Table 1.
Primer sequences for real time quantitative PCR studies of IL-17 pathway components and for optimisation of housekeeping genes.
Figure 1.
Expression of sulphated GAGs within archival small intestinal biopsies from two patients with coeliac disease during a biopsy challenge series following previous ESPGHAN guidelines.
The biopsies shown were taken from the children at initial diagnosis (top row), then on a gluten-free diet (centre row) and finally following gluten challenge (bottom row). The left hand column shows low power views (original magnification ×10) of case 1, and the right hand column high power (x40) views of epithelial staining in case 2. The specimens show decreased epithelial and increased lamina propria sulphated GAG expression in active coeliac disease compared to findings in the same patient on a gluten-free diet. Strong pericellular staining may be seen on aggregated lamina propria mononuclear cells. Similar findings were seen in 3 other cases.
Figure 2.
Staining density for glycosaminoglycans within (a) epithelium and (b) lamina propria, in coeliac disease mucosa in comparison to controls.
Sulphated GAGs, heparan sulphate proteoglycans (HSPG) and short chain heparan sulphate proteoglycan “stubs” (Δ-HSPG) are shown for both epithelial and lamina propria compartments, while the core protein for epithelial proteoglycans (syndecan-1) is shown for the epithelial compartment alone. Epithelial expression of heparan sulphate and sulphated GAGs is reduced in coeliac disease compared to controls, whereas the core protein syndecan-1 is maintained. Within the lamina propria the density of expression of heparan sulphate and sulphated GAGs is maintained similar to controls. Values shown represent means plus 95% confidence intervals. While the units displayed are derived from reciprocals of initial measurement minus background, statistical comparisons were made on unmodified initial measurements, as previously reported 21. Symbols: Significant difference between coeliac and controls: * p<0.05, ** p<0.01, *** p<0.001.
Figure 3.
Representative staining for sulphated GAGs, HSPG and Δ-HSPG stubs in control (top row) and coeliac cases (middle and bottom row).
All views are at same magnification (original ×40). In the control biopsies, there is strong expression of heparan sulphate and sulphated GAGs within the subepithelial basement membrane and on the basolateral surface of epithelial cells, most marked towards the villous tip. The coeliac biopsies show loss of HSPG and sulphated GAG expression within the basement membrane and from the basolateral epithelial surface. By contrast there is maintained expression within the lamina propria. Additional images from each of the patients studied can be seen in Figures S1 and S2. Quantitative staining intensity data derived from colour deconvoluted images (Figure S3) are shown in Figure 2.
Figure 4.
Syndecan-1 (CD138) expression in normal and coeliac mucosa using monoclonal MCA2459GA.
A. Expression within normal intestine, showing syndecan-1 localises predominantly to the basolateral epithelial surface (original magnification ×10). B. Higher power view (x40) of normal duodenum, showing similar epithelial localisation plus the presence of scattered syndecan-1+ plasma cells within the lamina propria. C,D. Low power view (x10) of 2 cases of coeliac disease, showing retention of epithelial staining similar to controls, plus additionally a dense confluent expression within the upper lamina propria. E,F. Higher power view (x40) of the subepithelial region in these cases. The lamina propria aggregates consist of cells with dense membrane staining (plasma cells), cells with recognisable nuclei and punctate cytoplasmic staining and scattered syndecan-1+ debris (shed ectodomains). G–J. Findings within the same case in two biopsies showing contrasting areas with containing areas with preserved villous architecture (G,H) and villous atrophy (I,J). G,I are at low power (x10), H,J at high power (x 40). The biopsy with villous atrophy shows dense subepithelial syndecan-1 syncytial expansion in comparison to that with preserved villous architecture. Mucosal syndecan-1 expression from all normal controls and coeliac patients studied is shown in Figure S4.
Figure 5.
a). IL-6 localisation by immunohistochemistry in 2 controls (top panel) and 4 cases of coeliac disease (lower 2 panels). The left column show low power (original magnification ×10) and the right column high power (x40) views. The coeliac patients showed increased numbers of IL-6+ mononuclear cells within the lamina propria but not epithelial compartments. b). Quantitative data for mucosal IL-6+ cell density in 9 coeliac and 9 control biopsies, expressed as IL-6+ cells per high power field. c). Mucosal IL-6 mRNA expression density normalised to the housekeeping gene GAPDH in matched biopsies from the same cases.
Figure 6.
Quantitative analysis of mRNA expression for IL-17α, IL-6, IL-23α and TGF-β1 in biopsies from 10 children with coeliac disease and 8 histologically normal controls.
For each cytokine, the left column represents results for coeliac disease and the right column results from controls. Il-17A and IL-6 showed significant increase in coeliac disease. Original data shown in Figure S7.