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Characterization of matrix metalloproteinases produced by rat alveolar macrophages
D F Gibbs1, R L Warner, S J Weiss
1Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0602, USA.
Abstract:
Evidence presented in the accompanying article (Gibbs, D. F., T. P. Shanley, R. L. Warner, H. S. Murphy, J. Varani, and K. J. Johnson. 1999. Role of matrix metalloproteinases in models of macrophage-dependent acute lung injury: evidence for alveolar macrophage as source of proteinases. Am. J. Respir. Cell Mol. Biol. 20:1145-1154) implicates alveolar macrophage matrix metalloproteinases (MMPs) in two models of acute lung inflammation in the rat. As a prerequisite to understanding which specific MMPs might be involved in the injury and how they might function, it was necessary to know the spectrum of enzymes present. To this end, alveolar macrophages were obtained from normal rat lungs by bronchoalveolar lavage, placed in culture with and without various agonists, and assessed by a variety of techniques for MMPs. The identification process involved characterization by gelatin, beta-casein, and kappa-elastin zymography, with confirmation of identity by Western blot/immunoprecipitation. Message levels of detected MMPs were assessed by Northern blot. Rat alveolar macrophages were found to produce a low constitutive level of MMP-2 (72-kD gelatinase A) that was only modestly upregulated following stimulation with phorbol myristate acetate, bacterial lipopolysaccharide, or immunoglobulin A-containing immune complexes. Although control cells were found to produce little or no MMP-9 (92-kD gelatinase B) or MMP-12 (metalloelastase), both enzymes were markedly upregulated upon stimulation. In the same stimulated macrophages there was little activity against type I collagen (associated with MMP-13 [collagenase-3] on the basis of Western blotting), no activity suggestive of stromelysin or matrilysin, and no measurable secretion of the serine proteinases, elastase and cathepsin G. These data demonstrate the ability of rat alveolar macrophages to elaborate certain MMPs under proinflammatory conditions, consistent with their possible involvement in the progression of acute inflammation.
Insights
Rat alveolar macrophages produce matrix metalloproteinases (MMPs), including MMP-2, MMP-9, and MMP-12, particularly when stimulated. These MMPs may play a role in acute lung inflammation progression.
Area of Science:
- Pulmonary Medicine
- Cell Biology
- Biochemistry
Background:
- Matrix metalloproteinases (MMPs) are implicated in acute lung injury.
- Alveolar macrophages are a potential source of proteinases in lung inflammation.
Purpose of the Study:
- To identify the spectrum of MMPs produced by rat alveolar macrophages.
- To investigate the regulation of MMP production by inflammatory stimuli.
Main Methods:
- Alveolar macrophages were isolated from rat lungs via bronchoalveolar lavage.
- MMP identification used gelatin, beta-casein, and kappa-elastin zymography, Western blot/immunoprecipitation.
- Message levels were assessed by Northern blot.
Main Results:
- Rat alveolar macrophages constitutively produce low levels of MMP-2.
- MMP-9 and MMP-12 were markedly upregulated upon stimulation with phorbol myristate acetate, LPS, or immune complexes.
- Little activity was detected for MMP-13, stromelysin, matrilysin, elastase, or cathepsin G.
Conclusions:
- Rat alveolar macrophages can produce specific MMPs under proinflammatory conditions.
- Upregulated MMPs, particularly MMP-9 and MMP-12, may contribute to acute lung inflammation.