The Effect of the Food Matrix on In Vivo Immune Responses to Purified Peanut Allergens
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《毒物学科学杂志》
Department of Immunotoxicology, Institute for Risk Assessment Sciences, Utrecht University, Utrecht, The Netherlands
Department of Experimental Immunology, TNO Quality of Life, Zeist, The Netherlands
Department of Protein Technology, TNO Quality of Life, Zeist, The Netherlands
ABSTRACT
There is little knowledge about the factors that determine the allergenicity of food proteins. One aspect that remains to be elucidated is the effect of the food matrix on immune responses to food proteins. To study the intrinsic immunogenicity of allergens and the influence of the food matrix, purified peanut allergens (Ara h 1, Ara h 2, Ara h 3, or Ara h 6) and a whole peanut extract (PE) were tested in the popliteal lymph node assay (PLNA) and in an oral model of peanut hypersensitivity. In the PLNA, peanut proteins were injected into the hind footpad of BALB/c mice; in the oral exposure experiments C3H/HeOuJ mice were gavaged weekly with PE or allergens in the presence of cholera toxin (CT). Upon footpad injection, none of the allergens induced significant immune activation. In contrast, PE induced an increase in cell number, cytokine production, and activation of antigen-presenting cells. Furthermore, the presence of a food matrix enhanced the immune response to the individual allergens. Oral exposure to the purified allergens in the presence of CT induced specific IgE responses, irrespective of the presence of a food matrix. These results suggest that purified peanut allergens possess little intrinsic immune-stimulating capacity in contrast to a whole PE. Moreover, the data indicate that the food matrix can influence responses to individual proteins and, therefore, the food matrix must be taken into account when developing models for allergenic potential assessment.
Key Words: peanut allergens; food matrix; mouse model; immunogenicity.
INTRODUCTION
Food allergy has emerged as a major health problem in westernized countries, and its prevalence is increasing (Sicherer et al., 2003). Type I food allergy is characterized by the production of protein-specific serum IgE antibodies that can reside on mast cells. Upon re-exposure to the offending food, cross-linking of the IgE antibodies provokes degranulation of mast cells and release of mediators, leading to a variety of symptoms.
In the past decade, considerable research has been conducted to characterize the food proteins that are responsible for allergic reactions. Knowledge of the general properties of proteins in relationship to their allergenicity is needed to assess the allergic potential of novel (including genetically modified) foods and foods produced by new processing techniques (Ladics et al., 2003; Maleki, 2004; Metcalfe, 2003).
Foods are composed of proteins, carbohydrates, lipids, and micronutrients. Major food allergens, in general, are glycoproteins ranging from 10 to 70 kDa that are abundant in the food and that possess multiple IgE binding epitopes (Bannon, 2004). To be able to induce allergic sensitization, allergens have to be absorbed through the gut epithelial barrier in an immunologically intact form (Taylor et al., 1987). Accordingly, food allergens are generally thought to be resistant to food-processing methods and digestion (Astwood et al., 1996). However, several food allergens have been identified that are sensitive to these processes; in addition, many non-allergenic proteins are as resistant to degradation as allergenic proteins (Fu, 2002). At present, the sum of properties that renders a food protein allergenic is unknown.
One aspect of food allergens that remains to be elucidated is the influence of the food matrix on the immune responses to food proteins. It has been hypothesized that the food body, consisting of fats, carbohydrates, and other proteins, may affect allergenic potential of proteins (Teuber, 2002). For example, high allergenic foods such as tree nuts and peanuts have medium protein concentrations but high levels of fat (Lehrer et al., 2002). The fats may protect proteins during the digestion process, or they may influence the activation of immune cells, which may result in enhanced allergenicity of a protein. These considerations prompted us to investigate the contribution of the food matrix to immune responses against peanut allergens.
Peanut allergy accounts for the majority of severe food allergic reactions; it tends to be persistent, and trace amounts of peanut can induce allergic responses (Al-Muhsen et al., 2003). Several peanut proteins belonging to different families of seed storage proteins have been identified by immunoblotting as major or minor allergens, and seven of them have been characterized and officially termed Ara h 1–7 (Kleber-Janke et al., 1999). We have recently shown that allergen-specific antibody and cytokine responses can be determined after oral or ip exposure to a whole peanut extract (PE) (van Wijk et al., 2004).
To study the intrinsic immunogenicity and adjuvanticity of purified peanut allergens versus a whole peanut protein extract, and to reveal the effect of the food matrix, two different peanut protein extracts (with or without fat), several purified major peanut allergens (Ara h 1, Ara h 2, and Ara h 3), and one minor allergen (Ara h 6) were used in the current experiments.
To determine the intrinsic immune-stimulating capacity of the purified allergens compared to the whole peanut extract, the popliteal lymph node assay (PLNA), an established model to test immune stimulation was used. After injection of 1 mg PE or purified allergen, PLN cell numbers, cytokine production, and expression of costimulatory molecules (CD80, CD86, and CD54), a measure for antigen-presenting cell (APC) activation, were determined. Data derived from this study show an important role of the PE food matrix (irrespective of the presence of fat) in activating immune cells, thereby driving immune responses to peanut proteins.
The effect of the food matrix on IgE responses against peanut allergens was also examined, because the elicitation of specific IgE responses is one of the absolute requirements for food allergens to induce immediate hypersensitivity responses. This effect was studied in an established mouse model of peanut hypersensitivity, using the relevant oral route of exposure in combination with a mucosal adjuvant, cholera toxin (CT). All purified peanut allergens induced IgE-specific serum antibody responses with only little additional effect of the food matrix. From these studies we concluded that purified peanut allergens possess little intrinsic adjuvanticity in contrast to whole PE. Furthermore, the results suggest that the food matrix can influence responses to individual proteins.
MATERIALS AND METHODS
Mice.
Female, specific pathogen-free BALB/c mice (6 weeks of age) and C3H/HeOuJ Ico mice (4 weeks of age), bred on a SM R/M-Z CRL VRF1 diet (ssniff Spezialdiaten GmbH, Germany) were purchased from Charles River (Lyon, France). Mice were maintained under barrier conditions in filter-topped Macrolon cages with wood chip bedding, at mean temperature of 23° ± 2°C, relative humidity of 50–55%, and a 12-h light/dark cycle. Drinking water and standard laboratory food pellets (rat and mouse breeder and grower, Special Diet Services, Essex, England) were provided ad libitum. No peanut proteins were present in the diets. The experiments were approved by the animal experiments committee of the faculty of veterinary medicine, Utrecht University.
Chemicals and reagents.
Chemicals were obtained from Sigma Aldrich (Zwijndrecht, The Netherlands) unless stated otherwise. For cytokine measurements, IL-2, IL-4, and IFN- capture and detecting antibodies, and an IL-10 ELISA kit were obtained from BD Pharmingen (Hamburg, Germany); and streptavidin–horseradish peroxidase (HRP) was from Sanquin (Amsterdam, The Netherlands). BD Pharmingen was the supplier of rat anti-mouse IgE, and the Digoxigenin (DIG)-coupling kit was obtained from Boehringer Mannheim (Mannheim, Germany); anti-DIG-peroxidase Fab fragments were from Roche Diagnostics (Mannheim, Germany). Alkaline phosphatase (AP)-conjugated goat anti-mouse, human adsorbed IgG1 and IgG2a were purchased from Southern Biotechnology Associates (Birmingham, AL). Campina Melkuni (Eindhoven, The Netherlands) was the supplier of milk powder used in blocking buffer. Medium used for the in vitro restimulation was complete RPMI 1640 with Glutamax-I (Life Technologies, Paisley, Scotland) supplemented with 10% fetal calf serum (ICN Pharmaceuticals, Costa Mesa, CA) and 2% penicillin-streptomycin.
Preparation of peanut extract and purification of allergens.
Peanuts from the Runner cultivar (Cargill, Dawson, GA) were kindly provided by Imko Nut Products (Doetinchem, The Netherlands). The PE was prepared as described by Koppelman et al. (2003). Briefly, peanuts were ground and the protein was extracted by mixing 25 g ground peanut with 200 ml 20 mM Tris buffer (pH 7.2). After this mixture was stirred for 2 h at room temperature, the aqueous fraction was collected by centrifugation (3000 x g for 30 min) and subsequently centrifuged at 10,000 g for 30 min to remove residual traces of fat and insoluble particles. The extract consisted of 4.3% protein, 0.2% fat, 0.5% carbohydrates, 0.2% ash, and 94.8% water, as determined by Kjehdahl analysis and acid hydrolysis. For the preparation of fatty PE (PE fat), fat was not removed after centrifugation, resulting in a PE–oil emulsion consisting of 3.6% protein, 3.5% fat, 0.4% carbohydrates, 0.2% ash, and 92.3% water. Protein concentration (PE 30 mg/ml and PE fat 25 mg/ml) was determined with a BCA protein assay kit (Pierce, Rockford, IL). Sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of PE and PE fat confirmed the presence of proteins migrating at the same molecular weights as the purified allergens Ara h 1 (14%), Ara h 2 (6.5%), Ara h 3 (50%), and Ara h 6 (4.5%), and the two extracts showed a similar protein pattern (data not shown). Their relative amounts (expressed as percentage of total protein) were quantified by densitometry as previously described (Koppelman et al., 2001). Purification of the allergens Ara h 1, Ara h 2, and Ara h 3 was performed according to previously described methods (de Jong et al., 1998; Koppelman et al., 2003), and Ara h 6 was similarly purified from a side-fraction. Endogenous endotoxin content was measured by Limulus Amebocyte Lysate assay (Bio Whittaker, Walkersville, MD) and was <0.02 μg LPS/mg protein (200 EU/mg protein) for all purified allergen and PE solutions.
PLNA protocol.
For the PLNA, a standard protocol was used as previously described (Bloksma et al., 1995). Pilot studies were performed to determine the optimal concentration of PE that also served as a positive control. Different doses of purified allergens (0.1 mg, 0.5 mg, and 1 mg) were tested, and no differences were found between the different dosing groups for all parameters measured. The findings for the 1-mg dose group are presented in the Results. Accordingly, BALB/c mice (n = 4) were subcutaneously injected into the hind footpad with 1 mg of protein (PE, PE fat, Ara h 1, Ara h 2, or Ara h 6) dissolved in 50 μl sterile NaCl, and control animals were injected with 50 μl vehicle. Ara h 3 could not be dissolved at this concentration and was therefore not included in the PLNA. Seven days after protein injection, mice were killed by cervical dislocation, and the draining popliteal lymph nodes (PLN) were excised. Popliteal lymph node single-cell suspensions were prepared under aseptic conditions in complete RPMI 1640 and adjusted to 2.5106 cells/ml.
Oral treatment protocol.
C3H/HeJ mice (n = 6) were orally exposed to the purified allergens Ara h 1, Ara h 2, Ara h 3, Ara h 6 (0.1 and 1 mg), PE (1 and 6 mg), or PE fat (6 mg) in the presence of the mucosal adjuvant CT. Oral exposure was performed by intragastric dosing of protein on 3 consecutive days, followed by weekly dosing (4 weeks). Cholera toxin (10 μg) was coadministered on days 1, 2, 3, 8, 15, and 21. At day 30, mice
Cell culture and cytokine measurement.
Popliteal lymph node single-cell suspensions (150 μl of 2.5106 cells/ml in complete RPMI 1640) were incubated in the presence of PE (200 μg/ml), Ara h 1 (200 μg/ml), Ara h 2 (200 μg/ml), Ara h 6 (200 μg/ml), Con A (5 μg/ml), or medium alone in 96-well plates (Costar, Cambridge, MA) for 96 h (Con A 24 h) at 37°C, 5% CO2. After centrifugation for 10 min at 150 x g, supernatant was collected and stored at –20°C until analysis.
In the culture supernatants, IL-2, IFN-, IL-4, and IL-10 levels were determined by sandwich ELISA. Plates (highbond 3590) were coated overnight with 1 μg/ml rat anti-mouse IL-2, IL-4, or IFN-, and the following day plates were blocked with PBS-Tween/3% milk powder for 4 h at room temperature. Samples and cytokine standards were added in several dilutions and incubated overnight at 4°C. Plates were incubated with 0.25 μg/ml rat anti-mouse IL-2, IL-4, or IFN- conjugate for 1 h at room temperature, followed by streptavidin-HRP incubation for 45 min. Finally, tetramethylbezidine(TMB)-substrate (0.1 mg/ml) was added, and the color reaction was stopped with 2 M H2SO4. Absorbance was measured at 450 nm. The IL-10 ELISA was performed in accordance with the manufacturer's instructions.
Flow cytometry.
For flow cytometric analysis, 2105 PLN cells were incubated with 50 μl of predetermined dilutions of fluorescein isothiocyanate (FITC)-, CyChrome (CY)-, and biotin-conjugated monoclonal antibodies in 96-well plates (30 min darkness at 4°C). Cells were characterized based on the following monoclonal antibodies: CD54 FITC (3E2), CD80 FITC (16–10A1), CD86 FITC (GL1), isotype controls for CD80 (hamster IgG2 FITC), and CD86 (Rat IgG2a FITC) and MHC-II biotin (NIMR-4), all obtained from BD Pharmingen. After incubation with biotin-conjugated monoclonal antibodies, cells were washed and incubated with streptavidin-CY (BD Pharmingen) in the same way. Finally, cells were washed, stored in formalin (0.1%), and analyzed within 18 h on a FACScan with standard FACSflow using CellQuest software (BD Biosciences, Franklin Lakes, NJ). Cells expressing CD80, CD86, or CD54 were checked for MHC-II expression to assure antigen-presenting capability.
Measurement of serum IgE antibodies.
Blood of orally exposed mice was collected at weekly intervals, and levels of PE and Ara h–specific IgE were measured by sandwich ELISA. Plates (highbond 3590; Costar, Cambridge, MA) were coated overnight with 1.5 μg/ml purified rat anti-mouse IgE in carbonate buffer (pH 9.6), followed by 1 h of blocking (37°C) with PBS-Tween/3% milk powder. Each test serum was titrated starting at 1:8 dilution and incubated for 2 h (37°C). A pre-sera pool was used as reference value (dilution 1:4). Subsequently a PE- or Ara h-DIG conjugate solution was added (1 h at 37°C). The coupling of DIG to PE or the Ara h's at an optimal labeling efficiency was performed according to the manufacturer's instructions. Briefly, the coupled proteins were separated on a Sephadex G-25 column, and the concentration of coupled protein was determined spectrophotometrically at 280 nm. After incubation (1 h at 37°C) with peroxidase-conjugated anti-DIG fragments, a TMB-substrate (0.1 mg/ml) solution was used, and the color reaction was stopped with 2 M H2SO4. Absorbance was measured at 450 nm using an ELISA reader ELX800 (BIOTEK Instruments, Winooski, VT). The reciprocal of the furthest test serum dilution resulting in an extinction higher than the reference value was read as a titer. Background values were <0.100 (OD).
Measurement of serum IgG1 and IgG2a antibodies.
Blood was collected at weekly intervals, and levels of PE-specific antibodies were measured by ELISA. Plates (highbond 3590; Costar, Cambridge, MA) were coated overnight with 20 μg/ml PE in carbonate buffer (pH 9.6), followed by 1 h of blocking (37°C) with PBS-Tween/3% milk powder. Each test serum was titrated starting at 1:8 dilution and incubated for 1 h (37°C). A pre-sera pool was used as reference value (dilution 1:4). After incubation, AP-conjugated antibodies were added (1 h at 37°C). Subsequently 1 mg/ml p-nitrophenylphosphate in diethanolamine buffer was used for the color reaction, which was stopped with a 10% EDTA solution, after which absorbance was measured at 405 nm using an ELISA reader ELX800 (BIOTEK Instruments, Winooski, VT). Background values were <0.100 (OD).
Statistics.
Multiple comparisons of group means were performed using one-way analyses of variance (ANOVA) with Bonferroni as a post hoc test. For PLN cell numbers, cytokine levels, and serum antibody levels, statistical analysis was performed following logarithmic transformation (to achieve normal distribution), whereas the levels of cell surface molecules were untransformed. A value of p < 0.05 was considered statistically significant.
RESULTS
Peanut Extract, But Not Purified Allergen, Induces PLN Cell Proliferation
To examine the intrinsic immune stimulating capacity and adjuvanticity of purified peanut allergens versus whole PE, the PLNA was used. Mice were injected with 1 mg of protein in the hind foodpad, and at day 7 total cell numbers of the draining PLN were determined (Fig. 1). After injection with PE fat or PE, total lymph node cell numbers increased significantly (fourfold), and no difference was found between the PE fat- and PE-treated groups. In contrast, no cell proliferation was observed after injection with the purified peanut allergens Ara h 1, Ara h 2, or Ara h 6.
Purified Allergen Treatment Fails to Induce Cytokine Production in Con A Restimulated PLN Cell Cultures
To reflect the general activation of T cells, PLN cells were cultured for 24 h in the presence of concanavalin A (Con A), and the concentrations of the Th2-associated cytokines IL-4 and IL-10, and of the Th1-associated cytokine IFN-, were measured in the supernatant (Fig. 2). Popliteal lymph node cells derived from PE- and PE fat-treated mice secreted similar high amounts of IL-4, IL-10, and IFN- compared to cells derived from control mice. No Th2 or Th1 bias was observed in either of the PE treatment groups.
With respect to the purified allergens, no significant IL-10 or IFN- cytokine production was found in PLN cell cultures of Ara h 1-, Ara h 2-, and Ara h 6-treated groups, and only Ara h 6-injection resulted in significant IL-4 production upon restimulation with Con A.
Cytokine Responses Induced by Restimulation with Purified Allergen Are Enhanced by the Presence of a Food Matrix at the Time of Injection
To examine antigen-specific T cell responses, PLN cells were obtained 7 days after the initial injection with 1 mg of PE, PE fat, or purified allergen and cultured for 96 h in the presence of PE, Ara h 1, Ara h 2, Ara h 6, or medium alone. Th1-associated (IL-2 and IFN-) and Th2-associated (IL-4 and IL-10) cytokine production was measured in the supernatant (Table 1).
Popliteal lymph node LN cell cultures derived from PE- and PE fat-treated mice restimulated with PE induced high levels of IL-2, IL-4, IL-10, and IFN-. No significant differences in cytokine concentration were found between the two groups, and a mixed Th1/Th2 cytokine response was observed.
Regarding the purified allergens, only Ara h 6 treatment resulted in a low but significantly increased cytokine production (IL-2, IL-4, IL-10, but not IFN-), whereas Ara h 1-and Ara h 2-injection failed to induce considerable cytokine production in PLN cell cultures when restimulated with the corresponding purified allergen.
Interestingly, cells derived from PE-treated or PE-fat–treated groups secreted higher levels of cytokines than cells derived from Ara h 1- and Ara h 6-treated animals when restimulated with Ara h 1 or Ara h 6. Restimulation with Ara h 2 only induced low IL-2 production, and levels of IL-2 were significantly higher in cell cultures of PE-treated and PE-fat–treated mice than in those of Ara h 2-treated mice. Popliteal lymph node cells cultured in the presence of medium alone showed no production of any of the cytokines examined (data not shown). These results indicate that T cell responses to purified allergens can be influenced by the food matrix.
Peanut Extracts But Not Purified Allergen Induces Expression of Co-stimulatory Molecules on APC
Activation of antigen-presenting cells (APC) is considered one of the requirements for initiating active immune responses. Therefore, the expressions of the activation markers CD80, CD86, and CD54 were determined on PLN APC, 7 days after protein injection (Fig. 3). Both PE fat and PE injection significantly augmented the percentages of CD80-, CD86-, and CD54-expressing cells in the PLN, whereas no increase was found after injection with Ara h 1, Ara h 2, or Ara h 6.
Together, these data indicate that purified peanut allergens, in contrast to the whole extract, lack intrinsic immune-stimulating capacity and thereby fail to induce significant immune responses in the PLNA.
Purified Allergens Induce Specific IgE Responses upon Oral Exposure, Irrespective of the Food Matrix
To further explore the role of the food matrix in immune responses to peanut proteins, an established oral model of peanut hypersensitivity was used. In this model, PE- and allergen-specific IgE responses can be determined using the relevant route of exposure to food proteins (van Wijk et al., 2004). Mice were dosed by gavage for 4 weeks with PE (1 and 6 mg), Ara h 1, Ara h 2, Ara h 3, or Ara h 6 (0.1 and 1 mg) in the presence of the mucosal adjuvant CT.
All purified allergens were able to induce specific IgE responses, but only for Ara h 1 treatment was a significant difference found between the 0.1 and 1 mg dosing groups (Fig. 4). Exposure to PE (1 mg or 6 mg) also resulted in Ara h-specific IgE responses, with levels comparable to those observed after purified allergen exposure. In the control group (oral exposure to PBS + CT) no Ara h-specific IgE responses were found (data not shown).
No Difference in Antibody Responses upon Oral Exposure to PE or PE Fat
To examine the effect of fat content on orally induced antibody responses to PE, mice were orally exposed to PE fat or PE in the presence or absence of the mucosal adjuvant CT. After 4 weeks of oral exposure, both Th1-associated (IgG2a) and Th2-associated (IgG1 and IgE) PE-specific antibodies were determined in the serum (Fig. 5). In the absence of CT, only low levels of IgG1 and IgG2a, and no IgE, were found, with no differences between the PE fat- and PE-treated group. In the presence of CT, similar high levels of IgG2a, IgG1, and IgE were found in both PE treatment groups. In line with the results found in the PLN, the presence of fat does not affect orally induced immune responses to PE.
DISCUSSION
No definitive characteristics of food allergens have been identified other than that they need to reach and stimulate immune cells in order to induce sensitization. It has been suggested that both properties may be influenced by the food matrix (Teuber, 2002). In the present study we have shown that purified peanut allergens, unlike a whole PE, possess little intrinsic immune-stimulating capacity and that the immune response to these allergens can be adjuvated by the presence of a food matrix. Furthermore, it was demonstrated that the purified allergens are stable enough to get into contact with the immune system after oral exposure, both in the presence and in the absence of a food matrix.
Although Ara h 1 and Ara h 2 are recognized as major peanut allergens, they were unable to induce PLN cell proliferation or cytokine production, suggesting that they possess little intrinsic immune-stimulating capacity. In contrast, PE induced a strong PLNA response (without causing inflammation in the paw) and was also able to adjuvate specific T cell responses against the allergens present in the extract. In line with these results, Ara h 1 and Ara h 2 appear to be poor IgG and IgE inducers in mice when injected ip (G. Ladics, Dupont, personal communication), whereas ip PE injection results in considerable Ara h 1–specific and Ara h 2–specific IgG and IgE responses (van Wijk et al., 2004).
The actual amount of individual allergens in PE and PE fat was always lower than the 1 mg of allergen injected in a purified form. This may raise the issue that the discrepancy in dose has affected the outcome of the immune response and that the high dose injection (1 mg) has induced non-responsiveness. However, injection of 0.1 and 0.5 mg of purified allergen also failed to induce an immune response in the PLNA (data not shown).
The difference in immune-stimulating capacity between the purified allergens and the extract may be explained by selective activation of APC. Generally considered, APC activation and the accompanying upregulation of costimulatory molecules is required for the induction of an active immune response. For contact allergens, which possess high intrinsic adjuvanticity due to their reactive groups (Kapsenberg, 1996), it has been shown that they selectively upregulate costimulatory molecules (e.g., CD54 and CD86) on dendritic cells in vitro (Rougier et al., 2000; Tuschl et al., 2000). Furthermore, drugs associated with immune-mediated hypersensitivity reactions were demonstrated to upregulate the expression of costimulatory molecules on APC in the PLNA (Nierkens et al., 2002; Popovic et al., 2004). Interestingly, none of the purified peanut allergens tested was able to increase expressions of costimulatory molecules on APC, whereas PE injection resulted in an upregulation of CD80, CD86, and CD54. Hence, in contrast to sensitizing chemicals, soluble peanut allergens do not possess intrinsic adjuvanticity, and they therefore need an accompanying adjuvant (in the case of PE provided by the food matrix) to be able to activate APC and to induce subsequent immune stimulation.
Peanut consists of proteins, carbohydrates and fatty acids, and all these different components—and their interactions—may be responsible for the adjuvant effect. For instance, it has been described that peanut oils can have an adjuvant effect in a vaccine model (Eghafona, 1996). Remarkably, in the PLNA, the presence of fat did not seem to play a major role in the induction of the immune response (PLN cell numbers, cytokine production, and APC activation). In addition to nutrients, non-nutritive compounds such as lectins (Watzl et al., 2001) and food contaminants such as LPS (Brix et al., 2004) and aflatoxins (Kocabas and Sekerel, 2003) may affect immune responses to food proteins. For future experiments, it would be interesting to test different PE components separately in order to elucidate their immune-modulating properties.
Concerning the relevant route of exposure, the food matrix has been suggested to affect the allergenic properties of orally administered proteins by providing adjuvant stimuli to the specialized gut mucosal immune system or by protecting them from digestion (Lehrer et al., 2002; Teuber, 2002), but to our knowledge no in vivo data are available. In general, oral administration (by feeding or gavage) of soluble food proteins to mice results in peripheral hyporesponsiveness (reviewed by Mayer et al., 2001; Nagler-Anderson et al., 2001) Administration of proteins with adjuvants induces a productive immune response to this normally tolerogenic form of antigen (Nagler-Anderson et al., 2001).We have previously shown that, in the present oral model, coadministration of the mucosal adjuvant CT is indispensable to the induction of antigen-specific IgE responses. Clearly, the intrinsic adjuvant activity of PE (i.e., without fat) is not sufficient to induce sensitization via the oral route (van Wijk et al., 2005). Consequently, it might be impossible to determine the adjuvant activity of the food matrix itself when CT is coadministered. Yet, in the current study, the presence of fat did not affect the magnitude or polarization (type 1 versus type 2) of the antibody responses against PE, irrespective of the presence of CT (Fig. 5). Fatty acids have been described as immune modulators following high-dose oral supplementation (Harbige and Fisher, 2001; Wu 2004), covalent coupling to a protein or peptide (Oliveira et al., 2002; BenMohamed et al., 2002), or the use of liposome-entrapped proteins (Sehra et al., 1998), but it remains to be elucidated whether fats present in a normal diet have similar immune-modulating properties.
Regarding the aspect of digestion, the present results indicate that peanut allergens are at least stable enough to reach the gut-associated lymphoid tissue, even without the presence of a protecting food matrix. Although it has been shown in vitro that polysaccharide–protein and protein–protein interactions may influence allergen digestion (Fremont et al., 2004; Maleki et al., 2003), these interactions seemed not to affect the immune response to allergens in the present in vivo model. Our data are in accordance with in vitro data showing that Ara h 1 and Ara h 2 are highly resistant to enzymatic digestion (Maleki et al., 2000; Sen et al., 2002). Furthermore, treatment of purified Ara h 1 and PE with pepsin resulted in the production of nearly identical sets of digestion products, suggesting an intrinsic stability of Ara h 1 (Kopper et al., 2004). The current results demonstrate that the major peanut allergens Ara h 1, Ara h 2, Ara h 3, and the minor allergen Ara h 6 are stable enough in vivo to elicit specific IgE responses.
In recent years there has been considerable interest in the development of experimental animal models to predict the allergenicity of proteins. Our data suggest that assays designed to measure immune stimulation, such as the PLNA, are clearly not suitable to identify allergenic proteins when injected in a purified form, because they do not appear to possess high intrinsic immune-stimulating capacity. Because PE injection did result in immune stimulation, further studies may reveal the feasibility of subcutaneous injection with whole food extract as a tool for identifying the adjuvating capacity of compounds within a food matrix.
After oral exposure, all purified peanut allergens were shown to be IgE inducers, which confirms that they posses IgE epitopes, and that they are able to reach the immune system of the gut in a relatively intact form, two qualities required for the induction of allergic sensitization. However, for the future use of this model as a screening tool, other allergens and especially non-allergens need to be tested. Recently, Gaudry et al. (2004) have examined several food allergens and non-allergens in a similar oral model and have shown that mice and humans respond similarly to major allergens and non-allergens. Although a broader panel of (non)allergens should be tested and the use of an adjuvant remains a topic of debate, the measurement of IgE antibody responses following oral exposure to proteins seems to be a promising tool for predicting allergenicity of these proteins. Furthermore, the oral route represents the most adequate route of exposure to study possible in vivo digestion and food matrix effects.
In summary, our results indicate that purified peanut allergens possess little intrinsic immune-stimulating capacity and that they probably need co-localization of an adjuvant compound to induce sensitization. Moreover, this study shows that the food matrix may affect immune responses to individual allergens; as a consequence, a possible influence of the food matrix has to be considered in the development of animal tests to predict allergenic potential.
REFERENCES
Al-Muhsen, S., Clarke, A. E., and Kagan, R. S. (2003). Peanut allergy: An overview. Can. Med. Assoc. J. 168, 1279–1285.
Astwood, J. D., Leach, J. N., and Fuchs, R. L. (1996). Stability of food allergens to digestion in vitro. Nat. Biotechnol. 14, 1269–1273.
Bannon, G. A. (2004). What makes a food protein an allergen Curr. Allergy Asthma Rep. 4, 43–46.
BenMohamed, L., Belkaid, Y., Loing, E., Brahimi, K., Gras-Masse, H., and Druilhe, P. (2002). Systemic immune responses induced by mucosal administration of lipopeptides without adjuvant. Eur. J. Immunol. 32, 2274–2281.
Bloksma, N., Kubicka-Muranyi, M., Schuppe, H. C., Gleichmann, E., and Gleichmann, H. (1995). Predictive immunotoxicological test systems: Suitability of the popliteal lymph node assay in mice and rats. Crit. Rev. Toxicol. 25, 369–396.
Brix, S., Kjaer, T. M., Barkholt, V., and Frokiaer, H. (2004). Lipopolysaccharide contamination of beta-lactoglobulin affects the immune response against intraperitoneally and orally administered antigen. Int. Arch. Allergy Immunol. 135, 216–220.
de Jong, E. C., Van Zijverden, M., Spanhaak, S., Koppelman, S. J., Pellegrom, H., and Penninks, A. H. (1998). Identification and partial characterization of multiple major allergens in peanut proteins. Clin. Exp. Allergy 28, 743–751.
Eghafona, N. O. (1996). Immune responses following cocktails of inactivated measles vaccine and Arachis hypogaea L. (groundnut) or Cocos nucifera L. (coconut) oils adjuvant. Vaccine 14, 1703–1706.
Fremont, S., Sanchez, C., Errahali, Y., Mouecoucou, J., Metche, M., Mejean, L., and Nicolas, J. P. (2004). [Food allergy: effect of proteins-lipids and proteins-polysaccharides interactions]. Allerg. Immunol. (Paris) 36, 82–87.
Fu, T. J. (2002). Digestion stability as a criterion for protein allergenicity assessment. Ann. N. Y. Acad. Sci. 964, 99–110.
Gaudry, P. A., Fernandes, J., Reish, R., Roux, K. H., Sathe, S. K., Teuber, S. S., Reese, G., and Lehrer, S. B. (2004). Murine IgE and IgG antibody responses to allergenic and non-allergenic foods: Potential model for allergenicity assessment of foods in man. J. Allergy Clin. Immunol. 113, 832 (Abstract).
Harbige, L. S., and Fisher, B. A. (2001). Dietary fatty acid modulation of mucosally-induced tolerogenic immune responses. Proc. Nutr. Soc. 60, 449–456.
Kapsenberg, M. L. (1996). Chemicals and proteins as allergens and adjuvants. Toxicol. Lett. 86, 79–83.
Kleber-Janke, T., Crameri, R., Appenzeller, U., Schlaak, M., and Becker, W. M. (1999). Selective cloning of peanut allergens, including profilin and 2S albumins, by phage display technology. Int. Arch. Allergy Immunol. 119, 265–274.
Kocabas, C. N., and Sekerel, B. E. (2003). Does systemic exposure to aflatoxin B(1) cause allergic sensitization Allergy 58, 363–365.
Koppelman, S. J., Knol, E. F., Vlooswijk, R. A., Wensing, M., Knulst, A. C., Hefle, S. L., Gruppen, H., and Piersma, S. (2003). Peanut allergen Ara h 3: Isolation from peanuts and biochemical characterization. Allergy 58, 1144–1151.
Koppelman, S. J., Vlooswijk, R. A., Knippels, L. M., Hessing, M., Knol, E. F., van Reijsen, F. C., and Bruijnzeel-Koomen, C. A. (2001). Quantification of major peanut allergens Ara h 1 and Ara h 2 in the peanut varieties Runner, Spanish, Virginia, and Valencia, bred in different parts of the world. Allergy 56, 132–137.
Kopper, R. A., Odum, N. J., Sen, M., Helm, R. M., Steve Stanley, J., and Wesley Burks, A. (2004). Peanut protein allergens: Gastric digestion is carried out exclusively by pepsin. J. Allergy Clin. Immunol. 114, 614–618.
Ladics, G. S., Holsapple, M. P., Astwood, J. D., Kimber, I., Knippels, L. M., Helm, R. M., and Dong, W. (2003). Workshop overview: Approaches to the assessment of the allergenic potential of food from genetically modified crops. Toxicol. Sci. 73, 8–16.
Lehrer, S. B., Ayuso, R., and Reese, G. (2002). Current understanding of food allergens. Ann. N. Y. Acad. Sci. 964, 69–85.
Maleki, S. J. (2004). Food processing: Effects on allergenicity. Curr. Opin. Allergy Clin. Immunol. 4, 241–245.
Maleki, S. J., Kopper, R. A., Shin, D. S., Park, C. W., Compadre, C. M., Sampson, H., Burks, A. W., and Bannon, G. A. (2000). Structure of the major peanut allergen Ara h 1 may protect IgE-binding epitopes from degradation. J. Immunol. 164, 5844–5849.
Maleki, S. J., Viquez, O., Jacks, T., Dodo, H., Champagne, E. T., Chung, S. Y., and Landry, S. J. (2003). The major peanut allergen, Ara h 2, functions as a trypsin inhibitor, and roasting enhances this function. J. Allergy Clin. Immunol. 112, 190–195.
Mayer, L., Sperber, K., Chan, L., Child, J., and Toy, L. (2001). Oral tolerance to protein antigens. Allergy 56, 12–15.
Metcalfe, D. D. (2003). Introduction: What are the issues in addressing the allergenic potential of genetically modified foods Environ. Health Perspect. 111, 1110–1113.
Nagler-Anderson, C., and Shi, H. N. (2001). Peripheral nonresponsiveness to orally administered soluble protein antigens. Crit. Rev. Immunol. 21, 121–131.
Nierkens, S., van Helden, P., Bol, M., Bleumink, R., van Kooten, P., Ramdien-Murli, S., Boon, L., and Pieters, R. (2002). Selective requirement for CD40-CD154 in drug-induced type 1 versus type 2 responses to trinitrophenyl-ovalbumin. J. Immunol. 168, 3747–3754.
Oliveira, F. M., Santos, E. M., Mota-Santos, T. A., Ruiz-De-Souza, V., and Gontijo, C. M. (2002). Covalent coupling of palmitate to ovalbumin inhibits and blocks the induction of oral tolerance. Scand. J. Immunol. 55, 570–576.
Popovic, M., Nierkens, S., Pieters, R., and Uetrecht, J. (2004). Investigating the role of 2-phenylpropenal in felbamate-induced idiosyncratic drug reactions. Chem. Res. Toxicol. 17, 1568–1576.
Rougier, N., Redziniak, G., Mougin, D., Schmitt, D., and Vincent, C. (2000). In vitro evaluation of the sensitization potential of weak contact allergens using Langerhans-like dendritic cells and autologous T cells. Toxicology 145, 73–82.
Sehra, S., Chugh, L., and Gangal, S. V. (1998). Polarized TH1 responses by liposome-entrapped allergen and its potential in immunotherapy of allergic disorders. Clin. Exp. Allergy 28, 1530–1537.
Sen, M., Kopper, R., Pons, L., Abraham, E. C., Burks, A. W., and Bannon, G. A. (2002). Protein structure plays a critical role in peanut allergen stability and may determine immunodominant IgE-binding epitopes. J. Immunol. 169, 882–887.
Sicherer, S. H., Munoz-Furlong, A., and Sampson, H. A. (2003). Prevalence of peanut and tree nut allergy in the United States determined by means of a random digit dial telephone survey: A 5-year follow-up study. J. Allergy Clin. Immunol. 112, 1203–1207.
Taylor, S. L., Lemanske, R. F., Jr., Bush, R. K., and Busse, W. W. (1987). Food allergens: Structure and immunologic properties. Ann. Allergy 59, 93–99.
Teuber, S. S. (2002). Hypothesis: The protein body effect and other aspects of food matrix effects. Ann. N. Y. Acad. Sci. 964, 111–116.
Tuschl, H., Kovac, R., and Weber, E. (2000). The expression of surface markers on dendritic cells as indicators for the sensitizing potential of chemicals. Toxicol. In Vitro 14, 541–549.
Watzl, B., Neudecker, C., Hansch, G. M., Rechkemmer, G., and Pool-Zobel, B. L. (2001). Dietary wheat germ agglutinin modulates ovalbumin-induced immune responses in Brown Norway rats. Br. J. Nutr. 85, 483–490.
van Wijk, F., Hartgring, S., Koppelman, S. J., Pieters, R., and Knippels, L. M. J. (2004). Mixed antibody and T cell responses to peanut and the peanut allergens Ara h1, Ara h3 and Ara h6 in an oral sensitization model. Clin. Exp. Allergy 34, 1422–1428.
van Wijk, F., Hoeks, S., Nierkens, S., Koppelman, S. J., van Kooten, P., Boon, L., Knippels, L. M., and Pieters, R. (2005). CTLA-4 signaling regulates the intensity of hypersensitivity responses to food antigens, but is not decisive in the induction of sensitization. J. Immunol. 174, 174–179.
Wu, D. (2004). Modulation of immune and inflammatory responses by dietary lipids. Curr. Opin. Lipidol. 15, 43–47.(F. van Wijk, S. Nierkens,)
Department of Experimental Immunology, TNO Quality of Life, Zeist, The Netherlands
Department of Protein Technology, TNO Quality of Life, Zeist, The Netherlands
ABSTRACT
There is little knowledge about the factors that determine the allergenicity of food proteins. One aspect that remains to be elucidated is the effect of the food matrix on immune responses to food proteins. To study the intrinsic immunogenicity of allergens and the influence of the food matrix, purified peanut allergens (Ara h 1, Ara h 2, Ara h 3, or Ara h 6) and a whole peanut extract (PE) were tested in the popliteal lymph node assay (PLNA) and in an oral model of peanut hypersensitivity. In the PLNA, peanut proteins were injected into the hind footpad of BALB/c mice; in the oral exposure experiments C3H/HeOuJ mice were gavaged weekly with PE or allergens in the presence of cholera toxin (CT). Upon footpad injection, none of the allergens induced significant immune activation. In contrast, PE induced an increase in cell number, cytokine production, and activation of antigen-presenting cells. Furthermore, the presence of a food matrix enhanced the immune response to the individual allergens. Oral exposure to the purified allergens in the presence of CT induced specific IgE responses, irrespective of the presence of a food matrix. These results suggest that purified peanut allergens possess little intrinsic immune-stimulating capacity in contrast to a whole PE. Moreover, the data indicate that the food matrix can influence responses to individual proteins and, therefore, the food matrix must be taken into account when developing models for allergenic potential assessment.
Key Words: peanut allergens; food matrix; mouse model; immunogenicity.
INTRODUCTION
Food allergy has emerged as a major health problem in westernized countries, and its prevalence is increasing (Sicherer et al., 2003). Type I food allergy is characterized by the production of protein-specific serum IgE antibodies that can reside on mast cells. Upon re-exposure to the offending food, cross-linking of the IgE antibodies provokes degranulation of mast cells and release of mediators, leading to a variety of symptoms.
In the past decade, considerable research has been conducted to characterize the food proteins that are responsible for allergic reactions. Knowledge of the general properties of proteins in relationship to their allergenicity is needed to assess the allergic potential of novel (including genetically modified) foods and foods produced by new processing techniques (Ladics et al., 2003; Maleki, 2004; Metcalfe, 2003).
Foods are composed of proteins, carbohydrates, lipids, and micronutrients. Major food allergens, in general, are glycoproteins ranging from 10 to 70 kDa that are abundant in the food and that possess multiple IgE binding epitopes (Bannon, 2004). To be able to induce allergic sensitization, allergens have to be absorbed through the gut epithelial barrier in an immunologically intact form (Taylor et al., 1987). Accordingly, food allergens are generally thought to be resistant to food-processing methods and digestion (Astwood et al., 1996). However, several food allergens have been identified that are sensitive to these processes; in addition, many non-allergenic proteins are as resistant to degradation as allergenic proteins (Fu, 2002). At present, the sum of properties that renders a food protein allergenic is unknown.
One aspect of food allergens that remains to be elucidated is the influence of the food matrix on the immune responses to food proteins. It has been hypothesized that the food body, consisting of fats, carbohydrates, and other proteins, may affect allergenic potential of proteins (Teuber, 2002). For example, high allergenic foods such as tree nuts and peanuts have medium protein concentrations but high levels of fat (Lehrer et al., 2002). The fats may protect proteins during the digestion process, or they may influence the activation of immune cells, which may result in enhanced allergenicity of a protein. These considerations prompted us to investigate the contribution of the food matrix to immune responses against peanut allergens.
Peanut allergy accounts for the majority of severe food allergic reactions; it tends to be persistent, and trace amounts of peanut can induce allergic responses (Al-Muhsen et al., 2003). Several peanut proteins belonging to different families of seed storage proteins have been identified by immunoblotting as major or minor allergens, and seven of them have been characterized and officially termed Ara h 1–7 (Kleber-Janke et al., 1999). We have recently shown that allergen-specific antibody and cytokine responses can be determined after oral or ip exposure to a whole peanut extract (PE) (van Wijk et al., 2004).
To study the intrinsic immunogenicity and adjuvanticity of purified peanut allergens versus a whole peanut protein extract, and to reveal the effect of the food matrix, two different peanut protein extracts (with or without fat), several purified major peanut allergens (Ara h 1, Ara h 2, and Ara h 3), and one minor allergen (Ara h 6) were used in the current experiments.
To determine the intrinsic immune-stimulating capacity of the purified allergens compared to the whole peanut extract, the popliteal lymph node assay (PLNA), an established model to test immune stimulation was used. After injection of 1 mg PE or purified allergen, PLN cell numbers, cytokine production, and expression of costimulatory molecules (CD80, CD86, and CD54), a measure for antigen-presenting cell (APC) activation, were determined. Data derived from this study show an important role of the PE food matrix (irrespective of the presence of fat) in activating immune cells, thereby driving immune responses to peanut proteins.
The effect of the food matrix on IgE responses against peanut allergens was also examined, because the elicitation of specific IgE responses is one of the absolute requirements for food allergens to induce immediate hypersensitivity responses. This effect was studied in an established mouse model of peanut hypersensitivity, using the relevant oral route of exposure in combination with a mucosal adjuvant, cholera toxin (CT). All purified peanut allergens induced IgE-specific serum antibody responses with only little additional effect of the food matrix. From these studies we concluded that purified peanut allergens possess little intrinsic adjuvanticity in contrast to whole PE. Furthermore, the results suggest that the food matrix can influence responses to individual proteins.
MATERIALS AND METHODS
Mice.
Female, specific pathogen-free BALB/c mice (6 weeks of age) and C3H/HeOuJ Ico mice (4 weeks of age), bred on a SM R/M-Z CRL VRF1 diet (ssniff Spezialdiaten GmbH, Germany) were purchased from Charles River (Lyon, France). Mice were maintained under barrier conditions in filter-topped Macrolon cages with wood chip bedding, at mean temperature of 23° ± 2°C, relative humidity of 50–55%, and a 12-h light/dark cycle. Drinking water and standard laboratory food pellets (rat and mouse breeder and grower, Special Diet Services, Essex, England) were provided ad libitum. No peanut proteins were present in the diets. The experiments were approved by the animal experiments committee of the faculty of veterinary medicine, Utrecht University.
Chemicals and reagents.
Chemicals were obtained from Sigma Aldrich (Zwijndrecht, The Netherlands) unless stated otherwise. For cytokine measurements, IL-2, IL-4, and IFN- capture and detecting antibodies, and an IL-10 ELISA kit were obtained from BD Pharmingen (Hamburg, Germany); and streptavidin–horseradish peroxidase (HRP) was from Sanquin (Amsterdam, The Netherlands). BD Pharmingen was the supplier of rat anti-mouse IgE, and the Digoxigenin (DIG)-coupling kit was obtained from Boehringer Mannheim (Mannheim, Germany); anti-DIG-peroxidase Fab fragments were from Roche Diagnostics (Mannheim, Germany). Alkaline phosphatase (AP)-conjugated goat anti-mouse, human adsorbed IgG1 and IgG2a were purchased from Southern Biotechnology Associates (Birmingham, AL). Campina Melkuni (Eindhoven, The Netherlands) was the supplier of milk powder used in blocking buffer. Medium used for the in vitro restimulation was complete RPMI 1640 with Glutamax-I (Life Technologies, Paisley, Scotland) supplemented with 10% fetal calf serum (ICN Pharmaceuticals, Costa Mesa, CA) and 2% penicillin-streptomycin.
Preparation of peanut extract and purification of allergens.
Peanuts from the Runner cultivar (Cargill, Dawson, GA) were kindly provided by Imko Nut Products (Doetinchem, The Netherlands). The PE was prepared as described by Koppelman et al. (2003). Briefly, peanuts were ground and the protein was extracted by mixing 25 g ground peanut with 200 ml 20 mM Tris buffer (pH 7.2). After this mixture was stirred for 2 h at room temperature, the aqueous fraction was collected by centrifugation (3000 x g for 30 min) and subsequently centrifuged at 10,000 g for 30 min to remove residual traces of fat and insoluble particles. The extract consisted of 4.3% protein, 0.2% fat, 0.5% carbohydrates, 0.2% ash, and 94.8% water, as determined by Kjehdahl analysis and acid hydrolysis. For the preparation of fatty PE (PE fat), fat was not removed after centrifugation, resulting in a PE–oil emulsion consisting of 3.6% protein, 3.5% fat, 0.4% carbohydrates, 0.2% ash, and 92.3% water. Protein concentration (PE 30 mg/ml and PE fat 25 mg/ml) was determined with a BCA protein assay kit (Pierce, Rockford, IL). Sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of PE and PE fat confirmed the presence of proteins migrating at the same molecular weights as the purified allergens Ara h 1 (14%), Ara h 2 (6.5%), Ara h 3 (50%), and Ara h 6 (4.5%), and the two extracts showed a similar protein pattern (data not shown). Their relative amounts (expressed as percentage of total protein) were quantified by densitometry as previously described (Koppelman et al., 2001). Purification of the allergens Ara h 1, Ara h 2, and Ara h 3 was performed according to previously described methods (de Jong et al., 1998; Koppelman et al., 2003), and Ara h 6 was similarly purified from a side-fraction. Endogenous endotoxin content was measured by Limulus Amebocyte Lysate assay (Bio Whittaker, Walkersville, MD) and was <0.02 μg LPS/mg protein (200 EU/mg protein) for all purified allergen and PE solutions.
PLNA protocol.
For the PLNA, a standard protocol was used as previously described (Bloksma et al., 1995). Pilot studies were performed to determine the optimal concentration of PE that also served as a positive control. Different doses of purified allergens (0.1 mg, 0.5 mg, and 1 mg) were tested, and no differences were found between the different dosing groups for all parameters measured. The findings for the 1-mg dose group are presented in the Results. Accordingly, BALB/c mice (n = 4) were subcutaneously injected into the hind footpad with 1 mg of protein (PE, PE fat, Ara h 1, Ara h 2, or Ara h 6) dissolved in 50 μl sterile NaCl, and control animals were injected with 50 μl vehicle. Ara h 3 could not be dissolved at this concentration and was therefore not included in the PLNA. Seven days after protein injection, mice were killed by cervical dislocation, and the draining popliteal lymph nodes (PLN) were excised. Popliteal lymph node single-cell suspensions were prepared under aseptic conditions in complete RPMI 1640 and adjusted to 2.5106 cells/ml.
Oral treatment protocol.
C3H/HeJ mice (n = 6) were orally exposed to the purified allergens Ara h 1, Ara h 2, Ara h 3, Ara h 6 (0.1 and 1 mg), PE (1 and 6 mg), or PE fat (6 mg) in the presence of the mucosal adjuvant CT. Oral exposure was performed by intragastric dosing of protein on 3 consecutive days, followed by weekly dosing (4 weeks). Cholera toxin (10 μg) was coadministered on days 1, 2, 3, 8, 15, and 21. At day 30, mice
Cell culture and cytokine measurement.
Popliteal lymph node single-cell suspensions (150 μl of 2.5106 cells/ml in complete RPMI 1640) were incubated in the presence of PE (200 μg/ml), Ara h 1 (200 μg/ml), Ara h 2 (200 μg/ml), Ara h 6 (200 μg/ml), Con A (5 μg/ml), or medium alone in 96-well plates (Costar, Cambridge, MA) for 96 h (Con A 24 h) at 37°C, 5% CO2. After centrifugation for 10 min at 150 x g, supernatant was collected and stored at –20°C until analysis.
In the culture supernatants, IL-2, IFN-, IL-4, and IL-10 levels were determined by sandwich ELISA. Plates (highbond 3590) were coated overnight with 1 μg/ml rat anti-mouse IL-2, IL-4, or IFN-, and the following day plates were blocked with PBS-Tween/3% milk powder for 4 h at room temperature. Samples and cytokine standards were added in several dilutions and incubated overnight at 4°C. Plates were incubated with 0.25 μg/ml rat anti-mouse IL-2, IL-4, or IFN- conjugate for 1 h at room temperature, followed by streptavidin-HRP incubation for 45 min. Finally, tetramethylbezidine(TMB)-substrate (0.1 mg/ml) was added, and the color reaction was stopped with 2 M H2SO4. Absorbance was measured at 450 nm. The IL-10 ELISA was performed in accordance with the manufacturer's instructions.
Flow cytometry.
For flow cytometric analysis, 2105 PLN cells were incubated with 50 μl of predetermined dilutions of fluorescein isothiocyanate (FITC)-, CyChrome (CY)-, and biotin-conjugated monoclonal antibodies in 96-well plates (30 min darkness at 4°C). Cells were characterized based on the following monoclonal antibodies: CD54 FITC (3E2), CD80 FITC (16–10A1), CD86 FITC (GL1), isotype controls for CD80 (hamster IgG2 FITC), and CD86 (Rat IgG2a FITC) and MHC-II biotin (NIMR-4), all obtained from BD Pharmingen. After incubation with biotin-conjugated monoclonal antibodies, cells were washed and incubated with streptavidin-CY (BD Pharmingen) in the same way. Finally, cells were washed, stored in formalin (0.1%), and analyzed within 18 h on a FACScan with standard FACSflow using CellQuest software (BD Biosciences, Franklin Lakes, NJ). Cells expressing CD80, CD86, or CD54 were checked for MHC-II expression to assure antigen-presenting capability.
Measurement of serum IgE antibodies.
Blood of orally exposed mice was collected at weekly intervals, and levels of PE and Ara h–specific IgE were measured by sandwich ELISA. Plates (highbond 3590; Costar, Cambridge, MA) were coated overnight with 1.5 μg/ml purified rat anti-mouse IgE in carbonate buffer (pH 9.6), followed by 1 h of blocking (37°C) with PBS-Tween/3% milk powder. Each test serum was titrated starting at 1:8 dilution and incubated for 2 h (37°C). A pre-sera pool was used as reference value (dilution 1:4). Subsequently a PE- or Ara h-DIG conjugate solution was added (1 h at 37°C). The coupling of DIG to PE or the Ara h's at an optimal labeling efficiency was performed according to the manufacturer's instructions. Briefly, the coupled proteins were separated on a Sephadex G-25 column, and the concentration of coupled protein was determined spectrophotometrically at 280 nm. After incubation (1 h at 37°C) with peroxidase-conjugated anti-DIG fragments, a TMB-substrate (0.1 mg/ml) solution was used, and the color reaction was stopped with 2 M H2SO4. Absorbance was measured at 450 nm using an ELISA reader ELX800 (BIOTEK Instruments, Winooski, VT). The reciprocal of the furthest test serum dilution resulting in an extinction higher than the reference value was read as a titer. Background values were <0.100 (OD).
Measurement of serum IgG1 and IgG2a antibodies.
Blood was collected at weekly intervals, and levels of PE-specific antibodies were measured by ELISA. Plates (highbond 3590; Costar, Cambridge, MA) were coated overnight with 20 μg/ml PE in carbonate buffer (pH 9.6), followed by 1 h of blocking (37°C) with PBS-Tween/3% milk powder. Each test serum was titrated starting at 1:8 dilution and incubated for 1 h (37°C). A pre-sera pool was used as reference value (dilution 1:4). After incubation, AP-conjugated antibodies were added (1 h at 37°C). Subsequently 1 mg/ml p-nitrophenylphosphate in diethanolamine buffer was used for the color reaction, which was stopped with a 10% EDTA solution, after which absorbance was measured at 405 nm using an ELISA reader ELX800 (BIOTEK Instruments, Winooski, VT). Background values were <0.100 (OD).
Statistics.
Multiple comparisons of group means were performed using one-way analyses of variance (ANOVA) with Bonferroni as a post hoc test. For PLN cell numbers, cytokine levels, and serum antibody levels, statistical analysis was performed following logarithmic transformation (to achieve normal distribution), whereas the levels of cell surface molecules were untransformed. A value of p < 0.05 was considered statistically significant.
RESULTS
Peanut Extract, But Not Purified Allergen, Induces PLN Cell Proliferation
To examine the intrinsic immune stimulating capacity and adjuvanticity of purified peanut allergens versus whole PE, the PLNA was used. Mice were injected with 1 mg of protein in the hind foodpad, and at day 7 total cell numbers of the draining PLN were determined (Fig. 1). After injection with PE fat or PE, total lymph node cell numbers increased significantly (fourfold), and no difference was found between the PE fat- and PE-treated groups. In contrast, no cell proliferation was observed after injection with the purified peanut allergens Ara h 1, Ara h 2, or Ara h 6.
Purified Allergen Treatment Fails to Induce Cytokine Production in Con A Restimulated PLN Cell Cultures
To reflect the general activation of T cells, PLN cells were cultured for 24 h in the presence of concanavalin A (Con A), and the concentrations of the Th2-associated cytokines IL-4 and IL-10, and of the Th1-associated cytokine IFN-, were measured in the supernatant (Fig. 2). Popliteal lymph node cells derived from PE- and PE fat-treated mice secreted similar high amounts of IL-4, IL-10, and IFN- compared to cells derived from control mice. No Th2 or Th1 bias was observed in either of the PE treatment groups.
With respect to the purified allergens, no significant IL-10 or IFN- cytokine production was found in PLN cell cultures of Ara h 1-, Ara h 2-, and Ara h 6-treated groups, and only Ara h 6-injection resulted in significant IL-4 production upon restimulation with Con A.
Cytokine Responses Induced by Restimulation with Purified Allergen Are Enhanced by the Presence of a Food Matrix at the Time of Injection
To examine antigen-specific T cell responses, PLN cells were obtained 7 days after the initial injection with 1 mg of PE, PE fat, or purified allergen and cultured for 96 h in the presence of PE, Ara h 1, Ara h 2, Ara h 6, or medium alone. Th1-associated (IL-2 and IFN-) and Th2-associated (IL-4 and IL-10) cytokine production was measured in the supernatant (Table 1).
Popliteal lymph node LN cell cultures derived from PE- and PE fat-treated mice restimulated with PE induced high levels of IL-2, IL-4, IL-10, and IFN-. No significant differences in cytokine concentration were found between the two groups, and a mixed Th1/Th2 cytokine response was observed.
Regarding the purified allergens, only Ara h 6 treatment resulted in a low but significantly increased cytokine production (IL-2, IL-4, IL-10, but not IFN-), whereas Ara h 1-and Ara h 2-injection failed to induce considerable cytokine production in PLN cell cultures when restimulated with the corresponding purified allergen.
Interestingly, cells derived from PE-treated or PE-fat–treated groups secreted higher levels of cytokines than cells derived from Ara h 1- and Ara h 6-treated animals when restimulated with Ara h 1 or Ara h 6. Restimulation with Ara h 2 only induced low IL-2 production, and levels of IL-2 were significantly higher in cell cultures of PE-treated and PE-fat–treated mice than in those of Ara h 2-treated mice. Popliteal lymph node cells cultured in the presence of medium alone showed no production of any of the cytokines examined (data not shown). These results indicate that T cell responses to purified allergens can be influenced by the food matrix.
Peanut Extracts But Not Purified Allergen Induces Expression of Co-stimulatory Molecules on APC
Activation of antigen-presenting cells (APC) is considered one of the requirements for initiating active immune responses. Therefore, the expressions of the activation markers CD80, CD86, and CD54 were determined on PLN APC, 7 days after protein injection (Fig. 3). Both PE fat and PE injection significantly augmented the percentages of CD80-, CD86-, and CD54-expressing cells in the PLN, whereas no increase was found after injection with Ara h 1, Ara h 2, or Ara h 6.
Together, these data indicate that purified peanut allergens, in contrast to the whole extract, lack intrinsic immune-stimulating capacity and thereby fail to induce significant immune responses in the PLNA.
Purified Allergens Induce Specific IgE Responses upon Oral Exposure, Irrespective of the Food Matrix
To further explore the role of the food matrix in immune responses to peanut proteins, an established oral model of peanut hypersensitivity was used. In this model, PE- and allergen-specific IgE responses can be determined using the relevant route of exposure to food proteins (van Wijk et al., 2004). Mice were dosed by gavage for 4 weeks with PE (1 and 6 mg), Ara h 1, Ara h 2, Ara h 3, or Ara h 6 (0.1 and 1 mg) in the presence of the mucosal adjuvant CT.
All purified allergens were able to induce specific IgE responses, but only for Ara h 1 treatment was a significant difference found between the 0.1 and 1 mg dosing groups (Fig. 4). Exposure to PE (1 mg or 6 mg) also resulted in Ara h-specific IgE responses, with levels comparable to those observed after purified allergen exposure. In the control group (oral exposure to PBS + CT) no Ara h-specific IgE responses were found (data not shown).
No Difference in Antibody Responses upon Oral Exposure to PE or PE Fat
To examine the effect of fat content on orally induced antibody responses to PE, mice were orally exposed to PE fat or PE in the presence or absence of the mucosal adjuvant CT. After 4 weeks of oral exposure, both Th1-associated (IgG2a) and Th2-associated (IgG1 and IgE) PE-specific antibodies were determined in the serum (Fig. 5). In the absence of CT, only low levels of IgG1 and IgG2a, and no IgE, were found, with no differences between the PE fat- and PE-treated group. In the presence of CT, similar high levels of IgG2a, IgG1, and IgE were found in both PE treatment groups. In line with the results found in the PLN, the presence of fat does not affect orally induced immune responses to PE.
DISCUSSION
No definitive characteristics of food allergens have been identified other than that they need to reach and stimulate immune cells in order to induce sensitization. It has been suggested that both properties may be influenced by the food matrix (Teuber, 2002). In the present study we have shown that purified peanut allergens, unlike a whole PE, possess little intrinsic immune-stimulating capacity and that the immune response to these allergens can be adjuvated by the presence of a food matrix. Furthermore, it was demonstrated that the purified allergens are stable enough to get into contact with the immune system after oral exposure, both in the presence and in the absence of a food matrix.
Although Ara h 1 and Ara h 2 are recognized as major peanut allergens, they were unable to induce PLN cell proliferation or cytokine production, suggesting that they possess little intrinsic immune-stimulating capacity. In contrast, PE induced a strong PLNA response (without causing inflammation in the paw) and was also able to adjuvate specific T cell responses against the allergens present in the extract. In line with these results, Ara h 1 and Ara h 2 appear to be poor IgG and IgE inducers in mice when injected ip (G. Ladics, Dupont, personal communication), whereas ip PE injection results in considerable Ara h 1–specific and Ara h 2–specific IgG and IgE responses (van Wijk et al., 2004).
The actual amount of individual allergens in PE and PE fat was always lower than the 1 mg of allergen injected in a purified form. This may raise the issue that the discrepancy in dose has affected the outcome of the immune response and that the high dose injection (1 mg) has induced non-responsiveness. However, injection of 0.1 and 0.5 mg of purified allergen also failed to induce an immune response in the PLNA (data not shown).
The difference in immune-stimulating capacity between the purified allergens and the extract may be explained by selective activation of APC. Generally considered, APC activation and the accompanying upregulation of costimulatory molecules is required for the induction of an active immune response. For contact allergens, which possess high intrinsic adjuvanticity due to their reactive groups (Kapsenberg, 1996), it has been shown that they selectively upregulate costimulatory molecules (e.g., CD54 and CD86) on dendritic cells in vitro (Rougier et al., 2000; Tuschl et al., 2000). Furthermore, drugs associated with immune-mediated hypersensitivity reactions were demonstrated to upregulate the expression of costimulatory molecules on APC in the PLNA (Nierkens et al., 2002; Popovic et al., 2004). Interestingly, none of the purified peanut allergens tested was able to increase expressions of costimulatory molecules on APC, whereas PE injection resulted in an upregulation of CD80, CD86, and CD54. Hence, in contrast to sensitizing chemicals, soluble peanut allergens do not possess intrinsic adjuvanticity, and they therefore need an accompanying adjuvant (in the case of PE provided by the food matrix) to be able to activate APC and to induce subsequent immune stimulation.
Peanut consists of proteins, carbohydrates and fatty acids, and all these different components—and their interactions—may be responsible for the adjuvant effect. For instance, it has been described that peanut oils can have an adjuvant effect in a vaccine model (Eghafona, 1996). Remarkably, in the PLNA, the presence of fat did not seem to play a major role in the induction of the immune response (PLN cell numbers, cytokine production, and APC activation). In addition to nutrients, non-nutritive compounds such as lectins (Watzl et al., 2001) and food contaminants such as LPS (Brix et al., 2004) and aflatoxins (Kocabas and Sekerel, 2003) may affect immune responses to food proteins. For future experiments, it would be interesting to test different PE components separately in order to elucidate their immune-modulating properties.
Concerning the relevant route of exposure, the food matrix has been suggested to affect the allergenic properties of orally administered proteins by providing adjuvant stimuli to the specialized gut mucosal immune system or by protecting them from digestion (Lehrer et al., 2002; Teuber, 2002), but to our knowledge no in vivo data are available. In general, oral administration (by feeding or gavage) of soluble food proteins to mice results in peripheral hyporesponsiveness (reviewed by Mayer et al., 2001; Nagler-Anderson et al., 2001) Administration of proteins with adjuvants induces a productive immune response to this normally tolerogenic form of antigen (Nagler-Anderson et al., 2001).We have previously shown that, in the present oral model, coadministration of the mucosal adjuvant CT is indispensable to the induction of antigen-specific IgE responses. Clearly, the intrinsic adjuvant activity of PE (i.e., without fat) is not sufficient to induce sensitization via the oral route (van Wijk et al., 2005). Consequently, it might be impossible to determine the adjuvant activity of the food matrix itself when CT is coadministered. Yet, in the current study, the presence of fat did not affect the magnitude or polarization (type 1 versus type 2) of the antibody responses against PE, irrespective of the presence of CT (Fig. 5). Fatty acids have been described as immune modulators following high-dose oral supplementation (Harbige and Fisher, 2001; Wu 2004), covalent coupling to a protein or peptide (Oliveira et al., 2002; BenMohamed et al., 2002), or the use of liposome-entrapped proteins (Sehra et al., 1998), but it remains to be elucidated whether fats present in a normal diet have similar immune-modulating properties.
Regarding the aspect of digestion, the present results indicate that peanut allergens are at least stable enough to reach the gut-associated lymphoid tissue, even without the presence of a protecting food matrix. Although it has been shown in vitro that polysaccharide–protein and protein–protein interactions may influence allergen digestion (Fremont et al., 2004; Maleki et al., 2003), these interactions seemed not to affect the immune response to allergens in the present in vivo model. Our data are in accordance with in vitro data showing that Ara h 1 and Ara h 2 are highly resistant to enzymatic digestion (Maleki et al., 2000; Sen et al., 2002). Furthermore, treatment of purified Ara h 1 and PE with pepsin resulted in the production of nearly identical sets of digestion products, suggesting an intrinsic stability of Ara h 1 (Kopper et al., 2004). The current results demonstrate that the major peanut allergens Ara h 1, Ara h 2, Ara h 3, and the minor allergen Ara h 6 are stable enough in vivo to elicit specific IgE responses.
In recent years there has been considerable interest in the development of experimental animal models to predict the allergenicity of proteins. Our data suggest that assays designed to measure immune stimulation, such as the PLNA, are clearly not suitable to identify allergenic proteins when injected in a purified form, because they do not appear to possess high intrinsic immune-stimulating capacity. Because PE injection did result in immune stimulation, further studies may reveal the feasibility of subcutaneous injection with whole food extract as a tool for identifying the adjuvating capacity of compounds within a food matrix.
After oral exposure, all purified peanut allergens were shown to be IgE inducers, which confirms that they posses IgE epitopes, and that they are able to reach the immune system of the gut in a relatively intact form, two qualities required for the induction of allergic sensitization. However, for the future use of this model as a screening tool, other allergens and especially non-allergens need to be tested. Recently, Gaudry et al. (2004) have examined several food allergens and non-allergens in a similar oral model and have shown that mice and humans respond similarly to major allergens and non-allergens. Although a broader panel of (non)allergens should be tested and the use of an adjuvant remains a topic of debate, the measurement of IgE antibody responses following oral exposure to proteins seems to be a promising tool for predicting allergenicity of these proteins. Furthermore, the oral route represents the most adequate route of exposure to study possible in vivo digestion and food matrix effects.
In summary, our results indicate that purified peanut allergens possess little intrinsic immune-stimulating capacity and that they probably need co-localization of an adjuvant compound to induce sensitization. Moreover, this study shows that the food matrix may affect immune responses to individual allergens; as a consequence, a possible influence of the food matrix has to be considered in the development of animal tests to predict allergenic potential.
REFERENCES
Al-Muhsen, S., Clarke, A. E., and Kagan, R. S. (2003). Peanut allergy: An overview. Can. Med. Assoc. J. 168, 1279–1285.
Astwood, J. D., Leach, J. N., and Fuchs, R. L. (1996). Stability of food allergens to digestion in vitro. Nat. Biotechnol. 14, 1269–1273.
Bannon, G. A. (2004). What makes a food protein an allergen Curr. Allergy Asthma Rep. 4, 43–46.
BenMohamed, L., Belkaid, Y., Loing, E., Brahimi, K., Gras-Masse, H., and Druilhe, P. (2002). Systemic immune responses induced by mucosal administration of lipopeptides without adjuvant. Eur. J. Immunol. 32, 2274–2281.
Bloksma, N., Kubicka-Muranyi, M., Schuppe, H. C., Gleichmann, E., and Gleichmann, H. (1995). Predictive immunotoxicological test systems: Suitability of the popliteal lymph node assay in mice and rats. Crit. Rev. Toxicol. 25, 369–396.
Brix, S., Kjaer, T. M., Barkholt, V., and Frokiaer, H. (2004). Lipopolysaccharide contamination of beta-lactoglobulin affects the immune response against intraperitoneally and orally administered antigen. Int. Arch. Allergy Immunol. 135, 216–220.
de Jong, E. C., Van Zijverden, M., Spanhaak, S., Koppelman, S. J., Pellegrom, H., and Penninks, A. H. (1998). Identification and partial characterization of multiple major allergens in peanut proteins. Clin. Exp. Allergy 28, 743–751.
Eghafona, N. O. (1996). Immune responses following cocktails of inactivated measles vaccine and Arachis hypogaea L. (groundnut) or Cocos nucifera L. (coconut) oils adjuvant. Vaccine 14, 1703–1706.
Fremont, S., Sanchez, C., Errahali, Y., Mouecoucou, J., Metche, M., Mejean, L., and Nicolas, J. P. (2004). [Food allergy: effect of proteins-lipids and proteins-polysaccharides interactions]. Allerg. Immunol. (Paris) 36, 82–87.
Fu, T. J. (2002). Digestion stability as a criterion for protein allergenicity assessment. Ann. N. Y. Acad. Sci. 964, 99–110.
Gaudry, P. A., Fernandes, J., Reish, R., Roux, K. H., Sathe, S. K., Teuber, S. S., Reese, G., and Lehrer, S. B. (2004). Murine IgE and IgG antibody responses to allergenic and non-allergenic foods: Potential model for allergenicity assessment of foods in man. J. Allergy Clin. Immunol. 113, 832 (Abstract).
Harbige, L. S., and Fisher, B. A. (2001). Dietary fatty acid modulation of mucosally-induced tolerogenic immune responses. Proc. Nutr. Soc. 60, 449–456.
Kapsenberg, M. L. (1996). Chemicals and proteins as allergens and adjuvants. Toxicol. Lett. 86, 79–83.
Kleber-Janke, T., Crameri, R., Appenzeller, U., Schlaak, M., and Becker, W. M. (1999). Selective cloning of peanut allergens, including profilin and 2S albumins, by phage display technology. Int. Arch. Allergy Immunol. 119, 265–274.
Kocabas, C. N., and Sekerel, B. E. (2003). Does systemic exposure to aflatoxin B(1) cause allergic sensitization Allergy 58, 363–365.
Koppelman, S. J., Knol, E. F., Vlooswijk, R. A., Wensing, M., Knulst, A. C., Hefle, S. L., Gruppen, H., and Piersma, S. (2003). Peanut allergen Ara h 3: Isolation from peanuts and biochemical characterization. Allergy 58, 1144–1151.
Koppelman, S. J., Vlooswijk, R. A., Knippels, L. M., Hessing, M., Knol, E. F., van Reijsen, F. C., and Bruijnzeel-Koomen, C. A. (2001). Quantification of major peanut allergens Ara h 1 and Ara h 2 in the peanut varieties Runner, Spanish, Virginia, and Valencia, bred in different parts of the world. Allergy 56, 132–137.
Kopper, R. A., Odum, N. J., Sen, M., Helm, R. M., Steve Stanley, J., and Wesley Burks, A. (2004). Peanut protein allergens: Gastric digestion is carried out exclusively by pepsin. J. Allergy Clin. Immunol. 114, 614–618.
Ladics, G. S., Holsapple, M. P., Astwood, J. D., Kimber, I., Knippels, L. M., Helm, R. M., and Dong, W. (2003). Workshop overview: Approaches to the assessment of the allergenic potential of food from genetically modified crops. Toxicol. Sci. 73, 8–16.
Lehrer, S. B., Ayuso, R., and Reese, G. (2002). Current understanding of food allergens. Ann. N. Y. Acad. Sci. 964, 69–85.
Maleki, S. J. (2004). Food processing: Effects on allergenicity. Curr. Opin. Allergy Clin. Immunol. 4, 241–245.
Maleki, S. J., Kopper, R. A., Shin, D. S., Park, C. W., Compadre, C. M., Sampson, H., Burks, A. W., and Bannon, G. A. (2000). Structure of the major peanut allergen Ara h 1 may protect IgE-binding epitopes from degradation. J. Immunol. 164, 5844–5849.
Maleki, S. J., Viquez, O., Jacks, T., Dodo, H., Champagne, E. T., Chung, S. Y., and Landry, S. J. (2003). The major peanut allergen, Ara h 2, functions as a trypsin inhibitor, and roasting enhances this function. J. Allergy Clin. Immunol. 112, 190–195.
Mayer, L., Sperber, K., Chan, L., Child, J., and Toy, L. (2001). Oral tolerance to protein antigens. Allergy 56, 12–15.
Metcalfe, D. D. (2003). Introduction: What are the issues in addressing the allergenic potential of genetically modified foods Environ. Health Perspect. 111, 1110–1113.
Nagler-Anderson, C., and Shi, H. N. (2001). Peripheral nonresponsiveness to orally administered soluble protein antigens. Crit. Rev. Immunol. 21, 121–131.
Nierkens, S., van Helden, P., Bol, M., Bleumink, R., van Kooten, P., Ramdien-Murli, S., Boon, L., and Pieters, R. (2002). Selective requirement for CD40-CD154 in drug-induced type 1 versus type 2 responses to trinitrophenyl-ovalbumin. J. Immunol. 168, 3747–3754.
Oliveira, F. M., Santos, E. M., Mota-Santos, T. A., Ruiz-De-Souza, V., and Gontijo, C. M. (2002). Covalent coupling of palmitate to ovalbumin inhibits and blocks the induction of oral tolerance. Scand. J. Immunol. 55, 570–576.
Popovic, M., Nierkens, S., Pieters, R., and Uetrecht, J. (2004). Investigating the role of 2-phenylpropenal in felbamate-induced idiosyncratic drug reactions. Chem. Res. Toxicol. 17, 1568–1576.
Rougier, N., Redziniak, G., Mougin, D., Schmitt, D., and Vincent, C. (2000). In vitro evaluation of the sensitization potential of weak contact allergens using Langerhans-like dendritic cells and autologous T cells. Toxicology 145, 73–82.
Sehra, S., Chugh, L., and Gangal, S. V. (1998). Polarized TH1 responses by liposome-entrapped allergen and its potential in immunotherapy of allergic disorders. Clin. Exp. Allergy 28, 1530–1537.
Sen, M., Kopper, R., Pons, L., Abraham, E. C., Burks, A. W., and Bannon, G. A. (2002). Protein structure plays a critical role in peanut allergen stability and may determine immunodominant IgE-binding epitopes. J. Immunol. 169, 882–887.
Sicherer, S. H., Munoz-Furlong, A., and Sampson, H. A. (2003). Prevalence of peanut and tree nut allergy in the United States determined by means of a random digit dial telephone survey: A 5-year follow-up study. J. Allergy Clin. Immunol. 112, 1203–1207.
Taylor, S. L., Lemanske, R. F., Jr., Bush, R. K., and Busse, W. W. (1987). Food allergens: Structure and immunologic properties. Ann. Allergy 59, 93–99.
Teuber, S. S. (2002). Hypothesis: The protein body effect and other aspects of food matrix effects. Ann. N. Y. Acad. Sci. 964, 111–116.
Tuschl, H., Kovac, R., and Weber, E. (2000). The expression of surface markers on dendritic cells as indicators for the sensitizing potential of chemicals. Toxicol. In Vitro 14, 541–549.
Watzl, B., Neudecker, C., Hansch, G. M., Rechkemmer, G., and Pool-Zobel, B. L. (2001). Dietary wheat germ agglutinin modulates ovalbumin-induced immune responses in Brown Norway rats. Br. J. Nutr. 85, 483–490.
van Wijk, F., Hartgring, S., Koppelman, S. J., Pieters, R., and Knippels, L. M. J. (2004). Mixed antibody and T cell responses to peanut and the peanut allergens Ara h1, Ara h3 and Ara h6 in an oral sensitization model. Clin. Exp. Allergy 34, 1422–1428.
van Wijk, F., Hoeks, S., Nierkens, S., Koppelman, S. J., van Kooten, P., Boon, L., Knippels, L. M., and Pieters, R. (2005). CTLA-4 signaling regulates the intensity of hypersensitivity responses to food antigens, but is not decisive in the induction of sensitization. J. Immunol. 174, 174–179.
Wu, D. (2004). Modulation of immune and inflammatory responses by dietary lipids. Curr. Opin. Lipidol. 15, 43–47.(F. van Wijk, S. Nierkens,)