Neutrophils are one of the predominant immune cells initially migrating to surgical wound edges. They produce mediators both associated with supporting (interleukin [IL]-1β, C5a) and reducing (opioid peptides) pain. Studies demonstrate neutrophil depletion/blockade reduces nociceptive sensitization after nerve injury and carrageenan administration, but enhance sensitization in complete Freund's adjuvant inflammation. This research identifies the contribution of infiltrating neutrophils to incisional pain and inflammation.


Antibody-mediated Gr1 neutrophil depletion preceded hind paw incisions. Sensitization to mechanical and thermal stimuli, effects on edema and local levels of IL-1β and C5a were measured. Local effects of C5a or IL-1 receptor antagonists PMX-53 and anakinra on sensitization after neutrophil depletion were examined. Groups of 4-8 mice were used.


Anti-Gr1 antibody depleted more than 90% of circulating and infiltrating skin neutrophils after incision. Neutrophil depletion did not change magnitude or duration of mechanical hypersensitivity in incised mice. However, paw edema was significantly reduced and heat hypersensitivity was slightly increased in depleted animals. In depleted animals IL-1β levels were half of controls 24 h after incision, whereas C5a levels were increased in both. Prominent IL-1β immunohistochemical staining of epidermis was seen in both groups. PMX-53 and anakinra reduced incisional mechanical and heat nociceptive sensitization to the same extent, regardless of neutrophil depletion.


Neutrophil-derived IL-1β and C5a do not appear to contribute critically to peri-incisional nociceptive signaling. Other sources of mediators, such as epidermal cells, may need to be considered. Controlling inflammatory activation of resident cells in epidermis/deeper structures may show therapeutic efficacy in reducing pain from surgical incisions.

  • Neutrophils responding to injury release mediators that enhance sensitization and pain after neuropathic injury in animals, but their role in incisional surgery is not known

  • In mice, depletion of neutrophils reduced paw edema and tissue interleukin-1β concentrations after paw incision, but failed to significantly alter mechanical hypersensitivity

  • Blockade of C5a and interleukin-1β signaling reduced hypersensitivity, suggesting that these factors are important to sensitization after incisional surgery, but are not dependent on local infiltration by neutrophils

PAIN after surgery remains problematic. Despite the heightened attention given to postoperative comfort, expanded use of patient-controlled analgesia devices, and increasing use of multimodal therapy, almost all patients experience some degree of postoperative pain, and 30–60% of patients undergoing surgery report moderate to severe pain levels.1,2On the other hand, progress has been made in understanding the mechanisms supporting this type of pain. Investigators have addressed a wide range of factors like wound dynamics, nociceptor sensitization, central nervous system changes, and patients' psychologic profiles to better understand postoperative pain. A good deal of attention has been focused on the liberation of local nociceptive mediators after incision, and the interaction of those mediators with primary afferent nerves.3,4The implicit hope of this research is that identification of key mediators and the sources of those mediators will further facilitate the development of specific therapeutic approaches.

One of the predominant immune cell types migrating to the injured tissue are neutrophils, which are present in wound edges within hours of incision, peak in abundance within 24 h, and then slowly decline in number. In addition to participating in fighting infection and regulating wound healing, these cells produce many known nociceptive mediators including cytokines, chemokines, proteinases, phospholipases, reactive oxygen species, and other molecules.5Cytokine-stimulated neutrophils can in turn activate additional incisional nociceptive mediators, such as complement system components.6Some of these mediators, namely interleukin (IL)-1β7,,9and the complement fragment C5a,10,11have been shown to support nociception in rodent incisional pain models.

Additional evidence suggests neutrophils regulate nociceptive sensitization in other pain models. For example, depletion of circulating neutrophils reduces nociceptive sensitization early after peripheral nerve injury.12Also, blockade of neutrophil infiltration using the migration inhibitor fucoidin resulted in reduced mechanical hyperalgesia after carrageenan injection in the plantar tissue of rat hind paws, suggesting that in this pain model neutrophils might contribute to mediator production and sensitization.13On the other hand, neutrophils produce endogenous opioid peptides such as met-enkephalin and β-endorphin, potentially reducing pain.14In the complete Freund's adjuvant model of inflammatory pain, opioid peptides derived from neutrophils reduce nociceptive sensitivity, whereas depletion of neutrophils does not alter baseline sensitization in this pain model.14,,16Thus, in some settings, neutrophils seem to provide a mechanism for endogenous peripheral analgesia.

We do not at this point understand whether the complex functions of neutrophils in incisional wounds lead to an overall enhancement, as would be suggested by mediator production, or reduction, as would be suggested by opioid peptide release, in nociceptive sensitization after incision. Furthermore, we do not understand for particular mediators already linked to sensitization in incisional wounds, such as IL-1β or C5a, whether local production by resident cells versus  neutrophil infiltration and release is the mechanism responsible for the observed inflammation and sensitization. In these experiments we utilized an antibody-mediated neutrophil depletion protocol combined with assessments of the local consequences of incision to address these questions.


All experimental protocols were approved by Veterans Affairs Palo Alto Healthcare System Institutional Animal Care and Use Committee (Palo Alto, California) before beginning the work. Male mice 10–14 weeks old of the C57Bl/6J strain obtained from Jackson Laboratories (Bar Harbor, MA) were kept in our facility a minimum of 1 week before initiating experiments. All mice were kept under standard conditions with a 12 h light/dark cycle and an ambient temperature of 22 ± 1°C. Animals were allowed food and water ad libitum . All procedures followed the guidelines of International Association for the Study of Pain for care and use of laboratory animals. All experiments were done with 4–8 mice per group, guided by power analyses based on pilot and previous experimentation.

Hind Paw Incision

The hind paw incision model modified for mice was used in a similar way as previous studies investigating nociceptive effects and cytokine level changes following incision.10,17,18Briefly, mice were anesthetized using isoflurane (AErrane; Baxter Healthcare Corporation, Deerfield, IL), and after sterile preparation, a 5-mm longitudinal incision was made on the plantar surface of one hind paw. The underlying plantaris muscle was incised longitudinally; the wound was then closed with a single 6-0 nylon suture and antibiotic ointment was applied.

In Vivo  Neutrophil Depletion

Treatment with functional grade purified antimouse Ly6G/Gr-1 (RB6-8C5 clone; eBioscience, San Diego, CA) or IgG2b control antibody (for controls) at 48 and 4 h before incision was carried out. Separate groups of mice received 4 mg/kg intraperitoneal injections of either antibody. Blood was collected by tail snip from anesthetized mice for neutrophil counts. For this purpose, blood samples were obtained before antibody treatment, before incision, and daily afterward for 5 days. Slides made using these samples were stained with Wright–Giemsa stain. Counting was completed manually.

Assessment of Mechanical Sensitivity

Mechanical sensitivity after incision was measured by using von Frey filaments following the “up-down” algorithm described by Chaplan et al.  10,18,19After acclimating mice on wire mesh platforms inside clear cylindrical plastic enclosures, sequential application of filaments lateral to the central wound edge was carried out. Hind paw withdrawal because of fiber application was scored as a response. By using a data-fitting algorithm, the mechanical withdrawal threshold was calculated and subjected to parametric statistical analysis.20These experiments were done by a single experienced experimenter not blinded to treatment groups.

Assessment of Heat Sensitivity

Heat sensitivity after incision was measured using a modified method described by Hargreaves.21,22Mice were acclimated on a temperature-controlled glass platform (23.5 to 24.0°C) in a plastic enclosure and the beam of light was applied to the area of hind paw incision. Withdrawal latency of the paw from the heat source was measured, and to prevent tissue damage, a 15-s cutoff was used. Three measurements were made per animal per test session, separated by several minutes. These experiments were done by a single experienced experimenter not blinded to treatment groups.

Assessment of Paw Edema

A laser sensor technique was used to determine the dorsal–ventral thickness of the hind paw, as previously described.23For laser measurements each mouse was briefly anesthetized with isoflurane and then held vertically so the hind paw rested on a table top below the laser. The paw was gently held flat on the table with a small metal rod applied to the top of the ankle joint. Using optical triangulation, a laser (4381 Precicura; Limab, Goteborg, Sweden) with a distance-measuring sensor (200-mm range, 0.01-mm resolution) was used to determine the distance to the table top and to the top of the hind paw, and the difference was used to calculate the dorsal–ventral paw thickness. Three measurements were made per paw, per animal.

Drug Administration

For some groups of mice, anakinra (Amgen, Thousand Oaks, CA), PMX-53 (Promix, Queensland, Australia), or saline vehicle was injected subcutaneously into the plantar skin of the hind paws of mice 2 h before incision and also daily 2 h ahead of behavioral testing. Preliminary experiments demonstrated this to be a point of maximal effect. For these injections mice were gently restrained. The injection volume was 15 μl administered through a 30-gauge needle, which raised a bleb similar to the length of the incisional wounds and approximately 1 mm of surrounding tissue.


The immunohistochemical analysis of mouse paw skin was done according to previously published methods.9,10Briefly, the primary and secondary antibodies used were IL-1β (H-153) rabbit polyclonal IgG, 1:50 (Santa Cruz Biotechnology, Santa Cruz, CA); fluorescein antirabbit IgG (H+L), 1:150 (Jackson ImmunoResearch Laboratories, West Grove, PA); rat antimouse neutrophil (allotypic marker) monoclonal antibody, 1:300 (AbD Serotec, Raleigh, NC); Texas red anti-rat IgG (H+L), 1:150 (Vector Lab Inc., Burlingame, CA); rat antimouse F4/80 antigen, 1:100 (AbD Serotec); fluorescein anti-rat IgG (H+L), 1:500 (Vector Lab Inc.); rabbit anti-β-endorphin, 1:100 (Peninsula Laboratories LLC, San Carlos, CA), and Texas red antirabbit IgG (H+L), 1:500 (Jackson ImmunoResearch Laboratories). Confocal laser-scanning microscopy was performed using Zeiss LSM 510 and LSM 510 META Laser Scanning Microscopes (Thornwood, NY). Control experiments included incubation of slices in primary and secondary antibody-free solutions, both of which led to low-intensity nonspecific staining patterns in preliminary experiments. After exposure with appropriate antibodies for neutrophils and macrophages, the number per high power field (x40) was counted by a blinded experimenter using SPOT Advanced software (SPOT, Sterling Heights, MI).

Cytokine (IL-1β) and C5a ELISA

An ovular patch of full-thickness skin providing 1 to 1.5 mm margins surrounding the hind paw incisions was collected rapidly after carbon dioxide asphyxiation of animals. These samples containing approximately 12 mg tissue per paw were placed immediately into ice-cold 0.9% NaCl containing a cocktail of protease inhibitors (Complete; Roche Applied Science, Indianapolis, IN). Samples were homogenized and centrifuged for 10 min at 12,000 x gravity at 4°C. An aliquot of the supernatant fractions was subjected to protein assay (DC Protein Assay; Bio-Rad Laboratories, Hercules, CA) and subsequently C5a and IL-1β protein levels were measured by a R&D Systems EIA kit (Minneapolis, MN), according to the manufacturer's protocol. The experimenter was blinded to the treatment groups.

Statistical Analysis

To compare time course and treatment effects for the behavior studies, a two-way ANOVA (time, treatment) with repeated measures for time was performed with Bonferroni correction for multiple comparisons. For data obtained from the peripheral blood and skin neutrophil determination experiments, a one-way ANOVA was performed with Bonferroni correction for multiple comparisons. All comparisons were run as two-tailed testing. All data are presented as means ± SEM, and differences were considered significant at P < 0.05 (Prism 4.0; GraphPad Software, San Diego, CA). No data were missing for any of the variables.

Time Course of Neutrophil Depletion after Anti-Ly6G/Gr-1 Antibody Treatment

The results of preliminary experiments showed that two injections of anti-Gr-1 antibody were required to achieve substantial depletion of circulating neutrophils. In figure 1A, the time course of antibody depletion and recovery is presented. Depletion of more than 90% of circulating neutrophils was achieved by the time of incision, and persisted for at least the first 24 h. Circulating neutrophil counts remained depressed for at least 3 days.

Anti-Ly6G/Gr-1 Antibody Treatment Significantly Depletes Peri-Incisional Neutrophils

In order to assess the efficacy of the above two-dose depletion regimen in preventing neutrophil accumulation after incision, immunohistchemical analysis of skin samples from separate groups of mice for neutrophils was next carried out. This was done as the circulating neutrophil levels may not reflect the actual tissue content of these cells after local injury. Figures 1B and C show that 24 h after incision, there is a marked reduction of infiltrating skin neutrophils in the anti-Gr-1 antibody group compared with controls. The neutrophils were mostly seen as densely packed cells within the superficial part of the incision site. At the same time-point, the macrophage numbers were modestly decreased by the antibody treatment. The macrophages were mostly seen in the superficial dermal layers.

Effects of Anti-Ly6G/Gr-1 Antibody Treatment on Incision-induced Mechanical and Heat Hypersensitivity

As the migration of neutrophils into injured tissue has been implicated with both the production of pronociceptive mediators and the release of opioid peptides, we next determined if a reduction in these immune cells affected pain sensitivity in the incisional model. Neutrophil depletion did not change the magnitude or duration of mechanical hypersensitivity in incised mice compared with controls (F1,50= 0.43, P = 0.528; fig. 2A). However, the neutrophil-depleted mice showed slightly increased heat hypersensitivity compared with controls 24 h after incision (F1,72= 5.84, P = 0.018). No difference was observed in the preincisional heat sensitivity or 2 h after surgery, or at later time-points between the neutrophil-depleted and mice treated with control antibody (fig. 2B).

Anti-Ly6G/Gr-1 Antibody Treatment Significantly Reduces Paw Edema

In order to determine the contribution of neutrophils to a separate index of the inflammatory response in incised animals, we measured paw thickness at time-points up to 72 h in control and anti-Gr1-antibody-treated mice. There was a significant difference between the neutrophil-depleted animals and controls in the measure of paw edema at the 2 and 24 h time-points (fig. 2C).

Effects of Anti-Ly6G/Gr-1 Antibody Treatment on Skin C5a and IL-1β Levels

The effects of neutrophil depletion on proinflammatory mediator IL-1β and complement fragment C5a was next determined. Peri-incisional levels of IL-1β increased sharply in skin after incision, but anti-Gr-1 mice displayed only approximately 50% of the levels achieved in control mice at 24 h after incision. At 72 h, control and neutrophil-depleted mice had the same level of cytokine elevation. On the other hand, levels of the pronociceptive complement fragment C5a were increased to the same extent in both control and neutrophil-depleted animals 24 h after incision, but were lower in the depleted animals 72 h after incision (fig. 3A–B). Thus levels of the two mediators were altered differentially after incision in the setting of neutrophil depletion.

As C5 is produced by organs such as the liver and only activated to C5a locally, whereas IL-1β is locally produced after trauma, we pursued complementary immunohistochemical experiments for IL-1β to further localize its source in the control and neutrophil-depleted mice. Figure 4demonstrates that neutrophils and dermal and epidermal cells produce IL-1β 24 h after incisions in control animals, and that the dermal and epidermal sources remain prominent in neutrophil-depleted mice.

Effects of Anti-Ly6G/Gr-1 Antibody Treatment on Neutrophil-derived β-endorphin

Double immunostaining for β-endorphin and neutrophils in peri-incisional skin demonstrated many positive cells (fig. 5). In intact skin, β-endorphin positive cells were absent (figs. 5A, C). After anti-Gr-1-antibody treatment, there was an overall decrease in peri-incisional β-endorphin-positive neutrophils (figs. 4E, F) compared with control antibody group (figs. 5C, D).

Effects of Treatment with IL-1R or C5a Antagonists on Nociceptive Hypersensitivity after Incision in AGr-1+-depleted Mice

Lastly, we examined the contribution of local IL-1β and C5a signaling in skin after incision both under control and neutrophil-depleted conditions. The selective complement fragment C5a receptor antagonist PMX-53 (30 mcg/paw) reduced both mechanical and heat nociceptive sensitization to the same extent regardless of neutrophil depletion during the 72-h period following incision (figs. 6A, B). Similarly, there was no significant difference in the effect of treatment with the IL-1R antagonist anakinra (1.5 mg/paw) between the depleted and control groups with respect to its ability to reverse mechanical or heat nociceptive sensitization (figs. 6C, D). Thus, although IL-1β and C5a both seemed to support sensitization, neutrophil-derived IL-1β and C5a did not appear to contribute critically to peri-incisional nociceptive signaling.

Investigations during the past several decades have identified many nociception-relevant components of the complex inflammatory soup generated after incision or tissue damage. Abundant evidence demonstrates that many of these mediators on their own or in combination can cause nociceptive sensitization. Some of these mediators, including IL-1β and C5a, two of the better examined nociceptive mediators in incisional pain models, can be generated by multiple cell types. Generally missing from these investigations have been experiments directed at understanding which sources of the mediators are most relevant to pain versus  other wound processes, such as healing or fighting infection. Such information both aids in our understanding of incision-related nociceptive mechanisms and helps to define the cellular targets when designing analgesic strategies.

These studies used antibody-mediated neutrophil depletion to examine their function in the early period surrounding hind paw incision in mice. The antibody used here provided profound reductions in the levels of wound-area neutrophils in the early postincision period, causing small but significant changes in heat sensitization in the 24-h period after hind paw incision and no changes in mechanical nociceptive sensitization. Meanwhile, neutrophil depletion was effective in reducing postincisional edema. Looking more closely, the levels of IL-1β were reduced in the peri-incisional skin of neutrophil-depleted animals 24 h after incision compared with controls, whereas complement fragment C5a was not reduced significantly in either group at this time-point. In both cases, they were still increased above baseline levels at a time-point when incisional sensitization was maximal. The local injection of the IL-1 receptor antagonist anakinra retained full effect in reducing mechanical allodynia even after neutrophil depletion, suggesting that the residual IL-1β production was sufficient to support nociceptive sensitization. It had been reported previously that the systemic administration of anakinra reduced nociceptive sensitization after hind paw incision.8Most IL-1β production in neutrophil-depleted mice appeared to be within the epidermal keratinocytes, shown previously to produce this cytokine after incision.24 

Evidence suggests that in some systems the conversion of C5 to C5a is supported by a proteolytic reaction on the surface of neutrophils or by the action of neutrophil-derived myeloperoxidase products.6,25,26In fact, a positive feedback loop has been hypothesized in which C5a-mediated neutrophil recruitment leads to further augmentation of C5a production.6In our experiments, C5a levels were decreased moderately and the selective C5a receptor antagonist PMX-53 administered locally reduced nociceptive sensitization to the same extent in control and neutrophil-depleted mice. This agent has been shown to reduce nociceptive changes in mice after incision, perhaps by a mechanism involving a reduction in primary afferent nerve fiber activity.11 

A strong circumstantial case can be made that infiltrating neutrophils support nociception in inflamed tissues. Activated neutrophils produce many mediators linked to nociception, including IL-1β and C5a, as well as reactive oxygen species, metalloprotinases, and other molecules.5The injection of exogenous IL-1β and C5a into rodent hind paw skin lowers nociceptive thresholds.11,27Moreover, neutrophils are recruited into paws after the injection of carrageenan model of inflammatory pain, and blocking the migration of neutrophils from the vasculature reduced mechanical hyperalgesia in these studies.13Conversely, complete Freund's adjuvant induced inflammation and hyperalgesia were unaffected by neutrophil depletion, despite reductions in IL-1β levels to a degree similar to that observed in our studies.16Importantly, the recruitment of activated neutrophils into hind paw tissues by virtue of local CXCL2/3 injection did not by itself cause hyperalgesia.16Moreover, neutrophils have been observed to infiltrate damaged nerves locally and at dorsal root ganglion level and have been linked to nociceptive sensitization in models of neuropathic pain.12,28,29Thus the apparent role of neutrophils in supporting nociception may depend both on the site and mechanism of inflammation and perhaps on the method of disruption of local neutrophil recruitment, i.e. , depletion versus  inhibition of migration.

Our results indicate that depending on the site of production in wounds, mediators like IL-1β and C5a might have different effects; we were able to reduce inflammation as measured by a reduction in paw thickness, and we observed a similar degree of nociceptive sensitization in neutrophil-depleted animals. Previous studies show that IL-1β and C5a can increase endothelial leak and edema,30,31suggesting that a reduction in neutrophil-derived mediators could reduce edema. However, previous studies suggest that the richly innervated epidermal layer may be an important source of IL-1β after hind paw incision and in inflamed tissues.24Unclear at this time is the mechanism by which in the absence of infection keratinocytes are stimulated to produce mediators like IL-1β, although recent evidence suggests that neuropeptides and adenosine triphosphate, which are present at increased levels in injured tissue, may be able to stimulate the assembly and activation of IL-1β-producing inflammasomes.32,33Once produced and released, peripheral sensory nerves penetrating the epidermis may be exposed to high local concentrations of IL-1β and other mediators.3This model of mediator-neuron interaction accommodates both the observations that neutrophil depletion affects sensitization minimally, and the observation that locally administered anakinra retains its ability to reduce sensitization even after neutrophil-generated IL-1β is eliminated.

Conversely, a significant number of studies examining the properties of endogenous opioids produced by leukocytes have been provided.34Leukocytes, including neutrophils, produce proopiomelanocortin and proenkephalin under inflammatory conditions.35,,37Also, the expression of opioid receptors on peripheral neurons is enhanced under inflammatory conditions.38,39Endogenous opioid peptides in sites of inflammation can be released spontaneously, though release is augmented by the presence of corticotropin-releasing factor or stress.40,,42Importantly, in models of inflammation and metastatic bone cancer pain, the local injection of opioid receptor antagonists or antibodies enhance nociceptive sensitization by blocking local opioid signaling.15,42,43Also, the duration of inflammation is related to the strength of the role of endogenous opioids in limiting sensitization.34It is therefore possible that under the relatively acute conditions employed in our studies, analgesic mechanisms supported by inflammation and neutrophilic infiltration had not developed to the degree that their effects were more prominent. Indeed, our previous report shows that naloxone administration after incision causes a worsening of the heat hyperalgesia and mechanical allodynia, which are most prominent a few days after incision.44There is further support for the type of injury determining the duration and extent of neutrophil-mediated proalgesic versus  analgesic mechanisms. In the setting of nerve injury, neutrophils steadily and continuously infiltrate endoneurium for 7 or 8 days, whereas they peak in 24 h after skin incision and decline rapidly to reach baseline levels in 3 days.12,45Neutrophil depletion attenuated nerve injury-induced hyperalgesia by potentially decreasing mediators responsible for induction of pain. However, local application of corticotropin-releasing factor to the site of nerve injury produced analgesia mediated by opioid-containing leukocytes.40Therefore, it seems that the neutrophils and opioid peptides produced by them are participating in an intensely localized inflammatory response. However, skin inflammatory response appears to be more diffused and involve many nonimmune cells. Keratinocytes and fibroblasts can produce and release proopiomelanocortin products, providing a potential nonimmunological origin for some portion of the peripheral opioids limiting sensitization after tissue injury.46,47Other immune sources of endogenous opioids, like memory T lymphocytes, which contain endorphin and migrate preferentially to inflamed tissues, could also contribute to limiting sensitization.48 

It needs to be acknowledged that the present study has the following limitations. Although depleting neutrophils using antibodies to surface antigens has been the preferred technique for causing depletion with profound effects seen in these and other experiments, the technique is not entirely selective. The monoclonal antibody RB6-8C5 against surface antigen granulocyte receptor (Gr-1) has been widely used, but may result in depletion of select subpopulations of monocytes and macrophages that participate in the inflammatory process.49,50The alternative 1A8 antibody ostensibly has a better selectivity in depleting circulating neutrophils, but possesses a similar profile to RB6-8C5 in terms of depleting the wound macrophages.51Here we observed a modest decrease in wound area macrophage numbers, which could be attributed to the RB6-8C5 antibody as well as to the decrease in neutrophil-mediated chemotactic signals. In addition, the present study has the limitation of having the behavior assessments being done by the experimenter not blinded to treatment groups, as experimenter bias could affect the pain tests' results.52 

Inflammatory mediators derive from multiple cell types in incisional wounds. Not all sources necessarily make the same contribution to nociceptive sensitization. Moreover, it should be kept in mind that any conclusions drawn concerning nociception may not apply to the source of mediators critical for wound healing or fighting of infection, which may rely more heavily on mediators derived from infiltrating immune cells. Likewise, our experiments were directed primarily at nociception deriving from skin and superficial tissues rather than deeper structures such as muscle and fascia, which may play a prominent role in pain-related behaviors observed in animals and pain scores reported in humans after incision.53,54Nevertheless, therapies directed at controlling the inflammatory activation of the epidermis may show efficacy in reducing pain from surgical incisions.

The authors thank Deyong Liang, Ph.D. (Anesthesiology Service, Veterans Administration Palo Alto Health Care System, Palo Alto, California, and Department of Anesthesia, Stanford University, Palo Alto, California), for valuable discussions throughout the preparation of the manuscript.

Apfelbaum JL, Chen C, Mehta SS, Gan TJ: Postoperative pain experience: Results from a national survey suggest postoperative pain continues to be undermanaged. Anesth Analg 2003; 97:534–40
Chung JW, Lui JC: Postoperative pain management: Study of patients' level of pain and satisfaction with health care providers' responsiveness to their reports of pain. Nurs Health Sci 2003; 5:13–21
Boulais N, Misery L: The epidermis: A sensory tissue. Eur J Dermatol 2008; 18:119–27
Jang JH, Liang D, Kido K, Sun Y, Clark DJ, Brennan TJ: Increased local concentration of complement C5a contributes to incisional pain in mice. J Neuroinflammation 2011; 8:80
Burg ND, Pillinger MH: The neutrophil: Function and regulation in innate and humoral immunity. Clin Immunol 2001; 99:7–17
Camous L, Roumenina L, Bigot S, Brachemi S, Frémeaux-Bacchi V, Lesavre P, Halbwachs-Mecarelli L: Complement alternative pathway acts as a positive feedback amplification of neutrophil activation. Blood 2011; 117:1340–9
Honore P, Wade CL, Zhong C, Harris RR, Wu C, Ghayur T, Iwakura Y, Decker MW, Faltynek C, Sullivan J, Jarvis MF: Interleukin-1αβ gene-deficient mice show reduced nociceptive sensitivity in models of inflammatory and neuropathic pain but not post-operative pain. Behav Brain Res 2006; 167:355–64
Hu Y, Liang D, Li X, Liu HH, Zhang X, Zheng M, Dill D, Shi X, Qiao Y, Yeomans D, Carvalho B, Angst MS, Clark JD, Peltz G: The role of interleukin-1 in wound biology. Part II: In vivo  and human translational studies. Anesth Analg 2010; 111:1534–42
Liang DY, Li X, Li WW, Fiorino D, Qiao Y, Sahbaie P, Yeomans DC, Clark JD: Caspase-1 modulates incisional sensitization and inflammation. ANESTHESIOLOGY 2010; 113:945–56
Clark JD, Qiao Y, Li X, Shi X, Angst MS, Yeomans DC: Blockade of the complement C5a receptor reduces incisional allodynia, edema, and cytokine expression. ANESTHESIOLOGY 2006; 104:1274–82
Jang JH, Clark JD, Li X, Yorek MS, Usachev YM, Brennan TJ: Nociceptive sensitization by complement C5a and C3a in mouse. Pain 2010; 148:343–52
Perkins NM, Tracey DJ: Hyperalgesia due to nerve injury: Role of neutrophils. Neuroscience 2000; 101:745–57
Cunha TM, Verri WA Jr, Schivo IR, Napimoga MH, Parada CA, Poole S, Teixeira MM, Ferreira SH, Cunha FQ: Crucial role of neutrophils in the development of mechanical inflammatory hypernociception. J Leukoc Biol 2008; 83:824–32
Brack A, Rittner HL, Machelska H, Leder K, Mousa SA, Schäfer M, Stein C: Control of inflammatory pain by chemokine-mediated recruitment of opioid-containing polymorphonuclear cells. Pain 2004; 112:229–38
Rittner HL, Hackel D, Yamdeu RS, Mousa SA, Stein C, Schäfer M, Brack A: Antinociception by neutrophil-derived opioid peptides in noninflamed tissue – role of hypertonicity and the perineurium. Brain Behav Immun 2009; 23:548–57
Rittner HL, Mousa SA, Labuz D, Beschmann K, Schäfer M, Stein C, Brack A: Selective local PMN recruitment by CXCL1 or CXCL2/3 injection does not cause inflammatory pain. J Leukoc Biol 2006; 79:1022–32
Pogatzki EM, Gebhart GF, Brennan TJ: Characterization of Aδ- and C-fibers innervating the plantar rat hindpaw one day after an incision. J Neurophysiol 2002; 87:721–31
Sahbaie P, Shi X, Guo TZ, Qiao Y, Yeomans DC, Kingery WS, Clark JD: Role of substance P signaling in enhanced nociceptive sensitization and local cytokine production after incision. Pain 2009; 145:341–9
Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL: Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods 1994; 53:55–63
Poree LR, Guo TZ, Kingery WS, Maze M: The analgesic potency of dexmedetomidine is enhanced after nerve injury: A possible role for peripheral α2-adrenoceptors. Anesth Analg 1998; 87:941–8
Hargreaves K, Dubner R, Brown F, Flores C, Joris J: A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 1988; 32:77–88
Li X, Angst MS, Clark JD: A murine model of opioid-induced hyperalgesia. Brain Res Mol Brain Res 2001; 86:56–62
Kingery WS, Davies MF, Clark JD: A substance P receptor (NK1) antagonist can reverse vascular and nociceptive abnormalities in a rat model of complex regional pain syndrome type II. Pain 2003; 104:75–84
Clark JD, Shi X, Li X, Qiao Y, Liang D, Angst MS, Yeomans DC: Morphine reduces local cytokine expression and neutrophil infiltration after incision. Mol Pain 2007; 3:28
Shingu M, Nonaka S, Nishimukai H, Nobunaga M, Kitamura H, Tomo-Oka K: Activation of complement in normal serum by hydrogen peroxide and hydrogen peroxide-related oxygen radicals produced by activated neutrophils. Clin Exp Immunol 1992; 90:72–8
Vogt W: Complement activation by myeloperoxidase products released from stimulated human polymorphonuclear leukocytes. Immunobiology 1996; 195:334–46
Li WW, Sabsovich I, Guo TZ, Zhao R, Kingery WS, Clark JD: The role of enhanced cutaneous IL-1β signaling in a rat tibia fracture model of complex regional pain syndrome. Pain 2009; 144:303–13
Morin N, Owolabi SA, Harty MW, Papa EF, Tracy TF Jr, Shaw SK, Kim M, Saab CY: Neutrophils invade lumbar dorsal root ganglia after chronic constriction injury of the sciatic nerve. J Neuroimmunol 2007; 184:164–71
Thacker MA, Clark AK, Marchand F, McMahon SB: Pathophysiology of peripheral neuropathic pain: Immune cells and molecules. Anesth Analg 2007; 105:838–47
Sedgwick JB, Menon I, Gern JE, Busse WW: Effects of inflammatory cytokines on the permeability of human lung microvascular endothelial cell monolayers and differential eosinophil transmigration. J Allergy Clin Immunol 2002; 110:752–6
Williams TJ: Vascular permeability changes induced by complement-derived peptides. Agents Actions 1983; 13:451–5
Li WW, Guo TZ, Liang D, Shi X, Wei T, Kingery WS, Clark JD: The NALP1 inflammasome controls cytokine production and nociception in a rat fracture model of complex regional pain syndrome. Pain 2009; 147:277–86
Yazdi AS, Ghoreschi K, Röcken M: Inflammasome activation in delayed-type hypersensitivity reactions. J Invest Dermatol 2007; 127:1853–5
Busch-Dienstfertig M, Stein C: Opioid receptors and opioid peptide-producing leukocytes in inflammatory pain–basic and therapeutic aspects. Brain Behav Immun 2010; 24:683–94
Chadzinska M, Maj M, Scislowska-Czarnecka A, Przewłocka B, Plytycz B: Expression of proenkephalin (PENK) mRNA in inflammatory leukocytes during experimental peritonitis in Swiss mice. Pol J Pharmacol 2001; 53:715–8
Rittner HL, Brack A: Leukocytes as mediators of pain and analgesia. Curr Rheumatol Rep 2007; 9:503–10
Mousa SA, Shakibaei M, Sitte N, Schäfer M, Stein C: Subcellular pathways of β-endorphin synthesis, processing, and release from immunocytes in inflammatory pain. Endocrinology 2004; 145:1331–41
Ballet S, Conrath M, Fischer J, Kaneko T, Hamon M, Cesselin F: Expression and G-protein coupling of μ-opioid receptors in the spinal cord and dorsal root ganglia of polyarthritic rats. Neuropeptides 2003; 37:211–9
Ji RR, Zhang Q, Law PY, Low HH, Elde R, Hökfelt T: Expression of μ-, δ-, and κ-opioid receptor-like immunoreactivities in rat dorsal root ganglia after carrageenan-induced inflammation. J Neurosci 1995; 15:8156–66
Labuz D, Schmidt Y, Schreiter A, Rittner HL, Mousa SA, Machelska H: Immune cell-derived opioids protect against neuropathic pain in mice. J Clin Invest 2009; 119:278–86
Machelska H: Functional evidence of pain control by the immune system. Adv Exp Med Biol 2003; 521:88–97
Stein EA, Fuller SA: Effects of opiate agonists and antagonists on cerebral metabolic activity in the conscious rat. NIDA Res Monogr 1990; 105:395–6
Baamonde A, Lastra A, Juárez L, García V, Hidalgo A, Menéndez L: Effects of the local administration of selective μ-, δ-and κ-opioid receptor agonists on osteosarcoma-induced hyperalgesia. Naunyn Schmiedebergs Arch Pharmacol 2005; 372:213–9
Li X, Angst MS, Clark JD: Opioid-induced hyperalgesia and incisional pain. Anesth Analg 2001; 93:204–9
Engelhardt E, Toksoy A, Goebeler M, Debus S, Bröcker EB, Gillitzer R: Chemokines IL-8, GROα, MCP-1, IP-10, and Mig are sequentially and differentially expressed during phase-specific infiltration of leukocyte subsets in human wound healing. Am J Pathol 1998; 153:1849–60
Slominski AT, Zmijewski MA, Zbytek B, Brozyna AA, Granese J, Pisarchik A, Szczesniewski A, Tobin DJ: Regulated proenkephalin expression in human skin and cultured skin cells. J Invest Dermatol 2011; 131:613–22
Teofoli P, Motoki K, Lotti TM, Uitto J, Mauviel A: Propiomelanocortin (POMC) gene expression by normal skin and keloid fibroblasts in culture: Modulation by cytokines. Exp Dermatol 1997; 6:111–5
Mousa SA, Zhang Q, Sitte N, Ji R, Stein C: β-Endorphin-containing memory-cells and μ-opioid receptors undergo transport to peripheral inflamed tissue. J Neuroimmunol 2001; 115:71–8
Hestdal K, Ruscetti FW, Ihle JN, Jacobsen SE, Dubois CM, Kopp WC, Longo DL, Keller JR: Characterization and regulation of RB6-8C5 antigen expression on murine bone marrow cells. J Immunol 1991; 147:22–8
Stirling DP, Liu S, Kubes P, Yong VW: Depletion of Ly6G/Gr-1 leukocytes after spinal cord injury in mice alters wound healing and worsens neurological outcome. J Neurosci 2009; 29:753–64
Daley JM, Thomay AA, Connolly MD, Reichner JS, Albina JE: Use of Ly6G-specific monoclonal antibody to deplete neutrophils in mice. J Leukoc Biol 2008; 83:64–70
Eisenach JC, Lindner MD: Did experimenter bias conceal the efficacy of spinal opioids in previous studies with the spinal nerve ligation model of neuropathic pain? ANESTHESIOLOGY 2004; 100:765–7
Xu J, Gu H, Brennan TJ: Increased sensitivity of group III and group IV afferents from incised muscle in vitro . Pain 2010; 151:744–55
Xu YM, Ge HY, Arendt-Nielsen L: Sustained nociceptive mechanical stimulation of latent myofascial trigger point induces central sensitization in healthy subjects. J Pain 2010; 11:1348–55