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Laboratory of Virology and Vaccinology, Division of Biomedical Research, National Institute of Biomedical Innovation, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan
Correspondence
Yasuko Mori
ymori{at}nibio.go.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). All human 
-herpesviruses, including human cytomegalovirus (HCMV), human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7), and most characterized 
-herpesviruses encode chemokine-receptor homologues (Gao & Murphy, 1994; Gompels et al., 1995; Nicholas, 2003; van Cleef et al., 2006). Most of these putative viral chemokine receptors have been shown to be dispensable for virus replication in vitro, including HCMV US28 (Vieira et al., 1998), HCMV UL33 (Margulies et al., 1996), mouse CMV (MCMV) M33 (Davis-Poynter et al., 1997), rat CMV (RCMV) R33 (Beisser et al., 1998) and HCMV UL78 (Michel et al., 2005). However, deletion of MCMV M78 reduces virus replication in cultured fibroblasts (Oliveira & Shenk, 2001), whilst RCMV R78 is required for efficient virus production in cell-culture systems (Beisser et al., 1999; Kaptein et al., 2003). Furthermore, deletion studies revealed that RCMV R33 and MCMV M33 exert profound effects on virus replication and pathogenesis in vivo (Beisser et al., 1998; Davis-Poynter et al., 1997) and RCMV R33 has been implicated in the induction of vascular sclerosis and effects on host cells (Streblow et al., 1999, 2005). Recent studies also showed that MCMV M33 is required for MCMV-induced vascular smooth muscle-cell migration (Melnychuk et al., 2005) and HHV-6 U51 regulates virus replication and enhances cell–cell fusion (Zhen et al., 2005). These findings provide a strong indication that virus-encoded chemokine receptors play important roles in the biology of these viruses (Rosenkilde, 2005).
In humans, 18 chemokine receptors have been identified and classified into four groups, CC, CXC, CX3C and C, and their ligand specificities have been defined (Murphy et al., 2000; Zlotnik & Yoshie, 2000). The expression of chemokine receptors on lymphocytes is dependent on their differentiation pathway and maturation. A large number of studies have shown that the migration of lymphocyte classes and subsets and their localization to particular tissue microenvironments, in accordance with their differentiation pathways and maturation stages, are regulated finely through the expression of specific sets of chemokine receptors. CCR4 is expressed in subsets of T cells at particular stages of differentiation and activation (Imai et al., 1997) and CCR7 is expressed at high levels in various lymphoid tissues and on peripheral blood T and B lymphocytes (Schweickart et al., 1994). Migration of several T cells from the blood into lymph nodes is dependent on the expression of CCR7 (von Andrian & Mempel, 2003).
HHV-7 was first isolated from the CD4+ T cells of healthy individuals in 1990 (Frenkel et al., 1990). HHV-7 infection is ubiquitous during childhood and widespread in the general population, and it can frequently be isolated from the saliva of healthy adults (Yoshikawa et al., 1993). The virus persists in T cells after the primary infection and can be reactivated at any time if the host immune system becomes impaired. CXCR4 expression is downregulated and CCR7 expression is induced in CD4+ T cells by HHV-7 infection (Hasegawa et al., 1994; Secchiero et al., 1998; Yasukawa et al., 1999). CD4+ T cells infected with HHV-7 may therefore be attracted into various lymphoid tissues through the cooperation of chemokines and chemokine receptors, and thus promote virus transmission.
The HHV-7 chemokine receptors exhibit the characteristic GPCR structure. HHV-7 U12 and U51 are positional and structural homologues of HHV-6 U12 and U51 and of HCMV UL33 and UL78, respectively (Isegawa et al., 1998; Menotti et al., 1999; Milne et al., 2000). We showed previously that HHV-7 U12 functions as a -chemokine receptor linked to a calcium-mobilizing signal-transduction pathway for CCL19 and CCL22. The cellular chemokine receptor CCR4 binds CCL17 and CCL22. The chemokines CCL19 and CCL21 are ligands for CCR7. We have shown previously that HHV-7 U51 functions as a calcium-mobilizing receptor in response to the binding of the same four chemokines (CCL17, CCL19, CCL21 and CCL22) (Nakano et al., 2003; Tadagaki et al., 2005). These results indicate that HHV-7 U12 and U51 act as functional -chemokine receptors.
HHV-7 infects cells expressing endogenous chemokine receptors. Here, we examined the potential involvement of U12 and U51 in human chemokine receptor activity. By analysing how different chemokine-receptor combinations regulated the cellular response to a specific chemokine, we found that co-expression of U12 with CCR4 or CCR7 in cells was associated with a calcium-mobilization response when exposed to CCL22 or CCL19, but cell migration could not be induced by either CCL22 or CCL19. Co-expression of U51 with CCR4 or CCR7 also induced a calcium-mobilization response when exposed to either CCL22 or CCL19, but again, cell migration was not observed.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Yoshida et al., 1998), were used for intracellular Ca2+ measurements and chemotaxis assay. These cells were designated L1.2-CCR4, L1.2-CCR7 and L1.2-vector, respectively. L1.2 cells were maintained in complete medium [RPMI 1640 medium supplemented with 10 % fetal calf serum (Life Technologies), 200 mM L-glutamine and 50 µM 2-mercaptoethanol].
Constructs and transfection.
The pCEP4-EF expression vectors for HHV-7 U12, U51, FLAG–U12 and FLAG–U51 were constructed as described previously (Tadagaki et al., 2005); the resulting constructs were designated pCEP4-EFU12, pCEP4-EFU51, pCEP4-EF-FLAG-U12 and pCEP4-EF-FLAG-U51 respectively. Plasmid DNA (2 µg) was transfected into 1x106 exponential-phase L1.2 cells by using a Nucleofector device and Nucleofector kit V (Amaxa Biosystems). Transfected cells were cultured in complete medium and, 48 h later, seeded at 105 cells ml–1 in complete medium containing 300 µg hygromycin B ml–1 and selected for 5 days. Subsequently, the L1.2 cells were maintained in complete medium with 250 µg hygromycin B ml–1 to produce L1.2 cells stably expressing U12 or U51. L1.2 cells were cultured as described above.
Intracellular calcium measurements.
L1.2-vector, -CCR4 or -CCR7 cells stably transfected with pCEP4-EF, pCEP4-EFU12 or pCEP4-EFU51 were used for intracellular Ca2+ measurements (Nakayama et al., 2002). The cells were washed twice in Hanks’ balanced salt solution (HBSS) containing 1 mg BSA ml–1 and 10 mM HEPES (pH 7.4). Next, the cells were suspended at 1x107 cells ml–1 and incubated for 1 h at 37 °C in the dark in 1 ml HBSS containing 1 mg BSA ml–1, 10 mM HEPES and 5 µM Indo-1 AM (Dojin Chemical Company). After being washed twice, the cells were suspended at 2.5x106 cells ml–1. Cell suspension (1 ml) was placed in a continuously stirred cuvette at 37 °C in a CAF-110 fluorimeter (Jasco). Fluorescence was monitored at an excitation wavelength of 355 nm and emission wavelengths of 405 and 485 nm; the data are presented as the relative ratio of fluorescence detected at 405 and 485 nm. Data were collected every 10 ms. CCL22 and CCL19 were purchased from PeproTech EC.
Chemotaxis assay.
The chemotaxis assay was carried out by using a chemotaxis chamber with a 5 µm pore size (Kurabo) (Nakayama et al., 2004; Yoshie et al., 2002). The chamber was precoated with type IV collagen (50 µg ml–1) for 3 h at 37 °C. Cells were suspended at 1x107 ml–1 in phenol red-free RPMI 1640 medium containing 1 mg BSA ml–1 (Nakalai Tesque) and 20 mM HEPES (pH 7.4) (chemotaxis assay medium). Cells were applied to the upper well of the chemotaxis chamber (100 µl per well) and chemotaxis assay medium (600 µl per well) with or without chemokines was applied to the lower well. After 4 h at 37 °C, cells that had migrated into the lower well were lysed with 0.1 % Triton X-100 and double-stranded DNA was quantified by using Picogreen (Molecular Probes). Cell migration was expressed as a percentage of input cells. All assays were done in triplicate.
Flow cytometry.
L1.2-vector, -CCR4 and -CCR7 cells stably transfected with pCEP4-EF, pCEP4-EF-FLAG-U12 or pCEP4-EF-FLAG-U51 were resuspended in fluorescence-associated cell sorting (FACS) buffer (1xPBS, 1 % BSA). The cells were fixed with cold acetone for permeabilization or 4 % paraformaldehyde for detection of cell-surface expression. After fixation, the cells were resuspended in 100 µl FACS buffer containing anti-FLAG–fluorescein isothiocyanate (FITC) mAb (40 µg ml–1; Sigma), incubated for 30 min on ice, washed twice and resuspended in 500 µl FACS buffer. The samples were analysed on a FACSCalibur instrument using CELLQuest software (BD Biosciences).
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RESULTS |
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). Stable L1.2 transfectants are used widely in functional studies of human chemokine receptors (Nakayama et al., 2004; Yoshida et al., 1998). We have therefore used L1.2 cells to examine the reciprocal roles of human and HHV-7-encoded chemokine receptors. As shown in Fig. 1(a), L1.2-vector cells stably transfected with pCEP4-EF alone did not respond to either CCL22 or CCL19. L1.2-CCR4 cells stably transfected with pCEP4-EF responded to CCL22, but not to CCL19. L1.2-CCR7 cells stably transfected with pCEP4-EF responded to CCL19, but not to CCL22. L1.2-vector cells stably transfected with U12 or U51 did not respond to either CCL22 or CCL19 (Fig. 1b, c). L1.2-CCR4 or -CCR7 cells stably transfected with U12 or U51 responded to stimulation with each of the non-active ligands for CCR4 or CCR7, CCL22 or CCL19 (Fig. 1b, c).
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). On the other hand, L1.2-CCR4 or -CCR7 cells transfected with U12 or U51 responded to both the first stimulation of each non-active ligand for CCR4 or CCR7 and a subsequent second stimulation with an equal amount of each active ligand for CCR4 or CCR7 (Fig. 1b, c). The results can be summarized as follows: although the active ligands for U12 or U51 were sensitive only to the first stimulation, the active ligands for CCR4 or CCR7 were sensitive to both the first and second stimulations. Treatment with buffer alone did not induce Ca2+ flux in L1.2 cells stably expressing CCR4, CCR7 or empty vector, or in L1.2-CCR4 or -CCR7 cells stably transfected with U12 or U51 (data not shown), indicating that non-specific stimulation did not occur.
Migration of L1.2-CCR4 or -CCR7 cells co-expressing U12 or U51
We next examined the chemotactic responses of L1.2-CCR4 or -CCR7 cells co-expressing U12 or U51. L1.2 cells stably expressing CCR4, CCR7 or vector alone were stably transfected with pCEP4-EF, U12 or U51, and the induction of migration in these cells toward CCL22 or CCL19 was examined (Fig. 2). Although L1.2-vector cells stably transfected with U12, U51 or pCEP4-EF did not respond to either CCL22 or CCL19, L1.2-CCR4 cells stably transfected with U12, U51 or pCEP4-EF responded to CCL22, but not to CCL19, and L1.2-CCR7 cells stably transfected with U12, U51 or pCEP4-EF responded to CCL19, but not to CCL22. These responses showed typical bell-shaped dose–response curves.
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). To analyse this further, the expression levels of U12 and U51 on the cell surface of L1.2-vector, -CCR4 or -CCR7 cells co-expressing FLAG–U12 or FLAG–U51 were examined both before and after cell permeabilization, by flow cytometry using an anti-FLAG–FITC antibody. The expression levels of FLAG–U12 and FLAG–U51 on the surface of the co-expressing cells were low (Fig. 3) and the levels among the total proteins (permeabilized cells) were also low (data not shown). Similar results were obtained when U12 or U51 was expressed alone (Fig. 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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A Ca2+ flux was induced in L1.2-CCR4 or -CCR7 cells co-expressing U12 and U51 by either CCL22 or CCL19, and not just by the active ligands for the cellular receptors, indicating that U12 or U51 was expressed on the cell surface in the presence of CCR4 or CCR7 and responded to each ligand for U12 or U51. Although the expression of FLAG–U12 and FLAG–U51 on the cell surface was not increased in the presence of CCR4 or CCR7 (Fig. 3), induction of the Ca2+ flux by ligand stimulation was increased in L1.2-CCR4 or -CCR7 cells co-expressing FLAG–U12 and FLAG–U51 (data not shown). The results indicate that the cellular receptors and U12 or U51 may be expressed close to each other on the cell surface or form heterodimers, and may accumulate in lipid rafts or other membrane microdomains to induce the conformational change of the receptors and subsequent signal transduction, dependent on ligand-induced stimulation. As a result, U12 or U51 may be able to bind its ligand with high affinity and signal transduction through U12 or U51 may occur via the intracellular domain of the cellular receptor, CCR4 or CCR7.
The induction of Ca2+ flux through U12 or U51 by ligand stimulation only occurred with the first stimulation, i.e. no Ca2+ flux induction by a second stimulation was observed, whereas induction through the cellular receptors was seen with both the first and second stimulations, indicating that downstream signalling through the cellular receptors may play an important role in the signalling of U12 or U51. Once the Ca2+ flux through the cellular receptor occurs, the signal-transduction pathway via the intracellular domain of the cellular receptor might be desensitized, possibly through the same routes and intracellular domains of the cellular receptor. Our findings suggest that the cellular chemokine receptors CCR4 and CCR7 affect U12- and U51-signalling events.
In recent years, oligomerization has been reported for four chemokine receptors, CCR2, CCR5, CXCR2 and CXCR4 (Springael et al., 2005). Recent studies have shown that CCR2 and CCR5 are able to form both homo- and heterodimers (Mellado et al., 2001). CXCR4 was also shown to form heterodimers with CCR2, but not with CCR5 (Babcock et al., 2003; Percherancier et al., 2005). Our study suggests that there may be some interaction between U12 or U51 with CCR4 or CCR7, possibly in terms of heterodimerization. Therefore, to investigate whether U12 or U51 and CCR4 or CCR7 form heterodimers, we co-expressed U12 or U51 and CCR4 or CCR7 in 293T cells, and the interactions between U12 or U51 and CCR4 or CCR7 were examined by immunoprecipitation followed by Western blot. However, an interaction between them was not found (data not shown). Nevertheless, the possibility of heterodimerization between the receptors cannot be excluded; further studies will be required to elucidate the mechanism by which the HHV-7 and cellular chemokine receptors interact functionally.
CCR4 or CCR7 induced the migration of the cells toward their recognized ligands, CCL22 or CCL19, respectively, but neither U12 nor U51 induced migration of the cells toward CCL22 or CCL19 (Fig. 2). In contrast to these results in murine cells, we showed previously that CCL19 induced migration in human Jurkat cells stably expressing U12, although neither CCL19 nor CCL22 induced migration in Jurkat cells stably expressing U51 (Tadagaki et al., 2005). The reason(s) for this apparent discrepancy is not clear. However, Jurkat cells express high levels of CXCR4 and low levels of CCR4, but are CCR7-negative (Yoshie et al., 2002), and may express other chemokine receptors. One possibility is that endogenous factors expressed in Jurkat cells, including chemokine receptors, may influence cell migration. Alternatively, differences in the expression, turnover and stability of the U12 proteins or in the intracellular environment could potentially impact on cell migration (Bradel-Tretheway et al., 2003).
Previously, stimulation of U12- or U51-expressing cells with either CCL19 or CCL22 was shown to elicit Ca2+ mobilization (Tadagaki et al., 2005). In this study, there were small Ca2+ peaks following initial stimulation (Fig. 1b, c). An explanation for the apparent discrepancy between the studies may also lie in the use of different cell types. Further experiments will be required to elucidate the reasons for the different cell responses.
Our study shows co-functioning of the cellular receptors CCR4 or CCR7 with HHV-7 U12 or U51 in host cells. Although the virus-encoded receptors apparently did not promote migration in the co-expressing cells, U12 and U51 appeared to contribute to the induction of Ca2+ flux by ligand stimulation in the cells. In conclusion, human chemokine receptors may be associated with expression on the cell surface and function of HHV-7 U12 and U51. The relationship of U12 and U51 with CCR4 and CCR7 needs to be investigated further. U12 and U51 may regulate signalling events in HHV-7-infected cells, given that signalling by virus-encoded GPCRs is involved in the pathogenesis of human disease (Couty & Gershengorn, 2005; Kirshner et al., 1999).
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ACKNOWLEDGEMENTS |
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Received 25 October 2006;
accepted 25 January 2007.
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) or CCL19 (
) and chemotaxis assays were performed in a Transwell chemotaxis chamber. The results are shown as the percentage of the total input cells that migrated and are representative of three independent experiments.