The effect of heavy metals on the immune system at low concentrations.

The present study describes the effect of cadmium on lymphokines that cannot be directly traced to an allergen, or antigen in order to be able to explain various immunological processes. Exposure to various environmental pollutants is known to induce epithelial and inflammatory changes, characterized by a release of cytokines and other soluble mediators. Heavy metals like CdCl2 can induce, or inhibit the synthesis and expression of the inflammatory cytokines IL-1beta, IL-4, IL-6, TNF-alpha, IFN-gamma and ICAM-1. Normal human peripherial blood mononuclear cells (PBMCs) were exposed for different periods of times (1 to 96 h) to 0, 5, 25 and 50 micromoles CdCl2, and mRNA for the above cytokines was quantified by RT-PCR. Highly purified blood B cells and PBMCs from healthy donors were stimulated with IL-4 and aCD40 mAb and incubated with non-toxic concentrations of cadmium chloride (0,1-10 micromol). Levels of IgG and IgE were measured in the supernatants. Proliferation and expression of surface markers were determined by measuring [3H]-thymidine incorporation and by flow cytometry. The study showed that the in vitro synthesis of IgE by purified B cells or PBMCs stimulated with IL-4/aCD40 is inhibited by Cd at doses as low as 0,1 microM. Cd was found to inhibit IL-4/aCD40 induced proliferation of purified B cells and PBMCs in a dose dependent fashion. Most strikingly, only IgE but not IgG synthesis of purified B cells was inhibited by Cd. These data suggest that inhibition of IgE synthesis in human B lymphocytes by Cd seems to be a selective effect on immunoglobulin synthesis.

In vitro cytokine mRNA expression in normal human peripheral blood mononuclear cells.

OBJECTIVE AND DESIGN: Normal human peripheral blood mononuclear cells (PBMC) were analyzed for basic profiles of mRNA expression of distinct genes during incubation in a standard cell culture system. MATERIAL: Human PBMC from healthy adult blood donors. METHODS: Steady-state mRNA expression was measured using a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay. RESULTS: Shortly after isolation and cell seeding, mRNA levels of monocyte-derived cytokines (IL-1alpha, IL-6, and TNF-alpha) were significantly increased, whereas lymphocyte-derived cytokines (IL-4, IFN-gamma) were not affected. Expression levels of monokines returned to basal within 24 h. At later stages of culture, the mRNA levels of all genes studied gradually increased and were significantly elevated after 96 h of incubation. CONCLUSIONS: Monokine and lymphokine mRNAs respond differently to cell culture even under control conditions. With regard to the enhanced mRNA expression of distinct cytokines in the early stages of culture, human PBMC should not be used for gene expression studies in vitro within 24 h of isolation.

Analysis of hsp70 mRNA levels in HepG2 cells exposed to various metals differing in toxicity.

Human hepatoma cells (HepG2) were exposed to several heavy metal salts and the induction of heat shock protein 70 (hsp70) mRNA was analysed by the reverse transcriptase-polymerase chain reaction (RT-PCR). Metals were added to the cell medium at concentrations ranging from 0.1 to 100 microM and incubation was continued for 4 h. In addition we analysed the time dependence of hsp70 induction by adding each metal at a certain concentration followed by an incubation for 0.5 to 24 h. CdCl2, NaAsO2, AgNO3 could be classified as very strong inducers (20-, 13- and 10-fold above control level) and they reached their maximum level of induction at 1-10 microM after 2 h. CuCl2, MnCl2, Pb(NO3)2, TlNO3, CoCl2 and NiCl2 were also strong inducing agents, giving a 4-6 fold induction at 10-100 microM after 4-8 h. ZnSO4, Hg(NO3)2 and AlCl3 were only weak inducers (1.5-2 fold at 50-100 microM after 4-8 h) of hsp70 mRNA. Cytotoxic effects (measured by release of lactate dehydrogenase) could only be detected for 100 microM Hg2+ after 4 h and when the cells were incubated with 5 microM Cd2+ for more than 8 h. We also tested a few combinations of these heavy metal salts for their hsp70-inducing ability. Zn2+ and Mn2+ were able to diminish Cd2+ induced hsp70 mRNA levels by 65%. Ag+ mediated induction was reduced by 40% when combined with Cu2+, whereas Hg2+ increased induction by Ag+ about 3-fold and led to a dramatic decrease in cell viability. In our study we were able to demonstrate that the analysis of hsp70 mRNA levels in chemically stressed HepG2 cells by RT-PCR can be a valuable tool for studying mechanisms of toxicity associated with elevated expression of hsp70.