EXOGENOUS GLUTAMINE REQUIREMENT IS CONFINED TO LATE EVENTS OF T-CELL ACTIVATION

Glutamine is required for the proliferation of lymphocytes, but quanti tative effects on discrete steps of activation remain unknown to date. Therefore the influence of glutamine (range: 0 mM-1 mM) on the in vit ro response of human peripheral blood mononuclear cells (PBMC) to a mi togenic anti-CD3 monoclonal antibody (mAb) was investigated. Expressio n of surface activation markers by flow cytometry, presence of mRNA of cytokine genes by polymerase chain reaction, release of cytokines by ELISA, and entering into the cell cycle by flow cytometry were sequent ially analyzed. Proliferation was measured by a 3H-thymidine incorpora tion assay. mRNA coding for IL-2, IL-2 receptor, IL-4, IL-5, GM-CSF, a nd IFN-gamma was detectable independently from exogenous glutamine pro vision; expression of the cell surface activation marker CD69 was also glutamine independent. In contrast, later activation events including the expression of the surface activation markers CD25, CD45RO, and CD 71 as well as the production of IFN-gamma were found to require exogen ous glutamine supply. In contrast, production of TNF-alpha could be ob served in the absence of glutamine and was increased to a limited exte nt by exogenous glutamine. The overall lymphocyte response as reflecte d by entering into the cell cycle and proliferation was directly corre lated with the glutamine concentration of the culture medium. Efficien t progression through the cell cycle was found to require at least 0.5 mM glutamine and an increase in glutamine concentration from 0.1 mM t o 1 mM enhanced proliferation by 50%. These results were supported by data obtained following anti-CD3 stimulation of a CD4+ T cell clone. A ltogether, these data underline that a complete cellular immune respon se depends on an exogenous glutamine supply. Regarding glutamine requi rements, they define early, glutamine-independent and late, glutamine- dependent lymphocyte activation stages. (C) 1993 Wiley-Liss, Inc.