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From www.bloodjournal.org by guest on August 3, 2017. For personal use only. Epstein-Barr Virus Transformed B Lymphocytes Produce Interleukin-5 By Cassandra C. Paul, Jonathan R. Keller, James M. Armpriester, and Michael A. Baumann Interleukin-5 (IL-5) has previously been isolated only as a product of T lymphocytes. We have found that EpsteinBarr virus transformed B lymphocytes produce large amounts of IL-5 activity in culture supernatants, inducing proliferation of murine BCL, cells, and supporting the selective growth of eosinophil colonies in semi-solid culture. Production of IL-5 messenger RNA by transformed B-cell lines was verified by Northern analysis using a 3.2- kilobase cloned DNA fragment containing the full-length human IL-5 gene, and immunoreactive IL-5 was detected in B-cell culture supernatants. These findings suggest a possible expanded role for the B cell in the induction of eosinophilia, and should serve as a focus for additional investigation into possible roles for IL-5 in human B-cell proliferation and differentiation. 0 1990 by The American Society of Hematology. I isolated after centrifugation of the buffy layer over Ficoll-Hypaque, and the cells were resuspended at 4 x 105/mL in complete medium consisting of RPMI-1640 containing 7.5% fetal calf serum, 2 mol/L mmol/L 1-glutamine, 1 mmol/L sodium pyruvate, 5 x 2-mercaptoethanol, and 40 pg/mL gentamicin. Suspension cultures were maintained in 75-cm’ tissue culture flasks and fed periodically by replacement of one third of the medium. Transformed B-cell1 lines were seen to emerge at about 6 weeks, after which time they rapidly became the only viable elements in the cultures. The identity of the emerging cells was confirmed by flow cytometric analysis for the B-cell specific antigens CD20 and CD19, and the EBV-induced antigen CD23. Presence of EBV in the cell lines was confirmed by immunofluorescent labeling with anti-EBNA (Chemicon International Inc, El Segundo, CA). All EBV-transformed cell lines studied for growth factor activity had been maintained continuously in culture for more than a year. The purity of the populations was verified by flow cytometric analysis demonstrating absence of cell types other than B cells. In some instances, immunoglobulin gene rearrangement studies were performed, documenting the presence after long-term culture of pure B-cell clones. Detection of IL-5 activity in B-cell culture supernatants. Culture supernatants were tested in two assays standardly used to detect IL-5 activity: the induction of proliferation of the murine B-cell line BCL,,’ and the support of selective growth of eosinophil colonies in semi-solid cultures of human bone marrow.8 These assays are quite specific for the detection of IL-5 activity, as no human growth factor other than IL-5 has been shown to stimulate BCL, cells, and other lymphokines capable of stimulating eosinophil colony growth, such as granulocyte-macrophage colony-stimulating factor and IL-3, also stimulate the growth of other hematopoietic lineages under the culture conditions used.’ BCL, stimulation assay. BCL, cells, freshly obtained after in vivo passage: were used for all assays. BCL, cells, 1 x lo5, were cultured in 96-well flat-bottomed tissue culture plates in either 0.2 mL complete medium alone or with the addition of 25% vol/vol medium conditioned by EBV-transformed B-cell lines. Quadruplicate cultures were incubated for 48 hours at 37OC before being labeled with 0.5 pCi ’H-thymidine for an additional 6 hours. The cultures were harvested onto glass fiber filters and the relative amount of radiolabel incorporation was determined by liquid scintillation counting. IL-5 activity present in B-cell supernatants was quantified by comparison to a reference curve constructed by culturing BCL, cells as above in varying concentrations of recombinant human IL-5 (generously provided by Steven Clark, Genetics Institute, Cambridge, MA). Eosinophil colony formation assay. Bone marrow from four normal volunteers was aspirated into heparinized syringes. The mononuclear cell fraction was cultured using standard methodology’ in Iscove’s Modified Dulbecco’s medium with 0.8% methylcellulose (final concentration), containing 20% heat-inactivated fetal calf serum, 5 x IO-’ mol/L 2-mercaptoethanol, and 2 mmol/L 1-glutamine. Colonies were scored on day 14 and were identified by characteristic morphology by inverted microscopy, with confirma- NTERLEUKIN-5 (IL-5) is a hematopoietic growth factor that was initially studied in murine systems, where it was found to induce proliferation and differentiation of eosinophil precursors and promote proliferation and antibody production by B cells.’ The gene coding for IL-5 was isolated from a murine T-cell hybridoma, molecularly cloned, and used to locate and clone the human IL-5 gene from a T-cell leukemia The murine and human IL-5 genes have 77% homology: and recombinantly produced human IL-5 has been found to induce the growth of human eosinophil colonies,’,*but has lacked stimulatory effects in established human B-cell functional assays.’ Infection of human B cells by the Epstein-Barr virus (EBV) results in a permanent state of activation, in which the B cell is induced to express a number of activation antigens and to secrete substances with apparent growth factor a ~ t i v i t y It . ~ is thought that an autocrine mechanism fuels the perpetual proliferation of EBV-transformed B cells in vitro.5 We have found that EBV transformation of human B cells consistently results in the induction of production and secretion of large amounts of IL-5. This phenomenon may have physiologic relevance, since it suggests a possible expanded role for the B cell in the promotion of eosinophilia during type 1 immune responses. In addition, these findings may serve as a focus for further investigation into possible activities of IL-5 on human B cells. MATERIALS AND METHODS Establishment of EBV-transformed B-cell lines. Peripheral blood was obtained after informed consent on several occasions from each of two subjects with known previous exposure to EBV. We had determined previously that spontaneous EBV-transformed B-cell lines would emerge from suspension cultures of peripheral blood mononuclear cells of these subjects.‘ The mononuclear fraction was From the Department of .Medicine, Wright State University School of Medicine and VA Medical Center. Dayton. OH: and Program Resources, Inc. Frederick Cancer Research Facility, MD. Submitted November 30,1989: accepted January 3.1990. Supported by a Merit Review Grant from the Veterans Administration, and by the American Heart Association. Ohio ABliate. Address reprint requests to Michael A . Baumann, MD, Department of Medicine 1 1 1 W , 4100 W Third St. Dayton, OH 45428. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with I8 U.S.C.section 1734 solely to indicate this fact. o I990 by The American Society of Hematology. 0006-4971/90/7507-0027$3.00/0 1400 Blood, Vol 75,No 7 (April l), 1990:pp 1400-1403 From www.bloodjournal.org by guest on August 3, 2017. For personal use only. 1401 8 CELLS PRODUCE IL-5 tion by Wright-Giemsa staining of colonies picked off the semi-solid culture and allowed to dry onto a glass microscope slide. Background colony numbers were obtained from cultures not supplied with any exogenous growth factor. Positive controls were incubated with 10 U of recombinant human (rh) IL-5. Experimental cultures contained 50 pL of sterile-filtered medium conditioned by EBV-transformed B-cell lines. All cultures were performed in quadruplicate, except unsupplemented cultures used for comparison. Northern analysis for IL-5 messenger R N A (mRNA) expression by B-cell lines. Northern analysis was performed using standard methodology." Cells were lysed in 4 mol/L guanidinium solution and the lysate was ultracentrifuged through 5.5 mol/L cesium chloride and 0.1 mol/L EDTA for 18 hours. The RNA pellet was resuspended in 10 mmol/L Tris.Cl (pH 7.4), 5 mmol/L EDTA, and 1% sodium dodecyl sulfate, ethanol-precipitated, and dissolved in diethylpyrocarbonate-treated water. Total cellular RNA (15 pg/ lane) was electrophoresed through a 1.3% denaturing agarose gel. Visualization of the 28s and 18s ribosomal RNA bands by ethidium bromide staining was used to ascertain that similar amounts of RNA were loaded in each lane before blotting to a nylon transfer membrane (Nytran, Schleicher & Schuell, Keene, NH). A 3.2kilobase (kb) cloned DNA fragment containing the full-length human IL-5 gene" (generously provided by Steven Clark) was )*P-labeled by the random oligonucleotide primer method,'* and hybridized with the blot. The blot was then washed to greater than 90% stringency and autoradiographed at -7OOC using an intensifying screen. Western analysis for IL-5. Separation by polyacrylamide gel electrophoresis was performed using the PhastSystem (Pharmacia, Inc, Piscataway, NJ), with an 8% to 25% gradient, followed by diffusion blotting at 7OoC of separated proteins to nitrocellulose. The filter was blocked in phosphate-buffered saline containing 20% fetal calf serum. The primary antibody was the rat monoclonal anti-IL-5, TRFK-5') (gift of Donna Rennick, DNAX, Palo Alto, CA). As this antibody is known to react preferentially with dimeric IL-5 (personal communication, Dr Michael Grace, Schering Corp, November 1989), electophoresis conditions were nonreducing. The secondary antibody was a goat anti-rat immunoglobulin (Ig) G alkaline phosphatase conjugate (BRL, Inc, Gaithersburg, MD), and the substrate was 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt and nitroblue tetrazolium chloride (BRL). B 3 - n x - a A C Fig 2. Removal of BCL, stimulation by preincubation of three EBV-B-cell supernatants with anti-IL-5. (A) BCL, cells alone; (B) BCL, cells B-cell supernatant; ( C ) BCL, cells antibodypreincubated B-cell supernatant. + + liferation of murine BCL, cells in short-term culture (Fig 1). In a repeat experiment, the activity of B-cell supernatants on BCL, cells was completely removed by preincubation of the supernatants for 2 hours at 22OC with the monoclonal IL-5 neutralizing antibody, TRFK-513 (Fig 2). Eosinophil colony formation assay. Eosinophil colonies were obtained in bone marrow cultures supplemented with EBV-transformed B-cell conditioned medium in numbers comparable with those obtained in cultures receiving 10 U rhIL-5. Only rare colony forming unit-granulocyte macrophage (CFU-GM) or CFU-M colonies were seen in cultures receiving no supplementation, and these colonies were present in similar numbers in supplemented cultures (Table 1). Northern analysis for ZL-5 mRNA. The DNA probe containing the full-length human IL-5 gene specifically hybridized to a 3.2-kb fragment of RNA in all four EBVtransformed B-cell lines tested (Fig 3). Western analysis for IL-5. Immunoreactive bands were found at a molecular weight of approximately 45 Kd, as has been reported for native human IL-5,I4 in culture supernatants of 3 of 4 EBV-transformed B-cell lines studied (Fig 4). RESULTS BCL, proliferation assay. Culture supernatants of five EBV-transformed B-cell lines markedly augmented the pro- DISCUSSION The finding that B cells can be induced to produce IL-5 by EBV infection is cause for re-evaluation of the interactions Table 1. Bone Marrow Colony Formation in Response to rhlL-5 and EBV-Transformed B-Cell Culture Supernatant Bone Marrow 1 2 3 4 BCLl ---- + B-Cell Supernatants--- Fig 1. Effect of EBV-transformed B-cell supernatants on proliferation of BCL, cells. Results graphed as mean 2 SD of four replicates. CPM, counts per minute. CFU-EO CFU-GM or CFU-EO CFU-GM or CFU-EO CFU-GM or CFU-EO CFU-GM or Unsupplemented 0 M M M M 1 0 2 0 2 0 2 + 10 U rhlL-5 +B-Cell CM 5.4,8.4 0.2,o. 1 8,7,6,9 1.2.1 ,o 5.8.6.8 1.0.0.0 5.4.5.3 0,l ,o,o 8,4.9.8 1,o, 1,o 8,10,10,12 1.0.0.2 9.11.15.10 0,2.0.0 6.8.8.10 1,o,o,o Cultures supplemented with rhlL-5 or E8V-transformed B-cell conditioned medium were performed in quadruplicate. Single unsupplemented cultures were done for comparison. Abbreviation: CM, conditioned medium. From www.bloodjournal.org by guest on August 3, 2017. For personal use only. PAUL ET AL 1402 a 20s b c d e D 3-2kb m 10s m ~ b. ' ' rk I. +2 1 4 14 Fig 4. W e " 0 ~ l y . hfor - i Culture W W M of h"n IL-6. (Lane 1) m p h m l " I - cdl'. (Lane 2, human IL-" and 4, Culture supermtant of two EBV-transformed B-cell lines. In addfihl of cell lines dawable IL-6. Fig 3. Northern amtysis for IL-6 mRNA showing apecitic bands a 3.2 kb for four distlnct EBV-transformed &cell lines (lanes b through e). (Lane a) WS1 human fibroblast cdls (neggaive control). (Right) Ethidiumbromidestainedgel showing comparable amounts of total cellular RNA loaded into each lane. that occur in the induction of eosinophilia and in B-cell activation, growth, and differentiation. Transformation of B cells by EBV results in the induction of expression of several cell-surface antigens. including the CD23 structure that functions as a low-affinity IgE receptor." It is now known that a variety of activation stimuli, including Stuphylmmcus uuteus Cowan strain I, TPA (12-0-tetra-decanoylphorbol 13-acetate), and IL-4 will induce normal B lymphocytes to express the CD23 activation antigen.I6 Thus, B-cell expression of an IgE receptor seems to be a general feature of B-cell activation, and may be coupled, a t least in EBV-transformed cells, with the production and secretion of large amounts of IL-5. If IL-5 production is found to be inducible in B cells by activation stimuli other than EBV, it will be necessary to consider the possibility that B cells may be capable of amplifying the IL-5 signal in the inductionof eosinophilia in type 1 immune responses. In murine systems, IL-5 has been shown to induce B cells to produce Ig and cause augmented proliferation of B cells costimulated with a mitogen such as dextran sulfate.' Murine B cells have been found to display an IL-5 receptor that is upregulated in response to lipopoiysaccharidestimulati~n.'~ Although no B a l l growth activity of IL-5 has yet been convincingly detected in human systems,' the finding that human B cells may be induced to produce and secrete IL-5 provides a focus for future investigations of the interaction of IL-5 with human B lymphocytes, including possible autocrine mechanisms operative during B-cell proliferation and differentiation. REFERENCES 1. Sanderson CJ, Campbell HD. Young I G Molecular and cellu- lar biology of eosinophil differentiation factor (interleukin-5) and its effects on human and mouse B cells. Immunol Rev 102:29.1988 2. Clutterbuck U.Hirst EMA. Sanderson CJ: Human interleukin-5 (IL-5) regulates the production of eosinophils in human bone m a w cultures: Comparisonand interaction with IL-I. IL-3. IL-6, and GM-CSF. Blood 73: 1504,1989 3. Azuma C. Tanabe T. Konishi M, Kinashi T, Noma T, Matsuda F. Yaoita Y. Takatsu K. Hammarstrtim L, Smith CIE. Severinson E. Honjo T Cloning of cDNA for human T e l l replacing factor (interleukin-5) and comparison with the murine homologue. Nucleic Acids Res 149149. 1986 4. Enokihara H. Furusawa S. Nakahbo H. Kajitani H. N a g shima s, Saito K. Shishido H. Hitoshi Y. Takatsu K. Noma T. Shimizu A. Honjo T T cells from eosinophilic patients produce interleukin-5 with interleukin-2 stimulation. Blood 73:1809,1989 5. Thorley-Lawson DA: Basic virological aspects of Epstein-Barr virus infection. Semin Hematol25:247. 1988 From www.bloodjournal.org by guest on August 3, 2017. For personal use only. B CELLS PRODUCE IL-5 6. Nilsson K: The nature of lymphoid cell lines and their relationship to the virus, in Epstein MA, Achong BG ( 4 s ) : The Epstein-Barr Virus. Berlin, Germany, Springer-Verlag, 1979, p 225 7. Swain SL, Dutton RW: Production of a B cell growthpromoting activity, (DL) BCGF, from a cloned T cell line and its assay on the BCLl B cell tumor. J Exp Med 156:1821, 1982 8. Metcalf D: Clonal culture of hemopoietic cells: Techniques and applications. Amsterdam, The Netherlands, Elsevier, 1984, p 32 9. Isakson PC, Krolick KA, Uhr JW, Vitetta ES: The effect of anti-immunoglobulin antibodies on the in vitro proliferation of normal and neoplastic murine B cells. J Immunol 125:886, 1980 10. Southern EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol98:503, 1975 11. Campbell HD, Tucker WQ, Hort Y, Martinson ME, Mayo G, Clutterbuck EJ, Sanderson CJ, Young IC: Molecular cloning, nucleotide sequence, and expression of the gene encoding human eosinophil differentiation factor (interleukin-5). Proc Natl Acad Sci USA 84:6629,1987 12. Feinberg AP, Vogelstein B: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6, 1983 1403 13. Schumacher JH, O’Garra A, Shrader B, van Kimmenade A, Bond MW, Mosmann TR, Coffman RL: The characterization of four monoclonal antibodies for mouse IL-5 and development of mouse and human 1L-5 enzyme-linked immunosorbent. J Immunol 141:1576,1988 14. Yokota T, Arai N, de Vries J, Spits H, Banchereau J, Zlotnik A, Rennick D, Howard M, Takebe Y, Miyatake S, Lee F, Arai K: Molecular biology of interleukin 4 and interleukin 5 genes and biology of their products that stimulate B cells, T cells and hemopoietic cells. Immunol Rev 102:137, 1988 15. Bentsson M, Gordon J, Flores-Romo L, Cairns JA, Smedmyr B, Oberg G, Simonsson B, Totterman TH: B-cell reconstitution after autologous bone marrow transplantation: Increase in serum CD23 (“IgE-binding factor”) precedes IgE and B-cell regeneration. Blood 73:2139, 1989 16. Gordon J, Guy GR: The molecules controlling B lymphocytes. Immunol Today 8:339,1987 17. Takatsu K, Tominaga A, Harada N, Mita S, Matsumoto M, Takahashi T, Kikuchi Y, Yamaguchi N: T cell-replacing factor (TRF)/interleukin 5 (IL-5): Molecular and functional properties. Immunol Rev 102:107,1988 From www.bloodjournal.org by guest on August 3, 2017. For personal use only. 1990 75: 1400-1403 Epstein-Barr virus transformed B lymphocytes produce interleukin-5 CC Paul, JR Keller, JM Armpriester and MA Baumann Updated information and services can be found at: http://www.bloodjournal.org/content/75/7/1400.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.