The eosinophilic granulocyte is a resident cell of gastrointestinal lamina propria, but to date, the precise function of this cell under resting or inflammatory conditions is not clearly understood.1,2 Eosinophils can be distinguished from other leukocytes by their unique granule populations, which include unicompartmental primary granules, bicompartmental secondary granules that consist of a matrix and a crystalloid core, and lipid bodies that are prominent organelles of the mature eosinophil.2 The only nonhematopoietic organ with resident eosinophils under resting conditions is the gastrointestinal tract.3 In humans that do not have alimentary tract disease, the cecal and appendiceal regions have the highest density of eosinophils and the esophagus is the only segment of the gastrointestinal tract without resident eosinophils.4 Within the gastrointestinal tract in humans, the eosinophil resides almost exclusively in the lamina propria compartment, close to the muscularis mucosae.1
Eosinophils develop from hematopoietic stem cells under the influence of IL-5, IL-3, and GM-CSF.5 Eotaxin-1 is the primary regulator of homing of eosinophils into the gastrointestinal tract. Homing occurs during the embryonic development and is independent of the presence of intestinal flora in mice.6 To date, many inflammatory mediators have been implicated in regulating eosinophil accumulation, but only IL-5 and eotaxin are somewhat specific for eosinophils.7 In horses, eotaxin-1 mRNA was found to be strongly expressed in the jejunum and colon,8 and the authors suggested that his could explain why the normal equine gastrointestinal mucosa had large numbers of resident eosinophils, compared with other equine tissues (eg, skin, lung, liver, spleen, and kidney).8
Eosinophils are proinflammatory leukocytes with a wide range of functions. In humans with eosinophilic gastroenteritis, eosinophils can be found in all levels of the affected mucosa9 and a correlation exists between degree of eosinophil accumulation and disease severity.10–12 Clinically affected horses with focal and diffuse eosinophilic gastroenteritis have been reported, and clinical signs include weight loss, diarrhea, signs of depression, acute and chronic colic, and hypoalbuminemia.13–16 The predominant histopathologic findings in these affected horses are severe mural inflammation in which eosinophilic leukocytes are the predominant inflammatory cell.17–19 The cause of eosinophil accumulation is unknown in these horses and has been attributed to immune-mediated processes, such as food allergy or parasite infection. Horses with focal eosinophilic enteritis often respond well to surgical removal of diseased intestinal segments.18,19 Furthermore, accumulation of eosinophils has been described for horses with experimentally induced acute colitis20 and in horses with experimentally induced ischemia and reperfusion injury.21 Eosinophil accumulation in the gastrointestinal mucosa can also be associated with parasitism.22 Another eosinophil-associated disease in horses is MEED.23–25 This disease is characterized by eosinophilic infiltrates in various tissues, most commonly the gastrointestinal tract, respiratory tract, and skin as well as the pancreas, biliary epithelium, and salivary glands.23,25 Clinical signs of MEED vary depending on the degree of involvement of these systems but often include weight loss and skin lesions.23,25 Multisystemic eosinophilic epitheliotropic disease affects mostly young horses between the ages of 3 to 13 years, and prognosis for survival is poor.24,25 The etiology of MEED is unknown, and parasitic, allergic, toxic, and viral causes have been suggested.24
To date, the authors have found no reference range information on the numbers of eosinophils and their distribution in the gastrointestinal mucosa of healthy horses. The goals of the study reported here were to establish these reference values for eosinophils and to describe the distribution of this cell within the equine gastrointestinal mucosa.
Materials and Methods
Specimen collection and grouping—Gastrointestinal mucosal specimens were collected from 17 horses euthanatized at the University of Illinois for reasons other than gastrointestinal disease. These horses were of both sexes, > 2 years old, of various breeds, and representative of the general hospital population. Although they had been on deworming programs, details of the deworming programs and the efficacy of parasite removal were unknown. At necropsy, none of these horses had any evidence of parasitism on gross inspection of the gastrointestinal mucosa and contents. In 3 horses, evidence of a parasite infection was found during histologic examination (ie, segments of parasites were seen in the intestinal mucosa). All mucosal specimens of these horses were excluded from further analysis, leaving specimens from 14 horses for further analysis. Specimens were grouped according to their anatomic regions of the gastrointestinal tract (ie, stomach, duodenum, jejunum, ileum, cecum, right ventral colon, left ventral colon, pelvic flexure, left dorsal colon, right dorsal colon, transverse colon, small colon, and rectum). For each anatomic region, specimens were taken approximately in the middle of the segment's length (eg, midjejunum).
Histologic evaluation—Mucosal specimens were fixed in 4% formalin and subsequently embedded in paraffin and cut into 5-μm-thick sections. Slides were stained with Luna's eosinophil stain26 to determine eosinophil accumulation and distribution. To perform Luna's eosinophil stain, slides are desiccated in xylene, stained with Biebrich scarlet-hematoxylin, differentiated in 1% acid alcohol, and subsequently stained with lithium carbonate. Eosinophil granules and erythrocytes will stain red-orange, Charcot-Leyden crystals stain red, and all nuclear elements are blue. One investigator (AKR) performed all histologic evaluations.
A computer-based programa was used for histomorphometric analysis of the images obtained by light microscopy. For tissues from the stomach, cecum, colon, and rectum, the mucosa was divided into 5 sections as follows: mean distance from the muscularis mucosae to the luminal surface was divided into successive quarters for sections M1 through M4, and the luminal surface of epithelial cells was section M5 (Figure 1) . For tissues from the small intestine, the mucosa was divided into 5 sections as follows: mean distance from the muscularis mucosae to the base of the villi was divided into 2 successive sections, M1 and M2; mean distance from the base of the villi to the tip of the villi was divided into 2 successive sections, M3 and M4; and the luminal surface of the epithelial cells was section M5 (Figure 2) . Absolute numbers of eosinophils were counted in 3 hpfs (650X magnification) for each section,21,27,28 and the mean number of eosinophils per section was calculated. These mean values were then used to calculate the percentage of eosinophils in each of the 5 sections relative to the total eosinophil count in all sections for each horse. Additionally, the number of eosinophils per square millimeter of mucosa was calculated as a measure of the eosinophil density.
Photomicrograph of a section of the right dorsal colon of a horse as an example of zones M1 to M5 used for counting of eosinophilic granulocytes (ht = height of mucosa) in tissues from the stomach, cecum, colon, and rectum. Luna's eosinophil stain, in which eosinophils are stained red and nuclear elements are blue; bar = 0.1 mm.
Citation: American Journal of Veterinary Research 69, 7; 10.2460/ajvr.69.7.874
Photomicrograph of a section of the small intestine of a horse as an example of zones M1 to M5 used for counting of eosinophilic granulocytes. Luna's eosinophil stain, in which eosinophils are stained red and nuclear elements are blue. Bar = 0.1 mm. See Figure 1 for key.
Citation: American Journal of Veterinary Research 69, 7; 10.2460/ajvr.69.7.874
Statistical analysis—Data were expressed as mean ± SD, and P < 0.05 was considered significant. Repeated-measures ANOVA and a commercially available statistical software programb were used for analysis. Whenever a significant F test for tissue (13 levels) or mucosal zone (5 levels) was present, appropriate Bonferroni-adjusted P values were used for each family of comparisons in order to identify significant differences. Specifically, the total eosinophil count for each tissue was compared with that for the other tissues. Likewise, the percent distribution of eosinophils within each of the 5 mucosal levels for 1 tissue was compared with that for all other tissues.
Results
Eosinophil accumulation—The number of eosinophils per square millimeter of mucosa in horses varied between sections of the gastrointestinal tract (Table 1). Lowest numbers were found in the stomach, and numbers increased in successive sections to the cecum, then decreased from the ascending colon (right ventral colon, left ventral colon, pelvic flexure, left dorsal colon, and right dorsal colon) to the small colon. Eosinophil counts in the cecum, all parts of the ascending colon, and the transverse colon were significantly higher than counts in the stomach, duodenum, jejunum, and small colon (P < 0.001 in all comparisons). Counts in the ileum were higher than those in the stomach (P = 0.003) and the duodenum (P = 0.002).
Eosinophil counts in gastrointestinal mucosa of healthy horses.
| Tissue | No. of horses | Eosinophil count (cells/mm2) | |
|---|---|---|---|
| Mean ± SD | Range | ||
| Stomach | 6 | 18 ± 22.4y,b | 0–71 |
| Duodenum | 13 | 100 ± 93y,b | 0–423 |
| Jejunum | 14 | 173 ± 142y | 0–552 |
| Ileum | 14 | 301.3 ± 282a | 0–1,173 |
| Cecum | 14 | 488 ± 285x | 100–1,338 |
| Right ventral colon | 14 | 432 ± 286x | 43–1,297 |
| Left ventral colon | 13 | 476 ± 285x | 9–1,327 |
| Pelvic flexure | 13 | 478 ± 301x | 65–1,216 |
| Left dorsal colon | 12 | 427 ± 253x | 16–1,041 |
| Right dorsal colon | 14 | 519 ± 417x | 7–1,556 |
| Transverse colon | 13 | 369 ± 254x | 0–948 |
| Small colon | 10 | 83 ± 99y | 0–321 |
| Rectum | 1 | 189 ± 86 | 138–288 |
Values with different superscript letters (ie, a vs b and x vs y) indicate values that are significantly (P < 0.05) different within the column.
Mucosal eosinophil distribution—In all gastrointestinal sections, most eosinophils were located in the basilar section M1 and most of the remaining eosinophils were located in section M2 (Table 2). Occasional eosinophils were found in the section closest to the lumen (M4), and an eosinophil on the luminal side of the epithelium (M5) was a rare finding. In the cecum and ascending colon, the distribution of eosinophils was closer to the surface than in the other sections of the gastrointestinal tract. A significantly higher percentage of eosinophils was observed in section M3 in the cecum and ascending colon and also in section M4 of the right dorsal colon, compared with sections of the small intestine.
Mucosal eosinophil percent distribution within the equine gastrointestinal mucosa.
| Tissue | M1 | M2 | M3 | M4 | M5 | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| Mean ± SD | Range | Mean ± SD | Range | Mean ± SD | Range | Mean ± SD | Range | Mean ± SD | Range | |
| Stomach | 58.4 ± 44.7 | 0–100 | 6.6 ± 12.4n | 0–40 | 8.9 ± 25.6 | 0–100 | 3.9 ± 12.4 | 0–50 | 0 | 0–0 |
| Duodenum | 64.7 ± 31.8 | 0–100 | 22.1 ± 22.7 | 0–66.9 | 4.5 ± 13.3y | 0–66.7 | 1.0 ± 5.4 | 0–33.3 | 0 | 0–0 |
| Jejunum | 62.6 ± 26.5 | 0–100 | 26.9 ± 19.3 | 0–66.7 | 3.2 ± 6.4n | 0–25 | 0.1±0.5b | 0–1.7 | 0 | 0–0 |
| Ileum | 66.3 ± 32.6 | 0–100 | 20.7 ± 21.7 | 0–84.6 | 0.9 ± 3b | 0–17.1 | 0.04 ± 0.3b | 0–1.7 | 0 | 0–0 |
| Cecum | 47.2 ±18.2 | 0–100 | 40.0 ± 14.6a,m,x | 0–79.6 | 11.8 ± 9.3a,m | 0–39.6 | 1.0 ± 2.7 | 0–14.1 | 0 | 0–0 |
| Right ventral colon | 56.4 ± 14.7 | 32–94.1 | 34.0 ± 12.3a,m | 0–58.3 | 8.8 ± 7.4a | 0–24.6 | 1.3 ±3.1 | 0–14.8 | 0.03 ± 0.2 | 0–1 |
| Left ventral colon | 57.8 ± 15.9 | 33.6–100 | 29.2 ± 12.1a | 0–50 | 10.3 ± 8.5a | 0–30 | 1.7 ±3.7 | 0–16.2 | 0 | 0–0 |
| Pelvic fexure | 50.1 ± 16.7 | 0–82.1 | 34.2 ± 12.2a,m | 10.7–64.7 | 13.04 ± 9.4a,m,x | 0–38.5 | 2.3 ± 4.3 | 0–19.2 | 0.2 ± 1 | 0–6.1 |
| Left dorsal colon | 54.8 ± 17 | 21.9–100 | 29.6 ± 11.2a,m | 0–50 | 12.8 ± 8.3a,m,x | 0–34.4 | 2.7 ± 4.2 | 0–14.1 | 0 | 0–0 |
| Right dorsal colon | 54.5 ± 21.1 | 28.8–100 | 29.11 ± 4.9a | 0–60 | 12.9 ± 8.6a,m | 0–29.2 | 3.5 ± 4.5a | 0–16.7 | 0 | 0–0 |
| Transverse colon | 64.1 ± 22.8 | 0–100 | 25.6 ± 16.7y | 0–52.6 | 6.6 ± 6.6 | 0–21.1 | 0.9 ± 1.8 | 0–6.5 | 0 | 0–0 |
| Small colon | 46.5 ± 38.8 | 0–100 | 12.7±15.5b | 0–51.5 | 6.8 ± 19.6 | 0–100 | 1.9 ± 5.6 | 0–20 | 0 | 0–0 |
| Rectum | 66.2 ± 13.6 | 52.8–80 | 32.9 ± 13.7 | 20–47 | 0.9 ± 1.5 | 0–2.6 | 0 | 0–0 | 0 | 0–0 |
Values with different superscript letters (ie, a vs n, and x vs y) indicate values that are significantly (P < 0.05) different within the column.
Discussion
In our study, distribution of eosinophils in the gastrointestinal tract of horses followed a consistent pattern similar to the distribution in humans.4 The highest numbers of eosinophils were found in the cecum and the ascending and transverse colon, and the lowest were found in the stomach and the small colon. These differences should be considered when examining biopsy specimens from equine gastrointestinal mucosa. In the right dorsal colon, an eosinophil count of 48 cells/650X hpf would be within reference range, whereas the same count in the jejunum would be approximately 4 times as high as expected. The distribution of eosinophils within the mucosal lamina propria also follows a consistent pattern that could enable these cells to react quickly to stimuli originating close to the luminal surface, without interfering with the normal functions of the mucosal epithelium. Most eosinophils were located in the basilar half of the mucosa in all sections of the gastrointestinal tract. The ascending colon had a slightly higher percentage of eosinophils in section M3 than did other sections of the gastrointestinal tract, indicating that care should be taken when interpreting the eosinophil distribution in this tissue. To the best of our knowledge, the mucosal distribution from base to surface that we found throughout the equine gastrointestinal tract has not been reported for other species. A previous evaluation of jejunum from clinically normal horses did reveal significantly greater numbers of resident eosinophils in the intestinal crypts than in the villous region,29 but only the jejunum was examined, and a profile from base to surface was not established.
To our knowledge, there has been no suggestion as to why the cecum, ascending colon, and transverse colon have higher numbers of eosinophils than other gastrointestinal segments. Because homing occurs during embryonic development and is independent of intestinal flora,6 eosinophil density throughout the gastrointestinal tract appears independent of intestinal contents in mice. The distribution profile for eosinophil numbers per segment peaked at the cecum and large colon in the study presented here, which does not coincide with peaks for dry-matter content.30 Water content of the equine gastrointestinal tract declines from proximal to distal,30 so that abrasion from dry matter or increasing concentrations of luminal antigens does not explain the eosinophil profile that we found. Gastrointestinal contents of ponies have a fairly constant pH from the cecum distally, but the concentration of volatile fatty acids peaks at the cecum and then declines distally31 in much the same way as eosinophil numbers. Total bacteria and viable bacteria are highest in the equine cecum, compared with the more proximal segments and the rectum,32 which does partly correlate with the eosinophil distribution in the equine gastrointestinal tract.
Eosinophils may have a regulatory role in protection against luminal contents that have greater access to the intestinal epithelium and lamina propria after mechanical epithelial damage. Cyathostomes are common equine intestinal parasites, and most identified cyathostomes are encysted larvae.33 Most are early third-stage larvae33 and are found in the cecum (48%) and ventral colon (40%), followed by the dorsal colon (12%). Adult cyathostomes are mainly in the ventral colon (64%), followed by the dorsal colon (27%) and cecum (9%).33 Therefore, the typical distribution of encysted cyathostomes is closely correlated with the peak of eosinophil counts in the current study. Because eosinophils have been described as part of the mucosal defense mechanism against intestinal parasites, the presence of a larger number of eosinophils in areas of the gastrointestinal tract prone to parasites, especially encysted parasites, would be an evolutionary advantage to the host. However, because the same pattern of eosinophil densities has been reported for the human gastrointestinal tract, the pattern that we found might be common for many mammals and might not be related to equine-specific diseases.
In a previous study,22 eosinophil and mast cell densities in the large intestinal mucosa were associated with cyathostome infection. In the same study,22 eosinophil counts were higher in horses > 2 years old, compared with younger horses, despite similar parasite burdens in both groups. That study22 also reported low numbers of intraepithelial eosinophils; however, comparisons with our study are difficult because tissue locations of cells were defined differently, the counting technique was different, and a stain specific for eosinophils was not used. Horses used in our study were representative of the general equine population that provides our hospital caseload and had been on deworming programs. None of the horses used in the study had clinical or pathologic evidence of parasitism, and those that did were excluded by our inclusion criteria. A previous study21 reported large numbers of resident eosinophils in the colon of horses that had been dewormed, and another study34 did not find any effect of parasitism with Strongylus vulgaris on tissue eosinophil numbers or focal distributions. Further studies are needed to evaluate the effect of current or transient exposure of parasites to the mucosal eosinophil numbers.
In an in vitro study27 of mucosa from equine right dorsal colon, damage to the mucosal surface epithelium with hypochlorous acid induced rapid migration of eosinophils toward the mucosal surface. Significant accumulation of eosinophils on the luminal surface was detected within 30 minutes of the injury.27 Eosinophils appeared to be degranulating in tissues treated with hypochlorous acid alone to a greater extent than they were in undamaged tissues, but this process was not statistically evaluated. Migration in vitro is of considerable interest because this model of gastrointestinal tract damage lacks any of the factors that are usually associated with eosinophil accumulation in the intestine, such as activation of the immune system by food allergens or parasites. More importantly, these findings may help to understand how eosinophils respond to injury to the surface epithelium by migrating toward the site of greatest cell damage, which is evidence that the mucosal location predominantly in M1 is more typical of the “resting” state for these cells. Further study is required to determine whether a similar migration occurs in response to intestinal parasites and other intestinal diseases.
In conclusion, eosinophils have a characteristic pattern of distribution within the gastrointestinal mucosa and between different sections of the equine gastrointestinal tract. Processes responsible for these findings and the timing of homing to these sites are unknown, but could be an evolutionary system for local defense at sites prone to various disease processes (eg, parasitism). Our results should provide reference values that can aid in the interpretation of eosinophil accumulation and distribution in the mucosa of horses with gastrointestinal tract disease.
ABBREVIATIONS
| GM-CSF | Granulocyte macrophage colony-stimulating factor |
| IL | Interleukin |
| MEED | Multisystemic eosinophilic epitheliotropic disease |
Image Pro Express, version 4.5, Media Cybernetics Inc, Bethesda, Md.
PROC MIXED, SAS, version 9.1, SAS Institute Inc, Cary, NC.
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