Increased expression of decay-accelerating factor (CD55) in the inflamed mucosa of patients with ulcerative colitis

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1 Pathophysiology 5 (1998) Increased expression of decay-accelerating factor (CD55) in the inflamed mucosa of patients with ulcerative colitis Akira Andoh *, Yoshihide Fujiyama, Toshio Kimura, Takao Saotome, Tomoyuki Tsujikawa, Masaya Sasaki, Shigeki Koyama, Tadao Bamba Department of Internal Medicine, Shiga Uni ersity of Medical Science, Seta Tukinowa, Otsu , Japan Received 8 October 1997; received in revised form 1 December 1997; accepted 8 December 1997 Abstract Decay-accelerating factor (DAF) protects host tissue from the attack of autologous complement activation. To evaluate the participation of complement-mediated immune responses in the pathogenesis of ulcerative colitis (UC), we performed an immunohistochemical examination of DAF expression in the colonic mucosa of patients with UC. In normal colonic mucosa, DAF expression was negligible on the epithelium. However, weak expression was sporadically detected on the epithelium at inactive lesion of UC patients. In the inflamed mucosa of active UC patients, DAF expression was markedly enhanced as compared with normal mucosa. DAF was detected at the luminal surface of the mucosa (apical surface of the epithelium). This expression was stronger at the upper part of the crypt than the lower part of the crypt. Our results indicate that activity to protect host tissues from complement activation is enhanced in the inflamed mucosa of UC patients, suggesting that complement-mediated immune responses may be involved in the pathogenesis of this chronic inflammatory bowel disease of unknown etiology Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Complement; Cytokine; DAF 1. Introduction * Corresponding author. Tel.: ; fax: ; andoh@belle.shiga-med.ac.jp Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) of unknown etiology. Previous studies have focused on evidence of local complement activation in the inflamed mucosa of UC patients [1,2], suggesting that complement activation may be involved in the pathogenesis of this chronic bowel disorder. Normal human cells are protected from damage by autologous complement activation through the activity of several cell-associated complement regulatory proteins [3]. Decay-accelerating factor (DAF; CD55) is one of these proteins which is anchored to the cell surfaces by a glycosyl phosphatidylinositol linkage. DAF prevents the assembly and accelerates the dissociation of autologous C3 (C4b2a and C3bBb) and C5 convertases (C4b2a3b and C3bBb3b) [3 5], thus efficiently interrupting the amplification steps of both the classical and alternative complement activation pathways on the host cell surface. In previous studies, it has been demonstrated that DAF is broadly distributed on the plasma membranes of all cell types, including peripheral blood cells and endothelial cells, which are in intimate contact with the plasma complement proteins [3 5]. Furthermore, DAF is expressed on the epithelial cells derived from various tissues lining extracellular compartments. In the human intestinal tract, DAF has been localized to the apical surfaces of epithelial cells [5,6]. As shown recently, the hepatobiliary system and the exocrine pancreas supply large amounts of active complement components into the intestinal tracts [7 9]. In addition, complement components are also locally synthesized and secreted by intestinal epithelial cells /98/$ - see front matter 1998 Published by Elsevier Science Ireland Ltd. All rights reserved. PII S (97)

2 106 A. Andoh et al. / Pathophysiology 5 (1998) Fig. 1. Expression of decay-accelerating factor (DAF) in normal colonic mucosa. DAF was detected in some leukocytes resident in the lamina propria, fibroblasts, and fibrillar structures particularly in the submucosa (A, 200; B, 200; C, 250).

3 A. Andoh et al. / Pathophysiology 5 (1998) [10 14]. So, DAF may protect intestinal epithelial cells from autologous complement activation at the luminal surfaces of the mucosa. With respect to the immunologic regulatory mechanisms of DAF expression in intestinal epithelial cells, we have recently found that interleukin-4 (IL-4) and tumor necrosis factor (TNF)- are potent inducers of DAF mrna and protein [15,16], suggesting that DAF expression may be modulated at the inflamed mucosa of UC patients. In this study, to evaluate the alteration of DAF protein expression in vivo, we performed an immunohistochemical examination of DAF expression in the diseased mucosa of UC patients. 2. Materials and methods Tissue samples tested were obtained endoscopically from 10 UC patients at the hospital of the Shiga University of Medical Science. We evaluated 10 biopsy samples from endoscopically active lesions and five samples from inactive lesions. Five normal control colorectal tissues were also obtained by endoscopic biopsy. Informed consent was obtained from each patient. Samples were fixed in periodate-lysin-paraformaldehyde fixative [17], and cryostat sections were stained by the indirect peroxidase-labeled antibody method. Mouse monoclonal anti-human DAF antibody (IA10; Pharmingen, San Diego, CA) was used as the first antibody. After incubation with the first antibody, the sections were reacted with 0.1% H 2 O 2 and 0.1% phenylhydralazine (Nakalai, Kyoto, Japan) in 0.1 M PBS to block endogenous peroxidase activity. Then, sections were reacted with biotin-conjugated rabbit anti-mouse IgG (Vector, Burlingame, CA), and with avidin-biotinperoxidase complex (ABC; Vector). Color development was performed with 0.05 M Tris HCl buffer containing 0.01% diaminobentidine, 1% ammonium nickel sulfate, and % H 2 O 2. positive for DAF (Fig. 3C). DAF expression was stronger in the upper parts of the crypt, as compared with expression in the lower parts (Fig. 3E). These observations were detected in all samples obtained from active lesions of UC patients. 4. Discussion Previous studies have demonstrated that local complement activation is involved in the pathogenesis of chronic inflammatory bowel diseases [1,2]. For example, epithelial deposition of activated complement components has been reported in UC patient [1]. However, to our knowledge, little is known about the changes in DAF expression in this disorder. Recently, we found that DAF expression in intestinal epithelial cell lines was markedly enhanced by the addition of pro- and 3. Results In normal colonic mucosa, DAF expression was negligible. After sufficient color development we detected DAF expression in some leukocytes resident in the lamina propria, fibroblasts, and fibrillar structures particularly in the submucosa (Fig. 1A, B, and C), but did not detect on epithelial cells. In contrast, DAF expression was weakly detectable in inactive lesions of UC patients, where DAF was expressed as small foci on the luminal surface of the epithelium (Fig. 2A and B). In the inflamed mucosa of active UC patients, DAF was strongly expressed on the luminal surfaces of mucosa (apical surface of epithelial cells) (Fig. 3A, B and D). Leukocytes composing the crypt abscess were weakly Fig. 2. DAF expression in inactive lesions of UC patients. DAF was expressed as small foci on the luminal surface of the epithelium (A, 200; B, 250).

4 108 A. Andoh et al. / Pathophysiology 5 (1998) Fig. 3. DAF expression in inflamed mucosa of active UC patients. DAF was strongly expressed on the luminal surfaces of mucosa (apical surface of epithelial cells) (A, 200; B, 250; C, 300; D, 200). Leukocytes composing the crypt abscess were weakly positive for DAF (C). DAF expression was stronger at the upper parts of the crypt as compared with the lower parts of the crypt (E, 200).

5 A. Andoh et al. / Pathophysiology 5 (1998) anti-inflammatory cytokines, such as TNF- and IL-4 [15,16]. These cytokines synergistically increased DAF protein expression as early as 24 h after the start of incubation [16]. Because the physiological importance of TNF- and IL-4 is evident in the development of IBD [18,19], these findings prompted us to speculate that DAF expression may be altered in the inflamed mucosa of IBD patients. In this study, we demonstrated that epithelial expression of DAF protein is strongly enhanced in the inflamed mucosa of UC patients. Although we do not have in vivo evidence of the exact role of either IL-4 or TNF- in DAF induction, it has become clear that DAF expression in intestinal epithelial cells can be up-regulated by various pathophysiological conditions, resulting in an increase in local complement defense activity. Local complement secretion in the human intestine has been clinically demonstrated by Ahrenstedt et al. [20]. With respect to local sites of complement biosynthesis, we and other investigators have reported the secretion of complement components by human intestinal epithelial cells [10 14]. Furthermore, the hepatobiliary system and the exocrine pancreas supply large amounts of active complement components into the intestinal tract [7 9]. Therefore, DAF protein expressed in intestinal epithelial cells may play an important role in the protection of intestinal epithelial cells from the activation of an autologous complement cascade at the luminal surface. Furthermore, in inflamed lesions of the intestinal mucosa, local activation of the complement cascade may be enhanced. In order to increase the anti-complement activity, the responses of epithelial cells to inflammatory stimuli such as cytokines may target the increased complement activation at the luminal surfaces. In the inflamed mucosa of UC patients, DAF expression was also detected in some leukocytes. It has been reported that DAF expression on leukocytes is altered in accordance with their differentiation stage, activation state and phenotypes [21]. However, the exact mechanism mediating DAF expression in leukocytes has not been identified. It is likely that several cytokines elevated in the inflamed mucosa also affected DAF expression in leukocytes. Since important roles of leukocytes, such as neutrophils, in the pathogenesis of UC have been reported, further studies are required to evaluate the correlation between DAF expression in leukocytes and the degree of mucosal inflammation. In conclusion, these data indicate that epithelial expression of DAF protein is markedly enhanced in the inflamed mucosa of patients with UC. Based on the results of our recent studies of cytokine regulation of DAF expression, it may be possible to explain some of the mechanisms by which DAF expression is induced. 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