Alveolar macrophages play a crucial role in the pathogenesis of inflammatory airway diseases. By the generation and release of different inflammatory mediators they contribute to both recruitment of different leukocytes into the lung and to airway remodeling. A potent stimulus for the release of inflammatory cytokines is ATP, which mediates its cellular effects through the interaction with different membrane receptors, belonging to the P2X and P2Y families. The aim of this study was to characterize the biological properties of purinoceptors in human alveolar macrophages obtained from bronchoalveolar lavages in the context of inflammatory airway diseases. The present study is the first showing that human alveolar macrophages express mRNA for different P2 subtypes, namely P2X1, P2X4, P2X5, P2X7, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y13, and P2Y14. We also showed that extracellular ATP induced Ca2+ transients and increased IL-1β secretion via P2X receptors. Furthermore, extracellular nucleotides inhibited production of IL-12p40 and TNF-α, whereas IL-6 secretion was up-regulated. In summary, our data further support the hypothesis that purinoceptors are involved in the pathogenesis of inflammatory lung diseases.
The alveolar macrophage is the predominant immune effector cell in the alveolar space and the conducting airways. Therefore, it is the first immune cell to meet inhaled pathogens such as microbiological agents, environmental toxins, or allergens (1, 2). By secreting different cytokines and chemokines they are involved in the recruitment of other immune cells, e.g., eosinophils or neutrophils, into the lung (3, 4, 5). Furthermore, they influence processes such as bronchial hyperresponsiveness, emphysema, or pulmonary fibrosis (6, 7, 8).
Extracellular nucleotides such as ATP, ADP, UTP, or UDP are released into the extracellular space from mechanically stressed endothelial and epithelial cells, specialized compartments of nerve terminals, activated platelets, and from LPS-stimulated monocytes (9, 10, 11, 12). The cellular effects of nucleotides are mediated through the interaction with P2 receptors. These plasma membrane receptors can be classified into two subfamilies: the metabotropic G protein-coupled P2Y receptors and P2X receptors that are ligand-gated ion channels. Cloned human P2Y subtypes include eight members: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14 (13, 14, 15). GPR80 (GPR99), temporarily designated as P2Y15 receptor (16), has turned out not to belong to the P2Y receptor family (17). Pharmacological data showed that P2Y1, P2Y11, P2Y12, and P2Y13 receptors interact with ATP and/or ADP (15, 18, 19, 20), whereas the P2Y2, P2Y4, and P2Y6 subtypes were responsive to uridine nucleotides. Although ATP and UTP activate P2Y2 with similar efficiency, UTP and UDP are the most potent agonists at P2Y4 and P2Y6, respectively. In addition, it has been shown that the P2Y14 is activated by UDP-glucose (UDP-glc)4 and related sugar nucleotides, but not by ATP, ADP, UTP, or UDP (14, 21, 22, 23, 24). In contrast to P2Y receptors the only naturally occurring ligand for P2X receptors is ATP. An intriguing member of the P2X subfamily is the P2X7 receptor. Upon activation this receptor forms large plasma membrane pores allowing transmembrane fluxes of Ca2+, Na+, K+, and small hydrophilic molecules (11). This receptor is involved in processes such as cell death, giant cell formation, or cytokine secretion (25, 26, 27).
Extracellular nucleotides have been shown to modulate a variety of different immune cells (11). They have chemotactic activity on eosinophils or immature dendritic cells (28, 29). Furthermore, they modulate cytokine secretion and expression of cell surface molecules in different cell types (4, 27, 30, 31, 32). We have recently shown that ATP levels were increased in the bronchoalveolar fluids of asthmatics and that ATP triggers and maintains allergic airway inflammation in an animal model of asthma (33). Although different studies on P2-mediated responses in macrophages are available, (34, 35, 36) expression and function of these membrane molecules in human alveolar macrophages hasn’t been studied yet and this would be important in the light of macrophage function in airway diseases.
Materials and Methods
Isolation and cultivation of human alveolar macrophages from bronchoalveolar lavage (BAL)
Healthy non-smoking volunteers underwent bronchoscopy with BAL as described in the guidelines of the American Thoracic Society. All subjects gave their written informed consent, and the study was approved by the local ethics committee. Alveolar macrophages were obtained from BAL fluids as previously described (37). Human alveolar macrophages were purified by adherence giving a purity of >98%. Thereafter, alveolar macrophages (5 × 105/ml) were cultured in RPMI 1640 medium (Life Technologies) containing 10% FCS (PAA Laboratories) and 1% gentamicin (Life Technologies). Cells were stimulated with the indicated concentrations of the different nucleotides (all purchased from Sigma-Aldrich) 1 h before overnight pulsing with LPS (10 ng/ml). At the end of this period, cell-free supernatants were harvested.
Analysis of mRNA expression
Total RNA was extracted by using Trizol reagent (Life Technologies) as described by the manufacturer. To obtain cDNA, 5 μg of total RNA were primed with oligo-dT primers (Hermann GbR) and reverse transcribed with StrataScript reverse transcriptase (Stratagene). Primers for the human P2 receptors were designed based to fit published sequence data (4, 29).
PCR was performed using 10 μl of iQ-Supermix (Bio-Rad), 7 μl of H2O, 1 μl of each primer (final concentration 0.5 μM each), and 1 μl of cDNA. An initial denaturation for 9 min at 95°C was followed by 40 cycles of amplification. Amplification conditions were as follows: 30 s of denaturation (94°C), 30 s of annealing, and 30 s of amplification (72°C). PCR products were resolved by electrophoresis on a 2.5% agarose gel.
Intracellular Ca2+ measurements
Ca2+ transients were measured in human alveolar macrophages loaded with the Ca2+ indicator fura-2/AM (Biotium) by using the digital fluorescence microscope unit Attofluor (Zeiss) (28). In brief, alveolar macrophages were incubated with 2 μM fura-2/AM for 30 min at 37°C in Hanks’ BSA solution. Cells were then rinsed twice and finally resuspended in Ca2+- and Mg2+-containing buffer (1.5 mM CaCl2 and 1.5 mM MgCl2) or in Ca2+-free, 0.5 mM EGTA-containing buffer. Traces were followed spectrofluorometrically, and Ca2+ transients were determined by multiple cell acquisitions with the 340/380 wavelength excitation ratio at an emission wavelength of 505 nm.
IL-1β, IL-10, IL-12p40, TNF-α, and IL-6 were measured by ELISA (R&D Systems) and performed according to the manufacturer’s recommendations. Samples were assayed in triplicate for each condition.
Unless otherwise stated, data are expressed as mean ± SEM. The statistical significance of differences between samples was calculated using the Mann-Whitney U test for unpaired data. Differences were considered significant if p < 0.05.
Human alveolar macrophages express mRNA for different P2 receptor subtypes
Expression of mRNA for different P2 receptor subtypes in human alveolar macrophages was analyzed by RT-PCR. Fig. 1,A shows that these cells express mRNA for the P2X1, P2X4, P2X5, and P2X7 subtypes, but not for P2X2 and P2X3 receptors. Furthermore, mRNA for the following P2Y subtypes: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y13, and P2Y14 were found (Fig. 1 B). However, no transcripts for P2Y12 receptors were detected in human alveolar macrophages. No products were obtained omitting reverse transcription (data not shown).
ATP stimulates Ca2+ transients in human alveolar macrophages
It has been reported that ATP induces Ca2+ changes in human and mouse monocytes and macrophages (34, 38). Therefore we evaluated the effects of ATP on intracellular Ca2+ signaling in human alveolar macrophages. Stimulation of human alveolar macrophages with ATP (100 μM) in Ca2+-containing medium induced a rapid and significant Ca2+ increase (Fig. 2,A, trace a) (control 0.82 ± 0.05 vs ATP 1.48 ± 0.07, ratios of intracellular Ca2+ were taken after 40 s p < 0.01, data are means ± SEM, n = 5). To discriminate between Ca2+ release from intracellular stores via activation of P2Y receptors and Ca2+ influx via P2X receptors, BAL macrophages were stimulated with ATP in Ca2+-free medium. As shown in Fig. 2,A, trace b, ATP still evoked a significant Ca2+ spike (Ca2+ ratios: control 0.78 ± 0.05 vs ATP 1.21 ± 0.06, p < 0.01, data are means ± SEM, n = 5), although with a lower amplitude, suggesting that Ca2+ increase was due to involvement of both P2Y and P2X receptors. To explore in a more detailed way responses mediated by P2Y subtypes, BAL macrophages were also stimulated with UTP, UDP, ADP, and UDP-glc (Fig. 2, B–E). Fig. 2,B shows that shape and amplitude of the Ca2+ peak triggered by UTP in the presence or absence of external Ca2+ are similar under both conditions, indicating that UTP-sensitive P2Y subtypes are expressed in BAL macrophages (Ca2+ ratio with/without external Ca2+, control 0.82 ± 0.03/0.80 ± 0.05 vs UTP 1.25 ± 0.05/1.21 ± 0.03; p < 0.01, data are means ± SEM, n = 5). In addition, dose-dependency experiments performed under Ca2+-free conditions showed that ATP and UTP were equipotent in increasing Ca2+, with a maximal response at a concentration of 100 μM (Fig. 2, A and B; data not shown).
To exclude participation of P2X subtypes in UDP, ADP, or UDP-glc-induced Ca2+ response, the following experiments were performed under Ca2+-free conditions. As shown in Fig. 2,C, UDP (at the optimal concentration of 100 μM) also induced an increase in intracellular Ca2+, although with a lower amplitude than UTP or ATP, suggesting that BAL macrophages express functional P2Y6 receptors (Ca2+ ratio: control 0.80 ± 0.04 vs UDP 1.17 ± 0.04; p < 0.01, data are means ± SEM, n = 5). To explore the role of P2Y1 and P2Y13 receptors in Ca2+ signaling, cells were stimulated with different concentrations of ADP (purified with hexokinase) in Ca2+-free conditions. As shown in Fig. 2,D, trace a, application of ADP induced a Ca2+ transient (Ca2+ ratio: control 0.80 ± 0.04 vs ADP 1.20 ± 0.04; p < 0.01, data are means ± SEM, n = 5), which was completely abrogated by preincubation of the cells with the P2Y1 antagonist MRS2179 (100 μM) (Fig. 2 D, trace b), indicating the involvement of the P2Y1 subtype in this response. Finally, in BAL macrophages from five of seven different donors, the P2Y14 agonist UDP-glc (100 μM) triggered a small Ca2+ spike, which was still present when cells were preincubated with apyrase (4 U/ml) to scavenge other contaminating nucleotides (4) (Ca2+ ratio: control 0.80 ± 0.04 vs UDP-glc 1.01 ± 0.05; p < 0.05, data are means ± SEM, n = 5). These data reveal that functional P2Y14 receptors are weakly expressed in some alveolar macrophages.
To evaluate the contribution of the different P2X subtypes, cells were stimulated in the presence or absence of extracellular Ca2+ with 2′3′-(4-benzoyl) benzoyl ATP (BzATP), which is active at the P2X1 and the P2X7 but also at the P2Y11 subtype. As shown in Fig. 2 F, BzATP was able to induce a Ca2+ peak only in the presence of extracellular Ca2+ (compare traces a and b), indicating that in BAL macrophages, BzATP only activated P2X subtypes (Ca2+ ratio in Ca2+-containing medium: control 0.81 ± 0.04 vs BzATP 1.21 ± 0.05; p < 0.01, data are means ± SEM, n = 5).
To investigate the role of the P2X7 subtype more directly, cells were also stimulated with ATP or BzATP in the presence of the selective P2X7 antagonist KN62. In brief, cells were incubated for 10 min with KN62 (25 nM) or vehicle in Ca2+-containing medium, before stimulation with nucleotides. As shown in Fig. 2,G, pretreatment of alveolar macrophages with KN62 only marginally affected the amplitude of ATP-induced Ca2+ increase (Ca2+ ratio with vs without KN62, ATP 1.39 ± 0.05 vs 1.24 ± 0.05; p < 0.05, data are means ± SEM, n = 5), while it completely abrogated that due to stimulation with BzATP (10 μM) (Fig. 2 H), indicating that P2X7 receptor was functional in human BAL macrophages.
Nucleotides enhance IL-1β secretion from LPS-primed human alveolar macrophages, via activation of the P2X7 subtype
It has been shown that ATP can modulate LPS-induced production of IL-1β by different cell types, including microglia, monocytes, monocyte-derived macrophages, and monocyte-derived dendritic cells (27). Therefore, we investigated whether extracellular ATP can also modulate LPS-induced IL-1β production in human alveolar macrophages. As shown in Fig. 3,A, ATP dose-dependently increased secretion of IL-1β. To elucidate the involvement of P2X and P2Y receptor subtypes, macrophages were also stimulated with increasing concentrations of ADP, UTP, UDP, and BzATP. As shown in Fig. 3,B, only BzATP enhanced LPS-primed IL-1β production in a dose-dependent manner, whereas the more P2Y-specific nucleotides UTP, UDP, ADP, and UDP-glc were ineffective. These results point to involvement of P2X subtypes in mediating this response. Indeed, it has been reported that the P2X7 subtype is responsible for ATP-induced IL-1β production in mouse macrophages (27). Therefore, we next investigated the effect of KN62 on ATP- and BzATP-mediated IL-1β secretion. Pretreatment of cells with KN62 completely abrogated ATP- and BzATP-induced IL-1β release (Fig. 3 C).
Extracellular nucleotides increase IL-6 secretion by human alveolar macrophages
IL-6 is a well-described proinflammatory cytokine involved in different lung diseases. As shown in Fig. 4, ATP, as well as UTP, dose-dependently increased IL-6 production in human LPS-primed alveolar macrophages. However, no significant changes in IL-6 secretion were observed after stimulation with UDP, UDP-glc, ADP, or BzATP, suggesting the involvement of the P2Y2R subtype in this cellular response.
Extracellular nucleotides inhibit LPS-induced IL-12p40 and TNF-α production
It has been previously demonstrated that secretion of IL-12p40 and TNF-α from human monocyte-derived dendritic cells is modulated by ATP (29). Therefore, we asked whether a similar response also occurs in human alveolar macrophages. As shown in Figs. 5 and 6, cells were stimulated with increasing concentrations of ATP, ADP, UTP, UDP, and BzATP. ATP (Figs. 5,A and 6,A), BzATP, and to a lesser extent ADP (Figs. 5,B and 6,B) reduced secretion of IL-12p40 and TNF-α from LPS-primed BAL macrophages in a dose-dependent manner. In contrast, application of UTP, UDP, and UDP-glc did not affect the release of IL-12p40 and TNF-α. Pretreatment with KN62 did not restore ATP-, ADP- or BzATP-induced inhibition of IL-12p40 or TNF-α secretion (data not shown). Thus, most likely ATP and BzATP suppress TNF-α and IL-12p40 secretion via the P2Y11 subtype. Though ADP has been reported to interact with the P2Y11 receptor as well (39), the effect of MRS2179 on ADP-mediated down-regulation point to an involvement of the P2Y1 subtype in mediating this response (Figs. 5,C and 6 C).
In the present study, we showed that human alveolar macrophages express mRNA for different P2 receptor subtypes, i.e., among P2X: P2X1, P2X4, P2X5, and P2X7, although except for P2Y12 all identified P2Y receptors i.e., P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y13, and P2Y14 were present in BAL macrophages. A similar pattern of expression has been described in mouse macrophages, human monocytes, and human monocyte-derived dendritic cells, although human monocyte-derived macrophages have been shown to express the mRNA for P2X1, P2X4, P2X5, P2X7, P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors (25, 40, 41, 42, 43).
Ca2+ experiments showed functionality of P2 receptors expressed by human alveolar macrophages. ATP elicited an increase in intracellular Ca2+ in these cells, which was higher in the presence than in the absence of extracellular Ca2+, showing: 1) functional expression of both P2Y and P2X subtypes; 2) that the extracellular ion plays a role in the ATP-induced Ca2+ signal. The ATP analogue BzATP, which has been shown to also activate the P2Y11 subtype, was only able to induce an intracellular Ca2+ increase in human alveolar macrophages in the presence of the external ion, indicating that its effect was due to stimulation of a receptor of the P2X subfamily, putatively P2X7. Further support to this conclusion was given by experiments performed with the selective P2X7 antagonist KN62 which completely abrogated BzATP-stimulated Ca2+ increase. In contrast, the ATP-triggered Ca2+ signal was only partly blocked by KN62, indicating that ATP was not only active at P2X7 receptors. These findings are in accordance with previous reports about P2X7 expression in rat alveolar macrophages (44, 45).
We also demonstrated that UTP a P2Y2 > P2Y4 ≫ P2Y6 agonist induced an intracellular Ca2+ response, which was comparable with that elicited by ATP in Ca2+-free medium, suggesting expression of P2Y2 receptors. This result is consistent with previous observations by our group on monocyte-derived dendritic cells, human eosinophils and, recently, human blood macrophages (28, 29, 34). In addition, elegant experiments performed in macrophages obtained from knockout mice for P2Y2, P2Y4 and P2Y2/P2Y4 receptors, showed the linkage between P2Y2 and ATP/UTP-induced Ca2+ mobilization (38).
We also observed that UDP, the most potent agonist at P2Y6 receptor, induced a Ca2+ transient, suggesting functionality of this subtype. Again, these results are in accordance with previous publication on human monocyte-derived dendritic cells and human blood macrophages (34, 46). In contrast, Bowler and colleagues did not detect P2Y6-mediated responses in rat alveolar macrophages (45). Furthermore, we found that the P2Y1/P2Y13 agonist ADP triggered intracellular Ca2+ changes most likely via activation of the Gq-coupled P2Y1 subtype, because the P2Y1 antagonist MRS2179 completely blocked this response. In addition, in five of seven experiments, activation of the P2Y14 subtype by UDP-glc was demonstrated. UDP-glc-induced Ca2+ increase was not abrogated by apyrase, indicating the functionality of this receptor subtype (4).
Alveolar macrophages, which are the most abundant cells in the lung, play a crucial role in the pathogenesis of chronic airway diseases, such as in chronic obstructive pulmonary disease or in bronchial asthma by releasing various proinflammatory cytokines (IL-1β, TNF-α, IL-12), chemokines, reactive oxygen metabolites, and tissue degrading enzymes (e.g., matrix metalloproteinases) that orchestrate chronic airway inflammation, tissue destruction, and contribute to the development of bronchial hyperresponsiveness (6, 7, 35, 37, 47, 48). Extracellular nucleotides have been shown to modulate cytokine/chemokine production by various cells (including dendritic cells, monocytes, and blood/peritoneal macrophages) via the activation of P2X and/or P2Y subtypes (32, 34, 46, 49).
In this study we showed that extracellular nucleotides potently modulated LPS-induced proinflammatory immune responses of human alveolar macrophages. ATP and the P2X7/P2Y11 agonist BzATP markedly enhanced LPS-induced production of IL-1β, whereas ADP, UTP, UDP, and UDP-glc had no significant effect. The effect of the P2X7-antagonist KN62 on ATP- and BzATP-mediated enhancement of IL-1β revealed the involvement of P2X7 receptors. In accord, several recent studies demonstrated that ATP and/or BzATP could enhance LPS-primed IL-1β secretion via activation of P2X7 receptor in human monocyte-derived dendritic cells, human blood monocytes/macrophages, and mouse macrophages and microglia cells (27, 50, 51, 52, 53). Increased IL-1β levels have been reported in the induced sputum (and BAL fluid) of COPD patient, as well as in the BAL fluid and serum of asthmatic individuals (54, 55). Additionally, it could be shown that induction of IL-1β production causes pulmonary inflammation, emphysema, and airway remodeling in mice (55). Furthermore, IL-1β has been found to be essential for allergen-specific Th2 cell activation and development of airway hypersensitivity response (56, 57). Our recent data demonstrating increased ATP levels in BAL fluids of asthmatic individuals allow the hypothesis that this could in turn induce secretion of IL-1β (33).
Furthermore, we showed that ATP strongly decreased LPS-induced IL-12p40 and TNF-α production, whereas IL-10 secretion was not significantly changed. KN62 had no effect on ATP-induced inhibition, pointing to an involvement of the P2Y subtype. Recent studies indicate that the inhibitory effect of ATP on IL-12 and/or TNF-α production in human monocyte-derived dendritic cells and blood monocytes is mediated by the P2Y11 subtype (58, 59, 60). Our findings confirm the role of P2Y11 in modulating TNF-α and IL-12p40 production. Whereas on the contrary, UTP, UDP, or UDP-glc had no effect, indicating that P2Y2, P2Y4, P2Y6, or P2Y14 receptors were not involved. Finally, the P2Y1 antagonist MRS2179 did not reduce the ability of ATP to inhibit LPS-primed production of TNF-α and IL-12p40. However, ADP was also able to reduce LPS-induced TNF-α and IL-12p40 secretion, pointing to a P2Y1- or P2Y13-mediated effect, which are both expressed by human alveolar macrophages. The observation that MRS2179 partly inhibited ADP-mediated down-regulation of TNF-α and IL-12p40 production suggests that P2Y1 was also involved. In keeping with these findings, we and others have previously reported that ADP can inhibit TNF-α, IL-12p40, IL-12p70, and MCP-1 secretion from LPS-primed human monocyte-derived dendritic cells (29, 58, 61). Furthermore, Hasko and colleagues demonstrated that ADP inhibits LPS-triggered IL-12p40 and TNF-α production in mouse peritoneal macrophages. However, in this study UTP and UDP were also able to suppress IL-12 and TNF-α production (62).
In this study ATP and UTP, but not ADP or UDP, were able to increase secretion of the cytokine IL-6. Because ATP and UTP equipotently up-regulated IL-6 secretion, the involvement of the P2Y2 receptor is likely. This is in accordance with previous studies showing up-regulation of IL-6 production by P2Y2 receptors in human airway epithelial cells (63). Additionally, P2Y1/P2Y2 knockout mice showed decreased IL-6 levels in BAL fluid following lung infection with Pseudomonas aeruginosa (64). IL-6 is associated with increased mucus production and airway remodeling (65, 66, 67). Therefore nucleotide-induced IL-6 secretion by alveolar macrophages might contribute to characteristic features of asthmatic airway inflammation.
Rather surprising are the findings that nucleotides on the one hand increase IL-1β and IL-6 release and on the other hand suppress TNF-α production by human alveolar macrophages, though these are well known as proinflammatory cytokines. However, this is in accordance with the observations that have been made in other cell types such as monocyte-derived dendritic cells (29, 68) or blood cells (69). Additionally, other mediators such as serotonin have been shown to increase IL-1β production and inhibit TNF-α secretion in different cell types as well (70, 71). ATP has been found to trigger and maintain asthmatic airway inflammation by activating dendritic cells, though TNF-α release of dendritic cells is suppressed by ATP (33). ATP is a potent chemoattractant for human eosinophils and a potent activator of the inflammasome (28, 72, 73). Furthermore ATP increases the capacity of dendritic cells to induce Th2 responses (74). Taken together there is good evidence to say that especially in Th2-dominated immune responses the effects of extracellular nucleotides are in summary proinflammatory.
In summary, this is the first study to show in detail the expression of different P2 receptor subtypes in human alveolar macrophages. Furthermore, we provided evidence that extracellular nucleotides via activation of selective P2 subtypes stimulate Ca2+ transients and modulate production and release of inflammatory mediators such as IL-1β, TNF-α, IL-12p40, and IL-6, thus contributing to the pathways leading to airway inflammation and asthma.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by a grant from the DFG (Deutsche Forschungsgesellschaft).
Abbreviations used in this paper: UDP-glc, UDP-glucose; BAL, bronchoalveolar lavage; BzATP, 2′3′-(4-benzoyl) benzoyl ATP.