Expression of Epstein-Barr Virus-Induced Gene 3, an Interleukin-12 p40-Related Molecule, throughout Human Pregnancy
2001; Elsevier BV; Volume: 159; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)63023-4
ISSN1525-2191
AutoresOdile Devergne, Aurore Coulomb‐L'Herminé, F. Capel, Marlène Moussa, Frédérique Capron,
Tópico(s)IL-33, ST2, and ILC Pathways
ResumoIn human pregnancy, trophoblasts are the only cells of fetal origin in direct contact with the maternal immune system: syncytiotrophoblasts are in contact with maternal blood, whereas extravillous trophoblasts are in contact with numerous maternal uterine natural killer (NK) cells. Therefore, trophoblasts are thought to play a key role in maternal tolerance to the semiallogeneic fetus, in part through cytokine production and NK cell interaction. Epstein-Barr virus-induced gene 3 (EBI3) encodes a soluble hematopoietin receptor related to the p40 subunit of interleukin-12. Previous studies indicated that EBI3 is expressed in the spleen and tonsils, and at high levels in full-term placenta. To investigate further EBI3 expression throughout human pregnancy, we generated monoclonal antibodies specific for EBI3 and developed an EBI3 enzyme-linked immunosorbent assay. Immunohistochemical experiments with EBI3 monoclonal antibody on first-, second-, and third-trimester placental tissues demonstrated that EBI3 was expressed throughout pregnancy by syncytiotrophoblasts and extravillous trophoblasts (cytotrophoblast cell columns, interstitial trophoblasts, multinucleated giant cells, and trophoblasts of the chorion laeve). EBI3 expression was also induced during in vitro differentiation of trophoblast cell lines. In addition, large amounts of secreted EBI3 were detected in explant cultures from first-trimester and term placentae. Consistent with these data, EBI3 levels were strongly up-regulated in sera from pregnant women and gradually increased with gestational age. These data, together with the finding that EBI3 peptide is presented by HLA-G, suggest that EBI3 is an important immunomodulator in the fetal-maternal relationship, possibly involved in NK cell regulation. In human pregnancy, trophoblasts are the only cells of fetal origin in direct contact with the maternal immune system: syncytiotrophoblasts are in contact with maternal blood, whereas extravillous trophoblasts are in contact with numerous maternal uterine natural killer (NK) cells. Therefore, trophoblasts are thought to play a key role in maternal tolerance to the semiallogeneic fetus, in part through cytokine production and NK cell interaction. Epstein-Barr virus-induced gene 3 (EBI3) encodes a soluble hematopoietin receptor related to the p40 subunit of interleukin-12. Previous studies indicated that EBI3 is expressed in the spleen and tonsils, and at high levels in full-term placenta. To investigate further EBI3 expression throughout human pregnancy, we generated monoclonal antibodies specific for EBI3 and developed an EBI3 enzyme-linked immunosorbent assay. Immunohistochemical experiments with EBI3 monoclonal antibody on first-, second-, and third-trimester placental tissues demonstrated that EBI3 was expressed throughout pregnancy by syncytiotrophoblasts and extravillous trophoblasts (cytotrophoblast cell columns, interstitial trophoblasts, multinucleated giant cells, and trophoblasts of the chorion laeve). EBI3 expression was also induced during in vitro differentiation of trophoblast cell lines. In addition, large amounts of secreted EBI3 were detected in explant cultures from first-trimester and term placentae. Consistent with these data, EBI3 levels were strongly up-regulated in sera from pregnant women and gradually increased with gestational age. These data, together with the finding that EBI3 peptide is presented by HLA-G, suggest that EBI3 is an important immunomodulator in the fetal-maternal relationship, possibly involved in NK cell regulation. Human pregnancy is a unique immune situation in which the semiallogeneic fetus avoids maternal rejection. Fetal trophoblast cells, which are in direct contact with maternal immune cells, are thought to play a key role in maternal tolerance. Trophoblasts are specialized epithelial cells and are distinguished into several types depending on their stage of differentiation and location at the maternal-fetal interface. Floating villi are composed of two trophoblast layers: an inner layer of mononucleated cytotrophoblast stem cells, and an outer layer of syncytiotrophoblasts resulting from the differentiation and cell fusion of cytotrophoblasts into syncytium. Syncytiotrophoblasts cover the entire surface of the villi and are in direct contact with maternal blood in the intervillous space. These cells, because of their specific location, play a key role in nutrient and gas exchange between the mother and the developing fetus, and may play an important role in peripheral tolerance. At selected sites in the villi, cytotrophoblasts can follow a second differentiation pathway by leaving the villous basement membrane, proliferating, and aggregating into the cytotrophoblast cell column characteristic of anchoring villi. Anchoring villi are attached to the uterine wall, and cytotrophoblasts from these anchoring villi invade the uterus wall. These invasive extravillous trophoblasts comprise a heterogeneous population of interstitial trophoblasts, multinucleated placental bed giant cells, and trophoblast cells that invade the uterine spiral arteries and adopt a vascular phenotype, as assessed by their expression of endothelial cell surface markers. Parallel to trophoblast invasion, the uterine endometrium decidualizes and is infiltrated by a large number of maternal immune cells. These cells are particularly abundant at the site of implantation, the decidua basalis, and are in close contact with invasive extravillous trophoblasts. Maternal uterine natural killer (NK) cells are particularly abundant accounting for up to 70% of the total decidual leukocyte population during early pregnancy.1Loke YW King A Immunological aspects of human implantation.J Reprod Fertil. 2000; 55: S83-S90Google Scholar, 2Le Bouteiller P Solier C Pröll J Aguerre-Girr M Fournel S Lenfant F Placental HLA-G protein expression in vivo: where and what for?.Human Reprod. 1999; 5: 223-233Crossref Scopus (102) Google Scholar The mechanisms by which the fetus escapes the maternal immune response are not fully understood. The expression by extravillous trophoblasts of a nonclassical major histocompatibility complex (MHC) class I antigen, HLA-G, may play a role by regulating the maternal NK response.2Le Bouteiller P Solier C Pröll J Aguerre-Girr M Fournel S Lenfant F Placental HLA-G protein expression in vivo: where and what for?.Human Reprod. 1999; 5: 223-233Crossref Scopus (102) Google Scholar, 3King A Hiby SE Gardner L Joseph S Bowen JM Verma S Burrows TD Loke YW Recognition of trophoblast HLA class I molecules by decidual NK cell receptors. A review.Placenta. 2000; 14: S81-S85Abstract Full Text PDF Scopus (139) Google Scholar The local secretion of soluble factors, including hormones and cytokines, may also be involved. Indeed, the expression at the maternal-fetal interface of various cytokines with a Th2 or immunosuppressive phenotype, such as interleukin (IL)-10, IL-13, or transforming growth factor-β, has been described in human pregnancy.4Roth I Corry DB Locksley RM Abrams JS Litton MJ Fisher SJ Human placental cytotrophoblasts produce the immunosuppressive cytokine interleukin 10.J Exp Med. 1996; 184: 539-548Crossref PubMed Scopus (312) Google Scholar, 5Dealtry GB Clark DE Sharkey A Charnock-Jones DS Smith SK Expression and localization of the Th2-type cytokine interleukin-13 and its receptor in the placenta during human pregnancy.Am J Reprod Immunol. 1998; 40: 283-290Crossref PubMed Scopus (52) Google Scholar, 6Selick CE Horowitz GM Gratch M Scott RT Navot D Hofmann GE Immunohistochemical localization of transforming growth factor-beta in human implantation sites.J Clin Endocrinol Metab. 1994; 78: 592-596Crossref PubMed Scopus (69) Google Scholar, 7Jokhi PP King A Loke YW Cytokine production and cytokine receptor expression by cells of the human first trimester placental-uterine interface.Cytokine. 1997; 9: 126-137Crossref PubMed Scopus (131) Google Scholar Previously, we have reported the identification of a novel homologue to IL-12 p40, Epstein-Barr virus (EBV)-induced gene 3 (EBI3).8Devergne O Hummel M Koeppen H Le Beau M Nathanson EC Kieff E Birkenbach M A novel interleukin-12 p40 related protein induced by latent Epstein-Barr virus infection in B lymphocytes.J Virol. 1996; 70: 1143-1153Crossref PubMed Google Scholar EBI3 is expressed at a high level by B cell lines transformed in vitro by EBV. In vivo, EBI3 is expressed in lymphoid organs such as tonsil and spleen, and at a very high level in term placenta. EBI3 has 27% amino acid identity to the IL-12 p40 subunit. Like p40, it lacks a membrane-anchoring motif and corresponds to the extracellular portion of a type I cytokine receptor. Also like p40, EBI3 can associate with the p35 subunit of IL-12 to form a heterodimeric hematopoietin, EBI3/p35.9Devergne O Birkenbach M Kieff E Epstein-Barr virus induced gene 3 and the p35 subunit of interleukin 12 form a novel heterodimeric hematopoietin.Proc Natl Acad Sci USA. 1997; 94: 12041-12046Crossref PubMed Scopus (279) Google Scholar Although EBI3 is structurally related to IL-12 p40, its biological activity is expected to be opposite to that of IL-12. IL-12 plays a key role in the cell-mediated immune response by driving the response toward a Th1 type, and by stimulating the activation and proliferation of cytotoxic T lymphocytes (CTL) and NK cells.10Trinchieri G Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity.Annu Rev Immunol. 1995; 13: 251-276Crossref PubMed Scopus (2243) Google Scholar, 11Szabo SJ Jacobson NG Dighe AS Gubler U Murphy KM Developmental commitment to the Th2 lineage by extinction of IL-12 signaling.Immunity. 1995; 2: 665-675Abstract Full Text PDF PubMed Scopus (415) Google Scholar, 12Magram J Connaughton SE Warrier RR Carvajal DM Wu CY Ferrante J Stewart C Sarimiento U Faherty DA Gately MK IL-12 deficient mice are defective in IFN-γ production and type 1 cytokine responses.Immunity. 1996; 4: 471-481Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar The high level of EBI3 expression observed in EBV-infected B lymphocytes and at the fetal-maternal interface, two situations in which a dampening of the cytotoxic cell-mediated immune response and of the Th1 response is required,13Rickinson AB Moss DJ Human cytotoxic T lymphocyte responses to Epstein-Barr virus infection.Annu Rev Immunol. 1997; 15: 405-431Crossref PubMed Scopus (637) Google Scholar, 14Wegmann TG Lin H Guilbert L Mossman TR Birectional cytokine interactions in the maternal-fetal relationship: is successful pregnancy a Th2 phenomenon?.Immunol Today. 1993; 14: 353-356Abstract Full Text PDF PubMed Scopus (2543) Google Scholar, 15Hill JA Polgar K Anderson DJ T-helper 1-type immunity to trophoblast in women with recurrent spontaneous abortion.J Am Med Assoc. 1995; 273: 1933-1936Crossref Scopus (502) Google Scholar, 16Piccinni MP Beloni L Livi C Maggi E Scarselli G Romagnani S Defective production of both leukemia inhibitory factor and type 2 T-helper cytokines by decidual T cells in unexplained recurrent abortions.Nat Med. 1998; 4: 1020-1024Crossref PubMed Scopus (579) Google Scholar, 17Hill JA Choi BC Maternal immunological aspects of pregnancy success and failure.J Reprod Fertil. 2000; 55: S91-S97Google Scholar is in favor of an immunosuppressive or Th2 function for EBI3 or EBI3/p35. Consistent with this hypothesis, analysis of EBI3 expression in human intestinal diseases has demonstrated that EBI3 and IL-12 display opposite patterns of expression.18Christ AD Stevens AC Koeppen H Walsh S Omata F Devergne O Birkenbach M Blumberg RS An interleukin 12-related cytokine is up-regulated in ulcerative colitis but not in Crohn's disease.Gastroenterology. 1998; 115: 307-313Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar To investigate further the expression and potential role of EBI3 in the placental-maternal relationship, we generated specific anti-EBI3 monoclonal antibodies (mAbs) that could be used in immunohistochemical studies on frozen and paraffin-embedded tissues, and developed an EBI3 sandwich enzyme-linked immunosorbent assay (ELISA). Using these antibodies, we analyzed EBI3 expression throughout human pregnancy, both in situ and in in vitro experiments. Plasmid pQE-EBI3 was constructed by polymerase chain reaction amplification of the human EBI3 cDNA amino acids 25 to 2298Devergne O Hummel M Koeppen H Le Beau M Nathanson EC Kieff E Birkenbach M A novel interleukin-12 p40 related protein induced by latent Epstein-Barr virus infection in B lymphocytes.J Virol. 1996; 70: 1143-1153Crossref PubMed Google Scholar using the 5′ primer (5′-ATCCGAGCTCCCCAGCAGCTCTGACACTG-3′) and the 3′ primer (5′-CCCAAGCTTCTACTTGCCCAGCGTCATT-3′). The polymerase chain reaction product was digested with SacI and HindIII and cloned into the SacI-HindIII sites of plasmid pQE-31 (Qiagen). The resulting plasmid encodes an N-terminal hexahistidine-tagged EBI3 protein deleted for its signal peptide. pSG5-EBI3, pSG5-EBI3-Flag, pSG5-p40-Flag, and pSG5-p35-Flag expression vectors have been previously described.8Devergne O Hummel M Koeppen H Le Beau M Nathanson EC Kieff E Birkenbach M A novel interleukin-12 p40 related protein induced by latent Epstein-Barr virus infection in B lymphocytes.J Virol. 1996; 70: 1143-1153Crossref PubMed Google Scholar, 9Devergne O Birkenbach M Kieff E Epstein-Barr virus induced gene 3 and the p35 subunit of interleukin 12 form a novel heterodimeric hematopoietin.Proc Natl Acad Sci USA. 1997; 94: 12041-12046Crossref PubMed Scopus (279) Google Scholar BJAB, BL41, and Ramos are EBV-negative Burkitt lymphoma cell lines. IB4 and NC37 are EBV-transformed human B cell lines. Jurkat, Molt-4, and CEM-T are human T cell leukemia lines, and U937 and HL-60 are human myelocytic and myeloblastic cell lines, respectively. BeWo and Jar are human choriocarcinoma cell lines. COS7 is a SV40-transformed monkey kidney cell line. Cell lines were grown in RPMI 1640-Glutamax (all cell lines except COS7) or Dulbecco's modified Eagle's medium-Glutamax (COS7) supplemented with 10% fetal calf serum and antibiotics. Approximately 107Jokhi PP King A Loke YW Cytokine production and cytokine receptor expression by cells of the human first trimester placental-uterine interface.Cytokine. 1997; 9: 126-137Crossref PubMed Scopus (131) Google Scholar BJAB cells or 4.106Selick CE Horowitz GM Gratch M Scott RT Navot D Hofmann GE Immunohistochemical localization of transforming growth factor-beta in human implantation sites.J Clin Endocrinol Metab. 1994; 78: 592-596Crossref PubMed Scopus (69) Google Scholar COS7 cells were transfected by electroporation on a Biorad electroporator at 210 V and 960 μF in 400 μl of RPMI medium containing 10% fetal calf serum. Phorbol 12-myristate 13-acetate (PMA) and forskolin were purchased from Sigma and Calbiochem, respectively. For recombinant 6His-EBI3 expression, M15/pREP4 cells (Qiagen) transformed with pQE-EBI3 were induced by incubation with 1 mmol/L isopropylthio-β-d-galactoside for 4 hours. The bacterial cell pellet was lysed in guanidine lysis buffer (6 mol/L guanidine-HCl, 0.1 mol/L NaH2PO4, 0.05 mol/L Tris, pH 8) and sonicated. Lysate was centrifuged at 13,000 × g for 20 minutes at 4°C to remove debris, and incubated with nickel-nitrilotriacetic acid (Ni-NTA) agarose beads (Qiagen) for 1 hour at room temperature. Beads were washed with washing buffer (8 mol/L urea, 0.1 mol/L NaH2PO4, 0.01 mol/L Tris-HCl, pH 6.3) and the His-tagged protein was eluted by adding elution buffer (8 mol/L urea, 0.1 mol/L NaH2PO4, 0.01 mol/L Tris-HCl, pH 4.5). Fractions containing 6HisEBI3 protein were dialyzed against phosphate-buffered saline (PBS), and 6HisEBI3 protein concentration was determined using the Pierce Micro bicinchoninic acid protein assay. Recombinant Flag-tagged EBI3 and Flag-tagged p40 were purified from the culture supernatant of COS7 cells transiently transfected with pSG5-EBI3-Flag or pSG5-p40-Flag, respectively. Sixty to 70 hours after transfection, COS7 cell supernatant was harvested, supplemented with protease inhibitors (1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 mmol/L phenylmethyl sulfonyl fluoride), and centrifuged to remove cell debris. Supernatants were then incubated for 3 hours at 4°C with M2 anti-Flag affinity gel (Sigma). Beads were washed twice with 1% Nonidet P-40 buffer (1% Nonidet P-40, 20 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 3% glycerol, 1.5 mmol/L ethylenediaminetetraacetic acid), twice with Tris-buffered saline (TBS: 10 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 7.4), and Flag-tagged proteins were eluted by addition of Flag peptide (250 μg/ml in TBS). p35-associated EBI3 was affinity-purified on M2 anti-Flag affinity gel under the same conditions from the culture supernatant of COS7 cells co-transfected with pSG5- EBI3 and pSG5-p35-Flag. Under these conditions, all of the purified EBI3 was associated with p35-Flag and most of the p35-Flag was associated with EBI3. The concentration of purified EBI3-Flag was determined by titration by M2 anti-Flag Western blot analysis using a C-terminal Flag-tagged BAP protein (Sigma) as a standard. The quantitative results obtained in this assay were similar to those obtained by titration by anti-EBI3 blot analysis using 6His-EBI3 as a standard. The concentration of purified p40-Flag was determined by IL-12 blot analysis using recombinant IL-12 (R&D Systems) as a standard. Soluble CNTF-Rα was purchased from R&D Systems. Mouse mAbs specific for human EBI3 were obtained by immunizing mice with a N-terminal hexahistidine-tagged EBI3 fusion protein (6His-EBI3) purified from bacteria. Mice immunization and hybridoma production were performed through Eurogentec (Seraing, Belgium) mAb production customer service. Briefly, four BALB/c mice were immunized by three injections of purified 6His-EBI3 (50 μg per injection) on days 0, 21, and 42. Sera of immunized mice collected on days 14, 35, and 56 after immunization were tested by indirect ELISA for reactivity against the immunogen. The best responding mouse was given a final Ag injection and selected for the fusion. Spleen lymphocytes were fused to the Sp2/O-Ag14 cell line using polyethylene glycol, and the hybridomas were seeded in hypoxanthine-aminopterin-thymidine medium in 96-well plates. IgG-positive hybridoma supernatants were screened by indirect ELISA for reactivity against the following antigens: a hexahistidine peptide (negative control), purified bacterial 6His-EBI3, and purified COS7 cell-derived EBI3-Flag. Hybridoma supernatants giving positive results in these assays were further tested by Western blotting and by indirect immunofluorescence staining of pSG5 or pSG5-EBI3-Flag-transfected BJAB cells. Clones of interest were then subcloned by limiting dilution, and the subclones were tested as described above for the parental clones. Two of the subclones, 2G4H6 and 1A1B2H3, were selected for this study. The 2G4H6 mAb was determined to be an IgG2a, kappa, and the 1A1B2H3 mAb an IgG1, kappa. Both hybridomas were submitted to ascite production in BALB/c mice, and mAbs were purified from ascite by chromatography on Protein G-Sepharose. The 2G4H6 mAb was biotinylated using the biotin labeling kit from Boehringer Mannheim, following the manufacturer's instructions. Microtitration plates (96-well, Linbro; Flow Laboratories Inc., Virginia) were coated with 100 μl of 1A1B2H3 mAb at 5 μg/ml in 0.05 mol/L carbonate buffer, pH 9.6, overnight at 4°C, washed with PBS-0.05% Tween 20 (PBS-T), and blocked with 200 μl of PBS-5% bovine serum albumin for 2 hours at room temperature. Test samples (100 μl) were added to the wells and incubated for 18 hours at 4°C. After washing with PBS-T, biotinylated 2G4H6 mAb was added at a 1:200 dilution in PBS-T (∼4 μg/ml) and incubated for 2 hours at room temperature. After washing with PBS-T, 100 μl of PBS containing streptavidin linked to horseradish peroxidase (Amersham) diluted 1:2000 was added and the plates incubated for 30 minutes at room temperature. Wells were further washed with PBS-T, and the reaction was developed by adding 200 μl of ortho-phenylenediamine dihydrochloride solution (Sigma). After 30 minutes, the reaction was stopped by adding 50 μl of 3 mol/L HCl and optical density was read at 490 nm. Recombinant Flag-tagged EBI3 purified from the culture supernatant of transfected COS7 cells was used as a standard. The sensitivity limit was ∼0.5 to 1 ng/ml and only values more than 1 ng/ml were considered as positive. Cell lines or transfected cells were washed in cold PBS and lysed for 1 hour on ice in ice-cold Nonidet P-40 lysis buffer (1% Nonidet P-40, 20 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 3% glycerol, 1.5 mmol/L ethylenediaminetetraacetic acid) supplemented with protease inhibitors (1 mmol/L phenylmethyl sulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml pepstatin). Cell debris was removed by centrifugation at 14,000 × g for 15 minutes at 4°C and total protein concentration was determined using the Pierce Micro bicinchoninic acid protein assay. For immunoprecipitation, cell lysates were precleared with GammaBindPlus-Sepharose beads (Pharmacia) for 1 to 2 hours at 4°C. Cleared lysates were incubated with M2 anti-Flag antibody (Sigma) or EBI3 mAbs for 2 hours at 4°C, and then with GammaBindPlus-Sepharose for 1 hour at 4°C. Beads were washed four times with 1 ml of lysis buffer and protein complexes were recovered by boiling in sodium dodecyl sulfate sample buffer. Immunoprecipitated material was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane for immunoblotting. On blots, EBI3 was detected using 2G4H6 mAb, and IL-12 p40 was detected using goat affinity-purified anti-IL-12 polyclonal antibodies (R&D Systems). Binding of antibodies was detected using horseradish peroxidase-conjugated sheep anti-mouse IgG antibodies (1:5000 dilution, Amersham), or horseradish peroxidase-conjugated donkey anti-goat antibodies (1:5000 dilution, Santa Cruz) and enhanced chemiluminescence reagents (Pierce). Eighteen placentae were analyzed by immunohistochemistry. For each placenta, frozen and/or formalin-fixed paraffin-embedded tissues were analyzed. Frozen tissues were collected from placentae at 6, 9, 10, 17, 29, 33, 36, and 41 weeks of pregnancy, and paraffin-embedded tissues were prepared from placentae collected at 4, 6, 7, 8, 10, 17, 20, 27, 33, 35, 38, and 39 weeks of pregnancy. Tissues collected at 17 and 33 weeks of gestation corresponded to placentae for which both frozen and paraffin-embedded tissues were prepared and analyzed. First-trimester placentae were obtained from voluntary pregnancy termination performed by vacuum suction (all but one case) or RU486 administration (4-week placenta). Second- and third-trimester placentae were obtained from therapeutic termination (n = 1), miscarriage (n = 1), cesarean section delivery (n = 2), or natural delivery (n = 6). In all cases, placentae were selected for the absence of abnormality on macroscopic and histological examination. Villi maturation was in accordance with gestational age, and no lesions, in particular no vascular lesions indicative of pre-eclampsia, and no infection were observed. Sera from 10 women with normal pregnancies, collected at various times during pregnancy (five to eight sera per individual) for serological diagnosis, were used in this study. Sera from nonpregnant women (n = 10) and men (n = 4) were used as controls. For placental explant culture, first-trimester placentae (n = 7) were obtained after 6 to 7.5 weeks of pregnancy by voluntary pregnancy termination initiated by RU486 administration and term placentae (n = 12) were obtained from cesarean section delivery. In all cases, placental villi were isolated, minced, washed three times in Hanks' balanced salt solution and a total of 3 g of placenta villi was cultured in 20 ml of RPMI 1640-Glutamax supplemented with 10% fetal calf serum, antibiotics, and 1% sodium bicarbonate. After various times of culture, culture supernatants were harvested, spun at 2000 ×g to remove debris and stored at −80°C. Immunochemistry was performed on either cytospin preparation, frozen tissue sections fixed in acetone, or formalin-fixed paraffin-embedded tissue sections. For cytospin preparation, transfected cells were washed in PBS, cytocentrifuged, air-dried, fixed in cold acetone/methanol (1:1), and stored at −80°C. For immunostaining of frozen sections or cytospin samples, slides were rehydrated in PBS, incubated for 5 minutes with a peroxidase-blocking solution (DAKO), washed, and saturated in PBS containing 10% normal goat serum and 20% normal human serum. Slides were then incubated with the primary mAb diluted in PBS-2% bovine serum albumin for 1 hour. Binding of mAbs was detected using an indirect avidin-biotin peroxidase kit (Biogenex). The peroxidase reaction was developed with 3-amino-9-ethylcarbazole (Sigma) and sections were counterstained with Mayer's hematoxylin. For immunostaining of paraffin sections, sections were first deparaffinized in xylene and rehydrated in successive ethanol baths and PBS. Ag was retrieved by microwave heat pretreatment in citrate buffer. Slides were saturated by incubation with PBS containing 5% normal goat serum (DAKO) for 10 minutes at room temperature. They were then incubated with the primary antibody for 30 minutes, followed by an indirect avidin-biotin peroxidase technique (DAKO StreptABComplex/horseradish peroxidase duet). The peroxidase reaction was developed with 3′-diaminobenzidine (Immunotech) and sections were counterstained with Harris hematoxylin. EBI3 was detected using 2G4H6 mAb at 2 μg/ml. IL-12 p35 was detected using G161-566 p35 mAb (IgG1, PharMingen) at 1.6 μg/ml. This mAb was verified to react specifically with p35 by immunostaining, immunoprecipitation, and Western blotting performed with transfected cells or cell lysates. CD56 mAb (IgG1, Novocastra Laboratories Ltd.) was used at a 1:25 dilution. Anti-cytokeratin mAb (KL1, IgG1; Immunotech) was used at a 1:50 dilution, anti-vimentin mAb (V9, IgG1; BioGenex) was used at a 1:100 dilution, and rabbit anti-human placental lactogen polyclonal antibodies (DAKO) were used at a 1:1000 dilution. RPC5 (IgG2a, kappa; Cappel, Durham, NC) and MOPC21 (IgG1, kappa; Sigma) were used as negative controls. Photomicrographs were taken on a Leica DMLB microscope using a 3 CCD color video camera (Sony Power HAD) and analyzed with Thunder software. Identical settings were used when scanning serial sections. Mouse mAbs specific for human EBI3 were obtained by immunizing mice with a N-terminal hexahistidine-tagged EBI3 fusion protein (6His-EBI3) purified from bacteria. Two of the hybridomas obtained, the 2G4H6 (IgG2a, kappa) and the 1A1B2H3 (IgG1, kappa) mAbs, were used for this study. As shown in Figure 1, both clones recognized Escherichia coli-derived 6HisEBI3, as well as COS7 cell-derived EBI3, in indirect ELISA, with 2G4H6 mAb showing a stronger reactivity than 1A1B2H3 mAb. The specificity of the two clones was further analyzed by Western blotting, immunoprecipitation, and immunostaining. First, two cell lines that do not express EBI3, the EBV-negative Burkitt lymphoma BJAB cell line and COS7 cells, were transiently transfected with pSG5-EBI3-Flag or pSG5 control vector, and cell lysates or cell culture supernatants were analyzed by immunoblotting with EBI3 mAbs. On blots, intracellular EBI3 is detected as a 33-kd protein together with smaller degradation products (26 to 29 kd), and the secreted form is detected as a 34-kd protein.8Devergne O Hummel M Koeppen H Le Beau M Nathanson EC Kieff E Birkenbach M A novel interleukin-12 p40 related protein induced by latent Epstein-Barr virus infection in B lymphocytes.J Virol. 1996; 70: 1143-1153Crossref PubMed Google Scholar, 9Devergne O Birkenbach M Kieff E Epstein-Barr virus induced gene 3 and the p35 subunit of interleukin 12 form a novel heterodimeric hematopoietin.Proc Natl Acad Sci USA. 1997; 94: 12041-12046Crossref PubMed Scopus (279) Google Scholar By 2G4H6 mAb immunoblotting, a strong EBI3 signal was detected in the cell lysate from EBI3-Flag-expressing BJAB cells, but no signal was observed in the lysate from control vector-transfected cells (Figure 2 A, left). Also, a major 34-kd EBI3-Flag protein, together with a 30-kd degradation product, were detected in the culture supernatant from pSG5-EBI3-Flag-transfected COS7 cells, whereas no signal was observed in the culture supernatant from vector-transfected cells (Figure 2A, right). 2G4H6 mAb recognized EBI3 with very high sensitivity because as little as 0.2 ng of secreted EBI3-Flag was readily detected by immunoblot analysis, and a faint signal was observed when 0.05 ng of EBI3 was used (Figure 2A, right). 2G4H6 mAb also recognized natural EBI3 in cell lysates of two EBV-transformed B cell lines, IB4 and NC37, but failed to give any signal in cell lysates of various cell lines that have been previously shown not to express EBI3 (Figure 2B).8Devergne O Hummel M Koeppen H Le Beau M Nathanson EC Kieff E Birkenbach M A novel interleukin-12 p40 related protein induced by latent Epstein-Barr virus infection in B lymphocytes.J Virol. 1996; 70: 1143-1153Crossref PubMed Google Scholar, 9Devergne O Birkenbach M Kieff E Epstein-Barr virus induced gene 3 and the p35 subunit of interleukin 12 form a novel heterodimeric hematopoietin.Proc Natl Acad Sci USA. 1997; 94: 12041-12046Crossref PubMed Scopus (279) Google Scholar By immunoblotting, 2G4H6 mAb did not cross-react with IL-12 p40 or soluble CNTF-R, the two type 1 cytokine receptors most homologous to EBI3 (Figure 2C). Also, 2G4H6 mAb specifically immunoprecipitated EBI3-Flag from the lysate of transfected COS7 cells, but did not immunoprecipitate IL-12 p40-Flag (Figure 2D). On immunoblots, 1A1B2H3 mAb also recognized EBI3,
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