International Journal of Molecular Sciences Article Recombinant Tissue Plasminogen Activator (r-tPA) Induces In-Vitro Human Neutrophil Migration via Low Density Lipoprotein Receptor-Related Protein 1 (LRP-1) 1,2, 2, 2 3 4 Luca Liberale y , Maria Bertolotto y, Silvia Minetti , Paola Contini , Daniela Verzola , Pietro Ameri 5,6, Giorgio Ghigliotti 5,6, Aldo Pende 5,7, Giovanni G. Camici 1, Federico Carbone 2,5 and Fabrizio Montecucco 2,5,* 1 Center for Molecular Cardiology, University of Zürich, Wagistrasse 12, 8952 Schlieren, Switzerland; [email protected] (L.L.); [email protected] (G.G.C.) 2 First Clinic of Internal Medicine, Department of Internal Medicine, University of Genoa, 6 Viale Benedetto XV, 16132 Genoa, Italy; [email protected] (M.B.); [email protected] (S.M.); [email protected] (F.C.) 3 Clinical Immunology, Department of Internal Medicine, University of Genoa, 6 viale Benedetto XV, 16132 Genoa, Italy; [email protected] 4 Division of Nephrology, Dialysis and Transplantation, Department of Internal Medicine, University of Genoa, 6 Viale Benedetto XV, 16132 Genoa, Italy; [email protected] 5 IRCCS Ospedale Policlinico San Martino Genoa—Italian Cardiovascular Network, 10 Largo Benzi, 16132 Genoa, Italy; [email protected] (P.A.); [email protected] (G.G.); [email protected] (A.P.) 6 Laboratory of Cardiovascular Biology, IRCCS Ospedale Policlinico San Martino & Department of Internal Medicine, University of Genoa, 16126 Genoa, Italy 7 Clinic of Emergency Medicine, Department of Emergency Medicine, University of Genoa, 16126 Genoa, Italy * Correspondence: [email protected]; Tel.: +39-010-353-8694; Fax: +39-010-353-8686 These authors equally contributed as first author. y Received: 9 September 2020; Accepted: 22 September 2020; Published: 23 September 2020 Abstract: Thrombolysis is the gold standard treatment for acute ischemic stroke. Besides its fibrinolytic role, recombinant tissue plasminogen activator (r-tPA) holds several non-fibrinolytic functions. Here, we investigated the potential role of r-tPA on human primary neutrophil migration in vitro. By means of modified Boyden chamber migration assay and checkerboard analysis we showed a dose-dependent chemotactic effect of r-TPA with a maximum effect reached by 0.03 mg/mL (0.003–1 mg/mL). Pre-incubation with MAP kinases inhibitors allowed the identification of PI3K/Akt, but not ERK1/2 as the intracellular pathway mediating the observed effects. Furthermore, by means of real-time PCR, immunocytochemistry and cytofluorimetry we demonstrated that the r-tPA receptor low density lipoprotein receptor-related protein 1 (LRP-1) is synthetized and expressed by neutrophils in response to r-tPA and TNF-α. Inhibition of LRP-1 by receptor-associated protein (RAP), prevented r-tPA-mediated F-actin polymerization, migration and signal through Akt but not ERK1/2. Lastly, also neutrophil degranulation in response to r-tPA seems to be mediated by LRP-1 under adhesion conditions. In conclusion, we show that r-tPA induces neutrophil chemotaxis through LRP-1/Akt pathway. Blunting r-tPA-mediated neutrophil activation might be beneficial as an adjuvant therapy to thrombolysis in this setting. Keywords: tissue plasminogen activator; neutrophil; inflammation Int. J. Mol. Sci. 2020, 21, 7014; doi:10.3390/ijms21197014 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 7014 2 of 14 1. Introduction Cerebro- and cardiovascular acute ischemic afflictions such as myocardial infarction and ischemic stroke benefit of early reperfusion to reduce organ damage. Together with interventional approaches, reperfusion strategies include the infusion of fibrinolytic agents such as recombinant tissue plasminogen activator (r-tPA). Currently, r-tPA represents the only medical therapy to improve outcomes in patients with acute ischemic stroke and its intravenous injection is recommended under specific conditions by international guidelines [1,2]. Yet, the late restoration of the blood flow in a previously ischemic tissue associates with a paradoxical exacerbation of inflammation and oxidative stress leading to cellular dysfunction and death, the so-called ischemia/reperfusion (I/R) injury [3]. Beside their important roles in host defence against infectious agents, neutrophils have been implicated in the pathogenesis of different conditions characterized by sterile inflammation such as I/R injury [4,5]. After blood flow re-establishment, circulating neutrophils are recruited early within the tissue and concur to its damage by different mechanisms including release of pro-inflammatory and pro-oxidant mediators such as interleukins and reactive oxygen species, degranulation of different proteases including matrix metalloproteases (MMPs), myeloperoxidase (MPO) and neutrophil elastase or formation of neutrophil extracellular traps [6]. Of interest, together with its direct action on vascular patency, r-tPA was also shown to contribute to I/R injury indirectly by inducing neutrophil degranulation both in-vitro and in-vivo [7]. Yet, the effect of r-tPA on neutrophil function remains only partially investigated, specifically the receptors and pathways involved in such process have not been fully identified. This information will be of utmost importance to counteract r-tPA-induced neutrophil activation potentially widening our interventional options in the setting of I/R injury and ischemic stroke. Accordingly, in this study, we investigated the role of r-tPA in human primary neutrophil migration. Furthermore, we characterized the membrane receptor and the intracellular pathways underlying the observed effect. 2. Results 2.1. r-tPA Induces Dose-Dependent Migration of Human Neutrophils Locomotory function of freshly isolated human neutrophils from healthy volunteers was tested in response to r-tPA employing the modified Boyden chamber assay with nitrocellulose filter. Concentrations of r-tPA herein employed are within the range observed in serum of patients undergoing thrombolysis [8]. Neutrophil migration toward different doses of r-tPA displayed the typical bell-shaped dose-response graphic of chemoattractants (Figure1A). The magnitude of neutrophil migration induced by 0.03 mg/mL r-tPA was similar to that generated by 1 nM chemokine (C-X-C motif) ligand 8 (CXCL8) (Figure1A), suggesting for r-tPA a powerful locomotion-inducing activity. To understand whether the migration was due to the induction of chemotaxis or to chemokinesis, we performed a checkerboard analysis with increasing concentrations of r-tPA above and below the filter (Figure1B). A directed migration vs. a gradient across the filter rather than random migration was observed indicating that r-tPA induces chemotaxis rather than random chemokinesis. Lastly, to investigate the signaling pathways involved in neutrophils migration towards r-tPA, we performed experiments with selective kinase inhibitors for PI3K and MEK1/2 (ERK1/2 activator). Figure1C shows that pre-incubation with 10 µM LY294002 (PI3K inhibitor) significantly reduced r-tPA-induced neutrophil migration. On the opposite, neutrophil pre-treatment with the MEK1/2 inhibitor U0126 had no effect on their migration (Figure1C). Int. J. Mol. Sci. 2020, 21, 7014 3 of 14 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 14 Figure 1.FigureRecombinant 1. Recombinant tissue tissue plasminogen plasminogen activator (r-tPA) (r-tPA) induces induces dose-dependent dose-dependent migration migrationin in humanhuman neutrophils. neutrophils. ( A(A)) The graphic graphic represents represents the migration the migration assay of neutrophils assay of neutrophilstowards control towards control mediummedium (CTL), (CTL), r-tPA r-tPA (0.003 (0.003 mg/mL mg up/mL to 1 up mg/mL) to 1 mgor 1/ mL)nM CXCL8 or 1 nMas positive CXCL8 control. as positive Data are control. Data areexpressed as asmean mean of migration of migration distance distance (µm) ± (µ1 m)SEM 1in SEMthe nitrocellulose in the nitrocellulose assay, n = 5. assay, (B) n = 5. ± (B) CheckerboardCheckerboard analysis analysis showing showing that neutrophil neutrophil migration migration depends depends on a r-tPA on gradient a r-tPA across gradient the across filter. Untreated monocytes were seeded with increasing concentrations of r-tPA in the upper well, the filter. Untreated monocytes were seeded with increasing concentrations of r-tPA in the upper increasing concentrations of r-tPA were also added to the lower well. Data are expressed as mean of well, increasingchemotaxis concentrations index in the polycarbonate of r-tPA were assay, also n = added5. (C) Pre-treatment to the lower with well. PI3K DataInhibitor are reduces expressed as mean of chemotaxisneutrophils migration index in towards the polycarbonate r-tPA. Human assay,neutrophilsn = 5.were (C )pr Pre-treatmente-treated for 1 h with with PI3Kcontrol Inhibitor reduces neutrophilsmedium (CTL) migration or 10 µM towardsLY294002 r-tPA.(PI3K inhibitor, Human neutrophilsLY) or 10 µM wereU0126 pre-treated(MEK 1/2 inhibitor). for 1 h withThen control medium (CTL)their chemotactic or 10 µM function LY294002 was (PI3K tested inhibitor,towards 0.03 LY) mg/mL or 10 r-tPAµM U0126or 1nM (MEKCXCL8 1as/2 positive inhibitor). control. Then their Data are expressed as mean of migration distance (µm) ±1 SEM in the nitrocellulose assay, n = 6. chemotactic function was tested towards 0.03 mg/mL r-tPA or 1nM CXCL8 as positive control. Data are expressed as mean of migration distance (µm) 1 SEM in the nitrocellulose assay, n = 6. ± 2.2. Low-Density Lipoprotein Receptor-Related Protein-1 (LRP-1) Expression