Michael J. McCabe

Immunomodulation by metals

Occupational or environmental exposure to metals is believed to affect human health adversely. One mechanism whereby metals can alter health is through modulation of immune homeostasis. Imbalances in immune regulation by metals can lead to inadequate or excessive production of inflammatory cytokines. Alternatively, metals can lead to inappropriate activation of lymphoid subsets involved in acquired immunity to specific antigens. Some resultant pathologies may include chronic inflammatory processes and autoimmune diseases. Metals may change the response repertoire by direct and indirect means by influencing expression of new antigens, new peptides, and/or antigen presentation by modifying the antigen-presenting complex. The differences in metal-induced immune responses between humans and the mechanisms of metal immunomodulation are discussed.

p53 Suppression of Arsenite-Induced Mitotic Catastrophe Is Mediated by p21CIP1/WAF1

Arsenic trioxide, an acute promyelocytic leukemia chemotherapeutic, may be an efficacious treatment for other cancers. Understanding the mechanism as well as genetic and molecular characteristics associated with sensitivity to arsenite-induced cell death is key to providing effective chemotherapeutic usage of arsenite. Arsenite sensitivity correlates with deficient p53 pathways in multiple cell lines. The role of p53 in preventing arsenite-induced mitotic arrest-associated apoptosis (MAAA), a form of mitotic catastrophe, was examined in TR9-7 cells, a model cell line with p53 exogenously regulated in a tetracycline-off expression system. Arsenite activated G1 and G2 cell cycle checkpoints independently of p53, but mitotic catastrophe occurred preferentially in p53(-) cells. Cyclin B/CDC2(CDK1) stabilization and caspase-3 activation persisted in arsenite-treated p53(-) cells consistent with MAAA/mitotic catastrophe. N-Benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a pan-caspase inhibitor, completely abolished arsenite-induced MAAA/mitotic catastrophe and greatly increased the mitotic index. WEE1 and p21CIP1/WAF1 inhibit cyclin B/CDC2 by CDC2 tyrosine-15 phosphorylation and direct binding, respectively. CDC2-Y15-P was transiently elevated in arsenite-treated p53(+) cells but persisted in p53(-) cells. Arsenite induced p53-S15-P and p21CIP1/WAF1 only in p53(+) cells. P21CIP1/WAF1-siRNA-treated p53(+) cells were similar to p53(-) cells in mitotic index and cell cycle protein levels. p53-inducible proteins GADD45α and 14-3-3σ are capable of inhibiting cyclin B/CDC2 but did not play a p53-dependent role in mitotic escape in TR9-7 cells. The data indicate that p53 mediates cyclin B/CDC2 inactivation and mitotic release directly via p21CIP1/WAF1 induction.

Blood Lead Level and Risk of Asthma

Asthma and lead poisoning are prevalent among urban children in the United States. Lead exposure may be associated with excessive production of immunoglobulin E, possibly increasing asthma risk and contributing to racial disparities. The objective of this study was to examine racial differences in the association of blood lead level (BLL) to risk of developing asthma. We established and followed a cohort prospectively to determine asthma onset, using patient encounters and drug claims obtained from hospital databases. Participants were managed care enrollees with BLL measured and documented at 1–3 years of age. We used multiple variable analysis techniques to determine the relationship of BLL to period prevalent and incident asthma. Of the 4,634 children screened for lead from 1995 through 1998, 69.5% were African American, 50.5% were male, and mean age was 1.2 years. Among African Americans, BLL ≥5 and BLL ≥10 μg/dL were not associated with asthma. The association of BLL ≥5 μg/dL with asthma among Caucasians was slightly elevated, but not significant [adjusted hazard ratio (adjHR) = 1.4; 95% confidence interval (CI), 0.7–2.9; p = 0.40]. Despite the small number of Caucasians with high BLL, the adjHR increased to 2.7 (95% CI, 0.9–8.1; p = 0.09) when more stringent criteria for asthma were used. When compared with Caucasians with BLL < 5 μg/dL, African Americans were at a significantly increased risk of asthma regardless of BLL (adjHR = 1.4–3.0). We conclude that an effect of BLL on risk of asthma for African Americans was not observed. These results demonstrate the need for further exploration of the complex interrelationships between race, asthma phenotype, genetic susceptibilities, and socioenvironmental exposures, including lead.

Sensitivity to Sodium Arsenite in Human Melanoma Cells Depends upon Susceptibility to Arsenite-Induced Mitotic Arrest

Arsenic induces clinical remission in patients with acute promyelocytic leukemia and has potential for treatment of other cancers. The current study examines factors influencing sensitivity to arsenic using human malignant melanoma cell lines. A375 and SK-Mel-2 cells were sensitive to clinically achievable concentrations of arsenite, whereas SK-Mel-3 and SK-Mel-28 cells required supratherapeutic levels for toxicity. Inhibition of glutathione synthesis, glutathione S-transferase (GST) activity, and multidrug resistance protein (MRP) transporter function attenuated arsenite resistance, consistent with studies suggesting that arsenite is extruded from the cell as a glutathione conjugate by MRP-1. However, MRP-1 was not overexpressed in resistant lines and GST-pi was only slightly elevated. ICP-MS analysis indicated that arsenite-resistant SK-Mel-28 cells did not accumulate less arsenic than arsenite-sensitive A375 cells, suggesting that resistance was not attributable to reduced arsenic accumulation but rather to intrinsic properties of resistant cell lines. The mode of arsenite-induced cell death was apoptosis. Arsenite-induced apoptosis is associated with cell cycle alterations. Cell cycle analysis revealed arsenite-sensitive cells arrested in mitosis whereas arsenite-resistant cells did not, suggesting that induction of mitotic arrest occurs at lower intracellular arsenic concentrations. Higher intracellular arsenic levels induced cell cycle arrest in the S-phase and G(2)-phase in SK-Mel-3 and SK-Mel-28 cells, respectively. The lack of arsenite-induced mitotic arrest in resistant cell lines was associated with a weakened spindle checkpoint resulting from reduced expression of spindle checkpoint protein BUBR1. These data suggest that arsenite has potential for treatment of solid tumors but a functional spindle checkpoint is a prerequisite for a positive response to its clinical application.

Inorganic mercury attenuates CD95-mediated apoptosis by interfering with formation of the death inducing signaling complex.

Inorganic mercury (Hg2+) modulates several lymphocyte signaling pathways and has been implicated as an environmental factor linked to autoimmune disease. From the standpoint that autoimmune diseases represent disorders of cell accumulation, in which dysregulated apoptosis may be one mechanism leading to the accumulation of autoreactive lymphocytes, we have been investigating the influences of Hg2+ on CD95-mediated apoptosis. We demonstrate here that low and noncytotoxic concentrations of Hg2+ impair CD95 agonist-induced apoptosis in representative Type-I and Type-II T cell lines. Hg2+ treatment blocks the CD95 agonist-induced activation of initiator and effector caspases as well as the association between CD95 and the signaling adaptor, FADD. CD95 multimerization does not appear to be affected by Hg2+. Thus, the Hg2+ sensitive step within the CD95 death pathway is localized to the level of the death inducing signaling complex (DISC). Disruption of proper DISC formation may be a biochemical mechanism whereby Hg2+ contributes to autoimmune disease.

Gene-environment interactions between CD14 C-260T and endotoxin exposure on Foxp3+ and Foxp3- CD4+ lymphocyte numbers and total serum IgE levels in early childhood

BACKGROUND Innate immune system stimuli, such as endotoxin, seem to affect allergy risk. Previously, we described gene-environment interactions between the endotoxin receptor polymorphism C-260T of the CD14 gene and endotoxin exposure on total serum IgE level; however, the mechanism of this interaction is not known. OBJECTIVE To examine whether this gene-environment interaction affects early CD4(+)Foxp3(-) or CD4(+)Foxp3(+) lymphocyte numbers. METHODS Participating children were part of a birth cohort in the Detroit metropolitan area. Participants were genotyped for the CD14 C-260T polymorphism. Endotoxin exposure was estimated from dust measured in the home when children were 6 months old. Intracellular Foxp3 protein expression, a regulatory T-cell marker, was used to characterize CD4(+) lymphocytes in blood samples collected at the age of 12 months; total serum IgE level was also measured at this time. Because race/ethnicity may confound or modify genetic associations, all analyses were stratified by race/ethnicity. RESULTS We observed a significant gene-environment interaction between CD14 C-260T genotype and endotoxin exposure on CD4(+) lymphocyte numbers, particularly CD4(+)Foxp3(-) lymphocytes. Stratified analyses suggest effect modification by race/ ethnicity on CD4(+)Foxp3(+) lymphocyte numbers, with a significant interaction in African American children but not in white children. The interaction between CD14 C-260T genotype and endotoxin exposure on total IgE levels was opposite that observed for CD4(+) lymphocyte numbers, suggesting reciprocal relationships. CONCLUSIONS A gene-environment interaction between endotoxin and CD14 C-260T genotype on IgE levels may be the result of an upstream, opposing effect on CD4(+)Foxp3(+) and CD4(+)Foxp3(-) lymphocyte numbers. Race/ethnicity may affect which of these cell populations is affected by this gene-environment interaction.

Exit from arsenite-induced mitotic arrest is p53 dependent.

Background Arsenic is both a human carcinogen and a chemotherapeutic agent, but the mechanism of neither arsenic-induced carcinogenesis nor tumor selective cytotoxicity is clear. Using a model cell line in which p53 expression is regulated exogenously in a tetracycline-off system (TR9-7 cells), our laboratory has shown that arsenite disrupts mitosis and that p53-deficient cells [p53(−)], in contrast to p53-expressing cells [p53(+)], display greater sensitivity to arsenite-induced mitotic arrest and apoptosis. Objective Our goal was to examine the role p53 plays in protecting cells from arsenite-induced mitotic arrest. Methods p53(+) and p53(−) cells were synchronized in G2 phase using Hoechst 33342 and released from synchrony in the presence or absence of 5 μM sodium arsenite. Results Mitotic index analysis demonstrated that arsenite treatment delayed exit from G2 in p53(+) and p53(−) cells. Arsenite-treated p53(+) cells exited mitosis normally, whereas p53(−) cells exited mitosis with delayed kinetics. Microarray analysis performed on mRNAs of cells exposed to arsenite for 0 and 3 hr after release from G2 phase synchrony showed that arsenite induced inhibitor of DNA binding-1 (ID1) differentially in p53(+)and p53(−) cells. Immunoblotting con-firmed that ID1 induction was more extensive and sustained in p53(+) cells. Conclusions p53 promotes mitotic exit and leads to more extensive ID1 induction by arsenite. ID1 is a dominant negative inhibitor of transcription that represses cell cycle regulatory genes and is elevated in many tumors. ID1 may play a role in the survival of arsenite-treated p53(+) cells and contribute to arsenic carcinogenicity.

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 μm) 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 μm 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.

T-cell receptor signaling is mediated by transient Lck activity, which is inhibited by inorganic mercury

Genetically susceptible rodents exposed to low nontoxic levels of inorganic mercury (Hg2+) develop idiosyncratic autoimmune disease associated with defective T‐cell function. However, the molecular mechanisms underlying this phenomenon remain mostly unexplained. Brief exposure of T cells to micromolar concentrations of Hg2+ leads to physiologically relevant nontoxic cellular mercury burdens, and as we have previously reported, attenuates T‐cell receptor (TCR) signal strength by ~50%. We have found this to be the result of an inadequate activation of the tyrosine kinase ZAP‐70, which is hypophosphorylated following TCR stimulation in Hg2+ burdened cells when compared to untreated controls. In T cells,ZAP‐70 phosphorylation is dependent on lymphocyte‐specific protein tyrosine kinase (Lck) activity, which in turn is either positively or negatively regulated by the phosphorylation of specific Lck tyrosine residues. In particular, the general belief is that Lck is negatively regulated by phosphorylation of tyrosine 192 (Y192). We now demonstrate by Western blotting that, in Jurkat T cells, TCR signal transduction (andZAP‐70 phosphorylation) was positively associated with a rapid transient phosphorylation of Y192, which was inhibited in cells that were briefly (5 min) exposed to 5 μM Hg2+. Thus, Hg2+ inhibits a critical activating role played by Lck Y192 during the most proximal events of the TCR‐induced cell signaling.—Ziemba, S.E., Menard, S.L., McCabe, Jr., M.J., Rosenspire, A.J. T‐cell receptor signaling is mediated by transient Lck activity, which is inhibited by inorganic mercury. FASEB J. 23, 1663–1671 (2009)

Reduction of myeloid suppressor cell derived nitric oxide provides a mechanistic basis of lead enhancement of alloreactive CD4+ T cell proliferation

The persistent environmental toxicant and immunomodulator, lead (Pb), has been proposed to directly target CD4(+) T cells. However, our studies suggest that CD4(+) T cells are an important functional, yet indirect target. In order to identify the direct target of Pb in the immune system and the potential mechanism of Pb-induced immunotoxicity, myeloid suppressor cells (MSCs) were evaluated for their ability to modulate CD4(+) T cell proliferation after Pb exposure. Myeloid suppressor cells regulate the adaptive immune response, in part, by inhibiting the proliferation of CD4(+) T cells. It is thought that the mechanism of MSC-dependent regulation involves the release of the bioactive gas, nitric oxide (NO), blocking cell signaling cascades downstream of the IL-2 receptor and thus preventing T cells from entering cell-cycle. In mixed lymphocyte culture (MLC), increasing numbers of MSCs suppressed T cell proliferation in a dose-dependent manner, and this suppression is strikingly abrogated with 5 microM lead (Pb) treatment. The Pb-sensitive MSC population is CD11b(+), GR1(+)and CD11c(-) and thus phenotypically consistent with MSCs described in other literature. Inhibition of NO-synthase (NOS), the enzyme responsible for the production of NO, enhanced alloreactive T cell proliferation in MLC. Moreover, Pb attenuated NO production in MLC, and exogenous replacement of NO restored suppression in the presence of Pb. Significantly, MSC from iNOS-/- mice were unable to suppress T cell proliferation. An MSC-derived cell line (MSC-1) also suppressed T cell proliferation in MLC, and Pb disrupted this suppression by attenuating NO production. Additionally, Pb disrupted NO production in MSC-1 cells in response to treatment with interferon-gamma (IFN-gamma) and LPS or in response to concanavalin A-stimulated splenocytes. However, neither the abundance of protein nor levels of mRNA for the inducible isoform of NOS (iNOS) were altered with Pb treatment. Taken together these data suggest that Pb abrogates an MSC-dependent suppression of alloreactive T cell proliferation by inhibiting the function, but not the expression of iNOS.