Hyaluronan Mediates Ozone-induced Airway Hyperresponsiveness in Mice

Abstract

Ozone is a common urban environmental air pollutant and significantly contributes to hospitalizations for respiratory illness. The mechanisms, which regulate ozone-induced bronchoconstriction, remain poorly understood. Hyaluronan was recently shown to play a central role in the response to noninfectious lung injury. Therefore, we hypothesized that hyaluronan contributes to airway hyperreactivity (AHR) after exposure to ambient ozone. Using an established model of ozone-induced airways disease, we characterized the role of hyaluronan in airway hyperresponsiveness. The role of hyaluronan in response to ozone was determined by using therapeutic blockade, genetically modified animals, and direct challenge to hyaluronan. Ozone-exposed mice demonstrate enhanced AHR associated with elevated hyaluronan levels in the lavage fluid. Mice deficient in either CD44 (the major receptor for hyaluronan) or inter-α-trypsin inhibitor (molecule that facilitates hyaluronan binding) show similar elevations in hyaluronan but are protected from ozone-induced AHR. Mice pretreated with hyaluronan-binding peptide are protected from the development of ozone-induced AHR. Overexpression of hyaluronan enhances the airway response to ozone. Intratracheal instillation of endotoxin-free low molecular weight hyaluronan induces AHR dependent on CD44, whereas instillation of high molecular weight hyaluronan protects against ozone-induced AHR. In conclusion, we demonstrate that hyaluronan mediates ozone-induced AHR, which is dependent on the fragment size and both CD44 and inter-α-trypsin inhibitor. These data support the conclusion that pulmonary matrix can contribute to the development of airway hyperresponsiveness. Ozone is a common urban environmental air pollutant and significantly contributes to hospitalizations for respiratory illness. The mechanisms, which regulate ozone-induced bronchoconstriction, remain poorly understood. Hyaluronan was recently shown to play a central role in the response to noninfectious lung injury. Therefore, we hypothesized that hyaluronan contributes to airway hyperreactivity (AHR) after exposure to ambient ozone. Using an established model of ozone-induced airways disease, we characterized the role of hyaluronan in airway hyperresponsiveness. The role of hyaluronan in response to ozone was determined by using therapeutic blockade, genetically modified animals, and direct challenge to hyaluronan. Ozone-exposed mice demonstrate enhanced AHR associated with elevated hyaluronan levels in the lavage fluid. Mice deficient in either CD44 (the major receptor for hyaluronan) or inter-α-trypsin inhibitor (molecule that facilitates hyaluronan binding) show similar elevations in hyaluronan but are protected from ozone-induced AHR. Mice pretreated with hyaluronan-binding peptide are protected from the development of ozone-induced AHR. Overexpression of hyaluronan enhances the airway response to ozone. Intratracheal instillation of endotoxin-free low molecular weight hyaluronan induces AHR dependent on CD44, whereas instillation of high molecular weight hyaluronan protects against ozone-induced AHR. In conclusion, we demonstrate that hyaluronan mediates ozone-induced AHR, which is dependent on the fragment size and both CD44 and inter-α-trypsin inhibitor. These data support the conclusion that pulmonary matrix can contribute to the development of airway hyperresponsiveness. Ozone is a commonly encountered urban air pollutant that significantly contributes to increased morbidity (1.Dockery D.W. 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It has been estimated that each year inhalation of ambient ozone contributes to 800 premature deaths, 4,500 hospital admissions, 900,000 school absences, and more than 1 million restricted activity days with an estimated $5 billion annual economic burden (5.Hubbell B.J. Hallberg A. McCubbin D.R. Post E. Environ. Health Perspect. 2005; 113: 73-82Crossref PubMed Scopus (132) Google Scholar). Population-based studies suggest that for each 10 ppb increase in 1-h daily maximum level of ozone there is an increase in mortality risk of 0.39–0.87%, especially in individuals with pre-existing respiratory disease (2.Bell M. McDermott A. Zeger S. Samet J. Dominici F. J. Am. Med. Assoc. 2004; 292: 2372-2378Crossref PubMed Scopus (884) Google Scholar, 3.Gryparis A. Forsberg B. Katsouyanni K. Analitis A. Touloumi G. Schwartz J. Samoli E. Medina S. Anderson H.R. Niciu E.M. Wichmann E. Kriz B. Kosnik M. Skorkovsky J. Vonk J.M. Dortbudak Z. Am. J. Respir. Crit. 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Short fragment hyaluronan (sHA) 2The abbreviations used are: sHA, short fragment hyaluronan; AHR, airway hyperreactivity; HABP, hyaluronan-binding protein; HA, hyaluronan; HAS2, hyaluronan synthase 2; IaI, inter-α-trypsin inhibitor; SBP, or scrambled binding protein; HMW-HA, high molecular weight hyaluronan; TK, tissue kallikrein. 2The abbreviations used are: sHA, short fragment hyaluronan; AHR, airway hyperreactivity; HABP, hyaluronan-binding protein; HA, hyaluronan; HAS2, hyaluronan synthase 2; IaI, inter-α-trypsin inhibitor; SBP, or scrambled binding protein; HMW-HA, high molecular weight hyaluronan; TK, tissue kallikrein. is released in the lung after sterile injury such as bleomycin instillation (8.Teder P. Vandivier R.W. Jiang D. Liang J. Cohn L. Pure E. Henson P.M. Noble P.W. Science. 2002; 296: 155-158Crossref PubMed Scopus (563) Google Scholar) or high tidal volume ventilation (9.Bai K.J. Spicer A.P. Mascarenhas M.M. Yu L. Ochoa C.D. Garg H.G. Quinn D.A. Am. J. Respir. Crit. Care Med. 2005; 172: 92-98Crossref PubMed Scopus (99) Google Scholar) and can modify the tissue response to injury. Furthermore, hyaluronan has been identified in airway secretions from asthmatics (10.Sahu S. Lynn W.S. Biochem. J. 1978; 173: 565-568Crossref PubMed Scopus (58) Google Scholar), and high molecular weight hyaluronan can attenuate the bronchoconstrictive response in exercise-induced asthma (11.Petrigni G. Allegra L. Pulm. Pharmacol. Ther. 2006; 19: 166-171Crossref PubMed Scopus (61) Google Scholar). We therefore hypothesized that hyaluronan may contribute to the biological response to airway injury after exposure to ozone. In this study, we provide evidence that hyaluronan mediates ozone-induced AHR. Mice exposed to ozone demonstrate elevated lung lavage fluid levels of hyaluronan, which is of low molecular weight. We observed that animals deficient in either CD44 (a hyaluronan surface receptor) or inter-α-trypsin inhibitor (IaI, which facilitates hyaluronan binding) are protected from ozone-induced AHR. Overexpression of hyaluronan by airway epithelia enhances ozone-induced AHR. Furthermore, pretreatment of mice with hyaluronan-binding peptide or high molecular weight hyaluronan protects animals from the development of ozone-induced AHR. Direct instillation of low molecular weight, but not high molecular weight, hyaluronan alone induces AHR. Our observations support a critical role for short fragment hyaluronan in the development of airway hyperresponsiveness after exposure to ozone. Mice—CD44-deficient mice were provided by T. Mak and backcrossed onto C57BL/6J for >10 generations (12.Schmits R. Filmus J. Gerwin N. Senaldi G. Kiefer F. Kundig T. Wakeham A. Shahinian A. Catzavelos C. Rak J. Furlonger C. Zakarian A. Simard J.J. Ohashi P.S. Paige C.J. Gutierrez-Ramos J.C. Mak T.W. Blood. 1997; 90: 2217-2233Crossref PubMed Google Scholar). Bikunin/IaI-deficient mice were backcrossed onto C57BL6J for >10 generations (13.Zhuo L. Yoneda M. Zhao M. Yingsung W. Yoshida N. Kitagawa Y. Kawamura K. Suzuki T. Kimata K. J. Biol. Chem. 2001; 276: 7693-7696Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). CC10-HAS2 transgenic animals were backcrossed onto C57BL/6J background for >5 generations (14.Jiang D. Liang J. Fan J. Yu S. Chen S. Luo Y. Prestwich G.D. Mascarenhas M.M. Garg H.G. Quinn D.A. Homer R.J. Goldstein D.R. Bucala R. Lee P.J. Medzhitov R. Noble P.W. Nat. Med. 2005; 11: 1173-1179Crossref PubMed Scopus (1120) Google Scholar). Experimental groups consisted of 10 male 6–8-week-old mice unless otherwise stated. Experimental protocols were approved by the Animal Care Committee at either the NIEHS, National Institutes of Health, or Duke University. Exposure Protocol—C57BL/6J, CD44-/-, IaI-/-, or HAS2 transgenic overexpressing mice were exposed to either Hepa-filtered air or ozone. Animals were housed in cages with low endotoxin bedding and given water and chow ad libitum. Ozone exposures were 2 ppm for 3 h. Our selection of ozone concentration levels in the mouse is based on similar biological response observed in human exposure studies and published deposition fraction data for O3 in rodent models (15.Wiester M.J. Tepper J.S. King M.E. Menache M.G. Costa D.L. Toxicol. Appl. Pharmacol. 1988; 96: 140-146Crossref PubMed Scopus (35) Google Scholar). Exposures were performed in a Hinner-style chamber. Air at 20–22 °C and 50–60% relative humidity was supplied at 20 exchanges/h. Ozone was generated by directing 100% O2 through a UV light generator and mixed with air supply to the chamber. Chamber ozone concentration was monitored continuously with a UV light photometer (1003AH, Dasibi, Glendale, CA). In some experiments, C57BL/6 mice were given 10 mg/kg subcutaneously hyaluronan-binding protein (HABP) or scrambled binding protein control (SBP) (16.Savani R. Hou G. Liu P. Wang C. Simons E. Grimm P. Stern R. Greenberg A. DeLisser H. Khalil N. Am. J. Respir. Cell Mol. Biol. 2000; 23: 475-484Crossref PubMed Scopus (65) Google Scholar) 1 h prior to exposure. In other experiments, IaI-/- mice were injected intraperitoneally with 1 ml of 0.5 mg/ml IaI (ProThera, East Providence, RI) or 1 ml of 1 mg/ml urinary trypsin inhibitor/bikunin (GenScript, Piscataway, NJ) 1 h prior to ozone exposure. HA Challenge—Sterile, endotoxin-free (0.00008 endotoxin units/ml) high molecular weight hyaluronan (HMW-HA) (Healon, AMO, Santa Ana, CA) was reconstituted at 0.5 mg/ml in 0.02 m acetate, 0.15 m sodium chloride, pH 6.0. For the production of low molecular weight hyaluronan (sHA), hyaluronan was sonicated on ice. Sizes were confirmed by gel electrophoresis (17.Lee H.G. Cowman M.K. Anal. Biochem. 1994; 219: 278-287Crossref PubMed Scopus (257) Google Scholar). In some experiments, 50 μl of HMW-HA, sHA, or vehicle were instilled oropharyngeally into isoflurane-anesthetized mice, and AHR was measured invasively 2–4 h later. In other experiments, 50 μl of HMW-HA or vehicle was instilled 1 h before and 23 h after acute ozone exposure, and AHR was measured invasively h after ozone exposure. after the exposure, of mice mg/kg were and and mice were and with a at a tidal volume of and a of 3 of was used for of respiratory airway and tidal volume data were generated by the of a volume with and mice were with at and the lung was with lung to to lung were at each and with the lung lavage was performed as D.L. Schwartz D.A. Am. J. Respir. Crit. Care Med. 2004; 170: PubMed Google Scholar). were at Hyaluronan hyaluronan levels were performed with a CA) as the For lavage fluid was with at °C for for 10 on and and the was in a were on a with HA, sonicated HA, and hyaluronan with in and in water and were in and with CD44 and hyaluronan-binding protein (HABP) of was used to hyaluronan. are as groups were identified by of and the unless otherwise stated. of was Ozone Exposure Hyaluronan in the that hyaluronan the response to ozone. C57BL/6 mice, mice, and mice were exposed to 2 ppm ozone for 3 h. The level of airway injury as measured by lavage protein was similar in groups and increased with mice Exposure to ozone increased the levels of hyaluronan in lavage fluid of of mice with the role of CD44 and IaI in of hyaluronan, we observed enhanced levels of HA in and IaI-/- with hyaluronan in the lavage fluid was of molecular (sHA) ozone exposure, the hyaluronan receptor CD44 was on both the airway epithelia and by Hyaluronan was in the there was increased hyaluronan deposition after ozone exposure Hyaluronan was whereas CD44 and hyaluronan on observations suggest that ozone exposure can hyaluronan in both the lavage fluid and hyaluronan and and CD44 after air or ozone exposure. and of hyaluronan and CD44 were identified in and and and mouse CD44-/-, CD44-/-, Hyaluronan is in the in mice but is more after ozone exposure CD44 is in and of hyaluronan and CD44 after ozone exposure. In a hyaluronan is to the epithelia as as of hyaluronan and CD44 after ozone exposure. C57BL/6J mice were exposed to either air or ozone and by for of CD44 and hyaluronan. after air exposure, for CD44 but not for hyaluronan after ozone exposure, hyaluronan and CD44 on an on HA for the of AHR after Ozone the role of of hyaluronan in the response to we characterized We that mice were protected from ozone-induced airway with C57BL/6J mice Furthermore, we the role of IaI, which facilitates L. A. R. N. J. M. N. Kimata K. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). with in CD44-/-, IaI-/- animals were protected from the response to ambient ozone IaI of which can hyaluronan, and a light urinary trypsin which is an but not hyaluronan L. Kimata K. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). We therefore the hyaluronan-binding of IaI was to ozone-induced AHR. of IaI but not urinary trypsin inhibitor/bikunin into mice reconstituted the response to ozone an role of IaI in the response to ozone and evidence for the role of hyaluronan in ozone-induced AHR. These observations demonstrate that the biological response to ozone in dependent on both CD44 and of is to in the of hyaluronan, the role of this in the response to ozone. HA the role of hyaluronan in airway hyperresponsiveness to C57BL/6J animals were with before ozone exposure. has been used to HA and attenuate the biological of this (16.Savani R. Hou G. Liu P. Wang C. Simons E. Grimm P. Stern R. Greenberg A. DeLisser H. Khalil N. Am. J. Respir. Cell Mol. Biol. 2000; 23: 475-484Crossref PubMed Scopus (65) Google Scholar). that significantly binding on after ozone exposure, with scrambled control peptide or was in the of lung injury as by lung lavage protein levels with either or on AHR We observed that of hyaluronan with significantly ozone-induced AHR similar to with either or mice These observations support the role of HA in the biological response to of hyaluronan ozone-induced AHR. with SBP, or hyaluronan-binding peptide not ozone-induced in protein in the lung with SBP, or not AHR in hyaluronan-binding but not scrambled significantly AHR after ozone exposure other groups Overexpression of Hyaluronan the role of increased levels of hyaluronan in the response to transgenic mice, which hyaluronan synthase 2 by airway epithelia in enhanced production of high molecular weight HA (14.Jiang D. Liang J. Fan J. Yu S. Chen S. Luo Y. Prestwich G.D. Mascarenhas M.M. Garg H.G. Quinn D.A. Homer R.J. Goldstein D.R. Bucala R. Lee P.J. Medzhitov R. Noble P.W. Nat. Med. 2005; 11: 1173-1179Crossref PubMed Scopus (1120) Google Scholar), were exposed to ozone. increased of hyaluronan, we observed a similar of lung injury as by lung lavage protein levels exposure to transgenic animals demonstrate a more and increased level of hyaluronan with was in AHR observed and animals with air exposure. ozone exposure, mice the enhanced response to after exposure to ozone. However, animals demonstrate an AHR response after ozone challenge observations suggest that elevated levels of high molecular weight HA alone are not to AHR and support that ozone-induced of HA are to AHR. Short HA to and IaI can each to hyaluronan, and evidence that mediates noninfectious lung injury (14.Jiang D. Liang J. Fan J. Yu S. Chen S. Luo Y. Prestwich G.D. Mascarenhas M.M. Garg H.G. Quinn D.A. Homer R.J. Goldstein D.R. Bucala R. Lee P.J. Medzhitov R. Noble P.W. Nat. Med. 2005; 11: 1173-1179Crossref PubMed Scopus (1120) Google Scholar). the role of in airway mice were by of hyaluronan in a We sonicated endotoxin-free high molecular weight HA to of similar size as hyaluronan in lung lavage fluid after ozone exposure of sHA, but not HMW-HA, into C57BL/6J mice AHR with vehicle The response to is We demonstrate increased response to high mg/ml or with low mg/ml or Furthermore, the surface receptor CD44 was for this AHR These data that in ozone-induced AHR. we not significant in or in the lung lavage fluid after instillation that the on AHR may not the of this we mice with HMW-HA before and after ozone HMW-HA can R. P. P. J.D. J. S. J. 1997; PubMed Scopus Google Scholar). We observed significant of AHR in mice with HMW-HA with ozone exposure demonstrate that can airway hyperresponsiveness and that HMW-HA has a role in ozone-induced AHR, that hyaluronan size is an in ozone-induced AHR. Cell into the on Hyaluronan through CD44 and and IaI can either or modify in the lung on the of lung as as the environmental in the lung has been associated with the of AHR. It was therefore to the of after exposure to ozone in and IaI-/- For both and mice, in lung lavage fluid were observed at h after exposure (the of maximum with C57BL/6 for of the observed of was in IaI-/- mice through of IaI but not is with the of that is dependent on hyaluronan CD44 and IaI been to binding to hyaluronan L. A. R. N. J. M. N. Kimata K. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar), and CD44 a role in of Z. S. A. Schwartz D.A. Am. J. Respir. Cell Mol. Biol. Scholar). In hyaluronan-binding experiments, we observed a significant on in the lavage fluid of mice but not mice we a in in HAS2 mice, with we not a significant in or in the after exposure to either high or low with vehicle not These observations support an role of CD44 and IaI in of to the lung after exposure to but suggest that the role of hyaluronan in airway hyperreactivity and is In this study, we demonstrate that hyaluronan mediates ozone-induced AHR binding to IaI and Furthermore, we demonstrate that hyaluronan size is for biological in ozone-induced AHR. HMW-HA instillation protected mice from the of whereas direct instillation of in mice AHR. we show that CD44 and IaI are for lung after ozone exposure. These support a role for hyaluronan in the of airway disease and provide a for ozone-induced airway hyperresponsiveness. Hyaluronan as a high molecular in extracellular with molecular HMW-HA is to and was shown to attenuate AHR in a model M. Y. R. Am. J. Respir. Crit. Care Med. 2001; PubMed Scopus Google Scholar). However, tissue injury can lead to of low molecular weight hyaluronan (sHA), which as a after bleomycin lung injury (8.Teder P. Vandivier R.W. Jiang D. Liang J. Cohn L. Pure E. Henson P.M. Noble P.W. Science. 2002; 296: 155-158Crossref PubMed Scopus (563) Google Scholar) or lung injury (9.Bai K.J. Spicer A.P. Mascarenhas M.M. Yu L. Ochoa C.D. Garg H.G. Quinn D.A. Am. J. Respir. Crit. Care Med. 2005; 172: 92-98Crossref PubMed Scopus (99) Google Scholar). Hyaluronan such as CD44 are on that can regulate the response in the such as airway epithelia Am. J. 2004; PubMed Scopus Google Scholar) and Chen B.J. K. Schwartz D.A. Am. J. Respir. Crit. Care Med. 2005; PubMed Scopus Google Scholar), that the to hyaluronan and In studies support that ozone and can in of hyaluronan J. 1994; PubMed Scopus Google Scholar), as observed in IaI is for binding of CD44 to hyaluronan L. A. R. N. J. M. N. Kimata K. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar), through the of hyaluronan. We demonstrate that the of both IaI and CD44 is for ozone-induced AHR. We that IaI can as an extracellular of hyaluronan binding to receptor Furthermore, is that is for of hyaluronan onto IaI H.G. J.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, D. E. C. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). We therefore that lead to a similar as we observed in We demonstrate that the biological of hyaluronan in of AHR are dependent on molecular weight. HMW-HA ozone-induced AHR, whereas induces AHR. These observations suggest that of hyaluronan may for receptor binding in hyaluronan of as as can by CD44, and extracellular hyaluronan can a size of there is an size of hyaluronan that may for CD44 Hyaluronan is a that is not to such as or after and therefore and concentration of hyaluronan may the of The and of the of high and low molecular weight hyaluronan remain However, is that HMW-HA is in the lung and that after by into or of there a hyaluronan It is that HAS2 transgenic mice not in increased AHR after ozone exposure, associated with in lavage fluid. We were to show a significant in of hyaluronan after ozone exposure in We a of hyaluronan However, suggest that ozone-induced AHR is at by which from ozone-induced of pre-existing We that HMW-HA with by of to that CD44 of the which dependent on hyaluronan Our are with which that HA can by binding and tissue M. Y. R. Am. J. Respir. Crit. Care Med. 2001; PubMed Scopus Google Scholar). We were to significant activity in lung lavage at h after ozone exposure, which may to the exposure model and h Therefore, we that the of HMW-HA is dependent on It was recently shown that the IaI light urinary trypsin but not the IaI is for the in human airways R. S.M. M.E. E. Am. J. Respir. Cell Mol. Biol. PubMed Scopus Google Scholar). In IaI, but not mediates AHR. It is therefore that the IaI and light on ozone-induced AHR. is to this of this was that CD44 and IaI were for into after ozone exposure. The of CD44 and IaI in the to hyaluronan has been recently shown L. A. R. N. J. M. N. Kimata K. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). The on the role of CD44 on with some studies that of CD44 to increased in (8.Teder P. Vandivier R.W. Jiang D. Liang J. Cohn L. Pure E. Henson P.M. Noble P.W. Science. 2002; 296: 155-158Crossref PubMed Scopus (563) Google Scholar, P. Noble P.W. Am. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) and other studies a S.M. B. Liu L. G. B. P. P. J. 2004; 173: PubMed Scopus Google Scholar). We that the may and In the of tissue such as lung injury or the of with of in the for tissue CD44 is for extracellular matrix and and CD44 is in of injury are to hyaluronan. by low such as in the of CD44 not and on the S.M. B. Liu L. G. B. P. P. J. 2004; 173: PubMed Scopus Google Scholar). However, of high the pulmonary binding to an for and to a by ozone exposure. this is for the bronchoconstrictive of hyaluronan We show that hyaluronan binding by both AHR and after ozone However, we not a in lavage after instillation or after HMW-HA instillation in mice to on AHR, and we demonstrate that HAS2 transgenic mice increased AHR but in the lavage fluid. has shown that production of HMW-HA by of transgenic of HAS2 a on epithelia after sterile lung injury (14.Jiang D. Liang J. Fan J. Yu S. Chen S. Luo Y. Prestwich G.D. Mascarenhas M.M. Garg H.G. Quinn D.A. Homer R.J. Goldstein D.R. Bucala R. Lee P.J. Medzhitov R. Noble P.W. Nat. Med. 2005; 11: 1173-1179Crossref PubMed Scopus (1120) Google Scholar). in the in after ozone exposure in the other hyaluronan is in the 2 and in direct to both epithelia and both of which CD44 It is therefore that after ozone exposure has a direct on airway or by of to regulate ozone-induced AHR Am. PubMed Scopus Google Scholar). Therefore, data suggest that AHR after ozone exposure is at in of The and which contribute to this an of In we demonstrate a central role of hyaluronan in ozone-induced AHR. Exposure to ozone in elevated levels of short fragment hyaluronan. The biological response to ozone is dependent on to hyaluronan, CD44 and We demonstrate hyaluronan has the lung dependent on the molecular weight. short of hyaluronan contribute to AHR, and high molecular weight hyaluronan ozone-induced AHR. is the short of hyaluronan contribute to AHR, which is through with CD44 and Our observations provide into the of environmental airways In conclusion, data a role for pulmonary matrix in lung and of hyaluronan as a for of airways We Santa Ana, for the of