Problems encountered in the preparation of migration inhibitory factor (MIF) and mitogenic factor (MIT) from phytomitogen-stimulated human peripheral lymphocytes and in the preparation of MIF from human lymphoid cell lines (LCL)

The induction by Concanavalin A (Con A) of MIF and MIT production in serum-free cultures of human peripheral lymphocytes was investigated. Optimum conditions were established, but even these did not consistently lead to factor production. Unless great care was taken residual Con A, at surprisingly low concentrations, affected both MIF- and MIT-assay results, but after studying its filtration and adsorption behavior, it could be eliminated from culture supernatants. It is possible that removal of Con A caused concomitant losses of factors. It is suggested that some of the activities ascribed in the literature to MIT and MIF were in fact due to residual Con A. Attempts to induce MIF production in peripheral lymphocytes with phytohaemagglutinin (PHA), gave rise to culture supernants that markedly inhibited the migration of test cells, but the inhibitory activity was lost on dialysis against fresh medium. Similarly, supernatants from human LCL cells were highly inhibitory, but this activity too was virtually lost on dialysis. These supernatants contained some macromolecular MIF (MW ~25,000) which, however, accounted for at most a few percent of the total inhibitory activity. Most of the activity was due to medium depletion and was related to the rate of metabolism of the various cells. It was partly abolished by the addition of glucose. Deliberate depletion of medium simulated MIF activity. It is unlikely that a dialysable migration inhibitor was involved. The findings suggest that some of the published data on MIT and MIF production should be treated with reserve.