Long-term exposure to methotrexate induces immunophenotypic changes, decreased methotrexate uptake and increased...

Methotrexate (MTX) treatment of rheumatoid arthritis may require increasing doses to maintain clinical efficacy. An overall plateau of clinical response is reached after only six months of treatment. To study the immunologic, biochemical and genetic effects of MTX on T cells, the Jurkat T cell line was made MTX-resistant by serial addition of methotrexate sodium into culture medium. Cells proliferated and divided successfully in MTX concentrations ranging to 15 microM. MTX resistance of Jurkat T cells in vitro was accompanied by significantly (P < 0.05) decreased expression of CD2, CD3, CD4, CD28, and CD69, IL-2 production, and MTX uptake assessed by cell association or disassociation of 3[H]-MTX or fluoresceinated MTX (FMTX), respectively. In addition, there was DHFR gene amplification and increased levels of DHFR in all resistant cell lines. Both permanent and transient phenotypic changes developed 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, whereas expression of CD26 and MTX uptake were significantly increased. Expression of CD2, CD3, CD69 and IL-2 production as well as the DHFR levels did not return to the parental phenotype after removal from MTX. We conclude that MTX-cultured cells express depressed levels of cell-surface markers vital for T cell function and activation. The return of enhancement of these cell-surface markers critical to T cell activation suggests a possible mechanism for the severe flares experienced by rheumatoid arthritis patients when drug treatment is discontinued.