A canine model for reaginic hypersensitivity and allergic bronchoconstriction

Immunization of neonatal dogs with a conjugate of 2,4-dinitrobenzene and ovalbumin (DNP₂-OA), using aluminum hydroxide as the adjuvant, elicited long-lasting (over 30 wk) anti-DNP and anti-OA IgE antibody responses of high titers as determined by homologous passive cutaneous anaphylaxis. Low antigen doses of 10 or 50 μg were more effective than the higher doses of 250 or 1,250 μg in inducing high IgE antibody levels. However, this method of immunization failed to elicit any detectable IgE antibody response in adult dogs. Bronchoprovocation with antigen of sensitized animals having IgE antibody titers in excess of 64 resulted in a marked increase in airflow resistance, which could be corrected by the administration of nebulized isoproterenol. On the other hand, sensitized animals with IgE antibody titers in the order of 64 did not manifest significant bronchoconstriction on inhalation challenge but developed anaphylaxis following intravenous injection of the antigen.     

Effects of tolerogenic conjugates in a canine model for reaginic hypersensitivity. I. suppression of hapten-specific IgE antibody response

Intraperitoneal administration to dogs of conjugates consisting of the 2,4-dinitrophenyl (DNP) groups coupled to nonimmunogenic macromolecules such as the copolymer of D-glutamic acid and D-lysine (DNP16-DGL) prior to sensitization with DNP2-ovalbumin led to the development of hapten-specific tolerance with respect to the IgE antibody response. Administration of these same conjugates to sensitized dogs resulted in complete abrogation of the ongoing anti-DNP IgE antibody production. A similar hapten-specific suppression of the ongoing anti-DNP response was also observed using the conjugates of DNP9-canine gamma globulins, and the tolerogenic effect was dose-dependent. The state of hapten-specific immunosuppression induced by these two types of tolerogenic conjugates was maintained despite repeated booster injections of the sensitizing antigens at biweekly inervals.     

Suppression of reaginic antibodies with modified allergens. IV. Induction of suppressor T cells by conjugates of polyethylene glycol (PEG) and monomethoxy PEG with ovalbumin

Administration of multiple injections of conjugates of ovalbumin (OA) and polyethylene glycol (PEG) or its monomethoxy derivative (mPEG) into mice which had been sensitized with 2,4-dinitrophenylated OA (DNP3-OA) abrogated both the anti-OA and anti-DNP IgE responses, in spite of additional injections of the sensitizing dose of DNP3-OA in the presence of A1(OH)3. Treatment of mice with OA-PEG in A1(OH)3 stimulated preferentially helper T cells, whereas injection of mice with OA-PEG in the absence of adjuvant elicited predominantly suppressor T cells. The unresponsive state of mice which had been treated 21 days earlier with OA-PEG could not be broken by the transfer of normal spleen cells and an additional sensitizing dose of DNP3-OA. Transfer of spleen cells from tolerized animals to normal mice dampened the capacity of the latter to mount both anti-DNP and anti-OA IgE responses; however, the suppressive effect of these cells was eliminated by treatment of the normal recipients with cyclophosphamide, which is a procedure known to inactive suppressor T cells, and hence it may be concluded that this effect was not due to the carryover of the tolerogen with the transferred cells. All these results provide strong support for the conclusion that the suppressor cells induced by the treatment of mice with OA-PEG and OA-mPEG conjugates belonged to a T cell subpopulation, and that the B cells of these mice were devoid of suppressive activity.     

The effect of hapten-specific suppression of IgE on antigen-induced histamine release from mouse peritoneal mast cells.

Subcutaneous injections of a mixture of dinitrophenylated ovalbumin (DNP3-OA) and dextran sulfate into Swiss-Webster mice elicited short-lived primary and long-lasting secondary IgE antibody responses to both DNP and OA. Histamine was released on in vitro challenge with antigen (OA or DNP22-BSA) of washed peritoneal mast cells (PMC) obtained from mice during a primary or a secondary IgE response. Administration of an intravenous injection of a tolerogenic conjugate of DNP8-mouse gamma-globulin, either prior to immunizationor during an ongoing IgE response, resulted in almost complete disappearance of circulating anti-DNP IgE antibody and in a very marked decrease in histamine release from PMC on challenge with DNP22-BSA. However, the IgE response to OA of these mice and the histamine release from their PMC on challenge with OA were not affected. Moreover, the PMC of mice, which had been tolerized to DNP, could be passively sensitized with serum containing DNP-specific IgE antibody for the release of histamine on DNP22-BSA challenge. The most significant finding of this study is the observation that the time course for the loss of reactivity of PMC to DNP22-BSA, after administration of the tolerogen during an ongoing secondary response, paralleled the decrease in circulating anti-GNP IgE antibody.     

Kinetics and drug sensitivity of the anti-hapten and anti-carrier IgG1 and IgG2 antibody production in guinea pigs

The influence of the hapten-protein ratio on the induction and kinetics of specific IgG1 and IgG2 anti-hapten and anti-carrier antibody synthesis, and the sensitivity of these reactions to cyclophosphamide (CY) and 6-mercaptopurine (6-MP), were studied following immunization with two different dinitrophenylated bovine gamma-globulin (DNP20-BGG and DNP47-BGG) conjugates in Freund's complete adjuvant (FCA) and drug treatment over the first 7 days after antigen injection. The DNP- and BGG-specific IgG1 and IgG2 serum antibody concentrations were determined weekly. Treatment by CY resulted in a complete suppression of the primary IgG1 and IgG2 anti-BGG antibody response, both in the DNP20-BGG and DNP47-BGG immunized guinea pigs. The anti-DNP response was completely suppressed only up to day 14 (DNP20-BGG immunized group) or day 21. This was followed by an increase that was significantly greater in the DNP20-BGG than in the DNP47-BGG immunized animals. The secondary IgG1 anti-BGG immune response induced by an injection of 1 mg BGG on day 86, was uninfluenced in animals immunized with DNP20-BGG but stimulated in the other group treated with DNP47-BGG. The IgG2 was completely suppressed in both groups. 6-MP, known to be less immunosuppressive in guinea pigs, led to very similar results. The finding that in animals immunized with DNP47-BGG the primary anti-BGG IgG2 antibody synthesis was uninfluenced by 6-MP, but the development of a memory was suppressed, would suggest that the primary antibody response and immunological memory are either separate mechanisms or have different sensitivities to 6-MP.     

In vivo effects of ovalbumin-conjugated muramyl peptides on the anti-ovalbumin IgE and IgG responses in mice

The effects of pretreatments of BALB/c mice with several conjugates of MDP and MDP-Lys to ovalbumin before immunization with ovalbumin (OA) were tested on the anti-OA IgE responses. Pretreatment with MDP-Lys-OA, but not with MDP-OA, induced an inhibition of the anti-OA primary and secondary responses, as measured by passive cutaneous anaphylaxis (PCA) and also by mast cell degranulation. The inhibition by pretreatment with MDP-Lys-OA was obtained whether it was administered in Freund's incomplete adjuvant (FIA) or in saline. This IgE suppression was accompanied by an enhancement of IgG_2a and IgG_2b anti-OA antibodies, with no change in the specific IgG₁ levels. Loss of antigenicity of OA, detected by the lack of degranulation of peritoneal mast cells sensitized by IgE anti-OA, was observed in the MDP-Lys-OA but not in the MDP-OA conjugates. This loss of antigenicity appears to correlate with the ability of the conjugate to induce suppression of the specific IgE response.     

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.     

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.     

Effect of epitope density on the induction of the IgE immune response in mice

The effect of hapten density on benzylpenicilloyl (BPO)-protein conjugates upon the induction of BPO-specific IgE immune response was investigated in BALB/c and C3H mice. Bovine gamma-globulin (BGG) and ovalbumin (OA) were employed as carrier proteins. In both strains of mice, moderately substituted conjugates (BPO19-BGG, and BPO-OA with an epitope density from 2 to 7.5) elicited a persistent BPO-specific IgE, while heavily substituted conjugates (BPO38-BGG) induced only a transient response. In contrast, lightly substituted conjugates (BPO3.2-, BPO9.5-BGG, and BPO1-OA) failed to produce BPO-specific IgE antibodies. There was no significant difference in carrier-specific IgE immune response among these various conjugates. These results suggest that moderately substituted hapten-protein conjugates are one of the favorable conditions for eliciting hapten-specific IgE immune response in mice.     

Suppression of immunoglobulin E-mediated anaphylactic reaction by Alpinia oxyphylla in rats

We investigated the effect of Alpinia oxyphylla water extract (AOWE) on immunoglobulin E (IgE)-mediated anaphylaxis activated by anti-dinitrophenyl (DNP) IgE antibody. AOWE dose-dependently suppressed passive cutaneous anaphylaxis (PCA) when intraperitoneally or orally administered. On the other hand, it showed weak suppressive activity when administered intravenously. AOWE dose-dependently suppressed anaphylactic histamine release from rat peritoneal mast cells (RPMC) activated by anti-DNP IgE antibody. However, AOWE had a significant augmenting effect on anti-DNP IgE antibody-induced tumor necrosis factor-alpha secretion from RPMC. These results indicated that AOWE may possess strong antianaphylactic action and also suggest that differential activity following administration routes may be caused by difference of bioavailability.     

Role of interferon-gamma in the modulation of the IgE response by 2,4-dinitrophenyl-Bordetella pertussis vaccine in the mouse

Immunization of mice with 2,4-dinitrophenyl-Bordetella pertussis (DNP-BP) failed to induce anti-DNP IgE responses. Administration of DNP-BP induced, however, the formation of anti-DNP IgE B memory cells, as demonstrated by adoptive transfer. Furthermore, mice pretreated with DNP-BP and primed with 2 micrograms DNP-ovalbumin (OA) in alum 2 weeks later produced high day-7 anti-DNP IgE levels. These subsided to near undetectable levels by day 12-14. The transient expression of serum IgE levels was accompanied by normal levels of anti-DNP IgG. The anti-OA response induced as a result of priming with DNP-OA in alum was not affected by pretreatment with DNP-BP. IgG subclass analysis revealed that mice pretreated with DNP-BP had elevated levels of IgG2a and reduced levels of IgG1 as compared to control (TNP-keyhole limpet hemocyanin-pretreated) mice. Treatment of mice with an anti-interferon-gamma monoclonal antibody, shortly after immunization with DNP-BP, not only reduced anti-DNP IgG2a levels, but prevented the sharp anti-DNP IgE decline that occurred after priming with DNP-OA in alum. These results suggest that DNP-BP-induced interferon-gamma production modulates Ig isotype expression in vivo in an anti-gen-specific manner.     

IgE-isotype-specific suppressor cells in the mouse: characterization using tetraparental chimera mice of high (DBA/2) and low (SJL) IgE responder embryos

Tetraparental chimera mice were developed by aggregation of IgE high responder (DBA/2) and IgE low responder (SJL) embryos. Anti-dinitrophenyl (DNP) IgE antibody response in such mice (SJL----DBA/2) upon challenge with DNP-keyhole-limpet hemocyanin (KLH) in alum was clearly suppressed, while anti-DNP IgG antibody response was not. High-titer anti-DNP IgE and IgG antibody response developed in F1 hybrid mice of SJL and DBA/2 (SDF1) mice. The experimental results suggest that high IgE antibody production is the dominant trait, and the IgE-specific suppressor gene in SJL mice is autosomal recessive. IgE-specific suppressor T cells in SJL mice actively suppressed IgE antibody formation by DBA/2 immuno-competent cells across the histocompatibility barrier. Hapten-specific B cells and carrier-specific T cells were prepared in SJL----DBA/2 and SDF1 mice by immunization with DNP-KLH or ovalbumin (OA) in alum and transferred to irradiated SDF1 mice followed by challenge with DNP-OA. Hapten-specific B cells and carrier-specific helper T cells clearly developed in SDF1 mice. Recipient mice transferred with DNP-KLH-primed SDF1 spleen cells and OA-primed SDF1 spleen cells showed high-titer anti-DNP IgE and IgG antibody responses. OA-primed SJL----DBA/2 spleen cells cotransferred with DNP-KLH-primed SDF1 spleen cells and OA-primed SDF1 spleen cells completely abolished secondary anti-DNP IgE antibody response. The data suggest that carrier-specific helper T cells for IgE and IgG antibody responses are distinct. The regulatory role of IgE-isotype-specific suppressor cells were considered to be the interference of cooperative cellular interaction between IgE B cells and carrier-specific, IgE-specific helper T cells.     

Suppression of IgE antibody response by the fatty acid-modified antigen

Ovalbumin (OA) of hens was chemically coupled with fatty acids (lauric acid, myristic acid, palmitic acid and stearic acid). These hydrophobically modified antigens were unable to react with mouse antiserum against native OA and were incapable of eliciting primary and secondary anti-OA antibody responses in BALB/c mice. Preadministration of these modified antigens, especially of palmitoyl OA (OA-pal), suppressed both primary and secondary anti-OA IgE antibody responses without affecting IgG antibody production. Administration of OA-pal after the primary immunization resulted in a rapid decrease of the ongoing anti-OA IgE antibody production and inhibited the anamnestic anti-OA IgE antibody response upon subsequent immunization with OA. The passive transfer of spleen cells from OA-pal-treated animals with OA-primed spleen cells suppressed the adoptive secondary anti-OA IgE antibody response in irradiated recipients. The suppressive effect was abrogated by treatment with an anti-T-cell antiserum indicating that suppressor T cells were primed by administration of hydrophobically modified antigens.     

Role of epitope density in the induction of immunity and tolerance with thymus-independent antigens. IV. Selective tolerance and IgE response by DNP-levan conjugates.

Tolerance induction of IgE antibody-forming cells by dinitrophenylated levan (DNP-LE) conjugates was investigated in CBA mice. Antibody production was measured by passive coutaneous anaphylaxis on rat skin. Specific antibody neutralization was obtained with conjugates of different substitution degrees. Specific inhibition of IgE antibody production was obtained only with highly substituted conjugates. However, the epitope density required for inducing tolerance to antibody synthesis of the IgE class was lower than for other classes of immunoglobulins. The tolerance induced by DNP-LE was shown to affect only the anti-DNP B cells of IgE class without T cell participation. These results demonstrate that B cell precursors of all Ig classes are susceptible to tolerance induction and not to triggering by T-independent antigens.     

The use of polyvinyl alcohols as tolerogenic carriers for suppression of anti-hapten IgE antibody responses

Conjugates of haptens [i.e. the dinitrophenyl (DNP) or benzylpenicilloyl (BPO) groups] with polyvinyl alcohols (PVA), with molecular weights in the range of 10–14 × 10³ daltons, were shown to suppress not only the primary anti-hapten IgE and hemagglutinating antibody responses of B₆D₂F₁ mice but, more importantly, also the ongoing antibody responses of presensitized mice. For this study, mice were immunized with the corresponding haptenated-ovalbumin (OA) conjugates (i.e. DNP₃-OA and BPO₄-OA) in presence of Al(OH)₃. In addition to the fact that the tolerogenic PVA-hapten conjugates did suppress exclusively hapten-specific responses, i.e. without affecting the anti-OA IgE antibody levels, their remarkable feature was that they were immunosuppressive even at the low average epitope density of about one. The immunosuppressive effectiveness of these tolerogenic conjugates was dose dependent, e.g. complete suppression of ongoing anti-hapten IgE responses was achieved with a single dose of 1 mg of the appropriate hapten-PVA conjugate.     

Chemical modification of allergen leading to changes in its epitopic activity

Modification of a model allergen ovalbumin (OA) with succinylation led to a decrease of its allergenicity measured by passive cutaneous anaphylaxis reaction, RAST inhibition assay and basophil histamine release. Modified OA stimulated OA-specific T-cell hybrid 3DO-548 to produce IL-2 at the same level as in case of non-modified OA. Modified OA did not induce anti-OA IgE, but did induce anti-OA IgG antibodies. This approach to chemical modification of allergen-selective blockade of B-cell epitopes while not affecting T-cell epitopes suggests new opportunities in creation of safe and effective allergovaccines.     

Global degranulation of rat mast cells stimulated with DNP-polystyrene.

Mediator release was studied in rat peritoneal mast cells sensitized with a mouse monoclonal anti-DNP IgE antibody, and stimulated with DNP-ornithine covalently attached to radio-derivatized polystyrene petri dishes. Cells releasing serotonin at maximal rates were investigated by transmission electron microscopy. Generalized exocytosis of granules could be observed, suggesting non-directional release of mediators, and non-compartmentalized action of second messengers in mast cells stimulated with polystyrene-bound DNP. Stimulation of sensitized mast cells by DNP covalently bound to the rigid polystyrene surface is consistent with extrinsic mechanisms proposed for Fc(epsilon)RI receptor action, and suggests that internalization of Fc(epsilon)RI is not needed for triggering cell degranulation.     

Inhibition of mast cell-dependent anaphylaxis by sodium salicylate

Sodium salicylate (NaSal) is a commonly used agent with a wide pharmacological spectrum. The objective of the present study was to investigate the effect of NaSal on anaphylaxis. NaSal (10-1 and 1 mm) significantly inhibited systemic anaphylaxis induced by compound 48/80 in rats. NaSal also significantly inhibited local anaphylaxis activated by anti-dinitrophenyl (DNP) immunoglobulin E (IgE). NaSal (10-1 and 1 mm) significantly inhibited histamine release from rat peritoneal mast cells (RPMC) activated by compound 48/80 or anti-DNP IgE. Northern-blot analysis demonstrated that a significantly reduced level of the mRNA of L-histidine decarboxylase was expressed in mast cells treated with NaSal, compared with that without NaSal. NaSal (10-2 and 10-1 mm) had a significant inhibitory effect on anti-DNP IgE-induced tumour necrosis factor-alpha secretion from RPMC. The level of cyclic AMP in RPMC, when NaSal (1 mm) was added, transiently and significantly increased about sixfold compared with that of basal cells. These results suggest a possible use of NaSal in managing mast cell-dependent anaphylaxis.     

Studies on immunity in hybridoma-bearing mice. A. Immune response to antigens. II. inhibition of production of anti-dinitrophenyl antibodies in mice which have rejected the B 53 anti-dinitrophenyl-producing IgE hybridoma

When BALB/c mice, which had rejected the anti-dinitrophenyl (DNP) IgE-producing hybridoma B 53, were immunized with DNP proteins, they produced much less anti-DNP antibodies than control (normal) mice. The anti-DNP plaque-forming cell (PFC) number was much less when spleen cells from mice immunized with DNP proteins were treated with sera of mice which had rejected the hybridoma B 53 than the PFC number from the same spleen cells not treated by the sera. The sera of mice which had rejected the hybridoma B 53 contained an inhibitor which was adsorbed and eluted from an anti-mouse immunoglobulin column and also a mouse anti-DNP IgG2a column. The inhibition of PFC was hapten-reversible. In Western blotting the eluates from the anti-DNP IgG2a column reacted as well with the blotted anti-DNP IgE B 53 as an anti-idiotypic antibody to anti-DNP IgE B 53. These criteria establish that the inhibitor in the sera of the mice which had rejected the B 53 tumor was an anti-idiotypic antibody of the type which mimics the epitope (DNP) of the immunizing antigen.     

Suppression of reaginic antibodies with modified allergens. II. Abrogation of reaginic antibodies with allergens conjugated to polyethylene glycol

The two immunogenic preparations, ovalbumin (OA) and the nondialysable constituents of the aqueous extract of ragweed pollen (RAG), were conjugated with polyethylene glycols of molecular weights of 6,000 and 20,000 (PEG6 and PEG20) with the aid of cyanuric chloride. The i.v. administration of OA-PEG6 or OA-PEG20 into normal mice or into mice sensitized to a state of immediate hypersensitivity to dinitrophenylated OA (DNP3-OA) resulted, respectively, in the suppression of the primary or secondary IgE responses to DNP and OA. Similarly, the administration of RAG-PEG6 abrogated the primary as well as the ongoing anti-RAG reaginic responses in mice sensitized to RAG. The unresponsiveness of spleen cells from animals which had received a tolerogenic dose of OA-PEG6 was also maintained after transfer into X-irradiated (550 rad) mice. The suppressive effects of the tolerogenic PEG conjugates of OA and RAG were shown to be immunologically specific. It is, therefore, suggested that PEG-modified allergens may prove useful for the abrogation of the IgE response to a variety of allergens responsible for conditions of common hypersensitivity in man.