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The Aml14 and Aml14.3D10 Cell Lines: A Long-Overdue Model for the Study of Eosinophils and More

Research Service, VA Medical Center and Division of Hematology/Oncology, Wright State University, Dayton, Ohio, USA

Key Words. Cell line • Eosinophil • Leukemia • Differentiation • Neutrophil • Macrophage

Dr. Michael A. Baumann, Hematology/Oncology (111W), VA Medical Center, 4100 W. Third Street, Dayton, OH 45428., USA.

	Abstract

Top Abstract Introduction Establishment and... Establishment of the AML14.3D10... Eosinophil Granule-Related... Transcriptional Regulation of... Characterization of the Eotaxin... Lineage Switching of... Autocrine GM-CSF Use Summary References

 
Over the past three decades, a number of myeloid cell lines have been established and have proven useful for the study of various aspects of normal and disordered hematopoiesis. However, one myeloid lineage for which a useful cell line model has been sorely lacking is the eosinophil. We review the characteristics of the recently developed AML14 and AML14.3D10 cell lines and summarize how they have been used to obtain important new information relevant to eosinophil biology. Observations regarding the apparent ability of the AML14.3D10 cell line to "switch" lineages and to produce and use GM-CSF in an autocrine fashion are also reviewed.

	Introduction

Top Abstract Introduction Establishment and... Establishment of the AML14.3D10... Eosinophil Granule-Related... Transcriptional Regulation of... Characterization of the Eotaxin... Lineage Switching of... Autocrine GM-CSF Use Summary References

 
The establishment of a continuous hematologic cell line is a rare event. In spite of this, a number of such lines have been established and have contributed much to our understanding of normal and malignant hematology. These cell lines have been established from leukemic populations and most commonly exhibit features analogous to those found in normal immature cells of the myeloid lineage. Some of these cell lines can be induced to a phenotypically more mature state through exposure to chemical or biologic agents. Such differentiation is usually along the neutrophilic or monocytic pathway [1-3], although a few cell lines have been described that will display some properties of erythroid or megakaryocytic differentiation [4-6].

Because eosinophils are scarce in normal blood, investigators desiring to study them have had to process large amounts of blood to obtain small numbers of short-lived eosinophils [7]. Until recently, the only widely available alternative to this has been the use of certain sublines of the HL60 cell line which can be induced to transiently exhibit some features of eosinophilic differentiation [8-10]. The AML14 cell line and its AML14.3D10 subclone were recently established and have rapidly become a standard cell line model for the study of eosinophils. This report will summarize their characteristics and how they have been used by several groups to make important new observations relevant to our understanding of the eosinophil and of normal and disordered hematopoiesis in general.

	Establishment and Characterization of the AML14 Cell Line

Top Abstract Introduction Establishment and... Establishment of the AML14.3D10... Eosinophil Granule-Related... Transcriptional Regulation of... Characterization of the Eotaxin... Lineage Switching of... Autocrine GM-CSF Use Summary References

 
The AML14 human myeloid leukemic cell line was established in 1992 from a 68-year-old man who presented with FAB M2 acute myeloid leukemia. The patient's circulating and bone marrow blasts displayed no evidence of eosinophilic differentiation, nor was there bone marrow eosinophilia. Cytogenetic studies were not performed on clinical specimens. The patient failed induction therapy and died soon after.

Freshly isolated peripheral blood blast cells were cultured in complete RPMI medium without cytokine support and proliferated vigorously from the outset, never experiencing a "crisis" period. Morphologically, the cells appeared quite undifferentiated ( Figs. 1A and 1B). Flow cytometric immunophenotypic studies demonstrated that the cells did not express CD34, but were positive for CD33, CD13, and weakly for CD14. A small subpopulation expressed CD11b. After about six months in culture, a cytogenetic study was obtained which revealed complex abnormalities including the presence of 1 to 21 double minute chromosomes per cell. When the blast-like AML14 cells were cultured for a prolonged period in a cocktail of cytokines, including interleukin 3 (IL-3), GM-CSF, and IL-5, a population of cells emerged after several weeks that exhibited phenotypic characteristics of advanced eosinophilic differentiation. By light microscopy, the cells appeared to achieve a level of differentiation equivalent to an eosinophilic myelocyte. Electron microscopic studies revealed the presence of eosinophil secondary granules that did not appear totally mature as they contained no crystalline cores ( Fig. 2). This phenomenon was reproducible, and mRNA studies demonstrated that the cells expressed the genes for the unique subunits of the receptors for IL-3, GM-CSF, and IL-5 as well as the ß receptor subunit common to all three [11]. Specific binding of fluorochrome-labeled GM-CSF and IL-5 was readily demonstrated, but we could not detect IL-3 binding by this method. Since the cells clearly could proliferate and differentiate in response to IL-3, we interpreted the lack of demonstrable binding as an indication that the cells expressed a very small number of IL-3 receptors [12]. We then went on to demonstrate that eosinophil-induced AML14 cells expressed mRNA and protein for all of the major eosinophil granule proteins (major basic protein [MBP], eosinophil derived neurotoxin [EDN], eosinophil cationic protein [ECP], eosinophil peroxidase [EPO]) and the cytosolic Charcot-Leyden crystal protein (CLC) ( Fig. 3) [13].

View larger version (476K): [in this window] [in a new window]   Figure 1. A) AML14 cells, Wright stain. B) AML14 cells, fast green stain. The cellular morphology of AML14 is blast-like, without evidence of cytoplasmic granules. C) AML14.3D10 cells, Wright stain. D) AML14.3D10 cells, fast green stain. Eosinophil granules are abundant in the cytoplasm of AML14.310 cells. Original magnifications x 1,000. Reprinted with modifications from [12, 14].

View larger version (127K): [in this window] [in a new window]   Figure 2. Electron micrograph of AML14 cells induced to eosinophilic differentiation with IL-3, GM-CSF, and IL-5. The level of maturity appears equivalent to an eosinophilic myelocyte. Completely dense, large, immature secondary granules fill the outer cytoplasm. Many of these have electron-lucent outer matrix compartments surrounding the electron-dense central core compartment. Magnification x 10,000. Reprinted with permission from [13].

View larger version (60K): [in this window] [in a new window]   Figure 3. A) Northern blots of mRNA isolated from undifferentiated and differentiated AML14 cells, and from RAMOS cells (negative control). Left lane, RAMOS cells; middle lane, undifferentiated AML14 cells; right lane, differentiated AML14 cells. Blots were rehybridized with a ß-actin probe to ensure that comparable amounts of RNA were present in each lane. B) Western blots of eosinophil granule proteins. Lane 1; RAMOS cells; lane 2, undifferentiated AML14 cells; lane 3, differentiated AML14 cells. Reprinted with permission from [13].

	Establishment of the AML14.3D10 Subclone

Top Abstract Introduction Establishment and... Establishment of the AML14.3D10... Eosinophil Granule-Related... Transcriptional Regulation of... Characterization of the Eotaxin... Lineage Switching of... Autocrine GM-CSF Use Summary References

We later found that it became more difficult to cause cytokine-induced eosinophilic differentiation of the parental AML14 cell line. We now believe that "cytokine inducibility" of AML14 was the result of the presence of a subpopulation of cells that we later "cloned out" as AML14.3D10. We repeated cytogenetic studies on the parental AML14 cells and on the AML14.3D10 subclone and found some important differences. Most notably, the double minute chromosomes were now found only in the AML14.3D10 subclone ( Fig. 4). It is possible that these structures are responsible for expressing genes that enable the maintenance of eosinophilic differentiation, although we have no direct evidence to support this conjecture. In fact, it is not clear what molecular mechanisms are responsible for the unusual ability of AML14.3D10 to maintain eosinophilic differentiation. Rearrangement of the genes coding for either subunit of the heterodimeric AML1 transcription factor is known to result in either spontaneous (inv16) [15] or IL-5-inducible [t(8;21)] [16] eosinophilic differentiation of fresh malignant blasts. Cytogenetic studies have not revealed these abnormalities in either AML14 or AML14.3D10, and we have also not been able to detect them by RT-PCR, using well-characterized primers [17, 18] (data not shown).

View larger version (62K): [in this window] [in a new window]   Figure 4. Cytogenetic studies of AML14 (A) and AML14.3D10 (B). AML14: 48-50, X,-Y,+add(1)(p31),del(5)(q23q34),dup(5) (q13q31),add(8)(q24.3),del(9)(q22),+13, add(14)(p13),-16,add(17) (p13),+18,-20,add(22)(q13),+3-5mar AML 14.3D10: 65-84 XY,+X,del(5)(q23q34)+6,+6,-7,add(8)(q24.3),del(9) (q22),add(13)(p13),add(14)(p13),add(14)(p13),+16,add(17) (p13),+19,-20,-21,add(22)(q13),+12-14mar. Multiple double minute chromosomes were present in all cells.

	Eosinophil Granule-Related Protein Synthesis and Purification

Top Abstract Introduction Establishment and... Establishment of the AML14.3D10... Eosinophil Granule-Related... Transcriptional Regulation of... Characterization of the Eotaxin... Lineage Switching of... Autocrine GM-CSF Use Summary References

 
The AML14.3D10 cell line produces huge amounts of eosinophil granule proteins, in some cases even more than normal eosinophils. The production of MBP has been measured by radioimmunoassay at 10 µg/cell [14]. This has permitted the detailed study of the post-translational processing of MBP [19], which is now known to be initially produced as a larger "pro-MBP" before cleavage to the mature, functional protein.

Charcot-Leyden crystal protein is produced in amounts that permitted use of the cell line for the determination of the crystallographic structure of the protein for the first time [20], revealing a structural topology having remarkable similarity to that of a family of carbohydrate-binding proteins termed galectins.

	Transcriptional Regulation of Expression of Eosinophil-specific Genes

Top Abstract Introduction Establishment and... Establishment of the AML14.3D10... Eosinophil Granule-Related... Transcriptional Regulation of... Characterization of the Eotaxin... Lineage Switching of... Autocrine GM-CSF Use Summary References

 
Using the HL-60-C15 subline, AML14 and cytokine-differentiated AML14, Ackerman and colleagues have obtained evidence for a myeloid (and possibly eosinophil) specific transcription factor that binds to a critical regulatory region in the IL-5 receptor subunit promoter/enhancer region. After determining the transcriptional start site, 561 bp immediately upstream of the start site was identified as containing a functionally active promoter region by transfection of promoterless reporter constructs containing the sequence into the cell lines. Significant promoter activity was found only in the above cell lines and not in nonmyeloid lines. The greatest activity was seen in eosinophil-induced AML14. Subsequent experiments with deletion mutants demonstrated that a 34 bp region was responsible for over 80% of promoter activity. Consensus sequences for known transcription factors were not found in the 34 bp region. Furthermore, gel shift experiments demonstrated the presence of a nuclear factor(s) that bound to the region only in myeloid cell lines [21, 22]. Using methylation interference, the sequence responsible for nuclear protein binding was determined to be GTTGCCTAGG, extending from bp -430 to -421. Mutation of this sequence abolished both nuclear factor binding and promoter activity. This 10-base sequence, which has been termed EOS1, acts as an enhancer, as it has been determined that it requires a basal promoter for full activity. Identification and cloning of the cognate transcription factor is being attempted.

Evidence for another possible eosinophil-specific transcription factor has been obtained by transfection of AML14.3D10 with a reporter construct containing a 10 bp mutation of a 30 bp sequence in the promoter region of the EPO gene known to bind proteins in gel shift experiments. It was found that the mutated construct reduced EPO promoter activity in AML13.3D10 by 80% when compared to a wild-type construct. Although sequence analysis of the 10 bp region showed two possible consensus sites for known transcription factors, supershift experiments suggested that other yet-to-be-identified proteins were responsible for the binding activity [23]. Using a similar strategy involving transient transfection of CLC enhancer-reporter constructs into AML14.3D10, gel shift experiments and methylation interference assays, a critical CTTCCT sequence was identified, which, when mutated to CTTGAT, lost 80% of promoter activity. Because the CTTCCT motif is identical to a murine ets factor-binding polyoma virus enhancer activator sequence (PEA3), it is possible that the CLC promoter may be activated by binding either the human eosinophil equivalent of PEA3 or a related ets-like factor [24].

	Characterization of the Eotaxin Receptor

Top Abstract Introduction Establishment and... Establishment of the AML14.3D10... Eosinophil Granule-Related... Transcriptional Regulation of... Characterization of the Eotaxin... Lineage Switching of... Autocrine GM-CSF Use Summary References

 
Eosinophils are recruited to sites of allergic inflammation and activated by certain chemoattractants, the most important of which are thought to be RANTES and the recently described eotaxin [25-27]. The identity of the putative eotaxin receptor was confirmed and further characterized by expressing the cDNA in AML14.3D10 [28]. The receptor belongs to the ß-chemokine receptor subset of the G protein-coupled receptor superfamily and has been designated CC CKR3. Although eosinophils and some other cell lines constitutively express the eotaxin receptor or are capable of low-level expression following transfection of an expression construct, the AML14.3D10 cell line apparently possesses the machinery to drive much higher-level expression, greatly facilitating study (Dr. Julie DeMartino, personal communication). Through analysis in AML14.3D10 cells, it was determined that CC CKR3 binds and is activated by eotaxin, RANTES, and MCP-3. Thus, CC CKR3 shares with other chemokine receptors the property of multiple ligand activation, but is activated by a unique set of ligands in comparison to the other known chemokine receptors.

	Lineage Switching of Hematopoietic Cells

Top Abstract Introduction Establishment and... Establishment of the AML14.3D10... Eosinophil Granule-Related... Transcriptional Regulation of... Characterization of the Eotaxin... Lineage Switching of... Autocrine GM-CSF Use Summary References

 
It has been assumed that once hematopoietic progenitor cells "commit" to a single lineage, the process is irreversible. Observations made using the AML14.3D10 cell line suggest that this may not be true. This cell line exists constitutively with a phenotype that approximates an eosinophilic myelocyte. Although the cells exhibit obvious advanced eosinophilic differentiation, they continue to maintain proliferative capacity. When these cells are exposed to all-trans retinoic acid (ATRA), a remarkable transformation takes place. Within 24 h, fast green staining has noticeably dissipated and is completely absent by 72 h. By five days, the cells phenotypically resemble neutrophils ( Fig. 5). During this transformation, the cells acquire surface CD16 (absent on normal eosinophils but present on neutrophils) ( Fig. 6), cease to express the genes for eosinophil-granule-associated proteins and the IL-5 receptor subunit, and newly express the G-CSF receptor gene ( Fig. 7) [14]. These events occur without significant cell death initially, but after about 14 days the neutrophil-like cells die and an eosinophilic population re-emerges. Although other cell lines, such as HL-60, may be induced to different myeloid lineages, this represents the selection of differing programs by a primitive cell [1]. In the case of AML14.3D10, cells that had already committed to one lineage appear to suppress that program and activate another. Although this concept may be considered hematologic heresy, there is precedence for lineage switching in other tissues. Lung cancers, for example, may be switched from neuroendocrine (small cell) to epithelial (adenocarcinoma) [29]. It should be noted that, as is usual with neutrophilic induction of leukemic cell lines, the process is incomplete. In spite of the striking neutrophil-like appearance of the ATRA-induced cells and the other neutrophil-associated features described above, there is no evidence that AML14.3D10 or other cell lines [30, 31] can be induced to synthesize neutrophil-specific secondary granule proteins.

View larger version (451K): [in this window] [in a new window]   Figure 5. A - C. AML14.3D10 cells during ATRA induction of neutrophilic transformation. A) Cells 24 h after exposure to ATRA have already lost most of their fast green granules (compare to Fig. 1D). B) By 5 days, they are completely fast green negative and morphologically appear neutrophilic. C) Wright stain 7 days following ATRA induction. D) alpha-naphthyl butyrate esterase stain of AML14.3D10 cells 48 h after induction of macrophage differentiation by vitamin D. All original magnifications x 1,000. Reprinted with modifications with permission from [14].

View larger version (42K): [in this window] [in a new window]   Figure 6. Flow cytometric analysis of lineage-related antigen changes of AML14.3D10 cells during ATRA (day 7) induction of neutrophilic transformation and Vitamin D (day 3) induction of macrophage differentiation. Graphs are plotted as fluorescence intensity (X) versus cell number (Y). Left and right graphs are from the same analysis, separated for clarity.

View larger version (154K): [in this window] [in a new window]   Figure 7. Changes in granule protein and cytokine receptor mRNA expression of AML14.3D10 cells during ATRA-induced neutrophilic transformation. Ethidium-stained gels of RT-PCR products showing: A) Suppression of expression of mRNA for eosinophil granule proteins during the "neutrophilic" period. Expression resumes later as the culture repopulates with eosinophilic cells. Lane A is the undifferentiated parental AML14 cell line, lane E is AML14.3D10 before ATRA exposure. B) Loss of expression of mRNA for the subunit of IL-5 and gain of expression of G-CSF receptor mRNA during neutrophilic transformation. GAPDH was used as a "housekeeping" gene for comparison. Reprinted with modifications with permission from [14].

	Autocrine GM-CSF Use

Top Abstract Introduction Establishment and... Establishment of the AML14.3D10... Eosinophil Granule-Related... Transcriptional Regulation of... Characterization of the Eotaxin... Lineage Switching of... Autocrine GM-CSF Use Summary References

 
We have determined that the AML14.3D10 cell line produces GM-CSF in large amounts [14]. Immunoassays of culture supernatant have detected concentrations of up to 113 pg/ml. The cell line apparently uses the GM-CSF to promote its proliferation, as neutralizing antibody or anti-sense constructs caused growth inhibition of the cells. No obvious effect of these treatments on eosinophilic differentiation was evident. We were further able to demonstrate activation by endogenously produced GM-CSF of components of the signal transduction pathway (receptor ß-subunit, JAK-2, p56 lyn) common to the IL-3, GM-CSF and IL-5 receptors [32]. Engagement of this system by endogenously produced GM-CSF can prevent access by exogenous GM-CSF or IL-5 ( Fig. 8).

View larger version (77K): [in this window] [in a new window]   Figure 8. Anti-phosphotyrosine Western blots of cell lysates of AML14.3D10 cells incubated for varying periods of time in receptor-saturating concentrations of GM-CSF, IL-3, and IL-5. Top: AML14.3D10 cells. Bottom: Cells that had been preincubated for 48 h in 80 ng/ml neutralizing anti-GM-CSF antibody. Neutralization of endogenously produced GM-CSF "frees" signaling pathways, allowing a response to exogenous GM-CSF and IL-5, as demonstrated by increased phosphorylation of a number of species evident on the bottom blot. No response is seen to IL-3 because AML14.3D10 does not express the IL-3 receptor. Reprinted with permission from [32].

	Summary

Top Abstract Introduction Establishment and... Establishment of the AML14.3D10... Eosinophil Granule-Related... Transcriptional Regulation of... Characterization of the Eotaxin... Lineage Switching of... Autocrine GM-CSF Use Summary References

 
In the few years since their establishment, the AML14 cell line and AML14.3D10 subclone have become standard tools for the study of many aspects of eosinophil biology. Future work should demonstrate their additional value for studies of the molecular basis of lineage decision making (or indecisiveness) and leukemic transformation.

	Acknowledgments

 
We are grateful to Dr. Gerald Hoeltge for performing cytogenetic studies, Ann Dvorak for electron microscopic studies, and Steven Ackerman for reagents and advice.

Supported by Merit Review funding from the Department of Veterans Affairs.

	References

Top Abstract Introduction Establishment and... Establishment of the AML14.3D10... Eosinophil Granule-Related... Transcriptional Regulation of... Characterization of the Eotaxin... Lineage Switching of... Autocrine GM-CSF Use Summary References

 

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