Combined effect of CCR5-Delta32 heterozygosity and the CCR5 promoter polymorphism -2459 A/G on CCR5 expression and resistance to human immunodeficiency virus type 1 transmission

Exposed seronegative individuals (ES) with persistent high-risk sexual behavior may be less susceptible to human immunodeficiency virus type 1 (HIV-1) infection because they carry the chemokine receptor (CR) gene alleles CCR5 open reading frame (ORF) Delta32, CCR5 promoter -2459G, or CCR2 ORF 64I (CCR2-64I), all of which have been found to diminish HIV-1 infectivity and/or disease progression. To investigate this, we determined the haplotypes for these three genetic loci in 93 ES and 247 low-risk control individuals. To test if protective haplotypes exert their effect by modulating CR expression, we measured the protein expression of CCR5 and CXCR4 on circulating CD4+ T cells and CD14+ monocytes in 71 ES and 92 controls. To avoid investigator bias, the analysis was performed without knowledge of each subject's risk and genotype. The CCR5 -2459G allele was significantly enriched in ES Caucasian men, who constituted the majority (84%) of the ES cohort, compared to the control Caucasian men (P = 0.02). This increase was mostly attributable to a higher frequency of the -2459 A/G versus the -2459 A/A genotype in individuals heterozygous for the delta32 allele (P = 0.012). No protective influence of the CCR2-64I allele was observed. The haplotypes CCR5 ORF delta32/CCR5 -2459A (in complete linkage disequilibrium) and CCR5 ORF wt/CCR5 -2459G had a cumulative negative effect on the expression of CCR5, since we measured significantly reduced CCR5 densities on both T-helper cells and monocytes only when both haplotypes were present. Densities of CCR5 on lymphocytes and monocytes were correlated (r = 0.59; P < 0.0001), indicating concordance of CCR5 expression patterns across different cell types. We conclude that the CCR5 ORF delta32/wt-CCR5 -2459 A/G genotype combination offers an advantage in resisting sexual HIV-1 transmission and that this effect is mediated by a relative paucity of CCR5 on potential target cells of HIV-1.     

Determination of the CCR2-64I/CCR5-59653T Haplotype Linkage Disequilibrium in a Sample of Polish Population*

It has been reported that different polymorphisms in the regulatory regions of CCR5 and in the CCR2 gene of the chemokine receptors are associated with AIDS. We decided to determine the congruence between CCR5-59653T and CCR2-64I alleles in a a group of 281 persons. Frequencies of combined CCR5-59653T and CCR2-64I haplotypes were examined in a group of 281 persons. Among 281 individuals 26 (9.3%) and 24 (8.5%) respectively were carriers of the CCR5-59653T and the CCR2-64I alleles. We also found that 24 persons (8.5%) were carriers of combined CCR2-64I/CCR5-59653T allele. The calculation of the congruence revealed that 92.0% of individuals exhibited the same genotype for both CCR2-64I and CCR5-59653T polymorphisms. Our results confirm that linkage between CCR5-59653T and CCR2-64I alleles is not absolute.     

Determination of the CCR2-64I/CCR5-59653T haplotype linkage disequilibrium in a sample of Polish population

It has been reported that different polymorphisms in the regulatory regions of CCR5 and in the CCR2 gene of the chemokine receptors are associated with AIDS. We decided to determine the congruence between CCR5-59653T and CCR2-64I alleles in a group of 281 persons. Frequencies of combined CCR5-59653T and CCR2-64I haplotypes were examined in a group of 281 persons. Among 281 individuals 26 (9.3%) and 24 (8.5%) respectively were carriers of the CCR5-59653T and the CCR2-64I alleles. We also found that 24 persons (8.5%) were carriers of combined CCR2-64I/CCR5-59653T allele. The calculation of the congruence revealed that 92.0% of individuals exhibited the same genotype for both CCR2-64I and CCR5-59653T polymorphisms. Our results confirm that linkage between CCR5-59653T and CCR2-64I alleles is not absolute.     

Reduced cell surface expression of CCR5 in CCR5Delta 32 heterozygotes is mediated by gene dosage, rather than by receptor sequestration

Macrophage tropic (M-tropic) human immunodeficiency virus (HIV) infection of primary human T cells and macrophages requires optimal cell surface expression of the chemokine receptor CCR5 in addition to CD4. Natural mutations of CCR5 that impair surface expression bestow in the homozygous state complete resistance to M-tropic HIV infection. ccr5Delta32 is the major prototype of such mutants. ccr5Delta32 heterozygosity is associated with delayed onset of AIDS and reduced risk of initial transmission, and this correlates with reduced levels of CCR5 and reduced infectability of CD4+ cells. In addition to gene dosage, sequestration of wild type (WT) CCR5 by mutant protein has been proposed as a mechanism to explain reduced surface expression of CCR5 in cells from ccr5Delta32 and CCR5-893(-) heterozygotes. However, here we demonstrate that a molar excess of ccr5Delta32 or related deletion mutants does not significantly impair the cell surface density of co-expressed WT receptor either in human epithelial cells or Jurkat T cells. Further, ligand-dependent signaling and M-tropic HIV usage of WT receptor are also unaffected. Nascent WT receptor does associate with ccr5Delta32 and related mutant proteins and with other unrelated CC and CXC chemokine receptors under transient labeling conditions. However, using confocal microscopy, we demonstrate that in the steady state, WT and truncated CCR5 proteins segregate into nonoverlapping subcellular compartments. These findings together with the observed and known variability in the cell surface density of CCR5 on quiescent PBLs lead us to conclude that reduced CCR5 gene dosage rather than receptor sequestration is the major determinant of reduced CCR5 expression in cells from ccr5Delta32 heterozygotes.     

Expression and characterization of the chemokine receptors CCR2 and CCR5 in mice

The chemokine receptors CCR2 and CCR5 play important roles in the recruitment of monocytes/macrophages and T cells. To better understand the role of both receptors in murine models of inflammatory diseases and to recognize potential problems when correlating these data to humans, we have generated mAbs against murine CCR2 and CCR5. In mice CCR2 is homogeneously expressed on monocytes and on 2--15% of T cells, closely resembling the expression pattern in humans. In contrast to humans, murine NK cells are highly CCR5 positive. In addition, CCR5 is expressed on 3--10% of CD4 and 10--40% of CD8-positive T cells and is weakly detectable on monocytes. Using a model of immune complex nephritis, we examined the effects of inflammation on chemokine receptor expression and found a 10-fold enrichment of CCR5(+) and CCR2(+) T cells in the inflamed kidneys. The activity of various chemokines and the antagonistic properties of the mAbs were measured by ligand-induced internalization of CCR2 and CCR5 on primary leukocytes. The Ab MC-21 (anti-CCR2) reduced the activity of murine monocyte chemotactic protein 1 by 95%, whereas the Ab MC-68 (anti-CCR5) blocked over 99% of the macrophage-inflammatory protein 1alpha and RANTES activity. MC-21 and MC-68 efficiently blocked the ligand binding to CCR2 and CCR5 with an IC(50) of 0.09 and 0.6--1.0 microg/ml, respectively. In good correlation to these in vitro data, MC-21 almost completely prevented the influx of monocytes in thioglycollate-induced peritonitis. Therefore, both Abs appear as useful reagents to further study the role of CCR2 and CCR5 in murine disease models.     

Fusion of monocytes and macrophages with HIV-1 correlates with biochemical properties of CXCR4 and CCR5

Human macrophages can be infected more efficiently by M-tropic than by T-tropic HIV-1 strains, despite surface expression of both CXCR4 and CCR5 co-receptors. Western blot analyses of total cell extracts and surface proteins from multiple sets of monocytes and macrophages demonstrated substantial differences between CXCR4 molecules. CXCR4 was mainly a monomer in monocytes, but was mainly a species of higher molecular weight (90 kDa) on the surface of macrophages. CCR5 was monomeric in both cell types. A constitutive association between CD4 and the co-receptors was seen in monocytes and macrophages. However, CD4 co-precipitated with CCR5 and CXCR4 monomers, but not with the high-molecular-weight forms of CXCR4, indicating that the high-molecular-weight CXCR4 species in macrophages are not available for association with CD4, which may contribute to the inefficient entry of T-tropic strains into mature macrophages.     

CCR5 promoter haplotypes differentially influence CCR5 expression on natural killer and T cell subsets in ethnically divergent HIV-1 uninfected South African populations

CCR5 plays a critical and central role in HIV-1 infection and, to date, a number of genetic mutations and haplotypes within the gene have been found to positively or negatively influence an individual’s susceptibility and rate of disease progression. In this study, we have evaluated the influence of CCR5 haplotypes, HHA, HHC, HHD, and HHE, on CCR5 expression in healthy HIV-1 uninfected individuals from two populations, South African Africans (SAA, n?=?22) and South African Caucasians (SAC, n?=?31). CCR5 haplotypes were determined through sequencing and real time polymerase chain reaction. Flow cytometry was used to quantitate CCR5 surface expression, as both CCR5 density and percentage of CCR5-expressing cells, on B, T, natural killer (NK) cells and monocytes. SAA individuals positive for the HHA haplotype had significantly lower percentages of CCR5-expressing CD8+ T cells in comparison to individuals without HHA (P?=?0.001). HHC+ SAC individuals had significantly higher CCR5 molecules per cell (density) on NK (CD56+) and CD16+ CD56+ NK cell subsets (P?=?0.030 and P?=?0.024, respectively) compared to HHC? SAC individuals. Haplotypes HHD and HHE had no impact on CCR5 expression. Overall, our data highlight that the protective effect of the HHC haplotype in Caucasians might be explained by higher density of CCR5 expression on NK cells that is not evident in HHC+ SAA individuals. Findings raise the question as to the role of CCR5-expressing cells other than CD4+ T cells in protection from HIV-1 acquisition and disease progression.     

Functional expression of the chemokine receptor CCR5 on virus epitope-specific memory and effector CD8+ T cells

Because the chemokine receptor CCR5 is expressed on Th1 CD4(+) cells, it is important to investigate the expression and function of this receptor on other T cells involved in Th1 immune responses, such as Ag-specific CD8(+) T cells, which to date have been only partially characterized. Therefore, we analyzed the expression and function of CCR5 on virus-specific CD8+ T cells identified by HLA class I tetramers. Multicolor flow cytometry analysis demonstrated that CCR5 is expressed on memory (CD28+CD45RA-) and effector (CD28-CD45RA- and CD28-CD45RA+) CD8+ T cells but not on naive (CD28+CD45RA+) CD8+ T cells. CCR5 expression was much lower on two effector CD8+ T cells than on memory CD8+ T cells. Analysis of CCR7 and CCR5 expression on the different types of CD8+ T cells showed that memory CD8+ T cells have three phenotypic subsets, CCR5+CCR7-, CCR5+CCR7+, and CCR5-CCR7+, while naive and effector CD8+ T cells have CCR5-CCR7+ and CCR5+CCR7- phenotypes, respectively. These results suggest the following sequence for differentiation of memory CD8+ T cells: CCR5-CCR7+-->CCR5+CCR7+-->CCR5+CCR7-. CCR5+CD8+ T cells effectively migrated in response to RANTES, suggesting that CCR5 plays a critical role in the migration of Ag-specific effector and differentiated memory CD8+ T cells to inflammatory tissues and secondary lymphoid tissues. This is in contrast to CCR7, which functions as a homing receptor in migration of naive and memory CD8+ T cells to secondary lymphoid tissues.     

Role of CCR5 in infection of primary macrophages and lymphocytes by macrophage-tropic strains of human immunodeficiency virus: resistance to patient-derived and prototype isolates resulting from the delta ccr5 mutation.

The alpha-chemokine receptor fusin (CXCR-4) and beta-chemokine receptor CCR5 serve as entry cofactors for T-cell (T)-tropic and macrophage (M)-tropic human immunodeficiency virus type 1 (HIV-1) strains, respectively, when expressed with CD4 in otherwise nonpermissive cells. Some M-tropic and dual-tropic strains can also utilize other beta-chemokine receptors, such as CCR2b and CCR3. A mutation of CCR5 (delta ccr5) was recently found to be common in certain populations and appears to confer protection against HIV-1 in vivo. Here, we show that this mutation results in a protein that is expressed intracellularly but not on the cell surface. Primary CD4 T cells from delta ccr5 homozygous individuals were highly resistant to infection with prototype M-tropic HIV-1 strains, including an isolate (YU-2) that uses CCR5 and CCR3, but were permissive for both a T-tropic strain (3B) and a dual-tropic variant (89.6) that uses CXCR-4, CCR5, CCR3, or CCR2b. These cells were also resistant to M-tropic patient isolates but were readily infected by T-tropic patient isolates. Primary macrophages from delta ccr5 homozygous individuals were also resistant to infection with M-tropic strains, including YU-2, but the dual-tropic strain 89.6 was able to replicate in them even though macrophages are highly resistant to CXCR-4-dependent T-tropic isolates. These data show that CCR5 is the essential cofactor for infection of both primary macrophages and T lymphocytes by most M-tropic strains of HIV-1. They also suggest that CCR3 does not function for HIV-1 entry in primary lymphocytes or macrophages, but that a molecule(s) other than CCR5 can support entry into macrophages by certain virus isolates. These studies further define the cellular basis for the resistance to HIV-1 infection of individuals lacking functional CCR5.     

Cutting edge: Human Th17 cells are identified as bearing CCR2+CCR5- phenotype

Recent reports have shown that IL-17-producing CD4+ T cells (Th17 cells) belong to a distinct helper T cell lineage and are critically involved in the pathogenesis of autoimmune diseases and allergies. However, the chemokine receptor profile of Th17 cells remains to be clarified. In this study, we report that human Th17 cells are identified as CCR2+CCR5- memory CD4+ T cells. Analysis of PBMC from healthy donors showed that CCR2+ cells produce much larger amounts of IL-17 than CCR2- cells, indicating the preferential expression of CCR2 on Th17 cells. Notably, CCR2+CCR5- memory CD4+ T cells produced a large amount of IL-17 and little IFN-gamma, whereas CCR2+CCR5+ cells reciprocally produced an enormous amount of IFN-gamma but little IL-17. Moreover, a higher expression of T-bet was seen in the CCR5+ memory T cells. These results indicate that absence of CCR5 distinguishes human Th17 cells from Th1 cells.     

Expression of CCR5 Increases during Monocyte Differentiation and Directly Mediates Macrophage Susceptibility to Infection by Human Immunodeficiency Virus Type 1

The stage of differentiation and the lineage of CD4⁺ cells profoundly affect their susceptibility to infection by human immunodeficiency virus type 1 (HIV-1). While CD4⁺ T lymphocytes in patients are readily susceptible to HIV-1 infection, peripheral blood monocytes are relatively resistant during acute or early infection, even though monocytes also express CD4 and viral strains with macrophage (M)-tropic phenotypes predominate. CCR5, the main coreceptor for M-tropic viruses, clearly contributes to the ability of CD4⁺ T cells to be infected. To determine whether low levels of CCR5 expression account for the block in infection of monocytes, we examined primary monocyte lineage cells during differentiation. Culturing of blood monocytes for 5 days led to an increase in the mean number of CCR5-positive cells from <20% of monocytes to >80% of monocyte-derived macrophages (MDM). Levels of CCR5 expression per monocyte were generally lower than those on MDM, perhaps below a minimum threshold level necessary for efficient infection. Productive infection may be restricted to the small subset of monocytes that express relatively high levels of CCR5. Steady-state CCR5 mRNA levels also increased four- to fivefold during MDM differentiation. Infection of MDM by M-tropic HIV-1_JRFL resulted in >10-fold-higher levels of p24, and MDM harbored >30-fold more HIV-1 DNA copies than monocytes. In the presence of the CCR5-specific monoclonal antibody (MAb) 2D7, virus production and cellular levels of HIV-1 DNA were decreased by >80% in MDM, indicating a block in viral entry. There was a direct association between levels of CCR5 and differentiation of monocytes to macrophages. Levels of CCR5 were related to monocyte resistance and macrophage susceptibility to infection because infection by the M-tropic strain HIV-1_JRFL could be blocked by MAb 2D7. These results provide direct evidence that CCR5 functions as a coreceptor for HIV-1 infection of primary macrophages.     

Functional expression of chemokine receptor CCR5 on CD4(+) T cells during virus-induced central nervous system disease

Intracranial infection of C57BL/6 mice with mouse hepatitis virus (MHV) results in an acute encephalomyelitis followed by a demyelinating disease similar in pathology to the human disease multiple sclerosis (MS). CD4(+) T cells are important in amplifying demyelination by attracting macrophages into the central nervous system (CNS) following viral infection; however, the mechanisms governing the entry of these cells into the CNS are poorly understood. The role of chemokine receptor CCR5 in trafficking of virus-specific CD4(+) T cells into the CNS of MHV-infected mice was investigated. CD4(+) T cells from immunized CCR5(+/+) and CCR5(-/-) mice were expanded in the presence of the immunodominant epitope present in the MHV transmembrane (M) protein encompassing amino acids 133 to 147 (M133-147). Adoptive transfer of CCR5(+/+)-derived CD4(+) T cells to MHV-infected RAG1(-/-) mice resulted in CD4(+)-T-cell entry into the CNS and clearance of virus from the brain. These mice also displayed robust demyelination correlating with macrophage accumulation within the CNS. Conversely, CD4(+) T cells from CCR5(-/-) mice displayed an impaired ability to traffic into the CNS of MHV-infected RAG1(-/-) recipients, which correlated with increased viral titers, diminished macrophage accumulation, and limited demyelination. Analysis of chemokine receptor mRNA expression by M133-147-expanded CCR5(-/-)-derived CD4(+) T cells revealed reduced expression of CCR1, CCR2, and CXCR3, indicating that CCR5 signaling is important in increased expression of these receptors, which aid in trafficking of CD4(+) T cells into the CNS. Collectively these results demonstrate that CCR5 signaling is important to migration of CD4(+) T cells to the CNS following MHV infection.     

Tat Protein Induces Human Immunodeficiency Virus Type 1 (HIV-1) Coreceptors and Promotes Infection with both Macrophage-Tropic and T-Lymphotropic HIV-1 Strains

Chemokine receptors CCR5 and CXCR4 are the primary fusion coreceptors utilized for CD4-mediated entry by macrophage (M)- and T-cell line (T)-tropic human immunodeficiency virus type 1 (HIV-1) strains, respectively. Here we demonstrate that HIV-1 Tat protein, a potent viral transactivator shown to be released as a soluble protein by infected cells, differentially induced CXCR4 and CCR5 expression in peripheral blood mononuclear cells. CCR3, a less frequently used coreceptor for certain M-tropic strains, was also induced. CXCR4 was induced on both lymphocytes and monocytes/macrophages, whereas CCR5 and CCR3 were induced on monocytes/macrophages but not on lymphocytes. The pattern of chemokine receptor induction by Tat was distinct from that by phytohemagglutinin. Moreover, Tat-induced CXCR4 and CCR5 expression was dose dependent. Monocytes/macrophages were more susceptible to Tat-mediated induction of CXCR4 and CCR5 than lymphocytes, and CCR5 was more readily induced than CXCR4. The concentrations of Tat effective in inducing CXCR4 and CCR5 expression were within the picomolar range and close to the range of extracellular Tat observed in sera from HIV-1-infected individuals. The induction of CCR5 and CXCR4 expression correlated with Tat-enhanced infectivity of M- and T-tropic viruses, respectively. Taken together, our results define a novel role for Tat in HIV-1 pathogenesis that promotes the infectivity of both M- and T-tropic HIV-1 strains in primary human leukocytes, notably in monocytes/macrophages.     

Adiponectin stimulates release of CCL2, -3, -4 and -5 while the surface abundance of CCR2 and -5 is simultaneously reduced in primary human monocytes

The adipokine adiponectin is well known to affect the function of immune cells and upregulation of CCL2 by adiponectin in monocytes/macrophages has already been reported. In the current study the effect of adiponectin on CCL2, -3, -4, and -5 and their corresponding receptors CCR1, CCR2, and CCR5 has been analyzed. Adiponectin elevates mRNA and protein of the CC chemokines in primary human monocytes. Simultaneously the surface abundance of CCR2 and CCR5 is reduced while CCR1 is not affected. Downregulation of CCR2 by adiponectin is blocked by a CCR2 antagonist although expression of the CCL2 regulated genes CCR2 and TGF-beta 1 is not altered in the adiponectin-incubated monocytes. CCL2, -3, and -5 concentrations measured in supernatants of monocytes of normal-weight (NW), overweight (OW), and type 2 diabetic (T2D) patients positively correlate with BMI and are increased in obesity and T2D. In contrast CCL4 is similarly abundant in the supernatants of all of these monocytes. The degree of adiponectin-mediated induction of the chemokines CCL3, -4, and -5 negatively correlates with their basal levels and upregulation of CCL3 and CCL5 is significantly impaired in OW and T2D cells. Serum concentrations of these chemokines are almost equal in the three groups and do not correlate with the levels in monocyte supernatants. In conclusion these data demonstrate that adiponectin stimulates release of CCL2 to CCL5 in primary human monocytes, and induction in cells of overweight probands is partly impaired. Adiponectin also lowers surface abundance of CCR2 and CCR5 and downregulation of CCR2 seems to depend on autocrine/paracrine effects of CCL2.     

A human immunodeficiency virus type 1 isolate from an infected person homozygous for CCR5Delta32 exhibits dual tropism by infecting macrophages and MT2 cells via CXCR4

The mechanisms of human immunodeficiency virus (HIV) infection of a man (VH) homozygous for the CCR5Delta32 mutation were investigated, and coreceptors other than CCR5 used by HIV type 1 (HIV-1) isolated from this individual were identified. In contrast to previous reports, this individual's rate of disease progression was not accelerated. Homozygosity for CCR5Delta32 mutation was demonstrated by PCR and DNA sequencing (R. Biti et al., Nat. Med. 3:252-253, 1997). CCR5 surface expression was absent on T lymphocytes and macrophages. HIV was isolated by coculture with peripheral blood mononuclear cells (PBMCs) from siblings who were homozygous (VM) or wild type (WT) for the CCR5Delta32 mutation. The virus demonstrated dual tropism for infection of MT2 cell line and primary macrophages. Sequencing of the full HIV genome directly from the patient's PBMCs revealed 21 nucleotide insertions in the V1 region of gp120. The VH envelope sequence segregated apart from both the T-cell-line-adapted tropic strains NL4-3 and SF2 and M-tropic strain JRFL or YU2 by phylogenetic tree analysis. VH was shown to utilize predominantly CXCR4 for entry into T lymphocytes and macrophages by HOS.CD4 cell infection assay, direct envelope protein fusion, and inhibition by anti-CXCR4 monoclonal antibody (12G5), SDF-1, and AMD3100. Microsatellite mapping demonstrated the separate inheritance of CXCR4 by both homozygote brothers (VH and VM). Our study demonstrates the ability of certain strains of HIV to readily use CXCR4 for infection or entry into macrophages, which is highly relevant to the pathogenesis of late-stage disease and presumably also HIV transmission.     

CCR5 deficiency aggravates crescentic glomerulonephritis in mice

The chemokine receptor CCR5 is predominantly expressed on monocytes and Th1-polarized T cells, and plays an important role in T cell and monocyte recruitment in inflammatory diseases. To investigate the functional role of CCR5 in renal inflammation, we induced a T cell-dependent model of glomerulonephritis (nephrotoxic serum nephritis) in CCR5(-/-) mice. Induction of nephritis in wild-type mice resulted in up-regulation of renal mRNA expression of the three CCR5 chemokine ligands, CCL5 (15-fold), CCL3 (4.9-fold), and CCL4 (3.4-fold), in the autologous phase of the disease at day 10. The up-regulated chemokine expression was paralleled by infiltration of monocytes and T cells, followed by renal tissue injury, albuminuria, and loss of renal function. Nephritic CCR5(-/-) mice showed a 3- to 4-fold increased renal expression of CCL5 (61.6-fold vs controls) and CCL3 (14.1-fold vs controls), but not of CCL4, in comparison with nephritic wild-type mice, which was accompanied by augmented renal T cell and monocyte recruitment and increased lethality due to uremia. Furthermore, CCR5(-/-) mice showed an increased renal Th1 response, whereas their systemic humoral and cellular immune responses were unaltered. Because the CCR5 ligands CCL5 and CCL3 also act via CCR1, we investigated the effects of the pharmacological CCR1 antagonist BX471. CCR1 blockade in CCR5(-/-) mice significantly reduced renal chemokine expression, T cell infiltration, and glomerular crescent formation, indicating that increased renal leukocyte recruitment and consecutive tissue damage in nephritic CCR5(-/-) mice depended on functional CCR1. In conclusion, this study shows that CCR5 deficiency aggravates glomerulonephritis via enhanced CCL3/CCL5-CCR1-driven renal T cell recruitment.     

Constitutive association of cell surface CCR5 and CXCR4 in the presence of CD4

Chemokine receptors CCR5 and CXCR4 are the major coreceptors of HIV-1 infection and also play fundamental roles in leukocyte trafficking, metastasis, angiogenesis, and embyogenesis. Here, we show that transfection of CCR5 into CXCR4 and CD4 expressing 3T3 cells enhances the cell surface level of CXCR4. In CCR5 high expressing cells, cell surface level of CXCR4 was incompletely modulated in the presence of the CXCR4 ligand CXCL12/SDF-1alpha. CCR5 was resistant to ligand-dependent modulation with the CCR5 ligand CCL5/RANTES. Confocal laser microscopy revealed that CCR5 was colocalized with CXCR4 on the cell surface. In CD4 expressing CCR5 and CXCR4 double positive NIH 3T3 cells, immunoprecipitation followed by Western blot analysis revealed that CCR5 was associated with CXCR4 and CD4. CXCR4 and CCR5 were not co-immunoprecipitated in cells expressing CCR5 and CXCR4 but without CD4 expression. Compared to NIH 3T3CD4 cells expressing CXCR4, the entry of an HIV-1 X4 isolate (HCF) into NIH 3T3CD4 expressing both CXCR4 and CCR5 was reduced. Our data indicate that chemokine receptors interact with each other, which may modulate chemokine-chemokine receptor interactions and HIV-1 coreceptor functions.     

Up-regulation of CCR5 and CCR6 on distinct subpopulations of antigen-activated CD4+ T lymphocytes.

Following infection, naive T cells are activated in the secondary lymphoid tissue, but then need to move to the infected tissue in the periphery to mediate their effector functions. The acquisition of inflammatory chemokine receptors, such as CCR5 and CCR6, may contribute to the efficient relocation of activated T cells to inflamed sites in the periphery. In keeping with this idea, the present study has demonstrated that CCR5 and CCR6 are up-regulated on CD4+ T cells upon activation in the MLR. The observed increase in expression correlated well with the acquisition of an activated/memory phenotype and was largely (CCR5) or completely (CCR6) separated temporally from the initiation of cell division. In contrast, the regulation of two other chemokine receptors, CXCR3 and CXCR4, occurred in close parallel with the cell division process. Increased mRNA levels are likely to contribute to the enhanced surface expression of CCR5 and CCR6, but in the case of CCR6, translocation of intracellular stores of protein to the cell surface may be an additional mechanism of regulation. The up-regulation of CCR5 was more extensive than that of CCR6, as only approximately half the activated CCR5+ T cells coexpressed CCR6. The increased expression of CCR5 resulted in enhanced chemotaxis toward the CCR5 ligand macrophage-inflammatory protein-1beta/CCL4, but up-regulation of CCR6 did not result in altered chemotactic responsiveness to macrophage-inflammatory protein-3alpha/CCL20, suggesting an alternative function for this receptor.     

CCR5 blockade is well tolerated and induces changes in the tissue distribution of CCR5+ and CD25+ T cells in healthy, SIV-uninfected rhesus macaques

Background CCR5 is a main co‐receptor for HIV, but also homes lymphocytes to sites of inflammation. We hypothesized that inhibition of CCR5 signaling would reduce HIV‐associated chronic immune activation. Methods To test this hypothesis, we administered an antagonistic anti‐CCR5 monoclonal antibody (HGS101) to five uninfected rhesus macaques (RMs) and monitored lymphocyte dynamics in blood and tissue. Results CCR5 blockade resulted in decreased levels of CCR5+ T cells in blood and, at later timepoints, in lymph nodes. Additionally, the levels of CD25+ T cells increased in lymph nodes, but decreased in blood, bone marrow, and rectal mucosa. Finally, a profile of gene expression from HGS101‐treated RMs revealed a subtle, but consistent, in vivo signature of CCR5 blockade that suggests a mild immune‐modulatory effect. Conclusions Treatment with anti‐CCR5 antibody induces changes in the tissue distribution of CCR5+ and CD25+ T cells that may impact on the overall levels of immune activation during HIV and SIV infection.