Toll-like receptor signaling alters the expression of regulator of G protein signaling proteins in dendritic cells: implications for G protein-coupled receptor signaling

Conserved structural motifs on pathogens trigger pattern recognition receptors present on APCs such as dendritic cells (DCs). An important class of such receptors is the Toll-like receptors (TLRs). TLR signaling triggers a cascade of events in DCs that includes modified chemokine and cytokine production, altered chemokine receptor expression, and changes in signaling through G protein-coupled receptors (GPCRs). One mechanism by which TLR signaling could modify GPCR signaling is by altering the expression of regulator of G protein signaling (RGS) proteins. In this study, we show that human monocyte-derived DCs constitutively express significant amounts of RGS2, RGS10, RGS14, RGS18, and RGS19, and much lower levels of RGS3 and RGS13. Engagement of TLR3 or TLR4 on monocyte-derived DCs induces RGS16 and RGS20, markedly increases RGS1 expression, and potently down-regulates RGS18 and RGS14 without modifying other RGS proteins. A similar pattern of Rgs protein expression occurred in immature bone marrow-derived mouse DCs stimulated to mature via TLR4 signaling. The changes in RGS18 and RGS1 expression are likely important for DC function, because both proteins inhibit G alpha(i)- and G alpha(q)-mediated signaling and can reduce CXC chemokine ligand (CXCL)12-, CC chemokine ligand (CCL)19-, or CCL21-induced cell migration. Providing additional evidence, bone marrow-derived DCs from Rgs1(-/-) mice have a heightened migratory response to both CXCL12 and CCL19 when compared with similar DCs prepared from wild-type mice. These results indicate that the level and functional status of RGS proteins in DCs significantly impact their response to GPCR ligands such as chemokines.     

Both CXCR3 and CXCL10/IFN-inducible protein 10 are required for resistance to primary infection by dengue virus

We examined the extent to which CXCR3 mediates resistance to dengue infection. Following intracerebral infection with dengue virus, CXCR3-deficient (CXCR3(-/-)) mice showed significantly higher mortality rates than wild-type (WT) mice; moreover, surviving CXCR3(-/-) mice, but not WT mice, often developed severe hind-limb paralysis. The brains of CXCR3(-/-) mice showed higher viral loads than those of WT mice, and quantitative analysis using real-time PCR, flow cytometry, and immunohistochemistry revealed fewer T cells, CD8(+) T cells in particular, in the brains of CXCR3(-/-) mice. This suggests that recruitment of effector T cells to sites of dengue infection was diminished in CXCR3(-/-) mice, which impaired elimination of the virus from the brain and thus increased the likelihood of paralysis and/or death. These results indicate that CXCR3 plays a protective rather than an immunopathological role in dengue virus infection. In studies to identify critical CXCR3 ligands, CXCL10/IFN-inducible protein 10-deficient (CXCL10/IP-10(-/-)) mice infected with dengue virus showed a higher mortality rate than that of the CXCR3(-/-) mice. Although CXCL10/IP-10, CXCL9/monokine induced by IFN-gamma, and CXCL11/IFN-inducible T cell alpha chemoattractant share a single receptor and all three of these chemokines are induced by dengue virus infection, the latter two could not compensate for the absence of CXCL10/IP-10 in this in vivo model. Our results suggest that both CXCR3 and CXCL10/IP-10 contribute to resistance against primary dengue virus infection and that chemokines that are indistinguishable in in vitro assays differ in their activities in vivo.     

RGS13 regulates germinal center B lymphocytes responsiveness to CXC chemokine ligand (CXCL)12 and CXCL13

Normal lymphoid tissue development and function depend upon directed cell migration. Providing guideposts for cell movement and positioning within lymphoid tissues, chemokines signal through cell surface receptors that couple to heterotrimeric G proteins, which are in turn subject to regulation by regulator of G protein signaling (RGS) proteins. In this study, we report that germinal center B lymphocytes and thymic epithelial cells strongly express one of the RGS family members, RGS13. Located between Rgs1 and Rgs2, Rgs13 spans 42 kb on mouse chromosome 1. Rgs13 encodes a 157-aa protein that shares 82% amino acid identity with its 159-aa human counterpart. In situ hybridization with sense and antisense probes localized Rgs13 expression to the germinal center regions of mouse spleens and Peyer's patches and to the thymus medulla. Affinity-purified RGS13 Abs detected RGS13-expressing cells in the light zone of the germinal center. RGS13 interacted with both Gialpha and Gqalpha and strongly impaired signaling through G(i)-linked signaling pathways, including signaling through the chemokine receptors CXCR4 and CXCR5. Prolonged CD40 signaling up-regulated RGS13 expression in human tonsil B lymphocytes. These results plus previous studies of RGS1 indicate the germinal center B cells use two RGS proteins, RGS1 and RGS13, to regulate their responsiveness to chemokines.     

Expression of Neuronal CXCL10 Induced by Rabies Virus Infection Initiates Infiltration of Inflammatory Cells,Production of Chemokines and Cytokines,and Enhancement of Blood-Brain Barrier Permeability

It has been shown that enhancement of blood-brain barrier (BBB) permeability is modulated by the expression of chemokines/cytokines and reduction of tight junction (TJ) proteins in the brains of mice infected with rabies virus (RABV). Since CXCL10 was found to be the most highly expressed chemokine, its temporal and spatial expression were determined in the present study. The expression of the chemokine CXCL10 was initially detected in neurons as early as 3 days postinfection (p.i.) in the brains of RABV-infected mice, after which it was detected in microglia (6 days p.i.) and astrocytes (9 days p.i.). Neutralization of CXCL10 by treatment with anti-CXCL10 antibodies reduced gamma interferon (IFN-γ) production and Th17 cell infiltration, as well as restoring TJ protein expression and BBB integrity. Together, these data suggest that it is the neuronal CXCL10 that initiates the cascade that leads to the activation of microglia/astrocytes, infiltration of inflammatory cells, expression of chemokines/cytokines, reduction of TJ protein expression, and enhancement of the BBB permeability.     

Mx1‐Cre mediated Rgs12 conditional knockout mice exhibit increased bone mass phenotype

Regulators of G‐protein Signaling (Rgs) proteins are the members of a multigene family of GTPase‐accelerating proteins (GAP) for the Galpha subunit of heterotrimeric G‐proteins. Rgs proteins play critical roles in the regulation of G protein couple receptor (GPCR) signaling in normal physiology and human diseases such as cancer, heart diseases, and inflammation. Rgs12 is the largest protein of the Rgs protein family. Some in vitro studies have demonstrated that Rgs12 plays a critical role in regulating cell differentiation and migration; however its function and mechanism in vivo is largely unknown. Here, we generated a floxed Rgs12 allele (Rgs12^flox/flox) in which the exon 2, containing both PDZ and PTB_PID domains of Rgs12, was flanked with two loxp sites. By using the inducible Mx1‐cre and Poly I:C system to specifically delete Rgs12 at postnatal 10 days in interferon‐responsive cells including monocyte and macrophage cells, we found that Rgs12 mutant mice had growth retardation with the phenotype of increased bone mass. We further found that deletion of Rgs12 reduced osteoclast numbers and had no significant effect on osteoblast formation. Thus, Rgs12^flox/flox conditional mice provide a valuable tool for in vivo analysis of Rgs12 function and mechanism through time‐ and cell‐specific deletion of Rgs12. genesis 51:201–209, 2013. © 2013 Wiley Periodicals, Inc.     

Severe disease, unaltered leukocyte migration, and reduced IFN-gamma production in CXCR3-/- mice with experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a CD4(+) Th1 T cell-mediated disease of the CNS, used to study certain aspects of multiple sclerosis. CXCR3, the receptor for CXCL10, CXCL9, and CXCL11, is preferentially expressed on activated Th1 T cells and has been proposed to govern the migration of lymphocytes into the inflamed CNS during multiple sclerosis and EAE. Unexpectedly, CXCL10-deficient mice were susceptible to EAE, leaving uncertain what the role of CXCR3 and its ligands might play in this disease model. In this study, we report that CXCR3(-/-) mice exhibit exaggerated severity of EAE compared with wild-type (CXCR3(+/+)) littermate mice. Surprisingly, there were neither quantitative nor qualitative differences in CNS-infiltrating leukocytes between CXCR3(+/+) and CXCR3(-/-) mice with EAE. Despite these equivalent inflammatory infiltrates, CNS tissues from CXCR3(-/-) mice with EAE showed worsened blood-brain barrier disruption and more von Willebrand factor-immunoreactive vessels within inflamed spinal cords, as compared with CXCR3(+/+) mice. Spinal cords of CXCR3(-/-) mice with EAE demonstrated decreased levels of IFN-gamma, associated with reduced inducible NO synthase immunoreactivity, and lymph node T cells from CXCR3(-/-) mice primed with MOG(35-55) secreted less IFN-gamma in Ag-driven recall responses than cells from CXCR3(+/+) animals. CXCR3(-/-) lymph node T cells also showed enhanced Ag-driven proliferation, which was reduced by addition of IFN-gamma. Taken with prior findings, our data show that CXCL10 is the most relevant ligand for CXCR3 in EAE. CXCR3 does not govern leukocyte trafficking in EAE but modulates T cell IFN-gamma production and downstream events that affect disease severity.     

TNFα-exposed Bone Marrow-derived Mesenchymal Stem Cells Promote Locomotion of MDA-MB-231 Breast Cancer Cells through Transcriptional Activation of CXCR3 Ligand Chemokines

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are often recruited to solid tumors, integrate into the tumor stroma, and contribute to tumor development. TNFα is a major inflammatory cytokine present in the tumor microenvironment and has a profound influence on the progression of tumor development. This study was aimed to investigate the role of BM-MSCs in tumor promotion in response to TNFα. Quantitative real-time PCR arrays show that diverse cytokines/chemokines were induced in TNFα-treated BM-MSCs; in particular, CXCR3 ligand chemokines, including CXCL9, CXCL10, and CXCL11, were potently induced. A serial and site-directed mutation analysis in the CXCL9, CXCL10, and CXCL11 promoters revealed that NF-κB binding elements were responsible for TNFα-induced promoter activation of CXCR3 ligand chemokines. TNFα stimulated NF-κB activity, and ectopic expression of NF-κB enhanced TNFα-induced promoter activities of the CXCR3 ligand chemokines. Gel shift and supershift assays showed that NF-κB was associated with CXCR3 ligand chemokine promoters in response to TNFα treatment. All three CXCR3 ligand chemokines enhanced the migration and invasive motility of MDA-MB-231 breast cancer cells expressing CXCR3. Treatment of MDA-MB-231 cells with CXCL10 activated small GTPase of Rho family proteins, such as RhoA and Cdc42. CXCL9-, CXCL10-, or CXCL11-induced invasive capability of MDA-MB-231 cells was completely abrogated in the presence of a neutralizing anti-CXCR3 antibody in the culture medium. Moreover, CXCL9, CXCL10, and CXCL11 stimulated the expression of MMP-9, but not MMP-2, in MDA-MB-231 cells. These results suggest that BM-MSCs promote the locomotion of breast cancer cells through CXCR3 ligand-mediated actin rearrangement by TNFα in the tumor microenvironment.     

Local production of chemokines and prostaglandin E2 in the acute, chronic and recovery phase of murine experimental colitis

Increased levels of chemokines and prostaglandins have been reported in patients with inflammatory bowel disease, although their changes during disease development are less understood. The aim of this study was to investigate the local production of nine selected chemokines and prostaglandin E(2) (PGE(2)) to elucidate their role in colitis progression in BALB/c and C57BL/6 mice exposed to dextran sulphate sodium. The acute inflammation in both strains was accompanied by a significant up-regulation of CXCL1, CXCL2/3, CXCL10, CCL2, CCL4 and CCL22 and a downregulation of PGE(2). In the recovery phase in BALB/c, one-week post-DSS, PGE(2) levels were significantly increased with a concomitant downregulation of CXCL1, CXCL2/3, CXCL10, CCL2, and CCL4. In contrast, in C57BL/6 mice CXCL1, CXCL2/3, CXCL10, CCL2, CCL3 and CCL4 production remained high during the chronic phase, without any up-regulation of PGE(2). In addition, CCL5 was significantly increased at d26 and 33 compared to d5. Interestingly, the number of macrophages was significantly increased during the acute phase, whereas T cells were significantly increased in both the acute and chronic phase in C57BL/6 mice. Thus, our results show that chemokines are produced in a dynamic manner during colitis progression.     

Cutting edge: Uniqueness of lymphoid chemokine requirement for the initiation and maturation of nasopharynx-associated lymphoid tissue organogenesis

CD3(-)CD4(+)CD45(+) inducer cells are required for the initiation of mucosa-associated organogenesis of both nasopharynx-associated lymphoid tissues (NALT) and Peyer's patches (PP) in the aerodigestive tract. CXCL13(-/-) mice and mice carrying the paucity of lymph node T cell (plt) mutation and lacking expression of CCL19 and CCL21 accumulate CD3(-)CD4(+)CD45(+) cells at the site of NALT but not of PP genesis. Although NALT was observed to develop in adult CXCL13(-/-) and plt/plt mice, the formation of germinal centers in CXCL13(-/-) mice was affected, and their population of B cells was much lower than in the NALT of CXCL13(+/-) mice. Similarly, fewer T cells were observed in the NALT of plt/plt mice than in control mice. These findings indicate that the initiation of NALT organogenesis is independent of CXCL13, CCL19, and CCL21. However, the expression of these lymphoid chemokines is essential for the maturation of NALT microarchitecture.     

Mechanisms of host defense following severe acute respiratory syndrome-coronavirus (SARS-CoV) pulmonary infection of mice

We describe a model of severe acute respiratory syndrome-coronavirus (SARS-CoV) infection in C57BL/6 mice. A clinical isolate of the virus introduced intranasally replicated transiently to high levels in the lungs of these mice, with a peak on day 3 and clearance by day 9 postinfection. Viral RNA localized to bronchial and bronchiolar epithelium. Expression of mRNA for angiotensin converting enzyme 2, the SARS-CoV receptor, was detected in the lung following infection. The virus induced production in the lung of the proinflammatory chemokines CCL2, CCL3, CCL5, CXCL9, and CXCL10 with differential kinetics. The receptors for these chemokines were also detected. Most impressively, mRNA for CXCR3, the receptor for CXCL9 and CXCL10, was massively up-regulated in the lungs of SARS-CoV-infected mice. Surprisingly Th1 (and Th2) cytokines were not detectable, and there was little local accumulation of leukocytes and no obvious clinical signs of pulmonary dysfunction. Moreover, beige, CD1-/-, and RAG1-/- mice cleared the virus normally. Infection spread to the brain as it was cleared from the lung, again without leukocyte accumulation. Infected mice had a relative failure to thrive, gaining weight significantly more slowly than uninfected mice. These data indicate that C57BL/6 mice support transient nonfatal systemic infection with SARS-CoV in the lung, which is able to disseminate to brain. In this species, proinflammatory chemokines may coordinate a rapid and highly effective innate antiviral response in the lung, but NK cells and adaptive cellular immunity are not required for viral clearance.     

CXCL17 Expression by Tumor Cells Recruits CD11b(+)Gr1(high)F4/80(-) Cells and Promotes Tumor Progression

Background

Chemokines are involved in multiple aspects of pathogenesis and cellular trafficking in tumorigenesis. In this study, we report that the latest member of the C-X-C-type chemokines, CXCL17 (DMC/VCC-1), recruits immature myeloid-derived cells and enhances early tumor progression.

Methodology/Principal Findings

CXCL17 was preferentially expressed in some aggressive types of gastrointestinal, breast, and lung cancer cells. CXCL17 expression did not impart NIH3T3 cells with oncogenic potential in vitro, but CXCL17-expressing NIH3T3 cells could form vasculature-rich tumors in immunodeficient mice. Our data showed that CXCL17-expressing tumor cells increased immature CD11b⁺Gr1⁺ myeloid-derived cells at tumor sites in mice and promoted CD31⁺ tumor angiogenesis. Extensive chemotactic assays proved that CXCL17-responding cells were CD11b⁺Gr1^highF4/80⁻ cells (∼90%) with a neutrophil-like morphology in vitro. Although CXCL17 expression could not increase the number of CD11b⁺Gr1⁺ cells in tumor-burdened SCID mice or promote metastases of low metastatic colon cancer cells, the existence of CXCL17-responding myeloid-derived cells caused a striking enhancement of xenograft tumor formation.

Conclusions/Significance

These results suggest that aberrant expression of CXCL17 in tumor cells recruits immature myeloid-derived cells and promotes tumor progression through angiogenesis.     

Rgs5 targeting leads to chronic low blood pressure and a lean body habitus

RGS5 is a potent GTPase-activating protein for G(ialpha) and G(qalpha) that is expressed strongly in pericytes and is present in vascular smooth muscle cells. To study the role of RGS5 in blood vessel physiology, we generated Rgs5-deficient mice. The Rgs5(-/-) mice developed normally, without obvious defects in cardiovascular development or function. Surprisingly, Rgs5(-/-) mice had persistently low blood pressure, lower in female mice than in male mice, without concomitant cardiac dysfunction, and a lean body habitus. The examination of the major blood vessels revealed that the aortas of Rgs5(-/-) mice were dilated compared to those of control mice, without altered wall thickness. Isolated aortic smooth muscle cells from the Rgs5(-/-) mice exhibited exaggerated levels of phosphorylation of vasodilator-stimulated phosphoprotein and extracellular signal-regulated kinase in response to stimulation with either sodium nitroprusside or sphingosine 1-phosphate. The results of this study, along with those of previous studies demonstrating that RGS5 stability is under the control of nitric oxide via the N-end rule pathway, suggest that RGS5 may balance vascular tone by attenuating vasodilatory signaling in vivo in opposition to RGS2, another RGS (regulator of G protein signaling) family member known to inhibit G protein-coupled receptor-mediated vasoconstrictor signaling. Blocking the function or the expression of RGS5 may provide an alternative approach to treat hypertension.     

Duffy antigen facilitates movement of chemokine across the endothelium in vitro and promotes neutrophil transmigration in vitro and in vivo

The Duffy Ag expressed on RBCs, capillaries, and postcapillary venular endothelial cells binds selective CXC and CC chemokines with high affinity. Cells transfected with the Duffy Ag internalize but do not degrade chemokine ligand. It has been proposed that Duffy Ag transports chemokines across the endothelium. We hypothesized that Duffy Ag participates in the movement of chemokines across the endothelium and, by doing so, modifies neutrophil transmigration. We found that the Duffy Ag transfected into human endothelial cells facilitates movement of the radiolabeled CXC chemokine, growth related oncogene-alpha/CXC chemokine ligand 1 (GRO-alpha/CXCL1), across an endothelial monolayer. In addition, neutrophil migration toward GRO-alpha/CXCL1 and IL-8 (IL-8/CXCL8) was enhanced across an endothelial monolayer expressing the Duffy Ag. Furthermore, GRO-alpha/CXCL1 stimulation of endothelial cells expressing the Duffy Ag did not affect gene expression by oligonucleotide microarray analysis. These in vitro observations are supported by the finding that IL-8/CXCL8-driven neutrophil recruitment into the lungs was markedly attenuated in transgenic mice lacking the Duffy Ag. We conclude that Duffy Ag has a role in enhancing leukocyte recruitment to sites of inflammation by facilitating movement of chemokines across the endothelium.     

Ablation of Arginylation in the Mouse N-End Rule Pathway: Loss of Fat,Higher Metabolic Rate,Damaged Spermatogenesis,and Neurological Perturbations

In the N-end rule pathway of protein degradation, the destabilizing activity of N-terminal Asp, Glu or (oxidized) Cys residues requires their conjugation to Arg, which is recognized directly by pathway''s ubiquitin ligases. N-terminal arginylation is mediated by the Ate1 arginyltransferase, whose physiological substrates include the Rgs4, Rgs5 and Rgs16 regulators of G proteins. Here, we employed the Cre-lox technique to uncover new physiological functions of N-terminal arginylation in adult mice. We show that postnatal deletion of mouse Ate1 (its unconditional deletion is embryonic lethal) causes a rapid decrease of body weight and results in early death of ∼15% of Ate1-deficient mice. Despite being hyperphagic, the surviving Ate1-deficient mice contain little visceral fat. They also exhibit an increased metabolic rate, ectopic induction of the Ucp1 uncoupling protein in white fat, and are resistant to diet-induced obesity. In addition, Ate1-deficient mice have enlarged brains, an enhanced startle response, are strikingly hyperkinetic, and are prone to seizures and kyphosis. Ate1-deficient males are also infertile, owing to defects in Ate1^−/− spermatocytes. The remarkably broad range of specific biological processes that are shown here to be perturbed by the loss of N-terminal arginylation will make possible the dissection of regulatory circuits that involve Ate1 and either its known substrates, such as Rgs4, Rgs5 and Rgs16, or those currently unknown.     

CXC chemokine ligand 10 controls viral infection in the central nervous system: evidence for a role in innate immune response through recruitment and activation of natural killer cells

How chemokines shape the immune response to viral infection of the central nervous system (CNS) has largely been considered within the context of recruitment and activation of antigen-specific lymphocytes. However, chemokines are expressed early following viral infection, suggesting an important role in coordinating innate immune responses. Herein, we evaluated the contributions of CXC chemokine ligand 10 (CXCL10) in promoting innate defense mechanisms following coronavirus infection of the CNS. Intracerebral infection of RAG1(-/-) mice with a recombinant CXCL10-expressing murine coronavirus (mouse hepatitis virus) resulted in protection from disease and increased survival that correlated with a significant increase in recruitment and activation of natural killer (NK) cells within the CNS. Accumulation of NK cells resulted in a reduction in viral titers that was dependent on gamma interferon secretion. These results indicate that CXCL10 expression plays a pivotal role in defense following coronavirus infection of the CNS by enhancing innate immune responses.     

Depletion of beta-arrestin-2 promotes tumor growth and angiogenesis in a murine model of lung cancer

Arrestins are adaptor/scaffold proteins that complex with activated and phosphorylated G protein-coupled receptor to terminate G protein activation and signal transduction. These complexes also mediate downstream signaling, independently of G protein activation. We have previously shown that beta-arrestin-2 (betaarr2) depletion promotes CXCR2-mediated cellular signaling, including angiogenesis and excisional wound closure. This study was designed to investigate the role of betaarr2 in tumorigenesis using a murine model of lung cancer. To that end, heterotopic murine Lewis lung cancer and tail vein metastasis tumor model systems in betaarr2-deficient mice (betaarr2(-/-)) and control littermates (betaarr2(+/+)) were used. betaarr2(-/-) mice exhibited a significant increase in Lewis lung cancer tumor growth and metastasis relative to betaarr2(+/+) mice. This correlated with decreased number of tumor-infiltrating lymphocytes but with elevated levels of the ELR(+) chemokines (CXCL1/keratinocyte-derived chemokine and CXCL2/MIP-2), vascular endothelial growth factor, and microvessel density. NF-kappaB activity was also enhanced in betaarr2(-/-) mice, whereas hypoxia-inducible factor-1alpha expression was decreased. Inhibition of CXCR2 or NF-kappaB reduced tumor growth in both betaarr2(-/-) and betaarr2(+/+) mice. NF-kappaB inhibition also decreased ELR(+) chemokines and vascular endothelial growth factor expression. Altogether, the data suggest that betaarr2 modulates tumorigenesis by regulating inflammation and angiogenesis through activation of CXCR2 and NF-kappaB.     

CXCL1/macrophage inflammatory protein-2-induced angiogenesis in vivo is mediated by neutrophil-derived vascular endothelial growth factor-A

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.