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Adenosine Receptor A1 Regulates Polymorphonuclear Cell Trafficking and Microvascular Permeability in Lipopolysaccharide-Induced Lung Injury This information is current as of October 1, 2021. Kristian-Christos Ngamsri, Rosalyn Wagner, Irene Vollmer, Stefanie Stark and Jörg Reutershan J Immunol 2010; 185:4374-4384; Prepublished online 20 August 2010;

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Adenosine Receptor A1 Regulates Polymorphonuclear Cell Trafﬁcking and Microvascular Permeability in Lipopolysaccharide-Induced Lung Injury

Kristian-Christos Ngamsri, Rosalyn Wagner, Irene Vollmer, Stefanie Stark, and Jo¨rg Reutershan

Extracellular adenosine and adenosine receptors are critically involved in various inflammatory pathways. Adenosine receptor A1 (A1AR) has been implicated in mediating transmigration of leukocytes to sites of inflammation. This study was designed to characterize the role of A1AR in a murine model of LPS-induced lung injury. LPS-induced transmigration of polymorphonuclear cells (PMNs) and microvascular permeability was elevated in A1AR2/2 mice. Pretreatment of wild-type mice with the specific A1AR agonist 29Me–2-chloro-N6-cyclopentyladenosine attenuated PMN accumulation in the interstitium and alveolar space as well as microvascular permeability. Lower PMN counts in the lungs of pretreated wild-type mice were associated with reduced amounts Downloaded from of the chemotactic cytokines TNF-a, IL-6, and CXCL2/3 in the bronchoalveolar lavage. Pretreatment was only effective when A1AR was expressed on hematopoietic cells as demonstrated in chimeric mice. These findings were confirmed by in vitro transmigration assays demonstrating that chemokine-induced transmigration of PMNs was reduced when PMNs but not when pulmonary endothelial or alveolar epithelial cells were pretreated. 29Me–2-chloro-N6-cyclopentyladenosine prevented pulmonary endothelial but not epithelial cells from LPS-induced cellular remodeling and cell retraction. Our data reveal what we believe to be a previously unrecognized distinct role of A1AR for PMN trafficking and endothelial integrity in a model of acute lung http://www.jimmunol.org/ injury. The Journal of Immunology, 2010, 185: 4374–4384.

cute lung injury (ALI) and acute respiratory distress inflammation and proved beneficial in patients with ALI/ARDS syndrome (ARDS) in their most severe forms are still (4–6). A major challenges in modern intensive care medicine that Excessive recruitment of PMNs into the lung is a key event in significantly contribute to morbidity and mortality of critically ill the early development of ALI and ARDS. A variety of experimental patients (1). ALI and ARDS develop as a result of direct lung studies implicate that modulation of PMN trafficking improves by guest on October 1, 2021 damage (e.g., pneumonia, aspiration, or lung trauma) or indirectly course and outcome of ALI (7–9). The significance of PMNs in in the course of remote or systemic inflammation (e.g., sepsis). ALI is highlighted by clinical studies showing that ARDS can Inflammatory response of the lungs is characterized by excessive deteriorate when patients recover from neutropenia (10). Lung infiltration of polymorphonuclear cells (PMNs), disruption of the function in ARDS patients negatively correlates with neutrophil alveolo-capillary barrier, and release of chemotactic cytokines (2, counts in the blood. Persisting pulmonary neutrophilia in ARDS is 3). Destruction of the pulmonary architecture results in interstitial correlated with poor outcome (11). PMN recruitment into the lung and alveolar edema and ultimately threatens pulmonary gas ex- occurs in a cascade-like sequence of activation, sequestration in change. Despite numerous experimental and clinical studies, mol- pulmonary vessels, transendothelial [from blood to the intersti- ecular mechanisms that regulate the inflammatory response of the tium (IS)], and transepithelial (from the IS to the alveolar air- lung have not been fully elucidated. Accordingly, specific thera- space) migration (8). Each migration step is regulated by distinct peutic options in ALI are not available. So far, only mechanical molecules, and the importance of investigating discrete steps of ventilation with low tidal volumes has been demonstrated to limit PMN migration in the lung has been emphasized (8, 12). Besides adhesion molecules (13), chemokines (14), and intracellular molecules (15), extracellular adenosine is an essential Department of Anesthesiology and Intensive Care Medicine, University Hospital of mediator of leukocyte trafficking to inflammatory sites. Adenosine Tu¨bingen, Tu¨bingen, Germany signals through four subtypes of G protein-coupled adenosine Received for publication February 9, 2010. Accepted for publication July 20, 2010. receptors (ARs; A1, A2a, A2b, and A3) that are ubiquitary ex- This work was supported by German Research Foundation Grant RE 1683/3-1 (to pressed on various hematopoietic and nonhematopoietic cells. J.R.). Activation of ARs induces a variety of cell responses through Address correspondence and reprint requests to Dr. Jo¨rg Reutershan, Department of Anesthesiology and Intensive Care Medicine, University of Tu¨bingen, Hoppe- changes in intracellular levels of cAMP, diacylglycerol, and ino- Seyler-Strasse 3, 72076 Tu¨bingen, Germany. E-mail address: joerg.reutershan@ sitoltriphosphate. Recent studies identified CD39 and CD73, both uni-tuebingen.de rate-limiting enzymes for the generation of extracellular adeno- 2 2 Abbreviations used in this paper: A1AR, adenosine receptor A1; A1AR / , A1AR sine, as critical mediators in pulmonary inflammation (16, 17). In knockout; ADA, adenosine deaminase; ALI, acute lung injury; AR, adenosine receptor; ARDS, acute respiratory distress syndrome; BAL, bronchoalveolar lavage; BM, bone addition, activation of ARs A2a and A2b has been demonstrated to marrow; BW, body weight; HMVEC-L, human lung microvascular endothelial cell; IS, attenuate organ damage in LPS- and ventilator-induced lung in- interstitium; IV, intravascular; 29Me-CCPA, 29Me-2-chloro-N6-cyclopentyladenosine; jury (18, 19). PFA, paraformaldehyde; PMN, polymorphonuclear cell. The role of AR A1 (A1AR) in mediating inflammatory pathways Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 has not been studied systematically. A1AR signals through Gi and www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000433 The Journal of Immunology 4375

+ Go and inhibits adenylyl cyclase, activates K channels, and in- LPS served as negative control. Total RNA samples were analyzed with hibits Ca2+ channels in various cells (20). Physiologic functions sense/antisense primers for wild-type mice (59-GTACATCTCGGCCTT- of A1AR signaling include bradycardia, reduced glomerular fil- CCAGG-39 and 59-GAG AATACCTGGCTGACTAG-39) and for A1 gene- deficient mice (59-ACAACAGACAATCGGCTGCTCTGATG-39 and 59- tration, antinociception, neuronal hyperpolarization, and ischemic TGCGCGCCTTGAGCCTGG CGAAC-39) using the iCycler iQ Real- preconditioning (21). A1AR is constitutively expressed in various Time Detection System (Bio-Rad, Hercules, CA). Values were determined tissues including the lung and brain (22). In the lung, A1AR is using iCycler iQ Real-Time Detection System Software version 3.1 (Bio- expressed on endothelial, airway, and alveolar epithelial cells and, Rad) and normalized to murine b-actin (59-ACATTGGCATGGCTTTGTTT- 39 and 59-GTTTGCTCCAACCAACTGCT-39). at high concentrations, on alveolar macrophages (23). The role of A1AR in pulmonary inflammation has been discussed controver- Western blotting sially. In an early study, pharmacologic blocking of A1AR at- LPS-induced expression of A1AR protein in whole lung tissue was eval- tenuated LPS-induced lung injury in cats (24). In more recent uated by Western blotting as previously described (15). Briefly, lungs were studies, protective effects of A1AR have been demonstrated in snap frozen and lysed in RIPA buffer [1% Nonidet P-40, 1% deoxycholate, models of ischemia reperfusion- and endotoxin-induced lung in- 0.1% SDS, 50 mM Tris (pH 7.4), 150 mM NaCl, protease, and phosphatase jury (25, 26). In adenosine deaminase (ADA)-deficient mice, which I and II inhibitors]. Lysates were cleared by centrifugation at 14,000 3 g exhibit elevated adenosine levels, genetic removal of the A1AR for 20 min, and protein concentration was determined using the DC protein assay (Bio-Rad). A total of 30 mg protein from each sample was resolved resulted in enhanced pulmonary inflammation and alveolar de- using SDS-PAGE, transferred to polyvinylidene difluoride membranes struction (23). These changes were associated with increased ex- (Bio-Rad), and blocked with 5% milk and 5% BSA in TBS plus 0.1% pression of chemokines and matrix metalloproteinases. A1AR- Tween-20. Blots were probed with anti-A1AR (Affinity BioReagents, Golden, dependent regulation of cytokines from human mononuclear cells CO). Proteins were labeled with HRP-conjugated secondary Abs and visu- alized by ECL (Amersham Biosciences, Piscataway, NJ). Downloaded from has been shown to be TLR4 dependent and highlights its impact in In separate experiments, A549 lung epithelial cells (American Type Cul- LPS-induced inflammation (27). However, whether A1AR is in- ture Collection, Manassas, VA), human lung microvascular endothelial cells volved in regulating LPS-induced neutrophil transmigration into the (HMVEC-Ls; Lonza, Cologne, Germany), or murine PMNs were exposed to different compartments of the lung has not been addressed yet. LPS and prepared for Western blotting as described above. In the current study, we sought to identify mechanisms by which PMN depletion A1AR regulates PMN trafficking in the lung. We hypothesized that activation of A1AR would reduce PMN infiltration by inhibiting To evaluate the impact of PMNs for A1-dependent inflammatory pathways, http://www.jimmunol.org/ the release of chemotactic cytokines into the alveolar space and circulating PMNs were depleted by injecting an anti-granulocyte Ab (RB6– 8C5, BD Pharmingen, San Diego, CA; 100 mg i.v. 24 h before the ex- reduce organ damage. In a murine model of LPS-induced lung periment) (30). Depletion reduced peripheral PMNs by .95% as con- injury, we determined the role of A1AR for the different migration firmed by peripheral blood smears. steps in the lung. We further determined the effects of A1AR for microvascular permeability and identified the target cell of a spe- Model of ALI cific A1AR agonist by creating chimeric mice. Wild-type and A1AR2/2 mice were exposed to aerosolized LPS (Salmo- nella enteritidis; Sigma-Aldrich, St. Louis, MO) in a cylindrical chamber (20 cm 3 9 cm) for 30 min connected to an air nebulizer (MicroAir, Materials and Methods Omron Healthcare, Vernon Hills, IL). As previously shown, this model by guest on October 1, 2021 Mice mimics several apects of ALI including migration of PMNs into all lung compartments (8). Control mice were exposed to aerosolized saline. Wild-type male C57BL/6 mice were obtained from Charles River Labo- ratories (Sulzfeld, Germany). A1AR knockout mice (A1AR2/2) were previously characterized and kindly provided by Ju¨rgen B. Schnermann PMN transmigration into the lung (National Institutes of Health, Baltimore, MD) (28). All animal experi- As previously described, we used a flow cytometry-based method to de- ments were approved by the Animal Care and Use Committee of the termine the number of PMNs in all compartments of the lung [intravascular University of Tu¨bingen, Tu¨bingen, Germany. Mice were 8–12 wk of age. space (IV), IS alveolar space] (8). Briefly, 24 h upon LPS exposure, IV Generation of chimeric mice PMNs were labeled by an i.v. injection of Alexa 633-labeled GR-1 (5 mg). After 5 min, mice were anesthetized, and their chests were opened. Sub- We transferred bone marrow (BM) between wild-type and A1AR2/2 mice sequently, the spontaneously beating right ventricle was cannulated and to generate chimeric mice that express A1AR either on hematopoietic or flushed with 10 ml PBS to remove nonadhaerent PMNs from the pulmo- nonhematopoietic cells (29). Recipient mice were irradiated in two doses nary vasculature. Tracheotomy was performed, and a 24-gauge insyte of 600 rad each (separated by 4 h). BM was harvested from both femora cannula was inserted into the trachea to obtain bronchoalveolar lavage and tibiae of donor mice, and ∼ 5 million cells were injected i.v. into the (BAL) fluid. Lungs were removed, minced, and digested in the presence of recipient mice immediately upon the second irradiation. BM trans- excess unlabeled anti–GR-1 to prevent possible binding of the injected Ab plantations were performed as follows: 1) BM from A1AR2/2 into wild- to extravascular PMNs. A cell suspension was prepared by passing the type recipients (chimeric mice express A1AR on nonhematopoietic cells digested lungs through a 70-mm cell strainer (BD Falcon, Bedford, MA). only); and 2) BM from wild-type into A1AR2/2 recipients (chimeric mice Total cells in BAL and lungs were counted, and the percentage of PMNs express A1AR only on hematopoietic cells). After complete reconstitution, was determined by flow cytometry. In the BAL, PMNs were identified by the animals were used for the experiments between 6 and 8 wk after BM their typical appearance in the forward/side scatter and their expression of transplantation. One control mouse per transplantation was not recon- CD45 (clone 30-F11), 7/4 (clone 7/4), and GR-1 (clone RB6-8C5). In stituted with BM and served as an indicator for an efficient radiation. the lung, the expression of GR-1 was used to distinguish IV (CD45+7/4+ 2 These sentinel animals died within 2 wk postradiation. GR-1+) from IS (CD45+7/4+GR-1 ) PMNs, which were not reached by the injected Ab (8). Differential blood cell counts We withdrew blood from the tail vein of wild-type and A1AR2/2 mice Microvascular leakage and obtained baseline differential blood counts to reveal possible differ- LPS-induced microvascular protein leakage into the lungs of wild-type and ences between both strains (Diff-Quik, Medion Diagnostics, Du¨dingen, A1AR2/2 mice was assessed by the Evans blue extravasation technique Switzerland). (31). Briefly, 6 h after LPS exposure, Evans blue (20 mg/kg; Sigma- A1AR mRNA expression Aldrich) was injected into the tail vein. After 30 min, mice were anesthetized, thoracotomy was performed, and the lungs were flushed through LPS-induced expression of A1AR in the lungs was evaluated by real-time the beating right ventricle to remove the IV dye. The lungs were removed RT-PCR. RNA from whole lung homogenates of wild-type mice was and homogenized, and Evans blue was extracted as described previously extracted 30, 120, and 180 min after LPS inhalation. Mice that did not inhale (32). Evans blue absorption of blood and lung was measured at 620 nm and 4376 A1AR IN ACUTE LUNG INJURY

corrected for the presence of heme pigments: A620 (corrected) = A620 2 Cytoskeletal remodeling (1.426 3 A750 + 0.030) and calculated against a standard curve (picograms Evans blue dye per gram lung) (12). In addition, LPS-induced protein To evaluate whether A1 is involved in cytoskeletal remodeling of pulmonary content in the BAL of wild-type and A12/2 mice was determined at in- endothelial or epithelial cells, we investigated the distribution of F-actin as dicated time points as previously described (33). described before (14). Briefly, HMVEC-L or A549 cells were allowed to adhere on gelatin-coated glass cover slips overnight (medium with 10% Chemokine release FBS). Cells were then serum-starved for 6 h and stimulated with LPS (100 ng/ml) in presence or absence of 29Me-CCPA (100 ng/ml). Untreated cells Release of TNF-a, IL-6, CXCL1 (keratinocyte-derived chemokine), and served as control. At different times, cells were washed, fixed (4% PFA), CXCL2/3 (MIP-2) into the alveolar space of wild-type and A1AR2/2 mice permeabilized (0.1% Triton X-100, Sigma-Aldrich), and stained with was measured by ELISA kits (R&D Systems, Minneapolis, MN) 3 h upon FITC-phalloidin and DAPI Nucleic Acid Stain (both Invitrogen/Life Tech- LPS inhalation. nologies). Cover slips were mounted on glass slides, and microscopy was performed on a confocal fluorescence microscope (LSM 510, Zeiss, Go¨t- Pharmacological activation of A1AR tingen, Germany).

2/2 Wild-type and A1AR mice were pretreated with the selective A1 ag- Statistical analysis onist 29Me–2-chloro-N6-cyclopentyladenosine (29Me-CCPA) (34). 29Me- CCPA was injected i.p. [0.1 mg/kg body weight (BW), 1 mg/kg BW, and Statistical analysis was performed by GraphPad Prism (version 5.0, La Jolla, 10 mg/kg BW] 30 min before LPS inhalation, and the effects on pulmo- CA) Statistical differences between the groups were evaluated by one-way nary PMN trafficking, microvascular permeability, and chemokine release ANOVA followed by a post hoc Tukey test. Significance was considered at were studied. p , 0.05, and data are presented as means 6 SD. In separate experiments, mice received either the A1 antagonist DPCPX or the A2b antagonist PSB1115 (both Sigma-Aldrich; 1 mg/kg BW) prior to treatment with 29Me-CCPA to reveal potential unspecific effects of 29Me- Results Downloaded from CCPA on the A2b receptor. Blood counts 2 2 Immunohistochemistry To reveal possible alterations in the blood counts of A1AR / mice 2 2 that may have interfered with the analysis of PMN trafficking, A1AR / and wild-type mice were euthanized 24 h after LPS exposure. Control mice did not receive LPS. The pulmonary circulation was perfused baseline blood counts were performed. Total leukocyte, PMN, and 2/2 free of blood, the trachea was cannulated, and the lung was inflated with lymphocyte counts were not different between A1AR mice and

4% paraformaldehyde (PFA) for 10 min at 25 cm H2O. The lungs were their appropriate wild-type controls (Table I). http://www.jimmunol.org/ subsequently removed and fixed in PFA for 24 h. Paraffin-embedded sections (5 mm) were stained for PMNs utilizing the avidin-biotin tech- LPS induces A1AR mRNA and protein in the lung nique (Vector Laboratories, Burlingame, CA) as previously described (35). Briefly, deparaffinized and rehydrated sections were incubated with avidin, Transcriptional responses of A1AR following LPS inhalation were 10% rabbit serum, and 0.5% fish skin gelatin oil for 1 h to block non- evaluated by real-time RT-PCR of lung homogenates at indicated specific binding. After washing with PBS, a specific Ab against mouse times. LPS exposure induced time-dependent A1AR mRNA ex- neutrophils (clone 7/4, Caltag Laboratories, Burlingame, CA) was added m pression. Three hours after LPS exposure, A1AR mRNA expres- (1 g/ml) and incubated overnight. Sections were then washed and in- , cubated with 5 mg/ml biotinylated rabbit anti-rat IgG (Vector Laboratories, sion was 2.7-fold (p 0.05; Fig. 1A). In addition, A1AR protein Burlingame, CA) for 1 h, followed by avidin–biotin–peroxidase complexes levels slightly increased upon LPS exposure (Fig. 1B). When by guest on October 1, 2021 (Vectastain Elite ABC kit, Vector Laboratories), washed with PBS, in- PMNs were depleted before LPS inhalation, induction of A1AR cubated with diaminobenzidine (DAB kit, Vector Laboratories), and co- was attenuated on both the mRNA (Fig. 1C) and protein level (Fig. unterstained with hematoxylin. Some sections were stained with the appropriate isotype and served as negative controls. 1D), suggesting that recruitment of PMNs largely contributes to In separate experiments, lung sections were stained for A1 (h-40, Santa the induction of A1AR in the inflamed lungs. A1AR protein was Cruz Biotechnology, Santa Cruz, CA) following the same protocol. also induced on LPS-exposed PMNs of wild-type but not of A12/2 mice (Fig. 1E). In vitro PMN transmigration We studied chemokine-induced in vitro transmigration of human PMNs A1AR depletion increases PMN migration into the lungs across a monolayer of HMVEC-Ls (Lonza) or epithelial cells. A549 lung We used a flow cytometry-based technique to detect PMNs in all epithelialcells(AmericanTypeCultureCollection)wereculturedincomplete Ham’s F-12 medium (Invitrogen/Life Technologies, Karlsruhe, Germany), three lung compartments (IV space, IS, and BAL space). LPS including 10% FCS (PAA Laboratories, Co¨lbe, Germany), 5% antibiotic inhalation resulted in a significant accumulation of PMNs in all 2 2 solution (Sigma-Aldrich), and 200 mM L-glutamin (Invitrogen/Life Tech- compartiments of the lungs of wild-type and A1AR / mice. PMN nologies). Cells were plated on the bottom of polycarbone filter inserts of recruitment to IS and BAL was significantly higher in A1AR2/2 a Transwell system (3.0 mm pore size, 6.5 mm diameter; Costar, Cambridge, 6 3 6 6 MA) and grown until confluent. Human PMNs were purified from healthy compared with wild-type mice (IS: 2.4 0.4 10 versus 1.5 6 6 donors (Percoll gradient, GE Healthcare Biosciences, Uppsala, Sweden). 0.4 3 10 ; p , 0.05; BAL: 3.4 6 0.4 3 10 versus 2.5 6 0.4 3 Epithelial cells, PMNs, or both were incubated with 29Me-CCPA for 30 min at indicated concentrations (10 ng/ml, 1 ng/ml, and 0.1 ng/ml). PMNs (1 3 106) were plated on the top of filters with or without epithelial cells and allowed to migrate toward CXCL2/3 (10 ng/ml; PeproTech, Hamburg, Germany). After 1 h, migrated cells were quantified by determination of Table I. Baseline cell counts myeloperoxidase in the bottom wells. Negative controls did not receive treatment with 29Me-CCPA and CXCL2/3. A1+/+ A12/2 p Value In separate experiments, HMVEC-Ls were cultured in EGM-2MV medium 6 6 (Lonza)andusedinthesameTranswellsystem, with the exception that cells Leukocytes 8.5 0.2 9.6 0.9 NS 6 6 were plated on the top of the filter inserts. Neutrophils 1.9 0.2 1.7 0.2 NS Lymphocytes 6.1 6 0.2 7.4 6 0.6 NS Monocytes 0.3 6 0.3 0.4 6 0.3 NS Oxidative burst of human neutrophils Neutrophils (%) 23.0 6 1.4 17.7 6 0.9 NS 6 6 LPS-induced oxidative burst of human neutrophils was determined as Lymphocytes (%) 71.7 3.3 77.0 1.6 NS 6 6 previously described (36). Briefly, PMNs of healthy volunteers were pre- Monocytes (%) 4.5 3.4 4.5 2.6 NS incubated with luminol (50 mmol/ml) and stimulated with LPS in presence Baseline differential cell counts (103/ml unless otherwise indicated) were per- or absence of 29Me-CCPA (1 ng/ml). Chemiluminescence was measured at formed in A1+/+ (wild-type) and A1 gene-deficient (A12/2) mice. indicated time points. Data are mean 6 SD of four mice. The Journal of Immunology 4377

FIGURE 1. LPS-induced A1AR mRNA (A) and protein (B) expression in the lungs of wild-type mice. LPS inhalation led to an induction of both A1AR mRNA and protein. When PMNs were depleted, increase of A1AR was attenuated (C, D). A1AR protein also Downloaded from increased in LPS-exposed PMNs of wild-type but not of A12/2 mice (E). A1AR mRNA levels were normalized to b-actin and presented as mean 6 SD of n = 4 samples. Representa- tive Western blots of n = 4 samples p , are shown. p 0.05 versus control http://www.jimmunol.org/ without LPS exposure. by guest on October 1, 2021

106; p , 0.05) (Fig. 2). LPS-induced adhesion of PMNs to the prior to treatment with 29Me-CCPA. We found that blocking A1 pulmonary endothelium did not differ between the two groups. but not A2b abolished the effect of 29Me-CCPA, supporting the Quantitative FACS data were illustrated by immunohistochemistry specificity of 29Me-CCPA (Fig. 5). as shown in Fig. 3. Pharmacologic activation of A1AR reduces PMN migration A1AR mediates LPS-induced increase in microvascular into the lungs permeability Treatment of wild-type mice with 29Me-CCPA prior to LPS ex- Disrupted alveolo-capillary barrier function is, in addition to in- posure resulted in a dose-dependent reduction of PMNs in the IS filtration of PMNs, another characteristic feature in acute pulmonary and alveolar space (Fig. 4). Transepithelial migration into the al- inflammation. We studied the role of A1AR for LPS-induced mi- veolar space was significantly reduced with two doses of 29Me- crovascular permeability by means of Evans blue extravasation. CCPA (10 and 1 mg/kg) (1.7 6 0.2 3 106 versus 2.5 6 0.3 3 106; LPS-induced increase in Evans blue extravasation was higher in p , 0.05; 1.9 6 0.1 3 106 versus 2.5 6 0.3 3 106; p , 0.05), A1AR2/2 than in wild-type mice (397 6 88 versus 271 6 66 mg/g whereas significant reduction of transendothelial migration into lung; p , 0.05) (Fig. 6A). Pretreatment with the A1 agonist 29Me- the IS space was only observed with the highest dose of 10 mg/kg CCPA (1 mg/kg BW) significantly reduced permeability in wild- (0.7 6 0.2 3 106 versus 1.3 6 0.4 3 106; p , 0.05). Pretreatment type but not A1AR2/2 mice (wild-type mice: 96 6 49 versus 271 6 did not alter adhesion of PMNs to the pulmonary endothelium. 66; p , 0.05; A1AR2/2 mice: 347 6 94 versus 397 6 88; p is NS), Although 29Me-CCPA did not reduce accumulation of PMNs in indicating a role of A1AR for maintaining endothelial integrity. A1AR2/2 mice significantly, a trend for decreased PMNs was LPS-induced increase in microvascular permeability was reduced observed in all lung compartments. To identify a potential in- when PMNs were depleted. In addition, pretreatment with 29Me- terference with the A2b receptor (that also regulates PMN traf- CCPA lost its effect when PMNs were depleted, indicating that in ficking and barrier function), we pretreated wild-type mice with our model, LPS-induced microvascular permeability is largely either the A1 antagonist DPCPX or the A2b antagonist PSB1115 PMN dependent. 4378 A1AR IN ACUTE LUNG INJURY

FIGURE 3. PMN infiltration into the lungs shown by immunohistochemistry. Wild-type and A1AR2/2 mice were exposed to LPS. Lung Downloaded from sections were stained with a specific neutrophil marker (7/4, brown cells; magnification 340). Images are representative of n = 4 experiments.

cells within the septal walls, consistent with an expression of A1 on PMNs, endothelial, and epithelial cells (Fig. 8A). As expected, lungs of A12/2 mice did not express A1AR. LPS-induced protein http://www.jimmunol.org/ by guest on October 1, 2021 FIGURE 2. PMN migration into all compartments of the lungs of wild- type (black bars) and A12/2 (white bars) mice. Accumulation of PMNs in the IV (A), the lung IS (B), and the BAL space (C) was analyzed. Data are means 6 SD of n = 4 samples. pp , 0.05 versus negative control without LPS; xp , 0.05 versus wild-type mice within the same treatment group.

In separate experiments, LPS-induced protein content in the BAL was determined. A12/2 mice exhibited significantly more protein at 12 h than wild-type mice (Fig. 6B). Activation of A1AR reduces alveolar release of chemokines Release of chemotactic cytokines into the alveolar space initiates recruitment of PMNs into the lungs. A1AR has been implicated in regulating chemokine release. We therefore measured concentrations of the relevant cytokines CXCL1, CXCL2/3, TNF-a, and IL-6. LPS inhalation induced significant production of all cytokines. Pretreatment with 29Me-CCPA (1 mg/kg BW) significantly reduced the amounts of CXCL2/3 (660 6 380 versus 3555 6 753; p , 0.05), TNF-a (550 6 450 versus 1532 6 664; p , 0.05), and IL-6 (505 6 450 versus 1640 6 261; p , 0.05) of wild-type but not of A12/2 mice (Fig. 7).

Hematopoietic versus nonhematopoietic effects of A1AR FIGURE 4. Effects of different doses of 29Me-CCPA on LPS-induced migration of PMNs into the different lung compartments of wild-type Next, we sought to characterize the cell types that contributed to A1- (black bars) and A12/2 (white bars) mice. Accumulation of PMNs in the dependent regulation of PMN trafﬁcking in our model. First, lung IV (A), the lung IS (B), and the BAL space (C) was analyzed. Values are 2/2 sections of LPS-treated wild-type and A1 mice were immu- means 6 SD of n = 4 experiments. #p , 0.05 versus LPS-treated mice nostained for A1AR. In wild-type mice, an ubiquitary distribu- without 29Me-CCPA; xp , 0.05 versus wild-type mice within the same tion of A1AR was found, with accumulation of A1-expressing treatment group. The Journal of Immunology 4379

FIGURE 5. To detect potential unspeciﬁc effects of 29Me-CCPA on the A2b receptor, wild-type mice were pretreated with either the A1 antagonist

DPCPX or the A2b antagonist PSB1115 prior to treatment with 29Me- Downloaded from CCPA. Accumulation of PMNs in the bronchoalveolar space was analyzed 24 h after LPS inhalation. Values are means 6 SD of n = 4 experiments. #p , 0.05 versus LPS-treated mice without 29Me-CCPA. expression of A1AR on pulmonary endothelial and alveolar

epithelial cells was conﬁrmed by Western blotting (Fig. 8B). http://www.jimmunol.org/ FIGURE 7. Effect of 29Me-CCPA on LPS-induced release of chemo- We then generated chimeric mice by transferring BM between 2/2 2 2 kines into the alveolar airspace of wild-type (black bars) and A1AR wild-type and A1AR / mice and determined LPS-induced PMN (white bars) mice. Data are means 6 SD of n = 4 samples. pp , 0.05 accumulation in the different lung compartments (Fig. 9). Lethally versus samples without LPS; #p , 0.05 versus LPS-treated mice without irradiated mice reconstituted with BM from the same genotype 29Me-CCPA.

served as controls. Controls exhibited similar phenotypes compared with wild-type or A1AR2/2 mice, respectively (data not

shown). As expected, LPS-induced accumulation of PMNs in the by guest on October 1, 2021

FIGURE 6. Effect of 29Me-CCPA and PMN depletion on LPS-induced microvascular permeability in the lungs of wild-type (black bars) and A12/2 (white bars) mice. Microvascular protein leakage was assessed by FIGURE 8. Distribution of A1AR in the lungs of wild-type and A12/2 Evans blue extravasation technique 6 h after LPS exposure (A) and by mice as shown by immunohistochemistry (A). Mice were exposed to LPS. measurement of protein content in the BAL at indicated times (B). Data are Lung sections were stained with a speciﬁc Ab to A1AR (brown cells). means 6 SD of n = 4 samples. pp , 0.05 versus negative control without Images are representative of n = 4 experiments. B, LPS-induced A1AR LPS; #p , 0.05 versus LPS-treated wild-type mice without 29Me-CCPA; protein expression on HMVEC-L and A549 cells. Representative Western xp , 0.05 versus wild-type mice within the same treatment group. blots of n = 4 samples are shown (original magniﬁcation 340). 4380 A1AR IN ACUTE LUNG INJURY

gration (10 ng/ml: 28,000 6 5,000 versus 164,000 6 44,000; 1 ng/ ml: 18,000 6 10,000 versus 86,000 6 2,000; 0.1 ng/ml: 19,000 6 10,000 versus 86,000 6 2,000; all p , 0.05) (Fig. 10). Simulta- neous activation of A3 on PMNs and endothelial cells did not show an additional effect. Pretreatment of endothelial cells alone did not reduce PMN migration. Similar results were obtained in appropriate chemotaxis experiments with A549 cells. Pharmacologic activation of A1AR on PMNs reduced transepithelial migration signiﬁcantly (10 ng/ml: 279,000 6 31,000 versus 741,000 6 5,000; 1 ng/ml: 321,000 6 22,000 versus 741,000 6 5,000; 0.1 ng/ml: 212,000 6 3,000 versus 741,000 6 5,000; all p , 0.05). Pretreatment of epithelial cells did not alter PMN migration. Simultaneous treatment of PMNs and epithelial cells appeared to reduce migration more than treatment of PMNs alone, but the difference failed statistical signiﬁcance (Fig. 11).

Cytoskeletal remodeling To evaluate the effects of A1AR on cytoskeletal remodeling, we Downloaded from stained F-actin in pulmonary endothelial (Fig. 12) and epithelial cells (Fig. 13). LPS induced formation of stress ﬁbers in both HMVEC-L and A549 cells. Pretreatment with 29Me-CCPA resulted in a substantial reduction of stress ﬁbers in HMVEC-Ls, consistent http://www.jimmunol.org/

FIGURE 9. Contribution of A1AR on hematopoietic and nonhematopoietic cells to LPS-induced migration of PMNs into the different

lung compartments as demonstrated by chimeric mice. Accumulation of by guest on October 1, 2021 PMNs in the IV (A), the lung IS (B), and the BAL space (C) is displayed. Some chimeric mice were pretreated with 29Me-CCPA to study its pref- erential target cell. Means 6 SD of n = 4 samples. pp , 0.05 versus negative control without LPS exposure; #p , 0.05 versus LPS-treated mice without 29Me-CCPA. pulmonary vasculature was not altered when A1AR was removed from either hematopoietic or nonhematopoietic cells (Fig. 9A). Transendothelial migration into the IS was high in mice that lacked A1AR on nonhematopoietic cells (2.4 6 0.2 3 106;Fig.9B). Mi- gratory activity in these mice was similar to A1AR2/2 mice (2.4 6 0.4 3 106). Similarly, transendothelial migration in mice that lacked A1AR on hematopoietic cells was not different from wild-type mice (1.9 6 0.4 3 106), indicating a distinct role of endothelial A1AR for migration of PMNs into the IS space. PMN counts in the BAL were comparable when A1AR was removed from either hematopoietic or nonhematopoietic cells. Pretreatment with 29Me-CCPA (1 mg/kg BW) reduced PMN migration into the BAL only when A1AR was expressed on hematopoietic cells (2.4 6 0.6 3 106 versus 0.7 6 0.1 3 106; p , 0.05; Fig. 9C). This indicates that the A1AR agonist acts through A1AR on BM-derived rather than through nonhematopoietic cells. Effects of 29Me-CCPA on in vitro transmigration of human FIGURE 10. In vitro effect of 29Me-CCPA on chemokine-induced mi- PMNs gration across a layer of HMVEC-Ls. Transmigration was significantly reduced when PMNs were pretreated with 29Me-CCPA (A). Pretreatment To extend the evidence for the distinct role of A1AR on hema- of HMVEC-Ls alone did not alter PMN migration (B). Simultaneous topoietic or nonhematopoietic cells, we studied chemokine- pretreatment of PMNs and endothelial cells did not further reduce mi- induced migration of human PMNs in an in vitro Transwell as- gration (C). Data are means 6 SD of n = 4 experiments (each in duplicate). say across an endothelial or epithelial layer. Activation of A3 on pp , 0.05 versus negative control without CXCL2/3; #p , 0.05 versus PMNs resulted in a significant decrease in transendothelial mi- positive control without 29Me-CCPA. The Journal of Immunology 4381 Downloaded from http://www.jimmunol.org/ FIGURE 12. A1AR-dependent formation of F-actin in HMVEC-Ls. LPS induced formation of stress fibers at indicated time points. Pre- treatment with 29Me-CCPA substantially reduced formation of F-actin. Images are representative of three experiments with similar results (original magnification 363). FIGURE 11. The effect of 29Me-CCPA on chemokine-induced transmigration across A549 cells was evaluated in vitro. Transmigration was significantly reduced when PMNs were pretreated with 29Me-CCPA (A). previously implicated in attenuating acute injury and inflammation in Pretreatment of epithelial cells alone did not alter PMN migration (B). models of ambient hypoxia (37, 38), cyclic-mechanical stretch (17), Simultaneous pretreatment of PMNs and A549 cells did not further reduce and LPS-induced lung injury (16). Adenosine signals through four by guest on October 1, 2021 migration (C). Data are means 6 SD of n = 4 experiments (each in duplicate). pp , 0.05 versus negative control without CXCL2/3; #p , 0.05 versus positive control without 29Me-CCPA.

with a speciﬁc role of A1AR in mediating microvascular permeability. In A549 cells, the effect of 29Me-CCPAwas less prominent. Oxidative burst The release of reactive oxygen species plays a critical role in the disturbance of the pulmonary integrity under inﬂammatory con- ditions. Activation of A1AR reduced LPS-induced oxidative burst of human PMNs and may represent an additional mechanism of the protective effects of 29Me-CCPA in our model (Fig. 14).

Discussion The present study was designed to elucidate the role of A1AR for pulmonary PMN trafficking in a model of LPS-induced lung injury. We found that A1AR on hematopoietic cells mediates transendothelial and transepithelial migration, most likely by inhibiting the release of chemotactic cytokines into the alveolar airspace. In addition, A1AR on endothelial cells is involved in regulating microvascular permeability and leukocyte transmigration. Both PMN infiltration and permeability can be controlled by the specific A1AR agonist 29Me-CCPA. Extracellular adenosine is an essential mediator of leukocyte traf- FIGURE 13. A1AR-dependent formation of F-actin in A549 cells. LPS ficking to inflammatory sites. Adenosine is generated by a cascade- induced formation of stress fibers at indicated time points. Pretreatment like enzymatic process involving the ectoapyrase (CD39, conversion with 29Me-CCPA reduced formation of F-actin. However, the effect was of ATP/ADP to AMP) and the ecto-59-nucleotidase (CD73, conver- less prominent than with endothelial cells. Images are representative of sion of AMP to adenosine). Both rate-limiting enzymes have been three experiments with similar results (original magnification 363). 4382 A1AR IN ACUTE LUNG INJURY

II cells to release chemokines, and it appears reasonable that chemokine production and release cannot be further increased (ceiling effect); and 2) LPS-induced release of adenosine into the alveolar airspace might not be sufficient to reduce chemokine release in wild-type animals. In addition, adenosine will have effects on all four ARs that might counteract each other. It is important to realize that AR-dependent production and release of chemokines from monocytes/macrophages requires specific TLR subtypes (27). In particular, A1AR-dependent inhibition of cytokine release is TLR4 dependent and highlights its specific relevance in LPS-induced inflammation. Interestingly, LPS itself binds to and activates A1AR on pulmonary endothelial cells (47). Whether LPS activates A1AR on alveolar epithelial cells and macrophages remains FIGURE 14. Respiratory burst in human polymorphonuclear leukocytes speculative but may represent a negative-feedback mechanism in with or without LPS and impact of A1AR on oxidative activity. Pre- our model. treatment with 29Me-CCPA (1 ng/ml) reduced the LPS-induced oxidative Data from our experiments with chimeric mice suggest that A1AR activity of human PMNs. on hematopoietic cells is critical for the regulation of leukocyte migration in vivo. Concordantly, recruited PMNs largely contributed to the induction of A1AR mRNA and protein in the inflamed lungs. subtypes of G protein-coupled ARs (A1, A2a, A2b, and A3) that are Moreover, A1AR protein in isolated PMNs was induced upon LPS Downloaded from ubiquitarily expressed on various hematopoietic and nonhematopoi- exposure, suggesting a particular role of A1AR on PMNs. However, etic cells. transendothelial migration was, at least in part, controlled by A1AR A1AR has been implicated as a potent anti-inflammatory me- on endothelial cells. In addition, activation of A1AR on pulmonary diator in various inflammatory models of other organ systems, endothelial but not epithelial cells prevented LPS-induced cellular including the kidney (39, 40), heart (41), liver (42), and brain (43). remodeling and cell retraction, indicating that endothelial A1AR Early upregulation of A1AR suggests that A1AR initiates an anti- is also involved in regulating microvascular permeability, which is http://www.jimmunol.org/ inflammatory cascade that also involves other ARs (44). largely controlled by the pulmonary endothelium. In vitro trans- The role of A1AR in pulmonary inflammation has, however, not migration was mediated by A1AR on PMNs but not on endothelial been studied systematically yet. A1AR is constitutively expressed or epithelial cells. It has been previously emphasized that micro- on various cells in the lung, including endothelium, airway, and vascular permeability and migration of leukocytes are independent alveolar epithelial cells and alveolar macrophages (23). The role of functions with distinct regulation and molecular requirements (49). A1AR in pulmonary inflammation has been discussed controver- In our study, LPS-induced microvascular permeability and PMN sially. In an early study, pharmacologic blocking of A1AR atten- transmigration was enhanced in A1AR2/2 compared with wild-type uated LPS-induced lung injury in cats (24), suggesting a pro- mice, suggesting that LPS-dependent activation of A1AR represents by guest on October 1, 2021 inflammatory role of A1AR. In two recent studies with rodents, an effective mechanism that limits the inflammatory response in the A1AR demonstrated protective effects in models of ischemia lung. reperfusion- and endotoxin-induced lung injury (25, 26). Both The present study on A1AR has to be placed in the context of studies focused on the effects of A1AR on the formation of lung other ARs that have been implicated in the regulation of ALI. Re- edema and are in line with our findings; leukocyte trafficking, search has particularly focused on the role of A2a and A2b in pul- however, was not considered in these studies. monary inflammation and revealed a protective effect of both The first evidence for an involvement of A1AR in regulating receptors in LPS- and ventilator-induced lung injury (18, 19). Pro- PMN trafficking in the lung derived from studies with ADA- tective properties of A1 and A2a seem particularly striking, as both deficient mice, which exhibit elevated adenosine levels. Genetic receptors exert opposing effects on intracellular cAMP levels. Ac- removal of the A1AR in these animals resulted in enhanced pul- tivation of A1AR results in decreased adenylate cyclase activity monary inflammation and alveolar destruction (22), highlighting through activation of pertussis toxin-sensitive Gi-proteins whereas the protective properties of A1AR. These changes were associated activation of A2a results in Gs- and Golf-dependent increase in in- with increased expression of chemokines and enhanced accumula- tracellular cAMP levels. However, A1 signaling also involves direct tion of all subtypes of leukocytes in the alveolar space. Baseline coupling to phospholipase C via Gbg subunits that may result in leukocyte counts were not different between wild-type and A1AR2/2 cAMP-independent regulation of inflammatory response (49, 50). A mice. This is consistent with our data (no difference in PMN counts recent study identified A2b as a key receptor in LPS-induced leu- at baseline without LPS) and indicates that A1AR requires activa- kocyte trafficking and barrier function in the lung (51). Pharmaco- tion in vivo. logical activation of both A2a and A2b receptors has been suggested In our model, LPS inhalation induces the release of relevant to be a promising approach in the therapy of pulmonary inflam- chemokines into the BAL. These chemokines are produced by mation. Interestingly, both receptors appear to exert their effects on alveolar macrophages and alveolar epithelial cells (45, 46) and are different cell types. Whereas A2a predominantly acts on inflam- critical for the recruitment of PMNs (14). We found that phar- matory leukocytes, expression of A2b on pulmonary endothelial and macologic activation of A1AR reduced the release of the che- epithelial cells seems to be pivotal for its effects in lung inflam- motactic cytokines CXCL2/3, IL-6, and TNF-a into the alveolar mation. In our model, A1AR on PMNs seemed to largely contribute space (Fig. 6). Inhibition of relevant chemokines may represent to LPS-induced lung damage. However, A1-dependent signaling on a pivotal mechanism of ARs that has also been demonstrated for endothelial cells was also involved. Do all ARs do the same thing in A2a (18). Despite the effect of 29Me-CCPA, LPS-induced release ALI? High levels of adenosine are associated with increased in- of chemokines did not differ between wild-type and A12/2 mice. flammatory response in patients with asthma (52). In addition, Two reasons may contribute to this phenomenon: 1) LPS inhalation ADA-deficient mice that exhibit excessive adenosine levels and die is a direct and strong stimulus for alveolar macrophages and type of severe respiratory distress shortly postbirth (53). In contrast, The Journal of Immunology 4383 pharmacological activation of A2b reverses these detrimental 19. Eckle, T., A. Grenz, S. Laucher, and H. K. Eltzschig. 2008. A2B adenosine effects in ADA-deficient mice. At present, the distinct role of each receptor signaling attenuates acute lung injury by enhancing alveolar fluid clearance in mice. J. Clin. Invest. 118: 3301–3315. AR in ALI (including redundant functions) remains speculative. 20. Linden, J. 2001. Molecular approach to adenosine receptors: receptor-mediated The expression pattern of ARs may be relevant when therapeutic mechanisms of tissue protection. Annu. Rev. Pharmacol. 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