Comparison of anti-inflammatory compounds in the carrageenan induced paw edema model and the reversed passive Arthus model utilizing the same animal

In order to better define antiinflammatory activity in new agents, a test was devised utilizing both carrageenan induced paw edema and the reversed passive Arthus reaction in the same animal. The model of carrageenan induced rat paw edema is a standard laboratory assay used to predict classical "aspirin-like" antiinflammatory molecules. The reversed passive cutaneous Arthus reaction involves precipitating antigen-antibody complexes, complement and infiltrating polymorphonuclear leukocytes (PMN's) and can be used to identify agents that affect one or more of these factors specifically. Antiinflammatory compounds were given orally one hour prior to the administration of carrageenan and goat anti-rat serum. Comparisons were made between several non-steroidal compounds and the steroid hydrocortisone. All of the compounds tested gave good carrageenan activity, but only hydrocortisone produced significant Arthus lesion inhibition in this assay.     

A specific antagonist of platelet-activating factor suppresses oedema formation in an Arthus reaction but not oedema induced by leukocyte chemoattractants in rabbit skin

The properties of a novel platelet-activating factor (PAF) antagonist, L-652731, on oedema responses in rabbit skin induced by exogenous inflammatory mediators and by mediators generated endogenously in a reversed passive Arthus reaction have been investigated. Oedema responses in the skin were measured by using the local accumulation of i.v. injected 125I-albumin. The antagonist, mixed with mediators before intradermal injection, caused a dose-dependent suppression of oedema responses to PAF. In contrast, responses induced by other directly acting mediators (bradykinin and histamine) and responses induced by PMN leukocyte-dependent mediators (C5a des Arg, N-formyl-methionyl-leucyl-phenylalanine, and leukotriene B4) were not suppressed. Thus, a secondary release of PAF does not appear to be involved in mediating the actions of these agents. In a reversed passive Arthus reaction, intradermal injection of L-652731 together with antibody resulted in a significant inhibition of the oedema formation measured for 2 hr after i.v. antigen challenge. In contrast, oedema responses induced by intradermal injection of preformed immune complexes were not affected by the antagonist. These results suggest that the endogenous production of PAF, in close proximity to microvascular endothelial cells, appears to be an important step in the development of an Arthus reaction. The cellular source of PAF is unknown, but one possibility is the PMN leukocyte, which releases PAF during phagocytosis of immune complexes.     

Stimulation of anaphylaxis in the mouse footpad by dietary fish oil fatty acids

The effect of fish oil-derived omega-3 (omega-3) fatty acids on anaphylaxis, Arthus and delayed type hypersensitivity reactions in mice has been investigated. Mice on a normal chow diet were fed eicosapentaenoic acid and docosahexaenoic acid at a dose of 500 and 333 mg/kg/day, respectively, by a gastric tube over a period of 61 days. Control groups were given water, safflower oil or oleic acid. Anaphylactic and Arthus type reactions were induced in the mouse footpad using bovine serum albumin as an antigen. Carrageenin was utilized to produce a delayed type hypersensitivity reaction. The animals fed omega-3 fatty acids induced a more anaphylactic foodpad reaction. There was no significant effect of the diet on Arthus and delayed type hypersensitivity responses. There was no effect of the fish oil-supplemented diet on production of antibodies to bovine serum albumin. Synthesis of prostaglandin E2 by peritoneal macrophages was significantly inhibited in the animals fed omega-3 fatty acid-enriched fish oil, while leukotriene B4 production was not affected. These results suggest that a diet enriched in omega-3 fatty acids modulates production of arachidonic acid metabolites and this may influence anaphylaxis, but not Arthus and cellular mediated hypersensitivity responses.     

Accumulation of platelets at sites of antigen-antibody-mediated injury: a possible role for IgE antibody and mast cells.

Circulating 51Cr-labeled platelets accumulate at skin sites in which a reversed passive Arthus reaction has been induced. The accumulation is biphasic in time and is accompanied by an increased vascular permeability. Increased permeability itself, however, will not produce localization of platelets. A similar platelet accumulation was observed upon injection of compound 48/80 or anti-IgE antibody into the skin and this was not altered in rabbits depleted of complement or neutrophils. Activation of skin mast cells and release of a platelet-activating factor (PAF) is suggested as a mechanism for the effect produced by anti-IgE and compound 48/80. The first phase of platelet accumulation in the Arthus reaction was also unaffected in rabbits depleted of neutrophils or complement, which may suggest a role for IgE antibody and mast cells. The second phase of accumulation was diminished in complement-depleted animals and abrogated in rabbits without neutrophils, suggesting a complement and neutrophil-mediated process but which still might be mediated through mast cell activation by neutrophil cationic protein.     

Pathways to inflammation induced by immune complexes: development of the Arthus reaction

The changes associated with inflammation induced by immune complexes (reversed passive Arthus reaction induced with egg albumin-anti-egg albumin) were quantitated and the kinetics of the various vascular phenomena were ascertained. Hyperemia, increase in vascular permeability, platelet accumulation, and polymorphonuclear (PMN) leukocyte accumulation occurred relatively early after induction of the inflammatory lesions, and peaked in 2-4 h. Hemorrhage peaked in 6-h-old lesions. Morphological studies confirmed that almost all infiltrating cells were PMN leukocytes and immunofluorescent tracer studies showed immune complexes in vessel walls as early as 15 min after i.v. injection of the fluoresceinated antigen and the intradermal injection of antibody. By 8 h the progression of the lesions had subsided and by 24 h there were signs of resolution. A pathway for the development of the inflammatory lesions induced with immune complexes is proposed.     

Polygonolide,an isocoumarin from Polygonum hydropiper possessing anti-inflammatory activity

A new isocoumarin polygonolide which inhibits the reversed passive Arthus reaction has been isolated from the methanol extract of the root of Polygonum hydropiper. The structure of polygonolide has been elucidated on the basis of spectroscopic data and confirmed to be 3,4-dimethyl-6-methoxy-8-hydroxyisocoumarin by total synthesis.     

Pathological study of Streptococcus faecalis antigen-induced arthritis in New Zealand white rabbits

The pathological examination of the rabbit knee joint with antigen-induced arthritis produced by heat-killed Streptococcus faecalis (Str. faec.) as antigen was carried out. Macroscopically, there were findings of acute inflammation about five hours after the injection. Histopathologically, very remarkable acute exudative inflammation was seen 48 hours later. This supported the picture of Arthus reaction. The Arthus reaction disappeared with time, and this supported the view of delayed-type hypersensitivity three weeks later. After that, an obvious chronic inflammation was admitted in 10 weeks. This resembled the histopathological feature of rheumatoid arthritis (RA) when these findings are summarized. It was suggested that arthritis produced by Str. faec. progresses from an acute condition to a one with time. As mentioned above, it thought that Arthus reaction of both immediate hypersensitivity and delayed-type hypersensitivity are necessary in the occurrence of this arthritis.     

Fibrinogen and plantar edema induced by lambda carrageenan

1. Batroxobine which induces fibrinogen consumption, accelerates the development of 48/80 and lambda carrageenan oedema: in the rat the various products released from fibrinogen and fibrin increase these inflammatory reactions. 2. Heparin and epsilon-aminocaproic acid have no influence on carrageenan oedema: the derivatives of fibrinogen do not take part in the constitution of the inflammatory reaction induced by carrageenan. 3. The anti-inflammatory action of this sulfated polygalactose does not depend on its anti-coagulant activity.     

Inhibition of carrageenan edema by carrageenan itself]

The influence of two kinin forming agents: iota carrageenan and ellagic acid, on the paw oedema induced by 48/80, an amino-liberator, or by carrageenan iota, has been studied, in the Rat. Ellagic acid and carrageenan, by intraperitoneal injection, reduce the paw oedema induced respectively by 48/80 and carrageenan itself. This inhibition depends on a non-specific "counter-irritation" and not on kininogen stores depletion. Ellagic acid, by intravenous injection, diminishes the oedema induced by carrageenan; swelling due to 48/80, is not affected. So kininogen activation plays some role in the inflammatory processes induced by iota carrageenan. Carrageenan by intravenous injection, suppresses his own inflammatory action but does not influence at all the similar action of 48/80. The anti-inflammatory effect of carrageenan does not exclusively depend on kininogen stores depletion.     

Ultrastructural evidence for lack of tissue damage in a local immune complex reaction: a study of a mild passive Arthus reaction.

An electron microscopic study of a mild reversed passive Arthus reaction (RPAR) was performed using a horeradish peroxidase (HRP)-anti-HRP system to demonstrate the biological usefulness of the process which exists to clear tissue of immune complexes. To disclose the antigen, HRP, the biopsy material was subjected to a peroxidase reaction. HRP was mainly detected within irregular electron-dense precipitates which were considered insoluable HRP-anti-HRP immune complexes. Neutrophils were found to phagocytose and digest the deposited immune complexes in a similar way as described previously. But there was no vascular and other tissue damage. This study provides morphological evidence that the process of clearing tissues of immune complexes need not be harmful to the host.     

The role of thromboxane A2 (TxA2) in allergic cutaneous reactions and the effect of (E)-3-[p-(1H-imidazol-1-ylmethyl) phenyl]-2-propenoic acid hydrochloride (OKY-046), a TxA2 synthetase inhibitor

To study the role of thromboxane A2 (TxA2) in cutaneous allergic reactions, the effect of (E)-3-[p-(1H-Imidazol-1-ylmethyl)phenyl]-2-propenoic acid hydrochloride (OKY-046), a selective TxA2 synthetase inhibitor, on cutaneous reactions in rats and mice was studied. Simultaneously, the effect of 9,11-methanoepoxy-prostaglandin H2 (U-46619), a stable analogue of TxA2, on capillary permeability in mouse and rat skin was investigated. Passive cutaneous anaphylaxis (PCA) in mouse ear was clearly inhibited by OKY-046 but not by indomethacin. The inhibitory action of OKY-046 was not influenced by pretreatment with indomethacin. Moreover, prostaglandin I2, which accumulated as a result of the inhibition of TxA2 synthetase, did not affect the PCA. But, the dye leakages caused by histamine, serotonin and leukotriene C4 in mouse ear were clearly inhibited by OKY-046. In addition, OKY-046 inhibited rat reversed cutaneous anaphylaxis, but its inhibitory action was not affected by pretreatment with indomethacin. Contrary to the above results, rat footpad passive Arthus reaction and mouse footpad tuberculin delayed hypersensitivity reaction were not affected by OKY-046. Additionally, U-46619 did not cause an increase of capillary permeability in either mouse and rat skin. These results suggest a slight role of TxA2 in cutaneous allergic reactions in mice and rats and the efficacy of OKY-046 on Type I and II reactions regardless of the inhibition of TxA2 synthetase activity.     

Involvement of steroids in anti-inflammatory effects of peripheral benzodiazepine receptor ligands

Mouse paw oedema induced by carrageenan is used to determine if glucocorticoids are involved in the anti-inflammatory effects of peripheral benzodiazepine receptor ligands. The anti-inflammatory responses elicited by i.p. treatment with 1-(2-chlorophenyl)-N-methyl-N (1-methyl-propyl)-3-isoquinoline carboxamide (PK11195) and 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2-H-1, 4-benzodiazepin-2 (Ro5-4864) were reversed by aminoglutethimide, an inhibitor of steroidal synthesis. Intraplantar injection into the ipsilateral paw of Ro5-4864, but not PK11195, inhibited the formation of paw oedema and this effect was reversed by aminoglutethimide. These results suggest that glucocorticoids are involved in the systemic and local anti-inflammatory effects of Ro5-4864 and only in the systemic response to PK11195.     

Antiphlogistic action of phenylbutazone in normal and adrenalectomized rats of different ages relation to the 5-hydroxytryptamine blood concentration.

The authors have attempted to demonstrate the effect of bilateral adrenalectomy on carrageenin-induced oedema and on the antiphlogistic action of phenylbutazone in relation to the changes of blood 5-hydroxtryptamine (5-HT) in rats of different ages (21 days, 42 days, 3 months and 18 months old). It was found that the influence of adrenalectomy on the antiphlogistic action of phenylbutazone and on the blood 5-HT concentration is related to the age of rats. The lowest antiphlogistic action of phenylbutazone was found in 21-day-old rats and highest in the 18-month-old ones. In adrenalectomized 21- and 42-day-old rats the antiphlogistic action is decreased and fully suppressed in rats 3 and 18 months old. Adrenalectomy does not influenced basal values of blood 5-HT concentration. Blood 5-HT in adrenalectomized rats with inflammationadrenalectomized rats 42 days and 3 months old with inflammation after injection of phenylbutazone an increase of 5-HT was observed, but in 18-month-old animals in which antiphlogistic action is highest a decrease of 5-HT was observed.     

Human recombinant soluble decay accelerating factor inhibits complement activation in vitro and in vivo.

Complement plays a role in activating the inflammatory response and has been implicated in the pathogenesis of some inflammatory diseases. With a view toward controlling unwanted C activation, we evaluated the C regulator, human decay accelerating factor (DAF). Three forms of recombinant DAF were purified from transfected Chinese hamster ovary cells: glycophosphatidylinositol (GPI)-linked membrane DAF (mDAF) extracted from cell membranes; spontaneously shed soluble DAF (sDAF) derived from mDAF; and a novel secreted protein (seDAF), generated by deletion of the signal for GPI attachment. We show that all three molecules inhibit both the classical and alternative pathways of C activation. The following observations indicate that mDAF extracted from Chinese hamster ovary cells reincorporates into RBC membranes via its GPI anchor: 1) cells that are preincubated with mDAF and then washed remain fully protected from C-mediated hemolysis; 2) incubation with phosphatidylinositol-specific phospholipase C abolishes this protection; and 3) sDAF and seDAF, which lack a GPI anchor, do not associate with cell membranes. mDAF is a more potent inhibitor of C-mediated hemolysis than either sDAF or seDAF, suggesting that incorporation into cell membranes greatly enhances the efficiency with which DAF inhibits C activation on the cell surface. In contrast, C activation in the fluid phase is inhibited by sDAF and seDAF, but not by mDAF, possibly due to interference by serum lipoproteins. A reversed passive Arthus reaction in guinea pigs was used to evaluate the ability of recombinant seDAF to inhibit C activation in vivo. When administered at dermal sites, seDAF reduced the severity of immune complex-mediated inflammatory reactions induced by a reversed passive Arthus reaction, as judged by both gross and histologic examination. These data indicate that seDAF may be useful as an anti-inflammatory therapeutic.     

Corneal test as a reliable method for detection of the delayed-type hypersensitivity reaction in mice

Antigen solution could be injected into the cornea of sensitized mice using a fine needle and a stereoscopic dissecting microscope. The resulting corneal reaction was shown to be a reliable method in the detection and estimation of delayed-type hypersensitivity in mice that had been immunized with a water-in-oil emulsion containing an ovalbumin and a cell wall adjuvant. Unlike the delayed skin reaction in the ear lobe, this corneal reaction was not affected by a coexisting Arthus reaction.     

The anti-inflammatory actions of tilorone hydrochloride.

Tilorone suppressed inflammation induced by immune (direct passive Arthus reaction) as well as by non-immune agents (carrageenam-induced paw edema and abscess formation), if the compound is given 24 hr prior to the proinflammatory agonists. The non-immune anti-inflammatory effect is independent of the adrenals. A surprising findings was that total serum hemolytic complement is markedly elevated 24 hr after a single dose of tilorone.     

The urokinase/urokinase receptor system mediates the IgG immune complex-induced inflammation in lung

Immune complex (IC) deposition induces an acute inflammatory response with tissue injury. IC-induced inflammation is mediated by inflammatory cell infiltration, a process highly regulated by the cell surface-specific receptor (uPAR), a binding partner for the urokinase-type plasminogen activator (uPA). We assessed the role of the uPA/uPAR system in IC-induced inflammation using the pulmonary reverse passive Arthus reaction in mice lacking uPA and uPAR compared with their corresponding wild-type controls. Both uPA-deficient C57BL/6J (uPA(-/-)) and uPAR-deficient mice on a mixed C57BL/6J (75%) x 129 (25%) background (uPAR(-/-)) demonstrated a marked reduction of the inflammatory response due to decreased production of proinflammatory mediators TNF-alpha and Glu-Leu-Arg (ELR)-CXC chemokine MIP-2. In uPAR(-/-) animals, the reduction of inflammatory response was more pronounced because of decreased migratory capacity of polymorphonuclear leukocytes. We show that the uPA/uPAR system is activated in lung of wild-type mice, particularly in resident alveolar macrophages (AM), early in IC-induced alveolitis. This activation is necessary for an adequate C5a anaphylatoxin receptor signaling on AM that, in turn, modulates the functional balance of the activating/inhibitory IgG FcgammaRs responsible for proinflammatory mediator release. These data provide the first evidence that the uPA/uPAR plays an important immunoregulatory role in the initiation of the reverse passive Arthus reaction in the lung by setting the threshold for C5a anaphylatoxin receptor/FcgammaR activation on AM. The findings indicate an important link between the uPA/uPAR system and the two main components involved in the IC inflammation, namely, complement and FcgammaRs.     

Accumulation of 111In-neutrophils in rabbit skin in allergic and non-allergic inflammatory reactions in vivo. Inhibition by neutrophil pretreatment in vitro with a monoclonal antibody recognizing the CD18 antigen

The mAb 60.3 recognizes the neutrophil CD18 Ag. We have investigated the effect of in vitro pretreatment of radiolabeled neutrophils with mAb 60.3 on their accumulation in vivo. Further, we have compared the in vivo effects of mAb 60.3 with its effects on neutrophil adherence in vitro. Neutrophil accumulation in vivo was measured in response to: 1) exogenous mediators FMLP, C5a des Arg, LTB4 and IL-1; 2) endogenous mediators generated in a non-allergic inflammatory reaction induced by zymosan; and 3) endogenous mediators generated in two allergic inflammatory reactions, a passive cutaneous anaphylactic reaction and a reversed passive Arthus reaction in rabbit skin. Pretreatment of neutrophils with mAb 60.3 inhibited their accumulation in all the responses. The results demonstrate that there is a common mechanism mediating neutrophil accumulation in these inflammatory reactions. Neutrophils pretreated with mAb 60.3 were also unresponsive to chemoattractants in in vitro adherence assays. However, the antibody-treated neutrophils responded normally to FMLP and C5a with respect to granular enzyme release. These results suggest that the basal expression of CD18 Ag is important for the adherence of neutrophils to microvascular endothelial cells stimulated by the local generation, or administration, of chemical mediators in vivo. Despite the fact that mediators such as FMLP can increase CD18 expression in vitro, it appears more likely that such mediators act in vivo by inducing a conformational change in the basally expressed neutrophil adhesive molecules.     

Delayed hypersensitivity and nonspecific cellular immunity. The role of mono- and polynuclear phagocytes

Differences in the influence produced by sensitization with BCG vaccine and Staphylococcus aureus and by the reaction of delayed hypersensitivity (DH) induced, respectively, by the injection of old tuberculin and staphylococcal phagolysate on the phagocytic activity of peritoneal macrophages and blood leukocytes in different animals were experimentally demonstrated. A considerable activation of the bactericidal and ingesting functions of macrophages was observed in animals showing a pronounced DH reaction (rabbits, guinea pigs and mice), while in Wistar rats no such activation was noted. The latter showed no DH reaction after sensitization with BCG vaccine and the injection of the specific antigen. Among different strains of mice, the activation of macrophages occurred in the animals with the most pronounced DH reaction. Sensitization with BCG vaccine led to an insignificant sensitization of macrophages, and sensitization with S. aureus even suppressed the phagocytic activity of macrophages. The treatment of mice with antimacrophagal preparations (carrageenan, silica and trypan blue, but T-lymphocyte antiserum) before and after the injection of the specific antigen into the sensitized animals abolished the stimulation of anti-infection immunity.     

Naloxone does not modify the anti-inflammatory effect of counter-irritation with turpentine

In the rat, previous injection of turpentine reduced carrageenan-induced oedema. Naloxone and nalorphine did not modify the anti-inflammatory effect of this kind of counter-irritation. Morphine had no influence on carrageenan oedema. These results suggest that endogenous endorphins take no part to the anti-inflammatory effect of counter-irritation by turpentine.