Citrulline can preserve proliferation and prevent the loss of CD3 ζ chain under conditions of low arginine

Background: Arginine depletion by the enzyme Arginase 1, decreases expression of the TCR zeta chain preventing T-cell activation and causing T-cell dysfunction. We hypothesized that citrulline could substitute for arginine under conditions of increased arginase expression. Thus, the goal was to establish a possible mechanism of how citrulline could overcome arginine depletion caused by arginase. Methods: Jurkat cells were cultured, with or without arginase, in media containing different amino-acid constituents: complete RPMI containing arginine (C-RPMI) (arginine), Arginine-Free-RPMI (Arg-Free RPMI) and Citrulline-containing RPMI (Cit RPMI). Incorporation of citrulline was measured via uptake of H-3-citrulline, whereas proliferation was measured via H-3-thymidine incorporation. zeta Chain was analyzed by 2-color flow cytometry. Argininosuccinate synthase (AS) and argininosuccinate lyase expression was detected using Northern blots, RT-PCR, and Western blots. Results: Jurkat cells exhibited a significant decrease in proliferation and zeta chain expression when cultured in the presence of arginase or in the absence of arginine. With citrulline, zeta chain expression and proliferation were maintained in the absence of arginine or in the presence of the enzyme arginase. Jurkat cells, cultured in the absence of arginine, were associated with a 5-fold increase in citrulline uptake. The absence of arginine was also associated with increased expression of AS. Conclusions: T cells exhibit the molecular capability of increasing citrulline membrane transport and up-regulating AS expression, thus exhibiting the necessary mechanisms for converting citrulline into arginine and escaping the ill effects of arginine depletion. Therefore, citrulline has the potential to be a substitute for supplemental arginine in diseases associated with arginase-mediated T cell dysfunction.