Rac, Cdc42, MEK1, and ERK control phagocytosis and granule localization as monitored by immunostaining of granzyme B in PMNs.

(A) Constitutive expression of granzyme B mRNA in PMNs. Total RNA (20 μg) was purified from PMNs cultured in 100 U/mL GM-CSF or medium for the indicated times and analyzed by Northern blotting for granzyme B mRNA (top and middle panels). The PMN lysates from 3 blood donors were Western blotted with antigranzyme B antibody to show the protein expression of granzyme B in neutrophils (bottom panel). Cell lysates from peripheral blood lymphocytes (PBLs) were used as a positive control for this experiment. (B) Percentages of inhibition of phagocytosis (■) or granzyme B (▪) mobilization by PD098059. PMNs pretreated with medium, DMSO, or 50 μM PD098059 were stimulated with C albicans for 0 to 5 minutes at 37°C, and the number of PMNs showing ingested C albicans of a total of 100 PMNs were counted and expressed as a percent of total PMNs. The number of PMNs with ingested C albicansthat also contained clearly visible granzyme B surrounding the phagosome were also counted and expressed as percent of the total PMNs. (C) Percentages of inhibition of phagocytosis (■) or granzyme B (▪) mobilization by dominant-negative Rac, Cdc42, Rho, and MEK1. PMNs expressing CD56, N17Rac, N17Cdc42, N17Rho, or MEK1 were also evaluated for phagocytosis and granule movement. (D) Freshly isolated PMNs were preincubated with DMSO or 50 μM PD098059 for 2 hours at 37°C. The cells were then mixed with fixed C albicans for 0 to 5 minutes at 37°C and cytospun onto microscope slides. Control PMNs stained with granzyme B–FITC (i), C albicans stained red with PKH67-GL (ii), PMNs incubated with C albicans at 0 minutes (iii), DMSO-treated (iv), PD098059-treated PMNs (v), PMNs expressing CD56 (vi), dominant-negative MEK1 (vi), N19Rho (viii), N17Rac (ix), or N17Cdc42 (x).