Spleen tyrosine kinase Syk is critical for sustained leukocyte adhesion during inflammation in vivo

Abstract

Background: During inflammation, beta2-integrins mediate leukocyte adhesion to the endothelium accompanied by the activation of the spleen tyrosine kinase Syk.

Results: We investigated leukocyte adhesion and rolling in cremaster muscle venules before and during stimulation with fMLP using mice with a Syk-/- hematopoietic system. In unstimulated venules, Syk-/- leukocytes adhered less efficiently than control leukocytes while rolling was similar between Syk-/- and control leukocytes. During fMLP-superfusion, control mice showed significantly increased adhesion accompanied by reduced rolling. For Syk-/- leukocytes, an increase in adhesion with a concomitant decrease in rolling was only observed during the first three minutes during fMLP stimulation, but not at later time points. We also investigated leukocyte spreading against the vessel wall during fMLP stimulation and found a significant impairment of spreading for Syk-/- leukocytes. Additional in vitro experiments revealed that the adhesion and spreading defect seen in Syk-/- chimeric mice was due to compromised beta2-integrin-mediated outside-in signaling.

Conclusion: We provide substantial evidence for an important role of Syk in mediating beta2-integrin dependent outside-in signaling leading to sustained leukocyte adhesion and spreading during the inflammatory response in vivo.

Figure 1

Leukocyte adhesion and rolling (mean ± SEM) in unstimulated cremaster muscle venules. Number of adherent leukocytes [adherent cells/mm² vessel surface area] (A), leukocyte adhesion efficiency [(adherent cells/mm² vessel surface area)/(systemic leukocyte count)] (B), and rolling flux fraction [%] (C) in Syk^-/- chimeric mice (gray bar, n = 4) and control mice (black bar, n = 6). * indicates significant difference (p < 0.05) between Syk^-/- chimeric and control mice.

Figure 2

Leukocyte adhesion and rolling (mean ± SEM) during local stimulation of cremaster muscle venules with fMLP. Leukocyte adhesion [number of adherent cells/mm²] (A) and leukocyte rolling flux fraction [%] (B) in Syk^-/- chimeric mice (gray bar, n = 4)) and control mice (black bar, n = 6) before and during 15 min local administration of fMLP (1 μM). * indicates significant difference (p < 0.05) between Syk^-/- chimeric and control mice.

Figure 3

Cell diameter changes of adherent leukocytes (mean ± SEM) during local stimulation of cremaster muscle venules with fMLP. Microphotographs of an adherent control leukocyte immediately after attachment (A, left panel) and during gradual stimulation (A, right panel) leading to flattening out of the cell with a concomitant decrease in cell diameter perpendicular to the vessel wall (white arrows). Diameters of adherent leukocytes from Syk^-/- chimeric mice (gray bar, n = 318 from 4 mice) and control mice (black bar, n = 419 from 6 mice) were measured perpendicular to the vessel wall before and during superfusion with fMLP (B). In addition, a cumulative frequency distribution of measured leukocyte diameters is given for Syk^-/- (gray lines) and control leukocytes (black lines) after 1 min (dashed lines) and 15 min (solid lines) fMLP superfusion (C). * in (B): significant difference (p < 0.05) between Syk^-/- chimeric and control mice. * in (C): significant difference (p < 0.05) in the distribution of control cell diameters at 15 min fMLP to all other groups.

Figure 4

Neutrophil adhesion and spreading (mean ± SD) on immobilized fibrinogen. Adhesion and spreading of isolated Syk^-/- (gray bar) or control neutrophils (black bar) with or without (w/o) addition of 1 mM Mn²⁺ at 37°C for 30 min. Adherent Syk^-/- (n = 7 mice) or control neutrophils (n = 5 mice) in percent of total cells added (A), microscopic images (B), increase of cell area (in μm², C) and frequency distribution of cell area (D) of adherent Syk^-/- (n = 400 from 4 mice) and control neutrophils (n = 400 from 4 mice) upon stimulation for 30 min at 37°C. * indicates significant difference (p < 0.05), n.s., not significant.