Cross-talk between TLR4 and FcgammaReceptorIII (CD16) pathways

Abstract

Pathogen-pattern-recognition by Toll-like receptors (TLRs) and pathogen clearance after immune complex formation via engagement with Fc receptors (FcRs) represent central mechanisms that trigger the immune and inflammatory responses. In the present study, a linkage between TLR4 and FcgammaR was evaluated in vitro and in vivo. Most strikingly, in vitro activation of phagocytes by IgG immune complexes (IgGIC) resulted in an association of TLR4 with FcgammaRIII (CD16) based on co-immunoprecipitation analyses. Neutrophils and macrophages from TLR4 mutant (mut) mice were unresponsive to either lipopolysaccharide (LPS) or IgGIC in vitro, as determined by cytokine production. This phenomenon was accompanied by the inability to phosphorylate tyrosine residues within immunoreceptor tyrosine-based activation motifs (ITAMs) of the FcRgamma-subunit. To transfer these findings in vivo, two different models of acute lung injury (ALI) induced by intratracheal administration of either LPS or IgGIC were employed. As expected, LPS-induced ALI was abolished in TLR4 mut and TLR4(-/-) mice. Unexpectedly, TLR4 mut and TLR4(-/-) mice were also resistant to development of ALI following IgGIC deposition in the lungs. In conclusion, our findings suggest that TLR4 and FcgammaRIII pathways are structurally and functionally connected at the receptor level and that TLR4 is indispensable for FcgammaRIII signaling via FcRgamma-subunit activation.

Figure 1. Association between TLR4 and FcRγIII.

Peritoneal PMNs and macrophages (3×10⁶ cells/ml) from Wt mice and FcRγ-subunit^−/− mice were incubated in vitro for 30 min with either IgG immune complexes (IgGIC; 100 µg/ml), LPS (20 ng/ml), or the combination. (A,B) Western blot analysis (IB) for FcγRIII of Wt PMN or macrophage lysates co-immunoprecipitated (IP) with anti-TLR4. (C,D) Reverse direction immunoprecipitation using anti-FcγRII/III IgG followed by Western blot analysis for TLR4. (E,F) Western blot analysis for FcγRIII of PMNs or macrophages from FcγRIII^−/− co-immunoprecipitated (IP) with anti-TLR4. (G,H) Samples were immunoprecipitated with anti-TLR6 IgG and probed for FcγRIII. (I,J) Immunoprecipitation with anti-CD23 followed by Western blots using anti-TLR4 IgG. (K) Western blots (IB) of cell lysates of Wt macrophages that were incubated for 30 min with BSA IgG immune complexes (IgGIC; 100 µg/ml), polymyxin-treated BSA IgG immune complexes (p.-t. BSA IC; 100 µg/ml) or peroxidase/anti-peroxidase IgGIC immune complexes (PAP IC, 100 µg/ml). IB for FcγRIII of Wt macrophage lysates co-immunoprecipitated (IP) with anti-TLR4. Corresponding loading controls are displayed in lower panels.

Figure 2. In vitro cytokine responses of elicited peritoneal PMNs and macrophages to LPS and IgGIC.

In vitro cytokine responses of elicited peritoneal PMNs (A–D) and macrophages (E,F). Cells (3×10⁶ cells/ml) from either Wt or TLR4 mut mice were incubated for 4 hr with LPS (20 ng/ml) or IgGIC; 100 µg/ml), respectively. In addition, macrophages were incubated with polymyxin-treated BSA IgG immune complexes (p.-t. BSA IC, 100 µg/ml) or peroxidase/anti-peroxidase IgGIC immune complexes (PAP IC, 100 µg/ml). (A) IL-6 release from PMNs after LPS stimulation. (B) TNFα levels after incubation of PMNs with LPS. (C) Concentration of IL-6 in supernatants when PMNs were exposed to IgGIC. (D) Production of TNFα by PMNs and macrophages in the presence of IgGIC. Ctrl = control levels of non-stimulated cells. (E) Release of IL-6 by macrophages into supernatant fluids after stimulation with LPS, IgGIC, p.-t. BSA IC, or PAP IC. (F) TNFα production by macrophages exposed to LPS, IgGIC, p.-t. BSA IC, or PAP IC. The experiments were performed in triplicates for each condition (each bar) with n≥3 donors of cells for each mouse strain, Wt or TLR4 mut. Differences between controls and stimulated cells were—if not otherwise noted—statistically significant (p<0.05).

Figure 3. Responsiveness of FcγRIII-deficient phagocytes to LPS.

Peritoneal PMNs (A,B) and macrophages (C,D) from Wt and FcγRIII^−/− mice were incubated to LPS (100 ng/ml) or Zymosan (300 µg/ml), or IgG immune complexes (IgGIC; 100 µg/ml; macrophages only), and supernatant fluids were analyzed for IL-6 and TNFα levels. Ctrl = control levels of non-stimulated cells. For each condition, n≥4. Differences between controls and stimulated cells were—if not otherwise noted—statistically significant (p<0.05).

Figure 4. Western blot analysis for tyrosine-phosphorylated (PY) FcRγ-subunit of PMN or macrophage lysates after in vitro incubation.

(A,B) 3×10⁶ cells/ml from either Wt or TLR4 mut mice were incubated for 5, 15, and 30 min with IgG immune complexes (IgGIC; 100 µg/ml). (C,D) The same protocol was used for stimulation with LPS (20 ng/ml). (E) Lysates from either Wt or TLR4 mut mice that were incubated with polymyxin-treated BSA immune complexes (100 µg/ml) under the same conditions as described above. Corresponding loading controls are displayed in the lower panels.

Figure 5. Parameters of acute lung injury in Wt and TLR4 mut mice.

(A) Lung injury (as measured by leak of ¹²⁵I-BSA into lung) in Wt, TLR4 mut, TLR4^+/+, and TLR4^−/− mice receiving LPS intratracheally. (B) Permeability indices in Wt, TLR4 mut, TLR4^+/+, and TLR4^−/− mice after intrapulmonary immune complex formation following administration of BSA (i.v.) and anti-BSA IgG (i.t.). (C) IL-6 levels in BAL fluids after IgG immune complex (IgGIC)- or LPS-induced lung injury using Wt and TLR4 mut mice. (D) TNFα in BAL fluids from the same mice described in frame (C). For each bar, n≥5. (E) Lung injury induced by IgG immune complexes (IgGIC) in Wt and TLR4 mut mice after endotoxin removal by polymyxin. (F) Lung permeability after intratracheal (i.t.) administration of anti-BSA IgG and intravenous (i.v.) injection of BSA, PBS i.t., and BSA i.v. or anti-BSA i.t. and PBS i.v. (G) IgGIC-induced lung injury in FcRγ-subunit^−/− mice in comparison to Wt mice (FcRγ-subunit^+/+). For each bar, n≥5.

Figure 6. Expression levels of FcγRII/III, FcRγ-subunit, and C5aR on phagocytes from Wt and TLR4 mut mice.

(A) Summary of flow cytometry analyses of FcγRII/III expression on blood PMNs. (B,C) Original flow cytometry results for FcγRII/III expression on the surface of PMNs from Wt (B) or TLR4 mut (C) mice. (D,E) Analysis of the expression of FcγRIII (upper bands) and FcRγ-subunit (middle bands) in cell lysates [(D), PMNs; (E), macrophages] from Wt or TLR4 mut mice by Western blotting. The lower bands represent the analysis for GAPDH as loading controls. (F) Surface expression of C5aR protein on PMNs from Wt or TLR4 mut mice as assessed by flow cytometry. MFI, mean fluorescence intensity. Studies were done in three separate and independent experiments, with each sample run in duplicates.