Figure 8.
Figure 8. SLE patients have increased FcgRIIA shedding related to neutrophil activation. Neutrophils (A) and monocytes (B) were analyzed for FcgRIIA shedding using a ratio between shed FcgRIIA (IV.3) and total FcgRIIA levels (FUN2) in healthy controls (HC, n = 5–7) and SLE patients (n = 19). Combined results are shown and compared using Mann-Whitney U test (Neutrophils, P < 0.0001; Monocytes, P < 0.0001). (C) Normal-density neutrophils (PMNs) and low-density granulocytes (LDGs) were analyzed for FcgRIIA shedding by flow cytometry. Combined results from six SLE patients are shown and compared using paired Student’s t test (P = 0.026). Neutrophil FcgRIIA shedding was correlated with neutrophil activation as measured by neutrophil CD11b (D) and CD66b expression (E) in SLE patients (n = 19). Combined results are analyzed using Spearman’s correlation (CD66b, r = −0.64, P = 0.0029; CD11b, r = −0.53, P = 0.021). (F) Correlation analysis for ex vivo monocyte and neutrophil (PMN) FcgRIIA shedding in SLE patients. Combined results are analyzed using Spearman’s correlation (r = 0.84; P < 0.0001). (G) Healthy control neutrophils were incubated with 10% serum from healthy controls (HC, n = 10) or SLE patients (n = 36) and analyzed for FcgRIIA shedding by flow cytometry as determined by the IV.3/FUN2 ratio. Combined results are shown and compared using Mann-Whitney U test (P < 0.0001). (H) Sera from 6 SLE patients, preincubated with either RNase, a pan-protease inhibitor (prot.inh.), or cytochalasin B (Cyto B; 5 µM) were added to neutrophils from a healthy individual and FcgRIIA shedding analyzed by flow cytometry. Combined results are shown and compared using paired Student’s t test (RNase: P = 0.012; prot.inh.: P = 0.0002; Cyto B: P < 0.0001). (I) Serum-mediated shedding of FcgRIIA on healthy control neutrophils were analyzed in SLE patients with (n = 7) or without (n = 6) anti-Sm/RNP antibodies. Combined results are compared using Mann-Whitney U test (P = 0.035). (J) Correlation between ex vivo FcgRIIA shedding observed on SLE neutrophils with the shedding ability by the serum obtained from the same SLE patients (n = 12). Combined results are shown and analyzed by Spearman’s correlation (r = 0.73; P = 0.0096). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

SLE patients have increased FcgRIIA shedding related to neutrophil activation. Neutrophils (A) and monocytes (B) were analyzed for FcgRIIA shedding using a ratio between shed FcgRIIA (IV.3) and total FcgRIIA levels (FUN2) in healthy controls (HC, n = 5–7) and SLE patients (n = 19). Combined results are shown and compared using Mann-Whitney U test (Neutrophils, P < 0.0001; Monocytes, P < 0.0001). (C) Normal-density neutrophils (PMNs) and low-density granulocytes (LDGs) were analyzed for FcgRIIA shedding by flow cytometry. Combined results from six SLE patients are shown and compared using paired Student’s t test (P = 0.026). Neutrophil FcgRIIA shedding was correlated with neutrophil activation as measured by neutrophil CD11b (D) and CD66b expression (E) in SLE patients (n = 19). Combined results are analyzed using Spearman’s correlation (CD66b, r = −0.64, P = 0.0029; CD11b, r = −0.53, P = 0.021). (F) Correlation analysis for ex vivo monocyte and neutrophil (PMN) FcgRIIA shedding in SLE patients. Combined results are analyzed using Spearman’s correlation (r = 0.84; P < 0.0001). (G) Healthy control neutrophils were incubated with 10% serum from healthy controls (HC, n = 10) or SLE patients (n = 36) and analyzed for FcgRIIA shedding by flow cytometry as determined by the IV.3/FUN2 ratio. Combined results are shown and compared using Mann-Whitney U test (P < 0.0001). (H) Sera from 6 SLE patients, preincubated with either RNase, a pan-protease inhibitor (prot.inh.), or cytochalasin B (Cyto B; 5 µM) were added to neutrophils from a healthy individual and FcgRIIA shedding analyzed by flow cytometry. Combined results are shown and compared using paired Student’s t test (RNase: P = 0.012; prot.inh.: P = 0.0002; Cyto B: P < 0.0001). (I) Serum-mediated shedding of FcgRIIA on healthy control neutrophils were analyzed in SLE patients with (n = 7) or without (n = 6) anti-Sm/RNP antibodies. Combined results are compared using Mann-Whitney U test (P = 0.035). (J) Correlation between ex vivo FcgRIIA shedding observed on SLE neutrophils with the shedding ability by the serum obtained from the same SLE patients (n = 12). Combined results are shown and analyzed by Spearman’s correlation (r = 0.73; P = 0.0096). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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