In Vivo Imaging of Inflamed Glomeruli Reveals Dynamics of Neutrophil Extracellular Trap Formation in Glomerular Capillaries
2016; Elsevier BV; Volume: 187; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2016.10.008
ISSN1525-2191
AutoresClare L V Westhorpe, James E. Bayard, Kim M. O’Sullivan, Pam Hall, Qiang Cheng, A. Richard Kitching, Michael J. Hickey,
Tópico(s)Immune cells in cancer
ResumoNeutrophil extracellular traps (NETs) have been documented in glomeruli of patients with glomerulonephritis. However, the dynamics of NET formation in the glomerulus and their functional contribution to acute glomerular injury are poorly understood. Herein, we used in vivo multiphoton microscopy to investigate NET formation in the acutely inflamed glomerulus. Glomerular inflammation was induced using an antibody against the glomerular basement membrane. After induction of inflammation, multiphoton microscopy revealed that approximately 20% of glomeruli contained structures composed of extracellular DNA within the capillaries. These structures were not seen in mice depleted of neutrophils, consistent with them being NETs. Most contained myeloperoxidase, as seen in NETs in other tissues, whereas intraglomerular NETs did not contain significant levels of the histone H2Ax or neutrophil elastase. In vivo imaging revealed that intraglomerular NETs were present only transiently, suggesting that NETs were susceptible to disruption under the high shear conditions in glomerular capillaries. Investigation of NETs under flow conditions in vitro supported this concept. Dissolution of NETs via DNase I did not alter anti–glomerular basement membrane antibody-induced glomerular injury, as assessed via albuminuria, although the degree of microscopic hematuria was reduced by this intervention. These data indicate that in this model of acute, neutrophil-dependent glomerulonephritis, NETs are generated in the glomerular capillaries, where they are short lived and make a modest contribution to glomerular injury. Neutrophil extracellular traps (NETs) have been documented in glomeruli of patients with glomerulonephritis. However, the dynamics of NET formation in the glomerulus and their functional contribution to acute glomerular injury are poorly understood. Herein, we used in vivo multiphoton microscopy to investigate NET formation in the acutely inflamed glomerulus. Glomerular inflammation was induced using an antibody against the glomerular basement membrane. After induction of inflammation, multiphoton microscopy revealed that approximately 20% of glomeruli contained structures composed of extracellular DNA within the capillaries. These structures were not seen in mice depleted of neutrophils, consistent with them being NETs. Most contained myeloperoxidase, as seen in NETs in other tissues, whereas intraglomerular NETs did not contain significant levels of the histone H2Ax or neutrophil elastase. In vivo imaging revealed that intraglomerular NETs were present only transiently, suggesting that NETs were susceptible to disruption under the high shear conditions in glomerular capillaries. Investigation of NETs under flow conditions in vitro supported this concept. Dissolution of NETs via DNase I did not alter anti–glomerular basement membrane antibody-induced glomerular injury, as assessed via albuminuria, although the degree of microscopic hematuria was reduced by this intervention. These data indicate that in this model of acute, neutrophil-dependent glomerulonephritis, NETs are generated in the glomerular capillaries, where they are short lived and make a modest contribution to glomerular injury. Glomerulonephritis is a key cause of end-stage renal failure, and in many forms of glomerulonephritis, leukocytes play critical roles in glomerular injury.1Devi S. Li A. Westhorpe C.L. Lo C.Y. Abeynaike L.D. Snelgrove S.L. Hall P. Ooi J.D. Sobey C.G. Kitching A.R. Hickey M.J. Multiphoton imaging reveals a novel leukocyte recruitment paradigm in the glomerulus.Nat Med. 2013; 19: 107-112Crossref PubMed Scopus (132) Google Scholar, 2Kitching A.R. Holdsworth S.R. Hickey M.J. Targeting leukocytes in immune glomerular diseases.Curr Med Chem. 2008; 15: 448-458Crossref PubMed Scopus (23) Google Scholar In acute glomerulonephritis, neutrophils accumulate in glomeruli and depletion of neutrophils or inhibition of their recruitment to the glomerulus reduces the level of injury, indicating a central role for neutrophils in this form of glomerular inflammation.1Devi S. Li A. Westhorpe C.L. Lo C.Y. Abeynaike L.D. Snelgrove S.L. Hall P. Ooi J.D. Sobey C.G. Kitching A.R. Hickey M.J. Multiphoton imaging reveals a novel leukocyte recruitment paradigm in the glomerulus.Nat Med. 2013; 19: 107-112Crossref PubMed Scopus (132) Google Scholar, 3Tipping P.G. Huang X.R. Berndt M.C. Holdsworth S.R. A role for P-selectin in complement-independent neutrophil-mediated glomerular injury.Kidney Int. 1994; 46: 79-88Abstract Full Text PDF PubMed Scopus (88) Google Scholar, 4Tang T. Rosenkranz A. Assmann K.J. Goodman M.J. Gutierrez-Ramos J.C. Carroll M.C. Cotran R.S. Mayadas T.N. A role for Mac-1 (CDIIb/CD18) in immune complex-stimulated neutrophil function in vivo: Mac-1 deficiency abrogates sustained Fcgamma receptor-dependent neutrophil adhesion and complement-dependent proteinuria in acute glomerulonephritis.J Exp Med. 1997; 186: 1853-1863Crossref PubMed Scopus (175) Google Scholar, 5Kuligowski M.P. Kitching A.R. Hickey M.J. 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 Despite these studies, the mechanisms whereby neutrophils are recruited to the unique microvasculature of the glomerulus, and their behavior after their recruitment, had remained poorly understood. To overcome this, we recently used in vivo multiphoton microscopy to examine neutrophil recruitment to acutely inflamed glomeruli of mice.1Devi S. Li A. Westhorpe C.L. Lo C.Y. Abeynaike L.D. Snelgrove S.L. Hall P. Ooi J.D. Sobey C.G. Kitching A.R. Hickey M.J. Multiphoton imaging reveals a novel leukocyte recruitment paradigm in the glomerulus.Nat Med. 2013; 19: 107-112Crossref PubMed Scopus (132) Google Scholar These studies demonstrated that in response to acute inflammatory stimulation, neutrophils are retained within the glomerular capillary lumen for prolonged periods, in some cases migrating extensively, and mediate glomerular injury without leaving the vasculature.1Devi S. Li A. Westhorpe C.L. Lo C.Y. Abeynaike L.D. Snelgrove S.L. Hall P. Ooi J.D. Sobey C.G. Kitching A.R. Hickey M.J. Multiphoton imaging reveals a novel leukocyte recruitment paradigm in the glomerulus.Nat Med. 2013; 19: 107-112Crossref PubMed Scopus (132) Google Scholar These and other studies have demonstrated that the effector mechanisms by which neutrophils mediate glomerular injury include generation of reactive oxygen species and release of proteases, including myeloperoxidase (MPO), neutrophil elastase, and cathepsin G.1Devi S. Li A. Westhorpe C.L. Lo C.Y. Abeynaike L.D. Snelgrove S.L. Hall P. Ooi J.D. Sobey C.G. Kitching A.R. Hickey M.J. Multiphoton imaging reveals a novel leukocyte recruitment paradigm in the glomerulus.Nat Med. 2013; 19: 107-112Crossref PubMed Scopus (132) Google Scholar, 6Boyce N.W. Holdsworth S.R. Hydroxyl radical mediation of immune renal injury by desferrioxamine.Kidney Int. 1986; 30: 813-817Abstract Full Text PDF PubMed Scopus (84) Google Scholar, 7Tipping P.G. Boyce N.W. Holdsworth S.R. Relative contributions of chemo-attractant and terminal components of complement to anti-glomerular basement membrane (GBM) glomerulonephritis.Clin Exp Immunol. 1989; 78: 444-448Google Scholar, 8Odobasic D. Kitching A.R. Semple T.J. Holdsworth S.R. Endogenous myeloperoxidase promotes neutrophil-mediated renal injury, but attenuates T cell immunity inducing crescentic glomerulonephritis.J Am Soc Nephrol. 2007; 18: 760-770Crossref PubMed Scopus (74) Google Scholar, 9Schrijver G. Schalkwijk J. Robben J.C. Assmann K.J. Koene R.A. Antiglomerular basement membrane nephritis in beige mice: deficiency of leukocytic neutral proteinases prevents the induction of albuminuria in the heterologous phase.J Exp Med. 1989; 169: 1435-1448Crossref PubMed Scopus (74) Google Scholar However, during the past decade, an additional neutrophil-derived effector mechanism has been identified—generation of neutrophil extracellular traps (NETs).10Brinkmann V. Reichard U. Goosmann C. Fauler B. Uhlemann Y. Weiss D.S. Weinrauch Y. Zychlinsky A. Neutrophil extracellular traps kill bacteria.Science. 2004; 303: 1532-1535Crossref PubMed Scopus (6111) Google Scholar, 11Brinkmann V. Zychlinsky A. Beneficial suicide: why neutrophils die to make NETs.Nat Rev Microbiol. 2007; 5: 577-582Crossref PubMed Scopus (689) Google Scholar, 12McDonald B. Urrutia R. Yipp B.G. Jenne C.N. Kubes P. Intravascular neutrophil extracellular traps capture bacteria from the bloodstream during sepsis.Cell Host Microbe. 2012; 12: 324-333Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar, 13Yipp B.G. Petri B. Salina D. Jenne C.N. Scott B.N. Zbytnuik L.D. Pittman K. Asaduzzaman M. Wu K. Meijndert H.C. Malawista S.E. de Boisfleury Chevance A. Zhang K. Conly J. Kubes P. Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo.Nat Med. 2012; 18: 1386-1393Crossref PubMed Scopus (721) Google Scholar, 14von Bruhl M.L. Stark K. Steinhart A. Chandraratne S. Konrad I. Lorenz M. Khandoga A. Tirniceriu A. Coletti R. Kollnberger M. Byrne R.A. Laitinen I. Walch A. Brill A. Pfeiler S. Manukyan D. Braun S. Lange P. Riegger J. Ware J. Eckart A. Haidari S. Rudelius M. Schulz C. Echtler K. Brinkmann V. Schwaiger M. Preissner K.T. Wagner D.D. Mackman N. Engelmann B. Massberg S. Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo.J Exp Med. 2012; 209: 819-835Crossref PubMed Scopus (1184) Google Scholar Little is known about the role of NETs in glomerular injury in acute glomerulonephritis. NETs are webs of DNA and intracellular proteins extruded by highly activated neutrophils.15Brinkmann V. Zychlinsky A. Neutrophil extracellular traps: is immunity the second function of chromatin?.J Cell Biol. 2012; 198: 773-783Crossref PubMed Scopus (698) Google Scholar These structures are coated with histones and proteolytic granule proteins, including MPO and neutrophil elastase, and are capable of capturing and killing microbes.10Brinkmann V. Reichard U. Goosmann C. Fauler B. Uhlemann Y. Weiss D.S. Weinrauch Y. Zychlinsky A. Neutrophil extracellular traps kill bacteria.Science. 2004; 303: 1532-1535Crossref PubMed Scopus (6111) Google Scholar, 16Urban C.F. Reichard U. Brinkmann V. Zychlinsky A. Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms.Cell Microbiol. 2006; 8: 668-676Crossref PubMed Scopus (707) Google Scholar Although NETs were initially associated with the response to bacterial infection, more recently they have been implicated in various forms of sterile inflammation, including anti–neutrophil cytoplasmic antibody–positive glomerulonephritis and lupus nephritis.14von Bruhl M.L. Stark K. Steinhart A. Chandraratne S. Konrad I. Lorenz M. Khandoga A. Tirniceriu A. Coletti R. Kollnberger M. Byrne R.A. Laitinen I. Walch A. Brill A. Pfeiler S. Manukyan D. Braun S. Lange P. Riegger J. Ware J. Eckart A. Haidari S. Rudelius M. Schulz C. Echtler K. Brinkmann V. Schwaiger M. Preissner K.T. Wagner D.D. Mackman N. Engelmann B. Massberg S. Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo.J Exp Med. 2012; 209: 819-835Crossref PubMed Scopus (1184) Google Scholar, 17Garcia-Romo G.S. Caielli S. Vega B. Connolly J. Allantaz F. Xu Z. Punaro M. Baisch J. Guiducci C. Coffman R.L. Barrat F.J. Banchereau J. Pascual V. Netting neutrophils are major inducers of type I IFN production in pediatric systemic lupus erythematosus.Sci Transl Med. 2011; 3: 73ra20Crossref PubMed Scopus (947) Google Scholar, 18Rossaint J. Herter J.M. Van Aken H. Napirei M. Doring Y. Weber C. Soehnlein O. Zarbock A. Synchronized integrin engagement and chemokine activation is crucial in neutrophil extracellular trap-mediated sterile inflammation.Blood. 2014; 123: 2573-2584Crossref PubMed Scopus (191) Google Scholar, 19Kaplan M.J. Radic M. Neutrophil extracellular traps: double-edged swords of innate immunity.J Immunol. 2012; 189: 2689-2695Crossref PubMed Scopus (732) Google Scholar, 20Chen G. Zhang D. Fuchs T.A. Manwani D. Wagner D.D. Frenette P.S. Heme-induced neutrophil extracellular traps contribute to the pathogenesis of sickle cell disease.Blood. 2014; 123: 3818-3827Crossref PubMed Scopus (222) Google Scholar, 21Kessenbrock K. Krumbholz M. Schonermarck U. Back W. Gross W.L. Werb Z. Grone H.J. Brinkmann V. Jenne D.E. Netting neutrophils in autoimmune small-vessel vasculitis.Nat Med. 2009; 15: 623-625Crossref PubMed Scopus (1148) Google Scholar, 22Villanueva E. Yalavarthi S. Berthier C.C. Hodgin J.B. Khandpur R. Lin A.M. Rubin C.J. Zhao W. Olsen S.H. Klinker M. Shealy D. Denny M.F. Plumas J. Chaperot L. Kretzler M. Bruce A.T. Kaplan M.J. Netting neutrophils induce endothelial damage, infiltrate tissues, and expose immunostimulatory molecules in systemic lupus erythematosus.J Immunol. 2011; 187: 538-552Crossref PubMed Scopus (842) Google Scholar, 23O'Sullivan K.M. Lo C.Y. Summers S.A. Elglass K.D. McMillan P.J. Longano A. Ford S.L. Gan P.Y. Kerr P.G. Kitching A.R. Holdsworth S.R. Renal participation of myeloperoxidase in antineutrophil cytoplsamic antibody (ANCA)-associated glomerulonephritis.Kidney Int. 2015; 88: 1030-1046Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar Components of NETs are capable of injuring endothelial cells, raising the possibility that the presence of these structures in glomeruli contributes to injury to the glomerular endothelium and other glomerular cells.24Xu J. Zhang X. Pelayo R. Monestier M. Ammollo C.T. Semeraro F. Taylor F.B. Esmon N.L. Lupu F. Esmon C.T. Extracellular histones are major mediators of death in sepsis.Nat Med. 2009; 15: 1318-1321Crossref PubMed Scopus (1049) Google Scholar, 25Saffarzadeh M. Juenemann C. Queisser M.A. Lochnit G. Barreto G. Galuska S.P. Lohmeyer J. Preissner K.T. Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones.PLoS One. 2012; 7: e32366Crossref PubMed Scopus (806) Google Scholar, 26Kumar S.V. Kulkarni O.P. Mulay S.R. Darisipudi M.N. Romoli S. Thomasova D. Scherbaum C.R. Hohenstein B. Hugo C. Muller S. Liapis H. Anders H.J. Neutrophil extracellular trap-related extracellular histones cause vascular necrosis in severe GN.J Am Soc Nephrol. 2015; 26: 2399-2413Crossref PubMed Scopus (124) Google Scholar This possibility is strengthened by reports that removal of NETs and histone neutralization reduce tissue injury and inflammation.12McDonald B. Urrutia R. Yipp B.G. Jenne C.N. Kubes P. Intravascular neutrophil extracellular traps capture bacteria from the bloodstream during sepsis.Cell Host Microbe. 2012; 12: 324-333Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar, 18Rossaint J. Herter J.M. Van Aken H. Napirei M. Doring Y. Weber C. Soehnlein O. Zarbock A. Synchronized integrin engagement and chemokine activation is crucial in neutrophil extracellular trap-mediated sterile inflammation.Blood. 2014; 123: 2573-2584Crossref PubMed Scopus (191) Google Scholar, 26Kumar S.V. Kulkarni O.P. Mulay S.R. Darisipudi M.N. Romoli S. Thomasova D. Scherbaum C.R. Hohenstein B. Hugo C. Muller S. Liapis H. Anders H.J. Neutrophil extracellular trap-related extracellular histones cause vascular necrosis in severe GN.J Am Soc Nephrol. 2015; 26: 2399-2413Crossref PubMed Scopus (124) Google Scholar Despite this, little is known about the dynamics of the generation of these structures in the glomerular microvasculature. The initial description of the dynamics of NET formation was based on in vitro analyses of neutrophils exposed to stimuli, including phorbol myristate acetate and live bacteria, and demonstrated that NET formation is a form of cell death (NETosis) in which neutrophils extrude DNA several hours after stimulation.10Brinkmann V. Reichard U. Goosmann C. Fauler B. Uhlemann Y. Weiss D.S. Weinrauch Y. Zychlinsky A. Neutrophil extracellular traps kill bacteria.Science. 2004; 303: 1532-1535Crossref PubMed Scopus (6111) Google Scholar However, recent imaging-based analyses of NET formation have shed interesting new light on the nature of this process in vivo.12McDonald B. Urrutia R. Yipp B.G. Jenne C.N. Kubes P. Intravascular neutrophil extracellular traps capture bacteria from the bloodstream during sepsis.Cell Host Microbe. 2012; 12: 324-333Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar, 13Yipp B.G. Petri B. Salina D. Jenne C.N. Scott B.N. Zbytnuik L.D. Pittman K. Asaduzzaman M. Wu K. Meijndert H.C. Malawista S.E. de Boisfleury Chevance A. Zhang K. Conly J. Kubes P. Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo.Nat Med. 2012; 18: 1386-1393Crossref PubMed Scopus (721) Google Scholar Studies of skin infections revealed that neutrophils in the skin can release NETs within minutes of encountering Gram-positive bacteria.13Yipp B.G. Petri B. Salina D. Jenne C.N. Scott B.N. Zbytnuik L.D. Pittman K. Asaduzzaman M. Wu K. Meijndert H.C. Malawista S.E. de Boisfleury Chevance A. Zhang K. Conly J. Kubes P. Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo.Nat Med. 2012; 18: 1386-1393Crossref PubMed Scopus (721) Google Scholar Moreover, neutrophils remained alive and retained the ability to migrate after NET generation, enabling wider spread of NETs within the skin. NETs can also be generated by neutrophils within the microvasculature. In a model of systemic endotoxemia, NETs were generated in the lumen of the hepatic sinusoids, where evidence indicated that they mediated hepatic injury.12McDonald B. Urrutia R. Yipp B.G. Jenne C.N. Kubes P. Intravascular neutrophil extracellular traps capture bacteria from the bloodstream during sepsis.Cell Host Microbe. 2012; 12: 324-333Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar Our recent studies indicate that neutrophils can mediate functional glomerular injury without leaving the glomerular capillaries, indicating that effector mechanisms generated within the microvasculature are critical to glomerular injury. However, the dynamics of NET formation in the glomerular microvasculature and the contribution of NETs to acute glomerular injury remain unclear. Therefore, the aim of this study was to investigate NET formation in the acutely inflamed glomerulus, using in vivo imaging to study the dynamics and nature of this process in the glomerulus. The results indicate that, in a model of in situ immune complex deposition, NETs are generated in glomerular capillaries, where they persist only briefly. In this model, removal of NETs does not affect albuminuria, the key readout of glomerular injury, although it does reduce the degree of microscopic hematuria. Male C57BL/6 mice were used for intravital microscopy. All experimental procedures were approved by the Monash University Animal Ethics Committee. Sheep anti-mouse glomerular basement membrane antibody (anti-GBM Ab) was prepared as described.5Kuligowski M.P. Kitching A.R. Hickey M.J. 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, 27Devi S. Kuligowski M.P. Kwan R.Y. Westein E. Jackson S.P. Kitching A.R. Hickey M.J. Platelet recruitment to the inflamed glomerulus occurs via an alphaIIbbeta3/GPVI-dependent pathway.Am J Pathol. 2010; 177: 1131-1142Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar As control Ab, normal sheep globulin (NSG) was prepared from nonimmune sheep serum using an identical protocol. Suppliers for reagents were as follows: Sytox Orange and DyLight conjugation kits (Life Technologies, Clayton, VIC, Australia); fluorescein isothiocyanate dextran (Sigma Aldrich, Castle Hill, NSW, Australia); anti–Gr-1 (RB6-8C5) conjugated to e450 or phycoerythrin (eBioscience, San Diego, CA); goat anti-mouse histone H2Ax and goat anti-mouse neutrophil elastase (Santa Cruz Biotechnology, Dallas, TX); mouse anti-mouse H2A-H2B (in house)10Brinkmann V. Reichard U. Goosmann C. Fauler B. Uhlemann Y. Weiss D.S. Weinrauch Y. Zychlinsky A. Neutrophil extracellular traps kill bacteria.Science. 2004; 303: 1532-1535Crossref PubMed Scopus (6111) Google Scholar, 23O'Sullivan K.M. Lo C.Y. Summers S.A. Elglass K.D. McMillan P.J. Longano A. Ford S.L. Gan P.Y. Kerr P.G. Kitching A.R. Holdsworth S.R. Renal participation of myeloperoxidase in antineutrophil cytoplsamic antibody (ANCA)-associated glomerulonephritis.Kidney Int. 2015; 88: 1030-1046Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar; DNase I (Roche Diagnostics, Dee Why, NSW, Australia); and Cl-amidine (Cayman Chemicals, Ann Arbor, MI). Anti-MPO used in in vivo imaging studies and mouse anti-mouse H2A-H2B were generated in house.10Brinkmann V. Reichard U. Goosmann C. Fauler B. Uhlemann Y. Weiss D.S. Weinrauch Y. Zychlinsky A. Neutrophil extracellular traps kill bacteria.Science. 2004; 303: 1532-1535Crossref PubMed Scopus (6111) Google Scholar, 28Kuligowski M.P. Kwan R.Y. Lo C. Wong C. James W.G. Bourges D. Ooi J.D. Abeynaike L.D. Hall P. Kitching A.R. Hickey M.J. Antimyeloperoxidase antibodies rapidly induce alpha-4-integrin-dependent glomerular neutrophil adhesion.Blood. 2009; 113: 6485-6494Crossref PubMed Scopus (40) Google Scholar NETs were detected via immunohistochemistry of kidney sections using a recently published technique.23O'Sullivan K.M. Lo C.Y. Summers S.A. Elglass K.D. McMillan P.J. Longano A. Ford S.L. Gan P.Y. Kerr P.G. Kitching A.R. Holdsworth S.R. Renal participation of myeloperoxidase in antineutrophil cytoplsamic antibody (ANCA)-associated glomerulonephritis.Kidney Int. 2015; 88: 1030-1046Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar Images were acquired with a Nikon C1 confocal microscope (Nikon Imaging, Sydney, NSW, Australia), and NETs were defined as areas of colocalization of citrullinated histone H3 (a NET marker)29Wong S.L. Demers M. Martinod K. Gallant M. Wang Y. Goldfine A.B. Kahn C.R. Wagner D.D. Diabetes primes neutrophils to undergo NETosis, which impairs wound healing.Nat Med. 2015; 21: 815-819Crossref PubMed Scopus (604) Google Scholar, MPO, and peptidyl arginine deiminase-4 (PAD4) associated with extracellular DNA (stained via DAPI). At least 20 glomeruli were assessed per section, and the data were displayed as NETs/glomerular cross section. In parallel experiments, sections were costained for H2A-H2B (using mouse anti-mouse H2A-H2B, 1 μg/mL) and MPO, and NETs were defined as areas of colocalization of H2A-H2B, MPO, and DNA. To prepare the kidney for glomerular intravital microscopy, mice underwent unilateral ureteric ligation, and imaging experiments were performed after 12 weeks, as previously described.5Kuligowski M.P. Kitching A.R. Hickey M.J. 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, 27Devi S. Kuligowski M.P. Kwan R.Y. Westein E. Jackson S.P. Kitching A.R. Hickey M.J. Platelet recruitment to the inflamed glomerulus occurs via an alphaIIbbeta3/GPVI-dependent pathway.Am J Pathol. 2010; 177: 1131-1142Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 28Kuligowski M.P. Kwan R.Y. Lo C. Wong C. James W.G. Bourges D. Ooi J.D. Abeynaike L.D. Hall P. Kitching A.R. Hickey M.J. Antimyeloperoxidase antibodies rapidly induce alpha-4-integrin-dependent glomerular neutrophil adhesion.Blood. 2009; 113: 6485-6494Crossref PubMed Scopus (40) Google Scholar The protocol for multiphoton microscopy imaging of the hydronephrotic kidney was as described previously, with minor modifications.5Kuligowski M.P. Kitching A.R. Hickey M.J. 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 The kidney was visualized with a Leica SP5 microscope (Leica Microsystems, Heidelberg, Germany) with a 20× 1.0 numerical aperture objective lens and a SpectraPhysics MaiTai pulsed infrared laser (NewSpec, Myrtle Bank, SA, Australia) tuned to 810 nm. Images were collected using a 3- to 6-μm Z-step size with a Z-depth of approximately 150 μm. Image stacks were acquired every 30 seconds. Recordings were analyzed using Imaris (Bitplane, Zurich, Switzerland). In some experiments, imaging of intact kidneys was performed, using 3-week-old mice weighing 10 to 12 g, as previously described.1Devi S. Li A. Westhorpe C.L. Lo C.Y. Abeynaike L.D. Snelgrove S.L. Hall P. Ooi J.D. Sobey C.G. Kitching A.R. Hickey M.J. Multiphoton imaging reveals a novel leukocyte recruitment paradigm in the glomerulus.Nat Med. 2013; 19: 107-112Crossref PubMed Scopus (132) Google Scholar In these experiments, the dose of anti-GBM Ab was adjusted according to the weight of the mice, whereas imaging was performed as described above. Mice were injected i.v. with either 15 or 20 mg of anti-GBM Ab, or NSG as control. For imaging experiments, mice received anti–Gr-1 (2 μg) and fluorescein isothiocyanate dextran (250 kDa) i.v. just before imaging. For assessment of NET protein markers, anti-MPO (5 μg), anti-H2Ax (5 μg), anti–H2A-H2B (10 μg), or anti–neutrophil elastase (2 μg), all conjugated to DyLight 650, were administered at the same time.12McDonald B. Urrutia R. Yipp B.G. Jenne C.N. Kubes P. Intravascular neutrophil extracellular traps capture bacteria from the bloodstream during sepsis.Cell Host Microbe. 2012; 12: 324-333Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar Kidneys were imaged over the subsequent 60 minutes, recording 10 separate fields containing three to five glomeruli each, for a 5-minute duration. As a result, 30 to 40 glomeruli were examined in each mouse, for a period of 5 minutes. To examine extracellular DNA release, at the end of 60 minutes of imaging, Sytox Orange (1 nmol/mouse i.v.) was administered and Z-stacks of approximately 10 fields containing two to three glomeruli each were recorded over the next 10 minutes. For neutrophil depletion, anti–Gr-1 (160 μg i.p.) was injected 24 hours before experiments.5Kuligowski M.P. Kitching A.R. Hickey M.J. 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 examine the functional role of NETs, mice received either the PAD4 inhibitor Cl-amidine (10 mg/kg i.p.) or DNase I (2000 U i.v.) 10 to 15 minutes before induction of the anti-GBM Ab model.12McDonald B. Urrutia R. Yipp B.G. Jenne C.N. Kubes P. Intravascular neutrophil extracellular traps capture bacteria from the bloodstream during sepsis.Cell Host Microbe. 2012; 12: 324-333Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar, 30Willis V.C. Gizinski A.M. Banda N.K. Causey C.P. Knuckley B. Cordova K.N. Luo Y. Levitt B. Glogowska M. Chandra P. Kulik L. Robinson W.H. Arend W.P. Thompson P.R. Holers V.M. N-alpha-benzoyl-N5-(2-chloro-1-iminoethyl)-L-ornithine amide, a protein arginine deiminase inhibitor, reduces the severity of murine collagen-induced arthritis.J Immunol. 2011; 186: 4396-4404Crossref PubMed Scopus (222) Google Scholar, 31Chumanevich A.A. Causey C.P. Knuckley B.A. Jones J.E. Poudyal D. Chumanevich A.P. Davis T. Matesic L.E. Thompson P.R. Hofseth L.J. Suppression of colitis in mice by Cl-amidine: a novel peptidylarginine deiminase inhibitor.Am J Physiol Gastrointest Liver Physiol. 2011; 300: G929-G938Crossref Scopus (148) Google Scholar, 32Knight J.S. Zhao W. Luo W. Subramanian V. O'Dell A.A. Yalavarthi S. Hodgin J.B. Eitzman D.T. Thompson P.R. Kaplan M.J. Peptidylarginine deiminase inhibition is immunomodulatory and vasculoprotective in murine lupus.J Clin Invest. 2013; 123: 2981-2993Crossref PubMed Scopus (281) Google Scholar Albuminuria was measured on 24-hour urine collections using a mouse albumin enzyme-linked immunosorbent assay quantification kit (Bethyl Laboratories, Montgomery, TX). Urine creatinine measurements were determined using an autoanalyzer-based alkaline picric acid method. Hematuria was assessed via dipstick. Mouse neutrophils were isolated from bone marrow via magnetic-based negative selection (Miltenyi-Biotec, Macquarie Park, NSW, Australia), and loaded into intercellular adhesion molecule-1–coated microfluidic devices (Microfluidic ChipShop, Jena, Germany) at 5 × 105/mL in Ca2+/Mg2+-containing Hanks' balanced salt solution. Neutrophils were allowed to adhere for 45 minutes, then the chambers were loaded with lipopolysaccharide (LPS; 1 μg/mL) and incubated at 37°C for 3 hours to stimulate NET formation.33Li P. Li M. Lindberg M.R. Kennett M.J. Xiong N. Wang Y. PAD4 is essential for antibacterial innate immunity mediated by neutrophil extracellular traps.J Exp Med. 2010; 207: 1853-1862Crossref PubMed Scopus (906) Google Scholar At the end of the incubation, Sytox Orange (1 μmol/L) and Alexa 488–conjugated anti–Gr-1 (1 μg/mL) were loaded into the chambers at low shear to stain the NETs and neutrophils. Chambers were examined using an API Deltavision imaging system (GE Healthcare Australia Pty. Ltd., Parramatta, NSW, Australia) on an IX71 Olympus microscope (Olympus Australia, Notting Hill, VIC, Australia), and images were recorded using a CoolSnapHQ camera (Photometrics, Tucson, AZ). Images of NETs were recorded while Hanks' balanced salt solution was perfused through the chamber either at 1 dyn/cm2 (sinusoidal shear) for 6 minutes or progressively at 1, 10, and 20 dyn/cm2 for 2 minutes each, with the latter two rates representing glomerular capillary shear. Intensity of NET staining (Sytox Orange) was
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