Neutrophils recruited by CXCR1/2 signalling mediate post-incisional pain

ORIGINAL ARTICLE

Neutrophils recruited by CXCR1/2 signalling mediatepost-incisional painE.U. Carreira1, V. Carregaro1, M.M. Teixeira2, A. Moriconi3, A. Aramini3, W.A. Verri Jr4, S.H. Ferreira1,F.Q. Cunha1, T.M. Cunha1

1 Department of Pharmacology, School of Medicine of Ribeirão Preto University of Sao Paulo, Brazil

2 Departamento de Bioquímica e Immunologia, Instituto Ciências Biológicas, Universidade Federal de Minas Gerais, Brazil

3 Dompé s.p.a., L’Aquila, Italy

4 Departamento de Patologia, Centro de Ciências Biológicas, Universidade Estadual de Londrina, Brazil

CorrespondenceThiago Mattar Cunha

E-mail: [email protected]

Funding sourcesThis work was supported by grants from the

Fundação de Amparo à Pesquisa do Estado

de São Paulo (FAPESP, Brazil), Conselho

Nacional de Pesquisa (CNPq, Brazil) and

Núcleo de Apoio a Pesquisa em Doenças

Inflamatórias (NAP-DIN). The American

Journal of Experts revised the language of the

article. Alessio Moriconi and Andrea Aramini

are employees of Dompé s.p.a., Italy. The

company has interests in the development of

ladarixin for the treatment of acute and

chronic inflammatory diseases.

Conflicts of interestNone declared.

Accepted for publication24 September 2012

doi:10.1002/j.1532-2149.2012.00240.x

Abstract

Background: Neutrophil recruitment mediated by the CXCL1/KCchemokine and its receptors CXCR1/CXCR2 plays a critical role in inflam-matory diseases. Recently, neutrophil migration and activation triggered byCXCL1-CXCR1/2 signalling was implicated in inflammatory nociception;however, their role in post-surgical pain has not been elucidated. In thisstudy, we addressed the function of neutrophils in the genesis of post-incisional pain in an experimental model of post-surgical pain.Methods: Mechanical hyperalgesia was determined with an electronicvon Frey test in a mouse hindpaw incisional model. Neutrophilaccumulation and the level of CXCL1/KC in the plantar tissue weredetermined by myeloperoxidase activity assay and enzyme-linkedimmunosorbent assay, respectively.Results: An incision in the mouse hindpaw produces long-lastingmechanical hyperalgesia that persists for at least 72 h after surgery.Following surgery, there was an increase in both neutrophil accumulationand the CXCL1/KC level in the incised paws. The depletion of the mouseneutrophils by vinblastine sulphate or anti-neutrophil antibody treatmentsreduced the mechanical hyperalgesia after paw incision. Furthermore, thetreatment of mice with ladarixin, an orally acting CXCR1/2 antagonist,also reduced both the mechanical hyperalgesia and the infiltration ofneutrophils in the incised paws.Conclusion: In conclusion, it appears that after surgical processes,neutrophils are recruited by CXCL1-CXCR1/2 signalling and participate inthe cascade of events, leading to mechanical hyperalgesia. These resultssuggest that blocking neutrophil migration through the inhibition ofCXCL1-CXCR1/2 signalling might be a target to control post-surgical pain.

1. Introduction

Post-operative pain is a common consequence of mostsurgeries. In fact, 30–40% of patients suffer moderateto severe pain during the post-operative period despitethe use of potent painkillers (Banik and Brennan,2004). To investigate the physiopathology of post-operative pain, several rodent models have beendeveloped, and the plantar incision model has been

extensively used. In this model, a small incision ismade in the plantar side of the mouse or rat hindpaw.After the plantar incision, animals exhibit long-lastinghyperalgesia to mechanical stimulation that persistsfor more than 3 days (Pogatzki et al., 2002; Xu andBrennan, 2011).

Underlying the mechanism involved in the genesis ofpost-operative pain, it was shown that incision in therat plantar hindpaw induced hyperexcitability of both

654 Eur J Pain 17 (2013) 654–663 © 2012 European Federation of International Association for the Study of Pain Chapters

primary afferent nociceptors and nociceptive neuronsin the spinal cord (Kang and Brennan, 2009). Interest-ingly, increased activity in the spinal dorsal hornneurons is reduced by treatment with local anaesthet-ics, suggesting that peripheral input is critical for centralsensitization (Pogatzki et al., 2002). With regard to theperipheral mechanisms of post-operative pain, there isevidence that the production and/or release of a varietyof inflammatory mediators at the wound site play animportant role. In fact, there are increased levels ofpronociceptive mediators, including interleukin (IL)-1ba, tumour necrosis factor-a, C5a, nerve growthfactor and substance P, in the tissue surrounding theincisions. Furthermore, inhibition of the productionand/or action of these mediators reduced pain hyper-sensitivity after surgery (Banik et al., 2005; Clark et al.,2006; Wu et al., 2007; Wolf et al., 2008; Sahbaieet al., 2009; Hu et al., 2010; Jang et al., 2011; Lianget al., 2012).

Neutrophils are highly motile phagocytic cells thatconstitute the first-line defence of the innate immunesystem against infection (Segal, 2005; Phillipson andKubes, 2011). However, uncontrolled migration ofneutrophils to the site of injury is involved in thepathophysiology of inflammatory/autoimmune dis-eases (Chou et al., 2010; Sadik et al., 2011). Neutro-phil migration is coordinated by inflammatorychemoattractant mediators that can be endogenouslygenerated by the host or released by bacteria (Foxmanet al., 1997; Sanz and Kubes, 2012). Among thesefactors, the CXCL1/KC chemokine and its receptor,CXCR1/2, which is highly expressed in neutrophils,are essential for the maximal recruitment of these cellsto the inflammatory site (Reutershan et al., 2006;Stillie et al., 2009).

We have shown that neutrophil migration driven byCXCL1/KC-CXCR1/2 signalling plays an essential rolein the sensitization of primary nociceptive neuronsand, consequently, in the genesis of inflammatorypain (Guerrero et al., 2008; Cunha et al., 2008a,2008c; Verri et al., 2009). However, there is no evi-

dence that these mechanisms contribute to post-operative pain. Therefore, in this study, we used theincisional mouse model to investigate whether neu-trophils triggered by the activation of CXCR1/2 par-ticipate in the genesis of post-operative pain. The roleof CXCR1/2 was assessed using ladarixin, a selectiveand orally acting antagonist of the CXCR1/2 receptors,previously disclosed with the name meraxin (Garauet al., 2006).

2. Methods

2.1 Drugs and cytokines

The following materials were obtained from thesources indicated: ladarixin was synthesized at theDompé s.p.a. Laboratories (L’Aquila, Italy); vinblas-tine sulphate and isotype mAb (rat IgG2b) were pur-chased from Sigma-Aldrich (St. Louis, MO, USA); ratanti-mouse Gr-1 mAb was purified from hybridomaRB6-8C clone; and the IL-1 receptor antagonist (IL-1ra) was a gift from Dr. Stephen Poole (National Insti-tute for Biological Standards and Control, SouthMimms, Hertfordshire, UK).

2.2 Animals

The pharmacological experiments were performed onmale C57BL/6J mice weighing between 20 and 30 g.The mice were housed in the animal care facility of theSchool of Medicine of Ribeirao Preto and moved to thetesting room at least 1 h before the experiments. Foodand water were available ad libitum. All behaviouraltests were performed between 9:00 a.m. and 5:00p.m., and the animals were used for experimentationonly once. Behavioural tests were performed onanimals in a randomized order in a double-blindfashion, in which the person who performed thesurgery was not the one who made the behaviouralassessment. Animal care and handling procedureswere in accordance with the guidelines of the Inter-national Association for the Study of Pain on the useof animals in pain research, and with the approval ofthe Ethics Committee of the School of Medicine ofRibeirao Preto, University of São Paulo (processnumber: 119/2009). For pharmacokinetic studies,C57BL/6J mice (Charles River, Calco, Lecco, Italy)were kept in a temperature-controlled environmentwith a 12-h light-dark cycle and allowed tap water andstandard mouse chow pellets ad libitum. All procedureswere performed in the animal facilities according tothe ethical guidelines for the conduct of animal

What’s already known about this topic?• Neutrophils seem to play a role in the genesis of

inflammatory and neuropathic pain.

What does this study add?• Described the involvement of neutrophils in the

genesis of post-operative pain and the mecha-nisms involved in their recruitment throughCXCR2 signalling.

E.U. Carreira et al. Role of neutrophils in incisional pain

655Eur J Pain 17 (2013) 654–663 © 2012 European Federation of International Association for the Study of Pain Chapters

research (Authorization Italian Ministry of Health No.271/95-B D.Lvo 116/92; Gazzetta Ufficiale dellaRepubblica Italiana No. 40, 18 February 1992; EECCouncil Directive 86/609 OJ L 358, 1, 12 December1987; NIH Guide for the Care and Use of LaboratoryAnimals, NIH Publication No. 85-23, 1985).

2.3 Pharmacokinetic studies

All animals were weighed (about 28–30 g) immedi-ately before the test. The appropriate dose volume ofladarixin, calculated for each animal according to bodyweight, was administered by oral gavage (5 mL/kg).Twelve animals were used for pharmacokineticstudies, and blood samples (about 220 mL) were col-lected by retro-orbital bleeding from each treatedanimal at 5 and 30 min, and 1, 2, 4, 8 and 24 h afteroral administration of ladarixin (50 mg/kg). Eachaliquot of withdrawn blood was collected in lithiumheparin tubes and centrifuged at 2000 g for 10 min.Plasma samples were stored at -80 °C until analysis.The bioanalytical method used for ladarixin detectionin plasma samples was described previously (Garauet al., 2006).

2.4 Surgical procedures

Mouse paw incisions were performed as describedpreviously (Jang et al., 2011). Briefly, mice wereanaesthetized with 1.5–2.0% isoflurane deliveredthrough a nose cone, and the right hindpaw of themouse was sterilized with 10% povidone-iodine.After sterile preparation of the hindpaw, a 0.5-cmlongitudinal incision was made through the skin andfascia of the plantar surface of the foot with a number11 scalpel blade. The incision started 0.2 cm from theproximal edge of the heel and extended distally. Theunderlying muscle was elevated with curved forceps,leaving the muscle origin and insertion intact. Afterwound haemostasis, the skin was closed with twosubcutaneous mattress sutures using 6-0 nylon andcovered with an antibiotic ointment. In some experi-ments, control mice underwent a sham procedurethat consisted of anaesthesia, antiseptic preparationand application of the antibiotic ointment without anincision.

2.5 Mechanical nociceptive paw test

Mechanical hyperalgesia was tested in mice as pre-viously reported (Cunha et al., 2004). Mice were

housed in acrylic cages (8 ¥ 8 ¥ 12 cm) with wire gridfloors 15–30 min before the start of testing. Thethreshold of nociceptive responsiveness to mechanicalstimuli applied to the right hindpaw was assessedusing a dynamic plantar aesthesiometer (model37400, Ugo Basile, Milan, Italy), an electronic andautomatic version of the von Frey test (Motta et al.,2009). The test consisted of evoking a hindpaw flexionreflex with an automatic force transducer adaptedwith a 0.5-mm2 polypropylene tip. The investigatorwas trained to apply the tip to the lateral area of theincised hindpaw with a gradual increase in pressure of2 g/s. The gradual increase in pressure was performedin blinded experiments. The upper limit of pressurewas 20 g. The endpoint was characterized by theremoval of the paw, followed by clear flinching move-ments. After paw withdrawal, the intensity of thepressure was automatically recorded, and the finalvalue for the response was obtained by averagingthree measurements. The animals were tested beforeand after treatments. The results are expressed as thewithdrawal threshold, in g. The withdrawal thresholdwas 8.1 � 0.2 g [mean � standard error of the mean(SEM); n = 30] before the paw incision.

2.6 Determination of neutrophil accumulationin the hindpaws

Myeloperoxidase (MPO) activity was used as an indexof neutrophil accumulation in the mouse plantartissues and was based on a kinetic-colorimetric assay,as previously described (Cunha et al., 2008b). Samplesof subcutaneous plantar tissue were collected at dif-ferent time points after paw incision. Samples werecollected in 50 mmol/L K2HPO4 buffer (pH 6.0) con-taining 0.5% hexadecyltrimethylammonium bromideand stored at -80 °C. Prior to the assay, the tissue washomogenized using a Polytron (PT3100, KinematicaAG, Lucerne, Switzerland) and centrifuged at 13,000 gfor 4 min. In our experimental conditions using lowpH, the MPO assay did not detect the activity of thisenzyme in mononuclear cells (data not shown). Toprepare the solution for analysis, 10 mL of supernatantwas mixed with 200 mL of 50 mmol/L phosphatebuffer (pH 6.0) containing 0.167 mg/mL O-dianisidinedihydrochloride and 0.0005% hydrogen peroxide.The solution was analysed by spectrophotometry(Spectra max) for MPO activity determination at450 nm, with three readings in 1 min. The MPO activ-ity was compared with a standard curve of mouseneutrophils. The results were presented as the numberof neutrophils ¥ 106/mg tissue.

Role of neutrophils in incisional pain E.U. Carreira et al.


2.7 CXCL1/KC chemokine measurements

At the indicated time points after paw incision, theanimals were terminally anaesthetized and the plantartissues were removed. The samples were triturated andhomogenized in 500 mL of phosphate-buffered salinecontaining 0.05% Tween 20, 0.1 mmol/L phenylmeth-ylsulfonyl fluoride, 0.1 mmol/L benzethonium chlo-ride, 10 mmol/L ethylenediaminetetraacetic acid and20 kallikrein international units of aprotinin A, fol-lowed by centrifugation at 2000 g for 10 min. Thesupernatants were stored at -70 °C until further analy-sis. The level of CXCL1/KC was evaluated using sand-wich enzyme-linked immunosorbent assays, asdescribed previously (Cunha et al., 2005). Briefly,microtitre plates were coated with an immunoaffinity-purified polyclonal sheep antibody against CXCL1/KC(1 mg/mL) and incubated overnight at 4 °C. Afterblocking the plates, recombinant murine CXCL1/KCstandards at various concentrations and the sampleswere added in duplicate and incubated overnight at4 °C. A rabbit biotinylated immunoaffinity-purifiedantibody against CXCL1/KC (0.2 mg/mL) was added,followed by incubation at room temperature for 1 h.Next, 50 mL of avidin-HRP (horseradish peroxidase)(1 : 5000 dilution; DAKO A/S, Glostrup, Denmark)was added to each well, and after 30 min, the plateswere washed and the colour reagent OPD (200 mg/well;Sigma) was added. After 15 min, the reaction wasstopped with 1 mol/L H2SO4 and the optical density at490 nm was measured. The results are expressed as pgof CXCL1/KC per paw. As a control, the concentrationof CXCL1/KC was determined in naïve mouse pawtissue (Cunha et al., 2008b).

2.8 Experimental protocols

The role of neutrophils in the genesis of incisionalnociception was investigated by depleting mouse neu-trophils using the following two protocols: (1) vinblas-tine sulphate-treated mice: Mice were treated withvinblastine sulphate (5 mg/kg, i.v.) 72 h prior to pawincision, and mechanical hyperalgesia was evaluatedfor 24 h subsequent to paw incision, followed byremoval of the paw tissue for evaluation of MPO activ-ity (Raeburn et al., 2002; Ting et al., 2008); and (2)anti-neutrophil antibody-treated mice: Mice weretreated intravenously with an antibody against GR-1(100 mg/mouse) 7 days and 24 h prior to paw incision(Guerrero et al., 2008). The specificity of this antibodywas demonstrated previously (Hestdal et al., 1991). Asa control group, animals were treated with rat IgG2b,the isotype control of the GR-1 antibody. Mechanical

hyperalgesia was evaluated for 24 h subsequent topaw incision, followed by removal of the paw tissuefor evaluation of MPO activity.

Ladarixin was used to evaluate the role of CXCR1/2in the genesis of neutrophil migration and mechanicalhyperalgesia after paw incision. It is a selective andorally acting antagonist of the CXCR1/2 receptors(Garau et al., 2006). Mice were treated orally once aday with ladarixin (50 mg/kg, dissolved in saline), andmechanical hyperalgesia was evaluated for 26 h sub-sequent to paw incision, followed by removal of thepaw tissue for evaluation of MPO activity.

In another set of experiments, the level ofCXCL1/KC was determined 6 h after paw incision inmice treated with the anti-neutrophil antibody, theisotype mAb, ladarixin or IL-1 receptor antagonist (IL-1ra; 30 mg/kg, i.v. 15 min prior to paw incision).

2.9 Statistical analyses

The results are presented as means � SEM and sub-mitted to statistical analyses using parametric tests.The results are representative of two separate experi-ments with five animals per group. The data obtainedin each experiment were not combined for statisticalanalysis, they were analysed individually and the sig-nificances were similar. Two-way analysis of variance(ANOVA) was used to compare the groups at all times(curves) when the hypernociceptive responses weremeasured at different times after the stimulus injec-tion. The factors analysed were treatment, time, andtime ¥ treatment interaction, followed by Bonferroni’stest for each time point. Alternatively, when thehypernociceptive responses were measured once afterthe stimulus injection, the differences betweenresponses were evaluated by one-way ANOVA, fol-lowed by Bonferroni’s t-test. Differences were consid-ered to be statistically significant at p < 0.05.

3. Results

3.1 Mechanical hyperalgesia is accompaniedby increased neutrophil infiltration in theincised mouse paw

Firstly, it was observed that a surgical incision in themouse hindpaw produces an intense and long-lastingdecrease in the mechanical nociceptive threshold asevaluated by the electronic and automatic von Freytest (Fig. 1A). The two-way ANOVA indicated a sig-nificant effect of time (F7,80 = 27.1; p < 0.0001), treat-ment (F1,80 = 701.8; p < 0.0001) and time ¥ treatment



interaction (F7,80 = 26.4; p < 0.0001). The mechanicalhyperalgesia caused by paw incision persisted for atleast 72 h after surgery and returned to the controllevel after 96 h (Fig. 1A). Furthermore, one-wayanalyses (F7,35 = 2.8) showed that mechanical hyperal-gesia was accompanied by a considerable increase(p < 0.02) in MPO activity in the incised paw, suggest-ing an infiltration of neutrophils (Fig. 1B).

Neutrophils are involved in mechanical hyperalge-sia after paw incision.

To investigate the role of neutrophils in the genesisof post-operative pain, two different approaches wereused to deplete the neutrophils. Firstly, neutrophilswere depleted by pretreating the mice with vinblastinesulphate. The two-way ANOVA indicated a significanteffect of time (F4,40 = 32.0; p < 0.0001), treatment(F1,40 = 128.1; p < 0.0001) and time ¥ treatment inter-action (F4,40 = 9.1; p < 0.0001). Vinblastine treatmentreduced the mechanical hyperalgesia produced by thepaw incision in all cases (Fig. 2A). One-way analyses(F2,11 = 80.9) showed that incision-induced neutrophilmigration to the mouse paw was also reduced(p < 0.001) by vinblastine treatment (Fig. 2B). Sec-ondly, neutrophils were depleted by treating the micewith an anti-neutrophil antibody. In this case, thetwo-way ANOVA indicated a significant effect of

time (F4,40 = 49.9; p < 0.0001), treatment (F1,40 = 87.8;p < 0.0001) and time ¥ treatment interaction (F4,40 =8.5; p < 0.0001). The anti-neutrophil antibody treat-ment also reduced the mechanical hyperalgesia afterthe paw incision in all cases (Fig. 3A). Moreover, one-way analyses (F2,12 = 76.2) indicated that neutrophilaccumulation in the incised paw was also significantly(p < 0.0001) inhibited by neutrophil depletion(Fig. 3B).

3.2 CXCL1/KC via CXCR1/2 receptors mediatesneutrophil migration and mechanicalhyperalgesia after paw incision

Next, we evaluated whether the CXCL1/KC chemok-ine, through binding of the CXCR1/2 receptors, isinvolved in the genesis of post-incisional pain. Firstly,the effect of the paw incision on the local levelof CXCL1/KC was evaluated. One-way ANOVA(F6,20 = 31.9) indicated that the paw incision produceda significant (p < 0.0001) increase in the level ofCXCL1/KC. The CXCL1/KC level peaked at least 2 hafter the paw incision and was still significant at least24 h after the paw incision (Fig. 4A). Treatment withthe anti-neutrophil antibody did not change the pawincision-induced CXCL1/KC production (F2,11 = 200.0;

A B Figure 1 Mechanical hyperalgesia induced by

paw incision is accompanied by local infiltra-

tion of neutrophils. Mice were subjected to

either hindpaw incision or sham procedure

(Control). Mechanical nociceptive thresholds

were determined prior to and up to 96 h after

surgery, and this was followed by collection of

the plantar hindpaw tissue for myeloperoxi-

dase (MPO) analysis (panels A and B, respec-

tively). Data are expressed as the

mean � standard error of the mean. *Indi-

cates statistical significance compared with

the control sham group. p < 0.05.

A B Figure 2 Effect of vinblastine sulphate on

mechanical hyperalgesia and neutrophil migra-

tion after paw incision. Mice were treated with

vinblastine sulphate (5 mg/kg, i.v.) or vehicle

(saline-Sal) 72 h prior to paw incision. Mechani-

cal nociceptive thresholds were measured

prior to and up to 24 h after surgery, and this

was followed by collection of the plantar

hindpaw tissue for myeloperoxidase (MPO)

analysis (panels A and B, respectively). Data

are expressed as the mean � standard error

of the mean. *Indicates statistical significance

compared with the vehicle-treated group.

p < 0.05.



p = 0.21; Fig. 4B). However, one-way analyses(F2,12 = 47.8) showed that the treatment of mice withIL-1ra significantly (p < 0.0001) reduced the CXCL/KCproduction after the paw incision (Fig. 4C).

Secondly, the incision-induced mechanical hyperal-gesia was reduced by treatment with ladarixin, a dualCXCR1/2 antagonist, at all evaluated times (Fig. 5A).The two-way ANOVA indicated a significant effect oftime (F5,46 = 21.9; p < 0.0001), treatment (F1,46 = 92.8;p < 0.0001) and time ¥ treatment interaction (F5,46 =3.4; p < 0.0001). One-way analyses (F2,12 = 90.5) indi-cated that ladarixin treatment also produced a signi-ficant (p < 0.0001) reduction in neutrophil accumula-tion in the incised mouse paw (Fig. 5B). Ladarixintreatment did not change the level of production ofCXCL1/KC (data not shown).

3.3 Pharmacokinetic studies of ladarixinin mice

The pharmacokinetic properties of the ladarixinpotassium salt were evaluated in mice plasmasamples. Ladarixin is highly soluble (>3 mg/mL), andthe salt was dissolved in a buffer solution for phar-macokinetic experiments. Ladarixin is highly bio-available after oral administration, with an oralbioavailability of 98%, a Cmax of 190 mg/mL and a Tmax

of approximately 3 h. The elimination of ladarixin ina mouse after i.v. administration was very slow, witha mean elimination half-life greater than 19 h.Ladarixin also had a mean total body clearance of6 mL/h/kg and a small distribution volume(Vss = 218 mL/kg).

A BFigure 3 Effect of anti-neutrophil antibody on

mechanical hyperalgesia and neutrophil migra-

tion after paw incision. Mice were treated with

either an antibody against neutrophils (100 mg/

mouse, i.v) or an isotype antibody (control Ab)

for 7 days and 24 h prior to paw incisions.

Mechanical nociceptive thresholds were mea-

sured prior to and up to 24 h after surgery,

followed by collection of the plantar hindpaw

tissue for MPO analysis (panels A and B,

respectively). Data are expressed as the



the vehicle-treated group. p < 0.05.

A B C

Figure 4 Local level of CXCL1/KC after paw incision. Mice were subjected to paw incision and the plantar hindpaw tissue was collected for CXCL1/KC

measurements up to 96 h after surgery (panel A). Mice were treated with either an antibody against neutrophils (100 mg/mouse, i.v) or an isotype antibody

(control Ab) for 7 days and 24 h prior to paw incisions. At 6 h after the paw incision, the plantar hindpaw tissue was collected for CXCL1/KC measurements

(panel B). Mice were treated with IL-1 receptor antagonist (IL-1ra; 30 mg/kg, i.v. 15 min prior to paw incision). At 6 h after the paw incision, the plantar

hindpaw tissue was collected for CXCL1/KC measurements (panel C). Data are expressed as the mean � standard error of the mean. *Indicates statistical

significance compared with the sham or vehicle-treated group. p < 0.05.



4. Discussion and conclusion

Neutrophils are the first-line defence of the immuno-logical system against a variety of pathogens. Despitethe importance of neutrophils to host defence, theyrepresent a potential cause of tissue damage in severalinflammatory diseases, such as rheumatoid arthritis.Indeed, at the site of inflammation, the interaction ofrecruited neutrophils with resident cells, local inflam-matory mediators and/or extracellular matrix maylead to the production of several other mediators,including cytokines/chemokines, degrading enzymes,oxygen and nitrogen species, and metalloproteases,which may further amplify the inflammatory responseand injure the surrounding tissue (Kasama et al.,2005; Eyles et al., 2006; Palmer et al., 2006). Recently,neutrophils were implicated in the genesis of inflam-matory pain (Cunha et al., 2008a). In fact, the prono-ciceptive role of cytokines was dependent on therecruitment and activation of neutrophils, and that, inturn, may produce inflammatory mediators that sen-sitize the primary nociceptive neurons that causehyperalgesia (Cunha et al., 2008a). Here, we extendthese observations and demonstrate that neutrophilstriggered by the activation of CXCR1/2 are involved inthe genesis of post-incisional pain in mice, an appro-priate model of post-operative pain in humans (Xuand Brennan, 2011).

The pronociceptive action of neutrophils was firstsuggested 30 years ago (Castro and Ferreira, 1979).For instance, neutrophils present in the joints of dogsenhanced the articular nociception promoted bylipopolysaccharide administration (Castro and Fer-reira, 1979). This indirect evidence was followed byseveral studies, showing that neutrophils participate inthe genesis of a large number of pathological types ofpain, including inflammatory (paw and joint), allergicand neuropathic pain (Levine et al., 1985; Perkins andTracey, 2000; Lavich et al., 2006; Guerrero et al., 2008;

Ting et al., 2008; Cunha et al., 2008b, 2008c; Verriet al., 2009; Pinto et al., 2010). However, to the best ofour knowledge, this is the first demonstration of neu-trophil mediation of post-operative pain. In this study,two pharmacological tools were used to deplete mouseneutrophils to investigate their role in the genesis ofpost-operative pain. Firstly, the treatment of mice withan acute dose of vinblastine sulphate reduced themechanical hyperalgesia after paw incision. It is note-worthy that in this acute protocol, vinblastine treat-ment caused a robust reduction of at least 78% in theblood neutrophil content and had only a modest effect(15 %) on mononuclear cells (data not shown).Animals that are neutrophil-depleted due to vinblas-tine sulphate treatment have been successfully used toinvestigate the role of neutrophils in the genesis ofinflammatory pain in rats (Ting et al., 2008). Forinstance, it was shown that vinblastine-treated ratspresent reduced inflammatory hyperalgesia inducedby carrageenan (Ting et al., 2008). The effect of vin-blastine on neutrophil counts could be verified by thefact that MPO activity was locally reduced after mousepaw incision. However, the reduction in mechanicalhyperalgesia after the incision was more prominentthan the reduction in neutrophil accumulation in themouse paw. Therefore, we could not disregard the factthat the reduction in the other cell populations otherthan the neutrophils could account for the effect of thevinblastine treatment.

To verify that neutrophil infiltration at the incisionsite does indeed mediate mechanical hyperalgesia,neutrophils were depleted using a specific anti-neutrophil antibody (Hestdal et al., 1991). Corroborat-ing the result obtained with vinblastine treatment, theanti-neutrophil antibody reduced mechanical hyper-algesia after the incision. It is important to mentionthat after treatment with the anti-neutrophil antibody,the blood leukocyte counts were reduced by approxi-mately 72% for the neutrophils compared with the

A B Figure 5 Effect of CXCR1/2 signalling block-

ade on mechanical hyperalgesia and neutro-

phil migration after paw incision. Mice were

treated with ladarixin (50 mg/kg, p.o.) or

vehicle (saline) once a day, starting 50 min

prior to paw incisions. Mechanical nociceptive

thresholds were measured prior to and up to

26 h after surgery, followed by collection of

the plantar hindpaw tissue for myeloperoxi-

dase (MPO) analysis (panels A and B, respec-

tively). Data are expressed as the



the vehicle-treated group. p < 0.05.



isotype mAb-treated animals, whereas the number oflymphocytes and monocytes did not change signifi-cantly (Guerrero et al., 2008). Although the anti-neutrophil antibody treatment caused a 72%reduction in the number of circulating neutrophils, itcaused more than 98% reduction in neutrophil migra-tion to the incised paws. This difference could beexplained by the fact that the fraction of the neutro-phils in the circulation is still not competent to migrateto the paws, most likely due to the continuous effect ofthe antibody.

Neutrophil migration is a multifactorial process inwhich a vast number of inflammatory mediators play arole (Williams et al., 2011; Sanz and Kubes, 2012). Wehave demonstrated that CXCL1/KC via CXCR1/2mediates inflammatory pain by triggering neutrophilaccumulation in the inflamed site (Cunha et al.,2008c). Here, we show that these mechanisms alsooperate in the genesis of post-incisional pain. In fact, adual CXCR1/CXCR2 antagonist inhibited the mechani-cal hyperalgesia induced by the paw incision. Ladarixinwas recently developed as a clinical candidate of a novelclass of CXCR1 and CXCR2 non-competitive allostericinhibitors (Garau et al., 2006). When dosed orally inmouse, ladarixin was completely absorbed, with anoral bioavailability of 98%, and showed plasma levelscomparable to the range of concentrations that result inthe maximal inhibition of CXCR1/CXCR2 activation invitro (Garau et al., 2006). This compound effectivelyreduced neutrophil migration in different models ofischaemia/reperfusion, and it also reduced experimen-tal angiogenesis in vivo (Garau et al., 2006; Bertiniet al., 2012). In agreement with pharmacological,behavioural and previously published data, CXCL1/KClevels increased significantly after paw incision(Sahbaie et al., 2009; Hu et al., 2010). There is evidencethat neutrophil, at the site of injury, might be involvedin the production of chemokines (Endlich et al., 2002).However, in our experimental model, ladarixin treat-ment, which reduced neutrophil recruitment, did notchange the production of CXCL1/KC. Thus, it is plau-sible to suggest that resident cells, such as macrophagesand keratinocytes, are the main source of CXCL1/KC(Clark et al., 2007). In agreement, it was previouslyshown that inhibition of neutrophil migration did notchange the production and/or release of CXCL1chemokine in the inflamed paws (Cunha et al., 2008c).

It is noteworthy that CXCR1/KC levels peaked at2 h after paw incision, reducing thereafter. However,this CXCL1/KC production does not correlate with thetime course of neutrophil migration, which peaks at24 h after paw incision. Nevertheless, some alterna-tives should be suggested to explain this contradiction.

Firstly, since in the present study an antagonist ofCXCR1/2 was used to explore the role of CXCL1/KCchemokines on neutrophil recruitment and nocicep-tion, we could not discard that other CXCR1/2 miceligands, such as CXCL2/MIP-2, could also be partici-pating in neutrophil recruitment in this model. In fact,it was demonstrated that there is a biphasic chemok-ine cascade after surgical injury and that functionallyequivalent chemokines, CXCL1/KC and CXCL2/MIP-2, exhibit very different temporal profiles ofexpression. For instance, CXCL1/KC is expressed atearlier time points, while CXCL2/MIP-2 predominatesduring the later phase (Endlich et al., 2002). Secondly,another possible explanation is that the earlier pro-duction of CXCL1/KC could be triggering a cascade ofother chemotactic mediators, which, in turn, cause amaximal recruitment of neutrophils (Grespan et al.,2008; Vieira et al., 2009). Another important periph-eral pronociceptive mediator involved in the genesis ofpost-incisional pain seems to be IL-1b (Wolf et al.,2008; Hu et al., 2010). Indeed, genetic or pharmaco-logical blockade of IL-1b decreased the nociceptiveresponse to an incisional wound. Here, we show thatthe production of CXCL1/KC in the incised mousepaw is triggered by IL-1b, confirming the results of aprevious study (Hu et al., 2010).

The results of our study lead us to ask: how doneutrophils contribute to post-operative pain? Ininflammatory pain models, neutrophils contribute tothe sensitization of primary nociceptive neurons bymediating the release of direct-acting hyperalgesicmediators, such as PGE2 (Guerrero et al., 2008; Cunhaet al., 2008a). Therefore, it is plausible that after pawincision, neutrophils could participate in the produc-tion of peripheral prostaglandins, which are involved inthe genesis of post-incisional pain. Neutrophils couldalso be implicated in other peripheral mechanisms ofpost-surgical pain, including the production of freeradicals, metalloproteases and hydrogen protons(Steen et al., 1992; Wang et al., 2004; Woo et al.,2004). Additionally, neutrophils can also be involved inthe generation of endothelins that could synergize withother hyperalgesic mediators, thus contributing to theincrease in nociceptor excitability and, consequently,the establishment of post-operative pain (Mujendaet al., 2007). For instance, there is evidence that met-alloprotease 9, which is expressed in neutrophils, isable to convert big ET-1 to ET-1 (Fernandez-Patronet al., 2001).

In conclusion, this study demonstrated the impor-tance of neutrophils for the genesis of post-operativepain using an appropriate mouse model. Neutrophilaccumulation in the incised paw of the mouse was



dependent on the activation of CXCR1/2 signalling bylocally produced CXCL1/KC. These results reinforcethe role of neutrophils in hyperalgesic states andsuggest that blockade of neutrophil migration throughinhibition of CXCL1/CXCR1/2 signalling may be anexcellent target for the control of post-operative pain.Nevertheless, one should be aware that although neu-trophils seem to play a role in post-operative pain,they might also be involved in wound healing or fight-ing against infection. Therefore, neutrophil-triggeredmechanisms of post-operative pain, including specificneutrophil-derived pronociceptive mediators, couldalso be considered during the development of noveltherapies.

Acknowledgements

The authors gratefully acknowledge the technical assistanceof Sérgio R. Rosa, Giuliana B. Francisco and Ieda ReginaSchivo.

Authors contribution

E.U.C., V.C., A.M., A.A., W.A.V. and T.M.C. had substantialcontributions to conception and design, or acquisition ofdata, or analysis and interpretation of data; T.M.C., A.M.,F.Q.C., M.M.T. and S.H.F. draft the article or revised it criti-cally for important intellectual content; T.M.C., A.M.,W.A.V., M.M.T., F.Q.C. and S.H.F. finally approved theversion to be published.

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Neutrophils recruited by CXCR1/2 signalling mediate post-incisional pain

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