Biomedical subjects
Steven D Shapiro
Publications and source records attributed to Steven D Shapiro.
Surfactant protein-D regulates soluble CD14 through matrix metalloproteinase-12.
Surfactant protein D (SP-D) and CD14 are important innate immune defense molecules that mediate clearance of pathogens and apoptotic cells from the lung. To test whether CD14 expression and function were influenced by SP-D, the surface expression of CD14 was assessed on alveolar macrophages from SP-D-/- mice. CD14 was reduced on alveolar macrophages from SP-D-/- mice and was associated with reduced uptake of LPS and decreased production of TNF-alpha after LPS stimulation. CD14 is proteolytically cleaved from the cell surface to form a soluble peptide. Soluble CD14 (sCD14) was increased in the bronchoalveolar lavage fluid from SP-D-/- mice. Because matrix metalloproteinase (MMP)-9 and -12 activities were increased in the lungs of SP-D-/- mice, the role of these metalloproteases in the production of sCD14 was assessed. sCD14 was decreased in both MMP(9-/-)/SP-D-/- and MMP12(-/-)/SP-D-/- mice demonstrating MMP-9 and MMP-12 contribute to proteolytic shedding of CD14. The increased sCD14 seen in SP-D-/- mice was dependent upon the activation of MMP-12 via an MMP-9-dependent mechanism. Supporting this observation, MMP-12 caused the release of sCD14 from RAW 264.7 cells in vitro. In conclusion, SP-D influences innate host defense, in part, by regulating sCD14 in a process mediated by MMP-9 and MMP-12.
The NIH Public Access Policy and ATS journals.
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Leukocyte-derived matrix metalloproteinase-9 mediates blood-brain barrier breakdown and is proinflammatory after transient focal cerebral ischemia.
Results of recent studies reveal vascular and neuroprotective effects of matrix metalloproteinase-9 (MMP-9) inhibition and MMP-9 gene deletion in experimental stroke. However, the cellular source of MMP-9 produced in the ischemic brain and the mechanistic basis of MMP-9-mediated brain injury require elucidation. In the present study, we used MMP-9-/- mice and chimeric knockouts lacking either MMP-9 in leukocytes or in resident brain cells to test the hypothesis that MMP-9 released from leukocytes recruited to the brain during postischemic reperfusion contributes to this injury phenotype. We also tested the hypothesis that MMP-9 promotes leukocyte recruitment to the ischemic brain and thus is proinflammatory. The extent of blood-brain barrier (BBB) breakdown, the neurological deficit, and the volume of infarction resulting from transient focal stroke were abrogated to a similar extent in MMP-9-/- mice and in chimeras lacking leukocytic MMP-9 but not in chimeras with MMP-9-containing leukocytes. Zymography and Western blot analysis from these chimeras confirmed that the elevated MMP-9 expression in the brain at 24 h of reperfusion is derived largely from leukocytes. MMP-9-/- mice exhibited a reduction in leukocyte-endothelial adherence and a reduction in the number of neutrophils plugging capillaries and infiltrating the ischemic brain during reperfusion; microvessel immunopositivity for collagen IV was also preserved in these animals. These latter results document proinflammatory actions of MMP-9 in the ischemic brain. Overall, our findings implicate leukocytes, most likely neutrophils, as a key cellular source of MMP-9, which, in turn, promotes leukocyte recruitment, causes BBB breakdown secondary to microvascular basal lamina proteolysis, and ultimately contributes to neuronal injury after transient focal stroke.
Normal lung development in RAIG1-deficient mice despite unique lung epithelium-specific expression.
RAIG1, 2, and 3 and GPCR5d represent a new subfamily of orphan G protein-coupled receptors. RAIG1 is expressed abundantly and specifically in the lung during development and in adult mice. During lung development, RAIG1 expression is initiated at E14.5 and gradually increases, reaching its highest levels at E18. High levels of expression are maintained in adult lungs. Given its abundant lung-specific expression and role in retinoic acid signaling, we hypothesized that RAIG1 plays a role in epithelial cell differentiation during lung development. To determine RAIG1 function and track endogenous RAIG1 spatial expression, a null allele of Raig1 was generated and the lacZ gene was "knocked-in." Although expression was detected in both proximal and distal epithelium during embryogenesis, it became restricted to type I and type II pneumocytes and the most distal bronchiolar cells in postnatal lungs. This is the first gene known to have this unique epithelial cell expression pattern. Despite this high level of expression, targeted inactivation of Raig1 did not cause significant developmental defects. Epithelial cell differentiation was normal and lung structure was intact. Analysis of other family members demonstrated some overlapping embryonic expression of RAIG3 mRNA that could have led to functional redundancy in the single RAIG1 null mutant mouse.
Induction of macrophage elastase (MMP-12) gene expression by statins.
The statins (including mevastatin and lovastatin) are a widely prescribed class of serum-cholesterol lowering drugs that function by inhibiting 3-hydroxymethylglutaryl coenzyme A (HMG CoA) reductase activity and cellular sterol synthesis. Statins are also widely being appreciated for their inhibitory effects upon inflammation, primarily mediated through direct regulation of inflammatory gene expression. Here we report that statins are also capable of increasing the expression of macrophage elastase (MMP-12). The induction of MMP-12 in mouse macrophages by statins is specific for HMG CoA reductase inhibition, rescued by mevalonate and not observed after inhibition of subsequent steps in the cholesterol biosynthetic pathway. Modulation of cholesterol metabolism may lead to changes in MMP-12 expression and subsequent impacts during physiological and pathophysiological states. We conclude that statins, in addition to their previously described anti-inflammatory properties, may promote the production of some proteinases from activated macrophages.
Serine protease cathepsin G regulates adhesion-dependent neutrophil effector functions by modulating integrin clustering.
The polymorphonuclear leukocyte (PMN)-derived serine proteases play a key role in immune complex (IC)-mediated inflammation. However, the mechanisms by which these proteases regulate inflammatory response remain largely undefined. Here, we show that IC-activated cathepsin G- and neutrophil elastase-deficient (CG/NE) PMNs adhered normally to IC-coated surfaces but did not undergo CD11b clustering and failed to initiate cytoskeletal reorganization and cell spreading. As a result, CG/NE-deficient PMNs exhibited severe defects in MIP-2 secretion and reactive oxygen intermediates production. Exogenously added CG, but not proteolytically inactive CG, was sufficient to restore these defects. These findings identify an important role for CG in integrin-dependent PMN effector functions that are separate from and downstream of integrin-dependent adhesion.
Polymers of Z alpha1-antitrypsin co-localize with neutrophils in emphysematous alveoli and are chemotactic in vivo.
The molecular mechanisms that cause emphysema are complex but most theories suggest that an excess of proteinases is a crucial requirement. This paradigm is exemplified by severe deficiency of the key anti-elastase within the lung: alpha(1)-antitrypsin. The Z mutant of alpha(1)-antitrypsin has a point mutation Glu342Lys in the hinge region of the molecule that renders it prone to intermolecular linkage and loop-sheet polymerization. Polymers of Z alpha(1)-antitrypsin aggregate within the liver leading to juvenile liver cirrhosis and the resultant plasma deficiency predisposes to premature emphysema. We show here that polymeric alpha(1)-anti-trypsin co-localizes with neutrophils in the alveoli of individuals with Z alpha(1)-antitrypsin-related emphysema. The significance of this finding is underscored by the excess of neutrophils in these individuals and the demonstration that polymers cause an influx of neutrophils when instilled into murine lungs. Polymers exert their effect directly on neutrophils rather than via inflammatory cytokines. These data provide an explanation for the accelerated tissue destruction that is characteristic of Z alpha(1)-antitrypsin-related emphysema. The transition of native Z alpha(1)-antitrypsin to polymers inactivates its anti-proteinase function, and also converts it to a proinflammatory stimulus. These findings may also explain the progression of emphysema in some individuals despite alpha(1)-antitrypsin replacement therapy.
An inhaled matrix metalloprotease inhibitor prevents cigarette smoke-induced emphysema in the mouse.
Inadequately regulated proteolytic activity is responsible for the chronic lung tissue degeneration and irreversible loss of pulmonary function that define emphysema. In this study, we show that an inhaled broad-spectrum matrix metalloprotease inhibitor, ilomastat, can provide protection against the development of emphysema in cigarette smoke-treated mice. Control animals were exposed to daily cigarette smoke for 6 months. As has been reported previously, cigarette smoke was seen to increase significantly the recruitment of macrophages into the lungs of these animals, leading to concomitant alveolar airspace enlargement and emphysema. In animals treated daily with nebulized ilomastat for 6 months, lung macrophage levels were greatly reduced, and neutrophil accumulation was also inhibited. Corresponding reductions in airspace enlargement of up to 96% were observed. These striking observations suggest that delivery of ilomastat directly into the lungs of smoke-treated mice can not only inhibit lung tissue damage mediated by metalloproteases, but may also reduce that component of tissue degeneration mediated by excess neutrophil-derived products. Our data also suggest that the matrix metalloprotease inhibitors may represent a class of drugs that, when delivered by inhalation, could be used practically to treat cigarette smoking-related chronic obstructive pulmonary disease by modifying the course of the disease.
The pathogenesis of chronic obstructive pulmonary disease: advances in the past 100 years.
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The unintended consequences of noble intentions to inform.
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The NIH Public Access Policy and ATS journals.
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Smoke and mirrors: Mouse models as a reflection of human chronic obstructive pulmonary disease.
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Membrane-bound matrix metalloproteinase-8 on activated polymorphonuclear cells is a potent, tissue inhibitor of metalloproteinase-resistant collagenase and serpinase.
Little is known about the cell biology or the biologic roles of polymorphonuclear cell (PMN)-derived matrix metalloproteinase-8 (MMP-8). When activated with proinflammatory mediators, human PMN release only approximately 15-20% of their content of MMP-8 ( approximately 60 ng/10(6) cells) exclusively as latent pro-MMP-8. However, activated PMN incubated on type I collagen are associated with pericellular collagenase activity even when bathed in serum. PMN pericellular collagenase activity is attributable to membrane-bound MMP-8 because: 1) MMP-8 is expressed in an inducible manner in both pro- and active forms on the surface of human PMN; 2) studies of activated PMN from mice genetically deficient in MMP-8 (MMP-8(-/-)) vs wild-type (WT) mice show that membrane-bound MMP-8 accounts for 92% of the MMP-mediated, PMN surface type I collagenase activity; and 3) human membrane-bound MMP-8 on PMN cleaves types I and II collagens, and alpha(1)-proteinase inhibitor, but is substantially resistant to inhibition by tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2. Binding of MMP-8 to the PMN surface promotes its stability because soluble MMP-8 has t(1/2) = 7.5 h at 37 degrees C, but membrane-bound MMP-8 retains >80% of its activity after incubation at 37 degrees C for 18 h. Studies of MMP-8(-/-) vs WT mice given intratracheal LPS demonstrate that 24 h after intratracheal LPS, MMP-8(-/-) mice have 2-fold greater accumulation of PMN in the alveolar space than WT mice. Thus, MMP-8 has an unexpected, anti-inflammatory role during acute lung injury in mice. TIMP-resistant, active MMP-8 expressed on the surface of activated PMN is likely to be an important form of MMP-8, regulating lung inflammation and collagen turnover in vivo.
Neutrophil elastase (NE)-deficient mice demonstrate a nonredundant role for NE in neutrophil migration, generation of proinflammatory mediators, and phagocytosis in response to zymosan particles in vivo.
Neutrophil elastase (NE) remains a controversial player in the process of leukocyte transmigration and much of this controversy stems from conflicting reports on the effects of NE inhibitors. The availability of NE-deficient mice (NE(-/-)) provides a clean and elegant tool for the study of leukocyte migration in vivo. In this study, NE(-/-) mice were used to investigate the role of NE in leukocyte migration through cremasteric venules, as observed by intravital microscopy, induced by locally administered cytokines IL-1beta and TNF-alpha and the particulate stimulus, zymosan. Although no defects in leukocyte responses induced by the cytokines were observed, zymosan-induced leukocyte firm adhesion and transmigration was suppressed in NE(-/-) mice. These responses were also inhibited in wild-type mice when zymosan was coinjected with a specific NE inhibitor. Quantification of inflammatory mediator levels in homogenates of zymosan-stimulated tissues indicated reductions in levels of IL-1beta, KC, and macrophage inflammatory protein-1alpha in NE(-/-) mice. Furthermore, phagocytosis of fluorescent zymosan particles, as observed by intravital microscopy, was diminished in NE-deficient animals. Collectively, the findings of this study indicate a nonredundant role for NE in zymosan-induced leukocyte firm adhesion and transmigration, and that this defect is associated with impaired generation of proinflammatory mediators as well as phagocytosis of zymosan particles in vivo.
Matrix metalloproteinase-12 (MMP-12) in osteoclasts: new lesson on the involvement of MMPs in bone resorption.
Osteoclasts require matrix metalloproteinase (MMP) activity and cathepsin K to resorb bone, but the critical MMP has not been identified. Osteoclasts express MMP-9 and MMP-14, which do not appear limiting for resorption, and the expression of additional MMPs is not clear. MMP-12, also called metalloelastase, is reported only in a few cells, including tissue macrophages and hypertrophic chondrocytes. MMP-12 is critical for invasion and destruction in pathologies such as aneurysm and emphysema. In the present study, we demonstrate that osteoclasts express MMP-12, although only in some situations. Northern blots show that highly purified rabbit osteoclasts in culture express MMP-12 at the same level as macrophages, whereas in situ hybridizations performed on rabbit bone do not show any MMP-12 expression in osteoclasts whatever the bone type. In contrast, in situ hybridizations performed on mouse bone show MMP-12 expression in osteoclasts in calvariae and long bones. We also demonstrate that recombinant MMP-12 cleaves the putative functional domains of osteopontin and bone sialoprotein, two bone matrix proteins that strongly influence osteoclast activities, such as attachment, spreading and resorption. Furthermore, we investigated the role of MMP-12 in bone resorption and osteoclast recruitment by comparing MMP-12 knockout and wild-type mice in specialized culture models known to depend on MMP activity, as well as in the ovariectomy model, and we did not find any indication for a limiting role of MMP-12 in these processes. In conclusion, we found that osteoclasts are able to express MMP-12, but MMP-12 did not appear critical for osteoclast recruitment or resorption. The fact that none of the MMPs identified so far in osteoclasts appears limiting for resorption, gives strength to the hypothesis that the critical MMP for bone solubilization is produced by non-osteoclastic cells.
Accelerated enlargement of experimental abdominal aortic aneurysms in a mouse model of chronic cigarette smoke exposure.
BACKGROUND: Cigarette smoking and pulmonary emphysema are strongly associated with abdominal aortic aneurysms (AAAs), but the biologic mechanisms linking these conditions are undefined. STUDY DESIGN: To determine if exposure to cigarette smoke influences formation and growth of experimental AAAs, 129/SvEv mice were acclimated to daily cigarette smoke exposure for 2 weeks followed by transient elastase perfusion of the abdominal aorta to induce aneurysmal degeneration. Smoking was continued for intervals of either 2 or 12 weeks (8 mice per group). Nonsmoking 129/SvEv controls (n = 29) underwent elastase perfusion and followup evaluation at the same time intervals. In all animals, abdominal aortic diameter (AD) was measured to determine interval increases in AD (Delta AD), with AAAs defined as a Delta AD > 100%. RESULTS: Preperfusion and immediate postperfusion ADs were not significantly different between experimental groups. Aneurysmal dilatation was present 2 weeks after elastase perfusion in both smoking mice and nonsmoking controls, with no significant difference in final AD (mean +/- SEM: smoking, 1.23 +/- 0.11 mm versus nonsmoking, 1.22 +/- 0.05 mm). There were also no differences in the overall extent of aortic dilatation (Delta AD smoking, 136 +/- 24% versus nonsmoking, 138 +/- 10%), or the incidence of AAAs (smoking, 75% versus nonsmoking, 79%). Although all animals had developed AAAs by 12 weeks after elastase perfusion, the overall extent of aortic dilatation was 50% greater in smoking mice compared with nonsmoking controls (Delta AD smoking, 204 +/- 23% versus nonsmoking, 135 +/- 17%; p < 0.05). CONCLUSIONS: Short-term exposure to cigarette smoke did not alter initial development of experimental AAAs, but chronic smoke exposure was associated with a substantial increase in the late progression of aneurysmal dilatation. This novel combination of in vivo experimental models offers a new approach to investigate mechanisms by which cigarette smoking promotes aneurysmal degeneration.
Role of metalloelastase in a model of allergic lung responses induced by cockroach allergen.
Our laboratory and others have shown an important role of metalloelastase (MMP-12) in the pathogenesis of acute and chronic lung injury. Because chronic asthma is characterized by airway inflammation and alterations in the airway extracellular matrix, we explored the role of metalloelastase in a model of allergic airway inflammation induced by cockroach antigen (CRA). Using MMP-12-deficient mice we found a significant reduction in CRA-induced inflammatory injury, as evidenced by fewer peribronchial leukocytes, significantly less protein in the bronchoalveolar lavage (BAL) fluid, and a significant reduction in the number of infiltrating neutrophils, eosinophils, and macrophages, relative to wild-type mice. Although we did not find a significant reduction in the number of T cells in the injured MMP-12-deficient animals as compared to controls, levels of the chemotactic factors interleukin-5, macrophage inflammatory protein-1 alpha, monocyte chemoattractant protein-1, thymus activation regulated chemokine, and the proinflammatory cytokine tumor necrosis factor-alpha were significantly reduced in the bronchoalveolar lavage fluid of CRA-challenged MMP-12-deficient mice, relative to CRA-challenged control animals. These studies indicate that MMP-12 plays an important proinflammatory role in the development of allergic inflammation in the CRA model. Alterations in the levels of chemotactic factors and other proinflammatory cytokines in the MMP-12-deficient mice may underlie the decrease in leukocyte recruitment into inflamed lungs.