The natural mediator for PMN emigration in inflammation: VI. Production of leucoegresin by inflammatory SH-dependent protease in active Arthus reactions in complement-depleted rabbits

A chemotactic factor (leucoegresin) specific for neutrophilic polymorphonuclear leucocytes was isolated from the site of active Arthus reactions in rabbits. This material was found to be associated with most of the chemotactic activity for the cells in the reactions. Its production by the neutral SH-dependent protease in the reactions was not related to the complement system. The effects of the protease and chemotactic factor were also unrelated to the complement system.

Most of the chemotactic activity for PMN leucocytes in active Arthus reactions in rabbits was associated with leucoegresin itself, because the chemotactic activity of inflammatory extracts was significantly absorbed by anti-leucoegresin. Production of this chemotactic substance by the inflammatory SH-dependent protease in the inflamed site was not related to the complement system, because a similar amount of leucoegresin was isolated from the same Arthus skin lesion or from the protease-induced skin lesion in rabbits whose serum complement was apparently depleted by an anti-complementary factor from cobra venom. The in vivo effects of the protease and leucoegresin were not influenced by such depletion of serum complement. It was thus indicated that leucoegresin played a significant and characteristic part in inflammatory leucotaxis. On the other hand, a slight decrease in the PMN emigration in active Arthus reactions in complement-depleted animals suggested the possible presence of a complement-derived chemotactic factor.

     

The natural mediator for PMN emigration in inflammation: I. Purification and characterization of leucoegresin from Arthus skin site

A chemotactic factor (leucoegresin) for polymorphonuclear leucocytes (PMN) of rabbit, guinea-pig, rat and mice was extracted in the pseudoglobulin fraction of Arthus skin lesions and then highly purified by chromatography using Sephadex G-50, DEAE-Sephadex A-50 and CM-Sephadex C-50 in this order. Chemotactic activity was estimated by Boyden's method during the process of purification. Leucoegresin behaved as a homogeneous substance on ultracentrifugation and on moving boundary electrophoresis. This factor was a protein free of nucleic acid and its molecular weight was approximately 140,000 when measured by gel filtration. The sedimentation coefficient of this substance was 6.58S and its isoelectric point was pH 5.0. It was relatively heat-stable.

Leucoegresin induced pronounced PMN migration, but did not increase vascular permeability; PMN emigration occurred only at the site of venules.

Leucoegresin satisfied such criteria for the natural mediator of Arthus leucotaxis as local availability, parallel between activity (amount) and time-course of the reaction, and histological resemblance to the Arthus reaction.

     

The natural mediator for PMN emigration in inflammation: IV. In vitro production of a chemotactic factor by papain from immunoglobulin G

In earlier work, a chemotactic factor (leucoegresin) specific for neutrophilic polymorphonuclear (PMN) leucocytes had been isolated from an inflamed site. The substance shared antigenic sites in common with IgG, and was produced in vitro from IgG by a purified neutral SH-dependent protease from inflammatory tissue.

A similar chemotactic factor was produced in vitro by papain from serum IgG of rabbit, mouse and man. The molecular size of the substance was approximately 140,000 when measured by gel filtration on Sephadex G-200, suggesting a minor structural change of the IgG molecule; it was indistinguishable from the molecular size of leucoegresin. Both Fab and Fc fragments from IgG showed no chemotactic activity. The chemotactic generation by the enzyme was found positive only for papain-resistant IgG; it included rabbit IgG with an electrophoretically fast mobility, human IgG₂ and IgG₄, and mouse IgG₁. Negative results were obtained with papain-sensitive IgG such as rabbit IgG with an electrophoretically slow mobility, human IgG₁ and IgG₃, and mouse IgG_2a and IgG_2b. The observations described suggest that the production of a chemotactic factor in inflammation may be associated with a structure specificity of IgG.

     

The natural mediator for PMN emigration in inflammation: III. In vitro production of a chemotactic factor by inflammatory SH-dependent protease from serum immunoglobulin G

In earlier work, a chemotactic factor (leucoegresin) specific for polymorphonuclear (PMN) leucocytes had been isolated from the sites of Arthus reactions or cutaneous burns. The substance shared antigenic sites with IgG.

The possible existence precursor of the chemotactic factor in the γ₂-globulin fraction of normal rabbit sera is suggested since the protein fraction on incubation with a purified neutral SH-dependent protease from inflammatory tissue became strongly chemotactic.

     

The natural mediator for PMN emigration in inflammation: II. Common antigenicity of leucoegresin with immunoglobulin G

A chemotactic factor (leucoegresin) for PMN was extracted from the inflamed skin of rabbits and purified. Agar immunoelectrophoresis and immunodiffusion using goat antiserum against rabbit serum and against rabbit IgG or Fc revealed that rabbit leucoegresin shared at least some antigenic determinants with rabbit IgG. Furthermore, the chemotactic activity of leucoegresin disappeared from the fluid phase when it reacted with goat antibody.     

Anti-inflammatory effects of methotrexate on reversed passive Arthus reactions in rats

The anti-inflammatory effects of methotrexate (MTX), an anti-rheumatic drug for treating rheumatoid arthritis, on acute inflammation were studied by using Arthus reactions induced in the pleural cavity and dorsal skin of rats. The effects were compared with those of dexamethasone (DEX), a synthetic analog of adrenocortical steroid, and of ketoprofen (KET), a nonsteroidal anti-inflammatory agent. In reversed passive Arthus (RPA) reactions induced in the pleural cavity by an anti-bovine-albumin serum, DEX significantly suppressed both neutrophil accumulation and plasma exudation at the sites of injection of an antibody, whereas MTX and KET had no effect. In the RPA reaction induced in the dorsal skin by an anti-ovalbumin serum, all three drugs inhibited exudation to the same level. However, DEX and MTX suppressed neutrophil accumulation, whereas KET did not. We found that the oral administration of MTX for 3 days significantly inhibited both neutrophil accumulation and exudation in the RPA reaction in the dorsal skin, suggesting that MTX is an effective anti-inflammatory agent. However, the manifestation of these anti-inflammatory effects might be restricted by differences in the inflammation models in rats.     

The natural mediator for PMN emigration in inflammation: V. The site of structural change in the chemotactic generation of immunoglobulin G by inflammatory SH-dependent protease

In earlier work, a chemotactic factor (leucoegresin) specific for neutrophilic polymorphonuclear (PMN) leucocytes had been isolated from the inflamed site of Arthus reactions or cutaneous burns. The substance shared common antigenic sites with IgG, and was produced in vitro from normal rabbit IgG and human IgG by a purified neutral SH-dependent protease from inflammatory tissue. A similar chemotactic factor was also produced in vitro by papain from papain-resistant IgG of rabbit, mouse and man.

The in vitro production of chemotactic factor by an inflammatory SH-dependent protease was similarly confirmed with rabbit antibody IgG specific for bovine serum albumin. The molecular size of the chemotactic factor was approximately 140,000, suggesting a minor structural change of the IgG molecule; it was indistinguishable from the molecular size of leucoegresin. The chemotactic generation was accompanied by a release of dialysable peptides from the IgG molecule without any release of fragments like Fab or Fc. The activity of the chemotactic factor was abolished by prolonged digestion with the SH-dependent protease, which was accompanied by an increased release of dialysable peptides. The SH-dependent protease did not produce Fab and Fc fragments even on such prolonged digestion. The minor structural change of the IgG molecule during chemotactic generation by the enzyme seemed to occur exclusively at the Fc portion. The prolonged digestion of antibody IgG with the enzyme did not affect its antigen-binding capacity.

     

Pathology of reversed passive Arthus reaction in the chicken

Sequential study of gross and microscopic changes in the chicken skin revealed that it was possible to induce a reversed passive Arthus reaction in 14- to 20-week-old chickens, using bovine serum albumin and anti-bovine serum albumin. However, high doses of immune reactants were required to elicit lesions of optimal intensity. The lesions were characterised by erythema, oedema, and the formation of thrombi in the vessels of the superficial dermis. Thrombosis, caused by the adherence of thrombocytes to vascular endothelium, induced widespread necrosis and haemorrhage. The inflammatory changes, which were confined to the deep dermis, included a necrotising vasculitis with infiltration of heterophils, monocytes and basophils. Phagocytosis of carbon particles by heterophils and basophils appeared to be sensitisation-dependent. The reaction was also characterised by the development of perivascular lymphoid foci. The findings indicate that in chickens the thrombocyte appears to be the principal cell involved in the induction of tissue damage.     

PMN chemotactic factor produced by glass-adherent cells in the acute inflammation caused by Paracoccidioides brasiliensis.

Intraperitoneal inoculation of BIO.A mice with P. brasiliensis induces an acute inflammatory infiltrate in which 40-50% of the cells are PMN leucocytes. Previous depletion of serotonin, prostaglandin, histamine and complement does not alter the course of inflammation. Complement-derived factors appear to have no active participation in the process since C5-deficient mice depleted or not by Cobra venom factor (CoF) show the same kind of cellular influx. On the other hand, peritoneal cells incubated (6 h) with the fungus release a soluble factor that induces in vivo an active chemotaxis of PMN cells when inoculated i.p. The factor has the following characteristics: a) it is produced by adherent cells; b) it is protein in nature; c) its production is inhibited by incubation of peritoneal cells with 10 micrograms/ml puromycin and d) it has a molecular weight less than 15 000 daltons, as determined by gel filtration through a Sephadex G-75 column.     

PMN chemotactic factor produced by glass-adherent cells in the acute inflammation caused by Paracoccidioides brasiliensis

Intraperitoneal inoculation of BIO.A mice with P. brasiliensis induces an acute inflammatory infiltrate in which 40-50% of the cells are PMN leucocytes. Previous depletion of serotonin, prostaglandin, histamine and complement does not alter the course of inflammation. Complement-derived factors appear to have no active participation in the process since C5-deficient mice depleted or not by Cobra venom factor (CoF) show the same kind of cellular influx. On the other hand, peritoneal cells incubated (6 h) with the fungus release a soluble factor that induces in vivo an active chemotaxis of PMN cells when inoculated i.p. The factor has the following characteristics: a) it is produced by adherent cells; b) it is protein in nature; c) its production is inhibited by incubation of peritoneal cells with 10 micrograms/ml puromycin and d) it has a molecular weight less than 15 000 daltons, as determined by gel filtration through a Sephadex G-75 column.     

Fluorescent protein tracer studies in allergic reactions: III. Distinguishing characteristics of direct and reversed passive Arthus reactions: The influence of size of antigen and of animal species

Direct and reversed passive Arthus reactions were provoked in various animal species (guinea-pigs, rats, mice), using different antigens (BSA, BGG, ferritin) labelled with lissamine rhodamine. Differences in localization of antigen—antibody precipitate and of granulocytic infiltration were evaluated statistically. The differences both between DPAR and RPAR and between these reactions when provoked with different antigen molecules are interpreted on the base of localization and concentration of antigen and antibody at the start of the reactions as a result of differences in the molecular size and mobility of the proteins involved.     

The mediation of tissue eosinophilia in hypersensitivity reactions. II. Separation of a delayed eosinophil chemotactic factor from macrophage chemotactic factors.

In anaphylactic cutaneous lesions induced by DNP-ascaris extract in the guinea-pig, the time-course of delayed tissue eosinophilia was found to parallel that of the macrophage reaction, reaching its peak in 24 h. Macrophages could be differentiated from lymphocytes by the numerous lysosomal granules which stained for acid phosphatase. Extracts from such skin lesions contained a delayed eosinophil chemotactic factor and two different macrophage chemotactic factors. Most of the delayed eosinophil chemotactic factor was separated from the two macrophage chemotactic factors by gel filtration on Sephadex G-100 and Sephadex G-200 in that order. The eosinophil chemotactic factor after re-chromatography on Sephadex G-I99 showed no or little chemotactic activity for macrophages.     

Role of anaerobic coryneforms in specific and non-specific immunological reactions: II. Production of a chemotactic factor specific for macrophages*

A chemotactic factor has been isolated from cultures and culture filtrates of many members of the group of anaerobic coryneform bacteria which includes various strains of Corynebacterium parvum. This factor attracts guinea-pig and mouse peritoneal exudate macrophages specifically and fails to attract either human blood neutrophils or guinea-pig peritoneal exudate neutrophils. Its activity is serum-independent. It is non-dialysable and destroyed by boiling. There is strong, positive correlation between the capacity of individual strains of anaerobic coryneforms to produce this macrophage chemotactic factor and the capacity of the same micro-organisms, when injected in vivo into mice, to enhance the clearance of carbon from the circulation.     

Role of leukotrienes in rat reversed passive Arthus pleurisy and the effect of AA-861, a 5-lipoxygenase inhibitor

In studies of the role of leukotrienes in inflammatory reactions, the induction of rat reversed passive Arthus pleurisy (a type III allergic reaction) was employed. Increases of exudate volume, vascular permeability, and migration of inflammatory cells in the pleural cavity were observed. The vascular permeability was enhanced biphasically during 0-30 min (early response) and during 3-6 h (late response) after induction of the pleurisy. The infiltration of inflammatory cells, mainly polymorphonuclear leukocytes, into the cavity increased and reached a maximum 6 h after the pleurisy was induced. Leukotriene B4 (LTB4), 5-monohydroxyeicosatetraenoic acid (5-HETE), and slow-reacting substance of anaphylaxis (SRS-A), consisting of LTC4, LTD4 and LTE4, were detected in the exudate by reversed-phase high-performance liquid chromatography during the early response. The contents of LTC4 reached a maximum 10 min after the challenge, followed by a rapid decrease within 1 h. The rise and decay of LTC4 correlated with the increase in vascular permeability during the early phase. AA-861, a 5-lipoxygenase inhibitor, given intrapleurally inhibited the increase in vascular permeability, cell migration, and generation of leukotrienes during the early phase of the pleurisy. These results indicate that products of the 5-lipoxygenase pathway, such as LTC4 and LTB4, may play an important role as chemical mediators in the inflammatory reaction.     

Platelet-activating factor: a mediator of pancreatic inflammation during cerulein hyperstimulation.

Hyperstimulation of the exocrine pancreas with cerulein causes acute pancreatitis, characterized by intensive interstitial edema, acinar vacuolization, leukocytic infiltration, and hyperamylasemia. Whereas the pathogenesis of cerulein-induced pancreatitis is not well-defined, a local inflammatory response may contribute to the full expression of acute pancreatitis. Platelet-activating factor (PAF) seems to be an important mediator of the inflammatory response. The present evidence includes: 1) pancreatic PAF levels increased in rats in which cerulein-induced pancreatitis was initiated, concomitant with an increase in calcium concentrations in the pancreatic tissue; 2) treatment of rats exposed to cerulein with WEB2170, a PAF receptor antagonist, was shown to reduce inflammatory injury, as demonstrated by decreases in pancreatic weight, Evan's blue extravasation, and myeloperoxidase activity and an improvement in pancreatic histology. In an idealized in vitro experiment mimicking cerulein-induced acute pancreatitis, in which pancreatic acini were employed, cerulein induced amylase release, an increase in [Ca2+]i, and an increase in PAF synthesis. Whereas amylase release was induced by low concentrations of cerulein (10(-11) mol/L), relatively high concentrations of cerulein (10(-9) mol/L) were required for the observed increases in PAF synthesis and the [Ca2+]i, indicating that these two responses may not occur under physiological conditions. The present study suggests that the pancreatic accumulation of PAF coupled with Ca2+ overload are important biochemical components of the pathophysiology of cerulein-induced acute pancreatitis. In fact, PAF production may serve as a primary mediator of inflammation observed during pancreatic hyperstimulation. This is an important observation that will allow a more detailed characterization of the molecular basis of cerulein-induced acute pancreatitis.     

A method for evaluating anti-allergic drugs by simultaneously induced passive cutaneous anaphylaxis and mediator cutaneous reactions.

Homologous passive cutaneous anaphylaxis (PCA) was induced by IgE antibody and, simultaneously, cutaneous reactions were induced by some allergic mediators such as histamine, serotonin and leukotriene (LT) C4 on rat back skin. Disodium cromoglycate and tranilast with inhibitory actions on mediator release inhibited PCA specifically, whereas antihistaminics, including ketotifen, azelastine, mequitazine and diphenhydramine, inhibited histamine- and serotonin-induced cutaneous reactions as well as PCA. Anti-slow-reacting substance of anaphylaxis drugs, KC-404 and FPL-55712, significantly inhibited PCA and histamine- and serotonin-induced reactions, but at the same doses they did not produce significant inhibition of the LTC4-induced reaction. All reactions tested were strongly inhibited dose dependently with the beta stimulants, salbutamol and isoproterenol, and a xanthine derivative, theophylline, which are known to increase the intracellular cyclic AMP level. We think that this method enables the determination of the properties of anti-allergic drugs.