Primary and malignant cholangiocytes undergo CD40 mediated Fas dependent Apoptosis, but are insensitive to direct activation with exogenous fas ligand

Introduction Cholangiocarcinoma is a rare malignancy of the biliary tract, the incidence of which is rising, but the pathogenesis of which remains uncertain. No common genetic defects have been described but it is accepted that chronic inflammation is an important contributing factor. We have shown that primary human cholangiocyte and hepatocyte survival is tightly regulated via co-operative interactions between two tumour necrosis family (TNF) receptor family members; CD40 and Fas (CD95). Functional deficiency of CD154, the ligand for CD40, leads to a failure of clearance of biliary tract infections and a predisposition to cholangiocarcinoma implying a direct link between TNF receptor-mediated apoptosis and the development of cholangiocarcinoma. Aims To determine whether malignant cholangiocytes display defects in CD40 mediated apoptosis. By comparing CD40 and Fas-mediated apoptosis and intracellular signalling in primary human cholangiocytes and three cholangiocyte cell lines. Results Primary cholangiocytes and cholangiocyte cell lines were relatively insensitive to direct Fas-mediated killing with exogenous FasL when compared with Jurkat cells, which readily underwent Fas-mediated apoptosis, but were extremely sensitive to CD154 stimulation. The sensitivity of cells to CD40 activation was similar in magnitude in both primary and malignant cells and was STAT-3 and AP-1 dependent in both. Conclusions 1) Both primary and malignant cholangiocytes are relatively resistant to Fas–mediated killing but show exquisite sensitivity to CD154, suggesting that the CD40 pathway is intact and fully functional in both primary and malignant cholangiocytes 2) The relative insensitivity of cholangiocytes to Fas activation demonstrates the importance of CD40 augmentation of Fas dependent death in these cells. Agonistic therapies which target CD40 and associated intracellular signalling pathways may be effective in promoting apoptosis of malignant cholangiocytes

cIAP-1 Controls Innate Immunity to C. pneumoniae Pulmonary Infection

The resistance of epithelial cells infected with Chlamydophila pneumoniae for apoptosis has been attributed to the induced expression and increased stability of anti-apoptotic proteins called inhibitor of apoptosis proteins (IAPs). The significance of cellular inhibitor of apoptosis protein-1 (cIAP-1) in C. pneumoniae pulmonary infection and innate immune response was investigated in cIAP-1 knockout (KO) mice using a novel non-invasive intra-tracheal infection method. In contrast to wildtype, cIAP-1 knockout mice failed to clear the infection from their lungs. Wildtype mice responded to infection with a strong inflammatory response in the lung. In contrast, the recruitment of macrophages was reduced in cIAP-1 KO mice compared to wildtype mice. The concentration of Interferon gamma (IFN-γ) was increased whereas that of Tumor Necrosis Factor (TNF-α) was reduced in the lungs of infected cIAP-1 KO mice compared to infected wildtype mice. Ex vivo experiments on mouse peritoneal macrophages and splenocytes revealed that cIAP-1 is required for innate immune responses of these cells. Our findings thus suggest a new immunoregulatory role of cIAP-1 in the course of bacterial infection

Therapeutic T cells induce tumor-directed chemotaxis of innate immune cells through tumor-specific secretion of chemokines and stimulation of B16BL6 melanoma to secrete chemokines

Background: The mechanisms by which tumor-specific T cells induce regression of established metastases are not fully characterized. In using the poorly immunogenic B16BL6-D5 (D5) melanoma model we reported that T cell-mediated tumor regression can occur independently of perforin, IFN-gamma or the combination of both. Characterization of regressing pulmonary metastases identified macrophages as a major component of the cells infiltrating the tumor after adoptive transfer of effector T cells. This led us to hypothesize that macrophages played a central role in tumor regression following T-cell transfer. Here, we sought to determine the factors responsible for the infiltration of macrophages at the tumor site. Methods: These studies used the poorly immunogenic D5 melanoma model. Tumor-specific effector T cells, generated from tumor vaccine-draining lymph nodes (TVDLN), were used for adoptive immunotherapy and in vitro analysis of chemokine expression. Cellular infiltrates into pulmonary metastases were determined by immunohistochemistry. Chemokine expression by the D5 melanoma following co-culture with T cells, IFN-gamma or TNF-alpha was determined by RT-PCR and ELISA. Functional activity of chemokines was confirmed using a macrophage migration assay. T cell activation of macrophages to release nitric oxide (NO) was determined using GRIES reagent. Results: We observed that tumor-specific T cells with a type 1 cytokine profile also expressed message for and secreted RANTES, MIP-1 alpha and MIP-1 beta following stimulation with specific tumor. Unexpectedly, D5 melanoma cells cultured with IFN-gamma or TNF-alpha, two type 1 cytokines expressed by therapeutic T cells, secreted Keratinocyte Chemoattractant (KC), MCP-1, IP-10 and RANTES and expressed mRNA for MIG. The chemokines released by T cells and cytokine-stimulated tumor cells were functional and induced migration of the DJ2PM macrophage cell line. Additionally, tumor-specific stimulation of wt or perforin-deficient (PKO) effector T cells induced macrophages to secrete nitric oxide (NO), providing an additional effector mechanism for T cell-mediated tumor regression. Conclusion: These data suggest two possible sources for chemokine secretion that stimulates macrophage recruitment to the site of tumor metastases. Both appear to be initiated by T cell recognition of specific antigen, but one is dependent on the tumor cells to produce the chemokines that recruit macrophages

Combinatorial Effect of Non-Steroidal Anti-inflammatory Drugs and NF-κB Inhibitors in Ovarian Cancer Therapy

Several epidemiological studies have correlated the use of non-steroidal anti-inflammatory drugs (NSAID) with reduced risk of ovarian cancer, the most lethal gynecological cancer, diagnosed usually in late stages of the disease. We have previously established that the pro-apoptotic cytokine melanoma differentiation associated gene-7/Interleukin-24 (mda-7/IL-24) is a crucial mediator of NSAID-induced apoptosis in prostate, breast, renal and stomach cancer cells. In this report we evaluated various structurally different NSAIDs for their efficacies to induce apoptosis and mda-7/IL-24 expression in ovarian cancer cells. While several NSAIDs induced apoptosis, Sulindac Sulfide and Diclofenac most potently induced apoptosis and reduced tumor growth. A combination of these agents results in a synergistic effect. Furthermore, mda-7/IL-24 induction by NSAIDs is essential for programmed cell death, since inhibition of mda-7/IL-24 by small interfering RNA abrogates apoptosis. mda-7/IL-24 activation leads to upregulation of growth arrest and DNA damage inducible (GADD) 45 α and γ and JNK activation. The NF-κB family of transcription factors has been implicated in ovarian cancer development. We previously established NF-κB/IκB signaling as an essential step for cell survival in cancer cells and hypothesized that targeting NF-κB could potentiate NSAID-mediated apoptosis induction in ovarian cancer cells. Indeed, combining NSAID treatment with NF-κB inhibitors led to enhanced apoptosis induction. Our results indicate that inhibition of NF-κB in combination with activation of mda-7/IL-24 expression may lead to a new combinatorial therapy for ovarian cancer

Trophic macrophages in development and disease

Specialized phagocytes are found in the most primitive multicellular organisms. Their roles in homeostasis and in distinguishing self from non-self have evolved with the complexity of organisms and their immune systems. Equally important, but often overlooked, are the roles of macrophages in tissue development. As discussed in this Review, these include functions in branching morphogenesis, neuronal patterning, angiogenesis, bone morphogenesis and the generation of adipose tissue. In each case, macrophage depletion impairs the formation of the tissue and compromises its function. I argue that in several diseases, the unrestrained acquisition of these developmental macrophage functions exacerbates pathology. For example, macrophages enhance tumour progression and metastasis by affecting tumour-cell migration and invasion, as well as angiogenesis

A potential role of macrophage activation in the treatment of cancer

One of the functions of macrophages is to provide a defense mechanism against tumor cells. In the last decades the mechanism of tumor cell killing by macrophages have been studied extensively. The tumor cytotoxic function of macrophages requires stimulation either with bacterial cell wall products such as lipopolysaccharide (LPS) or muramyldipeptide (MDP) or with cytokines such as interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Activated macrophages secrete several substances that are directly involved in tumor cell killing i.e. tumor necrosis factor (TNF) and nitric oxide (NO). On the other hand, substances are secreted that are able to stimulate tumor cell growth, depending on the stage and the nature of the tumor. Several clinical trials have been performed aiming at the activation of macrophages or dendritic cells, a subpopulation of the macrophages. In this review we will summarize and discuss experimental studies and clinical trials based on the activation of macrophages. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved