P2Y(6) nucleotide receptor mediates monocyte interleukin-8 production in response to UDP or lipopolysaccharide.

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 P2Y(6) regulates chemokine production and release in monocytes. We find that UDP, a selective P2Y(6) agonist, stimulates interleukin (IL)-8 release in human THP-1 monocytic cells whereas other nucleotides are relatively inactive. P2 receptor antagonists or P2Y(6) antisense oligonucleotides inhibit IL-8 release induced by UDP. Furthermore, UDP specifically activated IL-8 production in astrocytoma 1321N1 cells transfected with human P2Y(6). Since lipopolysaccharide has been suggested to activate P2 receptors via nucleotide release, we tested whether IL-8 production stimulated by lipopolysaccharide might result from P2Y(6) 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 P2Y(6) overexpression and inhibited by P2Y(6) antisense oligonucleotides. Thus, UDP activates IL-8 production via P2Y(6) in monocytic cells. Furthermore, lipopolysaccharide mediates IL-8 production at least in part by autocrine P2Y(6) activation. These findings indicate a novel role for P2Y(6) in innate immune defenses.

the most potent ligand at P2Y 4 and UDP selectively activates P2Y 6 (5-7). Many of these receptors have been cloned recently and their roles in immune responses are still poorly understood.
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 P2Y 6 transcripts in human spleen, placenta, thymus, small intestine, and leukocytes (neutrophils, lymphocytes, and monocytes) suggesting that P2Y 6 plays a role in immune defenses (7,10). However, the physiological responses mediated by UDP and P2Y 6 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, P2Y 6 mediates IL-8 production and IL-8 gene expression in response to UDP or LPS. Our studies demonstrate a new role for P2Y 6 in chemokine production and innate immune defenses.

MATERIALS AND METHODS
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 g/ml streptomycin (Life Technologies, Inc., Grand Island, NY), in a humid atmosphere containing 5% CO 2 . 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 * 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. This article must therefore be hereby marked "advertisement" in accordance with 18  units/ml penicillin, 100 g/ml streptomycin, and 400 g/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 g of DEAE-dextran (Amersham Pharmacia Biotech) and 7 g of DNA. In co-transfection experiments, total DNA was kept constant at 7 g using control vector and 1 g 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 CaCl 2 , 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 10 6 cells/ml. Aliquots of 500 l 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 ϫ 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.
THP-1 cells were washed twice in phosphate-buffered saline and resuspended in serum-free culture medium at 2 ϫ 10 6 /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% CO 2 for an additional 20 h. Fluorescein isothiocyanate-conjugated P2Y 6 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 ϫ 10 6 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 g 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 l of kinase buffer and the kinase reaction was started by addition of 20 M ATP, 100 Ci/ml [␥-32 P]ATP (PerkinElmer Life Sciences, Boston, MA), and 10 g of myelin basic protein (Sigma) (as substrate for ERK and p38) or 2 g 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.
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. luciferase reporter plasmid (Fig. 1B). ADP was also active but less potent (EC 50 : 20 M), 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 (EC 50 : 17 M). By contrast, UTP, ATP, and ADP had no effect at low concentrations (up to 1 M) 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 M, the optimal concentration. UDP preincubation for 1 h with 13 g/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 mol 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.

UDP Activates IL-8 Production in Monocytic Cells
P2Y 6 Mediates IL-8 Production in Monocytic Cells-We have previously shown that UDP is the most potent agonist of human P2Y 6 and that the anthraquinone-sulfonic acid derivative reactive blue 2 was a potent P2Y 6 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 M (IC 50 : 35 M) 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 (IC 50 : 5 M).
To specifically evaluate the role of P2Y 6 , THP-1 cells were incubated in the presence of various concentrations of P2Y 6 antisense S-oligonucleotides (up to 5 M) complementary to the P2Y 6 translation initiation region. Antisense S-oligos prevented IL-8 release by 40% when compared with cells pretreated with sense S-oligos (Fig. 2B, p Ͻ 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 P2Y 6 mediates IL-8 production in response to UDP, we also used 1321N1 astrocytoma cells stably transfected with a pcDNA3 vector encoding P2Y 6 . 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 (EC 50 : 2 M) whereas control cells did not respond to UDP (Fig. 2C). These results demonstrate that P2Y 6 mediates IL-8 gene expression in response to UDP in monocytic cells. (24). To explore the signal transduction mechanism whereby P2Y 6 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 M) 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 M) or the p38 inhibitor SB203580 (2 M) 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.

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).
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 g/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 Ϯ 16 pM to 4,500 Ϯ 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 P2Y 6 -Having demonstrated that extracellular nucleotides regulate IL-8 release induced by LPS we tested whether P2Y 6 might be involved. THP-1 cells exposure to P2Y 6 antisense S-oligos inhib-ited IL-8 production induced by LPS by 56% in comparison to cells exposed to sense S-oligos (Fig. 5A, p Ͻ 0.001). By contrast, IL-8 production induced by C. difficile toxin A or IL-1␤ was not inhibited. To confirm the involvement of P2Y 6 , we transiently overexpressed human P2Y 6 and an IL-8 luciferase reporter gene. P2Y 6 transfection significantly increased IL-8 gene expression induced by LPS (Fig. 5B; p ϭ 0.011). By contrast, P2Y 6 did not cause an up-regulation of IL-8 expression in response to toxin A or to IL-1␤. These results indicate that P2Y 6 both regulates IL-8 gene expression and IL-8 production in monocytic cells stimulated with LPS. DISCUSSION This study demonstrates that UDP stimulates IL-8 release via P2Y 6 in human monocytic cells. It also shows that P2Y 6 regulates IL-8 production induced by LPS. This is the first report that demonstrates a putative pathophysiological role for extracellular UDP involving the P2Y 6 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 P2Y 6 in inflammation and immune responses, P2Y 6 expression was found strongly increased in activated CD4 ϩ and CD8 ϩ 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 P2Y 6 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 EC 50 of 2, 3, and 17 M 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 ϳ44-fold less abundant than ATP (29). Since cytosolic [ATP] i averages 3 to 5 mM, intracellular UDP concentration should be ϳ90 M. In the event of cell membrane or tissue damage, passive diffusion of UDP may activate P2Y 6 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 [␥-32 P]ATP in extracellular medium resulted in the accumulation of [␥-32 P]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 P2Y 6 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 P2Y 6 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 P2Y 6 in monocytic cells. Furthermore, it shows that LPS-induced IL-8 production is at least in part mediated by autocrine P2Y 6 activation. These findings indicate a novel role for P2Y 6 in inflammation and innate immune defenses.