The detection of pain (nociception) following peripheral injury is regulated by central and peripheral nervous system pathways (1–4). In inflamed tissues, the enhancement of pain through the efferent activity of afferent neurons (the axon reflex of Lewis) may be partly responsible for the sensitization of peripheral nociceptors (5, 6). ATP may be released, together with substance P and calcitonin gene–related peptide, from some sensory nerves during “axon-reflex” activity, when antidromic impulses pass down collaterals supplying blood vessels (7); these neurotransmitters cause mast cells to degranulate, thereby producing further sensory and vasomotor effects (8). In addition, the site of injury is continuously invaded by immune cells that release cytokines, which further promote inflammation and pain transmission (9).
The possibility of pain relief induced by immune cells that have migrated to peripheral damaged tissues has been proposed (10). Thus, immunocytes, following selectin-induced migration to the inflamed hind paw of the rat, were shown to produce the opioid peptide β endorphin, which, by binding to opioid receptors on sensory nerves, was able to block the pain response (11). Such an effect was influenced by the presence of endogenous (stress) or exogenous (corticotropin-releasing factor) mechanisms. The existence of these local pain-regulatory factors prompted us to investigate the possibility of inducing local antihyperalgesic effects through other endogenous systems.
The release of ATP from sympathetic nerves innervating blood vessels acts to modulate pain at the level of the P2X subtype of P2 purinoceptors (12). Since P2X subunits are present in the cell body of nociceptive neurons, they may also occur in their peripheral terminals and thus be a target for extracellular ATP released from the cytoplasm of damaged cells (13). In this context, ATP has been proposed as a depolarizing agent of the sensory nerve terminals, with the ability to initiate a nociceptive signal. Experimental data have indicated that extracellular ATP possesses pronociceptive activity. In rats, acute nociception has been shown to be mediated by P2X receptor activation in the hind paw (14), and this is augmented by inflammation and inflammatory mediators (15). Distinct P2X receptors for ATP, which are present on pain-sensing and stretch-sensing neurons, have been identified (16).
Our studies have focused on the extracellular ATP receptor P2Z/P2X7, which mediates ATP cytolytic activity on macrophages (17). Activation of the P2Z/P2X7 receptor in microglial cells, by stimulation with bacterial endotoxin, has been shown to favor interleukin-1β release (18). This receptor is selectively blocked by periodate-oxidized ATP (oATP) (18, 19).
In our initial studies, we examined the effect of local administration of oATP into the rat hind paw on the peripheral nociceptive response following an inflammatory stimulus. We induced a unilateral inflammation in the rat hind paw by intraplantar injection of Freund's complete adjuvant (CFA), and showed that local treatment with oATP significantly reduced inflammatory pain (20). In the present study, we found that this antinociceptive effect is related to inhibition of the action of P2X7 receptors localized on nerve terminals and on endothelial cells, which become unable to release ATP. The effect was independent of the presence of immune cells. Such results demonstrate that ATP exerts a key role in the pathophysiology of peripheral pain related to inflammation, and that oATP may be effective in treating such inflammatory pain.
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- MATERIALS AND METHODS
Although many aspects of central pain modulation are understood, the control of peripheral nociceptive transmission by endogenous and exogenous mechanisms has still to be clarified (6). The control of peripheral inflammatory pain was previously related to the presence of immune cells that have migrated to injured sites (27). These immunocytes secrete β endorphins that activate peripheral opioid receptors, localized on sensory nerve terminals, to inhibit pain (28). Thus, pain is enhanced by measures that limit the migration of opioid-producing cells (10). Inflammatory pain is provoked by the release of many hyperalgesic mediators from migrant immune cells and from resident cells, such as prostaglandins, leukotrienes, and cytokines. In addition, neurotransmitters such as substance P and ATP are released by nerve endings of sensory fibers during “axon-reflex” activity (7), and these can further influence pain signaling.
We hypothesized that one approach to limit inflammatory pain could be to block ATP activity because of its cytolytic (17) and hyperalgesic (14, 15) effects. We used the same experimental model of inflammation in which the efficacy of peripheral opioid antinociception has been demonstrated, i.e., the intraplantar injection of CFA into the hind paw of the rat (10). We studied the antinociceptive function of oATP, a selective inhibitor of the P2X7 ATP receptor, and attempted to distinguish the role of P2X7 ATP receptors on migrating immunocytes compared with those on other sites (i.e., sensory nerve terminals).
We demonstrated, in the present study, that local treatment with oATP significantly increased PPT levels in inflamed paws, and that the antinociceptive effect (maximal at 4 hours following oATP injection) persisted for a long time (until 48 hours). Local treatment of noninflamed paws with oATP did not elicit significant changes in PPT values. P2X7 receptors are known to be present in nociceptive nerve endings (29). The presence of P2Z/P2X7 receptors has been demonstrated in lymphocytes (30) as well as in macrophages (31), microglial cells (18), and mast cells (32). Our histologic evaluations showed that nerve-ending fibers, peripheral nerves, and a few vessels expressed P2X7 receptors in samples obtained from inflamed tissues and from noninflamed controls. In samples obtained from hind paws treated with oATP, P2X7 receptor expression was reduced; it is possible that these receptors are not available to the antibody. However, ATP levels, which were significantly more elevated in inflamed tissues than in noninflamed tissues, were reduced by oATP treatment. This fact could indicate that oATP, by binding to P2X7 receptors, limits ATP production and release by cells bearing these receptors.
In our previous study (20), we have shown that a concentration of oATP higher than 168 μM induced a more elevated and more prolonged analgesic effect in inflamed rat paws. The results possibly depend on the level of saturation of P2X7 receptors; this saturation is progressively reached by increasing the oATP concentration. In addition, by increasing this concentration, the analgesic effect was also evident in noninflamed paws (Dell-Antonio G, et al: personal observations). It is known that inflamed tissues are more prone than noninflamed ones to the action of antinociceptive agents. So, treatment with antinociceptive molecules permits greater elevation of PPT values in inflamed rat paws compared with those observed in control, noninflamed paws.
We also determined whether the analgesic effect exerted by oATP was related to leukocyte migration in inflamed tissues and to their mediator release. We studied the influence exerted on peripheral antinociception by a block of selectin-dependent leukocyte extravasation into inflamed, peripheral subcutaneous tissues. Fucoidin, which binds to adhesion molecules of the selectin family and blocks the “rolling” of leukocytes, reduces leukocyte accumulation at inflammatory sites (10, 33–36). Our results showed that the presence or the absence of immune cells in inflamed tissues did not influence the antinociceptive activity of oATP. These findings are in accordance with the results of a previous study (10) which demonstrated that fucoidin did not affect hyperalgesia in inflamed tissues. Our results indicate that oATP does not act by favoring the release of mediators by recruited immune cells. Such observations suggest that oATP mainly inactivates nociceptive signals through binding of receptors, which are present on sensory nerves, as our histologic findings indicated.
Other data have suggested different ways to explain the analgesic effect due to oATP. Recently, it was shown that mice lacking P2X7 receptors were unable to release interleukin-1 in response to ATP from peritoneal macrophages (37). Impairment of the in vivo cytokine signaling cascade in P2X7-deficient mice suggests that the block of P2X7 receptors by oATP could reduce macrophage activation and the subsequent cytokine production, thereby limiting tissue damage. In addition, our histologic evaluations revealed the presence of P2X7 receptors at the level of capillaries. In endothelial cells, lipopolysaccharide-triggered ATP secretion, via P2X7 receptor activation, causing interleukin-1α release, has been demonstrated (38). We could hypothesize that the block of P2X7 receptors by oATP inhibits interleukin-1 as well as ATP release by endothelial cells. Recently, a role of E-selectin, but not of P-selectin, was demonstrated in the development of adjuvant-induced arthritis in the rat (39). E-selectin may contribute to the migration of antigen-reactive T cells to peripheral tissues.
Our data suggest that binding of oATP with receptors localized on many cells, and also on sensory nerve terminals, could competitively block the binding of extracellular ATP to the same structures, thus limiting ATP-related cytotoxicity and sensory-nerve activation and thereby inducing pain relief. Our results also indicate that oATP treatment of inflamed tissues limits further production of ATP by inflammatory and other cells, possibly through a block of their activation.