Experimental analysis of eosinophil-associated gastrointestinal diseases

Eosinophil infiltration into the gastrointestinal tract occurs in a wide range of diseases. However, the underlying cellular and molecular mechanisms involved in eosinophil migration and the role of eosinophils in disease pathogenesis are largely unknown. Recent studies using experimental models of eosinophil-associated gastrointestinal allergy have revealed differential roles for IL-5 and eotaxin in the modulation of eosinophil accumulation into various regions of the gastrointestinal tract. Furthermore, such studies have revealed a possible role for eosinophils in the pathogenesis of gastrointestinal disorders. The present review describes the clinical manifestations of various eosinophil-associated gastrointestinal disorders and the current understanding of the role of IL-5 and eotaxin in the allergic inflammatory response, and the participation of the eosinophilic granulocyte in the expression of disease.     

Th2-cell-mediated chemokine synthesis is involved in allergic airway inflammation in mice

Eosinophilic inflammation in the bronchial mucosa has been recognized as a prominent pathological feature of bronchial asthma. Th2 cells have been implicated in the local infiltration and activation of eosinophils. The migration of eosinophils as well as Th2 cells is controlled by chemokines, suggesting a crucial role of chemokines in the pathogenesis of bronchial asthma. To elucidate the mechanism by which Th2 cells induce eosinophilic inflammation, a Th2-cell-dependent murine model of asthma was employed in this study. Along with the infiltration of eosinophils and antigen-specific Th2 cells, CC chemokine receptor-3-active eotaxin, monocyte chemoattractant protein (MCP)-3 and RANTES, as well as CC chemokine receptor-3-inactive MCP-1 were produced in the lungs of Th2-cell-transferred mice after antigen provocation in vivo. On the other hand, differentiated antigen-specific Th2 cells produced MCP-3 and RANTES but not eotaxin or MCP-1 upon stimulation in vitro. Chemokines synthesized by Th2 cells and other cell types are involved in the development of eosinophilic inflammation in bronchial asthma.     

Eosinophilic gastrointestinal disorders (EGID)

Primary eosinophilic gastrointestinal disorders are defined as disorders that selectively affect the gastrointestinal tract with eosinophil-rich inflammation in the absence of known causes for eosinophilia (eg, drug reactions, parasitic infections, and malignancy). These disorders include eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic enteritis, and eosinophilic colitis and are occurring with increasing frequency. Significant progress has been made in elucidating that eosinophils are integral members of the gastrointestinal mucosal immune system and that eosinophilic gastrointestinal disorders are primarily polygenic allergic disorders that involve mechanisms that fall between pure IgE-mediated and delayed T(H)2-type responses. Preclinical studies have identified a contributory role for the cytokine IL-5 and the eotaxin chemokines, providing a rationale for specific disease therapy. An essential question is to determine the cellular and molecular basis for each of these clinical problems and the best treatment regimen, which is the main subject of this review.     

The pathology and causes of tissue eosinophilia in the gastrointestinal tract

Eosinophilic inflammation in the gastrointestinal tract may occur as a primary eosinophilic disorder or as a secondary response with other causes. Primary eosinophilic gastrointestinal disorders (EGIDs) are Th2‐mediated allergic diseases that overlap pathogenetically with atopic conditions involving other organs. The pathological diagnosis of primary EGIDs can be challenging, as the quantity of eosinophils considered to be ‘abnormal’ is difficult to define, and the diagnosis, by definition, requires exclusion of the far more common secondary causes. Our understanding of the basic biology and natural history of eosinophilic oesophagitis has advanced considerably over the last decade, whereas other EGIDs have proven more difficult to characterize; nonetheless, some recent advances have been made. This review summarizes current knowledge regarding the clinical presentation, diagnosis, natural history and treatment of EGIDs, including eosinophilic oesophagitis. We also draw attention to the numerous secondary causes of tissue eosinophilia in the gastrointestinal tract, and suggest a practical approach to the histological assessment, diagnosis and reporting of EGIDs.     

Eotaxin-1-deficient mice develop airway eosinophilia and airway hyperresponsiveness.

BACKGROUND: The accumulation of eosinophils in the lung is a hallmark of asthma. In addition to cytokines such as IL-5 which are essential, chemokines have been implicated in the recruitment of eosinophils to the airway. In particular, eotaxin has been shown to be a selective and potent eosinophil chemoattractant, important in the pathogenesis of allergic disease. The goal of the present study was to define the role of eotaxin-1 in the development of allergen-induced eosinophilic airway inflammation and airway hyperresponsiveness (AHR) to inhaled methacholine (MCh). METHODS: Eotaxin-1-deficient mice were sensitized and exposed to a single challenge with allergen. Airway function and airway and tissue as well as peripheral blood and bone marrow eosinophilia were examined 18 and 48 h after the last challenge. RESULTS: Following allergen sensitization and challenge, eotaxin-1-deficient mice developed levels of AHR to inhaled MCh at 18 and 48 h comparable to controls. Further, levels of bronchoalveolar lavage (BAL) and tissue eosinophilia at the same time points were comparable in the two strains of mice. Tissue eosinophilia, assessed by quantitating major basic protein staining cells, preceded BAL eosinophilia in a similar manner. Bone marrow and peripheral blood eosinophilia were unimpaired in deficient mice. CONCLUSION: The results demonstrate that the major eotaxin, eotaxin-1 is not essential for the development of airway eosinophilia or AHR, implying that other chemokines, alone or in combination, can overcome this deficiency.     

Molecular mechanisms of formation of blood eosinophilia under pulmonary tuberculosis

Molecular factors of pathogenesis of the eosinophilic blood reaction under pulmonary tuberculosis are analyzed in the article. It has been established that the key cytokine providing the development of hemic eosinophilia in patients with pulmonary tuberculosis is IL-5. IL-5 plasma concentration turned out to be increased only in patients with eosinophilia. Increase of eotaxin was determined in patients with tuberculosis despite of the presense of eosinophilia. One-directional nature of the defined changes in eotaxin concentration might be explained by dual properties of this chemokine: on the one hand, eotaxin mediates long-term presence of eosinophils in blood; on the other hand, it initiates the process of adhesion of eosinophilic leucocytes to vascular endothelium with their further migration to the focus of granulomatous inflammation. The established increase in number of IL-5R-positive eosinophils presents one more mechanism which explains the basis of long-term presence of eosinophils in peripheral blood in patients with pulmonary tuberculosis.     

Macrophages and cardiac fibroblasts are the main producers of eotaxins and regulate eosinophil trafficking to the heart

Cardiac manifestations are a major cause of morbidity and mortality in patients with eosinophil‐associated diseases. Eosinophils are thought to play a pathogenic role in myocarditis. We investigated the pathways that recruit eosinophils to the heart using a model of eosinophilic myocarditis, in which experimental autoimmune myocarditis (EAM) is induced in IFNγ^?/?IL‐17A^?/? mice. Two conditions are necessary for efficient eosinophil trafficking to the heart: high eotaxin (CCL11, CCL24) expression in the heart and expression of the eotaxin receptor CCR3 by eosinophils. We identified cardiac fibroblasts as the source of CCL11 in the heart interstitium. CCL24 is produced by F4/80⁺ macrophages localized at inflammatory foci in the heart. Expression of CCL11 and CCL24 is controlled by Th2 cytokines, IL‐4 and IL‐13. To determine the relevance of this pathway in humans, we analyzed endomyocardial biopsy samples from myocarditis patients. Expression of CCL11 and CCL26 was significantly increased in eosinophilic myocarditis compared to chronic lymphocytic myocarditis and positively correlated with the number of eosinophils. Thus, eosinophil trafficking to the heart is dependent on the eotaxin‐CCR3 pathway in a mouse model of EAM and associated with cardiac eotaxin expression in patients with eosinophilic myocarditis. Blocking this pathway may prevent eosinophil‐mediated cardiac damage.     

Gastrointestinal eosinophils

Summary: The gut‐associated lymphoid tissue (GALT) is composed of lymphocytes residing in Peyer's patches, lamina propria, and intraepithelial compartments. In addition to these features which distinguish GALT from other peripheral sites of the immune system, the gastrointestinal immune system is also composed of resident eosinophils. Eosinophils are generally considered to be peripheral blood leukocytes that have an important pro‐inflammatory role in various immune disorders. Although most research concerning this cell has focused on understanding its trafficking and function in the blood and lung, recent studies have also started to elucidate its regulation and function in the gastrointestinal tract. Interestingly, eosinophil numbers in the gastrointestinal tract are substantially higher than in other tissues. At baseline (healthy conditions), most eosinophils reside in the lamina propria in the stomach and intestine. Eosinophil homing to these sites occurs during embryonic development and their levels in perinatal mice are comparable to those in adults, indicating that their homing is not dependent upon the presence of intestinal flora. Furthermore, eosinophil localization to the lamina propria at baseline is critically regulated by eotaxin, a chemokine constitutively expressed throughout the gastrointestinal tract. Although eotaxin is required for eosinophil homing, its expression in the esophagus is not sufficient for eosinophil accumulation, since this organ is devoid of eosinophils at baseline. During Th2‐associated inflammatory conditions (e.g. interleukin (IL)‐5 overexpression or oral allergen challenge), marked increases of eosinophils occur not only in the lamina propria but also in Peyer's patches. The accumulation of Peyer's patch eosinophils, which mainly occurs in the outer cortex and interfollicular regions, is critically regulated by IL‐5 and less significantly by eotaxin, suggesting the involvement of other eosinophil chemokines in this lymphoid compartment. Preliminary investigations have shown that gastrointestinal eosinophils express the α4β7 integrin and that this molecule is responsible, in part, for eosinophil homing. In summary, eosinophils are resident cells of the gastrointestinal immune system whose levels can be induced by antigen exposure under Th2 conditions, in a manner that is critically regulated by eotaxin and IL‐5. 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. 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 wish to thank Drs. K. Frank Austen, Mitchell Cohen, Paul Foster, Glenn Furuta, and Nives Zimmermann for helpful discussions, as well as numerous other colleagues.     

The physiological and pathophysiological roles of eosinophils in the gastrointestinal tract

Eosinophils and the gastrointestinal tract interact in an intimate and enigmatic relationship. Under healthy conditions, the presence of eosinophils is limited almost exclusively to the digestive tract mucosa where they exert several effector and immunoregulatory functions. While their precise function in the gastrointestinal tract is not completely understood, it is likely that, together with different T cell subsets, eosinophils are involved in maintaining the immunologic homeostastis across the mucosal barrier under resting conditions. Eosinophils also play a role in several inflammatory conditions, such as intestinal infections, hypersensitivity reactions, primary eosinophilic inflammations and several other chronic intestinal disorders. Depending on the responsible trigger, their effects may be beneficial or detrimental. Here, we discuss the available information regarding the physiological and pathological functions of eosinophils within the gastrointestinal tract.     

Sensitization and tolerance

The mechanisms responsible for sensitization, in particular within the gastrointestinal tract, are IgE-mediated as well as of a non-IgE-mediated, immunological origin. The phenomenon that is the opposite of sensitization is the maintenance of tolerance and is exemplified by the phenomenon 'oral tolerance'. The cytokines transforming growth factor beta and interferon gamma have been shown to be key immunoregulatory cytokines in oral tolerance. A new experimental model of eosinophilic allergic gastroenteritis has been developed with the use of encapsulated dietary antigen. The model was used in mice with genetic deletions of the eosinophil chemokine eotaxin or of the cytokine IL-5, demonstrating the importance of eotaxin. In clinical allergy research, a major question has been to explain the global increase in asthma and allergy. The 'hygiene hypothesis' states that a lack of maturation of the infant immune system from a T helper 2 to a T helper 1 type of immune response may be caused by less microbial stimulation in Western societies. Several lines of data support this hypothesis. However, apart from the genetic constitution of the individual, breastfeeding in infancy may be the most important single determinant for the development of clinical tolerance.     

Infiltrating eosinophils and eotaxin: their association with idiopathic eosinophilic esophagitis.

BACKGROUND: Idiopathic eosinophilic esophagitis (IEE) is a very rare disease characterized by thickening and eosinophil infiltration of the esophagus. The most potent chemotactic factor for eosinophils is eotaxin, and its pathophysiologic significance in IEE needs to be elucidated. OBJECTIVE: To study the association between eotaxin and IEE. METHODS: We examined eotaxin expression in the esophagus of an IEE patient in comparison to controls by immunohistochemistry using a monoclonal antibody for human eotaxin. We also measured the free eotaxin level and the total (free and bound-form) eotaxin level in blood by enzyme-linked immunosorbent assay before and after the initiation of steroid therapy. RESULTS: Most of the infiltrating eosinophils in the affected esophageal tissue showed immunohistochemical staining with anti-eotaxin antibody. In blood samples, the free eotaxin level was slightly elevated before treatment, whereas the total eotaxin level was within the normal range. Unexpectedly, the total eotaxin level increased dramatically after the initiation of steroid therapy, whereas the increase of free eotaxin was modest. CONCLUSION: Infiltrating eosinophils that express eotaxin and the changes of blood eotaxin levels during steroid therapy suggest that eotaxin may be associated with IEE.     

Eotaxin expression by epithelial cells and plasma cells in chronic asthma

Chemoattractants such as eotaxin are believed to play an important role in the recruitment of eosinophils into the airways in asthma. We investigated expression of eotaxin in the airway wall in a model of chronic human asthma, in which systemically sensitized mice were exposed to low mass concentrations of aerosolized antigen for 6 weeks. In these animals, the number of intraepithelial eosinophils in the airways was significantly increased 3 hours after exposure and declined by 24 hours. In parallel, immunoreactivity for eotaxin was strikingly up-regulated in airway epithelial cells and in inflammatory cells in the lamina propria. The latter were identified as plasma cells by double immunofluorescent labeling. Increased expression of eotaxin by epithelial cells and plasma cells was also demonstrated in a case of fatal human asthma. In contrast, sensitized mice that received a single exposure to a high mass concentration of aerosolized antigen exhibited delayed eosinophil recruitment, which did not correlate with eotaxin expression. Furthermore, in sensitized chronically exposed interleukin-13-deficient mice there was virtually no recruitment of eosinophils into the airways, although eotaxin expression was greater than or equal to that in wild-type mice. These results indicate that there are striking differences between acute and chronic exposure models in the time course of eotaxin expression and eosinophil recruitment. Although high eotaxin levels alone are not sufficient to cause recruitment of eosinophils into the airways, recurrent exposure may generate or up-regulate additional signals required for eosinophil chemotaxis.     

Eotaxin in induced sputum of asthmatics: relationship with eosinophils and eosinophil cationic protein in sputum

BACKGROUND: Eosinophilic inflammation is a crucial aspect of allergic diseases such as bronchial asthma. An eosinophil-active chemokine, eotaxin, may play a role in the pathogenesis of the tissue eosinophilia accompanying asthma. METHODS: Induced sputa were obtained from 53 patients with atopic asthma and six healthy subjects, and the concentration of eotaxin in the sputum was measured by ELISA. We investigated whether the sputum content of eotaxin is related to 1) asthma status or corticosteroid therapy, and 2) other sputum indices, including percentage of eosinophils and concentration of eosinophil cationic protein (ECP). RESULTS: The patients with stable or unstable asthma showed significantly higher concentrations of sputum eotaxin than the normal controls. The level of sputum eotaxin demonstrated a positive correlation with the percentage of eosinophils in stable asthmatics not receiving corticosteroid therapy, but not in stable patients treated with corticosteroids, or in unstable patients. Sputum eotaxin demonstrated a positive correlation with ECP in asthmatic patients who were either in a stable state or not receiving steroid therapy. CONCLUSIONS: The elevated level of eotaxin detected in association with increased eosinophils and ECP in the sputum of asthmatics suggests that eotaxin is involved in the pathogenesis of eosinophilic airway inflammation. The relationship of eotaxin to airway eosinophilia may be modified by the stability status of asthma and corticosteroid therapy.     

Effect of HFA-flunisolide on peripheral lung inflammation in asthma

BACKGROUND: New hydrofluoroalkane (HFA) formulations of glucocorticoids have been shown to effectively control asthma. HFA glucocorticoids are deposited across all sizes of airways, including the small ones. However, it is not clear whether they can suppress peripheral airway inflammation. OBJECTIVE: We sought to determine whether HFA-flunisolide could suppress peripheral inflammation in asthma. METHODS: Twelve patients with mild to moderate asthma received HFA-flunisolide for 6 weeks. Transbronchial and endobronchial biopsy specimens were obtained before and after treatment, and spirometry was performed. Changes in inflammatory cells (eosinophils, neutrophils, lymphocytes, macrophages, basophils) and IL-5 and eotaxin were measured by using immunocytochemistry and in situ hybridization. RESULTS: Lung function significantly improved after treatment (P <.05). HFA-flunisolide significantly reduced eosinophils, IL-5, and eotaxin in both peripheral and central airways (P <.01). Neutrophils significantly increased after treatment in peripheral and central airways (P <.05). The numbers of lymphocytes remained unchanged. CONCLUSIONS: These results show that HFA-flunisolide effectively suppressed eosinophilic inflammation in peripheral and central airways. These changes were accompanied by improvement in lung function.     

Allergy-associated colitis. Characterization of an entity and its differential diagnoses

There is substantial evidence that allergic reactions exist in the gastrointestinal tract (GI). However, patients with food allergy-related enteropathy pose a diagnostic challenge to physicians because the clinical features are variable, unspecific, occur in other gastrointestinal disorders, and specific diagnostic tools are missing. Several recent studies and reviews have focused on the function of eosinophilic granulocytes in GI disease. The role of eosinophils in the pathophysiology of GI hypersensitivity reactions is poorly defined. However, some findings have been reported that imply an involvement of eosinophils in allergic reactions of the gut. The presumptive histology of allergy-associated colitis in colonic and ileal biopsies is based on prominent pure eosinophilic infiltration of a normal lamina propria, submucosa and epithelium with variable degrees of degranulation. An immunoperoxidase stain for eosinophilic peroxidase is supportive in establishing the diagnosis if suspected. Neutrophils or mononuclear infiltrates are not significantly increased and damage to the intestinal tissue is not prominent. Despite characteristic histologic changes in colonic biopsy specimens, a final diagnosis depends on careful clinical examination and exclusion of several differential diagnoses.     

Depletion of eosinophils in mice through the use of antibodies specific for C-C chemokine receptor 3 (CCR3).

We have generated rat monoclonal antibodies specific for the mouse eotaxin receptor, C-C chemokine receptor 3 (CCR3). Several anti-CCR3 mAbs proved to be useful for in vivo depletion of CCR3-expressing cells and immunofluorescent staining. In vivo CCR3 mAbs of the IgG2b isotype substantially depleted blood eosinophil levels in Nippostrongyus brasiliensis-infected mice. Repeated anti-CCR3 mAb treatment in these mice significantly reduced tissue eosinophilia in the lung tissue and bronchoalveolar lavage fluid. Flow cytometry revealed that mCCR3 was expressed on eosinophils but not on stem cells, dendritic cells, or cells from the thymus, lymph node, or spleen of normal mice. Unlike human Th2 cells, mouse Th2 cells did not express detectable levels of CCR3 nor did they give a measurable response to eotaxin. None of the mAbs were antagonists or agonists of CCR3 calcium mobilization. To our knowledge, the antibodies described here are the first mAbs reported to be specific for mouse eosinophils and to be readily applicable for the detection, isolation, and in vivo depletion of eosinophils.     

Elevated levels of thymus and activation-regulated chemokine (TARC) in pleural effusion samples from patients infested with Paragonimus westermani

To investigate the pathogenic mechanisms of eosinophilic pleural effusion in patients with paragonimiasis, we measured the levels of various chemokines including thymus and activation-regulated chemokine (TARC), eotaxin, RANTES and IL-8 in pleural effusion samples. Samples were obtained from 11 patients with Paragonimus westermani infection, six patients with pleural transudate, eight with tuberculous pleurisy and five with empyema. High percentages of eosinophils were detected in pleural fluid (range 9-100%, median 81%) of patients with paragonimiasis. TARC concentrations in pleural effusions of paragonimiasis were markedly higher than those of other groups. Eotaxin levels were also higher in pleural effusions of paragonimiasis patients, although significant difference was noted only against transudate samples. There was a significant correlation between TARC concentrations and percentages of eosinophils, and between TARC and eotaxin concentrations in pleural effusion. There were also significant correlations between TARC concentration and the titre of anti-P. westermani IgG and between eotaxin concentration and the titre of anti-P. westermani IgG. Our findings suggest that TARC contributes to the pathogenesis of eosinophilic pleural effusion in paragonimiasis.     

Eotaxin and RANTES enhance 5-oxo-6,8,11,14-eicosatetraenoic acid-induced eosinophil chemotaxis

BACKGROUND: The 5-lipoxygenase product 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a potent activator of human eosinophils and, among lipid mediators, is the most active chemoattractant for these cells. Studies have demonstrated the importance of 5-lipoxygenase products in allergen-induced pulmonary eosinophilia. Because CC chemokines such as eotaxin and RANTES also play critical roles in this phenomenon, it would seem likely that members of both classes of mediators contribute to this response. OBJECTIVE: The study was designed to directly compare the effects of 5-oxo-ETE on eosinophils with those of eotaxin and RANTES and to determine whether these chemokines could enhance the chemotactic response to 5-oxo-ETE. METHODS: Eosinophil chemotaxis was measured with microchemotaxis chambers. CD11b, L-selectin, and actin polymerization were measured by flow cytometry. Calcium mobilization was measured by fluorescence. RESULTS: 5-Oxo-ETE stimulated eosinophil chemotaxis with a potency between those of eotaxin and RANTES and a maximal response about 50% higher than that of eotaxin. Threshold concentrations of eotaxin and RANTES increased the chemotactic potency of 5-oxo-ETE by more than 4-fold. 5-Oxo-ETE and eotaxin were approximately equipotent in mobilizing cytosolic calcium in eosinophils. Eotaxin was more potent in inducing CD11b expression and actin polymerization, but the maximal responses to 5-oxo-ETE were about 50% higher. 5-Oxo-ETE strongly induced L-selectin shedding, whereas eotaxin elicited only a weak and variable response. CONCLUSION: 5-Oxo-ETE is a strong activator of human eosinophils with a chemotactic potency comparable to those of eotaxin and RANTES, both of wwhich enhance 5-oxo-ETE-induced chemotaxis. 5-Oxo-ETE and CC chemokines may combine to induce pulmonary eosinophilia in asthma.     

Rosmarinic acid in perilla extract inhibits allergic inflammation induced by mite allergen, in a mouse model

BACKGROUND: Perilla and its constituent rosmarinic acid have been suggested to have anti-allergic activity. However, few studies have examined the effects on allergic asthma. OBJECTIVE: The purpose of this study was to evaluate the effect of oral administration of perilla leaf extract, which contains high amount of rosmarinic acid, on a murine model of allergic asthma induced by house dust mite allergen. METHODS: C3H/He mice were sensitized by intratracheal administration of Dermatophagoides farinae (Der f). Mice were orally treated with rosmarinic acid in perilla extract (PE) (1.5 mg/mouse/day). RESULTS: Der f challenge of sensitized mice elicited pulmonary eosinophilic inflammation, accompanied by an increase in lung expression of IL-4 and IL-5, and eotaxin. Daily treatment with rosmarinic acid in PE significantly prevented the increases in the numbers of eosinophils in bronchoalveolar lavage fluids and also in those around murine airways. Rosmarinic acid in PE treatment also inhibited the enhanced protein expression of IL-4 and IL-5, and eotaxin in the lungs of sensitized mice. Der f challenge also enhanced allergen-specific IgG1, which were also inhibited by rosmarinic acid in PE. CONCLUSION: These results suggest that oral administration of perilla-derived rosmarinic acid is an effective intervention for allergic asthma, possibly through the amelioration of increases in cytokines, chemokines, and allergen-specific antibody.