A Novel Assay System for Macrophage-activating Factor Activity Using a Human U937 Cell Line

Results

In this study, phagocytic activity was assessed by the internalization of beads. Phagocytic activity of macrophages and the MAF activity were evaluated as the IBR and AR, respectively, at 30 min after MAF addition. Details of these calculations are described in Materials and Methods. We propose the IBR as a robust index of phagocytic activity.

We examined the culture conditions of macrophages just before adding GcMAF (Table I). In general, differentiated U937 macrophages are cultured with RPMI-1640 medium supplemented with 10% serum for 72 h (14). Initially, we examined the phagocytic activity of macrophages under these conditions with and without GcMAF. IBR values with and without GcMAF were 39.9±2.8% and 27.8±5.4%, respectively, and the AR was 1.5±0.2. When macrophages were incubated with serum-free medium for 72 h, IBR values with and without GcMAF were 27.5±6.3% and 6.8±1.7%, respectively. The AR was 4.1±0.2. Incubation with serum-free medium increased sensitivity to GcMAF. We added a 2-h incubation with serum-free medium after the 72 h serum-containing culture condition. Under these conditions, the IBR values with and without GcMAF were 39.5±9.0% and 7.1±1.9%, respectively. The AR was 5.5±0.8. The short incubation with serum-free medium effectively sensitized the differentiated U937 macrophages to GcMAF.

Using the sensitized macrophages, we evaluated the appropriateness of the IBR as an index of phagocytic activity and determined the ideal amount of beads for use in the assay protocol. Different amounts (30 to 120 μg) of beads were added to 5×10⁵ cells of U937 macrophages plated in 35-mm dishes. As shown in Figure 1A, IBN increased according to the amount of beads, suggesting that increasing the amount of beads elevated the chance of engulfment by macrophages. On the other hand, within this range of beads, IBRs converged to similar values (Figure 1B). This might have been because the chance of engulfment was offset by the chance of escaping from engulfment. The amount of beads in the assay protocol was adjusted to 90 μg.

In order to determine the effect of the number of macrophages, we compared the ARs between 5.0×10⁵ and 5.0×10⁶ cells in 35-mm dishes. As shown in Table II, although the IBR was increased when using higher cell numbers, the AR decreased from 5.4±2.2 to 2.2±1.0. Thus, the preferable number of macrophages in a 35-mm dish was 5.0×10⁵ cells.

The effect of GcMAF on differentiated U937 macrophages was verified by light microscopy (Figure 2). The spherical macrophage-like cells adhered to the bottom of the dish, and some extended lamellipodia. When U937 macrophages were cultured without GcMAF, only a few beads were internalized by each macrophage. In this situation, the beads that were not engulfed by macrophages were scattered on the bottom of the culture dish. On the other hand, when U937 macrophages were cultured with GcMAF, macrophages start engulfing beads only 10 min after addition of GcMAF. Sixty min after the addition of GcMAF, most of the beads had formed clusters within the macrophages. Although some clusters were formed within the flattened cell body and seemed to exist extracellularly, all of the clusters were formed within macrophages. Thus, it was easy to discriminate between internalized beads and beads that had escaped engulfment.

Finally, we analyzed the dose dependency of GcMAF in our novel MAF assay protocol. The MAF assay was carried-out in the presence of 0.005, 0.05, 0.5, 5, 50, or 500 μg/ml GcMAF. Over this concentration range, the IBR showed monotonic increase for at least 1 h (Figure 3A). The maximum AR was 5.8±0.5 at 5 μg/ml GcMAF (Figure 3B). These results showed the high sensitivity and relatively wide effective range of our MAF assay protocol. Thus, we propose this novel assay protocol for phagocytic activities as a suitable system for assessing MAF activity.