anti-OA IgE Research Articles

Development of a Novel Mouse Anaphylaxis Model Produced by Intermittent Intravenous Injections of Ovalbumin without Adjuvant

This study was performed to develop mouse anaphylaxis model using ovalbumin (OA) as an antigen, with no adjuvant, and an intravenous route of administration. OA at dose levels from 0.5 to 500 mg/kg was injected 3 times into the tail vein of female CD-1 (ICR) mice at 4-day intervals. Clinical signs and extravasation of Evans blue dye (vascular permeability) were observed, serum histamine levels and airway resistance were measured, and histopathological examinations were performed. Furthermore, to detect serum IgE and IgG, heterologous and homologous passive cutaneous anaphylaxis tests were performed. Additionally, strain differences in susceptibility to OA anaphylaxis was examined with 3 mouse strains: ICR, BALB/c and C57BL/6 mice. Shortly after the 3rd injection, most of the animals exhibited systemic anaphylactic signs including death, and this was accompanied by increases in vascular permeability, plasma histamine levels, and airway resistance. Four days after the 2nd injection, anti-OA IgE and IgG were detected in sera of mice injected with 0.5 and 5.0 mg/kg or higher doses of OA, respectively. Histological findings included: congestion in many tissues; detachment of mucosal epithelial cells in the gastrointestinal tract; enhanced death, decreased numbers of lymphocytes, and increased numbers of macrophages in the lymphoid tissues. ICR mice were shown to be most susceptible to the OA anaphylaxis of the 3 mouse strains examined. In conclusion, the results demonstrate that 3 intermittent intravenous injections of OA without adjuvant at 4-day intervals can induce anaphylactic shock in mice, in particular, most effectively in ICR mice, and that the anaphylaxis is characterized by specific IgE and IgG production.

The ex vivo study of synergistic effects of polycyclic aromatic hydrocarbon, benzo(a)pyrene with ovalbumin on systemic immune responses by oral route

The present study was undertaken in order to examine whether oral administration of soluble antigen together with one of polycyclic aromatic hydrocarbons (PAHs) which is present in diesel exhaust particles (DEPs) called benzo(a)pyrene (BP), induced the systemic immune response in mice or not. Mice were orally given 1 mg of ovalbumin (OA), a common food allergen, every 3 days over a period of 15 days. The results showed that oral administration of OA plus BP produced anti-OA IgE antibodies in serum, whereas either OA or BP alone failed to show the antigen-specific IgE antibody production. Production of anti-OA IgE antibody, which is dependent on Th2 CD4 + T cells, was seen in mice fed with combined OA and BP was significantly higher than that of other groups. The anti-OA antibody production was associated with marked secretion of the Th1 cytokines, IFN-γ and IL-12p70 as well as the Th2 cytokines IL-4, and IL-10. These results suggest that BP may act as a mucosal adjuvant in the gut enhancing systemic Th1 and Th2 immune responses and might play a role in oral immunization and food allergy.

The ex vivo study of synergistic effects of polycyclic aromatic hydrocarbon, benzo(a)pyrene with ovalbumin on systemic immune responses by oral route.

The present study was undertaken in order to examine whether oral administration of soluble antigen together with one of polycyclic aromatic hydrocarbons (PAHs) which is present in diesel exhaust particles (DEPs) called benzo(a)pyrene (BP), induced the systemic immune response in mice or not. Mice were orally given 1mg of ovalbumin (OA), a common food allergen, every 3 days over a period of 15 days. The results showed that oral administration of OA plus BP produced anti-OA IgE antibodies in serum, whereas either OA or BP alone failed to show the antigen-specific IgE antibody production. Production of anti-OA IgE antibody, which is dependent on Th2 CD4(+) T cells, was seen in mice fed with combined OA and BP was significantly higher than that of other groups. The anti-OA antibody production was associated with marked secretion of the Th1 cytokines, IFN-gamma and IL-12p70 as well as the Th2 cytokines IL-4, and IL-10. These results suggest that BP may act as a mucosal adjuvant in the gut enhancing systemic Th1 and Th2 immune responses and might play a role in oral immunization and food allergy.

Comparative Studies on Antigenicity and Allergenicity of Native and Denatured Egg White Proteins

The binding activities of IgG and IgE antibodies from egg-allergic patients to physically or chemically treated egg white proteins were examined and compared with those of rabbit anti-egg white IgG antibodies. The sera from eight patients and four rabbit antibodies were used in this study. The binding activities of human IgG antibody to partially denatured ovotransferrin (Tf), ovalbumin (OA), and lysozyme (Lys) forms were increased, whereas carboxymethylation (RCM) and heat treatment caused a dramatic decrease in the antigenicity of Tf and ovomucoid (OVM). Tf and OVM were major immunogenic antigens for the rabbit IgG response. Urea also caused Tf to exhibit greater rabbit IgG binding activity. In contrast, human and rabbit antibodies did not react with ovomucin. Partially denatured Tf and Lys also induced strong IgE binding activities. The allergenicity of Tf, OVM, and Lys was decreased by RCM, whereas OA retained its binding capacity. These results suggested that anti-OA IgE recognizes more sequential epitopes and that anti-OVM and Lys antibodies recognize both conformational and sequential epitopes. Tf and OVM were dominant allergens for the IgE antibodies of anaphylaxis patients, whereas IgE from atopic patients bound more strongly with OA and OVM.

THE FUNGAL CELL WALL COMPONENT b-1,3-GLUCAN HAS AN ADJUVANT EFFECT ON THE ALLERGIC RESPONSE TO OVALBUMIN IN MICE

The polyglucose b -1,3-D-glucan is a major structural component of the cell wall of yeasts and fungi. In the present study, the adjuvant activity of b-1,3-glucan from the fungus Sclerotinia sclerotiorum (SSG) on the response to the model allergen ovalbumin (OA) was studied, using the popliteal lymph node assay (PLNA) in BALB/c mice. The adjuvant activity on the local cellular response was determined by measuring the weight, cell number, and proliferation of the extracted PLNs. The levels of OA-specific immunoglobulin (Ig)E, IgG1, and IgG2a in serum were measured by enzyme-linked immunosorbent assay (ELISA). Groups of 8 mice were given either SSG + OA, SSG alone, or OA alone on d 0. Thereafter they were exsanguinated on d 20, or reinjected with OA on d 21, before exsanguination on d 26 or 33. Only on d 26 was SSG + OA found to significantly increase the PLN weight and cell numbers, but not cell proliferation (thymidine incorporation), compared with OA or SSG alone. SSG + OA was also found to significantly increase both the anti-OA IgE and IgG1 levels on d 20, 26, and 33 compared to OA alone. Compared to SSG alone, SSG + OA increased the OA-specific IgE and IgG1 levels significantly on d 26 and 33, but not on d 20. A similar increase was not found for IgG2a. Our results show that b -1,3-D-glucan provides a clear Th2-dependent (allergic) immune response to OA, indicated by elevated levels of IgE and IgG1 and not IgG2a, in the mouse model used.

Suppressive effects of Hochu-ekki-to, a traditional Chinese medicine, on IgE production and histamine release in mice immunized with ovalbumin.

We examined the effects of Bu-Zhong-Yi-Qi-Tang (Japanese name: Hochu-ekki-to, HET), a traditional Chinese medicine, on IgE production and histamine release in mice immunized intraperitoneally with a mixture of ovalbumin (OA) and aluminum hydroxide (alum adjuvant). Three groups of mice were orally administered 0, 1.7 or 17 mg of HET on day 13 after the first immunization with a mixture of 1 microg OA and 1 mg alum adjuvant. They were again immunized with the same dose of OA plus alum adjuvant on day 14. The immunological changes in mice treated with OA alone or OA plus HET were examined, and the following findings were obtained. In the HET-treated mice, the elevation of anti-OA IgE in serum, and histamine release from basophils in blood, were significantly suppressed. A significant suppression of interleukin-4 (IL-4) secretion and proliferation of splenic lymphocytes in primary culture was also observed. A tendency to suppress the elevation of anti-OA IgG1 in serum and interleukin-2 (IL-2) secretion from splenic lymphocytes was observed in the HET-treated mice. These findings suggest that oral administration of HET suppresses IgE antibody production and histamine release in type I allergic reaction in mice immunized with OA plus alum adjuvant; this shows the efficacy of HET in treating type I allergic diseases, such as asthma.

Inhalation of diesel exhaust enhances antigen-specific IgE antibody production in mice

To examine the effects of diesel exhaust (DE) inhalation on IgE antibody production, BALB/c mice were exposed to 0 (control), 3.0 and 6.0 mg/m3 DE inhalation for 3 weeks. Intranasal sensitization with ovalbumin (OA) three times at intervals of 3 weeks was conducted immediately before, immediately after and 3 weeks after DE inhalation. Body weight and thymus weight for the DE-exposed and control mice were essentially the same but spleen weight in mice exposed to 6 mg/m3 significantly increased. Anti-OA IgE antibody titers in the sera of mice exposed to 6 mg/m3 was significantly higher than the control. Total IgE and anti-OA IgG in sera for DE-exposed and control mice remained basically the same. To investigate cytokine production in mice exposed to 6 mg/m3, spleen cells from DE-exposed and control mice were stimulated with OA in vitro and cytokine production in the culture supernatants was measured by ELISA. In vitro antigen-stimulated interleukin-4 (IL-4) and -10 (IL-10) production in spleen cells of exposed mice significantly increased compared to the control. In vitro interferon (IFN)-γ production in spleen cells of exposed mice markedly decreased. DE inhalation is thus shown to have adverse effect on antigen-specific IgE antibody production in mice through alteration of the cytokine network.

IL-4 production in mediastinal lymph node cells in mice intratracheally instilled with diesel exhaust particulates and antigen

To clarify the relationship between air pollutants and IgE antibody production, interleukin 4 (IL-4) production was investigated in BALB/c mice intratracheally injected with diesel exhaust particulates (DEP) mixed with antigen (Ovalbumin (OA) or Japanese Ceder Pollen (JCP)). BALB/c mice were injected with DEP plus OA or OA alone three times with a 3-week interval. After the last instillation, proliferative response and lymphokine-producing activity of mediastinal lymph node cells (LNC) were examined in vitro. Proliferative response to OA in mediastinal LNC from mice injected with DEP plus OA was enhanced 4–17 times of that from control mice. IL-4-producing activity by OA stimulation also enhanced in mediastinal LNC from mice injected with DEP plus OA. A significantly larger amount of anti-OA IgE antibody was detected in sera from DEP- and OA-injected mice compared with those from control mice. The levels of IL-4, estimated by JCP antigen in mediastinal LNC, from mice injected with DEP plus JCP were two-fold higher than those from mice injected with JCP alone. These results suggest that intratracheal instillation of DEP affects antigen-specific IgE antibody responses via local T-cell activation, especially enhanced IL-4 production.

Modulation of IgE production in the mouse by beta 2-adrenoceptor agonist.

The present study examined the in vitro and in vivo effect of salbutamol on IgE production in the mouse. The present results show that salbutamol potentiates the in vitro interleukin 4 (IL-4)-induced IgE production from lipopolysaccharide-activated murine B lymphocytes. This effect is dose-dependent and is observed at concentrations above 10 nM. In vivo, when ovalbumin (OA)-sensitized BALB/c mice were treated with a daily injection of salbutamol, an increase of the anti-OA IgE levels in the serum was observed as compared to sensitized animals. Such an effect was observed at doses above 1 microgram/kg and was maximal at 10 micrograms/kg. Treatment of sensitized mice with salbutamol increased the ex vivo production of IL-4, IL-5, IL-6 and IL-10 from concanavalin A-activated splenocytes whereas no modification of IFN-gamma synthesis was noticed as compared to nontreated sensitized control mice. These results demonstrate that beta 2-adrenoceptor agonist stimulation results in an increase in IgE production both in vitro and in vivo in the mice. At least in vivo, they also suggest that the effect of this drug could be explained by an increase of the production of Th2-type lymphokines.

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.

IgE antibody-forming cells in rats infected with Nippostrongylus brasiliensis and immunized with antigens

The distribution of IgE antibody-forming cells was examined in rats infected with Nippostrongylus brasiliensis (Nb) or immunized with Nb antigen or with OA. The frequency of antigen-specific IgE antibody-forming cells was detected by a passive cutaneous anaphylactic (PCA) reaction using cell extract from lymphoid organs. In Nb-infected rats, anti-Nb and anti-4th stage larvae (L4) IgE-forming cells distributed mainly in the mesenteric and the bronchial lymph nodes (LN) near the parasite-harboring sites. After intraperitoneal (ip) immunization with Nb antigen mixed with Al(OH) 3 and Bordetella pertussis (Bp) as adjuvants, anti-Nb IgE antibodyforming cells were detected in the mesenteric and the bronchial LN. Anti-Nb or OA IgE antibody-forming cells after subcutaneous (sc) immunization were found in the inguinal and the axillary LN. An effect of Bp on the distribution of IgE antibody-forming cells seems to be ruled out. The distribution of IgG 2a antibody-forming cells was similar to that of IgE antibody-forming cells, indicating that the distribution of the IgE antibody-forming cells is not preferential. IgE antibody-forming cells were stimulated in the regional LN near the site of antigen administration. IgE antibody-forming cells induced by potentiated IgE antibody production were also examined. Rats were immunized ip or sc with OA and infected with Nb. Anti-OA IgE antibody-forming cells were found in all of the lymphoid organs and especially in the regional LN near the Nb parasite-harboring and antigen administration sites.

Diesel-exhaust particulates inoculated by the intranasal route have an adjuvant activity for IgE production in mice

Our previous study indicated that the IgE antibody responses in mice immunized with intraperitoneal injection of the antigens mixed with diesel-exhaust particulates (DEP) were higher than those in the animals immunized with the antigens alone. We examined the adjuvant activity of DEP inoculated by the intranasal route, i.e., the natural entrance of DEP. In 3-week interval immunization, the IgE antibody responses in mice immunized with intranasal inoculation of ovalbumin (OA) mixed with DEP were higher than responses in the animals immunized with OA alone. DEP had an adjuvant activity for anti-OA IgE antibody production, even in a small dose such as 1 micrograms administered with a 3-week interval. Also in 1-week interval immunization, the enhancing effect of DEP on anti-OA IgE antibody production was demonstrated when mice were immunized with intranasal inoculation of OA and DEP. The possibility cannot be excluded that DEP, which are kept buoyant in the environmental atmosphere of urban districts, may exert an adjuvant activity for IgE antibody production after being inhaled into the human body and have some relation to the mechanism of the outbreak of allergic rhinitis caused by pollens in Japan.

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

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

Induction of IgE antibody production to aerosolized ovalbumin in mice treated intratracheally with aluminum silicate.

Induction of IgE antibody production was studied in mice with aerosolized antigen exposure and intratracheal instillation of adjuvants. BALB/c mice were immunized intratracheally with 10 micrograms ovalbumin (OA) adsorbed with or without aluminum silicate as an adjuvant. A primary IgE antibody production was observed in mice immunized with OA plus adjuvant, but not with OA alone. In mice immunized with OA plus adjuvant, a secondary IgE antibody production was observed after a booster of OA aerosol exposure on day 28. Mice were instilled intratracheally with aluminum silicate or aluminum hydroxide (alum) and subsequently exposed to aerosolized OA for 30 min. Primary and secondary anti-OA IgE antibody productions were obtained in the groups instilled with the adjuvants. After the intratracheal instillation of alum, aluminum remained in the mice for a long period. These results suggest that the intratracheal instillation of adjuvants is effective in the induction of IgE antibody production by aerosolized antigen exposure.

Cyclosporin A-induced suppression of ongoing IgE antibody formation in the mouse.

Persistent anti-ovalbumin (OA) IgE antibody formation in the mouse was suppressed by oral administration of cyclosporin A (Cy A). Spontaneous anti-OA IgE antibody formation in vitro was also suppressed by Cy A added to the culture. Anti-OA IgG antibody responses in vivo and in vitro were less affected by Cy A. Cy A-induced immunosuppression was T cell-dependent since removal of T cells from the immune spleen cell suspension abolished the Cy A-induced suppression of antibody formation. Supplementing normal spleen T cells resulted in recovery of Cy A-induced suppression of spontaneous antibody formation in vitro. Cy A-induced suppressor T cells carried both Lyt 1 and Lyt 2 surface markers.

Cyclosporin A induced suppression of ongoing IgE antibody formation in the mouse.

Persistent anti-ovalbumin (OA) IgE antibody formation in the mouse was suppressed by oral administration of cyclosporin A (Cy A). Spontaneous anti-OA IgE antibody formation in vitro was also suppressed by Cy A added to the culture. Anti-OA IgG antibody responses in vivo and in vitro were less affected by Cy A. Cy A-induced immunosuppression was T cell-dependent since removal of T cells from the immune spleen cell suspension abolished the Cy A-induced suppression of antibody formation. Supplementing normal spleen T cells resulted in recovery of Cy A-induced suppression of spontaneous antibody formation in vitro. Cy A-induced suppressor T cells carried both Lyt 1 and Lyt 2 surface markers.

Adjuvant Effects of Aluminum Silicate on IgE and IgG1 Antibody Production in Mice

The adjuvant effects of aluminum silicate on IgE and IgG1 antibody production were investigated. BALB/c mice were immunized intraperitoneally with 10 micrograms ovalbumin (OA) adsorbed on 0.2, 2, or 20 mg aluminum silicate. The enhancement of anti-OA IgE antibody production was observed in the mice injected with aluminum silicate and antigen compared with the mice injected with antigen alone. Anti-OA IgE antibody production with 2 and 20 mg aluminum silicate was greater than that with aluminum hydroxide (alum) as an adjuvant. Similar adjuvant effects with 2 mg aluminum silicate or alum were observed in AKR and C57BL/6 mice using 10 micrograms OA, and in BALB/c mice using 2 micrograms DNP-KLH (dinitrophenyl keyhole limpet hemocyanin): IgE antibody production induced by aluminum silicate adjuvant persisted for weeks in these experiments. The enhancement of IgG1 antibody production to OA mixed with aluminum silicate was also demonstrated. However, no difference between aluminum silicate and alum was observed on the IgG1 antibody production.

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

The effects of pretreatments of BALB/c mice with several conjugates of MDP and MDP-Lys to ovalbumin before immunization with ovalbumin (OA) were tested on the anti-OA IgE responses. Pretreatment with MDP-Lys-OA, but not with MDP-OA, induced an inhibition of the anti-OA primary and secondary responses, as measured by passive cutaneous anaphylaxis (PCA) and also by mast cell degranulation. The inhibition by pretreatment with MDP-Lys-OA was obtained whether it was administered in Freund's incomplete adjuvant (FIA) or in saline. This IgE suppression was accompanied by an enhancement of IgG 2a and IgG 2b anti-OA antibodies, with no change in the specific IgG 1 levels. Loss of antigenicity of OA, detected by the lack of degranulation of peritoneal mast cells sensitized by IgE anti-OA, was observed in the MDP-Lys-OA but not in the MDP-OA conjugates. This loss of antigenicity appears to correlate with the ability of the conjugate to induce suppression of the specific IgE response.

Enhancement of IgE Antibody Production by Ovalbumin Aerosol in Mice

The effect of aerosolized ovalbumin (OA) on the induction of IgE antibody production was investigated in BALB/c mice. Enhancement of anti-OA IgE antibody production was obtained after the administration of 10 micrograms OA in mice preexposed to 1% aerosolized OA for 6 or 30 min, and after the administration of 1 microgram OA in mice preexposed to aerosolized OA for 30 min. IgE antibody production could also be induced by preexposure to aerosolized OA at a concentration as low as 0.1%. A carrier effect was observed by preexposure to aerosolized OA using a hapten-carrier system. In the latter system, whole body irradiation (200 R) could not enhance IgE response, suggesting that irradiation-sensitive suppressor T cells might be generated in insufficient amount or not at all by aerosolized OA. Preexposure to aerosolized dinitrophenol-OA could not enhance anti-dinitrophenol IgE response. These results suggest that the exposure to aerosolized antigen primed preferentially T helper cells.

Tolerization of B ϵ cells by conjugates of haptens and isologous γ-globulins

The anti-hapten IgE response induced in B 6D 2F 1 mice by an ip injection of 1 μg of dinitrophenylated ovalbumin (DNP 3-OA) with 1 mg A1(OH) 3 was abrogated by an iv injection of 1 mg of a conjugate consisting of the hapten coupled to isologous γ-globulins (DNP 8-MγG); the latter treatment did not affect the anti-OA IgE response induced by DNP 3-OA. On the other hand, administration of deaggregated OA into mice resulted in incapacitating their potential to produce both anti-DNP and anti-OA antibodies in response to a sensitizing dose of DNP 3-OA. Moreover, whereas the splenic cells of mice immunosuppressed by DNP 8-MγG did not mount an anti-DNP IgE response on challenge with either DNP 3-OA or the conjugate of DNP with constituents of Ascaris suum (DNP-ASC), the cells of mice tolerized with deaggregated OA were able to mount an anti-DNP IgE response on sensitization with DNP-ASC. In adoptive cell transfer experiments, it was demonstrated that (i) the anti-DNP IgE response was elicited with a mixture of thymus cells of tolerized mice and bone marrow cells of primed mice, but not by a mixture of thymus cells of primed mice and bone marrow cells of tolerized mice; (ii) the ability of tolerized spleen cells to produce anti-DNP IgE antibodies was restored when supplemented with bone marrow cells of primed mice, but not with thymus cells of primed mice; (iii) cooperation of splenic T cells of tolerized mice with splenic B cells of primed mice resulted in a marked anti-DNP IgE response, but the mixture of primed T cells and of tolerized B cells produced only a minimal response. From all these results, it is concluded that the immunosuppressive treatment resulting from the administration of DNP 8-MγG was due to tolerization of B ϵ cells.