Mercury intoxication disrupts tonic signaling in B cells, and may promote autoimmunity due to abnormal phosphorylation of STIM-1 and other autoimmunity risk associated phosphoproteins involved in BCR signaling.

Epidemiological studies link exposure to mercury with autoimmune disease. Unfortunately, in spite of considerable effort, no generally accepted mechanistic understanding of how mercury functions with respect to the etiology of autoimmune disease is currently available. Nevertheless, autoimmune disease often arises because of defective B cell signaling. Because B cell signaling is dependent on phosphorylation cascades, in this report, we have focused on how mercury intoxication alters phosphorylation of B cell proteins in antigen-non stimulated (tonic) mouse (BALB/c) splenic B cells. Specifically, we utilized mass spectrometric techniques to conduct a comprehensive unbiased global analysis of the effect of inorganic mercury (Hg2+) on the entire B cell phosphoproteome. We found that the effects were pleotropic in the sense that large numbers of pathways were impacted. However, confirming our earlier work, we found that the B cell signaling pathway stood out from the rest, in that phosphoproteins which had sites which were affected by Hg2+, exhibited a much higher degree of connectivity, than components of other pathways. Further analysis showed that many of these BCR pathway proteins had been previously linked to autoimmune disease. Finally, dose response analysis of these BCR pathway proteins showed STIM1_S575, and NFAT2_S259 are the two most Hg2+ sensitive of these sites. Because STIM1_S575 controls the ability of STIM1 to regulate internal Ca2+, we speculate that STIM1 may be the initial point of disruption, where Hg2+ interferes with B cell signaling leading to systemic autoimmunity, with the molecular effects pleiotropically propagated throughout the cell by virtue of Ca2+ dysregulation.

Low level exposure to inorganic mercury interferes with B cell receptor signaling in transitional type 1 B cells.

Mercury (Hg) has been implicated as a factor contributing to autoimmune disease in animal models and humans. However the mechanism by which this occurs has remained elusive. Since the discovery of B cells it has been appreciated by immunologists that during the normal course of B cell development, some immature B cells must be generated that produce immunoglobulin reactive to self-antigens (auto-antibodies). However in the course of normal development, the vast majority of immature auto-reactive B cells are prevented from maturing by processes collectively known as tolerance. Autoimmune disease arises when these mechanisms of tolerance are disrupted. In the B cell compartment, it is firmly established that tolerance depends in part upon negative selection of self-reactive immature (transitional type 1) B cells. In these cells negative selection depends upon signals generated by the B Cell Receptor (BCR), in the sense that those T1 B cells who's BCRs most strongly bind to, and so generate the strongest signals to self-antigens are neutralized. In this report we have utilized multicolor phosphoflow cytometry to show that in immature T1 B cells Hg attenuates signal generation by the BCR through mechanisms that may involve Lyn, a key tyrosine kinase in the BCR signal transduction pathway. We suggest that exposure to low, environmentally relevant levels of Hg, disrupts tolerance by interfering with BCR signaling in immature B cells, potentially leading to the appearance of mature auto-reactive B cells which have the ability to contribute to auto-immune disease.

Low concentrations of inorganic mercury inhibit Ras activation during T cell receptor-mediated signal transduction.

Mercury is widespread in the environment and consequently there are large populations that are currently exposed to low levels of mercury as a result of ubiquitous environmental factors. Whether these environmental levels of mercury are harmful is a matter of current debate, with epidemiological and animal studies suggesting detrimental effects on the immune and nervous systems. However, specific cellular effects of low concentrations of mercury have been hard to characterize. We now demonstrate that subtoxic concentrations of HgCl(2) can potently (maximal at 1 microM) increase Ras.GTP levels in Jurkat, a human T cell line. Remarkably, this activation of Ras occurs without a concomitant increase in MAP kinase activation, suggesting that mercury may direct Ras into a nonproductive state. In addition to its direct effect on Ras, concentrations of HgCl(2) as low as 0.6 microM inhibited the ability of the T cell receptor to activate Ras and MAP kinase. The inhibitory effect of mercury is selective, as activation of MAP kinase by phorbol diesters remain intact. Since the Ras/MAP kinase pathway is both highly conserved and central to signal transduction processes mediated by a myriad of diverse membrane receptor systems in a variety of cell types, these results suggest a mechanism for adverse health effects resulting from exposure to low levels of mercury. They also support a model for regulation of the Ras/MAP kinase pathway, whereby partial but unproductive activation of Ras can diminish signaling from cell surface receptors.

Pulsed DC electric fields couple to natural NAD(P)H oscillations in HT-1080 fibrosarcoma cells.

Previously, we have demonstrated that NAD(P)H levels in neutrophils and macrophages are oscillatory. We have also found that weak ultra low frequency AC or pulsed DC electric fields can resonate with, and increase the amplitude of, NAD(P)H oscillations in these cells. For these cells, increased NAD(P)H amplitudes directly signal changes in behavior in the absence of cytokines or chemotactic factors. Here, we have studied the effect of pulsed DC electric fields on HT-1080 fibrosarcoma cells. As in neutrophils and macrophages, NAD(P)H levels oscillate. We find that weak (approximately 10(-5) V/m), but properly phased DC (pulsed) electric fields, resonate with NAD(P)H oscillations in polarized and migratory, but not spherical, HT-1080 cells. In this instance, electric field resonance signals an increase in HT-1080 pericellular proteolytic activity. Electric field resonance also triggers an immediate increase in the production of reactive oxygen metabolites. Under resonance conditions, we find evidence of DNA damage in HT-1080 cells in as little as 5 minutes. Thus the ability of external electric fields to effect cell function and physiology by acting on NAD(P)H oscillations is not restricted to cells of the hematopoietic lineage, but may be a universal property of many, if not all polarized and migratory eukaryotic cells.

Differential screening and mass mapping of proteins from premalignant and cancer cell lines using nonporous reversed-phase HPLC coupled with mass spectrometric analysis.

Nonporous (NPS) RP-HPLC has been used to rapidly separate proteins from whole cell lysates of human breast cell lines. The nonporous separation involves the use of hard-sphere silica beads of 1.5-microm diameter coated with C18, which can be used to separate proteins ranging from 5 to 90 kDa. Using only 30-40 microg of total protein, the protein molecular weights are detectable on-line using an ESI-oaTOF MS. Of hundreds of proteins detected in this mass range, approxinately 75-80 are more highly expressed. The molecular weight profiles can be displayed as a mass map analogous to a virtual "1-D gel" and differentially expressed proteins can be compared by image analysis. The separated proteins can also be detected by UV absorption and differentially expressed proteins quantified. The eluting proteins can be collected in the liquid phase and the molecular weight and peptide maps determined by MALDI-TOF MS for identification. It is demonstrated that the expressed protein profiles change during neoplastic progression and that many oncoproteins are readily detected. It is also shown that the response of premalignant cancer cells to estradiol can be rapidly screened by this method, demonstrating significant changes in response to an external agent. Ultimately, the proteins can be studied by peptide mapping to search for posttranslational modifications of the oncoproteins accompanying progression.

Interferon-gamma and sinusoidal electric fields signal by modulating NAD(P)H oscillations in polarized neutrophils.

Metabolic activity in eukaryotic cells is known to naturally oscillate. We have recently observed a 20-s period NAD(P)H oscillation in neutrophils and other polarized cells. Here we show that when polarized human neutrophils are exposed to interferon-gamma or to ultra-low-frequency electric fields with periods double that of the NAD(P)H oscillation, the amplitude of the NAD(P)H oscillations increases. Furthermore, increases in NAD(P)H amplitude, whether mediated by interferon-gamma or by an oscillating electric field, signals increased production of reactive oxygen metabolites. Hence, amplitude modulation of NAD(P)H oscillations suggests a novel signaling mechanism in polarized cells.

Mercuric chloride damages cellular DNA by a non-apoptotic mechanism.

Mercury is a xenobiotic metal that is well known to adversely affect the immune system, however, little is known as to the molecular mechanism. Recently, it has been suggested that mercury may induce immune dysfunction by triggering apoptosis in immune cells. Here, we studied the effects of Hg(2+) (HgCl(2)) on U-937 cells, a human cell line with monocytic characteristics. We found that these cells continued to proliferate when exposed to low doses of mercury between 1 and 5 microM. Using the single cell gel electrophoresis (SCGE) or 'comet' assay, we found that mercury damaged DNA at these levels. Between 1 and 50 microM Hg(2+), comet formation was concentration-dependent with the greatest number of comets formed at 5 microM mercury. However, the appearance of mercury-induced comets was qualitatively different from those of control cells treated with anti-fas antibody, suggesting that although mercury might damage DNA, apoptosis was not involved. This was confirmed by the finding that cells treated with 5 microM mercury were negative for annexin-V binding, an independent assay for apoptosis. These data support the notion that DNA damage in surviving cells is a more sensitive indicator of environmental insult than is apoptosis, and suggests that low-concentrations of ionic mercury may be mutagenic.

Rapid screening of protein profiles of human breast cancer cell lines using non-porous reversed-phase high performance liquid chromatography separation with matrix-assisted laser desorption/ionization time-of-flight mass spectral analysis.

Non-porous reversed-phase (NP-RP) HPLC has been used to rapidly generate protein profiles of whole cell lysates of human breast cancer cell lines. The non-porous packing material used was silica coated with C18, which provided rapid separation with high collection efficiency of proteins from cell lysates. This method was used to study the differences in protein profiles among normal cells and fully malignant cells that share a common genetic background. The highly expressed proteins in each cell type were separated and collected in the liquid state where they were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to obtain the molecular weight of the proteins. The protein fractions were then subjected to tryptic digestion and analyzed by pulsed delay extraction (PDE)-MALDI-TOFMS to obtain the peptide maps. The expressed proteins were identified based upon the molecular weight and peptide map using database-searching procedures. It is shown that key cancer-related proteins can be detected and identified which may be potentially used as biomarkers for cancer detection.

Low and nontoxic levels of ionic mercury interfere with the regulation of cell growth in the WEHI-231 B-cell lymphoma.

WEHI-231 is a mouse B-cell line, which is a well-established model for studying signal transduction in B lymphocytes, normally responding to cross-linking of the B-cell receptor (BCR) complex by the rapid upregulation of protein tyrosine kinase activity, followed by increased intracellular calcium and activation of protein kinase C. In WEHI-231, activation of protein kinase C is functionally associated with downregulation of DNA synthesis, followed by the induction of apoptosis. We have found in WEHI-231, that at low and environmentally relevant exposure levels (0.1 microM) mercury is not toxic, but still interferes with signal transduction in that it attenuates the growth inhibitory effects of BCR cross-linking. The molecular target for mercury resulting in attenuation of the BCR-mediated growth inhibitory signal is likely proximal to activation of the BCR complex, as HgCl2 had no effect on the negative growth signal generated downstream by direct activation of protein kinase C with phorbol 12-myristate 13-acetate. Treatment of WEHI-231 cells with high and toxic concentrations of Hg results in a marked increase in protein tyrosine phosphorylation in a great many proteins; whereas treatment of WEHI-231 cells with 0.1 microM mercury is not toxic. Under these conditions mercury selectively perturbed BCR-mediated protein tyrosine phosphorylation of a 75 kDa protein, without grossly affecting tyrosine phosphorylation levels of most other proteins. These data suggest that low levels of mercury, which are not toxic, may still contribute to immune dysfunction by interfering with antigen-receptor-mediated and protein-kinase-dependent signal transduction in lymphocytes.

Protection against CD95-mediated apoptosis by inorganic mercury in Jurkat T cells.

Dysregulation of CD95/Fas-mediated apoptosis has been implicated as a contributing factor in autoimmune disorders. Animal studies clearly have established a connection between mercury exposure and autoimmune disease in rodents, while case reports have suggested a link between accidental mercury contamination and autoimmune disease in humans. The mechanism(s) for these associations are poorly understood. Using the Jurkat cell model, we have found that low levels (

Modeling the influence of ectodomain affinities on the spatial distribution of membrane receptors.

We have previously shown that the glycosylphosphatidyl-inositol (GPI)-linked urokinase-type plasminogen activator receptor (uPAR) reversibly associates with the integrins complement receptor type 3 (CR3; alphaMbeta2) and CR4 (alphaxbeta2) during leukocyte motility. These receptor-to-receptor interactions could potentially be accounted for by diffusion-controlled reactions or by directed transport phenomena. To address these alternatives, we have used computer simulation techniques. Our results show that a diffusion-controlled interaction between uPAR and CR4 during accumulation at lamellipodia is not physically reasonable. This suggests that a directed transport mechanism participates in establishing uPAR-integrin association.

Low levels of ionic mercury modulate protein tyrosine phosphorylation in lymphocytes.

The ability of ionic mercury to induce protein tyrosine phosphorylation in mouse spleen cells and in the mouse WEHI-231 B-cell lymphoma was investigated. We have confirmed previous studies which showed that exposure to high levels (several hundred microM) of mercury lead to very large increases in the level of protein tyrosine phosphorylation in these cell systems. However we have also demonstrated that low levels (in the order of 0.1 to 1.0 microM) of mercury also significantly upregulate protein tyrosine phosphorylation. Mercury induced protein tyrosine phosphorylation is inhibited by the mercury chelator penicillamine and by pretreating treating target cells with the sulfhydryl blocking reagent N-hydroxymaleimide. These results suggest that exposure to low levels of mercury could potentially interfere with lymphocyte signal transduction and so offer a possible explanation as to how mercury exposure could lead to immune cellular dysfunction. On a molecular level, the results suggest that the site(s) of action with respect to mercury dependent induction of protein tyrosine phosphorylation is likely a free disulfide group or groups located on the outer leaflet of the plasma membrane.

Monte Carlo simulation of ligand-receptor interactions on a cell surface.

In this paper we develop a compartmentalized, discrete simulation model for investigating the spatial distribution and dynamic properties of receptor crosslinking on the surface of a cell. Results generated by the model are compared with some of the major results of existing analytical models, and differences are discussed in relation to differences in the model assumptions. Finally, the model is used to evaluate the dynamic effects of a time-varying non-homogeneous ligand concentration.

CD4-independent inhibition of lymphocyte proliferation mediated by HIV-1 envelope glycoproteins.

The cytopathic effects of HIV-1 produced by direct infection of human T cells do not account for the disproportionate loss of CD4-positive lymphocytes during the course of HIV infection. Previous studies have demonstrated the inhibition of uninfected human T cell activation and proliferation by the HIV-1 envelope glycoproteins, presumably due to gp120-CD4 interactions. To examine the ability of HIV-1 to inhibit T cell proliferation in the absence of both direct infection and gp120-CD4 interactions, we tested the effect of HIV-1 on mouse T cell proliferation. Culture media containing HIV-1 released from infected cells inhibited T lymphocyte proliferation in response to interleukin-2 (IL-2). Studies to explore the mechanism of this inhibition suggested that the decrease in proliferation resulted from interactions between HIV-1 and the mouse cells, but did not involve IL-2/IL-2 receptor interactions. We used monoclonal antibodies to demonstrate that the HIV-1 envelope glycoproteins were required for the inhibition of murine T cell proliferation. Anti-gp120 antibodies completely restored proliferation, indicating that the surface protein gp120 was primarily required for the inhibition of proliferation. However, antibodies directed against the transmembrane protein of HIV-1 (gp41) also partially restored lymphocyte proliferation. The functional significance of the HIV-1 envelope protein epitopes recognized by the monoclonal antibodies is discussed.

G protein mediated signal transduction: membrane immunoglobulin associated phosphoproteins identified in octyl-beta-glucoside lysates of normal B cells.

Several recent studies have shown that the antigen receptors (mIgM and mIgD) on normal B cell surfaces are part of larger multimolecular complexes, as each is noncovalently coupled to at least two other proteins whose phosphorylation is inducible upon G protein activation. However, B cell receptor complexes appear to be extremely labile, so that extraction of membrane immunoglobulin (mIg) from cell lysates in physical association with noncovalently coupled proteins has been shown to be highly dependent on the choice of detergent. Recent progress has to a large extent been dependent upon the introduction of digitonin, a detergent in which B cell receptor complexes are at least partially stable, as an agent to solubilize B cell membranes. Studies presented here demonstrate that if another well known detergent, octyl-beta-D-glucopyranoside (octyl glucoside), is utilized in place of digitonin, mIg receptors are immunoprecipitated from B cell lysates in association with several newly identified proteins, whose phosphorylation is also G protein dependent. In particular we find three to four mIg associated phosphoproteins in the molecular weight range of 47 to 57 kDa, and at least two others at 82 and 112 kDa. Some of these proteins may correspond to polypeptides that have been functionally linked to mIg dependent signal transduction, but have not as yet been shown to be physically coupled to the receptor.

Membrane immunoglobulin: anti-immunoglobulin interactions mediate the phosphorylation of actin associated proteins in the B-lymphocyte.

The binding of membrane immunoglobulin (mIg) by anti-Ig antibodies is known to initiate a mitogenic signal in B lymphocytes. Because in many instances growth control appears to be correlated with phosphokinase activity, as well as with alterations in cytoskeletal architecture, we asked the question whether antibodies binding to mIg would also lead to the specific phosphorylation of lymphocyte actin-associated proteins. Utilizing a myosin affinity technique, we directly examined phosphoproteins that were associated with actin in the chicken B cell. We found that in a few instances the level of phosphorylation was indeed modulated by mIg:anti-Ig interactions. These actin-binding phosphoproteins may be important control elements in the lymphocyte cytoskeleton.

A unique polypeptide on avian antigen-specific suppressor T-cell radioiodinated in situ by antigen-lactoperoxidase conjugates.

Antigen-specific receptors of B- or T-cells were selectively radiolabeled among the spleen cells from either human gammaglobulin immunized normal or bursectomized agammaglobulinemic chickens. Selective in situ radioiodination was accomplished by employing lactoperoxidase (LPO) covalently linked to antigen (Ag). Ag-specific receptors on B- or T-cells were allowed to bind Ag-LPO conjugates via the Ag portion of the conjugates and then to be selectively catalyzed for iodination by the LPO portion of the bound Ag-LPO conjugates. Radioiodinated cells were either processed for autoradiography to detect Ag-binding cells directly under the microscope or solubilized with detergents for protein analysis with two-dimensional (2-D) gel electrophoresis. On a cellular level, Ag-binding B- and T-cells were selectively radiolabeled and clearly visualized via autoradiography. On a molecular level, selectively radiolabeled Ag-specific membrane immunoglobulin of B-cells was demonstrated on 2-D gel autoradiographs. Furthermore, a unique polypeptide of Ag-specific T-cells with a reduced apparent mol. wt of 27 K and an apparent pI of 5.5-5.7 was demonstrated on 2-D gel autoradiograms. The 27 K molecule appears to be a T-cell receptor component itself, or a closely associated molecule.

Disulfide linkages between antigen-binding receptors on chicken B-lymphocytes.

Membrane immunoglobulin receptors on chicken B-cells have been shown to display a heterogeneity with respect to interchain disulfide linkages. One fraction of the surface Ig (sIg) appears to display the traditional H2-L2 linkage. We also present evidence that this Ig is covalently bound via a disulfide linkage to actin. In this instance, the isolated Ig heavy chain, after reduction, has a mol. wt of 80 K. Perhaps more significantly, we show that another fraction of the sIg exists in a highly aggregated from that is stabilized by disulfide linkages. In contrast to the sIg found in the H2-L2 configuration, there is no evidence of actin within the aggregates and the sIg heavy chains isolated from these aggregates display a slightly faster mobility on SDS-PAGE under reducing conditions, running at about 77K. Furthermore, it appears that the Ig within the large aggregates may have a higher avidity with respect to antigen binding, and so this Ig structure may be the more relevant to antigen-induced receptor-mediated signaling in the B-cell.

A novel method for radiolabeling antigen-binding receptors of lymphocytes.

Antigen-binding receptor (ABR) molecules have been selectively radiolabeled and isolated from immunized chicken spleen cells. The specific radiolabeling of the receptors has been accomplished by utilizing a novel technique employing lactoperoxidase (LPO) covalently linked to antigen (Ag) for which human gammaglobulin was used. The cell surface ABRs were first bound to the Ag-LPO conjugates through specific recognition sites on the Ag portion of the conjugates. The bound LPO portions were then allowed to catalyze the radioiodination of the ABRs. After radiolabeling, cells were solubilized with detergents. ABRs still bound to Ag-LPO conjugates were directly isolated from the lysates via immunoaffinity chromatography utilizing an immunoaffinity reagent directed toward the antigen portion of the ABR-Ag-LPO complex. The radioactive materials isolated in this way were then analyzed via SDS-PAGE under reducing conditions. It appears that as expected, most of the specifically-labeled and isolated materials were immunoglobulin (Ig). Both the membrane-bound form of the heavy chain (mol. wt 77 K) as well as the secreted form (mol. wt 67 K) were detected, along with the light chain (mol. wt 25 K). An additional polypeptide of mol. wt 55 K was also selectively labeled and isolated along with the Ig. This may be a molecule closely associated with the membrane immunoglobulin on B-cell surface.

Binding of bacterial lipopolysaccharide to murine lymphocytes.

Does LPS activate lymphocytes by binding to a specific cell-surface receptor or by nonspecific hydrophobic interaction with the plasma membrane? We examined this question by detecting cell-bound LPS using immunofluorescence microscopy and radiobinding techniques. LPS binding to splenic lymphocytes from C3H/St mice has characteristics of specific binding: saturability with respect to dose and time, selectivity for a subclass of B-cells, and a correlation between binding and mitogenesis. 125I-labeled LPS bound to cells and analyzed quantitatively by SDS-PAGE separated into 3 major components: peaks 1, 2, and 3 (1 equals the fastest moving). Lymphocytes preferentially bound peak 1, murine RBC peaks 1 and 2, and macrophages peak 2. In contrast, specific antibody preferred peaks 2 and 3. Differential staining of gels suggested that peak 3 is carbohydrate-rich and peak 1 is lipid-rich. LPS was released from these cells at different rates. We conclude that selectivity of LPS binding may be reflected in preferential binding of LPS subunits of different size and/or composition, as well as differential retention of bound LPS.