beta-Adrenergic tachyphylaxis in the rat and its reversal and prevention by ketotifen.

A strong, long-lasting and reproducible tachyphylaxis was produced in rats by implantation of osmotic minipumps delivering isoprenaline continuously. The degree of tachyphylaxis was determined by measuring the inhibitory effect of isoprenaline on the passive cutaneous anaphylaxis (PCA). One dose of ketotifen given 1 h before the PCA test reversed this in vivo tachyphylaxis, as did dexamethasone given 24 h earlier. Implantation of a second minipump containing ketotifen prevented the development of tachyphylaxis. Weak tachyphylaxis was induced in guinea-pig trachea in vitro by incubation with a high concentration of isoprenaline, the effect being estimated by measuring the relaxation of carbachol-contracted trachea. Ketotifen partially restored the sensitivity of the trachea but this was considered to be a direct potentiation of isoprenaline effects rather than a reversal of tachyphylaxis since the same effect was seen in non-pretreated trachea. It is thought that the reversal of experimental beta-adrenergic tachyphylaxis by ketotifen could have implications for its use in the prophylactic treatment of asthma.

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.

Phagocytosis-stimulated macrophages. Production of prostaglandins and SRS-A, and prostaglandin effects on macrophage activation.

Quiescent mouse peritoneal macrophages which phagocytose, and which respond to phagocytosis with a sudden elevation in hexose monophosphate shunt activity, immediately release into the medium oxygen metabolites, arachidonic acid oxygenation products, and lysosomal hydrolases. Such cells subsequently differentiate and acquire the properties of inflammatory macrophages. The latter process appears to be under the control of prostaglandin E2 and possibly other cyclooxygenase products which are formed as a consequence of phagocytosis and seem to act as feed-back inhibitors.

Partial purification of collagenase and gelatinase from human polymorphonuclear leucocytes. Analysis of their actions on soluble and insoluble collagens.

The separation and further purification of human polymorphonuclear-leucocyte collagenase and gelatinase, using modifications of the method of Cawston & Tyler [(1979) Biochem J. 183, 647-656], are described. The final preparations yielded collagenase of specific activity 260 units/mg and gelatinase of specific activity 13 000 units/mg. Gelatinase was purified to apparent homogeneity in a latent form, and analysis of the activation of 125I-labelled latent enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel-filtration techniques suggested that no peptide material was lost on conversion into the active form. The purified natural inhibitors alpha 2-macroglobulin, tissue inhibitor of metalloproteinases ('TIMP') and amniotic-fluid inhibitor of metalloproteinases all inhibited the two polymorphonuclear-leucocyte metalloproteinases, but the last two inhibitors were slow to act and complete inhibition was difficult to attain. Collagenase degraded soluble types I and III collagen equally efficiently, but soluble type II collagen less well. Gelatinase alone had little activity on these substrates, although it enhanced the action of collagenase. Gelatinase was capable of degrading soluble types IV and V collagen at 25 degrees C, whereas collagenase was only active at higher temperatures when the collagens were susceptible to trypsin activity. By using tissue preparations of insoluble collagens (type I, II or IV) the activity of leucocyte collagenase was low and gelatinase activity was negligible, as measured by the solubilization of hydroxyproline-containing material. The two enzymes together were two or three times more effective in the degradation of these insoluble collagens.

Defective Aspergillus killing by neutrophil leucocytes in a case of systemic aspergillosis.

A persistent defect of Aspergillus killing was observed in the neutrophils of a 6-year-old patient with a systemic A. fumigatus infection which was highly refractory to anti-mycotic therapy. Aspergillus phagocytosis in vitro was normal, but nearly 80% of the ingested organisms (versus 30% in the controls) survived intracellularly during the 2-hr assay period. The patient's neutrophils showed a subnormal frequency of nitroblue tetrazolium reduction and a subnormal hexose monophosphate shunt activation in response to phagocytosis. The metabolic responsiveness, however, was clearly superior to that of chronic granulomatous disease neutrophils tested for comparison. The immune status of the patient and the following properties of his neutrophils were found to be normal: random and chemotactic motility, killing of S. aureus and C. albicans, and the contents of several granula enzymes. Our findings suggest the existence of neutrophil factors or functions which are required for killing Aspergillus, but not S. aureus and C. albicans.

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.

Phagocytosis stimulates the release of a slow reacting substance in cultured macrophages.

1 A slow-reacting substance (SRS) was released from non-elicited mouse peritoneal macrophages during phagocytosis of zymosan particles, whereas no detectable SRS was produced by resting cells. 2 The macrophage SRS induced a delayed and slow contraction of the guinea-pig ileum but not of the chick rectum. 3 The myotonic activity was antagonized by low concentrations of FPL 55712 (sodium 7-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)-2-hydroxypropoxy]-4-oxo-8-propyl-4H-1 -benzopyran-2 carboxylate) but was not affected by mepyramine or hyoscine, and was not associated with tachyphylaxis. 4 SRS release was increased by indomethacin and was abolished by the lipoxygenase and cyclooxygenase inhibitor, BW755C (3-amino-1-[m-(trifluoromethyl)-phenyl]-2-pyrazoline). 5 Addition of exogenous arachidonic acid or cysteine enhanced SRS production.

Cellular mechanisms of proteinase release from inflammatory cells and the degradation of extracellular proteins.

Neutrophils and macrophages produce, store and release large amounts of various acid and neutral proteinases. The two main proteinases of neutrophils are elastase and cathepsin G. They are localized in the azurophil granules, together with proteinase 3 and the acid cathepsins B and D. In addition neutrophils contain collagenase in the specific granules, acid proteinases in the C-particles and plasminogen activator in organelles with the characteristics of secretory vesicles. The granule-bound proteinases are released during phagocytosis while plasminogen activator is apparently secreted. In macrophages, the acid hydrolases are bound to lysosomes while the neutral proteinases are confined to secretory vesicles. The main mechanism of enzyme release in macrophages is secretion. Lysosomal hydrolases are also released by phagocytosis. Enzyme secretion is a characteristic property of activated or inflammatory macrophages. Macrophages become activated after phagocytosis of certain particles and the metabolic burst appears to be an initial event in the activation process. The action of neutrophils and of purified elastase or plasmin on cartilage was tested. These experiments indicate that neutrophil-mediated degradation of cartilage proteoglycans is largely dependent on elastase.

A volume adapter for use in a B-XIV zonal rotor.

A simple adapter for reducing the chamber volume of B-XIV-type rotors is described. The adapter consists of a disk-like body, which occupies the lower part of the rotor chamber, and an aluminum sleeve with four septa. The modified rotor has a volume of 272 ml and has proved very suitable for rate-sedimentation analysis of small amounts of biological material. The modified rotor is operated as is a standard B-XIV rotor.

The polymorphonuclear leukocyte.

Polymorphonuclear leukocytes (PMNs) are one of the main sources of enzymes responsible for tissue damage in inflammatory processes. These enzymes are stored in two types of cytoplasmic granules. Azurophil granules contain lysosomal hydrolases, neutral serine proteinases, and bactericidal elements (myeloperoxidase and lysozyme). Specific granules contain collagenase, lysozyme and lactoferrin but lack lysosomal hydrolases. PMNs store all four classes of tissue proteinases, carboxyl, thiol and serine proteinases in the azurophil granules, and metallo proteinases in the specific granules. Three serine proteinases have been identified, elastase, cathepsin G and a third enzyme, which together account for a large proportion of the protein of the azurophil granules. In the course of phagocytic events, all these enzymes are released extracellularly. The neutral proteinases degrade proteoglycans and collagen. In vitro, they stimulate B-lymphocytes, which suggests that they may have immuno-potentiating activity when they are released at sites of chronic inflammation.

Collagenase is a component of the specific granules of human neutrophil leucocytes.

Azurophil and specific granules were isolated from human polymorphonuclear neutrophil leucocytes. Collagenase was almost exclusively a component of the specific granules. This finding is in contrast with the distribution of other proteolytic enzymes, which are localized in the azurophil (or lysosomal) granules.

In vitro stimulation of lymphocytes by neutral proteinases from human polymorphonuclear leukocyte granules.

Two neutral proteinases, polymorphonuclear leukocyte (PMN) elastase and cathepsin G, were purified from azurophil granules of human PMN. Both enzymes were found to stimulate both human and mouse lymphocytes in vitro. Experiments with mouse cells showed that the PMN proteinases are B-cell stimulants. Stimulation appears to depend on direct proteolytic action on the lymphocyte surface and not to require the mediation of serum factors of the help of T-cells.

In vitro stimulation of lymphocytes by neutral proteinases from human polymorphonuclear leukocyte granules.

Two neutral proteinases from human polymorphonuclear leukocytes (PMN), an elastase and the chymotrypsin-like cathepsin G, were purified, and their actions on lymphocytes in culture were studied. Both PMN proteinases stimulate lymphocytes from human peripheral blood and from mouse spleen in vitro, but do not affect thymic cells from either normal or hydrocortisone-treated mice. In stimulated mouse spleen cell cultures, most of the developing blast cells bear surface immunoglobulins, and subsequently appear to engage in antibody synthesis. In their stimulatory action, the two PMN proteinases thus resemble the classic B-cell mitogen LPS and neutral pancreatic proteinases such as trypsin, chymotrypsin, and elastase. The effects of proteinase inhibitors indicate that lymphocyte stimulation is dependent on the proteolytic activity of the enzymes. This work suggests that PMN proteinases, which are released at sites of inflammation, may modulate the function of lymphocytes.

Subcellular localization and heterogeneity of neutral proteases in neutrophilic polymorphonuclear leukocytes.

The subcellular localization of elastase and of neutral proteases hydrolyzing histone and casein was determined in human and rabbit polymorphonuclear leukocytes using fractionation by isopycnic centrifugation. Granule-rich fractions obtained by this technique were extracted and analyzed by acrylamide gel electrophoresis, and proteolytic activity on the gels was demonstrated by staining with either N-acetyl-D,L-alanine alpha-naphthyl ester or naphthol AS-D acetate as substrate. In both species, all neutral proteases assayed were found to be localized exclusively in the azurophil granules. Specific activities were about 10-30 times higher in human than in rabbit preparations. In extracts of human azurophil granules up to 10 proteins exhibiting esterolytic activity could be demonstrated after electrophoretic separation. Three major and two or three minor components of these esterases were shown to possess elastase activity. Similar zymograms prepared with extracts from rabbit azurophil granules revealed only one major elastase band. The electrophoretic analysis further showed that the most strongly cationic proteins of both human and rabbit PMNs were also confined to the azurophil granules.

Biochemical and morphological characterization of azurophil and specific granules of human neutrophilic polymorphonuclear leukocytes.

Postnuclear supernates from homogenates of purified neutrophil polymorphonuclear leukocytes (PMNs) from human blood were fractionated by zonal sedimentation and isopycnic equilibration in sucrose gradients. The fractions were characterized biochemically by measuring protein content and the activities of eight enzymes. Selected fractions were further analyzed by electron microscopy. In both centrifugation systems, azurophil and specific granules could be resolved almost completely. Azurophil granules sediment three to four times faster than the specifics and have an average density of 1.23. They contain all the peroxidase of the cells, large portions of four lysosomal hydrolases, and about half of the total lysozyme, and therefore appear to be, in biochemical terms, very similar to the azurophil granules of rabbit PMNs. The specific granules, which have an average density of 1.19, contain the remaining half of the lysozyme but appear to be free of the other components of the azurophil granules, and of alkaline phosphatase. Isopycnic equilibration disclosed a minor lysosomal population, which strongly overlaps the specific granules, and made possible the identification of a membrane-fraction which is characterized by the presence of the thiol-sensitive acid 4-nitrophenyl phosphatase and of alkaline phosphatase.