Role of the advanced glycation end products receptor in Crohn’s disease inflammation

Figure 8 Functional assays. The in vitro apoptotic rates of lamina propria mononuclear cells (LPMC) incubated in the absence or presence of two different concentrations of the anti-receptor for the advanced glycation end products (RAGE) blocking antibody, and with or without the muramyl dipeptide (MDP) used as antigenic stimulation are given in panels A and B. The analysis was carried out by flow cytometry, and the mean percentage values ± SD of at least three experiments for each condition were the following: in the absence of MDP (A), LPMC from non-diseased mucosa (grey bars) showed a spontaneous apoptotic rate of 18.4 ± 3.1, and a value of 26.9 ± 2.8 and 32.7 ± 4.6 in the presence of 50 and 100 ng/mL concentration of the anti-RAGE blocking antibody, respectively; when using LPMC from diseased mucosa (black bars), a value of spontaneous apoptotic rate of 10.1 ± 4.2 was found, while when incubating with 50 and 100 ng/mL concentration of the anti-RAGE blocking antibody, values of 13.0 ± 6.2 and 26.1 ± 5.5 were found, respectively. In the presence of MDP (B), LPMC from non-diseased mucosa showed a spontaneous apoptotic rate of 15.6 ± 2.1, and values of 31.9 ± 3.8 and 33.3 ± 2.9 in the presence of 50 and 100 ng/mL concentration of the anti-RAGE blocking antibody, respectively; when using LPMC from diseased mucosa, a value of spontaneous apoptotic rate of 7.8 ± 2.6 was found, while when incubating with 50 and 100 ng/mL concentration of the anti-RAGE blocking antibody, values of 24.1 ± 2.2 and 28.0 ± 4.1, respectively, were found. The tumor necrosis factor (TNF)-α production of LPMC cultured in vitro in the absence or presence of two different concentrations of the anti-RAGE blocking antibody, and with or without the MDP as antigenic stimulation are given in the panels C and D. The analysis was carried out by ELISA assay on culture supernatants, and the mean values ± SD were as follows: in the absence of MDP (C), the cytokine level was 32 ± 14 pg/mL in the cultures with LPMC from non-diseased areas, and 124 ± 48 pg/mL in those with LPMC from diseased areas; when incubating with 50 and 100 ng/mL of the anti-RAGE blocking antibody, values of 41 ± 38 and 27 ± 21 pg/mL for LPMC from non-diseased areas, and of 102 ± 29 and 67 ± 11 pg/mL for LPMC from diseased areas, respectively, were observed. In the presence of MDP (D), the TNF-α level was 184 ± 49 pg/mL in the cultures with LPMC from non-diseased areas, and 307 ± 68 pg/mL in those with LPMC from diseased areas; when incubating with 50 and 100 ng/mL of the anti-RAGE blocking antibody, values of 138 ± 50 and 87 ± 41 pg/mL for LPMC from non-diseased areas, and of 198 ± 68 and 71 ± 47 pg/mL for LPMC from diseased areas, respectively, were observed.