Article

Production of Inflammatory Changes in the Microcirculation by Cationic Proteins Extracted from Lysosomes

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Abstract

Lysosomal granules of rabbit exudate polymorphonuclear (PMN) leucocytes were isolated and then lysed by freezing-thawing. Topical application of this material to rat and rabbit mesentery produced sticking and emigration of leucocytes, stasis of blood flow, and petechial hemorrhage. The granule-free, supernatant fraction of the homogenized leucocytes failed to produce any of these reactions. Cationic proteins extracted from these granules by weak acid and precipitated by ethanol at concentrations of 20 and 45 per cent, were also tested on heterologous, homologous, and autologous mesenteric vessels. The 20 per cent ethanol-precipitated fraction produced all of the aforementioned injury reactions, whereas the 45 per cent fraction was inactive. The intensity of inflammatory changes produced by the active cationic protein fraction was greater than that produced by lysed whole granules. Both the 20 per cent and 45 per cent ethanol fractions of cationic protein induced clumping of rabbit platelets, in vitro. The 20 per cent ethanol fraction also caused a slight acceleration in rate of swelling of isolated rabbit liver mitochondria. The active material proved to be non-pyrogenic in rabbits. This material exhibited no kinin-like effects when tested on isolated smooth muscle preparations (rabbit aorta and guinea pig ileum). In the rat, the protein produced a transient vasodepression which was inhibited by pretreatment of the animal with an antihistamine. Ultraviolet absorption data and ribose assays showed that the 20 per cent ethanol fraction contained only 4 per cent or less of ribonucleic acid. Upon electrophoresis in starch gel, using acid buffer, this fraction separated into at least three major components which migrated towards the cathode. Precipitation of one of the slowly migrating components by titration of the fraction to pH 10.5 greatly increased the inflammatory activity of the material. The inflammatory basic protein fraction was essentially devoid of acid phosphatase, beta glucuronidase, acid ribonuclease, lysozyme, and catalase activity. The non-inflammatory basic protein fraction contained appreciable quantities of acid ribonuclease and lysozyme. The foregoing data demonstrate that certain of the cationic proteins present in lysosomes of rabbit exudate PMN leucocytes can reproduce one of the cardinal features of the inflammatory response; namely, adhesion and emigration of leucocytes in the microcirculation. These findings offer fresh support for the role of lysosomes in the pathogenesis of tissue injury, and may help to account for the propagation of leucocyte emigration to peak numbers during inflammatory reactions.

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... Effect of M C F on Isolated Guinea Pig Ileum.--Strips of atropinized guinea pig ileum (see Materials and Methods) which responded to as little as 0.008 /~g per ml of histamine and bradykinin, gave no contractions (either immediate or delayed) when incubated with over 1 #g per ml of MCF (see Table III). This result is not surprising in view of earlier observations of a similar lack of smooth muscle stimulating activity in the whole ET20 fraction (12,31). In the present tests, responses of the ileum strip to control agonists (histamine and bradykinin) were measured both before and after incubation with the test agent (MCF). ...
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The metabolic reactions responsible for the release of endogenous pyrogen from rabbit granulocytes incubated in 0.15 M NaCl are specifically inhibited by the presence of K+ (and by related alkali metal ions, Rb+ and Cs+) in the medium. The inhibitory action of K+ apparently involves penetration of the cell membrane and is directly antagonized by the cardiac glycoside, ouabain. It is concluded, therefore, that the inhibition of pyrogen release by extracellular K+ is due to transport of K+ into the cell. Although the precise molecular mechanisms which are responsible for the release of pyrogen from granulocytes incubated in K-free saline have not been elucidated, further study of the process has revealed: (a) that it is preceded by the accumulation of pyrogen within the cell, (b) that it depends upon the catalytic action of one or more sulfhydryl-containing enzymes, (c) that it does not require energy, either from glycolysis or from reactions depending on molecular oxygen, and (d) that its inhibition by K+ and by arsenite is qualitatively similar to the depression caused by these same reagents on the release of other leucocytic proteins; i.e., lysozyme and aldolase.
Article
Rabbit leucocytes, if properly stimulated, will yield an endogenous or leucocytic pyrogen that is chemically and biologically distinct from exogenous pyrogens such as bacterial endotoxins. Just how the leucocytic pyrogen is produced is not yet known but the present study was undertaken in an effort to learn more of its chemical properties.Crude leucocytic pyrogen, obtained from sterile peritoneal exudates, was dialyzed against distilled water, freeze-dried, and applied first to a DEAE column to remove much of the protein, then to a phosphorylated cellulose column which adsorbed leucocytic pyrogen while allowing virtually all of the remaining protein to be eluted. The leucocytic pyrogen in turn was eluted by a stepwise increase in pH with a resulting thirty-fold increase in pyrogenic activity per gram of protein as compared with the crude pyrogen. This pyrogenic protein material is inactivated by 60°C for 5 minutes and by trypsin digestion. It is electrophoretically homogenous at pH 5.0 and 8.0 and yields at least nine amino acids on hydrolysis.The methods outlined above enable rabbit leucocytic pyrogen to be obtained in a relatively pure form so that further chemical characterization is now possible.
Article
1.1. A simple and useful method for the purification and fractionation of calf thymus histone is presented. Taking advantage of solubility differences in ethanol-water mixtures, two fractions, histones I and II, were obtained.2.2. Physicochemical and chemical properties of both fractions were studied and compared with each other. Histone I, the main fraction, markedly differed from histone II in several properties, i.e., molecular weight, electrophoretic behaviour, chemical composition, etc.3.3. Although each fraction showed a good degree of homogeneity in an ultracentrifuge, it was electrophoretically not homogeneous. Histone I contained at least three components and histone II showed mainly one component with three minor components. It was, therefore, concluded that there are more than four basic proteins present in calf thymus histone.
Article
A substance with pyrogenic properties appears in the blood streams of rabbits made febrile by the intravenous inoculation of the PR8 strain of influenza A and Newcastle disease viruses (NDV). By means of a technique involving passive transfer of sera from animals given virus to recipient rabbits, the titer of circulating pyrogen was found to be closely correlated with the course of fever produced by virus. Certain properties of the pyrogen are described which differentiate it from the originally injected virus and suggest that the induced pyrogen is of endogenous origin. These properties resemble those of endogenous pyrogens occurring in other forms of experimental fever. The source of virus-induced pyrogen is unknown. In vitro incubation of virus with various constituents of the circulation did not result in the appearance of endogenous pyrogen. Granulocytopenia induced by HN2 failed to influence either fever or the production of endogenous pyrogen in rabbits injected with NDV. Similarly, the intraperitoneal inoculation of NDV into prepared exudates did not modify the febrile response. These findings do not lend support to the possibility that the polymorphonuclear leukocyte is a significant source of endogenous pyrogen in virus-induced fever. It is concluded that the liberation of an endogenous pyrogen from some as yet undefined source is an essential step in the pathogenesis of fever caused by the influenza group of viruses.
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
A marked reduction in numbers of cytoplasmic granules in rabbit and human polymorphonuclear leucocytes takes place following ingestion of various microorganisms or of a yeast cell wall preparation. The degranulation occurs within 30 minutes of phagocytosis, and is directly related to the quantity of material engulfed. White cells completely degranulated following phagocytosis of large numbers of microorganisms remain viable for at least 1 hour. The granules of polymorphonuclear leucocytes contain the antimicrobial agent, phagocytin, and various digestive enzymes. These substances thus are released into the cytoplasm or into vacuoles following ingestion of foreign material. The granule system and granule lysis mechanism may well play a central role in the primary function of these specialized cells; namely, that of destroying invading microorganisms.
Article
1. Gentle stretching of the rabbit aortic strip produces rapid relaxation, and single doses can be applied at intervals as short as every 7 minutes. 2. Use of this preparation for a variety of purposes is described, including a 4-point bioassay of catecholamines and pharmacological studies, as exemplified by potentiation and inactivation.
Article
USING a new technique, Boyden1 has shown that rabbit serum after incubation in vitro with an antigen-antibody precipitate becomes strongly chemotactic to rabbit granulocytes. Prior heating of the serum to 56° for 30 min was found to prevent activation of the chemotactic principle which, once formed, was relatively heat-stable.
Article
complex ofthematrix, andthatliberation ofother lysosomal hydrolytic enzymesmightcausethecellular changes observed incartilage cultivated inthepresence of thevitamin; thishypothesis was supported by observations onthespecificity oftheaction ofthe vitamin, bothinorganculture andontheisolated lysosomes (Fell, Dingle& Webb,1962).Recent experiments ontheeffect ofthevitamin onthecell membraneoferythrocytes (Dingle & Lucy,1962), andonmitochondria (J.A.Lucy,M.Luscombe& J.T.Dingle, unpublished work),provide further evidence thatonesite ofvitamin A action isatthe lipoprotein membranes ofcells andtheir organelles. Thoughtheevidence sofarobtained favoured thishypothesis concerning themodeofaction of vitamin A oncartilage, threeimportant pieces of information werestill lacking. (1)Although normal cartilaginous rudiments hadbeenfoundtocontain a protease, withanacidpHoptimum, thatwhenfreed by treatmentwithhypo-osmotic solutions can degrade thematrix(Lucyetal.1961), andthough therewas increased lysis oftheplasmaclotby cartilaginous rudiments growninthepresence of excess ofvitamin A (Dingle, Lucy& Fell, 1961), the liberation ofanacidprotease bythevitamin Atreated explants had notbeenclearly demonstrated. (2)Itwasnotknownwhether thevitamin could release theenzymesfromintracellular particles isolated fromcartilage, thoughithadbeenshownto liberate aprotease fromlysosomes isolated fromrat liver (Dingle, 1961). (3)Itwasnotknownwhether acidhydrolases fromisolated intracellular particles coulddegrade cartilage rudiments. Thesethree points havenowbeeninvestigated.
Article
Induction of acute hypervitaminosis A in the guinea pig can substantially suppress delayed-type hypersensitivity and Arthus reactivity of moderate intensity and the inflammatory response to intradermal injection of diphtheria toxin. No effect of Vit. A was demonstrated on the clearance of bacteriophage φX 174 from the circulation or on antibody formation to the phage. The results suggest that acute hypervitaminosis A can suppress inflammatory responses of moderate intensity in the skin of guinea pigs, possibly as a consequence of an action on lysosomes.
Article
A lysosomal fraction from polymorphonuclear leukocytes has been directly analyzed for basic proteins by zone electrophoresis. Evidence for the presence of bactericidal basic proteins in polymorphonuclear leukocyte lysosomes is presented.
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
1. Phagocytosis promotes the release of endogenous pyrogen from polymorphonuclear leucocytes. 2. The release of pyrogen, though initiated by the phagocytic event, is not synchronous with it. 3. The postphagocytic release mechanism is not inhibited by sodium fluoride and, therefore, appears not to require continued production of energy by the cell. 4. The release process, on the other hand, is inhibited by arsenite, suggesting the participation of one or more sulfhydryl-dependent enzymes in the over-all reaction. 5. Particle for particle, the ingestion of heat-killed rough pneumococci causes the release of approximately 100 times as much pyrogen as the ingestion of polystyrene beads of the same size. 6. The pyrogen release mechanism of polymorphonuclear leucocytes separated directly from blood, unlike that of granulocytes in acute inflammatory exudates, is not readily activated by incubation of the cells in K-free saline. Despite this difference, both blood and exudate leucocytes following phagocytosis release large amounts of pyrogen, even in the presence of K(+). The fact that the postphagocytic reaction is uninhibited by the concentrations of K(+) which are present in plasma and extracellular fluids, suggests that this mechanism of pyrogen release may well operate in vivo. 7. As might be expected from the foregoing observations, the intravenous injection of a sufficiently large number of heat-killed pneumococci causes fever in the intact host. Intravenously injected polystyrene beads, on the other hand, are significantly less pyrogenic. Evidence is presented to support the conclusion that the fever in both instances is caused by pyrogen released from the circulating leucocytes which have phagocyted the injected particles. 8. The possible relationships of these findings to the pathogenesis of fevers caused by acute bacterial infections are discussed.
Article
A cationic protein fraction isolated from rabbit polymorphonuclear leukocyte lysosomes causes adhesion and emigration of leukocytes and petechial hemorrhage in the microcirculation of the rat and rabbit mesentery.
Article
The inhibition of the Shwartzman phenomenon by nitrogen mustard and benzene has been confirmed and investigated further. This inhibition can be correlated with the leucopenia induced by these agents. Evidence is presented indicating that polymorphonuclear leucocytes play an essential role in the preparation of the skin for the Shwartzman phenomenon.
Inhibition of local tissue injury by vitamin A, bacterial endotoxin
  • A Janoff
  • G Kaley
Janoff, A., and Kaley, G., Inhibition of local tissue injury by vitamin A, bacterial endotoxin, and 48/80, Fed. Proc., 22,433, (abstracts).
II. Effects on delayed -type hypersensitivity
. II. Effects on delayed -type hypersensitivity, Proc. Soc. Exp. Biol. and Med., 1963, 112, 287.
Unpublished observations reported at Federation of American Societies of
  • E S Golub
  • J K Spitznagel
Golub, E. S., and Spitznagel, J. K., Unpublished observations reported at Federation of American Societies of Experimental Biology Meeting, Chicago, 1964.