Disruption of the P2X7 purinoceptor gene abolishes chronic inflammatory

and neuropathic pain

Iain P. Chessella,*, Jonathan P. Hatchera, Chas Bountraa, Anton D. Michela, Jane P. Hughesb,Paula Greenb, Julie Egertonb, Melanie Murfina, Jill Richardsonb, Wendy L. Peckb,Caroline B.A. Grahamesc,1, Maria Anna Casulad, Yiangos Yiangoud, Rolfe Birche,

Praveen Anandd, Gary N. Buellf,2

aPain Research, N&GI CEDD, GlaxoSmithKline, Third Avenue, Harlow, Essex CM19 5AW, UKbNeuro-Cell Sciences, GlaxoSmithKline, Third Avenue, Harlow CM19 5AW, UK

cGlaxo Institute of Applied Pharmacology, Tennis Court Road, Cambridge CB2 5DH, UKdPeripheral Neuropathy Unit, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK

ePeripheral Nerve Injury Unit, Royal National Orthopaedic Hospital, Stanmore HA7 4LP, UKfGlaxo Institute of Molecular Biology, 14 Chemin Aulx, Plan Les Ouates, Geneva CH-1228, Switzerland

Received 20 August 2004; received in revised form 24 November 2004; accepted 5 January 2005

Abstract

The P2X7 purinoceptor is a ligand-gated cation channel, expressed predominantly by cells of immune origin, with a unique phenotype

which includes release of biologically active inflammatory cytokine, interleukin (IL)-1b following activation, and unique ion channel

biophysics observed only in this receptor family. Here we demonstrate that in mice lacking this receptor, inflammatory (in an adjuvant-

induced model) and neuropathic (in a partial nerve ligation model) hypersensitivity is completely absent to both mechanical and thermal

stimuli, whilst normal nociceptive processing is preserved. The knockout animals were unimpaired in their ability to produce mRNA for pro-

IL-1b, and cytometric analysis of paw and systemic cytokines from knockout and wild-type animals following adjuvant insult suggests a

selective effect of the gene deletion on release of IL-1b and IL-10, with systemic reductions in adjuvant-induced increases in IL-6 and

MCP-1. In addition, we show that this receptor is upregulated in human dorsal root ganglia and injured nerves obtained from chronic

neuropathic pain patients. We hypothesise that the P2X7 receptor, via regulation of mature IL-1b production, plays a common upstream

transductional role in the development of pain of neuropathic and inflammatory origin. Drugs which block this target may have the potential

to deliver broad-spectrum analgesia.

q 2005 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

Keywords: P2X7; Purinoceptor; Interleukin 1 beta; Neuropathic; Taqman

1. Introduction

The relationships governing expression and regulation of

key inflammatory and sensitising mediators are highly

0304-3959/$20.00 q 2005 International Association for the Study of Pain. Publi

doi:10.1016/j.pain.2005.01.002

* Corresponding author. Tel.: C44 1279875362.

E-mail address: iain.p.chessell@gsk.com (I.P. Chessell).1 Present address: AstraZeneca R&D Charnwood, Bakewell Road,

Loughborough, Leicestershire LE11 5RH, UK.2 Present address: Serono International, 15bis, Chemin des Mines,

CH-1211 Geneva 20, Switzerland.

complex with multiple redundancies. Identification of

upstream processes for intervention has proved to be

extremely challenging, either because discovery of suitable

molecular tools has hampered progress (e.g. interleukin-1bantagonists) or because disruption of a signalling process

has revealed multiple divergent roles (e.g. inducible nitric

oxide) (Aley et al., 1998; Budzinski et al., 2000). Chronic

pain is a huge global issue, affecting up to 80% of the

population (Clark, 2003), and the difficulty in identifying

pivotal processes manifests as significant shortcomings in

Pain 114 (2005) 386–396

www.elsevier.com/locate/pain

shed by Elsevier B.V. All rights reserved.

I.P. Chessell et al. / Pain 114 (2005) 386–396 387

the treatment of pain; currently no drug is capable of

adequately treating both inflammatory and neuropathic

pain, and drugs which are currently used to treat these pain

states suffer from poor responder rates, whilst more

efficacious therapies are often limited by tolerability and

safety concerns.

The P2X7 purinoceptor for ATP is a ligand-gated ion

channel with several unusual phenotypic features (Chessell

et al., 1998; Surprenant et al., 1996). Expressed predomi-

nantly on immune cells (Collo et al., 1997; see also Sim

et al., 2004), the receptor functions as a non-selective

cationic channel following brief activation, but prolonged or

repeated activation yields a pore dilation phenomenon.

Uniquely, activation of the P2X7 receptor is also associated

with release of mature, biologically active interleukin-1b(IL-1b; Perregaux and Gabel, 1994; Solle et al., 2001), a

potent inflammatory cytokine whose downstream effects

include induction of nitric oxide synthase (iNOS), cyclo-

oxygenase 2 (COX-2; Samad et al., 2001) and production of

superoxide products (Parvathenani et al., 2003) and tumour

necrosis factor a (TNF-a; Woolf et al., 1997), all of which

have well described roles in the generation or maintenance

of pain. Whilst currently used clinical pain therapies address

some of these targets individually (e.g. COX-2 inhibitors),

interruption of the pain cascade upstream may deliver a

broader analgesic profile than that achieved using current

therapies. Investigation of such a role of the P2X7 receptor

has been difficult as available antagonists lack selectivity,

although more selective molecules are now being developed

(Alcarez et al., 2004). Here we have used homologous

recombination to generate mice with a disruption of exon 1

of the mouse P2X7 gene to investigate the role of this

receptor in inflammatory and neuropathic hypersensitivity,

using recognised animal models of these pain states

(intraplantar Freund’s Complete Adjuvant (FCA), and

partial ligation of the sciatic nerve). Whilst genetically

modified animals may be subject to compensatory changes

in signalling or transduction, such phenotypic analysis

obtained in the absence of selective pharmacological tools

can be a useful predictor of potential drug action. We have

examined the effect of the knockout on the regulation of the

production of a number of inflammatory cytokines to further

elucidate the mechanism underlying the observed pheno-

type in the animals with the gene deletion. We have also

studied the expression of P2X7-like immunoreactivity in

painful injured human nerves and avulsed human dorsal root

ganglia to indicate whether this expression of this receptor is

changed in a human chronic pain state.

2. Methods

2.1. Targeting of P2X7 gene and generation of mutant mice

Mice carrying a targeted null mutation of the P2X7 gene were

generated according to published protocols (Conquet, 1995).

The P2X7 gene was isolated from a genomic library obtained

from 129/Sv mice. Partial sequencing of the 5 0 exons permitted

ligation of two fragments into the neomycin resistant ‘knock-out

vector’ pGN, a plasmid described previously (Le Mouellic et al.,

1990). Homologous recombination of the resulting plasmid DNA

into embryonic stem cells resulted in a disrupted P2X7 gene.

Germline chimaeras were crossed with C57Bl/6J females to

generate heterozygotes, and then intercrossed giving rise to overtly

healthy mutant offspring in the expected Mendelian ratio.

Successful targetting and transmission was confirmed by Southern

and Western analysis (Collo et al., 1997), by the polymerase chain

reaction, and by monitoring loss of channel function, using ATP-

activated YO-PRO-1 permeation of peritoneal macrophages

(Steinberg et al., 1987). As described for similar P2X7 mutants

(Solle et al., 2001), mice displayed altered cytokine production in

response to ATP and lipopolysaccharide. A further 6 backcrosses

onto the C57Bl/6J strain were performed before producing

homozygotes for study. Where animal numbers were limiting,

some studies were performed in one sex only.

2.2. Quantification of dorsal root ganglia neuronal populations

L4 and L5 dorsal root ganglia (DRG) were removed from

P2X7K/K and wild type litter mate controls (nZ5 animals per

group). Following fixation of DRG in 4% paraformaldehyde, DRG

were sectioned (12 mm) and stained for neurofilament L (1:50;

Novocastra, Newcastle, UK) and peripherin (1:1000; Chemicon,

Temicula, USA) to provide a total neuronal stain. Antibody

labelling was detected using standard fluorescent techniques. For

each animal, 4 whole DRG sections were analysed using KS300

image analysis software. The total numbers of neurones were

counted, and cell size determined for each neurone. The total

counts for the 5 animals in each group were then summed, and

distribution histograms generated and analysed using Stastistica

(Statsoft, Tulsa, USA).

2.3. Hot plate test

All animal studies were conducted under a UK Home Office

Licence in accordance with regulated guidelines. Mice of each sex

and genotype (nZ15) were tested in a random and blind fashion

for thermal withdrawal nociception using a hot plate (Harvard

Analgesia Meter, Harvard Instruments, USA) maintained at 50,

52.5 or 55 8C. Mice were observed for signs of nociception, i.e.

rapid fanning or licking of the paws. The response latency was

recorded and results analysed using 2-way analysis of variance in

Statistica (Statsoft Inc., Tulsa, USA) with sex, genotype and hot

plate temperature being used as independent variables. Follow up

analysis was carried out using Duncan’s test.

2.4. FCA-induced inflammation

Female mice (nZ15) were employed for these studies. Animals

were tested for changes in mechanical hypersensitivity using a

weight bearing averager. Following the establishment of baseline

latencies all mice received a sub plantar injection of 30 ml of

1 mg/ml FCA. Mice were re-tested 1 and 7 days later for changes

in weight bearing. Results were expressed as mean ipsilateral/con-

tralateral ratio for each test day and analysed using 2-way analysis

of variance in Statistica with genotype and days post FCA being

I.P. Chessell et al. / Pain 114 (2005) 386–396388

used as independent variables. Follow up analysis was carried out

using Duncan’s test.

2.5. Partial nerve ligation

Two separate studies were performed. For measurement of

thermal hyperalgesia, 20 mice of each sex and genotype were used,

whilst for investigation of mechanical hyperalgesia, 15 female

mice of each genotype were employed. For thermal studies, mice

were tested for paw withdrawal latency by aiming a localised heat

source (Plantar Test Apparatus, Ugo Basile, Italy) at the plantar

surface of the foot. For mechanical studies, withdrawal latencies

were assessed using a hand held electronic Von Frey hair (Life

Science Instruments, UK) onto the plantar surface of each hind

paw in turn until a withdrawal response was observed. On day 0 all

mice underwent surgery to partially ligate the sciatic nerve using a

method based on that described by Seltzer et al. (2000). After 3

days recovery, mice were tested for paw withdrawal latency, and

testing was repeated up to 28 days post operation. For the thermal

readout, results were expressed as mean withdrawal latency for the

ipsilateral paw and expressed as percentage of baseline reading.

For the mechanical readout, results were expressed as mean

percentage ipsilateral/contralateral ratio for each test day. Results

were analysed using 2-way analysis of variance in Statistica with

sex, genotype and days post op being used as independent

variables. Follow up analysis was carried out using Duncan’s test.

2.6. Profiling pro-IL1b gene expression

Tissues were isolated, dissected and stored at K80 8C prior to

extraction. RNA was then isolated from the animal tissues using

RNeasyw Lipid Tissue Mini Kit (Qiagen), following manufac-

turers’ instructions.

Reverse transcription and Taqmanw RT-PCR was performed as

described previously (MacDonald et al., 2001). The primer and

probe set (synthesized by Promega) were designed using Primer3

software and primer specificity was verified using FASTA

searches. Primer and probe sequences (forward primer, reverse

primer, Taqman probe): 5 0-TTGGGCCTCAAAGGAAAGAAT,

5 0-TCTCCAGCTGCAGGGTGG, 5-TATACCTGTCCTGTG-

TAATGAAAGACGGCACA. Analysis of covariance was per-

formed using Microsoftw Excel 97 (Microsoft, Redmond, USA)

with an add-in toolkit v2.1.2 for the analysis of real-time PCR data

(PRISM Training and Consultancy Ltd, Cambridge, UK).

Cyclophilin was used as covariate for normalization.

2.7. Cytokine analysis in P2X7C/C and P2X7

K/K animals

Studies to examine ex vivo, cellular regulation of IL-1b were

performed as previously described with modification (Grahames

et al., 1999). Briefly, in experiments using P2X7K/K animals,

peritoneal macrophages were elicited by injection of 1.5 ml

sterilised 4% thioglycolate into the peritoneal cavity of male F2

P2X7K/K mice (nZ6) or male C57Bl/6-DBA/1 F2 hybrid control

mice (nZ6). Macrophages were lavaged from the cavity 3 days

later using 5 ml DMEM media. Cells were plated in 24 well plates

and left for at least 24 h to adhere. Cells were maintained in

DMEM/10% foetal bovine serum/penicillin (100 U/ml) and

streptomycin (0.1 mg/ml) (Gibco, Paisley, UK). Similarly, follow-

ing seeding, THP-1 cells were maintained in RPMI/10% foetal

bovine serum (Gibco, Paisley, UK). Two days prior to experimen-

tation, THP-1 cells were differentiated using 0.5 mM phorbol-12-

myristate-13-acetate (PMA; Sigma, Poole, UK) for 3 h. Following

washing, all cells were replated at a density of 4!105 cells/well,

left for 24 h and treated with 10 mg/ml LPS for 24 h. Cells were

washed again in complete media immediately prior to experimen-

tation. For experiments examining peritoneal macrophage IL-1brelease, cells were incubated with various concentrations of 2 0- and

3 0-O-(4-benzoyl-benzoyl)-ATP (Bz-ATP; Sigma, Poole, UK) in a

sucrose-based buffer (Michel et al., 1999) at 37 8C and super-

natants removed 30 min following Bz-ATP application. For

experiments examining THP-1 IL-1b release, cells were incubated

in complete media with various concentrations of Bz-ATP and

reactions were terminated at 0, 4, 32 or 64 min. In some

experiments, apyrase (0.4 U/ml; Sigma, Poole, UK) was added to

the wells at 4 min following agonist application. Experiments were

performed in triplicate on at least 3 separate occasions.

Concentrations of IL-1b were performed using a Medgenix IL-

1b EASIA kit, according to the manufacturers instructions. The

monoclonal antibody supplied with this kit does not cross-react

with pro-IL-1b.

For cytometric analysis of concentrations of IL-10, IL-1b, IL-

6, MCP-1 and TNF-a following adjuvant treatment in P2X7K/K

and P2X7C/C animals, blood was collected into EDTA tubes and

subjected to centrifugation (13,000 rpm for 5 min), the resulting

plasma supernatants were harvested and their cytokine content

analysed by Luminex (Luminex Corp.) using a Bio-Plex

cytokine assay (Bio-Rad Laboratories, Inc.) as per manufacturers

instructions. Samples were collected at day 0, 1 and 7 following

FCA injection (nZ5 per group).

Paw samples were dissected and homogenised in PBS

containing ‘complete’ protease inhibitor cocktail (1 tablet/50 ml,

Boehringer Mannheim) using a Mixer Mill MM300 (Qiagen). The

homogenised samples were subjected to centrifugation

(13,000 rpm for 5 min) with the resulting supernatants analysed

for their cytokine content by Luminex (Luminex Corp.) using a

Bio-Plex cytokine assay (Bio-Rad Laboratories, Inc.) as per

manufacturers instructions. Samples were collected at day 0, 1

and 7 following FCA injection (nZ5 per group).

2.8. Immunochemical studies

Fully informed consent was obtained for all human tissues

collected, with approval of the Local Ethics Committee. Peripheral

nerves proximal to injury were obtained from patients with

associated maximum pain greater than 4/10 on a visual analogue

scale in the 24 h prior to surgery (nZ11; 5 male; age range 26–63,

delay from 7 weeks up to 8 y between injury and surgery): these

were used for immunohistochemical (IHC) studies. Extracts from

painful injured nerves (nZ15; 12 male; age range 15–62 y; delay

6 weeks-1 y) were available for Western blotting (WB). Control

nerves (nZ21; 19 male; age range 6–77 y; 13 for IHC and 8 for

WB) were obtained during limb amputation for a non-neurological

reason. Avulsed (central rhizotomy) DRG (nZ14; 13 male; age

range 15–44 y, delay 2 days up to 4.5 months) were obtained from

patients during brachial plexus reconstruction, 12 for IHC (11 male;

age range 15–44 y) and 8 for WB (8 male; age range 18–37 y).

Control post-mortem DRG (nZ13; 4 male; age range 34–98 yrs;

10 for IHC and 5 for WB) were obtained via the Netherlands Brain

Bank. Frozen post-fixed tissue sections (10 mm) were

I.P. Chessell et al. / Pain 114 (2005) 386–396 389

immunostained with primary antibodies to P2X7 (GlaxoSmithK-

line, Harlow, UK), GFAP (glial fibrillary acidic protein; Dako

Cytomation, High Wycombe, Bucks, UK), and neurofilaments

(Dako Cytomation, High Wycombe, Bucks, UK). Specificity of the

P2X7 antibody used has been demonstrated previously (Buell et al.,

1998). Sites of antibody attachment were revealed using a nickel-

enhanced ABC (immunoperoxidase; Vector Laboratories, High

Wycombe, Bucks., UK) method (Shu et al., 1988) or immunoalka-

line phosphatase (APAAP; DAKO Ltd, Ely, Cambs., UK) for

double staining co-localisation studies.

Computerized image analysis (Seescan, Cambs., UK) was used

to assess changes of immunoreactivity. Images were collected

from five microscopic fields (objective !40) from each specimen.

A fixed detection threshold was used for P2X7 and GFAP in

peripheral nerves, and the P2X7:GFAP ratio determined. For DRG,

in order to examine any changes in satellite cells, P2X7-

immunoreactivity was related to neuronal density, since neurons

and their associated satellite cells are not randomly distributed—all

neuronal profiles in each field were outlined and image analysed to

determine P2X7:neuronal area ratio.

For Western blotting, computerised image analysis and optical

density analysis of the P2X7 bands was performed as previously

described using 6% polyacrylamide gels (Yiangou et al., 2001).

3. Results

3.1. Quantification of DRG cell numbers

No significant differences (ANOVA comparison of

log transformed distribution data) were observed in

total cell counts, or distribution of cell size between the

P2X7K/K and P2X7

C/C animals (Fig. 1).

3.2. Neuropathic hypersensitivity

The partial nerve ligation model (Seltzer et al., 2000) is

an established preclinical model of neuropathic hypersensi-

tivity. Here, we have shown profound differences in both

thermal and mechanical sensitivity between P2X7C/C and

Fig. 1. Size distribution histogram for neuronal count against cross sectional

area for whole DRG sections from P2X7K/K (open bars) and P2X7

C/C (filled

bars) animals. No significant differences were observed between the cell

numbers or cross sectional area between the knockout and wild type

animals. NZ5 per group.

P2X7K/K mice. In mice lacking the P2X7 gene, development

of both thermal and mechanical hypersensitivity was

completely absent following nerve ligation, whereas the

P2X7C/C animals developed hyperalgesia in the expected

manner (Fig. 2). Thus, analysis of thermal hypersensitivity

in the P2X7C/C animals revealed development of significant

(P!0.001, ANOVA) hyperalgesia from days 3 to 21 post-

surgery, peaking at day 7 post surgery for both male and

female animals (5.1G0.1 and 4.4G0.4 s, male and female

withdrawal latencies, respectively, compared to pre-surgery

baselines of 10.5G0.7 and 11.6G0.8, respectively), and

showing some resolution in both males and females by day

28 post-surgery. Conversely, neither male nor female

P2X7K/K mice displayed any significant hyperalgesia

compared to pre-surgery baseline at any timepoint exam-

ined (Fig. 2a), with minima of 9.8G0.9 s (male, day 3 post-

surgery) and 10.1G0.9 s (female, day 7 post-surgery),

compared with baseline latencies of 11.6G0.8 and 11.1G0.6 s, respectively. Similarly, P2X7

K/K mice failed to

Fig. 2. Responses to noxious thermal and mechanical stimuli in P2X7C/C

and P2X7K/K mice following partial nerve ligation. (a) Development of

thermal hypersensitivity following nerve ligation in male and female

P2X7C/C and P2X7

K/K mice (nZ20 per group). Following ligation,

thermal hypersensitivity developed after 3 days in P2X7C/C animals, and

was significantly different from pre-surgery values for at least 21 days.

In P2X7K/K animals, hypersensitivity completely failed to develop. (b)

Development of mechanical hypersensitivity following nerve ligation in

male P2X7C/C and P2X7

K/K mice (nZ15 per group). P2X7C/C mice

developed significant mechanical hypersensitivity 3–7 days following

ligation, which was maintained until day 21. P2X7K/K mice completely

failed to display any significant hypersensitivity at any time point studied.

Fig. 3. (a) Effect of chronic inflammatory hypersensitivity on hindpaw

weight bearing in P2X7C/C and P2X7

K/K mice following intraplantar FCA

injection. Prior to FCA injection, weight was evenly distributed between

both hindpaws in both female P2X7C/C and female P2X7

K/K mice (nZ15

per group). One day following FCA injection, P2X7C/C mice displayed a

highly significant reduction in weight bearing on the ipsilateral (injected)

hindpaw (two asterisks, P!0.01 compared to pre-FCA injection values),

which persisted for the duration of the study (7 days). No differences in

weight bearing between hindpaws were observed at any timepoint in the

P2X7K/K mice. (b) Baseline nociceptive responses to hotplate stimulation.

No significant difference were observed between P2X7C/C and P2X7

K/K

animals at any temperature tested.

I.P. Chessell et al. / Pain 114 (2005) 386–396390

develop any mechanical hypersensitivity to static punctate

stimulation using an electronic Von Frey hair following

nerve ligation, compared with the development of signifi-

cant hyperalgesia in P2X7C/C animals (P!0.001, ANOVA;

Fig. 2b, e.g. ipsilateral:contralateral ratio of 0.99G0.04 vs

0.62G0.06, P2X7K/K vs P2X7

C/C animals respectively, 14

days post-ligation). Significant differences between with-

drawal thresholds were observed between P2X7C/C and

P2X7K/K animals from days 3 to 21 post-surgery (P!0.05,

Duncan’s test; Fig. 2b).

3.3. Inflammatory hypersensitivity

An exaggerated response to mechanical stimuli,

reflecting both allodynia and hyperalgesia, is associated

with chronic inflammation in both clinical disease such

as osteo- and rheumatoid arthritis and with preclinical

models. The established model of intraplantar Freund’s

Complete Adjuvant (FCA)-induced inflammation can be

used to examine the difference in sensitivity between

injected and uninjected paws, and here we have used

determination of relative weight distribution between

affected and unaffected hindpaws (Clayton et al., 1997)

as a measure of hypersensitivity. Following intraplantar

FCA injection, analysis of the ipsilateral/contralateral

weight bearing ratio revealed a significant effect of

genotype (P!0.001, ANOVA), where P2X7K/K mice

failed to develop significant hypersensitivity at any time

after FCA injection (e.g. injected paw:uninjected paw

weight bearing 7.9G0.6:8.9G0.6 g in P2X7K/K mice at

day 1 post-injection), whereas the P2X7C/C mice

displayed a significant reduction in ipsilateral/contralat-

eral ratios (e.g. 4.6G0.6:10.4G0.6 g in P2X7C/C mice at

day 1 post-injection) at both test points (24 h and 7d

post-FCA, P!0.01 in both cases; Fig. 3).

3.4. Baseline nociceptive behaviours

Mice were also tested for their sensitivity to noxious heat

in the absence of any neuropathic or inflammatory insult to

confirm that nociceptive processing in the absence of

pathological insult was normal. The latency of response to

an acute thermal stimulus was measured using a 50, 52.5 or

55 8C hotplate. Comparisons showed that withdrawal

latency for all test groups was reduced with increasing hot

plate temperature, with no significant differences between

P2X7C/C and P2X7

K/K animal groups (50 8C; 27.0G1.5 vs

25.4G1.1 s, 52.5 8C; 16.5G0.8 vs 16.8G0.9 s, 55 8C;

10.9G0.5 vs 9.4G0.5 s, P2X7C/C and P2X7

K/K, respect-

ively). Sub-analysis revealed a small but significant

difference between male P2X7C/C and male P2X7

K/K mice

at 55 8C (10.8G0.6 vs 8.2G0.6 s, P2X7C/C and P2X7

K/K,

respectively, P!0.05, Duncan’s test). No other significant

differences were found. Comparison of the response of the

genotypes to a noxious punctate stimulus (electronic Von

Frey hair; eVFH), in the absence of any induced

inflammatory insult, revealed no difference between with-

drawal latency in P2X7C/C and P2X7

K/K mice (10.7G0.5

and 10.6G0.5 g force to withdrawal, respectively, meanGs.e.mean). These data show that P2X7

K/K mice display a

largely normal nociceptive phenotype of withdrawal to both

thermal and mechanical noxious stimuli in the absence of

ongoing inflammatory or neuropathic hypersensitivity.

3.5. Pro-IL-1b gene expression

Pro-IL-1b gene expression levels in P2X7 knockout and

wild type paws were investigated using quantitative PCR.

Expression of pro-IL-1b mRNA was significantly (ANOVA

with LSD post-hoc test) higher in the P2X7K/K animals on days

0 and 1 (template copy numbers 95.5 [63.5–143.7] vs 31.5

[20.6–48.2], 10527.1 [6032.2–18371.2] vs 2387 [1590.3–

3585.0], days 0 and 1, P2X7K/K vs P2X7

C/C animals,

respectively, nZ5 per group, mean with 95% confidence

intervals), but was similar between the animal groups on

00

G6

.4

4.7

G2

71

.9

5.6

G6

4.9

*

G2

5.4

G3

.3

G4

.8

I.P. Chessell et al. / Pain 114 (2005) 386–396 391

day 7 post-FCA (1501.8 [998.9–2257.8] vs 2124.3 [1384.2–

3260.2] P2X7K/K vs P2X7

C/C animals, respectively, nZ5 per

group, mean with 95% confidence intervals).

TN

Fa

KO

wt

KO

0G

15

.31

00

G1

5.0

10

0G

3.1

1

7G

94

2.1

11

00

5.7

G1

03

0.3

99

9.4

G1

19

.01

42

5G

16

25.7

88

96

.1G

77

1.0

*1

22

4.8

G9

6.9

59

inb

old

ital

ics.

CP

-1T

NF

a

tK

Ow

tK

O

10

0G

8.5

10

0G

4.1

10

0G

12

.71

00

65

.0G

4.9

78

4.1

G6

.79

9.7

G2

.09

8.2

10

.6G

12

.08

9.7

G2

.91

05

.3G

3.0

84

.9

3.6. Cytokine profiling of P2X7K/K vs P2X7

C/C animals

In order to confirm abolition of P2X7-mediated IL-1brelease in P2X7

K/K animals, we examined lipopolysacchar-

ide (LPS)-induced IL-1b release in thioglycollate-elicited

macrophages obtained following peritoneal lavage from

P2X7K/K and control animals. Six hours following stimu-

lation of plated cells with 1 mg/ml LPS, cultures were

stimulated with the P2X agonist, Bz-ATP, and 30 min later,

concentrations of mature IL-1b were measured in cell

supernatants. Over the concentration range of Bz-ATP used,

no significant release of IL-1b was observed using cells

from P2X7K/K animals, with or without prior treatment with

LPS, whilst from LPS-treated cells from control animals,

IL-1b concentrations peaked at 700G115 pg/ml at

Fig. 4. (a) Mature IL-1b release from thioglycolate elicited peritoneal

macrophages obtained from P2X7K/K and control mice. Addition of agonist,

Bz-ATP, caused a dose-dependent release of IL-1b from control animal,

LPS-primed cells measured 30 min following agonist addition, but not from

LPS-primed cells from P2X7K/K animals or non-primed cells from control

or P2X7K/K animals. (b) Bz-ATP—induced release of mature IL-1b from

PMA-differentiated, LPS-primed THP-1 cells. Treatment with apyrase,

4 min following agonist addition where denoted, failed to halt the release of

mature IL-1b measured 28 and 60 min later. Tab

le1

Rel

ease

dcy

tok

ine

con

cen

trat

ion

sin

paw

sam

ple

sfr

om

P2

X7K

/Kan

dP

2X

7C/C

anim

als

Tim

ep

ost

FC

A(d

ays)

IL-1

0IL

-1b

IL-6

MC

P-1

wt

KO

wt

KO

wt

KO

wt

01

00

G3

8.8

10

0G

4.6

10

0G

31

.21

00

G1

1.4

10

0G

6.1

10

0G

15

.01

0

12

79

.7G

26

.81

00

.7G

4.1

*1

32

0.8

G8

4.5

51

9.7

G7

6.4

*1

79

.2G

21

.43

83

0.1

G7

17

.2*

90

97.

72

72

.6G

8.7

12

0.9

G7

.6*

73

4.2

G1

8.5

13

6.8

G1

1.3

*5

2.0

G4

.52

38

.1G

74

.7*

13

852

.

Dat

aar

eex

pre

ssed

as%

of

con

tro

lv

alues

GS

EM

.*

P!

0.0

5,

sig

nifi

can

tly

dif

fere

nt

fro

mW

Tv

alues

,S

tud

ent’

su

np

aire

dt-

test

,fi

gure

s

Tab

le2

Rel

ease

dcy

tok

ine

lev

els

inse

rum

sam

ple

sfr

om

P2

X7K

/Kan

dP

2X

7C/C

anim

als

Tim

e(d

ays)

IL-1

0IL

-1b

IL-6

M

wt

KO

wt

KO

wt

KO

w

01

00

G1

2.6

10

0G

8.0

10

0G

7.9

10

0G

9.2

10

0G

2.6

10

0G

16

.3

18

8.0

G1

.08

7.9

G1

.59

6.1

G2

.28

9.9

G4

.81

87

3.3

G4

40

.85

07

.1G

10

6.0

*1

79

2.5

G1

.69

0.4

G1

.28

8.5

G1

.48

9.2

G4

.46

1.5

G1

7.8

16

7.9

G5

0.4

*1

Dat

aar

eex

pre

ssed

as%

of

con

tro

lGS

EM

.*P

!0

.05

sig

nifi

can

tly

dif

fere

nt

from

WT

val

ues

,S

tud

ent’

su

np

aire

dt-

test

.

I.P. Chessell et al. / Pain 114 (2005) 386–396392

a Bz-ATP concentration of 160 mM. No significant IL-1brelease was observed from cells from normal animals which

were not treated with LPS (Fig. 4).

The natural ligand for the P2X7 receptor is ATP, and

whilst it has been determined that activation of this receptor

mediates release of IL-1b, experiments are often based on

prolonged contact of the ligand with the receptor; we wished

to confirm that a single, brief activation of the receptor

commits cells to release IL-1b at times after removal of

agonist, as this may be more representative of the pulsatile

nature of P2X7 receptor activation in vivo. Here we have

used LPS-treated, differentiated human monocytic cells,

THP-1, to examine Bz-ATP-induced IL-1b release, and the

agonist degrading enzyme, apyrase (Ferrari et al., 1997;

Grahames et al., 1999) to limit receptor activation time

(applied !4 min following agonist administration). In the

absence of apyrase addition, Bz-ATP (40 mM to 5 mM)

caused a concentration and time dependent release of IL-1b,

which reached a maximum at 32 min following Bz-ATP

addition (Fig. 4). Comparison of data obtained 4 min after

Bz-ATP addition with that obtained from cells treated with

apyrase after 4 min shows that removal of the agonist by

addition of the enzyme fails to halt the release of mature

IL-1b from the cells (Fig. 4).

In order to further elucidate a potential mechanism by

which deletion of the P2X7 gene abolishes FCA-induced

hypersensitivity, we profiled both local and systemic

released cytokine levels in P2X7K/K vs P2X7

C/C animals

0, 1 and 7 days post FCA-challenge using cytometric

analysis.

Profiling of the P2X7C/C paw samples revealed a sustained

increase of IL-1b levels in the P2X7C/C animals following

intraplantar FCA injection. This increase was significantly

reduced in the P2X7K/K animals (Table 1). Additional

cytokines upregulated following intraplanter FCA injection

included IL-10, MCP-1, TNF-a and in IL-6 (Table 1).

Interestingly, the increases in IL-10 following intraplantar

FCA injection are also significantly attenuated in the

P2X7K/K animals across the whole timecourse. In contrast,

the IL-6 response was enhanced in the P2X7K/K animals

suggesting potential compensatory changes.

Following FCA treatment, a transient increase (peaking

at 1 day post-FCA) in IL-6 and MCP-1 levels was observed

in the sera from the mice. The increase in IL-6 concentration

was significantly reduced in the P2X7K/K animals compared

to the wild-types on day 1, suggesting that this early

systemic inflammatory process following FCA-challenge is

attenuated in P2X7K/K animals. No significant increase was

observed in the levels of IL-10, TNFa or IL-1b in sera from

P2X7C/C mice post-FCA (Table 2).

3.7. P2X7 immunolocalisation in human tissue samples

To determine the significance of these findings in

human pain states, we have used a monoclonal antibody

raised to the extracellular loop of the human P2X7 receptor

(Buell et al., 1998) to examine its expression in tissues

obtained from patients suffering from persistent neuropathic

pain and hypersensitivity following trauma. In serial tissue

sections of painful injured nerves and matched control

nerves, P2X7 immunostaining (Fig. 5a) was observed and

correlated well with immunoreactivity for the glial/Schwann

cell marker, GFAP (Fig. 5b), but not with axonal marker,

neurofilament (Fig. 5c); co-localisation of P2X7 and GFAP

confirmed the presence of P2X7 in Schwann cells (Inset

Fig. 5a). Comparison of P2X7 and GFAP immunoreactivities

(P2X7:GFAP) by image analysis showed a significantly

increased ratio of P2X7:neurofilament immunostaining in

painful nerves [nZ11; median (range) 0.58 (0.26–1.78)]

compared to controls [nZ13; median (range) 0.45 (0.06–

0.58); Mann–Whitney U test; PZ0.032]. P2X7 immunos-

taining was not detected in sensory neurons of human dorsal

root ganglia (DRG) in both post-mortem controls and after

spinal root avulsion injury (i.e. central axotomy; Fig. 6a and

b), but was present in satellite cells surrounding neurons

(Inset Fig. 6a satellite cells red; neurons black), with

markedly increased density of these in the avulsed DRG

preparations. Image analysis of P2X7 immunoreactivity in

satellite cells of human DRG, expressed as a ratio to neuronal

cell body area (nZ10 control, nZ12 avulsed), showed a

significantly increased area of staining in avulsed DRG

[nZ12; median (range) 0.60 (0.17–0.76)], compared to

controls [nZ10; median (range) 0.38 (0.21–0.54); Mann–

Whitney U test; PZ0.045]. In control DRG, weak GFAP

immunostaining was observed in some fibres and cells

surrounding neurons (Fig. 6c). However, in avulsed DRG

there was a marked increase of both intensity and frequency

of GFAP-immunoreactive structures (Fig. 6d), in a very

similar fashion to that seen with P2X7. These findings in

DRG are similar to those reported in an animal model of

sciatic nerve injury, where neurotrophins such as NGF

expressed by reactive satellite cells contribute to sprouting of

sympathetic fibres. In our study, NGF immunoreactivity was

also detected in satellite cells of DRG, and appeared to be

increased after avulsion injury (data not shown). There was

no correlation between delay before surgery and P2X7

immunostaining in injured nerves or DRG. No P2X7

immunostaining was observed in human sympathetic

ganglion neurons, or in motoneurons in post-mortem

human spinal cord (data not shown).

3.8. P2X7 western blots from human tissue samples

Western blotting with the P2X7 monoclonal antibody in

nerve extracts showed a 70 kDa band in all samples

(Fig. 5d), and in some extracts a 65 kDa and larger,

approximately 140 kDa band. The relative optical density of

the 70 kDa band was significantly elevated in extracts of

painful injured nerve [median (range)Z36.0 (4.0–120)]

compared to controls [median (range)Z11.0 (6.0–34.0);

PZ0.018, Mann–Whitney U test]. There was a trend for

increased optical densities of the 70 kDa band in injured

I.P. Chessell et al. / Pain 114 (2005) 386–396 393

DRG [median (range), 18.5 (11.0–36.0)] compared to

controls [15.0 (7.0–36.0)]. The 65 kDa band may be an

unglycosylated form of the 70 kDa band and the larger

140 kDa band may be a 70 kDa dimer (Glass and Burnstock,

2001; Kim et al., 2001).

4. Discussion

These data indicate a clear effect of P2X7 gene deletion

upon responsiveness to noxious thermal or mechanical

stimuli in conditions of both chronic neuropathic and

inflammatory hypersensitivity. The gene deletion did not

affect DRG total cell numbers, the distribution of cell size,

or normal nociceptive responses to mechanical or thermal

stimuli. Given the role of the P2X7 receptor in triggering

post-translational processing of IL-1b, with the resulting

activation of an inflammatory cascade which includes

induction or release of cyclo-oxygenase 2 (Samad et al.,

2001), metalloproteinases (Flannery et al., 2003), inducible

nitric oxide synthase, superoxide products (Parvathenani

et al., 2003), and other cytokines, coupled with degenerative

changes that these mediators can induce, it appears that the

P2X7 receptor plays a pivotal role in initiating or

maintaining pathological changes as a result of inflamma-

tory or neuropathic insults. The locus of action may include

peripheral nerves, sensory neuronal cell bodies (Parvathe-

nani et al., 2003), or central pathways. Indeed, a cascade,

initiated by release of ATP following cellular perturbation

can be proposed in both the periphery and in the CNS. Thus,

in the periphery, it is likely that inflammation or a

neuropathic insult causes P2X7-dependent IL-1b release

Fig. 5. Serial sections of nerve immunostained with antibodies to P2X7 (a), GFA

GFAP (black) in Schwann cell]. (d) Representative P2X7 western blot using an an

polyacrylamide gel. Lanes 1–4 are extracts of human control nerve and lanes 5–8

from mononuclear, mast and Schwann cells (Colomar et al.,

2003), and in turn induces COX-2, nerve growth factor, and

other algogens (Woolf et al., 1996, 1997). Similarly, in the

CNS, maturation and release of IL-1b is able to induce

COX-2 (Samad et al., 2001) and other mediators, many of

which are thought to underlie increased neuronal excit-

ability, particularly in the spinal cord. ATP itself may play

a role in recruitment (Honda et al., 2001) and activation

(Verderio and Matteoli, 2001) of microglia or macrophages

which mediate plastic and degenerative changes, suggesting

that this transmitter is able to play a role in initiation of these

disease pathologies. Recent studies support initial findings

that P2X7 is expressed predominantly on cells of immune

origin (Sim et al., 2004), and functional studies suggest that

P2X7 is not expressed on neuronal structures (Kukley et al.,

2004), and thus P2X7 appears to play a critical role in glia-

neuronal signalling. The clinical relevance of these

proposals is supported by our findings of increased levels

of P2X7 receptor in injured nerve tissues from patients

suffering from chronic neuropathic pain. However, the

human tissue data needs to be interpreted with caution.

Further studies in greater numbers of patients with a range

of severity of pain symptoms, and in injured nerves from

patients who do not suffer from neuropathic pain, are

necessary to support a role for P2X7 in clinical pain

symptomatology. Moreover the lack of immunostaining in

sensory cell bodies, while consistent with current hypoth-

eses and finding, must be interpretated with caution, as this

may reflect a lack of sensitivity of the method.

In order to support the hypothesis that key actions of

activation of P2X7 are transduced via release of mature IL-1b,

we have also performed experiments in P2X7K/K animals to

P (b) or neurofilaments (c). [Inset in (a): Co-localisation of P2X7 (red) and

tibody dilution of 1:100,000 with human nerve extracts separated on a 6%

are extracts from painful injured nerve. Scale bars (a–c), 100 mm.

Fig. 6. P2X7-immunoreactivity in (a) control and (b) avulsion injured human DRG. [Inset in (a): Double immunostaining showing P2X7-immunoreactive

satellite/Schwann cells (red) surrounding a neuron and axons (neurofilament-black)]. GFAP-immunoreactivity in (c) control and (d) avulsion injured human

DRG. Scale bars (a–d), 50 mm.

I.P. Chessell et al. / Pain 114 (2005) 386–396394

demonstrate that, compared to controls, release of IL-1b is

absent following activation of the receptor. We have also

demonstrated that prior priming of cells using LPS is

necessary to achieve IL-1b release even in cells from normal

animals; LPS induces production of pro-IL-1b which is then

converted to mature IL-1b and released only following

stimulation of the P2X7 receptor (Colomar et al., 2003).

Previous experiments examining P2X7-stimulated IL-1brelease often utilise prolonged exposure to agonist—a

situation which seems unlikely to occur in vivo as ATP is

released in a pulsatile fashion from damaged or perturbed

cells. In addition, as part of delineation of the role of P2X7 in

physiological condition, it is important to establish a

relationship between receptor activation and IL-1b release in

a temporal manner; here we have shown that brief exposure of

cells to agonist commits those cells to process and release

IL-1b even after the agonist is removed—a phenomenon

likely to be relevant in vivo.

Cytometric analysis of cytokine concentrations in paws

and plasma of P2X7K/K and P2X7

C/C animals demonstrate

an attenuated cytokine response to FCA—challenge in the

animals with the gene deletion. Thus, the FCA-induced

release of IL-1b from P2X7K/K animal paws is significantly

reduced at both 1 and 7 days post FCA-injection compared

to wild type animals. However, there was no deficit in pro-

IL-1b mRNA expression in the P2X7K/K paws over the

time-course studied, indicating normal transcriptional

regulation of pro-IL-1b in these animals. As such, the

P2X7K/K animals appear to have a normal immune

capability at the transcriptional level but they possess a

deficiency in their ability to process pro-IL-1b in response

to an inflammatory stimulus. Interestingly, release of IL-10

was also reduced in the paws of the P2X7K/K animals. The

reason for the decrease in an ‘anti-inflammatory’ cytokine in

the P2X7K/K animals is not yet clear. However, studies in

macrophages derived from IL-10 deficient animals demon-

strated higher levels of TNF-alpha production upon

activation (Gazinelli et al., 2004), indicating an inverse

relationship as which is supported in the present study where

changes in concentrations of TNF-a in the paw samples

were temporally regulated (Table 1). It also raises the

interesting possibility that P2X7 may act directly upon IL-

10, as seen with IL-1b and IL-18 (Mehta et al., 2004).

In the sera, only IL-6 levels were significantly increased

in the P2X7C/C animals post-FCA. Differences in other

cytokine concentrations in sera from adjuvant-treated P2X7K

/K animals are perhaps anticipated given the established

relationships between IL-1b, IL-6 and MCP-1 (Sica et al.,

1990). However, eliciting an increase in IL-1beta levels

from blood samples may require re-stimulation in vitro

(Labasi et al., 2002) and it cannot be discounted that at 1 day

post inflammatory insult, any increases in ‘leaderless’

cytokines such as IL-1beta may have been missed. Another

interpretation is that inflammatory changes at the site if

action are not being translated systemically in this model.

Further studies to elucidate the inflammatory cascades

involved in initiating and mediating chronic pain are

ongoing.

In both clinical and preclinical settings there are marked

differences in the efficacy of agents to treat inflammatory

and neuropathic pain. Thus, while COX-2 inhibitors are

used successfully in the clinic for the treatment of chronic

inflammatory pain associated with osteo- and rheumatoid

arthritis, these agents are not generally effective in treating

I.P. Chessell et al. / Pain 114 (2005) 386–396 395

clinical neuropathic pain (Wallace, 2001). Similarly, drugs

used for the treatment of neuropathic pain, such as anti-

epileptic drugs and antidepressants, are ineffective in

adequately treating inflammatory pain (Jain, 2000).

Opioids, widely regarded as the most powerful analgesic

agents available, are effective in a broad range of pain states,

but chronic opiate use is associated with tolerability, side-

effect and addiction issues, which limits their use. Whilst

deletion of genes in animals provides important phenotypic

information, the possibility cannot be discounted that

compensatory changes, caused by persistent absence of a

receptor, may contribute to the phenotype in a way that

antagonism of the receptor after completion of development

may not. Efficacy of small molecule antagonists of P2X7

must be examined in these models of pain. However, the

data reported here strongly suggests that therapies based on

antagonism of this receptor may be capable of producing

broad-spectrum analgesia.

Acknowledgements

We thank Paul Facer for help with immunohistological

studies, Mr David Elliot for providing some of the nerve

specimens, and Ms Sarah Friel for assistance with the DRG

cell analysis.

References

Alcarez L, Baxter A, Bent J, Bowers K, Braddock M, Cladingboel D,

Donald D, Fagura M, Furber M, Laurent C, Lawson M, Martimore M,

McCormick M, Roberts N, Robertson M. Novel P2X7 receptor

antagonists. Bioorg Med Chem Lett 2004;13:4043–6.

Aley KO, McCarter G, Levine JD. Nitric oxide signaling in pain and

nociceptor sensitization in the rat. J Neurosci 1998;18:7008–14.

Budzinski M, Misterek K, Gumulka W, Dorociak A. Inhibition of inducible

nitric oxide synthase in persistant pain. Life Sci 2000;66:301–5.

Buell G, Chessell IP, Michel AD, Collo G, Salazzo M, Herren S,

Gretener D, Grahames CBA, Kaur R, Kosco-Vilbois M,

Humphrey PPA. Blockade of human P2X7 receptor function with a

monoclonal antibody. Blood 1998;92:3521–8.

Chessell IP, Simon J, Hibell AD, Michel AD, Barnard EA, Humphrey PPA.

Cloning and functional characterisation of the mouse P2X7 receptor.

Fed Eur Biolchem Soc Lett 1998;439:26–30.

Clark JD. Chronic pain prevalence and analgesic prescribing in a general

medical population. J Pain Sympt Manage 2003;23:131–7.

Clayton NM, Oakley I, Thompson S, Wheeldon A, Sargent RS, Bountra C.

Validation of the dual channel weight averager as an instrument for the

measurement of inflammatory pain. Br J Pharmacol 1997;120:219.

Collo G, Neidhart S, Kawashima E, Kosco-Vilbois M, North RA, Buell G.

Tissue distribution of the P2X7 receptor. Neuropharmacology 1997;36:

1277–83.

Colomar A, Marty V, Medina C, Combe C, Parnet P, Amedee T.

Maturation and release of interleukin-1b by lipopolysaccharide-primed

mouse Schwann cells require the stimulation of P2X7 receptors. J Biol

Chem 2003;278:30732–40.

Conquet F. Inactivation in vivo of metabotropic glutamate receptor 1 by

specific chromosomal insertion of a reported gene lacZ. Neurophar-

macology 1995;34:865–70.

Ferrari D, Chiozzi P, Falzoni S, Hanau S, Di Virgilio F. Purinergic

modulation of interleukin-1-beta release from microglial cells stimu-

lated with bacterial endotoxin. J Exp Med 1997;185:579–82.

Flannery CR, Little CB, Caterson B, Hughes CE. Effects of culture

conditions and exposure to catabolic stimulators (IL-1 and retinoic acid)

on the expression of matrix metalloproteinases (MMPs) and disintegrin

metalloproteinases (ADAMs) by articular cartilage chondrocytes.

Matrix Biol 2003;18:225–37.

Gazinelli RT, Wysocka M, Hieny S, Scharton-Kersten T, Cheever A, Kuhn R,

Muller W, Trinchieri G, Sher A. In the absence of endogenous IL-10, mice

acutely infected with Toxoplasma gondii succumb to a lethal immune

response dependent onCD4C T cells and accompanied by overproduction

of IL-12, IFN-gamma and TNF-alpha. J Immunol 2004;157:798–805.

Glass R, Burnstock G. Immunohistological identification of cells expres-

sing ATP-gated cation channels (P2X receptors) in the adult rat thyroid.

J Anat 2001;198:569–79.

Grahames CBA, Michel AD, Chessell IP, Humphrey PPA. Pharmacologi-

cal characterization of ATP- and LPS-induced IL-1b release in human

monocytes. Br J Pharmacol 1999;127:1915–21.

Honda S, Sasaki Y, Oshawa K, Imai Y, Nakamura Y, Inoue K, Kohsaka S.

Extracellular ATP or ADO induce chemotaxis of cultured microglia

through Gi/o-coupled P2Y receptors. J Neurosci 2001;21:1975–82.

Jain KK. A guide to drug evaluation for chronic pain. Emerg Drugs 2000;5:

241–57.

Kim M, Spelta V, North RA, Surprenant A. Differential assembly of rat

purinergic P2X7 receptor in immune cells of the brain and periphery.

J Biol Chem 2001;276:23232–67.

Kukley M, Stausberg P, Adelmann G, Chessell IP, Dietrich D. Ectonu-

cleotidases and nucleoside transporters mediate activation of adenosine

receptors by P2X7 receptor agonist 2 0-30-O-(4-benzoylbenzoyl)-ATP.

J Neurosci 2004;24:7128–39.

Labasi JM, Petrushova N, Donovan C, McCurdy S, Lira P, Payette MM,

Brissette W, Wicks JR, Audoly L, Gabel CA. Absence of the P2X7

receptor alters leukocyte function and attenuates an inflammatory

response. J Immunol 2002;168:6436–45.

Le Mouellic H, Lallemand Y, Brulet P. Targeted replacement of the

homeobox gene hox-3.1 by the escherichia-coli lacZ in mouse chimeric

embryos. Proc Natl Acad Sci USA 1990;87:4712–6.

MacDonald R, Bingham S, Bond BC, Parsons AA, Philpott KL. Determi-

nation of changes in mRNA expression in a rat model of neuropathic pain

by Taqman quantitative RT-PCR. Brain Res 2001;90:48–56.

Mehta VB, Hart J, Wewers MD. ATP-stimulated release of interleukin

(IL)-1beta and IL-18 requires priming by lipopolysaccharide and is

independent of caspase-1 cleavage. J Biol Chem 2004;276:3820–6.

MichelAD,Chessell IP,HumphreyPPA. Ioniceffectsonhumanrecombinant

P2X7 receptor function. N-S Arch Pharmacol 1999;359:102–9.

Parvathenani LK, Tertyshnikova S, Greco CR, Roberts SB, Robertson B,

Posmantur R. P2X7 mediates superoxide production in primary

microglia and is up-regulated in a transgenic mouse model of

Alzheimer’s disease. J Biol Chem 2003;278:13309–17.

Perregaux DG, Gabel CA. Interleukin-1b maturation and release in

response to ATP and nigericin. J Biol Chem 1994;269:15195–203.

Samad TA, Moore KA, Sapirstein A, Billet S, Allchorne A, Poole S,

Bonventre JV, Woolf CJ. Interleukin-1b-mediated induction of Cox-2

in the CNS contributes to inflammatory pain hypersensitivity. Nature

2001;410:471–5.

Seltzer Z, Dubner R, Shir Y. A novel behavioral model of neuropathic pain

disordersproducedinratsbypartial sciaticnerve injury.Pain2000;43:205–18.

Shu SY, Ju G, Fan LZ. The glucose oxidase-DAB-nickel method in

peroxidase histochemistry of the nervous system. Neurosci Lett 1988;

85:169–71.

Sica A, Wang JM, Colotta F, Dejana E, Mantovani A, Oppenheim JJ,

Larsen CG, Zachariae CO, Matsushima K. Monocyte chemotactic and

activating factor gene expression induced in endothelial cells by IL-1

and tumor necrosis factor. J Immunol 1990;144:3034–8.

Sim JA, Young MT, Sung H-Y, North RA, Surprenant A. Reanalysis of

P2X7 receptor expression in rodent brain. J Neurosci 2004;24:6307–14.

I.P. Chessell et al. / Pain 114 (2005) 386–396396

Solle M, Labasi J, Perregaux DG, Stam E, Petrushova N, Koller BH,

Griffiths RJ, Gabel CA. Altered cytokine production in mice lacking

P2X7 receptors. J Biol Chem 2001;276:125–32.

Steinberg TH, Newman AS, Swanson JA, Silverstein SC. ATP4K

permeabilizes the plasma membrane of mouse macrophages to

flourescent dyes. J Biol Chem 1987;262:8884–8.

Surprenant A, Rassendren F, Kawashima E, North RA, Buell G. The

cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor

(P2X7). Science 1996;272:735–8.

Verderio C, Matteoli M. ATP mediates calcium signalling between

astrocytes and microglia cells: modulation by IFN-g. J Immunol

2001;166:6383–91.

Wallace MS. Emerging drugs for neuropathic pain. Expert Opin Emerg

Drugs 2001;6:249–59.

Woolf CJ, Ma Q, Allchorne A, Poole S. Peripheral cell types contributing to

the hyperalgesic action of nerve growth factor in inflammation.

J Neurosci 1996;16:2716–23.

Woolf CJ, Allchorne A, Safieh-Garabedian B, Poole S. Cytokines,

nerve growth factor and inflammatory hyperlagesia: the

contribution of tumour necrosis factor a. Br J Pharmacol 1997;

121:417–24.

Yiangou Y, Facer P, Dyer NH, Chan CL, Knowles C, Williams NS,

Anand P. Vannilloid receptor 1 immunoreactivity in inflammed human

bowel. Lancet 2001;357:1338–9.