Platelet Recruitment to the Inflamed Glomerulus Occurs via an αIIbβ3/GPVI-Dependent Pathway
2010; Elsevier BV; Volume: 177; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2010.091143
ISSN1525-2191
AutoresSapna Devi, Michael Kuligowski, Rain Kwan, Erik Westein, Shaun P. Jackson, A. Richard Kitching, Michael J. Hickey,
Tópico(s)Renal Diseases and Glomerulopathies
ResumoRecruitment of leukocytes to glomeruli is fundamental to the pathogenesis of many forms of glomerulonephritis. In a model of glomerulonephritis induced by in situ immune complex deposition, we previously observed that, in addition to leukocytes, platelets accumulate in glomerular capillaries, where they contribute to leukocyte recruitment. However, the mechanisms of platelet recruitment and the role of platelet-expressed P-selectin in leukocyte recruitment require further investigation. We used intravital microscopy to examine the mechanisms of platelet and leukocyte recruitment to glomeruli of mice following administration of an antibody against the glomerular basement membrane (anti-GBM antibody). Platelet recruitment was initiated within five minutes of administration of anti-GBM antibody. This was unaltered by inhibition of platelet GPIbα but was prevented by the absence of platelet GPVI. Fibrinogen was deposited in glomerular capillaries via a partially intercellular adhesion molecule 1 (ICAM-1)–dependent mechanism, and inhibition of αIIbβ3, fibrinogen and ICAM-1 inhibited platelet recruitment. Notably, neutrophil depletion also reduced platelet accumulation, indicating a cooperative interaction underlying recruitment of platelets and neutrophils. Finally, using bone marrow chimeras to restrict expression of P-selectin to platelets or endothelial cells, platelet but not endothelial P-selectin was required for glomerular leukocyte recruitment. Together these data indicate that platelet recruitment in this model is dependent on the combined actions of GPVI and the αIIbβ3/fibrinogen/ICAM-1 pathway and that platelet P-selectin is crucial for subsequent leukocyte recruitment. Recruitment of leukocytes to glomeruli is fundamental to the pathogenesis of many forms of glomerulonephritis. In a model of glomerulonephritis induced by in situ immune complex deposition, we previously observed that, in addition to leukocytes, platelets accumulate in glomerular capillaries, where they contribute to leukocyte recruitment. However, the mechanisms of platelet recruitment and the role of platelet-expressed P-selectin in leukocyte recruitment require further investigation. We used intravital microscopy to examine the mechanisms of platelet and leukocyte recruitment to glomeruli of mice following administration of an antibody against the glomerular basement membrane (anti-GBM antibody). Platelet recruitment was initiated within five minutes of administration of anti-GBM antibody. This was unaltered by inhibition of platelet GPIbα but was prevented by the absence of platelet GPVI. Fibrinogen was deposited in glomerular capillaries via a partially intercellular adhesion molecule 1 (ICAM-1)–dependent mechanism, and inhibition of αIIbβ3, fibrinogen and ICAM-1 inhibited platelet recruitment. Notably, neutrophil depletion also reduced platelet accumulation, indicating a cooperative interaction underlying recruitment of platelets and neutrophils. Finally, using bone marrow chimeras to restrict expression of P-selectin to platelets or endothelial cells, platelet but not endothelial P-selectin was required for glomerular leukocyte recruitment. Together these data indicate that platelet recruitment in this model is dependent on the combined actions of GPVI and the αIIbβ3/fibrinogen/ICAM-1 pathway and that platelet P-selectin is crucial for subsequent leukocyte recruitment. In most tissues, leukocyte-endothelial cell interactions associated with inflammatory responses are restricted to postcapillary venules, where leukocytes undergo a well-characterized sequence of tethering, rolling, and arrest interactions before entering the tissue via transmigration.1Springer TA Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm.Cell. 1994; 76: 301-314Abstract Full Text PDF PubMed Scopus (6395) Google Scholar In general these interactions do not occur in capillaries, due to the minimal endothelial expression of specific adhesion molecules required, in particular, for the rolling step.2Jung U Ley K Regulation of E-selectin. P-selectin, and intercellular adhesion molecule 1 expression in mouse cremaster muscle vasculature.Microcirculation. 1997; 4: 311-319Crossref PubMed Scopus (97) Google Scholar, 3Ley K Gaehtgens P Endothelial, not hemodynamic, differences are responsible for preferential leukocyte rolling in rat mesenteric venules.Circ Res. 1991; 69: 1034-1041Crossref PubMed Scopus (212) Google Scholar However, in some specialized areas of the vasculature, particularly the glomerulus, capillaries can support leukocyte-endothelial cell interactions.4Kuligowski MP Kitching AR Hickey MJ Leukocyte recruitment to the inflamed glomerulus: a critical role for platelet-derived P-selectin in the absence of rolling.J Immunol. 2006; 176: 6991-6999Crossref PubMed Scopus (109) Google Scholar, 5Mizgerd JP Meek BB Kutkoski GJ Bullard DC Beaudet AL Doerschuk CM Selectins and neutrophil traffic: margination and Streptococcus pneumoniae-induced emigration in murine lungs.J Exp Med. 1996; 184: 639-645Crossref PubMed Scopus (150) Google Scholar, 6Wong J Johnston B Lee SS Bullard DC Smith CW Beaudet AL Kubes P A minimal role for selectins in the recruitment of leukocytes into the inflamed liver microvasculature.J Clin Invest. 1997; 99: 2782-2790Crossref PubMed Scopus (316) Google Scholar Indeed, leukocyte recruitment to the glomerulus is a key contributor to the pathogenesis of many forms of glomerulonephritis (GN).4Kuligowski MP Kitching AR Hickey MJ Leukocyte recruitment to the inflamed glomerulus: a critical role for platelet-derived P-selectin in the absence of rolling.J Immunol. 2006; 176: 6991-6999Crossref PubMed Scopus (109) Google Scholar, 7Holdsworth SR Tipping PG Leukocytes in glomerular injury.Semin Immunopathol. 2007; 29: 355-374Crossref PubMed Scopus (35) Google Scholar, 8Ikezumi Y Kanno K Karasawa T Han GD Ito Y Koike H Toyabe S Uchiyama M Shimizu F Kawachi H The role of lymphocytes in the experimental progressive glomerulonephritis.Kidney Int. 2004; 66: 1036-1048Crossref PubMed Scopus (27) Google Scholar, 9Zachem CR Alpers CE Way W Shankland SJ Couser WG Johnson RJ A role for P-selectin in neutrophil and platelet infiltration in immune complex glomerulonephritis.J Am Soc Nephrol. 1997; 8: 1838-1844PubMed Google Scholar To investigate the basis of this unusual form of leukocyte recruitment, we recently used intravital microscopy to visualize glomeruli undergoing an inflammatory response.4Kuligowski MP Kitching AR Hickey MJ Leukocyte recruitment to the inflamed glomerulus: a critical role for platelet-derived P-selectin in the absence of rolling.J Immunol. 2006; 176: 6991-6999Crossref PubMed Scopus (109) Google Scholar We found that recruitment of leukocytes to glomerular capillaries occurs via a process of immediate arrest, bypassing the conventional requirement for an initial rolling interaction. Intriguingly, this form of recruitment retained a role for the P-selectin/PSGL-1 pathway normally associated with leukocyte rolling. Moreover, platelets accumulated in glomeruli during this response, where they played key roles in leukocyte recruitment and resultant glomerular injury. Together these findings suggested the existence of a unique mechanism for leukocyte recruitment in this vascular bed. Clinical observations have reported platelet involvement in the pathogenesis of various forms of GN.10Barnes JL Platelets in glomerular disease.Nephron. 1997; 77: 378-393Crossref PubMed Scopus (29) Google Scholar, 11Zoja C Remuzzi G Role of platelets in progressive glomerular diseases.Pediatr Nephrol. 1995; 9: 495-502Crossref PubMed Scopus (32) Google Scholar, 12Cameron JS Platelets in glomerular disease.Annu Rev Med. 1984; 35: 175-180Crossref PubMed Scopus (43) Google Scholar In an immune complex model of GN, platelet depletion has been shown to reduce leukocyte infiltration and glomerular injury.13Ideura T Ogasawara M Tomura S Ida T Chida Y Kuriyama R Takeuchi J Motomiya T Yamazaki H Effect of thrombocytopenia on the onset of immune complex glomerulonephritis.Nephron. 1992; 60: 49-55Crossref PubMed Scopus (6) Google Scholar, 14Johnson RJ Alpers CE Pritzl P Schulze M Baker P Pruchno C Couser WG Platelets mediate neutrophil-dependent immune complex nephritis in the rat.J Clin Invest. 1988; 82: 1225-1235Crossref PubMed Scopus (80) Google Scholar However, little is known regarding the mechanisms of platelet recruitment to glomeruli. In addition to P-selectin, which is expressed upon platelet activation, platelets express a wide range of adhesive glycoprotein (GP) receptors with the potential to mediate platelet adhesion in the microcirculation. Platelet GPIbα can bind to von Willebrand factor (vWF) present on endothelial cells or the vascular wall and has also been shown to bind to P-selectin and Mac-1.15Simon DI Chen Z Xu H Li CQ Dong J McIntire LV Ballantye CM Zhang Li Furman MI Berndt MC Lopez JA Platelet Glycoprotein Ib-alpha is a Counterreceptor for the Leukocyte Integrin Mac-1 (CD11b/CD18).J Exp Med. 2000; 192: 193-204Crossref PubMed Scopus (509) Google Scholar, 16Romo GM Dong JF Schade AJ Gardiner EE Kansas GS Li CQ McIntire LV Berndt MC Lopez JA The glycoprotein Ib-IX-V complex is a platelet counterreceptor for P-selectin.J Exp Med. 1999; 190: 803-814Crossref PubMed Scopus (291) Google Scholar Platelet GPVI is a receptor for collagen,17Nieswandt B Brakebusch C Bergmeier W Schulte V Bouvard D Mokhtari-Nejad R Lindhout T Heemskerk JW Zirngibl H Fassler R Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen.EMBO J. 2001; 20: 2120-2130Crossref PubMed Scopus (443) Google Scholar and the αIIbβ3 integrin (GPIIb/IIIa) interacts with numerous ligands including fibrinogen, fibronectin, and thrombospondin.18Zarbock A Polanowska-Grabowska RK Ley K Platelet-neutrophil-interactions: linking hemostasis and inflammation.Blood Rev. 2007; 21: 99-111Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar, 19Andrews RK Shen Y Gardiner EE Berndt MC Platelet adhesion receptors and (patho)physiological thrombus formation.Histol Histopathol. 2001; 16: 969-980PubMed Google Scholar, 20Massberg S Enders G Matos FC Tomic LI Leiderer R Eisenmenger S Messmer K Krombach F Fibrinogen deposition at the postischemic vessel wall promotes platelet adhesion during ischemia-reperfusion in vivo.Blood. 1999; 94: 3829-3838Crossref PubMed Google Scholar, 21Savage B Saldivar E Ruggeri ZM Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von Willebrand factor.Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1009) Google Scholar, 22Massberg S Gawaz M Gruner S Schulte V Konrad I Zohlnhofer D Heinzmann U Nieswandt B A crucial role of glycoprotein VI for platelet recruitment to the injured arterial wall in vivo.J Exp Med. 2003; 197: 41-49Crossref PubMed Scopus (431) Google Scholar Examples of the contributions of these molecular pathways to platelet accumulation in the inflamed microvasculature include intestinal ischemia and reperfusion, in which platelet accumulation has been shown to be mediated via interaction with fibrinogen on the endothelial surface.20Massberg S Enders G Matos FC Tomic LI Leiderer R Eisenmenger S Messmer K Krombach F Fibrinogen deposition at the postischemic vessel wall promotes platelet adhesion during ischemia-reperfusion in vivo.Blood. 1999; 94: 3829-3838Crossref PubMed Google Scholar Platelets have also been shown to interact with the inflamed endothelium via both endothelial and platelet P-selectin.23Ishikawa M Cooper D Arumugam TV Zhang JH Nanda A Granger DN Platelet-leukocyte-endothelial cell interactions after middle cerebral artery occlusion and reperfusion.J Cereb Blood Flow Metab. 2004; 24: 907-915Crossref PubMed Scopus (127) Google Scholar, 24Cooper D Chitman KD Williams MC Granger DN Time-depenedent platelet-vessel wall interactions induced by intestinal ischaemia-reperfusion.Am J Physiol Gastrointest Liver Physiol. 2003; 284: G1027-G1033Crossref PubMed Scopus (59) Google Scholar, 25Massberg S Enders G Leiderer R Eisenmenger S Vestweber D Krombach F Messmer K Platelet-endothelial cell interactions during ischemia/reperfusion: the role of P-selectin.Blood. 1998; 92: 507-515Crossref PubMed Google Scholar, 26Frenette PS Johnson RC Hynes RO Wagner DD Platelets roll on stimulated endothelium in vivo: An interaction mediated by endothelial P-selectin.Proc Natl Acad Sci USA. 1995; 92: 7450-7454Crossref PubMed Scopus (408) Google Scholar However, in the glomerulus, capillary endothelial cells do not express P-selectin.2Jung U Ley K Regulation of E-selectin. P-selectin, and intercellular adhesion molecule 1 expression in mouse cremaster muscle vasculature.Microcirculation. 1997; 4: 311-319Crossref PubMed Scopus (97) Google Scholar, 27Rosenkranz AR Mendrick DL Cotran RS Mayadas TN P-selectin deficiency exacerbates experimental glomerulonephritis: a protective role for endothelial P-selectin in inflammation.J Clin Invest. 1999; 103: 649-659Crossref PubMed Scopus (106) Google Scholar, 28McEver RP Beckstead JH Moore KL Marshall-Carlson L Bainton DF GMP-140, a platelet alpha-granule membrane protein, is also synthesized by vascular endothelial cells and is localized in Weibel-Palade bodies.J Clin Invest. 1989; 84: 92-99Crossref PubMed Scopus (838) Google Scholar Moreover, we observed that P-selectin blockade did not abolish platelet recruitment in the inflamed glomerulus, indicating that P-selectin was not involved in this process.4Kuligowski MP Kitching AR Hickey MJ Leukocyte recruitment to the inflamed glomerulus: a critical role for platelet-derived P-selectin in the absence of rolling.J Immunol. 2006; 176: 6991-6999Crossref PubMed Scopus (109) Google Scholar This raises the possibility that one or more of the candidate platelet adhesion receptors described above mediates this response. The molecular basis of the contribution of platelets to glomerular leukocyte recruitment also requires further investigation. Platelets have been shown to support leukocyte recruitment in numerous tissues under a range of inflammatory conditions.25Massberg S Enders G Leiderer R Eisenmenger S Vestweber D Krombach F Messmer K Platelet-endothelial cell interactions during ischemia/reperfusion: the role of P-selectin.Blood. 1998; 92: 507-515Crossref PubMed Google Scholar, 29Carvalho-Tavares J Hickey MJ Hutchison J Michaud J Sutcliffe IT Kubes P A role for platelets and endothelial selectins in tumor necrosis factor-alpha-induced leukocyte recruitment in the brain microvasculature.Circ Res. 2000; 87: 1141-1148Crossref PubMed Scopus (211) Google Scholar, 30Asaduzzaman M Lavasani S Rahman M Zhang S Braun OO Jeppsson B Thorlacius H Platelets support pulmonary recruitment of neutrophils in abdominal sepsis.Crit Care Med. 2009; 37: 1389-1396Crossref PubMed Scopus (122) Google Scholar, 31Huo Y Schober A Forlow SB Smith DF Hyman MC Jung S Littman DR Weber C Ley K Circulating activated platelets exacerbate atherosclerosis in mice deficient in apolipoprotein E.Nat Med. 2003; 9: 61-67Crossref PubMed Scopus (853) Google Scholar In many cases, platelet-expressed P-selectin has been found to be critical to this response,32Fernandes LS Conde ID Wayne Smith C Kansas GS Snapp KR Bennet N Ballantyne C McIntire LV O'Brian Smith E Klem JA Mathew S Frangogiannis N Turner NA Maresh KJ Kleiman NS Platelet-monocyte complex formation: effect of blocking PSGL-1 alone, and in combination with alphaIIbbeta3 and alphaMbeta2, in coronary stenting.Thromb Res. 2003; 111: 171-177Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 33Zarbock A Singbartl K Ley K Complete reversal of acid-induced acute lung injury by blocking of platelet-neutrophil aggregation.J Clin Invest. 2006; 116: 3211-3219Crossref PubMed Scopus (489) Google Scholar raising the possibility of a similar role in glomerular leukocyte recruitment. We investigated this issue previously by transferring isolated platelets into P-selectin–deficient mice undergoing glomerular inflammation.4Kuligowski MP Kitching AR Hickey MJ Leukocyte recruitment to the inflamed glomerulus: a critical role for platelet-derived P-selectin in the absence of rolling.J Immunol. 2006; 176: 6991-6999Crossref PubMed Scopus (109) Google Scholar Leukocytes were recruited efficiently into glomeruli of mice that received wild-type platelets but not those that received P-selectin-deficient platelets, providing evidence that platelet-derived P-selectin was contributing to this response. However, under these experimental conditions, the majority of the circulating platelets remained of the recipient genotype. A more definitive approach would be to investigate bone marrow chimeras between wild-type and P-selectin–deficient mice, in which P-selectin expression can be restricted to either platelets or endothelial cells. Therefore, the aims of the present study were to investigate the mechanisms of platelet recruitment to the inflamed glomerulus and to clarify the role of platelet-derived P-selectin in glomerular leukocyte recruitment. This was achieved using intravital microscopy to examine leukocyte and platelet recruitment in the intact glomerulus. These experiments supported a role for platelet-derived P-selectin in glomerular leukocyte recruitment and demonstrated roles for αIIbβ3/fibrinogen/intercellular adhesion molecule 1 (ICAM-1), GPVI, and neutrophils in platelet recruitment to the inflamed glomerulus. Male C57BL/6 wild-type mice (supplied by Monash Animal Services, Clayton, Vic., Australia), weighing 27–35 g (aged 16 to 20 weeks), were used for intravital microscopy. Blood for platelet preparation was harvested from male wild-type C57BL/6 mice and FcRγ chain−/− mice (bred in-house).34Nieswandt B Bergmeier W Schulte V Rackebrandt K Gessner JE Zirngibl H Expression and function of the mouse collagen receptor glycoprotein VI is strictly dependent on its association with the FcRgamma chain.J Biol Chem. 2000; 275: 23998-24002Crossref PubMed Scopus (197) Google Scholar P-selectin−/− mice (bred in-house) were used for bone marrow chimera experiments. All experimental procedures were approved by the Monash University Animal Ethics Committee. Antibodies and reagents used for in vivo experiments were as follows: sheep anti-mouse-glomerular basement membrane (GBM) was prepared as described previously.4Kuligowski MP Kitching AR Hickey MJ Leukocyte recruitment to the inflamed glomerulus: a critical role for platelet-derived P-selectin in the absence of rolling.J Immunol. 2006; 176: 6991-6999Crossref PubMed Scopus (109) Google Scholar To serve as a control antibody, normal sheep globulin (NSG) was prepared from nonimmune sheep serum using an identical procedure. To inhibit αIIbβ3, GPI562 (4 mg/kg), a small molecule αIIbβ3 antagonist, was used.35Maxwell MJ Yuan Y Anderson KE Hibbs ML Salem HH Jackson SP SHIP1 and Lyn Kinase Negatively Regulate Integrin alpha IIb beta 3 signaling in platelets.J Biol Chem. 2004; 279: 32196-32204Crossref PubMed Scopus (70) Google Scholar, 36Choudhri TF Hoh BL Zerwes HG Prestigiacomo CJ Kim SC Connolly Jr, ES Kottirsch G Pinsky DJ Reduced microvascular thrombosis and improved outcome in acute murine stroke by inhibiting GP IIb/IIIa receptor-mediated platelet aggregation.J Clin Invest. 1998; 102: 1301-1310Crossref PubMed Scopus (207) Google Scholar Alboaggregin-B (280 μg/kg), a GPIbα-binding venom protein purified as a 25-kDa heterodimer from C.h. horridus (rattlesnake venom) as previously described (kindly provided by A/Prof. Rob Andrews, Australian Centre for Blood Diseases, Monash University, Australia), was used to inhibit GPIbα.37Andrews RK Kroll MH Ward CM Rose JW Scarborough RM Smith AI Lopez JA Berndt MC Binding of a novel 50-kilodalton alboaggregin from Trimeresurus albolabris and related viper venom proteins to the platelet membrane glycoprotein Ib-IX-V complex. Effect on platelet aggregation and glycoprotein Ib-mediated platelet activation.Biochemistry. 1996; 35: 12629-12639Crossref PubMed Scopus (80) Google Scholar Other inhibitory molecules used were as follows: goat polyclonal antibody against mouse fibrinogen (Nordic Immunology, Tiburg, Netherlands; 4 mg/kg)20Massberg S Enders G Matos FC Tomic LI Leiderer R Eisenmenger S Messmer K Krombach F Fibrinogen deposition at the postischemic vessel wall promotes platelet adhesion during ischemia-reperfusion in vivo.Blood. 1999; 94: 3829-3838Crossref PubMed Google Scholar; YN1/1.7.4, a rat monoclonal antibody against murine ICAM-1 (grown from hybridoma; 200 μg/mouse); RB6-8C5, a monoclonal antibody against Gr-1 (grown from hybridoma; 150 μg/mouse); and apyrase, an enzyme that metabolizes adenosine diphosphate (ADP), thereby limiting platelet activation (Sigma-Aldrich, Castle Hill, NSW, Australia; 0.2 U/g).38Sun D McNicol A James AA Peng Z Expression of functional recombinant mosquito salivary apyrase: a potential therapeutic platelet aggregation inhibitor.Platelets. 2006; 17: 178-184Crossref PubMed Scopus (47) Google Scholar For control experiments, isotype antibodies used included rat IgG2a and IgG2b (BD Biosciences, San Diego, CA). The actions of various antithrombotic agents were assessed in an in vivo ferric chloride thrombosis model.39Nesbitt WS Westein E Tovar-Lopez FJ Tolouei E Mitchell A Fu J Carberry J Fouras A Jackson SP A shear gradient-dependent platelet aggregation mechanism drives thrombus formation.Nat Med. 2009; 15: 665-673Crossref PubMed Scopus (646) Google Scholar In brief, in anesthetized C57BL/6 mice, FeCl3 was microinjected adjacent to a mesenteric arteriole, and platelet interactions were visualized with Differential Interference Contrast microscopy, using a Leica DMIRB inverted microscope (Leica Microsystems, Mt. Waverley), and a DAGE MTI charge-coupled device camera (SciTech, Preston, Vic., Australia). To assess the effect of alboaggregin-B, an in vitro flow chamber platelet adhesion assay was used.39Nesbitt WS Westein E Tovar-Lopez FJ Tolouei E Mitchell A Fu J Carberry J Fouras A Jackson SP A shear gradient-dependent platelet aggregation mechanism drives thrombus formation.Nat Med. 2009; 15: 665-673Crossref PubMed Scopus (646) Google Scholar Mouse blood was anticoagulated with hirudin and flowed over a collagen-coated glass microcapillary at 1800 s−1 for 3 minutes. Differential Interference Contrast microscopy images were taken at 4.5 minutes after Tyrode's buffer washout. To prepare kidneys for intravital microscopy, male mice (4 to 5 weeks old) underwent unilateral ureteric ligation as previously described.4Kuligowski MP Kitching AR Hickey MJ Leukocyte recruitment to the inflamed glomerulus: a critical role for platelet-derived P-selectin in the absence of rolling.J Immunol. 2006; 176: 6991-6999Crossref PubMed Scopus (109) Google Scholar, 40Kuligowski MP Kwan RY Lo C Wong C James WG Bourges D Ooi JD Abeynaike LD Hall P Kitching AR Hickey MJ Antimyeloperoxidase antibodies rapidly induce alpha-4-integrin-dependent glomerular neutrophil adhesion.Blood. 2009; 113: 6485-6494Crossref PubMed Scopus (40) Google Scholar Twelve weeks were then allowed for the kidney to undergo renal hydronephrosis. Intravital microscopy of the hydronephrotic kidney was performed as previously described.4Kuligowski MP Kitching AR Hickey MJ Leukocyte recruitment to the inflamed glomerulus: a critical role for platelet-derived P-selectin in the absence of rolling.J Immunol. 2006; 176: 6991-6999Crossref PubMed Scopus (109) Google Scholar, 40Kuligowski MP Kwan RY Lo C Wong C James WG Bourges D Ooi JD Abeynaike LD Hall P Kitching AR Hickey MJ Antimyeloperoxidase antibodies rapidly induce alpha-4-integrin-dependent glomerular neutrophil adhesion.Blood. 2009; 113: 6485-6494Crossref PubMed Scopus (40) Google Scholar Mice were anesthetized by i.p. injection of a cocktail of 150 mg/kg ketamine hydrochloride (Troy Lab, Smithfield, NSW, Australia) and 10 mg/kg xylazine (Pfizer, West Ryde, NSW, Australia). The jugular vein was then cannulated for administration of further anesthetic, platelets, and reagents. Mice were kept at a constant temperature of 37°C on a heating pad. The hydronephrotic kidney was exteriorized through a lateral skin incision, drained of urine using a 30G needle, and extended over a clear viewing platform using 4/0 silk tied to the kidney capsule. The kidney was superfused with bicarbonate-buffered saline at 37°C and covered with a coverslip. The renal microvasculature was observed with an intravital microscope (Axioplan 2 Imaging; Carl Zeiss, Australia) with a water immersion ×20 objective (×20/0.50 NA). Platelets were isolated using a previously described method.35Maxwell MJ Yuan Y Anderson KE Hibbs ML Salem HH Jackson SP SHIP1 and Lyn Kinase Negatively Regulate Integrin alpha IIb beta 3 signaling in platelets.J Biol Chem. 2004; 279: 32196-32204Crossref PubMed Scopus (70) Google Scholar Briefly, blood was withdrawn from the inferior vena cava of an anesthetized donor mouse and platelet-rich plasma was prepared via sequential centrifugation. Platelets were then pelleted from platelet-rich plasma and gently resuspended in Tyrode's buffer (10 mmol/L HEPES, 12 mmol/L NaHCO3, pH 7.4, 137 mmol/L NaCl, 2.7 mmol/L KCl, 5 mmol/L glucose). Platelets were allowed to rest in Tyrode's buffer for approximately 30 minutes at room temperature. Platelets isolated using this technique expressed minimal P-selectin, indicating that this protocol did not induce platelet activation. Subsequently, platelets (2 × 108) were labeled with 5, 6-carboxyfluorescein-succinimidyl-ester (CFSE) (5 mmol/L; Invitrogen, Mulgrave, Victoria, Australia) and slowly infused intravenously into hydronephrotic mice, which had been prepared for renal intravital microscopy. Most experiments used blood from C57BL/6 wild-type mice; however, in some experiments FcRγ chain−/− mice were used as platelet donors to provide platelets deficient in GPVI. To visualize leukocytes in experiments examining leukocyte recruitment, mice were injected with 50 μl of 0.05% rhodamine 6G (Sigma-Aldrich, Castle Hill, NSW, Australia) and the kidney was then examined via epifluorescence at 520–560 nm, using a 590-nm emission filter. Alternatively, CFSE-labeled platelets were observed by epifluorescence at 450–490 nm, using a 515-nm emission filter. Images were visualized using a low-light video camera (DAGE-MTI, IR1000; SciTech, Preston, Vic, Australia) and recorded for playback analysis using a digital video recorder. Three to four randomly chosen glomeruli were recorded at different time intervals throughout the experiment and subsequently analyzed for different parameters. For the analysis of leukocyte recruitment, leukocyte adhesion was measured by the number of rhodamine 6G-labeled leukocytes that remained adherent for more than 30 seconds. To assess platelet-endothelial cell interactions, platelets were defined as adherent if they remained stationary within the glomerulus for at least 30 seconds. In addition, primary platelet adhesion (tethering) was assessed. Platelet interactions were defined as primary when rapidly-moving platelets were observed to tether on to the endothelial surface and return to the bloodstream in less than 10s. These data were expressed as number of platelet tethers per glomerulus per 30 s. Mice chimeric for P-selectin were generated via irradiation and bone marrow transfer as described previously.41Ruth AJ Kitching AR Semple TJ Tipping PG Holdsworth SR Intrinsic renal cell expression of CD40 directs Th1 effectors inducing experimental crescentic glomerulonephritis.J Am Soc Nephrol. 2003; 14: 2813-2822Crossref PubMed Scopus (32) Google Scholar Briefly, mice were irradiated with two doses of 550 rad spaced three hours apart. Bone marrow (BM) cells were harvested from donor mice under sterile conditions, and recipient mice received 5 × 106 BM leukocytes within 24 hours of irradiation. Recipient mice were maintained under specific pathogen-free conditions for 8 weeks then used in intravital microscopy experiments. The following chimeric mice were generated: wild-type mice transplanted with P-selectin−/− BM (P-sel−/− into wild-type); P-selectin−/− mice transplanted with wild-type BM (wild-type into P-sel−/−). Additionally, wild-type into wild-type mice and P-sel−/− into P-sel−/− mice were generated as controls. To confirm BM reconstitution, platelets were isolated and assessed for expression of P-selectin under activating conditions via flow cytometry.42Tailor A Granger DN Hypercholesterolemia promotes P-selectin-dependent platelet-endothelial cell adhesion in postcapillary venules.Arterioscler Thromb Vasc Biol. 2003; 23: 675-680Crossref PubMed Scopus (79) Google Scholar To examine platelet recruitment in the inflamed glomerulus, platelets were infused and five minutes allowed for equilibration, then either anti-GBM antibody (20 mg per mouse) or an equivalent amount of NSG was administered intravenously. Subsequently, 30-second recordings were made at various time intervals over a period of 1 hour. In experiments investigating adhesion molecule function, Abs or various inhibitors were administered intravenously 5 minutes before infusion of CFSE-labeled platelets. To examine the effect of neutrophil depletion on platelet adhesion, mice were treated with the neutrophil-depleting antibody, RB6-8C5 (150 μg/mouse),4Kuligowski MP Kitching AR Hickey MJ Leukocyte recruitment to the inflamed glomerulus: a critical role for platelet-derived P-selectin in the absence of rolling.J Immunol. 2006; 176: 6991-6999Crossref PubMed Scopus (109) Google Scholar 24 hours before administration of fluorescently-labeled platelets. In a separate set of experiments, fibrinogen deposition in the inflamed glomerulus was investigated using Alexa Fluor 488-conjugated fibrinogen (Invitrogen; 17 mg/kg).20Massberg S Enders G Matos FC Tomic LI Leiderer R Eisenmenger S Messmer K Krombach F Fibrinogen deposition at the pos
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