60 mMKCl/30 mM sodium phosphate, pH 7.5, for 20 min at 37°C.The reaction mixture supernatant (0.03 ml) was added to 0.1ml of 1% bovine serum albumin, incubated with 0.5 ml of 15%trichloroacetic acid, and centrifuged at 10,000 X g for 10 min.The supernatant was extracted four times in ethyl ether andthe aqueous phase was measured for water-soluble inositolphosphate products of PLCyl activity by scintillation count-ing.

RESULTSOxidative Suppression of Intracellular Ca2+ Mobilization.

The inhibition of Ca2+ fluxes in Jurkat T cells by nonpolarmaleimides has been reported (7). To determine whetherNEM affected Ca2+ mobilization in normal PBL-derived Tcells, [Ca2+], was measured in CD20-negative PBLs afterCD3 stimulation following a 15-min treatment with variousconcentrations of NEM up to 40 ,M (Fig. 1A). Partialsuppression of intracellular Ca2+ mobilization was observedat 10 tM NEM, whereas complete inhibition was noted aftertreatment with 20-40 ,tM NEM. A slight but reproducibleincrease in basal [Ca2+], was seen at higher doses of NEM.

Previous results obtained by using activated neutrophils toinhibit hematopoietic cell function showed that the myeloper-oxidase (H202-generating) system of neutrophils suppressedmitogen stimulation ofT cells but not cell viability (9, 10). Tocompare chemically induced oxidative effects of NEM on[Ca2+], in T cells with a model for physiological oxidation,

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PBL-derived T cells were incubated with activated neutro-phils and assayed for intracellular Ca2" mobilization. A 50%reduction in [Ca2"], was observed in neutrophil-treated CD3-stimulated T cells (Fig. 1B), suggesting that NEM-inducedsulfhydryl oxidation induced a stronger but similar effect asactivated neutrophils on Ca2" mobilization in normal T cells.NEM treatment of the CEM T-cell line showed that Ca2+

mobilization was diminished at concentrations comparable tothat required to completely suppress normal T-cell Ca2+mobilization (Fig. 1C). Ca2+ fluxes induced by ligation ofeither CD3 and/or CD4 receptors were equally affected byNEM.

Inhibition of Tyrosine Phosphorylation by NEM. An earlybut transient tyrosine kinase activity precedes mobilizationof Ca2+ in CD3- and/or CD4-induced T cells (1). We thusaddressed whether the inhibition of intracellular Ca2+ mobi-lization by NEM correlated with a loss of tyrosine phosphor-ylation of cellular protein substrates. CEM cells were treatedwith NEM and stimulated with anti-CD4 mAb (Fig. 2A) orimmobilized anti-CD3 mAb (Fig. 2B), and total cellularproteins were immunoblotted with anti-phosphotyrosine an-tibodies. A reduction in the phosphotyrosine signal wasevident in several phosphoproteins after 20-40 AM NEMtreatment, including species of 125, 95, 65, and 35 kDa,following an increase in the phosphotyrosine signal with 10AM NEM. Further, a reduction in the phosphotyrosine signalwas noted in NEM-treated normal resting T cells stimulatedthrough co-ligation of CD3 and CD4 (Fig. 2C) or by ligationof either receptor alone (data not shown).

IL-2-induced tyrosine phosphorylation was also sensitiveto sulfhydryl oxidation by NEM (Fig. 2D). In addition,increased tyrosine phosphorylation of multiple protein sub-strates after NEM treatment was detected in control CEMcells (Fig. 2B) and after IL-2 stimulation of T-cell blastsinduced with phytohemagglutinin (Fig. 2D), but only aftersustained incubation at 40 AM NEM. This suggests thattyrosine phosphatases may be sensitive to increased NEM-induced oxidation.

Overall, NEM-induced inhibition of CD3- and/or CD4-stimulated tyrosine phosphorylation of protein substrates inT cells paralleled the observed suppression of Ca2+ mobili-zation, although [Ca2+]i appeared more sensitive to oxidationat low concentrations of NEM.

Effect of Oxidation on PLCyl Tyrosine Phosphorylation.PLCy1 is a substrate of receptor tyrosine kinases in fibro-blasts stimulated with epidermal growth factor and platelet-derived growth factor (27) and in T cells in response to CD3stimulation (28-30). The tyrosine phosphorylation of PLCy1correlates with enhanced enzymatic activity (31). In addition,

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FIG. 1. Oxidative suppression of intracellular Ca2+ mobilization. (A) Human PBLs were stained with anti-CD20 (mAb 1F5) and unstainedcells were gated and analyzed by indo-1 dye-binding assay. NEM was added at 10 ,uM (--- -), 20 uM (... ), or 40 ,M (- . -) or not added ( ),and the cells were then stimulated with anti-CD3 (mAb G19-4). (B) Human PBLs were incubated with (- - -) or without ( ) neutrophils andassayed for Ca2+ mobilization as in A. (C) CEM cells were treated with NEM at the indicated concentrations and analyzed for Ca2+ mobilization,and data are indicated as percent of maximal cytoplasmic Ca2+ measured for the specific stimulation indicated. Peak [Ca2+]: CD3, 4100 nM;CD3 x CD4, 4000 nM; CD4, 510 nM.

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the products of PLCyl catalytic activity are generated in Tcells stimulated through CD3 and CD4 (3-5). To determinewhether PLCyl tyrosine phosphorylation in CD3- and/orCD4-stimulated CEM cells was affected by NEM treatment,PLC-yl was immunoprecipitated and analyzed by anti-phosphotyrosine immunoblotting (Fig. 3A). Tyrosine phos-phorylation of PLCyl was readily detected after either CD3and CD3/CD4 receptor crosslinking. The level of phosphor-ylation increased slightly after 10 ILM NEM treatment, butwas strongly suppressed at 20-40 ,uM NEM. In addition,tyrosine phosphorylation of a 35-kDa protein (pp35/36) co-immunoprecipitating with PLCyl was inhibited coordinatelywith PLC-yl. In contrast, the level of expression of PLCylwas not altered either by receptor stimulation of cells or byNEM-induced oxidation (Fig. 3B). These data strongly sug-gest that thiol-sensitive tyrosine phosphorylation of PLC-ylplays a role in CD3- and CD4-mediated signal transduction.To address whether NEM was directly affecting PLC-yl

enzymatic activity rather than inhibiting its activity throughdephosphorylation, PLCyl was immunopurified and assayedin vitro afterNEM treatment of viable cells (Table 1). A rabbitantiserum to the carboxyl-terminal 23 amino acid residues ofPLCy1 was used for an in vitro immune-complex phospho-lipase assay. Incubation of labeled substrate ([3H]PIP2) withimmunoprecipitates of PLCy1 resulted in the formation ofdetectable [3H]inositol trisphosphate both before and afterstimulation of T cells through CD3 and CD4 (Table 1). Noeffect on PLCy1 catalytic activity was detected after thioloxidation ofT cells. PLCyl activity is negatively regulated incells by the actin-binding protein profilin (32). Profilin isbound to PIP2 in unstimulated cells and is then uncoupledafter tyrosine phosphorylation of PLCy1, thereby facilitatingthe catalytic activation of PLC-y1 (32). Since profilin is acomponent lacking in our in vitro system, we detectedcomparable unregulated activity in immunoprecipitates ofPLCy1 both before and after receptor crosslinking, regard-less of the tyrosine phosphorylation status of PLCy1. Hence,

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FIG. 2. Effect of NEM on tyrosinephosphorylation of cellular protein sub-strates. CEM cells (A and B), PBLs (C),or phytohemagglutinin-stimulated pri-mary T-cell blasts (D) were treated withNEM at the indicated concentrations,then stimulated by crosslinking (X-link)CD4 (mAb G17-2) (A), CD3 (B), or CD3and CD4 (CD3 x CD4) (C) or stimulatedwith soluble recombinant IL-2 (D). Totalcellular proteins (50 fIg) were subjectedto SDS/PAGE followed by immunoblot-ting with anti-phosphotyrosine. Arrowsindicate the time of avidin addition tocrosslink biotinylated mAbs. Sampleswere assayed at the time points indi-cated. In B, an additional 60-min controltime point was included (without stimu-lation by anti-receptor mAb) to deter-mine the long-term effects of NEM. Mo-lecular size markers are in kilodaltons.

regulation of receptor-induced PLCyl activity in T cellsoccurs through an oxidation-sensitive tyrosine kinase activ-ity, and PLCy1 is not directly inhibited by sulfhydryl oxida-tion.

Sulfhydryl Oxidation Uncouples CD4 and pp5611k. Mutagen-esis studies of CD4 and pp56Ick have shown that specificcysteine residues on both molecules are critical for theirassociation, suggesting that a zinc-finger motif may facilitatetheir interaction (33, 34). Due to the augmentation of CD4ligation on the tyrosine phosphorylation of PLC'yl, and sinceintracellular thiols are direct targets of nonpolar maleimides(7), we next addressed whether the CD4/pp561ck interactionwas sensitive to oxidation by NEM. T cells were treated withNEM and stimulated by crosslinking CD4 receptors, andCD4/ppS6Ick immune complexes were assayed for tyrosinekinase activity in vitro (Fig. 4). Prior to NEM treatment,autophosphorylation of CD4-associated pp56Ick was readilydetected in immune complexes (Fig. 4A). Although pp56lckwas detected in CD4 immunoprecipitates from cells treatedwith 10 AM NEM and increased slightly in cells treated with20 gM NEM, CD4/pp56Ick association was completely ab-rogated after 40 AM NEM treatment. The autophosphoryla-tion activity of total pp56lck in the cell was unaffected byNEM (Fig. 4B), suggesting that only the coupling ofCD4 withpp56Ick was sensitive to oxidation. To confirm that ppS6Ickprotein was present in CD4 immune complexes, immunopre-cipitates of CD4 were immunoblotted with anti-pp56Ick. Thepresence of pp56Ick protein correlated with the detection ofenzymatic activity (data not shown). These data indicate thatone mechanism of sulfhydryl-oxidative suppression of CD4-induced T-cell signaling may involve the uncoupling of CD4and pp56Ick, thereby limiting the tyrosine phosphorylation ofcritical protein substrates, including PLCy1. However, CD3-induced signaling appears to involve thiols sensitive to lowerdoses of NEM that affect receptor-coupled tyrosine kinaseactivity, which are most likely independent of the CD4/pp56Ick interaction.

302 Biochemistry: Kanner et al.

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FIG. 3. NEM inhibits receptor-induced tyrosine phosphorylationof PLCy1. CEM cells were treated with NEM at the indicatedconcentrations and stimulated by crosslinking surface receptors withmAb to CD3 or both CD3 and CD4. Immunoprecipitates of PLCy1were prepared from 2 x 107 cells and divided in half, and the twohalves were run in parallel SDS/8% polyacrylamide gels and immu-noblotted with either anti-phosphotyrosine (anti-p-Tyr) (A) or anti-PLCy1 (B). PLCy1 and the coimmunoprecipitating protein pp35/36are indicated at left. The bands at '50 kDa and -25 kDa are IgGheavy and light chains, respectively.

DISCUSSIONThe importance of protein-tyrosine kinase activity in T-cellsignal transduction has become clearer through studies ofprotein substrate phosphorylations (1, 2) and the effects oftyrosine kinase inhibitors (35). In this report, we have shownthat CD3- and CD4-induced protein-tyrosine kinase activitymeasured by substrate phosphorylation is sensitive to thioloxidation. The components of the T-cell accessory receptorCD4/ppS6 ck complex were uncoupled by 40 tLM NEM-induced sulfhydryl oxidation, raising the possibility thatppS6Ick activity promotes T-cell signaling through CD4-induced stimulation. Further, the tyrosine kinase pathway inCD3-activated T cells was also oxidation-sensitive at lowconcentrations of NEM, suggesting that a component of theCD3-coupled tyrosine kinase pathway requires interactivethiols.

Table 1. Effect of NEM on PLCy1 enzymatic activity in vitro

PIP2 hydrolysis, fmol

NEM, IAM Stimulation Preimmune Immune

0 - 28 396+ 26 329

10 451+ 416

20 593+ 488

40 344+ 351

NEM was incubated with viable cells for 15 min prior to stimu-lation. Cells were stimulated by co-ligation of CD3 and CD4 recep-tors with biotinylated mAbs and avidin. PIP2 hydrolysis was calcu-lated based on the average cpm of [3H]inositol trisphosphate gener-ated from two in vitro assays of PLCyl immunoprecipitates.

FIG. 4. Sulfhydryl oxidation dissociates CD4 and ppS61ck. CEMcells were treated with NEM at the indicated concentrations andstimulated by crosslinking (X-link) CD4 with mAb and anti-mouseimmunoglobulin. Immune complexes were collected and assayed forkinase activity in vitro. (A) Autophosphorylation of CD4-associatedppS61ck detected by in vitro kinase assay of CD4 receptor immuno-precipitates. (B) Total pp561ck autophosphorylation in immunopre-cipitates of pp56lck.

The regulation of both protein-tyrosine kinase activity andprotein phosphorylation in T cells appears to involve theactivity of protein-tyrosine-phosphatases. The transient na-

ture of tyrosine phosphorylation events in T cells stimulatedthrough surface receptor activation (1, 2) may be explainedby tightly modulated tyrosine phosphatases such as the CD45receptor (36). Previously, stimulation of protein tyrosinephosphorylation was demonstrated in a T-cell hybridomatreated with phenylarsine oxide, an oxidizing agent thatinhibits tyrosine phosphatases (37). Although phenylarsineoxide increases tyrosine phosphorylation in lymphocytes, itinhibits receptor-mediated signaling at concentrations lowerthan that required for phosphatase inhibition (unpublishedobservations). In experiments with NEM we saw evidencefor increased tyrosine phosphorylation after prolonged incu-bations at higher concentrations, suggesting that inhibition oftyrosine phosphatases requires more oxidative stress than isneeded for signal transduction uncoupling. Effects of thioloxidation with moderate levels ofNEM (20-40 ,uM) includeddissociation of the CD4/pp56Ick complex and loss of tyrosinephosphorylation of signaling-associated substrates, includingPLCyl. Responses ofT cells to even lower concentrations ofNEM (10 jLM) appear to involve slight increases in tyrosinephosphorylation of several protein substrates, although thespecific nature of these effects is not known. Perhaps at lowconcentrations of NEM, a specific subset of thiols are

alkylated, which may partially increase receptor-inducedtyrosine kinase activity, whereas at higher doses additionalsulfhydryl groups are oxidized, resulting in effects such as thedissociation of CD4 and ppS6Ick.Both CD3- and CD3/CD4-mediated tyrosine phosphory-

lation of PLCy1 were affected by NEM-induced thiol alky-lation. Although the CD4/pp561ck interaction was affected byhigh concentrations of NEM, this mechanism does not ex-

plain the suppression of CD3-induced signaling. Perhaps a

CD3-associated tyrosine kinase is also thiol sensitive. In-deed, pp59fyn has been shown to be associated with CD3 inimmune complexes (38). However, NEM treatment does not

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alter the functional integrity of the T-cell receptor (CD3/Ti)complex itself, i.e., CD3- and Ti-induced Ca2' signals wereequally sensitive to NEM (data not shown), indicating thatprotein-protein interactions of the T-cell receptor complexdo not appear to involve oxidation-sensitive thiols. Further,the kinetics of NEM activity on T-cell signaling correlatedwith targeting of early events. Addition ofNEM had no effecton IL-2 production or cell viability if added 30 min after T-cellstimulation (7), suggesting that oxidation-sensitive thiols donot play a significant role in late signaling events. In fact, only-'12% of total cellular thiols are sensitive to the oxidativeeffects ofNEM (7), indicating that specific residues involvedin receptor/protein-tyrosine kinase pathways may be criticalfor normal regulation.A recent report (39) indicated that CD4/pp56'ck complex

formation was not required for activation of pp56Ick in cellsstimulated with IL-2, although CD4-associated pp56lck wasactivated by IL-2 in CD4' cells. Thus, the role of CD4/pp561ck association in IL-2 responses by CD4' cells is notclear; however, further studies have shown that pp56 ck alsointeracts with the f3 chain of the IL-2 receptor (40). Ourresults indicated that IL-2-stimulated protein-tyrosine kinaseactivity was partially sensitive to thiol oxidation, suggestingthat interactive sulfhydryl groups potentially facilitating theinteraction between pp56ick and IL-2 receptor as well as CD4and pp56Ick were oxidized.The effects of chemical oxidants on T cells may mimic the

activity of the myeloperoxidase system of activated periph-eral blood neutrophils. Specifically, responsiveness to mito-gen stimulation is markedly inhibited in T cells exposed toopsonized particle-activated neutrophils (9, 10). The immu-nosuppressive effects of neutrophils have been attributed toH202 as a consequence of myeloperoxidase activity (10),which can be overcome by catalase addition or halide re-moval (9). The oxidative metabolism of neutrophils does notdiminish T-cell viability (10) but appears in our system topartially down-modulate early signaling events (i.e., a 50%reduction in intracellular Ca2' mobilization) similar to thatdemonstrated with the use of the thiol-alkylating reagent. Itis likely that further studies with neutrophils will help delin-eate the specific action of oxidative stress on T cells ininflammatory tissues and in tumor masses.

We thank S. G. Rhee for providing mAb to PLCy1; J. Blake forgenerating synthetic PLCy1 peptides; J. Deans and C. June forhelpful discussions on the effects of phenylarsine oxide on signalingin lymphocytes; T. Tsu, L. Grosmaire, and D. Hewgill for technicalassistance; M. West for editorial assistance; and J. Marth and T.Purchio for critical comments on the manuscript.

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