Protection Against CD95-Mediated Apoptosis
by Inorganic Mercury in Jurkat T Cells
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J Immunol 1999; 162:7162-7170; ;
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Copyright © 1999 by The American Association of
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References
Michael J. Whitekus, Ronald P. Santini, Allen J. Rosenspire
and Michael J. McCabe, Jr.
Protection Against CD95-Mediated Apoptosis by Inorganic
Mercury in Jurkat T Cells1
Michael J. Whitekus,* Ronald P. Santini,* Allen J. Rosenspire,† and Michael J. McCabe, Jr.2*
M
any 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 Ref. 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 1/1 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 –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.
*Institute of Chemical Toxicology, Detroit, MI 48201; and †Departments of Pediatrics 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 page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
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.
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: [email protected]
Copyright © 1999 by The American Association of Immunologists
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 Ref. 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 (11–14). Features of Hg-induced autoimmunity in rodent models include lymphoproliferation, generation of autoreactive CD41 T cells, T celldependent polyclonal B cell activation, hypergammaglobulinemia,
increased serum IgE, and the production of autoantibodies followed by immune complex-mediated tissue injury and glomerulonephritis (15–22). Progression to an autoimmune disease state in
mercury-exposed animal models is dependent on the genetic background (Refs. 17 and 18; reviewed in Ref. 10), in that both MHClinked 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
0022-1767/99/$02.00
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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 mM) 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-a-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. The
Journal of Immunology, 1999, 162: 7162–7170.
The Journal of Immunology
Materials and Methods
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 antipoly(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-a
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 mg/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 3 106 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.
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.
[3H]Thymidine incorporation assay
Quadruplicate samples consisting of 4 3 104 viable cells/0.1 ml/well were
cultured in 96-well flat-bottom plates in the presence or absence of 10 mM
HgCl2 and various concentrations of anti-CD95 (CH-11). Mercuric chloride was added to the appropriate wells in 0.1-ml volumes from a 23 stock
solution made in complete RPMI 1640. Culture wells were pulsed with 1
mCi 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-a were added
to the appropriate wells as 203 stock solutions diluted in media. At the
conclusion of the culture period, 20 ml 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 3 g, after which the
supernatants were removed by flicking, and 200 ml 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 3 106/ml) were cultured for 12 h in the presence or absence
of 5 mM 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 mg/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 3 106/ml) were incubated in the presence or absence of 10
mM 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 3 105
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 3 106/ml) were incubated in the presence or absence of 1
mM 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
mg/ml aprotinin, 1 mM PMSF, 1 mM sodium o-vanadate, and 10 mg/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 23 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 3 106/ml) were incubated in the presence or absence of 5
mM HgCl2 and/or 250 ng/ml CH-11 for 4 h at 37°C in HBSS 1 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 mg/ml) for 15 min,
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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 –32), protein kinase C activity (24, 33), and Ca21 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 – 41). Our investigations have established that noncytotoxic
concentrations of inorganic mercury (i.e., Hg21) 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.
7163
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INORGANIC MERCURY ATTENUATES CD95/Fas-MEDIATED APOPTOSIS
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 3 106 cells/ml) were incubated in the presence or absence
of 5 mM HgCl2 and/or 250 ng/ml CH-11 in HBSS 1 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 1 0.1% NaN3 1 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 3 106 cells/ml) were incubated in the presence or absence
of 5 mM 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 1 0.1% NaN3 1 1%
FBS, and then incubated for 30 min at 4°C in the dark with R-PE-conjugated anti-CD95 (DX2, mouse IgG1, k). 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
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. 1). Fig. 1 confirms that Jurkat cells are highly sensitive to CH-11, in that a significant de-
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 mM HgCl2, pulsed with [3H]TdR, and harvested as in Fig. 1. Statistical analysis (p , 0.01) was determined using ANOVA and the TukeyKrammer test. Values are means 6 SD of quadruplicate determinations for
a single experiment and represent three separate experiments. As in Fig. 1,
some error bars are not visible due to their relatively small magnitude.
crease 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 mM), 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 5 ;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).
The growth kinetics of Jurkat cells incubated in the presence and
absence of anti-CD95 6 HgCl2 were examined to establish that
anti-CD95-stimulated Jurkat cells survived and continued to proliferate in the presence of mercury (Fig. 2). 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 mM 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. 3).
While CH-11 is an accepted surrogate agonist for CD95, Fasligand (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
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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 mM HgCl2 for 48 h in 96-well plates. Analysis involved
exposure of Jurkat cells to a final 6-h pulse with 1 mCi/ml [3H]TdR. Statistical analysis (p, p , 0.01) was determined using ANOVA and the
Tukey-Krammer test. Values are means 6 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 Journal of Immunology
7165
ng/ml CD95-L 6 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 I).
Collectively, the data presented in Figs. 1–3 and in Table I 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 CD95mediated 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 6 HgCl2 for 12 h, and DNA
content was determined in the apoptotic and cycling cell populations (Fig. 4). 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
Table I. Inorganic mercury attenuates Fas/CD95-ligand mediated
apoptosisa
0 mM HgCl2
Control
358,040 6 12,239
Fas ligand
209,190 6 7,920
Potentiator
343,250 6 6,225
Fas ligand 1 potentiator 25,472 6 799
10 mM HgCl2
337,655 6 6,455
293,355 6 15,603
336,380 6 11,193
205,188 6 10,274
a
Jurkat cells (4 3 104 cells/well) were stimulated with 10 ng/ml Fas ligand 6 10
mM HgCl2 6 1.5 mg/ml potentiator for 72 h in 96-well plates. Wells were pulsed with
1 mCi [3H]TdR for the final 6 h of the culture period, harvested, and cpm determined
by liquid scintillation spectroscopy. Values represent the mean 6 SD from quadruplicate determinations and are representative of three separate experiments.
FIGURE 4. Inorganic mercury attenuates anti-CD95-induced DNA
fragmentation. Jurkat cells (2 3 106 cells/ml) were incubated with 250
ng/ml of anti-CD95 (CH-11) IgM mAb in the presence/absence of 5 mM
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.
displaying a sub-G1 peak for the untreated control was low (2.7 6
1.1%; Fig. 4, top left). Stimulation through CD95 resulted in the
appearance of a large sub-G1 peak, constituting 30.5 6 3.5% of the
total cells (Fig. 4, top right); however, in the presence of 5 mM
HgCl2, the appearance of the CD95-stimulated sub-G1 peak was
attenuated significantly, constituting only 13.6 6 3.6% of the total
cells (Fig. 4, 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 6 1.7%), and Hg treatment alone
did not affect the cell cycle kinetics of Jurkat cells (Fig. 4, lower
left).
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. 5, lane 1), which is cleaved to the 85-kDa
fragment upon stimulation with anti-CD95 (Fig. 5, lane 2). However, in the presence of 10 mM HgCl2, anti-CD95 agonist-induced
PARP cleavage was prevented (Fig. 5, lane 4), while treatment
with 10 mM HgCl2 alone had no effect on constitutive PARP expression or degradation (Fig. 5, lane 3).
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
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FIGURE 3. The concentration of inorganic mercury that attenuates antiCD95 is not toxic. Jurkat cells were exposed to 10 mM HgCl2 for the
indicated time intervals, and live cells were counted using trypan blue
exclusion dye. Values are means 6 SD of quadruplicate determinations for
a single experiment and represent three separate experiments. Some error
bars are not visible due to their small magnitude.
7166
INORGANIC MERCURY ATTENUATES CD95/Fas-MEDIATED APOPTOSIS
FIGURE 5. Inorganic mercury attenuates anti-CD95-induced degradation of PARP. Jurkat cells (2 3 106 cells/ml) were incubated for 4 h with
500 ng/ml anti-CD95 6 10 mM 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.
FIGURE 6. Inorganic mercury abrogates anti-CD95-induced PS externalization. Jurkat cells (2 3 106 cells/ml) were exposed to 250 ng/ml antiCD95 in the presence/absence of 5 mM 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. 7, lane 1) that is cleaved into a p17/p12
heterodimmer during apoptotic stimulation by anti-CD95 (Fig. 7,
lane 3) (43). CD95-dependent activation of caspase-3 was particularly sensitive to Hg21. In the presence of HgCl2 as low as 1 mM,
anti-CD95-induced caspase-3 activation was prevented (Fig. 7,
lane 4), yet treatment with Hg alone had no effect on constitutive
caspase-3 expression or cleavage (Fig. 7, 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.
Low level mercury has no effect on ceramide or TNF-a-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-a, 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. 8). A significant inhibition
of DNA synthesis was achieved at 15 mM ceramide-C6, and increasing ceramide to 50 mM nearly completely abolished Jurkat
cell proliferation. However, unlike the results obtained with antiCD95-induced growth inhibition, the growth inhibition induced by
ceramide-C6 treatment was not attenuated by Hg (Fig. 8). Thus,
inorganic mercury interferes with the CD95 death pathway by targeting a signaling component upstream of ceramide generation.
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(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.
6, top left), whereas stimulation with anti-CD95 resulted in an
;10-fold increase in the percentage of cells exhibiting increased
annexin-V binding (Fig. 6, top right). When Jurkat cells were stimulated with anti-CD95 in the presence of 5 mM HgCl2, PS translocation was reduced markedly, in comparison to cells stimulated
with anti-CD95 alone (Fig. 6, bottom right). Treatment of Jurkat
cells with 5 mM HgCl2 alone had no effect on PS externalization
(Fig. 6, bottom left). Separate experiments performed with necrotic
cells (i.e., PI1 cells) indicated that mercury does not interfere with
annexin-V binding to PS (data not shown).
FIGURE 7. Inorganic mercury substantially blocks caspase-3 activation. Jurkat cells (2 3 106 cells/ml) were incubated for 4 h with 500 ng/ml
anti-CD95 in the presence or absence of 1 mM 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 antirabbit 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.
The Journal of Immunology
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-a receptor,
the apoptotic pathways initiated by their ligands, CD95-L and
TNF-a, 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-a-mediated apoptosis was analyzed to determine whether Hg
could attenuate the TNF-a death pathway. The human promonocytic leukemia cell line (U-937), which responds well to TNF-a,
was used in place of the Jurkat cell line. This substitution was
made because Jurkat cells respond poorly to TNF-a-induced apoptosis, 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 resulted
in a significant, nearly 50% reduction in cell viability as measured
by the colorimetric MTT reduction assay (Fig. 9). Coincubation of
TNF-a-treated U-937 cells with 10 mM HgCl2 had no attenuating
effect on TNF-a-mediated cell death (Fig. 9). As was the case with
Jurkat cells, exposing U-937 cells to 10 mM HgCl2 alone had no
effect on cell viability (data not shown). Additionally, CD95-mediated cell death was attenuated by 10 mM HgCl2 in U-937 cells
(data not shown), indicating that the difference between the effects
of Hg on CD95- and TNF-a-mediated cell death was not attributable to differences in cell types.
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-a in the presence/absence of 10
mM HgCl2 in 96-well plates for 24 h, and cell viability was determined by
the MTT assay. Values are means 6 SD of quadruplicate determinations
for a single experiment and are representative of three separate
experiments.
agent (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).
Having established that Hg treatment did not interfere with antiCD95 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. 10, 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. 10, bottom). Since exposure to Hg for time periods that attenuate the CD95 death pathway
(e.g., see Figs. 5–7) did not decrease DX2 binding, this indicated
that Hg was not attenuating apoptosis by down-regulating CD95
expression.
Mercury does not interfere with CD95 agonist binding, nor does
it down-regulate CD95 expression
Discussion
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. 10, 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 re-
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 mM) 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
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FIGURE 8. Inorganic mercury has no protective effect on ceramideC6-mediated apoptosis, as measured by tritiated thymidine incorporation.
Jurkat cells were incubated with ceramide-C6 in the presence/absence of 10
mM HgCl2 for 48 h in 96-well plates. During the final 6 h, cells were pulsed
with [3H]TdR and harvested. Values are means 6 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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INORGANIC MERCURY ATTENUATES CD95/Fas-MEDIATED APOPTOSIS
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-Linduced cell death are diminished significantly in the presence of
HgCl2. Since Hg21 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 Hg21, 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.,
100
KCRRCRLCDE109) contain critical cysteine residues within
their primary sequences (49, 50). However, several lines of evidence from our studies suggest that Hg21 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. 1),
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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 (z z z z z), in the presence of 250 ng/ml anti-CD95 (CH-11) (—), or in the presence of 250 ng/ml
anti-CD95 and 5 mM HgCl2 (2 z z 2). 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. AntiCD95 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 (z z z z z), incubated alone and
stained with DX2 anti-CD95 conjugated to R-PE and IgG1 isotype (—), or
cultured in the presence of 5 mM HgCl2 and stained with DX2 anti-CD95
(2 z z2) 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.
suggesting that Hg21 does not behave as a competitive inhibitor of
anti-CD95 binding. Second, through a direct agonist binding assay
(Fig. 10), we showed that Hg21 does not interfere with CH-11
binding. Third, the primary sequence of the binding site for TNF-a
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-a-mediated apoptosis (Fig. 9), suggesting that it does not interfere with the TNF-a/
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). Hg21 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 Hg21 on the CD95 apoptotic death pathway (Figs. 4 –7).
This multiparameter approach firmly established that CD95-mediated apoptosis is attenuated by inorganic mercury. This attenuation
of apoptosis by Hg21 is in apparent contrast to several other reports suggesting that both inorganic and organic mercury compounds induce apoptosis in lymphoid and nonlymphoid cells (31,
51–56). 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 mM), we did not observe an
induction of apoptosis by HgCl2 alone. Clearly, concentration, as
well as distribution (i.e., extracellular vs intracellular), of Hg21 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 membranepermeable than Hg21, 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., Hg21) 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 Hg21 alters signaling
pathways through a process initiated by Hg21 binding to protein
SH-groups located within extracellular domains, we have reported
recently that Hg21-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 Hg21 is increased
markedly under culture conditions, such as 2-ME supplementation,
where the availability of Hg21 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
The Journal of Immunology
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.
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