Herkko Saari

Specificity of the anticollagenase action of tetracyclines: relevance to their anti-inflammatory potential.

The concentrations of doxycycline and 4-de-dimethylaminotetracycline required to inhibit 50% of collagenase activity were found to be 15 to 30 microM for human neutrophil and gingival crevicular fluid collagenases. Fibroblast collagenase was relatively resistant to inhibition by tetracyclines; the 50% inhibitory concentrations of doxycycline and 4-de-dimethylaminotetracycline were 280 and 510 microM, respectively.

Differential effects of reactive oxygen species on native synovial fluid and purified human umbilical cord hyaluronate

The ability of reactive oxygen species produced by triggered neutrophilic leukocytes, hypoxanthine/xanthine oxidase (HX/XAO), hydrogen peroxide, and hypochlorous acid/mycloperoxidase (HOC1/MPO) systems to degrade hyaluronate (HA) in human synovial fluid (SF) and purified umbilical cord HA was compared by measuring the molecular weight distribution of HA using high-performance liquid chromatography with a size-exclusion column. The exposure of noninflammatory SF to phorbol myristic acetate (PMA) -activated neutrophils or to hydrogen peroxide (H2O2) caused depolymerization of SF HA to the degree corresponding to that found in rheumatoid SFs. When HX/XAO was used as radical generator, the molecular weight of SF HA decreased from 3.42×106 to 1.40 × 104 daltons with concomitant decrease of SF viscosity to 36% from the original value. The HOC1/MPO system caused no depolymerisation of SF HA, even at very high unphysiological HOC1 concentrations that induced the precipitation of SF HA together with SF proteins. This effect was found to be comparable to conventional mucin clot formation in SF. However, purified human umbilical cord HA was easily depolymerized with HOC1/ MPO or with H2O2, but these effects were sensitive to the hydroxyl radical scavenger mannitol and iron chelator desferrioxamine, indicating that the formation of reactive hydroxyl radical (OH) is likely to participate in these reactions. Thus we conclude that in inflammatory SF HA is mainly depolymerized by OH produced by decomposition of H2O2 catalyzed by iron, free or locally bound to HA itself. In contrast to what has been reported earlier, HOC1/MPO only depolymerizes purified umbilical cord HA (in a hydroxyl radical-dependent manner) but does not depolymerize HA in SF. As a matter of fact, HOC1/MPO has a scavenging action on SF HA by consuming H2O2 and thus preventing the formation of reactive hydroxyl radicals.

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 bodys 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.

Substrate Specificity and Activation Mechanisms of Collagenase from Human Rheumatoid Synovium

Substrate specificity studies of collagenase extracted from human rheumatoid synovium suggest that synovial pannus tissue overlying articular cartilage may not be particularly active in degradation of cartilage type II collagen, which, considering the poor inherent healing capacity of the articular hyaline cartilage, may exert a protective function against inadvertant tissue damage. Rheumatoid synovial tissue was also used to establish synovial fibroblast cell lines. Treatment of these cells in monolayer cultures with IL-1 leads to collagenase gene activation, increased collagenase production and an almost complete autoactivation of secreted collagenase. Interleukin-1 also activated stromelysin gene suggesting this as a possible mechanism effecting autoactivation. Latent human fibroblast and macrophage collagenase purified from culture medium were efficiently activated by phenylmercuric chloride but also by gold thioglucose, gold sodium thiomalate and HCIO. These new observations support the Cys73 switch activation mechanism. In contrast to neutrophil collagenase, the activation by gold(I) compounds and HCIO was associated with a change in the apparent molecular weight of the fibroblast procollagenase. In addition, gold(I) compounds rendered collagenase more susceptible to thermal denaturation. Thus the fibroblast-type interstitial collagenase, probably derived from fibroblast- and macrophage-like synoviocytes, seems to provide the predominant collagenolytic potential in human rheumatoid synovial tissue. Furthermore, the conditions in synovitis tissue may be such as to favor at least initial activation of collagenase synthesized and secreted in situ.

Conformational changes in cytochrome aa3 and ATP synthetase of the mitochondrial membrane and their role in mitochondrial energy transduction.

Summary1.The thermodynamics and molecular basis of energy-linked conformational changes in the cytochromeaa3 and ATP synthetase complexes of the mitochondrial membrane have been studied with spectrophotometrical and fluorometrical techniques.2.Ferric cytochromeaa3 exists in two conformations, high spin and low spin, the equilibrium between these states being controlled by the electrical potential difference across the mitochondrial membrane. The conformational change is brought about by an electrical fielddriven binding of one proton peraa3 to the complex. At pH 7.2 the concentration of the two conformations is equal at a membrane potential of 170 mV corresponding to about 4 kcal/mole.3.The high to low spin transition in ferricaa3 is also induced by hydrolysis of ATP in which case two molecules ofaa3 are shifted per ATP molecule hydrolyzed. This is in accordance with translocation of two protons across the mitochondrial membrane coupled to hydrolysis of ATP as proposed in the chemiosmotic theory of oxidative phosphorylation.4.The conformational transition in cytochromeaa3 is not an expression of the formation of a ‘high-energy’ intermediate or reversal of the energy-transducing pathway of oxidative phosphorylation, but is presumably the basis of allosteric control of the activity of cytochrome oxidase by the energy state of the mitochondrion. This control is exerted by a regulatory mechanism in which the electrical potential difference controls the conformation and redox properties of the heme centres and thereby the rate of oxygen consumption.5.The synthesis of one molecule of ATP by oxidative phosphorylation is energetically equivalent to the work done in carrying two electrical charges across the entire mitochondrial membrane.6.Fluorescence changes of aurovertin bound to ATP synthetase reveal that the electrical membrane potential induces a conformational change in the F1 portion of the enzyme which is probably associated with dissociation of the natural F1 inhibitor protein. This conformational change is energetically equivalent to the work done in carrying one electrical charge across the mitochondrial membrane.7.A model is proposed for the mechanism of the electrical field-induced conformational changes in the cytochromeaa3 and ATP synthetase complexes, and the significance of these changes in the mechanism and control of mitochondrial energy conservation is discussed.

Interaction of Ca2+ and H+ with heme A in cytochrome oxidase

Ca2+ ions shift the absorption spectrum of reduced cytochromea in mitochondria by acting from the outside of the membrane. In isolated cytochrome oxidase the shift may be induced by either Ca2+ or H+, the apparent pK varying between 6.20 and 5.75 depending on the state of cytochromea3. Studies of the Soret band show that Ca2+ also shifts the spectrum of ferrocytochromea3 in isolated oxidase in contrast to the situation in mitochondria or isolated oxidase reconstituted into liposomes. Model studies with reduced bis-imidazole heme A reveals an analogous spectral shift induced by Ca2+. Esterification of the propionate carboxyls of heme A abolishes the spectral shift, suggesting that it is due to interaction of Ca2+ with these groups. When taken together with the data with intact mitochondria, this suggests that the propionate side chains of cytochromea are accessible to Ca2+ and H+ from the outside of the mitochondrial membrane. In the soluble enzyme both hemesa anda3 are accessible. Thus hemea may be located near the outside of the inner membrane whereas hemea3 experiences a different environment in which no Ca2+ shift occurs.

Relationship between neuropeptide immunoreactive nerves and inflammatory cells in adjuvant arthritic rats

The purpose of this study was to assess the relationship of neuropeptide nerves and inflammatory leukocytes in PVG rats with adjuvant-induced arthritis. Substance P- and calcitonin gene-related peptide (CGRP)-immunoreactive nerves and inflammatory leukocytes were studied, using peroxidase (ABC) and/or alkaline phosphatase (APAAP) staining. Inflamed synovial tissue proper was infiltrated with neutrophils, ED1 macrophages and focal accumulations of CD2 T lymphocytes. In such tissue, the relationship between peptide-immunoreactive nerves and inflammatory cells was such that substance P and CGRP nerves were absent in heavily infiltrated villous synovial tissue, whereas healthy synovial tissue and non-inflammatory areas in adjuvant arthritic rats were innervated by substance P and CGRP nerves close to normal synovial tissue resident cells. In order to elucidate an eventual mechanism for lost immunoreactivity, healthy synovial tissue was exposed to chymotrypsin or oxygen derived free radicals (ODFR) in vitro. The former treatment caused total loss of immunoreactivity. These findings suggest that neuropeptides and neuropeptide containing nerves may be destroyed by locally produced proteolytic enzymes and various reactive oxygen species in the vicinity of inflammatory cells.

Role of mesenchymal collagenase in the loosening of total hip prosthesis.

Fibroblast-type interstitial collagenase (E.C. 3.4.24.7) was associated with loosening of total hip prostheses in eight patients: there were four cemented stems and one cementless stem with the common type of loosening and two cemented stems and one cementless acetabular component with aggressive granulomatous lesions. The authors used a specific, well-characterized, heterologous, affinity-purified, polyclonal rabbit anti-human fibro-blast collagenase antiserum applied in avidin-biotin-peroxidase-complex (ABC) staining. In the aggressive granulomatous type of loosening, collagenase was found in most of the fibroblast- and macrophage like cells, including multinuclear giant cells and epitheloid cells in periprosthetic tissue. Collagenase-positive cells also were found in the periprosthetic tissue associated with common loosening. Collagenase was also found in capillary and postcapillary venule endothelial cells in the richly vascularized aggressive granulomatous tissue. Collagenase was extracted directly from the tissue samples and incubated with soluble Type I collagen. Collagen degradation products then were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and the three-fourths length degradation product quantitated by gel scanning densitometry. In both aggressive granulomatosis and the common type of loosening, ex-tractable collagenase was found in tissue. No significant differences between the sample groups were detected in respect to total measurable collagenase, however. The extractable collagenase was present in a latent form that could be activated by the organomercurial procollagenase activator, phenylmercuric chloride (PMC). It is likely that interstitial collagenase contributes to rapid growth of reactive infiltrative tissue, loosening of the prosthesis associated with aggressive granulomatosis, and the periprosthetic lytic process associated with the common type of hip prosthesis loosening.

Collagenase Reserves in Polymorphonuclear Neutrophil Leukocytes from Synovial Fluid and Peripheral Blood of Patients with Rheumatoid Arthritis

Degradation of cartilage in rheumatoid arthritis (RA) may be in part due to release of collagenase from specific granules of polymorphonuclear neutrophil leukocytes (PMNs) during degranulation. We decided to study, not synovial fluid (SF) collagenase, but PMN collagenase reserves. PMN were isolated from parallel SF and peripheral blood (PB) samples obtained from 7-arthritis patients. PMNs were stimulated in vitro by tetradecanoyl-phorbol-13-acetate (TPA). Collagenase activity in the supernatant without and with phenylmercuric chloride activation was studied. Compared to PB PMNs, there was no consistent decrease in the total collagenase reserves in the inflammatory SF PMNs. This suggests that the release of collagenase in the inflammatory synovial fluid does not deplete SF PMNs of the collagenase synthesized at the myelocyte stage. The role of PMN collagenase in pathogenesis of cartilage destruction would then seem to be more dependent on local release and autoactivation at cartilage surface by adherent PMNs and not excessive collagenase release from free floating SF PMNs at single cell level. Furthermore, under the experimental conditions used the proportion of collagenase released in active form was higher in SF PMN specimens than in PB PMN specimens (p less than 0.01). The predominant collagenous component of adult cartilage, native type II collagen, was degraded by PMN collagenase as fast as native type I collagen. These findings suggest an important role for this enzyme in destruction of the free cartilage surface in RA.

Salivary mucous glycoprotein MG1 in Sjögren's syndrome.

The aim of the study was to develop and apply a rapid method for the simultaneous analysis of the concentration and molecular weight of the human high-molecular weight mucin MG1 in small volumes of unprocessed saliva from healthy controls and from patients with Sjögrens syndrome (SS). In high performance liquid chromatography (HPLC) with a TSK 5000 PW size exclusion column, MG1 eluted with a retention time 10.6 min corresponding to a M(r) of 2 to 2.5 x 10(6). Molecular weight changes under various experimental conditions are compatible with the suggestion that the MG1 complex is composed of four 660 x 10(3) glycosylated subunits connected by disulphide bridges and associated with a 25-35 x 10(3) Da link protein. In SS the molecular weight of MG1 was normal and its concentration was high in resting (190 vs. 70 micrograms/ml, P = 0.001) but not in stimulated (46 vs. 48 micrograms/ml, P > 0.05) saliva; MG1 concentration in resting SS saliva did not vary in parallel with protein and the interindividual differences were considerable. Size exclusion HPLC is a rapid and reproducible method suitable for isolation and analysis of salivary MG1 from small volumes of unprocessed samples. The molecular weight or subunit structure of MG1 were not altered in SS. The high concentration of MG1 in resting saliva in SS, may be explained by the concentration effect, or alternatively by the low water retaining capacity, which may play an important pathogenic role in xerostomia of SS.