By Sadik Oez, Hanne Tittelbach, Raimund Fahsold, Roland Schaetzl, Christoph Buhrer, ..... Welte K, Platzer E, Lu L, Gabrilove JL, Levi E, Mertelsmann.
Establishment and Characterization of a Granulocyte-Macrophage Colony-Stimulating Factor-Dependent Human Myeloid Cell Line By Sadik Oez, Hanne Tittelbach, Raimund Fahsold, Roland Schaetzl, Christoph Buhrer, Jens Atzpodien, and Joachim R. Kalden A new human myeloid cell line has been established recently from the bone marrow cells of a patient with chronic myelogenous leukemia in blast crisis. The active proliferation and survival of the cells in RPMI 1640 medium containing fetal calf serum are clearly dependent on the presence of either natural or recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF). Despite permanent culturing in rhGM-CSF (100 U/mL), the cells do not differentiate and bear the myelomonocytic surface markers CD34, CD13, CD36, as well as HLA-DR,
but not CD3, CD7, CD10, CDllb, CD14, CD20, or CD42b. The predominant karyotype, apart from tetraploidy in several cells, is 45, XX, -9, -17, -19, -22, 7p-, 9q+ (der t[9;22]), der (13q). with three additional marker chromosomes, from which one was observed in the patient’s leukemic cells. On Bglll-digested DNA, Southern blot analysis with bcr 5’ as the probe detected two additional hybridizingrestriction fragments of 8.6 and 11.O kilobase pairs. 0 1990 by The American Society of Hematology.
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The disease had been diagnosed there primarily on the basis of clinical features of disease: white blood cell (WBC) count 228,000 (with 9% blasts, 3% promyelocytes, 18% myelocytes, 9% metamyelocytes, 19% band, 30% neutrophils, 6% eosinophils, 4% basophils, 2% lymphocytes); strongly decreased alkaline leukocyte phosphatase (3 score); as well as cytologic and histologic examination of the bone marrow specimen obtained through iliac crest puncture. Although an HLA typing had been performed, no karyotype analysis had been undertaken with respect to the Ph’ chromosome. Initially, the patient had been treated with busulfan orally until May 1987; but busulfan was ineffective, and was discontinued and replaced by hydroxyurea. The patient’s family doctor then took over attendance in June 1987, but again had to refer her to the hospital in December 1988 because of a blast increase in peripheral blood. Two days later, she was sent to our clinic. On examination, the patient appeared acutely ill and somnolent, with temperatures greater than 4OoC, dyspnea, and tachycardia. WBC count was 164,000 with 76% blasts, 16% neutrophils, 5% lymphocytes, and 3% monocytes, and the platelet count was 46,000. Phenotype examination of the peripheral blood leukocytes gave clear evidence that the blast cells bore myeloid antigens such as 5% CD3 (OKT3), 4% CD7 (3A1), 2% CD20 (Bl), 36% CD24 (BA-I), 16% CD13 (My7), and 20% CD33 (My9). Cytochemical staining of bone marrow cells showed that 25% of the blasts were positive for esterase but were negative for peroxidase and PAS. Despite treatment with antibiotics, the patient finally died of an overt pneumonia and sepsis on the third hospital day.
ELL LINES requiring specific factors for their survival, proliferation, or growth inhibition are excellent biologic tools for studying the actions of factors in question. Various cell lines have been reported from mouse or human origins for interleukin-2 (IL-2),’ tumor necrosis factor a (TNFa),’ IL-6,’ granulocyte colony-stimulating factor (GCSF): and granulocyte-macrophage C S F (GM-CSF).5s6 GM-CSF is one of the well-characterized hematopoietic growth factors that acts on myeloid progenitor cells at various maturation stages and induces proliferation, differentiation, and functional stimulation.’-’* The proliferative effect of GM-CSF is not absolutely restricted to myeloid cells, since lymphatic leukemia cell lines: colon adenocarcinoma cell lines,13 and human endothelial cells14 also have been reported to respond to GM-CSF. In this report we describe the characteristics of a GM-CSFdependent myeloid cell line, which has recently been derived from the bone marrow cells of a patient with chronic myelogenous leukemia (CML) in blast crisis. Although several aspects are still under investigation, we think that this newly established cell line, designated GM/SO, will contribute to understand the biologic action of GM-CSF on the cells. By using the effect of GM-CSF on this cell line, a very sensitive bioassay has already been developed for measuring the GM-CSF concentrations in cell culture supernatants or in human sera (Oez S, Buhrer C, Atzpodien J, Kalden JR, manuscript submitted). CASE REPORT
A 50-year-old female patient (G.M.) was referred to our clinic from another hospital due to a blast crisis of a CML, which had been diagnosed in February 1986.
From the Medizinische Klinik III and Institut fiir Humangenetik. Universitat Erlangen-Niirnberg; and Medizinische Hochschule Hannover. FRG. Submitted January 2, 1990; accepted April 3, 1990. Address reprint requests to Sadik Oez, MD. Medizinische Klinik III, Universitat Erlangen-Niirnberg, Krankenhausstr. 12, 8520 Erlangen, FAG. The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.section 1734 solely to indicate this fact. 0 1990 by The American Society of Hematology. 0006-4971/90/7603-00I4$3.00/0 578
MATERIALS AND METHODS
Bone marrow cells. With the patient’s prior informed consent, marrow cells were obtained by means of iliac crest puncture. Mononuclear cells were separated by density-gradient centrifugation on Ficoll (Nycomed AS, Oslo, Norway) and cultured in RPMI 1640 medium with 10% fetal calf serum (FCS) and growth factors. Part of the cells were preserved by freezing in liquid nitrogen with 10%dimethylsulfoxide (DMSO) and RPMI 1640 medium containing 40% FCS, and these were used later for karyotype analysis. Cytokines. Recombinant human G-CSF (rhG-CSF), rhGMCSF, rhIL-3, rhIL-4, and rhIL-6 were provided by L. Souza (AMGen, Thousand Oaks, CA), whereas rhIL-la and rhIL-2 were obtained from Boehringer-Mannheim (FRG). Antibodies. Rabbit anti-GM-CSF antiserum was also a gift from L. Souza (AMGen). Monoclonal antibodies (MoAbs) against CD34 (HPCA-I), CD7 (Leu 9), CDlO (CALLA), CD16 (Leu 1lb), CD20 (Leu 16), HLA-DR, and fluorescein-conjugated goat antimouse were purchased from Becton-Dickinson (Mountain View, CA); CD36 (OKM 5). C D l l b (OKM l ) , CD13 (OKM 13). CD14 (OKM 14) and CD3 (OKT 3) from Ortho-Diagnostic System (Neckargemiind, FRG); and CD42b (AN 51) from Dakopatts (Hamburg, FRG). B h d , VOI 76, NO 3 (August 1). 1990: PP 578-582
OM--DEPENDENT
HUMAN MYELOlD CELL LINE
579
h/lfemrlon asuy. After washing the cclb three times in phosphate-buffered saline (PBS), the celb (1 x lO'cells/well) were incubated for 72 hours in flat-bottom 96welI microtiter plates in a final volume of 200 rL RPMl 1640 medium containing 10% FCS and various cytokines. 'H-thymidine (Ammham. Braunxhweig. FRG) was added at I pCi (37 kBq) per well for 4 hours before the end of the culture. The cells were then harvestedon fiberglass filters and measured by liquid scintillation counting. All assays were performed in triplicate and data are expressed as the mean counts per minute of 'H-thymidine incorporated. Immunophenoryping. Cell surface antigens w m detected by standard indirect immunofluorescenceassny. I n brief. the cells were incubated at 4% for 30 minutes with mouse MoAbs. washed three t i m a by centrifugation with PBS. followed by adding FITCconjugated gost anti-mouse antibody for 30 minutes at 4%. Stained cells were then evaluated with a UV-microscope. The cells were considered positive for the antigen in question when the fluorescence intensity on intact cells was clearly visible in comparison with the cells treated only with FlTCconjugated goat anti-mouse antibody. Chromosome unulysis. Karyotyping of GM/SO cells and cryapreserved bone marrow cells was performed by standard techniques, including Giemsa-trypsin banding." Sourhem blor unulysis. High molecular weight DNA from celb was digested with &Ill. electrophoresedon a 0.8% agarose gel. and
A
B F b l . M#phdogymdmorphdoOicdl(hr.mC d o n of O M 1 8 0 dh QMISO colla growing In modhnneontoining100UlmL rhQM-CSF olocw (A). In c ~ r o s t.:ttroxpowro to odditiwl PMA (100 ng/mL) for 24 hours IB). Tho odhront cdls cukurod on stwilo s1id.r ond oir-drhd -0 staining IMay-GrGnmldl.
MI. B
blotted onto a nylon membrane (Gcnescrm Plus; NEN. d u h t de Nemoun, Bad Homburg, FRG). The &r-5' probe (provided by C.R. Bartram. Ulm. FRG) was labeled by nick-translation and hybridi 7 d according to standard protocols.'4" Sruiningfor micmmpy. May-GrtInwaldand ~ylochcmiilrtainings for PAS. myeloperoxidase. and a-naphtyl acetate esterase w m performed in our routine hematology laboratory.
RESULTS
Establishment and cellular characteristfcs. Initially. bone marrow mononuclear cells were cultured in the presenceof rhG-CSF. rhGM-CSF. and IL-3 (1.OOO U/mLcach) with the intention of studying the differentiation capacity of the blast cells. As usual, the culture medium used was RPMl 1640, supplemented with 10% FCS. 0.2 mmol/L Lglutamine, and antibiotics. Within the first 2 weeks there were no differences to be detected. either in proliferation rate or in maturation of the cells in the presence of these three hematopoietic growth factors. However, after scveral passages an active proliferation was sustained only by the cells exposod to rhGM-CSF. In subsequent steps. three stable subclones were established by using limiting dilution tech-
OEZ ET AL
580 5
ol''
0-0
T
0-
A-A
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hCU-CSF and anti-W-CSF antibody hlL-3
0-0
hGCSF
A-A
ML-2
e--*
hlL-4
0--0
0
.-
hCU-CSF
0
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Fig 2. Proliferative response of GM/SO cells t o rhGM-CSF, rhG-CSF, rhlL-la, rhlL-2, rhlL-3, rhlL4, or rhlL-6 either alone at concentrations between 1 and 1,OOO U/mL or in combination with 1 UlmL rhGM-CSF; measurement after 72 hours. The neutralizing antibody was tested in 1:40 final dilution, and the curves represent the mean values of four separate experiments.
1 u/ml hCU-CSF
+ hlL-1,
V-V
1 u/ml h W - C S F
+ hlL-6 I
n
1x10 10
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nique. These subclones differ in their morphology and generation time, but they still retain their requirement for external supply of rhGM-CSF. The dependency can be circumvented if the cells are cultured by carefully reducing GM-CSF for 2 or more weeks. As a consequence, cells that have once become independent do not require GM-CSF for their survival and proliferation, whereas they sustain to respond to GM-CSF with increased proliferation (data not shown). To avoid overly complex data, only the clone termed as G M / S O is discussed in this report.
2
1
(u/ml)
Although there is a considerable variation in cell size, the typical morphology of GM/SO cells in May-Grunwald staining is a large, round, or slightly irregular nucleus, three to six prominent nucleoli, and deep blue cytoplasm with a perinuclear pale zone (Fig 1A). There is no granula present and the cytochemical staining for PAS, myeloperoxidase, and cy-naphtyl acetate esterase are negative. Moreover, the cells growing in 100 U/mL rhGM-CSF (unless otherwise stimulated) retain their morphology and display no overt differentiation in liquid culture. However, a rapid change
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Fig 3. Stemline karyotype of line GM/SO 45, XX. -9, -17. -19, -22,7p-, chromosomes (Ml, M2, and M3) that could not be exactly classified.
22
9q+ der t(922) ( q M q l l ) , der (13q). plus three marker
GM-CSF-DEPENDENT HUMAN MYELOID CELL LINE
into a monocyte-macrophage morphology and increased adherence to plastic surface can be induced by the addition of phorbol myristatc acetate (PMA) (Sigma. St Louis. MO) at concentrations between 1 . 0 0 ng/ml. and I ng/ml. (Fig I R). lmmunophenotypeanalysis showed that the GM/SO cells clearly bear surface antigens of myelomonocytic cells CD34 (greater than 75%). CDI3 (greater than 968). and CD36 (greater than 92%). but not the other typical granulocytemacrophage antigens such as CDllb. CD14. or CD16. While HLA-DR (grater than 98%) i s strongly expressed on the cells. lymphocytic markers such as CD3. CD7. CDIO. CD2O. and thrombocytic marker CD42b are also definitely absent. As shown in Fig 2. the proliferative effect of GM-CSF on these cells is dose-dependent. and the saturatingdosis seems to be by I 0 0 U/mL. However. by using a poljvalent rabbit antiserum against GM-CSF at neutraliting titers, the proliferation could be completely abolished in these assays. This inhibition apparently is not due to any toxic effect in antiserum. since the neutralizing effect could in turn be entirely suppressed by e x c e ~of~ rhGM-CSF in culture medium. In addition, we simultaneously tested rhG-CSFand rhlL-3. as well as rhlL-In. rhlL-2. rhlL-4. and rhlL-6 to rule out any pssible effect on this cell line. As the given examples demonstrate. there was no detectable proliferative effect of these cytokines at concentrations between I and 1.000 U/mL. either alone or in combination with I U/mL rhGM-CSF. Cytogenetic and DNA a d y s i s . To find out whether GM/SO cells p o s s ~ sthe typical characteristics of a CML cell. eg. Ph' chromosome or kr-ah/ rcarranpment. we analyicd the karyotype with a G-banding technique and examined the DNA hybridiistion with a k r - 5 ' probe on Southern blots. The stemline karyotype was determined as 45. XX. -9. - 17. - 19. -22. 7p-. 9qc (der t[9;22] lq34;q I I]). der ( I 34). and three additional marker chromosomes (Fig 3). Since various numerical chromosomal anomalies were observed in the bone marrow blast cells. at least one of these markers (MI). together with the 9q c chromosome. seems to be characteristic for the patient's leukemic transformation because i t was also present in all metaphases of the bone marrow cells freshly frozen during the blast crisis. In the course of establishment. an increasing rate of tetraploid karyotypes was found in subsequent analyses. Southern blot analysis of &/I1 digested DlUA with bcr-5' as hybridiiation probe showed two &/lI restriction fragments of 8.6 and I 1 .O kilobase pair (kbp) s i x . givingclear evidence for a rearrangement typical for CML (Fig 4. lane A). Additionally. the signal of the 8.6 kbp fragment. i f compared to the 5.0 kbp. is many times stronger. and in this case indicates an amplification proms. The Occurrence of two aberrant fragments was observed in several blotting and hybridiistion experiments: thus. an artifact due to partial digestions i s excluded. DISCUSSION
The cell lines that were established in recent dccades have become an essential part of our contemporary biomedical research. Their contributions to discovering various new
58 1
Southern hybridlxmkn of DNA from OMIS0 4 I h EUh bmeontaicwd 6 ~ c of g d i g m d DNA from QMISO collr (A). K 6 8 2 c d l lino (6).borw morrow C d 8 of mnothor patiom with CML (C). and p.riph.rol blood mononuclrr cdlr of M k h y perm ( 0 ) . Fig 4.
whr0 ba-6."
wll
diagnostic and therapeutic agents can actually be exemplified with the development of MoAbs." purification of cytokines?" bioassays.'.'.m and so forth. In this context. the factor-dependent cell lines. being an ideal subject for studying the physiologic relevance of the factor in question. are undoubtedly of particular interest. In this report we describe a new human myeloid cell line GM/SO. of which survival and proliferation in vitro is primarily dependent on the presence of hGM-CSF. either natural or recombinant. Apart from excluding probable effec~sof rhlL-la. rhlL-2. rhlL-3. rhlL-4, rhlL-6. and rhG-CSF on GM/SO by testing them directly. we provided evidence by using a neutralizing antibody that the factor responsible for the detected proliferation is restricted to GM-CSF. In contrast to the reported synergistic effect of rhG-CSF." IL-6.:' and IL-3:' on leukemic blasts or normal CD34+ cells. respectively. we failed to observe such a synergism on GM/SO cells. rhM-CSFand IL-5 have not yet k e n assayed. In addition. we confirmed through immunophenotyping
582
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and differentiation induction that GM/SO cells possess exclusively myelogenous, but no lymphoid, features. This is indeed the major difference in comparison with the other cell lines reported to be dependent on the presence of external GM-CSF.5*6 Another characteristic to be noted is the additional second signal with bcr-5' probe in Southern blot analysis of D N A from GM/SO cells. This finding is actually consistent with the observation made by Bartram et al.24 They could also detect an additional novel 5' bcr-fragment in blast cells of a patient with CML, and they suggest that one
of the two Ph' had to be secondarily rearranged at the 5' bcr site. In summary, the data presented herein suggest that the GM/SO cell line possesses a functionally intact receptor for hGM-CSF as well as some characteristics of its normal counterpart. Apart from the use for bioassay, it is likely that prospective studies on this cell line may contribute to the understanding of the physiologic consequence of the interaction between GM-CSF and myelogenous cells.
REFERENCES
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