Chemokines comprise a large family of protein that are intimately involved in recruiting immune cells into peripheral tissues such as skin and in compartmentalizing them in certain regions of secondary lymphoid organs (e.g., lymph nodes). The goals of my laboratory are to uncover mechanisms by which chemokines and their receptors (1) influence the trafficking of immune cells into and out of the skin and (2) regulate cancer metastasis and progression. My laboratory has shown that skin dendritic cells express a variety of chemokine receptors that facilitate their migration into lymphatic vessels and discrete regions of LN. We have also shown that T cells express chemokine receptors that facilitate their adherence to endothelial cells. Based on the role of chemokine receptors in immune cell trafficking, we hypothesized that these receptors could play similarly important roles in the organ-selective homing or metastasis of cancer cells. Recently, we have demonstrated that human melanoma cells express a limited number of chemokine receptors. Our strategy has been to overexpress selected chemokine receptors in B16 murine melanoma by retroviral transduction. Overexpression of CCR7, a chemokine receptor critical to the migration of dendritic cells to regional lymph nodes, resulted in a 700-fold increase in metastasis of B16 cells from the skin to the draining lymph node in vivo. In contrast, transduction of B16 cells with CXCR4 resulted in a 10-fold increase (compared to a control B16 line) in lung metastases following inoculation of these cells intravenously into mice. Finally, CCR10-transduced B16 cells were able to form metastasizing tumors in the skin of mice in vivoa property that we demonstrated was dependent on the constitutive production of the CCR10 ligand known as CCL27 in the skin. In vitro in the presence of CCR10 ligand, CCR10-transduced B16 cells demonstrated increased resistance to apoptosis mediated by Fas-crosslinking and exposure to GP100-specific cytotoxic T cells, suggesting that selective expression of chemokine receptors may facilitate the survival of cancer cells and aid in their avoidance of the host immune response. Interestingly, treatment of the CCR10-B16-inoculated tumor site in the skin with neutralizing anti-CCL27 antibodies effectively prevented tumor formation, suggesting that anti-chemokine therapy may be useful in blocking cancer progression. CCL27 (the ligand for CCR1) is contstitutively produced in keratinocytes. Our studies indicate that CCL27 is slowly released under basal conditions and then accumulates in skin draining LN. When a topical contact sensitizer, DNFB, is applied to the skin, CCL27 is rapidly depleted from skin and accumulates to 10fold greater levels in skin-draining LN, where it stimulates recruitment of CCR10-postive T cells. This may be a novel mechanism for skin-associated chemokines to regulate the trafficking of T cells to skin-draining lymph node. In summary, we have shown selective LN, lung, and skin homing by B16 cells expressing the chemokine receptors CCR7, CXCR4, and CCR10, respectively, in vivo. Current work emphasizes translation of these results in preclinical models of metastasis prevention and treatment. For example, we have found that CXCR4 inhibitors effectively block pulmonary metastasis of CXCR4-expressing human melanoma cell lines in an IV injection model of lung metastasis in SCID mice. Moreover, we have found that inhibition of CXCR4 in conjunction with cyclophosphamide or anti-CTLA4 treatment synergistically reduces established melanoma metastases in the lung in a murine model. Of note, preliminary studies suggest that activation of CXCR4 in vitro does not protect B16 cells from cytoxicity due to treatment with common chemotherapeutic agents, including cisplatin. However, CXCR4 activation through SDF1 does dramatically protect B16 cells from apoptosis induced by cytolytic T cells (CD8+) specific for melanoma associated antigens, suggesting specificity of the protective effect of chemokine receptor activation. These studies should help us determine under which circumstances chemokine receptors antagonists will be useful in clinical anti-cancer therapy. We are beginning studies to designed to understand how chemokines expression is regulated in lymphatic endothelial cells and in skin-draining LN. Specifically, we now know that SLC, a major CCR7 ligand, is upregulated in lymphatic endothelial cells by oncostatin M, a cytokine produced by dendritic cells as well as other leukocytes. We hypothesize that oncostatin M contributes to high level expression of SLC in order to attract CCR7-expressing DC into lymphatic vessels prior to their migration to regional lymph nodes. We have also found that keratinocytes may synthesize a chemokine, CCL27, that is then transferred to draining LN via afferent lymphatic vessels. Interestingly the LN itself does not synthesize CCL27 at the mRNA level, suggesting that lymphatic transport of CCL27 may be a novel mechanism for regulating homing of T cells to the LN. We have already developed a sensitive ELISA assay that detects high levels of CCL27 in LN following application of a contact sensitizer (DNFB) to skin. We hypothesize that CCL27 in the LN may account for the large flux of skin-homing T cells that accumulate in LN with several days of application of topical haptens to skin
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