Scapini Patrizia, Morini Monica, Tecchio Cristina, Minghelli Simona, Di Carlo Emma, Tanghetti Elena, Albini Adriana, Lowell Clifford, Berton Giorgio, Noonan Douglas M, Cassatella Marco A
Department of Pathology, Section of General Pathology, University of Verona, Verona, Italy.
J Immunol. 2004 Apr 15;172(8):5034-40. doi: 10.4049/jimmunol.172.8.5034.
The angiogenic activity of CXC-ELR(+) chemokines, including CXCL8/IL-8, CXCL1/macrophage inflammatory protein-2 (MIP-2), and CXCL1/growth-related oncogene-alpha 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.
在体内基质胶海绵血管生成试验中，包括CXCL8/IL-8、CXCL1/巨噬细胞炎性蛋白-2（MIP-2）和CXCL1/生长相关癌基因-α在内的CXC-ELR(+)趋化因子的血管生成活性严格依赖于中性粒细胞，因为动物的中性粒细胞耗竭会完全消除血管生成反应。在本研究中，我们证明src家族激酶Hck和Fgr缺陷的小鼠（hck(-/-)fgr(-/-)）尽管对血管内皮生长因子-A（VEGF-A）反应正常，但对CXCL1/MIP-2无法产生血管生成反应。对从野生型或hck(-/-)fgr(-/-)小鼠分离的含CXCL1/MIP-2的基质胶植入物进行组织学检查，发现有广泛的中性粒细胞浸润，排除了中性粒细胞向基质胶海绵募集缺陷的可能性。因此，hck(-/-)fgr(-/-)小鼠的中性粒细胞在体外对CXCL1/MIP-2的反应中能正常迁移并释放明胶酶B，与野生型中性粒细胞相似。然而，与野生型中性粒细胞不同，hck(-/-)fgr(-/-)小鼠的中性粒细胞在受到CXCL1/MIP-2刺激时完全无法释放VEGF-A。此外，中和抗VEGF-A抗体消除了野生型小鼠对CXCL1/MIP-2的血管生成反应，并且CXCL1/MIP-2在鸡胚绒毛尿囊膜试验中诱导血管生成，这表明中性粒细胞衍生的VEGF-A是CXCL1/MIP-2诱导血管生成的主要介质。最后，体外激酶试验证实CXCL1/MIP-2可激活小鼠中性粒细胞中的Hck和Fgr。综上所述，这些数据表明CXCL1/MIP-2导致中性粒细胞募集，进而释放具有生物活性的VEGF-A，从而在体内引发血管生成。我们的观察结果描绘了一种CXCL1/MIP-2在体内诱导中性粒细胞依赖性血管生成的新机制。
