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Biomedical subjects

Z Werb

Publications and source records attributed to Z Werb.

At least 199 records · Page 11Linked to original sources
PubMed1980

Degradation of connective tissue matrices by macrophages. III. Morphological and biochemical studies on extracellular, pericellular, and intracellular events in matrix proteolysis by macrophages in culture.

We have shown that macrophages in culture degrade the glycoproteins and amorphous elastin of insoluble extracellular matrices. Ultrastructural observation of the macrophage-matrix interaction revealed that connective tissue macromolecules were solubilized from the matrix extracellularly. At least part of the matrix breakdown was localized to the immediate vicinity of the cells, as shown by morphological and biochemical studies, although the rate of degradation correlated closely with the secretion of proteinases by various inflammatory stimuli in vivo, by glucocorticoids, prostaglandin E2 or colchicine, or by phagocytosis of latex, zymosan, or cholesterol-albumin complexes in culture was reflected in altered rates of glycoprotein and elastin degradation by the macrophages. Alteration of endocytosis and lysosomal digestion by cytochalasin B, NH4Cl, and proteinase inhibitors did not decrease the overall rate of matrix solubilization, but reduced the processing of the matrix fragments to peptides. Therefore, extracellular, pericellular, and lysosomal events each contribute to degradation of extracellular matrix macromolecules by inflammatory macrophages.

Connective Tissue↗
PubMed1980

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.

Granulocytes↗
PubMed1980

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.

Animals↗
PubMed1978

Proteoglycan-degrading enzymes of rabbit fibroblasts and granulocytes.

A neutral proteinase secreted by rabbit synovial fibroblasts in parallel with specific collagenase was partially purified by ion-exchange chromatography. At pH 7.6 this proteinase degraded 35S-labelled bovine nasal proteoglycan and azo-casein. The enzymic activity was inhibited by EDTA, 1,10-phenanthroline and serum, whereas di-isopropyl phosphorofluoridate and soya-bean trypsin inhibitor had little effect. By gel filtration the apparent mol.wt. of the enzyme was 25000. The fibroblast neutral proteinase was compared with the proteoglycan-degrading neutral proteinases of rabbit polymorphonuclear-leucocyte granules. Two distinct activities were found in the granules: one was inhibited by soya-bean trypsin inhibitor and the other by EDTA. The proteoglycan-degrading proteinases of rabbit fibroblasts and polymorphonuclear leucocytes at acid pH also were examined. Both cathepsin D and a thiol-dependent proteinase contributed to the degradation of proteoglycan at pH 4.5.

Animals↗
PubMed1978

Interaction of glucocorticoids with macrophages. Identification of glucocorticoid receptors in monocytes and macrophages.

Glucocorticoid binding was measured in resident and thioglycollate-elicited mouse peritoneal macrophages, rabbit alveolar macrophages, and human monocytes. Two assays of binding were used--an assay with intact cells in suspension or monolayers, and an assay of cytosol and nuclear forms of glucocorticoid receptors. The mononuclear phagocytes contained approximately equal to 4--10 X 10(3) high affinity receptor sites per cell, with dissociation constants of approximately equal to 2--8 nM dexamethasone. The binding to the saturable sites was specific for steroids with glucocorticoid or antiglucocorticoid activity. Cortisol, corticosterone, and progesterone competed with dexamethasone for binding, whereas estradiol, dihydrotestosterone, and 11-epicortisol competed very little. Binding of dexamethasone to cytosol and nuclear forms of the receptor complex and temperature-sensitive translocation of cytosol forms to nuclear forms were shown. At 37 degrees C the predominant form of the hormone-receptor complex was nuclear. These results demonstrate that corticosteroids interact with macrophages at physiological concentrations.

Animals↗
PubMed1978

Biochemical actions of glucocorticoids on macrophages in culture. Specific inhibition of elastase, collagenase, and plasminogen activator secretion and effects on other metabolic functions.

The effects of glucocorticoids on biochemical functions of macrophages from man, mouse, rabbit, and guinea pig were examined. Secretion of plasminogen activator by human peripheral blood monocytes was decreased by 50% with 1 nM dexamethasone. Differentiation of murine monocytic and granulocytic colonies in agar from bone marrow precursors was decreased by 50% at 7 days with 20 nM dexamethasone. Secretion of elastase, collagenase, and plasminogen activator by resident and thioglycollate-elicited mouse peritoneal macrophages was decreased by dexamethasone, cortisol, and triamcinolone acetonide (1--1,000 nM), but not by progesterone, estradiol, and dihydrotestosterone (1,000 nM); in contrast, secretion of lysozyme was not affected by glucocorticoids or other steroids. The inhibition of macrophage secretion by dexamethasone was both time and dose dependent. Effects were detected within 1--6 h after addition of the glucocorticoids, became maximum by 24 h, and were reversed during a similar time period after removal of the hormones. The extent of inhibition of macrophage secretion increased with increasing glucocorticoid concentration. Half-maximum inhibition of secretion of elastase, collagenase, and plasminogen activator was seen at dexamethasone concentrations (1--10 nM) similar to those that half-saturated the specific glucocorticoid receptors in these cells. At high concentrations of dexamethasone (100--1,000 nM) the secretion of plasminogen activator was inhibited to a greater extent (greater than 95%) than the secretion of elastase (60--80%). Progesterone alone had no effect on secretion, but it blocked the inhibitory effects of dexamethasone and cortisol. Secretion of collagenase, neutral proteinases, and plasminogen activator by elicited rabbit alveolar macrophages was inhibited with glucocorticoids (0.1--100 nM) but not with progesterone or sex steroids. Secretion of a neutral elastinolytic proteinase by guinea pig alveolar macrophages was also inhibited by dexamethasone. These data support the regulatory role of glucocorticoids on macrophage functions at physiological concentrations.

Animals↗
PubMed1978

Cellular control of collagen breakdown in rheumatoid arthritis.

Remodelling of connective tissue and its destruction in rheumatoid arthritis is related to collagenolysis. Study of collagenase released by rheumatoid synovial cels has indicated that the enzyme is released as latent form from adherent synovial cells in culture. As a latent enzyme it is protected from complexing with alpha2 macroglobulin, the principal proteinase inhibitor. Activation in vivo is very likely caused by proteases which destroy or complex with a portion of collagenase responsible for its latency. Recent data suggest that the latent collagenase is an enzyme-inhibitor complex and not a true zymogen.

Arthritis, Rheumatoid↗
PubMed1978

Proteases induce secretion of collagenase and plasminogen activator by fibroblasts.

We have observed that treatment of rabbit synovial fibroblasts with proteolytic enzymes can induce secretion of collagenase (EC 3.4.24.7) and plasminogen activator (EC 3.4.21.-). Cells treated for 2-24 hr with plasmin, trypsin, chymotrypsin, pancreatic elastase, papain, bromelain, thermolysin, or alpha-protease but not with thrombin or neuraminidase secreted detectable amounts of collagenase within 16-48 hr. Treatment of fibroblasts with trypsin also induced secretion of plasminogen activator. Proteases initiated secretion of collagenase (up to 20 units per 10(6) cells per 24 hr) only when treatment produced decreased cell adhesion. Collagenase production did not depend on continued presence of proteolytic activity or on subsequent cell adhesion, spreading, or proliferation. Routine subculturing with crude trypsin also induced collagenase secretion by cells. Secretion of collagenase was prevented and normal spreading was obtained if the trypsinized cells were placed into medium containing fetal calf serum. Soybean trypsin inhibitor, alpha(1)-antitrypsin, bovine serum albumin, collagen, and fibronectin did not inhibit collagenase production. Although proteases that induced collagenase secretion also removed surface glycoprotein, the kinetics of induction of cell protease secretion were different from those for removal of fibronectin. Physiological inducers of secretion of collagenase and plasminogen activator by cells have not been identified. These results suggest that extracellular proteases in conjunction with plasma proteins may govern protease secretion by cells.

Blood Proteins↗
PubMed1978

Glucocorticoid receptors and glucocorticoid-sensitive secretion of neutral proteinases in a macrophage line.

A continuous line of mouse macrophages (P388D1) has been shown to secrete elastase, collagenase, and plasminogen activator at activities comparable to those of macrophages elicited by an inflammatory stimulus in vivo. At physiologic concentrations anti-inflammatory glucocorticoids selectively and reversibly inhibited secretion of the three proteinases but did not inhibit secretion of lysozyme, a constitutive enzyme produced by the P388D1 cells. The secretion of the neutral proteinases was inhibited 50% by 2 to 10 nM dexamethasone. Proliferation of the macrophages was also glucocorticoid sensitive. The P388D1 macrophages contained about 4000 saturable glucocorticoid-binding sites per cell. Concentrations of hormone saturating the high affinity receptor site (for dexamethasone the dissociation constant for steroid-receptor binding, Kd, was 4 nM) correlated well with concentrations inhibiting secretion of the proteinases. Only glucocorticoids and progesterone competed for binding to the specific receptors. Temperature-sensitive translocation of hormone-receptor complexes from "cytoplasm" to nucleus similar to that found with rat thymocytes was demonstrated. Thus, the interaction between glucocorticoids and the P388D1 cell line provides a model for the regulation of macrophage secretion of neutral proteinases under normal and stress conditions.

Cell Line↗
PubMed1977

Endogenous activation of latent collagenase by rheumatoid synovial cells. Evidence for a role of plasminogen activator.

To elucidate the mechanism of synovial damage in rheumatoid arthritis, we studied the activation of latent collagenases released from adherent rheumatoid synovial cells in culture. Latent enzyme was not complexed with alpha2 macroglobulin, the prinicpal proteinase inhibitor in serum, and could be activated by trypsin in the presence of alpha2 macroglobulin if sufficient proteinase was added to saturate inhibitor. Latent collagenase bound half as effectively to collagen fibrils as active enzyme. Plasmin was a threefold better activator of latent enzyme than trypsin and could be generated by addition of plasminogen to synovial-cell cultures. Production of both collagenase and plasminogen activator was inhibited by dexamethasone (10(-9) M). These studies emphasize in importance of control of activation in regulation collagenase activity, It is likely that rheumatoid synovium produces both latent collagenase and plasminogen activator; plasmin is activated from its zymogen, plasminogen, present in inflamed tissues, and in turn activates collagenase.

Arthritis, Rheumatoid↗
PubMed1976

Secretion of macrophage neutral proteinase is enhanced by colchicine.

We have studied the effect of colchicine and related compounds on secretion of enzymes by thioglycollated-elicited mouse peritoneal macrophages in culture. Colchicine stimulated secretion of inducible neutral proteinase activities of elastase (EC 3.4.21.11), collagenase (EC 3.4.24.3), gelatinase (pepsin B; EC 3.4.23.2), and azocaseinase 2- to 6-fold for a period of several days, but inhibited the production and release of lysozyme (mucopeptide N-acetylmuramoylhydrolase; EC 3.2.1.17), a noninducible macrophage secretory product. Parallel changes were observed in cell morphology and secretion after treatment with colchicine, Colcemid, and vinblastine, but not with lumicolchicine, and these effects could be gradually reversed by withdrawal of colchicine. Cytochalasin B also stimulated secretion of elastase 2- to 3-fold but did not influence release of lysozyme. These results demonstrate that tubulin-binding drugs may have opposite effects in macrophages than those usually reported for other experimental systems and also provide evidence for the nonparallel discharge of different macrophage secretion products.

Colchicine↗
PubMed1975

Secretion of a specific collagenase by stimulated macrophages.

Thioglycollate-stimulated mouse macrophages release a specific collagenase into their medium during in vitro culture. The macrophage collagenase has been characterized as a typical metal proteinase which catalyzes the cleavage of the native collagen molecule into three and one-quarter fragments. The extracellular accumulation and low activity in cell lysates suggest that collagenase is a secretion product of the stimulated macrophage. Prolonged secretion of the enzyme at a constant rate for more than 7 days in culture and its inhibition by cycloheximide provide evidence for biosynthesis in vitro. In contrast, secretion of collagenase is barely detectable from unstimulated macrophages which can, however, be stimulated to secret the enzyme by ingestion and intralysosomal storage of latex particles or dextran sulfate. Macrophages laden with latex, an undigestable particle, continue to release collagenase for at least 20 days. Several established mouse cell lines have also been examined for their capacity to secrete collagenase. Collagenase is one of a class of inducible neutral proteinases by which the activated macrophage can modify its extracellular environment.

Animals↗
PubMed1975

Elastase secretion by stimulated macrophages. Characterization and regulation.

Thioglycolate-stimulated mouse peritoneal macrophages secrete a Proteinase which degrades insoluble elastin. There is little elastase activity in cell lysates but the bulk of the enzyme accumulates extracellularly during culture in serum-free medium. The secretion of elastase is sustained for over 12 days in culture and continued secretion of elastase requires protein synthesis. Unstimulated macrophages secrete very little elastase activity but can be triggered to secrete higher levels of this enzyme by phagocytosis and intracellular storage of latex particles. The macrophages elastase is a distinctive proteinase differing from the elastases of pancreas and granulocytes and is distinct from the other secreted proteinases of macrophages, namely, collagenase and plasminogen activator. The macrophages elastase is a serine proteinase and is inhibited by di-isopropyl phosphoro-fluoridate, ovoinhibitor, EDTA, dithiothretiol, and serum. Its activity is little affected by soybean trypsin inhibitor, turkey ovomucoid and chloromethyl ketones derived from tosyl lysine, tosyl phenylalanine, and acetyltetra alanine. Hydrolysis by macrophage elastase of chromogenic ester substrates for pancreatic elastase could not be detected. Elastase secretion by stimulated macrophages exceeds that by primary and established fibroblast cell strains. It is likely that elastase secretion by macrophages plays a major role in the pathogenesis of chronic destructive pulmonary diseases such as emphysema.

Animals↗
PubMed1975

Collagenase and other proteinases in the cornea of the retinol-deficient rat.

1. Enzymes that may contribute to liquefaction of the cornea in retinol-deficient animals and in man have been studied using rat cornea. The established technique of culturing tissue fragments and determining the activity of collagenase (EC 3-4-24-3) and other enzymes in the medium after different periods of culture was used. 2. A collagenolytic system was detected in the media from cultures of rat corneas. This system probably consists of at least two enzymes, a collagenase and a neutral proteinase. 3. Both proteolytic and specific collagenolytic activity were greater in media from retinol-deficient rat corneas. The hydroxyproline level increased in parallel with the increase in enzyme activity. 4. In the final stages of retinol deficiency the cornea is invaded by granulocytes and other cells of the blood and we suggest that destruction of cornea collagen may be due largely to the activity of the enzymes from these cells.

Animals↗