TGF-β1 inhibits PMN interaction with ICAM-1, transendothelial migration, and in vivo intraluminal arrest. (A) Adhesion of WT PMNs to hIgG1 or ICAM-1-Fc coated on tissue culture dishes. PMNs were incubated with 20, 50, 100, or 200 ng/mL TGFβ-1 for 20 minutes at 37°C before stimulation with 100 ng/mL CXCL1 for 1 minute. (B) PMNs were incubated with ICAM-1-Fc (1.5 μg/1 × 10⁶ cells) and α-human IgG-APC antibody (0.75 μg/1 × 10⁶ cells) after preincubation, where indicated, for 20 minutes with TGFβ-1 (100 ng/mL) and/or with CXCL1 (100 ng/mL) for 3 minutes at 37°C. (C) Transendothelial migration of WT PMNs through a TNF-α–stimulated bEnd.5 cell monolayer in transwell filters along a CXCL1 chemotactic gradient. PMNs were incubated with TGF-β1 (100 ng/mL) or solute for 20 minutes before transmigration. (D) IVM of chemokine-induced leukocyte arrest in postcapillary venules of the cremaster muscle of WT mice. Fifteen-second movies were taken before and 1 minute after intra-arterial injection of CXCL1 (600 ng) every minute for 10 minutes. TGF-β1 (4 μg) or vehicle was intra-arterially injected 15 minutes before the experiment. (E-F) Binding of soluble ICAM-1-Fc (1.5 μg/1 × 10⁶ cells) after a 20 minute preincubation with TGF-β1 (100 ng/mL) and a 3 minute stimulation with CXCL1 (100 ng/mL) (where indicated) to ALK-5^lox/lox and ALK-5^LysMKO PMNs (E) and TGF-βRII^lox/lox and TGF-βRII^LysMKO PMNs (F). (G-I) Adhesion of PMNs (G) or THP-1 cells (H-I) to either ICAM-1-Fc or EM-Fc (G), or hVCAM-1-Fc or hIgG1 (H-I). Cells were preincubated with GW-788388 (200 nM) (G) or with 50 μg/mL anti-hTGF-βRII blocking antibody or control antibody (H) or both (I) for 10 minutes at RT before 20 minutes TGF-β1 incubation (100 ng/mL). Where indicated, CXCL1 or CCL2 (100 ng/mL) was added for 1 minute. Data are pooled from 4 (A,C,E-H), 3 (B,I), and 6 (D) independent experiments. Data are shown as mean ± SEM. *P < .05; **P < .01; ***P < .001. TEM, transendothelial migration; WT, wild-type.