Die Rolle der Leukozyten bei der Entstehung und Kontrolle von Entzündungsschmerz

Inflammatory pain is modulated by hyperalgesic and analgesic mediators. Hyperalgesic mediators include protons, cytokines, chemokines, and bradykinin produced by leukocytes at the site of inflammation. Simultaneously, leukocytes are known to produce analgesic mediators e.g. opioid peptides including beta- endorphin, met-enkephalin and endomorphins. Opioid peptides are secreted locally following exposure to stress or by local injection of releasing agents like corticotrophin releasing hormone (CRF). Following release, they bind to opioid receptors on sensory nerve terminals and confer peripherally mediated opioid analgesia. In this thesis we examined four aims in inflammatory pain induced by complete Freund’s adjuvant (CFA) in rats: i) role of granulocytes in the generation of inflammatory pain, ii) impact of inflammation on transcription of opioid receptor genes in the dorsal root ganglion (DRG), iii) peripherally mediated opioid analgesia by endomorphins, and iv) signaling pathways of opioid peptide release from granulocytes. The results show that i) granulocytes do not seem to confer pain, because selective recruitment of granulocytes by chemokines does not alter thermal or mechanical nociceptive thresholds. ii) Kappa-opioid receptor mRNA as well as protein in the DRG is upregulated in CFA inflammation mediated by IL-1beta iii) The newly discovered endomorphins can elicit potent analgesia in inflammation. Stress- as well as CRF-induced peripherally mediated analgesia is dependent on the release of classic opioid peptides but also on the release of endomorphins. iv) In granulocytes, opioid peptide release by is CXCR1/2 ligands dependent on Ca2+ from intracellular stores, phosphoinositol-3-kinase (PI3K) and p38 mitogen activated kinase (MAPK). In vivo, intraplantar injection of CXCR2 ligands elicits analgesia in inflammatory pain. Taken together, granulocytes contribute to peripherally mediated opioid analgesia in early inflammation while their role in the generation of inflammatory pain seems to be limited. Interference with granulocyte function using e.g. anti-inflammatory treatments like chemokine receptor antagonists or inhibitors of intracellular signaling pathways might inadvertently impair endogenous peripherally mediated opioid analgesia.Entzündungsschmerz wird durch algetische und analgetische Mediatoren moduliert. Zu den algetischen Mediatoren aus Leukozyten gehören z.B. Protonen, Zytokine, Chemokine und Bradykinin. Gleichzeitig werden aber von Leukozyten auch analgetische Mediatoren wie Opioidpeptide (Beta-Endorphin, Met-Enkephalin und Endomorphine produziert, die an Opioidrezeptoren an periphere sensorische Neuronen binden und so Analgesie auslösen. Stress oder bestimme Hormone wie Corticotrophin releasing factor (CRF) können Freisetzung von Opioidpeptiden auslösen. In der vorliegenden Arbeit wurden 4 Fragestellungen im Entzündungsschmerzmodell durch intraplantare Injektion von Freunds Adjuvans (CFA) untersucht: (i) Die Rolle von Granulozyten in der Schmerzentstehung, (ii) den Einfluss von Entzündung auf die Transkription von Opioidrezeptorgenen in Hinterwurzelganglien (DRG), (iii) periphere Opioidanalgesie durch Endomorphine und (iv) die Signalübertragungswege in der Freisetzung von Opioidpeptiden aus Granulozyten. Es wurde gezeigt, dass (i) Granulozyten per se keine Schmerzen auslösen, denn die selektive Rekrutierung durch Chemokine führt zu keiner Änderung der Schmerzschwellen. (ii) Kappa-Opioidrezeptor mRNA sowie Protein werden in der CFA Entzündung über IL-1beta hochreguliert. (iii) Die neu entdeckten Endomorphine können ebenfalls eine potente Analgesie auslösen. Stress und CRF induzierte Analgesie sind sowohl von der Ausschüttung klassischer Opioidpeptide als auch von Endomorphinen abhängig. (iv) Opioidpeptidfreisetzung aus Granulozyten nach CXCR1/2 Stimulation hängt von der intrzellulären Mobilisation von Kalzium sowie der Aktivierung von Phosphoinositol-3-Kinase (PI3K) und p38 mitogen activated kinase (MAPK) ab. In vivo können CXCR2 Liganden eine Analgesie in der entzündeten Pfote auslösen. Granulozyten spielen eine Rolle bei der peripheren Opioidanalgesie in der frühen Entzündungsphase während ihre Rolle bei der Schmerzentstehung begrenzt zu sein scheint. Anti-inflammatorische Medikamente, die auf Granulozytenfunktion oder die intrazellulären Signalkaskaden zielen, könnten unbeabsichtigter Weise daher die endogenen analgetischen Mechanismen des Körpers beeinträchtigen

The Connection of Monocytes and Reactive Oxygen Species in Pain

The interplay of specific leukocyte subpopulations, resident cells and proalgesic mediators results in pain in inflammation. Proalgesic mediators like reactive oxygen species (ROS) and downstream products elicit pain by stimulation of transient receptor potential (TRP) channels. The contribution of leukocyte subpopulations however is less clear. Local injection of neutrophilic chemokines elicits neutrophil recruitment but no hyperalgesia in rats. In meta-analyses the monocytic chemoattractant, CCL2 (monocyte chemoattractant protein-1; MCP-1), was identified as an important factor in the pathophysiology of human and animal pain. In this study, intraplantar injection of CCL2 elicited thermal and mechanical pain in Wistar but not in Dark Agouti (DA) rats, which lack p47phox, a part of the NADPH oxidase complex. Inflammatory hyperalgesia after complete Freund's adjuvant (CFA) as well as capsaicin-induced hyperalgesia and capsaicin-induced current flow in dorsal root ganglion neurons in DA were comparable to Wistar rats. Macrophages from DA expressed lower levels of CCR2 and thereby migrated less towards CCL2 and formed limited amounts of ROS in vitro and 4-hydroxynonenal (4-HNE) in the tissue in response to CCL2 compared to Wistar rats. Local adoptive transfer of peritoneal macrophages from Wistar but not from DA rats reconstituted CCL2-triggered hyperalgesia in leukocyte-depleted DA and Wistar rats. A pharmacological stimulator of ROS production (phytol) restored CCL2-induced hyperalgesia in vivo in DA rats. In Wistar rats, CCL2-induced hyperalgesia was completely blocked by superoxide dismutase (SOD), catalase or tempol. Likewise, inhibition of NADPH oxidase by apocynin reduced CCL2-elicited hyperalgesia but not CFA-induced inflammatory hyperalgesia. In summary, we provide a link between CCL2, CCR2 expression on macrophages, NADPH oxidase, ROS and the development CCL2-triggered hyperalgesia, which is different from CFA-induced hyperalgesia. The study further supports the impact of CCL2 and ROS as potential targets in pain therapy

Differential effects of everyday-life social support on chronic pain

Background: Social support is widely believed to positively influence pain symptoms, their intensity, and the ability to cope and influence pain. Social support is a multidimensional construct encompassing emotional support as well as pain-focused care and attention, also known as solicitous support. On the other hand, social support can be negative if it conflicts with the patient’s needs or even causes discomfort. How different types of social support influence pain is not very well understood especially because most of the present research originates from laboratory studies, raising uncertainties about its generalizability to everyday life of individuals with chronic pain. Methods: Here, we tested the effects of emotional, solicitous, and negative social support on pain intensity in everyday life. We collected data from 20 patients with acute complex regional pain syndrome using a smartphone-based Ecological Momentary Assessment with up to 30 survey prompts over a period of five consecutive days. Results: Our results showed that solicitous social support deceased pain, in particular in male patients. Emotional support was only beneficial on pain levels in women but not men. Conclusions: Together, these findings highlight the differential effects of every-day life social support on chronic pain

Selective blood-nerve barrier leakiness with claudin-1 and vessel-associated macrophage loss in diabetic polyneuropathy

Diabetic polyneuropathy (DPN) is the most common complication in diabetes and can be painful in up to 26% of all diabetic patients. Peripheral nerves are shielded by the blood-nerve barrier (BNB) consisting of the perineurium and endoneurial vessels. So far, there are conflicting results regarding the role and function of the BNB in the pathophysiology of DPN. In this study, we analyzed the spatiotemporal tight junction protein profile, barrier permeability, and vessel-associated macrophages in Wistar rats with streptozotocin-induced DPN. In these rats, mechanical hypersensitivity developed after 2 weeks and loss of motor function after 8 weeks, while the BNB and the blood-DRG barrier were leakier for small, but not for large molecules after 8 weeks only. The blood-spinal cord barrier remained sealed throughout the observation period. No gross changes in tight junction protein or cytokine expression were observed in all barriers to blood. However, expression of Cldn1 mRNA in perineurium was specifically downregulated in conjunction with weaker vessel-associated macrophage shielding of the BNB. Our results underline the role of specific tight junction proteins and BNB breakdown in DPN maintenance and differentiate DPN from traumatic nerve injury. Targeting claudins and sealing the BNB could stabilize pain and prevent further nerve damage

CCL2-induced hyperalgesia in Wistar, but not in Dark Agouti (DA) rats.

<p>Mechanical (<b>A, B</b>) and thermal (<b>C, D</b>) nociceptive thresholds were quantified before and after i.pl. injection of CCL2 (0.3 µg – open circle, 1 µg – filled triangle, 3 µg – open triangle) or 0.9 % NaCl (solvent  =  control – filled circle) into Wistar (<b>A, C</b>) and DA (<b>B, D</b>) rats (*p<0.05 versus time point 0 h, Two Way RM ANOVA, Student-Newman-Keuls, n = 6). Data are presented as means ± SEM of raw values (<b>A, B</b>) or % maximal possible effects (MPE) (<b>C, D</b>).</p

Establishment of CCL2-induced hyperalgesia following restoration of defective ROS generation in Dark Agouti (DA) rats.

<p>Peritoneal macrophages from Wistar and DA rats were isolated and treated for 1 h with 0.2 µg CCL2 or solvent control. ROS production was quantified by flow cytometry using the phagoburst assay. Representative histograms are shown for Wistar rats (<b>A</b>) and DA rats (<b>B</b>) with solvent (grey), CCL2 (black line) or positive assay control PMA (dotted line). (<b>C</b>) Results were analyzed statistically (n = 6/group, CCL2 i.pl. (white bars) or solvent (black bars), *p<0.05, t-test). (<b>D</b>) Wistar and DA rats were i.pl. injected with CCL2 i.pl. (lower panel) or solvent (upper panels) for 3 h, subcutaneous paw tissue was representative immunohistochemistry is shown for HNE (red), ED1⁺ macrophages (green) and merge of both together with nuclei (DAPI-blue). (<b>E</b>) Wistar and DA rats were i.pl. injected with CCL2 i.pl. (white bars) or solvent (black bars) for 3 h, subcutaneous paw tissue was prepared and HNE adducts were quantified by ELISA (n = 6/group, *p<0.05, t-test). (<b>F</b>) DA rats were s.c. injected with phytol for 5 days. Mechanical nociceptive thresholds were quantified before and after i.pl. injection of CCL2 (0.3 µg – open circle, 1 µg – filled triangle, 3 µg – open triangle). Control animals were treated 5 days with 0.9 % NaCl before i.pl. injection of 3 µg CCL2 (filled circle), (*p<0.05 versus time point 0 h, Two way RM ANOVA, Student-Newman-Keuls, n = 6). Data are presented as means ± SEM.</p

Inhibition of CCL2-induced mechanical hyperalgesia by ROS scavenging in Wistar rats.

<p>Mechanical nociceptive thresholds were quantified before and after (<b>A</b>) i.pl. injection of 1 µg apocynin with 3 µg CCL2 (filled circles) or 3 µg CCL2 alone and (<b>B</b>) i.pl. injection of 1 µg apocynin (filled circles) or its solvent 0.9 % NaCl (open circles) in rats with 4 d CFA inflammation. (<b>C–E</b>) Mechanical nociceptive thresholds were quantified before and after i.pl. injection of 3 µg CCL2 alone with the respective solvent (filled circle) or together with (<b>C</b>) SOD (3 U open circle, 30 U filled triangle, 300 U open triangle), (<b>D</b>) catalase (2–5 U – open circle, 20–50 U – filled triangle, 200–500 U – open triangle), or (<b>E</b>) tempol i.p. (1.5 mg – open circle, 7.5 mg – filled triangle, 15 mg – open triangle) (*p<0.05 versus time point 0 h, Two Way RM ANOVA, Student-Newman-Keuls, n = 6). Data are presented as means ± SEM.</p

Defective CCL2-induced chemotaxis of macrophages from Dark Agouti (DA) in comparison to Wistar rats.

<p>Wistar (<b>A</b>) and DA (<b>B</b>) rats were i.pl. injected with 3 µg CCL2 (light grey bars), with CFA (dark grey bars, positive control) or with 0.9% NaCl (black bars, solvent negative control). Single cell suspensions were prepared from paw tissue and the number of infiltrating ED1⁺ CD45⁺ macrophages was quantified by flow cytometry (n = 4, *p<0.05, One Way ANOVA, Student-Newman-Keuls). (<b>C</b>) CCR2 mRNA from peritoneal rat macrophages from Wistar (white bars) as well as DA (black bars) rats was measured with real time PCR (*p<0.05, t-test, n = 6). (<b>D</b>) The migratory capacity of macrophages from Wistar rats (white bars, n = 8) and DA rats (black bars, n = 5) was quantified in a Boyden chamber in response to CCL2 and solvent control (p<0.05, One way ANOVA, Dunns-Method).</p

Comparable nociceptive thresholds and DRG currents in Wistar and Dark Agouti (DA) rats.

<p>Rats received an i.pl. injection of CFA (<b>A–D</b>; ipsilateral injected paw  =  open circle, contralateral, non injected paw  =  filled circle). Mechanical (<b>A, B</b>) and thermal nociceptive (<b>C, D,</b>) thresholds were quantified (*p<0.05 versus time point 0 h, Two Way RM ANOVA, Student-Newman-Keuls, n = 6). Data are presented as means ± SEM of raw values (<b>A, B</b>) or % maximal possible effects (MPE) (<b>C, D</b>). (<b>E</b>) DRG-neurons from naïve DA rats were obtained and cultured for 48 h. Representative traces of whole cell recordings are shown after application of capsaicin (500, 1000 nM, left). Bar graph quantifies capsaicin-activated inward currents in DRG neurons from Wistar and DA rats (right) (n = 9, means ± SEM, p>0.05, t- test). In addition DA rats received i.pl. injection of capsaicin (<b>F</b>; solvent – filled circle, 30 µg capsaicin – open circle) and thermal nociceptive thresholds were quantified (*p<0.05 versus time point 0 h, Two Way RM ANOVA, Student-Newman-Keuls, n = 6). Data are presented as means ± SEM of % maximal possible effects (MPE).</p

Reconstitution of CCL2-induced hyperalgesia by adoptive transfer of macrophages derived from Wistar, but not from Dark Agouti (DA) rats.

<p>(<b>A</b>) Mechanical nociceptive thresholds were quantified after i.pl. injection of 3 µg CCL2 with prior leukocyte depletion by systemic injection of CTX (filled triangles), 3 µg CCL2 with prior systemic injection of solvent (cyclo-phosphamid (CTX) treatment (open circles), or solvent of CCL2 without CTX treatment (filled circles). CTX injections were performed 3 d with 10 mg/kg and 1d 50 mg/kg before cell and/or CCL2 treatment. (<b>B</b>) Representative dot blots (x-axis: forward scatter (FSC) – cell size; y-axis: sideward scatter (SSC) – cell granularity) for leukocyte subpopulations in peripheral blood of untreated (left) and CTX-treated Wistar rats (right) are shown by flow cytometry. Gates were set on beads (for quantification), neutrophils, lymphocytes and monocytes. (<b>C–F</b>) Wistar and DA rats were leukocyte-depleted by i.p. injection with CTX followed by i.pl. injection of different numbers of macrophages from either Wistar or DA rats, 30 min later, by i.pl. injection of 3 µg CCL2. Injection of 2×10⁶ macrophages without concomitant injection of CCL2 served as a negative control. (<b>C</b>) Wistar rats reconstituted with cells from Wistar rats. I.p. injections of solvent only served as a negative control for CTX treatment (open triangles) with the same injection pattern. (<b>D</b>) Wistar rats reconstituted with cells from DA rats. (<b>E</b>) DA rats reconstituted with cells from Wistar rats. (<b>F</b>) DA rats reconstituted with cells from DA rats. (*p<0.05 compared to time point 0 h, Two Way RM ANOVA, Student-Newman-Keuls, n = 6). Data are presented as means ± SEM.</p