PAK1 kinase is required for CXCL1-induced chemotaxis

The CXC subfamily of chemokines plays an important role in diverse processes, including inflammation, wound healing, growth regulation, angiogenesis, and tumorigenesis. The CXC chemokine CXCL1, or MGSA/GROalpha, is traditionally considered to be responsible for attracting leukocytes into sites of inflammation. To better understand the molecular mechanisms by which CXCL1 induces CXCR2-mediated chemotaxis, the signal transduction components involved in CXCL1-induced chemotaxis were examined. It is shown here that CXCL1 induces cdc42 and PAK1 activation in CXCR2-expressing HEK293 cells. Activation of the cdc42-PAK1 cascade is required for CXCL1-induced chemotaxis but not for CXCL1-induced intracellular Ca2+ mobilization. Moreover, CXCL1 activation of PAK1 is independent of ERK1/2 activation, a conclusion based on the observations that the inhibition of MEK-ERK activation by expression of dominant negative ERK or by the MEK inhibitor, PD98059, has no effect on CXCL1-induced PAK1 activation or CXCL1-induced chemotaxis.     

Optimal inhibition of X4 HIV isolates by the CXC chemokine stromal cell-derived factor 1 alpha requires interaction with cell surface heparan sulfate proteoglycans

The chemokine stromal cell-derived factor 1 (SDF-1) is the natural ligand for CXC chemokine receptor 4 (CXCR4). SDF-1 inhibits infection of CD4+ cells by X4 (CXCR4-dependent) human immunodeficiency virus (HIV) strains. We previously showed that SDF-1 alpha interacts specifically with heparin or heparan sulfates (HSs). Herein, we delimited the boundaries of the HS-binding domain located in the first beta-strand of SDF-1 alpha as the critical residues. We also provide evidence that binding to cell surface heparan sulfate proteoglycans (HSPGs) determines the capacity of SDF-1 alpha to prevent the fusogenic activity of HIV-1 X4 isolates in leukocytes. Indeed, SDF-1 alpha mutants lacking the capacity to interact with HSPGs showed a substantially reduced capacity to prevent cell-to-cell fusion mediated by X4 HIV envelope glycoproteins. Moreover, the enzymatic removal of cell surface HS diminishes the HIV-inhibitory capacity of the chemokine to the levels shown by the HS-binding-disabled mutant counterparts. The mechanisms underlying the optimal HIV-inhibitory activity of SDF-1 alpha when attached to HSPGs were investigated. Combining fluorescence resonance energy transfer and laser confocal microscopy, we demonstrate the concomitant binding of SDF-1 alpha to CXCR4 and HSPGs at the cell membrane. Using FRET between a Texas Red-labeled SDF-1 alpha and an enhanced green fluorescent protein-tagged CXCR4, we show that binding of SDF-1 alpha to cell surface HSPGs modifies neither the kinetics of occupancy nor activation in real time of CXCR4 by the chemokine. Moreover, attachment to HSPGs does not modify the potency of the chemokine to promote internalization of CXCR4. Attachment to cellular HSPGs may co-operate in the optimal anti-HIV activity of SDF-1 alpha by increasing the local concentration of the chemokine in the surrounding environment of CXCR4, thus facilitating sustained occupancy and down-regulation of the HIV coreceptor.     

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 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.     

Evidence for a role of glycosphingolipids in CXCR4-dependent cell migration

Chemotaxis induction is a major effect evoked by stimulation of the chemokine receptor CXCR4 with its sole ligand CXCL12. We now report that treatment of CHP-100 human neuroepithelioma cells with the glucosylceramide synthase (GCS) inhibitor DL-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol inhibits CXCR4-dependent chemotaxis. We provide evidence that the phenomenon is not due to unspecific effects of the inhibitor employed and that inhibition of GCS neither affects total or plasmamembrane CXCR4 expression, nor CXCL12-induced Ca(2+) mobilization. The effects of the GCS inhibitor on impairment of CXCL12-induced cell migration temporally correlated with a pronounced downregulation of neutral glycosphingolipids, particularly glucosylceramide, and with a delayed and more moderate downregulation of gangliosides; moreover, exogenously administered glycosphingolipids allowed resumption of CXCR4-dependent chemotaxis. Altogether our results provide evidence, for the first time, for a role glycosphingolipids in sustaining CXCL12-induced cell migration.     

Activating and inhibitory Ly49 receptors modulate NK cell chemotaxis to CXC chemokine ligand (CXCL) 10 and CXCL12

NK cells can migrate into sites of inflammatory responses or malignancies in response to chemokines. Target killing by rodent NK cells is restricted by opposing signals from inhibitory and activating Ly49 receptors. The rat NK leukemic cell line RNK16 constitutively expresses functional receptors for the inflammatory chemokine CXC chemokine ligand (CXCL)10 (CXCR3) and the homeostatic chemokine CXCL12 (CXCR4). RNK-16 cells transfected with either the activating Ly49D receptor or the inhibitory Ly49A receptor were used to examine the effects of NK receptor ligation on CXCL10- and CXCL12-mediated chemotaxis. Ligation of Ly49A, either with Abs or its MHC class I ligand H2-D(d), led to a decrease in chemotactic responses to either CXCL10 or CXCL12. In contrast, Ly49D ligation with Abs or H2-D(d) led to an increase in migration toward CXCL10, but a decrease in chemotaxis toward CXCL12. Ly49-dependent effects on RNK-16 chemotaxis were not the result of surface modulation of CXCR3 or CXCR4 as demonstrated by flow cytometry. A mutation of the Src homology phosphatase-1 binding motif in Ly49A completely abrogated Ly49-dependent effects on both CXCL10 and CXCL12 chemotaxis, suggesting a role for Src homology phosphatase-1 in Ly49A/chemokine receptor cross-talk. Ly49D-transfected cells were pretreated with the Syk kinase inhibitor Piceatannol before ligation, which abrogated the previously observed changes in migration toward CXCL10 and CXCL12. Piceatannol also abrogated Ly49A-dependent inhibition of chemotaxis toward CXCL10, but not CXCL12. Collectively, these data suggest that Ly49 receptors can influence NK cell chemotaxis within sites of inflammation or tumor growth upon interaction with target cells.     

Differential regulation of CXCR4-mediated T-cell chemotaxis and mitogen-activated protein kinase activation by the membrane tyrosine phosphatase,CD45

The chemokine receptor CXCR4 and its cognate ligand, stromal cell-derived factor-1alpha (CXCL12), regulate lymphocyte trafficking and play an important role in host immune surveillance. However, the molecular mechanisms involved in CXCL12-induced and CXCR4-mediated chemotaxis of T-lymphocytes are not completely elucidated. In the present study, we examined the role of the membrane tyrosine phosphatase CD45, which regulates antigen receptor signaling in CXCR4-mediated chemotaxis and mitogen-activated protein kinase (MAPK) activation in T-cells. We observed a significant reduction in CXCL12-induced chemotaxis in the CD45-negative Jurkat cell line (J45.01) as compared with the CD45-positive control (JE6.1) cells. Expression of a chimeric protein containing the intracellular phosphatase domain of CD45 was able to partially restore CXCL12-induced chemotaxis in the J45.01 cells. However, reconstitution of CD45 into the J45.01 cells restored the CXCL12-induced chemotaxis to about 90%. CD45 had no significant effect on CXCL12 or human immunodeficiency virus gp120-induced internalization of the CXCR4 receptor. Furthermore, J45.01 cells showed a slight enhancement in CXCL12-induced MAP kinase activity as compared with the JE6.1 cells. We also observed that CXCL12 treatment enhanced the tyrosine phosphorylation of CD45 and induced its association with the CXCR4 receptor. Pretreatment of T-cells with the lipid raft inhibitor, methyl-beta-cyclodextrin, blocked the association between CXCR4 and CD45 and markedly abolished CXCL12-induced chemotaxis. Comparisons of signaling pathways induced by CXCL12 in JE6.1 and J45.01 cells revealed that CD45 might moderately regulate the tyrosine phosphorylation of the focal adhesion components the related adhesion focal tyrosine kinase/Pyk2, focal adhesion kinase, p130Cas, and paxillin. CD45 has also been shown to regulate CXCR4-mediated activation and phosphorylation of T-cell receptor downstream effectors Lck, ZAP-70, and SLP-76. Our results show that CD45 differentially regulates CXCR4-mediated chemotactic activity and MAPK activation by modulating the activities of focal adhesion components and the downstream effectors of the T-cell receptor.     

Human B cell-attracting chemokine 1 (BCA-1; CXCL13) is an agonist for the human CXCR3 receptor

The CXC chemokine CXCL13, known as BCA-1 (B cell-attracting chemokine 1) or BLC (B-lymphocyte chemoattractant), has been identified as an efficacious attractant selective for B lymphocytes. The chemokine receptor BLR1 (Burkitt's lymphoma receptor 1)/CXCR5 expressed by all mature B cells has to date been identified as the only known receptor for BCA-1. As the loss of the BLR1/CXCR5 receptor is sufficient to disrupt organization of follicles in spleen and Peyer's patches, BCA-1 may act as a B cell homing chemokine. Nonetheless, BCA-1 has not been tested against all known chemokine receptors. In this study, we report that human BCA-1 competes with radiolabeled interferon gamma (IFN-gamma) inducible protein 10 (IP-10) for binding to the human CXCR3 receptor expressed in Ba/F3 and 293EBNA cell lines. Furthermore, human BCA-1 is an efficacious attractant for human CXCR3 transfected cells; BCA-1-induced chemotaxis is inhibited by a monoclonal antibody against human CXCR3. In these cells, as in human B lymphocytes expressing CXCR5, BCA-1 does not induce a calcium flux. Indeed, BCA-1 attenuates the calcium flux induced by IP-10. In addition, human BCA-1 is an agonist in stimulating GTP gamma S binding. Together these data suggest that human BCA-1 is a specific and functional G-protein-linked chemotactic ligand for the human CXCR3 receptor. The biological significance of this new finding is supported by our recent observation that human BCA-1 induces chemotaxis of activated T cells and the BCA-1-induced chemotaxis is inhibited by a monoclonal antibody against human CXCR3.     

Leukocyte-endothelium interaction promotes SDF-1-dependent polarization of CXCR4

Chemokine-driven migration is accompanied by polarization of the cell body and of the intracellular signaling machinery. The extent to which chemokine receptors polarize during chemotaxis is currently unclear. To analyze the distribution of the chemokine receptor CXCR4 during SDF-1 (CXCL12)-induced chemotaxis, we retrovirally expressed a CXCR4-GFP fusion protein in the CXCR4-deficient human hematopoietic progenitor cell line KG1a. This KG1a CXCR4-GFP cell line showed full restoration of SDF-1 responsiveness in assays detecting activation of ERK1/2 phosphorylation, actin polymerization, adhesion to endothelium under conditions of physiological flow, and (transendothelial) chemotaxis. When adhered to cytokine-activated endothelium in the absence of SDF-1, CXCR4 did not localize to the leading edge of the cell but was uniformly distributed over the plasma membrane. In contrast, when SDF-1 was immobilized on cytokine-activated endothelium, the CXCR4-GFP receptors that were present on the cell surface markedly redistributed to the leading edge of migrating cells. In addition, CXCR4-GFP co-localized with lipid rafts in the leading edge of SDF-1-stimulated cells, at the sites of contact with the endothelial surface. Inhibition of lipid raft formation prevents SDF-1-dependent migration, internalization of CXCR4, and polarization to the leading edge of CXCR4, indicating that CXCR4 surface expression and signaling requires lipid rafts. These data show that SDF-1, immobilized on activated human endothelium, induces polarization of CXCR4 to the leading edge of migrating cells, revealing co-operativity between chemokine and substrate in the control of cell migration.     

CXCL10/IP-10 in infectious diseases pathogenesis and potential therapeutic implications

C–X–C motif chemokine 10 (CXCL10) also known as interferon γ-induced protein 10 kDa (IP-10) or small-inducible cytokine B10 is a cytokine belonging to the CXC chemokine family. CXCL10 binds CXCR3 receptor to induce chemotaxis, apoptosis, cell growth and angiostasis. Alterations in CXCL10 expression levels have been associated with inflammatory diseases including infectious diseases, immune dysfunction and tumor development. CXCL10 is also recognized as a biomarker that predicts severity of various diseases. A review of the emerging role of CXCL10 in pathogenesis of infectious diseases revealed diverse roles of CXCL10 in disease initiation and progression. The potential utilization of CXCL10 as a therapeutic target for infectious diseases is discussed.     

CXCL14 is no direct modulator of CXCR4

C-X-C motif chemokine 12/C-X-C chemokine receptor type 4 (CXCL12/CXCR4) signaling is involved in ontogenesis, hematopoiesis, immune function and cancer. Recently, the orphan chemokine CXCL14 was reported to inhibit CXCL12-induced chemotaxis – probably by allosteric modulation of CXCR4. We thus examined the effects of CXCL14 on CXCR4 regulation and function using CXCR4-transfected human embryonic kidney (HEK293) cells and Jurkat T cells. CXCL14 did not affect dose–response profiles of CXCL12-induced CXCR4 phosphorylation, G protein-mediated calcium mobilization, dynamic mass redistribution, kinetics of extracellular signal-regulated kinase 1 (ERK1) and ERK2 phosphorylation or CXCR4 internalization. Hence, essential CXCL12-operated functions of CXCR4 are insensitive to CXCL14, suggesting that interactions of CXCL12 and CXCL14 pathways depend on a yet to be identified CXCL14 receptor.     

CXCL12 induces tyrosine phosphorylation of cortactin, which plays a role in CXC chemokine receptor 4-mediated extracellular signal-regulated kinase activation and chemotaxis

CXC chemokine receptor 4 (CXCR4) plays a role in the development of immune and central nervous systems as well as in cancer growth and metastasis. CXCR4-initiated signaling cascades leading to cell proliferation and chemotaxis are critical for these functions. The present study demonstrated that stimulation of CXCR4 by its ligand, CXCL12, induced transient translocation of cortactin from endosomal compartments to the cell periphery where it colocalized with CXCR4 followed by internalization of CXCR4 together with cortactin into endosomes. Cortactin was co-immunoprecipitated with CXCR4 in response to CXCL12 treatment in a time-dependent manner. Ligand stimulation induced phosphorylation of cortactin at tyrosine 421, and the phosphorylation was both c-Src- and dynamin-dependent. Cortactin overexpression promoted CXCR4 internalization and recycling. However, overexpression of a cortactin mutant in which tyrosine 421 was replaced with alanine (cortactin-Y421A) or knockdown of cortactin with RNA interference (RNAi) reduced CXCR4 internalization in response to CXCL12. CXCR4-mediated activation of extracellular signal-regulated kinases 1 and 2 was significantly prolonged by overexpression of wild-type cortactin but not by the cortactin-Y421A mutant and was inhibited by cortactin knockdown with RNAi. Moreover, CXCL12-induced chemotaxis was enhanced by cortactin overexpression, reduced by overexpression of the cortactin-Y421A mutant, and blocked by cortactin knockdown with RNAi. These data provide strong evidence for an important role of cortactin in CXCR4 signaling and trafficking as well in the receptor-mediated cell migration.     

Glycosaminoglycans and syndecan-4 are involved in SDF-1/CXCL12-mediated invasion of human epitheloid carcinoma HeLa cells

Background

In addition to their physiologic effects in inflammation and angiogenesis, chemokines are involved in cancer pathology. The CXC-chemokine stromal cell-derived factor-1 (SDF-1)/CXCL12 mediates its biological activities through activation of G protein-coupled receptor CXCR4 and binds to glycosaminoglycans (GAGs).

Methods

Using Bio-coat cell migration chambers, specific antagonists, flow cytometry and RNA interference, we evaluate the involvement of heparan sulfate proteoglycans (HSPG) in the SDF-1/CXCL12-induced invasion of human cervix epitheloid carcinoma HeLa cells.

Results

The SDF-1/CXCL12-induced cell invasion is dependent on CXCR4. Furthermore, Protein Kinase C delta (PKC δ) and c-jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) are implicated in this event, but not extracellular signal-regulated kinase (ERK) 1/2. Moreover, the invasion of HeLa cells induced by SDF-1/CXCL12 was dependent on matrix metalloproteinase-9 (MMP-9). The pre-incubation of HeLa cells with heparin or with anti-heparan sulfate antibodies or with β-d-xyloside inhibited SDF-1/CXCL12-mediated cell invasion. Furthermore, the down-regulation of syndecan-4, a heparan sulfate proteoglycan, decreased SDF-1/CXCL12-mediated HeLa cell invasion. GAGs, probably on syndecan-4, are involved in SDF-1/CXCL12-mediated cell chemotaxis.

General significance

These data suggest that targeting the glycosaminoglycan/chemokine interaction could be a new therapeutic approach for carcinomas in which SDF-1/CXCL12 is involved.     

CXCL12 is displayed by rheumatoid endothelial cells through its basic amino-terminal motif on heparan sulfate proteoglycans

The chemokine CXCL12 (also known as stromal cell-derived factor, SDF-1) is constitutively expressed by stromal resident cells and is involved in the homeostatic and inflammatory traffic of leukocytes. Binding of CXCL12 to glycosaminoglycans on endothelial cells (ECs) is supposed to be relevant to the regulation of leukocyte diapedesis and neoangiogenesis during inflammatory responses. To improve our understanding of the relevance of this process to rheumatoid arthritis (RA), we have studied the mechanisms of presentation of exogenous CXCL12 by cultured RA ECs. RA synovial tissues had higher levels of CXCL12 on the endothelium than osteoarthritis (OA) tissues; in both, CXCL12 colocalized to heparan sulfate proteoglycans (HSPGs) and high endothelial venules. In cultured RA ECs, exogenous CXCL12α was able to bind in a CXCR4-independent manner to surface HSPGs. Desulfation of RA EC HSPGs by pretreatment with sodium chlorate, or by replacing in a synthetic CXCL12α the residues Lys24 and Lys27 by Ser (CXCL12α-K2427S), decreased or abrogated the ability of the chemokine to bind to RA ECs. Ex vivo, synovial ECs from patients with either OA or RA displayed a higher CXCL12-binding capacity than human umbilical vein ECs (HUVECs), and in HUVECs the binding of CXCL12 was increased on exposure to tumor necrosis factor-α or lymphotoxin-α₁β₂. Our findings indicate that CXCL12 binds to HSPGs on ECs of RA synovium. The phenomenon relates to the interaction of HSPGs with a CXCL12 domain with net positive surface charge located in the first β strand, which encompasses a canonical BXBB HSPG-binding motif. Furthermore, we show that the attachment of CXCL12 to HSPGs is upregulated by inflammatory cytokines. Both the upregulation of a constitutive chemokine during chronic inflammation and the HSPG-dependent immobilization of CXCL12 in EC surfaces are potential sites for therapeutic intervention.     

Mucosal angiogenesis regulation by CXCR4 and its ligand CXCL12 expressed by human intestinal microvascular endothelial cells

Mice genetically deficient in the chemokine receptor CXCR4 or its ligand stromal cell-derived factor (SDF)-1/CXCL12 die perinatally with marked defects in vascularization of the gastrointestinal tract. The aim of this study was to define the expression and angiogenic functions of microvascular CXCR4 and SDF-1/CXCL12 in the human intestinal tract. Studies of human colonic mucosa in vivo and primary cultures of human intestinal microvascular endothelial cells (HIMEC) in vitro showed that the intestinal microvasculature expresses CXCR4 and its cognate ligand SDF-1/CXCL12. Moreover, SDF-1/CXCL12 stimulation of HIMEC triggers CXCR4-linked G proteins, phosphorylates ERK1/2, and activates proliferative and chemotactic responses. Pharmacological studies indicate SDF-1/CXCL12 evokes HIMEC chemotaxis via activation of ERK1/2 and phosphoinositide 3-kinase signaling pathways. Consistent with chemotaxis and proliferation, endothelial tube formation was inhibited by neutralizing CXCR4 or SDF-1/CXCL12 antibodies, as well as the ERK1/2 inhibitor PD-98059. Taken together, these data demonstrate an important mechanistic role for CXCR4 and SDF-1/CXCL12 in regulating angiogenesis within the human intestinal mucosa.     

Cutting edge: profile of chemokine receptor expression on human plasma cells accounts for their efficient recruitment to target tissues

We systematically examined the repertoire of chemokine receptors expressed by human plasma cells. Fresh bone marrow plasma cells and myeloma cells consistently expressed CXCR4, CXCR6, CCR10, and CCR3. Accordingly, plasma cells responded to their respective ligands in chemotaxis and very late Ag-4-dependent cell adhesion to fibronectin. Immobilized CXC chemokine ligand (CXCL)16, a novel transmembrane-type chemokine and CXCR6 ligand, also directly induced adhesion of plasma cells without requiring G(alpha i) signaling or divalent cations. Furthermore, we revealed consistent expression of CXCL12 (CXCR4 ligand), CXCL16 (CXCR6 ligand), and CC chemokine ligand 28 (CCR10 and CCR3 ligand) in tissues enriched with plasma cells including bone marrow, and constitutive expression of CXCL12, CXCL16, and CC chemokine ligand 28 by cultured human bone marrow stromal cells. Collectively, plasma cells are likely to be recruited to bone marrow and other target tissues via CXCR4, CXCR6, CCR10, and CCR3. CXCR6 may also contribute to tissue localization of plasma cells through its direct binding to membrane-anchored CXCL16.     

The chemokine receptors CXCR1 and CXCR2 couple to distinct g protein-coupled receptor kinases to mediate and regulate leukocyte functions

The chemokine receptors, CXCR1 and CXCR2, couple to Gαi to induce leukocyte recruitment and activation at sites of inflammation. Upon activation by CXCL8, these receptors become phosphorylated, desensitized, and internalized. In this study, we investigated the role of different G protein-coupled receptor kinases (GRKs) in CXCR1- and CXCR2-mediated cellular functions. To that end, short hairpin RNA was used to inhibit GRK2, 3, 5, and 6 in RBL-2H3 cells stably expressing CXCR1 or CXCR2, and CXCL8-mediated receptor activation and regulation were assessed. Inhibition of GRK2 and GRK6 increased CXCR1 and CXCR2 resistance to phosphorylation, desensitization, and internalization, respectively, and enhanced CXCL8-induced phosphoinositide hydrolysis and exocytosis in vitro. GRK2 depletion diminished CXCR1-induced ERK1/2 phosphorylation but had no effect on CXCR2-induced ERK1/2 phosphorylation. GRK6 depletion had no significant effect on CXCR1 function. However, peritoneal neutrophils from mice deficient in GRK6 (GRK6(-/-)) displayed an increase in CXCR2-mediated G protein activation but in vitro exhibited a decrease in chemotaxis, receptor desensitization, and internalization relative to wild-type (GRK6(+/+)) cells. In contrast, neutrophil recruitment in vivo in GRK6(-/-) mice was increased in response to delivery of CXCL1 through the air pouch model. In a wound-closure assay, GRK6(-/-) mice showed enhanced myeloperoxidase activity, suggesting enhanced neutrophil recruitment, and faster wound closure compared with GRK6(+/+) animals. Taken together, the results indicate that CXCR1 and CXCR2 couple to distinct GRK isoforms to mediate and regulate inflammatory responses. CXCR1 predominantly couples to GRK2, whereas CXCR2 interacts with GRK6 to negatively regulate receptor sensitization and trafficking, thus affecting cell signaling and angiogenesis.     

CXCR4 regulates migration of lung alveolar epithelial cells through activation of Rac1 and matrix metalloproteinase-2

Restoration of the epithelial barrier following acute lung injury is critical for recovery of lung homeostasis. After injury, alveolar type II epithelial (ATII) cells spread and migrate to cover the denuded surface and, eventually, proliferate and differentiate into type I cells. The chemokine CXCL12, also known as stromal cell-derived factor 1α, has well-recognized roles in organogenesis, hematopoiesis, and immune responses through its binding to the chemokine receptor CXCR4. While CXCL12/CXCR4 signaling is known to be important in immune cell migration, the role of this chemokine-receptor interaction has not been studied in alveolar epithelial repair mechanisms. In this study, we demonstrated that secretion of CXCL12 was increased in the bronchoalveolar lavage of rats ventilated with an injurious tidal volume (25 ml/kg). We also found that CXCL12 secretion was increased by primary rat ATII cells and a mouse alveolar epithelial (MLE12) cell line following scratch wounding and that both types of cells express CXCR4. CXCL12 significantly increased ATII cell migration in a scratch-wound assay. When we treated cells with a specific antagonist for CXCR4, AMD-3100, cell migration was significantly inhibited. Knockdown of CXCR4 by short hairpin RNA (shRNA) caused decreased cell migration compared with cells expressing a nonspecific shRNA. Treatment with AMD-3100 decreased matrix metalloproteinase-14 expression, increased tissue inhibitor of metalloproteinase-3 expression, decreased matrix metalloproteinase-2 activity, and prevented CXCL12-induced Rac1 activation. Similar results were obtained with shRNA knockdown of CXCR4. These findings may help identify a therapeutic target for augmenting epithelial repair following acute lung injury.     

Inhibition of chemokine receptor function by membrane cholesterol oxidation

Membrane cholesterol is required to maintain chemokine receptor conformation and function for CXCR4 and CCR5. We previously demonstrated that chemokines preferentially bind to receptors within lipid rafts, which are cholesterol- and sphingolipid-rich membrane microdomains. To further elucidate the role of cholesterol in chemokine receptor function, we examined the effects of membrane cholesterol oxidation by cholesterol oxidase (CO), which enzymatically converts cholesterol to 4-cholesten-3-one. Here, we demonstrate that CO treatment (0.25-2.0 U/ml) of human T cells inhibits CXCL12 (SDF-1alpha) and CCL4 (MIP-1beta) binding to cell surface CXCR4 and CCR5, respectively, resulting in the inhibition of chemokine-mediated intracellular calcium mobilization and chemotaxis. The effects were significantly enhanced by cotreatment with low-dose sphingomyelinase (SMase) (0.125 mU/ml), which produced little inhibitory effect by itself. CO and SMase treatment also inhibited HIV-1 infection through CXCR4, but not virus replication. Similar to the removal of membrane cholesterol, CO/SMase treatment induced conformation changes in the chemokine receptors as detected by differential loss in binding of epitope-specific monoclonal antibodies. We conclude that the native form of cholesterol with the hydroxyl group at C3 is critical to CXCR4 and CCR5 conformation and function.     

CXCL10 is the key ligand for CXCR3 on CD8+ effector T cells involved in immune surveillance of the lymphocytic choriomeningitis virus-infected central nervous system

IFN-gamma-inducible protein 10/CXCL10 is a chemokine associated with type 1 T cell responses, regulating the migration of activated T cells through binding to the CXCR3 receptor. Expression of both CXCL10 and CXCR3 are observed during immunopathological diseases of the CNS, and this receptor/ligand pair is thought to play a central role in regulating T cell-mediated inflammation in this organ site. In this report, we investigated the role of CXCL10 in regulating CD8(+) T cell-mediated inflammation in the virus-infected brain. This was done through analysis of CXCL10-deficient mice infected intracerebrally with lymphocytic choriomeningitis virus, which in normal immunocompetent mice induces a fatal CD8(+) T cell-mediated meningoencephalitis. We found that a normal antiviral CD8(+) T cell response was generated in CXCL10-deficient mice, and that lack of CXCL10 had no influence on the accumulation of mononuclear cells in the cerebrospinal fluid. However, analysis of the susceptibility of CXCL10-deficient mice to lymphocytic choriomeningitis virus-induced meningitis revealed that these mice just like CXCR3-deficient mice were partially resistant to this disease, whereas wild-type mice invariably died. Furthermore, despite marked up-regulation of the two remaining CXCR3 ligands: CXCL9 and 11, we found a reduced accumulation of CD8(+) T cells in the brain parenchyma around the time point when wild-type mice succumb as a result of CD8(+) T cell-mediated inflammation. Thus, taken together these results indicate a central role for CXCL10 in regulating the accumulation of effector T cells at sites of CNS inflammation, with no apparent compensatory effect of other CXCR3 ligands.