Muramyl dipeptide induces NOD2-dependent Ly6C(high) monocyte recruitment to the lungs and protects against influenza virus infection

Bacterial peptidoglycan-derived muramyl dipeptide (MDP) and derivatives have long-recognized antiviral properties but their mechanism of action remains unclear. In recent years, the pattern-recognition receptor NOD2 has been shown to mediate innate responses to MDP. Here, we show that MDP treatment of mice infected with Influenza A virus (IAV) significantly reduces mortality, viral load and pulmonary inflammation in a NOD2-dependent manner. Importantly, the induction of type I interferon (IFN) and CCL2 chemokine was markedly increased in the lungs following MDP treatment and correlated with a NOD2-dependent enhancement in circulating monocytes. Mechanistically, the protective effect of MDP could be explained by the NOD2-dependent transient increase in recruitment of Ly6C(high) "inflammatory" monocytes and, to a lesser extent, neutrophils to the lungs. Indeed, impairment in both Ly6C(high) monocyte recruitment and survival observed in infected Nod2-/- mice treated with MDP was recapitulated in mice deficient for the chemokine receptor CCR2 required for CCL2-mediated Ly6C(high) monocyte migration from the bone marrow into the lungs. MDP-induced pulmonary monocyte recruitment occurred normally in IAV-infected and MDP-treated Ips-1-/- mice. However, IPS-1 was required for improved survival upon MDP treatment. Finally, mycobacterial N-glycolyl MDP was more potent than N-acetyl MDP expressed by most bacteria at reducing viral burden while both forms of MDP restored pulmonary function following IAV challenge. Overall, our work sheds light on the antiviral mechanism of a clinically relevant bacterial-derived compound and identifies the NOD2 pathway as a potential therapeutic target against IAV.     

CCR7/CCL21 migration on fibronectin is mediated by phospholipase Cgamma1 and ERK1/2 in primary T lymphocytes

CCR7 binds to its cognate ligand, CCL21, to mediate the migration of circulating naive T lymphocytes to the lymph nodes. T lymphocytes can bind to fibronectin, a constituent of lymph nodes, via their β1 integrins, which is a primary mechanism of T lymphocyte migration; however, the signaling pathways involved are unclear. We report that rapid (within 2 min) and transient phosphorylation of ERK1/2 is required for T cell migration on fibronectin in response to CCL21. Conversely, prevention of ERK1/2 phosphorylation by inhibition of its kinase, MAPK/MEK, prevented T lymphocyte migration. Previous studies have suggested that phospholipase Cγ1 (PLCγ1) can mediate phosphorylation of ERK1/2, which is required for β1 integrin activation. Paradoxically, we found that inhibition of PLCγ1 phosphorylation by the general PLC inhibitor U73122 was associated with a delayed and reduced phosphorylation of ERK1/2 and reduced migration of T lymphocytes on fibronectin. To further characterize the relationship between ERK1/2 and PLCγ1, we reduced PLCγ1 levels by 85% using shRNA and observed a reduced phosphorylation of ERK1/2 and a significant loss of CCR7-mediated migration of T lymphocytes on fibronectin. In addition, we found that inhibition of ERK1/2 phosphorylation by U0126 resulted in a decreased phosphorylation of PLCγ1, suggesting a feedback loop between ERK1/2 and PLCγ1. Overall, these results suggest that the CCR7 signaling pathway leading to T lymphocyte migration on fibronectin is a β1 integrin-dependent pathway involving transient ERK1/2 phosphorylation, which is modulated by PLCγ1.     

Eotaxin (CCL11) induces in vivo angiogenic responses by human CCR3+ endothelial cells

Chemokines are attractants and regulators of cell activation. Several CXC family chemokine members induce angiogenesis and promote tumor growth. In contrast, the only CC chemokine, reported to play a direct role in angiogenesis is monocyte-chemotactic protein-1. Here we report that another CC chemokine, eotaxin (also known as CCL11), also induced chemotaxis of human microvascular endothelial cells. CCL11-induced chemotactic responses were comparable with those induced by monocyte-chemotactic protein-1 (CCL2), but lower than those induced by stroma-derived factor-1alpha (CXCL12) and IL-8 (CXCL8). The chemotactic activity was consistent with the expression of CCR3, the receptor for CCL11, on human microvascular endothelial cells and was inhibited by mAbs to either human CCL11 or human CCR3. CCL11 also induced the formation of blood vessels in vivo as assessed by the chick chorioallantoic membrane and Matrigel plug assays. The angiogenic response induced by CCL11 was about one-half of that induced by basic fibroblast factor, and it was accompanied by an inflammatory infiltrate, which consisted predominantly of eosinophils. Because the rat aortic sprouting assay, which is not infiltrated by eosinophils, yielded a positive response to CCL11, this angiogenic response appears to be direct and is not mediated by eosinophil products. This suggests that CCL11 may contribute to angiogenesis in conditions characterized by increased CCL11 production and eosinophil infiltration such as Hodgkin's lymphoma, nasal polyposis, endometriosis, and allergic diathesis.     

The C10/CCL6 chemokine and CCR1 play critical roles in the pathogenesis of IL-13-induced inflammation and remodeling

IL-13 is a potent stimulator of inflammation and tissue remodeling that plays a key role in the pathogenesis of a wide variety of human disorders. To further understand these responses, studies were undertaken to define the role(s) of the chemokine C10/CCL6 in the pathogenesis of IL-13-induced alterations in the murine lung. IL-13 was a very potent stimulator of C10/CCL6 mRNA and protein, and IL-13-induced inflammation, alveolar remodeling, and compliance alterations were markedly ameliorated after C10/CCL6 neutralization. Treatment with anti-C10/CCL6 decreased the levels of mRNA encoding matrix metalloproteinase-2 (MMP-2), MMP-9, and tissue inhibitor of metalloproteinase-4 (TIMP-4) in lungs from wild-type mice. C10/CCL6 neutralization also decreased the ability of IL-13 to stimulate the production of monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha, MMP-2, MMP-9, and cathepsins-K, -L, and -S and the ability of IL-13 to inhibit alpha1-antitrypsin. In accord with these findings, a targeted null mutation of CCR1, a putative C10/CCL6 receptor, also decreased IL-13-induced inflammation and alveolar remodeling and caused alterations in chemokines, proteases, and antiproteases comparable to those seen after C10/CCL6 neutralization. These C10/CCL6 and CCR1 manipulations did not alter the production of transgenic IL-13. These studies demonstrate that IL-13 is a potent stimulator of C10/CCL6 and highlight the importance of C10/CCL6 and signaling via CCR1 in the pathogenesis of the IL-13-induced pulmonary phenotype. They also describe a C10/CCL6 target gene cascade in which C10/CCL6 induction is required for optimal IL-13 stimulation of selected chemokines (monocyte chemoattractant protein-1 and MIP-1alpha) and proteases (MMP-2, MMP-9, and cathepsins-K, -L, and -S) and the inhibition of alpha1-antitrypsin.     

Ly6C induces clustering of LFA-1 (CD11a/CD18) and is involved in subtype-specific adhesion of CD8 T cells

Ly6C is a hemopoietic cell differentiation Ag found on a subset of CD8 T cells in the periphery. It is involved in target cell killing by CTLs, augments TCR-mediated activation of IL-2 and IFN-gamma production in CD8 T cells, and regulates CD8 T cell homing in vivo. In this study, we show that cross-linking of Ly6C causes clustering of LFA-1 (CD11a/CD18) on the surface of CD8 T cells via a mechanism dependent on reorganization of actin cytoskeleton and intracellular protease, calpain, but not the phosphatidylinositol 3-kinase pathway. In the capillary flow-adhesion assay, Ly6C cross-linking significantly augments lymphocyte adhesion to endothelium, and this is inhibited by an Ab that blocks LFA-1 function. Furthermore, upon in vitro cross-linking and during in vivo homing into lymph nodes, Ly6C is transiently lost from cell surface but becomes re-expressed on lymph node-resident CD8 T cells. The abilities of Ly6C to induce LFA-1 clustering and to be re-expressed after signaling-associated down-regulation may be important in regulating the homing of CD8 T cells into lymph nodes and in subsequent steps of CD8 T cell activation and effector function that again involve LFA-1.     

Activation of invariant NKT cells in early phase of experimental autoimmune encephalomyelitis results in differentiation of Ly6Chi inflammatory monocyte to M2 macrophages and improved outcome

Neuropathology in multiple sclerosis is closely linked to presence of macrophages in the CNS. Both M1 (inflammatory) and M2 (alternatively activated, noninflammatory) macrophages are found in the inflamed CNS and thought to differentiate from infiltrating monocytes. It is unclear whether the balance of M1 and M2 macrophages can be altered and whether this affects disease outcome. We show in this article that Ly6C(hi) inflammatory monocytes are the early and dominant infiltrating cells in the CNS during experimental autoimmune encephalomyelitis, a model for the acute phase of multiple sclerosis. Activation of invariant NKT (iNKT) cells reduced the frequency of Ly6C(hi) monocytes and increased the proportion of M2 macrophages in the CNS with associated improvement in neurologic impairment. In contrast, iNKT-deficient mice showed higher numbers of Ly6C(hi) monocytes, reduced M2, and much more severe disease. Adoptive transfer of M2-enriched cells to iNKT-deficient mice markedly improved neurologic impairment. In vitro and in vivo experiments showed that iNKT cells promote differentiation of monocytes to M2 macrophages in an IL-4 and CD1d-dependent process. These findings indicate that infiltrating Ly6C(hi) inflammatory monocytes are early players in acute neuroinflammation and that their frequency and differentiation can be influenced by activation of iNKT cells with resultant improvement in disease outcome.     

Esophageal cancer proliferation is mediated by cytochrome P450 2C9 (CYP2C9)

Cytochrome P450 epoxygenases (CYP450) have been recently shown to promote malignant progression. Here we investigated the mRNA and protein expression and potential clinical relevance of CYP2C9 in esophageal cancer. Highest expression was detected in esophageal adenocarcinoma (EAC; n=78) and adjacent esophageal mucosa (NEM; n=79). Levels of CYP2C9 in EAC and NEM were significantly higher compared to esophageal squamous cell carcinoma (ESCC; n=105). Early tumor stages and well-differentiated tumors showed a significantly higher CYP2C9 expression compared to progressed tumors. Moreover, CYP2C9 expression was correlated to high Ki-67 labeling indices in EAC and Ki-67 positive tumor cells in EAC and ESCC. Selective inhibition of CYP2C9 decreased tumor cell proliferation (KYSE30, PT1590 and OE19) in vitro, which was abolished by 11,12-epoxyeicosatrienoic acid (11,12-EET). Cell-cycle analysis using FACS revealed that inhibition of CYP2C9 leads to a G0/G1 phase cell-cycle arrest. CYP2C9 seems to be relevant for early esophageal cancer development by promoting tumor cell proliferation. Pharmacological inhibition of CYP2C9 might contribute to a more efficient therapy in CYP2C9 highly expressing esophageal cancers.     

Cyclooxygenase-2-derived prostaglandin D(2) is an early anti-inflammatory signal in experimental colitis

The ability of nonsteroidal anti-inflammatory drugs and cyclooxygenase-2 inhibitors to exacerbate inflammatory bowel disease suggests that prostaglandins are important anti-inflammatory mediators in this context. Prostaglandin D(2) has been suggested to exert anti-inflammatory effects. We investigated the possibility that prostaglandin D(2) derived from cyclooxygenase-2 plays an important role in downregulating colonic inflammation in rats. Colitis was induced by intracolonic administration of trinitrobenzene sulfonic acid. At various times thereafter (from 1 h to 7 days), colonic prostaglandin synthesis and myeloperoxidase activity (index of granulocyte infiltration) were measured. Prostaglandin D(2) synthesis was elevated >4-fold above controls within 1-3 h of induction of colitis, preceding significant granulocyte infiltration. Treatment with a selective cyclooxygenase-2 inhibitor abolished the increase in prostaglandin D(2) synthesis and caused a doubling of granulocyte infiltration. Colonic granulocyte infiltration was significantly reduced by administration of prostaglandin D(2) or a DP receptor agonist (BW-245C). These results demonstrate that induction of colitis results in a rapid increase in prostaglandin D(2) synthesis via cyclooxygenase-2. Prostaglandin D(2) downregulates granulocyte infiltration into the colonic mucosa, probably through the DP receptor.     

C(6)-ceramide inhibited Na(+) currents by intracellular Ca(2+) release in rat myoblasts

Ceramides are novel second messengers that may mediate signaling leading to apoptosis and the regulation of cell cycle progression. Moreover, ceramide analogs have been reported to directly modulate K(+) and Ca(2+) channels in different cell types. In this report, the effect of C(6)-ceramide on the voltage-gated inward Na(+) currents (I(Na)) in cultured rat myoblasts was investigated using whole-cell current recording and a fluorescent Ca(2+) imaging experiment. At concentrations of 1-100 microM, ceramide produced a dose-independent and reversible inhibition of I(Na). Ceramide also significantly shifted the steady-state inactivation curve of I(Na) by 16 mV toward the hyperpolarizing potential, but did not alter the steady-state activation properties. C(2)-ceramide caused a similar inhibitory effect on I(Na) amplitude. However, dihydro-C(6)-ceramide, the inactive analog of ceramide, failed to modulate I(Na). The effect of C(6)-ceramide on I(Na) was abolished by intracellular infusion of the Ca(2+)-chelating agent BAPTA, but was mimicked by application of caffeine. Blocking the release of Ca(2+) from the sarcoplasmic reticulum with xestospongin C or heparin, an inositol 1,4,5-trisphosphate (IP(3)) receptor blocker, induced a gradual increase in I(Na) amplitude and eliminated the effect of ceramide on I(Na). In contrast, ruthenium red, which is a blocker of the ryanodine-sensitive Ca(2+) receptor did not affect the action of C(6)-ceramide on I(Na). Intracellular application of the G-protein agonist GTPgammaS also induced a gradual decrease in I(Na) amplitude, while the G-protein antagonist GDPbetaS eliminated the effect of C(6)-ceramide on I(Na). Calcium imaging showed that C(6)-ceramide could give rise to a significant elevation of intracellular calcium. Our data show that increased calcium release through the IP(3)-sensitive Ca(2+) receptor, which probably occurred through the G-protein and phospholipase C pathway, may be responsible for C(6)-ceramide-induced inhibition of the I(Na) of rat myoblasts.     

Inhibition of voltage-gated Na(+) current by nanosecond pulsed electric field (nsPEF) is not mediated by Na(+) influx or Ca(2+) signaling

In earlier studies, we found that permeabilization of mammalian cells with nsPEF was accompanied by prolonged inhibition of voltage-gated (VG) currents through the plasma membrane. This study explored if the inhibition of VG Na(+) current (I(Na)) resulted from (i) reduction of the transmembrane Na(+) gradient due to its influx via nsPEF-opened pores, and/or (ii) downregulation of the VG channels by a Ca(2+)-dependent mechanism. We found that a single 300 ns electric pulse at 1.6-5.3 kV/cm triggered sustained Na(+) influx in exposed NG108 cells and in primary chromaffin cells, as detected by increased fluorescence of a Sodium Green Dye. In the whole-cell patch clamp configuration, this influx was efficiently buffered by the pipette solution so that the increase in the intracellular concentration of Na(+) ([Na](i)) did not exceed 2-3 mM. [Na](i) increased uniformly over the cell volume and showed no additional peaks immediately below the plasma membrane. Concurrently, nsPEF reduced VG I(Na) by 30-60% (at 4 and 5.3 kV/cm). In control experiments, even a greater increase of the pipette [Na(+)] (by 5 mM) did not attenuate VG I(Na), thereby indicating that the nsPEF-induced Na(+) influx was not the cause of VG I(Na) inhibition. Similarly, adding 20 mM of a fast Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) into the pipette solution did not prevent or attenuate the inhibition of the VG I(Na) by nsPEF. These findings point to possible Ca(2+)-independent downregulation of the VG Na(+) channels (e.g., caused by alteration of the lipid bilayer) or the direct effect of nsPEF on the channel.     

CD11b+Ly-6C(hi) suppressive monocytes in experimental autoimmune encephalomyelitis

Innate immune cells may regulate adaptive immunity by balancing different lineages of T cells and providing negative costimulation. In addition, CD11b(+)Gr-1(+) myeloid-derived suppressor cells have been described in tumor, parasite infection, and severe trauma models. In this study, we observe that splenic CD11b(+) cells markedly increase after experimental autoimmune encephalomyelitis (EAE) immunization, and they suppress T cell proliferation in vitro. Although >80% of CD11b(+) cells express varying levels of Gr-1, only a small population of CD11b(+)Ly-6C(high) inflammatory monocytes (IMC) can efficiently suppress T cell proliferation and induce T cell apoptosis through the production of NO. IFN-gamma produced by activated T cells is essential to induce IMC suppressive function. EAE immunization increases the frequencies of IMC in the bone marrow, spleen, and blood, but not in the lymph nodes. At the peak of EAE, IMC represent approximately 30% of inflammatory cells in the CNS. IMC express F4/80 and CD93 but not CD31, suggesting that they are immature monocytes. Furthermore, IMC have the plasticity to up-regulate NO synthase 2 or arginase 1 expression upon different cytokine treatments. These findings indicate that CD11b(+)Ly-6C(high) IMC induced during EAE priming are powerful suppressors of activated T cells. Further understanding of suppressive monocytes in autoimmune disease models may have important clinical implications for human autoimmune diseases.     

A high serum level of eotaxin (CCL 11) is associated with less radiographic progression in early rheumatoid arthritis patients

Introduction  

Prognosis in rheumatoid arthritis (RA) is difficult to assess. The aim of this study was to examine whether serum levels of a spectrum of cytokines were predictive of radiographic progression in early RA patients.     

Interleukin-4 induction of the CC chemokine TARC (CCL17) in murine macrophages is mediated by multiple STAT6 sites in the TARC gene promoter

Background  

Macrophages (Mθ) play a central role in the innate immune response and in the pathology of chronic inflammatory diseases. Macrophages treated with Th2-type cytokines such as Interleukin-4 (IL-4) and Interleukin-13 (IL-13) exhibit an altered phenotype and such alternatively activated macrophages are important in the pathology of diseases characterised by allergic inflammation including asthma and atopic dermatitis. The CC chemokine Thymus and Activation-Regulated Chemokine (TARC/CCL17) and its murine homologue (mTARC/ABCD-2) bind to the chemokine receptor CCR4, and direct T-cell and macrophage recruitment into areas of allergic inflammation. Delineating the molecular mechanisms responsible for the IL-4 induction of TARC expression will be important for a better understanding of the role of Th2 cytokines in allergic disease.     

CCL20/CCR6 feedback exaggerates epidermal growth factor receptor-dependent MUC5AC mucin production in human airway epithelial (NCI-H292) cells

Mucous hypersecretion is an important feature of obstructive airway diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. Multiple stimuli induce mucin production via activation of an epidermal growth factor receptor (EGFR) cascade, but the mechanisms that exaggerate mucin production in obstructive airway diseases remain unknown. In this study, we show that binding of CCL20, a G protein-coupled receptor (GPCR) ligand that is upregulated in the airways of subjects with obstructive airway diseases, to its unique GPCR CCR6 induces MUC5AC mucin production in human airway epithelial (NCI-H292) cells via metalloprotease TNF-α-converting enzyme (TACE)-dependent EGFR activation. We also show that EGFR activation by its potent ligand TGF-α induces reactivation of EGFR via binding of endogenously produced CCL20 to its receptor CCR6 in NCI-H292 cells but not in normal human bronchial epithelial (NHBE) cells, exaggerating mucin production in the NCI-H292 cells. In NCI-H292 cells, TGF-α stimulation induced two phases of EGFR phosphorylation (EGFR-P). The second EGFR-P was TACE-dependent and was responsible for most of the total mucin induced by TGF-α. Binding of endogenously produced CCL20 to CCR6 increased the second EGFR-P and subsequent mucin production induced by TGF-α. In NHBE cells, TGF-α-induced EGFR activation did not lead to significant CCL20 production or to EGFR rephosphorylation, and less mucin was produced. We conclude that NCI-H292 cells but not NHBE cells produce CCL20 in response to EGFR activation, which leads to a second phase of EGFR-P and subsequent exaggerated mucin production. These findings have potentially important therapeutic implications in obstructive airway diseases.     

The role of integrin alpha D beta2 (CD11d/CD18) in monocyte/macrophage migration

Integrin α_Dβ₂ (CD11d/CD18) is a multiligand macrophage receptor with recognition specificity identical to that of the major myeloid cell-specific integrin α_Mβ₂ (CD11b/CD18, Mac-1). Despite its prominent upregulation on inflammatory macrophages, the role of α_Dβ₂ in monocyte and macrophage migration is unknown. In this study, we have generated model and natural cell lines expressing different densities of α_Dβ₂ and examined their migration to various extracellular matrix proteins. When expressed at a low density, α_Dβ₂ on the surface of recombinant HEK293 cells and murine IC-21 macrophages cooperates with β₁/β₃ integrins to support cell migration. However, its increased expression on the α_Dβ₂-expressing HEK293 cells and its upregulation by PMA on the IC-21 macrophages result in increased cell adhesiveness and inhibition of cell migration. Furthermore, ligation of α_Dβ₂ with anti-α_D blocking antibodies restores β₁/β₃-driven cell migration by removing the excess α_Dβ₂-mediated adhesive bonds. Consistent with in vitro data, increased numbers of inflammatory macrophages were recovered from the inflamed peritoneum of mice after the administration of anti-α_D antibody. These results demonstrate that the density of α_Dβ₂ is critically involved in modulating macrophage adhesiveness and their migration, and suggest that low levels of α_Dβ₂ contribute to monocyte migration while α_Dβ₂ upregulation on differentiated macrophages may facilitate their retention at sites of inflammation.     

Metabolism of activated complement component C3 is mediated by the low density lipoprotein receptor-related protein/alpha(2)-macroglobulin receptor

Complement component 3 (C3) and alpha(2)-macroglobulin evolved from a common, evolutionarily old, ancestor gene. Low density lipoprotein-receptor-related protein/alpha(2)-macroglobulin receptor (LRP/alpha(2)MR), a member of the low density lipoprotein receptor family, is responsible for the clearance of alpha(2)-macroglobulin-protease complexes. In this study, we examined whether C3 has conserved affinity for LRP/alpha(2)MR. Ligand blot experiments with human (125)I-C3 on endosomal proteins show binding to a 600-kDa protein, indistinguishable from LRP/alpha(2)MR by the following criteria: it is competed by receptor-associated protein (the 39-kDa receptor-associated protein that impairs binding of all ligands to LRP/alpha(2)MR) and by lactoferrin and Pseudomonas exotoxin, other well known ligands of the multifunctional receptor. Binding of C3 is sensitive to reduction of the receptor and is Ca(2+)-dependent. All these features are typical for cysteine-rich binding repeats of the low density lipoprotein receptor family. In LRP/alpha(2)MR, they are found in four cassettes (2, 8, 10, and 11 repeats). Ligand blotting to chicken LR8 demonstrates that a single 8-fold repeat is sufficient for binding. Confocal microscopy visualizes initial surface labeling of human fibroblasts incubated with fluorescent labeled C3, which changes after 5 min to an intracellular vesicular staining pattern that is abolished in the presence of receptor-associated protein. Cell uptake is abolished in mouse fibroblasts deficient in LRP/alpha(2)MR. Native plasma C3 is not internalized. We demonstrate that the capacity to internalize C3 is saturable and exhibits a K(D) value of 17 nM. After intravenous injection, rat hepatocytes accumulate C3 in sedimentable vesicles with a density typical for endosomes. In conclusion, our ligand blot and uptake studies demonstrate the competence of the LRP/alpha(2)MR to bind and endocytose C3 and provide evidence for an LRP/alpha(2)MR-mediated system participating in C3 metabolism.     

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.