Suppression of reagin synthesis in rabbits by passively administered antibody

The formation of rabbit antibodies, capable of sensitizing homologous skin, (reagins), was completely inhibited by passive administration of serum containing large quantities of 7S antibody 24 hours before or after antigen injection. No evident effect on reagin formation was noted when passive antibody was administered 8 days after antigen injection although some suppression of agglutinating antibody synthesis was observed. In rabbits not treated with passive antibody the injection of haemocyanin resulted in the formation of reagins reaching maximum serum concentrations 1 and 3 weeks following antigen injection. Both the `early' and `late' reagins persisted for a long time in the skin of injected rabbits, they appeared to have similar molecular size and both were devoid of PCA activity when injected into decomplemented rabbits. There was some indication that the `early' reagins may be more heat-labile than the `late' ones. A secondary reagin response was obtained in several animals which had shown a primary reagin response, but not in rabbits with inhibited primary response. The reagins formed in response to secondary antigen stimulation disappeared rapidly from the circulation, simultaneously with the rise in agglutinating antibody titres.

The possible implications of the findings for the immunological treatment of allergic disorders is discussed.

     

Regulatory effects of antigen and antibody on the reagin response in rabbits

The influence of antigen dosage on reagin formation to haemocyanin in rabbits was studied. Using large doses of antigen, reagin formation was more readily elicited, the induction period was shorter and the persistence of reagins in serum was longer than when low antigen doses were employed. Reagin synthesis was readily induced after intravenous administration of antigen. Immunization with antigen mixed with varying doses of IgG antibody resulted in a suppressed formation of antibodies reactive in passive haemagglutination and in a total inhibition of reagin formation. The inhibition of reagin synthesis was long-lasting and booster injections of antigen did not result in the appearance of reagins. `Hyposensitization' with series of injections of antigen or antigen–antibody mixtures resulted in an initial decrease of the reagin titres, but restoration of reagin levels was observed already about 2 weeks following cessation of the treatment. The possible implications of the results for the specific immunologic therapy of allergic disorders are discussed.     

The effect of dose in tolerance induction on the subsequent response to a cross-reactive antigen

The amount of antibody produced by BSA-tolerant rabbits as a result of immunization with DNP—BSA is dependent upon the amount of BSA used to induce tolerance. Tolerance was induced by initial injection of 100 μg of antigen followed by progressively higher doses. Rabbits rendered tolerant with a maximum BSA dose of 1 mg had a mean serum anti-BSA antibody concentration of 0.39 mg/ml after immunization with DNP—BSA, whereas rabbits rendered tolerant with a maximum BSA dose of 100 mg had a mean serum anti-BSA concentration of 0.02 mg after the same course of immunization. This compares with a mean of 1.08 mg of anti-BSA antibody in normal rabbits immunized with DNP—BSA. There was a similar reduction in the concentration of anti-DNP antibodies and of conjugate-specific antibodies in the tolerant groups.

The results are discussed in terms of a thermodynamically-controlled induction of tolerance in individual precursor cells.

     

The adjuvant activity of saponin and aluminium hydroxide for promoting reagins

Saponin and aluminium hydroxide were compared as adjuvants for promoting serum reagins in cattle using BHK cell lysate as allergen and the passive cutaneous anaphylaxis test to demonstrate the antibodies. Saponin was the better adjuvant and provoked reagins in all the injected cattle while aluminium hydroxide did so in only 2 or 5 animals. The titres were also higher in the former case. A combination of both adjuvants stimulated reagin titres marginally higher than saponin alone while BHK cell lysate without adjuvant failed to provoke demonstrable reagins.     

Reagin synthesis in inbred strains of rats

Reaginic antibody synthesis after intraperitoneal immunization with ovalbumin in aluminium hydroxide gel (Al(OH)₃) has been evaluated in BN, F344 and ACI inbred strains of rats and compared to that in outbred Wistar rats. Regardless of the dose of antigen employed (0.1 mg, 0.01 mg, 0.001 mg), BN inbred and Wistar outbred rats produced comparable amounts of reaginic antibody 9 days after immunization and these reagins were detectable for at least 70 days. In addition mean serum reagin titres could be boosted by subsequent administration of antigen. There was no statistically significant difference in the response of males as compared to females. The use of Al(OH)₃ gel as an adjuvant yielded reaginic antibody titres in these animals that were comparable to those induced with antigen administered with saline extracts of Bordetella pertussis organisms. In contrast ACI inbred rats failed to generate detectable reaginic antibodies when immunized either singly or repetitively with the same doses of antigen in Al(OH)₃ gel as used in the BN and Wistar rats. Wistar, BN, and ACI strains produced IgGa-like antibodies after a single immunization; however, peak serum titres were somewhat greater in the Wistar and BN, as compared to the ACI strain. Haemagglutinating antibody titres were comparable and greater than 1:1000 in all the strains studied within 35 days after immunization. These studies have demonstrated another animal model to explore the genetic mechanisms involved in the synthesis of reaginic antibody.     

Studies on passive cutaneous anaphylaxis in the rat with disodium cromoglycate: II. A comparison of the rat anti-DNP 7Sγ2 and rat reagin induced cutaneous reactions

A study of the passive cutaneous anaphylactic reaction induced by anti-DNP 7Sγ₂ antibodies was undertaken in rats to compare this anaphylactic reaction with that induced by rat reagin. This study revealed that disodium cromoglycate was capable of inhibiting the 7Sγ₂ induced PCA reaction immediately following antigen challenge but not the slow developing late reaction. Using various doses of disodium cromoglycate on a series of rat reagin PCA reactions and on a series of early 7Sγ₂ induced PCA reactions, similarities in dose dependence were demonstrated suggesting that certain pathways might be common to both reactions. Studies using 48/80 treatment after sensitization revealed inhibition of both early and late 7Sγ₂ reactions. Concomitant sensitization with rat reagin and 7Sγ₂ at various concentrations demonstrated that either antibody was capable of inhibiting the reaction induced by the other antibody, suggesting similar sensitization sites might be involved. In actively sensitized rats with a high reagin titre we were unable to induce a 7Sγ₂ or rat reagin PCA reaction whereas a cutaneous reaction of the `Arthus' type was readily induced. It is concluded that passive sensitization by anti-DNP 7Sγ₂ antibodies has certain factors in common with sensitization by rat reagin and that the sensitized mast cell plays a vital role in the elicitation of both early and late 7Sγ₂ PCA reactions.     

Agglutinating and non-agglutinating antibodies in rabbits inoculated with a particulate antigen (Salmonella typhimurium)

Agglutinating and non-agglutinating anti-Salmonella typhimurium antibodies were specifically purified from the sera of immunized rabbits. Both types of antibody had the same electrophoretic mobility and were localized in the IgG fraction. It was not possible to find antigenic differences between agglutinating and non-agglutinating antibodies by immunodiffusion.

Agglutinating antibody activated the complement system, while non-agglutinating antibody lacked this capacity. Only the former increased clearance of antigen from the blood. When serum samples with different antibody titres determined by agglutination (agglutinating antibody) and Coombs test (non-agglutinating antibody) were injected in mice, clearance of antigen from the blood showed changes. These results were similar to those previously observed by us when different precipitating: co-precipitating antibody ratios were used, and indicated that competition of both antibodies for the antigen depends on their respective amounts.

When mice protection tests were set up by injection of agglutinating and non-agglutinating antibody before the inoculation of 10 LD₅₀ S. typhimurium, non-agglutinating antibody was found to be less effective than agglutinating antibody.

Non-agglutinating antibody was detectable during the whole course of immunization. Its serum concentration was higher than that of the agglutinating antibody.

Non-agglutinating antibody behaves in a similar way to co-precipitating antibody. The initially proposed hypothesis that such antibodies could interfere with immunity to certain chronic infections was extended to include the non-agglutinating antibodies demonstrated here.

     

The significance of circulating IgE: Correlation of amount of circulating reaginic antibody with cutaneous sensitivity in the rat

In order to determine the relationship between the level of circulating reaginic (IgE) antibody and degree of cutaneous hypersensitivity, rats producing different titres of reaginic antibody were assessed for skin test reactivity by means of the active cutaneous anaphylaxis test. Skin reaction diameters of ovalbumin-sensitive rats were similar whether they were producing low or markedly elevated levels of ovalbumin reagins. In rats infected with Nippostrongylus brasiliensis, skin test reactivity to N. brasiliensis antigen was demonstrable before reagin became detectable in the circulation. It increased to maximum shortly after the appearance of circulating reagin, and thereafter showed no significant increase commensurate with the further rise in reagin titre. The results show that in the rat there is no direct quantitative relationship between the size of skin test reactions and the level of specific circulating reaginic antibody.     

Suppression of IgE antibody production in sensitized mice and rats by tolerogenic conjugates of synthetic hydrophilic polymers with antigen or hapten: effect on antigen-induced histamine release from peritoneal mast cells

IgE antibodies were produced in mice and rats by immunization with ragweed pollen extract (RAG) or dinitrophenylated ovalbumin (DNP3-OA). Treatment of these animals with tolerogenic conjugates of (i) the antigen (RAG or OA) with monomethoxypolyethylene glycol (mPEG), or (ii) DNP with polyvinyl alcohol (DNP-PVA) resulted, within 7-14 days, in a fall in circulating IgE antibodies and in mast cell sensitivity, as assessed by the radioallergosorbent test (RAST) and in the in vitro antigen-induced histamine release (HR) test, respectively. The reduction in responsiveness was more marked in mice than rats; 10 days after a booster immunization, the IgE antibody titres in the RAG-mPEG-treated group of mice were approximately 10-fold lower than in the saline-treated group, with a 100-fold difference in cell sensitivity. DNP-PVA treatment of mice produced a more than 10-fold reduction in IgE anti-DNP titres with a substantial reduction in histamine release.     

Comparison of different antigenic preparations of Coxiella burnetii used for antibody detection in guinea pigs.

The dynamics of antibody response in guinea pigs infected with Coxiella burnetii was investigated by microagglutination (MA) and complement-fixation (CF) tests with different preparations of C. burnetii antigens. At the onset of antibody response the highest antibody titres were detected by the MA test with natural antigen 2, later on by the MA test with artificial antigen 2. Throughtout the 1-year period of observation, the CF antibody levels were usually lower and, with the exception of the highest infectious doses, the CF antibodies appeared later than agglutinating antibodies. There was no difference in the appearance of agglutinating and CF antibodies directed to antigen 1. Inactivation of the sera caused a marked decrease in antibody titres when tested with artificial antigen 2, whereas the antibody levels remained unchanged when tested with natural antigen 2.     

Immunomodulation with liposomes: the immune response elicited by liposomes with entrapped dichloromethylene-diphosphonate and surface-associated antigen or hapten

Macrophages in the murine spleen were eliminated by the intravenous administration of dichloromethylene-diphosphonate (DMDP) encapsulated in liposomes. The immune response against an antigen (bovine serum albumin, BSA) or hapten (dinitrophenyl, DNP) associated with the surface of the DMDP liposomes was studied. Significant effects of macrophage elimination on the primary (IgM), but not on the secondary (IgG), anti-BSA response were found. Dramatic effects on the response against liposome-associated DNP were observed in macrophage-depleted mice. The number of anti-DNP antibody-forming cells in the spleen decreased from 300 to 28 per section, and anti-DNP serum titres dropped to 12% of their normal values. Since a similar phenomenon was observed for TNP-Ficoll, a thymus-independent type-2 antigen (and not for thymus-dependent or thymus-independent type-1 antigens), we suggest that this response should be classified as thymus-independent type-2 on grounds of its in vivo behaviour. We conclude that adjuvant activity (and memory formation) of liposomes with antigen exposed on their surface occurs irrespective of the presence or absence of splenic macrophages, and that DMDP liposomes could be useful in drug targeting with antigenic liposomes.     

Effect of Preimmunization with Native and Methylated Carrier on Class-Specific Anti-Hapten and Anti-Carrier Antibody Response

Effects of priming with bovine serum albumin (BSA) and methylated USA (MBSA) on the primary anti-2,4-dinitrophenyl (DNP) response were studied in the chicken. A new radioimmunoassay of class-specific antibodies (RIACA) was used to quantitate IgM and IgG antibodies against BSA and DNP. The anti-DNP antibody response after priming with BSA was dose-dependent: large doses of USA suppressed and the smallest dose augmented it. In contrast, no dose dependence was observed between MBSA and the anti-hapten response. All doses of MBSA enhanced the primary anti-DNP antibody response of both immuno-globulin classes. No corresponding differences in anti-BSA antibodies were observed between BSA- and MBSA-primed groups. This suggests that the anti-carrier antibodies as such are not the agent suppressing the anti-hapten response when large doses of native carrier are used for priming. A more probable explanation of this phenomenon is a competition between carrier-specific B cell and hapten-specific B cells for common T cells. A less obvious explanation is the generation of more helper cells or less suppressor cells when priming with MBSA than with BSA.     

Human Reagins to Grass Pollens and Moulds: Their Purification and Physico-chemical Characterization

Chromatography of reaginic sera (to grass pollens and to moulds) on DEAE-cellulose leads to the concentration of reagins in three well separated fractions. They emerge (a) together with siderophilin, (b) with the early albumin-containing fractions, and (c) in association with strongly adsorbed proteins that require a comparatively high ionic strength buffer for their elution. The proportion of reagins in each of these fractions varies with different sera and with small alterations in the experimental procedure. All three reaginic fractions contain small amounts of γ globulins (referred to as R globulins) of mobilities slightly less than that of siderophilin; (c) contains much α_2M globulin and there are traces of α_2M in (a) and (b). Yet the bulk of the γ globulins are shown to be free from reaginic activity, and the same is true of the α_2M and the β_2M globulins, of siderophilin and of albumin. The purer the reaginic fractions are the smaller is the portion of the reagins that can be precipitated with the γ globulins by half saturation with ammonium sulphate. In contrast to the bulk of the γ globulins, R globulins and reagins appear to associate readily with other serum proteins, particularly with α_2M globulins. Fractionation with sodium sulphate produces three fractions of similar potency although they have little in common; one consists of the bulk of the γ globulins (0–15 per cent w/v), the most active fraction of the remaining globulins (15–18 per cent) and the third fraction (supernatant from the 18 per cent precipitate) of albumin containing some α globulins, but only a trace of γ globulin.

Ultracentrifugation studies on three main reaginic DEAE-fractions show that the bulk of the reagins are not macroglobulins although misinterpretation can arise from complex formation with α_2M globulin.

High agglutination titres (Stavitsky's method, Stavitsky and Arquilla, 1958) for pollen proteins are associated only with the unretarded non-reaginic γ globulins of post-treatment sera (which contain the blocking antibodies) although traces of agglutinating antibodies can be demonstrated in many fractions, including the reaginic fractions derived from the sera of untreated hay fever subjects.

     

Persistent formation of reagins in mice injected with low doses of ovalbumin

Immunization of mice with minute doses (0.1 μg) of ovalbumin in A1(OH)₃ gel induced persistent synthesis of high titres (1:160, 1:320) of reaginic and IgG1 antibodies. Immunization with high doses of ovalbumin (100 μg) induced high IgG1 titres but only weak and transient formation of reagins. Inbred mouse strains differ widely in their immune responsiveness to low doses of ovalbumin.     

Immunoglobulin E antibody formation in response to homologous rabbit albumin heavily substituted with dinitrophenol: effect of adjuvant.

Although much progess has been made in the detection and characterization of homocytotropic antibodies, identification of the factors which control their synthesis remains to be determined. To assess the influence of different adjuvants on anti-dinitrophenol (DNP) immunoglobulin E (IgE) antibody responses, rabbits were immunized with adjuvant plus homologous albumin (HRA) heavily substituted with DNP (DNP30-HRA). This antigen in rabbits has a B cell-reactive determinant (DNP) and weak non-B cell-reactive determinants (new antigenic determinants) which sensitize rabbits for delayed-type hypersensitivity reactions to DNP30-HRA. It was postulated that the anti-DNP IgE response to DNP30-HRA could be regulated if the immunogenicity of the weak non-B cell-reactive determinants (new antigenic determinants) in DNP30-HRA could be manipulated by adjuvants and dosage. Complete Freund adjuvant and incomplete Freund adjuvant increased the immunogenicity of the new antigenic determinants in DNP30-HRA (10 mg) much more than did alum. However, equivalent primary anti-DNP IgE responses were made by all rabbits sensitized with this dose, regardless of the adjuvant used. Larger doses of DNP30-HRA (25 mg) in alum sensitized rabbits for strong delayed-type hypersensitivity reactions to DNP30-HRA and also elicited enhanced and persistent primary anti-DNP IgE responses. Enhanced but transient primary anti-DNP IgE responses were elicited by 25 mg of DNP30-HRA in incomplete Freund adjuvant. In contrast, no primary anti-DNP IgE responses were made to 25 mg of DNP30-HRA in complete Freund adjuvant. Regardless of the adjuvant or dosage used for primary immunization, no secondary anti-DNP IgE responses to DNP30-HRA were detected.     

Characterization of the antibody response of the marmoset to sheep red blood cells.

The immune competence of two species of marmosets, S. fusciollis and S. oedipus, was evaluated by the intravenous (i.v.) and intramuscular (i.m.) injection of sheep red blood cells (SRBC). In S. fusciollis marmosets, 1 ml of a 50% suspension yielded titres of haemolysin and agglutinating antibodies equal to or greater than 1 ml of a 10% dose of antigen. In both species, the i.v. route, while resulting in formation of 19S and 7S agglutinins, yielded only 19S haemolysins, even after multiple antigen injections. Repeated i.v. injections resulted in a progressive decrease in peak titres, in contrast to the i.m. route, where booster inoculations gave a typical anamnestic response. Jerne plaque-forming cells (PFC) in the spleens of S. oedipus marmosets showed predominately 19S plaques after a primary i.v. challenge; only 19S PFC were detected in the spleen of an animal that had been given multiple inoculations, the type of antibody produced reflecting that found in the serum. 19S but not 7S haemolysins of both species were sensitive to heating at 56 degrees C for 1/2 hr. The serum titres and splenic PFC data from the marmosets suggest these animals, particularly S. oedipus, respond poorly to SRBC when a comparison is made to similar studies in mice and rats.     

Experimental humoral and cellular immunity to hog intrinsic factor

Humoral and cellular immunity to hog intrinsic factor (HIF) was studied in rabbits immunized with varying doses of HIF or HIF complexed to vitamin B₁₂. Animals immunized with large doses of HIF (1 mg) consistently produced high titres of blocking and binding antibodies to IF. At low dose immunization (10 μg), the humoral response was obviously blunted, with animals forming significantly reduced titres of antibodies to IF; several rabbits in this group made only binding antibodies and one rabbit produced neither blocking nor binding antibodies. No difference in humoral responsiveness was noted between those animals who received HIF or an equivalent amount of HIF complexed to vitamin B₁₂. By contrast, cellular immunity as measured by inhibition of leucocyte migration was readily induced to a similar degree in both high and low dose animals, including the rabbit which had no detectable antibodies to IF. When an intermediate dose of HIF (50 μg) was used as the immunogen, four rabbits gave positive leucocyte migration tests only with HIF—vitamin B₁₂ complex, suggesting that the interaction of HIF with vitamin B₁₂ may enhance its antigenicity in vitro. These data demonstrate that cellular and humoral immunity to HIF can be induced and partially dissociated from each other by varying the dose of immunogen. Although the mechanism responsible for this dissociation is not clear, one explanation would be differing sensitivities of lymphocyte-populations to different doses of antigen.     

Hypogammaglobulinaemia in nephrotic rats is attributable to hypercatabolism of IgG

The effect of the nephrotic syndrome induced by puromycin aminonucleoside (PA) in rats on specific antibody responses to 2,4 dinitrophenyl (DNP) conjugated to either spider crab haemocyanin (MSH), a T cell-dependent antigen, or hydroxyethyl starch (HES), a T cell-independent type 2 antigen were studied. The serum IgG anti-DNP levels following immunization with both antigens were reduced in nephrotic animals compared with controls while IgM anti-DNP antibody titres were higher. The half-life of IgG anti-DNP antibodies passively transferred into non-immunized nephrotic rats was markedly reduced while the half-life of anti-DNP antibodies of the IgM class was comparable to that in controls. Low serum IgG and elevated IgM levels were seen in nephrotic animals compared to controls. Antibody-forming cells specific for DNP were demonstrated by immunohistology on rat spleens and the numbers of both IgG and IgM-producing cells were found to be significantly increased (P less than 0.05) in nephrotic animals in response to both DNP-HES and DNP-MSH. These data indicate that in nephrotic rats the alteration seen in the serum immunoglobulin levels is not attributable to reduced antibody production but increased catabolism of serum IgG antibodies.     

Inhibition of allergic reactions due to competition for mast cell sensitization sites by two reagins

The ability of unrelated or incidental reagin to interfere competitively with mast cell sensitization by specific reagin has been examined using an experimental system whereby rats are induced to produce high levels of reaginic (IgE) antibody to two antigens. It is shown that passive sensitization by a reaginic antibody is inhibited (a) by simultaneous injection with a second reagin directed against another antigen, the degree of inhibition being related directly to the dose of the second serum or (b) if the test animal is actively producing unrelated reagin.

The skin test sensitivity of rats actively producing high levels of two reagins as opposed to only one is also discussed. The results are presented with particular reference to the interpretation of PCA and other skin tests and to the possible importance of interference effects in multiple allergies.

     

Passive cutaneous anaphylaxis in the rat, induced with two homologous reagin-like antibodies, and its specific inhibition with disodium cromoglycate

Rats immunized with egg albumin and Bordetella pertussis organisms produce a `mast cell sensitizing' antibody (MCSAb) which is thermolabile, a potent skin sensitizer and reagin in character. Similarly the immune response to Nippostrongylus brasiliensis in rats is closely associated with the formation of antibodies which also resembles human reagins. Homologous passive cutaneous anaphylactic (PCA) reactions induced by N. brasiliensis serum were found to be similar to those produced using the adjuvant induced antibody in that both were completely inhibited by, combined treatment with mepyramine and 2-bromo-D-lysergic acid diethylamide (BOL148), cyproheptadine or pretreatment with compound 48/80. In contrast, skin reactions involving passive sensitization of rats with rabbit hyperimmune antiserum were much less affected. Studies on mast cell disruption at the site of PCA reactions showed that such reactions using N. brasiliensis serum were accompanied by degranulation of mast cells, and confirmed that mast cell damage occurs in PCA induced with MCSAb. Both the PCA and the mast cell disruption were maximal 5 minutes after antigen challenge in both rat reagin systems. The skin reaction produced using rabbit hyperimmune antiserum was not primarily dependent on, or associated with, mast cell disruption, since it was still possible to induce skin reactions when the mast cells had been disrupted by compound 48/80, and skin reactions could be obtained without significant mast cell disruption.

Disodium cromoglycate, a new compound introduced for the treatment of asthma, was shown to inhibit both the PCA and mast cell disruption induced using both rat reagin antibodies but not the skin reactions produced with rabbit anti-serum. It was possible to obtain substantial inhibition of mast cell disruption induced by rat reagin, even when the PCA was inhibited only slightly. At higher doses the discharge of the mediators from mast cells was also prevented. This interference with mast cell permeability was not unspecific since disodium cromoglycate did not prevent the skin reaction or mast cell disruption produced by compound 48/80. As expected mepyramine was able to partially inhibit the skin reaction without affecting mast cell disruption induced by rat reagin or compound 48/80. It is suggested that disodium cromoglycate acts at some critical pathway in the events after the union of antigen with reagin antibody and that this critical pathway might be common to both the human and rat reagin systems.