Bactericidal/permeability-increasing protein promotes complement activation for neutrophil-mediated phagocytosis on bacterial surface

Abstract

The neutrophil bactericidal/permeability-increasing protein (BPI) has both bactericidal and lipopolysaccharide-neutralizing activities. The present study suggests that BPI also plays an important role in phagocytosis of Escherichia coli by neutrophils through promotion of complement activation on the bacterial surface. Flow cytometric analysis indicated that fluorescein-labelled E. coli treated with BPI were phagocytosed in the presence of serum at two- to five-fold higher levels than phagocytosis of the bacteria without the treatment. In contrast, phagocytosis of the fluoresceined bacteria with or without treatment by BPI did not occur at all in the absence of serum. The phagocytosis stimulated by BPI and serum was dose-dependent. The effect of BPI on phagocytosis in the presence of serum was not observed on Gram-positive bacteria (Staphylococcus aureus). Interestingly, the complement C3b/iC3b fragments were deposited onto the bacterial surface also as a function of the BPI concentration under conditions similar to those for phagocytosis. Furthermore, the BPI-promoted phagocytosis was blocked completely by anti-C3 F(ab')(2) and partially by anti-complement receptor (CR) type 1 and/or anti-CR type 3. These findings suggest that BPI accelerates complement activation to opsonize bacteria with complement-derived fragments, leading to stimulation of phagocytosis by neutrophils via CR(s).

Figure 1

Time course of phagocytosis of FITC-E. coli by neutrophils stimulated by BPI. FITC-E. coli were preincubated with buffer (○, □) or BPI (final 50 nm) (•, ▪) at 37° for various periods. The bacteria were then subjected to the phagocytosis assay in the absence (□, ▪) or presence of serum (final 10%) (○, •). Horizontal axis, incubation time of the bacteria with BPI; and vertical axis, relative amount of ingested bacteria. This result is representative of three independent experiments. For details, see the Materials and methods.

Figure 2

Dose-dependence of phagocytosis on serum (a) and BPI (b). (a) FITC-E. coli were preincubated with buffer (○) or BPI (final 50 nm) (•) at 37° for 5 min. The bacteria were then subjected to the phagocytosis assay in the presence of various concentrations of serum. Horizontal axis, the final concentration of serum added and vertical axis, relative amount of ingested bacteria. Results are expressed as the mean of three independent experiments ±SD. (b) FITC-E. coli were incubated with various concentrations of BPI at 37° for 5 min. The bacteria were then subjected to the phagocytosis assay in the presence of serum (final 10%). Horizontal axis, the final concentration of BPI added and vertical axis, relative amount of ingested bacteria. Results are expressed as the mean of three independent experiments ±SD. For details, see the Materials and methods.

Figure 5

Effect of anti-human C3 antibody on phagocytosis. FITC-E. coli were preincubated with buffer (□, ▪) or BPI (final 50 nm) (○, •) at 37° for 5 min. Serum and divalent cations were added to each bacterial suspension followed by incubation at 37°. After 5 min, the reaction mixtures were inseminated with various concentrations of F(ab′)₂ to mouse IgG (□, ○) or to human C3 (▪, •), and allowed to stand at 37° for 15 min. Neutrophils were then added and incubated at 37° for additional 1 hr. Horizontal axis, the final concentration of antibody added; and vertical axis, relative amount of ingested bacteria. Results are expressed as the mean of three independent experiments ±SD. For details, see the Materials and methods.

Figure 3

Fluorescence microscopy of neutrophils after ingestion of FITC-E. coli under various conditions. FITC-E. coli were preincubated with buffer (b, c) or BPI (final 50 nm) (d, e) at 37° for 5 min. The bacteria were then subjected to the phagocytosis assay in the absence (b, d) or presence of serum (final 2%) (c, e). (a) A view of neutrophils alone in the same buffer. Upper panel, phase contrast (200× magnification); and lower panel, fluorescence image of the same area as that of the upper image.

Figure 4

Western blot analysis to detect deposition of C3 fragments on the bacterial surface. Escherichia coli were preincubated with various concentrations of BPI at 37° for 5 min. Serum (final 10%) and divalent cations were then added to the bacterial suspension followed by additional incubation at 37° for 1 hr. After release of proteins deposited onto the bacteria by treatment with hydroxylamine, they were resolved by 12·5% SDS–PAGE under reducing conditions. C3-derived fragments in the sample were then detected by Western blotting using goat anti-human C3 antiserum. Molecular weights indicated at the left side are based on a pattern of the prestained protein ladder. Arrows at the right side indicate migrating positions of C3b- and iC3b-derived polypeptide chains, which were judged by those of the authentic C3b and iC3b. For details, see the Materials and methods.