Thrombin enhancement of interleukin-1 and tumor necrosis factor-α induced polymorphonuclear leukocyte migration

BACKGROUND: Cytokines such as IL-1α and tumor necrosis factor-α (TNF-α) activate vascular endothelium to express leukocyte adhesion molecules that promote polymorphonuclear leukocyte (PMNL) migration and to synthesize tissue factor, thus making the endothelium a procoagulant surface. α-Thrombin, generated during coagulation, also activates endothelial cells. Since all these processes are likely involved in inflammation, the effect of α- thrombin on PMNL interaction with cytokine activated endothelium was investigated. EXPERIMENTAL DESIGN: Human umbilical vein endothelium was grown on polycarbonate filters to investigate the effects interleukin-1α (IL-1α), TNF-α, and α-thrombin on PMNL transendothelial migration quantitated with ⁵¹Cr-labeled PMNL, and on endothelial monolayer permeability, quantitated with ¹²⁵I-labeled albumin (HSA). To evaluate the expression of endothelial-leukocyte adhesion molecules, enzyme-linked immunosorbent assay was performed on human umbilical vein endothelium monolayers. The effect of thrombin on PMNL accumulation and plasma exudation in inflammation was studied in a rabbit dermal model, using ⁵¹Cr-labeled blood leukocytes and [¹²⁵I]HSA respectively. RESULTS: On resting human umbilical vein endothelium, α-thrombin induced a transient increase (2.5- to 4-fold) in monolayer permeability lasting 30 minutes. Slight but significant transendothelial migration of ⁵¹Cr-labeled PMNL was induced by α-thrombin (7.4 ± 0.6% of cells added, unstimulated = 1.9 ± 0.4%), although this response was less than that induced by f-norLeu-Leu-Phe (17%), IL-1α (29%) or TNF-α (21%). α-Thrombin enhanced the initial rate of IL-1, TNF-α and f- norLeu-Leu-Phe induced PMNL transendothelial migration in an additive or supradditive manner (e.g., with IL-1α + α-thrombin, migration was 58% greater than additive at 15 to 30 minutes, p < 0.001). Catalytically inactivated α-thrombin, D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone and diisopropyl-fluorophosphate α-thrombin, did not enhance migration or permeability. In dermal inflammation in rabbits, α-thrombin (10 units/site) induced an increase in plasma protein exudation, with only a mild infiltration of PMNL. However, α-thrombin synergistically enhanced the PMNL infiltration induced by IL-1α, TNF-α, but not that induced by zymosan activated plasma (C5a) or IL-8 (neutrophil-activating peptide-1). These measurements were confirmed histologically. Investigations into the mechanisms of the enhancement of PMNL migration indicated that individually vascular permeability changes, prostaglandins, platelet activating factor, and P-selectin expression did not account for the observed effects. CONCLUSIONS: Alpha-thrombin may have a role in synergistically enhancing PMNL infiltration at sites of inflammation, in part via enzymatic action on the cytokine activated endothelium. The mechanisms involved in this effect are likely a complex interaction.