GASTROENTEROLOGY

SPECIAL REPORTS

AND REVIEWS

Gastrointestinal Food Hypersensitivity: Mechanisms of Pathophysiology SHEILA E. CROWE and MARY

Basic

H. PERDUE

Intestinal Disease Research Unit and Departments McMaster University, Hamilton, Ontario, Canada

of Medicine

Gastrointestinal symptoms occur in a large number of patients with food allergies. Immediate hypersensitivity mechanisms may give rise to the nausea, vomiting, abdominal pain, and diarrhea experienced by these patients. However, there are limited human data about the pathophysiological basis for these symptoms. Most of the available information comes from a variety of animal models. This article reviews the literature using models of intestinal food hypersensitivity, as well as human studies, that have contributed to our understanding of the pathophysiological mechanisms in gastrointestinal food hypersensitivity.

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mmunoglobulin (Ig) E-mediated immediate hypersensitivity to food and other antigens are involved in the pathogenesis of asthma, rhinitis, urticaria and eczema in certain individuals.‘-3 In allergic patients, foods may also induce gastrointestinal (GI) symptoms. Although the underlying pathophysiological mechanisms are poorly understood, recent studies in humans and animals implicate immediate hypersensitivity reactions in these adverse GI reactions to food. Immediate hypersensitivity reactions are a consequence of antigen exposure resulting in reaginic antibody (usually IgE but also IgG, in humans4) production and attachment to mast cells. Subsequent exposure to the antigen induces mast cell degranulation with release of stored and newly formed mediators.5 These mediators (Table 1)produce the physiological changes, including bronchoconstriction, mucous hypersecretion, and increased in vascular permeability, that underlie the typical manifestations of respiratory and dermatologic allergic reactions.” Similar mast cell mediator-induced pathophysiology has been shown in GI hypersensitivity. Released mast cell products recruit other immunocompetent and inflammatory cells (i.e., eosinophils and neutrophils), which in turn contribute to allergic reactions, in particular late-phase responses. De-

(Division of Gastroenterology)

and Pathology,

layed reactions are an aspect of allergic diseases’ that may also operate in the GI tract. Immunologic reactions to foods can involve mechanisms other than immediate hypersensitivity. Immune complex formations-l0 and complement deposition” have been documented in food allergy, particularly food protein enteropathies. Cell-mediated immune reactions to food antigens also play a role in these diseases (milk and soy protein enteropathies)2s’2 and in celiac disease.13’14 However, the focus of this review is immediate hypersensitivity and, in particular, the consequence of GI food antigen challenge in the sensitized host. The aim of this article is to first review the steps involved in sensitization and the role of intestinal mast cells and to then consider the clinical aspects of GI food allergy as well as information from animal models. Overview

of Food Sensitization

It is estimated that the human GI tract processes approximately 100 tons of food during a lifetime15; because only a small percentage of the population develops food allergy, it is clear that the GI system is uniquely designed to avoid potentially deleterious immune responses to foods. Protective factors such as gastric acid and proteolytic enzymes degrade food proteins while normal gut motility and mucous production help minimize mucosal contact of potentially antigenic substances.16 The epithelium acts as a barrier to prevent significant uptake of large molecules. When this barrier is damaged, as occurs in viral gastroenteritis or in inflammatory bowel disease (IBD), a greater uptake of macromolecules is observed.*’ Immaturity is also associated with increased gut uptake of macromo1ecules.‘6~‘g Local &A production may play a protective role in preventing food allergies by blocking antigen uptake, as sug0 1992by

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Table 1. Mast Cell Mediators Preformed: intragranular Amines Histamine Serotonin” Chemotactic factors Lysosomal enzymes Exoglycosidases Kininogenase Chymotrypsin/trypsin Peroxidase Superoxide dismutase Arylsulfatase, A and B Superoxide anions Proteoglycans Heparin Chondroitin sulfate Membrane derived (newly generated) Leukotrienes (LTB,, LTC,, LTD,, and LTE,) Prostaglandins (PGD,) Monohydroeicosatetraenoic acids Hydroperoxyeicosatetraenoic acids Thromboxanes Platelet-activating factor (PAF) Cytokinesb Interleukins 1-6 [IL-l-6) Interferon y (IFN-y) Tumour necrosis factor a (TNF-a) Transforming growth factor p (TGF-P) Macrophage inflammatory protein 1 (MIP-1) family Granulocyte-macrophage colony-stimulating

suppressor function, which normally develops around the time of weaningzB or to the transient IgA deficiency of the newborn state.ls In addition, enteric infections resulting in epithelial disruption could lead to the uptake of intact luminal antigen, IgE formation, and development of an allergic reaction upon secondary antigen exposure in atopic infants.” It is not clear at what site(s) B cells are stimulated to produce IgE, although it is unlikely that local intestinal IgE production is involved to any large extent because &E-positive B cells constitute ~2% of intestinal Ig-producing cells.2g*30Once produced, circulating IgE molecules can bind to receptors on basophils and mast cells (and possibly other cells). Secondary exposure to antigen causes cross-linkage of IgE and degranulation of these cells. Data from human and animal studies suggest that these events occur in the GI tract and lead to alterations of normal gut function. Intestinal

factor (GM-CSF)

‘Found in some rodent species. bMessenger RNA and/or product found in a variety of mast cell populations.31

gested by reports of IgA deficient patients having a greater incidence of food allergies.” In spite of these defenses, intestinal uptake of small quantities of immunologically intact macromolecules occurs in normal adults.21 Substances may traverse the epithelium through the intercellular space (paracellular, via tight junctions), epithelial cells (transcellular), or specialized epithelial cells (M cells) overlying Peyers’ patches.22*23 Generally only proteins of a molecular weight from 10,000 to 70,000 are capable of acting as antigens.24 For these proteins to stimulate immune responses, including antibody production, they must reach lymphocytes in the lamina propria, Peyers’ patches, spleen, lymph nodes, or circulation. In most food antigen exposures, oral tolerance occurs with suppression of local and systemic immune responses upon subsequent antigen challenge. This process is not fully understood but appears to involve T-suppressor cells and prevents over-reactivity (reviewed by Tomasiz5 and Mowat”). In some instances, particularly in genetically predisposed patients, ” food antigen exposure results in IgE production. This may be due to the lack of T-cell

Mast Cells

Mast cells are found in all layers of the GI tract but predominantly in the lamina propria and submucosa. These cells store or generate, upon activation, a host of potent chemical mediators (Table 1).5*31 Activation can be achieved by a number of different mechanisms including antigen or anti-&E crosslinking IgE bound to high-affinity surface receptors.32 Mast cells also have receptors for other homocytotropic antibodies4 and for anaphylatoxins generated during activation of the complement cascade (C,, and C,,).“” Mast cells can be activated during trauma and by certain chemicals (including neurotransmitter peptides). Mast cells are a heterogeneous population of cells that have been best studied in the rat. Here, intestinal mucosal mast cells (IMMCs) have been shown to possess different properties (Table 2) from connective tissue mast cells (CTMCs), isolated from the peritoneal cavity.35 IMMCs contain a soluble protease, rat mast cell protease type II (RMCP II), which is a convenient marker for activation because it is released into tissue fluids and serum and can be measured by sensitive immunoassays.3” IMMCs are susceptible to formalin fixation because the proteoglycan of the IMMC granule matrix is chondroitin di-B sulfate instead of heparin. Compared with CTMCs, IMMCs are much less sensitive to stimulation by various activators such as compound 48/80, bee venom, and neuropeptides.34 Antiallergenic compounds also show specificity of stabilizing action: drugs such as cromoglycate are effective in preventing histamine release from CTMCs but do not stabilize rat IMMCs, whereas doxantrazole prevents histamine release from activated mast cells of both categories. 37 The two populations of mast cells also

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Table 2. Rat

Mast Cell Heterogeneity Connective tissue mast cell

Mucosal mast cell

Nucleus Granules Granule constituents Protease Proteoglycans

Large (c. 20 pm), uniform Unilobed Many

Small (c. lo-12 pm), pleomorphic Unilobed or bilobed Few

Type I (RMCPI) Heparin

Histamine Serotonin IgE

215 pg/cell 11.5 pg/cell Surface

Type II (RMCPII) Chondroitin di-B sulfate 4 pg/cell 40 days Thymus independent

Blue 540 days Thymus dependent

++ ++ ++ +

++ ++ ++

0

0 ++

Note: Rough indications of the degree of effect of various compounds: + = some effect, +t = greater effect, and 0 = no effect. “Immature connective tissue mast cells stain blue. Modified and reprinted with permission from Stead et alZs’ Copyright CRC Press, Inc., Boca Raton, Florida.

differ in their dependence on growth factors; IMMCs require the T-cell cytokine interleukin 3 (IL-3), whereas CTMCs need fibroblast-derived growth factors.38 Elegant experiments injecting a single cell of a defined phenotype into specific sites in congenitally mast cell-deficient mice have shown phenotypic changes from IMMCs to CTMCs and vice versa, providing evidence for the importance of the microenvironment in phenotypic expression.3gp4o In humans, there is also evidence for heterogeneity of mast cells,414 although the situation is more complex than in the rodent. Even within the GI mucosa and muscle, two types of mast cells are present based on histochemical staining characteristics and protease content.45*4e One type similar to the IMMC is found mainly in the mucosa, contains a tryptase protease, and is depleted in immune-deficiency states (e.g., acquired immunodeficiency syndrome?‘) suggesting a dependence on T-cell cytokines. The other

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mast cell type contains both a tryptase and a chymase and is found in the submucosal/muscle layers.43 In formalin-fixed tissues, fewer mast cells are found, suggesting a fixation sensitivity similar to that in the rat.4* However, in humans, mediator-release and stabilization characteristics of mast cells do not appear to be as predictable from location and staining.42~44.4gIt is clear that it is not entirely feasible to extrapolate from studies of allergic reactions in other sites or species to those in the human intestinal tract. The Clinical Problem Although up to 45% of the population reports adverse reactions to food,50,51 the actual prevalence of immune-mediated food allergy is unknown5’ It is estimated to affect at least 2%-5% of the general population, with even higher values (up to 27%) in pediatric populations. 53 The incidence appears to be increasing, although this may reflect increased patient and physician reporting of allergic symptoms. Some of the difficulties in determining the actual incidence of food allergy result from problems in terminology and diagnosis. Accordingly, adverse reactions to foods have been categorized by the American Academy of Allergy and Immunology,54 with those involving immunological mechanisms, usually immediate hypersensitivity, classified as food allergy. Non-h&mediated immunologic reactions such as occur in celiac disease also fall into this category. Other categories of adverse reactions to food include idiosyncratic reactions (e.g., lactose intolerance), food toxicity or poisoning, pharmacological reactions (e.g., to caffeine or tyramine), and psychological reactions. The foods most frequently involved in food allergy include cow’s milk, wheat, eggs, corn, fish, seafood, and nuts.55 Symptoms are more common in atopic individuals who often have allergies to nonfood antigens such as pollens and molds56 and in young children who tend to “outgrow” their allergy.57 The process whereby children no longer react to the offending food(s) is unknown, because they continue to have in vivo (skin) and in vitro [radioallergosorbent test (RAST)] reactions to these foods and begin to develop airway reactivity to inhalant allergens as they grow older. In addition, age-based recovery appears to be food specific such that clinical reactions to eggs and milk are lost, whereas those to peanuts persist.58 The diagnosis of food allergy relies on historical documentation of reactions to specific foods that can be reproduced by ingestion of these foods. At present, the best method of confirming the diagnosis of a food allergy is the double-blind placebo-controlled food-ingestion challenge,2 a test in which the suspect

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food substance is disguised in a gelatin capsule or a drink so that it is not distinguishable from the placebo. Both the patient and physician are blinded with respect to the contents of the challenging vehicle. Reproducible reactions to the test food, but not placebo, constitute a positive test result. This test has been evaluated in adult5’ and pediatricW populations and found to correlate well with results of skin testing and RAST as well as basophil histamine release assays. ” However, most studies examiningthe prevalence of food allergy have not used these criteria. In those where suspected food allergies were tested by blinded challenge,5g~62-e4only 25%-45% of cases had positive results. The range of symptoms that occur in food allergies reflects the involvement of the upper and lower respiratory tracts (rhinitis and asthma), eyes (conjunctivitis), skin (urticaria and eczema), oropharynx (labial, lingual, and hypopharyngeal edema), and GI tract. Occasionally, severe and fatal anaphylaxis may result from accidental ingestion, particularly in peanut-sensitive patients. 58Arthritiss5 and migraine headaches” have also been attributed to food allergy, but the evidence for this is equivocal. Surindicate that 4O%-70%of food-allergic paveys 1,60,67 tients report GI symptoms, including nausea, vomiting, abdominal pain, bloating, and diarrhea. Although these symptoms can be significant, they are generally ignored by physicians because they are not life threatening, and available treatment options are directed at other target organs. Moreover, patients with primarily GI symptoms to foods may not be recognized by their physicians as having an allergic basis for their symptoms.

Human Studies The apparent disinterest in the GI aspects of food allergy by clinicians reflects in part, our limited understanding of the pathophysiology of this condition. The inaccessibility of the target organ, combined with a lack of objective tests, have resulted in largely descriptive studies of GI food hypersensitivity whereas relatively few reports have examined the basic mechanisms involved. Despite these limitations, significant information has been obtained from human studies. For example, some of the earliest work in this area showed that food-induced allergic reactions produced GI pathology. Passive sensitization of rectal mucosa in a nonallergic individual with serum from a peanut-allergic patient resulted in erythema, edema, and friability at the rectal site after the volunteer ingested peanuts.88 Similar experiments involving subjects with ileostomies and colostomies revealed that local allergic reactions also occurred at other GI sites.” Most recent studies have examined GI reactions in actual food-allergic pa-

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tients, rather than in artificially induced states of allergy, and these have usually relied on GI symptoms as a measurement index. In contrast to respiratory symptoms, which can be corroborated by changes in spirometry or skin reactions that are directly observable, GI reactions are more difficult to establish objectively. One early report using direct gastroscopic observation of the stomach illustrated that edema, erythema, petechiae, thick mucus, and reduced peristalsis occurred in response to food challenge.70 Barium radiography,71,72 fluoroscopy, and lactulose breath hydrogen testing73 suggest that GI motility may be altered as part of the response to specific foods in allergic individuals. Studies of allergic patients, mostly with atopic eczema, have examined intestinal permeability to a variety of probes: 51Cr-labeled ethylenediaminetetraacetic acid (EDTA),74 polyethylene glycol (PEG),75-77 and sugars (rhamnose, mannitol, and lactulose).76-*0 Results have been contradictory; some studies report increased permeability in the fasting, unchallenged 75*7g**o whereas others show no differences from state, contro1s.74*7”*78 However, following ingestion of specific foods there appears to be increased uptake of the probes.77,76*80In addition, enhanced uptake of a macromolecule, specifically egg albumin, has been reported in atopic children.” In contrast, serum levels of egg albumin were unchanged in children with food protein enterocolitis after food challenge.82 In vitro studies of jejunal biopsy specimens from children with cow’s milk allergy showed increased fluxes of the macromolecule horseradish peroxidase compared with control specimensE3 Increased serum levels of bovine serum albumin (BSA) and the milk protein (3-lactoglobulin (j3LG) have been observed after gluten challenge in celiac diseasesW Recently, increased leakage of plasma proteins has been described after intestinal inhalant allergen challenge in atopic patientsa Together these data suggest that the barrier function of the GI epithelium can be altered in some allergic individuals under baseline conditions and after specific antigen challenge. Several studies indicate that mast cell-dependent mechanisms occur in human GI allergic reactions. Selbekk et a1.86have shown that in vitro food antigen challenge of human jejunum, passively sensitized with serum from food-allergic patients, caused a decrease in the number of stained mast cells. This degranulation response was inhibited by the mast cellstabilizing drug sodium cromoglycate.*’ Symptoms of blinded intragastric food challenge in allergic patients correlated well with food-induced mast cell degranulation in mucosal biopsy specimens8* Other investigators have shown antigen-induced histamine release in duodenal biopsy specimens from

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children and adults with GI food allergies.8g~g0Recent experiments in which the jejuna of celiac patients were perfused with gliadin have indicated that both eosinophils and mast cells were activatedgl and that challenge was accompanied by loss of plasma proteins.g2 Investigators in Germanyg3sg4 and Polands5 have provided in vivo evidence for mast cell involvement in GI allergic reactions. Erythema and edema of the gastric mucosa were observed after direct endoscopic placement of antigens in allergic individuals. Reduced numbers of mast cells and decreased tissue histamine levels were found in biopsy specimens taken after antigen challenge, suggesting that mast cell degranulation had occurred to produce local tissue inflammation.94 The full significance of local IgE in GI food allergy has not been established. Elevated IgE levels have been found in small bowel aspirates of food-allergic adults,gs feces of allergic children,97 and intestinal washings of children with atopic eczema.g8 Increased IgE-bearing cells have been described in mucosal specimens from food-allergic patients by some groups99*‘oobut not by others.“l Moreover, reports of increased IgE-producing cells in the intestine of food-allergic patients should be viewed with caution, because the presence of IgE-bearing mast cells can bias the data.“’ The cornerstone of therapy in food allergy has been avoidance of the offending food(s).55 Oral or cutaneous desensitization has not proved useful in food agents inallergy. lo3 A variety of pharmacological tended to prevent mast cell degranulation (e.g., sodium cromoglycate and ketotifen3*‘“) or to inhibit mast cell mediators (e.g., antihistamineslo and cyclo-oxygenase inhibitors’05) has been tried in the management of food allergy. However, the evidence that currently available drugs are beneficial, particularly in GI hypersensitivity, is limited. Other GI Disorders IgE mast cell-mediated mechanisms may play a role in eosinophilic gastroenteritis, a disorder in which a history of food allergy and positive results of food skin tests and RASTs are often described.‘06’08 Because eosinophils have low-affinity receptors for food antigens could activate eosinophils to IgE, 109*110 release cytotoxic and inflammatory mediators including major basic protein (thought to be involved in the pathogenesis of this disease”‘). IgE-immune mechanisms may also be involved in the food protein enteropathies of childhood (cow’s milk and soy), although other immunological processes (e.g., cellmediated mechanisms and immune complexes) are felt to be more important in the pathogenesis of these disorders.12~“2

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The role of food allergies in a number of other human GI disorders is more speculative. It has been suggested that food allergies may mediate some of the symptoms of irritable bowel syndrome (IBS).l13 For example, 9 of 17 pediatric IBS patients with foodinduced intestinal hyperpermeability showed symptomatic improvement following elimination diets and cromoglycate treatment in some instances114 Similar results have been reported by other workers in this field.“5-“7 However, negative results with both food challenges and elimination diets in IBS patients have also been reported.“*-‘20 Moreover, in those studies suggesting a role for specific foods, it was not clear that IgE mast cell mechanisms were involved. Levels of rectal prostaglandin (PG) E, were elevated in IBS patients in one report,l15 suggesting that other cells were activated as mast cells release PGD, .121It has been proposed that infantile colic is caused by allergy to milk antigens,122 but again the evidence, although supportive, is inconclusive.‘23*‘24 Both IBS and colic represent heterogeneous disorders, and it is quite likely that in some cases allergic mechanisms may be involved. The question of whether allergy is related to the etiology or pathogenesis of IBD has been a moot point for many years.125 There are conflicting reports regarding the prevalence of atopy in both ulcerative colitis and Crohn’s disease, with some suggesting it is increased,‘26 whereas others have found no differences from control populations.12’ Elevated levels of circulating antifood antibodies’28-131 have been interpreted as evidence of food allergy but may simply reflect a secondary phenomenon in which greater uptake of food proteins, via damaged mucosa, stimulates an immune response. The beneficial responses observed with use of elemental diets or parenteral nutrition in IBD may be the result of improved nutrition, reducing the metabolic demands of digestion and decreasing antigenic stimulation.‘32r133 Maintenance of remission in Crohn’s disease has been observed in patients treated with elimination diets.‘34 A role for mast cells in IBD, especially in Crohn’s disease, has been suggested by studies showing increased mast cell numbers135 and intestinal (tissue or luminal) levels of mast cell mediators,136138 as well as evidence of enhanced mast cell degranulation. 139-141A recent report indicates that mast cell mediator release is enhanced in active ulcerative colitis. Some trials have found beneficial responses to sodium cromoglycate preparations in IBD,‘43*‘44although others have not.145*14sIt is difficult to draw any firm conclusions from the literature, but it is possible that food allergy and other mast cell-mediated mechanisms may play a role in the pathogenesis of IBD. Although unlikely to be the primary initiating event (as yet unknown), these mechanisms

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could well contribute to perpetuating the inflammatory process. Allergic mechanisms were proposed in the pathogenesis of peptic ulcer disease as early as 1931.‘47J48 Romanskig5 has shown that a large proportion of patients with peptic ulcer disease had reactions to antigens including foods by skin testing and by intragastric challenge. Increased numbers of &$-positive cells at the margins of peptic ulcers have been described.14’ Although this work is interesting, there is little other evidence to support IgE-mediated pathophysiology playing a role in human peptic ulcer disease. There are a variety of GI disorders that involve mast cells seemingly unrelated to the presence of food allergies. Well-known examples include systemic mastocytosis with involvement of the GI tract15’ and enteric parasitic infections. Significant improvement of both systemic and GI symptoms has resulted from treatment directed against mast cells or their mediators in mastocytosis.151 Elevated intestinal mast cell numbers have recently been described in microscopic colitis, in which improvement of diarrhea resulted after treatment with an H,-receptor antagonist. 15’In contrast to these clinical conditions, it is interesting to note that mast cell numbers are not increased in the intestine of patients with food allergies or in animal models of food hypersensitivity.153,154 Animal Studies Domestic animals can develop allergic reactions to food as evidenced by calves that react to artificial milk in a similar fashion to human infants drinking cow’s milk155,156and by the observation of hypersensitivity reactions in piglets’5”*‘57and dogs.15’ However, most comprehensive studies have, for obvious reasons, been carried out in smaller animals. Models of Gut Hypersensitivity Models of food hypersensitivity can involve either active or passive sensitization and have been developed mainly in rats, mice, and guinea pigs using chicken egg albumin or cow’s milk protein as antigens. Actively sensitized animals produce antigen-specific antibodies in response to food proteins. Subsequent exposure to that antigen, either in vivo or in isolated intestinal tissues from such animals (in vitro), is usually carried out approximately 2 weeks postsensitization. In passive sensitization, antibodycontaining serum is transferred into naive animals (in vivo) or added to isolated tissues (in vitro).159-‘61 Monoclonal antibodies directed against j3LG have also been used to produce sensitization in a mouse model.‘“’ Because mast cells have high-affinity re-

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ceptors for IgE, binding takes place quickly, and challenge can be conducted after 1-4 hours. Both types of sensitization have been used to study gut allergies and have provided interesting and complementary information. A novel approach to examine gut allergic reactions has used artificial “tissues” in which intestinal epithelial cells were cultured and combined with mast cells.‘s3~1s4If mast cells were passively sensitized, antigen presentation to such a system confirmed the findings in animal models.‘63 Studies using antibodies to IgE have also acted as models of antigen activation to further emphasize the role of mast cells and IgE in GI hypersensitivity reactions.*s5*166In addition, GI reactions to parasites or parasite antigens (see Russell and Castro167) have provided certain insights related to hypersensitivity reactions, and although these reactions are not the focus of this article, they will be referred to when appropriate. The production of large quantities of IgE antibodies may not be essential for a good model of GI allergy as certain IgG subclasses (IgG, in humans, IgG,, in rodents, and IgG, in guinea pigs) can also bind to and activate mast cells4 In addition, measurement of the level of circulating antibodies may not reflect antibody levels in gut tissues. However, in one study involving egg albumin-sensitized rats, serum titers of IgE measured by passive cutaneous anaphylaxis were proportional to the magnitude of antigen-induced physiological changes.lm IgE production is determined by a number of elements including host-related factors (genetics and “,16’ ) and antigen-associated factors (dose, route, age and presence of adjuvants’70-‘72). These factors have been evaluated most thoroughly in rodents, in whom it has been established that certain rat strains (e.g., Hooded Lister) have high levels of IgE under normal circumstances and produce significantly higher antibody titers following primary immunization compared with low-responder strains (e.g., Spragueit is worth noting that Dawley).16’ However, consistent physiological responses to antigens are observed in the GI tract of both high- and low-responder rats. IgE can be induced following oral administration of proteins,‘73~‘74 but tolerance to ingested antigens may occur with suppression of specific IgE with or without stimulation of 1gA.l” Because the most dependable stimulation of IgE production is observed when antigen is administered by the intravenous, intraperitoneal, subcutaneous, or intradermal most animal models of hypersensitivity route,‘75 have used these methods. Sensitization with food proteins alone does not usually result in significant production of IgE.“l Adjuvants commonly used, which stimulate production of antigen specific and

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nonspecific IgE (as well as homocytotropic IgG), are aluminum hydroxide (alum) and Bordetella pertussis vaccine.176~177 Little is known regarding the mechanisms by which these adjuvants stimulate specific antibody isotype production. Helminth parasite infection in rats also potentiates IgE responses to prior antigen exposure’7o and has been used in some egg albumin-sensitized models.“’ Occasionally other substances such as Freund’s complete adjuvant, silica gel, HgCl,, and lipopolysaccharide have been used for their adjuvant effect in animal models 171,179,160 Guinea pigs can be sensitized to raise IgE antibodies by injection of protein antigens alone or with adjuvantP (or by infection with helminth parasites.161~‘az Guinea pigs have also been used to develop a clinically relevant model of food hypersensitivity in which animals are sensitized by the oral route. After feeding guinea pigs with cow’s milk for approximately 3 weeks, challenge with whole milk or PLG (after 3 days without milk) results in an anaphylactic reaction.163 Isolated segments of intestine from these animals respond to antigen with an ionsecretory response,15g~*64 a response that can also be transferred with serum from immunized animals.‘5”161 However, in this particular model, GI reactions are IgG mediated.183’165 Responses to Antigen Challenge Although a variety of symptoms (diarrhea, abdominal pain, nausea, and vomiting) is reported in GI allergic reactions to food, the underlying mechanisms that give rise to these symptoms are not well understood. Animal models have afforded an excellent opportunity to examine the physiological consequences of short-term antigen challenge in vitro and in vivo and to elucidate the basic mechanisms involved. A consistent feature of such studies is the specificity of the response to challenge with the sensitizing antigen. Most experiments have described changes in the intestinal mucosa (mainly in the small bowel but also in the colon), either in terms of ion transport or permeability (measured by the uptake or excretion of marker molecules). More recently, studies have also examined changes in the stomach (motility, permeability, and gastric acid secretion) as well as alterations of intestinal motility following antigen challenge. Intestinal transport. Alterations of intestinal transport (enhanced secretion and/or decreased absorption) are an important mechanism in the pathogenesis of diarrhea166~“‘7 and may contribute to the symptoms of GI food allergy.‘*’ In vivo and in vitro experiments in sensitized animals showing changes in salt and water transport following oral or luminal challenge with specific antigens have provided in-

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sight into the mechanisms involved in these alterations. Hooded Lister rats sensitized to egg albumin showed a significantly reduced net absorption of Na+, Cl-, K+, and water within 40 minutes of adding egg albumin to a perfused segment of jejunum.lW These changes were associated with mucosal histamine depletion and increased histamine recovery in the perfusates”’ as well as villous edema (Figure l)."'Doxantrazole, but not cromoglycate, in the perfused buffer prevented these changes,lW implicating IMMCs in the response. Similar antigen-induced abnormalities were later identified in the colon in the same model.lgl Other in vivo studies using SpragueDawley rats sensitized to egg albumin have described fluid accumulation in the gut lumen and evidence of mast cell activation in response to an enterically administered antigen bolus.154~1g2~1g3 In mice passively sensitized with monoclonal IgE against PLG, gastric administration of antigen resulted in fluid accumulation in the small intestine and diarrhea.16’ In vitro studies in Ussing chambers have supplied further information regarding the driving force and mechanisms involved in the transport responses to antigen. In models of GI food hypersensitivity, addition of antigen to voltage-clamped intestinal tissues has produced transient increased in short-circuit current (Isc, a measure of net ion transport) due largely to secretion of chloride ion. It is worth noting that chloride secretion in vitro generally correlates with fluid secretion in vivo and when the magnitude of chloride and fluid secretion is great, it correlates with diarrhea in various disease states. In egg albumin-sensitized Hooded Lister rats, antigen challenge to the serosal side of isolated full-thickness jejunal segments resulted in a rapid increase in Isc, which

Figure I. Photomicrographs from a single section of rat jejunum (original magnification ~50) obtained from a sensitized rat after egg albumin challenge. (A)Normal morphology. (B)Mucosal edema. (Reprinted with permission.“)

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was inhibited by doxantrazole.‘94 Simultaneous experiments documented elevated cyclic adenosine monophosphate (CAMP) levels in the mucosa 2 minutes after egg albumin challenge. When antigen was added to the mucosal side, a minimal increase in Isc occurred 20-30 minutes after challenge. Together, this information suggested that IMMCs located in the subepithelial compartment were activated by antigen to release mediators that stimulated Cl- secretion from epithelial cells possibly by elevating intracellular CAMP levels. Subsequent studies showed a reduced Isc response following pretreatment with the HI-receptor antagonist diphenhydramine and the neural blocker tetrodotoxin’g5 or with the 5-hydroxytryptamine, (5-HT,)-receptor antagonist cinanserin.lgs Recent studies in this model have also implicated leukotrienes and platelet-activating factor in the secretory response to serosal antigen.lg7 Ion-transport abnormalities have also been reported in response to antigen in isolated musclestripped intestinal tissues from cow’s milk-sensitized guinea pigs. Preparations of duodenum, ileum, and colon all showed secretory (1s~)responses within minutes of adding antigen to the serosal side of tissue in the Ussing chamber.‘5Q*‘*4 In the colon, the response was abolished by indomethacin and reduced by the HI-receptor antagonist mepyramine, implicating eicosanoid production and histamine involvement in the response. 15QIn contrast, the only pharmacological agents with an effect on Isc responses in the ileum were serotonin antagonists, particularly the 5HT, antagonist ICS 205-930 and tetrodotoxin.“’ These drugs also inhibited colonic responses. These results suggest that 5-HT receptors on enteric nerves were involved in BLG-induced secretion but do not identify the source of 5HT or nerve type. Secretory responses to mucosal (luminal) antigen were also obtained in the ileum of cow’s milk-sensitized guinea pigs. These were smaller than the serosal responses and were inhibited if preceded by seroHowever, unlike serosal sal j3LG challenge.lw responses, these were not examined usingpharmacological agents. In experiments involving colonic tissues, responses to mucosal antigen were obtained only if the tissues were first “damaged” by radiation, detergent (bile salt), or incubation at room temperaAs in the ileum, mucosal responses in ture. 160~1QQ such pretreated colonic tissue were decreased by prior serosal antigen challenge, suggesting that the effector mast cell was in the subepithelial compartment most easily accessed from the serosal side. Passive sensitization of control guinea pig colon was achieved when serum from milk-drinking animals was applied to the serosal but not the apical epithelial (luminal) surface unless tissues were damaged as above.‘60 Antigen challenge of passively sensitized

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tissues resulted in Isc changes similar to those observed in actively sensitized animals. Recent studies in egg albumin-sensitized SpragueDawley rats have provided information with clinical relevance to gut hypersensitivity conditions in which antigens are found intraluminally rather than via the antiluminal (serosal) route. Cl--secretory responses similar to those observed in other food protein- or parasite-sensitizedzoo models were elicited in isolated jejunal tissues in response to serosal egg albumin challenge. However, unlike most othermodels, significant Isc responses were also found within 3 minutes of adding antigen to the luminal side in the absence of any pretreatment of tissues with damaging agents.“* Experiments with inhibitors once again implicated histamine, serotonin, prostaglandins, and enteric nerves in egg albumin-induced ion transport, but this time in response to luminal antigen (Table 3). In addition, neonatal capsaicin treatment, which depletes substance P-containing nerves, reduced secretion by half (Table 3). These results suggest that stimulation of Cl- secretion by the intestinal epithelium during intestinal anaphylaxis is caused by a combination of effects of mast cell mediators and neurotransmitters such as substance P. Interestingly, tissues from these sensitized rats in the absence of antigen also showed supramaximal Isc responses to the inflammatory mediators histamine and substance P compared with tissues from control rats.“* These findings suggest that intrinsic changes in intestinal tissues occurred following sensitization. Similar hypersecretory responses to secretagogues have been reported by Barrett in the coionic epithelial cell line T84 after coculture with intact cells or lysates of the rat mucosal mast celllike cell line RBL-2H3.1M The studies described up to this point suggest that Table 3. Effect of Pharmacological Agents on Ion Transport Responses to Luminal Antigen in Jejunum From Egg Albumin-Sensitized Rats Agent Doxantrazole Sodium cromoglycate Diphenhydramine Ketanserin Piroxicam Tetrodotoxin Neonatal cansaicin

Untreated 31.8 30.3 53.6 39.4 35.8 54.4 41.7

k + f f f f f

Treated

% control

5.6

17.0 f 2.5a

53.5

4.7

25.6 k 4.2 31.7 f 3.6b 27.7 f 3.2”

64.5 59.1 70.3

17.2 + 5.Zb 34.7 + 5.4a 19.6 f 3.6”

46.0 63.8 47.0

7.2 5.4 3.5 6.3 7.6

NOTE. Values (mean + SEM) represent the maximum increase in Isc current @A/cm’) after luminal antigen. Tissues were left untreated or treated by adding pharmacological agents to tissues at least 15 minutes before antigen. The third column shows results expressed as % of untreated control responses [n = 6-12 for each group). “P < 0.05, bP < 0.01 compared with untreated tissues. Modified from Crowe et al.“’ with permission.

September

GUT FOOD ALLERGY REVIEW

1992

mast cells are responsible for the ion transport abnormalities that occur during intestinal hypersensitivity responses to antigens. However, most of the evidence is indirect, based on inhibiting the production of or antagonizing the effects of mediators released from mast cells or, in some situations, by the use of mast cell-stabilizing drugs. Because the mediators implicated are also produced by other cell types, different approaches have been adopted to provide more direct information regarding the role of mast cells in intestinal anaphylaxis. In one approach, epithelial cells (human colonic adenocarcinoma cells) were cultured on filter supports and sandwiched with mast cells isolated from the peritoneal cavity of rats or guinea pigs actively sensitized to egg albumin or tissues” were then (3LG?“3 These “reconstructed mounted in Ussing chambers, where they were found to respond to antigen by an increase in Isc. Antigens were only effective when added to the mast cell (serosal) side of the preparation. As would be expected in these “tissues” without nerves, tetrodotoxin had no inhibitory effect on PLG-induced Isc responses. Another approach has used W/W” mice, which have a mutation involving the putative tyrosine kinase receptor, c-kit, resulting in abnormal m_ast cell precursors. 202~203 Adult mice have