Helen Theresa Caddick

Divergent antibody isotype responses induced in mice by systemic exposure to proteins: a comparison of ovalbumin with bovine serum albumin.

Whereas many foreign proteins are immunogenic, only a proportion is associated commonly with allergy, having the potential to induce the quality of immune response necessary for IgE antibody production and the development of immediate type hypersensitivity reactions in the gastrointestinal and/or respiratory tracts. In the context of toxicological evaluations there is a need to identify those properties that confer on proteins the ability to provoke allergic reactions. The characteristics of antibody responses induced in BALB/c strain mice following administration of ovalbumin (OVA), a significant human allergen, have been compared with those provoked by bovine serum albumin (BSA), a protein considered to have more limited allergenic potential. Intranasal or intraperitoneal (ip) administration of BSA or OVA elicited vigorous IgG and IgG1 antibody responses. Differential IgE antibody production was observed, however, with OVA stimulating relatively high IgE antibody titres at all doses tested whereas no or low titre IgE antibody was detected following exposure to BSA. Furthermore, a differential capacity for IgG2a antibody responses was observed, with only BSA provoking high titres of this IgG subclass. The relative quality of induced responses was equivalent following administration of these proteins via mucosal (in) tissue or via a non-mucosal (ip) route of exposure. IgG2a antibody production is promoted by the type 1 cytokine interferon gamma (IFN-gamma), whereas IFN-gamma and the type 2 cell product interleukin 4 exert reciprocal antagonistic effects on IgE antibody responses. Although cytokine expression patterns were not analysed in this series of experiments, the differential IgE and IgG subclass antibody responses induced by BSA and OVA are consistent with the preferential activation of T helper (Th) 1- and Th2-type cells, respectively. These data indicate that proteins can provoke in mice characteristic antibody (IgE and IgG) isotype profiles suggestive of discrete T lymphocyte responses and that such differences may be associated with variable allergenic activity.

Characterization of antibody responses induced in rodents by exposure to food proteins: influence of route of exposure.

There is a growing interest in the development of methods to characterize the allergenic properties of novel proteins, particularly those expressed by transgenic crop plants. Approaches to the direct evaluation of allergenic potential have focused generally on the ability of proteins to induce antibody (particularly IgE antibody) after systemic (intraperitoneal; i.p.) or gavage administration to high IgE responder strain rodents. To date there has been no systematic comparison of the reliability, sensitivity or selectivity of these approaches. We have, therefore, compared antibody (IgG and IgE) responses induced in Brown Norway (BN) rats by daily gavage administration and in BALB/c strain mice following intraperitoneal or gavage exposure to food proteins of varying allergenic potential. Animals were exposed to the allergens peanut agglutinin and ovalbumin (OVA) or to a crude potato protein extract (PPE) containing acid phosphatase activity, a common foodstuff which appears to be of low allergenicity. All test proteins were clearly immunogenic when administered by gavage to BN rats, with measurable, and in some cases very vigorous, IgG antibody responses recorded for all animals. Identical exposure of BALB/c strain mice also stimulated detectable IgG antibody responses, with particularly high titers recorded following treatment with peanut agglutinin and somewhat less vigorous responses induced by OVA and PPE. Despite these high titer IgG antibody responses, however, none of the proteins provoked detectable IgE antibody following gavage administration to BN rats. In contrast, in BALB/c mice oral exposure to peanut agglutinin elicited high titer IgE antibody, although IgE antibody responses to both OVA and PPE were much weaker. Parenteral (i.p.) treatment of BALB/c strain mice with each of the test materials induced relatively high titer IgG antibody and a differential potential to stimulate IgE antibody was observed. High titer IgE responses were provoked by i.p. administration of peanut agglutinin and OVA, whereas PPE stimulated little or no detectable IgE antibody. It would appear, therefore, that while it is possible to elicit robust IgE responses by gavage exposure of BALB/c strain mice to some protein allergens, such as peanut agglutinin, such responses are generally weaker and less consistent than those provoked by i.p. administration. Furthermore, gavage treatment failed to induce detectable IgE responses in the BN rat, suggesting that the ability these animals to mount IgE responses is somewhat variable. Following i.p. administration to BALB/c strain mice, these proteins displayed immunological properties consistent with what is known of their allergenic potential in humans, suggesting that, following further evaluation with a wider range of proteins, this method may provide one approach to the identification of potential protein allergens.

Evaluation of protein allergenic potential in mice: dose–response analyses

Background With the increasing interest in novel foods derived from transgenic crop plants, there is a growing need for the development of approaches for the characterization of the allergenic potential of proteins. Whereas immunogenicity is a common property of foreign proteins, including food proteins, relatively few are significant dietary allergens with the inherent capacity to provoke IgE antibody production and immediate‐type hypersensitivity responses.

Immunogenic properties of rapidly digested food proteins following gavage exposure of mice: a comparison of ovalbumin with a potato acid phosphatase preparation.

The ability of food proteins to resist digestion in simulated gastric fluid (SGF) correlates with allergenic potential. The purpose of the current investigations was to determine whether this association is due solely to the failure of unstable proteins to elicit an immune response when administered orally. We have examined immune responses induced in BALB/c mice by gavage administration of ovalbumin (OVA) and a crude potato protein extract (PPE) containing acid phosphatase activity. The stability of OVA and PPE in SGF was measured using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The ability of these proteins to stimulate specific IgG and IgE antibody production in mice following parenteral (intraperitoneal; ip) or oral (gavage) exposure was compared using enzyme-linked immunosorbent and homologous passive cutaneous anaphylaxis assays, respectively. Both OVA and PPE induced specific IgG antibody responses when administered either by gavage or by ip injection. Parenteral, but not gavage, exposure to OVA was associated with robust IgE antibody responses. Administration of PPE failed to stimulate strong IgE production via either route of exposure. Differential stability in SGF was observed, with PPE being digested extremely rapidly (within 1 min), whereas OVA was more resistant. The strong association reported by others between stability in SGF and allergenic potential is unlikely to be solely due to orally-ingested labile proteins failing to provoke immune responses due to degradation in the stomach.

Assessment of glycosylation-dependent cell adhesion molecule 1 as a correlate of allergen-stimulated lymph node activation

Early changes in gene expression have been identified by cDNA microarray technology. Analysis of draining auricular lymph node tissue sampled at 48 h following exposure to the potent contact allergen 2,4-dinitrofluorobenzene (DNFB) provided examples of up- and down-regulated genes, including onzin and guanylate binding protein 2, and glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1), respectively. Allergen-induced changes in these three genes were confirmed in dose-response and kinetic analyses using Northern blotting and/or reverse transcription-polymerase chain reaction techniques. The results confirmed that these genes are robust and relatively sensitive markers of early changes provoked in the lymph node by contact allergen. Upon further investigation, it was found that altered expression of the adhesion molecule GlyCAM-1 was not restricted to treatment with DNFB. Topical sensitization of mice to a chemically unrelated contact allergen, oxazolone, was also associated with a decrease in the expression of mRNA for GlyCAM-1. Supplementary experiments revealed that changes in expression of this gene are independent of the stimulation by chemical allergens of proliferative responses by draining lymph node cells. Taken together these data indicate that the expression of GlyCAM-1 is down-regulated rapidly following epicutaneous treatment of mice with chemical allergens, but that this reduction is associated primarily with changes in lymph node cell number, or some other aspect of lymph node activation, rather than proliferation.

Butyl benzyl phthalate: effects on immune responses to ovalbumin in mice

During recent decades the prevalence of IgE‐mediated (atopic) allergic diseases in Western Europe and the USA has been increasing dramatically. It has been suggested that one possible cause is the presence in the environment of chemicals that may act as adjuvants, enhancing immune and allergic responses. Certain commonly used phthalate plasticizers such as butyl benzyl phthalate (BBP) have been implicated in this way. In the current experiments, the impact of BBP, applied by a physiologically relevant exposure route, on the vigour of immune responses induced in BALB/c strain mice has been examined. Mice were immunized via subcutaneous injection with the reference allergen ovalbumin (OVA) and received concurrent topical treatment with doses of BBP that induced significant changes in liver weight. The generation of specific anti‐OVA IgE and IgG1 antibodies was measured by passive cutaneous anaphylaxis and by enzyme‐linked immunosorbant assays, respectively. Topical administration of BBP was without impact on anti‐OVA IgE antibody responses, regardless of whether BBP was applied locally or distant to the site of OVA immunization. However, same‐site treatment with high‐dose BBP (100 mg) did result in a modest elevation in anti‐OVA IgG1 antibody production, a subclass of antibody used as a surrogate marker of IgE responses. Taken together with human exposure data, these results suggest that the doses of phthalate encountered in the home environment are unlikely to be a major factor contributing to the increased incidence of asthma and allergy in the developed world. Copyright

Cytokine profiling of chemical allergens in mice: Measurement of message versus protein

Chemical respiratory allergy is an important occupational health problem, but there are currently available no validated methods for hazard identification. There has been interest for some time in the application of cytokine profiling for the characterization of chemical allergens. We have now examined whether these cytokine expression patterns are regulated at the level of mRNA and/or protein production. Mice (BALB/c strain) were exposed topically to the contact allergen 2,4-dinitrochlorobenzene (DNCB), or to the respiratory allergen trimellitic anhydride (TMA). Thirteen days after the initiation of exposure, a single cell suspension of draining (auricular) lymph node cells (LNC) was prepared. Cells were cultured for 24-120h and supernatants analyzed for cytokine protein by cytokine bead array. In parallel experiments total RNA was prepared from freshly isolated or cultured cells and cytokine gene expression was analyzed by ribonuclease protection assay (RPA) or by real time reverse transcription-polymerase chain reaction (RT-PCR). DNCB-activated LNC secreted high levels of the type 1 cytokines interferon (IFN)-gamma and IL-12 compared with TMA-stimulated LNC. The converse type 2 pattern was observed following treatment with TMA. Freshly isolated LNC from TMA-treated mice displayed a selective type 2 cytokine mRNA profile as measured by RPA. In contrast, RNA isolated from DNCB-activated LNC displayed a more mixed cytokine phenotype with relatively low levels of transcripts for both type 1 and type 2 cell products. When cytokine gene expression was measured by the more sensitive real time RT-PCR technique, more vigorous expression of IL-4 was recorded for TMA-activated LNC compared with DNCB-activated LNC but there was no evidence for elevated IFN-gamma transcripts for the latter treatment. The observation that IFN-gamma mRNA expression is not increased in DNCB-activated LNC despite robust secretion of this cytokine indicates that production is controlled mainly at the level of translation of previously transcribed mRNA or of protein secretion. Furthermore, the preferential cytokine profile recorded following TMA exposure was more clearly contrasting when measured at the level of protein secretion rather than at the level of mRNA expression. Experience to date suggests that the measurement of induced cytokine profiles shows promise for the hazard identification and characterization of chemical respiratory allergens, and that the end point best suited for this purpose is cytokine secretion rather than mRNA expression.

Cytokine profiling of chemical allergens in mice: impact of mitogen on selectivity of response

A major constraint in the development of methods for the identification of chemical respiratory allergens is the continuing uncertainty regarding the mechanisms of this disease and in particular the role of IgE antibody. There is, however, increasing evidence that respiratory sensitization is favoured by the induction of a selective type 2 cytokine response. The current investigations focus on the potential application of cytokine profiling to the identification of chemical respiratory sensitizers. The objective is to determine the optimal configuration of the method for discrimination between chemical contact and respiratory sensitizers. The reference contact sensitizer 2,4‐dinitrochlorobenzene (DNCB) and reference respiratory sensitizer trimellitic anhydride (TMA), which have been shown to induce type 1 and type 2 cytokine profiles, respectively, were utilized. Variables investigated included cell concentration, time in culture, dosing regimens (a 13 day and a truncated 8 day protocol) and the impact of restimulation in vitro with T cell mitogens. Cell culture conditions were critical for the selectivity of the response, with the addition of mitogen resulting in a less discriminatory pattern of cytokine expression, particularly for the type 1 cytokine interferon γ (IFN‐γ). Furthermore, a 13 day exposure period was required for vigorous expression of IFN‐γ by DNCB‐activated cells, whereas type 2 cytokine expression by TMA‐stimulated cells was recorded after 8 days. These data demonstrate that the most optimal method for cytokine profiling is a chronic (13 day) exposure regime followed by culture of lymph node cells at 107 cells ml−1 for120 h in the absence of mitogen. Copyright