ToxSci Advance Access originally published online on July 26, 2007
Toxicological Sciences 2007 99(2):512-521; doi:10.1093/toxsci/kfm188
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Low and Nontoxic Inorganic Mercury Burdens Attenuate BCR-Mediated Signal Transduction
,1
* Department of Environmental Medicine, University of Rochester, Rochester, NY
Department of Immunology and Microbiology, Wayne State University, Detroit, MI
Department of Pharmacology, Wayne State University, Detroit, MI
1 To whom correspondence should be addressed at the Department of Immunology and Microbiology, 7374 Scott Hall, Wayne State University School of Medicine, Detroit, MI 48201, Fax: (313) 577-1155. E-mail: arosenspire{at}wayne.edu.
Received May 18, 2007; accepted July 18, 2007
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
The ubiquitous environmental heavy metal contaminant mercury (Hg) is a potent immunomodulator that has been implicated as a factor contributing to autoimmune disease. However, the mechanism(s) whereby Hg initiates or perpetuates autoimmune responses, especially at the biochemical/molecular level, remain poorly understood. Recent work has established a relationship between impaired B-cell receptor (BCR) signal strength and autoimmune disease. In previous studies, we have shown that in mouse WEHI-231 B cells, noncytotoxic concentrations of inorganic mercury (Hg+2) interfered with BCR-mediated growth control, suggesting that BCR signal strength was impaired by Hg+2. Extracellular signal-regulated kinase (ERK) 1,2 mitogen-activated protein kinase (MAPK) is responsible for the activation of several transcription factors in B cells. Phosphorylation of ERK serves as an essential node of signal integration for the BCR. Thus, the magnitude of ERK activation serves as an operational metric for BCR signal strength. Using Western blotting and phospho-specific flow cytometry, we now show that the kinetics and magnitude of BCR-mediated activation of ERK-MAPK are markedly attenuated in WEHI-231 cells and splenic B cells that have been exposed to low and nontoxic burdens of Hg+2. However, Hg+2 does not seem to act directly on ERK-MAPK but rather on an upstream element or elements of the BCR signal transduction pathway, above the level of the key protein tyrosine kinase Syk. Our data suggest that the site of action of Hg+2 may very well be localized on the plasma membrane. These findings support a connection between Hg+2 and attenuated BCR signal strength in the etiology of autoimmune disease.
Key Words: mercury; B-cell receptor; ERK; Syk.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
While the specific etiologies of most autoimmune diseases are unknown, it is generally thought that they arise as a result of compromised immunoregulatory mechanisms after exposure of genetically predisposed individuals to selective environmental triggers (Perl, 2004
). The characteristic loss of tolerance to self-antigens is a complex process that is dependent on the interaction of multiple genetic loci with a large number of potential environmental factors (Brickman and Shoenfeld, 2001). While progress has been made in elucidating underlying genetic lesions that may contribute to the loss of tolerance to self-antigens (Lohr et al., 2005), in comparison little is known as to how environmental factors affect the immune system so as to give rise to autoimmune disease.
Epidemiological studies and case reports have suggested that exposure to low environmental mercury levels may be a factor that contributes to idiosyncratic autoimmune disease in humans (Mayes, 1999; Silbergeld et al., 2005). Additionally, animal studies have directly demonstrated that in (H-2) susceptible rodents, exposure to nontoxic levels of inorganic mercury gives rise to a systemic lupus erythematosus–like autoimmune disease characterized by lymphoproliferation, hyperglobulinemia, anti-nuclear antibody production, and systemic immune complex deposition leading to glomerulonephritis (reviewed in Rowley and Monestier, 2005). Furthermore in several autoimmune-prone mouse strains, nontoxic levels of mercury appear to exacerbate disease (Pollard et al., 1999, 2001). Significantly in animals not otherwise susceptible to mercury, or naturally autoimmune prone, low levels of mercury have been also been found to exacerbate disease in several models of induced autoimmunity (Hansson et al., 2005; Silbergeld et al., 2005; Via et al., 2003).
Recent findings suggest that the degree to which autoreactive B cells have been eliminated from the naive B-cell repertoire determines whether a nonspontaneous autoimmune host will develop autoimmune disease (Cappione et al., 2005; Wang et al., 2003). In B cells, the B-cell receptor (BCR) plays a central role in cell development, activation, survival, and apoptosis. It has been demonstrated that genetic alterations in the regulatory pathways governing BCR signal strength which lead to attenuated BCR signals are phenotypically characterized by aberrant survival and activation of autoreactive B cells, which then contribute to a naive BCR repertoire supporting autoimmune disease (Grimaldi et al., 2002, 2005).
We have previously reported that BCR functionality is impaired in B cells exposed to nontoxic concentrations of inorganic mercury (McCabe et al., 1999). In the experiments reported here, we directly demonstrate in B cells exposed to nontoxic levels of mercury that the strength of the BCR signal is reduced compared to cells that have not been exposed to mercury. Thus, evidence demonstrating that mercury is associated with autoimmune disease may in part be explained by impaired signal transduction (i.e., a reduction in BCR signal strength) in the B-cell compartment after mercury exposure. This suggests that attenuation of BCR signal strength, whether the result of a genetic lesion or exposure to environmental mercury, may be phenotypically characterized by aberrant survival and/or activation of autoreactive B cells.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
Reagents.
HgC12 was obtained from Aldrich Chemicals (St Louis, MO). All other reagents were obtained from Sigma Chemicals (St Louis, MO), unless otherwise indicated.
Cells.
WEHI-231 cells were obtained from the American Type Culture Collection (Manassas, VA). They were maintained in RPMI 1640 (Life Technologies, Rockville, MD), supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 2mM glutamine, and 100 U/ml penicillin, 100 µg/ml streptomycin in a humidified 5% CO2 atmosphere. Cells were passaged three times a week, and cells in logarithmic growth phase were used for all experiments. Spleen cells were teased from the spleens of 6- to 8-week-old BALB/c female mice and purified by density gradient centrifugation over ficoll-hypaque. Cell viability was monitored visually by Trypan Blue exclusion.
Antibodies.
Polyclonal goat affinity-purified antibody to mouse immunoglobulin was purchased from MP Biomedicals-Cappel, Solon, OH. Monoclonal antibody to phospholipase c-
(PLC) and rabbit polyclonal antibody to Syk were purchased from Santa Cruz Biotechnology, Santa Cruz, CA. Monoclonal antibody to dually phosphorylated extracellular signal-regulated kinase–mitogen-activated protein kinase (ERK-MAPK) was purchased from Sigma Chemicals or from BD Biosciences-Transduction Laboratories, Franklin Lakes, NJ. Anti-phospho horse radish peroxidase (HRP)–conjugated anti-phosphotyrosine antibody RC-20H and monoclonal antibodies to Ras and ERK were purchased from BD Biosciences-Transduction Laboratories. Fluorescently labeled antibodies used for flow cytometric analysis of phosphorylated ERK and Syk, as well as for B220 were also purchased from BD Biosciences-Transduction Laboratories.
Detection of Ras activation.
WEHI-231 were adjusted to 2 x 107 cells/ml. One-milliliter aliquots were then preincubated with the indicated concentrations of mercury prior to addition of agonist as described in the figure legends. The cells were collected by centrifugation and then each individual sample was lysed in 0.5 ml of a detergent buffer consisting of 50mM Hepes (pH 7.4), 150mM NaC1, 20mM MgCl, and 0.5% (wt/vol) Triton X-100. After centrifugation at maximum speed in a cold microcentrifuge (
12,000 x g), the pellets were discarded and sodium deoxycholate was added to 0.25% (wt/vol) of each lysate. The lysates were then precleared for 5 min with glutathione-agarose. For each sample, one-third of the cleared extract was precipitated with 10% cold trichloracetic acid, so that the total Ras content of all samples could be compared by Western blotting as shown in the figures. The remaining two-third of the cleared extracts were incubated for 30 min with glutathione-Sepharose beads that had been prebound with a fusion protein between glutathione-S-transferase and the Ras-binding domain of the Raf protein kinase (Raf:RBD, a gift from J. Downward, Imperial Research Cancer Fund, London). The beads were washed twice with lysis buffer and then boiled in Laemmli sample buffer for separation by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose, and Western blotting was done with a monoclonal antibody against Ras. The amount of Ras bound to the Raf:RBD construct is the fraction that was active and bound to GTP in the lysate. Western blots were developed with appropriate secondary antibodies coupled to HRP and enhanced chemiluminescence using Dura reagents (Pierce, Rockford, IL).
Detection of MAPK activation.
MAPK activity was measured in a manner similar to that described in Mattingly et al. (2001b). After incubation with anti-Ig, WEHI-231 cells were chilled, collected by centrifugation, and lysed in boiling Laemmli sample buffer. Proteins were resolved by SDS-PAGE and then transferred to nitrocellulose so that Western blotting with a monoclonal antibody against the active, diphosphorylated form of ERK (Gabay et al., 1997) could be accomplished. The membrane was then stripped for 30 min in 62.5mM Tris-HCl (pH 6.8), 2% (wt/vol) SDS, and 100mM ß-mercaptoethanol at 70°C and then extensively washed. Total MAPK was then assayed by Western blotting with an anti-ERK monoclonal antibody. Western blots were developed with appropriate secondary antibodies coupled to HRP and enhanced chemiluminescence reagents (Kirkland and Perry, Gaithersburg, MD).
Immunoprecipitation.
WEHI-231 cells were solubilized for 20 min on ice in detergent (1% Nonidet P-40 for Syk or 1% octylglucoside for PLC) at 5 x 107 cells/ml in Tris-Cl buffer (100mM Tris, 125mM NaCl, 10mM ethylenediaminetetraacetic acid, pH 7.5) in the presence of phosphatase and protease inhibitors composed of 100mM phenylmethylsulfonyl fluoride, 20mM NaF, and 200mM NaVO4. After solubilization, lysates were centrifuged at 12,000 x g for 15 min. The pellet was discarded and the supernatant was incubated with 10 µl anti-Syk or anti-PLC for 1 h on ice. The lysate was then transferred to an immunoabsorbant, either Protein A-Agarose (Oncogene Research Products, Boston, MA) or Pansorbin (Calbiochem, La Jolla, CA) for 1 h at 4°C in rotating motion. The immunoabsorbants were washed with lyaste buffer and then lyaste buffer with 0.1% detergent. Finally, the immunoprecipitated proteins were eluted with sample buffer and analyzed with SDS-PAGE and Western blotting.
Western blotting and detection of protein tyrosine phosphatase.
Proteins in sample eluates were directly resolved by SDS-PAGE in a mini-gel PAGE apparatus (BioRad, Hercules, CA), after which the gels were electrophoretically transferred to nitrocellulose membrane (Pall Life Sciences, Pensacola, FL), for 1 h at 100 V in a MiniGel Transfer Unit (Hoeffer, San Francisco, CA). Protein blots were probed for the presence of phosphotyrosine with the HRP-conjugated anti-phosphotyrosine reagent RC-20H. Blots were developed utilizing an enhanced chemiluminescent system (Pierce) and reflection film (Kodak, Rochester, NY).
Measurement of protein tyrosine phosphatase.
In some cases, blots were scanned and the results digitized and analyzed using a Fuji-Film LAS 1000 plus imaging system. Statistical analysis of the data was accomplished using the heteroscedastic t-test.
Phospho-specific flow cytometry.
Cells (WEHI-231 or spleen cells) were counted, washed with RPMI serum-free media, and resuspended at a concentration of 3 x 106 cells/ml in the presence or absence of 5µM HgCl2. Subsequently, 300 µl of cell suspension is aliquoted to individual 12 x 75–mm polypropylene flow cytometry tubes and placed in a humidified incubator containing 5% CO2 for 10 min. Tubes are then removed from the incubator and stimulated with the appropriate concentration of goat anti-IgM diluted in 5 µl of RPMI media per sample tube. Tubes are quickly vortexed and placed into the incubator for the specified period of time. At the end of the stimulation (0.5, 2, 5, 10, or 20 min later), tubes are removed and fixed for 5 min with 500 µl of 4% paraformaldehyde made fresh, pH 7.4. Tubes are pelleted at 900 x g and resuspended in 2 ml of 100% methanol to permeabilize the plasma membrane. After 10 min, cells are pelleted, methanol removed, and were washed twice with 1 ml phosphate-buffered saline for rehydration of the cells. Subsequently, cells were incubated in the presence of an AlexaFluor-488–conjugated antibody specific for the phosphophorylated residues of either ERK1/2 or Syk. After 20 min incubation, cells were washed, and WEHI-231 cells were immediately run on the BD LSR 2 flow cytometer. In the case of spleen cells, cells were counter-stained with a PerCt-conjugated antibody directed to B220 prior to flow cytometry. For all experiments, 10,000 viable events, as determined by forward scatter versus side scatter gating, are acquired per sample and data are analyzed by FloJo software. The percentage of p-ERK+ or p-Syk+ cells is determined by using the Overton multiple comparison algorithm for histograms. The Overton algorithm calculates the cumulative mean fluorescence intensity difference between overlaid experimental samples (stimulated cells) and control (unstimulated cells exposed to Hg).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
Hg+2 Attenuates ERK-MAPK Activity in BCR-Stimulated Cells
B cells recognize and respond to antigens in their environment as a result of antigen binding to membrane immunoglobulin on the cell surface. Binding of antigen to membrane immunoglobulin initiates a complex series of molecular interactions between members of the BCR and other B cell proteins and lipids, such that BCR signal transduction has been described as a labyrinth of interconnecting pathways which culminate in activation of transcription factors and the regulation of gene expression controlling cellular selection, maturation, survival, and antibody production (reviewed in Dal Porto et al., 2004
). Although B-cell signaling is a complex process encompassing many different molecular entities, an especially important integration point is ERK. ERK-MAPK is a dually threonine-tyrosine–activated serine kinase and a member of the MAPK family, which in B cells is directly responsible for the activation of several transcription factors, including Bcl-6, Erg-1, and Elk-1 (Dal Porto et al., 2004; Jacob et al., 2002).
It has been shown that several B-cell functions mediated by the BCR are disrupted, if during the signal transduction process the level of BCR-induced ERK activation is reduced (Dal Porto et al., 2004). We have previously shown that nontoxic levels of inorganic mercury disrupt BCR-mediated functions in B cells (McCabe et al., 1999). We have also shown that in T cells, T-cell receptor (TCR)–mediated activation of ERK-MAPK is attenuated in cells which have been exposed to Hg+2 (Mattingly et al., 2001a), suggesting that the ability of Hg+2 to interfere with BCR-mediated functions might be explained by an effect of Hg+2 on BCR-mediated ERK activation.
Therefore, in a series of experiments, we directly investigated whether Hg+2 interfered with BCR-mediated activation of ERK-MAPK. Figure 1 is a representative example of experiments (n = 3) where B cells were or were not exposed to mercury and then stimulated with anti-Ig. At timed intervals, cells were lysed and levels of activated ERK assessed by Western blot analysis for dually phosphorylated (activated) ERK-MAPK. We found in control cells that within 2 min of BCR stimulation, substantial activation of ERK-MAPK occurred. Activation peaked approximately 4–5 min later and remained well above background levels for at least 20 min. On the other hand, at all time points between 2 and 20 min, activation of ERK-MAPK in Hg+2-treated cells was uniformly suppressed with respect to cells which had not been exposed to Hg+2. In other experiments, we found that Hg+2 alone (in the absence of BCR signaling) had no effect on ERK-MAPK activation, although it did modestly activate Ras (Fig. 2).
|
|
These initial findings were expanded upon in experiments where we utilized phospho-specific flow cytometry (Irish et al., 2006
; Krutzik et al., 2004). These results are shown in Figure 3, where the basic experimental design was similar to that of Figure 1. As in Figure 1, cells were or were not incubated with Hg+2 and then stimulated for timed periods with goat anti-mouse Ig. They were then stained with fluorescently labeled anti–phospho-ERK 1,2 and analyzed by flow cytometry. First, we determined the percentage of phospho-ERK–expressing cells as a function of time after BCR stimulation by comparing the fluorescence signal of p-ERK at each time point to control cells which were neither stimulated nor exposed to Hg+2. As shown in Figure 3, BCR stimulation induced a marked increase in the number of p-ERK–expressing cells as rapidly as 0.5 min after stimulation, with nearly 80% of the cells expressing phosphorylated ERK by 5 min after stimulation. Preexposure of cells to Hg+2 resulted in a 25% decrease in the percentage of responding cells, and peak activation of ERK was delayed until 10 min after BCR stimulation (Fig. 3B). Moreover, mean fluorescence intensity of p-ERK, which is a measure of the magnitude of ERK phosphorylation, is reduced by more than 30% following exposure to Hg+2 (Fig. 3C). These data indicate that after BCR stimulation, Hg reduces the number of responding cells, and that within the responding cell population, exposure to Hg+2 leads to less phosphorylated ERK with respect to activated control cells.
|
The Kinetics of BCR-Induced Tyrosine Phosphorylation of Syk is Altered by Hg+2
Of obvious interest is the mechanism whereby Hg+2 attenuates activation of ERK. In the absence of any evidence for a direct interaction between Hg+2 and ERK, we considered that upstream signaling elements proximal to the BCR signalosome might be targeted by Hg+2. In particular, during BCR signal transduction, activation of ERK-MAPK is dependent on prior upstream phosphorylation and activation of the protein tyrosine kinase (PTK) Syk (Cornall et al., 2000
; Dal Porto et al., 2004). Therefore, we decided to determine if Hg+2 inhibition of ERK was associated with the suppression of Syk kinase activity in B cells during BCR-mediated signal transduction.
Accordingly, in a series of experiments, B cells were or were not pretreated with Hg+2 prior to the BCR being stimulated by the addition of anti-Ig. At timed intervals, cells were lysed and Syk was extracted from the lysates by immunoaffinity chromatography. Protein tyrosine phosphorylation, an overall indication of Syk activity (Keshvara et al., 1998), was then determined by Western blotting. Figure 4A is a representative example of the results of several experiments (n = 5), showing that in resting cells not treated with anti-Ig, little if any Syk is phosphorylated, and this is independent of whether or not the cells have been treated with mercury. Thus, mercury alone at nontoxic concentrations does not seem to have a direct effect on Syk activity. However, once the BCR is stimulated, measurable phosphorylation of Syk occurs within 30 s. In cells which have been treated with Hg+2 though, phosphorylation appears to be enhanced. This trend is continued at 2 min. At 5 min though, both the Hg+2-treated cells and controls have reached maximal phosphorylation and appear about equal in intensity. After 5 min, both Hg+2-treated and untreated populations decline to control levels.
|
-mouse IgG. At timed intervals, cells were lysed, and Syk was immunoprecipitated from the lysates. Tyrosine phosphorylation of Syk was determined by Western blotting. (A) A representative Western blot of phosphorylated Syk in cells exposed or not to Hg+2 (n = 10). (Lanes were normalized in the sense that loading of immunoprecipitated eluates were derived from equivalent cell numbers.) (B) Levels of Syk phosphorylation were then quantified by digitally scanning multiple Western blots of immunoprecipitated Syk. The results of 10 experiments were averaged and plotted (on a log scale to accentuate the early time points), with closed triangle representing cells exposed to Hg+2 and closed square representing cells not exposed to Hg+2. Error bars represent the SEM. The "*" and "**" indicate a statistically significant difference (as determined with the heteroscedastic t-test) between cells treated or not treated with Hg+2, with p < 0.005 and p < 0.001, respectively.Surprisingly, Figure 4A thus indicates that Hg+2 primarily affects the kinetics of Syk phosphorylation following BCR stimulation, in that it accelerates BCR-dependent tyrosine phosphorylation of Syk, but has little effect on maximal phosphorylation. This finding is put on a more quantitative footing in Figure 4B, where the basic experiment outlined in Figure 3A was repeated 9 additional times, but the resulting western blots for phospho-Syk (p-Syk) were quantified by densitometry. However, because it appeared that mercury was mostly having an effect on Syk phosphorylation at very early times, in these experiments time points prior to 30 s after BCR stimulation were assayed, in addition to later ones. The results are plotted in Figure 4B, where the time coordinates are plotted on a log scale so as to accentuate the early kinetics. The figure is consistent with Figure 4A in demonstrating that in the presence of Hg+2, the kinetics of Syk phosphorylation is altered. While (BCR induced) maximal phosphorylation of Syk in Hg+2-treated cells is similar to controls, the phosphorylation process is accelerated.
Hg+2 Alters BCR-Dependent Tyrosine Phosphorylation of Syk in Normal B Cells
As opposed to B-cell lines, normal B cells are a heterogeneous population of cells with polyclonal BCR specificities. Primary splenic B cells are also a mixed pool of cells with respect to maturational status (Chung et al., 2003). For these reasons, the analysis of BCR signaling profiles in normal B cells is more challenging than the analysis of signaling in B-cell lines. Single-cell phospho-specific flow cytometry represents a technical advance that is especially suited for this problem. In Figure 5, we have utilized phospho-specific flow cytometry to demonstrate that Hg+2 alters BCR-induced tyrosine phosphorylation of Syk in normal B cells.
|
Splenocytes were isolated from BALB/c mice, and as a positive control one aliquot was treated with H2O2. (H2O2 inactivates negative regulatory protein tyrosine phosphatases (PTPs.) This leads to hyperphosphorylation of proteins within the BCR signaling cascade, including Syk, as a result of unchecked endogenous PTK activity (Reth, 2002
). The remaining cells were preincubated with or without HgCl2, following which the BCR was stimulated with goat anti-IgM. At timed intervals up to 20 min after BCR stimulation, cells were stained for p-Syk. Cells exposed to H2O2 were also stained for p-Syk after 20 min. To provide a pan–B-cell marker, all cells were counter-stained with an anti-B220 antibody. Cells were then analyzed for p-Syk and B220 expression by two-color flow cytometry, and the results are shown in Figure 5. Rapid phosphorylation of Syk was detected in B220 cells within 0.5 min following BCR stimulation. The internal negative control confirmed lack of p-Syk in response to anti-Ig in the non–B cell (B220 negative) compartment in each of the plots in Figure 5A. Also shown is the positive control (Fig. 5A, lower left panel), where we find a robust Syk activation in (B220– and B220+) splenocytes following stimulation with H2O2. (Although B220– splenocytes do not express Syk, the anti–p-Syk reagent also recognizes a phospho-ZAP-70 epitope in T cells. Thus, the apparent p-Syk signal in the B220– cells shown in the lower left panel of Figure 5A likely represents phosphorylation of ZAP-70 in response to PTP inactivation in T cells.) Significantly, pretreatment of normal primary splenocytes with Hg+2 markedly increased the percentage of B220+ cells expressing phosphorylated Syk following BCR stimulation (right panels of Fig. 5A). Moreover, comparing the kinetics of Syk activation between BCR-stimulated control cells and Hg+2-intoxicated cells (Figs. 5B and 5C), we find that Hg+2 dramatically increases the magnitude of Syk phosphorylation within the B220+ compartment.
BCR-Induced Activation of PLC is Unaffected by Hg+2, Although its Binding to Syk is Accelerated
Aside from ERK, PLC is another important downstream effector of BCR signaling (Dal Porto et al., 2004). Upon BCR cross-linking, PLC is activated by the tyrosine phosphorylation of several residues in reactions catalyzed by the Syk and Btk kinases (Kim et al., 2004). Activated PLC then cleaves phosphatidylinositol biphosphate releasing IP3 and diacylglycerol (DAG). While IP3 is a positive regulator of the release of Ca2+ from intracellular stores, DAG is responsible for activating PKC and Ras. Since PLC and ERK are both downstream of Syk during BCR signal transduction, but not directly associated, we asked whether Hg+2 would depress PLC activation, as it does ERK.
In Figure 6, WEHI-231 B cells were or were not incubated with 5µM Hg+2 and as in Figure 4 stimulated with anti-Ig. At timed intervals, cells were lysed and PLC was isolated by immunoaffinity chromatography. Tyrosine phosphorylated PLC was then visualized by Western blotting with an anti-phosphotyrosine antibody. In control cells, phosphorylation of PLC is transient. We find that within 30 s, PLC is maximally phosphorylated but that by 20 min phosphorylation has returned to background levels. Significantly, we find no difference in tyrosine phosphorylation in cells which have been treated with Hg+2 and those which have not.
|
After the BCR is stimulated, aside from becoming phosphorylated, PLC
has been shown to translocate and bind to the phosphotyrosyl residues in the linker region of Syk (Law et al., 1996). In order to ascertain if Hg+2 had any effect on the binding of PLC to Syk, the blot was stripped and reprobed with an anti-Syk antibody to determine the amount of Syk coprecipitating with PLC. We find that under control conditions in the absence of mercury, Syk does not appreciably associate with PLC until 1 min after BCR signaling has begun. On the other hand, in cells which have been preincubated with Hg+2, maximum quantities of Syk have already bound to PLC by 30 s after BCR signaling is initiated. Hg+2 thus serves to accelerate Syk-PLC association during BCR signaling.
Inorganic Mercury Binds to but Does Not Cross to the External Leaflet of the Plasma Membrane
Although inorganic mercury under the exposure conditions that we have employed seems to have little effect on either ERK-MAPK or Syk by itself, it is possible that suppression of ERK phosphorylation, or accelerated activation of Syk after BCR stimulation, might still be the result of a direct action of the metal at the level of ERK and/or Syk. It is also possible that mercury might directly impact one or more of the several signaling intermediaries, such as Ras or the MAPK (mitogen-activated protein)/ERK kinase (MEK) 1/2 kinases, which lie between Syk and ERK in the BCR signal transduction network. However, ERK as well as all the signaling intermediates upstream to the level of Syk are cytoplasmic proteins. In order for inorganic mercury to directly interact with any of them, at the very least it would have to enter the cytoplasmic compartment. Therefore, we utilized cold vapor atomic absorption spectroscopy to determine to what extent inorganic mercury actually crossed the plasma membrane under the conditions utilized in Figures 1–6
.
In a series of experiments, WEHI-231 cells were incubated with from 0.2 to 10µM HgCl2, which we have previously established to be the highest nontoxic concentration of Hg+2 for these cells (McCabe et al., 1999). At timed intervals, cells were washed, and for each concentration of Hg+2, half were treated with heavy metal chelating agent British anti-lewisite (BAL) to remove mercury bound to cell surfaces but not internal mercury that has crossed the plasma membrane (Oehme, 1972). Cold vapor atomic absorption spectroscopy was then utilized to determine the cellular mercury burden of Hg+2 in BAL-treated and untreated cells. We found that for all concentrations of Hg+2, over 24 h of incubation, the mercury burden on cells not treated with BAL rose continuously. However, for all time points and for all mercury concentrations, the mercury burden for BAL-treated cells remained at baseline levels. This implies that within our detection limit all cell-associated mercury was bound to the cell surface, with little if any entering the cytoplasm. Figure 7 illustrates the results for 10µM, which was the highest mercury concentration tested. In other experiments, we have also determined that Hg+2 does not appreciably enter cells under conditions where the BCR has been cross-linked with anti-Ig (not shown).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
We have previously found that exposure of B cells to low and nontoxic concentrations of Hg+2 interfered with the ability of the BCR to respond to antigen (McCabe et al., 1999
). We now report that when the BCR is stimulated in the presence of nontoxic concentrations of Hg+2, normal activation of ERK-MAPK is attenuated (Figs. 1 and 3). Since it is known that the activation of several essential transcription factors regulating the B-cell response to antigen depend upon activated ERK (Dal Porto et al., 2004), it appears that mercury blocks or alters the B-cell response to antigen at least in part by attenuating BCR-mediated signal transduction at or above the level of ERK. But by what mechanism? It is unlikely that the effect of Hg+2 on ERK can be the result of a direct interaction with Hg+2. ERK is an intracellular protein, and we have found that (under the conditions we have employed) little, if any, Hg+2 crosses the plasma membrane (Fig. 7). Hg+2 initially must then directly interact with an element or elements of the B-cell signal transduction network which are upstream of ERK.
In B cells, as in other systems, activation of ERK is controlled by the upstream intracellular Raf-MEK kinase cascade which transmits signals from activated receptors to ERK. The cascade is controlled by Ras GTPase, which directly activates Raf (Dal Porto et al., 2004; Harding et al., 2005). Looking for an effect of Hg+2 on these upstream signaling elements, we found that although Ras is an intracellular protein, Hg+2 alone activated Ras, but not ERK (Fig. 2). This is likely explained by the fact that Ras functions as an integration point in many aspects of cellular signaling. Extracellular signaling pathways mediated by a variety of receptor-associated tyrosine kinases, including but not limited to the BCR complex, all utilize Ras, and Ras can potentially activate multiple downstream effectors in addition to Raf (Pouyssegur et al., 2002). Thus, at any particular time, only a fraction of Ras is associated with the Raf-MEK-MAPK cascade. Taken together, Figures 1 and 2 imply that by interfering with an upstream element of the BCR signaling cascade, Hg+2 attenuates BCR-induced activation of ERK. However, it also appears that at the same time Hg+2 activates membrane receptors which are linked to Ras but not to the Raf-MEK-ERK cascade.
In many respects, including activation of the Raf-MEK-ERK-MAPK cascade, BCR signaling is similar to TCR signaling. We have previously shown that in T cells, Hg+2 attenuates TCR signal transduction by interfering with TCR-induced activation of Ras and ERK (Mattingly et al., 2001a). In this case, we determined that interference with Ras activation resulted from Hg+2-dependent attenuation of ZAP-70 phosphorylation, the crucial tyrosine kinase upstream of Ras and the Raf-MEK-MAPK cascade in T cells (Ziemba et al., 2006).
In B cells, the ZAP-70 homologue Syk generally substitutes for ZAP-70 in antigen receptor–mediated signaling. Analogous to ZAP-70 during TCR signaling, Syk is phosphorylated on multiple tyrosines following B-cell activation, and prior activation of Syk is essential for Ras (and ERK) to be activated (Cornall et al., 2000). However, we have found that although Hg+2 clearly depresses activation of ERK after either BCR or TCR signaling, unlike ZAP-70 in Hg+2-exposed T cells, overall phosphorylation of Syk did not appear to be depressed in B cells which were preexposed to Hg+2. Rather, we found that in the presence of Hg+2, the rate of Syk phosphorylation was actually enhanced in WEHI-231 B cells and even more dramatically in normal splenic B cells responding to BCR signaling (Figs. 4 and 5). Nevertheless, signal transduction is still partially blocked at the level of ERK. We suggest that this results not because Hg+2 directly inhibits Syk activation but rather because the kinetics of Syk activation have been altered. If this is the case, then the effect of mercury on Syk activation kinetics and subsequent downstream attenuation of ERK activation must be understood as a network-dependent phenomenon, perhaps a consequence of subtle alterations in the timing of intermediate BCR signaling events.
Using a variety of genetic and biochemical techniques, including generation of dominant-negative mutants, coprecipitation protocols, etc, the hierarchical structure of the BCR signaling network has come more fully into focus (Dal Porto et al., 2004). Implicit in most discussions of B-cell signaling is the notion that signal transduction takes place in a stepwise fashion. That is, signaling elements can be more or less organized into a sequence, such that activation of any particular downstream element of the network depends primarily upon prior activation of an element or elements immediately upstream, surpassing some threshold. However, BCR signaling is also transient in nature. This arises because active signaling elements simultaneously transmit both positive and negative signals back to upstream elements as well as positive and negative signals to downstream elements.
It has been shown theoretically that while activation of downstream elements in such signaling networks generally depend upon activated upstream elements achieving threshold values, activity of downstream elements can also be modulated by the rate of change of activation in upstream elements (Ozaki et al., 2005). A central element controlling rate sensitivity in B cells is likely to be Syk. Although the functional consequences have perhaps not been fully appreciated, Syk has been previously determined to simultaneously facilitate aspects of both positive and negative signaling. Syk from (BCR) activated B cells is phosphorylated principally on six tyrosines. Tyrosine 130 is located between the tandem SH2 domains, but its function is not well defined. Tyrosines 519 and 520 are in the catalytic domain and are considered positive regulators of BCR signaling as their phosphorylation directly upregulates the enzymatic activity of the kinase (Keshvara et al., 1998). The phosphorylation of Tyr342 and Tyr346 in the Syk linker region is also considered to be positively associated with BCR signaling, as it has been shown that phosphorylation of these residues provides a binding site for PLC, another important mediator of BCR signaling (Hong et al., 2002; Law et al., 1996). On the other hand, Tyr317 also in the Syk linker region is functionally unique from the other tyrosines. As opposed to the others, phosphorylation of Tyr317 strongly dampens downstream BCR signaling (Hong et al., 2002).
Figure 6 demonstrates that BCR-induced phosphorylation of PLC is unaffected by Hg+2, as PLC appears maximally phosphorylated by 30 s after initiation of BCR signaling, whether or not the cells have been exposed to Hg+2. In contrast, after BCR stimulation, binding of PLC to Syk is accelerated by Hg+2. This is consistent with accelerated phosphorylation of Tyr342 and Tyr346 in the presence of Hg+2, as suggested by Figures 4 and 5. It is also consistent with an Hg+2-induced acceleration of signaling through the PLC pathway, at least to the extent that signaling through PLC is dependent on PLC binding to Syk. We should point out though, that while these results do not exclude a role of Syk in the phosphorylation and activation of PLC, they suggest that phosphorylation of PLC is independent of PLC binding to Tyr342 and Tyr346.
Accelerated phosphorylation of Syk during BCR signaling would be expected to alter negative as well as positive signals dependent on Syk, possibly altering the relative kinetics between the two. It is still not clear as to why Hg+2 should lead to accelerated phosphorylation of Syk during BCR signaling, but because it is an intracellular protein, it is unlikely that Hg+2 interacts directly with Syk. It is much more likely that in this instance Hg+2 directly interacts with an upstream member of the BCR signaling cascade, perhaps CD45, a transmembrane protein known to control BCR signal strength (Dal Porto et al., 2004).
It has been previously reported that in T lymphocytes, Hg+2 can directly aggregate multiple membrane receptors, leading to very high levels of activation of several intracellular tyrosine kinases (Nakashima et al., 1994). However, these experiments utilized very high (and toxic) levels of Hg+2 (0.5mM). Under the conditions of low Hg+2 burden utilized in our experiments, we have found no evidence of the massive tyrosine kinase activation characteristic of higher Hg+2 exposures (results not shown). In this case, Hg+2-induced tyrosine kinase activation is not likely to be a factor in Hg+2 interference with ERK activity after BCR stimulation, although it cannot be ruled out that Hg+2 influences on aggregation of specific membrane proteins, such as CD45 may be involved.
Finally, it cannot be ruled out that depending on cell type and specific exposure regimen, Hg+2 may interact with cellular systems in multiple ways. For instance, it has been reported that in mast cells, Hg+2 upregulated IL-4 gene expression by a mechanism involving oxidative stress-induced transcriptional upregulation (Wu et al., 2001). However, these results do not appear directly relevant to our B-cell experiments, in that higher Hg+2 exposure burdens (2 x 10–5M for 2 h) and longer time scales (2 h) were utilized. It has also been previously shown in T cells that moderate Hg+2 exposure increases intracellular calcium by stimulating influx through calcium channels in the plasma membrane (Badou et al., 1997; Tan et al., 1993). It is possible that in B cells, changes in intracellular calcium could affect Ras (and hence ERK) activity indirectly through an effect on PKC. On the other hand, there is no clear evidence that Syk activity is directly or indirectly affected by alterations in intracellular calcium.
|
FUNDING |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
National Institutes of Health (ES11000, ES12403, ES01247, and AI1007527).
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
Badou A, Savignac M, Moreau M, Leclerc C, Pasquier R, Druet P, Pelletier L. HgCl2-induced interleukin-4 gene expression in T cells involves a protein kinase C-dependent calcium influx through L-type calcium channels. J. Biol. Chem. (1997) 272:32411–32418.
Brickman CM, Shoenfeld Y. The mosaic of autoimmunity. Scand. J. Clin. Lab. Invest. (2001) 235:3–15.
Cappione A III, Anolik JH, Pugh-Bernard A, Barnard J, Dutcher P, Silverman G, Sanz I. Germinal center exclusion of autoreactive B cells is defective in human systemic lupus erythematosus. J. Clin. Invest. (2005) 115:3205–3216.[CrossRef][ISI][Medline]
Chung JB, Silverman M, Monroe JG. Transitional B cells: Step by step towards immune competence. Trends Immunol. (2003) 24:343–349.[ISI][Medline]
Cornall RJ, Cheng AM, Pawson T, Goodnow CC. Role of Syk in B-cell development and antigen-receptor signaling. Proc. Natl Acad. Sci. USA (2000) 97:1713–1718.
Dal Porto JM, Gauld SB, Merrell KT, Mills D, Pugh-Bernard AE, Cambier J. B cell antigen receptor signaling 101. Mol. Immunol. (2004) 41:599–613.[CrossRef][ISI][Medline]
Gabay L, Seger R, Shilo BZ. In situ activation pattern of Drosophila EGF receptor pathway during development. Science (1997) 277:1103–1106.
Grimaldi CM, Cleary J, Dagtas AS, Moussai D, Diamond B. Estrogen alters thresholds for B cell apoptosis and activation. J. Clin. Invest. (2002) 109:1625–1633.[CrossRef][ISI][Medline]
Grimaldi CM, Hicks R, Diamond B. B cell selection and susceptibility to autoimmunity. J. Immunol. (2005) 174:1775–1781.
Hansson M, Djerbi M, Rabbani H, Mellstedt H, Gharibdoost F, Hassan M, Depierre JW, Abedi-Valugerdi M. Exposure to mercuric chloride during the induction phase and after the onset of collagen-induced arthritis enhances immune/autoimmune responses and exacerbates the disease in DBA/1 mice. Immunology (2005) 114:428–437.[CrossRef][ISI][Medline]
Harding A, Tian T, Westbury E, Frische E, Hancock JF. Subcellular localization determines MAP kinase signal output. Curr. Biol. (2005) 15:869–873.[CrossRef][ISI][Medline]
Hong JJ, Yankee TM, Harrison ML, Geahlen RL. Regulation of signaling in B cells through the phosphorylation of Syk on linker region tyrosines. A mechanism for negative signaling by the Lyn tyrosine kinase. J. Biol. Chem. (2002) 277:31703–31714.
Irish JM, Czerwinski DK, Nolan GP, Levy R. Kinetics of B cell receptor signaling in human B cell subsets mapped by phosphospecific flow cytometry. J. Immunol. (2006) 177:1581–1589.
Jacob A, Cooney D, Pradhan M, Coggeshall KM. Convergence of signaling pathways on the activation of ERK in B cells. J. Biol. Chem. (2002) 277:23420–23426.
Keshvara LM, Isaacson CC, Yankee TM, Sarac R, Harrison ML, Geahlen RL. Syk- and Lyn-dependent phosphorylation of Syk on multiple tyrosines following B cell activation includes a site that negatively regulates signaling. J. Immunol. (1998) 161:5276–5283.
Kim YJ, Sekiya F, Poulin B, Bae YS, Rhee SG. Mechanism of B-cell receptor-induced phosphorylation and activation of phospholipase C-gamma2. Mol. Cell. Biol. (2004) 24:9986–9999.
Krutzik PO, Irish JM, Nolan GP, Perez OD. Analysis of protein phosphorylation and cellular signaling events by flow cytometry: Techniques and clinical applications. Clin. Immunol. (2004) 110:206–221.[CrossRef][ISI][Medline]
Law C-L, Chandran KA, Sidorenko SP, Clark EA. Phospholipase C-gamma 1 interacts with conserved phosphorylated residues in the linker region of Syk and is a substrate for Syk. Mol. Cell. Biol. (1996) 16:1305–1315.[Abstract]
Lohr J, Knoechel B, Nagabhushanam V, Abbas AK. T-cell tolerance and autoimmunity to systemic and tissue-restricted self-antigens. Immunol. Rev. (2005) 204:116–127.[CrossRef][ISI][Medline]
Mattingly RR, Felczak A, Chen CC, McCabe MJJ, Rosenspire AJ. Low concentrations of inorganic mercury inhibit Ras activation during T cell receptor-mediated signal transduction. Toxicol. Appl. Pharmacol. (2001a) 176:162–168.[CrossRef][ISI][Medline]
Mattingly RR, Milstein ML, Mirkin BL. Down-regulation of growth factor-stimulated MAP kinase signaling in cytotoxic drug-resistant human neuroblastoma cells. Cell. Signal. (2001b) 13:499–505.[CrossRef][ISI][Medline]
Mayes MD. Epidemiologic studies of environmental agents and systemic autoimmune diseases. Environ. Health Perspect. (1999) 107(Suppl. 5):743–748.[CrossRef][ISI][Medline]
McCabe MJ, Santini RP, Rosenspire AJ. Low and non-toxic levels of ionic mercury interfere with the regulation of cell growth in the WEHI-231 B cell lymphoma. Scand. J. Immunol. (1999) 50:233–241.[CrossRef][ISI][Medline]
Nakashima I, Pu M-Y, Nishizaki A, Rosila I, Ma L, Katano Y, Ohkusu K, Rahman SMJ, Isobe K, Hamaguchi M, et al. Redox mechanism as alternative to ligand binding for receptor activation delivering disregulated cellular signals. J. Immunol. (1994) 152:1064–1071.[Abstract]
Oehme FW. British anti-lewisite (BAL), the classic heavy metal antidote. Clin. Toxicol. (1972) 5:215–222.[ISI][Medline]
Ozaki Y, Sasagawa S, Kuroda S. Dynamic characteristics of transient responses. J. Biochem. (2005) 137:659–663.
Perl A. Pathogenesis and spectrum of autoimmunity. Methods Mol. Med. (2004) 102:1–8.[Medline]
Pollard KM, Pearson DL, Hultman P, Deane TN, Lindh U, Kono DH. Xenobiotic acceleration of idiopathic systemic autoimmunity in lupus-prone bxsb mice. Environ. Health Perspect. (2001) 109:27–33.[ISI][Medline]
Pollard KM, Pearson DL, Hultman P, Hildebrandt B, Kono DH. Lupus-prone mice as models to study xenobiotic-induced acceleration of systemic autoimmunity. Environ. Health Perspect. (1999) 107:729–735.[CrossRef][ISI][Medline]
Pouyssegur J, Volmat V, Lenormand P. Fidelity and spatio-temporal control in MAP kinase (ERKs) signalling. Biochem. Pharmacol. (2002) 64:755–763.[CrossRef][ISI][Medline]
Reth M. Hydrogen peroxide as second messenger in lymphocyte activation. Nat. Immunol. (2002) 3:1129–1134.[CrossRef][ISI][Medline]
Rowley B, Monestier M. Mechanisms of heavy metal-induced autoimmunity. Mol. Immunol. (2005) 42:833–838.[CrossRef][ISI][Medline]
Silbergeld EK, Silva IA, Nyland JF. Mercury and autoimmunity: Implications for occupational and environmental health. Toxicol. Appl. Pharmacol. (2005) 207:282–292.[CrossRef][Medline]
Tan XX, Tang C, Castoldi AF, Manzo L, Costa LG. Effects of inorganic and organic mercury on intracellular calcium levels in rat T lymphocytes. J. Toxicol. Environ. Health (1993) 38:159–170.[ISI][Medline]
Via CS, Nguyen P, Niculescu F, Papadimitriou J, Hoover D, Silbergeld EK. Low-dose exposure to inorganic mercury accelerates disease and mortality in acquired murine lupus. Environ. Health Perspect. (2003) 111:1273–1277.[ISI][Medline]
Wang C, Khalil M, Ravetch J, Diamond B. The naive B cell repertoire predisposes to antigen-induced systemic lupus erythematosus. J. Immunol. (2003) 170:4826–4832.
Wu Z, Turner DR, Oliveira DB. IL-4 gene expression up-regulated by mercury in rat mast cels: A role of oxidant stress in IL-4 transcription. Int. Immunol. (2001) 13:297–304.
Ziemba SE, Mattingly RR, McCabe MJ Jr, Rosenspire AJ. Inorganic mercury inhibits the activation of LAT in T-cell receptor-mediated signal transduction. Toxicol. Sci. (2006) 89:145–153.
CiteULike 
Connotea 
Del.icio.us What's this?