Dale K. Kobayashi

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.

Macrophage Elastase (Matrix Metalloproteinase-12) Suppresses Growth of Lung Metastases

Matrix metalloproteinases (MMP) have been implicated in virtually all aspects of tumor progression. However, the recent failure of clinical trials employing synthetic MMP inhibitors in cancer chemotherapy has led us to hypothesize that some MMPs may actually serve the host in its defense against tumor progression. Here we show that mice deficient in macrophage elastase (MMP-12) develop significantly more gross Lewis lung carcinoma pulmonary metastases than their wild-type counterparts both in spontaneous and experimental metastasis models. The numbers of micrometastases between the two groups are equivalent; thus, it seems that MMP-12 affects lung tumor growth, and not metastasis formation, per se. MMP-12 is solely macrophage derived in this model, being expressed by tumor-associated macrophages and not by tumor or stromal cells. The presence of MMP-12 is associated with decreased tumor-associated microvessel density in vivo and generates an angiostatic>angiogenic tumor microenvironment that retards lung tumor growth independent of the production of angiostatin. These data define a role for MMP-12 in suppressing the growth of lung metastases and suggest that inhibitors designed to specifically target tumor-promoting MMPs may yet prove effective as cancer therapeutics.

Immune modulation of metalloproteinase production in human macrophages. Selective pretranslational suppression of interstitial collagenase and stromelysin biosynthesis by interferon-gamma.

Interferon-gamma (IFN-gamma) is a lymphokine that activates mononuclear phagocytes. To test the hypothesis that IFN-gamma might have important effects upon the ability of human mononuclear phagocytes to degrade extracellular matrix, we have studied the action of this cytokine on the production of metalloproteinases and the counterregulatory tissue inhibitor of metalloproteinases (TIMP) by the human alveolar macrophage. We have found that IFN-gamma potently and selectively suppresses the lipopolysaccharide-induced production of two metalloproteinases--interstitial collagenase and stromelysin--by 50-90% at doses greater than or equal to 10 U/ml. The synthesis of TIMP and 92-kD type IV collagenase was also diminished by IFN-gamma, but these responses required 50- to 100-fold higher concentrations of the cytokine. All doses of IFN-gamma increased total and secreted protein synthesis slightly, indicating a highly specific effect on metalloenzyme biosynthesis. Inhibition of metalloproteinase expression occurred at a pretranslational level, as evidenced by parallel reductions in enzyme biosynthesis and collagenase-specific steady-state mRNA levels. Interestingly, the effect of IFN-gamma on metalloenzyme production was not readily reversible. Therefore, while IFN-gamma activates the macrophage and renders it tumoricidal, this enhanced function appears to be attained at the expense of the cells capacity to degrade extracellular matrix.

Regulation of the expression of tissue inhibitor of metalloproteinases and collagenase by retinoids and glucocorticoids in human fibroblasts.

The regulation of the expression of interstitial collagenase and tissue inhibitor of metalloproteinases (TIMP) was examined in response to both retinoid compounds and glucocorticoids. Effective retinoids induced a dose-dependent, specific increase in the production of TIMP of approximately two- to threefold by monolayer cultures of human fibroblasts derived from various tissues, while simultaneously causing a decrease in collagenase secretion of similar magnitude. These effects were apparent by 8-12 h in culture and disappeared within 24 h after the withdrawal of retinoid compounds. The retinoid effect on TIMP production was mediated via an increased biosynthesis of new inhibitor protein. Similarly, increased steady state levels of TIMP messenger RNA (mRNA) accompanied by decreased quantities of collagenase mRNA were demonstrated, suggesting transcriptional control of the retinoid action. The data suggest that retinoids co-regulate the expression of collagenase and TIMP, and do so in an inverse manner. Dexamethasone caused a dose-dependent, specific decrease in collagenase production without altering the biosynthesis of TIMP. These findings were paralleled by a marked reduction in collagenase mRNA, without any accompanying change in TIMP mRNA. Therefore, TIMP and collagenase expression appear to be independently modulated by glucocorticoids.

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).

Hyperpolarized 3He MRI of mouse lung

Hyperpolarized 3He images of mouse lung are presented. Ventilation images and measurements of 3He apparent diffusion coefficient (ADC) are reported in healthy mice, and preliminary studies of emphysema and lung cancer in mice are described using these techniques. The design and operation of an electronically controlled small‐animal ventilator to deliver the hyperpolarized gas and control animal respiration are described. Images are acquired using an asymmetric gradient echo imaging method to enhance the signal‐to‐noise ratio of the rapidly diffusing 3He. In mice with elastase‐induced emphysema, the whole‐lung average ADC is greater by approximately 25%, a statistically significant difference, compared to healthy animals. By contrast, mice exposed to cigarette smoke for up to 12 months reveal no statistically relevant increases in ADC, although emphysema was not confirmed in these mice. A study of lung cancer (melanoma) in mice is also presented. While tumors are shown to cause substantial ventilation defects in the lung, these defects appear confined to the cancerous regions and do not extend to large‐scale regions of the lung distal to the tumors. Magn Reson Med 52:1310–1317, 2004.

Cigarette Smoke Induces Nucleic-Acid Oxidation in Lung Fibroblasts

Oxidative stress is widely proposed as a pathogenic mechanism for chronic obstructive pulmonary disease (COPD), but the molecular pathway connecting oxidative damage to tissue destruction remains to be fully defined. We suggest that reactive oxygen species (ROS) oxidatively damage nucleic acids, and this effect requires multiple repair mechanisms, particularly base excision pathway components 8-oxoguanine-DNA glycosylase (OGG1), endonuclease III homologue 1 (NTH1), and single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1), as well as the nucleic acid-binding protein, Y-box binding protein 1 (YB1). This study was therefore designed to define the levels of nucleic-acid oxidation and expression of genes involved in the repair of COPD and in corresponding models of this disease. We found significant oxidation of nucleic acids localized to alveolar lung fibroblasts, increased levels of OGG1 mRNA expression, and decreased concentrations of NTH1, SMUG1, and YB1 mRNA in lung samples from subjects with very severe COPD compared with little or no COPD. Mice exposed to cigarette smoke exhibited a time-dependent accumulation of nucleic-acid oxidation in alveolar fibroblasts, which was associated with an increase in OGG1 and YB1 mRNA concentrations. Similarly, human lung fibroblasts exposed to cigarette smoke extract exhibited ROS-dependent nucleic-acid oxidation. The short interfering RNA (siRNA)-dependent knockdown of OGG1 and YB1 expression increased nucleic-acid oxidation at the basal state and after exposure to cigarette smoke. Together, our results demonstrate ROS-dependent, cigarette smoke-induced nucleic-acid oxidation in alveolar fibroblasts, which may play a role in the pathogenesis of emphysema.

Clarithromycin Prevents Smoke-induced Emphysema in Mice

RATIONALE Modulating the low-grade chronic inflammation in chronic obstructive pulmonary disease remains challenging. Clarithromycin (CAM), a macrolide antibiotic, reportedly ameliorates chronic inflammation via mechanisms independent of its antibacterial activity. OBJECTIVES The aim of this study was to examine whether CAM can prevent or reduce emphysema induced by chronic cigarette smoke exposure. METHODS Mice were exposed to cigarette smoke daily for 6 months and treated with orally administered CAM at doses of 25 to 100 mg/kg twice a day throughout the course of the experiment to test the preventive effects. The administration of CAM at 50 or 100 mg/kg was performed during the second half of a 6-month exposure period to assess the therapeutic effects. Histologic analysis was performed to evaluate the effect of CAM. MEASUREMENTS AND MAIN RESULTS CAM treatment for 6 months decreased airspace enlargement and the destruction of the alveolar walls and impaired the accumulation of macrophages in bronchoalveolar lavage fluid in a dose-related fashion. The administration of clarithromycin at 100 mg/kg in the therapeutic protocol reduced emphysema compared with the smoke-exposed group without treatment. An immunohistologic analysis revealed that CAM reduced the number of F4/80-positive macrophages in the lung parenchyma. In an in vitro test, CAM at 5 to 20 microM directly suppressed the activation of macrophages stimulated with tumor necrosis factor-alpha. CONCLUSIONS Our data demonstrated that CAM at a clinically achievable dose prevented cigarette smoke-induced emphysema by modulating lung inflammation. This study supports the possibility that low-dose CAM treatment might provide a new therapeutic strategy for chronic obstructive pulmonary diseases.

Inhaled Recombinant Alpha 1-Antitrypsin Ameliorates Cigarette Smoke-Induced Emphysema in the Mouse

In alpha 1-antitrypsin deficiency in humans, inadequately regulated activity of serine protease activity is responsible for the chronic lung tissue degeneration and irreversible loss of pulmonary function seen in those individuals with emphysema. Typically, disease symptoms in this patient population are exacerbated by cigarette smoke. Here we show that inhaled recombinant alpha 1-antitrypsin (rAAT) can provide significant protection against the development of emphysema in cigarette smoke-treated mice. As has been reported previously, cigarette smoke was seen to increase significantly the recruitment of neutrophils and macrophages into the lungs of these animals, leading to concomitant alveolar airspace enlargement and emphysema. In smoking animals treated for 6 months with inhaled rAAT, effects on lavage levels of neutrophils and macrophages were only moderate when compared with untreated animals. Furthermore, neutralizing antibodies to rAAT were generated in all rAAT-treated animals. Despite this, however, reductions in airspace enlargement of up to 73% were observed. These findings demonstrate that delivery of rAAT directly to the lungs of smoke-treated mice can inhibit lung tissue damage mediated by proteases, suggesting that rAAT inhalation therapy might represent a practical approach towards treating emphysema in humans, by modifying the course of the disease.