Carlo L. Mainardi

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 α2 macroglobulin, the principal proteinase inhibitor in serum, and could be activated by trypsin in the presence of α2, 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 the importance of control of activation in regulating 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. (N Engl J Med 296:1017–1023, 1977)

Human neutrophils contain a neutral metalloproteinase which degrades denatured collagens and potentiates the action of interstitial collagenase. This gelatinase is rapidly secreted from neutrophils stimulated with phorbol myristate acetate. The secreted enzyme has been purified by a combination of chromatography on DEAE-cellulose and gelatin-Sepharose. The purified enzyme was latent and had a specific activity of 24,000 units. Estimated molecular weight obtained by gel filtration was 150,000-180,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed three bands with relative molecular weights of 225,000, 130,000, and 92,000. Electrophoresis in the presence of a reducing agent revealed a single band of Mr = 92,000. All the proteins seen on the unreduced gel were found to contain proteolytic activity against gelatin and native type V collagen. Polyclonal antibodies were prepared against the Mr = 130,000 and 92,000 proteins. When analyzed by immunoblotting, both antibodies recognized all three proteins. Furthermore, the identical three proteins were identified by the antibodies when crude culture medium was immunoblotted. The purified enzyme was inhibited by EDTA and 1,10-phenanthroline but not by serine or thiol proteinase inhibitors, suggesting that the enzyme is a metalloendoproteinase. The enzyme had little or no activity against common protein substrates such as bovine serum albumin or casein. Native type I collagen was not cleaved under conditions where native type V collagen was extensively degraded.

We have identified and sequenced a cDNA encoding human neutrophil collagenase from a lambda gt11 cDNA library constructed from mRNA extracted from the peripheral leukocytes of a patient with chronic granulocytic leukemia. The library was screened with an oligonucleotide probe constructed from the putative zinc-binding region of fibroblast collagenase. Eleven positive clones were identified, of which the one bearing the largest insert (2.2 kilobases (kb)) was sequenced. From the nucleotide sequence of the 2.2-kb cDNA clone we have deduced a 467-amino acid sequence representing the entire coding sequence of the enzyme. The deduced protein was confirmed as neutrophil collagenase by conformity with the amino-terminal sequence analyses of three tryptic peptides of purified neutrophil collagenase. The cDNA clone hybridizes to a 3.3-kb mRNA present in RNA extracted from human bone marrow but did not hybridize with RNA isolated from U937 cells induced to differentiate with phorbol myristate acetate. Neutrophil collagenase was found to possess 57% identity with the deduced protein sequence for fibroblast collagenase with 72% chemical similarity. Certain regions of the molecule, including the putative zinc-binding region, are highly conserved. When compared with the published sequence for fibroblast collagenase, neutrophil collagenase contains four additional sites for glycosylation. Medium from COS-7 cells transfected with a pcDNA1 eucaryotic expression vector containing cDNA for neutrophil collagenase degraded type I collagen into the three-quarter, one-quarter fragments characteristic of mammalian interstitial collagenase activity. Thus, definitive evidence based on the cDNA sequence confirms the neutrophil collagenase is a distinct gene product and a member of the family of matrix metalloproteinases.

Interleukin 1 is a monokine that exerts biological effects on a variety of target cells in vitro. In this report, interleukin 1 has been found to be capable of stimulating collagenase production by cultured dermal fibroblasts. The concentrations of interleukin 1 that stimulate fibroblast collagenase production are similar to those that stimulate mouse thymocyte proliferation. Analyses by high performance liquid chromatography indicate that interleukin 1, rather than a contaminating monokine, is responsible for this effect on fibroblasts. Interleukin 1, released in vivo by macrophages infiltrating sites of tissue damage or inflammation, may function to stimulate the release of collagenase by connective tissue fibroblasts.

OBJECTIVE: To assess the presence of fibroblast collagenase (MMP-1), neutrophil collagenase (MMP-8), and collagenase 3 (MMP-13) in osteoarthritic (OA) cartilage, with particular emphasis on areas of macroscopic cartilage erosion. METHODS: Messenger RNA (mRNA) levels were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and Northern blot analysis. RESULTS: MMP-1 and MMP-13 were expressed at higher levels by OA chondrocytes than by normal chondrocytes. In addition, mRNA for MMP-8 was present in OA cartilage but not normal cartilage by PCR and Northern blot analyses. Chondrocytes from areas surrounding the OA lesion expressed greater quantities of MMP-1 and MMP-13 compared with normal chondrocytes, suggesting local modulation by mechanical and inflammatory factors. Tumor necrosis factor alpha stimulated the expression of all 3 collagenases. Retinoic acid, an agent which induces autodigestion of cartilage in vitro, stimulated only the expression of MMP-13. CONCLUSION: These findings suggest a key role of MMP-13 and MMP-8, as well as MMP-1 in osteoarthritis.