Extracellular ATP mediates numerous biological activities by interacting with plasma membrane P2 purinergic receptors. Recently, P2 receptors have been described on dendritic cells (DC), but their functional role remains unclear. Proposed functions include improved Ag presentation, cytokine production, chemotaxis, and induction of apoptosis. We investigated the effects of ATP and of other P2 receptor agonists on endocytosis, phenotype, IL-12 secretion, and T cell stimulatory capacity of human monocyte-derived DC. We found that in the presence of extracellular ATP, DC transiently increase their endocytotic activity. Subsequently, DC up-regulate CD86, CD54, and MHC-II; secrete IL-12; and exhibit an improved stimulatory capacity for allogeneic T cells. These effects were more pronounced when chemically modified ATP derivatives with agonistic activity on P2 receptors, which are resistent to degradation by ectonucleotidases, were applied. Furthermore, ATP and TNF-α synergized in the activation of DC. Stimulated with a combination of ATP and TNF-α, DC expressed the maturation marker CD83, secreted large amounts of IL-12, and were potent stimulators of T cells. In the presence of the P2 receptor antagonist suramin, the effects of ATP were completely abolished. Our results suggest that extracellular ATP may play an important immunomodulatory role by activating DC and by skewing the immune reaction toward a Th1 response through the induction of IL-12 secretion.

Dendritic cells (DC)3 initiate primary T cell-mediated immune responses by uptake and processing of Ag in peripheral tissues, followed by migration to the T cell-rich areas of lymphatic tissues, where they sensitize T cells (1). They interact with T cells via costimulatory molecules, adhesion molecules, MHC, as well as cytokines and chemokines. Inflammatory signals such as LPS, TNF-α, IL-1β, or IL-6 induce an activation process, referred to as maturation, upon which DC loose their phagocytic activity, up-regulate costimulatory and adhesion molecules, present Ags on MHC more effectively, and produce a number of cytokines, including TNF-α and IL-12 (2). These changes in phenotype and function lead to the capability of DC to potently activate T cells and to induce Ag-specific immune responses. However, the physiological signals for DC activation are still not fully understood.

The role of the intracellular adenine nucleotide ATP regarding energy metabolism in biological systems has been described by Lipmann and Kalckas as early as 1941 (3). Since then it has been recognized that nucleotides also represent a ubiquitous class of signaling molecules in many tissues, including the immune system (4). ATP is stored in the cytosol of most cells in a concentration of 5–10 mM and can be found in the 100 mM range in intracellular compartments. Due to its size and high density of charge, ATP cannot permeate membranes. However, release of ATP has been observed from a variety of cells, including tumor cells and lymphocytes, as well as from virtually all tissues under conditions of hypoxia, ischemia, inflammation, and cell necrosis (5, 6, 7, 8). The sources of released ATP include exocytotic granula from secretory cells, release from lysed cells, and nonlytic release from cytoplasmatic stores. Extracellular ATP mediates biological responses by activating P2 receptors (9). Two groups of P2 receptors have been characterized: P2X receptors, which are ligand-gated cation channels (10), and P2Y receptors coupled to G proteins (11). In the immune system, ATP triggers mainly proinflammatory reactions, such as the release of IL-1β, exocytosis of granula containing superoxide and reactive oxygen species, phagocytosis, giant cell formation, chemotaxis, cytolysis, and cell adhesion to endothelium (4, 12). Recently, it has been shown that DC express P2 receptors, but their physiological role is unclear. Proposed functions include improved Ag presentation (13), cytokine production (14), chemotaxis (15), and induction of apoptosis (16).

Based on the hypothesis, that ATP released at sites of cell stress, damage, or necrosis signals danger to the immune system, we investigated the effects of extracellular ATP on endocytosis, phenotype, IL-12 secretion, and T cell stimulatory capacity of human monocyte-derived DC.

All cell cultures were maintained in RPMI 1640 medium (Biochrom, Berlin, Germany) supplemented with 2% human pooled AB serum (BioWhittaker, Walkersville, MD), 2 mM l-glutamine (Life Technologies, Paisley, U.K.), 50 U/ml penicillin, and 50 μg/ml streptomycin (both from Sigma, Munich, Germany), hereafter referred to as complete medium.

Human rGM-CSF (Leucomax, sp. act., 1.11 × 107 U/mg) was purchased from Novartis (Basel, Switzerland), and IL-4 (sp. act., 1 × 107 U/mg) from Promega (Madison, WI). TNF-α (sp. act., 1.1 × 108 U/mg) was obtained from R&D Systems (Wiesbaden, Germany). ATP, α,β-methylene-ATP (αβmeATP), β,γ-methylene-ATP (βγmATP), adenosine-5′-O-(3-thio)triphosphate (ATPγS), P1,P5-di(adenosine-5′)pentaphosphate (AP5A), UTP, benzoylbenzoyl-ATP, 2-methylthio-ATP (2 MeSATP), ATP-2′,3′-dialdehyde (oxyATP), adenosine, PGE2, FITC-dextran (Mr = 40,000), and suramin were purchased from Sigma-Aldrich (Steinheim, Germany). All reagents were tested for endotoxin contamination with the sensitive Limulus amebocyte lysate assay (LAL assay; BioWhittaker), according to the manufacturer’s instructions, and were found to be negative (endotoxin content in stock solutions <1 pg/ml).

DC were generated from PBMC, as described elsewhere, with minor modifications (17). In brief, PBMC were isolated from healthy donors by standard density-gradient centrifugation on Ficoll separating solution (Biochrom), washed three times, and resuspended in complete medium. PBMC (5 × 106/ml) were allowed to adhere in 75-cm2 culture flasks for 60 min. Nonadherent cells were removed by pipetting. After an overnight incubation, the initially adherent cells were transferred into six-well plates (1.5 × 106 cells/2 ml) in fresh complete medium supplemented with 1000 U/ml GM-CSF and 500 U/ml IL-4. After 6 days of culture, DC were incubated with ATP or other P2R agonists in the presence or absence of TNF-α (1000 U/ml) for 48 h.

Mouse anti-human mAbs and the appropriate isotype controls were all obtained from PharMingen (San Diego, CA): TÜ36 (IgG2b, anti-HLA-DR, PE conjugated), BB1 (IgM, anti-CD80, PE conjugated), 2331/FUN-1 (IgG1, anti-CD86, PE conjugated), HB15e (IgG1, anti-CD83, FITC conjugated), HA58 (IgG1, anti-CD54, PE conjugated), G46-2.6 (IgG1, anti-HLA-ABC, FITC conjugated), and M5E2 (IgG2a, anti-CD14, FITC conjugated). For FACS analysis, 105 DC suspended in 100 μl PBS were incubated with 10 μl of the fluorochrome-labeled mAbs for 20 min on ice. After the staining procedure, the samples were washed once in PBS and measured immediately (FACS-Calibur; Becton Dickinson, Heidelberg, Germany). Data were analyzed using FlowJo software (version 2.7.8).

On day 6, DC were incubated for 48 h with various stimuli, and supernatants were collected for IL-12 measurements with a commercial ELISA kit (Endogen, Woburn, MA) that detects both IL-12 p40 and the bioactive p70 heterodimer.

On day 6, DC were incubated in the absence or presence of ATP, and endocytotic activity was assessed adding FITC-dextran (0.5 mg/ml) to the culture medium for 30 min at 37°C (control on ice). Thereafter, cells were extensively washed and analyzed by flow cytometry.

DC were harvested and cocultured in complete medium with a constant number of allogeneic nonadherent PBMC (2 × 105/200 μl) in 96-well round-bottom microtiter plates at ratios ranging from 1:20 to 1:320 in triplicates. On day 5, the cells were pulsed with [3H]thymidine (1 μCi/well; Amersham Buchler, Freiburg, Germany) and harvested after 18 h onto a filtermate. The amount of incorporated [3H]thymidine was analyzed in a liquid scintillation counter (Wallac, Turku, Finland).

Data were expressed as means ± SEM. Statistical significance was determined by the unpaired two-tailed Student’s t test. Significance at 95% confidence limits (asterisks in figures) is presented for individual experiments.

Immature DC have a high endocytotic activity that is down-regulated upon maturation (18). To test the influence of ATP on endocytosis, DC were incubated with ATP, and the ability to incorporate FITC-dextran was determined over the course of 2 days. Immature DC incubated with 100 μM ATP transiently up-regulated endocytosis (Fig. 1). This effect was only short-lived (∼1 h) and was followed by a long-lasting down-regulation. At higher ATP concentrations (500–1000 μM), the initial rise of dextran uptake was blunted (data not shown).

To test the hypothesis that extracellular adenine nucleotides activate DC, the expression of DC surface markers was analyzed after a 48-h stimulation period with ATP. As determined by flow cytometry, ATP induced the up-regulation of CD86 (B7-2), CD54 (ICAM-1), and MHC-II, but did not induce the neoexpression of the maturation marker CD83 on DC. This effect was most pronounced at concentrations of 250–500 μM, whereas 100 μM mainly enhanced CD86 expression (Fig. 2). Because ATP is rapidly hydrolyzed by ecto-ATP/ADPase (CD39), the chemically modified ATP derivatives αβmeATP, βγmeATP, ATPγS, or AP5A, which are resistant to hydrolyzation by this ectonucleotidase, were applied in subsequent experiments. At equimolar concentrations, these ATP derivatives were more potent in inducing the expression of activation markers than ATP (Fig. 2). Three of the ATP derivatives (αβmeATP, βγmeATP, and ATPγS) induced small subpopulations of CD83-positive DC (up to 10% of the total DC population).

DC have previously been shown to secrete IL-12, a heterodimeric cytokine composed of two covalently linked chains (p40 and p35), upon activation with LPS, with the combination of TNF-α and PGE2 or with CD40 ligation (19, 20, 21). To investigate the effects of extracellular nucleotides on the secretion of IL-12, DC were incubated with different concentrations of ATP, its derivatives, or UTP. After 48 h, the supernatants were harvested and IL-12 was quantified using a capture ELISA. Significant amounts of IL-12 were secreted by DC stimulated with 100 μM ATP (n = 11, p = 0.006). Fig. 3 demonstrates that extracellular ATP induced IL-12 production in a dose-dependent fashion. The highest IL-12 levels were recovered in the supernatants of DC stimulated with 500 μM ATP, whereas ATP concentrations of 1 mM or above were found to be toxic, as determined by the lack of trypan blue exclusion and positive propidium iodide staining (>30% dead cells after 48 h). Different P2 receptor agonists were tested for their potency to induce IL-12 secretion: βγmeATP, αβmeATP, ATPγS, and benzoylbenzoyl-ATP were clearly more potent than ATP, whereas AP5A and 2 MeSATP were not. The induction of IL-12 secretion was specific for adenine nucleotides, because UTP, a uracil nucleotide with agonistic activity on several P2Y receptor subtypes, and the ATP metabolite adenosine were ineffective (data not shown). To further test the hypothesis, that ATP-induced secretion of IL-12 by DC is mediated by the activation of P2 receptors, DC were incubated simultaneously with ATP (or its derivatives) and suramin (30 μM), a competitive inhibitor of P2 receptors. In the presence of suramin, IL-12 production of ATP-activated DC was completely abolished (Fig. 4). The specific P2X7 receptor antagonist oxyATP did not suppress IL-12 secretion (data not shown).

Activated DC are potent stimulators of T cell proliferation. The influence of ATP on the T cell stimulatory capacity of DC was assessed in a mixed lymphocyte proliferation assay. DC were cocultured with allogeneic nonadherent PBMC at ratios ranging from 1:20 to 1:320. DC exposed to 500 μM ATP 2 days before coculture were four times more potent in inducing a T cell proliferation than untreated DC, whereas 100 μM ATP had only a minor influence (Fig. 5). Again, this effect was more pronounced for the metabolically stable P2 receptor agonists and was abolished in the presence of suramin (data not shown).

The proinflammatory cytokine TNF-α is a well-known activator of DC. Several investigators have shown that activation by TNF-α can be enhanced in combination with other proinflammatory stimuli, such as IL-1β and IL-6 or PGE2 (21, 22). To determine whether TNF-α synergizes with ATP, DC were incubated with ATP (or its derivatives) and TNF-α (1000 U/ml) and were subsequently analyzed for IL-12 secretion, phenotype, and T cell stimulatory capacity. DC stimulated with TNF-α alone produced only small amounts of IL-12, whereas a single addition of 100 μM ATP synergistically increased IL-12 production (Fig. 6). When suramin was added, the synergistic effect of ATP and TNF-α was markedly reduced (Fig. 7). Suramin had no influence on the IL-12 secretion of DC stimulated with a combination of TNF-α and PGE2. As determined by flow cytometry, the combination of P2 receptor agonists and TNF-α induced the up-regulation of CD86, CD54, and MHC-II molecules as well as the neoexpression of the maturation marker CD83 (Fig. 8). Furthermore, these changes in phenotype and the increase of IL-12 secretion correlated with an increased T cell stimulatory capacity (Fig. 9).

The immunostimulatory properties of extracellular ATP have been recognized for several years, but only recently the influence of nucleotides on DC has been studied. Mutini et al. (13) demonstrated that murine DC express the P2X7 receptor and that DC clones lacking this receptor are poor stimulators of Ag-specific Th lymphocytes. Other investigators showed that murine DC activated with UTP released IL-1β, IL-6, IL-10, and IL-12 (14). First evidence for the expression of functional P2 receptors on human monocyte-derived dendritic cells came from receptor analyses by applying single cell Ca2+ imaging (15). The authors observed that DC exposed to ATP orientated their dendrites and cell shape toward an ATP gradient, suggesting chemokine effects of this nucleotide. Recently, it was shown that human DC express mRNA for several P2X and P2Y receptors and up-regulate costimulatory molecules when incubated with ATP (23). Taken together, limited published data about purinergic stimulation of DC point to a possible physiological relevance of nucleotides in DC-mediated immune responses.

In the present study, we demonstrate that human monocyte-derived DC exposed to ATP transiently enhance endocytosis, which is followed by the up-regulation of MHC-II, costimulatory molecules, adhesion molecules, IL-12 secretion, and an increased T cell stimulatory capacity. Furthermore, activation of DC is synergistically enhanced when ATP is combined with TNF-α. DC activation is mediated by P2 receptors, because ATP could be substituted by several other P2 receptor agonists and activation was completely abolished by suramin, a competitive antagonist of P2 receptors. The ATP derivatives αβmeATP and βγmeATP, which have substitutions of methylen bridges for normal oxygen bridges at the polyphosphates, as well as ATPγS, are superior to ATP. There are two possible explanations for this observation. First, these ATP derivatives are resistant to dephosphorylation by the membrane-bound enzymes ecto-ATP/ADPase (CD39) and ecto-5′-nucleotidase (CD73), which are expressed by DC and which in concert rapidly hydrolyze ATP via ADP and AMP to adenosine (23, 24). Adenosine has no activity on P2 receptors and, as we could demonstrate, DC are not activated by this nucleoside. Receptor activation by ATP derivatives may therefore be prolonged. Second, P2 receptor subtypes differ substantially in their affinities to different agonists. In fact, P2 receptor agonists are valuable pharmacological tools, because their rank order of potency can be used to discriminate P2 receptor subtypes (10, 25).

Different P2 receptor subtypes may be involved in DC activation. The P2Y family includes the selective purinoceptors P2Y1 and P2Y11, which are preferentially activated by ATP and ADP, nucleotide receptors that are responsive to both adenine and uracil nucleotides (P2Y2, P2Y8) as well as pyrimidinoceptors (P2Y3, P2Y4, P2Y6) (26). It has been shown that murine DC, but not human monocyte-derived DC, are activated by UTP. Thus, P2Y2, P2Y3, P2Y4, P2Y6, and P2Y8 are not involved in the activation of human monocyte-derived DC. Therefore, it is likely that the selective purinoceptors P2Y1 and P2Y11, which are both expressed by DC (23), mediate activation of human monocyte-derived DC. The rank order potency of ligands to activate the P2Y11 receptor is ATPγS > ATP > 2 MeSATP (26). Interestingly, this rank order was also found in our experiments. However, it is known that the P2Y1 receptor is not activated by the methylated ATP derivatives αβmeATP and βγmeATP, which were potent activators of DC in our study. To our knowledge, the P2Y11 has not been studied in this respect. The P2X receptor family comprises seven receptor subtypes, and αβmeATP is used to discriminate between them. P2X1 and P2X3 receptors are activated by both ATP and αβmeATP, whereas P2X2, P2X4, and P2X5 are not (27). A third type of receptor is the unselective pore-forming P2X7 receptor, which is activated by ATP in the millimolar range and selectively inhibited by oxyATP (28). P2X7 is involved in Ag presentation and apoptosis of murine DC (13, 16). Because oxyATP was not antagonistic in our experiments, the P2X7 receptor appears not to be involved in the activation of human DC. In summary, both P2X and P2Y receptors might mediate activation of human monocyte-derived DC by ATP, with the subtypes P2Y11, P2X1, and P2X3 being the most likely candidates. In the future, further efforts characterizing both P2 receptor expression and selective receptor activation would be helpful to delineate the differential role of P2 receptor subtypes in DC function.

Activation of DC is required for the initiation of many adoptive immune responses. In the self-nonself model by Janeway (29), the immune system responds to exogenous signals such as microorganisms. These signals include bacterial products such as LPS, nonmethylated CpG motifs in bacterial DNA and dsRNA of viral genomes (30). This view has been expanded by the danger model, which postulates that DC are activated by endogeneous mediators released from stressed or damaged cells (31, 32). In favor of this model is the observation that healthy or apoptotic cells do not activate DC, whereas stressed, virally infected, or necrotic cells can stimulate a primary immune response (33, 34). According to this theory, danger signals are either inducible or exist in a prepacked form only to be released in situations in which cells are threatened. To date, these danger signals have not been identified. We propose that the ubiquitous molecule ATP, which is closely linked to energy metabolism, is an ideal candidate for such a prepacked danger signal. This nucleotide is stored in the cytosol at a concentration of 5–10 mM. Therefore, it is not unlikely that large amounts of ATP accumulate in the interstitial space upon plasma membrane damage or acute cell death caused by inflammation, microbial invasion, or rapid tumor growth. Moreover, ATP can be released from vital cells, such as tumor cells (5), activated T cells (35), macrophages (36), and microglial cells (37), thereby enhancing the extracellular ATP concentration at inflammatory sites. Immediately after exposure to ATP, quiescent tissue DC would be rapidly activated to internalize Ags such as mannosilated glycoprotein precursors from necrotic cells, which are believed to be ligands for mannose receptors (38). This short-lived response to extracellular ATP would be followed by a second phase, in which activated DC migrate to the draining lymph nodes to present the captured Ags to T cells, inducing a Th1 immune response by secreting IL-12. In this respect, ATP could also amplify the effects of other activators of DC, such as TNF-α, which accumulate at sites of inflammation.

We conclude that ATP released in a proinflammatory environment might be a potent stimulus for the initiation of immune responses. ATP can be used to improve therapeutic strategies with ex vivo generated DC. Furthermore, stable ATP analogues might be useful in vaccination protocols in vivo.

1

Parts of this study have been presented at the Sixth International Symposium on Dendritic Cells, Port Douglas, Australia, May 26-June 1, 2000, and were published in abstract form.

3

Abbreviations used in this paper: DC, dendritic cell; AP5A, P1,P5-di(adenosine-5′)pentaphosphate; αβmeATP, α,β-methylene-ATP; βγmATP, β,γ-methylene-ATP; ATPγS, adenosine-5′-O-(3-thio)triphosphate; 2 MeSATP, 2-methylthio-ATP; oxyATP, ATP-2′,3′-dialdehyde.

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