Article

MEDIATORS OF INFLAMMATION IN LEUKOCYTE LYSOSOMES: II. MECHANISM OF ACTION OF LYSOSOMAL CATIONIC PROTEIN UPON VASCULAR PERMEABILITY IN THE RAT

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Abstract

The vascular permeability-increasing action of rabbit PMNL lysosomes has been studied in skin and cremaster muscle of the rat. Both an extract of frozen-thawed granules and a cathepsin-free cationic protein fraction of the granules (which had previously been demonstrated to cause leukocyte adhesion and emigration in vivo) induce increased vascular permeability in skin and muscle which resembles that produced by histamine or histamine-liberators with respect to the timing of the response and the predominant type of microvessel affected. Extracts of frozen-thawed lysosomes and the inflammatory lysosomal cationic protein both cause disruption of rat mesenteric mast cells in vitro, whereas a granule-free cytoplasmic fraction of PMN leukocytes and a non-inflammatory cationic protein fraction of the granules do not do so under identical test conditions. The mastocytolytic action of lysosomal materials in vitro is not inhibited in the presence of 10 kallikrein-inhibiting units of trasylol per ml. The mast cell rupturing fraction of PMNL granules (cationic protein) possesses no detectable peroxidase activity or acid-mucopolysaccharase activity. When compared with compound 48/80 on the basis of estimated molecular weight, the lysosomal cationic protein appears to be at least as active as the latter compound with respect to in vitro mastocytolytic potency. Chronic pretreatment of rats with an agent known to reduce tissue mast cell numbers causes marked suppression of the vascular permeability change normally induced in skin and muscle by lysosomal extracts and cationic protein. Similar results are obtained if lysosomal materials are tested in rats pretreated with an antihistaminic. These observations are discussed with respect to the mode of action of PMNL lysosomes in the early and late phases of local tissue-injury reactions.

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Chapter
Diabetic retinopathy is a prototypical microvascular disorder. Hyperglycemia causes a multiple pathological changes in the retinal vasculature. It has been suggested that apoptosis of pericytes due to high glucose levels plays a key role in the development of the earliest events during diabetic retinopathy. Advancement of the disease resulted in a progressive vessel leakage leading to edematous distortion of macula and increase in hypoxia inducing development of neovascularization with sight threatening complications. Four basis hypotheses explaining the hyperglycemia harmful effects were suggested: (1) increased glucose flux through the aldose reductase pathway, (2) overproduction of advanced glycation end products, (3) activation of protein kinase C isoforms, and (4) increased glucose flux via the hexosamine pathway. It was admitted as well that apoptosis of neurons and glial cell activation occur even earlier than vascular damage. Disturbance in glial cell functions leads to increase in metabolic abnormalities such as glutamate accumulation, promotion of inflammation, and oxidative stress resulting in neuron apoptosis and deterioration of vascular disorders. Clarification of significant biochemical mechanisms involving in the development of diabetic retinopathy can help to create new effective ways in diabetic retinopathy treatment.
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Previous hypotheses about the causes of venous ulceration are inconsistent with recently published data. In patients with chronic venous insufficiency the number of functioning capillary loops visible in the skin on microscopy fell after the legs had been dependent for 30 minutes. Another study had shown that leucocytes became trapped in the circulation in dependent legs. A new hypothesis linking these two findings proposes that the trapped white cells occlude the capillaries and result in ischaemia of the skin of the leg.
Article
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Article
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Article
Publisher Summary This chapter discusses the role of lysosomes in immune responses. Lysosomes are the central part of many studies related to the tissue injury, inflammation, and immunity. Lysosomes can also be considered as the important part of a vacuolar system or an “exoplasmic reticulum” concerned with the digestion of heterologous and autologous material. Although there is a considerable body of evidence implicating macrophages in the afferent limb of the immune response, their exact role is not clear. Macrophages may prevent excess antigen from paralyzing lymphocytes that act as convenient carriers of antigen throughout the body, concentrate and retain antigen over longer periods of time, present antigen to responsive cells, degrade antigen into active subunits, convert crude antigen into a better immunogen, translate antigen into specific information, or exert some nonspecific effect on antigen-reactive cells that allow them to perform properly. Trapping of antigens by reticular cells may not be the only mechanism responsible for their localization in follicles. Recently, there is a new subpopulation of lymphocytes that binds antigen–antibody complexes in the presence of modified complement. Because the bulk of this subpopulation, termed “complement receptor lymphocytes,” resides in the follicular areas of peripheral lymphoid tissue, it may seem that the lymphocytes contribute to antibody mediated retention of antigens by lymphoid follicles.
Article
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Article
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Article
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Article
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Article
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Article
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Article
Bei Hamstern wurde nach experimentell gesetzter steriler Peritonitis der Einflus von Trasylol und Solu Decortin auf Migration und Degranulation der ins Peritonealexsudat ausgewanderten neutrophilen Granulozyten untersucht.
Article
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Article
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Article
METHOD Experiments were carried out on 60 chinchilla rabbits (2.5-3.5 kg} and 70 Wistar rats (200-250 g} of both sexes. The leukocyte preparations were obtained by the method described previously [5] and injected intradermally into the experimental animals in 0.1 ml physiological saline. The dose of injected material was calculated per mg protein, determined by Lowry's method [14]. The dose of fragmented leukocytes injected varied from 67.103 to 870. 103/0.1 ml. The development of disturbances of permeability of the skin vessels 30 min after the injection served as index of the phlogogenic action of the lysosomes, granulocytic material, and destroyed leukocytes. Permeability of the skin capillaries was studied by the standard method [6]. The dye Evans' blue was injected in 1% solution in a dose of 20 mg/kg body weight. The phlogogenic activity of the leukocyte preparations in the experimental animals was determined after administration of various pharmacological agents. The antihistamine agent used was dimebolic (10 mg/kg intraperitoneally). Dimedrol (benadryl), combining antihistamine with antibradykinin properties [2], was injected in the same doses as dimebolin. Trasylol was injected intraperitoneall y in a dose of 2000 kallikrein units/kg as inhibitor of proteolysis. The phlogogenic activity of the leukocyte preparations was determined in control animals (20 rabbits and 20 rats} not receiving these pharmacological agents. The results were subjected to statistical analysis by Fisher's method for fourfield tables [4].
Article
The mature neutrophils in the circulation contain, besides the different proteases known for a long time, a recently discovered proteolytically inactive elastase homologue (HBP/CAP37/azurocidin). This homologue, which we have named HBP due to its strong affinity to heparin, is a chemoattractant for monocytes and has been shown to induce reversible detachment and contraction when added to monolayers of endothelial cells or fibroblasts. HBP may therefore play a pivotal role in leukocyte migration in response to inflammation. In this report a comparison of CH3O-Suc-Ala-Ala-Pro-Val-CH2Cl-inhibited elastase with HBP, its naturally occurring homologue selectively mutated in active serine and histidine, reveals that homotypic aggregation of monocytes and contraction of fibroblasts is specific for HBP. HBP induces thrombospondin secretion from monocytes four times as efficiently as the inhibited elastase, and the same molecule was found unable to compete for a specific saturable binding of HBP to monocytes with an apparent KD of 3 x 10(-8)M.
Article
Evidence is presented that serum from rats with adjuvant arthritis stabilizes the membranes of isolated normal rat liver lysosomes. The stabilizer retarded the release of the lysosomal enzymes, acid phosphatase and β-glucuronidase, but not the release of the mitochondrial enzyme, isocitric dehydrogenase. Stabilizing activity in serum paralleled adjuvant disease, being greatest when the inflammation was most intense and subsiding as the arthritis diminished. Activity could be removed from the serum by absorption with isolated membranes; adjuvant arthritis serum did not inhibit enzyme activities. The membrane reactant was neither a corticosteroid nor an antibody but was a large molecular weight substance with charge properties similar to an α-globulin.
Article
Although stroke has recently dropped to become the nation's fifth leading cause of mortality, it remains the top leading cause of morbidity and disability in the US. Recent advances in stroke treatment, including intravenous fibrinolysis and mechanical thromboembolectomy, allow treatment of a greater proportion of stroke patients than ever before. While intra-arterial fibrinolysis with recombinant tissue plasminogen is an effective for treatment of a broad range of acute ischemic strokes, endovascular mechanical thromboembolectomy procedures treat severe strokes due to large artery occlusions, often resistant to intravenous drug. Together, these procedures result in a greater proportion of revascularized stroke patients than ever before, up to 88% in 1 recent trial (EXTEND-IA). Subsequently, there is a growing need for neurointensivists to develop more effective strategies to manage stroke patients following successful reperfusion. Cerebral ischemic reperfusion injury (CIRI) is defined as deterioration of brain tissue suffered from ischemia that concomitantly reverses the benefits of re-establishing cerebral blood flow following mechanical or chemical therapies for acute ischemic stroke. Herein, we examine the pathophysiology of CIRI, imaging modalities, and potential neuroprotective strategies. Additionally, we sought to lay down a potential treatment approach for patients with CIRI following emergent endovascular recanalization for acute ischemic stroke.
Chapter
Various types of injury induced by complexes of antigen and anti-body are reviewed. These include reactions in which antibody reacts with antigens that are fixed in tissues or are soluble but localized at a particular site and reactions that occur following localization of circulating immunologic complexes in a particular tissue from the blood stream. Examples of each are reviewed. The role of mediation systems in the pathogenesis of immunologic disease is stressed. Two examples of mediation pathways are provided in which a sequence of interacting humoral and cellular reactants is activated by antigen-antibody complex and which is responsible for the injurious consequences.
Article
Several important discoveries of the past few decades have launched a renaissance of research into basophil and mast cell function. These include the discovery of IgE and its affinity for basophils and mast cells and the demonstration of the ability of these cells to elaborate and release not only histamine but also a variety of lowmolecular-weight mediators of immediate hypersensitivity. Moreover, the application of modern methods of tissue fixation and processing has permitted confident identification of basophils and mast cells with both the light and electron microscopes in diverse experimental and clinical situations, and has clearly implicated these cells in a variety of delayed-onset immunological phenomena where their participation had been entirely unexpected.
Chapter
This chapter discusses the intracellular neutral SH-dependent protease associated with inflammatory reactions. The events of inflammation fall naturally into two broad divisions involving the fluid and cellular phases of circulation. The fluid-phase reaction consists of transient vasoconstriction followed by substained dilation of arterioles, capillaries, and venules, during which blood flow is increased and subsequently decreased, and permeability to plasma protein is raised. The cellular response consists of changed function of tissue cells, migration of various types of leukocytes from blood vessels, and proliferation of migrated cells. Protease systems, which may be concerned with inflammatory reactions, fall naturally into two broad divisions, namely, the plasma protease system and the cellular protease system. The activation of the plasma protease system was first associated with anaphylaxis and in part with allergic inflammation. Neutral SH-dependent protease, which was termed an inflammatory protease, was inactivated by two types of inhibitors (serum and tissue). It was suggested that the balance between the enzyme and these inhibitors may control the intensity, extent, and duration of inflammatory reactions.
Article
Vascular basement membrane was disrupted in the presence of polymorphonuclear leukocytes (PMN's) during two immunologic reactions: The Arthus phenomenon and the reaction to locally injected antibody to vascular basement membrane. This disruption was evidenced by (a) the inability of the basement membrane to retain circulating carbon, by (b) loss of antigenic constituents, and by (c) electron microscopic observation showing actual gaps in the structure of the vascular basement membrane. The factors within PMN's responsible for damage to isolated glomerular basement membrane in vitro were found by isolation procedures to be cathepsins D and E. Cationic proteins of PMN's were separable from the cathepsins. While inducing vascular permeability upon injection, these basic proteins failed to inflict the severe damage to the basement membrane observed in Arthus and antibasement membrane reactions. It is concluded that the full expression of these immunologic lesions requires destruction of the basement membrane possibly brought about by cathepsins D and E. Some of the physicochemical properties of these pathologically active leukocytic factors are given.
Article
The number of satellites on D and G chromosomes in man may present in one of thirty-five combinations in individual cells. Blood obtained from volunteers on one or more occasions was cultured in one of two media. Satellites were counted on chromosome preparations grouped into three stages in the progression from early to late metaphase. The number of satellites varies with the individual, with the culture medium, and, less definitely, with the stage of cell division. The D/G ratio is the most useful variable for characterising an individual's chromosome complement.
Article
Cathepsin D activity has been studied by a fluoremetric assay in the urine of acute post-streptococcal glomerulonephritic (APSGN) patients aged from 3 to 14 years and has been found elevated when compared with four groups of controls. This activity cannot be accounted for by erythrocytes and/or leukocytes in the urine of these patients since haematuric and pyuric controls did not exhibit an amount of enzyme activity greater than the normal control group. Cathepsin D activity can be attributed to lysosomal enzymes released from polymorphonuclear leukocytes which are in close contact with glomerular basement membrane. Complement C3 levels in serum and cathepsin D activity in urine of these patients showed no correlation.
Article
The present study was performed in order to study the progressive growth of vessels in a regenerating tissue and to analylze the tissue events occurring outside and inside the mature micro-vessels immediately before sprout formation. Twenty, 10–20 months old female rabbits, weighing 2–3 kilos each were used. A modified Sandison's ear chamber was installed in each animal. Healing of a tissue defect was studied in a modified Leitz intravital microscope. Immediately after installation of the ear chamber, the center of the wound was practically devoid of cells. The venules of the bordering tissue showed signs of an acute inflammation. During the first hours there was an obvious leakage from the venules of the bordering tissue of an intravenously injected protein bound dye. Some hours later granular cells started to emigrate from the adjacent pre-existing venules into the defect space. Six to eight hours after surgery corpuscular and plasma flow was normal in all vessels in the region of the ear chamber. During the next 35–40 hours the protein network became denser. By the end of the second day most of the granular cells of the defect had lost their amoeboid properties. The nucleus and the granules of these cells became denser and more prominent. By 48–72 hours several of the rigid granular cells disintegrated and dispersed their cytoplasmic granules into the defect. A few hours after degranulation the corpuscular flow rate of the bordering vessels increased. Erythrocytes and later granulocytes penetrated the venular walls and entered the extra-vascular space. In the immediate vicinity of the vertices of the preformed venules, strings of red and white cells moved back and forth apparently creating well defined tubes bordered by a fibrillar structure. The cell movements seemed to open up and elongate the tubes. Between the moving cells and aggregated cells of the defect a layer of thin transparent cells could eventually be detected. In this situation there were also optically definable openings in the walls of the borderline venules and true communicationsestablished between the preexisting vessels and the newly formed.
Article
Injection of a small amount of medullasin protease (20 μg) into the skin of guinea pigs or rabbits caused inflammation characterized by severe injury to endothelial cells in venules and by infiltration of a large number of macrophages. Medullasin inhibited macrophage chemotaxis in vitro, stimulated granulocyte chemotaxis, and potentiated the production of superoxide by macrophages. These results indicate that medullasin in granulocytes plays an important role in the development of inflammation.
Article
Seven beagle dogs were used to study the gingivitis-inducing potenial of a topically applied neutrophil leukocyte extract and to compare the vascular and cellular reactions with those induced by plaque extract applied in an identical manner. In four dogs neutrophil extract was applied onto the marginal gingiva in one jaw quadrant while Gey's medium was applied to the contralateral gingival units. In the remaining three dogs plaque extract and Gey's medium, respectively, were applied in the same way. After 6.5 h of topical application colloidal carbon (1 ml/kg bodyweight) was injected into a leg vein and after a further 1.5 h biopsies were obtained from the experimental regions. The results show that topical application of an extract from neutrophils induces both an increase in the number of crevicular leukocytes and an enhanced vascular permeability. As in previous studies plaque extract was found to promote the emigration of leukocytes and to alter the permeability of the dentogingival vessels. The vascular and cellular reactions were, however, more pronounced after application of plaque extract than after neutrophil extract.
Article
1. The change in permeability of different regions of the microcirculatory system caused by drugs and toxins can be established by observing the distribution of carbon particles in the vessels of the cremaster muscle and skin of the rat. 2. Two groups of substances are found: Substances which have an effect on lipid structures of membranes (organic solvents, lysophosphatides, phospholipases) always affect both capillaries and postcapillary venoles. All other substances affect only venoles. To them belong compounds which affect the dynamics of the microcirculation (histamine, kinines, kininogenases) or those, which react with protein or polyanionic structural elements (basic peptides, polycations). 3. Pretreatment of rats with a histamine releaser, or with antihistamine, or with histidinedecarboxylase inhibitor does not affect the above results. Histamine can not be the principal cause of the results found in this report.
Article
Isolated lysosomal granules were prepared using the method of Hegner and lysed by Triton X 100. A crude peptide was isolated from concentrated extracts by precipitation with picric acid. Seven homogeneous peptides were separated by preparative polyvinylchloride block electrophoresis. The IEPs of these products were respectively about 2; 4.5; 6.5; 9.8; 11.2 and >12. The amino acid composition of two basic peptides (IEPs>12) was similar to arginine rich products found earlier in exudate leucocytes by Spitznagel and Zeya. MWs of 10,500 and 5,200 were estimated by gel filtration. These two arginine rich polypeptides were potent histamine liberators (0.5–1.0 μg/ml released more than 50% histamine from isolated peritoneal mast cells of the rat). Another basic polypeptide (MW=16,000–19,000, IEP=11.2) increased the permeability of venules in rabbit skin after i.c. injection of 0.2 μg. This peptide was found to be less potent in releasing histamine. Our observations were in agreement with those of Seegers and Janoff; Keller et al. In addition, not only lysosomes from leucocytes of exudates contain biologically active peptides, but also granules from PMN leucocytes of the circulating blood which store remarkable amounts of active basic products.
Article
Es wurde die Degranulation der zirkulierenden Thrombozyten und die Mobilisation von neutrophilen Granulozyten bei Goldhamstern whrend experimenteller steriler Peritonitis mit und ohne Trasylolbehandlung untersucht.Es wurde festgestellt, da 4 h nach Auslsung einer peritonealen Reaktion die Aktivitt von saurer Phosphatase und die Menge des Proteasen-Inhibitors in den zirkulierenden Thrombozyten vermindert ist. Gleichzeitig ist die Anzahl der zirkulierenden Granulozyten, die eine hhere alkalische Phosphataseaktivitt aufweisen als die Leukozyten vor der Auslsung des peritonealen Reizes, erhht.Bei Behandlung mit Trasylol ist die Abnahme der Aktivitt der sauren Phosphatase und der Menge des Proteasen-Inhibitors in den Thrombozyten verringert. Auch die leukozytre Reaktion ist abgeschwcht.In golden hamsters the degranulation of the circulating thrombocytes and the mobilization of neutrophil granulocytes in experimental sterile peritonitis with and without treatment by Trasylol was studied. Four hours after initiation of a peritoneal inflammation the activity of acid phosphatase and the amount of the protease inhibitors in the circulating thrombocytes is lowered. Simultaneously the number of the circulating granulocytes with enhanced alkaline phosphatase activity against the controls is augmented. After Trasylol treatment the decrease of activity of the acid phosphatase and of the amount of the protease inhibitor in the thrombocytes is lowered.
Article
Lysosomes catalyze a rapid disruption of mitochondrial structure and function in which oxidative phosphorylation is uncoupled and mitochondrial ATPase is activated. There is a concomitant irreversible high amplitude swelling of mitochondria which is independent of substrate availability. This swelling is not due to lipid peroxidation or digestion by proteolytic enzymes but closely parallels that produced by free fatty acids, in that it is inhibited by low levels of bovine serum albumin. The swelling factor, which is most active in the lysosomal membrane, is associated with the activity of lysosomal lipolytic enzymes. A phospholipid-hydrolyzing enzyme has been found in the lysosome, which liberates free fatty acids from lecithin. Disruption of mitochondrial membranes by these fatty acids, coupled with direct hydrolysis of the membranes by a lysosomal phospholipase, may initiate the digestion of mitochondria by lysosomes.
Article
Three types of response to the entry of foreign material into perivascular connective tissue are described. Under natural conditions this foreign material is usually microbial. Because of the great preponderance of polymorphonuclear cells in the blood, the majority of cells leaving the vessels are of this type. If the foreign material is digestible by these cells, it is disposed of by them. They release permeability factors which temporarily increase their numbers locally and they are equipped with such a broad spectrum of catabolic enzymes that they and the material they have ingested are reduced to diffusable products which are eventually absorbed or discharged.
Article
IN previous publications 1-3 I reported the production of immediate increases in vascular permeability in rat tissues treated with a basic protein fraction extracted from lysosomes of exudate polymorphonuclear (PMN) leucocytes. It was suggested that the permeability effect was caused by the release of vasotropic amines from tissue mast cells by a component of the lysosomes2,4. Part of the evidence cited in support of this mechanism was the significant inhibition of tissue reaction to the extract after treatment of rats with an antihistamine drug. This observation, however, was made in animals treated with a large dose of the antihistamine, and no consideration was given to non-specific effects of such a dose on vascular response to generalized inflammatory stimuli. Additional experiments with appropriate controls for non-specific effects of the antihistamine have now been performed. This communication reports results which confirm our earlier conclusion.
Article
Purified human lysozyme (LZM) in concentration 300–500 μg/ml significantly enhanced (p < 0.01) phagocytosis and the phagocytic index of normal human granulocytes (PMNs). However, LZM's enhancing activity was selective and did not correlate with susceptibility of microorganisms to its lytic activity. LZM enhanced phagocytic activity of PMN's from patients with collagen diseases, hypogammaglobulinemia or recurrent infections but did not influence immature cells from acute or chronic granulocytic leukemia. Rabbit band-2, another lysosomal cationic protein, also enhanced phagocytosis. Human cationic leukemia antigen (CLA) and lysosomal proteins with less cationic properties, namely RNAse and lactoferrin had no significant enhancing activity. Nuclear cationic proteins, histone and protamine and synthetic polyamino acids containing arginine or lysine enhanced phagocytosis and phagocytic index and increased intracellular killing (p < 0.01) of Gram (−)ve and Gram (+)ve bacteria. They also enhanced phagocytosis and phagocytic index of latex particles. Mastocytotropic compound enhanced phagocytosis and phagocytic index of various microorganisms but not of latex particles. The enhancing activity of cationic substances was abolished by washing PMNs after exposure to these substances. Preincubation of microorganisms with various cationic substances lead to their death and lysis to various degrees depending on the type and concentration of the substance added. When the phagocytic assays were performed at 40 °C, further enhancement of phagocytosis by nuclear but not by lysosomal cationic proteins was observed. To the contrary, reduction of pH to 4.4 enhanced phagocytosis by lysosomal but not by nuclear cationic proteins. Anionic substances such as heparin, DNA and endotoxin abolished enhancing activity of lysosomal, nuclear and synthetic cationic substances but not of the mastocytotropic compound . Cytochalasin B (CB) reduced, but not abolished, phagocytic activity of human PMNs and suppressed enhancing activity of cationic proteins. The most pronounced action of CB was inhibition of the intracellular killing of microorganisms by PMNs with or without cationic substances. CB did not influence viability of PMNs or of microorganisms and did not impair activity of lysostaphin. Phagocytic activity of human peripheral blood monocytes was not significantly influenced by cationic substances.
Article
Tissue injury of many types may be caused by deposited complexes of antigen and antibody. The circumstances under which the complexes form and deposit often determine the location and type of injury observed: If the complex forms in the circulation, deposition may occur in arterial walls and glomeruli, initiating lesions in those tissues. If the complex forms in the synovial tissues or spaces, then the reaction will develop at that point. Any local source of antigen will initiate these lesions once antibody is formed. If the source of antigen persists, antibody-forming cells soon establish themselves locally as they do in the active Arthus reaction, and injury will become chronic. When the antibody formed is capable of activating complement, polymorphonuclear leukocytes (PMNs, neutrophils) will accumulate, leading to release of injurious constituents. Such is the case in acute glomerulonephritis, arteritis, synovitis, and vasculitis. The ability of complement to attract the PMNs has been demonstrated as an in vitro phenomenon and as a clear possibility in vivo. The requirement of PMNs in the development of the lesions has been demonstrated. The process by which PMNs and other cells (platelets, mast cells, basophils, and macrophages) release injurious constituents is of great interest currently. The exocytosis of their cytoplasmic granules constitutes the major mechanism of release and involves a complicated series of events outlined in this review. The constituents of PMNs capable of injuring tissue in various ways is described, from peptides capable of increasing vascular permeability, to enzymes that indirectly bring more PMNs and other cells into the lesion, to proteolytic enzymes that hydrolyze vital structures in the tissues. These agents were most likely designed to rid the host of invaders; but at times they are unfortunately directed against the host's own tissues.
Article
The primary role of neutrophil leukocytes in inflammation is to ingest, kill, and degrade harmful microorganisms; a process recognized by Metchnikoff in 1891 (see Metchnikoff, 1968).
Article
Three cationic proteins from the granules of human neutrophil granulocytes were obtained in a high degree of purity be means of affinity chromatography on 4-phenylbutylamine-Sepharose. Together with lysozyme, the three cationic proteins exhibit the highest electrophoretic mobility toward the cathode in acrylamide gels at moderately acid pH, among the granule constituents that are solubilized in 0.1 M phosphate buffer, pH 7.0, containing 1 M NaCl. The three cationic proteins represent a group of "neutral proteases" distinct from elastase and collagenase. They hydrolyze casein, azocasein and the chymotrypsin substrate N-acetyl-L-tyrosine ethyl ester. Optimal activity is found at pH 7.4-7;5. The enzymes are inhibited by the specific chymotrypsin inhibitor N-tosyl-L-phenylalanylchloromethane and by the naturally occurring inhibitors alpha-antichymotrypsin, alpha-1-antitrypsin, as well as by the trypsin inhibitors from soy beans and limabeans.
Article
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Die anionischen Polysaccharide Chondroitinsulfat-Protein und Hyaluronat entfalten in wäßriger Lösung in Konzentrationen zwischen 10 und 20 mg/ml einen Molekularsiebeffekt, der in einer Beeinflussung der Diffusion gelöster Substanzen zum Ausdruck kommt und Analogien zu den in der Basalmembran von Blutkapillaren ablaufenden Permeationsvorgängen aufweist. Während Makromoleküle (Rinderserumalbumin, kolloidales Gold [ Die durch anionische Polysaccharide bewirkte Diffusionsbehinderung kann durch Polykationen (lysinreiches Polypeptid aus Kalbsthymus, Poly-L-Lysin, Oligo-N-methyl-morpholiniumpropylenoxid), ferner durch Trypsin, α-Chymotrypsin und Kallikrein, nicht jedoch durch basische Aminosäuren und Peptide (Lys, Arg, His, Arg-Gly-Arg) oder Histamin aufgehoben werden. Die wirksamen Polykationen beeinflussen Chondroitinsulfat-Protein und Hyaluronat durch Änderung ihrer makromolekularen Eigenschaften (Hydratation, Viskosität, Löslichkeit), in höheren Konzentrationen reagieren sie mit ihnen unter Bildung definierter wasserunlöslicher Komplexe (Präzipitate). Die Reaktion anionischer Polysaccharide mit biogenen basischen Polypeptiden wird als möglicher physiologischer Regelmechanismus zur Beeinflussung der Blutkapillar-Permeabilität aufgefaßt.
Article
1. A quantitative turbidimetric method for the estimation of hyaluronidase activity, based on the ability of the enzyme to decrease the capacity of the polysaccharide to precipitate acidified protein has been developed. Two units of hyaluronidase, by this method, are equivalent to one viscosity-reducing unit. 2. Hyaluronidase added to a phagocytic system containing defibrinated human blood, immune or non-immune, greatly increases the rate of phagocytosis of group A streptococci. Phagocytosis of Type I pneumococci is not affected by hyaluronidase under the same conditions. 3. The bactericidal activity of non-immune blood against group A streptococci is increased by hyaluronidase; the activity of immune blood is, however, somewhat inhibited by the enzyme. Killing of pneumococci is not affected by the presence of the enzyme. 4. Mice can be protected against group A streptococcal infection by frequent treatment with 200 turbidity-reducing units of hyaluronidase; the protective action of the enzyme is removed by heating at 60 degrees C. for 1 hour. Mice infected with Type I pneumococcus and treated with hyaluronidase die somewhat sooner than the untreated controls.
Article
EOSINOPHILS are phagocytic cells, and granules adjacent to the phagocytic vacuolo rupture1. Eosinophil granules contain a number of acid hydrolases together with basic protein and peroxidase2 and during phagocytosis these substances are discharged from the disrupted granules into, or alongside, the phagocytic vacuole. The acid hydrolases released from eosinophil granules probably have a digestive function, but the significance of the other granule components is not known. This work reports the presence in eosinophil granules of a substance capable of causing the disruption of mast cells. The active principle appears to be associated with the enzyme peroxidase.
Article
A method has been described for isolation of the specific cytoplasmic granules of rabbit polymorphonuclear leucocytes. Homogeneous suspensions of leucocytes were disrupted by lysis in 0.34 M sucrose. This procedure liberated the cytoplasmic contents of the cell and dissolved a considerable proportion of the nuclei. Following disruption, the sucrose lysate was separated into three fractions by differential centrifugation, i.e. 400 g or nuclear pellet, 8,200 g or granule pellet and the postgranule supernate. Microscopic examination revealed that the 8,200 g pellet was composed of intact granules as well as occasional mitochondria. The other two fractions were morphologically heterogeneous. Studies with isolated granules demonstrated their lysis by a variety of weak acids and surface-active agents. When buffered solutions were employed between the ranges of pH 2.0 and 9.0, granule lysis began at pH 5.5 and was complete at pH 4.0. Chemical analysis disclosed that the granule pellet contained protein and phospholipid with only traces of nucleic acids. Approximately 70 to 80 per cent of the total cellular antimicrobial agent phagocytin was present in the granule fraction. This material was liberated from the granules by acid (pH 5.0 or lower). Studies on selected enzymes showed that acid phosphatase, alkaline phosphatase, nucleotidase, ribonuclease, deoxyribonuclease, and beta glucuronidase were predominantly localized in the granule fraction. Approximately 50 per cent of total cellular lysozyme and cathepsin were also present in the 8,200 g pellet. Disruption of the granules was associated with the release of the majority of granule protein and enzymes in a non-sedimentable form. The properties and composition of rabbit polymorphonuclear leucocyte granules seem to be analogous to those of liver lysosomes.
Article
Preparation of the skin of rabbits by intradermal injection of the granule fraction obtained from peritoneal granulocytes, followed by intravenous injection of endotoxin, results in lesions of hemorrhagic necrosis which resemble the local Shwartzman phenomenon. An injection of granules into the skin enhances the reactivity of this area to the reversed passive Arthus phenomenon, and makes it possible to elicit this reaction in leucopenic animals. It is suggested that one or more of the acid hydrolases contained in granules may be implicated in the pathogenesis of vascular damage in the Shwartzman and Arthus reactions.
Article
Severe local Shwartzman reactions have been induced in rabbits by endo-toxin as well as by granules isolated from polymorphonuclear cells. Administration of polypeptide possessing potent antiprotease activity inhibited regularly and almost completely the Shwartzman reaction induced either by endotoxin or by polymorphonuclear granules. It is hypothesized that the tissue damage observed in the Shwartzman reaction is conditioned by release or activation of intracellular lysosomal enzymes contained in granulocytes.
Article
Material obtained from the in vitro incubation of granulocytes from saline-induced peritoneal exudates of rabbits has been shown to produce inflammation and fever in rabbits. The supernatant material from cells incubated in saline has been termed granulocytic substance (GS) and is heat-labile. Its production is temperature dependent, occurring at 37 degrees C but not at 4 degrees C, requires viable cells, and is inhibited by potassium ions. A similar material is liberated when cells are incubated in a more physiologic medium. Freezing and thawing of granulocytes does not release GS and the active principle cannot be obtained from the incubation of lymphocytes. GS produces a delayed inflammatory response as measured by leucocyte sticking and emigration in the rabbit ear chamber and the leakage of protein-conjugated dye at the site of intradermal injection. The former response can be accurately quantitated by calculation of the inflammatory index from reactions observed in the ear chamber. The inflammatory reaction and the properties of GS distinguish it from a variety of previously described mediators of inflammation, but GS appears to be identical with leucocytic pyrogen. The possible role of GS in delayed and protracted inflammation and its relationship to the pathogenesis of fever are discussed.
While it is an established fact that histamine and serotonin increase the permeability of blood vessels, the exact portion of the vascular tree which is so affected has not been conclusively demonstrated. The present study was undertaken to clarify this point. Our experiments were based on a method to which we refer as "vascular labeling," and which permits one to identify leaking vessels by means of visible accumulations of foreign particles within their walls. The mechanism of the labeling, elucidated by previous electron microscopic studies, is the following. Histamine and serotonin cause the endothelial cells of certain vessels to separate, and thus to create discrete intercellular gaps. Plasma escapes through these gaps, and filters through the basement membrane. If the plasma has been previously loaded (by intravenous injection) with colloidal particles of a black material such as carbon or mercuric sulfide, these particles-too large to pass through the basement membrane-will be retained and accumulate in visible amounts within the wall of the leaking vessel. This method is used to maximal advantage if the tissue is cleared and examined by transillumination in toto, so that leaking vessels can be accurately identified in their relationship to the vascular tree. As a test tissue we used the rat cremaster, a laminar striated muscle which can be easily excised with its vascular supply virtually intact. The rats were prepared with an intravenous injection of carbon or HgS, and a subcutaneous injection into the scrotum of histamine, serotonin, or NaCl (as a control). The injected drug diffused into the underlying cremaster and the vessels became labeled. One hour later, when the carbon had been cleared from the blood stream, the animal was killed. The cremaster was excised, stretched, fixed in formalin, cleared in glycerin, and examined by transillumination under a light microscope. The lesions induced by histamine and serotonin were identical. The leaking vessels, as indicated by the carbon deposits, always belonged to the venous side of the circulation. The heaviest deposits were found in venules 20 to 30 micra in diameter. The deposits decreased towards larger venules up to a maximum diameter of 75 to 80 micra, and towards the finer vessels until the caliber reached approximately 7 micra. Essentially spared by the deposits were the finest vessels, 4 to 7 micra in diameter, and constituting an extensive network oriented along the muscular fibers. By killing animals at varying intervals after the injections, it was found that the carbon particles were slowly removed from the vascular walls by the action of phagocytic cells. After 10 months there was still enough carbon locally to be recognized by the naked eye.
Effects of leukocyte lysosome fractions on rat mast cells in vitro
  • S Schaefer
Schaefer, S., and Janoff, A., Effects of leukocyte lysosome fractions on rat mast cells in vitro. M.S. thesis, to be published.
Leukocyte lysosomal basic protein as a mediator of inflammation
  • E S Golub
  • J K Spitznagel
Golub, E. S., and Spitznagel, J. K., Leukocyte lysosomal basic protein as a mediator of inflammation, submitted for publication.
The turbidometric assay of hyaluronidase
  • S Tolksdorf
  • M H Mccready
  • D R Mccullagh
  • E Schwenk
Tolksdorf, S., McCready, M. H., McCullagh, D. R., and Schwenk, E., The turbidometric assay of hyaluronidase, J. Lab. and Clin. Meg., 1949, 34, 74.