Regulation of enzyme release from human polymorphonuclear leukocytes: further evidence for the independent regulation of granule subpopulations

Addition of 5-20 mM LiCl to purified human polymorphonuclear leukocytes led to the release of lysozyme, the specific granule constituent, but not the release of elastase which is in azurophilic granules. In contrast, 2.5-10 micrograms cytochalasin D/mL induced the release of both lysozyme and elastase. Addition of lipopolysaccharide to leukocytes did not induce enzyme release but primed cells for enhanced release induced by cytochalasin D. Lipopolysaccharide also primed cells for enhanced release of lysozyme by either N-formylmethionylleucylphenylalanine (fMLP) or Li+ but did not prime cells for elastase release by these stimuli. In contrast, fMLP + cytochalasin D interacted synergistically, leading to enhanced elastase release but not lysozyme release from the cells. Additional experiments with combinations of secretagogues and lipopolysaccharide yielded results consistent with the hypothesis that specific granules and subpopulations of azurophilic granules are under separate regulation and, thus, may be influenced by separate elements of intracellular second messenger systems.     

Release of proteinases from stimulated polymorphonuclear leukocytes. Evidence for subclasses of the main granule types and their association with cytoskeletal components

The secretion of matrix-degrading proteinases and protein components involved in the production of cytotoxic metabolites is an important step in the sequence of defense reactions executed by polymorphonuclear leukocytes (PMNL) in response to stimulation. In the present report, we have analyzed degranulation of PMNL stimulated either with soluble synthetic peptides fLeu-Phe (fMet, formylmethionyl), or fAhx-Leu-Phe-Ahx-Tyr-Phe (Ahx, aminohexyl) which trigger chemotaxis and degranulation, or with opsonized zymosan which induces phagocytosis. The release of elastase, myeloperoxidase and lactoferrin-containing granules was not at all or only slightly (less than 6%) induced either by fAhx-Leu-Phe-Ahx-Tyr-Leu or by zymosan particles. In contrast, type-I collagenase and gelatinase were secreted in significant amounts after treatment with these agents. The disruption of microfilaments by cytochalasin B and subsequent stimulation of PMNL with the formyl-peptide led to the secretion of elastase, myeloperoxidase and lactoferrin, and enhanced the release of gelatinase. Disruption of microtubules by incubation with colcemid resulted in inhibition of fAhx-Leu-Phe-Ahx-Thyr-Leu and fAhx-Leu-Phe-Ahx-Tyr-Leu/cytochalasin-B-induced granule release. Furthermore, we found different patterns of enzyme distribution after fractionation by centrifugation: most (greater than 90%) type-I collagenase and gelatinase was measured in the supernatant whereas 60-90% of elastase, myeloperoxidase and lactoferrin had partitioned into the cytoskeleton-containing pellet. Our results suggest that the two main types of secretory vesicles identified in PMNL (specific and azurophilic granules) consist of subpopulations. The differential association of the various types of granules with cytoskeletal elements may serve to control their sequential discharge.     

Identification of elastase in human eosinophils: immunolocalization, isolation, and partial characterization.

Although an elastolytic activity in eosinophil-rich cell fractions from mice has been reported, this enzyme has not been purified and characterized as yet in any mammalian species. Eosinophilic elastase was isolated from human eosinophil fragments (cytosomes) obtained from normal and eosinophilic subjects. The enzyme was purified to apparent electrophoretic homogeneity by fast protein liquid chromatography. The enzyme shows the same physical properties of the major elastase isoenzyme of human neutrophils. In addition, like monocyte elastase, it reacts with a monoclonal antibody against human neutrophil elastase. The biochemical similarities observed between the above-mentioned enzymes and the immunolocalization findings strongly support the idea that human eosinophils and neutrophils contain the same enzyme activity. Eosinophils show immunoreactive material in both types of dense cytoplasmic granules. This observation supports the current hypothesis that the different types of eosinophilic granules represent successive morphological stages of maturation.     

Elastase and cathepsin G of human monocytes: heterogeneity and subcellular localization to peroxidase-positive granules

We used antibodies to human leukocyte ("neutrophil") elastase and cathepsin G to localize the corresponding antigens in human neutrophils, monocytes, and alveolar macrophages by immunohistochemistry. Furthermore, we combined immunogold localization with enzyme histochemistry to localize proteinase antigens and endogenous peroxidase activity in the same sections. As expected, all neutrophils contained both elastase and cathepsin G, and the proteinases localized to granules with peroxidase activity. In contrast, marked heterogeneity in monocyte staining for elastase, cathepsin G, and endogenous peroxidase was found. Sixty percent or more were unstained, while the remainder varied greatly in staining intensity. The elastase and cathepsin G in monocytes were localized by immunoelectron microscopy, combined with histochemistry, to cytoplasmic granules which had peroxidase activity. Alveolar macrophages were unstained. Therefore, a subpopulation of peripheral blood monocytes contains leukocyte elastase and cathepsin G in a cell compartment from which these enzymes may potentially be released into the extracellular space. The occurrence of peroxidase and neutral proteinases in the same granules in monocytes could permit the H2O2-myeloperoxidase-halide system and the neutral proteinases to act in concert in such functions as microbe killing and extracellular proteolysis.     

Variations in elastaselike esterase activities in human leucocytes during cell maturation.

Granules of human peripheral blood leucocytes contain four well-characterized elastase isozymes and one or two slow-moving elastaselike esterases (SE) which have not been as well characterized. SE are capable of hydrolyzing typical elastase synthetic sybstrates such as N-acetyl-dl-alanine-alpha-naphthyl ester (Ac-DL-Ala-1-ONap) and N-t-butyloxycarbonyl-L-alanine-p-nitrophenyl ester (Boc-Ala-ONp), but unlike the highly basic elastase isozymes, SE barely migrate into 13% acrylamide gels during cationic electrophoresis at pH 4.3. Hydrolysis of Ac-DL-Ala-1-ONap by SE requires the presence of Triton in the gel, and hydrolysis of Boc-Ala-ONp by the same enzyme(s) is also enhanced in the presence of the detergent. Triton is not required for these activities, in the case of the elastase isozymes. Diisopropylfluorophosphate (Dip-F) inactivates both SE and the elastase isozymes, whereas Ac-(Ala)2-Pro-AlaCH2Cl (a powerful inactivator of the leucocyte elastase isozymes at 10-4 M concentration) does not inactivate SE at the same concentration. Immunochemical studies revealed antigenic cross-reaction between the rapidly migrating leucocyte elastase isozymes and SE. Two preparations of leucocyte granules from nonleukemic bone marrow cells showed no activity of the rapidly migrating elastase isozymes, but did contain SE activity. SE may be a precursor or zymogen form of the elastase isozymes, present in immature cells and partly retained through later stages of development.     

Differential distribution of distinct forms of myeloperoxidase in different azurophilic granule subpopulations from human neutrophils

Myeloperoxidase (MPO), a characteristic enzyme of human polymorphonuclear neutrophils (PMN), is localized in specialized lysosomal or azurophilic granules, and can be resolved into three distinct forms (I, II, III) by ion-exchange chromatography. Granules were isolated from single donor PMN and fractionated with centrifugation into two different azurophilic subpopulations (high and low density) by banding in a continuous sucrose density gradient. Ion-exchange chromatography of granule extracts indicated that the lower density granules contained mainly MPO forms II and III while the higher density granules appeared to contain all three forms, but in much reduced amounts. Sodium dodecylsulfate polyacrylamide gel electrophoresis showed that, the mobilities of the heavy subunits of MPO appeared to be inversely related to the density of the granule population from which they were extracted. These observations suggest that the different forms of MPO may have distinct functional roles and/or are a possible reflection of maturational differences among the granule subpopulations.     

Some properties of neutral-acting proteases and other degradative enzymes in rat leucocytes

Granules from rat liver cells and peritoneal leucocytes were shown to contain a protease which attacks haemoglobin, a ‘trypsin-like’ enzyme, elastase, ribonuclease, muramidase and phospholipase. Most of these enzymes from either source had two pH optima, one between pH 3.5 and 5.5, and another near neutrality (pH 6.5 to 7.5). Exceptions were the liver protease which was not active at neutrality, and elastase and muramidase which were not active at acid pH. When the leucocytes were disrupted with Triton X-1oo over 85 per cent of protease, phospholipase and elastase active at near-neutral pH remained membrane-bound and sedimentable, whereas about half of the phospholipase active at pH 4 and phosphatase active at pH 5 and pH 10 were released. Most of the β-glucuronidase and all of the acid protease were found in the supernatant. Differential and isopycnic centrifugation of leucocyte granules showed that the protease active at pH 8 was associated mainly with granules heavier and denser than those associated with protease active at pH 4. Acid and alkaline phosphatases were associated with the lighter granules.     

Human neutrophil elastase releases a ligand-binding fragment from the 75-kDa tumor necrosis factor (TNF) receptor. Comparison with the proteolytic activity responsible for shedding of TNF receptors from stimulated neutrophils.

To localize the protease(s) involved in shedding of tumor necrosis factor receptors (TNF-R) from activated neutrophils (PMN) (Porteu, F., and C. Nathan (1990) J. Exp. Med. 172, 599-607), we tested subcellular fractions from PMN for their ability to cause loss of TNF-R from intact cells. Exposure of PMN to sonicated azurophil granules at 37 degrees C resulted in inhibition of 125I-TNF binding; 50% inhibition ensued when PMN were treated for approximately 1 min with azurophil granules equivalent to 2-3 PMN per indicator cell. The TNF-R-degrading activity in azurophil granules were identified as elastase by its sensitivity to diisopropyl fluorophosphate (DFP), alpha 1-antitrypsin and N-methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone (MSAAPV-CK), and by the ability of purified elastase to reproduce the effect of azurophil granules. Elastase preferentially acted on the 75-kDa TNF-R, reducing by 85-96% the binding of 125I-TNF to mononuclear cells expressing predominantly this receptor, while having no effect on endothelial cells expressing almost exclusively the 55-kDa TNF-R. Elastase-treated PMN released a 32-kDa soluble fragment of p75 TNF-R that bound TNF and reacted with anti-TNF-R monoclonal antibodies. In contrast, fMet-Leu-Phe-activated PMN shed a 42-kDa fragment from p75 TNF-R, along with similar amounts of a 28-kDa fragment from p55 TNF-R. Shedding of both TNF-Rs by intact activated PMN was more extensive than shedding caused by elastase and was completely resistant to DFP and MSAAPV-CK. Thus, the TNF-R-releasing activity of azurophil granules is distinct from that operative in intact stimulated PMN and could provide an additional mechanism for the control of cellular responses to TNF at sites of inflammation.     

Neurophysin heterogeneity. Difference between newly formed and aged neurosecretory granules

Neurosecretory granules from bovine neurohypophyses were isolated on iso-osmotic gradients. The content of the granules was analyzed by analytical and two-dimensional gel electrophoreses. The distributions in the gels of vasopressin precursor and neurophysins were detected by radioimmunoassays. Analytical gel electrophoresis of the content of a crude granule preparation showed the presence of different populations of neurophysin molecules. Further analysis demonstrated that vasopressin-neurophysin and oxytocin-neurophysin can be subdivided into molecules with different pI values. Whereas newly formed granules showed two main spots of neurophysin with pI of 5.0 and 5.6, aged granules contain in addition to these different populations of neurophysin-like material, some of which had a basic pI. Vasopressin precursor activity was detected in spots containing proteins with acidic pI and Mr approximately 18,000 and also in proteins of Mr = 8,000-10,000 migrating in the basic region of the gel. The results suggest that in the neural lobe there is an aging process which gives rise to several subpopulations of neurophysins. The different forms of vasopressin-associated bovine neurophysin and oxytocin-associated bovine neurophysin are only found in the granules which are not required for release.     

Discriminating between the activities of human neutrophil elastase and proteinase 3 using serpin-derived fluorogenic substrates

Human neutrophil elastase (HNE) has long been linked to the pathology of a variety of inflammatory diseases and therefore is a potential target for therapeutic intervention. At least two other serine proteases, proteinase 3 (Pr3) and cathepsin G, are stored within the same neutrophil primary granules as HNE and are released from the cell at the same time at inflammatory sites. HNE and Pr3 are structurally and functionally very similar, and no substrate is currently available that is preferentially cleaved by Pr3 rather than HNE. Discrimination between these two proteases is the first step in elucidating their relative contributions to the development and spread of inflammatory diseases. Therefore, we have prepared new fluorescent peptidyl substrates derived from natural target proteins of the serpin family. This was done because serpins are rapidly cleaved within their reactive site loop whether they act as protease substrates or inhibitors. The hydrolysis of peptide substrates reflects the specificity of the parent serpin including those from alpha-1-protease inhibitor and monocyte neutrophil elastase inhibitor, two potent inhibitors of elastase and Pr3. More specific substrates for these proteases were derived from the reactive site loop of plasminogen activator inhibitor 1, proteinase inhibitors 6 and 9, and from the related viral cytokine response modifier A (CrmA). This improved specificity was obtained by using a cysteinyl residue at P1 for Pr3 and an Ile residue for HNE and because of occupation of protease S' subsites. These substrates enabled us to quantify nanomolar concentrations of HNE and Pr3 that were free in solution or bound at the neutrophil surface. As membrane-bound proteases resist inhibition by endogenous inhibitors, measuring their activity at the surface of neutrophils may be a great help in understanding their role during inflammation.     

Human neutrophil elastase: degradation of basement membrane components and immunolocalization in the tissue

Human neutrophil elastase was purified to homogeneity as two isozymes named E1 and E2. The isozymes degraded Type IV collagen, laminin, fibronectin, and heparan sulfate proteoglycan similarly to each other. The degradation of such basement membrane components by elastase may assist the extravasation of neutrophils in the process of inflammation. Among the substrates tested, only type V collagen, which is susceptible to neutrophil gelatinase, was resistant to elastase. This broad substrate specificity of the enzyme may also contribute to tissue destruction at the sites of inflammation. We produced a monoclonal antibody against the purified enzyme and applied it to immunohistochemical studies. In bronchopneumonia and polyarteritis nodosa, elastase was associated with the cleaved elastic fibers, indicating that the enzyme really destroys tissue in vivo. In the exudates of rheumatoid joint, elastase was stained as diffuse fine granules. Immunohistochemical studies with the monoclonal antibody will provide a complementary way to disclose the mechanism of diseases related to neutrophil infiltration.     

Proteases from purulent sputum. Purification and properties of the elastase and chymotrypsin-like enzymes.

A procedure is described for the purification of the elastase and chymotrypsin-like enzymes from purulent sputum. This procedure permitted the isolation of 132 mg and 120 mg of the elastase and chymotrypsin-like enzymes, respectively, from 230 g of purulent sputum. The elastase enzymes consist of a family of five isozymes, and at least three isozymes comprise the chymotrypsin-like enzyme system. The elastases proved to be immunologically identical with the corresponding enzyme of human leukocytes. These enzymes were characterized with respect to molecular weight, amino acid and carbohydrate composition, several kinetic parameters, and inhibition by various synthetic and natural inhibitors. The properties so found were comparable to those which had been previously reported by others for the elastase and chymotrypsin-like enzymes isolated directly from leukocytic granules.     

Proteolytic cleavage by neutrophil elastase converts inactive storage proforms to antibacterial bactenecins.

Bac5 and Bac7, antibiotics of the bactenecin (proline/arginine-rich peptide) family, are stored as proforms in the large granules of bovine neutrophils [Zanetti, M., Litteri, L., Gennaro, R., Horstmann, H. and Romeo, D. (1990) J. Cell Biol. 111, 1363-1371]. These proforms have been purified to homogeneity from granule extracts by immunoaffinity and reverse-phase chromatography. While mature bactenecins efficiently kill Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium with minimal inhibitory concentrations of 6-12 micrograms/ml, proBac5 and proBac7 do not affect the growth of the same microorganisms, even at 500 micrograms/ml. Previous investigations have suggested that the conversion of probactenecins into mature antimicrobial peptides is catalyzed by a neutral serine protease stored in the azurophil granules. Purified proBac5 and proBac7 were thus treated with elastase, cathepsin G or proteinase 3, which constitute the pool of neutral serine proteases of the azurophils, and the reaction products were identified by Western blot analysis, mass spectrometry, and N-terminal sequence analysis. Of the three proteases, only elastase is able to catalyze the stepwise cleavage of probactenecins into the corresponding mature peptides, which have the same mass, N-terminal sequence and antibiotic activity of authentic Bac5 and Bac7. These results point to the importance of cooperation between azurophils and large granules in mounting a defense reaction.     

Localization of chymotrypsin-like cationic protein, collagenase and elastase in azurophil granules of human neutrophilic polymorphonuclear leukocytes.

The subcellular localization of granulocyte collagenase, elastase and chymotrypsin-like cationic protein was determined using velocity centrifugation of cytoplasmic granules of human polymorphonuclear leukocytes. The proteases were assayed by immunochemical and enzymatic methods. Measurements of lactoferrin and myeloperoxidase distinguish exactly between constituents of specific and azurophil granules. Collagenase, elastase and chymotrypsin-like cationic proteins showed an almost identical sharp and unimodal distribution. They co-sedimented with myeloperoxidase demonstrating that these enzymes are localized exclusively in the azurophil granules.     

Ectosomes released by human neutrophils are specialized functional units.

Here we show that human polymorphonuclear leukocytes (PMN) release ectosomes independently of complement attack during their activation both in vitro and at the site of inflammation in vivo. Patterns of biotinylated proteins on the surface of PMN and on PMN-derived ectosomes indicated a specific sorting of cell surface proteins into and out of ectosomes. Ectosomes expressed clusters of complement receptor 1 (CR1), which allowed them to bind efficiently to opsonized bacteria. Myeloperoxidase and human leukocyte elastase, both stored within the azurophilic granules of PMN, were found to colocalize on ectosomes with CR1. Furthermore, myeloperoxidase colocalized with human leukocyte elastase. In contrast, not present on CR1-expressing ectosomes were CD63, a selective marker for the azurophilic granules, and CD14, which is located within the same granules and the secretory vesicles as CR1. Of the other complement regulatory proteins expressed by PMN, only CD59 colocalized with CR1, while CD55 and CD46 were almost absent. Ectosomes released by activated PMN at the site of inflammation may function as a well organized element (ecto-organelle), designed to focus antimicrobial activity onto opsonized surfaces.     

The major fibrinolytic proteases of human leukocytes

The major fibrinolytic enzymes present in leukocyte granules and active at physiological pH have been identified. The fibrinolytic activity in extracts of leukocyte granules was bound to fibrinogen-Sepharose and eluted with 8.0 M urea. Two distinct zones of fibrinolytic activity were detected upon electrophoresis of leukocyte extracts on fibrinogen polyacrylamide gels, and both were qualitatively recovered in the 8.0 M urea eluate. Quantitatively, greater than 95% of the fibrinolytic activity was recovered in the urea eluate. Two major leukocyte proteases, elastase (EC 3.4.21.11) and cathepsin G (EC 3.4.21.-), were quantitatively recovered in the urea eluate. Both enzymes, when purified separately by affinity chromatography, were shown to: (a) possess fibrinolytic activity; (b) coincide in mobility and generate the two zones of fibrinolytic activity on fibrinogen polyacrylamide gels; and (c) quantitatively reconstitute the fibrinolytic activity of the leukocyte granules when combined at activity levels present in granular extracts. A highly significant correlation (r = 0.98) was found between the fibrinolytic activity and the sum of elastase and cathepsin G activity in leukocytes from five donors. Thus, elastase and cathepsin G are the major enzymes of the leukocyte fibrinolytic pathway, and fibrinogen-Sepharose chromatography may be used to obtain these enzymes.     

The latent collagenase and gelatinase of human polymorphonuclear neutrophil leucocytes.

Two metallo-proteinases of human neutrophil leucocytes, collagenase and gelatinase, were studied. Collagenase specifically cleaved native collagen into the TCA and TCB fragments, whereas gelatinase degraded denatured collagen, i.e. gelatin, and the TCA fragments produced by collagenase. On subcellular fractionation by zonal sedimentation, collagenase was found to be localized in the specific granules, separate from gelatinase, which was recovered in smaller subcellular organelles known as C-particles. Neither enzyme was present in the azurophil granules, which contain the two major serine proteinases of neutrophils, elastase and cathepsin G. Collagenase and gelatinase were separated by gel filtration from extracts of partially purified granules. Both enzymes were found to occur in latent forms and were activated either by trypsin or by 4-aminophenylmercuric acetate. Gelatinase was also activated by cathepsin G, which, however, destroyed collagenase. Both enzymes were destroyed by neutrophil elastase. Activation resulted in a decrease by 25 000 in the apparent mol. wt. of both latent metallo-proteinases.     

In cow anterior pituitary, growth hormone and prolactin can be packed in separate granules of the same cell

The ultrastructural localization of growth hormone and prolactin in cow anterior pituitary was studied by double immunocytochemical labeling using specific antibodies and protein A-gold particles of different sizes. The two hormones were found in specific somatotrophs and mammotrophs as well as in somatomammotropic cells which were multinucleated and predominantly arranged in clusters in the central area of the lobules. In these mixed cells the two hormones were packaged (a) in different granules of the same cell, (b) in the same granules where they were segregated in different portions of the granule content, or (c) in the same granules but evenly intermixed. The relative proportion of these three types of granules varied in somatomammotrophs of different animals. A single large Golgi complex was generally present in somatomammotrophs. Small, immature granules containing either growth hormone or prolactin or both hormones were found randomly distributed along Golgi stacks. This suggests that in these cells the two hormones are processed in the same Golgi cisternae and that mechanism(s) exist(s) to sort out the two hormones from each other.     

Constituents of connective tissue around the capillary of chick pecten oculi

The constituents of the connective tissues around the capillary of the chick pecten oculi were examined electron microscopically by HCl-collagenase and HCl-elastase methods. The basal lamina like membrane below the endothelial cell of the pecten capillary was digested by collagenases I, II and IV and elastase, and may be a false basal lamina. The basal lamina of cells with pigment granules which surround the capillary was digested by collagenase IV and elastase, and contained type IV collagen. Fibrils between the basal lamina like membrane of the pecten capillary endothelium and the basal lamina of the cells with pigment granules were digested by collagenases I, II and IV, and elastase. Thus, these fibrils are composed of many kinds of collagen. Elastase may be responsible for the breakdown of most collagens as well as elastin.     

On the properties of porcine elastase released from its complex with human alpha-1-antitrypsin by alkaline cleavage.

These investigations were made to determine how the elastase released from its complex with alpha-1-antitrypsin at high pH is modified. Most of the elastase component precipitates on returning the pH to neutral. The elastase component and the native enzyme subjected to the same conditions of pH and temperature reacted to approximately the same extent with radio-actively labeled diisopropyl fluorophosphate. There were about two moles of dehydroalanine per mole of enzyme either in the presence or absence of complex formation. Thus, the enzyme is either still capable of reacting with diisopropyl fluorophosphate or it is denatured and thus inactivated by partial conversion of cystine residues to dehydroalanine. Anhydroelastase is apparently not formed during cleavage of the complex.