Activation of neutrophil collagenase by cathepsin G

Collagenase is secreted from neutrophils as a latent or proenzyme. In an effort to understand the mechanism of collagenase activation in inflammation, human peripheral neutrophils (PMNs) were isolated and incubated with the tumor promotor, phorbol myristate acetate (PMA), which induces the neutrophils to degranulate and secrete proteinases. Neutrophil media were then treated with various activators or inhibitors of collagenase and other proteinases, and the collagenase activity was measured. A serine proteinase secreted from neutrophils, cathepsin G, was found to activate latent collagenase, but it was also found to require activation itself. Both hypochlorous acid (HOCl) and oxidized glutathione (GSSG) were tested for their coilagenase-activating ability and were found to be successful only in the presence of active cathepsin G. A specific cathepsin G inhibitor (0.5 mM Z-Gly-Leu-Phe-CH₂Cl) prevented the activation of latent collagenase by HOCl. To confirm these results, purified neutrophil cathepsin G was incubated with a neutrophil proteinase mixture which contained latent collagenase. The collagenase was shown to be activated upon incubation with purified cathepsin G. These results indicate that cathepsin G is a key mediator in neutrophil collagenase activation.     

Influence of human plasma kallikrein on lysosomal enzyme release from polymorphonuclear leukocytes

Stimulation of polymorphonuclear leukocytes with kallikrein demonstrated that enzyme acts selectively on the release of lysosomal enzymes of these cells. The release of collagenase, similarly to the release of lysozyme into the incubation medium increased proportionally to kallikrein concentration and the duration of incubation. Kallikrein had a small effect on -glucuronidase secretion. No effect on cytoplasm lactate dehydrogenase release was detected. These results suggest that kallikrein, as a soluble stimulus, predominantly induces degranulation of specific granules containing collagenase capable of degrading the connective tissue. Secretion of lysozyme and collagenase requires the presence of active kallikrein. Soybean trypsin inhibitor diminished the enzyme release.     

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.     

Activation of neutrophil collagenase in periodontitis

Neutrophil collagenase (matrix metalloproteinase 8 [MMP-8]) is an important mediator of tissue destruction in inflammatory diseases. Studies of anaerobic periodontal infections have shown that active MMP-8 in gingival crevicular fluid is associated with the degradation of periodontal tissues in progressive periodontitis whereas the latent enzyme is predominant in gingivitis. Since the activation of MMP-8 appears to be a crucial step in periodontitis, we have examined the activation of MMP-8 in gingival crevicular fluid samples by using a soluble biotinylated collagen substrate. Analysis of gingival crevicular fluid in periodontitis, gingivitis, and controls revealed sixfold (P < 0.001)-higher levels of active collagenase in periodontitis (n = 12) samples compared to gingivitis (n = 17) samples, which exhibited low levels of activity, while controls (n = 25) showed no activity. After gingival crevicular fluid was collected, no further activation of latent collagenase occurred in vitro. Although both MMP-1 and MMP-8, but not MMP-13, could be detected by immunoblots, blocking antibodies to MMP-1 showed that collagenase activity was largely contributed by MMP-8, which was localized to the matrix of diseased tissues. The MMP-8 in gingival crevicular fluid migrated primarily as a 60-kDa form with smaller amounts of a 78-kDa species, whereas MMP-8 isolated from peripheral neutrophils migrated at 70 and 89 kDa, corresponding to active and latent forms of the enzyme, respectively. Most of the MMP-8 in the 60- and 70-kDa bands selectively bound to tissue inhibitor of metalloproteinase 2 and collagen, indicating that most, but not all, of the enzyme in these bands was in an activated form. However, the amounts of the 78- and 60-kDa forms from gingival crevicular fluid in different samples did not correlate (r2 = 0.028) with the latent and active enzyme measured by collagenase assay. Collectively, these studies have identified distinct forms of latent and active MMP-8 in gingival crevicular fluid that appear to result from a unique activation mechanism that occurs in periodontitis. The complexity of MMP-8 activation is further indicated by the presence of latent, activated, and superactivated forms of MMP-8 in the 60- and 70-kDa bands obtained from gingival crevicular fluid and neutrophil samples, respectively.     

Effect of normal and atheromatous aortic tissue on platelet aggregation in vitro

An investigation was made into the platelet-aggregating ability of human aortic tissue. It was found that potent aggregating substances are present in the vascular wall but that the degree and character of platelet aggregation produced was dependent on the method employed in the preparation of the tissue for study.The supernatants of aortic tissue homogenates were found to produce potent irreversible platelet aggregation that occurred after a latent period of one to two minutes. The activity was heat labile, destroyed by collagenase, sedimented by ultracentrifugation, and was considered to be identical or similar to collagen. An extract obtained by boiling aortic tissue with saline was found to cause less powerful reversible platelet aggregation that occurred within 15 to 30 seconds' contact with platelets; the activity was not affected by heating, incubation with collagenase, or ultracentrifugation, and was considered to be due to adenosine diphosphate. Atheromatous aortic tissue when extracted by either method was found to contain less than one half of the potency of normal tissue in causing platelet aggregation.     

Antibody reactivity to human and vervet cytomegalovirus early antigen(s) in sera from patients with active cytomegalovirus infections and from asymptomatic donors.

Human fibroblasts were infected with vervet cytomegalovirus (VCMV) and cultured in medium containing 50 micrograms of cytosine arabinoside per ml for 72 h. Early antigens (EAg) were detected in the nuclei of infected cells by an indirect fluorescent antibody test with human sera having antibody to EAg of human cytomegalovirus (HCMV). Sera from patients with serological and/or virological evidence of active HCMV infection and from asymptomatic blood donors were examined for antibody activity with the HCMV and VCMV EAg's. The HCMV and VCMV EAg's were comparable in detecting levels of antibody activity and fluctuations in antibody titer of paired sera from patients. A total of 81% of patient sera reactive with HCMV EAg were also reactive with VCMV EAg. In contrast, only 14% of the asymptomatic donor sera reactive with HCMV EAg were also reactive with VCMV EAg. The VCMV EAg, therefore, appeared to differentiate latent from active infections in humans more effectively than did HCMV EAg.     

Mononuclear Cell Factors that Inhibit Fibroblast Collagen Synthesis

Supernatants harvested from peripheral blood mononuclear cells (PBMC) incubated either with the non-specific mitogen phytohaemagglutinin-P (PHA) or with the specific antigen tuberculin purified protein derivative for 72 h decreased collagen synthesis by dermal fibroblasts. PHA-induced mononuclear cell factors (PHA-MCF) responsible for decreases in collagen synthesis by dermal fibroblasts were localized by column chromatography on Sephacryl S-200 to fractions of 30,000-60,000 daltons. Proteolytic enzymes destroyed the activity of PHA-MCF, but after incubation with neuraminidase some activity of these factors remained. The activity of PHA-MCF was not inhibited by incubation with the monosaccharides L-fucose, L-rhamnose, N-acetylglucosamine, and alpha-methyl-D-mannoside but was partially destroyed by heating at 80 degrees C for 10 min. The factors were not mitogenic to PBMC. These factors did not appear to resemble any previously characterized factors produced by non-adherent mononuclear cells. The mechanism by which these factors decreased fibroblast collagen synthesis appeared complex. There was no detectable increase in the release of collagenase by fibroblasts, nor was a cytotoxic effect apparent. Increased PGE2 production by fibroblasts could not be related to the factor-induced decrease in fibroblast collagen synthesis.     

Polymorphonuclear leukocyte collagenase in carrageenin-induced inflammation: effect of nonsteroidal antiinflammatory drugs

Experimental pleurisy induced by carrageenin in rats caused an accumulation of PMN-leukocytes in the pleural cavity and changes in the blood leukocyte population. During the course of the inflammation involved, up to 90-98% activation of latent collagenase was observed in blood PMN-leukocytes and in leukocytes obtained from pleural exudate. The NSAIDs tested for their effect on this process had significant inhibitory effects on both exudate and leukocyte accumulation as well as on the activation of latent collagenase. Timegadine, medosan and sulindac were most effective in preventing collagenase activation, felden and naproxen were less effective, and peroxinorm was without an influence, whereas indomethacin destroyed collagenase activity. These results suggest that the protective effects of NSAIDs against the activation of latent collagenase could be one of their therapeutic actions.     

The effects of levamisole on some functions of mouse macrophages after in vitro and in vivo administration.

In vitro incubation of peritoneal macrophages from normal, peptone-stimulated mice with levamisole (1-100 microM) for 1 and 22 h had no effect on either phagocytosis of particulate material (sheep erythrocytes, zymosan) or cellular levels and release of lysosomal enzymes (beta-D-glucuronidase, cathepsin D). By contrast, levamisole 1 and 5 mM dramatically increased enzyme release while inhibiting phagocytosis. In some experiments, however, these high concentrations of levamisole caused an elevated cell mortality. When incubation was extended to 72 h, a decrease of both phagocytosis and enzyme release was observed. The catabolism of endocytosed antigens (sheep erythrocytes, human gammaglobulin) was not at all or only slightly modified depending upon the antigen. The cellular level of cyclic AMP remained unchanged in all experiments. In vivo exposure of macrophages to levamisole (2.5 and 10 mg/kg/day i.p. for 3 days) produced a dose-dependent increase in processing of endocytosed antigens as shown by an enhanced transfer of initially endocytosed material to the macrophage plasma membrane. The other parameters were not modified. The immunogenicity of erythrocytes, when endocytosed by levamisole-treated macrophages and transferred into unsensitized recipients, was increased in some in vivo experiments.     

Asbestos fibers induce release of collagenase by human polymorphonuclear leukocytes

The asbestos fibers chrysotile and crocidolite cause a dose-dependent release of specific granule collagenase by human polymorphonuclear leukocytes (PMNL). Release of azurophil granule elastase was induced by the asbestos fibers at higher concentrations, suggesting that asbestos fibers primarily cause the release of specific granule contents of human PMNL. Wollastonite, a fibrous silicate mineral, causes a weaker collagenase release and no elastase release. The collagenase was released in inactive, latent form. Carboxymethyl cellulose (CMC), an agent known to blunt chrysotile-induced hemolysis and production of reactive oxygen metabolites by human PMNL, specifically inhibits chrysotile-induced release of collagenase. Chrysotile asbestos was found to bind the PMNL serine proteinase cathepsin G. A role of collagenase release, production of reactive oxygen metabolites and cathepsin G binding by chrysotile for the perpetuation of the asbestos-induced alveolitis is suggested.     

Evidence that superoxide-anion, produced by PMA-activated human polymorphonuclear leukocytes, is the cytolytic agent for rabbit, but not for sheep red blood cells

The lysis of sheep and rabbit red blood cells (SRBC and RRBC, respectively) upon exposure to PMA-activated human polymorphonuclear leukocytes (PMN) was investigated. The lysis of these target cells, which was measured by the release of 51Cr, showed different kinetics and scavenger-sensitivity. The lysis of RRBC, which was already detectable within 45 min of incubation, was sensitive to superoxide dismutase (SOD), but was only poorly influenced by scavengers of hydroxyl radical formation, such as desferal or thiourea. In contrast, lysis of SRBC was first detectable after 90 to 135 min of incubation and sensitive to desferal and thiourea, but not to SOD. Finally, only RRBC were sensitive to the artificial superoxide-generating system hypoxanthine/xanthine oxidase. Taken together, these data point at a cytolytic activity of superoxide anion O2- towards RRBC. SRBC are relatively resistant to O2-, but are lysed by an H2O2- and hydroxyl radical-dependent process.     

Histamine, 5-hydroxytryptamine and compound 48/80 activate PMN-leucocyte collagenase of the rat

Experimental pleurisy in rat and intraperitoneal injection of histamine, serotonin and the histamine releaser compound 48/80 caused changes in blood leucocyte populations. An increased number of PMN-leucocytes was observed.The peripheral blood PMN-leucocytes of non-treated rats contained collagenase mainly in the latent form. During experimental pleurisy and after treatment with histamine, serotonin and compound 48/80 activation of latent collagenase of up to 90–98% was obtained. Mepyramine, cimetidine and timegadine inhibited activation of latent collagenase by compound 48/80. It is suggested that histamine is involved in the process of activation of leucocyte collagenase in the course of inflammation.     

Influence of suckling and feeding on insulin, gastrin, somatostatin and VIP levels in peripheral venous blood of lactating sows

Blood samples were collected in peripheral venous blood of seven lactating sows, when their piglets were suckling. In four of the experiments samples were also taken when the sows were fed a meal. Gastrin, insulin, somatostatin and VIP levels were measured by radioimmunoassay. Insulin levels increased by approximately 100% for about 10 min in response to suckling, in some experiments even before the suckling occurred, i.e. when the sows saw, heard and smelled their piglets. In four of the sows suckling caused a biphasic twofold increase in gastrin levels - one immediate peak which lasted for a few min and a second peak of longer duration (about 30-60 min), whereas gastrin levels remained unchanged in three animals. Somatostatin levels usually reflected gastrin levels in a reciprocal way. Thus, a biphasic decrease of somatostatin levels occurred in the high gastrin responders. In contrast, somatostatin levels increased in the experiments, in which gastrin levels did not change. Immediate and short-lasting (a few minutes long) increases of VIP levels were also induced by suckling. Large litters and long suckling periods appeared to be related to greater changes of the levels of all the peptides measured. Feeding influenced insulin, gastrin and somatostatin levels in the same way as did suckling from both a qualitative and a quantitative point of view. In contrast, VIP levels were not increased by feeding. The possible functional effects of the suckling-induced release of gastrointestinal hormones and possible mechanisms of their release are discussed.     

Endocytosis of cationized ferritin to vesicles in the Golgi region of the glomus cells dissociated from the adult rat carotid body

Glomus cells were dissociated from the carotid bodies of adult rats by enzymatic digestion with collagenase. The cells were then incubated at 37 degrees C for 30 minutes to 3 hours in the continuous presence of cationized ferritin (CF) as a membrane marker and extracellular tracer to study the intracellular route of endocytosis in this cell type. After 30 minutes of incubation with CF, occasional solitary CF-containing vesicles were observed at the cell periphery and also in the Golgi region. After 2-3 hours of incubation with CF, cell viability was still preserved and CF-labeled vesicles were abundant in the Golgi region. CF particles were also seen in some vesicles having a dense core. The core of these labeled vesicles appeared to be less electron-dense than that of typical secretory granules. It is suggested that the Golgi apparatus is involved in membrane recycling in glomus cells and that the membrane is then possibly further transported to an immature type of storage vesicle for reusage.     

Cleavage of membrane-bound C3b and C3bi by viable human neutrophils (PMN)

Cleavage of C3 by purified leukocyte enzymes and crude extracts of human polymorphonuclear leukocyte (PMN) granules has been reported. We demonstrate that viable PMN mediate the cleavage of erythrocyte-bound C3b and C3bi via cell-associated proteases. Greater than 50% of 125IC3(x) was released from EAC43bix during a 5-min incubation with viable PMN at 37 degrees C. More than a 30-min incubation was required for substantial release from EAC43bx. Culture fluids from PMN suspensions had limited cleaving ability; cleavage of cell-bound C3bx and C3bix was only partially reduced when PMN were preincubated with high levels of soluble C3 which completely blocked EAC43b rosettes. Thus, cell-to-cell contact between opsonized erythrocytes and viable PMN with surface-associated proteases are responsible for cleavage of these opsonic sites. The effect of defined protease inhibitors on PMN cleaving activity as well as on purified leukocyte elastase was examined. Phenylmethylsulfonyl fluoride (PMSF) and the leukocyte elastase inhibitor, methoxy-succinate-alanine-alanine-valine-chloromethyl ketone (MeO) each inhibited cleavage of C3b by 90% and C3bi by 60%. In contrast, the cathepsin-G inhibitor, benzyloxy-carbonyl-glycine-leucine-phenylalanine-chloromethyl ketone (Z) inhibited C3b and C3bi cleavage by less than 20 and less than 5%, respectively. Ethylenediaminetetra-acetate (EDTA), which had a minimal effect on soluble leukocyte elastase, also inhibited PMN-related release. Thus, elastase appeared to be the principle but not the only enzyme responsible for cleavage of C3b and C3bi. PMSF and MeO had a minimal effect on the activity of purified C3bINA (Factor I); and PMN-mediated release of C3b fragments was not inhibited by anti-Factor I and anti-beta 1H (Factor H) IgG and Fab. Thus, these control proteins are not involved in the PMN-mediated cleavage under study. PMN-mediated cleavage of C3b was also inhibited when PMSF- and MeO-treated PMN were washed to remove the fluid phase phase protease inhibitor before adding EAC43b. This suggests that proteases localized in the PMN membrane, prior to the adherence of EAC43b, are responsible for C3b cleavage. Normal human serum was effective in blocking PMN-mediated release activity, while serum from alpha 1 antitrypsin-deficient patients was minimally effective. This suggests a mechanism for the in vivo regulation of PMN-mediated release of C3b and C3bi from opsonized particles by the natural plasma protease inhibitors.     

The antigen-induced degranulation of basophil leukocytes from atopic subjects studied by electron microscopy.

Suspensions of washed leukocytes were prepared from the blood of atopic subjects and incubated with diluent, ragweed antigen E or rye grass group I antigen. Histamine release into the suspending medium was measured and directly correlated with changes in the ultrastructure of basophil leukocytes from the same tubes. Incubation of leukocytes with either diluent or an antigen to which the donor was not hypersensitive caused no significant histamine release and the morphology of the basophils was unaltered. By contrast, incubation of leukocytes with an intigen to which the donor was hypersensitive caused substantial histamine release and characteristic morphologic changes in the basophils, many of which underwent exocytotic degranulation. Degranulated human basophils showed the following features: (1) an irregular surface, to which platelets and leukocytes were often adherent; (2) reduction in the number of basophilic granules; (3) residual granular material, both in exocytotic cavities and at the cell surface; (4) coated vesicles and cisternae of smooth endoplasmic reticulum, frequently related to the plasma membrane at sites of exocytosis; (5) thin membrane-bounded granules appeared unaltered; (6) no consistent change was observed in centrioles, microtubules, 90 A filaments, mitochondria, or multivesicular bodies, and degranulated basophils appeared ultrastructurally viable; (7) in reaction cell suspensions, nondegranulated basophils often showed a more active contour than control cells; both reacting but nondegranulated basophils and degranulated basophils sometimes showed localized filamentous webs at their periphery.     

Immunogenicity and specificity of collagen: IX. Resistance of different antigenic determinants of calf collagen to proteolytic treatment

Serologically active collagen peptides obtained after trypsin or collagenase treatment were further digested by different proteases. The influence of degradation on the inhibiting activity was studied with collagen antibodies possessing three different restricted specificities. Structures responsible for general collagen specificity proved to be highly resistant to the action of different proteases except for collagenase. This suggests the location of this specificity in the helical apolar sequences of collagen. Quite in contrast species-specific structures appeared very susceptible to the further degradations used.     

Desensitization of beta2-adrenoceptor and hypersensitization to phosphodiesterase inhibitors elicited by beta2-agonists in guinea pig eosinophils

BACKGROUND: Although the existence of functional beta(2)-adrenoceptor on eosinophils has been reported, the effects of desensitization of beta(2)-adrenoceptors on eosinophils have not been well documented. OBJECTIVE: The effects of desensitization of beta(2)-adrenoceptors on the degranulation of guinea pig eosinophils were investigated. METHODS: Guinea pig eosinophils were stimulated with the calcium ionophore A23187, and eosinophil peroxidase (EPO) release was determined. Changes in intracellular cyclic adenosine monophosphate (cAMP) levels were also measured. RESULTS: A23187-induced EPO release from guinea pig eosinophils was inhibited in a concentration-dependent manner by pretreatment for 5 minutes with fenoterol, clenbuterol, and salbutamol. Such effects of beta(2)-agonists were abolished by pretreatment with KT5720, an inhibitor of protein kinase A. Desensitization of the inhibitory effects of beta(2)-agonists was observed when the incubation time was prolonged. Fenoterol (10(-6) mol/L) induced almost complete desensitization after 120 minutes of incubation, whereas clenbuterol did not bring about significant desensitization. The inhibitory effects of fenoterol and clenbuterol on A23187-induced EPO release were correlated with increases in the intracellular cAMP levels evoked by either compound. After incubation of eosinophils with 10(-6) mol/L fenoterol for 120 minutes to induce complete desensitization of beta(2)-adrenoceptors, the inhibitory effects of theophylline and rolipram were increased by about 100-fold in the desensitized cells, although the effects of forskolin and dibutyryl cAMP were not affected by beta(2)-adrenoceptor desensitization. CONCLUSIONS: Prolonged incubation with beta(2)-agonists induced desensitization of beta(2)-adrenoceptors. Also, we postulated that hypersensitization of phosphodiesterase to its inhibitors occurs in beta(2)-adrenoceptor-desensitized guinea pig eosinophils.     

In-vitro studies of the potential role of neutrophils in the process of menstruation

Significant numbers of neutrophils are found extravascularly within the endometrium only during the immediate premenstrual and menstrual phases of the cycle. In this study we investigated the effect of neutrophil products on the synthesis and activation of matrix metalloproteinases (MMP), enzymes considered to play a crucial role in the degradation of endometrial tissue that occurs at menstruation. Latent MMP-2, MMP-3 and MMP-9 released by endometrial stromal fibroblasts and peripheral blood neutrophils were activated when the two cell types were cultured together. Tissue inhibitors of metalloproteinases (TIMP) 1 and 2 were also degraded in this system. Neutralization studies identified a role for the serine protease, elastase, in the observed activation of MMP. Although cultured endometrial neutrophils behaved similarly to peripheral blood neutrophils in their ability to release latent MMP-9 and elastase, no active forms of MMP-2. MMP-3 and MMP-9 were detected in supernatant from co-cultures containing endometrial neutrophils and stromal fibroblasts. This appeared to be due to an alteration in the neutrophil production of elastase and inhibitors. e.g. alpha1-antitrypsin, in these cultures so that active elastase was not available. Our results demonstrate that any involvement of neutrophils in the tissue destruction occurring at menstruation may be tightly regulated by the focal concentration of degradative enzymes and their respective inhibitors.     

Serum and lymph-node collagenase in sarcoidosis. Comparison with angiotensin-converting enzyme.

Serum collagenase was significantly elevated in stage II, but not stage I sarcoidosis, in contrast to elevation of serum angiotensin-converting enzyme in stages I and II. Neither serum collagenase nor angiotensin-converting enzyme was elevated in active tuberculosis. Elevated serum collagenase and angiotensin-converting enzyme levels were decreased with steroid therapy. Serum collagenase and angiotensin-converting enzyme were significantly correlated but did not vary identically. Collagenase was not elevated in lymph nodes in sarcoidosis, in contrast to marked elevation of angiotensin-converting enzyme. The results suggest that serum angiotensin-converting enzyme is a more sensitive index of sarcoidosis activity than serum collagenase, which may have an ancillary role in assessment of the disease.