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Published ahead of print on June 28, 2007, doi:10.1165/rcmb.2006-0412OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 37, pp. 571-577, 2007
© 2007 American Thoracic Society
DOI: 10.1165/rcmb.2006-0412OC

Regulation of Functional Phenotypes of Cord Blood–Derived Eosinophils by {gamma}-Secretase Inhibitor

Jin Hyun Kang1, Da Hye Lee1, Hyemyung Seo1, Jong Sook Park2, Key Hyun Nam3, Soon Young Shin1, Choon-Sik Park2 and Il Yup Chung1

1 Division of Molecular and Life Sciences, College of Science and Technology, Hanyang University, Ansan, South Korea; 2 Genome Research Center for Allergy and Respiratory Diseases, Division of Allergy and Respiratory Medicine, and 3 Department of Obstetrics and Gynecology, Soonchunhyang University Hospital, Bucheon, South Korea

Correspondence and requests for reprints should be addressed to Il Yup Chung, Ph.D., Division of Molecular and Life Sciences, College of Science and Technology, Hanyang University, 1271 Sa-1-dong, Ansan, Gyeonggi-do 426-791, South Korea. E-mail: iychu{at}hanyang.ac.kr


   Abstract
Top
 Abstract
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Eosinophils develop from stem cells in the bone marrow under the influence of hematopoietic cytokines, particularly IL-5. Previously, we have demonstrated that blockage of Notch signaling by a {gamma}-secretase inhibitor (GSI) promotes the differentiation of umbilical cord blood (UCB)–derived eosinophils. These highly major basic protein (MBP)–positive eosinophils cultured in the presence of the inhibitor lack the migratory response to eotaxin, although their CCR3 levels are similar to those of eosinophils cultured without the inhibitor. We investigated the mechanism underlying the differential responses of differentiating eosinophils and their functionalities in response to eosinophil-active cytokines in the presence and absence of GSI. UCB cells cultured for 4 weeks with hematopoietic cytokines in the presence or absence of GSI were monitored for extracellular signal–regulated kinase (ERK) phosphorylation, MBP expression, and functionality. Eosinophil differentiation from UCB cells was accompanied by activation of the ERK1/2 pathway during the 4-week culture period. In particular, strong ERK1/2 phosphorylation was observed in eosinophils during the final stage of culture when GSI was present. Consistent with this finding, ERK inhibition nullified the effect of GSI on eosinophil differentiation. Eosinophils cultured with GSI resembled airway eosinophils rather than peripheral blood eosinophils based on reduced IL-5R{alpha} expression, blunted eosinophil cationic protein (ECP) degranulation, and decreased IL-13 and granulocyte macrophage–colony-stimulating factor production. These results suggest that Notch signaling regulates the terminal differentiation and subsequent effector phenotypes of eosinophils, partly through modulation of the ERK pathway. GSI has therapeutic potential for eosinophilic inflammatory diseases, such as asthma.

Key Words: CD44 • eosinophil • {gamma}-secretase inhibitor • IL-5R{alpha} • Notch

Eosinophils are granulocytes that function as the principal effectors and immunomodulatory cells of allergic inflammatory diseases, such as asthma (1). Eosinophil development is controlled by cytokines, which include IL-3, granulocyte macrophage–colony-stimulating factor (GM-CSF), and IL-5 (2, 3). CD34+ cells in the bone marrow (BM) are committed to the eosinophil lineage, which has the CD34+IL-5R{alpha}+ phenotype (4, 5). Eosinophils and their progenitor cells further differentiate in the BM, migrate into the blood, and are subsequently recruited into sites of allergic inflammation, where the eosinophil progenitor cells further undergo in situ differentiation (6–8). Although autocrine IL-5 production by CD34+ cells induces up-regulation of IL-5R{alpha} expression, IL-5R{alpha} is believed to be expressed as a result of commitment to the eosinophilic lineage (9), although the signals that induce IL-5R{alpha} expression are largely unknown.

The mitogen-activated protein kinase (MAPK) pathway plays central roles in eosinophil differentiation (10), survival (11), chemotaxis (12, 13), and degranulation (12). Pharmacologic approaches have demonstrated that extracellular signal–regulated kinase (ERK)1/2 is essential for the differentiation of BM-derived eosinophils (10). Consistent with this notion, a recent study has shown that Spred-1, which is an endogenous negative regulator of Ras-dependent ERK activation, suppresses IL-5–dependent eosinophil production by modulating IL-5 signaling (14).

The Notch pathway constitutes an evolutionarily conserved regulatory system that plays a key role in deciding the fate of diverse progenitor cells (15). Upon ligand binding, the Notch pathway is initiated by a series of proteolytic cleavages, including cleavage by {gamma}-secretase, which liberates the Notch intracellular domain (NICD). The released NICD translocates to the nucleus, where it forms a complex with RBP-J{kappa}, and this leads to the activation of Notch downstream signaling targets, such as Hes-1 (16). Within the hematopoietic system, different levels of Notch subfamily members are ubiquitously or selectively expressed, depending on hematopoietic cell type and differentiation stage (17). The Notch pathway modulates signaling induced by hematopoietic cytokines, often by delaying a particular pathway while permitting self-renewal or differentiation along an alternative pathway (18). In addition to having a role in cell fate decisions, emerging evidence reveals that Notch signaling influences functionalities of more mature blood cells, including activation, proliferation, and cytokine production (19, 20). However, with respect to the Notch pathway, little is known about the downstream signaling events; interactions with ubiquitous signaling pathways, such as the MAPK pathway; and the modulation of the resulting cellular functions.

Previously, we have demonstrated that treatment of umbilical cord blood (UCB) mononuclear cells with a {gamma}-secretase inhibitor (GSI) promotes eosinophil differentiation, as judged by the expression of major basic protein (MBP). The highly MBP-positive eosinophils lack the migratory response to eotaxin, although they have CCR3 levels that are similar to those of eosinophils cultured without GSI (21). This finding prompted us to investigate the mechanism underlying the differential response of terminally differentiating eosinophils to the eosinophil-active chemokine, and to analyze their functional characteristics.


   MATERIALS AND METHODS
Top
 Abstract
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Eosinophilic Differentiation of UCB Cells
Eosinophilic differentiation from UCB mononuclear cells has been described (21). Additional detail on the culture is provided in the online supplement. This study was approved by the Soonchunhyang University Hospital Institutional Review Board (protocols SCHBC-IRB-05-02 and SCHBC-IRB-06-04).

Treatment with Mitogen-Activated Protein Kinase Kinase Inhibitors
The mitogen-activated protein kinase kinase (MEK) inhibitors U0126 (Calbiochem, La Jolla, CA) and PD98059 (Tocris, Ballwin, MO) were dissolved in dimethyl sulfoxide. UCB cells that had been cultured with IL-5 and GSI for 21 days were washed with culture medium and incubated with U0126 or PD98059 or culture medium that contained 0.1% dimethyl sulfoxide in the presence of IL-5 and GSI for an additional 3 days. For the analysis of eosinophil differentiation, the cultured cells were stained with anti-human MBP antibody (Ab) (BD PharMingen, San Diego, CA).

Western Blot Analysis
Cells were lysed in RIPA with incubation on ice for 30 minutes. Twenty micrograms of the cell lysate were resolved by SDS-polyacrylamide gel electrophoresis, and then transferred to PVDF membranes. The membranes were blocked with 5% nonfat dry milk. The blots were probed with anti-NICD (Abcam, Cambridge, UK), anti–phospho-ERK1/2 (Cell Signaling Technology, Beverly, MA), and anti–phospho-Raf1 (Cell Signaling Technology), and were treated with anti-rabbit horseradish peroxidase–conjugated Abs (Cell Signaling Technology). Anti-ERK2 (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-GAPDH (Santa Cruz Biotechnology) Abs were used for loading controls. Immunostained proteins were detected with the enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech, Piscataway, NJ).

Flow Cytometry
Fresh and cultured UCB cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) for 15 minutes at 4°C, and washed twice with PBS that contained 2% bovine serum albumin (Sigma-Aldrich). Notch receptor and MBP expressions were analyzed as previously described (21). To determine IL-5R{alpha} expression, the cells were first stained with anti-human IL-5R{alpha} Ab (BD PharMingen) or anti-mouse IgG1 Ab, and then with PE-conjugated anti-mouse IgG1 Ab. Analysis of fluorescence staining was performed with a FACSCalibur flow cytometer and CellQuest software (Becton Dickinson, San Jose, CA).

Real-Time PCR
Real time quantitative PCR was carried out with SYBR Green dye. Additional detail is provided in the online supplement.

Apoptosis Assay
The cultured UCB cells at Day 28 were washed twice with cold PBS and incubated with or without IL-5 in fresh medium for 1, 3, 5, and 7 days. Apoptotic cells were assessed by staining with an Annexin V–FITC Apoptosis Detection Kit (BD PharMingen).

Measurement of Eosinophil Cationic Protein
The concentrations of eosinophil cationic protein (ECP) in the UCB culture supernatants were measured using a UniCAP ECP Fluoroenzymeimmunoassay Kit (Pharmacia Diagnostics AB, Uppsala, Sweden) according to the manufacturer's protocol.

Cytokine Assays
Cytokine concentrations were determined using enzyme-linked immunosorbent assay kits (BD Biosciences) to measure IL-4, IL-5, and GM-CSF, and using a kit (BioSource International, Camarillo, CA) to detect IL-13. The detection limits for IL-4, IL-5, IL-13, and GM-CSF were 7.8, 7.8, 7.8, and 4.7 pg/mL, respectively.

Statistical Analysis
All statistical analyses were performed using the Microsoft Excel data analysis program or the SPSS statistical analysis program for the Wilcoxon signed rank test. Differences with a P value of less than 0.05 were considered statistically significant. The results are expressed as mean ± SEM.


   RESULTS
Top
 Abstract
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
GSI Blocks the Generation of NICD in Differentiating UCB Cells
We have previously demonstrated that fresh UCB cells express high levels of the Notch 1 and Notch 2 mRNAs and proteins (21). To address the relevance of GSI L685,458 to Notch signaling in UCB cells, we performed an immunoblot analysis with an NICD-specific Ab. When the UCB cells were cultured with GSI, accumulation of the 80-kD NICD protein was considerably inhibited (Figure 1A). When UCB cells were cultured in the presence or absence of the inhibitor for 28 days, the levels of Notch 1 and Notch 2 mRNA, as evidenced by RT-PCR analysis, remained unchanged (data not shown). However, cell surface expression of Notch 1 and Notch 2 was reduced in the presence of the inhibitor (Figure 1B), which suggests that a post-transcriptional mechanism is involved, possibly in conjunction with endocytosis and subsequent protein degradation. Similarly, intracellular staining followed by flow cytometry analysis showed a reduction of NICD in cells cultured with the inhibitor (data not shown). In addition to Notch, the adhesion molecule CD44, which is expressed by activated eosinophils (22), has been shown to be a substrate for {gamma}-secretase (23). Like Notch, CD44 is cleaved by {gamma}-secretase to liberate the intracellular domain of CD44 (CD44ICD), which translocates to the nucleus and transactivates CD44 itself, the promoter of which contains a tetradecanoylphorbol acetate–responsive element (24). To confirm further the specificity of GSI, the cultured UCB cells were analyzed for CD44 expression. The CD44 mRNA and surface protein levels were much lower in the UCB cells cultured with GSI than in those cultured without the inhibitor (see Figure E1 in the online supplement). These data, together with our previous results showing that expression of Hes1, a transcriptional target of Notch, is decreased in differentiating UCB cells that are treated with GSI (21), validate the specificity of the inhibitor used in this study.


Figure 1
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  		Figure 1. {gamma}-Secretase inhibitor (GSI) prevents the accumulation of Notch intracellular domain (NICD) and reduces Notch receptors on the cell surfaces of differentiating umbilical cord blood (UCB) cells. (A) UCB cells (1x106 cells/ml) were cultured with a cytokine cocktail in the presence or absence of the GSI L685,458 (10 µM), for the indicated periods of time. The immunoblot was probed with an Ab specific for NICD. Twenty micrograms of protein were loaded on the gel, and GAPDH was used as the loading control. This blot is representative of three independent experiments. (B) UCB cells were cultured with a cytokine cocktail in the presence or absence of GSI for 14 and 28 days, and stained with Abs specific for the ectodomains of Notch 1 and Notch 2. Shaded areas represent staining with the specific Abs, and open areas represent staining with the isotype-control Ab (anti-rabbit IgG). The results presented are representative of three independent experiments.

 
ERK Activation during UCB-Derived Eosinophilic Differentiation
Since our previous study showed that the chemotactic responses of UCB cells cultured with the inhibitor for 28 days were significantly impaired, as compared with those in the absence of the inhibitor, despite comparable levels of CCR3 expression (21), we examined the possible cause of this phenotypic difference. In the absence of inhibitor, UCB cells cultured with the cytokine cocktail alone exhibited a small but steady increase in ERK1/2 phosphorylation. The cells cultured with GSI showed a similar pattern of ERK1/2 phosphorylation through Day 21, but displayed a large increase in ERK1/2 phosphorylation at Day 28 (Figure 2A), compared with cells grown without GSI. Consistent with our previous report (21), MBP expression, which is denoted in numbers to show a correlation with ERK1/2 phosphorylation, gradually increased over the culture period. Therefore, the robust phosphorylation of ERK1/2, especially at Day 28, was not necessarily commensurate with an increase in MBP expression. To determine the effects of ERK inhibitors on terminal eosinophil differentiation, UCB cells that had been cultured for 21 days in the presence of cytokine cocktail and GSI were washed extensively and treated with increasing concentrations of the MEK inhibitors U0126 and PD98059 in the presence of IL-5 (10 ng/ml) and GSI (10 µM). The treated cells were cultured for an additional 3 days, and then analyzed for MBP expression. The MEK inhibitors dose-dependently inhibited MBP expression (Figure 2B). In these experiments, MEK inhibitors minimally affect cell viability, as assessed by trypan blue exclusion. The highest concentration (50 µM) of both inhibitors with 3-day incubation caused cell death at more or less 10% (Figure 2B). Cell viability was maintained at 85% for even longer incubations (up to 7 d) of cultured UCB cells with the inhibitors (data not shown). These results suggest that MEK inhibitors antagonize the eosinophil differentiation–promoting effect of GSI.


Figure 2
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  		Figure 2. Involvement of extracellular signal–regulated kinase (ERK)1/2 in terminal eosinophilic differentiation. (A) Total cell lysates from differentiating UCB cells were subjected to Western blot analysis. The blot was probed with the anti–phospho-ERK1/2 Ab, and subsequently stripped and reprobed with anti-ERK2 Ab as an internal control. In parallel, a fraction of the differentiating UCB cells was harvested to measure major basic protein (MBP) expression, as denoted by the percentages below the blot. (B) UCB cells were cultured with a cytokine cocktail in the presence of L685,458 (10 µM) for 21 days, washed with culture medium, incubated with increasing concentrations of MEK inhibitor in the presence of IL-5 and L685,458 for an additional 3 days, and analyzed for MBP expression. Numbers displayed below antagonists' concentrations represent cell viability determined by tryphan blue exclusion. Data are expressed as mean ± SEM of five independent experiments (*P < 0.05 and **P < 0.01).

 
ERK Phosphorylation in Differentiating Eosinophils in Response to Eotaxin
Studies have demonstrated that ERK1/2 activation is required for the chemotactic responses of eosinophils to eotaxin (12, 13, 25). In the present study, UCB cells cultured with GSI for 28 days exhibited strong ERK1/2 activation (Figure 2A) and impaired chemotactic responses to eotaxin (21). Therefore, we hypothesized that cells treated with GSI have nearly full ERK1/2 activation and cannot produce any additional ERK1/2 activation in response to the chemotactic agonist. To examine this possibility, UCB cells cultured with or without the inhibitor for 28 days were washed extensively and assayed for ERK phosphorylation after treatment with eotaxin. The UCB cells cultured without the inhibitor exhibited eotaxin-induced ERK1/2 phosphorylation in a time-dependent manner. However, the UCB cells cultured with the inhibitor showed no further increase in ERK1/2 phosphorylation in response to eotaxin (Figure 3A). Raf1 phophorylation was accompanied by phosphorylation of ERK (Figure 3B). These results suggest that the lack of chemotactic response of the UCB cells cultured with the inhibitor for 28 days was partly due to the absence of newly activated ERK1/2.


Figure 3
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  		Figure 3. UCB cells cultured with GSI fail to carry out further phosphorylation of ERK1/2 in response to eotaxin. UCB cells that had been cultured with or without GSI for 28 days were washed extensively and stimulated with eotaxin (10 ng/ml) for 0, 5, 10, and 15 minutes. Immunoblots of the lysates from these cells were probed with anti–phospho-ERK1/2 (A) or anti–phospho-Raf1 (B). They were reprobed with anti-ERK2 or anti-GAPDH Abs. PC is the positive control supplied with the kit. The results shown are representative of two to three independent experiments.

 
Functional Characterization of Terminally Differentiating Eosinophils
We identified several phenotypes within the two eosinophil populations. Fresh UCB cells expressed low levels of cell surface IL-5R{alpha} (4–5%, n = 5). When the UCB cells were cultured with the cytokine cocktail in the absence or presence of GSI, IL-5R{alpha} cell surface expression increased at Day 7, and this level was maintained through Day 21 (Figure 4A). However, IL-5R{alpha} cell-surface expression was substantially increased at Day 28 in UCB cells cultured without the inhibitor, in comparison with cells that were treated with the inhibitor. Similarly, IL-5R{alpha} mRNA expression, as analyzed by real-time PCR, was consistent with its surface expression profile (Figure 4B). Therefore, at the terminal stage of eosinophil differentiation, GSI appears to affect IL-5R{alpha} expression. In addition, a lesser amount of IL-5 was detected at Day 28 in the culture supernatants from UCB cells cultured with IL-5 in the absence of GSI (UCB28 cells) compared to UCB cells cultured with IL-5 in the presence of GSI (UCB28i cells) (Figure 4C). This result suggests that exogenously added IL-5 is more vigorously taken up and used by UCB28 cells that express higher IL-5R{alpha} levels. The supernatants from UCB cells cultured for 28 days were collected, and the levels of cytokine production and ECP release were determined. The IL-13 and GM-CSF production levels were significantly higher in the culture supernatants of UCB28 cells than in those of UCB28i cells (Figure 5A), while the two populations produced minimal amounts of IL-4. However, the long-term incubation with GSI could affect degradation of the produced cytokines. Therefore, the cytokine measurement would probably be a result of both production and degradation of the cytokines in the presence of GSI. The ECP levels in the UCB28 cell culture supernatants were also significantly higher than the levels in the UCB28i cell culture supernatants. The UCB28 cells from three out of six donors produced appreciable amounts of ECP in their culture supernatants, whereas the UCB28i cells from all the donors exhibited virtually blunted ECP degranulation (Figure 5B). To examine the responsiveness of cultured UCB cells to IL-5, both UCB28 and UCB28i cells were washed extensively, re-stimulated with IL-5 or medium for 1 to 7 days, and ECP release and apoptosis were determined. Upon re-exposure to IL-5, the UCB28 cells significantly augmented ECP release. In contrast, the UCB28i cells showed no ECP release in response to IL-5 (Figure 6). Both types of cultured UCB cells were comparable to each other in the extent of apoptosis, but were, in general, highly refractory to apoptosis, regardless of the presence of IL-5 (< 20%) compared with peripheral blood eosinophils, which typically exhibited 90% and 50% annexin V–positive cells at Day 7 in the absence and presence of IL-5, respectively.


Figure 4
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  		Figure 4. The reduced expression of IL-5R{alpha} mRNA and surface protein in differentiating UCB cells cultured with GSI. (A) Surface expression of IL-5R{alpha}. The expression of IL-5R{alpha} on the surfaces of freshly isolated UCB cells and differentiating UCB cells was analyzed by flow cytometry. The average IL-5R{alpha} expression levels represent five to thirteen independent experiments (*P < 0.05). (B) IL-5R{alpha} mRNA expression. The differentiating UCB cells from five donors were pooled, and total RNA was extracted and analyzed for IL-5R{alpha} mRNA expression by real-time PCR. The GAPDH gene was used to normalize each analysis (**P < 0.01). (C) Levels of residual IL-5 at Day 28 in the UCB cell culture supernatant. UCB cells were routinely cultured with a cytokine cocktail including IL-5 in the presence or absence of GSI for 28 days. The culture supernatant was harvested and the residual amount of IL-5 was determined by enzyme-linked immunosorbent assay. The data represent mean ± SEM of twelve independent experiments. The amount of IL-5 per 106 cells is shown (*P < 0.05).

 

Figure 5
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  		Figure 5. Levels of cytokines and released eosinophil cationic protein (ECP) in UCB cell supernatants cultured with or without GSI. (A) IL-13, GM-CSF, and IL-4 production. UCB cells were cultured with a cytokine cocktail in the presence or absence of GSI for 28 days. The cell culture supernatant was harvested for cytokine measurement. The data represent mean ± SEM of twelve independent experiments (*P < 0.05 and **P < 0.01). (B) ECP release. ECP was measured in the culture supernatants from six donors (*P < 0.05). Bars indicate averages.

 

Figure 6
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  		Figure 6. Lack of ECP degranulation of eosinophils cultured with GSI. UCB cells that had been cultured with or without GSI for 28 days were washed extensively and stimulated with IL-5 for 1, 3, 5, and 7 days. The levels of ECP in the culture supernatants were measured. The data represent mean ± SEM for three donors (*P < 0.05 and **P < 0.01).

 

   DISCUSSION
Top
 Abstract
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
We have previously shown that GSI treatment of UCB cells promotes eosinophil development, and that these cells almost completely lose the chemotactic response to eotaxin. Nevertheless, these cells had CCR3 levels that were comparable with those of UCB cells cultured in the absence of GSI (21). Since eosinophil chemotaxis is closely associated with ERK1/2 activation (12, 13, 25), we tested whether the two differentiating eosinophil populations differed with respect to ERK1/2 phosphorylation. Our results show that ERK1/2 phosphorylation was modestly but gradually increased over the 4-week culture period in the presence of the hematopoietic cytokines. ERK1/2 phosphorylation was prominent at a relatively late stage (Day 21) of UCB-derived eosinophilic differentiation, at which time point MBP-immunoreactive cells were much evident (Figure 2). Our finding of increased ERK1/2 phosphorylation is in line with previous findings that ERK pathway activation is required for eosinophil (10, 14) and myeloid differentiation (26, 27). In the presence of GSI, a similar pattern of ERK1/2 phosphorylation was observed through Day 21, although the 28-day cultures with GSI had robust ERK1/2 phosphorylation. Although the presence of GSI enhanced the generation of MBP+ eosinophils throughout all culture stages, MBP-expressing cells increased slightly from Day 21 to Day 28 (from 87% to 94%, respectively), while in the absence of inhibitor, this population increased considerably during the same period (from 35% to 73%, respectively). Therefore, it is unlikely that the strong ERK1/2 phosphorylation seen at Day 28 in the presence of GSI was solely responsible for the augmentation of MBP-expressing eosinophils. Rather, this may have been linked to reduced IL-5R{alpha} expression, blunted degranulation, reduced cytokine production, and a lack of responsiveness to eotaxin and IL-5, all of which were manifested in the eosinophils cultured with inhibitor for 28 days (Figures 3–6GoGoGo). Our data suggest that modest ERK phosphorylation is required for eosinophilic differentiation, whereas eosinophilic terminal differentiation/maturation requires quantitatively higher levels of ERK phosphorylation.

Crosstalk between the Notch and MARK signaling pathways in cell fate determination has been demonstrated using the model system of Caenorhabditis elegans (28). During vulval development of C. elegans, MAPK signaling activation leads to down-regulation of Notch expression, which is partly due to endocytic clearance of Notch from the surfaces of vulval precursor cells. Recently, Kim and coworkers have demonstrated that ERK1/2 inhibits {gamma}-secretase activity by altering the pattern of phosphorylation (29), which suggests antagonism between Notch signaling and ERK activation. In the present study, UCB cells cultured with GSI for 28 days exhibited reductions in Notch 1 and Notch 2 surface expression (Figure 1B), while their transcription levels remained constant (data not shown). Furthermore, the persistent inhibition of Notch signaling by GSI resulted in ERK activation, which supports the idea of mutual antagonism between the two pathways. We speculate that cell-surface Notch expression is down-regulated via endocytosis triggered by ERK activation at a final stage of inhibitor-treated UCB cell differentiation.

Although Notch represents a primary target of {gamma}-secretase and its inhibitor blocks both NICD generation (Figure 1A) and Hes-1 mRNA expression in differentiating UCB cells (21), we cannot rule out the possibility of other effects of GSI on terminal eosinophilic differentiation. The adhesion molecule CD44 has recently been identified as a substrate for {gamma}-secretase (23). CD44 is also an activation marker of eosinophils (22) that is implicated in pulmonary eosinophil accumulation (30). Our results demonstrate that UCB cells cultured with GSI exhibits decreased CD44 expression. However, the contribution of CD44 signaling to eosinophil differentiation remains to be evaluated. It might also be possible that GSI induces a generalized, Notch-independent stress response that affects ERK activation.

To elucidate the functional activities of UCB cells cultured with or without GSI, we compared the levels of IL-5R{alpha} expression, ECP degranulation, and cytokine production, as well as IL-5 responsiveness. It is interesting that at the final stage (Day 28) of differentiation, UCB cells cultured with GSI failed to express IL-5R{alpha} mRNA (Figure 4B). This suggests that although CD34+ cells in the BM that are committed to the eosinophil lineage initially acquire surface IL-5R{alpha} (4, 5), further maturation requires additional signals, such as Notch signaling. These cells also exhibited reductions in ECP release and cytokine production (Figure 5). GSI is recently shown to attenuate NF-{kappa}B activity by blocking the nuclear import and the sustained nuclear activity of NF-{kappa}B, leading to the down-regulation of proinflammatory cytokine production (31). Therefore, such a GSI activity might account for the inhibition of IL-13 and GM-CSF production. Previous studies have shown that airway eosinophils have elevated oxygen species release (32), enhanced survival (32), increased ERK1/2 activation (33), and increased CD44 expression (34), compared with peripheral blood eosinophils. Airway eosinophils exhibit significantly decreased surface IL-5R{alpha} expression with a concomitant increase in soluble IL-5R{alpha}, as well as ablated degranulation of cationic proteins in response to IL-5 (35, 36). Thus, when eosinophils are recruited to the airway, the regulation of their functions becomes IL-5–independent. In terms of effector functions, the eosinophils generated by treatment with GSI are much similar to airway eosinophils, with the exception of decreased CD44 expression. Bates and colleagues have demonstrated that airway eosinophils display higher levels of surface expression of CD44 than peripheral blood eosinophils (34). Nonetheless, in our experimental setting, the reduced level of CD44 seen in the UCB cells cultured with GSI can be simply explained by the fact that CD44 is a target for {gamma}-secretase.

GSI promotes the development of eosinophil cells that strongly express MBP and that are nonfunctional. Our immunohistochemical analysis shows that Notch ligands Jagged1 and Delta1 are expressed abundantly, primarily on epithelial cells of both the mouse and human airways (unpublished observation). CD34+IL-5R{alpha}+ eosinophil progenitor cells, which have been identified in asthmatic lungs (7), might be able to express Notch receptors. It is possible that the developing eosinophils and their precursor cells may contact Notch ligands after recruitment to the lung, with consequent effects on functionality and in situ differentiation. Knowing whether GSI modulates eosinophilic inflammation in an animal model for asthma and is of therapeutic potential would be interesting.

In summary, we showed cross-talk between the Notch and MAPK pathways, especially during eosinophilic terminal differentiation. The treatment of differentiating eosinophils with GSI resulted in robust phosphorylation of ERK1/2 and concomitant promotion of Notch clearance from the cell surface. These cells were unable to induce further ERK1/2 phosphorylation in response to eotaxin and were refractory to degranulation and cytokine production. These results may be highly relevant to terminally differentiated eosinophils, especially in airways, and reflect the dependence of functional phenotypes on the leukocyte differentiation state.


   Acknowledgments
 
The authors thank Dr. Sung Hee Choi, Hanvit Clinic (Ansan) for helpful discussion.


   Footnotes
 
This work was supported by grants from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (A06-0097-AA1018-06N1-00010A to I.Y.C.), and the Research fund of Hanyang University (HY-2006-I to I.Y.C.). J.H.K. and D.H.L. were partially supported by the BK21 Korea Program.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1165/rcmb.2006-0412OC on June 28, 2007

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form November 3, 2006

Accepted in final form April 6, 2007


   References
Top
 Abstract
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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