IMMUNOPHENOTYPING OF LYMPHOCYTES IN LIVER-TISSUE OF PATIENTS WITH CHRONIC LIVER-DISEASES BY FLOW-CYTOMETRY

Immunological factors are important in the pathogenesis of a spectrum of hepatobiliary diseases. To characterize the nature of specific immunological responses in liver disease, we determined lymphocyte changes in liver tissue and in blood using flow cytometry. A total of 113 liver biopsy specimens was collected from patients with the following diseases: 19 chronic hepatitis B; 39 chronic non-A, non-B hepatitis; 27 alcoholic liver disease; 10 hepatic malignancy; 8 autoimmune hepatitis; 6 fatty liver and 4 primary biliary cirrhosis. The lymphocytes were isolated from the liver biopsy specimens by mechanical and enzymatic methods. The lymphocyte yield was 7,901 +/- 575 cells/mg of liver tissue. The viability of lymphocytes was 97.7% +/- 0.3%. Lymphocytes were stained with four pairs of two-color mixed fluorescein - conjugated monoclonal antibodies, including T4-T8 (CD4/CD8), T11-B1 (CD2-CD20), NKHl-T8 (CD56-CD8), IL-2R1-T11 (CD25-CD2), and the ratios were determined by an Epics Profile flow cytometer. Immunophenotyping of lymphocytes in whole blood samples was simultaneously analyzed. Variability in lymphocyte yield and different patterns of lymphocyte subsets were found in the liver biopsy specimens. The yields of lymphocytes from patients with chronic non-A, non-B and autoimmune hepatitis were highest, and the lowest yield was from patients with fatty liver. Patients with primary biliary cirrhosis, fatty liver and hepatic malignancy had relatively high ratios of CD4/CD8, CD56/CD8 and CD25/CD2; whereas patients with chronic hepatitis B, autoimmune hepatitis and non-A, non-B hepatitis had lower ratios of CD4/CD8, CD56/CD8 and CD25/CD2. No difference in lymphocyte ratios between the patients with cirrhotic and noncirrhotic alcoholic liver disease was found. The ratio of CD2/CD20 decreased, and the ratios of CD56/CD8 and CD25/CD2 increased in patients with chronic non-A, non-B hepatitis after 6 mo of interferon treatment, whereas no significant changes occurred in subsets in patients with chronic hepatitis B. The patterns of lymphocyte subsets in blood samples did not correlate with those found in the corresponding liver samples. These results demonstrate the feasibility of immunophenotyping lymphocyte subpopulations in small liver biopsy specimens using a novel approach. Such methodology is needed to establish the immunological mechanisms in liver damage and the effects of therapy.