ICAM-1 expression in a spontaneously transformed human keratinocyte cell line: characterization by a simple cell-ELISA assay.
Interferon gamma (IFN-gamma) is known to induce ICAM-1 on keratinocytes (KC) in vitro, and its expression in vivo is correlated with epidermal T-cell infiltration in various dermatoses. However, the mechanisms for this cytokine-mediated ICAM-1 expression are essentially unknown. We investigated the induction of ICAM-1 by IFN-gamma in HaCaT cells, a spontaneously transformed human KC cell line, using an immunoperoxidase-ELISA with the monoclonal antibody (MoAb) R6.5. HaCaT cells constitutively expressed low levels of ICAM-1, which were upregulated by IFN-gamma. The kinetics and dose response were similar to those published for primary KC, regardless of whether the HaCaT cells were cultured in low- or high-calcium medium. ICAM-1 expression was increased significantly at 4 h with 500 U/ml IFN-gamma, and reached a plateau (approximately 5 x greater than constitutive) by 24 h. At concentrations greater than 10 U/ml for 24 h, IFN-gamma induced ICAM-1 expression in a dose-dependent fashion (half maximal at 100 U/ml). TNF-alpha alone, and in synergistic combination with IFN-gamma, also upregulated the expression of HaCaT ICAM-1. IFN-gamma treatment of HaCaT cells increased the level of ICAM-1 mRNA and enhanced (approximately 3x) the adherence of fluorescently labeled (calcein) human T lymphoblasts, as determined by Northern blotting and an in vitro adhesion assay, respectively. Our findings suggest that HaCaT cells, in conjunction with a simple immunoperoxidase cell-ELISA, provide a reliable system for studying pharmacologic modulation of ICAM-1 on KC.
Winiski
A P
AP
Department of Dermatology, Sandoz Research Institute, Vienna, Austria.
Foster
C A
CA
D015152:Blotting, Northern; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000032:analysis* / Q000235:genetics; D002461:Cell Line, Transformed / Q000737:chemistry; D004357:Drug Synergism; D004797:Enzyme-Linked Immunosorbent Assay; D005453:Fluorescence; D006801:Humans; D018799:Intercellular Adhesion Molecule-1; D007371:Interferon-gamma / Q000494:pharmacology; D015603:Keratinocytes / Q000166:cytology*; D013601:T-Lymphocytes / Q000166:cytology; D015854:Up-Regulation / Q000187:drug effects
D015815:Cell Adhesion Molecules; D018799:Intercellular Adhesion Molecule-1; D007371:Interferon-gamma