A CCR2-V64I polymorphism affects stability of CCR2A isoform : AIDS (original) (raw)
Introduction
The chemokine receptor CCR2B has been regarded as a minor HIV-1 coreceptor because only a small number of HIV-1 strains has been shown to use CCR2B as an entry coreceptor [1–3]. Nevertheless, a polymorphism in the CCR2 gene, CCR2-64I, has been reported to be associated with delayed disease progression in HIV-1 infected individuals in several Caucasian cohorts [4–8]. This polymorphism, a G-to-A transition at position 190, changes CCR2B codon 64 from valine to isoleucine, introducing a conservative amino acid change into the first transmembrane domain. It was unclear why a single amino acid substitution in a minor coreceptor could affect HIV-1 disease progression, as there was no difference in HIV-1 co-receptor activity between the variant CCR2B-64I and CCR2B without the 64I substitution (CCR2B-64V) [9,10]. Furthermore, these studies also excluded the possibility that CCR2B-64I exerts a dominant-negative effect on the expression and activity of CCR5.
It is possible that the CCR2 polymorphism may be linked to other polymorphisms in genes that influence AIDS progression. The CCR2 gene is located approximately 15 kb from the 5′ end of the CCR5 gene, and the CCR2-64I allele is indeed linked to a certain CCR5 promoter haplotype [11]. However, experiments using promoter–reporter fusion constructs showed that the CCR5 promoter haplotype, which is in a strong linkage disequilibrium with CCR2-64I, did not affect transcriptional activity of the CCR5 promoter [10]. Thus, the mechanism underlying the protective effect of CCR2-64I against AIDS progression still remained to be elucidated.
Two alternatively spliced CCR2 isoforms, CCR2A and CCR2B, were reported to be present in freshly isolated human monocyte, THP-1, and MonoMac 6 leukaemia cell lines [12,13]. An open reading frame encoded in the chromosome corresponds to CCR2B, while alternatively spliced transcripts produce CCR2A. The two CCR2 isoforms differ only in their C-terminal cytoplasmic tails (Fig. 1). Therefore, an individual carrying the CCR2-64I allele also produces CCR2A molecules with isoleucine at position 64. Although the cytoplasmic tail spans less than one-fifth of the entire CCR2 molecule, this difference caused a drastic alteration in their localization in cells [13]. While CCR2B trafficked well to the cell surface, CCR2A was detected predominantly in the cytoplasm. A progressive truncation study of the C-terminal cytoplasmic tail indicated that a cytoplasmic retention signal(s) was located in the C-terminal cytoplasmic tail [13]. Nevertheless, CCR2A molecules that successfully trafficked to the cell surface could respond to the stimulation of monocyte chemoattractant protein (MCP)-1 in a similar fashion to CCR2B [14].
The structure of the CCR2A and CCR2B molecules. Outlined letters in grey circles denote amino acid residues present in CCR2B. Outlined letters in black circles denote amino acid residues present in CCR2A. A letter I in a large circle denotes a substitution at position 64.
As none of the previous studies investigated the effect of the 64I substitution on CCR2A molecules, we generated recombinant Sendai viruses (SeV) expressing either CCR2A-64V or CCR2A-64I. Here we show that the 64I substitution indeed affected the stability of CCR2A molecules in cells, and increased the ability of CCR2A to down-modulate the major HIV-1 co-receptor, CCR5.
Materials and methods
Generation of recombinant SeV
THP-1 cells were shown to possess both CCR2-64V and CCR2-64I alleles by using a standard genotyping method [15]. Therefore, CCR2A-64V, CCR2A-64I, CCR2B-64V, and CCR2B-64I cDNA were obtained by reverse transcription (RT)–PCR from mRNA extracted from THP-1 cells and then inserted to the _Not_I site of pSeV18+b(+). The entire coding regions in the resultant plasmids were verified for sequence authenticity as well as for the presence or absence of the 64I substitution. For generating CCR2A-64V and CCR2A-64I cDNA carrying a c-myc-tag (EQKLI SEEDL) at their C-termini, cloned CCR2A-64V and CCR2A-64I cDNA served as templates for PCR amplification using a primer containing a nucleotide sequence corresponding the c-myc-tag fused with the C-terminal portion of CCR2A. Recombinant SeV carrying CCR2A-64V, CCR2A-64I, CCR2B-64V, CCR2B-64I, or C-myc-tagged versions of CCR2A-64V and CCR2A-64I were recovered according to a previously described method [16]. The wild-type Z strain of SeV served as a control in all the experiments.
Generation of a recombinant vaccinia virus
For generating CCR5 cDNA carrying a HA tag (YPYDVPDYAA) at its C terminus, cloned CCR5 cDNA served as a template for PCR amplification by using a primer containing a haemagglutinin (HA) tag sequence fused with the C-terminal portion of CCR5. The resultant PCR products were then inserted into pNZ68K2-Not. The entire coding region of CCR5-HA was verified for sequence authenticity. A recombinant vaccinia virus (Vac) was recovered from the resultant plasmid according to previously described procedures [17].
Flow cytometric analysis
CV1 monkey kidney cells, U937 monocytic cells and Jurkat T cells were infected with recombinant SeV expressing CCR2A-64V, CCR2A-64I, CCR2B-64V, or CCR2B-64I. Five to 18 h after infection, cells were incubated with MAB150, a mouse monoclonal antibody (MAb) against CCR2 (R & D Systems, Minneapolis, Minnesota, USA). Antibodies bound to cells were detected using fluorescein-5-isothiocyanate (FITC)-conjugated goat antibody directed against mouse IgG (Cappel, Aurora, Ohio, USA). CV1 or H9 cells infected with SeV expressing CCR2A-64V, CCR2A-64I, CCR2B-64V, or CCR2B-64I were superinfected with a recombinant Vac expressing CCR5, CXCR4, or CD4 at 9 h after SeV infection. After incubation for 5 h at 37°C, cells were stained for CCR5 using T227 rat MAb against CCR5 [17] followed by FITC-conjugated goat anti-rat IgG; for CXCR4 using 12G5 mouse MAb (R & D systems) followed by FITC-conjugated goat anti-mouse IgG; or for CD4 using FITC-conjugated anti-human CD4, Leu3a (Becton Dickinson, San Jose, California, USA), and analysed by FACScan (Becton Dickinson).
Immunofluorescence microscopy
CV1 cells expressing CCR2A or CCR2B were fixed and permeabilized before being incubated with MAB150 antibody as described previously [17]. Bound antibodies were then detected using FITC-conjugated goat antibody against mouse IgG. Indirect immunofluorescence was visualized using a Lasersharp2000 Confocal Microscope System (Bio-Rad, Hercules, California, USA). Anti-Calnexin (Stressgen, San Diego, California, USA) or anti-Giantin (CRPinc, Berkeley, California, USA) rabbit polyclonal antibody was used with Cy5-conjugated goat antibody against rabbit IgG (Amersham Pharmacia Biotech, Piscataway, New Jersey, USA).
Chemotaxis assay
Chemotaxis assays were performed according to previously described methods [18]. Briefly, MCP-1 (PeproTech, Rockey Hill, New Jersey, USA) diluted at an indicated concentration of chemotaxis buffer (RPMI 1640 with 0.25% human serum albumin) was added to the bottom chamber of a 5-μm pore polycarbonate Transwell culture insert (Costar; Corning, New York, USA). Jurkat cells were infected with a SeV expressing CCR2A-64V or CCR2A-64I and incubated at 37°C for 4 h. Cells were then washed with RPMI1640 and re-suspended in chemotaxis buffer and added to the upper chamber of the insert. Transmigrated cells in 4 h at 37°C were counted using a FACScan.
Pulse-chase analyses of CCR2A and CCR5
CV1 or U937 cells were infected with a SeV expressing CCR2A-64V-myc or CCR2A-64I-myc. Nine hours after infection, cells were labelled with 500 kbq/ml of EXPRE35S35S[35S] protein labelling mix (> 37 Tbq/mmol; PerkinElmer (Boston, Massachusetts, USA) in amino acid-free medium for 30 min. For CCR5 analysis, cells were infected with a recombinant Vac expressing CCR5-HA, incubated at 37°C for 5 h and then labelled. Cells were then washed, fed with fresh medium and incubated for 0, 15, 30, 60, or 120 min at 37°C, chilled on ice, and lysed in lysis buffer (50 mM Tris–HCl pH7.5, 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate). CCR2A and CCR5 proteins in the lysates were precipitated with anti-myc mouse MAb (9B11; Cell Signaling, Beverly, Massachusetts, USA) and anti-HA high affinity rat MAb (Roche, Indianapolis, Indiana, USA), respectively, using a Protein G Immunoprecipitation Kit (Roche). Precipitated materials were subjected to SDS–PAGE on a 4–12% NuPAGE Bis–Tris gel (Invitrogen, Groningen, Netherlands), and the amount of radiolabel incorporated was visualized on a BAS Imager (Fujix, Kanagawa, Japan).
Gene reporter fusion assay
A recombinant Vac-based gene activation assay using a β-galactosidase gene as a reporter was performed as described previously [19]. Briefly, mouse fibroblast L cells were transfected with β-galactosidase reporter plasmid pGINT7β-gal and infected with a recombinant Vac expressing gp160 of an R5 HIV-1 strain SF162. At the same time, CV1 cells were infected with SeV expressing CCR2A-64V or CCR2A-64I and incubated at 37°C for 9 h. Cells were then superinfected with recombinant Vacs expressing T7 RNA polymerase, human CD4, and CCR5, detached by trypsinization, and cultured at 37°C for 5 h. Then, L and CV-1cells were mixed, incubated for 3 h, and β- galactosidase activities in the cell lysate were measured by using chlorophenol red-β-d-galactopyranoside as substrate.
HIV-1 productive infection
MT4 cells (4 × 105) were infected with SeV expressing CCR2A-64V, CCR2A-64I or parental Z strain of SeV at a multiplicity of infection (MOI) of 40 plaque forming unit (PFU)/cell mixed with SeV expressing CCR5 at an MOI of 10 PFU/cell and incubated at 37°C for 5 h. Cells were then superinfected with 60 ng p24 of an R5 HIV-1 strain SF162. The culture supernatants were collected periodically and p24 levels were measured.
Immunoprecipitation and western blot analysis
CV1 cells were infected with SeV expressing CCR2A-64V-myc or CCR2A-64I-myc, and incubated at 37°C for 9 h. Cells were then superinfected with a Vac expressing CCR5-HA and incubated at 37°C for 5 h and then lysed. CCR2A-64V-myc, CCR2A-64I-myc or CCR5-HA proteins were immunoprecipitated, and subjected to SDS–PAGE as described above. Proteins were then electrophoretically transferred to a PVDF membrane (Immobilon; Millipore, Bedford, Massachusetts, USA). Blots were blocked and probed with the antibodies overnight at 4°C and then incubated with peroxidase-conjugated anti-mouse (Kirkegaard & Perry Laboratories, Gaithersbug, Maryland, USA) or anti-rat IgG (American Qualex, San Clemente, California, USA) and developed using the Immun-Star HRP chemiluminescent kit (Bio-Rad).
Results
Expression of CCR2A and CCR2B
We generated a recombinant SeV expressing either CCR2A-64V or CCR2B-64V. Confocal microscopic observations (Fig. 2a) and flow cytometric analyses (Fig. 2b) confirmed the different subcellular localization of these two CCR2 isoforms. In CCR2B-64V expressing CV1 cells, fluorescent signals of CCR2 were observed mainly on the cell surface. In contrast, CCR2A-64V was localized predominantly to the cytoplasm, although a small portion of CCR2A was observed on the cell surface. In the cytoplasm, signals of an endoplasmic reticulum marker calnexin were only partially co-localized with CCR2A signals (Fig. 2a, left), whereas the majority of signals for the Golgi marker giantin overlapped with those of CCR2A (Fig. 2a, right). These results suggested that most CCR2A molecules were retained in the Golgi.
(a) Subcellular distribution of CCR2A-64V and CCR2B-64V in CV1 cells. SeV vector (SeV) was used to express the CCR2A-64V and CCR2B-64V molecules. Cells were fixed and permeabilized before staining with MAB150 anti-CCR2 mouse MAb followed by FITC-labelled anti-mouse IgG. Cells were then re-stained with anti-calnexin or anti-giantin rabbit polyclonal antibody followed by Cy5-labelled anti-rabbit IgG, and analysed by confocal laser microscopy. (b) Surface expression of CCR2A-64V (green) and CCR2A-64I (red) in U937, CV1 or Jurkat cells. Cells infected with the parental Z strain served as a negative control (black). In lower right panel, green and red indicates CCR2B-64V and CCR2B-64I, respectively. (c) The cell surface expression of CCR2A-64I (open circles), CCR2A-64I-myc (open squares), CCR2A-64V (filled circles), and CCR2A-64V-myc (filled squares) at 5, 9, 12 and 18 h after infection by SeV. MFI indicates mean fluorescence intensity of each sample. (d) Chemokine receptor activity of recombinant CCR2A-64V and CCR2A-64I. Jurkat cells infected with SeV expressing CCR2A-64V (closed circles) or CCR2A-64I (open circles) migrated in response to increasing concentration of MCP-1. Data points are means of triplicate determination with standard deviations.
To assess the effect of the 64I substitution on CCR2A expression, we generated a recombinant SeV expressing CCR2A-64I and compared levels of expression of CCR2A-64I with those of CCR2A-64V. As shown in Fig. 2b, CCR2A-64I showed slightly but significantly higher levels of expression than CCR2A-64V in various cell types, despite the same promoter being used. The mean fluorescence intensity (MFI) of CCR2A-64I and CCR2A-64V was 274 and 140 in CV1, 133 and 40 in U937 monocystic cells, and 29 and 21 in Jurkat T cells. The difference was greater in U937 cells than in Jurkat cells. The difference was also observed at 5, 12, and 18 h after infection of recombinant SeVs (Fig. 2c). Exactly the same result was obtained when recombinant SeV expressing C-myc-tagged versions of CCR2A-64V (CCR2A-64V-myc) and CCR2A-64I (CCR2A-64I-myc) were used (Fig. 2c). In contrast, we failed to detect any difference in the levels of expression between CCR2B-64V and CCR2B-64I (MFI 2698 and 2663, respectively; Fig. 2b), as had been described in the previous reports [9,10]. Northern blot analyses confirmed that there was no difference in the amount of CCR2 mRNA among cells expressing CCR2A-64V, CCR2A-64I, CCR2A-64V-myc, CCR2A-64I-myc, CCR2B-64V and CCR2B-64I (data not shown). These data clearly indicate that the substitution of valine to isoleucine affects levels of cell surface expression of CCR2A, but not of CCR2B.
Chemokine receptor activity of recombinant CCR2A-64V and CCR2A-64I
To determine whether or not CCR2A molecules expressed by a recombinant SeV fully retained chemokine receptor activity, we performed a chemotaxis assay. As shown in Fig. 2d, both cells expressing CCR2A-64V and CCR2A-64I migrate toward MCP-1. However, cells expressing CCR2A-64I migrated more efficiently than those expressing CCR2A-64V. These results are in good agreement with the observation that expression of CCR2A-64I is higher than that of CCR2A-64V.
CCR2A-64I is more stable than CCR2A-64V
Differential levels of expression between CCR2A-64V and CCR2A-64I prompted us to compare the rate of degradation of those proteins in pulse–chase experiments. For this purpose, we used recombinant SeV expressing CCR2A-64V-myc or CCR2A-64I-myc. Comparison of immunoprecipitated materials from 35S-labelled CV1 cells expressing CCR2A-64V-myc and CCR2A-64I-myc showed that almost identical levels of CCR2A-64V-myc and CCR2A-64I-myc proteins were synthesized during the 30-min labelling period (t = 0) (Fig. 3a). However, CCR2A-64V-myc proteins appeared to degrade more rapidly than CCR2A-64I-myc proteins. The half-life of CCR2A-64I-myc was approximately 90 min, whereas that of CCR2A-64V-myc was approximately 50 min in CV1 cells (Fig. 3b). More prominent results were obtained when we used U937 cells, as the half-life of CCR2A-64I-myc was approximately 60 min, whereas that of CCR2A-64V-myc was approximately 18 min in U937 cells. This finding is in a good agreement with the observation that the difference in cell surface expression levels between CCR2A-64V and CCR2A-64I was greater in U937 cells than in CV-1 cells (Fig. 2b). These results indicate that higher cell surface expression of CCR2A-64I was due to increased stability of CCR2A-64I. On the other hand, we failed to detect any significant difference in the half-life between CCR2B-64V and CCR2B-64I (data not shown).
CCR2A-64I is more stable than CCR2A-64V. CV1 cells were infected with SeV expressing CCR2A-64V-myc and CCR2A-64I-myc for 9 h. Cells were labelled for 30 min and then harvested following the chase time indicated. (a) Representative gels of pulse–chase analysis. (b) PhosphorImager analysis of the gels shown in (a). Open and closed circles denote cells infected with SeV expressing CCR2A-64V-myc and CCR2A-64I-myc, respectively. Data points are means of four independent experiments with standard deviations.
CCR5 but not CXCR4 expression was more severely blocked by co-expression of CCR2A-64I than by co-expression of CCR2A-64V
To determine whether or not CCR2A has a dominant-negative effect on the expression of major HIV-1 receptor molecules, we first inoculated SeV expressing CCR2A-64V or CCR2A-64I in CV1 cells and incubated the cells for 9 h at 37°C. The cells were then superinfected with recombinant Vac expressing CCR5, CXCR4, or CD4. Five hours after Vac infection, surface expression of CCR5, CXCR4, or CD4 were examined by flow cytometry. As shown in Fig. 4a, the CCR5 MFI of cells co-infected with parental Z strain of SeV was 391, while that of the cells co-infected with SeV expressing CCR2A-64V was 297, indicating that co-expression of CCR2A-64V significantly reduced levels of CCR5 expression on the cell surface. This dominant-negative effect on CCR5 expression was more prominent when SeV expressing CCR2A-64I were used (MFI, 145) than SeV expressing CCR2A-64V were used. The same results were obtained when we used recombinant SeV expressing CCR2A-64V-myc and CCR2A-64I-myc (MFI, 300 and 179, respectively). Similar results were obtained when CV1 cells were inoculated with Vac expressing CCR5 5 h after infection by SeV expressing CCR2A, as the CCR5 MFI on cells co-infected with Z, SeV expressing CCR2A-64V, and Sev expressing CCR2A-64I, was 299, 205, and 160, respectively. Furthermore, the dominant-negative effect of CCR2A on CCR5 expression was also observed when T cell line H9 was used. The CCR5 MFI on H9 cells co-infected with Z, SeV expressing CCR2A-64V, and SeV expressing CCR2A-64I was 263, 230 and 195, respectively. In contrast, the cell surface expression of CXCR4, another major co-receptor, as well as that of CD4, the main receptor of HIV-1, were not affected by CCR2A-64V or CCR2A-64I (Fig. 4a). In contrast with CCR2A, neither CCR2B-64V nor CCR2B-64I affected the surface expression of CCR5 (Fig. 4b).
(a) Effect of CCR2A-64V and CCR2A-64I on HIV-1 corecepter expression. Vac vectors were used to express CCR5, CXCR4 and CD4 in the CV1 cells inoculated with SeV expressing CCR2A-64V or CCR2A-64I. Z denotes the wild-type SeV. Five hours after infection, cells were stained with MAb against CCR5, CXCR4, or CD4. Flow cytometry was used to determine surface expression levels. The number in each panel indicates mean fluorescence intensity. (b) Effect of CCR2B-64V and CCR2B-64I on CCR5 expression.
HIV-1 coreceptor activity of CCR5 was more dramatically reduced by co-expression of CCR2A-64I than by co-expression of CCR2A-64V
To assess the effect of CCR2A-64I on HIV-1 infection, we examined the ability of cells expressing both CCR2A and CCR5 molecules to support CD4-dependent cell fusion mediated by an HIV-1 envelope protein of the R5 strain SF162. For this purpose, we prepared CV1 cells expressing both CCR5 and CCR2A as described in Fig. 4a, and mixed those cells with mouse L cells expressing HIV-1 envelope protein. As shown in Fig. 5a, the envelope-mediated cell fusion activity of CCR5 was more dramatically reduced by co-expression of CCR2A-64I than by that of CCR2A-64V.
(a) Coreceptor activity of CCR5 in CCR2A-64V or CCR2A-64I co-expressed cells. SeV vector was used to express CCR2A-64V or CCR2A-64I, and Vac vector was used to express CCR5 as described in Fig. 4. HIV-1 coreceptor activity of each sample was measured using the method described in Materials and methods. The wild-type Vac WR strain was used as a CCR5-negative control, and the wild-type SeV Z strain was used as the CCR2A-negative control. (b) MT4 cells were co-infected with SeV expressing CCR5 and SeV expressing CCR2A-64V (filled circles), CCR2A-64I (open circles), or parental Z strain (filled squares). Five hours after infection, cells were inoculated with an HIV-1 strain SF162. (c) Co-immunopresipitation of CCR2A and CCR5. Recombinant Vac expressing CCR5-HA or parental WR strain (–) was superinfected in CV1 cells infected with SeVs expressing CCR2A-64V-myc, CCR2A-64I-myc, or the parental Z strain (–). Immunoprecipitation and Western blot analysis were performed by using anti-HA or anti-myc antibody. An arrow indicates 37-kDa CCR5-HA molecules. (d) Pulse–chase analysis of CCR5 molecules. A recombinant Vac expressing CCR5-HA was inoculated into CV1 cells. An arrow indicates 37-kDa CCR5-HA molecules.
We also inoculated a live SF162 strain of HIV-1 into CD4 positive MT4 cells expressing both CCR5 and CCR2A. As shown in Fig. 5b, MT4 cells expressing CCR5 and CCR2A-64V supported SF162 replication better than those expressing CCR5 and CCR2A-64I.
Co-immunoprecipitation of CCR2A and CCR5
Many seven-transmembrane receptors, including chemokine receptors, have been reported to form homo-oligomers. CCR2A is highly homologous to CCR5 (68% at the amino acid level), and formation of heterodimers between CCR2B and CCR5 was reported previously [20]. The dominant-negative effect of CCR2A on CCR5 expression shown in Figs 4a, 5a and 5b raised the possibility of heterodimer formation between CCR2A and CCR5. To test this hypothesis, we used SeV expressing CCR2A-64V-myc or CCR2A-64I-myc, and Vac expressing HA-tagged version of CCR5 (CCR5-HA). Anti-myc and anti-HA immunoprecipitates from cell lysates were developed in Western blots by using anti-HA or anti-myc antibodies. As expected, CCR5-HA was detected by anti-HA antibody in anti-myc-derived immunoprecipitates from CCR5-HA and CCR2A-64V-myc co-expressed cell lysates as well as from CCR5-HA and CCR2A-64I-myc co-expressed cell lysates. At the same time, CCR2A-64V-myc and CCR2A-64I-myc were detected by anti-myc antibody in anti-HA-derived immunoprecipitates of CCR5-HA and CCR2A-64V-myc co-expressed cell lysates and in that of CCR5-HA and CCR2A-64I-myc co-expressed cell lysates (Fig. 5c). These results clearly indicate that CCR2A formed heterodimers with CCR5.
In CCR5-HA expressing cells, we consistently observed two types of CCR5-HA molecules with different electrophoretic mobility. When we used anti-HA antibody to precipitate CCR5-HA directly, most of the CCR5-HA molecules migrated at approximately 38 kDa. In contrast, most of the CCR5-HA molecules that co-precipitated with CCR2A-64V-myc or CCR2A-64I-myc migrated at 37 kDa. We speculated that the CCR5-HA of 38 kDa represented authentic CCR5 molecules and that of 37 kDa represented immature forms of CCR5. To verify the maturation process of CCR5, we labelled the cells infected with Vac expressing CCR5-HA by [35S]-methionine for 30 min and harvested those cells following chase periods ranging from 15 to 60 min. As shown in Fig. 5d, the 37-kDa CCR5-HA could be detected only after the labelling period (0 min). This result suggests that CCR2A binds to premature forms of CCR5 and interferes with the maturation process of CCR5 molecules in cytoplasm.
Discussion
Many independent cohort studies have affirmed the AIDS-delaying effects of the CCR2-64I allele [4–8], but the molecular mechanism of this protective effect had not yet been elucidated. In the present study, we demonstrated that a valine to isoleucine substitution at position 64 increased stability of CCR2A but not of CCR2B molecules in cells. When co-expressed with the major HIV-1 co-receptor CCR5, CCR2A-64I more severely interfered with cell surface expression as well as HIV-1 co-receptor activity of CCR5 than CCR2A-64V. Furthermore, CCR2A was shown to co-precipitate with immature form of CCR5. These results suggest that CCR2A binds to CCR5 in the cytoplasm and dominantly interferes with CCR5 maturation and surface expression. On the other hand, the 64I substitution did not affect the level of CCR2B expression, being consistent with results published previously [9,10]. We speculate that increased ability of CCR2A-64I to down modulate CCR5 expression might be a possible cause of delay in HIV-1 disease progression in patients with this allele. Alternatively, it is also possible that immune cell trafficking and/or signalling might be affected by CCR2A stabilization, leading to a delay in HIV-1 diseases.
Previously, Mellado et al. reported that CXCR4 could dimerize with CCR2B-64I variants but not with wild-type CCR2B-64V upon stimulation with SDF-1 and MCP-1. Based on this finding, they proposed that this ability of CCR2B-64I to heterodimerize with CXCR4 may cause a delay in AIDS progression [20]. However, several independent cohort studies have shown that the effects of the CCR2-64I allele were more pronounced in earlier stages of disease than in latter stages [5,8,21]. In a Dutch cohort, delay in HIV-1 disease progression was more pronounced before the emergence of X4 variants and was not observed after the emergence of X4 variants in individuals with the CCR2-64I allele [6]. Therefore, it is unlikely that CCR2B-64I/CXCR4 heterodimerization is the main cause of delay in AIDS progression in individuals with CCR2-64I.
Previous studies exploring the oligomerization of chemokine receptors also yielded controversial results. Rodrigues-Frade et al. reported that CCR2B forms homodimers upon stimulation by MCP-1 [22]. Other studies, however, have shown that CCR5 [23,24] and CXCR4 [25] can form homodimers without any stimulation by their ligands. Although we did not test whether or not stimulation with MCP-1 and/or RANTES increases hetero-oligomer formation between CCR2A and CCR5, our present results support the latter model that chemokine receptors may form oligomers without stimulation by their ligands.
In addition to AIDS pathogenesis, the CCR2-64I allele was reported to be associated with lower risks of coronary artery calcification [26] and acute rejection in renal transplantation [27]. Our present results shed light onto possible mechanisms of the association of this allele with such diverse human phenotypes. It is now widely accepted that monocyte attachment to cardiovascular wall is the first event implicated in atherogenesis of coronary arteries [28,29]. Since monocytes are known to express both CCR2A and CCR2B [13], an increased stability of CCR2A resulting from the 64I substitution may interfere with the function of CCR2B in monocytes, leading to decreased monocyte invasion to cardiovascular walls. With respect to acute rejection in renal transplantation, CCR5 is known to play an important role in both rejection of renal transplantation [30] and experimental graft-versus-host disease models [31]. Therefore, it is possible that an increased ability of CCR2A-64I to interfere with CCR5 expression can cause a decreased frequency of acute rejection after renal transplantation in recipients with this allele.
Previous studies have failed to show a statistically significant difference in levels of CCR5 expression on stimulated or non-stimulated peripheral blood mononuclear cells between CCR2-64I homozygotes and CCR2-64V homozygotes [9,10,32], although a slight reduction was noted in CCR2-64I homozygotes. In fact, we also failed to observe a statistically significant reduction of CCR5 levels on peripheral CD4 cells of homozygotes of CCR2-64I (data not shown). CCR2 is reported to be expressed on monocytes/macrophages [33], basophils [34,35], B cells [36], NK cells [37], dendritic cells [38,39], and a limited population of T cells [40]. Although we observed very few CCR2 cells in peripheral blood mononuclear cells, Bartoli et al. reported that numerous mononuclear cells in tonsil expressed CCR2A [41]. It may be possible that specific cell types expressing both CCR2A and CCR5 in tonsil or lymph nodes play an important role in AIDS pathogenesis and are responsible for the delay in HIV-1 diseases observed in patients with CCR2-64I.
Acknowledgements
pGIT7 beta-gal was kindly supplied by E. Berger. We thank D. Chao for critical discussion and S. Bando for technical assistance.
Sponsorship: Supported by grants from the Human Science Foundation, the Ministry of Education, Culture, Sports, Science, and Technology, and the Ministry of Health, Labour and Welfare, Japan.
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Keywords:
polymorphism; CCR2-64I; CCR2A; CCR5; stability
© 2004 Lippincott Williams & Wilkins, Inc.