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Originally published In Press as doi:10.1074/jbc.M505301200 on July 19, 2005

J. Biol. Chem., Vol. 280, Issue 37, 32459-32467, September 16, 2005
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"Host Tissue Damage" Signal ATP Promotes Non-directional Migration and Negatively Regulates Toll-like Receptor Signaling in Human Monocytes*{boxs}

Andreas Kaufmann{ddagger}, Boris Musset§, Sven H. Limberg§, Vijay Renigunta§, Rainer Sus§, Alexander H. Dalpke¶, Klaus M. Heeg¶, Bernard Robaye||, and Peter J. Hanley§1

From the {ddagger}Institute of Immunology, Marburg University, Robert-Koch-Strasse 17, 35037 Marburg, Germany, the §Institute of Physiology, Marburg University, Deutschhausstrasse 2, 35037 Marburg, Germany, Hygiene-Institut, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany, and ||Institute of Interdisciplinary Research, Institut de Biologie et de Médecine Moléculaires, Université Libre de Bruxelles, 6041 Gosselies, Belgium

Received for publication, May 13, 2005 , and in revised form, June 24, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The activation of Toll-like receptors (TLRs) by lipopolysaccharide or other ligands evokes a proinflammatory immune response, which is not only capable of clearing invading pathogens but can also inflict damage to host tissues. It is therefore important to prevent an overshoot of the TLR-induced response where necessary, and here we show that extracellular ATP is capable of doing this in human monocytes. Using reverse transcription-PCR, we showed that monocytes express P2Y1, P2Y2, P2Y4, P2Y11, and P2Y13 receptors, as well as several P2X receptors. To elucidate the function of these receptors, we first studied Ca2+ signaling in single cells. ATP or UTP induced a biphasic increase in cytosolic Ca2+, which corresponded to internal Ca2+ release followed by activation of store-operated Ca2+ entry. The evoked Ca2+ signals stimulated Ca2+-activated K+ channels, producing transient membrane hyperpolarization. In addition, ATP promoted cytoskeleton reorganization and cell migration; however, unlike chemoattractants, the migration was non-directional and further analysis showed that ATP did not activate Akt, essential for sensing gradients. When TLR2, TLR4, or TLR2/6 were stimulated with their respective ligands, ATP{gamma}S profoundly inhibited secretion of proinflammatory cytokines (tumor necrosis factor-{alpha} and monocyte chemoattractant protein-1) but increased the production of interleukin-10, an anti-inflammatory cytokine. In radioimmune assays, we found that ATP (or ATP{gamma}S) strongly increased cAMP levels, and, moreover, the TLR-response was inhibited by forskolin, whereas UTP neither increased cAMP nor inhibited the TLR-response. Thus, our data suggest that ATP promotes non-directional migration and, importantly, acts as a "host tissue damage" signal via the Gs protein-coupled P2Y11 receptor and increased cAMP to negatively regulate TLR signaling.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Innate immune cells express various surface membrane receptors, which enable them to respond to changes in their environment, such as invasion by pathogens. Among these receptors are the evolutionarily conserved Toll-like receptors (TLR2(s)), which recognize specific molecular patterns such as bacterial components. TLR4 recognizes lipopolysaccharide (LPS), a major structural component of the outer membrane of Gram-negative bacteria (1, 2). Stimulation of TLR4 by LPS, also known as endotoxin, can initiate a proinflammatory immune response, which serves to clear bacterial infection, but if the response is not sufficiently tamed it can also inflict damage to host tissues, culminating in life-threatening endotoxic shock (3). Accordingly, endotoxic shock can be simulated by exposing mice to LPS, whereas TLR4-/- (TLR4-deficient) mice are hyporesponsive to this ligand (4). TLR4 signaling is initiated when a complex of LPS and LPS-binding protein binds to the receptor CD14, which is strongly expressed in monocytes and also present in macrophages and dendritic cells. Alternatively, soluble CD14, a serum protein, can substitute for membrane-bound CD14 and initiate TLR4-signaling in cells lacking CD14, such as endothelial cells. The secreted protein MD-2, which binds to the extracellular domain of TLR4 is also essential for responsiveness to LPS, which is virtually absent in MD-2-/- mice (5, 6).

Although the molecular mechanisms by which LPS and other ligands stimulate TLRs leading to a specific pattern of gene expression are well understood (7), detailed information about its negative regulation is lacking. Stimulation of TLR4, as well as other TLRs, activates IRAKs (IL-1 receptor-associated kinases) via the adaptor protein MyD88 (myeloid differentiation primary-response protein 88) which leads to downstream activation of the IKK complex (inhibitor of I{kappa}B kinase complex), which consists of IKK-{alpha}, IKK-{beta}, and IKK-{gamma}. The upstream signaling has been confirmed with MyD88-/- and IRAK4-/- mice, which show little response to LPS (8, 9). The IKK complex phosphorylates the inhibitory protein I{kappa}B (inhibitor of (nuclear factor) {kappa}B), which leads to its subsequent degradation, allowing the release of NF-{kappa}B (nuclear factor {kappa}B), which translocates to the nucleus and promotes the expression of inflammatory cytokines. Following stimulation of TLRs in monocytes or macrophages, expression of IRAK-M increases, which has been shown to regulate negatively TLR signaling, as has up-regulation of single immunoglobulin IL-1 receptor-related molecule (SIGIRR) (10) and suppressor of cytokine signaling-1 (SOCS1) (11, 12). The latter, though, probably only dampens secondary (interferon-{beta}-dependent) pathways (13, 14). In any case, these negative regulators do not convey information about the state of host tissues.



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  		FIGURE 1.
Expression of P2Y and P2X receptors in monocytes and the effect of their activation on cAMP signaling and membrane potential. A and B, reverse transcription-PCR analyses performed using total RNA obtained from CD14+ monocytes. C, monocytes were treated with nucleotides or 30 µM forskolin (as indicated) for 15 min. cAMP was then quantified by radioimmune assay after acetylation. Data are means ± S.D. of triplicate measurements and are representative of two independent experiments. D, typical recording showing the effect of ATP (10 µM) application under whole-cell current-clamp conditions. Note that a transient depolarization precedes the hyperpolarization. E, application of 10 µM UTP under the same conditions. Note the absence of the transient depolarization. F, reverse transciption-PCR products obtained after using primers for the {beta}-subunits (KCNMB1–4) and {alpha}-subunit (KCNMA1) of BKCa channels (upper gel). The lower gel shows that IKCa (KCNN4) channels but not SKCa (KCNN1–3) channels were expressed.

 
Recent studies with human monocyte-derived dendritic cells suggest that extracellular ATP is capable of negatively regulating TLR-signaling via extracellular nucleotide receptors, which can be broadly divided into the P2Y and P2X receptor families. In dendritic cells, ATP has been shown to inhibit LPS-induced secretion of the proinflammatory cytokines TNF{alpha} and IL-12 (15–17), which raises the possibility that ATP may act, among other roles, as a "host tissue damage" signal for innate immune cells and inhibit TLR signaling in a negative feedback fashion. To test this possibility and to understand better the effect of extracellular ATP on innate immunity, we have investigated the effects of nucleotide receptor activation on the migration, second messenger signaling, and cytokine secretion of human monocytes in the absence and presence of various TLR ligands.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of Monocytes—Methods were performed essentially as recently described (18). In brief, human peripheral blood mononuclear cells were isolated from the blood of healthy volunteers by density centrifugation (Ficoll-Hypaque, 1.077 g/ml, Biochrom) and monocytes were enriched to a purity of >95% by counterflow centrifugation (19). Alternatively, monocytes of high purity (>95%) were isolated using anti-CD14 MicroBeads (Miltenyi Biotec, Germany) in a SuperMACS. Cells were plated in cell culture dishes containing Clicks/RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated fetal calf serum (Biochrom).

Analysis of Gene Expression—Total cellular RNA was prepared from CD14+ monocytes using a HighPure RNA extraction kit (Roche Diagnostics), which included DNase I digestion. After reverse transcription with a cDNA synthesis kit (MBI Fermentasm St. Leon-Rot, Germany), PCR analysis was performed using primers specific for P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, and P2Y13 and P2X1, P2X2, P2X3, P2X4, P2X5, and P2X7. The PCR primers have recently been published (18), except for P2Y13 which was TTGTTCTTCATCTCCCTGCCAAATA. Real time quantitative PCR was done using a Eurogentec qPCR kit, and measured values were normalized to the level of glyceraldehyde 3-phosphate dehydrogenase RNA expression.

Single Cell Ca2+ Imaging—A coverslip seeded with monocytes was sealed onto the bottom of a Perspex bath (volume, 100 µl) mounted on the stage of an inverted microscope (Nikon Diaphot 300). Cells were imaged via a x40 (1.4 numerical aperture) oil-immersion Nikon objective lens and superfused at 1 ml/min with solution containing 5% bovine serum albumin and 140 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 0.33 mM NaH2PO4, 5 mM HEPES, 1 mM CaCl2, 0.5 mM probenecid, and 10 mM glucose (pH 7.4). After a 15-min incubation with 10 µM fluo-3/AM (Molecular Probes), a single monocyte was selected and excited at 488 nm via a monochromator, and fluorescence was detected at 530 ± 15 nm. Only 1 cell/coverslip was used for experiments. The fluorescence signals were normalized with respect to the resting fluorescence intensity (F0) and expressed as F/F0.

Whole-cell Patch Clamp Measurements—Monocytes were superfused with solution containing 140 mM NaCl, 4.5 mM KCl, 1.2 mM NaH2PO4, 1.13 mM MgCl2, 1.6 mM CaCl2, 10 mM HEPES, 10 mM glucose, 2 mM Na-pyruvate, and 0.5% bovine serum albumin (pH 7.4) or with high K+ solution containing 140 mM KCl and 4.5 mM NaCl. The pipette solution contained 50 mM KCl, 65 mM K+ glutamate, 10 mM KH2PO4, 2 mM MgCl2, 1.9 mM K2ATP, 0.2 mM Na3-GTP, 0.1 mM EDTA, and 5 mM HEPES (pH 7.2). Data were acquired using an Axopatch 200B amplifier (Axon Instruments), an A/D converter (PCI-MIO 16-XR-10, National Instruments), and software (PC.DAQ1.1) developed in our laboratory. The sampling rate was 5 kHz (-3 dB at 1 kHz). All patch clamp and fluorescence measurements were performed at room temperature (~22 °C).



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  		FIGURE 2.
Ca2+ signaling induced by extracellular nucleotides. A, application of 10 µM ATP to a single monocyte induced a rapid increase in [Ca2+], which was followed by a slower secondary increase. UTP (10 µM) elicited a similar Ca2+ transient (inset). B, in Ca2+-free solution, an application of 10 µM ATP induced a single monotonic Ca2+ transient, whereas a "secondary increase" followed the introduction of Ca2+ to the external solution. The latter signal is consistent with Ca2+ influx via Ca2+ release-activated Ca2+ channels (store-operated Ca2+ entry), and the inset shows that it could be reproduced by treating the cells with thapsigargin (500 nM) and intermittently switching to Ca2+-free solution (indicated by solid bars). C, plot of ATP versus peak intracellular Ca2+. D, UTP was equally effective as ATP at mobilizing Ca2+ from internal stores.

 
Chemotaxis Assay—Monocyte migration was assayed in a 48-well microchemotaxis chamber (Neuro Probe Inc, Bethesda, MD) using polyvinylpyrrolidone-free polycarbonate filters with 5-µm diameter pores (20). The lower chamber was filled with standard RPMI 1640 medium, with or without (negative control) stimulus, and the upper chamber (50-µl volume) was filled with cell suspension (2 x 106 cells/ml). In parallel, we performed checkerboard analyses (used to distinguish chemotaxis from chemokinesis). The chemoattractant peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) was used as a positive control. After a 1-h incubation at 37 °C in air containing 5% CO2, cells were fixed with methanol and stained with hematoxylin (Sigma). A software-controlled imaging system (Vilber Lourmat; distributed by Fröbel, Wasserburg, Germany) was used to quantify the number of migrated monocytes per well. Results are expressed as specific chemotaxis, the number of stimulus-induced migrated cells after subtracting unspecific migration (negative control).

Time-lapse Microscopy—Images were acquired using a combination of differential interference contrast and time-lapse microscopy using an Olympus IX71 microscope equipped with a software-controlled (Image-Pro Plus 4.5, Media Cybernetics, Silver Spring, MD) Sensi-CamQE CCD camera (Chromaphor, Duisburg, Germany). Monocytes were plated on collagen type I-coated coverslips and imaged using a x60 (1.4 numerical aperture) Olympus objective lens and immersion oil.

Western Blot Analysis—Monocytes (5 x 106/well) were incubated for 20 h in 6-well tissue culture plates and then either left untreated or stimulated with 100 µM ATP, 100 µM ATP{gamma}S, or 0.1 µM fMLP. After 3 min, cells were lysed using a protein extraction reagent (M-PER, Pierce Biotechnology), supplemented with a protease inhibitor mixture (Complete, Roche Diagnostics). Phosphorylation of Akt was then assayed by Western blot as recently described (21). Equal loading was controlled by detecting total Akt.

cAMP Measurements—Monocytes were preincubated for 30 min in RPMI 1640/HEPES buffer containing 25 µM rolipram for 30 min and then treated with a nucleotide or forskolin in the same medium for 15 min. Activity was stopped by adding 0.1 M HCl, and cAMP was quantified by radioimmune assays after acetylation.

Cytokine Measurements—Secretion of cytokines into the culture supernatants was determined by sandwich enzyme-linked immunosorbent assay, as described previously (19, 20). In brief, 96-well microtiter plates (Maxisorp, Nunc, Wiesbaden, Germany) were coated with cytokine-specific monoclonal antibodies specific for TNF{alpha}, MCP-1, or IL-10 (Pharmingen). Plates were blocked with 2% bovine serum albumin in phosphate-buffered saline. Aliquots of culture supernatants (100 µl/well) were incubated at room temperature for 1 h, and then a specific biotinylated secondary antibody (Pharmingen) and a streptavidin-peroxidase complex was added. Conversion of the substrate ortho-phenylenediamine dihydrochloride was measured using a Dynatech MR7000 microplate reader. Sensitivities of the enzyme-linked immunosorbent assays were <3 pg/ml (IL-10), <20 pg/ml (MCP-1), and <100 pg/ml (TNF{alpha}).

Infection with Influenza A Virus—Influenza A/PR/8 (H1N1) virus was propagated and purified as previously described (21, 22). Viral titers were determined by plaque assay, and monocytes were infected with H1N1 virus at a multiplicity of infection of 2.

Cell Viability Assay—Cell viability was assessed using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After 16 h of incubation under control or treatment conditions, cells were washed twice with phosphate-buffered saline and further incubated for 2 h with 0.5 mg/ml MTT. After cell lysis and solubility of crystals with isopropanol, the reduction of MTT was determined by measuring absorption at 570 nm using a microplate reader. Cell viability, assessed by MTT reduction, was expressed as percent control.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
At least seven human P2Y receptors have been identified and characterized: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, and P2Y13. We found that monocytes express P2Y2 and P2Y11, similar to macrophages (18), as well as the recently described Gi-coupled P2Y13 receptor (23) (Fig. 1A). Weak signals for P2Y1 and P2Y4 were also detected. Furthermore, monocytes were found to express the ligand-gated ion channels P2X1, P2X4, and P2X7 (Fig. 1B). Most of the P2Y receptor subtypes are coupled to phospholipase C (PLC) and Ca2+ signaling; however, the P2Y11 receptor is additionally coupled to adenylate cyclase by the stimulatory G protein Gs (24), whereas the P2Y13 receptor is negatively coupled to this enzyme (23, 25). Because extracellular ATP can be hydrolyzed to ADP by ectonucleoside triphosphate diphosphohydrolase-1 (CD39), the major ectonucleotidase of monocytes, we tested whether or not ATP (10 and 100 µM) caused a net increase in cAMP levels. Indeed, ATP and its non-hydrolyzable analogue ATP{gamma}S both strongly increased cAMP in monocytes (Fig. 1C) suggesting that the P2Y11/Gs/adenylate cyclase pathway was functionally dominant in these cells.

In whole-cell current-clamp recordings, application of 10 µM ATP consistently induced a transient depolarization (n = 13), which was followed by hyperpolarization (Fig. 1D). The initial depolarization was probably due to activation of P2X receptors, because in 5 of 7 cells it was absent when UTP was substituted for ATP (Fig. 1E). The secondary hyperpolarization was almost certainly because of the opening of Ca2+-activated K+ (KCa) channels, which we have recently characterized in detail (18). Indeed, we found that monocytes express SK-4 (KCNN4) and BKCa (KCNMA1) channels (Fig. 1F), similar to macrophages.

We have also recently shown that ATP induces Ca2+ oscillations in human macrophages, which may regulate various enzymes and gene expression (18). Surprisingly, application of ATP to single human monocytes did not induce such an oscillatory release of Ca2+, but typically we observed a rapid Ca2+ transient consisting of 1–3 spikes followed by a slower secondary increase (Fig. 2A). The slower secondary increase, also observed with UTP (Fig. 2A, inset), was probably due to store-operated Ca2+ entry, because it was absent in Ca2+-free solution, and it could be seen when Ca2+ was subsequently introduced (Fig. 2B). Ca2+ influx consistent with store-operated Ca2+ entry could also be repeatedly observed when the sarcoendoplasmic reticular Ca2+-ATPase was blocked with thapsigargin (500 nM) and Ca2+ was intermittently introduced (Fig. 2B, inset). Fig. 2, C and D show that ATP and UTP equipotently increase Ca2+ with half-maximal response at ~0.1 µM. Using digital time-lapse microscopy, we observed that the application of either ATP (n = 10) or UTP (n = 4), within minutes, caused a strong retraction of pseudopodia (single frames shown in Fig. 3, A and B; see also time-lapse movie in supplemental data), even in the absence of extracellular Ca2+. This observation suggested that ATP may act as a chemoattractant.



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  		FIGURE 3.
ATP induces pseudopodia retraction and migration without phosphorylating Akt. A, differential interference contrast image of a monocyte before application of ATP (x-y dimensions of frame, 18 x 19 µm). B, the same monocyte 2–3 min after application of 10 µM ATP in Ca2+-free solution at 37 °C. Arrows have been added to indicate morphological changes (see also the supplemental movie). C, migration assays were performed in modified Boyden chambers with various concentrations of ATP in the lower well as indicated. fMLP was used as a positive control. D, Western blot analysis was performed using antibodies specific for total Akt or phosphorylated Akt (P-Akt).

 
We found in chemotaxis assays that ATP promoted cell migration in the concentration range 10–100 µM, whereas higher concentrations were inhibitory (Fig. 3C). In parallel, we performed checkerboard analyses (not shown), which revealed that ATP did not induce directional migration. That is, the stimulatory effect of ATP on migration was still observed when it was added at equal concentrations to both sides of the chamber. Although both P2Y and fMLP receptors are coupled via Gq to PLC, which hydrolyzes phosphatidylinositol 4,5-bisphosphate, yielding diacylglycerol and inositol 1,4,5-trisphosphate, this signaling pathway alone is not sufficient to induce migration along chemotactic gradients. The fMLP receptor is additionally coupled to phosphoinositide 3-kinase (PI3K), which through the generation of phosphatidylinositol 3,4,5-trisphosphate activates Akt (also known as protein kinase B), the activity of which is necessary for cell navigation (26–29). For example, in studies using PI3K{gamma}-/- mice, directional movement of neutrophils in fMLP gradients is severely impaired without affecting random movement (29). To test the hypothesis that ATP differs from fMLP in that it does not activate the PI3K-Akt pathway we performed an immunoblot analysis. Stimulation of monocytes with either ATP or ATP{gamma}S did not produce phosphorylation of Akt, whereas fMLP produced a strong "positive control" response (Fig. 3D).

We next investigated the effects of extracellular nucleotides on TLR-signaling. Monocytes were stimulated with 10 ng/ml LPS in the presence or absence of various concentrations of ATP{gamma}S or UTP for 16 h. ATP{gamma}S or UTP alone had no significant effect on TNF{alpha} production (Fig. 4A). However, ATP{gamma}S, but not UTP, inhibited LPS-induced production of TNF{alpha} in a dose-dependent fashion. These data indicate that ATP{gamma}S can potently regulate TLR signaling, and the lack of effect of UTP suggests that the neither P2Y2 nor P2Y4 receptors, where UTP is a full agonist (30), are involved. The P2Y1 and P2Y13 receptors are also unlikely to be involved because both of these receptors are activated preferentially by diphosphate adenine nucleotides. Moreover, the P2Y1 blocker MRS2179 (100 µM) did not reduce the ability of ATP{gamma}S to inhibit LPS-induced production of TNF{alpha} (Fig. 4B). It could be argued that hydrolysis-resistant ATP{gamma}S is slowly degraded to adenosine, which could confound the interpretation of data. However, the activity of CD73 (ecto-5'-nucleotidase), which catalyzes the hydrolysis of AMP to adenosine, is weak in monocytes (31) and, moreover, we found that TLR-signaling was similarly inhibited when experiments were repeated in the presence of 100 µM 8-(p-sulfophenyl)theophylline (8-p-SPT), an adenosine receptor blocker (not shown). Thus, P2Y11 is most likely the receptor through which ATP inhibits TLR signaling. Consistent with this conclusion, the potent P2Y11 (and P2X7) receptor agonist BzATP, as well as the weaker agonist ADP{beta}S (32), inhibited TLR signaling (Fig. 4B).



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  		FIGURE 4.
Various P2Y receptor agonists and forskolin, but not UTP, suppress LPS-induced TNF{alpha} production by monocytes. A, monocytes were treated with various concentrations of ATP{gamma}S or UTP either in the absence or presence of LPS stimulation as indicated. After 16 h, concentrations of TNF{alpha} in the culture supernatants were measured in duplicate by enzyme-linked immunosorbent assay. B, ATP{gamma}S (and ADP{beta}S) decreased TNF{alpha} production by LPS-stimulated monocytes in the presence or absence of the P2Y1 receptor blocker MRS2179. BzATP potently decreased LPS signaling. C, forskolin (100 µM) suppressed LPS-induced TNF{alpha} production, albeit less effectively than 10 or 100 µM ATP{gamma}S. D, the PKA blocker H89 (10 µM) did not abolish the inhibitory effect of ATP{gamma}S on LPS-induced TNF{alpha} production. E, quantitative PCR analysis. All of the above data are mean ± S.D., and values are representative of three independent experiments.

 



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  		FIGURE 5.
P2Y receptor agonists suppress LPS-induced MCP-1 production but augment IL-10 production. A, ATP{gamma}S alone had no effect on MCP-1 production, whereas ATP{gamma}S and, to a lesser extent, 100 µM forskolin inhibited LPS-induced production of MCP-1. B, the inhibitory effect of ATP{gamma}S on LPS-induced MCP-1 production was not reversed by either 100 µM 8-p-SPT or 10 µM H89. Like ATP{gamma}S, BzATP also inhibited LPS-induced MCP-1 production. C, ATP dose dependently synergized with LPS to increase IL-10 production. D, ADP{beta}S, in the absence or presence of MRS2179 (P2Y1 receptor blocker), also augmented IL-10 production induced by LPS. Finally, 100 µM 8-p-SPT (adenosine receptor blocker) did not block the synergistic effect of ATP{gamma}S on LPS-induced IL-10 release. In all cases, data are means ± S.D. of duplicate or triplicate determinations and are representative of at least three independent experiments.

 
Because the P2Y11 receptor is coupled to adenylate cyclase, we speculated that an increase in cAMP may inhibit the TLR signaling pathway. In support of this notion, direct activation of adenylate cyclase with 100 µM forskolin inhibited LPS-induced TNF{alpha} production but less effectively than ATP{gamma}S (Fig. 4C). When cells were pretreated with the PKA blocker H89 (10 µM), the inhibitory effect of ATP{gamma}S on LPS-induced TNF{alpha} production was still observed (Fig. 4D). Hence, based on this observation, cAMP probably inhibits the activation and/or action of NF-{kappa}B independent of PKA. To test whether ATP inhibits LPS-induced TNF{alpha} production at the pretranslational level we performed quantitative real time PCR. Fig. 4E shows that LPS-induced expression of TNF{alpha} mRNA was significantly inhibited by ATP{gamma}S.

ATP{gamma}S, but not UTP, also inhibited LPS-induced production of MCP-1 (Fig. 5A), which could be mimicked with forskolin. As was the case for TNF{alpha}, the inhibition of LPS-induced MCP-1 production by ATP{gamma}S was not blocked by either 8-p-SPT or H89 (Fig. 5B). Furthermore, BzATP was equally effective as ATP{gamma}S (Fig. 5B). Interestingly, ATP{gamma}S and ADP{beta}S did not inhibit LPS-induced production of IL-10, but rather, these agonists synergized with LPS to increase secretion of this anti-inflammatory cytokine (Fig. 5, C and D). This synergism was not blocked by either MRS2179 or 8-p-SPT (Fig. 5D).

Together, the above studies with the ligand LPS suggest that the P2Y receptor agonists such as ATP{gamma}S inhibit TLR4 signaling. We extended these studies and tested whether ATP{gamma}S can inhibit the proinflammatory response mediated by other TLR receptors. Indeed, the production of TNF{alpha} by monocytes stimulated with 100 pg/ml macrophage-activating lipopeptide-2 (MALP-2), a TLR2/6 ligand (1), or 1000 ng/ml lipoteichoic acid (LTA), a TLR2 ligand (33), was inhibited by ATP{gamma}S (Fig. 6, A and B). MCP-1 production induced by either MALP-2 (Fig. 6C) or LTA (Fig. 6D) was also inhibited by ATP{gamma}S. As was the case with LPS, ATP{gamma}S synergized with MALP-2 (Fig. 6E) or LTA (Fig. 6F) to increase IL-10 production.

The negative effect of ATP{gamma}S on inflammatory cytokine production induced by stimulating TLR2, TLR4, or TLR2/6 cannot be explained by global depression of cell function because IL-10 was increased in all cases. Moreover, ATP{gamma}S did not inhibit, but rather potentiated, MCP-1 production in monocytes stimulated by 10 units of interferon-{gamma}, which is not a TLR ligand (Fig. 7A). Similarly, ATP{gamma}S did not inhibit the MCP-1 response of cells infected with influenza A (H1N1) virus (Fig. 7B), which incidentally, as expected, decreased cell viability (Fig. 7C). Note that TLR3 recognizes viral double-stranded RNA and both TLR7 and TLR8 sense single-stranded RNA, and TLR3, TLR7, and TLR8 have all been implicated in the detection of influenza virus (34–36).



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  		FIGURE 6.
ATP{gamma}S is a negative regulator of proinflammatory TLR2/6 and TLR2 signaling. A, the TLR2/6 ligand MALP-2 (100 pg/ml) induced TNF{alpha} secretion by monocytes. This effect was inhibited by ATP{gamma}S. B, ATP{gamma}S also strongly inhibited TNF{alpha} production induced by the TLR2 ligand LTA (1000 ng/ml). C, inhibition of MALP-2-induced MCP-1 production by ATP{gamma}S. D, inhibition of LTA-induced MCP-1 production by ATP{gamma}S. E, ATP{gamma}S synergized with MALP-2 to increase secretion of the anti-inflammatory cytokine IL-10. F, IL-10 production stimulated by LTA was also augmented by ATP{gamma}S. Data are means ± S.D. of duplicate determinations and are representative of at least two independent experiments.

 
At high concentrations (>0.5 mM) ATP (or ATP{gamma}S) can induce cell apoptosis via P2X7 receptor activation (37, 38). However, at the concentrations used in this study (≤0.1 mM), ATP{gamma}S did not decrease cell viability (Fig. 7D). The apparent strong positive effect of LPS on cell viability (Fig. 7D), assessed by MTT reduction (an index of mitochondrial respiration), implies that TLR signaling increases cellular energy demand.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have shown that ATP promotes non-directional migration and acts as an extracellular regulator of TLR signaling in peripheral human monocytes. In chemotaxis assays we found that fMLP and ATP promoted migration, although checkerboard analyses revealed that monocytes could not sense ATP concentration gradients. The surface receptors for fMLP and ATP are each coupled to PLC, but recent studies with knock-out mice have suggested that the PLC pathway is not sufficient for chemotaxis (27), the recognition and response to chemoattractant gradients. Instead, work with neutrophils and peritoneal macrophages isolated from PI3K-/- mice have indicated that PI3K is essential for chemotaxis (26–28), probably by promoting phosphatidylinositol 3,4,5-trisphosphate accumulation and localization of Akt at a new leading edge (39). Consistent with this picture, we found by Western blot analysis that fMLP, but neither ATP nor ATP{gamma}S, increased Akt phosphorylation.

We also observed by means of time-lapse differential interference contrast microscopy that monocytes, plated on a collagen matrix, retracted their pseudopodia when ATP was applied, even in Ca2+-free solution. Hence, stimulation of P2Y receptors, and activation of PLC or other pathways, promotes release of contacts and rapid actin reorganization, a prerequisite for migration. We also found that the pattern of Ca2+ signaling induced by ATP in human monocytes was distinctly different to that recently observed in human monocyte-derived macrophages (18). In contrast to macrophages, ATP did not evoke prolonged Ca2+ oscillations in monocytes. Furthermore, the initial Ca2+ transient, which was independent of external [Ca2+], was followed by a second slower Ca2+ increase, which decayed slowly. This slower secondary Ca2+ transient was abolished in Ca2+-free solution, but it could be evoked by reintroduction of Ca2+, suggesting that it reflected the opening of Ca2+ release-activated Ca2+ channels rather than P2X channel activity. Although we detected the expression of several P2X receptors, our patch clamp recordings showed only a transient depolarization when ATP was applied. Because P2X1 desensitizes rapidly within 1–2 s (40), and P2X7 receptors are half-maximally activated by >300 µM ATP in the presence of physiological Ca2+ and Mg2+ concentrations (41, 42), we infer that P2X4 receptors were mainly responsible for this transient depolarization.



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  		FIGURE 7.
ATP{gamma}S does not inhibit MCP-1 production stimulated by interferon-{gamma} or H1N1 virus infection, and it does not decrease cell viability. A, interferon-{gamma} (10 units) stimulated MCP-1 production, which was not inhibited but rather augmented by ATP{gamma}S. B, MCP-1 production induced by H1N1 virus infection was not inhibited by ATP{gamma}S. C, H1N1 virus infection impaired cell viability. D, ATP{gamma}S and LPS did not decrease cell viability, as assessed by the MTT assay. Data are means ± S.D. of duplicate determinations and are representative of at least two independent experiments.

 
In addition to promoting migration, extracellular ATP potently inhibits the TLR-induced proinflammatory immune response of monocytes. Low micromolar concentrations of ATP{gamma}S markedly decreased LPS-induced production of TNF{alpha} and MCP-1, both of which are proinflammatory, whereas IL-10 secretion was augmented. In accord, several recent studies with DCs have shown that ATP can inhibit LPS-induced secretion of inflammatory cytokines (15–17). Aside from negatively regulating TLR4-signaling, we found using other TLR ligands that ATP{gamma}S also inhibited TLR2 and TLR2/6 signaling but not the cytokine response to viral infection, the nucleic acids of which are recognized by TLR3, TLR7, and TLR8. Unlike these receptors, TLR2, TLR4, and TLR6 use the adaptor molecule MyD88 to activate an IRAK4-dependent pathway, leading to release of NF-{kappa}B and increased production of inflammatory cytokines (7, 36). At present, we do not know the exact mechanism by which nucleotides inhibit this inflammatory signal pathway.

On one hand, we found that UTP had no effect on TLR signaling, implying that P2Y2 and P2Y4 receptors, and the PLC pathway, are probably not important for the negative regulation. On the other hand, the adenylate cyclase activator forskolin mimicked the inhibitory action of ATP on the LPS-induced proinflammatory response suggesting that cAMP may be a key regulator of TLR signaling. Consistent with this notion, we found that ATP and ATP{gamma}S increased cAMP levels, presumably via the activation of the unique P2Y11 receptor, unique in the sense that it is positively coupled to both adenylate cyclase and PLC. In concordance, activation of {beta}-receptors (which are also coupled to adenylate cyclase by Gs) and caffeine (a phosphodiesterase inhibitor) have been shown to increase cAMP concentrations and to inhibit LPS-induced TNF{alpha} production by innate immune cells (43–45). Moreover, selective activation of adenosine A2A receptors increases cAMP and exerts an anti-inflammatory action in various immune cells (46, 47). In human THP-1 monocytes, application of the membrane-permeable analogue dibutyryl-cAMP has been reported to inhibit LPS-induced expression of TNF{alpha} mRNA, without affecting translocation of NF-{kappa}B to the nucleus. Rather, further work with human umbilical vein endothelial cells, suggested that increased cAMP induced by forskolin inhibited NF-{kappa}B-mediated transcriptional activity (48). Hence, cAMP may target the ultimate step of the MyD88-dependent TLR signaling cascade.



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  		FIGURE 8.
Proposed model in which ATP acts via P2Y11 receptors and increased cAMP to inhibit the proinflammatory immune response evoked by TLR activation, thereby preventing excessive host tissue damage.

 
We cannot rule out a role for P2X receptor activation in modulating TLR signaling. Recent data obtained with murine RAW 264.7 macrophages suggest that P2X7 receptor activation may in fact enhance LPS-induced inflammatory cytokine production. Hu et al. (37) demonstrated that LPS-induced stimulation of NF-{kappa}B activity was reduced in cells pretreated with the P2X7 antagonist periodate-oxidized ATP. Moreover, NF-{kappa}B activity was increased more when macrophages were treated with both LPS and a P2X7 receptor agonist (BzATP) than with LPS alone (49). Hence, both P2Y and P2X receptors may regulate the innate immune response to LPS (50). One possibility is that ATP inhibits TLR-signaling at low concentrations (< 100 µM) via P2Y receptors, whereas at much higher concentrations it activates P2X7 receptors to boost inflammatory cytokine production. However, the regulation of innate immune responses by nucleotides may differ considerably between humans and mice because, for example, the mouse genome lacks P2Y11 receptors, and there is no known equivalent Gs-coupled P2Y receptor in this species.

In conclusion, our data suggest a model (shown in Fig. 8) whereby ATP acts as a host tissue damage signal for innate immune cells. That is, when the TLR-induced proinflammatory response to bacterial infection is excessively strong such that it damages host tissues, ATP is released into the extracellular space and negatively regulates the response by binding to P2Y11 receptors and increasing cytosolic cAMP, which either directly or indirectly, via cAMP-dependent PKA or possibly Epac (exchange protein directly activated by cAMP) (51), inhibits the production of inflammatory cytokines and concomitantly increases the expression of the anti-inflammatory cytokine IL-10. We cannot rule out a role for P2X receptors in this scheme, but at this stage there is no obvious mechanistic link. Independent of TLR signaling, ATP can act via P2Y receptors, which are coupled to PLC but not PI3K, to promote non-directional migration.


   FOOTNOTES
 
* This project was supported by a grant from the Kempkes-Stiftung (to P. J. H.). 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. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains a supplemental movie. Back

1 To whom correspondence should be addressed. Tel.: 49-6421-286-6546; Fax: 49-6421-286-8960; E-mail: hanley{at}mailer.uni-marburg.de.

2 The abbreviations used are: TLR, Toll-like receptor; ATP{gamma}S, adenosine 5'-O-(thiotriphosphate); LPS, lipopolysaccharide; IRAK, IL-1 receptor-associated kinase; IKK, inhibitor of I{kappa}B kinase; TNF, tumor necrosis factor; fMLP, N-formyl-methionyl-leucyl-phenylalanine; MTT, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide; PLC, phospholipase C; PI3K, phosphoinositide 3-kinase; 8-p-SPT, 8-(p-sulfophenyl)theophylline; BzATP, 3'-O-(4-benzoylbenzoyl) ATP; MALP-2, macrophage-activating lipopeptide-2; LTA, lipoteichoic acid. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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