Human intestinal epithelial cells secrete interleukin-1 receptor antagonist and interleukin-8 but not interleukin-1 or interleukin-6

Abstract

Background: There is growing evidence that intestinal epithelial cells (IECs) are involved in the mucosal immune system.

Aim: To assess the pattern of cytokines secreted by IECs and lamina propria mononuclear cells (LPMNCs). To achieve this, the expression and secretion of interleukin (IL)-1, IL-1 receptor antagonist (IL-1ra), IL-6, and IL-8 in human primary colonic and ileal IECs and LPMNCs from the same patient were studied.

Methods: IECs and LPMNCs were isolated from surgical specimens or endoscopic biopsy samples. mRNA expression was investigated by northern blot analysis. Secretion of IL-1beta, IL-6, IL-8, and IL-1ra was measured by enzyme linked immunosorbent assay.

Results: IL-1ra mRNA levels were higher in IECs than in LPMNCs in all probands. IL-8 mRNA was only present in low amounts in the IECs from two controls. In none of the specimens were IL-1beta and IL-6 mRNA present in IECs. Transcripts encoding IL-1beta, IL-1ra, IL-6, and IL-8 were identified in LPMNC preparations of all specimens. IECs from normal mucosa produced no detectable amounts of IL-1beta or IL-6, whereas LPMNCs did. IECs secreted some IL-8 (65 (9) pg/10(5) cells) but significantly more was generated by LPMNCs (408 (43) pg/10(5) cells, p<0.0001). However, IECs secreted more IL-1ra than did LPMNCs (120 (12) v 94 (11) pg/10(5) cells). In acute inflammation, IEC IL-1ra secretion was significantly increased. A correlation between secreted IL-1ra and the macroscopical degree of inflammation was found in Crohn's disease (r = 0.64, p<0.0001, n = 36) and ulcerative colitis (r = 0. 76, p<0.0001, n = 24).

Conclusions: IECs from normal mucosa express and secrete IL-1ra and low amounts of IL-8, but no IL-1 or IL-6. In inflamed mucosa the secretion of IL-1ra by IECs is slightly increased but may be not sufficient to antagonise the greatly increased production of proinflammatory cytokines by LPMNCs and the IECs themselves.

Figure 1

Northern blot analysis of interleukin (IL)-1 receptor antagonist (ra), IL-8, IL-1β, and IL-6 mRNA. RNA was extracted from intestinal epithelial cell (IEC) or lamina propria mononuclear cell (LPMNC) preparations from five control patients (CO), three patients with Crohn's disease (CD), and two patients with ulcerative colitis (UC). Total RNA (25 µg) extracted from IECs or LPMNCs was separated in an agarose gel containing 1% formaldehyde, transferred to nylon membranes, and hybridised with [³²P]dCTP labelled cDNA fragments from IL-1ra cDNA (A), IL-8 cDNA (B), IL 1β (C), and IL-6 cDNA (D). Methylene blue staining was performed to confirm equal loading of RNA (E). E, Epithelial cells; L, lamina propria mononuclear cells. Expression of IL-1ra mRNA was higher in IECs than LPMNCs and increased during inflammation. IL-8 mRNA was only expressed in two of five IEC preparations isolated from normal mucosa and was increased in mucosal inflammation but always to a lesser extent than in LPMNCs. IL-1β and IL-6 mRNA could not be found in the IEC preparation but were always present in LPMNCs.

Figure 2

Cytokine content of freshly isolated intestinal epithelial cells (IECs) and lamina propria mononuclear cells (LPMNCs) from normal and inflamed mucosa. Interleukin (IL)-1 receptor antagonist (ra) (A), IL-8 (B), IL-1β (C), and IL-6 (D) protein was determined from freshly isolated and homogenised IECs from 50 biopsy or surgical specimens of normal mucosa, from five patients with unspecific colonic inflammation (CO), 24 patients with Crohn's disease (CD), and 14 patients with ulcerative colitis (UC) by ELISA. In addition, cytokines were determined in LPMNC homogenates from 17 control patients, five patients with unspecific colitis, nine patients with Crohn's disease, and nine patients with ulcerative colitis. Results are mean (SEM) expressed as pg cytokine/10⁵ cells. 0 = not inflamed, 1 = slightly inflamed, 2 = moderately inflamed, 3 = severely inflamed. *p<0.05, ***p<0.001 v IECs; **p<0.01 inflamed v non-inflamed.

Figure 3

Secretion of interleukin (IL)-1 receptor antagonist (ra) and IL-8 by primary cultures of intestinal epithelial cells (IECs) after 24 hours. (A) IL-1ra was determined in culture medium after a 24 hour culture of primary human IECs from 72 biopsy or surgical specimens from control mucosa (CO (0)), 12 patients with unspecific colonic inflammation (CO (1−3)), 44 patients with Crohn's disease (CD), and 25 patients with ulcerative colitis (UC). The degree of inflammation was estimated endoscopically as described in Materials and methods: 0 = normal mucosa, 1 = low degree of inflammation, 2 = moderate inflammation, 3 = severe inflammation. (B) Comparison of IL-1ra secretion from IECs and LPMNCs after 24 hours of culture. In addition to IECs, LPMNCs were isolated from 29 control patients, five patients with unspecific colitis, 15 patients with Crohn's disease, and 12 patients with ulcerative colitis. (C, D) IL-8 secretion from 24 hour cultured IECs and LPMNCs. For details see (A) and (B). Results are mean (SEM) expressed as pg/10⁵ cells.

Figure 4

Comparison of interleukin (IL)-1 receptor antagonist (ra) and IL-8 secretion in isolated ileal (IEC) and colonic epithelial cells. Cytokines were determined in culture medium after 24 hours of culture. n = 7 for controls (CO), non-inflamed Crohn's disease (CD), and inflamed CD. Data are mean (SEM) expressed as pg cytokine/10⁵ cells. Differences between ileal and colonic epithelial cells did not reach significance.

Figure 5

Identification of interleukin (IL)-1 receptor antagonist (ra) isoforms in medium supernatants of peripheral blood mononuclear cells and intestinal epithelial cells (IECs). Cytosol of macrophages which is known to contain intracellular IL-1ra (18 kDa; icIL-1ra) and medium supernatants of macrophages known to contain secretory IL-1ra (22-25 kDa in its glycosylated form; sIL-ra) as well as recombinant human IL-1ra (non-glycosylated, 17 kDa; rh-IL1-ra) were used as positive controls. In the precipitated protein fraction of medium supernatants of primary IECs, only the 22-25 kDa sIL-1ra from could be detected after 24 hours of culture. Similar results were obtained for three different IEC cultures. TNF, tumour necrosis factor.