Inorganic Mercury Inhibits the Activation of LAT in T-Cell Receptor-Me
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《毒物学科学杂志》
Department of Immunology & Microbiology, Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201
Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
ABSTRACT
Little is known as to the molecular mechanisms involved with mercury intoxication at very low levels. Although the mechanism is not known, animal studies have nevertheless shown that low levels of mercury may target the immune system. Inorganic mercury (Hg2+) at very low (but non-toxic) levels can disrupt immune system homeostasis, in that genetically susceptible rodents develop idiosyncratic autoimmune disease, which is associated with defective T-cell function. T lymphocyte function is intimately coupled to the T-cell receptor. We have previously reported that on a molecular level, low concentrations of Hg2+ disrupt signaling from the T-cell receptor by interfering with activation of Ras and ERK MAP kinase. In this report we expand upon those results by showing that in T lymphocytes exposed to low concentration of Hg2+, Ras fails to become properly activated because upstream of Ras in the T cell signal transduction pathway, the important scaffolding element Linker for Activation of T Cells (LAT) fails to become properly phosphorylated. Hypo-phosphorylation of LAT occurs, because upstream of LAT, the LAT reactive tyrosine kinase ZAP-70 is also not properly activated in Hg2+ treated cells.
Key Words: LAT; T-cell receptor; mercury; signalosome.
INTRODUCTION
Over the past dozen years it has been shown that acute or chronic exposure to mercury, even at subtoxic doses alters the immune system by leading to increased susceptibility to either autoimmune or immunosuppressive syndromes (Bernier et al., 1995; Hansson and Abedi-Valugerdi, 2004; Hong, 1999; Kazantzis, 2002). Animal studies have shown that in rodent strains of susceptible MHC haplotypes, exposure to low and subtoxic doses of inorganic mercury (Hg2+) induces an autoimmune dysfunction characterized by the production of anti-nuclear antibodies, lymphoproliferation, and hyperglobulinemia (Bagenstose et al., 1998; Hu et al., 1999; Zheng and Monestier, 2003). On the other hand, in vitro studies in a number of systems have shown that mercury compounds often inhibit lymphocyte functions, including proliferation, expression of cell activation markers, and cytokine production (Moszczynski, 1997; Shen et al., 2001; Sweet and Zelikoff, 2001).
In T lymphocytes engagement of the T Cell Receptor (TCR) by antigen will activate the cell. Depending upon the subtype and maturity of the T cell, as well as the mix of available co-factors, a T cell may respond to antigen by proliferation, differentiation, secretion of cytokines, or perhaps undergo apoptosis. However one thing that all of these various functional outcomes between cells of different maturity and T-cell subtype have in common, is that fairly soon after TCR engagement of antigen, ERK MAP Kinase is phosphorylated and activated. The activation of ERK MAP Kinase holds a central role in lymphocyte biology because of its role in the activation of transcription factors, which so far as we know, are necessary prerequisites to alterations of gene expression associated with the various functional outcomes mediated by the TCR in all T cells, regardless of specific subtype or maturity. We have previously found that exposure of T lymphocytes to subtoxic concentrations of Hg2+ interferes with the ability of the TCR to activate ERK MAP Kinase (Mattingly et al., 2001). Consequently, it is likely that elucidating the mechanism behind this observation will be of general importance in understanding the immunotoxicity of mercury under a variety of diverse circumstances.
The activation of ERK MAP Kinase is controlled by the small GTPase Ras. We have previously found that the inability of T cells to properly activate ERK MAP Kinase after exposure to subtoxic concentrations of HgCl2 is a downstream consequence of Ras failing to become properly activated (Mattingly et al., 2001). These findings are supported by other investigators who have shown in vitro that exposure of CD4+ T (mouse) cells to 0.4 μM HgCl2 inhibits proliferation, as well as significantly decreases IL-2 and IL-3 cytokine production, (Shen et al., 2001). These cellular activities are initiated by the TCR and are associated with Ras activation through signaling pathways mediated by membrane-associated and cytosolic protein tyrosine kinases (PTKs). After antigen engagement the TCR complex, without tyrosine kinase activity of its own, serves as a docking port for Src family PTKs and the zeta chain-associated protein kinase of 70 kDa (ZAP-70), a Syk family PTK. This is a key event in the TCR-stimulated signaling cascade that eventually leads to Ras/ERK activation (Abraham and Weiss, 2004; Huang and Wange, 2004).
Signals generated by the activated TCR-ZAP-70 complex subsequently initiate the organization of a signaling complex referred to as the signalosome, which forms around a 38 kDa transmembrane docking protein called Linker for Activation of T cells (LAT). LAT like the protein subunits of the TCR is not a PTK itself, but when phosphorylated by ZAP-70 becomes the scaffold to which a number of other signaling proteins dock. PTK's and other proteins binding to LAT activate a complex web of signaling pathways radiating from the signalosome. The actions of these signaling cascades result in the generation of a number of intracellular signaling intermediates, including activated Ras (Brdicka et al., 1998; Finco et al., 1998; Zhang et al., 1999).
Toxicants are known to interfere with gene expression in targeted cells. However as we have seen, they need not directly act on transcription factors or DNA. They can potentially exert a powerful effect on gene expression and T-cell function by inhibiting the activation of transcription factors through interference with upstream signaling pathways. As already mentioned, we have previously found that exposure of T cells to low and non-toxic concentrations of inorganic mercury prior to TCR stimulation, suppressed activation of Ras and subsequent ERK MAP Kinase activation (Mattingly et al., 2001). Our attention was thus drawn upstream to the critical event of LAT activation, and the hypothesis that our findings of diminished Ras activity in the presence of Hg2+ might be explained by an upstream effect of Hg+2 on the ability of this critical linker molecule to form a signalosome.
MATERIALS AND METHODS
Reagents.
Solutions of mercuric chloride (HgCl2) were prepared in RPMI 1640 medium (HyClone, Logan, UT). Antibodies were obtained from Santa Cruz Technology, Santa Cruz, CA (-LAT, rabbit polyclonal IgG); Southern Biotechnology Associates, Birmingham, AL (-pTyr (PY20 clone)-HRP); goat -rabbit IgG-HRP); Pierce, Rockford, IL (goat -mouse IgG-HRP); BD-Transduction Labs/Pharmigen, San Diego, CA (mouse -ERK (pan ERK) and mouse -pERK (phospho-specific, T202/Y204)) and Cell Signaling Technology, Beverly, MA (-ZAP-70, rabbit -pZAP-70 (Tyr319), -pSYK (Tyr 352). -CD3 (OKT3) came from Ortho Pharmaceuticals (Raritan, NJ). SuperSignal West Pico chemiluminescent detection kit was purchased from Pierce (Rockford, IL). Reagents used for protein electrophoresis and transfer to nitrocellulose were prepared following published protocols, (Bollag, 1991; Firestone and Winguth, 1990). All reagents and chemicals used were obtained from Sigma-Aldrich, St. Louis, MO, or Fisher Scientific, Fair Lawn, NJ, and were of analytical grade.
Cell cultures.
The human Jurkat (clone E6) and HUT 78 T lymphocytes 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, Rockville, MD) and 10 g/ml gentamicin (Life Technologies) at 37°C in a humidified atmosphere of 5% CO2, passed every 3–4 days. Cell cultures used in assays reached average cell densities of 106 cells/ml and were at least 98% viable as determined by trypan blue dye exclusion after 4 days of incubation. The culture medium was removed by centrifugation and the cells suspended in an equal volume of fresh RPMI with supplements and incubated for an additional 24 h before experiments were performed.
Mercury exposure/TCR activation assays.
Cell culture aliquots (1 x 106 cells per aliquot) had their culture medium removed by centrifugation, were washed once with warmed RPMI and then suspended in 200 μl warmed, RPMI without or with 5 μM HgCl2. Aliquots were incubated with gentle agitation at 37°C for 10 min before -CD3 (2.5 μg OKT3 per aliquot) was added to activate the T cell receptors. Reactions were stopped by the addition (5 ml) of ice-cold phosphate-buffered saline (PBS), followed by immediate centrifugation to remove the cold liquid. Cell pellets were then quick-frozen in an iced (–25°C) ethanol bath and held frozen until processed for SDS-PAGE.
SDS-PAGE and Western blot analysis.
Thawed cell pellets were mixed (vortex) with 250 μM 2.5 x nonreducing electrophoresis sample buffer (NRSB: 6 ml 1 M Tris-HCl (pH 6.8), 50 ml 50% glycerol, 20 ml 10% SDS, 10 ml 1% bromophenol blue, 114 ml ddH20). This produced whole cell lysates with 6 x 104 cell equivalents per each 15 μl sample loaded onto a SDS-PAGE gel. Lysates were boiled at 100°C for 3 min and pulled through a 23 gauge needle 3–4 times to shear the DNA. 5 μl 2-mercaptoethanol was added to each lysate, all were vortexed briefly and boiled again for 2 min. Processed lysates were centrifuged (16,000 x g, 1–2 min) and samples taken from the top portion of each lysate preparation loaded onto 10% polyacrylamide gels. Proteins were resolved by SDS-PAGE on a mini-gel electrophoresis system (Hoefer, San Francisco CA). All electrophoretic separations were run at constant voltage (175 V) and at room temperature. Proteins were transferred to nitrocellulose membranes (Pall Life Sciences, Pensacola, FL) with an electroblotting system (BioRad Life Science Products, Hercules, CA) at constant voltage (100 V) for 60 min at 0–4°C. Transfer membranes were blocked 2–4 h at room temperature or overnight at 4°C with 1% bovine serum albumin (Sigma-Aldrich) Tris blocking buffer, then probed with specific antibodies as indicated. On occasion, membranes were re-probed with a different antibody. In these cases, immunoblots were washed in dH2O, then stripped for 10 min in 0.1M glycine with 0.1% SDS, pH 2.0 at room temperature. Stripped membranes were washed twice with dH2O, once in Tris buffer, and then blocked 2–3 h at room temperature before being probed with other antibodies.
LAT Immunoprecipitation assays.
In each experiment 2–4 x 106 cells were exposed to mercury, and then activated with 5–6 μg OKT3 for timed periods as previously described. The cell pellets were lysed on ice in 100 μl Tris buffer containing protease and phosphatase inhibitor cocktail (Sigma-Aldrich), and supernatants collected following 20 min centrifugation at 18,000 x g, 4°C. Following the addition of -LAT, supernatants were rotated 3 h at 4°C. Pansorbin (Calbiochem, San Diego, CA) was then added and rotation at 4°C continued overnight. Immunoprecipitates were then collected and processed for SDS PAGE and subsequent Western blotting (Firestone and Winguth, 1990).
Data acquisition and analysis.
Immunoblotted proteins were detected using appropriate primary or secondary antibodies coupled to horseradish perioxidase (HRP) and SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL). Kodak BioMax ML imaging films (Eastman Kodak Co., Rochester, NY) were used to collect the chemiluminescence data, which were then scanned and converted into digital image files. In selected experiments, Image J (available at http://rsb.info.nih.gov/ij/) and Microsoft Excel software were used to analyze the images.
RESULTS
Hg+2 Inhibits TCR Mediated Activation of MAP ERK Kinase as Well as Phosphorylation of a 38 kDa Protein
Figure 1 is a representative example of an experiment (n = 3), where Jurkat T cells were exposed to 5 μM Hg2+ for 10 min, prior to the TCR being cross-linked and stimulated by an -CD3 monoclonal antibody (OKT3), used as a surrogate T-cell antigen. At 1 or 2-min intervals after TCR stimulation, the cells were lysed, and lysate proteins resolved by SDS PAGE. Activated ERK was then assayed by Western blotting with an antibody specific to dually (threonine/tyrosine) phosphorylated ERK (pERK). Confirming our previous findings (Mattingly et al., 2001) it can be seen in control cells that within 1 min after TCR stimulation significant quantities of ERK are phosphorylated. In comparison at 1 min post TCR stimulation, ERK phosphorylation appears to be somewhat inhibited in cells which have been pre-incubated with Hg2+. However the effect is more apparent at 2 min post receptor stimulation. At this point TCR dependent ERK activation is further enhanced in control cells than at 1 min, and the inhibitory effect of mercury is more clearly pronounced in treated cells. After staining for pERK, as a control the membrane was stripped, and then re-probed for total ERK by Western blotting.
In T cells, the phosphorylation and activation of ERK MAP Kinase is dependent upon prior upstream activation of Ras. We have previously shown that the inhibition of ERK MAP Kinase by Hg2+ is due to the fact that in cells exposed to Hg2+, although Hg2+ seems to have little direct effect on Ras, Ras nevertheless fails to become properly activated by the TCR (Mattingly et al., 2001). The implication is that Hg2+ disrupts an element or elements of the TCR signal transduction pathway upstream of Ras. As is the case for ERK MAP Kinase activation, many of the earlier events in T-cell signal transduction are also mediated by phosphorylation of tyrosine residues. Therefore, in the second part of the experiment outlined in Figure 1, after probing for ERK the membrane was again stripped, and then re-probed for total tyrosine phosphorylated proteins with a phosphotyrosine specific antibody.
While multiple proteins become phosphorylated on tyrosine when the TCR is stimulated by -CD3, our attention was drawn to the most prominently phosphorylated band whose response to -CD3 was also clearly sensitive to mercury. In particular, we found that at 1 or 2 min post TCR stimulation, phosphorylation of a 38 kDa protein was greatly increased in control cells that had not been exposed to Hg2+. However in cells that had been pre-exposed to 5 μM Hg2+ for 10 min, phosphorylation of this protein still occurred in response to -CD3, but in comparison to controls, phosphorylation was decreased. In line with the results for the phosphorylation of ERK, the effect was marginal at 1 min, but fairly dramatic at 2 min post TCR stimulation.
Hg2+ Inhibits Phosphorylation of LAT after TCR Stimulation
LAT is a 38 kDa protein, whose phosphorylation on tyrosines has recently come to be recognized as a key early step in the TCR signal transduction pathway. Furthermore, since LAT activation is upstream of Ras activation and ERK phosphorylation, we felt it possible that the 38 kDa protein identified in figure 1 was LAT. Confirming this conjecture, Figure 2 is a representative example of a series of experiments (n = 3) where as in Figure 1, cells were or were not, exposed to 5 μM Hg2+ for 10 min before the TCR was stimulated by -CD3. At 30 s post stimulation, mercury treated cells and controls were lysed, and LAT isolated by immunoprecipitation as described in Material and Methods. Immunoprecipitated LAT was then resolved by SDS PAGE, and probed for phosphotyrosine by Western blotting. As a control the membrane was then stripped and re-probed for total LAT. Figure 2 demonstrates that within 30 s LAT responds to -CD3 stimulation of the TCR by increased tyrosine phosphorylation, and that this increase in LAT phosphotyrosine level is attenuated by Hg2+.
In order to more thoroughly describe the effect of Hg2+ on LAT activation, we returned to a direct analysis of whole cell lysates. Figure 3A is a representative example of a series of experiments (n = 10), where we looked in greater detail than in Figure 1 at the effect that Hg2+ has on TCR mediated phosphorylation of the 38 kDa (LAT) polypeptide. In these experiments we probed total tyrosine phosphorylation, beginning at 30 s, and out to 20 min, post TCR stimulation of the cells. Again as in Figures 1 and 2 cells were or were not, exposed to 5 μM Hg2+ for 10 min before the TCR was stimulated by -CD3, and at timed intervals after the addition of -CD3 cells were lysed. Lysate proteins were then resolved by SDS PAGE and Western blot analysis for phosphotyrosine performed. For control purposes, membranes were then stripped and blotted a second time with -LAT antibody.
The top row in Figure 3A shows the 38 kDa (LAT) tyrosine phosphorylation pattern as a function of time after TCR stimulation in cells either exposed to, or not exposed to mercury. The figure confirms that after the TCR is stimulated, total LAT phosphotyrosine very quickly increases, so that in as little as 30 s a substantial signal is observed whether or not the cells were exposed to Hg2+. In the absence of Hg2+, LAT phosphorylation seems to plateau out to one minute, and then decline. By 20 min post TCR stimulation, LAT phosphorylation is back close to control levels. On the other hand in cells which have been pre-treated with Hg2+, while we still find substantial LAT phosphorylation at 30 s post TCR stimulation, it is below the level of control cells which have not been exposed to Hg2+. This is also the case at one minute. Thus at these early time points the effect of Hg2+ is clearly to attenuate the TCR induced signal.
Unfortunately the situation at later time points is not quite so clear. While Figure 3A shows that lysates from Hg2+ exposed cells show enhanced LAT phosphorylation over controls at the later 5 and 10 min marks, upon further investigation we found that although the behavior at the 0 to 1 min time points was entirely consistent between all experiments (n = 10), in only about 1/2 of the experiments were we able to demonstrate enhanced phosphorylation of LAT at 5 and 10 min in Hg2+ treated cells. Thus when the results of the entire ensemble (n = 10) of independent experiments outlined in Figure 3A, were quantified and plotted in Figure 3B, differences at 1 and 2 min between Hg2+ treated cells and controls is seen to be reasonably significant, while overlapping error bars imply that those at 5 and 10 min are not.
Figure 3B was generated by first scanning and digitizing the film record from each of the 10 experiments. Then taking account of the fact that Figures 1 and 3A showed a single tyrosine phosphorylated 38 kDa band (identified as phospho-LAT) as both responsive to -CD3 and sensitive to Hg+2, in each lane the density of the 38 kDa band was integrated over the band area to arrive at a metric which we used to assess the degree of LAT phosphorylation. In order to normalize the results between different experiments, in each experiment the band with the absolute least density (minimum phosphorylation), was identified. The absolute amount of phosphorylation was somewhat variable. However this band was always at the zero time point, and depending on the particular experiment could be either from the Hg2+ treated cells, or the controls. In either event, the integrated density value for the band was taken as the background, or basal phosphorylation level. The band was assigned a relative value of zero, while the actual numerical density value was subtracted from the integrated density of the remaining bands to adjust for background signal. Next, the band with the greatest adjusted density (maximum phosphorylation) was assigned a value of 100%. The percentage of this maximum value was then determined for phospho-LAT in all other lanes of the same experiment. Finally, the percentage of maximum response for all time points in all ten experiments were then averaged and plotted. Viewed in this way, we find that mercury suppresses peak TCR dependent LAT phosphorylation by about 30% from control values.
Hg+2 Inhibits the Activation of ZAP-70
As phosphorylation of LAT is directly dependent upon ZAP-70, inhibition of LAT activity by Hg2+ need not be reflective of a direct effect of Hg+2 on LAT, but rather could be the result of an upstream effect of Hg2+ on ZAP-70 PTK activity. Following TCR engagement, ZAP-70 is known to be phosphorylated on several tyrosine residues. Therefore we decided to look directly for any effect of Hg2+ on ZAP-70 phosphorylation. As done previously, Jurkat T cells were or were not, pre-incubated with 5 μM Hg2+ for 10 min, and then incubated with OKT3 in order to stimulate the TCR. At timed intervals the cells were chilled, pelleted, and lysed. Proteins were resolved by SDS PAGE, and PTK activity determined by Western blotting. In 5 independent experiments, although we were able to show that the TCR signaled increased tyrosine phosphorylation at the 70 kDa level, we were unable to find consistent and convincing differences between Hg2+ treated and control cells (ns). However, while multiple tyrosine residues of ZAP-70 become phosphorylated upon TCR signaling (Watts et al., 1994), only phosphorylation of selected residues, including tyrosine-319 is associated with the positive regulation of ZAP-70 function, so that total phosphorylation is not necessarily a good measure of ZAP-70 activation (Di Bartolo et al., 1999; Williams et al., 1999).
Therefore membranes were stripped and explicitly probed a second time for activated ZAP-70, utilizing an antibody that specifically recognizes ZAP-70 which has been phosphorylated at tyrosine 319 (pZAP(Y319)). A representative example is shown in the top row of Figure 4A. We find that while Hg2+ alone may slightly enhance the basal level of ZAP-70 activation, peak TCR induced phosphorylation of ZAP-70 on tyrosine-319 is attenuated. To control for loading errors the membranes were then stripped again and probed for total ZAP-70, (bottom row in Figure 4A).
In order to quantify these findings, the film record of each independent experiment in the ensemble (n = 5) of independent experiments was scanned and digitized. Similar to the procedure outlined for Figure 3 in our analysis of LAT phosphorylation, on each Western blot the band representing pZAP(Y319) was integrated and then normalized. Next the average value of pZAP(Y319) was determined for each time point after TCR stimulation in cells exposed or not exposed to mercury. The results are shown in Figure 4B, and reinforce Figure 4A by demonstrating that while Hg+2 perhaps marginally augments basal levels of ZAP-70 activation, following TCR stimulation it clearly depresses peak ZAP-70 activation by about 40%.
Hg2+ Inhibits Activation of LAT and ZAP-70 in HUT 78 Cells
The experiments reported in Figures 1–4 were all performed in Jurkat T cells. To control for the possibility that the effects we have seen are cell line specific, we looked at the ability of Hg2+ to interfere with TCR activation of LAT and ZAP-70 in HUT 78, a different human T cell line. Accordingly in a series of experiments (n = 4), HUT 78 cells were treated or not treated with Hg2+ and then activated with OKT3 as was described for the Jurkat cells. Then as described for Jurkat, cells were lysed and lysates probed by Western blotting for phosphotyrosine, followed by probes for LAT, pZAP, and total ZAP. Figure 5 is a representative example of one such series of experiments. The region of the initial Western blot for phosphotyrosine between 35 and 75 kDa is shown in the central part of Figure 5, where phosphoproteins in the vicinity of 70 and 38 kDa, suggestive of ZAP-70 and LAT respectively, are clearly visible (indicated by the block arrows). The membrane was then stripped, and blotted with an antibody specific to LAT. This resulted in a band being identified, which was superimposable over the 38 kDa phosphoprotein detected in the first blot. The LAT-specific band is shown in the lower portion of the figure. Next, the membrane was stripped, and blotted a third time for phospho (p)-ZAP(Y319), then stripped and blotted a final time for total endogenous ZAP-70. The pZAP(Y319), and ZAP-70 blots were each super imposable over the 70 kDa band in the initial -phosphotyrosine blot. The bands from these immunoblots are shown at the top of Figure 5.
In total, Figure 5 demonstrates that TCR dependent tyrosine phosphorylation of both LAT and ZAP-70 is inhibited by Hg2+ in HUT 78 cells in a fashion similar to the inhibition seen in Hg2+-exposed Jurkat T cells. However there is one difference. In HUT 78 it appears that the basal level of ZAP-70 activation is slightly higher than in Jurkat. And it appears to be suppressed by Hg2+, while in Jurkat the opposite seems true.
DISCUSSION
In this study we confirm our previous findings that in Jurkat T cells, exposure to a low and non-toxic concentration of Hg2+ significantly decreases the amount of activated ERK MAP Kinase in the first minutes following stimulation of the T-cell antigen receptor. We then expand upon this result by showing that the amount of tyrosine phosphorylated LAT (phospho-LAT), a key early regulatory element in the TCR signal transduction pathway, that normally forms after the TCR is stimulated is similarly reduced in cells which have been exposed to 5 μM Hg2+. We have previously shown that in Jurkat cells, 5 μM Hg2+ is not toxic (Whitekus et al., 1999).
Recent findings show that phospho-LAT couples the activated TCR to downstream signaling, in that phospho-LAT is essential for the formation of a functional signalosome complex needed to create appropriate responses following TCR engagement with an activating ligand (Bonello et al., 2004; Horejsi et al., 2004; Zhang et al., 1999). Of particular importance here is that one of the signal transduction elements downstream of the signalosome is the important regulatory protein Ras. Ras, a 21 kDa GTPase anchored to the plasma membrane, lies at the head of an amplifying signal transduction cascade that among other things controls the activity of ERK MAP kinases (Moodie et al., 1993). We have previously found that Hg2+ weakly activates ERK MAP Kinase, but somewhat paradoxically inhibits the activation of ERK MAP Kinase after the TCR is stimulated. However because Hg2+ inhibits TCR-dependent activation of Ras, but does not affect Ras activation by phorbol 12-myristate 13-acetate (PMA), we suggested that Hg2+ was likely interfering with an element of the T-cell signal transduction pathway upstream of Ras (Mattingly et al., 2001).
In T lymphocytes, Ras can be directly activated by either of two upstream guanine nucleotide exchange factors, Son of sevenless (Sos) or Ras guanine nucleotide releasing protein (RasGRP). Although SOS and RasGRP are themselves each activated via distinct pathways, interestingly, phospho-LAT figures prominently in the activation pathway of each. It was initially found that following its phosphorylation, LAT, which is a transmembrane protein, recruits the adapter protein Grb2. Grb2 binds SOS through SH3 domains, in addition to binding specific phosphorylated tyrosine residues on the cytoplasmic domain of LAT. After translocating to the plasma membrane and binding to LAT thru Grb2, SOS directly activates Ras, and by way of Ras, ERK MAP kinase (Zhang et al., 1999).
Recently a second signaling pathway connecting phospho-LAT to Ras activation through RasGRP has been described. RasGRP is activated by diacylglycerol (DAG), which is a product of activated PLC- acting on membrane PIP2 (phosphatidylinositol 4,5 bisphosphate). However activation of PLC- only occurs when it and the Tec family kinase Itk are each complexed with LAT. This requires the adapter proteins Gads and SLP-76, and additionally that LAT be phosphorylated (Boerth et al., 2000; Horejsi et al., 2004). Thus LAT is an important node in the T-cell signaling pathway, and its phosphorylation is a critical event in the activation of Ras, and subsequently that of ERK MAP Kinase. Clearly, inhibition of LAT phosphorylation by Hg2+ is sufficient to explain our previous finding of inhibition of TCR mediated Ras and ERK MAP Kinase activation by Hg2+.
It is worth noting that in these experiments Hg2+ does not completely inhibit LAT phosphorylation. Rather we have found that LAT phosphorylation is depressed by about 30% after cells are exposed to 5 μM Hg2+. Likewise, we have previously determined that 5–10 μM Hg2+ diminishes, but does not completely inhibit Ras activation after TCR stimulation (Mattingly et al., 2001). While it is possible that exposure to concentrations of Hg2+ above 10 μM might give rise to greater inhibition, we have not explored higher mercury exposures, because it is likely that such exposures would also be toxic. Nevertheless, a relatively low and non-toxic level of Hg2+ is sufficient to significantly depress activation of LAT by about 30%. A 30% reduction in activation of LAT is likely significant because the TCR-ERK signaling pathway is characterized by a cascade of protein-protein interactions and activations. Pathways of this structure typically exhibit signal amplification, in the sense that small perturbations in upstream signaling elements lead to much larger changes in down stream elements (Li and Qian, 2003). Indeed we have previously found that on average, exposure of T cells to Hg2+ concentrations as low as 1 μM gave rise to an approximate 60% reduction in ERK MAP kinase activation after the TCR was stimulated (Mattingly et al., 2001).
We have also previously shown that the effect of low concentrations of Hg2+ on cell signaling could most likely be accounted for by its effect on cell surface thiols, as masking of membrane protein sulfhydryls with a membrane impermeable malemide inhibited the ability of Hg2+ to influence signaling (Rosenspire et al., 1998). More recently we have directly demonstrated, by cold-vapor atomic absorption, that virtually no Hg2+ penetrates the plasma membrane when Jurkat cells are incubated with micromolar concentrations of Hg2+ for periods as long as 4 h (McCabe, Jr., et al., 2005). While LAT is a transmembrane protein, it has a small extracellular domain of only 4 amino acids, none of which is cysteine (Zhang et al., 1998). As a result, it is unlikely that Hg2+ acts directly on LAT. Rather, Hg2+ most probably interferes with an element or elements of the T-cell signal transduction system upstream of LAT.
LAT was originally described as the ZAP-70 substrate responsible for linking the TCR to cell activation (Zhang et al., 1998). In fact, LAT can only organize a functional signalosome following its phosphorylation by ZAP-70. Therefore, as ZAP-70 is the PTK immediately upstream of LAT in the TCR signal transduction cascade, we looked for an effect that cell exposure to Hg2+ might have on its activity. ZAP-70 activation is associated with tyrosine phosphorylation at multiple sites (Watts et al., 1994). However while phosphorylation of some sites seems to be unnecessary for activation, phosphorylation of others including tyrosine 319 is strictly required for activation and for TCR-dependent signaling to proceed (Di Bartolo et al., 1999; Williams et al., 1999). We find that normally after the TCR is stimulated, increases in ZAP-70 phosphorylation at tyrosine 319 occur in concert with increases in LAT phosphorylation. But in cells treated with Hg2+, TCR induced phosphorylation of ZAP-70 is attenuated in concert with attenuation of LAT phosphorylation.
Our results thus suggest that TCR-induced generation of phospho-LAT is diminished after lymphocytes are exposed to low dose (5 μM) Hg2+, because upstream of LAT activation of ZAP-70 is inhibited. However while Hg2+ at low levels clearly interferes with TCR mediated signal transduction, the effect on basal activation levels of specific elements of the T-cell signal transduction pathway appear to be marginal. We have found that Hg2+ slightly increases background phosphorylation of ZAP-70-Tyr 319 in Jurkat cells. But on the other hand basal phosphorylation of these residues in HUT 78 seem to be reduced by Hg2+. However this finding might be reflective of the fact that basal phosphorylation levels of ZAP-70 phosphorylation in HUT 78 were found to be somewhat higher than those of Jurkat in the first place. It may be that in HUT 78 we were looking at ZAP-70 which was "endogenously activated" to start. Nevertheless in either event the effects are small, and as opposed to the much larger effect on TCR- mediated signal transduction, it is not clear how T-cell functionality is effected.
Returning to the question of TCR signaling, just as it is unlikely that Hg2+ interacts directly with LAT, it is unlikely that Hg2+ interacts directly with the cytoplasmic ZAP-70 protein. It is interesting to note that ZAP-70 is itself activated by the transmembrane phosphokinase p56Lck, which has been shown to have altered kinase activity following Hg2+ exposure (Lander et al., 1992). As the initial site of Hg2+ assault is most probably still upstream of ZAP-70 in the TCR-initiated signal transduction cascade, it may be that p56Lck is the proximal target of inorganic mercury in this system.
Because of its broad sulfhydryl reactivity, specific molecular targets of mercury are likely to be dependent upon both concentration and chemical form. Thus physiologically significant targets of inorganic mercury may be expected to differ from those of organic mercury species, and in either case be concentration dependent. In the experiments reported here, we have utilized inorganic mercury at 5 μM, a concentration which is low in the sense that it is non-toxic under in vitro culture conditions. Although it is often difficult to reconcile in vitro toxicant concentrations with in vivo exposures, it is likely that the mercury dose and exposure conditions used in these experiments (5 μM for 10 min), are relevant to modern occupational, and perhaps environmental exposures.
In order to rigorously demonstrate this, the parameter needed is cellular toxicant burden, determined for both in vitro and in vivo exposures. In preliminary studies we have determined by cold vapor atomic absorption spectroscopy that the conditions used in these experiments (10 min incubation of lymphocytes in tissue culture media with 5 μM Hg2+) results in a mercury burden of approximately 5 ng/106 cells. After an initial rapid uptake, the binding of additional Hg2+ to lymphocytes goes up quite slowly, so that this burden does not appreciably change over the 2–20 min time course of the experiments we have described. Significantly, it turns out that 5 ng/106 cells is also about the same mercury burden obtained if cells are incubated with 0.5 μM Hg2+ for 24 h (ns).
Unfortunately specific lymphocyte mercury burdens due to environmental and occupational exposures have not in general been reported. But based on reported whole blood mercury concentrations which have been reported, we can make reasonable estimates. For example, in Brazilian gold miners exposed to mercury vapor during gold refining operations, blood mercury is likely to be predominantly inorganic, and total mercury concentrations from 5 to as high as 100 μg/l have been reported (Silva et al., 2004). In the same study it was also found that there was an increased prevalence of antinucleolar antibodies and a positive interaction between Hg and malaria, suggesting that Hg2+ at these blood levels could function as a co-factor in autoimmune disease.
Admittedly, Hg2+ binding in vivo is considerably more complicated than binding in vitro, but considering that 100 μg/l mercury is equivalent to approximately 0.5 μM, it is not unreasonable as a first order estimate to assume that in these occupationally exposed individuals lymphocyte mercury burdens of at least 5 ng/106 cells may be achieved in some instances. If this is the case than our in vitro results are seen to be directly relevant to real world occupational exposures to inorganic mercury. Interference with TCR signal transduction might then be associated with autoimmunity seen in the Brazilian gold miners.
Finally, blood mercury levels in individuals consuming >4 fish meals a week, but otherwise characterized by no known mercury exposure risk factors can also approach 100 μg/l (Brune et al., 1991). Mercury uptake from fish is primarily organic, but as organic mercury is over time converted to inorganic mercury, our results may also be relevant to more common environmental exposures to inorganic mercury.
ACKNOWLEDGMENTS
This work was in part supported by NIH grants ES11000, ES12403, and ES01247.
REFERENCES
Abraham, R. T., and Weiss, A. (2004). Jurkat T cells and development of the T-cell receptor signalling paradigm. Nat. Rev. Immunol. 4, 301–308.
Bagenstose, L. M., Salgame, P., and Monestier, M. (1998). IL-12 down-regulates autoantibody production in mercury-induced autoimmunity. J. Immunol. 160, 1612–1617.
Bernier, J., Brousseau, P., Krzystyniak, K., Tryphonas, H., and Fournier, M. (1995). Immunotoxicity of heavy metals in relation to Great Lakes. Environ. Health Perspect. 103(Suppl. 9), 23–34.
Boerth, N. J., Sadler, J. J., Bauer, D. E., Clements, J. L., Gheith, S. M., and Koretzky, G. A. (2000). Recruitment of SLP-76 to the membrane and glycolipid-enriched membrane microdomains replaces the requirement for linker for activation of T cells in T cell receptor signaling. J. Exp. Med. 192, 1047–1058.
Bollag, A. G. (1991). Protein Methods. Wiley-Liss, New York.
Bonello, G., Blanchard, N., Montoya, M. C., Aguado, E., Langlet, C., He, H. T., Nunez-Cruz, S., Malissen, M., Sanchez-Madrid, F., Olive, D., et al. (2004). Dynamic recruitment of the adaptor protein LAT: LAT exists in two distinct intracellular pools and controls its own recruitment. J. Cell Sci. 117, 1009–1016.
Brdicka, T., Cerny, J., and Horejsi, V. (1998). T cell receptor signalling results in rapid tyrosine phosphorylation of the linker protein LAT present in detergent-resistant membrane microdomains. Biochem. Biophys. Res. Commun. 248, 356–360.
Brune, D., Nordberg, G. F., Vesterberg, O., Gerhardsson, L., and Wester, P. O. (1991). A review of normal concentrations of mercury in human blood. Sci. Total Environ. 100, 235–282.
Di Bartolo, V., Mege, D., Germain, V., Pelosi, M., Michel, F., Magistrelli, G., Isacchi, A., and Acuto, O. (1999). Tyrosine 319, a newly identified phosphorylation site of ZAP-70, plays a critical role in T cell antigen receptor signaling. J. Biol. Chem. 274, 6285–6294.
Finco, T. S., Kadlecek, T., Zhang, W., Samelson, L. E., and Weiss, A. (1998). LAT is required for TCR-mediated activation of PLCgamma1 and the Ras pathway. Immunity 9, 617–626.
Firestone, G. L., and Winguth, S. D. (1990). Immunoprecipitation of proteins. Methods Enzymol. 182, 688–700.
Hansson, M., and Abedi-Valugerdi, M. (2004). Mercuric chloride induces a strong immune activation, but does not accelerate the development of dermal fibrosis in tight skin 1 mice. Scand. J. Immunol. 59, 469–477.
Hong, R. (1999). Evaluation of toxic assault on the immune system. Mutat. Res. 428, 109–114.
Horejsi, V., Zhang, W., and Schraven, B. (2004). Transmembrane adaptor proteins: organizers of immunoreceptor signalling. Nat. Rev. Immunol. 4, 603–616.
Hu, H., Moller, G., and Abedi-Valugerdi, M. (1999). Mechanism of mercury-induced autoimmunity: both T helper 1- and T helper 2-type responses are involved. Immunology 96, 348–357.
Huang, Y., and Wange, R. L. (2004). T cell receptor signaling: Beyond complex complexes. J. Biol. Chem. 279, 28827–28830.
Kazantzis, G. (2002). Mercury exposure and early effects: An overview. Med. Lav. 93, 139–147.
Lander, H. M., Levine, D. M., and Novogrodsky, A. (1992). Stress stimuli-induced lymphocyte activation. Cell. Immunol. 145, 146–155.
Li, G., and Qian, H. (2003). Sensitivity and specificity amplification in signal transduction. Cell Biochem. Biophys. 39, 45–59.
Mattingly, R. R., Felczak, A., Chen, C. C., McCabe, M. J. J., and Rosenspire, A. J. (2001). Low concentrations of inorganic mercury inhibit Ras activation during T cell receptor-mediated signal transduction. Toxicol. Appl. Pharmacol. 176, 162–168.
McCabe, M. J., Jr., Eckles, K. G., Langdon, M., Clarkson, T. W., Whitekus, M. J., and Rosenspire, A. J. (2005). Attenuation of CD95-induced apoptosis by inorganic mercury: Caspase-3 is not a direct target of low levels of Hg2+. Toxicol. Lett. 155, 161–170.
Moodie, S. A., Willumsen, B. M., Weber, M. J., and Wolfman, A. (1993). Complexes of Ras.GTP with Raf-1 and mitogen-activated protein kinase kinase [see comments]. Science 260, 1658–1661.
Moszczynski, P. (1997). Mercury compounds and the immune system: A review. Int. J. Occup. Med. Environ. Health 10, 247–258.
Rosenspire, A. J., Bodepudi, S., Mathews, M., and McCabe, J. M. (1998). Low levels of ionic mercury modulate protein tyrosine phosphorylation in lymphocytes. Int. J. Immunopharmacol. 20, 697–707.
Shen, X., Lee, K., and Konig, R. (2001). Effects of heavy metal ions on resting and antigen-activated CD4(+) T cells. Toxicology 169, 67–80.
Silva, I. A., Nyland, J. F., Gorman, A., Perisse, A., Ventura, A. M., Santos, E. C., Souza, J. M., Burek, C. L., Rose, N. R., and Silbergeld, E. K. (2004). Mercury exposure, malaria, and serum antinuclear antibodies in amazon populations in Brazil: a cross-sectional study. Environ. Health 3, 11.
Sweet, L. I., and Zelikoff, J. T. (2001). Toxicology and immunotoxicology of mercury: A comparative review in fish and humans. J. Toxicol. Environ. Health B Crit. Rev. 4, 161–205.
Watts, J. D., Affolter, M., Krebs, D. L., Wange, R. L., Samelson, L. E., and Aebersold, R. (1994). Identification by electrospray ionization mass spectrometry of the sites of tyrosine phosphorylation induced in activated Jurkat T cells on the protein tyrosine kinase ZAP-70. J. Biol. Chem. 269, 29520–29529.
Whitekus, M. J., Santini, R. P., Rosenspire, A. J., and McCabe, M. J. J. (1999). Protection against CD95-mediated apoptosis by inorganic mercury in jurkat T cells. J. Immunol. 162, 7162–7170.
Williams, B. L., Irvin, B. J., Sutor, S. L., Chini, C. S., Yacyshyn, E., Wardenurg, J. B., Dalton, M., Chan, A. C., and Abraham, R. T. (1999). Phosphorylation of Tyr 319 in ZAP-70 is required for T-cell antigen receptor-dependent phospholipase C-1 and Ras activation. EMBO J. 18, 1832–1844.
Zhang, W., Irvin, B. J., Trible, R. P., Abraham, R. T., and Samelson, L. E. (1999). Functional analysis of LAT in TCR-mediated signaling pathways using a LAT-deficient Jurkat cell line. Int. Immunol. 11, 943–950.
Zhang, W., Sloan-Lancaster, J., Kitchen, J., Trible, R. P., and Samelson, L. E. (1998). LAT: The ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation. Cell 92, 83–92.
Zheng, Y., and Monestier, M. (2003). Inhibitory signal override increases susceptibility to mercury-induced autoimmunity. J. Immunol. 171, 1596–1601.(Stamatina E. Ziemba, Raymond R. Mattingl)
Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
ABSTRACT
Little is known as to the molecular mechanisms involved with mercury intoxication at very low levels. Although the mechanism is not known, animal studies have nevertheless shown that low levels of mercury may target the immune system. Inorganic mercury (Hg2+) at very low (but non-toxic) levels can disrupt immune system homeostasis, in that genetically susceptible rodents develop idiosyncratic autoimmune disease, which is associated with defective T-cell function. T lymphocyte function is intimately coupled to the T-cell receptor. We have previously reported that on a molecular level, low concentrations of Hg2+ disrupt signaling from the T-cell receptor by interfering with activation of Ras and ERK MAP kinase. In this report we expand upon those results by showing that in T lymphocytes exposed to low concentration of Hg2+, Ras fails to become properly activated because upstream of Ras in the T cell signal transduction pathway, the important scaffolding element Linker for Activation of T Cells (LAT) fails to become properly phosphorylated. Hypo-phosphorylation of LAT occurs, because upstream of LAT, the LAT reactive tyrosine kinase ZAP-70 is also not properly activated in Hg2+ treated cells.
Key Words: LAT; T-cell receptor; mercury; signalosome.
INTRODUCTION
Over the past dozen years it has been shown that acute or chronic exposure to mercury, even at subtoxic doses alters the immune system by leading to increased susceptibility to either autoimmune or immunosuppressive syndromes (Bernier et al., 1995; Hansson and Abedi-Valugerdi, 2004; Hong, 1999; Kazantzis, 2002). Animal studies have shown that in rodent strains of susceptible MHC haplotypes, exposure to low and subtoxic doses of inorganic mercury (Hg2+) induces an autoimmune dysfunction characterized by the production of anti-nuclear antibodies, lymphoproliferation, and hyperglobulinemia (Bagenstose et al., 1998; Hu et al., 1999; Zheng and Monestier, 2003). On the other hand, in vitro studies in a number of systems have shown that mercury compounds often inhibit lymphocyte functions, including proliferation, expression of cell activation markers, and cytokine production (Moszczynski, 1997; Shen et al., 2001; Sweet and Zelikoff, 2001).
In T lymphocytes engagement of the T Cell Receptor (TCR) by antigen will activate the cell. Depending upon the subtype and maturity of the T cell, as well as the mix of available co-factors, a T cell may respond to antigen by proliferation, differentiation, secretion of cytokines, or perhaps undergo apoptosis. However one thing that all of these various functional outcomes between cells of different maturity and T-cell subtype have in common, is that fairly soon after TCR engagement of antigen, ERK MAP Kinase is phosphorylated and activated. The activation of ERK MAP Kinase holds a central role in lymphocyte biology because of its role in the activation of transcription factors, which so far as we know, are necessary prerequisites to alterations of gene expression associated with the various functional outcomes mediated by the TCR in all T cells, regardless of specific subtype or maturity. We have previously found that exposure of T lymphocytes to subtoxic concentrations of Hg2+ interferes with the ability of the TCR to activate ERK MAP Kinase (Mattingly et al., 2001). Consequently, it is likely that elucidating the mechanism behind this observation will be of general importance in understanding the immunotoxicity of mercury under a variety of diverse circumstances.
The activation of ERK MAP Kinase is controlled by the small GTPase Ras. We have previously found that the inability of T cells to properly activate ERK MAP Kinase after exposure to subtoxic concentrations of HgCl2 is a downstream consequence of Ras failing to become properly activated (Mattingly et al., 2001). These findings are supported by other investigators who have shown in vitro that exposure of CD4+ T (mouse) cells to 0.4 μM HgCl2 inhibits proliferation, as well as significantly decreases IL-2 and IL-3 cytokine production, (Shen et al., 2001). These cellular activities are initiated by the TCR and are associated with Ras activation through signaling pathways mediated by membrane-associated and cytosolic protein tyrosine kinases (PTKs). After antigen engagement the TCR complex, without tyrosine kinase activity of its own, serves as a docking port for Src family PTKs and the zeta chain-associated protein kinase of 70 kDa (ZAP-70), a Syk family PTK. This is a key event in the TCR-stimulated signaling cascade that eventually leads to Ras/ERK activation (Abraham and Weiss, 2004; Huang and Wange, 2004).
Signals generated by the activated TCR-ZAP-70 complex subsequently initiate the organization of a signaling complex referred to as the signalosome, which forms around a 38 kDa transmembrane docking protein called Linker for Activation of T cells (LAT). LAT like the protein subunits of the TCR is not a PTK itself, but when phosphorylated by ZAP-70 becomes the scaffold to which a number of other signaling proteins dock. PTK's and other proteins binding to LAT activate a complex web of signaling pathways radiating from the signalosome. The actions of these signaling cascades result in the generation of a number of intracellular signaling intermediates, including activated Ras (Brdicka et al., 1998; Finco et al., 1998; Zhang et al., 1999).
Toxicants are known to interfere with gene expression in targeted cells. However as we have seen, they need not directly act on transcription factors or DNA. They can potentially exert a powerful effect on gene expression and T-cell function by inhibiting the activation of transcription factors through interference with upstream signaling pathways. As already mentioned, we have previously found that exposure of T cells to low and non-toxic concentrations of inorganic mercury prior to TCR stimulation, suppressed activation of Ras and subsequent ERK MAP Kinase activation (Mattingly et al., 2001). Our attention was thus drawn upstream to the critical event of LAT activation, and the hypothesis that our findings of diminished Ras activity in the presence of Hg2+ might be explained by an upstream effect of Hg+2 on the ability of this critical linker molecule to form a signalosome.
MATERIALS AND METHODS
Reagents.
Solutions of mercuric chloride (HgCl2) were prepared in RPMI 1640 medium (HyClone, Logan, UT). Antibodies were obtained from Santa Cruz Technology, Santa Cruz, CA (-LAT, rabbit polyclonal IgG); Southern Biotechnology Associates, Birmingham, AL (-pTyr (PY20 clone)-HRP); goat -rabbit IgG-HRP); Pierce, Rockford, IL (goat -mouse IgG-HRP); BD-Transduction Labs/Pharmigen, San Diego, CA (mouse -ERK (pan ERK) and mouse -pERK (phospho-specific, T202/Y204)) and Cell Signaling Technology, Beverly, MA (-ZAP-70, rabbit -pZAP-70 (Tyr319), -pSYK (Tyr 352). -CD3 (OKT3) came from Ortho Pharmaceuticals (Raritan, NJ). SuperSignal West Pico chemiluminescent detection kit was purchased from Pierce (Rockford, IL). Reagents used for protein electrophoresis and transfer to nitrocellulose were prepared following published protocols, (Bollag, 1991; Firestone and Winguth, 1990). All reagents and chemicals used were obtained from Sigma-Aldrich, St. Louis, MO, or Fisher Scientific, Fair Lawn, NJ, and were of analytical grade.
Cell cultures.
The human Jurkat (clone E6) and HUT 78 T lymphocytes 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, Rockville, MD) and 10 g/ml gentamicin (Life Technologies) at 37°C in a humidified atmosphere of 5% CO2, passed every 3–4 days. Cell cultures used in assays reached average cell densities of 106 cells/ml and were at least 98% viable as determined by trypan blue dye exclusion after 4 days of incubation. The culture medium was removed by centrifugation and the cells suspended in an equal volume of fresh RPMI with supplements and incubated for an additional 24 h before experiments were performed.
Mercury exposure/TCR activation assays.
Cell culture aliquots (1 x 106 cells per aliquot) had their culture medium removed by centrifugation, were washed once with warmed RPMI and then suspended in 200 μl warmed, RPMI without or with 5 μM HgCl2. Aliquots were incubated with gentle agitation at 37°C for 10 min before -CD3 (2.5 μg OKT3 per aliquot) was added to activate the T cell receptors. Reactions were stopped by the addition (5 ml) of ice-cold phosphate-buffered saline (PBS), followed by immediate centrifugation to remove the cold liquid. Cell pellets were then quick-frozen in an iced (–25°C) ethanol bath and held frozen until processed for SDS-PAGE.
SDS-PAGE and Western blot analysis.
Thawed cell pellets were mixed (vortex) with 250 μM 2.5 x nonreducing electrophoresis sample buffer (NRSB: 6 ml 1 M Tris-HCl (pH 6.8), 50 ml 50% glycerol, 20 ml 10% SDS, 10 ml 1% bromophenol blue, 114 ml ddH20). This produced whole cell lysates with 6 x 104 cell equivalents per each 15 μl sample loaded onto a SDS-PAGE gel. Lysates were boiled at 100°C for 3 min and pulled through a 23 gauge needle 3–4 times to shear the DNA. 5 μl 2-mercaptoethanol was added to each lysate, all were vortexed briefly and boiled again for 2 min. Processed lysates were centrifuged (16,000 x g, 1–2 min) and samples taken from the top portion of each lysate preparation loaded onto 10% polyacrylamide gels. Proteins were resolved by SDS-PAGE on a mini-gel electrophoresis system (Hoefer, San Francisco CA). All electrophoretic separations were run at constant voltage (175 V) and at room temperature. Proteins were transferred to nitrocellulose membranes (Pall Life Sciences, Pensacola, FL) with an electroblotting system (BioRad Life Science Products, Hercules, CA) at constant voltage (100 V) for 60 min at 0–4°C. Transfer membranes were blocked 2–4 h at room temperature or overnight at 4°C with 1% bovine serum albumin (Sigma-Aldrich) Tris blocking buffer, then probed with specific antibodies as indicated. On occasion, membranes were re-probed with a different antibody. In these cases, immunoblots were washed in dH2O, then stripped for 10 min in 0.1M glycine with 0.1% SDS, pH 2.0 at room temperature. Stripped membranes were washed twice with dH2O, once in Tris buffer, and then blocked 2–3 h at room temperature before being probed with other antibodies.
LAT Immunoprecipitation assays.
In each experiment 2–4 x 106 cells were exposed to mercury, and then activated with 5–6 μg OKT3 for timed periods as previously described. The cell pellets were lysed on ice in 100 μl Tris buffer containing protease and phosphatase inhibitor cocktail (Sigma-Aldrich), and supernatants collected following 20 min centrifugation at 18,000 x g, 4°C. Following the addition of -LAT, supernatants were rotated 3 h at 4°C. Pansorbin (Calbiochem, San Diego, CA) was then added and rotation at 4°C continued overnight. Immunoprecipitates were then collected and processed for SDS PAGE and subsequent Western blotting (Firestone and Winguth, 1990).
Data acquisition and analysis.
Immunoblotted proteins were detected using appropriate primary or secondary antibodies coupled to horseradish perioxidase (HRP) and SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL). Kodak BioMax ML imaging films (Eastman Kodak Co., Rochester, NY) were used to collect the chemiluminescence data, which were then scanned and converted into digital image files. In selected experiments, Image J (available at http://rsb.info.nih.gov/ij/) and Microsoft Excel software were used to analyze the images.
RESULTS
Hg+2 Inhibits TCR Mediated Activation of MAP ERK Kinase as Well as Phosphorylation of a 38 kDa Protein
Figure 1 is a representative example of an experiment (n = 3), where Jurkat T cells were exposed to 5 μM Hg2+ for 10 min, prior to the TCR being cross-linked and stimulated by an -CD3 monoclonal antibody (OKT3), used as a surrogate T-cell antigen. At 1 or 2-min intervals after TCR stimulation, the cells were lysed, and lysate proteins resolved by SDS PAGE. Activated ERK was then assayed by Western blotting with an antibody specific to dually (threonine/tyrosine) phosphorylated ERK (pERK). Confirming our previous findings (Mattingly et al., 2001) it can be seen in control cells that within 1 min after TCR stimulation significant quantities of ERK are phosphorylated. In comparison at 1 min post TCR stimulation, ERK phosphorylation appears to be somewhat inhibited in cells which have been pre-incubated with Hg2+. However the effect is more apparent at 2 min post receptor stimulation. At this point TCR dependent ERK activation is further enhanced in control cells than at 1 min, and the inhibitory effect of mercury is more clearly pronounced in treated cells. After staining for pERK, as a control the membrane was stripped, and then re-probed for total ERK by Western blotting.
In T cells, the phosphorylation and activation of ERK MAP Kinase is dependent upon prior upstream activation of Ras. We have previously shown that the inhibition of ERK MAP Kinase by Hg2+ is due to the fact that in cells exposed to Hg2+, although Hg2+ seems to have little direct effect on Ras, Ras nevertheless fails to become properly activated by the TCR (Mattingly et al., 2001). The implication is that Hg2+ disrupts an element or elements of the TCR signal transduction pathway upstream of Ras. As is the case for ERK MAP Kinase activation, many of the earlier events in T-cell signal transduction are also mediated by phosphorylation of tyrosine residues. Therefore, in the second part of the experiment outlined in Figure 1, after probing for ERK the membrane was again stripped, and then re-probed for total tyrosine phosphorylated proteins with a phosphotyrosine specific antibody.
While multiple proteins become phosphorylated on tyrosine when the TCR is stimulated by -CD3, our attention was drawn to the most prominently phosphorylated band whose response to -CD3 was also clearly sensitive to mercury. In particular, we found that at 1 or 2 min post TCR stimulation, phosphorylation of a 38 kDa protein was greatly increased in control cells that had not been exposed to Hg2+. However in cells that had been pre-exposed to 5 μM Hg2+ for 10 min, phosphorylation of this protein still occurred in response to -CD3, but in comparison to controls, phosphorylation was decreased. In line with the results for the phosphorylation of ERK, the effect was marginal at 1 min, but fairly dramatic at 2 min post TCR stimulation.
Hg2+ Inhibits Phosphorylation of LAT after TCR Stimulation
LAT is a 38 kDa protein, whose phosphorylation on tyrosines has recently come to be recognized as a key early step in the TCR signal transduction pathway. Furthermore, since LAT activation is upstream of Ras activation and ERK phosphorylation, we felt it possible that the 38 kDa protein identified in figure 1 was LAT. Confirming this conjecture, Figure 2 is a representative example of a series of experiments (n = 3) where as in Figure 1, cells were or were not, exposed to 5 μM Hg2+ for 10 min before the TCR was stimulated by -CD3. At 30 s post stimulation, mercury treated cells and controls were lysed, and LAT isolated by immunoprecipitation as described in Material and Methods. Immunoprecipitated LAT was then resolved by SDS PAGE, and probed for phosphotyrosine by Western blotting. As a control the membrane was then stripped and re-probed for total LAT. Figure 2 demonstrates that within 30 s LAT responds to -CD3 stimulation of the TCR by increased tyrosine phosphorylation, and that this increase in LAT phosphotyrosine level is attenuated by Hg2+.
In order to more thoroughly describe the effect of Hg2+ on LAT activation, we returned to a direct analysis of whole cell lysates. Figure 3A is a representative example of a series of experiments (n = 10), where we looked in greater detail than in Figure 1 at the effect that Hg2+ has on TCR mediated phosphorylation of the 38 kDa (LAT) polypeptide. In these experiments we probed total tyrosine phosphorylation, beginning at 30 s, and out to 20 min, post TCR stimulation of the cells. Again as in Figures 1 and 2 cells were or were not, exposed to 5 μM Hg2+ for 10 min before the TCR was stimulated by -CD3, and at timed intervals after the addition of -CD3 cells were lysed. Lysate proteins were then resolved by SDS PAGE and Western blot analysis for phosphotyrosine performed. For control purposes, membranes were then stripped and blotted a second time with -LAT antibody.
The top row in Figure 3A shows the 38 kDa (LAT) tyrosine phosphorylation pattern as a function of time after TCR stimulation in cells either exposed to, or not exposed to mercury. The figure confirms that after the TCR is stimulated, total LAT phosphotyrosine very quickly increases, so that in as little as 30 s a substantial signal is observed whether or not the cells were exposed to Hg2+. In the absence of Hg2+, LAT phosphorylation seems to plateau out to one minute, and then decline. By 20 min post TCR stimulation, LAT phosphorylation is back close to control levels. On the other hand in cells which have been pre-treated with Hg2+, while we still find substantial LAT phosphorylation at 30 s post TCR stimulation, it is below the level of control cells which have not been exposed to Hg2+. This is also the case at one minute. Thus at these early time points the effect of Hg2+ is clearly to attenuate the TCR induced signal.
Unfortunately the situation at later time points is not quite so clear. While Figure 3A shows that lysates from Hg2+ exposed cells show enhanced LAT phosphorylation over controls at the later 5 and 10 min marks, upon further investigation we found that although the behavior at the 0 to 1 min time points was entirely consistent between all experiments (n = 10), in only about 1/2 of the experiments were we able to demonstrate enhanced phosphorylation of LAT at 5 and 10 min in Hg2+ treated cells. Thus when the results of the entire ensemble (n = 10) of independent experiments outlined in Figure 3A, were quantified and plotted in Figure 3B, differences at 1 and 2 min between Hg2+ treated cells and controls is seen to be reasonably significant, while overlapping error bars imply that those at 5 and 10 min are not.
Figure 3B was generated by first scanning and digitizing the film record from each of the 10 experiments. Then taking account of the fact that Figures 1 and 3A showed a single tyrosine phosphorylated 38 kDa band (identified as phospho-LAT) as both responsive to -CD3 and sensitive to Hg+2, in each lane the density of the 38 kDa band was integrated over the band area to arrive at a metric which we used to assess the degree of LAT phosphorylation. In order to normalize the results between different experiments, in each experiment the band with the absolute least density (minimum phosphorylation), was identified. The absolute amount of phosphorylation was somewhat variable. However this band was always at the zero time point, and depending on the particular experiment could be either from the Hg2+ treated cells, or the controls. In either event, the integrated density value for the band was taken as the background, or basal phosphorylation level. The band was assigned a relative value of zero, while the actual numerical density value was subtracted from the integrated density of the remaining bands to adjust for background signal. Next, the band with the greatest adjusted density (maximum phosphorylation) was assigned a value of 100%. The percentage of this maximum value was then determined for phospho-LAT in all other lanes of the same experiment. Finally, the percentage of maximum response for all time points in all ten experiments were then averaged and plotted. Viewed in this way, we find that mercury suppresses peak TCR dependent LAT phosphorylation by about 30% from control values.
Hg+2 Inhibits the Activation of ZAP-70
As phosphorylation of LAT is directly dependent upon ZAP-70, inhibition of LAT activity by Hg2+ need not be reflective of a direct effect of Hg+2 on LAT, but rather could be the result of an upstream effect of Hg2+ on ZAP-70 PTK activity. Following TCR engagement, ZAP-70 is known to be phosphorylated on several tyrosine residues. Therefore we decided to look directly for any effect of Hg2+ on ZAP-70 phosphorylation. As done previously, Jurkat T cells were or were not, pre-incubated with 5 μM Hg2+ for 10 min, and then incubated with OKT3 in order to stimulate the TCR. At timed intervals the cells were chilled, pelleted, and lysed. Proteins were resolved by SDS PAGE, and PTK activity determined by Western blotting. In 5 independent experiments, although we were able to show that the TCR signaled increased tyrosine phosphorylation at the 70 kDa level, we were unable to find consistent and convincing differences between Hg2+ treated and control cells (ns). However, while multiple tyrosine residues of ZAP-70 become phosphorylated upon TCR signaling (Watts et al., 1994), only phosphorylation of selected residues, including tyrosine-319 is associated with the positive regulation of ZAP-70 function, so that total phosphorylation is not necessarily a good measure of ZAP-70 activation (Di Bartolo et al., 1999; Williams et al., 1999).
Therefore membranes were stripped and explicitly probed a second time for activated ZAP-70, utilizing an antibody that specifically recognizes ZAP-70 which has been phosphorylated at tyrosine 319 (pZAP(Y319)). A representative example is shown in the top row of Figure 4A. We find that while Hg2+ alone may slightly enhance the basal level of ZAP-70 activation, peak TCR induced phosphorylation of ZAP-70 on tyrosine-319 is attenuated. To control for loading errors the membranes were then stripped again and probed for total ZAP-70, (bottom row in Figure 4A).
In order to quantify these findings, the film record of each independent experiment in the ensemble (n = 5) of independent experiments was scanned and digitized. Similar to the procedure outlined for Figure 3 in our analysis of LAT phosphorylation, on each Western blot the band representing pZAP(Y319) was integrated and then normalized. Next the average value of pZAP(Y319) was determined for each time point after TCR stimulation in cells exposed or not exposed to mercury. The results are shown in Figure 4B, and reinforce Figure 4A by demonstrating that while Hg+2 perhaps marginally augments basal levels of ZAP-70 activation, following TCR stimulation it clearly depresses peak ZAP-70 activation by about 40%.
Hg2+ Inhibits Activation of LAT and ZAP-70 in HUT 78 Cells
The experiments reported in Figures 1–4 were all performed in Jurkat T cells. To control for the possibility that the effects we have seen are cell line specific, we looked at the ability of Hg2+ to interfere with TCR activation of LAT and ZAP-70 in HUT 78, a different human T cell line. Accordingly in a series of experiments (n = 4), HUT 78 cells were treated or not treated with Hg2+ and then activated with OKT3 as was described for the Jurkat cells. Then as described for Jurkat, cells were lysed and lysates probed by Western blotting for phosphotyrosine, followed by probes for LAT, pZAP, and total ZAP. Figure 5 is a representative example of one such series of experiments. The region of the initial Western blot for phosphotyrosine between 35 and 75 kDa is shown in the central part of Figure 5, where phosphoproteins in the vicinity of 70 and 38 kDa, suggestive of ZAP-70 and LAT respectively, are clearly visible (indicated by the block arrows). The membrane was then stripped, and blotted with an antibody specific to LAT. This resulted in a band being identified, which was superimposable over the 38 kDa phosphoprotein detected in the first blot. The LAT-specific band is shown in the lower portion of the figure. Next, the membrane was stripped, and blotted a third time for phospho (p)-ZAP(Y319), then stripped and blotted a final time for total endogenous ZAP-70. The pZAP(Y319), and ZAP-70 blots were each super imposable over the 70 kDa band in the initial -phosphotyrosine blot. The bands from these immunoblots are shown at the top of Figure 5.
In total, Figure 5 demonstrates that TCR dependent tyrosine phosphorylation of both LAT and ZAP-70 is inhibited by Hg2+ in HUT 78 cells in a fashion similar to the inhibition seen in Hg2+-exposed Jurkat T cells. However there is one difference. In HUT 78 it appears that the basal level of ZAP-70 activation is slightly higher than in Jurkat. And it appears to be suppressed by Hg2+, while in Jurkat the opposite seems true.
DISCUSSION
In this study we confirm our previous findings that in Jurkat T cells, exposure to a low and non-toxic concentration of Hg2+ significantly decreases the amount of activated ERK MAP Kinase in the first minutes following stimulation of the T-cell antigen receptor. We then expand upon this result by showing that the amount of tyrosine phosphorylated LAT (phospho-LAT), a key early regulatory element in the TCR signal transduction pathway, that normally forms after the TCR is stimulated is similarly reduced in cells which have been exposed to 5 μM Hg2+. We have previously shown that in Jurkat cells, 5 μM Hg2+ is not toxic (Whitekus et al., 1999).
Recent findings show that phospho-LAT couples the activated TCR to downstream signaling, in that phospho-LAT is essential for the formation of a functional signalosome complex needed to create appropriate responses following TCR engagement with an activating ligand (Bonello et al., 2004; Horejsi et al., 2004; Zhang et al., 1999). Of particular importance here is that one of the signal transduction elements downstream of the signalosome is the important regulatory protein Ras. Ras, a 21 kDa GTPase anchored to the plasma membrane, lies at the head of an amplifying signal transduction cascade that among other things controls the activity of ERK MAP kinases (Moodie et al., 1993). We have previously found that Hg2+ weakly activates ERK MAP Kinase, but somewhat paradoxically inhibits the activation of ERK MAP Kinase after the TCR is stimulated. However because Hg2+ inhibits TCR-dependent activation of Ras, but does not affect Ras activation by phorbol 12-myristate 13-acetate (PMA), we suggested that Hg2+ was likely interfering with an element of the T-cell signal transduction pathway upstream of Ras (Mattingly et al., 2001).
In T lymphocytes, Ras can be directly activated by either of two upstream guanine nucleotide exchange factors, Son of sevenless (Sos) or Ras guanine nucleotide releasing protein (RasGRP). Although SOS and RasGRP are themselves each activated via distinct pathways, interestingly, phospho-LAT figures prominently in the activation pathway of each. It was initially found that following its phosphorylation, LAT, which is a transmembrane protein, recruits the adapter protein Grb2. Grb2 binds SOS through SH3 domains, in addition to binding specific phosphorylated tyrosine residues on the cytoplasmic domain of LAT. After translocating to the plasma membrane and binding to LAT thru Grb2, SOS directly activates Ras, and by way of Ras, ERK MAP kinase (Zhang et al., 1999).
Recently a second signaling pathway connecting phospho-LAT to Ras activation through RasGRP has been described. RasGRP is activated by diacylglycerol (DAG), which is a product of activated PLC- acting on membrane PIP2 (phosphatidylinositol 4,5 bisphosphate). However activation of PLC- only occurs when it and the Tec family kinase Itk are each complexed with LAT. This requires the adapter proteins Gads and SLP-76, and additionally that LAT be phosphorylated (Boerth et al., 2000; Horejsi et al., 2004). Thus LAT is an important node in the T-cell signaling pathway, and its phosphorylation is a critical event in the activation of Ras, and subsequently that of ERK MAP Kinase. Clearly, inhibition of LAT phosphorylation by Hg2+ is sufficient to explain our previous finding of inhibition of TCR mediated Ras and ERK MAP Kinase activation by Hg2+.
It is worth noting that in these experiments Hg2+ does not completely inhibit LAT phosphorylation. Rather we have found that LAT phosphorylation is depressed by about 30% after cells are exposed to 5 μM Hg2+. Likewise, we have previously determined that 5–10 μM Hg2+ diminishes, but does not completely inhibit Ras activation after TCR stimulation (Mattingly et al., 2001). While it is possible that exposure to concentrations of Hg2+ above 10 μM might give rise to greater inhibition, we have not explored higher mercury exposures, because it is likely that such exposures would also be toxic. Nevertheless, a relatively low and non-toxic level of Hg2+ is sufficient to significantly depress activation of LAT by about 30%. A 30% reduction in activation of LAT is likely significant because the TCR-ERK signaling pathway is characterized by a cascade of protein-protein interactions and activations. Pathways of this structure typically exhibit signal amplification, in the sense that small perturbations in upstream signaling elements lead to much larger changes in down stream elements (Li and Qian, 2003). Indeed we have previously found that on average, exposure of T cells to Hg2+ concentrations as low as 1 μM gave rise to an approximate 60% reduction in ERK MAP kinase activation after the TCR was stimulated (Mattingly et al., 2001).
We have also previously shown that the effect of low concentrations of Hg2+ on cell signaling could most likely be accounted for by its effect on cell surface thiols, as masking of membrane protein sulfhydryls with a membrane impermeable malemide inhibited the ability of Hg2+ to influence signaling (Rosenspire et al., 1998). More recently we have directly demonstrated, by cold-vapor atomic absorption, that virtually no Hg2+ penetrates the plasma membrane when Jurkat cells are incubated with micromolar concentrations of Hg2+ for periods as long as 4 h (McCabe, Jr., et al., 2005). While LAT is a transmembrane protein, it has a small extracellular domain of only 4 amino acids, none of which is cysteine (Zhang et al., 1998). As a result, it is unlikely that Hg2+ acts directly on LAT. Rather, Hg2+ most probably interferes with an element or elements of the T-cell signal transduction system upstream of LAT.
LAT was originally described as the ZAP-70 substrate responsible for linking the TCR to cell activation (Zhang et al., 1998). In fact, LAT can only organize a functional signalosome following its phosphorylation by ZAP-70. Therefore, as ZAP-70 is the PTK immediately upstream of LAT in the TCR signal transduction cascade, we looked for an effect that cell exposure to Hg2+ might have on its activity. ZAP-70 activation is associated with tyrosine phosphorylation at multiple sites (Watts et al., 1994). However while phosphorylation of some sites seems to be unnecessary for activation, phosphorylation of others including tyrosine 319 is strictly required for activation and for TCR-dependent signaling to proceed (Di Bartolo et al., 1999; Williams et al., 1999). We find that normally after the TCR is stimulated, increases in ZAP-70 phosphorylation at tyrosine 319 occur in concert with increases in LAT phosphorylation. But in cells treated with Hg2+, TCR induced phosphorylation of ZAP-70 is attenuated in concert with attenuation of LAT phosphorylation.
Our results thus suggest that TCR-induced generation of phospho-LAT is diminished after lymphocytes are exposed to low dose (5 μM) Hg2+, because upstream of LAT activation of ZAP-70 is inhibited. However while Hg2+ at low levels clearly interferes with TCR mediated signal transduction, the effect on basal activation levels of specific elements of the T-cell signal transduction pathway appear to be marginal. We have found that Hg2+ slightly increases background phosphorylation of ZAP-70-Tyr 319 in Jurkat cells. But on the other hand basal phosphorylation of these residues in HUT 78 seem to be reduced by Hg2+. However this finding might be reflective of the fact that basal phosphorylation levels of ZAP-70 phosphorylation in HUT 78 were found to be somewhat higher than those of Jurkat in the first place. It may be that in HUT 78 we were looking at ZAP-70 which was "endogenously activated" to start. Nevertheless in either event the effects are small, and as opposed to the much larger effect on TCR- mediated signal transduction, it is not clear how T-cell functionality is effected.
Returning to the question of TCR signaling, just as it is unlikely that Hg2+ interacts directly with LAT, it is unlikely that Hg2+ interacts directly with the cytoplasmic ZAP-70 protein. It is interesting to note that ZAP-70 is itself activated by the transmembrane phosphokinase p56Lck, which has been shown to have altered kinase activity following Hg2+ exposure (Lander et al., 1992). As the initial site of Hg2+ assault is most probably still upstream of ZAP-70 in the TCR-initiated signal transduction cascade, it may be that p56Lck is the proximal target of inorganic mercury in this system.
Because of its broad sulfhydryl reactivity, specific molecular targets of mercury are likely to be dependent upon both concentration and chemical form. Thus physiologically significant targets of inorganic mercury may be expected to differ from those of organic mercury species, and in either case be concentration dependent. In the experiments reported here, we have utilized inorganic mercury at 5 μM, a concentration which is low in the sense that it is non-toxic under in vitro culture conditions. Although it is often difficult to reconcile in vitro toxicant concentrations with in vivo exposures, it is likely that the mercury dose and exposure conditions used in these experiments (5 μM for 10 min), are relevant to modern occupational, and perhaps environmental exposures.
In order to rigorously demonstrate this, the parameter needed is cellular toxicant burden, determined for both in vitro and in vivo exposures. In preliminary studies we have determined by cold vapor atomic absorption spectroscopy that the conditions used in these experiments (10 min incubation of lymphocytes in tissue culture media with 5 μM Hg2+) results in a mercury burden of approximately 5 ng/106 cells. After an initial rapid uptake, the binding of additional Hg2+ to lymphocytes goes up quite slowly, so that this burden does not appreciably change over the 2–20 min time course of the experiments we have described. Significantly, it turns out that 5 ng/106 cells is also about the same mercury burden obtained if cells are incubated with 0.5 μM Hg2+ for 24 h (ns).
Unfortunately specific lymphocyte mercury burdens due to environmental and occupational exposures have not in general been reported. But based on reported whole blood mercury concentrations which have been reported, we can make reasonable estimates. For example, in Brazilian gold miners exposed to mercury vapor during gold refining operations, blood mercury is likely to be predominantly inorganic, and total mercury concentrations from 5 to as high as 100 μg/l have been reported (Silva et al., 2004). In the same study it was also found that there was an increased prevalence of antinucleolar antibodies and a positive interaction between Hg and malaria, suggesting that Hg2+ at these blood levels could function as a co-factor in autoimmune disease.
Admittedly, Hg2+ binding in vivo is considerably more complicated than binding in vitro, but considering that 100 μg/l mercury is equivalent to approximately 0.5 μM, it is not unreasonable as a first order estimate to assume that in these occupationally exposed individuals lymphocyte mercury burdens of at least 5 ng/106 cells may be achieved in some instances. If this is the case than our in vitro results are seen to be directly relevant to real world occupational exposures to inorganic mercury. Interference with TCR signal transduction might then be associated with autoimmunity seen in the Brazilian gold miners.
Finally, blood mercury levels in individuals consuming >4 fish meals a week, but otherwise characterized by no known mercury exposure risk factors can also approach 100 μg/l (Brune et al., 1991). Mercury uptake from fish is primarily organic, but as organic mercury is over time converted to inorganic mercury, our results may also be relevant to more common environmental exposures to inorganic mercury.
ACKNOWLEDGMENTS
This work was in part supported by NIH grants ES11000, ES12403, and ES01247.
REFERENCES
Abraham, R. T., and Weiss, A. (2004). Jurkat T cells and development of the T-cell receptor signalling paradigm. Nat. Rev. Immunol. 4, 301–308.
Bagenstose, L. M., Salgame, P., and Monestier, M. (1998). IL-12 down-regulates autoantibody production in mercury-induced autoimmunity. J. Immunol. 160, 1612–1617.
Bernier, J., Brousseau, P., Krzystyniak, K., Tryphonas, H., and Fournier, M. (1995). Immunotoxicity of heavy metals in relation to Great Lakes. Environ. Health Perspect. 103(Suppl. 9), 23–34.
Boerth, N. J., Sadler, J. J., Bauer, D. E., Clements, J. L., Gheith, S. M., and Koretzky, G. A. (2000). Recruitment of SLP-76 to the membrane and glycolipid-enriched membrane microdomains replaces the requirement for linker for activation of T cells in T cell receptor signaling. J. Exp. Med. 192, 1047–1058.
Bollag, A. G. (1991). Protein Methods. Wiley-Liss, New York.
Bonello, G., Blanchard, N., Montoya, M. C., Aguado, E., Langlet, C., He, H. T., Nunez-Cruz, S., Malissen, M., Sanchez-Madrid, F., Olive, D., et al. (2004). Dynamic recruitment of the adaptor protein LAT: LAT exists in two distinct intracellular pools and controls its own recruitment. J. Cell Sci. 117, 1009–1016.
Brdicka, T., Cerny, J., and Horejsi, V. (1998). T cell receptor signalling results in rapid tyrosine phosphorylation of the linker protein LAT present in detergent-resistant membrane microdomains. Biochem. Biophys. Res. Commun. 248, 356–360.
Brune, D., Nordberg, G. F., Vesterberg, O., Gerhardsson, L., and Wester, P. O. (1991). A review of normal concentrations of mercury in human blood. Sci. Total Environ. 100, 235–282.
Di Bartolo, V., Mege, D., Germain, V., Pelosi, M., Michel, F., Magistrelli, G., Isacchi, A., and Acuto, O. (1999). Tyrosine 319, a newly identified phosphorylation site of ZAP-70, plays a critical role in T cell antigen receptor signaling. J. Biol. Chem. 274, 6285–6294.
Finco, T. S., Kadlecek, T., Zhang, W., Samelson, L. E., and Weiss, A. (1998). LAT is required for TCR-mediated activation of PLCgamma1 and the Ras pathway. Immunity 9, 617–626.
Firestone, G. L., and Winguth, S. D. (1990). Immunoprecipitation of proteins. Methods Enzymol. 182, 688–700.
Hansson, M., and Abedi-Valugerdi, M. (2004). Mercuric chloride induces a strong immune activation, but does not accelerate the development of dermal fibrosis in tight skin 1 mice. Scand. J. Immunol. 59, 469–477.
Hong, R. (1999). Evaluation of toxic assault on the immune system. Mutat. Res. 428, 109–114.
Horejsi, V., Zhang, W., and Schraven, B. (2004). Transmembrane adaptor proteins: organizers of immunoreceptor signalling. Nat. Rev. Immunol. 4, 603–616.
Hu, H., Moller, G., and Abedi-Valugerdi, M. (1999). Mechanism of mercury-induced autoimmunity: both T helper 1- and T helper 2-type responses are involved. Immunology 96, 348–357.
Huang, Y., and Wange, R. L. (2004). T cell receptor signaling: Beyond complex complexes. J. Biol. Chem. 279, 28827–28830.
Kazantzis, G. (2002). Mercury exposure and early effects: An overview. Med. Lav. 93, 139–147.
Lander, H. M., Levine, D. M., and Novogrodsky, A. (1992). Stress stimuli-induced lymphocyte activation. Cell. Immunol. 145, 146–155.
Li, G., and Qian, H. (2003). Sensitivity and specificity amplification in signal transduction. Cell Biochem. Biophys. 39, 45–59.
Mattingly, R. R., Felczak, A., Chen, C. C., McCabe, M. J. J., and Rosenspire, A. J. (2001). Low concentrations of inorganic mercury inhibit Ras activation during T cell receptor-mediated signal transduction. Toxicol. Appl. Pharmacol. 176, 162–168.
McCabe, M. J., Jr., Eckles, K. G., Langdon, M., Clarkson, T. W., Whitekus, M. J., and Rosenspire, A. J. (2005). Attenuation of CD95-induced apoptosis by inorganic mercury: Caspase-3 is not a direct target of low levels of Hg2+. Toxicol. Lett. 155, 161–170.
Moodie, S. A., Willumsen, B. M., Weber, M. J., and Wolfman, A. (1993). Complexes of Ras.GTP with Raf-1 and mitogen-activated protein kinase kinase [see comments]. Science 260, 1658–1661.
Moszczynski, P. (1997). Mercury compounds and the immune system: A review. Int. J. Occup. Med. Environ. Health 10, 247–258.
Rosenspire, A. J., Bodepudi, S., Mathews, M., and McCabe, J. M. (1998). Low levels of ionic mercury modulate protein tyrosine phosphorylation in lymphocytes. Int. J. Immunopharmacol. 20, 697–707.
Shen, X., Lee, K., and Konig, R. (2001). Effects of heavy metal ions on resting and antigen-activated CD4(+) T cells. Toxicology 169, 67–80.
Silva, I. A., Nyland, J. F., Gorman, A., Perisse, A., Ventura, A. M., Santos, E. C., Souza, J. M., Burek, C. L., Rose, N. R., and Silbergeld, E. K. (2004). Mercury exposure, malaria, and serum antinuclear antibodies in amazon populations in Brazil: a cross-sectional study. Environ. Health 3, 11.
Sweet, L. I., and Zelikoff, J. T. (2001). Toxicology and immunotoxicology of mercury: A comparative review in fish and humans. J. Toxicol. Environ. Health B Crit. Rev. 4, 161–205.
Watts, J. D., Affolter, M., Krebs, D. L., Wange, R. L., Samelson, L. E., and Aebersold, R. (1994). Identification by electrospray ionization mass spectrometry of the sites of tyrosine phosphorylation induced in activated Jurkat T cells on the protein tyrosine kinase ZAP-70. J. Biol. Chem. 269, 29520–29529.
Whitekus, M. J., Santini, R. P., Rosenspire, A. J., and McCabe, M. J. J. (1999). Protection against CD95-mediated apoptosis by inorganic mercury in jurkat T cells. J. Immunol. 162, 7162–7170.
Williams, B. L., Irvin, B. J., Sutor, S. L., Chini, C. S., Yacyshyn, E., Wardenurg, J. B., Dalton, M., Chan, A. C., and Abraham, R. T. (1999). Phosphorylation of Tyr 319 in ZAP-70 is required for T-cell antigen receptor-dependent phospholipase C-1 and Ras activation. EMBO J. 18, 1832–1844.
Zhang, W., Irvin, B. J., Trible, R. P., Abraham, R. T., and Samelson, L. E. (1999). Functional analysis of LAT in TCR-mediated signaling pathways using a LAT-deficient Jurkat cell line. Int. Immunol. 11, 943–950.
Zhang, W., Sloan-Lancaster, J., Kitchen, J., Trible, R. P., and Samelson, L. E. (1998). LAT: The ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation. Cell 92, 83–92.
Zheng, Y., and Monestier, M. (2003). Inhibitory signal override increases susceptibility to mercury-induced autoimmunity. J. Immunol. 171, 1596–1601.(Stamatina E. Ziemba, Raymond R. Mattingl)