Diane G. Kelley

Neutrophil Elastase Contributes to Cigarette Smoke-Induced Emphysema in Mice

To address the role of neutrophil elastase in pulmonary emphysema, neutrophil elastase-deficient mice and wild-type littermate controls were exposed to long-term cigarette smoke. Compared to wild-type littermates, mice that were deficient in neutrophil elastase were significantly protected (59%) from the development of emphysema. Previously, we demonstrated complete protection from emphysema in the absence of macrophage elastase. Further analysis revealed several interactions between these two elastases. Each elastase inactivated the endogenous inhibitor of the other, with neutrophil elastase degrading tissue inhibitor of metalloproteinase-1, and macrophage elastase degrading alpha-1-antitrypsin. Cigarette smoke-induced recruitment of both neutrophils and monocytes was impaired in the absence of neutrophil elastase. Moreover, there was less macrophage elastase activity secondary to decreased macrophage accumulation in neutrophil elastase-deficient mice. This study demonstrates a direct role for neutrophil elastase in emphysema and highlights the interdependence of the proteinases and inflammatory cells that mediate lung destruction in response to cigarette smoke.

Elastin fragments drive disease progression in a murine model of emphysema

Mice lacking macrophage elastase (matrix metalloproteinase-12, or MMP-12) were previously shown to be protected from the development of cigarette smoke-induced emphysema and from the accumulation of lung macrophages normally induced by chronic exposure to cigarette smoke. To determine the basis for macrophage accumulation in experimental emphysema, we now show that bronchoalveolar lavage fluid from WT smoke-exposed animals contained chemotactic activity for monocytes in vitro that was absent in lavage fluid from macrophage elastase-deficient mice. Fractionation of the bronchoalveolar lavage fluid demonstrated the presence of elastin fragments only in the fractions containing chemotactic activity. An mAb against elastin fragments eliminated both the in vitro chemotactic activity and cigarette smoke-induced monocyte recruitment to the lung in vivo. Porcine pancreatic elastase was used to recruit monocytes to the lung and to generate emphysema. Elastin fragment antagonism in this model abrogated both macrophage accumulation and airspace enlargement.

Dipeptidyl peptidase I activates neutrophil-derived serine proteases and regulates the development of acute experimental arthritis.

Leukocyte recruitment in inflammation is critical for host defense, but excessive accumulation of inflammatory cells can lead to tissue damage. Neutrophil-derived serine proteases (cathepsin G [CG], neutrophil elastase [NE], and proteinase 3 [PR3]) are expressed specifically in mature neutrophils and are thought to play an important role in inflammation. To investigate the role of these proteases in inflammation, we generated a mouse deficient in dipeptidyl peptidase I (DPPI) and established that DPPI is required for the full activation of CG, NE, and PR3. Although DPPI(-/-) mice have normal in vitro neutrophil chemotaxis and in vivo neutrophil accumulation during sterile peritonitis, they are protected against acute arthritis induced by passive transfer of monoclonal antibodies against type II collagen. Specifically, there is no accumulation of neutrophils in the joints of DPPI(-/-) mice. This protective effect correlates with the inactivation of neutrophil-derived serine proteases, since NE(-/-) x CG(-/-) mice are equally resistant to arthritis induction by anti-collagen antibodies. In addition, protease-deficient mice have decreased response to zymosan- and immune complex-mediated inflammation in the subcutaneous air pouch. This defect is accompanied by a decrease in local production of TNF-alpha and IL-1 beta. These results implicate DPPI and polymorphonuclear neutrophil-derived serine proteases in the regulation of cytokine production at sites of inflammation.

Polymers of Z α1-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.

The Role of Matrix Metalloproteinase-9 in Cigarette Smoke–induced Emphysema

RATIONALE Matrix metalloprotease (MMP)-9 is an elastolytic endopeptidase produced by activated macrophages that may be involved in the development of human pulmonary emphysema and could be inhibited with existing compounds. Mouse models have demonstrated that excess MMP-9 production can result in permanent alveolar destruction. OBJECTIVES To determine if MMP-9 causes cigarette smoke-induced emphysema using MMP-9 knockout mice and human samples. METHODS Mouse lungs were analyzed for inflammation and airspace enlargement using a mainstream smoke-exposure model. Human macrophage mRNA was isolated from subjects with emphysema by laser capture microdissection. Human blood monocyte mRNA was isolated from subjects with greater than 30 pack-year smoking history. Human gene expression was determined by quantitative polymerase chain reaction and compared with emphysema severity determined by automated computed tomography analysis. Plasma Clara cell secretory protein and surfactant protein-D were quantified to measure ongoing lung injury. MEASUREMENTS AND MAIN RESULTS Mice deficient in MMP-9 develop the same degree of cigarette smoke-induced inflammation and airspace enlargement as strain-matched controls. Macrophages are the predominant source of MMP-9 production in human emphysema specimens and similar quantities of macrophage MMP-9 mRNA is present in areas of lung with and without emphysema. Circulating monocytes produce more MMP-9 in individuals with advanced emphysema severity despite no correlation of MMP-9 with markers of ongoing lung damage. CONCLUSIONS These results suggest that MMP-9 in humans who smoke is similar to smoke-exposed mice, where MMP-9 is present in emphysematous lung but not correlated with the emphysema. To the degree that the mechanisms of emphysema in humans who smoke resemble the mouse model, these data suggest specific inhibition of MMP-9 is unlikely to be an effective therapy for cigarette smoke-induced emphysema. Clinical trial registered with www.clinicaltrials.gov (NCT 00757120).

Neutrophil Elastase Cleaves Laminin-332 (Laminin-5) Generating Peptides That Are Chemotactic for Neutrophils

Proteolytic processing of laminin-332 by matrix metalloproteinase (MMP)-2 and MMP-14 has been shown to yield fragments that are promigratory for epithelial cells. During acute and chronic inflammation, proteases are elaborated by neutrophils and macrophages that can degrade basement membranes. We investigated the susceptibility of laminin-332 to degradation by the following neutrophil and macrophage proteases: neutrophil elastase (NE), cathepsin G, proteinase-3, and MMPs-2, -8, -9, and -12. Protease-specific differences were seen in the capacity to cleave the individual chains of laminin-332. NE and MMP-12 showed the greatest activity toward the γ2 chain, generating a fragment similar in size to the γ2x fragment generated by MMP-2. The digestion pattern of laminin-332 by degranulated neutrophils was nearly identical to that generated with NE alone. Digestion by supernatants of degranulated neutrophils was blocked by an inhibitor of NE, and NE-deficient neutrophils were essentially unable to digest laminin-332, suggesting that NE is the major neutrophil-derived protease that degrades laminin-332. In vivo, laminin γ2 fragments were found in the bronchoalveolar lavage fluid of wild-type mice treated with lipopolysaccharide, whereas that obtained from NE-deficient mice showed a different cleavage pattern. In addition, NE cleaved a synthetic peptide derived from the region of human laminin γ2 containing the MMP-2 cleavage site, suggesting that NE may generate laminin-332 fragments that are also promigratory. Both laminin-332 fragments generated by NE digestion and NE-digested laminin γ2 peptide were found to be chemotactic for neutrophils. Collectively, these data suggest that degradation of laminin-332 by NE generates fragments with important biological activities.

Clara cell adhesion and migration to extracellular matrix

BackgroundClara cells are the epithelial progenitor cell of the small airways, a location known to be important in many lung disorders. Although migration of alveolar type II and bronchiolar ciliated epithelial cells has been examined, the migratory response of Clara cells has received little attention.MethodsUsing a modification of existing procedures for Clara cell isolation, we examined mouse Clara cells and a mouse Clara-like cell line (C22) for adhesion to and migration toward matrix substrate gradients, to establish the nature and integrin dependence of migration in Clara cells.ResultsWe observed that Clara cells adhere preferentially to fibronectin (Fn) and type I collagen (Col I) similar to previous reports. Migration of Clara cells can be directed by a fixed gradient of matrix substrates (haptotaxis). Migration of the C22 cell line was similar to the Clara cells so integrin dependence of migration was evaluated with this cell line. As determined by competition with an RGD containing-peptide, migration of C22 cells toward Fn and laminin (Lm) 511 (formerly laminin 10) was significantly RGD integrin dependent, but migration toward Col I was RGD integrin independent, suggesting that Clara cells utilize different receptors for these different matrices.ConclusionThus, Clara cells resemble alveolar type II and bronchiolar ciliated epithelial cells by showing integrin mediated pro-migratory changes to extracellular matrix components that are present in tissues after injury.

Distal Airways in Mice Exposed to Cigarette Smoke Nrf2-Regulated Genes Are Increased in Clara Cells

Cigarette smoke (CS) is the main risk factor for chronic obstructive pulmonary disease (COPD). Terminal bronchioles are critical zones in the pathophysiology of COPD, but little is known about the cellular and molecular changes that occur in cells lining terminal bronchioles in response to CS. We subjected C57BL/6 mice to CS (6 d/wk, up to 6 mo), looked for morphologic changes lining the terminal bronchioles, and used laser capture microdissection to selectively isolate cells in terminal bronchioles to study gene expression. Morphologic and immunohistochemical analyses showed that Clara cell predominance remained despite 6 months of CS exposure. Since Clara cells have a role in protection against oxidative stress, we focused on the expression of antioxidant/detoxification genes using microarray analysis. Of the 35 antioxidant/detoxification genes with at least 2.5-fold increased expression in response to 6 months of CS exposure, 21 were NF-E2-related factor 2 (Nrf2)-regulated genes. Among these were cytochrome P450 1b1, glutathione reductase, thioredoxin reductase, and members of the glutathione S-transferase family, as well as Nrf2 itself. In vitro studies using immortalized murine Clara cells (C22) showed that CS induced the stabilization and nuclear translocation of Nrf2, which correlated with the induction of antioxidant and detoxification genes. Furthermore, decreasing Nrf2 expression by siRNA resulted in a corresponding decrease in CS-induced expression of several antioxidant and detoxification genes by C22 cells. These data suggest that the protective response by Clara cells to CS exposure is predominantly regulated by the transcription factor Nrf2.

Characterization of mouse alveolar epithelial cell monolayers.

We investigated the influence of extracellular matrix on transport properties of mouse alveolar epithelial cell (AEC) monolayers (MAECM) and transdifferentiation of isolated mouse alveolar epithelial type II (AT2) cells into an alveolar epithelial type I (AT1) cell-like phenotype. Primary mouse AT2 cells plated on laminin 5-coated polycarbonate filters formed monolayers with transepithelial resistance (R(T)) and equivalent short-circuit current (I(EQ)) of 1.8 kOmega.cm(2) and 5.3 microA/cm(2), respectively, after 8 days in culture. Amiloride (10 microM), ouabain (0.1 mM), and pimozide (10 microM) decreased MAECM I(EQ) to 40%, 10%, and 65% of its initial value, respectively. Sequential addition of pimozide and amiloride, in either order, revealed that their inhibitory effects are additive, suggesting that cyclic nucleotide-gated channels contribute to amiloride-insensitive active ion transport across MAECM. Ussing chamber measurements of unidirectional ion fluxes across MAECM under short-circuit conditions indicated that net absorption of Na(+) in the apical-to-basolateral direction is comparable to net ion flux calculated from the observed short-circuit current: 0.38 and 0.33 microeq.cm(-2).h(-1), respectively. Between days 1 and 9 in culture, AEC demonstrated increased expression of aquaporin-5 protein, an AT1 cell marker, and decreased expression of pro-surfactant protein-C protein, an AT2 cell marker, consistent with transition to an AT1 cell-like phenotype. These results demonstrate that AT1 cell-like MAECM grown on laminin 5-coated polycarbonate filters exhibit active and passive transport properties that likely reflect the properties of intact mouse alveolar epithelium. This mouse in vitro model will enhance the study of AEC derived from mutant strains of mice and help define important structure-function correlations.

A Chemotactic Peptide from Laminin α5 Functions as a Regulator of Inflammatory Immune Responses via TNFα-mediated Signaling

Tissue injury triggers inflammatory responses that may result in release of degradation products or exposure of cryptic domains of extracellular matrix components. Previously, we have shown that a cryptic peptide (AQARSAASKVKVSMKF) in the α-chain of laminin-10 (α5β1γ1), a prominent basement membrane component, is chemotactic for both neutrophils (PMNs) and macrophages (Mφs) and induces matrix metalloproteinase-9 (MMP-9) production. To determine whether AQARSAASKVKVSMKF has additional effects on inflammatory cells, we performed microarray analysis of RNA from RAW264.7 Mφs stimulated with AQARSAASKVKVSMKF. Several cytokines and cytokine receptors were increased >3-fold in response to the laminin α5 peptide. Among these were TNF-α and one of its receptors, the p75 TNFR (TNFR-II), increasing 3.5- and 5.7-fold, respectively. However, the peptide had no effect on p55 TNFR (TNFR-I) expression. Corroborating the microarray data, the protein levels of TNF-α and TNFR-II were increased following stimulation of RAW264.7 cells with AQARSAASKVKVSMKF. In addition, we determined that the production of TNF-α and TNFR-II in response to AQARSAASKVKVSMKF preceded the production of MMP-9. Furthermore, using primary Mφs from mice deficient in TNFR-I, TNFR-II, or both TNF-α receptors (TNFRs), we determined that AQARSAASKVKVSMKF induces MMP-9 expression by Mφs through a pathway triggered by TNFR-II. However, TNF-α signaling is not required for AQARSAASKVKVSMKF-induced PMN release of MMP-9 or PMN emigration. These data suggest that interactions of inflammatory cells with basement membrane components may orchestrate immune responses by inducing expression of cytokines, recruitment of inflammatory cells, and release of proteinases.