Degradation of connective tissue matrices by macrophages. I. Proteolysis of elastin, glycoproteins, and collagen by proteinases isolated from macrophages.

We have investigated the ability of neutral and lysosomal enzymes of mouse macrophages to degrade the insoluble extracellular matrices secreted by smooth muscle cells, endothelial cells, and fibroblasts. Matrices produced by smooth muscle cells contained glycoproteins, elastin, and collagens, but matrices of endothelial cells and fibroblasts contained no elastin. Sequential enzyme digestion of residual matrix revealed that plasmin, a product of macrophage plasminogen activation, degraded 50-70% of the glycoprotein in the matrices but did not degrade the elastin or the collagens. Purified macrophage elastase degraded glycoprotein and elastin components but had no effect on the collagens. The rate of elastin degradation by macrophage elastase was decreased in the presence of the glycoproteins. In contrast, human granulocyte elastase effectively degraded the matrix glycoproteins, elastin, and, to a lesser extent, collagens, Mammalian collagenase degraded only collagens. Conditioned medium from resident and inflammatory macrophages, containing mixtures of the secreted proteinases, degraded the glycoprotein and elastin components of the matrices. However, conditioned medium was less effective in degrading matrix than comparable amounts of purified macrophage elastase because > 90% of the elastase in the medium was in a latent form. Inclusion of plasminogen in the assays accelerated degradation. In the presence of plasminogen, glycoproteins were degraded readily by medium from P388D1, pyran copolymer-, thioglycollate-, and periodate-elicited macrophages and, to a lesser extent, by medium from endotoxin-elicited and resident macrophages; medium from P388D1, thioglycollate-, and periodate-elicited macrophages was most effective in elastin degradation, and resident, endotoxin-elicited and pyran copolymer-elicited macrophages degraded almost no elastin. The macrophage cathepsins D and B degraded all the matrix components at an optimum pH of 5.5 and acted with the secreted neutral proteinases to degrade the connective tissue macromolecules to amino acids and oligopeptides. These data indicate that macrophages at inflammatory sites contain and secrete proteolytic enzymes that could degrade the extracellular matrix.

Limited proteolysis by macrophage elastase inactivates human alpha 1-proteinase inhibitor.

Inflammatory mouse peritoneal macrophages secrete a metalloproteinase that is not inhibited by alpha 1-proteinase inhibitor. This proteinase, macrophage elastase, recognizes alpha 1-proteinase inhibitor with macrophage elastase does not involve a stable proteinase-inhibitor complex and results in the proteolytic removal of a peptide of apparent molecular weight 4,000-5,000 from the inhibitor. After degradation by macrophage elastase, alpha 1-proteinase inhibitor is no longer able to inhibit human granulocyte elastase, a serine proteinase implicated in the pathogenesis of emphysema. Macrophage elastase apparently does not degrade human granulocyte elastase-alpha 1-proteinase inhibitor complexes or release active granulocyte elastase from these complexes. The ability of macrophage elastase to degrade alpha 1-proteinase inhibitor is inhibited by EDTA and alpha 2-macroglobulin.

Selective proteolysis of immunoglobulins by mouse macrophage elastase.

Mouse macrophage elastase, a metalloproteinase secreted by inflammatory macrophages, catalyzed the limited proteolysis of selected subclasses of mouse immunoglobulins, including monomeric IgG2a, IgG3, and some forms of IgG2b. Mouse IgG1 was resistant to elastase degradation; however, human IgG1 was degraded. IgG3 in immune complexes was cleaved in a manner similar to that of monomeric IgG3. Degradation by macrophage elastase was limited to the heavy chain, resulting in products that did not compete for binding to the macrophage Fc receptor. Macrophage elastase usually produced a pepsin-like rather than a papain-like pattern of proteolysis, resulting in the release of F(ab')2 and Fc' subfragments. This degradation of IgG differed from the papain-like cleavage of IgG by granulocyte elastase. Macrophage elastase degraded papain-generated Fc fragments of IgG2a into multiple fragments. Therefore, macrophage elastase at concentrations found in culture medium has the potential to regulate some aspects of cellular events associated with immunoglobulins.

The inhibitory complex of human alpha 1-proteinase inhibitor and human leukocyte elastase is a neutrophil chemoattractant.

An inhibitor-proteinase complex consisting of human alpha 1-PI and human leukocyte elastase is chemotactic for human neutrophils. The chemotactic activity is optimal at 1 nM and is associated only with the alpha 1-PI portion of the complex. Neither HLE in the complex, free HLE, nor native alpha 1-PI possesses chemotactic activity for human neutrophils. alpha 1-PI in complex is hydrolyzed at the Met-358-Ser-359 bond. The chemotactic activity is associated with the Mr 4,200 fragment of alpha 1-PI that has Ser-359 as its NH2 terminus. The region of the HLE-alpha 1-PI complex that stimulates chemotaxis appears to be the same as that of the Mr 4,200 fragment generated by hydrolysis of the Pro-357-Met-358 bond during proteolytic inactivation of alpha 1-PI. The data suggest the presence of a neutrophil surface receptor bound by alpha 1-PI after the formation of a complex with HLE or after proteolytic degradation. This receptor may play a role in clearance of these modified alpha 1-PI molecules.

Wound macrophages express TGF-alpha and other growth factors in vivo: analysis by mRNA phenotyping.

The presence of macrophages is required for the regeneration of many cell types during wound healing. Macrophages have been reported to express a wide range of mitogenic factors and cytokines, but none of these factors has been shown in vivo to sustain all the wound-healing processes. It has been suggested that transforming growth factor-alpha (TGF-alpha) may mediate angiogenesis, epidermal regrowth, and formation of granulation tissue in vivo. Macrophages isolated from a wound site, and not exposed to cell culture conditions, expressed messenger RNA transcripts for TGF-alpha, TGF-beta, platelet-derived growth factor A-chain, and insulin-like growth factor-1. The expression of these transcripts was determined by a novel method for RNA analysis in which low numbers of mouse macrophages were isolated from wound cylinders, their RNA was purified and reverse-transcribed, and the complementary DNA was amplified in a polymerase chain reaction primed with growth factor sequence-specific primers. This single-cell RNA phenotyping procedure is rapid and has the potential for quantification, and mRNA transcripts from a single cell or a few cells can be unambiguously demonstrated, with the simultaneous analysis of several mRNA species. Macrophages from wounds expressed TGF-alpha antigen, and wound fluids contained TGF-alpha. Elicited macrophages in culture also expressed TGF-alpha transcripts and polypeptide in a time-dependent manner after stimulation with modified low-density lipoproteins and lipopolysaccharide endotoxin, which are characteristic of the activators found in injured tissues.

Oxygen tension regulates the expression of angiogenesis factor by macrophages.

When cultured in a hypoxic environment similar to that found in the center of a wound, macrophages secreted active angiogenesis factor into the medium. Under conditions similar to those of well-oxygenated tissue, macrophages did not secrete active angiogenesis factor. Macrophages that secreted the factor at hypoxic conditions stopped secreting it when returned to room air. Thus the control of angiogenesis in wound healing may be the result of macrophages responding to tissue oxygen tension without the necessity of interacting with other cell types or biochemical signals.

Regulation of alpha 1 proteinase inhibitor function by rabbit alveolar macrophages. Evidence for proteolytic rather than oxidative inactivation.

Rabbit alveolar macrophages were cultured in an environment conducive to the secretion of both reactive oxygen and proteinases, so that the relative importance of proteolytic and oxidative inactivation of alpha 1-proteinase inhibitor by alveolar macrophages could be evaluated. The inactivation of alpha 1-proteinase inhibitor was proportional to its proteolysis, and there was no detectable inactivation in the absence of proteolysis. Although the live macrophages were capable of secreting reactive oxygen, they did not inactivate alpha 1-proteinase inhibitor by oxidation. The inactivation of alpha 1-proteinase inhibitor by proteolysis was proportional to the secretion of elastinolytic activity by the alveolar macrophages. The inability of the alveolar macrophages to oxidize alpha 1-proteinase inhibitor was attributed to the methionine in the macrophages, in secreted proteins, and in the culture medium competing for oxidants. The data suggest that proteolytic inactivation of alpha 1-proteinase inhibitor may be important in vivo and that the methionine concentration in vivo may protect alpha 1-proteinase inhibitor from significant oxidative inactivation.

Interaction of mouse macrophage elastase with native and oxidized human alpha 1-proteinase inhibitor.

Native and oxidized alpha 1-proteinase inhibitor (alpha 1-PI) were compared as substrates for the metalloproteinase macrophage elastase. At substrate concentrations at which native alpha 1-PI was readily degraded by macrophage elastase, oxidized alpha 1-PI was hardly degraded at all. Incubation of macrophage elastase with oxidized alpha 1-PI before the addition of native alpha 1-PI showed that oxidized alpha 1-PI was not an inhibitor of macrophage elastase. Competition experiments with up to twofold excess oxidized alpha 1-PI did not interfere with the degradation of native alpha 1-PI by macrophage elastase. Sequence analysis of amino acids in degraded native alpha 1-PI showed that macrophage elastase attacked a single peptide bond between Pro-357 and Met-358, the latter representing the P1 reactive-site residue of alpha 1-PI. In oxidized alpha 1-PI, Met-358 was converted to methionine sulfoxide and macrophage elastase hydrolyzed the bond between Phe-352 and Leu-353. These data suggest that methionine may be the primary cleavage site for macrophage elastase and not leucine, as previously thought.

Expression of metalloproteinases and metalloproteinase inhibitors by fetal astrocytes and glioma cells.

Metalloproteinases have been implicated as important factors mediating the tissue migration of a variety of normal and transformed cells. The conditioned medium (CM) of fetal human astrocytes and five glioma cell lines did not degrade azocoll in suspension, but several proteolytic activities, inhibitable by 1,10-phenanthroline, were detected on sodium dodecyl sulfate-polyacrylamide gels containing gelatin. Both cell types secreted three major proteolytic species (Mr 65,000, 57,000, and 52,000). Two of the glioma lines secreted an additional proteinase (Mr 92,000). After treatment with 12-O-tetradecanoylphorbol-13-acetate, the secretion of the Mr 92,000, 57,000, and 52,000 proteinases was induced or enhanced in all of the cells. The Mr 92,000 and 65,000 proteinases bound specifically to a gelatin affinity column. When purified by preparative gel electrophoresis, the Mr 65,000 proteinase was found to degrade type IV procollagen. The Mr 57,000 and 52,000 species were precipitated by anticollagenase IgG. Tissue inhibitor of metalloproteinases was detected in the CM of all of the cells by substrate gel analysis and immunoprecipitation of [35S]methionine-labeled proteins with anti-tissue inhibitor of metalloproteinases IgG. The glioma lines also secreted various amounts of two smaller inhibitors of metalloproteinases (IMPs), also seen in rabbit brain capillary endothelial cell CM (IMP-1 at Mr 22,000 and IMP-2 at Mr 19,000), and an inhibitor not previously identified (IMP-3 at Mr 16,500). 12-O-Tetradecanoylphorbol-13-acetate stimulated the secretion of tissue inhibitor of metalloproteinases in all of the cells and induced IMPs in some of the glioma lines. When gel filtration chromatography of concentrated CM was used to resolve inhibitors from proteinases, the isolated proteinases had activity against azocoll and the glycoprotein and collagen components of an in vitro model of the extracellular matrix. The secretion of a battery of metalloproteinases by astrocytes may be important in facilitating astrocytic migration during development and in pathological conditions such as inflammation or local invasion of astrocytic neoplasms.

Elastases and elastin degradation.

The metabolic turnover of mature elastin fibers in adult animals is relatively slow. Although only small amounts of elastin are degraded normally, increased degradation and fragmentation of elastic fibers may play a significant role in disease processes. Elastinolytic enzymes are found in microorganisms, snake venoms, and in a number of mammalian cells and tissues, including pancreas, polymorphonuclear leukocytes, and macrophages. Elastinolytic enzymes fall into all 4 classes of proteinases (aspartic, cysteine, serine, and metallo) and show a spectrum of different specificities. All elastases studied to date have catalytic activity against protein and peptide substrates other than elastin. The presence of elastase activity is a virulence factor associated with the pathogenicity of Pseudomonas and other bacteria, dermatophytic fungi, and necrosis by rattlesnake venoms. Only elastinolytic enzymes are capable of inducing experimental pulmonary emphysema. Elastin degradation mediated by living macrophages and trophoblasts is confined to the immediate pericellular environment. Destruction of mature elastin by other mammalian elastases is probably the result of an imbalance in the normal inhibitor-proteinase ratio. The major plasma inhibitors contributing to the regulatory balance are alpha 1-proteinase inhibitor and alpha 2-macroglobulin.

Type I collagen degradation by invasive oral squamous cell carcinoma.

Expression of extracellular matrix-degrading proteases is required for tumor cell invasion. In the present study we examined the production of type I collagen-degrading matrix metalloproteinases (MMPs) in the invasive oral squamous cell carcinoma-derived cell line HSC-3. In the absence of serum or exogenous growth factors, HSC-3 cells displayed no collagen degradation activity. Addition of serum slightly increased collagen proteolysis. However, addition of epidermal growth factor (EGF) resulted in nearly complete degradation of the collagen matrix. Zymography showed that MMP-2 and -9 are secreted by HSC-3 cells. EGF stimulated secretion of an additional gelatinase with a molecular weight similar to that of MMP-1. Immunoblotting of conditioned medium confirmed that EGF and, to a lesser degree type I collagen, increased production of MMP-1. Finally, in situ hybridization revealed intense expression of MMP-1 in oral squamous cell carcinoma specimens. Together, these results indicate that MMP-1 is expressed, induced by EGF, and required for type I collagen degradation in oral squamous cell carcinoma.

12-O-tetradecanoylphorbol-13-acetate (TPA) induces sister-chromatid exchanges and delays in cell progression in Chinese hamster ovary and human cell lines.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induced a 20-45% increase in sister-chromatid exchange (SCE) frequency in Chinese hamster ovary cells (CHO) and in 2 SV40-transformed human fibroblast cell lines (GM637 and XP12RO) at concentrations up to 1 microgram/ml. The increase was independent of the time at which the cells were fixed after treatment and was not due to an impurity in the TPA preparation or to increased incorporation of bromodeoxyuridine into the DNA. There was no synergistic effect on SCE induction when CHO cells were simultaneously exposed to TPA and the carcinogens mitomycin C or ultraviolet light, but there was when TPA and benzo[a]pyrene were used. In addition to its weak SCE-inducing effects in CHO cells, TPA caused slight delays in cell cycle progression and greatly enhanced the cell cycle delay induced by benzo[a]pyrene.

Transforming growth factor beta-1 decreases interstitial collagenase in healing human fetal skin.

Fetal dermal wounds heal without scarring. Because wound repair requires extracellular matrix turnover, the authors hypothesized that fetal skin would have increased levels of proteinases responsible for matrix degradation compared with adult skin. It was further hypothesized that transforming growth factor beta-1 (TGF-beta1) induces scarring in fetal skin by altering proteinase synthesis. A model of human fetal skin transplanted subcutaneously onto immunodeficient mice was used to study the role of matrix metalloproteinases in healing human fetal skin. In this model, transplanted second trimester fetal skin heals without scarring; addition of TGF-beta1 induces scarring. Proteinases were detected by immunohistochemistry in untransplanted fetal skin, untransplanted adult skin, TGF-beta1-treated fetal skin grafts, and sucrose-treated control grafts. In untransplanted fetal skin, interstitial collagenase, stromelysin-1, and gelatinase A were found in dermal cells and keratinocytes, and around vascular structures. Proteinases were detected in adult skin at similar locations but stained less intensely. Addition of TGF-beta1 decreased interstitial collagenase in fetal skin, but detection of gelatinase A and stromelysin-1 was unchanged. The authors conclude that matrix metalloproteinases are present in midgestation human fetal skin and that more proteinase-containing cells are found in fetal skin than in adult skin. Manipulation of fetal skin with TGF-beta1 is accompanied by a decrease in interstitial collagenase. These data suggest that the increased matrix metalloproteinases found in fetal skin contribute to scarless healing and that the fibrotic effects of TGF-beta1 on fetal skin may be mediated in part by decreasing the synthesis of interstitial collagenase.

Binding of tissue inhibitor of metalloproteinases 2 to two distinct sites on human 72-kDa gelatinase. Identification of a stabilization site.

We have identified a binding site for tissue inhibitors of metalloproteinases 2 (TIMP-2) on human 72-kDa gelatinase that is distinct from the active site. 72-kDa progelatinase is found in a complex with TIMP-2 in the medium of cultured cells and can be activated with organomercurial compounds to yield a gelatinolytic proteinase that remains bound to TIMP-2. Removal of TIMP-2 from 72-kDa progelatinase by reverse-phase high performance liquid chromatography, followed by reconstitution of the progelatinase in neutral pH buffer, results in autocatalytic activation. When samples of autoactivated gelatinase were blotted onto nitrocellulose, then probed with 125I-TIMP-2, we found a 29-kDa peptide that was capable of binding TIMP-2. We isolated this fragment and identified it as the region of gelatinase from amino acid 414 to the carboxyl terminus in the primary amino acid sequence of progelatinase. This portion of the molecule does not contain the putative zinc- or gelatin-binding sites and is proteolytically inactive. Incubation of 125I-TIMP-2 with 72-kDa progelatinase-TIMP-2 complexes resulted in a concentration-dependent exchange of labeled TIMP-2 with unlabeled TIMP-2, in both the presence and absence of the metalloproteinase inhibitor 1,10-phenanthroline. Saturation binding kinetics for the active site of 72-kDa gelatinase were measured in pools of the 43-kDa active fragment that results from the autoactivation of 72-kDa progelatinase; this fragment has no carboxyl-terminal TIMP-2 binding capability. Binding of 125I-TIMP-2 to the active site was completely inhibited by 1,10-phenanthroline. Binding kinetics for the putative stabilization site were determined with isolated 72-kDa progelatinase. In the presence of 1,10-phenanthroline, 72-kDa progelatinase bound 125I-TIMP-2 but not 125I-TIMP-1. Scatchard analysis yielded an approximate dissociation constant (Kd) of 0.72 nM for the active site and 0.42 nM for the stabilization site.

Neutrophil elastase processing of gelatinase A is mediated by extracellular matrix.

Gelatinase A (72-kDa type IV collagenase) is a metalloproteinase that is expressed by many cells in culture and is overexpressed by some tumor cells. It has been suggested that the serine proteinase neutrophil elastase might play a role in the posttranslational processing of gelatinase A and that noncatalytic interactions between gelatinase A and components of the extracellular matrix might alter potential processing pathways. These questions were addressed with the use of gelatin substrate zymography, gelatinolytic activity assays, and amino acid sequence analysis. We found that neutrophil elastase does proteolytically modify gelatinase A by cleaving at a number of sites within gelatinase A. Sequential treatment of gelatinase A with 4-aminophenylmercuric acetate (APMA) and neutrophil elastase yielded an active gelatinase with a 4-fold increase in gelatinolytic activity. The increased gelatinolytic activity correlated with that of a 40-kDa fragment of gelatinase A. Matrix components altered the proteolytic modifications in gelatinase A that were mediated by neutrophil elastase. In the absence of gelatin, neutrophil elastase destructively degraded gelatinase A by hydrolyzing at least two bonds within the fibronectin-like gelatin-binding domain of gelatinase A. In the presence of gelatin, these two inactivating cleavage sites were protected, and cleavage at a site within the hemopexin-like carboxyl-terminal domain resulted in a truncated yet active gelatinase. The results suggest a regulatory role for extracellular matrix molecules in stabilizing gelatinase A fragments and in altering the availability of sites susceptible to destructive proteolysis by neutrophil elastase.

Mouse macrophage elastase. Purification and characterization as a metalloproteinase.

Macrophage elastase was purified from tissue-culture medium conditioned by inflammatory mouse peritoneal macrophages. Characterized as a secreted neutral metalloproteinase, this enzyme was shown to be catalytically and immunochemically distinct from the mouse pancreatic and mouse granulocyte elastases, both of which are serine proteinases. Inhibition profiles, production of nascent N-terminal leucine residues and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of degraded elastin indicated that macrophage elastase is an endopeptidase, with properties of a metalloproteinase, rather than a serine proteinase. Macrophage elastase was inhibited by alpha 2-macroglobulin, but not by alpha 1-proteinase inhibitor. Macrophage elastase was resolved into three chromatographically distinct forms. The predominant form had mol.wt. 22 000 and was purified 4100-fold. Purification of biosynthetically radiolabelled elastase indicated that this form represented less than 0.5% of the secreted protein of macrophages. Approx. 800% of the starting activity was recovered after purification. Evidence was obtained for an excess of an endogenous inhibitor masking more than 80% of the secreted activity.

Wound fluid angiogenesis factor stimulates the directed migration of capillary endothelial cells.

During wound healing, new capillaries grow into the wound site. An angiogenesis factor isolated from wound fluid stimulates the movement of capillary endothelial cells in a filter migration assay. Experiments were carried out to determine whether the movement seen in the assay was chemokinetic or chemotactic. Capillary endothelial cells were plated onto a collagen-coated coverslip and inverted over a visualization apparatus. Cells exposed to a constant concentration of wound fluid angiogenesis factor (WAF) were more mobile than cells not exposed to WAF, and this movement was chemokinetic. When exposed to a gradient of WAF, the cells translocated toward the higher concentration; this directional movement was chemotactic. Cells in a gradient of WAF morphologically aligned with the gradient. These data support the idea that wound healing angiogenesis is regulated by the chemotaxis of capillary endothelial cells.

Cell interactions in post-traumatic fibrosis.

Models of reparative fibrosis, or wound healing, disclose a basic spatial relationship between inflammatory cells, fibroblasts, dividing fibroblasts and angiogenic endothelial cells. Many components of the extracellular fluid, especially steep gradients of PO2, PCO2, pH, glucose and lactate, have been measured. After components of coagulation play out their role in the first few days after injury, macrophages sense excitatory substances and signs of respiratory distress, and secrete chemoattractants for endothelial cells and fibroblasts. If excitatory substances (such as silica) are eliminated, active fibrosis ends with the restoration of the energy supply. Some of the mitogens and chemoattractants acting as intercellular messengers between these cells have been isolated and identified.