Adenosine activates Gαs proteins and inhibits C3a-induced activation of human mast cells

Accepted Manuscript Adenosine activates Gαs proteins and inhibits C3a-induced activation of human mast cells Narcy Arizmendi, Marianna Kulka PII: DOI: Reference:

S0006-2952(18)30331-9 https://doi.org/10.1016/j.bcp.2018.08.011 BCP 13244

To appear in:

Biochemical Pharmacology

Received Date: Accepted Date:

14 May 2018 8 August 2018

Please cite this article as: N. Arizmendi, M. Kulka, Adenosine activates Gαs proteins and inhibits C3a-induced activation of human mast cells, Biochemical Pharmacology (2018), doi: https://doi.org/10.1016/j.bcp.2018.08.011

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Adenosine activates Gαs proteins and inhibits C3a-induced activation of human mast cells

Narcy Arizmendia and Marianna Kulkaa, b a

Nanotechnology Research Centre, National Research Council Canada, Edmonton, AB, Canada.

b

Department of Medical Microbiology and Immunology, Faculty of Medicine, University of

Alberta, Edmonton, AB, Canada `

Running title: A2A inhibits C3aR-mediated mast cell activation

Correspondence: Dr. Marianna Kulka Nanotechnology Research Centre National Research Council Canada 11421 Saskatchewan Drive, Edmonton, Alberta, Canada T6G 2M9 Telephone: 780-641-1687 Fax: 780-641-1601 E-mail: [email protected]

Abstract Anaphylatoxin C3a and adenosine receptors (AR) are implicated in the inflammatory process associated with allergic rhinitis and asthma by modifying mast cell (MC) responses. Possible interactions between these G-protein coupled receptor (GPCR) pathways in MCs has not yet been demonstrated. LAD2 human MC were stimulated with C3a in the presence or absence of AR agonists and antagonists and their adhesion, chemotaxis and mediator release were measured. The pan-specific AR agonist, 5'-N-Ethylcarboxamidoadenosine (NECA) inhibited C3a-induced LAD2 cell migration, adhesion, degranulation, production of CCL2, and ERK1/2 phosphorylation. The selective A2A receptor agonist CGS 21680 inhibited C3a-mediated degranulation, while the A2B and A3 receptor agonists BAY 60-6583 and IB-MECA, respectively, had no effect. Moreover, an A2A receptor antagonist SCH 58261 blocked the inhibitory effect of NECA on C3a-induced degranulation, suggesting that inhibition of degranulation was mediated through the A2A receptor. NECA increased intracellular cAMP in C3a-activated mast cells, suggesting that, Gs protein signals are required for adenosine-induced inhibition of C3a-mediated human mast cell activation. The adenylyl cyclase inhibitor SQ 22536 attenuated the inhibitory effect of NECA on C3a-activated degranulation, and the A2A agonist CSG 21680 potentiated the inhibition of mast cell activation mediated by the A2A receptor. Our results suggest that adenosine inhibits C3a-mediated activation of human mast cells, possibly through a Gαs protein-dependent pathway.

Key words: Allergic inflammation; adenosine; adenosine receptors; complement; G proteins; mast cells

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1.

Introduction Tissue inflammation is a complex process involving the activation of many inflammatory

and structural cells, and the interactions between immunological mediators released by these cells (1). Mast cells (MC) play a significant role in the pathogenesis of allergic and non-allergic inflammation through the release of stored mediators (degranulation) and de novo production of lipid mediators and cytokines (2-4). In allergen-driven inflammation, activation of MC is initiated by cross linking of the high affinity receptors for IgE (FcRI) by antigen. However, in some forms of urticaria (5, 6), pseudo-allergic reactions (7), diabetes (8), and metabolic diseases (9), MC are activated through G protein-coupled receptors (GPCRs) (10). These receptors are activated by basic secretagogues, biolipids, antimicrobial peptides, complement, adenine nucleotides and adenosine (11, 12). Adenosine (ADO), an endogenous purine nucleoside, is a signaling molecule produced by cells in response to stress by dephosphorylation of adenosine triphosphate (ATP) (13). Extracellular concentrations of adenosine can rise from the nM to µM range in response to inflammation, ischemia, hypoxia, or trauma (14, 15). Adenosine has long been implicated in asthma, possibly due to its ability to enhance IgE-induced mediator release from MC and induce mast cell-dependent bronchoconstriction upon inhalation or intravenous administration in asthmatic patients. However, the inability of adenosine to elicit similar responses in healthy subjects challenges this hypothesis (16-18) and some experimental data has demonstrated that adenosine and its analogues exert protective and anti-inflammatory effects in murine models of asthma. Furthermore, adenosine differentially regulates lung inflammation and tissue repair (1922). Thus, the role of adenosine in inflammation appears to be complex and likely dependent

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upon many factors such as adenosine concentration, duration of exposure and tissue microenvironment. Adenosine receptors exhibit high sequence homology but differ in affinity for adenosine, tissue distribution and effector coupling. Therefore, each receptor subtype has a unique pharmacological profile (23, 24). Adenosine functions through binding to the A1, A2A, A2B and A3 GPCRs. A1 and A3 receptors signal through Gαi proteins to decrease cyclic AMP (cAMP) levels, while A2A and A2B signal through Gαs proteins to increase cAMP levels (25). Accordingly, binding of adenosine to its various receptor subtypes can elicit differential responses in MC (25). For example, adenosine potentiates IgE-mediated degranulation of murine bone marrow-derived MC (BMMC) (26, 27), rat basophilic leukemia MC (RBL-2H3) (28), rat pleural MC (29), and human lung MC via Gαi proteins (30). In rat serosal MC and guinea-pig lung, adenosine binding to Gαs protein-coupled adenosine receptors can elevate cAMP levels, resulting in enhanced IgE- and calcium ionophore-induced mediator release (30). Adenosine alone stimulates the production of interleukin (IL)-4 and IL-8 in the human MC line HMC-1, which expresses A2A and A2B receptors (31). In clear contrast, adenosine inhibits IgE-induced degranulation of human skin MC (32) and dispersed human lung MC (33) through Gαs-coupled adenosine receptors. Similar to A1 and A3 receptors, P2Y receptors are Gαi-coupled purinergic receptors (34). Treatment with native P2Y agonists ATP, ADP, UDPG, and the more selective P2Y14R agonist MRS2690 significantly enhances anaphylatoxin C3a receptor (C3a) -induced degranulation of human MC (35). Complement components C3a and C5a activate MC through their cognate GPCRs: C3aR, C5aR1 or C5aR2 (36). Complement-mediated activation of MC establishes an inflammatory response in tissue which may in turn lead to increased concentrations of adenosine (37). Thus, it 4

is likely that these signaling pathways interact and influence immune cell responses; a hypothesis supported by emerging data. Theophylline, a non-selective adenosine receptor antagonist, inhibits C5a-induced degranulation of human eosinophils and its interaction with adenosine, possibly via the A3 receptor (38). Adenosine blocks C5a-initiated actin polymerization and motility of human polymorphonuclear leucocytes (PMN) through the A2A receptor (39). Our preliminary studies clearly suggested that ligation of the adenosine A2A receptor inhibited C3amediated activation of primary human cultured mast cells (HuMC) and human mast cell line (LAD2) cells (40). Given that both adenosine and C3a contribute to the pathophysiology associated with asthma, and as a follow up from our previous studies, we hypothesized that adenosine directly modulated the effect of C3a on human MC through the A1, A2A, A2B and/or A3 GPCRs. We tested the interaction of these two GPCR signaling pathways in a human mast cell model (LAD2) known to express adenosine receptors using IC50-appropriate concentrations of receptor agonists and antagonists.

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2.

Materials and Methods

2.1 Cell culture LAD2 (Laboratory of Allergic Diseases 2) human MC, originally isolated from the bone marrow of a 44-year-old male patient with mastocytoma (41), were cultured in StemPro-34 SFM medium (Life Technologies, Burlington, Canada) supplemented with 2 mM L-glutamine (Life Technologies, Burlington, Canada), 100 U/ml penicillin, 100 ug/ml streptomycin (Life Technologies, Burlington, Canada), and 100 ng/ml stem cell factor (SCF; Peprotech, Rocky Hill, NJ, USA). Cell suspensions were seeded at a density of 0.1 × 106 cells/ml and maintained at 37°C and 5% CO2, and periodically tested for the expression of CD117 and FcεRI by flow cytometry. Cell culture medium was hemi-depleted every week with fresh medium. The HMC-1 cell line, derived from peripheral blood of a patient with mast cell leukaemia, exhibit a phenotype similar to that of human mast cells and expresses several mast cell‐ related markers (42). HMC-1.1 and HMC-1.2 exhibit different phenotypes defined by the location of specific mutations in the c-kit proto-oncogene. HMC-1.1 presents a point mutation in codon 560 (Gly560Val), HMC-1.2 has mutations in codons 560 (Gly560Val) and 816 (Asp816Val) of the CKIT surface receptor, being the second mutation located in the intracellular kinase domain. These mutations on the C-KIT proto-oncogene cause constitutive tyrosine phosphorylation and activation of the KIT receptor, rendering HMC-1 cells independent of SCF for growth and survival (43). HMC-1.1 and HMC-1.2 cell lines were maintained in Iscove’s Modified Dulbecco’s Medium (Gibco, Carlsbad, USA) containing 10% FBS, 100 U/ml penicillin and 100 ug/ml streptomycin, and maintained at 37C and 5% CO2.

2.2 Real-time PCR 6

Total RNA was isolated using the Tri Reagent method (Sigma-Aldrich Canada, Oakville, Canada). cDNA was synthesized by reverse transcription of 1 μg of total cellular RNA using oligo dT primers (Integrated DNA Technologies, Coralville, USA) and M-MLV Reverse Transcriptase (Life Technologies, Burlington, Canada) in a 20-μl reaction mix according to the manufacturer’s recommendation. Gene expression was analyzed using real-time qPCR on a StepOnePlus system (Applied Biosystems, Foster City, USA). For each qPCR assay, a total of 20 ng of cDNA was used, and all reactions were performed in triplicate for 40 cycles as per the manufacturer’s recommendation. TaqMan probes, IDT primers, and Gene Expression Master Mix (Life Technologies, Burlington, Canada) were utilized. All data were normalized to glyceraldehyde 3-phospahte dehydrogenase (GAPDH) internal control, and are reported as ratio of GAPDH expression.

2.3 Flow cytometry analysis LAD2 cells were washed with 0.1% bovine serum albumin (BSA)-phosphate-buffered saline (PBS), resuspended at 0.25 × 106 cells per well fixed with 5 % formalin neutral Buffered solution (Sigma-Aldrich Canada, Oakville, Canada) for 5 min at RT, and blocked with 3% BSA/PBS for 10 min on ice. Cells were subsequently stained with either phycoerythrin (PE)conjugated anti-C3aR antibody or isotype control antibody (R&D Systems, Minneapolis, USA) for 1 h in the dark at 4°C. For intracellular A2A receptor staining, cells were stained with anti-A2A receptor (clone 7F6) monoclonal antibody (Millipore Sigma, Oakville, CA, USA), in 3% milk/0.1 % saponin/PBS for 1 h on ice, washed twice, followed by 1 h incubation with PEconjugated goat anti-mouse IgG (Southern Biotech, Birmingham, AL, USA). LAD2 cells were incubated with the A1, A2B (Millipore Sigma, Oakville, CA, USA), or A3 receptor antibodies 7

(Chemicon International, Temekula CA, USA), for 1 hr on ice, washed twice, followed by incubation with goat anti-rabbit allophycocyanin (APC)-secondary antibody (Invitrogen, Carlsbad CA, USA), and were also evaluated by flow cytometry analysis. Cells were then washed twice with 0.1% BSA-PBS and analyzed on a CytoFLEX flow cytometer (Beckman Coulter, USA). Cell analysis was performed using the FlowJo V10 LLC software (Ashland, USA). Results were expressed as the median of fluorescence intensity (MFI) ± SEM.

2.4 Degranulation assay LAD2 cells were washed with 0.4% BSA-HEPES buffer and suspended at 0.5 × 106 cells/ml. Fifty µl of cell suspension was added to each well of a 96-well plate, incubated with various concentrations of adenosine receptor agonists or antagonists, and adenylyl cyclase inhibitor

SQ22536

(9-(Tetrahydro-2-furanyl)-9H-purin-6-amine),

or

NECA

(5'-N-

(ethylcarboxamido)adenosine); Tocris bioscience, Bristol, UK, our results showed batch to batch variability with NECA, we found that the results obtained with batch No. 3 were more consistent, this batch was used throughout the study) for the indicated time, followed by stimulation with 0.01 µM C3a (Calbiochem, Billerica, MA), for 30 min at 37°C and 5% CO2. βhexosaminidase released into the supernatants and in total cell lysates solubilized with 0.1% Triton X-100 was quantified by hydrolysis of p-nitrophenyl N-acetyl-β-D-glucosamide (SigmaAldrich Canada, Oakville, Canada) in 0.1 M sodium citrate buffer (pH 4.5) for 90 min at 37°C, using a multiskan Ascent Plate reader. Results are reported as the percentage of intracellular hexosaminidase that was released into the medium after correction for spontaneous release.

2.5 Histamine release assay 8

HMC-1.1 and HMC-1.2 cells were washed, suspended in BSA-free HEPES at 0.1 × 106 cells per well and incubated with 10 μM CGS 21680 (4-[2-[[6-Amino-9-(N-ethyl-β-Dribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid),

for 30 min,

followed by stimulation with 0.01 μM C3a for 30 min at 37°C and 5% CO2. Histamine (SigmaAldrich Canada, Oakville, Canada) working standards of 2000 ng/ml to 7.8 ng/ml were freshly prepared using two-fold serial dilution. O-phthalaldehyde (OPT; Sigma-Aldrich Canada, Oakville, Canada) was dissolved in acetone-free methanol and kept in the dark at 4°C. Histamine standards and cell-free supernatants were transferred to a flat bottom 96 black well microplate and mixed with 1M NaOH and OPT (10 mg/ml) followed by 3M HCl to stop the reaction. Fluorescence intensity was measured using a 355 nm excitation and a 460 nm emission filters using a Synergy H1 microplate reader (Biotek Instruments, Inc., Winooski, USA). Histamine release was expressed as a percentage of the total cellular histamine content calculated by the formula: (histamine in supernatant/histamine in supernatant and pellet) x 100 and corrected for spontaneous histamine release.

2.6 Intracellular calcium mobilization LAD2 cells were washed with 0.4% BSA-HEPES and resuspended to a total cell number of 4 × 106 cells. Cells were loaded with 1 μM Fura-2 AM (Life Technologies) in the presence of 30 µM NECA for 30 min at 37°C and 5% CO2, followed by washing and 15 min incubation in BSA-free HEPES at 37°C. Cells were then placed in poly-D lysine (Sigma-Aldrich Canada, Oakville, Canada)-coated glass-bottom dish under an inverted microscope (Axiovert 200, Carl Zeiss Canada Ltd., Canada) and excited at 340 and 380 nm. Florescence measurements were performed and Ca2+ responses were recorded at 100 ms intervals using SlideBook for Stallion, 9

version 4.26.04 software (Intelligent Imaging Innovations, Denver, USA). In each experiment, 0.01 µM C3a was added at the time point 70 s and 1 μM ionomycin (Sigma-Aldrich Canada, Oakville, Canada) at the time point 256 s. Ca2+ responses of 20 randomly selected cells were analyzed for each experiment and plotted as 340/380 ratio vs. time.

2.7 Chemokine production and measurement LAD2 cells were washed, resuspended at 1 × 106 cells/ml in fresh medium, incubated with 30 µM NECA for 30 min, followed by 24 h stimulation with 0.01 µM C3a at 37°C and 5% CO2. Cell-free supernatants were harvested and analyzed for CCL2 using the human CCL2 ELISA Ready-SET-Go kit (eBioscience, San Diego, USA) according to the manufacter’s instructions.

2.8 Adhesion assay A maxisorp 96-well NUNC plate (NUNC, Naperville, IL) was coated with 10 μg/ml human fibronectin (Sigma-Aldrich Canada, Oakville, Canada) for 16 h at 4°C, washed three times with PBS, blocked with 3% BSA-HEPES for 1 h at 37°C and washed three times with PBS. LAD2 cells were washed with 0.4% BSA-HEPES, resuspended at 1 × 106 cells/ml and stained with 5 μM calcein-AM (Life Technologies, Burlington, Canada) for 30 min in the presence of 30 µM NECA at 37°C and 5% CO2. Fifty µl of cell suspension and 50 µl C3a (0.01 µM) were added to fibronectin-coated wells, and the plate was incubated for 2 h at 37°C and 5% CO2. After incubation, wells were rinsed with warm HEPES to remove cells that had not adhered and filled with 100 µl 0.4% BSA-HEPES. Fluorescence emission was measured at 530 nm (485

10

nm excitation) using a Synergy H1 microplate reader (Biotek Instruments, Inc., Winooski, USA). The results are reported as percent cell adhesion.

2.9 Chemotaxis assay LAD2 cells were incubated overnight in SCF-free media, washed with 0.4% BSAHEPES, resuspended at 0.25 × 106 cells/ml and incubated with 30 µM NECA for 30 min at 37°C and 5% CO2. C3a (0.01 µM; 150 µl) was added to the wells of lower chamber of a 96-well monocyte cell migration plate (Calbiochem, Billerica, USA) and 100 µl cell suspension was added to the wells on insert and incubated for 6 h at 37°C and 5% CO2. After incubation, the insert was rinsed twice with warm 0.4% BSA-HEPES, and the cells were labeled with 5 μM calcein-AM for 30 min at 37°C and 5% CO2. After labeling, cells that had migrated across the insert membrane were detached using 0.01% Triton X-100. One hundred µl of cell suspension were transferred to a 96-well white plate, and fluorescence intensity was measured using a 485 nm excitation filter and a 530 nm emission filter using a Synergy H1 microplate reader (Biotek Instruments, Inc., Winooski, USA). Results are reported as cell chemotaxis percent.

2.10 Western blot analysis for ERK 1/2 LAD2 cells were harvested, washed, resuspended in 0.4 % BSA-HEPES at a density of 1x106 cells per ml, and treated for 30 min with 30 M NECA prior stimulation with 0.01 M C3a for the indicated time period. In some experiments, cells were left untreated or stimulated with C3a only. The reactions were terminated by adding ice-cold 2X lysis Buffer containing 1% Triton X-100 (Sigma-Aldrich Canada, Oakville, Canada) in TBS containing a mixture of commercially available protease inhibitors, including Complete protease inhibitor cocktail 11

(Roche Diagnostics, Indianpolis, IN, USA), Sigma protease inhibitor cocktail (Sigma-Aldrich Canada, Oakville, Canada), 50 g/ml 3,4 dichloroisocoumarin (Roche Diagnostics, Indianpolis, IN, USA), 1 mM benzamidine (Sigma-Aldrich Canada, Oakville, Canada), 1 mM sodium orthovanadate (Sigma-Aldrich Canada, Oakville, Canada), 5.4 mM sodium pyrophosphate (Sigma-Aldrich Canada, Oakville, Canada), and 50 mM sodium fluoride (Sigma-Aldrich Canada, Oakville, Canada). Protein content in the cell lysates was quantified using a Bicinchoninic acid assay (Sigma-Aldrich Canada, Oakville, Canada). The lysates were heated for 10 min at 70C with 4X Bolt LDS sample Buffer (Life Technologies, Burlington, Canada) containing 10X Bolt sample reducing agent (Life Technologies, Burlington, Canada). Samples were then separated on 4-12% Bolt Plus Bis-Tris gels (Life Technologies, Burlington, Canada), and transferred onto 0.45 m Immobilon-FL PVDF membranes (Life Technologies, Burlington, Canada). The membranes were blocked with a 1:2 Odyssey Blocking Buffer (LI-COR Biotechnology, Lincoln, NE, USA) and TBS for 1 hr at RT, and then probed with primary antibodies against phospho-p44/42 MAPK (ERK 1/2) (Thr202/Tyr204) (1:2000, Cell signaling Technologies, Danvers, MA, USA), or p44/42 MAPK (ERK 1/2) antibodies (1:1000, Cell signaling Technologies, Danvers, MA, USA) in TBS-0.1% Tween 20 (Fisher Biotech, Fair Lawn, NJ, USA)-4% BSA (Sigma-Aldrich Canada, Oakville, Canada) overnight at 4C. The membranes were washed with TBS-0.1% Tween 20 four times, and then incubated 1 h with IRDye 680 goat anti-rabbit IgG (H+L) (LI-COR Biotechnology, Lincoln, NE, USA) and Dylight 800 donkey anti-mouse IgG (Rockland Immunochemicals Inc., Pottstown, PA, USA), dilution 1;10000, respectively. The membranes were washed with TBS-0.1% Tween 20 four times, scanned and analyzed using the Odyssey Infrared Imaging System analyzer (LI-COR Biosciences, Lincoln, NE, USA). 12

2.11 cAMP EIA LAD2 cells were resuspended at 1 × 106 cells/ml in 0.4% BSA-HEPES, incubated for 10 min with 30 µM NECA or ADO, and then treated with 0.01 µM C3a for 30 min at 37°C. Reactions were stopped with the addition of 0.1 M HCl for 20 min at room temperature followed by cell dissociation with a pipette. Cell-free supernatants were isolated by 10 min centrifugation at 1000 x g, cAMP was measured using a commercial competitive immunoassay (Cayman Chemicals, Ann Arbor, USA), with 3 pmol/ml minimum detection limit. The results are expressed as percent increase in cAMP concentration over untreated cells.

2.12 Statistical analysis Experiments were performed at least 3 separate times and values are expressed as mean ± standard error of the mean (SEM). Data were analyzed using one-way ANOVA test with Tukey’s post hoc analysis for multiple comparisons, or two-way ANOVA test with Bonferroni post test to compare replicate means by row. All data were corrected for vehicle (DMSO effects and significance were defined as a P value less than 0.5). All statistical analyses were performed using GRAPH PAD PRISM statistical (GraphPad, Sand Diego, USA).

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3.

Results

3.1 Human mast cells express C3a and adenosine receptors MC responses to complement and adenosine can vary depending on MC phenotype, likely due to differences in C3aR and adenosine receptor expression. Therefore, we first analyzed the expression of adenosine receptors A1, A2A, A2B, A3, and C3aR on our human MC by flow cytometry (Fig. 1). LAD2 cells expressed C3aR on their surface (Figs. 1E and J) as expected. Flow cytometry analysis of permeabilized cells indicated that LAD2 cells expressed intracellular pools of A2A, A2B and A3 receptors. Although the A1 antibody utilized in this analysis indicated some minor binding to the LAD cell surface, subsequent analysis of mRNA expression by qPCR (Fig. 1K) indicated that the LAD2 did not express mRNA for A1 receptors. LAD2 expressed mRNA for A2A, A2B and A3 receptors (Fig. 1K). The mRNA expression of the A2A receptor was the highest among the four subtypes tested (A2A > A2B > A3; n=4).

3.2 Adenosine inhibits C3a-induced human mast cell degranulation To examine if NECA, a non-selective adenosine receptor agonist modified the metabolic activity of human mast cells, LAD2 cells were treated with different concentrations of NECA for 24h, and cell viability was evaluated by the MTT assay. We observed that cell viability remained unaltered after NECA treatment (Fig. 2A, n=3). To determine the effect of adenosine on human MC degranulation, LAD2 cells were treated with 0-30 M of NECA, and some cells were subsequently activated with C3a. NECA alone had no effect on degranulation. LAD2 cells pretreated with NECA for 30 min showed up to a 70 % decrease in degranulation (Fig. 2B; n=3) and this effect was dose-dependent. Therefore, we used 30 M of NECA for subsequent experiments, unless otherwise stated. Longer exposure to NECA (24 h) was more effective at 14

inhibiting degranulation such that 3 M of NECA inhibited 50% degranulation (Fig. 2C, n=3). For comparison, we also examined the effect of adenosine (ADO) on C3a activation of MC degranulation. ADO significantly decreased human MC degranulation when added 30 min (25 to 45% inhibition; Fig. 2D, n=3), or 24 h (68% inhibition; Fig. 2E, n=3), prior to activation with C3a, in a dose-dependent fashion.

3.3 Modulation of intracellular calcium Since MC degranulation is calcium dependent (44), we examined the effect of NECA on intracellular calcium levels when added 30 min prior to stimulation with C3a. LAD2 cells activated with C3a showed a rapid increase in intracellular calcium, measured by Fura-2 AM fluorescence intensity, reaching a peak within 70 s following C3a exposure. Ionomycin was used as a positive control. Additionally, we found that NECA pre-treatment prevented intracellular calcium flux after C3a stimulation (Figs. 2F, and 2G; n=3).

3.4 Inhibition of chemokine production C3a has been shown to stimulate LAD2 cells to preferentially produce chemokines CCL2 and CCL5 (45). To test our hypothesis that adenosine receptor activation blocks C3a-mediated MC activation, we evaluated the ability of NECA to inhibit C3a-mediated production of CCL2. C3a-treated LAD2 cells released 44 ± 4 pg/mL of CCL2, but when they were exposed to NECA for 30 min, C3a-actived LAD2 released only 14 ± 2 pg/mL of CCL2 (68% inhibition; Fig. 3A, n=3).

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3.5 Inhibition of mast cell adhesion and chemotaxis C3a stimulates chemotaxis and adhesion of MC, facilitating their recruitment and subsequent activation at sites of inflammation (46). Since NECA inhibited mast cell mediator release, we assessed its effect on mast cell migration and adhesion. Comparable with SCF, a positive control, C3a activated LAD2 cells to adhere to fibronectin, an extracellular matrix protein. Pretreatment with NECA for 30 min abolished adhesion of LAD2 cells to fibronectin (Fig. 3B, n=3). In addition to its promotion of adhesion, C3a is also a potent chemoattractant for human mast cells (47, 48). C3a activated LAD2 cell chemotaxis but this was blocked by 55 ± 2% by NECA pre-treatment (Fig. 3C, n=3). Stem cell factor (100 ng/ml) was used as positive control in both adhesion and chemotaxis analyses.

3.6 Downregulation of ERK phosphorylation The biological effects of C3aR are mediated via the activation of extracellular signalregulated kinases (ERK) (45). C3a causes a transient ERK phosphorylation in LAD2 cells, and ERK signaling is necessary for C3a-induced chemokine production. Therefore, we investigated the effect of NECA on ERK phosphorylation in LAD2 cells. While C3a caused rapid ERK1/2 phosphorylation (≤5 min), the response was substantially attenuated in NECA-pretreated cells (Fig. 3D).

3.7 Role of adenosine receptor subtypes To determine the adenosine receptor subtype(s) responsible for the inhibitory effects of NECA, we investigated the effects of specific agonists and antagonists on LAD2 cell degranulation. The A2A receptor agonist CGS 21680 reduced C3a-induced degranulation by 16

more than 35% (Fig. 4A). The A2B agonist, BAY 60-6583 (2-[[6-Amino-3,5-dicyano-4-[4(cyclopropylmethoxy)phenyl]-2-pyridinyl]thiol]-acetamide); Fig. 4B), and A3 agonist, IBMECA,(1-Deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-β-Dribofuranuronamide); Fig 4C) are less potent, even when using higher agonist concentrations (~10% inhibition at 10 M in both cases). We next blocked A2A receptors by pretreating LAD2 cells for 30 min with different concentrations of the highly selective A2A antagonist, SCH 58261 (2-(2-Furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine); 0.5-50 M), and treated with NECA and C3a. As shown in Fig. 4D, NECA was unable to inhibit C3a-induced degranulation in LAD2 cells pretreated with SCH 58261. Pretreating LAD2 cells with different concentrations of either A2B or A3 antagonists, MRS 1754 (N-(4-Cyanophenyl)-2[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]-acetamide);

and MRS

1334 (1,4-Dihydro-2-methyl-6-phenyl-4-(phenylethynyl)-3,5-pyridinedicarboxylic acid 3-ethyl5-[(3-nitrophenyl)methyl] ester); 0.5-50 M respectively) had no effect on NECA reduction of activation (Figs. 4E and 4F, n=3). Given that ligation of A2A receptors led to inhibition of MC degranulation, it was possible that changes in adenylyl cyclase activity were involved (49-51). Thus, we preincubated LAD2 cells with an adenylyl cyclase inhibitor (SQ 22536, 10 M) for 30 min, followed by C3a activation, and assessed its effect on MC degranulation. Our results showed that SQ 22536 reduced C3a-activated -hexosaminidase release by 30-35%, confirming that increasing intracellular cAMP inhibited the C3a signaling pathway. Next, LAD2 cells were pretreated with the A2A agonist followed by the adenylyl cyclase inhibitor for 30 min, and finally activated by C3a. This sequential treatment resulted in up to 46% inhibition of degranulation, revealing an additive effect (Fig 5A, n=3). Furthermore, when LAD2 were pretreated with the adenylyl 17

cyclase inhibitor, followed by NECA, and activated with C3a, we observed a 70% decrease in degranulation, showing a potentiating effect. Gαs-coupled receptors mediate their effect through activation or inhibition of adenylyl cyclase (25), which in turn activates or inhibits the accumulation of cAMP in cells, affecting various cascade of enzymes. In this context, we measured cAMP levels in LAD2 cells pretreated with NECA, using a competitive cAMP ELISA. Our results showed that NECA treatment elevated cAMP in LAD2 cells. Furthermore, NECA pretreatment also increased the cAMP levels in C3a-stimulated LAD2 cells (Fig. 5B). Collectively, these findings suggest that Gαs-mediated intracellular cAMP may be required for inhibition of MC activation by adenosine.

3.8 Adenosine receptor A2A inhibits C3a-induced activation of other mast cell lines by activating Gs. Considering the possibility that the observed effects extended only to LAD2 cells, we sought to confirm our findings using another mast cell line, HMC-1, derived from a patient with MC leukemia (52). To evaluate if the adenosine receptor A2A inhibited C3a-induced activation by activating Gs in other cell lines, we evaluated the inhibition of activation of HMC-1.1 and HMC-1.2 cells by measuring histamine release after 30 min treatment with 10 M CGS 21680, followed by stimulation with C3a for 30 min. A2A blocked histamine release of HMC1.1 and HMC1.2 exhibiting a significant reduction of 69 ± 1.4% (Fig. 6A) and 51 ± 2.3% (Fig. 6B, n=3) respectively.

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4.

Discussion Adenosine has been implicated in asthma due to its ability to enhance IgE-dependent MC

degranulation (18). Intriguingly, in vitro studies with human lung MC have reported robust inhibition, rather than potentiation, of IgE-dependent degranulation at relatively high concentrations of adenosine, such as those found in asthmatic lungs. Existing conflicting evidence indicates that adenosine may play different roles depending on micro environmental concentration and its interactions with different adenosine receptor subtypes. Consistent with these observations, we found that NECA inhibited C3a-mediated degranulation of human MC at a higher concentration of 30 μM, within the range observed in vivo in inflamed tissues, where adenosine levels range from 10 – 100 μM, but lower than the ~200 μM found in the bronchoalveolar lavage fluid from asthmatic lungs (32). Leung et al. published contrasting results reporting that NECA enhances IgE and C3astimulated degranulation of LAD2 cells (53). The differences may be due to technical differences in the assays, as the authors used much higher concentrations of both anti-IgE (5 μg/mL vs. 0.1 μg/mL) and C3a (0.2 μM vs. 0.01 μM) to induce MC degranulation. Moreover, these studies were performed using a relatively low adenosine concentration (1 μM). Our studies showed that NECA had no effect on degranulation by itself. Pre-treatment with NECA (30 M) followed by C3a activation showed that NECA was able to decrease up to 70 % MC degranulation. To evaluate the interaction of anaphylatoxin C3a signaling controlled by adenosine receptors in human mast cells on more physiological conditions, LAD2 cells were expose to adenosine (ADO), as stimulant instead of NECA. Our results showed that 30 min treatment with 30 M ADO, generated a 40% MC degranulation inhibition and that longer ADO treatment (24 h), exhibited a 55% inhibition. This effect confirm the results observed by Gomez et al., (32), 19

where it was showed that higher concentrations of adenosine inhibited mast cell degranulation triggering the A2A receptor. Other researchers have shown that ADO modulates mast cell degranulation, in this context, ADO at 10 -6-10-5 M enhanced IgE-dependent degranulation, and it was inhibited at 10-5-10-3M (32, 54-58). Earlier studies by Hughes (33) have shown that adenosine and NECA have dual capacity inhibiting or potentiating IgE-dependent histamine release by human lung mast cells depending upon time and dose concentration and this effects involving the interaction of adenosine with cell surface A2 receptors associated with stimulation of adenylate cyclase and rise in cyclic AMP. MC activation via FcεRI is a critical event in allergic inflammation and asthma (3) , and there is growing evidence that MC activation via cell-surface GPCRs also plays a crucial role in inflammation (11). GPCRs can mediate MC degranulation, either directly or through the modulation of FcεRI-mediated degranulation (59, 60). For example, activation by C3a and sphingosine-1-phosphate can induce degranulation directly;

however,

adenosine and

prostaglandin E2 alone cannot directly induce degranulation, but they potentiate IgE-induced MC degranulation via their cognate GPCRs (59). Furthermore, cross-talk between GPCRs has also been acknowledged to modulate MC responses (59, 61). In this study, we present evidence for a novel role for adenosine as a negative regulator of C3a-dependent human MC activation. Our study shows that the non-hydrolyzable adenosine analog NECA inhibited C3a-induced degranulation of human MC, and that this inhibitory effect of NECA was abolished by the treatment of MC with the A2A receptor specific antagonist SCH 58261. While treatment of MC with the A2A receptor specific agonist CGS 21680 had similar inhibitory effect, treatment with A2B and A3 receptor specific agonists BAY 60-6583 and IB-MECA did not. Overall, this

20

supports our hypothesis that adenosine receptors modulate complement-mediated activation of human MC, specifically the A2A receptor. The contrasting effects of adenosine on MC and in asthma have been linked to the differential activation of adenosine receptors (25). We found that LAD2 cells expressed adenosine receptors A2A, A2B, A3, but not A1. A2A receptor had the highest expression levels compared to A2B and A3. This pattern of adenosine receptor expression was different from that shown for BMMC, RBL-2H3, skin MC, and murine primary lung MC, where A3 receptor is predominantly expressed. Adenosine can exert a direct effect on degranulation of these MC types directly (62-64) or enhance IgE-induced degranulation through a Gαi protein-dependent pathway (26-29), perhaps due to the abundance of A3 receptor transcripts in these cells. Similarly, adenine nucleotides ATP and UDP can potentiate IgE-dependent degranulation of human MC via Gαi protein-coupled P2Y receptors (35). Thus, it seems that most, if not all, GPCRs that mediate degranulation are coupled to Gαi proteins. NECA alone did not degranulate LAD2 MC. Studies using selective adenosine receptor agonists and antagonists suggested that the activation of the A2A receptor could inhibit C3a-induced degranulation and mediator release. Furthermore, this inhibition of degranulation was associated with decrease in intracellular Ca 2+ that was possibly mediated through Gαs protein-dependent pathway. Previous studies have shown that A1, A2B or A3 receptors elicit pro-inflammatory effects in asthma through the activation of histamine and bronchoconstrictive substances released from MC, mucus secretion and bronchoconstriction (25). A2A receptors are believed to almost always elicit anti-inflammatory signals in asthma by inhibiting histamine and tryptase release from MC in vitro (33, 65-67). In vivo administration of an A2A receptor agonist attenuates airway inflammation in ovalbumin–sensitized and –challenged rats (68). Genetic ablation of the A2A 21

receptor augments airway inflammation and hyperresponsiveness in a murine model of allergic airway inflammation (69). Anti-inflammatory effects of the A2A receptor are also linked to suppression of inflammatory cell recruitment. Adenosine inhibits chemotaxis of neutrophils by inhibiting the expression of adhesion molecules, and suppresses recruitment of T cells and macrophages to the lungs (70). Accordingly, we found that adenosine inhibited chemotaxis and adhesion of human MC that had been activated with C3a. Since migration of MC is an important mechanism towards their accumulation at sites of inflammation, the activity of adenosine further adds to its anti-inflammatory role in complement-mediated inflammation. Stimulation of A2A receptor leads to the accumulation of intracellular cAMP, which is a second messenger generated by adenylyl cyclase enzyme from AMP. cAMP can abolish IgEdependent MC degranulation by inhibiting store-operated calcium channels in MC through a protein kinase A-dependent pathway. Therefore, raising intracellular cAMP levels inhibits MC signaling pathways, including those initiated by Gαs protein-coupled receptors (71). Indeed, activation of A2A and A2B receptors has been shown to increase cAMP levels in MC (25, 72). In contrast, engagement of the C3aR on immune cells inhibits the adenylyl cyclase pathway and reduces intracellular levels of cAMP, further suggesting that C3a exerts its pro-inflammatory function through Gαi protein-coupled C3aR (73). Intracellular cAMP has inhibitory effects on pro-inflammatory cytokine production such as tumor necrosis factor (TNF) and IL-12, but has a stimulatory effect on anti-inflammatory IL10 production by immune cells (70). This is related to cAMP-mediated modulation of NF-κB and MAPK (JNK, ERK) signaling pathways. A2A receptor activation can not only stimulate but inhibit ERK phosphorylation (74). On the other hand, stimulation of C3aR mediates activation of ERK1/2 and subsequent production of pro-inflammatory cytokines and chemokines (45). We 22

found that adenosine was able to inhibit C3a-induced CCL2 production from human MC, as well as ERK1/2 phosphorylation. As cAMP causes smooth muscle relaxation, decreases of cAMP level in airway induced by adenosine are of particular interest in the treatment of asthma, especially for asthma attack or severe asthma patients. In this context, substances such as adenosine, bradykinin and neuropeptides, acting as indirect stimuli, can evoke bronchoconstriction in the pathophysiology of asthmatic patients, activating various cell types including mast cells, vascular smooth muscle cells, vascular endothelial cells, and/or airway nerves (75-77). Adenosine release under conditions of cellular stress in asthmatic airways can, therefore, produce bronchoconstriction and inflammation, largely depending on the receptor they target. On one hand, activation of high affinity A2A receptor on inflammatory cells suppresses the release of proinflammatory cytokines and mediators. The activation of A2A receptor coupled to Gs and adenylate cyclase may lead to bronchial smooth muscle relaxation via the cAMP-PKA (cyclic adenosine monophosphateprotein kinase A) pathway (78). In contrast, binding of adenosine to the low-affinity Adenosine A2B receptor causes degranulation of mast cells, releasing histamine, which in turn induces bronchoconstriction by binding to histamine receptors on airway smooth muscle cells (33, 65, 79, 80). Our results show a role for cAMP in mediating the effect of adenosine on C3a-induced MC function. Treatment with NECA significantly augmented the cAMP levels in C3a activated human MC. Furthermore, the adenylyl cyclase inhibitor SQ 22536 blocked the effect of C3a, and MC pre-treatment with the A2 A agonist (CGS 21680), followed by the adenylyl cyclase agonist SQ 22536 and C3a activation potentiate the inhibitory effect, clearly indicating that A2A mediate their C3a activation effects through Gαi and Gαs proteins, respectively. Overall, these data 23

confirm that adenosine inhibits C3a-mediated activation of human MC through a Gαs proteindependent pathway, demonstrating that A2A-Gαs signaling may be of critical importance in regulation of MC activation in inflammation. Forskolin also exerts its biological activities by direct stimulation of adenylyl cyclase, thereby increasing cellular concentrations of cAMP. Forskolin potentiation of cAMP in turn inhibits basophils and MC degranulation and histamine release (81). We therefore utilized forskolin (10 M) to further confirm the role of constitutive cAMP in regulation of MC functions and found 70% inhibition of C3a-mediated MC degranulation (data not shown). In summary, our study provides strong evidence that activation of the Gαs-coupled A2A adenosine receptor reduces the stimulating effect of Gαi-coupled C3aR on human MC. This provides a critical balance between MC activation and mediator release in host defense and inflammation, while mitigating cellular damage. In case of unregulated MC activation via a GPCR, for instance by C3aR, targeting inhibitory A2A adenosine receptor might prove beneficial in limiting harmful effects of hypersensitivity. Thus, adenosine receptors offer novel therapeutic targets in allergic inflammation mediated by mast cells. Acknowledgements This work was funded by intramural funds from the National Research Council Canada. We would like to thank Dr. Ramses Ilarraza for his revisions to this manuscript; and Sara Stredulinsky for her laboratory technical assistance.

Disclosure Statement The authors declare no conflict of interest.

24

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Figure Legends Fig. 1. Expression of C3a and adenosine receptors by human mast cells. (A-D) Intracellular A1 (A), A2A (B), A2B (C), and A3 (D) receptor expression on LAD2 cells by flow cytometry, (A, C, D) APC-conjugated goat anti-rabbit antibody (dark histograms). Rabbit polyclonal IgG-APC antibody -treated LAD2 cells were included as controls (light grey histograms). (B) PE-conjugated A2A receptor (dark histogram), or PE-conjugated mouse IgG isotype control (light grey histogram). (E) Expression of C3aR-PE on LAD2 cell surface, (dark grey histogram), PE-conjugated mouse IgG, as isotype control (light grey histogram). (F-J) MFI (median fluorescence intensity) results of the flow cytometric analyses from A1, (F), A2A (G), A2B (H), and A3 (I) intracellular receptor expression, as well as C3a surface expression (J), (n=3, **P<0.01, ***P<0.001). (K)

Total RNA was isolated from LAD2 cells and A1, A2A, A2B, and A3 receptors mRNA

expression was examined by real time qPCR. The data was normalized to GAPDH, and is reported as ratio of GAPDH expression (n=4).

Fig. 2. Adenosine treatment inhibits human mast cell degranulation and modulates intracellular calcium. (A)

Effect of NECA on mast cell viability. Data represent average viability of LAD2 cells

exposed to different concentrations of NECA (0 – 30 M) for 24h, metabolic activity was measured by the MTT assay, and expressed as percentage with respect to control untreated cells (n=3 ± standard error of the mean, SEM). Treatment of LAD2 cells with NECA did not impact

35

cell viability up to a concentration of 30 M. Hence we used 30M of NECA for subsequent experiments, unless otherwise stated. (B)

β-hexosaminidase released by LAD2 cells treated with NECA (0 – 30 µM, closed bar) for

30 min and stimulated with C3a (0.01 µM, open bar) for 30 min. LAD2 cells in presence of C3a, were also evaluated (n=3, values are presented as mean ±SEM, ***P<0.001, analyzed by a oneway ANOVA test with Tukey’s posthoc). The inhibitory effect of LAD2 cells cultured in the presence of NECA and C3a stimulation was not due to changes in cell viability since the metabolic activity of LAD2 cells remained unaltered after NECA treatment. (C)

LAD2 cells were treated with NECA (0 - 30 µM) for 24 h, then stimulated with C3a

(0.01 µM) for 30 min and β-hexosaminidase release was measured. LAD2 cells in presence of C3a, were included (n=3, values are presented as mean ±SEM, ***P<0.001, analyzed by a oneway ANOVA test with Tukey’s posthoc). (D)

LAD2 cells were treated with adenosine (ADO, 0 – 30 µM, closed bar) for 30 min,

followed by C3a (0.01 µM, open bar) for 30 min, and β-hexosaminidase release was measured. LAD2 cells in presence of C3a, were also evaluated (n=3, values are presented as mean ±SEM, ***P<0.001, analyzed by a one-way ANOVA test, with Tukey’s posthoc). (E)

LAD2 cells were treated with ADO (0 – 30 µM) for 24 h, followed by C3a (0.01 µM)

stimulation for 30 min, and β-hexosaminidase release was measured (n=3, values are presented as mean ±SEM, ***P<0.001, analyzed by a one-way ANOVA test, with Tukey’s posthoc). (F)

Representative calcium flux response of LAD2 cells loaded with 1 M Fura-2 AM and

treated with NECA (30 M, --) for 30 min, or cells incubated with NECA followed by C3a treatment (0.01 µM, --), changes in fluorescence measurements are indicated with an arrow. Intracellular calcium effects were observed within 70 s following C3a treatment; ionomycin (1 36

μM) was added as positive control at 256 s. Data was recorded at 100 ms intervals. Values are reported as 340/380 ratio vs time. (G)

Quantification of the calcium flux responses from (F). Untreated LAD2 cells and cells in

presence of ionomycin were included as negative and positive calcium movement controls and plotted independently (n=3, values are reported as mean ± SEM *P<0.05, **P<0.01, ***P<0.001, analyzed by a two-way ANOVA, with Bonferroni post tests).

Fig. 3. Adenosine inhibits C3a-induced chemokine production, mast cell adhesion, chemotaxis and ERK phosphorylation. (A)

LAD2 cells were treated with NECA (30 µM) for 30 min, then stimulated with C3a (0.01

µM) for 24 h, and CCL2 production was analyzed in cell free supernatants by ELISA. Data represent mean CCL2 levels measured in control untreated (open bar), C3a (checkered bar), and NECA+C3a (horizontal bar) treated LAD2 cells (n=3, values are presented as mean ±SEM, ***P<0.001, analyzed by a two way ANOVA test, with Turkey’s posthoc). (B)

LAD2 cells were treated with NECA (30 µM) for 30 min, allowed to adhere to

fibronectin-coated wells in the presence of C3a (0.01 µM) for 2 h, and the percentage of cell adhesion over untreated cells was assessed, SCF (vertical bar) was included as positive control (n=3, values are presented as mean ±SEM, *P<0.05, ***P<0.001, analyzed by a two way ANOVA test, with Turkey’s posthoc). (C)

LAD2 cells were treated with NECA (30 µM) for 30 min, allowed to migrate towards

C3a (0.01 µM) for 6 h and the percentage of cell chemotaxis over untreated cells was assessed, SCF was included as positive control (n=3, values are presented as mean ±SEM*P<0.05, **P<0.01, analyzed by a two way ANOVA test, with Turkey’s posthoc). 37

(D)

Cell lysates of LAD2 cells treated with NECA (30 µM) for 30 min, and stimulated with

C3a at different time points were analyzed for phospho-p44/42 MAPK (ERK 1/2) (Thr202/Tyr204) and p44/42 MAPK (ERK 1/2) expression by Western blot. Data shown is representative of four independent experiments.

Fig. 4. Effects of adenosine receptor agonists and antagonists on C3a-induced degranulation. C3a-induced degranulation was evaluated by β-hexosaminidase released in cell-free supernatants of LAD2 cells that were treated for 30 min with (0-10 M of A2A agonist CGS 21680 (panel A); A2B agonist BAY 60-6583 (panel B); or A3 agonist IB-MECA (panel C), followed by 30 min stimulation with C3a (0.01 M, closed bars). -hexosaminidase released by cell-free supernatants of LAD2 cells in presence of C3a only (closed bar), treated with NECA (30 M) for 30 min, and stimulated with C3a (0.01 M, open bar), or with increasing concentrations (0-50 M) of adenosine receptor antagonists SCH 58261 (A2A -horizontal bar-, panel D); MRS 1754 (A2B -hatched bar-, panel E); or MRS 1334 (A3 -shingle bar-, panel F), for 30 min, then treated with NECA (30 µM) for 30 min, and stimulated with C3a (0.01 µM) for 30 min, was also evaluated at each condition. NECA was unable to inhibit C3a-induced degranulation in LAD2 cells pretreated with the highly selective A2A antagonist SCH 58261 (n=3, values are presented as mean ±SEM, *P<0.05, **P<0.01, ***P<0.001, analyzed by a two way ANOVA test, with Turkey’s posthoc).

38

Fig. 5. Effects of adenosine are Gαs-dependent. (A)

LAD2 cells were treated either with the A2A agonist CGS 21680 (10 µM), or with

adenylyl cyclase inhibitor SQ 22536 (10 µM) for 30 min, and stimulated with C3a (0.01 µM) for 30 min. In addition, LAD2 were preincubated with A2A agonist CGS 21680 (10 µM) for 30 min, then treated with the adenylyl cyclase inhibitor SQ 22536 (10 µM) for 30 min, followed by 30 min C3a stimulation. Cell-free supernatants were collected and analyzed by β-hexosaminidase release. -hexosaminidase values of LAD2 cells treated with NECA for 30 min, followed by 30 min incubation with C3a, as well as cells treated with SQ 22536 for 30 min, incubated with NECA for 30 min and stimulated with C3a are also included. (n=3, values are presented as mean ±SEM, ***P<0.001, analyzed by a two way ANOVA test, with Turkey’s posthoc). (B)

LAD2 cells were treated with NECA (30 µM) for 30 min, then stimulated with C3a (0.01

µM) for 30 min and cAMP content was measured by EIA (n=3, values are presented as mean ±SEM, ***P<0.001, analyzed by a two way ANOVA test, with Turkey’s posthoc).

Fig 6. Effects of A2A on C3a-induced activation of other mast cell lines by activating Gs To confirm the effects of the adenosine receptor A2A on other human mast cell lines, HMC-1.1 (A), and HMC1.2 (B), were treated with CGS 21680 (10 M, closed bars) for 30 min and then stimulated with C3a (0.01 M, open bars) for 30 min, and histamine release was evaluated. Histamine release values obtained with HMC1.1 and HMC-1.2 in the absence of CSG 21680 are also showed (n=3, values are presented as mean ±SEM, **P<0.01, analyzed by a two way ANOVA test, with Turkey’s posthoc).

39

Fig. 1

A)

B)

C)

D)

E)

F)

G)

H)

I)

J)

2000 1000 0 Isotype Control

A1 receptor

10000

0

Isotype Control

A2A receptor

8000 6000 4000 2000 0

Isotype A2B receptor Control

20000 C3a receptor expression (MFI)

3000

20000

***

10000 A3 receptor expression (MFI)

A2A receptor expression (MFI)

A1 receptor expression (MFI)

4000

***

10000 A2B receptor expression (MFI)

30000

5000

8000 6000 4000 2000 0

Isotype Control

A3 receptor

**

15000 10000 5000 0

Isotype C3a receptor Control

K) Ratio of GAPDH expression

1.0

0.8

0.6

0.4

0.2

0.0

A1

A2A A2B

A3

40

50

NECA

40 30 20 10

% Metabolic activity

A)

B) 100

50 0

*** 0.3 ***

0

3

***

40

0 0

0.3

3

30

NECA ( M)

***

NECA NECA + C3a

***

30

D ***

0 0

***

**0.3

3

30

ADO

NECA (M)

ADO + C3a

20 10 0

0

3 NECA (M)

-hexosaminidase (% release)

E)

F)

***

ADO ADO + C3a

30

20 0

***

***

20

ADO ADO + C3a

***

0

3

0 0

3

3

30

5 4

+ NECA

Ionomycin

3 C3a

2

0

100

200

300

- NECA

Ratio 340/380

+ NECA 3

***

* 1 0 Untreated

C3a

5 4 3 2

0

400

**

2

0

1

5 4

10

- NECA

Time (s)

ADO (M)

20

F

30

ADO (M)

0

0

30

30

1

10

40

ADO ( M)

10

E

30

***

40

0

G)

***

1030

***

0

10

2040

Ratio 340/380

-hexosaminidase (% release)

40

D)

10

NECA + C3a

30

C)

20

30

40

NECA (M)

30

NECA

C 20

50

40

30

-hexosaminidase (% release)

Fig. 2

NECA + C3a

-hexosaminidase (% release)

B

Ratio 340/380

-hexosaminidase (% release) (% release)-hexosaminidase (% release)-hexosaminidase (% release) -hexosaminidase

A

Ionomycin

41

Unt

Fig. 3

42

Fig. 4

B)

***

30 20 10 0 0.1

1

80

**

40 30 20 10 0

10

0

CGS 21680 (M)

60

*** ***

40

20

0 C3a + SCH 58261 (M) 0 + NECA + C3a

0.5 +

1 +

5 +

1

40 20 0

10

0

0.1

10 +

50 +

1

10

IB-MECA (M)

F)

E)

*** *** *** ***

0.1

* 60

BAY 60-6583 (M)

-hexosaminidase (% release)

-hexosaminidase (% release)

D)

-hexosaminidase (% release)

**

40

0

C) 50

**

60

-hexosaminidase (% release)

50

-hexosaminidase (% release)

-hexosaminidase (% release)

A)

*** 40

20

0 C3a + MRS 1754 (M) 0 + NECA + C3a

0.5 +

1 +

5 +

10 +

50 +

***

60

***

*

*

40

20

0 C3a + MRS 1334 (M) 0 + NECA+C3a

0.5 +

1 +

5 +

10 +

50 +

43

Fig. 5

A)

B)

*** *** *** *** ***

80

*** 40

*** 30 20 10

cAMP (pg/mL)

-hexosaminidase (% release)

50

***

60 40 20 0

0 C3a CGS 21680 SQ22536 CGS 21680 NECA SQ22536 +C3a + C3a +SQ22536 + C3a +NECA + C3a + C3a

C3a

NECA

NECA+C3a

44

Fig. 6

CGS CGS + C3a

Histamine (ng/mL)

** 40

20

0

B) 80

CGS CGS + C3a

** Histamine (ng/mL)

A) 60

60 40 20 0

0

10

CGS 21680 ( M)

0

10

CGS 21680 ( M)

45

Graphical abstract. A2A inhibits C3aR-mediated mast cell activation.

46