Matrix metalloproteinase-9 promotes neutrophil migration and alveolar capillary leakage in pancreatitis-associated lung injury in the rat

doi:10.1053/gast.2002.30348

Gastroenterology

Volume 122, Issue 1, January 2002, Pages 188-201

https://doi.org/10.1053/gast.2002.30348 Get rights and content

Abstract

Background & Aims: In pancreatitis-associated lung injury, neutrophils (PMN) access the lung by migration through endothelial basement membranes. We hypothesize that degeneration of the basement membrane by specific PMN-produced matrix metalloproteinases (MMPs) may facilitate this process. Methods: Mild or severe pancreatitis was induced in rats and the consequent pulmonary injury characterized. MMP-2 and MMP-9 activity in supernatant of PMN cultures and homogenates of lungs were assessed by zymography and Western blot. Congruence of PMN and MMP expression in lung tissue was evaluated by neutrophil depletion and fluorescent immunohistochemistry (IHC). The contribution of MMPs to PMN transmigration and lung injury was tested with the MMP inhibitor batimastat (BB-94) in vitro (PMN transmigration across matrigel chambers) and in vivo (myeloperoxidase activity and Evans blue in broncho-alveolar lavage fluid). Results: MMP-9 was highly expressed in lungs and supernatant of neutrophil cultures in severe pancreatitis, and, to a lesser degree, in mild pancreatitis. Lung IHC showed colocalization of MMP-9 and PMN. PMN depletion simultaneously reduced neutrophil infiltration and MMP-9 levels in lung tissue. Trypsin, interleukin 1β, and tumor necrosis factor (TNF)-α all potently stimulated MMP-9 release from PMN. BB-94 significantly reduced TNF-α–induced PMN transmigration across matrigel and ameliorated transendothelial PMN migration and protein leak in severe pancreatitis. Conclusions: MMP-9 secretion by PMN can be stimulated by trypsin and proinflammatory cytokines and increases in pancreatitis in proportion to its severity. MMP inhibition reduces PMN transmigration and reduces resultant alveolar-capillary leakage. These findings suggest an important role for MMP-9 from PMN in the pathogenesis of pancreatitis-associated lung injury.

GASTROENTEROLOGY 2002;122:188-201

Section snippets

Animals and reagents

Male Sprague-Dawley rats (300 ± 50 g body mass [BM]) were obtained from Charles River Laboratories (Wilmington, MA). Care was provided in accordance to the procedures outlined in the Guide for Care and Use of Laboratory Animals (DHHS Publication No. (NIH) 85-23, revised 1985, Office of Science and Health Reports, Bethesda, MD). The study was approved by the subcommittee on animal research at our institution. Rats were housed individually in hanging cages at standard conditions (12-hour

MMPs are expressed in lung in acute pancreatitis

We first evaluated the expression of MMPs in lungs from animals with acute pancreatitis. MMP activity was evaluated on zymograms and MMP protein expression on Western blots with specific antibodies against MMP-2 and MMP-9. As illustrated in the Western blots and zymograms in Figures 1 and 2, MMP-2 activity and protein expression were present and at the same levels in the lungs of normal control animals, animals with mild pancreatitis, and those with severe acute pancreatitis.

. Gelatin zymogram of

Discussion

Pancreatitis-associated lung injury is clinically and pathologically indiscernible from ARDS consequent to other pathogenetic factors such as sepsis.36, 37, 38 Diffuse endothelial injury leading to large molecule extravasation and protein leakage into the alveolar space has been shown in animal models18, 39, 40 and in patients with ARDS.⁴¹ The fundamental pathophysiologic process causing that injury is neutrophil-dependent and protease- and oxygen-radical—mediated.⁴² Neutrophil activation and

Acknowledgements

The authors thank Gerald Waneck, Ph.D. for assistance and advice with the confocal microscope, Paul Leavis, Ph.D. for his expertise with the mass spectroscopy of BB-94 in rat serum, and British Biotech for kindly providing BB-94.

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^*: Address requests for reprints to: A. L. Warshaw, M.D., Surgeon-in-Chief, W. Gerald Austen Professor of Surgery, Massachusetts General Hospital, Harvard Medical School, 55 Fruit Street WHT 506, Boston, Massachusetts 02114. e-mail [email protected]; fax: (617) 726-7593.

^**: Supported by the Deutsche Forschungsgemeinschaft (KE 763/3-1; to T.K.), a grant by the National Pancreas Foundation, and the Edward D. Churchill, M.D. Resident Research Fellowship, Department of Surgery, Massachusetts General Hospital (to J.H.B.).

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Basic ResearchMatrix metalloproteinase-9 promotes neutrophil migration and alveolar capillary leakage in pancreatitis-associated lung injury in the rat*,**

Abstract

Section snippets

Animals and reagents

MMPs are expressed in lung in acute pancreatitis

Discussion

Acknowledgements

Gastroenterology

Gastroenterology

J Surg Res

Anal Biochem

Anal Biochem

J Immunol Methods

Gastroenterology

Methods Enzymol

Surgery

Am J Surg

J Biol Chem

FEBS Lett

Etiology and pathophysiology of acute pancreatitis

Multicentre audit of death from acute pancreatitis

Br J Surg

Arterial hypoxia in acute pancreatitis

Br J Surg

Respiratory complications in acute pancreatitis

Ann Surg

Early respiratory insufficiency in acute pancreatitis

Ann Surg

Quantitative measurement of P- and E-selectin adhesion molecules in acute pancreatitis: correlation with distant organ injury

Ann Surg

Specific therapy for local and systemic complications of acute pancreatitis with monoclonal antibodies against ICAM-1

Ann Surg

Polymorphonuclear leukocyte migration through human amnion membrane

J Cell Biol

An electron microscopic study on leukocytic emigration and vascular permeability in rat skin

Aust J Exp Biol Med Sci

Electron microscopy of leukocytic margination and emigration in acute inflammation in dog pancreas

Am J Pathol

Alveolar basement membrane breaks down in diffuse alveolar damage: an immunohistochemical study

Pathol Int

Degradation of human glomerular basement membrane by stimulated neutrophils. Activation of a metalloproteinase(s) by reactive oxygen metabolites

J Clin Invest

Disruption of the subendothelial basement membrane during neutrophil diapedesis in an in vitro construct of a blood vessel wall

J Clin Invest

Tissue destruction by neutrophils

N Engl J Med

The effects of neutrophil depletion on a completely noninvasive model of acute pancreatitis-associated lung injury

Int J Pancreatol

Matrix metalloproteinases. Matrix degradation and more

Am J Respir Cell Mol Biol

Matrix metalloproteinases in immunity

J Immunol

Partial purification of collagenase and gelatinase from polymorphonuclear leukocytes. Analysis of their actions on soluble and insoluble collagens

Biochem J

The basement membrane in pathology

Lab Invest

A better model of acute pancreatitis for evaluating therapy

Ann Surg

Matrix metalloproteinase inhibitor BB-94 (batimastat) inhibits human colon tumor growth and spread in a patient-like orthotopic model in nude mice

Cancer Res

Basic Research
Matrix metalloproteinase-9 promotes neutrophil migration and alveolar capillary leakage in pancreatitis-associated lung injury in the rat*,**