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Background: Specific IgG antibodies are frequently observed in food-allergic patients. However, the allergen-fraction specificity of IgG antibodies in relation to IgE antibodies is not well defined. Our aim was to determine the IgE and IgG antibody profile to major cow's milk and peanut antigen fractions in food-allergic patients and tolerant individuals.
Methods: Sera were collected from 10 patients allergic to cow's milk and 10 patients allergic to peanuts, as well as from 20 control subjects. Cow's milk and peanut proteins were migrated on SDS–PAGE and immunoblotted for IgE, IgG, and IgG4 antibodies. Food-specific IgE concentrations were measured by CAP System FEIA™, and IgG and IgG4 concentrations by ELISA.
Results:In food-allergic children, similar fraction-specific IgE, IgG, and IgG4 antibody-binding profiles to the major cow's milk or peanut antigens were found. In nonallergics, the presence of fraction-specific IgG antibodies was mostly dependent on regular ingestion of the food. The presence of specific antibody on immunoblots correlated with their quantitative measurement. The mean value for specific IgE in cow's milk-allergic patients was 450±1326 IU/ml, and 337±423 IU/ml in peanut allergic patients. Specific IgG antibody values in milk-allergic patients were not different (median OD 1.5, range 0.3–2.3) from controls (median OD 1, range 0.2–1.8). However, in peanut-allergic patients, IgG concentrations were significantly higher than in controls (OD 1.2 [0.5–1.3] vs 0.5 [0.3–0.7]; P<0.01).
Conclusions: Similar fraction-specific IgE and IgG antibody profiles in allergic individuals suggest a common switching trigger involving both isotypes. Intrinsic allergenicity might explain identical IgG antibody fraction specificity in nonallergics and in allergics. The presence of IgG antibodies in nonallergics was related to regular ingestion of the food.
Cow's milk and peanut are two of the most prevalent sources of allergens in IgE-mediated food allergy. The major antigens of cow's milk, caseins, β-lactoglobulin (BLG), and α-lactalbumin (ALA), have been well characterized ( 1), and their implication in clinical reactions has been established ( 2). Similarly, Ara h 1, Ara h 2, and Ara h 3 were found to be the major peanut allergens ( 3–5). Initial identification of these allergens was obtained by IgE antibody (Ab) immunoblotting, but only a few studies have examined IgG Abs in relation to fraction-specific IgE Abs. de Jong et al. found similar IgE and IgG Ab-binding patterns on peanut protein immunoblots with pooled sera from allergic patients ( 6). In a study on soy allergy, Burks et al. identified in eight patients a similar binding pattern of IgE and IgG Abs to two major antigens ( 7). Other investigators found similar results in wheat allergy ( 8). It has been speculated that IgG Abs may play a role in the pathogenesis of food allergy ( 9, 10). However, the presence of specific IgG Abs to a food regularly ingested may represent a normal response of the immune system ( 11–13).
Extensive knowledge has been acquired recently on the mechanisms involved in Ab production. Immunoglobulin isotype switching can result in different types of Abs, but with a similar antigen specificity. In mice, IL-4 has been found to promote predominantly IgG1 and IgE, while interferon-γ induces IgG2a Ab switching. In man, prolonged exposure to an antigen favors an IgG4 response ( 14). Other studies suggest a sequential isotype switching for antigen-specific Abs in experimental models ( 15–18). However, little is known about the presence of food-specific IgE and IgG Abs in man in relation to clinical reactivity and to food consumption. In this study, we investigated the antigen-fraction specificity of IgG Abs in relation to IgE Abs in milk- or peanut-allergic patients. Antibody-fraction specificity was also determined in nonallergic subjects, and food-specific IgG and IgE Ab values were compared in both groups.
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We examined the fraction specificity of different Ab isotypes to major peanut and milk allergens. Serum IgE Abs from most allergic patients did bind to the major allergenic fractions already described. Therefore, the study population corresponded to a representative sample of patients with IgE-mediated milk or peanut allergy. Ab-binding patterns were specific to each individual patient, and Ab binding to minor antigens was found in a few patients only. Furthermore, intraindividual analysis of IgG or IgG4 Ab-binding profiles to either milk or peanut fractions was similar to IgE ( Table 2).
Several studies have examined IgG and IgE antibody specificity to foods. Spuergin et al. foundsimilar epitope binding of αs1-casein by IgE and IgG Abs ( 22). Wheat- or soy-specific IgE and IgG Abs recognize the same antigenic fractions ( 7, 8). de Jong et al. found similar results with peanut-specific IgE and IgG immunoblots ( 6). However, the use of pooled sera did not allow these authors to analyze individual immunoblot profiles. In our study, the specificity of IgE and IgG Abs to similar fractions in individual peanut- or cow's milk-allergic patients suggests a common antigen-specific secretion trigger in both isotypes. Most of the allergics had avoided the food well before serum was obtained, and had specific IgG Abs. Thus, the presence of IgG in the sera of milk allergics cannot be attributed to an immunologic response due to a constant exposure to a food antigen. As previously shown in experimental models with human cells (15–18), sequential or cyclic isotype switching to IgG and IgE Abs triggered by IL-4 seems to be a more convincing explanation. It should be emphasized that the presence of IgG Abs to major allergenic fractions of peanut or cow's milk does not prove a pathogenic role. However, controversy continues in the literature ( 9, 10).
Immunoblots with identical IgG Ab profiles were found in tolerant children regularly ingesting cow's milk, while control subjects occasionally ingesting peanuts had low or no detectable specific IgG Abs. These results tend to confirm the hypothesis that the presence of IgG Abs in nonallergics is largely dependent on a regular ingestion of the food ( 11, 12). IgG4 was correlated to regular exposure to a food in a previous study ( 14). However, the low amount of peanut-specific total IgG found in nonallergics might explain undetectable peanut-specific IgG4. The role of regular exposure to a food in IgG Ab production was confirmed by the absence of cow's milk-specific IgG Abs in patients with cow's milk-induced enterocolitis syndrome, a non-IgE-mediated disease ( 23), who were on a milk-free diet. Levels of specific IgG Abs might also be influenced by early feedings, as children exposed early in life to cow's milk proteins have higher levels of specific IgG Abs ( 12, 13). Thus, no food-specific IgE Abs were detectable in nonallergics, suggesting an inhibiting stage for immunoglobulin between IgG and IgE switching. Several in vitro studies with human cells have shown inhibition of IgE switching by IFN-γ ( 24, 25). Furthermore, it has been shown that immunotherapy with Hymenoptera venom induces a Th1-type switch with increased secretion of IFN-γ ( 26), associated with a progressive decrease of specific IgE, but an increase of IgG4 levels ( 27). It remains unknown whether a similar cytokine dysbalance toward Th2 cytokines can explain the pathogenesis of food allergy. Interestingly, the intensity of IgG binding to different protein fractions on the blots from nonallergics did not correspond to the relative amount of the protein in the food; for example, BLG represents 9% of total cow's milk proteins, but most nonallergics had a strong signal for IgG Abs to the BLG fraction. Whether intrinsic allergenicity of specific fractions plays a role in the intensity of the immune response in nonallergics remains to be determined.
The IgG4 Ab subtype has been the focus of several studies on food allergy. Høst et al. found elevated BLG-specific IgG4 Abs in sera from cow's milk-allergic children ( 11), and Morgan et al. found elevated specific IgG4 Abs in shrimp-allergic patients ( 28). James et al. followed IgG4 and IgE Ab titers in patients “growing out” of their sensitivity to cow's milk. They found lower IgE/IgG4 and IgE/IgG1 Ab ratios in patients becoming tolerant ( 29). In our study, most patients with food-specific IgG Abs also had IgG4 Abs, confirming that the sequential Ab switching mechanism to IgE includes IgG4.
In summary, most food-allergic patients had specific IgG Ab and IgE Abs to a similar fraction. These findings suggest an IgE switching mechanism, including IgG production, in cow's milk- and peanut-allergic individuals. The presence of food-specific IgG Abs in tolerant controls can be correlated with the regular ingestion of a food. In nonallergics, specific IgG Abs are found to the same major allergenic fractions, but in the absence of IgE Abs. Whether food allergy results from a defective regulatory mechanism with decreased IFN-γ levels remains to be determined.