©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification and Characterization of a Functional Promoter Region in the Human Eosinophil IL-5 Receptor Subunit Gene (*)

(Received for publication, August 4, 1994; and in revised form, October 14, 1994)

Zijie Sun (§) Donald A. Yergeau Tania Tuypens Jan Tavernier Cassandra C. Paul Michael A. Baumann Daniel G. Tenen (¶) Steven J. Ackerman (**)

From the Divisions of Infectious Diseases and Hematology-Oncology, Department of Medicine, Beth Israel Hospital and Harvard Medical School, Boston, Massachusetts 02215, Roche Research Gent, Hoffman-La Roche, Gent, Belgium, and Research Service, Dayton Veterans Administration Medical Center and Hematology/Oncology Division, Wright State University Medical School, Dayton, Ohio 45435

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The molecular basis for the commitment of multipotential myeloid progenitors to the eosinophil lineage, and the transcriptional mechanisms by which eosinophil-specific genes are subsequently expressed and regulated during eosinophil development are currently unknown. Interleukin-5 (IL-5) is a T cell and mast cell-derived cytokine with actions restricted to the eosinophil and closely related basophil lineages in humans. The high affinity receptor for IL-5 (IL-5R) is composed of an alpha subunit (IL-5Ralpha) expressed by the eosinophil lineage, that associates with a beta(c) subunit shared with the receptors for IL-3 and granulocyte-macrophage colony stimulating factor (GM-CSF). As a prerequisite to studies of the transcriptional regulation of the IL-5Ralpha subunit gene, we used three different methods, including primer extension, RNase protection, and 5`-RACE to precisely map the transcriptional start site to a position 15 base pairs (bp) upstream of the 5` end of the published sequence of IL-5Ralpha exon 1. To initially identify the IL-5Ralpha promoter, 3.5 kilobases (kb) and 561 bp of the 5` sequence flanking the transcriptional start site were subcloned into the promoterless pXP2-luciferase vector. Transient transfection of these constructs into an eosinophil-committed HL-60 subline, clone HL-60-C15, induced the expression of 240-fold greater luciferase activity than the promoterless vector, identifying a strong functionally active promoter region within the 561 bp of sequence proximal to the transcriptional start site and with activity equivalent to pXP2 constructs containing the entire 3.5 kb of upstream sequence. To more precisely localize the cis-acting regulatory elements in this region important for promoter activity, a series of 5` deletion mutants of the 561-bp region were generated in the pXP2-luciferase vector. Deletion of the region between bp -432 and -398 reduced promoter activity by more than 80% in the HL-60-C15 cell line. Further analyses of the activity of the IL-5Ralpha promoter constructs in various other eosinophil, myeloid, and non-myeloid cell lines indicated that the promoter was relatively myeloid and eosinophil lineage-specific in its expression. Consensus sequences for known transcription factor binding sites were not present in the 34-bp region of the promoter required for maximal activity, suggesting unique myeloid- and possibly eosinophil-specific regulatory elements. Using electrophoretic mobility shift assays, we have identified a nuclear factor(s) that binds to the 34-bp functional region of the the promoter and that is expressed in the myeloid and eosinophilic cell lines in which the promoter is active, but not in non-myeloid or non-hematopoietic lines. This functional promoter segment likely serves as the binding site for a myeloid- and possibly eosinophil-specific transcription factor(s). Further study of the IL-5Ralpha promoter should elucidate unique transcriptional features of this gene whose expression is essential to the commitment and differentiation of multipotential myeloid progenitors to the eosinophil lineage and to the functional activation of the mature cell.


INTRODUCTION

Interleukin-5 (IL-5), (^1)produced primarily by activated T cells (1) and mast cells(2, 3) , stimulates the proliferation and differentiation of murine activated B cells and regulates the production of eosinophils(4, 5, 6) . The proliferation, differentiation, and maturation of eosinophils in the bone marrow and their post-mitotic functional activation in tissues occurs in response to a number of cytokines in addition to IL-5, including GM-CSF and IL-3 (7, 8, 9) . Both IL-3 and GM-CSF have activities on other hematopoietic lineages, whereas IL-5 is more eosinophil-specific and plays a crucial role in regulating the differentiation and development of the eosinophil lineage(10) . Although IL-3 and GM-CSF participate in the proliferation and commitment of progenitors to the eosinophil lineage, IL-5 is both necessary and sufficient for eosinophil development to proceed(10, 11) . In humans, the high affinity receptor for IL-5 is apparently restricted to eosinophils and hematopoietically related basophils(12) ; in contrast to murine B cells, the activity of IL-5 on human B cells is controversial (10, 13) and is still being delineated (14) . Thus, the expression of the high-affinity receptor for IL-5 is an important prerequisite and very early lineage-specific event in the hematopoietic program for these granulocytes. Of interest, IL-5 is active in vitro both in the production of eosinophils from bone marrow and umbilical cord blood progenitors as well as in the priming, activation, and enhanced survival of mature eosinophils. Overexpression of IL-5 is observed in many eosinophil-associated diseases(15, 16, 17) , and IL-5 transgenic mice develop profound eosinophilia(18, 19) , indicating that IL-5 plays important roles in promoting the production and function of eosinophils in vivo.

Prior studies based on binding and cross-linking experiments on murine B cell lines suggested a two-chain model for the IL-5 receptor (IL-5R): a unique alpha subunit (60-kDa component) corresponding to the low affinity IL-5 binding site (K = 10M), and a beta(c) subunit (130-kDa component) that is shared with the IL-3 and GM-CSF receptors and associates with the alpha chain to form the high affinity receptor (K = 5 times 10M) (20, 21, 22) . Only the high affinity binding site is generated upon induction of eosinophilic sublines of human promyelocytic HL-60 cells with butyric acid(23, 24) . Recently, it has been found that the high affinity IL-5R requires both alpha and beta(c) subunits (25) for optimal signaling, and that the intracellular cytoplasmic portion of the alpha chain is essential to this process (64) . (^2)The isolated alpha subunit if the GM-CSFR has likewise been shown to participate in signaling, albeit via a phosphorylation independent pathway(26) . To clarify the role of these two components of the IL-5 receptor in eosinophil differentiation and biologic function, Tavernier et al.(27) and Murata et al.(28) have explored the characteristics of the human IL-5 receptor alpha (IL-5Ralpha) gene. The gene encoding the IL-5Ralpha subunit is located on chromosome 3 in the region 3p26(29) . The organization of this gene reflects the functional domains of this protein and shares many characteristics with other members of the cytokine/hemopoietin receptor gene family(9, 24, 29) . Several alternatively spliced transcripts have been identified in the mRNA and reflect the membrane versus soluble isoforms(30) . Aside from its ability to bind IL-5 in vitro(31) , the in vivo function(s) of the soluble form of the IL-5R have not been elucidated.

Like other hematopoietic genes, expression of the cytokine receptor genes are likely regulated in part at the transcriptional level in a lineage-specific and temporal, developmental manner. Regulation of IL-5R expression is extremely pertinent to an understanding of the processes involved in the commitment and differentiation of multipotential hematopoietic progenitors to the eosinophil lineage. However, the mechanisms for the transcriptional control and tissue-specific expression of human cytokine receptor genes such as IL-5Ralpha are currently unknown. To investigate the regulation of human IL-5Ralpha expression, we first isolated the 5` upstream region of the gene and mapped the transcriptional start site. We have identified a functional promoter region upstream of the transcriptional start site that is highly active in eosinophil-inducible myeloid leukemic cell lines and is active in a myeloid- and eosinophil-specific manner, and we have localized the minimum cis-acting sequence required for promoter activity to a 34-bp region between bp -432 and -398 of the gene.


EXPERIMENTAL PROCEDURES

Genomic Cloning Procedure

Two human genomic libraries, prepared from placental DNA in the FIX II phage vector (Stratagene), were screened as described previously(29) . A KpnI fragment from the ghIL5Ralpha-2 clone (29) covering the 5` upstream region of the gene was subcloned into the pGEM7ZF(-) plasmid (Promega) and sequenced on both strands using the Sanger dideoxy method with Sequenase 2.0 (U. S. Biochemical Corp.).

Cell Culture

An eosinophil-committed subline of the HL-60 promyelocytic leukemia cell line, HL-60-C15(32) , was maintained in RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented with 10% fetal bovine serum (Hyclone Laboratories), 2 mML-glutamine, 100 units of penicillin, 100 µg of streptomycin (Life Technologies Inc.) and passaged twice weekly. HL-60-C15 cells (2 times 10^5/ml) were induced with 0.5 mM butyric acid (Sigma), and total RNA was isolated after 1, 3, and 5 days of culture. Other myeloid and non-myeloid cell lines utilized in these studies including the promyelocytic HL-60 line (ATCC CCL 240), monocytic U937 line (ATCC CRL1593), BJA-B B lymphocytic cells (33) , REX T lymphocytic cells, and the cervical carcinoma line, HeLa (ATCC CCL 2), were maintained by passage twice weekly in RPMI 1640 (Life Technologies Inc.) (HL-60, U937, BJA/B, REX) or Dulbecco's modified Eagle's medium (HeLa) supplemented with 10% fetal bovine serum (Hyclone) and 2 mML-glutamine as described previously(34, 35) . In addition, two recently described acute myeloid leukemia cell lines, AML14, a line committed to the eosinophil lineage(36) , and AML14.eos, a cytokine-induced fully differentiated eosinophilic myelocyte subline of AML14 that continues to proliferate and maintain a differentiated phenotype with cytokine supplementation every 4-6 weeks(37) , were cultured in RPMI 1640 (BioWhittaker) with 8% fetal bovine serum (Life Technologies, Inc.), 2 mML-glutamine, 5 times 10M beta-mercaptoethanol, and 1 mM sodium pyruvate as described previously(36, 37) .

RNA Isolation and RT-PCR Assay

Total RNA was prepared from HL-60-C15 cells, HeLa cells, and peripheral blood eosinophils obtained from a patient with hypereosinophilic syndrome (HES) as described previously(38) . The reverse transcription polymerase chain reaction (RT-PCR) method was used to investigate whether there was an additional exon upstream of exon 1 of the IL-5Ralpha cDNA. Briefly, 2 µg of total RNA was treated with RNase-free DNase I. After removal of the DNase I by extracting twice with phenol, cDNA was synthesized with 9 units of avian myeloblastosis virus reverse transcriptase (Promega) using 0.1 µM oligo(dT) primer in a total volume of 20 µl. Three to five µl of cDNA was added to a standard PCR mixture containing a 1 µM concentration of each primer. The PCR reaction was performed on a thermal cycler using 40 cycles of 1 min, 45 s at 94 °C, 30 s at 48 °C, and 45 s at 72 °C. The final polymerization step was extended an additional 10 min at 72 °C. Two upstream primers were used for this experiment: one forward primer, 5`-GTCTTTTGAAAGGATCT-3`, was identical in sequence with the 5` end of the IL-5Ralpha cDNA as described by Murata et al.(28) , and the second, 5`-CCGCATTTCTCAGGCCAG-3`, spanned the exon 1-2 boundary. The reverse primer for this reaction, 5`-GGGAGAAGTGAAATCTTTTCATC-3`, spanned the exon 4-5 boundary.

Primer Extension Assay

Primer extension was performed essentially as described previously(39) . Briefly, 100 ng of a reverse primer spanning the exon 4-5 boundary (see above) was labeled by T4 polynucleotide kinase. The labeled primer was hybridized with 20 µg of RNA from either HL-60-C15 or HeLa cells at 33 °C overnight. After precipitation with 100% ethanol, the sample was dried and resuspended in 10 µl of H(2)O. The extension reaction was performed with 40 units of avian myeloblastosis virus reverse transcriptase under standard conditions at 42 °C for 90 min. The specific cDNA fragments were analyzed on a 6% polyacrylamide, 7 M urea sequencing gel.

RNase Protection Assay

We generated a hybrid cDNA/genomic DNA probe by PCR which consisted of a fusion between part of exon 2 (95 bp), all of exon 1, and 194 bp of upstream genomic sequence. This 325-bp DNA fragment was cloned into a pGEM-T-vector (Promega). The complementary sense RNA (cRNA) probe for RNase protection was synthesized with T7 polymerase using linearized plasmid. Twenty µg of DNase I-treated total RNA from HL-60-C15 and HeLa cell lines were hybridized overnight at 46 °C with the [P]UTP-labeled cRNA probe in 40 µl of 80% formamide, 40 mM PIPES, pH 6.5, containing 400 mM NaCl and 1 mM EDTA. After hybridization, unhybridized cRNA probe was removed by digestion for 20 min at 37 °C with 40 µg/ml RNase A and 1 µg/ml RNase T1 in 350 µl of 10 mM Tris-HCl, pH 7.5, containing 300 mM NaCl and 5 mM EDTA, with 5 µg of carrier RNA added. Protected fragments were ethanol-precipitated and analyzed on a 6% polyacrylamide sequencing gel as above.

Rapid Amplification of 5` cDNA End Assay

The IL-5Ralpha cDNA was reverse-transcribed from 10 µg of total RNA isolated from the butyrate-induced HL-60-C15 cell line using the same exon 4-5 boundary primer used for primer extension. The cDNA was diluted to 1 ml with water, and excess primers were removed by two rounds of centrifugal ultrafiltration on a Centricon 100 filter (Amicon) for 20 min at 1,000 times g. The retentate was further reduced to 10 µl by evaporation and treated with 30 units of terminal transferase (Life Technologies, Inc.) and 200 µM dATP in a final volume of 20 µl at 37 °C for 13 min, followed by incubation at 67 °C for 5 min. The tailed cDNA was diluted to 300 µl with 10 mM Tris buffer, pH 7.5, with 1 mM EDTA, and 10 µl were amplified by PCR as described above, using 7 pmol of an oligo(dT)-adapter primer and 25 pmol of both the short adapter primer and a specific reverse primer, 5`-GGCGAGGACCGTGTCTGTCGTGTCTAT-3`, from exon 2 of the IL-5Ralpha sequence. The PCR products were cloned into a pBluescript-T-vector and sequenced with T3 or T7 universal primers.

Construction of Plasmids for Promoter Analysis

For analysis of the putative promoter region of the human IL-5Ralpha subunit gene, three reporter constructs in the promoterless pXP2-luciferase vector were prepared as shown in Fig. 6; a 2.9-kb BamHI/KpnI DNA fragment from the ghIL5Ralpha-2 clone(29) , a 612-bp fragment covering all of exon 1 and 561 bp of upstream sequence, and a 3.5-kb fragment including both the 2.9-kb and 612-bp regions were cloned into the promoterless pXP2-luciferase vector in the BamHI/KpnI, KpnI/XhoI, and BamHI/XhoI sites, respectively (Fig. 6A). Deletion mutants from the 5` end of the -561/IL-5Ralpha-pXP2 plasmid were generated using PCR amplification of the appropriate region with oligonucleotide primers, cloning into a pBluescript-derived T-vector, followed by subcloning into the pXP2-luciferase vector at BamHI and XhoI sites. Deletion mutants containing 561, 517, 469, 432, 398, 179, and -561/-377 bp of upstream sequence were prepared; all deletion mutants were sequenced in their entirety to identify potential PCR-generated errors and to confirm the 5` end of each mutant.


Figure 6: Functional activity of the IL-5Ralpha promoter. A, construction of the IL-5Ralpha promoter constructs in the promoterless pXP2 luciferase expression vector. A 3.5-kb fragment, comprised of 2.9 and 0.6 kb of the 5`-flanking region of IL-5Ralpha including all of exon 1, was subcloned into pXP2 and analyzed for functional activity (see B and Fig. 7). B, functional activity of the longest IL-5Ralpha-pXP2 promoter constructs. Constructs of IL-5Ralpha upstream sequence (A) were transiently transfected along with a cytomegalovirus-human growth hormone plasmid (CMV-hGH) as transfection control into uninduced HL-60-C15 cells by electroporation. Luciferase activity was measured by luminometry in cell lysates prepared 5 h post-transfection. Relative light units (RLU) were corrected based on the concentration of hGH (ng/ml) released into the culture supernatants. The mean ± S.D. for three or more replicate experiments is shown.




Figure 7: Activity of IL-5Ralpha promoter deletion mutants in uninduced HL-60-C15 cells. 5` deletion mutants of the IL-5Ralpha promoter in the pXP2-luciferase vector were generated as shown on the left and co-transfected with a CMV-hGH control plasmid as in Fig. 6B. Relative promoter activity of each mutant is shown in comparison to the wild type, -561-bp IL-5Ralpha/pXP2 plasmid (100%).



Transient Transfections of Leukemic Cell Lines

The promoterless luciferase plasmid pXP2 was used for all promoter studies (40) . A cytomegalovirus-human growth hormone (CMV-hGH) plasmid, for use as an internal control in transfections, was provided by Dr. Leonard Zon (Children's Hospital and Harvard Medical School, Boston, MA). Plasmid DNA was prepared, and cells were transfected as described previously using 1-1.5 times 10^7 cells per transfection with minor modifications(34, 35, 41) . Transfection of plasmid DNA was carried out by electroporation (35) using 5 µg of test plasmid, 25 µg of carrier plasmid, and 0.5-1 µg of the reference plasmid (CMV-hGH) expressing human growth hormone. The HL-60-C15 subline and parental HL-60 cells were electroporated at 310 and 300 V, 960 µF, respectively, and the AML14.eos subline and AML14 parental cells at 350 V, 960 µF, conditions optimized for these cell lines. The U937, BJA-B, REX, and HeLa lines were electroporated at 300, 220, 220, and 150 V, 960 µF, respectively, optimal conditions previously utilized for these lines(34, 41, 42, 43, 44) . Luciferase activity in cell lysates prepared 5 h post-transfection was measured as relative light units (RLU) using a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA) as described previously(34, 41) ; cell extracts were prepared in 500 µl of 1% Triton X-100, 25 mM Gly-Gly, 15 mM MgSO(4), 4 mM EGTA, 1 mM dithiothreitol, and 100 µl of the extract (equivalent to 3 times 10^6 cells) was analyzed for luciferase activity. The CMV-hGH co-transfection provided for standardization among the different myeloid and non-myeloid lines, different plasmid DNA preparations, and individual transfection experiments(43) , and the carrier DNA decreased variability due to differences in the DNA preparations for the individual IL-5Ralpha promoter-pXP2 constructs(45) . An enhancer-containing vector, CMV-pXP2, was used as a positive control for each transfection as well. Growth hormone production was measured by assaying the culture supernatants from transfected cells with a commercially available radioimmunoassay kit (Nichols Institute Diagnostics, San Juan Capistrano, CA). The RLU from individual transfections were normalized for transfection efficiency based on the number of nanograms of growth hormone produced per ml of culture supernatant. Individual transfection experiments were repeated a minimum of three times with at least two different preparations of plasmid DNA, and the results are reported as mean RLU/ng/ml hGH (± S.E.) or as the mean percentage (± S.E.) of the activity of the -561/luc construct in each experiment.

Nuclear Extracts

Nuclear extracts were prepared from HL-60, HL-60-C15, AML-14, AML-14.eos, U937, BJA-B, and HeLa cell lines, essentially according to the method of Dignam et al.(46) with minor modifications. Since nuclear extracts were prepared from protease-rich promyelocytic and eosinophilic cell lines, we included two protease inhibitors, diisopropylphosphofluoridate (Sigma) and phenylmethylsulfonyl fluoride (Sigma), at final concentrations of 1 mM in the preparation of all nuclear extracts.

Gel Mobility Shift Assay

The probe for mobility shift analysis in this study was a 93-bp fragment located in the bp -469 to -377 region of IL-5Ralpha promoter. This fragment was generated by PCR amplification. Prior to amplification, one of the primers was labeled with T4 polynucleotide kinase and [-P]ATP. The PCR fragments were separated from free primer by gel electrophoresis. The gel shift assay was performed essentially as described by Singh et al.(47) . Each binding reaction contained 2 µg of crude nuclear extract with 4 µg of poly(dI-dC) and 1 times 10^4 cpm of P-labeled DNA fragment in 20 µl of a buffer containing 20 mM Hepes (pH 7.9), 50 mM KCl, 5 mM EDTA, 1 mM dithiothreitol, 3 mM MgCl(2), and 5% glycerol. After a 20-min incubation at room temperature, the binding reaction was separated on a native 4% polyacrylamide gel (19:1 bisacrylamide) in 0.25 times TBE buffer at 5 V/cm for 4 h at room temperature. The gels were dried prior to autoradiography using Kodak X-Omat XAR film and Lightning Plus enhancing screen. Competition for nuclear factor binding to the radiolabeled probe was performed by adding a 50-fold molar excess of cold competitor DNA probe to the binding reaction prior to adding the P-labeled DNA fragment, followed by analysis on nondenaturing, native gels as above.


RESULTS

RT-PCR Analysis of a Putative Exon 0 in the IL-5Ralpha mRNA

The cDNA sequence and genomic structure of the human IL-5Ralpha chain gene has been reported previously (27, 29) (Fig. 1). However, the structure of the 5`-untranslated and upstream regulatory regions of the IL-5Ralpha gene was not completely characterized. The 196-bp 5`-untranslated region of the mRNA is encoded by exons 1-3 and 3 bp of exon 4(27, 29) . However, Murata et al.(28) reported an additional 18-bp sequence 5` of exon 1 (referred to here as ``putative exon 0'' for clarity) that was not identified in the cDNAs cloned by Tavernier et al.(27) , as well as the absence of exon 3 in their cDNA sequence(28) . These conflicting findings made the precise localization and characterization of the transcriptional start site a prerequisite for further analysis of the regulatory promoter region of the gene. To address this issue, we first isolated the 5`-untranslated sequence by RT-PCR using RNA isolated from both butyrate-induced HL-60-C15 cells and blood eosinophils purified from a patient with HES. Oligonucleotide primers designed to hybridize to either the boundary of exon 1 to 2 or the entire putative exon 0 sequence (Fig. 1) were used in combination with a reverse primer spanning the exon 4-5 boundary in RT-PCR (Fig. 2). After 40 cycles of amplification, no specific PCR DNA products were detectable using the putative exon 0 primer. In contrast, only the exon 1 primer was able to generate an appropriately sized 252-bp fragment. Sequence analysis of this 252-bp fragment amplified from both of the RNA samples tested from butyrate-induced HL-60-C15 cells or HES patient eosinophils showed it to be identical with that reported previously (27, 28) , except for the absence of exon 3. A 3.5-kb fragment from the ghIL5Ralpha-2 genomic clone (29) covering the entire upstream region of the gene was also screened with the 18-bp putative exon 0 oligonucleotide primer by both Southern blotting and sequencing; no homologous sequence could be detected by either method (data not shown). These findings indicated that the putative exon 0 sequence was not present in either the 5`-untranslated region of the IL-5Ralpha mRNA or 3.5 kb of upstream genomic sequence.


Figure 1: Structure of the 5` upstream region of the IL-5Ralpha gene. Oligonucleotide primers and the primer extension, RNase protection and 5`-RACE strategies, and anticipated fragment sizes for mapping of the transcriptional start site are indicated. Exons 1-3 encode the 261-bp 5`-untranslated region. The 15-bp dashed line for the primer extension and 5`-RACE procedures refers to the additional 15 bp of upstream sequence identified by these techniques. The ``putative exon 0'' represents the 18 bp of 5`-untranslated sequence published by Murata et al.(28) , which was not identifiable in the IL-5Ralpha cDNA by RT-PCR using oligonucleotides A and D or by 5`-RACE nor present in 3.5 kb of upstream genomic DNA by Southern blotting. The 5`-RACE utilized oligonucleotide D for the initial reverse transcription step and the internal oligonucleotide C for subsequent amplification of the resultant cDNAs.




Figure 2: RT-PCR analysis of mRNA for the IL-5Ralpha subunit for detection of putative exon 0 in mRNA from eosinophils purified from a patient with HES or from HL-60-C15 cells induced with butyrate for 5 days. Reverse transcription used oligo(dT) priming and PCR amplification for 40 cycles with primers identical in sequence with putative exon 0 or the exon 1/2 boundary and an exon 4/5 boundary reverse primer (primers A, B, and D, Fig. 1). 10 µl of the PCR products were electrophoresed on a 2% agarose gel and stained with ethidium bromide. A 252-bp PCR fragment was obtained with the exon 1-2 boundary primer, but not the putative exon 0 primer.



Mapping of the Transcriptional Start Site

To identify the functional promoter region that regulates IL-5Ralpha gene expression, we have carefully mapped the transcriptional start site using three different techniques: primer extension, RNase protection, and 5`-RACE. For primer extension, a specific exon 4-5 boundary reverse primer (Fig. 1) was labeled and hybridized to the RNA templates, and IL-5Ralpha cDNA was synthesized using avian myeloblastosis virus reverse transcriptase. Two different transcripts were identified in the RNA from butyrate-induced HL-60-C15 cells (Fig. 3), with no transcripts identified when total RNA from HeLa cells was used as a control. Two cDNA fragments were obtained including a major species of 291 bp and a minor one of 332 bp in length. Expression of both transcripts was up-regulated in response to butyrate-induced eosinophilic differentiation of the HL-60-C15 cell line (Fig. 3). According to the published cDNA sequence(27) , the distance between the starting point of the reverse primer and the first nucleotide of exon 1 should be 276 bp when exon 3 is skipped. This size was in close agreement with the 291-bp size of the major transcript shown in the primer extension analysis, but suggested that the previous cDNA sequence was not full-length and contained additional sequence upstream of the 5`-most end of exon 1. These extra nucleotides were either from an additional short exon or part of the genomic (``intronic'') sequence immediately upstream of exon 1. To distinguish these possibilities, we performed an RNase protection analysis, as well as cloned and sequenced the 5` end of the IL-5Ralpha mRNA by RACE. For RNase protection, we generated a hybrid cDNA/genomic DNA probe consisting of a fusion between part of exon 2 (95 bp), all of exon 1, and 194 bp of upstream genomic sequence. The RNA samples from butyrate-induced HL-60-C15 cells protected a series of fragments from this ``cRNA'' that clustered around 148 bp in length relative to the DNA standards (Fig. 4). No protected fragments were found when RNA from HeLa cells was used as a negative control. The protected size of the longer fragment precisely matched the major start site identified in the primer extension analysis, which was 15 bp upstream of the first nucleotide of exon 1. An additional minor protected species was also observed which was about 24 bp shorter than the major 148-bp fragment. Since primer extension and RNase protection did not provide the exact sequence of the transcriptional start site, the 5` end of the mRNA was reverse-transcribed from total RNA of butyrate-induced HL-60-C15 cells (5-day induction), and the cDNA was amplified and subcloned into pBluescript for sequencing using the 5`-RACE procedure. Plasmid DNA from 7 individual clones was isolated and purified for sequence analysis; sequences from 4 of these clones localized the transcriptional start site to a position 15 bp upstream of exon 1, a perfect match to the major transcriptional start site suggested by both primer extension and RNase protection. No longer species were found even though 5 additional clones were analyzed (data not shown). However, 3 shorter species were found; 1 started at the middle of exon 1, 1 at the first nucleotide of exon 2, and 1 at the fifth nucleotide of exon 2 (Fig. 5). Taken together, the data from the above experiments unequivocally localize the major transcriptional start site of the hIL-5Ralpha gene to a position 15 bp upstream of the published end of exon 1 ( Fig. 1and Fig. 5).


Figure 3: Primer extension analysis; IL-5Ralpha mRNA from HL-60-C15 cells induced toward eosinophil differentiation with butyrate for 5 days. The primer for reverse transcription, located at the exon 4-5 boundary region of the gene (primer D, Fig. 1), was kinased using [alpha-P]dATP. The primer was hybridized to the RNA samples, and cDNA was synthesized with avian myeloblastosis virus reverse transcriptase. The major 291-bp product (arrow) corresponds to a transcriptional start site 15 bp upstream of the published exon 1 sequence (Fig. 1).




Figure 4: RNase protection to locate the 5`-most end of the IL-5Ralpha transcript. A plasmid clone containing a 325-bp hybrid cDNA/genomic DNA fragment (Fig. 1) was generated and transcribed with T7 RNA polymerase and [alpha-P]UTP. The probe was hybridized to total RNA from butyrate-induced HL-60-C15 cells and HeLa cells and treated with RNase, and protected fragments were analyzed on a 6% polyacrylamide sequencing gel. The major, largest protected fragment of 148 bp (arrow) corresponds to a transcriptional start site consistent with the primer extension analysis ( Fig. 1and 3).




Figure 5: Nucleotide sequence of the IL-5Ralpha promoter. Open boxes show the positions of exon 1 and part of exon 2 as previously reported. The transcriptional start sites as identified by primer extension, RNase protection, and 5`-RACE are indicated. The consensus start site based on results from all three methods is labeled as +1. Several consensus sequences are indicated for putative TFIID/TBP, and other potential transcription factor binding sites are boxed and shaded. Functionally active promoter regions ( Fig. 7and Fig. 8) are boldly underlined. This genomic sequence has been deposited in the GenBank data base under Accession No. U18373.




Figure 8: Lineage specificity of IL-5Ralpha promoter activity. The -3.5-kb and -561-bp IL-5Ralpha pXP2-luciferase constructs were transfected into eosinophil-inducible (HL-60-C15, AML-14, AML-14.eos), myeloid (HL-60), lymphoid (BJA-B B-cell, REX T-cell), and non-myeloid (HeLa) cell lines. Luciferase activities for each construct and cell line from at least two experiments were measured, and values were corrected using hGH levels to control for differences in transfection efficiency among the various cell lines.



Sequence of the IL-5Ralpha Promoter Region

Two human genomic placental DNA libraries in the FIX II phage vector were screened as described previously(29) . A 4.3-kb KpnI fragment from the ghIL5Ralpha-2 clone (29) was subcloned into the pGEM7ZF(-) plasmid (Promega), and the region from -561 to +51 relative to the transcription start site was sequenced in its entirety on both strands (Fig. 5). The consensus position of the major transcriptional start site as mapped by primer extension, RNase protection, and sequencing of the 5`-RACE products is indicated as +1. Two potential TATA boxes (TFIID/TBP binding sites) were identified by searching the Ghosh transcription factors' data base(48) , as were a number of putative consensus binding sites for GATA-1 (49) immediately upstream of the transcriptional start site. Putative binding sites (consensus sequences) for AP-1 (50) and GATA-1 (51) were likewise identified in regions further upstream, some of which are in regions of the promoter that are functionally active in experiments characterizing the promoter region of the gene (see below).

Localization and Characterization of Functionally Active Promoter Regions

To identify the regulatory sequence elements required for expression of this gene in the eosinophil lineage, we first analyzed promoter activity using transient transfections of reporter (luciferase) gene constructs in the eosinophil-committed HL-60-C15 cell line (Fig. 6). Three constructs were initially prepared in the promoterless pXP2 luciferase (luc) expression vector (Fig. 6A). Transient transfection of the IL-5Ralpha promoter construct containing 3.5 kb of upstream sequence directed the expression of 240-fold greater luciferase activity than the promoterless pXP2 plasmid alone in HL-60-C15 cells (Fig. 6B). This marked promoter activity was essentially ablated when the sequence from bp -561 to +51 was deleted. In contrast, the sequence from bp -561 to +51 reproducibly showed equal or greater promoter activity than the longest 3.5-kb fragment analyzed in the HL-60-C15 cell line. Transient transfection of the series of 5` deletion mutants localized the most functionally active element of the IL-5Ralpha promoter to the 34-bp region between bp -432 and -398, such that 78% of activity was lost when this sequence was deleted (Fig. 7). Of interest, the bp -561 to -377 promoter fragment itself expressed only 20% of the activity of the full-length promoter in the absence of sequence more proximal to the transcriptional start site, suggesting that the proximal region is required for full promoter activity (Fig. 7).

Analysis of the Myeloid and Eosinophil Lineage Specificity of the IL-5Ralpha Promoter

The specificity of the IL-5Ralpha promoter for the eosinophil and other myeloid lineages was assessed by transiently transfecting the -3.5 kb/luc and -561 bp/luc promoter constructs into a variety of leukemic cell lines committed to the eosinophil (HL-60-C15, AML14, AML14.eos), lymphoid (BJA-B, REX), myeloid (HL-60), and non-myeloid (HeLa) lineages as shown in Fig. 8. Both promoter constructs were significantly less active in the parental multipotential HL-60 line than in the eosinophil-committed HL-60-C15 subline. Among the various other cell lines tested, the IL-5Ralpha promoter showed the greatest activity in the AML14.eos acute myeloid leukemia subline that has been differentiated into eosinophils by culture with IL-3, IL-5, and GM-CSF, is comprised of eosinophilic myelocytes and mature eosinophils(36, 37) , and expresses markedly increased amounts of IL-5Ralpha mRNA. (^3)Both IL-5Ralpha promoter constructs were also active in the undifferentiated AML14 parental line, which likewise expresses mRNA for the IL-5Ralpha subunit and a functional IL-5 receptor(36) . The IL-5Ralpha promoter constructs showed significantly reduced activity that was comparable to the parental HL-60 line, in both lymphoid (T and B) and non-myeloid (HeLa cervical carcinoma) cell lines, indicative of basal promoter activity. These results suggest that the region of the IL-5Ralpha gene required for maximal expression of promoter activity possesses both myeloid and possibly eosinophil lineage specificity.

The Region between bp -432 and -377 of the IL-5Ralpha Promoter Binds a Nuclear Factor(s) Present in Myeloid and Eosinophilic, but Not Non-myeloid Cell Lines

As indicated above, the majority of promoter activity was localized to the region between bp -432 and -398. This promoter activity is likely derived from the interaction between DNA binding cis-elements in this region and lineage-specific transcriptional activators expressed by the myeloid and eosinophilic cell lines, but not the non-myeloid or non-hematopoietic lines. To identify the transcription factors in these lines that may form DNA-protein complexes with this region of the promoter, we performed gel mobility shift assays by using a P-end-labeled 93-bp DNA fragment which encompasses the region from bp -469 to -377. Two major DNA-protein complexes, termed C1 and C2, were detected in all nuclear extracts isolated from myeloid and eosinophil lineage cells, including the promyelocytic HL-60 parental line, undifferentiated eosinophil-committed AML-14, eosinophil-committed HL-60-C15, fully differentiated AML-14.eos, and myeloid U937. In contrast, these two complexes were not detected using nuclear extracts from the non-myeloid BJA-B B cells and non-hematopoietic HeLa lines (Fig. 9). In addition, a third complex (termed C3) was detected using nuclear extracts from all the myeloid cell lines tested, from non-hematopoietic HeLa, but not from the BJA-B line (Fig. 9). Formation of all three DNA-protein complexes could be specifically inhibited by excess unlabeled DNA probe. In addition, a longer 184-bp probe (bp -561 to -377) and a shorter 55-bp probe (bp -432 to -377) both produced the identical pattern obtained with the 93-bp probe used in Fig. 9(data not shown). These results suggest that the sequence element(s) involved in DNA binding is likely located in the region between bp -432 and -377.


Figure 9: Gel mobility shift analysis of the IL-5Ralpha promoter sequence using eosinophil, myeloid, and non-myeloid nuclear extracts. A 93-bp PCR-generated probe from bp -469 to -377 of the IL-5Ralpha promoter was labeled with T4 polynucleotide kinase and used for gel shift analysis. Two µg of crude nuclear extract from the indicated cell lines were mixed with 4 µg of poly(dI-dC) and 1 times 10^4 cpm of P-labeled DNA fragment and incubated 20 min at room temperature. Two DNA-protein complexes (C1 and C2, arrows) were identified using nuclear extracts from HL-60-C15, HL-60, AML14, AML14.eos, and U937 cell lines, but not with extracts from BJA-B or HeLa cells. A third specific complex (C3, arrow) was identified using nuclear extracts from all cell lines including HeLa, with the exception of BJA-B. Labeled free DNA probe is indicated (F). A 50-fold molar excess of the identical unlabeled DNA probe added as cold competitor completely inhibited the formation of the C1, C2, and C3 complexes.




DISCUSSION

Little is currently known regarding the mechanisms by which human cytokine and growth factor receptor genes are expressed and regulated during the commitment and differentiation of hematopoietic progenitors to the myeloid lineages in general or the eosinophil lineage in particular. In addition to genes encoding the murine (52) and human IL-5Ralpha subunits, the promoters for a number of other hematopoietic growth factor receptor genes including the M-CSF (CSF-1)(53, 55, 65) , G-CSF(56, 57, 66) , and GM-CSFalpha (58, 67) receptors are currently being analyzed. Our isolation of the 5` upstream region of the human IL-5Ralpha gene and identification of functional promoter sequences provides an opportunity to elucidate the cis-acting regulatory elements and possibly unique trans-acting factors that regulate tissue- and differentiation-specific transcription in the eosinophil as opposed to other granulocyte or macrophage/monocyte myeloid lineages.

Prior studies indicate that IL-5 is a late-acting cytokine that demonstrates maximum activity on an eosinophil progenitor pool expanded by the earlier-acting, multipotential cytokines such as IL-3 or GM-CSF (10) . These observations suggest that IL-5, as a tissue-specific cytokine, plays a crucial role in regulating the development of the eosinophil lineage, and that the IL-5Ralpha subunit, as an essential and specific component of this differentiation pathway, is expressed very early in response to GM-CSF, IL-3, or possibly IL-5 itself. (^4)For these reasons, analysis of how expression of the IL-5Ralpha gene is regulated, and what inducible and tissue-specific transcription factors are involved in this process, is extremely pertinent to understanding the mechanisms regulating eosinophil development. Since eosinophil differentiation, maturation, and activation are all regulated in part by IL-5, regulation of the gene encoding the IL-5Ralpha subunit is likewise pertinent to the multiple activities of this and other cytokines on eosinophil function (59) , especially with regard to the mechanisms for specific receptor-mediated signal transduction pathways in eosinophil activation (9) . Studies of the IL-5Ralpha promoter will hopefully lead to the identification of transcription factors uniquely expressed by the eosinophilic granulocyte and should add to our understanding of the molecular basis for the complex cytokine- and growth factor-mediated processes that occur during the commitment of multipotential myeloid progenitors to the eosinophil lineage and subsequent eosinophil development and maturation. These regulatory mechanisms are also likely to be important both to the development of eosinophilia and to the functional activation of eosinophils in tissues in eosinophil-associated allergic, parasitic, inflammatory, and other diseases.

The cDNA sequence and genomic structure of the human IL-5Ralpha subunit gene were reported previously(27, 28, 29) . However, the complete sequence of the 5`-untranslated region, transcriptional start site, and promoter region had not been determined. The 5`-untranslated region of the gene was originally reported to be derived in its entirety from exons 1-3 (29) . In contrast, Murata et al.(28) reported an additional 18-bp sequence at the 5`-most end of their eosinophil-derived cDNA that was not present in our own HL-60-C15-derived cDNA clones(27) , as well as alternative splicing which removes exon 3 in all cDNA clones we obtained from RNA of HES patient eosinophils or HL-60-C15 cells. These conflicting results made the identification of the transcriptional start site an imperative prior to any attempt to localize and characterize the promoter region of the gene, especially since there was a possibility of the existence of an additional exon (putative exon 0). To resolve this issue, we first isolated the IL-5Ralpha 5`-untranslated sequence from RNA samples obtained from both HL-60-C15 cells and eosinophils purified from a patient with HES using RT-PCR. These experiments showed that only an oligonucleotide primer identical in sequence with the 5`-most end of exon 1 (27, 29) and not putative exon 0 (28) was able to generate an appropriately sized DNA fragment. In addition, we also failed to detect any homology of the putative exon 0 within 3.5 kb of the upstream sequence of the IL-5Ralpha genomic DNA. These findings are consistent with the conclusion that there is no putative exon 0 sequence in the 5`-untranslated region of the IL-5Ralpha gene as transcribed in either the butyrate-induced eosinophilic HL-60-C15 cell line or HES patient eosinophils. Sequence analyses also indicated that exon 3 was missing entirely from the 5`-untranslated region in mRNA samples from both of these sources. Tavernier and co-workers (29) also noted a cDNA variant from butyrate-induced HL-60-C15 cells in which there was an absence of exon 3; however, only 1 in 8 of the fully characterized cDNA clones isolated from their butyrate-induced HL-60-C15 library was missing this exon(27) . One potential explanation for these observations may be variability in the copy number of the exon 3-containing RNA isoform originally reported (27) or differences in the patterns of alternative splicing in HL-60-C15 cells maintained and induced with butyrate in different laboratories.

We have carefully mapped the transcriptional start site of the IL-5Ralpha gene using three complementary techniques. Primer extension produced two cDNA fragments of 291 bp and 332 bp in length. Both species were consistent with additional sequences 5` of the published end of exon 1 (27) although the longer 332-bp fragment was likely a primer extension artifact, perhaps resulting from the 3` end of the cDNA forming a loop by folding back on its own template and serving as a primer for (limited) second strand synthesis. These extra nucleotides were either from an additional short exon further upstream or from the genomic sequence immediately upstream of exon 1. To distinguish these possibilities, we performed both RNase protection and 5`-RACE; RNase protection used an artificial probe containing half of exon 2, all of exon 1, and the genomic sequence immediately upstream of exon 1 (Fig. 1). Results from RNase protection were consistent with an additional 15 bp of sequence upstream of the published end of exon 1, as suggested by primer extension. However, these results could not exclude the possibility of an additional small exon further upstream in the gene. To address this issue, we used RACE to specifically amplify the 5` ends of IL-5Ralpha cDNAs prepared from RNA of butyrate-induced HL-60-C15 cells. Sequences from the 4 longest clones obtained by the RACE procedure were identical with the 15 bp of genomic sequence immediately upstream of the published end of exon 1, providing confirmation of the major transcriptional start site suggested by both primer extension and RNase protection; no longer cDNA species were found. Taken together, these results map the major transcriptional start site to a position approximately 15 bp upstream of exon 1, albeit at a site that lacks a canonical CANYYY CAP site(60) .

Activity of the promoter for the IL-5Ralpha gene was analyzed in the eosinophil-inducible HL-60-C15 cell line. Transient transfections with the IL-5Ralpha promoter constructs suggested that most of the sequence elements required for maximum promoter activity were located within a 561-bp region immediately upstream of the transcriptional start site. A series of smaller, 5` deletions in this region were generated using the -561/luc plasmid to more closely map the minimal sequence elements required for promoter activity. Results from transient transfection of these mutants in HL-60-C15 cells indicated that the region between -561 and -398 bp contributed 75% of maximal promoter activity. Of interest, when a bp -561 to -377 fragment was cloned into the promoterless pXP2-luciferase vector, it expressed only 20% of total promoter activity suggesting that both the distal and proximal regions of the promoter are required for full promoter activity. Two potential TATA boxes (TFIID/TBP binding sites) located in the bp -23 to -79 region (Fig. 5) were identified by searching the transcription factor data base(48) ; a TFIID site is critical for RNA II polymerase activity (61, 62) . Since the distal region of the IL-5Ralpha promoter contains the DNA sequences required for functional activity, it is likely that the promoter requires transactivation by transcription factors which bind either directly or indirectly to both this and the more proximal TFIID binding regions independently(62) . In this regard, the functional cis-elements in the distal region of the promoter are likely present within the small 34-bp region from bp -432 to -398 (Fig. 7), for which the nucleotide sequence does not contain any previously identified transcription factor binding sites(48) . Using electrophoretic mobility shift assays, we identified a nuclear factor or factors that bind specifically to this region of the promoter and that are present in the myeloid and eosinophilic cell lines in which the promoter was functionally active, but absent in the non-myeloid and non-hematopoietic lines for which the promoter was significantly less active. Thus, this functional segment likely serves as a binding site for lineage-specific transcription factor(s) required for optimal expression of the IL-5Ralpha gene. Whether the two DNA-protein complexes consistently observed in electrophoretic mobility shift experiments with this promoter region (Fig. 9) represent distinct nuclear factors, proteolytic cleavage or post-translational modification of a single factor, or binding by a heterodimer is unclear pending further biochemical characterization or cloning.

An analysis of the lineage specificity of the IL-5Ralpha promoter in eosinophilic and other myeloid and non-myeloid leukemic cell lines (Fig. 8) suggests that the 561-bp upstream region of the gene required for maximal expression of promoter activity possesses both myeloid and possibly eosinophil specificity. Among the various eosinophil cell lines tested, the IL-5Ralpha promoter showed greatest activity in AML14.eos, a subline of AML14 that has been differentiated into eosinophilic myelocytes and mature eosinophils by induction with a combination of IL-3, IL-5, and GM-CSF(37) , expresses increased amounts of IL-5Ralpha mRNA, and continues to proliferate in culture and maintain the differentiated phenotype with cytokine supplementation.^3 Similarly, the IL-5Ralpha promoter showed greater activity in the eosinophil-inducible HL-60-C15 line (in which 5-20% of the cells spontaneously become granulated in culture (38) ) than the undifferentiated parental HL-60 line (Fig. 8). Of interest, the IL-5Ralpha promoter also showed high levels of activity in U937, a myelomonocytic cell line, with mean levels of 13,631 ± 4,700 RLU/ng/ml hGH. Other myeloid promoters analyzed thus far in our laboratories including those for the eosinophil peroxidase (35) and CLC protein (34) genes have likewise shown particularly high levels of activity in the U937 line for reasons that are as yet unclear but may relate to a particularly high transfection efficiency for this monocytoid leukemic cell line. Further, the U937 cell line contained the same nuclear factor(s) that formed specific protein-DNA complexes with the IL-5Ralpha promoter in the gel shift analyses (Fig. 9).

The structure of the murine IL-5Ralpha subunit gene was recently published(52) . In contrast to the human IL-5Ralpha subunit's unique expression and activity in only the eosinophil lineage(10) , the murine gene is also expressed and functional in B cells(52) . Comparison of the promoter regions and transcription factors regulating the differential expression of the human versus murine genes in eosinophil only versus eosinophil and B cell lineages(10, 63) , respectively, could provide insights into their respective mechanisms of expression and regulation. While the upstream, 5`-flanking region of the murine IL-5Ralpha gene contains consensus sequences for Ap1, AP-1, GATA-1, and PU.1, these sequences have not as yet been analyzed for functional activity(52) . In the limited functional analysis of the murine promoter published thus far(52) , 256 bp of the 5`-flanking region (-96 to +160 bp) exhibited minimal promoter activity in a pCAT vector in fibroblast (NIH3T3), FDC-P1, and IL-5-dependent pre-B cell (Y16) lines. Of interest, analysis of a larger 1.5-kb construct (-1371 to +160) to find a region that directs B cell-specific expression, failed to detect any promoter activity in any of the murine lines tested, suggesting suppressive elements in this region, in marked contrast to the results we have obtained for the human IL-5Ralpha promoter with up to 3.5 kb of upstream sequence. The murine gene also contains a 52-bp AC-rich Z-type DNA sequence (-445 to -394), which is also found in the IL-2Rbeta upstream region, but is absent in the human IL-5Ralpha gene in the first 576 bp of upstream DNA we have sequenced thus far. Alignments we have performed on the human and murine upstream regions did not show any significant sequence similarities within the functionally defined region of the human IL-5Ralpha promoter, i.e. bp -561 to +51. A comparison of the human IL-5Ralpha upstream sequence to the 5`-flanking sequences of the human GM-CSFRalpha gene (58) also failed to identify any regions of significant similarity in the functionally active segments of the IL-5Ralpha promoter. These observations indicate important differences in the regulatory regions of the human versus murine IL-5Ralpha and human GM-CSFRalpha genes that are likely relevant to their differential expression in eosinophil versus B cell lineages in the two species and granulocyte/macrophage lineages in humans, respectively. Finally, the promoters for a number of other genes preferentially expressed in the myeloid series, including CD11b(54) , M-CSF(53) , and CD14 (44) have been shown to require either PU.1 and/or Sp1 (44) binding for myeloid-specific expression. However, the functionally active region of the IL-5Ralpha promoter we have identified lacks consensus PU.1 or Sp1 binding sites, and the bp -561 to +51 region of the promoter does not show any binding of in vitro-transcribed PU.1 transcription factor in electrophoretic mobility shift assays. (^5)However, these analyses do not exclude the presence of a nonconsensus, weak, but functionally significant PU.1 binding site in the promoter.

Our current studies have served to identify a functionally active promoter region for the human IL-5Ralpha gene that is myeloid- and relatively eosinophil lineage-specific in its expression in eosinophil-inducible leukemic cell lines in vitro. In addition, we have shown that this region binds a nuclear factor(s) expressed in the same myeloid and eosinophilic lines in which the promoter is functionally active. Studies to more precisely map the functional elements in this region and to characterize and clone the cognate transcription factors that bind to this region of the IL-5Ralpha promoter and are required for optimal and eosinophil-specific activity in vitro and in vivo are currently in progress.


FOOTNOTES

*
This work was supported in part by NIAID Grant AI33043 (to S. J. A.), an American Lung Association research grant (to Z. J. S.), NCI Grant CA41456 (to D. G. T.), and Veterans Administration merit review grants (to C. C. P. and M. A. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Edward Livingston Trudeau Scholar of the American Lung Association.

Scholar of the Leukemia Society of America.

**
To whom correspondence and reprint requests should be addressed: Infectious Disease Division, RE219, Beth Israel Hospital, 330 Brookline Ave., Boston, MA 02215. Tel.: 617-735-4355; Fax: 617-735-3299.

(^1)
The abbreviations used are: IL, interleukin; GM-CSF, granulocyte-macrophage colony stimulating factor; IL-5Ralpha, IL-5 receptor alpha subunit; CLC, Charcot-Leyden crystal protein; RLU, relative light units; bp, base pair(s); kb, kilobase(s); RT, reverse transcription; PCR, polymerase chain reaction; WT, wild type; hGH, human growth hormone; CMV, cytomegalovirus; HES, idiopathic hypereosinophilic syndrome.

(^2)
J. Tavernier, unpublished results.

(^3)
C. C. Paul and M. A. Baumann, unpublished observations.

(^4)
Z. Sun, D. G. Tenen, and S. J. Ackerman, unpublished results.

(^5)
Z. Sun, D. A. Yergeau, D. G. Tenen, and S. J. Ackerman, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. René Devos for scientific input and editorial suggestions and Mary Singleton for secretarial and administrative assistance in the preparation of this manuscript.


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