Fig 2: Csk is involved in integrin-mediated neutrophil slow rolling, chemokine-induced arrest, and neutrophil recruitment in vivo.(A–D) Intravital microscopy of postcapillary venules in the murine cremaster muscle 2 hours after intrascrotal TNF injection. (A) Rolling velocities of neutrophils from Cskfl/flLyz2wt/wt and Cskfl/flLyz2cre/wt mice. (B) Adherent cells per square millimeter and (C) the number of extravasated cells per 1.5 × 104 μm2 tissue area surrounding postcapillary venules. (D) Representative reflected light oblique transillumination microscopy photographs. White circles represent transmigrated neutrophils within the tissue, boxes indicate the analyzed tissue area. Scale bars: 50 μm. (E and F) Chemokine-induced arrest of neutrophils in postcapillary venules of Cskfl/flLyz2wt/wt and Cskfl/flLyz2cre/wt mice before and following CXCL1 injection. (E) Number of adherent cells per mm2. (F) Representative images of postcapillary venules of Cskfl/flLyz2wt/wt and Cskfl/flLyz2cre/wt mice following CXCL1 injection. White circles represent adherent neutrophils within the vessel. Scale bars: 50 μm. (G–I) Carotid cannulas were placed in Cskfl/flLyz2wt/wt and Cskfl/flLyz2cre/wt mice and connected to autoperfused flow chambers. Average rolling velocity of neutrophils on (G) E-selectin and E-selectin/ICAM-1 and (H) P-selectin and P-selectin/ICAM-1. (I) Number of adherent cells on P-selectin/ICAM-1– and P-selectin/ICAM-1/CXCL1–coated flow chambers. n as indicated; n = 3 for E–H, mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by 2-tailed Student’s t test (A–C), 2-way-ANOVA with Šídák’s multiple-comparison test (E), or 2-way ANOVA with Tukey’s multiple-comparison test (G–I).

Fig 3: Csk regulates the activity of Src kinases through a cAMP-dependent pathway.(A) cAMP levels in cell lysates of murine bone marrow–derived WT neutrophils after stimulation with CXCL1 (2 minutes) or E-selectin (5 minutes). (B and C) Blood-perfused flow chambers coated with E-selectin or E-selectin/ICAM-1 were used to analyze rolling velocities. Rolling velocity of human neutrophils isolated from whole blood (B) and neutrophils isolated from Cskfl/flLyz2wt/wt and Cskfl/flLyz2cre/wt mice (C) after incubation with different concentrations of 8-CPT-cAMP, a cAMP analog and selective activator of the cAMP-dependent PKA. (D) Chemokine-induced arrest of neutrophils in postcapillary venules of Cskfl/flLyz2wt/wt and Cskfl/flLyz2cre/wt mice before and following CXCL1 injection, 30 minutes after intraarterial injection of the specific Src family kinase inhibitor PP2 or the inactive control PP3. Significant differences within the Cskfl/flLyz2wt/wt between PP2 and PP3 are marked in black; significant differences within the Cskfl/flLyz2cre/wt between PP2 and PP3 are marked in gray. (E–G) Bone marrow–derived neutrophils were left untreated or were stimulated with CXCL1 for 1 minute, E-selectin for 5 minutes, or serum-opsonized K. pneumoniae for 1 minute. Lysates were immunoblotted with an Ab against total Src (tSrc) and p-Src Y416 or Y529. (E–G) Representative Western blots of total lysates of Cskfl/flLyz2wt/wt and Cskfl/flLyz2cre/wt neutrophils showing the phosphorylation of Src Y416 and Y529 and total amounts of Src. n as indicated; n = 3–4 for D, mean ± SEM. *P < 0.05; **P < 0.01; ****P < 0.0001 by 1-way ANOVA with Tukey’s multiple-comparison test (A) or 2-way ANOVA with Šídák’s multiple-comparison test (B–D).