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 CD40 on the surface of B 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 , 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, .

Communication between members of the hematopoietic On the other hand, CD40L is expressed on CD4+ Tcells system is mediated by 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 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 (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 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 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). Cells were cultured with RA for 2 days and then with IFN-ƒÁ (•œ) or GM-CSF (•¡) lysed in lysis buffer (see "MATERIALS AND METHODS") and was analyzed flow-cytometrically. Mean values of relative fluores supernatants were subjected to immunoblotting . As a positive control cence intensity at each period are plotted. Results for naive HL60 for CD40, cell lysate of B cell line RPMI 1788 was employed (lane 1) . cells cultured with IFN-ƒÁ(•Z) or GM-CSF (• ) are also shown. Molecular weights of marker proteins are indicated on the right.

J. Biochem. CD4O in HL6O Cells 537

Fig.3. Flow cytometrical analysis of various cell surface proteins on HL60 cells. HL60 cells were cultured with or without RA, IFN-y, GM-CSF, or their combination as described in the legend to Fig.1. Expression of CD15, CD14, CD16, and cytochrome b558 were analyzed flow cytometrically as described in "MATE RIALS AND METHODS."

Vol. 118, No. 3, 1995 538 T. Shinagawa et al.

Fig. 6. Detection of CD40mRNA in HL60 cells by Northern blotting. RNA was extracted from the cells cultured under the following conditions and total RNA (20ƒÊg/lane) was subjected to Northern blotting. The Northern blots were hybridized with a probe Fig.5. Immunoblot analysis of CD40 protein in culture for CD40 and ƒÀ-actin (hybridization control) as described in "MATE medium of HL60 cells. Culture media of HL60 cells were changed to RIALS AND METHODS." The culture conditions were; with (lane 3) serum-free medium after cultivation under the following conditions. or without IFN-ƒÁ(lane 2) for 2 days, with GM-CSF (lane 4) for 2 The cells (1•~106/ml) in serum-free medium were cultured for a days, with 1ƒÊM RA for 3 days (lane 5), with RA for 3 days and then further 2 days. Proteins from the culture supernatant (1.2ml either with 150 JRU/ml IFN-ƒÁ (lane 6), or 1ng/ml GM-CSF (lane 7). equivalent) were subjected to immunoblotting. Initial culture condi B cell line RPMI 1788 cells were used as a positive control (lane 1). tions were for 3 days with (lane 3) or without 1ƒÊM RA (lane 2), with RA for 3 days and then either with 150 JRU/ml IFN-ƒÁ(lane 4), or 1 ng/ml GM-CSF (lane 5). The stained band in lane 1 exhibits the membrane form of CD40 in cell lysate of a B cell line, RPMI 1788. The days and also in the cells cultured with RA and then with immunostained bands found in culture media have a slightly smaller IFN-ƒÁ or GM-CSF for 2 days. No detectable band around molecular mass than that of membrane form of CD40 in the lysate of 50kDa was observed in naive HL60 cells. In addition, a RPMI 1788. wide band with a molecular mass slightly smaller than that of the membrane form was detected in the medium of HL60 cultured with RA for 3 days, suggesting that the cells generated a soluble form of CD40 protein. Such a band was not detected in the culture medium of naive HL60 cells RESULTS (Fig.5). The band was also detected in the medium of HL60 Flow-Cytometrical Analysis of CD40 and Other Surface cells cultured with RA and then with either IFN-ƒÁ or Antigens on HL60 Cells Cultured with Retinoic Acid and/ GM-CSF. Thus, both soluble and membrane forms of CD40 or with Various Cytokines-Although naive HL60 cells did seem to be induced in HL60 cells during development. not express CD40, we found that the cells cultivated with Levels of CD40 mRNA in HL60 Cells Cultured under RA for 3-5 days expressed it on their surface (Fig. 1). As Various Conditions-We then investigated mRNA levels of shown in Fig. 2A, the epitope of CD40 appeared on the cells CD40 in HL60 cells cultured with RA and/or various after 3 days of cultivation and its level increased slightly cytokines. As shown in Fig.6, no detectable amount of during prolonged cultivation with RA. During the first 3 CD40mRNA was found in naive HL60 cells, whereas the days, the levels of CD15, a marker for granulocytes, and highest amount of its mRNA was detected in the cells cytochrome b558, a component of the superoxide-generating cultured with RA. The level of the mRNA was slightly system in phagocytes, were increased (Fig.3). CD16, a reduced during further cultivation with IFN-ƒÁ or GM-CSF marker for mature granulocytes was not detected and the for 2 days. A low level of the mRNA was found in the cells increase of CD14, a marker for monocytes, was slight. cultured with IFN-ƒÁ or GM-CSF for 2 days. These results indicate that CD40 is expressed on HL60 cells during development toward granulocytes. DISCUSSION When the RA-treated cells were cultured for a further 2 days in the presence of either GM-CSF or IFN-ƒÁ, the In this paper, we have shown that RA induced CD40 expression of CD40 was enhanced, whereas no enhance expression in HL60 cells. Although surface expression of ment was observed with IL-3 or G-CSF (Fig.1). The CD40 was not so high in such cells, the level of its mRNA enhancement with GM-CSF or IFN-ƒÁ was apparent after 2 was highest among the cells cultured with the various days and its level slightly increased up to 4 days (Fig.2B). inducers used. It was shown that HL60 cells differentiated However, the level of CD14 also increased with these to granulocyte-like cells when cultured with RA (5, 9). In treatments and the level of CD15 decreased slightly (Fig. fact, CD15, a granulocyte marker, was enhanced on the 3). When naive HL60 cells were cultured with IFN-y but cells under the culture conditions employed, although not with the other cytokines used above, CD40 was induced CD16, a mature granulocyte marker, did not appear. We (Figs.1 and 2B) together with CD14 (Fig.3). CD15 did not could not detect CD40mRNA in myelocytes from bone appear under these conditions (Fig. 3). These results marrow or granulocytes from peripheral blood (unpub lished observation). It is possible indicate that IFN-ƒÁ transforms the cells to monocyte-like , therefore, that CD40 is cells, which express both CD14 and CD40. transiently expressed on the cells during differentiation of Immunoblot Analysis of CD40 in Naive and Cultured myeloid cells. It was shown that CD40 is expressed in HL60 Cells and in Culture Media-To examine the expres myeloid progenitor cells (CD34+CD33+cells) (34). The sion of CD40 in terms of protein in HL60 cells cultured possibility remains, however, that a malignant cell line under various conditions, we performed immunoblotting such as HL60 has a different regulatory mechanism for assays using antibody against CD40. As shown in Fig.4, a CD40 expression from normal ontogeny . wide band with a molecular mass of around 50 kDa was When RA-treated HL60 cells were further cultured in labeled with ƒ¿-CD40 in the cells cultured with RA for 3 the presence of IFN-ƒÁ or GM-CSF , these cytokines en-

J. Biochem. CD4O in HL6O Cells 539

hanced the expression of CD40 . The cells express both Grabstein, K.H., Cosman, D., and Spriggs, M.K. (1992) Molecu CD14 and CD15, indicating that the cells exhibited the lar and biological characterization of a murine ligand for CD40. character of both granulocytes and monocytes. When HL60 Nature 357, 80-82 cells were cultured, from the beginning 4. Aruffo, A., Farrington, M., Hollenbaugh, D., Li, X., Milatovich, , with IFN-y, a A., Nonoyama, S., Bajorath, J., Grosmaire, L.S., Stenkamp, R., that converts the cells to monocyte-like cells , C Neubauer, M., Roberts, R.L., Noelle, R.J., Ledbetter, J.A., D4O and CD14 were well expressed . In contrast, IL-3, Francke, U., and Ochs, D. (1993) The CD40 ligand, gp39, is which was reported to up-regulate CD40 expression in defective in activated T cells from patients with X-linked monocytes (1), did not enhance CD40 expression on HL60 hyper-IgM syndrome. Cell 72, 291-300 cells cultured with RA. The monocyte-like cells induced by 5. Breitman, T.R., Collins, S.J., and Keene, B.R. (1980) Replace IFN-ƒÁ may differ from peripheral monocytes in cytokine ment of serum by insulin and transferrin supports growth and differentiation of the human promyelocytic cell line, HL-60. Exp. responses. It is interesting that surface expression of CD40 Cell Res. 126, 494-498 was enhanced in RA-treated cells when they were cultured 6. Caux, C., Massacrier, C., Vanbervliet, B., Dubois, B., Kooten, further with IFN-ƒÁ or GM-CSF, whereas the level of C.V., Durand, I., and Banchereau, J. (1994) Activation of human mRNA for CD40 was the same or even decreased during dendritic cells through CD40 cross-linking. J. Exp. Med. 180, cultivation with the cytokines. The enhanced surface 1263-1272 expression of CD40 by these cytokines may be regulated at 7. Cayabyab, M., Phillips, J.H., and Lanier, L.L. (1994) CD40 a posttranslational level. preferentially costimulates activation of CD4+ T lymphocytes. J. Immunol. 152, 1523-1531 As mentioned above, the level of CD40mRNA in HL60 8. Clark, E.A. and Ledbetter, J.A. (1986) Activation of human B cells cultured with RA was highest among the cells cultured cells mediated through two distinct cell surface differentiation with various cytokines or other inducers used, whereas antigens, Bp35 and Bp50. Proc. Natl. Acad. Sci. USA 83, 4494- surface expression of CD40 was low. The discrepancy may 4498 be explained by the presence of a soluble form of CD40 in 9. Collins, S.J., Ruscetti, F.W., Gallagher, R.E., and Gallo, R.C. the culture medium. We found that a wide band (indicative (1979) Normal functional characteristics of cultured human of glycoprotein) that reacted with ƒ¿-CD40, and whose promyelocytic leukemia cells (HL-60) after induction of differentiation by dimethylsulfoxide. J. Exp. Med. 149, 969-974 molecular mass was slightly smaller than that of the 10. DiSanto, J.P., Bonnefoy, J.Y., Gauchat, J.F., Fischer, A., and de membrane form of CD40, was present in the culture Saint Basile, G. (1993) CD40 ligand mutations in X-linked medium. Such a band was also detected in media of HL60 immunodeficiency with hyper-IgM. Nature 361, 541-543 cells cultured with the combination of RA and GM-CSF. 11. DiStefano, P.S. and Johnson, E.J. (1988) Identification of a The existence of the soluble form of CD40 in B cell lines truncated form of the nerve growth factor receptor. Proc. Natl. was reported before (22). It is possible, therefore, that the Acad. Sci. USA 85, 270-274 band was that of a soluble form of CD40. Soluble-form 12. Freudenthal, P.S. and Steinman, R.M. (1990) The distinct surface of human blood dendritic cells, as observed after an receptors are known to be present in other members of the improved isolation method. Proc. Natl. Acad. Sci. USA 87, NGF receptor family, including NGF-R (11, 38), TNF-R 7698-7702 (30, 33, 36), CD27 (17), and CD30 (20). In the case of 13. Fuleihan, R., Ramesh, N., Loh, R., Jabara, H., Rosen, R.S., CD27, which belongs to the same receptor family as CD40, Chatila, T., Fu, S.M., Stamenkovic, I., and Geha, R.S. (1993) the soluble receptor was proposed to be derived from the Defective expression of the CD40 ligand in X -linked transmembrane form by proteolysis (27). Soluble-form immunoglobulin deficiency with normal or elevated IgM. Proc. Natl. Acad. Sci. USA 90, 2170-2173 CD4O may also be generated from the membrane receptor 14. Galy, A.H. and Spits, H. (1992) CD40 is functionally expressed by proteolysis. It has been proposed that the release of on human thymic epithelial cells. J. Immunol. 149, 775-782 soluble-form CD40 could prevent further signaling via the 15. Gordon, J., Millsum, M.J., Guy, G.R., and Ledbetter, J.A. CD40-CD40L pathway upon direct interaction between T (1988) Resting B lymphocytes can be triggered directly through and B lymphocytes (22). If CD40 is, in fact, transiently the CDw40 (Bp50) antigen. A comparison with IL-4-mediated expressed on myeloid progenitor cells during differentia signaling. J. Immunol. 140, 1425-1430 tion toward granulocytes, and soluble form of CD40 is 16. Gruber, M.F., Bjorndahl, J.M., Nakamura, S., and Fu, S.M. released under such conditions, its role may be to prevent (1989) Anti-CD45 inhibition of human B cell proliferation depends on the nature of activation signals and the state of B cell further interaction of such cells with CD40L-positive cells. activation. A study with anti-IgM and anti-CDw40 antibodies. J. Immunol. 142, 4144-4152 We thank Dr. M. Shimazu, Department of Genetics, Mitsubishi Yuka 17. Hintzen, R.Q., de Jong, R., Hack, C.E., Chamuleau, M., de Vries, Bio-clinical Laboratories, Inc., Tokyo, for providing plasmid pHSG E.F.R., ten Berge, I.J.M., Borst, J., and van Lier, R.A.W. (1991) 298-CD40 and Ms. T. Okazaki for assistance. A soluble form of the human differentiation antigen CD27 is released after triggering of the TCR/CD3 complex. J. Immunol. 147,29-35 REFERENCES 18. Hollenbaugh, D., Grosmaire, L.S., Kullas, C.D., Chalupny, N.J., 1. Alderson, M.R., Armitage, R.J., Tough, T.W., Strockbine, L., Braesch, A.S., Noelle, R.J., Stamenkovic, I., Ledbetter, J.A., Fanslow, W.C., and Spriggs, M.K. (1993) CD40 expression by and Aruffo, A. (1992) The human T cell antigen gp39, a member human monocytes: Regulation by cytokines and activation of of the TNF gene family, is a ligand for the CD40 receptor: monocytes by the ligand for CD40. J. Exp. Med. 178, 669-674 Expression of a soluble form of gp39 with B cell co-stimulatory 2. Allen, R.C., Armitage, R.J., Conley, M.E., Rosenblatt, H., activity. EMBO J. 11, 4313-4321 Jenkins, N.A., Copeland, N.G., Bedell, M.A., Edelhoff, S., 19. Jabara, H. H., Fu, S. M., Geha, R.S., and Vercelli, D. (1990) CD40 Disteche, C.M., Simoneaux, D.K., Faslow, W.C., Belmont, J., and IgE: Synergism between anti-CD40 monoclonal antibody and and Spriggs, M.K. (1993) CD40 ligand gene defects responsible in the induction of IgE synthesis by highly purified for X-linked hyper-IgM syndrome. Science 259, 990-993 human B cells. J. Exp. Med. 172, 1861-1864 3. Armitage, R.J., Fanslow, W.C., Strockbine, L., Sato, T.A., 20. Josimovic, A.O., Durkop, H., Schwarting, R., Backe, E., Stein, Clifford, K.N., Macduff, B.M., Anderson, D.M., Gimpel, S.D., H., and Diamantstein, T. (1989) Ki-1 (CD30) antigen is released Davis, S.T., Maliszewski, C.R., Clark, E.A., Smith, C.A., by Ki-1-positive tumo cells in vitro and in vivo. I. Partial

Vol. 118, No. 3, 1995 540 T. Shinagawa et al.

characterization of soluble Ki-1 antigen and detection of the 22- to 23-Kd subunit of cytochrome b,58 at the surface of human antigen in cell culture supernatants and in serum by an enzyme peripheral phagocytes. Blood 72, 1550-1552 linked immunosorbent assay. Eur. J. Immunol. 19, 157-162 30. Nophar, Y., Kemper, 0., Brakebusch, C., Englemann, H., 21. Kobayashi, S., Imajo-Ohmi, S., Nakamura, M., and Kanegasaki, Zwang, R., Aderka, D., Holtmann, H., and Wallach, D. (1990) S. (1990) Occurrence of cytochrome b558in B-cell lineage of Soluble forms of tumor necrosis factor receptors (TNF-Rs). The human lymphocytes. Blood 75, 458-461 cDNA for the type I TNF-R, cloned using amino acid sequence 22. Kooten, C.V., Gaillard, C., Galizzi, J.-P., Hermann, P., Fosseiz, data of its soluble form, encodes both the cell surface and a F., Banchereau, J., and Blanchard, D. (1994) B cells regulate soluble form of the receptor. EMBO J. 9, 3269-3278 expression of CD40 ligand on activated T cells by lowering the 31. Paulie, S., Ehlin, H.B., Mellstedt, H., Koho, H., Ben, A.H., and mRNA level and through the release of soluble CD40. Eur. J. Perlmann, P. (1985) A p50 surface antigen restricted to human Immunol. 24, 787-792 urinary bladder carcinomas and B lymphocytes. Cancer Immunol. 23. Korthauer, U., Graf, D., Mages, H.W., Briere , F., Padayachee, Immunother. 20, 23-28 M., Malcolm, S., Ugazio, A.G., Notarangelo, L.D., Levinsky, 32. Paulie, S., Rosen, A., Ehlin, H.B., Braesch, A.S., Jakobson, E., R.J., and Kroczek, R.A. (1993) Defective expression of T-cell Koho, H., and Perlmann, P. (1989) The human B lymphocyte and CD40 ligand causes X-linked immunodeficiency with hyper-IgM. carcinoma antigen, CDw40, is a phosphoprotein involved in Nature 361, 539 541 growth . J. Immunol. 142, 590-595 24. Laemmli, U.K. (1970) Cleavage of structural proteins during the 33. Porteu, F. and Nathan, C. (1990) Shedding of tumor necrosis assembly of the head of bacteriophage T4. Nature 227, 680-685 factor receptors by activated human . J. Exp. Med. 25. Lin, C.S., Ng, S.Y., Gunning, P., Kedes, L., and Leavitt, J. 172,599-607 (1985) Identification and order of sequential mutations in 34. Saeland, S., Duvert, V., Caux, C., Pandrau, D., Favre, C., Valle, beta-actin genes isolated from increasingly tumorigenic human A., Durand, I., Charbord, P., de Vries, J., and Banchereau, J. fi broblast strains. Proc. Natl. Acad. Sci. USA 82, 6995-6999 (1992) Distribution of surface-membrane molecules on bone 26. Liu, Y.J., Joshua, D.E,, Williams, G.T., Smith, C.A., Gordon, J., marrow and cord blood CD34+ hematopoietic cells. Exp. and MacLennan, I.C. (1989) Mechanism of antigen-driven selec Hematol. 20, 24-33 tion in germinal centres. Nature 342, 929-931 35. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Moleculer 27. Loenen, W.A.M., de Vries, E., Gravestein, L.A., Hintzen, R.Q., Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory van Lier, R.A.W., and Borst, J. (1992) The CD27 membrane Press, Cold Spring Harbor, NY receptor, a lymphocyte-specific member of the nerve growth 36. Spinas, G.A., Keller, U., and Brockhaus, M. (1992) Release of factor receptor family, gives rise to a soluble form by protein soluble receptors for tumor necrosis factor (TNF) in relation to processing that does not involve receptor endocytosis. Eur. J. circulating TNF during experimental endotoxinemia. J Clin. Immunol. 22, 447-455 Invest. 90, 533-536 28. Mizuno, T., Kaibuchi, K., Ando, S., Musha, T., Hiraoka, K., 37. Stamenkovic, I., Clark, E.A., and Seed, B. (1989) A B-lympho Takaishi, K., Asada, M., Nunoi, H., Matsuda, I., and Takai, Y. cyte activation molecule related to the nerve growth factor (1992) Regulation of the superoxide-generating NADPH oxidase receptor and induced by cytokines in carcinomas. EMBO J. 8, by a small GTP-binding protein and its stimulatory and inhibi 1403-1410 tory GDP/GTP exchange proteins. J. Biol. Chem. 267, 10215 38. Zupan, A.A., Osborne, P.A., Smith, C.E., Siegel, N.R., Leim - 10218 gruber, R.M., and Johnson, E.J. (1989) Identification, purifica 29. Nakamura, M., Sendo, S., van Zwieten, R., Koga, T., Roos, D., tion, and characterization of truncated forms of the human nerve and Kanegasaki, S. (1988) Immunocytochemical discovery of the growth factor receptor. J. Biol. Chem. 264, 11714-11720

J. Biochem.