FcgammaRIIb balances efficient pathogen clearance and the cytokine-mediated consequences of sepsis

Abstract

The immune response to infection must be controlled to ensure it is optimal for defense while avoiding the consequences of excessive inflammation, which include fatal septic shock. Mice deficient in FcgammaRIIb, an inhibitory immunoglobulin G Fc receptor, have enhanced immune responses. Therefore, we examined whether FcgammaRIIb controls the response to Streptococcus pneumoniae. Macrophages from FcgammaRIIb-deficient mice showed increased antibody-dependent phagocytosis of pneumococci in vitro, and consistent with this infected FcgammaRIIb-deficient mice demonstrated increased bacterial clearance and survival. In contrast, previously immunized FcgammaRIIb-deficient mice challenged with large inocula showed reduced survival. This correlated with increased production of the sepsis-associated cytokines tumor necrosis factor alpha and interleukin 6. We propose that FcgammaRIIb controls the balance between efficient pathogen clearance and the cytokine-mediated consequences of sepsis, with potential therapeutic implications.

Figure 1.

Anti-pneumococcal antibody production and phagocytosis in FcγRIIb-deficient mice. (A) Anti-pneumococcal polysaccharide IgG3 titres in control BALB/c (□) and FcγRIIb^−/− (▪) mice 14 and 21 d after immunization with 1 μg Pneumovax II. Each point represents data from an individual mouse expressed relative to a positive control. The horizontal bar is the mean. (B–E) The effect of FcγRIIb on the phagocytosis of S. pneumoniae in vitro. The RAW-297 macrophage cell line (B and C) or peritoneal macrophages (D and E) were incubated with FITC-labeled S. pneumoniae opsonized with heat-inactivated serum, followed by flow cytometric analysis. Antibody-dependent phagocytosis is expressed as percent FITC⁺ cells relative to nonopsonized sample (see Fig. S2). (B) Serum from unimmunized control (□) and FcγRIIb^−/− (▪) mice provides equivalent opsonization, whereas (C) serum from immunized FcγRIIb^−/− mice enhances uptake. (D) FcγRIIb^−/− peritoneal macrophages show increased phagocytosis of opsonized S. pneumoniae. Phagocytosis was assessed as above but using peritoneal macrophages from control BALB/c (□) and FcγRIIb^−/− (▪) mice, and serum from control mice only. (E) Macrophages from FcγRIIb^−/− mice phagocytose a larger number of bacteria per macrophage than control mice, estimated by the geometric mean fluorescence of FITC⁺ cells (see Fig. S2). (B–E) Values represent mean of triplicates, the experiments shown are representative of two, and p-values were obtained using an unpaired Student's t test. (F) 24 h after inoculation with S. pneumoniae, tail bleeds were performed on C57BL/6 control (n = 11) and FcγRIIb^−/− (n = 13) mice and blood cultured for bacterial growth. Fewer FcγRIIb^−/− mice were bacteremic (results from two experiments combined; Chi-square test).

Figure 2.

Survival after S. pneumoniae infection. Unimmunized FcγRIIb^−/− or control mice were inoculated with S. pneumoniae type 2 i.p. Both C57BL/6 (10⁶ CFU) and BALB/c (10⁷ CFU) FcγRIIb^−/− mice have increased survival (P = 0.027 and P = 0.026, respectively). When unimmunized mice were challenged with higher doses of S. pneumoniae (10⁷ in C57BL/6, 10⁸ in BALB/c), both FcγRII^−/− and control mice succumbed to infection. FcγRIIb^−/− and control mice were immunized with 1 μg pneumococcal polysaccharide (Pneumovax II) s.c. and 21–28 d later mice were challenged with an intermediate dose of S. pneumoniae type 2 or a high dose of S. pneumoniae type 2 (gray shading). At intermediate doses, both strains were protected by immunization. However, at high doses of S. pneumoniae, both C57BL/6 and BALB/c FcγRIIb^−/− mice showed increased mortality (P = 0.017 and P = 0.012, respectively). Each experiment shown is representative of at least two, and p-values were obtained with a log-rank test.

Figure 3.

Proinflammatory cytokine production in response to S. pneumoniae in control and FcγRIIb^−/− mice. (A) Peritoneal macrophages from C57BL/6 and FcγRIIb^−/− mice were cultured for 12 h alone, with unopsonized, heat-killed S. pneumoniae, or with heat-killed S. pneumoniae opsonized with heat-inactivated immune serum. TNF-α levels, measured using a cytometric bead assay, and were higher in FcγRIIb^−/− culture supernatant in all conditions, but particularly when opsonized bacteria were used. The experiment shown is representative of two. (B) Serum TNF-α levels were higher in FcγRIIb^−/− mice whether unimmunized (left), 7 h after inoculation with S. pneumoniae (middle), and in particular, in mice immunized and subsequently inoculated with S. pneumoniae (right, gray shading). (C) IL-6 levels in peritoneal macrophage culture supernatant. (D) Serum IL-6 levels in control (□) and FcγRIIb^−/− (▪) mice, uninfected or 7 h after inoculation with S. pneumoniae either with (gray shading) or without prior immunization. Values are from individual mice, experiments shown are representative of two, and p-values were obtained using an unpaired Student's t test.

Figure 4.

The physiological role of FcγRIIb appears to be to control cytokine release, antibody production, and phagocytosis to balance the inflammatory response to infection to optimize survival in different circumstances. Factors that determine the level of expression of FcγRIIb include the cytokine milieu and naturally occurring FcγRIIb promoter polymorphisms (references and 30).