Inhibition of Pyk2 blocks lung inflammation and injury in a mouse model of acute lung injury

Respiratory Research

Xiangdong Zhu²  Alan R Leff¹  Angelo Y Meliton²  Jonathan Learoyd²  Yingli Duan² 

[1] Departments of Anesthesia and Critical Care, Neurobiology and Pediatrics, and Committees on Clinical Pharmacology, Cell Physiology and Molecular Medicine, Division of the Biological Sciences, The University of Chicago, Chicago, IL 60637;Section of Pulmonary and Critical Care Medicine, Department of Medicine, The University of Chicago, Chicago, IL 60637

关键词: Pyk2;    neutrophils;    lung;    lipopolysaccharide;    inflammation;   

Others  :  796757
DOI  :  10.1186/1465-9921-13-4

 received in 2011-10-18, accepted in 2012-01-18,  发布年份 2012

【 摘 要 】

Background

Proline-rich tyrosine kinase 2 (Pyk2) is essential in neutrophil degranulation and chemotaxis in vitro. However, its effect on the process of lung inflammation and edema formation during LPS induced acute lung injury (ALI) remains unknown. The goal of the present study was to determine the effect of inhibiting Pyk2 on LPS-induced acute lung inflammation and injury in vivo.

Methods

C57BL6 mice were given either 10 mg/kg LPS or saline intratracheally. Inhibition of Pyk2 was effected by intraperitoneal administration TAT-Pyk2-CT 1 h before challenge. Bronchoalveolar lavage analysis of cell counts, lung histology and protein concentration in BAL were analyzed at 18 h after LPS treatment. KC and MIP-2 concentrations in BAL were measured by a mouse cytokine multiplex kit. The static lung compliance was determined by pressure-volume curve using a computer-controlled small animal ventilator. The extravasated Evans blue concentration in lung homogenate was determined spectrophotometrically.

Results

Intratracheal instillation of LPS induced significant neutrophil infiltration into the lung interstitium and alveolar space, which was attenuated by pre-treatment with TAT-Pyk2-CT. TAT-Pyk2-CT pretreatment also attenuated 1) myeloperoxidase content in lung tissues, 2) vascular leakage as measured by Evans blue dye extravasation in the lungs and the increase in protein concentration in bronchoalveolar lavage, and 3) the decrease in lung compliance. In each paradigm, treatment with control protein TAT-GFP had no blocking effect. By contrast, production of neutrophil chemokines MIP-2 and keratinocyte-derived chemokine in the bronchoalveolar lavage was not reduced by TAT-Pyk2-CT. Western blot analysis confirmed that tyrosine phosphorylation of Pyk2 in LPS-challenged lungs was reduced to control levels by TAT-Pyk2-CT pretreatment.

Conclusions

These results suggest that Pyk2 plays an important role in the development of acute lung injury in mice and that pharmacological inhibition of Pyk2 might provide a potential therapeutic strategy in the pretreatment for patients at imminent risk of developing acute lung injury.

【 授权许可】

   
2012 Duan et al; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

【 参考文献 】

• [1]Erickson SE, Martin GS, Davis JL, Matthay MA, Eisner MD: Recent trends in acute lung injury mortality: 1996-2005. Crit Care Med 2009, 37(5):1574-1579.
• [2]Phua J, Badia JR, Adhikari NK, Friedrich JO, Fowler RA, Singh JM, Scales DC, Stather DR, Li A, Jones A, et al.: Has mortality from acute respiratory distress syndrome decreased over time?: A systematic review. Am J Respir Crit Care Med 2009, 179(3):220-227.
• [3]Kabir K, Gelinas JP, Chen M, Chen D, Zhang D, Luo X, Yang JH, Carter D, Rabinovici R: Characterization of a murine model of endotoxin-induced acute lung injury. Shock 2002, 17(4):300-303.
• [4]Chignard M, Balloy V: Neutrophil recruitment and increased permeability during acute lung injury induced by lipopolysaccharide. Am J Physiol Lung Cell Mol Physiol 2000, 279(6):L1083-1090.
• [5]Munoz NM, Meliton AY, Meliton LN, Dudek SM, Leff AR: Secretory group V phospholipase A2 regulates acute lung injury and neutrophilic inflammation caused by LPS in mice. Am J Physiol Lung Cell Mol Physiol 2009, 296(6):L879-887.
• [6]Nonas S, Miller I, Kawkitinarong K, Chatchavalvanich S, Gorshkova I, Bochkov VN, Leitinger N, Natarajan V, Garcia JG, Birukov KG: Oxidized phospholipids reduce vascular leak and inflammation in rat model of acute lung injury. Am J Respir Crit Care Med 2006, 173(10):1130-1138.
• [7]Poynter ME, Irvin CG, Janssen-Heininger YM: A prominent role for airway epithelial NF-kappa B activation in lipopolysaccharide-induced airway inflammation. J Immunol 2003, 170(12):6257-6265.
• [8]Lipinski CA, Loftus JC: Targeting Pyk2 for therapeutic intervention. Expert Opin Ther Targets 2010, 14(1):95-108.
• [9]Loftus JC, Yang Z, Tran NL, Kloss J, Viso C, Berens ME, Lipinski CA: The Pyk2 FERM domain as a target to inhibit glioma migration. Mol Cancer Ther 2009, 8(6):1505-1514.
• [10]Kamen LA, Schlessinger J, Lowell CA: Pyk2 is required for neutrophil degranulation and host defense responses to bacterial infection. J Immunol 2011, 186(3):1656-1665.
• [11]Di Cioccio V, Strippoli R, Bizzarri C, Troiani G, Cervellera MN, Gloaguen I, Colagrande A, Cattozzo EM, Pagliei S, Santoni A, Colotta F, Mainiero F, Bertini R: Key role of proline-rich tyrosine kinase 2 in interleukin-8 (CXCL8/IL-8)-mediated human neutrophil chemotaxis. Immunology 2004, 111(4):407-415.
• [12]Han H, Fuortes M, Nathan C: Critical role of the carboxyl terminus of proline-rich tyrosine kinase (Pyk2) in the activation of human neutrophils by tumor necrosis factor: separation of signals for the respiratory burst and degranulation. J Exp Med 2003, 197(1):63-75.
• [13]Wang L, Learoyd J, Duan Y, Leff AR, Zhu X: Hematopoietic Pyk2 regulates migration of differentiated HL-60 cells. J Inflamm (Lond) 2010, 7(1):26.
• [14]Xu J, Gao XP, Ramchandran R, Zhao YY, Vogel SM, Malik AB: Nonmuscle myosin light-chain kinase mediates neutrophil transmigration in sepsis-induced lung inflammation by activating beta2 integrins. Nat Immunol 2008, 9(8):880-886.
• [15]Okigaki M, Davis C, Falasca M, Harroch S, Felsenfeld DP, Sheetz MP, Schlessinger J: Pyk2 regulates multiple signaling events crucial for macrophage morphology and migration. Proc Natl Acad Sci USA 2003, 100(19):10740-10745.
• [16]Guinamard R, Okigaki M, Schlessinger J, Ravetch JV: Absence of marginal zone B cells in Pyk-2-deficient mice defines their role in the humoral response. Nat Immunol 2000, 1(1):31-36.
• [17]Duan Y, Learoyd J, Meliton AY, Clay BS, Leff AR, Zhu X: Inhibition of Pyk2 blocks airway inflammation and hyperresponsiveness in a mouse model of asthma. Am J Respir Cell Mol Biol 2010, 42(4):491-497.
• [18]Szarka RJ, Wang N, Gordon L, Nation PN, Smith RH: A murine model of pulmonary damage induced by lipopolysaccharide via intranasal instillation. J Immunol Methods 1997, 202(1):49-57.
• [19]Osier M, Oberdorster G: Intratracheal inhalation vs intratracheal instillation: differences in particle effects. Fundam Appl Toxicol 1997, 40(2):220-227.
• [20]Zhu X, Boetticher E, Wang L, Duan Y, Learoyd J, Leff AR: Proline-rich tyrosine kinase 2 regulates spreading and migration of eosinophils after beta2-integrin adhesion. Am J Respir Cell Mol Biol 2008, 39(3):263-269.
• [21]Moitra J, Sammani S, Garcia JG: Re-evaluation of Evans Blue dye as a marker of albumin clearance in murine models of acute lung injury. Transl Res 2007, 150(4):253-265.
• [22]Myou S, Leff AR, Myo S, Boetticher E, Tong J, Meliton AY, Liu J, Munoz NM, Zhu X: Blockade of inflammation and airway hyperresponsiveness in immune-sensitized mice by dominant-negative phosphoinositide 3-kinase-TAT. J Exp Med 2003, 198(10):1573-1582.
• [23]Reutershan J, Ley K: Bench-to-bedside review: acute respiratory distress syndrome - how neutrophils migrate into the lung. Crit Care 2004, 8(6):453-461.
• [24]Abraham E, Carmody A, Shenkar R, Arcaroli J: Neutrophils as early immunologic effectors in hemorrhage- or endotoxemia-induced acute lung injury. Am J Physiol Lung Cell Mol Physiol 2000, 279(6):L1137-1145.
• [25]Zemans RL, Colgan SP, Downey GP: Transepithelial migration of neutrophils: mechanisms and implications for acute lung injury. Am J Respir Cell Mol Biol 2009, 40(5):519-535.
• [26]Breslin JW, Sun H, Xu W, Rodarte C, Moy AB, Wu MH, Yuan SY: Involvement of ROCK-mediated endothelial tension development in neutrophil-stimulated microvascular leakage. Am J Physiol Heart Circ Physiol 2006, 290(2):H741-750.
• [27]DiStasi MR, Ley K: Opening the flood-gates: how neutrophil-endothelial interactions regulate permeability. Trends Immunol 2009, 30(11):547-556.
• [28]Li XY, Donaldson K, MacNee W: Lipopolysaccharide-induced alveolar epithelial permeability: the role of nitric oxide. Am J Respir Crit Care Med 1998, 157(4 Pt 1):1027-1033.
• [29]Rossi JL, Velentza AV, Steinhorn DM, Watterson DM, Wainwright MS: MLCK210 gene knockout or kinase inhibition preserves lung function following endotoxin-induced lung injury in mice. Am J Physiol Lung Cell Mol Physiol 2007, 292(6):L1327-1334.
• [30]Perlman CE, Lederer DJ, Bhattacharya J: Micromechanics of alveolar edema. Am J Respir Cell Mol Biol 2011, 44(1):34-39.
• [31]Allingham MJ, van Buul JD, Burridge K: ICAM-1-mediated, Src- and Pyk2-dependent vascular endothelial cadherin tyrosine phosphorylation is required for leukocyte transendothelial migration. J Immunol 2007, 179(6):4053-4064.
• [32]Cain RJ, Vanhaesebroeck B, Ridley AJ: The PI3K p110alpha isoform regulates endothelial adherens junctions via Pyk2 and Rac1. J Cell Biol 2010, 188(6):863-876.
• [33]Kalyanaraman M, Heidemann SM, Sarnaik AP: Macrophage inflammatory protein-2 predicts acute lung injury in endotoxemia. J Investig Med 1998, 46(6):275-278.
• [34]Anand AR, Cucchiarini M, Terwilliger EF, Ganju RK: The tyrosine kinase Pyk2 mediates lipopolysaccharide-induced IL-8 expression in human endothelial cells. J Immunol 2008, 180(8):5636-5644.
• [35]Nick JA, Young SK, Brown KK, Avdi NJ, Arndt PG, Suratt BT, Janes MS, Henson PM, Worthen GS: Role of p38 mitogen-activated protein kinase in a murine model of pulmonary inflammation. J Immunol 2000, 164(4):2151-2159.
• [36]Arndt PG, Young SK, Lieber JG, Fessler MB, Nick JA, Worthen GS: Inhibition of c-Jun N-terminal kinase limits lipopolysaccharide-induced pulmonary neutrophil influx. Am J Respir Crit Care Med 2005, 171(9):978-986.
• [37]Novogrodsky A, Vanichkin A, Patya M, Gazit A, Osherov N, Levitzki A: Prevention of lipopolysaccharide-induced lethal toxicity by tyrosine kinase inhibitors. Science 1994, 264(5163):1319-1322.
• [38]Ruetten H, Thiemermann C: Effects of tyrphostins and genistein on the circulatory failure and organ dysfunction caused by endotoxin in the rat: a possible role for protein tyrosine kinase. Br J Pharmacol 1997, 122(1):59-70.
• [39]Lowell CA, Berton G: Resistance to endotoxic shock and reduced neutrophil migration in mice deficient for the Src-family kinases Hck and Fgr. Proc Natl Acad Sci USA 1998, 95(13):7580-7584.
• [40]Lee HS, Moon C, Lee HW, Park EM, Cho MS, Kang JL: Src tyrosine kinases mediate activations of NF-kappaB and integrin signal during lipopolysaccharide-induced acute lung injury. J Immunol 2007, 179(10):7001-7011.
• [41]Baruzzi A, Caveggion E, Berton G: Regulation of phagocyte migration and recruitment by Src-family kinases. Cell Mol Life Sci 2008, 65(14):2175-2190.
• [42]Collins M, Tremblay M, Chapman N, Curtiss M, Rothman PB, Houtman JC: The T cell receptor-mediated phosphorylation of Pyk2 tyrosines 402 and 580 occurs via a distinct mechanism than other receptor systems. J Leukoc Biol 2010, 87(4):691-701.