In companion animal gastroenterology, IBD is the term used to describe patients affected with signs of persistent or recurrent GI tract disease and histopathologic evidence of intestinal inflammation without underlying causes (eg, infection, endocrine disease, or neoplasia).1,2 Mucosal inflammation is the main histopathologic feature of IBD in dogs, and variants of IBD are classified on the basis of the predominant type of inflammatory cells present and site of affected intestine.1,3 The primary forms of small intestinal IBD consist of lymphoplasmacytic enteritis and EE. With regard to the large intestine, 4 main forms of IBD are recognized in dogs, comprising lymphoplasmacytic colitis, eosinophilic colitis, histiocytic ulcerative colitis, and regional granulomatous colitis.3 After lymphoplasmacytic enteritis, EE is the second most frequently diagnosed form of IBD in dogs; it is characterized by a mixed infiltration of inflammatory cells, with eosinophils predominating.4,5 Clinical signs of EE (eg, diarrhea, weight loss, and signs of abdominal pain) are similar to those for other forms of IBD, although mucosal erosion or ulceration may also be seen along with hematemesis and melena.1 Eosinophilic infiltration, as judged by evaluation of specimens stained with H&E stain, is considered a histologic feature of IBD.1,6 Because of the various criteria used by pathologists with respect to the extent of eosinophilic infiltration, diagnosis of EE has been problematic.1 However, standards established by the World Small Animal Veterinary Association suggest clearly defined criteria for the diagnosis of EE.7 One persisting limitation with routine use of H&E stain is that it readily stains intact eosinophils but not degranulated eosinophils.
Eosinophils typically are present in the intestinal mucosa, albeit in low numbers.1,6 Eosinophils can serve as major effector cells in tissues because of their capacity to release several highly cytotoxic preformed granule proteins, including eosinophil cationic protein, EPX, eosinophil-derived neurotoxin, and major basic protein, during conditions of inflammation, which can lead to tissue damage and dysfunction.8–10 Human patients with GI disorders such as food allergy and eosinophilic gastroenteritis have increased numbers of activated eosinophils in the intestinal mucosa, where they are implicated as the main effector cells.11,12 Several studies9,13–15 have indicated that eosinophil granule proteins (eosinophil cationic protein or major basic protein) released by activated eosinophils contribute to tissue damage and inflammation associated with GI disorders. Although studies of affected humans and of animals with experimentally induced disease have increased our current understanding of the role of eosinophils and eosinophil granule proteins in intestinal inflammation, the authors are not aware of any studies conducted to investigate the prevalence of degranulated eosinophils and potential implication of extracellular granule proteins in the small intestine of dogs with IBD. Presence of degranulated eosinophils in target tissues is representative of the activation state of eosinophils and potentially the pathogenic state of disease. However, degranulated eosinophils are extremely difficult to identify by use of routine H&E staining, the method currently used for histologic evaluation of canine GI tissue.16 An mAb specific for EPX, an eosinophil secondary granule protein, was developed and found to specifically interact with eosinophils as well as enable identification of degranulated eosinophils in formalin-fixed specimens from humans17 and dogs.18 The objective of the study reported here was to assess whether this antibody could be used for the accurate detection and quantification of tissue-infiltrating intact and degranulated eosinophils in dogs with IBD and serve as a prognostic marker of disease.
Materials and Methods
Dogs
Dogs with IBD were identified by searching a database that contained information regarding demographics, clinical history, and results of a necropsy report (gross and histologic examinations). A diagnosis of IBD was established if there were clinical signs consistent with IBD (anorexia, weight loss, vomiting or diarrhea, hematochezia, or mucus in the feces), persistence of clinical signs for > 3 weeks, histopathologic evidence of GI inflammation or morphological changes, and absence of a specific underlying cause. Formalin-fixed, paraffin-embedded tissue blocks containing stomach, duodenum, jejunum, and colon of dogs with IBD that were submitted to the University of Minnesota Veterinary Diagnostic Laboratory between 2008 and 2015 for necropsy and histologic examination were used. Dogs with no evidence of GI disease and that had not received immunosuppressive treatment were used as control animals; biopsy specimens were collected from the control dogs at the time of necropsy. Dogs were excluded from the study if they had evidence of any neoplastic condition or infectious agent identified in the GI tract.
The study comprised 3 groups: dogs with IBD that were treated with prednisolone (1 to 2 mg/kg for > 3 weeks) for immunosuppression (n = 5), dogs with IBD that were not treated with prednisolone (11), and control dogs without evidence of IBD (8). Treated IBD dogs (3 females and 2 males; mean ± SD age, 9.0 ± 3.5 years) consisted of 2 mixed-breed dogs, 1 German Shepherd Dog, 1 Golden Retriever, and 1 Norwich Terrier. Untreated IBD dogs (4 females and 7 males; mean age, 11.4 ± 1.7 years) consisted of 2 German Shepherd Dogs, 2 Bernese Mountain Dogs, 1 Golden Retriever, 1 Australian Shepherd, 1 Chinese Shar-Pei, 1 Labrador Retriever, 1 Mastiff, 1 Yorkshire Terrier, and 1 mixed-breed dog; 2 other dogs were excluded because of sudden death suspected to have been caused by poinsettia ingestion. Untreated IBD dogs had mild to moderate lymphocytic plasmacytic inflammation involving the lamina propria. Control dogs (3 females and 5 males; mean age, 5.6 ± 2.8 years) consisted of 2 Golden Retrievers, 1 Bichon Frise, 1 Boxer, 1 Pekingese, 1 Pug, 1 Welsh Corgi, and 1 mixed-breed dog.
Tissue preparation and staining
Serial 4-μm-thick sections were cut from each paraffin-embedded block. One section was stained with H&E stain for assessment of inflammatory cell populations, and 2 other sections were immunostained with EPX mAba and mouse IgG (negative control treatment).b
For immunohistochemical analysis, sections were deparaffinized by immersion in xylene, rehydrated in a descending series of ethanol solutions, and rinsed in water. Slides were placed in 10mM sodium citrate buffer and heated in a microwave oven for 5 to 10 minutes until the buffer solution boiled, after which slides were maintained at 95°C for an additional 20 minutes. Slides were then allowed to sit undisturbed at room temperature (22°C) for 30 minutes. Slides were incubated with proteinase Kb (200 μL/slide) for 7 minutes and then rinsed in TBS solution (3 rinses/slide; 5 min/rinse). Nonspecific peroxidase activity was blocked by immersing the slides in 3% hydrogen peroxide. Slides were rinsed 3 times with TBS solution, and sections were blocked by incubation in blocking bufferc (200 μL/slide) in a moist chamber at room temperature for 60 minutes. Slides were then incubated overnight with EPX mAb (200 μL of a 1 mg/mL solution diluted 1:500 in blocking buffer) at 4°C. Mouse IgG at the same concentration as the EPX mAb was used as a negative control sample. After incubation with the primary antibody, slides were warmed to room temperature, maintained at room temperature for 15 minutes, and rinsed with TBS solution (3 rinses/slide; 5 min/rinse). Detection was performed by use of a commercial immunoperoxidase detection kitc with biotinylated secondary antibodies and avidin-biotin horseradish peroxidase complex followed by 3-amino-9-ethylcarbazole peroxidase substrate according to the manufacturer's recommendation. Slides were rinsed several times with tap water, counterstained with hematoxylin, and mounted in a glycerol gelatin aqueous slide mounting medium.b Stained sections were evaluated by use of a microscope,d and images were recorded with a digital microscope camera.e
Quantification of stained eosinophils
Only jejunum was present in all tissue blocks; therefore, results pertaining to only the jejunum were reported. Stained intact and degranulated eosinophils in consecutive microscopic fields (400× magnification) of the upper (villus tips) and lower (between the muscularis mucosae and crypts) regions of the lamina propria of the jejunum were counted by use of a light microscope.f Degranulated eosinophils were identified on the basis of staining with EPX in the extracellular space, which corresponded to an adjacent nucleus that fit the morphological criteria for an eosinophil (hyperchromatic bilobed nucleus) as described elsewhere.19 All measurements and cell counts were obtained by 1 investigator (IB) who did not have knowledge of patient history or study group. Several randomly selected sections were evaluated separately by other investigators (NAR and XNG) to ensure consistency in quantitation.
Statistical analysis
Mean number of eosinophils per microscopic field was calculated for each region-type combination for each dog. This value was used for all subsequent analyses. Visual inspection indicated that the data were not normally distributed, with substantial skew and several outlying points. To compare treatment groups with regard to each region-type combination, pairwise Wilcoxon tests were performed, with P values corrected for multiple comparisons within each region-type combination by use of a Bonferroni-Holm correction. Tests were considered significant at values of P ≤ 0.05.
Results
H&E staining
A mixed inflammatory cell infiltrate was evident in the lamina propria of the jejunum, with eosinophils predominating followed by lesser numbers of lymphocytes and plasma cells in untreated IBD dogs. Neutrophils were rarely seen in untreated IBD dogs. No abnormal cellular infiltrate was detected in the jejunum of the control and treated IBD dogs.
EPX staining
Evaluation of EPX mAb-stained sections of the jejunum indicated that there was eosinophilic infiltration in the lamina propria in all groups. Results for EPX staining indicated intact eosinophils with prominent intracellular staining within membrane-bound secondary granules as well as degranulated eosinophils with extracellular granule staining that was more diffuse in appearance (Figure 1). However, there were differences in the number of eosinophils (intact and degranulated) among the groups (Figures 2 and 3). The number of degranulated eosinophils was significantly higher in both the lower and upper regions of the lamina propria of untreated IBD dogs, compared with numbers for the control and treated IBD dogs (Figure 4). No significant differences in numbers of intact eosinophils in the lower region of the lamina propria were detected among groups. However, in the upper region of the lamina propria, untreated IBD dogs had a significantly higher number of intact eosinophils than did the control and treated IBD dogs. No differences in eosinophil numbers were detected in the upper or lower region of the lamina propria between the control and treated IBD dogs.
Photomicrographs of H&E-stained (A) and EPX mAb-stained (B) tissue sections of the jejunum showing the distribution of eosinophils in the upper (villous tips) and lower (between the muscularis mucosae and crypts) regions of the lamina propria from an untreated dog with IBD. In panel A, notice that there is a high density of eosinophils in the tissues. In panel B, notice that EPX mAb staining improves the ability to detect the eosinophil distribution and to identify intact eosinophils (black arrow) and degranulated eosinophils (red arrow). Bar = 200 μm.
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.36
Photomicrographs of H&E-stained (A) and EPX mAb-stained (B) tissue sections of the jejunum showing the distribution of eosinophils in the upper (villous tips) and lower (between the muscularis mucosae and crypts) regions of the lamina propria from an untreated dog with IBD. In panel A, notice that there is a high density of eosinophils in the tissues. In panel B, notice that EPX mAb staining improves the ability to detect the eosinophil distribution and to identify intact eosinophils (black arrow) and degranulated eosinophils (red arrow). Bar = 200 μm.
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.36
Photomicrographs of H&E-stained (A) and EPX mAb-stained (B) tissue sections of the jejunum showing the distribution of eosinophils in the upper (villous tips) and lower (between the muscularis mucosae and crypts) regions of the lamina propria from an untreated dog with IBD. In panel A, notice that there is a high density of eosinophils in the tissues. In panel B, notice that EPX mAb staining improves the ability to detect the eosinophil distribution and to identify intact eosinophils (black arrow) and degranulated eosinophils (red arrow). Bar = 200 μm.
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.36
Photomicrographs of tissue sections of the upper region of the lamina propria of the jejunum of a healthy control dog (A and B), an untreated dog with IBD (C and D), and a dog with IBD treated with prednisolone (E and F) depicting the distribution of eosinophils. Notice the difference in appearance of the eosinophils between sections prepared with H&E stain (A, C, and E) and with EPX mAb (B, D, and F). Bar = 20 μm for panels A, B, C, and E and 50 μm for panels D and F.
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.36
Photomicrographs of tissue sections of the upper region of the lamina propria of the jejunum of a healthy control dog (A and B), an untreated dog with IBD (C and D), and a dog with IBD treated with prednisolone (E and F) depicting the distribution of eosinophils. Notice the difference in appearance of the eosinophils between sections prepared with H&E stain (A, C, and E) and with EPX mAb (B, D, and F). Bar = 20 μm for panels A, B, C, and E and 50 μm for panels D and F.
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.36
Photomicrographs of tissue sections of the upper region of the lamina propria of the jejunum of a healthy control dog (A and B), an untreated dog with IBD (C and D), and a dog with IBD treated with prednisolone (E and F) depicting the distribution of eosinophils. Notice the difference in appearance of the eosinophils between sections prepared with H&E stain (A, C, and E) and with EPX mAb (B, D, and F). Bar = 20 μm for panels A, B, C, and E and 50 μm for panels D and F.
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.36
Photomicrographs of jejunal tissue sections obtained from an untreated dog with IBD and stained with H&E stain (A) and EPX mAb (B). In panel A, eosinophils are evident. In panel B, fine detail of the eosinophil granules is evident within the cytoplasm of eosinophils (black arrows) and also in the extracellular matrix, which indicates degranulation (red arrows). Bar = 20 μm.
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.36
Photomicrographs of jejunal tissue sections obtained from an untreated dog with IBD and stained with H&E stain (A) and EPX mAb (B). In panel A, eosinophils are evident. In panel B, fine detail of the eosinophil granules is evident within the cytoplasm of eosinophils (black arrows) and also in the extracellular matrix, which indicates degranulation (red arrows). Bar = 20 μm.
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.36
Photomicrographs of jejunal tissue sections obtained from an untreated dog with IBD and stained with H&E stain (A) and EPX mAb (B). In panel A, eosinophils are evident. In panel B, fine detail of the eosinophil granules is evident within the cytoplasm of eosinophils (black arrows) and also in the extracellular matrix, which indicates degranulation (red arrows). Bar = 20 μm.
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.36
Box-and-whisker plots of the number of degranulated (A and B) and intact (C and D) eosinophils in the lower (A and C) and upper (B and D) regions of the lamina propria of the jejunum of healthy control dogs (n = 8), dogs with IBD that were treated with prednisolone (5), and dogs with IBD that were untreated (11). Cells were immunohistochemically stained with EPX mAb and eosinophils were then quantitated in consecutive microscopic fields (400× magnification) by use of a light microscope. Each box represents the first and third quartiles, the horizontal line in each box represents the median, the whiskers represent the most extreme data point < 1.5 times the interquartile range from the first and third quartiles, and the circles represent outliers. Notice that the scale on the y-axis differs between degranulated and intact eosinophils. *Median value of this group differs significantly from the values for the other 2 groups of dogs.
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.36
Box-and-whisker plots of the number of degranulated (A and B) and intact (C and D) eosinophils in the lower (A and C) and upper (B and D) regions of the lamina propria of the jejunum of healthy control dogs (n = 8), dogs with IBD that were treated with prednisolone (5), and dogs with IBD that were untreated (11). Cells were immunohistochemically stained with EPX mAb and eosinophils were then quantitated in consecutive microscopic fields (400× magnification) by use of a light microscope. Each box represents the first and third quartiles, the horizontal line in each box represents the median, the whiskers represent the most extreme data point < 1.5 times the interquartile range from the first and third quartiles, and the circles represent outliers. Notice that the scale on the y-axis differs between degranulated and intact eosinophils. *Median value of this group differs significantly from the values for the other 2 groups of dogs.
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.36
Box-and-whisker plots of the number of degranulated (A and B) and intact (C and D) eosinophils in the lower (A and C) and upper (B and D) regions of the lamina propria of the jejunum of healthy control dogs (n = 8), dogs with IBD that were treated with prednisolone (5), and dogs with IBD that were untreated (11). Cells were immunohistochemically stained with EPX mAb and eosinophils were then quantitated in consecutive microscopic fields (400× magnification) by use of a light microscope. Each box represents the first and third quartiles, the horizontal line in each box represents the median, the whiskers represent the most extreme data point < 1.5 times the interquartile range from the first and third quartiles, and the circles represent outliers. Notice that the scale on the y-axis differs between degranulated and intact eosinophils. *Median value of this group differs significantly from the values for the other 2 groups of dogs.
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.36
Discussion
Eosinophils are present in variable numbers in the small intestine of healthy dogs.20,21 Although their physiologic function at this site remains poorly understood, a study22 of mice suggested that activated eosinophils may play a role in intestinal adaptive immunity by activating dendritic cells via release of granule proteins. Activated eosinophils are extremely difficult to identify by use of H&E stain, and any degranulation observed severely underrepresents the actual amount.16 Immunohistochemical analysis with EPX mAb of human mucosal biopsy specimens has revealed prominent granule staining of eosinophils that makes morphological identification (degranulated and intact) more reliable.17 Investigators of 1 study18 found that EPX mAb can specifically detect eosinophils in canine skin and is more effective than the use of H&E stain to detect eosinophils with extracellular eosinophil granules or granule proteins. In the present study, EPX mAb-based immunohistochemical staining was used to determine, for the first time, the presence and activation state of eosinophils in dogs with IBD relative to results for dogs without IBD.
Enumeration of eosinophils after EPX mAb immunostaining indicated that degranulated eosinophils were more prevalent in the upper and lower regions of the lamina propria of the jejunum in untreated IBD dogs than in control and treated IBD dogs, which suggested that activated eosinophils may be important participants in the inflammatory process of IBD in dogs. Increasingly, it has been suggested that activated eosinophils can cause damage to intestinal tissues through the release of various inflammatory mediators and granule proteins.10 Patients with eosinophilic gastritis have an accumulation of activated eosinophils, compared with results for control patients.12 Furthermore, amounts of mucosal-released eosinophil granule proteins (indicative of eosinophil activation) are much higher in patients with colitis and proctitis, compared with amounts in healthy control subjects.23 Similarly, results for a study24 of rats with induced gastric ulcers indicated substantial infiltration of activated degranulated eosinophils at the margin of newly formed ulcers that were believed to contribute to the inflammatory process. Overall, results of these studies suggest an important role for activated eosinophils in promoting or mediating inflammation in the intestinal mucosa.
In the present study, we detected differences in the number of activated eosinophils among the 3 groups; however, there were no differences in the prevalence of intact nonactivated eosinophils in the lower region of the lamina propria among the 3 groups. These nonactivated cells may represent resident cells constitutively present in healthy small intestine. Interestingly, the upper region of the lamina propria in untreated IBD dogs had a significantly larger number of intact eosinophils, compared with the number in control and treated IBD dogs. This may have been linked, in part, to elevated concentrations of chemokines (eg, eotaxin-1 and chemokine [C-C motif] ligand 5) that are known to be associated with eosinophilic GI disease in humans and mice.25,26 During inflammatory conditions, elevated concentrations of these chemokines can induce migration of eosinophils to the villi, where they can degranulate and cause tissue damage.27 In a study28 of mice with antigen-induced eosinophilic GI allergy, control mice had few eosinophils that primarily localized to the base of the villi, compared with results for allergen-challenged mice, which had an elevated number of eosinophils distributed throughout the lamina propria (mucosa and villi). Furthermore, for eotaxin-deficient mice in that study,28 the number and distribution of eosinophils were similar between control and allergen-challenged mice, which validated the role of eotaxin in recruitment of eosinophils to the GI tract.
Overall, the present study indicated the utility of EPX mAb-based immunohistochemical staining as a strategy for the quantitative assessment of eosinophils (intact and degranulated) in the jejunal lamina propria in small intestinal biopsy specimens obtained from dogs with IBD. Furthermore, we identified the importance of detecting and quantitating degranulated eosinophils in the lamina propria by use of EPX mAb immunostaining, which may be underrepresented by use of H&E staining alone. On the basis of known cytotoxic effects of eosinophilic granule proteins, prevalence of degranulated eosinophils in the upper and lower regions of the lamina propria may be a characteristic feature of IBD and may serve as an important indicator of disease severity.
The study reported here was limited by the small number of treated IBD dogs, compared with the number of untreated IBD dogs, and the lack of complete information about clinical response of the treated dogs to treatment. Furthermore, although it would be expected that prednisolone treatment reduced the number of eosinophils, there was a lack of information about eosinophil numbers before treatment in the treated IBD dogs; thus, additional studies with this antibody on a larger study population are needed to identify associations between eosinophil numbers or activation state and the disease status and severity, as well as age, breed, and sex of dogs, to improve the diagnosis and treatment of IBD. Future studies are needed to determine whether the current World Small Animal Veterinary Association grading system should be modified to take into account novel findings pertaining to eosinophils.
Results of the present study suggested that EPX mAb staining may serve as a potential diagnostic method for identifying IBD, monitoring disease activity, and evaluating the success of treatment in dogs. It may constitute a first step toward understanding the role of degranulated eosinophils in the pathogenesis of IBD in dogs.
Acknowledgments
Supported in part by University of Minnesota College of Veterinary Medicine funds (Robinson) and GAR funds (grant No. MIN-62-059 [Wasabau]).
The authors declare that there were no conflicts of interest.
The authors thank the University of Minnesota Veterinary Diagnostic Laboratory for providing archived tissue samples.
ABBREVIATIONS
| EE | Eosinophilic enteritis |
| EPX | Eosinophil peroxidase |
| GI | Gastrointestinal |
| IBD | Inflammatory bowel disease |
| mAb | Monoclonal antibody |
| TBS | Tris-buffered saline |
Footnotes
Mayo Clinic, Scottsdale, Ariz.
Sigma-Aldrich Corp, St Louis, Mo.
Vectastain ABC kit, Vector Laboratories, Burlingame, Calif.
Olympus BX53 microscope, Olympus Scientific Solutions Americas Inc, Waltham, Mass.
Olympus DP73 digital microscope camera, Olympus Scientific Solutions Americas Inc, Waltham, Mass.
Leica DME microscope, Leica Microsystems Inc, Buffalo Grove, Ill.
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