Neutrophil degranulation: coactivation of chemokine receptor(s) is required for extracellular nucleotide-induced neutrophil degranulation

Medical Hypotheses (2001) 57(3), 306–309 © 2001 Harcourt Publishers Ltd doi: 10.1054/mehy.2000.1313, available online at http://www.idealibrary.com on

Neutrophil degranulation: coactivation of chemokine receptor(s) is required for extracellular nucleotide-induced neutrophil degranulation S. Kannan Department of Physiology, School of Medicine, Temple University, Philadelphia, Pennsylvania, USA

Summary Extracellular nucleotide-induced stimulation of leukocytes and subsequent adhesion to endothelium plays a critical role in inflammatory diseases. The extracellular nucleotides stimulate a P2Y receptor on human PMN with the pharmacological profile of the P2Y2 receptor. Followed by generation of arachidonic acid, subsequently metabolized by 5 lipoxygenase forming the leukotrienes (LT). Of the several LTs generated, LTB4 is a potent chemokine and upon its release binds to the PMN in an autocrine manner leading to the PMN degranulation. It is known that LTB4 causes neutrophil degranulation through its receptor specific binding while the molecular mechanism remains not known at present. However, it is not known whether any LTB4 receptor exists in cytoplasm in any given cell type and also, the existence of any other signaling cascade for the extracellular nucleotide-induced neutrophil degranulation. Based on the few direct experimental and numerous circumstantial evidence, it is conceivable that the extracellular nucleotides require LT generation, as an essential intermediate for mediating neutrophil degranulation. © 2001 Harcourt Publishers Ltd Abbreviations: A3P5P, adenosine-3’-phosphate’5’-phosphate, LTB4, leukotriene B4; LTB4R, leukotriene B4 receptor; fMLP, formyl-methionine-leucine-phenylalanine; P2TAC, platelet ADP receptor coupled to inhibition of adenylyl cyclase; P2Y1R, platelet ADP receptor coupled to stimulation of phospholipase C; AA, arachidonic acid; 5-LO 5 lipoxygenase; N, nucleus; PLC, phospholipase C; PLA2, phospholipase A2; PKC, protein kinase C; PMN, polymorphonuclear leukocytes; Gi, heterotrimeric GTP-binding protein which inhibits adenylyl cyclase; Gq, heterotrimeric GTP-binding protein which stimulates phospholipase C. INTRODUCTION Inflammation is a physiological process by which the soluble mediators and cellular components act in concert to contain and eliminate the signals causing inflammatory distress in the vascularized tissue (1). The most significant response in acute inflammation is leukocyte recruitment to the focus of inflammatory activity. As a first step, neutrophils role along the periphery of the blood vessel and subsequently adhere to vasculature and migrate into

Received 11 October 2000 Accepted 9 January 2001 Correspondence to: S. Kannan MTech, PhD, Department of Physiology, School of Medicine, Temple University, Philadelphia, PA 19140, USA. Phone/Fax:+1 610 352 4612; E-mail: [email protected]

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the tissues via endothelial cell junction and basement membrane (1). Several chemotactic agents, such as leukotriene B4 (LTB4) and formyl-Met-Leu-Phe (fMLP) attract neutrophils to the site of inflammation. At the site of inflammation neutrophils degranulate and proteolytic enzymes, such as elastase, thus released, digest the pathogenic material (2). The factors that initiate and regulate the inflammatory response are referred to as soluble inflammatory factor (3). EXTRACELLULAR NUCLEOTIDES AND THEIR ROLE IN NEUTROPHIL STIMULATION Upon vascular injury, nucleotides released from damaged cells stimulate platelets and other peripheral blood leukocytes and endothelial cells. In addition, activated platelets release ATP and ADP from dense granules. Extracellular

Neutrophil degranulation

nucleotides cause degranulation of neutrophils resulting in the release of proteolytic enzymes, whereas pretreatment of neutrophils with ATP enhances chemotactic peptide induced superoxide radical release (4–9). The receptors through which extracellular nucleotides elicit their physiological effects are referred to as P2 receptors. They are divided into two subclasses: P2X receptors are intrinsic ion channels and P2Y receptors are metabotropic and coupled to heterotrimeric G protein. These receptor subtypes are numbered in the order of cloning. To date several subtypes of P2X and P2Y receptors have been cloned: human PMN express P2Y2, P2Y4, and P2Y6 receptors but not the P2Y1 receptor (10–12). Although ATP has been shown to increase intracellular calcium concentrations and stimulate PKC, through activation of phospholipase C (13), the molecular mechanisms of ATPinduced leukocyte stimulation have not been elucidated. ATP or UTP mediate human PMN stimulation with an EC50 of ~1 µM. ADP does not cause the release of elastase in PMN (9). This agonist profile is similar to that of the characteristics of P2Y2 receptor mediated response in PMN (14). P2Y2 receptor was demonstrated in myeloid leukocytes like PMN, monocytes, macrophages and myeloid progenitor cells (15–17). P2Y2 receptors are primarily expressed in bone marrow, restricted myeloid progenitor cells and also peripheral blood neutrophils and monocytes (18). Lack of PMN responses to ADP is consistent with the fact that the P2Y1 receptor, to which ADP is an agonist, is not expressed in human PMN (15). Although, PMN express the P2Y4 and P2Y6 receptors, their contribution to extracellular nucleotide-induced neutrophil degranulation cannot be ascertained during the activation of P2Y2 receptor which is activated by lower concentrations of ATP or UTP. The lack of availability of selective P2Y2 receptor antagonists precludes the possibility of investigating the role of the P2Y4 or P2Y6 receptors in PMN degranulation. Mice lacking the P2Y2 receptor were developed recently and it would be interesting to study the nucleotide-induced responses in the PMN from these mice (19).

EVIDENCE FOR THE ROLE OF LEUKOTRIENES IN EXTRACELLULAR NUCLEOTIDE INDUCED NEUTROPHIL-DEGRANULATION A G protein-coupled receptor referred as P2Y7 was isolated, and identified to have sequence homology to P2 receptors and chemokine receptors (20). P2Y7 receptor was originally, identified to be functionally coupled to phospholipase C in COS-7 cells transiently expressing this receptor (20). Later, P2Y7 receptor was identified to be the LTB4 receptor by Yokomizo and coworkers (21) and its lack of second messenger signaling responses were © 2001 Harcourt Publishers Ltd

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characterized in 1321N1 cells by heterologous expression (22). Nevertheless, P2Y7 transfected astrocytoma cells, devoid of any P2 receptors, did not result in inositol phosphate formation upon treatment with nucleotides. The most plausible explanation for these observations in the transiently transfected cells is that the action of extracellular nucleotides on P2Y receptors in COS-7 cells might have generated LTB4, which was acting on the transfected receptor in an autocrine fashion. It is known that extracellular nucleotides cause liberation of arachidonic acid, the precursor of LTs in endothelial cells (23), in promyelocytic HL-60 cells (24). These studies led to a hypothesis that at least some of the effects (degranulation, chemotaxis, and increased adhesion molecules expression) of extracellular nucleotides on leukocytes are mediated by LTs. In order to demonstrate that the LT intermediates are required, mice deficient in 5-lipoxygenase enzyme is a valuable tool. In neutrophils obtained from the 5lipoxygenase deficient mice the extracellular nucleotides should fail to result the degranulation. Also, it would be prudent to demonstrate the role of LT by utilizing the LT receptor specific antagonists (e.g. U75302 a LTB4 receptor antagonist). Utilization of blocking agents such as MK-886, to block 5-lipoxygenase activating protein (FLAP) binding to 5 lipoxygenase to generate LTs would prove a valuable asset. In testing this hypothesis, in PMN, elastase release could be utilized as a marker for extracellular nucleotide-induced degranulation.

ROLE OF SECRETARY MOLECULES FROM ACTIVATED PLATELETS IN NEUTROPHILS DEGRANULATION It is of physiological significance to point out that, ATP could be converted to ADP by ectonucleotidases on the cell surface (15) and ADP thus formed could activate contaminating platelets. This activation could result in the release of chemokines/factors (such as platelet activating factor) that stimulate PMN in a paracrine fashion and cause PMN degranulation. This possibility should be tested by blocking ADP-induced platelet activation with established antagonists of the platelet P2 receptors (25). In order to rule out this possibility, the effect of adenosine 3′-phosphate-5′-phosphate (1 mM), a P2Y1 receptor antagonist, and AR-C 66096 (1 µM), a P2TAC receptor antagonist, which completely block ADP-induced platelet activation (26), should be shown to have no effect on nucleotide-induced elastase release in PMN. Thus, these results will be invaluable in ruling out the contribution of secretary molecules released from contaminating platelets (activated ) to extracellular nucleotide induced PMN degranulation. Medical Hypotheses (2001) 57(3), 306–309

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Sec. LTB4 / [LTB4 ]0

LTB4 4

4

LTB4R

ATP or UTP

1 PLA2

P2Y2R

PLC

2 5-LO Free AA

LTB4R

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3 LTB4

LTB4 7

5

1

6

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ATP or UTP

PLA2

P2Y2R

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5-LO 2 Free AA

5

6

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N

Fig. 1

Fig. 2

CHEMOKINE-INDUCED NEUTROPHIL ACTIVATION

thromboxane A2 generation to cause granule release in platelets (12). Obviously, LTB4 (and thromboxane A2 in platelets) activates signaling cascades in PMN of which the nucleotides are not capable. Hence it can be viewed that this phenomenon is another example of complexity in signaling for granule release. Subsequent to the activation of neutrophils by extracellular nucleotides the arachidonic acid is released from the membrane bound phospholipids (2); subsequently metabolized by 5-lipoxygenase pathway forming the leukotrienes (3). LTB4 a known byproduct of the 5-lipoxygenase pathway released from PMN binds to the LTB4 receptor and activated the PMN (4). It is known that LTB4 cause the PMN degranulation (5,6). It remains unclear, whether any cytoplasm LTB4 receptor exists in any given cell type (7). Furthermore, it is far from clear that any other alternative-signaling cascade exists for the direct nucleotide induced PMN degranulation (8). As shown in Figure 2, the extracellular nucleotides stimulate a P2 receptor (1) on human PMN with the pharmacological profile of the P2Y2 receptor. Followed by generation of arachidonic acid (2) subsequently metabolized by 5 lipoxygenase forming the leukotrienes (3). LTB4 a potent chemokine upon its release binds to the PMN in an autocrine manner leading to the PMN degranulation (4). On the other hand it is known that LTB4 causes neutrophil degranulation through its receptor specific binding (5,6). However, it is not known whether any cytoplasm LTB4 receptor exists in any given cell type (7). At present it is not known, whether any other signaling cascade exists for the nucleotide induced PMN degranulation (8).

Of the several LTs, LTB4 is a potent stimulator of the PMNs. The presence of LTB4 receptor on human PMN was previously demonstrated (10). Using a receptor selective antagonist like U-75302, a known LTB4 receptor antagonist (27), to test the hypothesis that the LTs are intermediates in extracellular nucleotide-induced PMN degranulation is LTB4. If this antagonist abolished the elastase release mediated by ATP or UTP in PMN, then probably the extracellular nucleotide-induced release of elastase from PMN is mediated through the formation of LTB4 as an intermediate. This fact should be substantiated by observations that the P2Y1 receptor antagonist, A3P5P, should not have any or minimum effect on nucleotide-induced PMN degranulation. COACTIVATION OF NUCLEOTIDE AND LT RECEPTORS IN NEUTROPHIL DEGRANULATION As shown in Figure 1, the activation of either P2Y2 receptor or LTB4 receptor results in stimulation of phospholipase C and an increase in intracellular calcium (1). This response is pertussis toxin sensitive, indicating a role for Gi/Go heterotrimeric G proteins. The conceptual basis for the LTB4 generation needed for nucleotide-induced neutrophil degranulation can be derived from the following facts. Cowen et al. (5) demonstrated that the activation of P2Y2 (P2U) receptors on PMN or HL-60 cells results in an increase in intracellular calcium through activation of phospholipase C. However, Verghese et al. (28) showed that, although P2Y2 (P2U) receptor activation on human neutrophils leads to the chemotactic response, increase in intracellular calcium is not sufficient to induce chemotaxis. An analogy can be drawn from platelet biology, wherein although both ADP and thromboxane A2 cause intracellular calcium mobilization, ADP depends on Medical Hypotheses (2001) 57(3), 306–309

ACKNOWLEDGMENTS The constructive criticism, critical appraisal and valuable friendship bestowed upon me, by Dr Satya P. Kunapuli, is gratefully acknowledged. The kindness and cooperation of Drs J. Jin, S. Kim, V. V. Reddy, Sr J. Manns, Mrs C. Fisher, Mrs C. Dangelmaier, Mrs M. Merrick are gratefully acknowledged. © 2001 Harcourt Publishers Ltd

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Medical Hypotheses (2001) 57(3), 306–309