Figure 2.
Figure 2. TLR7/8 activation induces shedding of FcgRIIA. (A) Neutrophils were activated with R848 and cell surface expression of FcgRs analyzed by flow cytometry. The results are presented as FcgR levels as compared with no R848 added (percentage of control). The experiment was repeated 5 (FcgRI), 7 (FcgRIII), and 25 (FcgRIIA) times; combined results are shown and compared using paired Student’s t test (FcgRIIA, P < 0.0001; FcgRI, P = 0.027; FcgRIII, P = 0.0044). (B) Neutrophils were activated with the TLR7/8 agonist R848 and analyzed for FcgRIIA at different time-points and concentrations. The experiment was repeated four (concentration) and six (kinetics) times; combined results are shown and compared using paired Student’s t test (30 min, P = 0.0158; 60 min, P < 0.0001; 120 min, P = 0.0003; 0.125 µg/ml, P = 0.0071; 0.25 µg/ml, P = 0.0058; 0.5 µg/ml, P = 0.0008; 1 µg/ml, P < 0.0001; 2 µg/ml, P < 0.0001). (C) Neutrophils were activated with TLR ligands (LPS, 1 µg/ml, PAM3CSK4 (5 µg/ml), CpG DNA (2 µg/ml), Loxoribine (0.1 mM), CL075 (2.5 µg/ml), or R848 (2 µg/ml) for 60 min or (D) 4 h and analyzed for (C) FcgRIIA, (D) FcgRIII, or (E) CD11b (black bars) and CD66b (gray bars) cell surface expression by flow cytometry. For C, the experiment was repeated 6 (LPS, P = 0.0008), 8 (CpG DNA, P = 0.035; Loxoribine, P < 0.0001; CL075, P < 0.0001), 10 (PAM3CSK4, P < 0.0001), and 40 times (R848, P < 0.0001); combined results are shown and compared using paired Student’s t test. For D and E, the experiment was repeated four (D) and eight (E) times; combined results are shown and compared using paired Student’s t test (CD11b: R848, P < 0.0001; LPS, P = 0.0002; PAM, P < 0.0001; CpG DNA, P < 0.0001; CD66b: R848, P < 0.0001; LPS, P < 0.0001; PAM, P < 0.0001; CpG DNA, P = 0.014). F) Neutrophils were activated with R848 and FcgRIIA levels analyzed in permeabilized cells by flow cytometry. The experiment was repeated five times and compared using paired Student’s t test (P = 0.0075). (G) Cartoon illustrating the FcgRIIA receptor with the binding site for the IV.3 antibody (aa 132–137), potential cleavage site of FcgRIIA, and likely binding site of FUN2 indicated. (H) FcgRIIA cell surface expression was analyzed by flow cytometry using two antibodies, FUN2 and IV.3, in nonstimulated and R848-stimulated neutrophils. The experiment was repeated six times; combined results are shown and compared using paired Student’s t test (P < 0.0001). (I) Neutrophils were labeled with FITC-conjugated IV.3 anti-FcgRIIA or anti–FUN-2 antibodies and the shed antibody-FcgRIIA complex quantified by fluorimetry after R848 stimulation with or without prior addition of a pan-protease inhibitor. The experiment was repeated 4 (FUN2), 6 (IV.3 R848+prot.inh.), or 14 (IV.3 R848) times; combined results are shown and compared using paired Student’s t test (IV.3: R848, P < 0.0001; R848+prot.inh., P = 0.0001). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

TLR7/8 activation induces shedding of FcgRIIA. (A) Neutrophils were activated with R848 and cell surface expression of FcgRs analyzed by flow cytometry. The results are presented as FcgR levels as compared with no R848 added (percentage of control). The experiment was repeated 5 (FcgRI), 7 (FcgRIII), and 25 (FcgRIIA) times; combined results are shown and compared using paired Student’s t test (FcgRIIA, P < 0.0001; FcgRI, P = 0.027; FcgRIII, P = 0.0044). (B) Neutrophils were activated with the TLR7/8 agonist R848 and analyzed for FcgRIIA at different time-points and concentrations. The experiment was repeated four (concentration) and six (kinetics) times; combined results are shown and compared using paired Student’s t test (30 min, P = 0.0158; 60 min, P < 0.0001; 120 min, P = 0.0003; 0.125 µg/ml, P = 0.0071; 0.25 µg/ml, P = 0.0058; 0.5 µg/ml, P = 0.0008; 1 µg/ml, P < 0.0001; 2 µg/ml, P < 0.0001). (C) Neutrophils were activated with TLR ligands (LPS, 1 µg/ml, PAM3CSK4 (5 µg/ml), CpG DNA (2 µg/ml), Loxoribine (0.1 mM), CL075 (2.5 µg/ml), or R848 (2 µg/ml) for 60 min or (D) 4 h and analyzed for (C) FcgRIIA, (D) FcgRIII, or (E) CD11b (black bars) and CD66b (gray bars) cell surface expression by flow cytometry. For C, the experiment was repeated 6 (LPS, P = 0.0008), 8 (CpG DNA, P = 0.035; Loxoribine, P < 0.0001; CL075, P < 0.0001), 10 (PAM3CSK4, P < 0.0001), and 40 times (R848, P < 0.0001); combined results are shown and compared using paired Student’s t test. For D and E, the experiment was repeated four (D) and eight (E) times; combined results are shown and compared using paired Student’s t test (CD11b: R848, P < 0.0001; LPS, P = 0.0002; PAM, P < 0.0001; CpG DNA, P < 0.0001; CD66b: R848, P < 0.0001; LPS, P < 0.0001; PAM, P < 0.0001; CpG DNA, P = 0.014). F) Neutrophils were activated with R848 and FcgRIIA levels analyzed in permeabilized cells by flow cytometry. The experiment was repeated five times and compared using paired Student’s t test (P = 0.0075). (G) Cartoon illustrating the FcgRIIA receptor with the binding site for the IV.3 antibody (aa 132–137), potential cleavage site of FcgRIIA, and likely binding site of FUN2 indicated. (H) FcgRIIA cell surface expression was analyzed by flow cytometry using two antibodies, FUN2 and IV.3, in nonstimulated and R848-stimulated neutrophils. The experiment was repeated six times; combined results are shown and compared using paired Student’s t test (P < 0.0001). (I) Neutrophils were labeled with FITC-conjugated IV.3 anti-FcgRIIA or anti–FUN-2 antibodies and the shed antibody-FcgRIIA complex quantified by fluorimetry after R848 stimulation with or without prior addition of a pan-protease inhibitor. The experiment was repeated 4 (FUN2), 6 (IV.3 R848+prot.inh.), or 14 (IV.3 R848) times; combined results are shown and compared using paired Student’s t test (IV.3: R848, P < 0.0001; R848+prot.inh., P = 0.0001). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal