Lewis L. Lanier,§ Takashi Saito,¶ and Hisashi Arase2*†
Human and rodent CD200 are recognized by the inhibitory CD200R, and these molecules play an important role in the regulationof the immune system. Several viruses, such as human herpesvirus-6 (HHV-6), HHV-7, and HHV-8, possess a CD200 homologue,suggesting that these viruses regulate the immune response via CD200R. In this study, we analyzed the effect of human CD200 andthe viral CD200 homologues on human CD200R-expressing cells. We found that human CD200R is predominantly expressed onbasophils in amounts higher than on other human peripheral blood leukocytes. Furthermore, the viral CD200 homologues as wellas human CD200 were recognized by human CD200R, and the activation of basophils was down-regulated by these CD200proteins. These results suggested that CD200R is an important regulatory molecule of basophil activation. In addition, thepresence of CD200 homologues on several viruses suggests a potentially unique relationship between basophil function and viralinfection. The Journal of Immunology, 2005, 175: 4441–4449.
I mmune cells express inhibitory receptors to prevent damageto self. Many of the inhibitory receptors recognize broadlyexpressed self-proteins, such as MHC class I (1, 2). CD200R
(3) is an inhibitory receptor that possesses a tyrosine phosphatase-recruiting inhibitory motif in its cytoplasmic domain (3, 4).CD200R is primarily expressed on leukocytes of the myeloid lin-eage, and it recognizes CD200, which is broadly expressed on avariety of cell types (3, 5, 6). In mice, disruption of the geneencoding CD200 led to expansion of the macrophage and granu-locyte populations in the spleen and the macrophage population inmesenteric lymph nodes (7). Furthermore, CD200-deficient miceshowed rapid onset of experimental autoimmune encephalomyeli-tis (7). Administration of CD200-Ig fusion protein, which func-tions as an agonist for the inhibitory CD200R, suppressed allograftrejection and collagen-induced arthritis in animal models (8, 9).These data suggested that recognition of CD200 by an inhibitoryCD200R plays an important role in regulating immune responses.
Several viruses that persistently infect the host have acquiredligands for inhibitory receptors, presumably to suppress an im-mune response against these pathogens. For example, murineCMV (MCMV)3 has evolved m157, which functions as a ligand
for an inhibitory Ly49 receptor in certain MCMV-susceptiblemouse strains (10). Similarly, human CMV acquired UL18 as aligand for the inhibitory CD85J (leukocyte inhibitory receptor/Ig-like transport) receptor (11). These decoy ligands for inhibitoryreceptors may play an important role, allowing the viruses to evadethe immune system and potentially cause persistent infectious dis-eases (12). Interestingly, herpesviruses, such as human herpesvi-rus-6 (HHV-6), HHV-7, and HHV-8 (13–15), and poxviruses, in-cluding myxoma virus and shope fibroma virus, possess genesencoding proteins with homology to CD200 (16, 17). Althoughamino acid homologies between human CD200 and these viralCD200 homologues are �40%, this level of similarity is sufficientto permit binding to the inhibitory CD200R. In fact, the CD200homologue of HHV-8 (HHV-8 CD200) has been described as adecoy ligand for CD200R (18, 19). However, the effect of theHHV-8 CD200 on CD200R-expressing human cells has remainedunclear, and there are conflicting reports in the literature (18, 19).
In the present study we show that basophils are a majorCD200R-positive population in human peripheral blood. Further-more, we show that not only the HHV-8 CD200, but also theCD200 homologues of HHV-6 (HHV-6 CD200) and HHV-7(HHV-7 CD200), are recognized by CD200R and that activation ofbasophils is down-regulated by recognizing human CD200 andHHV-8 CD200. These findings suggest that CD200R regulatesbasophil activation, and the viral CD200 homologues are involvedin the regulation of basophil-dependent immune responses.
Materials and MethodscDNA preparation
CD200 homologues of HHV-7 (U85) and HHV-8 (K14) were cloned usingPCR from genomic DNA of HHV-7 and HHV-8 (provided by Dr. L.Coscoy, University of California, Berkeley, CA). The CD200 homologueof HHV-6 (U85) was cloned using PCR directly from supernatant contain-ing HHV-6 virus (strain SF; provided by Dr. L. Coscoy). The GenBank/EMBL/DDBJ accession numbers of HHV-6, HHV-7, and HHV-8 areAB190768, HHU43400, and AF367765, respectively. Human CD200 andCD200R were cloned using PCR from cDNA generated from humanPBMC-derived poly(A)� RNA. Mouse CD200R was cloned from cDNAgenerated from mouse spleen-derived poly(A)� RNA.
*Department of Immunochemistry, Research Institute for Microbial Diseases, OsakaUniversity, Osaka, Japan; †Precursory Research for Embryonic Science and Tech-nology, Japan Science and Technology Agency, Saitama, Japan; ‡Department of Al-lergy and Rheumatology, Graduate School of Medicine, University of Tokyo, Tokyo,Japan; §Department of Microbiology and Immunology and the Cancer Research In-stitute, University of California, San Francisco, CA 94143; ¶Laboratory for Cell Sig-naling, RIKEN Research Center for Allergy and Immunology, Kanagawa, Japan
Received for publication May 24, 2005. Accepted for publication July 20, 2005.
The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by a Grant-in-Aid for Scientific Research from the Min-istry of Education, Science, and Culture, Japan (to T.S. and H.A.), and the UeharaMemorial Foundation (to H.A.). L.L.L. is an American Cancer Society ResearchProfessor and is supported by National Institutes of Health Grant CA89294.2 Address correspondence and reprint requests to Dr. Hisashi Arase, Department ofImmunochemistry, Research Institute for Microbial Diseases, Osaka University, 3-1Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail address: [email protected] Abbreviations used in this paper: MCMV, murine CMV; HHV, human herpesvirus;HHV-6 CD200, CD200 homolog of HHV-6; HHV-7 CD200, CD200 homolog ofHHV-7; HHV-8 CD200, CD200 homolog of HHV-8; PIPES-A, PIPES-Albumin.
cDNA fragments corresponding to the extracellular domains of humanCD200R (aa residues 25–267), mouse CD200R (aa residues 26–238), hu-man CD200 (aa residues 56–263), and HHV8-CD200 (aa residues 25–226)were cloned into a unique XhoI site of a modified pME18S expressionvector carrying the human CD150 leader segment and the Fc segment ofhuman IgG1 (20). COS-7 cells were transiently transfected by using293fectin (InvitroGen) with these expression vectors to generate Ig fusionproteins. After 72 h, the culture supernatants were collected, and theamounts of Ig fusion protein were measured using standard ELISA meth-ods. For some experiments, human CD200-Ig and HHV-8-CD200-Ig fu-sion proteins were further purified using protein A-coupled Sepharose col-umns (Amersham Biosciences) by standard methods.
Cells and transfectants
FLAG-tagged human CD200, HHV-6-CD200, HHV7-CD200, and HHV-8-CD200 were transfected into Ba/F3 cells using the pMx-neo retrovirusvector, which contains a human CD8� signal sequence and a FLAG tag atthe N terminus of the mature protein (20). These transfectants were stainedusing anti-FLAG mAb (clone M2; Sigma-Aldrich), and FLAG-positivecells were isolated by using a MACS purification system (Miltenyi Biotec).Human CD200 and the open reading frame of the HHV-8 CD200 were alsotransfected into 721.221 cells using the pMx-IRES-GFP retrovirus vector,and GFP-positive cells were purified using a flow cytometer (FACSVan-tage; BD Biosciences). Human CD200R was also transfected into the hu-man NK cell line NKL (21) using the pMx-puro retrovirus vector, and cellsstained by human CD200-Ig fusion protein were purified using flow cy-tometry (FACSVantage). BC1, a human primary effusion lymphoma cellline harboring HHV-8, was provided by Dr. K. Ueda (Osaka University,Osaka, Japan). We recloned BC1 cells and selected cells that were highlystained with human CD200R-Ig fusion protein after induction of lytic re-activation by PMA (20 ng/ml) for 24 h. All cells were cultured in RPMI1640 medium containing 10% FCS, except for NKL cells, in which case400 U/ml human IL-2 (PeproTech) was added to the culture medium.
Human basophils used for the degranulation assay were purified fromEDTA-treated (to prevent coagulation) venous blood using Percoll densitycentrifugation (Amersham Biosciences) (22). Basophils were further puri-fied using a MACS basophil isolation kit (Miltenyi Biotech) for measure-ment of CD200R expression. The purities of Percoll- and MACS-purifiedbasophils determined by Alcian blue staining (23) were 8–30 and �95%,respectively. Human CD3-, CD14-, CD19-, and CD56-positive lympho-cytes were purified from human PBMC using a MACS purification system(Miltenyi Biotech), and the purity of each population was �90%.
Flow cytometry
Cells were incubated with saturating concentrations of various Ig fusionproteins or mAbs for 30 min on ice, followed by incubation with F(ab�)2
of PE-conjugated goat anti-human IgG or anti-mouse IgG (Jackson Immu-noResearch Laboratories) for 30 min. For analyses of CD200R expressionon human PBMC, we generated a PE-labeled Ig fusion protein complex bymixing 10 �g/ml Ig fusion protein with 4 �g/ml PE-conjugated goat anti-human IgG (Jackson ImmunoResearch Laboratories) for 30 min on ice,followed by the addition of 30 �g/ml normal human IgG (Cappel Labo-ratories) for 30 min. Human PBMC were pretreated with 15 �g/ml normalhuman IgG for 15 min on ice and stained with the PE-labeled Ig fusioncomplexes and various FITC-conjugated mAbs for 30 min. CD11b expres-sion on basophils was analyzed by staining with PE-conjugated anti-human-CD11b mAb (Bear1; Coulter Immunotech) and FITC-conjugatedanti-human IgE Ab (BioSource International) for 60 min on ice. Stainedcells were analyzed using a FACSCalibur (BD Biosciences), and the me-dian values of fluorescence intensity were converted to the number of mol-ecules of equivalent soluble fluorochrome units using a quantum fluores-cent microbead standard for PE (Sigma-Aldrich), as described previously(24). FITC-conjugated anti-CD3 (BW264/56), FITC-conjugated anti-CD14(TUK4),FITC-conjugatedanti-CD16(VEP13),FITC-conjugatedanti-CD19 (LT19), and FITC-conjugated anti-CD123 (AC145) mAbs were pur-chased from Miltenyi Biotec. FITC-conjugated anti-CD56 (MEM188) andanti-HLA-A,B,C (G46-2.6) mAbs were purchased from BD Pharmingen.
Real-time PCR analysis
cDNA generated from total RNA of human CD-3, CD14-, CD19-, andCD56-positive leukocytes and MACS-purified human basophils, eosino-phils, and neutrophils (25) were used as templates. Real-time PCR analysiswas performed using a SYBR Green PCR kit (Applied Biosystems) and anABI PRISM 7900 HT instrument (Applied Biosystems). Primers used foramplification were as follows: �-actin: sense primer, 5�-TCTACAAT
GAGCTGCGTGTG-3�; antisense primer, 5�-CGTAGATGGGCACAGTGTGG-3�; GAPDH: sense primer, 5�-ATGCTGGCGCTGAGTACGTC-3�; antisense primer, 5�-CAGGGGTGCTAAGCAGTTGGT-3�; and humanCD200R: sense primer, 5�-CTTCCTGTTCCAGGTGCCAAA-3�; anti-sense primer, 5�-GCCTCAGATGCCTTCACCTTG-3�. �-Actin andGAPDH were used to standardize the relative amounts of CD200 tran-scripts in the different cell populations. Comparable results were obtainedwhen either �-actin or GAPDH was used for normalization of the data.
Histamine release assay
Histamine release from basophils was analyzed as previously described(22). Purified basophils (2.0–4.0 � 105) were incubated with 10 �g/mlhuman CD200-Ig, HHV-8 CD200-Ig, or control-Ig fusion proteins inPIPES-Albumin (PIPES-A) buffer containing 25 mM PIPES, 119 mMNaCl, 5 mM KCl, and 0.03% human serum albumin (pH 7.4) for 30 minon ice. Cells were then washed in ice-cold PIPES-A and incubated with 5�g/ml F(ab�)2 of goat anti-human IgG Fc� Ab (Jackson ImmunoResearchLaboratories) in PIPES-A for 30 min on ice. Basophils were then washedin ice-cold PIPES-A, resuspended at 4.0–8.0 � 104 basophils/ml inPIPES-A buffer containing 2 mM Ca2� and 0.5 mM Mg2� (PIPES-ACM),and stimulated with 1 �g/ml mouse anti-human Fc�RI mAb (CRA1,Kyokuto Pharmaceutical Industry) or 300 pM human rIL-3 (donated byKirin Brewery) for 45 min at 37°C. For coculture experiments, purifiedbasophils (2.0–4.0 � 105) were suspended with 1 � 106 of mock-trans-fected or CD200-transfected 721.221 cells in PIPES-ACM and were cen-trifuged at 4°C, followed by incubation for 30 min on ice. Cells were thenresuspended at 4.0–8.0 � 104/ml in PIPES-ACM containing 1 �g/mlmouse anti-human Fc�RI mAb or 300 pM human rIL-3 and then centri-fuged at 4°C, followed by incubation for 45 min at 37°C. The concentrationof histamine in supernatants was measured using an automated fluoromet-ric analyzer. Histamine release was calculated as a percentage of the totalhistamine content, as previously described (26).
Cytotoxic assays51Cr-labeled, mock-transfected or CD200-transfected 721.221 cells (1 �105/well) were cocultured with various numbers of mock-transfected orCD200R-transfected NKL cells in 96-well, round-bottom plates for 4 h.For Ab-blocking experiments, CD200-transfected 721.221 cells were in-cubated with anti-human CD200 mAb (MRC OX-104; BD Pharmingen) oran isotype-matched control mAb for 30 min on ice before the coculturewith effector cells. Specific lysis (percentage) was analyzed by 51Cr releaseassays using standard methods (20).
Cytokine production
We cocultured 3 � 105 mock-transfected or CD200R-transfected NKLcells with various numbers of mock-transfected or CD200-transfected721.221 cells in 96-well, round-bottom plates. Culture supernatants werecollected after 24 h, and concentrations of IFN-� in the supernatants weredetermined using a human IFN-� ELISA kit (BD Pharmingen). To analyzethe response against HHV-8-infected cells, BC1 cells were stimulated with20 ng/ml PMA for 24 h. Thereafter, PMA-stimulated BC1 cells werewashed four times and cocultured with 3 � 105 of mock-transfected orCD200R-transfected NKL in 96-well, round-bottom plates for 24 h.
ResultsSpecific binding of soluble CD200R to viral CD200 homologuesof HHV-6, -7, and -8
Some herpesviruses and poxviruses possess CD200 homologues(13–15, 17), for example, HHV-6, -7, and -8. These viral CD200homologues are thought to serve as decoy ligands for the inhibi-tory CD200R (19). To analyze the function of the CD200 homo-logues of HHV-6, -7, and -8, we stably transduced these viralgenes into mouse pro-B Ba/F3 cells using retroviruses. We addeda FLAG epitope tag at the N terminus of the viral CD200 homo-logues to permit detection of these proteins. Ba/F3 cells stablyexpressing human CD200 or the viral CD200 homologues on thecell surface were purified using Ab-coupled magnetic beads. Fig.1A shows the levels of FLAG-tagged human CD200 or viralCD200-like molecules expressed on these transfectants.
We then analyzed whether Ig fusion proteins of human andmouse CD200R recognize these viral CD200 homologues. Asshown in Fig. 1B, human and mouse CD200R-Ig specifically
bound to cells expressing human CD200. The fluorescence inten-sity of cells stained with mouse CD200R-Ig was 1/10th the fluo-rescent intensity of cells stained with human CD200R-Ig. Thisresult is consistent with findings of a prior study that demonstrateda lower affinity of mouse CD200R compared with human CD200Rfor human CD200 measured using a Biacore (Biacore International)(6). When cells transduced with the viral CD200 homologues wereanalyzed, all transductants expressing the viral CD200 ho-mologues were stained with human CD200R-Ig. In contrast,mouse CD200R-Ig did not recognize these viral CD200 homo-logues despite the fact that the homologies of human and mouseCD200 to the viral CD200 homologues are almost the same(amino acid homologies of the viral CD200 proteins of HHV-6, -7,and -8 to human CD200 are 24, 24, and 31%, respectively,whereas those to mouse CD200 are 16%, 24, and 32%, respec-tively). In addition, mouse CD200R-Ig recognized not only mouseCD200, but also human CD200 (Fig. 1B and data not shown).Anti-CD200 mAb recognized human CD200, but not the viralCD200 homologues (Fig. 1B). These results suggested that thesehuman herpesviruses might have acquired CD200 homologues asligands for the human inhibitory CD200R.
Specific expression of CD200R on basophils in humanperipheral blood
Recent studies have suggested that CD200R is expressed on mosthuman leukocytes, including T cells, monocytes, granulocytes, anda small subset of NK cells (6). However, the expression level ofCD200R on these populations was relatively low, and the functionof CD200R on these cells has not been evaluated (6). To study thecells expressing CD200R, we stained peripheral blood leukocyteswith CD200-Ig and HHV-8 CD200-Ig fusion proteins in combi-nation with various lineage-specific mAbs. As shown in Fig. 2A,�1% of the cells were brightly stained with the CD200-Ig fusionprotein. Most cells binding to the CD200-Ig fusion protein werecostained with anti-CD123 mAb, but not with anti-CD3, -CD14,-CD16, -CD19, or -CD56 mAb. Similar results were obtainedwhen cells were stained with the HHV-8 CD200-Ig fusion protein.
This suggested that T cells, B cells, NK cells, and monocytes ex-press lower levels of CD200R than the CD123-positive population.Because basophils are the main population strongly expressingCD123 in peripheral blood leukocytes, we purified basophils andstained them with the human CD200-Ig and HHV-8 CD200-Igfusion proteins (Fig. 2B). As predicted, all basophils were stainedwith the CD200-Ig fusion protein. In contrast, eosinophils and neu-trophils were not stained by the CD200-Ig fusion protein (Fig. 2B).Granulocytes have been reported previously to express CD200R,as detected using an anti-human CD200R mAb (6), indicating thatthe CD200-Ig fusion and HHV-8 CD200-Ig fusion proteins prob-ably stain cells with only the highest amounts of CD200R. Anti-human CD200R mAb was not available for a direct comparisonwith the fusion protein staining; however, the mAb almost cer-tainly has a higher affinity than the fusion proteins and would becapable of detecting cells expressing lower levels of CD200R.Nonetheless, because CD200-Ig and HHV-8 CD200-Ig specifi-cally bind to CD200R, these data suggested that basophils repre-sent a major CD200R-positive cell type in human peripheral blood.Next, to confirm that CD200R is expressed on basophils, we pu-rified each leukocyte population and analyzed the expression ofCD200R transcripts by real-time PCR. As shown in Fig. 2C, theexpression of CD200R transcripts in basophils was substantiallyhigher than that in other populations. When the nucleic acid se-quence of the CD200R cDNA expressed in basophils was ana-lyzed, there was no difference in the sequence from that of theCD200R cDNA previously reported (GenBank accession no.AY284975; data not shown). These data indicated that basophilsare a major population in peripheral blood expressing CD200R.
Human CD200- and HHV-8 CD200-expressing cells suppresseffector functions of basophils through inhibitory CD200R
Degranulation of basophils is correlated with the pathogenesis ofallergic disorders. Engagement of Fc�RI with anti-Fc�RI mAb in-duces CD11b up-regulation and histamine release from basophils(27). We analyzed the function of CD200R on basophils by cross-linking the receptor with human CD200-Ig and HHV-8 CD200-Ig.
FIGURE 1. Recognition of viral CD200 homologuesby human inhibitory CD200R. FLAG-tagged CD200homologues of HHV-6, -7, and -8, as well as humanCD200, were transduced into Ba/F3 cells. Parental cellsand transduced cells were stained with anti-FLAG mAb(A), human CD200R-Ig fusion protein, mouseCD200R-Ig fusion protein, and anti-human CD200mAb (solid line; B). Histograms of cells stained with acontrol mAb or a control-Ig fusion protein were overlaid(dotted line).
As shown in Fig. 3, cross-linking CD200R with human CD200-Ig,but not with control-Ig, clearly suppressed both CD11b up-regu-lation and histamine release of basophils induced by the engage-ment of Fc�RI. Cross-linking CD200R with HHV-8 CD200-Igalso suppressed Fc�RI-dependent CD11b up-regulation and hista-mine release. In contrast, IL-3 induced up-regulation of CD11bexpression, but not histamine release (27, 28). Human CD200-Ig
and HHV-8 CD200-Ig showed negligible effects against IL-3-de-pendent CD11b up-regulation on basophils.
Next, we analyzed then function of CD200R on basophils usingCD200 transfectants. We transfected human CD200 or the HHV-8CD200 into human B lymphoblastoid cell line 721.221 cells. Al-though 721.221 cells express low amounts of endogenous CD200on the cell surface, the level of CD200 expression was greatly
FIGURE 2. Expression of CD200R on basophils. A, PBMC were costained with control Ig, human CD200-Ig, or HHV-8 CD200-Ig (vertical axis) incombination with anti-CD3, -CD14, -CD16, -CD19, -CD56, and -CD123 mAbs (horizontal axis). The proportion of each population is indicated in thefigure. B, Neutrophil- and eosinophil-enriched human blood leukocytes (Neu/Eos) and MACS-purified basophils were stained with control-Ig (dotted line),human CD200-Ig, or HHV-8 CD200-Ig (solid line). C, Transcripts of CD200R in purified leukocyte populations were analyzed by real-time PCR.Transcription levels of CD200R were normalized by comparison with those of �-actin (or GAPDH, not shown), and the relative amounts of CD200Rtranscripts are shown. Representative data from four different healthy blood donors are shown.
FIGURE 3. Suppression of basophil activation byhuman CD200 and HHV-8 CD200. Basophils were in-cubated with human CD200-Ig, HHV-8 CD200-Ig, orcontrol-Ig fusion proteins, followed by addition ofF(ab�)2 of goat anti-human IgG Ab. Cells were thenstimulated with anti-human Fc�RI mAb or human IL-3,and CD11b expression on basophils and concentrationsof histamine in the supernatants were measured. Dataare shown as the mean � SD.
increased when these cells were transduced with CD200 (Fig. 4A).The human CD200-transfected 721.221 cells were stained brightlyby both CD200R-Ig fusion protein and anti-human CD200 mAb.721.221 cells transfected with the HHV-8 CD200 were alsostained brightly by the CD200R-Ig fusion protein, but not by anti-human CD200 mAb. Basophils were cocultured with humanCD200 or HHV-8 CD200-transfected 721.221 human B lympho-blastoid cells, and Fc�RI on basophils was cross-linked with ananti-Fc�RI mAb (Fig. 4B). Both CD11b up-regulation and hista-mine release induced by engagement of Fc�RI were significantlydown-regulated in the presence of human CD200-transfected721.221 cells and HHV-8 CD200-transfected 721.221 cells, butwere not down-regulated in the presence of mock-transfected721.221 cells. These data suggested that the CD200 homologue ofHHV-8 as well as human CD200 suppress Fc�RI-dependent acti-vation of basophils through CD200R.
HHV-8 CD200 down-regulates activation of CD200R-transfected NK cells
To analyze the function of CD200R, we used a human NK cellline, NKL, as a model system to validate the inhibitory function ofthe CD200R when it is engaged by its human CD200 or viralCD200 ligands. We transduced human CD200R into NKL, whichlacks endogenous expression of CD200R (Fig. 5A). We then an-alyzed the cytolytic activity of NKL or human CD200R-trans-duced NKL cells against CD200-transfected 721.221 target cells.Mock-transfected NKL cells showed high and equivalent levels ofcytotoxicity against 721.221 and the CD200 transfectants (Fig.5B). By contrast, human CD200R-transfected NKL cells showeddiminished cytolytic activity against parental 721.221 cells (prob-ably due to expression of endogenous CD200) and negligible cy-totoxicity against 721.221 cells transfected to express highamounts of human CD200 or the HHV-8 CD200 (Fig. 5B). Cyto-
toxicity mediated by human CD200R-expressing NKL cellsagainst parental 721.221 cells was augmented when anti-CD200mAb was added to the assay, indicating that endogenous CD200expressed by 721.221 is functional (Fig. 5C). Moreover, CD200R-transfected NKL cells did not kill human CD200-transfected721.221 cells expressing high amounts of CD200 (Fig. 5B); how-ever, cytotoxicity was restored when anti-CD200 mAb was addedto the assay (Fig. 5C). This indicated that interactions betweenCD200R on the effector NK cells and CD200 on the target cellsdown-regulated the lytic function of NKL cells. Similarly,CD200R-transfected NKL cells showed negligible cytotoxicityagainst 721.221 cells transfected with HHV-8 CD200 (Fig. 5B).However, addition of anti-human CD200 mAb did not restore thecytotoxicity mediated by human CD200R-transfected NKL cellsbecause the anti-human CD200 mAb does not recognize HHV-8CD200 (Fig. 5C). Essentially identical data were obtained whenIFN-� production by NKL was analyzed (Fig. 5D). HumanCD200R-expressing NKL cells did not express substantialamounts of cytokine in response to human CD200 or HHV-8CD200-transfected target cells. These data indicated that humanCD200 and the HHV-8-CD200 expressed on the target cells down-regulated the effector functions of CD200R-expressing effectorcells.
HHV-8 infected cells down-regulate activation of CD200R-transduced NKL cells
We analyzed the function of HHV-8 CD200 using the HHV-8persistently infected cell line, BC1. BC1 cells express only limitedHHV-8 viral gene products. However, BC1 cells express severalviral proteins when lytic reactivation is induced by PMA stimula-tion (29). As shown in Fig. 6A, nonstimulated BC1 cells were onlyweakly stained by human CD200R-Ig, and BC1 cells were notstained by anti-human CD200 mAb. By contrast, when BC1 cells
FIGURE 4. Suppression of basophil activation byhuman and HHV-8 CD200-expressing cells. A, HumanCD200 and HHV-8 CD200 were transduced into721.221 B lymphoblastoid cells. Mock or CD200 trans-fectants were stained with human CD200R-Ig fusionprotein or anti-human CD200 mAb (solid line) and acontrol mAb or control-Ig fusion protein (dotted line).B, Basophils were cocultured with mock, humanCD200, or HHV-8 CD200-transduced 721.221 cells andstimulated with anti-human Fc�RI mAb or human IL-3.CD11b expression on basophils and concentrations ofhistamine in the supernatants were measured. Represen-tative data from more than three independent experi-ments using different healthy blood donors are pre-sented. Data are shown as the mean � SD.
were stimulated with PMA, most BC1 cells were stained withCD200R-Ig, but not with anti-human CD200 mAb, 1 d after stim-ulation. The K3 and K5 proteins of HHV-8 are well known todown-regulate cell surface expression of MHC class I proteins (30,31). Indeed, the amount of MHC class I expression on BC1 cellswas significantly decreased 2 d after stimulation. These data sug-gested that HHV-8 CD200 was expressed on the cell surface afterPMA-induced reactivation.
We cocultured parental (i.e., CD200R-negative) NKL cells withnonstimulated or PMA-stimulated BC1 cells and analyzed IFN-�production. Because lytic reactivation was observed after 1-d stim-ulation with PMA (Fig. 6A), we stimulated BC1 cells with PMAfor 1 d, washed the cells extensively, and cocultured them withparental NKL cells for an additional day. NKL did not produceIFN-� when these cells were cocultured with unstimulated BC1cells (Fig. 6B); however, NKL produced a large amount of IFN-�
when they were cocultured with PMA-stimulated BC1 cells. Cul-ture supernatants of PMA-stimulated BC1 cells did not activateNKL cells. In addition, BC1 itself did not produce IFN-� uponPMA stimulation (data not shown). These data suggested thatPMA-stimulated BC1 cells directly activated NKL cells. However,the activation of NKL was not specific to HHV-8-infected BC1cells, because other B lymphoblastoid cell lines, such as the EBV-transformed 721.221 and C1R cell lines or the EBV-negativeBJAB cell line, also stimulated NKL after PMA stimulation ofthese B cell lines (data not shown). Nevertheless, this system issuitable to analyze the function of the HHV-8 CD200 expressed onHHV-8-infected cells, because NKL seemed to be activated bydirect recognition of PMA-stimulated BC1 cells.
We analyzed IFN-� production by mock-transduced orCD200R-transduced NKL cells upon coculture with PMA-stimu-lated BC1 cells. Mock-transduced NKL cells produced significant
FIGURE 5. HHV-8 CD200 down-regulates activa-tion of CD200R-transduced NK cells. A, Mock-trans-fected or human CD200R-transduced NKL cells werestained with a control-Ig (dotted line) or humanCD200-Ig fusion protein (solid line). B, The cytotoxicityof mock-transduced (E) or human CD200R-transduced(F) NKL cells against mock-transduced (left), humanCD200-transduced (middle), or HHV-8 CD200-trans-duced (right) 721.221 cells at the indicated E:T cell ratiois shown. C, The cytotoxicity of CD200R-transfectedNKL cells against CD200-transfected 721.221 in thepresence of anti-human CD200 mAb (F) or a controlmAb (E) is shown. D, Mock-transduced (E) or humanCD200R-transduced (F) NKL cells were coculturedwith CD200-transduced 721.221 at the indicated cellnumber for 24 h. Concentrations of IFN-� in the culturesupernatants are shown. Representative data from morethan three independent experiments are shown.
amounts of IFN-� after coculture with PMA-stimulated BC1 cells,but not after culture with unstimulated BC1 cells (Fig. 6C). Pro-duction of IFN-� by CD200R-expressing NKL cells after cocul-ture with PMA-stimulated BC1 cells was significantly lower thanthat by mock-transfected NKL cells. There were no significantdifferences in cytokine production between the mock and CD200Rtransfectants when these transfectants were directly stimulatedwith PMA (data not shown). Because BC1 cells do not expressCD200, these data suggested that the CD200 homologue ofHHV-8 expressed on PMA-stimulated BC1 cells down-regulatesthe activation of CD200R-expressing effector cells.
DiscussionIn the present study we found that basophils are a major populationexpressing CD200R in human peripheral blood. Recently,CD200R has been reported to be expressed on various populations,including granulocytes, macrophages, and dendritic cells, as de-tected using an anti-CD200R mAb (6, 32, 33). However, we couldnot detect CD200R expression on peripheral blood cells other thanbasophils using CD200-Ig fusion protein, most likely because thefusion protein has a lower affinity than the anti-CD200R mAb andtherefore detects only cells with the highest amounts of CD200R.Although our studies indicate that basophils express higher levelsof CD200R than other PBL, the broad distribution of CD200Rindicates that CD200R may regulate the immune response of many
different hemopoietic cell populations. Furthermore, the amountsof CD200R on leukocytes in tissues and organs other than periph-eral blood need to be evaluated to determine the potential role forthis inhibitory receptor in regulating immune responses.
We showed that engagement of CD200R by its ligand resultedin the down-regulation of Fc�RI-dependent activation of basophils.These findings suggest that CD200R may play an important role inthe regulation of basophil function. Recently, the inhibitoryCD200R has been reported to regulate the activation of mousebone marrow-derived mast cells and human cultured mast cells (4,33). Although mast cells are similar to basophils in their array ofinflammatory mediators and cell surface expression of Fc�RI, theirtissue distributions and developmental pathways are quite differ-ent. Indeed, mature mast cells do not exist in blood leukocytes, incontrast to basophils. Although basophils bear a closer develop-mental relationship to eosinophils than to mast cells, eosinophilsdo not stain with CD200-Ig or HHV-8 CD200-Ig fusion proteins(Fig. 4B). Therefore, basophils are a unique leukocyte subpopula-tion in human peripheral blood brightly expressing CD200R. Incontrast, because basophils can migrate into tissues during an al-lergic response, CD200R expressed on basophils as well as onresident mast cells might function as a negative regulator of anallergic response in local tissue sites. Therefore, analysis of thefunction of CD200R expressed on cells at the site of an allergicresponse would be informative.
FIGURE 6. HHV-8-infected cells down-regulate ac-tivation of CD200R-expressing effector cells via a viralCD200 homologue. A, Expression of HHV-8 CD200 onHHV-8-infected cells. Lytic reactivation was inducedby stimulation of BC1 cells with PMA. Nonstimulated(day 0) or PMA-stimulated (days 1 and 2) BC1 cellswere stained with anti-human CD200 mAb; anti-humanHLA-A, -B, -and -C; or human CD200R-Ig fusion pro-tein (solid line) or with a control-IgG (dotted line). B,NKL cells were cocultured with unstimulated BC1 cells(E) or with PMA-stimulated BC1 cells (F) at the indi-cated cell number for 24 h. NKL cells were also culturedin the presence of culture supernatants of PMA-stimu-lated BC1 cells (1:1 dilution; gray circles). C, Mock-transduced (E) or human CD200R-transduced (F) NKLcells were cocultured with unstimulated BC1 cells (left)or PMA-stimulated BC1 cells (right) at the indicatedcell number for 24 h. Concentrations of IFN-� producedin the culture supernatants are shown. All data are pre-sented as the mean � SD (nanograms per milliliter).Representative data from three independent experimentsare shown.
Not only herpesviruses, but also several other viruses, such aspoxviruses, possess viral CD200 homologues. However, the func-tions of the CD200 homologues of most viruses have not beenestablished. In the present study, we demonstrate that the CD200homologues of HHV-6, -7, and -8 are ligands for the human in-hibitory CD200R. Furthermore, we show that viral CD200 as wellas human CD200 down-regulate the activation of basophils andCD200R-transduced NKL cells. We have shown that not onlyHHV-8 CD200-transfected cells but also HHV-8-infected cellsdown-regulate the activation of CD200R-bearing effector cells.These functional analyses suggested that these viral CD200 ho-mologues might play an important role in down-regulating an an-tiviral immune response. In other words, these data suggested thatHHV-6, -7, and -8 might have acquired CD200 viral homologuesto regulate the function of basophils in addition to other CD200R-bearing immune cells.
Regarding the function of the CD200 homologue of HHV-8,two conflicting papers have been published. Chung et al. (18) pre-pared soluble HHV-8 CD200 homologue as a GST fusion proteinand showed that the soluble HHV-8 CD200 homologue activatedhuman monocytes. In contrast, Foster-Cuevas et al. (19) reportedthat transfectants expressing the HHV-8 CD200 suppressed theactivation of monocytes, similar to our findings presented in thisstudy. This discrepancy might be due to the manner in which theHHV-8 CD200 protein was prepared in the different studies. Pro-duction of HHV-8 CD200 by transfection in mammalian cells maybetter mimic the structure of the protein in virus-infected cells,because it will be properly folded and glycosylated. In contrast, theGST fusion protein would not be glycosylated and could be con-taminated with bacterial products, which might inadvertently ac-tivate macrophages.
A number of recent reports have suggested that drug-inducedhypersensitivity syndrome, characterized by serious adverse sys-temic reactions in addition to skin rash, is associated with thereactivation of human herpesviruses, mainly HHV-6 (34, 35). In-deed, in this case, viral infection exacerbates allergic diseases.However, because basophils highly express inhibitory CD200Rand are actively involved in the pathogenesis of allergic disordersin concert with eosinophils and mast cells, at least in part, herpes-virus might have acquired CD200 homologues to suppress the in-flammatory response mediated by these effector cells. Therefore,CD200R might be used as a unique therapeutic target to manageallergic disorders, including pathologies caused by herpesvirusinfections.
We have recently proposed a hypothesis that paired receptors,which consist of related activating and inhibitory receptors, play animportant role in the regulation of viral infections (10, 36). Them157 protein of MCMV, which is structurally related to MHCclass I proteins, is recognized by the inhibitory Ly49I receptor incertain MCMV-susceptible mouse strains, whereas m157 is rec-ognized by the activating Ly49H receptor in an MCMV-resistantmouse strain (10). This suggested that MCMV acquired m157 asa ligand for inhibitory receptors, and the immune system evolvedthe activating Ly49H to protect against viral infection. These ob-servations suggest that the virus and the paired receptors evolvedtogether, allowing for coexistence of host and virus. Interestingly,the mouse CD200R family consists of one inhibitory receptor andthree activating receptors (6). These activating CD200R are ex-pressed on various populations, including mouse basophils andactivated NK cells (37) (I. Shiratori, M. Yamaguchi, M. Su-zukawa, K. Yamamoto, L. L. Lanier, T. Saito, and H. Arase, un-published observation). According to our hypothesis, the activat-ing mouse CD200R may have evolved to protect against virulentviruses that had acquired CD200 homologues. However, as yet, we
have not identified a mouse virus encoding a CD200-like protein totest this concept. In contrast, although the human genome containsa linked gene that encodes a putative activating CD200R, the ac-tivating human CD200R is not expressed on the cell surface due tothe lack of a cysteine residue that is required to form its Ig-likedomain (6). Whether this activating CD200R is still in the stagesof being selected in the human population or is being eliminatedbecause it is no longer necessary for protection against an extinctviral pathogen encoding a CD200 homologue is not known. How-ever, it is interesting to note that HHV-6, -7, and -8 do not causelethal infectious diseases in healthy individuals, although theyshow persistent infection. Nonetheless, the presence of a CD200Rin the human genome with the capacity to generate an activatingreceptor by mutation might be important in the future to acquireresistance to an emerging virulent virus that possesses a CD200homologue. Individuals with a polymorphism generating a func-tional activating CD200R may, in theory, survive such a viral in-fection. Therefore, identification of viruses that are recognized bypaired activating and inhibiting receptors may serve to support aphysiological role of paired receptors in host defense.
AcknowledgmentsWe thank Drs. L. Coscoy, K. Ueda, Y. Mori, and K. Yamanishi for pro-viding HHV-6, -7, and -8, and A. Sakamoto, R. Hirohata, and M. Matsu-moto for technical assistance.
DisclosuresThe authors have no financial conflict of interest.
References1. Long, E. O. 1999. Regulation of immune responses through inhibitory receptors.
Annu. Rev. Immunol. 17: 875–904.2. Ravetch, J. V., and L. L. Lanier. 2000. Immune inhibitory receptors. Science 290:
84–89.3. Nathan, C., and W. A. Muller. 2001. Putting the brakes on innate immunity: a
regulatory role for CD200? Nat. Immunol. 2: 17–19.4. Zhang, S., H. Cherwinski, J. D. Sedgwick, and J. H. Phillips. 2004. Molecular
mechanisms of CD200 inhibition of mast cell activation. J. Immunol. 173:6786–6793.
5. Wright, G. J., M. J. Puklavec, A. C. Willis, R. M. Hoek, J. D. Sedgwick,M. H. Brown, and A. N. Barclay. 2000. Lymphoid/neuronal cell surface OX2glycoprotein recognizes a novel receptor on macrophages implicated in the con-trol of their function. Immunity 13: 233–242.
6. Wright, G. J., H. Cherwinski, M. Foster-Cuevas, G. Brooke, M. J. Puklavec,M. Bigler, Y. Song, M. Jenmalm, D. Gorman, T. McClanahan, et al. 2003. Char-acterization of the CD200 receptor family in mice and humans and their inter-actions with CD200. J. Immunol. 171: 3034–3046.
7. Hoek, R. M., S. R. Ruuls, C. A. Murphy, G. J. Wright, R. Goddard,S. M. Zurawski, B. Blom, M. E. Homola, W. J. Streit, M. H. Brown, et al. 2000.Down-regulation of the macrophage lineage through interaction with OX2(CD200). Science 290: 1768–1771.
8. Gorczynski, R. M., Z. Chen, K. Yu, and J. Hu. 2001. CD200 immunoadhesinsuppresses collagen-induced arthritis in mice. Clin. Immunol. (Oxf.) 101:328–334.
9. Gorczynski, R. M., M. S. Cattral, Z. Chen, J. Hu, J. Lei, W. P. Min, G. Yu, andJ. Ni. 1999. An immunoadhesin incorporating the molecule OX-2 is a potentimmunosuppressant that prolongs allo- and xenograft survival. J. Immunol. 163:1654–1660.
10. Arase, H., E. S. Mocarski, A. E. Campbell, A. B. Hill, and L. L. Lanier. 2002.Direct recognition of cytomegalovirus by activating and inhibitory NK cell re-ceptors. Science 296: 1323–1326.
11. Cosman, D., N. Fanger, L. Borges, M. Kibin, W. Chin, L. Peterson, and M.-L.Hsu. 1997. A novel immunoglobulin superfamily receptor for cellular and viralMHC class I molecules. Immunity 7: 273–282.
12. Orange, J. S., M. S. Fassett, L. A. Koopman, J. E. Boyson, and J. L. Strominger.2002. Viral evasion of natural killer cells. Nat. Immunol. 3: 1006–1012.
13. Russo, J. J., R. A. Bohenzky, M. C. Chien, J. Chen, M. Yan, D. Maddalena,J. P. Parry, D. Peruzzi, I. S. Edelman, Y. Chang, et al. 1996. Nucleotide sequenceof the Kaposi sarcoma-associated herpesvirus (HHV8). Proc. Natl. Acad. Sci.USA 93: 14862–14867.
14. Nicholas, J. 1996. Determination and analysis of the complete nucleotide se-quence of human herpesvirus. J. Virol. 70: 5975–5989.
15. Gompels, U. A., J. Nicholas, G. Lawrence, M. Jones, B. J. Thomson,M. E. Martin, S. Efstathiou, M. Craxton, and H. A. Macaulay. 1995. The DNAsequence of human herpesvirus-6: structure, coding content, and genome evolu-tion. Virology 209: 29–51.
16. Cameron, C. M., J. W. Barrett, L. Liu, A. R. Lucas, and G. McFadden. 2005.Myxoma virus M141R expresses a viral CD200 (vOX-2) that is responsible fordown-regulation of macrophage and T-cell activation in vivo. J. Virol. 79:6052–6067.
17. Seet, B. T., J. B. Johnston, C. R. Brunetti, J. W. Barrett, H. Everett, C. Cameron,J. Sypula, S. H. Nazarian, A. Lucas, and G. McFadden. 2003. Poxviruses andimmune evasion. Annu. Rev. Immunol. 21: 377–423.
18. Chung, Y. H., R. E. Means, J. K. Choi, B. S. Lee, and J. U. Jung. 2002. Kaposi’ssarcoma-associated herpesvirus OX2 glycoprotein activates myeloid-lineage cellsto induce inflammatory cytokine production. J. Virol. 76: 4688–4698.
19. Foster-Cuevas, M., G. J. Wright, M. J. Puklavec, M. H. Brown, andA. N. Barclay. 2004. Human herpesvirus 8 K14 protein mimics CD200 in down-regulating macrophage activation through CD200 receptor. J. Virol. 78:7667–7676.
20. Shiratori, I., K. Ogasawara, T. Saito, L. L. Lanier, and H. Arase. 2004. Activationof natural killer cells and dendritic cells upon recognition of a novel CD99-likeligand by paired immunoglobulin-like type 2 receptor. J. Exp. Med. 199:525–533.
21. Robertson, M. J., K. J. Cochran, C. Cameron, J.-M. Le, R. Tantravahi, and J. Ritz.1996. Characterization of a cell line, NKL, derived from an aggressive humannatural killer cell leukemia. Exp. Hematol. 24: 406–415.
22. Iikura, M., M. Yamaguchi, T. Fujisawa, M. Miyamasu, T. Takaishi, Y. Morita,T. Iwase, I. Moro, K. Yamamoto, and K. Hirai. 1998. Secretory IgA inducesdegranulation of IL-3-primed basophils. J. Immunol. 161: 1510–1515.
23. Gilbert, H. S., and L. Ornstein. 1975. Basophil counting with a new stainingmethod using Alcian blue. Blood 46: 279–286.
24. Yoshimura-Uchiyama, C., M. Yamaguchi, H. Nagase, T. Fujisawa, C. Ra,K. Matsushima, T. Iwata, T. Igarashi, K. Yamamoto, and K. Hirai. 2003. Com-parative effects of basophil-directed growth factors. Biochem. Biophys. Res. Com-mun. 302: 201–206.
25. Nagase, H., S. Okugawa, Y. Ota, M. Yamaguchi, H. Tomizawa, K. Matsushima,K. Ohta, K. Yamamoto, and K. Hirai. 2003. Expression and function of Toll-likereceptors in eosinophils: activation by Toll-like receptor 7 ligand. J. Immunol.171: 3977–3982.
26. Hirai, K., Y. Morita, Y. Misaki, K. Ohta, T. Takaishi, S. Suzuki, K. Motoyoshi,and T. Miyamoto. 1988. Modulation of human basophil histamine release byhemopoietic growth factors. J. Immunol. 141: 3958–3964.
27. Bochner, B. S., and S. A. Sterbinsky. 1991. Altered surface expression of CD11and Leu 8 during human basophil degranulation. J. Immunol. 146: 2367–2373.
28. Bochner, B. S., A. A. McKelvey, S. A. Sterbinsky, J. E. Hildreth, C. P. Derse,D. A. Klunk, L. M. Lichtenstein, and R. P. Schleimer. 1990. IL-3 augmentsadhesiveness for endothelium and CD11b expression in human basophils but notneutrophils. J. Immunol. 145: 1832–1837.
29. Jenner, R. G., M. M. Alba, C. Boshoff, and P. Kellam. 2001. Kaposi’s sarcoma-associated herpesvirus latent and lytic gene expression as revealed by DNA ar-rays. J. Virol. 75: 891–902.
30. Coscoy, L., and D. Ganem. 2000. Kaposi’s sarcoma-associated herpesvirus en-codes two proteins that block cell surface display of MHC class I chains byenhancing their endocytosis. Proc. Natl. Acad. Sci. USA 97: 8051–8056.
31. Ishido, S., J. K. Choi, B. S. Lee, C. Wang, M. DeMaria, R. P. Johnson,G. B. Cohen, and J. U. Jung. 2000. Inhibition of natural killer cell-mediatedcytotoxicity by Kaposi’s sarcoma-associated herpesvirus K5 protein. Immunity13: 365–374.
32. Fallarino, F., C. Asselin-Paturel, C. Vacca, R. Bianchi, S. Gizzi, M. C. Fioretti,G. Trinchieri, U. Grohmann, and P. Puccetti. 2004. Murine plasmacytoid den-dritic cells initiate the immunosuppressive pathway of tryptophan catabolism inresponse to CD200 receptor engagement. J. Immunol. 173: 3748–3754.
33. Cherwinski, H. M., C. A. Murphy, B. L. Joyce, M. E. Bigler, Y. S. Song,S. M. Zurawski, M. M. Moshrefi, D. M. Gorman, K. L. Miller, S. Zhang, et al.2005. The CD200 receptor is a novel and potent regulator of murine and humanmast cell function. J. Immunol. 174: 1348–1356.
34. Mitani, N., M. Aihara, Y. Yamakawa, M. Yamada, N. Itoh, N. Mizuki, andZ. Ikezawa. 2005. Drug-induced hypersensitivity syndrome due to cyanamideassociated with multiple reactivation of human herpesviruses. J. Med. Virol. 75:430–434.
35. Kano, Y., M. Inaoka, K. Sakuma, and T. Shiohara. 2005. Virus reactivation andintravenous immunoglobulin (IVIG) therapy of drug-induced hypersensitivitysyndrome. Toxicology 209: 165–167.
36. Arase, H., and L. L. Lanier. 2004. Specific recognition of virus-infected cells bypaired NK receptors. Rev. Med. Virol. 14: 83–93.
37. Voehringer, D., D. B. Rosen, L. L. Lanier, and R. M. Locksley. 2004. CD200receptor family members represent novel DAP12-associated activating receptorson basophils and mast cells. J. Biol. Chem. 279: 54117–54123.
