Carlo L. Mainardi

Secretion of Collagenolytic Enzymes by Human Polymorphonuclear Leukocytes

The polymorphonuclear leukocyte contains three proteinases capable of degrading the collagenous components of the connective tissue matrix. These proteinases, gelatinase, interstitial collagenase and elastase, were found to be rapidly secreted by the neutrophil in response to soluble stimuli with maximal accumulation of the gelatinase and interstitial collagenase occurring during a 20 minute incubation. When neutrophils were stimulated with the chemotactic peptide, formyl-met-leu-phe, gelatinase was the predominant collagenolytic enzyme detected. Stimulation of neutrophils with increasing doses of the Ca++ ionophore A23187 lead to a sequential release of collagenolytic proteinases. Gelatinase release was detected at Ca++ ionophore concentrations of .05-.1 microM, while significant release of interstitial collagenase required 0.5-1 microM A23187. Elastolytic activity was detected only at high concentrations of A23187 (5-10 microM). No release of lactic acid dehydrogenase was detected indicating that the enzyme release was not due to cell death. These studies suggest that the neutrophil may modulate its collagenolytic potential by selective release of collagenolytic proteinases.

Stimulation of interstitial collagenase in co-cultures of rat hepatocytes and sinusoidal cells

Although the fibrosis observed during chronic liver injury is the result of a complex process, the striking accumulation of collagen in end stage liver disease has provoked interest in the mechanisms that regulate both collagen production and degradation in the diseased liver. The present studies have examined the cell interactions that may be important in the regulation of collagen degradation. Although minimal amounts of interstitial collagenase activity were noted in cultures of normal hepatocytes and sinusoidal cells, the co-cultures of these cells in the presence of lipopolysaccharide showed a substantial increase in collagenase activity. When the hepatocytes were obtained from rats that had been treated with carbon tetrachloride in vivo, the enhanced activity seen in the co-cultures did not require the addition of lipopolysaccharide. Further characterization of this interaction suggested that the increase in collagenolytic activity was partially due to the elaboration of soluble factors by the hepatocyte, which stimulated collagenase production by the sinusoidal cell population. Elaboration of collagenase activity by the sinusoidal cells was inhibited by cycloheximide, suggesting that protein synthesis was required. The proteolytic activity was abrogated by inhibitors of metalloproteinases but not by serine or thiol proteinase inhibitors. The degradation products of type I collagen were typical of the expected products seen with vertebrate collagenases. Thus, it appears that the increased collagenolytic activity detected in this co-culture system is attributable to the production of interstitial collagenase by the sinusoidal cell population. Such cell-cell interactions may play an important role in the maintenance of normal connective tissue structure of the liver during disease processes.

7S Domain Constitutes the Amino-Terminal End of Type IV Collagen: an Immunochemical Study

The collagenous material from bovine kidney cortex was prepared by pepsin digestion of cortex homogenates. The 7S collagen was isolated after bacterial collagenase digestion of collagenous basement membrane material and was further purified by molecular sieve and ion exchange chromatography. The antibodies were raised in rabbits against a purified 7S collagen preparation. The pepsin-derived collagenous fragments alpha 1(IV)130K, 95K and alpha 2(IV)120K prepared from bovine cortex were tested for their cross-reactivity with the antiserum. The fragment alpha 1(IV)130K was found to be significantly cross-reactive. The results show the presence of common structural antigenic determinants between 7S collagen and alpha 1(IV)130K fragment, thus indicating that 7S collagen is located at the amino-terminal end of type IV collagen.

Effect of dexamethasone, indomethacin, and lipopolysaccharide on the secretion of interstitial collagenase and type V collagenase by cultured macrophages

The rabbit alveolar macrophage secretes at least two collagenolytic metalloproteinases in vitro including an interstitial collagenase and a type V collagenase. Using assays previously shown to discriminate between these two activities, the secretion of these two enzyme activities was investigated. Both enzyme activities accumulated in culture over 11 days and the release of both were similarly inhibited by cycloheximide. Collagenolytic activity was negligible in cell lysates. The interstitial collagenase was found in a latent form but the type V collagenase activity was active in the culture medium. When cultured in the presence of dexamethasone, the secretion of both the enzymes were identically inhibited in a dose-dependent manner. Indomethacin was an effective inhibitor of secretion of both collagenases at a concentration of 10(-5) M but not at lower concentrations. Finally, bacterial lipopolysaccharide stimulated the secretion of both type V and interstitial collagenase by these cells. These studies indicate that, like the interstitial collagenase, the type V collagenase is released from the cell as synthesized and is not stored intracellularly. Protein synthesis is necessary for the release of both these collagenases. Furthermore, the release of type V collagenase responded to dexamethasone, indomethacin, and lipopolysaccharide in a manner identical to the secretion of the interstitial collagenase suggesting that synthesis and secretion of these two enzymes are regulated in a similar manner.

Role of the conserved histidine and aspartic acid residues in activity and stabilization of human gelatinase B: An example of matrix metalloproteinases

Gelatinase B (MMP-9), a member of the matrix metalloproteinase family, is a zinc- and calcium-dependent endopeptidase that is known to play a role in tumor cell invasion and in destruction of cartilage in arthritis. It contains a conserved sequence400His-(X)3-His-(X)28-Asp-Asp-(X)2-436Gly, the function of which is under investigation. The conserved Asp-432 and Asp-433 residues were individually replaced with Gly; these substitutions reduced the gelatinolytic activity of the enzyme to 23% and 0%, respectively. Replacing Asp-433 with Glu, however, decreased the gelatinolytic activity of the enzyme by 93% and proteolytic activity of the enzyme for the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 substrate by 79%. The wild-type and D432G and D433E mutant enzymes had similarKm values for the synthetic substrate and similarKi values for the competitive inhibitor, GM6001. Thekcat/Km values for D432G and D433E mutant enzymes, however, were reduced by a factor of ∼4 and their KaCa values were increased by four- and sixfold, respectively. The significance of His-400 in the activity of the enzyme was assessed by replacing this residue with Ala and Phe. Both H400A and H400F mutants were inactive toward gelatin substrate. These data demonstrate that Asp-432, Asp-433, and His-400 residues are important for the activity of gelatinase B. His-400 may act as a zinc-binding ligand similar to the His-197 in interstitial collagenase (MMP-7) and Asp-432 and Asp-433 residues are probably involved in stabilization of the active site of the enzyme. The His-400 and Asp-433 residues are conserved in all members of the MMP family. Therefore, our results are relevant to this group as a whole.

Antibody to Rabbit Macrophage Type V Collagenase/Gelatinise and its Use to Further Characterize the Enzyme

A highly specific antiserum was raised against purified rabbit alveolar macrophage type V collagenase/gelatinase. The reactivity of the antiserum was tested in ELISA and was detected at a high titer. Competitive inhibition ELISA demonstrated a linear inhibition of reactivity by pure antigen if either pure antigen or crude culture medium was used as the solid phase antigen. In the latter case, greater than 90% inhibition was achieved indicating a high degree of specificity of the antibody. Immobilized antibody was successful in absorbing the type V collagenolytic activity. Immunoblot analysis of crude culture medium revealed a single immunoreactive band which correlated with both the stained gel of the purified antigen as well as the only zone of gelatinolysis in a gelatin/acrylamide gel. This monospecific antibody will be a useful tool in the further characterization of this enzyme as well as the delineation of its relative role in chronic inflammation.

Secretion and Glycosylation of Rabbit Macrophage Type V Collagenase

Cultures of freshly isolated rabbit alveolar macrophages were used to study the synthesis, secretion, and glycosylation of type V collagenase. Cells were pulse-labeled with [35S-]methionine for 15 minutes followed by a chase with cold methionine for various time periods. Type V collagenase was identified in the culture supernatants and cell lysates by immunoprecipitation with a specific antiserum. Within 10 minutes of chase, an 82-kDa protein was found in the cell lysates. This protein was subsequently processed to a 92-kDa protein without identifiable intermediate forms. By 60 minutes of chase, intracellular radioactivity was no longer detectable. The larger protein could be detected within 20 minutes in the culture supernatants and accumulated in the medium for 60 minutes of chase time. Only the 92-kDa form was seen in the supernatants and the proteinase was secreted without intracellular storage or membrane association. Treatment of the 92-kDa proteinase with an enzyme which specifically removes N-linked carbohydrates resulted in an apparent reduction in molecular mass of approximately 10 kDa. Deglycosylation of the proteinase did not result in an apparent loss of activity. Thus, it was concluded that macrophage type V collagenase is synthesized as an 82-kDa polypeptide which is glycosylated by N-linkage and secreted.

Collagen disease: A new perspective

These includethe interstitial collagens (types I, II and Ill), basement membrane collagen (type IV), type V collagen which is a minor component of many tissues and has been localized in the pericellular space, a newly described high molecular weight, cystine-rich complex of collagen chains, and an unusual collagen which is a minor component of cartilage. The specific function of each of these collagens is incompletely understood at the present time.