Development of Human Keratinocyte Colonies for Confocal Microscopy and for Study of Calcium Effects on Growth Differentiation and Sulfur Mustard Lesions

Abstract

One of the most fascinating questions about sulfur mustard (HD) injuries is the unanswered issue of why basal cells are specific targets for vesicating lesions. What mechanism sets basal cells apart from all other cells of the epidermis and causes their interface with the basement membrane to be the primary site for blister formation? In recent studies with confocal microscopy, Werrlein et al. (1) reported that increased extracellular calcium affected the morphology and in vitro response of human epidermal keratinocytes (HEK) to HD. The image changes observed were consistent with discoveries by Hennings et al. (2–4) that small increases in extracellular calcium (as little as 0.01 mM) can cause fast-acting, dramatic responses in the differentiation of keratinocytes. Kruszewski et al. (5) demonstrated that keratinocytes can respond to small incremental calcium changes (of 0.1–0.12 mM) within 60 s, but noted that the capacity for transient response to calcium was lost as keratinocytes differentiated. Others have demonstrated that calcium-induced terminal differentiation can alter cell-surface receptors (6) and epidermal protein markers (7,8). Complementary studies (9–11) have demonstrated that calcium gradients exist in the epidermis of mice and humans, with the lowest calcium concentrations residing at the basal layer. Each has determined that basal cells exist in an environment that is relatively low in calcium when compared to keratinocytes located in the suprabasal layers of the epidermis. Absolute calcium concentrations within the gradient have not been determined. It is known, however, that epidermal gradients can be disrupted by certain chemicals, e.g., acetone and lovastatin (12),by diseases like psoriasis (13,14), and by essential fatty acid deficiencies (12). Based on the evidence accumulated, we have proposed that exposure to HD may disrupt extracellular calcium gradients or programmed responses to calcium, which would be especially disruptive to basal cells because they are more sensitive to changes in calcium concentration than are differentiated keratinocytes. To test this hypothesis, we have initiated development of primary and first-passage keratinocyte cultures from human skin resections that will allow us to use confocal microscopy to determine how calcium concentration affects HEK proliferation, differentiation, and basal cell response(s) to sulfur mustard toxicity. The system will subsequently be used for testing the efficacy of prophylactic and therapeutic compounds.