ToxSci Advance Access originally published online on April 26, 2006
Toxicological Sciences 2006 92(1):103-114; doi:10.1093/toxsci/kfj212
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Exposure to a Metabolite of the Environmental Toxicant, Trichloroethylene, Attenuates CD4+ T Cell Activation-Induced Cell Death by Metalloproteinase–Dependent FasL Shedding
,1
* Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205; and Arkansas Children's Hospital Research Institute, Little Rock, Arkansas 72202
1 To whom correspondence should be addressed at Arkansas Children's Hospital Research Institute, Little Rock, AR 72202. Fax: (501) 364-3599. E-mail: blossomsarah{at}uams.edu.
Received February 17, 2006; accepted April 7, 2006
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Long-term exposure to the environmental contaminant trichloroethylene (TCE) in drinking water has been shown to promote autoimmune disease in association with the expansion of activated CD4+ T cells. The effects of TCE on CD4+ T cells were linked in the present study to the ability of TCE metabolite, trichloroacetaldehyde hydrate (TCAH), to inhibit activation-induced cell death (AICD) in CD4+ T cells. TCAH attenuated AICD in CD4+ T cells by decreasing FasL (CD178) expression but not by altering Fas (CD95) expression or by interfering with Fas-signaling events following direct engagement of the Fas receptor. The TCAH-induced decrease in FasL expression did not appear to be mediated at the transcriptional level but was instead due to increased shedding of FasL from the surface of the CD4+ T cells. The ability of TCAH to cleave FasL and thereby decrease AICD appeared to be mediated by metalloproteinases and correlated with a TCAH-induced increase in matrix metalloproteinase-7. Thus, this study presents the novel finding that the environmental contaminant TCE works via its metabolite TCAH to attenuate AICD by increasing metalloproteinase activity that cleaves FasL from CD4+ T cells. This represents a mechanism by which an environmental trigger inhibits AICD in CD4+ T cells and may thereby promote CD4+ T cell–mediated autoimmune disease.
Key Words: apoptosis; autoimmunity; T cells; metalloproteinase; trichloroethylene.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The risk factors that promote the onset and progression of autoimmune disease are only partially defined but clearly include genetics. However, since the concordance rate for most autoimmune diseases in monozygotic twins is less than 50%, certain nongenetic triggers including xenobiotics may also contribute to disease development. One such xenobiotic is the industrial solvent trichloroethylene (TCE). Several human epidemiologic studies have focused on evaluating the health effects related to occupational and environmental TCE exposure (ATSDR, 1997
). Consequently, an association between TCE exposure and the development of lupus- and scleroderma-like disease has been established (for reviews, see He et al., 2001; Kimber and Dearman, 2002; Parks and Cooper, 2005).
Approximately 3.5 million people are occupationally exposed to TCE annually in the United States (Wu and Schaum, 2000). Because of its widespread commercial use and improper disposal, TCE has also become a major environmental pollutant. TCE is the most frequently reported organic contaminant in groundwater (ATSDR, 1997), the source of 93% of public water systems. In addition, TCE is found in air emissions, soil, and a wide variety of food in the United States. TCE is highly lipophilic and is readily absorbed into the circulation via oral, dermal, or inhalation exposure. Considering the multiple routes of exposure, it is perhaps not surprising that the Third National Health and Nutrition Examination Survey (using samples collected between 1988 and 1994) revealed that about 10% of the general, nonoccupationally exposed, population in the United States had detectable levels of TCE in the blood (Ashley et al., 1994). Others have reported that chronic exposure to a domestic water supply contaminated with TCE was associated with lupus-like symptoms including increased numbers of T cells and increased serum levels of antinuclear antibodies (Abs) (Gist and Burg, 1995; Kilburn and Warshaw, 1992; Nietert et al., 1998). Taken together, these results provide a strong circumstantial evidence that exposure to TCE, either occupationally or environmentally, can alter immune function in a manner that promotes autoimmunity.
Previous studies designed to examine a direct effect of TCE on autoimmune disease development demonstrated that TCE promoted autoantibody production and autoimmune disease pathology in female MRL+/+ mice following chronic exposure to occupationally relevant concentrations of the toxicant in drinking water (Gilbert et al., 1999; Griffin et al., 2000b,c). TCE-induced autoimmunity was associated with the expansion of an activated (CD62Llo) population of CD4+ T cells with an increased capacity to secrete IFN-
. Since the immunotoxicological effects of TCE appeared to require its metabolism (Griffin et al., 2000a), recent studies have focused on one of the major metabolic intermediates of TCE, namely trichloroacetaldehyde hydrate (TCAH). Subsequently, in vitro studies demonstrated that TCAH formed a Schiff base with CD4+ T cell–surface proteins in association with increased CD4+ T cell signaling (i.e., mitogen-activated protein kinase activation) (Gilbert et al., 2004). Perhaps, even more relevant was the finding that MRL+/+ mice treated for 4 weeks with TCAH in the drinking water exhibited the same expansion of activated (CD62Llo), IFN-–producing CD4+ T cells observed in TCE-treated mice (Blossom et al., 2004). In the same study, the CD4+ T cells from the mice exposed in vivo to TCAH in the drinking water were also shown to resist activation-induced cell death (AICD) following their restimulation in vitro, even in the absence of the addition of exogenous TCAH to cultures.
AICD is said to occur when repeatedly stimulated T cells coexpress death receptors such as Fas (CD95) as well as the ligand for the death receptor (e.g., FasL; CD178) (for reviews, see Green et al., 2003). FasL cross-links Fas on the CD4+ T cell surface, promoting a series of intracellular signaling events leading to the release of active caspase-8 and initiating apoptosis. In contrast, AICD in CD8+ T cells is predominantly induced through a second death domain, i.e., the receptor for the cytokine TNF-
(tumor necrosis factor-) (Zheng et al., 1995). AICD of CD4+ T lymphocytes is essential for controlling the normal immune response against foreign antigens. In addition, it is believed that the removal of autoreactive T lymphocytes is dependent upon Fas-mediated AICD (Van Parijs et al., 1998). Thus, it is not surprising that defects in Fas-dependent apoptosis are found in several idiopathic autoimmune diseases in both humans and mice (Bona et al., 2003; Kovacs et al., 1996; Le Deist et al., 1996; Semra et al., 2002; Sneller et al., 1997; Zhang et al., 2001). Along this line, therapies that facilitate Fas-mediated T cell apoptosis can ameliorate autoimmune disease (Hong et al., 1998; Jin et al., 2004; Nishimura-Morita et al., 1997; Zhou et al., 1999). Aberrant, Fas-dependent AICD leading to autoimmune disease was shown to encompass impaired Fas or FasL expression (e.g., mutations in Fas or FasL in humans [Fisher et al., 1995] or mice [Takahashi et al., 1994; Watanabe-Fukunaga et al., 1992]). In addition, defects in the Fas-signaling pathway as evidenced by defective caspase-8 activation or NF-
B activity (Kuhtreiber et al., 2003; Zhang et al., 2001) have been described in autoimmune disease models. The present study was designed to examine the effects of TCAH on Fas-dependent AICD in order to better understand the mechanism by which this toxicant inhibits CD4+ T cell apoptosis. Such information could provide an important mechanistic link between toxicant exposure and the development of autoimmune disease.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Mice.
Six- to eight-week-old female C3H/HeJ mice and female C57BL/6
ß TCR transgenic mice were purchased from Jackson Laboratories (Bar Harbor, ME). The animals were housed in polycarbonate, ventilated cages (Animal Care Systems, Littleton, CO) and provided standard lab chow and drinking water ad libitum. All mice were maintained according to the National Research Council guidelines.
Reagents.
TCAH (purity > 99%) was obtained from Sigma (St Louis, MO) and resuspended in deionized water as a stock solution and diluted with 1x PBS. Concoravalin A (Con A) type V and VI from Canavalia ensiformis) and dexamethasone were obtained from Sigma. Recombinant mouse IL-2 (rIL-2) was purchased from R&D Systems (Minneapolis, MN). For inhibition of the Fas/FasL interaction, recombinant murine Fas-Fc fusion protein (R&D Systems) was added to cultures of cells activated to undergo AICD. The recombinant mouse Fas-Fc is a disulfide-linked homodimeric protein that contains the extracellular domain of mouse Fas that is fused to the carboxy-terminal Fc region of human IgG via a linker peptide. This fusion protein has been used by others to inhibit the Fas/FasL interaction in vitro (Hur et al., 2004). Recombinant soluble human FasL (rshFasL) (Alexis Biochemicals, San Diego, CA), a fusion protein that consists of the extracellular domain of FasL fused at the N-terminus to a linker peptide, was used to stimulate AICD directly via the Fas receptor. Abs purchased from BD Biosciences (San Jose, CA) included purified anti-CD3
(hamster IgG1 clone 145-2C11), neutralizing anti–TNF- (clone MP6-XT3, rat IgG1), PE–anti-CD4 (clone GK1.5, rat IgG2b), purified anti-FasL (clone MF14, Armenian hamster IgG3), purified anti-Fas (clone Jo2, Armenian hamster IgG2), purified anti-mouse TNF- (clone MP6-XT3, rat IgG1), FITC-labeled anti-hamster IgG (H&L), and biotinylated anti-V2 (clone B20.1). FITC–Annexin V and PE-streptavidin were also purchased from BD Biosciences. Rabbit anti–matrix metalloproteinase-7 (MMP-7) Ab was obtained from Calbiochem (La Jolla, CA).
Analysis of cell viability, induction of AICD, and measurement of FasL and Fas on CD4+ T cells.
All cells were cultured in standard RPMI medium supplemented with 2mM L-glutamine, 1mM, nonessential amino acids, 1mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, 5 x 10–5M 2-ME, and 10% FCS. For the viability experiments, cells harvested from the spleens of C3H/HeJ mice were cultured in medium alone or with Con A (5 µg/ml) and IL-2 (5 ng/ml) for 5 days in the presence or absence of increasing concentrations of TCAH. Approximately 1 x 106 cells from triplicate cultures were harvested at the given time points, and 10,000 events were collected and analyzed by flow cytometry using a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA). The percentage of nonviable cells was determined based on low forward and side scatter. AICD was induced by activating spleen cells from C3H/HeJ mice with Con A (5 µg/ml) and IL-2 (5 ng/ml) for 4 days in standard RPMI medium. Viable cells were isolated by passage over Ficoll-Hypaque, counted using trypan blue exclusion and light microscopy, and then stimulated with immobilized anti-CD3 Ab (5 µg/ml) for 18 h as described in detail (Blossom et al., 2004). rshFasL was used instead of anti-CD3 Ab in some experiments to induce apoptosis by direct engagement of the Fas receptor. TCAH was added at the initiation of cultures and again during the 18-h stimulation with immobilized anti-CD3 Ab or rshFasL. In some cases, attempts were made to block AICD by adding neutralizing anti–TNF- Ab (Guevara Patino et al., 2000) or recombinant murine Fas-Fc fusion protein during the final 18 h of culture with anti-CD3 Ab.
For dexamethasone-mediated apoptosis, spleen cells were activated with Con A and IL-2 as described above in the presence or absence of TCAH. Following passage over Ficoll-Hypaque, viable cells were treated for 18 h with different concentrations of dexamethasone within a range shown previously to induce apoptosis in vitro (Zacharchuk et al., 1990). To demonstrate apoptosis mediated by IL-2 removal, spleen cells activated for 4 days with Con A and IL-2 in the presence or absence of TCAH were then stimulated with 5 ng/ml IL-2 or incubated in medium alone for an additional 2 days ± TCAH.
After stimulating the cells to undergo Fas-dependent or -independent apoptosis, the cells were stained with PE–anti-CD4 in conjunction with FITC-labeled Annexin V as described previously (Blossom et al., 2004). The T cells induced to undergo AICD were also examined for their expression of FasL and Fas. Cells were incubated with PE–anti-CD4 Ab and either purified anti-FasL or purified anti-Fas followed by FITC-labeled, anti-hamster IgG (H&L). The analysis of 10,000 CD4 events per sample was carried out using a FACSCalibur flow cytometer (Becton Dickinson), and the data were presented as histograms of CD4+ or as dot plots. Data analysis was performed with the use of WinMDI software. For all groups tested, staining with isotype Ig controls was also examined to visualize background staining. The results in each of the histograms are expressed as the percentage of positive CD4+ T cells that were determined by gating outside the staining region observed with the isotype control Ig.
AICD in TCR transgenic mice.
C57BL/6 ß TCR transgenic mice, constructed originally from a C57BL/6 strain (H-2b), possess a TCR that pairs with the CD4 coreceptor that is specific for chicken Ovalbumin323–339 peptide (OVA peptide) in the context of I-Ab. To activate TCR transgenic T cells to undergo AICD, TCR transgenic lymph node cells (2.5 x 105/ml) were cocultured medium in 24-well plates with 2 x 106/ml irradiated spleen cells (2000R) that were isolated from syngeneic nontransgenic C57BL/6 mice for use as a source of antigen-presenting cells. The cultures were then stimulated in the presence or absence of 0.2µM OVA peptide (Peptide International, Louisville, KY) in the presence or absence of either 0.25 or 0.5mM TCAH. rIL-2 (5 ng/ml) was added to the cultures to keep cells cycling and facilitate Fas-dependent apoptosis as described previously (Lenardo, 1991; Van Parijs et al., 1999). After primary stimulation for 3 days at 37°C, viable cells (90–95% V2 TCR) were isolated by Ficoll density centrifugation and cultured with fresh, irradiated C57BL/6 spleen cells in the presence or absence of OVA peptide (0.2, 0.8, 4.0, or 20µM) ± 0.25 or 0.5mM TCAH. After 24 h, the cells were harvested and two color stained with FITC–Annexin V and biotinylated anti-V2 TCR mAb followed by PE-conjugated streptavidin and subjected to flow cytometry as described above.
Reverse transcription–polymerase chain reaction.
For the detection of Fas and FasL mRNA, spleen cells from the C3H/HeJ mice were left untreated (resting) or stimulated with Con A and IL-2 as described above in the presence or absence of 0.05 or 0.5mM TCAH. The Con A– and IL-2–activated T cell blasts were then passed over Ficoll-Hypaque and the CD4+ T cells purified using anti-CD4–coated magnetic beads per manufacturer's instructions (Invitrogen, Carlsbad, CA). The purified CD4+ T cells (2 x 106/ml; > 95% purity) were activated with immobilized anti-CD3 mAb for 0, 2, 4, 8, and 20 h in the presence or absence of 0.05 or 0.5mM TCAH. Total RNA was extracted using the TRIzol reagent (Invitrogen). One microgram of RNA was then reverse transcribed according to the Superscript first-strand synthesis system for RT-PCR protocol (Invitrogen). The cDNAs were amplified with specific primers. Amplification of GAPDH was used as a control. The oligonucleotide primers for amplification of Fas and FasL PCRs were custom ordered from Integrated DNA Technology (Coralville, IA) and have the following sequence—Fas: sense, 5'-ATGCTGTGGATCTGGGCT-3' and antisense, 5'-TCACTCGACACATTGTCC-3'; FasL: sense, 5'-ACTGGACAGATATGGGCCCAC-3' and antisense, 5'-GCCTCTGTGAGGTAGTAAGTAG-3'; and GAPDH: sense, 5'-TGAAGGTCGGTGTGAACGGATTTGGC-3' and antisense, 5'-CATGTAGGCCATGAGGTCCACCAC-3'. One-fourth of the RT reaction mixture was utilized in standard PCRs containing 1x PCR buffer, 1mM MgCl2, a 0.5µM concentration of each oligonucleotide primer, 0.2mM deoxynucleoside triphosphates, and 1 U of Taq polymerase (Amersham Pharmacia Biotech, Piscataway, NJ). The amplification cycle (45 s at 94°C, 45 s at 54–60°C, and 2 min at 72°C) was repeated 20–30 times, depending on the primers used, and followed by a single 7-min period at 72°C. Aliquots were removed at the end of cycles 20 through 30 in order to determine the minimum amplification necessary for detection. PCR amplification was performed in a thermal cycler (BioRad icycler). Following amplification, PCR samples were subjected to standard gel electrophoresis in 1.5–2.0% agarose and the images scanned with the Versadoc Imaging System (BioRad, Hercules, CA). In some experiments, RNA was extracted from the Con A/IL-2 blasts that were stimulated with anti-CD3 Ab for 0, 6, and 18 h and subjected to RT-PCR as described above using the following primers—MMP-7: sense, 5'-ACAGGTGCAGCTCAGGAAGG-3' and antisense, 5'-GTGAAGGACGCAGGAGTGAAC-3'; and MMP-9: sense, 5'-TCAGGAACTTCCAGTACCAACCGT-3' and antisense, 5'-GCGACCACATCGAACTTCGACACT-3'.
Soluble FasL ELISA.
Spleen cells from C3H/HeJ mice were treated with Con A and IL-2 for 4 days as described above. Viable cells were purified by Ficoll-Hypaque gradient centrifugation, counted using trypan blue and light microscopy, and activated with 5 µg of plate-bound anti-CD3 mAb for 6 or 18 h in the presence or absence of 0.5mM TCAH. The resulting culture supernatants were tested for the presence of soluble FasL (sFasL) using the Quantikine M mouse FasL ELISA kit (R&D Systems) according to manufacturer's instructions.
Western blotting analysis.
The anti-CD3–treated T cell blasts were lysed with buffer containing 10mM KCl, 10mM HEPES, 1.5mM MgCl2, 0.5% Nonidet P-40, 1mM NaVO4, aprotinin (10 mg/ml), leupeptin (10 mg/ml), and 0.5mM PMSF. Protein concentrations of the lysates were determined using a Pierce BCA Protein Assay Kit (Rockford, IL). Equal protein amounts of the cell lysates (200 µg) were separated by SDS-PAGE using a 12% Tris-HCl polyacrylamide gel and transferred to nitrocellulose. The nitrocellulose was then immunoblotted with Abs specific for MMP-7. The appropriate HRP-labeled secondary Ab was then applied. The bands were detected using SuperSignal West Pico Chemiluminescent Substrate (Pierce). To test for appropriate protein loading, a blot was stripped using the western Blot Recycling Kit (Alpha Diagnostic International, San Antonio, TX) and reprobed with an anti-actin Ab.
Statistical analysis.
For each in vitro experiment, triplicate cultures were established for each condition, and equal numbers of cells for each replicate culture were then pooled to conduct each assay as described above. All experiments were independently conducted at least twice. Unless stated otherwise, all statistical comparisons were conducted between independent experiments. Paired groups were compared by the Student t test. Values of p < 0.05 were considered statistically significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Effects of TCAH on Lymphocyte Viability in 5-Day Cultures
In an earlier study, TCAH stimulated T cells in vitro in short-term cultures (< 24 h) with concentrations in the range of 0.04–1mM (Gilbert et al., 2004
). However, it was not known whether similar doses of TCAH would diminish cell viability following the 5-day culture period required to induce AICD. As shown in Figure 1A, the addition of 1.0mM, but not lower doses of TCAH, significantly increased the cell death observed over that of medium-only cultures in the presence of Con A and IL-2 stimulation by 48 h. It is likely that the improvement of cell viability observed by 120 h was due to that fact that the T cell population in splenocytes stimulated under similar conditions is known to double by 96 h (Hedfors and Brinchmann, 2003). As expected, all the cells that were cultured under static conditions were nonviable by 120 h (Fig. 2B). Taken together, these results demonstrated that concentrations of TCAH lower than 1.0mM were not likely to affect cell viability in long-term cultures under conditions required to induce AICD.
|
|
TCAH Attenuated AICD of CD4+ T Lymphocytes
The goal of this study was to investigate how TCAH inhibited CD4+ T cell AICD using an established in vitro model of AICD induction. This model, used by this laboratory and others, involves lymphocyte activation in the presence of IL-2 followed by CD3 receptor engagement under long-term (4–7 day) culture conditions (Blossom et al., 2004
; Delgado and Ganea, 2000; Schmitz et al., 2003; Tucek-Szabo et al., 1996). Different concentrations of TCAH (0.03–0.5mM) were tested for their ability to alter AICD induced in CD4+ T cells activated with Con A and IL-2 and then restimulated with anti-CD3 Ab. Figure 2A showed that TCAH inhibited AICD in CD4+ T cells in a dose-dependent manner, becoming measurable at a concentration of 0.2mM (18% inhibition) and maximal at the 0.5mM dose (42% inhibition). A representative histogram from the data presented in Figure 2A showed the protective effect of TCAH (0.5mM) on AICD in CD4+ T cells (Fig. 2B). It was found that TCAH needed to be present throughout the early activation period in order to inhibit AICD of CD4+ T cells; if TCAH was added only during the final 18-h stimulation with anti-CD3 Ab, CD4+ T cell AICD was not attenuated (data not shown). This finding makes it unlikely that the decrease in Annexin V staining observed when TCAH was instead present throughout the 5-day culture period was due to a nonspecific blocking effect on Annexin V binding. Apoptosis in the in vitro model of AICD used here was blocked most effectively by the addition of a 1.0-µg/ml, soluble Fas-Fc fusion protein (Fig. 2C) but not by a neutralizing Ab specific for TNF- (Fig. 2D). Consequently, the protocol used here to examine the effects of TCAH on apoptosis appeared to be mediated by Fas/FasL rather than TNF-/TNF-R interactions. Taken together, the results showed that continuous exposure to TCAH inhibited Fas-mediated AICD in CD4+ T cells.
TCAH Inhibited Apoptosis of Antigen-Activated T Cells
The effects of TCAH on antigen-induced CD4+ T cell AICD were also investigated in vitro using peptide-specific T cells from TCR transgenic mice. Lymph node T cells from mice expressing a transgenic TCR with specificity for the OVA peptide were activated in vitro with OVA peptide in the presence or absence of TCAH. As expected, increasing concentrations of OVA peptide induced a dose-dependent increase in AICD in transgenic T cells (Fig. 3A). However, Annexin V staining was decreased in the presence of TCAH, especially at the 4.0 and 20µM dose of OVA peptide. The results of two independent experiments showed that 0.5mM TCAH induced a dose-related, statistically significant decrease in Annexin V staining of TCR transgenic T cells restimulated with 20µM OVA peptide by 56% in comparison to control cultures (Fig. 3B). Thus, similar to the Con A/IL-2 and anti-CD3 model of AICD, TCAH also attenuated CD4+ T cell AICD in an antigen-specific manner, a model of AICD that also involves Fas/FasL interactions (Van Parijs et al., 1998). These results provided further evidence that TCAH attenuated AICD by acting on some aspect of the Fas/FasL pathway.
|
TCAH Did Not Protect CD4+ T Cells from Fas-Independent Apoptosis
In order to determine whether the ability of TCAH to inhibit Fas-mediated AICD represented a generalized effect on apoptosis, TCAH was next tested for its ability to suppress forms of CD4+ T cell apoptosis that do not require Fas/FasL signaling, i.e., apoptosis mediated by the glucocorticoid dexamethasone or by IL-2 withdrawal (Hieronymus et al., 2000
; Tamura and Yui, 1995). Unlike anti-CD3–induced AICD, CD4+ T cell apoptosis induced by different doses of dexamethasone was unaltered by CD4+ T cell exposure to 0.5mM TCAH (Fig. 4A). Similarly, CD4+ T cell apoptosis induced by withdrawal of IL-2 was not susceptible to TCAH inhibition (Fig. 4B). These findings suggest that the antiapoptotic effect of TCAH is specific for Fas-mediated AICD.
|
TCAH Does Not Inhibit AICD Induced by Direct Engagement of the Fas Receptor
Defects in the susceptibility of CD4+ T cells to Fas-mediated AICD can encompass faulty expression of either Fas or FasL as well as impaired Fas signaling. All three possibilities were examined to determine the mechanism by which TCAH inhibited AICD. rshFasL is capable of cross-linking Fas on the surface of mouse T cells and triggering Fas-mediated apoptosis (Lens et al., 2002
; Tanaka et al., 1997). Con A– and IL-2–activated blasts treated with rshFasL in the presence of 0.5mM TCAH underwent the same degree of apoptosis as CD4+ T cells treated with rshFasL in the absence of TCAH (Fig. 5). Thus, TCAH treatment of CD4+ T cells in vitro did not appear to inhibit AICD in CD4+ T cells by blocking signaling through the Fas molecule.
|
TCAH Inhibited the Expression of FasL, but not Fas, on the Surface of Activated CD4+ T Cells
Since TCAH did not alter Fas signaling, the next set of experiments tested the possibility that TCAH attenuated CD4+ T cell AICD by inhibiting the expression of Fas or FasL. Exposure to TCAH had no effect on the expression of Fas by CD4+ T cells stimulated with anti-CD3 Ab to undergo AICD (Fig. 6). In contrast, TCAH treatment inhibited the expression of FasL on the CD4+ T cells stimulated to undergo AICD by 51% (79 vs. 39%). Therefore, the ability of TCAH to inhibit anti-CD3 Ab–mediated AICD was linked to downregulation of FasL but was not correlated with a decreased expression of Fas or suppression of Fas signaling.
|
Additional experiments were conducted to examine the mechanism for the decrease in FasL expression on the TCAH-treated CD4+ T cells. Using RT-PCR it was shown that stimulation of purified Con A– and IL-2–activated CD4+ T cells with anti-CD3 Ab increased the expression of both Fas and FasL mRNA by 8 h (Fig. 7A). In a separate experiment, Figure 7B demonstrated that the levels of FasL transcription observed at 8 h begin to decrease after 20 h in culture. As expected, levels of FasL were either undetectable or low in resting spleen cells or purified Con A– and IL-2–activated CD4+ T cells, respectively. However, treatment with TCAH in either experiment did not affect this expression; control and TCAH-treated CD4+ T cells contained similar levels of message for both FasL and Fas following activation to undergo AICD. Thus, TCAH did not appear to regulate expression of FasL at the transcriptional level.
|
TCAH Increases Release of FasL from CD4+ T Cells in a Metalloproteinase-Dependent Manner
Since TCAH did not appear to affect FasL gene expression, the next experiment tested whether TCAH instead decreased FasL expression on CD4+ T cells by cleaving it from the cell surface. The culture supernatants from spleen cells simulated to undergo AICD in the presence of TCAH were examined for the presence of sFasL. As shown in Figure 8, very little sFasL was detected in the culture supernatants of Con A– and IL-2–treated blasts incubated for an additional 6 or 18 h in medium alone. If, however, the blasts were incubated with immobilized anti-CD3 Ab for 18 h, sFasL was released into the culture supernatant. The level of sFasL was significantly increased if TCAH was also added to the cultures. TCAH similarly increased the levels of sFasL detected in culture supernatants collected as soon as 6 h after stimulation with anti-CD3 Ab. Although not conclusive, this result suggested that TCAH inhibited AICD in CD4+ T cells by promoting cleavage of FasL from the surface of the CD4+ T cells.
|
Since FasL cleavage is mediated by metalloproteinases, additional experiments examined the functional importance of these enzymes in the TCAH-induced decrease in AICD. As expected, the presence of TCAH decreased AICD as detected by Annexin V staining (Figs. 9A and 9B) and FasL expression in CD4+ T cells (Fig. 9A). The general metalloproteinase inhibitor GM6001 suppressed the ability of TCAH to inhibit FasL and Annexin V staining in CD4+ T cells. GM6001 had no effect on Fas expression, which was also unaffected by exposure to TCAH. Thus, TCAH appeared to use a metalloproteinase-dependent mechanism to cleave FasL from CD4+ T cells, thereby inhibiting AICD in these cells.
|
TCAH Increased MMP-7 Expression
In order to further investigate the role of metalloproteinases in mediating the antiapoptotic effects of TCAH, experiments were initiated to determine whether TCAH increased expression of MMP-7, the metalloproteinases shown to cleave FasL (Vargo-Gogola et al., 2002
). In a representative experiment, MMP-7 mRNA was found in activated lymphocytes stimulated with Con A and IL-2 in the presence of 0.5mM TCAH (Fig. 10A). Expression of MMP-7 mRNA remained high in TCAH-treated blasts 6 h following stimulation with anti-CD3 Ab and then decreased by 18 h poststimulation. The results from two independent experiments clearly demonstrated a statistically significant increase of MMP-7 expression over that of medium-only cultures at the 0-h time point (Fig. 10B). In contrast, lymphocytes stimulated with Con A and IL-2 in the absence of TCAH contained no detectable MMP-7 mRNA. Although not significant, TCAH induced higher levels of MMP-7 expression after 6 and 18 h of culture with anti-CD3 Ab. Further stimulation of Con A/IL-2–activated blasts with anti-CD3 Ab in the absence of TCAH induced only low levels of detectable MMP-7 mRNA by 18 h. Unlike MMP-7, MMP-9 was not increased by exposure to TCAH.
|
The ability of 0.5mM TCAH to increase MMP-7 was also observed at the protein level; Con A/IL-2–activated blasts stimulated with anti-CD3 Ab for 18 h in the presence of TCAH contained the proenzyme form of MMP-7 (Fig. 10C). In contrast, MMP-7 could not be detected in CD4+ T cells stimulated with anti-CD3 Ab in the absence of TCAH. Thus, TCAH increased expression of the FasL-cleaving metalloproteinase MMP-7 at both the mRNA and protein level in Con A/IL-2–activated blasts.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The present study focused on exploring the mechanism of how the TCE metabolite TCAH attenuated CD4+ T cell AICD in vitro. The process of AICD in lymphocytes is crucial in controlling the immune response. Thus, any event that disrupts the ability to limit the expansion of activated CD4+ T lymphocytes could have important implications for the development of autoimmunity in humans. TCAH appeared to inhibit Fas-dependent AICD in CD4+ T cells by suppressing expression of FasL. FasL expression on CD4+ T cell surface in response to activation is a transient event that is regulated at the level of transcription (Brunner et al., 1996
; Monleon et al., 2001). FasL expression on the T cell surface can also be regulated by the cleavage of membrane-bound FasL into sFasL by MMP-7 (Vargo-Gogola et al., 2002). Because sFasL is much less efficient than membrane-bound FasL at inducing Fas-mediated apoptosis in T cells, a mechanism that promotes FasL shedding can decrease Fas-mediated AICD (Oyaizu et al., 1997). The association between FasL shedding and autoimmunity is supported by the finding that sFasL levels are increased in the sera of patients with autoimmune diseases, such as multiple sclerosis, Graves' disease, and systemic lupus erythematosus, and in joints of patients with rheumatoid arthritis (Sakai et al., 1999; Tomokuni et al., 1999; Wang et al., 2002). Thus, it is possible that the ability of the TCE metabolite TCAH to promote FasL shedding and inhibit AICD in CD4+ T contributes to the development of TCE-induced autoimmune hepatitis.
The experiments examining the effects of TCAH on AICD were conducted in vitro. Although Fas-mediated AICD in CD4+ T cells has been well documented in vitro, its role in immune tolerance in vivo is somewhat more contentious. The results of experiments in which Fas- or FasL-deficient mice were treated with superantigens or anti-CD3 Ab led some investigators to conclude that polyclonal activation–induced elimination of mature T lymphocytes in vivo was Fas dependent (Mountz et al., 1994; Renno et al., 1996; Scott et al., 1993), while others concluded that it was Fas independent (Desbarats et al., 1998; Hildeman et al., 2002). More recently, antigen-specific AICD has been examined in adoptively transferred TCR transgenic CD4+ T cells. Exposure to large amounts or repeated doses of peptide induced Fas-mediated cell deletion of activated, peptide-specific peripheral T cells (Boissonnas and Combadiere, 2004; Pinkoski et al., 2002; Sytwu et al., 1996). Similarly, a study revealing a lack of cell death in Fas-deficient T cells undergoing lymphopenia-induced proliferation led the authors to conclude that Fas mediates deletion of T cells undergoing homeostatic proliferation (Fortner and Budd, 2005). Taken together, it appears that the death of activated T cells in vivo can be induced by more than one major route, one of which is often called the extrinsic pathway and involves death receptors such as Fas (Green et al., 2003; Marrack and Kappler, 2004; Tripathi and Hildeman, 2004). Fas-mediated apoptosis is thought to be particularly important for the in vivo deletion of T cells stimulated with autoantigens (Van Parijs et al., 1998). Thus, since TCAH inhibits AICD in vitro, it seems likely that it performs the same function in vivo for autoreactive CD4+ T cells.
Presumably, the ability of TCAH to inhibit activation-induced CD4+ T cell death in vivo would be concentration dependent as it is in vitro. Humans can be exposed to TCAH as a result of occupational or environmental exposure to TCE. Environmental exposures to TCE are generally low, however; there have been instances where environmental contamination of TCE has been significant. For example, the ATSDR estimates that dating back 30 years from 1985, somewhere between 50,000 and 200,000 people living in Marine Camp Lejeune in North Carolina were drinking from wells contaminated with TCE at levels almost 300 times the level now considered safe (ATSDR, 1997). Although environmental exposures are obviously a great health concern, the most significant exposures to TCE occur in the workplace. The Occupational Safety and Health Administration states that the 8-h permissible exposure limit for TCE should not exceed a time-weighted average of 100 or 300 ppm for 5 min in any 2-h period (ATSDR, 1997). Human occupational exposure to 100 ppm of TCE results in blood levels of TCE at 1.5 µg/ml (11µM) (Pleil et al., 1998). Accidental acute exposure in the workplace to an unknown concentration of TCE has yielded TCE levels of 84 µg/ml in the blood (640µM) (Coopman et al., 2003). Assuming that over 85% of TCE is metabolized through TCAH (Davidson and Beliles, 1991), occupational exposure to 100–300 ppm TCE could result in systemic levels of TCAH in the micromolar range. Local levels of TCAH in the liver where most TCE metabolism occurs may be even higher. Aside from exposure to TCE, humans can also encounter TCAH directly through the use of the sedative chloral hydrate. Exposure to a single, nonsedative dose of 200 ppm TCAH was shown to yield blood concentrations of 202µM TCAH in mice (Beland, 1999). Since the concentration of TCAH in plasma was found to decrease by about 50% during storage, the authors of that study state that this peak concentration may have been underestimated by a factor of 2. In any case, it seems likely that exposure to TCAH either directly or indirectly via TCE can generate blood concentrations of TCAH that fall within the range of those used in the present in vitro study. In addition, the cumulative effects associated with chronic in vivo exposure may be achieved at lower concentrations of TCE than those needed to demonstrate an effect in a short-term, in vitro assay using TCE metabolite TCAH. Because mice chronically exposed to occupationally relevant levels of TCE (0.5 and 2.5 mg/ml or 100–400 mg/kg/day) over a period of 32 weeks developed autoimmune disease (Griffin et al., 2000c), the in vivo functional relevance of the results described here should not be disqualified based on the concentrations of TCAH used.
The ability of TCAH to inhibit activation-induced death of CD4+ T cells appeared to be related to an increase in metalloproteinase activity. Metalloproteinases are not constitutively expressed in most normal tissue but can be induced by a variety of stimuli in a tissue-specific manner. With regard to T cells, metalloproteinase expression is just beginning to be described. It has been shown that T cell stimulation with mitogens such as phorbol ester or with certain cytokines such as IL-2 can increase expression of metalloproteinases (Bar-Or et al., 2003; Goetzl et al., 1996). Metalloproteinase activity can also be induced in T cells by engaging adhesion molecules on accessory cells such as endothelial cells (Van Themsche et al., 2004). The current study provided the novel finding that an environmental toxicant promoted metalloproteinase activity in Con A/IL-2–activated blasts. However, in this study, MMP-7 expression was not examined in purified CD4+ T cells isolated from the Con A/IL-2–activated lymphocytes. Although it is tempting to speculate that the TCAH-treated, activated CD4+ T cells themselves are producing MMP-7, it is possible that some other cell type within the Con A/IL-2–activated population (i.e., macrophages, B lymphocytes or CD8+, T lymphocytes) is responsible for producing MMP-7, and thereby facilitating FasL shedding of CD4+ T cells via a bystander effect. Experiments to determine the cellular source of the MMP-7 in the Con A/IL-2–activated population are currently underway in our laboratory.
The factors that regulate metalloproteinase gene transcription in T cells are not known. However, the promoter for MMP-7 has binding sites for the activator protein-1 (AP-1) transcription factor (Lynch et al., 2004), and AP-1 has been shown to be required for expression of MMP-7 in colon cancer cells (Yamamoto et al., 1995). TCAH exposure of CD4+ T cells promotes the phosphorylation of activating transcription factor 2 and c-Jun, two components of AP-1 (Gilbert et al., 2004). The possibility that TCAH increases metalloproteinase activity in CD4+ T cells via activation of AP-1 is currently being investigated.
The ability of TCAH to regulate T cell AICD by inhibiting FasL expression represents a mechanism by which an environmental stressor may contribute to autoimmune disease etiology. Little is known about the manner in which other environmental contaminants affect AICD in CD4+ T cells. One exception is the documented ability of inorganic mercury to inhibit Fas-dependent AICD in the human T cell lines (Whitekus et al., 1999). Mercury was subsequently shown to block the association between CD95 and the signaling adapter protein Fas-associated death domain (FADD) (McCabe Jr et al., 2003). The finding that TCAH inhibits the AICD by regulating FasL expression represents another mechanism by which xenobiotics can alter Fas-mediated apoptosis. This novel finding underscores the immunoregulatory capacity of certain environmental toxicants and has important implications regarding the etiology of autoimmunity as well as other diseases where defective AICD is known to play an important role.
|
ACKNOWLEDGMENTS |
|---|
We thank Susan Panozzo for technical assistance. This work was made possible by funds from the Arkansas Children's Hospital Research Institute Lyon New Scientist Development Award (to S.J.B.), the Environmental Protection Agency (MA0223) (to K.M.G.), and the Arkansas Biosciences Institute (to K.M.G.)
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Agency for Toxic Substances and Disease Registry (ATSDR) (1997). Public Health Statement for Trichloroethylene. Available at: http://www.atsdr.cdc.gov. Accessed 2006.
Ashley, D. L., Bonn, M. A., Cardinali, F. L., McGraw, J. M., and Wooten, J. V. (1994). Blood concentrations of volatile organic compounds in a non-occupationally exposed US population and in groups with suspected exposure. Clin. Chem. 40, 1401–1404.
Bar-Or, A., Nuttall, R. K., Duddy, M., Alter, A., Kim, H. J., Ifergan, I., Pennington, C. J., Bourgoin, P., Edwards, D. R., and Yong, V. W. (2003). Analyses of all matrix metalloproteinase members in leukocytes emphasize monocytes as major inflammatory mediators in multiple sclerosis. Brain 126, 2738–2749.
Beland, F. A. (1999). NTP technical report on the toxicity and metabolism studies of chloral hydrate (CAS No. 302-17-0). Administered by gavage to F344/N rats and B6C3F1 mice. Toxic. Rep. Ser. 59, 1–66, A1–E7.[Medline]
Blossom, S. J., Pumford, N. R., and Gilbert, K. M. (2004). Activation and attenuation of apoptosis of CD4(+) T cells following in vivo exposure to two common environmental toxicants, trichloroacetaldehyde hydrate and trichloroacetic acid. J. Autoimmun. 23, 211–220.[CrossRef][ISI][Medline]
Boissonnas, A., and Combadiere, B. (2004). Interplay between cell division and cell death during TCR triggering. Eur. J. Immunol. 34, 2430–2438.[CrossRef][ISI][Medline]
Bona, G., Defranco, S., Chiocchetti, A., Indelicato, M., Biava, A., DiFranco, D., Dianzani, I., Ramenghi, U., Corrias, A., Weber, G., et al. (2003). Defective function of Fas in T cells from paediatric patients with autoimmune thyroid diseases. Clin. Exp. Immunol. 133, 430–437.[CrossRef][ISI][Medline]
Brunner, T., Yoo, N. J., Griffith, T. S., Ferguson, T. A., and Green, D. R. (1996). Regulation of CD95 ligand expression: A key element in immune regulation? Behring Inst. Mitt. 97, 161–174.[Medline]
Coopman, V. A., Cordonnier, J. A., De Letter, E. A., and Piette, M. H. (2003). Tissue distribution of trichloroethylene in a case of accidental acute intoxication by inhalation. Forensic Sci. Int. 134, 115–119.[CrossRef][ISI][Medline]
Davidson, I. W. F., and Beliles, R. P. (1991). Consideration of the target organ toxicity of trichloroethylene in terms of metabolite toxicity and pharmacokinetics. Drug Metab. Rev. 23, 493–599.[ISI][Medline]
Delgado, M. and Ganea, D. (2000). Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibit antigen-induced apoptosis of mature T lymphocytes by inhibiting Fas ligand expression. J. Immunol. 164, 1200–1210.
Desbarats, J., Duke, R. C., and Newell, M. K. (1998). Newly discovered role for Fas ligand in the cell-cycle arrest of CD4+ T cells. Nat. Med. 4, 1377–1382.[CrossRef][ISI][Medline]
Fisher, G. H., Rosenberg, F. J., Straus, S. E., Dale, J. K., Middleton, L. A., Lin, A. Y., Strober, W., Lenardo, M. J., and Puck, J. M. (1995). Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome. Cell 81, 935–946.[CrossRef][ISI][Medline]
Fortner, K. A. and Budd, R. C. (2005). The death receptor Fas (CD95/APO-1) mediates the deletion of T lymphocytes undergoing homeostatic proliferation. J. Immunol. 175, 4374–4382.
Gilbert, K. M., Griffin, J. M., and Pumford, N. R. (1999). Trichloroethylene activates CD4+ T cells: Potential role in an autoimmune response. Drug Metab. Rev. 31, 901–916.[CrossRef][ISI][Medline]
Gilbert, K. M., Whitlow, A. B., and Pumford, N. R. (2004). Environmental contaminant and disinfection by-product trichloroacetaldehyde stimulates T cells in vitro. Int. Immunopharmacol. 4, 25–36.[CrossRef][ISI][Medline]
Gist, G. L. and Burg, J. R. (1995). Trichloroethylene—A review of the literature from a health effects perspective. Toxicol. Ind. Health 11, 253–307.[ISI][Medline]
Goetzl, E. J., Banda, M. J., and Leppert, D. (1996). Matrix metalloproteinases in immunity. J. Immunol. 156, 1–4.[Abstract]
Green, D. R., Droin, N., and Pinkoski, M. (2003). Activation-induced cell death in T cells. Immunol. Rev. 193, 70–81.[CrossRef][ISI][Medline]
Griffin, J. D., Gilbert, K. M., and Pumford, N. R. (2000a). Inhibition of CYP2E1 reverses CD4+ T cell alterations in trichloroethylene-treated MRL+/+ mice. Toxicol. Sci. 54, 384–389.
Griffin, J. M., Blossom, S. J., Jackson, S. K., Gilbert, K. M., and Pumford, N. R. (2000b). Trichloroethylene accelerates an autoimmune response in association with Th1 T cell activation in MRL+/+ mice. Immunopharmacology 46, 123–137.[CrossRef][ISI][Medline]
Griffin, J. M., Gilbert, K. M., Lamps, L. W., and Pumford, N. R. (2000c). CD4+ T cell activation and induction of autoimmune hepatitis following trichloroethylene treatment in MRL+/+ mice. Toxicol. Sci. 57, 345–352.
Guevara Patino, J. A., Ivanov, V. N., Lacy, E., Elkon, K. B., Marino, M. W., and Nikolic-Zugic, J. (2000). TNF-alpha is the critical mediator of the cyclic AMP-induced apoptosis of CD8+4+ double-positive thymocytes. J. Immunol. 164, 1689–1694.
He, X. S., Ansari, A. A., and Gershwin, M. E. (2001). Xenobiotic considerations for the development of autoimmune liver diseases: Bad genes and bad luck. Rev. Environ. Health 16, 191–202.[Medline]
Hedfors, I. A., and Brinchmann, J. E. (2003). Long-term proliferation and survival of in vitro-activated T cells is dependent on interleukin-2 receptor signalling but not on the high-affinity IL-2R. Scand. J. Immunol. 58, 522–532.[CrossRef][ISI][Medline]
Hieronymus, T., Blank, N., Gruenke, M., Winkler, S., Haas, J. P., Kalden, J. R., and Lorenz, H. M. (2000). CD 95-independent mechanisms of IL-2 deprivation-induced apoptosis in activated human lymphocytes. Cell Death Differ. 7, 538–547.[CrossRef][ISI][Medline]
Hildeman, D. A., Zhu, Y., Mitchell, T. C., Bouillet, P., Strasser, A., Kappler, J., and Marrack, P. (2002). Activated T cell death in vivo mediated by proapoptotic bcl-2 family member bim. Immunity 16, 759–767.[CrossRef][ISI][Medline]
Hong, N. M., Masuko-Hongo, K., Sasakawa, H., Kato, T., Shirai, T., Okumura, K., Nishioka, K., and Kobata, T. (1998). Amelioration of lymphoid hyperplasia and hypergammaglobulinemia in lupus-prone mice (gld) by Fas-ligand gene transfer. J. Autoimmun. 11, 301–307.[CrossRef][ISI][Medline]
Hur, Y. G., Yun, Y., and Won, J. (2004). Rosmarinic acid induces p56lck-dependent apoptosis in Jurkat and peripheral T cells via mitochondrial pathway independent from Fas/Fas ligand interaction. J. Immunol. 172, 79–87.
Jin, Y., Qu, A., Wang, G. M., Hao, J., Gao, X., and Xie, S. (2004). Simultaneous stimulation of Fas-mediated apoptosis and blockade of costimulation prevent autoimmune diabetes in mice induced by multiple low-dose streptozotocin. Gene Ther. 11, 982–991.[CrossRef][ISI][Medline]
Kilburn, K. H. and Warshaw, R. H. (1992). Prevalence of symptoms of systemic lupus erythematosus (SLE) and of fluorescent antinuclear antibodies associated with chronic exposure to trichloroethylene and other chemicals in well water. Environ. Res. 57, 1–9.[Medline]
Kimber, I., and Dearman, R. J. (2002). Immunologic basis for autoimmunity and the potential influences of xenobiotics. Toxicol. Lett. 127, 77–81.[CrossRef][ISI][Medline]
Kovacs, B., Vassilopoulos, D., Vogelgesang, S. A., and Tsokos, G. C. (1996). Defective CD3-mediated cell death in activated T cells from patients with systemic lupus erythematosus: Role of decreased intracellular TNF-alpha. Clin. Immunol. Immunopathol. 81, 293–302.[CrossRef][ISI][Medline]
Kuhtreiber, W. M., Hayashi, T., Dale, E. A., and Faustman, D. L. (2003). Central role of defective apoptosis in autoimmunity. J. Mol. Endocrinol. 31, 373–399.[Abstract]
Le Deist, F., Emile, J. F., Rieux-Laucat, F., Benkerrou, M., Roberts, I., Brousse, N., and Fischer, A. (1996). Clinical, immunological, and pathological consequences of Fas-deficient conditions. Lancet 348, 719–723.[CrossRef][ISI][Medline]
Lenardo, M. J. (1991). Interleukin-2 programs mouse alpha beta T lymphocytes for apoptosis. Nature 353, 858–861.[CrossRef][Medline]
Lens, S. M. A., Kataoka, T., Fortner, K. A., Tinel, A., Ferrero, I., MacDonald, R. H., Hahne, M., Beermann, F., Attinger, A. F., Orbea, H. A., et al. (2002). The caspase 8 inhibitor c-FLIP(L) modulates T-cell receptor-induced proliferation but not activation-induced cell death of lymphocytes. Mol. Cell. Biol. 22, 5419–5433.
Lynch, C. C., Crawford, H. C., Matrisian, L. M., and McDonnell, S. (2004). Epidermal growth factor upregulates matrix metalloproteinase-7 expression through activation of PEA3 transcription factors. Int. J. Oncol. 24, 1565–1572.[ISI][Medline]
Marrack, P., and Kappler, J. (2004). Control of T cell viability. Annu. Rev. Immunol. 22, 765–787.[CrossRef][ISI][Medline]
McCabe, M. J., Jr., Whitekus, M. J., Hyun, J., Eckles, K. G., McCollum, G., and Rosenspire, A. J. (2003). Inorganic mercury attenuates CD95-mediated apoptosis by interfering with formation of the death inducing signaling complex. Toxicol. Appl. Pharmacol. 190, 146–156.[CrossRef][ISI][Medline]
Monleon, I., Martinez-Lorenzo, M. J., Monteagudo, L., Lasierra, P., Taules, M., Iturralde, M., Pineiro, A., Larrad, L., Alava, M. A., Naval, J., et al. (2001). Differential secretion of Fas ligand- or APO2 ligand/TNF-related apoptosis-inducing ligand-carrying microvesicles during activation-induced death of human T cells. J. Immunol. 167, 6736–6744.