CC chemokine receptor-3 as new target for age-related macular degeneration

CC chemokine receptor-3 (CCR3) is involved in angiogenic processes. Recently, CCR3 was accounted to participate in choroidal neovascularization (CNV) and CCR3 targeting was reported to be superior to standard antivascular endothelial growth factor-A (VEGF-A) administration when tested in an artificially induced CNV in animals. As human CCR3 studies are lacking in age-related macular degeneration (AMD) patients we sought to determine if CCR3 has any association with inflammatory processes that occur in CNV. A total of 176 subjects were included on the basis of inclusion criteria. Real time PCR was used to analyze the single nucleotide polymorphism in CCR3 of AMD (115) and normal controls (n = 61). Genotype frequency was adjusted for possible confounders like cigarette smoking, alcohol, meat consumption and other risk factors. Chi-square test was used for analysis of polymorphism. The genotype distribution of CCR3 (rs3091250) polymorphism was significantly different in AMD patients in the Indian population. GT (heterozygous) and TT (homozygous) at the rs3091250 SNP increased risk of AMD as compared to the GG genotypes (OR = 4.8, CI 95% = 2.2–10.8 and OR = 4.1, CI 95% = 1.6–10.1 respectively). Subgroup analysis of AMD patients in wet and dry revealed no significant differences. There was no significant difference for rs3091312 in AMD and control group. A significant association between AMD and CCR3 (rs3091250) polymorphism localized on chromosome 3p21.3 was detected. The results suggest the possible contribution of rs3091250, a new predisposing allele in AMD.     

VEGF Receptor Blockade Markedly Reduces Retinal Microglia/Macrophage Infiltration into Laser-Induced CNV

Although blocking VEGF has a positive effect in wet age-related macular degeneration (AMD), the effect of blocking its receptors remains unclear. This was an investigation of the effect of VEGF receptor (VEGFR) 1 and/or 2 blockade on retinal microglia/macrophage infiltration in laser-induced choroidal neovascularization (CNV), a model of wet AMD. CNV lesions were isolated by laser capture microdissection at 3, 7, and 14 days after laser and analyzed by RT-PCR and immunofluorescence staining for mRNA and protein expression, respectively. Neutralizing antibodies for VEGFR1 or R2 and the microglia inhibitor minocycline were injected intraperitoneally (IP). Anti-CD11b, CD45 and Iba1 antibodies were used to confirm the cell identity of retinal microglia/macrophage, in the RPE/choroidal flat mounts or retinal cross sections. CD11b(+), CD45(+) or Iba1(+) cells were counted. mRNA of VEGFR1 and its three ligands, PlGF, VEGF-A (VEGF) and VEGF–B, were expressed at all stages, but VEGFR2 were detected only in the late stage. PlGF and VEGF proteins were expressed at 3 and 7 days after laser. Anti-VEGFR1 (MF1) delivered IP 3 days after laser inhibited infiltration of leukocyte populations, largely retinal microglia/macrophage to CNV, while anti-VEGFR2 (DC101) had no effect. At 14 days after laser, both MF1 and DC101 antibodies markedly inhibited retinal microglia/macrophage infiltration into CNV. Therefore, VEGFR1 and R2 play differential roles in the pathogenesis of CNV: VEGFR1 plays a dominant role at 3 days after laser; but both receptors play pivotal roles at 14 days after laser. In vivo imaging demonstrated accumulation of GFP-expressing microglia into CNV in both CX3CR1^gfp/gfp and CX3CR1^gfp/+ mice. Minocycline treatment caused a significant increase in lectin⁺ cells in the sub-retinal space anterior to CNV and a decrease in dextran-perfused neovessels compared to controls. Targeting the chemoattractant molecules that regulate trafficking of retinal microglia/macrophage appears to be a compelling therapeutic strategy to control CNV and treat wet AMD.     

NLRP3 has a protective role in age-related macular degeneration through the induction of IL-18 by drusen components

Age-related macular degeneration (AMD) is the leading cause of central vision loss worldwide. Drusen accumulation is the major pathological hallmark common to both dry and wet AMD. Although activation of the immune system has been implicated in disease progression, the pathways involved are unclear. Here we show that drusen isolated from donor AMD eyes activates the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome, causing secretion of interleukin-1b (IL-1b) and IL-18. Drusen component C1Q also activates the NLRP3 inflammasome. Moreover, the oxidative-stress-related protein-modification carboxyethylpyrrole (CEP), a biomarker of AMD, primes the inflammasome. We found cleaved caspase-1 and NLRP3 in activated macrophages in the retinas of mice immunized with CEP-adducted mouse serum albumin, modeling a dry-AMD–like pathology. We show that laser-induced choroidal neovascularization (CNV), a mouse model of wet AMD, is exacerbated in Nlrp3(-/-) but not Il1r1(-/-) mice, directly implicating IL-18 in the regulation of CNV development. These findings indicate a protective role for NLRP3 and IL-18 in the progression of AMD.     

Inhibitory role of adiponectin peptide I on rat choroidal neovascularization

Age-related macular degeneration (AMD) is a leading cause of central blindness in the elderly population. The wet type of AMD is characterized by extensive growth of new vessels. One of the effective strategies to treat wet AMD is to limit the choroidal neovascularization (CNV). We studied the effects of adiponectin peptide I (APNpI) on new vessel growth in laser-induced rat model of wet AMD and on rat choroidal endothelial cell (CEC) culture. CNV size and vessel density were investigated by microscopy. Immunohistochemical staining (IHC) for von Willebrand Factor (vWF), APN, APN receptors 1 (AdipoR1), 2 (AdipoR2), VEGF, VEGF receptor 2 (VEGF-R2), proliferating cell nuclear antigen (PCNA) was performed in CNV area. The mRNA expression of VEGF and VEGF-R2 in RPE-choroid was investigated by RT-PCR and real-time PCR. APNpI inhibited area of CNV by 4 fold, number of vWF positive vessels by 99% and area of subretinal tissue by 40%. The expression of VEGF and VEGF-R2 at mRNA and protein levels decreased after APNpI treatment in vivo. Proliferative index (PCNA) was 5 folds less in laser spots of APNpI treated rats compared to controls. In conclusion, APNpI inhibited formation of new vessels in rat model of CNV by decreasing VEGF, VEGF-R2 expression and cell proliferation. Thus, APNpI may have potential therapeutic use for AMD treatment since it significantly inhibited CNV.     

Pulmonary epithelial CCR3 promotes LPS-induced lung inflammation by mediating release of IL-8

Interleukin (IL)-8 from pulmonary epithelial cells has been suggested to play an important role in the airway inflammation, although the mechanism remains unclear. We envisioned a possibility that pulmonary epithelial CCR3 could be involved in secretion and regulation of IL-8 and promote lipopolysaccharide (LPS)-induced lung inflammation. Human bronchial epithelial cell line NCI-H292 and alveolar type II epithelial cell line A549 were used to test role of CCR3 in production of IL-8 at cellular level. In vivo studies were performed on C57/BL6 mice instilled intratracheally with LPS in a model of acute lung injury (ALI). The activity of a CCR3-specific inhibitor (SB-328437) was measured in both in vitro and in vivo systems. We found that expression of CCR3 in NCI-H292 and A549 cells were increased by 23% and 16%, respectively, 24 h after the challenge with LPS. LPS increased the expression of CCR3 in NCI-H292 and A549 cells in a time-dependent manner, which was inhibited significantly by SB-328437. SB-328437 also diminished neutrophil recruitment in alveolar airspaces and improved LPS-induced ALI and production of IL-8 in bronchoalveolar lavage fluid. These results suggest that pulmonary epithelial CCR3 be involved in progression of LPS-induced lung inflammation by mediating release of IL-8. CCR3 in pulmonary epithelia may be an attractive target for development of therapies for ALI.     

Decreased VEGF-A and sustained PEDF expression in a human retinal pigment epithelium cell line cultured under hypothermia

Background

Previous reports have described a decrease in retinal temperature and clinical improvement of wet age-related macular degeneration (AMD) after vitrectomy. We hypothesized that the retinal temperature decrease after vitrectomy plays a part in the suppression of wet AMD development. To test this hypothesis, we evaluated the temperature dependence of the expression of vascular endothelial growth factor-A (VEGF-A) and in vitro angiogenesis in retinal pigment epithelium (RPE).

Results

We cultured ARPE-19 cells at 37, 35, 33 and 31°C and measured the expression of VEGF-A, VEGF-A splicing variants, and pigment epithelium–derived factor (PEDF). We performed an in vitro tube formation assay. The dehydrogenase activity was also evaluated at each temperature. Expression of VEGF-A significantly decreased with decreased temperature while PEDF expression did not. VEGF165 expression and in vitro angiogenesis also were temperature dependent. The dehydrogenase activity significantly decreased as the culture temperature decreased.

Conclusions

RPE cultured under hypothermia that decreased cellular metabolism also had decreased VEGF-A and sustained PEDF expression, creating an anti-angiogenic environment. This mechanism may be associated with a beneficial effect after vitrectomy in patients with wet AMD.     

Inhibition of new vessel growth in mouse model of laser-induced choroidal neovascularization by adiponectin peptide II

We have investigated the effect of adiponectin (APN) peptide II on new vessel growth in mouse model of choroidal neovascularization (CNV) or wet type age-related macular degeneration (AMD). Mice were injected intraperitoneally with APN peptide II, control peptide, or PBS on day 1-7 or day 5-14. APN, AdipoR1, PCNA, and VEGF localization was investigated using confocal microscopy, immunohistochemistry, and RT-PCR. APN peptide II decreased the relative area of FITC-dextran perfused vessels by 4-fold, PCNA expression by 3-fold, and the number of PCNA stained HUVEC and MAVEC cells by 38 and 46%, respectively. We concluded that APN peptide II inhibits CNV size on days 7 and 14 by inhibiting the proliferation of endothelial cells in vivo and in vitro. APN peptide II may have therapeutic potential to inhibit CNV or wet AMD.     

年龄相关性黄斑变性患者黄斑区脉络膜新生血管特征分析

目的:探讨年龄相关性黄斑变性黄斑区脉络膜新生血管(CNV)的特征。方法:对41例(41只眼)年龄相关性黄斑变性患者CNV进行荧光素眼底血管造影(FFA)和相干光断层扫描(OCT)检查,并对两种不同的图像检查结果进行对比分析。结果:FFA发现典型CNV29眼,FFA早期黄斑区可见边界清晰的高荧光区,FFA晚期荧光扩大并增强;隐匿型CNV10眼,FFA早期可见边界欠清的强荧光,FFA晚期持续为荧光素渗漏或染色,边界多不清晰。OCT发现典型CNV33眼,显示CNV多位于视网膜神经上皮层下,呈边界清晰的纺锤形或圆形强反射,隐匿型CNV8眼,表现为视网膜色素上皮层平面不规则增强紊乱的反射信号。两种检查对黄斑区CNV的检出率对比差异无统计学意义(P0.05)。结论:结合FFA和OCT的图像特征对年龄相关性黄斑变性黄斑区脉络膜新生血管形态学改变进行对比分析,有助于明确病变的性质,为临床诊疗提供帮助。     

A dual-targeting PDGFRβ/VEGF-A molecule assembled from stable antibody fragments demonstrates anti-angiogenic activity in vitro and in vivo

Targeting angiogenesis is a promising approach to the treatment of solid tumors and age-related macular degeneration (AMD). Inhibition of vascularization has been validated by the successful marketing of monoclonal antibodies (mAbs) that target specific growth factors or their receptors, but there is considerable room for improvement in existing therapies. Combination of mAbs targeting both the VEGF and PDGF pathways has the potential to increase the efficacy of anti-angiogenic therapy without the accompanying toxicities of tyrosine kinase inhibitors and the inability to combine efficiently with traditional chemotherapeutics. However, development costs and regulatory issues have limited the use of combinatorial approaches for the generation of more efficacious treatments. The concept of mediating disease pathology by targeting two antigens with one therapeutic was proposed over two decades ago. While mAbs are particularly suitable candidates for a dual-targeting approach, engineering bispecificity into one molecule can be difficult due to issues with expression and stability, which play a significant role in manufacturability. Here, we address these issues upstream in the process of developing a bispecific antibody (bsAb). Single-chain antibody fragments (scFvs) targeting PDGFRβ and VEGF-A were selected for superior stability. The scFvs were fused to both termini of human Fc to generate a bispecific, tetravalent molecule. The resulting molecule displays potent activity, binds both targets simultaneously, and is stable in serum. The assembly of a bsAb using stable monomeric units allowed development of an anti-PDGFRB/VEGF-A antibody capable of attenuating angiogenesis through two distinct pathways and represents an efficient method for rapid engineering of dual-targeting molecules.     

Blocking the monocyte chemoattractant protein-1/CCR2 chemokine pathway induces permanent survival of islet allografts through a programmed death-1 ligand-1-dependent mechanism

Islet allografts are subject to rapid rejection through host cellular immune responses involving mononuclear cell recruitment and tissue injury. Interruption of leukocyte recruitment through chemokine receptor targeting is of therapeutic benefit in various experimental models, but little is known about the contribution of chemokine pathways to islet allograft rejection. We found that murine islets produce monocyte chemoattractant protein-1 (MCP-1; CCL2) in vitro and that islet allograft rejection was associated with intragraft expression of MCP-1 and its receptor, CCR2. We therefore investigated whether MCP-1 and CCR2 are required for the rejection of fully MHC-disparate islet allografts. Wild-type mice treated with blocking anti-MCP-1 mAb plus a brief, subtherapeutic course of rapamycin had long-term islet allograft survival, in contrast to the effect of treatment with either mAb or rapamycin alone. CCR2(-/-) mice treated with rapamycin also maintained islet allografts long-term. Both MCP/CCR2- and rapamycin-sensitive signals were required for maximal proliferation of alloreactive T cells, suggesting that MCP-1/CCR2 induce rejection by promoting alloreactive T cell clonal expansion and homing and migration. Prolonged islet allograft survival achieved by blockade of the MCP-1/CCR2 pathway plus rapamycin therapy was accompanied by a mononuclear cell infiltrate expressing the inhibitory receptor, programmed death-1 (PD-1), and its ligand (PD-L1, B7-H1), and prolongation of islet allograft survival was abrogated by anti-PD-L1 mAb therapy. These data show that the blockade of MCP-1 binding to CCR2 in conjunction with subtherapeutic immunosuppression can have profound effects on islet allograft survival and implicate the expression of the PD-1/PD-L1 pathway in the regulation of physiologic responses in vivo.     

Increased efficacy of HIV-1 neutralization by antibodies at low CCR5 surface concentration

It has been observed that some antibodies, including the CD4-induced (CD4i) antibody IgG X5 and the gp41-specific antibody IgG 2F5, exhibit higher neutralizing activity in PBMC-based assays than in cell line based assays [J.M. Binley, T. Wrin, B. Korber, M.B. Zwick, M. Wang, C. Chappey, G. Stiegler, R. Kunert, S. Zolla-Pazner, H. Katinger, C.J. Petropoulos, D.R. Burton, Comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency virus type 1 monoclonal antibodies, J. Virol. 78 (2004) 13232-13252]. It has been hypothesized that the lower CCR5 concentration on the surface of the CD4 T lymphocytes compared to that on cell lines used for the neutralization assays could be a contributing factor to the observed differences in neutralizing activity. To test this hypothesis and to further elucidate the contribution of CCR5 concentration differences on antibody neutralizing activity, we used a panel of HeLa cell lines with well-defined and differential surface concentrations of CCR5 and CD4 in a pseudovirus-based assay. We observed that the CCR5 cell surface concentration but not the CD4 concentration had a significant effect on the inhibitory activity of X5 and several other CD4i antibodies including 17b and m9, as well as that of the gp41-specifc antibodies 2F5 and 4E10 but not on that of the CD4 binding site antibody (CD4bs), b12. The 50% inhibitory concentration (IC50) decreased up to two orders of magnitude in cell lines with low CCR5 concentration corresponding to that in CD4 T cells used in PBMC-based assays (about 10(3) per cell) compared to cell lines with high CCR5 concentration (about 10(4) or more). Our results suggest that the CCR5 cell surface concentration could be a contributing factor to the high neutralizing activities of some antibodies in PBMC-based-assays but other factors could also play an important role. These findings could have implications for development of vaccine immunogens based on the epitopes of X5 and other CD4i antibodies, for elucidation of the mechanisms of HIV-1 neutralization by antibodies, and for design of novel therapeutic approaches.     

Impact of IGF-I/IGF-IR circuit on the angiogenetic properties of Ewing's sarcoma cells.

The insulin-like growth factor receptor I (IGF-I)-mediated circuit is a major autocrine loop for Ewing's sarcoma (ES) cells, and plays a role the pathogenesis and malignancy of this tumor. IGF-I receptor (IGF-IR) has emerged as a good therapeutic site for ES patients. In this study, we analyzed the impact of strategies targeting the IGF-IR on the regulation of VEGFs, which are of fundamental importance in angiogenesis, and TGFbeta, CTGF and Cyr61, which are factors primarily involved in skeletal growth control and angiogenesis. IGF-I increases expression of VEGF-A, TGFbeta, CTGF and Cyr61 mRNA. However, only the modulation of VEGF-A expression appears to be mediated by IGF-IR. Functional assays on endothelial cells indicate a strict correlation between survival and proliferation of HUVECs and VEGF-A levels, confirming a major role for this factor in angiogenesis. Blockage of IGF-IR functions by neutralizing antibody or antisense strategies significantly reduced the expression and secretion of VEGF-A by ES cells, and supernatants of treated cells were unable to sustain the survival and proliferation of HUVECs. Analysis of the signaling mechanisms involved in constitutive or IGF-induced expression and secretion of VEGF-A indicated that PI3-K and MAPK signaling pathways are both required for VEGF expression and production in ES cells. Selective inhibitors LY294002 or PD98059 were highly effective in reducing the ability of ES cells to produce VEGF-A and stimulate survival and proliferation of HUVECs. Taken together, these findings add a new activity to the IGF-I repertoire in ES and highlight how disruption of IGF-IR functions may constitute an effective tool for the control of neovascularization in this tumor.     

Transforming growth factor-beta induces expression of vascular endothelial growth factor in human retinal pigment epithelial cells: involvement of mitogen-activated protein kinases

Vascular endothelial growth factor (VEGF) is a major agent in choroidal and retinal neovascularization, events associated with age-related macular degeneration (AMD) and diabetic retinopathy. Retinal pigment epithelium (RPE), strategically located between retina and choroid, plays a critical role in retinal disorders. We have examined the effects of various growth factors on the expression and secretion of VEGF by human retinal pigment epithelial cell cultures (HRPE). RT-PCR analyses revealed the presence of three isoforms of mRNA corresponding to VEGF 121, 165, and 189 that were up regulated by TGF-beta1. TGF-beta1, beta2, and beta3 were the potent inducers of VEGF secretion by HRPE cells whereas bFGF, PDGF, TGF-alpha, and GM-CSF had no effects. TGF-beta receptor type II antibody significantly reversed induction of VEGF secretion by TGF-beta. In contrast activin, inhibin and BMP, members of TGF-beta super family, had no effects on VEGF expression in HRPE. VEGF mRNA levels and protein secretion induced by TGF-beta were significantly inhibited by SB203580 and U0126, inhibitors of MAP kinases, but not by staurosporine and PDTC, protein kinase C and NF-kappaB pathway inhibitors, respectively. TGF-beta also induced VEGF expression by fibroblasts derived from human choroid of eye. TGF-beta induction of VEGF secretion by RPE and choroid cells may play a significant role in choroidal neovascularization (CNV) in AMD. Since the secretion of VEGF by HRPE is regulated by MAP kinase pathways, MAP kinase inhibitors may have potential use as therapeutic agents for CNV in AMD.     

Upregulated expression of CCR3 in osteoarthritis and CCR3 mediated activation of fibroblast-like synoviocytes

ObjectivesUpregulated expression of CC chemokine receptor (CCR)3 was observed in osteoarthritis (OA) cartilage and chondrocytes, but expression of CCR3 on synovial tissue of OA remains unknown. Fibroblast-like synoviocyte (FLS) invasion in synovium appears one of the features of OA, but expression and function of CCR3 on FLS remain uninvestigated. We therefore explored them in the present study.MethodsEnzymatically dispersed synovial tissue cells were analyzed by flowcytometry. Primary cultured FLS isolated from OA synovium were challenged and the expression of CCR3, eotaxin-1 and matrix metalloproteinase (MMP)-9 was determined by quantitative real-time PCR (qPCR) and ELISA.ResultsApproximately 4.5% dispersed OA synovial tissue cells are CCR3+ cells. Among them, 58.4% cells are CD90+CD14−CD3− cells (representing FLS) and 36.7% are CD8+ cells, indicating that FLS are major population of CCR3+ cells in the synovial tissue. Levels of eotaxin-1 and MMP-9 in OA synovial fluid (SF) were greater than that in OA plasma and in healthy control (HC) plasma. Eotaxin-1 induced up to 5.8 and 7.2-fold increases in the expression of MMP-9 mRNA and protein, respectively following 12 h incubation with FLS, which was inhibited by antagonist of CCR3 SB328437 and an inhibitor of ERK U0126, indicating that action of eotaxin-1 on FLS seemed via CCR3 and ERK signaling pathway. IL-1β and TNF-α was found to elicit release of eotaxin-1 from OA FLS.ConclusionFLS via eotaxin-1 and its receptor CCR3 plays an important role in the pathogenesis of OA, which strengthen the concept that OA is likely an inflammation related disease.     

Mitochondrial haplogroups and control region polymorphisms in age-related macular degeneration: a case-control study

Background

Onset and development of the multifactorial disease age-related macular degeneration (AMD) are highly interrelated with mitochondrial functions such as energy production and free radical turnover. Mitochondrial dysfunction and overproduction of reactive oxygen species may contribute to destruction of the retinal pigment epithelium, retinal atrophy and choroidal neovascularization, leading to AMD. Consequently, polymorphisms of the mitochondrial genome (mtDNA) are postulated to be susceptibility factors for this disease. Previous studies from Australia and the United States detected associations of mitochondrial haplogroups with AMD. The aim of the present study was to test these associations in Middle European Caucasians.

Methodology/Principal Findings

Mitochondrial haplogroups (combinations of mtDNA polymorphisms) and mitochondrial CR polymorphisms were analyzed in 200 patients with wet AMD (choroidal neovascularization, CNV), in 66 patients with dry AMD, and in 385 controls from Austria by means of multiplex primer extension analysis and sequencing, respectively. In patients with CNV, haplogroup H was found to be significantly less frequent compared to controls, and haplogroup J showed a trend toward a higher frequency compared to controls. Five CR polymorphisms were found to differ significantly in the two study populations compared to controls, and all, except one (T152C), are linked to those haplogroups.

Conclusions/Significance

It can be concluded that haplogroup J is a risk factor for AMD, whereas haplogroup H seems to be protective for AMD.     

Polyethylene glycol (PEG)-induced mouse model of choroidal neovascularization

In this study, we describe a new method for inducing choroidal neovascularization (CNV) in C57BL/6 mice, an animal model of wet age-related macular degeneration (AMD). AMD is a disease that causes central blindness in humans. We injected PEG-8 subretinally in different doses (0.125-2 mg) to induce CNV. After PEG-8 injection, we examined CNV at several time points (days 3-42). We also used Western blotting, immunohistochemistry, and ELISA to examine the complement component C3 split products, C9, VEGF, TGF-β2, and basic FGF. As early as day 1 after treatment, we found that a single subretinal injection of 1 mg of PEG-8 increased the C3 split products and the C9, TGF-β2, and basic FGF levels in the retinal pigment epithelium-choroid tissue. By day 3 after PEG-8 injection, the intraocular activation of the complement system caused induction and progression of CNV, including new vessels penetrating the Bruch's membrane. At day 5 after PEG-8 injection, we observed a fully developed CNV and retinal degeneration. Thus, in this study, we present a new, inexpensive, and accelerated mouse model of CNV that may be useful to study AMD.     

Inhibition of human immunodeficiency virus replication by a dual CCR5/CXCR4 antagonist

Here we report that the N-pyridinylmethyl cyclam analog AMD3451 has antiviral activity against a wide variety of R5, R5/X4, and X4 strains of human immunodeficiency virus type 1 (HIV-1) and HIV-2 (50% inhibitory concentration [IC(50)] ranging from 1.2 to 26.5 microM) in various T-cell lines, CCR5- or CXCR4-transfected cells, peripheral blood mononuclear cells (PBMCs), and monocytes/macrophages. AMD3451 also inhibited R5, R5/X4, and X4 HIV-1 primary clinical isolates in PBMCs (IC(50), 1.8 to 7.3 microM). A PCR-based viral entry assay revealed that AMD3451 blocks R5 and X4 HIV-1 infection at the virus entry stage. AMD3451 dose-dependently inhibited the intracellular Ca(2+) signaling induced by the CXCR4 ligand CXCL12 in T-lymphocytic cells and in CXCR4-transfected cells, as well as the Ca(2+) flux induced by the CCR5 ligands CCL5, CCL3, and CCL4 in CCR5-transfected cells. The compound did not interfere with chemokine-induced Ca(2+) signaling through CCR1, CCR2, CCR3, CCR4, CCR6, CCR9, or CXCR3 and did not induce intracellular Ca(2+) signaling by itself at concentrations up to 400 microM. In freshly isolated monocytes, AMD3451 inhibited the Ca(2+) flux induced by CXCL12 and CCL4 but not that induced by CCL2, CCL3, CCL5, and CCL7. The CXCL12- and CCL3-induced chemotaxis was also dose-dependently inhibited by AMD3451. Furthermore, AMD3451 inhibited CXCL12- and CCL3L1-induced endocytosis in CXCR4- and CCR5-transfected cells. AMD3451, in contrast to the specific CXCR4 antagonist AMD3100, did not inhibit but enhanced the binding of several anti-CXCR4 monoclonal antibodies (such as clone 12G5) at the cell surface, pointing to a different interaction with CXCR4. AMD3451 is the first low-molecular-weight anti-HIV agent with selective HIV coreceptor, CCR5 and CXCR4, interaction.