Citrulline Can Preserve Proliferation and Prevent the Loss of CD3 ζ Chain Under Conditions of Low Arginine

Abstract

Background: Arginine depletion by the enzyme Arginase I, decreases expression of the TCR ζ 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 ³H-citrulline, whereas proliferation was measured via ³H-thymidine incorporation. ζ 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 ζ chain expression when cultured in the presence of arginase or in the absence of arginine. With citrulline, ζ 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.

Arginase 1, an enzyme that metabolizes arginine, causes arginine deficiency and potential T cell dysfunction after injury. Citrulline provides an alternative route for the generation of arginine, escaping arginine destruction by arginase. This paper demonstrates that role of citrulline in maintaining T cell function in the presence of arginase.