What Do Eosinophils Tell Us in Biopsies of Patients With Inflammatory Bowel Disease?

When viewing intestinal mucosal biopsies of patients suspected of having chronic inflammatory bowel disease (IBD), the most critical information includes evidence of chronicity (distorted mucosal architecture is of particular importance), the activity of the inflammation (usually represented by the intensity of the lymphoplasmacytic infiltrate and of neutrophil infiltration into epithelia or crypts), the presence of granulomas (suggestive of Crohn's disease [CD]), and the presence of dysplasia or carcinoma. The classic histologic appearance of IBD is mucosal damage: architectural distortion with erosions, ulceration, and occasionally granuloma formation, and with an inflammatory cell infiltrate containing neutrophils, lymphocytes, plasma cells, macrophages, eosinophils, and mast cells. Additional features such as mucin depletion, basal plasmacytosis, and Paneth cell and pseudopyloric metaplasia may also be seen. The quantity and composition of the inflammatory infiltrate can vary markedly over time. Neutrophil infiltration is simplistically referred to as “active” inflammation, whereas mononuclear cell infiltration (including lymphocytes, plasma cells, and macrophages) is referred to as “chronic” inflammation, although the notion that lymphoplasmacytic infiltrates in the mucosa are `inactive' does not bear scrutiny. Of course, a chronic inflammatory infiltrate (lymphocytes and plasma cells) is a normal feature of the intestinal mucosa and alone does not suggest IBD. Eosinophil and mast cell infiltration is a prominent feature of allergic inflammation, certain parasitic infestations, and autoimmune disorders. The pathogenesis of both ulcerative colitis (UC) and CD is commonly thought to involve autoimmune mechanisms and hypersensitivity reactions initiated by exogenous factors in genetically susceptible individuals. What do the eosinophils in biopsies of IBD patients tell us about disease activity, chronicity, and etiology? Eosinophils are a subgroup of granulocytes that are produced in bone marrow, circulate in peripheral blood, and migrate into tissues as a normal component of the host immune system. 1,2 The eosinophil is a potent proinflammatory cell that contains several highly cytotoxic proteins, such as major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), and eosinophil protein X (EPX), which is also referred to as eosinophil-derived neurotoxin (EDN). 3 These cytotoxic proteins make up the eosinophilic cytoplasmic granules, the distinguishing morphologic feature of this cell type. Ultrastructurally, eosinophil granules exhibit a distinctive feature, the crystalloid, an electron-dense central core surrounded by an electron-lucent matrix (Fig. 1A). Major basic protein is a major component of crystalloid, and together with three other major eosinophil proteins (ECP, EPO and EPX/EDN) comprises the matrix of the eosinophilic granules. 3 The activation of eosinophils by allergens or parasites results in their degranulation and the release of granule proteins (Fig. 1B). 1–3 Major basic protein and ECP are both directly cytotoxic to parasites and cells. The proinflammatory effects of eosinophils are further elicited by the generation by EPO of reactive oxygen metabolites that are cytotoxic to microbes and possibly result in tissue damage. In addition, the production of leukotriene C4 by activated eosinophils induces increased vascular permeability, mucus secretion, and smooth muscle contraction. 4 Activated eosinophils produce a wide range of cytokines, including interleukin (IL)-1, IL-3, IL-4, IL-5, IL-13, IL-16, GM-CSF, TGF-α/β, TNF-α, RANTES, macrophage inflammatory protein-1α, vasoactive intestinal peptide, substance P, and eotaxin. 4 These inflammatory mediators and cytokines may be involved in multiple functional aspects of immunomodulation and the inflammation-caused tissue damage, regeneration, and repair processes. Besides their ability to destroy parasites and microbes, eosinophils might contribute to the extensive tissue damage associated with the accumulation of these cells in specific disease states. 1–4FIG. 1.: Electron micrographs of eosinophils showing (A) distinctive granules containing crystalloids, and (B) degranulating eosinophil.In this issue of the Journal, Carvalho et al. 5 expand our knowledge of eosinophils in IBD by quantitatively analyzing the accumulation of eosinophils and IL-5–producing cells in samples from patients with IBD and controls. The authors have taken advantage of the histochemical stain Sirius Red (which stains eosinophil granules) and immunohistochemical staining with antibody EG1 (which reacts with both stored and secreted forms of ECP), antibody EG2 which reacts with both ECP during secretion (and apparently not the stored form) and EPX, and an antibody to IL-5. Using these stains, Carvalho et al. 5 observed increased numbers of eosinophils in inflamed colonic mucosa of patients with UC, and in both inflamed and noninflamed regions of the colon in patients with CD, compared with normal controls. In CD, the number of eosinophils was greater in inflamed areas than in noninflamed regions. Differences in the numbers of cells staining with antibodies EG1 and EG2 suggest that there is more activation of eosinophils in CD than in UC, whereas IL-5 staining was greater in UC than in CD, suggesting a more predominant Th2 response in UC than in CD. The accumulation of eosinophils in the intestinal mucosa of patients with IBD appears to be a common event, as shown in Figure 2A–D, and has been noted qualitatively for many years. 6–20 The study by Carvalho et al. 5 confirms this quantitatively, and raises the question of whether the presence of eosinophils is of clinical significance, and if so, at what levels.FIG. 2.: Micrographs of eosinophil-rich ulcerative colitis showing (A) transepithelial eosinophil migration, (B) free eosinophilic granules and eosinophils in the lamina propria, (C) crypt abscess with many eosinophils, and (D) mixed neutrophil and eosinophil infiltration into crypt epithelia.QUANTITATION OF EOSINOPHILS IN THE INTESTINAL MUCOSA Methodologic Considerations Eosinophils, because of their distinctive appearance in conventional stains, including hematoxylin and eosin, are readily noted in routine histopathology. Although they are normal constituents of the mucosa of the gastrointestinal tract, there has been no definitive determination of the number of eosinophils that can be defined as “normal.” They are not distributed uniformly along the length of the bowel, and in the large intestine, it is well recognized that they are usually more numerous in the cecum and ascending colon than elsewhere. 21–23 Increased eosinophil numbers have previously been found in association with eosinophilic gastroenteritis, allergic colitis, drug-induced colitis, acute gastrointestinal radiation injury, eosinophilic cryptitis, and certain parasitic infections. 21–25 Quantitation of inflammatory cells in the gastrointestinal mucosa poses several problems for the microscopist, and most pathologists use qualitative measures based on experience to decide whether eosinophils are increased in number. Attempts to generate more quantitative data are diverse. The time-honored method of counting the number of cells per high power field, which is used by many pathologists, including those in Carvalho's study, 5 suffers from the problem that the area of high-power fields varies greatly these days, depending upon the microscope and its objective and eyepiece lenses, some microscopes having high-power fields (×40 objective) almost twice the area of others in our experience. Carvalho et al. 5 avoid this problem appropriately by reporting their results per mm2. However, there remains the problem of the relative proportion of the high-power field occupied by glandular elements, which can vary wildly in mucosal injury in IBD, when the aim is to count cells that are located in the lamina propria. Counting the number of cells in the mucosa overlying a specified length of muscularis mucosae is an alternative, but suffers from the problem that if there is mucosal injury with distortion of architecture resulting in disturbance of the relative areas occupied by glands and lamina propria, as is usual in IBD, interpretation of the results will be difficult. This approach also depends on uniform handling and fixation of the tissues, so that contraction of the muscularis mucosae is uniform. A further problem in quantitating eosinophils is that of identifying degranulated cells when using stains that specifically illuminate granule contents, as in the study of Carvalho et al. 5 Ideally, stains should be used that allow the numbers of both granulated and degranulated eosinophils to be recorded: in similar studies of mast cells, the limitations of using granule stains have been clearly demonstrated by use of antibodies to mast cell tryptase and kinase which stain cells with and without granules. 26 In functional terms, the presence of degranulated eosinophils, which remain undetected by currently used stains, may be of much greater significance than that of the readily counted granulated cells. In addition to the actual counting methods, factors that could influence the data in studies of biopsies from patients with IBD include disease activity and the nature of the therapy being used to control it. 27,28 Disease activity in both UC and CD waxes and wanes and is variable both geographically and temporally. Drug therapy, in particular the use of steroids and other antiinflammatory agents, and therapeutic regimens (such as elemental diets) may influence eosinophil numbers, either down-regulating them or resulting in an increase, as has been postulated in the case of NSAIDs. Peripheral eosinophilia is not commonly seen in patients with IBD, and if present, merits further investigation. These factors are difficult to control for in clinical studies, and add another layer of difficulty to interpretation of quantitative results. MARKERS OF EOSINOPHIL ACTIVATION The functional role of eosinophils in IBD remains inconclusive: whether activated eosinophils are involved in active IBD is a point of debate. The findings by Carvalho et al. 5 provide support for the concept that eosinophils participate in active inflammation in IBD patients. These authors observed an increased proportion of EG1-positive cells in the noninflamed mucosa of CD patients as compared with the inflamed mucosa of the same patient and of UC patients. Interestingly, they found that “all sections stained with EG2 showed numerous positive cells and also an intense red granular background in the lamina propria, particularly in the inflamed mucosa of UC.” The suggestion is that this staining is indicative of the degranulation or activation of eosinophils. Recently, several antibodies against different eosinophilic granule proteins have been used as markers not only for eosinophil identification but also for eosinophil activity. 6,8,9 These antibodies are specific for eosinophil granule proteins, such as MBP, ECP, and EPO. EG1 antibody recognizes both stored and secreted forms of ECP, and EG2 is thought to be specific for secretory products of activated eosinophils (ECP and EPX during secretion). Even though some studies have shown that EG2-positivity may not be an indication of eosinophil activation, 29,30 the presence of EG2-positive eosinophils or decreased ratio of EG1- to EG2-positive eosinophils has been reported to be an indicator of the involvement of activated eosinophils in IBD. 8 A significantly decreased ratio of EG1/EG2 was found in IBD patients compared with healthy controls, and in inflamed mucosa compared with noninflamed mucosa. A study using improved immunostaining protocols (combining antibodies for EG1, EG2, and MBP) further supports the idea that increased numbers of eosinophils are involved in the active, fulminant inflammation of both UC and CD and is indicative of active rather than quiescent IBD. 6 The findings from an immunohistochemical study using anti-EPO antibody are also consistent with those of the previously mentioned reports. 9 These findings suggest that activated eosinophils are involved in active IBD. Degranulation and release of cytotoxic proteins are indicators of eosinophil activation. 1–4 Increased levels of the eosinophil granule proteins such as ECP and EPX/EDN have been found in feces, gut perfusates, and gut lavages from patients with UC and CD. 31–34 The presence of eosinophil proteins in the feces could be the result of eosinophil degranulation in the tissues, but may also result from intraluminal release from dead eosinophils that have transmigrated through the intestinal epithelium. Ultrastructurally, degranulation of eosinophils has been observed in the intestinal tissues of patients with IBD. 35 However, there is evidence that eosinophil degranulation occurs in the normal gastrointestinal tract and that nonactivated eosinophils are also capable of degranulation. 36 Ultrastructural analysis has demonstrated the presence of degranulated eosinophils in the inflamed mucosa, but epithelial cells in the vicinity of eosinophils did not exhibit injury during the early recurrence of CD. Discovering a reliable means of identifying functionally activated eosinophils is important in exploring the pathogenic role of eosinophils in IBD. EOSINOPHIL RECRUITMENT AND INTERACTION WITH OTHER INFLAMMATORY CELLS IN IBD The involvement of eosinophils in IBD may not be restricted to the release of cytotoxic proteins. Activated eosinophils can be involved in the inflammatory process by directly mediating cytotoxic effects, or by recruiting other inflammatory cells through the release of inflammatory mediators and cytokines. Several chemoattractants and cytokines are involved in eosinophil recruitment, both in disease and under physiological conditions. 37,38 These factors, including IL-3, IL-4, IL-5, IL-8, IL-16, GM-CSF, RANTES, PAF (platelet activating factor), and eotaxin, can be produced by lymphocytes, mast cells, intestinal epithelial cells, or eosinophils themselves. Eotaxin is an eosinophil-specific chemoattractant that functions mainly in eosinophil recruitment under physiological conditions. 37 IL-5 is the most potent modulator of eosinophil accumulation under disease conditions. 38 This cytokine enhances eosinophil recruitment by increasing adhesion, transendothelial migration, and mucosal infiltration. Overexpression of IL-5 is also crucial for delaying eosinophil apoptosis in many disease states. 39 In the absence of inflammation, the life span of terminally differentiated eosinophils in the tissue is of short duration due to programmed cell death. IL-5 induces the expression of Bcl-2 in eosinophils, resulting in resistance to apoptosis. Reduced cell death is an important mechanism of eosinophil accumulation in tissues. Carvalho et al. 5 showed that increased numbers of IL-5–expressing cells, which they considered “were probably activated eosinophils,” were detectable in the inflamed mucosa from UC and CD patients as compared with the noninflamed mucosa from CD and normal controls. Unfortunately, the authors did not confirm the nature of these cells. Since IL-5 is produced by CD4 T lymphocytes and mast cells, as well as eosinophils, 40–43 identification of the cells producing it is of critical importance, as the observation that eosinophils in the inflamed mucosa of IBD patients produce IL-5 would be evidence that these cells are functionally active. A comprehensive profile of the types of infiltrating inflammatory cells in the pathogenesis of IBD is needed to assess the functional interaction of the different cell types. The cytokine network performs a pivotal function in inflammatory cell recruitment. 37–39 Although different cell types require different signals for their recruitment, much overlap exists among these signals, as summarized in Figure 3. Tumor necrosis factor (TNF), IL-1, IL-8, and PAF are involved in inducing neutrophil infiltration. They are also potent eosinophil-recruiting factors. Simultaneous increases in neutrophil and eosinophil numbers are known to occur in the actively inflamed mucosa of IBD. It is uncertain whether these different inflammatory cell types perform cooperative or synergistic functions in the inflammatory process, or if some cell types are bystanders brought to the site of inflammation due to redundancy in the recruitment signals.FIG. 3.: Interaction of inflammatory cells and cytokines in IBD.ACCUMULATION OF EOSINOPHILS IN IBD Possible Significance Early clinical investigations attempted to explore whether IBD was an allergic disease, but this possibility has not been established. A recent study by D'Arienzo et al. 7 analyzed the association between allergen exposure and mucosal eosinophil accumulation in UC patients. Using standardized allergenic preparations, it was found that the degree of colonic tissue eosinophilia was higher in the presence of skin reactivity to food allergens, suggesting an association between UC, tissue eosinophilia, and type-I allergy. This raises the possibility that some acute exacerbations of IBD might result from an allergic response to a dietary component or components, superimposed on the underlying chronic disease. If this proves to be the case, increased numbers of eosinophils in mucosal biopsies of patients with IBD may be an indication for clinically evaluating exposure to dietary allergens. As far as we are aware, eosinophil infiltration has not been associated with the dysplasia-adenocarcinoma sequence in IBD. However, inflammation-induced oxidative stress plays a central role in the development of cancer. 44–46 Since the eosinophil granule protein, EPO is an important enzyme in the production of reactive oxygen metabolites it is theoretically possible that eosinophil accumulation could contribute to carcinogenesis by an inflammatory pathway. The relationship between tissue eosinophil accumulation and the risk for IBD-associated tumor development remains to be explored.