Chemokine-like factor 1 is a functional ligand for CC chemokine receptor 4 (CCR4)

Chemokine-like factor 1 (CKLF1) exhibits chemotactic effects on leukocytes. Its amino acid sequence shares similarity with those of TARC/CCL17 and MDC/CCL22, the cognate ligands for CCR4. The chemotactic effects of CKLF1 for CCR4-transfected cells could be desensitized by TARC/CCL17 and markedly inhibited by PTX. CKLF1 induced a calcium flux in CCR4-transfected cells and fully desensitized a subsequent response to TARC/CCL17, and TARC/CCL17 could partly desensitize the response to CKLF1. CKLF1 caused significant receptor internalization in pCCR4-EGFP transfected cells. Taken together, CKLF1 is a novel functional ligand for CCR4.     

Crystal structure and biophysical characterisation of Helicobacter pylori phosphopantetheine adenylyltransferase

CKLF1, a human cytokine that is a functional ligand for CCR4, is upregulated in various inflammation and autoimmune diseases. CKLF1 contains at least two secreted forms, the C-terminal peptides C19 and C27. Chemically synthesized C19 and C27 can interact with CCR4 and attenuate allergic inflammation. In this study, we found C19 and C27 could inhibit SDF-1-induced CXCR4-mediated chemotaxis and promote CXCR4 internalization. The inhibitory effect was due to desensitization of CXCR4, which was mediated by CCR4. Further experiments confirmed that CXCR4 desensitization required activation of PI3K/PKC pathway. Altogether our data elucidate the mechanism of C19- and C27-induced CXCR4 desensitization.     

Identification of a novel chemokine (CCL28), which binds CCR10 (GPR2)

We report the identification and characterization of a novel CC chemokine designated CCL28 and its receptor CCR10, known previously as orphan G-protein-coupled receptor GPR2. Human and mouse CCL28 share 83% identity at the amino acid and 76% at the nucleic acid levels. We also identified the mouse homologues of CCL28 and of CCR10, which map to mouse chromosomes 13 and 11, respectively. CCL28 is expressed in a variety of human and mouse tissues, and it appears to be predominantly produced by epithelial cells. Both human and mouse CCL28 induce calcium mobilization in human and mouse CCR10-expressing transfectants. CCL28 desensitized the calcium mobilization induced in CCR10 transfectants by CCL27, indicating that these chemokines share this new chemokine receptor. In vitro, recombinant human CCL28 displays chemotactic activity for resting CD4 or CD8 T cells.     

Differential CCR1-mediated chemotaxis signaling induced by human CC chemokine HCC-4/CCL16 in HOS cells

Human CC chemokine-4 (HCC-4)/CCL16 is a chemoattractant for monocytes and lymphocytes. Although HCC-4 binds to multiple CC chemokine receptors, the receptor-mediated signal transduction pathway induced by HCC-4 has not been characterized. Human osteogenic sarcoma cells stably expressing CCR1 were used to investigate HCC-4-mediated chemotaxis signaling events via CCR1. The chemotactic activity of HCC-4 as well as those of other CCR1-dependent chemokines including MIP-1alpha/CCL3, RANTES/CCL5, and Lkn-1/CCL15 was inhibited by the treatment of pertussis toxin, an inhibitor of Gi/Go protein, U73122, an inhibitor of phospholipase C (PLC), and rottlerin, a specific inhibitor of protein kinase Cdelta (PKCdelta). These results indicate that HCC-4-induced chemotaxis signaling is mediated through Gi/Go protein, PLC, and PKCdelta. SB202190, an inhibitor of p38 mitogen activated protein kinase, only blocked the chemotactic activity of HCC-4, but not those of other CCR1-dependent chemokines. SB202190 inhibited HCC-4-induced chemotaxis in a dose-dependent manner (P < 0.01). HCC-4 induces p38 activation in both a time and dose-dependent manner. However, such p38 activation was not induced by other CCR1-dependent chemokines. To further investigate the differential effect of HCC-4, the Ca2+ mobilization was examined. HCC-4 induced no intracellular Ca2+ flux in contrast to other CCR1-dependent chemokines. These results indicate that HCC-4 transduces signals differently from other CCR1-dependent chemokines and may play different roles in the immune response.     

Rac1 plays a role in CXCL12 but not CCL3-induced chemotaxis and Rac1 GEF inhibitor NSC23766 has off target effects on CXCR4

Cell migration towards a chemotactic stimulus relies on the re-arrangement of the cytoskeleton, which is triggered by activation of small G proteins RhoA, Rac1 and Cdc42, and leads to formation of lamellopodia and actin polymerisation amongst other effects. Here we show that Rac1 is important for CXCR4 induced chemotaxis but not for CCR1/CCR5 induced chemotaxis. For CXCL12-induced migration via CXCR4, breast cancer MCF-7 cells are reliant on Rac1, similarly to THP-1 monocytes and Jurkat T-cells. For CCL3-induced migration via CCR1 and/or CCR5, Rac1 signalling does not regulate cell migration in either suspension or adherent cells. We have confirmed the involvement of Rac1 with the use of a specific Rac1 blocking peptide. We also used a Rac1 inhibitor EHT 1864 and a Rac1-GEF inhibitor NSC23766 to probe the importance of Rac1 in chemotaxis. Both inhibitors did not block CCL3-induced chemotaxis, but they were able to block CXCL12-induced chemotaxis. This confirms that Rac1 activation is not essential for CCL3-induced migration, however NSC23766 might have secondary effects on CXCR4. This small molecule exhibits agonistic features in internalisation and cAMP assays, whereas it acts as an antagonist for CXCR4 in migration and calcium release assays. Our findings strongly suggest that Rac1 activation is not necessary for CCL3 signalling, and reveal that NSC23766 could be a novel CXCR4 receptor ligand.     

Differential desensitization, receptor phosphorylation, beta-arrestin recruitment, and ERK1/2 activation by the two endogenous ligands for the CC chemokine receptor 7

Many members of the chemokine receptor family of G protein-coupled receptors utilize multiple endogenous ligands. However, differences between the signaling properties of multiple chemokines through a single receptor have yet to be well characterized. In this study we investigated the early signaling events of CCR7 initiated by its two endogenous ligands, CCL19 and CCL21. Both CCL19 and CCL21 induce G protein activation and calcium mobilization with equal potency. However, only activation by CCL19, not CCL21, promotes robust desensitization of endogenous CCR7 in the human T cell lymphoma cell line H9. Desensitization occurs through the induction of receptor phosphorylation and beta-arrestin recruitment (shown in HEK293 cells expressing CCR7-FLAG). The sites of CCL19-induced phosphorylation were mapped by mutating to alanines the serines and threonines found within kinase phosphorylation consensus sequences in the carboxyl terminus of CCR7. A cluster of sites, including Thr-373-376 and Ser-378 is important for CCL19-mediated phosphorylation of the receptor, whereas residues serine 356, 357, 364, and 365 are important for basal receptor phosphorylation by protein kinase C. Activation of CCR7 by both ligands leads to signaling to the ERK1/2 mitogen-activated protein kinase pathway. However, CCL19 promotes 4-fold more ERK1/2 phosphorylation than does CCL21. The mechanism by which CCL19 activates ERK1/2 was determined to be beta-arrestin-dependent, because it is reduced both by depletion of beta-arrestin-2 with small interfering RNA and by elimination of the phosphorylation sites in the tail of the receptor. Taken together, these findings demonstrate that CCL19 and CCL21 place CCR7 in functionally distinct conformations that are independent of their G protein-coupling potency: one that allows the efficient desensitization of the receptor and activation of ERK1/2, and another that is impaired in these functions.     

Arrestin 3 mediates endocytosis of CCR7 following ligation of CCL19 but not CCL21

Internalization of ligand bound G protein-coupled receptors, an important cellular function that mediates receptor desensitization, takes place via distinct pathways, which are often unique for each receptor. The C-C chemokine receptor (CCR7) G protein-coupled receptor is expressed on naive T cells, dendritic cells, and NK cells and has two endogenous ligands, CCL19 and CCL21. Following binding of CCL21, 21 +/- 4% of CCR7 is internalized in the HuT 78 human T cell lymphoma line, while 76 +/- 8% of CCR7 is internalized upon binding to CCL19. To determine whether arrestins mediated differential internalization of CCR7/CCL19 vs CCR7/CCL21, we used small interfering RNA (siRNA) to knock down expression of arrestin 2 or arrestin 3 in HuT 78 cells. Independent of arrestin 2 or arrestin 3 expression, CCR7/CCL21 internalized. In contrast, following depletion of arrestin 3, CCR7/CCL19 failed to internalize. To examine the consequence of complete loss of both arrestin 2 and arrestin 3 on CCL19/CCR7 internalization, we examined CCR7 internalization in arrestin 2(-/-)/arrestin 3(-/-) murine embryonic fibroblasts. Only reconstitution with arrestin 3-GFP but not arrestin 2-GFP rescued internalization of CCR7/CCL19. Loss of arrestin 2 or arrestin 3 blocked migration to CCL19 but had no effect on migration to CCL21. Using immunofluorescence microscopy, we found that arrestins do not cluster at the membrane with CCR7 following ligand binding but cap with CCR7 during receptor internalization. These are the first studies that define a role for arrestin 3 in the internalization of a chemokine receptor following binding of one but not both endogenous ligands.     

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.     

Inhibition of generation of cytotoxic T lymphocyte activity by a CCL19/macrophage inflammatory protein (MIP)-3beta antagonist

Chemokines constitute a group of over 40 secreted peptides that are important for the control of leukocyte migration both during homeostasis and inflammation. Recent studies have implicated the ligands CCL19 and CCL21 and their receptor, CCR7, in the specific migration of na?ve lymphocytes and mature dendritic cells to secondary lymphoid organs during immune homeostasis. However, the role that these molecules play during immune priming is not well understood. In this study, using CCL19((8-83)), a novel N-terminal truncation mutant, we have investigated the role of CCL19 in a primary allogeneic immune response, a response of particular relevance to transplant rejection. This antagonist specifically inhibited wild type CCL19-induced chemotaxis and intracellular calcium mobilization without affecting that of CCL21. The treatment of mice with CCL19((8-83)) did not globally inhibit the recruitment of cells into lymph nodes; however, it inhibited the generation of cytotoxic T lymphocytes toward allogeneic dendritic cells. This is the first evidence that CCL19 plays a role in immune priming.     

Proinflammatory chemokines, such as C-C chemokine ligand 3, desensitize mu-opioid receptors on dorsal root ganglia neurons

Pain is one of the hallmarks of inflammation. Opioid receptors mediate antipain responses in both the peripheral nervous system and CNS. In the present study, pretreatment of CCR1: mu-opioid receptor/HEK293 cells with CCL3 (MIP-1alpha) induced internalization of mu-opioid receptors and severely impaired the mu-opioid receptor-mediated inhibition of cAMP accumulation. Immunohistochemical staining showed that CCR1 and mu-opioid receptors were coexpressed on small to medium diameter neurons in rat dorsal root ganglion. Analysis of ligand-induced calcium flux showed that both types of receptors were functional. Pretreatment of neurons with CCL3 exhibited an impaired [D-Ala(2),N-MePhe(4),Gly-o15]enkephalin-elicited calcium response, indicative of the heterologous desensitization of mu-opioid receptors. Other chemokines, such as CCL2, CCL5, and CXCL8, exhibited similar inhibitory effects. Our data indicate that proinflammatory chemokines are capable of desensitizing mu-opioid receptors on peripheral sensory neurons, providing a novel potential mechanism for peripheral inflammation-induced hyperalgesia.     

Plectin regulates the signaling and trafficking of the HIV-1 co-receptor CXCR4 and plays a role in HIV-1 infection

The CXC chemokine CXCL12 and its cognate receptor CXCR4 play an important role in inflammation, human immunodeficiency virus (HIV) infection and cancer metastasis. The signal transduction and intracellular trafficking of CXCR4 are involved in these functions, but the underlying mechanisms remain incompletely understood. In the present study, we demonstrated that the CXCR4 formed a complex with the cytolinker protein plectin in a ligand-dependent manner in HEK293 cells stably expressing CXCR4. The glutathione-S-transferase (GST)-CXCR4 C-terminal fusion proteins co-precipitated with the full-length and the N-terminal fragments of plectin isoform 1 but not with the N-terminal deletion mutants of plectin isoform 1, thereby suggesting an interaction between the N-terminus of plectin and the C-terminus of CXCR4. This interaction was confirmed by confocal microscopic reconstructions showing co-distribution of these two proteins in the internal vesicles after ligand-induced internalization of CXCR4 in HEK293 cells stably expressing CXCR4. Knockdown of plectin with RNA interference (RNAi) significantly inhibited ligand-dependent CXCR4 internalization and attenuated CXCR4-mediated intracellular calcium mobilization and activation of extracellular signal regulated kinase 1/2 (ERK1/2). CXCL12-induced chemotaxis of HEK293 cells stably expressing CXCR4 and of Jurkat T cells was inhibited by the plectin RNAi. Moreover, CXCR4 tropic HIV-1 infection in MAGI (HeLa-CD4-LTR-Gal) cells was inhibited by the RNAi of plectin. Thus, plectin appears to interact with CXCR4 and plays an important role in CXCR4 signaling and trafficking and HIV-1 infection.     

Small molecule antagonists of CCR8 inhibit eosinophil and T cell migration

In this study, we demonstrate that in addition to T lymphocytes, human naïve eosinophils and the differentiated eosinophil-like cell line, AML14.3D10 express CCR8 and respond to CCL1 through CCR8 engagement. The responsiveness of cells was dependent on maturation stage, since CCL1 induced pronounced chemotaxis only in differentiated CCR8 positive AML14.3D10 cells. Despite the low CCR8 surface expression, human naïve eosinophils respond with a chemotaxis to high concentration CCL1. We further describe that Th2 clones in a maturation dependent fashion produce autocrine CCL1, which renders them unresponsive to further stimulation. An innovative method to enrich primary CCR8 reactive T cells was developed which demonstrates that primary peripheral CCR8 expressing T cells respond significantly to CCL1.We have developed novel small molecule CCR8 antagonists that are effective in inhibiting calcium mobilization and chemotaxis in differentiated AML cells as well as in human primary CCR8 positive T cells. Importantly, we demonstrate that the compounds can be divided into two subgroups: (i) compounds that are functional agonists for calcium mobilization and chemotaxis (ii) compounds that are pure antagonists. We demonstrate that agonism of these compounds does not correlate with their antagonistic potency. Taken together, we have identified a novel set of CCR8 compounds with antagonistic properties that inhibit CCL1 driven chemotaxis in both CCR8 expressing eosinophils as well as primary human T cells.     

Chemokine-Like Factor 1-Derived C-Terminal Peptides Induce the Proliferation of Dermal Microvascular Endothelial Cells in Psoriasis

Psoriasis is an inflammatory disease characterized by the abnormal proliferation of skin cells, including dermal microvascular endothelial cells. Recently, chemokine-like factor 1 (CKLF1) was found to participate in the local inflammation and cell proliferation. To explore its role in the pathogenesis of psoriasis, the expression of both CKLF1 and its receptor (CCR4) was determined in the psoriatic lesions. Also, the effect of the C-terminal peptides (C19 and C27) of CKLF1 on the proliferation of human umbilical vein endothelial cells was studied in vitro. By immunohistochemistry and immunofluorescence, the expression of both CKLF1 and CCR4 was determined in the psoriatic lesions. The effect of C-terminal peptides on human umbilical vein endothelial cells (HUVECs) was studied in vitro by the evaluation of cell proliferation and apoptosis. The in vivo assessment was performed accordingly through the subcutaneous injection peptides on BALB/c mice. The results showed that, by immunohistochemistry, both CKLF1 and CCR4 were increasingly expressed in psoriatic lesions as compared to normal skins. Moreover, the primary umbilical vein endothelial cells exhibited higher proliferation ratio under the C19 or C27 stimulation, which was even enhanced by the addition of psoriatic sera or TNF-α. Furthermore, the enhancement of peptide simulation was accompanied with the activation of ERK1/2-MAPKs pathway. In addition, such effect of C19 and C27 was mirrored by the hyperproliferation of cutaneous microvessels in BALB/c mice that were subcutaneously injected with the two peptides. Therefore, we concluded that CKLF1 plays a role in the pathogenesis of psoriasis by promoting the proliferation of microvascular endothelial cells that possibly correlates with ERK1/2-MAPKs activation.     

Molecular mechanism of desensitization of the chemokine receptor CCR-5: receptor signaling and internalization are dissociable from its role as an HIV-1 co-receptor.

The chemokine receptor, CCR-5, a G protein-coupled receptor (GPCR) which mediates chemotactic responses of certain leukocytes, has been shown to serve as the primary co-receptor for macrophage-tropic human immunodeficiency virus type 1 (HIV-1). Here we describe functional coupling of CCR-5 to inhibition of forskolin-stimulated cAMP formation via a pertussis toxin-sensitive G(i) protein mechanism in transfected HEK 293 cells. In response to chemokines, CCR-5 was desensitized, phosphorylated and sequestered like a prototypic GPCR only following overexpression of G protein-coupled receptor kinases (GRKs) and beta-arrestins in HEK 293 cells. The lack of CCR-5 desensitization in HEK 293 cells in the absence of GRK overexpression suggests that differences in cellular complements of GRK and/or beta-arrestin proteins could represent an important mechanism determining cellular responsiveness. When tested, the activity of CCR-5 as an HIV-1 co-receptor was dependent neither upon its ability to signal nor its ability to be desensitized and internalized following agonist stimulation. Thus, while chemokine-promoted cellular signaling, phosphorylation and internalization of CCR-5 may play an important role in regulation of chemotactic responses in leukocytes, these functions are dissociable from its HIV-1 co-receptor function.     

CP-481,715, a potent and selective CCR1 antagonist with potential therapeutic implications for inflammatory diseases

The chemokines CCL3 and CCL5, as well as their shared receptor CCR1, are believed to play a role in the pathogenesis of several inflammatory diseases including rheumatoid arthritis, multiple sclerosis, and transplant rejection. In this study we describe the pharmacological properties of a novel small molecular weight CCR1 antagonist, CP-481,715 (quinoxaline-2-carboxylic acid [4(R)-carbamoyl-1(S)-(3-fluorobenzyl)-2(S),7-dihydroxy-7-methyloctyl]amide). Radiolabeled binding studies indicate that CP-481,715 binds to human CCR1 with a Kd of 9.2 nm and displaces 125I-labeled CCL3 from CCR1-transfected cells with an IC50 of 74 nm. CP-481,715 lacks intrinsic agonist activity but fully blocks the ability of CCL3 and CCL5 to stimulate receptor signaling (guanosine 5'-O-(thiotriphosphate) incorporation; IC50 = 210 nm), calcium mobilization (IC50 = 71 nm), monocyte chemotaxis (IC50 = 55 nm), and matrix metalloproteinase 9 release (IC50 = 54 nm). CP-481,715 retains activity in human whole blood, inhibiting CCL3-induced CD11b up-regulation and actin polymerization (IC50 = 165 and 57 nm, respectively) on monocytes. Furthermore, it behaves as a competitive and reversible antagonist. CP-481,715 is >100-fold selective for CCR1 as compared with a panel of G-protein-coupled receptors including related chemokine receptors. Evidence for its potential use in human disease is suggested by its ability to inhibit 90% of the monocyte chemotactic activity present in 11/15 rheumatoid arthritis synovial fluid samples. These data illustrate that CP-481,715 is a potent and selective antagonist for CCR1 with therapeutic potential for rheumatoid arthritis and other inflammatory diseases.     

Liver-expressed chemokine/CC chemokine ligand 16 attracts eosinophils by interacting with histamine H4 receptor

Liver-expressed chemokine (LEC)/CCL16 is a human CC chemokine that is constitutively expressed by the liver parenchymal cells and present in the normal plasma at high concentrations. Previous studies have shown that CCL16 is a low-affinity ligand for CCR1, CCR2, CCR5, and CCR8 and attracts monocytes and T cells. Recently, a novel histamine receptor termed type 4 (H4) has been identified and shown to be selectively expressed by eosinophils and mast cells. In this study, we demonstrated that CCL16 induced pertussis toxin-sensitive calcium mobilization and chemotaxis in murine L1.2 cells expressing H4 but not those expressing histamine receptor type 1 (H1) or type 2 (H2). CCL16 bound to H4 with a K(d) of 17 nM. By RT-PCR, human and mouse eosinophils express H4 but not H3. Accordingly, CCL16 induced efficient migratory responses in human and mouse eosinophils. Furthermore, the responses of human and mouse eosinophils to CCL16 were effectively suppressed by thioperamide, an antagonist for H3 and H4. Intravenous injection of CCL16 into mice induced a rapid mobilization of eosinophils from bone marrow to peripheral blood, which was also suppressed by thioperamide. Collectively, CCL16 is a novel functional ligand for H4 and may have a role in trafficking of eosinophils.     

PLP2/A4 interacts with CCR1 and stimulates migration of CCR1-expressing HOS cells

Multiple CC chemokines bind to CCR1, which plays important roles in immune and inflammatory responses. To search for proteins involved in the CCR1 signaling pathway, we screened a yeast two-hybrid library using the cytoplasmic tail of CCR1 as the bait. One of the positive clones contained an open reading frame of 456bp, of which the nucleotide sequence was identical to that of proteolipid protein 2 (PLP2), also known as protein A4. Mammalian two-hybrid and coimmunoprecipitation analyses demonstrated the association of PLP2/A4 with CCR1. Indirect immunofluorescence analysis revealed that PLP2/A4 was predominantly located in plasma membrane and colocalized with CCR1 in transfected human HEK293 cells. In addition, focal staining of CCR1 appeared on the periphery of the membrane upon short exposure to Leukotactin-1(Lkn-1)/CCL15, a CCR1 agonist, and was costained with PLP2/A4 on the focal regions. PLP2/A4 mRNAs were detected in various cells such as U-937, HL-60, HEK293, and HOS cells. Overexpression of PLP2/A4 stimulated a twofold increase in the agonist-induced migration of HOS/CCR1 cells, implicating a functional role for PLP2/A4 in the chemotactic processes via CCR1.     

Human NK cells express CC chemokine receptors 4 and 8 and respond to thymus and activation-regulated chemokine, macrophage-derived chemokine, and I-309

NK cells respond to various chemokines, suggesting that they express receptors for these chemokines. In this paper, we show that IL-2-activated NK (IANK) cells express CC chemokine receptor 4 (CCR4) and CCR8, as determined by flow cytometric, immunoblot, and RNase protection assays. Macrophage-derived chemokine (MDC), the ligand for CCR4, induces the phosphorylation of CCR4 within 0.5 min of activating IANK cells with this ligand. This is corroborated with the recruitment of G protein-coupled receptor kinases 2 and 3 and their association with CCR4 in IANK cell membranes. Also, CCR4 is internalized between 5 and 45 min but reappears in the membranes after 60 min of stimulation with MDC. MDC, thymus and activation-regulated chemokine (TARC), and I-309 induce the chemotaxis of IANK cells, an activity that is inhibited upon pretreatment of these cells with pertussis toxin, suggesting that receptors for these chemokines are coupled to pertussis toxin-sensitive G proteins. In the calcium release assay, cross-desensitization experiments showed that TARC completely desensitizes the calcium flux response induced by MDC or I-309, whereas both MDC and I-309 partially desensitize the calcium flux response induced by TARC. These results suggest that TARC utilizes CCR4 and CCR8. Our results are the first to show that IL-2-activated NK cells express CCR4 and CCR8, suggesting that these receptors are not exclusive for Th2 cells.     

Human CC chemokine CCL23, a ligand for CCR1, induces endothelial cell migration and promotes angiogenesis

A number of chemokines induce angiogenesis and endothelial cells express several chemokine receptors. To date, only a limited number of CC chemokines for CCR1 have been reported to induce angiogenic responses. We investigated the ability of CCL23 (also known as MPIF-1, MIP-3, or CKbeta8) to promote angiogenesis, which induces chemotaxis of immune cells through CCR1. CCL23 promoted the chemotactic migration and differentiation of endothelial cells, and neovascularization in the chick chorioallantoic membrane. An N-terminal truncated form of CCL23 was at least 100-fold more potent than its intact form and was comparable to that of FGF in the angiogenic activities. Treatment with either pertussis toxin or anti-CCR1 antibody completely inhibited the CCL23-induced endothelial cell migration, indicating that endothelial cell migration was mediated through CCR1. CCL23 didn't promote the migration of HT1080 human fibrosarcoma cells that did not express CCR1. Our results suggest a role of CCL23 in angiogenesis in vitro as well as in vivo.     

HIV type 1 glycoprotein 120 inhibits human B cell chemotaxis to CXC chemokine ligand (CXCL) 12, CC chemokine ligand (CCL)20, and CCL21

We analyzed the modulation of human B cell chemotaxis by the gp120 proteins of various HIV-1 strains. X4 and X4/R5 gp120 inhibited B cell chemotaxis toward CXCL12, CCL20, and CCL21 by 40-50%, whereas R5 gp120 decreased inhibition by 20%. This gp120-induced inhibition was strictly dependent on CXCR4 or CCR5 and lipid rafts but not on CD4 or V(H)3-expressing BCR. Inhibition did not impair the expression or ligand-induced internalization of CCR6 and CCR7. Our data suggest that gp120/CXCR4 and gp120/CCR5 interactions lead to the cross-desensitization of CCR6 and CCR7 because gp120 does not bind CCR6 and CCR7. Unlike CXCL12, gp120 did not induce the activation of phospholipase Cbeta3 and PI3K downstream from CXCR4, whereas p38 MAPK activation was observed. Similar results were obtained if gp120-treated cells were triggered by CCL21 and CCL20. Our results are consistent with a blockade restricted to signaling pathways using phosphatidylinositol-4,5-bisphosphate as a substrate. X4 and X4/R5 gp120 induced the cleavage of CD62 ligand by a mechanism dependent on matrix metalloproteinase 1 and 3, CD4, CXCR4, Galpha(i), and p38 MAPK, whereas R5 gp120 did not. X4 and X4/R5 gp120 also induced the relocalization of cytoplasmic CD95 to the membrane and a 23% increase in CD95-mediated apoptosis. No such effects were observed with R5 gp120. The gp120-induced decrease in B cell chemotaxis and CD62 ligand expression, and increase in CD95-mediated B cell apoptosis probably have major deleterious effects on B cell responsiveness during HIV infection and in vaccination trials.