A functional IFN-gamma-inducible protein-10/CXCL10-specific receptor expressed by epithelial and endothelial cells that is neither CXCR3 nor glycosaminoglycan.

Interferon-gamma-inducible protein-10 (IP-10)/CXCL10 is a CXC chemokine that attracts T lymphocytes and NK cells through activation of CXCR3, the only chemokine receptor identified to date that binds IP-10/CXCL10. We have found that several nonhemopoietic cell types, including epithelial and endothelial cells, have abundant levels of a receptor that binds IP-10/CXCL10 with a Kd of 1-6 nM. Surprisingly, these cells expressed no detectable CXCR3 mRNA. Furthermore, no cell surface expression of CXCR3 was detectable by flow cytometry, and the binding of 125I-labeled IP-10/CXCL10 to these cells was not competed by the other high affinity ligands for CXCR3, monokine induced by IFN-gamma/CXCL9, and I-TAC/CXCL11. Although IP-10/CXCL10 binds to cell surface heparan sulfate glycosaminoglycan (GAG), the receptor expressed by these cells is not GAG, since the affinity of IP-10/CXCL10 for this receptor is much higher than it is for GAG, its binding is not competed by platelet factor 4/CXCL4, and it is present on cells that are genetically incapable of synthesizing GAG. Furthermore, in contrast to IP-10/CXCL10 binding to GAG, IP-10/CXCL10 binding to these cells induces new gene expression and chemotaxis, indicating the ability of this receptor to transduce a signal. These high affinity IP-10/CXCL10-specific receptors on epithelial cells may be involved in cell migration and, perhaps, in the spread of metastatic cells as they exit from the vasculature. (All of the lung cancer cells we examined also expressed CXCR4, which has been shown to play a role in breast cancer metastasis.) CXCR3-negative endothelial cells may also use this receptor to mediate the angiostatic activity of IP-10/CXCL10, which is also expressed by these cells in an autocrine manner.     

CXCR3 and heparin binding sites of the chemokine IP-10 (CXCL10)

The chemokine IP-10 (interferon-inducible protein of 10 kDa, CXCL10) binds the G protein-coupled receptor CXCR3, which is found mainly on activated T cells and NK cells, and plays an important role in Th1-type inflammatory diseases. IP-10 also binds to glycosaminoglycans (GAGs), an interaction thought to be important for its sequestration on endothelial and other cells. In this study, we performed an extensive mutational analysis to identify the CXCR3 and heparin binding sites of murine IP-10. The mutants were characterized for heparin binding, CXCR3 binding, and the ability to induce chemotaxis, Ca(2+) flux, and CXCR3 internalization. Double mutations neutralizing adjacent basic residues at the C terminus did not lead to a significant reduction in heparin binding, indicating that the main heparin binding site of IP-10 is not along the C-terminal alpha helix. Alanine exchange of Arg-22 had the largest effect on heparin binding, with residues Arg-20, Ile-24, Lys-26, Lys-46, and Lys-47 further contributing to heparin binding. A charge change mutation of Arg-22 resulted in further reduction in heparin binding. The N-terminal residue Arg-8, preceding the first cysteine, was critical for CXCR3 signaling. Mutations of charged and uncharged residues in the loop regions of residues 20-24 and 46-47, which caused reduced heparin binding, also resulted in reduced CXCR3 binding and signaling. CXCR3 expressing GAG-deficient Chinese hamster ovary cells revealed that GAG binding was not required for IP-10 binding and signaling through CXCR3, which suggests that the CXCR3 and heparin binding sites of IP-10 are partially overlapping.     

Activation of p38(MAPK) mediates the angiostatic effect of the chemokine receptor CXCR3-B

Chemokines binding the CXCR3 receptor have been shown to inhibit angiogenesis via the CXCR3-B isoform, but the underlying molecular mechanisms are unknown. Aim of this study was to elucidate the effects of CXCR3-B on activation of members of the mitogen-activated protein kinase family, and to explore the relevance of defined signaling pathways to the angiostatic effects of CXCR3-B ligands. Human embryonic kidney (HEK) 293 cells were transfected with expression vectors encoding for CXCR3-A or CXCR3-B. In cells expressing CXCR3-A, CXCL10 (IP-10) at nanomolar concentrations induced activation of ERK, Akt, and Src, as previously described in human vascular pericytes. In HEK-293 cells expressing CXCR3-B, exposure to CXCL10 in the micromolar concentration range led to activation of the p38(MAPK) pathway, as indicated by phosphorylation of p38(MAPK) itself, and of MKK3/6 and MAPKAPK-2, that lie upstream and downstream of p38(MAPK), respectively. Similar results were obtained in cells stimulated with CXCL4 (PF4), a specific ligand of CXCR3-B. In contrast, CXCL4 was unable to activate p38(MAPK) in mock-transfected HEK-293 cells. Only a modest induction of ERK or JNK was observed upon CXCR3-B activation. In human microvascular endothelial cells, which selectively express CXCR3-B, in a cell cycle-dependent fashion, CXCL10 and CXCL4 increased the enzymatic activity of p38(MAPK). Pharmacologic inhibition of p38(MAPK) by SB302580 resulted in a significant increase in DNA synthesis and in reversal of the inhibitory action of CXCL10. In conclusion, the p38(MAPK) pathway is a downstream effector of CXCR3-B implicated in the angiostatic action of this chemokine receptor.     

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient''s blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis¹. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.     

Oligomerization of CXCL10 is necessary for endothelial cell presentation and in vivo activity

The chemokine IFN-gamma-inducible protein of 10 kDa (IP-10; CXCL10) plays an important role in the recruitment of activated T lymphocytes into sites of inflammation by interacting with the G protein-coupled receptor CXCR3. IP-10, like other chemokines, forms oligomers, the role of which has not yet been explored. In this study, we used a monomeric IP-10 mutant to elucidate the functional significance of oligomerization. Although monomeric IP-10 had reduced binding affinity for CXCR3 and heparin, it was able to induce in vitro chemotaxis of activated T cells with the same efficacy as wild-type IP-10. However, monomeric IP-10 was unable to induce recruitment of activated CD8+ T cells into the airways of mice after intratracheal instillation. Use of a different IP-10 mutant demonstrated that this inability was due to lack of oligomerization rather than reduced CXCR3 or heparin binding. Molecular imaging demonstrated that both wild-type and monomeric IP-10 were retained in the lung after intratracheal instillation. However, in vitro binding assays indicated that wild-type, but not monomeric, IP-10 was retained on endothelial cells and could induce transendothelial chemotaxis of activated T cells. We therefore propose that oligomerization of IP-10 is required for presentation on endothelial cells and subsequent transendothelial migration, an essential step for lymphocyte recruitment in vivo.     

IFN-gamma-inducible protein-10 (CXCL10) is hepatoprotective during acute liver injury through the induction of CXCR2 on hepatocytes.

IFN-gamma-inducible protein-10 (IP-10/CXCL10) is a non-ELR-CXC chemokine that is present during various forms of acute and chronic liver injury. The purpose of this study was to explore the role of IP-10 during acute liver injury induced by acetaminophen (APAP). After a 400 mg/kg APAP challenge in fasted CD-1 mice, immunoreactive levels of IP-10 were dramatically elevated in the serum within 8 h. CXCR3, the receptor for IP-10, was up-regulated in the liver. Mice that received an i.v. injection of rIP-10 10 h after APAP challenge exhibited a dramatic reduction in alanine aminotransferase 8 h later. Histologic analysis confirmed that the delayed IP-10 therapy dramatically improved the appearance of the liver when examined 48 h after APAP. The therapeutic effect of IP-10 was associated with a marked increase in CXCR2 expression on hepatocytes. Neutralization of CXCR2 during IP-10 therapy resulted in an abrogation of the hepatoprotective effect of IP-10. Furthermore, IP-10 treatment of cultured hepatocytes stimulated a CXCR2-dependent proliferative response. In conclusion, IP-10 has a hepatoregenerative effect in a murine model of acute liver injury that is dependent on its up-regulation of CXCR2 on hepatocytes.     

Dysregulated expression of MIG/CXCL9, IP-10/CXCL10 and CXCL16 and their receptors in systemic sclerosis

Introduction  

Systemic sclerosis (SSc) is characterized by fibrosis and microvascular abnormalities including dysregulated angiogenesis. Chemokines, in addition to their chemoattractant properties, have the ability to modulate angiogenesis. Chemokines lacking the enzyme-linked receptor (ELR) motif, such as monokine induced by interferon-γ (IFN-γ) (MIG/CXCL9) and IFN-inducible protein 10 (IP-10/CXCL10), inhibit angiogenesis by binding CXCR3. In addition, CXCL16 promotes angiogenesis by binding its unique receptor CXCR6. In this study, we determined the expression of these chemokines and receptors in SSc skin and serum.     

The CXCR3 binding chemokine IP-10/CXCL10: structure and receptor interactions

The structure of IP-10 was solved by NMR spectroscopy and represents the first structure from the class of agonists toward the receptor CXCR3. CXCR3 binding chemokines are unique in their ability to bind receptors from both the CC and CXC classes of chemokine receptors. An unusual structural feature of IP-10 was identified that may provide the basis for the ability of IP-10 to bind both CXCR3 and CCR3. The surface of IP-10 that interacts with the N-terminus of CXCR3 was defined by monitoring changes in the NMR spectrum of IP-10 upon addition of a CXCR3 N-terminal peptide. These studies indicated that the interaction involves a hydrophobic cleft, formed by the N-loop and 40s-loop region of IP-10, similar to the interaction surface observed for other chemokines such as IL-8. An additional region of interaction was observed that consists of a hydrophobic cleft formed by the N-terminus of IP-10 and 30s-loop of IP-10.     

Overexpression of IFN-induced protein 10 and its receptor CXCR3 in myasthenia gravis

Myasthenia gravis (MG) and its animal model, experimental autoimmune MG (EAMG), are autoimmune disorders in which the acetylcholine receptor (AChR) is the major autoantigen. Microarray technology was used to identify new potential drug targets for treatment of myasthenia that would reduce the need for the currently used nonspecific immunosuppression. The chemokine IFN-gamma-inducible protein 10 (IP-10; CXCL10), a CXC chemokine, and its receptor, CXCR3, were found to be overexpressed in lymph node cells of EAMG rats. Quantitative real-time PCR confirmed these findings and revealed up-regulated mRNA levels of another chemoattractant that activates CXCR3, monokine induced by IFN-gamma (Mig; CXCL9). TNF-alpha and IL-1beta, which act synergistically with IFN-gamma to induce IP-10, were also up-regulated. These up-regulations were observed in immune response effector cells, namely, lymph node cells, and in the target organ of the autoimmune attack, the muscle of myasthenic rats, and were significantly reduced after suppression of EAMG by mucosal tolerance induction with an AChR fragment. The relevance of IP-10/CXCR3 signaling in myasthenia was validated by similar observations in MG patients. A significant increase in IP-10 and CXCR3 mRNA levels in both thymus and muscle was observed in myasthenic patients compared with age-matched controls. CXCR3 expression in PBMC of MG patients was markedly increased in CD4(+), but not in CD8(+), T cells or in CD19(+) B cells. Our results demonstrate a positive association of IP-10/CXCR3 signaling with the pathogenesis of EAMG in rats as well as in human MG patients.     

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.     

Discovery of a pyrazole derivative promoting angiogenesis through modulating reactive oxygen species and interferon-inducible protein 10 levels

Human umbilical cord vascular endothelial cells (HUVECs) cultured without serum and fibroblast growth factor-2 is an in vitro model of ischemic conditions. Our previous study showed that ethyl 3-(o-chlorophenyl)-5-methyl-1-phenyl-1H-pyrazole-4-carboxylate (MPD) could inhibit apoptosis of HUVECs in this model. In this study, we investigated the effect of MPD on angiogenesis and the possible mechanisms. Capillary-like tube formation assay on Matrigel and cell migration assay were performed to investigate the effect of MPD on angiogenesis. The reactive oxygen species (ROS) and interferon-inducible protein 10 (IP-10) levels were respectively evaluated by intracellular ROS assay and western blot analysis. MPD at 5 and 10 ??M promoted vascular structure formation and HUVEC migration in an in vitro ischemic model. MPD promoted angiogenesis through elevating ROS levels and depressing IP-10 level. ROS seemed to be necessary for angiogenesis, and a high level of IP-10 inhibited angiogenesis in ischemic state. ROS provide clues for seeking new key factors involved in angiogenesis. IP-10 may become a new target for future therapeutic intervention. MPD is a good tool for investigating the mechanism of angiogenesis, and MPD might be useful in the development of new drugs in therapy of ischemic diseases.     

CXCR4/CXCL12 axis promotes VEGF-mediated tumor angiogenesis through Akt signaling pathway

CXC chemokine receptor 4 (CXCR4) has been shown to play a critical role in chemotaxis and homing, which are key steps in cancer metastasis. There is also increasing evidence that links this receptor to angiogenesis; however, its molecular basis remains elusive. Vascular endothelial growth factor (VEGF), one of the major angiogenic factors, promotes the formation of leaky tumor vasculatures that are the hallmarks of tumor progression. Here, we investigated whether CXCR4 induces the expression of VEGF through the PI3K/Akt pathway. Our results showed that CXCR4/CXCL12 induced Akt phosphorylation, which resulted in upregulation of VEGF at both the mRNA and protein levels. Conversely, blocking the activation of Akt signaling led to a decrease in VEGF protein levels; blocking CXCR4/CXCL12 interaction with a CXCR4 antagonist suppressed tumor angiogenesis and growth in vivo. Furthermore, VEGF mRNA levels correlated well with CXCR4 mRNA levels in patient tumor samples. In summary, our study demonstrates that the CXCR4/CXCL12 signaling axis can induce angiogenesis and progression of tumors by increasing expression of VEGF through the activation of PI3K/Akt pathway. Our findings suggest that targeting CXCR4 could provide a potential new anti-angiogenic therapy to suppress the formation of both primary and metastatic tumors.     

CXCR3 expression and activation of eosinophils: role of IFN-gamma-inducible protein-10 and monokine induced by IFN-gamma

CXC chemokine receptor 3 (CXCR3), predominately expressed on memory/activated T lymphocytes, is a receptor for both IFN-gamma-inducible protein-10 (gamma IP-10) and monokine induced by IFN-gamma (Mig). We report a novel finding that CXCR3 is also expressed on eosinophils. gamma IP-10 and Mig induce eosinophil chemotaxis via CXCR3, as documented by the fact that anti-CXCR3 mAb blocks gamma IP-10- and Mig-induced eosinophil chemotaxis. gamma IP-10- and Mig-induced eosinophil chemotaxis are up- and down-regulated by IL-2 and IL-10, respectively. Correspondingly, CXCR3 protein and mRNA expressions in eosinophils are up- and down-regulated by IL-2 and IL-10, respectively, as detected using flow cytometry, immunocytochemical assay, and a real-time quantitative RT-PCR technique. gamma IP-10 and Mig act eosinophils to induce chemotaxis via the cAMP-dependent protein kinase A signaling pathways. The fact that gamma IP-10 and Mig induce an increase in intracellular calcium in eosinophils confirms that CXCR3 exists on eosinophils. Besides induction to chemotaxis, gamma IP-10 and Mig also activate eosinophils to eosinophil cationic protein release. These results indicate that CXCR3-gamma IP-10 and -Mig receptor-ligand pairs as well as the effects of IL-2 and IL-10 on them may be especially important in the cytokine/chemokine environment for the pathophysiologic events of allergic inflammation, including initiation, progression, and termination in the processes.     

Angiostatic factors in the pulmonary endarterectomy material from chronic thromboembolic pulmonary hypertension patients cause endothelial dysfunction

Chronic thromboembolic pulmonary hypertension (CTEPH) is a rare disease with persistent thrombotic occlusion or stenosis of the large pulmonary arteries resulting in pulmonary hypertension. Surgical removal of the neointimal layer of these vessels together with the non-resolved thrombus consisting of organized collagen-rich fibrotic areas with partly recanalized regions is the treatment of choice (pulmonary endarterectomy, PEA). The present study investigates endothelial cells isolated from such material as well as factors present in the surgical PEA material, which may contribute to impairment of recanalization and thrombus non-resolution. We observed muscularized vessels and non-muscularized vessels in the PEA material. The isolated endothelial cells from the PEA material showed significantly different calcium homeostasis as compared to pulmonary artery endothelial cells (hPAECs) from normal controls. In the supernatant (ELISA) as well as on the tissue level (histochemical staining) of the PEA material, platelet factor 4 (PF4), collagen type I and interferon-gamma-inducible 10 kD protein (IP-10) were detected. CXCR3, the receptor for PF4 and IP-10, was particularly elevated in the distal parts of the PEA material as compared to human control lung (RT-PCR). PF4, collagen type I and IP-10 caused significant changes in calcium homeostasis and affected the cell proliferation, migration and vessel formation in hPAECs. The presence of angiostatic factors like PF4, collagen type I and IP-10, as recovered from the surgical PEA material from CTEPH patients, may lead to changes in calcium homeostasis and endothelial dysfunction.     

Anti-tumor activities of the angiogenesis inhibitors interferon-inducible protein-10 and the calreticulin fragment vasostatin

Tumor growth depends upon an adequate supply of oxygen and nutrients achieved through angiogenesis and maintenance of an intact tumor vasculature. Therapy with individual agents that target new vessel formation or existing vessels has suppressed experimental tumor growth, but rarely resulted in the eradication of tumors. We therefore tested the combined anti-tumor activity of vasostatin and interferon-inducible protein-10 (IP-10), agents that differently target the tumor vasculature. Vasostatin, a selective and direct inhibitor of endothelial cell proliferation, significantly reduced Burkitt tumor growth and tumor vessel density. IP-10, an "angiotoxic" chemokine, caused vascular damage and focal necrosis in Burkitt tumors. When combined, vasostatin plus IP-10 reduced tumor growth more effectively than each agent alone, but complete tumor regression was not observed. Microscopically, these tumors displayed focal necrosis and reduction in vessel density. Combination therapy with the inhibitors of angiogenesis vasostatin and IP-10 is effective in reducing the rate of tumor growth but fails to induce tumor regression, suggesting that curative treatment may require supplemental drugs targeting directly the tumor cells.     

CXCL10 Can Inhibit Endothelial Cell Proliferation Independently of CXCR3

CXCL10 (or Interferon-inducible protein of 10 kDa, IP-10) is an interferon-inducible chemokine with potent chemotactic activity on activated effector T cells and other leukocytes expressing its high affinity G protein-coupled receptor CXCR3. CXCL10 is also active on other cell types, including endothelial cells and fibroblasts. The mechanisms through which CXCL10 mediates its effects on non-leukocytes is not fully understood. In this study, we focus on the anti-proliferative effect of CXCL10 on endothelial cells, and demonstrate that CXCL10 can inhibit endothelial cell proliferation in vitro independently of CXCR3. Four main findings support this conclusion. First, primary mouse endothelial cells isolated from CXCR3-deficient mice were inhibited by CXCL10 as efficiently as wildtype endothelial cells. We also note that the proposed alternative splice form CXCR3-B, which is thought to mediate CXCL10''s angiostatic activity, does not exist in mice based on published mouse CXCR3 genomic sequences as an in-frame stop codon would terminate the proposed CXCR3-B splice variant in mice. Second, we demonstrate that human umbilical vein endothelial cells and human lung microvascular endothelial cells that were inhibited by CXL10 did not express CXCR3 by FACS analysis. Third, two different neutralizing CXCR3 antibodies did not inhibit the anti-proliferative effect of CXCL10. Finally, fourth, utilizing a panel of CXCL10 mutants, we show that the ability to inhibit endothelial cell proliferation correlates with CXCL10''s glycosaminoglycan binding affinity and not with its CXCR3 binding and signaling. Thus, using a very defined system, we show that CXCL10 can inhibit endothelial cell proliferation through a CXCR3-independent mechanism.     

CXCR3 requires tyrosine sulfation for ligand binding and a second extracellular loop arginine residue for ligand-induced chemotaxis

CXCR3 is a G-protein-coupled seven-transmembrane domain chemokine receptor that plays an important role in effector T-cell and NK cell trafficking. Three gamma interferon-inducible chemokines activate CXCR3: CXCL9 (Mig), CXCL10 (IP-10), and CXCL11 (I-TAC). Here, we identify extracellular domains of CXCR3 that are required for ligand binding and activation. We found that CXCR3 is sulfated on its N terminus and that sulfation is required for binding and activation by all three ligands. We also found that the proximal 16 amino acid residues of the N terminus are required for CXCL10 and CXCL11 binding and activation but not CXCL9 activation. In addition, we found that residue R216 in the second extracellular loop is required for CXCR3-mediated chemotaxis and calcium mobilization but is not required for ligand binding or ligand-induced CXCR3 internalization. Finally, charged residues in the extracellular loops contribute to the receptor-ligand interaction. These findings demonstrate that chemokine activation of CXCR3 involves both high-affinity ligand-binding interactions with negatively charged residues in the extracellular domains of CXCR3 and a lower-affinity receptor-activating interaction in the second extracellular loop. This lower-affinity interaction is necessary to induce chemotaxis but not ligand-induced CXCR3 internalization, further suggesting that different domains of CXCR3 mediate distinct functions.     

Cell surface heparan sulfate participates in CXCL1-induced signaling

The CXC subfamily of chemokines plays an important role in diverse processes, including inflammation, wound healing, growth regulation, angiogenesis, and tumorigenesis. The ELR-CXC chemokine, CXCL1 or MGSA/GROalpha, is traditionally considered to attract neutrophils to sites of inflammation. The non-ELR-CXC chemokine, CXCL10 or IP-10, is chemotactic for monocytes, B cells, and activated T lymphocytes. In addition to its role in leukocyte migration, CXCL10 inhibits the angiogenic functions of the ELR-CXC chemokines as well as bFGF and VEGF. Heparan sulfate proteoglycans (HSPGs) are required for the interaction of bFGF and vEGF ligands and their receptors. However, the role of HSPGs in regulating the ELR-chemokines signaling and biological functions is poorly understood. We show here that the CXCL1 maximal binding to CXCR2 expressed on HEK293 and CHO-K1 cells is dependent on the presence of cell surface HSPGs. The cell surface HSPGs on cells are required for CXCL1-induced PAK1 activation. Moreover, CXCL10 can inhibit CXCL1-induced PAK1 and ERK activation as well as the CXCL1-induced chemotaxis through decreasing CXCL1 binding to cell surface heparan sulfate. These data indicate that HSPGs are involved in modulating CXCL1-induced PAK1 activation and chemotaxis through regulating CXCL1 binding activity to CXCR2 receptor. CXCL10 inhibits CXCL1-induced PAK1 activation and chemotaxis by interfering with appropriate binding of CXCL1 to CXCR2 receptor.