Cutting Edge: Neutrophil Complement Receptor Signaling Is Required for BAFF-Dependent Humoral Responses in Mice

Abstract

T cell-independent (TI) B cell responses to nonprotein Ags involve multiple cues from the innate immune system. Neutrophils express complement receptors and activated neutrophils can release BAFF, but mechanisms effectively linking neutrophil activation to TI B cell responses are incompletely understood. Using germline and conditional knockout mice, we found that TI humoral responses involve alternative pathway complement activation and neutrophil-expressed C3a and C5a receptors (C3aR1/C5aR1) that promote BAFF-dependent B1 cell expansion and TI Ab production. Conditional absence of C3aR1/C5aR1 on neutrophils lowered serum BAFF levels, led to fewer Peyer's patch germinal center B cells, reduced germinal center B cells IgA class-switching, and lowered fecal IgA levels. Together, the results indicate that sequential activation of complement on neutrophils crucially supports humoral TI and mucosal IgA responses through upregulating neutrophil production of BAFF.

Fig 1.. T-cell independent responses require alternative complement pathway and C3aR/C5aR on neutrophils.

Mice were injected with 50μg TNP-LPS i.p, sacrificed on d10 after immunization. A-B. Peritoneal B1 cell counts (top) and serum concentration of anti-TNP IgM antibodies (bottom) were analyzed in (A) WT, C3aR1^−/−, C5aR1^−/−, C3aR1^−/−C5aR1^−/− and (B) WT, C3^−/−, CR2^−/−, fB^−/− mice. D. Kinetics of DAF expression on peritoneal B1 cells (MFI, mean fluorescence intensity via flow cytometry); n=4/experiment. D. Donor peritoneal B1 cell count (IgMa⁻CD19⁺B220^lowCD11b⁺) and concentration of anti-TNP IgM in MD4⁺ mice d10 after transfer of 4×10⁷ WT or C3aR1^−/−C5aR1^−/− B-cells and TNP-LPS immunization. E-F. Peritoneal B1 cell count (E) and serum anti-TNP IgM concentration (F) in naïve and TNP-LPS immunized groups of mice as indicated. Graphs show mean ± SEM from ≥2 independent experiments with *p<0.05, **p<0.01, ***p<0.005, ****p<0.001, and n.s.; not significant, by non-parametric ANOVA with Bonferroni post-test (A-C, E) or Student’s t-test (D). In E-F, responses in immunized mice greater than in naïve controls (p<0.001 for each) unless otherwise indicated.

Fig 2.. Neutrophil C3aR1/C5aR1 ligations drive BAFF-dependent IgM responses to TNP-LPS

A. Kinetics of intra-peritoneal C5a (pg/ml × ml per lavage), neutrophil (CD11b⁺Ly6G⁺) influx and serum BAFF levels following TNP-LPS immunization of WT mice (n=3–4/experiment). B-C. Intracellular BAFF expression in neutrophils (MFI, flow cytometry), representative histograms, quantification (B, top) and serum BAFF concentration (C, bottom) 12h after TNP-LPS immunization. D-E. Neutrophil intracellular BAFF MFI, representative histogram (D), quantification (E, top) and serum BAFF levels (E, bottom) 12h after 5μg rmC3a and rmC5a, i.p. (F) Concentration of anti-TNP IgM on d10 after TNP-LPS immunization of groups of mice injected with 100ng recombinant mBAFF daily for 5 days. Graphs show mean ± SEM from ≥2 independent experiments with *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 and n.s.: not significant, by non-parametric ANOVA with Bonferroni post-test (B-F).

Fig 3.. Normal IgA mucosal responses require neutrophil C3aR1/C5aR1 activation and can be rescued by exogenous BAFF.

A-C. %IgA⁺ GCB cells (top row), absolute GCB cell count in Peyer’s patches (middle row) and concentration of fecal IgA (bottom row) in (A) WT, C3^−/−, fB^−/− mice, (B) WT, C3aR1^−/−, C5aR1^−/−, C3aR1^−/−C5aR1^−/− mice and (C) C3aR1/C5aR1^fl/fl, C3aR1/C5aR1^ΔLySM, C5aR1^ΔS100 mice. Mice in (C) were injected daily for 5 days with PBS or 100ng mBAFF as indicated and PP % IgA⁺ GCB (top) and GCB number (bottom) were quantified on d6. D. Intracellular BAFF expression in neutrophils from Peyer’s patches of C3aR1/C5aR1^fl/fl and C3aR1/C5aR1^ΔLySM mice quantified as MFI by flow cytometry, representative histograms. All graphs show mean ± SEM from ≥3 independent experiments with ***p<0.005, ****p<0.001 and n.s.: not significant, by non-parametric ANOVA with Bonferroni post-test (A-C).