Modulation of eotaxin-3 (CCL26) in alveolar type II epithelial cells

Modulation of eotaxin-3 (CCL26) in alveolar type II epithelial cells

www.elsevier.com/locate/issn/10434666 Cytokine 36 (2006) 237–244 Modulation of eotaxin-3 (CCL26) in alveolar type II epithelial cells Barack O. Abony...

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www.elsevier.com/locate/issn/10434666 Cytokine 36 (2006) 237–244

Modulation of eotaxin-3 (CCL26) in alveolar type II epithelial cells Barack O. Abonyo b, Kimberly D. Lebby b, Jessica H. Tonry Munir Ahmad a, Ann S. Heiman a,*

c,1

,

a b

Florida A & M University, College of Pharmacy and Pharmaceutical Sciences, Tallahassee, FL 32307, USA Florida A & M University, College of Arts and Sciences, Department of Biology, Tallahassee, FL 32307, USA c Hawaii Biotech, Inc. 99-193 Aiea Heights Drive #200, Aiea, HI 96701, USA Received 20 October 2006; received in revised form 20 December 2006; accepted 16 January 2007

Abstract Airway epithelial inflammation associated with emphysema, chronic bronchitis, chronic obstructive pulmonary disease (COPD) and asthma is regulated in part by alveolar type II cell chemokine signaling. Data suggest that resident lung cells use CCR3, CCR5 and CCR2 chemokine receptor/ligand systems to regulate the profile of leukocytes recruited in disease-associated inflammatory conditions. Thus studies were designed to test whether alveolar type II cells possess a Th1-activated CCR5-ligand system that modulates the Th2-activated CCR3/eotaxin-2 (CCL24), eotaxin-3 (CCL26) chemokine systems. The A549 alveolar type II epithelial-like cell culture model was used to demonstrate that alveolar type II cells constitutively express CCR5 which may be upregulated by MIP-1a (CCL3) whose expression was induced by the Th1 cytokines IL-1b and IFN-c. Selective down-regulation of CCL26, but not CCL24, was observed in CCL3 and IL-4/CCL3 stimulated cells. Down-regulation was reversed by anti-CCR5 neutralizing antibody treatment. Thus, one mechanism through which Th1-activated CCCR5/ligand pathways modulate Th2-activated CCR3/ligand pathways is the differential down-regulation of CCL26 expression. Results suggest that the CCR3 and CCR5 receptor/ligand signaling pathways may be important targets for development of novel mechanism-based adjunctive therapies designed to abrogate the chronic inflammation associated with airway diseases.  2007 Elsevier Ltd. All rights reserved. Keywords: Alveolar epithelial cells; CCR5 receptors; CCL3; CCR3 receptors; Eotaxins

1. Introduction In addition to their specifically defined pathologies, one pulmonary problem common to emphysema, chronic bronchitis, chronic obstructive pulmonary disease (COPD), asthma and cigarette smoke exposure is underlying and complex lower airway inflammation regulated by chemokine receptor/ligand signaling. Airway structural or constituent cells that include fibroblasts, myofibroblasts, smooth muscle cells and airway epithelial cells are impor*

Corresponding author. Fax: +1 850 599 3323. E-mail addresses: [email protected] (B.O. Abonyo), kimberly @lebbyimaging.com (K.D. Lebby), [email protected] (J.H. Tonry), [email protected] (M. Ahmad), [email protected] (A.S. Heiman). 1 Fax: +1 808 792 1343. 1043-4666/$ - see front matter  2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2007.01.001

tant sources of cytokines and chemokines that play pivotal roles in the perpetuation of the underlying airway inflammatory responses [1–3]. The chemokines are a large group of signaling peptides divided into CC, CXC and CXXC families based upon sequence considerations. Chemokines exert bioactivities with complex agonist and antagonistic molecular interactions with multiple chemokine receptors through which they activate homeostatic or pathological inflammatory cell responses [4,5]. In addition to their surfactant synthesizing, xenobiotic metabolic and replacement of alveolar type I cell functions, alveolar type II cells are recognized as rich sources of chemokines. In a recent report, basal release chemokines that were upregulated by treatment of primary human alveolar type II cells with LPS and a Th1 cytokine mix (TNF-a, IL-1b and IFN-c) included IL-8 (CXCL8), MCP-1 (CCL2), RANTES (CCL5), MIP-1a (CCL3) and GRO-a (CXCL1) [6].

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Treatment of alveolar type II-like epithelial cells in culture with the Th2 cytokines IL-4/IL-13 increases release of the eotaxin chemokines, particularly eotaxin-2 (CCL24) and eotaxin-3 (CCL26) [9,10]. Interestingly, these Th1 and Th2 stimulated chemokines share receptors. Both CXCL1 and CXCL8 recruit neutrophils through the CD128 type 2 receptor (IL8RB) also used by members of the macrophage selective MIP-2 family of CC chemokines. CCL3, involved in acute neutrophilic inflammation, recruitment of T-lymphocytes, monocytes, dendritic cells and activation of granulocytes and macrophages [11], and CCL5 which is chemotactic for T-lymphocytes, eosinophils and basophils are ligands for CCR5. MCP-2 signals chemotaxis of monocytes via CCR2. The three eotaxins, CCL11, CCL24 and CCL26 selectively signal chemotaxis and activation of eosinophils through the CCR3 receptor, to which CCL5 and other members of the monocyte chemotactic peptide MCP family also bind [12]. This suggests that alveolar type II cells have the potential to orchestrate the number and profile of all classes of leukocytes recruited under basal, Th1 and Th2 proinflammatory conditions through CCR3, CCR5, CCR2 and IL8RB receptor/ligand signaling. CCR3 is expressed on basophils, mast cells, eosinophils, subsets of Th2 lymphocytes, dendritic cells and bronchial epithelial and alveolar type II epithelial cells [7,11–13]. These locations are suggestive of a role for CCR3-ligand interactions in the underlying inflammation associated with airway diseases. Recent experimental results from this laboratory suggest that CCL11, CCL24 and CCL26 may contribute to pathogenesis at sites of Th2 phenotypic inflammation not only through eotaxin receptor CCR3-ligand signal transduction pathways in eosinophils but modulation of the airway epithelium through ligand-CCR3 receptor feedback mechanisms [7,8]. The human CCR5 receptor has been the focus of intensive research following discovery of its role as a fusion cofactor for the macrophage-tropic human immunodeficiency virus-1. In this context, biological functions and mechanisms which regulate its signal transduction and cell surface expression have been reported [14]. Human CCR5 is a G protein-coupled receptor expressed on peripheral blood-derived dendritic cells, memory and effector Th1 lymphocytes and macrophages, capillary endothelial cells and vascular smooth muscle cells. In many of these cell types, CCR5 functional roles have not been fully established [15]. Though CCR5 has been closely associated with Th1 cells, its presence on Th2 lymphocytes of chronic hypereosinophilia have been reported [16]. Using a murine model, pulmonary expression of CCR5 has been shown to play a role in IFN-c-induced and cigarette smoke-induced emphysema [17]. A number of CC chemokines, namely, CCL3, MIP-1b (CCL4), CCL5 and MCP-2 (CCL8) are considered CCR5 high-potency agonists [18]. Of these, evidence of CCL3 and CCL5 in the pathogenesis of airway inflammation has been reported [19,20].

Many cells express multiple chemokine receptors and experience a microenvironment containing many diverse ligands. As shown for the CCR3 and CCR5 receptor/ligand families, chemokines directly influence and modify cell bioactivities through multiple chemokine receptor binding by acting as an agonist for one receptor, an antagonist for another receptor or may dock and sequester at yet another receptor. CCL11 exhibits agonist activity through CCR5 while, in contrast, CCL26 is considered a natural antagonist for CCR5 while CCL24 did not compete for CCR5 binding [21,22]. CCL3, a high affinity agonist for CCR5 did not compete for binding to CCR3 [23], while, in contrast, CCL5 exerts agonist activities through CCR3 and CCR5 [24]. Thus, in underlying inflammation of diseased airways, the alveolar airway epithelium is actively involved in orchestrating the number and profile of leukocytes through cytokine and chemokine networks. Involvement of the Th2-activated CCR3/ligand network in chemoattraction and activation of leukocytes which perpetuate underlying airway inflammation is known. These present studies have been designed to test the hypothesis that alveolar type II cells possess a Th1-activated CCR5-ligand system that may modulate the Th2-activated CCR3/CCL24, CCL26 chemokine systems. These findings will assist in establishing the appropriate targets for design of adjunctive treatments to control progression of alveolar inflammation in airway diseases. 2. Materials and methods 2.1. Culture and stimulation of airway epithelial cells Human A549 alveolar type II epithelium-like cells (ATCC CCL-185) were purchased from American Type Culture Collection and handled as previously described [7,8]. Briefly, cells were cultured in RPMI1640/F12K (50/ 50 v/v) supplemented with 10% fetal calf serum, penicillin (100 U/ml) and streptomycin (100 lg/ml) in a humidified atmosphere of 5% carbon dioxide at 37 C. Prior to stimulation, cells were incubated in serum-free medium for 3 h then stimulated in fresh serum-free medium with indicated concentrations and combinations of chemokines and/or cytokines (Atlanta Biologicals, Atlanta, GA, USA). 2.2. Detection of CCR5 in airway epithelial cells by immunocytochemistry Human A549 cells (1 · 104) were cultured in Labtek coverslip chambers for 24 h and treated with 100 ng/ml IL-4, CCL3 or CCL5. Cells were washed three times with PBS and fixed in 4% paraformaldehyde for 20 min. Fixed cells were permeabilized with 0.2% Triton-X-100 for 5 min, blocked in 10% normal goat serum then incubated overnight with or without 10 lg/ml mouse Texas Red-conjugated monoclonal human anti-CCR5 (Medical & Biological Laboratories Co., Nagoya, Japan) or Texas

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Red-conjugated mouse IgG (Santa Cruz Biotechnology, CA) isotype control. Slides were mounted with slow fade containing DAPI and the expression of CCR5 protein was visualized and captured with an Olympus fluorescent microscope (10· objective) fitted with an Olympus DP70 camera. Pictures were documented using Adobe Photoshop. 2.3. Detection of airway epithelial cell CCR5 receptors by flow cytometry

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IgG-horseradish peroxidase (Santa Cruz Biotechnology, CA) in PBST overnight at 4 C. Following another three washes in PBST and two rinses in PBS, immunoblot images were obtained using a Fluor-s Max Multimager (Bio-Rad Laboratories, Hercules, CA). Protein loading was monitored in each gel lane by probing the membranes with anti-GAPDH antibodies (R&D Systems). Lane density data were acquired with UN-SCAN-IT gel software (Silk Scientific, Inc., Orem, UT). 2.6. Data handling and analysis

A549 airway epithelial cells (0.5 · 106 cells/ml) were treated for 12 h with 1–100 ng/ml of the indicated cytokines in serum free medium, detached with 0.5 mM EDTA in PBS and centrifuged at 500g, 4 C for 5 min. Cell pellets were washed twice in cold FACSflow buffer (BD Biosciences, San Jose, CA) containing 2% FBS and resuspended to a final concentration of 5 · 106 cells/ml. Cells were then Fc-blocked by treatment with 1 lg human IgG/105 cells for 15 min. Aliquots (25 ll) of cells were stained with 10 ll mouse anti-human PE-conjugated CCR5 or mouse PE-IgG isotype control and 10 ll rat anti-human FITCconjugated CCR3 or rat FITC-IgG isotype control (R&D Systems, Minneapolis, MN) for 30 min in the dark at 4 C. After two washes in cold FACSflow buffer, stained cells were maintained at 4 C then subjected to flow cytometry on a FACSCalibur (BD Biosciences, San Jose, CA). Data were analyzed using CellQuest software (BD Biosciences, San Jose, CA) [8]. 2.4. CCL24 and CCL26 detection by specific ELISA Cells were treated with human recombinant cytokines, chemokines or anti-human CCR5 clone 45631 (R& D Systems, Minneapolis, MN, USA) for the times indicated. Supernatants and lysates of stimulated cells were prepared as previously described [7] and assessed for presence of each of the chemokines by specific CCL24, CCL26 or CCL3 ELISAs (R&D Systems, Minneapolis, MN, USA). Secreted and synthesized chemokines were quantified with a Power Wave X 340 microplate reader equipped with KC4 v3.0 PowerReports software (Bio-Tek Instruments, Winooski, VT, USA). 2.5. Western immunoblotting of the CCR5 receptor Treated airway epithelial cell lysates were prepared as previously described [7]. Cell lysates were then separated by electrophoresis on 10% SDS-polyacrylamide gels (5 lg protein/lane) and transferred to Immobilon-P membranes (Millipore Co, Bedford, MA). Blots were blocked at 4 C overnight in 5% Carnation Instant Milk in phosphate-buffered saline with 0.05% Tween 20 in PBS (PBST) and then incubated overnight at 4 C with a 0.75 lg/ml rabbit antihuman CCR5 affinity purified antibody (Imgenex., San Diego, CA). Membranes were then washed three times with 5% instant milk and incubated with 1:500 goat anti-rabbit

Unless otherwise stated, experiments were conducted in triplicate and repeated on at least three separate occasions (flow cytometer experiments were performed in duplicate on three different occasions). Data shown as mean ± SEM is the mean of three experiments with the average of duplicates or triplicates from one experiment serving as one observation. When indicated, one-way analysis of variance (ANOVA) followed by either the Tukey multiple comparisons or Dunnett post-test, as appropriate, was applied to experimental results to determine statistical significance (p < 0.05) between indicated groups. 3. Results 3.1. A549 alveolar type II epithelial-like cells express CCR5 and its high affinity ligand CCL3 Data suggest that resident lung cells use CCR3, CCR5 and CCR2 receptor/ligand systems to orchestrate the number and profile of leukocytes recruited in disease-associated inflammatory conditions. Since CCR3/ligand pathways with multiple ligand binding to CCR5 and CCR2 are known, probing for expression of CCR5 and CCR2 was of interest. Immunocytochemistry results of permeabilized cells allowing visualization of both surface and internalized receptors are shown in Fig. 1. Results demonstrate that A549 alveolar type II cells do not possess CCR2 receptors (data not shown), but do constitutively express CCR5 receptors. Respiratory syncytial virus infection of lower airway epithelial cells, including A549 alveolar type II cells, has been shown to induce CCL5 and CCL3 mRNA expression and protein secretion [25,26]. Since the alveolar type II cells possess CCR5, it was of interest to determine expression of the CCR5 ligand CCL3 and delineate cytokine signals which elicit its synthesis and release. Cells were stimulated with cytokines and culture supernatants and cell lysates tested for presence of CCL3 by specific ELISA. IL-4, IL-13 and IL-10 did not stimulate expression of any detectable amounts of the chemokine (data not shown). Results following stimulation with IL-1b, IFN-c or TNF-a, either alone or in combination, are shown in Fig. 2. In contrast to IFN-c or TNF-a, IL-1b alone stimulated a concentrationdependent release of CCL3 up to 187 pg/ml. Stimulation of cells with IFN-c (10 ng/ml) together with IL-1b

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Fig. 1. CCR5 is constitutively expressed in A549 alveolar type II epithelial cells. A549 cells (1 · 105/100 ll) were cultured in RPMI1640/F12K plus 10% FBS for 24 h then stimulated with indicated cytokines (100 ng/ml) for 24 h. The monolayers were washed three times in PBS then fixed in 0.4% paraformaldehyde for 20 min. Fixed cells were permeabilized with 0.2% TX-100 in PBS for 5 min. Nonspecific binding was blocked by treatment with 10% normal goat serum for 1 h, then cells were incubated with 10 lg/ ml of Texas Red-conjugated mouse anti-human CCR5 or mouse IgG. Cells were washed three times with PBS and images captured at 10· magnification using a fluorescent microscope. Documented images are representative of several pictures taken from duplicates of three separate experiments.

(0.1–10 ng/ml), resulted in a potentiation of CCL3 release. These results suggest that alveolar type II cells possess a CCR5-ligand system that may be involved in regulation of chemokine expression and release. 3.2. Constitutive expression of CCR5 is enhanced by CCL3 Suggestive increases in fluorescence intensity of cells treated with the CCR5 agonists CCL3 and CCL5 led to flow cytometric explorations of surface expression of CCR5. Cells were treated for 12 h with the CCR5 high affinity agonist and surface CCR5 stained for flow cytometry. Results are depicted in Fig. 3. Density plots of dual-stained anti-CCR3-FITC versus anti-CCR5-PE fluorescence intensity indicate that stimulation of alveolar type II cells with CCL3 increased cell surface CCR5 receptors. Semi-quantitative analyses of density plots of unstimulated, dual-stained cells demonstrated increased fluorescence of CCR5 in 43% of cells. Stimulation with CCL3 (1 ng/ ml) enhanced CCR5 fluorescence in 66% of cells. Following treatment with 100 ng/ml CCL3, 97% of cells showed

Fig. 2. A549 alveolar type II cells synthesize and release CCL3 when stimulated with IL-1b or IL-1b with INF-c. A549 airway epithelial cells (5 · 105 cells/well in 24-well cluster plates) were allowed to attach overnight, bathed in serum free medium then treated under serum free conditions with 0.1–10 ng/ml IL-1b, IFN-c, TNF-a, IFN-c (10 ng/ml) with either IL-1b (0.1–10 ng/ml) or TNF-a (0.1–10 ng/ml) for 24 h. Resulting supernatants were immediately assessed for CCL3 by specific ELISA. Unstimulated controls released 4.6 ± 0.33 pg/ml CCL3. Data presented are the average ± SEM of three separate experiments each conducted in triplicate. Release from cells treated with IL-1b + IFN-c was significantly different from controls at all concentrations tested, and release from cells treated with IL-1b alone was significantly different from controls at 0.3–10 ng/ml. Asterisks indicate significant differences between IL-1b alone and same concentrations of IL-1 b in presence of 10 ng/ml IFN-c at p < 0.05.

increased fluorescence for CCR5 accompanied by increased fluorescence for CCR3 in 77% of A549 cells. These results indicate that CCL3 upregulates its receptor and suggests an interaction between the CCR5/ligand and CCR3/ligand pathways. Experiments were then carried out to assess treatment effects on A549 alveolar type II cell CCR5 protein expression. Results are shown in Fig. 4. Protein expression in A549 cells stimulated with IL-4 (1 or 10 ng/ml) and CCL3 (1–100 ng/ml) is shown in Fig. 4a. Results of densitometry studies of multiple experiments, shown in Fig. 4b, demonstrate that CCL3 treatments significantly increased CCR5 protein expression in a concentration dependent manner. Taken together, these results suggest that CCR5 surface expression is modulated by the CCL3 by autoregulation or perhaps in response to monocytic cell-derived CCL3. 3.3. CCL3 inhibits synthesis of CCL26 As previously reported, IL-4 upregulates CCR3 surface expression and also expression and release of the CCR3 high affinity ligands CCL24 and CCL26 in A549 type II alveolar cells. Pretreatment of cells with the CCR3 antagonist SB328437 significantly inhibited secretion and synthesis of CCL26, but had little effect on CCL24 [7]. To explore interaction of the CCR5 and CCR3 ligand/receptor systems, cells were treated with CCL3 and IL-4 alone and in

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Fig. 3. CCL3 increases surface CCR5 receptor expression on A549 alveolar type II epithelial cells. A549 airway epithelial cells (0.5 · 106 cells/well in 6-well cluster plates) were treated with vehicle (medium), or CCL3 (1 or 100 ng/ml) for 12 h. A549 cells were then detached with PBS/0.5 mM EDTA, washed and stained with PE-IgG isotype and FITC-IgG isotype controls (a) or anti-human PE-conjugated CCR5 antibody and anti-human FITCconjugated CCR3 antibody (b–d). Treatments of cells depicted in density plots were as follows: (a) isotype controls, (b) unstimulated cells, (c) CCL3 at 1 ng/ml and (d) CCL3 at 100 ng/ml. The data presented are representative of three separate experiments each conducted in duplicate.

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combination. Expression and secretion of CCL24 and CCL26 were then quantified. Results with CCL3 alone showed a concentration dependent decrease of 40% in CCL26 expression when compared with untreated controls as shown in Fig. 5a. The addition of 10 ng/ml IL-4 to CCL3 treated cells increased CCL26 synthesis but not to the level seen with IL-4 as the sole stimulating agent. Conversely, when cells were stimulated with 1–100 ng/ml IL-4 in the presence of CCL3 (100 ng/ml) CCL26 synthesis was significantly inhibited implying that the CCR5 agonist CCL3 modulates IL-4 dependent CCL26 expression. In data not shown, it was determined that there were no significant differences in CCL24 synthesis in cells treated with CCL3 or CCL5 alone or in combination with IL-4. To establish involvement of CCL3 in CCL26 gene expression, proteins were extracted from A549 alveolar type II cells treated with 1–100 ng/ml CCL3 alone or in the presence of 1–100 ng/ml IL-4 for 24 h. Western blot results, shown in Fig. 5b, corroborate the ELISA data.

Fig. 4. CCR5 expression is enhanced by CCL3. Cells were cultured as described in Fig. 2, then treated with indicated concentrations of cytokines. (a) Representative CCR5 and GAPDH western blot. (b) Densitometry of three western blot experiments each performed in duplicate with untreated control CCR5/GAPDH ratio set equal to 1. Asterisks indicate those groups which differed from controls at p < 0.05.

3.4. Blocking CCR5 modulates IL-4 and IL-4/CCL3 induced effects on CCL26 To confirm the role of the CCR5/CCL3 involvement in CCR3/CCL26 regulation, cells were pretreated with a

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anti-CCR5 neutralizing antibody. These results suggest that one mechanism through which Th1-activated CCCR5/ligand pathways modulate Th2-activated CCR3/ ligand pathways is down-regulation of CCL26 expression. This interplay of the CCR3/CCL26 and CCR5/agonist pathways may play an important modulating role in leukocyte recruitment to sites of inflammation in airway diseases.

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Fig. 5. The CCR5 agonist CCL3 down-regulates CCL26 protein expression in A549 alveolar type II epithelial cells. A549 cells (5 · 105/500 ll) were cultured as described in Fig. 2. Cells were then treated as indicated with 1–100 ng/ml of CCL3 or IL-4 alone, IL-4 (1–100 ng/ml) with 100 ng/ ml CCL3 or CCL3 (1–100 ng/ml) in presence of 10 ng/ml IL-4 in serum free media for 24 h. (a) Cell lysates, prepared from treated, washed cells, were diluted 10· in PBS and 100 lg protein used to quantify cellular CCL26 by specific ELISA. The data presented are an average of four separate experiments each conducted in duplicate. Asterisks indicate those groups which differed from controls at p < 0.05. (b) Representative CCR5 and GAPDH western blot.

bioactivity neutralizing human anti-CCR5 monoclonal antibody [27], then stimulated with IL-4 and/or CCL3. Results with IL-4 alone indicate that antibody pretreatment significantly suppresses CCL26 release up to 40% (Fig. 6). When cells were stimulated with both IL-4 and CCL3, CCL26 release, reduced by 45%, was reversed in a concentration dependent manner by treatments with 2000 IL-4 + CCL3

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Anti-CCR5 Fig. 6. Human anti-CCR5 reverses the CCL3-induced modulation of CCL26 secretion. A549 airway epithelial cells, cultured as described in Fig. 2, were treated under serum free conditions with 0–10 lg/ml antiCCR5 for 30 min followed by IL-4 (10 ng/ml) alone or in combination with CCL3 (100 ng/ml) for 24 h. Culture supernatants (100 ll) were used to detect released CCL26 by specific ELISA. Data shown are the mean ± SEM of three experiments each conducted in triplicate. Asterisks indicate groups that differed significantly from cells stimulated with IL-4 in the absence of anti-CCR5 at p < 0.05.

Chemokine ligand/receptor pathways are emerging as critical events in the orchestration of the underlying inflammatory conditions in airway diseases. Evidence suggests that alveolar type II cells have the potential to orchestrate leukocyte recruitment in Th1 and Th2 proinflammatory conditions through CCR3, CCR5, CCR2 and IL8RB receptor/ligand signaling. While Th2-activated CCR3/ligand chemoattraction and activation of leukocytes by alveolar type II cells has been reported, these other pathways have not been explored. Thus, the present studies were carried out to investigate the presence of an alveolar type II cell Th1-activated CCR5-ligand system that may modulate the Th2-activated CCR3/CCL24, CCL26 chemokine pathways. Results of these investigations demonstrate that alveolar type II cells do not express CCR2 but constitutively express CCR5. CCR5 was upregulated by CCL3 whose expression was induced by Th1 cytokines suggesting that alveolar epithelial cells have the potential to engage in target/effector responses via regulatory and autoregulatory CCR5-ligand pathways. Selective down-regulation of CCL26, but not CCL24, was evident in CCL3 and IL-4/ CCL3 stimulated cells. Reversal of the CCR5/CCL3-induced effect by an anti-CCR5 neutralizing antibody was shown. These studies demonstrate that one mechanism through which Th1-activated CCCR5/ligand pathways modulate Th2-activated CCR3/ligand pathways is the differential down-regulation of CCL26 expression. Differential roles of the three eotaxins, particularly CCL26, are emerging. For example, bronchial epithelial cells, alveolar type II cells, airway smooth muscle cells can be stimulated to synthesize and release large amounts of CCL26. In contrast to CCL11, a sustained cytokine-induced production of CCL26 has been reported for these cells. A 10-fold difference in the potency of IL-13 and IL4-mediated induction of CCL26 mRNA expression was noted in human bronchial epithelial cells where inhibition of NF-jB abrogated Th2 cytokine CCL11 induction but stimulated CCL26 expression [28,29]. In BEAS-2B bronchial epithelial cells, it was found that IFN-c enhanced Th2 cytokine induced CCL26 mRNA and sustained protein expression [30]. This suggests that airway Th1-type events may affect Th2 driven inflammation by a sustained upregulation of CCL26. In recent comparative clinical studies of asthmatics it was found that the elevated prechallenge CCL11 and CCL24 mRNA levels did not increase following allergen challenge. In contrast, CCL26

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expression was dramatically upregulated (to 100-fold) in asthmatics 24 hours following allergen exposure [31]. Numbers of biopsy cells expressing CCL26 and CCL24, but not CCL11, correlate significantly with the magnitude of the late asthmatic response [32]. Results of the present investigations also support differential regulation of CCL26 and CCL24. CCR5/ligand signaling was not significantly involved in the expression or release of CCL24. Interestingly, in bronchial epithelial cells it is recently been reported that CCL24 accelerated epithelial wound closure and induced epithelial cell proliferation and chemotaxis that could be abrogated by treatment with a CCR3 antagonist [33]. Collectively, these results imply that CCL26 is regulated differently than the other two eotaxins, that multiple receptor/ligand pathways are involved in CCL26 signaling and modulation, release of CCL26 from activated cells is robust and sustained, and that CCL26 may signal leukocyte recruitment in an organized and coordinated manner to elicit specific disease stage-specific bioactivities. The underlying lower inflammation in airway diseases is chronic and complex. In contrast to the eosinophilic responses measured in asthmatics, neutrophils and macrophages have been implicated in development of COPD which exhibits overlap with chronic bronchitis and emphysema. In severe emphysema, however, significant increases in macrophages, neutrophils, lymphocytes and eosinophils are seen [34]. In contrast to asthma, COPD and emphysema are associated with high percentages of activated Th1 phenotype lymphocytes that express the chemokine receptors CCR5 and CXCR3 and secrete IL-2 and IFN-c [35]. In the transgenic murine pulmonary IFN-c overexpression model, IFN-c caused a mononuclear and neutrophil-rich tissue inflammatory response, pulmonary emphysema and alveolar destruction with concomitant increases in CCL3, CCL4 and CCL5 and their CCR5 receptor. Interventions used to neutralize or abrogate CCR5 diminished IFN-c-induced inflammatory, cell death and remodeling responses in the murine lung [17,36]. Collectively, results of these studies demonstrate contributions of CCR5/ligand signaling as well as CCR3/ligand involvement in lower airway inflammatory and remodeling responses taking place in COPD and the chronically smoke-exposed lung. Studies on the chemokine production by primary human alveolar type II cells are very limited. It was recently shown that chemokines released by resting primary human alveolar type II cells included CXCL8, CCL2, CCL5, CCL3 and CXCL1. Treatment with LPS and a cytokine mix (TNF-a, IL-1b and IFN-c) upregulated release [6]. Human cytokine antibody membrane array studies carried out in this laboratory have revealed basal release of these same chemokines from A549 alveolar type II epithelial-like cells cultured in serum free conditions (unpublished data). Stimulated release of CCL3 from primary human alveolar type II cells was quantitatively very similar to CCL3 amounts released by A549 cells stimulated with IL-1b alone or in combination with IFN-c. This suggests that alveolar type

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II cells have the potential to orchestrate the number and profile of leukocytes recruited under both basal and proinflammatory type Th1 or Th2 environments. Results also suggest that A549 cells are a suitable surrogate model for alveolar type II cell chemokine receptor/ligand investigations. In summary, alveolar type II cells are a rich source of chemokines and play a role in orchestrating the chronic underlying inflammation associated with COPD, emphysema, chronic bronchitis and asthma disease stages. It is understood that the alveolar epithelium is exposed to a complex microenvironment of autocrine-regulated as well as paracrine-regulated chemokines produced by resident and emigrated leukocytes within the airways. Within results of the present investigations and cited studies, an alveolar type II cell chemokine receptor/ligand scheme operative in the inflamed airways can be envisioned as follows. Alveolar type II cells have the ability to respond to either Th1 or Th2 dominant inflammatory environments by inducing chemotaxis of all classes of leukocytes accomplished predominantly via CCR3 and CCR5 chemokine receptor/ligand signaling. In a Th2driven inflammatory response, IL-4/IL-13 stimulate release of eotaxins that act as chemoattractants and/or activating agents for CCR3 bearing eosinophils, basophils, dendritic cells, lung parenchyma mast cells and subsets of Th2 lymphocytes. In a Th-1 dominated inflammatory response, T cell mediators such as IFN-c stimulate upregulation of CCR5 and release of CCL3 and CCL5 which stimulate emigration of monocytes, T lymphocytes, eosinophils and basophils. Constitutive alveolar type II cell release of CXCL1 and CXCL8 serve as constant recruiting signals for neutrophils. The absence of alveolar type II cell CCR2 receptors circumvents autoregulation of CCL2 which is constitutively released as a signaling chemokine for monocytes. Stimulated alveolar type II cells release large and sustained amounts of CCL26 whose differential down-regulation may be accomplished by modulation of Th2-activated CCR3/CCL26 pathways by CCR5/ligand signaling. Ultimately, the close functional relationship between the alveolar epithelium, other resident cells and leukocytes recruited into the inflamed airway may result in alterations observed in the COPD, emphysemic and asthmatic lung. Thus, it should be viewed as a target cell for development of novel anti-inflammatory therapeutics. Specifically, the CCR3 and CCR5 receptor/ligand signaling pathways may be important targets for development of novel mechanism-based adjunctive therapies designed to interrupt the underlying chronic inflammation associated with diseases of the airways. Acknowledgments Support for this research was provided in part by NIH grants RR08111, RR03020 and the Pharmaceutical Research Center NIH/NCRR1 C06-RR12512-01.

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