anti-DNP IgE Antibody Research Articles

Mechanism of allergic cross-reactions—II. Cross-stimulation, by chemically unrelated ligands, of rat basophilic leukemia cells sensitized with an anti-DNP IgE antibody

Serotonin release from rat basophilic leukemia (RBL) cells, sensitized with a DNP-binding monoclonal IgE, was stimulated with solid surface (polystyrene)-bound DNP-amino acids. The stimulatory potency of DNP-amino acids was dependent on the structure of amino acid attached to DNP. Generally, DNP-amino acids with high affinities to the sensitizing IgE (I(50) < 10 μM) were stimulatory in polystyrene-bound form; DNP-amino acids with lower affinities (Pro, Cys, Trp), and aliphatic/aromatic DNP-amino acid derivatives were inactive. In addition to structural analogues of DNP, lymecycline, that is chemically unrelated to DNP but was found to have high affinity to IgE(aDNP), was also stimulatory in this system. This drug, and various quinones (e.g. acenaphthene-quinone) in BSA-conjugated forms also stimulated serotonin release from RBL cells sensitized with IgE(aDNP). These studies suggest that (1) There is a threshold of intrinsic ligand binding affinities at approximately I(50) = 10–100 μM; ligands with lower affinities do not stimulate mediator release even if they are presented in multivalent forms; (2) The above affinity threshold for mediator cell stimulation is valid for various ligands, irrespective of their chemical similarity to the immunogen; (3) Multispecific stimulation of mediator release may contribute to the frequently observed allergic cross-reactions, false positive tests for allergies, and anaphylactic reactions to drugs upon first exposure.

Mechanism of allergic cross-reactions—I. Multispecific binding of ligands to a mouse monoclonal anti-DNP IgE antibody

A recently developed solid-phase binding assay was used to investigate the specificity of ligand binding to a mouse monoclonal anti-dinitrophenyl IgE [IgE(aDNP)]. All DNP-amino acids, that were tested, inhibited the binding of radio-labeled IgE(aDNP) to DNP covalently attached to polystyrene microtiter plates; however, the concentration for 50% inhibition varied within four orders of magnitude, DNP- l-serine being the most, DNP-proline the least potent inhibitor. In addition to DNP analogues a large number (2074) of drugs and other compounds were tested for their ability to compete with DNP for the binding site of IgE(aDNP). At the concentrations used for screening 59% of the compounds had no significant inhibition; 19% inhibited the binding of IgE(aDNP) more than 50%. Several families of compounds (tetracyclines, polymyxines, phenotiazines, salicylates and quinones) of effective competitors were found. Within these families change in the functional groups attached to the “family stem” had major effects on the affinity of ligand binding. The occurrence frequencies of interactions of ligands with IgE(aDNP) is in good agreement with a semi-empirical model for multispecific antibody-ligand interactions.

Production of Tumor Necrosis Factor with IgE Receptor Triggering from Sensitized Lung Tissue

Mast cells and basophils have been known to play a central role in allergic inflammation through the release of chemical mediators by cross-linkage of IgE receptors. The IgE receptor triggering and calcium ionophore A23187 have also been shown to induce gene expression and production of tumor necrosis factor (TNF) by rat basophilic leukemia cells. In the present study, we examined whether IgE receptor triggering could induce gene expression and production of TNF in rat lung tissue. The lung tissue released not only histamine but also cytotoxic activity on L929 cells 2 and 4 h after incubation with dinitrophenyl conjugated to ovalbumin (DNP-OVA) following passive sensitization with anti-DNP monoclonal rat IgE antibody, whereas neither DNP-OVA nor anti-DNP IgE antibody could induce the cytotoxic activity when used solely. Calcium ionophore A23187 also could induce both histamine release and cytotoxic activity. These activities induced by IgE receptor triggering, A23187, and lipopolysaccharide were completely neutralized by preincubation with anti-mouse TNF-rabbit serum, but not with normal rabbit serum. Northern blot analysis using cDNA probe of mouse TNF demonstrated expression of TNF gene as early as 2 h after IgE receptor triggering. These data demonstrating that IgE receptor triggering induced gene expression and production of TNF in lung tissue suggest the participation of TNF in the pathogenesis of late asthmatic response through its biologic activities such as the attraction and activation of neutrophils and eosinophils.

Induction of Eczematous Skin Reaction in Experimentally Induced Hyperplastic Skin of Balb/C Mice by Monoclonal Anti-DNP IgE Antibody: Possible Implications for Skin Lesion Formation in Atopic Dermatitis

Biphasic skin reaction with peak response at 1 and 24 h with prominent mast cell degranulation was induced by intravenous application of a monoclonal anti-DNP IgE antibody and subsequent skin test. This reaction was hapten-specific and mast-cell-dependent because no reaction was observed when oxasolone was used as an elicitation antigen or skin test was elicited in genetically mast-cell-deficient mice (W/Wvv). A partial spongiotic reaction and mononuclear cell infiltration into the epidermis were observed in mice with hyperplastic epidermis induced by topical retinoic acid. Cotransfer of DNFB-sensitized lymph node cells with anti-DNP IgE antibodies failed to enhance the skin test reaction in unsensitized mice. These results suggest that, to some degree, IgE antibody may play some role in the development of eczematous skin lesions in the rodent system without the involvement of cellular hypersensitivity.

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.

Sensitivity of passive cutaneous anaphylaxis in rats. II. Suppression of passive cutaneous anaphylactic reactions in rats infected with Nippostrongylus brasiliensis.

The sensitivity of passive cutaneous anaphylactic (PCA) reaction was examined in rats infected with the nematode parasite, Nippostrongylus brasiliensis (Nb). PCA reactions with anti-DNP IgE antibody were remarkably suppressed by Nb infection already 10 days after infection and persisted for at least 28 weeks. 10 days after infection, no anti-Nb IgE antibody was present in the sera of infected rats, whereas 18 or 28 weeks after infection, circulating anti-Nb IgE antibody was present. It was concluded that two different mechanisms might explain the suppression. 10 days after infection, nonspecific IgE induced by potentiated IgE production inhibited passive sensitization with anti-DNP IgE antibody by occupying the IgE receptors on mast cells, whereas 18 or 28 weeks after infection, the mast cell IgE receptors were occupied by IgE antibody specific for the Nb antigen. The reactivity through non-immunological processes (skin sensitivity to compound 48/80) was not modified by Nb infection at any time after infection.

A canine model for the study of hapten-specific suppression of IgE-mediated bronchoconstriction and anaphylaxis.

Newborn mongrel dogs were sensitized with conjugates of ovalbumin (OA) and 2,4-dinitrophenol (OA-DNP3) in the presence of Al(OH)3 to produce high levels of anti-OA and anti-DNP IgE antibody. At 4-6 months of age, when anti-DNP and anti-OA antibody levels reached titers of 64 by passive cutaneous anaphylaxis, the dogs underwent separate inhalation and intravenous challenges with conjugates of DNP and bovine gamma globulin (DNP15-BGG) and OA. Inhalation challenge with DNP15-BGG and OA resulted in 5- and 10-fold increases in airflow resistance, respectively. Intravenous challenge with either DNP15-BGG or OA produced profound anaphylaxis with 60-80% decreases in blood pressure, cardiac output and regional blood flows in the carotid, superior mesenteric and renal arteries, and the distal aorta. Treatment of sensitized dogs with 5 doses of 20 mg of conjugates of DNP and polyvinyl alcohol (DNP2-PVA) on alternate days resulted in suppression of anti-DNP IgE antibody production; abrogation of established airway and vascular anaphylactic sensitivities; no change in regional blood flows, and no effect on sensitivities to challenge with OA.

Immunosuppressive effects of glycosylation inhibiting factor on the IgE and IgG antibody response.

Glycosylation inhibiting factor (GIF) was purified from culture filtrates of a T cell hybridoma, 23A4, by affinity chromatography on anti-lipomodulin Sepharose. The factor exhibited phospholipase inhibitory activity upon dephosphorylation. Immunization of BDF1 mice with aluminum hydroxide gel (alum)-absorbed dinitrophenyl derivatives of ovalbumin (DNP-OA) resulted in persistent IgE and IgG antibody formation. However, repeated injections of the affinity-purified GIF into the DNP-OA-primed mice beginning on the day of priming prevented the primary anti-hapten antibody responses of both the IgE and the IgG1 isotypes. Treatment with GIF also diminished on-going IgE antibody formation in the DNP-OA-primed mice. The treatment changed the nature of IgE-binding factors formed by BDF1 spleen cells. Incubation of spleen cells from OA + alum-primed mice with OA resulted in the formation of IgE-potentiating factor, whereas spleen cells of OA-primed, GIF-treated mice formed IgE-suppressive factor upon antigenic stimulation. It was also found that Lyt-2+ T cells in the OA-primed, GIF-treated mouse spleen cells released GIF, which had affinity for OA and bore I-Jb determinant(s). Transfer of a Lyt-1+ cell-depleted fraction of the OA-primed, GIF-treated mouse spleen cells into naive syngeneic animals resulted in suppression of the primary anti-DNP IgE antibody response of the recipients to alum-absorbed DNP-OA, but failed to affect the anti-DNP antibody response to DNP-keyhole limpet hemocyanin. The results indicate that GIF treatment during the primary response to OA facilitated the generation of antigen-specific suppressor T cells.

Clearance of passively transferred IgE antibody from peripheral blood of mast cell-deficient W/Wv mice.

The plasma decay curve of intravenously injected murine monoclonal or conventional anti-DNP IgE antibody was determined in mast cell-deficient W/Wv and their normal littermate +/+ mice. The results show that the clearance of passively transferred IgE was not altered by mast cell deficiency. Possible sites of clearance and/or catabolism of IgE are discussed.

Monoclonal IgE-dependent eosinophil cytotoxicity to haptenated schistosomula of Schistosoma japonicum: enhancement of the cytotoxicity and expression of Fc receptors for IgE by Nippostrongylus brasiliensis infection.

To obtain direct evidence for the involvement of IgE antibodies in eosinophil-mediated killing of schistosomula of S. japonicum, dinitrophenylated (DNP) schistosomula pretreated with mouse monoclonal IgE antibodies were co-cultured with purified rat peritoneal eosinophils. It was found that the eosinophil-mediated adherence and damage to haptenated schistosomula were dependent on a monoclonal anti-DNP IgE antibody, but not on monoclonal anti-ovalbumin IgE antibody. Moreover, eosinophils from N. brasiliensis-infected rats demonstrated an enhanced ability in the IgE-dependent damage to DNP-schistosomula as compared with the cells from normal rats. The enhancement was associated with an increase in the proportion of eosinophils expressing Fc receptors for IgE.

Enhancement of IgE-dependent eosinophil cytotoxicity to dinitrophenylated schistosomula by a nematode infection.

In order to obtain direct evidence for the involvement of IgE in the eosinophil-mediated cytotoxicity, dinitrophenylated (DNP) schistosomula of Schistosoma japonicum coated with monoclonal IgE antibodies were examined as to whether they were killed by rat peritoneal eosinophils. The results indicated that the cytotoxicity of the eosinophils was dependent on a mouse monoclonal anti-DNP IgE antibody, but not on another monoclonal antibody specific to ovalbumin. Moreover, an enhanced cytotoxicity was observed when eosinophils were obtained from rats 4 weeks after infection with Nippostrongylus brasiliensis.

Studies on immunity in hybridoma-bearing mice. A. Immune response to antigens. I. Role of subcutaneously injected IgE-producing hybridoma on antibody production of different isotypes against immunizing antigens.

When immunized with ovalbumin (OVA), mice bearing subcutaneously injected anti-dinitrophenyl (DNP) IgE-secreting hybridoma cells do not produce anti-OVA IgE antibodies as long as anti-DNP IgE is present in abundance in their sera. As soon as the titer of anti-DNP IgE antibody falls, anti-OVA IgE antibody appears. This inhibition of production of anti-OVA IgE is attributed to Fc epsilon-bearing T cells which have a suppressive action of limited duration. Other isotypes of anti-OVA antibodies are produced in similar amounts as in controls which were immunized with OVA but not injected with hybridoma cells.

Suppression of IgE antibody production in SJL mice: V. Effect of irradiation and adult thymectomy on the suppression of IgE antibody production in SJL mice

Anti-DNP IgE antibody production was low and transient in SJL mice which were immunized with 1 μg DNP-Nb and 1 mg A1(OH) 3. The immunized SJL mice were irradiated (60–540 R) 1 day after challenge. A dose higher than 180 R induced enhancement of anti-DNP IgE antibody production as compared to nonirradiated control mice, suggesting the existence of irradiation-sensitive suppressor cells. Anti-DNP IgE antibody production was suppressed when immunized and irradiated SJL mice were injected with spleen cells from adult-thymectomized SJL mice. The donors of the spleen cells were thymectomized 2 or 4 months previously, and this suggests that the suppressor cells from unprimed mice are long-living T cells.

Half-Life of Murine IgE Antibodies in the Mouse

The half-life of intravenously injected murine anti-DNP IgE antibody was determined in recipient mice by sequential bleeding and was found to be 12 h. Intradermally injected IgE antibody persisted much longer, the half-life was 6 days.

Regulation of immune response by preadministration of cells briefly pulsed with antigen in vitro. I. Suppression of IgE antibody response by antigen pulsed spleen cells.

The intravenous administration of syngeneic spleen cells (SPCs) briefly pulsed with antigen in vitro, results in a profound state of IgE antibody unresponsiveness. In Balb/c mice, the primary response of anti-DNP, anti-beef insulin and anti-ovalbumin IgE antibody is completely suppressed by the administration of antigen-pulsed spleen cells, 1 X 10(7), 5 X 10(7) and 1 X 10(8), respectively. This suppression is antigen specific and effects both primary and secondary immune responses. Furthermore, the immune response to dinitrophenylated Keyhole limpet hemocyanin (DNP-KLH) is most extensively suppressed by DNP-KLH pulsed SPCs, intermediately suppressed by KLH-pulsed SPCs and minimally suppressed by dinitrophenylated mouse gamma globulin or dinitrophenylated mouse serum albumin pulsed SPCs. Suppressing directly cells specific for hapten and carrier, hapten carrier protein pulsed SPCs would caused the additive suppressive effect. The suppression is induced strongly by the intravenous administration of antigen pulsed spleen cells, slightly by the subcutaneous administration and is not induced by the intravenous administration of antigen solution in phosphate buffer saline. This suppression may be mediated by either of two different mechanisms: one of them is responsible for the immediate tolerance which is induced without any suppressor cells 1 day after the administration of antigen pulsed SPCs, and the other is responsible for the suppression transferred by suppressor cells or factors to normal mice 7 days after the administration of antigen pulsed SPCs. This method in which IgE antibody response is suppressed by the administration of cells briefly pulsed in vitro with antigen, provides a powerful tool to analyze the first step of antigen specific suppression developed in vivo by conventional antigens.

Induction of Anti-DNP IgE Response by DNP-Coupled BCG in Mice

We have demonstrated that intraperitoneal, subcutaneous, or intravenous immunization with a large dose of 2,4-dinitrophenylated (DNP) bacillus Calmette-Guérin induced anti-DNP IgE antibodies in C3H/He, C57BL/6, Balb/c and DBA/1 mice but not in SJL mice, although the use of Freund’s complete adjuvant has been considered ineffective in producing reagin antibodies.

Immunological properties of Fc receptor on lymphocytes. 9. Functional analysis of FcR gamma+ and FcR gamma- lymphocytes in IgE antibody responses.

The present study was undertaken to clarify the function of FcR gamma+ and FcR gamma- lymphocytes in anti-DNP IgE antibody responses. The helper activity was observed predominantly in FcR gamma- T cells, but poorly in FcR gamma+ T cells, as in the case of IgG antibody responses, indicating that the absence of FcR gamma on T cell surface is a common surface characteristic to helper T cells, and that there is no close relationship between the determination of class specificity of Ig and T cell subsets divided by the presence or absence of FcR gamma. Furthermore, IgE antibody responses were more dependent upon helper T cells than IgG antibody responses. The data presented in this paper also demonstrated that the precursors of anti-DNP IgE antibody-forming cells were equally contained in both FcR gamma+ and FcR gamma- B cells, when they were transferred into recipients with helper T cells. This is a striking contrast to IgM/IgG antibody responses, in which only FcR gamma- B cells could differentiate into antibody-forming cells. Moreover, the precursor activity of FcR gamma+ and FcR gamma- B cells for IgE antibody-forming cells was not affected by the treatment of cells with suppressive B cell factor released from FcR gamma+ B cells after binding of antigen-antibody (IgG) complexes and known to suppress the proliferation of FcR gamma- IgM/IgG antibody-forming cell precursors. Thus, IgE B cells seem to be more heterogeneous than IgM/IgG B cells with respect to the FcR gamma marker and insensitive to the regulatory effect of FcR gamma+ B cells. IgE B cells are regulated differently from IgG B cells in B-B cell interactions.

Fine specificity and idiotype analysis of the IgE response to the synthetic terpolymer l-Glutamic acid 60- l-Alanine 30- l-Tyrosine 10 (GAT) and its dinitrophenyl conjugate (DNP-GAT)

In three mouse strains, BALB/c, CBA and C3H/He, specific IgE antibodies could be induced by DNP-GAT and GAT. The presence of both anti-GAT and anti-DNP IgE antibodies was measured, using both heterologous PCA in rats and specific degranulation of peritoneal mast cell tests. The fine specificity of the IgE anti-GAT antibodies elicited by both DNP-GAT and GAT was similar to the specificity of anti-GAT IgG antibodies. The IgE anti-GAT antibodies cross reacted with l-glutamic acid 50- l-tyrosine 50 (GT) copolymer, but not with l-glutamic acid 60- l-alanine (GA) copolymer. Anti-idiotypic serum was found to be able to degranulate specifically peritoneal mast cells from GAT and DNP-GAT immunized animals, indicating that IgG and IgE anti-GAT antibodies share common idiotypic determinants.

Suppression of the IgE antibody response to ovalbumin in mice with a conjugate of ovalbumin and isologous γ-globulins

Injection of a conjugate of ovalhumin (OA) with mouse γ-globulins (MγG) into normal B 6D 2F 1 mice, or into mice preimmunized with DNP 3-OA and Al(OH) 3, markedly depressed the capacity of these two groups of mice to mount, respectively, primary or secondary anti-DNP and anti-OA IgE antibody responses. The immunosuppressive effect of OA-Mγ G conjugates was specific for both the antigenic and carrier functions of OA, i.e., it did not affect the ability of the mice to mount anti-DNP and anti-ASC IgE responses upon immunization with DNP-ASC in Al(OH) 3, Injection of unmodified OA or of conjugates of OA with rabbit γ-globulins, on the other hand, interfered only in a transient manner with the full expression of the primary anti-OA and anti-DNP IgE responses elicited by immunization with DNP 3-OA and AI(OH) 3. In experiments designed to elucidate the mechanism underlying this mode of immunosuppression, 3 × 10 7 spleen cells from mice, which had received four repeated ip injections of OA-MγG 4 weeks before sacrifice, were injected ip into normal nonirradiated syngeneic recipients, which received two injections of DNP 3-OA and Al(OH) 3 4 hr and 4 weeks after cell transfer. The primary and secondary anti-DNP and anti-OA IgE responses of the recipients were profoundly suppressed by comparison with the responses of recipients of cells from normal or immunized mice. Moreover, transfer of spleen cells from normal or immune animals into mice which had been immunosuppressed with OA-MγG did not restore the ability of the recipients to mount anti-OA and anti-DNP reaginic responses. These results suggest, therefore, that the immunosuppression is at least in part due to suppressor cells which are induced by conjugates of isologous γ-globulins with a protein antigen.

Nonspecific enhancement of mouse antihapten IgE antibody response: Involvement of a T-cell subpopulation and its product for the potentiation

A concomitant administration of Nippostrongylus brasiliensis saline extract (Nb) with dinitrophenylated ovalbumin (DNP-Ov) significantly enhanced the anti-DNP IgE antibody response in mice which had been irradiated and given a combination of spleen and mesenteric lymph node cells from syngeneic, infected donors and cells from DNP-keyhole limpet hemocyanin (DNP-KLH)-primed donors. The treatment of lymphocytes from infected mice with anti-mouse brain-associated θ serum and C abrogated the enhancing activity. The potentiation occurred in mice receiving nylon wool-nonadherent cells but not in mice receiving adherent cells. Challenge with Nb plus DNP-Ov failed to induce potentiation in C3H mice which are known as nonresponders to low doses of Ov, whereas challenge with Nb plus DNP-bovine gamma globulin (BGG) potentiated the response. However, further increase of the enhanced response was not obtained by adding carrier (BGG or Ov)-primed cells to the transferred lymphocyte populations. When a T-independent antigen, DNP-Ficoll, was used for challenge concomitantly with antigen Nb, no potentiation occurred, even though DNP-Ficoll did not give any tolerogenic or suppressive effect on the IgE antibody response to DNP-Nb. An enhancing activity on the IgE class of antibody response but not on the IgG class was observed in supernatants of in vitro culture of lymphocytes from infected mice upon stimulation of the cells with 10 to 50 μg Nb. These results indicate that the potentiation is mediated by Nb specific T cells via a soluble factor(s) that enhances specifically the IgE class of antibody responses but nonspecifically in terms of antigens used for immunization. The results also suggest that the potentiating factor displays its activity in the presence of other T cells reactive to carrier determinants of the challenging antigen but not of cells which already have committed themselves to the carrier and differentiated as helper cells.