- Email: [email protected]
Immunosuppressive effect of budesonide on human lamina propria lymphocytes
Immunopharmacology 38 Ž1998. 279–285
Immunosuppressive effect of budesonide on human lamina propria lymphocytes Yoram Elitsur a
a,)
, Steven N. Lichtman b, Cheryl Neace a , Julie Dosescu c , Jeffrey A. Moshier c
Department of Pediatrics, Pediatric GI DiÕision, Marshall UniÕersity School of Medicine, 1801 Sixth AÕenue, Huntington, WV 25701-0195, USA b DiÕision of Pediatric GI, North Carolina UniÕersity at Chapell Hill, Chapell Hill, NC, USA c VA Medical Research, Wayne State UniÕersity, Detroit, MI, USA Received 28 May 1997; revised 5 August 1997; accepted 5 August 1997
Abstract Budesonide, a beta-adreno-receptor agonist, is comparable to corticosteroid in the treatment of patients with inflammatory bowel disease with the advantage of minimal side effect. Although the immunomodulatory effects of budesonide on the circulatory and respiratory mucosal immune system have been reported, its effect on the human gut immune system has not been published. In this study, the effect of budesonide on the human gut immune system was compared to methyl-prednisolone. The cellular immune function was measured in-vitro by DNA synthesis, ornithine decarboxylase ŽODC. activity and TNFalpha secretion. We found that both drugs have a comparable inhibitory effect on DNA synthesis, ODC activity and suppression of TNFalpha secretion. Exogenous addition of IL-2, did not restore the antiproliferative effect of both drugs. We conclude that budesonide has a comparative suppressive effect to methyl-prednisolone on the gut immune system which is not related to IL-2 secretion. The antiproliferative response may explain the therapeutic effect of budesonide on patients with inflammatory bowel disease. q 1998 Elsevier Science B.V. Keywords: Budesonide; Mucosal immunity; Methyl-prednisolone; Gut immune system
1. Introduction The significant clinical side effects observed in patients treated with corticosteroid led to the introduction of corticosteroid analogues. Budesonide, a beta-adreno-receptor agonist, is one of these analogues which has been successfully used as an alternative to corticosteroid treatment in patients with )
Corresponding author. Tel.: q1-304-6967065; fax: q1-3046967067.
acute asthma ŽKraan et al., 1985; Connett et al., 1993.. More recently, in control clinical trials budesonide was found to be as effective as corticosteroid in patients with inflammatory bowel disease ŽRutgeerts et al., 1994; Greenberg et al., 1996; Lofberg et al., 1996.. Previous studies have shown the immunomodulatory effect of budesonide on the circulatory and the respiratory mucosal immune system ŽBergstrand et al., 1990; Broden and McTavish, 1992; Linden, 1992; Evans et al., 1993; Oddera et al., 1995..
0162-3109r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 2 - 3 1 0 9 Ž 9 7 . 0 0 0 9 0 - 8
280
Y. Elitsur et al.r Immunopharmacology 38 (1998) 279–285
Although the clinical efficacy of budesonide was comparable to that of corticosteroid in patients with inflammatory disease ŽRutgeerts et al., 1994; Lofberg et al., 1996., the mechanism behind this effect is yet unknown. Moreover, the interaction between budesonide and the mucosal immune system, especially with gut immunity, has not been established. The aim of this study was to compare the in vitro effect of budesonide and corticosteroid, methyl-prednisolone, on the human gut immune system as represented by the human colonic lamina propria lymphocyte ŽLPL. DNA synthesis Žthymidine incorporation., ornithine decarboxylase activity ŽODC. and TNFalpha secretion.
2. Materials and methods 2.1. Chemicals and reagents Tissue culture media, fetal calf serum and supplies were purchased from GIBCO ŽGrand Island, NY.. Collagenase, dithiothreitol ŽDTT., EDTA, trypan blue, methyl-prednisolone and other chemicals were purchased from Sigma ŽSt. Louis, MO.. Phytohemagglutinin ŽPHA. was purchased from ICN Immuno Biological ŽLisle, IL.. Recombinant human IL2 was purchased from Genzyme ŽCambridge, MA.. Budesonide was a gift from Astra Draco, Lund. Radioactive materials Žtritiated thymidine Ž83 mCirmmol. and L-14C-ornithine Ž50 mCirmmol.. were purchased from NEN DuPont ŽBoston, MA.. TNFalpha kits were purchased from Genzyme ŽCambridge, MA.. 2.2. Human colon specimens Colon specimens were obtained from patients who underwent bowel resection for colon cancer in two teaching hospitals located in Huntington, WV. Macroscopically and histologically normal colon sections, located at least 5 cm from the diseased area, were used in this study. Colon specimens were transferred in ice-cold RPMI-1640 medium to the laboratory and were processed within 30 min after surgical resection. The study was approved by the local Institutional Human Investigation Committee.
2.3. Mononuclear lymphocyte isolation Human colonic lamina propria lymphocytes were isolated according to Bull and Bookman with modification as previously described ŽBull and Bookman, 1977.. Briefly, colonic mucosa was separated from the muscular layer, cut into small pieces and washed in calcium–magnesium free Hank’s balanced salt solution ŽCMF-HBSS.. The tissue fragments were then treated in dithiothreitol Ž1 mM. and EDTA solution until all epithelial cells were removed. Tissue fragments were washed and treated with RPMI1640 medium containing 2 mM L-glutamine, 25 mM HEPES buffer, antibiotics Ž1% vrv., 15% fetal calf serum and collagenase for overnight digestion Ž378C.. LPL were isolated over Ficoll-Hypaque gradient. Cells were then resuspended in the standard culture solution containing RPMI-1640, antibiotics Ž1% vrv. and 15% AB human serum. LPL preparation was assayed for purity and viability by light microscopy and trypan blue exclusion. In all experiments viability and purity were above 90%. 2.4. LPL proliferation assay PHA Ž20 m grml.-stimulated colonic LPL Ž10 6 cellsrml. in standard culture solution were incubated Ž378C, 5% CO 2 . in a 96-well micro titer plate Ž0.2 mlrwell. in the presence or absence of different concentrations of budesonide and methyl-prednisolone. Control cultures did not contain the tested drugs. Cells were then pulsed with 3 H-thymidine Ž0.5 m Cirwell. and were harvested over glass paper by a cell harvester machine ŽInotech, Biosystem Int... Radioactivity was quantitated in liquid scintillation fluid by beta-counter. Results were expressed as a fraction of control according to the following formula: fraction of control s cpm Ž test. y cpmŽbackground.rcpmŽcontrol. y cpmŽbackground.. Background was defined as the radioactivity count Žcpm. of LPL culture alone, control cpm referred to the radioactivity count of PHA-stimulated LPL alone and test cpm indicated the radioactivity count of PHA-stimulated LPL in the presence of the tested drug. Viability of LPL tested by trypan blue exclusion was above 85% after five days of incubation. In other experiments, exogenous recombinant cytokine IL-2 Ž60 Urml., was added to PHA-stimu-
Y. Elitsur et al.r Immunopharmacology 38 (1998) 279–285
281
lated LPL cultures which were incubated for 5 days. Previous data have suggested that this is the optimal concentrations for this cytokine in LPL proliferation studies ŽElitsur et al., 1995.. Cultures were then pulsed with 3 H-thymidine and were harvested. Radioactive measurement and calculations were performed according to the protocol as described above. In different experiments, we incubated PHAstimulated LPL cultures with budesonide or methylprednisolone Žconc. 10y7 M. for 2 h. Drugs were then washed and the cultures were resuspended in standard culture media with PHA Ž20 m grml. and incubated for 5 days. Cell were then pulsed and harvested over glass filter paper and radioactivity was measured as described above.
The TNF assay was measured in the culture supernatant with a commercial ELISA kit which measures human TNFalpha ŽGenzyme, Cambridge, MA. using a standard sandwich technique. The range of detection is 25 to 1000 pgrml and the coefficient of variance is 10%. Color changes at 405 nm were measured using a Dynetec ELISA reader ŽChantilly, VA..
2.5. Ornithine decarboxylase studies
2.9. Statistical analysis
We have previously shown that ODC activity in stimulated LPL culture peaks after 24 h of incubation ŽElitsur et al., 1990a.. In these experiments PHA-stimulated LPL Ž5 = 10 6r2 ml. in a standard culture medium were incubated Ž378C, 5% CO 2 . in the presence or absence of budesonide and methylprednisolone Žconc. 10y5 M, 10y11 M. for one day. Control cultures did not contain the tested drugs. Cells were then pelleted and stored at y708C until analyzed.
Statistical analysis was done by a two-tailed unpaired Student’s t-test for comparison between the test group and the control. p - 0.05 was considered significant.
2.6. Ornithine decarboxylase assay
Budesonide and methyl-prednisolone have comparable suppressive effects on PHA-stimulated LPL DNA synthesis ŽFig. 1.. Both drugs demonstrated dose–response curves. Results are expressed as a fraction of control as described in Section 2. The data represent the mean " SD of 4–7 different experiments, each done by triplicate determinations. Short time drug-exposure studies Ž2 h exposure. showed a similar inhibitory effect of both drugs on LPLthymidine incorporation Žsuppression of 37% " 0.03 and 46% q 0.33 for budesonide and methyl-prednisolone at concentration 10y7 M, respectively.. The effect of budesonide or methyl-prednisolone on LPL-thymidine incorporation in the presence of exogenous IL-2 is shown in Fig. 2. Results showed a significant suppressive effect on LPL-thymidine incorporation by both drugs irrespective of rIL-2 presence. The data represent the mean " SD of 3–4 different experiments, each done in triplicate. Results
The ODC activity was determined according to a micro-method of Beaven et al. Ž1978., as previously reported by our laboratory ŽElitsur et al., 1993.. Briefly, the cell pellet was homogenized in buffer solution, centrifuged at 100,000 g for 60 min and the supernatant was collected. The ODC activity was measured in the supernatant by a CO 2 releasing assay as previously described elsewhere ŽElitsur et al., 1993.. 2.7. TNF secretion studies Previous experiments showed that TNF secretion from PHA-stimulated LPL peaked at 24 h Ždata not shown.. Accordingly, PHA-stimulated LPL Ž1 = 10 6 cellsrml. in RPMI-1640 culture medium were incubated Ž378C, 5% CO 2 . in culture tubes for 24 h with
the presence of budesonide and methyl-prednisolone Žconc. 10y9 M.. Control cultures did not contain any drug. Cells were then pelleted and the supernatant was stored at y708C until analyzed. 2.8. TNF assay
3. Results 3.1. The effect of budesonide and methyl-prednisolone on LPL proliferation
282
Y. Elitsur et al.r Immunopharmacology 38 (1998) 279–285 Table 1 The effect of Bud.rMP on LPL-ODC activity
Fig. 1. The effect of Bud.rMP on LPL thymidine incorporation. PHA-stimulated LPL were incubated in the presence or absence of Bud.rMP as described in Section 2. Results are expressed as a fraction of control and represent the mean"SD of 4–7 different experiments, each done in triplicate. ) p- 0.05, ) ) p- 0.01.
are presented as a fraction of control as described in Section 2. 3.2. The effect of budesonide and methyl-prednisolone on LPL ODC actiÕity Budesonide and methyl-prednisolone had comparable effects on LPL ODC activity ŽTable 1.. Both
Concentration ŽM.
ODC Žpmol 14 CO 2 r mg proteinrh.
Exp. 1 Control Bud Bud MP MP
0 10y6 10y7 10y6 10y7
1694"131 818"47 1476"117 975"21 909"8
0.001 0.06 0.001 0.001
Exp. 2 Control Bud Bud MP MP
0 10y5 10y1 1 10y5 10y1 1
618"131 221"26 674"21 20"3 1065"55
0.003 0.54 0.000 0.002
Exp. 3 Control Bud Bud MP MP
0 10y5 10y1 1 10y5 10y1 1
607"107 53"18 494"43 16"11 559"33
0.003 0.55 0.001 0.147
p-valuea
Stimulated LPL cultures were incubated for 24 h in the presence or absence of the budesonide or methyl-prednisolone. Control cultures did not contain the tested drugs. ODC activity was measured as described in Section 2. a Compared to control.
drugs significantly suppressed LPL ODC activity at concentrations 10y5 and 10y6 M Ž p - 0.003., but not at lower concentrations Ž10y7 and 10y11 M.. Except for concentration 10y7 M, no differences in ODC suppression were found between both drugs. Results represent the mean " SD of three separate experiments, each done by triplicate determinations.
3.3. The effect of budesonide and methyl-prednisolone on LPL TNF secretion Fig. 2. The effect of Bud.rMP on thymidine incorporation of LPL culture in the presence of exogenous rIL-2. PHA-stimulated LPL were incubated in the presence or absence of Bud.rMP and rIL-2 Ž60 Urml. as described in Section 2. Results are expressed as fraction of control and represent the mean"SD of 3–4 different experiments, each done in triplicate. ) p- 0.05 compared to control.
Budesonide and methyl-prednisolone significantly suppressed TNFalpha secretion from LPL cultures ŽFig. 3.. Approximately 50% suppression was seen by both drugs at concentration 10y9 M. No significant difference was found in TNFalpha inhibition between
Y. Elitsur et al.r Immunopharmacology 38 (1998) 279–285
Fig. 3. The effect of Bud.rMP on LPL TNFalpha secretion. PHA-stimulated LPL were incubated in the presence or absence of Bud.rMP for 24 h. Cells were then pelleted and TNFalpha level was measured in the supernatant as described in Section 2. Results represent the mean"SD of 6 different experiments, each done in duplicate. The data are expressed as the percentage of TNFalpha suppression compared to control. The mean TNFalpha level for control, Bud. Žconc. 10y9 M. and MP Žconc. 10y9 M. were 375, 135 and 230 pgrml, respectively. ) p- 0.001.
the two drugs. Results are the mean " SD of 6 different experiments, each done in duplicate.
4. Discussion Budesonide has been recently shown to have a therapeutic effect similar to that of the standard corticosteroid treatment in adult patients with inflammatory bowel disease ŽRutgeerts et al., 1994; Lofberg et al., 1996.. Previous immunological studies of these drugs in asthmatic patients have suggested that the therapeutic effects of both drugs are closely related to their anti-inflammatory effect on the circulatory or the local mucosal immune system ŽBroden and McTavish, 1992; Linden, 1992; Evans et al., 1993.. We have previously reported that steroids inhibited DNA synthesis into human gut LPL ŽElitsur et al., 1990b.. Others ŽMcKay et al., 1996. have shown that budesonide significantly suppressed 3 Hthymidine incorporation into human peripheral blood mononuclear cells. To our knowledge, there have been no reported data on the effect of budesonide on the gut mucosal immune system. In this study we
283
report that budesonide has a suppressive effect on LPL proliferation comparative to that of methyl-prednisolone and that this effect was not abolished by the presence of exogenous rIL-2. Moreover, exposure of LPL culture to these drugs for 2 h resulted in significant inhibition of DNA synthesis, suggesting that this effect is a receptor-mediated phenomenon located on the cell membrane. Previous data have suggested that corticosteroid inhibited the secretion of IL-2 from stimulated human peripheral Tlymphocytes ŽSnijdewint et al., 1995.. Moreover, exogenous addition of rIL-2 in these experiments partially restored the corticosteroid inhibitory effect suggesting that IL-2, directly or indirectly, participates in the inhibitory mechanism. In this study, we were unable to confirm the ‘rescue’ effect of rIL-2 on stimulated LPL. The difference in the experimental methods may explain this discrepancy. ODC is the first-limiting enzyme in the polyamine biosynthesis pathway ŽRassell and Durie, 1978.. ODC has been found in all eukaryotic cells and has been used as a marker for cell proliferation in normal and premalignant conditions ŽLamuraglia et al., 1986; Porter et al., 1987; Elitsur et al., 1992.. We ŽElitsur et al., 1993. and others ŽScott et al., 1985., have previously reported the importance of ODC enzyme on peripheral blood lymphocytes as well as LPL proliferations. In this study we found that parallel to the drugs suppressive effect on LPL-DNA synthesis, budesonide and methyl-prednisolone significantly decrease ODC activity. Although a non-significant difference was observed between both drugs, similar to clinical studies, methyl-prednisolone had a higher suppressive effect on our in-vitro culture as well as a better reduction of the clinical symptoms of Crohn’s disease ŽRutgeerts et al., 1994.. Such a difference was not observed in LPL-ODC activity in the present in-vitro study. TNFalpha is a macrophage-derived, proinflammatory cytokine, that plays an important role in the initiation, propagation and regulation of the immune response, especially in inflammatory bowel disease ŽBreese et al., 1994; Murata et al., 1995.. Previous data have shown that TNFalpha may be a serologic marker for inflammatory bowel disease activity ŽMurch et al., 1991; Koutroubakis et al., 1995.. Moreover, the specific anti-TNF mononuclear antibody ŽcA2. found to reduce Crohn’s disease activity
284
Y. Elitsur et al.r Immunopharmacology 38 (1998) 279–285
and healed ulcers in patients with steroid-resistant disease ŽVan-Dullemen et al., 1996.. The specific effect of budesonide on this cytokine may thus be important to explain its therapeutic efficacy in patients with inflammatory bowel disease. In this study we found that budesonide and methyl-prednisolone had a similar inhibitory effect on the secretion of TNFalpha from LPL cultures. Others ŽMcKay et al., 1996. have shown that budesonide decrease TNFalpha production from human peripheral mononuclear lymphocyte cultures. These data support the hypothesis that the therapeutic effect of both drugs is partially related to their ability to reduce TNFalpha production from LPL. The therapeutic efficacy of a drug is closely related to its pharmacokinetics and clinical potency. The systemic bioavailability of budesonide is lower than prednisolone ŽEdsbacker et al., 1993. but in contrast, is sustained longer and achieves a higher concentration in the intestinal mucosal ŽMiller-Larsson et al., 1995.. Previous studies in human and animal models have demonstrated a serum level of prednisolone and budesonide at approximately 6 m grml ŽFrey et al., 1994. and 1.2–2.1 nmolrl ŽDanielsson et al., 1993., respectively. The tissue level of prednisolone in the colon and small intestine of monkeys was reported between 0.2–0.8 m grg tissue ŽFrey et al., 1994.. Others ŽVan Den Bosch et al., 1993. reported a budesonide level in human lung from 2.1–8.9 nmolrkg tissue. We report a significant suppressive effect of both drugs at concentrations 10y5 , 10y7 and 10y1 1 M. These concentrations correspond to 3.7 mgrl, 37 m grl and 37 pgrl for prednisolone and 4.3 mgrl, 43 m grl and 43 pgrl for budesonide, respectively. Although these drug levels are in the therapeutic range reported in human, to our knowledge, the gastrointestinal mucosal levels of these drugs in humans have not been published. Accordingly, direct correlation between our data and the clinical response of patients to similar drug concentrations cannot be done. In conclusion, we showed in-vitro that budesonide is comparable to methyl-prednisolone by the anti-inflammatory effect on human LPL as demonstrated by its effect on LPL DNA synthesis, ODC activity, and TNFalpha production. We hypothesize that this data may explain the mechanism behind the thera-
peutic effect of budesonide observed in patients with inflammatory bowel disease. References Beaven, M.A., Wilcox, G., Trepstra, G.K., 1978. A microprocedure for the measurement of 14 CO 2 release from w14 Cxcarboxyl-labeled amino acid. Ann. Biochem. 84, 638. Bergstrand, H., Bjornson, A., Blaschke, E., Brattsand, R., Eklund, A., Larsson, K., Linden, M., 1990. Effects of an inhaled corticosteroid, budesonide, on alveolar macrophage function in smokers. Thorax 45, 362–368. Breese, E.J., Michie, C.A., Nicholls, S.W., Murch, S.H., Williams, C.B., Domizio, P., Walker-Smith, J.A., 1994. Tumor necrosis factor alpha-producing cells in the intestinal mucosa of children with inflammatory bowel disease. Gastroenterology 106, 1455–1466. Broden, R.N., McTavish, D., 1992. Budesonide: An updated review of its pharmacological properties and therapeutic efficacy in asthma and rhinitis. Drugs 44, 375–407. Bull, D.M., Bookman, M.A., 1977. Isolation and functional characterization of human intestinal mucosal lymphoid cells. J. Clin. Invest. 59, 966–974. Connett, G.J., Warde, C., Wooler, E., Lenney, W., 1993. Use of budesonide in severe asthmatics aged 1–3 years. Arch. Dis. Child. 69, 351–355. Danielsson, A., Edsbacker, S., Lofberg, R., Nilsson, A., NymanPantelidis, M., Olsson, O., Suhr, O., Willen, R., 1993. Pharmacokinetics of budesonide enema in patients with distal ulcerative colitis or proctitis. Aliment. Pharmacol. Ther. 7, 403–407. Edsbacker, S., Wollmer, P., Nilsson, A., Nilsson, M., 1993. Pharmacokinetics and gastrointestinal transit of budesonide controlled ileal release ŽCIR. capsule. Gastroenterology 104 ŽSuppl.., A695, Abstract. Elitsur, Y., Bull, A.W., Luk, G.D., 1990a. Modulation of human colonic lamina propria lymphocyte proliferation: Effect of bile acids and oxidized fatty acids. Dig. Dis. Sci. 35, 212–220. Elitsur, Y., Freedland, C.P., Luk, G.D., 1990b. Inhibition of DNA synthesis in human peripheral and lamina propria lymphocytes by 5-aminosalicylic acid and hydrocortisone. Reg. Immunol. 3, 56–61. Elitsur, Y., Moshier, J.A., Murthy, R., Barbish, A., Luk, G.D., 1992. Polyamine levels, ornithine decarboxylase ŽODC. activity, and, ODC mRNA expression in normal and cancerous human colonocytes. Life Sci. 50, 1417–1424. Elitsur, Y., Strom, J., Luk, G.D., 1993. Inhibition of ornithine decarboxylase ŽODC. activity decreases polyamines and suppresses, DNA synthesis in human colonic lamina propria lymphocytes. Immunopharmacology 25, 253–260. Elitsur, Y., Liu, X., Dosescu, J., Moshier, J.A., 1995. FK-506 and cyclosporine ŽCsA. immunomodulation of the human mucosal immune system. Dig. Dis. Sci. 40, 1934–1940. Evans, P.M., O’Conner, B.J., Fuller, R.W., Barnes, P.J., Chung, K.F., 1993. Effect of inhaled corticosteroid on peripheral
Y. Elitsur et al.r Immunopharmacology 38 (1998) 279–285 blood eosinophil counts and density profiles in asthma. J. Allergy Clin. Immunol. 91, 643–650. Frey, F.J., Escher, G., Frey, B.M., 1994. Pharmacology of 11beta-hydroxysteroid dehydrogenase. Steroids 59, 74–79. the Canadian Inflammatory Bowel Disease Study Group, Greenberg, G.R., Feagan, B.G., Martin, F., Sutherland, L.R., Thomson, A.B.R., Williams, C.N., Nilsson, L.G., Persson, T., 1996. Oral budesonide as maintenance treatment for Crohn’s disease: A placebo-controlled, dose-ranging study. Gastroenterology 110, 45–51. Koutroubakis, I., Crusius, J.B.A., Pena, A.S., 1995. Review: Immunogenetics of cytokines, relevance for future research on inflammatory bowel disease. Scand. J. Gastroenterol. 30, 1139–1146. Kraan, J., Koeter, G.H., Van-Der Mark, T.W., Sluiter, H.J., De Vries, K., 1985. Changes in bronchial hyper reactivity induced by 4 weeks of treatment with anti-asthmatic drugs in patients with allergic asthma: A comparison between budesonide and terbutaline. J. Allergy Clin. Immunol. 76, 628–636. Lamuraglia, G.M., Lacaine, R., Malt, R.A., 1986. High ornithine decarboxylase activity and polyamine levels in human colorectal neoplasia. Ann. Surg. 204, 89–93. Linden, M., 1992. The effects of beta-adrenoceptor agonists and corticosteroid, budesonide, on the secretion of inflammatory mediators from monocytes. Br. J. Pharmacol. 107, 156–160. Lofberg, R., Danielsson, A., Suhr, O., Nilsson, A., Schioler, R., Nyberg, A., Hultcrantz, R., Kollberg, B., Gillberg, R., Willen, R., Persson, T., Salde, L., 1996. Oral budesonide versus prednisolone in patients with active extensive and left-sided ulcerative colitis. Gastroenterology 110, 1713–1718. McKay, D.M., Brattsand, R., Wieslander, E., Perdue, M.H., 1996. Budesonide inhibits immune cell activation and epithelial pathophysiology in co-culture studies. Gastroenterology 108, A873, Abstract. Miller-Larsson, A., Gustafsson, B., Brattsand, R., Persson, C.G.A., Draco, A., 1995. Mucosal uptake and retention of steroids in rat ileum: Possible explanation of topical efficacy in inflammatory bowel diseases. Gastroenterology 108, A876. Murata, Y., Ishiguro, Y., Itoh, J., Munakata, A., Yoshida, Y.,
285
1995. The role of proinflammatory and immunoregulatory cytokines in the pathogenesis of ulcerative colitis. J. Gastroentrol. 8, 56–60. Murch, S.H., Lamkin, V.A., Savage, M.O., Walker-Smith, J.A., MacDonald, T.T., 1991. Serum concentrations of tumor necrosis factor alpha in childhood chronic inflammatory bowel disease. Gut 32, 913–917. Oddera, S., Silvestri, M., Sacco, O., Lantero, S., Morelli, M.C., Rossi, G.A., 1995. Effects of ‘systemic’ budesonide concentrations on in vitro allergen-induced activation of blood mononuclear cells isolated from asthmatic patients. Allergy 50, 397– 404. Porter, C.W., Herrera-Ornelas, L., Pera, P., Petrelli, N.F., Mittelman, A., 1987. Polyamine biosynthetic activity in normal and neoplastic human colorectal tissues. Cancer 60, 1275–1281. Rassell, D.H., Durie, B.G.M. ŽEds.., 1978. Polyamines as Biochemical Markers of Normal and Malignant Growth. Raven Press, New York, pp. 43–58. Rutgeerts, P., Lofberg, R., Malchow, H., Lamers, C., Olaison, G., Jewell, D., Danielsson, A., Goebell, H., Thomsen, O., Lorenz-Meyer, H., Hodgson, H., Persson, T., Seidegard, C., 1994. A comparison of budesonide with prednisolone for active Crohn’s disease. N. Engl. J. Med. 331, 842–845. Scott, I.G., Poso, H., Akerman, K.E.O., Andersson, L.C., 1985. Rapid activation of ornithine decarboxylase by mitogenic Žbut not by non-mitogenic. ligands in human T-lymphocytes. Eur. J. Immonol. 15, 783–787. Snijdewint, F.G., Kapsenberg, M.L., Wauben-Penris, P.J., Bos, J.D., 1995. Corticosteroid class-dependently inhibit in vitro Th1- and Th2-type cytokine production. Immunopharmacology 29, 93–101. Van Den Bosch, J.M.M., Westermann, C.J.J., Aumann, J., Edsbacker, S., Tonnesson, M., Selroos, 1993. Relationship between lung tissue and blood plasma concentrations of inhaled budesonide. Biopharm. Drug Dispos. 14, 455–459. Van-Dullemen, H.M., Van Deventer, S.J.H., Hommes, D.W., Bul, H.A., Jansen, J., Tytgat, G.N.J., Woody, J., 1996. Treatment of Crohn’s disease with anti-tumor necrosis factor chimeric monoclonal antibody ŽcA2.. Gastroenterology 109, 129–135.
Immunosuppressive effect of budesonide on human lamina propria lymphocytes Yoram Elitsur a
a,)
, Steven N. Lichtman b, Cheryl Neace a , Julie Dosescu c , Jeffrey A. Moshier c
Department of Pediatrics, Pediatric GI DiÕision, Marshall UniÕersity School of Medicine, 1801 Sixth AÕenue, Huntington, WV 25701-0195, USA b DiÕision of Pediatric GI, North Carolina UniÕersity at Chapell Hill, Chapell Hill, NC, USA c VA Medical Research, Wayne State UniÕersity, Detroit, MI, USA Received 28 May 1997; revised 5 August 1997; accepted 5 August 1997
Abstract Budesonide, a beta-adreno-receptor agonist, is comparable to corticosteroid in the treatment of patients with inflammatory bowel disease with the advantage of minimal side effect. Although the immunomodulatory effects of budesonide on the circulatory and respiratory mucosal immune system have been reported, its effect on the human gut immune system has not been published. In this study, the effect of budesonide on the human gut immune system was compared to methyl-prednisolone. The cellular immune function was measured in-vitro by DNA synthesis, ornithine decarboxylase ŽODC. activity and TNFalpha secretion. We found that both drugs have a comparable inhibitory effect on DNA synthesis, ODC activity and suppression of TNFalpha secretion. Exogenous addition of IL-2, did not restore the antiproliferative effect of both drugs. We conclude that budesonide has a comparative suppressive effect to methyl-prednisolone on the gut immune system which is not related to IL-2 secretion. The antiproliferative response may explain the therapeutic effect of budesonide on patients with inflammatory bowel disease. q 1998 Elsevier Science B.V. Keywords: Budesonide; Mucosal immunity; Methyl-prednisolone; Gut immune system
1. Introduction The significant clinical side effects observed in patients treated with corticosteroid led to the introduction of corticosteroid analogues. Budesonide, a beta-adreno-receptor agonist, is one of these analogues which has been successfully used as an alternative to corticosteroid treatment in patients with )
Corresponding author. Tel.: q1-304-6967065; fax: q1-3046967067.
acute asthma ŽKraan et al., 1985; Connett et al., 1993.. More recently, in control clinical trials budesonide was found to be as effective as corticosteroid in patients with inflammatory bowel disease ŽRutgeerts et al., 1994; Greenberg et al., 1996; Lofberg et al., 1996.. Previous studies have shown the immunomodulatory effect of budesonide on the circulatory and the respiratory mucosal immune system ŽBergstrand et al., 1990; Broden and McTavish, 1992; Linden, 1992; Evans et al., 1993; Oddera et al., 1995..
0162-3109r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 2 - 3 1 0 9 Ž 9 7 . 0 0 0 9 0 - 8
280
Y. Elitsur et al.r Immunopharmacology 38 (1998) 279–285
Although the clinical efficacy of budesonide was comparable to that of corticosteroid in patients with inflammatory disease ŽRutgeerts et al., 1994; Lofberg et al., 1996., the mechanism behind this effect is yet unknown. Moreover, the interaction between budesonide and the mucosal immune system, especially with gut immunity, has not been established. The aim of this study was to compare the in vitro effect of budesonide and corticosteroid, methyl-prednisolone, on the human gut immune system as represented by the human colonic lamina propria lymphocyte ŽLPL. DNA synthesis Žthymidine incorporation., ornithine decarboxylase activity ŽODC. and TNFalpha secretion.
2. Materials and methods 2.1. Chemicals and reagents Tissue culture media, fetal calf serum and supplies were purchased from GIBCO ŽGrand Island, NY.. Collagenase, dithiothreitol ŽDTT., EDTA, trypan blue, methyl-prednisolone and other chemicals were purchased from Sigma ŽSt. Louis, MO.. Phytohemagglutinin ŽPHA. was purchased from ICN Immuno Biological ŽLisle, IL.. Recombinant human IL2 was purchased from Genzyme ŽCambridge, MA.. Budesonide was a gift from Astra Draco, Lund. Radioactive materials Žtritiated thymidine Ž83 mCirmmol. and L-14C-ornithine Ž50 mCirmmol.. were purchased from NEN DuPont ŽBoston, MA.. TNFalpha kits were purchased from Genzyme ŽCambridge, MA.. 2.2. Human colon specimens Colon specimens were obtained from patients who underwent bowel resection for colon cancer in two teaching hospitals located in Huntington, WV. Macroscopically and histologically normal colon sections, located at least 5 cm from the diseased area, were used in this study. Colon specimens were transferred in ice-cold RPMI-1640 medium to the laboratory and were processed within 30 min after surgical resection. The study was approved by the local Institutional Human Investigation Committee.
2.3. Mononuclear lymphocyte isolation Human colonic lamina propria lymphocytes were isolated according to Bull and Bookman with modification as previously described ŽBull and Bookman, 1977.. Briefly, colonic mucosa was separated from the muscular layer, cut into small pieces and washed in calcium–magnesium free Hank’s balanced salt solution ŽCMF-HBSS.. The tissue fragments were then treated in dithiothreitol Ž1 mM. and EDTA solution until all epithelial cells were removed. Tissue fragments were washed and treated with RPMI1640 medium containing 2 mM L-glutamine, 25 mM HEPES buffer, antibiotics Ž1% vrv., 15% fetal calf serum and collagenase for overnight digestion Ž378C.. LPL were isolated over Ficoll-Hypaque gradient. Cells were then resuspended in the standard culture solution containing RPMI-1640, antibiotics Ž1% vrv. and 15% AB human serum. LPL preparation was assayed for purity and viability by light microscopy and trypan blue exclusion. In all experiments viability and purity were above 90%. 2.4. LPL proliferation assay PHA Ž20 m grml.-stimulated colonic LPL Ž10 6 cellsrml. in standard culture solution were incubated Ž378C, 5% CO 2 . in a 96-well micro titer plate Ž0.2 mlrwell. in the presence or absence of different concentrations of budesonide and methyl-prednisolone. Control cultures did not contain the tested drugs. Cells were then pulsed with 3 H-thymidine Ž0.5 m Cirwell. and were harvested over glass paper by a cell harvester machine ŽInotech, Biosystem Int... Radioactivity was quantitated in liquid scintillation fluid by beta-counter. Results were expressed as a fraction of control according to the following formula: fraction of control s cpm Ž test. y cpmŽbackground.rcpmŽcontrol. y cpmŽbackground.. Background was defined as the radioactivity count Žcpm. of LPL culture alone, control cpm referred to the radioactivity count of PHA-stimulated LPL alone and test cpm indicated the radioactivity count of PHA-stimulated LPL in the presence of the tested drug. Viability of LPL tested by trypan blue exclusion was above 85% after five days of incubation. In other experiments, exogenous recombinant cytokine IL-2 Ž60 Urml., was added to PHA-stimu-
Y. Elitsur et al.r Immunopharmacology 38 (1998) 279–285
281
lated LPL cultures which were incubated for 5 days. Previous data have suggested that this is the optimal concentrations for this cytokine in LPL proliferation studies ŽElitsur et al., 1995.. Cultures were then pulsed with 3 H-thymidine and were harvested. Radioactive measurement and calculations were performed according to the protocol as described above. In different experiments, we incubated PHAstimulated LPL cultures with budesonide or methylprednisolone Žconc. 10y7 M. for 2 h. Drugs were then washed and the cultures were resuspended in standard culture media with PHA Ž20 m grml. and incubated for 5 days. Cell were then pulsed and harvested over glass filter paper and radioactivity was measured as described above.
The TNF assay was measured in the culture supernatant with a commercial ELISA kit which measures human TNFalpha ŽGenzyme, Cambridge, MA. using a standard sandwich technique. The range of detection is 25 to 1000 pgrml and the coefficient of variance is 10%. Color changes at 405 nm were measured using a Dynetec ELISA reader ŽChantilly, VA..
2.5. Ornithine decarboxylase studies
2.9. Statistical analysis
We have previously shown that ODC activity in stimulated LPL culture peaks after 24 h of incubation ŽElitsur et al., 1990a.. In these experiments PHA-stimulated LPL Ž5 = 10 6r2 ml. in a standard culture medium were incubated Ž378C, 5% CO 2 . in the presence or absence of budesonide and methylprednisolone Žconc. 10y5 M, 10y11 M. for one day. Control cultures did not contain the tested drugs. Cells were then pelleted and stored at y708C until analyzed.
Statistical analysis was done by a two-tailed unpaired Student’s t-test for comparison between the test group and the control. p - 0.05 was considered significant.
2.6. Ornithine decarboxylase assay
Budesonide and methyl-prednisolone have comparable suppressive effects on PHA-stimulated LPL DNA synthesis ŽFig. 1.. Both drugs demonstrated dose–response curves. Results are expressed as a fraction of control as described in Section 2. The data represent the mean " SD of 4–7 different experiments, each done by triplicate determinations. Short time drug-exposure studies Ž2 h exposure. showed a similar inhibitory effect of both drugs on LPLthymidine incorporation Žsuppression of 37% " 0.03 and 46% q 0.33 for budesonide and methyl-prednisolone at concentration 10y7 M, respectively.. The effect of budesonide or methyl-prednisolone on LPL-thymidine incorporation in the presence of exogenous IL-2 is shown in Fig. 2. Results showed a significant suppressive effect on LPL-thymidine incorporation by both drugs irrespective of rIL-2 presence. The data represent the mean " SD of 3–4 different experiments, each done in triplicate. Results
The ODC activity was determined according to a micro-method of Beaven et al. Ž1978., as previously reported by our laboratory ŽElitsur et al., 1993.. Briefly, the cell pellet was homogenized in buffer solution, centrifuged at 100,000 g for 60 min and the supernatant was collected. The ODC activity was measured in the supernatant by a CO 2 releasing assay as previously described elsewhere ŽElitsur et al., 1993.. 2.7. TNF secretion studies Previous experiments showed that TNF secretion from PHA-stimulated LPL peaked at 24 h Ždata not shown.. Accordingly, PHA-stimulated LPL Ž1 = 10 6 cellsrml. in RPMI-1640 culture medium were incubated Ž378C, 5% CO 2 . in culture tubes for 24 h with
the presence of budesonide and methyl-prednisolone Žconc. 10y9 M.. Control cultures did not contain any drug. Cells were then pelleted and the supernatant was stored at y708C until analyzed. 2.8. TNF assay
3. Results 3.1. The effect of budesonide and methyl-prednisolone on LPL proliferation
282
Y. Elitsur et al.r Immunopharmacology 38 (1998) 279–285 Table 1 The effect of Bud.rMP on LPL-ODC activity
Fig. 1. The effect of Bud.rMP on LPL thymidine incorporation. PHA-stimulated LPL were incubated in the presence or absence of Bud.rMP as described in Section 2. Results are expressed as a fraction of control and represent the mean"SD of 4–7 different experiments, each done in triplicate. ) p- 0.05, ) ) p- 0.01.
are presented as a fraction of control as described in Section 2. 3.2. The effect of budesonide and methyl-prednisolone on LPL ODC actiÕity Budesonide and methyl-prednisolone had comparable effects on LPL ODC activity ŽTable 1.. Both
Concentration ŽM.
ODC Žpmol 14 CO 2 r mg proteinrh.
Exp. 1 Control Bud Bud MP MP
0 10y6 10y7 10y6 10y7
1694"131 818"47 1476"117 975"21 909"8
0.001 0.06 0.001 0.001
Exp. 2 Control Bud Bud MP MP
0 10y5 10y1 1 10y5 10y1 1
618"131 221"26 674"21 20"3 1065"55
0.003 0.54 0.000 0.002
Exp. 3 Control Bud Bud MP MP
0 10y5 10y1 1 10y5 10y1 1
607"107 53"18 494"43 16"11 559"33
0.003 0.55 0.001 0.147
p-valuea
Stimulated LPL cultures were incubated for 24 h in the presence or absence of the budesonide or methyl-prednisolone. Control cultures did not contain the tested drugs. ODC activity was measured as described in Section 2. a Compared to control.
drugs significantly suppressed LPL ODC activity at concentrations 10y5 and 10y6 M Ž p - 0.003., but not at lower concentrations Ž10y7 and 10y11 M.. Except for concentration 10y7 M, no differences in ODC suppression were found between both drugs. Results represent the mean " SD of three separate experiments, each done by triplicate determinations.
3.3. The effect of budesonide and methyl-prednisolone on LPL TNF secretion Fig. 2. The effect of Bud.rMP on thymidine incorporation of LPL culture in the presence of exogenous rIL-2. PHA-stimulated LPL were incubated in the presence or absence of Bud.rMP and rIL-2 Ž60 Urml. as described in Section 2. Results are expressed as fraction of control and represent the mean"SD of 3–4 different experiments, each done in triplicate. ) p- 0.05 compared to control.
Budesonide and methyl-prednisolone significantly suppressed TNFalpha secretion from LPL cultures ŽFig. 3.. Approximately 50% suppression was seen by both drugs at concentration 10y9 M. No significant difference was found in TNFalpha inhibition between
Y. Elitsur et al.r Immunopharmacology 38 (1998) 279–285
Fig. 3. The effect of Bud.rMP on LPL TNFalpha secretion. PHA-stimulated LPL were incubated in the presence or absence of Bud.rMP for 24 h. Cells were then pelleted and TNFalpha level was measured in the supernatant as described in Section 2. Results represent the mean"SD of 6 different experiments, each done in duplicate. The data are expressed as the percentage of TNFalpha suppression compared to control. The mean TNFalpha level for control, Bud. Žconc. 10y9 M. and MP Žconc. 10y9 M. were 375, 135 and 230 pgrml, respectively. ) p- 0.001.
the two drugs. Results are the mean " SD of 6 different experiments, each done in duplicate.
4. Discussion Budesonide has been recently shown to have a therapeutic effect similar to that of the standard corticosteroid treatment in adult patients with inflammatory bowel disease ŽRutgeerts et al., 1994; Lofberg et al., 1996.. Previous immunological studies of these drugs in asthmatic patients have suggested that the therapeutic effects of both drugs are closely related to their anti-inflammatory effect on the circulatory or the local mucosal immune system ŽBroden and McTavish, 1992; Linden, 1992; Evans et al., 1993.. We have previously reported that steroids inhibited DNA synthesis into human gut LPL ŽElitsur et al., 1990b.. Others ŽMcKay et al., 1996. have shown that budesonide significantly suppressed 3 Hthymidine incorporation into human peripheral blood mononuclear cells. To our knowledge, there have been no reported data on the effect of budesonide on the gut mucosal immune system. In this study we
283
report that budesonide has a suppressive effect on LPL proliferation comparative to that of methyl-prednisolone and that this effect was not abolished by the presence of exogenous rIL-2. Moreover, exposure of LPL culture to these drugs for 2 h resulted in significant inhibition of DNA synthesis, suggesting that this effect is a receptor-mediated phenomenon located on the cell membrane. Previous data have suggested that corticosteroid inhibited the secretion of IL-2 from stimulated human peripheral Tlymphocytes ŽSnijdewint et al., 1995.. Moreover, exogenous addition of rIL-2 in these experiments partially restored the corticosteroid inhibitory effect suggesting that IL-2, directly or indirectly, participates in the inhibitory mechanism. In this study, we were unable to confirm the ‘rescue’ effect of rIL-2 on stimulated LPL. The difference in the experimental methods may explain this discrepancy. ODC is the first-limiting enzyme in the polyamine biosynthesis pathway ŽRassell and Durie, 1978.. ODC has been found in all eukaryotic cells and has been used as a marker for cell proliferation in normal and premalignant conditions ŽLamuraglia et al., 1986; Porter et al., 1987; Elitsur et al., 1992.. We ŽElitsur et al., 1993. and others ŽScott et al., 1985., have previously reported the importance of ODC enzyme on peripheral blood lymphocytes as well as LPL proliferations. In this study we found that parallel to the drugs suppressive effect on LPL-DNA synthesis, budesonide and methyl-prednisolone significantly decrease ODC activity. Although a non-significant difference was observed between both drugs, similar to clinical studies, methyl-prednisolone had a higher suppressive effect on our in-vitro culture as well as a better reduction of the clinical symptoms of Crohn’s disease ŽRutgeerts et al., 1994.. Such a difference was not observed in LPL-ODC activity in the present in-vitro study. TNFalpha is a macrophage-derived, proinflammatory cytokine, that plays an important role in the initiation, propagation and regulation of the immune response, especially in inflammatory bowel disease ŽBreese et al., 1994; Murata et al., 1995.. Previous data have shown that TNFalpha may be a serologic marker for inflammatory bowel disease activity ŽMurch et al., 1991; Koutroubakis et al., 1995.. Moreover, the specific anti-TNF mononuclear antibody ŽcA2. found to reduce Crohn’s disease activity
284
Y. Elitsur et al.r Immunopharmacology 38 (1998) 279–285
and healed ulcers in patients with steroid-resistant disease ŽVan-Dullemen et al., 1996.. The specific effect of budesonide on this cytokine may thus be important to explain its therapeutic efficacy in patients with inflammatory bowel disease. In this study we found that budesonide and methyl-prednisolone had a similar inhibitory effect on the secretion of TNFalpha from LPL cultures. Others ŽMcKay et al., 1996. have shown that budesonide decrease TNFalpha production from human peripheral mononuclear lymphocyte cultures. These data support the hypothesis that the therapeutic effect of both drugs is partially related to their ability to reduce TNFalpha production from LPL. The therapeutic efficacy of a drug is closely related to its pharmacokinetics and clinical potency. The systemic bioavailability of budesonide is lower than prednisolone ŽEdsbacker et al., 1993. but in contrast, is sustained longer and achieves a higher concentration in the intestinal mucosal ŽMiller-Larsson et al., 1995.. Previous studies in human and animal models have demonstrated a serum level of prednisolone and budesonide at approximately 6 m grml ŽFrey et al., 1994. and 1.2–2.1 nmolrl ŽDanielsson et al., 1993., respectively. The tissue level of prednisolone in the colon and small intestine of monkeys was reported between 0.2–0.8 m grg tissue ŽFrey et al., 1994.. Others ŽVan Den Bosch et al., 1993. reported a budesonide level in human lung from 2.1–8.9 nmolrkg tissue. We report a significant suppressive effect of both drugs at concentrations 10y5 , 10y7 and 10y1 1 M. These concentrations correspond to 3.7 mgrl, 37 m grl and 37 pgrl for prednisolone and 4.3 mgrl, 43 m grl and 43 pgrl for budesonide, respectively. Although these drug levels are in the therapeutic range reported in human, to our knowledge, the gastrointestinal mucosal levels of these drugs in humans have not been published. Accordingly, direct correlation between our data and the clinical response of patients to similar drug concentrations cannot be done. In conclusion, we showed in-vitro that budesonide is comparable to methyl-prednisolone by the anti-inflammatory effect on human LPL as demonstrated by its effect on LPL DNA synthesis, ODC activity, and TNFalpha production. We hypothesize that this data may explain the mechanism behind the thera-
peutic effect of budesonide observed in patients with inflammatory bowel disease. References Beaven, M.A., Wilcox, G., Trepstra, G.K., 1978. A microprocedure for the measurement of 14 CO 2 release from w14 Cxcarboxyl-labeled amino acid. Ann. Biochem. 84, 638. Bergstrand, H., Bjornson, A., Blaschke, E., Brattsand, R., Eklund, A., Larsson, K., Linden, M., 1990. Effects of an inhaled corticosteroid, budesonide, on alveolar macrophage function in smokers. Thorax 45, 362–368. Breese, E.J., Michie, C.A., Nicholls, S.W., Murch, S.H., Williams, C.B., Domizio, P., Walker-Smith, J.A., 1994. Tumor necrosis factor alpha-producing cells in the intestinal mucosa of children with inflammatory bowel disease. Gastroenterology 106, 1455–1466. Broden, R.N., McTavish, D., 1992. Budesonide: An updated review of its pharmacological properties and therapeutic efficacy in asthma and rhinitis. Drugs 44, 375–407. Bull, D.M., Bookman, M.A., 1977. Isolation and functional characterization of human intestinal mucosal lymphoid cells. J. Clin. Invest. 59, 966–974. Connett, G.J., Warde, C., Wooler, E., Lenney, W., 1993. Use of budesonide in severe asthmatics aged 1–3 years. Arch. Dis. Child. 69, 351–355. Danielsson, A., Edsbacker, S., Lofberg, R., Nilsson, A., NymanPantelidis, M., Olsson, O., Suhr, O., Willen, R., 1993. Pharmacokinetics of budesonide enema in patients with distal ulcerative colitis or proctitis. Aliment. Pharmacol. Ther. 7, 403–407. Edsbacker, S., Wollmer, P., Nilsson, A., Nilsson, M., 1993. Pharmacokinetics and gastrointestinal transit of budesonide controlled ileal release ŽCIR. capsule. Gastroenterology 104 ŽSuppl.., A695, Abstract. Elitsur, Y., Bull, A.W., Luk, G.D., 1990a. Modulation of human colonic lamina propria lymphocyte proliferation: Effect of bile acids and oxidized fatty acids. Dig. Dis. Sci. 35, 212–220. Elitsur, Y., Freedland, C.P., Luk, G.D., 1990b. Inhibition of DNA synthesis in human peripheral and lamina propria lymphocytes by 5-aminosalicylic acid and hydrocortisone. Reg. Immunol. 3, 56–61. Elitsur, Y., Moshier, J.A., Murthy, R., Barbish, A., Luk, G.D., 1992. Polyamine levels, ornithine decarboxylase ŽODC. activity, and, ODC mRNA expression in normal and cancerous human colonocytes. Life Sci. 50, 1417–1424. Elitsur, Y., Strom, J., Luk, G.D., 1993. Inhibition of ornithine decarboxylase ŽODC. activity decreases polyamines and suppresses, DNA synthesis in human colonic lamina propria lymphocytes. Immunopharmacology 25, 253–260. Elitsur, Y., Liu, X., Dosescu, J., Moshier, J.A., 1995. FK-506 and cyclosporine ŽCsA. immunomodulation of the human mucosal immune system. Dig. Dis. Sci. 40, 1934–1940. Evans, P.M., O’Conner, B.J., Fuller, R.W., Barnes, P.J., Chung, K.F., 1993. Effect of inhaled corticosteroid on peripheral
Y. Elitsur et al.r Immunopharmacology 38 (1998) 279–285 blood eosinophil counts and density profiles in asthma. J. Allergy Clin. Immunol. 91, 643–650. Frey, F.J., Escher, G., Frey, B.M., 1994. Pharmacology of 11beta-hydroxysteroid dehydrogenase. Steroids 59, 74–79. the Canadian Inflammatory Bowel Disease Study Group, Greenberg, G.R., Feagan, B.G., Martin, F., Sutherland, L.R., Thomson, A.B.R., Williams, C.N., Nilsson, L.G., Persson, T., 1996. Oral budesonide as maintenance treatment for Crohn’s disease: A placebo-controlled, dose-ranging study. Gastroenterology 110, 45–51. Koutroubakis, I., Crusius, J.B.A., Pena, A.S., 1995. Review: Immunogenetics of cytokines, relevance for future research on inflammatory bowel disease. Scand. J. Gastroenterol. 30, 1139–1146. Kraan, J., Koeter, G.H., Van-Der Mark, T.W., Sluiter, H.J., De Vries, K., 1985. Changes in bronchial hyper reactivity induced by 4 weeks of treatment with anti-asthmatic drugs in patients with allergic asthma: A comparison between budesonide and terbutaline. J. Allergy Clin. Immunol. 76, 628–636. Lamuraglia, G.M., Lacaine, R., Malt, R.A., 1986. High ornithine decarboxylase activity and polyamine levels in human colorectal neoplasia. Ann. Surg. 204, 89–93. Linden, M., 1992. The effects of beta-adrenoceptor agonists and corticosteroid, budesonide, on the secretion of inflammatory mediators from monocytes. Br. J. Pharmacol. 107, 156–160. Lofberg, R., Danielsson, A., Suhr, O., Nilsson, A., Schioler, R., Nyberg, A., Hultcrantz, R., Kollberg, B., Gillberg, R., Willen, R., Persson, T., Salde, L., 1996. Oral budesonide versus prednisolone in patients with active extensive and left-sided ulcerative colitis. Gastroenterology 110, 1713–1718. McKay, D.M., Brattsand, R., Wieslander, E., Perdue, M.H., 1996. Budesonide inhibits immune cell activation and epithelial pathophysiology in co-culture studies. Gastroenterology 108, A873, Abstract. Miller-Larsson, A., Gustafsson, B., Brattsand, R., Persson, C.G.A., Draco, A., 1995. Mucosal uptake and retention of steroids in rat ileum: Possible explanation of topical efficacy in inflammatory bowel diseases. Gastroenterology 108, A876. Murata, Y., Ishiguro, Y., Itoh, J., Munakata, A., Yoshida, Y.,
285
1995. The role of proinflammatory and immunoregulatory cytokines in the pathogenesis of ulcerative colitis. J. Gastroentrol. 8, 56–60. Murch, S.H., Lamkin, V.A., Savage, M.O., Walker-Smith, J.A., MacDonald, T.T., 1991. Serum concentrations of tumor necrosis factor alpha in childhood chronic inflammatory bowel disease. Gut 32, 913–917. Oddera, S., Silvestri, M., Sacco, O., Lantero, S., Morelli, M.C., Rossi, G.A., 1995. Effects of ‘systemic’ budesonide concentrations on in vitro allergen-induced activation of blood mononuclear cells isolated from asthmatic patients. Allergy 50, 397– 404. Porter, C.W., Herrera-Ornelas, L., Pera, P., Petrelli, N.F., Mittelman, A., 1987. Polyamine biosynthetic activity in normal and neoplastic human colorectal tissues. Cancer 60, 1275–1281. Rassell, D.H., Durie, B.G.M. ŽEds.., 1978. Polyamines as Biochemical Markers of Normal and Malignant Growth. Raven Press, New York, pp. 43–58. Rutgeerts, P., Lofberg, R., Malchow, H., Lamers, C., Olaison, G., Jewell, D., Danielsson, A., Goebell, H., Thomsen, O., Lorenz-Meyer, H., Hodgson, H., Persson, T., Seidegard, C., 1994. A comparison of budesonide with prednisolone for active Crohn’s disease. N. Engl. J. Med. 331, 842–845. Scott, I.G., Poso, H., Akerman, K.E.O., Andersson, L.C., 1985. Rapid activation of ornithine decarboxylase by mitogenic Žbut not by non-mitogenic. ligands in human T-lymphocytes. Eur. J. Immonol. 15, 783–787. Snijdewint, F.G., Kapsenberg, M.L., Wauben-Penris, P.J., Bos, J.D., 1995. Corticosteroid class-dependently inhibit in vitro Th1- and Th2-type cytokine production. Immunopharmacology 29, 93–101. Van Den Bosch, J.M.M., Westermann, C.J.J., Aumann, J., Edsbacker, S., Tonnesson, M., Selroos, 1993. Relationship between lung tissue and blood plasma concentrations of inhaled budesonide. Biopharm. Drug Dispos. 14, 455–459. Van-Dullemen, H.M., Van Deventer, S.J.H., Hommes, D.W., Bul, H.A., Jansen, J., Tytgat, G.N.J., Woody, J., 1996. Treatment of Crohn’s disease with anti-tumor necrosis factor chimeric monoclonal antibody ŽcA2.. Gastroenterology 109, 129–135.