Sympathetic nerves and abnormal motility in inflammatory bowel disease (IBD)

CONT TM HP

ACH

COLON +TI'X 5-HT

+TTX

76:t:1

35±5

91±1

50±1

6

*

5

4*

80-22 5 75±1 5

7_+5¢~

75±1 6 96±2 3

86±1 5~ 8~+2 2

40~2

2726

ACH

INTESTINE +TTX 5-HT

+TTX

95±6

69~1

32±8

13±4.

35±4

33_+8~

1~6 •

14±6

The Effect of NO-donorson NOS-expressingMyenteric Neurons Michele Zandecki, Pieter Vanden Berghe, Leen Thielemans, Petra Raeymaekers,Jozef Janssens, Jan Tack, Ctr for gastroenterological research, Leuven Belgium

8*

115± 13 4+2*

35~1

Recently, we demonstrated that intestinal inflammation leads to a post-inflammatory loss of nitrergic myenteric neurons and motility disturbances, suggesting a loss of inhibition (Demerits '00, Kelles '00). During inflammation, upregulation of iNOS generates vast amounts of NO. We aimed to investigate whether high NO concentrations could be responsible for a decrease in nNOS neurons. Methods: Myenteric neuron cultures were prepared from guinea pig small intestine and grown in neuron specific medium (9 days). The NO-donors sodium nitroprusside (SNP) or 3-rnorpbolinosydnonimine (SIN-l) were added during 4-24 hours at 10~ - 10.3M. To revealthe mechanism of action, we studied the effect of the NO-scavengersoxyhaemoglobin and 2-phenyl-4,4,5,5,-tetramethyllimidazoline-3-oxyde-l-oxyl (PTIO), the 02 -scavenger supamxyde dismutase (SOD) and the guanylute cyclase blocker 1H-[1,2,4]oxadiazolo[4,3a]quinoxalin-l-oue (ODQ), incubated alone or simultaneously with SNP. After fixation (4% paraformaldebyde), NOS neurons ware stained by a NADPH-diaphorasereaction and counted par well. The effect of SNP on total neuronal outgrowth and survival was assessed with a semi-quantitative ELISA using an antibody against neuron specific enolase (NSE, 1/2000). All results were normalized and expressed as percentage -+ SEM of control condition. Results: Incubation with SNP for 24 hrs induced no alterations when applied at 10-6, 10.5 M. At higher concentrations, SNP reduced the number of NADPH-d positive cells in a concentrationdependent manner (65_+10, 49_+7, 11_+7 & 1_+1% resp. for 5"10"5, 10-4, 5"10"4 & 10.3M SNP; p<0.01). SIN-1 caused a significant reduction at 10.3 M (18-+8%; p<0,01). Shorter incubation with SNP (4 & 12 hr) did not cause a significant reduction (74-+11 & 84_+14%; NS). The NSE-ELISA revealed no reduction in overall neuronal survival with 10-s & 10" M SNP (107_+5 & 97_+4% respectively; NS). The effect of SNP persisted in the presence of oxyhaemoglobin (10.5 M), PTIO (10~ M), SOD (250 U/ml) or ODQ (10.5M; NS). Incubation with ODQ alone increased the number of NADPH-d neurons (158_+17; p<0.01). Conclusion: The NO-donore SNP and SIN-1 decrease the number of cultured NOS-expressing myenteric neurons in a time and concentration dependent manner, without affecting total neuronal survival. The underlying mechanism dues not seem to involve 02 generation or guaflylate cyclase activation, although a role for the latter in controlling NOS expression in basal conditions is suggested.

0~

0~0~

*p<0.05 vs CONT;@p<0.05vs CONT+TTX

2724 Interleron-a Inhibits Gastric Secretion Through Central NervousSvstom CRF Iw Rats. Gabor Suto, Jozsef Czimmer, Agnes Kiraly, Boglarka Falusi, Sarolta Uodi, Gyula Mozsik, First Dept of Medicine, Univ of P6cs, Pecs Hungary There is an interaction betweenthe immune, endocrine and central nervous system to control gastric functions. Interferon-a (IFN-c~)is a cytokine having antiviral and antitumour properties, and reduces food intake and gastric emptying. Cytokines release brain CRF to alter different gastric functions. Aims were to investigate: (1.) the influence of IFN-a injected intracistemally (i.c.) or subcutaneously (s.c.) on gastric secretion, (2.) the role of central CRF in the inhibition of gastric secretion by i.c. IFN-a. Methods: Pylorus iigation was done under short ether anesthesia in rats (CFY rats, 220-250 g) and the volume (ml) and acid content (mEq/2h) of gastric secretion were measured 2 h later. (1.) IFN-a was injected i.c. (10, 100, 1000 IU/rat) or s.c. (1000, 10.000, 100,000 lU/rat) immediately before pylorus lioation. (2.) IFN-a (100 lU/rat, i.c.) was injected 60, 120 or 360 min before gastric emptying test. (3.) a-belical CRF 9-41 (20/~g/rat, i.c.) was injected immediately before IFN-a (100 iU/rat, i.c.). The volume of injections were 0.1 ml i.v. 5-10 p,I i.c. The control groups were treated by the respective vehicles. Results: (1.) IFN-rx dose-dependently inhibited gastric secretion. The ED~owas 100 IU/rat i.c. and 10.000 lU/rat s.c. (2.) The inhibitory action of IFN-a (100 no/rat, i.c.) on gastric secretion was observed for 120 rain after i.c. injection. (3.) n-belical CRF 9-41 (20 p,g/rat, i.c.) prevents the inhibition of gastric secretion by IFN-a. Conclusions: The inhibition of gastric secretion by IFN-e is mediated by central CRF. These findings provide new insight into the immune-regulation of gastric functions. The work was supported by the grant OTKAT034432.

2727

DNA Mismanay Analysis Reveals Variable Immune And Neuroendocrine 6ene Exprecaine In Chronically Inflamed 6aoglineis Bowel Of Hirschsprung's Disease. Anthony Stallion, Tznyung O. Kou, Kelly A. Miller, Enrique R. Grisoni, David L. Dudgeon, Alan D. Levins, Case Western Reserve Univ Sch of Medicine, Cleveland, OH

Effect of or-helicalCRFg-41on the inhibi~onof gastric secretionby IFN*(x

Volume (ml) Acid output (mEq/2h)

veh+veh

veh+IFN,,,~

CRFA~'FN-a CRFA+veh

4.05±0.46 0.53_+0.04

2.19±0.21+ 0.33_+0.07+

4.57±0.52+ 0 47±0.06+

Hirschspmng's disease is characterized by aganglionosis of various lengths in the distal colon, resulting in chronic obstruction and enterocolitis. A subset of patients also develops enterocol~s after surgical intervention. Our previous report showed differential expression of mRNAs coding for genes involved in mucosal bamer function and neuroendocrine activity, including mucin, intestinal trefoil factor, and vasoactive intestinal protein (VIP). Since a complete analysis of the neuroendocrine system and the immune response in Hirschsprung s disease before and after surgical intervention is unavailable, we initiated a genome-wide scan of Hirschspmng s patients using the microarray assay. METHODS:Aganglionic and ganglionic tissue samples (n = 10) were obtained from patients with Hirschsprung's disease at the time of rectal biopsy or definitive surgical repair. Controls (n = 11) were from patients undergoing rectal biopsy without Hirschsprung's disease,Biotinylated cRNAwas preparedfrom pooled samples for hybridization to oligonucleotide HuGene FI sets (GeneChips,Affymetrix). GeneCltips were analyzed (Fluidics Station, GeneChip3.1 Analysis Suite, Affymetrix) and changes >4-fold over control are reported. RESULTS:Expressionof the heat shock protein gene family was greatest in ganglionic bowel and was significantly increased in aganglionic samples when compared to controls. Expression of vasnactive intestinal peptide and secretograoin geue families was elevated in ganglionic bowel when compared to aganglionic and control bowel. In particular, VIP expression was significantly decreasedin aganglionic samples. Immunoglobulin A (IgA) gene expression was also significantly elevated in both ganglionic and aganglionic samples when compared to controls. CONCLUSIONS:These findings, in need of confirmation, support the presence of ongoing inflammation in the ganglionic bowel, which may lead to increased expression of mucosal protective IgA and repair promoting secretogranin. The decreasedexpression of vasoactive intestinal peptides in aganglionic bowel is consistent with abnormal neumdevelopment of Hirschsprung s segment of bowel. Expansion of these findings will advanceour understanding of the etiology of Hirschsprung's enterocolitis.

4.90-/-080 0.57±0 13

Means±SEM,+=P_<0.05v.s. respectivecontrolgroup, CRFA=cz-helicalCRFg-4120pg/rat i.c., IFN-c~100 IU/rati.c., veh.: vehicle

2726 Sympathetic Nerves end Abnormal Motility in Inflammatory Bowel Disease (IBD) Scott Rehrig, Steve Lawson, Waiter Reed Army Medical Ctr, Washington, DC; Aiping Zhao, Sherry Fleming, USUHS, Bethesda, MD; Daniel Otchy, Waiter Reed Army Medical Ctr, Bethesda, MD; Terez Shea-Donohue, USUHS, Bethesda, MD IBD is a chronic condition with periods of quiescenceinterrupted by disease relapse characterized by abnormal gut motility. Ablation of intestinal sympathetic nervesattenuatesinflammation in animal models of IBD; therefore, we hypothesized that sympathetic nerves contribute to abnormal smooth muscle function observed in IBD patients. Aim: To determine the adrenergic control of smooth muscle contractility in IBD patients. Methods: A prospective non-randomized study was conducted w th eft colon specimens (IBD, n = 3; control, n = 4). Mucosa free smooth muscle strips were suspended in organ baths in the circular axis to measure spontaneous contractions (amplitude and frequency) and smooth muscle responses to nerve stimulation (EFS; 1-20 Hz, 0.1 ms duration, 80V) + / - inhibitor of nitric oxide synthase (L-NNA) or the and/~ adrenergic antagonists, phentolamine and propranolol (P + P). KCL (60 mM) challenge was performed to assess non-receptor mediated smooth muscle contractility. Results: Amplitude was similar in both groups (control= 1.0_+0.1; IBD =0.8-+0.1 N/cm2) and was unaltered by L-NNA or P+P. In controls, frequency was unaltered by L-NNA or P+P. In IBD, the frequency of spontaneous contractions (VEH) was elevated. P+P, but not L-NNA, further increased the frequency in IBD only. IBD decreased responses to EFS. P + P, but not L-NNA, increased EFS responses in IBD to control levels. IBD had no effect on responses to KCI (4+-1 vs 3_+1 N/cm2). Conclusions: In controls and IBD, inhibition of nitrergic nerves had no effect on spontaneous contractions or nerve stimulation. In contrast, inhibition of adrenergic nerves enhanced the frequency of spontaneous contractions and the response to nerve stimulation in IBD only. IBD exhibits upregulation of adrenergic nerves that may be central to the maintenance of abnormal motility in IBD. Spontaneous contractions (FREQ) VEH L-NNA P+P Control IBD

10 ± 5 3 5 ± 7*

16 ± 8 28 ± 8

7± 3 59 ± 6'#t

2728 Lnegterm Immune-Sllmulntion of Enteric Glial Ceils: Planticily and AntiInffammalop/Potential of the Enteric Nervous System (ENS) Anne Ruhl, Simone Franzke, Wolfgang Stremmel, Univ Hospitals, Heidelberg Germany BACKGROUND: Enteric glial cells (EGC) appear to play a central role in the inflammatory response of the neuromuscular layers of the intestinal wall. in our previous work, we have demonstrated that short-term immune-stimulation of EGC induces synthesis and secretion of the key inflammatory cytokine IL-6. However, it has recently been reported that ablation of EGC results in a serious hemorrhagic jejune-ileitis (Bush e.a., Cell 1998) which argues for an anti-intlammatory role of EGC in the intestine. METHODS:To further study the response of EGC in an immune-stimulated environment, adult rat myenteric plexus preparations were enzymatically dissociated, EGCpurified by complement-mediated cytolysis and grown in tissue culture. Cultured cells were stimulated with rat recombinant IL-1/~ (1, 10 and lOOng/mL) for 24 or 168 h followed by extraction of total cellular RNA from half of the cultures. IL-6 mRNA expression was assessed by semiquantitative RT-PCR using GAPDH as house-keeping gene. The remaining cells were rastimulated for 24 or 168 h after 1 or 3 weeks followed by semiquantitative determination of IL-6 mRNA expression. RESULTS:Acute stimulation of EGC with IL-lp for 24 h results in a marked upreguletion of IL-6 expression. In contrast, extended

Nerve Stimulation (EFS) VEH L-NNA P+P 3.4±0.8 1.3±0.3

4.5t-0.9 2.0-I-0.3"

4.1:LO.5 3.9±0..5~

Valuesare means± SEM; EFS is tensionin N/crn2;FREQ= #/10rain; EFS = maximumtensionto 5 Hz; *p<0.05vs CONT;¢ p<0.05 CONTVEH

A-536