RESUMO
Exposure to environmental heavy metals has been reported to affect the immune system. Here, we tested the hypothesis that Hg(+2), acting through membrane proteins, disrupts metabolic dynamics and downstream cell functions in human neutrophils. We found that HgCl(2) inhibited: (1) polarization and (2) immunoglobulin (Ig)G-mediated phagocytosis of sheep erythrocytes in a dose-dependent manner from 2.5 to 10 microM. Because these activities have been linked with pro-inflammatory signalling, we also studied the effects of HgCl(2) on intracellular signalling by measuring protein tyrosine phosphorylation. HgCl(2) at doses = 1 microM increased tyrosine phosphorylation. We also studied the effect of HgCl(2) on neutrophil metabolism by measuring NAD(P)H autofluorescence as an indicator of intracellular NAD(P)H concentration. After HgCl(2) treatment, we found that normal sinusoidal NAD(P)H oscillations became incoherent. We recently reported that the NAD(P)H oscillation frequency is affected by cell migration and activation, which can in turn be regulated by integrin-mediated signalling. Therefore, we examined the effects of HgCl(2) on cell surface distribution of membrane proteins. After exposure to environmentally relevant concentrations of HgCl(2) we found that CR3, but not other membrane proteins (e.g. uPAR, Fc gamma RIIA and the formyl peptide receptor), became clustered on cell surfaces. We suggest that HgCl2 disrupts integrin signalling/functional pathways in neutrophils.
Assuntos
Cloreto de Mercúrio/farmacologia , Neutrófilos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Relógios Biológicos/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Depressão Química , Relação Dose-Resposta Imunológica , Eritrócitos , Humanos , Imunoglobulina G/imunologia , Integrinas/efeitos dos fármacos , Proteínas de Membrana/efeitos dos fármacos , Cloreto de Mercúrio/toxicidade , Modelos Biológicos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , NADP/metabolismo , Neutrófilos/metabolismo , Fagocitose/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Agregação de Receptores/efeitos dos fármacos , Receptores de IgG/efeitos dos fármacos , OvinosRESUMO
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.
