JAK-STAT1/3-induced expression of signal sequence-encoding proopiomelanocortin mRNA in lymphocytes reduces inflammatory pain in rats

To identify potential regulators of POMC gene expression, we compared the expression profile of inflammatory cytokines in lymph nodes draining normal vs. inflamed paws 2 h following intraplantar (i.pl.) injection of Complete Freund’s Adjuvant (CFA). Among nineteen cytokines analyzed, only IL-1β and IL-4 were significantly up-regulated in comparison to lymph node lysates from healthy animals (Table 1). Stimulation of lymph node-derived naïve lymphocytes with 5 ng/ml interleukin (IL)-1β for 2 h in vitro did not significantly elevate POMC exon 2–3 mRNA transcript levels over unstimulated controls (Figure 1A). Dose-dependent increases of these mRNA transcripts were observed after incubation with IL-4; a significant elevation over control levels was obtained with 10 ng IL-4/ml (Figure 1B). No differences were detectable between untreated and IL-2-, MIP-3α-, MCP-1- (data not shown), or ConA-treated cells (Figure 1C).

The pan-JAK inhibitor pyridon 6 reduced the IL-4-induced elevation of POMC mRNA. This inhibition was significant at concentrations of 0.3 and 0.6 μM (Figure 1D). The JAK1/3 inhibitor A771726 (125 μM), but not the STAT6 inhibitor cyclic pifithrin-alpha, significantly decreased the IL-4-induced elevation of POMC mRNA (Figure 1E, F). IL-4-induced POMC mRNA levels were significantly attenuated by STAT1/3 but not by STAT5 or −6 decoy oligonucleotides (Figure 1G, H, I). After exposure of naïve cells to IL-4, cell lysates were analyzed using Western Blotting. STAT1, STAT3, and STAT5 showed strong Tyrosine-phosphorylations, which were abolished by pyridon 6 pretreatment (see Figures. 2B and C). Significant differences were observed between treatment groups for STAT3 and STAT5 phosphorylation (Friedman test, p < 0.05). Post hoc comparison using Dunn’s test revealed significant differences for Tyrosine-phosphorylation of STAT3 between IL-4 and IL-4 plus 0.16 and 0.66 μM pyridon 6 treated cells. For STAT5 phosphorylation the post hoc comparison remained insignificant. Phosphorylation of STAT3 at Serine 727 was not observed (Figure 2A), while slight STAT3 acetylation at Lysine 685 was observed in unstimulated, IL-4-treated, and pyridon 6 pretreated cells and appeared to be unaffected by the cell treatments (Figure 2C, Friedman test, p > 0.05). Akt phosphorylation at Serine 473 was present after IL-4-stimulation and absent in cells pretreated with pyridon 6 (Figure 2B, Friedman test, p < 0.05). However, post hoc comparison for Akt phosphorylation remained insignificant. Phosphorylation of extracellular-signal regulated kinase (ERK) 2 (p42) and of mitogen-activated protein kinase p38 remained largely unaffected by IL-4-stimulation (Figure 3) but phosphorylation of both kinases tended to increase in IL-4 plus pyridon 6 treated cells. Significant differences between treatment groups were observed for p42-phosphorylation (Friedman test, p < 0.05). Post hoc comparison using Dunn’s test revealed significant differences for p42-phosphorylation between untreated controls and IL-4 plus 0.66 μM pyridon 6 treated cells.

Cellular amounts of immunoreactive beta-endorphin did not change in naïve lymphocytes stimulated with IL-4 for 24 h (Figure 4A). To mimick cell activation, naïve lymphocytes were incubated for 24 h with ConA, which had no effect on the cellular levels of immunoreactive beta-endorphin (Figure 4A). However, the combined stimulation with ConA and IL-4 significantly increased contents of immunoreactive beta-endorphin (Figure 4A). Vesicular release was induced by ionomycin-treatment. Extracellular levels of immunoreactive beta-endorphin were significantly higher than controls when cells were pre-stimulated with combined but not separate ConA and IL-4 (Figure 4B). Ionomycin-induced release of immunoreactive beta-endorphin from ConA/IL-4 stimulated cells was not significantly influenced by up to 1 μM pyridon 6 pretreatment (Figure 4B).

Four days after i.pl. injection of Complete Freund’s Adjuvant (CFA), paw pressure thresholds (PPT) in inflamed (ipsilateral) paws were significantly lower than in noninflamed (contralateral) paws of rats immunosuppressed by cyclophosphamide (CTX, Figure 5). Intraplantar transfer of unstimulated or stimulated cells did not change the reduced PPT (hyperalgesia) in inflamed paws in comparison to the baseline levels (Figure 5). However, i.pl. injection of 1.5 ng CRF completely reversed hyperalgesia in paws injected with ConA/IL-4-stimulated T cells compared to all other groups, such that PPT were similar to contralateral noninflamed paws (Figure 5). CRF-induced increases of ipsilateral PPT values were significantly higher in animals receiving 1×10⁵ (63.8 ± 4.4 g) and 5×10⁵ (65.0 ± 7.3 g) ConA/IL-4 treated cells in comparison to 10×10⁵ cells (53.8 ± 8.0 g) (One-Way ANOVA and Bonferroni’s Test, P < 0.05). Therefore, subsequent experiments were performed with the lowest cell number. Four days after i.pl. CFA, PPT were analyzed in immunosuppressed (Figure 6A) and in immunocompetent (Figure 6B) animals pretreated with s.c. NLX prior to CRF injection. PPT in immunosuppressed versus immunocompetent rats were slightly but significantly lower in both contralateral (64.4 ± 1.0 g versus 68.1 ± 1.0 g, respectively; unpaired t-test, P < 0.05, 8 rats per group) and in inflamed paws (29.4 ± 1.2 g versus 36.04 ± 0.8 g, respectively, unpaired t-test, P < 0.05, 8 rats per group). Baseline PPT in inflamed paws were not influenced by NLX. In recipients of ConA/IL-4-stimulated cells, NLX completely reversed CRF-induced increases of PPT (Figure 6A). In immunocompetent rats, CRF-induced PPT elevations in inflamed paws were significantly higher than in contralateral paws. This effect was abolished by NLX (Figure 6B).

At 1 and 2 h after induction of CFA-inflammation in vivo, phosphorylated STAT6 was undetectable in cells isolated from ipsi- and contralateral popliteal lymph nodes (Figure 7A). Tyrosine-phosphorylation of STAT3 was observed in cells from ipsi- and contralateral nodes (Figure 7B). Densitometry showed that at both 1 and 2 h post CFA-injection, this phosphorylation was significantly stronger in the ispilateral than in the contralateral node cells (Wilcoxon signed rank test, P < 0.05). Serine-phosphorylation of STAT3 and Tyrosine-phosphorylation of STAT1 were not detectable in cells from ipsi- or contralateral nodes (Figure 7B). Despite background, Threonine-phosphorylation of Akt was detectable in cells from the ipsi- and contralateral nodes, but no reliable densitometric analysis could be performed.

Experiments were approved by the animal care committee of the State of Berlin and strictly followed the guidelines of the International Association for the Study of Pain [52]. Male Wistar rats (225–300 g, Charles River Breeding Laboratories) received an i.pl. injection of 0.15 ml Complete Freunds’ Adjuvant (CFA, Calbiochem, La Jolla, CA, USA) into the right hind paw under brief isoflurane (Rhodia Organic Fine Ldt., Bristol, UK) anesthesia. The inflammation remained confined to that paw throughout the observation period.

Popliteal lymph nodes draining normal and inflamed hind paws (2 h post CFA) were dissected, homogenized, and lyzed. Expression of cytokines was analyzed using RayBio^TM Rat Cytokine Antibody Array 1.1 kits (RayBiotech, Inc., Norcross, GA, USA) following the manufacturer’s instructions. Array-membranes carrying antibodies for the detection of nineteen cytokines and anti-rat IgG (loading control) were incubated with the lymph node lysates (500 μg protein/membrane) for 2 h. After washing, the membranes were incubated for 2 h with a mixture of biotin-conjugated antibodies, followed by peroxidase-conjugated streptavidin. Immunoreactive dots were subsequently visualized using an enhanced chemiluminescence (ECL) system, and membranes were exposed to autoradiograph hyperfilms for 10 to 30 seconds.

Healthy rats were sacrificed by isoflurane overdose. Popliteal, axillary, and inguinal lymph nodes were dissected and pooled. Our previous flow cytometry analyses had shown that about 95% of cells residing in naïve nodes express the hematopoietic cell marker CD45, 70-80% are CD3⁺ T lymphocytes, and 20-25% are IgG kappa light chain⁺ B lymphocytes [21]. Lymphocytes were dissociated from surrounding tissue using 40 μm mesh cell strainers and were cultured in RPMI-1640 medium containing 1% penicillin/streptomycin under standard culturing conditions (37°C and 5% CO₂) unless otherwise stated.

Naïve lymphocytes were stimulated for 2 or 24 h with 0.1 – 10 ng/ml of rat recombinant cytokines and chemokines (IL-1β, -2, -4, -10, MIP-3α, or MCP-1, all from R&D Systems, Wiesbaden, Germany), and with the mitogen ConA (1 μg/ml, Sigma-Aldrich). After stimulation, cells were collected on ice, centrifuged, and pellets were stored at −80°C.

Cells were pretreated with permeable small molecule inhibitors (Calbiochem, EMD Chemicals Inc., an Affiliate of Merck KgaA, Darmstadt, Germany) prior to the addition of the stimulants. Pyridon 6, a pan-JAK inhibitor, was applied at concentrations of 0.16, 0.3, 0.6 and 1 μM for 30 min [53]. To inhibit JAKs 1 and 3, the active metabolite of leflunomide, A771726, was added for 120 min (75 and 125 μM) [54]. The STAT6 inhibitor cyclic pifithrin-α was applied for 30 min (0.1, 0.5, 1, 5, 10 and 50 μM) [55].

Double-stranded decoy oligonucleotides (TIBMOLBIOL, Berlin, Germany) providing binding motifs for STAT6 (5^′-gATCCTACTTCATggAAgAAT-3^′), STAT1/3 (5^′- gATCgAgTTTACgAgAACTC-3^′), or STAT5 (5^′-gATCgCATTTCggAgAAgACg-3^′) were prepared as described without chemical modifications [56]. Cells were diluted in antibiotic-free culture medium containing 10% serum and were plated for transfection on 6-well-plates (10 cm² surface area) using the BLOCKiT^TM Transfection Kit (Invitrogen GmbH, Karlsruhe, Germany) according to the manufacturer’s instructions. The transfection reagent Lipofectamine 2000^TM (5 μl/well) was mixed with 50, 100 or 200 pmol decoy oligonucleotide solutions to allow complex formation prior to addition of the mixture to the cells. Cells were then incubated at 37°C and 5% CO₂. A non-target, fluorescein-labeled double-stranded RNA oligomer (BLOCKiT^TM fluorescent oligo) was used as an indicator of transfection efficiency. Uptake of the BLOCKiT oligo was observed already at 6 h post transfection and persisted for at least 24 h in the cells, as assessed using a fluorescence microscope. Accordingly, medium was replaced 24 h after transfection by pure RPMI-1640 medium, then cytokine was added and cells were incubated for another 2 h at 37°C and 5% CO₂. Thereafter, cells were collected on ice, centrifuged, and pellets were stored at −80°C until POMC exon 2–3 mRNA was assayed using qRT-PCR.

Cellular content of beta-endorphin was determined by measuring immunoreactive beta-endorphin in cell lysates using a rat radioimmunoassay (RIA) kit according to the manufacturer’s instructions (Phoenix Peptides Inc., Burlingame, CA, USA) and as previously described [21, 57]. Briefly, lymphocytes were lyzed by sonication (15 sec, 1 impulse/sec) at a concentration of approximately 3 × 10⁶ cells per 100 μl assay buffer and beta-endorphin immunoreactivity was determined in 100 μl of the lysates in duplicate.

The release of beta-endorphin was determined in cell supernatants using a human/rat-fluorescent EIA kit according to the manufacturer’s instructions (Phoenix Peptides Inc.) as previosly described [7] Briefly, release was induced by incubation of approximately 6× 10⁶ cells/120 μl RPMI-1640 medium containing 10 μM ionomycin (Sigma-Aldrich). Cells were then incubated for 7 min at 37°C and 600 rpm in a thermal heating block, chilled on ice, and centrifuged for 10 min at 450 × g and 4°C. Wells of EIA plates were loaded with 50 μl of the supernatants each; beta-endorphin immunoreactivity was assessed in duplicate.

Western Blotting was performed as previously described [46]. Briefly, cells were sonicated and homogenized in RIPA buffer [50 mM Tris–HCl (pH 8.0); 150 mM NaCl; 1% Nonidet P-40 (v/v); 0.5% Desoxycholat (w/v); 0.1% SDS (w/v)] in the presence of protease and phosphatase inhibitors (Complete mini and PhosSTOP tablets, Roche). Proteins (30–50 μg/sample) were subjected to polyacrylamide gel-electrophoresis, the gels were composed of an upper stacking and a lower resolving part according to the method of Laemmli [58]. After separation, proteins were transferred at 350 mA/60 min to Immobilon-P membranes (Millipore Corperation, Billerica, MA, USA). Membranes were blocked in Tris buffered saline containing 2.5% bovine serum albumin and 0.1% Tween-20 for at least 30 min at room temperature. After blocking, blots were sequentially probed with the following polyclonal rabbit antibodies overnight at 4°C: anti-phospho-STAT1 (Tyrosine 701, 84/91 kDa), anti-STAT1 (84/91 kDa), anti-phospho-STAT3 (Tyrosine 705, 79/86 kDa), anti-phospho-STAT3 (Serine 727, 86 kDa), anti-acetyl-STAT3 (Lysine 685, 79/86 kDa), anti-STAT3 (79/86 kDa), anti-phospho-STAT5 (Tyrosine 694, 90 kDa), anti-STAT5 (90 kDa), anti-phospho-STAT6 (Tyrosine 641, 110 kDa), anti-STAT6 (110 kDa), anti-phospho-Akt (Threonine 308, 60 kDa), anti-phospho-Akt (Serine 473, 60 kDa), anti-Akt (60 kDa), anti-phospho-p44/42 (recognizes p42 when phosphorylated at Threonine 183/Tyrosine 185 in the rat, 42 kDa), anti-p44/42 (42/44 kDa), as well as with anti-phospho-p38 (Threonine 180/Tyrosine 182, 38 kDA) and anti-p38 (38 kDA). All antibodies were diluted 1/1000 in blocking buffer and purchased from Cell Signaling Technologies (Danvers, Massachusetts, USA). After incubation with peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG and rabbit anti-mouse IgG purchased from Jackson ImmunoResearch Europe Ltd., Suffolk, UK) diluted 1/5000 in blocking buffer, immunoreactive bands were visualized using an ECL system. Peroxidase-conjugated anti-beta-actin (42 kDa) was purchases from Sigma-Aldrich and diluted 1:50,000 in blocking buffer; this antibody renders the use of a secondary antibody before overlay of the blot with ECL solution unnecessary. Exposure time of the blots to autoradiograph hyperfilms was 10 to 120 s. Bound antibodies were removed by stripping for 15 min at 50°C in 62.5 mM Tris–HCl containing 100 mM beta-Mercaptoethanol and 2% SDS. Controls included reprobing upon omission of primary or secondary antibodies.

Primers were designed to amplify POMC and ribosomal protein L19 mRNA transcripts using OLIGO Primer Analysis Software Version 5.0 for Windows. Oligodeoxynucleotides were synthesized and purified by TIBMOLBIOL. Real-time PCR assays were performed using the Fast start DNA Master SYBR Green I assay (Roche) according to the instructions of the manufacturer in a LightCycler 1.5 instrument including melting curve analyses. Positive controls contained pituitary cDNA; negative controls contained double-distilled H₂O or RT^- cDNA. Amplification was performed as detailed in Sitte et al. [21], all samples but positive and negative controls were run in duplicate. For some measurements, sensitivity for POMC mRNA amplification was enhanced using a semi-nested real-time PCR protocol as previously described [21]. The amount of POMC exon 2–3 transcripts (sense primer: 5^′-CCCTCCTGCTTCAGACCTCCA-3^′, antisense primer: 5^′-TCTCTTCCTCCGCACGCCTCT-3^′) was normalized to the expression levels of ribosomal protein L19 using exon 2–5 spanning primers (sense primer: 5^′-AATCGCCAATGCCAACTCTCG-3^′, antisense primer: 5^′-TGCTCCATGAGAATCCGCTTG-3^′), treatment effects were evaluated by applying the delta-delta CP method as detailed below.

Rats were handled daily for 4 days. They were treated thrice at intervals of 48 h intraperitoneally (i.p.) with cyclophosphamide (CTX, Baxter Oncology) to induce depletion of immune cells as previously described [49]; a single i.pl. CFA injection into the right hind paw was given 72 h after the first CTX-injection. At 96 h post CFA-inoculation, immunosuppressed rats received purified T lymphocytes into inflamed paws (i.pl.); control animals were injected with vehicle (PBS). These T cells were obtained from pooled axilliary and inguinal lymph nodes of healthy donor rats as detailed above. Cells were treated for 24 h with/without ConA (1 μg/ml), IL-4 (10 ng/ml), or ConA plus IL-4 ex vivo. Then cell suspensions were depleted of MHC class II receptor⁺ and CD45RA⁺ cells (dendritic cells, monocytes/macrophages, and B lymphocytes) using magnetic cell sorting columns, anti-rat MHC class II receptor and anti-rat CD45RA beads (Miltenyi Biotec, Bergisch Gladbach, Germany), similar to Sitte et al. 2007 [21]. This procedure revealed > 95% pure T cell suspensions that were reconstituted at 1× 10⁵, 5× 10⁵ and 10× 10⁵ cells per 50 μl PBS for i.pl. injections. In the first set of experiments, animals received i.pl. CRF (1.5 ng/50 μl) to induce opioid peptide release 10 min after i.pl. T cell administration. In the second experiment, naloxone-methiodide (NLX; 10 mg/kg) or vehicle (saline) were injected subcutaneously (s.c.) 5 min after i.pl. T cell administration. Another 5 min later the animals received i.pl. CRF (1.5 ng/50 μl). Another group of immunocompetent rats received s.c. NLX or vehicle, followed by i.pl. CRF.

Mechanical hyperalgesia was tested by measuring paw pressure thresholds (PPT) (modified Randall–Selitto test; Ugo Basile) as previously described [59]. Measurements were performed immediately before (baseline) and 7 min after T cell transfer, as well as 5 min post CRF injection. Three consecutive trials, separated by 10 s intervals each, were conducted and the average was calculated. The sequence of left and right paws was alternated between animals to avoid bias. The experimenter was blind to the treatment.

POMC and ribosomal protein L19 qRT-PCR data were analyzed using the LightCycler software 3.5 (Roche). Levels of transcripts were assessed as crossing points (CP) when specific amplification exceeded background fluorescence using the Second Derivative Maximum analysis method of the system. Average PCR efficiencies were 1.89 for rpL19 and 1.82 for POMC exon 2–3 transcripts. All data were subsequently extrapolated using MS Excel 2003. Differences between treated and untreated cells are shown as mean POMC exon 2–3 mRNA ratios ± SEM and were calculated by applying the delta-delta method: Ratio = (PCR efficiency of POMC)^delta CP_{POMC(control-sample)}/(PCR efficiency of rpL19)^delta CP_{rpL19(control-sample)}. Based on this equation, enhanced POMC mRNA expression gives ratios > 1, decreased POMC mRNA expression gives ratios < 1. The cytokine array and western blot hyperfilms were scanned at 400 dpi and inverted for analysis by optical densitometry using Image J software 1.37v (Wayne Rasband, National Institute of Mental Health, Bethesda, Maryland, USA). Data are presented as mean % expression of loading control ± SEM after background correction.

All data were analyzed with GraphPad Prism Version 4.01 for Windows (GraphPad Software, USA). Normalized cytokine array data were analyzed using unpaired t-test with Welch’s correction to account for the number of experiments performed. Statistical analysis was performed on normalized CP values (e.g. CP_POMC-CP_rpL19) in case of the qRT-PCR data; beta-endorphin immunoreactivity values (cellular content as well as amount in the supernatant in pg) were normalized to one million cells. Statistical significance with respect to qRT-PCR data was calculated using the non-parametric Wilcoxon signed rank test if two groups were compared. Multiple comparisons of matched qRT-PCR, RIA, EIA, and Western Blot data were performed using the non-parametric Friedman Test; post-hoc comparisons were performed by Dunn’s test. Behavioral data (average PPT values) were analyzed by Two-Way repeated measures (RM) ANOVA and Bonferroni correction for multiple comparisons. For all tests, statistical significance was considered if P < 0.05.