Figure 5.
Figure 5. FcgRIIA shedding differentially regulates neutrophil phagocytosis and NETosis. (A) Neutrophils were incubated with CMK (25 µM), before addition of stimuli and phagocytosis analyzed by flow cytometry. The experiment was repeated five times; combined results are shown and compared using paired Student’s t test (P = 0.0032). (B) Neutrophils were incubated with CMK before the addition of RNP-ICs and cell surface levels of CD11b and CD66b analyzed by flow cytometry. The results are expressed as CD11b or CD66b (percentage of control), as compared with nonstimulated cells. The experiment was repeated 15 times; combined results are compared using paired Student’s t test (CD11b: RNP-IC; P < 0.0001; RNP-IC+CMK, P = 0.0002; CD66b: RNP-IC; P < 0.0001; RNP-IC+CMK, P < 0.0001). (C) Neutrophils, pretreated with CMK, were activated with RNP-ICs and the ability to release NETs analyzed by fluorimetry. The experiment was repeated six times; combined results are compared using paired Student’s t test (P = 0.0001). (D) RNP-ICs were treated with RNases before addition to neutrophils and NET formation analyzed by fluorimetry. The experiment was repeated seven times; combined results are shown and compared using paired Student’s t test (P < 0.0001). (E) SmRNP, NETs, dsDNA, or ssRNA were degraded by human RNase without (E and F) or with (G) presence of autoantibodies, and analyzed by fluorimetry over time. The experiment was repeated three (G), four (E), or eight (F) times. (H) NET formation was analyzed upon preincubation with different amounts of beads. The experiment was repeated three times; combined results are shown and compared using paired Student’s t test (1 µl, P = 0.0135; 5 µl, P = 0.0031). (I) Neutrophil uptake of RNP-ICs was analyzed upon pretreatment with beads. The experiment was repeated four times; combined results are shown and compared using paired Student’s t test (P = 0.018). (J and K) Neutrophils from healthy individuals (n = 12) were analyzed for baseline FcgRIIA IV.3/FUN2 ratio in relation to IC-mediated NETosis (J) and phagocytosis (K). The combined results are shown and analyzed using Spearman’s correlation. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FcgRIIA shedding differentially regulates neutrophil phagocytosis and NETosis. (A) Neutrophils were incubated with CMK (25 µM), before addition of stimuli and phagocytosis analyzed by flow cytometry. The experiment was repeated five times; combined results are shown and compared using paired Student’s t test (P = 0.0032). (B) Neutrophils were incubated with CMK before the addition of RNP-ICs and cell surface levels of CD11b and CD66b analyzed by flow cytometry. The results are expressed as CD11b or CD66b (percentage of control), as compared with nonstimulated cells. The experiment was repeated 15 times; combined results are compared using paired Student’s t test (CD11b: RNP-IC; P < 0.0001; RNP-IC+CMK, P = 0.0002; CD66b: RNP-IC; P < 0.0001; RNP-IC+CMK, P < 0.0001). (C) Neutrophils, pretreated with CMK, were activated with RNP-ICs and the ability to release NETs analyzed by fluorimetry. The experiment was repeated six times; combined results are compared using paired Student’s t test (P = 0.0001). (D) RNP-ICs were treated with RNases before addition to neutrophils and NET formation analyzed by fluorimetry. The experiment was repeated seven times; combined results are shown and compared using paired Student’s t test (P < 0.0001). (E) SmRNP, NETs, dsDNA, or ssRNA were degraded by human RNase without (E and F) or with (G) presence of autoantibodies, and analyzed by fluorimetry over time. The experiment was repeated three (G), four (E), or eight (F) times. (H) NET formation was analyzed upon preincubation with different amounts of beads. The experiment was repeated three times; combined results are shown and compared using paired Student’s t test (1 µl, P = 0.0135; 5 µl, P = 0.0031). (I) Neutrophil uptake of RNP-ICs was analyzed upon pretreatment with beads. The experiment was repeated four times; combined results are shown and compared using paired Student’s t test (P = 0.018). (J and K) Neutrophils from healthy individuals (n = 12) were analyzed for baseline FcgRIIA IV.3/FUN2 ratio in relation to IC-mediated NETosis (J) and phagocytosis (K). The combined results are shown and analyzed using Spearman’s correlation. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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