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Role of the advanced glycation end products receptor in Crohn’s disease inflammation
Rachele Ciccocioppo, Alessandro Vanoli, Catherine Klersy, Venerina Imbesi, Vincenzo Boccaccio, Rachele Manca, Elena Betti, Giuseppina Cristina Cangemi, Elena Strada, Roberta Besio, Antonio Rossi, Colomba Falcone, Sandro Ardizzone, Paolo Fociani, Piergiorgio Danelli, Gino Roberto Corazza
Rachele Ciccocioppo, Venerina Imbesi, Vincenzo Boccaccio, Elena Betti, Giuseppina Cristina Cangemi, Gino Roberto Corazza, Department of Internal Medicine, Fondazione IRCCS Policlinico San Matteo, University of Pavia, 27100 Pavia, Italy
Alessandro Vanoli, Rachele Manca, Department of Human Pathology, Fondazione IRCCS Policlinico San Matteo, University of Pavia, 27100 Pavia, Italy
Catherine Klersy, Biometry and Clinical Epidemiology Unit, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy
Elena Strada, Endoscopy Unit, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy
Roberta Besio, Antonio Rossi, Department of Molecular Medicine, University of Pavia, 27100 Pavia, Italy
Colomba Falcone, Center of Research in Molecular Medicine-CIRMC, University of Pavia, 27100 Pavia, Italy
Sandro Ardizzone, IBD Unit, Chair and Division of Gastroenterology, Luigi Sacco Hospital, University of Milan, 20157 Milan, Italy
Paolo Fociani, Department of Pathology, Luigi Sacco Hospital, University of Milan, 20157 Milan, Italy
Piergiorgio Danelli, Department of Surgery, Luigi Sacco Hospital, University of Milan, 20157 Milan, Italy
Author contributions: Ciccocioppo R was responsible for the conception and design of the study, the diagnostic and therapeutic management of the B series and control patients, and wrote the manuscript; Vanoli A and Manca R developed the method for the study of RAGE expression by immunohistochemistry, evaluated all histological preparations in a blind manner, took the pictures, and helped in drafting the manuscript; Klersy C planned and carried out the statistical analysis of the histological data, and helped in drafting the manuscript; Boccaccio V helped in the clinical management of the B series and control patients, developed the grading system for the quantization of the RAGE expression on histological preparations, collected the literature, and helped in drafting the manuscript; Betti E and Cangemi GC developed and carried out the western blot analysis and the functional experiments; Imbesi V collected and analyzed all the clinical data and histological samples of the A series patients, and contributed to the writing-up of the manuscript; Strada E performed the endoscopic examinations of all B series and control patients, and helped in drafting the manuscript; Besio R and Rossi A carried out the densitometric analysis of the western blot preparations, and helped in drafting the manuscript; Falcone C helped in the conception of the study and in drafting the manuscript; Ardizzone S was clinically responsible for the A series patients, and critically revised the article for important intellectual content; Fociani P helped in collecting the histological samples of the A series patients, evaluated all histological preparations in a blind manner, and critically revised the article for important intellectual content; Danelli P carried out all surgical procedures of the A series patients, and critically revised the article for important intellectual content; Corazza GR critically revised the article for important intellectual content; all the authors reviewed and approved the final version to be published.
Supported by A grant from Direzione Scientifica, Fondazione IRCCS Policlinico San Matteo-Progetto di Ricerca Corrente, code 08061307/11
Correspondence to: Rachele Ciccocioppo, MD, Department of Internal Medicine, Center for the Study and Cure of Inflammatory Bowel Disease, Fondazione IRCCS Policlinico San Matteo, University of Pavia, Piazzale Golgi 19, 27100 Pavia, Italy. rachele.ciccocioppo@unipv.it
Telephone: +39-382-502786 Fax: +39-382-502618
Received: April 17, 2013
Revised: June 21, 2013
Accepted: August 4, 2013
Published online: December 7, 2013
AIM: To investigate the level of mucosal expression and the involvement of the receptor for the advanced glycation end products (RAGE) in delayed apoptosis and tumor necrosis factor (TNF)-α production in Crohn’s disease (CD).
METHODS: Surgical and endoscopic specimens from both inflamed and non-inflamed areas of the ileum and/or colon were collected from 20 and 14 adult CD patients, respectively, and used for the assessment of RAGE expression by means of immunohistochemistry and western blotting analysis. Normal tissues from 21 control subjects were used for comparison. The same polyclonal anti-human RAGE antibody (R and D System) was used in all experimental conditions. RAGE staining was quantized by a score including both the amount of positive cells and intensity of immunoreactivity; cellular pattern was also described. The effects of RAGE blocking on apoptotic rate and TNF-α production were investigated on immune cells freshly isolated from CD mucosa and incubated both with and without the muramyl dipeptide used as antigenic stimulus. Statistical analysis was performed via the test for trend, with regression models to account for intra-patient correlations. A 2-sided P < 0.05 was considered significant.
RESULTS: In inflamed areas, RAGE expression in both the epithelial and lamina propria compartments was higher than control tissues (P = 0.001 and 0.021, respectively), and a cluster of positive cells were usually found in proximity of ulcerative lesions. Similar results were obtained in the lamina propria compartment of non-inflamed areas (P = 0.025). The pattern of staining was membranous and granular cytosolic at the epithelial level, while in the lamina propria it was diffuse cytosolic. When evaluating the amount of protein expression by immunoblotting, a significant increase of both surface area and band intensity (P < 0.0001 for both) was observed in CD inflamed areas compared to control tissue, while in non-inflamed areas a significant increase was found only for band intensity (P < 0.005). Moreover, a significantly lower expression in non-inflamed areas in comparison with inflamed areas was found for both surface area and band intensity (P < 0.0006 for both). Finally, RAGE blocking largely affects both the apoptotic rate of mucosal cells (towards an increase in both non-inflamed and inflamed areas of P < 0.001 and < 0.0001, respectively) and TNF-α secretion (towards a decrease in both non-inflamed and inflamed areas of P < 0.05 and < 0.01, respectively), mainly in the presence of antigenic stimulation.
CONCLUSION: RAGE is up-regulated in CD, especially in inflamed areas, and it appears to play a role in the mechanisms involved in chronic inflammation.
Core tip: Receptor for the advanced glycation end products (RAGE) is a multiligand transmembrane receptor whose activation sustains chronic inflammation. The inhibition of RAGE-ligand interaction has proved successful in an experimental model of Crohn’s disease (CD). Our work shows an up-regulation of RAGE expression in both inflamed and non-inflamed mucosa of CD patients in comparison to healthy tissue from control subjects. Moreover, RAGE blocking significantly affects both the apoptotic rate and tumor necrosis factor-α secretion of mucosal immune cells, which are considered to be leading mechanisms in the chronic inflammation of CD. These findings pave the way for a possible use of RAGE blocking agents as a new therapeutic tool in this disabling condition.
