Elastin Degradation by Mononuclear Phagocytes" STEVEN D. SHAPIRO,~EDWARD J. CAMPBELL,' HOWARD G. WELGUS,dAND ROBERT M. SENIORb.' Divisions of Respiratory and Critical Care" and Dermatologyd Department of Medicine Jewish Hospital at Washington University Medical Center St. Louis, Missouri 631 10 "Division of Respiratory, Critical Care and Occupational Pulmonary Medicine Department of Medicine University of Utah Health Science Center Salt Lake City. Utah 84132
Macrophages participate in a variety of phagocytic, immune, and inflammatory processes. By secreting proteinases, proteinase inhibitors, and cytokines that affect proteinase production by other cells, macrophages also play a role in the degradation and remodeling of the extracellular matrix. I Cigarette smoking is associated with a marked increase in the number of alveolar macrophages recovered from bronchoalveolar lavage,2and histologic examination of smokers' lungs demonstrates infiltration of macrophages within and surrounding the small airways where destructive changes in emphysema are most p r ~ m i n e n t These .~ observations and the close association between smoking and emphysema suggest that human alveolar macrophages play a role in the pathogenesis of pulmonary emphysema. Because degradation of lung parenchymal elastic fibers appears to be a central feature in the development of emphysema, interest has become directed at the elastolytic activity of alveolar macro phage^.^,^ This article reviews information relating to the potential of macrophages and macrophage precursors to promote elastin degradation through the production and release of elastolytic enzymes. It should be recognized, however, that alveolar macrophages can affect the elastase-antielastase balance of the lungs in diverse ways (TABLE1). ELASTASE EXPRESSION DURING HUMAN MONONUCLEAR PHAGOCYTE DEVELOPMENT Human mononuclear phagocytes synthesize and release proteinases capable of degrading extracellular matrix. The spectrum of such proteinases varies with the developmental stage of the c e k 6 We used human promonocyte-like cells, U937, to study the profile of elastases and other matrix-degrading proteinases
This work was supported by USPHS grant HL29594. Address for correspondence: Robert M. Senior, M.D.,Respiratory and Critical Care Division, Jewish Hospital at Washington University Medical Center, 216 S. Kingshighway, St. Louis, Missouri 631 10. a
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produced during mononuclear phagocyte differentiation. Upon exposure to phorbol esters, such as 12-o-tetradecanoyl-phorbol-13-acetate (TPA), U937 cells acquire characteristics of more mature mononuclear phagocytes.' We found that U937 cells contain neutrophil elastase and cathepsin G; however, after exposure to TPA, there is a delayed (12-16 hour) but sharp decline in the cellular content of these serine proteinases, with minimal activity after 72 hours (FIG.I). These findings reflect changes in gene expression. Nuclear runoff assays in basal U937 cells demonstrate active transcription of cathepsin G; exposure to TPA markedly down-regulates transcription, probably by stimulating the synthesis of a suppressor protein.8 In contrast to their production of serine proteinases, undifferentiated U937 cells do not produce interstitial collagenase and they secrete only small amounts of the tissue inhibitor of metalloproteinases (TIMP), whereas TPA-differentiated U937 cells show high levels of synthesis of collagenase and TIMP following a 1216-hour lag time.9 These changes in production of collagenase and TIMP are matched by similarly delayed increases in steady-state mRNA. Production of 92kD type IV collagenase, a metalloenzyme with potent gelatinolytic activity in TABLE 1. Effects of Alveolar Macrophages on Elastase-Antielastase Balance"
Increased elastase activity Release of elastase activity: Inactivation of a,-antiproteinase: Recruitment of neutrophils: Stimulation of elastase release from neutrophils: Decreased elastase activity Release of elastase inhibitors: Receptor-mediated endocytosis of elastases:
Metalloelastase, cysteinyl elastase (cathepsin L). serine (neutrophil) elastase Oxidation by activated reactive oxygen species, proteolysis by metalloelastase and cathepsin L Secretion of chemoattractants Secretion of neutrophil secretagogue activity Alpha,-antiproteinase, alpha2-macroglobulin,tissue inhibitor of metalloproteinases (TIMP), cystatin C Neutrophil elastase. elastase-alphaz-rnacroglobulin complexes
Adapted from Senior and Kuhms
addition to its capacity to degrade basement membrane collagen, also increases markedly with TPA-induced differentiation of U937 cells.I0 Thus, the differentiated U937 cells have a profile of metalloproteinase production similar to that of normal alveolar macrophages. Takahashi et al." studied the expression of the neutrophil elastase gene in HL60 cells, a human myelomonocytic cell line that can be induced to differentiate along monocytic or neutrophilic pathways. In the basal state, HL-60 cells were found to express neutrophil elastase mRNA, and cells induced along the myelocytic lineage with dimethyl sulfoxide expressed increased amounts of neutrophil elastase steady-state mRNA. Maturation along the monocytic lineage with phorbol esters, however, resulted in decreased neutrophil elastase mRNA levels. N o elastase mRNA was detected in mature neutrophils, monocytes, or macrophages. Campbell et al. I 2 found that normal peripheral blood monocytes contain immunoreactive neutrophil elastase and cathepsin G, which are rapidly released in response to degranulating agents. The amount of each enzyme is approximately 6% of that in neutrophils (FIG.2). Further analysis revealed that a small subpopu-
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Cathemin G t
-
TIME (hours) FIGURE 1. Time course of U937 cell elastase and cathepsin G activities after incubation with TPA (thick lines, 0)or no treatment; control (rhin lines, i3. Elastase and cathepsin G activities were determined by measuring radioactivity following exposure of cell extracts at neutral pH with I4C-labeled insoluble elastin36 and succinyl-alanyl-alanyl-prolyl-phenylalanyl-p-nitr~anilide,~’ respectively. (Reproduced from Senior ef U I . with ~ permission.)
-3 -4
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0.10
1 .o
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0.5
v)
c1
8
8 5
s
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CG MONOCYTES
HLE
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FIGURE 2. lmmunoreactive human leukocyte elastase (HLE) and cathepsin G (CG) in extracts of monocytes and neutrophils. Whole cells were extracted with 10 mM phosphate, 0.02% Triton X-100,1 .O M NaCI, p H 7.4, and subjected to competitive binding ELISA. Data are the means of triplicate assays of lysates from each donor: for monocytes and neutrophils, n = 23 and 19 donors, respectively. The heights of the bars represent mean values SEM. Note that the amounts of HLE and CG were similar in each cell type; the monocyte contents of HLE and CG were 6.2% and 5.2% of neutrophil content, respectively. (Reproduced from Campbell ef a/.” with permission from J . Immunol.)
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lation of monocytes (15-20% of total) accounts for the enzymes. Most of the cells contain little or none of the enzymes. Overall, these results with monocyte precursors and monocytes demonstrate that the genes for the serine proteinases neutrophil elastase and cathepsin G are expressed transiently during monomyelocytic development in cells destined to be monocytes, and that a subpopulation of monocytes resemble neutrophils in having granules containing neutrophil elastase and cathepsin G that can be released from the cells on stimulation.
Macrophage Elastase Activities Mature human macrophages do not contain serine proteinases, but they have the capacity to secrete several metalloproteinases capable of degrading extracellular matrix (TABLE2). Despite the broad substrate specificity of these metalloenzymes, however, none has been shown to possess substantial elastolytic activity. In fact, the ability of human macrophages to degrade elastin at all has been controversial for years. In the following section we summarize information about a well-characterized macrophage elastase, the metalloelastase produced by murine macrophages. 22-kD M u r k Metdoelasrase Werb and Gordon13 were the first to identify a macrophage-derived elastase. They used mouse peritoneal macrophages to generate conditioned media and demonstrated elastase activity using elastin-agar plates. The inhibition profile of the elastase activity did not fit with a serine proteinase, and it exhibited a narrower range of substrate specificity than did serine elastases. Banda and Werb14 subsequently isolated the enzyme, confirmed it to be a metalloproteinase that requires zinc for activity, and found that it has a molecular weight of 22 kD. Kettner and Shawl5 determined its peptide bond specificity using the beta-chain of insulin as a reference substrate. The enzyme was shown to be an endopeptidase that cleaves peptide bonds with leucine contributing to the amino group. Similar macrophage metalloelastase activity has been found in varying degrees in other species including the cow,I6 monkey,17 and rabbit,13 but not in humans. In addition to its direct elastolytic activity, murine macrophage 22-kD metalloelastase might augment elastin degradation indirectly by catalytically degrading and inactivating alphal-antiproteinase. the major physiologic inhibitor of neutrophi1 elastase. l 8 The cleavage affects a single peptide bond between Pro357and Met358of alphal-antiproteinase, resulting in 4.2-kD and 48-kD fragments that remain associated but alter the structure of the protein.I9 The 4.2-kD fragment is chemotactic for neutrophils, further suggesting that metalloelastase might promote elastin degradation in multiple ways.*O We have extended the characterization of murine metalloelastase using enzyme purified from media conditioned by P388D 1 murine macrophage-like cells (Senior, Griffin, and Welgus, unpublished observations). The amino acid sequence of the amino-terminus shows similarity with three human metalloproteinases: interstitial collagenase. 72-kD type IV collagenase, and stromelysin.*' Also, similar to other metalloenzymes, this proteinase is secreted as a zymogen. By casein-PAGE analysis of freshly harvested conditioned medium, the proenzyme has a molecular mass of about 36 kD, and the proenzyme can be converted
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to the 22-kD active form by treatment with trypsin. It is less effectively converted by organomercurials.
HUMAN MACROPHAGE-MEDIATED ELASTOLYSIS Internalized Neutrophil Elastase
In contrast to murine macrophages, detecting and characterizing elastase activity with human macrophages have been problematic. Early studies consisting of assays of cell extracts gave negative results. Later approaches using macrophage-conditioned medium led to the finding of elastase activity, but characterization of the elastase has proven difficult. Rodriguez ef a/.*? found elastolytic activity in conditioned media from alveolar macrophages obtained from bronchoalveolar lavage of smokers but not nonsmokers. The source of the elastase activity was unknown; however, its capacity to attack the synthetic peptide substrate succinyl-L-alanyl-L-alanyl-L-alanine-p-nitroanilide (SLAPN) indicated
TABLE 2.
Macrophage Metalloproteinases Proenzyme Molecular Weight
Proteinase
(kD)
Interstitial collagenase
52,57
Stromelysin
57.60
Type IV collagenase/ gelatinase Elastase (murine)
92.12 36
Substrates Collagens types 1-111, VII, X , alphal-antiproteinase Proteoglycans. laminin. fibronectin, alphal-antiproteinase Collagen types IV, V, denatured collagens, fibronectin Elastin, proteoglycans, type IV collagen. fibronectin. alphalantiproteinase
that it was likely to be a serine proteinase. Other investigators have shown that the elastase activity of conditioned media from human alveolar macrophages has a similar profile to that of neutrophil elastase, and that elastolysis is not inhibited by cy~loheximide.?~ Moreover, additional studies indicated that macrophages can internalize neutrophil elastase via receptor-mediated binding and subsequently release the enzyme still in an active f ~ r m . ~Therefore, ~.*~ neutrophil elastase internalized by macrophages could be responsible for macrophage-mediated elastolysis without invoking the synthesis of an elastase by macrophages. Potential Role of Cathepsin L
Problems in detecting elastase activity in the early studies of cultured human alveolar macrophages may have been a result of proteinase inhibitors simultaneously secreted into the conditioned media employed. To circumvent this potential complication, Chapman and associate^^^^" used an experimental system con-
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sisting of living alveolar macrophages cultured in contact with adherent radiolabeled elastin. They showed that human alveolar macrophages can degrade insoluble elastin and that the elastin-degrading activity persists in serum-containing media. These investigators also demonstrated that human macrophages synthesize cathepsin L. a cysteine proteinase that cleaves collagen, proteoglycans, and elastin. On the basis of inhibition by the synthetic inhibitor Z-phenylalaninephenylalanine-diazomethylketone,cathepsin L appeared to be at least partly responsible for the observed elastolysis.28Cathepsin L is synthesized as a proenzyme of 29 kD and stored in lysosomes, where it is converted to a 25-kD species that is active at an acid pH. Although newly synthesized enzyme molecules are directed into lysosomes, the chaneling process is imperfect because small amounts of the precursor forms of the enzyme are released into the media by human alveolar macrophages in culture.29Like metalloelastase, cathepsin L cleaves alpha,-antipr~teinase.~~ Indirect Role of Plasminogen and Plasminogen Activator
Human alveolar macrophages exhibited minimal elastolytic activity when cultured with a more complex, elastin-rich matrix deposited by rat smooth muscle cells in uitro. The addition of plasminogen markedly augmented elastolytic activit^.^' Presumably, tissue plasminogen activator acted on plasminogen to form plasmin, which in turn degraded fibrin and glycoproteins that covered the elastin substrate. However, activation of proelastases is another possible mechanism. These findings corroborate work by Jones and Werb3?with mouse macrophages and elastin-containing matrices. The synergism between macrophage elastases and other proteinases to degrade complex matrices more efficiently suggests that, unlike neutrophil elastase, the spectrum of substrates attacked by macrophage elastase(s) may be limited and that elastin degradation in extracellular matrix by macrophages requires combined activity of a number of proteinases. Metalloelastase Activity
To further investigate solubilization of elastin by human macrophages we adopted the procedures of Chapman and associate^.^^ Accordingly, human alveolar macrophages and murine macrophages (P388D1 cell line) were incubated on radiolabeled elastin substrate for varying lengths of time, and elastin degradation was measured by release of [3H] elastin pep tide^.^^ During the first 24 hours in culture, elastin degradation by either human or murine macrophages was minimal; however, the increase in elastolysis over the next 48 hours was large (FIG. 3). Despite the elastolysis, elastase activity was not found in the conditioned medium. Elastin degradation required close association between the cell and elastin substrate. When cells were cultured on micropore filters placed between the cells and the labeled substrate, or when plates were only partially coated with elastin and cells were plated adjacent to the substrate, elastolysis was minimal compared to that of cells plated directly on elastin (FIG. 4).These findings suggest that direct contact of cells with the substrate effectively excluded inhibitors, and they raise the possibility that contact with elastin has an inductive effect on elastase production by macrophages. Elastin degradation produced by human macrophages and P388D1 cells had similar inhibitor profiles (FIG. 5A), consistent with metalloelastase activity. They
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TIME (hours) FIGURE 3. Time course of degradation of radiolabeled elastin adherent to tissue culture wells by human alveolar macrophages (open circles) and P388D I cells (closed circles). Human alveolar macrophages were recovered by saline bronchoalveolar lavage from healthy smokers.38Cells were washed twice in Hanks' solution and plated in Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum. P388DI murine macrophagelike cells were also cultured in DMEM with 10% fetal bovine serum. Plates contained I3H] elastin dried on the inside bottom of the wells. Elastin degradation was measured as release of ['HI into the conditioned media.39Human alveolar macrophages were collected from five separate bronchoalveolar lavages, and six separate experiments were performed on P388DI cells. Each experiment was performed in triplicate. Values are mean f SEM. (Reproduced from Senior ei with permission from Am. Rev. Respir. Dis.)
hAM
P388D1
hAM
P388D1
FIGURE 4. Effects of cell contact with elastin on the degradation of radiolabeled elastin by human alveolar macrophages and P388DI cells. (Left panel) Cells were plated on Millicell inserts, which were then placed into tissue culture wells coated with elastin (solid c o l i ~ r n n s ) , or plated directly on elastin-coated wells (hatched c d u m n s ) ; four experiments. (Righi panel) Cells were plated on tissue culture wells that were partially coated with elastin, with the cells plated either on the elastin (cross-hatched c d r t r n n s ) or adjacent to it (haiched columns); three experiments. Each experiment was done in triplicate. Values are mean -+ with permission from Am. Rev. Respir. Dis.) SEM. (Reproduced from Senior et
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0
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ce! 0.125 p g h L
I
0
.
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I
I
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do
ELASTIN DEGRADED (% control)
FIGURE 5. Effects of proteinase inhibitors (panel A), dexamethasone (Dex), cycloheximide (Chx), and lipopolysaccharide (LPS) (panel B) on the degradation of radiolabeled elastin by human alveolar macrophages (harched columns) and P388D I cells (solid columns). Each inhibitor was tested in triplicate at the indicated concentration in four to eight separate experiments from separate lavages. Values are mean 2 SEM, with the results expressed as the percentage of degradation observed in parallel control cultures. (Reproduced from Senior er with permission from Am. Rev. Respir. Dis.)
were both inhibited in a dose-dependent manner by TIMP, but inhibitors of cysteine proteinases including cystatin C, epoxysuccinyl-leucyl-agmatine(E-64), CBZ-phe-phe-CHN2, and CBZ-phe-ala-CHN2 had little effect. The serine proteinase inhibitor eglin C was completely ineffective. Of note, not only was elastase activity not found in the medium, but also the medium was found capable of inhibiting isolated murine metalloelastase. Because nontoxic concentrations of
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cycloheximide drastically reduced elastolytic activity (FIG.5B), elastin degradation appeared to be dependent on protein synthesis. Dexamethasone also diminished elastin degradation (FIG.5B), a finding perhaps explained by steroidinduced suppression of elastase synthesis, because with other metalloproteinases, dexamethasone has been shown to block tran~cription.’~ Thus, human alveolar macrophages plated on elastin appear to synthesize and secrete a metalloenzyme similar to the murine metalloelastase. These results agree with the findings of Chapman rt ul.?’ that human macrophages can degrade elastin, but differ in that our work demonstrated a prominent role for metalloproteinase activity rather than for cysteine proteinase activity. The reason for the discrepancy is not clear; however, it may be important that Chapman ct d. studied elastolytic activity only during the first 24 hours of in uitro culture.
SUMMARY During their development, mononuclear phagocytes express a changing profile of proteinases that may participate in the degradation of elastin and other extracellular matrix components. Neutrophil elastase is produced and stored in azurophil-like granules in immature mononuclear phagocytes. Monocytes contain small amounts of neutrophil elastase but do not synthesize the enzyme. Macrophages neither synthesize nor contain neutrophil elastase, but they can internalize and secrete scavenged neutrophil elastase. Human alveolar macrophages synthesize cysteine proteinases including cathepsin L, a lysosomal enzyme with elastolytic activity at an acidic pH. Macrophages from several animal species synthesize an approximately 22-kD metalloelastase that, in the mouse, is secreted as a zymogen of about 36 kD. In addition to its direct elastolytic properties, this metalloelastase may also promote elastolysis by cleaving alpha,-antiproteinase and thus protecting neutrophil elastase from inhibition. A human counterpart of this enzyme has not yet been purified; however, the elastolytic activity of human macrophages appears to depend predominantly on the activity of one or more metalloproteinases. Because elastin is intertwined with other matrix components in natural matrices, degradation of elastin in uiuo probably involves cooperation of multiple proteinases to uncover macromolecules that mask the elastic fibers. Degradation of matrix may be localized to pericellular sites, where proteinases are protected from inhibitors and where potentially surface-bound enzymes may be concen~ r a t e d . ~Complete ’ breakdown of matrix may be completed within the cells after partially cleaved molecules are internalized. Growth and remodeling of the extracellular matrix must involve highly coordinated interactions between cells, cytokines, proteinases, proteinase activators and inhibitors, as well as the matrix itself. The intrapulmonary process resulting in emphysema probably involves equally complex interactions. Mononuclear phagocytes accumulate in large numbers in the lung in response to cigarette smoking, and they may play a role in the pathogenesis of the alveolar septa1 injury that characterizes pulmonary emphysema. Note added in proof: In recent studies, we have found that the 92-kD type IV collagenase released by alveolar macrophages is elastolytic (Senior et ul. 1990. J .
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Cell Biol. 111: 16a). These data suggest that this enzyme accounts for at least part of the metalloelastase activity of human macrophages.
ACKNOWLEDGMENT The authors express appreciation to Gail L. Griffin for invaluable assistance in the unpublished studies cited. REFERENCES I. 2. 3. 4.
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36.
37. 38. 39.
Macrophages participate in a variety of phagocytic, immune, and inflammatory processes. By secreting proteinases, proteinase inhibitors, and cytokines that affect proteinase production by other cells, macrophages also play a role in the degradation and remodeling of the extracellular matrix. I Cigarette smoking is associated with a marked increase in the number of alveolar macrophages recovered from bronchoalveolar lavage,2and histologic examination of smokers' lungs demonstrates infiltration of macrophages within and surrounding the small airways where destructive changes in emphysema are most p r ~ m i n e n t These .~ observations and the close association between smoking and emphysema suggest that human alveolar macrophages play a role in the pathogenesis of pulmonary emphysema. Because degradation of lung parenchymal elastic fibers appears to be a central feature in the development of emphysema, interest has become directed at the elastolytic activity of alveolar macro phage^.^,^ This article reviews information relating to the potential of macrophages and macrophage precursors to promote elastin degradation through the production and release of elastolytic enzymes. It should be recognized, however, that alveolar macrophages can affect the elastase-antielastase balance of the lungs in diverse ways (TABLE1). ELASTASE EXPRESSION DURING HUMAN MONONUCLEAR PHAGOCYTE DEVELOPMENT Human mononuclear phagocytes synthesize and release proteinases capable of degrading extracellular matrix. The spectrum of such proteinases varies with the developmental stage of the c e k 6 We used human promonocyte-like cells, U937, to study the profile of elastases and other matrix-degrading proteinases
This work was supported by USPHS grant HL29594. Address for correspondence: Robert M. Senior, M.D.,Respiratory and Critical Care Division, Jewish Hospital at Washington University Medical Center, 216 S. Kingshighway, St. Louis, Missouri 631 10. a
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produced during mononuclear phagocyte differentiation. Upon exposure to phorbol esters, such as 12-o-tetradecanoyl-phorbol-13-acetate (TPA), U937 cells acquire characteristics of more mature mononuclear phagocytes.' We found that U937 cells contain neutrophil elastase and cathepsin G; however, after exposure to TPA, there is a delayed (12-16 hour) but sharp decline in the cellular content of these serine proteinases, with minimal activity after 72 hours (FIG.I). These findings reflect changes in gene expression. Nuclear runoff assays in basal U937 cells demonstrate active transcription of cathepsin G; exposure to TPA markedly down-regulates transcription, probably by stimulating the synthesis of a suppressor protein.8 In contrast to their production of serine proteinases, undifferentiated U937 cells do not produce interstitial collagenase and they secrete only small amounts of the tissue inhibitor of metalloproteinases (TIMP), whereas TPA-differentiated U937 cells show high levels of synthesis of collagenase and TIMP following a 1216-hour lag time.9 These changes in production of collagenase and TIMP are matched by similarly delayed increases in steady-state mRNA. Production of 92kD type IV collagenase, a metalloenzyme with potent gelatinolytic activity in TABLE 1. Effects of Alveolar Macrophages on Elastase-Antielastase Balance"
Increased elastase activity Release of elastase activity: Inactivation of a,-antiproteinase: Recruitment of neutrophils: Stimulation of elastase release from neutrophils: Decreased elastase activity Release of elastase inhibitors: Receptor-mediated endocytosis of elastases:
Metalloelastase, cysteinyl elastase (cathepsin L). serine (neutrophil) elastase Oxidation by activated reactive oxygen species, proteolysis by metalloelastase and cathepsin L Secretion of chemoattractants Secretion of neutrophil secretagogue activity Alpha,-antiproteinase, alpha2-macroglobulin,tissue inhibitor of metalloproteinases (TIMP), cystatin C Neutrophil elastase. elastase-alphaz-rnacroglobulin complexes
Adapted from Senior and Kuhms
addition to its capacity to degrade basement membrane collagen, also increases markedly with TPA-induced differentiation of U937 cells.I0 Thus, the differentiated U937 cells have a profile of metalloproteinase production similar to that of normal alveolar macrophages. Takahashi et al." studied the expression of the neutrophil elastase gene in HL60 cells, a human myelomonocytic cell line that can be induced to differentiate along monocytic or neutrophilic pathways. In the basal state, HL-60 cells were found to express neutrophil elastase mRNA, and cells induced along the myelocytic lineage with dimethyl sulfoxide expressed increased amounts of neutrophil elastase steady-state mRNA. Maturation along the monocytic lineage with phorbol esters, however, resulted in decreased neutrophil elastase mRNA levels. N o elastase mRNA was detected in mature neutrophils, monocytes, or macrophages. Campbell et al. I 2 found that normal peripheral blood monocytes contain immunoreactive neutrophil elastase and cathepsin G, which are rapidly released in response to degranulating agents. The amount of each enzyme is approximately 6% of that in neutrophils (FIG.2). Further analysis revealed that a small subpopu-
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Cathemin G t
-
TIME (hours) FIGURE 1. Time course of U937 cell elastase and cathepsin G activities after incubation with TPA (thick lines, 0)or no treatment; control (rhin lines, i3. Elastase and cathepsin G activities were determined by measuring radioactivity following exposure of cell extracts at neutral pH with I4C-labeled insoluble elastin36 and succinyl-alanyl-alanyl-prolyl-phenylalanyl-p-nitr~anilide,~’ respectively. (Reproduced from Senior ef U I . with ~ permission.)
-3 -4
0.15
1.5
0.10
1 .o
0.05
0.5
v)
c1
8
8 5
s
0.00
CG MONOCYTES
HLE
0.0
PMN
FIGURE 2. lmmunoreactive human leukocyte elastase (HLE) and cathepsin G (CG) in extracts of monocytes and neutrophils. Whole cells were extracted with 10 mM phosphate, 0.02% Triton X-100,1 .O M NaCI, p H 7.4, and subjected to competitive binding ELISA. Data are the means of triplicate assays of lysates from each donor: for monocytes and neutrophils, n = 23 and 19 donors, respectively. The heights of the bars represent mean values SEM. Note that the amounts of HLE and CG were similar in each cell type; the monocyte contents of HLE and CG were 6.2% and 5.2% of neutrophil content, respectively. (Reproduced from Campbell ef a/.” with permission from J . Immunol.)
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lation of monocytes (15-20% of total) accounts for the enzymes. Most of the cells contain little or none of the enzymes. Overall, these results with monocyte precursors and monocytes demonstrate that the genes for the serine proteinases neutrophil elastase and cathepsin G are expressed transiently during monomyelocytic development in cells destined to be monocytes, and that a subpopulation of monocytes resemble neutrophils in having granules containing neutrophil elastase and cathepsin G that can be released from the cells on stimulation.
Macrophage Elastase Activities Mature human macrophages do not contain serine proteinases, but they have the capacity to secrete several metalloproteinases capable of degrading extracellular matrix (TABLE2). Despite the broad substrate specificity of these metalloenzymes, however, none has been shown to possess substantial elastolytic activity. In fact, the ability of human macrophages to degrade elastin at all has been controversial for years. In the following section we summarize information about a well-characterized macrophage elastase, the metalloelastase produced by murine macrophages. 22-kD M u r k Metdoelasrase Werb and Gordon13 were the first to identify a macrophage-derived elastase. They used mouse peritoneal macrophages to generate conditioned media and demonstrated elastase activity using elastin-agar plates. The inhibition profile of the elastase activity did not fit with a serine proteinase, and it exhibited a narrower range of substrate specificity than did serine elastases. Banda and Werb14 subsequently isolated the enzyme, confirmed it to be a metalloproteinase that requires zinc for activity, and found that it has a molecular weight of 22 kD. Kettner and Shawl5 determined its peptide bond specificity using the beta-chain of insulin as a reference substrate. The enzyme was shown to be an endopeptidase that cleaves peptide bonds with leucine contributing to the amino group. Similar macrophage metalloelastase activity has been found in varying degrees in other species including the cow,I6 monkey,17 and rabbit,13 but not in humans. In addition to its direct elastolytic activity, murine macrophage 22-kD metalloelastase might augment elastin degradation indirectly by catalytically degrading and inactivating alphal-antiproteinase. the major physiologic inhibitor of neutrophi1 elastase. l 8 The cleavage affects a single peptide bond between Pro357and Met358of alphal-antiproteinase, resulting in 4.2-kD and 48-kD fragments that remain associated but alter the structure of the protein.I9 The 4.2-kD fragment is chemotactic for neutrophils, further suggesting that metalloelastase might promote elastin degradation in multiple ways.*O We have extended the characterization of murine metalloelastase using enzyme purified from media conditioned by P388D 1 murine macrophage-like cells (Senior, Griffin, and Welgus, unpublished observations). The amino acid sequence of the amino-terminus shows similarity with three human metalloproteinases: interstitial collagenase. 72-kD type IV collagenase, and stromelysin.*' Also, similar to other metalloenzymes, this proteinase is secreted as a zymogen. By casein-PAGE analysis of freshly harvested conditioned medium, the proenzyme has a molecular mass of about 36 kD, and the proenzyme can be converted
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to the 22-kD active form by treatment with trypsin. It is less effectively converted by organomercurials.
HUMAN MACROPHAGE-MEDIATED ELASTOLYSIS Internalized Neutrophil Elastase
In contrast to murine macrophages, detecting and characterizing elastase activity with human macrophages have been problematic. Early studies consisting of assays of cell extracts gave negative results. Later approaches using macrophage-conditioned medium led to the finding of elastase activity, but characterization of the elastase has proven difficult. Rodriguez ef a/.*? found elastolytic activity in conditioned media from alveolar macrophages obtained from bronchoalveolar lavage of smokers but not nonsmokers. The source of the elastase activity was unknown; however, its capacity to attack the synthetic peptide substrate succinyl-L-alanyl-L-alanyl-L-alanine-p-nitroanilide (SLAPN) indicated
TABLE 2.
Macrophage Metalloproteinases Proenzyme Molecular Weight
Proteinase
(kD)
Interstitial collagenase
52,57
Stromelysin
57.60
Type IV collagenase/ gelatinase Elastase (murine)
92.12 36
Substrates Collagens types 1-111, VII, X , alphal-antiproteinase Proteoglycans. laminin. fibronectin, alphal-antiproteinase Collagen types IV, V, denatured collagens, fibronectin Elastin, proteoglycans, type IV collagen. fibronectin. alphalantiproteinase
that it was likely to be a serine proteinase. Other investigators have shown that the elastase activity of conditioned media from human alveolar macrophages has a similar profile to that of neutrophil elastase, and that elastolysis is not inhibited by cy~loheximide.?~ Moreover, additional studies indicated that macrophages can internalize neutrophil elastase via receptor-mediated binding and subsequently release the enzyme still in an active f ~ r m . ~Therefore, ~.*~ neutrophil elastase internalized by macrophages could be responsible for macrophage-mediated elastolysis without invoking the synthesis of an elastase by macrophages. Potential Role of Cathepsin L
Problems in detecting elastase activity in the early studies of cultured human alveolar macrophages may have been a result of proteinase inhibitors simultaneously secreted into the conditioned media employed. To circumvent this potential complication, Chapman and associate^^^^" used an experimental system con-
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sisting of living alveolar macrophages cultured in contact with adherent radiolabeled elastin. They showed that human alveolar macrophages can degrade insoluble elastin and that the elastin-degrading activity persists in serum-containing media. These investigators also demonstrated that human macrophages synthesize cathepsin L. a cysteine proteinase that cleaves collagen, proteoglycans, and elastin. On the basis of inhibition by the synthetic inhibitor Z-phenylalaninephenylalanine-diazomethylketone,cathepsin L appeared to be at least partly responsible for the observed elastolysis.28Cathepsin L is synthesized as a proenzyme of 29 kD and stored in lysosomes, where it is converted to a 25-kD species that is active at an acid pH. Although newly synthesized enzyme molecules are directed into lysosomes, the chaneling process is imperfect because small amounts of the precursor forms of the enzyme are released into the media by human alveolar macrophages in culture.29Like metalloelastase, cathepsin L cleaves alpha,-antipr~teinase.~~ Indirect Role of Plasminogen and Plasminogen Activator
Human alveolar macrophages exhibited minimal elastolytic activity when cultured with a more complex, elastin-rich matrix deposited by rat smooth muscle cells in uitro. The addition of plasminogen markedly augmented elastolytic activit^.^' Presumably, tissue plasminogen activator acted on plasminogen to form plasmin, which in turn degraded fibrin and glycoproteins that covered the elastin substrate. However, activation of proelastases is another possible mechanism. These findings corroborate work by Jones and Werb3?with mouse macrophages and elastin-containing matrices. The synergism between macrophage elastases and other proteinases to degrade complex matrices more efficiently suggests that, unlike neutrophil elastase, the spectrum of substrates attacked by macrophage elastase(s) may be limited and that elastin degradation in extracellular matrix by macrophages requires combined activity of a number of proteinases. Metalloelastase Activity
To further investigate solubilization of elastin by human macrophages we adopted the procedures of Chapman and associate^.^^ Accordingly, human alveolar macrophages and murine macrophages (P388D1 cell line) were incubated on radiolabeled elastin substrate for varying lengths of time, and elastin degradation was measured by release of [3H] elastin pep tide^.^^ During the first 24 hours in culture, elastin degradation by either human or murine macrophages was minimal; however, the increase in elastolysis over the next 48 hours was large (FIG. 3). Despite the elastolysis, elastase activity was not found in the conditioned medium. Elastin degradation required close association between the cell and elastin substrate. When cells were cultured on micropore filters placed between the cells and the labeled substrate, or when plates were only partially coated with elastin and cells were plated adjacent to the substrate, elastolysis was minimal compared to that of cells plated directly on elastin (FIG. 4).These findings suggest that direct contact of cells with the substrate effectively excluded inhibitors, and they raise the possibility that contact with elastin has an inductive effect on elastase production by macrophages. Elastin degradation produced by human macrophages and P388D1 cells had similar inhibitor profiles (FIG. 5A), consistent with metalloelastase activity. They
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0
24
75
72
48
TIME (hours) FIGURE 3. Time course of degradation of radiolabeled elastin adherent to tissue culture wells by human alveolar macrophages (open circles) and P388D I cells (closed circles). Human alveolar macrophages were recovered by saline bronchoalveolar lavage from healthy smokers.38Cells were washed twice in Hanks' solution and plated in Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum. P388DI murine macrophagelike cells were also cultured in DMEM with 10% fetal bovine serum. Plates contained I3H] elastin dried on the inside bottom of the wells. Elastin degradation was measured as release of ['HI into the conditioned media.39Human alveolar macrophages were collected from five separate bronchoalveolar lavages, and six separate experiments were performed on P388DI cells. Each experiment was performed in triplicate. Values are mean f SEM. (Reproduced from Senior ei with permission from Am. Rev. Respir. Dis.)
hAM
P388D1
hAM
P388D1
FIGURE 4. Effects of cell contact with elastin on the degradation of radiolabeled elastin by human alveolar macrophages and P388DI cells. (Left panel) Cells were plated on Millicell inserts, which were then placed into tissue culture wells coated with elastin (solid c o l i ~ r n n s ) , or plated directly on elastin-coated wells (hatched c d u m n s ) ; four experiments. (Righi panel) Cells were plated on tissue culture wells that were partially coated with elastin, with the cells plated either on the elastin (cross-hatched c d r t r n n s ) or adjacent to it (haiched columns); three experiments. Each experiment was done in triplicate. Values are mean -+ with permission from Am. Rev. Respir. Dis.) SEM. (Reproduced from Senior et
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0
100
50
ce! 0.125 p g h L
I
0
.
50
I
I
100
I
do
ELASTIN DEGRADED (% control)
FIGURE 5. Effects of proteinase inhibitors (panel A), dexamethasone (Dex), cycloheximide (Chx), and lipopolysaccharide (LPS) (panel B) on the degradation of radiolabeled elastin by human alveolar macrophages (harched columns) and P388D I cells (solid columns). Each inhibitor was tested in triplicate at the indicated concentration in four to eight separate experiments from separate lavages. Values are mean 2 SEM, with the results expressed as the percentage of degradation observed in parallel control cultures. (Reproduced from Senior er with permission from Am. Rev. Respir. Dis.)
were both inhibited in a dose-dependent manner by TIMP, but inhibitors of cysteine proteinases including cystatin C, epoxysuccinyl-leucyl-agmatine(E-64), CBZ-phe-phe-CHN2, and CBZ-phe-ala-CHN2 had little effect. The serine proteinase inhibitor eglin C was completely ineffective. Of note, not only was elastase activity not found in the medium, but also the medium was found capable of inhibiting isolated murine metalloelastase. Because nontoxic concentrations of
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cycloheximide drastically reduced elastolytic activity (FIG.5B), elastin degradation appeared to be dependent on protein synthesis. Dexamethasone also diminished elastin degradation (FIG.5B), a finding perhaps explained by steroidinduced suppression of elastase synthesis, because with other metalloproteinases, dexamethasone has been shown to block tran~cription.’~ Thus, human alveolar macrophages plated on elastin appear to synthesize and secrete a metalloenzyme similar to the murine metalloelastase. These results agree with the findings of Chapman rt ul.?’ that human macrophages can degrade elastin, but differ in that our work demonstrated a prominent role for metalloproteinase activity rather than for cysteine proteinase activity. The reason for the discrepancy is not clear; however, it may be important that Chapman ct d. studied elastolytic activity only during the first 24 hours of in uitro culture.
SUMMARY During their development, mononuclear phagocytes express a changing profile of proteinases that may participate in the degradation of elastin and other extracellular matrix components. Neutrophil elastase is produced and stored in azurophil-like granules in immature mononuclear phagocytes. Monocytes contain small amounts of neutrophil elastase but do not synthesize the enzyme. Macrophages neither synthesize nor contain neutrophil elastase, but they can internalize and secrete scavenged neutrophil elastase. Human alveolar macrophages synthesize cysteine proteinases including cathepsin L, a lysosomal enzyme with elastolytic activity at an acidic pH. Macrophages from several animal species synthesize an approximately 22-kD metalloelastase that, in the mouse, is secreted as a zymogen of about 36 kD. In addition to its direct elastolytic properties, this metalloelastase may also promote elastolysis by cleaving alpha,-antiproteinase and thus protecting neutrophil elastase from inhibition. A human counterpart of this enzyme has not yet been purified; however, the elastolytic activity of human macrophages appears to depend predominantly on the activity of one or more metalloproteinases. Because elastin is intertwined with other matrix components in natural matrices, degradation of elastin in uiuo probably involves cooperation of multiple proteinases to uncover macromolecules that mask the elastic fibers. Degradation of matrix may be localized to pericellular sites, where proteinases are protected from inhibitors and where potentially surface-bound enzymes may be concen~ r a t e d . ~Complete ’ breakdown of matrix may be completed within the cells after partially cleaved molecules are internalized. Growth and remodeling of the extracellular matrix must involve highly coordinated interactions between cells, cytokines, proteinases, proteinase activators and inhibitors, as well as the matrix itself. The intrapulmonary process resulting in emphysema probably involves equally complex interactions. Mononuclear phagocytes accumulate in large numbers in the lung in response to cigarette smoking, and they may play a role in the pathogenesis of the alveolar septa1 injury that characterizes pulmonary emphysema. Note added in proof: In recent studies, we have found that the 92-kD type IV collagenase released by alveolar macrophages is elastolytic (Senior et ul. 1990. J .
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Cell Biol. 111: 16a). These data suggest that this enzyme accounts for at least part of the metalloelastase activity of human macrophages.
ACKNOWLEDGMENT The authors express appreciation to Gail L. Griffin for invaluable assistance in the unpublished studies cited. REFERENCES I. 2. 3. 4.
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