Ongoing IgE Responses Research Articles

Effect of filgrastim (recombinant human granulocyte colony stimulating factor) on IgE responses in human asthma (48.1)

Abstract Rationale: The role of peripheral blood progenitor cell mobilization on IgE responses has not been studied. Methods: Distributions of blood lymphocytes (CD4+, CD8+, CD8+CD60+, CD19+, CD23+, CD16/56+, CD25, CD45RA+, CD45RO+, CD34+), and levels of serum immunoglobulins (IgM, IgG, IgA, IgE) were studied in an allergic asthmatic serum IgE+ (181 IU/mL) adult (m/45 y/o) donor undergoing routine stem cell mobilization protocol (American Society of Hematology) before (day -30), during (day 4), and after (1 wk post last dose) filgrastim (subcutaneous, 480 mcg, 2qd) treatment (flow cytometry, nephelometry, UniCAP Total IgE Fluoroenzymeimmunoassay). Results: On day 4 of filgrastim treatment, numbers of CD8+CD60+ T cells and CD23+ blood cells dramatically increased (>2 fold); CD34+ cell numbers also increased. One week after treatment, numbers of these cells decreased to pre-treatment levels. On day 4 of treatment, serum IgE levels decreased (>50%). However, one week after treatment, IgE levels approached pre-treatment levels. Conclusions. Filgrastim treatment transiently increases numbers of CD8+CD60+ T cells known to regulate human IgE responses (Smith-Norowitz, et al., JI, 2008), while also transiently suppressing ongoing IgE responses. These results suggest that filgrastim might be useful in modulating allergic responses.

Induction of Specific Th1 Responses and Suppression of IgE Antibody Formation by Vaccination with Plasmid DNA Encoding Cyn d 1

Background: DNA vaccines encoding allergens have been developed to prevent or to treat specific IgE responses. Objective: To evaluate the potential preventive and therapeutic effect of DNA vaccines encoding Cyn d 1 alone or combined with different adjuvants on specific allergies. Methods: Recombinant plasmid Cyn d 1 (pCyn d 1) was constructed by insertion of Cyn d 1 cDNA into the vector pcDNA3. BALB/c mice were injected with pCyn d 1 alone or plus adjuvants such as bupivacaine, bestatin, liposome, or CpG. Control mice were treated with pcDNA3 or PBS. They were boosted 3 weeks later and then sensitized twice with recombinant Cyn d 1 and alum. Their serum antibody responses and cytokine profiles of spleen cells were studied. Adoptive transfer of spleen cells of pCyn d 1-vaccinated mice was also performed. Results: Vaccination of mice with pCyn d 1 induced Th1 responses characterized by IgG2a responses and spleen cell secretion of interferon-γ. Vaccination with pCyn d 1 not only prevented the induction of specific IgE responses but also suppressed ongoing IgE responses. The mice receiving untreated, CD4+- or CD8+-depleted spleen cells from pCyn d 1-vaccinated mice all had suppression of IgE responses. Conclusion: This study confirms the prophylactic and therapeutic effects of DNA vaccines encoding Bermuda grass pollen allergen Cyn d 1 on specific IgE responses. Both CD4+ and CD8+ T cells are crucial for the immunomodulatory effect of pCyn d 1 on specific IgE responses.

Effect of Tetracyclines on IgE Allergic Responses and Asthma

There are no current therapies that specifically target IgE production in human allergic disease. We found that tetracyclines and chemically modified tetracyclines (CMT) that lack antibiotic activity prevent IgE production, making them ideal candidates for anti-allergy therapy. This is based on our findings that minocycline treatment of allergic asthmatic humans significantly improves their asthma symptoms, reduces their oral steroid requirements, and strongly suppresses their ongoing IgE responses. Tetracyclines and CMT also suppress ongoing IgE responses of BPO-KLH sensitized mice in vivo and in vitro and humans IgE responses in vitro. We also found that highly increased levels of phosphorylated p38 MAP kinase, but not phosphorylated JNK or ERK, are expressed by blood T and B cells and monocytes of allergic asthmatic humans. Levels of phosphorylated p38 MAP kinase, but not ERK or JNK, correlated with their IgE. Tetracyclines significantly suppressed expression of phosphorylated p38 MAP kinase by CD4+ and CD8+ T cells but not B cells or monocytes in vivo and in vitro. Our findings open the door to development of new drugs and patents, especially for CMTs that lack antibiotic activity (US 7649113), for treatment of human allergic disease.

Non-antibiotic properties of tetracyclines as anti-allergy and asthma drugs

All available therapies for human allergic disease target IgE mediated pathologic responses after IgE has been produced. We are developing tetracyclines as anti-allergy drugs to prevent IgE production, based on our findings that minocycline or doxycycline treatment of allergic asthmatic humans significantly improves their asthma symptoms, reduces their oral steroid requirements, and strongly suppresses their ongoing IgE responses (ELISA, mast cell mediated cutaneous late phase responses); the tetracyclines also strongly suppress peak IgE responses of BPO-KLH sensitized mice (ELISPOT assay, ELISA, skin tests). The antibiotic activity of the tetracyclines is not required for suppression of IgE responses; inclusion of minocycline or doxycycline in sterile culture prevents anti-CD40/IL-4 mediated induction of memory IgE responses by PBMC of allergic asthmatic patients (ELISA), and induction of specific memory IgE responses by spleen cells of BPO-KLH sensitized mice (ELISPOT assay, ELISA). The tetracyclines affect an epsilon specific pathway because IgM, IgG and IgA responses did not decrease. Further, in humans, DTH responses to recall antigens did not decrease. In related studies, we found that two distinct T cell subsets: CD4+CD60 negative and CD8+CD60+ (CD60 is a ganglioside) (humans) and CD4+ Asialo GM1 ganglioside negative and CD8+Asialo GM1 ganglioside+ (mice), both are required for induction of memory IgE responses. Phosphorylated (phos) p38 MAP kinase, but not phos ERK or phos JNK expression by CD4+ and CD8+, including CD8+CD60+, T cells is increased in allergic asthmatic humans, as is IL-4 and IL-10 production. The tetracyclines appear to target T cell pathways to induce suppression of IgE responses because they suppress phos p38 MAP kinase expression by both CD4+ and CD8+, including CD8+CD60+, T cell subsets, and IL-4 and IL-10, while upregulating IL-2 and IFN gamma, and suppressing IgE responses. Our finding that tetracyclines do not require antibiotic activity to suppress IgE responses opens the door to development of new tetracycline-based and other therapeutics for human allergic disease.

Isotype-selective abrogation of established IgE responses

Chemically modified allergens have been extensively studied in an attempt to develop materials of increased efficacy and improved safety for use in the immunotherapy of allergic disease. Most of the strategies that have been developed yield products that strongly inhibit de novo IgE responses but have only marginal impact on ongoing IgE responses. We report the virtual abrogation of pre-established murine anti-ovalbumin IgE responses using a glutaraldehyde-polymerized ovalbumin preparation (OA-POL) of Mr 3.5 x 10(7). Secondary IgE responses are inhibited by 97-99% over a period of at least 8 months following three i.p. courses of OA-POL treatment. Administration of five additional ovalbumin [Al(OH)3] booster immunizations over this period fails to alter this unresponsive state. The inhibition of antigen-specific IgE responses is isotype specific.

IgE peptide-specific CTL inhibit IgE production: A transient IgE suppression model in wild-type and HLA-A2.1 transgenic mice

Effect of IgE peptide-specific CTL on IgE antibody production was studied in mouse models. CTL elicited in B6.A2Kb tg mice against a human IgE peptide nonamer, pWV, lysed human IgE-secreting U266 myeloma cells and inhibit IgE production by these cells. U266 transfected with mouse A2Kb transgene (U266-A2Kb) were optimally lysed by these CTL, because the α3 domain of A2Kb interacts well with the CD8 co-receptors. The CTL generated were more effective in inhibiting IgE production by U266-A2Kb cells than lysing these cells. IgE production by and progression of U266 myeloma were suppressed in B6.A2Kb tg mice rendered tolerant to these cells and vaccinated with pWV along with CpG. We also studied the CTL response elicited in wild-type mice by a mouse nonameric IgE peptide, PI-1, along with CpG. This treatment caused a transient suppression of the IgE response in mice previously sensitized to an antigen. In mice treated with this regimen repeatedly, the IgE response was fully recovered 20 days after each treatment. Notably, while IgE peptide/CpG-treated mice remained unresponsive to antigen challenge in vivo, antigen-specific IgE production can be elicited by antigen in cultured splenocytes from these mice. Moreover, IgE peptide/CpG also inhibited an on-going IgE response, including IgE production by bone marrow cells. Taken together, these observations indicate that a CTL-based IgE peptide vaccine targeting IgE-secreting B/plasma cells may be safely employed as a therapeutic approach for suppressing IgE production.

Generation of hypoallergenic DNA vaccines by forced ubiquitination: Preventive and therapeutic effects in a mouse model of allergy

Hypoallergenic immunotherapy of type I allergies aims at inducing T-cell immunity while avoiding cross-linking of pre-existing IgE. DNA-based immunotherapy depends on the recruitment of antigen-specific T(H)1 cells and therefore has to provide the whole repertoire of T-cell epitopes. Ubiquitination offers a general approach for the production of hypoallergenic DNA vaccines. A DNA-based vaccine encoding the major birch pollen allergen Bet v 1 stably linked to ubiquitin was evaluated for its antiallergic potential in a BALB/c mouse model of allergy. Plasmid DNA was applied to mice before (preventive) or after (therapeutic) sensitization with recombinant Bet v 1. In the preventive setting, mice were exposed to aerosolized allergen in addition. Cytokine production was monitored via ELISPOT and Luminex. IgG(1), IgG(2a), and IgE subclass antibody titers were determined by ELISA. In vitro antigen-specific cross-linking of IgE was measured in a degranulation assay. Bronchoalveolar lavages were analyzed for leukocyte subsets as well as for IFN-gamma and IL-5, and paraffin sections of lungs were examined for mucus production and endothelial damage. Prevaccination with ubiquitinated Bet v 1-stimulated T(H)1-biased immune responses with concomitant suppression of functional IgE, reduction of eosinophil counts in bronchoalveolar lavages, and alleviation of lung pathology, and could also suppress an ongoing IgE response in a therapeutic setting. The data clearly demonstrate that hypoallergenic DNA vaccines encoding ubiquitin fusion constructs induce effective antiallergic immune responses. Ubiquitination of allergen gene vaccines eliminates the risk of IgE cross-linking, thereby meeting the safety requirements for clinical applications.

Induction of specific Th1 responses and suppression of IgE antibody formation by vaccination with plasmid DNA encoding Der f 11

DNA vaccines encoding low-molecular-weight allergens have been used to prevent IgE responses. A high-molecular-weight mite allergen Der f 11 that was hardly to be purified for immunotherapy was used to develop a DNA vaccine here. Vaccination of mice with plasmid DNA encoding Df11 (pDf11) induced Th1 responses characterized by IgG2a responses and spleen cell secretion of IFN-γ. In contrast, sensitization with recombinant Der f 11 (rDf11) and alum induced Th2 responses characterized by IgE responses and spleen cell secretion of IL-4 and IL-5. Vaccination with pDf11 prevented the induction of IgE responses. Moreover, it could inhibit on-going IgE responses. The debate whether CD4 + or CD8 + T cells were the regulatory cells to inhibit IgE responses by DNA vaccination was also examined. First, sensitization of pDf11-vaccinated mice after depletion of CD8 + T cells still showed suppression of IgE responses. Secondly, adoptive transfer of either CD4- or CD8-depleted spleen cells from pDf11-vaccinated mice suppressed IgE responses. In conclusion, this is the first report to confirm the therapeutic effect of a DNA vaccine encoding a strong allergen on specific IgE responses. Both CD4 + and CD8 + T cells are crucial for the immunomodulation of IgE responses by pDf11.

CpG oligodeoxynucleotide vaccination suppresses IgE induction but may fail to down-regulate ongoing IgE responses in mice.

Antigen-specific IgE plays an important role in the pathogenesis of allergic disorders. Immunostimulatory CpG motifs (CpG) in bacterial DNA or synthesized oligodeoxynucleotides (ODN) are gaining recognition as potential immunomodulators for switching on protectiveT(h)1-mediated immunity and preventing or potentially inhibiting T(h)2-dependent allergic responses. To date, allergic models used in CpG ODN studies have been established by immunization of mice with allergen in the presence of adjuvant. This, in addition to failure to assess specific IgE production in most of the studies, has limited understanding of the role of CpG ODN vaccination in allergic responses. Here, we examine the effects of synthesized CpG ODN on both developing and ongoing IgE responses in mice sensitized using a recombinant mosquito salivary antigen (rAed a 2) without adjuvant. Pretreatment of mice with CpG ODN mixed with rAed a 2 successfully inhibited subsequent induction of serum rAed a 2-specific IgE (but not IgG1) and antigen-induced IL-4 and IL-5 production in spleen cells. This was associated with an increase of serum IgG2a and IL-12, and increased IFN-gamma and IL-12 production by spleen cells. In this model, however, co-administration of CpG ODN with rAed a 2 to presensitized mice failed to down-regulate ongoing IgE responses despite significant up-regulation of serum IL-12 and specific IgG2a. Strikingly, a transient skin delayed-type hypersensitivity reaction occurred in CpG ODN-treated mice. These observations provide a new insight into the potential therapeutic application of CpG ODN to allergic disorders.

232 Prevention of airways eosinophilia in mice receiving subsequent sensitization and in mice with ongoing IgE responses using CpG oligodeoxynucleotide

232 Prevention of airways eosinophilia in mice receiving subsequent sensitization and in mice with ongoing IgE responses using CpG oligodeoxynucleotide

335 Effect of CpG oligodeoxynucleotice vaccination on ongoing IgE response to rAed a 2 in mice

335 Effect of CpG oligodeoxynucleotice vaccination on ongoing IgE response to rAed a 2 in mice

B- and T-cell epitopes of tropomyosin, the major shrimp allergen.

The major crustacean allergen characterized from different species of shrimp is the muscle protein tropomyosin. Two shared epitopes corresponding to 47-63 and 150-158 of the deduced amino-acid sequence of the brown shrimp, M. ensis, were identified as IgE-binding B-cell epitopes. A 21-mer peptide spanning the amino-acid residues 261-281 was identified as a putative T-cell epitope capable of reducing ongoing tropomyosin-specific IgG and IgE responses in a mouse model. These observations suggest that peptide immunotherapy may also be effective in the treatment of food hypersensitivity.

Administration of IL-12 during ongoing immune responses fails to permanently suppress and can even enhance the synthesis of antigen-specific IgE.

The synthesis of antibodies of the IgE isotype in mice largely depends on IL-4, a cytokine that is released by T lymphocytes of the Th2 subtype. IL-12 is a cytokine considered to direct Th cell development into a Th1 direction and to suppress Th2 responses including the synthesis of IgE. Here we report about the influence of IL-12 on the IgE responses of mice immunized with protein antigens adsorbed to aluminum hydroxide. To avoid problems with the detection of IgE caused by an excess of competitive IgG antibodies produced in IL-12-treated mice, serum IgE was first extracted from the serum by plate-bound anti-IgE mAb and then determined either as total IgE or as antigen-specific IgE by using biotinylated anti-IgE or biotinylated antigen. Depending on the strain of mice and the dose of IL-12 injected together with the antigen, IL-12 can either temporarily suppress or augment the synthesis of (antigen-specific) IgE antibodies. This applies for CBA/J mice immunized six times in biweekly intervals with minute (0.1 micrograms/injection) or three-times with large (5 micrograms/injection) amounts of the bee venom allergen phospholipase A2 (PLA2). Under both conditions the antibody response is characterized by the production of predominantly IgG1 as well as IgE but very little IgG2a, IgG2b and IgG3 antibodies. Simultaneous application of low doses of IL-12 (1 or 10 ng/day) led to a 2- to 4-fold enhancement of IgE production (PLA2-specific IgE or total IgE). Only a high dose of 1 micrograms IL-12/day resulted in a 3- to 10-fold reduction of the IgE response. This suppression was not stable, however, because the synthesis of IgE antibodies was stimulated to a high level when these mice subsequently received a second course of immunizations in the absence of IL-12. Likewise, the synthesis of IgE was only temporarily suppressed by IL-12 treatment in CBA/J mice immunized with keyhole limpet hemocyanin (KLH) as antigen. However, application of low (10 ng/day) or high (1 microgram/day) doses of IL-12 during the primary course of immunizations of CBA/J mice with KLH suppressed the IgE response slightly or strongly respectively. In striking contrast, the KLH-specific IgE response of BALB/c mice was upregulated even when high doses of IL-12 (1 microgram/day) were injected simultaneously with the immunizations. Thus, these results demonstrate a great variability regarding the influence of IL-12 treatment on ongoing IgE responses in vivo.

Modulation of systemic cytokine levels by implantation of alginate encapsulated cells

The availability of cell lines that are transfected with IL-4, IL-5 and IFN-γ 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 cells was shown to be equivalent to that of injecting purified antibodies.

Efficacy of liposome entrapped allergen in down regulation of IgE response in mice.

The effect of liposome entrapped allergen on primary, secondary and ongoing IgE response was studied in mice. It was observed that mice primed with liposome entrapped allergen maintained significantly lower levels of serum specific IgE as compared to that of controls primed with allergen. Although alum adsorbed allergen could induce IgE synthesis in mice primed with liposome entrapped allergen the increase in serum specific IgE levels was lower than the animals primed and challenged with alum adsorbed allergen. On the contrary when BALB/c mice were primed with alum adsorbed allergen to induce IgE synthesis subsequent challenge by liposome entrapped allergen could down regulate the serum specific IgE response. Serum specific IgG response did not show any significant difference between the two groups. There was an increase in T suppressor cell subpopulation as measured by immunofluorescence technique in mice injected liposome entrapped allergen as compared to that in controls. The results indicate that liposome entrapped allergens may prove to be safe and effective in immunotherapy by reducing the allergenic response of the allergen at the same time increasing the antigenicity.

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.

Regulation of in vivo IgE Biosynthesis in Mice with Complete Freund’s Adjuvant

Complete Freund's adjuvant (CFA) administered before sensitization dampened the normal and cyclophosphamide-enhanced response of high and moderate IgE responder phenotype mice (CAF1 and C57B1/6J, respectively). CFA-induced suppression of IgE biosynthesis was effective in reducing anaphylactic histamine release from approximately 2,900 ng histamine per milliliter to background levels (less than 100 ng/ml). CFA-induced ascites fluid was able to reduce the cyclophosphamide-enhanced IgE response of low-responder phenotype SJL mice from 1:320 to less than 1:5 as determined by passive cutaneous anaphylaxis. Muramyl dipeptide, a mycobacterial cell wall component capable of eliciting effects similar to those seen with CFA, was shown to induce suppression of IgE production if incorporated in incomplete Freund's adjuvant. Muramyl dipeptide administered in saline was ineffective, while incomplete Freund's adjuvant alone had some immunoregulatory properties. Ongoing IgE responses were less susceptible to regulation. CFA administered to sensitized C57B1/6J mice was ineffective in inducing IgE suppression when animals were challenged with antigen.

The introduction of one or two 3 beta-cholestanyl residues into benzylpenicilloyl-eicosa-L-lysines greatly potentiates their tolerogenicity for anti-benzylpenicillol IgE antibody formation.

BALB/c mice were repeatedly immunized with microgram doses of benzylpenicilloylated Ascaris protein(s) (BPO9Asc) in alum. At different stages of the immune response, BPO21 eicosa-L-lysine or two analogs containing one or two hydrophobic p-oxymethylbenzyl-3 beta-cholestanyl succinate (OSuco) groups were injected. When injected early in the immune response, the anti-BPO IgE antibody formation was much more strongly and permanently suppressed by the lipophilic conjugates than by the hydrophilic BPO21 eicosa-L-lysine. A similar, but less marked, suppressive effect was observed on the anti-BPO IgG1 response. By adoptive cell transfer experiments, it was found that the OSuco-containing derivatives induce and act via suppressor T lymphocytes, since this cell-mediated suppression was sensitive to cyclophosphamide or to treatment with anti-Lyt-2.2 antibody plus complement. When these compounds were injected into repeatedly immunized mice producing late ongoing antibody responses no differences in suppression between hydrophilic and hydrophobic derivatives were observed. In this case, the IgE response was suppressed by about 50%, while the IgG1 response was not affected. These results are compatible with the suggestion that early IgE responses are most sensitive to T cell-mediated suppression and that T suppressor cells are better induced by lipophilic than by hydrophilic antigens. The late ongoing IgE response, on the other hand, is less amenable to T cell-induced suppression and tolerogenic effects brought about by plurivalent BPO antigens operate directly on hapten-specific IgE-bearing B cells, regardless of their lipophilic character.

Induction of immunological tolerance to the penicilloyl antigenic determinant—iv. The effect of BPO-oligolysines and cholestanol-bearing BPO-oligolysines on murine IgE responses

Penta-, deca- and eicosalysine carriers were synthesized in solution and conjugated with benzylpenicillin to give BPO-conjugates of high haptenic density. Each oligolysine conjugate was prepared in two forms—with a free C-terminus and with an esterified C-terminus carrying via a benzylester bridge in essence a lipophilic cholestanol moiety [ p-moxymethylbenzylcholestan-3β-yl succinate (OSuco group)]. Decalysines that carried a single haptenic BPO group and succinyl groups on the other amino functions were also prepared. Suppression of IgE responses was studied in BALB/c mice. It was found that BPO-specific suppression could be induced by injecting OSuco-bearing deca- or eicosalysine conjugates before immunization with BPO-Asc in A1(OH) 3. The pentalysine conjugate was only slightly effective as were all OSuco-deficient conjugates. Ongoing IgE responses were only slightly suppressed and OSucobearing conjugates were not more effective than OSuco-deficient derivatives. When the monohaptenic OSuco-bearing decalysine, which exhibited weak tolerogenic effects on primary as well as on ongoing responses, was applied under conditions that favour suppressor T-cell induction, a pronounced unresponsiveness resulted. Direct evidence for suppressor T-cell involvement in the abrogation of anti-BPO responses by OSuco-bearing BPO-conjugates was obtained from cell transfer experiments. The study shows that relatively small haptenic conjugates, the lower limit of effectiveness being approximately represented by decalysine conjugates, may be effective tolerogens depending on the immune status.

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