Contrasting effects of fluoroquinolone antibiotics on the expression of the collagenases, matrix metalloproteinases (MMP)-1 and -13, in human tendon-derived cells.

OBJECTIVES: Fluoroquinolone antibiotics may cause tendon pain and rupture. We reported previously that the fluoroquinolone ciprofloxacin potentiated interleukin (IL)-1beta-stimulated expression of matrix metalloproteinases (MMP)-3 and MMP-1 in human tendon-derived cells. We have now tested additional fluoroquinolones and investigated whether they have a similar effect on expression of MMP-13. METHODS: Tendon cells were incubated for two periods of 48 h with or without fluoroquinolones and IL-1beta. Total ribonucleic acid (RNA) was assayed for MMP messenger RNA by relative quantitative reverse transcriptase polymerase chain reaction, with normalization for glyceraldehyde-3-phosphate dehydrogenase mRNA. Samples of supernatant medium were assayed for MMP output by activity assays. RESULTS: MMP-13 was expressed by tendon cells at lower levels than MMP-1, and was stimulated typically 10- to 100-fold by IL-1beta. Ciprofloxacin, norfloxacin and ofloxacin each reduced both basal and stimulated expression of MMP-13 mRNA. In contrast, ciprofloxacin and norfloxacin increased basal and IL-1beta-stimulated MMP-1 mRNA expression. Both the inhibition of MMP-13 and the potentiation of MMP-1 expression by fluoroquinolones were accompanied by corresponding changes in IL-1beta-stimulated MMP output. The non-fluorinated quinolone nalidixic acid had lesser or no effects. CONCLUSIONS: Fluoroquinolones show contrasting effects on the expression of the two collagenases MMP-1 and MMP-13, indicating specific effects on MMP gene regulation.

Ciprofloxacin reduces the stimulation of prostaglandin E(2) output by interleukin-1beta in human tendon-derived cells.

OBJECTIVE: Fluoroquinolone antibiotics such as ciprofloxacin can induce tendon pathology and have various effects on tendon-derived cells in culture. We are investigating whether ciprofloxacin modifies signalling responses in tendon cells. METHODS: Human Achilles tendon-derived cells were preincubated with or without ciprofloxacin (50 mug/ml) and were then challenged with interleukin-1beta (IL-1beta, 1 ng/ml) for up to 48 h. Prostaglandin E2 (PGE2) output was assayed by ELISA. The expression of cyclooxygenase-2 (COX-2) was examined by Western blotting. RESULTS: IL-1beta stimulated a substantial and prolonged increase in the output of PGE2. Preincubation with ciprofloxacin reduced IL-1beta-induced PGE2 output at all times tested; the reduction at 48 h was 69% (99% confidence interval 59-79%; 15 experiments). Norfloxacin and ofloxacin also reduced PGE2 output. However, ciprofloxacin did not affect the induction of COX-2 by IL-1beta, measured at 4 or 48 h. CONCLUSIONS: Ciprofloxacin reduces IL-1beta-induced PGE2 output in tendon-derived cells. The reduction in PGE2 output could modulate various cellular activities of IL-1beta, and may be implicated in fluoroquinolone-induced tendinopathy.

The role of oncostatin M in animal and human connective tissue collagen turnover and its localization within the rheumatoid joint.

OBJECTIVE: To study the interaction of interleukin-1alpha (IL-1alpha) and oncostatin M (OSM) in promoting cartilage collagen destruction. METHODS: Bovine, porcine, and human cartilage and human chondrocytes were studied in culture. The levels of collagenase (matrix metalloproteinase 1 [MMP-1]) and tissue inhibitor of metalloproteinases 1 (TIMP-1) were measured by bioassay and enzyme-linked immunosorbent assay (ELISA). The levels of OSM in rheumatoid synovial fluid were measured by ELISA. RESULTS: When combined with OSM, IL-1alpha, IL-1beta, and tumor necrosis factor alpha released proteoglycan and collagen from cartilage. OSM was the only member of the IL-6 family to have this effect. Human tendon also responded to IL-1alpha and OSM. OSM increased the production of MMP-1 and TIMP-1 but when combined with IL-1alpha, synergistically promoted MMP-1 production in human chondrocytes and synovial fibroblasts. High levels of OSM were found in human rheumatoid synovial fluids, and confocal microscopy showed that OSM was produced by macrophages in rheumatoid synovial tissue. CONCLUSION: These results highlight an important new mechanism by which there is irreversible loss of collagen from cartilage.

Stabilisation of purified human collagenase by site-directed mutagenesis.

During purification, human fibroblast collagenase breaks down into two major forms, an N-terminal 22000/25000-Mr fragment and a C-terminal 27000-Mr fragment; the most likely mechanism being autolysis. The cleavage site has been identified (Pro269- Ile270) and in an attempt to obtain full-length human collagenase (i.e., Mr 42570), this cleavage site and another potential cleavage site (Ala258- Ile259) have been mutated by PCR- directed mutagenesis: Ile270Ser and Ile259Leu. The mutated cDNA was then cloned into the expression vector, pGEX2T, and expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (GST). After cleavage with factor Xa, the mutated collagenase was purified on a peptide hydroxamic acid affinity column. The mutated recombinant collagenase is stable, remains full length and retains the ability to cleave collagen.

Structure of full-length porcine synovial collagenase reveals a C-terminal domain containing a calcium-linked, four-bladed beta-propeller.

BACKGROUND: The collagenases are members of the family of zinc-dependent enzymes known as the matrix metalloproteinases (MMPs). They are the only proteinases that specifically cleave the collagen triple helix, and are important in a large number of physiological and pathological processes. Structures are known for the N-terminal catalytic' domain of collagenases MMP-1 and MMP-8 and of stromelysin (MMP-3). This catalytic domain alone, which comprises about 150 amino acids, has no activity against collagen. A second domain, of 200 amino acids, is homologous to haemopexin, a haem-binding glycoprotein. RESULTS: The crystal structure of full-length MMP-1 at 2.5 A resolution gives an R-factor of 21.7%. Two domains are connected by an exposed proline-rich linker of 17 amino acids, which is probably flexible and has no secondary structure. The catalytic domain resembles those previously observed, and contains three calcium-binding sites. The haemopexin-like domain contains four units of four-stranded antiparallel beta sheet stabilized on its fourfold axis by a cation, which is probably calcium. The domain constitutes a four-bladed beta-propeller structure in which the blades are scarcely twisted. CONCLUSIONS: The exposed linker accounts for the difficulty in purifying full-length collagenase. The C-terminal domain provides a structural model for haemopexin and its homologues. It controls the specificity of MMPs, affecting both substrate and inhibitor binding, although its role remains obscure. These structural results should aid the design of site-specific mutants which will reveal further details of the specificity mechanism.

The prevention of collagen breakdown in bovine nasal cartilage by TIMP, TIMP-2 and a low molecular weight synthetic inhibitor.

Interleukin-1 stimulated bovine nasal cartilage fragments were cultured in the presence and absence of various metalloproteinase inhibitors. Tissue inhibitor of metalloproteinases (TIMP) and tissue inhibitor of metalloproteinases-2 (TIMP-2) completely blocked the release of collagen from the cartilage but were unable to prevent the release of proteoglycan. Similarly, a low molecular weight synthetic inhibitor (BB87) inhibited collagen release in a dose dependent manner, but was unable to inhibit proteoglycan release at the same concentrations. Significantly greater concentrations of inhibitor than those required to block collagen release did, however, block proteoglycan release. These results indicate that the therapeutic use of naturally occurring or synthetic inhibitors may provide a means of modifying the destruction of connective tissue proteins occurring in the arthritides and other connective tissue pathologies.

Large inhibitor of metalloproteinases (LIMP) contains tissue inhibitor of metalloproteinases (TIMP)-2 bound to 72,000-M(r) progelatinase.

Connective-tissue cells in culture produce a family of metalloproteinases which, once activated, can degrade all the components of the extracellular matrix. These potent enzymes are all inhibited by the tissue inhibitor of metalloproteinases (TIMP), and it was thought that this inhibitor was solely responsible for the inhibition of these enzymes within connective tissue. However, other inhibitors have recently been described, including large inhibitor of metalloproteinases (LIMP) present in the culture medium of human foetal lung fibroblasts. Here we show that a large proportion of the inhibitory activity of LIMP consists of 72,000-M(r)-progelatinase bound to TIMP-2, a recently discovered low-M(r) metalloproteinase inhibitor closely related to TIMP. The physiological implications of the secretion of a complex of 72,000-M(r) progelatinase and TIMP-2 are discussed, and the separation of the complex in 6 M-urea is described.

Identification of a new metalloproteinase inhibitor that forms tight-binding complexes with collagenase.

Connective-tissue cells produce a family of metalloproteinases which, once activated, can degrade all the components of the extracellular matrix. These potent enzymes are all inhibited by the tissue inhibitor of metalloproteinases (TIMP), and it was thought that the levels of this inhibitor controlled the extracellular activity of these enzymes. We recently detected a new metalloproteinase inhibitor present in culture media of WI-38 fibroblasts. The inhibitor, named 'large inhibitor of metalloproteinases' (LIMP), can be separated from TIMP by gel filtration on Ultrogel AcA 44, where it is eluted with an apparent Mr of 76,000. A portion of this inhibitor-containing peak binds to concanavalin A-Sepharose, indicating that at least some of the inhibitor contains carbohydrate. LIMP inhibits collagenase (MMP-1), stromelysin (MMP-3) and gelatinase (MMP-2) in a dose-dependent fashion. Collagenase forms tight-binding complexes with LIMP, which can be separated from free collagenase on gel-filtration columns. The complex is eluted with Mr 81,600 (AcA 44) or Mr 60,000 (Superose 12). This complex is larger than that formed between collagenase and TIMP, which has Mr 52,800 (Aca 44) or 41,000 (Superose 12). Polyclonal antibody to TIMP does not recognize LIMP by immunoblotting, and will not block the inhibition of collagenase by LIMP, showing that LIMP is not a multimeric form of TIMP. The role of this new inhibitor in connective-tissue breakdown studies and its relationship to previously described inhibitors of metalloproteinases is discussed.

Purification to homogeneity of pig leucocyte catabolin, a protein that causes cartilage resorption in vitro.

Catabolin, a protein that causes proteoglycan resorption in explants of living cartilage, was purified to homogeneity from culture medium conditioned by culturing buffy-coat leucocytes from 60 litres of pig blood in the presence of concanavalin A. The purification steps were (1) gel filtration of concentrated medium, (2) chromatofocusing, (3) hydroxyapatite chromatography, (4) anion-exchange chromatography (Mono Q), (5) reversed-phase high-pressure liquid chromatography (h.p.l.c.) (Zorbax ODS). These achieved approx. 9000-fold purification from the starting material. The purified protein when reduced ran as a single band on sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis with Mr 21000. On isoelectric focusing its pI was 4.8-5.0, and there was evidence of micro-heterogeneity. The protein co-migrated with active material on h.p.l.c., isoelectric focusing and SDS gels (15 and 12.5% acrylamide) under both reducing and non-reducing conditions. The pure protein caused proteoglycan release from cultured bovine nasal cartilage at 20pM. Its possible identity with interleukin 1 is discussed.

The effect on fertility of vaccinating female sheep with biochemically defined fractions of ram spermatozoa.

Sixty sheep were vaccinated with six biochemically defined fractions of ram spermatozoa obtained by differential extraction procedures with 0.1% Triton X-100 and 2 M MgCl2, followed by ion-exchange chromatography. No one group showed a significant reduction in fertility as compared with the controls, but there was some evidence that MgCl2 extracts were not more potent infertility-inducing agents. Some of the mechanisms by which the immune response could influence fertility are also discussed.