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. 2000 Sep;47(3):387–396. doi: 10.1136/gut.47.3.387

Neurokinin-1 receptor expression in inflammatory bowel disease: molecular quantitation and localisation

T Goode 1, J O'Connell 1, P Anton 1, H Wong 1, J Reeve 1, G O'Sullivan 1, J Collins 1, F Shanahan 1
PMCID: PMC1728039  PMID: 10940277

Abstract

BACKGROUND—Substantial evidence implicates the neuropeptide substance P (SP) in mucosal immunoinflammatory responses. Autoradiographic studies have suggested a disturbance in SP receptor expression in inflammatory bowel disease (IBD).
AIMS—Because of technical limitations such as poor cellular resolution with autoradiography, we used molecular methods to specifically localise the cellular expression of the neurokinin-1 receptor (NK-1R) in IBD colon, and to quantitate NK-1R mRNA expression levels therein.
METHODS—In situ hybridisation and immunohistochemistry were used to localise NK-1R mRNA and protein, respectively, in normal, ulcerative colitis (UC), and Crohn's disease (CD) colonic resections. NK-1R mRNA expression levels of normal, UC, and CD mucosal biopsies were quantitated by competitive reverse transcription-polymerase chain reaction.
RESULTS—NK-1R expression was localised to lamina propria mononuclear cells, epithelium, submucosal vasculature, smooth muscle, and myenteric plexus of normal and IBD colon. No ectopic NK-1R expression was observed in IBD. However, we found increased numbers of NK-1R expressing lymphoid cells in IBD tissue, aberrant negative epithelial expression of NK-1R in UC, and increased expression of NK-1R in CD myenteric plexus. Quantitation of NK-1R mRNA expression in IBD colonic mucosal biopsies revealed marked upregulation of NK-1R mRNA levels compared with non-inflamed mucosal expression levels (p<0.01).
CONCLUSIONS—This report demonstrates the strategic localisation and upregulation of NK-1R expression in IBD colon, and thereby suggests the involvement of substance P in the pathophysiological symptoms of IBD.


Keywords: substance P; neurokinin-1 receptor; inflammatory bowel disease

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Figure 1  .

Figure 1  

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Localisation of neurokinin-1 receptor (NK-1R) mRNA expression in non-inflamed colon (A, B), in active ulcerative colitis (UC) colon (C, D), and in active Crohn's disease (CD) colon (E, F) by in situ hybridisation. (A) NK-1R mRNA (purple) is detected in lamina propria mononuclear cells (LPMC), and in surface and crypt epithelium of normal colon. (C) NK-1R mRNA expression (purple) by LPMC and epithelial cells of UC colon. Note the "patchy" positivity of surface epithelium. (E) Expression of NK-1R mRNA (purple) by LPMC and crypt epithelium of CD colon. Note the dense inflammatory infiltrate of NK-1R mRNA expressing cells. (B, D, F) Control hybridisation of consecutive sections of normal (B), UC (D), and CD (F) colon, with a 10-fold excess of unlabelled riboprobe. Signal intensity is dramatically reduced, confirming the specificity of hybridisation.

Figure 2  .

Figure 2  

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Immunohistochemical localisation of neurokinin-1 receptor (NK-1R) protein expression in non-inflamed colon (A, B), in active ulcerative colitis (UC) colon (C, D), and in active Crohn's disease (CD) colon (E, F). Sections are consecutive to those processed for in situ hybridisation in fig 1 and are shown at higher magnification. (A) NK-1R immunoperoxidase staining (brown) is detected in lamina propria mononuclear cells (LPMC), and in surface (top right of figure) and crypt epithelium of normal colon. (C) NK-1R expression (brown) is present in LPMC and in surface epithelium of UC colon. Note the crypt epithelium is negative for NK-1R expression (blue). (E) Expression of NK-1R protein (brown) by LPMC and crypt epithelial cells of CD colon. (B, D, F) Control staining of consecutive sections of normal (B), UC (D), and CD (F) colon. Specificity of antibody binding was confirmed, as preincubation of the primary antibody with the NK-1R immunising peptide inhibited staining. All sections were counterstained with haematoxylin (blue).

Figure 3  .

Figure 3  

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Localisation of neurokinin-1 receptor (NK-1R) mRNA expression in active ulcerative colitis (UC) colon by in situ hybridisation. (A) NK-1R mRNA expression (purple) is detected in lamina propria mononuclear cells (LPMC) and in a lymphoid follicle of UC colon. Crypt epithelial cells negative for NK-1R mRNA are present. (C) Expression of NK-1R mRNA by submucosal arterioles and venules of UC colon. mRNA expression (purple) is evident in the endothelium and muscularis of blood vessels. (E) High magnification view of crypt epithelial cells negative for NK-1R mRNA expression, surrounded by NK-1R positive (purple) LPMC in UC colon. (B, D, F) Control hybridisation of consecutive sections of UC colon, with a 10-fold excess of unlabelled riboprobe. Signal intensity is dramatically reduced, confirming specificity of hybridisation.

Figure 4  .

Figure 4  

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Localisation of neurokinin-1 receptor (NK-1R) expression in non-inflamed (A, C, and E), and inflamed Crohn's disease (CD) (B, D, F) myenteric plexus by in situ hybridisation (A-D), and immunohistochemistry (E, F). (A) NK-1R mRNA (purple) is detected in enteric neurones of the myenteric plexus of normal colon. (C) Higher magnification view of (A). (E) NK-1R protein (brown) staining is evident in the myenteric plexus of normal colon. Section was counterstained with haematoxylin (blue). (B) Expression of NK-1R mRNA by enteric neurones of the myenteric plexus of CD colon. Note the NK-1R positive cells of lymphoid morphology. (D) Higher magnification view of (B). (F) Immunohistochemical detection of NK-1R protein expression (brown) in the myenteric plexus of CD colon. Section was counterstained with haematoxylin (blue).

Figure 5  .

Figure 5  

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Quantitation of neurokinin-1 receptor (NK-1R) mRNA expression in a mucosal biopsy from active ulcerative colitis (UC) colon. (A) Gel shows a representative NK-1R mRNA quantitation using qcRT-PCR. Competitive standards were spiked into the aliquoted biopsy RNA sample at concentrations ranging from 2.6×104 molecules to 2.6×102 molecules (lanes 1-6; 2.5-fold dilution series). Equivalence (*) is seen at 4.1×103 NK-1R mRNA molecules, in which target (295 bp) and competitive standard (228 bp) PCR products are of equal band intensity (lane 3). When adjusted for the amount of total RNA in the assay, this represents a level of expression of 4.1×104 NK-1R mRNA transcripts/µg RNA in this particular UC biopsy sample. HaeIII digested ϕX174 DNA size markers (M) were used. (B) Confirmation of heteroduplex formation. A third band of slower electrophoretic mobility (~360 bp) than the target (295 bp) or competitor (228 bp) PCR products was observed when the concentration of both was relatively high (lane 3). This was absent when competitor or target were amplified separately (lanes 1 and 2, respectively). To confirm that the 360 bp band consisted of a heteroduplex between one strand each of target and competitor PCR products, the PCR products from lane 3 were subjected to digestion with the single stranded DNA specific nuclease, S1 (lane 4). This treatment eliminated the 360 bp band, confirming the presence of a single stranded "loop out" amenable to S1 digestion, which is characteristic of the predicted heteroduplex.

Figure 6  .

Figure 6  

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Neurokinin-1 receptor (NK-1R) mRNA levels in mucosal biopsies from normal colon (n=9), active ulcerative colitis (UC) colon (n=10), and active Crohn's disease (CD) colon (n=10). The bar indicates mean NK-1R mRNA expression in each group. *p<0.01.

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