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Objective: Neutrophil infiltration of the postischemic tissue considerably contributes to organ dysfunction on ischemia/reperfusion injury. Beyond its established role in fibrinolysis, tissue-type plasminogen activator (tPA) has recently been implicated in nonfibrinolytic processes. The role of this serine protease in the recruitment process of neutrophils remains largely obscure., Approach and Results: Using in vivo microscopy on the postischemic cremaster muscle, neutrophil recruitment and microvascular leakage, but not fibrinogen deposition at the vessel wall, were significantly diminished in tPA(-/-) mice. Using cell transfer techniques, leukocyte and nonleukocyte tPA were found to mediate ischemia/reperfusion-elicited neutrophil responses. Intrascrotal but not intra-arterial application of recombinant tPA induced a dose-dependent increase in the recruitment of neutrophils, which was significantly higher compared with stimulation with a tPA mutant lacking catalytic activity. Whereas tPA-dependent transmigration of neutrophils was selectively reduced on the inhibition of plasmin or gelatinases, neutrophil intravascular adherence was significantly diminished on the blockade of mast cell activation or lipid mediator synthesis. Moreover, stimulation with tPA caused a significant elevation in the leakage of fluorescein isothiocyanate dextran to the perivascular tissue, which was completely abolished on neutrophil depletion. In vitro, tPA-elicited macromolecular leakage of endothelial cell layers was abrogated on the inhibition of its proteolytic activity., Conclusions: Endogenously released tPA promotes neutrophil transmigration to reperfused tissue via proteolytic activation of plasmin and gelatinases. As a consequence, tPA on transmigrating neutrophils disrupts endothelial junctions allowing circulating tPA to extravasate to the perivascular tissue, which, in turn, amplifies neutrophil recruitment through the activation of mast cells and release of lipid mediators., (© 2014 American Heart Association, Inc.)

In vitro studies suggest that leukocytes locomote in an ameboid fashion independently of pericellular proteolysis. Whether this motility pattern applies for leukocyte migration in inflamed tissue is still unknown. In vivo microscopy on the inflamed mouse cremaster muscle revealed that blockade of serine proteases or of matrix metalloproteinases (MMPs) significantly reduces intravascular accumulation and transmigration of neutrophils. Using a novel in vivo chemotaxis assay, perivenular microinjection of inflammatory mediators induced directional interstitial migration of neutrophils. Blockade of actin polymerization, but not of actomyosin contraction abolished neutrophil interstitial locomotion. Multiphoton laser scanning in vivo microscopy showed that the density of the interstitial collagen network increases in inflamed tissue, thereby providing physical guidance to infiltrating neutrophils. Although neutrophils locomote through the interstitium without pericellular collagen degradation, inhibition of MMPs, but not of serine proteases, diminished their polarization and interstitial locomotion. In this context, blockade of MMPs was found to modulate expression of adhesion/signaling molecules on neutrophils. Collectively, our data indicate that serine proteases are critical for neutrophil extravasation, whereas these enzymes are dispensable for neutrophil extravascular locomotion. By contrast, neutrophil interstitial migration strictly relies on actin polymerization and does not require the pericellular degradation of collagen fibers but is modulated by MMPs.