Conditions for generating and assaying human leukocyte inhibitory factor (LIF) induced by purified protein derivative (PPD)

Abstract

This report examines a variety of experimental conditions for the production of human leukocyte inhibitory factor (LIF). The data indicate that the production of LIF as measured in the indirect capillary-tube migration inhibition assay using human polymorphonuclear indicator cells accurately reflects delayed hypersensitivity to purified protein derivative (PPD) as detected by skin-test reactivity. Mononuclear cells and semipurified lymphocytes separated from whole blood by sedimentation in Ficoll-Hypaque were able to generate LIF in response to PPD. The quantity of cells responsible for LIF production were standardized and as little as 1 × 106 mononuclear cells were required for detectable LIF production. LIF produced by mononuclear cells required only temporary exposure to PPD, thus eliminating the necessity for a control to which antigen was added at the end of culture to antigen-free supernatants. In addition, the removal of antigen after a brief exposure helps to avoid the possible toxic effect of some antigens on the migrating polymorphonuclear leukocytes (PMNL) population. LIF production did not require extraneous serum protein (i.e., fetal bovine serum). Further, kinetic studies indicated t that LIF was detected as early as 8 hr following a 2 hr-exposure to PPD and that even a 1 hr-exposure was sufficient to generate measurable detectable quantities of LIF. After 48 hr of culture, supernatants were found to contain considerable amounts of LIF, with reactivity at dilutions as high as 1:40.