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Keywords:

  • allergic asthma;
  • GW501516;
  • GW9578;
  • rosiglitazone

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References
  10. Supporting Information

Peroxisome proliferator-activated receptor (PPAR) agonists have been suggested as novel therapeutics for the treatment of inflammatory lung disease, such as allergic asthma. Treatment with PPAR agonists has been shown to inhibit airway eosinophilia in murine models of allergic asthma, which can occur through several mechanisms including attenuated generation of chemoattractants (e.g. eotaxin) and decreased eosinophil migrational responses. In addition, studies report that PPAR agonists can inhibit the differentiation of several cell types. To date, no studies have examined the effects of PPAR agonists on interleukin-5 (IL-5) -induced eosinophil differentiation from haemopoietic progenitor cells. Non-adherent mononuclear cells or CD34+ cells isolated from the peripheral blood of allergic subjects were grown for 2 weeks in Methocult® cultures with IL-5 (10 ng/ml) and IL-3 (25 ng/ml) in the presence of 1–1000 nm PPARα agonist (GW9578), PPARβ/δ agonist (GW501516), PPARγ agonist (rosiglitazone) or diluent. The number of eosinophil/basophil colony-forming units (Eo/B CFU) was quantified by light microscopy. The signalling mechanism involved was assessed by phosphoflow. Blood-extracted CD34+ cells cultured with IL-5 or IL-5 + IL-3 formed Eo/B CFU, which were significantly inhibited by rosiglitazone (100 nm, P < 0·01) but not GW9578 or GW501516. In addition, rosglitazone significantly inhibited IL-5-induced phosphorylation of extracellular signal-regulated kinase 1/2. We observed an inhibitory effect of rosiglitazone on eosinophil differentiation in vitro, mediated by attenuation of the extracellular signal-regulated kinase 1/2 signalling pathway. These findings indicate that the PPARγ agonist can attenuate tissue eosinophilia by interfering with local differentiative responses.


Abbreviations
Eo/B CFU

eosinophil/basophil colony-forming units

ERK1/2

extracellular signal-regulation kinase-1

IL-3

interleukin-3

IL-5

interleukin-5

IL-5Rα

interleukin-5 receptor-α

JAK/STAT

Janus kinase/signal transducer and activator of transcription

MAPK

mitogen-activated protein kinase

NAMNCs

non-adherent mononuclear cells

PPAR

peroxisome proliferator-activated receptor

SMFI

specific minus isotype geometric mean fluorescence intensity

STAT

signal transducer and activator of transcription

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References
  10. Supporting Information

Asthma therapy is focused on reducing the associated symptoms and frequency of exacerbation through modification of the mechanisms of lung inflammation (corticosteroids) or bronchoconstriction (β2-adrenergic receptor agonist), or both. Most asthma research is centred on developing new drugs that target the inflammatory pathways that lead to bronchoconstriction and airway damage. In addition to the mature inflammatory component, there is a role for the development of inflammation through haemopoietic progenitor cells. Inflammation is associated with the ability of the bone marrow and affected tissues to support the mobilization, proliferation and in situ differentiation of haemopoietic progenitors. Haemopoietic myeloid progenitors represent an important therapeutic target because of their contribution to the ongoing recruitment of pro-inflammatory cells, such as eosinophils and basophils, to target tissue sites in allergic diseases.

In culture, blood from allergic subjects grows greater numbers of eosinophil/basophil colony-forming units (Eo/B CFU) compared with controls.[1] There are also increased numbers of circulating CD34+ progenitor cells in the peripheral blood of atopic subjects compared with non-atopic.[2] An increase in Eo/B CFU was observed in the peripheral blood of asthmatic patients during an exacerbation and the numbers decreased upon recovery from symptoms.[3] A significant increase in circulating Eo/B CFU and CD34+ cells is observed 24-hr after whole lung allergen challenge in atopic asthmatic subjects who develop a dual response (early-phase and late-phase bronchoconstriction).[4, 5] This is also associated with the up-regulation of interleukin-5 receptor α chain (IL-5Rα) expression on CD34+ cells in the bone marrow.[6]

In bronchial biopsies an increase in CD34+ cell numbers is observed in asthmatic and atopic non-asthmatic subjects.[7] In contrast, CD34+ IL-5Rα mRNA+ cells increase in asthmatic subjects only.[7] Furthermore, CD34+ IL-5Rα mRNA+ cells correlate with eosinophil numbers.[7] When stimulated with allergen or recombinant human IL-5, the number of major basic protein immunoreactive cells (eosinophils) increases in cultured human nasal mucosa that was obtained from individuals with seasonal allergic rhinitis.[8] These findings indicate that a subset of tissue eosinophils arise from the local IL-5-driven outgrowth of progenitor cells, a process termed in situ differentiation.[8]

More recent studies have shown that airway smooth muscle stimulates eosinophil differentiation and maturation of progenitor cells. This suggests that lung structural cells can promote local eosinophilia by stimulating in situ differentiation from lineage committed progenitor cells.[9] As IL-5 is the principal regulatory cytokine for the differentiation of eosinophils[10] and eosinophils represent a major effector cell in asthma,[11] CD34+ haemopoietic progenitor cells may be an important therapeutic target for the treatment of allergic asthma.

Treatment with mepolizumab, anti-IL-5 therapy, is ineffective at completely attenuating eosinophil numbers in bronchial biopsies from asthmatic subjects, which is the site of disease pathology.[12] However, treatment improves asthma control and significantly reduces the prednisone dose required by asthmatic patients with persistent eosinophilic bronchitis.[13] Researchers continue to search for a more effective strategy to completely ablate eosinophils at the site of disease, including investigations into drugs that target eosinophil progenitor cells.[14, 15] This may lead to a more effective therapy for controlling asthma.

Several reviews suggest that peroxisome proliferator-activated receptors (PPARs) are novel anti-inflammatory targets[16, 17] and PPARα as well as PPARγ play a role in chronic inflammatory disease.[18] Treatment with PPAR agonists inhibits airway eosinophilia in murine models of allergic asthma,[19] which occurs through several mechanisms including a decrease cytokine/chemoattractant (IL-5/eotaxin) release, a decrease in eosinophil migration and/or a decrease in eosinophil differentiation within the tissue. Previously, we have shown that PPAR agonists inhibit eosinophil chemotaxis in vitro.[20] The anti-inflammatory capability of PPARγ agonists could also include inhibitory effects on differentiation.

PPARγ is expressed in adipose tissue and is critical for adipocyte differentiation.[21, 22] PPARγ is also expressed in haemopoietic tissues, such as bone marrow stromal cells and CD34+ progenitor cells.[23] PPARγ agonists suppress the proliferation and differentiation phenotype of cell lines for human B lymphocytic and erythroid leukaemia.[24, 25] Canines treated with thiazolidinedione have reduced bone marrow erythropoiesis.[26] Furthermore, troglitazone inhibits proliferation, without inducing apoptosis, and delayed the maturation of erythroid colony-forming cells purified from human peripheral blood.[27] These previous studies show inhibition of differentiation in several cell types, including erythroid cells.[27-29] However, no studies address the effects of PPARs on IL-5-induced eosinophilopoiesis.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References
  10. Supporting Information
Subjects

Forty allergic non-smoking subjects (18–65 years) donated blood for the study. Allergies were determined by skin-prick testing. Subjects used no regular medication other than an inhaled β2-agonist infrequently (less than twice weekly) as required to treat their symptoms. All medications were withheld for at least 48 hr before the blood draw. The study was approved by the FHS/HHS Research Ethics Board, and subjects gave informed consent to participate.

Flow cytometry for PPARγ expression

Peripheral blood mononuclear cells were stained for PPARγ expression using mouse anti-human allophycocyanin-conjugated CD45, Peridinin chlorophyll protein-conjugated CD34, phycoerythrin-conjugated IL5-Rα (Becton-Dickinson Biosciences, Mississauga, ON, Canada) and intracellular FITC-conjugated PPARγ (E-8; Cayman Chemicals, Ann Arbor, MI), as well as the appropriate isotype control antibodies. Cells were permeabilized with BD Cytoperm™ buffer to internalize the PPARγ antibody (BD Biosciences). Using standard FACS methods, the isotype control was set at 2% positive. A minimum of 3000 CD45+ CD34+ cells were collected for analysis. Two parameters were determined from this gating strategy, the per cent stained and the specific minus isotype geometric mean fluorescence intensity (SMFI). Cells were acquired with an LSR II flow cytometer (Becton Dickinson Instrument Systems; Becton-Dickinson) using the FACSDiva software program (Becton-Dickinson Biosciences).

Progenitor cell purification

Peripheral blood (100 ml) was collected into sodium heparin vacutainers, diluted 1 : 1 with McCoy's 5A (Invitrogen Canada Inc, Burlington, ON, Canada) and mononuclear cells were purified by centrifugation on an accuprep™ density gradient (Accurate Chemical & Scientific Corporation, Westbury, NY). Monocytes were depleted by adherence to plastic (2-hr, 5% CO2 and 37°). CD34+ progenitor cells were enriched by positive selection using MACS immunomagnetic beads (Miltenyi Biotec, Auburn, CA). Following isolation, the cell population obtained had a purity of 95% (± 1 SEM) and all cells were > 95% viable (see Supporting information, Fig. S1).

Progenitor cell cultures

CD34-containing non-adherent mononuclear cells (NAMNCs) or purified CD34+ progenitor cells were resuspended in Iscove's 2+ (Iscove's modified Dulbecco's medium with 1% penicillin/streptomycin and 1% 2-mercaptoethanol) and placed in Methocult® cultures (Stemcell Technologies, Vancouver, BC, Canada) in the presence of 16% fetal bovine serum and 10 ng/ml IL-5 or IL-5 and IL-3 (25 ng/ml).

The NAMNC cells were cultured at a concentration of 0·25 × 106 cells/ml and the CD34+ cells were cultured at a concentration of 0·016 × 106 cells/ml. The cells were co-cultured, with diluent (Iscove's 2+ with 0·04% DMSO), or with 1–1000 nm of a PPARα agonist (GW9578; Cayman Chemical, Ann Arbor, MI), PPARβ/δ agonist (GW501516; Axxora LLC, San Diego, CA) or PPARγ agonist (rosiglitazone; Cayman Chemical), for 2 weeks at 5% CO2, with high humidity at 37°. The number of Eo/B CFU was quantified in duplicate plates using an inverted light microscope at 40× magnification (see Supporting information, Fig. S2). Quantification methods were based on those previously published.[2] A colony was defined as a cluster of eosinophils/basophils with a minimum density of 40 cells, which could be verified by positive staining for chromotrope 2R (Fig. S2). The average number of CFU per plate was determined.

Measurement of phosphorylated of extracellular signal-regulated kinase 1/2 in progenitor cells

Purified CD34+ progenitor cells were resuspended in complete RPMI-1640 in the presence of IL-5 (10 ng/ml). The cells were co-cultured in the presence of diluent (complete RPMI with 0·04% DMSO), or 1000 nm of the PPARγ agonist (rosiglitazone) for 15 min in 5% CO2 with high humidity and at 37°. Timing was optimized using IL-5 and various incubation times (0, 2, 5, 15, 30 and 45 min). Cells were then fixed with BD Phosflow fix buffer (Becton-Dickinson Biosciences) for 15 min at 37°. BD Phosflow Perm buffer III (Becton-Dickinson Biosciences) at −20° was slowly added to permeabilize the cells (30 min on ice) and they were resuspended in mouse block buffer (5% mouse serum, 5% human serum in FACS buffer) for 15 min. Cells were stained with mouse anti-human CD34-Pacific Blue (eBioscience, San Diego, CA), CD45-FITC and phos-extracellular signal-regulated kinase 1/2 (ERK1/2)-phycoerythrin (Becton-Dickinson Biosciences), or the isotype control antibody. The gating strategy used to determine the CD34+ progenitor population was previously developed.[5] Three thousand CD45+ CD34+ cells were acquired with an LSR II flow cytometer (Becton Dickinson Instrument Systems; Becton-Dickinson) using the FACSDiva software program (Becton-Dickinson Biosciences).

Statistical analysis

All data are expressed as mean ± standard error of the mean unless otherwise stated. Analysis of variance with repeated measures was used to compare PPAR agonist treatments versus diluent at the various concentrations, with post hoc Tukey tests for pre-specified comparisons. For data not normally distributed, the statistics were performed on the log-transformed data. Statistically significant differences were accepted at P < 0·05.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References
  10. Supporting Information

Subjects

Subjects recruited for the study were 32 ± 2·9 years old with 16 male and 24 female volunteers. All subjects had a positive skin-prick test.

Expression of PPARγ in CD34+ cell populations

PPARγ was expressed in haemopoietic progenitor cells (CD45+ CD34+) and eosinophil lineage committed progenitor cells (CD45+ CD34+ IL5Rα+) at baseline in cells isolated from the peripheral blood of allergic subjects (Table 1).

Table 1. The expression of peroxisome proliferator-activated receptor γ (PPAR-γ) on CD45+ CD34+ and CD45+ CD34/IL5Rα+ cells. Data are arithmetic mean ± standard error of the mean
 % of CD45+ CD34+SMFI
  1. SMFI, specific minus isotype geometric mean fluorescence intensity.

CD45+ CD34+ PPARγ+20 ± 101807 ± 258
CD45+ CD34+ IL5Rα+ PPARγ+22 ± 132004 ± 347

The effects of rosiglitazone on NAMNC cultures

NAMNCs grew significantly higher numbers of Eo/B CFU in response to IL-5 compared with a negative control (11·6 ± 1·8 versus 2·5 ± 0·4 Eo/B CFU per 250 000 NAMNCs, P < 0·001). Incubation with 10–1000 nm rosiglitazone significantly inhibited IL-5-induced Eo/B CFU formation [rosiglitazone (10 nm) 6·6 ± 1·0 versus diluent 11·6 ± 1·8 Eo/B CFU per 250 000 NAMNCs, P < 0·01; Fig. 1a]. The maximum inhibition of Eo/B CFU was 42·8% at the 10 nm dose. Troglitazone, another PPARγ agonist, was also examined to determine the robustness of the inhibitory response. Troglitazone treatment significantly decreased Eo/B CFU at a 2000 nm dose compared with diluent (6·7 ± 0·9 versus 11·1 ± 1·4 Eo/B CFU per 250 000 NAMNCs, P < 0·01, Fig. 1b).

image

Figure 1. The effect of peroxisome proliferator-activated receptor γ (PPARγ) agonists, rosiglitazone (a) and troglitazone (b), on interleukin-5 (IL-5) -induced eosinophil differentiation of non-adherent mononuclear cells isolated from the peripheral blood of atopic subjects (= 15). *P < 0·01 compared with diluent control (stimulation with IL-5 in the absence of PPAR agonist). δ P < 0·001 compared with negative control (stimulation with PBS in the absence of PPAR agonist). Data are shown as arithmetic mean ± standard error of the mean.

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The effects of PPAR agonists on CD34+ progenitor cultures

An enriched population of CD34+ progenitor cells cultured with IL-5 formed significantly more Eo/B CFU compared with a negative control (4·9 ± 1·00 versus 0·05 ± 0·02 Eo/B CFU per 16 000 CD34+ cells, P < 0·001). Incubation with 100–1000 nm rosiglitazone significantly inhibited IL-5-induced Eo/B CFU formation [rosiglitazone (100 nm) 2·7 ± 0·6 versus diluent 4·9 ± 1·0 Eo/B CFU per 16 000 CD34+ cells, P < 0·01, Fig. 2a]. The maximum inhibition was at the 100 nm dose (45·9% decrease, P < 0·01). Conversely, there was no effect of GW9578 or GW501516 on IL-5-induced differentiation (Fig. 2b, c). In CD34+ progenitor cell cultures we demonstrated significantly higher Eo/B CFU in response to co-stimulation with IL-5 plus IL-3 compared with negative control (12·50 ± 4·0 versus 0·5 ± 0·05 Eo/B CFU per 16 000 CD34+ cells, P < 0·05). Incubation with 1 and 100 nm rosiglitazone significantly inhibited IL-5 + IL-3 induced Eo/B CFU formation [rosiglitazone (1 nm) 5·8 ± 1·7 versus diluent 13·0 ± 4·0 Eo/B CFU, P < 0·05, Fig. 3]. The maximum inhibition was 50·8% at the 100 nm dose of rosiglitazone. The effects of GW9578 and GW501516 on IL-5 + IL-3 co-cultures were not examined.

image

Figure 2. The effect of rosiglitazone [peroxisome proliferator-activated receptor γ (PPARγ) agonist] (a), GW9578 (PPARα agonist) (b), and GW501516 (PPARβ/δ agonist) (c) on IL-5-induced eosinophil differentiation of CD34+ cells isolated from peripheral blood of atopic subjects (= 15). *P < 0·01 compared with diluent control (stimulation with IL-5 in the absence of PPAR agonist). δ P < 0·001 compared with negative control (stimulation with PBS in the absence of PPAR agonist). Data are shown as arithmetic mean ± standard error of the mean.

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image

Figure 3. The effect of rosiglitazone [peroxisome proliferator-activated receptor γ (PPARγ) agonist] on interleukin-5 (IL-5) + IL-3-induced eosinophil differentiation in CD34+ cells isolated from peripheral blood of atopic subjects (= 10) *P < 0·05 compared with diluent control (stimulation with IL-5 + IL-3 in the absence of PPAR agonist). δ P < 0·05 compared with negative control (stimulation with PBS in the absence of PPAR agonist). Data are shown as arithmetic mean ± standard error of the mean.

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The effects of PPAR agonists on phosphorylation of ERK1/2

To examine the pathways involved in PPAR agonist inhibition of IL-5-induced eosinophil differentiation, ERK1/2 phosphorylation was measured. Phosphorylation was measured following stimulation with IL-5 (10 ng/ml) at varying incubation times (0, 1, 2, 5, 15, 30 45, 60 min, and expressed as the ratio of SMFI from pre-stimulation baseline. A significant increase in phospho-ERK1/2 was detected at 15 min post-stimulation Fig. 4) and this time-point was selected to examine the effect of rosiglitazone on IL-5-induced ERK1/2 stimulation. Interleukin-5 stimulation for 15 min induced a significant increase in ERK1/2 phosphorylation compared with a negative control (157·4 ± 19·2 versus 115·9 ± 16·7 SMFI, P < 0·05) which was inhibited by rosiglitazone [rosiglitazone (1000 nm) 119·7 ± 10·5 versus diluent 157·4 ± 19·2 SMFI, P < 0·05, Fig. 5]. Upon expanding the time–course to evaluate the effect of PPAR agonists on IL-5 stimulation at later time-points, we observed no effect of rosiglitazone (Fig. 6) suggesting that the effect of rosiglitazone is not likely to be the result of delayed responses. Treatment with rosiglitazone (1000 nm) had no effect on viability (> 98% viable). We examined alternative IL-5 signalling pathways, but using a flow cytometry assay we could not observe significant phosphorylation of signal transducer and activator of transcription (STAT5) or p38 by IL-5 stimulation (Fig. 7). Although other mechanisms may be involved, these results show that rosiglitazone inhibits IL-5 signalling through the ERK1/2 pathway.

image

Figure 4. Time–course of extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in CD34+ cells after interleukin-5 (IL-5) stimulation (= 8). Data are the ratio of the specific minus isotype geometric mean fluorescence intensity (SMFI) from baseline and shown as arithmetic mean ± standard error of the mean.

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image

Figure 5. The effect of rosiglitazone [a peroxisome proliferator-activated receptor γ (PPARγ) agonist] on interleukin-5 (IL-5) -induced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in CD34+ cells isolated from peripheral blood of atopic subjects (= 8). *P < 0·05. Data are arithmetic mean ± standard error of the mean. SMFI, specific minus isotype geometric mean fluorescence intensity.

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image

Figure 6. The effect of rosiglitazone [a peroxisome proliferator-activated receptor γ (PPARγ) agonist] on the time–course of interleukin-5 (IL-5)-induced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in CD34+ cells isolated from the peripheral blood of atopic subjects (= 8). Data are the specific minus isotype geometric mean fluorescence intensity (SMFI) ratio from baseline and shown as arithmetic mean ± standard error of the mean.

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image

Figure 7. Time–course for interleukin-5 (IL-5)-induced signal transducer and activator of transcription 5 (STAT5) and p38 phosphorylation in CD34+ cells isolated from the peripheral blood of atopic subjects (= 8). Data are the specific minus isotype geometric mean fluorescence intensity (SMFI) ratio from baseline and shown as arithmetic mean ± standard error of the mean.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References
  10. Supporting Information

This is the first study to show that a PPARγ agonist inhibits eosinophil differentiation in vitro when added to cultures of NAMNCs and purified CD34+ progenitor cells collected from the peripheral blood of allergic subjects. We demonstrated that rosiglitazone treatment was effective in reducing the number of Eo/B CFU in cultures of NAMNCs and CD34+ progenitor cells grown with IL-5, and in cultures with IL-5 and IL-3 co-stimulation. We have described an attenuation of IL-5-activated ERK1/2 phosphorylation after rosiglitazone treatment in CD34+ progenitor cells. STAT5, ERK1/2 and p38 pathways have all been implicated in eosinophil differentiation.[30-33] During IL-5 and IL-3 co-stimulation rosiglitazone was still able to inhibit eosinophil differentiation, showing its continued effectiveness in the presence of other growth factors. Furthermore, the p38 pathway plays a greater role than ERK1/2 during IL-3-induced differentiation,[32] which suggests that rosiglitazone may inhibit more than just the ERK1/2 pathway. Although CD34+ cells used in these experiments were shown to express PPARγ, we cannot rule out the possibility that the effect of rosiglitazone on eosinophil progenitors is acting indirectly through contaminating cells. Differentiation of progenitor cells into eosinophils is an important mechanism for expansion of the eosinophil population in inflammatory lung disease.

It has been shown that bone marrow progenitor commitment to the Eo/B lineage is regulated by IL-3, IL-5 and granulocyte–macrophage colony-stimulating factor.[34, 35] Interleukin-5 induces eosinophil differentiation from the Eo/B-committed progenitor in vivo and when co-cultured with IL-3 there is an additive effect to the number of colonies.[36, 37] Intravenous IL-5 increases the number of progenitor cells in the peripheral blood and increases the expression of CCR3 on mature eosinophils.[38] Hence IL-5 increases the opportunity for peripheral eosinophilia to occur.[38] Inhaled IL-5 caused a decrease in the number of CD34+ IL-5Rα+ cells and an increase in the number of eosinophils in the bronchial mucosa. This suggests that IL-5 is important for eosinophil differentiation in the tissue as well as the peripheral blood.[39] Mepolizumab, an anti-IL-5 therapy, caused a decrease in CD34+ IL-5Rα+ cells in the bronchial mucosa,[40] again, suggesting a role for IL-5 in peripheral tissue eosinophil differentiation and its importance in the development of peripheral eosinophilia.

CD34+ cells have been observed in different airway compartments in humans, including mucosa of the upper and lower airway.[7, 8] Increased progenitor cells have also been observed in nasal polyps from asthmatic subjects versus healthy controls.[41] When compared with normal controls, the number of progenitor cells also increased in the bronchial mucosa of asthmatics.[7] Furthermore, the number of progenitor cells increased in the sputum after allergen challenge.[41] The increased number of progenitors in the upper and lower airways could lead to increased eosinophilia.

Several studies have described the relationship between progenitor cells and the differentiation of eosinophils. The number of CD34+ cells correlates with increased Eo/B-CFU cultured from the blood of atopic subjects.[2] Furthermore, asthmatic subjects that developed airway eosinophilia expressed higher levels of the IL-5Rα on CD34+ progenitors compared with subjects without eosinophilia.[5, 42] In ex vivo cultures of nasal mucosa, IL-5 or allergen decreased the number of CD34+ cells and this coincided with an increase in major basic protein-positive cells, which could be a subset of eosinophils.[8] The link between progenitor cells and the development of eosinophils has guided researchers to examine the impact of treatment on this relationship.

The effect of budesonide treatment on progenitor cells has been examined in vivo and has been observed to inhibit the turnover of a subpopulation of bone marrow-derived progenitors in allergic asthmatics.[43] Budesonide treatment reduced the number of eosinophils in the blood and the number of granulocyte–macrophage colony-stimulating factor-induced Eo/B CFU grown in culture.[44] However, in vitro studies have described a stimulatory effect of steroids on IL-5-induced colony formation.[45] This phenomenon is observed in specific in vivo models, such as an acute stress model.[46] Cyr et al. have shown that in the presence of budesonide, there was increased production of Eo/B CFU in response to IL-5.[45] This budesonide-induced increase was a result of an increase in GATA-1 transcription factor expression. This suggests that the development of eosinophilia may be less responsive to steroid therapy because of ineffective blockade of IL-5-induced eosinophil differentiation. PPAR agonists might represent an alternative therapy to inhibit the formation of eosinophils from its progenitor cell.

We have shown by flow cytometry that rosiglitazone inhibits eosinophil/basophilopoiesis through inhibition of ERK1/2 phosphorylation, and confirmation by Western blot would be ideal in systems not limited by low cell numbers. ERK1/2 is part of the mitogen-activated protein kinase (MAPK) pathway, which is involved in multiple cellular functions, such as proliferation, differentiation, survival and locomotion.[47] There are four other MAPK pathways including, c-Jun N-terminal kinase (JNK), the p38 MAPK, ERK3, and ERK5. ERK1/2 is activated by growth factors, such as platelet-derived growth factor, which is crucial for mitogenesis.[48] Conversely, JNK and p38 are usually activated by stress or cytokines.[49, 50] Furthermore, JNK and p38 can be also be activated by haemopoietic growth factors.[51] Interleukin-5 can stimulate ERK1/2 as well as p38 in eosinophils.[52, 53, 9] The MAPK inhibitor, PD98059 can inhibit the number of eosinophils as well as total cells grown in culture.[32] In contrast, the p38 inhibitor, SB202190, specifically inhibited eosinophil differentiation.[32] We have shown that rosiglitazone inhibits the ERK pathway, which may represent the influence of PPAR on the normal mitogenic development of eosinophils and basophils. We were unable to determine the effects of PPAR agonists on phosphorylation of STAT5 or p38 following IL-5 stimulation because of an inability to detect changes in activation. This may simply be a limitation of the assay used.

In summary, the PPARγ agonist, rosiglitazone inhibited eosinophil differentiation of NAMNCs isolated from the peripheral blood of allergic subjects. It also inhibited eosinophil differentiation of isolated CD34+ progenitor cells. The inhibition of eosinophil differentiation was specific for the PPARγ agonist, as there was no effect of PPARα or PPARβ/δ agonists. In light of our findings that a selective PPARγ agonist can inhibit eosinophil differentiation at a concentration of 10–1000 nm and inhibit eosinophil migration at similar doses,[20] PPARγ agonists have a promising therapeutic potential as an anti-inflammatory therapy, when used within the specified dosage.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References
  10. Supporting Information

This study was supported by a grant from the Ontario Thoracic Society and by a Canadian Allergy and Immune Disease Advanced Training Initiative Award from the AllerGen Networks of Centres of Excellence Incorporated.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References
  10. Supporting Information
FilenameFormatSizeDescription
imm12280-sup-0001-SupportingInformation.docxWord document602K

Figure S1. Representative FlowJo® plot that demonstrates the effectiveness of the gating strategy and purity of the CD34+ cells isolated from the peripheral blood of allergic subjects.

Figure S2. A: Eo/B-CFU after 2-week incubation with IL-5 in Methocult® at 200× magnification under an inverted light microscope. B: Eo/B CFU cells stained with chromotrope 2R at 200× magnification under a light microscope.

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