Activated factor XI inhibits chemotaxis of polymorphonuclear leukocytes

Abstract

PMN leukocytes are the most abundant leukocytes in the circulation and play an important role in host defense. PMN leukocyte recruitment and inflammatory responses at sites of infection are critical components in innate immunity. Although inflammation and coagulation are known to have bidirectional relationships, little is known about the interaction between PMN leukocytes and coagulation factors. Coagulation FXI participates in the intrinsic coagulation pathway upon its activation, contributing to hemostasis and thrombosis. We have shown previously that FXI-deficient mice have an increased survival and less leukocyte accumulation into the peritoneum in severe polymicrobial peritonitis. This result suggests a role for FXI in leukocyte trafficking and/or function. In this study, we characterized the functional consequences of FXIa binding to PMN leukocytes. FXIa reduced PMN leukocyte chemotaxis triggered by the chemokine, IL-8, or the bacterial-derived peptide, fMLP, perhaps as a result of the loss of directed migration. In summary, our data suggest that FXIa modulates the inflammatory response of PMN leukocytes by altering migration. These studies highlight the interplay between inflammation and coagulation and suggest that FXIa may play a role in innate immunity.

Figure 1.. Human PMN leukocyte binding to FXIa.

Purified human PMN leukoctyes (1×10⁶/ml) were placed on protein-coated surfaces for 30 min at 37°C and imaged using 10× phase microscopy for quantification. (A) Representative images of phase-bright PMN leukoctyes on BSA (2% w/v), FXIa (50 μg/ml), or fibronectin (FN; 50 μg/ml)-coated surfaces. (B) Adherent PMN leukoctyes on immobilized BSA, FXIa, FXII (50 μg/ml), PK (50 μg/ml), polyRGD (pRGD; 20 μg/ml), or fibronectin (50 μg/ml) were calculated and are reported as mean cell count/mm² ± sem. Results were obtained from at least three independent experiments.

Figure 2.. Characterization of PMN leukoctye–FXIa interaction.

Purified human PMN leukoctyes (1×10⁶/ml) were incubated over surfaces of FXIa (50 μg/ml) for 30 min at 37°C in the presence of vehicle (PMN leukoctye buffer; –), EDTA (5 mΜ), AnnexinV (AnxV; 10 μg/ml), or serine protease inhibitor PPACK (40 μΜ). In selected experiments, cells were treated with Zn²⁺ (25 μΜ) and HK (42 nΜ). Results are reported as mean cell count/mm² ± sem of three independent experiments. P < 0.05 compared with adhesion of vehicle-treated cells in the absence (*) or presence (**) of Zn²⁺/HK.

Figure 3.. FXIa attenuates fMLP-induced Ca²⁺mobilization.

Human PMN leukoctyes were loaded with fluo-4 AM (2 μΜ) and plated on a fibronectin surface. Intracellular Ca²⁺ spikes were monitored for 15 min. Vehicle (PMN leukoctye buffer; dashed line) or FXIa (10 μg/ml; solid line) was added at 5 min (first arrow), followed by the addition of fMLP (10 nΜ) at 10 min (second arrow). Data are presented as mean intensity of five PMN leukoctyes in a field of view from each treatment.

Figure 4.. Inhibitory effects on fMLP-driven PMN leukocyte chemotaxis.

(A) Human PMN leukoctyes, pretreated with vehicle (PMN leukoctye buffer; –), FXIa (1, 5, 10, 20 μg/ml), PK (20 μg/ml), or TNF-α (10 ng/ml), were placed on fibronectin (5 μg/ml)-coated transwell inserts and allowed to migrate toward the fMLP (10 nΜ)-containing lower chamber for 90 min at 37°C. (B) PMN leukoctyes pretreated with vehicle, FXIa (10 μg/ml), or PK (10 μg/ml) were allowed to migrate toward the IL-8 (100 ng/ml)-containing lower chamber for 90 min at 37°C. The results are presented as mean percentage ± sem of the migratory population for each treatment relative to chemoattractant-driven chemotaxis. *P < 0.05 compared with the migration of vehicle-treated cells to the indicated chemoattractants.

Figure 5.. FXIa inhibits directed migration.

Adherent human PMN leukoctyes on fibronectin-coated coverslips were pretreated with vehicle (PMN leukoctye buffer) or FXIa (10 μg/ml) and placed on an Insall chamber. Chemotaxis was induced by adding fMLP (10 nΜ) in the outer well of the chamber. Time-lapse images were taken every 10 s for 30 min. (A) Time-lapse images at the first 0, 5, and 10 min with cell paths (white lines). (B) The PMN leukoctye migration was analyzed using the ImageJ plugin MTrackJ and quantified using the CircStat toolbox for MATLAB. Spider plots indicate the paths of individual cells (upper panels), and rose plots indicate the angular histograms associated with the trajectories (lower panels). The mean resultant vectors are shown in rose plots (arrows).