Human Molecular Genetics Advance Access originally published online on April 6, 2006
Human Molecular Genetics 2006 15(10):1667-1679; doi:10.1093/hmg/ddl090
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Neuropeptide S and G protein-coupled receptor 154 modulate macrophage immune responses
1Department of Medical Genetics, Biomedicum Helsinki and 2Department of Pathology, University of Helsinki, Helsinki, Finland, 3Finnish Institute of Occupational Health, Helsinki, Finland, 4Swiss Institute of Allergy and Asthma Research, Davos, Switzerland, 5Department of Allergy, Helsinki University Central Hospital, Helsinki, Finland, 6Department of Medicine, Helsinki University Central Hospital, Helsinki, Finland, 7Department of Biosciences and Nutrition at Novum and Clinical Research Centre, Karolinska Institutet, 14157 Huddinge, Sweden and 8GeneOS Ltd, Helsinki, Finland
* To whom correspondence should be addressed. Tel: +46 86089158; Fax: +46 87745538; Email: juha.kere{at}biosci.ki.se
Received January 25, 2006; Accepted March 29, 2006
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ABSTRACT |
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G protein-coupled receptor 154 (GPR154) is a recently discovered asthma susceptibility gene upregulated in the airways of asthma patients. We previously observed increased pulmonary mRNA expression of the murine ortholog Gpr154 in a mouse model of ovalbumin (OVA)-induced inflammation. However, the expression profile of GPR154 in leukocytes and the cellular functions of the receptor and its endogenous agonist neuropeptide S (NPS) have remained unidentified. Here, we characterized the mRNA expression of NPS and GPR154 by using real-time RT–PCR in fractionated human blood cells and in peripheral blood mononuclear cells (PBMCs) with monocyte or T cell activation. The expression of GPR154 in leukocytes was further confirmed by immunoblotting experiments and immunohistochemical staining of human sputum samples. Additionally, we characterized the expression of GPR154 in the lung tissue samples and in the bronchoalveolar lavage (BAL) fluid of OVA sensitized and challenged BALB/c mice. In human blood and sputum cells, monocyte/macrophages and eosinophils were identified as GPR154-positive cells. In PBMCs, monocyte activation with LPS but not T cell activation with anti-CD3/CD28 antibodies resulted in increased NPS and GPR154 expression. In the lung tissue samples and in the BAL fluid of OVA-challenged mice, GPR154 expression was upregulated in alveolar macrophages in comparison to controls. In the mouse macrophage RAW 264.7 cell line, NPS-stimulated G
s- and Gq-dependent phagocytosis of Escherichia coli. The results show that GPR154 is upregulated in macrophages after antigen challenge and that NPS is capable of inducing phagocytosis of unopsonized bacteria. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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We have recently identified a novel susceptibility gene, GPR154 (G protein-coupled receptor 154 also known as GPRA, PGR14, VRR1 and NPSR), on human chromosome 7p15–p14 that associates with asthma and high serum total immunoglobulin E (IgE) concentrations (1
,2). The findings were recently replicated in large European cohorts in two independent studies, where the associated haplotypes could be allocated into risk and non-risk groups in agreement with the original results (3,4). There are several options how GPR154 could have an influence on the disease mechanism of asthma. First, the polymorphisms might affect the signaling, alternative splicing or the allele-specific expression of the two main GPR154 isoforms, A and B. These mechanisms should result in dysregulation of GPR154-mediated signaling in a small fraction of patients. Second, the complex disease mechanisms of asthma might also directly affect to GPR154 pathway, resulting in abnormal function of the receptor in a large proportion of patients. However, evidence concerning the functions of GPR154 in the airway wall is only beginning to accumulate.
A proteomic screening for endogenous ligands of GPR154 resulted in the discovery of a novel linear 20-residue peptide that activated the receptor increasing both intracellular cAMP and Ca2+ levels (5). The agonist was named neuropeptide S (NPS). It is highly expressed in the brain and has pharmacological effects related to locomoter activity and anxiolytic-like effects in mice (6). So far, at least, three groups have studied the NPS–GPR154 (NPSR) pathway, and all published evidence (5–8) as well as our unpublished microarray data (J. Vendelin et al., in preparation) support the idea of a specific ligand–receptor pathway. Interestingly, NPS and GPR154 mRNA are co-localized in the bronchial and colon epithelia, revealing the resemblance to the cell profile of the innate immunity and suggesting that GPR154 may be activated by an autocrine or paracrine mechanism (8).
A mouse model of lung inflammation induced by repeated intraperitoneal sensitization with ovalbumin (OVA) has many similarities to human asthma, including elevated total and specific IgE as well as inflammation characterized by infiltration of eosinophils (9). Furthermore, mice sensitized to OVA and then challenged with OVA aerosol exhibit increased airway hyperreactivity (AHR) to inhaled methacholine, a hallmark of asthma. In our recent work, the mRNA levels of the murine ortholog Gpr154 were significantly upregulated in a mouse model of OVA-induced inflammation (2). Remarkably, public mouse genome databases do not appear to include the sequence for the B isoform. Nevertheless, a GPR154 transcript was detected in human peripheral blood leukocytes (2,6), further suggesting a role for the GPR154 pathway in inflammation. In this study, we characterized the expression of GPR154 in human leukocytes and analyzed the expression of GPR154 in a mouse model of bronchial inflammation at cellular level. Our results implicate a novel role for macrophage-derived GPR154 in the cellular response during antigen challenge.
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RESULTS |
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GPR154 and NPS mRNA expression in human leukocytes
In order to characterize the expression of GPR154 and its agonist in leukocytes, we analyzed GPR154-A, -B and NPS mRNA expression (Fig. 1A–C, respectively) in fractionated cell types and compared the corresponding expression levels with the epithelial and smooth muscle cell lines known previously to express GPR154 (8
). GPR154-A mRNA expression in peripheral blood mononuclear cells (PBMCs) was at the same level as in bronchial epithelial (BEAS) and bronchial smooth muscle cells (BSMCs), whereas the expression of GPR154-B mRNA was 3-fold when compared with BEAS cells. In agreement with our previous results (2,8), GPR154-B mRNA expression in BSMCs was very weak. Among fractionated leukocytes, monocytes and eosinophils expressed both GPR154-A and GPR154-B mRNA, whereas CD4+ T cells expressed mainly GPR154-B mRNA. NPS mRNA expression in blood cells was strong in comparison to the expression levels in BEAS cells and resembled the expression profile of GPR154. This supports the idea that NPS may activate GPR154 by an autocrine or paracrine mechanism (Fig. 1C).
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Next, we analyzed the effects of LPS-induced monocyte activation on GPR154 and NPS expression and compared the results with those of T cell activation. After a 6 h lag period, the expression of GPR154-A, -B and NPS mRNA was upregulated in PBMCs 16 and 28 h after LPS stimulation. T-cell activation with anti-CD3 and anti-CD28 antibodies decreased the expression of GPR154-A, -B and NPS mRNA 28 h after challenge (Fig. 1D–F, respectively).
GPR154 protein expression in human leukocytes
The changes of GPR154 mRNA levels after LPS stimulation were confirmed at protein level. In western-blotting analysis of PBMC lysates, detection with both anti-GPR154-A and GPR154-B antibodies resulted in one major polypeptide band at 
50 kDa (Fig. 2A and B). This is in agreement with the calculated full-length molecular weights of the GPR154-A and GPR154-B proteins (42.7 and 43.1, respectively). Consistent with the results of the mRNA expression studies, the amount of both GPR154 isoforms was increased in LPS-treated PBMCs 48 h after challenge in comparison to unstimulated samples. Quantification of the band intensities demonstrated that the increases in GPR154-A and -B expression were 1.4-fold and 1.6-fold, respectively (Fig. 2A and B). T cell activation did not modulate GPR154-A or -B expression. The high amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in T cell-activated PBMCs refers to the proliferation of T cells and validates the activation protocol used in the study (Fig. 2C). Blocking experiments with 10 times molar excess of free peptide as a competitor were used to verify the correct polypeptide bands in immunoblotting.
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Specificities of the anti-GPR154 antibodies in recombinant GPR154 constructs
For further verification of the antibody specificities, anti-GPR154 antibodies were tested in western-blotting analysis of bacterial lysates with the corresponding polypeptide sequences expressed in E. coli. The cytosolic C-terminal tail of the human A isoform was expressed as a dihydrofolate reductase (DHFR) fusion polypeptide (DHFR-GPR154-A), whereas the cytosolic C-terminal tail of the GPR154-B isoform and the third cytoloop and the N-terminus of GPR154 were expressed as glutathione-S-transferase fusion polypeptides [glutathione-S-transferase (GST)–GPR154-B, GST–GPR154-CL3 and GST–GPR154-N, respectively]. Epitope-specific signals were detected with no cross-reactivity between the constructs or with the DHFR or GST construct alone (Fig. 3A–C). Because anti-GPR154-B and anti-GPR154-B-N antibodies detected also shorter GPR154 variants produced as a result of protein disruption, full-length GST-constructs were purified from the lysates with the aid of glutathione affinity chromatography. Immunoblotting of the purified GST-constructs with the corresponding antibodies resulted in detection of a single band as expected (Fig. 3D).
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Expression of GPR154-A and -B in human sputum samples
In order to demonstrate the expression of GPR154 in leukocytes obtained from asthmatic patients, human sputum samples were studied. In immunoblotting analysis, detection with both anti-GPR154-A and GPR154-B antibodies resulted in one major polypeptide band at
50 kDa with an additional weaker band at 60 kDa (Fig. 2D). The 60 kDa band may represent a glycosylated form of the receptor, which is a typical post-translational modification for GPCRs. Omission of the primary antibodies and the negative control experiments with blocking peptides resulted in no signal (data not shown). In qualitative immunocytochemical analysis of human sputum samples, macrophages and eosinophils expressed GPR154-A and -B (Fig. 4A and B). The anti-GPR154-A antibodies recognize intracellular epitopes of the receptor and thus membrane-associated GPR154 staining in the permeabilized cells could not be properly visualized in the color reaction-based immunohistochemistry setting. However, membrane-associated staining was detectable in macrophages and eosinophils by using non-permeabilized samples stained with the N-terminus-specific antibodies, detecting extracellular epitopes of the receptor (Fig. 4C). The characteristic cell population profile with high eosinophil count in asthma patients indicates that the cell population in the sputum samples reflects the physiological status of the corresponding study subject (Supplementary Material, Table E1). The proportion of T cells in human sputum was <1% of total cell count. No staining was observed in T cells and they were excluded from the final analysis. In asthmatic patients, GPR154-A and -B positive eosinophils were detected only in one of the non-atopic subjects, whereas macrophages expressed GPR154-A and -B in the atopic and in four and five of the six non-atopic patients, respectively. In non-asthmatics, GPR154-A and -B positive eosinophils were found in two of the six atopic and two and one of the nine non-atopic subjects, respectively. Macrophages expressed GPR154-A and -B in all of the non-asthmatic subjects except for in one non-atopic subject (Supplementary Material, Table E2). Stainings with the non-immune IgG and omitting the primary antibodies resulted in no signal (2D).
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Immunohistochemical analysis of GPR154 in mouse lung tissue samples
To further examine the expression kinetics of GPR154 during inflammation, lung tissue samples of classical OVA-induced mouse model of bronchial inflammation were analyzed by immunohistochemistry. Previously characterized polyclonal anti-GPR154-A antibodies (2
,8) raised against the human intracellular sequence CREQRSQDSRMTFRERTER were used to detect the corresponding murine epitope CKMQRSQDSRMTYRERSER (Fig. 5).
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In the lung tissue samples of naïve and unsensitized but OVA-challenged mice, GPR154-A was expressed in the epithelial cells of the bronchioles as expected from the previous studies (Fig. 5A and B). In the OVA-challenged mice without sensitization, non-existent to moderate immunostaining was also observed in alveolar macrophages (AMs) (Fig. 5B). In the OVA-sensitized and -challenged mice, GPR154-A expression was decreased in the epithelial cells of the bronchioles, whereas the expression was increased in macrophages when compared with naïve and unsensitized control groups (Fig. 5C and H). The staining of the corresponding sections with the GPR154-A preimmune sera did not result in any immunoreactivity (Fig. 5D).
The sequence of the human and mouse GPR154 differ from their N-terminal parts and thus human antibodies detecting extracellular epitopes of the receptor could not be used in the mouse model. However, the specificity of the immunostaining was further verified with the GPR154-CL3 antibodies, detecting intracellular third cytoloop common to all receptor isoforms. The corresponding murine epitope is identical to the human sequence except for the first amino acid. Staining of the corresponding sections with the GPR154-CL3 antibodies resulted in identical immunoreactivity in overlapping locations to anti-GPR154-A stainings, confirming the results (Fig. 5E–G).
Immunohistochemical analysis of GPR154 in murine BAL samples
In order to confirm the findings obtained from immunohistochemical stainings of the mouse lung tissue samples, we analyzed the expression of GPR154 in bronchoalveolar lavage (BAL) samples from the corresponding study groups. In the BAL fluid of naïve control mice, GPR154 expression in AMs varied between non-existent to very low (Fig. 5I). On the other hand, moderate GPR154 expression was observed in macrophages of the unsensitized control mice with OVA challenge (Fig. 5J). In the asthma model of OVA-sensitized and -challenged mice, GPR154 staining in macrophages varied from mild to strong (Fig. 5K). Positive GPR154 staining was also detected in eosinophils. These results confirmed the findings indicating upregulated GPR154 expression in tissue infiltrated macrophages upon allergen challenge.
The effect of NPS on macrophage phagocytosis
As the results suggested earlier that inhalation of allergens induces GPR154 expression in macrophages, we studied whether NPS is able to modulate macrophage phagocytosis. Mouse macrophage cell line RAW 264.7 was found to express GPR154 at mRNA and protein levels (data not shown) as assessed by real-time RT–PCR (Supplementary Material) and immunohistochemistry and was used as a model.
Prestimulation with NPS resulted in a dose-dependent increase in phagocytosis of the fluorescein-labeled E. coli (Fig. 6A). Incubation with 1 µM NPS resulted in up to 10.8-fold increase (P<0.001) in phagocytosis, which was similar to the results obtained with the positive control tuftsin (Fig. 6A). When visualized by fluorescence microscopy, prestimulation with NPS resulted in increased cellular uptake of the fluorescent E. coli in the intracellular compartments in comparison to the untreated control cells (Fig. 6C and D). The nuclei of the cells did not emit any fluorescence as expected.
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P<0.05).In order to identify the specific signaling pathways involved in the NPS-mediated phagocytosis, specific inhibitors for intracellular Ca2+ and PKC involved with the G
q pathway and PKA involved with the Gs pathway were used. Although NPS has been previously shown to activate both intracellular pathways (5,6), we first verified that NPS increases intracellular Ca2+ mobilization and cAMP accumulation in our study setting as well. The 293H cell clones stably overexpressing GPR154-A (8) were used as a specific model for the NPS/GPR154 pathway. For measurements of cytoplasmic Ca2+ levels, the cells were labeled with Fluo-3 AM as an indicator. NPS (1 µM) stimulation increased fluorescence levels 10–15 s after injection, indicating release of Ca2+ to the cytoplasm of the GPR154-A overexpressing cells (Fig. 7). No change in Ca2+ levels was detected in parental 293H cells (data not shown). In an ELISA-based assay, 20 min stimulation with 1 µM NPS resulted in a 36–40% increase in basal cAMP levels (from 48 fmol/10 000 cells to 76–81 f/mol/10 000 cells) in GPR154-A overexpressing cells but not in parental 293H cells (Fig. 7). These findings indicate that NPS is able to activate its cognate receptor GPR154.
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In RAW 264.7 cells, incubation with each of the inhibitors for intracellular Ca2+, protein kinase C (PKC) and protein kinase A resulted in a decrease in NPS-mediated phagocytosis when compared with the cells treated with NPS alone (Fig. 6B). However, treatment with none of the inhibitors alone inhibited GPR154-mediated phagocytosis totally to the baseline level of the control cells. The most effective inhibition was obtained with bisindolylmaleimide I (PKC inhibitor), which decreased the NPS-mediated phagocytosis for
70% (P<0.05). Use of the inhibitors did not decrease phagocytosis in cells not treated with NPS (data not shown). The results indicate that NPS-stimulated phagocytosis is dependent on both Gs and Gq pathways.
The effect of NPS in macrophage adhesion, cell migration and chemotaxis
In the mouse lung tissue and BAL samples, GPR154 expression correlated with the tissue infiltration of macrophages. Directed cellular movement is characterized by a dynamic control of both attachment and detachment of the cell surface adhesive receptors with their extracellular matrix ligands. Therefore, we first studied whether NPS modulates cell adhesion in RAW 264.7 cell line. Cells were plated onto fibronectin, collagen type I or poly-L lysine coated wells and incubated in the presence or absence of 1 µM NPS. The amount of the cells attached was assessed after 50 min of incubation. NPS slightly but significantly (P=0.013) decreased macrophage adhesion onto immobilized fibronectin, indicating integrin-mediated events in NPS signaling (Fig. 8A). Poly-L-lysine-mediated adhesion resulted in no differences in cell adhesion. For further demonstration of the importance of the immobilized matrix, collagen type I did not support adhesion of RAW 264.7 cells (data not shown).
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As detected by a quantitative fluorescence-based cell assay, 2 h stimulation with NPS induced a 2-fold increase (P=0.007) in RAW cell migration up to the same level of the positive control MCP-1 (Fig. 8B). To confirm the results, random migration of the confluent RAW cells was measured with a wound-healing assay. As shown in Figure 8C and D, 24 h stimulation with NPS (1 µM) increased cell migration onto the denuded culture dish when compared with the untreated control cells. This further supports the finding that NPS is a chemotactic agent to macrophages in vitro.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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The results of the present study suggest that in addition to the polymorphisms in the GPR154 gene that influence on asthma susceptibility, the GPR154 pathway is likely to play a wider role in the pathogenesis of asthma. The findings in human blood and sputum cells as well as the induced expression of GPR154 in the AMs of the OVA-challenged mice suggest GPR154 to participate in the modulation of macrophage immune response after antigen challenge.
In the current study, several lines of evidence indicated an active role for macrophage-derived GPR154 during inflammation. In blood CD4+ T cells and especially in eosinophils, the expression of GPR154 and NPS mRNA was relatively strong, but none of the subsequent findings addressed a comprehensible role for GPR154 derived from these cells. Direct T-cell activation with anti-CD3 and anti-CD28 antibodies resulted in reduced GPR154 and NPS mRNA expression in PBMCs as late as 28 h after stimulation. Therefore, the decreased expression may result from induced cytokine production. Accordingly, the expression of GPR154 in PBMCs was not immediately increased upon LPS challenge but was upregulated at later timepoints. Thus, the expression of GPR154 correlates to the maturation of monocytes to macrophages. The mRNA expression profile of NPS was similar to GPR154, suggesting that NPS may activate GPR154 by an autocrine or paracrine mechanisms.
In the mouse model of asthma, the additive effect of OVA sensitization on macrophage GPR154 expression was small when compared with the OVA challenged mice. Although commercial OVA preparations may contain trace amounts of LPS, the reagent used in this study was of high quality grade and has been used similarly in previous studies (2,10). Hence, the increased levels of GPR154 after OVA challenge may indicate to non-T-cell-mediated events and suggest that inhalation of foreign substance itself may be sufficient to upregulate GPR154 expression.
According to the results of the present study, we propose that autocrine or paracrine secretion of NPS may activate macrophages to increase migration and phagocytosis resulting in more rapid clearance of tissue invading pathogens. The slightly decreased cell adhesion of RAW 264.7 cells after NPS challenge is more likely to reflect the dynamics of chemotactic events in the cytoskeleton of macrophages upon activation. We suggest that activation of both Gs and Gq-initiated pathways are linked to the NPS-mediated phagocytosis, as the use of inhibitors for Ca2+, protein kinase A and C decreased the enhancement obtained upon NPS stimulation. Although the use of inhibitors was not observed to decrease phagocytosis in cells not stimulated with NPS, the inhibitors are common to a variety of G protein-coupled receptors (GPCRs) and might decrease the NPS-induced phagocytosis through a GPCR other than GPR154. In our cell line stably overexpressing GPR154-A, NPS increased both intracellular Ca2+ mobilization and cAMP accumulation suggesting that NPS activates GPR154 in a manner typical for GPCRs. Because the specificity of the NPS/NPSR pathway has been confirmed in GPR154 overexpressing cells, future knock-down experiments and the development of GPR154 antagonists will finally help in validating the physiological effects of NPS on endogenous GPR154 expression. In a recent study, NPS induced a dose-dependent proliferation of Colo205 human colon cancer cells endogenously expressing GPR154 and stimulated MAPK phosphorylation in a dose-dependent manner in HEK cells stably expressing GPR154 (7). These findings implicate the potential of NPS to induce functional effects relevant to asthma pathogenesis.
The upregulated GPR154 expression in macrophages upon allergen challenge probably represents a normal response to inhaled allergens. Thus, a defect in GPR154 function might lead to altered phagocytosis of inhaled particles. This is in good agreement with previous studies, suggesting a link between decreased phagocytic function and asthma (11,12). AMs from human subjects with asthma exhibit impaired Fc
receptor-mediated phagocytosis associated with reduced surface expression of Fc receptor I (12). The clearance of apoptotic cells prevents the dying cells from releasing pro-inflammatory compounds into surroundings. A decrease in LPS-responsiveness of AMs in severe asthma is manifested by defective apoptotic cell uptake and reduced secretion of inflammatory mediators such as prostaglandin E2 and 15-hydroxyeicosatetraenoic acid (13). Furthermore, complement component C3 mediates its effect via a cognate G protein-coupled receptor C3a and plays a major role in phagocytosis and airway inflammation. Interestingly, the gene variants of C3 and its receptor are associated with asthma and related phenotypes (14,15). Furthermore, alleles of the pattern-recognition receptor NOD1 on chromosome 7p14 are associated with asthma and high IgE, suggesting that recognition of bacterial products may contribute to the presence of asthma (16).
In addition to the asthma phenotype, GPR154 is also associated with increased total and specific serum IgE as well as bronchial hyperreactiveness (2–4,17) but not with atopic dermatitis (18). This underlines the different etiopathogenesis of these conditions and the potential effects of allergen inhalation in asthma. Overexpression of GPR154-B in the asthmatic airways points to a potential function in airway hyperresponsiveness (1,8). The expression pattern of GPR154 in macrophages fits well to the hypothetical role of GPR154 in AHR as well. Animals with immunologic memory for an allergen, depleted of their AMs, show highly elevated IgE response and a large infiltration of T cells in the airways, as well as an increase in IgE-secreting B cells in draining lymph nodes following challenge with aerosolized allergen (19–21). In a recent experiment, AMs from Sprague–Dawley rats were able to confer resistance to antigen-induced AHR when transferred into the lungs of otherwise susceptible Brown Norway rats (22). This highlights the importance of AMs in preventing the development of AHR in sensitized animals. This could result from induction of mediators that suppress pathogenic components contributing to AHR. The activation of such a pathway might require cell–cell interactions such as intake of apoptotic cells. Thus, a defect in phagocytic function of macrophages could lead to increased AHR and other symptoms of asthma. Future studies with high throughput technologies might provide new insights to the interaction of inflammatory signalling and GPR154.
Taken together, our results suggest a link between GPR154 and innate immunity-induced adaptive immunity in the pathogenesis of asthma. Epithelium-derived NPS may activate underlining macrophages to increase migration and phagocytosis to engulf the invading allergens.
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MATERIALS AND METHODS |
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Additional details of the methods are provided online in Supplementary Material.
PBMCs and eosinophils
Peripheral blood cells were isolated from healthy volunteers or buffy coat donors by Ficoll density gradient centrifugations (Biochrom KG, Berlin, Germany). From the granulocyte fraction, erythrocytes were first lysed and eosinophils were further separated by depletion of neutrophils using MACS CD16 Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of the eosinophil fraction was confirmed by microscopic evaluation of cytospin samples and was detected to be 99%. Eosinophils were cultured for 24 h with 25 ng/ml GM-CSF in complete RPMI 1640 medium supplemented with 2 mM L-glutamine, 1 mM sodium puryvate, 1% MEM non-essential amino acids and vitamins, 100 U/ml penicillin, 100 µg/ml streptomycin (all from Life Technologies, Basel, Switzerland) and 10% fetal bovine serum (Sera-Lab, Sussex, UK). PBMCs were cultured in complete RPMI 1640 over night and stimulated then for 6, 16, 28 or 48 h either with 10 ng/ml LPS or plate-bound anti-CD3 (1 µg/ml, clone OKT3, ATCC) and anti-CD28 (2 µg/ml, PeliCluster clone 15E8, Sanguin, Amsterdam, The Netherlands). CD4+ T cells were isolated from PBMCs using Dynal CD4 Positive Isolation Kit (Dynal, Hamburg, Germany) and rested o/n in serum free AIM-V medium (Life Technologies, Basel, Switzerland). Monocytes were isolated by incubating freshly separated PBMCs in complete RPMI 1640 medium in cell culture bottle for 10–20 min. Unattached cells were washed out and the adherent monocytes were trypsinized, washed and rested o/n in complete RPMI 1640.
Cell lines
Bronchial epithelial cell line BEAS-2B was cultured in DMEM medium supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin (Life Technologies) and 10% fetal bovine serum (Sera-Lab). BSMC was cultured in SmGM-2 medium (Clonetics, Cambrex Bio Science Walkersville, Inc., Walkersville, MD, USA). Mouse macrophage cell line, RAW 264.7 (ATCC, Rockville, MD, USA), was cultured as described previously (23). All cultures were grown in six-well plates and cells were lysed for mRNA extraction before reaching confluence.
Human sputum samples
Two atopic and six non-atopic patients with asthma diagnosed according to ATS guidelines (24) and six atopic and eight non-atopic, non-asthmatic subjects were recruited for the study approved by the Ethics Committee, Department of Medicine, Helsinki University Hospital. Sputum processing was done as previously described (25). May-Grünwald-Giemsa staining was used for cell population counting.
Experimental animal model
BALB/c female mice, age 6–8 weeks, free of specific pathogens, were obtained from M&B, Ry, Denmark. The mice were housed under pathogen-free conditions and maintained on OVA-free diet. All experiments conducted were approved by the Animal Experimental Committee of the State Provincial Office of Southern Finland. The study groups consisted of six naïve, nine unsensitized but OVA-challenged and nine OVA-sensitized and -challenged BALB/c female mice. Sensitization was performed intraperitoneally on days 1 and 10 with 20 µg grade V OVA (Sigma-Aldrich, St Louis, MO, USA) adsorbed to 2 mg of alum adjuvant (Pierce Biotechnology Inc., Rockford, IL, USA) diluted in saline. Unsensitized groups received 200 µg of saline-diluted alum intraperitoneally. OVA-challenge consisted of a 40-min exposure to 1% (wt/vol) OVA in saline on days 20, 21 and 22. All mice were exposed to aerosol in an exposure chamber connected to the outlet of a six-jet disperser that delivered an aerosol of particles with a mean diameter of 0.3 µm (TSI Inc., St Paul, MN, USA). The paraffin-embedded lung samples and BAL samples were collected and prepared as described previously (10). All experiments were approved by the Animal Experimental Committee of the State Provincial Office of Southern Finland.
Quantitative real-time PCR
Total RNA was isolated with RNeasy Mini Kit from 0.1 to 5x106 cells lysed and stored frozen in RNeasy lysis buffer, according to the manufacturer's instructions (Qiagen, Hamburg, Germany). cDNA was synthesized with TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA, USA) and random hexamer or oligo dT primers. Quantitative real-time PCR for human samples was performed with ABI PRISM 7500 Sequence Detection System applying SybrGreen chemistry (Applied Biosystems). The primers amplifying specifically the human NPS and GPR154-A and -B isoforms were designed with the Primer Express software version 1.2 (Applied Biosystems). The specificities of the GPR154 primers were tested with cDNA derived from stable cell lines overexpressing GPR154-A and -B (8). The primer sequences were GPR154-A forward 5'-CCTGCAGGGAGCAAAGATCA-3', GPR154-A reverse 5'-AATCTGCATCTCATGCCTCTCA-3', GPR154-B forward 5'-CCTCAACGAGAGAACTGGAAG-3' and GPR154-B reverse 5'-AGAGCTGTCACCTTGGAAGAG-3', NPS forward 5'-CAAAATGATTAGCTCAGTAAAACTCAATC-3' and NPS reverse 5'-GGAACTGGATAACACCAAAACACA-3'. Elongation factor 1 (EF-1) was amplified as an endogenous reference gene with forward primer 5'-CTGAACCATCCAGGCCAAAT-3' and reverse primer 5'-GCCGTGTGGCAATCCAAT-3'. The relative gene expression differences were calculated with the comparative 
CT method.
For mouse RAW 264.7 samples, real-time quantitative RT–PCR assay was performed according to the manufacturer's (Applied Biosystems) instructions as described previously (23). Mouse GPR154-specific oligonucleotides were 5'-GGCTCATCTCTAAGGCAAAAATCA-3' (forward), 5'-ACGCTCCTTGGTGTCTGGAA-3' (reverse) and 6-FAM-5'-CGTCATAATCCTTGCTTTCATCTGCTGCTG-3'-TAMRA (probe).
Antibodies
The specific polyclonal goat antibodies against the N-terminus of GPR154 and the rabbit antibodies against the third cytoloop and the two alternative carboxy terminals of GPR154 have been described previously (2,8). Affinity-purified antibodies were further analyzed by immunoblotting of recombinant GPR154 fragments expressed with the pGEX 4T-3 GST fusion expression vector (Amersham Biosciences, Buckinhamshire, UK) as GST-fusion protein in E. coli except for the C-terminal tail of the A isoform, which was expressed as a DHFR fusion protein (Qiagen). Glutathione Sepharose was used for protein purification according to the manufacturer's (Amersham Biosciences) instructions.
The goat polyclonal GAPDH antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) and the horseradish peroxidase-conjugated rabbit anti-goat secondary antibodies from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA). The non-immune rabbit IgG fraction was obtained from DAKO (Glostrup, Denmark).
Western blot
For immunoblotting of human sputum samples, 50 µg of protein from cell pellet or supernatant was lysed into standard sample buffer. For detection of PBMC samples, 1x106 cells were lysed into sample buffer. Western-blot analysis with the rabbit GPR154-A and -B antibodies has been described earlier (8). Omitting the primary antibodies was used as a negative control (data not shown). Blocking experiments using 10 times molar excess of free peptide as a competitor were also performed to demonstrate antibody specificity in immunoblotting.
The relative expression of GPR154-A and -B was quantified after normalizing with the relative expression of GAPDH used as an endogenous reference gene. Syngene GeneTools software (Synoptics Ltd, Cambridge, UK) was used for detecting differences between intensities of the corresponding lanes. The unstimulated cells were assigned a relative band intensity value of 1.0.
Immunohistochemistry
Immunohistochemical staining of paraffine-embedded sections has been described previously (2,8). Omission of primary antibody and staining with the preimmune sera did not result in any immunoreactivity. Imunoreactivity was scored from 0–3 (0=non-existent, 1=weak, 2=moderate, 3=strong) by two different observers. The consensus of the values between the observers was used for final analysis, where the proportion of positive cells and the intensity of the stain in positive cells were multiplied to a final score value.
For immunocytochemical analysis of human sputum and mouse BAL and RAW cells, cytospin samples were fixed with 3.5% paraformaldehyde and stained with the Avidin and Biotinylated horseradish peroxidase macromolecular complex method (Vector Laboratories, Burlingame, CA, USA) with 3-amino-9-ethylcarbazole (Vector Laboratories) as a substrate. For detection of intracellular epitopes, the samples were permeabilized with 0.5% Triton X-100 in PBS for 5 min. The samples were not permeabilized for detection of the extracellular epitopes with the N-terminal GPR154 antibodies. Staining with the non-immune rabbit IgG fraction did not result in any immunoreactivity.
Phagocytosis assay
Phagocytotic activity of RAW 264.7 cells was measured with Vybrant Phagocytosis Assay Kit (Molecular Probes, Eugene, OR, USA) according to the manufacturer's instructions. Briefly, 1x105 cells were seeded to the 96 well plates and incubated in RPMI for 1 h in the presence or absence of 100 nM and 1 µM NPS followed by 2 h incubation with the fluorescein-labeled Escherichia coli. The fluorescence from non-internalized E. coli was then quenched by the addition of Trypan blue, and the samples were assayed with a Victor2 microplate reader. Tutftsin (Sigma-Aldrich, St Louis, MO, USA) was used as a positive control at a concentration of 1 µg/ml.
In order to identify the specific signaling pathways involved in the NPS-mediated phagocytosis, the specific inhibitors BAPTA-AM, bisindolylmaleimide I and H-89 (Calbiochem, Madison, WI, USA) for intracellular Ca2+, PKC and protein kinase A, respectively, were incubated with the respective suboptimal concentrations of 50 µM, 1 µM and 1 µM in the presence of 1 µM NPS. The inhibitors were diluted into DMSO (Sigma-Aldrich).
The results were verified by fluorescence microscopy under a magnification of 6300. Briefly, 4x105 cells were seeded onto cover slips and incubated for 1 h in the presence or absence of 1 µM NPS followed by 2 h incubation with the fluorescein-labeled E. coli. In order to avoid cell detachment and bad image quality, the cells were not treated with Trypan blue. After two washes with PBS, the cells were fixed with 3.5% paraformaldehyde in PBS, mounted and visualized by fluorescence microscopy.
Measurements of intracellular Ca2+ mobilization and cAMP accumulation
The previously described cell line stably overexpressing GPR154-A (8) was used to study NPS-mediated signaling events. Cell clones were divided into serum-free SFM II medium at a density of 2.5x105 cells/ml. The cells were loaded with 4 µM Fluo-3 AM (Molecular Probes) for 15 min at 37°C followed by a 20 min incubation at RT. After two washes, 50 000 cells were seeded into 96-well plates and Ca2+-dependent fluorescence traces (1 data-point/s, exitation at 488 nm) were measured by Fluostar Optima (BMG Labtechnologies, Offenburg, Germany) microplate reader. NPS (1 µM) was added prior to measurement at 535 nm.
For cyclic adenosine monophosphate (cAMP) assay, the GPR154 overexpressing cell clones and 293H parental cells were divided into cell culture medium at a density of 5x104 cells/well and incubated for 3 h at 37°C. Thereafter, increasing concentrations (0.1–10 µM) of NPS were added to the cells for 20 min. The cells were lysed with cAMP detection lysis reagent for 10 min and stored at –80°C. Before storing, viability of the cells was confirmed with Trypan blue exclusion method. Total cellular cAMP was detected with cAMP Biotrak Enzymeimmunoassay System (Amersham Biosciences) according to the manufacturer's instructions. The reactions were stopped with 1 M H2SO4 and the absorbance values were measured at 450 nm. Duplicate wells were studied and half of the total end volume of 200 µl was used for measurements.
Cell attachment assay
For the cell attachment assay, microtiter wells were coated overnight with 20 ng/ml of fibronectin, 5 ng/ml of collagen or 100 ng/ml of poly-L-lysine at +37°C. The wells were blocked for 60 min with 0.5% BSA in PBS. RAW 264.7 cells were harvested by scraping, washed and resuspended into RPMI medium at a density of 3.5x105 cells/ml. The cells were preincubated in suspension in the presence or absence of 1 µM NPS for 30 min on ice. Subsequently, 3.5x104 cells were seeded onto the wells, and the plates were incubated for 50 min at 37°C. Cells were washed three times with PBS and the relative number of attached cells was measured using CyQuant reagent (Molecular Probes) and Victor2 plate reader (Perkin-Elmer, Wellesley, MA, USA) using excitation/emission wavelengths of 485/530 nm.
Cell migration assays
Chemotactic migration of the RAW 264.7 cell line was investigated using 24-well transwell culture chambers (Corning, NY, USA) and polystyrene cell culture inserts with 6 µm pore size. Transwell chambers were coated overnight with fibronectin (20 ng/ml) at +4°C. 105 cells were seeded onto the cell culture inserts in serum-free RPMI. Lower chambers contained 200 ng/ml MCP-1 (R&D Systems) or 1 µM NPS in serum-free RPMI as a chemoattractant, whereas media alone was added to the control chambers. The cells were allowed to migrate for 2 h at +37°C. The upper side of the membrane was wiped off and the migrated cells in the lower side were lysed into 400 µl of CyQuant reagent (Molecular Probes). Fluorescence was measured with the Victor2 plate reader using excitation/emission wavelengths of 485/530 nm. In ‘wound healing assay’ demonstrating random cell migration, RAW 264.7 cells were grown to confluency for 14 days on a 24-well plate (Costar, Bethesda, MD, USA). Pipette tips were used to scrape a cell free zone with constant width in the middle of the wells. Cells were washed with PBS and 1 µM NPS in serum-free RPMI was added to the wells to induce cell migration, whereas media alone was added to the control wells. The cells were allowed to migrate for 24 h at +37°C and cell migration was analyzed by light microscopy. The experiment was repeated three times using six parallel wells.
Statistical analysis
All data are expressed as mean ±SEM unless stated otherwise. Student's t-test and analysis of variance were used for parametric comparisons, whereas Mann–Whitney and Kruskal–Wallis tests were used for non-parametric comparisons.
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Supplementary Material is available at HMG Online.
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ACKNOWLEDGEMENTS |
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We would like to thank Marina Leino for providing the mouse lung tissue samples and Tuula Lahtinen, Virpi Päivinen and Riitta Känkänen for their skillful laboratory work. Juhani Lahdenperä is acknowledged for technical assistance. This study has been supported by the Finnish National Technology Agency Tekes, Academy of Finland, Sigrid Juselius Foundation, Päivikki and Sakari Sohlberg Foundation, GeneOS Ltd, The Maud Kuistila Memorial Foundation, Jalmari and Rauha Ahokas Foundation, The Pulmonary Association Heli, Sirpa and Markku Jalkanen Foundation, The Research and Science Foundation of Farmos and Finnish Cultural Foundation.
Conflict of Interest statement. None declared.
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