Lung cancer-derived bombesin-like peptides down-regulate the generation and function of human dendritic cells

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Abstract

Development of tumors is regulated by tumor-derived neuroendocrine factors, including bombesin-like peptides (BLP). We have evaluated neuroendocrine regulation of dendritic cell (DC) maturation and function by both tumor-derived and purified bombesin (BOM), neuromedin B (NMB), gastrin-releasing peptide (GRP), and a BOM antagonist d-Phe-bombesin (DPB). BOM, NMB and GRP dose-dependently inhibited maturation of DC assessed as down-regulation of CD40, CD80 and CD86 expression on DC. BOM and GRP also inhibited interleukin-12 (IL-12) production by DC and their ability to activate T cells. DPB partly abrogated immunosuppressive effect of tumor cells on DC. These data are a first evidence for the role of BLP in the regulation of DC maturation and function, demonstrating that BLP inhibit DC maturation and longevity in the lung cancer microenvironment. This suggests a new mechanism of tumor escape and provides new targets for the immunopharmacological correction of immune effectors in cancer.

Introduction

Dendritic cells (DC) are the most potent antigen presenting cells that recognize, process and present MHC-restricted antigens to naı̈ve T lymphocytes (Banchereau and Steinman, 1998). Because of unique ability of DC to present antigens to naı̈ve T cells, they play a key role in the host defense mechanisms against bacterial and viral infections and are responsible for antitumor immunity and limiting the progression of cancer (Shurin, 1996). Immature DC reside in non-lymphoid tissues, including respiratory, gastrointestinal and reproductive systems, where they constantly sample their environment for antigens by phagocytosis, macropinocytosis and highly efficient receptor-mediated pinocytosis (Hart, 1997). After capturing antigens, DC migrate to secondary lymphoid organs and undergo alteration of phenotype and function, referred to as maturation. Mature DC lose their capacity to process antigen but increase their immunostimulatory potential and antigen presentation capacity Banchereau and Steinman, 1998, Hart, 1997. These specialized functions of DC permit the generation and maintenance of adaptive immune responses to antigens and induction of antigen-specific immune responses, including antitumor immunity (Becker, 1992).

It is well documented that different tumor-derived factors such as TGF-β, VEGF, interleukin-10 (IL-10), gangliosides and PGE2 could exhibit immunomodulatory properties and play an important role in the regulation of tumor growth and escape from immune recognition and elimination Gabrilovich et al., 1998, Marincola et al., 2000, Shurin et al., 1999. Many tumors have evolved mechanisms that alter different functions of DC. For instance, recent data from this laboratory indicate that the generation, maturation, function and longevity of DC are all markedly inhibited in cancer Aalamian et al., 2001, Esche et al., 1999, Katsenelson et al., 2001, Shurin et al., 2001a, Shurin et al., 2001b. It has been also demonstrated that DC directly purified from the tumor site showed a decreased expression of MHC class II antigens and costimulatory molecules (Shurin and Gabrilovich, 2001). In addition, the decreased infiltration of many tumors by DC was shown to correlate with a worse clinical prognosis Becker, 1992, Shurin et al., 2003, Shurin and Gabrilovich, 2001.

Neuropeptides are factors that are produced by tumor cells and might be also involved in the regulation of DC differentiation and functions. It has been observed that cancer cell lines, as well as primary human tumors, synthesize a number of hormones and neuropeptides (Viallet and Sausville, 1996). These peptides act in autocrine fashion to stimulate the growth of the tumor cells and enhance their ability to metastasize (Iishi et al., 2001). For instance, many neuroendocrine tumors, including tumors in the lung, overexpress bombesin-like peptides (BLP), which correlates with poor prognosis. BLP, such as bombesin (BOM), gastrin-releasing peptide (GRP) and neuromedin B (NMB), have been shown to have mitogenic activity in small cell lung carcinomas (SCLC) and non-small cell lung carcinomas and to be produced by a large variety of human tumors, including prostate cancer, breast carcinomas and renal cell carcinomas Cuttitta et al., 1985, Konety and Nelson, 2001, Markwalder and Reubi, 1999, Nelson et al., 1991, Pansky et al., 2000, Siegfried et al., 1999. Recently, the production of GRP was detected in 16 out of 32 lung cancer cell lines, and NMB was detected in 23 out of 33 lung cancer cell lines (Giaccone et al., 1992). In fact, cigarette smoking, a major cause of SCLC, is associated with hyperplasia of pulmonary neuroendocrine cells and increased levels of GRP and NMB in the lower respiratory tract (Aguayo et al., 1990). On the other hand, BLP regulate various functions of immunocompetent cells. BOM, GRP and NMB were shown to be chemotactic for human monocytes and lymphocytes, and to modulate adherence, chemotaxis and phagocytosis of human peripheral and murine peritoneal monocytes De la Fuente et al., 1991, Meloni et al., 1996, Ruff et al., 1985. Furthermore, BLP decreases the capacity of monocytes/macrophages to support Concanavalin A-mediated T cell proliferation and inhibit mucosal immunity (Elitsur et al., 1990).

Thus, BLP act as growth factors in normal and neoplastic tissues and have been implicated in the regulation of proliferation and functional activity of various cell types, including antigen-presenting cells, monocytes and B cells. However, the effect of BLP on DC, the most potent antigen-presenting cells, has not yet been investigated. The aim of this study was to evaluate the role of neuroendocrine regulation of DC maturation and DC function by using both tumor-derived and purified BOM, NMB and GRP and the highly specific BOM antagonist d-Phe-bombesin (DPB). We have demonstrated that BOM, NMB and GRP dose-dependently inhibit maturation of DC and the functional ability of DC to stimulate proliferation of allogeneic T cells. BLP also down-regulate the ability of DC to produce IL-12 upon CD40L stimulation that correlates with a marked inhibition of CD40 expression on BLP-treated DC. DPB, when added to DC culture treated by lung cancer-conditioned medium, partly abrogated the immunosuppressive effect of tumor cells on dendropoiesis. This study provides the first evidence for the role of BLP in the regulation of DC maturation and function. Together, our data suggest a new mechanism of tumor escape from immune recognition and might provide a new target for the immunopharmacological correction of inhibited immune effector cells in cancer.

Section snippets

Tumor cell lines

Human SCLC lines CRL-5815 and H345 were obtained from American Type Culture Collection (ATCC, Rockville, MD) and maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 U/ml penicillin, 100 μg/ml streptomycin, 0.2 mM l-glutamine, 1 mM sodium pyruvate and 0.1 mM HEPES (GIBCO, Grand Island, NY).

Human DC cultures

Human DC cultures were initiated from CD14+ adherent peripheral blood monocytes as described earlier (Shurin, 2003). Peripheral blood was obtained from healthy

Human DC express gastrin-releasing preferring receptor mRNA and CRL5815 tumor cells express neuromedin B mRNA

It has been previously reported that many lung cancer cell lines produce BLP (Giaccone et al., 1992). To confirm that CRL5815 tumor cells are able to synthesize BLP, such as NMB or GRP, the corresponding mRNA expression levels were determined by RT-PCR. Fig. 1A demonstrates that CRL5815 cells express NMB mRNA. The band of PCR product was similar to the band obtained from H345 cell line, which served as a positive control (Siegfried et al., 1999). These results are in agreement with others

Discussion

The neuroendocrine and immune systems are now recognized as intimately linked and involved in bidirectional communication. There is an increasing body of evidence during the last several years suggesting that the neuroendocrine system plays an important role in the regulation of function and survival of immune cells, including DC (Dunzendorfer and Wiedermann, 2001). Dysfunction of this regulation has been implicated in the pathogenesis of a number of human disease states including inflammatory

Acknowledgements

This work was supported by RO1 CA80126 (M.R.S.), RO1 CA84270 (M.R.S.) and NCI Lung Cancer SPORE grant P50 CA090440 (J.M.S.).

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