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The Journal of Immunology, 1998, 160: 4427-4432.
Copyright © 1998 by The American Association of Immunologists

Selective Inhibition of IL-5 Receptor {alpha}-Chain Gene Transcription by IL-5, IL-3, and Granulocyte-Macrophage Colony-Stimulating Factor in Human Blood Eosinophils

Peng Wang1, Ping Wu, Boonlert Cheewatrakoolpong, Joyce G. Myers, Robert W. Egan and M. Motasim Billah

Allergy Department, Schering-Plough Research Institute, Kenilworth, NJ 07033


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
High affinity receptor for IL-5 (IL-5R), a predominant eosinophil maturation factor, is composed of an IL-5-binding {alpha}-chain (IL-5R{alpha}) and a signal-transducing ß-chain that is shared by IL-3 and granulocyte-macrophage CSF (GM-CSF) receptors (IL-3R and GM-CSFR). By Northern blot analysis of mRNAs obtained from normal human blood eosinophils, we show in this report that the hematopoietic cytokines IL-5, IL-3, and GM-CSF down-regulate IL-5R{alpha} mRNA while up-regulating {alpha}-chain mRNAs for both IL-3R and GM-CSFR as well as the ß-chain mRNA. More detailed characterization reveals that the down-regulation of IL-5R{alpha} mRNA is specific to IL-3, IL-5, and GM-CSF; occurs very rapidly (reaching maximum inhibition within 2 h); is cytokine dose dependent; and does not require protein synthesis. Nuclear run-on and mRNA stability experiments demonstrate that cytokine-induced inhibition of IL-5R{alpha} mRNA accumulation occurs at the level of IL-5R{alpha} gene transcription, whereas enhanced accumulation of mRNAs for IL-3R{alpha} and the ß-chain results from reduced mRNA degradation. We suggest from these experiments that in human blood eosinophils, IL-5R{alpha} gene transcription and IL-5R{alpha} mRNA metabolism can be regulated by mechanisms that are distinct from those used for IL-3R{alpha} and GM-CSFR{alpha}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin-5, a cytokine produced by several cell types including Th2 cells, mast cells, and eosinophils, is a potent differentiation and activation factor for eosinophilic lineage. IL-5 can act either alone or in conjunction with other hematopoietic cytokines such as granulocyte-macrophage CSF (GM-CSF)2 and IL-3 to stimulate the proliferation and differentiation of eosinophilic progenitors to produce eosinophils in the bone marrow. For mature eosinophils, IL-5 causes cell migration into tissues and release of granule proteins and supports cell survival (for review, see Refs. 1 and 2). Eosinophils are important inflammatory cells, particularly in asthma and other allergic diseases (3, 4, 5). Therefore, IL-5 is of great interest in terms of therapeutic intervention in asthma and other inflammatory conditions (6, 7).

The differentiation of eosinophils in the bone marrow requires several cytokines, in addition to IL-5, including IL-3 and GM-CSF (1, 2, 8). Both IL-3 and GM-CSF stimulate various lineages of hematopoietic cells (9, 10), whereas IL-5 is mainly an eosinophil lineage-specific factor (1, 2, 6, 7). In humans, the high affinity receptor for IL-5 is apparently restricted to eosinophils and hematopoietically related basophils (1). This restricted expression of the high affinity receptor for IL-5 (IL-5R) determines the lineage specificity of IL-5 (11). Because the IL-5R{alpha} subunit is restricted to only eosinophil/basophil lineages, its expression would seem to be tightly regulated, perhaps by cell type-specific transcriptional mechanisms.

The high affinity IL-5R consists of two subunits: a unique {alpha} subunit (IL-5R{alpha}) binding the cytokine with a low affinity and a ß subunit shared by IL-3 and GM-CSF receptors (1). Although the ß-chain does not bind the cytokines by itself, it interacts with the {alpha}-chains to form high affinity receptors and acts as a signal transducer. Because of the sharing of the signal transducer, IL-5, IL-3, and GM-CSF show a similar pattern of functional responses (1). Like many other cytokines, IL-5 binding to its receptor results in the activation of JAK-STAT pathway, and more specifically, IL-5 activates JAK2 and STAT1{alpha} in human eosinophils (12, 13). Recently, evidence for a signaling role of IL-5R {alpha}-chain has also been presented (14), adding further complexity to IL-5R signaling mechanisms.

Human IL-5R{alpha} cDNAs have been isolated (15, 16, 17), and the intron-exon organization of IL-5R{alpha} gene has been published (18). Recently, human eosinophil IL-5R{alpha} gene promoter has been isolated and characterized (19). Several alternatively spliced transcripts have been identified that reflect the membrane-bound vs soluble isoforms (17). One of the mRNA species for the soluble forms of receptor is the major transcript expressed in human eosinophilic HL-60 cells and in eosinophils grown from human cord blood (15, 16). The soluble IL-5R binds IL-5 and neutralizes the biologic activity of the cytokine in vitro (20, 21), suggesting that the soluble receptor may play a role in the immunoregulation of eosinophilia in vivo.

Our objective in the present study was to identify the mechanisms by which IL-5R{alpha} gene expression is regulated in normal human blood eosinophils. To our knowledge, no study on the regulation of IL-5R{alpha} gene expression in blood eosinophils has been reported. This lack of data on IL-5R{alpha} mRNA regulation might well have been due to the fact that it is difficult to obtain large quantities of eosinophils from normal human blood. We have established a method for the preparation of normal human blood eosinophils in large quantities from healthy donors and have investigated, in the present report, the effects of various cytokines on the expression of IL-5R{alpha} mRNA by using Northern blot analysis. Of various cytokines tested, including IL-5, IL-3, GM-CSF, IL-1, TNF, IFN-{gamma}, granulocyte CSF (G-CSF), and stem cell factor (SCF), none was found to up-regulate the level of IL-5R{alpha} mRNA in eosinophils. Interestingly, however, the hematopoietic cytokines IL-5, IL-3, and GM-CSF caused a specific down-regulation of the IL-5R{alpha} mRNA through a mechanism involving IL-5R{alpha} gene transcription.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of human blood eosinophils

Normal human blood eosinophils were prepared from leukocyte-rich buffy coat of blood from healthy donors as described by Cramer et al. (22) with some modifications. Briefly, after the removal of erythrocytes by sedimentation, the buffy coat was diluted with an equal volume of PBS-BSA-citrate solution and centrifuged. The cell pellet was resuspended in 40 ml of a Percoll (Pharmacia, Piscataway, NJ) solution with a density of 1.085 g/ml. Ten milliliters of this cell suspension was stratified between a 2.5-ml cushion of a 1.1 g/ml Percoll solution and a 1.5-ml overlay of PBS-BSA-citrate in a 16 x 100-mm polyethylene tube. After centrifugation at 1000 x g for 20 min at 20°C, eosinophils at the interface of the two Percoll solutions were recovered, and the remaining erythrocytes were removed by hypotonic lysis. Eosinophil preparations thus obtained were more than 90% pure, and most of the contaminating cells were neutrophils. Typically, we obtained approximately 150–200 x 106 eosinophils from one preparation using 15 units of buffy coats.

Cell stimulation

Cells were suspended at a density of 1 x 106/ml in RPMI 1640, which was supplemented with 1% penicillin, 1% streptomycin, and glutamine (all from Life Technologies, Grand Island, NY) and with 10% heat-inactivated FCS (HyClone, Logan, UT). The cell suspension (approximately 30 x 106 per sample) was incubated with appropriate cytokines at 37°C in a humidified atmosphere of 5% CO2/95% air for specified periods. Human recombinant IL-3, IL-5, and GM-CSF were obtained from Upstate Biotechnology (Lake Placid, NY). IFN-{gamma} was from Boehringer Mannheim (Indianapolis, IN). Other cytokines were from R&D Systems (Minneapolis, MN).

Northern blot analysis

Steady state levels of receptor mRNAs in cells after various treatments were determined by Northern blot analysis as described previously (23). cDNA probes for IL-5R{alpha} and the common ß-chain were obtained from Drs. Maria Wiekowski and Chuan-chu Chou (Schering-Plough Research Institute, Kenilworth, NJ), and those for IL-3R{alpha} and GM-CSFR{alpha} were from Dr. Robert Kastelein (DNAX Research Institute, Palo Alto, CA). A sample containing approximately 10 µg of total RNA (obtained from about 30 x 106 cells) was subjected to Northern blot analysis. Autoradiographic exposure was for 2 to 5 days at -70°C with two intensifying screens.

mRNA stability analysis

mRNA stability analysis was performed as described previously (23).

Nuclear run-on assay

Nuclear run-on gene transcription assays were performed as described previously (23).

IL-5 binding assay

Human IL-5 (Schering-Plough Research Institute) was labeled with 125I by the Iodogen method (24). The average specific activity of 125I-labeled IL-5 was approximately 190 µCi/µg, and free iodide was less than 5%. The IL-5 binding assay was performed by using the MultiScreen system (Millipore, Bedford, MA). Cells suspended in RPMI 1640 containing 5% heat-inactivated FCS and 0.02% sodium azide were transferred to a 96-well MultiScreen-DV filtration plate (0.4 x 106 cells in 0.2 ml/well) and incubated at 4°C for 2 h with various doses of 125I-labeled IL-5. To measure nonspecific binding, a parallel series of wells containing a 100-fold excess of cold IL-5 was included. The binding was terminated by applying vacuum to the filter plate. Cells trapped on filter were washed four times, each with 200 µl of ice-cold PBS-BSA-citrate solution. Filter discs were dried, punched out, and counted in a gamma counter.

Data presentation

Each set of experiments was performed at least twice, and the data presented are from representative experiments. For the sake of clarity of presentation, some data of Northern blotting are shown as plotted graphs of densitometric quantification of the autoradiographic signals as normalized to ß-actin (control) signals.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Selective reduction of IL-5R{alpha} mRNA in human blood eosinophils by IL-5, IL-3, and GM-CSF

When freshly isolated normal human blood eosinophils from healthy donors were subjected to Northern blot analysis without being stimulated by any cytokine, mRNAs for both IL-5R {alpha}- and ß-chains were detected (Fig. 1GoA). This observation was consistent with the constitutive expression of high affinity IL-5R on human eosinophils (see below and 25 . Two IL-5R{alpha} mRNA bands with m.w.s of about 5 kb and 1.4 kb were detected in human eosinophils. The larger transcript, which accounted for the majority of the message, represents the membrane-bound form, and the smaller one encodes the soluble receptor (15, 16, 17).



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  		FIGURE 1. Effects of various cytokines on levels of IL-5R{alpha}, IL-3R{alpha}, GM-CSF-R{alpha}, and ß-chain mRNAs. Human blood eosinophils were incubated with various cytokines, as specified, at a concentration of 10 ng/ml for 4 h. Total RNA was extracted and subjected to Northern blot analysis. Details are described in Materials and Methods. Three separate experiments (A, B, and C) are presented to show results with various cytokines tested. In each of the experiments, the blot was hybridized sequentially with various cDNA probes, as specified. The differences observed in IL-3R{alpha} mRNA levels of unstimulated eosinophils between experiments shown in A and B may have been due to the use of different cell preparations.

 
When eosinophils were cultured in the presence of specific cytokines known to be involved in eosinophil differentiation and/or activation, IL-5R{alpha} mRNA level was dramatically reduced by IL-3 but not by the others (Fig. 1GoA). Contrary to IL-5R{alpha}, the level of mRNA for the common ß-chain was increased significantly by IL-3 as well as by IFN-{gamma} (Fig. 1GoA). Subsequent experiments revealed that IL-5 and GM-CSF also induced identical response profiles: down-regulation of IL-5R{alpha} mRNA (Fig. 1GoB) and up-regulation of ß-chain mRNA (Fig. 1GoC).

Because IL-3 and GM-CSF are very similar to IL-5 in eliciting biologic activities in eosinophils, the effects of these cytokines on the expression of IL-3R{alpha} and GM-CSFR{alpha} mRNAs were also investigated. As shown in Figure 1Go, A and B, IL-3R{alpha} mRNA level was, in contrast with IL-5R{alpha}, enhanced by treatment of eosinophils with either IL-5, IL-3, or GM-CSF, but not with any of the other cytokines tested. Similar results were obtained for GM-CSFR{alpha} (Fig. 1GoC).

Characteristics of IL-3-induced reduction of IL-5R{alpha} mRNA

To further characterize the cytokine-induced reduction of IL-5R{alpha} mRNA in human eosinophils, IL-3 was used as a stimulus. IL-3 reduced IL-5R{alpha} mRNA level in a dose-dependent manner (Fig. 2Go). Substantial reduction was still observed at an IL-3 concentration as low as 0.01 ng/ml.



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  		FIGURE 2. Effects of IL-3 concentrations on IL-5R{alpha} mRNA level. Cells were incubated with various concentrations, as specified, of IL-3 for 4 h. Total RNA was extracted and then subjected to Northern blot analysis. Details are described in Materials and Methods.

 
IL-3-induced reduction of IL-5R{alpha} mRNA level was rapid. Significant reduction was observed at 2 h after IL-3 addition (Fig. 3Go). Although the mRNA level decreased gradually from 2 h to 6 h in untreated cells (Fig. 3Go), the extent of the decrease due to cytokine treatment remained similar for up to 14 h (data not shown).



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  		FIGURE 3. Time course for change of IL-5R{alpha} mRNA level. Cells were incubated without ({circ}) or with (•) 10 ng/ml of IL-3 for various time periods, as specified. Total RNA was extracted and then subjected to Northern blot analysis. Details are described in Materials and Methods. For the sake of clarity of presentation, autoradiographic signals were quantified by a Bio Image Analyzer (Millipore, Ann Arbor, MI), normalized to ß-actin signals, and then plotted.

 
Treatment of human eosinophils with IL-3 or GM-CSF decreases IL-5 binding sites

Receptor binding experiments showed that there were 337 ± 32 (n = 6) IL-5Rs per cell in our eosinophil preparations and that the binding affinity (kDa) was 122 ± 35 pM (n = 6). These values are very similar to those published by others (25). To examine whether the down-regulation of IL-5R{alpha} mRNA correlates with decrease in IL-5 binding to the cells, human blood eosinophils were treated with IL-3 or GM-CSF overnight, and then the IL-5 binding assay was performed. As shown in Fig. 4Go, 125I-labeled IL-5 binding to cells treated with either IL-3 or GM-CSF decreased by about 70%. This decrease cannot be attributed to the competition of IL-3 or GM-CSF with IL-5 for binding, because GM-CSF or IL-3 at a 300-fold molar excess inhibited 125I-labeled IL-5 binding by only 50% or 15%, respectively (data not shown). Thus, the observed decrease in IL-5 binding sites likely reflected reduced synthesis of IL-5R{alpha} protein. In a separate set of experiments, IL-5R turnover was assessed by measuring 125I-labeled IL-5 binding to human eosinophils that were pretreated with cycloheximide to block new receptor synthesis. The binding was reduced in a time-dependent manner with a half-life of approximately 6 h (data not shown), suggesting that in resting eosinophils, IL-5R does undergo metabolic turnover.



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  		FIGURE 4. Effects of IL-3 and GM-CSF treatment on specific IL-5 binding. Cells were incubated without any cytokine ({circ}), with 10 ng/ml of IL-3 (•), or with 10 ng/ml of GM-CSF ({blacksquare}) for 14 h. After the treatment, cells were washed to remove the cytokines, and then the IL-5 binding assay was performed. Details are described in Materials and Methods. Specific binding was determined after subtraction of nonspecific binding from total binding.

 
Cytokine-induced down-regulation of IL-5R{alpha} mRNA in eosinophils occurs at the transcriptional level

When human blood eosinophils were incubated with actinomycin D at concentrations of 2.5 to 5 µg/ml for 2 h, the IL-5R{alpha} mRNA accumulation was completely inhibited (Fig. 5Go), indicating that IL-5R{alpha} mRNA has a high turnover rate. Treatment of eosinophils with cycloheximide (5 µg/ml) had no effect on IL-3-induced reduction of IL-5R{alpha} mRNA (Fig. 5Go), suggesting that protein synthesis is not required for cytokine-induced down-regulation of IL-5R{alpha} mRNA.



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  		FIGURE 5. Effects of actinomycin D on IL-5R{alpha} mRNA level and of cycloheximide on IL-3 inhibition of IL-5R{alpha} mRNA accumulation. Cells were incubated without any agent (lane 1) or with 10 ng/ml of IL-3 (lane 2), 5 µg/ml of actinomycin D (lane 3), 2.5 µg/ml of actinomycin D (lane 4), 5 µg/ml of cycloheximide (lane 5), or 5 µg/ml of cycloheximide plus 10 ng/ml of IL-3 (lane 6) for 2 h. Total RNA was extracted and then subjected to Northern blot analysis. Details are described in Materials and Methods. A, Raw data of Northern blot analysis; B, Autoradiographic signals were quantified by the Bio Image Analyzer, normalized to ß-actin signals, and then plotted.

 
To determine whether the down-regulation of IL-5R{alpha} mRNA occurs at the level of transcriptional inhibition, nuclear run-on gene transcription assays were performed. Nuclear preparations from unstimulated cells produced IL-5R{alpha} mRNA (Fig. 6Go), confirming that IL-5R{alpha} gene is constitutively expressed in human eosinophils. This constitutive gene transcription was inhibited by IL-3. On the other hand, IL-3R{alpha} gene transcription, which also occurs constitutively, was not affected by IL-3 stimulation (Fig. 6Go). Thus, IL-3-induced reduction of IL-5R{alpha} mRNA involves transcriptional inhibition.



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  		FIGURE 6. Effects of IL-3 on the transcription of IL-5R{alpha} and IL-3R{alpha} genes. Cells were incubated without (lane 1) or with (lane 2) 10 ng/ml of IL-3 for 1 h. Nuclei were isolated, and in vitro nuclear run-on transcription assays were performed as described in Materials and Methods. A, Raw data of nuclear run-on assay; B, Autoradiographic signals were quantified by the Bio Image Analyzer, normalized to ß-actin signals, and then plotted. This assay was repeated twice, with an average inhibition rate of 64%.

 
To determine whether IL-3 treatment affects IL-5R{alpha} mRNA degradation, mRNA levels at 0, 1, and 2 h after actinomycin D addition were quantified. IL-3 was added 1 h before actinomycin D. There was no major effect of IL-3 treatment on IL-5R{alpha} mRNA disappearance, whereas the disappearance of ß-chain mRNA was significantly inhibited in the presence of IL-3 (Fig. 7Go). Thus, IL-3-induced reduction of IL-5R{alpha} mRNA is mediated through transcriptional suppression and does not involve mRNA degradation.



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  		FIGURE 7. Effects of IL-3 on the degradation of IL-5R{alpha} and ß-chain mRNAs. Cells were incubated without ({circ}) or with (•) 10 ng/ml of IL-3 for 1 h before adding actinomycin D at a final concentration of 5 µg/ml to stop RNA synthesis. At 1 and 2 h after actinomycin D addition, total RNA was extracted and then subjected to Northern blot analysis. Autoradiographic signals were quantified by the Bio Image Analyzer. The plotted values, normalized to ß-actin signals, represent the percentage of mRNA present at the time of actinomycin D addition.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates that hematopoietic cytokines, including IL-5, IL-3, and GM-CSF, that transduce their signals through a common ß-chain down-regulate IL-5R{alpha} mRNA in freshly isolated mature human eosinophils while up-regulating IL-3R{alpha}, GM-CSFR{alpha}, and ß-chain mRNAs. Several other cytokines that are potent eosinophil activators (IL-1, TNF, and IFN-{gamma}; Refs. 26–28) or are important differentiation factors for early stage eosinophilic progenitors (SCF and G-CSF; Refs. 29–33) do not elicit these responses. The distinct behavior of the IL-5R{alpha} gene to common signals generated by the activated ß-chain suggests that IL-5R{alpha} gene in eosinophils is regulated differently from IL-3R{alpha} and GM-CSFR{alpha} genes. This suggestion is consistent with the fact that IL-5R{alpha} gene expression is restricted to eosinophils, whereas IL-3R{alpha} and GM-CSFR{alpha} genes are expressed by various hematopoietic cells. Furthermore, a recent study has observed remarkable similarities between IL-3R{alpha} and GM-CSFR{alpha} genes, which, however, differed significantly from the IL-5R{alpha} gene (34), further underscoring the uniqueness of IL-5R{alpha}. Nevertheless, the molecular basis of the differential regulation between IL-5 and IL-3/GM-CSF receptor {alpha} genes remains to be elucidated.

It is clear from nuclear run-on assays and mRNA stability analyses that cytokine-induced down-regulation of IL-5R{alpha} mRNA is mediated through transcriptional inhibition. The rapidity with which the response takes place and lack of cycloheximide effect on this response are consistent with an involvement of pre-existing transcription factors, such as STAT. Hematopoietic cytokines activate STAT (35), but the recently identified IL-5R{alpha} gene promoter has no consensus sequence for STAT (19). Nevertheless, our present results define a paradigm that may allow identification of novel components of IL-5 gene transcription machinery. As to IL-3R{alpha} (probably also GM-CSFR{alpha}) and the ß-chain, cytokine stimulation of mature blood eosinophils does not affect gene transcription but enhances the stability of the messages formed constitutively (Figs. 6Go and 7Go). Similar up-regulation of the ß-chain mRNA through message stability has been previously reported in IFN-{gamma}-stimulated human blood monocytes (36).

Consistent with our current finding in eosinophils is the observation that in HL-60 eosinophilic cells, IL-5R number decreases upon stimulation with GM-CSF, IL-3, or IL-5 (37, 38). In the same cells, we also find that IL-5, IL-3, or GM-CSF down-regulates IL-5R{alpha} mRNA level (P. Wang et al., unpublished observation). However, in erythroleukemic TF-1 cells and eosinophils from hypereosinophilic patients, similar stimulation causes either no change or up-regulation of IL-5R (39, 40). Reasons for this discrepancy are not clear. It is, however, noteworthy in this context that our present eosinophil preparation from healthy donors produces the mRNA encoding the membrane-bound {alpha} subunit in large excess over the mRNA encoding the soluble form and that the latter mRNA predominates in TF-1 erythroleukemia cells and in eosinophils from hypereosinophilia patients (15). Thus, normal eosinophils differ from abnormal populations in ways they handle IL-5R{alpha} gene transcriptional and posttranscriptional processes.

Down-regulation of cytokine receptors by their natural ligands has been widely reported. For example, it was shown that receptors for SCF in human myeloid leukemia cells (41, 42, 43, 44) and CD34+ cells (44), GM-CSF and IL-3 in human CD34+ cells (44), IL-1 in human articular chondrocytes (45), and IL-6 in mouse myelomonocytic leukemic cells (46) and human monocytes (47) were down-regulated by the respective cytokines. In addition, many cytokine receptors are down-regulated by nonligand cytokines that mimic natural ligands in eliciting biologic responses. Examples include SCFR down-regulation by GM-CSF in human myeloid leukemia cells (43), IL-1R down-regulation by TNF in human articular chondrocytes (45), IL-2R down-regulation by IL-4 in mouse T and B cell lines (48), IL-6R down-regulation by leukemia inhibitory factor in mouse myelomonocytic leukemic cells (46), G-CSFR down-regulation by GM-CSF or IL-3 (49), and macrophage CSF receptor down-regulation by GM-CSF or IL-3 (50) in mouse bone marrow cells. The molecular basis and biologic role of this intriguing phenomenon remain to elucidated.

As has been proposed for the IL-6/IL-6R system (46), the IL-5R down-regulation in human blood eosinophils by hematopoietic cytokine receptor system may prevent unnecessary ligand binding, thereby ensuring preservation and efficient use of IL-5. Despite drastic reduction of IL-5R expression, eosinophils remain fully responsive to IL-5, suggesting that only a limited number of the receptors is sufficient for the mediation of IL-5 action. In view of the crucial role of IL-5 in the terminal differentiation of eosinophils, a minor component of the total leukocyte population, it may be speculated that IL-5R down-regulation might be involved in the maintenance of eosinophil homeostasis.

IL-3R, in contrast to IL-5R, is up-regulated by IL-3, IL-5, and GM-CSF in human eosinophils, although it was reported to be down-regulated by IL-3 in human CD34+ cells (44). In fact, similar differential regulation in different cell types has been observed with some other cytokine receptors. For example, IL-4R was down-regulated in human renal cell carcinoma cells (51) but up-regulated in T and B lymphocytes (52) by IL-4. Thus, one cytokine receptor can be up- as well as down-regulated by its ligand, depending on the particular cell type.

In conclusion, the present study shows in normal human blood eosinophils that hematopoietic cytokines IL-3, IL-5, and GM-CSF down-regulate IL-5R{alpha} mRNA while up-regulating IL-3R{alpha} and GM-CSFR{alpha} mRNAs and that the IL-5R{alpha} mRNA down-regulation occurs through a mechanism involving transcriptional inhibition. One important implication of these data is that IL-5R{alpha} gene transcription and IL-5R{alpha} mRNA metabolism are regulated by mechanisms that are distinct from those used for IL-3R{alpha} and GM-CSFR{alpha}.


   Acknowledgments
 
We thank Drs. Maria Wiekowski and Chuan-chu Chou (Schering-Plough Research Institute) for cDNA probes of IL-5R{alpha} and the common ß-chain and Dr. Robert Kastelein (DNAX Research Institute) for cDNA probes of IL-3R{alpha} and GM-CSFR{alpha}.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Peng Wang, Schering-Plough Research Institute, K-15-1600, 2015 Galloping Hill Road, Kenilworth, NJ 07033. E-mail address: Back

2 Abbreviations used in this paper: GM-CSF, granulocyte-macrophage colony-stimulating factor; G-CSF, granulocyte colony-stimulating factor; SCF, stem cell factor. Back

Received for publication July 18, 1997. Accepted for publication January 7, 1998.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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