Abstract 342: Coagulation Pathways Drive Substantial Leukocyte Recruitment Relative to Solitary TLR4-mediated Pathways
Recruitment of leukocytes is critical for controlling infections. However, chronic leukocyte activity is deleterious in a variety of pathological conditions. As such, leukocyte behavior is an attractive pharmacological target, although knowledge is lacking on the exact triggers driving leukocyte migration. We explored the contribution of coagulation pathways versus TLR4 signaling on the leukocyte recruitment by mimicking non-sterile and sterile leukocyte activation. A sterile inflammatory milieu was generated by spontaneous ex-vivo coagulation of human whole blood, a non-sterile milieu by incubating human whole blood with LPS (at the maximal effective dose), inducing TLR-4 mediated cytokines. The chemoattractant activity of supernatant obtained from these conditions was compared by assessment of primary human leukocyte recruitment in transwell cultures, microscopically and by flow cytometry. Human recombinant IL-8 (100 ng/mL) was used as control chemoattractant.
Ex-vivo coagulation resulted in a strong release of IL-8 (119±58 ng/mL), being 5-fold higher than LPS-induced IL-8 release. Leukocyte recruitment (mainly identified as granulocytes) towards the sterile milieu was greater than TLR4-driven chemoattraction. Comparison of recombinant IL-8-induced and coagulation-induced chemotaxis indicated that chemotaxis towards the coagulated milieu was driven by more factors than IL-8 alone. Additional experiments indicated that fibrinogen cleavage product fibrinopeptide B may be involved, as this dose-dependently increased leukocyte migration.
Coagulation activation drives substantial neutrophil recruitment, primarily mediated by IL-8. For this ex-vivo coagulation model, a stronger and differently mediated leukocyte response was observed compared to TLR4-driven chemoattraction. The model can be used to study the involvement of extracellular coagulation pathways in specific inflammatory pathways that differ distinctly from pathways involved in non-sterile inflammatory conditions. This model, when further optimized, may be helpful as a methodological tool for early evaluation of new treatment modalities for chronic inflammatory diseases.
- © 2013 by American Heart Association, Inc.