Secondary IgE Responses Research Articles

Regulation of murine IgE responses: Induction of long-lived inhibition of allergen-specific responses is genetically restricted

We previously reported that administration of high M r, glutaraldehyde-polymerized ovalbumin (OA-POL) 10 days prior to OA Al(OH) 3 immunization results in 85 to 99% inhibition of primary and secondary anti-OA IgE responses and 10 2- to 10 4-fold increases in OA-specific IgG 2a production in each of 14 inbred and 1 outbred murine strains tested. Administration of unmodified OA under the same conditions fails to inhibit IgE synthesis and yields only minor increases in IgG 2a production. In the present report, the genetic restrictions placed on the capacity of this modified allergen to elicit long-lived reciprocal regulation of specific IgE and IgG 2a responses were examined. Virtually permanent, antigen-specific inhibition of IgE production (>90% for >22 months) was elicited in C57B1/6 mice following administration of a single course of OA-POL. This inhibition was paralleled by substantial (250-fold) increases in specific IgG 2a production and was dependent on the activity of extremely long-lived regulatory CD4 T cells. In contrast, BALB/c mice failed to maintain an IgE unresponsive state beyond 10–12 weeks and exhibited transient and less intense increases in IgG 2a production. Examination of MHC and Igh congenic strains revealed that the induction of long-term split tolerance by these modified allergens is under multigenic control and is not solely attributable to MHC, Igh, or background genes.

Antigen-specific inhibition of ongoing murine IgE responses. II. Inhibition of IgE responses induced by treatment with glutaraldehyde-modified allergens is paralleled by reciprocal increases in IgG2a synthesis.

Administration of high m.w. glutaraldehyde-polymerized OVA (termed OVA-POL) before OVA-[A1(OH)3] immunization of C57BL/6 mice markedly impairs their capacity to generate OVA-specific IgE responses, while simultaneously resulting in striking enhancement of Ag-specific IgG2a responses. We demonstrate here that treatment with this class of chemically modified allergen also results in pronounced inhibition of ongoing IgE responses in vivo. The abrogation of well established murine IgE responses that is elicited after treatment with OVA-POL (i) is potent (97%), (ii) is long lived, and (iii) reflects reciprocal regulation of Ag-specific IgE and IgG2a responses in vivo. Moreover, the capacity of OVA-POL-treated mice to generate secondary IgE responses remains strongly decreased for at least 260 days and six subsequent immunizations with native allergen, despite there being no further treatment with modified allergen. These changes in IgE and IgG2a responsiveness are Ag specific and T cell dependent.

Elicitation of antigen-induced primary and secondary murine IgE antibody responses in vitro

Described herein are methods for eliciting and quantitating primary and secondary murine IgE antibody responses in vitro, and the important role of antigen concentration in determining the level of IgE produced during an immune response. The methods for quantitating IgE antibody levels in culture supernatant fluids and in serum by ELISA are presented in detail. The specificity of such methods was confirmed in that (1) no other isotype of antibody registered in the IgE-ELISA, and (2) parallel determinations of IgE antibody concentrations could be obtained by independent analysis using FcϵRI-dependent basophil degranulation. We examined various parameters of cell donor immunization and lymphoid cell culture which allow for optimal in vitro primary and secondary IgE responses. High relative antigen doses result in diminished IgE antibody responses in experimental animals, a finding confirmed herein. High antigen concentrations in vitro also result in relative suppression of IgE antibody synthesis. This was also true for in vitro production of IgG1 and IgA antibodies. Conversely, IgM and IgG2a responses were elicited at both low and high antigen concentrations; IgG2b and IgG3 were not produced under the conditions of priming and culture used herein. Finally, production of IgE in vitro depended on the presence of carrier-primed CD4 + T cells and hapten-specific B cells. Generation of maximal IgE antibody secretion, and hence elicitation of an allergic reaction, is thus dependent on the amount of antigen acting as stimulant for the immune response.

IL-4 requirements for the generation of secondary in vivo IgE responses.

IL-4 has been shown to induce B lymphocytes to switch from the expression of membrane IgM to the expression of membrane IgE and to be required for the generation of primary polyclonal and secondary Ag-specific IgE responses in mice. To further define the role of IL-4 in the generation of memory IgE responses, we investigated the ability of a combination of anti-IL-4 and anti-IL-4R mAb to block the generation of secondary IgE responses induced by: 1) a second infection with the nematode parasites Nippostrongylus brasiliensis or Heligmosomoides polygyrus; or 2) injection of anti-IgD antibody-primed mice with anti-IgE antibody. The latter stimulus was designed to induce intrinsic membrane IgE-expressing B cells to differentiate into IgE-secreting cells. Although the IgE responses induced by a second nematode infection were completely inhibited by the combination of anti-IL-4 and anti-IL-4R mAb, anti-IgE antibody-induced IgE responses in anti-IgD primed mice were not inhibited by these antibodies to a large degree. Additional experiments demonstrated that the anti-IgE antibody-induced memory IgE response was dependent on CD4+ T cells but did not involve the low affinity B cell Fc epsilon RII. Taken together, these observations provide evidence that IL-4 is required for virgin B lymphocytes to develop into IgE-expressing cells, but is not required for B cells that express intrinsic membrane IgE to differentiate into IgE-secreting cells in a T-dependent response. Furthermore, these data suggest that secondary IgE responses in the parasite models that we have studied develop from B cells that had not previously switched to the expression of IgE.

Antigen Dose-Dependent Regulation of Bε-Memory Cell Expression

The data presented in this study document that the phospholipase A2 (PLA2)-specific IgE antibody response in high responder CBA/J mice is solely dependent on the antigen dose used for immunization. Repeated injections of minute doses (MD) of antigen (0.1 microgram/mouse) induce a persisting high level of PLA2-specific IgE antibody titer, whereas large doses (LD) (10 micrograms/mouse) induce a persisting low level of IgE. The IgG antibody titers are the same under both conditions. The low level IgE immune status induced by repeated LD is irreversible and cannot be boosted by MD. In contrast a single LD primes for a secondary IgE response which can be recalled by MD. A high level of PLA2-specific IgE antibodies induced by MD can be downregulated by a single intervening LD of antigen. A low level IgE immune status can be transferred with spleen cells of mice immunized with LD into naive syngeneic recipients, which then fail to mount a high level IgE response upon injection of MD of antigen. The experiments reveal two countercurrent processes, induction of B epsilon-memory cells after a single LD and additional activation of a persisting IgE-specific cellular suppression mechanism after repeated LD of antigen. These properties make the system suitable for the analysis of cellular interactions and of potential desensitization protocols.

New Concepts of IgE Regulation

B cell switch to IgE expression is mediated by IL-4 and is regarded as a T helper cell-related phenomenon. In this overview we describe that IgE switch can also be induced by mast cell/basophil like cells (from splenic non-B, non-T cells), activated by IgE receptor cross-linking and/or IL-3 which results in IL-4 production by these cells. Furthermore, activated mast cells produce their own growth factors, IL-3 and GM-CSF. Thus, activation of mast cells can provoke an ongoing local allergic reaction as long as antigen confrontation is maintained, a process which is sustained by further IgE production as well as renewal of mast cells. It is furthermore demonstrated that in certain established immune situations the IgE response may become independent of IL-4, namely in the spontaneous in vitro IgE expression of cells from atopic individuals as well as in an in vitro antigen-induced secondary IgE response of spleen cells derived from previously immunized mice. Thus, IgE-switched B cells may persist in vivo and may represent a pool of potentially IgE-producing cells. Finally, a selective inhibition of the IgE response is described in vitro and in vivo by the use of so-called non-anaphylactic monoclonal anti-IgE antibodies. Such antibodies bind to surface IgE+ B cells, but not to IgE-sensitized mast cells, and thereby inhibit IgE responses. Non-anaphylactic antibodies blocked the binding of allergen-specific IgE to mast cells by competing with the Fc epsilon on these cells. As a consequence they do not induce but rather prevent allergen-induced mediator release by mast cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Lymphokine Control of In Vivo Immunoglobulin Isotype Selection

Several specific conclusions can be drawn from these studies: 1. IL-4 is required for the generation of both primary polyclonal and secondary antigen-specific IgE responses in vivo. 2. IL-4 is required to maintain established, ongoing, antigen-specific and polyclonal IgE responses. 3. Most, but not all, polyclonal IgE production during a secondary immune response is IL-4-dependent. Memory B cells that have already switched to IgE at the DNA level may no longer require stimulation with IL-4 to be induced to secrete IgE. 4. The generation of a secondary IgE response is not dependent upon the presence of IL-4 during primary immunization. However, if IL-4 is not present during primary immunization, it is required during secondary immunization for the generation of an IgE response. 5. IL-4 does not appear to be required for the generation of in vivo IgG1 responses, and in at least some instances, does not contribute significantly to the generation of IgG1 responses in vivo. 6. A late-acting form of T-cell help other than IL-4 appears to be required for the generation of an IgE, but not an IgG1 response. 7. An antibody that inhibits IL-4 binding to IL-4 receptors affects Ig isotype selection in the same way as an antibody that neutralizes IL-4. 8. IFN-gamma can act in both spontaneous and induced immune responses to suppress IgE production. 9. IFN-gamma can also suppress IgG1 production and stimulate IgG2a production. However, IFN-gamma appears to suppress polyclonal IgG1 responses more than antigen-specific IgG1 responses, and it enhances, but is not required for, the generation of IgG2a responses. 10. IFN-alpha appears to resemble IFN-gamma in its ability to inhibit IgE and enhance IgG2a responses in GaM delta-injected mice, but it requires the presence of IFN-gamma to suppress IgG1 production in these mice. 11. Both IFN-alpha and IFN-gamma appear to be able to decrease IgE production in some human patients. 12. There is no direct evidence that IL-5 contributes to the generation of in vivo antibody responses. Two general conclusions may also be drawn.(ABSTRACT TRUNCATED AT 400 WORDS)

IL-4 is required to generate and sustain in vivo IgE responses.

Antibodies of the IgE isotype play a predominant role in immediate hypersensitivity reactions. IL-4, a T cell-derived lymphokine that stimulates increased Ia expression by resting B cells and increased IgG1 secretion by LPS-activated B cells in vitro, has also been shown to regulate in vitro and in vivo polyclonal IgE responses. We report that large quantities of a purified anti-IL-4 mAb inhibit primary in vivo polyclonal IgE responses by 99% in mice infected with Nippostrongylus brasiliensis or injected with anti-IgD antibodies, and totally inhibit secondary Ag-specific IgE responses to TNP-keyhole limpet hemocyanin without effect on either IgG1 or IgG2a responses to these stimuli. The lack of effect of anti-IL-4 antibody on IgG1 secretion cannot be explained simply by inadequate neutralization of IL-4, inasmuch as the doses of anti-IL-4 antibody used blocked an N. brasiliensis-induced increase in B cell Ia expression by more than 85%, whereas in vitro studies indicate that enhancement of B cell Ia expression requires less IL-4 than induction of IgG1 secretion. In addition to demonstrating that IL-4 plays a necessary role in the generation of an in vivo IgE response, we show that IL-4 has an important role in sustaining established IgE responses, because anti-IL-4 antibody, when given at the peak of an N. brasiliensis- or TNP-keyhole limpet hemocyanin-induced IgE response, accelerates the declines in total serum IgE and in IgE anti-TNP antibody levels, respectively. These observations suggest that the effects of IL-4 on in vivo immune responses may be more specific than might have been predicted from in vitro observations, and that regulation of IL-4 production or action may be useful for the prevention or therapy of immediate hypersensitivity disorders.

The role of L3T4+ and Lyt-2+ T cells in the IgE response and immunity to Nippostrongylus brasiliensis.

The role of L3T4+ and Lyt-2+ T cells in protective immunity to Nippostrongylus brasiliensis (Nb) was studied in BALB/c mice that were depleted of either the L3T4+ or Lyt-2+ T cell population by injection with rat mAb specific for the appropriate determinant. Host responses to Nb infection including spontaneous elimination of adult worms, development of intestinal mucosal mast cell hyperplasia and the generation of a polyclonal IgE response were all completely blocked by 0.5 mg anti-L3T4 antibody administered simultaneously with Nb inoculation. However, administration of 0.5 mg of anti-Lyt-2 antibody at the same time and 7 days after inoculation with Nb had no effect on any of these responses. Injection of anti-L3T4 antibody as late as 9 days after Nb inoculation interfered with spontaneous cure of Nb infection and anti-L3T4 antibody injection 11 days after Nb inoculation inhibited serum IgE levels measured on day 13 by 50%. In addition, administration of anti-L3T4 antibody at the time of the peak serum IgE response, 13 days after Nb inoculation, accelerated the decline in serum IgE levels. Injection of previously Nb-infected mice with anti-L3T4 antibody at the time of a second Nb inoculation prevented the development of a secondary IgE response but did not affect immunity to Nb infection based on finding no adult worms in the intestines of these mice. These data indicate that 1) L3T4+ T cells are required for spontaneous cure of Nb infection, development of intestinal mucosal mast cell hyperplasia, and the generation and persistence of an IgE response during primary infection with Nb and 2) L3T4+ T cells are required for a considerable time after inoculation for optimal development of these responses. However, L3T4+ T cells are not required for all protective responses in immune mice. In contrast, our data indicate that considerable depletion of the Lyt-2+ T cell population has no significant effect on either worm expulsion or the generation of serum IgE responses.

Induction of secondary IgE antibody response in rats immunized with X-irradiated metacercariae of Paragonimus ohirai.

Rats reinfected with Paragonimus ohirai (P.o.) elicited little secondary IgE response to adult P.o. antigen. In contrast, rats immunized with X-irradiated (2-10 krad) metacercariae elicited not only a marked primary IgE response comparable to that in normally infected rats, but also a marked secondary IgE response after challenge infection. Rats immunized with 2 krad X-irradiated metacercariae yielded a higher secondary IgE response than with 5 or 10 krad irradiated ones, and elicited a secondary response lasting at least 1 year. An IgE response to antigen of newly excysted juvenile parasites (NEJ) was clearly different from the IgE response to adult P.o. antigen. The former IgE response was weak after infection or immunization, but after challenge infection a marked secondary response occurred at a level similar to the IgE response to adult P.o. antigen. The secondary IgE responses to adult P.o. and NEJ antigens were also elicited by reinoculation with X-irradiated metacercariae. Cross-absorption experiments confirmed that there were adult type and NEJ type allergens.

Differential effect of cyclosporin A (CyA) on IgE response: role of CyA-induced suppressor cells

Administration of cyclosporin A (CyA, 30 mg/kg) for the five days following immunization of Brown Norway rats with DNP 14-OVA in alum abolished the primary total and specific IgE response, whereas this treatment had no significant effect on the secondary IgE response. On the contrary, with a single injection of CyA on the day of priming, the secondary IgE response only is abolished. The inhibition of the secondary IgE response could be attributed to the induction by a single dose of CyA of nylon wool-adherent spleen cells, as shown by passive transfer experiments. CyA might thus affect the IgE response variously depending on single or repeated administrations.

The role of macrophages in anti-idiotypic antibody and T suppressor factor induction of timothy grass pollen antigen B-specific T suppressor cells.

Cultures of normal spleen cells with anti-idiotypic antibody (anti-Id) or antigen B (AgB)-specific T suppressor factor (Tsf1) in mini-Marbrook chambers for 4 days at 37 degrees C lead to the in vitro induction of AgB-specific T suppressor (TS) cells. These TS cells significantly suppress a secondary AgB-specific IgE response, but they do not affect a secondary AgB-specific IgG response. Depletion of both B cells and macrophages from normal spleen cells by panning on anti-Ig-coated petri dishes provides an enriched T cell population. These enriched T cells when cultured with anti-Id or Tsf1 in mini-Marbrook chambers do not produce AgB-specific TS cells, and mice treated with cells harvested from the mini-Marbrook chambers have normal secondary AgB-specific IgG and IgE responses. The addition of as few as 1000 bone marrow-derived macrophages (BMDM) to cultures of the enriched T cells with anti-Id, or Tsf1 restores the ability of these cultures to produce significant levels of AgB-specific TS cells. Further studies reveal that the macrophage population must be histocompatible and express a cell surface I-J antigen. Attempts to pulse BMDM with anti-Id or Tsf1 at 4 degrees C and to culture in mini-Marbrook chambers 10(3) pulsed BMDM with enriched T cells were unsuccessful in producing AgB-specific TS cells. However, pulsing BMDM with anti-Id or Tsf1 at 37 degrees C, and adding 10(3) of these pulsed BMDM to enriched T cells in culture led to the formation of significant levels of AgB-specific TS cells.

Suppression of IgE antibody response against ovalbumin by the chemical conjugate of ovalbumin with a polyaspartic acid derivative.

Ovalbumin was modified with alpha,beta-poly[(2-hydroxyethyl)-DL-aspartamide] which had been developed as a nontoxic plasma expander. Preadministration of the modified ovalbumin suppressed both primary and secondary anti-ovalbumin IgE responses.

Inhibition of primary and secondary IgE-response by a schistosome-derived inhibitory factor.

Schistosome-derived inhibitory factor (SDIF) previously shown to inhibit lymphocyte proliferation, markedly decreased the primary IgE response of rats immunized with dinitrophenylated ovalbumin (DNP-OVA) when injected either simultaneously or shortly after antigen administration. No effect however was observed when SDIF was injected before the immunization. An inhibition of non-IgE anti-DNP antibodies was also found in SDIF-treated rats although the decrease was lower than with IgE antibody. IgE responses of both low and high IgE responder rats were reduced but a lower dose of SDIF was required in the case of high IgE responder Brown Norway rats. When SDIF was only given at the time of priming, the secondary IgE response was no longer modified. However, the administration of SDIF together with the second injection of the antigen induced marked decrease in the secondary IgE response. The effects of SDIF on primary and secondary IgE responses could be attributed to the inhibitory activity of the parasite-derived factor on lymphocyte proliferation. The observed inhibition of secondary IgE antibody responses confers to SDIF a pharmacological interest in allergic diseases.

Antigen- and IgE class-specific suppression mediated by T suppressor cells of mice treated with glutaraldehyde-polymerized ovalbumin.

Various size polymers are obtained following glutaraldehyde treatment of native ovalbumin (OA). OA-POL, approximately 35 X 10(6) daltons, was prepared at the isoelectric point of OA. Treatment of CBA mice with microgram amounts of OA-POL led to efficient antigen-specific suppression of IgE responses. IgG anti-OA antibodies were not suppressed. Transfer of cells from OA-POL-treated donors into normal, unprimed recipients interfered with the ability of these animals to mount a primary or secondary IgE response. In addition, cotransfer of spleen cells from OA-POL-treated mice along with OA (in alum)-primed cells, into irradiated syngeneic recipients resulted in IgE class-specific suppression that was abrogated by treatment of OA-POL donor cells with monoclonal anti-Thy 1.2 + complement. The presence or absence of T cells in the OA-POL population had no effect on IgG levels in the recipients. Analysis of the antigenic properties of OA-POL revealed 5-15% cross-reactivity with native OA as perceived by IgG or IgE antibodies. In contrast, OA-POL was highly cross-reactive at the T cell level as shown functionally by its potent induction of OA-specific, IgE-selective suppressor T cells. The results suggest that the beneficial effects of glutaraldehyde-modified allergens, recently introduced in the immunotherapy of atopic individuals may be due to the preferential exposure on the polymerized protein, of antigenic determinants generating T suppressor cells and to the selective loss of B cell-reactive determinants.

Immunomodulating effects of 13-cis-Retinoic acid on the IgE response of BALB/c mice.

The results reported here are an extension of our previously reported studies on the adjuvant effects of 13-cis-retinoic acid (13-cRA). Previous results, as well as those reported herein, show the lack of a primary IgE response with elevated and sustained secondary IgE responses in animals treated with 13-cRA. It was determined that this lack of a primary IgE response was not due to overt immunotoxicity by 13-cRA, suggesting that 13-cRA is priming a population of memory cells; however, either the formation of suppressor cells or the lack of an appropriate stimulus at the time of primary immunization also prevented a demonstrable primary IgE antibody response. It was also demonstrated that the absence of a primary response was not due to a sensitivity of the immune system to the timing of the administration of 13-cRA relative to immunization, as all animals given 13-cRA at either day -2, -1, 0, 1, 2 or 3 relative to primary immunization showed no primary response but high secondary IgE responses. These experiments, which closely mimic the clinical treatment regimen, indicate that patients receiving retinoid therapy should be closely monitored for immunological side effects, such as immediate hypersensitivity to environmental allergens.

The effect of a low protein diet on the immunogenic activity of murine peritoneal macrophages.

The activity of resident and exudate peritoneal macrophages from mice maintained on a low protein diet (4% casein initiated at weaning) was assessed in a variety of assay systems which examined aspects of macrophage 'immunogenic' function in vivo and in vitro. Antigen-pulsed macrophages from malnourished mice triggered secondary IgE and IgG responses following intraperitoneal inoculation into pre-immunized syngeneic recipients. Similarly, intraperitoneal transfer of low numbers of these cells successfully primed immunologically naive syngeneic mice; in both cases, the activity of macrophages from malnourished mice could not be distinguished from these derived from normal animals. Examination of uptake and breakdown of radio-labelled antigen revealed normal rates of phagocytosis, and a brief lag in the early phase of degradation, relative to control macrophages. Antigen-pulsed macrophages from malnourished mice were severely impaired in their capacity to trigger the proliferation of antigen-primed T cells in vitro.

Primary, secondary and late ongoing IgE responses differ in their susceptibility to suppression

Primary, secondary and late ongoing IgE responses differ in their susceptibility to suppression

Induction of adjuvant-independent IgE responses in inbred mice: primary, secondary, and persistent IgE responses to ovalbumin and ovomucoid.

Immunization of a number of inbred mouse strains intraperitoneally with low levels (1-100 micrograms) of ovalbumin (OA) or ovomucoid (OM) in the absence of adjuvant, revealed marked strain differences in IgE responsiveness. High-OA-responder strains such as Balb/c exhibited PCA titre up to 256 in the primary response, and up to 1,280 in the secondary, while non-responders such as SJL and NZB remained PCA negative; similar variations were found in the response of the strains to OM. Repeated immunization of high-OA-responder mice with low doses of antigen without adjuvant elicited anamnestic second and tertiary HA antibody responses. In contrast, IgE responses exhibited anamnestic secondary responses, but were not further boosted by tertiary stimulation. Instead, a 'persistent' high IgE response of the type previously associated with the use of specialized adjuvants, developed in these mice. Fractionation of the OA antigen by gel filtration yielded molecular species (dimers pentamers) of considerably greater IgE-immunogenicity than either monomeric OA or very highly aggregated forms. Immunization of high-OA-responder mice with the former permitted lowering of the threshold for induction of adjuvant-independent primary OA-IgE responses to a single 1.0 microgram dose, and the threshold for priming to 0.1 microgram.

Induction of immunological tolerance to the penicilloyl antigenic determinant. III. Suppression of benzylpenicilloyl-specific IgE antibody formation by cleavable penicilloylated dextran.

The suppressive effect of cleavable penicilloylated dextran (BPO-DEX), whose directly bound penicilloyl groups undergo hydrolytic cleavage within 3 days under physiological conditions, on murine IgE antibody formation against the benzylpenicilloyl (BPO) determinant was investigated in BALB/c and C3H mice. Intraperitoneal administration of BPO-DEX during either primary or secondary IgE responses to BPO ascaris produced a reversible suppression of BPO-specific IgE, while not affecting carrier-specific IgE antibody formation. Suppression of longer duration, at least 10 weeks, was achieved, however, by repeated administrations of BPO-DEX. BPO-DEX itself did not generate detectable BPO-specific IgE antibodies. These results suggest that BPO-DEX might be one of the promising tolerogens in the prevention of penicillin allergy.