Animal protocols were approved by the animal care committees (Landesamt für Arbeitsschutz, Gesundheitsschutz und Technische Sicherheit, Senate of Berlin, und Regierung von Unterfranken, Germany), and are in accordance with the International Association for the Study of Pain . Male Wistar rats weighing 180–220 g were treated as described below under brief isoflurane anesthesia. Animals were sacrificed using intracardial injection of a solution of T61 (embutramide, mebezonium and tetracaine) under isoflurane anesthesia according to national guidelines.
Nociceptive thresholds and paw volume
Mechanical nociceptive thresholds were determined using the paw pressure algesiometer (modified Randall-Selitto test; Ugo Basile, Comerio, Italy) as described before . A blunt piston onto the dorsal surface of the hind paw applied the pressure. The pressure required to elicit paw withdrawal was defined as the paw pressure threshold. The average of three measurements was calculated. Treatments were randomized and blinded. A decrease in the paw pressure threshold was interpreted as pain (hyperalgesia) whereas a rise in the paw pressure threshold was interpreted as analgesia (antinociception).
Thermal nociceptive thresholds were measured by the Hargreaves test as previously described . The latency (time; s) required to elicit paw withdrawal was measured with an electronic timer (IITC Inc/Life Science, Woodland Hills, CA, USA) after application of radiant heat to the plantar surface of a hind paw from underneath the glass floor with a high-intensity light bulb. The stimulus intensity was adjusted to 20 s for the paw withdrawal latency in non-inflamed paws, and a cutoff of 30 s was set to avoid tissue damage. The average of two measurements taken with 20 s intervals was calculated. A decrease in paw withdrawal latency was interpreted as pain (hyperalgesia) whereas a rise in paw withdrawal latency was interpreted as analgesia (antinociception)
Paw volume was measured by submerging the hind paw till the tibiotarsal joint inside the water-filled Perspex cell of a plethysmometer at the same time points (37140, Ugo Basile, Comerio, Italy) .
Rats were intraplantarly (i.pl.) injected into the right hind paws with 150 μl CFA (Calbiochem, Darmstadt, Germany) for 4 d. Separate groups of rats were treated with i.pl. injection of LPS, naloxone (NLX) (both Sigma-Aldrich, St. Louis, MO, USA) or TAK-242, resatorvid, a small molecule TLR4 inhibitor (MedChem Express, HY-11109, Princeton, NJ, USA). Doses were chosen according to pilot experiments or previous experiments [50, 52].
For immunodepletion rats were treated with cyclophosphamide (CTX) (Baxter, Unterschleissheim, Germany) according to an established protocol , because selective monocyte/macrophages depletion using clodronate has previously been unsuccessful . Animals were injected i.p. 3 d and 1 d before the experiment with 100 mg/kg and 50 mg/kg of cyclophosphamide as well as on day two after CFA i.pl. This treatment results in over 90% reduction in circulating leukocytes . No change in basal nociceptive thresholds after this treatment has been observed before .
Monocyte isolation and macrophage differentiation
Human monocytes were used because of lack of sufficient quantity and purity of rat blood monocytes to be used for in vitro stimulation [53, 54]. Monocytes were isolated from leukocyte apheresis filters of healthy donors by magnetic cell separation using anti-CD14-microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) or a combined purification using anti-CD16-microbeads immediately after harvest. Due to anonymous collection from left over apheresis filters, no consent from donors was necessary in line with the ethical committee of the institution. Human monocytes were cultured in RPMI (Invitrogen, Darmstadt, Germany) supplemented with 10% fetal calf serum (PAA Laboratories, Cölbe, Germany), 100 μg/ml streptomycin and 100 units/ml penicillin (both Biochrom AG, Berlin, Germany).
CD14+ human monocytes were cultured in a density of 2 × ∁105 cells/cm2 in CellBIND surface cell culture flasks for 6–7 days with 50 ng/ml GM-CSF to generate GM- or M1-macrophages and 50 ng/ml M-CSF and 25 ng/ml IL-10 to generate M- or M2-macrophges according to the literature .
Opioid peptide content and release
1×107 CD14+ monocytes or 0.5×107 M1 or M2 macrophages were stimulated with LPS (1–10 μg/ml, Sigma-Aldrich), Pam3CSK4 (1–10 μg/ml, InvivoGen, San Diego, USA) or ionomycin (10 μM) after preincubation with cytochalasin B (dissolved in dimethyl sulfoxide, final 5 μg/ml) for 5 min in Hank’s balanced salt solution containing the proteinase inhibitors bestatin (40 μg/ml), aprotinin (10 μg/ml) and thiorphan (dissolved in dimethyl sulfoxide, final 25 μg/ml, all Sigma-Aldrich) . Doses of LPS, Pam3CSK4 and ionomycin were based on pilot experiments and the literature . In some experiments, cells were concomitantly incubated with inhibitors: 1, 2-Bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM, dissolved in dimethyl sulfoxide, Calbiochem, Merck4Biosciences, Darmstadt, Germany) is a cell-permeant Ca2+ chelator used to control the level of intracellular Ca2+. 2-Aminoethoxydiphenyl borate (2-APB, dissolved in dimethyl sulfoxide, Calbiochem) inhibits IP3 receptors. In previous experiments the solvent dimethyl sulfoxide (maximal final concentration 1%) did not induce significant release . Supernatants were obtained after 15–120 min stimulation in accordance with the rapid effect of these mediators in pain behavior tests.
For the measurement of opioid peptide content 1×106 M1 and M2 macrophages were resuspended in ELISA buffer followed by five freeze (10–20 s) - thaw (90 s) cycles using liquid nitrogen and a 37°C water bath as well as subsequent vortexing as described before . After centrifugation to remove unsolvable material the supernatants containing opioid peptides were obtained. Supernatants were stored at −20°C until further analysis by radioimmunoassay using commercially available kits for human β-END (Phoenix Pharmaceuticals, Burlingame, USA).
Cellular staining of monocytes or macrophages was performed as previously described . Single cell suspensions were incubated with the following primary and secondary antibodies (all antibodies by BD Biosciences, Heidelberg, Germany unless stated otherwise): Macrophages were preincubated with Fc block (mouse anti-rat CD32) for 5 min at 4°C, then the first antibody anti-human, PM-2 K (10 μg/ml, AbD Serotec, Oxford, Great Britain), anti-human 25 F9 Biotin (10 μg/ml, ebioscience, Frankfurt, Germany) or isotype control antibodies were added for 30 min at room temperature. After washing cells were incubated with the second antibody for 30 min (anti-mouse IgG1 FITC or Streptavidin PE-Cy5). TLRs were labeled on human monocytes or macrophages using PE conjugated anti-human-TLR2, anti-human-TLR4 (ebioscience) or isotype control antibodies according to manufacturer’s instructions. CD14 and CD16 were stained with FITC-conjugated anti-CD14 and PE-conjugated anti-CD16 antibodies for 15 min (both from Miltenyi Biotec, Bergisch Gladbach, Germany).
For tissue analysis, subcutaneous paw tissue was obtained and digested as described before [26, 56]. Cell suspension were stained with a mouse monoclonal anti-rat-CD45-PerCP-Cy5, anti-rat-RP-1-PE (both BD bioscience), anti-rat-CD68-FITC and anti-rat-CD163-FITC (both AbD Serotec) and mouse monoclonal anti-rat-TLR4-PE (Thermo Scientific Pierce Product, Bonn, Germany). For RP-1 and CD68 double staining cells were first fixed in paraformaldehyde and permeabilized in saponin as previously described [26, 56].
The FACS Scan acquired 10.000-20.000 FACS events. Data analysis was performed by CellQuest software (all BD Biosciences).
CD14+ human monocytes were preincubated for 5 min with cytochalasin B followed by stimulation with 10 mM ionomycin for 15 min. Cells were harvested and centrifugated. Cell pellets of monocytes were reconstituted in 5 ml PBS, and 50,000 monocytes in suspension were then centrifuged by a Shandon Cytospin 3 (Thermo Shandon, Pittsburgh, PA) at 20 g for 3 min on glass slides. Monocytes were fixed for 30 min and confocal analysis was carried out as previously described [14, 57]. Briefly, monocytes cytospins were incubated with rabbit polyclonal antibodies against β-END (1:1000, Peninsula Laboratories, Belmont, CA, USA) and subsequently with a Texas red-conjugated goat anti-rabbit antibody. Thereafter, cytospins were washed with PBS and mounted in vectashield. Images were acquired on a Zeiss LSM510META confocal laser scanning system (Zeiss AIM; Jena, Germany) using a 63×/1.4 Plan-Apochromat or 40×/1.3 Plan-Neofluar oil immersion objective in a series of optical sections of about 1 μm thickness. Each experiment was repeated three times. To demonstrate specificity of staining, the following controls were included as mentioned in detail elsewhere: (1) preabsorption of diluted antibody against β-END with purified β-END (Peninsula laboratories-Bachem) and (2) omission of either the primary or the secondary antibodies.
Data are presented as raw values (mean ± SEM). Data were tested for normality and for equal variance. Normally distributed data were analyzed by t-test or paired t-test for samples before and after treatment. If aliquots of one sample were exposed to different conditions or repeated measurements from one animal were taken, One or Two Way repeated measurement (RM) ANOVA was used. The post hoc comparisons were performed by Student-Newman-Keuls. In case of not normally distributed data the test was performed on ranks. Differences were considered significant if p < 0.05.