CD46 of Protein-Tyrosine Phosphatase SHP-1 to Nitric Oxide

Functional Modulation of Human Macrophages Through CD46 (Measles Virus Receptor): Production of IL-12 p40 and Nitric Oxide in Association with Recruitment This information is current as of Protein-Tyrosine Phosphatase SHP-1 to of September 25, 2021. CD46 Mitsue Kurita-Taniguchi, Aya Fukui, Kaoru Hazeki, Akiko Hirano, Shoutaro Tsuji, Misako Matsumoto, Michiko

Watanabe, Shigeharu Ueda and Tsukasa Seya Downloaded from J Immunol 2000; 165:5143-5152; ; doi: 10.4049/jimmunol.165.9.5143 http://www.jimmunol.org/content/165/9/5143 http://www.jimmunol.org/

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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 © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Functional Modulation of Human Macrophages Through CD46 (Measles Virus Receptor): Production of IL-12 p40 and Nitric Oxide in Association with Recruitment of Protein-Tyrosine Phosphatase SHP-1 to CD461

Mitsue Kurita-Taniguchi,* Aya Fukui,*† Kaoru Hazeki,* Akiko Hirano,§ Shoutaro Tsuji,*‡ Misako Matsumoto,*‡ Michiko Watanabe,¶ Shigeharu Ueda,¶ and Tsukasa Seya2*†‡

Human CD46, formerly membrane cofactor protein, binds and inactivates complement C3b and serves as a receptor for measles virus (MV), thereby protecting cells from homologous complement and sustaining systemic measles infection. Suppression of ␾

cell-mediated immunity, including down-regulation of IL-12 production, has been reported on macrophages (M ) by cross-linking Downloaded from their CD46. The intracellular events responsible for these immune responses, however, remain unknown. In this study, we found that 6- to 8-day GM-CSF-treated peripheral blood monocytes acquired the capacity to recruit protein-tyrosine phosphatase SHP-1 to their CD46 and concomitantly were able to produce IL-12 p40 and NO. These responses were induced by stimulation with mAbs ؅ F(ab )2 against CD46 that block MV binding or by a wild-type MV strain Kohno MV strain (KO; UV treated or untreated) that was reported to induce early phase CD46 down-regulation. Direct ligation of CD46 by these reagents, but not intracellular MV

replication, was required for these cellular responses. Interestingly, the KO strain failed to replicate in the 6- to 8-day GM-CSF- http://www.jimmunol.org/ cultured M␾, while other MV strains replicated to form syncytia under the same conditions. When stimulated with the KO strain, rapid and transient dissociation of SHP-1 from CD46 was observed. These and previous results provide strong evidence that CD46 serves as a signal modulatory molecule and that the properties of ligands determine suppression or activation of an innate immune system at a speciﬁc maturation stage of human M␾. The Journal of Immunology, 2000, 165: 5143–5152.

uman membrane cofactor protein (MCP;3 CD46) was and Neisseria gonorrhoeae (13, 14). Many ligands therefore bind ﬁrst identiﬁed as a complement regulatory protein the extracellular domains of CD46. widely expressed on nucleated cells (1, 2). Its main role The extracellular portion of CD46 consists of four SCRs and a

H by guest on September 25, 2021 was to protect host cells from homologous complement attack serine/threonine-rich (ST) domain (15, 16). The intracellular do- (3–7) by acting as a cofactor for protease factor I to irreversibly main is made up of a transmembrane region and a cytoplasmic tail inactivate C3b/C4b, both pivotal effectors of complement (8). (CYT). Polymorphism of the ST, transmembrane region, and CYT Later, other functions of CD46 were also reported in addition to domains, but not SCRs results in multiple isoforms of CD46 (17, the known complement regulatory function. CD46 serves as a 18). The composition of ST domains affects the degree of O-gly- measles virus (MV) entry receptor (9, 10) for most laboratory- cosylation, which controls efficacy of ligand binding to the SCR adapted strains and some wild-type strains. H protein of MV serves domains (19). CD46 functions were blocked with the mAbs, M75 as a ligand that binds to the N-terminal portion (SCR1 and SCR2) and M177, which recognize the SCR2 region (11, 12). Since the of CD46 (11, 12). CD46 also serves as a receptor for streptococci protein polymorphism of CD46 is caused by alternative mRNA splicing, intracellular domains of each isoform possess different primary structures (16–18). However, no physiological signifi- *Department of Immunology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Higashinari-ku, Osaka, Japan; †Nara Institute of Science and Technology, cance had been suggested for the intracellular heterogeneity of Ikoma, Nara, Japan; ‡Organization for Pharmaceutical Safety and Research, Tokyo, human CD46. Japan; §Department of Microbiology, University of Washington School of Medicine, Seattle, WA 98195; and ¶Department of Neurovirology, Research Institute for Mi- Recent studies indicate that CD46 may possess signaling func- crobial Diseases, Osaka University, Suita, Osaka, Japan tions that modulate cellular responses. Cross-linking CD46 on hu- Received for publication January 4, 2000. Accepted for publication July 26, 2000. man macrophages (M␾) with either MV, dimerized C3b, or CD46- The costs of publication of this article were defrayed in part by the payment of page specific mAbs leads to inhibition of IL-12 production in response charges. This article must therefore be hereby marked advertisement in accordance to LPS or Staphylococcus aureus Cowan (20). Cross-linking CD46 with 18 U.S.C. Section 1734 solely to indicate this fact. on human astrocytoma cells with a CD46-specific Ab increases 1 This work was supported in part by grant-in-aids from the Ministry of Culture, Technology, and Sciences, the Uehara Memorial Foundation, and PROBRAIN. production of IL-6 (21). Cross-linking of CD46 synergizes with 2 Address correspondence and reprint requests to Dr. Tsukasa Seya, Department of IL-4 to enhance IgE class switching in the human Ramos B cell Immunology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Hi- line (22). In human dendritic cells (DC), MV causes increased gashinari-ku, Osaka 537-8511, Japan. E-mail address: [email protected] expression of costimulators and IL-12, but results in a failure to 3 Abbreviations used in this paper: MCP, membrane cofactor protein; CAM, a mea- induce lymphocyte proliferation (23). Binding of Neisseria gon- sles vaccine strain; CYT, cytoplasmic tail; DC, dendritic cells; ED, Edmonston MV strain; KO, Kohno MV strain; MV, measles virus; M␾, macrophages; moi, multi- orrhoeae to human epithelial cells induces a transient increase in plicity of infection; NV, Nagahata MV strain; PAMP, pathogen-associated molecular intracellular calcium (14). These results suggest that ligand bind- pattern; p.i., postinfection; SAPK, stress-activated protein kinase; SCR, short consensus repeat; SHP-1, a protein-tyrosine phosphatase; ST, serine/threonine-rich ing to the consensus SCRs of CD46 results in intracellular signal- domain. ing, which is propagated through CYT.

In fact, Hirano et al. (24, 25) showed that mouse M␾-like cell The viruses were prepared as described previously (11). No contami- lines expressing human CD46 produce higher levels of NO upon nating LPS (Ͻ0.2 pg/ml) or a mycoplasma lipoprotein (Ͻ10 pg/ml) in the infection by MV in the presence of IFN-␥. This response is de- virus preparations was checked in our laboratory (data not shown). mAbs were obtained as follows: CD4, CD14, CD40, and CD64 from pendent upon the CD46 CYT, denoted CYT1. Previous reports PharMingen (San Diego, CA); CD71 from Dako (Copenhagen, Denmark), suggested that human M␾ and DC are primary targets for MV to and CD80 and CD86 from Ancell (Bayport, MN). CD14 microbeads were modulate costimulator levels and IL-12 production (20, 23, 26, obtained from Miltenyi Biotec (Bad Gladbach, Germany); FITC-conju- Ј 27). These findings are thought to be connected to the immune gated goat anti-mouse IgG F(ab )2 was purchased from Cappel (West Chester, PA); HRP-labeled goat anti-rabbit IgG was obtained from Bio- suppression seen after MV infection. It is possible that modulation Rad (Hercules, CA); anti-SHP-1, anti-Rac 1, and anti-Cdc42 were obtained of immune responses through CYT of CD46 in M␾/DC could be from Santa Cruz Biotechnology (Santa Cruz, CA); anti-IQGAP was a gift a major cause of the MV-mediated immune suppression. from S. Kuroda (Nara Institute of Science and Technology, Nara, Japan) However, it is known that immune activation is another pheno- (31); and nonimmune IgG was purchased from Sigma (St. Louis, MO). type seen with measles (28, 29). The lymphocyte proliferation mAbs against human CD46, M177, M75 (which recognize the SCR2 of ␥ human CD46 and block MV receptor activity of CD46); M160 (which markers, soluble IL-2 and IFN- , are increased in the early phase recognizes the SCR3 of human CD46 without blocking MV receptor ac- of MV infection (28, 29). In addition, soluble CD8, reflecting CTL tivity); and CD35 (CR1), 243R, were established in our laboratory (11, 32). Ј induction, is elevated Ͼ1 wk after the measles rash (28). These F(ab )2 of these mAbs were prepared as described previously (33). Anti- findings can be interpreted to indicate that CD46 signaling governs MV-H mAbs that block MV infection were obtained from Research Insti- tute for Microbial Diseases (Osaka University) and a generous gift from D. more than simple suppression of innate immune responses in hu- Gerlier (UniversiteĆlaude Bernard, Lyon, France) (30). man M␾/DC. The CYT of CD46 might involve a signaling path- ␾ way related to innate immune activation in M␾/DC even if im- Isolation of monocytes and their differentiation into M Downloaded from mune suppression is a typical or final phenotype in measles. The A monocyte-rich fraction was prepared from 400 ml of CPD-supplemented molecular mechanisms connecting structural variations of CD46 human blood by methylcellulose sedimentation and the Ficoll-Paque CYT with reported M␾/DC responses, however, are currently method (Pharmacia Biotech, Uppsala, Sweden) as described previously unknown. (34). The monocyte-rich fraction was treated with microbeads bearing anti- CD14 mAb and subjected to a MACS system (Miltenyi Biotec). Mono- Here, we found that the two mAbs that block the MV receptor cytes (1–5 ϫ 107) were collected with Ͼ90% purity from 400 ml of blood.

function of CD46, and a wild-type MV strain Kohno (KO) lead to Cells were pelleted, washed, plated in 10-cm plastic tissue culture dishes http://www.jimmunol.org/ the activation of M␾. The results demonstrated that cross-linking (no. 25020; Corning, Corning, NY) at 5 ϫ 106 cells/dish, and cultured for CD46 on human M␾ with either the KO strain or F(abЈ) of the 3–12 days at 37°C in 5% CO2 in 10 ml of RPMI 1640 (Life Technologies, 2 Grand Island, NY) containing 100 U/ml GM-CSF (PeproTech, London, mAbs enhanced IL-12 p40 secretion and/or NO production only at U.K.) and 10% FCS (BioWhittaker, Walkersville, MD). Morphological the time point in the maturation process when intracellular tyrosine changes were examined under a microscope (IX-70; Olympus, New Hyde phosphatase SHP-1 is recruited to CD46. Furthermore, these ef- Park, NY) (34). fector responses accompanied a rapid and transient dissociation of Determination of IL-12 p40 and p70, and IL-18 SHP-1 from the tail of CD46. We propose that CD46 can serve as a signal regulatory molecule for innate immune responses. A sandwich ELISA was performed to determine levels of IL-12 p40, IL-12

p70 (Genzyme, Cambridge, MA), and IL-18 type 1 (MBL, Nagoya, Japan) by guest on September 25, 2021 according to the manufacturer’s protocol. The level of IL-18 type 2 was Materials and Methods determined in our laboratory as previously described (35). The absorbance Cells, viruses, and Abs at 490 nm (A490 nm) was measured with a microplate photometer (MTP- 120, Corona Electric, Tokyo, Japan). The human monocytic cell line THP-1, the mouse erythroblastoid cell line MEL, the Chinese hamster ovarian tumor cell line (CHO), and the African Determination of NO green monkey kidney cell line Vero were purchased from American Type ␾ Culture Collection (Manassas, VA). Human monocytes were prepared The amount of NO in the culture medium produced by human M was from fresh human blood as described below. determined by measuring its end product NO in triplicate using a fluoro- ␾ The MV strains (30), Nagahata (NV) and Kohno (KO), were obtained metric method for mouse M NO (36) with modification. Briefly, equal ␮ from the Institute for Microbial Diseases, Osaka University (Osaka, Japan), volumes (100 l) of supernatant of phenol red-free culture medium and 10 ␮ and the National Institutes of Health, Japan, respectively. A CAM vaccine l of 2,3-diaminonaphthalene (dissolved in 0.62 N HCl; Calbiochem, La strain was purchased from Tanabe (Tokyo, Japan). The Edmonston (ED) Jolla, CA) were mixed in a 96-well plate at room temperature for 10 min. ␮ strain was obtained from A. Hirano. The wild-type strain PB was isolated The reaction was terminated by the addition of 5 l of 2.8 N NaOH, and in 1998 at Osaka University. KO (30) could be amplified in Vero cells the fluorescence signal was measured at 365 nm on a spectrophotometer (expressing Vero CD46 as the MV receptor). Amplification of KO was (BioSpec-1600, Shimazu, Tokyo, Japan). A standard curve was obtained Ј for each experiment using known concentrations of sodium nitrite. completely blocked with the mAb against CD46 (F(ab )2 of M75) (30). KO induced severe down-regulation of CD46 within 60 min in CHO cells Flow cytometric analysis expressing human CD46, although other strains did not (30). The known properties and the amino acid conversions in the KO H protein are shown Flow cytometry was performed as described previously (32). The in Table I. FACSCalibur program was used for measurement of the mean fluorescence intensities and comparative analysis. Immunoprecipitation and immunoblotting Table I. Summary of the properties of the MV strains tested Immunoprecipitation of CD46 and other proteins (CD4, CD35, etc.) was performed as described previously (33). In some experiments a cross-linker Amino Acidsa IL-12 p40 (M␾) Early-Phase (0.75 mM dithiobis-succinimidyl propionate (DSP) purchased from Pierce, CD46 Down- Rockford, IL) was used for linking proteins to CD46. For immunoblotting Strain 451 473 481 Untreated LPS-treated Regulation analysis, cells (2 ϫ 106 to 1 ϫ 107 cells) were washed with PBS (pH 7.4) and solubilized with 100 ␮l of 1% Triton X-100 containing 137 mM NaCl, ED V I Y 3n Ϫ 2 mM EDTA, 1 mM Na VO , 5 mM NaF, and 1 mM PMSF. After cen- NV ⅐⅐N 3n Ϫ 3 4 trifugation (10,000 ϫ g for 10 min), aliquots of the supernatants were KO A L N 11 ϩ subjected to SDS-PAGE under nonreducing or reducing conditions (33). PB ⅐⅐N 3n Ϫ Proteins were transferred onto nitrocellulose membranes. The blots were CAM ⅐⅐N 3n Ϫ incubated with 10 ml of 10% of skim milk (Morinaga, Tokyo, Japan) for a The amino acid conversions seen in the number of 430–500 of the H proteins are 1 h at 37°C, followed by addition of 10 ␮g of mAb. One hour later, the shown. The dots mean Val451 and Ile473. membranes were washed extensively with PBS containing 0.05% Tween The Journal of Immunology 5145

20 and then incubated with HRP-conjugated goat anti-mouse IgG or goat Results anti-rabbit IgG Abs (Bio-Rad) for1hat37°C. After thorough washing, Recruitment of SHP-1 to CD46 in 7-day cultured human M␾ proteins were detected with an ECL kit (Amersham). Human monocytes were cultured with GM-CSF to induce dif- ␾ Determination of phosphatase activities in M ferentiation to M␾. During the process of maturation, we de- CD46-associated phosphatase activity was determined using p-nitrophenyl tected an intracellular phosphatase SHP-1 coprecipitating with phosphate as a substrate. The assay was performed at 30°C for 30 min in the MV receptor CD46 on day 7 (Fig. 1A). SHP-1 reproducibly ␮ 20 l of reaction mixture (20 mM HEPES (pH 7.4), 1 mM EDTA, 10 mM coprecipitated with CD46 during days 6–8. The CD46-SHP-1 DTT, 10 mM p-nitrophenyl phosphate, and CD46 immunoprecipitates). ␾ The reactions were terminated by adding 100 ␮l of 1 N NaOH. The reac- association was specific in a certain activation stage of M , tion product p-nitrophenolate was quantified by measuring absorbance at since 1) M-CSF (substituted for GM-CSF) did not allow the 405–500 nm. recruitment of SHP-1 to CD46 in 7-day cultured M␾ (data not shown); 2) no SHP-1 was coprecipitated with an Ig superfamily MV infection assay protein CD4 (Fig. 1A); and 3) the CD46-SHP-1 association was The levels of messages for MV-H and -N proteins of the KO strain were observed in a human M␾-like cell line, THP-1 (Fig. 1B). M␾ determined by quantitative RT-PCR, as KO does not form syncytia in Vero express another SCR protein CR1 (CD35) concomitantly with cells or human cells. The amount of MV for other strains was expressed as cell differentiation, and CR1 may coprecipitate SHP-1, notably the multiplicity of infection (moi) and then confirmed by quantitative RT- ␾ PCR (see below). not during days 6–8 but after day 9 M (Fig. 1A). The SHP-1 Cells were cultured at 70% confluence in 24-well plates (Corning) for recruitment to CD46 is again specific to day 6–8 M␾. CD46 is 15 h and were infected with MV at an moi of 0.12, which was determined

known to bind the cytoplasmic protein moesin (40, 41), which Downloaded from ␾ through dose-response studies for optimal IL-12 p40 production by M . is associated with the small G protein Rho involved in cell The syncytia formed were observed 2–8 days postinfection (p.i.) (37). Cells were photographed under an Olympus microscope (IX-70) (38). motility (41–43). Other G protein-related molecules, including Cdc42, Rac1, and IQGAP, failed to be connected to CD46 in RT-PCR and quantitative RT-PCR any stage of M␾ (data not shown) or THP-1 even using a cross- Total RNA was isolated from either MV-infected or noninfected cells ac- linker (Fig. 1B). cording to a standard protocol using guanidium-HCl and acid-phenol/chlo- http://www.jimmunol.org/ roform (39). cDNA was synthesized with SuperScript II RNase H-Reverse Transcriptase (Life Technologies). The primers for detection of MV-H Effects of MV strains or mAbs binding to CD46 on cellular (upstream, 5Ј-TCAGTAATGATCTCAGCAACTG-3Ј; downstream, 5Ј- responses in M␾ TTCAATGGTGCCCCACTCGGGA-3Ј) were designed to amplify a 369-bp segment. As a control for the presence of ampliﬁable RNA, During M␾ differentiation, the cellular responses occurring GAPDH primers were used to amplify a 249-bp segment as previously through CD46 were investigated. CD46 of monocytes/M␾ was described (34). Ampliﬁed PCR products were analyzed by agarose gel cross-linked with either F(abЈ) of mAbs M177, M75 (data not (2.0%) containing ethidium bromide (39). 2 The quantitative RT-PCR assay was performed essentially according to shown), or M160 or with the MV strains ED, NV, PB, CAM, or the manufacturer’s guide book. The upstream primer (5Ј-GATGACAAGT KO at each differentiation step. M␾ responses, including IL-12 and

TGCGAATGGAGA-3Ј) and the downstream primer (5Ј-GACAAGAC NO production, were analyzed after the addition of these by guest on September 25, 2021 CCCGTATGAAGGAA-3Ј) were used for PCR amplification of MV-H stimulators. messages. Similarly, the upstream primer (5Ј-ACATTAGCATCTGAACTC GGTATCAC-3Ј) and the downstream primer (5Ј-TTTTCGCTTTGAT Within 24 h the addition of the wild-type strain KO strongly CACCGTGTA-3Ј) were used for N protein messages. The TaqMan probes induced the production of IL-12 p40 in 6- to 8-day GM-CSF- for quantitative analysis for H and N proteins were 5Ј-CCCGAGT cultured M␾ (Fig. 2A). Other strains, including ED and NV, did GGGCACCATTGAAGGATAA-3Ј and 5Ј-CCGAGGATGCAAGGCTT not induce IL-12 p40. Similar results were obtained with mAbs. GTTTCAGA-3Ј, respectively. The 5Ј ends were labeled with a fluorescein Ј Ј The F(ab )2 of M177 and M75 preferentially induced IL-12 p40 derivative (as a reporter), and the 3 ends were labeled with a rhodamine ␾ Ј derivative (as a quencher). Three micrograms of total RNA and 200 U of from the 6- to 8-day GM-CSF-cultured M , whereas the F(ab )2 of reverse transcriptase were used for one assay. RT was performed for 2 min M160 did not (Fig. 2B). Thus, the mAbs that block MV receptor at 50°C followed by 10 min at 95°C for activation of AmpliTaq Gold function of CD46 induced similar IL-12 p40 production as the KO (Takara, Tokyo, Japan). PCR was performed for 50 cycles of denaturation strain. IL-12 p70 as well as either type of IL-18, however, were for 15 s at 95°C and annealing and extension for 1 min at 60°C using an ABI PRISM 7700 (PE Biosystems, Foster City, CA). barely detected under the conditions tested (data not shown). Other factors produced by M␾ during maturation appeared not to be Expression of MV-H mutants on MEL cells and coculture assay involved in IL-12 production, since changing the medium had no with M␾ effect on IL-12 levels during each step of M␾ maturation. Thus, ␾ MV-H and MV-F cDNA of the NV strain placed in pME18s mammalian CD46-mediated IL-12 p40 induction depends on the M matura- expression vectors were prepared as described previously (38). Mutations tion step characterized by SHP-1 recruitment to CD46 and on the of single nucleotides to generate either V451A or I473L and both were ligands with which CD46 is stimulated. introduced into this NV MV-H-based cDNA by site-directed mutagenesis NO production in human M␾ was assessed in monocytes and (QuikChange, Stratagene). These two mutations resulted in the conversion day 3, day 7, and day 12 GM-CSF-cultured M␾. Unexpectedly, of the H protein sequence of the NV strain to that of the KO strain. Various rodent cells (39) were transfected with the MV-H cDNA of monocytes responded to the CAM vaccine strain, resulting in the NV, those with V451A or I473L single mutations or that of KO using release of a moderate amount of NO into the culture medium (Fig. Lipofectamine (38). Vector only was used as the control. Cells with high 3). In contrast, virtually no NO was detected at any stage of M␾ expression levels were screened through FACS Vantage using MV-H mAb maturation with any MV strain other than KO despite the addition as a marker. These cells were additionally transfected with MV-F cDNA in ␥ some experiments (38). Cells were cultured for 48 h at 37°C in RPMI of IFN- . A high level of NO production was induced again by the 1640/10% FCS. Of the cell lines tested, MEL and CHO cells were found KO strain only in 7-day GM-CSF-cultured M␾ (Fig. 3), although to express MV-H and -F with minimal cell damage. there was sample-to-sample variation in NO production by M␾. 6 MEL cells (5 ϫ 10 /well) expressing MV-H and -F or only MV-H were Interestingly, this response occurred at the same time as SHP-1 poured over human M␾ (5 ϫ 105/well) prepared as described above and centrifuged at 600 rpm for 3 min. Cells were cocultured for 24 h at 37°C recruitment to CD46. In contrast, cross-linking CD46 with M177 Ј in 0.5 ml of phenol red-free RPMI 1640/10% FCS. The levels of NO in the F(ab )2 did not result in NO production in 7-day GM-CSF-cultured supernatants were determined as described above. M␾ (data not shown). 5146 MEASLES VIRUS-MEDIATED CD46 SIGNALING Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021 FIGURE 1. Molecular association between CD46 and SHP-1 in human M␾. A, Recruitment of SHP-1 to CD46 in M␾ cultured with GM-CSF. Peripheral blood monocytes were cultured with GM-CSF (100 U/ml) for the indicated period. Cells (2 ϫ 106) were then solubilized, and the supernatants were collected by centrifugation. These samples were immunoprecipitated with M177 (top panel), 243R (center panel), and anti-CD4 (bottom panel) and run on SDS-PAGE followed by immunoblotting with a polyclonal anti-SHP-1 (upper column in each panel) or each specific Ab (lower column in each panel). The day 9 M␾ produced a relatively large amount of CR1 compared with the day 6–8 M␾ and concomitantly appeared to recruit a small amount of SHP-1 (exposed for Ͼ30 min). An arrow indicates the band of SHP-1 (upper columns). B, Specific molecular association between CD46 and SHP-1. THP-1 cells (2 ϫ 107) were treated with a cross-linker (indicated as ϩ) or with buffer alone (indicated as Ϫ), then solubilized. Soluble (Sol.) and insoluble (Insol.) fractions were separated by centrifugation. Insoluble fractions were further solubilized with PBS containing 0.05% SDS. These samples were immunoprecipitated with M177 and analyzed as described in a. Polyclonal Abs against various intracellular molecules (indicated over the panels) were used as probes. Although positive controls are not shown, all these Abs detected corresponding proteins by immunoblotting (data not shown). The nonspecific 30-kDa band was observed in all panels. Membranes were then stripped and reprobed with the rabbit polyclonal anti-CD46 Ab (lower panel). Molecular markers are shown. C, Kinetics of phosphatase activity and amounts of CD46-bound SHP-1 in GM-CSF-treated M␾. Phosphatase activity of the CD46 immunoprecipitates from M␾ (see Materials and Methods) was determined after the indicated culture periods. The same lots of cells were solubilized, and CD46-associated SHP-1 was evaluated as described in A. The amounts of SHP-1 were assessed by densitometer.

Alteration of costimulator levels on M␾ by MV stimulation irradiated MV strains were used to stimulate 7-day GM-CSF-cul- ␾ The expression levels of APC-related molecules were next as- tured M . The UV-irradiated KO strain retained the ability to ␾ sessed by flow cytometry. Surface levels of CD14, CD40, CD46, induce IL-12 p40 in M , although somewhat less potently (Fig. 4). CD71, CD80, and CD86 on monocytes were compared with those The replication ability of MV was completely abolished by UV on M␾ cultured with GM-CSF for 7 days (Table II). Significant treatment. One day p.i., IL-12 was again undetectable after the elevations of CD86 and CD80 were detected in 7-day-cultured M␾ addition of another MV strain regardless of UV irradiation. Thus, after the addition of KO (underlined in Table II), but not in mono- direct stimulation of CD46 with KO caused the induction of IL-12 cytes. In comparing day 3, day 6, and day 12 cultured M␾, the day p40 independently of viral replication. Induction of IL-12 p40 by 6 cultured M␾ were found to exhibit the most significant alter- direct binding of mAb against CD46 also supports this interpreta- ations (data not shown), again consistent with the time frame of tion and parallels the previous finding that the receptor for KO is SHP-1 recruitment to CD46. CD46 (30).

MV replication does not contribute to M␾ cellular responses Virus replication and syncytium formation To rule out the possibility that products, including dsRNA, result- We then analyzed the phenotypes of M␾ infected with the NV and ing from MV replication contribute to IL-12 p40 production, UV- KO strains. MV ampliﬁcation in 7-day GM-CSF-cultured M␾ was The Journal of Immunology 5147

FIGURE 3. NO production by various maturation stages of human M␾ stimulated with KO. Human M␾ cultured with GM-CSF for increasing Downloaded from times were incubated with various MV strains for 24 h at 37°C. NO production was determined by measuring nitrite in the culture medium. The bar atop each column represents the SD determined from three independent measurements. Although there was a sample-to-sample variation, a similar tendency was observed in two additional experiments. http://www.jimmunol.org/

The cytopathic effect is shown in Fig. 6. Monocytes were cultured for 3 and 6 days with or without GM-CSF, then inoculated with the NV, KO, or ED strain of MV (moi ϭ 0.12 or its equivalent), and morphological changes were followed under a phase-shift microscope. M␾ formed syncytia 2–3 days p.i., while monocytes were highly resistant to syncytium formation. In Fig. 6, MV-mediated syn-

cytia were observed in day 3 and day 6 cultured M␾ when either the by guest on September 25, 2021 NV or ED strain was added. Cells infected with KO would not form any syncytium under any of these conditions. Instead, KO induced FIGURE 2. IL-12 p40 production from 7-day GM-CSF-cultured M␾ a scattered pattern in both monocytes and GM-CSF-treated M␾ Ј after stimulation with various MV strains or F(ab )2 of mAbs against (Fig. 6, left column). This tendency was also seen in day 6 cultured human CD46. A, IL-12 production by MV strains. Monocytes (5 ϫ 105) M␾ (Fig. 6, right column). GM-CSF accelerated M␾ proliferation were cultured in a 24-well plate with GM-CSF for the indicated periods. and attachment to the dish (top row, middle and right columns of Medium was changed 2 days before use. The MV strains were added, Fig. 6). Infection by ED or NV resulted in cells with similar mor- and 24 h later the level of IL-12 p40 in the medium was determined by phological features, while KO infection produced elongated cells. sandwich ELISA. Six experiments were performed, and a representative ϫ 5 Infection with KO through CD46 suppressed viral ampliﬁcation one is shown. B, IL-12 production by mAbs. Monocytes (5 10 ) were ␾ cultured in a 24-well plate with 5 ␮g of the indicated regents for the and induced a unique phenotype in human M , rather than typical time periods shown. Medium was changed 2 days before use. mAbs syncytium formation. Ј (F(ab )2) against CD46 were added, and 24 h later the level of IL-12 p40 in the medium was determined by sandwich ELISA. The experiments were performed twice. Kinetics of SHP-1 recruitment to CD46 in human M␾ SHP-1 is involved in the negative regulation of immune responses, evaluated4hto7days p.i. (moi ϭ 0.12 or equivalent) using as it is a major phosphatase in lymphocytes and M␾ (44). As quantitative RT-PCR. MV-H mRNA was detected at 18 h and shown in Fig. 7A (Panels A and B), KO input induced a rapid and peaked at 48 h for the NV strain (Fig. 5). Similar results were transient dissociation of SHP-1 from CD46 in day 7 cultured M␾. obtained after infection with ED and PB (data not shown), but not After addition of NV (Fig. 7A) and ED (data not shown) strains, with KO (Fig. 5). The MV-H message for KO was slightly in- however, a much more gradual reduction was seen in the amount creased at 24 h, yet decreased at 48 h p.i. and was not present on of SHP-1 associated with CD46 over a 30-min period. Repetitive day 7 p.i. Even at the maximal time point, the relative level of viral analysis suggested that SHP-1 dissociates from CD46 15–20 min mRNA was about 500 times lower in KO-infected M␾ than in M␾ after KO input in M␾. Kinetics of CD46-associated phosphatase infected with other viral strains. With the KO strain, again the N activity paralleled the amounts of CD46-bound SHP-1 (Fig. 7C). protein message of MV failed to be detected in 7-day GM-CSF- Total phosphorylated proteins were slightly increased concomi- cultured M␾ after 48 h p.i. (data not shown). Unlike other strains, tantly with dissociation of SHP-1 in KO-infected M␾ (Fig. 7A, ampliﬁcation of KO was severely suppressed in M␾. Thus, only Panel D). In the human monocytic cell line THP-1, KO induced the KO strain activates M␾ to induce IL-12 p40 and NO and to SHP-1 dissociation from CD46 within 15 min, with reassociation up-regulate surface levels of costimulators, yet, unlike other MV at 30 min (Fig. 7B). This early response was not observed in strains, it failed to replicate or even survive in the targeted M␾. THP-1 cells with NV. 5148 MEASLES VIRUS-MEDIATED CD46 SIGNALING

Table II. Flow cytometric proﬁles of costimulators on 7-day GM-CSF- cultured M␾ before and after MV treatmenta

Monocytes 7-Day GM-CSF-Cultured M␾

Molecules Control NV KO Control NV KO

CD14 64.37 50.02 57.09 4.13 3.23 3.66 CD40 5.14 6.68 6.46 22.35 33.67 29.46 CD46 21.15 25.96 21.53 32.98 20.74 13.61 CD64 4.65 3.48 4.22 12.41 15.78 12.8 CD71 0 0.05 0.14 17.82 24.57 20.5 CD80 0.85 0.61 1.06 0.98 0.5 2.39b CD86 7.20 7.63 8.25 10.72 12.34 25.60b

a Three experiments were performed and one representative result is shown. b See the text for details.

MV-H of KO, but not NV, induces immune responses by M␾ FIGURE 5. Virus mRNA replication in 7-day GM-CSF-cultured M␾. To confirm that MV-H of KO is responsible for M␾ cellular re- ␾ Message levels of MV-H for the NV and KO strains were determined at the sponses, we performed coculture studies in which human M were indicated times by quantitative RT-PCR. The y-axis indicates the comput- Downloaded from incubated with MEL transfectants expressing MV-H of a KO-type er-processed values for relative levels of MV-H mRNA. The relative mutant, chimeric mutants (both generated from MV-H of NV), or amount of viral mRNA is about 500 times lower in KO-infected cells than intact MV-H of NV (Fig. 8). The construct of KO-type MV-H was in cells infected with other viral strains. The RT-PCR profiles of MV-H made by the addition of two mutations to the construct of MV-H mRNA for NV and KO are shown in the inset. An amplified fragment of of NV (which conferred the same amino acid sequence as KO GAPDH (249 bp) was tested as a control for the presence of amplifiable MV-H) (30). MEL cells expressing MV-H with the two mutations RNA (data not shown). Similar results were obtained with MV-N message http://www.jimmunol.org/ potentiated NO production by human M␾. Other mutant- and in- (data not shown). tact NV H-expressing cells barely induced augmentation of NO production by M␾. Similar results were obtained with cells expressing both H and F. Although the background NO levels were toplasmic tail of CD46 possesses motifs involved in protein tar- high, presumably due to species difference between M␾ and stim- geting and recruitment (9, 45, 46). In the cytoplasm, CD46 actually ulant cells, significant enhancement of NO production was repro- binds moesin (9, 40, 41), the phosphorylated version of which may ducibly observed by stimulation with KO-type H protein. A similar tendency was observed with IL-12 p40 (data not shown). Virtually, no enhancement of NO and IL-12 production was observed with by guest on September 25, 2021 monocytes and Ͻ3-day GM-CSF-cultured cells (data not shown). Hence, the M␾ cellular responses are attributable to the specific sequence of H protein and the maturation stage of M␾.

Discussion There is increasing evidence suggesting that CD46 is responsible for cellular responses (20, 24, 25). For most responses, Ab cross- linking of CD46 can replace MV infection. Furthermore, the cy-

FIGURE 6. Cytopathic effect induced by various MV strains on monocytes and GM-CSF-cultured M␾. Left column, Monocytes were incubated with NV, KO, or ED (moi ϭ 0.12) for 3 days. Rapid cell growth was observed in cells infected with NV and ED, but not in those infected with KO. KO induced elongated cells that adhered tightly. Center column, Monocytes were incubated with GM-CSF for 3 days, and then the indicated MV strains (moi ϭ 0.12) were added. After 3 days, syncytia were observed FIGURE 4. IL-12 p40 production from 7-day GM-CSF-cultured M␾ by in cells infected with NV or ED. Cells infected with KO show no syncytia, stimulation with UV-treated or untreated MV strains. MV strains (moi ϭ but several elongated cells are observed. Right column, Monocytes were 0.12 or equivalent), either UV treated (ϩUV) or untreated (ϪUV), were incubated with GM-CSF for 6 days, then the indicated MV strains (moi ϭ added to 7-day-cultured M␾. IL-12 p40 was determined 24 h p.i. as de- 0.12) were added. Again, syncytia were observed in cells infected with NV scribed in Fig. 2A. MV-H message was assessed by RT-PCR 24 h p.i. and or ED. Cells infected with KO show a more pronounced elongation proﬁle is depicted in the inset. One of two experiments is shown. with no syncytia. The Journal of Immunology 5149

act as a Rho-mediated signal transducer (42, 43). Direct evidence for CD46 signaling, however, has remained elusive. This study demonstrated that CD46 allows signaling in human M␾. An intracellular phosphatase, SHP-1, was found to be stage speciﬁcally recruited to CD46 in M␾. After stimulation with ligand, SHP-1 transiently dissociated from the tail complex of CD46. Interestingly, these responses were induced in 6- to 8-day GM-CSF-cultured M␾ stimulated with the wild-type MV strain KO or with the mAbs that block the MV receptor function of CD46. Examples of cellular responses propagated by M␾ CD46 include induction of IL-12 p40, NO production, and up-regulation of the surface levels of costimulators. A scattered morphology was also induced in M␾ after CD46 cross-linking, similar to that reported for early phase down-regulation of CD46 (30). These ﬁnd- ings suggest that SHP-1, moesin, and presumably unknown phosphatase substrates are involved in an intracellular complex with CD46, and that ligand stimulation of CD46 during virus infection plays a major role in signal transmission and cellular responses.

However, the participation of another MV receptor or other virus- Downloaded from binding molecules (47) in the observed immune responses cannot be ruled out. MV infection induces transient suppression of host immunity, leading to secondary infections that are a major cause of death in measles patients (48, 49). MV-infected APCs can suppress the

proliferation of unaffected lymphocytes, most likely through cell- http://www.jimmunol.org/ to-cell contact (23, 26, 27). Temporal lymphopenia and loss of delayed-type hypersensitivity reaction were found in measles patients, which cannot be fully explained by MV susceptibility of lymphocytes or MV-mediated cytolysis (48, 49). Recently, the mechanism of immune suppression has been investigated in association with CD46 function in APC (20, 23, 26, 27). The present data suggest, however, that in some cases M␾ are activated through CD46, as measured by levels of surface markers, cytokines, and NO production. Our results demonstrating the in- by guest on September 25, 2021 ability of the KO strain to grow in the CD46-prestimulated M␾ may in part agree with the previous idea that MV infection produces low levels of viral proteins and virtually no infectious virus in M␾ (37). An attractive hypothesis is that APC serve as reser- voirs of infectious materials and their CD46 CYT may essentially activate M␾/DC to eliminate invading materials. Indeed, immune activation profiles have been observed in the early phase of infection in measles patients (28, 29). In addition, Schnorr et al. (23) showed that human DC precursors are matured by infection with MV, particularly the wild-type WTF strain, resulting in the up- regulation of HLA-DR, CD83, and CD86 as well as IL-12 syn- thesis. Our DC analysis with the KO strain is currently in good agreement with these reports, supporting the presence of another FIGURE 7. Dissociation of SHP-1 from CD46 by MV stimulation. A, The CD46 functional pathway, immune activation. Although M␾ and 7-day GM-CSF-cultured M␾ (5 ϫ 106 in each lane) were stimulated with NV DC have distinct properties, these results with DC parallel our or KO for the indicated intervals at 37°C. The M␾ were solubilized, and the results with M␾. MV strains free from immune suppression would supernatants were immunoprecipitated with M177. No cross-linker was used be useful for the development of measles vaccine and therapeutic in this experiment. The precipitates were analyzed by SDS-PAGE followed by vectors. immunoblotting with an anti-SHP-1 Ab as a probe. Two independent data are Three cross-linkers of CD46, including mAbs, C3b dimer, and shown (A and B). The total amounts of SHP-1 and phosphorylated proteins in each sample are shown in C and D, respectively. The amounts of SHP-1 in MV (ED), are all reported to lead to the suppression of IL-12 ␾ each sample were not affected by MV stimuli. The arrow indicates the SHP-1 production in human activated M (20). Unlike M75 and M177, band. Cont., untreated cells. The m.w. markers are shown to the left. The other mAbs tested could not inhibit MV infection (20). The bind- 69 experiments were performed six times, and similar results were obtained. B, ing sites on CD46 for M75 and M177 contain Arg (11, 50, 51), THP-1 cells (1 ϫ 107 in each lane) were stimulated with NV or KO, solubi- which is not shared with the epitopes of other mAbs. The primary lized, immunoprecipitated, and analyzed as described in A. The arrow indicates structure of the H protein of the KO strain differs from those of the the SHP-1 band. The bands around 30 kDa are nonspecific contaminants. ED and NV strains (30). The CAM vaccine strain also possesses a Cont., Untreated cells. C, Kinetics of phosphatase activity and amounts of unique H protein (52) that may cause the unidentified monocyte- ␾ CD46-bound SHP-1 in KO-stimulated M . Phosphatase activity and amounts stimulating activity (Fig. 3). We confirmed that MV strains other of the CD46 immunoprecipitates from M␾ were measured after the indicated than KO and the mAb M160, which did not block the MV receptor stimulation periods as described in Fig. 1C. function of CD46, tended to suppress the stimulation-dependent 5150 MEASLES VIRUS-MEDIATED CD46 SIGNALING Downloaded from

FIGURE 8. Establishment of MV-H-expressing MEL cells and enhancement of NO production by M␾ cocultured with MEL cells expressing KO type H protein. Left panel, Flow cytometric analysis for assessment of the expression levels of various types of MV-H. The MV-H-positive transfectants were sorted by FACS Vantage and cultured for 48 h. Cells were again stained with anti-MV-H mAb and FITC-labeled second Ab and analyzed by FACS. The y-axis shows the relative cell number; the x-axis shows levels of MV-H. The types of MV-H transfected are indicated in the inset. Of note, introduction http://www.jimmunol.org/ of the two mutations to the construct of MV-H of NV resulted in the construct of MV-H of KO (30). Right panel, NO levels produced by M␾ cocultured with MEL transfectants expressing various MV-H or H plus F. M␾ were incubated with 10-fold more stimulant cells for 48 h at 37°C as described in Materials and Methods. The supernatants were collected, and the levels of NO were determined. The types of MV-H are shown in the figure. When the stimulant cells expressed NV-type MV-H and -F proteins, NO was barely detected, since human M␾ were largely killed by apoptosis for the reason described previously (38). production of IL-12 p40 (data not shown). Immune-suppressive that M␾ produce C3, which, in turn, is activated by M␾ surface signal may be a common feature compared with activation signal proteases (57, 58). The activated C3b can then bind back to the by guest on September 25, 2021 in CD46 cross-linking (20). M␾ in an autocrine fashion (57). The C3b binding to CD46 may One possible explanation for the functioning of CD46 in both also be involved with the SHP-1-dependent regulatory mechanism the suppression and activation of cellular responses is the existence of M␾. of multiple signaling pathways, involving the various isoforms of We have not yet identified the mechanism of SHP-1 recruitment CD46 with their differing cytoplasmic tails. Additionally, the prop- to CD46. The presence of two SH2 domains in SHP-1 provides a erties of the ligands for CD46 appear to be critical in the activation structural prerequisite for the diverse range of molecular interac- of M␾. The two amino acids Val451 and Ile473 in the H protein of tions in which this phosphotyrosine phosphatase appears to par- the NV strain were converted to Ala451 and Leu473 in the H protein ticipate (59). The VxYxxL sequence in the CD46 juxtamembrane of the KO strain (30). These differences are localized within (or domain (60) may be too close to the inner surface of the plasma near) the region reported to be involved in H binding to CD46 membrane to allow access of SHP-1 to this sequence. There are no (53–56). Thus, an alternative interpretation is that the occurrence immunoreceptor tyrosine-based inhibitory motif present in any of immune suppression or activation depends on the portion of isoform of the CD46 molecule (61). Thus, it remains unclear CD46 that is accessible to mAbs or ligands. IL-12 regulation, for whether the effect is mediated directly by receptor phosphorylation example, depends on the properties of the ligands and signaling. or indirectly by phosphorylation of receptor-associated proteins or Additionally, our study demonstrates that the ligand-binding por- protein tyrosine kinases. Recently, IL-12 p40 production was re- tion of CD46, but not MV replication, is important for determining ported to be regulated through the stress-activated protein kinase positive or negative cellular responses. Other factors, including the (SAPK) activation pathway (62). The family of mitogen-activated maturation stage of the M␾, may contribute to the final outcome of protein kinase kinase proteins is about 40 kDa and may be phos- the response. phorylated during SAPK activation (62). Indeed, our preliminary It is surprising, however, that only a two-amino acid difference data suggest that a CD46-associated 40-kDa protein can be de- in the H protein can cause opposite responses. The altered amino tected concomitantly with dissociation of SHP-1 from CD46 by in acids are near the Tyr481/Asn, which may determine CD46-medi- vitro kinase assay in the 7-day GM-CSF-cultured M␾ only when ated down-regulation and CD46 binding capacity (53–55). We the M␾ were treated with the KO strain. Studies of motheaten mice have made the cDNA constructs encoding the H protein of KO by (me/me), known to be homozygous for loss-of-function mutations site-directed mutagenesis. The relevant point was in part confirmed in the SHP-1 gene (63), as well as human CD46 transgenic mice by stimulating M␾ with murine cells expressing various mutant H may give some insight into these possibilities. proteins but no CD46 counterpart. M␾/DC are the main effectors of the innate immune system. CD46 was initially identified as a C regulator that was ubiqui- They express receptors for foreign material, i.e., bacteria, fungi, tously expressed on human nucleated cells (2, 18) and has not been and viruses, and mature into active phenotypes through stimulation analyzed as a signal-transducing receptor. Previous reports suggest with ligand. The ligands for these receptors are known to contain The Journal of Immunology 5151 pathogen-associated molecular pattern (PAMP) (64, 65). Recently, 19. Iwata, K., T. Seya, H. Ariga, S. Ueda, and S. Nagasawa. 1994. Modulation of representatives of the PAMP receptors were recognized as Toll- complement regulatory function and measles virus receptor function by the serine-threonine-rich domains of membrane cofactor protein (CD46). Bio- like receptors (66–69). It has been found that many proteins with chem. J. 304:169. SCR also serve as bacteria and virus receptors (70). We hypoth- 20. Karp, C. L., M. Wysocka, L. M. Wahl, J. M. Ahearn, P. J. Cuomo, B. Sherry, G. Trinchieri, and D. E. Griffin. 1996. Mechanism of suppression of cell- esize that both SCRs and leucine-rich repeats in Toll-like receptors mediated immunity by measles virus. Science 273:228. are PAMP recognition motifs that modulate innate immune func- 21. Ghali, M., and J. Schneider-Schaulies. 1998. Receptor (CD46)- and replica- tions. The responses via these receptors lead to the activation or tion-mediated interleukin-6 induction by measles virus in human astrocytoma cells. J. Neurovirol. 4:521. suppression of the immune system. In this study, we show that a 22. Imani, F., D. Proud, and D. E. Griffin. 1999. Measles virus infection syner- SCR protein, CD46, which was previously identified as a C reg- gizes with IL-4 in IgE class switching. J. Immunol. 162:1597. ulator and an MV receptor, also has a novel signal regulatory func- 23. Schnorr, J. J., S. Xanthakos, P. Keikavoussi, E. Kampgen, V. ter Meulen, and ␾ S. Schneider-Schaulies. 1997. Induction of maturation of human blood den- tion in M . dritic cell precursors by measles virus is associated with immunosuppression. Proc. Natl. Acad. Sci. USA 94:5326. Acknowledgments 24. Hirano, A., Z. Yang, Y. Katayama, J. Korte-Sarfaty, and T. C. Wong. 1999. Human CD46 enhances nitric oxide production in mouse macrophages in We are grateful to Drs. K. Toyoshima and H. Akedo (Osaka Medical Cen- response to measles virus infection in the presence of ␥ interferon: depen- ter, Osaka, Japan) for support of this work, and to Drs. O. Hazeki, dence on the CD46 cytoplasmic domains. J. Virol. 73:4776. N. A. Begum, and M. Nomura (Osaka Medical Center) for invaluable 25. Wong, T. C., S. Yant, B. J. Harder, J. Korte-Sarfaty, and A. Hirano. 1997. The discussions. Generous gifts of reagents from Dr. Kuroda (Nara Institute of cytoplasmic domains of complement regulatory protein CD46 interact with multiple kinases in macrophages. J. Leukocyte Biol. 62:892. 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