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The chemokine receptor CCR3 participates in tissue remodeling during atopic skin inflammation

      Abstract

      Background

      Recent studies provided insights into the recruitment and activation pathways of leukocytes in atopic dermatitis, however, the underlying mechanisms of tissue remodeling in atopic skin inflammation remain elusive.

      Objective

      To identify chemokine-mediated communication pathways regulating tissue remodeling during atopic skin inflammation.

      Methods

      Analysis of the chemokine receptor repertoire of human dermal fibroblasts using flow cytometry and immunofluorescence. Quantitative real-time polymerase chain reaction and immunohistochemical analyses of chemokine expression in atopic vs. non-atopic skin inflammation. Investigation of the function of chemokine receptor CCR3 on human dermal fibroblasts through determining intracellular Ca2+ mobilization, cell proliferation, migration, and repair capacity.

      Results

      Analyses on human dermal fibroblasts showed abundant expression of the chemokine receptor CCR3 in vitro and in vivo. Among its corresponding ligands (CCL5, CCL8, CCL11, CCL24 and CCL26) CCL26 demonstrated a significant and specific up-regulation in atopic when compared to psoriatic skin inflammation. In vivo, epidermal keratinocytes showed most abundant CCL26 protein expression in lesional atopic skin. In structural cells of the skin, TH2-cytokines such as IL-4 and IL-13 were dominant inducers of CCL26 expression. In dermal fibroblasts, CCL26 induced CCR3 signaling resulting in intracellular Ca2+ mobilization, as well as enhanced fibroblast migration and repair capacity, but no proliferation.

      Conclusion

      Taken together, findings of the present study suggest that chemokine-driven communication pathways from the epidermis to the dermis may modulate tissue remodeling in atopic skin inflammation.

      Abbreviations:

      AD (atopic dermatitis), BrdU (5-bromo-2-deoxyuridine), FSP (fibroblast surface protein), NHDF (normal human dermal fibroblast), qPCR (quantitative real-time polymerase chain reaction)

      Keywords

      1. Introduction

      Remodeling of the skin is an important pathophysiologic feature of chronic atopic dermatitis (AD). It is characterized by structural changes of the skin [
      • Leung D.Y.
      • Bieber T.
      Atopic dermatitis.
      ,
      • Leung D.Y.
      Immunopathology of atopic dermatitis.
      ,
      • Katoh N.
      • Hirano S.
      • Suehiro M.
      • Ikenaga K.
      • Yasuno H.
      Increased levels of serum tissue inhibitor of metalloproteinase-1 but not metalloproteinase-3 in atopic dermatitis.
      ]. The underlying mechanisms of tissue remodeling in AD, however, are not well understood.
      In AD patients proinflammatory cytokines are responsible for the initiation and maintenance of skin inflammation [
      • Homey B.
      • Steinhoff M.
      • Ruzicka T.
      • Leung D.Y.
      Cytokines and chemokines orchestrate atopic skin inflammation.
      ]. These cytokines may also contribute to the pathogenesis of skin remodeling. Fibrogenic and fibrosis-associated cytokines (TGF-β1, IL-11 and IL-17) are increased in chronic AD lesions compared with non-lesional AD, or healthy skin [
      • Toda M.
      • Leung D.Y.
      • Molet S.
      • Boguniewicz M.
      • Taha R.
      • Christodoulopoulos P.
      • et al.
      Polarized in vivo expression of IL-11 and IL-17 between acute and chronic skin lesions.
      ]. Besides, matrix metallopoteinases and their inhibitors have been shown to contribute to inflammation-induced tissue destruction and remodeling in atopic skin inflammation [
      • Katoh N.
      • Hirano S.
      • Suehiro M.
      • Ikenaga K.
      • Yasuno H.
      Increased levels of serum tissue inhibitor of metalloproteinase-1 but not metalloproteinase-3 in atopic dermatitis.
      ]. Phipps et al. reported the relationship between allergen-induced tissue eosinophilia and markers of remodeling in human atopic skin [
      • Phipps S.
      • Ying S.
      • Wangoo A.
      • Ong Y.E.
      • Levi-Schaffer F.
      • Kay A.B.
      The relationship between allergen-induced tissue eosinophilia and markers of repair and remodeling in human atopic skin.
      ].
      Next to cytokines, members of the chemokine superfamily have recently been shown to regulate atopic skin inflammation [
      • Homey B.
      • Alenius H.
      • Muller A.
      • Soto H.
      • Bowman E.P.
      • Yuan W.
      • et al.
      CCL27–CCR10 interactions regulate T cell-mediated skin inflammation.
      ,
      • Pivarcsi A.
      • Gombert M.
      • Dieu-Nosjean M.C.
      • Lauerma A.
      • Kubitza R.
      • Meller S.
      • et al.
      CC chemokine ligand 18, an atopic dermatitis-associated and dendritic cell-derived chemokine, is regulated by staphylococcal products and allergen exposure.
      ,
      • Gombert M.
      • Dieu-Nosjean M.C.
      • Winterberg F.
      • Bunemann E.
      • Kubitza R.C.
      • Da Cunha L.
      • et al.
      CCL1–CCR8 interactions: an axis mediating the recruitment of T cells and langerhans-type dendritic cells to sites of atopic skin inflammation.
      ]. Chemokines are small cytokine-like proteins that regulate leukocyte trafficking under homeostatic and inflammatory conditions [
      • Luther S.A.
      • Bidgol A.
      • Hargreaves D.C.
      • Schmidt A.
      • Xu Y.
      • Paniyadi J.
      • et al.
      Differing activities of homeostatic chemokines CCL19, CCL21, and CXCL12 in lymphocyte and dendritic cell recruitment and lymphoid neogenesis.
      ,
      • Homey B.
      • Bunemann E.
      Chemokines and inflammatory skin diseases.
      ]. Recently, a defined set of chemokines (CCL1-5, CCL11, CCL13, CCL17–18, CCL20, CCL22, CCL26–27 and CX3CL1) was identified to initiate and perpetuate atopic skin inflammation [
      • Homey B.
      • Steinhoff M.
      • Ruzicka T.
      • Leung D.Y.
      Cytokines and chemokines orchestrate atopic skin inflammation.
      ,
      • Homey B.
      • Meller S.
      • Savinko T.
      • Alenius H.
      • Lauerma A.
      Modulation of chemokines by staphylococcal superantigen in atopic dermatitis.
      ], however, no report demonstrates their contribution to the pathogenesis of cutaneous remodeling during atopic skin inflammation. Notably, serum levels of CCL11, CCL17, CCL22, CCL26, CCL27 and CX3CL1 are also directly correlated with disease activity and severity suggesting an important role in the immunopathogenesis of AD [
      • Homey B.
      • Alenius H.
      • Muller A.
      • Soto H.
      • Bowman E.P.
      • Yuan W.
      • et al.
      CCL27–CCR10 interactions regulate T cell-mediated skin inflammation.
      ,
      • Homey B.
      • Meller S.
      • Savinko T.
      • Alenius H.
      • Lauerma A.
      Modulation of chemokines by staphylococcal superantigen in atopic dermatitis.
      ].
      Beside leukocytes an increasing number of non-hematopoietic cells have been shown to express chemokine receptors and respond to their corresponding ligands. Hence, the aim of the present study was to identify chemokine-mediated communication pathways regulating tissue remodeling during atopic skin inflammation. Here, we systematically analyzed the chemokine receptor repertoire of human dermal fibroblasts and demonstrate expression of functional CCR3 on their cell surface. Moreover, we showed that among all known CCR3 ligands, the chemokine CCL26 is most abundantly expressed in atopic skin inflammation, and that the keratinocytes were the predominant source of CCL26 inducing enhanced migratory responses and repair activity in human fibroblasts. Findings of the present study point to a role for CCL26–CCR3 interactions regulating tissue remodeling in AD.

      2. Materials and methods

      2.1 Patients

      Six-millimeter punch biopsies were taken from healthy individuals (n = 29), non-lesional (n = 9) and lesional (n = 37) psoriasis, non-lesional (n = 29) and lesional (n = 65) chronic AD, and prurigo nodularis (n = 46) patients. AD was identified according to the criteria defined by Hanifin and Rajka [
      • Hanifin J.M.
      • Rajka G.
      Diagnostic criteria of atopic dermatitis.
      ]. All clinical diagnoses were confirmed by histological evaluation. Atopy patch tests were performed with house dust mite preparations containing a mixture of Dermatophagoides farinae (10%) and Dermatophagoides pteronyssimus (10%) antigens in petrolatum (Chemotechnique Diagnostics) as described previously [
      • Gombert M.
      • Dieu-Nosjean M.C.
      • Winterberg F.
      • Bunemann E.
      • Kubitza R.C.
      • Da Cunha L.
      • et al.
      CCL1–CCR8 interactions: an axis mediating the recruitment of T cells and langerhans-type dendritic cells to sites of atopic skin inflammation.
      ]. A total of 17 patients with atopic dermatitis and positive house dust mite prick tests were selected for the study. Tests were performed with Finn Chambers (Epitest) on healthy appearing dorsal skin. Of those tested, seven patients reacted positively and were chosen for the study. Patients were studied as follow: after 2, 6, and 48 h of application, the patches were removed and test results were determined. Patch sites and untreated normal skin were biopsied, snap frozen in liquid nitrogen and stored at −70 °C. All 7 patients included showed positive reactions to repeated atopy patch tests and patch testing with petrolatum alone was uniformly negative in all patients. The studies were approved by the local ethics committee.

      2.2 Quantitative real-time RT-PCR (TaqMan) analysis

      Quantitative real-time RT-PCR analyses were performed, as previously described [
      • Homey B.
      • Alenius H.
      • Muller A.
      • Soto H.
      • Bowman E.P.
      • Yuan W.
      • et al.
      CCL27–CCR10 interactions regulate T cell-mediated skin inflammation.
      ]. Briefly skin biopsies specimens were homogenized in liquid nitrogen using Mikro-DismembratorU (Braun Biotech, San Diego, CA) and RNA was extracted using TRIzol (Invitrogen Life Technologies). Total RNA of 4 μg was treated with DNase I (Boehringer Mannheim) and reverse transcribed. The cDNA was subjected to qPCR analyses (ABI PRISM 7000 Sequence Detection Systems (Applied Biosystems) continuously during 40 cycles) of chemokine and receptor expression. Target gene expression was normalized to 18S RNA expression. Primers and probes specific for chemokines and their receptors were obtained from Applied Biosystems.

      2.3 Cell cultures, hemopoietic factors and reagents

      Human primary epidermal keratinocytes, and normal human dermal fibroblasts (NHDF) were cultured in keratinocyte (KGM-2), or fibroblast (FGM-2) growth medium (all Clonetics, San Diego, CA) as described previously [
      • Homey B.
      • Wang W.
      • Soto H.
      • Buchanan M.E.
      • Wiesenborn A.
      • Catron D.
      • et al.
      Cutting edge: the orphan chemokine receptor G protein-coupled receptor-2 (GPR-2, CCR10) binds the skin-associated chemokine CCL27 (CTACK/ALP/ILC).
      ]. Cultured cells were either left untreated or stimulated for different period of time with either TNF-α (100 ng/ml) plus IL-1β (5 ng/ml), or IFN-γ (50 ng/ml), or IL-13 (10 ng/ml), or IL-4 (50 ng/ml), or hGM-CSF (50 ng/ml, Schering-Plough Research Institute, Kenilworth, NJ). All other reagents were purchased from R&D Systems Inc. (Minneapolis, MN).

      2.4 Histology and immunofluorescence

      To determine tissue remodeling and changes within the dermal compartment, skin specimens from healthy volunteers (n = 10) or AD patients (n = 37) with chronic lesions were fixed in 4% formaldehyde in 0.075 M phosphate buffer, dehydrated in ethanol, and infiltrated with paraffin at 60 °C. Sections of 5 μm thickness were cut. Slides were stained by elastic van Giesson, and Masson's trichrome. Immunofluorescence analyses of CCL26 protein expression in normal skin of healthy volunteers (n = 9) and lesional skin of AD patients (n = 10) was performed using acetone fixed cryo-sections. Sections were blocked with PBS ± 2% donkey serum and stained overnight with a goat anti-human CCL26 or isotype controls antibodies, respectively (goat IgG; Santa Cruz Biotechnology, Heidelberg, Germany; Jackson Immunoresearch, West Grove, PA). Subsequently, sections were stained with an AlexaFluor 555-labeled donkey anti-goat antibody (R&D Systems, Mineapolis, MN) and DAPI (Invitrogen, Karlsruhe, Germany). Images were captured using a Zeiss Cell Observer System (Zeiss, Oberkochen, Germany). CCL26 protein expression was quantified using the measurement module of the ImageJ-Tool (NIH, Bethesda, MD, USA).

      2.5 Flow cytometric analyses

      NHDFs (106 cells) were washed in PBS, then incubated and stained with phycoerythrin-conjugated rat anti-human anti-CCR3 (IgG2a) monoclonal antibody (BD Pharmingen, San Diego, CA), or appropriate isotype control. Finally, samples were fixed in 1% paraformaldehyde and subsequently washed in PBS. Fluorescence was quantified by FACScan and CellQuest software (Becton Dickinson Biosciences, San Jose, CA).

      2.6 Immunofluorescence of cultured normal human dermal fibroblasts

      NHDFs were cultured in chamber slides and fixed (equal amount of acetone + ethanol), then preprocessed with H2O2. Staining was performed with primary antibodies against human CCR3 (goat IgG; Santa Cruz Biotechnology, Heidelberg, Germany), and goat IgG (Jackson Immunoresearch, West Grove, PA) as isotype control (2 h, room temperature). Binding was detected by rabbit anti-goat fluorescent secondary antibodies (1:200; Molecular Probes, Eugene, OR). For double immunofluorescent staining in healthy (n = 10), and lesional AD (n = 10) skin, frozen sections were fixed with acetone. Fibroblast surface protein (FSP) was stained by anti-FSP (mouse IgG; Sigma, Taufkirchen, Germany), and then with fluorescent secondary anti-mouse IgG antibody (R&D Systems, Mineapolis, MN). Afterwards CCR3 was detected by anti-CCR3 (goat IgG; Santa Cruz Biotechnology, Heidelberg, Germany), and fluorescent secondary anti-goat IgG antibody (R&D Systems, Mineapolis, MN). DAPI (Invitrogen, Karlsruhe, Germany) was used for nuclear staining. Cells and samples were examined with Zeiss Axiovert2 MOT microscope, Zeiss Axiocam MRc, Zeiss AxioVision 4.6 Software.

      2.7 In vitro repair

      NHDFs were cultured to 80% confluence, and a defined single path scratch was made with a sterile pipette tip through the intact cell layer. Medium was removed, and cells were washed with PBS, and either medium or 100 ng/ml CCL26 was added to each well. For neutralizing experiments cells were treated for 30 min with mouse anti-human CCR3 (2 μg/ml) before addition of CCL26 (both R&D Systems Inc., Minneapolis, MN). The experiment was performed in triplicates. The status of the single path wound was monitored for 15 h by time-lapse video microscopy (Inverse Leitz Microscope with Incubator, Zeiss AxioCam HRc, Zeiss AxioVision Software, Carl Zeiss, Oberkochen, Germany).

      2.8 Fibroblast migration in 3D collagen matrix

      To gain spheroids, NHDFs (750 cells) were plated into 1% agarose gel-coated wells (Agarose TypVII Low Gelling, Sigma, Taufkirchen, Germany), and cultured for 24 h. Fibroblast spheroids (1 spheroid/well) were embedded in 300 μl/cm2 Matrigel (BD Bioscience, Erembodegem, Belgium) in 24-well plate, into which 100 ng/ml CCL26 was added, or was left untreated. The experiment was performed in quadruplicates. To gain migratory activity of cells, the area of spheroids was determined at day 0 and day 6. Motility of individual cells was also tracked. In the measurement and analyses digital camera system (Olympus, Hamburg, Germany), and Zeiss AxioVision Software (Carl Zeiss, Oberkochen, Germany) were used.

      2.9 Calcium imaging with confocal microscopy

      Changes in intracellular calcium ([Ca2+]i) upon drug applications were detected by fluorimetric Ca2+ imaging. Cells were seeded on glass coverslips at pre-confluent density one day before measurements. Cells were incubated in Hank's solution (136.8 mM NaCl, 5.4 mM KCl, 0.34 mM Na2HPO4, 0.44 mM KH2PO4, 0.81 mM MgSO4, 1.26 mM CaCl2, 5.56 mM glucose, 4.17 mM NaHCO3, pH 7.2, all from Sigma, Taufkirchen, Germany) containing 1% bovine serum albumin and 2.5 mM Probenecid (both from Sigma, Taufkirchen, Germany) with 2 μM of the cytoplasmic calcium indicator Fluo-4 AM (Invitrogen, Karlsruhe, Germany) at 37 °C for 40 min. The cells were washed four times and finally incubated in Hank's solution described above for 30 min at 37 °C. The coverslips were then placed in a custom-made mount, and imaged using an LSM 510 laser scanning confocal microscope (Zeiss, Oberkochen, Germany). Cells were treated with 100 ng/ml CCL26 (R&D Systems Inc., Minneapolis, MN). For neutralization mouse anti-human CCR3 (2 μg/ml) was used. A total of 200 images were taken during one treatment, with one image captured every 987 ms, using a 20× objective. Fluo-4 was excited with an argon ion laser (at 488 nm; 10% laser intensity) and emitted light was collected through a bandpath filter and digitized at 12 bits. These image series were then converted to .avi format with the Zeiss LSM Image Browser version 4.2.0.121 (Zeiss, Oberkochen, Germany). The relative fluorescence intensity of responding cells was then determined with the ImageJ program (NIH, Bethesda MD, USA) in each frame.

      2.10 BrdU assay

      NHDFs were cultured in standard 96-well microtiter plates (200 μl medium/well) in the presence, or absence of either 10 ng/ml, or 100 ng/ml, or 1000 ng/ml CCL26. 24 h after seeding and incubating, 20 μl BrdU was added to each well. After incubation for 20 h DNA synthesis was assayed with Cell Proliferation ELISA, BrdU (Roche Molecular Biochemicals, Indianapolis, IN) using colorimetric detection according to the manufacturer's instructions. Newly synthesized BrdU-DNA was determined by ELISA-reader (BioRad, 450).

      2.11 Statistical analysis

      To determine statistical significance of experiments, the F test was performed to analyze variances, and subsequently the student's t-test was applied using the Excel® software (Microsoft Corp., Redmond, WA). p-Values < 0.05 were considered to be statistically significant (*p < 0.05; **p < 0.01; ***p < 0.005).

      3. Results

      3.1 Tissue remodeling during atopic skin inflammation

      To determine changes in matrix deposition during chronic atopic skin inflammation histochemical analyses were performed in specimens of lesional skin of chronic AD patients (n = 37) and that of healthy volunteers (n = 10). Elastica van Giesson staining showed marked decrease of normal elastic fibers in AD patients (Fig. 1B ) compared to healthy skin (Fig. 1A). Furthermore, Masson's trichrome staining showed that the amount of collagen bundles in the papillary dermis is markedly increased in chronic atopic skin inflammation (Fig. 1D) compared to normal skin (Fig. 1C). These findings indicate that tissue remodeling is a prominent feature of chronic atopic skin inflammation.
      Figure thumbnail gr1
      Fig. 1Elastic van Giesson (A and B), and Masson's trichrome (C and D) staining of healthy (A and C) and atopic skin (B and D). Flow cytometry (E) shows CCR3 staining of NHDFs (black line-histogram; isotype control: gray histogram). Immunofluorescence microscopy: CCR3 in cultured NHDFs (G) (F: isotype control); fibroblasts express CCR3 in human lesional AD [H-fibroblast (FSP, green); I-CCR3 (red); J-overlay].

      3.2 Human fibroblasts express CCR3 in vitro and in vivo

      Recently, chemokines have been shown to play a crucial role in the initiation and amplification of atopic skin inflammation. Next we sought to investigate whether chemokines directly interact with human dermal fibroblasts and contribute to tissue remodeling. Systematic flow cytometric analyses of the chemokine receptor repertoire of cultured NHDFs (Supplementary Table 1) showed that fibroblasts abundantly express CCR3 on their cell surface (Fig. 1E). Immunofluorescence analyses confirmed the expression of CCR3 on NHDFs in vitro (Fig. 1F and G), also on FSP-positive dermal fibroblasts in vivo (Fig. 1H–J).

      3.3 CCR3 and its ligand CCL26 are significantly induced in atopic dermatitis

      Although some information on the chemokine signature of AD was recently reported, no systematic analysis of the expression of CCR3 and its ligands in chronic inflammatory and autoimmune skin diseases in the context of tissue remodeling exists, to date. Hence, we aimed to compare the expression profiles of CCR3, CCL5, CCL8, CCL11, CCL24 and CCL26 in lesional (n = 65) and non-lesional (n = 29) skin of AD patients with lesional (n = 37) or non-lesional skin (n = 37) of psoriatic patients. Moreover, we investigated expressions in prurigo nodularis (n = 46), a skin disease characterized by severe pruritus and development of erythematous nodules showing inflammatory infiltrate with characteristic fibrosis. Normal skin of healthy individuals (n = 29) was also examined. The qPCR analyses showed that CCL26 represents the most AD-specific CCR3 ligand (Fig. 2E ). On average CCL26 was expressed 150-fold higher in lesional AD skin compared with skin samples from non-lesional AD and healthy skin, and more than 15 times higher when compared with lesional psoriatic skin (**p < 0.01). CCL26 was also significantly up-regulated in prurigo nodularis when compared with lesional psoriatic skin (***p < 0.005), or healthy skin (**p < 0.01). Interestingly, CCL11, the most extensively studied CCR3 ligand, did not show a dominant regulation during atopic versus psoriatic skin inflammation. Although CCL11 was 4-fold higher expressed in lesional AD compared to healthy skin and to non-lesional atopic skin and 3-fold higher than in lesional psoriatic skin the results did not meet statistical significance (Fig. 2C, p > 0.05). Overall, CCL24 transcript levels were low but showed significant up-regulation in inflamed AD skin compared with non-lesional AD or lesional psoriatic skin (Fig. 2D, ***p < 0.005). In contrast, CCL5 and CCL8 were predominantly expressed in psoriatic skin and down-regulated in lesional AD skin (Fig. 2A and B). Their corresponding receptor CCR3 showed highest expression in lesional AD followed by prurigo nodularis. CCR3 transcripts were significantly induced in lesional AD compared with non-lesional atopic, lesional psoriatic or healthy skin (Fig. 2F). Taken together, our findings identify CCL26 among the ligands of CCR3 as the chemokine showing the strongest regulation and association with AD.
      Figure thumbnail gr2
      Fig. 2Quantitative real-time PCR analysis of CCL5 (A), CCL8 (B), CCL11 (C), CCL24 (D), CCL26 (E), and CCR3 (F) expression in healthy skin, in lesional and non-lesional skin of patients with psoriasis or atopic dermatitis, and lesional skin of patients with prurigo nodularis. Values are expressed in relative units (RU). Measurements of individual samples and mean + SD are shown.
      To obtain insights into the cellular origin of CCL26 in healthy skin of normal volunteers and lesional skin of atopic dermatitis patients, we performed immunofluorescence analyses using specific antibodies directed against CCL26 or isotype controls (Fig. 3A and B ). CCL26 displayed abundant expression in the epidermis of lesional AD skin (Fig. 3B). Computer-assisted image analyses of skin specimens of healthy volunteers (n = 9) and lesional skin of atopic dermatitis patients (n = 10) revealed that CCL26 protein is significantly overexpressed within the epidermal compartment during atopic skin inflammation (Fig. 3C, ***p < 0.005; Student's t-test). During atopic skin inflammation, keratinocytes of basal and suprabasal layers of the epidermis represented the dominant source of CCL26 protein (Fig. 3B). Furthermore, endothelial cells of the superficial dermal plexus displayed immunoreactivity for CCL26 protein (Fig. 3A and B). Isotype controls were uniformly negative (data not shown). Hence, the epidermal as well as the dermal compartment of the skin represent sources of CCL26 production or deposition during atopic skin inflammation.
      Figure thumbnail gr3
      Fig. 3CCL26 protein expression in skin specimens of healthy volunteers (A) and lesional skin of atopic dermatitis patients (B) (immunofluorescence analyis of representative specimens, red: CCL26, blue: DAPI; magnification: 400×). (C) Computer-assisted quantification of CCL26 protein expression in healthy volunteers (n = 9) and atopic dermatitis patients (n = 10). ***p-Value ≤ 0.005; Student's t-test. Quantitative real-time PCR analysis of CCL11 (D and G), CCL24 (E and H), and CCL26 (F and J) in untreated or treated cultured human epidermal keratinocytes for different time periods (D–F) and NHDFs (24 h stimulation) (G–J). Values are expressed in relative units (RU).
      To further investigate the regulation of CCL26 and its receptor during atopic skin inflammation in vivo, we determined their expression kinetics during atopy patch test reactions. In patients with a history of house dust mite allergy and positive atopy patch tests (n = 7), CCL26 and CCR3 mRNA expression were determined 2, 6, and 48 h post relevant allergen exposure and compared with untreated skin (Supplementary Fig. 1). Six out of seven patients showed a more than 2-fold upregulation of CCL26 expression during the course of the atopy patch test reaction (Supplementary Fig. 1). Overall, highest CCL26 mRNA expression was observed at early time points after allergen exposure (2–6 h; three patients). Patients showing peak expression at 48 h post allergen challenge showed on average lower mRNA expression levels (Supplementary Fig. 1). With regard to CCR3 mRNA, in four out of six patients peak CCR3 expression followed CCL26 transcription indicating an orchestrated program of transcription in sensitized patients post allergen exposure (Supplementary Fig. 1).

      3.4 TH2 effector cytokines (IL-4 and IL-13) regulate CCL26 expression in primary keratinocytes and NHDFs

      Immunofluorescence analyses suggested that keratinocytes are the dominant source of CCL26 protein within the skin (Fig. 3B). To gain insights into the regulatory pathways of CCL26 and other eotaxin family members (CCL11 and CCL24), we treated human primary epidermal keratinocytes and NHDFs with proinflammatory cytokines (TNF-α/IL-1β), and TH cell-derived effector cytokines (IFN-γ, IL-4 and IL-13) that are known to play a crucial role in the initiation or amplification of atopic skin inflammation [
      • Leung D.Y.
      • Bieber T.
      Atopic dermatitis.
      ]. The qPCR analyses showed that keratinocytes constitutively express low levels of CCL26 that can be significantly induced by IL-4 and IL-13 following stimulation for 6 or 24 h (Fig. 3F). Direct comparison with other eotaxin family members indicated that CCL26 was expressed at more than 100-fold higher levels in keratinocytes compared to CCL11 or CCL24 indicating its dominant role as CCR3 ligand expressed in the epithelial compartment. Although CCL26 expression was very low in NHDFs, 24-h stimulation with IL-4 and IL-13 markedly induced CCL26 expression (Fig. 3J). Similar to CCL26, resting NHDFs expressed low levels of CCL11 (Fig. 3G). Interestingly, cultured keratinocytes expressed no CCL11 transcripts under homeostatic conditions indicating that CCL11 might be dominant CCR3 ligand in fibroblasts (Fig. 3D). Both cell-types showed a marked up-regulation of CCL11 expression following IL-4 but not IL-13 stimulation and fibroblasts produced approximately 400-fold higher levels of CCL11 compared to keratinocytes. Direct comparison of the expression of eotaxin family members in NHDF indicated that CCL11 is homeostatically expressed and both CCL11 and CCL26 are markedly inducible by TH2 cytokines. Overall CCL24 was expressed at negligible levels in keratinocytes and fibroblast and some minor induction of CCL24 was observed in NHDFs after IL-4 and IL-13 stimulation (Fig. 3E and H). Hence, a picture emerges indicating that TH2 cytokines drive CCL11 and CCL26 expression in structural cells of the skin and that CCL26 represent the major CCR3 ligand present within the epidermal compartment.

      3.5 CCR3 ligands induce intracellular Ca2+ mobilization, migration and repair processes in dermal fibroblasts

      To investigate whether the chemokine receptor CCR3 is functionally active on NHDFs, we performed Ca2+ mobilization assays and observed a steady rise in cytosolic Ca2+ after the addition of CCL26 (100 ng/ml), confirming that calcium flux occurs following chemokine stimulation of primary cells (Fig. 4A , upper panel). The pretreatment with a neutralizing anti-CCR3 antibody inhibited the stimulatory effect of CCL26 in fibroblasts, indicating that CCR3 is functionally active on human dermal fibroblasts (Fig. 4A, lower panel).
      Figure thumbnail gr4
      Fig. 4Functional effects of CCL26 on NHDF. Calcium mobilization after CCL26 stimulation (A, upper panel) (A, lower panel: pre-treatment with anti-CCR3 antibody). Effects on proliferation (B). Effects on repair activity (G) and motility (H) in the presence (E and F) or absence (C and D) of CCL26 and anti-CCR3. Migratory effects (M and N) on spheroids in the presence (K and L) or absence (I and J) of CCL26.
      After having established that CCR3 is expressed and functionally active on NHDFs, we sought to investigate the biological consequences of CCR3 signaling. To this point, we aimed to study the role of CCR3 in the modulation of fibroblast proliferation and migration (Fig. 4B–N). Since fibroblast proliferation is an important step during tissue remodeling, we determined if CCL26 has an enhancing effect on NHDF proliferation in vitro. Interestingly, increasing concentration of CCL26 stimulation did not result in differences of fibroblast proliferation (Fig. 4B); however, significant effects were observed in migratory responses of NHDFs. In vitro-wound repair assays using NHDF layers and inducing a linear injury indicated that CCL26 markedly increased the repair capacity of fibroblasts from 40.37 ± 1.84% (unstimulated controls) to 70.06 ± 12.86% (CCL26, 100 ng/ml; *p < 0.05) in a 15-h time period (Fig. 4C–H). Pretreatment of NHDFs with a neutralizing anti-CCR3 antibody completely inhibited CCL26-induced in vitro wound repair indicating that this process critically depends on CCR3 signaling (Fig. 4G).
      To further support these findings, we performed migration assays of NHDF spheroids in 3D collagen matrix in the presence or absence of CCL26. The chemokine enhanced the migration of fibroblasts from the spheroids. The total area of fibroblast spheroids (36.5% increase; **p < 0.01; Fig. 4M) and the mean motility of the cells from the focal point (95.7% increase; ***p < 0.005; Fig. 4N) were significantly augmented when stimulated with 100 ng/ml CCL26 compared to untreated spheroids (Fig. 4I–N). Our results are in line with the notion that CCL26 directly affects fibroblast migration.

      4. Discussion

      Remodeling is an important pathophysiologic feature of chronic atopic skin inflammation; however, the underlying mechanisms remain largely elusive [
      • Katoh N.
      • Hirano S.
      • Suehiro M.
      • Ikenaga K.
      • Yasuno H.
      Increased levels of serum tissue inhibitor of metalloproteinase-1 but not metalloproteinase-3 in atopic dermatitis.
      ,
      • Toda M.
      • Leung D.Y.
      • Molet S.
      • Boguniewicz M.
      • Taha R.
      • Christodoulopoulos P.
      • et al.
      Polarized in vivo expression of IL-11 and IL-17 between acute and chronic skin lesions.
      ]. Current concepts suggest the involvement of inflammatory cells, in particular eosinophils, the alteration of microvascular structures, as well as the recruitment and activation of resident mesenchymal cells [
      • Trojanowska M.
      • LeRoy E.C.
      • Eckes B.
      • Krieg T.
      Pathogenesis of fibrosis: type 1 collagen and the skin.
      ]. Complex communication pathways within the skin may be important to regulate eosinophil–fibroblast interactions that finally lead to dermal remodeling.
      In this study, we aimed to unravel the chemokine-driven communication pathways between epithelial, stromal and inflammatory cells within atopic skin. So far, no systematic analysis of the chemokine receptor repertoire of human dermal fibroblasts exists. In 2005, Carulli et al. reported that dermal fibroblasts of patients suffering diffuse cutaneous systemic sclerosis (SSc) strongly expressed CCR2 protein both in vitro and in vivo and possessed a profibrotic phenotype in the early stage of the disease, while fibroblasts of healthy individuals did not express the receptor [
      • Carulli M.T.
      • Ong V.H.
      • Ponticos M.
      • Shiwen X.
      • Abraham D.J.
      • Black C.M.
      • et al.
      Chemokine receptor CCR2 expression by systemic sclerosis fibroblasts: evidence for autocrine regulation of myofibroblast differentiation.
      ]. The authors also showed that CCR2 signaling in SSc fibroblasts increased the expression of CCL2 protein, suggesting an autocrine profibrotic regulation pathway.
      Chemokine signaling in general leads to biological responses such as migration, invasion, proliferation and gene expression [
      • Homey B.
      • Steinhoff M.
      • Ruzicka T.
      • Leung D.Y.
      Cytokines and chemokines orchestrate atopic skin inflammation.
      ,
      • Homey B.
      • Bunemann E.
      Chemokines and inflammatory skin diseases.
      ]. Our findings show that NHDFs express functionally active forms of CCR3 in vitro and in vivo. Chemokine ligand-induced stimulation of the CCR3 receptor results in intracellular calcium mobilization in NHDFs. Moreover we demonstrate that CCR3 on resident dermal fibroblasts is directly activated by CCL26, resulting in strong, and selective activation of cell migration, invasion, and repair activity. However, we did not detect any significant modulation of cell proliferation. Recent studies by Puxeddu et al. support our findings showing CCR3 expression on human lung fibroblast (HLF). Complementary to our findings, the authors demonstrated that CCR3 signaling in HLF also increased cell proliferation and collagen production [
      • Puxeddu I.
      • Bader R.
      • Piliponsky A.M.
      • Reich R.
      • Levi-Schaffer F.
      • Berkman N.
      The CC chemokine eotaxin/CCL11 has a selective profibrogenic effect on human lung fibroblasts.
      ,
      • Kohan M.
      • Puxeddu I.
      • Reich R.
      • Levi-Schaffer F.
      • Berkman N.
      Eotaxin-2/CCL24 and eotaxin-3/CCL26 exert differential profibrogenic effects on human lung fibroblasts.
      ]. Hence, the present study demonstrates for the first time that CCR3-driven pathways may also play a role in fibroblast activation and tissue remodeling within the skin.
      CCR3 binds a variety of different ligands. Here we demonstrate for the first time a comprehensive overview of the expression of CCR3 ligands in chronic inflammatory and autoimmune skin diseases and show that among these ligands, keratinocyte-derived CCL26 proves significant, strong, and selective expression in atopic when compared to psoriatic skin inflammation, or to healthy skin. We suggest that CCL26 is an inflammatory and atopy-associated chemokine induced during atopic skin inflammation playing an important role in the pathogenesis of AD. Indeed previous studies described that CCL26 is overexpressed in lesional AD skin [
      • Owczarek W.
      • Paplinska M.
      • Targowski T.
      • Jahnz-Rozyk K.
      • Paluchowska E.
      • Kucharczyk A.
      • et al.
      Analysis of eotaxin 1/CCL11, eotaxin 2/CCL24 and eotaxin 3/CCL26 expression in lesional and non-lesional skin of patients with atopic dermatitis.
      ], and serum levels of CCL26 are significantly up-regulated in patients with AD and directly correlated with disease activity [
      • Kagami S.
      • Kakinuma T.
      • Saeki H.
      • Tsunemi Y.
      • Fujita H.
      • Nakamura K.
      • et al.
      Significant elevation of serum levels of eotaxin-3/CCL26, but not of eotaxin-2/CCL24, in patients with atopic dermatitis: serum eotaxin-3/CCL26 levels reflect the disease activity of atopic dermatitis.
      ].
      Another major hallmark in the pathogenesis of AD is tissue eosinophilia [
      • Homey B.
      • Steinhoff M.
      • Ruzicka T.
      • Leung D.Y.
      Cytokines and chemokines orchestrate atopic skin inflammation.
      ]. These cells are also known to produce profibrogenic signals that are crucial for fibroblast activation in skin tissue remodeling. Phipps et al. demonstrated that eosinophilia, provoked by intradermal allergen challenge in atopic patients, induced the activation of fibroblasts, the formation of myofibroblast-like cells, and the increase of the expression of matrix proteins in skin [
      • Phipps S.
      • Ying S.
      • Wangoo A.
      • Ong Y.E.
      • Levi-Schaffer F.
      • Kay A.B.
      The relationship between allergen-induced tissue eosinophilia and markers of repair and remodeling in human atopic skin.
      ]. Other findings suggest that CCR3-driven pathways are essential for the recruitment of eosinophils to sites of atopic skin inflammation [
      • Yawalkar N.
      • Uguccioni M.
      • Scharer J.
      • Braunwalder J.
      • Karlen S.
      • Dewald B.
      • et al.
      Enhanced expression of eotaxin and CCR3 in atopic dermatitis.
      ]. Hence CCR3 ligands direct eosinophils to the atopic skin inflammation and in turn eosinophils support skin remodeling.
      Next to eosinophils CCR3 signaling also recruits TH2 cells to the sites of atopic inflammation [
      • Sallusto F.
      • Mackay C.R.
      • Lanzavecchia A.
      Selective expression of the eotaxin receptor CCR3 by human T helper 2 cells.
      ,
      • Wang C.R.
      • Chen P.C.
      • Liu M.F.
      Increased circulating CCR3+ type 2 helper T cells in house dust mite-sensitive Chinese patients with allergic diseases.
      ]. Our results show that TH2 effector cytokines, such as IL-4 and IL-13, represent major regulators of CCL26 expression in vitro. Moreover, these cytokines support remodeling by serving as profibrogenic signals in collagen homeostasis [
      • Oriente A.
      • Fedarko N.S.
      • Pacocha S.E.
      • Huang S.K.
      • Lichtenstein L.M.
      • Essayan D.M.
      Interleukin-13 modulates collagen homeostasis in human skin and keloid fibroblasts.
      ]. Furthermore, IL-13 levels in bleomycin-induced systemic sclerosis murine model are markedly increased parallel with the progression of cutaneous fibrosis [
      • Matsushita M.
      • Yamamoto T.
      • Nishioka K.
      Upregulation of interleukin-13 and its receptor in a murine model of bleomycin-induced scleroderma.
      ].
      Taken together, within the complex puzzle of mediators regulating leukocyte trafficking and tissue remodeling during atopic skin inflammation, a concept emerges suggesting an orchestrated process of chemokine-driven communication that leads to the formation of a profibrogenic microenvironment or “niche” created by keratinocytes, eosinophils, TH2 cells, and fibroblasts (Fig. 5). Atopy-associated, keratinocyte-derived CCL26 will support TH2 and eosinophil recruitment to sites of atopic skin inflammation. Moreover, CCL26 will support the recruitment and migratory activation of CCR3-expressing resident mesenchymal cells. In turn, TH2 effector cytokines will locally enhance the production of CCR3 ligands such as CCL11 and CCL26 perpetuating the process that in concert with eosinophil-derived TGF-β production will lead to atopy-associated cutaneous remodeling.
      Figure thumbnail gr5
      Fig. 5CCR3–CCL26 chemokine receptor–ligand interaction in atopic skin inflammation. A chemokine-driven communication pathway leads to the formation of a profibrogenic microenvironment or “niche” created by keratinocytes, eosinophils, TH2 cells, and fibroblasts. Atopy-associated, keratinocyte-derived CCL26 supports TH2 and eosinophil recruitment to sites of atopic skin inflammation. Moreover, CCL26 supports the recruitment and migratory activation of CCR3-expressing resident mesenchymal cells. In turn, TH2 effector cytokines locally enhance the production of CCR3 ligands such as CCL11 and CCL26 perpetuating the process that in concert with eosinophil-derived TGF-β production leads to atopy-associated cutaneous remodeling.

      Acknowledgments

      Supported by Contract QLRK4-CT-2001-00366 Chemokines and Atopy from the European Union (to B.H., A.I.L., H.A., L.K., and M.-C.D.-N.). K.G. and A.S. were supported by National Research Grant ( OTKA K81381 ), and TAMOP 4.2.2.A-11/1/KONV-2012-0023 “VÉD-ELEM” (implemented by the New Hungary Development Plan co-financed by European Social Fund and European Regional Developmet Fund ). J.F. was supported by SFB728, TP C6 . B.H. was supported through following grants of the German Research Foundation SFB503/C9 , Ho 2092/3-1 and Ho 2092/4-1 .

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