FIG 6.

Complement C5a-C5aR signaling induces enhanced expression of CD11b on the surface of neutrophils. (A) Purified neutrophils (1 × 10⁶) were incubated with C. neoformans (Cn) H99 expressing GFP (1 × 10⁵ cells) for 10, 20, 30, or 60 min in 1 ml RPMI 1640 containing 40% wild-type mouse serum and then placed on ice. The cells were then fixed in 1% paraformaldehyde and stained with APC–anti-Ly6G and PE–anti-CD11b. The surface expression of CD11b on neutrophils was analyzed by using flow cytometry. Gray-filled histogram, naive neutrophils; solid line, stimulated neutrophils (without phagocytosis/binding of the fungus); dotted line, stimulated neutrophils (with phagocytosis/binding of the fungus). (B) Surface expression of CD11b on neutrophils incubated with C. neoformans in the presence of wild-type or C5^−/− mouse serum with or without the addition of rC5a. Gray-filled histogram, naive neutrophils; dotted line, C. neoformans with C5^−/− serum; solid line, C. neoformans with wild-type serum; dashed line, C5^−/− serum with the addition of rC5a. (C) Surface expression of CD11b on neutrophils incubated with C. neoformans in the presence of wild-type serum with or without the addition of anti-C5aR MAb. Gray filled histogram, naive neutrophils; dotted line, C. neoformans with wild-type serum with the addition of anti-C5aR MAb; solid line, C. neoformans with wild-type serum. Data are representative of results from 3 independent experiments.