APO-1 is a 48-kDa cell-membrane protein identical to the Fas antigen now designated CD95. It is a member of the NGF/TNF receptor superfamily. Anti-APO-1 monoclonal antibody induces apoptosis in a variety of cell types expressing this antigen. We immunohistochemically investigated APO-1 expression in normal colon mucosa, 20 adenomas, 258 colon carcinomas and 10 liver metastases and carried out in vitro studies using a panel of colon-carcinoma cell lines. Immunohistochemically, APO-1 was regularly expressed at the basolateral membrane of normal colon epithelia. In a minor fraction of colon adenomas and in 39.1% of colon carcinomas APO-1 expression was diminished and in 48.1% of carcinomas, predominantly of the non-mucinous type, APO-1 expression was completely abrogated. The normal level of APO-1 in carcinomas was correlated with the mucinous type. Reduced/lost APO-1 expression was more frequent in rectal carcinomas. Complete loss of APO-1 was more frequent in tumors that had already metastasized. APO-1 expression in liver metastases essentially corresponded to that of the primary tumors. Comparative analysis with data from previous studies revealed that the mode of APO-1 expression is correlated with that of HLA-A,B,C./beta 2m, HLA-DR, HLA-D-associated invariant chain and of the secretory component. Surface expression of APO-1 was heterogeneous in colon-carcinoma cell lines; SW480 expressed considerable amounts of APO-1 on all cells, while HT-29 constitutively did less so and only in a minority of cells. Surface density of APO-1 and the fraction of positive cells in HT-29 was enhanced by interferon-gamma (IFN-gamma) and, additively, by tumor necrosis factor-alpha (TNF-alpha), whereas in SW480 APO-1 expression was not modulated by these cytokines. We conclude that neoplastic transformation of colon epithelium often leads to a loss of the physiologic, high level of surface APO-1 by giving rise either to a stable lack of APO-1 or to an IFN-gamma/TNF-alpha-sensitive phenotype of inducible APO-1 expression.

Summary Decay-accelerating-factor (DAF, CD55), a phosphatidyl-inositol anchored glycoprotein, is a member of the cell membrane bound complement regulatory proteins that inhibit autologous complement cascade activation. DAF was found expressed on cells that are in close contact with serum complement proteins, but also on cells outside the vascular space and on tumour cells. Using CD55(BRIC110) and CD55(143-30) we show here that DAF(CD55) is only sporadically expressed on the luminal surface of normal colonic epithelium. However, 5/20 adenomas expressed DAF(CD55) on the cell surface of all tumour cells, 5/20 adenomas were completely negative, 10/20 adenomas expressed DAF(CD55) in various amounts. DAF(CD55) was expressed in various intensities on almost all tumour cells of the colon carcinoma cell line HT29. In 5/88 colorectal carcinomas DAF(CD55) was localised on the apical cell surface of all tumour cells, 31/88 were completely negative, 52/88 expressed DAF(CD55) in parts of their neoplastic populations. There was no correlation between the tumour grading, staging and location and the mode of DAF(CD55) expression, but DAF(CD55) was found more often in mucinous carcinomas (P= 0.007).Although the mode of DAF(CD55) expression is not correlated with tumour prognostic parameters, the upregulation of DAF(CD55) in a subset of adenomas and carcinomas needs further investigation concerning protection of tumour cells against complement cytotoxity.During the complement cascade activation complement fragments can be deposited on autologous cells that are not the desired target (Davitz et al., 1986). In order to avoid the destruction of cells by complement, there are several membrane bound regulatory proteins that inhibit the complement cascade activation (Kinoshita, 1991). One regulatory protein of the C3/C5 convertase is the 'decay accelerating factor' (DAF) (Lublin & Atkinson, 1989). DAF is a phosphatidylinositol anchored cell membrane protein (Low, 1987) with a Mr of 70 kDa on erythrocyte membranes (Nicholson-Weller et al., 1982). The DAF gene is located in the complementregulatory locus on the long arm of chromosome 1 (Lublin et al., 1987). DAF blocks the C3 and C5 convertases of the classical and alternative complement pathway inhibiting the formation and promoting the catabolism of the C3 (C4b2a, C3bBb) and C5 complex (C4b2a3b, C3bBb3b) on autologous cells (Medof et al., 1984;Kinoshita et al., 1985;1986;Fujita et al., 1987;Mold et al., 1990). DAF was first isolated from erythrocyte membranes (Nicholson-Weller et al., 1982) and was also found on cells with close contact to serum complement proteins, on leucocytes, monocytes, platelets (Nicholson-Weller et al., 1985;Berger & Medof, 1987;Davis et al., 1988) and endothelial cells (Asch et al., 1986). Recently DAF was also found in extravascular cells and tissues , in the myocard (ZimInermann et al., 1990), in mesothelial cells, in the epithelium of the urogenital tract (Quigg et al., 1989) and gastrointestinal tract and fibrillar structures of the extracellular matrix (W...

SummaryCD59 (protectin) and CD46 (membrane cofactor protein, MCP) are membrane-bound complement regulator proteins which inhibit complement-mediated cytolysis of autologous cells. CD59, a phosphatidyl-inositol-anchored glycoprotein, inhibits the formation of the terminal membrane attack complex (MAC) of complement and was found to be a second ligand for CD2 contributing to T-cell activation. In 20 colorectal normal mucosa samples, in ten adenomas, 71 carcinomas and in ten liver metastases derived thereof, CD59 was inconsistently expressed in the epithelial compartment. In carcinomas CD59 expression in the whole neoplastic compartment was more often found in well-and moderately differentiated tumours. By contrast, focal expression or even complete lack of CD59 was more often found in poorly differentiated tumours (P = 0.021). In addition, carcinomas without metastases at the time of operation (Dukes A/B) more often expressed CD59 in the entire neoplastic population compared to those carcinomas which had already metastasised (P = 0.018). There was no correlation between the mode of CD59 expression in colorectal carcinomas and the tumour type or location. CD46 has C3b/C4b binding and factor-I dependent cofactor activity and is broadly expressed in various cells and tissues. In the epithelial compartment of normal colorectal mucosa, of all adenomas, carcinomas and their liver metastases, CD46 was expressed throughout the epithelial compartment.Since CD46 was consistently expressed in colorectal carcinomas the low expression or even lack of CD59 in a subset of tumours might not lead to critical complement-mediated attack of CD59-negative tumour cells. Regarding CD59 as a natural T-cell ligand involved in cognate T-cell -target-cell interaction, however, loss of CD59 might well be a selection advantage, provided that tumour antigen-mediated T-cell toxicity in colorectal carcinoma exists.Membrane-bound regulatory proteins protect autologous cells from complement mediated cytotoxicity when fragments of the complement cascade are deposited on host cells which are not the desired target (Davitz, 1986). These proteins regulate the coordinating points in the classic and alternative pathways, the formation of the C3 convertases and the assembly of the terminal membrane attack complex (MAC) (Kinoshita, 1991). One of these proteins which aids in regulating the latter is protectin or CD59 (Hadam, 1989a), an 18-20 kD phosphatidyl-inositol anchored glycoprotein (Meri et al., 1990;Ratnoff et al., 1992). CD59 restricts homologous lysis by binding to the C8 and C9 molecules during the MAC formation, thus disturbing the C8: C9 ratio in the MAC complex (Meri et al., 1990;Lachmann, 1991 Asghar, 1992) where it functions as a second ligand for CD2 (Hahn et al., 1992). One protein which helps regulate the formation of the C3 convertases on autologous cells (Kinoshita, 1991) and the C5 convertase of the alternative complement pathway (Seya et al., 1991) is the 45-70 kD membrane cofactor protein (MCP) (Lublin & Atkinson, 1989) or CD46 (H...

Abstract. Ligation of CD95 (APO-1/Fas) cell surface receptors induces death in apoptosis-sensitive cells. Induction of apoptosis in adherent gamma interferonstimulated HT-29 and COLO 205 colon carcinoma cells by cross-linking CD95 with anti-APO-1 monoclonal antibody resulted in detachment of the cells from hyaluronate starting about i h after antibody exposure. Loss of adhesion was paralleled by a substantial reduction of the multifunctional cell surface adhesion molecule CD44. As evidenced by cycloheximide treatment, this effect was not caused by impaired protein synthesis. Depletion of surface CD44 was also not due to membrane blebbing, since cytochalasin B failed to inhibit ascension from hyaluronate. Instead, ELISA and time kinetics showed increasing amounts of soluble CD44 in the supernatant of CD95-triggered cells. SDS-PAGE revealed that soluble CD44 had an apparent molecular mass of about 20 kD less than CD44 immunoprecipitated from intact cells. Thus, CD95-triggering induced shedding of CD44. Shedding is a novel mechanism operative in early steps of CD95-mediated apoptosis. Shedding surface molecules like CD44 might contribute to the active disintegration of dying epithelial cells in vivo.PoPVos]s is an irreversible intracellular program induced by a variety of internal and external stimuli and which leads to the cell's extinction. Apoptosis does not cause any tissue reaction except phagocytosis (Cohen and Duke, 1992). An early feature of cells undergoing apoptosis in tissues is detachment from neighboring cells or the basement membrane (Searle et al., 1982). This also applies to colonic epithelium where senescent enterocytes undergo apoptosis, detach from the basement membrane, and are released into the gut lumen (Gavrieli et al., 1992; Str~iter et al., 1995). There is good evidence that this phenomenon is maintained in colonic carcinoma cell lines in vitro. Induction of apoptosis in HT-29 cells by factors like transforming growth factor [3, etoposide, or teniposide leads to detachment from the substratum, and cells are found in the supernatant (Oberhammer et al., 1993;Desjardins and MacManus, 1995).CD95 (APO-1/Fas) is a 48-kD cysteine-rich type I transmembrane glycoprotein and is a member of the tumor necrosis factor receptor family (Itoh et al., 1991;Oehm et al., 1992). Upon cross-linking of CD95 by antibody (Trauth et al., 1989;Dhein et al., 1992) or by its natural ligand, CD95-positive cells undergo apoptosis, provided the cells are sensitive. It has been shown that CD95 is expressed at high levels on colonic epithelial cells along the crypt axis and at the mucosal surface (M611er et al., 1994). CD95 is often down-modulated in colon carcinoma in situ and on colon carcinoma cell lines (MOller et al., 1994). Stimulation of cells by gamma interferon (IFN-~), 1 however, increases CD95-expression (MOller et al., 1994) and sensitizes cells for CD95-induced cell death (Yonehara et al., 1989).CD95 ligand is a 40-kD type II membrane protein belonging to the tumor necrosis factor family of cytokines (Suda e...

SUMMARYTransport of major histocompatibility complex (MHC ) class II molecules to the endocytic route is directed by the associated invariant chain (Ii). In the endocytic pathway, Ii is proteolytically cleaved and, upon removal of residual Ii fragments, class II ab dimers are charged with antigenic peptide and recognized by CD4+ T cells. Although distinct peptide-loading compartments such as MIIC (MHC class II loading compartment) and CIIV (MHC class II vesicles) have been characterized in different cells, there is growing evidence of a multitude of subcellular compartments in which antigenic peptide loading takes place. We employed a physiological cellular system in which surface Ii (CD74) and surface human leucocyte antigen (HLA)-DR were induced either alone or in combination. This was achieved by transient exposure of HT-29 cells to recombinant interferon-c (rIFN-c). Using distinct cellular variants, we showed that: (i) the majority of Ii molecules physically associate on the cell membrane with class II dimers to form DR ab5Ii complexes; (ii) the presence of surface Ii is a prerequisite for the rapid uptake of HLA-DRspecific monoclonal antibodies into early endosomes because only the surface DR+/Ii+ phenotype, and not the DR+/Ii− variant, efficiently internalizes; and (iii) the HLA-DR:Ii complexes are targeted to early endosomes, as indicated by co-localization with the GTPase, Rab5, and endocytosed bovine serum albumin. Internalization of HLA-DR5Ii complexes, accommodation of peptides by DR ab heterodimers in early endosomes and recycling to the cell surface may be a mechanism used to increase the peptide repertoire that antigen-presenting cells display to MHC class II-restricted T cells.