Matrix metalloproteinases generate angiostatin: effects on neovascularization.

Angiostatin, a cleavage product of plasminogen, has been shown to inhibit endothelial cell proliferation and metastatic tumor cell growth. Recently, the production of angiostatin has been correlated with tumor-associated macrophage production of elastolytic metalloproteinases in a murine model of Lewis lung cell carcinoma. In this report we demonstrate that purified murine and human matrix metalloproteinases generate biologically functional angiostatin from plasminogen. Macrophage elastase (MMP-12 or MME) proved to be the most efficient angiostatin-producing MMP. MME was followed by gelatinases and then the stomelysins in catalytic efficiency; interstitial collagenases had little capacity to generate angiostatin. Both recombinant angiostatin and angiostatin generated from recombinant MME-treated plasminogen inhibited human microvascular endothelial cell proliferation and differentiation in vitro. Finally, employing macrophages isolated from MME-deficient mice and their wild-type littermates, we demonstrate that MME is required for the generation of angiostatin that inhibits the proliferation of human microvascular endothelial cells.

Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice.

To determine which proteinases are responsible for the lung destruction characteristic of pulmonary emphysema, macrophage elastase-deficient (MME-/-) mice were subjected to cigarette smoke. In contrast to wild-type mice, MME-/- mice did not have increased numbers of macrophages in their lungs and did not develop emphysema in response to long-term exposure to cigarette smoke. Smoke-exposed MME-/- mice that received monthly intratracheal instillations of monocyte chemoattractant protein-1 showed accumulation of alveolar macrophages but did not develop air space enlargement. Thus, macrophage elastase is probably sufficient for the development of emphysema that results from chronic inhalation of cigarette smoke.

Hydrolysis of a broad spectrum of extracellular matrix proteins by human macrophage elastase.

Macrophage elastase (ME) was originally named when metal-dependent elastolytic activity was detected in conditioned media of murine macrophages. Subsequent cDNA cloning of the mouse and human enzyme demonstrated that ME is a distinct member of the matrix metalloproteinase family. To date, the catalytic parameters that describe the hydrolysis of elastin by ME have not been quantified and its activity against other matrix proteins have not been described. In this report, we have examined the action of purified recombinant human ME (rHME), produced in Escherichia coli, on elastin and other extracellular matrix proteins. On a molar basis, rHME is approximately 30% as active as human leukocyte elastase in solubilizing elastin. rHME also efficiently degrades alpha1-antitrypsin (alpha1-AT), the primary physiological inhibitor of human leukocyte elastase. In addition, rHME efficiently degrades fibronectin, laminin, entactin, type IV collagen, chondroitan sulfate, and heparan sulfate. These results suggest that HME may be required for macrophages to penetrate basement membranes and remodel injured tissue during inflammation. Moreover, abnormal expression of HME may contribute to destructive processes such as pulmonary emphysema and vascular aneurysm formation. To further understand the specificity of HME, the initial cleavage sites in alpha1-AT have been determined. In addition, the hydrolysis of a series of synthetic peptides with different P'1 residues has been determined. rHME can accept large and small amino acids at the P'1 site, but has a preference for leucine.

Metalloelastase is required for macrophage-mediated proteolysis and matrix invasion in mice.

Macrophages secrete a variety of proteinases that are thought to participate in remodeling of the extracellular matrix associated with inflammatory processes. We have eliminated expression of the macrophage metalloelastase (MME) gene by targeted disruption to assess the role of this protein in macrophage-mediated proteolysis. We found that the macrophages of MME-deficient (MME-/-) mice have a markedly diminished capacity to degrade extracellular matrix components. In addition, MME-/- macrophages are essentially unable to penetrate reconstituted basement membranes in vitro and in vivo. MME is therefore required for macrophage-mediated extracellular matrix proteolysis and tissue invasion.

Human macrophage metalloelastase. Genomic organization, chromosomal location, gene linkage, and tissue-specific expression.

Human macrophage metalloelastase (HME) is a recent addition to the matrix metalloproteinase (MMP) family that was initially found to be expressed in alveolar macrophages of cigarette smokers. To understand more about HME expression, analysis of the structure and location of the gene was performed. The gene for HME is composed of 10 exons and 9 introns, similar to the stromelysins and collagenases, and HME shares the highly conserved exon size and intron-exon borders with other MMPs. The 13-kilobase (kb) HME gene has been localized by fluorescence in situ hybridization to chromosome 11q22.2-22.3, the same location of the interstitial collagenase and stromelysin genes. We determined that HME and stromelysin 1 genes are physically linked within 62 kb utilizing pulse-field gel electrophoresis. The promoter region of the HME gene contains several features common to other MMP genes including a TATA box 29 bp upstream to the transcription initiation site, an AP-1 motif, and a PEA3 element. HME mRNA is not detectable in normal adult tissues but is induced in rapidly remodeling tissues such as the term placenta. In situ hybridization and immunohistochemistry of placental tissue demonstrated HME mRNA and protein expression in macrophages and stromal cells. Cell-specific expression and response to inflammatory stimuli such as endotoxin is conferred within 2.8 kb of the HME 5'-flanking sequence as demonstrated by HME promoter-CAT expression constructs. Knowledge of the genomic organization and chromosomal location of HME may allow us to further define mechanisms responsible for cell- and tissue-specific expression of HME.

Cloning and characterization of a unique elastolytic metalloproteinase produced by human alveolar macrophages.

Human alveolar macrophages have the capacity to degrade elastin. As an approach to define proteinases responsible for this activity, we recently cloned a murine macrophage elastase cDNA and demonstrated that it is a member of the matrix metalloproteinase gene family (Shapiro, S. D., Griffin, G. L., Gilbert, D. J., Jenkins, N. A., Copeland, N. G., Welgus, H. G., Senior, R. M., and Ley, T. J. (1992) J. Biol. Chem. 267, 4664-4671). We now report that there is a human orthologue of murine macrophage metalloelastase that we call human macrophage metalloelastase (HME). The full-length HME cDNA spans 1.8 kilobases and contains an open reading frame of 1410 base pairs; the predicted molecular mass of the HME proenzyme is 54 kDa. HME mRNA and protein were detected in human alveolar macrophages. Similar to murine macrophage metalloelastase, HME readily undergoes NH2- and COOH-terminal processing to a mature 22-kDa form. Both recombinant HME expressed in Escherichia coli and native HME derived from human alveolar macrophage-conditioned media degraded insoluble elastin. HME is a unique human metalloproteinase that possesses elastolytic activity and is expressed in alveolar macrophages; it is therefore a candidate molecule for the causation of diseases characterized by damage to the extracellular matrix.

Induction of macrophage metalloproteinases by extracellular matrix. Evidence for enzyme- and substrate-specific responses involving prostaglandin-dependent mechanisms.

Many cellular properties are influenced by the surrounding environment of extracellular matrix. To better define the interaction between mononuclear phagocytes and the extracellular matrix components they contact, we studied the effect of various matrices on the biosynthesis and secretion of metalloenzymes and the tissue inhibitor of metalloproteinases in human alveolar macrophages. We found that native and denatured collagen types I and III markedly augmented production of interstitial collagenase (> 25-fold) and increased tissue inhibitor of metalloproteinases to a lesser degree (2.5-fold). In contrast, the biosynthesis of another major secreted macrophage metalloproteinase, 92-kDa gelatinase, was unaffected by contact with extracellular matrices. Furthermore, other matrix components (i.e. type IV collagen, laminin, fibronectin, elastin) failed to induce collagenase production. Maximal stimulation of macrophage collagenase production was achieved with 1-5 micrograms/ml (3-15 x 10(-9) M) denatured collagen in contact with cells for 2 h. Increased biosynthesis of collagenase was detected within 24 h of cell contact with native or denatured collagen and was accompanied by marked induction of collagenase mRNA levels. Our studies of signal transduction mechanisms demonstrated that indomethacin decreased gelatin-induced collagenase production by 90%, with enzyme levels completely restored by the addition of exogenous prostaglandin E2. Prostaglandin E2 was only effective when added within the first 2 h after indomethacin treatment. These results indicate that extracellular matrix can directly influence its remodeling and repair via regulation of the production of metalloenzymes by resident inflammatory cells. Furthermore, matrix-metalloproteinase inductive interactions are both enzyme- and matrix-specific, and are mediated, at least in part, by a prostaglandin-dependent mechanism.

Identification of TIMP-2 in human alveolar macrophages. Regulation of biosynthesis is opposite to that of metalloproteinases and TIMP-1.

We have identified the metalloproteinase inhibitor TIMP-2 as a secreted product of human alveolar macrophages. In contrast to human fibroblasts, TIMP-2 was released from macrophages free of any apparent complexed metalloproteinases. Also in marked distinction to fibroblasts, TIMP-2 secretion from mononuclear phagocytes was subject to modulation by a variety of agents. TIMP-2 was synthesized by macrophages placed in culture under basal conditions in amounts approximately 30% of those secreted by fibroblasts on a per cell basis. The additions of lipopolysaccharide, denatured type I collagen, and zymosan to culture medium each resulted in a dose-dependent and profound decrease in macrophage TIMP-2 protein production and steady-state mRNA levels. In contrast, all of these agents markedly enhanced the biosynthesis of macrophage interstitial collagenase and TIMP-1 as assessed by analysis of identical cell and conditioned media samples. In human fibroblasts, TIMP-2 biosynthesis was unaffected by interleukin-1, tumor necrosis factor-alpha, platelet-derived growth factor, and phorbol ester despite the massive collagenase stimulation induced by each of these agents. We conclude that TIMP-2 is a potentially important mononuclear phagocyte product whose biosynthesis is regulated in a distinct and completely opposite manner to that of collagenase and TIMP-1.

Dexamethasone selectively modulates basal and lipopolysaccharide-induced metalloproteinase and tissue inhibitor of metalloproteinase production by human alveolar macrophages.

To define the capacity of glucocorticoids to regulate tissue damage associated with inflammation more clearly, we have studied the effects of dexamethasone on human alveolar macrophage secretion of both a variety of metalloproteinases and also the counter-regulatory tissue inhibitor of metalloproteinases (TIMP). We found that dexamethasone selectively and coordinately inhibited expression of the following human metalloproteinases: interstitial collagenase, stromelysin, and the 92-kDa type IV collagenase, as well as TIMP. Both basal and LPS-stimulated cells exhibited similar degrees of inhibition, with greater than 50% decrease in secretion of all enzymes and TIMP observed at dexamethasone concentrations of greater than or equal to 10(-8) M in serum-containing medium. The effects of dexamethasone were mediated at a pretranslational level. In summary, our results indicate that glucocorticoids suppress the matrix-degrading phenotype that is characteristic of mature human mononuclear phagocytes, and block the effects of the most potent known signal for upregulation of metalloproteinase secretion. Similar actions in vivo would serve to limit tissue damage associated with the inflammatory response.

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 cell's 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 collagen substrate specificity of rat uterus collagenase.

The collagen substrate specificity of rat uterus collagenase was studied as a function of both collagen type and species of substrate origin. For each collagen examined, values for the basic kinetic parameters, Km and Vmax (kcat), were determined on collagen in solution at 25 degrees C. In all cases, Lineweaver-Burk plots were linear and rat uterus collagenase behaved as a normal Michaelis-Menten enzyme. Collagen types I, II, and III of all species tested were degraded by rat uterus collagenase. Collagen types IV and V were resistant to enzymatic attack. Both enzyme-substrate affinity and catalytic rates were very similar for all susceptible collagens (types I-III). Values for Km ranged from 0.9 to 2.5 X 10(-6) M. Values for kcat varied from 10.7 to 28.1 h-1. The homologous rat type I collagen was no better a substrate than the other animal species type I collagens. The ability of rat uterus collagenase to degrade collagen types I, II, and III with essentially the same catalytic efficiency is unlike the action of human skin fibroblast collagenase or any other interstitial collagenase reported to date. The action of rat uterus collagenase on type I collagen was compared to that of human skin fibroblast collagenase, with regard to their capacity to cleave collagen as solution monomers versus insoluble fibrils. Both enzymes had essentially equal values for kcat on monomeric collagen, yet the specific activity of the rat uterus collagenase was 3- to 6-fold greater on collagen fibrils than the skin fibroblast enzyme. Thus, in spite of their similar activity on collagen monomers in solution, the rat uterus collagenase can degrade collagen aggregated into fibrils considerably more readily than can human skin fibroblast collagenase.