anti-CCR5 Antibody Research Articles

Identification of human monoclonal antibodies specific for CCR5 from an antibody library derived from HIV-infected long-term non-progressors

It has been reported that about one quarter of long-term nonprogressors had anti-CCR5 antibodies and such antibodies were not found in disease progressing HIV-1 positive individuals [1]. To identify such antibodies we panned an HIV immune library constructed from bone marrow of three long-term nonprogressors against a synthetic sulfated N-terminal CCR5 peptide (2–15) (R5Nt). Twenty anti-CCR5 monoclonal antibodies were selected that bind to the R5Nt and cell-associated CCR5. Sequence analysis revealed that eighteen clones have VHs that were derived from the same germline sequence. Preliminary data showed that these antibodies inhibited primary HIV-1 isolates from clade B and E as tested in a pseudovirus assay. Identification of these anti-CCR5 human monoclonal antibodies indicates possible contribution to mechanisms determining the slow progression or lack of disease in long-term nonprogresssors. The selected anti-CCR5 antibodies may have potentials as therapeutics and/or prophylactics in combination with other HIV-1 neutralizing antibodies and antiretroviral drugs.

Modulation of eotaxin-3 (CCL26) in alveolar type II epithelial cells

Airway epithelial inflammation associated with emphysema, chronic bronchitis, chronic obstructive pulmonary disease (COPD) and asthma is regulated in part by alveolar type II cell chemokine signaling. Data suggest that resident lung cells use CCR3, CCR5 and CCR2 chemokine receptor/ligand systems to regulate the profile of leukocytes recruited in disease-associated inflammatory conditions. Thus studies were designed to test whether alveolar type II cells possess a Th1-activated CCR5-ligand system that modulates the Th2-activated CCR3/eotaxin-2 (CCL24), eotaxin-3 (CCL26) chemokine systems. The A549 alveolar type II epithelial-like cell culture model was used to demonstrate that alveolar type II cells constitutively express CCR5 which may be upregulated by MIP-1α (CCL3) whose expression was induced by the Th1 cytokines IL-1β and IFN-γ. Selective down-regulation of CCL26, but not CCL24, was observed in CCL3 and IL-4/CCL3 stimulated cells. Down-regulation was reversed by anti-CCR5 neutralizing antibody treatment. Thus, one mechanism through which Th1-activated CCCR5/ligand pathways modulate Th2-activated CCR3/ligand pathways is the differential down-regulation of CCL26 expression. Results suggest that the CCR3 and CCR5 receptor/ligand signaling pathways may be important targets for development of novel mechanism-based adjunctive therapies designed to abrogate the chronic inflammation associated with airway diseases.

The strength of the chemotactic response to a CCR5 binding chemokine is determined by the level of cell surface CCR5 density

We have shown that the intensity of expression of the C-C chemokine receptor CCR5 at the single CD4(+) cell level strongly determines the efficiency of its function as a coreceptor for human immunodeficiency virus type 1. By analogy, we examined if the number of CCR5 molecules at the cell surface might determine its chemotactic response to CCR5 ligands. To test this hypothesis, we measured by flow cytometry the migration of primary human T cells towards the CCR5-binding chemokine CCL5 in vitro. First, we observed a dose-dependent blockage of this migration exerted by an anti-CCR5 monoclonal antibody. Second, we sorted peripheral blood mononuclear cells into five subpopulations expressing various cell surface CCR5 densities, and observed a correlation between the intensity of migration towards CCL5 and the level of CCR5 expression on these subpopulations. Third, we transduced CCR5(+) peripheral blood mononuclear cells with the CCR5 gene, and observed that the CCR5 over-expression induced an over-migration towards CCL5. Finally, we observed in healthy donors a correlation between the chemotactic response of peripheral blood CD8(+) T cell to CCL5 and their level of surface CCR5 expression. T-cell surface CCR5 density, which is constant over time for a given individual, but varies drastically among individuals, might therefore be an important personal determinant of T-cell migration in many biological situations where CCR5-binding chemokines play a role, such as graft rejection, T helper 1-mediated auto-immune diseases, and infectious diseases involving CCR5. Moreover, our data highlight the therapeutic potential of CCR5 antagonists in these situations.

Expression Levels of CXC Chemokine Receptors 3 Are Associated with Clinical Phenotype of Type 1 Diabetes

Type 1 diabetes is recognized as one of T helper 1 cell (Th1)-mediated diseases. The purpose of this article was to investigate the expression levels of CXC chemokine receptor 3 (CXCR3) and CC chemokine receptor 5 (CCR5) on CD4 T cells as Th1 markers in Japanese patients with type 1 diabetes and control subjects. A total of 72 patients with type 1 diabetes and 24 healthy subjects were enrolled. Their peripheral mononuclear cells were obtained and stained with anti-CXCR3, anti-CCR5, and anti-CD4 monoclonal antibodies. Flow-cytometric analysis was performed and patients were classified according to their onset pattern as fulminant, typical, or slow onset. Statistical analysis was performed using ANOVA. CXCR3 expression on CD4 T cells in patients with a fulminant pattern of onset was significantly lower than that in the other groups, and that in patients with a typical pattern of onset was significantly higher than that in the other groups. CCR5 expression on CD4 T cells was not different among the three clinical phenotypes. CXCR3 expression level is associated with the onset pattern of type 1 diabetes. Further studies are needed to clarify the role of chemokines in type 1 diabetes.

CCR5 Usage by CCL5 Induces a Selective Leukocyte Recruitment in Human Skin Xenografts In Vivo

CCR5 is one of the major inflammatory chemokine receptors with potential therapeutical applications in humans. However, the redundancy of chemokines and their receptors, and the species specificity of chemokine receptor antagonists pose challenges to understanding of the role they play in pharmacological situations. To address this question, we used a humanized severe combined immunodeficient mouse model grafted with human skin and autologous leukocytes, and evaluated the effect of a blocking antibody against human CCR5, on CCL5-induced cutaneous leukocyte recruitment in vivo. At baseline, CCL5 induced a significant recruitment of T cells mainly of the memory phenotype, of monocytes/macrophages, eosinophils, and IFN-gamma(+) but not IL-4(+) and IL-5(+) cells. In vivo, anti-CCR5 antibody was able to almost completely inhibit the recruitment of monocytes/macrophages and T-helper (Th)1-type cells to inhibit partially the attraction of memory T cells, but had no effect on eosinophil infiltration, although all these cell types express other CCL5 binding chemokine receptors than CCR5. These results indicate that the in vivo environment regulates target cell specificity of CCL5 leading to differential cell recruitment, suggesting that antagonizing CCR5 receptor may be of therapeutic value in diseases such as acquired immuno deficiency syndrome, where CCL5/CCR5, monocytes, and Th1-type cells play a predominant role.

Long-lasting CCR5 internalization by antibodies in a subset of long-term nonprogressors: a possible protective effect against disease progression

AbstractExposure to HIV-1 does not necessarily result in infection and progression toward disease, thus suggesting that the control of viral infection may be achieved. Antibodies to CCR5 have been detected in HIV-exposed but uninfected subjects (ESNs); thus, these antibodies could be involved in HIV protection. To assess whether anti-CCR5 antibodies may also contribute to slow HIV disease progression, we searched for anti-CCR5 antibodies in 497 subjects, including 85 long-term nonprogressors (LTNPs), 70 progressors, 135 HIV+ patients treated with highly active antiretroviral therapy (HAART), and 207 seronegative donors. We found anti-CCR5 antibodies in a fraction of the LTNPs(23.5%) but not in the other populations studied (P < .001). These antibodies recognized a conformational epitope within the first extramembrane loop of CCR5, and they induced a stable and long-lasting downregulation of CCR5 on the surface of T lymphocytes, which inhibited HIV entry. In addition, CD4+ lymphocytes from LTNPs having anti-CCR5 antibodies are resistance to R5 strains of HIV-1. Follow-up studies showed that the loss of anti-CCR5 antibodies occurred in some subjects, and this loss was significantly associated with a progression toward disease, whereas subjects who retained anti-CCR5 Abs maintained their LTNP status. Induction of anti-CCR5 Abs could be relevant to vaccine design and therapeutics.

An anti-CCR5 monoclonal antibody and small molecule CCR5 antagonists synergize by inhibiting different stages of human immunodeficiency virus type 1 entry

HIV-1 coreceptors are attractive targets for novel antivirals. Here, inhibition of entry by two classes of CCR5 antagonists was investigated. We confirmed previous findings that HIV-1 isolates vary greatly in their sensitivity to small molecule inhibitors of CCR5-mediated entry, SCH-C and TAK-779. In contrast, an anti-CCR5 monoclonal antibody (PA14) similarly inhibited entry of diverse viral isolates. Sensitivity to small molecules was V3 loop-dependent and inversely proportional to the level of gp120 binding to CCR5. Moreover, combinations of the MAb and small molecules were highly synergistic in blocking HIV-1 entry, suggesting different mechanisms of action. This was confirmed by time course of inhibition experiments wherein the PA14 MAb and small molecules were shown to inhibit temporally distinct stages of CCR5 usage. We propose that small molecules inhibit V3 binding to the second extracellular loop of CCR5, whereas PA14 preferentially inhibits subsequent events such as CCR5 recruitment into the fusion complex or conformational changes in the gp120-CCR5 complex that trigger fusion. Importantly, our findings suggest that combinations of CCR5 inhibitors with different mechanisms of action will be central to controlling HIV-1 infection and slowing the emergence of resistant strains.

Chemokine Receptor CCR5 Expression in Conjunctival Epithelium of Patients With Dry Eye Syndrome

To characterize chemokine receptor CCR5 expression on the conjunctival epithelium in dry eye syndromes. Conjunctival impression cytology samples were obtained from normal subjects (n = 15) and patients with dry eye syndrome (n = 45). Cells were harvested from impression cytology samples, and flow cytometry was performed to quantitatively analyze the cell surface expression of chemokine receptor CCR5. Characterization of CCR5-positive cells was done by 2-color flow cytometry using fluorescein-conjugated anti-CCR5 and phycoerythrin-conjugated anti-CD45 antibodies (where CD45 is a marker for bone marrow-derived cells). To study CCR5 messenger RNA transcripts, real-time polymerase chain reaction was done on RNA isolated from the impression cytology samples of normal subjects (n = 5) and patients with dry eye syndrome (n = 14). We found significant up-regulation in cell surface expression of CCR5 in patients with both aqueous tear-deficient and evaporative forms of dry eye syndrome (P<.001). The real-time polymerase chain reaction results (for messenger RNA) corroborated the flow cytometry data (for protein). The majority of the cells expressing CCR5 were non-bone marrow-derived resident epithelial cells of the conjunctiva. Our findings suggest that CCR5 up-regulation is significantly associated with dry eye syndrome-associated ocular surface disease. Clinical Relevance Chemokine receptor CCR5 or its ligands may serve as useful targets for modulation of tissue immunoinflammatory responses in dry eye syndromes.

Transient Chemokine Receptor Blockade does not Prevent, but may Accelerate Type 1 Diabetes in Prediabetic NOD Mice

The influx of autoreactive lymphocytes into the site of an autoimmune inflammation is mediated by certain chemokines. Autoimmune insulitis in type 1 diabetes is viewed as the result of destructive Th-1-cells and their corresponding antigen-presenting cells infiltrating the pancreatic islets. Blocking the chemokine receptors that mediate a Th-1-reaction has been shown to reduce autoimmunity in other experimental autoimmune disorders. We used the NOD mouse model to investigate the potency of anti-CCR2 and anti-CCR5 antibodies to inhibit the influx of Th-1-cells into the pancreatic islets, thus preventing diabetes onset. Eleven-week-old female NOD mice were treated with 500 microg of a monoclonal anti-CCR5 or anti-CCR2 or an isotype control antibody every third day over two weeks. We did not observe any preventive effect in either treatment group, but accelerated diabetes onset in the anti-CCR5 treated group. The number of autoantigen-specific Th-1-cells detected in the two treated groups was not reduced, but increased in the anti-CCR5 group. Redundancy within the chemokine system may account for this lack of prevention, or the intervention may have come too late in the disease process. Furthermore, blocking Th-1 chemokine receptors in the late autoimmune process may also inhibit regulatory T-cells, thus accelerating rather than preventing the disease.

Interleukin 12 Induces T-Cell Recruitment Into the Atherosclerotic Plaque

CD4 T cells, through the release of cytokines as well as direct effector functions, have been implicated in promoting inflammation of the atherosclerotic plaque. Plaque-infiltrating CD4 T cells include a specialized subset of (CD4+)CD28- T cells that express a unique profile of regulatory receptors and are responsive to novel microenvironmental cues. Here we report that (CD4+)CD28- T cells, either isolated from the plaque tissue or from the blood of patients with acute coronary syndrome (ACS), spontaneously express interleukin (IL)-12 receptors, even in the absence of antigenic stimulation. (CD4+)CD28- IL-12R+ cells responded to IL-12 stimulation with the upregulation of the chemokine receptor CCR5 and the C-type lectin receptor CD161, both implicated in regulating tissue homing of effector T cells. IL-12 treatment of (CD4+)CD28- T cells enhanced their chemotaxis and transendothelial migration toward the chemokine CCL5. In vivo relevance for the role of IL-12 in regulating the recruitment of (CD4+)CD28- T cells into the atheroma was examined in human atheroma-SCID mouse chimeras. Exposure of nonstimulated (CD4+)CD28- T cells to IL-12 was sufficient to amplify T-cell accumulation within the inflamed plaque, and coadministration of anti-CCR5 antibodies blocked T-cell recruitment into the plaque. Thus, (CD4+)CD28- T cells functionally resemble NK cells, which have proinflammatory activity even in the unprimed state and respond to any IL-12-inducing host infection with a shift in tissue trafficking and accrual in inflammatory lesions.

Virus Isolates during Acute and Chronic Human Immunodeficiency Virus Type 1 Infection Show Distinct Patterns of Sensitivity to Entry Inhibitors

We studied the effect of entry inhibitors on 58 virus isolates derived during acute and chronic infection to validate these inhibitors in vitro and to probe whether viruses at early and chronic disease stages exhibit general differences in the interaction with entry receptors. We included members of all types of inhibitors currently identified: (i) agents that block gp120 binding to CD4 (CD4-IgG2 and monoclonal antibody [MAb] IgG1b12), (ii) compounds that block the interaction with CCR5 (the chemokine RANTES/CCL5, the small-molecule inhibitor AD101, and the anti-CCR5 antibody PRO 140), (iii) the fusion inhibitor enfuvirtide (T-20), and (iv) neutralizing antibodies directed against gp120 (MAb 2G12) and gp41 (MAbs 2F5 and 4E10). No differences between viruses from acute and chronic infections in the susceptibility to inhibitors targeting the CD4 binding site, CCR5, or fusion or to MAb 2G12 were apparent, rendering treatment with entry inhibitors feasible across disease stages. The notable exceptions were antibodies 2F5 and 4E10, which were more potent in inhibiting viruses from acute infection (P = 0.0088 and 0.0005, respectively), although epitopes of these MAbs were equally well preserved in both groups. Activities of these MAbs correlated significantly with each other, suggesting that common features of the viral envelope modulate their potencies.

Induction of Murine Mucosal CCR5-Reactive Antibodies as an Anti-Human Immunodeficiency Virus Strategy

The genital mucosa is the main site of initial human immunodeficiency virus type 1 (HIV-1) contact with its host. In spite of repeated sexual exposure, some individuals remain seronegative, and a small fraction of them produce immunoglobulin G (IgG) and IgA autoantibodies directed against CCR5, which is probably the cause of the CCR5-minus phenotype observed in the peripheral blood mononuclear cells of these subjects. These antibodies recognize the 89-to-102 extracellular loop of CCR5 in its native conformation. The aim of this study was to induce infection-preventing mucosal anti-CCR5 autoantibodies in individuals at high risk of HIV infection. Thus, we generated chimeric immunogens containing the relevant CCR5 peptide in the context of the capsid protein of Flock House virus, a presentation system in which it is possible to engineer conformationally constrained peptide in a highly immunogenic form. Administered in mice via the systemic or mucosal route, the immunogens elicited anti-CCR5 IgG and IgA (in sera and vaginal fluids). Analogous to exposed seronegative individuals, mice producing anti-CCR5 autoantibodies express significantly reduced levels of CCR5 on the surfaces of CD4+ cells from peripheral blood and vaginal washes. In vitro studies have shown that murine IgG and IgA (i) specifically bind human and mouse CD4+ lymphocytes and the CCR5-transfected U87 cell line, (ii) down-regulate CCR5 expression of CD4+ cells from both humans and untreated mice, (iii) inhibit Mip-1beta chemotaxis of CD4+ CCR5+ lymphocytes, and (iv) neutralize HIV R5 strains. These data suggest that immune strategies aimed at generating anti-CCR5 antibodies at the level of the genital mucosa might be feasible and represent a strategy to induce mucosal HIV-protective immunity.

Macaque trophoblast migration is regulated by RANTES

In human and non-human primates, migratory trophoblasts penetrate the uterine epithelium, invade the endometrium, enter the uterine vasculature, and migrate within the arteries. The mechanisms that regulate this directional migration are unknown. We have used early gestation macaque trophoblasts to test the hypothesis that trophoblast migration is regulated by the chemokine, Regulated on Activation T-Cell Expressed and Secreted (RANTES). Immunohistochemical analysis of cryosections of endometrial tissue showed expression of RANTES by stromal cells and vascular cells. Isolated endothelial cells expressed RANTES as determined by immunocytochemistry and RT-PCR analyses. Immunohistochemical analysis of endometrial cryosections showed that the RANTES receptor, CCR5, was expressed by trophoblasts on anchoring villi and by cells within the trophoblastic shell. Cytokeratin-positive/CCR5-positive cells, consistent with trophoblasts, were also found scattered within the stroma and were often clustered around blood vessels. Isolated trophoblast cells expressed CCR5 as determined by immunocytochemistry and RT-PCR analyses. Isolated trophoblasts migrated towards RANTES when cultured in migration chambers and migration was reduced in the presence of anti-CCR5 antibody. When trophoblasts were cultured on dishes coated with recombinant RANTES, expression of β1 integrin was increased. The RANTES-induced increase in β1 integrin expression was inhibited by pertussis toxin. These data suggest a role for RANTES and CCR5 in the regulation of trophoblast migration within the endometrium and within the uterine vasculature.

Soluble chemokine CCR5 receptor is present in human plasma

In view of the natural resistance to infection by HIV and occasional delayed clinical manifestation of the disease, as also the fact that the virus is able to enter only cells that express CD4 and a co-receptor, we initiated a search for a soluble co-receptor that might compete with its membrane counterpart. Using a sandwich ELISA system, a soluble human CCR5 receptor (sCCR5) was indeed detected in the circulation. Immunoprecipitation of sCCR5-positive plasma samples from Israelis of Ethiopian and non-Ethiopian origin with mAb 2D7, a conformation-dependent anti-CCR5 antibody, revealed the presence of a ∼22 kDa protein. A panel of antibodies directed against the membrane receptor was used to characterize the structure of the soluble CCR5: mAb CTC8, recognizing the N-terminal sequence of the protein, 10YDIN 13; “multidomain” mAbs FAB181B and FAB183B that are dependent upon the presence of Q 93 and D 95 in ECL1 and K 171 and E 172 in ECL2A, and mAb FAB182B, recognizing the stretch 184YSQYQF 189, which spans the C-terminal part of the second extracellular loop. The presence of short soluble CCR5 in human plasma has not been previously described. Among HIV-negative non-Ethiopian Israelis, 20.4% were sCCR5-positive, as against only 10.5% in HIV-positives. However, 7.1% of HIV-negative Ethiopian Israelis were sCCR5 positive, as were 5.6% HIV-positives. Plasma concentrations of MIP-1β, the CCR5 agonist, were twice as high in sCCR5-positives (140.8 ± 25.8 pg/ml) as in the sCCR5-negatives (77.6 ± 11.0 pg/ml, P = 0.0157). A significant positive correlation between plasma levels of sCCR5 and MIP-1β was found (Fig. 4, r = 0.8, P < 0.0001).

Chemokine CCL5 signals required for survival during viral infection

Rationale Inducible expression of the CCL5 (RANTES) gene is an extremely sensitive indicator of viral infection, but the functional role of CCL5 in anti-viral defense still needs to be defined. We therefore studied newly developed mice with null mutations of the CCL5 gene in a model of viral bronchiolitis/pneumonia. Methods CCL5-null and WT cells (especially T cells and macrophages) isolated from these mice were monitored after inoculation with mouse parainfluenza type I (Sendai) virus. Studies were also extended to chemokine receptor CCR5-null mice and human cells treated with anti-CCR5 antibody and infected with respiratory syncytial virus (RSV). Results CCL5- and CCR5-null mice were immune compromised to the point of delayed viral clearance, excessive airway inflammation, and respiratory death compared to control mice. Inflammation was characterized by accumulation of virus-infected, apoptotic macrophages in the airway mucosa. Isolated CCL5- and CCR5-null macrophages (as well as human macrophages treated with anti-CCR5 antibody) were also more susceptible to virus-induced apoptosis. This susceptibility was rescued by CCL5 add-back in CCL5-null mouse macrophages and was reproduced by inhibition of G-protein/PI3K/Akt or MEK/ERK1/2 signaling in mouse and human macrophages. These same Akt and ERK pathways were resistant to endogenous activation in CCL5-null (but not wild-type) mice after viral infection in vivo. Conclusions Inducible expression of CCL5 is critical for host survival during viral infection, most likely because CCL5 protects macrophages from virus-induced apoptosis and thereby allows for effective clearance of cellular corpses that would otherwise cause excessive airway inflammation and obstruction.

Leukocyte attraction through the CCR5 receptor controls progress from insulitis to diabetes in non-obese diabetic mice.

Lymphocyte infiltration to pancreatic islets is associated to chemoattraction, as are other inflammatory autoimmune processes. We examined whether development of insulitis and diabetes depends on chemoattraction of lymphocytes via the CCR5 chemokine receptor. In non-obese diabetic (NOD) mice, a substantial fraction of peripheral T cells and virtually all B cells expressed high CCR5 levels. CCR5 expression characterized the effector T cell phenotype, suggesting their potential involvement in disease development. In view of these findings and the CCL5 (RANTES, the CCR5 ligand) expression by pancreatic islets, we treated NOD mice with a neutralizing anti-CCR5 antibody. This did not influence peri-insulitis advancement, but inhibited beta-cell destruction and diabetes. These data demonstrate a role of CCR5-dependent chemoattraction in insulitis progression to islet destruction, suggesting the potential value of therapeutic intervention by CCR5 targeting.

Analysis of the mechanism by which the small-molecule CCR5 antagonists SCH-351125 and SCH-350581 inhibit human immunodeficiency virus type 1 entry.

Human immunodeficiency virus type 1 (HIV-1) entry is mediated by the consecutive interaction of the envelope glycoprotein gp120 with CD4 and a coreceptor such as CCR5 or CXCR4. The CCR5 coreceptor is used by the most commonly transmitted HIV-1 strains that often persist throughout the course of infection. Compounds targeting CCR5-mediated entry are a novel class of drugs being developed to treat HIV-1 infection. In this study, we have identified the mechanism of action of two inhibitors of CCR5 function, SCH-350581 (AD101) and SCH-351125 (SCH-C). AD101 is more potent than SCH-C at inhibiting HIV-1 replication in primary lymphocytes, as well as viral entry and gp120 binding to cell lines. Both molecules also block the binding of several anti-CCR5 monoclonal antibodies that recognize epitopes in the second extracellular loop of CCR5. Alanine mutagenesis of the transmembrane domain of CCR5 suggests that AD101 and SCH-C bind to overlapping but nonidentical sites within a putative ligand-binding cavity formed by transmembrane helices 1, 2, 3, and 7. We propose that the binding of small molecules to the transmembrane domain of CCR5 may disrupt the conformation of its extracellular domain, thereby inhibiting ligand binding to CCR5.

Cholesterol is essential for macrophage inflammatory protein 1β binding and conformational integrity of CC chemokine receptor 5

The chemokine receptor, CCR5, is used as a human immunodeficiency virus coreceptor in combination with CD4 during transmission and early infection. CCR5 has been shown to be palmitoylated and targeted to cholesterol- and sphingolipid-rich membrane microdomains termed "lipid rafts." However, the role of cholesterol and lipid rafts on chemokine binding and signaling through CCR5 remains unknown. We found that cholesterol extraction by hydroxypropyl-beta-cyclodextrin (BCD) significantly reduced the binding and signaling of macrophage inflammatory protein 1 beta (MIP-1 beta) using CCR5-expressing CEM-NKR T cells. Reloading treated cells with cholesterol but not 4-cholesten-3-one, an oxidized form of cholesterol, restored MIP-1 beta binding to BCD-treated cells. Antibodies specific for distinct CCR5 epitopes lost their ability to bind to the cell surface after cholesterol extraction to varying degrees. Moreover, cells stained with fluorescently labeled MIP-1 beta extensively colocalized with the GM1 lipid raft marker while using anti-CCR5 antibodies; most of CCR5 on these cells only partially colocalized with GM1, suggesting that active ligand binding facilitates receptor association with lipid rafts or that raft association promotes a higher affinity conformation of CCR5. Together, these data demonstrate that cholesterol and lipid rafts are important for the maintenance of the CCR5 conformation and are necessary for both the binding and function of this chemokine receptor.

The chemoattraction of lymphocytes by rheumatoid arthritis - synovial fluid is not dependent on the chemokine receptor CCR5.

The objective was to study the potential role of the chemokine receptor CCR5 in the chemoattraction of lymphocytes by rheumatoid arthritis synovial fluid (RA-SF). The expression of the CCR5 receptor was studied by flow cytometry. Chemotaxis of peripheral blood lymphocytes in response to RA-SF was analyzed on transmigration chambers. Chemotaxis of immortalized lymphocytes from individuals homozygous for the Delta32 deletion of the CCR5 gene (CCR5-/-) was analyzed. The effect of a neutralizing anti-CCR5 antibody on the migration of CCR5+/+ cells was also studied. We confirmed an increase in the proportion of CCR5-expressing lymphocytes in RA-SF and a preferential migration of CCR5+ lymphocytes toward RA-SF in vitro. CCR5-/- lymphocytes showed decreased chemotactic responses to the chemokine MIP-1beta but not to RA-SF. The chemotactic responses of CCR5+/+ lymphocytes to RA-SF were not modified by anti-CCR5 neutralizing antibody. We confirm a preferential accumulation of CCR5-expressing lymphocytes into RA-SF. However, the chemotactic responses of lymphocytes to RA-SF were not dependent on a functional CCR5 receptor, suggesting that CCR5 is a marker of a lymphocyte subset rather than a specific mediator of chemotactic responses to chemokines in RA-SF.

Multiple active states and oligomerization of CCR5 revealed by functional properties of monoclonal antibodies.

CC-chemokine receptor 5 (CCR5) is the principal coreceptor for macrophage-tropic strains of human immunodeficiency virus type 1 (HIV-1). We have generated a set of anti-CCR5 monoclonal antibodies and characterized them in terms of epitope recognition, competition with chemokine binding, receptor activation and trafficking, and coreceptor activity. MC-4, MC-5, and MC-7 mapped to the amino-terminal domain, MC-1 to the second extracellular loop, and MC-6 to a conformational epitope covering multiple extracellular domains. MC-1 and MC-6 inhibited regulated on activation normal T cell expressed and secreted (RANTES), macrophage inflammatory polypeptide-1beta, and Env binding, whereas MC-5 inhibited macrophage inflammatory polypeptide-1beta and Env but not RANTES binding. MC-6 induced signaling in different functional assays, suggesting that this monoclonal antibody stabilizes an active conformation of CCR5. Flow cytometry and real-time confocal microscopy showed that MC-1 promoted strong CCR5 endocytosis. MC-1 but not its monovalent isoforms induced an increase in the transfer of energy between CCR5 molecules. Also, its monovalent isoforms bound efficiently, but did not internalize the receptor. In contrast, MC-4 did not prevent RANTES binding or subsequent signaling, but inhibited its ability to promote CCR5 internalization. These results suggest the existence of multiple active conformations of CCR5 and indicate that CCR5 oligomers are involved in an internalization process that is distinct from that induced by the receptor's agonists.