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J. Biol. Chem., Vol. 276, Issue 28, 26051-26056, July 13, 2001

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P2Y₆ Nucleotide Receptor Mediates Monocyte Interleukin-8 Production in Response to UDP or Lipopolysaccharide^*

Michel Warny^§, Samer Aboudola, Simon C. Robson, Jean Sévigny, Didier Communi^¶, Stephen P. Soltoff, and Ciarán P. Kelly

From the  Gastroenterology Divison and  Signal Transduction Division, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215 and the ^¶ Institute of Interdisciplinary Research, Medical School, Université Libre de Bruxelles, Route de Lennik 808, 1070 Brussels, Belgium

Received for publication, March 21, 2001, and in revised form, May 8, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Extracellular nucleotides are autocrine and paracrine cellular mediators that signal through P2 nucleotide receptors. Monocytic cells express several P2Y receptors but the role of these G protein-coupled receptors in monocytes is not known. Here, we present evidence that P2Y₆ regulates chemokine production and release in monocytes. We find that UDP, a selective P2Y₆ agonist, stimulates interleukin (IL)-8 release in human THP-1 monocytic cells whereas other nucleotides are relatively inactive. P2 receptor antagonists or P2Y₆ antisense oligonucleotides inhibit IL-8 release induced by UDP. Furthermore, UDP specifically activated IL-8 production in astrocytoma 1321N1 cells transfected with human P2Y₆. Since lipopolysaccharide has been suggested to activate P2 receptors via nucleotide release, we tested whether IL-8 production stimulated by lipopolysaccharide might result from P2Y₆ activation. P2 antagonists or apyrase, an enzyme which hydrolyzes nucleotides including UDP, inhibit IL-8 production induced by lipopolysaccharide but not by other stimuli. Furthermore, IL-8 gene expression activated by lipopolysaccharide is enhanced by P2Y₆ overexpression and inhibited by P2Y₆ antisense oligonucleotides. Thus, UDP activates IL-8 production via P2Y₆ in monocytic cells. Furthermore, lipopolysaccharide mediates IL-8 production at least in part by autocrine P2Y₆ activation. These findings indicate a novel role for P2Y₆ in innate immune defenses.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Extracellular nucleotides induce a wide range of cellular responses mediated by P2 nucleotide receptors. P2 nucleotide receptors include two distinct subtypes classified as P2X and P2Y receptors. P2X are ligand-gated ion channels permeable to Na⁺ and K⁺ whereas P2Y are G protein-coupled receptors. Cloned human P2 receptors include seven P2X (P2X_1-7) and six P2Y (P2Y_{1,2,4,6,11,12}) (1-4). P2 receptors exhibit a wide tissue distribution and display a very complex pharmacology. For example, ATP activates P2Y₁, P2Y₂, and all P2X; ADP activates P2Y₁₂ and is a potent agonist of P2Y₁ receptor; UTP is the most potent ligand at P2Y₄ and UDP selectively activates P2Y₆ (5-7). Many of these receptors have been cloned recently and their roles in immune responses are still poorly understood.

Monocytic cell line and primary monocytes express several P2X (P2X₁, P2X₇) as well as P2Y (P2Y₁, P2Y₂, P2Y₄, and P2Y₆) receptors (8-10). Many studies of P2 receptors in monocytes have focused on P2X₇ that is mainly expressed on monocytic cells (11). In monocytic cells or in LPS¹-primed monocytes, extracellular ATP regulates pro-inflammatory signaling pathways including caspase-1 activation, IL-1 release, and nitric-oxide synthase via P2X7 (12-14). Little is known about the role of the other P2 receptors.

Previous studies have suggested a role for extracellular nucleotides in regulating cellular responses to lipopolysaccharide (LPS). For instance, extracellular signal regulated kinase (ERK) activation by LPS in macrophages can be inhibited by P2 nucleotide antagonists (15). Moreover, LPS was shown to activate IL-1 secretion via ATP release and autocrine stimulation (16, 17). Monocytes exposed to LPS also synthesize large amounts of IL-8, a potent chemoattractant for neutrophils and monocytes (18). Whether LPS-induced IL-8 release is also regulated by extracellular nucleotides is not known.

Previous studies by us and others have reported the presence of P2Y₆ transcripts in human spleen, placenta, thymus, small intestine, and leukocytes (neutrophils, lymphocytes, and monocytes) suggesting that P2Y₆ plays a role in immune defenses (7, 10). However, the physiological responses mediated by UDP and P2Y₆ are not known.

Our initial experiments showed that UDP activated IL-8 release by monocytic cells and led us to explore this new pathway of monocyte activation. Here, we show that in monocytic cells, P2Y₆ mediates IL-8 production and IL-8 gene expression in response to UDP or LPS. Our studies demonstrate a new role for P2Y₆ in chemokine production and innate immune defenses.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Lines-- Human monocytic THP-1 cells (ATCC) were grown in RPMI 1640 supplemented with 5% fetal bovine serum, 10 mM Hepes, 50 units/ml penicillin G, and 50 µg/ml streptomycin (Life Technologies, Inc., Grand Island, NY), in a humid atmosphere containing 5% CO₂. 1321N1 astrocytoma cells were stably transfected with a pcDNA3 expression vector encoding the human P2Y6 receptor and selected as described previously (7). Transfected 1321N1 astrocytoma cells were cultured in Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 400 µg/ml G418. Mouse peritoneal macrophages isolated as previously described (19).

IL-8 Protein Measurement-- IL-8 concentration was determined using a double-ligand enzyme-linked immunosorbent assay and a goat anti-human IL-8 antibody (R & D Systems, Minneapolis, MN) as previously described (20).

Luciferase IL-8 Reporter Gene Assay-- THP-1 cells were transiently transfected as described previously (20). The IL-8 reporter construct was a generous gift from Andrew C. Keates, Ph.D. Briefly, THP-1 cells were suspended in 1 ml of Tris-buffered saline containing 80 µg of DEAE-dextran (Amersham Pharmacia Biotech) and 7 µg of DNA. In co-transfection experiments, total DNA was kept constant at 7 µg using control vector and 1 µg of reporter plasmid DNA was transfected. Following transfection, cells were cultured for 48 h before stimulation. Cell lysis and luciferase assay were performed using the Luciferase Assay System (Promega) following the manufacturer's instructions.

NTPDase Activity Measurement-- Enzyme activity was determined on the protein fractions as previously described (21). Briefly, enzyme activity was tested in 150 mM NaCl, 8 mM CaCl₂, 100 mM Tris, pH 7.4. Apyrase was added to the reaction buffer and was preincubated at 37 °C for 3 min. The reaction was started by addition of 0.3 mM substrate (ATP, ADP, UTP, or UDP as indicated), then terminated at 5-15 min with 0.25 ml of malachite green reagent. P_i release was measured as previously described (22). Protein concentration was measured using the bicinchoninic acid assay (BCA, Pierce, Rockford, IL).

ATP Release-- THP-1 cells (500,000 cells/ml) were washed in phosphate-buffered saline and resuspended in serum-free cuture medium at 10⁶ cells/ml. Aliquots of 500 µl were dispensed in prewarmed 1.5-ml Ependorf tubes and incubated for 30 min or more until extracellular ATP concentration is below 1 nM. Then, cells were stimulated for various periods of time with LPS (1-100 ng/ml) or IL-1 (10 ng/ml) as control. Stimulation was stopped by centrifugation (200 × g, 30 s) and the supernatant was transferred into sterile and ATP-free tubes (Eppendorf Biopur, Westbury, NY). Pipetting and other procedures such as shaking were avoided since they stimulate ATP release. ATP in supernatant fluids was immediately measured by a luciferase assay (Enliten kit, Promega) following the instructions of the manufacturer.

Antisense Oligonucleotides to P2Y₆-- Phosphorothioate-modified oligonucleotides (S-oligos) were synthesized and purified by high performance liquid chromatography by Sigma Genosys (The Woodlands, Texas). 19-Mer sequences including the translation initiation site of human P2Y₆ were as follows: sense: 5'-GGCATGGAATGGGACAATG, antisense: 5'-CATTGTCCCATTCCATGGC-3'. The sequence was checked for uniqueness using NCBI's Blast.

THP-1 cells were washed twice in phosphate-buffered saline and resuspended in serum-free culture medium at 2 × 10⁶/ml. Cells were supplemented with 2.5% LipofectAMINE (Life Technologies, Inc.) and various concentrations of oligonucleotides and were incubated for 4 h. Then, 4 volumes of complete culture medium were added and THP-1 cells were cultured at 37 °C and 5% CO₂ for an additional 20 h. Fluorescein isothiocyanate-conjugated P2Y₆ antisense S-oligos (Sigma Genosys) were used to quantify cellular nucleotide uptake by THP-1 cells in the same conditions.

MAP Kinase Assays-- MAP kinase assays were performed as previously described (20). THP-1 cells were cultured overnight in serum-free culture medium. Stimulation (5 × 10⁶ cells per conditions) was stopped by adding ice-cold phosphate-buffered saline and cells were lysed in 1 ml of lysis buffer. MAP kinases were immunoprecipitated with 2 µg of rabbit specific IgG to ERK2 (sc-154), p38 (sc-535), or JNK1 (sc-571) and protein G-Sepharose (Santa Cruz Biotechnology). After washing, immunopellets were resuspended in 40 µl of kinase buffer and the kinase reaction was started by addition of 20 µM ATP, 100 µCi/ml [-³²P]ATP (PerkinElmer Life Sciences, Boston, MA), and 10 µg of myelin basic protein (Sigma) (as substrate for ERK and p38) or 2 µg of glutathione S-transferase c-Jun-(1-79) (Stratagene, La Jolla, CA) (as substrate for JNK1). Samples were subjected to SDS-polyacrylamide gel electrophoresis (12%) and analyzed by autoradiography.

Endotoxin Assay-- Endotoxin was measured using amebocyte lysate (QCL-1000 kit, Biowittaker, Walkersville, MD) following the manufacturer's protocol and standard.

Statistical Analyses-- Results are represented as means and S.D. Statistical analyses were performed using the SIGMA-STAT software (Jandel Scientific, San Raphael, CA). Analysis of variance (ANOVA) with protected t test was used for intergroup comparison.

	RESULTS

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UDP Activates IL-8 Production in Monocytic Cells-- To investigate the role of extracellular nucleotides in chemokine production by monocytic cells, THP-1 cells were exposed to various concentrations of ATP, ADP, UTP, or UDP for 5 h. UDP strongly activated IL-8 release (EC₅₀: 3 µM) (Fig. 1A) and IL-8 gene expression in THP-1 cells transiently transfected with a luciferase reporter plasmid (Fig. 1B). ADP was also active but less potent (EC₅₀: 20 µM), whereas ATP and UTP were inactive even at high concentrations. A similar dose-response curve was observed with another commercial source of UDP (Roche Molecular Biochemicals) (data not shown). In human peripheral blood monocytes, UDP activated IL-8 production in a dose-dependent fashion (EC₅₀: 17 µM). By contrast, UTP, ATP, and ADP had no effect at low concentrations (up to 1 µM) and inhibited spontaneous IL-8 production at higher concentrations (Fig. 1C). Monocytes exposed to 1 mM ATP exhibited cell swelling and a translucent cytoplasm that are typical features of necrosis (23), whereas these alterations were absent in the other conditions (data not shown). We found undetectable levels of endotoxin in UDP when diluted at 100 µM, the optimal concentration. UDP preincubation for 1 h with 13 µg/ml potato apyrase III inhibited IL-8 response by 88% (Fig. 1D). In separate experiments, we found that apyrase hydrolyzes UDP at the rate of 13.7 µmol of UDP/min/mg (data not shown). Heat denaturation (95°, 5 min) abolished apyrases effect. These data indicate that UDP is a potent activator of IL-8 release and IL-8 gene expression both in THP-1 cells and human primary monocytes.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   UDP induces IL-8 production and IL-8 gene expression in THP-1 cells. A, THP-1 cells (106/ml) were suspended in complete culture medium and stimulated for 5 h with various concentrations of UDP, UTP, ADP, or ATP. IL-8 concentration was measured by enzyme-linked immunosorbent assay in the supernatant. Means and S.D. of three independent experiments are shown. B, THP-1 cells were transiently transfected with a luciferase reporter gene carrying the IL-8 promotor region. After culture for 48 h, cells (106/ml) were stimulated for 5 h with UDP (0-100 µM). Means and S.D. of two independent experiments run in triplicate are shown. C, human peripheral blood monocytes were cultured in complete culture medium and stimulated for 5 h with various concentrations of UDP, UTP, ADP, or ATP. IL-8 concentration was measured by enzyme-linked immunosorbent assay in the supernatant. Means and S.D. of two independent experiments run in triplicate. D, THP-1 cells (106/ml) were incubated in culture medium and stimulated for 6 h with 100 µM UDP in the absence or presence of native or heat-inactivated (95 °C for 3 min) apyrase (2 units/ml). IL-8 concentration was measured in the supernatant. Mean and S.D. of two independent experiments run in triplicate are shown.

P2Y₆ Mediates IL-8 Production in Monocytic Cells-- We have previously shown that UDP is the most potent agonist of human P2Y₆ and that the anthraquinone-sulfonic acid derivative reactive blue 2 was a potent P2Y₆ antagonist (7). Therefore, we tested whether reactive blue could inhibit UDP-induced IL-8 production in THP-1 cells. As shown in Fig. 2A, reactive blue inhibited IL-8 production by 74% at 100 µM (IC₅₀: 35 µM) but did not prevent IL-1 induced-IL-8 release. Similarly, the P2 nucleotide antagonist suramin caused selective inhibition of UDP-induced IL-8 release (IC₅₀: 5 µM).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   P2Y6 mediates IL-8 production in monocytic cells. A, THP-1 cells (106/ml) were preincubated for 15 min in culture medium alone or supplemented with reactive blue (10, 50, or 100 µM) a competitive antagonist active on P2Y6. Then cells were stimulated for 5 h with IL-1 (10 ng/ml) or UDP (100 µM). IL-8 concentration was measured in the supernatant. Mean and S.D. are shown (n = 3). B, THP-1 cells were incubated in the presence of various concentrations of P2Y6 antisense S-oligonucleotides for 4 h in RPMI containing of 2.5% LipofectAMINE. Cells were then cultured for 24 h in regular culture medium. Finally, cells were washed and stimulated with UDP (100 µM) or toxin A (100 nM) for 5 h and IL-8 was measured in the supernatant. Mean and S.D. are shown (n = 3). C, 1321N1 astrocytoma cells were stably transfected with a pcDNA3 vector encoding human P2Y6. Parental and P2Y6 expressing 1321N1 cells were stimulated for 5 h with UDP (up to 1 mM) and IL-8 concentration was measured in the supernatant. Mean and S.D. of three independent experiments are shown.

To specifically evaluate the role of P2Y₆, THP-1 cells were incubated in the presence of various concentrations of P2Y₆ antisense S-oligonucleotides (up to 5 µM) complementary to the P2Y₆ translation initiation region. Antisense S-oligos prevented IL-8 release by 40% when compared with cells pretreated with sense S-oligos (Fig. 2B, p < 0.001). By contrast, no inhibition was observed when cells were stimulated with Clostridium difficile toxin A, a 308-kDa protein that activates IL-8 production in human monocytes (20).

To test further whether human P2Y₆ mediates IL-8 production in response to UDP, we also used 1321N1 astrocytoma cells stably transfected with a pcDNA3 vector encoding P2Y₆. Nontransfected 1321N1 cells have been shown not to respond to UDP (7). In transfected 1321N1 stimulated for 5 h, UDP (up to 1 mM) induced a dose-dependent increase in IL-8 concentration (EC₅₀: 2 µM) whereas control cells did not respond to UDP (Fig. 2C). These results demonstrate that P2Y₆ mediates IL-8 gene expression in response to UDP in monocytic cells.

UDP-Induced IL-8 Production Is Mediated by ERK-- Previous studies by others and us have shown that P2Y receptors signal through MAP kinase (24). To explore the signal transduction mechanism whereby P2Y₆ mediates IL-8 gene expression, we investigated the involvement of mitogen-activated protein kinases (MAP), ERK, p38, and JNK. In THP-1 cells, UDP (100 µM) caused rapid and strong ERK activation whereas, p38 and JNK activation was minimal (Fig. 3A). To test whether ERK and p38 were involved in IL-8 gene expression induced by UDP, cells were preincubated in the presence of the MEK1/2 inhibitor PD98059 (20 µM) or the p38 inhibitor SB203580 (2 µM) for 60 min. PD98059 prevented UDP-induced IL-8 production by 50% (Fig. 3B) and blocked ERK activation by UDP (Fig. 3C). By contrast, SB203580 did not prevent IL-8 release in response to UDP. However, it decreased IL-8 release induced by toxin A by 71% (Fig. 3B). Thus, ERK activation by UDP mediates IL-8 production in THP-1 cells.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   ERK activation by UDP mediates IL-8 release in THP-1 cells. A, THP-1 cells were stimulated with UDP for 5 and 15 min. After cell lysis, ERK, p38, and JNK were immunoprecipitated and their kinase activity was measured using exogenous substrates. B, THP-1 cells (106 cells/ml) were preincubated in complete culture medium with the MEK1/2 inhibitor PD98059 (20 µM) or the p38 inhibitor SB203580 (2 µM) for 60 min. Then cells were stimulated for 6 h with C. difficile toxin A (50 nM) or UDP (100 µM). IL-8 concentration was measured in the supernatant. Means and S.D. of three independent experiments are shown. C, THP-1 cells were preincubated with PD98059 (20 µM) and then stimulated for 5 min with UDP (100 µM). MAP kinase activity was measured as in A.

LPS-induced IL-8 Production Is Modulated by Extracellular Nucleotides-- Previous studies have implicated nucleotide receptor signaling as a component of monocyte/macrophage activation by LPS (15, 16, 17). Therefore, we investigated whether extracellular nucleotides might regulate IL-8 production induced by LPS in THP-1 cells. First, we tested whether nucleotide receptor antagonists might prevent IL-8 production specifically induced by LPS. We found that suramin and reactive blue inhibited IL-8 release induced by LPS (100 ng/ml) but not by IL-1 (Fig. 4A).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   LPS-induced IL-8 production is modulated by extracellular nucleotides. A, the nucleotide receptor antagonists suramin and reactive blue inhibited IL-8 production induced by LPS but not by IL-1. THP-1 cells (106/ml) were preincubated for 15 min in culture medium alone or supplemented with suramin (10 µM) or reactive blue (50 µM). Then cells were stimulated for 5 h with 10 ng/ml IL-1 or 100 µM LPS. IL-8 concentration was measured in the supernatant. Mean and S.D. are shown (n = 3). B, THP-1 cells (106/ml) were incubated in culture medium and stimulated for 6 h with 10 nM C. difficile toxin A, 10 ng/ml IL-1 , or 100 ng/ml Escherichia coli LPS. Cells were stimulated in the absence or presence of extracellular potato apyrase grade III (2 units/ml). IL-8 concentration was measured in the supernatant. Mean and S.D. are shown (n = 3). C, THP-1 cells were transiently transfected with an IL-8 luciferase reporter gene and were stimulated for 5 h with LPS (100 ng/ml) in the absence or presence of apyrase (2 units/ml). Mean and S.D. of two independent experiments run in triplicate are shown.

Next, we observed that the enzyme potato apyrase grade III (2 units/ml) that degrades tri- and diphosphate nucleotides, inhibited LPS-induced IL-8 production by 70% and IL-8 gene expression by 71%. By contrast, apyrase had no effect on IL-8 production stimulated by C. difficile toxin A or IL-1  (Fig. 4B). However, apyrase failed to significantly inhibit IL-8 production in the presence of very high LPS concentrations (10 µg/ml; data not shown).

Finally, we tested whether LPS stimulates the release of extracellular nucleotides in the system tested. Unfortunately, UDP release from monocytic cells could not be assessed because of a lack of an appropriately sensitive method. However, in THP-1 cells, we found that LPS (100 ng/ml) induced a 12-fold increase in extracellular ATP (from 150 pM up to 1,940 pM) at 5 min of stimulation after which ATP levels returned progressively to control value within 60 min (data not shown). In mouse peritoneal macrophages, LPS (100 ng/ml) induced a 28-fold increase in extracellular ATP within 10 min (from 160 ± 16 pM to 4,500 ± 140 pM).

These data strongly suggest that extracellular nucleotides are rapidly released from monocytic cells following LPS stimulation and regulate IL-8 release.

LPS-induced IL-8 Release Is Mediated by P2Y₆-- Having demonstrated that extracellular nucleotides regulate IL-8 release induced by LPS we tested whether P2Y₆ might be involved. THP-1 cells exposure to P2Y₆ antisense S-oligos inhibited IL-8 production induced by LPS by 56% in comparison to cells exposed to sense S-oligos (Fig. 5A, p < 0.001). By contrast, IL-8 production induced by C. difficile toxin A or IL-1 was not inhibited. To confirm the involvement of P2Y₆, we transiently overexpressed human P2Y₆ and an IL-8 luciferase reporter gene. P2Y₆ transfection significantly increased IL-8 gene expression induced by LPS (Fig. 5B; p = 0.011). By contrast, P2Y₆ did not cause an up-regulation of IL-8 expression in response to toxin A or to IL-1. These results indicate that P2Y₆ both regulates IL-8 gene expression and IL-8 production in monocytic cells stimulated with LPS.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   LPS-induced IL-8 release is mediated by P2Y6. A, THP-1 cells were incubated in the presence of 5 µM P2Y6 antisense or sense S-oligonucleotides for 2 h in RPMI containing of 2.5% LipofectAMINE. Cells were then cultured for 24 h in regular culture medium and were stimulated with UDP (100 µM) for 5 h. Means and S.D. are shown (n = 3). B, THP-1 cells were transiently co-transfected with an expression vector encoding human P2Y6 and an IL-8 luciferase reporter gene. After culture for 48 h, cells (106/ml) were stimulated for 5 h with 100 nM C. difficile toxin A, 10 ng/ml IL-1 , or 100 ng/ml LPS. Mean and S.D. are shown (n = 3).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

This study demonstrates that UDP stimulates IL-8 release via P2Y₆ in human monocytic cells. It also shows that P2Y₆ regulates IL-8 production induced by LPS. This is the first report that demonstrates a putative pathophysiological role for extracellular UDP involving the P2Y₆ receptor. Previous studies have shown that ATP and UTP activate various pathways in monocytic cells. For instance, ATP can stimulate IL-1 maturation, arachidonic acid mobilization, intracellular calcium increase, and mobilizes nuclear factor of activated T-cells (12, 22, 25, 26). UTP can enhance inducible nitric-oxide synthase in macrophages (27). However, ATP and UTP were unable to induce IL-8 gene expression in THP-1 cells. Thus diphosphate nucleotides and especially UDP play a specific role in IL-8 production by monocytic cells.

In support for a role of P2Y₆ in inflammation and immune responses, P2Y₆ expression was found strongly increased in activated CD4⁺ and CD8⁺ T cells infiltrating intestinal mucosa in active inflammatory bowel disease (28). Whether this increase is specific for inflammatory bowel disease is not known but would be unlikely. The fact that UDP activates monocytes and modulates LPS effect, suggests that P2Y₆ is involved in various immune responses. Whether UDP also stimulates cytokine production by T cells has not been reported.

In monocytic cells, UDP exhibited an EC₅₀ of 2, 3, and 17 µM in THP-1, 1321N1 cells, and human peripheral blood monocytes, respectively. Whether this concentration can be achieved by autocrine release is not known. In mouse peritoneal macrophages, total cellular UDP is ~44-fold less abundant than ATP (29). Since cytosolic [ATP]_i averages 3 to 5 mM, intracellular UDP concentration should be ~90 µM. In the event of cell membrane or tissue damage, passive diffusion of UDP may activate P2Y₆ receptors on neighboring cells and macrophages to stimulate leukocyte recruitment. Moreover, there is preliminary evidence that UDP as well as ATP and UTP are released from several cell types in vitro and interconverted (30-32). UDP release was demonstrated by the observation that addition of [-³²P]ATP in extracellular medium resulted in the accumulation of [-³²P]UTP, presumably via by membrane diphosphokinase. However, identification of the mechanisms that regulate nucleotide release in basal conditions or in response to LPS will help to better understand the physiological role of UDP.

Pericellular nucleotide concentration is also regulated by ecto-apyrases including CD39, the prototype nucleoside triphosphate diphosphohydrolase (or NTPDase-1) (33, 34). This enzyme is the major NTPDase expressed by monocytes-macrophages and is expressed on THP-1 cells.² In endothelial cells, CD39 overexpression can block ATP release induced by LPS and prevent subsequent maturation and release of IL-1 (17). Since CD39 hydrolyzes diphosphonucleotides, this enzyme may regulate inflammatory responses mediated by UDP in monocytes. Another potential player in regulating UDP and P2Y₆ activity is CD39-L4 (or NTPDase 5), a CD39 analogue (35). In contrast to CD39, CD39-L4 is secreted and its role is not known. Interestingly, CD39-L4 transcripts were found only in macrophages. In addition, this enzyme was specific for diphosphate nucleotides and exhibited a maximal activity for UDP (35). Therefore, CD39-L4 might regulate inflammation and UDP-induced chemokine release.

Our finding that LPS triggers ATP release from THP-1 cells and primary macrophages is consistent with previous studies in microglial cells (16), HUVEC cells (17), and Raw 264.7 macrophagic cells (13). However, two other studies did not detect ATP release from a murine macrophage cell line (36) or from THP-1 cells (37). The reasons for these discrepancies are unclear, but in our experiments we have excluded artifactual ATP release by physical stimuli (medium change, shaking, or pipetting).

This study extends previous reports implicating extracellular nucleotides in the regulation of intracellular responses induced by LPS in monocytes/macrophages (15, 16, 27). Moreover, it is now well established that Toll-like receptor 4 (TLR4) mediates LPS signaling. In concert with CD14 and MD-2, TLR4 plays a key role in mediating IL-8 production in response to LPS (38, 39). In vivo, TLR4 mutations are associated with hyporesponsiveness to LPS in mice and humans (40, 41). However, TLR4 has not been shown to be the only signaling mechanism for LPS. Our finding that IL-8 production induced by LPS can be partially inhibited by nucleotidases or by P2Y₆ antisense S-oligos suggest that UDP and perhaps other extracellular nucleotides act synergistically with TLR4. Whether TLR4 or CD14 might be implicated in nucleotide release is not known.

In summary, this study demonstrates that UDP activates IL-8 gene expression and IL-8 release via P2Y₆ in monocytic cells. Furthermore, it shows that LPS-induced IL-8 production is at least in part mediated by autocrine P2Y₆ activation. These findings indicate a novel role for P2Y₆ in inflammation and innate immune defenses.

	ACKNOWLEDGEMENT

We thank Andrew C. Keates Ph.D. for providing the IL-8 luciferase construct.

	FOOTNOTES

^* This work was supported by a Fellowship Award from the Crohn's and Colitis Foundation of America (to M. W.), National Institutes of Health Grants RO1DK58858 and RO1DK54920 (to C. P. K.) and RO1HL57307 and RO1HL63972 (to S. C. R.), the American Liver Foundation, and the Canadian Institutes of Health Research (to J. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: Gastroenterology Div., Dana Bldg., Rm. 501, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave., Boston, MA 02215. Tel.: 617-667-1944; Fax: 617-667-2767; E-mail: mwarny@caregroup.harvard.edu.

Published, JBC Papers in Press, May 10, 2001, DOI 10.1074/jbc.M102568200

² M. Warny, S. C. Robson, and J. Sévigny, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: LPS, lipopolysaccharide; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; NTPDase, nucleoside triphosphate diphosphohydrolase; TLR, Toll-like receptor; IL, interleukin; MAP, mitogen-activated protein kinase.

	REFERENCES

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