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Originally published online as doi:10.1189/jlb.0805452 on March 7, 2006

Published online before print March 7, 2006
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(Journal of Leukocyte Biology. 2006;79:1022-1032.)
© 2006 by Society for Leukocyte Biology

Selective local PMN recruitment by CXCL1 or CXCL2/3 injection does not cause inflammatory pain

Heike L. Rittner, Shaaban A. Mousa, Dominika Labuz, Karin Beschmann, Michael Schäfer, Christoph Stein and Alexander Brack1

Klinik für Anaesthesiologie und operative Intensivmedizin, Charité–Universitätsmedizin Berlin, Campus Benjamin Franklin, Germany

1Correspondence: Klinik für Anaesthesiologie und operative Intensivmedizin, Charité–Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, D-12200 Berlin, Germany. E-mail: alexander.brack{at}charite.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polymorphonuclear cells (PMN) are recruited in early inflammation and are believed to contribute to inflammatory pain. However, studies demonstrating a hyperalgesic role of PMN did not examine selective PMN recruitment or did not document effective PMN recruitment. We hypothesized that hyperalgesia does not develop after chemokine-induced PMN selective recruitment and is independent of PMN infiltration in complete Freund’s adjuvant (CFA)-induced, local inflammation. PMN were recruited by intraplantar injection of CXC chemokine ligand 1 (CXCL1; keratinocyte-derived chemokine), CXCL2/3 (macrophage inflammatory protein-2), or CFA, with or without preceding systemic PMN depletion. Chemokine inoculation resulted in dose (0–30 µg)- and time (0–12 h)-dependent, selective recruitment of PMN as quantified by flow cytometry. CXCL2/3, but not CXCL1, was less effective at high doses, probably as a result of significant down-regulation of CXC chemokine receptor 2 expression on blood PMN. Neither chemokine caused mechanical or thermal hyperalgesia as determined by the Randall-Selitto and Hargreaves test, respectively, despite comparable expression of activation markers (i.e., CD11b, CD18, and L-selectin) on infiltrating PMN. In contrast, CFA injection induced hyperalgesia, independent of PMN recruitment. c-Fos mRNA and immunoreactivity in the spinal cord were increased significantly after inoculation of CFA-independent of PMN-migration but not of CXCL2/3. Measurement of potential hyperalgesic mediators showed that hyperalgesia correlated with local prostaglandin E2 (PGE2) but not with interleukin-1ß production. In summary, hyperalgesia, local PGE2 production, and spinal c-Fos expression occur after CFA-induced inflammation but not after CXCL1- or CXCL2/3-induced, selective PMN recruitment. Thus, PMN seem to be less important in inflammatory hyperalgesia than previously thought.

Key Words: chemokine • CXCR2 • neutrophil • hyperalgesia • complete Freund’s adjuvant


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pain is a cardinal symptom of inflammation. In the early phase, inflammation is accompanied by recruitment of polymorphonuclear cells (PMN) and monocytes. As pain and leukocyte recruitment commonly occur simultaneously, leukocytes are thought to contribute to the generation of pain. Indeed, PMN and other leukocytes have been shown to contain and release numerous hyperalgesic (i.e., pain-enhancing) mediators such as cytokines [e.g., interleukin (IL)-1ß], protons, nerve growth factor (NGF), prostaglandins (e.g., PGE2), and leukotrienes (LTs; reviewed in refs. [1 , 2 ]). However, PMN and other leukocytes also contain potent antinociceptive (i.e., pain-reducing) mediators such as opioid peptides (reviewed in refs. [1 , 3 ]), indicating that leukocyte recruitment might as well counteract and not necessarily cause pain.

Previous studies demonstrated that PMN could be recruited locally by mediators such as formyl-methionine-leucine-phenylalanine (fMLP), LTB4, complement C5a, and NGF [4 5 6 7 ]. Injection of these mediators induced pain (i.e., mechanical or thermal hyperalgesia) and was attenuated by PMN depletion. However, the receptors for these mediators (i.e., formylated peptide receptor, LTB4 receptor 1, C5a receptor, and trkA, respectively) are not expressed exclusively on PMN but also on other leukocyte subpopulations [8 9 10 ]. This raises the question of whether selective PMN recruitment indeed causes pain.

In fact, other studies do not support a prominent role for PMN in the development of pain. For example, local glycogen injection induces PMN recruitment without an accompanying hyperalgesia [5 ]. In a model of acetic acid-induced peritoneal irritation, visceral pain was reduced by intraperitoneal glycogen injection, and this was abolished by prior PMN depletion [11 ]. In our own studies, mechanical hyperalgesia was neither influenced by PMN depletion nor by enhancement of PMN recruitment in local complete Freund’s adjuvant (CFA)-induced inflammation [12 13 14 ].

Selective PMN recruitment in rats can be achieved by CXC chemokine ligand 1 [CXCL1; keratinocyte-derived chemokine (KC)] and CXCL2/3 (macrophage inflammatory protein-2 [15 , 16 ]). These chemokines bind to the same chemokine receptor {CXC chemokine receptor 2 (CXCR2) [17 , 18 ]}, which is expressed widely during embryonic development [19 ] in the central nervous system [20 ] and on leukocytes (i.e., PMN and monocytes). Although CXCR2 ligands are clearly chemotactic for PMN, monocyte chemotaxis is controversial [21 22 23 24 ]. Studies in CXCR2-deficient mice showed that PMN recruitment in inflammation is consistently abolished, and effects on monocyte recruitment are contradictory [25 26 27 ].

We have previously examined the role of leukocytes and their chemokine-mediated recruitment in opioid-mediated peripheral pain control (i.e., antinociception). In CFA-induced inflammation, PMN are the predominant, opioid-containing leukocyte subpopulation in early inflammation [28 ], and their recruitment is mediated by CXCR2 ligands. Selective PMN depletion or local blockade of CXCL1 and CXCL2/3 inhibits antinociception significantly [12 , 29 ]. It is important that opioid-mediated antinociception needs to be elicited specifically (e.g., local injection of a releasing agent such as corticotrophin-releasing factor or exposure to cold water swim stress in rats [30 ] or postoperative stress in humans [31 ]). Taken together, PMN can produce antinociception by the triggered release of opioid peptides.

Noxious stimulation leads to the activation of specialized, primary, afferent neurons (A{delta} and C fibers). Electrical impulses are conducted to the dorsal horn of the spinal cord (mainly to Laminae I and II) and further to the brain. Moreover, noxious stimuli and inflammatory pain induce transcription and translation of the immediate early gene c-Fos in Laminae I and II [32 , 33 ]. c-Fos expression, thus, allows for an indirect determination of painful activation and provides a biochemical approach to support behavioral testing.

In this study, we examined whether PMN recruitment to a local, subcutaneous (s.c.) site induces hyperalgesia (i.e., pain) in the absence of an inflammatory stimulus (i.e., CFA). Specifically, we tested whether injection of CXCL1 or CXCL2/3 induces dose- and time-dependent, selective PMN recruitment and whether this leads to concomitant augmentation of pain sensitivity (hyperalgesia). We further compared CXCL2/3-induced PMN recruitment with CFA-induced inflammation to analyze whether hyperalgesia, PMN activation in the paw, local production of potential hyperalgesic mediators (IL-1ß and PGE2), and c-Fos expression in the dorsal horn of the spinal cord are affected differentially under these conditions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Animal protocols were approved by the Animal Care Committee of the Senate of Berlin (Landesamt für Arbeitsschutz, Gesundheitsschutz und Technische Sicherheit, Germany) and are in accordance with the International Association for the Study of Pain [34 ]. Male Wistar rats weighing 160–200 g received intraplantar (i.pl.) or intravenous (i.v.) injections under brief isoflurane anesthesia as described in the experimental protocols. Experiments were conducted at 2 h after the injections unless stated otherwise.

Measurement of hyperalgesia and of paw volume
Mechanical hyperalgesia was assessed using the paw pressure algesiometer (modified Randall-Selitto test, Ugo Basile, Comerio, Italy) [35 ]. Rats were handled once per day for 4 days before testing. On the day of testing, rats were held under paper wadding, and incremental pressure was applied via a wedge-shaped, blunt piston onto the dorsal surface of the hind paw by means of an automated gauge. The pressure required to elicit paw withdrawal, the paw pressure threshold, was determined. An arbitrary cut-off was set at 250 g. Three trials, separated by 10 s intervals, were conducted, and the average was calculated. The same procedure was performed on the contralateral paw; the sequence of paws was alternated between subjects to preclude order effects. A decrease in paw pressure threshold (i.e., a lower weight tolerance) was interpreted as mechanical hyperalgesia.

Thermal hyperalgesia was measured by the Hargreaves test [36 ]. Animals were acclimatized to the testing apparatus. Radiant heat was applied to the plantar surface of a hind paw from underneath the glass floor using a high-intensity light bulb, and paw withdrawal latency was measured with an electronic timer (IITC Inc./Life Science, Woodland Hills, CA). The average of two measurements taken with 20 s intervals was calculated. The stimulus intensity was adjusted to yield a paw withdrawal latency of 9–10 s in noninflamed paws, and the cutoff was 20 s to avoid tissue damage. A decrease in paw withdrawal latency (i.e., an earlier withdrawal of the paw to a standardized heat beam) was interpreted as thermal hyperalgesia.

Paw volume was measured with a plethysmometer (Ugo Basile). Two measurements were performed, and the average was calculated.

Fluorescence-activated cell sorting (FACS) and staining
Antibodies
All hematopoietic cells, with the exception of red blood cells, were stained by mouse anti-rat CD45 CyChrome monoclonal antibody (mAb; Clone OX-1, identifies the leukocyte common antigen, 4 µg/ml, BD Biosciences, Heidelberg, Germany) [37 ]. PMN (but not monocytes/macrophages) were identified by mouse anti-rat RP-1 phycoerythrin (PE) mAb (12 µg/ml) [38 39 40 41 ]. Monocytes/macrophages (but not PMN) were stained by mouse anti-rat CD68 fluorescein isothiocyanate (FITC) mAb (formerly named ED1; 2 µg/ml, Serotec, Oxford, UK) [39 , 42 , 43 ]. Lymphocytes were identified by mouse anti-rat CD3 PE mAb (4 µg/ml, BD Biosciences). The following describes adhesion molecules and chemokine receptor: hamster anti-rat CD62 ligand (CD62L; L-selectin; Clone HRL 1) FITC mAb, mouse anti-rat CD11b FITC mAb or mouse anti-rat CD18 FITC mAb (all 20 µg/ml, BD Biosciences), and rabbit anti-rat CXCR2 polyclonal antibody (pAb; 4 µg/ml, K-19, raised against the intracellular C terminus of the receptor, Santa Cruz Biotechnology, CA). The following describes secondary antibodies: goat anti-rabbit FITC pAb (13.5 µg/ml, Biosource, Camarillo, CA).

Leukocyte sorting
Heparinized blood was fixed, and erythrocytes were lysed using FACS lysing solution as described by the manufacturer (BD Biosciences). PMN were then sorted by characteristic forward-/side-scatter expression as performed by others [44 45 46 ]. Subsequently, intracellular staining of sorted PMN with RP-1 PE was performed as described below.

Leukocyte staining
Heparinized blood was obtained by cardiac puncture 2 h after i.pl. injection of saline or chemokines. Blood aliquots (100 µl) were incubated with the following monoclonal antiadhesion mAb to assess PMN activation as described previously [47 ]: Aliquots were fixed and lysed with FACS lysing solution as described by the manufacturer (BD Biosciences). For intracellular stains, fixed cells were permeabilized by saponin buffer [0.5% saponin, 0.5% bovine serum albumin (BSA), 0.05% NaN3 in phosphate-buffered saline (PBS), all from Sigma Chemicals, Deisenhofen, Germany]. Permeabilized cells were incubated with primary and secondary antibodies for 15 min: Replacement of the primary antibodies with isotype-matched, irrelevant antibodies was used for negative controls [12 , 47 ]. Data were analyzed using CellQuest software (BD Biosciences).

Cell suspensions from paw tissue were prepared and stained as described previously [28 ]. Briefly, s.c. paw tissue was enzymatically digested and was pressed through a 70-µm nylon filter (BD Biosciences). For intracellular stains, cells were fixed with 1% paraformaldehyde and permeabilized with saponin buffer as described above. Permeabilized cells were incubated with primary and secondary antibodies: Replacement of the primary antibodies with isotype-matched, irrelevant antibodies was used for negative controls [47 ].

To calculate absolute numbers of cells per paw, the stained cell suspension was transferred to a TruCOUNT® tube containing a known number of fluorescent beads. FACS events from the fluorescent TruCOUNT® beads and stained cells were collected simultaneously. Numbers of CD45+ cells per tube were calculated in relation to the known number of fluorescent TruCOUNT® beads and extrapolated for the whole paw. For quantification, 70,000 FACS events were acquired. Data were analyzed using CellQuest software (all BD Biosciences).

Enzyme-linked immunosorbent assay (ELISA)
All experiments were performed 2 h after i.pl. injection of NaCl, chemokines, or CFA. To obtain serum, blood was withdrawn by cardiac puncture, incubated at 4°C overnight, and centrifuged (8000 g for 10 min at 4°C). Supernatants (i.e., serum) were stored at –20°C until measurement. Paw tissue was retrieved, the skin was removed, and s.c. tissue was cut into small pieces and processed as described previously [12 ]. For PGE2 determination, paws were injected with indomethacin (2 µg in 100 µl 0.9% NaCl, ALPHARMA-ISIS, Langenfeld, Germany) before tissue retrieval to block PGE2 production during tissue processing. Also, the sample buffer contained 20 µg/ml indomethacin.

Chemokines, IL-1ß, and PGE2 were measured by commercially available mouse (CXCL1) and rat (CXCL2/3, IL-1ß, and PGE2) ELISA kits according to manufacturers’ instructions (R&D Systems, Minneapolis, MN, and Biosource International, Nivelles, Belgium). As described previously, the mouse CXCL1 ELISA kit can be used together with rat CXCL1 peptide standard (PeproTech, London, UK) for quantification of rat CXCL1 [12 , 48 ]. Optical density was measured by Spectra Max (Molecular Devices, Ismaning, Germany). Data were analyzed by the Softmax program. The manufacturers of both chemokine ELISA kits state that no significant cross-reactivity to other chemokines exists.

Quantitative polymerase chain reaction (PCR)
The spinal cord was removed at 2 h post-i.pl. injection of solvent or chemokines. The posterior quadrant of the lumbar segments L3-5 on the side of i.pl. injection was dissected, frozen in liquid nitrogen, and homogenized in Trizol (Invitrogen, Karlsruhe, Germany) as described previously [13 , 49 ]. To synthesize cDNA, RNA was annealed to 0.125 µm oligodesoxythymidine for 3 min at 65°C and transcribed in the presence of reverse-transcriptase (RT) buffer, 0.01 mM dithiothreitol (DTT), 1 mM deoxynucleoside triphosphate, 10 U avian myeloblastosis virus RT, and 4 U RNase inhibitor (all from Roche Diagnostics, Mannheim, Germany, except DTT, Sigma Chemicals) at 42°C for 2 h. PCR was performed with DNA Sybr Green following the manufacturer’s instructions (Roche Diagnostics) using 10 µM sense and antisense primers (synthesized by TibMolbiol, Berlin, Germany; c-Fos sense: 5' GGG ACA GCC TTT CCT ACT ACC A; c-Fos antisense: 5' CGT GGG GAT AAA GTT GGC AC). cDNA was amplified in a four-step-per-cycle PCR consisting of 95°C for 30 s, annealing at 59°C for 5 s, elongation at 72°C for 5 s, and a fluorescence-detection [temperature (Tm) 86°C] step. In pilot experiments, the Tm was determined just below the product-specific melting Tm, and this Tm was incorporated into the PCR program for detection of fluorescence. c-Fos mRNA was quantified using duplicate samples and a standard dilution with a known number of copies for a specific PCR product as described previously [13 , 49 ].

Immunohistochemical staining
Rats were perfused with 4% paraformaldehyde/PBS [28 , 50 ]. The spinal cord was removed, postfixed for 30 min at 4°C, and cryoprotected overnight at 4°C in 10% sucrose/PBS. The tissue was embedded in tissue-Tek compound and frozen (optical cutting Tm compound, Miles Inc., Elkhart, IN). Sections (40 µm) were cut on cryostat at the level of the lumbar spinal cord (L3-6) and collected in PBS (floating sections).

c-Fos staining was performed on floating sections with a vectastain avidin-biotin peroxidase complex kit (Vector Laboratories, Burlingame, CA) as described previously in detail [50 , 51 ]. Endogenous peroxidase was blocked by incubation with 0.3% H2O2 and 10% methanol in PBS for 45 min. To reduce nonspecific binding, sections were covered with 0.3% Triton X-100, 1% BSA, 4% goat serum, and 4% horse serum. The sections were incubated in four steps with rabbit anti-rat c-Fos pAb at 4°C overnight (1:2000 dilution, Oncogene, San Diego, CA), biotinylated goat anti-rabbit antibody (Vector Laboratories) for 60 min, avidin/biotinylated peroxidase complex for 45 min, and 3',3'-diamino-benzidine tetrahydrochloride (Sigma Chemicals) containing 0.01% H2O2 in 0.05 M Tris-buffered saline (pH 7.6, all Vector Laboratories) for 3–5 min. Sections were washed with PBS after each incubation step. After the last incubation step, sections were washed with tap water, then mounted onto gelatin-coated slides, dehydrated in alcohol, cleared in xylene, and mounted in dibutylphtalate polystyrene xylene (Merck Eurolab, Darmstadt, Germany). The following controls were included: omission of primary antibody, secondary antibody, or avidin-biotin complex. c-Fos-positive cells were counted in Laminae I-II of the dorsal horn by a blinded observer using a Zeiss microscope. Cell numbers were determined in 15 sections per animal and five animals per group.

Experimental protocols
Chemokine injection—dose-dependency
Separate groups of rats (n=6–7 per group) were injected i.pl. with 0–30 µg rat CXCL1 or rat CXCL2/3 (PeproTech), dissolved in 100 µl NaCl 0.9%. Control animals were injected with 100 µl solvent. Experiments were performed at 2 h after i.pl. injection. This time-point was chosen based on pilot data showing maximum recruitment after 2 h.

Chemokine injection—time course
Separate groups of rats (n=6–7 per group) were injected i.pl. with 3 µg CXCL1 or CXCL2/3, dissolved in 100 µl NaCl 0.9%. The dose was based on pilot data with a maximum of recruitment at 3 µg. Control animals were injected with 100 µl solvent. Experiments were performed at 0, 1, 2, 4, 6, and 12 h after injection.

CFA-induced inflammation
Rats (n=6–7 per group) were injected i.pl. with 150 µl CFA (Calbiochem, La Jolla, CA) and developed an inflammation confined to the inoculated paw.

PMN depletion
Rats (n=6–7 per group) were injected i.v. with rabbit anti-rat PMN serum (Accurate Chemical and Scientific Corp., Westbury, NY) or nonimmune control rabbit serum (Sigma Chemicals) as published previously [12 ]. Anti-PMN serum (80 µl) was diluted with 420 µl NaCl 0.9% and was injected 18 h before CFA.

Statistical analysis
Data are presented as raw values (mean±SEM). Normally distributed data were analyzed by t-test. Multiple measurements were analyzed by one-way ANOVA or by one-way ANOVA on ranks in case of data not normally distributed. The post-hoc comparisons were performed by Student-Newman-Keuls, Dunnett’s, or Dunn’s method, respectively. Differences were considered significant if P< 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Local injection of CXCL1 and CXCL2/3 results in dose-dependent PMN recruitment but does not induce mechanical hyperalgesia
CXCL1 and CXCL2/3 injection into noninflamed paws caused a significant recruitment of PMN (P<0.05, ANOVA) but not of monocytes/macrophages (Fig. 1A and 1B ) or lymphocytes (data not shown, P>0.05 ANOVA) after 2 h. PMN recruitment after CXCL2/3 injection was decreased at high doses (Fig. 1A) .


Figure 1
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  		Figure 1. Dose-dependent effects of locally injected CXCL1 and CXCL2/3 on leukocyte subpopulations in the paw and on hyperalgesia. PMN (A) and monocytes/macrophages (B) in the paw were quantified by flow cytometry 2 h after i.pl. 0.9% saline (NaCl, solid bars), CXCL2/3 (open bars), or CXCL1 (hatched bars; n=67/group; *, P<0.05, ANOVA vs. 0.9% NaCl control). In a separate group of rats, paw pressure threshold was quantified in the injected (C) and noninjected, contralateral hind paw (D), according to the protocol described above (n=6/group; P>0.05 ANOVA). Data are presented as mean ± SEM. n.s., Not significant.

 
High doses of CXCL1 and CXCL2/3 (10 and 30 µg) yielded significant increases in serum levels of both chemokines (Fig. 2A ). To examine adhesion molecule expression, PMN were identified in whole blood by forward-/side-scatter characteristics or by expression of the rat granulocyte-specific marker RP-1 for intracellular stains. Sorted PMN identified by forward-/side-scatter characteristics expressed RP-1 in >92% of cells (Fig. 2B) . Blood PMN showed increased expression of CD11b and CD18, partial shedding of CD62L, and decreased expression of CXCR2 after i.pl. chemokine injection (Fig. 2C 2D 2E 2F) . Changes in adhesion molecule and in CXCR2 expression were significantly more pronounced after 30 µg CXCL2/3 than after 30 µg CXCL1 inoculation (Fig. 2C 2D 2E 2F ; mean fluorescence intensity CD11b: saline 43±3, CXCL1 81±5, CXCL2/3 125±12; CD18: saline 68±3, CXCL1 89±2, CXCL2/3 105±2; CD62L: saline 31±1, CXCL1 27±2, CXCL2/3 20±1; CXCR2: saline 228±13, CXCL1 121±4, CXCL2/3 57±2, n=5–6 per group; all P<0.05 ANOVA, P<0.05 Student-Newman-Keuls test for all subgroup comparisons).


Figure 2
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  		Figure 2. Effects of locally injected CXCL1 and CXCL2/3 on serum chemokines and on adhesion molecule as well as CXCR2 expression in blood PMN. (A) Serum concentrations of CXCL1 (•) and CXCL2/3 ({circ}) were measured by ELISA 2 h post-i.pl. chemokine inoculation (n=6/group, 0–30 µg; *, P<0.05, ANOVA on Ranks vs. 0.9% NaCl control; data are presented as mean±SEM). (B) Rat PMN were sorted based on characterstic size and granularity [forward-/side-scatter (FSC/SSC); data not shown] and were stained for RP-1 [RP-1 PE (bold line); isotype-matched control antibody PE (isotype control, thin line)]. (C–F) Rats were injected as described in A (CXCL1 and CXCL2/3 at 30 µg i.pl. for 2 h). Expression of adhesion molecules was determined by flow cytometry on blood PMN identified by forward-/side-scatter characteristics (C–E). CXCR2 expression was quantified in RP-1+ PMN [F; isotype-matched control antibody (isotype control, thin line), 0.9% NaCl (----), CXCL1 (...), CXCL2/3 (bold line); representative histograms are shown].

 
Neither CXCL1 nor CXCL2/3 injections significantly altered the nociceptive threshold at any dose on the side of inoculation (Fig. 1C) or on the contralateral side (Fig. 1D) .

CXCL1- and CXCL2/3-induced PMN recruitment does not cause mechanical or thermal hyperalgesia within 12 h after injection
Local injection of an effective dose of CXCL1 and CXCL2/3 (3 µg) produced a significant recruitment of PMN with peak effects occurring at 2–4 h (Fig. 3A ). Modest increases in the number of monocytes/macrophages were observed at 4–12 h (Fig. 3B) . Neither CXCL1 nor CXCL2/3 altered mechanical or thermal nociceptive thresholds on the side of injection (Fig. 3C and 3D) or on the noninjected, contralateral side (data not shown).


Figure 3
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  		Figure 3. Time-dependent effects of locally injected CXCL1 and CXCL2/3 on leukocyte subpopulations in the paw and on hyperalgesia. PMN (A) and monocytes/macrophages (B) were analyzed by flow cytometry following i.pl. injection of 0.9% NaCl (solid bars), CXCL1 (hatched bars; 3 µg), and CXCL2/3 (open bars; 3 µg, n=6–7/group, *, P<0.05, ANOVA vs. saline control). Paw pressure threshold (C) and paw withdrawal latency (D) were measured on the side of inoculation in rats injected according to the same protocol (n=6/group; P>0.05 ANOVA). Data are presented as mean ± SEM.

 
Comparison of hyperalgesia, edema formation, and potential hyperalgesic mediators in CXCL2/3- and CFA-treated rats
i.pl. inoculation with CXCL2/3 or CFA resulted in a significant recruitment of PMN (Fig. 4A ). Prior systemic PMN depletion abolished the recruitment of PMN to the CFA-inoculated paw (Fig. 4A) . Increases in paw volume as a marker of inflammation were detected in CFA-inoculated paws but not after CXCL2/3 injection (Fig. 4B) . Mechanical and thermal hyperalgesia (i.e., a decrease in paw pressure threshold and paw withdrawal latency, respectively) was induced significantly and similarly in all CFA-inoculated paws, irrespective of systemic PMN depletion (Fig. 4C and 4D) . CXCL2/3 alone did not alter nociceptive thresholds on the side of injection (Fig. 4C and 4D) . Nociceptive thresholds and paw volume were unchanged in all groups in the noninjected, contralateral hind paws. IL-1ß content in the paw was increased significantly in comparison with controls in CXCL2/3 as well as CFA-injected paws, irrespective of prior PMN depletion (Fig. 4E) . PGE2 production in the paw was elevated significantly in CFA-treated rats, irrespective of PMN depletion but not in CXCL2/3-inoculated rats (Fig. 4F) .


Figure 4
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  		Figure 4. Comparison between locally injected CXCL2/3 and CFA on PMN recruitment, paw volume, and hyperalgesia. (A) PMN were quantified by flow cytometry 2 h after i.pl. 0.9% saline (NaCl), CXCL2/3 (3 µg), CFA, and CFA following prior systemic PMN depletion (aPMN, n=6–7/group; *, P<0.05, vs. 0.9% saline control). Paw volume (B), paw pressure threshold (C), and paw withdrawal latency (D) were determined in rats injected according to the same protocol [n=6 rats/group; ipsilateral paw (open bars), P<0.05; contralateral paw (cross-hatched bars), P>0.05, all ANOVA]. IL-1ß (E) and PGE2 (F) were quantified by ELISA in paw tissue homogenates of rats injected as described above (n= 6–8/group; *, P<0.05, vs. 0.9% saline control, ANOVA on ranks). Data are presented as mean ± SEM.

 
CXCL2/3 and CFA recruit PMN with equal expression patterns of activation markers
Adhesion molecule expression was analyzed in CD45+ leukocytes in the paw after i.pl. injection of CXCL2/3 or CFA (Fig. 5 ). In all groups, a lack of expression of CD62L and a similar intensity of CD11b and CD18 staining was observed. Samples after solvent injection or after systemic PMN depletion were not analyzed as a result of the low number of infiltrating CD45+ leukocytes.


Figure 5
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  		Figure 5. Adhesion molecule expression on PMN in the paw following local injection of CXCL2/3 and CFA. Surface expression of CD62L, CD11b, and CD18 was examined on CD45+ leukocytes of the injected paw [CD62L, CD11b, CD18 (solid line); isotype-matched control antibody (dotted line), n=4/group] 2 h after i.pl. injection of CXCL2/3 (upper row) or CFA (lower row). Representative histograms are shown.

 
c-Fos is activated in the spinal cord following CFA-induced inflammation but not after CXCL2/3-mediated PMN recruitment
In comparison with solvent injection, i.pl. inoculation of CFA and CFA after PMN depletion, but not of CXCL2/3 alone, resulted in significant increases of c-Fos mRNA transcription (Fig. 6A ). Likewise, a fivefold increase in the number of c-Fos immunoreactive cells was seen in Laminae I + II (Fig. 6B and 6C) . On the noninjected, contralateral side, no changes in c-Fos transcription or in c-Fos immunoreactive cells were detected (data not shown). c-Fos expression was only observed in CFA but not in CXCl2/3 or NaCl-treated rats and was independent of the number of infiltrating PMN.


Figure 6
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  		Figure 6. c-Fos transcription and c-Fos immunoreactive cells in the spinal cord following local injection of CXCL2/3 or CFA. c-Fos mRNA copy numbers (A) in the posterior quadrant of the spinal cord (L3-6) were qunatified following ipsilateral injection of 0.9% saline (NaCl, solid bar), CXCL2/3 (open bar), CFA (striped bar), and CFA preceeded by systemic PMN depletion (cross-hatched bar; *, P<0.05, ANOVA vs. saline control). Immunohistochemical staining of c-Fos immunoreactive cells (B, black cells) was performed in the dorsal horn of the spinal cord (L3-6) after i.pl. 0.9% saline (a), CXCL2/3 (b), CFA (c), and CFA following PMN depletion (d). Representative tissue sections are shown. The number of c-Fos immunoreactive cells was quantified (C; *, P<0.05, ANOVA vs. saline control; bars are as described in A). Data are presented as mean ± SEM.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we now demonstrated that CXCL1- and CXCL2/3-mediated selective recruitment of PMN to s.c. paw tissue does not induce pain in the absence of inflammatory agents such as CFA. Furthermore, hyperalgesia, PGE2 production, or c-Fos expression in the dorsal horn of the spinal is not induced by CXCL2/3-mediated PMN recruitment alone. In CFA-induced inflammation, hyperalgesia, PGE2 production, and c-Fos expression are independent of infiltrating PMN.

Low doses of CXCL1 and CXCL2/3 resulted in a time-dependent, selective PMN recruitment without relevant changes in monocytes/macrophages or lymphocytes (Figs. 1A and 1B and , 3A and 3B ). At high doses, CXCL2/3 but not CXCL1 induced less PMN recruitment. To elucidate this unexpected finding, we examined chemokine concentrations in serum and adhesion molecule and CXCR2 expression on circulating PMN. Serum chemokine concentrations increased proportionally to their local doses (Fig. 2A) . Both chemokines altered adhesion molecule expression {L-selectin shedding and up-regulation of CD11b/CD18 (Fig. 2) [52 53 54 ]}. Although changes in adhesion molecule expression did not explain the differences in recruiting efficacy of CXCL1 and CXCL2/3, CXCL2/3 induced a more pronounced loss of CXCR2 expression in blood PMN (Fig. 2F) . In line with this finding, CXCR2 ligands were detected in serum of septic patients with CXCR2 down-regulation [55 ], impaired PMN migration in rats [56 , 57 ], and induced lysosomal degradation of CXCR2 in transfected cells [58 ]. Taken together, in our model, CXCL1 and CXCL2/3 caused selective PMN recruitment. The recruiting efficacy was decreased at high doses of CXCL2/3, most probably as a result of the more pronounced down-regulation of CXCR2.

Despite significant CXCL1 and CXCL2/3-mediated PMN recruitment (Figs. 1A and 3A) , we did not observe any mechanical or thermal hyperalgesia in injected or contralateral paws (Figs. 1C and 3 , C and D), and hyperalgesia was independent of PMN infiltration in CFA-induced inflammation (Fig. 4A 4C and 4D ). The role of chemokines in the induction of hyperalgesia has been examined in several previous studies. i.pl. injection of CXCL1 (formerly cytokine-induced chemoattractant-1; KC) and CXCL8 (formerly IL-8) induced hyperalgesia in noninflamed paws at doses that were several orders of magnitude lower than ours (10–100 pg) [59 , 60 ]. These concentrations are significantly lower than required for PMN recruitment (Figs. 1A and 3A) [52 , 53 , 61 ]; pain behavioral testing involved a highly painful stimulus that induced sympathetic activation with a freezing reaction and apnea [59 , 60 , 62 ], and we determined the detection threshold. Other groups showed that chemokines, in general, induce hyperalgesia by direct activation of chemokine receptors on sensory neurons [63 ] or by sensitization of the transient receptor potential vanilloid subfamily member 1 (TRPV1) channel [64 ]. However, CXCL8 was the only CXCR1/2 ligand examined in these studies; it did not evoke neuropeptide release [65 ], and it was not tested in vivo for the induction of hyperalgesia. Taken together, although some chemokines can activate nociceptive neurons directly, our own and other studies suggest that CXCR2 ligand-mediated, selective PMN recruitment does not induce hyperalgesia.

Several previous studies claimed that mediators such as fMLP, complement factor C5a, LTB4, or NGF cause hyperalgesia, which is mediated by PMN [4 , 5 , 7 ]. However, although we used CXCR2 ligands, which selectively recruit PMN (Figs. 1A and 1B and 3A and 3B ; refs. [21 , 66 ]), all the other agents (fMLP, LTB4, complement C5a, and NGF) also affect the migration and function of monocytes/macrophages [10 , 67 68 69 70 ]. In support of our results, local injection of glycogen was shown to selectively recruit PMN without causing hyperalgesia [5 , 11 ], and this recruitment is mediated by CXCR2 ligands [71 , 72 ]. In addition, several studies that proposed a role for PMN in hyperalgesia used nonselective leukocyte depletion [4 5 6 ] or detected significant differences in nociceptive thresholds only at a time-point when monocytes/macrophages were also significantly depleted [7 , 70 ]. In summary, the previously demonstrated hyperalgesic effects of fMLP, LTB4, C5a, and NGF cannot be attributed exclusively to PMN, and PMN accumulation per se did not induce hyperalgesia.

The absence of hyperalgesia after CXCL1 and CXCL2/3 injection might be related to a lack of inflammation, as we did not observe any changes in paw volume (Fig. 4B) . However, although edema and hyperalgesia develop in parallel in most models of inflammatory pain [73 , 74 ], it can occur in the absence of edema [75 , 76 ]. Furthermore, it might be argued that local chemokine injection results in poorly activated PMN in comparison with an inflammatory model. Tissue PMN recruited by CXCL2/3 were also fully activated (i.e., high surface expression of CD11b and CD18 and by down-regulation ("shedding") of L-selectin (CD62L; Fig. 5 ) [53 , 77 , 78 ]. Taken together, neither the absence of edema formation (i.e., paw volume changes) nor insufficient activation of PMN explains the lack of mechanical and thermal hyperalgesia in our present study.

Why does hyperalgesia occur after CFA-induced inflammation but not after CXCR2 ligand-mediated PMN recruitment? Previous studies demonstrated that CXCL1 induced hyperalgesia is mediated by a cascade-like induction of IL-1ß and PGE2 [62 , 79 ]. Neither IL-1ß nor PGE2 activates peripheral nociceptive neurons directly [80 ], but both can sensitize nociceptive neurons to noxious heat or chemical mediators [81 , 82 ]. In our study, local IL-1ß production was elevated in CXCL2/3- and CFA-injected rats but did not correlate with hyperalgesia. In contrast, PGE2 production was increased in CFA- but not in CXCL2/3-injected animals, and it correlated with hyperalgesia (Fig. 4C 4D 4E 4F) . This suggests that PGE2 might contribute to hyperalgesia in our model. However, in addition to cytokines and prostaglandins, numerous other mediators from infiltrating immune or resident cells (e.g., monocytes/macrophages, mast cells, fibroblasts, or endothelial cells) are produced during inflammation (e.g., adenosine 5'-triphosphate, protons, bradykinin), and these can also activate nociceptive transducers on peripheral neurons {e.g., purinergic receptor (P2X3), acid-sensing ion channel, Nav 1.8/1.9, TRPV1 [2 , 80 , 83 ]}. Any one or more likely, any combination of these could contribute to the differences between models of inflammation and CXCR2 ligand-induced PMN recruitment.

To corroborate our behavioral experiments, we sought to examine an additional parameter of neuronal activation by nociceptive stimuli. Expression of c-Fos in the spinal cord has been shown to identify activity in spinal neurons in response to noxious stimulation (reviewed in ref. [84 ]). First, there is good correspondence between c-Fos and electrophysiological and tract-tracing methods. A{delta} and C primary afferent noxious fibers project to Lamina I and the outer portion of Lamina II as well as Laminae V and VI at the base of the dorsal horn. Second, c-Fos activation is found in the laminae in response to noxious but not to non-noxious (e.g., tactile) stimulation, and antinociceptive treatment suppresses c-Fos activation. Spinal c-Fos expression has, nonetheless, several limitations: Nociceptive stimulation has to be intense and prolonged; not all neurons express c-Fos; and the absence of c-Fos expression does not always identify the absence of neuronal activation. Despite these limitations, c-Fos is the most extensively studied marker available. In line with previous studies [33 , 85 , 86 ], we observed that inflammation induces hyperalgesia as well as c-Fos transcription and increases in the number of c-Fos immunoreactive cells in Laminae I and II in the dorsal horn (Fig. 6) , and these changes were also detectable following PMN depletion. In contrast, CXCL2/3 injection led to PMN accumulation but did not alter c-Fos expression (Fig. 6) . Together, spinal c-Fos expression correlates with hyperalgesia detected in behavior experiments, and both are observed after CFA-induced inflammation, with or without PMN depletion, but not after CXCL2/3-induced, local PMN recruitment.

In conclusion, we have now shown that selective recruitment of PMN by CXCL1 or CXCL2/3 does not induce activation of nociceptive, second-order neurons in the spinal cord or pain. Furthermore, pain in the CFA model seems to be independent of the number of PMN at the site of inflammation. In contrast, our previous studies showed that opioid-mediated, local antinociception is critically dependent on PMN in early CFA-induced inflammation [12 ]. These findings suggest that PMN seem to have a more prominent role in the counteraction rather than in the induction of inflammatory hyperalgesia. Furthermore, our data indicate that a reduction of PMN in inflammatory diseases might not be sufficient for pain relief.


   ACKNOWLEDGEMENTS
 
This study was supported by the Deutsche Forschungsgemeinschaft (KFO 100/1) and by the European Society of Anaesthesiology (ESA). The authors thank Susanne Kotré and Katharina Hopp, Berlin, for expert technical assistance.

Received August 13, 2005; revised December 27, 2005; accepted January 5, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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