The increased mucosal mRNA expressions of complement C3 and interleukin-17 in inflammatory bowel disease

Abstract

Recent studies have demonstrated that the complement system participates in the regulation of T cell functions. To address the local biosynthesis of complement components in inflammatory bowel disease (IBD) mucosa, we investigated C3 and interleukin (IL)-17 mRNA expression in mucosal samples obtained from patients with IBD. The molecular mechanisms underlying C3 induction were investigated in human colonic subepithelial myofibroblasts (SEMFs). IL-17 and C3 mRNA expressions in the IBD mucosa were evaluated by real-time polymerase chain reaction. The C3 levels in the supernatant were determined by enzyme-linked immunosorbent assay. IL-17 and C3 mRNA expressions were elevated significantly in the active lesions from ulcerative colitis (UC) and Crohn's disease (CD) patients. There was a significant positive correlation between IL-17 and C3 mRNA expression in the IBD mucosa. IL-17 stimulated a dose- and time-dependent increase in C3 mRNA expression and C3 secretion in colonic SEMFs. The C3 molecules secreted by colonic SEMFs were a 115-kDa alpha-chain linked to a 70-kDa beta-chain by disulphide bonds, which was identical to serum C3. The IL-17-induced C3 mRNA expression was blocked by p42/44 mitogen-activated protein kinase (MAPK) inhibitors (PD98059 and U0216) and a p38 MAPK inhibitor (SB203580). Furthermore, IL-17-induced C3 mRNA expression was inhibited by an adenovirus containing a stable mutant form of I kappaB alpha. C3 and IL-17 mRNA expressions are enhanced, with a strong correlation, in the inflamed mucosa of IBD patients. Part of these clinical findings was considered to be mediated by the colonic SEMF response to IL-17.

Fig. 1

Complement C3 mRNA expression in the inflammatory bowel disease (IBD) mucosa. Total RNA was extracted from biopsy samples, and the C3 mRNA expression was evaluated by real-time polymerase chain reaction (PCR) analyses. Data from the real-time PCR were normalized versusβ-actin for human C3. All values are expressed as means ± standard deviation. *P < 0·05, **P < 0·01.

Fig. 2

Interleukin (IL)-17 mRNA expression in the inflammatory bowel disease (IBD) mucosa. Total RNA was extracted from biopsy samples, and the IL-17 mRNA expression was evaluated by real-time polymerase chain reaction (PCR) analyses. The data from the real-time PCR were normalized versusβ-actin for human IL-17. All values are expressed as means ± standard deviation. *P < 0·05.

Fig. 3

Correlation between C3 and interleukin (IL)-17 mRNA expression in the mucosa. Data from the real-time polymerase chain reaction analyses were normalized versusβ-actin for human IL-17 and C3. A significant positive correlation was observed between the C3 and IL-17 mRNA levels (Spearman's correlation R = 0·71, P < 0·001, n = 70).

Fig. 4

Interleukin (IL)-17 expression in colonic subepithelial myofibroblasts (SEMFs). (a) Dose-dependent effects of IL-17 on C3 mRNA expression. Colonic SEMFs were incubated for 12 h with increasing concentrations of IL-17. The levels of C3 mRNA expression were determined by real-time polymerase chain reaction (PCR). The data were normalized versusβ-actin for C3.All values are expressed as means ± standard deviation (s.d.) (n = 5). *P < 0·05, **P < 0·01; a significant difference from the values for medium alone. (b) Kinetics of C3 mRNA expression. Colonic SEMFs were stimulated with IL-17 (100 ng/ml) for the predetermined times, and then the C3 mRNA levels were determined by real-time PCR. The data were normalized versusβ-actin for C3. All values are expressed as means ± s.d. (n = 5). *P < 0·05, **P < 0·01; a significant difference from the values of culture start. (c) Dose-dependent effects of IL-17 on C3 secretion. Colonic SEMFs were incubated for 24 h with increasing concentrations of IL-17. The levels of C3 protein levels in supernatants were determined by enzyme-linked immunosorbent assay (ELISA). All values are expressed as means ± s.d. (n = 5). *P < 0·05, **P < 0·01; a significant difference from the values for medium alone. (d) Kinetics of C3 secretion. Colonic SEMFs were stimulated with IL-17 (100 ng/ml) for the predetermined times, and then the C3 levels in the supernatant were determined by ELISA. All values are expressed as means ± s.d. (n = 5).

Fig. 5

C3 molecules secreted by colonic subepithelial myofibroblasts (SEMFs). C3 molecules in the supernatant from SEMFs were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing conditions and Western blotting. Serum C3 was used as the control.

Fig. 6

(a) Effects of mitogen-activated protein kinase (MAPK) inhibitors on C3 mRNA expression in colonic SEMFs. The cells were pretreated with 5 µM MAPK inhibitors (SB203580, PD098059 or U02016) for 15 min, and then stimulated with interleukin (IL)-17 (100 ng/ml) for 12 h. The C3 mRNA expression was then determined by real-time polymerase chain reaction (PCR). All values are expressed as means ± standard deviation (s.d.) (n = 5). *P < 0·05, **P < 0·01; a significant difference from the values for IL-17 stimulation. (b) Effects of a recombinant adenovirus expressing a stable mutant form of IκBα (Ad-IκBΔN), a dominant negative mutant of c-Jun (Ad-DN-c-Jun) and β-galactosidase cDNA (Ad-LacZ). Forty-eight hours after infection with the adenovirus, colonic SEMFs were stimulated with IL-17 (100 ng/ml) for 12 h. The C3 mRNA expression was then determined by real-time PCR. The data were normalized versusβ-actin for human C3. All values are expressed as means ± s.d. (n = 5). *P < 0·05, **P < 0·01; a significant difference from the values for IL-17 stimulation.