Src Family Kinase-Mediated Vesicle Trafficking Is Critical for Neutrophil Basement Membrane Penetration
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Granulocyte Biology & its Disorders ARTICLE Src family kinase-mediated vesicle trafficking is critical for neutrophil basement membrane Ferrata Storti Foundation penetration Ina Rohwedder,1 Angela R.M. Kurz,1 Monika Pruenster,1 Roland Immler,1 Robert Pick,1 Tanja Eggersmann,1 Sarah Klapproth,1 Jennifer L. Johnson,2 Sergi Masgrau Alsina,1 Clifford A. Lowell,3 Attila Mócsai,4 Sergio D. Catz2 and Markus Sperandio1 1Walter-Brendel-Center of Experimental Medicine, Institute of Cardiovascular Physiology Haematologica 2020 and Pathophysiology, Klinikum der Universität, Ludwig-Maximilians-University Munich, Volume 105(7):1845-1856 Planegg-Martinsried, Germany; 2Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USA; 3Department of Laboratory Medicine, University of California, San Francisco, CA, USA and 4Department of Physiology, Semmelweis University School of Medicine, Budapest, Hungary ABSTRACT EUKOCYTE RECRUITMENT INTO INFLAMED TISSUE IS HIGHLY DEPENDENT ON THE ACTIVATION AND BINDING OF INTEGRINS TO THEIR RESPECTIVE LIGANDS, FOLLOWED LBY THE INDUCTION OF VARIOUS SIGNALING EVENTS WITHIN THE CELL REFERRED TO AS OUTSIDE-IN SIGNALING. SRC FAMILY KINASES (SFK) ARE THE CENTRAL PLAYERS IN THE OUTSIDE-IN SIGNALING PROCESS, ASSIGNING THEM A CRITICAL ROLE FOR PROPER IMMUNE CELL FUNCTION. OUR STUDY INVESTIGATED THE ROLE OF SFK ON NEUTROPHIL RECRUIT- MENT in vivo USING Hck-/- Fgr-/- Lyn-/- MICE, WHICH LACK SFK EXPRESSED IN NEU- TROPHILS. WE SHOW THAT LOSS OF SFK STRONGLY REDUCES NEUTROPHIL ADHESION AND POST-ARREST MODIFICATIONS IN A SHEAR FORCE DEPENDENT MANNER. ADDITIONALLY, WE FOUND THAT IN THE ABSENCE OF SFK, NEUTROPHILS DISPLAY IMPAIRED RAB27A- DEPENDENT SURFACE MOBILIZATION OF NEUTROPHIL ELASTASE, VLA3 AND VLA6 CON- TAINING VESICLES. THIS RESULTS IN A DEFECT IN NEUTROPHIL VASCULAR BASEMENT Correspondence: MEMBRANE PENETRATION AND THUS STRONGLY IMPAIRED EXTRAVASATION. TAKEN MARKUS SPERANDIO TOGETHER, WE DEMONSTRATE THAT SFK PLAY A ROLE IN NEUTROPHIL POST-ARREST MOD- [email protected] IFICATIONS AND EXTRAVASATION DURING ACUTE INFLAMMATION. THESE FINDINGS MAY SUPPORT THE CURRENT EFFORTS TO USE SFK-INHIBITORS IN INFLAMMATORY DISEASES Received: April 29, 2019. WITH UNWANTED NEUTROPHIL RECRUITMENT. Accepted: November 5, 2019. Pre-published: November 7, 2019. Introduction doi:10.3324/haematol.2019.225722 CHRONIC INFLAMMATORY DISEASES ARE AN INCREASING PROBLEM IN WESTERN INDUSTRIAL- IZED COUNTRIES. A HALLMARK OF THESE DISORDERS IS THE RECRUITMENT OF LEUKOCYTES FROM Check the online version for the most updated THE CIRCULATION INTO AFFECTED TISSUES. THIS PROCESS FOLLOWS A WELL-DEFINED CASCADE OF information on this article, online supplements, ACTIVATION AND ADHESION EVENTS STARTING WITH THE INITIAL CAPTURE OF LEUKOCYTES FROM and information on authorship & disclosures: 1 www.haematologica.org/content/105/7/1845 THE BLOOD STREAM, FOLLOWED BY ROLLING ALONG INFLAMED ENDOTHELIUM. BOTH STEPS ARE mediated by selectins interacting with glycosylated ligands on leukocytes.2 THROUGH BINDING OF THE LEUKOCYTE SPECIFIC INTEGRIN LFA1 ( L 2) TO ICAM-1 ON α β ©2020 Ferrata Storti Foundation ENDOTHELIAL CELLS, LEUKOCYTES SLOW DOWN THEIR ROLLING VELOCITY, AND IN COMBINATION WITH CHEMOKINE STIMULATION, FIRMLY ARREST ON THE ENDOTHELIAL SURFACE. THIS IS FOL- Material published in Haematologica is covered by copyright. LOWED BY POST-ARREST MODIFICATIONS, A PROCESS CHARACTERIZED BY CELL SPREADING, All rights are reserved to the Ferrata Storti Foundation. Use of published material is allowed under the following terms and CYTOSKELETON REARRANGEMENTS AND CRAWLING ALONG THE ENDOTHELIUM, THAT IS CRITICAL conditions: FOR TIGHT ADHESION TO THE SUBSTRATE AND ALLOWS AN APPROPRIATE SPOT FOR EXTRAVASATION https://creativecommons.org/licenses/by-nc/4.0/legalcode. INTO TISSUE. TRANSMIGRATION INVOLVES THE CROSSING OF THE VENULAR WALL AND THE UNDER- Copies of published material are allowed for personal or inter- LYING VASCULAR BASEMENT MEMBRANE (BM), TWO STEPS WHICH ARE STILL INCOMPLETELY nal use. Sharing published material for non-commercial pur- poses is subject to the following conditions: UNDERSTOOD. SEVERAL REPORTS SUGGEST A ROLE FOR THE INTEGRINS VLA3 (α3β1) AND VLA6 https://creativecommons.org/licenses/by-nc/4.0/legalcode, 3-5 (α6β1) ALONG WITH NEUTROPHIL ELASTASE (NE) IN THIS PROCESS. RECENTLY, OUR GROUP HAS sect. 3. Reproducing and sharing published material for com- SHOWN THAT NEUTROPHILS TRANSLOCATE VLA3, VLA6 AND NE FROM INTERNALLY STORED mercial purposes is not allowed without permission in writing VESICLES TO THE CELL SURFACE, TO SUBSEQUENTLY CROSS THE VASCULAR BM.6 THE RELEASE OF from the publisher. THESE VESICLES IS INITIATED BY INTERACTIONS OF NEUTROPHILS WITH THE INFLAMED ENDOTHE- LIUM IN A PECAM-1/ICAM-1- AND CXCL1-DEPENDENT MANNER. VESICLE TRANSPORT IS haematologica | 2020; 105(7) 1845 I. Rohwedder et al. MAINLY REGULATED BY RAB GTPASES AND THEIR EFFECTOR PRO- USA). ISOLATED NEUTROPHILS FROM WILDTYPE AND SFK-KO ANIMALS TEINS WITH RAB27A BEING THE MAIN RAB MOLECULE INVOLVED WERE LABELED WITH CELLTRACKER™ GREEN CMFDA DYE AND IN THE SECRETORY MACHINERY OF NEUTROPHILS.7 RAB27A FUNC- CELLTRACKER™ RED CMTPX DYE (THERMO FISHER, WALTHAM, MA, TION IS MEDIATED BY THE TWO EFFECTOR MOLECULES SYNAPTO- USA), RESPECTIVELY, AND DISTRIBUTED INTO THE UPPER CHAMBER COM- TAGMIN-LIKE PROTEIN 1 (JFC1, ENCODED BY SYTL1 IN MICE) AND PARTMENT IN A 1:1 RATIO. TIME-LAPSE MICROSCOPY WITH AN INTERVAL OF MUNC13-4 (UNC13D).8 ALTHOUGH MOST OF THESE PROCESSES 50 SECONDS WAS PERFORMED USING AN UPRIGHT SPINNING-DISK CONFOCAL RELY ON INTEGRIN SIGNALING, INTEGRINS THEMSELVES LACK ENZY- MICROSCOPE (EXAMINER; ZEISS) EQUIPPED WITH A CONFOCAL SCANNER MATIC ACTIVITY. THEREFORE, NUMEROUS PROTEINS ARE RECRUITED UNIT CSU-X1 (YOKOGAWA ELECTRIC CORPORATION, JAPAN), AN EMCCD TO THEIR INTRACELLULAR TAILS, AMONG THOSE SRC FAMILY KINASES CAMERA (EVOLVE; PHOTOMETRICS), AND A X20/1.0 NA WATER IMMER- (SFK). THEIR EARLY RECRUITMENT DURING INTEGRIN ACTIVATION SION OBJECTIVE (PLAN APOCHROMAT; ZEISS). 3D IMAGES (70 Z-STACKS ASSIGNS SFK A CRITICAL ROLE IN THE SO-CALLED OUTSIDE-IN SIGNAL- WITH A STEP SIZE OF 2µM) WERE ACQUIRED PER TIME POINT AND ANA- ING PROCESS AND THUS REGULATION OF CENTRAL SIGNALING PATH- LYZED BY GENERATING MAXIMUM Z-PROJECTIONS OVER TIME USING WAYS DOWNSTREAM OF INTEGRIN-RECEPTOR LIGATION.9 SLIDEBOOK 6.0.8 SOFTWARE (3I) AND IMAGEJ. INTERACTION STRENGTH WAS NEUTROPHILS EXPRESS THREE MEMBERS OF THIS FAMILY, NAMELY ANALYZED WITH THE MOSAICIA INTERACTION PLUGIN OF FIJI. HCK, FGR AND LYN.10 THEIR FUNCTION HAS BEEN INTENSIVELY FUNCTIONAL in vitro AND in vivo EXPERIMENTS, INCLUDING INTRAVITAL STUDIED IN SFK SINGLE, DOUBLE (Hck-/-Fgr-/-), AND TRIPLE KNOCK- MICROSCOPY,19 PERITONITIS EXPERIMENTS, FLOW CHAMBER EXPERIMENTS, OUT (KO) (Hck-/-Fg-/-Lyn-/-) MICE,11 DEMONSTRATING A ROLE FOR VESICLE TRAFFICKING, FLOW CYTOMETRY, WESTERN BLOT ANALYSIS AND STA- SFK IN INTEGRIN ACTIVATION12 AND THEIR DOWNSTREAM SIGNAL- TISTICS ARE DESCRIBED IN DETAIL IN THE Online Supplementary Methods. ING. NEUTROPHILS ISOLATED FROM Hck-/-Fgr-/- MICE SHOWED POOR SPREADING, INDICATING THAT INTEGRIN OUTSIDE-IN SIGNAL- Data sharing statement ING IS IMPAIRED.13 ADDITIONALLY, NEUTROPHIL MIGRATION INTO ORIGINAL DATA ARE AVAILABLE ON REQUEST. THE LIVER OF Hck-/-Fgr-/- MICE IN AN in vivo ENDOTOXEMIA MODEL IS SEVERELY REDUCED.14 FURTHERMORE, KOVáCS et al. RECENTLY SHOWED THAT NEUTROPHILS EXHIBIT REDUCED CAPABIL- Results ITY TO CREATE A MICROINFLAMMATORY ENVIRONMENT, RESULTING IN DIMINISHED NEUTROPHIL EXTRAVASATION IN A MODEL OF Src family kinase depletion reduces neutrophil AUTOANTIBODY-INDUCED ARTHRITIS AND INFLAMMATORY BLISTER- adhesion and extravasation in inflamed postcapillary ING SKIN DISEASE.15 venules in vivo OUR STUDY AIMED TO INVESTIGATE THE EFFECT OF SFK ON WE FIRST INVESTIGATED HOW LOSS OF NEUTROPHIL-EXPRESSED LEUKOCYTE RECRUITMENT IN AN in vivo SETTING OF ACUTE INFLAM- SFK INFLUENCES NEUTROPHIL ADHESION IN TNFα-STIMULATED MATION, FOCUSING ON POST-ARREST MODIFICATIONS AND THE CREMASTER MUSCLE POSTCAPILLARY VENULES USING INTRAVITAL MOLECULAR MECHANISM OF VASCULAR BM PENETRATION. WE MICROSCOPY. INTERESTINGLY, THE ABSOLUTE NUMBER OF ADHER- SHOW THAT, IN THE GENETIC ABSENCE OF SFK, THESE TWO STEPS ENT NEUTROPHILS WAS STRONGLY INCREASED COMPARED TO WILD- ARE STRONGLY IMPAIRED. ADHERENT Hck-/-Fgr-/-Lyn-/- NEUTROPHILS TYPE CONTROL MICE (Online Supplementary Figure S1A). ARE UNABLE TO WITHSTAND SHEAR FORCES AND DISPLAY DIMIN- HOWEVER, AFTER ADJUSTING THE NUMBER OF ADHERENT NEU- ISHED LFA1 CLUSTERING WITH REDUCED PHOSPHORYLATION LEVELS TROPHILS TO THE CIRCULATING NEUTROPHIL COUNT, WHICH WAS SIG- OF PAXILLIN, CORTACTIN AND SYK, SUGGESTING THAT SFK DEPLE- NIFICANTLY HIGHER IN SFK-KO MICE COMPARED TO WILDTYPE TION RESULTS IN SEVERELY IMPAIRED ADHESION STRENGTHENING. MICE (Online Supplementary Figure S1B and C), THE NEU- IN ADDITION, WE SHOW THAT SFK ARE CRITICAL FOR CROSSING THE TROPHIL ADHESION EFFICIENCY CALCULATED AS NUMBER OF ADHER- VASCULAR BM DURING NEUTROPHIL EXTRAVASATION BY FACILITAT- ENT CELLS/MM2 DIVIDED BY THE SYSTEMIC NEUTROPHIL COUNT ING TRANSLOCATION OF VLA3-, VLA6- AND NE-CONTAINING WAS SIGNIFICANTLY REDUCED IN SFK-KO MICE (0.48±0.04) COM- VESICLES TO THE CELL SURFACE. THEREFORE, SFK ARE NOT ONLY PARED TO WILDTYPE MICE (1.04±0.08) (FIGURE 1A). THIS WAS IMPORTANT MEDIATORS OF NEUTROPHIL POST-ARREST MODIFICA- NOT DUE TO ALTERED SURFACE EXPRESSION OF ROLLING AND ADHE- TIONS, BUT ARE ALSO REQUIRED TO BREACH THE VASCULAR BM. SION RELEVANT SURFACE PROTEINS INCLUDING CD18, CD11A, CD11B, CD62L,