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BackgroundTumor-associated macrophages (TAMs) constitute a significant part of infiltrating inflammatory cells that are frequently correlated with progression and poor prognosis of a variety of cancers. Tumor cell-produced human β-defensin-3 (hBD-3) has been associated with TAM trafficking in oral cancer; however, its involvement in tumor-related inflammatory processes remains largely unknown.MethodologyThe relationship between hBD-3, monocyte chemoattractant protein-1 (MCP-1), TAMs, and CCR2 was examined using immunofluorescence microscopy in normal and oral carcinoma in situ biopsy specimens. The ability of hBD-3 to chemoattract host macrophages in vivo using a nude mouse model and analysis of hBD-3 on monocytic cell migration in vitro, applying a cross-desensitization strategy of CCR2 and its pharmacological inhibitor (RS102895), respectively, was also carried out.Conclusions/FindingsMCP-1, the most frequently expressed tumor cell-associated chemokine, was not produced by tumor cells nor correlated with the recruitment of macrophages in oral carcinoma in situ lesions. However, hBD-3 was associated with macrophage recruitment in these lesions and hBD-3-expressing tumorigenic cells induced massive tumor infiltration of host macrophages in nude mice. HBD-3 stimulated the expression of tumor-promoting cytokines, including interleukin-1α (IL-1α), IL-6, IL-8, CCL18, and tumor necrosis factor-α (TNF-α) in macrophages derived from human peripheral blood monocytes. Monocytic cell migration in response to hBD-3 was inhibited by cross-desensitization with MCP-1 and the specific CCR2 inhibitor, RS102895, suggesting that CCR2 mediates monocyte/macrophage migration in response to hBD-3. Collectively, these results indicate that hBD-3 utilizes CCR2 to regulate monocyte/macrophage trafficking and may act as a tumor cell-produced chemoattractant to recruit TAMs. This novel mechanism is the first evidence of an hBD molecule orchestrating an in vivo outcome and demonstrates the importance of the innate immune system in the development of tumors.

Cells of the mononuclear phagocyte system develop from precursors in the bone marrow (1), but little is known about their pathway of differentiation or its control in vivo. The development of morphology (2) (8), which are found on mouse m~ but not on other hemopoietic or unrelated cells, provide new markers to define stages in the differentiation of m~b and to characterize and isolate their precursors.In this paper, we analyze the expression of Ag F4/80 on cells of the m~ lineage from progenitors in bone marrow to mature ceils obtained after culture in the presence of L cell-conditioned medium (LCM). F4/80 is also used in a clonal approach to examine mq~ subset heterogeneity. Materials and MethodsAnimals. Swiss mice and CBA T6T6 mice of both sexes were bred in this department. Similar results were obtained using either sex or strain.Cells. Bone marrow (BM) and resident peritoneal cells were obtained as described (7,8). Mass liquid cultures of BM were prepared in a BM culture medium (BMCM) consisting of Dulbecco's modification of minimal essential medium (DMEM) (high glucose), 15% heatinactivated (HI) horse serum (Flow Laboratories, Inc., Irvine, Scotland), 1% HI fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, 100 #g/ml streptomycin, I00/xg/ml kanamycin, and 10-20% LCM. BM 2 cells were cultured in 75-or 175-cm tissue culture flasks (Falcon Labware, Div. of Becton, Dickinson & Co., Oxnard, Calif., and Nunclon, Gibco, Middlesex, England.) 25 or 50 ml, respectively, at 8 × 10 n nucleated cells/ml. Flasks were coated with gelatin as described (7).Cultures were divided into a nonadherent cell (NAC) fraction, usually after 3 d, and adherent

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