Studies on cutaneous allergy: Cytophilic antibodies, passive cutaneous anaphylaxis and delayed type allergy

In guinea-pigs a delayed type allergy against sheep erythrocytes (SE) or ovalbumin without detectable circulating and macrophage-cytophilic antibodies was produced by immunization with antigen—antibody complexes. In these animals skin reactions to SE were not enhanced by admixture of macrophages, neither when the macrophages had been sensitized with cytophilic antibodies in vitro, nor when the SE had been (partially) ingested within the macrophages after incubation with opsonizing antibody in vitro.

Also in these animals no enhanced delayed skin reactions to SE were obtained if before the injection of the antigen the skin had been infiltrated with cytophilic antibody or passive cutaneous anaphylaxis (PCA) antibody.

Thus, no enhancing effect of cytophilic antibody on delayed cutaneous hypersensitivity reactions could be obtained.

Furthermore, in guinea-pigs with delayed type allergy to ovalbumin, no enhancing effect of delayed allergy on passive cutaneous anaphylaxis was observed.

     

In vitro studies of delayed hypersensitivity: inhibition of macrophage spreading in rats sensitive to tuberculin and diphtheria toxoid

Wistar rats were sensitized by footpad injection of BCG in adjuvant, or Mycobacterium butyricum in adjuvant, or diphtheria toxoid in Freund's incomplete adjuvant. It was found that the cell population of the peritoneal washings contained approximately 57 per cent macrophages, 22 per cent lymphocytes, 11 per cent granulocytes and 8 per cent mast cells. The lymphocyte count was significantly reduced and the granulocyte count increased after sensitization. The animals sensitized to M. butyricum exhibited delayed skin reactivity to tuberculin and the spreading of macrophages in vitro was significantly inhibited with the same antigen. On the contrary, the spreading of macrophages obtained from animals sensitized to BCG was not inhibited by tuberculin and there was no cutaneous reactivity. Spreading of macrophages obtained from rats sensitized by diphtheria toxoid was significantly inhibited in the presence of diphtheria toxoid, but not in the presence of tuberculin. These animals displayed delayed cutaneous hypersensitivity to diphtheria toxoid. Spreading of macrophages from normal rats was unaffected by serum antibodies. This was true either when the peritoneal cells were treated with antiserum prior to contact with antigen, or when the antigen—antibody reaction took place in the chamber containing the macrophages ready to spread. These results indicate that the technique of macrophage spreading inhibition is able to detect specifically hypersensitivity of delayed type and offers a convenient method for the in vitro study of delayed hypersensitivity.     

Induction of thyroiditis in guinea-pigs by intravenous injection of rabbit antiguinea-pig thyroglobulin serum: I. Light microscopic study

An inflammatory reaction in the thyroid of guinea-pigs, induced by intravenous injection of rabbit anti-guinea-pig thyroglobulin serum, has been studied at time intervals ranging from ¼ hour to 20 days after injection. Specific staining methods for eosinophils have been used to demonstrate that the inflammatory infiltrate mainly consisted of eosinophil granulocytes, but that neutrophil granulocytes were also present at the earliest time intervals. PAS-staining revealed that the granulocytes contained rounded droplets of material with a PAS-reactivity comparable to that of the colloid. This was most clearly seen 12 and 24 hours after injection. The possibility that this material represents phagocytosed thyroglobulin perhaps in an antigen—antibody complex has been suggested. The number of mast cells was counted and degranulation, which was apparently most extensive 12 hours before maximum granulocyte infiltration, was observed. Possible mechanisms involved in eosinotaxis and uptake of antigen—antibody complexes in the granulocytes have been discussed.     

Antibody production in mice: II. The mechanism of antigenic stimulation in the secondary immune response

To study the mechanism of antigenic stimulation in the secondary immune response, primed lymphoid cells were stimulated with antigen in various ways in vitro. The effect of antigenic stimulation was assessed by the antibody titres obtained after in vivo culture of primed cells in X-irradiated recipients.

Primed cells were much more effectively stimulated by antigen—antibody (Ag—Ab) complex than by free antigen. Primed cells could also be stimulated by spleen or lymph node cells from normal mice which had been exposed to free antigen or Ag—Ab complex in vitro or in vivo and thoroughly washed. Under these conditions, Ag—Ab complex was again much more effective than free antigen. When the cells were incubated with Ag—Ab complex, the dose of antigen bound to the cells was somewhat increased. But this increased binding of antigen could not solely account for the increase in immunogenicity.

It is suggested that the ingestion of antigen by macrophages is facilitated by the presence of antibody and that the macrophages mediate the effective immune stimulus to memory cells.

The effect of antibody in increasing the immunogenicity of antigen was lost completely when antibody was digested with pepsin. Thus, the Fc portion of antibody seemed to be important for this effect. However, it was demonstrated that antibody does not operate by becoming attached to macrophages as cytophilic antibody, and that complement is not involved in this process. The augmenting mechanism of antibody on the antigenic stimulation mediated by macrophages was discussed.

     

Antigen–antibody induced cutaneous eosinophilia in complement deficient guinea-pigs

Decomplementation with cobra venom factor had no effect on eosinophil accumulation into the site of IgG₁-mediated PCA reactions in the guinea-pig. Eosinophil and neutrophil accumulation also followed PCA reactions in animals partially or totally deficient in C4.

The effect of intradermal injections of pre-formed antigen–antibody complexes prepared from guinea-pig IgG₁ or IgG₂ were not affected by decomplementation with cobra venom. The lesions produced with either immunoglobulin complex were similar in appearance in decomplemented and normal animals and were followed by comparable tissue accumulation of eosinophils and neutrophils.

     

Studies on eosinophil leucocyte migration. I. Eosinophil and neutrophil accumulation following antigen–antibody reactions in guinea-pig skin

Eosinophil leucocytes migrated into the site of PCA reactions mediated by those fractions of 7S guinea-pig IgG containing IgG₁; neutrophils were associated with IgG₁ and IgG₂. Maximal eosinophil infiltration is seen at 12 hr and was associated with degranulation.

Intradermal histamine was not eosinophilotactic in guinea-pigs.

Preformed antigen–antibody complexes of IgG₁ and IgG₂ both promoted eosinophil and neutrophil migration in guinea-pig skin but slightly more eosinophils were seen following injections of complexes containing IgG₁. Local eosinophilia and PCA activity were mediated by a relatively heat-stable element since these effects were demonstrable even after prolonged heating of fractions containing IgG₁. Eosinophils were seen following injections of Compound 48/80 and this was accompanied by low mast cell counts; however, there was also some associated tissue destruction.

     

Preparation of anti-β1C antisera and their use in fluorescent antibody techniques

Antisera against components of guinea-pig complement were raised in rabbits by:

(1) Using as antigen a suspension of killed Proteus species bacteria which had been allowed to combine with their homologous antiserum in the presence of guinea-pig complement in optimum proportions.

(2) By injection of the β_1C component of guinea-pig complement adsorbed to zymosan particles. The antisera raised by the two methods contained antibodies mainly against the β_1C component of complement.

When coupled with FITC both antisera were found useful in detecting sites of complement fixation. The fluorescence anti-complement technique was found four times more sensitive than the indirect method for detecting antigen—antibody reactions in the presence of diminishing concentrations of antigen. It was only twice as sensitive for detecting antigen—antibody reactions in the presence of diminishing concentrations of antibody. The comparisons were based upon both visual assessment and photometric measurement.

Coupled antisera raised by the first method gave brighter specific fluorescence than antisera raised by the second method when used in the highest concentration which did not give non-specific staining.

The usefulness for detection of viral antigens of sera raised by both methods is discussed.

     

Quantitative changes in the basophil cells of guinea-pig bone marrow following the administration of Ascaris body fluid

A quantitative study of basophil cells of bone marrow, blood and peritoneal fluid was carried out in seventy-two mixed strain male guinea-pigs, following daily subcutaneous injection of Ascaris body fluid (ABF).

An increase of marrow basophils was observed 4–10 days after one and the last of three daily injections of ABF. The degree of increase appeared to be related to the quantity of the antigen, but the duration of increase was about 2 weeks in both the singly and the repeatedly injected animals.

It is suggested that the protein in ABF may not be the sole agent responsible for the basophil production.

An allergic or antigen—antibody reaction may develop following the re-injection of ABF in the sensitized animals.

A great number of basophils and eosinophils was discharged from the bone marrow into the blood stream 1 day after the subcutaneous injection of ABF and following re-injection of the antigen.

The antigen administered subcutaneously also caused a cellular reaction in the peritoneal fluid.

     

β Toxin of Clostridium welchii : Cytotoxic Effects Produced by the • on Guinea-Pig Monocytes

The cytopathic effects of Cl. welchii β toxin on guinea-pig monocytes in vitro have been studied using the uptake of eosin-Y^* as evidence of cell death.

The experiments show that these effects are due to antigen—antibody reaction probably on the cell surface, and that these reactions are not dependent on the presence of complement. Monocytes can be actively sensitized in vivo, and passively sensitized in vitro. The serum used to passively sensitize the monocytes need not possess a precipitating antitoxin titre.

Comparable experiments using an ovalbumin antigen—antibody system produced the same cytopathic effects on the monocytes as those which occurred in the β toxin—cellular system.

     

The effect of antibody on the degradation of a polysaccharide by the reticulo-endothelial system

A galactan isolated from gum arabic was shown to be non-antigenic in mice. However, O-acetylated derivatives of this polysaccharide were antigenic and also gave rise to antibodies which reacted with the galactan.

The degradation of the galactan and its O-acetylated derivatives in vivo by the liver and lungs of normal mice has been followed.

It was found that, whereas, both the liver and lung degraded the galactan, O-acetylated derivatives containing 10–20 per cent by weight O-acetyl groups were degraded only by the lung.

The breakdown of the acetylated polysaccharide by the lung was enhanced by added antibody under conditions where the antigen was in excess. These conditions also led to an enhanced antibody response. In contrast both degradation and antibody formation were inhibited when animals received antigen—antibody complexes in antibody excess. The significance of these results in relation to the anamnestic response is discussed.

     

Studies on eosinophil leucocyte migration. II. Factors specifically chemotactic for eosinophils and neutrophils generated from guinea-pig serum by antigen–antibody complexes

Chemotactic activity for eosinophils and neutrophils has been studied using guinea-pig serum activated by preformed antigen–antibody complexes.

Rabbit complexes or complexes prepared either with guinea-pig IgG₂ or IgG₁ were equally capable of generating a heat stable activity from guinea-pig serum that was chemotactic for guinea-pig eosinophils and for neutrophils of both the guinea-pig and the rabbit. This was distinguished from a relatively heat-labile chemotactic activity present in untreated guinea-pig serum.

The heat-stable complex-mediated chemotactic activity was thought to be complement dependent since chemotaxis for eosinophils or neutrophils could not be generated from heated serum, ammonia treated serum or from serum treated with complexes in the presence of 0·01 M EDTA.

Guinea-pig sera, activated either with rabbit, guinea-pig IgG₁ or IgG₂ complexes, was fractionated by sucrose-density gradient ultracentrifugation and by Sephadex chromatography. In all experiments two peaks of cell specific chemotactic activity could be separated. The peak of activity for guinea-pig neutrophils was approximately 4·5S and in the fractionation range of proteins having a molecular weight of between 65,000 and 85,000. The peak of guinea-pig eosinophil chemotactic activity was 1·5S–2S and in the molecular weight range of between 15,000 and 20,000. Those fractions which were chemotactic for guinea-pig neutrophils did not attract rabbit neutrophils. Rabbit neutrophils migrated towards those fractions of guinea-pig serum chemotactic for guinea-pig eosinophils; therefore, the properties associated with guinea-pig eosinophil chemotactic activity were similar to previously published data for a fragment cleaved from the 5th component of complement.

     

Prolongation and enhancement of Nippostrongylus infection in the laboratory rat by a heterologous antiserum to rat peritoneal cells—A possible role for pharmacologically-active cells in immunity

A rabbit anti-rat peritoneal cell (APCS) serum significantly suppresses (P<0.001) the immune response to Nippostrongylus brasiliensis infection in the rat and the infection can be prolonged by such an antiserum. The antiserum appeared to contain antibodies directed against the surface antigens of pharmacologically-active cells such as the mast cell. Prolongation of infection following treatment with APCS was accompanied by a reduction in the number of eosinophils, basophils and neutrophils in the peripheral circulation. The number of circulating peripheral lymphocytes was not significantly affected by the antiserum, nor did the antiserum affect the homograft response. This antiserum, however, suppressed the homologous PCA reaction and the response to an intradermal injection of histamine, and also provoked the complement-dependent release of histamine from mast cells in vitro up to a dilution of 1:512.

It was estimated that the average rat mast cell contains 19.8 pg of histamine. These results provide evidence for the importance of eosinophils, basophils and mast cells, i.e. pharmacologically-active cells, in allowing the host to mount a homocytotropic antibody-allergen mediated reaction as part of the immune response.

     

Antigen—antibody complexes and the weal and erythema skin responses

The smallest amount of purified antibody capable of positive weal and erythema response is of the order of 4 μg N.

Antigen—antibody complexes are capable of producing specific weal and erythema reactions with antigen—antibody ratio of 1:1 or above. In these instances antibodies occupied at one or both sites are responsible for this response. Preformed antigen—antibody complexes are capable of mediating this response.

     

Antigen-induced histamine release from peritoneal mast cells of mice producing reagin-like antibody

The time course of production in the mouse circulation, after different schemes of immunization, of reagin-like and γ₁ antibodies, as measured by the ability of the antisera to induce thermolabile long-latence and thermostable short-latence PCA reactions, has been studied. γ₁ antibody was obtained in all schemes of immunization and its amount in antiserum increased with time. Reagin-like antibody appeared on the 7th day after immunization with sufficient dose of ovalbumin and adjuvant, and on later days with less efficient antigens such as DNP—BSA and DNP—BGG. Maximal amounts of the reagin-like antibody were reached in the early days after immunization and its presence in the circulation had a somewhat transient character; however, it persisted in the circulation for periods of more than 60 days, on occasion.

The time course of antigen-induced histamine release from washed peritoneal mast cells of the immunized mice was also studied. A correlation was observed between the histamine release and the production of reagin-like antibody. It is suggested that the antigen-induced histamine release from washed peritoneal mast cells may be taken as an indication of the production of reagin-like antibody.

Some quantitative aspects of the PCA and histamine release reactions were also studied. PCA reactions were affected similarly by varying either dilution or volume of the antiserum intradermally injected. Histamine release from peritoneal mast cells did not seem to be affected specifically by excess of antigen.

     

Glucocorticoid inhibition of antigen-evoked histamine release from human skin

Prednisolone causes a dose-related inhibition of antigen-evoked histamine release from IgE-sensitized human skin in vitro. The effective concentrations are of the same order as are achieved in plasma therapeutically.

Analysis of prednisolone inhibition shows that it acts on the second histamine release stage, antigen—antibody combination being unaffected.

In contrast with the traditional view, our results show that, at least in human skin, glucocorticoids can inhibit antigen-evoked histamine release.

     

Analysis in vitro of tolerance

The generation in vitro of cells synthesizing haemolytic antibody to sheep erythrocytes (plaque forming cells—PFC) was shown to be due to the combined activity of two distinct populations of cells—macrophages and lymphocytes.

Suspensions of cells from mice rendered tolerant to sheep erythrocytes by injection of a large dose of antigen and cyclophosphamide showed a greatly reduced capacity to generate PFC if tested within the first 17 days after treatment.

This loss of capacity to mount an immune response in vitro was restored by suspensions of lymphocytes from spleens of normal mice. The lymphocytes from recently paralysed mice failed to generate PFC when incubated with preparations of normal macrophages. The macrophages from paralysed mice were as active as normal macrophages in generating PFC. Tolerance in this model is, therefore, associated with a loss of function in vitro of the lymphocytes in the spleens of such animals.

Most spleens tested 3 weeks after induction of paralysis, when recovery in the intact animal is incomplete, had a normal capacity to generate PFC in vitro.

     

The relationship between circulating soluble antigen—antibody complexes and the production of chronic glomerulonephritis in the rabbit

Twenty-three rabbits were injected over long periods of time with isotopically labelled bovine serum albumin. Varying injection schedules and primary and secondary responses were studied, but all schedules were designed to preclude the presence of free circulating antibody at any time. All animals became tolerant of injected antigen after a period of 60–80 days. During the intervening period (from 10 to 60 days) large amounts of antigen—antibody complex were shown to be circulating.

Proteinuria was intermittent in all animals. Histological examination of the kidneys from 50 days on showed only very minor evidence of renal damage; no animal developed chronic renal disease.

     

Cytotoxicity of immune guinea-pig cells: II. The mechanism of macrophage cytotoxicity

Macrophages from immune guinea-pigs, as well as macrophages from non-immune animals in the presence of specific antibody, were cytotoxic to chicken erythrocytes. Elution from macrophages produced a γ₂-globulin which enabled non-immune macrophages to form rosettes with chicken erythrocytes, to phagocytose, and to show cytotoxicity towards such cells. This antibody was target-cell specific; it also rendered spleen cells cytotoxic. Isoelectric focusing of antiserum gave a peak of cytophilic antibody at pH 6.8–7.6 which proved to be in the γ₂ region.

The significance of phagocytosis for the cytotoxic process was examined with the aid of cytochalasin B which inhibited macrophage—phagocytosis, but produced enhanced cytotoxicity in this sytem. At the same time there was greatly increased release of lysosomal enzymes from macrophages in the presence of target cells. It is considered that, although phagocytosis is of significance for macrophage cytotoxicity, target cell destruction can also occur by the liberation of lysosomal enzymes from the plasma membrane into the target cell when this is in close surface contact.

     

The action of soluble antigen—antibody complexes in perfused guinea-pig lung

When soluble antigen—antibody complexes, prepared in antigen excess, were injected into the perfused pulmonary artery of the unsensitized guinea-pig lung, they produced a bronchoconstrictor response which was associated with the appearance of histamine and a slow-reacting substance in the effluent. This activity was inhibited by the presence of normal rabbit serum or rabbit globulin; rabbit albumin and bovine γ-globulin produced no inhibition. Insoluble antigen—antibody complexes prepared at equivalence were at least 125 times less active than soluble complexes prepared in antigen excess. On comparing the activity of different solutions of soluble complex prepared in antigen excess ranging from 2½ to 160 times that required for equivalence, all were found to be equal. The properties of soluble antigen—antibody complexes are consistent with the possibility that circulating antibody may participate in in vivo anaphylaxis, if reached by an amount of antigen greater than that required for equivalence.     

The use of antigen antibody precipitates for specific detection of antigens in tissue sections

Antigen—antibody precipitates, prepared either in solution in antibody excess or by cutting precipitin lines from immunoelectrophoretic plates have been shown to bind specifically to the homologous antigen in tissue sections.

This finding has been used as the basis of an immunofluorescent technique in which antigen—antibody precipitates are applied to a section and then stained with a fluorescein-conjugated antiglobulin reagent.

The technique allows the localization of antigens in tissue even where they are not characterized or purified and where monospecific antisera are not available.