We examined the in vivo effect of nicotine on the synthesis of (pro-)inflammatory mediators by mouse colonic mucosa. The synthesis of lipid mediators such as the prostanoids prostaglandin E2, 6-keto-prostaglandin F1α and thromboxane B2, the 5-lipoxygenase products leukotriene B4 and leukotriene C4 and the platelet activating factor was not affected, whereas the synthesis of the pro-inflammatory cytokines interleukin-1 β and tumor necrosis factor α was completely abolished. The beneficial effects of smoking and nicotine in ulcerative colitis could be attributed to this inhibition

Dijk, J.P.M. van

Keuskamp, D.H.G.

Madretsma, G.S. (Guno)

Zijlstra, F.J. (Freek)

Erasmus University Digital Repository

ejp ELSEVIER European Journal of Pharmacology 278 (19951 R1 I-RI2 Rapid communication Nicotine inhibits cytokine synthesis by mouse colonic mucosa Jeanette P.M. Van Dijk, G. Stanley Madretsma, Zosca J. Keuskamp, Freek J. Zijlstra Department ofPharmacoloj,,y, Erasmus Unicersi<v Rotterdam, Rotterdam, Netherlands Received 3April 1995; accepted 4 April 1995 Abstract We examined the in vivo effect of nicotine on the synthesis of (pro-)inflammatory mediators by mouse colonic mucosa. The synthesis of lipid mediators uch as the prostanoids prostaglandin E 2, 6-keto-prostaglandin Fl~~ and thromboxane B2, the 5-1ipoxygenasc products leukotriene B4 and leukotriene C4 and the platelet activating factor was not affected, whereas the synthesis of the pro-inflammatory cytokines interleukin-1/3 and tumor necrosis factor a was completely abolished. The beneficial effects of smoking and nicotine in ulcerative colitis could be attributed to this inhibition. Keywords: Nicotine; Colonic mucosa; Cytokine During the last decade it has become clear that cigarette smoking not only increases the risk of devel- oping cancer or cardiovascular disease, but surpris- ingly, also has beneficial effects on acne (Mills et al., 1993) and chronic diseases uch as Alzheimer disease (Jones et al., 1987) and ulcerative colitis (Rhodes and Thomas, 1994). The mechanisms underlying the benefi- cial effects are unknown. Nicotine is the most impor- tant pharmacologically active substance in tobacco smoke and it therefore seems logical to assume it to be responsible for most of the immunoregulatory effects of cigarette smoke. Treatment of patients with active ulcerative colitis with nicotine patches proved to have a beneficial effect. Symptoms significantly improved in these patients (Pullan et al., 19941. The effects of nicotine on lipid mediators uch as prostaglandins are still controversial. In rabbits prostaglandins were re- duced by nicotine, which showed an inverse dose de- pendence, whereas in ferrets no significant effects were observed (Zijlstra et al., 19941. This study was approved by the Animal Experimen- tal Committee of the Erasmus University of Rotterdam (protocol 118.94.17). Colonic mucosa was obtained from * Corresponding author. Department of Pharmacology, Faculty of Medicine and Health Sciences, Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, Netherlands. Tel (+)31 10 4087550, fax (+)31 10 4366839. 01114-2999/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0(114-2999(95)00211-1 BALB/c  female mice (19-21 g). Nine mice were used as controls, six mice were treated with nicotine. Ini- tially mice received nicotine in drinking water ad libi- turn: 2 days 25 /xg/ml, 2 days 50 /xg/ml, 2 days 100 p~g/ml and 2 days 200 p.g/ml. Thereafter nicotine was dissolved in saline and given during 14 days subcuta- neously by an Alzet osmotic mini-pump with an infu- sion rate of 200 /xg/day. Controls received tap water and an osmotic pump filled with saline. Plasma nico- tine and cotinine concentrations reflected the dose of nicotine given and were measured by Dr. C. Feyer- abend, Nicotine Laboratory, Poisons Unit, New Cross Hospital London, UK. By the third week plasma nico- tine and cotinine concentrations were 3.6 + 0.61 and 0.3 _+ 0.05 ng/ml  in controls and 42 _+ 3.0 and 105 _+ 11 ng/ml  in nicotine treated mice respectively. These data were comparable to plasma levels of cotinine (102_+ 52 ng/ml)  in patients treated with nicotine patches with a release of 15 mg nicotine per day, reflecting 15 cigarettes per day (Pullan et al., 1994). After the mice were killed by cervical dislocation the colon from hepatic flexure to rectum was removed immediately. The colon was opened in a transverse direction and faeces was removed. The weight of the colon was determined and 2 ml Krebs-Henseleit buffer added, after which the tissue (approx. 200 rag) was fragmented using an Ultra-Turrax (Polytron, Switzer- land) and inflammatory mediators were measured. The amount of protein per mg wet homogenized tissue was R12 J.P.M. Van Dijk et al. / European Journal of Pharmacology 278 (1995) RI1-R12 Table 1 Formation of lipid mediators and cytokines by mouse colonic mucosa fter nicotine treatment Mediator Controls Nicotine Protein 6-Keto-prostaglandin F1, ~ Prostaglandin E 2 Thromboxane B 2 Leukotriene B 4 Leukotriene C 4 Platelet activating factor Interleukin-I/3 Tumor necrosis factor a (~g/mg) (pg/mg) (pg/rag) (pg/mg) (pg/mg) (pg/mg) (pg/mg) (pg/mg) (pg/mg) 11.8_+ 0.69 12.5 ± 0.52 3620 +_428 3500 +341 1020 _+157 1170 ± 73 416 _+ 53 415 ± 32 3.4_+ 0.42 2.9± 0.28 1.5± 0.27 1.8± 0.39 54 + 20 48 ± 14 1.3 ± 0.60 <0.1 ~ 120 ± 58 13 ±13 a P < 0.05 compared with controls. determined by a micro-scale method (Instruchemie, Netherlands). Eicosanoids were determined by ra- dioimmunoassay, using antibodies from Advanced Magnetics (MA, USA), tritiated compounds from Amersham (UK) and standards from Sigma Co. (USA). Platelet activating factor was determined by radioim- munoassay (PAF-SPA system, Amersham, UK) and cytokines by enzyme-linked immunosorbent assay: tu- mor necrosis factor a from Genzyme Corp. (USA) and interleukin-1/3 from Cistron Biotechnology (Euro- genetics, Belgium). The synthesis of the lipid mediators was unaffected, whereas the production of pro-inflammatory c tokines was completely abolished (Table 1). This is, as far as we know, the first paper in which the direct effect of nicotine on the synthesis of pro-in- flammatory cytokines was investigated and shown to be of a strong inhibitory nature. Recently the effect of exposure to cigarette smoke was investigated in alveo- lar macrophages in rats (Pessina et al., 1993). As ex- pected tumor necrosis factor a was increased in alveo- lar macrophages, but surprisingly decreased in peri- toneal macrophages 8 h after the smoking session. In that study peritoneal macrophages were taken as inter- nal control cells and the authors failed to conclude that this unexpected effect could be due to circulating nico- tine. Both tumor necrosis factor a and interleukin-1/3 are thought o be the most important cytokines involved in the initial events preceding the further development ofinflammation i  the bowel. Although tumor necrosis factor a and interleukin-1/3 are mainly produced by macrophages (Pruimboom et al., 1994), other cells also secrete tumor necrosis factor a. It was clearly shown that tumor necrosis factor a secreting cells are in- creased in the mucosa of inflamed intestine (Breese et al., 1994). Our results offer a possible explanation for the beneficial effects of nicotine in ulcerative colitis. References Breese, E.J., C.A. Michie, S.W. Nicholls, S.H. Murch, C.B. Williams, P. Domizio, J.A. Walker-Smith and T.T. MacDonald, 1994, Tu- mor necrosis factor u-producing cells in the intestinal mucosa of children with inflammatory bowel disease, Gastroenterology 106, 1455. Jones, G.M.M., M. Reith, M.P. Philpot and B.J. Sahakian, 1987, Smoking and dementia of Alzheimer type, J. Neurol. Neurosurg. Psychiatry 50, 1383. Mills, C.M., S.A. Hill and R. Marks, 1993, Altered inflammatory responses in smokers, Br. Med. J. 307, 911. Pessina, G.P., L. Paulesu, F. Corradeschi, E. Luzzi, M. Tanzini, C. Aldinucci, A. Di Stefano and V. Bocci, 1993, Chronic cigarette smoking enhances spontaneous release of turnout necrosis factor- o~ from alveolar macrophages of rats, Mediators Inflamm. 2, 423. Pullan, R.D., J. Rhodes, S. Ganesh, V. Man±, J.S. Morris, G.T. Williams, R.G. Newcombe, M.A.H. Russell, C. Feyerabend, G.A.O. Thomas and U. Siiwe, 1994, Transdermal nicotine for active ulcerative colitis, New Engl. J. Med. 330, 811. Pruimboom, W.M., A.P.M. Van Dijk, C.J.A.M. Tak, I.L. Bonta, J.H.P. Wilson and F.J. Zijlstra, 1994, Interactions between eicosanoids and cytokines: a study using human peritoneal macrophages, Immunol. Lett. 41, 255. Rhodes, J. and G.O.A. Thomas, 1994, Smoking: good or bad for inflammatory bowel disease?, Gastroenterology 106, 807. Zijlstra, F.J., E.D. Srivastava, M. Rhodes, A.P.M. Van Dijk, F. Fogg, H.J. Samson, M. Copeman, M.A.H. Russell, C. Feyerabend, G.T. Williams, R.D. Pullan, G.O.A. Thomas, M. Van Blankenstein, J.H.P. Wilson, A. Allen and J. Rhodes, 1994, Effect of nicotine on rectal mucus and mucosal eicosanoids, Gut 35, 247. 

Nicotine inhibits cytokine synthesis by mouse colonic mucosa

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