Molecular mechanism of nucleotide-induced primary granule release in human neutrophils: role for the P2Y2 receptor

John Meshki, Florin Tuluc, Ovidiu Bredetean, Zhongren Ding, and Satya P. Kunapuli

Department of Physiology, Temple University Medical School, Philadelphia, Pennsylvania 19140

Submitted 7 July 2003 ; accepted in final form 16 October 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nucleotides are released during vascular injury from activated platelets and broken cells, which could stimulate human neutrophils. In this study, we characterized the P2Y receptors and investigated the functional effects of extracellular nucleotides on human neutrophils. Pharmacological characterization using selective agonists and pertussis toxin revealed that human neutrophils express only functional P2Y2 receptors. However, P2Y2 receptor agonists ATP or uridine triphosphate (UTP) caused intracellular Ca2+ increases in isolated human neutrophils with an EC50 of 1 µM but failed to cause release of primary granules from human neutrophils. ATP and UTP were equally potent in causing elastase release from human neutrophils in the presence of exogenous soluble fibrinogen, whereas ADP and UDP were without effect. We investigated whether nucleotides depend on generated arachidonic acid metabolites to cause degranulation. However, phenidone and MK-886, inhibitors of the 5-lipoxygenase pathway, failed to block nucleotide-induced intracellular calcium mobilization and elastase release. ATP and UTP caused activation of p38 MAPK and ERK1/2 in human neutrophils. In addition, the inhibitors of the MAPK pathway, SB-203580 and U-0126, inhibited nucleotide-induced elastase release. We conclude that fibrinogen is required for nucleotide-induced primary granule release from human neutrophils through the P2Y2 receptor without a role for arachidonic acid metabolites. Both ERK1/2 and p38 MAPK play an important role in nucleotide-induced primary granule release from human neutrophils.

elastase release; fibrinogen; extracellular nucleotides; uridine triphosphate; adenosine triphosphate


POLYMORPHONUCLEAR LEUKOCYTES are terminally differentiated phagocytes that act as the host's first line of defense against invading microorganisms (8). Neutrophils are responsible for bringing plasma molecules and other leukocytes to the sites of tissue damage or infection. Neutrophils normally circulate in a quiescent state but respond rapidly to inflammatory stimuli such as chemoattractants and migrate into tissue where they engulf invading pathogens and necrotic debris. In the tissue, neutrophils degranulate, releasing proteolytic enzymes, including elastase, in an effort to destroy any invading pathogens.

It has been well established that extracellular nucleotides are released from damaged platelets and endothelial cells (26, 41). Thus at the site of vascular injury, the concentrations of extracellular nucleotides can become elevated. Nucleotides have been shown to cause mobilization of intracellular calcium (15), upregulation of Mac-1 (CD11b/CD18) (1), degranulation (11), and chemotaxis in human neutrophils (52). In addition, pretreatment of neutrophils with nucleotides has been shown to enhance formyl-Met-Leu-Phe (fMLP)-induced oxidative burst (30, 54) and cause elastase release (3, 21). The increase in intracellular Ca2+ is mediated through the activation of a pertussis toxin-sensitive G protein, which in turn activates the phosphoinositide phospholipase C cascade (11, 15, 39, 56). The Ca2+ response is similar to that observed with the chemotactic factor fMLP (51).

Nucleotides bind to P2 receptors, which are classified into the two distinct subtypes P2X and P2Y receptors. P2X are ligand-gated ion channels, whereas the P2Y members are G protein-coupled receptors (GPCR). So far, several subtypes of P2X and P2Y receptors have been cloned and their pharmacological profile determined (44). P2Y receptors are characterized by their responses to some selective agonists and the rank order of agonist potencies. The P2Y2 receptor is activated equally by both adenine and uridine triphosphates (ATP = UTP). The P2Y4 receptor is potently activated by UTP and less by ATP but is not activated by UDP (UTP > ATP), whereas the P2Y6 receptor is highly activated by UDP, mildly activated by UTP, and weakly activated by ADP (UDP > UTP > ADP) (37). Except for the P2Y1 receptor, there are no specific antagonists for the P2Y receptors. RT-PCR analysis revealed that human neutrophils express P2Y2, P2Y4, P2Y6, and P2Y11 receptors (9, 13, 14, 24, 27, 33, 50). P2Y1, P2Y4, and P2Y6 couple to Gq to activate phospholipase C, causing formation of inositol-1,4,5-triphosphate and mobilization of intracellular Ca2+ (15, 18).

Neutrophils are known to have several subsets of granules, which include the primary, secondary, and tertiary granules as well as secretory vesicles (8). Signaling events between activation of GPCRs and degranulation are largely unknown. In addition to intracellular calcium increases (15), members of the MAPK family, which include extracellular signal-regulated kinase 1 and 2 (ERK1/2), the c-Jun NH2-terminal kinases, and p38 MAPK, have been shown to mediate fMLP-induced primary granule exocytosis as well as other functional responses in neutrophils (10, 16, 36).

Our aim, in the present study, was to characterize the functional P2Y receptor subtypes and understand molecular mechanisms of the nucleotide-mediated functional responses in human neutrophils. Previously, several investigators (11, 12, 30, 53) identified a P2U receptor on neutrophils that causes intracellular calcium increases in response to ATP and UTP in a pertussis toxin-sensitive manner. However, subsequent work in the P2 receptor field has identified other P2Y receptors that can be stimulated by ATP and/or UTP (eg., P2Y4, P2Y6, P2Y11) (37). Here we demonstrate for the first time that human neutrophils express functional P2Y2 receptors but not functional P2Y1, P2Y4, P2Y6, or P2Y11 receptors and that the P2Y2 receptor subtype mediates the primary granule release from human neutrophils. We further show that nucleotide-induced elastase release depends on the presence of fibrinogen and does not involve arachidonic acid metabolites. Finally, we demonstrate that MAP kinases play an important role in the nucleotide-induced elastase release in human neutrophils.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents. ATP, ADP, UTP, fMLP, N-(methoxysuccinyl)-Ala-Ala-Pro-Val p-nitroanilide, SB-203580, U-0126, and BSA (fraction V) were obtained from Sigma (St. Louis, MO). Dextran T500 and Ficol-Paque were from Amersham Biosciences (Piscataway, NJ). Phenidone and MK-886 were obtained from Biomol (Plymouth Meeting, PA). Fura-2 AM was from Molecular Probes (Eugene, OR). Hexokinase was from Hoffmann-La Roche (Nutley, NJ). Polyclonal anti-phospho-p44/42 MAP kinase (Thr202/Tyr204), anti-p44/442 MAP kinase, anti-p38 MAP kinase (Thr180/Tyr182) antibodies, and anti-p38 MAP kinase were from Cell Signaling Technology (Beverly, MA). CDP-star was from Tropix (Bedford, MA). Nucleotide diphosphates (ADP and UDP) were incubated with hexokinase (10 U/ml) and glucose (200 µM)for 1 h to remove the contaminating nucleotide triphosphates as previously described by Nicholas et al. (38).

Human astrocytoma cells 1321N1 stably expressing P2Y4 were kindly provided by Dr. Kendall T. Harden, University of North Carolina (Chapel Hill, NC). Generation of 1321N1 astrocytoma cells expressing the P2Y2 receptor was achieved in our laboratory, as others have done previously (31, 40).

Neutrophil isolation. Venous blood was collected from healthy subjects in polypropylene tubes containing ACD anticoagulant (in %: 1.5 citric acid, 2.5 sodium citrate, 2 dextrose). Blood was mixed with an equal volume of 3% dextran T500 in saline. Erythrocytes were allowed to sediment for 20 min, and the leukocyte-rich plasma was then subjected to centrifugation on Ficol-Paque at 400 g for 45 min. The pellet was collected and the contaminating erythrocytes were removed by hypotonic lysis. Isolated neutrophils were resuspended in HBSS containing BSA 0.2%. Neutrophils were counted by using a Reichert-Jung hemacytometer (Hausser Scientific, Horsham, PA). Cell viability was checked by the Trypan blue exclusion method and was routinely found >96%.

Measurement of intracellular Ca2+ concentration. Isolated neutrophils were incubated for 45 min at room temperature with 1 µM fura-2 AM (Molecular Probes). Cells were washed three times and then resuspended at a concentration of 3 x 106/ml in HBSS containing 0.5 mM EDTA and 0.2% BSA. Aliquots of 0.5 ml were placed in a quartz cuvette maintained at 37°C. The intracellular calcium concentrations were assayed during agonist stimulation by using excitation wavelengths of 340 and 380 nm and the emission was monitored at 510 nm by using an AB2 spectrofluorometer (Spectronic Instruments, Rochester, NY). The cytoplasmic concentrations of calcium were calculated according to Tsien's ratiometric method (24).

Western blot analysis. Neutrophils at 6 x 106 cells/ml were preincubated at 37°C with or without fibrinogen (2 mg/ml) for 10 min. After incubation, 2 mM CaCl2 and 1 mM MgCl2 were added to the cells. Aliquots of 0.5 ml of cells were then stimulated with agonist for 3 min at 37°C. The reaction was terminated by the addition of 0.5 ml of cold lysis buffer (in mM: 25 Tris·HCl, 150 NaCl, 5 EDTA, 1 sodium vanadate, and 1% Triton X-100, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 0.1 µM caliculyn B). Lysis was allowed to occur for 20 min on ice. Lysates were centrifuged for 5 min at 14,000 g. Proteins from the supernatants were separated by SDS-PAGE, transferred to PVDF membrane, and incubated for 1 h in Tris-buffered saline (TBS; 20 mM Tris·HCL, 150 mM NaCl, 0.05% Tween 20, pH 7.5) containing 1% BSA. Primary antibodies were diluted in TBS containing 1% BSA and membranes were incubated overnight at 4°C in the presence of the indicated primary antibody. Membranes were washed three times for 5 min at room temperature. The appropriate secondary antibody conjugated with alkaline phosphatase was diluted (1:5,000) in TBS containing 1% BSA. Membranes were incubated with the secondary antibody for 1 h at room temperature. Chemiluminescent reagents were used to visualize the reactive proteins on a Fuji LAS1000 digital camera.

Elastase release. Neutrophils at 6 x 106 cells/ml were preincubated at 37°C with 10 µM cytochalasin B and with or without fibrinogen (2 mg/ml) for 10 min. After incubation, 2 mM calcium chloride and 1 mM magnesium chloride were added to the cells. In the case inhibitors, cells were incubated with inhibitors or vehicle for 30 min at 37°C. Aliquots of 150 µl of cell suspension were stimulated with agonists for 20 min at 37°C in a polypropylene 96-well plate. The plate was centrifuged for 3 min at 250 g, supernatants were then collected, and the elastase activity was assayed by using N-(methoxysuccinyl)-Ala-Ala-Pro-Val p-nitroanilide as a chromogenic substrate. The absorbance for each sample was determined at 405 nM every 5 min by using a Polarstar Galaxy microplate reader (BMG Labtechnologies, Bristol, RI). Elastase activity was expressed as the rate of cleavage of the chromogenic substrate and normalized for the degranulating effect induced by 100 µM ATP. One unit is defined as the amount of elastase that releases one nanomole of p-nitrophenol per second from BOC-L-alanine p-nitrophenyl ester at pH 6.5 at 37°C.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Characterization of the P2Y receptor subtypes in human neutrophils. With the exception of the P2Y12 and P2Y13 receptors, P2Y receptor subtypes stimulate phospholipase C and increase intracellular calcium levels. On the basis of the differences in agonist profiles and the rank order of agonists, we can largely characterize the functional P2Y receptor subtypes on a cell. Hence we used nucleotide-induced intracellular calcium increases in human neutrophils to identify the functional P2Y receptors. Addition of nucleotides to fura-2 AM-loaded human neutrophils led to increases in intracellular Ca2+ concentrations (Fig. 1). Of the nucleotides tested, only UTP and ATP increased intracellular calcium levels (Fig. 1A), whereas hexokinase-treated ADP and UDP were inactive (Fig. 1B). Moreover, 2-methylthioadenosine triphosphate and 2-methylthioadenosine diphosphate (Fig. 1C) failed to elicit any calcium response. The EC50 values for UTP and ATP were 1.20 ± 0.6 µM and 1.95 ± 0.5 µM, respectively (Fig. 1A). Thus the rank order of potency for nucleotides was UTP = ATP.



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Fig. 1. Nucleotide-induced intracellular Ca2+ increase in isolated human neutrophils. Isolated human neutrophils were loaded with fura-2 and the intracellular Ca2+ was measured as described in MATERIALS AND METHODS. A: concentration-response curves were constructed for ATP and uridine triphosphate (UTP). Peak values of increases were normalized for the maximum response caused by UTP in the presence of fibrinogen. Results are means ± SE of at least 3 independent experiments. B: intracellular Ca2+ tracings obtained after neutrophil stimulation with ADP (a) or UDP (b). Tracings were obtained by stimulating neutrophils with nucleotide diphosphates incubated with hexokinase and glucose (black trace) or with glucose alone (gray trace). C: intracellular Ca2+ tracings obtained after neutrophil treatment with 2-methylthioadenosine triphosphate (a) or 2-methylthioadenosine diphosphate (b). Tracings are representative for at least 3 independent experiments.

 

Because the agonist profile indicates that the P2Y2 receptor might be involved in the calcium mobilization(37), we further investigated the role of the P2Y2 receptor in neutrophils. Previous studies (27) have shown the presence of mRNA for P2Y2, P2Y4, and P2Y6 receptors in human neutrophils. However, a lack of response to UDP indicated that the P2Y6 receptor is not functional in human neutrophils (Fig. 1B). Furthermore, previous studies (37, 40) have shown that the P2Y2 receptor activates a pertussis toxin-sensitive G protein to cause PLC activation. Hence we investigated the effect of pertussis toxin on the P2Y2 and P2Y4 receptors at which UTP is an agonist (37). Astrocytoma cell lines stably expressing the P2Y4 receptors were pretreated with pertussis toxin and agonist-induced intracellular calcium mobilization was measured. As shown in Fig. 2A, pertussis toxin inhibited ATP- or UTP-induced intracellular calcium mobilization in P2Y2 receptor expressing cells. However, pertussis toxin had no significant effect on UTP-induced calcium increases in P2Y4 receptor-expressing cells (Fig. 2B). When pertussis toxin is used on human neutrophils, ATP- or UTP-induced calcium mobilization was inhibited to the same extent (Fig. 2C). Quantitation of this inhibition revealed that ATP- or UTP-induced intracellular calcium increases were inhibited by pertussis toxin to the same extent in the human neutrophils and in the cells expressing only P2Y2 receptor.



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Fig. 2. Effect of pertussis toxin on nucleotide-induced intracellular Ca2+ increase. Human astrocytoma cells 1321N1 stably expressing P2Y2 (A) or P2Y4 (B) receptors or isolated human neutrophils (C) were loaded with fura-2 and the intracellular Ca2+ was measured as described in MATERIALS AND METHODS. Intracellular Ca2+ tracings were obtained after neutrophil stimulation with ATP (a) or UTP (b). Cells were stimulated with agonists after treatment with 0.2 µg/ml pertussis toxin (black traces) or solvent (gray traces). Representative traces for at least 3 independent experiments are shown. Bar graphs are means ± SE of intracellular calcium increases measured in at least 3 independent experiments. Filled bars represent cells treated with pertussis toxin and empty bars represent controls.

 

Effect of fibrinogen addition on nucleotide-induced elastase release. Based on previous studies demonstrating that nucleotides can cause elastase release in human neutrophils, we evaluated the effects of P2Y2 receptor agonists on human neutrophil primary granule release. ATP or UTP (<=100 µM) were unable to cause significant increases in the release of elastase in human neutrophils (Fig. 3). With the addition of soluble human fibrinogen (2 mg/ml), UTP and ATP caused concentration-dependent increases in elastase release from human neutrophils (Fig. 3). At the maximum concentration, UTP and ATP released 96 ± 1.5 and 104 ± 1.1 mU/ml, respectively, whereas fMLP (10 nM) caused 306 ± 5.5 mU/ml per 6 x 106 cells/ml. The EC50 for elastase release were 1.9 µM for UTP and 6.7 µM for ATP.



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Fig. 3. Effect of fibrinogen on nucleotide-induced elastase release from human neutrophils. Isolated human neutrophils were incubated for 10 min at 37°C in the presence of buffer ({circ}) or 2 mg/ml fibrinogen ({bullet}). Cells were stimulated for 20 min at 37°C with different concentrations of ATP (A) or UTP (B). Supernatants were collected after centrifugation of the cell suspensions and elastase activity was detected by using a specific chromogenic substrate. Results were normalized for the maximal response elicited by UTP in the presence of 2 mg/ml fibrinogen. Values are means ± SE for 3 or more experiments performed in duplicate.

 

Effect of 5-lipoxygenase pathway inhibitors on nucleotide-induced degranulation. We investigated the requirement for fibrinogen in nucleotide-induced primary granule release from neutrophils. In platelets, nucleotide-induced effects are dependent on fibrinogen for arachidonic acid release (27). Arachidonic acid metabolites, particularly LTB4, cause elastase release from neutrophils (43). To eliminate the possibility that arachidonic acid metabolites produced by the neutrophils in the presence of fibrinogen are involved in the nucleotide-induced elastase release, we treated the cells with two known inhibitors of the 5-lipoxygenase pathway, MK-886 and phenidone. Incubation of the neutrophils for 30 min at 37°C with inhibitors (Fig. 4) had no significant effect on the amount of elastase released (Fig. 4A) or on intracellular calcium increases (Fig. 4B) by 30 µM UTP in the presence of 2 mg/ml fibrinogen. These results indicate that arachidonic acid metabolites do not play any significant role in nucleotide-induced responses in human neutrophils.



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Fig. 4. Effect of 5-lipoxygenase inhibitors phenidone and MK-886 on elastase release from human neutrophils. A: isolated human neutrophils were incubated for 30 min at 37°C with buffer (open bars), 100 nM MK-886 (filled bars) or 100 µM phenidone (hatched bars). Fibrinogen (2 mg/ml) or buffer was added, and cells were incubated for 10 min at 37°C in the presence of cytochalasin B (10 µM). UTP (30 µM) or buffer was then added, and cells were further incubated for 20 min at 37°C. Supernatants were collected after centrifugation of the cell suspensions and elastase activity was detected by using a specific chromogenic substrate. Results were normalized for the maximal response elicited by UTP in the presence of 2 mg/ml fibrinogen. Values are means ± SE for 3 or more experiments performed in duplicate. B: intracellular Ca2+ tracings obtained after neutrophil stimulation with 30 µM UTP. Cells were stimulated with agonists after treatment with 100 nM MK-886 or 100 µM phenidone. Representative tracings for at least three independent experiments are shown.

 

Effect of nucleotides on MAP kinase activation in human neutrophils. ERK1/2 and p38 MAP kinase have been shown to play an important role in fMLP-induced elastase release (36). To identify the signaling molecules downstream of P2Y2 receptor activation, we investigated whether ATP and UTP cause activation of MAP kinases. UTP or ATP was able to cause phosphorylation of ERK1/2 even in the absence of exogenous fibrinogen, whereas fibrinogen (2 mg/ml) alone was ineffective. Similarly, ATP or UTP activated p38 MAPK, whereas fibrinogen did not (Fig. 5).



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Fig. 5. Effects of nucleotides and fibrinogen on p38 and p44/42-MAPK phosphorylation. Isolated neutrophils were stimulated for 3 min with 10 µM of nucleotides in the presence or absence of human fibrinogen (2 mg/ml). Cells were lysed for 20 min on ice in the presence of protease and phosphatase inhibitors. Samples were run on SDS-PAGE, and the proteins were transferred on PVDF membranes. Membranes were probed with the appropriate antibodies. Chemiluminescent reagents were used to visualize the reactive proteins.

 

Effect of MAP kinase inhibitors on nucleotide-induced elastase release in human neutrophils. Because nucleotides caused phosphorylation of MAP kinases, we studied their role in UTP-induced elastase release. Treatment of neutrophils with SB-203580 (an inhibitor of p38 MAPK) and U-0126 (an inhibitor of the mitogen-activated protein kinase kinase MEK1/2) had inhibitory effects on UTP-induced elastase release, shifting the concentration-response curves to the right (Fig. 6, A and B). When U-0126 (10 µM) was added to the neutrophils, phosphorylation of ERK1/2 was abolished (data not shown). Blocking the Erk pathway with U-0126 caused a marked inhibition of elastase release (Fig. 6A). The inhibitor of p38 MAPK, SB-203580, also caused partial inhibition of elastase release (Fig. 6B).



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Fig. 6. Effect of MAPK inhibitors on UTP-induced elastase release. Isolated human neutrophils were incubated for 30 min at 37°C with (A) 10 µM SB-203580 or (B) 10 µM U-0126. Fibrinogen (2 mg/ml; {bullet}) or buffer ({circ}) was added, and cells were incubated for 10 min at 37°C. UTP 30 µM was added and cells were further incubated for 20 min at 37°C. Supernatants were collected after centrifugation of the cell suspensions and elastase activity was detected by using a specific chromogenic substrate. Results were normalized for the maximal response elicited by UTP in the presence of 2 mg/ml fibrinogen. Values are means ± SE for 3 or more experiments performed in duplicate.

 


    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Extracellular nucleotides have been shown to have a wide range of effects in human neutrophils and in many other cell types (23). Specifically in neutrophils, these effects include enhancing [o2] generation (30, 47), inducing chemotaxis (52), increasing intracellular calcium (2, 15), and aggregation (48). Nucleotides have also been shown to enhance degranulation caused by fMLP (48). Despite these functional effects, the P2 receptor subtypes on human neutrophils mediating these responses have not been fully characterized. Previously, several investigators have identified a P2U receptor that causes intracellular calcium increases in response to ATP and UTP in a pertussis toxin-sensitive manner (11, 12, 30, 53). However, subsequent work in the P2 receptor field has identified other P2Y receptors that can be stimulated by ATP and/or UTP (eg., P2Y4, P2Y6, P2Y11) (37). Molecular characterization of P2Y receptor subtypes in human neutrophils and differentiated HL-60 cell line by RT-PCR analysis revealed that granulocytic cells express P2Y2, P2Y4, P2Y6, and P2Y11 but not the P2Y1 receptor (9, 13, 14, 24, 27, 33, 50). We undertook the characterization of the P2Y receptor subtypes in human neutrophils by functional approaches by using intracellular calcium increases as an early signaling event downstream of P2Y receptor activation.

Nucleotide diphosphates ADP and UDP have been initially shown to stimulate the P2Y2 and P2Y4 receptors. However, elegant studies from Harden and coworkers (38) by using hexokinase have shown that ADP and UDP do not activate P2Y2 or P2Y4 receptors. Hexokinase treatment converts any contaminating nucleotide triphosphate in the preparation to nucleotide diphosphate (38). The lack of response to 2Me-SADP and hexokinase-treated ADP shows that P2Y1 receptor is not functionally expressed in human neutrophils, because these are potent agonists at this receptor subtype. The agonists UDP and 2MeSATP have been shown to be selective at the P2Y6 and P2Y11 receptors, respectively (44). On the basis of the observation that hexokinase-treated UDP and 2MeSATP failed to cause any intracellular calcium mobilization, we conclude that the P2Y6, and P2Y11 receptor subtypes are not functional on human neutrophils. The agonist profile of ATP = UTP indicates the expression of P2Y2 receptor, but not P2Y4 receptor (UTP » ATP). We further confirmed the functional expression of the P2Y2 receptor and ruled out the P2Y4 receptor by the use of pertussis toxin. As shown in Fig. 2, pertussis toxin inhibited the responses of P2Y2 but not those of P2Y4 subtype. Similar to the observations of other investigators (20), we also found that pertussis toxin does not completely inhibit the responses mediated by P2Y2 receptor. The reason for incomplete inhibition could be explained by the ability of P2Y2 receptor to couple to pertussis toxin-insensitive G16 in addition to pertussis toxin-sensitive Gi (4). The extent of inhibition of both ATP- and UTP-induced intracellular calcium increases by pertussis toxin was similar in human neutrophils and in cells stably expressing the P2Y2 receptor subtype. Under the conditions of P2Y2 receptor signaling blockade by pertussis toxin, UTP failed to cause higher intracellular calcium increase than ATP in human neutrophils, as expected, if a P2Y4 receptor subtype would also be functional. On the basis of these observations, we believe that human neutrophils functionally express only the P2Y2 receptor subtype. However, final confirmation of the lack of functional expression of the P2Y4 receptor should come from studies on neutrophils isolated from P2Y2 receptor-deficient mice (17, 25). Hence we used the P2Y2 receptor selective agonists, UTP and ATP, in the subsequent studies in human neutrophils.

There are conflicting reports regarding the ability of nucleotides to cause primary granule release. Different groups of investigators have shown that nucleotides, even at concentration of 100 µM, do not cause {beta}-glucuronidase secretion (3, 48), whereas others (11, 12, 57) have shown nucleotide-induced primary granule release. In our hands, ATP or UTP failed to cause elastase release. However, when physiological concentrations of fibrinogen were added, ATP or UTP caused elastase release in a concentration-dependent manner with an agonist profile and EC50 values consistent with the P2Y2 receptor. Fibrinogen has been shown to promote neutrophil activation, enhancing phagocytosis and antibody-dependent cellular cytotoxicity (45, 46). In addition, fibrinogen has potentiating effects on fMLP-induced IL-8 synthesis in human neutrophils (29). Removal of fibrinogen from the circulation in rats resulted in decreased leukocyte-mediated tissue damage during nephrotoxic nephritis (55). Also, mice subjected to defibrination experienced a reduction in the degree of intraabdominal abscess formation (34). This would strongly support the role of fibrinogen in modulating neutrophil stimulation and in inflammation. Fibrinogen has been shown to be deposited in tissues under many pathological states, including atherosclerotic lesions (5). By acting in concert with nucleotides at the sites of injury to cause the release of proteolytic enzymes, fibrinogen may aid in the inflammatory process. It is thought that neutrophil interaction with fibrinogen is through {beta}2-integrins, demonstrated by the fact that {alpha}M{beta}2-deficient mice had reduced fibrinogen-dependent inflammatory reactions (32). It has been recently shown that the P2Y2 receptor contains an RGD sequence on the first extracellular loop and interacts with the thrombospondin receptor CD47, and this interaction is required for intracellular signaling downstream the P2Y2 receptor (19). Soluble fibrinogen contains two exposed RGD sequences that are available for binding to integrins. Fibrinogen can also potentiate fMLP-induced elastase release (unpublished observations from our lab). Thus the role of the RGD motif in primary granule release requires further investigation

Because fibrinogen is required for nucleotide-induced elastase release, it would appear that other signaling events are necessary for nucleotide-induced primary degranulation. In platelets, signaling events downstream of a fibrinogen receptor are required for nucleotide-induced arachidonic acid liberation, but not for subsequent metabolite formation (28). In addition, ADP causes dense granule release in human platelets depending on the generation of thromboxane A2 an arachidonic acid metabolite (35). Hence, we investigated whether nucleotides depend on the generation of arachidonic acid metabolites, such as leukotrienes, to cause elastase release. Because the 5-lipoxygenase pathway inhibitors MK-886 (22) and phenidone (6) failed to affect either calcium mobilization or elastase release mediated by UTP, we conclude that nucleotide-induced responses in human neutrophils are independent of arachidonic acid metabolites.

MAP kinases are known to mediate fMLP-induced degranulation (36, 42), and we investigated whether MAP kinases are activated by nucleotides independently of fibrinogen. Our studies show that both ERK1/2 and p38 MAP kinase are activated by nucleotides even in the absence of fibrinogen. These data are consistent with previous studies (7, 49) demonstrating Erk activation by P2Y2 receptor in PC12 cells. Furthermore, blocking ERK1/2 by using the MEK1/2 inhibitor U-0126 or blocking p38 MAP kinase with SB-203580 significantly inhibited nucleotide-induced elastase release in human neutrophils (Fig. 6).

In conclusion, we have shown that human neutrophils express functional P2Y2 receptors and all the nucleotide responses are mediated by P2Y2 receptor subtype without a role for arachidonic acid metabolites. We further conclude that fibrinogen is required for nucleotide-induced primary granule release from human neutrophils. Both ERK1/2 and p38 MAPK play an important role in nucleotide-induced degranulation in human neutrophils.


    ACKNOWLEDGMENTS
 
We thank Dr. Todd Quinton for critically reviewing the manuscript and Drs. Steve Driska, James Daniel, and Raul DeLaCadena for helpful discussions.

GRANTS

This work was supported by a National Heart, Lung, and Blood Institute Grant HL-63933.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. P. Kunapuli, Dept. of Physiology, Temple Univ. Medical School, 3420 N. Broad St., Philadelphia, PA, 19140 (E-mail: spk{at}temple.edu).

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.


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