Macrophages have important roles in the induction and resolution of inflammation, but the intracellular

pathways from inflammatory signals to pain response remain unclear. a recent study demonstrates that
the P2X4 receptor mediates inflammatory pain by inducing formation of the potent lipid mediator
prostaglandin E
2
.
Introduction
In the complex environment of an inflammatory site such as the arthritic joint, little is known
about the relative contributions or the exact nature of the various receptorsToll-like receptors,
cytokine receptors, formyl peptide receptors and othersthat mediate the intracellular events
that lead to the forma tion of prostaglandins and to the perception of pain. in a recent publication,
ulmann et al.
[1]
demonstrate the importance of the ATP-gated calcium channel P2X4, and its
expression on macrophages, for noci ception via up regulation of prosta glandin E
2
(PGE
2
).
P2X4 belongs to a family of seven cation-permeable ligand-gated ion channels that open in
response to the binding of extra-cellular ATP. The functions of the P2X family of receptors have
previously been associated with pain
[2,3]
but the downstream mechanisms by which pain occurs
are poorly understood. unique insights into their structure have, however, been provided by
crystallization of the zebrafish P2X4 ortholog.
[4]
P2X receptors are trimers, with each subunit
composed of two trans membrane -helices and a large extra cellular domain. ATP binds to a
specific pocket on the extracellular domain away from the actual channel.
[4]

Perhaps undervalued as a mediator of inflammation, elevated levels of extra cellular atP have
nonetheless been observed in the synovial fluid of patients with arthritis.
[5]
thus, the finding by
ulmann et al.
[1]
that mice deficient in P2X4 were protected from induced pain is very interesting
and the upstream and downstream mechanisms quite intriguing. the researchers first demon
strated that local hypersensitivity was substantially diminished in P2X4 receptor knockout mice
treated with formalin, carrageenan or complete Freund's adjuvant (which induced acute chemical
pain, acute inflammatory pain and subacute inflammation, respectively, in the injected paw). as
expected, the level of PGE
2
in paw tissue increased several-fold in wild-type mice treated with
carrageenan or complete Freund's adjuvant; by contrast, this increase was abrogated in the
knockout animals, in which, however, the basal levels of PGE
2
remained almost unaffected.
Importantly, the source of P2X4 receptor in the affected paw tissue was shown to be
predominantly resident macrophages.
Using peritoneal macrophages, which were also demonstrated to express P2X4 receptor, the
authors showed that the level of intra cellular calcium rose substantially upon challenge with
extracellular ATP, whereas this rise was clearly diminished in P2X4 knockout macrophages.
Calcium is probably the most important intra cellular second messenger involved in triggering
the arachidonic acid cascade, which prompted the researchers to demonstrate that P2X4
deficiency indeed prevented the release of arachidonic acid as well as PGE
2
in knockout
macrophages stimulated with exo genous ATP. Consistent with this theory, the effect of ATP on
the release of PGE
2
and arachidonic acid from wild-type cells was potentiated by co-stimulation
with ivermectin, a positive allosteric modulator of P2X4 receptor. PGE
2
biosynthesis was
prevented if the incubation of wild-type cells was performed in the presence of inhibitors of p38
mitogen-activated protein kinase or cyclooxygenase-2, or if extracellular calcium was removed
from the incubation buffer. as final proof of a role for macrophages in nociception, transfer
experiments were performed by injecting wild-type or knockout macrophages, treated in vitro
with ATP and ivermectin, into the paw of naive mice: only the stimulated wild-type
macrophages elicit ed a transient pain response.
As calcium is such a versatile intracellular second messenger, one must question how selective
P2X4 signaling could be. For example, taking only the arachidonic acid cascade into
consideration, both the 5-lipoxygenase and cyclooxygenase pathways are regulated by calcium
(Figure 1). Nevertheless, it seems that the inflammatory pain in these models of acute and
subacute inflammation was elicited primarily via formation of PGE
2
and not via other common
mediators of pain such as substance P, endocannabinoids, bradykinin, adenosine, nitric oxide or
prostacyclin.
[6]
on the other hand, the role of P2X4 in inflammation merits further investigation
because it is likely that this receptor will do more than just cause pain in the inflamed paw; PGE
2

evokes many downstream events other than pain via each of the four dedicated e-type
prostaglandin receptors.
[7]


The results in the study by Ulmann et al.
[1]
suggest that antagonizing the peripheral and, as it
seems, macrophage-specific P2X4 receptor could become a very interesting new principle in
treating inflammatory pain associated with arthritis and perhaps osteoarthritis. NSAIDs,
including cyclooxygenase-2-specific drugs, are effective against pain and inflammation but their
use has been hampered by common adverse effects on the gastrointestinal tract and serious,
although less common, adverse effects on the cardiovascular system. as a consequence, new anti-
inflammatory and analgesic drugs are urgently needed, and targeting the PGE
2
pathway has the
potential to deliver such drugs. PGE
2
causes pain, as shown for instance by Portanova et al.,
[8]

and PGE
2
produced via the cyclooxygenasemicrosomal prostaglandin E synthase 1 pathway
also gives rises to inflammation and pain in experimental models of arthritis.
[9]
Prostacyclin and,
to a lesser extent, leukotriene B
4

[10]
also elicit pain responses (Figure 1). a selective P2X4
antagonist would probably shut down the entire arachidonic acid cascade, similar to the effect of
an inhibitor of cytosolic phospholipase A
2
, but as the cellular expression profile of P2X4 seems
narrow any putative adverse effects should, it is hoped, be limited. Selectively downregulating
the arachidonic acid cascade by modulating calcium influx in tissue-resident macrophages
seems, therefore, to be a promising approach to treating inflammation and the concomitant pain.
One major difficulty will certainly be to accomplish selective antagonism of this particular
receptor. Furthermore, several questions remain about the underlying mechanisms of pain
perception: PGE
2
might be induced in the spinal cord and, likewise, several of the P2X
receptorsincluding P2X4are expressed in the central nervous system by microglia and
neurons, which suggests that cells other than tissue-resident macrophages might also mediate
pain perception via the P2X4 pathway. thus, peripheral inflammation starts many events at
different levels of the sensory nervous system that subsequently lead to pain. Blocking P2X4
might stop the initial peripheral events but perhaps not at later stages, when the pain and
inflammation have become manifest.