J. Comp. Path. 2011, Vol. 145, 359e366 Available online at www.sciencedirect.com

www.elsevier.com/locate/jcpa

SPONTANEOUSLY ARISING DISEASE

CD11c+ Cells are Significantly Decreasedin the Duodenum, Ileum and Colon of Dogs

with Inflammatory Bowel Disease

Cor

002

doi

A. Kathrani*, S. Schmitz*, S. L. Priestnall†, K. C. Smith†, D. Werling†,O. A. Garden* and K. Allenspach*

*Department of Veterinary Clinical Sciences and †Department of Pathology and Infectious Diseases, Royal Veterinary

College, University of London, London, UK

resp

1-99

:10.1

Summary

CD11c serves as a marker for human and murine dendritic cells (DCs) and cells expressing this marker havebeen shown to have similar morphological and functional characteristics in the canine immune system. Theaim of this study was to quantify CD11c+ cells in the duodenum, ileum and colon of healthy dogs and dogswith inflammatory bowel disease (IBD). Endoscopic biopsies from the duodenum (n¼ 12 cases), ileum(n¼ 8 cases) and colon (n¼ 12 cases) were obtained from dogs diagnosed with IBD. Intestinal tissue from10 healthy beagle dogs was used as control. Immunofluorescence microscopy was carried out using an anti-canine CD11cmonoclonal antibody. Labelled cells were recorded as cells per 120,000 mm2. The canine chronicenteropathy clinical activity index (CCECAI) was calculated for all dogs with IBD. In addition, sections fromall dogs with IBD were evaluated according to the guidelines of the World Small Animal Veterinary Associ-ation Gastrointestinal Standardization Group. The number of CD11c+ cells in the duodenum, ileum andcolon of dogs with IBD was significantly reduced compared with controls (P< 0.01, P< 0.01 and P< 0.05,respectively). There was a significant negative correlation between the number of CD11c+ cells in the colonof dogs with IBD and the CCECAI (P¼ 0.044, r2¼�0.558). Chronic inflammation in canine IBD appearsto involve an imbalance in the intestinal DCpopulation. Future studies will determine whether reduced expres-sion of CD11c could be a useful marker for the diagnosis and monitoring of canine IBD.

� 2011 Elsevier Ltd. All rights reserved.

Keywords: CD11c; dendritic cell; dog; inflammatory bowel disease

Introduction

The healthy gut is able to mount an inflammatoryresponse to pathogenic bacteria while maintainingthe ability to tolerate commensal and dietary antigens(Rakoff-Nahoum et al., 2004; Abreu et al., 2005;Magalhaes et al., 2007). This important distinction isthought to occur via the action of dendritic cells (DCs).

Inflammatory bowel disease (IBD) occurs in manand dogs and results from an inappropriate immuneresponse to commensal and dietary antigens (Abreuet al., 2005). The two main types of human IBD are

ondence to: K. Allenspach (e-mail: kallenspach@rvc.ac.uk).

75/$ - see front matter

016/j.jcpa.2011.03.010

Crohn’s disease (CD) and ulcerative colitis (UC),but in dogs IBD includes a spectrum of chronic gastro-intestinal (GI) diseases (Jergens et al., 1992; Xavierand Podolsky, 2007). Accumulating evidencesuggests that DCs may be responsible for thebreakdown of tolerance that occurs in human IBDand murine models of IBD. For example, manystudies have demonstrated an increase in thenumber of DCs in inflamed gut mucosa in bothpeople and mice, and studies have also demonstratedthat DCs isolated from IBD tissue are phenotypicallymore mature and produce more pro-inflammatorycytokines than cells isolated from control tissue(Bernstein et al., 1998; Krajina et al., 2003; Sandset al., 2005; Baumgart et al., 2009; Strauch et al., 2010).

� 2011 Elsevier Ltd. All rights reserved.

360 A. Kathrani et al.

Canine IBD is similar to human IBD in that bothare chronic relapsing conditions with underlyingexcessive immune responsiveness and inappropriateinflammation (Cerquetella et al., 2010). DCs mayalso play a role in the breakdown of tolerance seenin dogs with this disease. Investigating the numberand phenotype of DCs in dogs with IBD may helpwith future diagnostic and therapeutic interventions.

The first aim of the present study was to determinewhether there is a significant difference in the numberof DCs in the duodenum, ileum and colon of dogs withIBD compared with healthy controls. CD11c is a b-integrin that is used as a cell surface marker for theidentification of DCs in mice (Henri et al., 2001).This marker is also used to identify human DCs, butis commonly combined with other markers as it isknown to be expressed also by human macrophagesand other cell types (Springer, 1994; Petty andTodd, 1996; Ihanus et al., 2007). Canine cells thatexpress CD11c have a phenotype and functionconsistent with DCs (Isotani et al., 2006; Mielcareket al., 2007; Wang et al., 2007). Unlike in man,CD11c is more widely associated with canine DCsthan tissue macrophages (Danilenko et al., 1992).Therefore, for the purpose of this study, CD11c wasused as a marker for canine DCs.

The World Small Animal Veterinary Association(WSAVA) Gastrointestinal Standardization Grouphas recently published guidelines for the evaluationand scoring of canine intestinal inflammation (Dayet al., 2008; Washabau et al., 2010); however, furtherstudies have suggested that this system correlatespoorly with the severity of clinical activity(Allenspach et al., 2010; Willard et al., 2010). Thesecond aim of the present study was therefore todetermine whether there is a correlation betweenCD11c expression, the canine chronic enteropathyclinical activity index (CCECAI) and the WSAVAintestinal histopathology score in dogs with IBD. Ifsuch a correlation existed, CD11c expression mightserve as a potential marker of the severity of canineIBD.

Materials and Methods

Case Material

Endoscopically-derived tissue samples of duodenum(n¼ 12 cases), ileum (n¼ 8 cases) and colon (n¼ 12cases) were collected from dogs during the diagnosticinvestigation of IBD. The samples from duodenum,ileum and colon generally did not come from thesame dogs. Only one dog with IBD had all three sitessampled. One dog with IBD had both duodenal andileal biopsy tissues collected and five dogs had both

ileal and colonic biopsies collected. Therefore, a totalof 26 dogs with IBD were investigated in this study.

All cases had a detailed medical history taken at thetime of first examination and at subsequent re-exam-inations, haematological and serum biochemicalanalyses, ultrasonography and histopathologicalexamination of biopsy samples. Known causes of GIinflammation were ruled out by these examinationsand by measurement of the serum concentration oftrypsin-like immunoreactivity. Faecal flotation,including zinc flotation to detect Giardia spp., andculture of faeces for Salmonella spp., Campylobacter

spp., Clostridium spp. and Yersinia spp. was carriedout in all dogs. Definitive diagnosis of IBD was basedon microscopical evidence of an inflammatory infil-trate within the lamina propria of endoscopically-ob-tained samples of intestinal mucosa. None of the dogsrecruited into the study had prior glucocorticoid orother anti-inflammatory medication within 3 monthsof being diagnosed. The CCECAI was calculated forall dogs with IBD (Allenspach et al., 2007).

Control tissues comprised full-thickness and endo-scopic samples from the duodenum, ileum and colonof 10 healthy beagle dogs. These dogs were humanelydestroyed for reasons unrelated to the present study.The health of these dogs had been monitored daily(including evaluation of faecal consistency). Necropsyexaminations were carried out while harvesting theintestinal tissues and no gross lesions were visible inany of the dogs.

Histology and Immunofluorescence Microscopy

Formalin-fixed and paraffin wax-embedded tissuesfrom each dog with IBDwere used to prepare sectionsstained by haematoxylin and eosin (HE). Thesesections were reviewed and scored according to theWSAVA guidelines by a board-certified veterinarypathologist. As there are guidelines only for the stom-ach, duodenum and colon, ileal sections were notscored. Sections of intestinal tissue from the healthybeagle dogs were also assessed.

Tissues for immunofluorescence (IF) microscopywere embedded in optimal cutting temperature(OCT) compound (Miles Inc., Elkhart, Indiana,USA) and frozen in liquid nitrogen, followed by stor-age at -80�C until further use. Frozen tissue blockswere warmed to -20�C and serial sections(8e10 mm) were prepared and stored at -80�C in air-tight boxes until further use.

Slides were removed from storage and warmed toroom temperature. Sections were fixed for 10 min incold (-20�C) acetone and the tissues circled with a hy-drophobic pen and rehydrated with 600 ml of Tris-buffered saline (TBS) per section for 10 min. Sections

CD11c+ Cells in Canine Inflammatory Bowel Disease 361

were then blocked with 50% rabbit serum (Serotec,Kidlington, UK) for 60 min and incubated with200 ml of specific or control antibody for 60 min.The specific antibody was mouse monoclonal anti-canine CD11c (isotype IgG1; Serotec) diluted 1 in50 in TBS and the control reagent was purified mouseIgG1 (Serotec) diluted 1 in 100 in TBS. Sections werethen washed in TBS and incubated with secondaryrabbit anti-mouse IgG Fab2 antibody conjugated tofluorescein isothiocyanate (FITC; Serotec) at a dilu-tion of 1 in 5,000 for 1 h at room temperature in thedark. The sections were then washed in TBS, driedand cover slips applied with Vectashield� (VectorLaboratories, Peterborough, UK) and stored in thedark at 4�C. Analysis was performed using a fluores-cent microscope (Olympus BX60, Essex, UK).

Fluorescent cells were quantified in the intestinalsections as described by German et al. (2001). Countsweremade in six areas, eachmeasuring 400� 300 mm.These areas included the central area of the tip of thevillus, the central area of the mid-villus, the left villustip, the left mid-villus, the right villus tip and the rightcentral villus. Themean number of cells was expressedas cells per 120,000 mm2. Counts weremade by two in-dependent assessors in a blinded fashion and the re-sults were averaged.

Statistical Analysis

Statistical comparison of the number of CD11c+ cellsbetween cases with and without IBD was performedusing the ManneWhitney U test. In addition, theManneWhitneyU test was used to determine if therewas a significant difference between the number ofCD11c+ cells in the mid-villus and villus tip in theduodenum, ileum and colon of healthy dogs and thosewith IBD. Spearman’s rank correlation coefficientwas used to assess the relationship between CD11c+

cells in dogs with IBD and the CCECAI or WSAVAscore for the appropriate intestinal section. Spear-man’s rank correlation coefficient was also used to as-sess the correlation between the numbers of CD11c+

cells in the different areas of the intestine in healthybeagle dogs and in dogs with IBD where more thanone site had been sampled.

Results

Clinical Findings

Endoscopic biopsy samples of the duodenum werecollected from 12 dogs with IBD. These includedtwo Labrador retrievers, two German shepherddogs, two West Highland white terriers and oneeach of Jack Russell terrier, Yorkshire terrier, Borderterrier, Lhasa apso, collie cross and cross-bred dog.

Four dogs were male, two were neutered males, onewas female and five were neutered females. The me-dian age was 5 years and 9 months (range 1 year to10 years and 4 months). HE-stained sections of fixedtissue from the duodenum of all 12 cases had lympho-plasmacytic infiltration and in four of these cases therewas also an eosinophilic component to the inflamma-tory reaction.

Endoscopic biopsy samples of the ileum were col-lected from eight dogs with IBD. These dogs werea Scottish terrier, a beagle, a greyhound, a lurcher,a German shepherd dog, a border terrier, an Ameri-can bulldog and a cross-bred dog. Two of the dogswere male, two were neutered males and four wereneutered females. The median age of these dogs was7 years and 7 months (range 2 years and 3 monthsto 11 years and 6 months). HE-stained sections offixed tissue from the ileum of all eight cases had lym-phoplasmacytic infiltration and in two of these casesthere was also an eosinophilic component to the in-flammatory reaction.

Endoscopic biopsy samples of the colon were col-lected from 12 dogs with IBD. These included threecross-bred dogs, three German shepherd dogs, andone each of Labrador retriever, English bull terrier,beagle, Hungarian vizla, greyhound and Scottish ter-rier. Three dogs were male, four were neutered males,one was female and four were neutered females.Median age was 3 years and 9 months (range 1 yearand 5 months to 11 years and 6 months). HE-stainedsections of fixed tissue fromthe colonof 11 caseshad lym-phoplasmacytic infiltration, with one of these cases hav-ing an additional neutrophilic component. One casehad only lymphocytic infiltration of the colonicmucosa.

Full-thickness biopsy samples were taken from theduodenum, ileum and colon of 10 healthy beagledogs immediately after death. Six of these dogs werefemale and four were male; their median age was 5years and 1 month (range 2 years and 11 months to6 years and 1 month). No microscopical abnormali-ties were detected on examination of the HE-stainedsections of these tissues.

Immunofluorescence Microscopy

CD11c+ cells were detected in frozen sections of intes-tinal tissue from healthy dogs and those with IBD.Two distinct cell types were seen to express CD11c:a minor population of small round cells that werethought to be granulocytes or monocytes and a majorpopulation of much larger, irregularly-shaped cellsinterpreted as being DCs (Fig. 1). Only the latter cellswere quantified. Examples of CD11c+ cells in the du-odenum, ileum and colon from healthy dogs and dogswith IBD are presented in Figs. 2e4. In healthy dogs

Fig. 1. Immunofluorescence labelling of two morphologically distinct CD11c+ cells. (A) Small round cells, which are few in number.(B) and (C) Larger, irregular cells, which are more abundant in the lamina propria. Bar, 50 mm.

362 A. Kathrani et al.

the majority of the CD11c+ cells were found in thelamina propria of the mucosa, with few or no cellsin the submucosa or muscularis propria.

There was no significant difference in the numberof CD11c+ cells between the villus tip and mid-villusin any of the sections of duodenum, ileum or colonfrom healthy dogs (two-tailed P> 0.05). However,the one-tailed P-value approached significance fora greater number of CD11c+ cells at the villus tipcompared with the mid-villus in the ileum of healthydogs (P¼ 0.053). There was no significant correlationbetween the number of CD11c+ cells in the duode-num, ileum and colon of healthy beagle dogs (duode-num and ileum, r2¼ 0.045, ileum and colon,r2¼ 0.063, duodenum and ileum, r2¼ 0.0.384).

Fig. 2. Immunofluorescence labelling of CD11c+ cells in duodenal ssection from a healthy beagle dog incubated with negative ca healthy beagle dog labelled with mouse anti-canine CD11from a healthy beagle dog labelled with mouse anti-canine CDsample from a dog with IBD labelled with mouse anti-canine

There were significantly fewer CD11c+ cells in themucosa of the duodenum, ileum and colon of dogswith IBD compared with healthy controls (two-tailedP< 0.01, P< 0.01 and P< 0.05, respectively)(Fig. 5). There was no significant difference in thenumber of CD11c+ cells between the villus tip andmid-villus in any of the sections from the duodenum,ileum or colon of dogs with IBD (two-tailedP> 0.05). There was no significant correlation be-tween the number of CD11c+ cells in the ileum andcolon from the five dogs in which tissue was collectedfrom both sites (r2¼ 0.133). In addition, there was nosignificant correlation between the WSAVA histopa-thology score for the ileum and colon from these samefive dogs (r2¼ 0.004).

amples from control dogs and dogs with IBD. (A) Full-thicknessontrol mouse IgG. Bar, 100 mm. (B) Full-thickness section fromc. Bar, 100 mm. (C) Mid-mucosal region of full-thickness section11c. Bar, 100 mm. (D) Mid-mucosal region of endoscopic biopsy

CD11c. Bar, 100 mm.

Fig. 3. Immunofluorescence labelling ofCD11c+ cells in ileal samples fromcontrol dogs anddogswith IBD. (A)Endoscopic biopsy sectionfrom a healthy beagle dog incubated with negative control mouse IgG. Bar, 100 mm. (B) Endoscopic biopsy section from a healthybeagle dog labelled with mouse anti-canine CD11c. Bar, 100 mm. (C) Endoscopic biopsy section from dog with IBD labelled withmouse anti-canine CD11c. Bar, 100 mm.

CD11c+ Cells in Canine Inflammatory Bowel Disease 363

Correlation between CD11c+ Cells and the World Small

Animal Veterinary Association Score

Five of the 12 duodenal sections were considered inad-equate for scoring as the biopsy samples were eithertoo superficial or too few in number. Similarly, forthe colon, one sample was deemed inadequate forthe same reasons. The remaining sections were all ofadequate quality and only these were used in the sta-tistical analysis. There was no significant correlationbetween the number of CD11c+ cells and theWSAVA histopathology score in the duodenum andcolon of dogs with IBD (Spearman rank correlationcoefficient, P> 0.05 duodenum and colon; r2¼ 0.327[duodenum] and 0.052 [colon]).

Correlation between CD11c+ Cells and the Canine Chronic

Enteropathy Clinical Activity Index

Spearman’s rank correlation coefficient showed no sig-nificant correlation between the number of CD11c+

cells in the duodenum and ileum and the CCECAI(P> 0.05, r2¼ 0.300 [duodenum], �0.315 [ileum],respectively). However, there was a significant linearinverse correlation between CD11c+ cells in the colonand the CCECAI (P¼ 0.044, r2¼�0.588).

Discussion

The results of the present study have shown fewerCD11c+ cells in the duodenum, ileum and colon of

Fig. 4. Immunofluorescence labelling of CD11c+ cells in colonic samsection from a healthy beagle dog incubated with negativefrom a healthy beagle dog labelled with mouse anti-caninewith IBD labelled with mouse anti-canine CD11c. Bar, 100 mm

dogs with IBD compared with healthy control dogs.This finding suggests that the expression of CD11cmay play a role in the development of canine IBD.

CD11c is a member of the leucocyte integrin family(Stewart et al., 1995). Integrins are receptors presenton the cell surface and enable cell-to-cell or extracel-lular matrix attachment (Stewart et al., 1995). CD11cis found on different cell types, depending on the spe-cies. In mice, it is preferentially expressed on DCs(Henri et al., 2001). Inman, it is expressed by a varietyof cells and is often used in combination with othermarkers to detect DCs (Springer, 1994; Petty andTodd, 1996; Ihanus et al., 2007). In dogs, CD11chas been shown to be expressed by DCs, monocytesand granulocytes. In contrast to man, canineCD11c was thought to be more specific for DCsthan macrophages (Danilenko et al., 1992). However,a recent study has shown that CD11c is also abun-dantly expressed by macrophages and other cellsand thus should not be used as a sole marker forDCs in dogs (Ricklin Gutzwiller et al., 2010). There-fore, we cannot exclude the possibility that cells otherthan DCs (e.g. monocytes/macrophages and granulo-cytes) were labelled for expression of CD11c in thisstudy and may have been counted as DCs. However,cells resembling granulocytes and monocytes werefew in number and were easy to detect morphologi-cally and were thus excluded from analysis. Detectionof CD11c+ cells using immunohistochemistry would

ples from control dogs and dogs with IBD. (A) Endoscopic biopsycontrol mouse IgG. Bar, 100 mm. (B) Endoscopic biopsy sectionCD11c. Bar, 100 mm. (C) Endoscopic biopsy section from a dog.

Fig. 5. Total number of CD11c+ cells in the mucosa of duodenum, ileum and colon from normal dogs and dogs with IBD. The meannumber of CD11c+ cells for each group is represented by the blue bars. The number of CD11c+ cells in the duodenum, ileumand colon of healthy beagle dogs is significantly higher than for dogs with IBD (*P< 0.01, †P< 0.05). Controls (n¼ 10), duode-num from dogs with IBD (n¼ 12), ileum from dogs with IBD (n¼ 8) and colon from dogs with IBD (n¼ 12). Results are expressedas mean� standard deviation of the number of CD11c+ cells.

364 A. Kathrani et al.

have allowed for better morphological characteriza-tion of these cells, but although both the immunoper-oxidase and phosphatase methods were attempted inorder to detect CD11c+ cells, due to the high endog-enous enzyme activity in the frozen intestinal sections,these methods could not be utilized.

Significant depletion of DCs has been reported to oc-cur in the colon of paediatric patients with CD com-pared with controls (Silva et al., 2004). This may beresponsible in part for the loss of physiological immunetolerance towards commensal and dietary antigens inthis disease. Interestingly, in the present study,CD11c+ cells were found in discrete clustersintimately associatedwith the overlying epithelium, es-pecially in the ileum of healthy dogs. In the inflamedileum, where there was a significant reduction in thenumber of CD11c+ cells, these clusters were not appar-ent. It could be speculated that this depletionmay sim-ilarly represent loss of tolerance, leading to the chronicinflammation that characterizes canine IBD(Duchmann et al., 1995; Landers et al., 2002; Stagget al., 2003; Sydora et al., 2003). In addition, thepresent study has shown a significant negativecorrelation between the CD11c+ cell number in thecolon and the CCECAI, suggesting that the numberof these cells in the colon is directly related to theseverity of clinical signs and may in the future beuseful in grading the severity of the disease.

In contrast, some studies have shown a significantincrease in DCs in the inflamed mucosa of IBD pa-tients (Bernstein et al., 1998; Baumgart et al., 2009),while others have shown no significant difference(Malizia et al., 1991). This discrepancy might be dueto differences in patient selection, varying techniquesof quantification and selection ofmarkers used to char-acterize DCs. Studies in mice have revealed more

consistent results, showing an increase in CD11c+

DCs in the inflamed colon (Krajina et al., 2003;Strauch et al., 2010). These results are in contrast tothose seen in the present study; however, as DCs area heterogeneous group of cells, these results may bedue to an increase in pro-inflammatory DC subsets.DCs isolated from inflamed tissue in mice show up-regulation of maturation markers such as CD80 andCD86 and have increased production of pro-inflammatory cytokines (Krajina et al., 2003). Thispro-inflammatoryDCphenotype has also been shownin human IBD, where intestinal DCs from patientswith active IBD had increased production of interleu-kin (IL)-12 and IL-6 and increased expression ofCD40 (Sands et al., 2005). Therefore, it is likely thatanti- and pro-inflammatoryDCs exist in the intestinalmucosa of patients with IBD tissue and that theimbalance of the two subsets is responsible for the in-appropriate inflammation. Better phenotypic charac-terization of the different DC subsets in man and dogswill aid in clarifying their role in IBD.

There was a statistically significant negative corre-lation between the number of CD11c+ cells in the co-lon and the CCECAI; however, this was not apparentwhen the number of labelled cells in the duodenumand ileum were analyzed. This finding may havebeen due to other factors in the duodenum and ileumthat contribute to the severity of clinical signs, such asthe number or nature of luminal bacteria, the intesti-nal transit time or local immune responses. In addi-tion, larger number of samples may be needed toascertain if there is a statistically significant correla-tion between the CCECAI and CD11c+ cells in theduodenum and ileum.

The number of CD11c+ cells in the duodenum, il-eum and colon of dogs with IBD was not statistically

CD11c+ Cells in Canine Inflammatory Bowel Disease 365

correlated with the WSAVA intestinal histopathol-ogy score. This may have been due to the small studysize or it may be that histopathological changes lagbehind alterations in the number of CD11c+ cells. Al-though it may be that there is no true correlation, itcould also be argued that as canine IBD is amultifocaldisease, it may be difficult to detect a correlation dueto the small number of samples taken for diagnosis, es-pecially as it has been shown that there is poor agree-ment between histopathological findings fromdifferent regions in the intestinal tract (Casamian-Sorrosal et al., 2010). However, several studies havefailed to reveal any significant correlation betweenhistopathology and severity of clinical signs in canineIBD (Craven et al., 2004; Allenspach et al., 2007;Schreiner et al., 2008). The WSAVA guidelines wereintroduced in an attempt to reduce inter-pathologistvariation in the interpretation of endoscopic biopsysamples (Willard et al., 2002; Washabau et al.,2010). However, it has been shown that variationbetween pathologists still exists when using thesenew guidelines, and that clinical severity does notcorrelate with the WSAVA histology score(Allenspach et al., 2010; Willard et al., 2010).

There are many potential limitations to the inter-pretation of the data presented in the present study.As CD11c is expressed by other cell types in dogs,when conducting future studies other markers (e.g.CD40, CD1c) should also be evaluated in parallel(Ricklin Gutzwiller et al., 2010) to enable discrimina-tion of DCs from macrophages and other cells.

In summary, the present study has shown signifi-cantly fewer CD11c+ cells in the duodenum, ileumand colon of dogs with IBD compared with healthycontrol dogs, and this may play a role in the break-down in mucosal tolerance seen in this disease. AsCD11c+ cell numbers in the colon were correlatedwith clinical severity, estimation of these cell numbersusing immunohistochemistry may provide a newmarker of clinical severity in canine IBD. Larger stud-ies, including a higher number of dogs with biopsysamples taken from all intestinal segments and fromdogs with chronic diarrhoea other than due to IBD,will help to investigate the specificity of this change.

References

Abreu MT, Fukata M, Arditi M (2005) TLR signaling inthe gut in health and disease. Journal of Immunology,174, 4453e4460.

AllenspachK,HouseA, SmithK,McNeill FM,HendricksAet al. (2010) Evaluation of mucosal bacteria and histopa-thology, clinical disease activity and expression of Toll-like receptors in German shepherd dogs with chronicenteropathies. Veterinary Microbiology, 146, 326e335.

Allenspach K, Wieland B, Grone A, Gaschen F (2007)Chronic enteropathies in dogs: evaluation of risk factorsfor negative outcome. Journal of Veterinary Internal Medi-

cine, 21, 700e708.BaumgartDC,Thomas S, Przesdzing I,MetzkeD,BieleckiC

et al. (2009) Exaggerated inflammatory response ofprimary humanmyeloid dendritic cells to lipopolysaccha-ride in patients with inflammatory bowel disease. Clinicaland Experimental Immunology, 157, 423e436.

Bernstein CN, Sargent M, Gallatin WM (1998) Beta-2integrin/ICAM expression in Crohn’s disease. ClinicalImmunology and Immunopathology, 86, 147e160.

Casamian-Sorrosal D, Willard MD, Murray JK, Hall EJ,Taylor SS et al. (2010) Comparison of histopathologicfindings in biopsies from the duodenum and ileum ofdogs with enteropathy. Journal of Veterinary Internal Med-

icine, 24, 80e83.Cerquetella M, Spaterna A, Laus F, Tesei B, Rossi G et al.

(2010) Inflammatory bowel disease in the dog: differ-ences and similarities with humans.World Journal of Gas-

troenterology, 16, 1050e1056.CravenM, Simpson JW, Ridyard AE, ChandlerML (2004)

Canine inflammatory bowel disease: retrospective analy-sis of diagnosis and outcome in 80 cases (1995e2002).Journal of Small Animal Practice, 45, 336e342.

Danilenko DM,Moore PF, Rossitto PV (1992) Canine leu-kocyte cell adhesion molecules (LeuCAMs): character-ization of the CD11/CD18 family. Tissue Antigens, 40,13e21.

DayMJ, Bilzer T,Mansell J,Wilcock B,Hall EJ et al. (2008)Histopathological standards for the diagnosis of gastroin-testinal inflammation in endoscopic biopsy samples fromthe dog and cat: a report from the World Small AnimalVeterinary Association Gastrointestinal StandardizationGroup. Journal of Comparative Pathology, 138, S1eS43.

Duchmann R, Kaiser I, Hermann E, Mayet W, Ewe Ket al. (1995) Tolerance exists towards resident intestinalflora but is broken in active inflammatory bowel disease(IBD). Clinical and Experimental Immunology, 102,448e455.

German AJ, Hall EJ, DayMJ (2001) Immune cell popula-tions within the duodenal mucosa of dogs with enterop-athies. Journal of Veterinary Internal Medicine, 15, 14e25.

Henri S, Vremec D, Kamath A, Waithman J, Williams Set al. (2001) The dendritic cell populations of mouselymph nodes. Journal of Immunology, 167, 741e748.

Ihanus E, Uotila LM, Toivanen A, Varis M,Gahmberg CG (2007) Red-cell ICAM-4 is a ligand forthe monocyte/macrophage integrin CD11c/CD18:characterization of the binding sites on ICAM-4. Blood,109, 802e810.

Isotani M, Katsuma K, Tamura K, Yamada M,Yagihara H et al. (2006) Efficient generation of caninebonemarrow-derived dendritic cells. Journal of VeterinaryMedical Science, 68, 809e814.

Jergens AE, Moore FM, Haynes JS, Miles KG (1992) Idi-opathic inflammatory bowel disease in dogs and cats: 84cases (1987e1990). Journal of the American VeterinaryMed-

ical Association, 201, 1603e1608.

366 A. Kathrani et al.

Krajina T, Leithauser F, Moller P, Trobonjaca Z,Reimann J (2003) Colonic lamina propria dendriticcells in mice with CD4+ T cell-induced colitis. EuropeanJournal of Immunology, 33, 1073e1083.

Landers CJ, Cohavy O, Misra R, Yang H, Lin YC et al.(2002) Selected loss of tolerance evidenced by Crohn’sdisease-associated immune responses to auto- and mi-crobial antigens. Gastroenterology, 123, 689e699.

Magalhaes JG, Tattoli I, Girardin SE (2007) The intestinalepithelial barrier: how to distinguish between the micro-bial flora and pathogens. Seminars in Immunology, 19,106e115.

Malizia G, Calabrese A, Cottone M, Raimondo M,Trejdosiewicz LK et al. (1991) Expression of leukocyteadhesion molecules by mucosal mononuclear phago-cytes in inflammatory bowel disease. Gastroenterology,100, 150e159.

Mielcarek M, Kucera KA, Nash R, Torok-Storb B,McKenna HJ (2007) Identification and characteriza-tion of canine dendritic cells generated in vivo. Biologyof Blood and Marrow Transplantation, 13, 1286e1293.

Petty HR, Todd RF (1996) Integrins as promiscuous signaltransduction devices. Immunology Today, 17, 209e212.

Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F,Edberg S,Medzhitov R (2004) Recognition of commen-sal microflora by toll-like receptors is required for intes-tinal homeostasis. Cell, 118, 229e241.

Ricklin Gutzwiller ME, Moulin HR, Zurbriggen A,Roosje P, Summerfield A (2010) Comparative analysisof canine monocyte- and bone-marrow-derived den-dritic cells. Veterinary Research, 41, 40e51.

Sands BE, Abreu MT, Ferry GD, Griffiths AM,Hanauer SB et al. (2005) Design issues and outcomesin IBD clinical trials. Inflammatory Bowel Diseases,11(Suppl. 1), S22eS28.

Schreiner NM, Gaschen F, Grone A, Sauter SN,Allenspach K (2008) Clinical signs, histology, andCD3-positive cells before and after treatment of dogswith chronic enteropathies. Journal of Veterinary InternalMedicine, 22, 1079e1083.

Silva MA, Lopez CB, Riverin F, Oligny L, Menezes J et al.(2004) Characterization and distribution of colonic den-dritic cells in Crohn’s disease. Inflammatory Bowel Diseases,10, 504e512.

Springer TA (1994) Traffic signals for lymphocyte recircu-lation and leukocyte emigration: the multistep para-digm. Cell, 76, 301e314.

Stagg AJ, Hart AL, Knight SC, Kamm MA (2003) Thedendritic cell: its role in intestinal inflammation andrelationship with gut bacteria. Gut, 52, 1522e1529.

Stewart M, Thiel M, Hogg N (1995) Leukocyte integrins.Current Opinion in Cell Biology, 7, 690e696.

Strauch UG, Grunwald N, Obermeier F, Gurster S,Rath HC (2010) Loss of CD103+ intestinal dendriticcells during colonic inflammation. World Journal of Gas-

troenterology, 16, 21e29.Sydora BC, Tavernini MM, Wessler A, Jewell LD,

Fedorak RN (2003) Lack of interleukin-10 leads to in-testinal inflammation, independent of the time at whichluminal microbial colonization occurs. Inflammatory

Bowel Diseases, 9, 87e97.Wang YS, Chi KH, Liao KW, Liu CC, Cheng CL et al.

(2007) Characterization of canine monocyte-deriveddendritic cells with phenotypic and functional differen-tiation. Canadian Journal of Veterinary Research, 71,165e174.

Washabau RJ, Day MJ, Willard MD, Hall EJ, Jergens AEet al. (2010) Endoscopic, biopsy, and histopathologicguidelines for the evaluation of gastrointestinal inflam-mation in companion animals. Journal of Veterinary Inter-nal Medicine, 24, 10e26.

Willard MD, Jergens AE, Duncan RB, Leib MS,McCracken MD et al. (2002) Interobserver variationamong histopathologic evaluations of intestinal tissuesfrom dogs and cats. Journal of the American Veterinary Med-

ical Association, 220, 1177e1182.Willard MD, Moore GE, Denton BD, Day MJ, Mansell J

et al. (2010) Effect of tissue processing on assessment ofendoscopic intestinal biopsies in dogs and cats. Journalof Veterinary Internal Medicine, 24, 84e89.

Xavier RJ, Podolsky DK (2007) Unravelling the pathogen-esis of inflammatory bowel disease.Nature, 448, 427e434.

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eceived, December 3rd, 2010

ccepted, March 15th, 2011

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