Characterization of the stromal cell-derived factor-1alpha-heparin complex

The binding of chemokines to glycosaminoglycans is thought to play a crucial role in chemokine functions. It has recently been shown that stromal cell-derived factor-1alpha (SDF-1alpha), a CXC chemokine with potent anti-human immunodeficiency virus activity, binds to heparan sulfate through a typical consensus sequence for heparin recognition (BBXB, where B is a basic residue KHLK, amino acids 24-27). Calculation of the accessible surface, together with the electrostatic potential of the SDF-1alpha dimer, revealed that other amino acids (Arg-41 and Lys-43) are found in the same surface area and contribute to the creation of a positively charged crevice, located at the dimer interface. GRID calculations confirmed that this binding site will be the most energetically favored area for the interaction with sulfate groups. Site-directed mutagenesis and surface plasmon resonance-based binding assays were used to investigate the structural basis for SDF-1alpha binding to heparin. Among the residues clustered in this basic surface area, Lys-24 and Lys-27 have dominant roles and are essential for interaction with heparin. Amino acids Arg-41 and Lys-43 participate in the binding but are not strictly required for the interaction to take place. Direct binding assays and competition analysis with monoclonal antibodies also permitted us to show that the N-terminal residue (Lys-1), an amino acid critical for receptor activation, is involved in complex formation. Binding studies with selectively desulfated heparin, heparin oligosaccharides, and heparitinase-resistant heparan sulfate fragments showed that a minimum size of 12-14 monosaccharide units is required for efficient binding and that 2-O- and N-sulfate groups have a dominant role in the interaction. Finally, the heparin-binding site was identified on the crystal structure of SDF-1alpha, and a docking study was undertaken. During the energy minimization process, heparin lost its perfect ribbon shape and fitted the protein surface perfectly. In the model, Lys-1, Lys-24, Lys-27, and Arg-41 were found to have the major role in binding a polysaccharide fragment consisting of 13 monosaccharide units.     

Matrix metalloproteinase activity inactivates the CXC chemokine stromal cell-derived factor-1

Chemokines provide directional cues for leukocyte migration and activation that are essential for normal leukocytic trafficking and for host responses during processes such as inflammation, infection, and cancer. Recently we reported that matrix metalloproteinases (MMPs) modulate the activity of the CC chemokine monocyte chemoattractant protein-3 by selective proteolysis to release the N-terminal tetrapeptide. Here we report the N-terminal processing, also at position 4-5, of the CXC chemokines stromal cell-derived factor (SDF)-1alpha and beta by MMP-2 (gelatinase A). Robustness of the MMP family for chemokine cleavage was revealed from identical cleavage site specificity of MMPs 1, 3, 9, 13, and 14 (MT1-MMP) toward SDF-1; selectivity was indicated by absence of cleavage by MMPs 7 and 8. Efficient cleavage of SDF-1alpha by MMP-2 is the result of a strong interaction with the MMP hemopexin C domain at an exosite that overlaps the monocyte chemoattractant protein-3 binding site. The association of SDF-1alpha with different glycosaminoglycans did not inhibit cleavage. MMP cleavage of SDF-1alpha resulted in loss of binding to its cognate receptor CXCR-4. This was reflected in a loss of chemoattractant activity for CD34(+) hematopoietic progenitor stem cells and pre-B cells, and unlike full-length SDF-1alpha, the MMP-cleaved chemokine was unable to block CXCR-4-dependent human immunodeficiency virus-1 infection of CD4(+) cells. These data suggest that MMPs may be important regulatory proteases in attenuating SDF-1 function and point to a deep convergence of two important networks, chemokines and MMPs, to regulate leukocytic activity in vivo.     

Dual role of H-Ras in regulation of lymphocyte function antigen-1 activity by stromal cell-derived factor-1alpha: implications for leukocyte transmigration

We investigated the role of H-Ras in chemokine-induced integrin regulation in leukocytes. Stimulation of Jurkat T cells with the CXC chemokine stromal cell-derived factor-1alpha (SDF-1alpha) resulted in a rapid increase in the phosphorylation, i.e., activation of extracellular signal receptor-activated kinase (ERK) but not c-Jun NH(2)-terminal kinase or p38 kinase, and phosphorylation of Akt, reflecting phosphatidylinositol 3-kinase (PI3-K) activation. Phosphorylation of ERK in Jurkat cells was enhanced and attenuated by expression of dominant active (D12) or inactive (N17) forms of H-Ras, respectively, while N17 H-Ras abrogated SDF-1alpha-induced Akt phosphorylation. SDF-1alpha triggered a transient regulation of adhesion to intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 mediated by lymphocyte function antigen-1 (LFA-1) and very late antigen-4 (VLA-4), respectively, and a rapid increase in LFA-1 binding to soluble ICAM-1.Ig, which was inhibited by D12 but not N17 H-Ras. Both D12 and N17 H-Ras abrogated the regulation of LFA-1 but not VLA-4 avidity, and impaired LFA-1-mediated transendothelial chemotaxis but not VLA-4-dependent transmigration induced by SDF-1alpha. Analysis of the mutant Jurkat J19 clone revealed LFA-1 with constitutively high affinity and reduced ERK phosphorylation, which were partially restored by expression of active H-Ras. Inhibition of PI3-K blocked the up-regulation of Jurkat cell adhesion to ICAM-1 by SDF-1alpha, whereas inhibition of mitogen-activated protein kinase kinase impaired the subsequent down-regulation and blocking both pathways abrogated LFA-1 regulation. Our data suggest that inhibition of initial PI3-K activation by inactive H-Ras or sustained activation of an inhibitory ERK pathway by active H-Ras prevail to abolish LFA-1 regulation and transendothelial migration induced by SDF-1alpha in leukocytes, establishing a complex and bimodal involvement of H-Ras.     

The potential of statin and stromal cell-derived factor-1 to promote angiogenesis

Angiogenesis requires the mobilization of progenitor cells from the bone marrow (BM) and homing of progenitor cells to ischemic tissue. The cholesterol lowering drug Statins can stimulate angiogenesis via mobilization of BM derived endothelial progenitor cells (EPCs), promoting EPC migration, and inhibiting EPC apoptosis. The chemokine stromal cell-derived factor-1 (SDF-1) augments EPC chemotaxis, facilitates EPC incorporation into the neovasculature. The combined use of a statin to mobilize EPCs and local overexpression of SDF-1 to augment EPC homing to ischemic muscle resulted in superior angiogenesis versus use of either agent alone. Their effects are through augmenting EPC mobilization, incorporation, proliferation, migration and tube formation while inhibiting EPC apoptosis. Statin and SDF-1 therefore display synergism in promoting neovascularization by improving reperfusion of ischemic muscle, increasing progenitor cell presentation and capillary density in ischemic muscle, and diminishing apoptosis. These results suggest that the combination of statin and SDF-1 may be a new therapeutic strategy in the treatment of limb ischemia.Key words: angiogenesis, endothelial progenitor cells, statin, SDF-1, migrationAngiogenesis is the process by which new vessels form in ischemic tissue. The cytokine Stromal Cell Derived Factor-1 (SDF-1) is released into the circulation in response to ischemia and is an initiating signal in the angiogenesis process. SDF-1 mobilizes bone marrow cells (BMC) by binding to the cell surface receptor CXCR4. BMCs then enter the circulation and migrate to the ischemic site following the SDF-1 gradient. On arrival, BMCs promote angiogenesis by providing cellular elements such as endothelial cells (EC) and perivascular cells and also by secreting signaling proteins that mature the angiogenesis process. BMC surface CXCR4 expression and the SDF-1/CXCR4 interaction are essential for BMC to home to the injured site.Cell-based strategies to improve neovascularization of ischemic tissue have been achieved by injecting mononuclear cells derived from either BM¹ or peripheral blood, directly into ischemic muscle,² or by mobilizing BM-MNC with cytokines³ or other drugs such as statins.^4–6Statins are 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors and are primarily used to lower circulating cholesterol levels. In addition to reducing cholesterol synthesis, inhibition of the mevalonate pathway prevents synthesis of isoprenoid intermediates including geranylgeranylpyrophosphate. Geranylgeranylation is important in the posttranslational modification of intracellular signaling proteins, including Rho GTPases. This mechanism underlies many of the pleiotropic effects including the ability of statins to stabilize endothelial nitric oxide synthase mRNA and increase nitric oxide biosynthesis. In fact, statins have been shown to protect against ischemic injury of the heart and stimulate angiogenesis in ischemic limbs of normocholesterolemic animals.^7,8 The mechanism of action of statins has been demonstrated via mobilization of BM endothelial progenitor cells (EPCs) and facilitation of EPC incorporation into the neovasculature through a phosphoinositide-3 (PI-3) kinase-dependent pathway.^4–6 Statins have also been reported to enhance EPC migration, augment EPC chemotaxis and inhibit EPC apoptosis both in vitro and in vivo.^4,9,10SDF-1, an 89-amino acid polypeptide, is a member of the chemokine CXC subfamily originally isolated from murine bone marrow stromal cells.¹¹ SDF-1 was initially identified as a potent chemoattractant for lymphocytes and monocytes, and as an enhancer of B cell proliferation. SDF-1 is considered to be a key regulator of hematopoietic stem cell trafficking between BM and the peripheral circulation. SDF-1 is highly expressed in ischemic tissues.^12,13 Elevation of SDF-1 levels in peripheral blood results in BMC mobilization to the peripheral circulation with a concurrent decrease within the BM.¹⁴ SDF-1 not only mobilizes progenitor cells in BM but also directs them to the ischemic site by promoting cell migration and proliferation.^3,15 SDF-1 may generate a gradient similar to developmental morphogens during ischemia that provides the cues and directions for progenitor cell mobilization into peripheral blood and homing to ischemic tissues.^16,17 Furthermore, SDF-1 also reduces EPC apoptosis and enhances survival of the progenitor cells.^3,18 SDF-1, either delivered locally in its protein form,^3,19,20 or generated in situ via plasmid and viral vector-mediated gene expression,^10,21,22 enhances neovascularization by augmenting EPC recruitment into ischemic tissues.SDF-1 binding to its receptor CXCR4 on the cell surface provides essential signals for mobilization and homing of EPCs to the injured site.^23–25 SDF-1 binding with CXCR4 triggers internalization of CXCR4. This SDF-1/CXCR4 interaction results in elevation of cytoplasmic Ca²⁺ levels²⁶ and phosphorylation of PI-3 kinase and other protein kinases, e.g. Akt,²¹ MEK/ERK^27,28 and Janus kinase (JAK)-2.²⁹ Activation of Akt protein kinase further upregulates the activity of eNOS by increasing both eNOS expression and phosphorylation, which in turn catalyzes the production of nitric oxide (NO), an important signal molecule for vascular protection and remodeling.^21,26 Disruption of SDF-1/CXCR4 interaction impaired incorporation of EPC into sites of ischemia, and disturbed ischemic limb neo-vascularization.³⁰To explore if the combined use of a statin to mobilize BM EPCs and local overexpression of SDF-1 to augment EPC homing to ischemic muscle will result in superior angiogenesis versus use of either agent alone, we used the murine hindlimb ischemia model to determine the effects of Fluvastatin and SDF-1 on angiogenesis.¹⁰ Fluvastatin (5 mg/kg) was injected intra-peritoneally into the mice daily for 7 days to mobilize progenitor cells prior to ischemia-inducing surgery. NIH 3T3 cells transduced with the retroviral vector carrying SDF-1 gene were injected I.M. into the ischemic limb after surgery to locally deliver SDF-1 to ischemic muscle.²² The number of circulating EPCs increased 9–18 fold seven days post statin/SDF-1 treatment.Our data of single treatment with Fluvastatin are consistent with the previous reports that statins not only augment mobilization of progenitor cells by increasing circulating EPC originated from BM,^4,31 but also modulate their differentiation. We further give a new insight view of the mechanism for statin induced EPC mobilization. We found that statin induced activation of matrix metalloproteinases (MMP)-2 and -9 in EPC. The increased MMP activity could result in degradation of extracellular matrix.¹⁷ Progenitor cells will be such mobilized into circulation when the cellular attachment is reduced within the bone marrow niches. We show that statin alone can enhance the phosphorylation of Akt, promote EPC proliferation, migration and inhibit cell apoptosis in vitro. The proangiogenic effects of statin are also illustrated in vivo using a murine hind-limb ischemia model. In this model, Fluvastatin treatment results in more EPC in circulation, more BM derived progenitor cells in ischemic muscle, more cell proliferation, enhanced capillary formation, and diminished cell apoptosis; these effects end up in improved reperfusion versus control. The beneficial effects of statin on angiogenesis are independent of cholesterol since the total serum cholesterol level is not changed by Fluvastatin treatment under these experimental conditions.To be noted, the effect of statins on EPCs was found to be concentration dependent. EPC proliferation, migration and the inhibition of apoptosis are enhanced at low statin concentrations (10 nM and 100 nM) but are significantly inhibited at a higher statin concentration (1,000 nM). The toxic effect of statin at high concentration cannot be compensated by addition of SDF-1, indicating that Statin causes apoptosis in a pathway different from the pathway that SDF-1 uses to prevent EPC apoptosis. Increased apoptosis at the higher statin concentration could explain the reversed effect of stain in angiogenesis. These findings are consistent with the reports in which statins were found to have proangiogenic effects at low therapeutic concentrations but angiostatic effects at high concentrations, the latter effect being reversible by geranylgeranyl pyrophosphate.^32,33Combined statin and SDF-1 treatment significantly enhanced angiogenesis versus treatment with either reagent alone. More cell proliferation and less apoptosis were observed both in vitro and in vivo, along with increased cell migration and tube formation in vitro, and enhanced progenitor cell incorporation and higher capillary density in ischemic tissue in vivo. It is interesting to note that neither statin nor SDF-1 alone promotes EPC tube formation, but combined treatment results in significant EPC tube formation. These results suggest that SDF-1 and statin have different mechanisms of action with regards to the promotion of neovascularization. It is possible that each drug affects a specific subset of progenitor cells.The facilitative effect of both statin and SDF-1 on EPC proliferation and migration is involved with Akt phosphorylation and endothelial nitric oxide synthase (eNOS) activation. The mechanism by which statins promote angiogenesis is through, at least partly, improved nitric oxide bioavailability. Statins have been reported to induce eNOS mRNA stability³⁴ and eNOS activity through a PI3k/Akt dependent pathway.^31,35–37 However, neither eNOS mRNA/protein expression nor EPCs are reported to be essential for the therapeutic effect of Fluvastatin on hypoxia-induced pulmonary hypertension; Fluvastatin improved eNOS phosphorylation by a mechanism independent of Akt activation.³⁸ Our data favor a mechanism involving Akt phosphorylation since phosphorylated Akt is increased when EPCs are cultured in the presence of statin, and statin-enhanced EPC proliferation and migration were inhibited by the PI3K/Akt inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002.The angiogenic effects of SDF-1 also involve increased production of NO²⁶ as NO is essential for EC migration and angiogenesis. SDF-1α gene transfer has been shown to enhance eNOS activity.²¹ Our in vitro data confirmed the involvement of Akt and eNOS in SDF-1 mediated cell migration.¹⁰ Phosphorylated Akt is increased when EPCs are cultured in the presence of SDF-1. The facilitative effect of SDF-1 on EPC migration is blocked by both the Akt inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and the eNOS inhibitor L-NMMA. In contrast, L-NMMA does not reverse the inhibitory effect of SDF-1 on apoptosis, indicating that the inhibitory effect of SDF-1 on apoptosis is not mediated through NO.²²We also show that the expression of MMP-2 and MMP-9 was increased when EPCs were cultured in the presence of statin or SDF-1. MMPs are a family of proteolytic enzymes that degrade components of the extracellular matrix (ECM). Degradation of ECM is an essential step for cell mobilization and migration. Our data indicate that the novel effect of statin and SDF-1 on migration is through enhancement of MMP-2 and MMP-9 activity, resulting in ECM degradation, thus promoting progenitor cell mobilization and migration. Both Akt phosphorylation and expression of MMP-2 and MMP-9 in EPCs are further enhanced by combined treatment with statin and SDF-1. This result indicates that treatment of EPCs with either statin or SDF-1 as monotherapy results in a sub-maximal angiogenic response. The effects of statin partially overlap with that of SDF-1; and the combined use of two factors appears to have an optimal effect on progenitor cells (Fig. 1).Open in a separate windowFigure 1Effect of statins and SDF-1 on promoting angiogenesis. Statin enhances the phosphorylation of Akt with a yet undefined mechanism. SDF-1 binding with the G-protein coupled membrane receptor CXCR4 results in phosphorylation of protein kinases like PI3 kinase and Akt. Activation of Akt then upregulates the activities of MMPs and eNOS. NOS catalyze the synthesis of NO which is essential for the EPC migration. MMPs degrade extracellular matrix to initiate cell migration. Activation of Akt also prevents cell apoptosis. These reactions promote cell migration and proliferation and enhance EPC survival. EPCs from bone marrow are thus mobilized into circulation. The circulating EPC are homed into ischemia area in lure of SDF-1. EPCs contribute to neovascularisation, either directly by incorporation into endothelium and differentiation into endothelial cells or indirectly by differentiating into perivascular cells that provide physical support and secrete signaling proteins and structural enzymes enabling the angiogenesis process. The effects of statin partially overlap with that of SDF-1; and the combined use of two factors appears to have an additive/synergistic effect on progenitor cells.In summary, the combination of progenitor cell mobilization with statin and targeted recruitment into the ischemic bed by SDF-1 leads to improved blood flow in the ischemic limb versus treatment with either agent alone. Statin and SDF-1 therefore display synergism in promoting neovascularization. This result suggests that the combination of statin and SDF-1 may be a new therapeutic strategy in the treatment of limb ischemia. However, the use of statins as a clinical modifier of angiogenesis is still unproven. A great number of patients have been treated with these drugs and if they were potently proangiogenic, one might expect to see an increased risk of tumors. However, there is no evidence that these drugs encourage tumor development. Likewise, there is no definitive evidence for an antiangiogenic, tumor-modulating action of statins. We await further studies with interest.     

The potential of statin and stromal cell-derived factor-1 to promote angiogenesis

Angiogenesis requires the mobilization of progenitor cells from the bone marrow (BM) and homing of progenitor cells to ischemic tissue. The cholesterol lowering drug Statins can stimulate angiogenesis via mobilization of BM derived endothelial progenitor cells (EPCs), promoting EPC migration, and inhibiting EPC apoptosis. The chemokine stromal cell-derived factor-1 (SDF-1) augments EPC chemotaxis, facilitates EPC incorporation into the neovasculature. The combined use of a statin to mobilize EPCs and local over-expression of SDF-1 to augment EPC homing to ischemic muscle resulted in superior angiogenesis versus use of either agent alone. Their effects are through augmenting EPC mobilization, incorporation, proliferation, migration, and tube formation while inhibiting EPC apoptosis. Statin and SDF-1 therefore display synergism in promoting neovascularization by improving reperfusion of ischemic muscle, increasing progenitor cell presentation and capillary density in ischemic muscle, and diminishing apoptosis. These results suggest that the combination of statin and SDF-1 may be a new therapeutic strategy in the treatment of limb ischemia.     

The signaling pathway of stromal cell-derived factor-1 and its role in kidney diseases

Abstract

The chemokine stromal cell-derived factor-1 (SDF-1) regulates the trafficking of progenitor cell (PGC) during embryonic development, cell chemotaxis, and postnatal homing into injury sites. SDF-1 also regulates cell growth, survival, adhesion and angiogenesis. However, in different tissues/cells, the role of SDF-1 is different, such as that it is increased in most of the tumors and associated with cancer metastasis, whereas it is essential for the development of vasculature. For kidney diseases, its role remains controversial. Signaling pathways might be very important in the pathogenesis of kidney diseases. We performed this review to provide a relatively complete signaling pathway flowchart for SDF-1 to the investigators who were interested in the role of SDF-1 in the pathogenesis of kidney diseases. Here, we reviewed the signal transduction pathway of SDF-1 and its role in the pathogenesis of kidney diseases.     

Adhesion regulation of stromal cell-derived factor-1 activation of ERK in lymphocytes by phosphatases

We have investigated whether chemokine signaling to the extracellular-signal-regulated kinase (ERK) was regulated by beta 1-integrin-mediated adhesion in B- and T-cell lines. Activation of ERK by the chemokine SDF-1 can be regulated by adhesion to beta 1-integrin substrates in the T-cell lines MOLT-3, Jurkat, and H9 and in the Daudi B-cell line. In Jurkat T-cells, adhesion to the immobilized alpha 4 beta 1-integrin ligand VCAM-1 or to the alpha 5 beta 1-integrin ligand fibronectin regulated stromal-cell derived factor-1 (SDF-1) activation of ERK. Adhesion control of SDF-1 signaling was a rapid event, occurring as early as 10 min after adhesion, and loss of signaling occurred within 10 min of deadhesion. In contrast, SDF-1 activation of the ERK kinase MEK was independent of adhesion. Partial restoration of signaling to ERK in suspension was accomplished by pretreatment with pharmacological inhibitors of serine/threonine or protein-tyrosine phosphatases. In addition, we used a non-radioactive phosphatase assay using phosphorylated ERK as the substrate to determine relative ERK dephosphorylation in whole cell extracts. These results showed greater relative ERK dephosphorylation in extracts from Jurkat cells treated in suspension, as compared with adherent cells. Therefore, these data suggest that adhesion influences SDF-1 activation of ERK by regulating the activity of ERK phosphatases. This identifies a novel locus of adhesion regulation of the ERK cascade.     

Pro-inflammatory properties of stromal cell-derived factor-1 (CXCL12) in collagen-induced arthritis

CXCL12 (stromal cell-derived factor 1) is a unique biological ligand for the chemokine receptor CXCR4. We previously reported that treatment with a specific CXCR4 antagonist, AMD3100, exerts a beneficial effect on the development of collagen-induced arthritis (CIA) in the highly susceptible IFN-γ receptor-deficient (IFN-γR KO) mouse. We concluded that CXCL12 plays a central role in the pathogenesis of CIA in IFN-γR KO mice by promoting delayed type hypersensitivity against the auto-antigen and by interfering with chemotaxis of CXCR4⁺ cells to the inflamed joints. Here, we investigated whether AMD3100 can likewise inhibit CIA in wild-type mice and analysed the underlying mechanism. Parenteral treatment with the drug at the time of onset of arthritis reduced disease incidence and modestly inhibited severity in affected mice. This beneficial effect was associated with reduced serum concentrations of IL-6. AMD3100 did not affect anti-collagen type II antibodies and, in contrast with its action in IFN-γR KO mice, did not inhibit the delayed type hypersensitivity response against collagen type II, suggesting that the beneficial effect cannot be explained by inhibition of humoral or cellular autoimmune responses. AMD3100 inhibited the in vitro chemotactic effect of CXCL12 on splenocytes, as well as in vivo leukocyte infiltration in CXCL12-containing subcutaneous air pouches. We also demonstrate that, in addition to its effect on cell infiltration, CXCL12 potentiates receptor activator of NF-κB ligand-induced osteoclast differentiation from splenocytes and increases the calcium phosphate-resorbing capacity of these osteoclasts, both processes being potently counteracted by AMD3100. Our observations indicate that CXCL12 acts as a pro-inflammatory factor in the pathogenesis of autoimmune arthritis by attracting inflammatory cells to joints and by stimulating the differentiation and activation of osteoclasts.     

The chemokine stromal cell-derived factor-1/CXCL12 activates the nigrostriatal dopamine system

We recently demonstrated that dopaminergic (DA) neurons of the rat substantia nigra constitutively expressed CXCR4, receptor for the chemokine stromal cell-derived factor-1 (SDF-1)/CXCL12 (SDF-1). To check the physiological relevance of such anatomical observation, in vitro and in vivo approaches were used. Patch clamp recording of DA neurons in rat substantia nigra slices revealed that SDF-1 (10 nmol/L) induced: (i) a depolarization and increased action potential frequency; and (ii) switched the firing pattern of depolarized DA neurons from a tonic to a burst firing mode. This suggests that SDF-1 could increase DA release from neurons. Consistent with this hypothesis, unilateral intranigral injection of SDF-1 (50 ng) in freely moving rat decreased DA content and increased extracellular concentrations of DA and metabolites in the ipsilateral dorsal striatum, as shown using microdialysis. Furthermore, intranigral SDF-1 injection induced a contralateral circling behavior. These effects of SDF-1 were mediated via CXCR4 as they were abrogated by administration of a selective CXCR4 antagonist. Altogether, these data demonstrate that SDF-1, via CXCR4, activates nigrostriatal DA transmission. They show that the central functions of chemokines are not restricted, as originally thought, to neuroinflammation, but extend to neuromodulatory actions on well-defined neuronal circuits in non-pathological conditions.     

Involvement of phosphatidylinositol 3-kinase in stromal cell-derived factor-1 alpha-induced lymphocyte polarization and chemotaxis.

The role of phosphatidylinositol 3-kinase (PI3-kinase), an important enzyme involved in signal transduction events, has been studied in the polarization and chemotaxis of lymphocytes induced by the chemokine stromal cell-derived factor-1 alpha (SDF-1 alpha). This chemokine was able to directly activate p85/p110 PI3-kinase in whole human PBL and to induce the association of PI3-kinase to the SDF-1 alpha receptor, CXCR4, in a pertussis toxin-sensitive manner. Two unrelated chemical inhibitors of PI3-kinase, wortmannin and Ly294002, prevented ICAM-3 and ERM protein moesin polarization as well as the chemotaxis of PBL in response to SDF-1 alpha. However, they did not interfere with the reorganization of either tubulin or the actin cytoskeleton. Moreover, the transient expression of a dominant negative form of the PI3-kinase 85-kDa regulatory subunit in the constitutively polarized Peer T cell line inhibited ICAM-3 polarization and markedly reduced SDF-1 alpha-induced chemotaxis. Conversely, overexpression of a constitutively activated mutant of the PI3-kinase 110-kDa catalytic subunit in the round-shaped PM-1 T cell line induced ICAM-3 polarization. These results underline the role of PI3-kinase in the regulation of lymphocyte polarization and motility and indicate that PI3-kinase plays a selective role in the regulation of adhesion and ERM proteins redistribution in the plasma membrane of lymphocytes.     

Syndecan-4 is a signaling molecule for stromal cell-derived factor-1 (SDF-1)/ CXCL12

Stromal cell-derived factor-1 (SDF-1)/CXCL12, the ligand for CXCR4, induces signal transduction. We previously showed that CXCL12 binds to high- and low-affinity sites expressed by primary cells and cell lines, and forms complexes with CXCR4 as expected and also with a proteoglycan, syndecan-4, but does not form complexes with syndecan-1, syndecan-2, CD44 or beta-glycan. We also demonstrated the occurrence of a CXCL12-independent heteromeric complex between CXCR4 and syndecan-4. However, our data ruled out the glycosaminoglycan-dependent binding of CXCL12 to HeLa cells facilitating the binding of this chemokine to CXCR4. Here, we demonstrate that CXCL12 directly binds to syndecan-4 in a glycosaminoglycan-dependent manner. We show that upon stimulation of HeLa cells by CXCL12, CXCR4 becomes tyrosine phosphorylated as expected, while syndecan-4 (but not syndecan-1, syndecan-2 or beta-glycan) also undergoes such tyrosine phosphorylation. Moreover, tyrosine-phosphorylated syndecan-4 from CXCL12-stimulated HeLa cells physically coassociates with tyrosine phosphorylated CXCR4. Pretreatment of the cells with heparitinases I and III prevented the tyrosine phosphorylation of syndecan-4, which suggests that the heparan sulfate-dependent binding of SDF-1 to this proteoglycan is involved. Finally, by reducing syndecan-4 expression using RNA interference or by pretreating the cells with heparitinase I and III mixture, we suggest the involvement of syndecan-4 and heparan sulfate in p44/p42 mitogen-activated protein kinase and Jun N-terminal/stress-activated protein kinase activation by action of CXCL12 on HeLa cells. However, these treatments did not modify the calcium mobilization induced by CXCL12 in these cells. Therefore, syndecan-4 behaves as a CXCL12 receptor, selectively involved in some transduction pathways induced by SDF-1, and heparan sulfate plays a role in these events.     

Plerixafor induces the rapid and transient release of stromal cell-derived factor-1 alpha from human mesenchymal stromal cells and influences the migration behavior of human hematopoietic progenitor cells

The interaction between the stromal cell-derived factor-1 alpha (SDF-1α, CXCL12) and its chemokine receptor CXCR4 has been reported to regulate stem cell migration, mobilization and homing. The CXCR4 antagonist plerixafor is highly efficient in mobilizing hematopoietic progenitor cells (HPCs). However, the precise regulatory mechanisms governing the CXCR4/SDF-1α axis between the bone marrow niche and HPCs remain unclear. In this study, we quantify the impact of plerixafor on the interaction between human bone marrow derived mesenchymal stromal cells (MSCs) and human CD34+ HPCs. An assessment of SDF-1α levels in the supernatant of MSC cultures revealed that exposure to plerixafor led to a transient increase but had no long-term effect. In Transwell experiments, we observed that the addition of SDF-1α significantly stimulated HPC migration; this stimulation was almost completely antagonized by the addition of plerixafor, confirming the direct impact of the CXCR4/SDF-1α interaction on the migration capacity of HPCs. We also developed a new microstructural niche model to determine the chemotactic sensitivity of HPCs. Time-lapse microscopy demonstrated that HPCs migrated actively along an SDF-1α gradient within the microchannels and the quantitative assessment of the required minimum gradient initiating this chemotaxis revealed a surprisingly high sensitivity of HPCs. These data demonstrate the fine-tuned balance of the CXCR4/SDF-1α axis and the synergistic effects of plerixafor on HPCs and MSCs, which most likely represent the key mechanisms for the consecutive mobilization of HPCs from the bone marrow niche into the circulating blood.     

Butanol-extracted lipoteichoic acid induces in vivo leukocyte adhesion

Lipoteichoic acid (LTA), an immunostimulatory component of the cell walls of Gram positive bacteria, has pro-inflammatory effects in vitro and in vivo. However, one in vivo study concluded that LTA had no noticeable effects on leukocyte recruitment. In this study we investigated the effects of highly purified LTA, prepared by butanol extraction (Bu-LTA) at room temperature, on in vivo leukocyte adhesion. Using intravital microscopy we measured adhesion of leukocytes in mesenteric post-capillary venules of rats and mice. Topical superfusion of Bu-LTA (1 μg/ml) in rats significantly (p < 0.05) increased adhesion within 30 min. By contrast, hot phenol-extracted LTA did not increase adhesion. Alkaline hydrolysis of Bu-LTA removed alanine residues and prevented adhesion. Also, pre-administration of anti-rat β₂-integrin antibody abolished Bu-LTA-induced adhesion. Finally, intraperitoneal injection of Bu-LTA (100 μg/ml) into mice also significantly (p < 0.01) increased leukocyte adhesion measured at 60 min. In conclusion, Bu-LTA with intact alanine residues promotes β₂-integrin-dependent leukocyte adhesion in vivo.     

Structural and functional basis of CXCL12 (stromal cell-derived factor-1 alpha) binding to heparin

CXCL12 (SDF-1alpha) and CXCR4 are critical for embryonic development and cellular migration in adults. These proteins are involved in HIV-1 infection, cancer metastasis, and WHIM disease. Sequestration and presentation of CXCL12 to CXCR4 by glycosaminoglycans (GAGs) is proposed to be important for receptor activation. Mutagenesis has identified CXCL12 residues that bind to heparin. However, the molecular details of this interaction have not yet been determined. Here we demonstrate that soluble heparin and heparan sulfate negatively affect CXCL12-mediated in vitro chemotaxis. We also show that a cluster of basic residues in the dimer interface is required for chemotaxis and is a target for inhibition by heparin. We present structural evidence for binding of an unsaturated heparin disaccharide to CXCL12 attained through solution NMR spectroscopy and x-ray crystallography. Increasing concentrations of the disaccharide altered the two-dimensional (1)H-(15)N-HSQC spectra of CXCL12, which identified two clusters of residues. One cluster corresponds to beta-strands in the dimer interface. The second includes the amino-terminal loop and the alpha-helix. In the x-ray structure two unsaturated disaccharides are present. One is in the dimer interface with direct contacts between residues His(25), Lys(27), and Arg(41) of CXCL12 and the heparin disaccharide. The second disaccharide contacts Ala(20), Arg(21), Asn(30), and Lys(64). This is the first x-ray structure of a CXC class chemokine in complex with glycosaminoglycans. Based on the observation of two heparin binding sites, we propose a mechanism in which GAGs bind around CXCL12 dimers as they sequester and present CXCL12 to CXCR4.     

Cutting edge: stromal cell-derived factor-1 is a costimulator for CD4+ T cell activation

Stromal cell-derived factor (SDF)-1 is a chemoattractant for T cells, precursor B cells, monocytes, and neutrophils. SDF-1alpha was also found to up-regulate expression of early activation markers (CD69, CD25, and CD154) by anti-CD3-activated CD4+ T cells. In addition, SDF-1alpha costimulated proliferation of CD4+ T cells and production of IL-2, IFN-gamma, IL-4, and IL-10. Stimulation with SDF-1alpha alone did not induce activation marker expression, proliferation, or cytokine production by the CD4+ T cells. SDF-1alpha-mediated costimulation was blocked by anti-CXC chemokine receptor-4 mAb. RANTES also increased activation marker expression by anti-CD3-stimulated peripheral CD4+ T cells, but less effectively than SDF-1alpha did, and did not up-regulate IL-2 production and proliferation. These results indicate that SDF-1 and CXC chemokine receptor-4 interactions not only play a role in T cell migration but also provide potent costimulatory signals to Ag-stimulated T cells.     

NMR studies of active N-terminal peptides of stromal cell-derived factor-1. Structural basis for receptor binding

Stromal cell-derived factor 1 (SDF-1), a member of the CXC chemokine family, is the only chemokine to bind to the receptor CXCR4. This receptor is also a co-receptor for syncytia-inducing forms of HIV in CD4(+) cells. In addition, SDF-1 is responsible for attracting mature lymphocytes to the bone marrow and can therefore contribute to host versus graft rejection in bone marrow transplantation. Clearly, by manipulating SDF-1 activity, we could find a possible anti-viral AIDS treatment and aid in bone marrow transplantation. SDF-1 binds to CXCR4 primarily via the N terminus, which appears flexible in the recently determined three-dimensional structure of SDF-1. Strikingly, short N-terminal SDF-1 peptides have been shown to have significant SDF-1 activity. By using NMR, we have determined the major conformation of the N terminus of SDF-1 in a 17-mer (residues 1-17 of SDF-1) and a 9-mer dimer (residues 1-9 of SDF-1 linked by a disulfide bond at residue 9). Residues 5-8 and 11-14 form similar structures that can be characterized as a beta-turn of the beta-alphaR type. These structural motifs are likely to be interconverting with other states, but the major conformation may be important for recognition in receptor binding. These results suggest for the first time that there may be a link between structuring of short N-terminal chemokine peptides and their ability to activate their receptor. These studies will act as a starting point for synthesizing non-peptide analogs that act as CXCR4 antagonists.     

The physiology of leukocyte recruitment: an in vivo perspective

The mechanisms of leukocyte recruitment have been studied extensively in vitro and have shed light on the basic molecular structure-function relationship of adhesion and signaling molecules involved in this essential immune response. This review will summarize how these in vitro observations extend to leukocyte behavior in inflamed blood vessels in the microcirculation. We highlight physiological results that might not have been predicted from in vitro systems. Special attention is placed on the physiology of rolling, adhesion, and intralumenal crawling in blood vessels. The importance of the glycocalyx, secondary tethers, shear, and the microenvironment are discussed. Docking structures forming rings of adhesion molecules together with a novel endothelial dome-like structure in vivo during transmigration are highlighted. Transcellular and paracellular emigration out of inflamed blood vessels is also discussed. The last section highlights leukocyte recruitment in some organs that do not always follow the accepted paradigm of leukocyte recruitment.