The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2008, 181, 7689 -7698
Copyright © 2008 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ueki, S.
Right arrow Articles by Chihara, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ueki, S.
Right arrow Articles by Chihara, J.

Retinoic Acids Are Potent Inhibitors of Spontaneous Human Eosinophil Apoptosis

Shigeharu Ueki,1 Gulixian Mahemuti,1 Hajime Oyamada, Hikari Kato, Junko Kihara, Masako Tanabe, Wataru Ito, Takahito Chiba, Masahide Takeda, Hiroyuki Kayaba, and Junichi Chihara2

Department of Clinical and Laboratory Medicine, Akita University School of Medicine, Akita, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Retinoic acids (RAs), which are active metabolites of vitamin A, are known to enhance Th2-type immune responses in vitro, but the role of RAs in allergic inflammatory cells remains unclear. In this study, we demonstrated that purified peripheral blood eosinophils expressed nuclear receptors for RAs at the mRNA and protein levels. Eosinophils cultured with all-trans RA (ATRA) and 9-cis-RA showed dramatically induced cell survival and nuclear hypersegmentation, and the efficacy of RAs (10–6M) was similar to that of IL-5 (1 ng/ml), the most critical cytokine for eosinophil activation. Pharmacological manipulation with receptor-specific agonists and antagonists indicated that the antiapoptotic effect of RAs was mediated through ligand-dependent activation of both retinoid acid receptors and retinoid X receptors (mainly retinoid acid receptors). Furthermore, using a gene microarray and a cytokine Ab array, we discovered that RAs induced vascular endothelial growth factor, M-CSF, and MCP-1 secretion, although they were not involved in eosinophil survival. RA-induced eosinophil survival appears to be associated with down-regulation of caspase 3 and inhibition of its enzymatic activity. These findings indicate an important role of RAs in homeostasis of granulocytes and provide further insight into the cellular and molecular pathogenesis of allergic reactions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Tissue eosinophilia is one of the hallmarks of allergic diseases and Th2-type immune responses. Eosinophils contribute to tissue injury, vascular leakage, mucus secretion, and airway contraction by releasing cytotoxic granule proteins, reactive oxygen species, and lipid mediators (1). In addition, eosinophils can produce several chemokines, growth factors, and Th2-type cytokines such as IL-4 and IL-13 (2). The evidence suggests the potential of eosinophils to not only act as terminal effecter cells but also to possess immunoregulatory activities.

Prolonged eosinophil survival at an allergic inflammatory site is essential for them to exert their function sufficiently and is associated with pathological conditions. IL-5 is one of the key regulators of eosinophil differentiation and survival. Eosinophils cultured in the absence of IL-5 undergo rapid apoptosis, which is physiological cell death characterized by morphological changes (3). However, treating patients with anti-IL-5 mAb only partially reduced eosinophilia in airway tissue and bone marrow, suggesting that other factors contributed to eosinophil survival in these tissues (4, 5). Therefore, investigation of the mechanism and factors involved in the survival prolongation of eosinophils would be beneficial in the treatment of allergic diseases.

Retinoids, particularly retinoic acids (RAs),3 are active metabolites of vitamin A that are known to regulate cell differentiation, proliferation, and apoptosis in a variety of cell types (6, 7). The biological actions of retinoids are exerted via two families of nuclear receptors, RA receptors (RARs) and retinoid X receptors (RXRs) each of which has three isoforms, i.e., {alpha}, β, and {gamma} (8). The RARs and RXRs are members of the steroid receptor superfamily and they function as ligand-dependent transcriptional regulators. Among the natural retinoids, all-trans RA (ATRA) and 9-cis-RA are high-affinity ligands for RARs and 9-cis-RA additionally binds RXRs (8). RARs require the promiscuous partner RXRs to form receptor heterodimers. After ligand binding, these receptors form homodimers or heterodimers and function as transcriptional regulators.

A considerable number of studies have indicated the important roles of vitamin A in the immune system. Vitamin A deficiency increases mortality from common childhood infections and, conversely, vitamin A supplementation reduces the mortality among children in developing countries (9, 10, 11). This accumulated evidence presumably suggests that vitamin A deficiency is involved in the impairment of host defense mechanisms. As for Th2-type responses, an early study by Carman et al. (12) showed that parasitic helminth infection in vitamin A-deficient mice strikingly reduced IL-4 and IL-5 production and bone marrow eosinophilia. In line with this, recent studies demonstrated that supplemental treatment with vitamin A or RA decreased IFN-{gamma} production and increased IL-4, IL-5, IL-10, and IL-13 production (13, 14, 15). Interestingly, RA also exerted direct in vitro effects on T cells to suppress Th1 and enhance Th2 development (16). Despite these results that suggest that vitamin A biases the response in a Th2 direction, the influence of RA on allergic disease is limited and appears contradictory. For instance, supplementation with a natural source of vitamin A had a protective effect against exercise-induced asthma in some patients (17). Several epidemiological studies suggested that vitamin A intake had no association with or protective effect on asthma (18). In contrast, RA can induce eosinophilia and exacerbation of asthma under certain conditions (19). Therefore, it is of paramount importance to clarify the regulation of immune responses by RA and its underlying cellular and molecular mechanisms.

The direct effect of RA on allergic inflammatory cells, especially eosinophils, is less well understood. In the present study, we investigated the functional roles of RA on purified human eosinophils. We found that peripheral blood eosinophils expressed receptors for RAs and, interestingly, ATRA and 9-cis-RA dramatically inhibited spontaneous eosinophil apoptosis. The effect exerted by RA was parallel with the down-regulation of caspase 3 transcription and its enzymatic activity. Together, our experiments showed that RAs activated eosinophils to produce several proinflammatory cytokines.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Materials

9-cis-RA and ATRA were from Sigma-Aldrich. The synthetic RAR agonist [(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid (TTNPB) and the synthetic RAR{alpha} antagonist Ro-41-5253 were purchased from BioMol Research Laboratories. RXR agonist HX630 was a gift from Dr. H. Kagechika (University of Tokyo, Japan) (20). All retinoids were dissolved in DMSO (Sigma-Aldrich) at 5–25 mM stock solutions, protected from light, and stored at –70°C.

Cell preparation

Peripheral venous blood was obtained from subjects with mild eosinophilia. Informed consent was obtained from each subject, and the study protocol was approved by the Ethics Committee of Akita University School of Medicine. Eosinophils were isolated by sedimentation with 6% dextran followed by centrifugation on 1.088 Percoll (Pharmacia) density gradients (21, 22). The cells were further purified by negative selection using anti-CD16 immunomagnetic beads and a MACS system (Miltenyi Biotec). The eosinophils (>98% purity) were then suspended in HBSS with 1% FCS in tubes coated with 3% human serum albumin. Mononuclear cells were isolated from peripheral venous blood by Ficoll-Paque (Pharmacia) density gradient centrifugation. The cells were washed twice in cold PBS and monocytes were purified using a MACS system according to the manufacturer’s instructions.

RT-PCR

Total RNA was extracted with the use of Ultraspec RNA (Biotex Laboratories) from eosinophils. The total RNA was reverse transcribed with 3 µg of RNA using an Omniscript reverse transcriptase (RT) kit (Qiagen) according to the manufacturer’s protocol. One microliter of the cDNA synthesis reaction was used as a template for PCR amplification with AmpliTaq DNA polymerase (PerkinElmer). The sequences of the 5' sense primers and the 3' antisense primers synthesized based on published sequence data (23) and used in this study were as follows: RAR{alpha}, 5'-TGGGTGGACTCTCCCCGCCA-3' (sense) and 5'-CCCACCTCCGGCGTCAGCGTG-3' (antisense) (product size 438bp); RARβ, 5'-CACTGGCTTGACCATCGCAGACC-3' (sense) and 5'-GAGAGGTGGCATTGATCCAGG-3' (anti-sense) (product size 400bp); RAR{gamma}, 5'-GGCCTGGGCCAGCCTGACCTC-3' (sense) and 5'-CAGCCCCAGATCCAGCTGCACG-3' (antisense) (product size 537 bp); RXR{alpha}, 5'-ATGGCTGCCCCCTCGCTGCAC-3' (sense) and 5'-GGCGCAGATGTGCTTGGTG-3' (antisense) (product size 327 bp); RXRβ, 5'-ATGCCACCCCCGCCACTGGGC-3' (sense) and 5'-GCCTCCAGGATCCTGTCCACAGGC-3' (antisense) (product size 552 bp); and RXR{gamma}, 5'-CCCCTGGTCACACTGGCTCGACG-3' (sense) and 5'-CACCAGAGACCCAGGGCTGGTGG-3' (anti-sense) (product size 351 bp). The PCR cycle consisted of 45-s denaturation at 94°C; 45-s annealing at 60°C (RARβ, RXR{gamma}, β-actin), 62°C (RXRβ), and 64°C (RAR{alpha}, RAR{gamma}, RXR{alpha}); and 1-min extension at 72°C for 30–35 cycles. The PCR products were separated by electrophoresis on a 12.5% PhastGel and the gel was stained using a PhastGel DNA Silver Staining Kit (Amersham Pharmacia Biotech).

Immunoblotting

Eosinophils or monocytes (2 x 106 cells) were lysed in a lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM Na3VO4, 1 mM NaF, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 1% Triton X-100, 10% glycerol, 1 mg/ml aprotinin, leupeptin, and pepstatin). After 20 min on ice, detergent-insoluble materials were removed by centrifugation at 4°C at 12,000 x g. The supernatants were mixed with SDS sample buffer and boiled for 4 min. SDS-polyacrylamide (10%) gels (Ready Gel J) were obtained from Bio-Rad. The electrophoresed gel was blotted onto Hybond ECL membranes (Amersham). Excess binding sites were blocked by incubation with 10% BSA in TBS-T buffer (20 mM Tris-HCl, 137 mM NaCl, and 0.05% Tween 20, pH7.6) for 1 h followed by incubation in the primary Ab overnight at 4°C (rabbit polyclonal anti-RAR{alpha}, β, or {gamma} and RXR{alpha}, β, or {gamma} Abs) from Santa Cruz Biotechnology. After washing three times with TBS-T, the membrane was incubated with the secondary Ab (0.04 mg/ml HRP-conjugated goat anti-rabbit IgG Ab from Santa Cruz Biotechnology) for 30 min. The blots were visualized by an ECL system (Amersham) according to the manufacturer’s instructions.

Flow cytometric analysis for retinoid receptors

Intercellular staining was performed to detect nuclear retinoid receptors in eosinophils using a Fix and Perm Cell Permeabilization Kit (Caltag Laboratories). The purified eosinophils were washed twice with PBS and then stained with 1/500 diluted rabbit polyclonal anti-RAR{alpha}, β, or {gamma} and RXR{alpha} or β Abs (Santa Cruz Biotechnology) and PE-conjugated, affinity-purified anti-rabbit IgG (Rockland). Rabbit IgG Ab (DakoCytomation) was used as an isotype-matched control. The stained cells were analyzed using a flow cytometer (FACScan; BD Immunocytometry Systems).

Cell culture

Purified eosinophils were resuspended at 0.5 x 106 cells/ml in RPMI 1640 medium (Life Technologies) with 10% FCS and incubated with or without indicated concentrations of 9-cis-RA, ATRA, TTNPB, HX630, 0.1% DMSO (vehicle), IL-5 (Genzyme), or GM-CSF (Genzyme) at 37°C in humidified air with 5% CO2 for 4–72 h. In some experiments, eosinophils were preincubated with or without neutralizing anti-IL-5 or anti-GM-CSF Ab (Genzyme). The RPMI 1640 was supplemented with 2 mM L-glutamine, penicillin (100 IU/ml), and streptomycin (100 µg/ml). Eosinophils were then examined for each analysis. We confirmed that the effect of DMSO was negligible at the concentration used in each experiment.

Determination of cell death and apoptosis by flow cytometry and morphological analysis

An apoptosis detection kit (Medial Biological Laboratories) was used to quantitatively determine eosinophils undergoing apoptosis by virtue of their ability to bind to annexin V and propidium iodide (PI). Briefly, harvested eosinophils were washed twice in cold PBS and stained with annexin V and PI according to the manufacturer’s instructions. Eosinophil apoptosis and viability were analyzed using a BD Biosciences FACScan cytometer (22). To evaluate apoptosis and nuclear hypersegmentation, cytospins of eosinophils recovered from different cultures were prepared and then stained with Diff-Quick staining. Evidence of apoptotic morphology such as decreased cell size, nuclear condensation, and anucleation was assessed by light microscopy in a blind fashion. Nonapoptotic eosinophils were also assessed for the number of nuclear segmentations. Two hundred eosinophils were counted, and the percentage of eosinophils in each morphological change was calculated.

Microarray hybridization

After incubation with vehicle, 10–6 M 9-cis-RA or ATRA in 1% human serum albumin-coated plates, each 4 x 106 cells, for 4 h, eosinophils were lysed, and total RNA was isolated using Isogen (Nippon Gene) as per the manufacturer’s instructions. RNA was repurified with phenol-chloroform extraction and ethanol precipitation. For each cell culture condition, the total RNA from three donors was pooled, enabling us to acquire adequate amounts of RNA for microarray analysis. RNA quality was assessed using a microcapillary electrophoretic analyzer (Agilent Technologies). The 28S/18S rRNA intensity was >1.4. The gene array procedures were performed at Mitsubishi Kagaku BCL using a protocol described elsewhere (24, 25). Differences of 747 genes (Genomessage version 2; Japan Genome Solutions) between RAs and vehicle control were determined using ImaGene software (BioDiscovery). The sample and reference amplified sample RNAs were labeled with Cy3-dUTP and Cy5-dUTP (PerkinElmer), respectively, using a SuperScript II kit (Invitrogen) along with random hexamers (TaKaRa). RNA mixture ({lambda} DNA, baculovirus glycoprotein gene, and Renilla luciferase gene) employed as external control. Competitive hybridization of Cy3-labeled sample and Cy5-labeled reference cDNA on the microarray was conducted according to a previous report (26). The Cy3 (treated sample)/Cy5 (reference sample) ratio for each mRNA signal was calculated according to global Lowess normalization (27). Slides were scanned five times with five different power ranges using a ScanArray 5000 (PerkinElmer). For statistical analysis, the data were converted from tiff image data to signals using ImaGene software (BioDiscovery). The microarray data were deposited in the public Gene Expression Omnibus database (www.ncbi.nlm.nih.gov/geo/) under accession no. GSE12926.

Detection of cytokines in cell culture supernatants

Culture samples were analyzed with a cytokine Ab array by using a RayBio Human Cytokine Ab Array (RayBiotech) according to the manufacturer’s instructions. Concentrations of IL-5, IL-8, GM-CSF, vascular endothelial growth factor (VEGF), M-CSF, and MCP-1 were measured using an ELISA kit (R&D Systems), and colorimetric measurements were performed according to the manufacturer’s protocol.

RT-PCR confirmation of gene microarray data

For confirmation of expression patterns indicated by microarray analysis, 4 µg of total RNA was subjected to first-strand cDNA synthesis as described in Microarray hybridization. RT-minus controls were included for each RT reaction. Primers and TaqMan probes for target genes were purchased from Applied Biosystems TaqMan Gene Expression Assays. Quantification of target cDNA and an internal reference gene (GAPDH) was performed in 96-well plates on the Applied Biosystems PRISM 7700 Sequence Detection System for data collection, and analyses were performed using the machine software. PCR was conducted in a final volume of 25 µl containing cDNA equivalent to 10–100 ng of total RNA, 12.5 µl of 1x TaqMan Universal PCR Master Mix, and 1.25 µl of 20x TaqMan Expression Assay reagent. Each sample was analyzed in triplicate. Thermal cycler conditions were 50°C for 2 min and 95°C for 10 min, followed by 50 cycles at 95°C for 15 s and 60°C for 1 min. The comparative cycle threshold method of data analysis was used to analyze the data.

Measurement of caspase 3 activity

After treatment of eosinophils with 9-cis-RA and ATRA for 18 h, caspase 3 activity in these cells was assayed with an APOPCYTO colorimetric assay kit (MBL) according to the manufacturer’s instructions.

Statistical analysis

For comparison of groups, ANOVA was used. If the ANOVA was significant, post hoc pairwise comparisons were conducted using Tukey’s test, with the level of statistical significance taken as p < 0.05. The results are expressed as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Retinoid receptor profile of human peripheral blood eosinophils

To provide a baseline for the interpretation of our studies, we first investigated the gene expression of all known RAR subtypes in human peripheral blood eosinophils using RT-PCR. The expression profile was compared with freshly isolated human monocytes. We found that human eosinophils expressed mRNA for all RAR subtypes. In contrast, RARβ was undetectable in human monocytes, consistent with a previous report (28). A similar pattern of RXR mRNA expression was observed between monocytes and eosinophils: RXR{alpha} and RXRβ, but not RXR{gamma}, were expressed (Fig. 1A). On the basis of these results, the protein expression of RARs and RXRs on eosinophils was studied. Using Western blotting, we demonstrated that all three RAR isoforms, RXR{alpha}, and RXRβ were present in the lyses of eosinophils from two different donors (Fig. 1B). We also confirmed the protein expression by flow cytometry analysis on permeabilized cells. Similarly to the Western blotting, RAR{alpha}, RARβ, RAR{gamma}, RXR{alpha}, and RXRβ were expressed in human eosinophil preparations (Fig. 1C). Because RXR{gamma} mRNA was not detected by PT-PCR, RXR{gamma} protein expression was not studied further.


Figure 1
View larger version (36K):
[in this window]
[in a new window]

  		FIGURE 1. The expression of RARs and RXRs in human eosinophils. A, The mRNA expression profile was compared with freshly isolated human monocytes using RT-PCR. B, Western blot analysis for protein expression of RARs and RXRs in eosinophils from two different donors. C, Flow cytometric analysis confirming the findings of Western blotting. Purified human peripheral blood eosinophils were permeabilized and then stained with indicated Abs. Histograms show the isotype-matched control (open histograms) and receptor expression (filled histograms). Results are representative of n = 3.

 
9-cis-RA and ATRA markedly delay eosinophil apoptosis

It has been reported that RAs (10–6 M) regulate survival in several types of cells. To investigate the functional role of RAs, we first examined the capacity to modulate eosinophil apoptosis that spontaneously occurs after culturing these cells. Eosinophil apoptosis was determined by flow cytometry; Annexin V was used to stain the early phase apoptotic cells, while PI was used to stain the late phase cells. Purified human blood eosinophils were incubated with the natural ligand for RARs and RXRs (9-cis-RA and ATRA, 10–6M) or vehicle control (0.1% DMSO) for 48 h. The percentage of cells undergoing apoptosis was dramatically decreased in eosinophils incubated with 9-cis-RA or ATRA compared with vehicle control (Fig. 2A). 9-cis-RA and ATRA delayed eosinophil apoptosis in a dose-dependent manner and occurred around their physiological concentrations (10–9–10–8 M) (Ref. 29 and Fig. 2B). Of note, their ability to delay apoptosis almost equaled that of IL-5 (1 ng/ml), a critical cytokine mediating eosinophil survival. As shown in Fig. 2C, eosinophils incubated with RAs displayed almost 80% viability at 72 h after incubation (vehicle, 12.0 ± 4.1%; 9-cis-RA, 81.9 ± 3.5%; and ATRA, 78.0 ± 3.8%).


Figure 2
View larger version (29K):
[in this window]
[in a new window]

  		FIGURE 2. The effect of RAs on eosinophil survival. A, After incubation with IL-5 (1 ng/ml), 9-cis-RA (10–6 M), or ATRA (10–6 M) 48 h, eosinophil apoptosis was determined by flow cytometry; Annexin V was used to stain the early phase apoptotic cells and PI was used to stain the late phase cells. Concentration-dependent response (B) and time course (C) of eosinophil survival (n = 4–5). Eosinophil viability was assessed by the percentage of annexin V (–) and PI (–) cells. *, p < 0.05 vs vehicle control.

 
Morphological appearance of eosinophils incubated with RAs

To assess the morphological evidence of apoptosis and cell activation, eosinophils were cultured for 48 h with vehicle alone, IL-5 (1 ng/ml), 9-cis-RA (10–6 M), or ATRA (10–6 M) and were observed by light microscopy (Fig. 3). Reflecting spontaneous apoptosis, eosinophils with apoptotic morphology (cytoplasmic and nuclear chromatin condensation, and anucleation) were seen after they were cultured with vehicle alone. In contrast, eosinophils cultured with IL-5, 9-cis-RA, or ATRA avoided apoptotic changes and, interestingly, several nonapoptotic cells had more than three nuclear lobes. Because nuclear hypersegmentation is induced by eosinophiliopoietic cytokines and indicates the increased life span (30), the number of nuclear segmentations besides apoptotic change was quantified and is shown in Table I. In line with the flow cytometric studies, after culture for 48 h, apoptotic eosinophils were decreased by IL-5 or RAs compared with vehicle alone. Of note, incubation with IL-5 or RAs induced a significant increase in the number of eosinophils with hypersegmented nuclei, while most of the freshly purified peripheral blood eosinophils or vehicle-cultured live eosinophils showed bisegmented nuclei. These data provide multiple lines of evidence demonstrating prolonged survival induced by RAs.


Figure 3
View larger version (48K):
[in this window]
[in a new window]

  		FIGURE 3. The morphological appearance of eosinophils incubated with IL-5 (1 ng/ml), 9-cis-RA (10–6 M), or ATRA (10–6 M) for 48 h was confirmed by light microscopy (original magnification, x400; Diff-Quick staining). Black arrows indicate the apoptotic cells with morphological changes; cytoplasmic and nuclear chromatin condensation. Open arrows indicate eosinophils with hypersegmented nuclei.

 

View this table:
[in this window]
[in a new window]

  		Table I. Number of nuclear segmentationsa

 
Effect of pharmacological agonists and antagonists on eosinophil survival

To determine which of the two types of RARs is involved in the antiapoptotic effect, we used the specific agonists for each receptor: RAR-selective agonist TTNPB (31) and RXR-selective agonist HX630 (32). Eosinophils were cultured in the presence of either TTNPB or HX630 for 48 h, and cell viability was assessed by flow cytometry staining with annexin V and PI. Interestingly, both TTNPB and HX630 delayed the eosinophil apoptosis, although HX630 had a less potent effect (Fig. 4A). The cell viability of vehicle, TTNPB (10–6 M), and HX630 (10–6 M) was 15.2 ± 5.5%, 73.4 ± 3.4%, and 28.8 ± 5.7%, respectively. These results indicate that both RARs and RXRs (mainly RARs) are involved in the effect of RAs to delay eosinophil apoptosis. Next, we examined the effect of receptor blockade on RA-induced eosinophil survival. As shown in Fig. 4B, pretreatment with RAR{alpha}-specific antagonist Ro41-5253 nearly completely inhibited the effect of ATRA at the concentration of 10–5 M (70.1% inhibition). In contrast, the antiapoptotic effect of 9-cis-RA was partially blocked by Ro41-5253 at 10–5 M (26.9% inhibition). Therefore, it is suggested that RAR{alpha} activation is important for RA-induced eosinophil survival, although RXR activation can partially compensate for the effect.


Figure 4
View larger version (21K):
[in this window]
[in a new window]

  		FIGURE 4. The effect of receptor agonists and antagonists on eosinophil survival. A, After incubation with increased concentrations of RAR-selective agonist TTNPB and RXR-selective agonist HX630 for 48 h, eosinophil viability was determined by flow cytometry with the percentage of annexin V (–) and PI (–) cells (n = 4). *, p < 0.05 vs vehicle control. B, The effect of RAR{alpha} antagonist Ro41-5253 on 9-cis-RA- or ATRA-induced eosinophil survival (n = 4). Purified eosinophils were preincubated with indicated concentrations of Ro41-5253 for 45 min, following the stimulation with 9-cis-RA or ATRA (each 10–7 M) for 48 h, after which viability was assessed. *, p < 0.05 vs each RA alone (without preincubation of Ro41-5253).

 
Identification of RA-dependent transcriptional targets by gene microarray

In an attempt to identify genes that are associated with RA-induced eosinophil survival, we next determined the expression levels of eosinophil transcripts using a microarray following stimulation with vehicle, 9-cis-RA (10–6 M), or ATRA (10–6 M) for 4 h. Additional longer time points were prohibited by the difficulty of obtaining subjects to harvest the required amount of RNA for the microarray. Approximately 444 genes were detected on the chip containing 747 genes (raw data are available at the Gene Expression Omnibus site as cited in Materials and Methods). There was little variation between the samples treated by 9-cis-RA and ATRA. The main results are indicated in Table II. The comparison between vehicle control and 9-cis-RA, or vehicle control and ATRA, identified an increase (>2-fold change over control) in 19 and 11 transcripts, respectively. The number of decreased transcripts (<0.5-fold) resulting from 9-cis-RA or ATRA exposure was 19 and 8 transcripts, respectively. In these transcripts, the expression changes relative to vehicle control were smaller in with ATRA compared with 9-cis-RA, suggesting that 9-cis-RA induced gene transcription more effectively than ATRA. Of note, among the 10 genes with the biggest decrease relative to vehicle control, we found an apoptosis-related gene, caspase 3 (9-cis-RA, 0.33-fold decrease and ATRA, 0.39-fold decrease).


View this table:
[in this window]
[in a new window]

  		Table II. RA induced genes >2-fold over vehicle control samples and RA reduced genes <0.5-folda

 
RAs induced production of M-CSF, VEGF, and MCP-1, but they were not involved in antiapoptotic effect

RAs affect cytokine, chemokine, and growth factor production from several types of cells. Because of the strong efficacy of RAs and the potential of eosinophil to produce IL-5 and GM-CSF, critical promoters of eosinophil survival (2), we examined whether RAs prolong survival by autocrine production of IL-5 or GM-CSF using neutralizing Abs. However, cotreatment of neutralizing anti-IL-5 or anti-GM-CSF Abs failed to reverse the effect of RAs (Fig. 5). We also found that IL-3, an eosinophil survival-promoting cytokine (2), was not involved in RA-induced cell survival using a neutralizing anti-IL-3 Ab (n = 3; data not shown). Moreover, neither IL-5 nor GM-CSF in the supernatants of eosinophils cultured with RAs for 24 h was detected by ELISA. These results indicate that the antiapoptotic effect of RAs was not dependent on autocrine production of IL-3, IL-5 or GM-CSF.


Figure 5
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 5. The effect of neutralizing anti-IL-5 or anti-GM-CSF Abs on RA-induced eosinophil survival. Eosinophils were stimulated with 10–6M RA along with neutralizing anti-IL-5 or anti-GM-CSF (20 µg/ml) Abs for 48 h, and eosinophil viability was then determined by flow cytometry with the percentage of annexin V (–) and PI (–) cells (n = 4). Eosinophil survival was significantly prolonged by culturing with IL-5 (1 ng/ml) or GM-CSF (0.1 ng/ml) and these effects were completely blocked by each neutralizing Ab. In contrast, neutralizing anti-IL-5 or anti-GM-CSF (20 µg/ml) Abs failed to reverse the effect of RAs. *, p < 0.05.

 
To further establish the profiles of cytokine, chemokine, and growth factor release from eosinophils exposed to 9-cis-RA (10–6 M) and ATRA (10–6 M), Ab arrays were used (Fig. 6). Among the 23 cytokines and chemokines, a significant amount of IL-8 was detected in the supernatants of the cultured eosinophils. However, the production did not differ from that after stimulation with vehicle and RAs, and this was confirmed by ELISA (vehicle control, 129.1 ± 44.8 pg/ml; 9-cis-RA, 97.1 ± 38.5 pg/ml; and ATRA, 97.7 ± 38.1 pg/ml, each value is not statistically significant, n = 3). Remarkably, we found that both 9-cis-RA and ATRA could activate eosinophils to markedly induce the release of MCP-1. Based on this finding, the MCP-1 concentration was also measured by ELISA, revealing increased secretion in the supernatants by stimulation with 9-cis-RA or ATRA (Table III). In addition, because the gene microarray data showed increased M-CSF and VEGF expression, secretion of these was measured by ELISA. Consistent with previous report by Horiuchi et al. (33), IL-5 induced VEGF production. As we expected, M-CSF and VEGF secretion in the supernatants was increased by stimulation with 10–6M 9-cis-RA or ATRA. However, stimulation with each cytokine (100 pg/ml) or combination of them for 24 h did not affect eosinophil survival (vehicle control, 65.1 ± 7.1%; M-CSF, 62.2 ± 7.1%; VEGF, 63.6 ± 7.4%; MCP-1, 65.5 ± 8.5%; and combination of M-CSF, VEGF, and MCP-1, 63.2 ± 8.4%; n = 3). These results indicated that RAs triggered eosinophils to produce M-CSF, VEGF, and MCP-1, but they were not involved in inhibition of spontaneous eosinophil apoptosis.


Figure 6
View larger version (61K):
[in this window]
[in a new window]

  		FIGURE 6. Eosinophils were cultured with 9-cis-RA (10–6 M) or ATRA (10–6 M) for 48 h. The cell culture supernatants were then subjected to a membrane array containing 23 different cytokine/chemokine Abs. 9-cis-RA and ATRA induced eosinophils to release MCP-1 (arrows). Results are representative of n = 2.

 

View this table:
[in this window]
[in a new window]

  		Table III. MCP-1, M-CSF, and VEGF production after stimulation with RAsa

 
RAs reduced mRNA expression and enzymatic activity of caspase 3

Caspases, a family of cysteine proteases, are activated in the interior of cells during the process to apoptosis. Among them, caspase 3 is the final effector caspase whose targets include the DNA nuclease responsible for the characteristic nuclear degradation of apoptosis (34). Because gene microarray data revealed markedly decreased caspase 3 gene expression by RAs, we next confirmed this observation using quantitative real-time RT-PCR. As shown in Fig. 7A, in comparison to the vehicle controls, we found around one-quarter the mRNA levels in RA-stimulated eosinophils. Caspase 3 activity, measured colorimetrically in arbitrary units as the cleavage of the tetrapeptide N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNA), was increased in eosinophils cultured with vehicle for 18 h. In agreement with the initial description of Zangrilli et al. (35), IL-5 (1 ng/ml) reduced activation of caspase 3, and we also observed that 9-cis-RA or ATRA reduced caspase 3 activation to nearly the same extent as IL-5 (Fig. 7B). These results suggested that RAs inhibited caspase 3 expression following enzymatic activity, which resulted in resistance to spontaneous apoptosis.


Figure 7
View larger version (15K):
[in this window]
[in a new window]

  		FIGURE 7. A, The effect of RAs on caspase 3 mRNA expression. Expression levels of the caspase 3 message in eosinophils were assessed by quantitative real-time RT-PCR after incubation with 9-cis-RA (10–6 M) or ATRA (10–6 M) for 4 h. The results are expressed as relative transcript levels with a value of 1 for the respective control (n = 4). RAs markedly reduced caspase 3 mRNA expression in eosinophils. *, p < 0.05 vs vehicle control. B, The effect of RAs on caspase 3 activity. Eosinophils were treated with IL-5 (1 ng/ml), 9-cis-RA (10–6 M), or ATRA (10–6 M) for 18 h, followed by measurement of caspase 3 activity using a colorimetric assay kit (n = 5–6). *, p < 0.05 vs vehicle control (18 h).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Recent accumulating evidence has indicated the important roles of vitamin A in the immune system, especially Th2-type reactions. In the present study, we report the expression of retinoid receptors in human peripheral blood eosinophils and that RAs strikingly inhibited eosinophil spontaneous apoptosis. This novel activity appears to be associated with down-regulation of caspase 3 and inhibition of its enzymatic activity. Additionally, RAs also activated eosinophils to produce proinflammatory cytokines. To our knowledge, this is the first report indicating the functional existence of vitamin A derivatives on human eosinophils. Our study adds additional information on the potential role of vitamin A in regulating immune responses.

Nuclear hormone receptors mediate the important biological function on eosinophils. We previously reported that human eosinophils expressed peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), a heterodimer partner of RXR, and that stimulation of eosinophils with a synthetic PPAR{gamma} agonist inhibited IL-5-induced eosinophil survival by leading apoptosis (22). Based on previous reports that the PPAR{gamma} agonist-induced apoptotic effect was increased by cotreatment with the agonist for RXR (36) or with 9-cis-RA (37), we initially speculated that PPAR{gamma}/RXR activation by each agonist synergistically induced eosinophil apoptosis. However, contrary to our expectation, a preliminary study revealed that cotreatment of eosinophils with 9-cis-RA and a synthetic PPAR{gamma} agonist prolonged cell survival. In this study, because of the strong efficacy of RAs to inhibit eosinophil apoptosis comparable to that of eosinophiliopoietic cytokines such as IL-3, IL-5, and GM-CSF, we investigated the ability of RAs on eosinophils to trigger antiapoptotic protein secretion in cell culture supernatants, although they were not involved in the effect of RAs. Furthermore, the intracellular expression of Bcl-2 and Bcl-xL, which are involved in the process of cytokine-induced inhibition of eosinophil apoptosis (38), were not altered by stimulation with 9-cis-RA or ATRA (our unpublished data). Taken together with the pharmacologic manipulations with the specific agonists and antagonists, the evidence could support the hypothesis that RAs directly inhibit eosinophil spontaneous apoptosis through activation of both RARs and RXRs.

Because little is known about the mechanism behind the antiapoptotic effect of activated nuclear hormone receptors on eosinophils, we used a gene microarray to search for RA-responsive antiapoptotic targets. Among the several transcripts responsive to both 9-cis-RA and ATRA, down-regulation of caspase 3 was screened as the most likely candidate responsible for the eosinophil survival-promoting activity. Spontaneous eosinophil apoptosis involves Bax translocation to the mitochondria, cytochrome c release, and perturbation of the mitochondrial membrane followed by activation of caspases (39). In the case of IL-5-induced eosinophil survival, it is mediated by inhibition of Bax translocation and the consequent activation of the caspase pathway. We were able to show that the inhibition of spontaneous caspase 3 activation was nearly the same extent as that of eosinophiliopoietic cytokine IL-5. Taken together, our data suggest that the caspase 3-mediated spontaneous apoptotic process is, at least in part, involved in RA-induced prolonged eosinophil survival. In contrast, we cannot rule out the possibility that the effects of RAs are mediated by an additional molecular target(s), as indicated in the results from the gene array. For example, peroxiredoxin 6, ranked as the fifth most-increased transcript both in 9-cis-RA- and ATRA-stimulated eosinophils, uses glutathione to catalyze the reduction of reactive oxygen species and protects from reactive oxygen species-induced cytotoxicity in different cell types (40). The second most-decreased transcript, fos, is known to be an AP-1 transcription factor and is involved in the regulation of myeloid differentiation (41), but it is not clear whether it also has other effects on human eosinophils. Thus, further studies are necessary to clarify the involvement of these molecules and/or alternative mechanisms, but such studies will definitely provide interesting insights into the physiological importance of RAs and these molecules on eosinophil survival.

RARs are known to be important regulators of granulopoiesis. Dietary vitamin A-deficient mice exhibited abnormal expansion of myeloid cells probably due to impaired spontaneous apoptosis (42), and mice genetically deficient in both RAR{alpha} and RAR{gamma} displayed an in vitro block in granulocyte differentiation (43). Thus, it is now widely accepted that ATRA is a clinically useful compound that induces terminal granulocyte differentiation and apoptosis of most acute promyelocytic leukemia cells. In addition, retinoids can alter lineage commitment: ATRA suppresses erythroid maturation of normal pluripotent progenitor cells, forcing differentiation toward the neutrophil lineage (44). It was reported that ATRA inhibited eosinophil differentiation of CD34+ cord blood cells (45) and bone marrow mononuclear cells obtained from the hypereosinophilic syndrome (46). In line with these observations, previous studies with eosinophil-committed cell lines such as HL-60 (47) and AML14.3D10 (48) have also indicated the inhibition of eosinophil differentiation by RAs. In contrast to these data that point out the inhibition of survival and differentiation in immature eosinophils by RAs, here we clearly demonstrated that RAs prolong survival in terminal differentiated eosinophils in vitro. This effect was quite reproducible, as it was consistently observed using eosinophils from many different donors. One explanation for this difference is that the effect of RAs is cell type specific and could be changed according to the stage of differentiation. The plasma concentration of RAs has been reported to reach 10–8 M (29), which is sufficient to stimulate eosinophil survival, as shown in our study; hence, RAs may play a role in the regulation of eosinophil homeostasis.

This work also led us to consider the potential role of RAs in allergic inflammatory diseases, namely, bronchial asthma. The majority of the body’s vitamin A is stored in the liver; however, many other organs including the lung have a large concentration of vitamin A (6). Vitamin A derivatives are known to influence the development, maintenance, differentiation, and regeneration of lung epithelial cells (49); hence, it may play a central role in the development of airway diseases. Interestingly, Shoseyov et al. (50) showed that asthmatic rats increased vitamin A utilization by repeated allergen inhalation. Furthermore, it was reported that the serum vitamin A concentration in asthmatic patients in both developing and developed countries was significantly lower than that in normal controls (51, 52). Thus, these studies suggest increased vitamin A utilization in allergic airway inflammation.

Eosinophils contribute to persistent airway inflammation, which leads to airway structural changes called airway remodeling, including subepithelial fibrosis, hyperplasia of mucus glands, myofibroblast and smooth muscle proliferation, and vascular changes (2). In the course of the screening procedure with a gene microarray and a cytokine array, we discovered that RAs induced VEGF and MCP-1 production in cell culture supernatants. In the asthmatic airway, they are produced in abundance and are thought to be involved in the allergic inflammatory processes by activation of their target cells, especially vascular endothelial cells and monocytes/macrophages (53, 54). Another intriguing observation was that RAs induced hypersegmentation of eosinophil nuclei, a morphological abnormality often observed in cells from bronchoalveolar lavage fluid in eosinophilic pneumonia (30, 55). This evidence raises the possibility of RAs as a significant contributor to sustaining the heightened eosinophilic inflammatory response and of anti-RARs as a novel therapeutic strategy in asthma and other allergic inflammatory diseases. Efforts are currently under way to clarify the pathophysiological significance of RAs using an in vivo model.

In conclusion, our current data provide novel insights into the roles of vitamin A in eosinophil homeostasis. Physiological levels of RAs have a potent inhibitory effect on eosinophil spontaneous apoptosis and might facilitate sustainment of allergic inflammation. Our finding is potentially of therapeutic importance, as an anti-RAR/RXR strategy would not only be the antiapoptotic effect on eosinophils but also the action of removing the potential role of RA on producing proinflammatory cytokines.


   Acknowledgments
 
We are grateful to Dr. Hiroyuki Kagechika for the gift of RXR agonist HX630, Yoko Nakamura and Yumiko Kamada for technical assistance, Yoko Oyama for secretary assistance, and Kenji Matsumoto for helpful discussions about gene microarray analysis.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 S.U. and G.M. contributed equally to this study. Back

2 Address correspondence and reprint requests to Dr. Junichi Chihara, Department of Clinical and Laboratory Medicine, Akita University School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan. E-mail address: chihara{at}hos.akita-u.ac.jp Back

3 Abbreviations used in this paper: RA, retinoic acid; ATRA, all-trans RA; MFI, mean fluorescence intensity; PPAR{gamma}, peroxisome proliferator-activated receptor {gamma}; RAR, RA receptor; RXR, retinoid X receptor; PI, propidium iodide; VEGF, vascular endothelial growth factor; RT, reverse transcriptase; TTNPB, [(E)-2–2(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid. Back

Received for publication April 9, 2008. Accepted for publication September 17, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Kita, H., C. R. Adolphson, G. J. Gleich. 2003. Biology of eosinophils. N. F. Adkinson, and J. W. Yunginger, and W. W. Busse, and B. S. Bochner, and S. T. Holgate, and F. E. Simons, eds. Middleton’s Allergy: Principles and Practice 305-332. Mosby, Philadelphia.
  2. Lacy, P., R. Moqbel. 2000. Eosinophil cytokines. Chem. Immunol. 76: 134-155. [Medline]
  3. Simon, H. U.. 2001. Regulation of eosinophil and neutrophil apoptosis-similarities and differences. Immunol. Rev. 179: 156-162. [Medline]
  4. Flood-Page, P. T., A. N. Menzies-Gow, A. B. Kay, D. S. Robinson. 2003. Eosinophil’s role remains uncertain as anti-interleukin-5 only partially depletes numbers in asthmatic airway. Am. J. Respir. Crit. Care Med. 167: 199-204. [Abstract/Free Full Text]
  5. Bartemes, K. R., K. M. Cooper, K. L. Drain, H. Kita. 2005. Secretory IgA induces antigen-independent eosinophil survival and cytokine production without inducing effector functions. J. Allergy Clin. Immunol. 116: 827-835. [Medline]
  6. De Luca, L. M.. 1991. Retinoids and their receptors in differentiation, embryogenesis, and neoplasia. FASEB J. 5: 2924-2933. [Abstract]
  7. Altucci, L., H. Gronemeyer. 2001. Nuclear receptors in cell life and death. Trends Endocrinol. Metab. 12: 460-468. [Medline]
  8. Chambon, P.. 1996. A decade of molecular biology of retinoic acid receptors. FASEB J. 10: 940-954. [Abstract]
  9. Villamor, E., W. W. Fawzi. 2000. Vitamin A supplementation: implications for morbidity and mortality in children. J. Infect. Dis. 182: (Suppl. 1):S122-S133. [Medline]
  10. Sommer, A., I. Tarwotjo, G. Hussaini, D. Susanto. 1983. Increased mortality in children with mild vitamin A deficiency. Lancet 2: 585-588. [Medline]
  11. Glasziou, P. P., D. E. Mackerras. 1993. Vitamin A supplementation in infectious diseases: a meta-analysis. BMJ 306: 366-370. [Abstract/Free Full Text]
  12. Carman, J. A., L. Pond, F. Nashold, D. L. Wassom, C. E. Hayes. 1992. Immunity to Trichinella spiralis infection in vitamin A-deficient mice. J. Exp. Med. 175: 111-120. [Abstract/Free Full Text]
  13. Stephensen, C. B., R. Rasooly, X. Jiang, M. A. Ceddia, C. T. Weaver, R. A. Chandraratna, R. P. Bucy. 2002. Vitamin A enhances in vitro Th2 development via retinoid X receptor pathway. J. Immunol. 168: 4495-4503. [Abstract/Free Full Text]
  14. Dawson, H. D., G. Collins, R. Pyle, M. Key, A. Weeraratna, V. Deep-Dixit, C. N. Nadal, D. D. Taub. 2006. Direct and indirect effects of retinoic acid on human Th2 cytokine and chemokine expression by human T lymphocytes. BMC Immunol. 7: 27[Medline]
  15. Cantorna, M. T., F. E. Nashold, C. E. Hayes. 1994. In vitamin A deficiency multiple mechanisms establish a regulatory T helper cell imbalance with excess Th1 and insufficient Th2 function. J. Immunol. 152: 1515-1522. [Abstract]
  16. Iwata, M., Y. Eshima, H. Kagechika. 2003. Retinoic acids exert direct effects on T cells to suppress Th1 development and enhance Th2 development via retinoic acid receptors. Int. Immunol. 15: 1017-1025. [Abstract/Free Full Text]
  17. Neuman, I., H. Nahum, A. Ben-Amotz. 1999. Prevention of exercise-induced asthma by a natural isomer mixture of β-carotene. Ann. Allergy Asthma Immunol. 82: 549-553. [Medline]
  18. McKeever, T. M., J. Britton. 2004. Diet and asthma. Am. J. Respir. Crit. Care Med. 170: 725-729. [Free Full Text]
  19. Mawson, A. R.. 2001. Could bronchial asthma be an endogenous, pulmonary expression of retinoid intoxication?. Front Biosci. 6: D973-D985. [Medline]
  20. Umemiya, H., H. Fukasawa, M. Ebisawa, L. Eyrolles, E. Kawachi, G. Eisenmann, H. Gronemeyer, Y. Hashimoto, K. Shudo, H. Kagechika. 1997. Regulation of retinoidal actions by diazepinylbenzoic acids: retinoid synergists which activate the RXR-RAR heterodimers. J. Med. Chem. 40: 4222-4234. [Medline]
  21. Kobayashi, Y., S. Ueki, G. Mahemuti, T. Chiba, H. Oyamada, N. Saito, A. Kanda, H. Kayaba, J. Chihara. 2005. Physiological levels of 15-deoxy-{Delta}12,14-prostaglandin J2 prime eotaxin-induced chemotaxis on human eosinophils through peroxisome proliferator-activated receptor-{gamma} ligation. J. Immunol. 175: 5744-5750. [Abstract/Free Full Text]
  22. Ueki, S., T. Adachi, J. Bourdeaux, H. Oyamada, Y. Yamada, K. Hamada, A. Kanda, H. Kayaba, J. Chihara. 2003. Expression of PPAR{gamma} in eosinophils and its functional role in survival and chemotaxis. Immunol. Lett. 86: 183-189. [Medline]
  23. Rosewicz, S., F. Brembeck, A. Kaiser, Z. V. Marschall, E. O. Riecken. 1996. Differential growth regulation by all-trans retinoic acid is determined by protein kinase C{alpha} in human pancreatic carcinoma cells. Endocrinology 137: 3340-3347. [Abstract]
  24. Hayashida, K., A. Daiba, A. Sakai, T. Tanaka, K. Kaji, N. Inaba, S. Ando, N. Kajiyama, H. Terasaki, A. Abe, et al 2005. Pretreatment prediction of interferon-{alpha} efficacy in chronic hepatitis C patients. Clin. Gastroenterol. Hepatol. 3: 1253-1259. [Medline]
  25. Daiba, A., N. Inaba, S. Ando, N. Kajiyama, H. Yatsuhashi, H. Terasaki, A. Ito, M. Ogasawara, A. Abe, J. Yoshioka, et al 2004. A low-density cDNA microarray with a unique reference RNA: pattern recognition analysis for IFN efficacy prediction to HCV as a model. Biochem. Biophys. Res. Commun. 315: 1088-1096. [Medline]
  26. Khodursky, A. B., B. J. Peter, N. R. Cozzarelli, D. Botstein, P. O. Brown, C. Yanofsky. 2000. DNA microarray analysis of gene expression in response to physiological and genetic changes that affect tryptophan metabolism in Escherichia coli. Proc. Natl. Acad. Sci. USA 97: 12170-12175. [Abstract/Free Full Text]
  27. Yang, Y. H., S. Dudoit, P. Luu, D. M. Lin, V. Peng, J. Ngai, T. P. Speed. 2002. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res. 30: e15[Abstract/Free Full Text]
  28. Fritsche, J., T. J. Stonehouse, D. R. Katz, R. Andreesen, M. Kreutz. 2000. Expression of retinoid receptors during human monocyte differentiation in vitro. Biochem. Biophys. Res. Commun. 270: 17-22. [Medline]
  29. van Vliet, T., E. Boelsma, A. J. de Vries, H. van den Berg. 2001. Retinoic acid metabolites in plasma are higher after intake of liver paste compared with a vitamin A supplement in women. J. Nutr. 131: 3197-3203. [Abstract/Free Full Text]
  30. Chihara, J., D. Kurachi, T. Yamamoto, H. Yamada, N. Hayashi, S. Nakajima. 1995. Possible involvement of mononuclear cells stimulated with specific allergen from asthmatic patients in ICAM-1 expression on the eosinophilic cell line. Immunol. Lett. 46: 241-244. [Medline]
  31. Newcomer, M. E., R. S. Pappas, D. E. Ong. 1993. X-ray crystallographic identification of a protein-binding site for both all-trans- and 9-cis-retinoic acid. Proc. Natl. Acad. Sci. USA 90: 9223-9227. [Abstract/Free Full Text]
  32. Umemiya, H., H. Fukasawa, M. Ebisawa, L. Eyrolles, E. Kawachi, G. Eisenmann, H. Gronemeyer, Y. Hashimoto, K. Shudo, H. Kagechika. 1997. Regulation of retinoidal actions by diazepinylbenzoic acids: retinoid synergists which activate the RXR-RAR heterodimers. J. Med. Chem. 40: 4222-4234. [Medline]
  33. Horiuchi, T., P. F. Weller. 1997. Expression of vascular endothelial growth factor by human eosinophils: upregulation by granulocyte macrophage colony-stimulating factor and interleukin-5. Am. J. Respir. Cell Mol. Biol. 17: 70-77. [Abstract/Free Full Text]
  34. Enari, M., H. Sakahira, H. Yokoyama, K. Okawa, A. Iwamatsu, S. Nagata. 1998. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391: 43-50. [Medline]
  35. Zangrilli, J., N. Robertson, A. Shetty, J. Wu, A. Hastie, J. E. Fish, G. Litwack, S. P. Peters. 2000. Effect of IL-5, glucocorticoid, and Fas ligation on Bcl-2 homologue expression and caspase activation in circulating human eosinophils. Clin. Exp. Immunol. 120: 12-21. [Medline]
  36. Schlezinger, J. J., G. J. Howard, C. H. Hurst, J. K. Emberley, D. J. Waxman, T. Webster, D. H. Sherr. 2004. Environmental and endogenous peroxisome proliferator-activated receptor {gamma} agonists induce bone marrow B cell growth arrest and apoptosis: interactions between mono(2-ethylhexyl)phthalate, 9-cis-retinoic acid, and 15-deoxy-{Delta}12,14-prostaglandin J2. J. Immunol. 173: 3165-3177. [Abstract/Free Full Text]
  37. Schlezinger, J. J., B. A. Jensen, K. K. Mann, H. Y. Ryu, D. H. Sherr. 2002. Peroxisome proliferator-activated receptor {gamma}-mediated NF-{kappa} B activation and apoptosis in pre-B cells. J. Immunol. 169: 6831-6841. [Abstract/Free Full Text]
  38. Dibbert, B., I. Daigle, D. Braun, C. Schranz, M. Weber, K. Blaser, U. Zangemeister-Wittke, A. N. Akbar, H. U. Simon. 1998. Role for Bcl-xL in delayed eosinophil apoptosis mediated by granulocyte-macrophage colony-stimulating factor and interleukin-5. Blood 92: 778-783. [Abstract/Free Full Text]
  39. Dewson, G., G. M. Cohen, A. J. Wardlaw. 2001. Interleukin-5 inhibits translocation of Bax to the mitochondria, cytochrome c release, and activation of caspases in human eosinophils. Blood 98: 2239-2247. [Abstract/Free Full Text]
  40. Kümin, A., C. Huber, T. Rülicke, E. Wolf, S. Werner. 2006. Peroxiredoxin 6 is a potent cytoprotective enzyme in the epidermis. Am. J. Pathol. 169: 1194-1205. [Abstract/Free Full Text]
  41. Baltus, B., T. B. van Dijk, E. Caldenhoven, E. Zanders, J. A. Raaijmakers, J. W. Lammers, L. Koenderman, R. P. de Groot. 1998. An AP-1 site in the promoter of the human IL-5R{alpha} gene is necessary for promoter activity in eosinophilic HL60 cells. FEBS Lett. 434: 251-254. [Medline]
  42. Kuwata, T., I. M. Wang, T. Tamura, R. M. Ponnamperuma, R. Levine, K. L. Holmes, H. C. Morse, L. M. De Luca, K. Ozato. 2000. Vitamin A deficiency in mice causes a systemic expansion of myeloid cells. Blood 95: 3349-3356. [Abstract/Free Full Text]
  43. Labrecque, J., D. Allan, P. Chambon, N. N. Iscove, D. Lohnes, T. Hoang. 1998. Impaired granulocytic differentiation in vitro in hematopoietic cells lacking retinoic acid receptors {alpha}1 and {gamma}. Blood 92: 607-615. [Abstract/Free Full Text]
  44. Labbaye, C., M. Valtieri, U. Testa, A. Giampaolo, E. Meccia, P. Sterpetti, I. Parolini, E. Pelosi, D. Bulgarini, Y. E. Cayre, C. Peschie. 1994. Retinoic acid downmodulates erythroid differentiation and GATA1 expression in purified adult-progenitor culture. Blood 83: 651-656. [Abstract/Free Full Text]
  45. Upham, J. W., R. Sehmi, L. M. Hayes, K. Howie, J. Lundahl, J. A. Denburg. 2002. Retinoic acid modulates IL-5 receptor expression and selectively inhibits eosinophil-basophil differentiation of hemopoietic progenitor cells. J. Allergy Clin. Immunol. 109: 307-313. [Medline]
  46. Robert, C., L. Delva, N. Balitrand, S. Nahajevszky, T. Masszi, C. Chomienne, B. Papp. 2006. Apoptosis induction by retinoids in eosinophilic leukemia cells: implication of retinoic acid receptor-{alpha} signaling in all-trans-retinoic acid hypersensitivity. Cancer Res. 66: 6336-6344. [Abstract/Free Full Text]
  47. Leber, B. F., J. A. Denburg. 1997. Retinoic acid modulation of induced basophil differentiation. Allergy 52: 1201-1206. [Medline]
  48. Paul, C. C., S. Mahrer, M. Tolbert, B. L. Elbert, I. Wong, S. J. Ackerman, M. A. Baumann. 1995. Changing the differentiation program of hematopoietic cells: retinoic acid-induced shift of eosinophil-committed cells to neutrophils. Blood 86: 3737-3744. [Abstract/Free Full Text]
  49. Harik-Khan, R. I., D. C. Muller, R. A. Wise. 2004. Serum vitamin levels and the risk of asthma in children. Am. J. Epidemiol. 159: 351-357. [Abstract/Free Full Text]
  50. Shoseyov, D., H. Bibi, H. Biesalski, R. Reifen. 2002. Repeated allergen challenge in rats increases vitamin A consumption. Chest 122: 1407-1411.
  51. Mizuno, Y., T. Furusho, A. Yoshida, H. Nakamura, T. Matsuura, Y. Eto. 2006. Serum vitamin A concentrations in asthmatic children in Japan. Pediatr. Int. 48: 261-264. [Medline]
  52. Arora, P., V. Kumar, S. Batra. 2002. Vitamin A status in children with asthma. Pediatr. Allergy Immunol. 13: 223-226. [Medline]
  53. Simcock, D. E., V. Kanabar, G. W. Clarke, B. J. O'Connor, T. H. Lee, S. J. Hirst. 2007. Proangiogenic activity in bronchoalveolar lavage fluid from patients with asthma. Am. J. Respir. Crit. Care Med. 176: 146-153. [Abstract/Free Full Text]
  54. Tillie-Leblond, I., H. Hammad, S. Desurmont, J. Pugin, B. Wallaert, A. B. Tonnel, P. Gosset. 2000. CC chemokines and interleukin-5 in bronchial lavage fluid from patients with status asthmaticus: potential implication in eosinophil recruitment. Am. J. Respir. Crit. Care Med. 162: 586-592. [Abstract/Free Full Text]
  55. Fujimura, M., M. Yasui, S. Shinagawa, M. Nomura, T. Matsuda. 1998. Bronchoalveolar lavage cell findings in three types of eosinophilic pneumonia: acute, chronic and drug-induced eosinophilic pneumonia. Respir. Med. 92: 743-749. [Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
J. Carlens, B. Wahl, M. Ballmaier, S. Bulfone-Paus, R. Forster, and O. Pabst
Common {gamma}-Chain-Dependent Signals Confer Selective Survival of Eosinophils in the Murine Small Intestine
J. Immunol., November 1, 2009; 183(9): 5600 - 5607.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ueki, S.
Right arrow Articles by Chihara, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ueki, S.
Right arrow Articles by Chihara, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS