Expression of Cytokines, Inducible Nitric Oxide Synthase, and Matrix Metalloproteinases in Pouchitis: Effects of Probiotic Treatment Salvatore Ulisse, Ph.D., Paolo Gionchetti, M.D., Simona D’Alo `, M.D., F. Paola Russo, Ph.D., Isabella Pesce, B.Sc., Gianluca Ricci, B.Sc., Fernando Rizzello, M.D., Ulf Helwig, M.D., M. Grazia Cifone, Ph.D., Massimo Campieri, M.D., and Claudio De Simone, M.D. Department of Experimental Medicine, University of L’Aquila, L’Aquila; and Department of Internal Medicine and Gastroenterology, University of Bologna, Bologna, Italy OBJECTIVE: The efficacy of probiotic organisms in the treat- ment of pouchitis has been reported. In the present study, we evaluated the tissue levels of pro- and anti-inflammatory cytokines, nitric oxide synthase, and matrix metalloprotein- ases in control and inflamed pouches before and after anti- biotic and probiotic treatment of patients with acute pou- chitis. METHODS: Pouch biopsy samples were obtained from seven patients with pouchitis before and after antibiotic and pro- biotic treatment. Tissue samples from five patients with normal pouches were used as controls. Cytokines were determined by ELISA, matrix metalloproteinase activity was evaluated by zymograms, and nitric oxide synthase activity was determined by measuring arginine to citrulline conversion. RESULTS: Tissue levels of tumor necrosis factor  increased (p  0.01) in pouchitis relative to uninflamed pouches and reduced after antibiotic and probiotic treatment. Also, inter- feron  and interleukin 1 (IL-1) augmented in pouchitis, but their increase did not reach statistical significance. The latter, however, were lower (p  0.05) after treatment with the antibiotics and probiotics. Tissue levels of IL-4 and IL-10 were unchanged in inflamed pouches and unaffected by antibiotic treatment. However, IL-10 increased (p  0.05) after probiotic treatment. Moreover, inflamed pouches had higher levels of inducible nitric oxide synthase and gelatinase activities, which decreased after treatment. CONCLUSIONS: The ability of antibiotic and probiotic treat- ments to increase tissue levels of IL-10, at a higher level than those observed in control pouches, and to decrease, to levels present in control pouches, proinflammatory cyto- kine, inducible nitric oxide synthase, and matrix metallo- proteinase activity may suggest a mechanism of action to explain the efficacy of this therapeutic regime in pouchitis. (Am J Gastroenterol 2001;96:2691–2699. © 2001 by Am. Coll. of Gastroenterology) INTRODUCTION Restorative proctocolectomy with ileal pouch anal anasto- mosis (IPAA) represents the procedure of choice for the surgical management of ulcerative colitis (UC) and familial adenomatous polyposis (FAP) (1–3). This approach responds to patient desire for preservation of transanal defecation and improved postsurgical quality of life with respect to standard surgical approaches such as Brooke ileostomy or the Kock pouch. However, patients operated on with IPAA show a high risk of developing a nonspecific inflammation of the ileal reservoir, known as pouchitis (4, 5). Indeed, the cumulative frequency of pouchitis varies from 15–18% to 46 –50% after 1 and 10 yr, respectively, from IPAA (6 – 8). Clinical symptoms associated with pouchitis include abdominal cramping, diarrhea, fecal urgency, rectal bleeding, malaise, and fever (3, 4). A mucosal biopsy from normal functioning pouches after IPAA shows villous atrophy, crypt hyperplasia, and change in mucin type from sialomucin (produced in the small bowel) to sulfomucin (produced by the large bowel) (9). These changes in the ileal mucosal pouch may be interpreted as an adaptation of the ileal mucosa to the luminal changes (9). Endoscopic analysis of the inflamed pouch shows the presence of edema, granularity, loss of vascular pattern, mucosal hemorrhage, mucus exudate, and ulceration, whereas on histological evaluation of the inflamed pouch biopsy the presence of neutrophil infiltration is evident (9). Recently, in mucosal biopsy specimens from inflamed pouches, an increased CD4:CD8 ratio; increased expression of T-cell activation markers, such as CD25; and an in- creased number of interferon –producing cells, relative to uninflamed pouches or normal ileal mucosa, have been demonstrated (10). In addition, different groups have dem- onstrated, by different experimental approaches, an in- creased expression of proinflammatory cytokines, such as interleukin 1 (IL-1), IL-6, IL-8, and tumor necrosis factor  (TNF-), in the ileal reservoir of patients with pouchitis relative to uninflamed pouches (11, 12). THE AMERICAN JOURNAL OF GASTROENTEROLOGY Vol. 96, No. 9, 2001 © 2001 by Am. Coll. of Gastroenterology ISSN 0002-9270/01/$20.00 Published by Elsevier Science Inc. PII S0002-9270(01)02701-0