350 Gut 2000;46:350–358 Human intestinal epithelial cells secrete

-1 receptor antagonist and Gut: first published as 10.1136/gut.46.3.350 on 1 March 2000. Downloaded from interleukin-8 but not interleukin-1 or interleukin-6

R Daig, G Rogler, E Aschenbrenner, D Vogl, W Falk, V Gross, J Schölmerich, T Andus

Abstract The classical functions of the intestinal epithe- Background—There is growing evidence lium are absorption and secretion of fluids, that intestinal epithelial cells (IECs) are electrolytes, and nutrients. Furthermore, the involved in the mucosal immune system. intestinal epithelial cells (IECs) form the Aim—To assess the pattern of primary physiological barrier against poten- secreted by IECs and lamina propria tially pathogenic microorganisms and a multi- mononuclear cells (LPMNCs). To achieve tude of antigens in the gut lumen. Damage of this, the expression and secretion of inter- the epithelial barrier is followed by bacterial leukin (IL)-1, IL-1 receptor antagonist invasion and inflammation of the mucosa. (IL-1ra), IL-6, and IL-8 in human pri- In addition, during the last few years more mary colonic and ileal IECs and LPMNCs and more data have accumulated showing that from the same patient were studied. IECs play an active role in the intestinal Methods—IECs and LPMNCs were iso- immune system. They express HLA-DR after lated from surgical specimens or endo- stimulation with -ã.1–5 After stimula- scopic biopsy samples. mRNA expression tion with interferon or interleukin (IL)-1, they was investigated by northern blot analysis. show enhanced expression of adhesion Secretion of IL-1â, IL-6, IL-8, and IL-1ra molecules—for example, intercellular adhesion was measured by enzyme linked immuno- molecule-16 and lymphocyte function associ- sorbent assay. ated antigen (CD58).7 Intercellular adhesion Results—IL-1ra mRNA levels were higher molecule-1 could be enhanced in HT-29 and in IECs than in LPMNCs in all probands. Caco-2 cells by incubation with inflammatory

IL-8 mRNA was only present in low cytokines; however, the responsiveness to http://gut.bmj.com/ amounts in the IECs from two controls. In interferon-á, tumour necrosis factor (TNF)-á, none of the specimens were IL-1â and IL-6 or IL-1 treatment was diVerent in the two cell mRNA present in IECs. Transcripts en- lines.8 Furthermore, evidence was found that coding IL-1â, IL-1ra, IL-6, and IL-8 were epithelial cells are able to respond to damage or identified in LPMNC preparations of all bacterial invasion by secreting cytokines.9–18 specimens. IECs from normal mucosa The cytokines reported to be secreted by IECs produced no detectable amounts of IL-1â are important inflammatory mediators such as or IL-6, whereas LPMNCs did. IECs IL-1, IL-6, and IL-8.19–21 To characterise on September 28, 2021 by guest. Protected copyright. secreted some IL-8 (65 (9) pg/105 cells) further the participation in the mucosal but significantly more was generated by immune system and the immunomodulatory LPMNCs (408 (43) pg/105 cells, p<0.0001). function of the intestinal epithelium, in vitro However, IECs secreted more IL-1ra than studies were performed. production did LPMNCs (120 (12) v 94 (11) pg/105 has been studied in human and rat epithelial cells). In acute inflammation, IEC IL-1ra cell lines such as Caco-2, HT-29, and 18 22–24 secretion was significantly increased. A IEC-6. T84, HT-29,and SW620 cell lines secreted substantial amounts of IL-8 after Department of correlation between secreted IL-1ra and Internal Medicine I, the macroscopical degree of inflammation stimulation with IL-1â, TNF-á or interferon-ã, University of was found in Crohn’s disease (r = 0.64, but Caco-2 cells responded only to IL-1â.In Regensburg, Germany p<0.0001, n = 36) and ulcerative colitis (r = addition to theses cytokines, bacterial lipopoly- R Daig 0.76, p<0.0001, n = 24). saccharide was eYcient in stimulating IL-8 G Rogler Conclusions—IECs from normal mucosa release in SW620 and HT29 cells, whereas E Aschenbrenner T84 and Caco-2 cells were almost unrespon- D Vogl express and secrete IL-1ra and low 23 W Falk amounts of IL-8, but no IL-1 or IL-6. In sive to it. We showed that the synthesis of IL-8 V Gross in HT-29 cells was stimulated by IL-1â or inflamed mucosa the secretion of IL-1ra 22 J Schölmerich by IECs is slightly increased but may be TNF-á. These data indicate that the use of T Andus not suYcient to antagonise the greatly diVerent IEC lines may lead to diVerent results Correspondence to: increased production of proinflammatory Professor T Andus, cytokines by LPMNCs and the IECs Department of Internal themselves. Abbreviations used in this paper: IEC, intestinal Medicine I, University of ( 2000; :350–358) Regensburg, 93042 Gut 46 epithelial cell; LPMNC, lamina propria mononuclear Regensburg, Germany cell; IL, interleukin; IL-1ra, IL-1 receptor antagonist; Keywords: intestinal epithelial cells; lamina propria ELISA, enzyme linked immunosorbent assay; TNF, Accepted for publication mononuclear cells; mucosa; cytokines; interleukin; tumour necrosis factor; IBD, inflammatory bowel 9 September 1999 immune system disease; PBS, phosphate buVered saline. Intestinal epithelial cells in mucosal immune system 351

and diVerent interpretations of their immuno- and friability of the mucosa in ulcerative colitis; logical function. single small aphthous lesions in Crohn’s Several studies with epithelial cell lines have disease); ++ = moderate inflammation (mu- shown IL-6 secretion by IECs. Experiments cous membranes, spontaneous bleeding, and Gut: first published as 10.1136/gut.46.3.350 on 1 March 2000. Downloaded from with the rat-derived IEC-6 cell line suggested small ulcers in ulcerative colitis; multiple that IECs could represent an important source aphthous lesions, and small ulcers in Crohn’s of IL-6 in inflammatory responses at the intes- disease); +++ = severe inflammation (large tinal mucosa and that transforming growth ulcers in ulcerative colitis; large ulcerous factor-â could potentiate this function.24 A lesions in Crohn’s disease). recent study showed IL-1 induced and NF-êB Colitis was regarded as non-specific when mediated IL-6 production in Caco-2 cells.25 histological examination showed inflammatory Salmonella typhi has been shown to stimulate infiltrates in the mucosa that were not typical of IL-6 secretion in the small intestine epithelial IBD, and treatment with antibiotics led to cell line Int407.26 However, in a study using in complete restitution. Stool cultures from these situ hybridisation of human mucosa, IL-6 patients did not show any specific pathogen mRNA was not found in the epithelial cells and were negative for Clostridium diYcile toxin. except in one patient.27 In addition, in murine The study was approved by the ethics intestine, IL-6 mRNA was not located in the committee of the University of Regensburg and epithelial cell layer.28 performed in accordance with the declaration Recently we and others showed an imbal- of Helsinki. ance of the IL-1 system in colonic mucosa dur- 29 30 ing inflammation. IL-1á and IL-1â were ISOLATION OF COLONIC AND ILEAL EPITHELIAL significantly increased in the inflamed mucosa CELLS from patients with inflammatory bowel disease A method for the isolation of viable epithelial (IBD) and also in that from non-IBD patients cell preparation has been developed based on compared with healthy controls.29 The ratio of standard protocols and is described in more IL-1(á+â) to IL-1 receptor antagonist (IL-ra) detail elsewhere.32 In brief, mucosa was was significantly decreased, leading to a change stripped from submucosa within 30 minutes of in the balance of proinflammatory and anti- bowel resection and rinsed several times with inflammatory cytokines. IL-8 mRNA detected phosphate buVered saline (PBS). The mucus by in situ hybridisation was also increased in was removed by treatment with 1 mM inflamed colonic mucosa.31 The signal for IL-8 dithiothreitol (Serva, Heidelberg, Germany) could only be found in the lamina propria, for 15 minutes. After being washed with PBS, indicating intestinal as the major the mucosa was placed in 1.5 mM EDTA in source of IL-8. Therefore we concluded that Hanks balanced salt solution without calcium IL-8 is mainly produced in inflammatory cells and magnesium and tumbled for 10 minutes at http://gut.bmj.com/ in the lamina propria of the colon in IBD and 37°C. This supernatant containing debris and correlates with mucosal inflammation.31 These mainly villus cells was discarded. The mucosa results were in contrast with our findings in was incubated again with EDTA for 10 HT-29 cells, which responded to stimulation minutes at 37°C. The supernatant was col- with IL-8 secretion.22 lected into a 15 ml tube. Then the remaining As cell lines dediVerentiate in culture, we mucosa was vortex-mixed in PBS and this developed a culture system for primary cul- supernatant was also collected. It contained 32 tures of human IECs. Using this new system, complete crypts, some single cells, and a small on September 28, 2021 by guest. Protected copyright. we were able for the first time to compare the amount of debris. To separate IECs (crypts) expression and secretion of cytokines by IECs from contaminating non-epithelial cells, the and the corresponding lamina propria mono- suspension was allowed to sediment for 15 nuclear cells (LPMNCs) from the same minutes. The cells (mainly complete crypts) patient. were collected and washed twice with PBS. The procedure to isolate IECs from mucosal Materials and methods biopsy specimens was identical except that the PATIENTS dithiothreitol treatment was omitted. The Colonic tissue was obtained from 12 patients number and viability of the cells were deter- undergoing surgical resection for colorectal mined by 0.1% trypan blue exclusion. The carcinoma. The tissue was taken at least 5 cm purity of the epithelial cell preparation was distant from the tumour. In addition, surgical checked by FACS analysis with the EP4 specimens from three patients with Crohn’s antibody (Dako, Hamburg, Germany), show- disease and two with ulcerative colitis were ing more than 90% of EP4 positive cells. The obtained. Colonic mucosal biopsy specimens amount of macrophages (CD33 positive) or (six per patient) were taken from 63 patients lymphocytes (CD3 or CD19 positive) was less and ileal biopsy specimens from 20 patients than 4% each. Colonic epithelial cell cultures during colonoscopy for diVerent reasons. from surgical specimens were used for extrac- The mucosa from the control patients was tion of RNA, which required larger cell macroscopically normal. None of the patients numbers. had received corticosteroids, mesalamine, sul- phasalazine, or immunosuppressive drugs that CULTURE OF COLONIC AND ILEAL EPITHELIAL would aVect cytokine secretion. CELLS The degree of inflammation of the speci- The cells ((1–5) × 105) were resuspended in mens was graded endoscopically31:+=low 400 µl minimal essential medium supple- degree of inflammation (increased granularity mented with Earle’s salts, 20% fetal calf serum, 352 Daig, Rogler, Aschenbrenner, et al

ITS (5 µg/ml insulin, 5 µg/ml transferrin, 5 NORTHERN BLOT ANALYSIS ng/ml selenious acid; Becton Dickinson, Hei- IECs and LPMNCs freshly isolated from delberg, Germany), 2 mM glutamine, 100 surgical specimens were homogenised with a U/ml penicillin, 100 µg/ml streptomycin, 100 sonifier (Bandelin) at 50% of maximal power Gut: first published as 10.1136/gut.46.3.350 on 1 March 2000. Downloaded from µg/ml gentamicin, and 2.5 µg/ml fungisone for five seconds. Isolation of total RNA was (JRH Bioscience, Lenexa, Kansas, USA). based on guanidinium isothiocyanate accord- Medium with stabilised glutamine was pur- ing to standard procedures. RNA (25 µg) was chased from Biochrom (Berlin, Germany). separated in a formaldehyde-containing agar- Other supplements were obtained from Sigma ose gel, transferred to a nylon membrane (Deisenhofen, Germany). The IECs were (Appligene, Illkirch, France), and cross linked by exposure to ultraviolet light using a seeded into collagen A (Biochrom) coated Stratalinker (Stratagene, Heidelberg, Ger- Millicell-CM cell culture plate inserts suitable many). The transfer was controlled by staining for 24 well culture plates with a translucent and the membrane with methylene blue. Hybridisa- permeable membrane at the bottom (Milli- tion was carried out with cDNA probes pore, Eschborn, Germany). The cells were labelled with [32P]dCTP by random priming ° 32 incubated at 37 C in air with 10% CO2. (Stratagene) (190 bp fragment of IL-1, 455 bp fragment of IL-1ra, 587 bp fragment of IL-6, ISOLATION OF LPMNCs and 244 bp fragment of IL-8). Agarose gel After removal of IECs, the mucosa was electrophoresis, RNA transfer, and hybridisa- tion of the membrane were performed as digested enzymically by incubation with 1 22 mg/ml collagenase (collagenase type I; Sigma), described previously. The membranes were 0.3 mg/ml DNAse I (Boehringer Mannheim, exposed to x ray films with intensifying screens (Hyperfilm MP; Amersham, Braunschweig, Mannheim, Germany), and 2 mg/ml hyaluro- Germany) for 1–2 days at −70°C. nidase (Sigma) at 37°C for 30 minutes. The digest was filtered and then centrifuged in DETERMINATION OF IL-1, IL-1ra, IL-6, AND IL-8 Ficoll-Paque (Pharmacia, Freiburg, Germany) for 20 minutes at 700 g. LPMNCs showed a Supernatants of IEC and LPMNC cultures viability of >90% as assessed by 0.1% trypan were collected after 24 hours. Cytokines were blue exclusion. measured by enzyme linked immunosorbent assay (ELISA; Biotrak-Amersham, Braun- CO CD UC schweig, Germany) according to the manufac- ___ _ turer’s protocol. The results were expressed as ++ ++ pg cytokine/105 cells. ELEELEELCell type http://gut.bmj.com/ WESTERN BLOTTING FOR IL-1ra Peripheral blood monocytes and IECs cultured A IL–1ra for 24 hours with or without 10 ng/ml TNF were lysed in a buVer containing 4 mM Hepes, 320 mM sucrose, 1 mM EDTA, 0.1 mM dithiothreitol, 1 mM phenylmethanesulphonyl B IL–8 fluoride, 1.5 mM pepstatin, 2 mM leupeptin,

0.7 mM aprotinin, and 0.1% CHAPS on ice on September 28, 2021 by guest. Protected copyright. for 20 minutes. After sonification for five seconds, protein extracts were centrifuged for C IL–1 10 minutes at 15 000 U/min. Protein concen- tration in the supernatant was determined using the bicinchoninic acid (BCA) test from Bio-Rad. Medium were precipitated D IL–6 by incubation with ice cold acetone (8:1, v/v) for one hour on ice. Centrifugation was performed for 30 minutes at 6000 g in a Heraeus Megafuge 1.0R at 4°C. Proteins were resuspended in reducing sample buVer. Then Methylene 80 µg of total protein from each sample was E blue loaded on a sodium dodecyl sulphate/15% polyacrylamide gel. Recombinant human IL- 1ra (R&D Systems, Wiesbaden, Germany) was Figure 1 Northern blot analysis of interleukin (IL)-1 receptor antagonist (ra), IL-8, used as a positive control. After electrophore- IL-1â, and IL-6 mRNA. RNA was extracted from intestinal epithelial cell (IEC) or sis, protein was transferred to a nitrocellulose lamina propria mononuclear cell (LPMNC) preparations from five control patients (CO), membrane. Blots were blocked with 5% 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 non-fat milk in PBS and probed with 1:3000 containing 1% formaldehyde, transferred to nylon membranes, and hybridised with dilutions of IL-1ra monoclonal antibody (R&D [32P]dCTP labelled cDNA fragments from IL-1ra cDNA (A), IL-8 cDNA (B), IL 1â Systems) in 5% non-fat milk/PBS. Immunob- (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 lots were then processed with 1:5000 diluted IL-1ra mRNA was higher in IECs than LPMNCs and increased during inflammation. horseradish peroxidase conjugated anti-goat IL-8 mRNA was only expressed in two of five IEC preparations isolated from normal IgG (Dianova, Hamburg, Germany) using the mucosa and was increased in mucosal inflammation but always to a lesser extent than in LPMNCs. IL-1â and IL-6 mRNA could not be found in the IEC preparation but were enhanced chemiluminescence plus (ECL Plus) always present in LPMNCs. western blotting detection system kit (Amer- Intestinal epithelial cells in mucosal immune system 353

sham Pharmacia Biotech). The blots were As the epithelial cell preparations contained exposed to Hyperfilm (ECL, Amersham) at some (<10%) non-epithelial cells, RNA from room temperature. LPMNCs was isolated from the same surgical specimens for comparison. Gut: first published as 10.1136/gut.46.3.350 on 1 March 2000. Downloaded from STATISTICAL ANALYSIS Data are expressed as mean (SEM). The IL-1ra statistical analysis was performed by the Low expression of IL-1ra was found in the Kruskal-Wallis H test or the Mann-Whitney IECs from all control patients and also in the rank sum test for non-parametric data. DiVer- uninflamed mucosa of patients with Crohn’s ences withapvalue of <0.05 were considered disease (fig 1, lane A). It was higher in inflamed significant. regions of patients with IBD. In the IECs of all patients, IL-1ra mRNA levels were as high as, Results or higher than, in LPMNCs (fig 1, lane A). The integrity of the extracted RNA and gel loading EXPRESSION OF CYTOKINE mRNA Total RNA extracted from freshly isolated was confirmed by methylene blue staining of IECs and LPMNCs from five colonic surgical the nylon membrane (fig 1, lane E). specimens without inflammation (carcinoma patients), two surgical specimens from pa- IL-8 tients with ulcerative colitis, and three speci- IL-8 mRNA was only present in low amounts mens from patients with Crohn’s disease was in IECs from two of the five control patients. subjected to northern blot analysis to screen IL-8 expression was also low in uninflamed for IL-1â, IL-1ra, IL-6, and IL-8 expression. areas of the specimens from patients with

IEC 1600 LPMNC 600 A B NS *** 1400 ** ** 500

1200 ** ** ** 400

cells 1000 5 cells

* 5 *** NS 800 300 *** *** 600 pg IL–8/10 http://gut.bmj.com/ pg IL–1ra/10 NS 200 NS 400 *** 100 *** 200 *** ***

0 0 CO CO CD CD UC UC CO CO CD CD UC UC (0) (1–3) (0) (1–3) (0) (1–3) (0) (1–3) (0) (1–3) (0) (1–3) on September 28, 2021 by guest. Protected copyright.

40 C D 60 35

50 30

cells 40 25 cells 5 5

/10 20 30

15 pg IL–6/10 pg IL–1 20 10

10 5

0 0 CO CO CD CD UC UC CO CO CD CD UC UC (0) (1–3) (0) (1–3) (0) (1–3) (0) (1–3) (0) (1–3) (0) (1–3) 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-1â (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. 354 Daig, Rogler, Aschenbrenner, et al

Table 1 Interleukin (IL) 1 receptor antagonist (ra), IL-8, IL-1â, and IL-6 content of freshly isolated intestinal epithelial cells (IECs) and lamina propria mononuclear cells (LPMNCs) normalised to cell protein

IL-1ra IL-8 IL-1â IL-6 Gut: first published as 10.1136/gut.46.3.350 on 1 March 2000. Downloaded from

IEC LPMNC IEC LPMNC IEC LPMNC IEC LPMNC

CO (0) 7397 (1039) 3199 (375) 137 (19) 1144 (145) 1.8 (0.6) 97 (14) 1.4 (0.2) 42 (5.6) CO (1–3) 8973 (1570) 4325 (380) 187 (17) 1836 (133) 9.8 (2.1) 263 (46) 2.9 (1.3) 100 (18) CD (0) 5706 (1056) 3836 (154) 122 (8.7) 1853 (436) 9.9 (2.9) 305 (45) 3.1 (1.0) 86 (1.9) CD (1–3) 9301 (2188) 5440 (748) 216 (29) 2005 (194) 30 (4.4) 348 (80) 8.8 (1.6) 185 (34) UC (0) 5914 (1777) 4253 (533) 181 (69) 2283 (197) 11 (5.9) 339 (39) 13 (1.6) 99 (12) UC (1–3) 17856 (2013) 10216 (348) 1162(206) 6624 (1812) 29 (6.8) 697 (24) 17 (2.2) 422 (32)

Cytokines were measured in homogenised, freshly isolated IECs from 50 control patients (CO, 0), five patients with unspecific intestinal inflammation (CO, 1–3), 24 patients with Crohn’s disease (CD), and 14 patients with ulcerative colitis (UC). Cytokines were also measured in freshly isolated LPMNCs from 17 control patients, five patients with unspecific intestinal inflammation, nine patients with Crohn’s disease, and nine patients with ulcerative colitis. Data (mean (SEM) are expressed as pg cytokine/mg total protein. 0, mucosa not inflamed; 1, slightly inflamed; 2, moderately inflamed; 3, severely inflamed. Crohn’s disease. It was higher in IECs from IL-1â and IL-6 inflamed mucosa from patients with ulcerative In contrast with IL-1ra and IL-8 mRNA, in colitis and Crohn’s disease (fig 1, lane B). In none of the specimens was IL-1â (fig 1, lane C) LPMNCs from control patients, IL-8 expres- and IL-6 (fig 1, lane D) mRNA present in sion was low, but higher than in IECs. In patients IECs. IL-1â expression in LPMNCs from con- with IBD, there was an obvious increase in IL-8 trol patients was low but was obviously expression by LPMNCs. In all patients investi- increased in patients with IBD (fig 1, lane C). gated, IL-8 expression was higher in LPMNCs Already in the control specimens, IL-6 was than in IECs (fig 1, lane B). expressed in the LPMNC fraction. This

IEC 500 LPMNC 350 AB 300 400 250 cells

300 5 200 epithelial cells 5 150 200 http://gut.bmj.com/

pg IL–1ra/10 100 100

pg IL–1ra/10 50

0 0 CO CD UC CO CO CD CD UC UC 0 1 2 3 0 1 2 3 0 1 2 3 (0) (1–3) (0) (1–3) (0) (1–3)

5000 on September 28, 2021 by guest. Protected copyright. 700 C D

600 4500

500 4000 cells 5 400 3500 epithelial cells 5 300 500 pg IL–8/10

pg IL–8/10 200 250 100

0 0 CO CD UC CO CO CD CD UC UC 0 1 2 3 0 1 2 3 0 1 2 3 (0) (1–3) (0) (1–3) (0) (1–3) 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. Intestinal epithelial cells in mucosal immune system 355

expression was obviously increased in Crohn’s A disease, but not in ulcerative colitis. 250 IEC Colonic epithelial cells Gut: first published as 10.1136/gut.46.3.350 on 1 March 2000. Downloaded from CYTOKINE CONTENT OF FRESHLY ISOLATED IECs AND LPMNCs 200 To exclude post-transcriptional diVerences in processing of the mRNAs leading to a

misinterpretation of the northern blot findings, cells

5 150 we determined the cytokine content of freshly isolated colonic IECs and LPMNCs. 100 IL-1ra In freshly isolated IECs from non-inflamed pg IL–1ra/10 mucosa, IL-1ra concentrations were higher in 50 control patients than in those with Crohn’s disease and ulcerative colitis, but these diVer-

ences did not reach significance (fig 2A). 0 IL-1ra levels in IECs from control patients CO CD CD increased slightly when inflammation occurred (0) (0) (1+2) (not significant compared with non-inflamed B controls). However, the increase caused by 600 inflammation was significantly higher in IECs from patients with IBD (p = 0.04 for Crohn’s disease and p = 0.01 for ulcerative colitis versus 500 non-inflamed IECs) (fig 2A). IL-1ra levels in freshly isolated LPMNCs 400 cells

from control patients with and without inflam- 5 mation were significantly lower than in the cor- 300 responding IECs (p = 0.0002 and p = 0.003 125 respectively) (fig 2A). In addition, freshly 100 isolated LPMNCs from non-inflamed mucosa pg IL–8/10 from patients with Crohn’s disease or ulcera- 75 tive colitis contained less IL-1ra than the corresponding IECs. During acute inflamma- 50 tion, IL-1ra increased significantly (p = 0.01) 25 in the LPMNCs in Crohn’s disease and ulcera- 0 http://gut.bmj.com/ tive colitis but did not reach the levels of the CO CD CD corresponding IECs (fig 2A). (0) (0) (1+2) Figure 4 Comparison of interleukin (IL)-1 receptor IL-8 antagonist (ra) and IL-8 secretion in isolated ileal (IEC) Freshly isolated IECs from non-inflamed areas and colonic epithelial cells. Cytokines were determined in culture medium after 24 hours of culture. n=7forcontrols of specimens from patients with IBD and con- (CO), non-inflamed Crohn’s disease (CD), and inflamed trol patients contained comparable amounts of CD. Data are mean (SEM) expressed as pg cytokine/105

IL-8. The content of IL-8 in freshly isolated cells. DiVerences between ileal and colonic epithelial cells did on September 28, 2021 by guest. Protected copyright. IECs from controls with colonic inflammation not reach significance. or Crohn’s disease was still low but significantly increased compared with controls without 4.6 (0.9) pg IL-1â/105 cells. In macroscopically inflammation (p = 0.02 and p = 0.04 respec- uninvolved mucosa of patients with Crohn’s tively). In inflamed areas of patients with disease (p = 0.008) and ulcerative colitis (p = ulcerative colitis, IL-8 levels were also signifi- 0.04), IL-1â levels were significantly higher cantly increased (p = 0.016) (fig 2B). than in controls (fig 2C). During inflamma- IL-8 protein concentration was significantly tion, IL-1â was increased in LPMNCs from higher in LPMNCs than in the corresponding control patients (p = 0.005), patients with IECs in all patient groups (p<0.001). It was Crohn’s disease (not significant), and patients also increased in LPMNCs in control patients with ulcerative colitis (p = 0.04) (fig 2C). during inflammation (fig 2B). In LPMNCs As found for IL-1â, IL-6 levels in freshly iso- from non-inflamed areas of Crohn’s disease lated IECs were at the detection limit of the mucosa, IL-8 levels were already significantly ELISA in all patient groups (0.5 pg IL-6) (fig increased to 200 (51) pg/105 cells and in 2D). A low amount of IL-6 was found in ulcerative colitis to 228 (25) pg/105 cells. Dur- LPMNCs from controls, and higher levels were ing macroscopical inflammation, there was an found in non-inflamed mucosa from patients increase in Crohn’s disease to 285 (50) pg/105 with Crohn’s disease or ulcerative colitis (fig cells and in ulcerative colitis to 501 (83) pg/105 2D). During inflammation, IL-6 levels in cells (fig 2B). LPMNCs were significantly increased in con- trol patients (p = 0.001), patients with Crohn’s IL-1â and IL-6 disease (p = 0.01), and patients with ulcerative In freshly isolated IECs from all patient groups, colitis (p = 0.03) (fig 2D). IL-1â protein was at the detection limit of the Comparable results were obtained when ELISA (0.5 pg). Freshly isolated LPMNCs cytokine levels were normalised to cellular pro- from controls without inflammation contained tein content (table 1). 356 Daig, Rogler, Aschenbrenner, et al

CYTOKINE SECRETION IN PRIMARY CULTURES OF inflamed IBD mucosa (Crohn’s disease, IECs AND LPMNCs p = 0.0003; ulcerative colitis, p = 0.0016) but To investigate whether the cytokine levels in also more than inflamed mucosa from control freshly isolated cells corresponded to secreted patients (fig 3C). Gut: first published as 10.1136/gut.46.3.350 on 1 March 2000. Downloaded from and thereby bioactive cytokines, we cultured Compared with IECs, LPMNCs isolated IECs and LPMNCs for 24 hours and measured from the same patients secreted significantly cytokine levels in the culture medium. more IL-8 (p<0.0001) (fig 3D). In control patients, there was no increase in IL-8 IL-1ra secretion in LPMNCs during inflammation. In After 24 hours of culture, IECs from normal LPMNCs from non-inflamed mucosa from mucosa secreted 120 (12) pg IL-1ra/105 cells patients with Crohn’s disease, the amount of (fig 3A). When there was acute inflammation in IL-8 secretion was in the same range as for patients without IBD—for example, patients control patients. In inflammation, IL-8 secre- with diverticulitis—secretion increased, but tion increased but only to 491 (75) pg/105 cells, this eVect was not significant (p = 0.56). IECs which is in the same range as shown for the from non-inflamed mucosa from patients with IECs. The secretion of IL-8 by LPMNCs in IBD showed lower secretion of IL-1ra than ulcerative colitis even in non-inflamed mucosa controls (Crohn’s disease, 62 (24) pg/105 cells; was much higher (3522 (365) pg/105 cells); in ulcerative colitis, 77 (24) pg/105 cells). When inflamed mucosa it increased to 4154 (488) IL-1ra secretion by IECs from macroscopically pg/105 cells (fig 3D). inflamed mucosa was analysed, there was a sig- nificantly higher secretion than from cells from IL-1â and IL-6 an area with a low degree of inflammation IL-1â was not detectable in supernatants from (Crohn’s disease, p = 0.0013; ulcerative colitis, IECs from all patient subgroups. Also IL-6 was p = 0.0002) (fig 3A). There was a correlation only present in very low amounts and was between the secreted amounts of IL-1ra and probably due to contamination with LPMNCs, the macroscopical degree of inflammation supporting the results obtained from northern (Crohn’s disease, r = 0.64, p<0.001; ulcerative blot analysis and freshly isolated IECs (data colitis, r = 0.76, p<0.0001). In all groups, IECs not shown). secreted more IL-1ra than did LPMNCs after 24 hours of culture (fig 3B). CYTOKINE SECRETION OF ILEAL AND COLONIC IECs To investigate further whether there is a diVer- IL-8 ence in cytokine secretion between ileal and The secretion of IL-8 in supernatants of IECs colonic IECs, we cultured IECs isolated from cultured for 24 hours was low (fig 3C). IECs both locations for 24 hours and determined from unspecific inflamed mucosa showed a

cytokine release into the medium. The values http://gut.bmj.com/ significant increase (p = 0.008) (fig 3C). There in controls, non-inflamed Crohn’s disease, and was also low IL-8 secretion by IECs from non- inflamed Crohn’s disease were not diVerent for inflamed regions of IBD mucosa, which was IL-1ra (fig 4A) and IL-8 (fig 4B), except for not significantly diVerent from normal IECs IL-8 secretion from inflamed Crohn’s disease (fig 3C). IECs from patients with macroscopi- ileal cells. However, this diVerence may be due cally severely inflamed mucosa secreted signifi- to severe terminal ileitis in these patients. cantly more IL-8 than those from non- on September 28, 2021 by guest. Protected copyright. Monocytes Monocytes IEC IL-1ra FORMS SECRETED BY IECs cytosol medium medium rh IL–1ra As there are two intracellular and one secreted form of IL-ra which are supposed to have diVerent functions, we performed western blots to determine which isoform was found in the medium of primary human IECs after 24 hours of culture. Only the glycosylated 22–25 kDa secretory form of IL-1ra could be detected in the medium of IECs (fig 5). Similarly, in the medium of cultured peripheral blood mononu- 30 kDa clear cells, the 22–25 kDa form was predomi- sIL–1ra nant, but the smaller intracellular IL-1ra form could also be detected (fig 5). Stimulation of icIL–1ra the cells with 10 ng/ml TNF for 24 hours did 17 kDa not change these findings.

Discussion 10 ng/ml 10 ng/ml 10 ng/ml In this study we show that IECs from normal TNF TNF TNF mucosa produce no detectable amounts of Figure 5 Identification of interleukin (IL)-1 receptor antagonist (ra) isoforms in medium IL-1â or IL-6, whereas LPMNCs do. IECs supernatants of peripheral blood mononuclear cells and intestinal epithelial cells (IECs). produce and secrete very low amounts of IL-8, 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 but in non-inflamed mucosa, much more was in its glycosylated form; sIL-ra) as well as recombinant human IL-1ra (non-glycosylated, generated by the corresponding LPMNCs. 17 kDa; rh-IL1-ra) were used as positive controls. In the precipitated protein fraction of However, IL-8 production in IECs can be 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 diVerent IEC cultures. induced upon stimulation. IECs secrete more TNF,tumour necrosis factor. IL-1ra than do the corresponding LPMNCs. Intestinal epithelial cells in mucosal immune system 357

Therefore the pattern of cytokine secretion by mucosa. However, IL-8 secretion could be IECs is a more anti-inflammatory one than that measured after 24 hours of culture of cells iso- of LPMNCs, which secrete all the cytokines lated from normal mucosa. We assume that the investigated. isolation procedure for IECs may represent an Gut: first published as 10.1136/gut.46.3.350 on 1 March 2000. Downloaded from Several lines of evidence indicate that the ‘injury to the ’ inducing IL-8 data are reliable. No mRNA for IL-1 or IL-6 production in vitro. was seen in the IEC preparation, indicating Our data showing lack of IL-1â production that potential contamination with macro- by IECs are in accordance with in vivo phages (less than 4% in FACS analysis) was findings, as IL-1â protein and mRNA have not relevant to the analysis. In addition, the been found in LPMNCs but not in IECs by IEC preparation did not contain any relevant most authors.27 38–40 Only in rabbit immune amounts of fibroblasts, as, in another series of complex colitis41 and acute acetic acid induced experiments, we showed that fibroblasts secrete colitis in rats42 was IL-1 production found in IL-6 even under unstimulated conditions. enterocytes. Elevated levels of IL-1ra had been previously In vivo results on IL-6 production by IECs reported in human IBD.29 32 33 In addition, it had are contradictory, as mentioned in the intro- been shown that IECs contribute to the IL-1ra duction. Our study clearly shows that, in pool of the intestinal mucosa.32 The role of humans, IL-6 is not produced by epithelial IL-1ra in intestinal inflammation has been cells. In earlier investigations, contamination of shown in animal models. Increased IL-1ra levels the cultures with fibroblasts or macrophages have been reported in peptidoglycan- may have influenced the results. Interestingly, polysaccharide induced colitis in rats.34 Treat- even without inflammatory stimuli, macro- ment with recombinant IL-1ra attenuated the phages express high levels of IL-6 mRNA, mucosal inflammation in this model.34 Vice especially when compared with IL-1â or IL-8. versa, inhibition of endogenous IL-1ra with a The physiological role of this basal IL-6 secre- neutralising antibody exacerbated formalin in- tion remains to be elucidated. duced complex colitis in rabbits.35 IL-1ra knock There is no major diVerence in the cytokine out mice show an increased susceptibility to response between epithelial cells from ileal and endotoxaemia.36 Dextran sodium sulphate in- colonic mucosa. The response of IECs during duced colitis was followed by a large increase in inflammation is not dependent on the type and mortality compared with normal IL-1ra ex- whether acute or chronic mucosal inflamma- pressing mice.37 These data indicate an impor- tion is present. Therefore our study does not tant role for IL-1ra in the balance of the mucosal provide any evidence for any special “behav- immune system. Our data show that IECs are iour” of IECs during IBD. the major source of IL-1ra in the mucosa. The present study clarifies some important Primary IECs were able to secrete sIL-1ra in the points about the function of IECs which have http://gut.bmj.com/ 24 hour culture model. This could mean that been controversial in the past. Human IECs do loss of epithelial cells from certain areas of the not produce IL-1â and do not therefore have mucosa could be followed by localised imbal- any direct proinflammatory potential, as sug- ance of proinflammatory and anti-inflammatory gested by studies with the IEC-6 cell line. This is cytokines. Previous studies from our laboratory very important for the interpretation of data have shown that the imbalance of the mucosal derived from murine colitis models, as murine immune system is not specific to IBD, but can epithelial cells may show diVerent behaviour

also be found in other types of colonic from human IECs. Human IECs do not on September 28, 2021 by guest. Protected copyright. inflammation.29 The degree of inflammation was produce IL-6 in an acute phase response. This the most important factor for the production of again indicates that studies with transformed IL-1á, IL-1â, and IL-1ra. cell lines have to be interpreted very carefully. The diVerence between IL-1ra secretion Human IECs are able to produce and secrete from IECs and LPMNCs was greatest in IL-8 upon stimulation. This may be important patients with Crohn’s disease with or without for the recruitment of granulocytes during acute inflammation (fig 3B). It is noteworthy that the or chronic mucosal inflammation. Human IECs ratio between IL-1ra and IL-1 was also lowest are the major source of IL-1ra in the human in inflamed mucosa in Crohn’s disease in our mucosa. Upon inflammation they respond with previous investigations29 and that the IL-1ra upregulation of IL-1ra production. levels in homogenates of mucosal biopsy speci- In conclusion, expression and secretion of mens were lower in Crohn’s disease than in cytokines by IECs from normal mucosa ulcerative colitis.29 Impaired induction or even displayed a more anti-inflammatory pattern downregulation of IL-ra secretion in LPMNCs than LPMNCs. In inflamed mucosa, secretion could worsen the imbalance of the mucosal of the anti-inflammatory secretory IL-1ra by immune system in Crohn’s disease. IECs is increased but may be antagonised by There have been conflicting data on the pro- production of proinflammatory cytokines, such duction of IL-8 by IECs. Whereas IL-8 as IL-8 by the IECs themselves or IL-1 by secretion could be clearly induced in colonic LPMNCs. epithelial cell lines HT-29and Caco-2 by IL-1â and TNFá22, in situ hybridisation showed IL-8 This work was supported by the Deutsche Forschungsgemein- schaft (An 168/3-1). Parts of this manuscript were presented at mRNA only in the lamina propria of inflamed the Digestive Disease Week 1997, Washington and at the Diges- mucosa.30 Under our in vitro conditions, LPM- tive Disease Week 1998, New Orleans. NCs produced significantly more IL-8 than 1 Selby WS, Poulter LW, Hobbs S, et al. Expression of IECs. Only very low amounts of IL-8 mRNA HLA-DR antigens by colonic epithelium in inflammatory were present in cells isolated from normal bowel disease. Clin Exp Immunol 1981;44:453–8. 358 Daig, Rogler, Aschenbrenner, et al

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