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. 2021 Feb 26;118(9):e2017527118. doi: 10.1073/pnas.2017527118

Fig. 1.

Fig. 1.

Sema7A controls the macrophage inflammatory phenotype and regulates human macrophage chemotaxis and chemokinesis. Human PBMCs were stimulated with GM-CSF, M-CSF, or Sema7ASL4cd for 7 d, and Sema7A transcript expression in differentiated M1 and M2 MΦs was quantified by RT-PCR (n = 14 to 16) (A). (B) Cell morphology was analyzed by phase contrast images and measurements of the cell shape, length, and perimeter (magnification 200×). (Scale bar: 20 μm.) n = 210. (C) The expression levels of key genes that contribute to M2 differentiation, Arg1 and CD163, and central genes of M1 differentiation, STAT-1 and CD80, were analyzed (n = 11). (D and E) Phenotypic polarization of macrophages: M1 MΦs were sequentially challenged with Sema7ASL4cd and TNF-α or vehicle for 24 h. The gene expression levels of M1 polarization markers (including STAT-1, CD40, CD80, IL-1β, and IL-6), key genes of M2 polarization (such as Arg1, CD163, CD206, and IL-10) and the ALX/FPR2 and GPR32 receptors were quantified by RT-PCR (n = 13 to 20). (F) Schematic model of the microfluidic migration chamber. A chemoattractive gradient with a monocyte chemotactic protein (MCP-1) and Sema7ASL4cd was established between eight peripheral wells and a central cell loading well. M1 MΦ chemotaxis was evaluated using a Casy TT cell counter (Omni Life Science) (n = 10). (G) The rate of MΦ clearance of fluorescence-labeled ZyA particles and human apoptotic PMNs was assessed photometrically (n = 5 to 9). (H and I) MΦs were transfected with integrin α1-, integrin αv-, or integrin β1-siRNA or treated with an anti-Plexin C1 and then stimulated with Sema7ASL4cd antibody. MΦ clearance of fluorescence-labeled ZyA particles was then assessed photometrically (n = 4 to 10). (J) The colocalization of integrin heterodimers and integrins with Sema7A was assessed with the Similarity feature of the ImageStreamx mkII system. The results are representative of three to eight independent experiments and are expressed as the mean ± SEM; significance was determined by one-way ANOVA with Bonferroni correction (B–I) or the unpaired two-tailed Student’s t test (A). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.