Tissue plasminogen activator promotes postischemic neutrophil recruitment via its proteolytic and nonproteolytic properties.
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.


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.
From the Walter Brendel Centre of Experimental Medicine (B.U., G.Z., D.P.-W., M.P., M.R., M.F., M. Hessenauer, M. Holzer, F.K., C.A.R.), Department of Otorhinolaryngology, Head and Neck Surgery (G.Z., M. Hessenauer, M. Holzer, C.A.R.), Department of Surgery, Klinikum der Universität München (A.K.), and Department of Pharmacy (R.F., S.Z.), Ludwig-Maximilians-Universität München, Munich, Germany; and Institute of Pharmaceutical Biology, Biocenter, Goethe-University Frankfurt/Main, Frankfurt am Main, Germany (R.F.).
Bernd
B
Uhl
From the Walter Brendel Centre of Experimental Medicine (B.U., G.Z., D.P.-W., M.P., M.R., M.F., M. Hessenauer, M. Holzer, F.K., C.A.R.), Department of Otorhinolaryngology, Head and Neck Surgery (G.Z., M. Hessenauer, M. Holzer, C.A.R.), Department of Surgery, Klinikum der Universität München (A.K.), and Department of Pharmacy (R.F., S.Z.), Ludwig-Maximilians-Universität München, Munich, Germany; and Institute of Pharmaceutical Biology, Biocenter, Goethe-University Frankfurt/Main, Frankfurt am Main, Germany (R.F.).
Gabriele
G
Zuchtriegel
From the Walter Brendel Centre of Experimental Medicine (B.U., G.Z., D.P.-W., M.P., M.R., M.F., M. Hessenauer, M. Holzer, F.K., C.A.R.), Department of Otorhinolaryngology, Head and Neck Surgery (G.Z., M. Hessenauer, M. Holzer, C.A.R.), Department of Surgery, Klinikum der Universität München (A.K.), and Department of Pharmacy (R.F., S.Z.), Ludwig-Maximilians-Universität München, Munich, Germany; and Institute of Pharmaceutical Biology, Biocenter, Goethe-University Frankfurt/Main, Frankfurt am Main, Germany (R.F.).
Daniel
D
Puhr-Westerheide
From the Walter Brendel Centre of Experimental Medicine (B.U., G.Z., D.P.-W., M.P., M.R., M.F., M. Hessenauer, M. Holzer, F.K., C.A.R.), Department of Otorhinolaryngology, Head and Neck Surgery (G.Z., M. Hessenauer, M. Holzer, C.A.R.), Department of Surgery, Klinikum der Universität München (A.K.), and Department of Pharmacy (R.F., S.Z.), Ludwig-Maximilians-Universität München, Munich, Germany; and Institute of Pharmaceutical Biology, Biocenter, Goethe-University Frankfurt/Main, Frankfurt am Main, Germany (R.F.).
Marc
M
Praetner
From the Walter Brendel Centre of Experimental Medicine (B.U., G.Z., D.P.-W., M.P., M.R., M.F., M. Hessenauer, M. Holzer, F.K., C.A.R.), Department of Otorhinolaryngology, Head and Neck Surgery (G.Z., M. Hessenauer, M. Holzer, C.A.R.), Department of Surgery, Klinikum der Universität München (A.K.), and Department of Pharmacy (R.F., S.Z.), Ludwig-Maximilians-Universität München, Munich, Germany; and Institute of Pharmaceutical Biology, Biocenter, Goethe-University Frankfurt/Main, Frankfurt am Main, Germany (R.F.).
Markus
M
Rehberg
From the Walter Brendel Centre of Experimental Medicine (B.U., G.Z., D.P.-W., M.P., M.R., M.F., M. Hessenauer, M. Holzer, F.K., C.A.R.), Department of Otorhinolaryngology, Head and Neck Surgery (G.Z., M. Hessenauer, M. Holzer, C.A.R.), Department of Surgery, Klinikum der Universität München (A.K.), and Department of Pharmacy (R.F., S.Z.), Ludwig-Maximilians-Universität München, Munich, Germany; and Institute of Pharmaceutical Biology, Biocenter, Goethe-University Frankfurt/Main, Frankfurt am Main, Germany (R.F.).
Matthias
M
Fabritius
From the Walter Brendel Centre of Experimental Medicine (B.U., G.Z., D.P.-W., M.P., M.R., M.F., M. Hessenauer, M. Holzer, F.K., C.A.R.), Department of Otorhinolaryngology, Head and Neck Surgery (G.Z., M. Hessenauer, M. Holzer, C.A.R.), Department of Surgery, Klinikum der Universität München (A.K.), and Department of Pharmacy (R.F., S.Z.), Ludwig-Maximilians-Universität München, Munich, Germany; and Institute of Pharmaceutical Biology, Biocenter, Goethe-University Frankfurt/Main, Frankfurt am Main, Germany (R.F.).
Maximilian
M
Hessenauer
From the Walter Brendel Centre of Experimental Medicine (B.U., G.Z., D.P.-W., M.P., M.R., M.F., M. Hessenauer, M. Holzer, F.K., C.A.R.), Department of Otorhinolaryngology, Head and Neck Surgery (G.Z., M. Hessenauer, M. Holzer, C.A.R.), Department of Surgery, Klinikum der Universität München (A.K.), and Department of Pharmacy (R.F., S.Z.), Ludwig-Maximilians-Universität München, Munich, Germany; and Institute of Pharmaceutical Biology, Biocenter, Goethe-University Frankfurt/Main, Frankfurt am Main, Germany (R.F.).
Martin
M
Holzer
From the Walter Brendel Centre of Experimental Medicine (B.U., G.Z., D.P.-W., M.P., M.R., M.F., M. Hessenauer, M. Holzer, F.K., C.A.R.), Department of Otorhinolaryngology, Head and Neck Surgery (G.Z., M. Hessenauer, M. Holzer, C.A.R.), Department of Surgery, Klinikum der Universität München (A.K.), and Department of Pharmacy (R.F., S.Z.), Ludwig-Maximilians-Universität München, Munich, Germany; and Institute of Pharmaceutical Biology, Biocenter, Goethe-University Frankfurt/Main, Frankfurt am Main, Germany (R.F.).
Andrej
A
Khandoga
From the Walter Brendel Centre of Experimental Medicine (B.U., G.Z., D.P.-W., M.P., M.R., M.F., M. Hessenauer, M. Holzer, F.K., C.A.R.), Department of Otorhinolaryngology, Head and Neck Surgery (G.Z., M. Hessenauer, M. Holzer, C.A.R.), Department of Surgery, Klinikum der Universität München (A.K.), and Department of Pharmacy (R.F., S.Z.), Ludwig-Maximilians-Universität München, Munich, Germany; and Institute of Pharmaceutical Biology, Biocenter, Goethe-University Frankfurt/Main, Frankfurt am Main, Germany (R.F.).
Robert
R
Fürst
From the Walter Brendel Centre of Experimental Medicine (B.U., G.Z., D.P.-W., M.P., M.R., M.F., M. Hessenauer, M. Holzer, F.K., C.A.R.), Department of Otorhinolaryngology, Head and Neck Surgery (G.Z., M. Hessenauer, M. Holzer, C.A.R.), Department of Surgery, Klinikum der Universität München (A.K.), and Department of Pharmacy (R.F., S.Z.), Ludwig-Maximilians-Universität München, Munich, Germany; and Institute of Pharmaceutical Biology, Biocenter, Goethe-University Frankfurt/Main, Frankfurt am Main, Germany (R.F.).
Stefan
S
Zahler
From the Walter Brendel Centre of Experimental Medicine (B.U., G.Z., D.P.-W., M.P., M.R., M.F., M. Hessenauer, M. Holzer, F.K., C.A.R.), Department of Otorhinolaryngology, Head and Neck Surgery (G.Z., M. Hessenauer, M. Holzer, C.A.R.), Department of Surgery, Klinikum der Universität München (A.K.), and Department of Pharmacy (R.F., S.Z.), Ludwig-Maximilians-Universität München, Munich, Germany; and Institute of Pharmaceutical Biology, Biocenter, Goethe-University Frankfurt/Main, Frankfurt am Main, Germany (R.F.).
Fritz
F
Krombach
From the Walter Brendel Centre of Experimental Medicine (B.U., G.Z., D.P.-W., M.P., M.R., M.F., M. Hessenauer, M. Holzer, F.K., C.A.R.), Department of Otorhinolaryngology, Head and Neck Surgery (G.Z., M. Hessenauer, M. Holzer, C.A.R.), Department of Surgery, Klinikum der Universität München (A.K.), and Department of Pharmacy (R.F., S.Z.), Ludwig-Maximilians-Universität München, Munich, Germany; and Institute of Pharmaceutical Biology, Biocenter, Goethe-University Frankfurt/Main, Frankfurt am Main, Germany (R.F.). christoph.reichel@med.uni-muenchen.de.
Christoph A
CA
Reichel
D000818:Animals; D002199:Capillary Permeability; D002478:Cells, Cultured; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D005341:Fibrinolysin / Q000378:metabolism; D018093:Gelatinases / Q000378:metabolism; D006439:Hemodynamics; D006801:Humans; D008297:Male; D008407:Mast Cells / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008833:Microcirculation; D055806:Microvessels / Q000378:metabolism / Q000503:physiopathology; D009132:Muscles / Q000098:blood supply*; D009154:Mutation; D020556:Neutrophil Infiltration / Q000187:drug effects; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology; D011994:Recombinant Proteins / Q000008:administration & dosage; D015427:Reperfusion Injury / Q000201:enzymology* / Q000235:genetics / Q000276:immunology / Q000503:physiopathology; D013997:Time Factors; D010959:Tissue Plasminogen Activator / Q000008:administration & dosage / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D057705:Transendothelial and Transepithelial Migration
D011994:Recombinant Proteins; D005340:Fibrinogen; C067262:PLAT protein, human; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin; D018093:Gelatinases
fibrinolysis; inflammation; leukocytes; microvascular permeability; neutrophils; reperfusion injury; tissue plasminogen activator