Bacillus subtilis alpha-amylase: the rate limiting step of secretion is growth phase-independent.

When Bacillus subtilis alpha-amylase was expressed under the control of sacR in a degU32(Hy) strain, the production of exoenzyme occurred during both the exponential and stationary phases of growth. In each phase, pulse-chase experiments showed that the rate-limiting step of the secretion process was the release of the processed form of the protein in each physiological context. The rate of this event was slightly slower (t(1/2) = 3.2 min) during the stationary phase than during the exponential phase (t(1/2) = 2 min). The effectors which possibly control the efficiency of the release stage, the level of PrsA or the calcium binding properties of the cell wall, remained unchanged throughout growth phases.

High levels of expression of collagenase-3 (MMP-13) in pathological conditions associated with a foreign-body reaction.

A foreign-body-type host response can contribute to the induction and release of collagenolytic tissue-destructive enzymes of pathogenetic significance. Our aim was to analyse collagenase-3 in two conditions with putative involvement of foreign-body reactions. Synovial membrane-like tissue samples were obtained from cases of aseptic loosening of a total hip replacement (THR) and osteoarthritis (OA). The reverse transcription polymerase chain reaction (RT-PCR) disclosed that all the samples from patients contained collagenase-3 mRNA compared with only three out of ten control samples. The identity of the RT-PCR amplification product was confirmed by nucleotide sequencing. Immunohistochemical staining showed that collagenase-3 was present in endothelial cells, macrophages and fibroblasts, including those found in the synovial lining. This finding was confirmed by avidin-biotin-peroxidase complex-alkaline phosphatase-anti-alkaline phosphatase double staining and the specificity of the staining by antigen preabsorption using recombinant human collagenase-3. Collagenase-3 was released into the extracellular space and thus found in the synovial fluid in all patient samples as shown by Western blotting. The similar extent of collagenase-3 expression in aseptic loosening and OA compared with the low expression in control synovial membrane suggests involvement of a similar, foreign-body-based pathogenetic component in both. Comparative analysis of collagenase-3 and of foreign particles indicates that paracrine factors rather than phagocytosis per se are responsible for the induction of collagenase-3. We suggest that due to its localisation and substrate specificity, collagenase-3 may play a significant pathogenetic role in accelerating tissue destruction in OA and in aseptic loosening of a THR.

Anti-collagenolytic mechanism of action of doxycycline treatment in rheumatoid arthritis.

Tetracyclines exert, independently of their antimicrobial activity, anti-collagenolytic effects by inhibiting activities of human interstitial collagenases and by preventing the oxidative activation of latent pro-collagenases. We tested the clinical response to a 3-month doxycycline in concert with collagenase activity in 12 rheumatoid arthritis (RA) patients. Patients received 150 mg/day of doxycycline for 3 months. Clinical assessments at zero, six and 12 weeks comprised classification of the functional class, joint score index, Hb, CRP, ESR, health assessment questionnaire, visual analogue scale (VAS) of pain, pain disability index, comprehensible psychopathological rating scale (CPRS), SDS-PAGE laser densitometric collagenase activity measurements and Western blots. Significant reductions were seen in joint score index (P < 0.01), pain VAS (P < 0.05) and some CPRS parameters. Furthermore, collagenase activities measured from saliva by quantitative SDS-PAGE electrophoresis were significantly reduced during the 12-week intervention (P < 0.01). Western blots demonstrated intact 75-80 kDa enzyme protein (classic neutrophil collagenase), but also a newly discovered mesenchymal, less glycosylated 40-55 kDa MMP-8 subtype of fibroblast/chondrocytic origin. These results indicate that the documented favourable clinical response may in part be due to in vivo inhibition of classic neutrophil and mesenchymal collagenase/MMP-8 activities produced by doxycycline. This anti-collagenolytic doxycycline effects is mediated through inhibition of the enzyme activity and not through degradation of the enzyme, which may have contributed to the reportedly reduced tissue destruction, as has been seen in clinical studies concerning RA as well as reactive arthritis.

Matrix metalloproteinase 13 (collagenase 3) in human rheumatoid synovium.

OBJECTIVE: To show the eventual presence and extent of production of matrix metalloproteinase 13 (MMP-13, or collagenase 3) in rheumatoid synovial tissue samples and extracts, and to assess the inhibition characteristics of recombinant MMP-13. METHODS: Immunohistochemical avidin-biotin-peroxidase complex staining/morphometry was used to analyze MMP-13-positive cells in situ. Neutral salt extraction of synovial tissue, electrophoresis of the extract in different buffer systems, and Western blotting were also used. The inhibitory properties of doxycycline, clodronate, pamidronate, and D-penicillamine for recombinant enzyme were determined with a soluble type II collagen assay. RESULTS: MMP-13 was detected in fibroblast- and macrophage-like mononuclear cells in the synovial lining and stroma and in vascular endothelial cells. The overall expression of MMP-13 in these cells in the synovial stroma was high in rheumatoid arthritis (86 +/- 12%) compared with osteoarthritis (17 +/- 5%) patient samples (P = 0.0027). In a high-pH native electrophoresis gel, immunoreactivity to anti-MMP-1 and anti-MMP-13 were clearly separated, with anti-MMP-13-immunoreactive material migrating faster than anti-MMP-1-immunoreactive material. Finally, in contrast to MMP-1 and MMP-8, MMP-13 was found to be relatively resistant to the inhibitory effects of doxycycline and clodronate in vitro. CONCLUSION: Due to its localization in synovial tissue, its substrate profile, increased expression, and relative resistance to known MMP inhibitors, MMP-13 is suggested to play a major role in the pathogenesis of tissue destruction in rheumatoid arthritis.

Cathepsin G and elastase in synovial fluid and peripheral blood in reactive and rheumatoid arthritis.

The purpose of the study was to evaluate the involvement of serine proteinases cathepsin G and elastase on pathomechanisms in synovial fluid (SF) of patients with reactive (ReA) and rheumatoid, (RA) arthritis. Cathepsin G, elastase, and their endogenous inhibitors alpha1-antichymotrypsin (alpha1-ACT) and alpha1-proteinase inhibitor (alpha1-PI) were identified immunohistochemically from SF and peripheral blood (PB) of patients with ReA and RA. Cathepsin G and elastase activities in SF and PB were measured spectrophotometrically. Dot-immunostaining was used to identify cathepsin G, elastase, but also alpha1-ACT and alpha1-PI from SF and PB. Cathepsin G and elastase-like activities (IU/I) were slightly elevated in ReA SF compared to the corresponding peripheral blood values (11.4 +/- 9.2 vs 4.8 +/- 1.7, NS, and 5.1 +/- 2.8 vs 2.3 +/- 2.2, NS), which was similar to what was seen in RA (16.4 +/- 6.2 vs 0.53 +/- 0.4, p < 0.05, and 6.51 +/- 1.8 vs 1.22 +/- 0.58, p < 0.05). Although some samples did not contain cathepsin G and/or elastase-like activities, all samples contained immunoreactive enzyme, but also alpha1-ACT and alpha1-PI. In ReA SF, in contrast to monocytes, all polymorphonuclear (PMN) cells contained cathepsin G and elastase. Cathepsin G and elastase activities correlated with each other (r = 0.78, p < 0.05) suggesting PMN / primary granules as their likely source. There was a closer association between the cathepsin G or elastase and SF leukocyte count in ReA than in RA. In ReA and RA SF elevated cathepsin G and elastase activities are detected compared to activity levels in PB suggesting local production mainly from PMNs. The co-existence of highly cellular SF and cathepsin G and elastase activity in the documented presence of endogenous inhibitors in ReA SF together with the, known, usually self-remitting clinical course of ReA, suggest a brisk and even exaggerated local PMN serine proteinase release; sparing of joints does not seem to be due to lack or inhibition of PMN responses but rather to a successful down-regulation or cessation of the responses initially elicited.

Collagenase in Sjögren's syndrome.

OBJECTIVE: To study collagenase production in labial salivary glands in patients with Sjögren's syndrome (SS). METHODS: Collagenases were localised in labial salivary glands by immunohistochemistry. Collagenase activity against triple helical type I collagen monomers in stimulated saliva was measured using sodium dodecyl sulphate polyacrylamide gel electrophoresis and laser densitometry; tissue inhibitor metalloproteinase (TIMP) was measured by enzyme linked immunosorbent assay. RESULTS: Cells containing collagenase of matrix metalloproteinase (MMP)-1 type were more frequent and more intensely staining in SS than in healthy glands. Only SS saliva contained functional enzyme (11.7 (6.8) x 10(-6) IU/1). Cells containing MMP-8 type neutrophil collagenase were not found in situ, which was in accordance with sialochemical findings/doxycycline inhibition studies. TIMP was found in both SS and normal saliva. CONCLUSIONS: Fibroblast, but not neutrophil type, collagenase is synthesised, secreted, and subsequently activated, but is not inhibited by TIMP in labial salivary glands or saliva in SS. Collagenase may destroy glandular and salivary duct tissue and perturb factors influencing the morphogenetic extracellular matrix.

Gelatinase/type IV collagenases in the loosening of total hip replacement endoprostheses.

Gelatinase/Type IV collagenases, namely, 72 kDa matrix metalloproteinase (MMP)-2 type and 92 kDa MMP-9 type, were analyzed to evaluate the role of extracellular matrix degradation in 17 cases of loosening of total hip endoprostheses. Zymographic and densitometric analyses revealed elevated production of MMP-2 and induction of MMP-9 in tissue extracts from both the interface tissues between bone and implants and the pseudocapsular tissues around the loose endoprostheses when compared with those of 8 control noninflammatory knee synovial tissues. The level of MMP-9 was higher in the interface tissues than in the pseudocapsular tissues. MMP-9 activity was not detected in the control samples. Although differences in the type of prosthetic fixation (cemented versus cementless) or the type of alloy (cobalt-chromium-molybdenum versus titanium-aluminum-vanadium) existed, they shared a similar potential to stimulate tissues to produce MMP-2 and MMP-9. These findings suggest a role for MMP-2 and MMP-9 type gelatinase/Type IV collagenases in the degradation of extracellular matrix of periprosthetic tissues, where they may cause weakening of the connective tissue bed and the loosening of total hip replacement endoprostheses. The pseudocapsular tissues could contribute to the loosening via the production and release of matrix metalloproteinases into the synovial fluid. Alternatively, induction of matrix metalloproteinases in such tissues may reflect remodeling of the pseudocapsular connective tissues.

Multiple forms of gelatinases/type IV collagenases in saliva and gingival crevicular fluid of periodontitis patients.

The aim of the present study was to characterize the eventual presence and molecular forms of gelatinase/type IV collagenase activities in gingival crevicular fluid (GCF) and saliva in different forms of periodontitis; patients with clinically healthy periodontium served as controls. Enzyme activities were monitored electrophoretically by zymography using gelatin and type IV collagen as substrates and analyzed visually and/or densitometrically. Both saliva and GCF collected from adult periodontitis, localized juvenile periodontitis and type II diabetes mellitus periodontitis patients contained species moving identically with gelatinase isolated from human neutrophils or MMP-9 (mean 98 kD), and species with mobility similar to gelatinase in fibroblast cell culture supernatants or MMP-2 (mean 76 kD). Hitherto, undescribed high molecular weight forms (mean 128 kD), were found, possibly representing polymerized or complexed enzyme active/activated in situ in the gel matrix. Small molecular forms of gelatinases (mean 51 kD and 46 kD), unable to cleave type IV collagen, were also found, most likely representing in vivo proteolytically activated, truncated enzymes. Although multiple forms of gelatinases/type IV collagenases in saliva and GCF may take part in the tissue destruction in periodontitis, their profile judged according to molecular weights does not differentiate between different forms of periodontitis.

Salivary collagenase, elastase- and trypsin-like proteases as biochemical markers of periodontal tissue destruction in adult and localized juvenile periodontitis.

The profile of salivary proteases and their cellular origin, with special reference to polymorphonuclear leukocytes and bacteria, was studied in localized juvenile periodontitis and compared with adult periodontitis and healthy controls. General proteolytic activity in saliva as well as collagenase, elastase-like and trypsin-like activity was measured. In addition, the sensitivity of salivary collagenase of patients with localized juvenile periodontitis to doxycycline inhibition was studied. The saliva of localized juvenile periodontitis patients contained low amounts of collagenase compared with adult periodontitis saliva, and almost all salivary collagenase was found to exist in endogenously active form, as was found to be the case also in adult periodontitis patients and healthy controls. The salivary collagenase of localized juvenile periodontitis patients was relatively insensitive to 100 mumol/l doxycycline but was completely inhibited by 600 mumol/l doxycycline, reflecting rather matrix metalloproteinase-1 (fibroblast-type) than matrix metalloproteinase-8 (polymorphonuclear leukocyte) enzyme. The saliva of localized juvenile periodontitis patients also contained low amounts of elastase-like activity compared with the saliva of untreated adult periodontitis patients. Scaling and root planing caused a significant decrease in elastase-like activity in the saliva of adult periodontitis patients. General proteolytic and trypsin-like activities were also low in the saliva of localized juvenile periodontitis patients. Furthermore, the reducing agent beta-mercaptoethanol did not activate or inhibit the salivary trypsin-like activity of localized juvenile periodontitis or adult periodontitis patients, although the reductant readily activated partially purified Porphyromonas gingivalis trypsin-like protease in a characteristic manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Doxycycline in the protection of serum alpha-1-antitrypsin from human neutrophil collagenase and gelatinase.

The concentration of doxycycline required to inhibit 50% (50% inhibitory concentration for serpinase activity) of alpha-1-antitrypsin degradation by purified neutrophil collagenase was found to be approximately 20 microM, a value similar to the 50% inhibitory concentration of doxycycline required to inhibit collagen degradation by neutrophil collagenase. Doxycycline also efficiently inhibited phorbol myristate acetate-triggered neutrophil-mediated degradation of alpha-1-antitrypsin. This suggests that doxycycline can protect alpha-1-antitrypsin from collagenase and gelatinase in the presence of other proteases and biologically active molecules that are released by triggered neutrophils. The protection of a body's alpha-1-antitrypsin shield from serpinolytic activity of collagenase and matrix metallproteinases can result in inhibition of serine proteases such as neutrophil elastase. Tetracyclines may thus protect matrix constituents from a wider spectrum of neutral proteases than previously recognized, not just from the matrix metalloproteinases collagenase and gelatinase.

Tetracycline inhibition and the cellular source of collagenase in gingival crevicular fluid in different periodontal diseases. A review article.

Tetracyclines have recently been shown to inhibit the activity of some but not all mammalian matrix metalloproteinases believed to mediate periodontal destruction. However, the specificity of this effect, which could have significant therapeutic implications for different periodontal diseases, has not been examined in detail. Doxycycline and 4-de-dimethylaminotetracycline (CMT-1) have been tested in vitro for their ability to inhibit human neutrophil and fibroblast interstitial collagenases and collagenase in human gingival crevicular fluid (GCF). The GCF samples were obtained from systemically healthy and insulin-dependent diabetic adult periodontitis patients and from localized juvenile periodontitis (LJP) patients. The concentrations of these 2 tetracyclines required to inhibit 50% of the collagenase activity (IC50) were found to be 15 to 30 microM for human neutrophil collagenase and for collagenase in GCF of systemically healthy and diabetic adult periodontitis patients. These concentrations approximate the tetracycline levels observed in vivo during treatment with these drugs. In contrast, human fibroblast collagenase and GCF collagenase from LJP patients were both relatively resistant to tetracycline inhibition; the IC50 for doxycycline and CMT-1 for these 2 sources of collagenase were 280 and 500 microM, respectively. Based on these and other findings, we propose the following: 1) that systemic levels of tetracycline may inhibit connective tissue breakdown by inhibiting neutrophil collagenase; 2) that tetracyclines do not inhibit fibroblast-type collagenase, which may help explain their lack of effect on normal connective tissue remodeling; 3) that tetracycline inhibition of collagenases may serve to identify the cellular origin of the enzyme; and 4) that tetracyclines can also prevent the oxidative activation of latent human procollagenases.(ABSTRACT TRUNCATED AT 250 WORDS)

Doxycycline protects serum alpha-1-antitrypsin from human neutrophil collagenase.

Interstitial collagenases, members of the matrix metalloproteinase family, are key initiators of collagen destruction during various disorders such as rheumatoid arthritis. Recently interstitial collagenases were found to efficiently degrade an additional non-collagenous substrate, the serum alpha-1-antitrypsin (AAT also called alpha-1-proteinase inhibitor or serpin). Serpins are major endogenous inhibitors of serine proteinases, particularly neutrophil elastase. Of relevance to neutrophil-mediated collagen degradation, the tetracycline family of antibiotics are now known to inhibit inhibit mammalian collagenases by a mechanism unrelated to their antimicrobial activity. This study identifies an additional mechanism by which tetracyclines may retard tissue breakdown during inflammatory diseases. Doxycycline, added to the reaction mixture as in concentrations as low as 10 microM, which correspond to levels of the drug readily achieved in vivo, produced detectable inhibition of serpinase activity of neutrophil collagenase, although levels of 50-100 microM or greater were required to reduce AAT degradation more than 75%. The concentration of doxycycline to inhibit 50% (IC50 of serpinase activity) of AAT degradation by neutrophil collagenase was found to approximate 20 microM, a value similar to the IC50 for doxycycline required to inhibit collagen degradation by neutrophil collagenase. Doxycycline was also found to inhibit at cell level neutrophil-mediated degradation of AAT. The protection of bodies' AAT-shield from serpinolytic activity of collagenase would result in inhibition of serine proteinases such as neutrophil elastase. Tetracyclines may thus protect matrix constituents from a wider spectrum of neutral proteases than previously recognized, not just from the matrix metalloproteinases collagenase and gelatinase.

Identification of proteases from periodontopathogenic bacteria as activators of latent human neutrophil and fibroblast-type interstitial collagenases.

Activation of latent human fibroblast-type and neutrophil interstitial procollagenases as well as degradation of native type I collagen by supra- and subgingival dental plaque extracts, an 80-kDa trypsinlike protease from Porphyromas gingivalis (ATCC 33277), a 95-kDa chymotrypsinlike protease from Treponema denticola (ATCC 29522), and selected bacterial species commonly isolated in periodontitis was studied. The bacteria included were Prevotella intermedia (ATCC 25261), Prevotella buccae (ES 57), Prevotella oris (ATCC 33573), Porphyromonas endodontalis (ES 54b), Actinobacillus actinomycetemcomitans (ATCC 295222), Fusobacterium nucleatum (ATCC 10953), Mitsuokella dentalis (DSM 3688), and Streptococcus mitis (ATCC 15909). None of the bacteria activated latent procollagenases; however, both sub- and supragingival dental plaque extracts (neutral salt extraction) and proteases isolated from cell extracts from potentially periodontopathogenic bacteria P. gingivalis and T. denticola were found to activate latent human fibroblast-type and neutrophil interstitial procollagenases. The fibroblast-type interstitial collagenase was more efficiently activated by bacterial proteases than the neutrophil counterpart, which instead preferred nonproteolytic activation by the oxidative agent hypochlorous acid. The proteases were not able to convert collagenase tissue inhibitor of metalloproteinase (TIMP-1) complexes into active form or to change the ability of TIMP-1 to inhibit interstitial collagenase. None of the studied bacteria, proteases from P. gingivalis and T. denticola, or extracts of supra- and subgingival dental plaque showed any significant collagenolytic activity. However, the proteases degraded native and denatured collagen fragments after cleavage by interstitial collagenase and gelatinase. Our results indicate that proteases from periodontopathogenic bacteria can act as direct proteolytic activators of human procollagenases and degrade collagen fragments. Thus, in concert with host enzymes the bacterial proteases may participate in periodontal tissue destruction.

Collagenase in synovitis of rheumatoid arthritis.

There are two types of collagenases, products of two distinct genes, called MMP-1 (matrix metalloproteinase 1 or "fibroblast-type collagenase") and MMP-8 ("neutrophil collagenase"). In synovial fluid, MMP-8 is stored as latent proenzyme in polymorphonuclear neutrophils. MMP-8 is activated by hypochlorous acid produced by myeloperoxidase from hydrogen peroxide and chloride ion and by the hydroxyl radical produced in Haber Weiss reaction fed by superoxide produced by, eg, NADPH (reduced nicotinamide adenine dinucleotide) oxidase and xanthine oxidase. In addition to activation upon secretion, oxidatively modified MMP-8 is susceptible to a subsequent proteolytic attack and activation by cathepsin G. The authors suggest that activation of neutrophil-derived MMP-8 involves oxidative, nonproteolytic activation upon secretion and a more slowly progressive proteolytic activation by cathepsin G (or chymases and tryptases), and that these oxidative and proteolytic activation mechanisms act in concert. In contrast to MMP-8, MMP-1 is synthesized de novo and secreted immediately after synthesis by fibroblasts, macrophages, and some epithelial cells. Human rheumatoid synovial tissue contains mainly fibroblast-type MMP-1 collagenase as assessed by collagenase extracted from synovial tissue and by MMP-1 and MMP-8 immunostaining. It is suggested that in vivo, MMP-1 in synovitis tissue is activated by a plasminogen activator/plasminogen/prostromelysin (alternatively tryptases)/proMMP-1 cascade. In conclusion, MMP-8 and MMP-1 show type-specific compartmentalization and modes of activation in rheumatoid synovial fluid and tissue.

Tetracycline inhibition identifies the cellular origin of interstitial collagenases in human periodontal diseases in vivo.

Mammalian interstitial collagenases (E.C.3.4.24.7) are considered as key initiators of collagen degradation in periodontal diseases. However, the cellular sources of collagenases present in gingival crevicular fluid have not been completely clarified. Resident fibroblasts and epithelial cells as well as infiltrating neutrophils and monocyte/macrophages are potential sources of the enzymes. We have recently found significant differences in tetracycline inhibition between human neutrophil and fibroblast interstitial collagenases. To address the cellular source of collagenase present in gingival crevicular fluid in 2 distinct periodontal diseases, we studied the tetracycline inhibition of collagenase in gingival crevicular fluid of patients with localized juvenile periodontitis and adult periodontitis. Gingival crevicular fluid samples were collected from deep (greater than 5 mm) periodontal pockets and assayed for collagenase in the presence of 0-1000 microM doxycycline as well as a chemically modified tetracycline devoid of antimicrobial activity (4-de-dimethylaminotetracycline). The drug concentration required to inhibit 50% of collagenase activity (IC50) in localized juvenile periodontitis gingival crevicular fluid was 280 microM for doxycycline and 470 microM for 4-de-dimethylaminotetracycline. Significantly lower values, 10-20 microM, were obtained for collagenase in gingival crevicular fluid of patients with adult periodontitis. We propose that systemic tetracycline levels are efficient inhibitors of collagenase in gingival crevicular fluid in affected sites of patients with adult periodontitis but not of patients with localized juvenile periodontitis and that the fibroblast type interstitial collagenase is the predominant collagenase type in gingival crevicular fluid in affected sites of patients with localized juvenile periodontitis and the neutrophil collagenase in adult periodontitis gingival crevicular fluid.(ABSTRACT TRUNCATED AT 250 WORDS)

The anticollagenolytic potential of lymecycline in the long-term treatment of reactive arthritis.

OBJECTIVE: We sought to determine the antiinflammatory properties of lymecycline in the long-term treatment of reactive arthritis (ReA). METHODS: Quantitative assay of collagenase activity by densitometry after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. RESULTS: Therapeutic levels of lymecycline do not directly inhibit the activity of human neutrophil interstitial collagenase, but can prevent the oxidative activation of latent human neutrophil collagenase. CONCLUSION: This non-antimicrobial, anticollagenolytic property of lymecycline may contribute to its therapeutic efficacy in the treatment of patients with ReA.

Reactive oxygen species as regulators of human neutrophil and fibroblast interstitial collagenases.

The effects of various reactive oxygen species on latent human neutrophil and fibroblast-type interstitial collagenases were studied. Latent human neutrophil collagenases (proMMP-8) was efficiently activated by hypochlorous acid and hydrogen peroxide and less efficiently by the serine proteinases trypsin and chymotrypsin. Human plasmin and plasma kallikrein did not activate latent human neutrophil collagenase. The activation of latent human neutrophil collagenase by hypochlorous acid and hydrogen peroxide corresponded to the activation obtained with the other known non-proteolytic activators phenylmercuric chloride and gold thioglucose. The activation by hydrogen peroxide was inhibited by mannitol and desferoxamine, suggesting a localized Fenton-type reaction to be responsible for the generation of hydroxyl radical and/or hydroxyl radical-like reactive oxygen pathway of neutrophil procollagenase does not involve plasmin and plasma kallikrein, which are efficient proteolytic activators of latent fibroblast-type procollagenase (proMMP-1). Fibroblast procollagenase was also slightly activated by hypochlorous acid and gold thioglucose. Thus neutrophil procollagenase seems to prefer non-proteolytic means of activation and reactive oxygen species can be regarded as potent activators in vivo. Synovial-fluid neutrophils from rheumatoid arthritis patients were found to release collagenase in 30% active form when compared to same patients' peripheral blood neutrophils, which released collagenase in completely latent form. This may indicate that the triggering of neutrophil at the site of inflammation in vivo involves initial oxidative activation of collagenase upon the degranulation process.

Tetracycline inhibition identifies the cellular sources of collagenase in gingival crevicular fluid in different forms of periodontal diseases.

Tetracyclines have recently been shown to inhibit the activity of mammalian matrix metalloproteinases, i.e. type I collagenase (MMP-1) and type IV collagenase/gelatinase (MMP-2). The specificity of this effect, however, has not been examined in detail. In the present study, doxycycline (a clinically widely used commercial tetracycline) and 4-de-dimethylaminotetracycline (CMT-1, a chemically modified non-antimicrobial tetracycline) were tested, at a wide range of concentrations, for their ability to inhibit human neutrophil and fibroblast interstitial collagenases, which are distinct gene products, as well as collagenase in human gingival crevicular fluid (an inflammatory exudate in periodontal lesions) obtained from adult, juvenile and diabetic adult periodontitis patients. The concentrations of these two tetracyclines, required to inhibit 50% of the collagenase activity (IC50), were found to be 15-30 microM for purified human neutrophil collagenase as well as collagenase in gingival crevicular fluid of adult periodontitis patients and diabetic adult periodontitis patients, thus approximating in vivo therapeutic tetracycline levels. In contrast, the fibroblast collagenase and collagenase in gingival crevicular fluid of patients with juvenile periodontitis were relatively resistant to tetracycline inhibition: the IC50 for doxycycline and CMT-1 were 280 and 500 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)