Models of lineage switching in hematopoietic development - Nature

Leukemia (1998) 12, 1430–1439  1998 Stockton Press All rights reserved 0887-6924/98 $12.00 http://www.stockton-press.co.uk/leu

Models of lineage switching in hematopoietic development: a new myeloidcommitted eosinophil cell line (YJ) demonstrates trilineage potential Y Yamaguchi1, H Nishio1, T Kasahara2, SJ Ackerman3, H Koyanagi4 and T Suda1 1

Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, Kumamoto; Department of Biochemistry, Kyoritsu College of Pharmacy, Tokyo, Japan; 3Department of Biochemistry and Molecular Biology, University of Illinois, Chicago, IL, USA; and 4Department of Pediatrics, Jichi Medical School, Tochigi, Japan

2

A new human leukemia cell line with an eosinophilic phenotype, designated YJ, was established from the peripheral blood cells of a patient with chronic myelomonocytic leukemia (CMMoL) with eosinophilia. When cultured in RPMI 1640 medium containing 10% fetal bovine serum, most YJ cells were myeloblastoid with a small number of the cells having eosinophilic granules. Cell surface markers in the YJ cells were positive for CD33 and were negative for CD34, CD16 and CD23. The eosinophilic characteristics of YJ cells were confirmed by histochemical staining with Fast-Green/Neutral-Red and by the expression of mRNAs for eosinophil-associated granule proteins, eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), eosinophil peroxidase (EPO), and major basic protein (MBP), and for the Charcot–Leyden crystal (CLC) protein. The YJ cells could be induced towards monocytic differentiation by stimulation with phorbol 12-myristate 13-acetate (PMA). The monocytic characteristics of YJ cells treated with PMA were confirmed by morphological analysis with ␣-naphthyl butyrate esterase staining, by CD14 expression, and by increased expression of Egr-1 mRNA. Furthermore, YJ cells could be differentiated towards the neutrophil lineage by stimulation with all-trans retinoic acid (RA). YJ cells treated in vitro with 2 ␮M RA differentiated into metamyelocytes and band neutrophils, and increased the number of nitroblue tetrazolium (NBT)-positive cells and increased gp91phox mRNA expression. Thus, the YJ cell line exhibited eosinophilic characteristics, but was able to differentiate to the monocytic or neutrophilic lineages in response to PMA or RA, respectively. The expression of genes for transcription factors involved in myeloid differentiation was evaluated by Northern blot analysis. Increased expression of Egr-1 was observed with macrophage differentiation. In contrast, increased expressions of C/EBP␤ and MZF1 mRNA occurred with neutrophilic differentiation. The YJ cell line should be useful for elucidating the molecular mechanisms governing lineage switching from the eosinophil to monocytic or neutrophil lineages. Keywords: eosinophils; neutrophils; macrophages; cell line; transcription factors

Introduction A large number of continuous cell lines from patients with myeloblastic leukemia have been established and many cases of eosinophilic leukemia have been reported.1–3 To date, however, only one eosinophil leukemia cell line, EoL, from a patient with eosinophilic leukemia has been established.4 Other eosinophilic cell lines have been subcloned from the HL-60 promyelocytic leukemia line, including HL-60-C155,6 and HL-60-3+C5.7 Recently, an acute myelogenous leukemia (AML) cell line capable of terminally differentiating to eosinophils in response to IL-5, IL-3 and GM-CSF has been reported.8 Correspondence: Y Yamaguchi, Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, Honjo 2-2-1, Kumamoto, 860-0811 Japan; Fax: 81 96 373 5332 Received 19 September 1997; accepted 30 April 1998

Eosinophil differentiation from hematopoietic stem cells is regulated by a number of different cytokines, including IL-3, GM-CSF and IL-5.9–11 However, the molecular basis for the commitment of progenitors to the eosinophil lineage remains unknown. One of the reasons why it has been difficult to analyze transcription factors that may be involved in the differentiation of myeloid progenitors to the eosinophil lineage has been the absence of eosinophilic cell lines suitable for elucidating the molecular basis of eosinophilic differentiation. We describe the establishment of a new eosinophilic leukemia cell line, YJ, capable of differentiating to the monocytic lineage with PMA stimulation or to the neutrophil lineage with all-trans retinoic acid (RA). YJ cells may provide a potentially useful model for analyzing the molecular basis for lineage switching among the eosinophil, macrophage and neutrophil hematopoietic programs. In addition, this cell line may be suitable to isolate the transcription factor(s) involved in eosinophil differentiation using the subtraction or the differential display methods. Materials and methods

Case report HO, a 69-year-old Japanese male, was admitted to the Hematology Division of Jichi Medical School on 15 June 1994, for the treatment of intense breathing difficulty and high fever. The history of his illness began in 1988 with the onset of anemia. At that time, a diagnosis of myelodysplastic syndrome (MDS) with refractory anemia, was made according to the classification of the French–American–British (FAB) nomenclature. The patient had been well except for eosinophilia and easy fatiguability due to anemia until June 1994, when dyspnea and high fever developed. The eosinophilia and splenomegaly had developed gradually. At the time of admission, hepatosplenomegaly was observed and no lymphadenopathy was found. Hematological findings showed a hemoglobin of 6.3 g/dl, a leukocyte count of 35.2 × 109/l, and a platelet count of 47.3 × 109/l. The leukocyte differential count revealed 5.4% blasts, 1.2% promyelocytes, 12% myelocytes, 3.4% metamyelocytes, 52% mature myelocytes, 12% eosinophils, 4.2% monocytes and 7% lymphocytes. The bone marrow was hypocellular and myelofibrotic, with cytogenetic studies of bone marrow showing 47XY, 1qh+, +8, del(12)(q15 q23) (100%). After admission, a diagnosis of myeloproliferative disorder with eosinophilia was made and the patient was treated with cytosine arabinoside and prednisolone, and there was a striking clinical improvement. Three months after chemotherapy, leukocytosis with increased blast cells and marked splenomegaly developed. Hematological findings in peripheral blood were: 166 × 109/l leukocytes with 7% blast cells, 8.6% eosinophils, 11% neutrophils, 2% myelocytes and metamyelocytes, 60% monocytoid cells, 6.2 g/dl hemoglobin

Models of lineage switching in hematopoietic development Y Yamaguchi et al

and 60 × 109/l platelets. Bone marrow aspirate was normocellular with 90% myeloid cells including 6.2% blast cells, 20% neutrophils, 10.4% eosinophils, and 50% monocytoid cells. The serum and urinary lysozyme levels were increased. A final diagnosis of blastic crisis of chronic myelomonocytic leukemia (CMMoL) with eosinophilia was made. Despite additional chemotherapy, the patient died of internal bleeding.

Cell culture and cloning Heparinized peripheral blood was obtained following informed consent from the patient. The polymorphonuclear (PMN) leukocyte fraction, composed mostly of cells of eosinophilic lineage including immature cells, was prepared by a discontinuous Percoll centrifugation method. The PMN cells were cultured in tissue culture flasks at 5 × 105 cells/ml in RPMI 1640 supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), 2 mM L-glutamine, 50 IU/ml of penicillin and 50 ␮g/ml streptomycin, and the supernatant (10% v/v) of X63Ag8.653 plasmacytoma cells transfected with BMGNeo murine IL-5 cDNA as a source of IL-5, in a humidified atmosphere of 5% CO2.12 The cultures were fed once a week, but the cells cultured in the absence of IL-5 did not proliferate. Cultured cells were subcloned by the in vitro colony formation method as described previously.10

Reagents and cytokines The cytokines used were human rIL-1␣, human rTNF-␣ (kindly donated by Dainippon Pharmaceutical, Suita, Osaka, Japan), rhIL-2 (specific activity 1 × 107 U/mg, provided by Shionogi Pharmaceutical, Osaka, Japan), rhIL-3 (specific activity, 21 900 U/ml, provided by Genentics Institute, Cambridge, MA, USA), rhIL-4 (specific activity, 1.3 × 107 U/ml, provided by DNAX Research Institute, Palo Alto, CA, USA), human rIL-5 (donated by Suntory, Tokyo, Japan), rhIL-6 (specific activity, 3.9 × 109 U/mg, provided by Ajinomoto, Kawasaki, Japan), rhIL-8 (a half-maximal neutrophil chemotactic activity, 0.5–1.0 ng/ml, provided by Dr Matsushima, Cancer Research Institute, Kanazawa, Japan), rhGM-CSF (specific activity, 1 × 109 U/ng, provided by Sumitomo Pharmaceutical, Osaka, Japan), rhRANTES (specific activity assessed as effective chemotaxis on human monocytes detected at 10 to 100 ng/ml, purchased from Biosource, CA, USA), human rIFN-␥ (Takeda Pharmaceutical, Osaka, Japan), and human recombinant adult T cell leukemia-derived factor/thioredoxin (rADF/TRX) (Ajinomoto, Tokyo, Japan). C5a, formylmethionyl-leucyl-phenylalanine (fMLP), lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), and all-trans retinoic acid (RA) were purchased from Sigma Chemical (St Louis, MO, USA). All these stimulants were used at optimal concentrations.

Measurement of IL-8 Cytochemical staining The cells were stained with Wright–Giemsa, Fast-Green/ Neutral-Red, and naphthol AS-D chloracetate/␣-naphthyl butyrate esterase.

Northern blot analysis Total cellular RNA was isolated from HL-60-C15 cells, YJ cells, and PMA- or RA-induced YJ cells using the single step RNA isolation method,13 followed by poly(A)+ mRNA selection using oligotex-dT30 (Takara Shuzo, Kyoto, Japan). The poly(A)+ mRNA (4 ␮g per lane) was denatured in formamideformaldehyde and then subjected to electrophoresis in 1% agarose-formaldehyde gels. The RNA was then transferred to Biodyne nylon membranes (Pall BioSupport, East Hills, NY, USA) and the blots probed sequentially with radiolabeled cDNAs encoding human eosinophil peroxidase (EPO),14 human major basic protein (MBP),15 human eosinophil cationic protein (ECP),16 human eosinophil-derived neurotoxin (EDN),17 human Charcot–Leyden crystal (CLC) protein,18 neutrophil elastase,19 gp91phox,20 myeloperoxidase (MPO),21 CD14,22 GATA-123 and GATA-2,24 C/EBP␣,25 C/EBP␤,26 Egr1,27 MZF-1,28 PU.129 and 18S ribosomal RNA30 to confirm equivalent RNA loading. All hybridizations were performed with random-primed cDNA probes at 42°C for 18 h in 50% formamide, 6X SSC, 0.2% Ficoll-polyvinylpyrrolidone (PVP), and 0.1% sodium dodecyl sulfate (SDS). Filters were washed twice in 2X SSC containing 0.2% SDS at 53°C for 30 min and twice in 0.2% SSC containing 0.2% SDS at 55°C for 30 min. Autoradigraphy was performed at −80°C with Kodak XAR-5 film.

IL-8 was measured by a sensitive ELISA method established as described previously.31 The minimum detection level of IL8 by this ELISA was approximately 20 pg/ml.

Fluorescence-activated cell sorting (FACS) analysis YJ cells were collected and resuspended in PBS with bovine serum albumin (4%), adjusted to a final concentration of 5 × 105 cells/100 ␮l, and incubated for 30 min at 4°C with monoclonal antibodies against CD16 (PharMingen, San Diego, CA, USA), CD23 (Nichirei, Tokyo, Japan), CD33 (Dako, Glostrup, Denmark), CD34 (Nichirei), and CD14 (Dako) conjugated with fluorescein isothiocyanate or phycoerythrin.

Assay of MBP concentration The concentration of MBP in the supernatants of YJ cell suspensions was measured by ELISA as described previously.32 The YJ cells were washed twice with HBSS/2ME and resuspended in the same medium at 5 × 104 cell/ml. Aliquots of cell suspensions of 0.2 ml were added to the Eppendorf tubes. Cell suspensions were incubated for 2 h at 37°C in the presence of various reagents. Two hours after incubation, the concentration of MBP in the supernatants of each sample was assayed.

Nitroblue Tetrazolium (NBT) Reduction Test The NBT assay was performed on RA-stimulated YJ cells as described by Baehner and Nathan.33

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Statistical analysis Results were expressed as means ± s.d. Statistical significance was evaluated using Student’s t-test.

teristics of YJ cells were readily apparent by FastGreen/Neutral-Red staining (Figure 1d).

Chromosome analysis Results

Establishment of YJ cell line For the first month of culture, cells did not increase in number. However, by 2 months of culture the proliferation rate of the cultured cells had increased considerably. After culturing the cell line for 5 months and confirming its proliferation in the absence of IL-5, the line was designated as YJ cells. Once the line was established, IL-5, GM-CSF, and IL-3 had no effect on the proliferation or differentiation of YJ cells (data not shown). This YJ cell line has a doubling time of 24 h.

Morphologic and cytochemical characteristics of YJ cells Most YJ cells were myeloblastic with a small number (about 10–15%) of the cells having eosinophilic granules by Wright– Giemsa staining (Figure 1a). As well, the eosinophilic charac-

The chromosomal analysis of YJ cells was very complex, exhibiting a 45, X, −Y, −2, add(3)(q27), del(3)(p13), add(4)(q34), −5, add(6)(q25), −9, del(10)(p12), add(11)(p15), add(13)(q32), −14, −14, −15, −16, add(16)(q23), −17, del(18)(q22), +m1-m8. This was significantly different from that of the patient’s bone marrow cells at the time of admission.

Expression of mRNAs for eosinophil granule proteins in the YJ cells Morphologic and cytochemical studies suggested that the YJ cells possessed eosinophilic characteristics. To determine whether YJ cells expressed cellular markers of the eosinophil lineage, we performed Northern blot analysis to investigate the expression of mRNAs for the eosinophil-associated granule proteins (ECP, EDN, EPO and MBP) and cytosolic CLC protein. As shown in lane 2 of Figure 2, the level of mRNA

a

b

c

d

Figure 1 Morphological appearance of YJ cells treated with PMA or RA. Wright–Giemsa-stained cytospin preparations of YJ cells treated not at all (a), or with 10 nM PMA for 2 days (b), 2 ␮M RA for 4 days (c), or Fast Green/Neutral Red stains of uninduced YJ cells (d) (original magnification ×1000).


secretion from YJ cells stimulated with cytokines (IL-1␣, IL-5, TNF␣, IFN␤), chemokines (RANTES), or other activating agents (ADF/TRX, C5a, fMLP, LPS, PMA, A23187) are summarized in Table 1. The levels of secreted IL-8 were assayed 24 h after culture in the presence of various stimulants. Whereas the amount of IL-8 secreted from YJ cells cultured without stimulants was 0.32 ng/ml, in cells cultured with PMA or TNF␣ for 24 h, this value increased 100-fold and six-fold, respectively. No significant increase in the IL-8 level was observed for YJ cells cultured with any of the other stimulants.

YJ cells differentiate to the monocytic lineage in response to PMA

Figure 2 Northern blot analysis for eosinophil-associated proteins (CLC, ECP, EDN, EPO, and MBP), neutrophil elastase, gp91phox, MPO, CD14, and 18S rRNA mRNA expression in HL-60-C15 (lane 1), uninduced YJ cells (lane 2), YJ cells induced with 10 nM PMA for 2 days (lane 3), and YJ cells induced with 2 ␮M RA for 4 days (lane 4).

expression in the YJ cells for the eosinophil granule cationic proteins, ECP, EDN, EPO and MBP, as well as CLC protein, were similar to those expressed in an eosinophilic subline of HL-60, HL-60-C15 cells. These findings demonstrate the eosinophil characteristics of the YJ cell line.

Flow cytometric analysis of surface antigens expressed on YJ cells To characterize surface antigen expression on YJ cells, FACS analysis was performed. As shown in Figure 3, YJ cells were mainly CD33-positive and contained a small number of CD14-positive cells. No expression of CD16 (Fc␥RIII), CD23 (FC⑀RII), or CD34 (stem cell antigen) antigens was observed in YJ cells. It has been reported that B cells and myeloid cells can be generated from a single progenitor cell.34 Accordingly, we analyzed YJ cells for the expression of CD19 and CD20. However, YJ cells were negative for both of these B cell markers (data not shown). These results indicate that YJ cells do not express characteristics of more mature myeloid cells or B lymphocytes.

IL-8 production from YJ cells in response to various stimulants To determine whether YJ cells respond to various cytokines or other activating stimuli, we measured the amount of IL-8, which is mainly produced by myeloid cells including eosinophils,35,36 using a sensitive ELISA method.31 Results for IL-8

Because it was observed that YJ cells produced a considerable amount of IL-8 following stimulation with PMA, we analyzed whether there was a morphological change in YJ cells after treatment with PMA. The YJ cells became more adherent and aggregative after treatment with PMA. Wright–Giemsa cytospin preparations demonstrated that PMA induced YJ cell differentiation towards the macrophagic lineage. PMA-induced YJ cells showed a significant decrease in the nuclear-cytoplasmic ratio, and an increase in cytoplasmic vesicles (Figure 1b). The ␣-naphthyl butyrate esterase reaction was positive in the PMA-induced macrophage-differentiated YJ cells (data not shown). To investigate further the monocytic differentiation of YJ cells induced by PMA, expression of Egr-1 mRNA by Northern blot analysis and expression of CD14 surface antigen was analyzed by FACS. In the same cells, expression of CD14 was significantly increased (Figure 4), with increasing duration of exposure to PMA (data not shown).

YJ cells differentiate to the neutrophilic lineage in response to RA It is well known that the human promyelocytic leukemia cell line, HL-60, differentiates into neutrophilic cells following treatment with RA. We tested whether YJ cells have the capacity to differentiate towards the neutrophilic lineage in response to RA, even though this cell line was not derived from a patient with acute promyelocytic leukemia. Surprisingly, RA-induced YJ cells differentiated morphologically into mature neutrophils (Figure 1c). YJ cells were cultured with various concentrations of RA for 4 days and their ability to reduce NBT was measured. As shown in Figure 5, 2 ␮M RA induced about 85% of YJ cells to become NBT-positive. Also, as shown in Figure 2, the RA-induced YJ cells showed expression of mRNA for gp91phox and CD14 and decreased expression for MPO. Importantly, mRNA expression for the eosinophil-associated CLC and MBP genes was not observed, in YJ cells induced with RA towards the neutrophil lineage, suggesting a loss of eosinophilic characteristics.

Induction of genes for transcription factors in the differentiation of eosinophil-committed cells to macrophages or neutrophils Northern blot analysis showed that the expression of mRNA for the eosinophil-associated proteins CLC, ECP, EDN, EPO, and MBP was either decreased (ECP, EDN, EPO) or no longer evident (CLC, MBP) in YJ cells cultured with PMA for 2 days or with RA for 4 days. These findings support a loss of eosino-

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Figure 3 Flow cytometric analysis of surface antigens expressed on YJ cells. The expression of CD16, CD23, CD33, CD34 and CD14 on YJ cells is shown. Uninduced YJ cells were clearly positive for CD33 and marginally positive for CD14.

Table 1

IL-8 production in YJ cells treated with various reagents

Stimulants

None IL-1␣ IL-5 TNF-␣ IFN-␥ RANTES ADF C5a fMLP LPS PMA A23187

IL-8 ng/ml

(20 ng/ml) (1 ␮g/ml) 20 ng/ml) (103 U/ml) (100 ng/ml) (200 ng/ml) (5 × 10−7 M) (5 × 10−7 M) (20 ␮g/ml) (20 nM) (100 ng/ml)

0.32 ± 0.09 0.49 ± 0.02 0.27 ± 0.04 1.84 ± 0.20 0.48 ± 0.03 0.37 ± 0.05 0.39 ± 0.02 0.48 ± 0.03 0.44 ± 0.10 0.43 ± 0.08 33.7 ± 3.6 1.2 ± 0.3

Cells (5 × 105 cells/ml in 1% FCS-RPMI 1640) were incubated with the indicated stimulants for 24 h. IL-8 content in the culture supernatant was determined by ELISA.

philic characteristics by YJ cells in the process of lineage switching to the macrophage or neutrophil programs. Hematopoietic differentiation is regulated by the expression of transcription factors that activate lineage-specific genes. It was therefore of interest to analyze the expression of transcription factors by YJ cells in their differentiation from the eosinophil to the macrophage or neutrophil lineages. Among transcription factors known to be involved in myeloid differentiation, the expression of GATA-1, GATA-2, C/EBP␣, C/EBP␤, Egr-1, MZF-1 and PU.1 transcripts were analyzed (Figure 6). The expression of GATA-1, which is involved in the differentiation of erythroid/megakaryocyte, eosinophil, and mast cell lineages, did not change with macrophage or neutrophil differentiation induced by PMA or RA, respectively. Expression of GATA-2 also remained unchanged with macrophage or neutrophil differentiation. No GATA-3 transcript was observed in all the cells, HL-60-C15, YJ, YJ induced with PMA, and YJ induced with RA (data not shown). In contrast, expression of C/EBP␣ decreased slightly, with both macrophage and neutrophil differentiation, whereas expression of C/EBP␤ significantly increased. These results suggest that


Discussion

Figure 4 Expression of CD14 in YJ cells induced with 10 nM PMA for 2 days. Cells were incubated with biotinylated CD14 and FITCconjugated streptavidin. (A) unstained YJ cells, (B) YJ cells cultured without PMA, (C) YJ cells cultured with 10 nM PMA for 2 days.

C/EBP␣ plays an important role in immature myeloid cells and that C/EBP␤ may regulate the cellular functions of more mature myeloid cells, macrophages and neutrophils. The expression of Egr-1, which apparently plays a role in the development of hematopoietic cells along the macrophage lineage, was induced in YJ cells with PMA stimulation and macrophage differentiation. However, the Egr-1 transcript was also increased with RA-induced neutrophil differentiation. The level of MZF-1 mRNA, which is an apparently necessary factor for granulocytic differentiation,37 was also increased somewhat in YJ cells differentiated with RA towards the neutrophil lineage. PU.1, the product of the Spi-1 oncogene and a member of the ets family, is essential for myeloid cell development.38 Expression of PU.1 mRNA was increased slightly after RA treatment, but remained unchanged in response to PMA. These results suggest that the Egr-1 and C/EBP␤ transcription factors accumulate during differentiation of YJ cells to both macrophage and neutrophil lineages.

Activity of various stimuli on the release of eosinophil granule MBP from YJ cells To determine whether YJ cells respond to various cytokines, chemokines, or other activating stimuli in a manner equivalent to mature human eosinophils, we examined the effect of ADF/TRX, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, GM-CSF, and RANTES on MBP secretion by YJ cells. As shown in Figure 7, rADF, IL-3, IL-5, GM-CSF, RANTES, and IL-4 significantly increased MBP release from YJ cells compared to a control in which YJ cells were cultured in the absence of cytokines. Among the cytokines examined, IL-4 (40 U/ml) augmented the release of MBP most potently. These results suggest that the YJ cell line may be useful for analyzing the mechanisms of eosinophil activation, signal transduction, or degranulation.

In this paper, we report the establishment of a new human eosinophilic cell line (YJ) derived from the peripheral blood of a patient with chronic myelomonocytic leukemia (CMMoL) with eosinophilia. For the first 3 months of culture, the polymorphonuclear (PMN) fraction of cells from the peripheral blood required IL-5 for maintenance of proliferation, after which they became factor-independent. This suggests that only the cells capable of proliferating in response to IL-5 developed the ability to grow autonomously. The proliferation of the YJ cell line may therefore have been due to an autocrine mechanism involving IL-5. Human eosinophilic cell lines established to date include the following: HL-60-C15 cells,5,6 HL-60-3+C5 cells,7 EoL cells,4 and AML 14 cells.8 The HL-60-C15 and HL-60-3+C5 eosinophil sublines were derived from the acute promyelocytic leukemia cell line HL-60, and the EoL cell line was derived from an eosinophilic leukemia. The AML14 cell line was derived from an acute myelocytic leukemia, M2.8 The YJ line reported here was derived from CMMoL, and the YJ cell line is the only eosinophilic cell line to be derived from a myelodysplastic syndrome (MDS) patient among the eosinophilic cell lines established to date. Table 2 shows the chromosomal abnormalities reported in eosinophilic cell lines established to date. One chromosomal abnormality in all cell lines involves chromosome 9. In human neoplasia, 5.6% of the cytogenetic aberrations described involve chromosome 9,39 most of which are the Ph1 chromosome. Sreekantaiah et al40 have reported five patients with acute nonlymphocytic leukemia (ANLL) with chromosomal aberrations involving bands 9q21-q22. The diagnoses were ANLL FAB type M4 (myelomonocytic leukemia), M4 with eosinophilia (M4eo), M4, M5 (monocytic leukemia), and M2 (myelocytic leukemia), respectively. The report by Sreekantaiah et al40 suggests that abnormalities in chromosome 9 may be associated with the differentiation of myelocytic or monocytic lineages. Recently, it has been shown that homozygous deletion of the cyclin-dependent-4 inhibitor gene (CD4I; p16), which is mapped to chromosome 9p21, is one of the tumor-suppressor genes, that may result in immortalization of human leukemia cells.41 Cytogenetic study of our patient’s BM cells did not show deletion of chromosome 9, but YJ cells did. This may indicate that only those cells with deletion of p16 were capable of becoming immortalized or that the YJ cell deletion of p16 developed in long-term culture of the leukemic cells. We have previously reported the presence of precursor cells in bone marrow which are able to respond to both G-CSF and IL-5 and form trilineage (neutrophils, macrophages, and eosinophils) colonies.42 In addition, it was reported that large eosinophil colonies derived from single cells contain a very small number of neutrophils or macrophages.43 For the present studies, we speculate that YJ cells are induced towards the monocyte/macrophage or neutrophil lineages from blast cells capable of differentiating the eosinophil lineage in response to PMA or RA stimulation (Figure 8). From the evidence described above, the developmental stage of YJ cells might be slightly advanced still toward the eosinophil lineage at a stage which can nevertheless differentiate to all three myeloid lineages (eosinophils, macrophages and neutrophils) depending on the inducing stimulus (Figure 8). Paul et al44 reported that a cloned subline of the AML14 cell line (AML14.3D10), which was capable of spontaneously differentiating to eosinophilic myelocytes in the absence of cytokine stimulation, could change in the differentiation program to the

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Figure 5 NBT reduction test in YJ cells treated with various concentrations of RA. YJ cells were cultured without or with 20, 200 and 2000 nM RA for 4 days. (a) Only neutrophilic cells with a large black deposit were classified as NBT positive. (b) Quantitation of the NBT reduction test of cells shown in (a).

Figure 7 Effect of various agonists on MBP release from YJ cells. YJ cells (5 × 104 cells/ml) were incubated with medium alone, ADF (10 ␮g/ml), IL-1␣ (20 U/ml), IL-2 (20 U/ml), IL-3 (50 ng/ml), IL-4 (40 U/ml), IL-5 (50 ng/ml), IL-6 (50 ng/ml), IL-8 (50 ng/ml), GM-CSF (10 ng/ml), or RANTES (10 nM) for 2 h at 37°C. After incubation, the amount of MBP in the culture supernatants was measured by ELISA. The mean ± s.d. for triplicate determinations is shown. *P ⬍ 0.05

Figure 6 Northern blot analysis for transcription factors associated with myeloid differentiation. HL-60-C15 cells (lane 1), untreated YJ cells (lane 2), YJ cells differentiated towards macrophage (lane 3) or neutrophil (lane 4) lineages. Lane 1, Eosinophil-committed cell line, HL-60-C15 cells; lane 2, uninduced YJ cells; lane 3, YJ cells treated with 20 nM PMA for 2 days; lane 4, YJ cells treated with 2 ␮M RA for 4 days. Each lane contained 4 ␮g of poly(A)+ mRNA. The blot was probed sequentially with each cDNA probe and an 18S rRNA probe to control for equivalent loading.

neutrophil lineage after exposure to RA. They suggest that the lineage switch occurs at least in part by a change in the differentiation program of cells that already show advanced evidence of eosinophilic differentiation. Accordingly, it can not be excluded that the differentiation of YJ cells to monocyte/macrophage or neutrophil lineages do not develop from the blast-like progenitors in YJ cells, but from the promyelocytic stage of eosinophils in YJ cells. It has been reported that GATA-1 mRNA is expressed in not only erythroid and megakaryocyte but also eosinophil and mast cell lineages. We have previously demonstrated


Table 2

Cell line HL-60-C15 EoL-1 EoL-2 EoL-3 AML 14 AML14.3D 10 YJ

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Chromosomal abnormalities in eosinophilic cell lines

Cytogenetic analysis −5, −9, −10, −16, −16,−17, +?18, der(5)t(5:17)(q13;q12), +der(9)t(9;?)(q34.3;?), +der(10)t(10;13)(p11.2; q12.1), +der(16)t(16;?)(q22;?), +der(16)t(16;?)(123;?), del(9)(q13) 48XY, +6, +8, 9q49XY, +8, 9q-, +12, +13 49XY, +4, +8, 9q-, +13 −5, +8, −9, +13, −14, −18, −21, +der(3), del(5q) 65-84XY, +X, del(5)(q23;q34), +6, +6, −7, add(8)(Q24.3), del(9)(q22), add(13)(p13), add(14)(p13), add(14)(p13), +16, add17(p13), +19, −20, −21, add(22)(q13), +12–14 mar X, Y, −2, −5, −9, −14, −14, −15, −16, −17, add(3)(q27), del(3)(p13), add(4)(q34), add(6)(q25), del 10(p12), add(11)(p15), add13(q32), add16(q23), del(18)(q22)

Figure 8 The YJ cell line is composed of blastoid progenitor cells which undergo a constitutive eosinophilic differentiation. These blastoid progenitors can differentiate towards the macrophage or neutrophil lineages in response to PMA or RA, respectively.

increased expression of mRNA for the GATA-binding proteins GATA-1, GATA-2, and GATA-3 during eosinophil differentiation of myeloid leukemic cell lines and constitutive expression in mature eosinophils and basophils.45 Furthermore, we have shown a major positive regulatory role for GATA-1 and negative regulatory role for GATA-2 in the transcription of eosinophil major basic protein (MBP) gene.46 In addition, as shown in Figure 6, expression of GATA-1 mRNA was very little changed in uninduced YJ cells, YJ cells induced with PMA, and YJ cells induced with RA. These results indicate that GATA-1 may play an important role not only in the differentiation of erythroid/megakaryocyte but also in the functions of myeloid cells including eosinophils, macrophages and neutrophils. We have provided evidence that the YJ cell line is able to produce high levels of IL-8 in response to PMA and TNF-␣, but not other agents, including IL-1␣, IL-5 and C5a. Recently, it has been reported that human mature eosinophils are able to produce IL-8 in response to C5a or calcium ionophore.35,47

In addition, it has been shown that eosinophil MBP itself stimulates the production of IL-8 by human eosinophils.48 Our results indicate, however, that the YJ eosinophil cell line is a poor producer of IL-8, but did turn out to be a good producer when differentiated to the monocyte/macrophage lineage by PMA treatment. To date, the transcription factors shown to be involved in monocyte/macrophage differentiation include Egr-1 and AP1.49,50 In YJ cells, macrophage differentiation induced by PMA was accompanied by an up-regulation of Egr-1 mRNA level (Figure 6) and protein at an early phase (data not shown). Mollinedo et al50 have reported that expression of AP-1 is not required for the induction of HL-60 cells towards granulocytes, whereas induction of monocytic differentiation was correlated with an increase in AP-1 activity. Although there is some evidence that AP-1 and Egr-1 play a role in monocyte differentiation, the mechanism by which transcription factors such as AP-1 and Egr-1 act during monocytic differentiation remains unknown. In contrast, the paper has also been reported that Egr-1 is not required for macrophage differentiation or activation.51 Accordingly, the role of Egr-1 on the differentiation along the macrophage lineage is controversial. In addition, the effect of Egr-1 on the differentiation or activation of myeloid lineage remains to be elucidated. In these regards, the YJ cell line may be useful for elucidating the molecular mechanisms that govern lineage switching of myeloid progenitors amongst the eosinophil, monocyte/macrophage and neutrophil hematopoietic programs.

Acknowledgements

This work was supported by Grants-in-Aid from the Ministry of Education, Science, and Culture of Japan (YY). SJA is supported by Grants AI 33043 and AI 25230 from the National Institute of Health, USA. We thank Mr Masayuki Sonoyama for performing the chromosomal analysis of this cell line, Dr Motohiro Hashiyama for the flow cytometric analysis. We also thank Dr Ronald G Crystal, Dr Michio Nakamura, Dr Shunsuke Yamamoto, Dr Masayuki Yamamoto, Dr Kleanthis G Xanthopoulos, Dr Shizuo Akira, Dr Takashi Yokota, Dr Robert Hromas, and Dr Franc¸oise Moreau-Gachelin for providing the neutrophil elastase, gp91phox, CD14, GATA-1 and -2, C/EBP␣, C/EBP␤, Egr-1, MZF-1, and PU.1 cDNAs, respectively.


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