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by Inorganic Mercury in Jurkat T CellsProtection Against CD95-Mediated Apoptosis

and Michael J. McCabe, Jr.Michael J. Whitekus, Ronald P. Santini, Allen J. Rosenspire

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Protection Against CD95-Mediated Apoptosis by InorganicMercury in Jurkat T Cells 1

Michael J. Whitekus,* Ronald P. Santini,* Allen J. Rosenspire,† and Michael J. McCabe, Jr.2*

Dysregulation of CD95/Fas-mediated apoptosis has been implicated as a contributing factor in autoimmune disorders. Animalstudies clearly have established a connection between mercury exposure and autoimmune disease in rodents, while case reportshave suggested a link between accidental mercury contamination and autoimmune disease in humans. The mechanism(s) for theseassociations are poorly understood. Using the Jurkat cell model, we have found that low levels (<10 mM) of inorganic mercury(i.e., HgCl2) attenuated anti-CD95-mediated growth arrest and markedly enhanced cell survival. Several biochemical assays forapoptosis, including DNA degradation, poly(ADP-ribose) polymerase degradation, and phosphatidylserine externalization, di-rectly verified that HgCl 2 attenuated anti-CD95-mediated apoptosis. In an attempt to further characterize the effect of mercuryon CD95-mediated apoptosis, several signaling components of the CD95 death pathway were analyzed to determine whether HgCl2

could modulate them. HgCl2 did not modulate CD95 expression; however, it did block CD95-induced caspase-3 activation. HgCl2

was not able to attenuate TNF-a-mediated apoptosis in U-937 cells, or ceramide-C6-mediated apoptosis in Jurkat cells, suggestingthat mercury acts upstream of, or does not involve, these signals. Thus, inorganic mercury specifically attenuates CD95-mediatedapoptosis likely by targeting a signaling component that is upstream of caspase-3 activation and downstream of CD95.TheJournal of Immunology,1999, 162: 7162–7170.

M any systemic autoimmune diseases are associated withan abnormal accumulation of autoreactive lympho-cytes resulting from defects in the termination of lym-

phocyte activation and growth via apoptosis (1, 2). CD95 (Fas) isa transmembrane protein belonging to the TNF receptor superfam-ily, some members of which are death receptors associated withthe initiation of apoptosis (reviewed in Ref. 3). The importance ofCD95 in immunoregulation has been best described in certain micecarrying a homozygous defect in CD95, thelpr defect (4, 5). WhileMRL 1/1 mice are prone to an autoimmune proliferative syn-drome characterized by massive lymphadenopathy and lupus-likeimmunopathology, the disease process is more severe and accel-erated in MRLlpr/lpr mice. Similarly, CD95 mutations, resultingin an lpr-like autoimmune lymphoproliferative syndrome and de-fects in the molecular components of the CD95 death pathwaydownstream of CD95 have been described in patients sufferingfrom a variety of autoimmune diseases (6–9). Thus, defects in theapoptotic signaling pathway seem to be linked to autoimmune dis-ease susceptibility. While genetic defects have received consider-able attention, dysregulation of apoptosis by environmental agentsand its association with autoimmune abnormalities have receivedcomparatively less consideration.

Exposure to various forms of mercury has been reported to in-duce an autoimmune disease in animal models that is similar tosystemic lupus erythematosus (reviewed in Ref. 10). Additionally,case reports of accidental mercury exposure and studies of occu-pationally exposed mercury workers show a link with immunesystem dysfunction and autoimmune abnormalities (11–14). Fea-tures of Hg-induced autoimmunity in rodent models include lym-phoproliferation, generation of autoreactive CD41 T cells, T cell-dependent polyclonal B cell activation, hypergammaglobulinemia,increased serum IgE, and the production of autoantibodies fol-lowed by immune complex-mediated tissue injury and glomeru-lonephritis (15–22). Progression to an autoimmune disease state inmercury-exposed animal models is dependent on the genetic back-ground (Refs. 17 and 18; reviewed in Ref. 10), in that both MHC-linked and non-MHC-linked genes contribute to the immunopa-thology. Thus, similar to the MRL model, progression to anautoimmune state in Hg-mediated autoimmunity is dependent onthe genetic background, although the specific contributing genesare likely different. With this in mind, we thought it useful toexplore the idea that disruption of the CD95 pathway by Hg maybe a contributing factor in Hg-mediated autoimmunity.

In the mercury model of autoimmunity in rodents, the poly-clonal B cell activation ultimately responsible for the immunopa-thology is widely believed to be due to a selective stimulation ofTh2 cells (23). In this model, up-regulation of IL-4 expression hasbeen shown in response to mercury treatment both in vivo and invitro (24, 25). However, the importance of an imbalance of Th1and Th2 in the susceptibility to Hg-mediated autoimmunity hasrecently been called into question (26), and Hg-induced autoim-mune disease and IL-4 production have been dissociated from eachother (27, 28). Hence, despite well-established literature support-ing the view that Hg-induced systemic autoimmunity is a proto-typic Th2-mediated disease, the cellular immune mechanisms un-derlying the disease process are not as clearly understood as theywere previously thought to be. Furthermore, while some progresshas been made in understanding the biochemical signaling mech-anisms mediating the effects of mercury on Th2 cells (23), the

*Institute of Chemical Toxicology, Detroit, MI 48201; and†Departments of Pediat-rics and Biological Sciences, Wayne State University, Detroit, MI 48201

Received for publication January 4, 1999. Accepted for publication April 6, 1999.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by National Institutes of Health Grants R29 ES07365 andP30 ES06639, and by an Interdisciplinary Research Seed Fund from Wayne StateUniversity. Performance of this work was facilitated by the Imaging and CytometryFacility Core of the Environmental Health Sciences center in Molecular and CellularToxicology with Human Applications.2 Address correspondence and reprint requests to Dr. Michael J. McCabe, Jr., Instituteof Chemical Toxicology, 2727 Second Avenue, Detroit, MI 48201-2654. E-mail ad-dress: m.j.mccabe.jr@wayne.edu

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00

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molecular components directly or indirectly targeted by mercurythat provoke IL-4 expression are not known.

A growing collection of studies has shown effects of mercury onglobal biochemical signaling events, such as tyrosine phosphory-lation (29–32), protein kinase C activity (24, 33), and Ca21 sig-naling (34); however, the modulation of these signaling processesby mercury has not been well correlated with any biological effectsin lymphoid cell systems. Therefore, given that 1) mercury is animmunomodulator associated with autoimmune disease, 2) impair-ments of the apoptotic program have been linked with the accu-mulation of autoreactive lymphocytes, and 3) large segments of thepopulation are exposed to low levels of this toxicant through ubiq-uitous environmental and occupational sources (35), we hypothe-sized that noncytotoxic concentrations of mercury could dysregu-late the CD95 death pathway, which might contribute toautoimmune disease susceptibility. To test this hypothesis, weused the Jurkat T cell line as a model system because it is wellcharacterized and has been used extensively to establish the mo-lecular components and the epistasis of the CD95 death pathway(36–41). Our investigations have established that noncytotoxicconcentrations of inorganic mercury (i.e., Hg21) significantly at-tenuate CD95 agonist-induced apoptotic cell death. Furthermore,using the strategy of molecular ordering, we have established thatthe molecular component either directly or indirectly targeted byHg is localized upstream of caspase-3 activation and downstreamof CD95 itself. This report is the first that we are aware of todemonstrate dysregulation of the CD95 death pathway by an en-vironmental toxicant, and it represents a framework in which tofurther elucidate the molecular mechanisms whereby mercury mod-ulates peripheral tolerance leading to autoimmune dysfunction.

Materials and MethodsReagents

A stock solution of mercuric chloride (i.e., HgCl2), obtained from AldrichChemical (Milwaukee, WI), was prepared in endotoxin-free distilled H2O(Life Technologies, Grand Island, NY) and was filter-sterilized before ad-dition to cell culture media. Anti-CD95 mAb (CH-11) and rabbit anti-poly(ADP-ribose) polymerase (PARP)3 were obtained from Upstate Bio-technology (Lake Placid, NY). Fas ligand and potentiator were alsopurchased from Upstate Biotechnology. The R-PE conjugates of anti-CD95(clone DX2) and anti-trinitrophenol (TNP) (negative staining control) werepurchased from PharMingen (San Diego, CA). FITC-conjugated an-nexin-V, rabbit anti-caspase-3, and FITC-conjugated goat anti-mouse Igwere all purchased from PharMingen. Alkaline phosphatase-conjugatedanti-rabbit IgG was from Tropix (Bedford, MA). Immuno-Star chemilu-minescence detection kit was purchased from Bio-Rad (Hercules, CA).Ceramide-C6 was purchased from Calbiochem (San Diego, CA). TNF-awas obtained from Collaborative Biomedical Products (Bedford, MA).[3H]TdR (6.7 Ci/mmol) was purchased from NEN Research Products (Bos-ton, MA). All other reagents and chemicals used were obtained from com-mercial sources and were of analytical grade.

Cell culture

The human Jurkat T cell line (clone E6-1) and the human promonocyticleukemia cell line, U-937, were obtained from the American Type CultureCollection (Manassas, VA). Cells were maintained in RPMI 1640 medium(HyClone, Logan, UT) supplemented with 10% FBS (HyClone), 2 mML-glutamine (Life Technologies), and 10mg/ml gentamicin (Life Technol-ogies). Cells were grown at 37°C in a humidified atmosphere consisting of5% CO2. Cells were passaged three times weekly and maintained at adensity between 0.2 and 13 106 cells/ml. Cells used for all experimentswere in logarithmic growth phase, and the medium used for experimentshad the same constituents as that used for cell passage, unless otherwiseindicated.

[ 3H]Thymidine incorporation assay

Quadruplicate samples consisting of 43 104 viable cells/0.1 ml/well werecultured in 96-well flat-bottom plates in the presence or absence of 10mMHgCl2 and various concentrations of anti-CD95 (CH-11). Mercuric chlo-ride was added to the appropriate wells in 0.1-ml volumes from a 23 stocksolution made in complete RPMI 1640. Culture wells were pulsed with 1mCi of [3H]TdR for the final 6 h of 24-, 48-, or 72-h incubations. Sampleswere harvested using a Skatron (Sterling, VA) cell harvester, and radioac-tivity was determined by liquid scintillation spectroscopy.

MTT assay

Jurkat or U-937 cells were cultured and exposed to HgCl2, as indicatedabove for the [3H]TdR incorporation assay. CH-11 or TNF-a were addedto the appropriate wells as 203 stock solutions diluted in media. At theconclusion of the culture period, 20ml of a 5-mg/ml MTT stock solutionwas added to each culture well, and the plates were incubated at 37°C foran additional 2 h. Plates were centrifuged at 2003 g, after which thesupernatants were removed by flicking, and 200ml of DMSO (Sigma, St.Louis, MO) was added to each well. Absorbance was read at 540 nm in amicroplate reader.

Flow cytometric analysis of DNA content

Jurkat cells (23 106/ml) were cultured for 12 h in the presence or absenceof 5 mM HgCl2 and/or 250 ng/ml CH-11. The method used to evaluateDNA fragmentation was essentially the same as that described by Nicolettiet al. (42). Briefly, cells were harvested from culture, washed twice withPBS, and then incubated overnight at 4°C in a hypotonic staining solutionconsisting of 0.1% sodium citrate, 0.1% Triton X-100, and 50mg/ml pro-pidium iodide (Sigma). Nuclei stained with propidium iodide were ana-lyzed by flow cytometry on a FACScalibur (Becton Dickinson, San Jose,CA) using doublet discrimination. Propidium iodide fluorescence was col-lected on FL2 (585/42 nm) using linear amplification.

PARP degradation

Jurkat cells (23 106/ml) were incubated in the presence or absence of 10mM HgCl2 and/or 500 ng/ml CH-11 for 4 h at37°C in HBSS supplementedwith 10 mM HEPES. After the treatment period, cells were collected,washed with PBS, and lysed in a buffer consisting of 62.5 mM Tris (pH6.8), 6 M urea, 10% glycerol, 2% SDS, 0.003% bromphenol blue, and 5%2-ME, as previously described (43). Cell lysates, representing 3.23 105

cell equivalents, were separated on 7.5% SDS-PAGE and transblotted to apolyvinylidene difluoride (PVDF) membrane (Bio-Rad). The membranewas probed with a rabbit anti-human PARP Ab (1:2,000 dilution) followedby alkaline phosphatase-conjugated goat anti-rabbit IgG (1:50,000). Blotswere developed utilizing the Immun-Star chemiluminescent protein detec-tion system (Bio-Rad) and BioMax ML imaging film (Sigma). Digitizedimages of the films were captured using an IS1000 gel documentation andimage analysis system (Alpha Innotech, San Leandro, CA).

Caspase-3 activation

Jurkat cells (23 106/ml) were incubated in the presence or absence of 1mM HgCl2 and/or 500 ng/ml CH-11 for 4 h at37°C in HBSS. At the endof the treatment period, samples were pelleted rapidly and snap frozen ina dry-ice bath. Cell pellets were lysed on ice for 30 min in a buffer con-sisting of 300 mM NaCl, 50 mM Tris (pH 7.6), 0.5% Triton X-100, 2mg/ml aprotinin, 1 mM PMSF, 1 mM sodiumo-vanadate, and 10mg/mlleupeptin. Cell lysates were centrifuged at top speed in an Eppendorf re-frigerated microfuge for 15 min, then the supernatants were diluted with anequal volume of 23sample dilution buffer (125 mM Tris (pH 6.8), 4%SDS, 10% bromphenol blue, and 20% glycerol). Proteins representing 106

cell equivalents were electrophoretically separated on 12.5% SDS-PAGE,transferred to a PVDF membrane, and probed with rabbit anti-caspase-3(1:2,000) followed by alkaline phosphatase-conjugated goat anti-rabbit IgG(1:20,000). Bands were detected and imaged as described above for thePARP degradation assay.

Phosphatidylserine (PS) externalization

Jurkat cells (23 106/ml) were incubated in the presence or absence of 5mM HgCl2 and/or 250 ng/ml CH-11 for 4 h at37°C in HBSS1 10 mMHEPES. PS externalization on apoptotic cells was determined followingthe recommendations detailed by van Engeland et al. (44). At the end of theincubation period, cells were collected, washed twice in PBS, resuspendedin annexin-V binding buffer (i.e., 0.01 M HEPES/NaOH (pH 7.4), 0.14mM NaCl, 2.5 mM CaCl2), followed by staining at room temperature in thedark with annexin-V-FITC and propidium iodide (5mg/ml) for 15 min,

3 Abbreviations used in this paper: PARP, poly(ADP ribose) polymerase; PS, phos-phatidylserine; FADD, Fas-associated death domain; PVDF, polyvinylidene difluo-ride; TNP, trinitrophenol; CD95-L, CD95 ligand; PI, propidium iodide; PI 3-kinase,phosphoinositide 3-kinase.

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according to the supplier’s (PharMingen) protocol. Green fluorescence(FL1 530/30 nm) indicative of annexin-V-FITC binding and red fluores-cence (FL2 585/42 nm) indicative of propidium iodide (PI) uptake by dam-aged cells were collected using logarithmic amplification and electroniccompensation for spectral overlap.

Anti-CD95 agonist binding assay

Jurkat cells (23 106 cells/ml) were incubated in the presence or absenceof 5 mM HgCl2 and/or 250 ng/ml CH-11 in HBSS1 10 mM HEPES at37°C for 0.5 or 4 h. At the end of the incubation period, the cells werewashed twice in PBS followed by staining at 4°C for 30 min with anFITC-conjugated goat anti-mouse Ig (PharMingen) in a buffer consisting ofPBS1 0.1% NaN3 1 1% FBS. Green fluorescence indicative of CH-11binding in this indirect immunofluorescence assay was collected using log-arithmic amplification on FL1 (530/30 nm) of cells gated on forward andside scatter.

Cell surface CD95 density

Jurkat cells (23 106 cells/ml) were incubated in the presence or absenceof 5 mM HgCl2 in HBSS at 37°C for 0.5 or 4 h. At the end of the incubationperiod, cells were collected, washed twice in PBS1 0.1% NaN3 1 1%FBS, and then incubated for 30 min at 4°C in the dark with R-PE-conju-gated anti-CD95 (DX2, mouse IgG1,k). R-PE-conjugated anti-TNP wasused as an isotype control for nonspecific binding. Red fluorescence wascollected using logarithmic amplification on FL2 (585/42 nm) of cellsgated on forward and side scatter.

Statistical analysis

Data were analyzed using ANOVA and the Tukey-Krammer test.

ResultsInorganic mercury attenuates anti-CD95-mediated growthinhibition and cell death

Jurkat cells, which are a well-established model for the study of theCD95 apoptotic death pathway, were incubated in the presence ofanti-CD95 agonist (CH-11) to trigger growth inhibition and celldeath. Exposing cells to increasing concentrations of anti-CD95resulted in a dose-dependent inhibition of DNA synthesis as mea-sured by [3H]TdR incorporation (Fig. 1). Fig. 1 confirms that Ju-rkat cells are highly sensitive to CH-11, in that a significant de-

crease in [3H]TdR incorporation was observed at the lowestconcentration (i.e., 2 ng/ml) of anti-CD95 used, and [3H]TdR in-corporation was almost completely abolished at;50 ng/ml. In thepresence of what we have found to be a noncytotoxic concentra-tion of inorganic mercury (i.e., 10mM), anti-CD95-mediatedgrowth inhibition was attenuated significantly, in that Hg shiftedthe ED50 for CH-11 by approximately two orders of magnitude(i.e., ED50 in the absence of Hg5 ;3.2 ng/ml vs;200 ng/ml inthe presence of Hg). Furthermore, at 50 ng/ml of anti-CD95, themaximum inhibitory concentration of agonist for control cells, co-incubation with Hg resulted in an;18-fold attenuation of growthinhibition. To confirm these results, cell proliferation/survival wasdirectly assessed by measuring changes in cell density using thecolorimetric MTT assay. Results obtained by the MTT assay gen-erally paralleled the [3H]TdR incorporation data with respect to theCH-11 dose response and attenuation of the response by mercury(data not shown).

The growth kinetics of Jurkat cells incubated in the presence andabsence of anti-CD956 HgCl2 were examined to establish thatanti-CD95-stimulated Jurkat cells survived and continued to pro-liferate in the presence of mercury (Fig. 2). Over the time courseof 3 days, untreated control Jurkat cells continued to incorporate[3H]TdR, while [3H]TdR incorporation was reduced markedly inCH-11-treated Jurkat cells. Coincubation with Hg significantly at-tenuated the antiproliferative effect of anti-CD95, resulting in arate of [3H]TdR incorporation approaching that of the untreatedcontrol cells. Exposure of Jurkat cells to 10mM HgCl2 alone hadno significant effect on [3H]TdR incorporation (data not shown),the MTT assay (data not shown), or cell viability, as measured byequivalent cell growth (Fig. 3).

While CH-11 is an accepted surrogate agonist for CD95, Fas-ligand (i.e., CD95-L) is the natural biological agonist that triggersthe CD95 death pathway. Therefore, the ability of inorganic mer-cury to attenuate CD95-L-induced growth arrest was tested. Insome cases the assay was performed in the presence of Fas poten-tiator, a cross-linking Ab directed against soluble Fas ligand,which enhances the ability of soluble Fas ligand to induce celldeath. Thymidine incorporation by Jurkat cells incubated with 10

FIGURE 1. Inorganic mercury attenuates the effects of CD95 stimula-tion, as measured by DNA synthesis. Jurkat cells were stimulated with theindicated concentrations of anti-CD95 agonist (CH-11) in the presence andabsence of 10mM HgCl2 for 48 h in 96-well plates. Analysis involvedexposure of Jurkat cells to a final 6-h pulse with 1mCi/ml [3H]TdR. Sta-tistical analysis (p,p , 0.01) was determined using ANOVA and theTukey-Krammer test. Values are means6 SD of quadruplicate determi-nations for a single experiment and represent three separate experiments.Some error bars are not visible because, on the scale of the figure, theywere smaller than the symbols.

FIGURE 2. Temporal response of Jurkat cells to anti-CD95 stimulation,as measured by DNA synthesis. Jurkat cells were stimulated with 50 ng/mlanti-CD95 (CH-11), cultured for 1, 2, or 3 days in the presence and absenceof 10 mM HgCl2, pulsed with [3H]TdR, and harvested as in Fig. 1. Sta-tistical analysis (p, 0.01) was determined using ANOVA and the Tukey-Krammer test. Values are means6 SD of quadruplicate determinations fora single experiment and represent three separate experiments. As in Fig. 1,some error bars are not visible due to their relatively small magnitude.

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ng/ml CD95-L6 potentiator was significantly reduced. However,as was the case when CH-11 was used as agonist, inorganic mer-cury significantly attenuated the growth inhibitory effects ofCD95-L, irrespective of the presence of the Fas potentiator Ab(Table I).

Collectively, the data presented in Figs. 1–3 and in Table I es-tablish that, in the presence of a noncytotoxic concentration ofinorganic mercury, Jurkat cells survive CD95 stimulation and con-tinue to proliferate. Inasmuch as anti-CD95 is an inducer of apo-ptosis in Jurkat cells, this suggests that mercury blocks the CD95-mediated apoptotic death pathway. To directly test this hypothesis,we investigated the effect of mercury on CD95-stimulated cellsusing several complementary assays specific for apoptosis.

Mercury attenuates anti-CD95-mediated apoptosis in Jurkatcells

As a result of endonuclease activation late in the apoptotic process,a fraction of low m.w. DNA leaks from the nuclei of apoptoticcells, resulting in a diminished PI fluorescence intensity, whichappears on DNA histograms as a subdiploid peak having less thanG0/G1 DNA content (42). In a first approach to establish that mer-cury attenuates CD95-mediated apoptotic death, flow cytometricanalysis of DNA degradation using PI was performed. Jurkat cellswere stimulated with anti-CD956 HgCl2 for 12 h, and DNAcontent was determined in the apoptotic and cycling cell popula-tions (Fig. 4). Flow cytometric analysis of the DNA content of theuntreated Jurkat cell population revealed normal cell cycle kineticsfor a cell line in logarithmic growth, and the percentage of cells

displaying a sub-G1 peak for the untreated control was low (2.761.1%; Fig. 4,top left). Stimulation through CD95 resulted in theappearance of a large sub-G1 peak, constituting 30.56 3.5% of thetotal cells (Fig. 4,top right); however, in the presence of 5mMHgCl2, the appearance of the CD95-stimulated sub-G1 peak wasattenuated significantly, constituting only 13.66 3.6% of the totalcells (Fig. 4, lower right). Treatment with HgCl2 alone did notprovoke DNA degradation, as indicated by the low percentage ofcells within the sub-G1 peak (3.66 1.7%), and Hg treatment alonedid not affect the cell cycle kinetics of Jurkat cells (Fig. 4,lowerleft).

As is the case with oligosomal degradation of nuclear DNA,degradation of the death substrate PARP is another biochemicalmarker and nuclear event associated with apoptotic cell death.Constitutively expressed as a 116-kDa protein in the cell nucleusand intimately involved with DNA repair, PARP is cleaved to an85-kDa fragment during apoptosis by the activation of caspases(43). Jurkat cells constitutively express the 116-kDa protein foundin nuclear extracts (Fig. 5,lane 1), which is cleaved to the 85-kDafragment upon stimulation with anti-CD95 (Fig. 5,lane 2). How-ever, in the presence of 10mM HgCl2, anti-CD95 agonist-inducedPARP cleavage was prevented (Fig. 5,lane 4), while treatmentwith 10 mM HgCl2 alone had no effect on constitutive PARP ex-pression or degradation (Fig. 5,lane 3).

A feature of apoptotic cell death that is distinct from necroticcell death is that apoptotic cells undergo cell surface modificationswithout losing plasma membrane integrity. One such modificationis the externalization of the membrane phospholipid PS, which isnormally confined to the inner leaflet of the plasma membrane.Translocation of PS to the outer leaflet of the bilayer serves as arecognition signal for phagocytic removal of apoptotic cells (45).In contrast to DNA and PARP degradation, which occur relativelylate during apoptosis, measurable changes in PS externalizationoccur early during apoptosis and often can be detected before anyof the nuclear changes characteristic of apoptosis have taken place

FIGURE 3. The concentration of inorganic mercury that attenuates anti-CD95 is not toxic. Jurkat cells were exposed to 10mM HgCl2 for theindicated time intervals, and live cells were counted using trypan blueexclusion dye. Values are means6 SD of quadruplicate determinations fora single experiment and represent three separate experiments. Some errorbars are not visible due to their small magnitude.

Table I. Inorganic mercury attenuates Fas/CD95-ligand mediatedapoptosisa

0 mM HgCl2 10 mM HgCl2

Control 358,0406 12,239 337,6556 6,455Fas ligand 209,1906 7,920 293,3556 15,603Potentiator 343,2506 6,225 336,3806 11,193Fas ligand1 potentiator 25,4726 799 205,1886 10,274

a Jurkat cells (43 104 cells/well) were stimulated with 10 ng/ml Fas ligand6 10mM HgCl2 6 1.5mg/ml potentiator for 72 h in 96-well plates. Wells were pulsed with1 mCi [3H]TdR for the final 6 h of theculture period, harvested, and cpm determinedby liquid scintillation spectroscopy. Values represent the mean6 SD from quadru-plicate determinations and are representative of three separate experiments.

FIGURE 4. Inorganic mercury attenuates anti-CD95-induced DNAfragmentation. Jurkat cells (23 106 cells/ml) were incubated with 250ng/ml of anti-CD95 (CH-11) IgM mAb in the presence/absence of 5mMHgCl2 for 12 h. Cells were lysed and stained with PI, and the nuclei wereanalyzed by flow cytometry. Values are means of three separate determi-nations. Pie charts within each panel indicate the percentages of cells ineach cell cycle phase.

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(44, 45). Changes in the external PS content can be monitored byflow cytometry using fluorochrome-conjugated annexin-V, whichhas high affinity for PS (44). Using the annexin-V-FITC bindingassay, control Jurkat cells displayed little PS externalization (Fig.6, top left), whereas stimulation with anti-CD95 resulted in an;10-fold increase in the percentage of cells exhibiting increasedannexin-V binding (Fig. 6,top right). When Jurkat cells were stim-ulated with anti-CD95 in the presence of 5mM HgCl2, PS trans-location was reduced markedly, in comparison to cells stimulatedwith anti-CD95 alone (Fig. 6,bottom right). Treatment of Jurkatcells with 5mM HgCl2 alone had no effect on PS externalization(Fig. 6,bottom left). Separate experiments performed with necroticcells (i.e., PI1 cells) indicated that mercury does not interfere withannexin-V binding to PS (data not shown).

Inorganic mercury inhibits CD95-mediated apoptosis upstreamof caspase-3 activation

DNA degradation, PARP degradation, and PS externalization areall downstream consequences of caspase-3 activation (43, 45, 46).Thus, the effect of HgCl2 on anti-CD95-mediated caspase-3 acti-vation was analyzed in an effort to establish whether the molecularcomponent of the CD95 death pathway targeted by Hg was up-stream or downstream of caspase-3. Caspase-3 is constitutivelyexpressed in Jurkat cells appearing on immunoblots as an inactive32-kDa precursor (Fig. 7,lane 1) that is cleaved into a p17/p12heterodimmer during apoptotic stimulation by anti-CD95 (Fig. 7,lane 3) (43). CD95-dependent activation of caspase-3 was partic-ularly sensitive to Hg21. In the presence of HgCl2 as low as 1mM,anti-CD95-induced caspase-3 activation was prevented (Fig. 7,lane 4), yet treatment with Hg alone had no effect on constitutivecaspase-3 expression or cleavage (Fig. 7,lane 2). Thus, Hg atten-uates the CD95 apoptotic death pathway by directly or indirectlytargeting a molecular component upstream of caspase-3 orcaspase-3 itself.Low level mercury has no effect on ceramide or TNF-a-inducedapoptosis.To test whether Hg-inhibitable apoptosis was specificfor the CD95 pathway and to further refine the molecular orderingof the Hg-inhibitable steps of the CD95 death pathway, the atten-uating effect of Hg on two other apoptotic-inducing stimuli, cer-amide and TNF-a, was examined. Ceramide, a plasma membranesphingolipid metabolite, has been shown to induce apoptosis in avariety of cell lines, including Jurkat, and it has been implicated asa second messenger upstream of caspase-3 in the CD95 death path-way (39, 47). The influence of Hg treatment on ceramide-C6-me-diated apoptosis was analyzed to determine whether it could at-tenuate cell death induced by this second messenger. IncubatingJurkat cells with increasing concentrations of ceramide-C6 for 48 hresulted in a dose-dependent inhibition of DNA synthesis, as mea-sured by [3H]TdR incorporation (Fig. 8). A significant inhibitionof DNA synthesis was achieved at 15mM ceramide-C6, and in-creasing ceramide to 50mM nearly completely abolished Jurkatcell proliferation. However, unlike the results obtained with anti-CD95-induced growth inhibition, the growth inhibition induced byceramide-C6 treatment was not attenuated by Hg (Fig. 8). Thus,inorganic mercury interferes with the CD95 death pathway by tar-geting a signaling component upstream of ceramide generation.

FIGURE 5. Inorganic mercury attenuates anti-CD95-induced degrada-tion of PARP. Jurkat cells (23 106 cells/ml) were incubated for 4 h with500 ng/ml anti-CD956 10 mM HgCl2. Total cell lysates were prepared,and PARP and degraded PARP were detected by Western blotting. Lysateswere first run on 7.5% SDS-PAGE, transblotted to a PVDF membrane, andprobed with anti-human PARP followed by alkaline phosphatase-conju-gated goat anti-rabbit Ab. Protein bands were visualized using the Immun-Star chemiluminescence detection system. The data presented here are rep-resentative of three experiments.

FIGURE 6. Inorganic mercury abrogates anti-CD95-induced PS exter-nalization. Jurkat cells (23 106 cells/ml) were exposed to 250 ng/ml anti-CD95 in the presence/absence of 5mM HgCl2 for 4 h. Cells were washed,stained with annexin-V and PI (to exclude dead cells), and analyzed byflow cytometry. Results shown are representative of three separateexperiments.

FIGURE 7. Inorganic mercury substantially blocks caspase-3 activa-tion. Jurkat cells (23 106 cells/ml) were incubated for 4 h with 500 ng/mlanti-CD95 in the presence or absence of 1mM HgCl2, and caspase-3 anddegraded caspase-3 were detected by Western blotting. Lysates were firstrun on 12% SDS-PAGE, transblotted to a PVDF membrane, and probedwith anti-caspase-3 followed by alkaline phosphatase-conjugated goat anti-rabbit Ab. Protein bands were visualized using the ImmunStar chemilu-minescence detection system. Arrows inlane 3indicate the p17/p12 cleav-age products. The data presented here are from a single experiment that isrepresentative of three experiments.

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Fas/CD95 is a member of the TNF receptor superfamily, a subsetof which constitutes the recently described death receptors (3). Inaddition to the homology between CD95 and the TNF-a receptor,the apoptotic pathways initiated by their ligands, CD95-L andTNF-a, respectively, share many of the same downstream deatheffectors (3, 36, 48). For example, both apoptotic agonists recruitFas-associated death domain (FADD), activate caspase-8, andstimulate ceramide generation. Therefore, the effect of HgCl2 onTNF-a-mediated apoptosis was analyzed to determine whether Hgcould attenuate the TNF-a death pathway. The human promono-cytic leukemia cell line (U-937), which responds well to TNF-a,was used in place of the Jurkat cell line. This substitution wasmade because Jurkat cells respond poorly to TNF-a-induced ap-optosis, due to low TNF-a receptor expression on Jurkat cells (36,48). Incubating U-937 cells with 20 ng/ml TNF-a for 24 h resultedin a significant, nearly 50% reduction in cell viability as measuredby the colorimetric MTT reduction assay (Fig. 9). Coincubation ofTNF-a-treated U-937 cells with 10mM HgCl2 had no attenuatingeffect on TNF-a-mediated cell death (Fig. 9). As was the case withJurkat cells, exposing U-937 cells to 10mM HgCl2 alone had noeffect on cell viability (data not shown). Additionally, CD95-me-diated cell death was attenuated by 10mM HgCl2 in U-937 cells(data not shown), indicating that the difference between the effectsof Hg on CD95- and TNF-a-mediated cell death was not attribut-able to differences in cell types.

Mercury does not interfere with CD95 agonist binding, nor doesit down-regulate CD95 expression

It is possible that Hg might block the activation of the CD95 deathpathway by simply interfering with the binding of the anti-CD95(CH-11) agonist to CD95. To determine whether or not Hg inhib-ited CH-11 binding, Jurkat cells were incubated with CH-11 (IgMisotype) in the presence or absence of HgCl2, followed by stainingwith an FITC-conjugated anti-mouse IgM reagent to label boundCH-11. As shown in Fig. 10,top, HgCl2 treatment did not preventCH-11 from binding to Jurkat cells. Jurkat cells incubated withCH-11 in the presence or absence of HgCl2 for 0.5 (top left) or 4 h(top right) at 37°C had essentially identical mean fluorescenceintensities upon staining with the FITC-conjugated anti-IgM re-

agent (i.e., at either the 0.5 or 4 h time points, the two peaksoverlap and are nearly indistinguishable from each other). Controlcells not treated with the CH-11 agonist had background levels offluorescence upon staining with the anti-IgM-FITC reagent (dottedlines). Similar experiments, yielding identical results, were con-ducted at 4°C (data not shown).

Having established that Hg treatment did not interfere with anti-CD95 agonist binding, another possibility we considered was thatHg might down-regulate the plasma membrane expression ofCD95. N-Acetyl-L-cysteine, a thiolreactive compound, has beenshown previously to down-regulate CD95 expression on Jurkatcells and to consequently decrease T cell sensitivity to CD95-me-diated apoptosis (40). LikeN-acetyl-L-cysteine, inorganic mercuryis also thiol reactive (35); therefore, we thought it important todetermine whether Hg could down-regulate cell surface CD95 ex-pression. To detect changes in CD95 expression, Jurkat cells wereincubated in the presence and absence of Hg followed by stainingwith a PE-conjugated anti-CD95 reagent (i.e., DX2) (Fig. 10,bot-tom). R-PE-conjugated anti-TNP (IgG1, same isotype as DX2)was used as a negative control (dotted lines). Jurkat cells treatedwith or without Hg for 0.5 or 4 h had equivalent mean fluorescenceintensities upon staining with DX2 (Fig. 10,bottom). Since expo-sure to Hg for time periods that attenuate the CD95 death pathway(e.g., see Figs. 5–7) did not decrease DX2 binding, this indicatedthat Hg was not attenuating apoptosis by down-regulating CD95expression.

DiscussionStudies using animal models, as well as observations stemmingfrom exposed human populations, strongly support an associationbetween mercury intoxication and the development of a lupus-likesystemic autoimmune disease process. Given the fact that geneticdefects in the CD95 death pathway also have been linked to thedevelopment of systemic autoimmune diseases in both animals andhumans, we sought to determine whether low concentrations ofmercuric chloride (1–10mM) could interfere with the CD95-me-diated apoptogenic signaling pathway. The two major findings ofthis study are that, indeed, Hg does attenuate CD95-mediatedapoptotic cell death, and that the molecular target for Hg, either

FIGURE 8. Inorganic mercury has no protective effect on ceramide-C6-mediated apoptosis, as measured by tritiated thymidine incorporation.Jurkat cells were incubated with ceramide-C6 in the presence/absence of 10mM HgCl2 for 48 h in 96-well plates. During the final 6 h, cells were pulsedwith [3H]TdR and harvested. Values are means6 SD of quadruplicatedeterminations for a single experiment and represent three separate exper-iments. On the scale of the figure, some error bars are not visible as theywere smaller than the symbols.

FIGURE 9. Inorganic mercury had no protective effect on TNF-medi-ated apoptosis, as measured by the MTT assay. U-937 cells were incubatedwith the indicated concentrations of TNF-a in the presence/absence of 10mM HgCl2 in 96-well plates for 24 h, and cell viability was determined bythe MTT assay. Values are means6 SD of quadruplicate determinationsfor a single experiment and are representative of three separateexperiments.

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directly or indirectly, is localized downstream of agonist binding toCD95 and upstream of caspase-3 activation.

Of paramount importance are the observations that Jurkat cellscultured in the presence of a CD95 agonist do not growth arrestand die in the presence of noncytotoxic concentrations of HgCl2,but rather survive and continue to proliferate. Coincubation withinorganic mercury attenuates apoptosis, irrespective of the CD95agonist employed, in that both anti-CD95-mediated and CD95-L-induced cell death are diminished significantly in the presence ofHgCl2. Since Hg21 has high affinity for free protein sulfhydryls(35), one explanation that was considered for the marked attenu-ation of CD95-mediated apoptosis observed in the presence ofHgCl2 was that Hg21, by binding to critical thiols on CD95, mightinhibit agonist binding and subsequent signal transduction events.In fact, the CD95 death receptor possesses cysteine-rich sequencesin its extracellular domain (3), and the binding sites for bothCH-11 (i.e., 126KCRCKPNFFC135) and the CD95-L (i.e.,100KCRRCRLCDE109) contain critical cysteine residues withintheir primary sequences (49, 50). However, several lines of evi-dence from our studies suggest that Hg21 does not attenuateCD95-mediated apoptosis by interfering with agonist binding.First, the attenuating effects of Hg could not be overcome with a.100-fold excess concentration of the anti-CD95 agonist (Fig. 1),

suggesting that Hg21 does not behave as a competitive inhibitor ofanti-CD95 binding. Second, through a direct agonist binding assay(Fig. 10), we showed that Hg21 does not interfere with CH-11binding. Third, the primary sequence of the binding site for TNF-aon the TNF receptor (i.e., VCGCRKNQYR) contains homologouscysteines to the corresponding binding site for CH-11 on CD95,yet inorganic mercury does not attenuate TNF-a-mediated apopto-sis (Fig. 9), suggesting that it does not interfere with the TNF-a/TNF-R interaction. The functional importance of the cysteine-richsequences in the extracellular domains of both CD95 and theTNF-R is believed to lie in receptor trimerization, which is neededfor the proper propagation of the death signal (3). Hg21 may at-tenuate the CD95 death pathway by interfering with receptor tri-merization; however, this remains to be addressed in further detail.

Several assays, including flow cytometric analysis of DNA frag-mentation, PS externalization, and immunoblot analysis of deathsubstrate (i.e., PARP) degradation and death protease (i.e.,caspase-3) activation, were employed to specifically assess the ef-fects of Hg21 on the CD95 apoptotic death pathway (Figs. 4–7).This multiparameter approach firmly established that CD95-medi-ated apoptosis is attenuated by inorganic mercury. This attenuationof apoptosis by Hg21 is in apparent contrast to several other re-ports suggesting that both inorganic and organic mercury com-pounds induce apoptosis in lymphoid and nonlymphoid cells (31,51–56). The most obvious difference between these reports and ourresults is that our experimental design employed lower, noncyto-toxic concentrations of inorganic mercury. At the concentrationsemployed in our studies (i.e.,#10 mM), we did not observe aninduction of apoptosis by HgCl2 alone. Clearly, concentration, aswell as distribution (i.e., extracellular vs intracellular), of Hg21 isan important variable to consider in evaluating the effects of mer-cury compounds on lymphocyte function. Not surprisingly, orga-nomercurials, such as methylmercury, which is more membrane-permeable than Hg21, are more toxic by an order of magnitude tolymphocytes (35), and the mechanisms responsible for this toxicityare likely to be quite different from those mediating the attenuationof the CD95 death pathway by inorganic mercury. Our view issimilar to that proposed by Nakashima et al. (29), where bivalentinorganic mercury (i.e., Hg21) binds to multiple cell surface re-ceptors via free sulfhydryl groups, resulting in nonspecific receptorclustering, dysregulated signal transduction, and disorders of cel-lular functions. In support of this view that Hg21 alters signalingpathways through a process initiated by Hg21 binding to proteinSH-groups located within extracellular domains, we have reportedrecently that Hg21-stimulated tyrosine phosphorylation in lym-phocytes is prevented by preincubation of the cells withN-hy-droxymaleimide, a plasma membrane impermeable thiol maskingagent (32). Furthermore, the cytotoxicity of Hg21 is increasedmarkedly under culture conditions, such as 2-ME supplementation,where the availability of Hg21 to intracellular targets is likely fa-cilitated (57).

A second difference between our study and others reporting in-duction of apoptosis by mercurials is the cell type used. We em-ployed the Jurkat T cell lymphoma because it is a well-establishedmodel for studying signal transduction pathways and, in particular,the molecular components and molecular ordering of the CD95apoptotic death pathway. Jurkat cells more closely represent acti-vated lymphocytes, which for a variety of reasons, including in-creased intracellular glutathione levels (58), may be more resistantto the cytotoxic/apoptogenic effects of mercury. However, it isprecisely in these activated lymphocytes that CD95-mediated pe-ripheral tolerance is of key immunoregulatory importance (59). Inany case, interference with apoptosis by mercury fits well with

FIGURE 10. Inorganic mercury does not influence anti-CD95 agonistbinding (top) nor down-regulate CD95 levels (bottom). Jurkat cells (top)were incubated at 37°C in HBSS for 0.5 or 4 h alone (z z z z z), in the pres-ence of 250 ng/ml anti-CD95 (CH-11) (—), or in the presence of 250 ng/mlanti-CD95 and 5mM HgCl2 (2 z z 2). The flow cytometer peaks of cellstreated with anti-CD95 alone and anti-CD95/HgCl2 share the same meanfluorescence intensity and are indistinguishable from one another. Anti-CD95 agonist binding was assessed by using an Ig Ab conjugated to FITCthat binds IgM isotypes (the CH-11 Ab is IgM). Jurkat cells (bottom) wereincubated for 0.5 or 4 h inHBSS and stained with anti-TNP conjugated toR-PE, an IgG1 nonspecific binding control (z z z z z), incubated alone andstained with DX2 anti-CD95 conjugated to R-PE and IgG1 isotype (—), orcultured in the presence of 5mM HgCl2 and stained with DX2 anti-CD95(2 z z2) to assess CD95 density. The flow cytometer peaks of cells treatedwith and without HgCl2 share the same mean fluorescence intensity and areindistinguishable from each other. The data presented here are representa-tive of three experiments.

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findings showing that autoimmune diseases are often disorderscaused by a failure to delete autoreactive lymphocytes (60).

As discussed above, treatment with mercury attenuated DNAfragmentation, PARP degradation, and PS externalization, all ofwhich are apoptotic processes controlled by the effector protease,caspase-3 (43, 45, 46). Thus, a pivotal observation made in ourinvestigation was that anti-CD95-induced caspase-3 activation wasimpaired by mercury (Fig. 7). In addition to corroborating thatHg21 attenuates CD95-mediated apoptosis, the impairment ofcaspase-3 activation by Hg21 localizes the molecular target forHg21 within the CD95 death pathway upstream of caspase-3.Since caspase-3, a cysteine protease, contains a critical cysteinylresidue in its active site (43), and since Hg21 binds free sulfhy-dryls with high affinity, the notion that caspase-3 itself may be amolecular target for Hg21 is attractive. Nevertheless, we believethat caspase-3 is not directly targeted by inorganic mercury. It isour view that Hg21 initiates its effects on signaling pathways bybinding to free SH-groups on membrane proteins and nonspecifi-cally cross-linking receptors. We think it unlikely that inorganicmercury gains access to the intracellular compartment where thecaspase-3 proenzyme is located. Furthermore, the death pathwaysfor CD95 and for TNF-a have activation of caspase-3 as a com-mon feature (3, 36, 48), but our data indicate that TNF-a-inducedapoptosis is not impaired by mercury. Since mercury does notattenuate TNF-a-mediated apoptosis (Fig. 9), it appears unlikelythat Hg21 directly impairs any caspases common to both path-ways, even if Hg21 does gain access to the cytosolic compartment.This result eliminates caspase-3 as a possible target (direct or in-direct) for Hg21 modulation, implying that the target for Hg21

within the CD95 death pathway is likely upstream of capase-3, andpossibly a component of the CD95 death-inducing signaling com-plex (DISC). Whether inorganic mercury interferes with the for-mation of the DISC (i.e., CD95/FADD/caspase-8), a membrane-proximal event that initiates the CD95 death pathway (3) remainsto be addressed.

We and others have reported that low levels of inorganic mer-cury stimulate the phosphorylation of a great many proteins ontyrosine residues (29–32). Activation of specific kinase cascadesupon treatment with Hg21 may be mechanistically linked to theattenuation of apoptosis by this agent. In keeping with this notion,Holmstrom et al. (37) have reported that activation of the mitogen-activated protein kinase (MAPK) cascade negatively regulatesCD95-mediated apoptosis in Jurkat cells by targeting a purportedphosphoprotein upstream of caspase-3 activation. Given the sim-ilarities between the work of these investigators and the presentstudy, we tested whether pharmacological inhibition of the MAPKpathway could reverse the attenuation of CD95-mediated apopto-sis by inorganic mercury. Attenuation of CD95-mediated apoptosisby Hg21 was unaffected by the MAPK inhibitor PD098059, sug-gesting that activation of the MAPK pathway is not the underlyingbasis for our observations (M. J. Whitekus et al., manuscript inpreparation). Likewise, recent reports have implicated the phos-phoinositide 3-kinase (PI 3-kinase) pathway in the negative regu-lation of the CD95 death pathway upstream of caspase-3 activation(61, 62). Despite the prominent appearance of tyrosine-phospho-rylated p85 and p110, which respectively may be the regulatoryand catalytic subunits of PI 3-kinase, on immunoblots obtainedfrom Jurkat cells stimulated with low concentrations of HgCl2 (M.J. Whitekus et al., manuscript in preparation), pharmacologicalinhibitors of PI 3-kinase (i.e., wortmannin and LY294002) did notabrogate the attenuating effects of mercury on CD95-mediated ap-optosis (M. J. Whitekus et al., manuscript in preparation). Never-theless, the linkage between the ability of inorganic mercury to

stimulate tyrosine phosphorylation and attenuate CD95-mediatedapoptosis is currently under investigation in our laboratories.

This paper presents a novel mechanism whereby inorganic mer-cury interacts with the immune system resulting in its dysregula-tion and possibly leading to autoimmune disease. Many studies upto the present time have implicated mercury exposure as a poten-tial environmental agent linked to the development and/or exacer-bation of autoimmune disease processes; however, the underlyingbasis for mercury-mediated autoimmunity has not been elucidated.The present study represents a framework on which to build futurein vitro and in vivo studies aimed at better understanding mecha-nisms by which environmental factors contribute to autoimmunedisease.

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