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Characterization of gelatinases in bronchoalveolar lavage fluid and gelatinases produced by alveolar macrophages isolated from healthy calves

Jeffrey LakritzDepartment of Veterinary Medicine and Surgery, University of Missouri, Columbia, MO 65211.
Department of Veterinary Pathobiology, University of Missouri, Columbia, MO 65211.
Present address is the Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.

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Antoinette E. MarshDepartment of Veterinary Pathobiology, University of Missouri, Columbia, MO 65211.

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Mary CockrellDepartment of Veterinary Medicine and Surgery, University of Missouri, Columbia, MO 65211.

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Michael F. SmithCollege of Veterinary Medicine, and the Department of Animal Science, College of Agriculture, Food and Natural Resources, University of Missouri, Columbia, MO 65211.

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Jeff W. TylerDepartment of Veterinary Medicine and Surgery, University of Missouri, Columbia, MO 65211.
Department of Veterinary Pathobiology, University of Missouri, Columbia, MO 65211.

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Abstract

Objective—To characterize gelatinases in bronchoalveolar lavage fluid (BALF) and gelatinases produced by alveolar macrophages of healthy calves.

Sample Population—Samples of BALF and alveolar macrophages obtained from 20 healthy 2-month-old calves.

Procedure—BALF was examined by use of gelatin zymography and immunoblotting to detect gelatinases and tissue inhibitor of metalloproteinase (TIMP)-1 and -2. Cultured alveolar macrophages were stimulated with lipopolysaccharide (LPS), and conditioned medium was subjected to zymography. Alveolar macrophage RNA was used for reverse transcriptasepolymerase chain reaction assay of matrix metalloproteinases (MMPs), cyclooxygenase-2, and inducible nitric oxide synthase.

Results—Gelatinolytic activity in BALF was evident at 92 kd (14/20 calves; latent MMP-9) and 72 kd (18/20; latent MMP-2). Gelatinolytic activity was evident at 82 kd (10/20 calves; active MMP-9) and 62 kd (17/20; active MMP-2). Gelatinases were inhibited by metal chelators but not serine protease inhibitors. Immunoblotting of BALF protein and conditioned medium confirmed the MMP-2 and -9 proteins. Endogenous inhibitors (ie, TIMPs) were detected in BALF from all calves (TIMP-1) or BALF from only 4 calves (TIMP-2). Cultured alveolar macrophages expressed detectable amounts of MMP-9 mRNA but not MMP-2 mRNA.

Conclusions and Clinical Relevance—Healthy calves have detectable amounts of the gelatinases MMP-2 and -9 in BALF. Endogenous inhibitors of MMPs were detected in BALF (ie, TIMP-1, all calves; TIMP-2, 4 calves). Lipopolysaccharide-stimulated alveolar macrophages express MMP-9 but not MMP-2 mRNA. The role of proteases in the pathogenesis of lung injury associated with pneumonia has yet to be determined. (Am J Vet Res 2004;65:163–172)

Abstract

Objective—To characterize gelatinases in bronchoalveolar lavage fluid (BALF) and gelatinases produced by alveolar macrophages of healthy calves.

Sample Population—Samples of BALF and alveolar macrophages obtained from 20 healthy 2-month-old calves.

Procedure—BALF was examined by use of gelatin zymography and immunoblotting to detect gelatinases and tissue inhibitor of metalloproteinase (TIMP)-1 and -2. Cultured alveolar macrophages were stimulated with lipopolysaccharide (LPS), and conditioned medium was subjected to zymography. Alveolar macrophage RNA was used for reverse transcriptasepolymerase chain reaction assay of matrix metalloproteinases (MMPs), cyclooxygenase-2, and inducible nitric oxide synthase.

Results—Gelatinolytic activity in BALF was evident at 92 kd (14/20 calves; latent MMP-9) and 72 kd (18/20; latent MMP-2). Gelatinolytic activity was evident at 82 kd (10/20 calves; active MMP-9) and 62 kd (17/20; active MMP-2). Gelatinases were inhibited by metal chelators but not serine protease inhibitors. Immunoblotting of BALF protein and conditioned medium confirmed the MMP-2 and -9 proteins. Endogenous inhibitors (ie, TIMPs) were detected in BALF from all calves (TIMP-1) or BALF from only 4 calves (TIMP-2). Cultured alveolar macrophages expressed detectable amounts of MMP-9 mRNA but not MMP-2 mRNA.

Conclusions and Clinical Relevance—Healthy calves have detectable amounts of the gelatinases MMP-2 and -9 in BALF. Endogenous inhibitors of MMPs were detected in BALF (ie, TIMP-1, all calves; TIMP-2, 4 calves). Lipopolysaccharide-stimulated alveolar macrophages express MMP-9 but not MMP-2 mRNA. The role of proteases in the pathogenesis of lung injury associated with pneumonia has yet to be determined. (Am J Vet Res 2004;65:163–172)