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IgE plays a central role in allergic reactions. Some anti-IgE antibodies (HMK-12, 6HD5) inhibit the binding of IgE to the FcepsilonRI of mast cells/basophilic leukemia cells (PT-18, RBL/2H3), but less inhibition is seen with the anti-allotypic JKS-6 and the anti-idiotypic Eb-1. Anti-IgE HMK-12 can detach bound IgE molecules from the FcepsilonRI. When mast cells or basophils were incubated with monoclonal anti-DNP-IgE SPE-7, washed and treated with anti-IgE HMK-12, anti-IgE/IgE complexes were found in the supernatant. Similar results were obtained with the Fab fragment of HMK-12. Mice injected with anti-DNP-IgE SPE-7 and later with DNP-BSA had the typical systemic anaphylactic shock. However, if they were injected with the anti-IgE antibody (HMK-12) before the challenge, they did not get an anaphylactic shock. In the sera of mice injected with monoclonal IgE SPE-7 and anti-IgE antibody (HMK-12), IgE/anti-IgE complexes were detected. No passive cutaneous anaphylaxis occurred if the rats were injected with anti-IgE antibodies before the challenge. In summary, anti-IgE antibodies can remove IgE antibodies from the FcepsilonRI; anti-IgE/IgE complexes can be detected in vitro and in vivo, and anti-IgE antibodies can inhibit IgE-mediated systemic or local anaphylactic reactions., (Copyright 2002 S. Karger AG, Basel)

The existence of memory B cells for IgE was investigated using spleen cells harvested from mice primed and boosted with dinitrophenylated keyhole limpet hemocyanin. Using the polymerase chain reaction, it was found that mRNA for IgE was already demonstrable in spleen cells taken 1 day after boost. Spleen cells taken from mice 1 day after boost and cultured in vitro without the addition of antigen also produced anti-dinitrophenylated (DNP) IgE antibody. In contrast, mRNA for IgE in spleen cells from mice primed 4 weeks previously, but not boosted, was not demonstrable. Anti-DNP IgE was not produced in cultures of spleen cells from mice primed but not boosted. As mRNA for IgE was demonstrable already 1 day after boost we concluded that mice produce memory B cells not only for IgM, but also for IgE specific for the immunizing antigen.

Isotype- and allotype-specific IgE determinations by sandwich ELISA with monoclonal antibodies to Igh-7 (6HD5, HMK-12), Igh-7a (No. 297) and Igh-7b /JKS-6) were used to qualitate antibodies of the IgE class. The sensitivity of the method is 0.1 ng/ml. Serum IgE levels were much higher in mice infected with Nippostrongylus brasiliensis. In sera of BALB/c, AKR and C3H/JCL mice only IgEa, in sera of C57BL/6 and CB-20 only IgEb, in sera of (BALB/c x C57BL/6) F1 mice, both allotypes were detected, as expected. In mice with autoimmune diseases, such as NZB, NZW, (NZB x NZW) F1, MRL/Ipr and MRL/n strains which all belong to the IgEa allotype group, IgE and IgEa were very high. In the sera of BXSB mice, both IgEa and IgEb were detected. Both IgEa and IgEb from donor and host were increased in the sera of mice with graft-versus-host disease after transplantation but only the IgEa (from the donor) was increased in mice with graft-versus-host disease after bone marrow transplantation. In the former, both B cells (from donor and host) secreted IgE, whereas in the latter only the donor cells did. These are the first observations showing the importance of IgE allotype secretion as an indication of graf-versus-host disease.

Various side effects have been associated with the clinical use of contrast media. Immunological mechanisms have been proposed but there have been very few experimental studies with animal models. We have attempted to develop murine models to determine whether or not anaphylactic antibodies such as IgE and IgG1 against hapten (DNP) were enhanced with contrast medium (iopamidol) as an adjuvant or if the contrast medium itself produced antibodies of the IgE class. The results showed that anti-hapten IgE and IgG1 production was greatly enhanced with immunogen plus contrast medium. Anti-contrast medium antibodies of the IgE class could not be detected by PCA reactions. The enhancement of IgE and IgG1 production for hapten was associated with IL-4 release by the neutralization test used by monoclonal anti-IL-4 antibodies. This is the first observation to show that contrast media may have a strong adjuvant effect for the production of IgE and IgG1. This murine model demonstrates a possible immunological function of contrast media in vivo.

Anti-dinitrophenyl IgG1 and IgE antibody production were examined in vitro. Splenic B cells from mice immunized in vivo at various intervals with dinitrophenylated proteins were cultured in vitro with splenic T cells and splenic antigen-presenting cells from mice immunized previously (2 weeks) with keyhole limpet hemocyanin. Anti-dinitrophenyl IgG1 and IgE antibodies were determined in the supernatants by ELISA. Without T cells and antigen-presenting cells or without antigen, no anti-dinitrophenyl antibodies were detected. When the B cells were separated by panning with anti-murine IgG1 antibody, only the cells adhering to anti-IgG1 produced anti-dinitrophenyl IgG1 antibodies. Anti-dinitrophenyl IgE production was observed from cells adhering and from cells not adhering to the anti-IgG1-coated plates, as expected. However, anti-dinitrophenyl IgE was also produced from cells which adhered to the anti-IgG1-coated plates, but did not have surface IgE antibodies. Therefore, cells without surface IgE adhering to anti-IgG1 were capable of producing anti-dinitrophenyl IgE antibodies.

When B cells from BALB/c mice were cultured with lipopolysaccharide (LPS) and interleukin-4 (IL-4), a large amount of IgE was detected in the culture supernatants. The IgE production from unseparated spleen cells cultured with LPS and IL-4 was less than the amount of IgE obtained from separated B cells. When syngeneic T cells were added to separated B cells cultures, which were subsequently stimulated with LPS and IL-4, less IgE was produced, as compared to cultures without T cells. The hypothesis that T cells, or factors secreted by these cells, inhibit IgE production is supported by the fact that the degree of suppression of IgE production paralleled the number of T cells added. CD8(+)-enriched T cells were slightly more suppressive than CD4(+)-enriched T cells. Addition of exogenous IL-3 was only partially suppressive. These observations suggest that IL-4 added to unseparated spleen cells in vitro stimulates B cells for IgE production and also stimulates T cells. Lymphokines secreted by these stimulated T cells may in turn act on B cells. Some of these lymphokines, such as interferon-gamma and IL-2, may have a suppressive action on IgE production.

Participation of IgE in protective immunity against Schistosoma japonicum was examined by comparing congenital IgE-deficient SJA/9 and IgE-producing SJL/J mice. Mice immunized with 100 irradiated cercariae 7 weeks previously were infected with 50 live cercariae. In SJL/J mice at 40 days after infection, a 3-to 4-fold increase of total IgE levels and anti-S, japonicum egg IgE antibody production were observed with no significant difference between immunized and nonimmunized mice. IgE was not detected in SJA/9 mice throughout the experiments. Protective immunity evaluated by recovery of adult worms was found in SJA/9 mice and was comparable to that of SJL/J mice. These results suggest that acquired immunity in mice with irradiated cercariae of S. japonicum was not dependent on IgE in these strains of mice.

IgE production was obtained from B cells of BALB/c or nude mice when these cells were cultured with IL-4 plus LPS. IL-2 added to these cultures at the start (day 0), 1 or 2 days later completely suppressed the production of IgE. The production of IgG1 was also inhibited, but only if IL-2 was added on day 0. The production of other isotypes (IgM, IgG2a, IgG2b) was only slightly decreased by addition of IL-2. No suppression of IgE or IgG1 production was observed if monoclonal anti-IL-2 was added, whereas anti-IFN-gamma had no effect on the suppression of the production of these isotypes. The expression of CD23 on the third day of culture on B cells stimulated with LPS and IL-4 was markedly decreased when IL-2 was added to the cultures on day 0. Addition of monoclonal anti-IL-2 suppressed all effects produced by IL-2, whereas addition of anti-IFN-gamma had no effect. These results show that the suppression by IL-2, at least for the first signaling processes, are different from the suppression produced by IFN-gamma.

The production of anti-trinitrophenyl (TNP) antibodies of different isotypes from in vivo primed B cells was studied using the plaque-forming cell method. It was shown that these B cells secrete anti-trinitrophenyl antibodies of different isotypes only in the presence of Th2 cells specific for keyhole limpet hemocyanin (KLH) and the hapten-carrier conjugate TNP-KLH. Lipopolysaccharide-stimulated primed B cells without cells from the Th2 clone did not produce anti-TNP-specific IgG1 or IgE antibodies even in the presence of the hapten-carrier antigen TNP-KLH. Supernatants from these Th2 clones cultured with antigen-presenting cells and the complete antigen were unable to activate primed B cells for antibody secretion. Cognate interaction between primed B cells and carrier-specific Th2 cells is a prerequisite for hapten-specific IgG1 or IgE production. Anti-IL-4 antibody inhibited secretion of anti-hapten IgE antibody. Therefore, for production of anti-hapten antibody of the IgE isotype IL-4 is also necessary.

A low number of highly irradiated spleen or lymph node cells from reticulum cell sarcoma (RCS)-bearing SJL mice brings about suppression of IgE antibody production in adequately immunized and sublethally irradiated recipients, probably by known proliferative action of RCS cells on the few Ly-1 T cells which escaped from the irradiation.

To investigate the involvement of the heart in acute allergic reactions in a system immunologically analogous to that of humans, a model of cardiac anaphylaxis mediated by IgE antibodies was developed in the guinea pig. Hearts obtained from guinea pigs, passively sensitized with homologous antidinitrophenyl IgE antibodies, were perfused and challenged in vitro with antigen. Challenge resulted in sinus and ventricular tachycardia, atrioventricular conduction block and substantial histamine release. The results demonstrate that IgE antibodies can sensitize the heart and that the severity of cardiac dysfunction, which follows challenge with specific antigen, directly correlates with the magnitude of histamine released. Since myocardial ischemia and similar arrhythmias occur during immediate hypersensitivity reactions in humans, this experimental model will be helpful in the investigation of cardiac involvement in acute allergic reactions.

Nippostrongylus brasiliensis infection was found to be highly effective in inducing a carrier-specific enhancing effect on primary and secondary antihapten IgE antibody response in the rat, when animals were immunized i.p. with 10 mug of dinitrophenylated N. brasiliensis protein (DNP-Nb) plus 10 mg Al(OH)3 2 weeks after the infection. The carrier effect by the infection was much greater than that obtained by any other supplementary immunization with the carrier (Nb) plus adjuvant, so far examined, in terms of the IgE antibody response. The results, together with our previous observation in the mouse, provide an explanation for the reason why antiworm IgE antibodies are so easily detectable in helminth infections.

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.

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.

The half-life of IgE in the mouse was investigated by using radiolabeled and unlabeled monoclonal antibodies of the IgE class. Quantitative serologic assays were used for the unlabeled antibodies. IgE was cleared rapidly upon i.v. inoculation; after 48 hr, less than 0.2% of the initial concentration remained in the serum. The IgE was cleared initially with a half-life of 1 to 2 hr, attaining a relatively constant value of 5 to 8 hr. The corresponding values for IgG1, determined as a control, were 11 to 12 hr and 9 to 11 days, respectively. The initial stage probably reflects equilibration with extravascular spaces. This is supported by experiments with mice in which IgE-secreting tumors were implanted and then resected; IgE was cleared from such mice with an average initial half-life of about 5 hr. The rates of clearance of inoculated IgE were approximately the same in mice bearing an IgE-secreting tumor and in normal mice. This suggests that the initial rapid clearance of IgE from normal mice is not due to adherence of IgE to saturable sites; such sites might be expected to be occupied in mice expressing high serum concentrations of IgE. This conclusion was supported by experiments in which 1-mg quantities of IgE were inoculated i.v. into normal mice daily for 6 days. Additional IgE injected on day 7 was cleared normally. The results obtained with tumor-bearing mice indicate that the reported failure to elicit an IgE response to an antigen in mice bearing IgE-secreting hybridomas cannot be attributed to rapid clearance of newly synthesized IgE in such mice, as compared with normal mice.

Anti-DNP IgE antibody production was low and transient in SJL mice which were immunized with 1 microgram 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.

A/J and DBA/1 mice were infected with 750 third-stage larvae of Nippostrongylus brasiliensis and immunized with 1 mug dinitrophenylated N. brasiliensis extract (DNP-Nb) with 1 mg Al(OH)3 to produce high titers of anti-hapten IgG1 and IgE antibody. Partial tolerance to the production of anti-hapten IgG1 and IgE antibody could be induced by DNP-Ficoll from 5 weeks before to 1 week after the DNP-Nb immunization. The tolerized state persisted through the duration of the experiments. However, no tolerizing effect could be demonstrated on secondary antihapten IgE antibody production induced by DNP-Nb. Moreover, DNP-Ficoll failed to evoke anti-hapten IgG1 or IgE antibody production.

To elucidate the roles of IgE antibody in host responses to helminth infection, selective IgE-deficient SJA/9 mice were infected with the nematode parasites Nippostrongylus brasiliensis, which elicits remarkable potentiated IgE production, and Trichinella spiralis, which induces strong anti-Trichinella IgE antibody production in normal mice. The kinetics of blood eosinophilia, worm burden after primary infection, and resistance to secondary infection in SJA/9 mice were the same in both infections as those in congenic SJL/J mice used as an IgE-producing control. These results indicate that the host responses examined here operate under IgE-independent mechanisms.

High titer and persistent antihapten IgE production in SJL mice can be obtained using appropriate immunization and radiation. Nonirradiated mice rapidly terminate this antihapten IgE production. Radiation was not necessary to prolong antihapten IgE production in other strains of mice. Termination can be obtained even in irradiated SJL mice by transferring normal SJL spleen cells. That the suppressor cells are T cells is shown by using thymocytes or cells treated with anti-Thy 1.2 and complement. No appreciable suppressive effect by normal spleen cells could be demonstrated on IgG1 production in SJL mice. The characteristic of low and transient IgE antibody response in SJL mice is inherited as a recessive trait controlled by a single Mendelian autosomal gene and is not linked to the H-2-gene complex. This characteristic does not depend on the infectivity of Nippostrongylus brasiliensis, the effect of anticarrier antibody, or the recognition of antigen.

To investigate why SJA9 mice do not produce IgE antibody, we took advantage of the adoptive transfer method and the carrier effect. Antibody-producing B cells (donor I) were primed with 2,4-dinitrophenylated ovalbumin (DNP-OVA) and carrier-specific T cells (donor II) were primed with keyhole limpet hemocyanin (KLH). The recipients were challenged with DNP-KLH. When 0.2 micrograms of DNP-OVA was used and the donor II cells were from SJL mice the recipient produced anti-DNP IgE. When donor I cells were from the SJL strain and donor II cells were from the SJA9 strain the recipient produced anti-DNP IgE. This pointed to a T-cell defect (absent or functionally inactive T epsilon o cell) in the SJA9 strain, which could be corrected either by injecting unprimed T cells from the SJL strain into the SJA9 donor I or by infection with Nippostrongylus brasiliensis, a hookworm, after priming. It seems that in the SJA9 strain in the absence of functioning T epsilon o cells isotype-specific (IgE) suppression, described previously in the SJL strain, is more pronounced, because when the SJA9 donor I was primed with 1 microgram of DNP-OVA only trace amounts of anti-DNP IgE were produced with SJL donor II cells.

Mouse and rabbit IgE antibody-containing sera do not lose sensitivity capacity when stored at -20 degrees C even for 2 years or when lyophilized.

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.

Homologous skin sensitizing antibodies in the rabbit can be differentiated by their requirement for complement in expressing the passive cutaneous anaphylactic reaction as complement dependent (CD) or complement independent (CI). CI antibodies are representative of the IgE class, while the CD antibodies belong to a proposed new subclass of IgG designated IgGa (a = anaphylaxis). The following are characteristics of CI (IgE) antibodies: elution at 0.05 M on DEAE at pH 8.1 migration ahead of 7S in ultracentrifugation, molecular weight 215,000 daltons by Sephadex G-200, fast gamma-electrophoretic mobility, isoelectric point, pI = 4.98 (5.68-4.58), 85-95% heat sensitivity, sensitivity to reduction plus alkylation and option at 0.01 M on DEAE, 7S in ultracentrifugation, MW 138,000 daltons, slow gamma-electrophoretic mobility pI = 6.90 (8.30-5.70), resistance to heating and to reduction plus alkylation and 1-day optimal SP. CI antibodies could be absorbed by anti-Fab, anti-epsilon, the homologous antigen and were not absorbed by anti-gamma, anti-alpha, or heterologous antigens. The CD antibodies were absorbed by anti-Fab, anti-gamma and not absorbed by anti-epsilon, anti-alpha, or heterologous antigens.

When dinitrophenylated keyhole limpet hemocyanin (DNP-KLH) primed AKR mice were injected with dinitrophenylated Nippostrongylus brasiliensis extract (DNP-Nb), an enhancement of IgE antibody production was obtained by nematode parasite, N. brasiliensis, infection or by Nb antigen injection 2 weeks before DNP-Nb injection. A preferential enhancement of IgE production was observed in infected mice compared with Nb-injected mice. Anti-hapten IgE antibody response was suppressed in AKR mice after appropriate immunization. No suppression of IgE response was observed after whole body irradiation (180-540 R) at an early stage of immunization. High and persistent IgE response was observed in (AKR X DBA/1)F1 mice of both sexes, therefore, the suppressive effect on IgE response in AKR mice was inherited as an autosomal recessive trait. The similarity of IgE class specific suppression in AKR and SJL mice is discussed.

Anti-2,4-dinitrophenyl (DNP) IgE producing hybridoma B 53 when injected subcutaneously is established equally well in syngeneic BALB/c (heavy-chain allotype a) and congenic CB20 (heavy-chain allotype b) mice. However, secretion of anti-DNP IgE monoclonal antibody is greatly suppressed in CB20 mice. B 53 cells taken from the subcutaneous tumors of CB20 mice produce anti-DNP IgE in vivo in BALB/c mice and in vitro. No difference was observed in IgE production between these cells and the controls taken from BALB/c mice. The suppression of IgE production was due to T cells and/or their product(s) of CB20 mice.

The adjuvant effects of an amorphous silica (Aerosil) and of A1(OH)3 on primary and secondary IgE and IgG1 immune responses to small doses of OA were studied comparatively in BALB/c, C57BL, DBA/1, AKR, DBA/2 and SJL inbred mouse strains. During the primary response, all mouse strains produced both IgG1 and IgE antibodies when antigen was given with A1(OH)3, whereas only DBA/1, BALB/c and DBA/2 mice produced IgG1 and/or IgE antibodies when Aerosil was used as the adjuvant. A booster effect, higher than that obtained by A1(OH)3, was induced by Aerosil in all mouse strains. The adjuvant effect of Aerosil was more selectively directed to the production of IgE antibody.

Anti-dinitrophenyl (DNP) IgE antibody was produced in guinea pigs (GP) by immunization and repeated boosters with 1 mug DNP-Ascaris extract in A1 (OH)3. The isoelectric point was determined to be 6.48 and the molecular weight to be 185,000 daltons. In absorbtion chromatography, rabbit anti-GPIgG1 could not absorb the anaphylactic activity of the IgE. The activity of IgE was destroyed by heating at 56 degrees C for 1 h. IgE persisted in the skin for at least 3 weeks.

The antigenicity of Trichomonas vaginalis to elicit IgE antibody was examined in mice. The soluble antigen was prepared by sonication of in vitro cultured T. vaginalis lines. BALB/c mice were immunized by various doses of the antigen with alum followed by a booster injection on day 14 after primary injection of the protozoan antigen. The highest IgE antibody titer was found in mice immunized with 30 micrograms of the antigen. The IgE antibody response to Trichomonas antigen is regulated by at least two immune response genes: one localized in the I region (K to I-B subregions) of chromosome 17, the other in a not yet identified chromosome.

IgE levels after Nippostrongylus brasiliensis infestation were high in all strains of mice examined except in the SJA/9 strain. However, surface IgE levels on B cells were not different from those observed in the SJL strain. Most surface IgE-bearing cells also had surface IgM molecules. We suggest that the surface IgE-bearing cells are precursors of IgE-producing cells. In adoptive transfer, hapten-specific IgE antibody was produced when hapten-primed cells from SJA/9 mice were transferred together with cells from SJL donors infested with N. brasiliensis but not when the cells were from SJA/9 donors. It is suggested that the lack of collaboration between hapten-primed B cells and cells from infested mice is a genetic trait of the SJA/9 strain.

IgE anti-DNP antibody-secreting hybridomas were obtained by polyethylene glycol fusion of murine myeloma cells with spleen cells from mice, primed with DNP-KLH and challenged with DNP-Nippostrongylus brasiliensis extract. The in vivo and in vitro continuously growing hybridomas are producing high amounts of monoclonal IgE anti-DNP antibodies, which are heat-labile, hapten-specific and have rat mast cell sensitizing capacity.

Two alloantisera against hybridoma-derived IgE detected allotypic determinants expressed on the murine epsilon chain. An antiserum raised in BALB/c mice against monoclonal IgE of C57BL/6 origin reacted exclusively with IgE of strains having Igh-1b (IgG2 a) allotype. The second antiserum, C57BL/6 anti-BALB/c monoclonal IgE, reacted with IgE of strains having Igh-1a, Igh-1d, Igh-1e and Igh-1j allotypes. The genetic studies of (BALB/c x C57BL/6)F1 and backcross F2 animals indicated that the locus controlling the IgE allotype is linked to the Igh-1 locus. This was further confirmed by the possession of respective IgE allotypes by Igh-C congenic mice, BALB/c and BAB-14, C3H.SW/Hz and CWB/Hz. Thus, the allotype detected on the epsilon chain is controlled by the seventh murine immunoglobulin allotype locus, and should be designated as the Igh-7 allotype.

The mechanism of selective anti-hapten IgE antibody production was studied in SJL mice. Using an adoptive transfer method of spleen cells into syngeneic recipients irradiated with a sublethal dose of 600 rads, it was demonstrated that for the suppression of anti-dinitrophenyl (DNP) IgE antibody production the interaction of two subsets of T cells is necessary. DNP-primed B cells and carrier-primed T helper cells are taken from donors primed with small amounts of DNP-carrier conjugates. Without injection of other cells, high titer and persistent anti-DNP antibodies are produced in the recipients. The two subsets of T cells that are active in suppression of IgE are taken from two types of donors: one donor is immunized (hyperprimed) with larger amounts of carrier protein twice, the other is an unprimed donor. The carrier for hyperpriming the first type of donor may be unrelated to the carrier used for priming the helper T cells. To bring about anti-DNP IgE suppression it is necessary that the animals should be challenged with the same DNP-carrier conjugate used for priming the B and T helper cells. If the hyperprimed donors were immunized with a heterologous, unrelated carrier, then this heterologous unconjugated carrier must also be injected together with the homologous DNP-carrier conjugate. In these conditions, anti-DNP IgE antibody production is suppressed, but the production of anti-DNP IgG1 antibody is not diminished.

Since it has been proposed that T cells with receptors for the Fc portion of IgE (T epsilon) in rats, mice and humans are IgE-specific regulatory cells, it was investigated whether mice with an IgE-secreting hybridoma might be a source of large numbers of T epsilon cells. BALB/c mice with ascitic tumors of the IgE hybridoma B53 (epsilon, kappa, anti-DNP) developed high serum concns of monoclonal IgE which was followed by the appearance of large numbers of Lyt1-2+ L3T4- T epsilon cells. In contrast, mice bearing a non-IgE producing variant of B53 failed to develop T epsilon cells. Mice infused with cell-free ascites fluid (containing high levels of monoclonal IgE) from the IgE-secreting B53 hybridoma developed high serum IgE levels and also developed high numbers of T epsilon cells. Mice infused with cell-free ascites obtained from the non-IgE-secreting variant ENP-1 (containing very low amounts of IgE) did not develop either high serum IgE levels or T epsilon cells. These findings suggest that high serum IgE concns induce large numbers of T cells that express phenotypic markers of suppressor cells and have surface IgE-Fc receptors. These studies extend to IgE the principle that hybridomas and plasmacytomas induce large numbers of immunoregulatory T cells that express Fc receptors specific for the heavy chain class of the secreted monoclonal immunoglobulin. Since T epsilon cells are normally present only in small numbers, their marked increase in mice with IgE-secreting hybridomas identifies a ready source of large numbers of T epsilon cells that can be used to investigate their regulatory properties.

An intrastrain cross-reactive idiotype, CRIA, is associated with a large proportion of the anti-p-azophenylarsonate (anti-Ar) antibodies of A/J mice. The present experiments, in which the methods of direct and reverse passive cutaneous anaphylaxis (RPCA) were used, indicate that IgE anti-Ar antibodies are produced in A/J mice upon stimulation with a protein-Ar conjugate and that a large proportion of these antibodies express CRIA. The use of monoclonal anti-CRIA for RPCA eliminated the strong nonspecific reactions previously observed. The results provide a basis for studying factors that regulate the switch to IgE biosynthesis during an immune response.

Serum IgE levels were determined in different strains of mice with enzyme-linked immunosorbent assay by using rat monoclonal anti-murine IgE antibodies in normal and in Nippostrongylus brasiliensis-infected mice. After infection, serum IgE levels were high in BALB/c and CB-20, low in SJL/J and SJA/20 mice, and not detected at all in SJA/9 and nude mice. Surface IgE-positive cells were greatly increased in BALB/c and SJL/J mice after infection, but not in SJA/9 and nude mice. Most surface IgE-positive spleen cells were also surface IgM- and surface IgD-positive. When spleen cells from SJA/9 or nude mice were stimulated in vitro with lipopolysaccharide and recombinant interleukin 4 (formerly B cell-stimulating factor 1), IgE was produced and detected in the supernatants of these cultures. In addition, surface IgE-positive cells could be detected in these cultures. Most of the surface IgE-positive cells were surface IgM- and surface IgD-negative, unlike those seen in the spleens of Nippostrongylus-infected BALB/c and SJL/J mice. These observations show that SJA/9 and nude mice have IgE-producing precursor B cells, and after appropriate stimulation interleukin 4 can induce them to secrete IgE.

SJL mice were immunized with 1 microng dinitrophenylated keyhole limpet hemocyanin in 1 mg Al(OH)3. The mice were infected 21 days later with 750 third stage larvae of Nippostrongylus brasiliensis. On day 35, 14 days after infection, they were injected with 1 microng DNP-N, brasiliensis extract (Nb) in 1 mg Al(OH)3. In order to obtain high titer and persistent anti-DNP IgE antibody the mice were irradiated (540 R) 1 day after injection of DNP-Nb. Suppression of anti-DNP IgE antibody production was induced by spleen cells from normal SJL mice. Suppression of IgE antibody response is also obtained by an extract from normal SJL spleen cells. The suppressor substance from normal SJL spleen cell extract is a heat-labile protein, and is not absorbed by anti-mouse immunoglobulin. The mol wt of this substance is larger than 300,000 daltons as determined by gel filtration on Sephadex G-200, but after ultracentifugation, the supernate still has suppressive activity on IgE antibody production.

Mouse IgE was titrated in rats. The sensitization period was 2 h. The results were consistent and corresponded to the titers obtained in young adults SJL mice using a sensitization period of 48 h. With longer sensitization periods in rats, higher antibody titers were obtained. The optimum sensitization period in rats was found to be 48 h. Old CFW mice are inadequate for mouse IgE titration. IgG1 will not give a PCA reaction in rats. IgG1 titers are higher in SJL or A/J mice than in young CFW mice, and markedly higher than in old CFW mice.

Under appropriate conditions of immunization combined with irradiation, SJL/J mice show a high and persistent anti-DNP IgE antibody response. Spleen cells transferred from normal untreated SJL mice suppress this response. Elimination of Ly-1+ cells, but not of Ly-2+ cells, abolished the capacity of spleen cells to suppress the IgE response. Thus of the three T cell Ly subclasses presently identified, Ly-1, Ly-2,3, and Ly-1,2,3, the normal SJL spleen cell which suppresses the IgE response of irradiated-immunized SJL mice belongs to the Ly-1 set. It is not known whether this Ly-1 cell suppresses the IgE response directly or by helping another cell in the recipient. The carrier-specific helper cell activity for IgE and probably IgG1 antibody response belongs to Ly-1 subclass in the SJL strain also.

The suppression of the production of IgE antibody was investigated with the Biozzi 'High' and 'Low' lines of mice. Using the adoptive transfer method formerly experimented with SJL mice (donor I immunized with 0.2 microgram dinitrophenylated ovalbumin, donor II hyperimmunized with 10 micrograms keyhole limpet hemocyanin and donor III being unimmunized mice) IgE suppression was observed in recipients irradiated with 600 R only with the 'Low' line.

IgE and IgG2a antibody-forming cells in the lymphoid organs of rats were detected by passive cutaneous anaphylaxis (PCA) reaction using the extract from the tissue. The amount of antigen-specific IgE and IgG2a antibodies in the tissue extract could be expressed as PCA titers in which extract from 10(8) cells/ml was used as starting dilution. The total amount of antigen-specific IgE antibody produced in mouse IgE-producing hybridoma cells was calculated by maximum dilution of the extract to the minimal amount of IgE antibody inducing PCA reaction, i.e., 4 ng/ml. In the case of the extract from mouse IgE antibody-forming hybridomas, the minimal number of hybridoma cells to be detected by this method proved to be 2-3 X 10(4) cells/ml.