C3-mediated opsonophagocytosis of S. aureus in elevated glucose. (A) C3 binding to S. aureus in 2%, 5%, or 10% NHS in PBS with 3 mmol/l glucose or 17 mmol/l glucose assayed by Western blot. (B) Optical densitometry measurements of the forms of C3 (C3, black bars; C3b, grey bars; iC3b, white bars) present on the S. aureus surface in 10% NHS for 3 mM or 17 mM glucose. Alpha-chain densitometry was normalized to the beta-chain and shown as a total percent of C3 forms. Data are mean ± SE for 4 independent experiments. (C) C3a generation by S. aureus in varying serum and glucose concentrations (3 mmol/l, grey bars and 17 mmol/l, black bars) was assayed by dot blot. Data are mean ± SE for 3 independent experiments. (D) Purified C3 incubated with purified factor H (fH) and factor I (fI) shows no significant cleavage in 3 or 17 mmol/l glucose, whereas expected cleavage occurs for purified C3b in these conditions. (E) For S. aureus incubated in various concentration of NHS, C5a generation was increased in 3 mmol/l glucose (grey bars) compared with 17 mmol/l glucose (black bars). Data are mean ± SE for 4 independent experiments. (F, G) S. aureus opsonized in 5% NHS in 3 mmol/l glucose (grey bars) showed increased phagocytosis by human neutrophils compared with bacteria opsonized in 17 mmol/l glucose (black bars). The number of bacteria phagocytized by 100 PMN and the number of neutrophils phagocytizing bacteria were counted by fluorescence microscopy. Data are mean ± SE for 3 independent experiments.