FcγRIIB/III mediate IVIG-induced inhibition of leukocyte recruitment and RBC interactions. A, Experimental scheme. WT mice (n=11–14 per group) were injected with either mAb FcγRIIB/III or isotype rat IgG2b (1 mg/kg) followed by IVIG or control albumin administration (800 mg/kg) 3 hours after administration of TNF-α (0.5 μg). Leukocyte behaviors were analyzed in cremasteric venules for 1 hour. B, Blood from WT mice was collected after an intravital microscopy experiment, and surface expression of FcγRIIB/III on the neutrophil population gated on the basis of side- and forward-scatter properties was examined by flow cytometry after PE-conjugated anti-FcγRIIB/III staining. C, Numbers of circulating leukocytes. D, Percentages of monocytes, neutrophils and lymphocytes. E, Adherent leukocytes in venules. F, Number of circulating RBC-adherent leukocyte interactions per minute. Bars represent mean±SEM. **P<0.01, ***P<0.001 versus albumin.
FcγRIII but not FcγRIIB is expressed on neutrophils. A, Circulating leukocytes from WT mice were stained for FcγRIIIB/III, CD115, and CD3 expression after RBC lysis. Neutrophils (blue) were gated as CD115low, monocytes (red) as CD115hi, and T-cell population (black) as CD3pos. B, Blood was collected from WT (green, n=3) and Fcgr3−/− (blue, n=6) mice and then stained for Gr-1, CD115, and FcγRIIB/III expression. FcγRIIB/III expression was analyzed by gating on the neutrophil population with Gr-1hi and CD115lowand monocyte population with Gr-1low-hi and CD115hi. Representative histogram of FcγRIIB/III expression and control isotype (gray, left panel) and quantification of the geometric mean of fluorescence (GMF) (right panel) are shown. C, Representative expression of FcγRIIIB/III (left panel) on neutrophil population (Gr-1hi/ CD115low) from TNF-α–treated mice deficient in the FcγRIIB, FcγRIII or control WT mice (n=2–3 per group) 1 hour after administration of IVIG or control albumin. Gray histograms represent isotype control. Quantification of FcγRIIIB/III expression levels from WT, Fcgr2−/−, and Fcgr3−/− mice (right panel) is shown.
FcγRIII mediates IVIG-induced inhibition of leukocyte adhesion and Mac-1 activity. IVIG or control albumin (800 mg/kg) was administrated 3 hours after TNF-α injection (0.5 μg) in WT, Fcgr2b−/−, and Fcgr3−/−mice (n=5–7 per group). Cremasteric venules were analyzed by intravital microscopy for 1 hour. A, Adherent leukocytes in venules. B, Number of circulating RBC-adherent leukocyte interactions per minute. Bars represent mean±SEM. *P<0.05, **P<0.01 versus albumin. C, Representative images of fluosphere bound to leukocytes in venules from WT mice. Images were acquired in the bright-field and FITC channels. Asterisks represent RBCs that interact with adherent leukocytes and arrows indicate the direction of flow. Scale bar, 20 μm. D, Binding of albumin-coated fluospheres to leukocytes in WT, Fcgr2b−/−, and Fcgr3−/−mice. Mice prepared for intravital microscopy were intravenously injected with 109 albumin-coated fluospheres 10 minutes after administration of either IVIG or control albumin. Each dot represents the average number of fluospheres bound per leukocyte within individual venules (n=35–45 venules from 4 mice per group). *P<0.05, ***P<0.001, Mann-Whitney test.
Abrogation of IVIG-induced anti-inflammatory activity in meν/meν mice. A, SHP-1 association with FcγRIIB/III in response to IVIG in neutrophils. Bone marrow neutrophils isolated from control WT mice (n=3) were incubated with IVIG or albumin (6.7 mg/mL) at 37°C for 15 minutes, and then lysates were prepared. Lysates were immunoprecipitated (IP) with anti-FcγRIIB/III or control isotype rat IgG2b followed by immunoblotting (IB) with anti-SHP-1/2 ab. B, Adherent leukocytes in venules. C, Number of circulating RBC-adherent leukocyte interactions per minute. Bars represent mean±SEM. *P<0.05, ***P<0.001 versus albumin. D, Binding of albumin-coated flurospheres to leukocytes10 minutes after either IVIG or control albumin administration in control chimeric WT or meν/meν mice. Each dot represents the average number of fluospheres bound per leukocyte within individual venules (n=20–28 venules from 4 mice per group). ***P<0.001 versus albumin, Mann-Whitney test.
Blockade of FcγRIIB and III abrogates the protective actions of IVIG against acute vaso-occlusion in SCD mice. A, Experimental scheme. Three hours after administration of TNF-α (0.5 μg), SCD mice (n=8 per group) were injected with either anti-FcγRIIB/III monoclonal antibody (mAb) or isotype rat IgG2b, (1 mg/kg) before administration of IVIG or an equivalent volume of human albumin (800 mg/kg). B, Blood from SCD mice was collected after an intravital experiment, and surface expression of FcγRIIB/III on the neutrophils was examined with PE-conjugated anti-FcγRIIB/III mAb by flow cytometry. C, Adherent leukocytes in venules. D, Number of circulating sickle RBC-adherent leukocyte interactions per minute. E, Blood flow rates. Bars represent mean±SEM. *P<0.05, **P<0.01, ***P<0.001 versus albumin. F, Representative images of each group after IVIG or control albumin administration showing leukocyte recruitment and heterotypic interactions. Scale bars, 20 μm. G, Kaplan-Meier survival curves for individual SCD mice. P=0.03, log-rank test, IVIG versus albumin in IgG2b-treated group; P=0.93, log-rank test, IVIG versus albumin in anti-FcγRIIB/III mAb-treated group.
By Jung-EunJang, AndrésHidalgo and Paul S.Frenette
Hemodynamic Parameters After IVIG or Control Albumin Administration Following Injection of Anti-FcγRIIB/III mAb or IgG2b Control in TNF-α–Treated SCD...Show More
Hemodynamic Parameters After IVIG or Control Albumin Administration Following Injection of Anti-FcγRIIB/III mAb or IgG2b Control in TNF-α–Treated SCD MiceShow Less
FcγRIIB/III mediate IVIG-induced inhibition of leukocyte recruitment and RBC interactions. A, Experimental scheme. WT mice (n=11–14 per group) were injected with either mAb FcγRIIB/III or isotype rat IgG2b (1 mg/kg) followed by IVIG or control albumin administration (800 mg/kg) 3 hours after administration of TNF-α (0.5 μg). Leukocyte behaviors were analyzed in cremasteric venules for 1 hour. B, Blood from WT mice was collected after an intravital microscopy experiment, and surface expression of FcγRIIB/III on the neutrophil population gated on the basis of side- and forward-scatter properties was examined by flow cytometry after PE-conjugated anti-FcγRIIB/III staining. C, Numbers of circulating leukocytes. D, Percentages of monocytes, neutrophils and lymphocytes. E, Adherent leukocytes in venules. F, Number of circulating RBC-adherent leukocyte interactions per minute. Bars represent mean±SEM. **P<0.01, ***P<0.001 versus albumin.
FcγRIII but not FcγRIIB is expressed on neutrophils. A, Circulating leukocytes from WT mice were stained for FcγRIIIB/III, CD115, and CD3 expression after RBC lysis. Neutrophils (blue) were gated as CD115low, monocytes (red) as CD115hi, and T-cell population (black) as CD3pos. B, Blood was collected from WT (green, n=3) and Fcgr3−/− (blue, n=6) mice and then stained for Gr-1, CD115, and FcγRIIB/III expression. FcγRIIB/III expression was analyzed by gating on the neutrophil population with Gr-1hi and CD115lowand monocyte population with Gr-1low-hi and CD115hi. Representative histogram of FcγRIIB/III expression and control isotype (gray, left panel) and quantification of the geometric mean of fluorescence (GMF) (right panel) are shown. C, Representative expression of FcγRIIIB/III (left panel) on neutrophil population (Gr-1hi/ CD115low) from TNF-α–treated mice deficient in the FcγRIIB, FcγRIII or control WT mice (n=2–3 per group) 1 hour after administration of IVIG or control albumin. Gray histograms represent isotype control. Quantification of FcγRIIIB/III expression levels from WT, Fcgr2−/−, and Fcgr3−/− mice (right panel) is shown.
