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A Novel Proteolytic Cascade Generates an Extracellular Matrix-Derived Chemoattractant in Chronic Neutrophilic Inflammation^1,2

Amit Gaggar^3,*,,, Patricia L. Jackson, Brett D. Noerager^,¶, Philip J. O’Reilly^*, D. Brent McQuaid^*,, Steven M. Rowe^*,,,, J. P. Clancy^, and J. Edwin Blalock^,

^* Department of Medicine, Department of Pediatrics, Department of Physiology and Biophysics, and Gregory Fleming James CF Center, University of Alabama at Birmingham, Birmingham, AL 35294; and ^¶ Department of Biology, Chemistry, and Mathematics, University of Montevallo, Montevallo, AL 35115

Chronic neutrophilic inflammation is a manifestation of a variety of lung diseases including cystic fibrosis (CF). There is increasing evidence that fragments of extracellular matrix proteins, such as collagen and elastin, play an important role in inflammatory cell recruitment to the lung in animal models of airway inflammation. Unfortunately, the association of these peptides with human disease and the identification of therapeutic targets directed toward these inflammatory pathways have remained elusive. In this study, we demonstrate that a novel extracellular matrix-derived neutrophil chemoattractant, proline-glycine-proline (PGP), acts through CXC receptors 1 and 2 on neutrophils, similar to N-acetylated proline-glycine-proline (N--PGP). We describe the specific multistep proteolytic pathway involved in PGP generation from collagen, involving matrix metalloproteases 8 and 9 and prolyl endopeptidase, a serine protease for which we identify a novel role in inflammation. PGP generation correlates closely with airway neutrophil counts after administration of proteases in vivo. Using CF as a model, we show that CF sputum has elevated levels of PGP peptides and that PGP levels decline during the course of CF inpatient therapy for acute pulmonary exacerbation, pointing to its role as a novel biomarker for this disease. Finally, we demonstrate that CF secretions are capable of generating PGP from collagen ex vivo and that this generation is significantly attenuated by the use of inhibitors directed toward matrix metalloprotease 8, matrix metalloprotease 9, or prolyl endopeptidase. These experiments highlight unique protease interactions with structural proteins regulating innate immunity and support a role for these peptides as novel biomarkers and therapeutic targets for chronic, neutrophilic lung diseases.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by the University of Alabama Clinical Investigator Fellowship Award, the Cystic Fibrosis Foundation (Grant GAGGA07A0 to A.G.). S.M.R. is funded through the National Institutes of Health (Grant 1K23DK075788). J.P.C. is funded through The Thrasher Award. J.E.B. is funded through the Cystic Fibrosis Foundation (Grant R464-CR02) and National Institutes of Health (Grants HL07783 and HL090999). Funds for the purchase of mass spectrometers and the operation of the Mass Spectrometry Shared Facility came from the following National Institutes of Health grants to the University of Alabama at Birmingham: S10 RR19231, P30 CA13148, P50 AT00477, U54 CA100949, P30AR050948, and P30 DK74038. This project was supported in part by grants from the National Heart, Lung, and Blood Institute.

² The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Heart, Lung, and Blood Institute or the National Institutes of Health.

³ Address correspondence and reprint requests to Dr. Amit Gaggar, Department of Medicine, University of Alabama, 1900 University Boulevard, THT 422, Birmingham, AL 35294. E-mail address: agaggar{at}uab.edu

⁴ Abbreviations used in this paper: COPD, chronic obstructive pulmonary disease; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; ECM, extracellular matrix; ELR, glutamate-leucine-arginine; MS, mass spectrometry; ESI LC-MS/MS, electrospray ionization liquid chromatography-MS/MS; FEV1, forced expiratory volume 1 second; FVC, forced vital capacity; GRO, growth-related oncogene; HNE, human neutrophil elastase; PGP, proline-glycine-proline; N--PGP, N-acetylated PGP; PE, prolyl endopeptidase; PMN, polymorphonuclear leukocytes or neutrophils; pNA, paranitroaniline; MMP, matrix metalloprotease.

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