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The objective of this study was to identify possible roles of the class II antigen-associated, electrophoretically invariant chain (Ii) in class II antigen functions through analysis of the kinetics of synthesis, processing, and turnover of Ii in various cellular systems of Ii hyperexpression. Using [35S]methionine metabolic labeling of microsomal membrane proteins and analysis in one-dimensional SDS polyacrylamide gradient gels and two-dimensional electrophoretic gels, enhanced expression of Ii was demonstrated in leukemic cells of a subset of patients with hairy cell leukemia (HCL), in normal peripheral blood cells freshly transformed with EBV, and in Burkitt's lymphoma cell lines treated with n-butyrate. As part of an initial effort to identify leukemic cell subset-defining, membrane-associated molecules which might then be tested as targets for, or regulators of, the anti-leukemic cell immune response, subsets of HCL patients were identified on the basis of leukemic cell enhanced expression of 35,000 and 15,000 dalton proteins (p35 and p15). A similar enhanced expression of the p35 molecule was demonstrated in peripheral blood or cord blood lymphocytes freshly transformed with Epstein-Barr virus (EBV), as compared to long-term cultured EBV cell lines. Further structural characterization of the HCL p35 by one-dimensional SDS-PAGE and two-dimensional gel electrophoresis of HCL total microsomal membrane proteins, anti-class II antigen immunoprecipitates, and anti-Ii immunoprecipitates showed that the HCL p35 molecule was the human class II antigen-associated Ii chain. In order to determine the functional significance of altered Ii expression on class II antigen function, the relative kinetics of Ii synthesis, processing, and turnover was examined in an in vitro model system of regulated Ii synthesis. Treatment of the Burkitt's lymphoma cell line, Jijoye, and its class II antigen-negative, mutant, daughter cell line, P3HR-1, with 4 mM n-butyrate for 48 hr, enhanced the rate of synthesis of the Ii chain. One-dimensional SDS-PAGE and two-dimensional gel, electrophoretic analysis of anti-Ii and anti-class II antigen immunoprecipitates isolated from pulse-/pulse-chase-/or continuously labeled, control and butyrate-treated P3HR-1 and Jijoye cells demonstrated enhanced levels of synthesis of the dominant, most basic form of Ii in the butyrate-treated samples (to a greater degree in Jijoye cells). The butyrate enhancement of Ii synthesis occurred in the presence or absence of detectable class II alpha and beta chains, as did the relative turnover, although the terminal processing of Ii to an electrophoretically slower and more acidic form was dependent upon its association with class II antigens. The level of the dominant, most basic form of Ii expressed in the hairy leukemic cells equaled that in butyrate-treated Jijoye cells. However, hairy leukemic cell, class II antigen-associated Ii was not terminally processed. The results of these experiments were consistent with the following. In lymphoblastoid cells, two pathways for Ii turnover might exist. One is through association with class II antigen complexes and progressive carbohydrate-chain terminal processing, and a second is not associated with class II antigens and such processing. Class II antigens may direct the processing of Ii towards some function (rather than vice versa). Butyrate treatment rather uniquely enhances the synthesis of Ii in some lymphoid cells in the presence or absence of class II antigens. Hairy leukemic cells demonstrate an inability to terminally process Ii which might be relevant to the function of Ii and/or class II antigens on those cells. |
