Release Of Gelatinase Research Articles

Intracellular localization of a leukocyte adhesion glycoprotein family in the tertiary granules of human neutrophils

Stimulation of human neutrophils by the chemoattractant formyl-methionyl-leucyl-phenylalanine or the calcium ionophore A23187 induced increments in the cell surface expression of the α subunits of Mol (CD11b) and gp150, 95 (CD11c) and their common β subunit (CD18). These increases showed a good correlation with the extracellular release of gelatinase, a marker for tertiary granules in human neutrophils. Conversely, the cell surface expression of the α subunit of the lymphocyte function-associated antigen-1 or LFA-1 (CD11a) was not altered upon cell activation. By subcellular fractionation and immunoprecipitation studies, we have found that CD11b, CD11c and CD18 molecules were mainly localized in the membranes of tertiary granules, which were resolved from the specific and azurophilic granules as well as from the plasma membrane fraction.

Neutrophil C3bi receptors: formation of membrane clusters during cell triggering requires intracellular granules.

Video-intensification fluorescence microscopy has been used to study the cell surface distribution of the complement receptor (CR) for C3bi (CR3) on human neutrophils. Fluorescein- or rhodamine-labeled monoclonal IgG or Fab fragments of antireceptor antibody were used as probes of receptor localization. C3bi receptors are uniformly distributed on untreated cells. Glass coverslips were coated with lipopolysaccharide (LPS) and serum was added; the serum deposits complement components, including C3bi, on the surface. When neutrophils were adherent to these coverslips, receptors were found in large clusters, and a fraction of the fluorescence remained uniform. Double-labeling studies were conducted by first labeling with anti-CR3 followed by attachment to LPS/serum-treated slides. This, in turn, was followed by labeling with the antibody conjugated to a second fluorophore. These studies revealed that the CR3 clusters were predominantly new antigenic sites exposed after attachment to the LPS/serum-treated slides. To determine the contribution of granule-associated CR3, we have studied neutrophils defective in receptor up-regulation, neutrophil cytoplasts, and a stimulator of granule release, A23187. Neutrophils from a patient with specific granule deficiency were found to be defective in granular CR3 and did not form clusters on C3-modified surfaces. The patient's neutrophils were defective in CR3 up-regulation and enzyme release as shown by fluorescence flow cytometry and gelatinase release, respectively. Cytoplasts also failed to show CR3 clusters on LPS/serum-treated coverslips. Furthermore, neutrophils treated with A23187 demonstrated numerous CR3 clusters. We suggest that formation of CR3 membrane domains during immune recognition requires the participation of intracellular granules. We speculate that these domains are formed by fusion of CR3-bearing granules at local sites of adhesion.

Inhibition by nonsteroidal anti-inflammatory drugs of superoxide production and granule enzyme release by polymorphonuclear leukocytes stimulated with immune complexes or formyl-methionyl-leucyl-phenylalanine

The effects of nonsteroidal anti-inflammatory agents on superoxide production and granule enzyme release by human polymorphonuclear leukocytes stimulated with either formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)) or immune complexes were investigated. Cytochrome c reduction and the release of lysozyme, β-glucuronidase, myeloperoxidase and gelatinase were measured. Auranofin, phenylbutazone, sulfasalazine and the phospholipase A 2 inhibitor, 4-bromophenacyl bromide, strongly inhibited these responses in fMet-Leu-Phe stimulated cells, at concentrations below 50 μM. Indomethacin, piroxicam, mefenamic acid, primaquine and quinacrine at 50–250 μM were inhibitory. Up to 1 mM ibuprofen and chloroquine inhibited superoxide production but had little effect on degranulation. With cells stimulated by IgG aggregates (immune complexes), up to 1 mM ibuprofen, mefenamic acid and piroxicam did not inhibit either response. Indomethacin, phenylbutazone, sulfasalazine and primaquine inhibited, but considerably higher concentrations were required than with fMet-Leu-Phe. Quinacrine inhibited superoxide production equally well with both stimuli but inhibited enzyme release only with fMet-Leu-Phe. Only auranofin, 4-bromophenacyl bromide, and the weakly effective chloroquine exerted approximately the same effect with both stimuli. d-Penicillamine did not affect enzyme release with either stimulus and interfered in the superoxide assay. Gelatinase release induced by fMet-Leu-Phe was affected to the same extent, or slightly more, than release of the other granule enzymes. With immune complexes, there was only modest inhibition of gelatinase release by any of the drugs at 250–1000 μM. Our results reinforce previous observations that many anti-inflammatory drugs affect neutrophil functions, but their effects vary with stimulus. The relative insensitivity of immune complex-induced responses to most of the drugs must be taken into account when considering their mode of action.

Inhibition of neutrophil-mediated degradation of isolated basement membrane collagen by nonsteroidal antiinflammatory drugs that inhibit degranulation.

The ability of nonsteroidal antiinflammatory drugs to inhibit neutrophil-mediated degradation of type IV collagen in an in vitro tissue injury model using glomerular basement membrane (GBM) containing immune complexes was investigated. Auranofin (2.5-10 microM), phenylbutazone (50-250 microM), sulfasalazine (250-1,000 microM), and 4-bromophenacyl bromide (5-20 microM) each inhibited up to 70% of the collagen degradation, in parallel with almost complete inhibition of the release of azurophil and specific granule enzymes. These drugs had much less an effect on gelatinase release. Indomethacin and the antimalarials, which inhibited the neutrophil oxidative burst but not degranulation, had little effect on GBM collagen degradation. Our results do not necessarily imply that inhibition of neutrophil-mediated degradation of connective tissue is relevant to the action of nonsteroidal antiinflammatory drugs in vivo; however, using the GBM model system, we have shown that when a drug inhibits granule enzyme release, there is an associated decrease in collagen degradation, whereas inhibition of the oxidative burst has relatively little effect.

Intracellular organelle motility and membrane fusion processes in human neutrophils upon cell activation

Release subcellular fractionation experiments indicate that fusion of a novel tertiary granule with the plasma membrane is concomitant with human neutrophil activation. Phorbol 12-myristate 13-acetate (PMA) induced a respiratory burst in human neutrophils as well as a high release of gelatinase, a marker of the tertiary granule. Preincubation of neutrophils with cytochalasin E induced a partially activated or ‘primed’ state, in which cells were unable to generate superoxide anion, but showed a reduced latency period for this activity. Fusion of tertiary granules with the cell surface also occurred during priming, although to a lesser extent than in PMA stimulation. The rapid tertiary granule degranulation, preceding that of specifics azurophilics, seems to play an important role in the functionality and secretory properties of human neutrophils.

Platelet-activating factor as a stimulus of exocytosis in human neutrophils

The properties of platelet-activating factor (PAF-acether) 42–48ulus of exocytosis in human neutrophils have been re-investigated with particular attention to effects on cells that were not pretreated with cytochalasin B. Release of gelatinase, the most sensitive marker of exocytosis, was determined in addition to that of vitamin B-12-binding protein and β-glucuronidase. Superoxide production was assayed as a measure of the respiratory burst. The effects of PAF-acether were compared to those of leukotriene B 4 and N-formylmethionylleucylphenylalanine (fMet-Leu-Phe). Our results show that PAF-acether elicits marked secretion in untreated human neutrophils, and refute the prevalent view that cytochalasin B treatment is required for responsiveness. PAF-acether induced abundant release of gelatinase, increasing on average from 20% at 10 nM to 35% at 1 μM. This release was very rapid, i.e., almost complete after 2 min. fMet-Leu-Phe induced the same maximum response already at 0.1 μM, but release was considerably slower. Leukotriene B 4 was less potent with a maximum release of 20%. Exocytosis of gelatinase was always paralleled by liberation of smaller but significant amounts of vitamin B 12-binding protein from the specific granules. In contrast to their effect on exocytosis, PAF-acether and leukotriene B 4 were very weak stimuli of the respiratory burst when compared with fMet-Leu-Phe.

The activation of the human neutrophil respiratory burst occurs only at temperatures above 17°C: Evidence that activation requires membrane fusion

By considering the effects of temperature on the respiratory burst activity of human neutrophils stimulated by phorbol myristate acetate, we have found that activation only occurs at 17°C and above. Between 20 and 30°C, the rates of oxygen consumption rose dramatically in correspondence with cellular release of gelatinase and lactoferrin. Inasmuch as these mark tertiary and specific granules, respectively, a necessity for membrane fusion of one or both granules with the plasma membrane during triggering of the respiratory burst is very probable.

Secretion of Collagenolytic Enzymes by Human Polymorphonuclear Leukocytes

The polymorphonuclear leukocyte contains three proteinases capable of degrading the collagenous components of the connective tissue matrix. These proteinases, gelatinase, interstitial collagenase and elastase, were found to be rapidly secreted by the neutrophil in response to soluble stimuli with maximal accumulation of the gelatinase and interstitial collagenase occurring during a 20 minute incubation. When neutrophils were stimulated with the chemotactic peptide, formyl-met-leu-phe, gelatinase was the predominant collagenolytic enzyme detected. Stimulation of neutrophils with increasing doses of the Ca++ ionophore A23187 lead to a sequential release of collagenolytic proteinases. Gelatinase release was detected at Ca++ ionophore concentrations of .05-.1 μM, while significant release of interstitial collagenase required 0.5-1 μM A23187. Elastolytic activity was detected only at high concentrations of A23187 (5-10 μM). No release of lactic acid dehydrogenase was detected indicating that the enzyme release was not due to cell death. These studies suggest that the neutrophil may modulate its collagenolytic potential by selective release of collagenolytic proteinases.

Release of gelatinase from a novel secretory compartment of human neutrophils.

Gelatinase is a metallo-proteinase that acts specifically on denatured collagen. In human neutrophils, this enzyme is localized in small, morphologically still unidentified storage organelles that are resolved from the specific and the azurophil granules upon subcellular fractionation by differential sedimentation. When neutrophils isolated from freshly drawn blood are exposed to soluble stimuli such as N-formyl-methionyl-leucyl-phenylalanine, zymosan-activated serum, phorbol myristate acetate, or the calcium ionophore A 23187, or are induced to phagocytose opsonized zymosan, they rapidly release gelatinase in large amounts (30-70% of the cellular content in 10 min). When neutrophils from donor blood, which had been stored for 24 h at 4 degrees C are used, extensive release even occurs without added stimuli by simply warming to 37 degrees C. Gelatinase release appears to occur by secretion because it is not dependent on phagocytosis. It is paralelled by the release of specific granule contents (vitamin B(12)-binding protein), but is more rapid and much more extensive. It is, however, dissociated from the discharge of azurophil granules (as assessed by beta-glucuronidase). In addition, it was found that gelatinase release does not depend on the activation of the respiratory burst, although the two responses are often observed in parallel. Release is not due to cell damage as the cytoplasmic enzyme lactate dehydrogenase is fully retained. The distinct subcellular distribution and kinetics of release of gelatinase reported in this paper uncover a novel, truly secretory compartment of human neutrophils, which is highly responsive to stimulation. Gelatinase and possibly other enzymes stored in this secretory organelle may be involved in the early events of neutrophil mobilization, the response to chemotactic signals and diapedesis.