Leukocyte chemotaxis assay development and studies of leukocyte chemotaxis modulation by metallothionein

Date of Completion

January 2006

Keywords

Biology, Cell|Health Sciences, Immunology

Degree

Ph.D.

Abstract

In this work, we have first developed a novel method, ECIS/taxis, for monitoring cell movement in response to chemotactic factors. In ECIS/taxis, cells migrate under-agarose, and their positions are monitored using the electric cell substrate impedance sensor (ECIS) technology to measure the resistance change at a target electrode, that is lithographed onto the substrate, as the cells reach the target electrode. ECIS/taxis could detect chemotaxis of Dictyostelium to folate, and was further optimized for leukocyte chemotaxis detection. This assay system was found compatible with measurements of leukocyte movement and should be valuable in the assessment of both agonists and antagonists of cell movement. ^ Secondly, we have examined the effects of metallothionein (MT), (a stress-induced protein), on thioglycollate-induced acute peritoneal inflammation, and on the function of inflammatory leukocytes using three different mouse strains including wild type strain (WT-2 copies of Mt1 and Mt2 genes), MT-null strain (KO-0 copies of Mt1 and Mt2), and MT transgenic strain (TGN-114 copies of Mt1 and 2 copies of Mt2). We have found that at 24h post-injection, the number of neutrophils in KO mice was higher than in WT and TGN mice suggesting that MT may influence the in vivo recruitment of inflammatory cells during inflammation. Moreover, assessment of the chemotactic response of inflammatory neutrophils to C5a in vitro indicated that 4h and 24h-elicited TGN cells were faster than WT and KO cells. At 4h post inflammation, TGN hepatic MT levels were the highest although MT increased significantly in both WT and TGN mice. At 24h post inflammation, although WT hepatic MT decreased to naïve levels, TGN MT decreased also but remained higher than WT levels. Taken together, the in vitro chemotaxis and hepatic MT data may suggest that leukocytes taken from their in vivo environment may become exposed to environmental or intracellular oxidants, which then affect the chemotactic response. The presence of MT may confer an antioxidative capacity that is absent in KO cells. The results from these studies support a role of metallothionein in altering the development of the inflammation and the function of the recruited inflammatory leukocytes. ^

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