The core domain of chemokines binds CCR5 extracellular domains while their amino terminus interacts with the transmembrane helix bundle

CCR5 is a functional receptor for various inflammatory CC-chemokines, including macrophage inflammatory protein (MIP)-1alpha and RANTES (regulated on activation normal T cell expressed and secreted), and is the main coreceptor of human immunodeficiency viruses. The second extracellular loop and amino-terminal domain of CCR5 are critical for chemokine binding, whereas the transmembrane helix bundle is involved in receptor activation. Chemokine domains and residues important for CCR5 binding and/or activation have also been identified. However, the precise way by which chemokines interact with and activate CCR5 is presently unknown. In this study, we have compared the binding and functional properties of chemokine variants onto wild-type CCR5 and CCR5 point mutants. Several mutations in CCR5 extracellular domains (E172A, R168A, K191A, and D276A) strongly affected MIP-1alpha binding but had little effect on RANTES binding. However, a MIP/RANTES chimera, containing the MIP-1alpha N terminus and the RANTES core, bound to these mutants with an affinity similar to that of RANTES. Several CCR5 mutants affecting transmembrane helices 2 and 3 (L104F, L104F/F109H/F112Y, F85L/L104F) reduced the potency of MIP-1alpha by 10-100 fold with little effect on activation by RANTES. However, the MIP/RANTES chimera activated these mutants with a potency similar to that of MIP-1alpha. In contrast, LD78beta, a natural MIP-1alpha variant, which, like RANTES, contains a proline at position 2, activated these mutants as well as RANTES. Altogether, these results suggest that the core domains of MIP-1alpha and RANTES bind distinct residues in CCR5 extracellular domains, whereas the N terminus of chemokines mediates receptor activation by interacting with the transmembrane helix bundle.     

A non-peptide functional antagonist of the CCR1 chemokine receptor is effective in rat heart transplant rejection

Chemokines like RANTES appear to play a role in organ transplant rejection. Because RANTES is a potent agonist for the chemokine receptor CCR1, we examined whether the CCR1 receptor antagonist BX471 is efficacious in a rat heterotopic heart transplant rejection model. Treatment of animals with BX471 and a subtherapeutic dose of cyclosporin (2.5 mg/kg), which is by itself ineffective in prolonging transplant rejection, is much more efficacious in prolonging transplantation rejection than animals treated with either cyclosporin or BX471 alone. We have examined the mechanism of action of the CCR1 antagonist in in vitro flow assays over microvascular endothelium and have discovered that the antagonist blocks the firm adhesion of monocytes triggered by RANTES on inflamed endothelium. Together, these data demonstrate a significant role for CCR1 in allograft rejection.     

CXC and CC chemokine receptors on coronary and brain endothelia

BACKGROUND: Chemokine receptors on leukocytes play a key role in inflammation and HIV-1 infection. Chemokine receptors on endothelia may serve an important role in HIV-1 tissue invasion and angiogenesis. MATERIALS AND METHODS: The expression of chemokine receptors in human brain microvascular endothelial cells (BMVEC) and coronary artery endothelial cells (CAEC) in vitro and cryostat sections of the heart tissue was determined by light and confocal microscopy and flow cytometry with monoclonal antibodies. Chemotaxis of endothelia by CC chemokines was evaluated in a transmigration assay. RESULTS: In BMVEC, the chemokine receptors CCR3 and CXCR4 showed the strongest expression. CXCR4 was localized by confocal microscopy to both the cytoplasm and the plasma membrane of BMVEC. In CAEC, CXCR4 demonstrated a strong expression with predominantly periplasmic localization. CCR5 expression was detected both in BMVEC and CAEC but at a lower level. Human umbilical cord endothelial cells (HUVEC) expressed strongly CXCR4 but only weakly CCR3 and CCR5. Two additional CC chemokines, CCR2A and CCR4, were detected in BMVEC and CAEC by immunostaining. Immunocytochemistry of the heart tissues with monoclonal antibodies revealed a high expression of CXCR4 and CCR2A and a low expression of CCR3 and CCR5 on coronary vessel endothelia. Coronary endothelia showed in vitro a strong chemotactic response to the CC chemokines RANTES, MIP-1alpha, and MIP-1beta. CONCLUSIONS: The endothelia isolated from the brain display strongly both the CCR3 and CXCR4 HIV-1 coreceptors, whereas the coronary endothelia express strongly only the CXCR4 coreceptor. CCR5 is expressed at a lower level in both endothelia. The differential display of CCR3 on the brain and coronary endothelia could be significant with respect to the differential susceptibility of the heart and the brain to HIV-1 invasion. In addition, CCR2A is strongly expressed in the heart endothelium. All of the above chemokine receptors could play a role in endothelial migration and repair.     

Neutralizing Nanobodies Targeting Diverse Chemokines Effectively Inhibit Chemokine Function

Chemokine receptors and their ligands play a prominent role in immune regulation but many have also been implicated in inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, allograft rejection after transplantation, and also in cancer metastasis. Most approaches to therapeutically target the chemokine system involve targeting of chemokine receptors with low molecular weight antagonists. Here we describe the selection and characterization of an unprecedented large and diverse panel of neutralizing Nanobodies (single domain camelid antibodies fragment) directed against several chemokines. We show that the Nanobodies directed against CCL2 (MCP-1), CCL5 (RANTES), CXCL11 (I-TAC), and CXCL12 (SDF-1α) bind the chemokines with high affinity (at nanomolar concentration), thereby blocking receptor binding, inhibiting chemokine-induced receptor activation as well as chemotaxis. Together, we show that neutralizing Nanobodies can be selected efficiently for effective and specific therapeutic treatment against a wide range of immune and inflammatory diseases.     

The role of the CC chemokine, RANTES, in acute lung allograft rejection

Lung transplantation is a therapeutic option for patients with end-stage lung disease. Acute allograft rejection is a major complication of lung transplantation and is characterized by the infiltration of activated mononuclear cells. The specific mechanisms that recruit these leukocytes have not been fully elucidated. The CC chemokine, RANTES, is a potent mononuclear cell chemoattractant. In this study we investigated RANTES involvement during acute lung allograft rejection in humans and in a rat model system. Patients with allograft rejection had a 2.3-fold increase in RANTES in their bronchoalveolar lavages compared with healthy allograft recipients. Rat lung allografts demonstrated a marked time-dependent increase in levels of RANTES compared with syngeneic control lungs. RANTES levels correlated with the temporal recruitment of mononuclear cells and the expression of RANTES receptors CCR1 and CCR5. To determine RANTES involvement in lung allograft rejection, lung allograft recipients were passively immunized with either anti-RANTES or control Abs. In vivo neutralization of RANTES attenuated acute lung allograft rejection and reduced allospecific responsiveness by markedly decreasing mononuclear cell recruitment. These experiments support the idea that RANTES, and the expression of its receptors have an important role in the pathogenesis of acute lung allograft rejection.     

Differential role of CCR2 in islet and heart allograft rejection: tissue specificity of chemokine/chemokine receptor function in vivo

Chemokines have a pivotal role in the mobilization and activation of specific leukocyte subsets in acute allograft rejection. However, the role of specific chemokines and chemokine receptors in islet allograft rejection has not been fully elucidated. We now show that islet allograft rejection is associated with a steady increase in intragraft expression of the chemokines CCL8 (monocyte chemoattractant protein-2), CCL9 (monocyte chemoattractant protein-5), CCL5 (RANTES), CXCL-10 (IFN-gamma-inducible protein-10), and CXCL9 (monokine induced by IFN-gamma) and their corresponding chemokine receptors CCR2, CCR5, CCR1, and CXCR3. Because CCR2 was found to be highly induced, we tested the specific role of CCR2 in islet allograft rejection by transplanting fully MHC mismatched islets from BALB/c mice into C57BL/6 wild-type (WT) and CCR2-deficient mice (CCR2-/-). A significant prolongation of islet allograft survival was noted in CCR2-/- recipients, with median survival time of 24 and 12 days for CCR2-/- and WT recipients, respectively (p < 0.0001). This was associated with reduction in the generation of CD8+, but not CD4+ effector alloreactive T cells (CD62L(low)CD44(high)) in CCR2-/- compared with WT recipients. In addition, CCR2-/- recipients had a reduced Th1 and increased Th2 alloresponse in the periphery (by ELISPOT analysis) as well as in the grafts (by RT-PCR). However, these changes were only transient in CCR2-/- recipients that ultimately rejected their grafts. Furthermore, in contrast to the islet transplants, CCR2 deficiency offered only marginal prolongation of heart allograft survival. This study demonstrates the important role for CCR2 in early islet allograft rejection and highlights the tissue specificity of the chemokine/chemokine receptor system in vivo in regulating allograft rejection.     

I-TAC is a dominant chemokine in controlling skin intragraft inflammation via recruiting CXCR3 cells into the graft

Chemokines play a critical role in the acute transplant rejection. In order to provide an overview of the chemokine expression during the course of acute allograft rejection, the intragraft expression profile of 11 chemokines representative of all four chemokine subfamilies was analyzed in a murine skin transplantation model of acute rejection. It was found that RANTES/CCL5, TARC/CCL17 and FKN/CX₃CL1 were expressed at equivalent levels in iso- and allografts. However, the other eight chemokines expression was up-regulated to some extent in allograft compared with that in isograft. The levels of MIP-1α/CCL3, MIP-3α/CCL20 and CTACK/CCL27 were progressively increased from early stage (day 3 post-transplantation) to late stage (day 11). Mig/CXCL9, IP-10/CXCL10, I-TAC/CXCL11, CXCL16 and LTN/XCL1 expression was elevated at middle stage (day 7), and peaked at late stage. Among the up-regulated chemokines, I-TAC was the most obviously elevated chemokine. Therefore, the effect of I-TAC on the skin acute allograft rejection was evaluated. Block of I-TAC by the intradermal injection of anti-I-TAC monoclonal antibody (mAb) reduced the number of CXCR3⁺ cells in skin allograft and significantly prolonged the skin allograft survival. The mAb treatment did not influence the proliferation of the intragraft infiltrating cells in response to the allogeneic antigens, but significantly decreased the number of the infiltrating cells and consequently lowered the secretion of IFN-γ and TNF-α. These data indicate I-TAC might be a dominant chemokine involved in the intradermal infiltration and I-TAC-targeted intervening strategies would have potential application for the alleviation of acute transplant rejection.     

Reduced chemokine and chemokine receptor expression in spinal cords of TCR BV8S2 transgenic mice protected against experimental autoimmune encephalomyelitis with BV8S2 protein

The perivascular transmigration and accumulation of macrophages and T lymphocytes in the CNS of mice with experimental autoimmune encephalomyelitis (EAE) may be partly regulated by low m.w. chemotactic cytokines. Using the RNase protection assay and ELISA, we quantified expression of chemokines and chemokine receptors in the spinal cord (SC), brain, and lymph nodes of BV8S2 transgenic mice that developed or were protected from EAE by vaccination with BV8S2 protein. In paralyzed control mice, the SC had increased cellular infiltration and strong expression of the chemokines RANTES, IFN-inducible 10-kDa protein, and monocyte chemoattractant protein-1 and the cognate chemokine receptors CCR1, CCR2, and CCR5, with lower expression of macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta, and MIP-2; whereas brain had less infiltration and a lower expression of a different pattern of chemokines and receptors. In TCR-protected mice, there was a decrease in the number of inflammatory cells in both SC and brain. In SC, the reduced cellular infiltrate afforded by TCR vaccination was commensurate with profoundly reduced expression of chemokines and their cognate chemokine receptors. In brain, however, TCR vaccination did not produce significant changes in chemokine expression but resulted in an increased expression of CCR3 and CCR4 usually associated with Th2 cells. In contrast to CNS, lymph nodes of protected mice had a significant increase in expression of MIP-2 and MIP-1beta but no change in expression of chemokine receptors. These results demonstrate that TCR vaccination results in selective reduction of inflammatory chemokines and chemokine receptors in SC, the target organ most affected during EAE.     

Examination of the function of RANTES, MIP-1alpha, and MIP-1beta following interaction with heparin-like glycosaminoglycans

Chemokines are a group of small proteins that have a variety of functions, including the activation and recruitment of immune cells during episodes of inflammation. In common with many cytokines, it has been observed that chemokines have the potential to bind heparin-like glycosaminoglycan molecules, which are normally expressed on proteoglycan components of the cell surface and extracellular matrix. The significance of this interaction for chemokine activity remains a subject of debate. In this study, Chinese hamster ovary cells were transfected separately with the human chemokine receptors CCR1 and CCR5, and these receptors were shown to induce an intracytoplasmic Ca(2+) flux and cellular chemotaxis following stimulation with the natural CC chemokine ligands (MIP-1alpha, RANTES (regulated on activation normal T cell expressed), and MIP-1beta). In further experiments, mutant CHO cells, with a defect in normal glycosaminoglycan (GAG) expression, were also transfected with, and shown to express similar levels of, CCR1 and CCR5. Although these receptors were functional, it was found that the mutant cells required exposure to higher concentrations of ligands than the wild-type cells in order to produce the same intracytoplasmic Ca(2+) flux. Radioligand binding experiments demonstrated that specific chemokine receptors expressed by wild-type cells had a significantly greater affinity for MIP-1alpha than similar receptors expressed by GAG-deficient mutants. However, there was no significant difference between these cells in their affinity for RANTES or MIP-1beta. In conclusion, it has been demonstrated clearly that GAG expression is not necessary for the biological activity of the chemokines MIP-1alpha, RANTES, or MIP-1beta. However, the presence of cell surface GAGs does enhance the activity of low concentrations of these chemokines by a mechanism that appears to involve sequestration onto the cell surface.     

LD78beta, a non-allelic variant of human MIP-1alpha (LD78alpha), has enhanced receptor interactions and potent HIV suppressive activity.

Chemokines play diverse roles in inflammatory and non-inflammatory situations via activation of heptahelical G-protein-coupled receptors. Also, many chemokine receptors can act as cofactors for cellular entry of human immunodeficiency virus (HIV) in vitro. CCR5, a receptor for chemokines MIP-1alpha (LD78alpha), MIP-1beta, RANTES, and MCP2, is of particular importance in vivo as polymorphisms in this gene affect HIV infection and rate of progression to AIDS. Moreover, the CCR5 ligands can prevent HIV entry through this receptor and likely contribute to the control of HIV infection. Here we show that a non-allelic isoform of human MIP-1alpha (LD78alpha), termed LD78beta or MIP-1alphaP, has enhanced receptor binding affinities to CCR5 (approximately 6-fold) and the promiscuous beta-chemokine receptor, D6 (approximately 15-20-fold). We demonstrate that a proline residue at position 2 of MIP-1alphaP is responsible for this enhanced activity. Moreover, MIP-1alphaP is by far the most potent natural CCR5 agonist described to date, and importantly, displays markedly higher HIV1 suppressive activity than all other human MIP-1alpha isoforms examined. In addition, while RANTES has been described as the most potent inhibitor of CCR5-mediated HIV entry, MIP-1alphaP was as potent as, if not more potent than, RANTES in HIV-1 suppressive assays. This property suggests that MIP-1alphaP may be of importance in controlling viral spread in HIV-infected individuals.     

Chemokines as natural HIV antagonists

The unexpected encounter between the fields of HIV and chemokines has opened new perspectives for understanding the mechanisms of AIDS pathogenesis, as well as for the development of effective therapies and vaccines. Selected chemokines act as potent natural inhibitors of HIV infection, as they bind and downmodulate chemokine receptors that serve as critical coreceptors for HIV to gain access into cells. The differential usage of the two major HIV coreceptors, CCR5 and CXCR4, determines the biological diversity among HIV variants. Most primary HIV strains use CCR5 as a coreceptor and thereby are sensitive to inhibition by the CCR5-ligand chemokines, RANTES, MIP-1alpha and MIP-1beta. The high level of expression of these proinflammatory chemokines in HIV-infected secondary lymphoid tissues may help to explain the inherently slow course of HIV disease. The crucial role played by CCR5 in the physiology of HIV infection is further attested by the near-complete resistance to HIV infection in people carrying a homozygous 32 bp deletion within the CCR5 gene (CCR5-delta32). A smaller proportion of HIV isolates, commonly emerging in concomitance with the clinical progression toward AIDS, uses CXCR4 as a coreceptor and is inhibited by the CXCR4 ligand, SDF-1. The high level of expresion of SDF-1 in the genital mucosa may help to explain the inefficient transmission of CXCR4-tropic HIV. Although chemokines or derivative-molecules could be exploited as therapeutic agents against HIV, the risk of inducing inflammatory side-effects or of interfering with the physiology of the homeostatic chemokine system represents a potential limitation. However, the ability of chemokines to block HIV infection can be uncoupled from their receptor-mediated signaling activity, thus providing a theoretical foundation for the rational design of safe and effective chemokine receptor inhibitors.     

Permanent survival of fully MHC-mismatched islet allografts by targeting a single chemokine receptor pathway

Chemokine receptor blockade can diminish the recruitment of host effector cells and prolong allograft survival, but little is known of the role of chemokine receptors in promoting host sensitization. We engrafted fully allogeneic islets into streptozotocin-treated normal mice or mice with the autosomal recessive paucity of lymph node T cell (plt) mutation; the latter lack secondary lymphoid expression of the CCR7 ligands, secondary lymphoid organ chemokine (CCL21) and EBV-induced molecule-1 ligand chemokine (CCL19). plt mice showed permanent survival of islets engrafted under the kidney capsule, whereas controls rejected islet allografts in 12 days (p < 0.001), and consistent with this, plt mice had normal allogeneic T cell responses, but deficient migration of donor dendritic cell to draining lymph nodes. Peritransplant i.v. injection of donor splenocytes caused plt recipients to reject their allografts by 12 days, and sensitization at 60 days posttransplant of plt mice with well-functioning allografts restored acute rejection. Finally, islet allografts transplanted intrahepatically in plt mice were rejected approximately 12 days posttransplant, like controls, as were primarily revascularized cardiac allografts. These data show that the chemokine-directed homing of donor dendritic cell to secondary lymphoid tissues is essential for host sensitization and allograft rejection. Interruption of such homing can prevent T cell priming and islet allograft rejection despite normal T and B cell functions of the recipient, with potential clinical implications.     

Expression and Use of Human Immunodeficiency Virus Type 1 Coreceptors by Human Alveolar Macrophages

Human immunodeficiency virus type 1 (HIV-1) requires, in addition to CD4, coreceptors of the CC or CXC chemokine families for productive infection of T cells and cells of the monocyte-macrophage lineage. Based on the hypothesis that coreceptor expression on alveolar macrophages (AM) may influence HIV-1 infection of AM in the lung, this study analyzes the expression and utilization of HIV-1 coreceptors on AM of healthy individuals. AM were productively infected with five different primary isolates of HIV-1. Levels of surface expression of CCR5, CXCR4, and CD4 were low compared to those of blood monocytes, but CCR3 was not detectable. mRNA for CCR5, CXCR4, CCR2, and CCR3 were all detectable, but to varying degrees and with variability among donors. Expression of CCR5, CXCR4, and CCR2 mRNA was downregulated following stimulation with lipopolysaccharide (LPS). In contrast, secretion of the chemokines RANTES, MIP-1alpha, and MIP-1beta was upregulated with LPS stimulation. Interestingly, HIV-1 replication was diminished following LPS stimulation. Infection of AM with HIV-1 in the presence of the CC chemokines demonstrated blocking of infection. Together, these studies demonstrate that AM can be infected by a variety of primary HIV-1 isolates, AM express a variety of chemokine receptors, the dominant coreceptor used for HIV entry into AM is CCR5, the expression of these receptors is dependent on the state of activation of AM, and the ability of HIV-1 to infect AM may be modulated by expression of the chemokine receptors and by chemokines per se.     

The effect of MCP-1 depletion on chemokine and chemokine-related gene expression: evidence for a complex network in acute inflammation

The expression of chemokines has been suggested to involve an interdependent network, with the absence of a single chemokine affecting the expression of multiple other chemokines. Monocyte chemoattractant protein (MCP-1), a member of C-C chemokine superfamily, plays a critical role in the recruitment and activation of leukocytes during acute inflammation. To examine the effect of the loss of MCP-1 on expression of the chemokine network, we compared the mRNA expression profiles of MCP-1(-/-) and wild type mice during the acute inflammatory phase of excisional wounds. Utilizing a mouse cDNA array containing 514 chemokine and chemokine related genes, the loss of MCP-1 was observed to cause a significant upregulation of nine genes (Decorin, Persephin, IL-1beta, MIP-2, MSP, IL1ra, CCR5, CCR3, IL-11) and significant downregulation of two genes (CCR4 and CD3Z) in acute wounds. The array data was confirmed by semi-quantitative RT-PCR. The effect of MCP-1 deletion on chemokine expression was further examined in isolated macrophages. Compared to wild type, LPS-stimulated peritoneal macrophages from MCP-1(-/-) mice showed a significant increase in the expression of RANTES, MIP-1beta, MIP-1alpha and MIP-2 mRNA. The data suggest that loss of a single chemokine perturbs the chemokine network not only in the setting of acute inflammation but even in an isolated inflammatory cell, the macrophage.     

Genetic Subtype-Independent Inhibition of Human Immunodeficiency Virus Type 1 Replication by CC and CXC Chemokines

We have studied the breadth and potency of the inhibitory actions of the CC chemokines macrophage inhibitory protein 1α (MIP-1α), MIP-1β, and RANTES against macrophage-tropic (M-tropic) primary isolates of human immunodeficiency virus type 1 (HIV-1) and of the CXC chemokine stromal cell-derived factor 1α against T-cell-tropic (T-tropic) isolates, using mitogen-stimulated primary CD4⁺ T cells as targets. There was considerable interisolate variation in the sensitivity of HIV-1 to chemokine inhibition, which was especially pronounced for the CC chemokines and M-tropic strains. However, this variation was not obviously dependent on the genetic subtype (A through F) of the virus isolates. Peripheral blood mononuclear cell donor-dependent variation in chemokine inhibition potency was also observed. Among the CC chemokines, the rank order for potency (from most to least potent) was RANTES, MIP-1β, MIP-1α. Some M-tropic isolates, unexpectedly, were much more sensitive to RANTES than to MIP-1β, whereas other isolates showed sensitivities comparable to those of these two chemokines. Down-regulation of the CCR5 and CXCR4 receptors occurred in cells treated with the cognate chemokines and probably contributes to anti-HIV-1 activity. Thus, for CCR5, the rank order for down-regulation was also RANTES, MIP-1β, MIP-1α.     

Induction of RANTES and CCR5 through NF-κB Activation via MAPK Pathway in Aged Rat Gingival Tissues

Chemokine and chemokine receptor expression in gingival tissues plays a central role in periodontal disease during aging. In the present study, we explored the modulation of chemokines and chemokine receptors expression in aging rat gingival tissues. In the 24-month-old (Old) rat gingival tissues, RANTES and CCR5 mRNA and protein levels were 2–4 fold increased over those of the 6-month-old (Young) rats. The Old rats had considerable enhancement of all three of the studied MAPK activities: extracellular signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. These results suggest that age-related increases in RANTES and CCR5 expression are associated with increased IκBα, nuclear NF-κB, and MAPK activity in gingival tissues.     

Endocytosis and recycling of the HIV coreceptor CCR5

The chemokine receptor CCR5 is a cofactor for the entry of R5 tropic strains of human immunodeficiency viruses (HIV)-1 and -2 and simian immunodeficiency virus. Cells susceptible to infection by these viruses can be protected by treatment with the CCR5 ligands regulated on activation, normal T cell expressed and secreted (RANTES), MIP-1alpha, and MIP-1beta. A major component of the mechanism through which chemokines protect cells from HIV infection is by inducing endocytosis of the chemokine receptor. Aminooxypentane (AOP)-RANTES, an NH(2)-terminal modified form of RANTES, is a potent inhibitor of infection by R5 HIV strains. AOP-RANTES efficiently downmodulates the cell surface expression of CCR5 and, in contrast with RANTES, appears to prevent recycling of CCR5 to the cell surface. Here, we investigate the cellular basis of this effect.Using CHO cells expressing human CCR5, we show that both RANTES and AOP-RANTES induce rapid internalization of CCR5. In the absence of ligand, CCR5 shows constitutive turnover with a half-time of 6-9 h. Addition of RANTES or AOP-RANTES has little effect on the rate of CCR5 turnover. Immunofluorescence and immunoelectron microscopy show that most of the CCR5 internalized after RANTES or AOP-RANTES treatment accumulates in small membrane-bound vesicles and tubules clustered in the perinuclear region of the cell. Colocalization with transferrin receptors in the same clusters of vesicles indicates that CCR5 accumulates in recycling endosomes. After the removal of RANTES, internalized CCR5 recycles to the cell surface and is sensitive to further rounds of RANTES-induced endocytosis. In contrast, after the removal of AOP-RANTES, most CCR5 remains intracellular. We show that these CCR5 molecules do recycle to the cell surface, with kinetics equivalent to those of receptors in RANTES-treated cells. However, these recycled CCR5 molecules are rapidly reinternalized. Our results indicate that AOP-RANTES-induced changes in CCR5 alter the steady-state distribution of the receptor and provide the first evidence for G protein-coupled receptor trafficking through the recycling endosome compartment.     

Involvement of both the V2 and V3 regions of the CCR5-tropic human immunodeficiency virus type 1 envelope in reduced sensitivity to macrophage inflammatory protein 1alpha

To determine whether C-C chemokines play an important role in the phenotype switch of human immunodeficiency virus (HIV) from CCR5 to CXCR4 usage during the course of an infection in vivo, macrophage inflammatory protein (MIP)-1alpha-resistant variants were isolated from CCR5-tropic (R5) HIV-1 in vitro. The selected variants displayed reduced sensitivities to MIP-1alpha (fourfold) through CCR5-expressing CD4-HeLa/long terminal repeat-beta-galactosidase (MAGI/CCR5) cells. The variants were also resistant to other natural ligands for CCR5, namely, MIP-1beta (>4-fold) and RANTES (regulated upon activation, normal T-cell expressed and secreted) (6-fold). The env sequence analyses revealed that the variants had amino acid substitutions in V2 (valine 166 to methionine) and V3 (serine 303 to glycine), although the same V3 substitution appeared in virus passaged without MIP-1alpha. A single-round replication assay using a luciferase reporter HIV-1 strain pseudotyped with mutant envelopes confirmed that mutations in both V2 and V3 were necessary to confer the reduced sensitivity to MIP-1alpha, MIP-1beta, and RANTES. However, the double mutant did not switch its chemokine receptor usage from CCR5 to CXCR4, indicating the altered recognition of CCR5 by this mutant. These results indicated that V2 combined with the V3 region of the CCR5-tropic HIV-1 envelope modulates the sensitivity of HIV-1 to C-C chemokines without altering the ability to use chemokine receptors.