Macrophages From Cancer Patients: Analysis of TRAIL, TRAIL Receptors, and Colon Tumor Cell Apoptosis

Jean-Philippe Herbeuval 0 1 Claude Lambert 0 1 Odile Sabido 0 1 Michle Cottier 0 1 Pierre Fournel 0 1 Michel Dy 0 1 Christian Genin ) 0 1 0 Affiliations of authors: J.-P. Herbeuval, C. Lambert, C. Genin (Groupe Im- munite des Muqueuses et Agents Pathogenes [GIMAP]), O. Sabido (Department of Flow Cytometry), M. Cottier (Department of Cytology), P. Fournel (Depart- ment of Pneumology), Jean Monnet University , Saint-Etienne , France; M. Dy , Unite Mixte de Recherche, Hopital Necker , Paris, France. cine Jacques Lisfranc, 15 rue A. Pare, 42023 Saint-Etienne Cedex 2 , France ( 1 Journal of the National Cancer Institute , Vol. 95, No. 8, April 16, 2003 Background: Tumor-infiltrating macrophages secrete cytokines, including Fas ligand, tumor necrosis factor- (TNF- ), and TNF-related apoptosis-inducing ligand (TRAIL). TRAIL induces apoptosis in tumor cells but not in normal cells; however, regulation of TRAIL and its receptors in cancer patients is relatively uncharacterized. We investigated whether macrophages from cancer patients produce TRAIL and whether apoptosis in cultured colon adenocarcinoma cells involves TRAIL and its receptors. Methods: Macrophages isolated from pleural effusions of nine cancer patients and five control patients with congestive heart failure (whose effusions contained no tumor cells) were cultured. Levels of TRAIL, TNF- , interferon , and Fas ligand in conditioned medium were measured by enzyme-linked immunosorbent assays. Apoptosis of human colon adenocarcinoma cell lines, including Colo 205, was determined by the Annexin V method and terminal deoxynucleotidyltransferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL). Cell-surface TRAIL receptors were measured by flow cytometry. Results: Conditioned culture medium from macrophages isolated from pleural effusions containing 1%-5% tumor cells (CM-A) contained TRAIL at 9801300 pg/mL, whereas that from macrophages from pleural effusions containing more than 50% tumor cells or containing no tumor cells (CM-B) contained TRAIL at 0-50 pg/mL. When cultured with medium containing 50% CM-A, 40% (95% confidence interval [CI] = 30% to 50%) of Colo 205 cells underwent apoptosis; when cultured with 50% CM-B, 8% (95% CI = 3% to 13%) underwent apoptosis. When Colo 205 cells were cultured with 50% CM-A, cell-surface expression of TRAIL death receptors DR5 and DR4 increased 13fold and sixfold, respectively, compared with that of untreated Colo 205 cells. Recombinant TRAIL induced 90% (95% CI = 85% to 95%) of Colo 205 cells to undergo apoptosis and acted synergistically with TNF- to induce apoptosis. Conclusion: Macrophages from cancer patients appear to be activated by tumor cells to produce TRAIL and to increase the expression of TRAIL death receptors DR4 and DR5 on tumor cells. [J Natl Cancer Inst 2003;95:611-21] - Tumor infiltration by macrophages directly influences cancer cell growth in vitro and in vivo (13). Tumor-infiltrating macrophages appear to be able to distinguish between malignant and normal cells and can attack neoplastic cells in patients through contact-dependent and cytotoxin-mediated mechanisms (4); the presence of tumor-infiltrating macrophages is associated with good prognosis for patients with colorectal adenocarcinoma (5). However, the story is complicated because such macrophages can secrete a variety of cytokines and thus can promote or inhibit tumor growth, depending on which cytokines are secreted (6). When cytokines of the tumor necrosis factor- (TNF-) family are secreted, as is frequently observed in macrophages that have been exposed to human lung cancer cells (7) or colorectal cancer cells (810), apoptosis is induced. TNF- and Fas ligand (FasL), members of the TNF- family, induce apoptosis by binding to their corresponding cell-surface receptors. The receptor for TNF- is TNF-R1, and the receptor for FasL is Fas. These receptors contain intracytoplasmic domains that, after ligand binding, induce apoptosis in tumor cells (11). TNF-related apoptosis-inducing ligand (TRAIL), another member of the TNF- family (12), does not induce apoptosis in normal cells (13) but does induce apoptosis in several human tumor cell lines (14). Several melanoma cell lines (e.g., WM9, WM35, WM981, WM164, WM793, WM1205-Ln, WM1791-C, and WM3211) are resistant to apoptosis induced by FasL or TNF- but are sensitive to TRAIL-mediated apoptosis (15,16). The following five TRAIL receptors have been described: TRAIL-R1 or death receptor 4 (DR4), TRAIL-R2 or DR5, TRAIL-R3 or decoy receptor 1 (DcR1), TRAIL-R4 or DcR2, and osteoprotegerin (OPG) (17). DR4 and DR5 contain cytoplasmic death domains and induce apoptosis after ligand binding (18,19); DcR1, DcR2, and OPG lack such death domains and do not induce apoptosis (20,21). TRAIL is secreted by immune cells, including lymphocytes, natural killer cells (22), monocytes, dendritic cells (23), and macrophages (24). The expression of decoy receptors for TRAIL in normal tissues, but not in many tumor cell lines, may account for the resistance of normal tissues and for the broad sensitivity of tumor cell lines to TRAIL-induced apoptosis (25). However, the molecular mechanisms involved in the production and regulation of TRAIL and its receptors in vivo remain to be elucidated. An inverse association between the number of macrophages in pleural effusions from cancer patients and the extent of malignant disease has been reported (2628). Our objective in this study was to investigate the cytotoxic effects of macrophages on human tumor cells. We sampled pleural effusions from nine patients with cancer and from five control patients with congestive heart failure to obtain large numbers of macrophages that had been in contact with large or small numbers of tumor cells in pleural effusions from the cancer patients or no tumor cells in pleural effusions from patients with congestive heart failure. We then used human Colo 205, Colo 320, and Caco-2 colon adeno Tumor and Control Cell Collection Pleural fluids containing tumor cells and leukocytes were collected by drainage thoracocentesis from nine patients with effusions related to various cancers (three with pleura mesothelioma, four with lung adenocarcinoma, one with breast adenocarcinoma, and one with colon adenocarcinoma). All cancer patients had stage IV metastatic disease. As a tumor-unrelated control, pleural fluids were also collected from five patients with congestive heart failure, as previously described (29). This in situ control was considered appropriate because the macrophages had not been in contact with tumor cells. Although in vitro adherence of macrophages to a culture dish might have induced the macrophages to produce TRAIL, we did not detect any TRAIL after a 48-hour culture of macrophages from control donors, whereas we did detect the production of TRAIL by macrophages from cancer patients after a 48-hour culture. If the pleural effusion was bloody, red cells (which represented less than 1 (...truncated)