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The Journal of Immunology, 1999, 162: 7162-7170.
Copyright © 1999 by The American Association of Immunologists

Protection Against CD95-Mediated Apoptosis by Inorganic Mercury in Jurkat T Cells1

Michael J. Whitekus*, Ronald P. Santini*, Allen J. Rosenspire{dagger} and Michael J. McCabe, Jr.2,*

* Institute of Chemical Toxicology, Detroit, MI 48201; and {dagger} Departments of Pediatrics and Biological Sciences, Wayne State University, Detroit, MI 48201


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dysregulation of CD95/Fas-mediated apoptosis has been implicated as a contributing factor in autoimmune disorders. Animal studies clearly have established a connection between mercury exposure and autoimmune disease in rodents, while case reports have suggested a link between accidental mercury contamination and autoimmune disease in humans. The mechanism(s) for these associations are poorly understood. Using the Jurkat cell model, we have found that low levels (<=10 µM) of inorganic mercury (i.e., HgCl2) attenuated anti-CD95-mediated growth arrest and markedly enhanced cell survival. Several biochemical assays for apoptosis, including DNA degradation, poly(ADP-ribose) polymerase degradation, and phosphatidylserine externalization, directly verified that HgCl2 attenuated anti-CD95-mediated apoptosis. In an attempt to further characterize the effect of mercury on 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-{alpha}-mediated apoptosis in U-937 cells, or ceramide-C6-mediated apoptosis in Jurkat cells, suggesting that mercury acts upstream of, or does not involve, these signals. Thus, inorganic mercury specifically attenuates CD95-mediated apoptosis likely by targeting a signaling component that is upstream of caspase-3 activation and downstream of CD95.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Many systemic autoimmune diseases are associated with an abnormal accumulation of autoreactive lymphocytes resulting from defects in the termination of lymphocyte activation and growth via apoptosis (1, 2). CD95 (Fas) is a transmembrane protein belonging to the TNF receptor superfamily, some members of which are death receptors associated with the initiation of apoptosis (reviewed in 3). The importance of CD95 in immunoregulation has been best described in certain mice carrying a homozygous defect in CD95, the lpr defect (4, 5). While MRL +/+ mice are prone to an autoimmune proliferative syndrome characterized by massive lymphadenopathy and lupus-like immunopathology, the disease process is more severe and accelerated in MRL lpr/lpr mice. Similarly, CD95 mutations, resulting in an lpr-like autoimmune lymphoproliferative syndrome and defects in the molecular components of the CD95 death pathway downstream of CD95 have been described in patients suffering from a variety of autoimmune diseases (6, 7, 8, 9). Thus, defects in the apoptotic signaling pathway seem to be linked to autoimmune disease susceptibility. While genetic defects have received considerable attention, dysregulation of apoptosis by environmental agents and its association with autoimmune abnormalities have received comparatively less consideration.

Exposure to various forms of mercury has been reported to induce an autoimmune disease in animal models that is similar to systemic lupus erythematosus (reviewed in 10). Additionally, case reports of accidental mercury exposure and studies of occupationally exposed mercury workers show a link with immune system dysfunction and autoimmune abnormalities (11, 12, 13, 14). Features of Hg-induced autoimmunity in rodent models include lymphoproliferation, generation of autoreactive CD4+ T cells, T cell-dependent polyclonal B cell activation, hypergammaglobulinemia, increased serum IgE, and the production of autoantibodies followed by immune complex-mediated tissue injury and glomerulonephritis (15, 16, 17, 18, 19, 20, 21, 22). Progression to an autoimmune disease state in mercury-exposed animal models is dependent on the genetic background (Refs. 17 and 18; reviewed in 10), in that both MHC-linked and non-MHC-linked genes contribute to the immunopathology. Thus, similar to the MRL model, progression to an autoimmune state in Hg-mediated autoimmunity is dependent on the genetic background, although the specific contributing genes are likely different. With this in mind, we thought it useful to explore the idea that disruption of the CD95 pathway by Hg may be a contributing factor in Hg-mediated autoimmunity.

In the mercury model of autoimmunity in rodents, the polyclonal B cell activation ultimately responsible for the immunopathology is widely believed to be due to a selective stimulation of Th2 cells (23). In this model, up-regulation of IL-4 expression has been shown in response to mercury treatment both in vivo and in vitro (24, 25). However, the importance of an imbalance of Th1 and Th2 in the susceptibility to Hg-mediated autoimmunity has recently been called into question (26), and Hg-induced autoimmune disease and IL-4 production have been dissociated from each other (27, 28). Hence, despite well-established literature supporting the view that Hg-induced systemic autoimmunity is a prototypic Th2-mediated disease, the cellular immune mechanisms underlying the disease process are not as clearly understood as they were previously thought to be. Furthermore, while some progress has been made in understanding the biochemical signaling mechanisms mediating the effects of mercury on Th2 cells (23), the molecular components directly or indirectly targeted by mercury that provoke IL-4 expression are not known.

A growing collection of studies has shown effects of mercury on global biochemical signaling events, such as tyrosine phosphorylation (29, 30, 31, 32), protein kinase C activity (24, 33), and Ca2+ signaling (34); however, the modulation of these signaling processes by mercury has not been well correlated with any biological effects in lymphoid cell systems. Therefore, given that 1) mercury is an immunomodulator associated with autoimmune disease, 2) impairments of the apoptotic program have been linked with the accumulation of autoreactive lymphocytes, and 3) large segments of the population are exposed to low levels of this toxicant through ubiquitous environmental and occupational sources (35), we hypothesized that noncytotoxic concentrations of mercury could dysregulate the CD95 death pathway, which might contribute to autoimmune disease susceptibility. To test this hypothesis, we used the Jurkat T cell line as a model system because it is well characterized and has been used extensively to establish the molecular components and the epistasis of the CD95 death pathway (36, 37, 38, 39, 40, 41). Our investigations have established that noncytotoxic concentrations of inorganic mercury (i.e., Hg2+) significantly attenuate CD95 agonist-induced apoptotic cell death. Furthermore, using the strategy of molecular ordering, we have established that the molecular component either directly or indirectly targeted by Hg is localized upstream of caspase-3 activation and downstream of CD95 itself. This report is the first that we are aware of to demonstrate dysregulation of the CD95 death pathway by an environmental toxicant, and it represents a framework in which to further elucidate the molecular mechanisms whereby mercury modulates peripheral tolerance leading to autoimmune dysfunction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

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

Cell culture

The human Jurkat T cell line (clone E6-1) and the human promonocytic leukemia cell line, U-937, were obtained from the American Type Culture Collection (Manassas, VA). Cells were maintained in RPMI 1640 medium (HyClone, Logan, UT) supplemented with 10% FBS (HyClone), 2 mM L-glutamine (Life Technologies), and 10 µg/ml gentamicin (Life Technologies). Cells were grown at 37°C in a humidified atmosphere consisting of 5% CO2. Cells were passaged three times weekly and maintained at a density between 0.2 and 1 x 106 cells/ml. Cells used for all experiments were in logarithmic growth phase, and the medium used for experiments had the same constituents as that used for cell passage, unless otherwise indicated.

[3H]Thymidine incorporation assay

Quadruplicate samples consisting of 4 x 104 viable cells/0.1 ml/well were cultured in 96-well flat-bottom plates in the presence or absence of 10 µM HgCl2 and various concentrations of anti-CD95 (CH-11). Mercuric chloride was added to the appropriate wells in 0.1-ml volumes from a 2x stock solution made in complete RPMI 1640. Culture wells were pulsed with 1 µCi of [3H]TdR for the final 6 h of 24-, 48-, or 72-h incubations. Samples were harvested using a Skatron (Sterling, VA) cell harvester, and radioactivity was determined by liquid scintillation spectroscopy.

MTT assay

Jurkat or U-937 cells were cultured and exposed to HgCl2, as indicated above for the [3H]TdR incorporation assay. CH-11 or TNF-{alpha} were added to the appropriate wells as 20x stock solutions diluted in media. At the conclusion of the culture period, 20 µl of a 5-mg/ml MTT stock solution was added to each culture well, and the plates were incubated at 37°C for an additional 2 h. Plates were centrifuged at 200 x g, after which the supernatants were removed by flicking, and 200 µl of DMSO (Sigma, St. Louis, MO) was added to each well. Absorbance was read at 540 nm in a microplate reader.

Flow cytometric analysis of DNA content

Jurkat cells (2 x 106/ml) were cultured for 12 h in the presence or absence of 5 µM HgCl2 and/or 250 ng/ml CH-11. The method used to evaluate DNA fragmentation was essentially the same as that described by Nicoletti et al. (42). Briefly, cells were harvested from culture, washed twice with PBS, and then incubated overnight at 4°C in a hypotonic staining solution consisting of 0.1% sodium citrate, 0.1% Triton X-100, and 50 µg/ml propidium iodide (Sigma). Nuclei stained with propidium iodide were analyzed by flow cytometry on a FACScalibur (Becton Dickinson, San Jose, CA) using doublet discrimination. Propidium iodide fluorescence was collected on FL2 (585/42 nm) using linear amplification.

PARP degradation

Jurkat cells (2 x 106/ml) were incubated in the presence or absence of 10 µM HgCl2 and/or 500 ng/ml CH-11 for 4 h at 37°C in HBSS supplemented with 10 mM HEPES. After the treatment period, cells were collected, washed with PBS, and lysed in a buffer consisting of 62.5 mM Tris (pH 6.8), 6 M urea, 10% glycerol, 2% SDS, 0.003% bromphenol blue, and 5% 2-ME, as previously described (43). Cell lysates, representing 3.2 x 105 cell equivalents, were separated on 7.5% SDS-PAGE and transblotted to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). The membrane was probed with a rabbit anti-human PARP Ab (1:2,000 dilution) followed by alkaline phosphatase-conjugated goat anti-rabbit IgG (1:50,000). Blots were developed utilizing the Immun-Star chemiluminescent protein detection system (Bio-Rad) and BioMax ML imaging film (Sigma). Digitized images of the films were captured using an IS1000 gel documentation and image analysis system (Alpha Innotech, San Leandro, CA).

Caspase-3 activation

Jurkat cells (2 x 106/ml) were incubated in the presence or absence of 1 µM HgCl2 and/or 500 ng/ml CH-11 for 4 h at 37°C in HBSS. At the end of the treatment period, samples were pelleted rapidly and snap frozen in a dry-ice bath. Cell pellets were lysed on ice for 30 min in a buffer consisting of 300 mM NaCl, 50 mM Tris (pH 7.6), 0.5% Triton X-100, 2 µg/ml aprotinin, 1 mM PMSF, 1 mM sodium o-vanadate, and 10 µg/ml leupeptin. Cell lysates were centrifuged at top speed in an Eppendorf refrigerated microfuge for 15 min, then the supernatants were diluted with an equal volume of 2x sample 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 the PARP degradation assay.

Phosphatidylserine (PS) externalization

Jurkat cells (2 x 106/ml) were incubated in the presence or absence of 5 µM HgCl2 and/or 250 ng/ml CH-11 for 4 h at 37°C in HBSS + 10 mM HEPES. PS externalization on apoptotic cells was determined following the recommendations detailed by van Engeland et al. (44). At the end of the incubation period, cells were collected, washed twice in PBS, resuspended in annexin-V binding buffer (i.e., 0.01 M HEPES/NaOH (pH 7.4), 0.14 mM NaCl, 2.5 mM CaCl2), followed by staining at room temperature in the dark with annexin-V-FITC and propidium iodide (5 µg/ml) for 15 min, according to the supplier’s (PharMingen) protocol. Green fluorescence (FL1 530/30 nm) indicative of annexin-V-FITC binding and red fluorescence (FL2 585/42 nm) indicative of propidium iodide (PI) uptake by damaged cells were collected using logarithmic amplification and electronic compensation for spectral overlap.

Anti-CD95 agonist binding assay

Jurkat cells (2 x 106 cells/ml) were incubated in the presence or absence of 5 µM HgCl2 and/or 250 ng/ml CH-11 in HBSS + 10 mM HEPES at 37°C for 0.5 or 4 h. At the end of the incubation period, the cells were washed twice in PBS followed by staining at 4°C for 30 min with an FITC-conjugated goat anti-mouse Ig (PharMingen) in a buffer consisting of PBS + 0.1% NaN3 + 1% FBS. Green fluorescence indicative of CH-11 binding in this indirect immunofluorescence assay was collected using logarithmic amplification on FL1 (530/30 nm) of cells gated on forward and side scatter.

Cell surface CD95 density

Jurkat cells (2 x 106 cells/ml) were incubated in the presence or absence of 5 µM HgCl2 in HBSS at 37°C for 0.5 or 4 h. At the end of the incubation period, cells were collected, washed twice in PBS + 0.1% NaN3 + 1% FBS, and then incubated for 30 min at 4°C in the dark with R-PE-conjugated anti-CD95 (DX2, mouse IgG1, {kappa}). R-PE-conjugated anti-TNP was used as an isotype control for nonspecific binding. Red fluorescence was collected using logarithmic amplification on FL2 (585/42 nm) of cells gated on forward and side scatter.

Statistical analysis

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


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Inorganic mercury attenuates anti-CD95-mediated growth inhibition and cell death

Jurkat cells, which are a well-established model for the study of the CD95 apoptotic death pathway, were incubated in the presence of anti-CD95 agonist (CH-11) to trigger growth inhibition and cell death. Exposing cells to increasing concentrations of anti-CD95 resulted in a dose-dependent inhibition of DNA synthesis as measured by [3H]TdR incorporation (Fig. 1Go). Fig. 1Go confirms that Jurkat cells are highly sensitive to CH-11, in that a significant decrease in [3H]TdR incorporation was observed at the lowest concentration (i.e., 2 ng/ml) of anti-CD95 used, and [3H]TdR incorporation was almost completely abolished at ~50 ng/ml. In the presence of what we have found to be a noncytotoxic concentration of inorganic mercury (i.e., 10 µM), anti-CD95-mediated growth inhibition was attenuated significantly, in that Hg shifted the ED50 for CH-11 by approximately two orders of magnitude (i.e., ED50 in the absence of Hg = ~3.2 ng/ml vs ~200 ng/ml in the presence of Hg). Furthermore, at 50 ng/ml of anti-CD95, the maximum inhibitory concentration of agonist for control cells, coincubation with Hg resulted in an ~18-fold attenuation of growth inhibition. To confirm these results, cell proliferation/survival was directly assessed by measuring changes in cell density using the colorimetric MTT assay. Results obtained by the MTT assay generally paralleled the [3H]TdR incorporation data with respect to the CH-11 dose response and attenuation of the response by mercury (data not shown).



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  		FIGURE 1. Inorganic mercury attenuates the effects of CD95 stimulation, as measured by DNA synthesis. Jurkat cells were stimulated with the indicated concentrations of anti-CD95 agonist (CH-11) in the presence and absence of 10 µM HgCl2 for 48 h in 96-well plates. Analysis involved exposure of Jurkat cells to a final 6-h pulse with 1 µCi/ml [3H]TdR. Statistical analysis (*, p < 0.01) was determined using ANOVA and the Tukey-Krammer test. Values are means ± SD of quadruplicate determinations for a single experiment and represent three separate experiments. Some error bars are not visible because, on the scale of the figure, they were smaller than the symbols.

 
The growth kinetics of Jurkat cells incubated in the presence and absence of anti-CD95 ± HgCl2 were examined to establish that anti-CD95-stimulated Jurkat cells survived and continued to proliferate in the presence of mercury (Fig. 2Go). Over the time course of 3 days, untreated control Jurkat cells continued to incorporate [3H]TdR, while [3H]TdR incorporation was reduced markedly in CH-11-treated Jurkat cells. Coincubation with Hg significantly attenuated the antiproliferative effect of anti-CD95, resulting in a rate of [3H]TdR incorporation approaching that of the untreated control cells. Exposure of Jurkat cells to 10 µM HgCl2 alone had no significant effect on [3H]TdR incorporation (data not shown), the MTT assay (data not shown), or cell viability, as measured by equivalent cell growth (Fig. 3Go).



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

 


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  		FIGURE 3. The concentration of inorganic mercury that attenuates anti-CD95 is not toxic. Jurkat cells were exposed to 10 µM HgCl2 for the indicated time intervals, and live cells were counted using trypan blue exclusion dye. Values are means ± SD of quadruplicate determinations for a single experiment and represent three separate experiments. Some error bars are not visible due to their small magnitude.

 
While CH-11 is an accepted surrogate agonist for CD95, Fas-ligand (i.e., CD95-L) is the natural biological agonist that triggers the CD95 death pathway. Therefore, the ability of inorganic mercury to attenuate CD95-L-induced growth arrest was tested. In some cases the assay was performed in the presence of Fas potentiator, a cross-linking Ab directed against soluble Fas ligand, which enhances the ability of soluble Fas ligand to induce cell death. Thymidine incorporation by Jurkat cells incubated with 10 ng/ml CD95-L ± potentiator was significantly reduced. However, as was the case when CH-11 was used as agonist, inorganic mercury significantly attenuated the growth inhibitory effects of CD95-L, irrespective of the presence of the Fas potentiator Ab (Table IGo).


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  		Table I. Inorganic mercury attenuates Fas/CD95-ligand mediated apoptosis1

 
Collectively, the data presented in Figs. 1–3GoGoGo and in Table IGo establish that, in the presence of a noncytotoxic concentration of inorganic mercury, Jurkat cells survive CD95 stimulation and continue to proliferate. Inasmuch as anti-CD95 is an inducer of apoptosis 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 cells using several complementary assays specific for apoptosis.

Mercury attenuates anti-CD95-mediated apoptosis in Jurkat cells

As a result of endonuclease activation late in the apoptotic process, a fraction of low m.w. DNA leaks from the nuclei of apoptotic cells, resulting in a diminished PI fluorescence intensity, which appears on DNA histograms as a subdiploid peak having less than G0/G1 DNA content (42). In a first approach to establish that mercury attenuates CD95-mediated apoptotic death, flow cytometric analysis of DNA degradation using PI was performed. Jurkat cells were stimulated with anti-CD95 ± HgCl2 for 12 h, and DNA content was determined in the apoptotic and cycling cell populations (Fig. 4Go). Flow cytometric analysis of the DNA content of the untreated Jurkat cell population revealed normal cell cycle kinetics for a cell line in logarithmic growth, and the percentage of cells displaying a sub-G1 peak for the untreated control was low (2.7 ± 1.1%; Fig. 4Go, top left). Stimulation through CD95 resulted in the appearance of a large sub-G1 peak, constituting 30.5 ± 3.5% of the total cells (Fig. 4Go, top right); however, in the presence of 5 µM HgCl2, the appearance of the CD95-stimulated sub-G1 peak was attenuated significantly, constituting only 13.6 ± 3.6% of the total cells (Fig. 4Go, lower right). Treatment with HgCl2 alone did not provoke DNA degradation, as indicated by the low percentage of cells within the sub-G1 peak (3.6 ± 1.7%), and Hg treatment alone did not affect the cell cycle kinetics of Jurkat cells (Fig. 4Go, lower left).



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  		FIGURE 4. Inorganic mercury attenuates anti-CD95-induced DNA fragmentation. Jurkat cells (2 x 106 cells/ml) were incubated with 250 ng/ml of anti-CD95 (CH-11) IgM mAb in the presence/absence of 5 µM HgCl2 for 12 h. Cells were lysed and stained with PI, and the nuclei were analyzed by flow cytometry. Values are means of three separate determinations. Pie charts within each panel indicate the percentages of cells in each cell cycle phase.

 
As is the case with oligosomal degradation of nuclear DNA, degradation of the death substrate PARP is another biochemical marker and nuclear event associated with apoptotic cell death. Constitutively expressed as a 116-kDa protein in the cell nucleus and intimately involved with DNA repair, PARP is cleaved to an 85-kDa fragment during apoptosis by the activation of caspases (43). Jurkat cells constitutively express the 116-kDa protein found in nuclear extracts (Fig. 5Go, lane 1), which is cleaved to the 85-kDa fragment upon stimulation with anti-CD95 (Fig. 5Go, lane 2). However, in the presence of 10 µM HgCl2, anti-CD95 agonist-induced PARP cleavage was prevented (Fig. 5Go, lane 4), while treatment with 10 µM HgCl2 alone had no effect on constitutive PARP expression or degradation (Fig. 5Go, lane 3).



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  		FIGURE 5. Inorganic mercury attenuates anti-CD95-induced degradation of PARP. Jurkat cells (2 x 106 cells/ml) were incubated for 4 h with 500 ng/ml anti-CD95 ± 10 µM HgCl2. Total cell lysates were prepared, and PARP and degraded PARP were detected by Western blotting. Lysates were first run on 7.5% SDS-PAGE, transblotted to a PVDF membrane, and probed with anti-human PARP followed by alkaline phosphatase-conjugated goat anti-rabbit Ab. Protein bands were visualized using the ImmunStar chemiluminescence detection system. The data presented here are representative of three experiments.

 
A feature of apoptotic cell death that is distinct from necrotic cell death is that apoptotic cells undergo cell surface modifications without losing plasma membrane integrity. One such modification is the externalization of the membrane phospholipid PS, which is normally confined to the inner leaflet of the plasma membrane. Translocation of PS to the outer leaflet of the bilayer serves as a recognition signal for phagocytic removal of apoptotic cells (45). In contrast to DNA and PARP degradation, which occur relatively late during apoptosis, measurable changes in PS externalization occur early during apoptosis and often can be detected before any of the nuclear changes characteristic of apoptosis have taken place (44, 45). Changes in the external PS content can be monitored by flow cytometry using fluorochrome-conjugated annexin-V, which has high affinity for PS (44). Using the annexin-V-FITC binding assay, control Jurkat cells displayed little PS externalization (Fig. 6Go, top left), whereas stimulation with anti-CD95 resulted in an ~10-fold increase in the percentage of cells exhibiting increased annexin-V binding (Fig. 6Go, top right). When Jurkat cells were stimulated with anti-CD95 in the presence of 5 µM HgCl2, PS translocation was reduced markedly, in comparison to cells stimulated with anti-CD95 alone (Fig. 6Go, bottom right). Treatment of Jurkat cells with 5 µM HgCl2 alone had no effect on PS externalization (Fig. 6Go, bottom left). Separate experiments performed with necrotic cells (i.e., PI+ cells) indicated that mercury does not interfere with annexin-V binding to PS (data not shown).



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  		FIGURE 6. Inorganic mercury abrogates anti-CD95-induced PS externalization. Jurkat cells (2 x 106 cells/ml) were exposed to 250 ng/ml anti-CD95 in the presence/absence of 5 µM HgCl2 for 4 h. Cells were washed, stained with annexin-V and PI (to exclude dead cells), and analyzed by flow cytometry. Results shown are representative of three separate experiments.

 
Inorganic mercury inhibits CD95-mediated apoptosis upstream of caspase-3 activation

DNA degradation, PARP degradation, and PS externalization are all downstream consequences of caspase-3 activation (43, 45, 46). Thus, the effect of HgCl2 on anti-CD95-mediated caspase-3 activation was analyzed in an effort to establish whether the molecular component of the CD95 death pathway targeted by Hg was upstream or downstream of caspase-3. Caspase-3 is constitutively expressed in Jurkat cells appearing on immunoblots as an inactive 32-kDa precursor (Fig. 7Go, lane 1) that is cleaved into a p17/p12 heterodimmer during apoptotic stimulation by anti-CD95 (Fig. 7Go, lane 3) (43). CD95-dependent activation of caspase-3 was particularly sensitive to Hg2+. In the presence of HgCl2 as low as 1 µM, anti-CD95-induced caspase-3 activation was prevented (Fig. 7Go, lane 4), yet treatment with Hg alone had no effect on constitutive caspase-3 expression or cleavage (Fig. 7Go, lane 2). Thus, Hg attenuates the CD95 apoptotic death pathway by directly or indirectly targeting a molecular component upstream of caspase-3 or caspase-3 itself.



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  		FIGURE 7. Inorganic mercury substantially blocks caspase-3 activation. Jurkat cells (2 x 106 cells/ml) were incubated for 4 h with 500 ng/ml anti-CD95 in the presence or absence of 1 µM HgCl2, and caspase-3 and degraded caspase-3 were detected by Western blotting. Lysates were first run on 12% SDS-PAGE, transblotted to a PVDF membrane, and probed with anti-caspase-3 followed by alkaline phosphatase-conjugated goat anti-rabbit Ab. Protein bands were visualized using the ImmunStar chemiluminescence detection system. Arrows in lane 3 indicate the p17/p12 cleavage products. The data presented here are from a single experiment that is representative of three experiments.

 
Low level mercury has no effect on ceramide or TNF-{alpha}-induced apoptosis. To test whether Hg-inhibitable apoptosis was specific for the CD95 pathway and to further refine the molecular ordering of the Hg-inhibitable steps of the CD95 death pathway, the attenuating effect of Hg on two other apoptotic-inducing stimuli, ceramide and TNF-{alpha}, was examined. Ceramide, a plasma membrane sphingolipid metabolite, has been shown to induce apoptosis in a variety of cell lines, including Jurkat, and it has been implicated as a second messenger upstream of caspase-3 in the CD95 death pathway (39, 47). The influence of Hg treatment on ceramide-C6-mediated apoptosis was analyzed to determine whether it could attenuate cell death induced by this second messenger. Incubating Jurkat cells with increasing concentrations of ceramide-C6 for 48 h resulted in a dose-dependent inhibition of DNA synthesis, as measured by [3H]TdR incorporation (Fig. 8Go). A significant inhibition of DNA synthesis was achieved at 15 µM ceramide-C6, and increasing ceramide to 50 µM nearly completely abolished Jurkat cell proliferation. However, unlike the results obtained with anti-CD95-induced growth inhibition, the growth inhibition induced by ceramide-C6 treatment was not attenuated by Hg (Fig. 8Go). Thus, inorganic mercury interferes with the CD95 death pathway by targeting a signaling component upstream of ceramide generation. Fas/CD95 is a member of the TNF receptor superfamily, a subset of which constitutes the recently described death receptors (3). In addition to the homology between CD95 and the TNF-{alpha} receptor, the apoptotic pathways initiated by their ligands, CD95-L and TNF-{alpha}, respectively, share many of the same downstream death effectors (3, 36, 48). For example, both apoptotic agonists recruit Fas-associated death domain (FADD), activate caspase-8, and stimulate ceramide generation. Therefore, the effect of HgCl2 on TNF-{alpha}-mediated apoptosis was analyzed to determine whether Hg could attenuate the TNF-{alpha} death pathway. The human promonocytic leukemia cell line (U-937), which responds well to TNF-{alpha}, was used in place of the Jurkat cell line. This substitution was made because Jurkat cells respond poorly to TNF-{alpha}-induced apoptosis, due to low TNF-{alpha} receptor expression on Jurkat cells (36, 48). Incubating U-937 cells with 20 ng/ml TNF-{alpha} for 24 h resulted in a significant, nearly 50% reduction in cell viability as measured by the colorimetric MTT reduction assay (Fig. 9Go). Coincubation of TNF-{alpha}-treated U-937 cells with 10 µM HgCl2 had no attenuating effect on TNF-{alpha}-mediated cell death (Fig. 9Go). As was the case with Jurkat cells, exposing U-937 cells to 10 µM HgCl2 alone had no effect on cell viability (data not shown). Additionally, CD95-mediated cell death was attenuated by 10 µM HgCl2 in U-937 cells (data not shown), indicating that the difference between the effects of Hg on CD95- and TNF-{alpha}-mediated cell death was not attributable to differences in cell types.



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  		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 10 µM HgCl2 for 48 h in 96-well plates. During the final 6 h, cells were pulsed with [3H]TdR and harvested. Values are means ± SD of quadruplicate determinations for a single experiment and represent three separate experiments. On the scale of the figure, some error bars are not visible as they were smaller than the symbols.

 


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  		FIGURE 9. Inorganic mercury had no protective effect on TNF-mediated apoptosis, as measured by the MTT assay. U-937 cells were incubated with the indicated concentrations of TNF-{alpha} in the presence/absence of 10 µM HgCl2 in 96-well plates for 24 h, and cell viability was determined by the MTT assay. Values are means ± SD of quadruplicate determinations for a single experiment and are representative of three separate experiments.

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

It is possible that Hg might block the activation of the CD95 death pathway by simply interfering with the binding of the anti-CD95 (CH-11) agonist to CD95. To determine whether or not Hg inhibited CH-11 binding, Jurkat cells were incubated with CH-11 (IgM isotype) in the presence or absence of HgCl2, followed by staining with an FITC-conjugated anti-mouse IgM reagent to label bound CH-11. As shown in Fig. 10Go, top, HgCl2 treatment did not prevent CH-11 from binding to Jurkat cells. Jurkat cells incubated with CH-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 fluorescence intensities upon staining with the FITC-conjugated anti-IgM reagent (i.e., at either the 0.5 or 4 h time points, the two peaks overlap and are nearly indistinguishable from each other). Control cells not treated with the CH-11 agonist had background levels of fluorescence upon staining with the anti-IgM-FITC reagent (dotted lines). Similar experiments, yielding identical results, were conducted at 4°C (data not shown).



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  		FIGURE 10. Inorganic mercury does not influence anti-CD95 agonist binding (top) nor down-regulate CD95 levels (bottom). Jurkat cells (top) were incubated at 37°C in HBSS for 0.5 or 4 h alone (· · · · ·), in the presence of 250 ng/ml anti-CD95 (CH-11) (—), or in the presence of 250 ng/ml anti-CD95 and 5 µM HgCl2 (- · · -). The flow cytometer peaks of cells treated with anti-CD95 alone and anti-CD95/HgCl2 share the same mean fluorescence intensity and are indistinguishable from one another. Anti-CD95 agonist binding was assessed by using an Ig Ab conjugated to FITC that binds IgM isotypes (the CH-11 Ab is IgM). Jurkat cells (bottom) were incubated for 0.5 or 4 h in HBSS and stained with anti-TNP conjugated to R-PE, an IgG1 nonspecific binding control (· · · · ·), incubated alone and stained with DX2 anti-CD95 conjugated to R-PE and IgG1 isotype (—), or cultured in the presence of 5 µM HgCl2 and stained with DX2 anti-CD95 (- · ·-) to assess CD95 density. The flow cytometer peaks of cells treated with and without HgCl2 share the same mean fluorescence intensity and are indistinguishable from each other. The data presented here are representative of three experiments.

 
Having established that Hg treatment did not interfere with anti-CD95 agonist binding, another possibility we considered was that Hg might down-regulate the plasma membrane expression of CD95. N-Acetyl-L-cysteine, a thiolreactive compound, has been shown previously to down-regulate CD95 expression on Jurkat cells and to consequently decrease T cell sensitivity to CD95-mediated apoptosis (40). Like N-acetyl-L-cysteine, inorganic mercury is also thiol reactive (35); therefore, we thought it important to determine whether Hg could down-regulate cell surface CD95 expression. To detect changes in CD95 expression, Jurkat cells were incubated in the presence and absence of Hg followed by staining with a PE-conjugated anti-CD95 reagent (i.e., DX2) (Fig. 10Go, bottom). R-PE-conjugated anti-TNP (IgG1, same isotype as DX2) was used as a negative control (dotted lines). Jurkat cells treated with or without Hg for 0.5 or 4 h had equivalent mean fluorescence intensities upon staining with DX2 (Fig. 10Go, bottom). Since exposure to Hg for time periods that attenuate the CD95 death pathway (e.g., see Figs. 5–7GoGoGo) did not decrease DX2 binding, this indicated that Hg was not attenuating apoptosis by down-regulating CD95 expression.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Studies using animal models, as well as observations stemming from exposed human populations, strongly support an association between mercury intoxication and the development of a lupus-like systemic autoimmune disease process. Given the fact that genetic defects in the CD95 death pathway also have been linked to the development of systemic autoimmune diseases in both animals and humans, we sought to determine whether low concentrations of mercuric chloride (1–10 µM) could interfere with the CD95-mediated apoptogenic signaling pathway. The two major findings of this study are that, indeed, Hg does attenuate CD95-mediated apoptotic cell death, and that the molecular target for Hg, either directly or indirectly, is localized downstream of agonist binding to CD95 and upstream of caspase-3 activation.

Of paramount importance are the observations that Jurkat cells cultured in the presence of a CD95 agonist do not growth arrest and die in the presence of noncytotoxic concentrations of HgCl2, but rather survive and continue to proliferate. Coincubation with inorganic mercury attenuates apoptosis, irrespective of the CD95 agonist employed, in that both anti-CD95-mediated and CD95-L-induced cell death are diminished significantly in the presence of HgCl2. Since Hg2+ has high affinity for free protein sulfhydryls (35), one explanation that was considered for the marked attenuation of CD95-mediated apoptosis observed in the presence of HgCl2 was that Hg2+, by binding to critical thiols on CD95, might inhibit agonist binding and subsequent signal transduction events. In fact, the CD95 death receptor possesses cysteine-rich sequences in its extracellular domain (3), and the binding sites for both CH-11 (i.e., 126KCRCKPNFFC135) and the CD95-L (i.e., 100KCRRCRLCDE109) contain critical cysteine residues within their primary sequences (49, 50). However, several lines of evidence from our studies suggest that Hg2+ does not attenuate CD95-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. 1Go), suggesting that Hg2+ does not behave as a competitive inhibitor of anti-CD95 binding. Second, through a direct agonist binding assay (Fig. 10Go), we showed that Hg2+ does not interfere with CH-11 binding. Third, the primary sequence of the binding site for TNF-{alpha} on the TNF receptor (i.e., VCGCRKNQYR) contains homologous cysteines to the corresponding binding site for CH-11 on CD95, yet inorganic mercury does not attenuate TNF-{alpha}-mediated apoptosis (Fig. 9Go), suggesting that it does not interfere with the TNF-{alpha}/TNF-R interaction. The functional importance of the cysteine-rich sequences in the extracellular domains of both CD95 and the TNF-R is believed to lie in receptor trimerization, which is needed for the proper propagation of the death signal (3). Hg2+ may attenuate the CD95 death pathway by interfering with receptor trimerization; however, this remains to be addressed in further detail.

Several assays, including flow cytometric analysis of DNA fragmentation, PS externalization, and immunoblot analysis of death substrate (i.e., PARP) degradation and death protease (i.e., caspase-3) activation, were employed to specifically assess the effects of Hg2+ on the CD95 apoptotic death pathway ( Figs. 4–7GoGoGoGo). This multiparameter approach firmly established that CD95-mediated apoptosis is attenuated by inorganic mercury. This attenuation of apoptosis by Hg2+ is in apparent contrast to several other reports suggesting that both inorganic and organic mercury compounds induce apoptosis in lymphoid and nonlymphoid cells (31, 51, 52, 53, 54, 55, 56). The most obvious difference between these reports and our results is that our experimental design employed lower, noncytotoxic concentrations of inorganic mercury. At the concentrations employed in our studies (i.e., <=10 µM), we did not observe an induction of apoptosis by HgCl2 alone. Clearly, concentration, as well as distribution (i.e., extracellular vs intracellular), of Hg2+ is an important variable to consider in evaluating the effects of mercury compounds on lymphocyte function. Not surprisingly, organomercurials, such as methylmercury, which is more membrane-permeable than Hg2+, are more toxic by an order of magnitude to lymphocytes (35), and the mechanisms responsible for this toxicity are likely to be quite different from those mediating the attenuation of the CD95 death pathway by inorganic mercury. Our view is similar to that proposed by Nakashima et al. (29), where bivalent inorganic mercury (i.e., Hg2+) binds to multiple cell surface receptors via free sulfhydryl groups, resulting in nonspecific receptor clustering, dysregulated signal transduction, and disorders of cellular functions. In support of this view that Hg2+ alters signaling pathways through a process initiated by Hg2+ binding to protein SH-groups located within extracellular domains, we have reported recently that Hg2+-stimulated tyrosine phosphorylation in lymphocytes is prevented by preincubation of the cells with N-hydroxymaleimide, a plasma membrane impermeable thiol masking agent (32). Furthermore, the cytotoxicity of Hg2+ is increased markedly under culture conditions, such as 2-ME supplementation, where the availability of Hg2+ to intracellular targets is likely facilitated (57).

A second difference between our study and others reporting induction of apoptosis by mercurials is the cell type used. We employed the Jurkat T cell lymphoma because it is a well-established model for studying signal transduction pathways and, in particular, the molecular components and molecular ordering of the CD95 apoptotic death pathway. Jurkat cells more closely represent activated lymphocytes, which for a variety of reasons, including increased intracellular glutathione levels (58), may be more resistant to the cytotoxic/apoptogenic effects of mercury. However, it is precisely in these activated lymphocytes that CD95-mediated peripheral tolerance is of key immunoregulatory importance (59). In any case, interference with apoptosis by mercury fits well with findings showing that autoimmune diseases are often disorders caused by a failure to delete autoreactive lymphocytes (60).

As discussed above, treatment with mercury attenuated DNA fragmentation, PARP degradation, and PS externalization, all of which are apoptotic processes controlled by the effector protease, caspase-3 (43, 45, 46). Thus, a pivotal observation made in our investigation was that anti-CD95-induced caspase-3 activation was impaired by mercury (Fig. 7Go). In addition to corroborating that Hg2+ attenuates CD95-mediated apoptosis, the impairment of caspase-3 activation by Hg2+ localizes the molecular target for Hg2+ within the CD95 death pathway upstream of caspase-3. Since caspase-3, a cysteine protease, contains a critical cysteinyl residue in its active site (43), and since Hg2+ binds free sulfhydryls with high affinity, the notion that caspase-3 itself may be a molecular target for Hg2+ is attractive. Nevertheless, we believe that caspase-3 is not directly targeted by inorganic mercury. It is our view that Hg2+ initiates its effects on signaling pathways by binding to free SH-groups on membrane proteins and nonspecifically cross-linking receptors. We think it unlikely that inorganic mercury gains access to the intracellular compartment where the caspase-3 proenzyme is located. Furthermore, the death pathways for CD95 and for TNF-{alpha} have activation of caspase-3 as a common feature (3, 36, 48), but our data indicate that TNF-{alpha}-induced apoptosis is not impaired by mercury. Since mercury does not attenuate TNF-{alpha}-mediated apoptosis (Fig. 9Go), it appears unlikely that Hg2+ directly impairs any caspases common to both pathways, even if Hg2+ does gain access to the cytosolic compartment. This result eliminates caspase-3 as a possible target (direct or indirect) for Hg2+ modulation, implying that the target for Hg2+ within the CD95 death pathway is likely upstream of capase-3, and possibly a component of the CD95 death-inducing signaling complex (DISC). Whether inorganic mercury interferes with the formation of the DISC (i.e., CD95/FADD/caspase-8), a membrane-proximal event that initiates the CD95 death pathway (3) remains to be addressed.

We and others have reported that low levels of inorganic mercury stimulate the phosphorylation of a great many proteins on tyrosine residues (29, 30, 31, 32). Activation of specific kinase cascades upon treatment with Hg2+ may be mechanistically linked to the attenuation of apoptosis by this agent. In keeping with this notion, Holmström et al. (37) have reported that activation of the mitogen-activated protein kinase (MAPK) cascade negatively regulates CD95-mediated apoptosis in Jurkat cells by targeting a purported phosphoprotein upstream of caspase-3 activation. Given the similarities between the work of these investigators and the present study, we tested whether pharmacological inhibition of the MAPK pathway could reverse the attenuation of CD95-mediated apoptosis by inorganic mercury. Attenuation of CD95-mediated apoptosis by Hg2+ was unaffected by the MAPK inhibitor PD098059, suggesting that activation of the MAPK pathway is not the underlying basis for our observations (M. J. Whitekus et al., manuscript in preparation). Likewise, recent reports have implicated the phosphoinositide 3-kinase (PI 3-kinase) pathway in the negative regulation of the CD95 death pathway upstream of caspase-3 activation (61, 62). Despite the prominent appearance of tyrosine-phosphorylated p85 and p110, which respectively may be the regulatory and catalytic subunits of PI 3-kinase, on immunoblots obtained from Jurkat cells stimulated with low concentrations of HgCl2 (M. J. Whitekus et al., manuscript in preparation), pharmacological inhibitors of PI 3-kinase (i.e., wortmannin and LY294002) did not abrogate the attenuating effects of mercury on CD95-mediated apoptosis (M. J. Whitekus et al., manuscript in preparation). Nevertheless, the linkage between the ability of inorganic mercury to stimulate tyrosine phosphorylation and attenuate CD95-mediated apoptosis is currently under investigation in our laboratories.

This paper presents a novel mechanism whereby inorganic mercury interacts with the immune system resulting in its dysregulation and possibly leading to autoimmune disease. Many studies up to the present time have implicated mercury exposure as a potential environmental agent linked to the development and/or exacerbation of autoimmune disease processes; however, the underlying basis for mercury-mediated autoimmunity has not been elucidated. The present study represents a framework on which to build future in vitro and in vivo studies aimed at better understanding mechanisms by which environmental factors contribute to autoimmune disease.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants R29 ES07365 and P30 ES06639, and by an Interdisciplinary Research Seed Fund from Wayne State University. Performance of this work was facilitated by the Imaging and Cytometry Facility Core of the Environmental Health Sciences center in Molecular and Cellular Toxicology with Human Applications. Back

2 Address correspondence and reprint requests to Dr. Michael J. McCabe, Jr., Institute of Chemical Toxicology, 2727 Second Avenue, Detroit, MI 48201-2654. E-mail address: Back

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

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


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hunig, T., A. Schimpl. 1997. Systemic autoimmune disease as a consequence of defective lymphocyte death. Curr. Opin. Immunol. 9:826.[Medline]
  2. Mountz, J. D., J. Wu, J. Cheng, T. Zhou. 1994. Autoimmune disease: a problem of defective apoptosis. Arthritis Rheum. 37:1415.[Medline]
  3. Ashkenazi, A., V. M. Dixit. 1998. Death receptors: signaling and modulation. Science 281:1305.[Abstract/Free Full Text]
  4. Watanabe-Fukunaga, R., C. I. Brannan, N. G. Copeland, N. A. Jenkins, S. Nagata. 1992. Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 356:314.[Medline]
  5. Takahashi, T., M. Tanaka, C. I. Brannan, N. A. Jenkins, N. G. Copeland, T. Suda, S. Nagata. 1994. Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand. Cell 76:969.[Medline]
  6. Dianzani, U., M. Bragardo, D. DiFranco, C. Alliaudi, P. Scagni, D. Buonfiglio, V. Redoglia, S. Bonissoni, A. Correra, I. Dianzani, U. Ramenghi. 1997. Deficiency of the Fas apoptosis pathway without Fas gene mutations in pediatric patients with autoimmunity/lymphoproliferation. Blood 89:2871.[Abstract/Free Full Text]
  7. Le Deist, F., J.-F. Emile, F. Rieux-Laucat, M. Benkerrou, I. Roberts, N. Brousse, A. Fischer. 1996. Clinical, immunological, and pathological consequences of Fas-deficient conditions. Lancet 348:719.[Medline]
  8. Fisher, G. H., F. J. Rosenberg, S. E. Straus, J. K. Dale, L. A. Middleton, A. Y. Lin, W. Strober, M. J. Lenardo, J. M. Puck. 1995. Dominant interfering Fas gene mutations impair apoptosis in a human autoimune lymphoproliferative syndrome. Cell 81:935.[Medline]
  9. Puck, J. M., M. C. Sneller. 1997. ALPS: an autoimmune human lymphoproliferative syndrome associated with abnormal lymphocyte apoptosis. Semin. Immunol. 9:77.[Medline]
  10. Lawrence, D. A., Jr M. J. McCabe. 1995. Immune modulation by toxic metals. R. A. Goyer, and C. D. Klaassen, and M. P. Waalkes, eds. Metal Toxicology 305. Academic Press, New York.
  11. Dantas, D. C. M., M. L. S. Queiroz. 1997. Immunoglobulin E and autoantibodies in mercury-exposed workers. Immunopharmacol. Immunotoxicol. 19:383.[Medline]
  12. Röger, J., D. Zillikens, G. Burg, E. Gleichmann. 1992. Systemic autoimmunity in a patient with longstanding exposure of mercury. Eur. J. Dermatol. 2:168.
  13. Schrallhammer-Benkler, K., J. Ring, B. Przybila, M. Meurer, M. Landthaler. 1992. Acute mercury intoxication with lichenoid drug eruption followed by mercury contact allergy and development of antinuclear antibodies. Acta Derm. Venereol. 72:294.[Medline]
  14. Stonard, M. D., B. V. Chater, D. P. Duffield, J. J. O’Sullivan, C. M. Lockwood. 1983. Circulating immune complexes in individuals occupationally exposed to mercury vapor. S. S. Brown, and J. Savory, eds. Chemical Toxicology and Clinical Chemistry of Metals 135. Academic Press, New York.
  15. Al-Balaghi, S., E. Möller, G. Möller, M. Abedi-Valugerdi. 1996. Mercury induces polyclonal B cell activation, autoantibody production and renal immune complex deposits in young (NZB x NZW) F1 hybrids. Eur. J. Immunol. 26:1519.[Medline]
  16. Pollard, K. M., D. K. Lee, C. A. Casiano, M. Blüthner, M. M. Johnston, E. M. Tan. 1997. The autoimmunity-inducing xenobiotic mercury interacts with the autoantigen fibrillarin and modifies its molecular and antigenic properties. J. Immunol. 158:3521.[Abstract]
  17. Hultman, P., L. J. Bell, S. Eneström, K. M. Pollard. 1993. Murine susceptibility to mercury. II. Autoantibody profiles and renal immune deposits in hybrid, backcross, and H-2d congenic mice. Clin. Immunol. Immunopathol. 68:9.[Medline]
  18. Hultman, P., L. J. Bell, S. Eneström, K. M. Pollard. 1992. Murine susceptibility to mercury. I. Autoantibody profiles and systemic immune deposits in inbred, congenic and intra-H-2 recombinant strains. Clin. Immunol. Immunopathol. 65:98.[Medline]
  19. Monestier, M., M. J. Losman, K. E. Novick, J. P. Aris. 1994. Molecular analysis of mercury-induced antinucleolar antibodies in H-2s mice. J. Immunol. 152:667.[Abstract]
  20. Pelletier, L., R. Pasquier, F. Hirsch, C. Sapin, P. Druet. 1986. Autoreactive T cells in mercury-induced autoimmune disease: in vitro demonstration. J. Immunol. 137:2548.[Abstract]
  21. Reuter, R., G. Tessars, H.-W. Vohr, E. Gleichmann, R. Lührmann. 1989. Mercuric chloride induces autoantibodies against U3 small nuclear ribonucleoprotein in susceptible mice. Proc. Natl. Acad. Sci. USA 86:237.[Abstract/Free Full Text]
  22. Jiang, Y., G. Möller. 1995. In vitro effects of HgCl2 on murine lymphocytes. I. Preferable activation of CD4+ T cells in a responder strain. J. Immunol. 154:3138.[Abstract]
  23. Mathieson, P. W.. 1995. Mercury: god of Th2 cells?. Clin. Exp. Immunol. 102:229.[Medline]
  24. Badou, A., M. Savignac, M. Moreau, C. Leclerc, R. Pasquier, P. Druet, L. Pelletier. 1997. HgCl2-induced interleukin-4 gene expression in T cells involves a protein kinase C-dependent calcium influx through L-type calcium channels. J. Biol. Chem. 272:32411.[Abstract/Free Full Text]
  25. Prigent, P., A. Saoudi, C. Pannetier, P. Graber, J.-Y. Bonnefoy, P. Druet, F. Hirsch. 1995. Mercuric chloride, a chemical responsible for T helper cell (Th)2-mediated autoimmunity in Brown Norway rats, directly triggers T cells to produce interleukin-4. J. Clin. Invest. 96:1484.
  26. Kono, D. H., D. Balomenos, D. L. Pearson, M. S. Park, B. Hildebrandt, P. Hultman, K. M. Pollard. 1998. The prototypic Th2 autoimmunity induced by mercury is dependent on IFN-{gamma} and not Th1/Th2 imbalance. J. Immunol. 161:234.[Abstract/Free Full Text]
  27. Biancone, L., G. Andres, H. Ahn, A. Lim, C. Dai, R. Noelle, H. Yagita, C. DeMartino, I. Stamenkovic. 1996. Distinct regulatory roles of lymphocyte costimulatory pathways on T helper type 2-mediated autoimmune disease. J. Exp. Med. 183:1473.[Abstract/Free Full Text]
  28. Bagenstose, L. M., P. Salgame, M. Monestier. 1998. Mercury-induced autoimmunity in the absence of IL-4. Clin. Exp. Immunol. 114:9.[Medline]
  29. Nakashima, I., M.-y. Pu, A. Nishizaki, I. Rosila, L. Ma, Y. Katano, K. Ohkusu, S. M. J. Rahman, K. Isobe, M. Hamaguchi, K. Saga. 1994. Redox mechanism as alteranative to ligand binding for receptor activation delivering dysregulated cellular signals. J. Immunol. 152:1064.[Abstract]
  30. Rahman, S. M. J., M.-y. Pu, M. Hamaguchi, T. Iwamoto, K. Isobe, I. Nakashima. 1993. Redox-linked ligand-independent cell surface triggering for extensive protein tyrosine phosphorylation. FEBS Lett. 317:35.[Medline]
  31. Akhand, A. A., M. Kato, H. Suzuki, T. Miyata, I. Nakashima. 1998. Level of HgCl2-mediated phosphorylation of intracellular proteins determines death of thymic T-lymphocytes with or without DNA fragmentation. J. Cell. Biochem. 71:243.[Medline]
  32. Rosenspire, A. J., S. Bodepudi, M. Mathews, Jr M. J. McCabe. 1998. Low levels of ionic mercury modulate protein tyrosine phosphorylation in lymphocytes. Int. J. Immunopharmacol. 20:697.[Medline]
  33. Lison, D., F. Raguzzi, R. Lauwerys. 1990. Comparison of the effects of auranofin, heavy metals and retinoids on protein kinase C in vitro and on a protein kinase C mediated response in macrophages. Pharmacol. Toxicol. 67:239.[Medline]
  34. Tan, X., C. Tang, A. F. Castoldi, L. Manzo, L. G. Costa. 1993. Effects of inorganic and organic mercury on intracellular calcium levels in rat T lymphocytes. J. Toxicol. Environ. Health 38:159.[Medline]
  35. Clarkson, T. W.. 1997. The toxicology of mercury. Crit. Rev. Clin. Lab. Sci. 34:369.[Medline]
  36. Totpal, K., S. Singh, R. Lapushin, B. B. Aggarwal. 1996. Qualitative and quantitative differences in the cellular responses mediated through Fas antigen and tumor necrosis factor receptor. J. Interferon Cytokine Res. 16:259.[Medline]
  37. Holmström, T. H., S. C. Chow, I. Elo, E. T. Coffey, S. Orrenius, L. Sistonen, J. E. Eriksson. 1998. Suppression of Fas/APO-1-mediated apoptosis by mitogen-activated kinase signaling. J. Immunol. 160:2626.[Abstract/Free Full Text]
  38. Cuvillier, O., D. S. Rosenthal, M. E. Smulson, S. Spiegel. 1998. Sphingosine 1-phosphate inhibits activation of caspases that cleave poly(ADP-ribose) polymerase and lamins during Fas- and ceramide-mediated apoptosis in Jurkat T lymphocytes. J. Biol. Chem. 273:2910.[Abstract/Free Full Text]
  39. Gamen, S., I. Marzo, A. Anel, A. Pineiro, J. Naval. 1996. CPP32 inhibition prevents Fas-induced ceramide generation and apoptosis in human cells. FEBS Lett. 390:232.[Medline]
  40. Delneste, Y., P. Jeannin, E. Sebille, J.-P. Aubry, J.-Y. Bonnefoy. 1996. Thiols prevent Fas (CD95)-mediated T cell apoptosis by down-regulating membrane Fas expression. Eur. J. Immunol. 26:2981.[Medline]
  41. Greidinger, E. L., D. K. Miller, T.-T. Yamin, L. Casciola-Rosen, A. Rosen. 1996. Sequential activation of three distinct ICE-like activities in Fas-ligated Jurkat cells. FEBS Lett. 390:299.[Medline]
  42. Nicoletti, I., G. Migliorati, M. C. Pagliacci, F. Grignani, C. Riccardi. 1991. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods 139:271.[Medline]
  43. Tewari, M., L. T. Quan, K. O’Rourke, S. Desnoyers, Z. Zeng, D. R. Beidler, G. G. Poirier, G. S. Salvesen, V. M. Dixit. 1995. Yama/CPP32ß, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell 81:801.[Medline]
  44. van Engeland, M., L. J. W. Nieland, F. C. S. Ramaekers, B. Schutte, C. P. M. Reutelingsperger. 1998. Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry 31:1.[Medline]
  45. Fadok, V. A., D. R. Voelker, P. A. Campbell, J. J. Cohen, D. L. Bratton, P. H. Henson. 1992. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. Immunol. 148:2207.[Abstract]
  46. Enari, M., H. Sakahira, H. Yokoyama, K. Okawa, A. Iwamatsu, S. Nagata. 1998. A caspase-activated Dnase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391:43.[Medline]
  47. Cifone, M. G., R. De Maria, P. Roncaioli, M. R. Rippo, M. Azuma, L. L. Lanier, A. Santoni, R. Testi. 1993. Apoptotic signaling through CD95 (Fas/Apo-1) activates an acidic sphingomyelinase. J. Exp. Med. 177:1547.
  48. Clement, M.-V., I. Stamenkovic. 1994. Fas and tumor necrosis factor receptor-mediated cell death: similarities and distinctions. J. Exp. Med. 180:557.[Abstract/Free Full Text]
  49. Fadeel, B., J. Lindberg, A. Achour, F. Chiodi. 1998. A three dimensional model of the Fas/APO1 molecule: crossreactivity of antiFas antibodies explained by structural mimicry of antigenic sites. Int. Immunol. 10:131.[Abstract/Free Full Text]
  50. Fadeel, B., J. Thorpe, F. Chiodi. 1995. Mapping of the linear site on the Fas/Apo-1 moleculae targeted by the prototypic anti-Fas mAb. Int. Immunol. 7:1967.[Abstract/Free Full Text]
  51. Aten, J., P. Prigent, P. Poncet, C. Blanpied, N. Claessen, P. Druet, F. Hirsch. 1995. Mercuric chloride-induced programmed cell death of a murine T cell hybridoma. I. Effect of the proto-oncogene Bcl-2. Cell. Immunol. 161:98.[Medline]
  52. Prigent, P., P. Poncet, J. Aten, C. Blanpied, A. Chand, M. Février, P. Druet, F. Hirsch. 1995. Mercuric chloride-induced programmed cell death of a murine T cell hybridoma. II. Opposite effect of interleukin-2 and interleukin-4. Cell. Immunol. 161:107.[Medline]
  53. Duncan-Achanzar, K. B., J. T. Jones, M. F. Burke, D. E. Carter, H. E. Laird. 1996. Inorganic mercury chloride-induced apoptosis in the cultured porcine renal cell line LLC-PK1. J. Pharmacol. Exp. Ther. 277:1726.[Abstract/Free Full Text]
  54. InSug, O., S. Datar, C. Koch, I. M. Shapiro, B. J. Shenker. 1997. Mercuric compounds inhibit human monocyte function by inducing apoptosis: evidence for formation of reactive oxygen species, development of mitochondrial membrane permeability transition and loss of reductive reserve. Toxicology 124:211.[Medline]
  55. Shenker, B. J., S. Datar, K. Mansfield, I. M. Shapiro. 1997. Induction of apoptosis in human T cells by organomercuric compounds: a flow cytometric analysis. Toxicol. Appl. Pharmacol. 143:397.[Medline]
  56. Guo, T. L., M. A. Miller, S. Datar, I. M. Shapiro, B. J. Shenker. 1998. Inhibition of poly(ADP-ribose) polymerase rescues human T lymphocytes from methylmercury-induced apoptosis. Toxicol. Appl. Pharmacol. 152:397.[Medline]
  57. M. J. McCabe, Jr., and A. J. Rosenspire. 1999. Mercury represses membrane Ig-mediated signal transduction in the WEHI-231 B cell lymphoma. Scand. J. Immunol. In press.
  58. Messina, J. P., D. A. Lawrence. 1989. Cell cycle progression of glutathione-depleted human peripheral blood mononuclear cells is inhibited at S phase. J. Immunol. 143:1974.[Abstract]
  59. Abbas, A. K.. 1996. Die and let live: eliminating dangerous lymphocytes. Cell 84:655.[Medline]
  60. Thompson, C. B.. 1995. Apoptosis in the pathogenesis and treatment of disease. Science 267:1456.[Abstract/Free Full Text]
  61. Häusler, P., G. Papoff, A. Eramo, K. Reif, D. A. Cantrell, G. Ruberti. 1998. Protection of CD95-mediated apoptosis by activation of phosphatidylinositide 3-kinase and protein kinase B. Eur. J. Immunol. 28:57.[Medline]
  62. Berra, E., M. T. Diaz-Meco, J. Mosca. 1998. The activation of p38 and apoptosis by the inhibition of Erk is antagonized by the phosphoinositide 3-kinase/Akt pathway. J. Biol. Chem. 273:10792.[Abstract/Free Full Text]



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