A new sensitive assay for measurement of cell-mediated cytotoxicity to intact layers of cultured human keratinocytes.
A cytotoxicity assay for sensitive measurement of cell-mediated lympholysis (CML) of human cultured keratinocytes (cK) is described. The usage of 51Cr-labeled keratinocytes in intact layers as target cells in this assay allows objective and accurate determination of lysis of keratinocytes which have not undergone trypsin- and suspension-induced membrane changes. Furthermore, the problem of high spontaneous 51Cr release values encountered with suspended keratinocytes is overcome. The assay was applied to study antigen-specific CML of cK by cloned cytotoxic T cells (CTL) and to determine the effect of IFN-gamma on the susceptibility of cK to lysis. The results showed that HLA-A2 specific CTLs could reproducibly lyse cK of HLA-A2 positive healthy skin donors both with and without incubation of cK with IFN-gamma. Applications of this keratinocyte cytotoxicity assay lie in determining the antigenic expression of human cK, in analysis of effector cell/keratinocyte interactions in CML and of the modulatory effects of cytokines on these mechanisms. The assay thus may provide a helpful tool in gaining insight into the role of CML of keratinocytes in the destruction of inflamed skin.
De Bueger
M M
MM
Department of Immunohaematology and Bloodbank, University Hospital Leiden, The Netherlands.
Van Els
C A
CA
Kempenaar
J
J
Ponec
M
M
Goulmy
E
E
D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000502:physiology; D002478:Cells, Cultured; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods*; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D015603:Keratinocytes / Q000276:immunology*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology
D015815:Cell Adhesion Molecules; D007371:Interferon-gamma