Mechanisms of cell activation by heavy metal ions.

Heavy metal ions can be released by corroding metallic implants into the surrounding tissue. When they enter blood vessels some of them are carried by proteins like albumin and can be taken up by endothelial cells lining the vessels. To study their involvement in the inflammatory response we investigated heavy metal ion induced effects in cultured human vascular endothelial cells (HUVECs). NiCl2 and CoCl2 upregulate, especially in concentrations of 1 mM, the expression of adhesion molecules (e.g., E-selectin and intercellular adhesion molecule-1), as well as the cytokines IL-6 and IL-8, as shown by enzyme immunoassay and Northern blot analysis. In addition, possible signal transduction mechanisms were elucidated. The HUVECs were treated with various selective inhibitory drugs followed by the incubation of metal ions before measuring the expression of the above-mentioned endothelial factors. Two protein kinase inhibitors (H-7 and H-8) strongly repressed Ni2+ and Co2+ enhanced expression, as did the phospholipase A2 inhibitor quinacrine. Other selective inhibitors of protein kinases C or A, or cGMP-dependent protein kinases, as well as calcium antagonists like 1,2-bis(2-aminophenoxy)ethan-N,N,N',N'-tetraacetic acid and 3,4,5-trimethoxybenzosaure 8-(diethylamino)-octylester and inhibitors of receptor mediated endocytosis (primary amines), had no influence. We showed that NiCl2 and CoCl2 activate the translocation of the transcription factor nuclear factor (NF)-kappaB into the cell nucleus and enhance its binding to a NF-kappaB consensus sequence as shown by mobility shift analysis. Furthermore, we demonstrated the activation of AP-1. Despite the repression of heavy metal induced adhesion molecule synthesis, we did not detect any inhibition of NF-kappaB translocation by H-7 or H-8. Therefore, it must be concluded that heavy metal ions like Ni2+ and Co2+ activate two or more signal transduction pathways in endothelial cells. We clearly showed that there is one pathway in which H-7 and H-8 sensitive protein kinases are involved and a second pathway leading to NF-kappaB activation, which is insensitive to H-7 and H-8. Our results demonstrate that heavy metal ions induce mechanisms of gene activation in endothelial cells as do proinflammatory mediators, indicating that corroding metal ion containing biomaterials can provoke inflammatory reactions by known, as well as by yet unknown, intracellular signaling pathways.