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In humans, respiratory viral infections lead to increased airway responsiveness and exacerbations of asthma. In the present study, the role of interleukin-5 (IL-5) in virus-induced airway hyperresponsiveness and inflammation was examined in guinea pigs. In animals treated with control antibody, parainfluenza-3 virus significantly potentiated (219%) the histamine-induced increase in lung resistance compared with vehicle treatment. In addition, viral infection significantly increased (130 to 450%) the responsiveness of isolated tracheal segments to histamine in animals treated with control antibody. In guinea pigs treated with control antibody, the numbers of eosinophils (226%), neutrophils (1,380%), and monocytes (626%) in bronchoalveolar lavage fluid were significantly increased after viral infection. The level of major basic protein in bronchoalveolar lavage fluid was not altered after viral infection. In addition, electron microscopic examination of eosinophils in airway tissue and alveolar lumen did not point to increased degranulation after viral infection. In guinea pigs treated with antibody to IL-5 the virus-induced airway hyperresponsiveness to histamine both in vivo and in vitro was almost completely inhibited. In guinea pigs treated with anti-IL-5, viral infection significantly increased the numbers of eosinophils (234%), neutrophils (1,255%), and monocytes (617%) in bronchoalveolar lavage fluid. These data suggest that IL-5 plays an important role in airway hyperresponsiveness to histamine but not in the infiltration of eosinophils after respiratory viral infection.

We have recently developed a model in which mice were treated with IL-4 after primary immunization, resulting in elevated total serum IgG1 and IgE levels, but decreased antigen-specific levels and memory formation for these isotypes. In this report, we describe that these effects of IL-4 are mediated at the B cell and not the T-cell level. Major changes occurred in the gamma 1 epsilon-double positive B-cell population which is increased as a result of IL-4 treatment. Moreover, it is shown that gamma 1 epsilon-double positive B cells can develop in vitro out of gamma 1-positive primed B cells and that these double positive cells can differentiate into IgG1- and IgE-secreting cells. The existence of gamma 1 epsilon-double positive memory B cells can explain the differences in cytokine dependence of TNP-specific memory IgG1 and IgE responses found after adoptively transferring primed spleen cells into irradiated naive recipients. Whereas the IL-4 independent TNP-specific memory IgG1 responses could be blocked efficiently by neutralizing IL-5 and IL-6, TNP-specific memory IgE responses were virtually not susceptible to such treatment. These IgE responses were also not susceptible to IFN-gamma, used in doses that could inhibit the primary IgE response. Inhibition of the TNP-specific memory IgG1 response by neutralizing IL-5 and IL-6 is accompanied by a 10-fold increase of the IL-4 independent TNP-specific IgE memory response. These data indicate that secondary IgE responses primarily result from B cells that are either switched to IgG1, or are double positive for IgG1 and IgE, thereby suggesting a minor role for epsilon-single positive B cells in secondary IgE responses.

The effects of rat anti-CD3 and anti-CD4 moAb, of the IgG2a as well as of the IgG2b subclass, on the development of lethal graft-versus-host disease (GVHD) in a fully allogeneic mouse strain combination were compared in vivo. After treatment with these moAb, mice recovered from an initial loss of body weight. Moreover, their survival significantly improved. A single dose of 200 micrograms moAb resulted in a complete and long-term survival, which was not the case after treatment with anti-CD4 IgG2a moAb. A dose of at least 1 mg anti-CD4 IgG2a was necessary to induce a tolerant state. Mice effectively treated were fully repopulated with donor-type cells. Flow cytometric analysis of the recipient spleen cells demonstrated that the moAb caused depletion, modulation or coating of T cells or a combination of these. The moAb with the highest depleting capacity appeared to be anti-CD4 IgG2b moAb. Anti-CD3 IgG2a as well as IgG2b treatment resulted in a strong modulation of CD3 surface proteins, which was found on all days examined. Modulation of CD4 surface antigens did not occur in the case of anti-CD4 IgG2a moAb treatment. Anti-CD4 IgG2b moAb treatment, on the other hand, not only caused some CD4 modulation, but also, quite unexpectedly, a significant modulation of the CD3 molecule. Coating was only observed after treatment with anti-CD4 IgG2a moAb and lasted at least 1 week.(ABSTRACT TRUNCATED AT 250 WORDS)

IL-4 is obligatory for primary IgE responses, whereas primary IgG1 and secondary IgE responses are partially IL-4 independent. To investigate the effect of IL-4 on the antigen-specific memory formation for these isotypes, BALB/c mice were treated after primary TNP-KLH immunization with recombinant IL-4 for a period fo 4 months. This prolonged presence of a high IL-4 level resulted in increased serum levels of total IgG1 and IgE, whereas total IgG2a did not change. The expression of CD23, but not I-Ad, increased on the splenic B cells. IL-4 treatment did not affect the IL-4 production by Con A stimulated spleen cells, whereas it did decrease the IFN-gamma production. In the same mice the TNP-specific IgG1 and IgE serum levels, however, were decreased. Similar results were found when the antigen was continuously present during the IL-4 treatment. Furthermore, it was shown that IL-4 decreased the formation of IgG1 and IgE memory cells. These results point to different effects of IL-4 in regulating antigen-specific and bystander responses.

A number of rat hybridomas were adapted to grow in RPMI containing either 5% IgG-depleted FCS or 1% serum-free Nutridoma. Alternatively, protein-free Ultradoma PF was used. Growth in these media allowed purification procedures to be used that are based on tangential ultrafiltration in combination with affinity chromatography on gels linked to protein G or anti-rat L chain coupled antibodies. The isolated antibody preparations were found to be pure and to consist of monomeric intact IgG. The yield and recovery of mAb using this procedure were found to be consistently high. These antibody preparations were analyzed for endotoxin contamination. Whereas during isolation endotoxin contamination increased, the endotoxin content per mg purified protein did not. Affinity chromatography on Detoxi-gel resulted in the efficient removal of this contamination and using this protocol the antibody preparations obtained were found to be of sufficient purity, activity and low endotoxin content to permit their in vivo use in animal models of immunotherapy.

The crucial role of interleukin (IL)-4 in the induction of murine IgG1 and IgE responses, which are coupled through the process of sequential isotype switching, has been well documented. Whereas IL-4 is obligatory for the induction of IgE responses, it enhances IgG1 responses. In this study, using neutralizing antibodies, we provide evidence that, besides IL-4, also IL-6 is required for obtaining peak IgG1 responses. The mRNA levels of these two cytokines are coordinately expressed in the spleen of mice immunized with trinitrophenol-keyhole limpet hemocyanin (TNP-KLH). No IL-6 requirement was observed for peak IgE responses. The IL-6 dependence of IgG1 responses was found for both antigen-specific and polyclonal responses. Moreover, it was noted using TNP-KLH and goat anti-mouse (GAM) IgD as antigen that polyclonal IgG1 responses are more dependent on IL-6 than antigen-specific responses. In vitro experiments revealed that exogenous IL-6 neither enhanced nor inhibited the IgG1 and IgE production by naive B cells, suggesting that IL-6 did not interfere with the IL-4-induced isotype switch potential. Primary and memory IgG1 responses were both similarly dependent on IL-6. These observations point to a role of IL-6 in the terminal differentiation of B cells switched to IgG1. Neutralization of IL-6 did not inhibit either antigen-specific or polyclonal IgE responses. Therefore, it was concluded that IL-6 is not involved in the terminal differentiation of B cells switched to IgE. These findings thus provide a distinct role for IL-6, besides IL-4, in regulating murine IgG1 responses. The formation of IgE, however, is completely dependent on IL-4 alone.

The availability of cell lines that are transfected with IL-4, IL-5 and IFN-gamma cytokine genes permits the prolonged in vivo delivery of functional cytokines in relatively large doses for the modulation of specific immune responses. Often the transfected cells are xenogeneic or allogeneic to the experimental animal and have to be encapsulated in such a way that no cellular response by the host will be induced. Alginate has proven to be a simple matrix for encapsulating cells under mild conditions suitable for in vivo implantation. Encapsulated cells express the transfected IL-4 gene for at least 14 days after in vivo implantation and were shown to be functional during that period by modulating ongoing IgE responses. The application of adherent growing transfected cells permits dose-response titrations and provides an easy method for local and systemic cytokine delivery. Alternatively, hybridoma cells can be encapsulated and the secreted antibody monitored in the serum. It was found that no host immune response was triggered by alginate encapsulated cells. The efficiency of treatment by encapsulated hybridoma cells was shown to be equivalent to that of injecting purified antibodies.

Lethal GVHD in the fully allogeneic BALB/c (donor)-(C57BL x CBA)F1 (recipient) mouse strain combination could be prevented by a single dose of IgG2b monoclonal antibodies (moAb) directed to T cells. The influence of the time of administration of this moAb after GVHD induction and the effect of anti-T cell subset moAb on the development of GVHD was investigated in this study. Moreover, the state of tolerance in the mice that had become long-term chimeras was examined. Anti-Thy-1 treatment of the recipients 1 day before, 2 h before or 1 day after reconstitution almost completely prevented lethal GVHD. A single dose of 100 micrograms of anti-Thy-1 was as effective as four daily doses of 25 micrograms each. Treatment with a single dose of 25 micrograms or with intervals of 4 days between doses of 25 micrograms was statistically significantly less effective. We injected the recipients with moAb directed to the CD4+ or CD8+ T cells subsets. Using a dose of 100 micrograms moAb, anti-CD4 treatment appeared to be less effective than anti-Thy-1 treatment whereas anti-CD8 treatment was not effective at all. A double dose of anti-CD4 was equally effective as anti-Thy-1 treatment. All mice that became long term survivors remained free of signs of GVHD and were > 99% repopulated with donor type cells. Injection of spleen cells from these BALB/c into (C57BL x CBA)F1 chimeric mice was used to reconstitute lethally irradiated BALB/c, BALB.K and (C57BL x CBA)F1 recipients. Lethal GVHD developed in the BALB.K and (C57BL x CBA)F1 recipients but not in the BALB/c recipients.(ABSTRACT TRUNCATED AT 250 WORDS)

Adjuvants are considered to play an important role in directing the isotype and amount of antibodies produced upon immunization by conducting the development of either Th-1 or Th-2 cells upon T-cell stimulation. This is based on the different cytokine production patterns that were observed after in vitro resttmulation of T cells isolated from mice immunized with antigen either adsorbed on alum or emulsified in complete Freund adjuvant (CFA). However, other studies suggest that primarily the type of antigen determines which isotypes are produced and to what extent. In these studies, however, IgE was not determined. Therefore, this study examined whether alum and CFA influenced the amount and/or ratio of IgG(1), IgE and IgG(2a) produced after TNP-KLH immunization. Similar levels of IgG(1), IgE and IgG(2a) antibodies were found upon immunization with TNP-KLH either adsorbed on alum or emulsified in CFA. Moreover, administration of IFN-gamma in combination with TNP-KLH adsorbed on alum did not increase the amount of IgG(2a) produced. IFN-gamma treatment resulted in an increased IL-6 and decreased IFN-gamma production by spleen cells upon Con A stimulation, whereas it did not change the IL-4 production in similar conditions. The presented results suggest that upon immunization with TNP-KLH high IL-4 levels are produced, resulting in an antibody response that is dominated by IgG(1), independent of the adjuvant employed. The IL-4 inducing property of TNP-KLH is substantiated by the finding that repeated immunization of mice with TNP-KI, without adjuvant, increases the serum total IgE level. The presented data suggest that the carrier part of TNP-KLH preferentially results in Th-2 cell activity after which the adjuvant merely enhances the antibody responses generated.

Allograft rejection is a complex process, which requires interactions between different cell types and a variety of soluble factors, such as cytokines. In this review we discuss the role of cytokines in the induction and effector phases of the rejection process and in the induction and maintenance of allospecific graft tolerance. Furthermore, we discuss the feasibility of clinical graft function monitoring by measuring cytokines and the possibilities for intervention in the cytokine network in order to inhibit graft rejection and eventually obtain graft acceptance.

A murine model for acute lethal graft vs. host disease (GVHD) was used to study the role that a number of cytokines play in the development of lethal GVHD. In this study we focused on the role of IL-1, IL-2, IL-4, IL-6, IFN-gamma and TNF-alpha. Lethally irradiated (C57BL x CBA)F1 mice were reconstituted either with 10(7) allogeneic BALB/c spleen cells or with a similar number of syngeneic cells, as a control. A significant rise in serum levels of IL-6, TNF-alpha and IFN-gamma levels was found in allogeneically reconstituted mice. This is in contrast to the syngeneic control group in which no rise was seen. Serum IL-2 and IL-4 levels were below the detection limit. In the supernatant of Con A stimulated spleen cells from allogeneically reconstituted mice IL-6, IFN-gamma and TNF-alpha concentrations were increased. The expression of mRNA for cytokines as detected by reverse transcription PCR was studied in spleen cells. In the allogeneic reconstituted mice the mRNA expression of IL-1alpha, IL-2, IL-6, IFN-gamma and TNF-alpha displayed faster kinetics compared with that in syngeneic reconstituted mice. The effect of treatment with recombinant cytokines, antibodies to cytokines and to cytokine receptors on the development of GVHD was investigated. Administration of recombinant IL-2 to allogeneically reconstituted mice strongly increased the morbidity and mortality whereas injection of IL-1alpha and TNF-alpha did not influence survival. Administration of antibodies against IL-2 or the IL-2 receptor decreased the morbidity and mortality. Anti-IL-6, anti-IFN-gamma, and anti-TNF-alpha mAB, on the other hand, did not affect the morbidity and mortality of GVHD. The results of this study suggest successive waves of cytokine-secreting cell populations consistent with the induction of an inflammatory response in the development of acute GVH disease.

Interleukin-5-producing CV-1 cells were encapsulated in alginate and injected i.p. in guinea pigs (4 x 10(6)/animal). These cells produced approximately 8 ng interleukin-5 per 4 x 10(6) cells per day. Airway hyperresponsiveness to histamine in vivo was observed 3 and 7 days after administration. The increase in lung resistance after intravenous administration of histamine to guinea pigs was significantly potentiated, by approximately 70 to 90% in interleukin-5-treated animals. In animals treated with antibody to interleukin-5, the administration of interleukin-5-producing CV-1 cells did not induce hyperresponsiveness. The percentage of eosinophils in broncho-alveolar lavage fluid was increased by 100% at 7 days but not at 3 days after administration of interleukin-5-producing CV-1 cells. Antibody to interleukin-5 prevented the broncho-alveolar lavage eosinophilia at 7 days after interleukin-5 administration. It can be concluded that interleukin-5 induces broncho-alveolar lavage eosinophilia and airway hyperresponsiveness and that these phenomena do not occur simultaneously. These data suggest a role for interleukin-5 in the development of airway hyperresponsiveness in bronchial asthma.

Chronic ovalbumin challenge of sensitized guinea pigs induces bronchoalveolar lavage (BAL) eosinophilia, neutrophilia, and tracheal hyperreactivity. In the present study, the influence of monoclonal antibody to murine interleukin-5 (anti-IL-5) on these phenomena is examined. In ovalbumin-sensitized guinea pigs treated with isotype-matched control antibody and challenged daily with ovalbumin for 8 days, the number of BAL eosinophils and neutrophils is increased significantly six- and fivefold, respectively, compared with saline-challenged animals. The maximal contractions of tracheal rings to histamine and arecoline in ovalbumin-challenged animals are enhanced significantly to 155% compared with saline-challenged animals. In sensitized guinea pigs treated with anti-IL-5, the BAL eosinophil number is markedly inhibited compared with control antibody treatment in both saline- and ovalbumin-challenged animals. In contrast, the number of neutrophils is not affected by anti-IL-5 treatment. In guinea pigs treated with anti-IL-5, the development of hyperreactivity to histamine and arecoline after ovalbumin challenge is completely inhibited. The contractions to histamine and arecoline of tracheal rings isolated from guinea pigs treated with recombinant murine IL-5 for 3 or 7 days are enhanced significantly to approximately 140% compared with controls. Treatment with IL-5 for 7 days tends to increase the number of eosinophils in BAL fluid. It can be concluded that IL-5 is involved in airway eosinophilia and in the development of hyperreactivity in this animal model, but other cytokines may contribute. Development of IL-5 synthesis inhibitors and/or receptor antagonists could provide another therapeutic class of anti-asthma drugs.

Graft-versus-host disease (GVHD) is caused by donor T lymphocytes that recognize foreign antigens on host tissues. This leads to T cell activation, which involves a cascade of events including the transcription of genes for cytokines and their receptors and the production of cytokines. One of the first cytokines to appear is interleukin 2 (IL-2). IL-2 production enhances the IL-2 receptor expression and leads to T cell proliferation. As a further step, differentation of T cells occurs, which results in the production of a certain pattern of cytokines. These cytokines influence the expression of cell surface antigens and adhesion molecules, and are able to activate other cell types such as cytotoxic T cells, macrophages and natural killer cells, which might act as effector cells in tissue destruction. Insight into the sequential expression of the various cytokines involved might enable a more effective treatment of GVHD. Therefore, we investigated the occurrence of cytokines in a murine model for acute GVHD. We addressed in particular the period early after allogeneic reconstitution.

Delayed-type hypersensitivity responses against recipient's histocompatibility antigens can occur early in the course of a graft-versus-host reaction in lethally irradiated allogeneically reconstituted mice. This reactivity could be suppressed by a single host-specific blood transfusion to the prospective donors of allogeneic spleen cells. Maximum suppression was found when the blood transfusion was given 4 or 5 days before the mice were used to reconstitute lethally irradiated hosts. Whole blood and purified white blood cells were capable of inducing suppression, whereas purified red blood cells, plasma, and serum were not. Suppression was already detectable after administration of 1 microliters of whole blood and virtually complete at a dose of 1.0 ml. Irradiation of the blood reduced but did not abrogate its capacity to induce suppression. Purified B and purified T lymphocytes appeared equally effective in inducing suppression. Two helper T cells clones, a Th1 and a Th2 clone, were able to induce suppression as well. A high dose of recombinant IL-2, injected daily for 5 days after reconstitution, did not abrogate or reduce the suppression. Suppression could be induced by H-2 as well as non-H-2 alloantigens, separately or together. A pure H-2-incompatible transfusion was more effective in inducing suppression than a pure non-H-2-incompatible one. Suppression appeared to be a dominant phenomenon and was mediated by a Thy-1+, CD4+, CD8- spleen cell population. This T cell population had its origin in the transfused donor, which excludes the possible involvement of blood-derived "veto-cells."

Helper T-lymphocytes tightly regulate the proliferation and antibody production of B-lymphocytes by a variety of molecules called lymphokines. Molecular biology has provided the tools to produce large amounts of these regulatory molecules in highly purified "recombinant" form. Furthermore, monoclonal antibodies have been produced that selectively neutralize each of these lymphokines. This enables thorough investigation of the regulation of human and murine immunoglobulin E (IgE) production. This paper reviews recent data on the regulation of murine IgE production by lymphokines produced by distinct helper T-cell clones. Special attention is paid to the IgE enhancing activity of interleukin-4 (IL-4) and the IgE inhibiting activity of interferon-gamma (IFN-gamma). Evidence is presented that these lymphokines also play a crucial role in genetically determined IgE "high responder" and IgE "low responder" mouse strains.

SJA/9 mice are unable to produce significant serum IgE responses to either primary or secondary challenge with protein Ag or helminth parasites. Previous work has shown that this defect resides the T cell rather than with the B cells. These experiments test several possible explanations for the lack of IgE production in these mice. SJA/9 mice do not appear to overproduce IFN-gamma or any other antagonist of IL-4 function. Helminth parasite infection induces normal levels of IL-5-dependent eosinophilia, suggesting that there is not a complete absence of a Th2 response in these mice. The defect, however can be explained by a substantial reduction in IL-4 production in vivo and can be corrected by infusion with IL-4. The reason for this lack of IL-4 production is not yet clear and, paradoxically, SJA/9 T cells can be stimulated in vitro to produce IL-4 levels comparable to T cells from other strains.

Several cytokines have been shown to regulate the expression of specific isotypes by affecting either the frequency of isotype switching or allowing maturation of precommited precursors. IL-4 enhances the production of IgG1 and is required for IgE production in vitro and in vivo. Evidence has been provided that Il-4 acts by enhancing switching to IgE, and perhaps IgG1. IFN-gamma was shown to stimulate the secretion of IgG2a in concentrations at which it inhibits IL-4 induced production of IgG1 and IgE. IL-5, on the other hand, appears to stimulate maturation. Recent evidence suggests that transforming growth factor-beta induces switching to IgA, despite the inhibitory activity on other isotypes.

Several different grades of T-system impairment were studied for their effects on the total serum IgE concentration in BALB/c mice. Homozygous athymic nu/nu mice and their heterozygous nu/+ littermates were compared for serum IgE levels while kept under either barrier-maintained or conventional conditions. The results show a paradox between the T-cell dependency of the IgE immune response and the increased levels of serum IgE in the absence of T cells. Both barrier-maintained and conventionalized nu/nu mice have at least twofold increased serum IgE levels as compared to nu/+ mice. With age, IgE levels increased faster and reached higher plateau values in nu/nu than nu/+ mice. Moreover, after adult thymectomy of BALB/c mice the serum IgE levels increased up to 15-fold at 4 months of age, while infusion of immunocompetent T cells in nude mice resulted in a 2- to 5-fold decrease of the IgE level.

H-2 heterologous T cell hybridomas were used to study the genetic control of dual, anti-nominal antigen and anti-self H-2 specificity of H-2 restricted T cell factors. Each of four hybridoma clones produced two helper factors. One was restricted for the Ia type of the normal T cell partner (H-2b), whereas the other was restricted for the ia type of the lymphoma partner (H-2k) of the somatic hybrid. This was shown by affinity separation on parental type spleen cells and on monoclonal anti-I-A-Sepharose. Both factors had carrier (chicken gamma globulin; CGG)-specific helper effect, and both bound to anti-VH-315-Sepharose. Because the lymphoma (BW-5147) partner could not contribute a CGG-specific locus, the H-2k-restricted, CGG-specific factor had to be the product of segregating anti-nominal and anti-self loci. This suggests that dual specificity is due to two independent loci and support the validity of dual recognition concepts. Anti-self specificity was associated with homologous Ia alloantigens in the individual factors. Therefore, Ia and anti-self might be linked. Implications of the major histocompatibility complex or VH nature of anti-self receptors and the relationship of T cell factors and receptors was discussed.

A simple Terasaki tray-based ELISA technique with a fluorescent detecting system has been used to determine the affinity of murine IgE antibodies. The system was shown to be sensitive enough to measure affinities in the range of 10(-6)-10(-10) M as well as detect IgE antibodies down to a limit of 0.1 ng/ml. The results, expressed as arbitrary fluorescence units (AFU), were compared with those obtained using equilibrium dialysis for several DNP-specific IgE monoclonal antibodies of known affinities yielding KD values. The relationship between KAFU and KD established a conversion factor which could then be used to compute KD from KAFU, provided the detection system remained identical. Based on the equations proposed, an alternative method for the quantitation of murine IgE is described which is independent of the affinity of IgE for the coated antigen.

A rapid and efficient procedure is described for the coupling of proteins (protein A, ovalbumin, albumin and chicken gamma globulin) to sheep red blood cells (SRBC) to be used in antigen-specific or protein A plaque assays. This modification of the original procedure has three distinct features: prewash of the red cells with a low concentration of essentially freshly prepared CrCl3, use of a relatively high concentration of CrCl3 in the reaction mixture and a coupling time of only 4 min. Protein A plaque assays performed with such target cells have the same sensitivity as those employing red cells coupled with protein A according to the original procedure. Studies with hybridomas secreting antibody specific for a protein antigen showed that antigen-specific plaque assays employing target red cells coupled with the protein antigen according to the modified procedure have the same sensitivity as the protein A plaque assay. The modified procedure greatly facilitates cellular studies on antibody formation after immunization with protein antigens.

IL-4 is able to preferentially enhance murine IgE levels in the supernatant of LPS-stimulated T cell-depleted splenic B cell cultures. Clonal and quantitative analysis of this response revealed that this is due partly to a 14-fold increased IgE precursor frequency and partly to a three-fold increased clone size of IgE-secreting cells. IL-4 increased the precursor frequency and the clone size of IgM-secreting cells not more than twofold. Both the IgM and IgE response in LPS-stimulated B cells were completely inhibited by the addition of anti-IgM mAb (M41) to the cultures, indicating that the IgE-secreting clones developed as subclones from precursors that express IgM. These cells lacked expression of membrane-bound IgE up to day 5 of the culture. Application of feeder cells in these cultures resulted in an increased precursor frequency of IgE-secreting clones among LPS-reactive B cells that is due, partially, to IL-4 produced by the feeder cells.

Nonresponder SJL mice produce low levels of antigen-specific IgE after immunization, compared to responder strains. Young athymic BALB/c nude mice are unable to produce antigen-specific or total IgE in their serum. These mice also have very numbers of background IgE-secreting cells in their lymphoid organs. High-responder BALB/c mice do have substantial numbers of background IgE-secreting cells while low-responder AKR mice show intermediate numbers. Similar differences were found when analyzing lipopolysaccharide (LPS)-reactive B cells in cell suspensions of spleen and bone marrow in limiting dilution cultures. Limiting dilution analysis of T cell-depleted splenic B cell cultures revealed that the defective IgE production in SJL mice is not due to an intrinsic B cell defect. This defect can be substantially overcome by addition of exogenous interleukin 4 (IL4) to these cultures. Furthermore, it was shown in limiting dilution cultures that SJL thymocyte feeder cells were able to suppress IgE production by LPS-activated high-responder BALB/c B cells. The addition of IL4 or neutralizing antibodies against IL4 or interferon-gamma to these cultures helped to overcome this suppressive effect to a large extent. We conclude that different IgE responder types are caused, at least in part, by a defective IL4 production or by a defect in the TH2 system that is functionally detectable at the level of thymocytes.

In order to develop an ELISA for the quantitative determination of murine IgE, antisera specific for murine IgE were prepared in the goat and rabbit. As immunogen, monoclonal IgE antibody mixtures of several allotypically different hybridomas were used. Before use, these antibodies were purified employing procedures that allow maximum recovery of binding activity. The goat and rabbit mouse epsilon chain-specific antisera were adsorbed on normal mouse serum. The purified antisera were found to be free of allotypic activity. However, immunoadsorption on NMS could not always remove contaminating anti-idiotypic antibodies. Repeated adsorptions with monoclonal antibodies of different isotypes carrying a similar idiotype were necessary to remove all detectable anti-idiotypic activity. Only after these precautions were the antisera suitable for detecting IgE molecules on nitrocellulose blots as well as for quantitating circulating IgE antibodies (ELISA) and IgE-secreting cells (plaque assay and reverse ELISA plaque assay). The purity and reactivity of several commercially available anti-IgE preparations were tested in similar types of specificity assays. Since the specificity of antibodies used in ELISA determines the monospecificity of the assay, retesting for contaminating cross-reactivities in commercial preparations was shown to be necessary.

A Terasaki tray-based ELISA system was developed for the quantitative measurement of antigen-specific and total IgE antibodies in 5 microliter samples of mouse serum dilutions. The assay was based upon non-competitive binding of mouse IgE antibodies between the immobilized appropriate antigen or capture antibodies and the detecting rabbit antibodies. A conjugate of protein A-labelled beta-galactosidase and the fluorigenic substrate methylumbelliferyl-beta-D-galactoside were used as a detecting system. The resulted fluorescence could be measured rapidly and automatically using an inverted micro-fluorimeter. These measurements were automatically transformed into absolute concentrations by a microprocessor-based program using a four-parameter logistic function and an absolute IgE standard. The assay was shown to have a detection limit of 0.04 ng/ml and a range of linearity of 0.04-20 ng/ml, which is sufficient to measure IgE concentrations in mouse serum.