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Characterization of the IgG Antibody Response to Timothy Grass Pollen Antigens
Abstract
The IgG and IgE antibody responses against timothy grass pollen antigen B (AgB) in several mouse strains were determined. Considerable variability in both responses was demonstrated, and (CBA/J X C57BL/6)F1 mice were selected for use in subsequent experiments. Anti-idiotypic-antibody-induced T suppressor cells almost completely suppressed AgB-specific IgE, but the IgG response was not altered. Further studies with photooxidized AgB (Ox-AgB) indicated that the IgG response was directed against an antigenic determinant expressed on both native antigen and Ox-AgB. Our data indicates that AgB possesses two distinct antigenic determinants, one that induces an IgE response, and one that induces an IgG response.
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.
Liposomes are non-toxic, biodegradable and weakly immunogenic lipid vesicles which can be used as immunomodulating agents. In the present study, multilamellar vesicles (MLV) and small unilamellar vesicles (SUV) were used to incorporate an allergenic protein from Artemisia scoparia pollen. MLV incorporated more allergenic protein than SUV. To assess the immunomodulating effect of allergen entrapped in liposomes, Swiss strain mice (made IgE responders) were injected with either free allergen or liposome-entrapped allergen (LEA) and their immune response was measured in terms of specific IgG and specific IgE levels. Results indicated that specific IgE response was significantly lower in mice injected LEA (P less than 0.02) than in mice injected free allergenic protein. Although specific IgG response was higher in mice injected LEA, there was no statistically significant difference between the two groups. Potential use of liposomes as non-immunogenic biocompatible vehicle for antigen presentation in immunotherapy will be discussed.
Liposomes are non-toxic, biodegradable and feebly immunogenic lipid vesicles made from natural and synthetic lipids. They are known to act as immunopotentiating agents and can be used to formulate sustained release preparation by encapsulation. In the present study, liposome entrapped allergen and free allergen were used to inject in Balb/C mice at different time intervals and their immune response in terms of specific IgG and specific IgE levels was quantitated by ELISA (Enzyme Linked Immuno sorbent Assay). The results indicated that specific IgE response was significantly higher in mice injected free allergen as compared to that of mice given liposome entrapped allergen. However, the specific IgG response was not statistically significant. Experiments carried out with liposome entrapped allergen and liposome coupled allergen showed no statistically significant difference in specific IgE and specific IgG titre between the two groups of mice. This type of immunomodulatory effect of liposomes in reducing IgE levels and without affecting IgG levels may be useful in Type I allergic disorders.
The immune system of offspring mice from mothers immunized with photo-oxidized timothy grass pollen antigen B (OX-AgB) or Trinitrophenyl-bovine gamma globulin (TNP-BGG) during their pregnancy was examined. Offspring mice immunized 6 or 8 weeks after delivery with the same antigen administered to their mothers have completely suppressed primary responses and greater than 80% suppressed secondary responses. The observed immunosuppression in offspring mice appears to persist until about 16 weeks after delivery, and is antigen-specific. Adoptive transfer studies show that spleen cells from adult OX-AgB primed mice injected i.v. into lethally X-irradiated (800 rads) syngeneic recipients and challenged with antigen produce significant levels of AgB-specific IgG antibody. Spleen cells (5 x 10(6] from offspring mice of mothers immunized with OX-AgB during their pregnancy were added to spleen cells from adult OX-AgB primed mice and injected i.v. into lethally X-irradiated syngeneic recipients and challenged with antigen. These recipients showed a significantly (greater than 85%) immunosuppressed secondary response. The observed immunosuppression appears to be mediated by CD4+ and CD8+ T cells suggesting a requirement for both T suppressor inducer and effector cell populations. The reported findings are discussed in relation to possible mechanisms to explain the immunosuppression obtained in the offspring of mothers immunized during pregnancy.
The IgG subclass response was evaluated by a sensitive allergen- and subclass-specific solid phase immunoradiometric assay during a 1-year placebo-controlled, double-blind study of immunotherapy with Cladosporium herbarum in 22 adult asthmatics. The IgG response was mainly restricted to subclasses 1 and 4 but a few patients were IgG2 and IgG3 responders. An intense and early IgG1 response was observed during the first clusters of injection followed by a levelling down of the titer. The IgG4 response had a later onset and showed slowly increasing levels during the 12 months of immunotherapy. The graded clinical efficacy estimated by symptom-medication score was significantly correlated to the preseasonal IgG1 value, with high values indicating a deleterious response of immunotherapy (deterioration of disease activity). Likewise, the fold increase of IgG1 and IgG4 after two clusters of immunotherapy (i.e. after 4 weeks) was significantly related to the clinical outcome. Little or no increase of IgG1 and IgG4 was associated with improvement, i.e. decrease in symptom-medication score. The magnitude of the IgG1 response during the dose-increase phase was directly correlated to the number of systemic side effects. No relation of IgG1, IgG4 or IgG4/IgG1 ratio to changes in the IgE-mediated parameters (skin prick test, bronchial challenge and circulating specific IgE) was observed. Our data, which are based on few patients and only one allergen system, do not support the hypothesis of IgG acting as blocking antibody being the immunologic mechanism of immunotherapy. The association between high IgG4 values and a deleterious efficacy of immunotherapy might be caused by IgG4 acting as sensitizing antibodies. This explanation, however, is opposed by the lack of relation to systemic side effects.
The effects of free and encapsulated allergens of Artemisia scoparia pollen on lymphocyte proliferation and immunoglobulin production in BALB/c mice were investigated. Splenic lymphocytes from mice immunized with liposome entrapped allergen (LEA) elicited a marked proliferative response upon in vitro stimulation with both free and encapsulated allergen in comparison to mice immunized with free allergen (FA). The serum immunoglobulin profile of mice administered LEA revealed a predominance of IgG1 antibodies concomitant with an enhancement of IgG2a, IgG2b, IgG3 and IgM responses and suppression of IgE responses. However immunization with FA resulted in significant production of IgE responses and low levels of IgG antibodies. The differential ability of free and encapsulated allergens to selectively induce immunoglobulin isotypes suggests that different presentation and T cell differentiation pathways may be followed by FA and LEA in the immune system. Proliferation studies involving macrophage depletion demonstrated that macrophages play an obligatory role in the processing of LEA. Analysis of cytokine production in sera of immunized mice (FA/LEA) revealed that LEA induced significant IFN-gamma responses and lower IL-4 responses than mice immunized with FA. The results of the present study indicate that liposomes synergise the proliferation by the antigen incorporated in it and polarizes the response towards Th1 type of cytokine production. The immunoadjuvant and immunomodulation property of liposomes make it an efficient vehicle for effective immunotherapy.
The conventional immunotherapy used for treating allergic individuals may at times lead to varying degrees of anaphylactic reaction. Liposomes have been proposed as a vehicle for safe and effective allergen-specific immunotherapy as multiple injections of liposome-entrapped allergen (LEA) have been shown to reduce specific IgE response and induce specific IgG response in BALB/c mice.
To elucidate the effect of LEA on polarization of T-cell responses, its effect on the relative production of TH1/TH2 type cytokines (namely IL-2, IL-4 and IFN-gamma by commercially available ELISA kits and immunoglobulin profile as measured by ELISA) were studied. Histamine release on challenge of immunized mice was measured to examine the efficacy of LEA in preventing anaphylactic reactions.
Measurement of cytokine levels in serum and spleen cell culture supernatants of BALB/c mice injected repeatedly with either free allergen (FA) or LEA (three mice per group) indicate that LEA preferentially induces a TH1-type of response dominated by IFN-gamma and IL-2 production. Further, it was also shown that immunization of mice with FA or LEA and subsequent challenge with a large dose of the sensitizing allergen leads to fatal systemic reactions in 50-65% of the animals treated with FA, whereas no mortality was observed in mice injected with LEA. Analysis of IgG subclasses in sera of mice immunized with LEA revealed a sixfold higher production of IgG1 antibodies than mice immunized with FA. Serum IgG2a, IgG2b, IgG3 and IgM responses were also enhanced in the group of mice immunized with LEA in comparison with mice injected with FA.
The results indicate that LEA confers protection against anaphylaxis to mice due to their ability to induce a high IFN-gamma:IL-4 ratio which may lead to decreased synthesis of IgE and reduced histamine release on challenge with FA.
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