CXCL1/Macrophage Inflammatory Protein-2-Induced Angiogenesis In Vivo Is Mediated by Neutrophil-Derived Vascular Endothelial Growth Factor-A

Abstract

The angiogenic activity of CXC-ELR+ chemokines, including CXCL8/IL-8, CXCL1/macrophage inflammatory protein-2 (MIP-2), and CXCL1/growth-related oncogene-α in the Matrigel sponge angiogenesis assay in vivo, is strictly neutrophil dependent, as neutrophil depletion of the animals completely abrogates the angiogenic response. In this study, we demonstrate that mice deficient in the src family kinases, Hck and Fgr (hck−/−fgr−/−), are unable to develop an angiogenic response to CXCL1/MIP-2, although they respond normally to vascular endothelial growth factor-A (VEGF-A). Histological examination of the CXCL1/MIP-2-containing Matrigel implants isolated from wild-type or hck−/−fgr−/− mice showed the presence of an extensive neutrophil infiltrate, excluding a defective neutrophil recruitment into the Matrigel sponges. Accordingly, neutrophils from hck−/−fgr−/− mice normally migrated and released gelatinase B in response to CXCL1/MIP-2 in vitro, similarly to wild-type neutrophils. However, unlike wild-type neutrophils, those from hck−/−fgr−/− mice were completely unable to release VEGF-A upon stimulation with CXCL1/MIP-2. Furthermore, neutralizing anti-VEGF-A Abs abrogated the angiogenic response to CXCL1/MIP-2 in wild-type mice and CXCL1/MIP-2 induced angiogenesis in the chick embryo chorioallantoic membrane assay, indicating that neutrophil-derived VEGF-A is a major mediator of CXCL1/MIP-2-induced angiogenesis. Finally, in vitro kinase assays confirmed that CXCL1/MIP-2 activates Hck and Fgr in murine neutrophils. Taken together, these data demonstrate that CXCL1/MIP-2 leads to recruitment of neutrophils that, in turn, release biologically active VEGF-A, resulting in angiogenesis in vivo. Our observations delineate a novel mechanism by which CXCL1/MIP-2 induces neutrophil-dependent angiogenesis in vivo.

The Src Family Kinases Hck and Fgr Regulate Neutrophil Responses to N-Formyl-Methionyl-Leucyl-Phenylalanine

“…Notably, triple mutant (hck Ϫ/Ϫ fgr Ϫ/Ϫ lyn Ϫ/Ϫ ) PMNs do not exhibit alteration in their migratory ability both in Transwell assays in vitro, and in a chemical peritonitis model in vivo (38). Additionally, hck Ϫ/Ϫ fgr Ϫ/Ϫ PMNs displayed the same migratory response to CXCL1/MIP-2 as WT cells both in vitro and in vivo (23). We therefore asked whether the decrease in actin polymerization we found in hck Ϫ/Ϫ fgr Ϫ/Ϫ PMNs resulted in alteration in cell migration in response to fMLP (Fig.…”

“…Both in vitro and in vivo studies excluded the fact that neutrophil chemotaxis toward chemoattractants or chemokines requires Src family kinases (23,28,38). However, in the LPS-induced systemic inflammatory reaction, hck Ϫ/Ϫ fgr Ϫ/Ϫ PMNs accumulate in the blood and are impaired in their capability to migrate into the liver (67).…”

“…Among cytoplasmic tyrosine kinases, members of the Src (13)(14)(15)(16)(17)(18) and the Tec families (19) in PMNs, and Pyk2 in a granulocytic cell line (20) have been implicated in signal transduction by fMLP. Additionally, signaling by receptors for either CXC and CC chemokines were reported to implicate Src family tyrosine kinases in granulocytes (21)(22)(23). Support for a role of tyrosine kinases in signal transduction by fMLP receptors derives from the evidence that inhibitory drugs, including some displaying significant specificity for Src or Tec family kinases, inhibit PMN responses to fMLP (16 -18, 24, 25).…”

“…Importantly, whereas Src family kinase-deficient PMNs display a marked reduction in the degranulation response to fMLP (17,18) or MIP-2 (23), syk Ϫ/Ϫ cells respond as wild-type (WT) cells to stimulation with fMLP and other chemoattractants (26). The defective degranulation response to fMLP and chemokines (17,18,21,23) by Src family kinase-deficient PMNs might not necessarily reflect alterations in upstream G␣i-signaling, but simply the role played by these kinases in more distal signaling events regulating exocytosis, a possibility consistent with the physical association of Hck and Fgr with primary and secondary granule (13,27). Indeed, very recent results showed that hck Ϫ/Ϫ fgr Ϫ/Ϫ neutrophils and fgr Ϫ/Ϫ dendritic cells manifest a more robust signaling and functional responses to several murine-derived chemokines (28).…”

The Multifaceted Roles Neutrophils Play in the Tumor Microenvironment

“…On the other hand, CCL2 prevents apoptosis of neutrophils [245] and activates neutrophils in the pre-metastatic lung towards an anti-tumor phenotype where they produce H 2 O 2 to kill disseminated tumor cells [32,66], suggesting a dual role of this chemokine. Also, CXCL1 was shown to promote tumor cell proliferation [246], tumor angiogenesis [247], invasion and migration [248], in addition to its ability to recruit and activate neutrophils [249]. CXCL1 was shown to be involved in a paracrine network mediating both metastatic progression and chemoresistance [250].…”