Expression of the activation-induced CD18 fragment is suppressed by protease inhibitors.

(A) PBLs (as shown in Figure 1) were first preincubated with the indicated protease inhibitors, then activated with PMA, stained with MEM-148 or standard CD18 mAb (MEM-48), and analyzed by flow cytometry. Results from 3 experiments are represented as MFI ± SD values (arbitrary units) calculated from geometric means of fluorescence intensity for gated neutrophils and monocytes after subtraction of irrelevant isotype control. Other protease inhibitors tested (TLCK, TPCK, aprotinin, PMSF, bestatin, EDTA, or EGTA) showed less than 10% inhibition as detected by flow cytometry; actually, further increase of the MEM-148 epitope was observed when the PMA treatment was performed in the presence of 5 mM EDTA or EGTA (not shown). (B) Detergent lysates of the cells treated with the indicated protease inhibitors and PMA (as in panel A) were analyzed by SDS-PAGE and immunoblotting (staining with CD18 mAb MEM-48). The last lane shows PBLs treated with PMA for 2 hours and then exposed for 2 minutes at 0°C to pH 3.5 to wash away any noncovalently associated and nonmembrane-anchored protein fragments. Control low pH sensitive protein β₂-microglobulin was fully dissociated from the cell surface under these experimental conditions (not shown). Open and closed arrowheads indicate the zones corresponding to the uncleaved CD18 and the fragment, respectively.