When tuberculin-sensitive peritoneal exudate cells are incubated in a culture flask with tuberculin purified protein derivative, macrophage inhibition factor and other lymphokines are released into the culture medium. We have described how, if incubation is carried out in a stationary conical culture tube, intercellular contact between the peritoneal exudate cells is facilitated as the cells sediment into a pellicle at the bottom of the tube. This results in augmented release of inhibitory lymphokines into the supernatant culture medium with titers up to 10
9
times greater than those obtained by conventional culture methods using a flatbottomed culture dish or flask. When such high-titered inhibitory supernatants were subjected to fractionation by sequential Amicon ultrafiltration, two clearly distinct macrophage-inhibitory lymphokines were found. The first was present, after fractionation, in a titer of 10
12
, had a molecular weight in the range of 50,000 to 100,000, and was heat stable at 56°C for 1 h. This moiety is probably identical to guinea pig macrophage inhibition factor. Unexpectedly, a second heat-labile inhibitory substance with a molecular weight between 500 and 1,000 was found in a titer of 10
4
after fractionation. This low-molecular-weight, heat-labile material may represent a new lymphokine with a direct inhibitory action on macrophage migration. Theoretically, the data are also consistent with the possibility that it could act as a chemical immunotransmitter which stimulates amplified production of macrophage inhibition factor by lymphocytes within the cell pellicle and leads indirectly to inhibition of macrophage migration.
Langerhans' cells, veiled cells, and interdigitating cells in the mouse recognized by a monoclonal antibody.
