Tenidap enhances P2Z/P2X7 receptor signalling in macrophages

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Abstract

Tenidap is an anti-inflammatory drug whose mechanism of action is not fully understood. It has been shown to block plasma membrane anion transport and to decrease release of interleukin-1β, probably via the inhibition of interleukin-1β converting enzyme. In the present study we showed that: (a) tenidap increases the sensitivity of mouse macrophages to cytotoxic effects mediated by extracellular ATP; (b) tenidap increases lucifer yellow uptake through the macrophage ATP receptor; (c) pretreatment with oxidised ATP, a blocker of the P2Z/P2X7 receptor, inhibits cytotoxicity and lucifer yellow uptake due to the combined effects of ATP and tenidap; (d) macrophages lacking the P2Z/P2X7 receptor are resistant to the synergistic effect of tenidap and ATP. The results suggest that tenidap synergises with extracellular ATP for activation of the P2Z/P2X7 receptor.

Introduction

The ability to modulate cytokine release is a major goal in the therapy of inflammatory diseases. Attention has been devoted to a novel compound, tenidap, that has shown powerful in vivo anti-inflammatory activity (Blackburn et al., 1991; Sipe et al., 1992; Pelletier et al., 1993; McNiff et al., 1994; Laliberte et al., 1994). While the cellular basis of this anti-inflammatory activity is not well understood, in vitro experiments demonstrated that, in human macrophages, tenidap blocks the release of interleukin-1β caused by several agonists such as bacterial lipopolisaccharide, exogenous ATP and cytotoxic T lymphocytes (Laliberte et al., 1994; Perregaux et al., 1996). The mechanism is not clear, but it may involve the activity of tenidap as inhibitor of plasma membrane anion transport (McNiff et al., 1994).

A novel plasma membrane receptor of immune cells has attracted interest as a potential modulator of interleukin-1β release from mononuclear phagocytes, the P2Z/P2X7 purinoceptor (Perregaux and Gabel, 1994; Ferrari et al., 1996, Ferrari et al., 1997). This receptor, that selectively binds extracellular ATP, very likely in its fully dissociated form (ATP4−), was originally described in macrophages by Steinberg and Silverstein (1987), extensively characterized in our laboratory (Murgia et al., 1992; Falzoni et al., 1995; Di Virgilio, 1995) and was cloned by Surprenant et al. (1996). P2Z/P2X7 belongs to the subfamily of ligand-gated P2X receptors and is structurally related to a newly identified family of plasma membrane molecules with two transmembrane hydrophobic domains (Surprenant et al., 1995). Other members of this family are Mec-4 of Caenorhabditis elegans, mscL of Escherichia coli, Kir of β pancreatic acini and amiloride-sensitive epithelial Na+ channels (North, 1996). Although the physiological function of the P2Z/P2X7 receptor is unknown, some interesting cellular responses are triggered by its activation: apoptotic or necrotic cell death (Murgia et al., 1992; Molloy et al., 1994), release of mature interleukin-1β (Perregaux and Gabel, 1994; Ferrari et al., 1997) and loss of l-selectins (Jamieson et al., 1996). Furthermore, it has also been suggested that this receptor might be involved in the formation of multinucleated giant cells during chronic granulomatous inflammation (Falzoni et al., 1995). These observations point to the P2Z/P2X7 receptor as a potential target for immunomodulation.

We now show that tenidap strongly potentiates the cytotoxic effect of ATP in the J774 mouse macrophage cell line and suggest that this is due to synergism at the P2Z/P2X7 receptor.

Section snippets

Cells

The J774 mouse macrophage cell line was grown in Dulbecco' s modified Eagle medium supplemented with 10% heat-inactivated horse serum, penicillin (100 μg/ml) and streptomycin (100 μg/ml). ATP-resistant J774 cells were selected by repeated rounds of incubation in the presence of 5 mM ATP followed by cloning by limiting dilution. In lucifer yellow uptake experiments, the medium was replaced by a Na+ saline solution containing 125 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM Na2HPO4, 5.5 mM glucose, 5 mM

Results

Fig. 1A shows the ATP dose dependence of lactic dehydrogenase release from mouse macrophages in the presence or absence of two different concentrations of tenidap. As documented from previous studies (Steinberg and Silverstein, 1987; Murgia et al., 1992; Laliberte et al., 1994), a 5-h incubation with ATP caused a cytolytic effect that was clearly detectable at concentrations above 2 mM (closed circles). The simultaneous addition of 50 μM tenidap together with ATP caused a dramatic shift to the

Discussion

Immune cells express plasma membrane receptors for extracellular ATP that have been grouped into two broad families, P2Y and P2X purinoceptors (Abbracchio and Burnstock, 1994; Burnstock, 1996). P2Y are typical seven membrane-spanning receptors coupled via G-protein to inositol trisphosphate generation and Ca2+ release from intracellular stores. P2X on the contrary are ligand-gated channels that exhibit broad ionic selectivity. So far, three P2X receptors have been described in immune cells: P2X1

Acknowledgements

This work was supported by grants from the National Research Council of Italy (target project Clinical Applications of Cancer Research, ACRO), the Ministry of Scientific Research (MURST), the Italian Association for Cancer Research (AIRC), the IX AIDS Project, the II Tuberculosis Project and Telethon of Italy. JMS is supported by a fellowship awarded by the Spanish Ministry of Science and Education. We thank Pfizer (Groton, CT, USA) for the kind gift of tenidap and Dr. Chris Gabel (Pfizer) for

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