key: cord-0857387-obgk7b35
authors: Coutelier, Jean‐Paul; Godfraind, Catherine; Dveksler, Gabriela S.; Wysocka, Maria; Cardellichio, Christine B.; Noël, Henri; Holmes, Kathryn V.
title: B lymphocyte and macrophage expression of carcinoembryonic antigen‐related adhesion molecules that serve as receptors for murine coronavirus
date: 2005-12-01
journal: Eur J Immunol
DOI: 10.1002/eji.1830240622
sha: 4e2b2def7cc16262328f9d84a05238ee49326ead
doc_id: 857387
cord_uid: obgk7b35

The expression of carcinoembryonic antigen (CEA)‐related glycoproteins that have been associated with intercellular adhesion and that serve as receptors for mouse hepatitis virus (MHV) was analyzed in cells from the immune system of BALB/c mice using immunolabeling and RNA polymerase chain reaction amplification of receptor transcripts. These glycoproteins, which are called biliary glycoproteins, were highly expressed in B lymphocytes, including cells of the B‐la (CD5(+)) lineage, and in macrophages, but were not detectable in resting T lymphocytes. Similarly, murine cell lines of B cell and macrophage origin expressed messenger RNA encoding CEA‐related molecules, while the corresponding mRNA was only slightly detectable in a T cell line. These CEA‐related cell adhesion glycoproteins were also expressed in endothelial cells. Therefore, their specific interaction with their so far unknown ligand may be of functional importance in cellular interactions in the immune response. Monoclonal antibody directed against these glycoproteins blocked MHV‐A59 infection of the B cell‐derived SP20 cell line. Thus, the functional receptors for MHV on B lymphocytes, like those on murine fibroblasts, are isoforms of CEA‐related glycoproteins. Treatment of B cells with anti‐receptor antibody also blocked B cell‐mediated cytotoxicity against MHV‐A59‐infected fibroblasts, indicating that this phenomenon is mediated by interaction of viral attachment protein on the infected target cells with specific CEA‐related receptor glycoproteins on the effector B cells.

Efficiency of the immune system relies on the development of an appropriate communication network between various cell types. Such a communication system involves both soluble lymphokines that can reach their target cells at some distance and molecules that allow direct recognition between different cell types. It has indeed been demonstrated that adhesion molecules belonging to three distinct families, namely the Ig superfamily, the integrin family and the selectins, play a crucial role in various immune func-

tions [l] . In the Ig superfamily, different mouse isoforms of glycoproteins related to the biliary glycoprotein (BGP) group in the carcinoembryonic antigen (CEA) family have been shown to promote adhesion between transfected fibroblasts [2, 31. The homologous rat molecule, cell-CAM 105, mediates adhesion between hepatocytes [4] , and human CEA is a cell adhesion molecule for colon cells and for transfected cells [5, 61. Receptors for mouse hepatitis virus (MHV)-A59, a virus with known lymphotropism, have been identified as glycoproteins in the CEA-related family of BGP , and clones, oligonucleotides and antibodies specific for these molecules are now available. BALB/c and SJL/J mice differ markedly in susceptibility to h4HV infection [12] , and their receptor glycoproteins, called MHVR and mmCGM2, respectively, differ significantly in the N-terminal Ig-like domain that is the site of virus binding [13] . Splicing of transcripts occurs in various murine tissues, resulting in isoforms with either two or four Ig-like domains, and with either long or short cytoplasmic tails.

Using anti-MHVR antibodies and PCR to analyze expression of the isoforms of this molecule in BALBlc immune cells, we found that virus receptors were highly expressed in B lymphocytes and macrophages, but that little or no receptor was expressed in T lymphocytes. Expression of these BGP glycoproteins on B cells correlated with functional virus receptor activity and with the MHV-specific, B cell-mediated cytotoxicity previously described [14, 151 . This is the first report of the expression and function of CEA-related molecules on murine lymphocytes and macrophages.

Specific pathogen-free female BALB/c mice were bred at the Ludwig Institute for Cancer Research by Dr. G. Warnier or purchased from Iffa Credo (EArbresle, France) or Jackson Labs (Bar Harbor, ME) and were used for experiments at the age of 6-8 weeks. Animals were infected by i.p. injection of approximately lo4 tissue culture infectious doses (TCIDso) of the A59 strain of MHV (MHV-A59) grown in NCTC 1469 cells. Cell cultures were infected with MHV-A59 grown in 17 CI 1 cells [lo].

esceinated streptavidin for 10 min at 4 "C and MACS biotin microbeads (5 pl in 500 pl PBS containing 5 mM EDTA and 1 YO BSA), for 5 min at 4°C. Cells were then loaded in a   magnetic cell sorter on a cooled B2 column through a   22-gauge needle, washed with 20 ml PBS containing 1 YO   BSA using a 19-gauge needle and eluted outside the  magnetic sorter with 40ml PBS with BSA. For Tcell  purification, an additional treatment with anti-Mac-1, anti-IAd and anti-B220 antibodies, followed by complement was performed before the MACS separation. The purity of the cell preparations was checked after labeling with biotinylated anti-B220 or anti-Thy-1 mAb, followed by alkaline phosphatase streptavidine and naphtol-AS-MX-phosphate. B cell preparations contained 87-97 % B and approximately 2 YO T lymphocytes and T cell preparations contained 90-94 YO T and 2-3 YO B lymphocytes.

Mouse anti-MHV antibody was obtained 34 days after infection of BALB/c mice with MHV-A59. Polyclonal 2.4 Tissue preparation and immuno,abeling mouse anti-receptor antibody was raised by immunization of SJL/J mice with deoxycholate-solubilized intestinal brush border membranes from adult BALB/c mice [16] . This serum recognized the 100-120-kDa receptor for MHV-A59 in Western blots, immunoprecipitated this and the 58 kDa isoform, recognized the MHVR glycoprotein expressed in hamster cells from cloned MHVRl cDNA, and protected mouse fibroblasts from infection with MHV-A59. Polyclonal rabbit anti-receptor antibody (#655) was obtained after immunization of New Zealand White rabbits with immunoaffinity-purified receptor from Swiss Webster mouse liver [9] . Anti-receptor mAb (MAb-CC1) was derived from SJL/J mice immunized with protein extracted from BALB/c intestinal brush border membranes [7] . MAb-CC1 reacts with the N-terminal domain of MHVR and blocks binding of MHV-A59 to MHVR glycoproteins [13, 161. MAb-CC1 will be made available for research purposes upon request to K. V. Holmes.

WEHI-3, a macrophage-like cell line of BALBlc origin, [17] (ATCC, Rockville, MD) was grown in RPMI medium with 10 YO FCS. SP207 (gift from J. Van Snick), a cloned cell line from the BALB/c myeloma SP2/0-Ag14 [18] , was maintained in Iscove's medium containing 10 % FCS. TB6D2 (gift from A. Vink) was derived from a BALB/c plasmacytoma [19] . TSlC3 is a subclone of TS1, that was derived from TUC2.15, a T helper cell line of C57BL/6 origin [20] (gift from C. Uyttenhove). It was cultured in Dulbecco's modified Eagle's medium supplemented with 10 % FCS, 50 pM 2-mercaptoethanol, 0.55 mM L-arginine, 0.24 mM L-asparagine and 1.25 mM L-glutamine. The 17 C1 1 line of spontaneously transformed BALB/c 3T3 cells was used to propagate MHV-A59 and as a target for cytotoxicity studies [ 141 and the L2 cell line was used for plaque assays.

Total B lymphocytes were purified either by panning on plates coated with anti-mouse Ig antibody, as described previously [21] or by magnetic cell sorting (MACS, Miltenyi Biotec, Bergisch-Gladbach, Germany). After depletion of macrophages by two sequential incubations in tissue culture dishes, lo* spleen cells were incubated for 10 min at 4°C with biotinylated anti-B220 rat mAb, followed with fluor-Tissues were prepared for the ultracryosection technique of Tokuyasu [22] . 

After lysis of erythrocytes in 0.83 YO NH4CI, spleen cells were incubated for 60 rnin at 4 "C in HAFA buffer (137 mM NaCl, 5 mM KCI, 0.4 mM MgS04, 0.3 mM MgC12, 5 mM glucose, 4 mM NaHC03, 1 mM EDTA, 1 mM phosphate, 20 mM NaN3, 100 U/ml penicillin, 100 pg/ml streptomycin, pH 7.4, supplemented with 3 YO FCS) with control rabbit serum or rabbit anti-receptor antiserum #655, followed by fluoresceinated swine anti-rabbit Ig antibody (Dakopatts, Copenhagen, Denmark). After fixation in 0.62 ' 1' 0 paraformaldehyde, fluorescence was analyzed with an ATC3000 flow cytometer (ODAM, Wissembourg, France). Cell subpopulations were analyzed by simultaneous labeling with biotinylated mAb specific for surface markers followed by streptavidin-conjugated RED613 (Gibco BRL, Gaithersburg, MD). For triple labeling, cells were incubated with fluoresceinated, biotinylated and phycoerythrin-conjugated antibodies, followed by Cy-chrome-labeled streptavidin (PharMingen, San Diego, CA).

Primers from conserved regions that recognize either the MHVR isoforms or mmCGM2 isoforms of murine BGP were used to determine which splice variants were transcribed in the cells of the BALBk immune system. The antisense primer (5'-CTGAATAAACTGTTTCTGTGG-an irrelevant antigen. Cells were then washed and used as effector cells to lyse MHV-A59-infected 17 CL 1 cells prelabeled with 51Cr, followed by incubation for 4 h at 37 "C in a C02 incubator as described previously [14, 15, 25] .The specific 51Cr release was calculated as follows:

% specific 51Cr release = C-3') maps to the 3' untranslated region and the sense primer (5 '-A ATTCGA AGA AGCTAGCAG GCAG G-3') maps to the 5' untranslated region of both isoforms. Using 2 pg of RNA isolated from cells by the guanidine isothiocyanate method [23] with the antisense primer and 0.5 mM of each of the four nucleotide triphosphates, cDNA was synthesized with 200 U of Moloney murine leukemia virus RNase H reverse transcriptase (Life Technologies) at 42 "C for 1 h, after which the reaction was incubated for an additional 10min with 2 U of E. coli RNase H. The reaction was terminated by heating at 95°C for 10 min. Controls without template RNA were included. The cDNAs were amplified for 30 cycles with AmpliTaq (Perkin-Elmer Cetus) in a 100-pl reaction with the two primers.The PCR products were analyzed by Southern blot hybridization with 32P-labeled oligonucleotides (5'-ACAC-TACGGCTATAGACAAAG-3' and 5'-AGACTACAAC-AGGGCCTG-3') that hybridize to the first domain of MHVR or mmCGM2 isoforms, respectively [ 101. The sizes of the PCR products indicated whether the transcripts encoded BGP with two or four Ig-like domains. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) expression was used as an internal control to test the quality of the cDNA [24] .

SP20 cells were incubated for 60 min at 37 "C with 2 ml of a 1 : 2 dilution of MAb-CC1 culture supernatant or with a control IgGl mAb, the challenged with MHV-A59. After virus adsorption for 1 h at 37"C, cells were washed twice with 5 ml of medium. At 1 h after virus addition (p. i.), control and infected cells were attached to coverslips with poly-L-lysine. At 8 h p.i., cells were fixed with acetone at -25 "C, and immunolabeled with mouse anti-MHV Ab, followed by rhodamine-conjugated goat anti-mouse Ab. To determine the effect of anti-receptor Ab upon the yield of MHV-A59, aliquots of 5 x lo6 SP20 cells were incubated in 2 ml of a 1 : 2 dilution of MAb-CC1 or control mAb for 1 h at 4 7 T , then pelleted and resuspended in 2 ml of medium with 2 x lo7 PFU of MHV-A59 and incubated at 37 "C for 1 h. Cells were then washed three times and resuspended in a final volume of 5 ml of medium and plated in 60-mm tissue culture dishes. Supernatant medium was harvested at 1, 18,25 and 50 h pi., and the infectivity of released virus was analyzed by plaque assay in L2 cells.

(experimental cpmspontaneous cpm) 1 x 1 0 .

-.

L (total cpmspontaneous cpm) 1

Spontaneous release and total release were determined from target cells incubated with medium alone or with 5 % triton detergent, respectively.

To determine whether CEA-related glycoproteins were expressed in the immune system, 1-pm sections of BALB/c spleen were analyzed after immunolabeling with anti-MHVR antibody #655. Large clusters of lymphoid cells that may correspond to follicles, as well as vascular endothelial cells were strongly stained by this antibody (Fig. 1A) . Higher magnification showed that the receptor was distributed in small dots on the membranes (Fig. 1C) . Lympocytes in adjacent areas of the spleen were not labeled by anti-receptor antibody, suggesting that the receptor glycoproteins were only expressed on particular subsets of spleen cells. No reaction with spleen cells occurred with control normal rabbit serum (Fig. 1B) .

CEA-related glycoproteins were also detected on spleen cells by flow cytometry analysis. As shown in Fig. 2 , approximately 44% of these cells were recognized by anti-MHVR MAb-CC1, confirming the expression of these glycoproteins in cells of the immune system. Similar results were obtained with polyclonal antibody #655 (data not shown). 

Spleen cell subpopulations that expressed CEA-related glycoproteins were identified by flow cytometry analysis by double labeling with antibody #655 and mAb to different cell markers. B lymphocytes and macrophages bound anti-MHVR antibody ( Fig. 3B and F) , while most T lymphocytes, identified either by anti-Thy-1 mAb or by CD4 and CD8 subset markers,were not significantly labeled with antibody #655 (Fig. 3C-E) . Similar results were obtained when cells were incubated with anti-receptor MAb-CC1 (data not shown).

Expression of CEA-related glycoproteins on macrophageand B lineage-derived cell lines correlated with expression on B cells and macrophages isolated from the spleen. Anti-MHVR antibody #655 reacted strongly with WEHI-3, a macrophage-like cell line of BALB/c origin, and SP207, a BALB/c myeloma ( Fig. 4A and B) , but only minimally with TSlC3 cells derived from a C57BV6 T helper line (Fig. 4C) . Because antibody #655 labeled spleen cells from C57BY6 and BALB/c mice similarly (data not shown), the differences in staining of the cell lines could not be attributed to the strain from which the lines originated and probably reflected the types of cells studied.

RNA PCR of cell populations isolated from BALB/c spleen and of lymphocyte and macrophage cell lines also showed that CEA-related glycoproteins were expressed in B cells and macrophages, but barely in T cells (Fig. 5) . In some experiments, with an increased number of amplification cycles, MHVR expression was detected in T cells but at much lower levels than in B cells or in macrophages (data not shown). However, it is not clear whether this observation was due to very low expression of the CEA-related glycoprotein message in T lymphocytes or to minor contamination of the samples with a few B lymphocytes or macrophages. The levels of CEA-related glycoprotein expressed in TB6D2 cells varied from one experiment to another, possibly reflecting effects on expression of CEArelated proteins or modifications in cell conditions (data not shown). The sizes of the PCR products indicated that the B cells, macrophages, WEHI-3 and SP207 cells all produced transcripts that would encode proteins with two and four Ig-like domains. Blotting with oligonucleotides specific for either the MHVR or mmCGM2 alleles showed that these BALBk or C57B1/6 cells and cell lines expressed only transcripts of MHVR (Fig. 5) , and not mmCGM2 (data not shown).

CD5+ B lymphocytes constitute a separate lineage of B cells, called B-la, that is particularly abundant in neonatal mice and in the NZB strain and may play an important role in autoimmune responses [26, 27] . Expression of MHVR glycoproteins was analyzed in this B-la cell subpopulation by flow cytometry after triple labeling of spleen cells from 1-week-old BALBk mice with anti-B220, anti-CD5 and anti-MHVR antibodies. As shown in Fig. 6 , expression of CEA-related glycoproteins was as strong on CD5+ B lymphocytes as on CD5-B cells (Fig. 6C-D) , while CEA-related glycoproteins were not detected on MHV receptor on B lymphocytes and macrophages 1387 B220-CD5+ T lymphocytes (Fig. 6B) . Similar results were obtained with NZB mice and on B-la cells identified with anti-IgM and anti-CDS antibodies (data not shown).

One of the functions of the murine CEA-related BGP proteins is as a receptor for mouse coronavirus MHV. We therefore tested whether that CEA-related glycoprotein expressed in mouse B cells could serve as a receptor for MHV. Pretreatment of SP20 cells with anti-MHVR MAb-CC1 blocked infection of the cells by MHV-A59 (Fig. 7) . At 8 h p i , viral antigens developed in the cytoplasm of cells pretreated with medium alone or with irrelevant antibody (Fig. 7b and c) , but no infected cells were observed in cultures pretreated with anti-receptor MAb-CC1 (Fig. 7d) . Table 1 shows that pretreatment of cells with MAb-CC1 inhibited the replication of virus in SP20 cells. Thus, anti-MHVR MAb-CC1 blocked virus receptor activity on SP20 cells of the B cell lineage, showing that the CEA-related glycoproteins expressed on these cells serve as functional receptors for MHV-A59.

To determine whether this ability of MHV-A59 to infect B lymphocytes would cause lysis of large numbers of B cells in vivo, spleen cell subpopulations were isolated from BALB/c mice at different times after virus inoculation and analyzed by flow cytometry. As shown in Table 2 , only minor changes in total number of spleen cells and no change in the relative proportions of the various cell subpopulations were observed.

Mouse fibroblasts infected with MHV-A59 can serve as target cells for an unusual, rapid, MHV-specific, non-MHC restricted cytotoxicity mediated by B cells from naive BALB/c mice which have not been exposed to MHV [14, 15, 251 . We previously showed that treatment of the target cells with antibody directed against the viral spike glycoprotein prior to addition of the effector cells could block this cytotoxic reaction and postulated that the effect was mediated by interaction of virus glycoproteins on the target cell with receptors on the effector cell [14, 151 . We pretreated the effector B cells from BALBlc mice with polyclonal mouse anti-receptor antibody or normal SJL/J mouse serum or with anti-MHVR mAb-CC1 or control mAb to determine whether blocking of virus receptors on the B cells could prevent the B cell-mediated cytotoxicity or "virus-killer'' (VK) activity. Table 3 shows that both polyclonal mouse anti-receptor antibody and MAb-CC1 prevented MHV-specific cytotoxicity mediated by spleen cells of BALB/c mice, while neither control antibody had any effect. SJL/J spleen cells did not induce B cellmediated cytotoxicity [14] . The values of specific T r release in this assay when antibody-treated, serum-treated or untreated SJL/J spleen cells were used as effectors varied between 0 and 3 % (data not shown). Thus, as previously demonstrated, spleen cells from SJLlJ mice could not serve as effectors for this cytotoxicity assay. Interestingly, SP20 cells could also serve as effectors of B cell-mediated, MHV-specific cytotoxicity, and this function was also blocked by polyclonal anti-receptor antibody ( Table 3) .

Using immunohistochemistry, flow cytometry, RNA PCR and immunoblotting (data not shown), we observed expression of CEA-related glycoproteins in mouse macrophages and B lymphocytes, including cells of the B-la lineage. These molecules were also detected in endothelial cells (Fig. l) , as well as in thymic epithelial cells (data not shown), but not in T cells. The splenic B lymphocytes and macrophages, as well as the WEHI-3 BALBlc macrophagelike cell line and the TB6D2 and SP207 BALBlc myeloma cell lines, expressed transcripts that would encode both isoforms with two or four Ig-like domains. As expected, the transcripts were of the MHVR allele of the murine BGP, and not the mmCGM2 allele which has only been detected in SJL/J and outbred mice [7, 10, 28] . Because in other systems, mouse CEA-related molecules, as well as rat cell-CAM 105 and human CEA, have been shown to participate in intercellular adhesion [2, 3, 5, 291 , their expression on the membrane of cells which all require close contact with T lymphocytes for physiological functions, suggests that these glycoproteins play a role in recognition and binding to Tcells. If so, then a ligand for these molecules might be found on T lymphocytes. infected with MHV-A59, we found no loss of B cells from the spleen, which contrasts with the splenolysis resulting from MHV-3 infection of C57BY6 B lymphocytes [59, 601. Additional studies will therefore be needed to show whether MHVR glycoproteins are functional virus receptors on B cells in vivo as well as in cell lines.

Our data add to the increasing evidence of the expression of a variety of CEA-related glycoproteins in immune cells from many species. CEA expression has been observed in human B cell lymphomas and in Epstein-Barr immortalized B cell lines [30] and a related protein, CD66, is expressed in human neutrophils and epithelial cells, but not in lymphocytes or monocytes [31] . A cell adhesion molecule homologous to human CD22 that was recently identified in murine B lymphocytes shows limited homology to MHV receptors and to CEA [32] . CD22 has seven Ig-like domains and is considerably larger than the MHVR-related glycoproteins which have either two or four domains. CD22 mediates binding of B lymphocytes to monocytes, T cells and B cells . It may facilitate antigen recognition [33] , regulate T cell receptor/CD3-induced signaling [36] and promote B cell activation and proliferation in association with tyrosine phosphorylation [37- [53] . One might therefore expect that cells bearing these adhesion molecules can be infected, or that their function can be altered, when exposed to the viruses which recognize these molecules as receptors. Thus, it has been known for many years that MHV strains can infect macrophages [54] , so it was not surprising to find that these cells express the CEA-related glycorpoteins that serve in vivo as receptors for MHV in fibroblasts [55] In adult BALB/c animals A second biological function of the CEA-related MHVR glycoproteins on the B cell membrane, that of B cellmediated, MHV-specific cytotoxicity, is also associated with the virus receptor activity of murine BGP. MHV-A59-infected murine fibroblasts are killed rapidly and with high efficiency by incubation with splenocytes from BALBlc mice that have never been infected with MHVThis VK activity is not restricted by MHC and, surprisingly, the effector cells were shown to be B lymphocytes [15] . We previously showed that SJL/J B cells did not bind MHV-A59 virus or serve as effectors for VK activity and that treatment of MHV-A59-infected fibroblasts that serve as target cells with antibody directed against the viral spike glycoprotein blocked theVK activity [15] .This paper shows that treatment of the effector BALB/c spleen cells with anti-receptor antibody also blocked this VK activity, and that SP20 cells, which express MHVR, can serve as effector cells for VK activity.VK activity is associated with fusion of the virus-infected target cell with the effector B lymphocyte [25] , and MHV-induced fusion depends upon specific interaction of the viral spike glycoprotein with glycoprotein receptors related to MHVR. Thus, VK activity depends upon specific interaction of the virus glycoproteins with specific CEA-related glycoprotein receptors on B cell membranes. It is not yet clear whether VK activity plays any role in MHV infections in vivo.

Finally, MHV infection of mice can induce IgG2a-restricted hypergammaglobulinemia and enhancement of concomitant antibody responses [61-641. Whereas Ig production is most probably a T-dependent phenomenon, binding of virions to receptor glycoproteins in the membrane of B lymphocytes could possibly be required to trigger proliferation of these cells. Similarly, in vitro activation of B lymphocytes results from influenza virus binding to the membrane of these cells [65, 66] .

Expression of CEA-related glycoproteins in the BGP subfamily in macrophages and B lymphocytes has been demonstrated using specific antibodies and RNA PCR.The roles of these glycoproteins in the numerous cell adherence, activation and proliferation processes in the normal immune system can now be explored.The expression of the virus receptor glycoproteins in immune cells can explain some, but probably not all, of the changes of the immune system that are induced by MHV infection. Virus strain differences and differential expression of various receptor isoforms may explain the variable effects of MHV infection on the immune system of different inbred mouse strains.

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