51Cr-labeled human hepatocytes as target cells for cytotoxicity mediated by freshly isolated liver-infiltrating lymphocytes.
Previous studies on lymphocyte cytotoxicity against autologous human hepatocytes had been studied using Terasaki plates, in which dead hepatocytes after incubation with lymphocytes were counted visually. No studies with 51Cr-labeled human hepatocytes as targets have been reported, although it can give us more objective results. In the present study, we established procedures for labeling human hepatocytes with 51Cr and for measuring cytotoxicity of freshly isolated liver-infiltrating lymphocytes (LIL) against 51Cr-labeled human autologous hepatocytes. Hepatocytes were isolated from diseased and 'normal' liver tissues, cultured overnight, and labeled with 51Cr 'in situ' in the wells of 96-well round bottom plates. Human hepatocytes isolated from either 'normal' or diseased liver tissues labeled well with 51Cr, and mean spontaneous 51Cr release was less than 15% in the presence of 5% fetal bovine serum or human AB serum. Freshly isolated LIL obtained from chronic viral hepatitis but not from other liver diseases showed cytotoxicity against 51Cr-labeled autologous hepatocytes in 4 h 51Cr release assays, and the percent specific lysis was linearly related to the E:T cell ratio. LIL from viral hepatitis were able to mediate natural killer (NK) activity against K562 targets, lymphokine-activated killer-like activity against Daudi cells, and lectin-dependent cellular cytotoxicity. Two-color flow cytometry analysis showed that these LIL contained both CD3+ T cells and CD3-CD56+ NK cells. This is the first report which examined the cytotoxicity of liver-infiltrating lymphocytes against 51Cr-labeled human hepatocytes, and it will be useful in assessing local immune response against autologous hepatocytes in chronic liver diseases.
Shimizu
Y
Y
Department of Internal Medicine, Toyama Medical and Pharmaceutical University, Japan.
Hata
K
K
Herberman
R B
RB
Whiteside
T L
TL
D015703:Antigens, CD / Q000032:analysis; D002478:Cells, Cultured; D002860:Chromium Radioisotopes; D003601:Cytotoxicity Tests, Immunologic; D003602:Cytotoxicity, Immunologic; D006801:Humans; D016130:Immunophenotyping; D066298:In Vitro Techniques; D008099:Liver / Q000166:cytology / Q000276:immunology*; D008107:Liver Diseases / Q000276:immunology*; D008214:Lymphocytes / Q000276:immunology*
D015703:Antigens, CD; D002860:Chromium Radioisotopes