Gastrointestinal Eosinophils in Health and Disease

ADVANCES IN IMMUNOLOGY, VOL. 78

Gastrointestinal Eosinophils in Health and Disease MARC E. ROTHENBERG,* ANIL MISHRA, ERIC B. BRANDT, AND SIMON P. HOGAN Division of Pulmonary Medicine, Allergy and Clinical Immunology, Department of Pediatrics, Children’s Hospital Medical Center, Cincinnati, Ohio 45229-3039

I. Introduction

Eosinophils are multifunctional proinflammatory leukocytes implicated in the pathogenesis of numerous inflammatory processes, especially allergic disorders (Gleich and Adolphson, 1986; Weller, 1991); in addition, it has been recently recognized that eosinophils may have a physiological role in organ morphogenesis (e.g., postgestational mammary gland development) (Gouon-Evans et al., 2000). Eosinophils express numerous receptors (for cytokines, immunoglobulin, and complement proteins) that when engaged lead to eosinophil activation, resulting in several processes, including the release of toxic secondary granule proteins (Fig. 1) (Rothenberg, 1998). The secondary granule contains a crystalloid core composed of major basic protein (MBP) and a granule matrix which is mainly composed of eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), and eosinophil peroxidase (EPO). These proteins elicit potent cytotoxic effects on a variety of host tissues at concentrations similar to those found in biological fluid from patients with eosinophilia. The cytotoxic effects of eosinophils may be elicited through multiple mechanisms, including degrading cellular ribonucleic acid, since ECP and EDN have substantial functional and structural homology to a large family of ribonuclease genes (Rosenberg et al., 1995; Slifman et al., 1986). ECP also inserts ion-nonselective pores into the membranes of target cells, which may allow the entry of the cytotoxic proteins (Young et al., 1986). Further proinflammatory damage is caused by the generation of unstable oxygen radicals formed by the respiratory burst oxidase apparatus and EPO. Furthermore, direct degranulation of mast cells and basophils is triggered by MBP. In addition to being cytotoxic, MBP directly increases smooth muscle reactivity by causing dysfunction of vagal muscarinic M2 receptors (Jacoby et al., 1993). Activation of eosinophils also leads to the generation of large amounts of leukotriene (LT)C4, which induces increased vascular permeability, mucous secretion, and smooth muscle constriction (Lewis et al., 1990). Additionally, activated eosinophils generate a wide range of cytokines, including interleukin (IL)-1, IL-3, IL-4, IL-5, IL-13, GM-CSF, TGF-/, TNF-, RANTES, macrophage inflammatory protein (MIP)-1, vasoactive intestinal ∗ To whom correspondence should be addressed. Telephone: (513) 636-7210; Fax: (513) 636-3310;

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FIG. 1. Schematic diagram of an eosinophil and its diverse properties. Eosinophils are bilobed granulocytes that respond to diverse stimuli including allergens, helminths, viral infections, allografts, and nonspecific tissue injury. The secondary granules contain four primary cationic proteins designated eosinophil peroxidase (EPO), major basic protein (MBP), eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN). All four proteins are cytotoxic molecules; in addition, ECP and EDN are ribonucleases. In addition to releasing their preformed cationic proteins, eosinophils can also release a variety of cytokines (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-12, TGF/, GM-CSF, TNF, and IFN ), chemokines (e.g., eotaxin, RANTES, and MIP-1), neuromediators (vasoactive intestinal peptide [VIP] and substance P), and can generate large amounts of leukotriene (LT)C4. Lastly, eosinophils can be induced to express MHC class II and costimulatory (e.g., B7.2) molecules, and may be involved in propagating immune responses by presenting antigen to T cells.

peptide, substance P, and eotaxin, indicating that they have the potential to sustain or augment multiple aspects of the immune response, inflammatory reaction, and tissue repair process (Kita, 1996). Finally, eosinophils have the capacity to initiate antigen-specific immune responses by acting as antigen-presenting cells. Consistent with this, eosinophils express relevant costimulatory molecules (CD40, CD28, CD86, B7) (Ohkawara et al., 1996; Woerly et al., 1999), secrete cytokines capable of inducing T cell proliferation and maturation (IL-2, IL-4, IL-6, IL-10, IL-12) (Kita, 1996; Lacy et al., 1998; Lucey et al., 1989), and can be induced to express major histocompatibility complex (MHC) class II molecules (Lucey et al., Weller, 1989). Interestingly, experimental adoptive transfer of antigen-pulsed eosinophils induces antigen-specific T cell responses in vivo (Shi et al., 2000). Increased levels of eosinophils are seen in the gastrointestinal tissue obtained from patients with a variety of disorders (Table I). In some of these diseases (e.g., eosinophilic gastroenteritis), eosinophils are believed to be the principal

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TABLE I EOSINOPHIL-ASSOCIATED GASTROINTESTINAL DISEASESa Primary Eosinophil Disorders Eosinophilic colitis Eosinophilic esophagitis Eosinophilic gastroenteritis Gastrointestinal Disorders Allergic colitis Inflammatory bowel disease Food allergy Gastroesophageal reflux Protein-sensitive enteropathy Systemic Disorders Idiopathic hypereosinophilic syndrome Parasitic infections (helminthic) a

These exclude primary diseases of the biliary tract.

effector cell, whereas in other diseases (e.g., inflammatory bowel disease and gastroesophageal reflux), the finding of eosinophils in tissues is an enigma, since eosinophils do not always appear to be degranulating, implying that they may have a regulatory function (Furuta et al., 1995; Gleich and Adolphson, 1986; Rothenberg, 1998; Weller, 1991). Understanding the processes that regulate eosinophil trafficking in the gastrointestinal tract is not only important in clinical diseases but may also have important implications in further understanding the role of eosinophils in innate immune responses and in immune surveillance of healthy tissues. Whereas numerous reviews have recently been written concerning eosinophils (Weller, 1991), their regulation (Hirai et al., 1997; Walsh, 1997), and role in allergic respiratory diseases (Capron and Desreumaux, 1997; Desreumaux and Capron, 1996; Gleich, 2000; Seminario and Gleich, 1994), this article is a comprehensive review focused on the properties and role of gastrointestinal eosinophils in health and disease. II. Gastrointestinal Eosinophils in Healthy States

Eosinophils have been noted to be present at low levels in numerous tissues. When a large series of biopsy and autopsy specimens were analyzed, the only organs that demonstrated tissue eosinophils (at substantial levels) were the gastrointestinal tract, spleen, lymph nodes, and thymus (Kato et al., 1998). Interestingly, eosinophil infiltrations were only associated with eosinophil degranulation in the gastrointestinal tract. For the purpose of systematically characterizing eosinophils, recent analyses have been primarily conducted in mice, since this species allows unique experimental manipulation. Identification of eosinophils

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has been facilitated by development of antiserum against murine MBP, since this reagent allows for eosinophil-specific recognition (Larson et al., 1995; Mishra et al., 1999). Interestingly, similar to human tissues, eosinophils predominantly reside in the hematopoietic organs, gastrointestinal tract, and thymus of mice (Matthews et al., 1998). A. LAMINA PROPRIA EOSINOPHILS Eosinophils throughout the gastrointestinal tract of conventional healthy mice (untreated mice maintained under pathogen-free conditions) are present in the lamina propria of the stomach, small intestine, cecum, and colon. No eosinophils are detected in the esophagus or tongue, which is a pattern of distribution that has been observed in humans (Furuta et al., 1995; Kato et al., 1998). Eosinophils are at similar levels in all mouse strains analyzed (BALB/c, C57BL/6, 129 SvEv, and Black Swiss Webster) and are predominantly localized in the submucosa and not in the mucosal or serosal layers of the gastrointestinal tract (Mishra et al., 1999). The distribution of eosinophils varies in different regions of the small intestine. For example, in the duodenum, eosinophils are primarily detected through the entire length of villi. Whereas in the jejunum and ileum, most eosinophils are at the base of villi in the region of the crypt of Lieberkuhn ¨ (Mishra et al., 1999). B. PEYER’S PATCH AND INTEREPITHELIAL EOSINOPHILS The identification of eosinophils as resident cells of the gastrointestinal lamina propria during normal healthy states (Matthews et al., 1998; Mishra et al., 1999) prompted the question whether eosinophils also resided in intestinal Peyer’s patches. B and T lymphocytes recirculate between three predominant compartments in the gastrointestinal tract (lamina propria, Peyer’s patches, and interepithelial regions). Using immunohistochemical techniques with the anti-MBP antibody, eosinophils were barely detected in any region of the Peyer’s patches or in the interepithelial compartment; whereas, the same mice had detectable eosinophils within the lamina propria (Matthews et al., 1998; Mishra et al., 1999). Thus, in contrast to lymphocytes, gastrointestinal eosinophils reside only in the lamina propria during healthy conditions. C. EOSINOPHILS IN PERINATAL MICE AND THE ROLE OF ENDOGENOUS FLORA Early studies have demonstrated a significant reduction in the number of lymphoid cells in the gastrointestinal tract of perinatal mice (Ferguson and Parrott, 1972a, 1972b). Lymphocytes and plasma cells do not appear in the lamina propria of the gastrointestinal tract until 3 weeks after birth (i.e., around the time of weaning) and only reach adult levels by 6 weeks of age in mice. Similarly, mast cells home into the gastrointestinal tract primarily after the first month of life in

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rats (Watkins et al., 1976; Woodbury and Neurath, 1978). An exception to this homing pattern is seen with  /-T cells; these cells home to their interepithelial location in the absence of intestinal flora (Bandeira et al., 1990). Thus, it was of interest to determine whether eosinophils migrate into the gastrointestinal tract as a normal developmental process during gestation or if they migrate during the postnatal period in response to an extrinsic stimulus. Migration during the postnatal period would suggest that factors such as gastrointestinal colonization with bacteria are involved in initiating eosinophil homing. The absence of resident neutrophils in the gastrointestinal tract at baseline, suggested that granulocyte homing was unlikely to be a mere consequence of intestinal flora, since neutrophils would be expected to respond to this stimulus. We therefore examined the number of intestinal eosinophils in the mice 1 day prior to birth (embryonic Day 19) and during the first 2 weeks postpartum. Prenatal mice had readily detectable eosinophils located in similar regions as observed in adults (Mishra et al., 1999). In order to make comparisons between the experimental groups (e.g., to control for the growing size of the gastrointestinal tract during this time period), eosinophil numbers were normalized per unit area of the lamina propria and their concentrations were found to be similar at all ages (Fig. 2). Staining for CD45, a pan-leukocyte marker, assessed the presence

FIG. 2. Gastrointestinal eosinophil numbers in embryonic and perinatal mice. The number of eosinophils (A) and total leukocytes measured by CD45+ cells (B) residing in the small intestine of wild-type mice was determined in Day 19 embryos and postnatal mice during the first 2 weeks of life and in adult mice. Cell levels were normalized to the unit area (mm2) of the lamina propria. The mean ±SEM for littermate 129 SvEv mice (n = 5–12 mice in each group) is shown. In (A), there was no significant difference between any of the groups (p > 0.05). Reprinted in part with permission (Mishra et al., 1999).

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of total leukocytes in the gastrointestinal tract. CD45+ cells were substantially reduced in the perinatal mice compared with adult mice (Fig. 2), indicating that eosinophil homing was unique compared to most other gastrointestinal leukocytes. In an attempt to identify the stimulus for eosinophil homing, the role of endogenous intestinal bacterial flora on eosinophil homing into the gastrointestinal tract was examined by analyzing eosinophil levels in germ-free mice. Germ-free mice have never come in contact with viable bacteria and have been shown to have decreased levels of lymphocytes in the lamina propria (Ferguson, 1976). However, germ-free mice were found to have eosinophil levels that were similar to control colonized mice (Mishra et al., 2000b). Taken together, these results suggest that eosinophil homing into the gastrointestinal tract occurs prenatally, is independent of the presence of viable bacterial flora, and appears to be regulated by mechanisms distinct from those regulating other gastrointestinal leukocytes (e.g., mast cells and lymphocytes). D. ROLE OF CONSTITUTIVE EOTAXIN Numerous inflammatory mediators have been implicated in regulating eosinophil accumulation including IL-1, IL-3, IL-4, IL-5, IL-13, and granulocyte/ macrophage-colony stimulating factor (GM-CSF) and the chemokines RANTES, monocyte chemoattractant protein (MCP)-3, MIP-1, and eotaxin (Silberstein, 1995; Teixeira et al., 1995). Interleukin-3 and GM-CSF, in association with IL-5, have been shown to enhance eosinophil development, migration, and effector function, while IL-1, IL-4, IL-13, and tumor necrosis factor (TNF)- regulate eosinophil trafficking by promoting adhesive interactions with the endothelium (Bochner et al., 1995; Bochner and Schleimer, 1994). In collaboration with IL-5, chemokines and lipid mediators [platelet-activating factor (PAF) and leukotriene B4] induce eosinophil trafficking by promoting chemoattraction. Of the mediators implicated in modulating eosinophil accumulation, only the recently described subfamily of chemokines, termed eotaxin, is specific for eosinophils (Rothenberg, 1999). Eotaxin was originally described as the chief eosinophil chemoattractant generated in a guinea pig model of allergic lung disease (Jose et al., 1994). Subsequently, several other chemokines (of the CC family) have been shown to be eosinophil-selective and have been termed eotaxin-2 (identified in mice and humans) and eotaxin-3 (identified only in humans) (Forssmann et al., 1997; Kitaura et al., 1999; Shinkai et al., 1999; Zimmermann et al., 2000). It is important to note that although several chemokines have activity on eosinophils, eotaxin is the only chemokine that was discovered based on a biological assay designed to identify eosinophil-specific chemoattraction, attaching further biological significance to this molecule. When the eotaxin cDNA from guinea

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pigs, mice, and humans was identified, the mRNA for this chemokine was noted to be constitutively expressed in a variety of tissues (Garcia-Zepeda et al., 1996; Rothenberg et al., 1995a,b). A finding in all species was that the intestine expressed relatively high levels of eotaxin mRNA. Examination of multiple segments of the gastrointestinal tract of mice for expression of eotaxin mRNA indicated that this chemokine was ubiquitously expressed, at variable levels, in all segments from the tongue to the colon. Notably, the constitutive expression of eotaxin was distinct from the expression of related chemokines (MCP-1, MCP-2, MCP-3, MIP-1), which were not readily detectable. Only RANTES and eotaxin-2 were detectable, but they were not ubiquitously expressed (Mishra et al., 1999; Zimmermann et al., 2000). The expression patterns of eosinophil-active chemokines at baseline indicated that eotaxin may be involved in the selective regulation of eosinophil homing in the gastrointestinal tract. Furthermore, eotaxin mRNA expression in the small intestine is localized to mononuclear cells that reside in the lamina propria, the region where most gastrointestinal eosinophils reside (Matthews et al., 1998). In order to test the role of eotaxin in regulating eosinophil homing to the gastrointestinal tract, the number of eosinophils in the small intestine of mice deficient in eotaxin (through gene targeting) (Rothenberg et al., 1997) was shown to be significantly lower when compared with wild-type mice (Matthews et al., 1998). For example, the duodenum, jejunum, and ileum of wildtype mice had 6.2 ± 1.0, 2 ± 0.4, and 1.7 ± 0.5 eosinophils/villus in comparison to eotaxin-deficient mice which had 0.52 ± 0.14, 0.09 ± 0.02, and 0.05 ± 0.03 eosinophils/villus, respectively. Other gastrointestinal segments were also examined in order to determine if eotaxin had a similar role in other regions of the gastrointestinal tract. Eosinophils were only rarely encountered in any segment of the gastrointestinal tract of eotaxin-deficient mice. The eosinophil numbers (mean ± SEM) in the stomach, cecum, and colon of wild-type mice were 36.7 ± 7.5, 69.3 ± 14, and 39 ± 10 cells/mm2, respectively, whereas eotaxin gene-targeted mice had 2.6 ± 0.6, 8.9 ± 2.9, and 7.4 ± 2.8 cells/mm2, respectively. No significant differences were observed in the level of bone marrow and peripheral blood eosinophils in these two groups of mice, indicating that eotaxin was having a tissue specific effect rather than a primary effect on eosinophil hematopoiesis (Mishra et al., 1999). To further support the hypothesis that eotaxin had a critical role in regulating eosinophil recruitment to the gastrointestinal tract, the level of gastrointestinal eosinophils in mice that were deficient in another eosinophil-active CC chemokine gene, MIP-1, was also examined. These gene-targeted mice did not have an alteration in the level of gastrointestinal eosinophils compared to wild-type mice (Mishra et al., 1999). Collectively, these data suggest that eotaxin has a key role in the modulation of eosinophil accumulation in the gastrointestinal tract and that the effect is primarily tissue specific.

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E. EOSINOPHIL HEMATOPOIETINS Interleukin-3, IL-5, and GM-CSF have been shown to be critically involved in the proliferation and accumulation of eosinophils in response to allergic stimuli (Lopez et al., 1986; Owen et al., 1987; Rothenberg et al., 1988, 1989). These three cytokines also regulate the postmitotic differentiation of eosinophils, including their survival, activation, and responsiveness to other signals (Silberstein et al., 1989). Additionally, activated eosinophils and eosinophils exposed to tissue components such as fibronectin have been shown to produce these cytokines, in particular GM-CSF (Anwar et al., 1993). It has been hypothesized that an autocrine process may be responsible, at least in part, for eosinophil tissue survival. The physiological role of IL-5 and the related cytokine, GM-CSF, was therefore investigated for their involvement in the regulation of baseline levels of gastrointestinal eosinophils. The IL-5 deficient mice but not the GM-CSF deficient mice had ∼50% reduction in gastrointestinal eosinophils compared with age, sex, and background controlled mice (Mishra et al., 1999). Mice that were deficient in the functional receptor for IL-5 and GM-CSF (c-gene-targeted mice) were also examined (Mishra et al., 1999). The c-deficient mice (C57BL/6) had ∼80% reduction in gastrointestinal eosinophils compared with control mice. The effect of IL-5 and c was not restricted to the gastrointestinal tract because mice deficient in these molecules had a ∼75% reduction in circulating levels of eosinophils. These data suggest that GM-CSF and IL-5 have a combined effect in regulating eosinophil levels in the gastrointestinal tract; however, this effect appears to be secondary to their effect on regulating the pool of circulating eosinophils. F. REGULATION BY LYMPHOCYTES T cells and their products (e.g., IL-5) are known to regulate the development of peripheral blood and pulmonary eosinophilia following allergen challenge (Gavett et al., 1994; Hogan et al., 1998b). It was therefore of interest to analyze the role of T cells in the maintenance of gastrointestinal eosinophil levels at baseline. Athymic nude mice are deficient in the majority of T cells and have impaired eosinophil responses to allergens and parasites (Hamelmann et al., 1997b). However, these mice have no difference in the level of eosinophils in the jejunum (1.7 ± 0.3 vs. 1.07 ± 0.15 [mean ± SEM, n = 8]) for control and athymic mice, respectively (Mishra et al., 1999). To investigate the role of residual T cells (e.g., intraepithelial  /-T cells) and B cells in regulating eosinophil accumulation in the gastrointestinal tract, mice deficient in the recombinase gene-1 (RAG-1) were examined (Mombaerts et al., 1992). These mice also had a ∼50% reduction in jejunum eosinophils compared with strain matched controls. Analysis of blood and bone marrow levels of eosinophils in the RAG-1 deficient and athymic nude mice revealed significant increases in eosinophils

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(Mishra et al., 1999). These data indicate that in contrast to their critical role in regulating eosinophils during inflammatory conditions, lymphocytes do not have a major role in maintaining homeostatic levels of eosinophils in the gastrointestinal tract. III. Gastrointestinal Eosinophils in Disease States

The accumulation of eosinophils in the gastrointestinal tract is a common feature of numerous disorders such as drug reactions (Rothenberg, 1998), helminth infections (Behm and Ovington, 2000), idiopathic hypereosinophilic syndrome (Assa’ad et al., 2000; Bauer et al., 1996; Weller, 1994), eosinophilic esophagitis (Kelly, 2000), eosinophilic gastroenteritis (Katz et al., 1984; Keshavarzian et al., 1985; Torpier et al., 1988), allergic colitis (Hill and Milla, 1990; Odze et al., 1995; Sherman and Cox, 1982), inflammatory bowel disease (Dvorak, 1980; Sarin et al., 1978; Walsh and Gaginella, 1991), and gastroesophageal reflux (Brown et al., 1984; Liacouras et al., 1998; Winter et al., 1982) (Table I). A subset of these disorders may comprise a distinct set of hypersensitivity disorders that lies in the middle of a spectrum ranging from anaphylaxis to celiac disease (Fig. 3) (Moon and Kleinman, 1995; Saavedra-Delgado and Metcalfe, 1985; Sampson, 1999). Although the underlying causes of eosinophilic gastrointestinal disorders are not yet understood, several investigations have demonstrated an association between atopy and eosinophil-associated gastrointestinal disorders (Furuta et al., 1995; Sampson, 1999). For example, nearly half of the patients with eosinophilic gastrointestinal disorders are atopic as defined by elevated levels of

FIG. 3. The spectrum of inflammatory disorders of the gastrointestinal tract associated with eosinophil accumulation. Increased levels of eosinophils in the gastrointestinal tract occur in a wide variety of primary gastrointestinal disorders. These diseases vary in spectrum from strong dependence (e.g., food allergy) to low dependence (e.g., celiac disease) on IgE. Diseases in the intermediate spectrum are characterized by specific organ inflammation primarily associated with eosinophil accumulation (e.g., eosinophilic esophagitis, eosinophilic gastroenteritis, and eosinophilic colitis). In the latter set of diseases, increased levels of IgE have been associated with the disorders in a subset of patients, but the etiological role of IgE is not clear.

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total IgE or food-specific IgE (Caldwell et al., 1975; Cello, 1979; Furuta et al., 1995; Iacono et al., 1996; Sampson, 1997; Scudamore et al., 1982). In addition, IgE-mediated mast cell degranulation in eosinophilic gastroenteritis has been demonstrated (Oyaizu et al., 1985). Nevertheless, food-induced IgE-mediated reactions have been shown to be critical in only a minority of patients (Sampson, 1999; Talley et al., 1990). The accumulation of gastrointestinal eosinophils can occur in all regions of the gastrointestinal tract and can involve all depths of the tissue, including the mucosa, muscularis, and/or serosal layers (Kelly, 2000). In vitro studies have shown that eosinophil granule constituents are toxic to a variety of tissues including heart (Tai et al., 1984), brain (Venge et al., 1980), bronchial epithelium (Frigas et al., 1980), and intestinal epithelium (Gleich et al., 1979). Clinical investigations have demonstrated extracellular deposition of MBP and ECP in the small bowel of patients with eosinophilic gastroenteritis (Dvorak, 1980; Keshavarzian et al., 1985; Tajima and Katagiri, 1996; Talley et al., 1990; Torpier et al., 1988) and have shown a correlation between the level of eosinophils and disease severity (Desreumaux et al., 1996; Talley et al., 1990). Electron microscopy studies have revealed ultrastructural changes in the secondary granules (indicative of eosinophil degranulation and mediator release) in duodenal samples from patients with eosinophilic gastroenteritis (Torpier et al., 1988). Furthermore, Charcot–Leyden crystals, remnants of eosinophil degranulation, are commonly found on microscopic examination of stools obtained from patients with eosinophilic gastroenteritis (Cello, 1979; Klein et al., 1970). Despite these clinical findings, there is currently only a limited understanding of the biological and pathological significance of eosinophils in the gastrointestinal tract. Most studies on eosinophils in vivo have concentrated on trafficking and activation of these cells in the lung. It remains to be determined if the mechanisms involved in the regulation of eosinophil recruitment in the lung are conserved in the gastrointestinal tract. Therefore, elucidating the properties of gastrointestinal eosinophils and the molecular mechanisms of their tissue localization has important implications in further understanding this cell type and the pathogenesis of various gastrointestinal disorders. A. EOSINOPHIL-ASSOCIATED GASTROINTESTINAL DISEASES In classic food allergy, IgE-mediated hypersensitivity initially leads to mast cell activation leading to immediate clinical responses. In other forms of food allergy, an adverse immunologic response to food leads to a delayed clinical response, and eosinophils have been shown to be elevated in various inflamed segments of the gastrointestinal tract (Moon and Kleinman, 1995). Another set of patients have eosinophilic gastroenteritis, an idiopathic disease characterized by selective infiltration of eosinophils into the stomach, with variable involvement of the esophagus and/or large intestine (Katz et al., 1984; Keshavarzian et al., 1985;

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Torpier et al., 1988). Eosinophilic gastroenteritis encompasses multiple disease entities and can occur independent of peripheral blood eosinophilia ∼50% of the time, indicating the potential significance of gastrointestinal-specific mechanisms for regulating eosinophil levels. The disease has been divided, based on the degree of histological involvement. Most patients have mucosal involvement, whereas others have eosinophil infiltration in the muscularis and/or serosal layers. Atopy and food hypersensitivity are common in patients who have the mucosal pattern of the disease. In the atopic subset, IgE levels are elevated, and food-specific IgE has been detected. In another disorder, eosinophilic colitis, eosinophil accumulation occurs in the first few months of life and is a frequent cause of bloody diarrhea (Hill and Milla, 1990; Odze et al., 1995; Sherman and Cox, 1982). In these patients, eosinophils predominantly accumulate in the colon in the presence or absence of peripheral blood eosinophilia. The eosinophil infiltration appears to be triggered by cow’s milk protein hypersensitivity and improves upon withdrawal of the allergic triggers from the diet (Hill and Milla, 1990; Maluenda et al., 1984; Saavedra-Delgado and Metcalfe, 1985). In patients with gastroesophageal reflux, a common disorder than affects nearly 50% of the population, eosinophils are often found in the esophagus and are part of the diagnostic criteria for the disease (Brown et al., 1984). The magnitude of esophageal eosinophilia has been proposed to be a negative prognostic indicator (Liacouras et al., 1998; Winter et al., 1982) and adversely predicts response to conventional antigastroesophageal reflux medication (Ruchelli et al., 1999). It remains unclear if the trigger responsible for initiating the eosinophil accumulation in this disorder is secondarily related to the reflux of acidic gastric contents into the esophagus or if eosinophils themselves are pathogenically involved. In at least a subset of refractory patients, the severity of the gastroesophageal inflammation is reversed by institution of an allergen-free diet (Kelly et al., 1995). Lastly, patients with inflammatory bowel disease also have an accumulation of eosinophils in the gastrointestinal tract. Both forms of inflammatory bowel disease, Crohn’s disease and ulcerative colitis, are characterized by gastrointestinal eosinophilia; however, eosinophils usually represent only a small percentage of the infiltrating leukocytes (Desreumaux et al., 1999; Walsh and Gaginella, 1991). Interestingly, both diseases are associated with overproduction of eotaxin-1; however, there is controversy concerning whether IL-5 is overproduced in both disorders (Fuss et al., 1996; Hankard et al., 1997). Crohn’s disease is thought to be a predominantly T helper1 (Th1) and TNF- associated response, whereas ulcerative colitis is predominantly a Th2-associated process accompanied by IL-5 overproduction. The level of eosinophils in inflammatory bowel disease lesions has been proposed to be a negative prognostic indicator (Desreumaux et al., 1999; Nishitani et al., 1998).

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IV. Experimental Dissection of Eosinophilic Gastrointestinal Inflammation

It is currently thought that eosinophils may augment and sustain gastrointestinal inflammatory responses through the release of proinflammatory mediators and/or granule cationic proteins that are toxic to the mucosa (Dvorak et al., 1993; Furuta et al., 1995; Kato et al., 1998; Sampson, 1999). However, it should be noted that these conclusions are primarily based on the recent exploitation of murine models of asthma and on evaluation of clinical tissue from patients with a variety of eosinophil-associated gastrointestinal disorders. Although substantial progress has been made in elucidating the inflammatory mechanisms involved in allergic responses in the lung, there has been limited progress in understanding the pathogenesis of allergic disorders of the gastrointestinal tract. The development of experimental models of allergy has provided important insights into the immunological mechanisms regulating systemic (e.g., anaphylaxis) and pulmonary (e.g., asthma) allergic diseases. Collectively, these studies have identified a central role for cytokines (e.g., IL-4, IL-5, and IL-13), CD4+ T cells, mast cells, and in particular, eosinophils, in the induction and sustainment of allergic inflammatory responses (Drazen et al., 1996; Wills-Karp, 1999). While there have been recent advances in modeling some allergic gastrointestinal disease processes (e.g., IgE-mediated anaphylaxis responses), there have been only limited models of eosinophil-associated gastrointestinal allergy, and the precise mechanisms regulating gastrointestinal eosinophilia and the immunopathological role of this leukocyte in gastrointestinal disorders remain an enigma (Furuta et al., 1995; Kelly, 2000; Sampson, 1999). In order to elucidate these processes, murine models of eosinophil-associated gastrointestinal allergy have recently been developed. A. EXPERIMENTAL EOSINOPHILIC GASTROENTERITIS One of the complexities of inducing allergic inflammation of the gastrointestinal tract is the ineffectiveness of orally administered soluble protein antigens in promoting hypersensitivity responses; rather, oral antigens generally promote immunological tolerance (Miller et al., 1992). The poor immune response and induction of oral tolerance is thought to be associated, at least in part, with gastric digestion of soluble protein antigens, which leads to the formation of nonimmunogenic peptides (Mestecky et al., 1978; Michael, 1989). In order to overcome immunological tolerance associated with oral administration of soluble antigens (Mayer et al., 1996; Weiner and Mayer, 1996), our group has used an encapsulated soluble protein antigen in order to protect against gastric digestion (Litwin et al., 1998). The encapsulated biodegradable antigen particles are resistant to degradation at gastric pH (pH 2.5); however, they are susceptible to degradation at pH 5.5 which facilitates the delivery and release of the allergen in a preserved native conformational state to the small intestine (Litwin

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et al., 1998). Extensive previous investigations have demonstrated that the particles may overcome immunological tolerance associated with oral administration of antigens and possess adjuvant and immunostimulatory activity promoting antigen-specific antibody production (Challacombe et al., 1997). Mice were intraperitoneally injected with the egg antigen ovalbumin (OVA) in the presence of adjuvant (alum) and subsequently challenged with oral OVAparticles after 12 and 15 days (Hogan et al., 2000). Administration of oral allergen to sensitized mice induced peripheral blood eosinophilia and allergen-specific IgE and IgG1 as well as expansion of a Th2-biased immune response. Histological examination of the jejunum revealed vascular congestion, edema, and a prominent cellular infiltrate in the oral allergen-challenged mice as compared to placebo-challenged mice (Hogan et al., 2000). The cellular infiltrate was predominantly localized to the mucosa and lamina propria throughout the small intestine and was primarily composed of eosinophils. The infiltrating eosinophils were observed interspersed throughout the reticular connective tissue of the lamina propria and mucosa and throughout the length of the lamina propria of the villi. Electron microscopic analysis revealed that the eosinophils were physically associated with damaged axons (Hogan et al., 2001). Placebo-challenged mice had low levels of eosinophils predominantly localized to the base of the villus in the region of the crypt of Lieberkuhn ¨ and occasional cells within the lamina propria of the villus. This distribution is similar to the location of eosinophils at baseline (na¨ıve mice), indicating that placebo challenge alone did not significantly affect eosinophil trafficking (Mishra et al., 1999). Morphometric analysis revealed that the level of eosinophils in oral allergen-challenged mice was significantly higher than that observed in placebo-challenged mice. Since mast cells have also been implicated in the pathogenesis of various gastrointestinal hypersensitivity responses, their participation in oral-antigen induced eosinophil-associated gastrointestinal allergy was preliminarily examined by determining their levels. Histological analysis of the jejunum revealed no significant difference in the level of mast cells and their degranulation status within the lamina propria and villus between placebo-challenged and oral allergen-challenged mice, suggesting that mast cells were not involved in propagating eosinophilic gastrointestinal inflammation. B. ORAL ALLERGEN-INDUCED LAMINA PROPRIA EOSINOPHILS ARE CRITICALLY REGULATED BY EOTAXIN Elevated levels of eotaxin and eosinophils have been associated with various human inflammatory disorders of the respiratory tract, and increased levels correlate with disease severity (Garcia-Zepeda et al., 1996; Lamkhioued et al., 1997; Ying et al., 1999). In addition, elevated levels of eotaxin mRNA are seen in the lesions of patients with inflammatory bowel disease (Garcia-Zepeda et al., 1996). Thus, it was critical to determine the role of eotaxin in gastrointestinal allergic

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inflammation. In particular, it was of interest to determine if the reduced level of lamina propria eosinophils, normally seen in non-allergen-exposed eotaxindeficient mice, was increased following oral allergen exposure. Oral allergen challenge of eotaxin-deficient mice induced a marked increase in peripheral blood eosinophils compared with placebo-challenged eotaxin-deficient mice. Interestingly, the peripheral blood eosinophilia was significantly higher than that of oral allergen-challenged wild-type mice. We hypothesized that the elevated level of eosinophils in the peripheral blood of oral allergen-challenged eotaxindeficient mice was due to the failure to recruit eosinophils to the gastrointestinal tract, thus preventing the transmigration of circulating eosinophils into the site of inflammation. To test this hypothesis, the level of eosinophils in the lamina propria of the small intestine of oral allergen-challenged eotaxin-deficient mice was examined. Histological analysis of the intestinal tissue from oral allergenchallenged mice revealed no significant morphological changes to the small intestine structural integrity in the absence of eotaxin. Morphometric analysis of anti-MBP stained tissue revealed that in contrast to wild-type mice, oral allergen challenge of eotaxin-deficient animals induced no significant increase in the level of eosinophils as compared to placebo-challenged eotaxin-deficient mice. The level of eosinophils in oral allergen-challenged eotaxin-deficient mice was markedly reduced compared to wild-type mice (P < 0.001). This indicates that the reduced baseline level of gastrointestinal lamina propria eosinophils in eotaxin-deficient mice is not increased by allergen challenge (Matthews et al., 1998). The reduction of intestinal inflammation in the absence of eotaxin was not due to the failure to develop allergen-specific lymphocyte responses, since eotaxin-deficient mice produced marked levels of allergen-specific IgE, IgG1, and Th2 cytokines. C. THE ROLE OF IL-5 It was of interest to define the role of IL-5 in regulating eosinophil-associated gastrointestinal allergy in the small intestine since this cytokine is a pivotal modulator of eosinophil trafficking during allergic airways inflammation (Foster et al., 1996; Hamelmann et al., 1997a; Hogan et al., 1998a; Nakajima et al., 1992). Interleukin-5 has been shown to mobilize eosinophils from the bone marrow into the circulation and to promote eosinophil tissue trafficking during allergic airway inflammation (Foster et al., 1996; Mould et al., 1997; Palframan et al., 1998). We therefore compared oral allergen-induced gastrointestinal allergy in IL-5 deficient and wild-type mice (Hogan et al., 1999). In marked contrast to wild-type mice, allergen challenge of IL-5 deficient mice did not promote a peripheral blood eosinophilia. Interestingly, after the second allergen challenge (Day 19), the level of eosinophils in the blood of these mice was significantly lower than placebo-challenged IL-5 deficient mice (p < 0.05). Levels of allergen-specific IgE and IgG1 resembled those present in wild-type mice.

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These data suggest that the limited number of circulating eosinophils present in IL-5 deficient animals were being recruited into the intestine following allergen challenge, thereby depleting the level of peripheral blood eosinophils. Morphometric analysis revealed a three-fold increase in the level of eosinophils in the intestine of allergen-challenged mice. The level of eosinophil recruitment was still lower than in allergen-challenged wild-type mice. These studies also indicated that the baseline production of eosinophils, which occurs independently of known eosinophil hematopoietins (IL-3, IL-5, and GM-CSF) (Mishra et al., 1999), provides a sufficient number of eosinophils for the development of tissue eosinophilia. In addition, combining the results of the studies conducted with eotaxin and IL-5 deficient mice allows us to propose a model to explain the dichotomy often seen between peripheral blood and tissue eosinophilia (Fig. 4). We propose that when eotaxin is overproduced relative to IL-5 (e.g., IL-5 genetargeted mice), then eosinophils predominantly accumulate in tissue locations (e.g., eosinophilic esophagitis and/or gastroesophageal reflux); whereas when IL-5 is overproduced relative to eotaxin (e.g., eotaxin gene-targeted mice), then eosinophils predominantly accumulate in the blood compartment (e.g., drug reaction). As a corollary, when IL-5 and eotaxin are both increased, then eosinophils accumulate in both the tissue and blood compartments (e.g., idiopathic hypereosinophilic syndrome). D. EFFECT OF T CELL OVEREXPRESSION ON LAMINA PROPRIA EOSINOPHILS Transgenic mice that overexpress IL-5 under the control of the T cell promoter CD2 were demonstrated to have a ∼10- to 20-fold increase in the number of eosinophils in the hematopoietic organs (Dent et al., 1990). Because T cells reside in the gastrointestinal tract, and local overexpression of IL-5 can promote eosinophil accumulation in the lung (Lee et al., 1997), we hypothesized that T cell-driven IL-5 transgenic mice would have increased levels of gastrointestinal eosinophils. Eosinophils in the lamina propria of the small intestine of IL-5 transgenic mice were 4-fold higher compared with wild-type mice (Mishra et al., 1999) (Fig. 5). Interestingly, IL-5 transgenic mice also had increased eosinophils in the esophagus (wild-type and IL-5 transgenic mice had 0.44 ± 0.23 and 21.6 ± 6.9 eosinophils/mm2, respectively [mean ± SEM, n = 3–4]), as will be discussed later. In light of known synergistic effects between eotaxin and IL-5 (Collins et al., 1995; Mould et al., 1997; Rothenberg et al., 1996), it was important to examine the relationship between eotaxin and IL-5 in regulating gastrointestinal eosinophil levels. In order to test the dependency of gastrointestinal eosinophil levels on eotaxin in the presence of elevated levels of IL-5, mice that were transgenic for IL-5 and deficient in eotaxin were generated (Mishra et al., 1999). These IL-5 transgenic/eotaxin-deficient mice had a significantly reduced level (∼10%)

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FIG. 4. Proposed model for the dichotomy between peripheral blood and tissue eosinophilia. Eosinophil accumulation can occur in the peripheral blood and/or tissue depending upon the relative expression of the eosinophil chemoattractant (eotaxin) in the tissue and the systemic level of IL-5. In the top panel, a model for peripheral blood and tissue eosinophilia (e.g., idiopathic hypereosinophilic syndrome) involving overexpression of both eotaxin and IL-5 is presented. In the middle panel, a model for peripheral blood eosinophilia in the absence of tissue eosinophilia is presented. In these disorders (drug induced peripheral blood eosinophilia), there is a relative overexpression of IL-5 compared with eotaxin (as exemplified in eotaxin deficient mice). Finally, in the bottom panel, a model for tissue eosinophilia in the absence of circulating eosinophilia is presented. In these disorders (e.g., eosinophilic gastroenteritis, eosinophilic esophagitis), a relatively higher level of eotaxin is expressed in the tissue compared with IL-5 (as seen in IL-5 deficient mice).

of eosinophils within the jejunum as compared with IL-5 transgenic wild-type eotaxin mice (Fig. 5). Furthermore, the IL-5 transgenic mice that were deficient in eotaxin had > 2-fold higher level of circulating eosinophils compared to IL-5 transgenic mice that had wild-type eotaxin. Collectively, these data suggest that the elevation in circulating eosinophils was a consequence of an inhibition of

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FIG. 5. Gastrointestinal eosinophils in mice carrying a combination of the CD2 IL-5 transgene and the wild-type or eotaxin gene-targeted allele. The level of eosinophils in the jejunum of littermate mice transgenic (Tg) or wild-type (WT) for IL-5, and carrying the wild-type (+/+) or homozygous deletion (−/−) of eotaxin is shown. Each data point represents an individual mouse. The horizontal line is the mean value. Reprinted in part with permission (Mishra et al., 1999).

eosinophil recruitment into the gastrointestinal tract in the absence of eotaxin. Furthermore, these data indicate that eotaxin, even in the presence of high levels of IL-5 and circulating eosinophils, is critical for maintaining gastrointestinal eosinophil levels. E. PEYER’S PATCH EOSINOPHILS Since IL-5 overexpression was associated with eosinophil accumulation in the lamina propria of the small intestine (Mishra et al., 1999), it was of interest to determine if eosinophils also accumulated in Peyer’s patches under conditions in which IL-5 is overproduced. Eosinophil levels were determined in the Peyer’s patches of mice transgenic for IL-5 under the control of the T cell promoter CD2 (Mishra et al., 2000). These transgenic mice were found to have a marked increase in the number of eosinophils in the Peyer’s patches as compared to wild-type mice. For example, the eosinophil levels in the Peyer’s patches of wild-type and IL-5 transgenic mice were 0.043 ± 0.025 (n = 8) and 9.71 ± 1.56 (mean ± SEM, n = 12) MBP/area (%), respectively. Interestingly, the number of eosinophils in the lamina propria of the small intestine of the same IL-5 transgenic and wild-type control mice, measured in parallel, exhibited a smaller increase in eosinophils (only 5-fold higher) (Mishra et al., 1999). Characterization of the distribution of eosinophils in IL-5 transgenic mice revealed that these cells were predominantly distributed in the outer cortex and interfollicular regions of Peyer’s patches, the T cell-rich regions.

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FIG. 6. Histological analysis of eosinophils in Peyer’s patches of wild-type mice following oral allergen treatment. OVA sensitized wild-type mice were subjected to two treatments with (A) oral saline or (B) OVA (in the form of enteric beads) (Hogan et al., 2000), and the presence of eosinophils in the Peyer’s patches was determined 3 days after the last challenge. Eosinophils in the Peyer’s patches were determined by anti-MBP immunostaining, which results in black staining of eosinophils (depicted with arrows). Representative Peyer’s patches (designated PP) are shown with eosinophils present in the outer cortex and interfollicular regions only following allergen challenge (B). Eosinophils are also readily detectable in the lamina propria following allergen challenge. Following control saline treatment, eosinophils in Peyer’s patches remain at low baseline levels (data not shown) (Mishra et al., 2000b). (See color insert.)

To determine the relationship between IL-5 and eotaxin in regulating eosinophil trafficking to Peyer’s patches, IL-5 transgenic mice that were either genetically wild-type or deficient in eotaxin were evaluated for the presence of eosinophils in Peyer’s patches (Mishra et al., 2000). The level of eosinophils in Peyer’s patches was markedly increased in IL-5 transgenic mice and reduced in eotaxin-deficient IL-5 transgenic mice. In the absence of the eotaxin, there was a ∼3-fold reduction in the number of eosinophils in Peyer’s

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patches compared to IL-5 transgenic mice. Eosinophil levels were 9.7 ± 1.6 (mean ± SEM, n = 11) and 3.7 ± 1.9% MBP/area (mean ± SEM, n = 8) in IL-5 transgenic mice and eotaxin-deficient IL-5 transgenic mice, respectively. However, the level of eosinophils in eotaxin-deficient IL-5 transgenic mice was significantly higher than that observed in wild-type mice. Collectively, these data indicate the occurrence of IL-5 mediated eotaxin-dependent and -independent pathways for eosinophil trafficking to Peyer’s patches. It was also of interest to determine if eosinophils migrated into Peyer’s patches during Th2-associated conditions induced by experimental challenge with mucosal allergens. Mice were subjected to two distinct models of mucosal allergeninduced eosinophilic inflammation (Mishra et al., 2000). In the first approach, a well-accepted model of allergic airway disease using repeated doses of intranasal Aspergillus fumigatus antigen was employed (Kurup et al., 1992; 1994). Mice exposed to nine doses of intranasal antigen developed marked increases (>50 fold) in eosinophils in the peripheral blood and lung (Mishra et al., 1999). When the Peyer’s patches from these mice were examined, they were found to have increased levels of eosinophils following the allergen challenge. Eosinophils in the allergen-challenged mice were predominantly located in the outer cortex and interfollicular regions similar to their location in IL-5 transgenic mice. Morphometric analysis of eosinophil levels confirmed a marked increase in eosinophil levels in the Peyer’s patches for Aspergillus fumigatus antigen-challenged mice compared to placebo-challenged mice. Aspergillus antigen challenge did not increase eosinophil levels in the lamina propria following allergen challenge (Mishra et al., 1999), indicating that discrete mechanisms regulate Peyer’s patch and the lamina propria compartments. Eosinophil trafficking to Peyer’s patches was also examined in the experimental model of oral antigen-induced Th2associated allergic hypersensitivity responses of the gastrointestinal tract (as described earlier) (Hogan et al., 2000). Exposure of OVA-sensitized mice to enteric-coated OVA beads induces Th2-associated accumulation of eosinophils in the peripheral blood and gastrointestinal lamina propria of the small intestine as compared to mice challenged with placebo beads (Hogan et al., 2000). Similar to the lamina propria, oral allergen-challenged animals had elevated levels of eosinophils in the Peyer’s patches compared to placebo bead treated mice (Fig. 6). These results demonstrated that eosinophils traffic to Peyer’s patches during Th2 responses following mucosal allergen exposure. It was of interest to determine next the role of eotaxin in allergen-induced recruitment of eosinophils into Peyer’s patches. Eotaxin-deficient and wild-type mice were subjected to experimental allergic airway inflammation (Mishra et al., 2000). As expected, allergen challenge of wild-type mice promoted eosinophil accumulation in the blood, lung, and Peyer’s patches (Mishra et al., 2000). In the absence of eotaxin, there was reduced accumulation of eosinophils in Peyer’s patches and the lung compared with wild-type mice. However, in the absence

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of eotaxin, allergen challenge still induced eosinophil infiltration into Peyer’s patches compared with mice that were challenged with placebo alone. In particular, wild-type mice subjected to placebo or allergen challenge had 0.014 ± 0.01 and 1.53 ± 0.46 MBP/area (%) (mean ± SEM, n = 7), respectively. Eotaxindeficient mice subjected to placebo or allergen challenge had 0.008 ± 0.004 and 0.89 ± 0.16 MBP/area (%) (mean ± SEM, n = 5), respectively. This indicates that eosinophil trafficking to Peyer’s patches during mucosal allergen exposure occurs by eotaxin-dependent and -independent pathways. The localization of eosinophils in T cell-rich regions of the Peyer’s patches, and the recent demonstration that eosinophils can present antigen to T cells, provides evidence that eosinophils may have a critical role in the induction of antigen-specific T cell responses. F. COLONIC EOSINOPHILS Eosinophils accumulate in the colon of patients with a variety of disorders, including eosinophilic colitis, allergic colitis, and inflammatory bowel disease (Furuta et al., 1995). Some of these disorders are very common; for example, allergic colitis, which is often due to milk protein hypersensitivity, is the most common cause of bloody diarrhea in the first year of life (Chong et al., 1986; Hill and Milla, 1990; Machida et al., 1994). In an attempt to dissect the immunological mechanisms involved in eosinophil-associated gastrointestinal disorders of the large intestine, a murine model of antigen-induced colitis was developed (Kweon et al., 2000). Mice were systemically sensitized to OVA in the presence of Complete Freund’s Adjuvant and subsequently challenged with repeated doses of intragastric OVA. After nine doses of antigen over 3 weeks, the mice developed diarrhea accompanied by a dramatic infiltration of eosinophils, mast cells, and CD4+ Th2 cells into the large but not small intestine. Similar experimental analysis in mice with the targeted deletion of signal transducers and activators of transcription (STAT)6 completely eliminated the colonic eosinophils and the diarrhea. Adoptive transfer experiments showed that systemically primed splenic CD4+ T cells were preferentially recruited to the large but not small intestine upon oral allergen challenge. These results indicate that eosinophilassociated inflammation of the large intestine appears to be critically regulated by Th2 cells that specifically home to the colon. While eotaxin has been shown to be essential for the baseline homing of eosinophils to the colon (Mishra et al., 1999), its importance in inflammatory disorders of the colon has yet to be evaluated. G. ESOPHAGEAL EOSINOPHILS Eosinophil infiltration into the esophagus is a commonly observed medical problem in patients with diverse diseases including gastroesophageal reflux, eosinophilic esophagitis, and allergic gastroenteritis (Furuta et al., 1995; Kelly,

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2000; Sampson, 1999). As mentioned earlier, the murine and human esophagus, even though they express eotaxin, are devoid of resident eosinophils at baseline (Kato et al., 1998; Mishra et al., 1999). This indicates that eotaxin expression is not sufficient for eosinophil homing into these segments of the gastrointestinal tract. In an effort to understand the mechanisms and significance of eosinophil accumulation in the esophagus in diseased states, we have developed a murine model for antigen-induced esophagitis (Mishra et al., 2001). Mice were repeatedly challenged with intransal Aspergillus fumigatus allergen (under conditions which promote allergic airway inflammation) and were found to develop ∼100to 125-fold increase in their levels of esophageal eosinophils and epithelial hyperplasia. In contrast, exposure to repeated doses of oral or intragastric soluble allergen did not promote esophageal inflammation. Since allergen challenge is associated with Th2 immune responses, the role of IL-5, a Th2 cell-derived cytokine, was examined for its role in regulating eosinophil accumulation in the esophagus. Intranasal allergen challenge of IL-5 gene-targeted mice resulted in the complete loss of eosinophil recruitment to the esophagus and the onset of epithelial hyperplasia. In contrast, in the absence of eotaxin, allergen-induced esophageal eosinophils were only partially reduced (∼2-fold). Consistent with the important role of Th2 cells and their cytokines, a clinical study of patients with eosinophilic esophagitis has demonstrated elevated levels of IL-4 secreting T cells in esophageal lesions (Nicholson et al., 1997). These findings have several implications: (1) they implicate aeroallergens and eosinophils in the etiology of eosinophilic esophagitis; (2) they suggest that esophageal eosinophilic inflammation is mechanistically associated with pulmonary inflammation; and (3) they suggest that targeting IL-5 (e.g., with the humanized anti-IL-5 reagent that is currently being evaluated for asthma) may be a useful strategy for patients with eosinophilic esophagitis and/or refractory gastroesophageal reflux. In addition, they suggest that distinct mechanisms regulate eosinophil accumulation in the esophagus compared with other components of the gastrointestinal tract such as the small intestine. H. GASTROINTESTINAL EOSINOPHILS IN ENTERIC HELMINTH INFECTIONS Early work indicated that the hematopoietic expansion of eosinophils but not neutrophils during helminth infections (e.g., Schistosoma mansoni, Nippostrongyloides brasiliensis, Onchocerca volvulus, and Trichinella spiralis) was reduced in athymic mice (Hsu et al., 1976; Ruitenberg et al., 1977). Subsequently, mice treated with anti-IL-5 monoclonal antiserum (or mice containing a disruption of the IL-5 gene) were shown to have dramatically reduced systemic and local eosinophilia following infection with Nippostrongyloides brasiliensis (Behm and Ovington, 2000; Coffman et al., 1989); taken together these studies identified IL-5 as the chief T cell factor responsible for mediating

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helminth-induced eosinophilia (Maizels et al., 1993; Sanderson, 1992). Experimental infection of animals with helminths has provided an opportunity to examine the mechanisms of eosinophil trafficking to the gastrointestinal tract. For example, following Trichinella spiralis infection, eosinophils accumulate in the lamina propria of the jejunum, in the mesenteric lymph nodes, and in the spleen (Friend et al., 2000). In the jejunum, eosinophils are located in the lamina propria and not in the interepithelial location (in contrast to mast cells) (Friend et al., 2000). During the recovery phase, the fate of lamina propria eosinophils was assessed by the TUNEL assay, a method for identifying apoptotic cells. Apoptotic eosinophils were rarely found in the lamina propria, but were readily detected in mesenteric lymph nodes. Additionally, many macrophages in the lymph nodes were demonstrated to be actively phagocytosing apoptotic eosinophils. Thus, although only examined in the context of Trichinella spiralis infection, gastrointestinal eosinophils may be destined for trafficking to local lymph nodes, where they undergo clearance by apoptosis. The role of adhesion molecules in the regulation of eosinophil trafficking to the gastrointestinal tract has also been evaluated following Trichinella spiralis infection. Eosinophils express several families of adhesion molecules, including members of the selectin and integrin families (summarized in Fig. 7). In brief, reversible interactions between eosinophils and endothelial cells are primarily mediated by selectins. Eosinophils express the ligands (e.g., sialylated LewisX antigen) for E- and P-selectins. In addition, eosinophils express L-selectin, but its exact ligand on endothelial cells is not known. Eosinophils have recently been demonstrated to selectively express a sialic acid binding immunoglobulinlike lectin designated Siglec-8 (Floyd et al., 2000; Kikly et al., 2000). The ligand for Siglec-8 has not yet been identified, but other members of the Siglec family of adhesion molecules have been shown to be important signaling receptors, employing immunomodulatory inhibitory motifs to interact with tyrosine phosphatases (e.g., SHP-1). The integrins expressed by eosinophils include members of the 1 (e.g., very late antigen (VLA)-4), 2 (e.g., CD18 family of molecules), and 7 families (e.g., 47 molecule) (Bochner and Schleimer, 1994) (Fig. 7). The CD18 family of molecules includes lymphocyte function antigen (LFA)-1 and Mac-1 that both interact with endothelial cells via intercellular adhesion molecule (ICAM); the VLA-4 integrin (which is not expressed by neutrophils) binds to vascular cell adhesion molecule (VCAM)-1. These adhesion interactions have been shown to be important for eosinophil recruitment into the lung and skin, but their role in eosinophil recruitment to the gastrointestinal tract has not yet been evaluated. The 47 molecule, which is coexpressed on lymphocytes and eosinophils, is perhaps the most important integrin for gastrointestinal eosinophils. This integrin binds to mucosal addressin cell adhesion molecule-1 (MAdCAM-1), a major adhesion molecule expressed on high endothelial venules in the intestinal lamina propria, lymph nodes, and Peyer’s patches. Mice with targeted disruption of the 7 gene have been examined for

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FIG. 7. Schematic diagram of eosinophil adhesion molecules and their ligands. Eosinophils express several classes of adhesion molecules, including members of the selectin and integrin families [more extensively reviewed in other publications (Bochner and Schleimer, 1994)]. Depicted are eosinophil ligands (e.g., sialylated Lewis-X antigen) for E- and P-selectins, which mediate reversible interactions between eosinophils and endothelial cells. In addition, eosinophils express L-selectin, but its exact ligand on endothelial cells is not known. Eosinophils have recently been demonstrated to selectively express a sialic acid binding immunoglobulin-like lectin designated Siglec-8. The ligand for Siglec-8 has not yet been identified, but other members of the Siglec family of adhesion molecules have been shown to be important signaling receptors, employing immunomodulatory inhibitory motifs to interact with tyrosine phosphatases (e.g., SHP-1). The integrins expressed by eosinophils include members of the 1 (e.g., very late antigen (VLA)-4), 2 (e.g., CD18 family of molecules), and 7 (e.g., 47) families of molecules. The CD18 group of receptors includes lymphocyte function antigen (LFA)-1 and Mac-1 that both interact with endothelial cells via intercellular adhesion molecule (ICAM); the VLA-4 integrin (which is not expressed by neutrophils) binds to vascular cell adhesion molecule (VCAM)-1. The 47 molecule, which is coexpressed on lymphocytes and eosinophils, binds to mucosal addressin cell adhesion molecule-1 (MAdCAM-1), a major adhesion molecule expressed on high endothelial venules in the intestinal lamina propria, lymph nodes, and Peyer’s patches.

inflammatory responses to helminth infection. These studies have demonstrated a delayed influx and reduced magnitude of intestinal eosinophilia in response to Trichinella spiralis (Artis et al., 2000). Consistent with this, we have found reduced eosinophil recruitment into the jejunum of 7-deficient mice following oral allergen challenge compared with wild-type mice (unpublished results). In

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contrast, during infection with Trichuris muris, a helminth that infects the large intestine, no difference in the level of eosinophils in the colon is observed (Artis et al., 2000). Thus, similar to the important role of 47 in lymphocyte homing to the gastrointestinal tract (Berlin et al., 1993), eosinophil recruitment to the small intestine also appears to be regulated by this integrin. Although eosinophils have been shown to elicit powerful antihelminthic cytotoxicity in vitro (Butterworth, 1977, 1984), studies in vivo have not consistently demonstrated an important role for eosinophils in this process, with the exception of immunity against select organisms such as Strongyloides venezuelensis (Behm and Ovington, 2000; Korenaga et al., 1991), as will be discussed later. V. Function of Eosinophils

A. PROINFLAMMATORY ROLE The function of eosinophils in inflammatory states in the gastrointestinal tract has been largely derived from studies investigating the properties of these cells in vitro and by analysis of their involvement in allergic respiratory disorders. A variety of studies (primarily utilizing antibody neutralization of IL-5 and IL-5 gene deficient mice) have demonstrated a strong association of eosinophilic airway inflammation and the development of lung damage (e.g., airway hyperresponsiveness) (Hogan et al., 1998c). However, recent studies have shown that the mere presence of eosinophils in the lung is not sufficient for airway hyperresponsiveness (Mould et al., 2000), indicating that multiple signals are necessary for eosinophil activation and/or airway hyperresponsiveness. As stated earlier, eosinophils generate a variety of mediators that augment inflammatory responses. Most prominent among these molecules are the secondary granule constituents (MBP, ECP, EPO, and EDN) that are specific to eosinophils (Fig. 1). These mediators induce a variety of proinflammatory effects, including toxicity to multiple tissues (including host cells), induction of mast cell degranulation, and dysfunction of vagal muscarinic M2 receptors (Gleich and Adolphson, 1986). Further proinflammatory damage can be caused by the generation of unstable oxygen radicals by the respiratory burst oxidase apparatus and EPO. The eosinophil also generates large amounts of LTC4 (Lewis et al., 1990) which is metabolized to LTD4 and LTE4, potent smooth muscle constrictors. Additionally, activated eosinophils generate a wide range of inflammatory cytokines (IL-1, IL-3, IL-4, IL-5, IL-6, GM-CSF, TGF-/, TNF-, and eotaxin) which can be preformed or generated de novo following cellular activation. Eosinophils also produce neuroactive mediators (e.g., substance P and vasoactive intestinal peptide [VIP]) (Metwali et al., 1994). Interestingly, specimens from patients with eosinophilic gastroenteritis often display eosinophils undergoing marked degranulation near nerves, suggesting that they may indeed be involved in promoting inflammatory changes to neurons (Stead, 1992; Dvorak et al., 1993).

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Interestingly, the gastric dysmotility during experimental oral antigen-induced gastrointestinal inflammation is associated with eosinophils in the proximity of damaged nerves, suggesting a causal role for eosinophils in nerve dysfunction (Hogan et al., 2001). Additionally, experimental eosinophil accumulation in the gastrointestinal tract is associated with the development of weight loss, which is attenuated in eotaxin-deficient mice (Hogan et al., 2001). Taken together with the clinical data in patients with gastroesophageal reflux, where the level of eosinophils directly correlates with the severity of the disease, and lack of responsiveness to medical management (Ruchelli et al., 1999), these results suggest a proinflammatory detrimental role for eosinophils in the pathogenesis of gastrointestinal disorders. B. ANTIHELMINTH IMMUNITY The beneficial function of eosinophils has been primarily attributed to their ability to defend the host against parasitic helminths. This is based on several lines of evidence, including (1) the ability of eosinophils to mediate antibody (or complement) dependent cellular toxicity against helminths in vitro (Butterworth, 1977, 1984); (2) the observation that eosinophil levels increase during helminth infections and that eosinophils aggregate and degranulate in the local vicinity of damaged parasites in vivo; and (3) the results in experimental parasite-infected mice that have been depleted of eosinophils by IL-5 neutralization and/or gene targeting (Behm and Ovington, 2000). However, it should be noted that murine studies are particularly problematic since mice are not the natural hosts of many of the experimental parasites. Nevertheless, in some primary infection models, a role for IL-5 (and hence eosinophils) in protective immunity has been suggested following infection with Strongyloides venezuelensis, Strongyloides ratti, Nippostrongyloides brasiliensis, and Heligmosomoides polygyrus (Behm and Ovington, 2000; Korenaga et al., 1991). Most recently, a role for eosinophils in the encystment of larvae in Trichinella spiralis infection has been demonstrated (D. S. Friend, Harvard Medical School, personal communication). In this study, a markedly reduced level of gastrointestinal eosinophils was found in Trichinella spiralis-infected CCR3 gene-targeted mice compared with control infected mice that contained abundant degranulating eosinophils. The reduced level of eosinophils correlated with a greater number of intact encysted larvae. Thus, although the debate continues, it seems likely that eosinophils participate in the protective immunity against selected helminths. C. INTERACTIONS WITH T LYMPHOCYTES The localization of gastrointestinal eosinophils in juxtaposition with lymphocytes (e.g., lamina propria and Peyer’s patches) suggests a functional interaction between these two leukocytes. While most studies have focused on the role of T cells in the regulation of eosinophils (e.g., through IL-5), it is likely

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that eosinophils may also regulate lymphocytes. Consistent with this finding, eosinophils are known to express the necessary cellular machinery for antigen presentation such as H-2 class II and costimulatory molecules (e.g., B7-1) (Lucey et al., 1989; Tamura et al., 1996; Woerly et al., 1999). Eosinophils are also known to express a variety of cytokines that can induce the proliferation and/or maturation of T cells (e.g., IL-2, IL-4, IL-12). Furthermore, preliminary investigations with human eosinophils in vitro have shown that eosinophils have the capacity to present antigen to T cells (Lucey et al., 1989; Tamura et al., 1996). Recent studies have shown that eosinophils isolated from the mouse lung can present antigen to T cells when adoptively transferred to na¨ıve animals (Shi et al., 2000). In addition, it has been proposed that lymph node eosinophils in patients with Hodgkin’s disease may provide cellular ligands for TNF superfamily receptors and CD30, thereby transducing proliferation and antiapoptotic signals (Pinto et al., 1996, 1997). Additional support for an interaction between eosinophils and T cells has recently been derived from analysis of thymic eosinophils. As mentioned earlier, the thymus is a primary site for eosinophils under healthy conditions. In young mice, thymic eosinophils are primarily located in the corticomedullary region, express IL-4 and IL-13, and are CD11b and CD11c positive (similar to dendritic cells) (Throsby et al., 2000). In adult mice, eosinophils traffic to the medulla under the regulation of eotaxin (Matthews et al., 1998). During experimental induction of tolerance, the level of thymic eosinophils increases, and their location correlates with areas of active T cell apoptosis. Taken together, these studies indicate that regulation of T cell responses is one of the physiological functions of eosinophils. D. DEVELOPMENTAL BIOLOGY The finding that eosinophils home into the gastrointestinal tract during gestational development (Mishra et al., 1999) suggests that eosinophils may have a role in tissue or organ development. A role for eosinophils in developmental processes in the gastrointestinal tract has not yet been identified. However, a physiological function for eosinophils in postnatal mammary gland development has been recently proposed (Gouon-Evans et al., 2000). In this investigation, F4/80-positive leukocytes were identified to be present in the developing mammary gland, primarily in the region of the terminal end buds. Surprisingly, on close histological examination, the F4/80-positive cells were identified as macrophages and eosinophils. The important role for leukocytes in mammary gland development was demonstrated by depleting hematopoietic precursors by whole-body  -irradiation. Following  -irradiation, ductal outgrowth was impaired, and this abnormality was reversed by bone marrow transplantation. Interestingly, the level of eotaxin and eosinophils in the mammary gland was shown to increase with the development of the mammary gland during puberty. Furthermore, eotaxin-deficient mice had a near complete loss of mammary gland

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eosinophils, and this was associated with a decreased number of ductal branches and a defect in terminal end bud formation. Taken together, these data establish that eosinophils are critically involved in the branching morphogenesis of the mammary gland. The presence of constitutive eotaxin and eosinophils in other endocrine organs (e.g., uterus) (Hornung et al., 2000; Salamonsen and Lathbury, 2000; Zhang et al., 2000), as well as in the gastrointestinal tract, suggests that the involvement of tissue eosinophils in developmental processes is not likely to be restricted to the mammary gland. VI. Summary and Concluding Remarks

Eosinophils are well known as proinflammatory leukocytes that account for a small subset of circulating blood cells. The recent studies outlined in this review have identified eosinophils as constituents of the mucosal immune system in the gastrointestinal tract residing in the lamina propria and Peyer’s patches, and have identified complex regulatory pathways and characteristics of these cells (summarized in Fig. 8). Under baseline (healthy) conditions, gastrointestinal eosinophils predominantly reside in the lamina propria in the stomach and intestine, and their numbers in these organs are substantially higher than in hematopoietic tissues. Interestingly, eosinophils migrate to the gastrointestinal tract during embryonic development, and their concentrations in perinatal mice are comparable to those in adults, indicating that eosinophil homing occurs independent of intestinal flora. The chemokine eotaxin, an eosinophil-selective chemoattractant that is constitutively expressed throughout all segments of the gastrointestinal tract, is required for eosinophil homing to the lamina propria. During Th2-associated inflammatory conditions (e.g., IL-5 overexpression or allergen challenge), marked increases of eosinophils occur in the lamina propria in an eotaxin-dependent manner. In addition, allergen challenge promotes eosinophil migration to the outer cortex and interfollicular regions of Peyer’s patches, and this process is critically regulated by IL-5 and less significantly by eotaxin, suggesting the involvement of other eosinophil chemokines in this lymphoid compartment. Furthermore, following mucosal allergen challenge, eosinophils under the regulation of IL-5 accumulate in the esophagus, an organ normally devoid of eosinophils at baseline. Preliminary investigations have shown that gastrointestinal eosinophils express the 47 integrin and that this molecule is responsible, in part, for eosinophil homing. In summary, eosinophils are resident cells of the gastrointestinal immune system, their levels are increased by antigen exposure under Th2-associated conditions, and eotaxin and IL-5 differentially regulate their trafficking in a tissue-specific manner. We propose that eosinophils are integral members of the gastrointestinal immune system and are likely to be important in innate, regulatory, and inflammatory immune responses.

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FIG. 8. Schematic representation of eosinophil trafficking to the intestine. Eosinophils develop in the bone marrow where they differentiate from hematopoietic progenitor cells into mature eosinophils. Factors that control this process have not been fully defined; however, IL-3, IL-5, and GM-CSF are important in eosinophil expansion during conditions of hypereosinophilia. Eosinophil migration out of the bone marrow into the circulation is primarily regulated by IL-5. Circulating eosinophils subsequently interact with the endothelium by processes involving rolling, adhesion, and diapedesis. In the gastrointestinal tract, the adhesion 47 ligand on eosinophils interacts with the endothelial receptor mucosal vascular addressin MAdCAM-1. Eosinophils are mobilized into the lamina propria in response to a chemotactic gradient primarily established by the chemokine eotaxin liberated from mononuclear cells in the crypts. Additionally, eosinophils are mobilized into the interfollicular and paracortical regions of Peyer’s patches. The chemotactic response is enhanced by IL-5, an important eosinophil cytokine for eosinophil priming and survival. Depending upon the chemokine concentration gradient, gastrointestinal eosinophils can also migrate into the villi, residing in proximity to lymphocytes, and have the potential to degranulate, resulting in tissue damage. (See color insert.)

In view of the ability of eosinophils to participate in antigen presentation and to secrete cytokines, which induce T cell proliferation and maturation, the finding of eosinophils as part of gut-associated lymphoid tissue (GALT) seems logical since most lymphocytes also reside in the gastrointestinal tract. However, both cell types reside in distinct locations and are regulated by unique processes (Table II). At baseline, eosinophils only reside in the lamina propria; however, during inflammatory conditions these cells can be located in several other compartments shared with T cells (lamina propria, Peyer’s patches, and interepithelial regions). Eosinophil localization to the gastrointestinal tract is not

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TABLE II CHARACTERISTICS OF GASTROINTESTINAL T CELLS AND EOSINOPHILS

Location—baseline Peyer’s patch Lamina propria Interepithelial Location—diseased state Peyer’s patch Lamina propria Interepithelial Role for 47 Eotaxin IL-5 Gut flora a

T Cells

Eosinophils

+ + +

– + –

+ + +

+ + +

+ – – +a

+ + + –

Excluding  /-T cells.

simply a response to the “proinflammatory” environment of the gastrointestinal tract, since prenatal mice (free of exogenous flora), as well as germ-free mice (free of viable intestinal flora) have normal levels of eosinophils. While some of the mechanisms involved in leukocyte homing to the gastrointestinal tract appear to be conserved between lymphocytes and eosinophils (e.g., utilization of 47 integrin), gastrointestinal eosinophils are critically regulated by local eotaxin, and less significantly by IL-5. Recent studies that have begun to uncover functional properties of eosinophils indicate that these cells are more than just pro inflammatory leukocytes. Rather, they are likely to have diverse physiological functions involved in critical arms of the immune system, including innate responses, tolerance, and antigen presentation; in addition, eosinophils may have a role in tissue morphogenesis. The accumulation of eosinophils in the gastrointestinal tract in diverse medical diseases is often associated with serious medical consequences (e.g., weight loss, malabsorption, architectural changes of the intestine such as blunting of the villi), but the role of eosinophils in the pathogenesis of these diseases has been debated. Recent progress in experimental modeling of eosinophil-associated gastrointestinal diseases has been instrumental in determining that gastrointestinal eosinophils can be directly increased by mucosal allergen challenge. These models have been useful in identifying the critical role for eotaxin and in suggesting a causal association of eosinophilic inflammation with clinical and pathological changes to the gastrointestinal tract. These findings parallel the large number of mechanistic studies concerning eosinophilic allergic inflammation in the lung (e.g., asthma). Interestingly, patients with inflammatory gastrointestinal disorders

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have been found to have an altered proallergic mucosa in the lung, including occult airway hyperresponsiveness (Louis et al., 1999; Mansi et al., 2000). Likewise, patients with asthma have been demonstrated to contain increased levels of eosinophils in the gastrointestinal tract (Wallaert et al., 1995). Taken together, it is likely that common mucosal immune responses are operational in patients with allergic disorders, perhaps controlled by trafficking T cells. It is hoped that examination of gastrointestinal eosinophils with the same scrutiny that has been applied to investigate pulmonary eosinophils and gastrointestinal lymphocytes may soon uncover the role of eosinophils in health and disease. ACKNOWLEDGMENTS This work was supported in part by the National Health Medical Research Council (Australia) C. J. Martin Post-doctoral Fellowship (S. P. H.), the Jaffe Family Fund of the American Academy of Allergy, Asthma, and Immunology (S. P. H.), NIH Grant R01 AI45898 (M. E. R.), and the Human Frontier Science Program (M. E. R.). The authors thank Drs. K. Frank Austen, Mitchell Cohen, Susan Wert, Paul Foster, Glenn Furuta, and Nives Zimmermann for helpful discussions, numerous other instrumental colleagues, and Andrea Lippelman for expert editorial assistance.

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