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Induction of CD40 in Promyelocytic HL60 Cells Cultured with Retinoic Acid And/Or Various Cytokines1

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Induction of CD40 in Promyelocytic HL60 Cells Cultured with Retinoic Acid And/Or Various Cytokines1 J. Biochem. 118, 534-540 (1995) Induction of CD40 in Promyelocytic HL60 Cells Cultured with Retinoic Acid and/or Various Cytokines1 Toshie Shinagawa,2 Hiroyuki Nunoi, Shigeaki Nonoyama,3 and Shiro Kanegasaki4 The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108 Received for publication, February 27, 1995 The antigenic protein CD40 on the surface of B lymphocytes plays an important role in their proliferation, immunoglobulin class switching, and rescue from apoptosis in the germinal center through interaction with T lymphocytes expressing CD40 ligand. The protein is also found on the cell surface of other antigen-presenting cells such as monocytes, dendritic cells, and thymic epithelium cells, but its presence in other myeloid cells has not been reported. We show here that CD40 protein is induced in promyelocytic HL60 cells, when cultured with retinoic acid, a vitamin that converts them to granulocyte-like cells. The cultured cells also expressed CD15, a marker for granulocytes, and cytochrome b558, an essential component of the superoxide-generating system in phagocytes, on their surface. No detectable amount of mRNA for CD40 was found in naive HL60 cells, whereas a large amount of the message was induced in the cells cultured with the vitamin. Although CD40 expression was enhanced when the cells were further cultured with GM-CSF or IFN-ƒÁ, expression of CD14, a marker for monocytes, was also enhanced. HL60 cells, therefore, express CD40 protein during differentiation not only toward monocytes but also toward granulocytes, at least transiently. Key words: CD40, CD40L, granulocyte, HL60 cell line, monocyte. Communication between members of the hematopoietic On the other hand, CD40L is expressed on CD4+ Tcells system is mediated by cytokines and by cell surface only when they are activated (3). It was shown that murine receptors that engage with membrane-bound counter-re transfectants expressing CD40 augmented proliferation of ceptors on other cells. CD40 and CD40 ligand (CD40L) a-CD3-treated CD4+ T cells that expressed CD40L (7). were identified as a pair of counter-receptors involved in B The reciprocal communication between APC and Tlympho and T lymphocyte interactions (3, 18). CD40 is a 50-kDa cytes through CD40-CD40L, therefore, seems to affect not transmembrane glycoprotein expressed on antigen-pre only APC functions but also clonal expansion and effector senting cells (APC) such as B lymphocytes (8, 31) mono functions of CD4+ T cells. cytes (1), and dendritic cells (12) and on thymic epithelium The in vivo importance of the CD40-CD40L activation (14, 32). This surface protein is a member of the nerve pathways was recognized in patients with X-linked hyper growth factor receptor (NGF-R) family that includes the IgM syndrome (2, 4, 10, 13, 23). In these patients, a TNF receptors, OX-40, CD27, CD30, and Fas (37). Cross functional CD40L protein is not present on the activated T linking of CD40 withƒ¿-CD40 mAb induces B cell prolifera lymphocytes, resulting in the lack of isotype switching of B tion (15, 16) and isotype switching in concert with IL-4 or lymphocytes and the inability to form germinal centers. IL- 10 (19), and it prevents germinal center B lymphocytes The patients suffer from recurrent infections not only due from undergoing apoptosis (26). The a-CD40 antibody also to a lack of B lymphocyte activation, but also due to defect activates monocytes and dendritic cells (1, 6). of T lymphocyte functions. In addition, neutropenia is 1 This investigation was supported in part by a Grant-in-Aid for frequently seen. The relationship between neutropenia and Scientific Research from the Ministry of Education, Science and T lymphocyte dysfunction is not known. We are interested Culture of Japan. in whether or not myeloid cells express CD40 during the Present addresses: 2Department of Veterinary Public Health, School maturation process and if so, whether their numbers are of Veterinary Medicine, Obihiro University of Agriculture and regulated by CD40-CD40L interaction . Veterinary Medicine, Obihiro, Hokkaido 080; 3Department of In this paper, we show that transmembrane and soluble Pediatrics, School of Medicine, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113. forms of CD40 are induced in promyelocytic HL60 cells , 4 To whom correspondence should be addressed. when cultured with retinoic acid (RA), a vitamin that is Abbreviations: BSA, bovine serum albumin; BCIP, 5-bromo-4 known to convert the cells to granulocyte-like cells . chloro-3-indolyl phosphate; CD40L, CD40 ligand; EBV, Epstein-Barr virus; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; IFN-y, interferon-y; JRU, Japan research unit; NGF-R, nerve MATERIALSAND METHODS growth factor receptor; NBT, nitro blue tetrazolium; PMSF, phenyl. methylsulfonyl fluoride; PBS, phosphate-buffered saline; PVDF, Cells and Culture Conditions-Promyelocytic leukemia polyvinylidene difluoride; RA, retinoic acid; SDS, sodium dodecyl cell line HL60 was provided by Dr . M. Asada (28). HL60 sulfate. and an EBV-transformed B lymphoma cell line RPMI 1788 534 J. Biochem. CD40 in HL60 Cells 535 Fig. 1. Flow-cytometrical analysis of CD40 ex pression on HL60 cells. HL60 cells were cultured with 1 ƒÊM RA for 0 (a), 3 (b) or 5 days (g), cultured with 150 JRU/ml IFN-ƒÁ (c), 1ng/ml GM-CSF (d), 1 ng/ml IL-3 (e), or 1ng/ml G-CSF (f) for 3 days, or cultured with RA for 3 days and then either with 150 JRU/ml IFN-y (h), 1ng/ml GM-CSF (i), 1ng/ml IL-3 (j), or 1ng/ml G-CSF (k) for 2 days. The cells were stained with a ƒ¿-CD40 mAb or with control IgG1 antibodies. Vol. 118, No. 3, 1995 536 T. Shinagawa et al. (21) were cultured in RPMI 1640 medium containing 10% them in lysis buffer (1% Nonidet P-40, 10mM Tris-Cl, pH FCS, 100U/ml penicillin, 100ƒÊg/ml streptomycin , and 2 8.0, and 2mM PMSF) at 4•Ž for 30 min. Each tube mM glutamine. HL60 cells were cultured in the presence or containing lysed cells was centrifuged at 10,000•~g for 10 absence of 1ƒÊM RA, 150 JRU/ml IFN-y, 1ng/ml GM min to remove debris. The supernatant was mixed with CSF, 1ng/ml IL-3, 1ng/ml G-CSF, or their combination. sample buffer and proteins from 1•~106 cells were separat The cells were characterized flow-cytometrically using ed on 10% sodium dodecyl sulfate (SDS)-polyacrylamide antibodies against various surface antigens. Antibodies gel (24) under non-reducing conditions. They were electro used for characterization of the cells were 7D5, mAb phoretically transferred to a Immobilon PVDF transfer against cytochrome b558 (29), ƒ¿-CD14 (MY4, mouse membrane (Millipore). The membrane was incubated with IgG2b; Coulter Clone), ƒ¿-CD15 (anti Leu-Ml, mouse IgM; 3% bovine serum albumin in TBS for 1 h at room tempera Becton Dickinson), ƒ¿-CD16 (MG-38, mouse IgGl; Nichi ture, and then incubated with ƒ¿-CD40 mAb, BB20. After rei), and isotype-matched control mouse IgG or IgM washing, alkaline phosphatase-conjugated goat anti-mouse (DACO A/S, Denmark). CD14, 15, and 16 are differentia IgG (Promega) was added as a second antibody. Immune tion antigens on the leukocyte surface. reactive bands were visualized using Problot NBT and Flow Cytometry•|HL60 cells were harvested and sus BCIP color development system (Promega). pended in PBS(•|) containing 0.1% bovine serum albumin For the detection of the soluble form of CD40 protein, and 1mg/ml human ƒÁ-globulin. ƒ¿CD40 (BB20, mouse cells were cultured at a concentration of 1•~106 cells/ml in IgG1; Serotec, UK) or any of the antibodies against the serum-free medium (Cellgrosser-P; Sumitomo) for 48 h other surface antigens was added to the cell suspension and and culture supernatants were collected. Proteins were the mixture was incubated for 30 min on ice. The cells were precipitated with 1/2 volume of saturated ammonium washed and incubated further with FITC-conjugated goat sulfate and analyzed for soluble CD40 by immunoblotting anti-mouse Igs (TACO) for 30 min on ice. The cells were as described above. washed twice with PBS(-), and examined in a FACScan Analysis of CD40 mRNA-RNA blots were performed (Becton Dickinson). according to the method described by Sambrook et al. (35). Immunoblot Analysis-Cells were lysed by incubating Briefly, RNA was extracted with guanidinium/cesium chloride from HL60 cells cultured in the presence of RA, IFN-ƒÁ (150 JRU/ml), GM-CSF (1ng/ml), or their combi nation. Total RNA (20 jig/lane) was electrophoresed, transferred onto a Hybond nylon membrane (Amersham) and hybridized with a 32P-labeled CD40 cDNA probe (approximately 800 by fragment from XbaI-Pstl digestion of pHSG 298-CD40; a kind gift from Dr. M. Shimazu of the Department of Genetics, Mitsubishi Yuka Bio-clinical Laboratories, Tokyo). After washing, the filter was exposed for autoradiography. For control hybridization, the filter was boiled in water to remove CD40 specific probe, fol lowed by rehybridization with a human ƒÀ-actin cDNA probe (a kind gift from Dr. T. Tsuchiya) (25). Fig.2. Kinetics of CD40 induction in HL60 cells during culti vation. (A) CD40 expressed on HL60 cells cultured in the presence Fig.4. Analysis of CD40 in HL60 cells by immunoblotting. (•œ) or absence of RA (•Z) for the indicated periods was analyzed HL60 cells were cultured with (lane 3) or without (lane 2) 1ƒÊM RA flow -cytometrically. Mean values of relative fluorescence intensity at for 3 days or cultured with 1ƒÊM RA for 3 days and then either with each period are plotted. (B) Expression of CD40 on HL60 cells 150 JRU/ml IFN-y (lane 4) or 1ng/ml GM-CSF (lane 5).
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