Soluble factors produced by macrophages/monocytes inhibit lymphokine-activated killer activity in rat splenocyte cultures

In the present study we investigated the inhibition of interleukin-2(IL-2)-induced lymphokine-activated killer (LAK) activity in rat splenocyte cultures in relation to the presence of 2-mercaptoethanol and macrophages/monocytes. The presence of 2-mercaptoethanol is necessary for induction of LAK activity in rat splenocyte cultures. Removal of macrophages/monocytes from rat splenocytes by plastic or nylon-wool adherence, or iron ingestion resulted in LAK induction by IL-2 in the absence of 2-mercaptoethanol. The effect of macrophages/monocytes on LAK activity was also studied in transwell co-cultures. In the absence of 2-mercaptoethanol, the induction of LAK activity was very low in macrophage/monocyte-depleted splenocytes with macrophages/monocytes in the upper compartment of a transwell culture. In contrast, in the presence of 2-mercaptoethanol a high level of LAK activity was induced in these transwell cultures, showing that 2-mercaptoethanol abolished the LAK-inhibiting capacity of macrophages/monocytes. In addition, established LAK activity was strongly inhibited when, after LAK induction, splenocytes were cultured with supernatant of unfractionated splenocytes, which were cultured with IL-2 but in the absence of 2-mercaptoethanol. Addition of 2-mercaptoethanol abrogated the inhibiting effect of the supernatant completely. These experiments demonstrate that rat macrophages/monocytes produce 2-mercaptoethanolsensitive soluble LAK-inhibiting factors. Ultrafiltration of conditioned culture medium of macrophages/monocytes revealed the presence of LAK-inhibiting factors larger than 10 kDa. We concluded that 2-mercaptoethanol-sensitive soluble factors produced by macrophages/monocytes determine the level of LAK induction in rat splenocyte cultures.