Abstract
AM J RESPIR CRIT CARE MED 1999;160:S29−S32.Macrophages are the predominant defense cell in the normal lung and during conditions associated with chronic inflammation such as COPD. However, the role of the macrophage in the development of COPD has been controversial. Scientific evidence from a variety of sources is emerging that supports a primary role for the macrophage in regulating the inflammatory response and tissue destruction associated with COPD. The role of macrophage proteinases in the development of emphysema, a major component of COPD, will be discussed. Shapiro SD. The macrophage in chronic obstructive pulmonary disease.
Pulmonary emphysema is a major component of the morbidity and mortality of chronic obstructive pulmonary disease (COPD), a condition that afflicts more than 14 million persons in the United States and has become the fourth leading cause of death. Furthermore, COPD accounts for 13% of hospital admissions in our country and evidence suggests that its incidence is rising, particularly in women. Given the large increase in smoking in many foreign countries, COPD will become a larger worldwide problem in the ensuing years (1).
Emphysema is defined as enlargement of peripheral airspaces of the lung, including respiratory bronchioles, alveolar ducts, and alveoli, accompanied by destruction of the walls of these structures. Inherited deficiency of α1-antitrypsin (α1-AT), the primary inhibitor of neutrophil elastase, predisposes individuals to early onset emphysema, and intrapulmonary instillation of elastolytic enzymes in experimental animals causes emphysema. Together, these findings led to the elastase:antielastase hypothesis of the pathogenesis of emphysema, which was proposed more than 30 years ago and remains the prevailing concept today (2).
The pathogenesis of garden-variety emphysema associated with cigarette smoking can be dissected into three interrelated events: chronic exposure to cigarette smoke may lead to (1) inflammatory cell recruitment within the terminal airspaces of the lung; (2) these inflammatory cells release elastolytic proteinases, which damage the extracellular matrix of the lung; and (3) ineffective repair of elastic fibers and perhaps other extracellular matrix components.
In healthy nonsmokers, macrophages comprise the major host defense cell in the lower airspace. Cigarette smoking is associated with a more than fivefold increase in total cells recovered by bronchoalveolar lavage (BAL), with macrophages comprising 95–98%. Moreover, macrophages are prominent in the respiratory bronchioles of cigarette smokers, where emphysematous changes are first manifest. Other immune and inflammatory cells including neutrophils, T lymphocytes, eosinophils, and mast cells are likely also to contribute to proteolytic injury. Resident cells within the lung such as fibroblasts and alveolar type II cells may be induced by cigarette smoke to synthesize chemokines attracting and potentially activating inflammatory cells. Resident cells may also produce proteolytic enzymes in response to cigarette smoking. Most likely, complex interaction between resident and immune/inflammatory cells results in release of proteolytic enzymes capable of destroying lung tissue and leading to emphysema.
Defining the cells and proteinases responsible for destruction of lung extracellular matrix associated with cigarette smoking will be required for development of appropriate proteinase inhibitors for application in COPD. Different cell types generate different classes of proteinases with differing modes of gene regulation. For example, neutrophils are short-lived and package active serine proteinases in azurophil (primary) granules and matrix metalloproteinases (MMP-8, MMP-9) in specific (secondary/tertiary) granules. Specific granule components are readily released in response to a variety of stimuli, and Liou and Campbell have shown that neutrophil migration in culture is associated with quantum release of primary granules (3). In mice, to date, deletion of individual MMPs and serine proteinases has not led to altered neutrophil mobilization, tissue invasion, or migration ([4]; and T. J. Ley and R. M. Senior, unpublished observations]. Use of neutrophil elastase (NE)-deficient mice (4) has demonstrated that NE plays a role in intracellular killing of gram-negative, but not gram-positive, bacteria. It is tempting to postulate that NE is meant to function within the cell, or perhaps on the cell surface, and release of NE into the extracellular space is pathologic. This could occur during inefficient apoptosis or inability of macrophages to clear dead neutrophils from the lung. Thus, another key interaction preventing lung destruction would be macrophage- mediated neutrophil apoptosis in the lungs of cigarette smokers.
Peripheral blood monocytes resemble neutrophils in that they contain HNE and CG in peroxidase-positive granules that are similar to the azurophil granules of neutrophils. These proteinases are synthesized by monocyte precursors in the bone marrow. Circulating monocytes are able to synthesize significant amounts of matrilysin (MMP-7) (5) but either none (MMP-3 [stromelysin] and MMP-12 [macrophage elastase]) or small amounts (MMP-1 [collagenase 1] and MMP-9 [gelatinase B]) of other MMPs. Interestingly, expression of both serine proteinases and matrilysin is limited to a subset of “proinflammatory” monocytes (∼ 15% of total) (6). When monocytes differentiate to macrophages they lose their serine proteinase armamentarium but acquire the capacity to synthesize and secrete several matrix metalloproteinases (MMPs) (7). Human alveolar macrophages are capable of producing several MMPs including macrophage elastase (MMP-12), collagenase 1 (MMP-1), gelatinase B (MMP-9), MT-1-MMP (S. D. Shapiro and H. G. Welgus, unpublished), and smaller amounts of stromelysin 1 (MMP-3) and matrilysin (MMP-7). Macrophage metalloproteinase expression is highly regulated by inflammatory cytokines, matrix fragments, and other agents. Thus, unlike neutrophils and monocytes, which store proteinases potentially for rapid release, macrophages monitor and respond to their environment; properties that could allow for tissue remodeling and possibly control of other inflammatory events. Dysregulated expression of macrophage MMPs either directly or indirectly by cigarette smoke exposure could lead to the lung destruction characteristic of emphysema. Macrophages also have the capacity to produce elastolytic cysteine proteinases including cathepsins K, L, and S. If these proteinases are secreted from the cell, particularly in an acidic environment, they could cause significant lung destruction. Mice have a similar macrophage proteinase profile, with two exceptions: (1) they do not appear to express collagenase 1 and (2) macrophage elastase is the predominant mouse macrophage MMP.
Macrophage elastase (MMP-12) was first detected in 1975 when Werb and Gordon identified elastolytic activity in mouse peritoneal macrophage-conditioned medium. Subsequently, in 1981, Banda and Werb purified a 22-kD metal-dependent proteinase that was responsible for this activity (8). Cloning of the murine cDNA from a murine macrophage (P388D1) library demonstrated that macrophage elastase was a distinct member of the MMP family, with 33–49% amino acid homology with other MMPs (9). The human ortholog of macrophage elastase was then cloned from a cDNA library derived from human alveolar macrophages of a cigarette smoker (10). The cDNAs for human and murine macrophage elastase have 74% homology; there is 64% identity between the enzymes at the amino acid level.
MMP-12 shares many features typical of MMPs including its domain structure, chromosomal location within the MMP gene cluster on human chromosome 11q22, and its capacity to degrade extracellular matrix (ECM) components. MMP-12 is unique with respect to its predominantly macrophage-specific pattern of expression and the ability to readily shed its C-terminal domain on processing. With respect to proteolytic activity, MMP-12 has the capacity to hydrolyze a broad spectrum of extracellular matrix components, excluding interstitial collagens. MMP-12, like other MMPs, also cleaves a variety of non-ECM proteins such as plasminogen (11) and latent tumor necrosis factor α (TNF-α) (12), resulting in angiostatin and active TNF-α, respectively.
Macrophages of MMP-12−/− mice have a markedly diminished capacity to degrade extracellular matrix components (13). MMP-12−/− macrophages are essentially unable to penetrate reconstituted basement membranes both in vitro and in vivo. The prominent and specific expression of MMP-12 in mouse macrophages has made MMP-12−/− mice an excellent model system for determining the role of extracellular macrophage-mediated proteolysis in a variety of (patho)biological processes.
Finley and colleagues (14) cultured macrophages of patients with and without COPD and performed reverse transcriptase-polymerase chain reaction (RT-PCR) for several MMPs. They found that expression of collagenase 1 (MMP-1) and gelatinase B (MMP-9) mRNA was enhanced in macrophages from patients with COPD. Although not explicitly stated in that study, macrophage elastase (MMP-12) was minimally detected in nonsmokers and induced in both smokers and patients with COPD. Other correlative studies of MMP expression in sputum, BAL, and lung tissue are emerging. The chronicity, general absence of smoking late in the disease, and the static nature of these types of studies contribute to the complexity of correlating the presence of proteinases with dynamic changes in lung architecture. With the large number of potential candidates capable of causing lung destruction in pulmonary emphysema, it is imperative to determine directly the relative contribution of these proteinases in smoking-associated emphysema. The most direct methods available to establish protein functions are to perform gain of function and loss of function experiments in complex biological organisms (mammals). Investigators are beginning to take advantage of transgenic technology to overexpress and delete specific proteinases in mice.
Overexpression of a human collagenase 1 (MMP-1) transgene driven by the haptoglobin reporter unexpectedly resulted in lung-specific expression in several independent founder lines of mice (15). These mice developed enlarged airspaces characteristic of emphysema. This was the first demonstration that an MMP could directly cause emphysema. Also, since MMP-1 is inactive against mature elastin, this result suggested that collagen degradation was sufficient to cause emphysema. However, it is still not certain whether the alveolar pathology in these animals was due to destruction of collagen in mature lung tissue or whether expression of the transgene during growth and development interfered with normal elastic fiber assembly, perhaps through destruction of the elastic fiber microfibrillar scaffold. Use of cell-specific inducible promoters could circumvent this problem. The role of collagen turnover in emphysema is intriguing. If the earliest lesions in emphysema consist of increased number and size of alveolar pores (16), then collagen destruction must be involved. Collagen must also be lost late in the disease process as entire alveolar units are lost. However, both older and more recent studies indicate increased collagen per unit volume of airspace wall in emphysematous lungs from smokers (17) and in experimental animals subjected to chronic cigarette smoke inhalation (18). Thus, collagen turnover is complex, with areas of increased deposition in small airways and depletion in alveolar walls. However, compared with elastin, collagen synthesis and assembly may be more readily achieved after injury. Given this complex scenario, more work needs to be done to determine whether collagenase inhibition in emphysema will be beneficial or harmful.
Loss of function (underexpression) models achieved by targeted mutations in genes in embryonic stem cells allow the creation of strains of mice deficient in specific proteins. The great advantage of this technology is that it allows the performance of controlled experiments in mammals. Strains of mice deficient in individual candidate proteinases can be compared to determine their contribution to the development of emphysema in response to cigarette smoke. The usefulness of these studies in dissecting the pathogenesis of human disease is directly related to the similarities between human and mouse (patho)biology. Ultimately, as we learn more about mouse and human biology, differences may be as informative as similarities in determining biological pathways. With respect to emphysema, mouse and human airspaces are quite similar. Mice have less airway branching and lack respiratory bronchioles. Nevertheless, in response to long-term cigarette smoke exposure (2 cigarettes/d, 6 d/wk) several strains develop macrophage-predominant inflammation and airspace enlargement similar to humans (19).
Macrophage elastase (MMP-12), nearly undetectable in normal macrophages, is expressed in the human alveolar macrophages of cigarette smokers. MMP-12 may also be detected by immunohistochemistry and in situ hybridization in the macrophages of patients with emphysema, but not in normal lung tissue (20). To determine directly the contribution of macrophage elastase to emphysema we generated macrophage elastase-deficient (MMP-12−/−) mice by gene targeting (13), and subjected MMP-12−/− mice and wild-type (MMP-12+/+) littermates to chronic cigarette smoke exposure (19). In contrast to MMP-12+/+ mice, mice lacking macrophage elastase (MMP-12−/−) did not develop emphysema in response to long-term cigarette smoke exposure. Surprisingly, MMP-12−/− mice also failed to recruit macrophages into their lungs in response to cigarette smoke. Monthly intratracheal instillation of monocyte chemoattractant protein 1 to smoke-exposed MMP-12−/− mice resulted in recruitment of MMP-12−/− alveolar macrophages but failed to cause airspace enlargement. Thus, macrophage elastase is required for both macrophage accumulation and emphysema resulting from chronic inhalation of cigarette smoke. Our current working model is that cigarette smoke induces constitutive macrophages to produce macrophage metalloelastase (MME), which cleaves elastin-generating fragments chemotactic for monocytes (Figure 1). This positive feedback loop perpetuates macrophage accumulation and lung destruction. This concept is supported by earlier studies demonstrating that elastin fragments are chemotactic for monocytes (21, 22).
Fig. 1. Mechanism of metalloelastase-mediated monocyte recruitment in response to cigarette smoke. Inhaled cigarette smoke induces constitutive lung macrophages to produce macrophage metalloelastase (MME). MME proteolytically generates a chemotactic gradient, presumably by cleaving a protein into a chemotactic fragment [chemokine(s) X] that attracts blood monocytes into the lung parenchyma.
[More] [Minimize]Clearly, there are several proteinases expressed in association with emphysema. The major lesson to be gained from the murine model of cigarette smoke-induced emphysema is not that MMP-12 is the sole proteinase responsible for human emphysema, but that macrophages have the capacity to cause emphysema with recruitment and activation by cigarette smoke. This study also unmasked a proteinase-dependent mechanism of inflammatory cell recruitment that may have broader biological implications. In mice, the predominant macrophage proteinase is MMP-12; human macrophages have a broader spectrum of MMPs. Moreover, serine proteinases and MMPs may interact; MMPs degrade α1-AT and NE degrades TIMPs, potentiating proteinase activity. Also, human COPD involves small airway changes, and is complicated by recurrent airway infections. These infections are associated with neutrophil recruitment, whose proteinases (as well as bacterial proteinases) may also contribute to destructive changes.
While altered proteinase:antiproteinase balance may partially explain why only 15–20% of cigarette smokers develop clinically significant emphysema, susceptibility might also rest on the capacity for matrix repair after predictable proteolytic injury. In fact, if we are ever to actually reverse this disease process, we must begin to focus on the process of extracellular matrix repair.
| Enzyme Class | Enzyme | Molecular Mass*(kD) | Cell of Origin | Matrix Substrates Other Than Elastin | Relative Elastolytic Activity, pH 7.5 (%) | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| Serine proteinase | Neutrophil | 27–31 | Neutrophil | bm components† | 100 | |||||
| elastase | (monocyte) | |||||||||
| Proteinase 3 | 28–34 | Neutrophil | bm components† | 40 | ||||||
| (monocyte) | ||||||||||
| Cathepsin G | 27–32 | Neutrophil | bm components† | 20 | ||||||
| (monocyte, | ||||||||||
| mast cell) | ||||||||||
| Matrix metallopro- | 92-kD gelatinase | 92–95 | Macrophage | Denatured collagens, | 30 | |||||
| teinase | neutrophil, | types IV, V, and VII | ||||||||
| eosinophil | collagen | |||||||||
| Macrophage | 54 | Macrophage | bm components† | 35 | ||||||
| elastase | ||||||||||
| Cysteine proteinase | Cathepsin L | 29 | Macrophage | (Inactive at pH 7.5) | 0‡ | |||||
| Cathepsin S | 28 | Macrophage | (Unknown) | 80‡ | ||||||
| T cell |
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