CD200 Human CD200 and Human Herpesvirus-8 Down-Regulation Of

Down-Regulation of Basophil Function by Human CD200 and Human Herpesvirus-8 CD200

This information is current as Ikuo Shiratori, Masao Yamaguchi, Maho Suzukawa, of September 26, 2021. Kazuhiko Yamamoto, Lewis L. Lanier, Takashi Saito and Hisashi Arase J Immunol 2005; 175:4441-4449; ; doi: 10.4049/jimmunol.175.7.4441

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Down-Regulation of Basophil Function by Human CD200 and Human Herpesvirus-8 CD2001

Ikuo Shiratori,*† Masao Yamaguchi,‡ Maho Suzukawa,‡ Kazuhiko Yamamoto,‡ 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 regulation of 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 and the viral CD200 homologues on human CD200R-expressing cells. We found that human CD200R is predominantly expressed on basophils in amounts higher than on other human peripheral blood leukocytes. Furthermore, the viral CD200 homologues as well as human CD200 were recognized by human CD200R, and the activation of basophils was down-regulated by these CD200 proteins. These results suggested that CD200R is an important regulatory molecule of basophil activation. In addition, the Downloaded from presence of CD200 homologues on several viruses suggests a potentially unique relationship between basophil function and viral infection. The Journal of Immunology, 2005, 175: 4441–4449.

mmune cells express inhibitory receptors to prevent damage for an inhibitory Ly49 receptor in certain MCMV-susceptible to self. Many of the inhibitory receptors recognize broadly mouse strains (10). Similarly, human CMV acquired UL18 as a expressed self-proteins, such as MHC class I (1, 2). CD200R ligand for the inhibitory CD85J (leukocyte inhibitory receptor/Ig- I http://www.jimmunol.org/ (3) is an inhibitory receptor that possesses a tyrosine phosphatase- like transport) receptor (11). These decoy ligands for inhibitory recruiting inhibitory motif in its cytoplasmic domain (3, 4). receptors may play an important role, allowing the viruses to evade CD200R is primarily expressed on leukocytes of the myeloid lin- the immune system and potentially cause persistent infectious dis- eage, and it recognizes CD200, which is broadly expressed on a eases (12). Interestingly, herpesviruses, such as human herpesvi- variety of cell types (3, 5, 6). In mice, disruption of the gene rus-6 (HHV-6), HHV-7, and HHV-8 (13–15), and poxviruses, in- encoding CD200 led to expansion of the macrophage and granu- cluding myxoma virus and shope ﬁbroma virus, possess genes locyte populations in the spleen and the macrophage population in encoding proteins with homology to CD200 (16, 17). Although mesenteric lymph nodes (7). Furthermore, CD200-deﬁcient mice amino acid homologies between human CD200 and these viral

showed rapid onset of experimental autoimmune encephalomyeli- by guest on September 26, 2021 CD200 homologues are Ͻ40%, this level of similarity is sufficient tis (7). Administration of CD200-Ig fusion protein, which func- to permit binding to the inhibitory CD200R. In fact, the CD200 tions as an agonist for the inhibitory CD200R, suppressed allograft homologue of HHV-8 (HHV-8 CD200) has been described as a rejection and collagen-induced arthritis in animal models (8, 9). These data suggested that recognition of CD200 by an inhibitory decoy ligand for CD200R (18, 19). However, the effect of the CD200R plays an important role in regulating immune responses. HHV-8 CD200 on CD200R-expressing human cells has remained Several viruses that persistently infect the host have acquired unclear, and there are conflicting reports in the literature (18, 19). ligands for inhibitory receptors, presumably to suppress an im- In the present study we show that basophils are a major mune response against these pathogens. For example, murine CD200R-positive population in human peripheral blood. Further- CMV (MCMV)3 has evolved m157, which functions as a ligand more, we show that not only the HHV-8 CD200, but also the CD200 homologues of HHV-6 (HHV-6 CD200) and HHV-7 (HHV-7 CD200), are recognized by CD200R and that activation of *Department of Immunochemistry, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan; †Precursory Research for Embryonic Science and Tech- basophils is down-regulated by recognizing human CD200 and nology, Japan Science and Technology Agency, Saitama, Japan; ‡Department of Al- HHV-8 CD200. These findings suggest that CD200R regulates lergy and Rheumatology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan; §Department of Microbiology and Immunology and the Cancer Research In- basophil activation, and the viral CD200 homologues are involved stitute, University of California, San Francisco, CA 94143; ¶Laboratory for Cell Sig- in the regulation of basophil-dependent immune responses. 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 page charges. This article must therefore be hereby marked advertisement in accordance Materials and Methods with 18 U.S.C. Section 1734 solely to indicate this fact. cDNA preparation 1 This work was supported by a Grant-in-Aid for Scientific Research from the Min- CD200 homologues of HHV-7 (U85) and HHV-8 (K14) were cloned using istry of Education, Science, and Culture, Japan (to T.S. and H.A.), and the Uehara PCR from genomic DNA of HHV-7 and HHV-8 (provided by Dr. L. Memorial Foundation (to H.A.). L.L.L. is an American Cancer Society Research Professor and is supported by National Institutes of Health Grant CA89294. Coscoy, University of California, Berkeley, CA). The CD200 homologue of HHV-6 (U85) was cloned using PCR directly from supernatant contain- 2 Address correspondence and reprint requests to Dr. Hisashi Arase, Department of Immunochemistry, Research Institute for Microbial Diseases, Osaka University, 3-1 ing HHV-6 virus (strain SF; provided by Dr. L. Coscoy). The GenBank/ Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail address: [email protected] EMBL/DDBJ accession numbers of HHV-6, HHV-7, and HHV-8 are u.ac.jp AB190768, HHU43400, and AF367765, respectively. Human CD200 and 3 CD200R were cloned using PCR from cDNA generated from human Abbreviations used in this paper: MCMV, murine CMV; HHV, human herpesvirus; ϩ HHV-6 CD200, CD200 homolog of HHV-6; HHV-7 CD200, CD200 homolog of PBMC-derived poly(A) RNA. Mouse CD200R was cloned from cDNA ϩ HHV-7; HHV-8 CD200, CD200 homolog of HHV-8; PIPES-A, PIPES-Albumin. generated from mouse spleen-derived poly(A) RNA.

Ig fusion proteins GAGCTGCGTGTG-3Ј; antisense primer, 5Ј-CGTAGATGGGCACAGT GTGG-3Ј; GAPDH: sense primer, 5Ј-ATGCTGGCGCTGAGTACGTC- cDNA fragments corresponding to the extracellular domains of human 3Ј; antisense primer, 5Ј-CAGGGGTGCTAAGCAGTTGGT-3Ј; and human CD200R (aa residues 25–267), mouse CD200R (aa residues 26–238), hu- CD200R: sense primer, 5Ј-CTTCCTGTTCCAGGTGCCAAA-3Ј; anti- man CD200 (aa residues 56–263), and HHV8-CD200 (aa residues 25–226) sense primer, 5Ј-GCCTCAGATGCCTTCACCTTG-3Ј. ␤-Actin and were cloned into a unique XhoI site of a modified pME18S expression GAPDH were used to standardize the relative amounts of CD200 tran- vector carrying the human CD150 leader segment and the Fc segment of scripts in the different cell populations. Comparable results were obtained human IgG1 (20). COS-7 cells were transiently transfected by using when either ␤-actin or GAPDH was used for normalization of the data. 293fectin (InvitroGen) with these expression vectors to generate Ig fusion proteins. After 72 h, the culture supernatants were collected, and the Histamine release assay amounts of Ig fusion protein were measured using standard ELISA methods. For some experiments, human CD200-Ig and HHV-8-CD200-Ig fu- Histamine release from basophils was analyzed as previously described sion proteins were further purified using protein A-coupled Sepharose col- (22). Purified basophils (2.0–4.0 ϫ 105) were incubated with 10 ␮g/ml umns (Amersham Biosciences) by standard methods. human CD200-Ig, HHV-8 CD200-Ig, or control-Ig fusion proteins in PIPES-Albumin (PIPES-A) buffer containing 25 mM PIPES, 119 mM Cells and transfectants NaCl, 5 mM KCl, and 0.03% human serum albumin (pH 7.4) for 30 min on ice. Cells were then washed in ice-cold PIPES-A and incubated with 5 FLAG-tagged human CD200, HHV-6-CD200, HHV7-CD200, and HHV- ␮g/ml F(abЈ)2 of goat anti-human IgG Fc␥ Ab (Jackson ImmunoResearch 8-CD200 were transfected into Ba/F3 cells using the pMx-neo retrovirus ␣ Laboratories) in PIPES-A for 30 min on ice. Basophils were then washed vector, which contains a human CD8 signal sequence and a FLAG tag at in ice-cold PIPES-A, resuspended at 4.0–8.0 ϫ 104 basophils/ml in the N terminus of the mature protein (20). These transfectants were stained PIPES-A buffer containing 2 mM Ca2ϩ and 0.5 mM Mg2ϩ (PIPES-ACM), using anti-FLAG mAb (clone M2; Sigma-Aldrich), and FLAG-positive and stimulated with 1 ␮g/ml mouse anti-human Fc⑀RI mAb (CRA1, cells were isolated by using a MACS purification system (Miltenyi Biotec).

Kyokuto Pharmaceutical Industry) or 300 pM human rIL-3 (donated by Downloaded from Human CD200 and the open reading frame of the HHV-8 CD200 were also Kirin Brewery) for 45 min at 37°C. For coculture experiments, purified transfected into 721.221 cells using the pMx-IRES-GFP retrovirus vector, basophils (2.0–4.0 ϫ 105) were suspended with 1 ϫ 106 of mock-trans- and GFP-positive cells were purified using a flow cytometer (FACSVan- fected or CD200-transfected 721.221 cells in PIPES-ACM and were cen- tage; BD Biosciences). Human CD200R was also transfected into the hu- trifuged at 4°C, followed by incubation for 30 min on ice. Cells were then man NK cell line NKL (21) using the pMx-puro retrovirus vector, and cells resuspended at 4.0–8.0 ϫ 104/ml in PIPES-ACM containing 1 ␮g/ml stained by human CD200-Ig fusion protein were purified using flow cy- mouse anti-human Fc⑀RI mAb or 300 pM human rIL-3 and then centri- tometry (FACSVantage). BC1, a human primary effusion lymphoma cell fuged at 4°C, followed by incubation for 45 min at 37°C. The concentration line harboring HHV-8, was provided by Dr. K. Ueda (Osaka University, of histamine in supernatants was measured using an automated fluoromet- http://www.jimmunol.org/ Osaka, Japan). We recloned BC1 cells and selected cells that were highly ric analyzer. Histamine release was calculated as a percentage of the total stained with human CD200R-Ig fusion protein after induction of lytic re- histamine content, as previously described (26). activation by PMA (20 ng/ml) for 24 h. All cells were cultured in RPMI 1640 medium containing 10% FCS, except for NKL cells, in which case Cytotoxic assays 400 U/ml human IL-2 (PeproTech) was added to the culture medium. Human basophils used for the degranulation assay were purified from 51Cr-labeled, mock-transfected or CD200-transfected 721.221 cells (1 ϫ EDTA-treated (to prevent coagulation) venous blood using Percoll density 105/well) were cocultured with various numbers of mock-transfected or centrifugation (Amersham Biosciences) (22). Basophils were further puri- CD200R-transfected NKL cells in 96-well, round-bottom plates for 4 h. fied using a MACS basophil isolation kit (Miltenyi Biotech) for measure- For Ab-blocking experiments, CD200-transfected 721.221 cells were in- ment of CD200R expression. The purities of Percoll- and MACS-purified cubated with anti-human CD200 mAb (MRC OX-104; BD Pharmingen) or basophils determined by Alcian blue staining (23) were 8–30 and Ͼ95%, an isotype-matched control mAb for 30 min on ice before the coculture by guest on September 26, 2021 respectively. Human CD3-, CD14-, CD19-, and CD56-positive lympho- with effector cells. Specific lysis (percentage) was analyzed by 51Cr release cytes were purified from human PBMC using a MACS purification system assays using standard methods (20). (Miltenyi Biotech), and the purity of each population was Ͼ90%. Cytokine production Flow cytometry We cocultured 3 ϫ 105 mock-transfected or CD200R-transfected NKL Cells were incubated with saturating concentrations of various Ig fusion cells with various numbers of mock-transfected or CD200-transfected Ј proteins or mAbs for 30 min on ice, followed by incubation with F(ab )2 721.221 cells in 96-well, round-bottom plates. Culture supernatants were of PE-conjugated goat anti-human IgG or anti-mouse IgG (Jackson Immu- collected after 24 h, and concentrations of IFN-␥ in the supernatants were noResearch Laboratories) for 30 min. For analyses of CD200R expression determined using a human IFN-␥ ELISA kit (BD Pharmingen). To analyze on human PBMC, we generated a PE-labeled Ig fusion protein complex by the response against HHV-8-infected cells, BC1 cells were stimulated with mixing 10 ␮g/ml Ig fusion protein with 4 ␮g/ml PE-conjugated goat anti- 20 ng/ml PMA for 24 h. Thereafter, PMA-stimulated BC1 cells were human IgG (Jackson ImmunoResearch Laboratories) for 30 min on ice, washed four times and cocultured with 3 ϫ 105 of mock-transfected or followed by the addition of 30 ␮g/ml normal human IgG (Cappel Labo- CD200R-transfected NKL in 96-well, round-bottom plates for 24 h. ratories) for 30 min. Human PBMC were pretreated with 15 ␮g/ml normal human IgG for 15 min on ice and stained with the PE-labeled Ig fusion Results complexes and various FITC-conjugated mAbs for 30 min. CD11b expres- Specific binding of soluble CD200R to viral CD200 homologues sion on basophils was analyzed by staining with PE-conjugated anti- human-CD11b mAb (Bear1; Coulter Immunotech) and FITC-conjugated of HHV-6, -7, and -8 anti-human IgE Ab (BioSource International) for 60 min on ice. Stained Some herpesviruses and poxviruses possess CD200 homologues cells were analyzed using a FACSCalibur (BD Biosciences), and the me- dian values of fluorescence intensity were converted to the number of mol- (13–15, 17), for example, HHV-6, -7, and -8. These viral CD200 ecules of equivalent soluble fluorochrome units using a quantum fluores- homologues are thought to serve as decoy ligands for the inhibi- cent microbead standard for PE (Sigma-Aldrich), as described previously tory CD200R (19). To analyze the function of the CD200 homo- (24). FITC-conjugated anti-CD3 (BW264/56), FITC-conjugated anti- logues of HHV-6, -7, and -8, we stably transduced these viral CD14(TUK4),FITC-conjugatedanti-CD16(VEP13),FITC-conjugatedanti- genes into mouse pro-B Ba/F3 cells using retroviruses. We added CD19 (LT19), and FITC-conjugated anti-CD123 (AC145) mAbs were purchased from Miltenyi Biotec. FITC-conjugated anti-CD56 (MEM188) and a FLAG epitope tag at the N terminus of the viral CD200 homo- anti-HLA-A,B,C (G46-2.6) mAbs were purchased from BD Pharmingen. logues to permit detection of these proteins. Ba/F3 cells stably expressing human CD200 or the viral CD200 homologues on the Real-time PCR analysis cell surface were purified using Ab-coupled magnetic beads. Fig. cDNA generated from total RNA of human CD-3, CD14-, CD19-, and 1A shows the levels of FLAG-tagged human CD200 or viral CD56-positive leukocytes and MACS-purified human basophils, eosino- CD200-like molecules expressed on these transfectants. phils, and neutrophils (25) were used as templates. Real-time PCR analysis was performed using a SYBR Green PCR kit (Applied Biosystems) and an We then analyzed whether Ig fusion proteins of human and ABI PRISM 7900 HT instrument (Applied Biosystems). Primers used for mouse CD200R recognize these viral CD200 homologues. As amplification were as follows: ␤-actin: sense primer, 5Ј-TCTACAAT shown in Fig. 1B, human and mouse CD200R-Ig specifically The Journal of Immunology 4443

FIGURE 1. Recognition of viral CD200 homologues by human inhibitory CD200R. FLAG-tagged CD200 homologues of HHV-6, -7, and -8, as well as human CD200, were transduced into Ba/F3 cells. Parental cells and transduced cells were stained with anti-FLAG mAb (A), human CD200R-Ig fusion protein, mouse CD200R-Ig fusion protein, and anti-human CD200 mAb (solid line; B). Histograms of cells stained with a control mAb or a control-Ig fusion protein were overlaid (dotted line). Downloaded from http://www.jimmunol.org/ bound to cells expressing human CD200. The fluorescence inten- This suggested that T cells, B cells, NK cells, and monocytes ex- sity of cells stained with mouse CD200R-Ig was 1/10th the fluo- press lower levels of CD200R than the CD123-positive population. rescent intensity of cells stained with human CD200R-Ig. This Because basophils are the main population strongly expressing result is consistent with findings of a prior study that demonstrated CD123 in peripheral blood leukocytes, we purified basophils and a lower affinity of mouse CD200R compared with human CD200R stained them with the human CD200-Ig and HHV-8 CD200-Ig for human CD200 measured using a Biacore (Biacore International) fusion proteins (Fig. 2B). As predicted, all basophils were stained (6). When cells transduced with the viral CD200 homologues were with the CD200-Ig fusion protein. In contrast, eosinophils and neu- by guest on September 26, 2021 analyzed, all transductants expressing the viral CD200 ho- trophils were not stained by the CD200-Ig fusion protein (Fig. 2B). mologues were stained with human CD200R-Ig. In contrast, Granulocytes have been reported previously to express CD200R, mouse CD200R-Ig did not recognize these viral CD200 homo- as detected using an anti-human CD200R mAb (6), indicating that logues despite the fact that the homologies of human and mouse the CD200-Ig fusion and HHV-8 CD200-Ig fusion proteins prob- CD200 to the viral CD200 homologues are almost the same ably stain cells with only the highest amounts of CD200R. Anti- (amino acid homologies of the viral CD200 proteins of HHV-6, -7, human CD200R mAb was not available for a direct comparison and -8 to human CD200 are 24, 24, and 31%, respectively, with the fusion protein staining; however, the mAb almost cer- whereas those to mouse CD200 are 16%, 24, and 32%, respec- tainly has a higher affinity than the fusion proteins and would be tively). In addition, mouse CD200R-Ig recognized not only mouse capable of detecting cells expressing lower levels of CD200R. CD200, but also human CD200 (Fig. 1B and data not shown). Nonetheless, because CD200-Ig and HHV-8 CD200-Ig specifi- Anti-CD200 mAb recognized human CD200, but not the viral cally bind to CD200R, these data suggested that basophils repre- CD200 homologues (Fig. 1B). These results suggested that these sent a major CD200R-positive cell type in human peripheral blood. human herpesviruses might have acquired CD200 homologues as Next, to confirm that CD200R is expressed on basophils, we pu- ligands for the human inhibitory CD200R. rified each leukocyte population and analyzed the expression of CD200R transcripts by real-time PCR. As shown in Fig. 2C, the Specific expression of CD200R on basophils in human expression of CD200R transcripts in basophils was substantially peripheral blood higher than that in other populations. When the nucleic acid se- Recent studies have suggested that CD200R is expressed on most quence of the CD200R cDNA expressed in basophils was ana- human leukocytes, including T cells, monocytes, granulocytes, and lyzed, there was no difference in the sequence from that of the a small subset of NK cells (6). However, the expression level of CD200R cDNA previously reported (GenBank accession no. CD200R on these populations was relatively low, and the function AY284975; data not shown). These data indicated that basophils of CD200R on these cells has not been evaluated (6). To study the are a major population in peripheral blood expressing CD200R. cells expressing CD200R, we stained peripheral blood leukocytes with CD200-Ig and HHV-8 CD200-Ig fusion proteins in combi- Human CD200- and HHV-8 CD200-expressing cells suppress nation with various lineage-specific mAbs. As shown in Fig. 2A, effector functions of basophils through inhibitory CD200R ϳ1% of the cells were brightly stained with the CD200-Ig fusion Degranulation of basophils is correlated with the pathogenesis of protein. Most cells binding to the CD200-Ig fusion protein were allergic disorders. Engagement of Fc⑀RI with anti-Fc⑀RI mAb in- costained with anti-CD123 mAb, but not with anti-CD3, -CD14, duces CD11b up-regulation and histamine release from basophils -CD16, -CD19, or -CD56 mAb. Similar results were obtained (27). We analyzed the function of CD200R on basophils by cross- when cells were stained with the HHV-8 CD200-Ig fusion protein. linking the receptor with human CD200-Ig and HHV-8 CD200-Ig. 4444 REGULATION OF BASOPHIL FUNCTION BY CD200 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. Expression of CD200R on basophils. A, PBMC were costained with control Ig, human CD200-Ig, or HHV-8 CD200-Ig (vertical axis) in combination with anti-CD3, -CD14, -CD16, -CD19, -CD56, and -CD123 mAbs (horizontal axis). The proportion of each population is indicated in the figure.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 CD200R transcripts are shown. Representative data from four different healthy blood donors are shown.

As shown in Fig. 3, cross-linking CD200R with human CD200-Ig, and HHV-8 CD200-Ig showed negligible effects against IL-3-de- but not with control-Ig, clearly suppressed both CD11b up-regu- pendent CD11b up-regulation on basophils. lation and histamine release of basophils induced by the engage- Next, we analyzed then function of CD200R on basophils using ment of Fc⑀RI. Cross-linking CD200R with HHV-8 CD200-Ig CD200 transfectants. We transfected human CD200 or the HHV-8 also suppressed Fc⑀RI-dependent CD11b up-regulation and hista- CD200 into human B lymphoblastoid cell line 721.221 cells. Al- mine release. In contrast, IL-3 induced up-regulation of CD11b though 721.221 cells express low amounts of endogenous CD200 expression, but not histamine release (27, 28). Human CD200-Ig on the cell surface, the level of CD200 expression was greatly

FIGURE 3. Suppression of basophil activation by human CD200 and HHV-8 CD200. Basophils were incubated with human CD200-Ig, HHV-8 CD200-Ig, or control-Ig fusion proteins, followed by addition of Ј F(ab )2 of goat anti-human IgG Ab. Cells were then stimulated with anti-human Fc⑀RI mAb or human IL-3, and CD11b expression on basophils and concentrations of histamine in the supernatants were measured. Data are shown as the mean Ϯ SD. The Journal of Immunology 4445 increased when these cells were transduced with CD200 (Fig. 4A). toxicity mediated by human CD200R-expressing NKL cells The human CD200-transfected 721.221 cells were stained brightly against parental 721.221 cells was augmented when anti-CD200 by both CD200R-Ig fusion protein and anti-human CD200 mAb. mAb was added to the assay, indicating that endogenous CD200 721.221 cells transfected with the HHV-8 CD200 were also expressed by 721.221 is functional (Fig. 5C). Moreover, CD200R- stained brightly by the CD200R-Ig fusion protein, but not by anti- transfected NKL cells did not kill human CD200-transfected human CD200 mAb. Basophils were cocultured with human 721.221 cells expressing high amounts of CD200 (Fig. 5B); how- CD200 or HHV-8 CD200-transfected 721.221 human B lympho- ever, cytotoxicity was restored when anti-CD200 mAb was added blastoid cells, and Fc⑀RI on basophils was cross-linked with an to the assay (Fig. 5C). This indicated that interactions between anti-Fc⑀RI mAb (Fig. 4B). Both CD11b up-regulation and hista- CD200R on the effector NK cells and CD200 on the target cells mine release induced by engagement of Fc⑀RI were signiﬁcantly down-regulated the lytic function of NKL cells. Similarly, down-regulated in the presence of human CD200-transfected CD200R-transfected NKL cells showed negligible cytotoxicity 721.221 cells and HHV-8 CD200-transfected 721.221 cells, but against 721.221 cells transfected with HHV-8 CD200 (Fig. 5B). were not down-regulated in the presence of mock-transfected However, addition of anti-human CD200 mAb did not restore the 721.221 cells. These data suggested that the CD200 homologue of cytotoxicity mediated by human CD200R-transfected NKL cells HHV-8 as well as human CD200 suppress Fc⑀RI-dependent acti- because the anti-human CD200 mAb does not recognize HHV-8 vation of basophils through CD200R. CD200 (Fig. 5C). Essentially identical data were obtained when IFN-␥ production by NKL was analyzed (Fig. 5D). Human HHV-8 CD200 down-regulates activation of CD200R- CD200R-expressing NKL cells did not express substantial transfected NK cells amounts of cytokine in response to human CD200 or HHV-8 Downloaded from To analyze the function of CD200R, we used a human NK cell CD200-transfected target cells. These data indicated that human line, NKL, as a model system to validate the inhibitory function of CD200 and the HHV-8-CD200 expressed on the target cells down- the CD200R when it is engaged by its human CD200 or viral regulated the effector functions of CD200R-expressing effector CD200 ligands. We transduced human CD200R into NKL, which cells. lacks endogenous expression of CD200R (Fig. 5A). We then an- HHV-8 infected cells down-regulate activation of CD200R- alyzed the cytolytic activity of NKL or human CD200R-trans- http://www.jimmunol.org/ duced NKL cells against CD200-transfected 721.221 target cells. transduced NKL cells Mock-transfected NKL cells showed high and equivalent levels of We analyzed the function of HHV-8 CD200 using the HHV-8 cytotoxicity against 721.221 and the CD200 transfectants (Fig. persistently infected cell line, BC1. BC1 cells express only limited 5B). By contrast, human CD200R-transfected NKL cells showed HHV-8 viral gene products. However, BC1 cells express several diminished cytolytic activity against parental 721.221 cells (prob- viral proteins when lytic reactivation is induced by PMA stimula- ably due to expression of endogenous CD200) and negligible cy- tion (29). As shown in Fig. 6A, nonstimulated BC1 cells were only totoxicity against 721.221 cells transfected to express high weakly stained by human CD200R-Ig, and BC1 cells were not amounts of human CD200 or the HHV-8 CD200 (Fig. 5B). Cyto- stained by anti-human CD200 mAb. By contrast, when BC1 cells by guest on September 26, 2021

FIGURE 4. Suppression of basophil activation by human and HHV-8 CD200-expressing cells. A, Human CD200 and HHV-8 CD200 were transduced into 721.221 B lymphoblastoid cells. Mock or CD200 transfectants were stained with human CD200R-Ig fusion protein or anti-human CD200 mAb (solid line) and a control mAb or control-Ig fusion protein (dotted line). B, Basophils were cocultured with mock, human CD200, or HHV-8 CD200-transduced 721.221 cells and stimulated with anti-human Fc⑀RI mAb or human IL-3. CD11b expression on basophils and concentrations of histamine in the supernatants were measured. Represen- tative data from more than three independent experiments using different healthy blood donors are presented. Data are shown as the mean Ϯ SD. 4446 REGULATION OF BASOPHIL FUNCTION BY CD200

FIGURE 5. HHV-8 CD200 down-regulates activation of CD200R-transduced NK cells. A, Mock-transfected or human CD200R-transduced NKL cells were

stained with a control-Ig (dotted line) or human Downloaded from CD200-Ig fusion protein (solid line). B, The cytotoxicity of mock-transduced (E) or human CD200R-transduced (F) NKL cells against mock-transduced (left), human CD200-transduced (middle), or HHV-8 CD200-transduced (right) 721.221 cells at the indicated E:T cell ratio is shown. C, The cytotoxicity of CD200R-transfected

NKL cells against CD200-transfected 721.221 in the http://www.jimmunol.org/ presence of anti-human CD200 mAb (F) or a control mAb (E) is shown. D, Mock-transduced (E) or human CD200R-transduced (F) NKL cells were cocultured with CD200-transduced 721.221 at the indicated cell number for 24 h. Concentrations of IFN-␥ in the culture supernatants are shown. Representative data from more than three independent experiments are shown. by guest on September 26, 2021

were stimulated with PMA, most BC1 cells were stained with when they were cocultured with PMA-stimulated BC1 cells. Cul- CD200R-Ig, but not with anti-human CD200 mAb, 1 d after stim- ture supernatants of PMA-stimulated BC1 cells did not activate ulation. The K3 and K5 proteins of HHV-8 are well known to NKL cells. In addition, BC1 itself did not produce IFN-␥ upon down-regulate cell surface expression of MHC class I proteins (30, PMA stimulation (data not shown). These data suggested that 31). Indeed, the amount of MHC class I expression on BC1 cells PMA-stimulated BC1 cells directly activated NKL cells. However, was significantly decreased 2 d after stimulation. These data sug- the activation of NKL was not specific to HHV-8-infected BC1 gested that HHV-8 CD200 was expressed on the cell surface after cells, because other B lymphoblastoid cell lines, such as the EBV- PMA-induced reactivation. transformed 721.221 and C1R cell lines or the EBV-negative We cocultured parental (i.e., CD200R-negative) NKL cells with BJAB cell line, also stimulated NKL after PMA stimulation of nonstimulated or PMA-stimulated BC1 cells and analyzed IFN-␥ these B cell lines (data not shown). Nevertheless, this system is production. Because lytic reactivation was observed after 1-d stim- suitable to analyze the function of the HHV-8 CD200 expressed on ulation with PMA (Fig. 6A), we stimulated BC1 cells with PMA HHV-8-infected cells, because NKL seemed to be activated by for 1 d, washed the cells extensively, and cocultured them with direct recognition of PMA-stimulated BC1 cells. parental NKL cells for an additional day. NKL did not produce We analyzed IFN-␥ production by mock-transduced or IFN-␥ when these cells were cocultured with unstimulated BC1 CD200R-transduced NKL cells upon coculture with PMA-stimu- cells (Fig. 6B); however, NKL produced a large amount of IFN-␥ lated BC1 cells. Mock-transduced NKL cells produced significant The Journal of Immunology 4447

FIGURE 6. HHV-8-infected cells down-regulate activation of CD200R-expressing effector cells via a viral CD200 homologue. A, Expression of HHV-8 CD200 on HHV-8-infected cells. Lytic reactivation was induced by stimulation of BC1 cells with PMA. Nonstimulated (day 0) or PMA-stimulated (days 1 and 2) BC1 cells were stained with anti-human CD200 mAb; anti-human HLA-A, -B, -and -C; or human CD200R-Ig fusion protein (solid line) or with a control-IgG (dotted line). B, NKL cells were cocultured with unstimulated BC1 cells E F

( ) or with PMA-stimulated BC1 cells ( ) at the indi- Downloaded from cated cell number for 24 h. NKL cells were also cultured in the presence of culture supernatants of PMA-stimulated BC1 cells (1:1 dilution; gray circles). C, Mock- transduced (E) or human CD200R-transduced (F) NKL cells were cocultured with unstimulated BC1 cells (left) or PMA-stimulated BC1 cells (right) at the indicated

cell number for 24 h. Concentrations of IFN-␥ produced http://www.jimmunol.org/ in the culture supernatants are shown. All data are presented as the mean Ϯ SD (nanograms per milliliter). Representative data from three independent experiments are shown. by guest on September 26, 2021

amounts of IFN-␥ after coculture with PMA-stimulated BC1 cells, different hemopoietic cell populations. Furthermore, the amounts but not after culture with unstimulated BC1 cells (Fig. 6C). Pro- of CD200R on leukocytes in tissues and organs other than periph- duction of IFN-␥ by CD200R-expressing NKL cells after cocul- eral blood need to be evaluated to determine the potential role for ture with PMA-stimulated BC1 cells was significantly lower than this inhibitory receptor in regulating immune responses. that by mock-transfected NKL cells. There were no significant We showed that engagement of CD200R by its ligand resulted differences in cytokine production between the mock and CD200R in the down-regulation of Fc⑀RI-dependent activation of basophils. transfectants when these transfectants were directly stimulated These findings suggest that CD200R may play an important role in with PMA (data not shown). Because BC1 cells do not express the regulation of basophil function. Recently, the inhibitory CD200, these data suggested that the CD200 homologue of CD200R has been reported to regulate the activation of mouse HHV-8 expressed on PMA-stimulated BC1 cells down-regulates bone marrow-derived mast cells and human cultured mast cells (4, the activation of CD200R-expressing effector cells. 33). Although mast cells are similar to basophils in their array of inflammatory mediators and cell surface expression of Fc⑀RI, their Discussion tissue distributions and developmental pathways are quite differ- In the present study we found that basophils are a major population ent. Indeed, mature mast cells do not exist in blood leukocytes, in expressing CD200R in human peripheral blood. Recently, contrast to basophils. Although basophils bear a closer develop- CD200R has been reported to be expressed on various populations, mental relationship to eosinophils than to mast cells, eosinophils including granulocytes, macrophages, and dendritic cells, as de- do not stain with CD200-Ig or HHV-8 CD200-Ig fusion proteins tected using an anti-CD200R mAb (6, 32, 33). However, we could (Fig. 4B). Therefore, basophils are a unique leukocyte subpopula- not detect CD200R expression on peripheral blood cells other than tion in human peripheral blood brightly expressing CD200R. In basophils using CD200-Ig fusion protein, most likely because the contrast, because basophils can migrate into tissues during an al- fusion protein has a lower affinity than the anti-CD200R mAb and lergic response, CD200R expressed on basophils as well as on therefore detects only cells with the highest amounts of CD200R. resident mast cells might function as a negative regulator of an Although our studies indicate that basophils express higher levels allergic response in local tissue sites. Therefore, analysis of the of CD200R than other PBL, the broad distribution of CD200R function of CD200R expressed on cells at the site of an allergic indicates that CD200R may regulate the immune response of many response would be informative. 4448 REGULATION OF BASOPHIL FUNCTION BY CD200

Not only herpesviruses, but also several other viruses, such as have not identified a mouse virus encoding a CD200-like protein to poxviruses, possess viral CD200 homologues. However, the func- test this concept. In contrast, although the human genome contains tions of the CD200 homologues of most viruses have not been a linked gene that encodes a putative activating CD200R, the ac- established. In the present study, we demonstrate that the CD200 tivating human CD200R is not expressed on the cell surface due to homologues of HHV-6, -7, and -8 are ligands for the human in- the lack of a cysteine residue that is required to form its Ig-like hibitory CD200R. Furthermore, we show that viral CD200 as well domain (6). Whether this activating CD200R is still in the stages as human CD200 down-regulate the activation of basophils and of being selected in the human population or is being eliminated CD200R-transduced NKL cells. We have shown that not only because it is no longer necessary for protection against an extinct HHV-8 CD200-transfected cells but also HHV-8-infected cells viral pathogen encoding a CD200 homologue is not known. How- down-regulate the activation of CD200R-bearing effector cells. ever, it is interesting to note that HHV-6, -7, and -8 do not cause These functional analyses suggested that these viral CD200 ho- lethal infectious diseases in healthy individuals, although they mologues might play an important role in down-regulating an an- show persistent infection. Nonetheless, the presence of a CD200R tiviral immune response. In other words, these data suggested that in the human genome with the capacity to generate an activating HHV-6, -7, and -8 might have acquired CD200 viral homologues receptor by mutation might be important in the future to acquire to regulate the function of basophils in addition to other CD200R- resistance to an emerging virulent virus that possesses a CD200 bearing immune cells. homologue. Individuals with a polymorphism generating a func- Regarding the function of the CD200 homologue of HHV-8, tional activating CD200R may, in theory, survive such a viral in- two conflicting papers have been published. Chung et al. (18) pre- fection. Therefore, identification of viruses that are recognized by pared soluble HHV-8 CD200 homologue as a GST fusion protein paired activating and inhibiting receptors may serve to support a Downloaded from and showed that the soluble HHV-8 CD200 homologue activated physiological role of paired receptors in host defense. human monocytes. In contrast, Foster-Cuevas et al. (19) reported that transfectants expressing the HHV-8 CD200 suppressed the Acknowledgments activation of monocytes, similar to our findings presented in this We thank Drs. L. Coscoy, K. Ueda, Y. Mori, and K. Yamanishi for pro- study. This discrepancy might be due to the manner in which the viding HHV-6, -7, and -8, and A. Sakamoto, R. Hirohata, and M. Matsu-

HHV-8 CD200 protein was prepared in the different studies. Pro- moto for technical assistance. http://www.jimmunol.org/ duction of HHV-8 CD200 by transfection in mammalian cells may better mimic the structure of the protein in virus-infected cells, Disclosures because it will be properly folded and glycosylated. In contrast, the The authors have no financial conflict of interest. GST fusion protein would not be glycosylated and could be con- taminated with bacterial products, which might inadvertently ac- References tivate macrophages. 1. Long, E. O. 1999. Regulation of immune responses through inhibitory receptors. A number of recent reports have suggested that drug-induced Annu. Rev. Immunol. 17: 875–904. 2. Ravetch, J. V., and L. L. Lanier. 2000. Immune inhibitory receptors. Science 290: hypersensitivity syndrome, characterized by serious adverse sys- 84–89. temic reactions in addition to skin rash, is associated with the 3. Nathan, C., and W. A. Muller. 2001. Putting the brakes on innate immunity: a by guest on September 26, 2021 reactivation of human herpesviruses, mainly HHV-6 (34, 35). In- regulatory role for CD200? Nat. Immunol. 2: 17–19. 4. Zhang, S., H. Cherwinski, J. D. Sedgwick, and J. H. Phillips. 2004. Molecular deed, in this case, viral infection exacerbates allergic diseases. mechanisms of CD200 inhibition of mast cell activation. J. Immunol. 173: However, because basophils highly express inhibitory CD200R 6786–6793. and are actively involved in the pathogenesis of allergic disorders 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 OX2 in concert with eosinophils and mast cells, at least in part, herpes- glycoprotein recognizes a novel receptor on macrophages implicated in the con- virus might have acquired CD200 homologues to suppress the in- trol of their function. Immunity 13: 233–242. 6. Wright, G. J., H. Cherwinski, M. Foster-Cuevas, G. Brooke, M. J. Puklavec, flammatory response mediated by these effector cells. Therefore, M. Bigler, Y. Song, M. Jenmalm, D. Gorman, T. McClanahan, et al. 2003. Char- CD200R might be used as a unique therapeutic target to manage acterization of the CD200 receptor family in mice and humans and their inter- allergic disorders, including pathologies caused by herpesvirus actions with CD200. J. Immunol. 171: 3034–3046. 7. Hoek, R. M., S. R. Ruuls, C. A. Murphy, G. J. Wright, R. Goddard, infections. S. M. Zurawski, B. Blom, M. E. Homola, W. J. Streit, M. H. Brown, et al. 2000. We have recently proposed a hypothesis that paired receptors, Down-regulation of the macrophage lineage through interaction with OX2 which consist of related activating and inhibitory receptors, play an (CD200). Science 290: 1768–1771. 8. Gorczynski, R. M., Z. Chen, K. Yu, and J. Hu. 2001. CD200 immunoadhesin important role in the regulation of viral infections (10, 36). The suppresses collagen-induced arthritis in mice. Clin. Immunol. (Oxf.) 101: m157 protein of MCMV, which is structurally related to MHC 328–334. class I proteins, is recognized by the inhibitory Ly49I receptor in 9. Gorczynski, R. M., M. S. Cattral, Z. Chen, J. Hu, J. Lei, W. P. Min, G. Yu, and J. Ni. 1999. An immunoadhesin incorporating the molecule OX-2 is a potent certain MCMV-susceptible mouse strains, whereas m157 is rec- immunosuppressant that prolongs allo- and xenograft survival. J. Immunol. 163: ognized by the activating Ly49H receptor in an MCMV-resistant 1654–1660. 10. Arase, H., E. S. Mocarski, A. E. Campbell, A. B. Hill, and L. L. Lanier. 2002. mouse strain (10). This suggested that MCMV acquired m157 as Direct recognition of cytomegalovirus by activating and inhibitory NK cell re- a ligand for inhibitory receptors, and the immune system evolved ceptors. Science 296: 1323–1326. the activating Ly49H to protect against viral infection. These ob- 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 viral servations suggest that the virus and the paired receptors evolved MHC class I molecules. Immunity 7: 273–282. together, allowing for coexistence of host and virus. Interestingly, 12. Orange, J. S., M. S. Fassett, L. A. Koopman, J. E. Boyson, and J. L. Strominger. the mouse CD200R family consists of one inhibitory receptor and 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, three activating receptors (6). These activating CD200R are ex- J. P. Parry, D. Peruzzi, I. S. Edelman, Y. Chang, et al. 1996. Nucleotide sequence pressed on various populations, including mouse basophils and of the Kaposi sarcoma-associated herpesvirus (HHV8). Proc. Natl. Acad. Sci. USA 93: 14862–14867. activated NK cells (37) (I. Shiratori, M. Yamaguchi, M. Su- 14. Nicholas, J. 1996. Determination and analysis of the complete nucleotide se- zukawa, K. Yamamoto, L. L. Lanier, T. Saito, and H. Arase, un- quence of human herpesvirus. J. Virol. 70: 5975–5989. published observation). According to our hypothesis, the activat- 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 DNA ing mouse CD200R may have evolved to protect against virulent sequence of human herpesvirus-6: structure, coding content, and genome evolu- viruses that had acquired CD200 homologues. However, as yet, we tion. Virology 209: 29–51. The Journal of Immunology 4449

16. Cameron, C. M., J. W. Barrett, L. Liu, A. R. Lucas, and G. McFadden. 2005. 27. Bochner, B. S., and S. A. Sterbinsky. 1991. Altered surface expression of CD11 Myxoma virus M141R expresses a viral CD200 (vOX-2) that is responsible for and Leu 8 during human basophil degranulation. J. Immunol. 146: 2367–2373. down-regulation of macrophage and T-cell activation in vivo. J. Virol. 79: 28. Bochner, B. S., A. A. McKelvey, S. A. Sterbinsky, J. E. Hildreth, C. P. Derse, 6052–6067. D. A. Klunk, L. M. Lichtenstein, and R. P. Schleimer. 1990. IL-3 augments 17. Seet, B. T., J. B. Johnston, C. R. Brunetti, J. W. Barrett, H. Everett, C. Cameron, adhesiveness for endothelium and CD11b expression in human basophils but not J. Sypula, S. H. Nazarian, A. Lucas, and G. McFadden. 2003. Poxviruses and neutrophils. J. Immunol. 145: 1832–1837. immune evasion. Annu. Rev. Immunol. 21: 377–423. 29. Jenner, R. G., M. M. Alba, C. Boshoff, and P. Kellam. 2001. Kaposi’s sarcoma- 18. Chung, Y. H., R. E. Means, J. K. Choi, B. S. Lee, and J. U. Jung. 2002. Kaposi’s associated herpesvirus latent and lytic gene expression as revealed by DNA ar- sarcoma-associated herpesvirus OX2 glycoprotein activates myeloid-lineage cells rays. J. Virol. 75: 891–902. to induce inflammatory cytokine production. J. Virol. 76: 4688–4698. 30. Coscoy, L., and D. Ganem. 2000. Kaposi’s sarcoma-associated herpesvirus en- 19. Foster-Cuevas, M., G. J. Wright, M. J. Puklavec, M. H. Brown, and codes two proteins that block cell surface display of MHC class I chains by A. N. Barclay. 2004. Human herpesvirus 8 K14 protein mimics CD200 in down- enhancing their endocytosis. Proc. Natl. Acad. Sci. USA 97: 8051–8056. regulating macrophage activation through CD200 receptor. J. Virol. 78: 31. Ishido, S., J. K. Choi, B. S. Lee, C. Wang, M. DeMaria, R. P. Johnson, 7667–7676. G. B. Cohen, and J. U. Jung. 2000. Inhibition of natural killer cell-mediated 20. Shiratori, I., K. Ogasawara, T. Saito, L. L. Lanier, and H. Arase. 2004. Activation cytotoxicity by Kaposi’s sarcoma-associated herpesvirus K5 protein. Immunity of natural killer cells and dendritic cells upon recognition of a novel CD99-like 13: 365–374. ligand by paired immunoglobulin-like type 2 receptor. J. Exp. Med. 199: 32. Fallarino, F., C. Asselin-Paturel, C. Vacca, R. Bianchi, S. Gizzi, M. C. Fioretti, 525–533. G. Trinchieri, U. Grohmann, and P. Puccetti. 2004. Murine plasmacytoid den- 21. Robertson, M. J., K. J. Cochran, C. Cameron, J.-M. Le, R. Tantravahi, and J. Ritz. dritic cells initiate the immunosuppressive pathway of tryptophan catabolism in 1996. Characterization of a cell line, NKL, derived from an aggressive human response to CD200 receptor engagement. J. Immunol. 173: 3748–3754. natural killer cell leukemia. Exp. Hematol. 24: 406–415. 22. Iikura, M., M. Yamaguchi, T. Fujisawa, M. Miyamasu, T. Takaishi, Y. Morita, 33. Cherwinski, H. M., C. A. Murphy, B. L. Joyce, M. E. Bigler, Y. S. Song, T. Iwase, I. Moro, K. Yamamoto, and K. Hirai. 1998. Secretory IgA induces S. M. Zurawski, M. M. Moshrefi, D. M. Gorman, K. L. Miller, S. Zhang, et al. degranulation of IL-3-primed basophils. J. Immunol. 161: 1510–1515. 2005. The CD200 receptor is a novel and potent regulator of murine and human 23. Gilbert, H. S., and L. Ornstein. 1975. Basophil counting with a new staining mast cell function. J. Immunol. 174: 1348–1356. method using Alcian blue. Blood 46: 279–286. 34. Mitani, N., M. Aihara, Y. Yamakawa, M. Yamada, N. Itoh, N. Mizuki, and Downloaded from 24. Yoshimura-Uchiyama, C., M. Yamaguchi, H. Nagase, T. Fujisawa, C. Ra, Z. Ikezawa. 2005. Drug-induced hypersensitivity syndrome due to cyanamide K. Matsushima, T. Iwata, T. Igarashi, K. Yamamoto, and K. Hirai. 2003. Com- associated with multiple reactivation of human herpesviruses. J. Med. Virol. 75: parative effects of basophil-directed growth factors. Biochem. Biophys. Res. Com- 430–434. mun. 302: 201–206. 35. Kano, Y., M. Inaoka, K. Sakuma, and T. Shiohara. 2005. Virus reactivation and 25. Nagase, H., S. Okugawa, Y. Ota, M. Yamaguchi, H. Tomizawa, K. Matsushima, intravenous immunoglobulin (IVIG) therapy of drug-induced hypersensitivity K. Ohta, K. Yamamoto, and K. Hirai. 2003. Expression and function of Toll-like syndrome. Toxicology 209: 165–167. receptors in eosinophils: activation by Toll-like receptor 7 ligand. J. Immunol. 36. Arase, H., and L. L. Lanier. 2004. Specific recognition of virus-infected cells by

171: 3977–3982. paired NK receptors. Rev. Med. Virol. 14: 83–93. http://www.jimmunol.org/ 26. Hirai, K., Y. Morita, Y. Misaki, K. Ohta, T. Takaishi, S. Suzuki, K. Motoyoshi, 37. Voehringer, D., D. B. Rosen, L. L. Lanier, and R. M. Locksley. 2004. CD200 and T. Miyamoto. 1988. Modulation of human basophil histamine release by receptor family members represent novel DAP12-associated activating receptors hemopoietic growth factors. J. Immunol. 141: 3958–3964. on basophils and mast cells. J. Biol. Chem. 279: 54117–54123. by guest on September 26, 2021