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CCL26 EXPRESSION: MODULATION BY CYTOKINES AND GLUCOCORTICOIDS
doi:10.1006/cyto.2002.1021, available online at http://www.idealibrary.com on
REGULATION OF HUMAN EOTAXIN-3/CCL26 EXPRESSION: MODULATION BY CYTOKINES AND GLUCOCORTICOIDS Miles Edwin Banwell,1,2 Neil Samuel Tolley,2 Timothy John Williams,1 Tracey Jane Mitchell1 Eotaxin-3 (CCL26) is a CC chemokine that signals exclusively via the CCR3 receptor and has eosinophil-selective chemoattractant activity. Comparison of Eotaxin-1 (CCL11) and Eotaxin-2 (CCL24), demonstrates differences in their expression profiles, cell specificity and effector kinetics, implying distinct biological actions. But little data in this regard have been reported for Eotaxin-3. We aimed to analyse the effect of Th2 cytokines and glucocorticoids on Eotaxin-3 mRNA expression in human lung epithelial cells and dermal fibroblasts; cells implicated in the pathogenesis of allergic asthma and allergic dermatitis respectively. Eotaxin-3 mRNA levels in primary dermal fibroblasts and NCI-H727 lung epithelial cells were determined by Northern hybridization. In contrast to Eotaxin-1, Eotaxin-3 mRNA expression was not detected in unstimulated cells. The Th2 cytokines IL-4 and IL-13 induced Eotaxin-3 expression in a time and dose dependent manner, with IL-4 demonstrating a 100-fold greater potency. Unlike Eotaxin-1, Eotaxin-3 mRNA expression was not induced by either tumour necrosis factor (TNF)- or interleukin (IL)-1 alone. Both IL-4 and IL-13 acted synergistically with TNF- in superinducing Eotaxin-3 mRNA expression. Dexamethasone pre-treatment diminished induction of Eotaxin-3 mRNA expression. We conclude that modulation of Eotaxin-3 mRNA expression by Th2 cytokines is different from that of Eotaxin-1 and Eotaxin-2, further supporting a distinct biological role for Eotaxin-3. 2002 Elsevier Science Ltd. All rights reserved.
Allergic asthma and allergic dermatitis are chronic inflammatory diseases with clear similarities in their pathogenesis. In particular, a marked tissue eosinophilia associated with a Th2 skewed immune phenotype is a predominant feature of both conditions.1 Eotaxin-1, a highly potent eosinophil chemoattractant, was originally purified and sequenced as a CC- chemokine from the bronchoalveolar lavage fluid of allergen-challenged guinea pigs.2 Guinea pig and
From the 1Leukocyte Biology Section, Biomedical Sciences Division, Imperial College School of Medicine, South Kensington, London SW7 2AZ, UK; 2Department of Otolaryngology/Head & Neck Surgery, St Mary’s Hospital NHS Trust, London W2 1NY, UK Correspondence to: Miles Banwell, Leukocyte Biology Section, Biomedical Sciences Division, Imperial College School of Medicine, South Kensington, London SW7 2AZ, UK. E-mail: [email protected] Present address: T. J. Mitchell’s, Skin Tumour Unit, St. Johns Institute of Dermatology, St. Thomas’ Hospital, Lambeth Palace Road, London SE1 7EH, UK Received 25 September 2001; accepted for publication 23 January 2002 1043–4666/02/$-see front matter 2002 Elsevier Science Ltd. All rights reserved. KEY WORDS: eotaxin-3/CCL26/Th2/epithelial/fibroblast CYTOKINE, Vol. 17, No. 6 (21 March), 2002: pp 317–323
human Eotaxin-1 genes were cloned subsequently.3,4 Eotaxin-1 and the closely related MCP chemokines are clustered together on human chromosome 17q11. Subsequently, two further genes encoding for CC chemokines with eosinophil-selective chemoattractant activity, designated Eotaxin-2 and Eotaxin-35–7 have been identified on chromosome 7. However, they are only approximately 30% identical in sequence to Eotaxin-1. The Eotaxins signal exclusively via CCR3, a receptor highly expressed on eosinophils and also on other cells involved in allergic reactions, including basophils, mast cells and a subpopulation of Th2 cells.5,8–10 Other chemokines can also signal via CCR3, for example RANTES (regulated upon activation in normal T cells, expressed and secreted/CCL5) and MCP (monocyte chemoattractant proteins)-2,3,4 (CCL8/7/13), but they are non-selective and act on other chemokine receptors.11,12 Th2 cells regulate eosinophil recruitment suggesting that Th2 derived cytokines may regulate Eotaxin gene expression.1,12,13 The ability of cytokines and glucocorticoids to modulate in vitro expression of Eotaxin-1 and Eotaxin-2 mRNA and protein in both human lung 317
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epithelial and dermal fibroblast cell lines has been well documented; consistent findings are that TNF- and IL-1 induce Eotaxin-1 and Eotaxin-2 expression, as do the Th2 cytokines IL-4 and IL-13.14–16 Furthermore, TNF- in combination with either IL-4 or IL-13 has a synergistic effect on expression. The glucocorticoid dexamethasone diminishes cytokine induced Eotaxin-1 and Eotaxin-2 expression, an effect not altered by pre-treatment with the protein synthesis inhibitor cyclohexamide.14 Such work has suggested a mechanism linking inflammatory cytokine secretion to eosinophil recruitment and recent in vivo evidence supports this concept. IL-4 and IL-13 administered intranasally to naı¨ve mice potently induced airway epithelial cell Eotaxin-1 expression in association with lung eosinophilia.17 IL-4 knockout mice in an allergic dermatitis model had reduced eosinophilic infiltrate and an associated reduction in Eotaxin-1 mRNA.18 Similarly IL-13 transgenepositive mice (expressing IL-13 selectively in the lung) developed a mononuclear inflammatory response around small and large airways comprising significant numbers of eosinophils as well as large quantities of Eotaxin-1 protein and mRNA.19 In humans, Eotaxin-1 has been detected at elevated levels in the bronchial epithelium of patients with asthma, as well as in lesional skin biopsies of allergic dermatitis sufferers.20,21 Recent evidence has described differential expression patterns of Eotaxin-1 and Eotaxin-2. For example lung fibroblasts produce Eotaxin-1 protein in response to in vitro Th2 cytokine stimulation, however no Eotaxin-2 is detected in such circumstances, even at mRNA level.22 Furthermore, in human atopic subjects undergoing allergen induced late-phase allergic skin reactions, results suggested that Eotaxin-1 has a role in the early (6 h) recruitment of eosinophils, whilst Eotaxin-2 is more involved with the later (24 h) eosinophil infiltration.23 Hence the implication is that the Eotaxins all have different expression profiles with regard to cell specificity, kinetics and eosinophil effector function. Indeed Berkman and co-workers24 have provided evidence that at mRNA level Eotaxin-1 and Eotaxin-2 may be co-expressed in human lungs of non-challenged asthmatic patients, whilst Eotaxin-3 is expressed following allergen challenge. However, little additional data on Eotaxin-3 expression have been reported. Eotaxin-3 was described first by Shinkai et al., who stimulated vascular endothelial cells with IL-4 before subjecting the cDNA to differential display analysis.7 The novel CC chemokine detected was designated Eotaxin-3 as it demonstrated in vitro chemotaxis of eosinophils, it acted on cell lines transfected with CCR3 and furthermore when the protein was injected in vivo into primates, local tissue eosinophilia was noted at injection sites. The Th2 derived
CYTOKINE, Vol. 17, No. 6 (21 March, 2002: 317–323)
cytokines IL-4 and IL-13 were shown to upregulate Eotaxin-3 expression in a endothelial cell line, conversely IL-1 and TNF- had no effect7 Eotaxin-3 was cloned also by Kitaura and co- workers6, who reported constitutive Eotaxin-3 mRNA expression in human heart and ovary tissue. Eotaxin-3 was also found to be 10-fold less potent as a CCR3 ligand than Eotaxin-1. Recently it was reported that the three eotaxins exhibit distinct activity profiles with respect to CCR3 binding and the release of toxic reactive oxygen species from human eosinophils.25 In this report we describe Eotaxin-3 mRNA expression in NCI-H727 lung epithelial cells and primary dermal fibroblasts (foreskin-derived). Specifically we demonstrate modulation of its expression by the Th2 cell derived cytokines IL-4 and IL-13 as well as by TNF-, IL-1 and dexamethasone. The data reported here indicate a distinct expression profile and response to cytokine stimulation for the three Eotaxin family members. This study represents a further step towards elucidating the specific role of the latest addition to the Eotaxin family, particularly with reference to chronic allergic inflammatory diseases such as allergic asthma and allergic dermatitis.
RESULTS Eotaxin-3 expression in primary dermal fibroblasts and NCI-H727 lung epithelial cells In unstimulated cells Northern analyses did not detect Eotaxin-3 mRNA expression in either primary dermal fibroblasts or the lung epithelial cell line (Figs 1A and B). However, Eotaxin-3 mRNA was detected by RT-PCR (data not shown), which suggests that levels are too low for detection by Northern blotting rather than totally absent. The Th2 cytokines IL-4 and IL-13 are potent inducers of Eotaxin-1 mRNA expression and they also induce expression of Eotaxin-3 mRNA (Figs 1A and B). Whilst TNF- alone strongly induces Eotaxin-1 mRNA expression, it had minimal effect on Eotaxin-3 mRNA expression. Yet TNF- did act synergistically with both IL-4 and IL-13 in upregulating Eotaxin-3 mRNA expression, similar to the effect observed on Eotaxin-1. IL-1 is a potent Eotaxin-1 inducer, but did not induce Eotaxin-3 mRNA expression in either primary dermal fibroblasts or the lung epithelial cell line. Eotaxin-2 mRNA was not detected in either cell type (data not shown).
Dose response for Eotaxin-3 expression in NCI-H727 lung epithelial cells stimulated with either IL-4 or IL-13 The addition of increasing concentrations of either IL-4 or IL-13 resulted in upregulation of Eotaxin-3
Cytokine modulation of Eotaxin-3 expression / 319
Figure 1B. cells.
Eotaxin-3 (Eo-3) expression in NCI-H727 lung epithelial
Total RNA was isolated following a 24 h (apart from IL-1 which maximally induced at 4 h) cytokine stimulation (10 ng/ml). The unstimulated (no cytokine) control is marked U/S. Eotaxin-3 mRNA levels were determined by Northern hybridization (B). Representative data from one experiment of four is shown. Above each blot a quantitative comparison (A) is made for each cytokine stimulation between the eotaxin-3 specific signal and the GAPDH-specific signal (Eotaxin-3 is 500 bp and GAPDH is 1.1 kb).
Eotaxin-3 expression in NCI-H727 lung epithelial cells stimulated with either IL-4 or IL-13 over a 48 h time course Figure 1A. Eotaxin-1 (Eo-1) and Eotaxin-3 (Eo-3) expression in primary dermal fibroblast cells. Total RNA was isolated following a 24 h (apart from IL-1 which maximally induced at 4 h) cytokine stimulation (10 ng/ml). The unstimulated (no cytokine) controls are annotated U/S. mRNA levels were determined by Northern hybridization (B). Representative data from one experiment of four is shown. Above each blot a quantitative comparison (A) is made for each cytokine stimulation between the eotaxin or eotaxin-3 specific signal and the GAPDHspecific signal (Eotaxin-1 mRNA is 800 bp, Eotaxin-3 is 500 bp and GAPDH is 1.1 kb).
mRNA expression in a dose-dependent manner (Fig. 2). Following IL-4 stimulation, induction was apparent at 0.1 ng/ml compared to 10 ng/ml for the IL-13 stimulated cells. This indicates that at equivalent concentrations, IL-4 is a 100-fold more powerful inducer of Eotaxin-3 mRNA expression than IL-13. Similar effects were observed after stimulation of human dermal fibroblasts (data not shown).
Treatment of both cell types with either IL-4 or IL-13, at 10 ng/ml demonstrated time-dependent induction of Eotaxin-3 mRNA expression (Fig. 3). The concentration of 10 ng/ml was chosen following the dose response experiment; at this concentration both cytokines are potent inducers of Eotaxin-3 mRNA expression. In the IL-4 stimulated cells Eotaxin-3 mRNA was detected earlier than in the IL-13 group (2 h and 8 h respectively). With both cytokines mRNA levels continued to increase throughout the 48 h time course.
Modulation of Eotaxin-3 mRNA expression in NCI-H727 lung epithelial cells by pre-treatment with dexamethasone Pretreatment of cells with 1 M dexamethasone was associated with a similar percentage reduction in both IL-4 and IL-13 induced Eotaxin-3 mRNA expression (Fig. 4). However, at 10 ng/ml IL-4 more potently induces Eotaxin-3 mRNA production than IL-13; this
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Figure 2.
CYTOKINE, Vol. 17, No. 6 (21 March, 2002: 317–323)
Dose response for Eotaxin-3 (Eo-3) expression in NCI-H727 lung epithelial cells stimulated with IL-4 or IL-13.
The unstimulated (no cytokine) controls are annotated U/S. Total RNA was isolated at 24 h and Eotaxin-3 mRNA levels determined by Northern hybridization (B). Representative data from one experiment of four is shown. Above each blot a quantitative comparison (A) is made at each dose between the Eotaxin-3 specific signal and the GAPDH-specific signal (Eotaxin-3 is 500 bp and GAPDH is 1.1 kb).
is reflected in the greater (4-fold) induction of mRNA detected in the dexamethasone treated IL-4 group compared to the IL-13 group. Eotaxin-3 mRNA accumulation was superinduced when the cells were pretreated with cyclohexamide before either IL-4 or IL-13 stimulation.
DISCUSSION Eotaxin-3 has been suggested to have a role in mediating the tissue eosinophilia characteristic of chronic allergic disease; a role which is biologically distinct from that of Eotaxin-1 or Eotaxin-2 (24). We investigated Eotaxin-3 gene expression in response to the main Th2 cytokines. Dermal fibroblasts and lung epithelial cells are important cells in the pathogeneses of allergic dermatitis and allergic asthma respectively. Our data demonstrates that Eotaxin-3 mRNA expression in these cell types is strongly influenced by the Th2 cytokines IL-4 and IL-13, but with a profile that is different to that observed for the similar experiments with Eotaxin-1. Furthermore IL-1, a potent stimulator of Eotaxin-1 mRNA expression, did not influence Eotaxin-3 mRNA expression. The therapeutic use of glucocorticoids in the treatment of allergic asthma action is associated with
widespread improvements in disease pathology, including a reduction in airway eosinophilia, although its mechanism of action in this regard is uncertain. It has been reported previously that dexamethasone treatment of airway epithelial cells, results in the downregulation of both Eotaxin-1 mRNA and protein.14 Our data demonstrates that dexamethasone is also a potent inhibitor of Eotaxin-3 mRNA expression in both airway epithelial cells (Fig. 4) and primary dermal fibroblasts (data not shown). This lends further support to the hypothesis that dexamethasone achieves a reduction in tissue eosinophilia through interrupting chemokine action. In the absence of cytokine stimulation, Eotaxin-3 mRNA could not be detected on Northern analysis in either cell population despite pre-treatment with the protein synthesis inhibitor cyclohexamide. In contrast low levels of Eotaxin-1 mRNA have been reported following similar cyclohexamide treatment.14 However, Eotaxin-3 mRNA accumulation was superinduced when the cells were pretreated with cyclohexamide before either IL-4 or IL-13 stimulation. It may be that a protein exists that negatively regulates Eotaxin-3 mRNA transcription or Eotaxin-3 protein expression. Therefore loss of such a protein’s activity (through inhibition via cyclohexamide) would account for the augmented induction of Eotaxin-3 mRNA by the
Cytokine modulation of Eotaxin-3 expression / 321
Figure 3.
Eotaxin-3 (Eo-3) expression in NCI-H727 lung epithelial cells stimulated with either IL-4 or IL-13 (10 ng/ml) over a 48 h time course.
The unstimulated (no cytokine) controls are annotated U/S. Total RNA was isolated and Eotaxin-3 mRNA levels determined (B). Representative data from one experiment of four is shown. Above each blot a quantitative comparison (A) is made at each time point between the Eotaxin-3 specific signal and the GAPDH-specific signal (Eotaxin-3 is 500 bp and GAPDH is 1.1 kb).
cytokines IL-4 and IL-13. Such mechanisms have been proposed to account for the similar results seen with Eotaxin-1.14 However, if this is the case, then the absence of Eotaxin-3 mRNA in unstimulated cells pretreated with cyclohexamide would suggest that unlike Eotaxin-1, there is little or no basal Eotaxin-3 mRNA production, and that for production to begin there is first a requirement for the appropriate cytokine stimulation. Our data suggests that Eotaxin-3 mRNA is expressed under distinct cytokine conditions and with different kinetics and cell specificity to Eotaxin-1. This complements the evidence already reported that describes a distinct profile of activity for Eotaxin-3 with respect to its binding to CCR3 and the release of toxic oxygen species.6,24 We have shown that Eotaxin-3 expression is augmented by the Th2 cytokine axis and diminished by corticosteroids, adding further to the evidence suggesting that Eotaxin-3 is important in the allergic disease process.24 It has been recently reported that in addition to its ligands, airway epithelial cells express functional CCR3.26 This co-expression of both ligand and receptor raises the possibility that airway epithelial cells may respond in an autoregulatory or juxtaregulatory fashion to CCR3 ligands (such as the Eotaxins). If this is the case then
distinct expression profiles for each of the eotaxins, as reported here, assumes further potential functional significance. Experiments aimed at modifying Eotaxin-3 protein activity both in-vitro and in-vivo represent the next step in elucidating its specific function as regards airway epithelial cell homeostasis as well as in assessing its contribution to the local tissue eosinophilia characteristic of chronic allergic disease.
MATERIALS AND METHODS Cell culture The human lung epithelial cell line NCI-H727 was obtained from the European Collection of Cell Cultures (Wiltshire, UK). Primary human dermal fibroblasts were obtained from human foreskins obtained following circumcision. Cells were cultured in Dulbecco’s Modified Eagle Medium with GlutaMAX1 , 4500 mg/l D-glucose, 110 mg/l sodium pyruvate (Gibco, Paisley, Scotland), supplemented with 10% fetal calf serum (Gibco). Prior to cytokine stimulation the medium was changed and replaced with medium containing the appropriate cytokine, at the concentration and time indicated. Cytokines were purchased from Peprotech, UK.
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CYTOKINE, Vol. 17, No. 6 (21 March, 2002: 317–323)
Figure 4. Modulation of Eotaxin-3 (Eo-3) mRNA expression in NCI-H727 lung epithelial cells by pre-treatment with dexamethasone (1 M) and/or cyclohexamide (10 g/ml). Total RNA was isolated following a 24 h stimulation with either IL-4 or IL-13 (10 ng/ml). The unstimulated (no cytokine) controls are annotated U/S. Eotaxin mRNA levels were determined by Northern hybridization (B). Representative data from one experiment of four is shown. Above each blot a quantitative comparison (A) is made between the Eotaxin-3 specific signal and the GAPDH-specific signal (Eotaxin-3 is 500 bp and GAPDH is 1.1 kb).
RNA isolation and Northern blotting RNA was isolated from cultured cell lines using RNAzolB (Biogenesis, UK) following the manufacturers instructions. Twenty micrograms of total RNA per lane was electrophoresed through formaldehyde gels, and then transferred onto nylon membranes (Duralon , Stratagene, UK) according to standard protocols. Membranes were hybridized in ExpressHyb (Clontech, UK) hybridization solution with radiolabelled gene specific probes, according to the manufacturers instructions. Membranes were washed at high stringency and exposed to X-ray film for an appropriate length of time. Gene specific cDNA probes were as follows: eotaxin 1 366 bp (position 63 to 427 in the eotaxin 1 cDNA); eotaxin 2 328 bp (position 33 to 360 in the eotaxin 2 cDNA); eotaxin 3 223 bp (position 105–327 in the eotaxin 3 cDNA); GAPDH full length (1.1 kb) cDNA.
Analysis of autoradiographs Autoradiographs of Northern blots were scanned with a Umax Astra image analyser using VistaScan V3.1 software. Densitometry was performed with National Institutes of Health IMAGE 1.52 software. As a control for RNA loading all membranes were stripped and reprobed with a
GAPDH probe. Signals were normalized against the GAPDH control.
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REGULATION OF HUMAN EOTAXIN-3/CCL26 EXPRESSION: MODULATION BY CYTOKINES AND GLUCOCORTICOIDS Miles Edwin Banwell,1,2 Neil Samuel Tolley,2 Timothy John Williams,1 Tracey Jane Mitchell1 Eotaxin-3 (CCL26) is a CC chemokine that signals exclusively via the CCR3 receptor and has eosinophil-selective chemoattractant activity. Comparison of Eotaxin-1 (CCL11) and Eotaxin-2 (CCL24), demonstrates differences in their expression profiles, cell specificity and effector kinetics, implying distinct biological actions. But little data in this regard have been reported for Eotaxin-3. We aimed to analyse the effect of Th2 cytokines and glucocorticoids on Eotaxin-3 mRNA expression in human lung epithelial cells and dermal fibroblasts; cells implicated in the pathogenesis of allergic asthma and allergic dermatitis respectively. Eotaxin-3 mRNA levels in primary dermal fibroblasts and NCI-H727 lung epithelial cells were determined by Northern hybridization. In contrast to Eotaxin-1, Eotaxin-3 mRNA expression was not detected in unstimulated cells. The Th2 cytokines IL-4 and IL-13 induced Eotaxin-3 expression in a time and dose dependent manner, with IL-4 demonstrating a 100-fold greater potency. Unlike Eotaxin-1, Eotaxin-3 mRNA expression was not induced by either tumour necrosis factor (TNF)- or interleukin (IL)-1 alone. Both IL-4 and IL-13 acted synergistically with TNF- in superinducing Eotaxin-3 mRNA expression. Dexamethasone pre-treatment diminished induction of Eotaxin-3 mRNA expression. We conclude that modulation of Eotaxin-3 mRNA expression by Th2 cytokines is different from that of Eotaxin-1 and Eotaxin-2, further supporting a distinct biological role for Eotaxin-3. 2002 Elsevier Science Ltd. All rights reserved.
Allergic asthma and allergic dermatitis are chronic inflammatory diseases with clear similarities in their pathogenesis. In particular, a marked tissue eosinophilia associated with a Th2 skewed immune phenotype is a predominant feature of both conditions.1 Eotaxin-1, a highly potent eosinophil chemoattractant, was originally purified and sequenced as a CC- chemokine from the bronchoalveolar lavage fluid of allergen-challenged guinea pigs.2 Guinea pig and
From the 1Leukocyte Biology Section, Biomedical Sciences Division, Imperial College School of Medicine, South Kensington, London SW7 2AZ, UK; 2Department of Otolaryngology/Head & Neck Surgery, St Mary’s Hospital NHS Trust, London W2 1NY, UK Correspondence to: Miles Banwell, Leukocyte Biology Section, Biomedical Sciences Division, Imperial College School of Medicine, South Kensington, London SW7 2AZ, UK. E-mail: [email protected] Present address: T. J. Mitchell’s, Skin Tumour Unit, St. Johns Institute of Dermatology, St. Thomas’ Hospital, Lambeth Palace Road, London SE1 7EH, UK Received 25 September 2001; accepted for publication 23 January 2002 1043–4666/02/$-see front matter 2002 Elsevier Science Ltd. All rights reserved. KEY WORDS: eotaxin-3/CCL26/Th2/epithelial/fibroblast CYTOKINE, Vol. 17, No. 6 (21 March), 2002: pp 317–323
human Eotaxin-1 genes were cloned subsequently.3,4 Eotaxin-1 and the closely related MCP chemokines are clustered together on human chromosome 17q11. Subsequently, two further genes encoding for CC chemokines with eosinophil-selective chemoattractant activity, designated Eotaxin-2 and Eotaxin-35–7 have been identified on chromosome 7. However, they are only approximately 30% identical in sequence to Eotaxin-1. The Eotaxins signal exclusively via CCR3, a receptor highly expressed on eosinophils and also on other cells involved in allergic reactions, including basophils, mast cells and a subpopulation of Th2 cells.5,8–10 Other chemokines can also signal via CCR3, for example RANTES (regulated upon activation in normal T cells, expressed and secreted/CCL5) and MCP (monocyte chemoattractant proteins)-2,3,4 (CCL8/7/13), but they are non-selective and act on other chemokine receptors.11,12 Th2 cells regulate eosinophil recruitment suggesting that Th2 derived cytokines may regulate Eotaxin gene expression.1,12,13 The ability of cytokines and glucocorticoids to modulate in vitro expression of Eotaxin-1 and Eotaxin-2 mRNA and protein in both human lung 317
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epithelial and dermal fibroblast cell lines has been well documented; consistent findings are that TNF- and IL-1 induce Eotaxin-1 and Eotaxin-2 expression, as do the Th2 cytokines IL-4 and IL-13.14–16 Furthermore, TNF- in combination with either IL-4 or IL-13 has a synergistic effect on expression. The glucocorticoid dexamethasone diminishes cytokine induced Eotaxin-1 and Eotaxin-2 expression, an effect not altered by pre-treatment with the protein synthesis inhibitor cyclohexamide.14 Such work has suggested a mechanism linking inflammatory cytokine secretion to eosinophil recruitment and recent in vivo evidence supports this concept. IL-4 and IL-13 administered intranasally to naı¨ve mice potently induced airway epithelial cell Eotaxin-1 expression in association with lung eosinophilia.17 IL-4 knockout mice in an allergic dermatitis model had reduced eosinophilic infiltrate and an associated reduction in Eotaxin-1 mRNA.18 Similarly IL-13 transgenepositive mice (expressing IL-13 selectively in the lung) developed a mononuclear inflammatory response around small and large airways comprising significant numbers of eosinophils as well as large quantities of Eotaxin-1 protein and mRNA.19 In humans, Eotaxin-1 has been detected at elevated levels in the bronchial epithelium of patients with asthma, as well as in lesional skin biopsies of allergic dermatitis sufferers.20,21 Recent evidence has described differential expression patterns of Eotaxin-1 and Eotaxin-2. For example lung fibroblasts produce Eotaxin-1 protein in response to in vitro Th2 cytokine stimulation, however no Eotaxin-2 is detected in such circumstances, even at mRNA level.22 Furthermore, in human atopic subjects undergoing allergen induced late-phase allergic skin reactions, results suggested that Eotaxin-1 has a role in the early (6 h) recruitment of eosinophils, whilst Eotaxin-2 is more involved with the later (24 h) eosinophil infiltration.23 Hence the implication is that the Eotaxins all have different expression profiles with regard to cell specificity, kinetics and eosinophil effector function. Indeed Berkman and co-workers24 have provided evidence that at mRNA level Eotaxin-1 and Eotaxin-2 may be co-expressed in human lungs of non-challenged asthmatic patients, whilst Eotaxin-3 is expressed following allergen challenge. However, little additional data on Eotaxin-3 expression have been reported. Eotaxin-3 was described first by Shinkai et al., who stimulated vascular endothelial cells with IL-4 before subjecting the cDNA to differential display analysis.7 The novel CC chemokine detected was designated Eotaxin-3 as it demonstrated in vitro chemotaxis of eosinophils, it acted on cell lines transfected with CCR3 and furthermore when the protein was injected in vivo into primates, local tissue eosinophilia was noted at injection sites. The Th2 derived
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cytokines IL-4 and IL-13 were shown to upregulate Eotaxin-3 expression in a endothelial cell line, conversely IL-1 and TNF- had no effect7 Eotaxin-3 was cloned also by Kitaura and co- workers6, who reported constitutive Eotaxin-3 mRNA expression in human heart and ovary tissue. Eotaxin-3 was also found to be 10-fold less potent as a CCR3 ligand than Eotaxin-1. Recently it was reported that the three eotaxins exhibit distinct activity profiles with respect to CCR3 binding and the release of toxic reactive oxygen species from human eosinophils.25 In this report we describe Eotaxin-3 mRNA expression in NCI-H727 lung epithelial cells and primary dermal fibroblasts (foreskin-derived). Specifically we demonstrate modulation of its expression by the Th2 cell derived cytokines IL-4 and IL-13 as well as by TNF-, IL-1 and dexamethasone. The data reported here indicate a distinct expression profile and response to cytokine stimulation for the three Eotaxin family members. This study represents a further step towards elucidating the specific role of the latest addition to the Eotaxin family, particularly with reference to chronic allergic inflammatory diseases such as allergic asthma and allergic dermatitis.
RESULTS Eotaxin-3 expression in primary dermal fibroblasts and NCI-H727 lung epithelial cells In unstimulated cells Northern analyses did not detect Eotaxin-3 mRNA expression in either primary dermal fibroblasts or the lung epithelial cell line (Figs 1A and B). However, Eotaxin-3 mRNA was detected by RT-PCR (data not shown), which suggests that levels are too low for detection by Northern blotting rather than totally absent. The Th2 cytokines IL-4 and IL-13 are potent inducers of Eotaxin-1 mRNA expression and they also induce expression of Eotaxin-3 mRNA (Figs 1A and B). Whilst TNF- alone strongly induces Eotaxin-1 mRNA expression, it had minimal effect on Eotaxin-3 mRNA expression. Yet TNF- did act synergistically with both IL-4 and IL-13 in upregulating Eotaxin-3 mRNA expression, similar to the effect observed on Eotaxin-1. IL-1 is a potent Eotaxin-1 inducer, but did not induce Eotaxin-3 mRNA expression in either primary dermal fibroblasts or the lung epithelial cell line. Eotaxin-2 mRNA was not detected in either cell type (data not shown).
Dose response for Eotaxin-3 expression in NCI-H727 lung epithelial cells stimulated with either IL-4 or IL-13 The addition of increasing concentrations of either IL-4 or IL-13 resulted in upregulation of Eotaxin-3
Cytokine modulation of Eotaxin-3 expression / 319
Figure 1B. cells.
Eotaxin-3 (Eo-3) expression in NCI-H727 lung epithelial
Total RNA was isolated following a 24 h (apart from IL-1 which maximally induced at 4 h) cytokine stimulation (10 ng/ml). The unstimulated (no cytokine) control is marked U/S. Eotaxin-3 mRNA levels were determined by Northern hybridization (B). Representative data from one experiment of four is shown. Above each blot a quantitative comparison (A) is made for each cytokine stimulation between the eotaxin-3 specific signal and the GAPDH-specific signal (Eotaxin-3 is 500 bp and GAPDH is 1.1 kb).
Eotaxin-3 expression in NCI-H727 lung epithelial cells stimulated with either IL-4 or IL-13 over a 48 h time course Figure 1A. Eotaxin-1 (Eo-1) and Eotaxin-3 (Eo-3) expression in primary dermal fibroblast cells. Total RNA was isolated following a 24 h (apart from IL-1 which maximally induced at 4 h) cytokine stimulation (10 ng/ml). The unstimulated (no cytokine) controls are annotated U/S. mRNA levels were determined by Northern hybridization (B). Representative data from one experiment of four is shown. Above each blot a quantitative comparison (A) is made for each cytokine stimulation between the eotaxin or eotaxin-3 specific signal and the GAPDHspecific signal (Eotaxin-1 mRNA is 800 bp, Eotaxin-3 is 500 bp and GAPDH is 1.1 kb).
mRNA expression in a dose-dependent manner (Fig. 2). Following IL-4 stimulation, induction was apparent at 0.1 ng/ml compared to 10 ng/ml for the IL-13 stimulated cells. This indicates that at equivalent concentrations, IL-4 is a 100-fold more powerful inducer of Eotaxin-3 mRNA expression than IL-13. Similar effects were observed after stimulation of human dermal fibroblasts (data not shown).
Treatment of both cell types with either IL-4 or IL-13, at 10 ng/ml demonstrated time-dependent induction of Eotaxin-3 mRNA expression (Fig. 3). The concentration of 10 ng/ml was chosen following the dose response experiment; at this concentration both cytokines are potent inducers of Eotaxin-3 mRNA expression. In the IL-4 stimulated cells Eotaxin-3 mRNA was detected earlier than in the IL-13 group (2 h and 8 h respectively). With both cytokines mRNA levels continued to increase throughout the 48 h time course.
Modulation of Eotaxin-3 mRNA expression in NCI-H727 lung epithelial cells by pre-treatment with dexamethasone Pretreatment of cells with 1 M dexamethasone was associated with a similar percentage reduction in both IL-4 and IL-13 induced Eotaxin-3 mRNA expression (Fig. 4). However, at 10 ng/ml IL-4 more potently induces Eotaxin-3 mRNA production than IL-13; this
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Figure 2.
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Dose response for Eotaxin-3 (Eo-3) expression in NCI-H727 lung epithelial cells stimulated with IL-4 or IL-13.
The unstimulated (no cytokine) controls are annotated U/S. Total RNA was isolated at 24 h and Eotaxin-3 mRNA levels determined by Northern hybridization (B). Representative data from one experiment of four is shown. Above each blot a quantitative comparison (A) is made at each dose between the Eotaxin-3 specific signal and the GAPDH-specific signal (Eotaxin-3 is 500 bp and GAPDH is 1.1 kb).
is reflected in the greater (4-fold) induction of mRNA detected in the dexamethasone treated IL-4 group compared to the IL-13 group. Eotaxin-3 mRNA accumulation was superinduced when the cells were pretreated with cyclohexamide before either IL-4 or IL-13 stimulation.
DISCUSSION Eotaxin-3 has been suggested to have a role in mediating the tissue eosinophilia characteristic of chronic allergic disease; a role which is biologically distinct from that of Eotaxin-1 or Eotaxin-2 (24). We investigated Eotaxin-3 gene expression in response to the main Th2 cytokines. Dermal fibroblasts and lung epithelial cells are important cells in the pathogeneses of allergic dermatitis and allergic asthma respectively. Our data demonstrates that Eotaxin-3 mRNA expression in these cell types is strongly influenced by the Th2 cytokines IL-4 and IL-13, but with a profile that is different to that observed for the similar experiments with Eotaxin-1. Furthermore IL-1, a potent stimulator of Eotaxin-1 mRNA expression, did not influence Eotaxin-3 mRNA expression. The therapeutic use of glucocorticoids in the treatment of allergic asthma action is associated with
widespread improvements in disease pathology, including a reduction in airway eosinophilia, although its mechanism of action in this regard is uncertain. It has been reported previously that dexamethasone treatment of airway epithelial cells, results in the downregulation of both Eotaxin-1 mRNA and protein.14 Our data demonstrates that dexamethasone is also a potent inhibitor of Eotaxin-3 mRNA expression in both airway epithelial cells (Fig. 4) and primary dermal fibroblasts (data not shown). This lends further support to the hypothesis that dexamethasone achieves a reduction in tissue eosinophilia through interrupting chemokine action. In the absence of cytokine stimulation, Eotaxin-3 mRNA could not be detected on Northern analysis in either cell population despite pre-treatment with the protein synthesis inhibitor cyclohexamide. In contrast low levels of Eotaxin-1 mRNA have been reported following similar cyclohexamide treatment.14 However, Eotaxin-3 mRNA accumulation was superinduced when the cells were pretreated with cyclohexamide before either IL-4 or IL-13 stimulation. It may be that a protein exists that negatively regulates Eotaxin-3 mRNA transcription or Eotaxin-3 protein expression. Therefore loss of such a protein’s activity (through inhibition via cyclohexamide) would account for the augmented induction of Eotaxin-3 mRNA by the
Cytokine modulation of Eotaxin-3 expression / 321
Figure 3.
Eotaxin-3 (Eo-3) expression in NCI-H727 lung epithelial cells stimulated with either IL-4 or IL-13 (10 ng/ml) over a 48 h time course.
The unstimulated (no cytokine) controls are annotated U/S. Total RNA was isolated and Eotaxin-3 mRNA levels determined (B). Representative data from one experiment of four is shown. Above each blot a quantitative comparison (A) is made at each time point between the Eotaxin-3 specific signal and the GAPDH-specific signal (Eotaxin-3 is 500 bp and GAPDH is 1.1 kb).
cytokines IL-4 and IL-13. Such mechanisms have been proposed to account for the similar results seen with Eotaxin-1.14 However, if this is the case, then the absence of Eotaxin-3 mRNA in unstimulated cells pretreated with cyclohexamide would suggest that unlike Eotaxin-1, there is little or no basal Eotaxin-3 mRNA production, and that for production to begin there is first a requirement for the appropriate cytokine stimulation. Our data suggests that Eotaxin-3 mRNA is expressed under distinct cytokine conditions and with different kinetics and cell specificity to Eotaxin-1. This complements the evidence already reported that describes a distinct profile of activity for Eotaxin-3 with respect to its binding to CCR3 and the release of toxic oxygen species.6,24 We have shown that Eotaxin-3 expression is augmented by the Th2 cytokine axis and diminished by corticosteroids, adding further to the evidence suggesting that Eotaxin-3 is important in the allergic disease process.24 It has been recently reported that in addition to its ligands, airway epithelial cells express functional CCR3.26 This co-expression of both ligand and receptor raises the possibility that airway epithelial cells may respond in an autoregulatory or juxtaregulatory fashion to CCR3 ligands (such as the Eotaxins). If this is the case then
distinct expression profiles for each of the eotaxins, as reported here, assumes further potential functional significance. Experiments aimed at modifying Eotaxin-3 protein activity both in-vitro and in-vivo represent the next step in elucidating its specific function as regards airway epithelial cell homeostasis as well as in assessing its contribution to the local tissue eosinophilia characteristic of chronic allergic disease.
MATERIALS AND METHODS Cell culture The human lung epithelial cell line NCI-H727 was obtained from the European Collection of Cell Cultures (Wiltshire, UK). Primary human dermal fibroblasts were obtained from human foreskins obtained following circumcision. Cells were cultured in Dulbecco’s Modified Eagle Medium with GlutaMAX1 , 4500 mg/l D-glucose, 110 mg/l sodium pyruvate (Gibco, Paisley, Scotland), supplemented with 10% fetal calf serum (Gibco). Prior to cytokine stimulation the medium was changed and replaced with medium containing the appropriate cytokine, at the concentration and time indicated. Cytokines were purchased from Peprotech, UK.
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Figure 4. Modulation of Eotaxin-3 (Eo-3) mRNA expression in NCI-H727 lung epithelial cells by pre-treatment with dexamethasone (1 M) and/or cyclohexamide (10 g/ml). Total RNA was isolated following a 24 h stimulation with either IL-4 or IL-13 (10 ng/ml). The unstimulated (no cytokine) controls are annotated U/S. Eotaxin mRNA levels were determined by Northern hybridization (B). Representative data from one experiment of four is shown. Above each blot a quantitative comparison (A) is made between the Eotaxin-3 specific signal and the GAPDH-specific signal (Eotaxin-3 is 500 bp and GAPDH is 1.1 kb).
RNA isolation and Northern blotting RNA was isolated from cultured cell lines using RNAzolB (Biogenesis, UK) following the manufacturers instructions. Twenty micrograms of total RNA per lane was electrophoresed through formaldehyde gels, and then transferred onto nylon membranes (Duralon , Stratagene, UK) according to standard protocols. Membranes were hybridized in ExpressHyb (Clontech, UK) hybridization solution with radiolabelled gene specific probes, according to the manufacturers instructions. Membranes were washed at high stringency and exposed to X-ray film for an appropriate length of time. Gene specific cDNA probes were as follows: eotaxin 1 366 bp (position 63 to 427 in the eotaxin 1 cDNA); eotaxin 2 328 bp (position 33 to 360 in the eotaxin 2 cDNA); eotaxin 3 223 bp (position 105–327 in the eotaxin 3 cDNA); GAPDH full length (1.1 kb) cDNA.
Analysis of autoradiographs Autoradiographs of Northern blots were scanned with a Umax Astra image analyser using VistaScan V3.1 software. Densitometry was performed with National Institutes of Health IMAGE 1.52 software. As a control for RNA loading all membranes were stripped and reprobed with a
GAPDH probe. Signals were normalized against the GAPDH control.
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