LONG-TERM EXPOSURE TO METHOTREXATE INDUCES IMMUNOPHENOTYPIC CHANGES, DECREASED METHOTREXATE UPTAKE AND INCREASED DIHYDROFOLATE GENE COPY NUMBER IN JURKAT T-CELLS

Methotrexate (MTX) treatment of rheumatoid arthritis may require incre asing doses to maintain clinical efficacy. An overall plateau of clini cal response is reached after only six months of treatment. To study t he immunologic, biochemical and genetic effects of MTX on T cells, the Jurkat T cell line was made MTX resistant by serial addition of metho trexate sodium into culture medium. Cells proliferated and divided suc cessfully in MTX concentrations ranging to 15 mu M. MTX resistance of Jurkat T cells in vivo was accompanied by significantly (P < 0.05) dec reased expression of CD2, CD3, CD4, CD28, and CD69, IL-2 production, a nd MTX uptake assessed by cell association or disassociation of (3)[H] -MTX or fluoresceinated MTX (FMTX), respectively. In addition, there w as DHFR gene amplification and increased levels of DHFR in all resista nt cell lines. Both permanent and transient phenotypic changes develop ed in resultant cell lines exposed to increasing concentrations of MTX in vitro. Expression of CD4 and CD25 and sensitivity to MTX returned to near-parental levels after removal of MTX from culture medium, wher eas expression of CD26 and MTX uptake were significantly increased. Ex pression of CD2, CD3, CD69 and IL-2 production as well as the DHFR lev els did not return to the parental phenotype after removal from MTX. W e conclude that MTX-cultured cells express depressed levels of cell-su rface markers vital for T cell function and activation. The return of enhancement of these cell-surface markers critical to T cell activatio n suggests a possible mechanism for the severe flares experienced by r heumatoid arthritis patients when drug treatment is discontinued. (C) 1998 Published by Elsevier Science Ltd on behalf of the International Society for Immunopharmacology.