Table 1.
Details of antibodies used in the study.
Table 2.
Characteristics of patients with refractory IBD and controls.
Figure 1.
Quantification of Eosinophil localization to bowel wall in patients with IBD.
Graph (A) shows the average number of eosinophils per mm2 in the entire wall of the colon in patients with refractory IBD compared with controls. Graph (B) shows the average number of eosinophils per mm2 within the mucosal layer of patients with refractory IBD and therapeutically responsive IBD compared with controls. It demonstrates that eosinophilic infiltrate correlates with disease activity in IBD. Graph (C) shows the average number of eosinophils per mm2 within the smooth muscle layer of patients with refractory IBD compared with controls. The quantification was performed as outlined in the materials and methods section. Control patients n = 8; responsive IBD n = 11; refractory CD n = 8; refractory UC n = 7.The data are expressed as mean ± SEM, *p<0.05.
Figure 2.
Quantification of Eosinophil localization to bowel wall in patients with IBD.Quantification of eosinophil localization to mucosal nerves in patients with refractory IBD.
Eosinophils localize to nerves in the colonic mucosa of patients with refractory IBD. The number of MBP immmunoreactive eosinophils in association with nerves/mm2 is shown for controls and subjects with refractory (unresponsive) CD and UC (A). Control patients n = 8; CD n = 8; UC n = 7.The data are expressed as mean ± SEM, *p<0.05. The photomicrograph shown in (B) represents a section taken from a patient with refractory CD. The boxed areas from (B) are shown in higher magnification in (C) and (D). Eosinophils, stained red were detected with anti-MBP antibody, while nerves, stained brown were detected with anti-S100 antibody. There is significant accumulation of eosinophils in these sections compared to the control section shown in photomicrograph (I). Photomicrographs (E) and (F) demonstrate neuroeosinophilic co-localization in a refractory CD section in more detail. Photomicrographs (G) and (H) represent sections from a patient with refractory ulcerative colitis. The arrows in (C-H) demonstrate MBP stained eosinophils in contact or in close association with S100 stained nerves. In all sections note the absence of detectable extra-cellular MBP deposition.
Figure 3.
Quantification of Eosinophil localization to bowel wall in patients with IBD.Quantification of eosinophil localization to mucosal nerves in patients with refractory IBD.Eosinophils localize to the muscle layer in IBD and specifically to nerves in CD.
The graph (A) shows the total number of eosinophil per mm2 localizing to nerves in the muscle layer of patients with refractory IBD. The data are from 8 control patients, 8 patients with CD and 7 with UC. Values are mean ± SEM, * p<0.05. The photomicrograph shown in (B) shows eosinophil localization to nerves in the smooth muscle layer of patients with refractory CD. Photomicrograph (C) demonstrates neuroeosinophilic association in closer detail. Photomicrograph (D) shows a section from a control patient. The arrows in (B,C) demonstrate MBP stained eosinophils in contact or in close association with S100 stained nerves.
Figure 4.
Eosinophil phenotype and nerve subspecies in IBD.
There is a considerable eosinophil accumulation and deposition of extra-cellular MBP in patients with therapeutically responsive IBD. Photomicrographs (A and B) are representative sections from patients with clinically active responsive UC and arrowed areas demonstrate extracellular MBP. Photomicrograph (B) is also immune-stained with anti-S100 to demonstrate neural tissue. Photomicrograph (C) is taken from a patient with clinically quiescent therapeutically responsive UC. The arrows in photomicrographs (A-C) illustrate examples extracellular MBP. Note that extracellular MBP deposition is less prominent in the clinically quiescent state. (D) is a representative section from a control patient. Photomicrographs (E) and (F) demonstrate eosinophil co-localization to Substance P and ChAT immunoreactive nerves respectively in patients with IBD. Eosinophils did not co-localize to nNOS immunoreactive nerves (G). The arrows in (E, F) demonstrate MBP stained eosinophils in association with nerve subspecies.
Figure 5.
Quantification of Eosinophil localization to bowel wall in patients with IBD.Quantification of eosinophil localization to mucosal nerves in patients with refractory IBD.Eosinophils localize to the muscle layer in IBD and specifically to nerves in CD.Up-regulation of eotaxin-3 and ICAM-1 expression in patients with refractory IBD.
In graphs A and B, ICAM-1 and eotaxin-3 expression are shown as the fold increase of gene product normalized for β-actin from the same cDNA sample above control (normal bowel tissue). Controls n = 5; UC n = 4; CD n = 3, *p<0.05.
Figure 6.
Eosinophilic expression of TGFβ-1.
Photomicrographs (A-D) are paired serial sections taken from patients with active (A, B) and quiescent (C, D) UC. (A) and (C) were stained with an anti-MBP antibody while (B) and (D) were stained with an anti-TGFβ-1 antibody. An illustratation of TGFβ-1 immunoreactive eosinophil localization to neural tissue is arrowed in (E). Photomicrographs (F-H) represent a section from a patient with quiescent UC which was double immunofluorescently stained with anti-MBP and anti-TGFβ1. In (F) MBP positively staining cells confirms that these cells are eosinophils. In (G) the same section demonstrates TGFβ1 expression. (H) is a merged image of (F) and (G) confirming that these are TGFβ1 expressing eosinophils. (I) represents the results of the quantitative analysis of eosinophilic TGFβ1 expression. Eosinophilic TGFβ1expression is markedly reduced in patients with refractory IBD (12%) compared to controls (58%) and patients with therapeutically responsive IBD (>40%).