Sequential release of cytokines, lipid mediators and nitric oxide in experimental colitis

The object of this study was to establish whether different pro- and anti-inflammatory mediators were formed in colonic tissue from experimental colitis depending on the course of the disease. Concentrations of mediators of inflammation were examined in colonic tissue in dextran induced colitis in mice. Initial inflammation was produced by 5 days treatment of 10% dextran sodium sulfate (DSS) in drinking water, followed by a further 9 day period of 2% DSS in an attempt to produce a milder chronic inflammation. The degree of inflammation was scored by a standardized macroscopic and histological examination. Initially, a 60% maximum inflammation score was observed at day 4. At this time inflammation was associated with the release of interleukin-lβ (IL-1β) and tumour necrosis factor-α (TNFα), whereas both prostaglandins 6kPGF1α and PGE2 and nitric oxide (NO) markedly decreased. Then a 25% inflammation score was reached which coincided with an increased production of platelet-activating factor (PAF). No significant changes were observed in leukotriene B4 and C4 formation. In conclusion, pro-inflammatory cytokines IL-1β and TNFα are considered to be primary mediators, whereas PAF, eicosanoids and NO may reflect secondary mediators in experimental colitis.


Introduction
bioregulator of eicosanoid and cytokine production. 1 Since damage of the endothelial cell can Several experimental models have been be a prime event leading to mucosal injury, this described which resemble human inflammatory could reflect a diminished release of the endobowel disease (IBD), including ulcerative colitis thelial derived mediators prostacyclin (PGI2) and (UC) and Crohn's disease (CD). These animal nitric oxide (NO). On the contrary, we have models have been used to examine the producfound high amounts of the 15-1ipoxygenase tion of inflammatory mediators by intestinal product 15-hydroxy-eicosatetraenoic acid (15-mucosa and to determine the effects of various HETE) in human colonic mucosa. 1 This subdrugs on inflammation and on individual mediastance is synthesized both by endothelial cells tots. 2 '3 The customary drugs used in IBD--cortand macrophages. The minor anti-inflammatory icosteroids and 5-aminosalicylates--are thought effects of 15-H(P)ETE could be ascribed to to act by interfering with eicosanoid producinhibition of the pro-inflammatory mediator tion. 4 In IBD, the synthesis of leukotriene B4 LTB4. 2 It has been suggested that cytokines (LTB4) in colonic mucosa has been shown to such as turnout necrosis factor (TNFa) and be enhanced compared with its production in interleukin-I (IL-1) are primary triggers of the normal tissue. 5 However, specific 5-1ipoxygenase initial inflammation, 3 whereas platelet-activating inhibitors such as Zileuton have not been factor (PAF)is considered to be involved as a shown to have marked beneficial effects on secondary trigger, resulting in a chronic phase inflammation in man, although LTB4 levels in of the inflammation. 4 This hypothesis implies dialysates were decreased. 6 Prostaglandin E2 that the choice of drug therapy for IBD might (PGE2) is also increased in dialysates 7 and depend on the stage of the disease. In the mucosal tissue s of patients with UC and CD.
search for a suitable model for UC resembling However, the anti-inflammatory effects of this acute, and chronic inflammation of colonic substance are well documented, 9 and it is theremucosa, we modified the dextran sodium sulfate fore likely that PGE2 mainly acts as a feed-back (DSS) induced colitis in mice and measured a wide range of mediators of inflammation during be done at the same time in one assay. the course of the disease.
Microscope evaluation of the colonic tissue was assessed as follows: cell infiltration of the Materials and Methods tissue; depletion of goblet cells; loss of the tissue architecture; and the formation of crypt absces-Induction of colitis: Colitis was induced in BALB/ ses (score 0-4). Each of the given parameters on c female mice (20-22 g) by adding high-molemacroscopic and microscopic evaluation was cular dextran sodium sulfate (DSS; Pharmacia, subdivided into a three-scaled point system (+, Sweden) to their drinking water, which was given + + and + + + ), resulting in a total inflammato the mice ad libitum from the first day of the tion score range of 0-33 points, which served as treatment. DSS, given orally, causes a diffuse an objective measure of the severity of inflammainflammation of the mucosa of the entire gut. tion of the colonic tissue. 12 The mechanism by which this inflammation is induced is not entirely clear, but bacterial flora in Measurement of mediators: The amount (mg) of the gut appear necessary for the process. 15 protein per g wet homogenized tissue was deter-During the treatment schedule, the concentration mined in the supematant by a micro-scale of DSS in the water was altered. For the first 5 method (Instruchemie, Hilversum, The Netherdays 10% (w/v) DSS was administered via the lands) using an ELISA reader (Biorad, USA) at drinking water in an attempt to provide a model 600 nm. Myeloperoxidase (MPO) in these for the acute and initial phase of inflammation, samples was determined by means of a double During the last 9 days of the schedule, the con-antibody radioimmunoassay (RIA, Pharmacia, centration was lowered to 2% (w/v) DSS, in Sweden) in which bound and free MPO were order to create a more chronic and milder separated by addition of a second antibody inflammation in the mucosa of the colon, immunosorbent and 125I was counted.
The concentrations of different mediators of Treatment schedule: In this study 29 mice were inflammation were measured in duplicate in the randomly divided into five groups. The first group liquid samples described above. The nitric oxide consisted of nine mice and served as a control metabolites, nitrate (after reduction to nitrite) + group throughout the investigation period. The nitrite, were measured colometrically after the other 20 mice were divided into four groups of Griess reaction, as described by Phizackerly and five mice each. The animals were killed by cervi-A1-Dabbagh. 16 Briefly, the following modifications cal dislocation after 2, 4, 8 and 14 days. were applied: 7 samples were deproteinized by adding 1M NaOH and 1.3 M ZnSO4; nitrate was Experimental procedure and inflammation converted to nitrite by the addition of Klebsiellascore: After the mice were killed, the colon from pneumonia suspension, 0.2 M N-tris-(hydroxyflexure to rectum was removed immediately. A methyl)-methyl-2-amino-ethane sulfonic acid small piece was cut off and stored in form-(TES) and 0.5 M sodium formate; after anaerobic aldehyde, and subsequently prepared for histoincubation, water was added and nitrite was logical examination. The colon was opened in assayed in supernatants. Nitrite was estimated in a transverse direction and examined macro-deproteinized samples, mixed with 1% sulfanilscopically using a 10 x magnification. Signs of amide in 15% phosphoric acid; after the addition inflammation and changes in the appearance of of 0.1% N-(1-naphthyl)ethylenediamine, the the colonic mucosa were evaluated and the absorption was determined at 540 nm by means severity of inflammation (erythema, haemor-of an ELISA reader (Biorad). The linear detection rhages, mucus, oedema, strictures, diarrhoea) range was between 1 and 250 IM. was scored (0-6). Faeces were also examined for Cytokines, eicosanoids and PAF were deterthe presence of occult blood (score 0-1). After mined in the samples of the colonic tissue by macroscopic assessment, the colon was incu-means of ELISAs and RIAs. For the assay of bated in 5 ml 0.9% w/v saline and then shaken TNEc, a specific mouse TNFz ELISA kit was lightly for 5 min. This suspension was centrifuged obtained from Genzyme Corp., using a hamster (2800 x g, 10 min, 4C) and the supernatant monoclonal antibody specific for murine TNF, a was stored at -80C. The colon was weighed goat polyclonal anti-murine TNF antibody, and, and 2 ml KREBS buffer added. The colonic tissue as third antibody, horsedish peroxidase-conwas fragmented in this solution using the Ultrajugated donkey anti-goat Ig. The peroxidase Turrax (Polytron, Switzerland), after which this enzyme reacted with peroxide substrate, after suspension was centrifuged (10 000 x g, 10 min, which the colour intensity was measured at 492 4C) and the supernatant stored at -80C until nm. Detection range was between 0.05 and 1. using a high sensitivity IL-lJ3 assay from Cistron Biotechnology (Eurogenetics, Belgium), bound to a solid phase monoclonal antibody, after which in the second stage polyclonal rabbit anti-IL-113 was added, followed by goat anti-rabbit IgG conjugated to horseradish peroxidase enzyme which reacts with peroxide substrate. The end product was measured at 450 nm. Detection range was between 4 and 250 pg/ml. Eicosanoids were determined by RIA. Specific antibodies were obtained from Advanced Magnetics Inc. (MA, USA), tritiated compounds from Amersham (UK) and standards from Sigma Co. (USA). Assays were performed for a 16 h competitive binding period, using standard curves from 0-500 pg/tube; sensitivities were: PGE2, 2 pg/ tube; 6kPGFI=, LTB4 and LTC4, 5 pg/tube at 98% B/Bo. Finally, PAF was measured using a SPA-RIA kit assay system of Amersham (UK). The standard curve was prepared from 0 to 1280 pg/tube and a sensitivity of 20 pg/tube. The concentrations of the mediators produced during inflammation were expressed per mg wet colonic tissue. shown in Fig. 1. In the control group, which contained mice that had not been treated with DSS, no inflammatory activity was observed ( Fig.  2(a)).
The group treated for 2 days with 10% DSS solution and then killed at the second day of the treatment schedule, showed a significant increase in the inflammation score. The maximum inflammation score was seen after 4 days treatment with 10% DSS (Fig. 2(b)). The inflammation score of the fourth and fifth group, in which the DSS concentration in the drinking water was reduced to 2% (w/v) on the fifth day of the treatment schedule, showed a significant decrease of the inflammation score compared with groups which had been treated with 10% DSS for 2 or 4 days. Compared with the control group, however, inflammatory activity was still present (Fig. 2(c)). The production of NO, 6kPGFla and PGE2 in 100 mg tissue decreased significantly during the initial phase (d2-d4) of the inflammation (Fig.   3). The production of LTB 4 and LTC4 was extremely low in comparison with PGs (1-4 vs. 500-3500 pg/mg wet tissue, data not shown) and did not change significantly. The production of the cytokines TNFa and IL-1[3 was only increased in the first two days (initial phase) of DSS treatment. The production of PAF was unchanged during the initial phase, whereas a marked increase of PAF production was observed (d8) after the concentration of DSS was reduced (Fig.  4).

Discussion
Dextran sodium sulfate induced a diffuse inflammatory response in the colonic mucosae of the mice when given according to the method described by Okayasu et a/. 5 21 In our observations, the elevated release of MPO on day 2 indeed precedes the diminished elevation of NO. Endothelial cells could be depleted and unable to generate these bioactive substances. The formation of neutrophil aggregates has been described in PAF-induced gastrointestinal damage, 22 whereas it has been demonstrated that NO can prevent 23 neutrophil adhesion to the endothelium. A significan increase of NO was observed after 8 days of inflammation. This could be due to a secondary influx of activated macrophages which may contribute to this phenomenon. Corticosteroids are known to inhibit the production of both eicosanoids 7 and the pro-inflammatory IL-I. 24 These non-selective inhibitory actions could explain why both the initial and the chronic phase of inflammation are successfully treated by this group of compounds. Sulfasalazine derived compounds such as 5-amino-salicylates (5-ASA) non-selectively inhibit eicosanoid formation in dextran sulfate induced colitis in mice, 25 whereas we previously found that 5-ASA increases rather than inhibits PAF synthesis. 16 This effect of 5-ASA on PAF might explain the fact that exacerbations during chronic colitis are well treated by 5-ASA compounds, but total remission is not achieved in many cases. Further investigations with specific synthesis inhibitors and soluble receptor antagonists are needed to gain more insight in the importance of the sequential release of pro-and anti-inflammatory mediators during acute and chronic colitis.