Synergistic action of CCL2 and CXCL16 on angiogenesis

Abstract

Gr-1+CD11b+ cells can suppress innate and adaptive immunity, and the functional immunosuppressive characteristics of these cells can be modulated by the tumor microenvironment. Since Gr-1+CD11b+ cells are also involved in tumor-associated angiogenesis, we hypothesized that the angiogenic nature of Gr-1+CD11b+ cells could be regulated by the tumor milieu. To address this hypothesis, we imitated a tumor microenvironment by exposing Gr-1+CD11b+ cells isolated from spleen of 4T1 mammary carcinoma-bearing mice to tumor-conditioned medium. Supernatants from tumor-conditioned Gr-1+CD11b+ cells significantly induced capillary-like tube formation and migration of human umbilical vein endothelial cells (HUVECs) compared to naive Gr-1+CD11b+ cells. Incubation of Gr-1+CD11b+ cells with tumor-conditioned medium induced production of pro-angiogenic chemokines CCL2 and CXCL16. Osteopontin induced the production of CCL2 and CXCL16 in Gr-1+CD11b+ cells. Pretreatment with an anti-CCL2 antibody, but not an anti-CXCL16 antibody, suppressed the angiogenic effects of tumor-conditioned Gr-1+CD11b+ cells on HUVECs. Simultaneous neutralization of CCL2 and CXCL16 significantly inhibited tube formation and migration of HUVECs compared to the sole neutralization against CCL2. Supernatants from tumor-conditioned Gr-1+CD11b+ cells induced phosphorylation of ERK1/2 in HUVECs, and inhibition of the ERK pathway blocked angiogenic effects. ERK pathway activity was partially abrogated by neutralization of CCL2 and more suppressed by simultaneous neutralization of CCL2 and CXCL16. These results collectively indicate that CCL2 and CXCL16 chemokines produced by tumor-conditioned Gr-1+CD11b+ myeloid cells synergistically induce angiogenesis in vitro by stimulating the ERK1/2 signaling pathway. Thus, regulation of Gr-1+CD11b+ cells in the tumor microenvironment may contribute to angiogenesis through the secretion of pro-angiogenic chemokines.