Figure 3.
Sema7A binds to neutrophil PlexinC1 and influences neutrophil chemotaxis. Stained neutrophils isolated from saline (NaCl)- or LPS–treated WT and Sema7A−/− mice 4 hours after incubation. (A) Expression of Sema7A (red) and CD29 (green) on PMNs harvested from WT mice treated with NaCl or LPS (scale bar, 10 μm). (B) Expression of Sema7A (red) and EGFR (green) on PMNs harvested from WT mice treated with NaCl or LPS. No protein colocalization was visible in the merged pictures in either condition (scale bar, 10μm). (C) Expression of Sema7A (red) and PlexinC1 (green) on PMNs harvested from WT mice treated with NaCl or LPS. Sema7A expression is highly increased in LPS–treated mice, and the merged pictures show a strong interaction between Sema7A and PlexinC1 (scale bar, 10 μm). (D) Surface PMN expression of Sema7A (red) and PlexinC1 (green) in Sema7A−/− mice after the injection of exogenous recombinant Sema7A or IgG-Fc (control) after LPS or NaCl (controls) inhalation. Strong binding of exogenous Sema7A to Sema7A−/− PMNs was observed in LPS inhalation group. Multiple acquisitions of stained cells were analyzed from independently performed triplicate experiments (scale bar, 10 μm). (E) Human PMNs were subjected to different stimuli in bidirectional chemotactic chambers. Acquired time lapse videos over a 3-hour period were analyzed. Representative plots of PMN chemotactic tracks toward NaCl (control; red), fMLP (green), recombinant human SEMA7A (recSema7A; blue), or recSema7A together with antibodies against human PlexinC1 (anti-PLEXINC1; gray). (F-I) Comparison of the chemotaxis parameters forward migration index (FMI), Euclidean distance under the aspect of the direction, PMN velocity, and accumulated PMN distance. (J) PMN binding affinity was indicated by APC−labeled fibrinogen on the surface of Ly6G-positive PMNs, as analyzed by FACS. The EDTA group was the internal negative control to measure the baseline autofluorescence, the untreated group was the fibrinogen-negative control, TNF-α was used as a potent PMN stimulator, and treatment of PMNs with recombinant SEMA7A before APC−labeled fibrinogen represented the fibrinogen binding target group of interest. The fibrinogen-APC MFI was normalized and is displayed as a percentage. (K) PMN binding affinity was indicated by PerCP-labeled ICAM-1 on the surface of Ly6G-positive PMNs by FACS. The untreated group was the ICAM-1–negative control, TNF-α was used as a potent PMN stimulator, and treatment of PMNs with recombinant SEMA7A before PerCP-labeled ICAM-1 represented the ICAM-1 binding target group of interest. CD11b antibody treatment was used as a control for the inactivation of ICAM-1 binding. The ICAM-1 PerCP MFI was normalized and is displayed as a percentage. In (F-K), all group comparisons were performed by unpaired 2-tailed Student t tests (the data are the mean ± SD); ∗P < .05; ∗∗P < .01; ∗∗∗P < .001 as indicated. MFI, Mean Fluorescence Intensity.

Sema7A binds to neutrophil PlexinC1 and influences neutrophil chemotaxis. Stained neutrophils isolated from saline (NaCl)- or LPS–treated WT and Sema7A−/− mice 4 hours after incubation. (A) Expression of Sema7A (red) and CD29 (green) on PMNs harvested from WT mice treated with NaCl or LPS (scale bar, 10 μm). (B) Expression of Sema7A (red) and EGFR (green) on PMNs harvested from WT mice treated with NaCl or LPS. No protein colocalization was visible in the merged pictures in either condition (scale bar, 10μm). (C) Expression of Sema7A (red) and PlexinC1 (green) on PMNs harvested from WT mice treated with NaCl or LPS. Sema7A expression is highly increased in LPS–treated mice, and the merged pictures show a strong interaction between Sema7A and PlexinC1 (scale bar, 10 μm). (D) Surface PMN expression of Sema7A (red) and PlexinC1 (green) in Sema7A−/− mice after the injection of exogenous recombinant Sema7A or IgG-Fc (control) after LPS or NaCl (controls) inhalation. Strong binding of exogenous Sema7A to Sema7A−/− PMNs was observed in LPS inhalation group. Multiple acquisitions of stained cells were analyzed from independently performed triplicate experiments (scale bar, 10 μm). (E) Human PMNs were subjected to different stimuli in bidirectional chemotactic chambers. Acquired time lapse videos over a 3-hour period were analyzed. Representative plots of PMN chemotactic tracks toward NaCl (control; red), fMLP (green), recombinant human SEMA7A (recSema7A; blue), or recSema7A together with antibodies against human PlexinC1 (anti-PLEXINC1; gray). (F-I) Comparison of the chemotaxis parameters forward migration index (FMI), Euclidean distance under the aspect of the direction, PMN velocity, and accumulated PMN distance. (J) PMN binding affinity was indicated by APC−labeled fibrinogen on the surface of Ly6G-positive PMNs, as analyzed by FACS. The EDTA group was the internal negative control to measure the baseline autofluorescence, the untreated group was the fibrinogen-negative control, TNF-α was used as a potent PMN stimulator, and treatment of PMNs with recombinant SEMA7A before APC−labeled fibrinogen represented the fibrinogen binding target group of interest. The fibrinogen-APC MFI was normalized and is displayed as a percentage. (K) PMN binding affinity was indicated by PerCP-labeled ICAM-1 on the surface of Ly6G-positive PMNs by FACS. The untreated group was the ICAM-1–negative control, TNF-α was used as a potent PMN stimulator, and treatment of PMNs with recombinant SEMA7A before PerCP-labeled ICAM-1 represented the ICAM-1 binding target group of interest. CD11b antibody treatment was used as a control for the inactivation of ICAM-1 binding. The ICAM-1 PerCP MFI was normalized and is displayed as a percentage. In (F-K), all group comparisons were performed by unpaired 2-tailed Student t tests (the data are the mean ± SD); ∗P < .05; ∗∗P < .01; ∗∗∗P < .001 as indicated. MFI, Mean Fluorescence Intensity.

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