Amplified lymphokine production phenomenon confirmed by a macrophage 2-D-[3H]deoxyglucose transport assay.

The amplified lymphokine production phenomenon was confirmed by using an improved macrophage 2-d-[(3)H]deoxyglucose uptake assay as an indicator of lymphokine activity. Amplified lymphokine titers were determined in supernatants derived from tuberculin-sensitive, antigen (purified protein derivative)-stimulated, guinea pig peritoneal exudate and spleen cell suspensions after the cells were allowed to sediment into a pellicle in a conical culture tube. The deoxyglucose uptake assay, which probably measured an effect on the macrophage cell membrane, was easy to perform, and the prozone phenomenon observed with other lymphokine assay systems did not occur. The deoxyglucose uptake-enhancing moiety was stable at 56 degrees C for 1 h and had a molecular weight of between 50,000 and 100,000, as defined by Amicon ultrafiltration. Exposure of macrophages to the lymphokine-containing supernatants did not increase macrophage deoxyglucose uptake significantly until after 9 h of incubation had elapsed. The effect on deoxyglucose uptake was to increase the V(max) without changing the K(m) value. Deoxyglucose uptake also involved a stereospecific carrier-facilitated transport system both in the presence and in the absence of lymphokine. The increased deoxyglucose transport induced by the lymphokine-containing supernatants was reversible. A migration inhibitory factor activity of similar molecular weight and heat stability was also present in these supernatants, but in titers lower than the titers of the deoxyglucose uptake-enhancing activity. Consequently, in the absence of a complete biochemical characterization, the two effects cannot be ascribed to the same molecular species at this time. Such a characterization, along with studies of lymphokine production and action, should be facilitated greatly by the availability of very high-titer supernatants derived by this geometric culture method.