RESUMO
The endothelial chemokine CXC motif ligand 16 (CXCL16) is involved in the recruitment and firm adhesion of CXCR6+ cells to the atherosclerosis-prone aortic vessel wall. Recently we showed that CXCR6+ platelets from flowing blood attach to CXCL16 expressed by activated endothelium on the luminal side of the blood vessel. With this study we supplement these findings with the observation that platelets bound to the inflamed endothelium are presenting CXCR6 to CXCL16-positive peripheral blood mononuclear cells (PBMCs) and, thus, are mediating an increased adhesion of PBMCs to the arterial wall. Furthermore we identified endothelial CXCL16 as an important adhesion molecule promoting the firm adhesion of CXCR6-positive PBMCs to inflamed endothelium. Our results demonstrate that endothelial CXCL16 as well as platelet CXCR6 are acting as potent PBMC-adhesion ligands, inducing PBMC-adhesion to the atherosclerosis-prone vessel wall and thus promoting the progression of atherosclerosis.
Assuntos
Aterosclerose/metabolismo , Moléculas de Adesão Celular/sangue , Adesão Celular , Quimiocina CXCL16/sangue , Endotélio Vascular/metabolismo , Leucócitos Mononucleares/metabolismo , Receptores CXCR6/sangue , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Artérias/metabolismo , Buffy Coat/metabolismo , Plaquetas/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Imuno-Histoquímica , Inflamação/metabolismo , LigantesRESUMO
Platelets are well known for their role in hemostasis and are also increasingly recognized for their roles in the innate immune system during inflammation and their regulation of macrophage activation. Here, we aimed to study the influence of platelets on the production of inflammatory mediators by monocytes and macrophages. Analyzing cocultures of platelets and murine bone marrow-derived macrophages or human monocytes, we found that collagen-activated platelets release high amounts of prostaglandin E2 (PGE2) that leads to an increased interleukin- (IL-) 10 release and a decreased tumor necrosis factor (TNF) α secretion out of the monocytes or macrophages. Platelet PGE2 mediated the upregulation of IL-10 in both cell types via the PGE2 receptor EP2. Notably, PGE2-mediated IL-10 synthesis was also mediated by EP4 in murine macrophages. Inhibition of TNFα synthesis via EP2 and EP4, but not EP1, was mediated by IL-10, since blockade of the IL-10 receptor abolished the inhibitory effect of both receptors on TNFα release. This platelet-mediated cross-regulation between PGE2 and cytokines reveals one mechanism how monocytes and macrophages can attenuate excessive inflammatory responses induced by activated platelets in order to limit inflammatory processes.
Assuntos
Citocinas/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Animais , Plaquetas/metabolismo , Humanos , Inflamação/metabolismo , Interleucina-10/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Prostaglandina E Subtipo EP2/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
FTY720 (fingolimod) is, after its phosphorylation by sphingosine kinase (SPHK) 2, a potent, non-selective sphingosine-1-phosphate (S1P) receptor agonist. FTY720 has been shown to reduce the nociceptive behavior in the paclitaxel model for chemotherapy-induced neuropathic pain through downregulation of S1P receptor 1 (S1P1) in microglia of the spinal cord. Here, we investigated the mechanisms underlying the antinociceptive effects of FTY720 in a model for trauma-induced neuropathic pain. We found that intrathecal administration of phosphorylated FTY720 (FTY720-P) decreased trauma-induced pain behavior in mice, while intraplantar administered FTY720-P had no effect. FTY720-P, but not FTY720, reduced the nociceptive behavior in SPHK2-deficient mice, suggesting the involvement of S1P receptors. Fittingly, intrathecal administration of antagonists for S1P1 or S1P3, W146 and Cay10444 respectively, abolished the antinociceptive effects of systemically administered FTY720, demonstrating that activation of both receptors in the spinal cord is necessary to induce antinociceptive effects by FTY720. Accordingly, intrathecal administration of S1P1 receptor agonists was not sufficient to evoke an antinociceptive effect. Taken together, the data show that, in contrast to its effects on chemotherapy-induced neuropathy, FTY720 reduces trauma-induced neuropathic pain by simultaneous activation of spinal S1P1 and S1P3 receptor subtypes.
Assuntos
Analgésicos/farmacologia , Lisofosfolipídeos/metabolismo , Neuralgia/etiologia , Organofosfatos/farmacologia , Receptores de Lisoesfingolipídeo/metabolismo , Esfingosina/análogos & derivados , Ferimentos e Lesões/complicações , Anilidas/farmacologia , Animais , Hibridização In Situ , Masculino , Camundongos , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Organofosfatos/uso terapêutico , Organofosfonatos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Receptores de Lisoesfingolipídeo/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Esfingosina/metabolismo , Esfingosina/farmacologia , Esfingosina/uso terapêuticoRESUMO
BACKGROUND: Prostacyclin (PGI2) is known to be an important mediator of peripheral pain sensation (nociception) whereas little is known about its role in central sensitization. METHODS: The levels of the stable PGI2-metabolite 6-keto-prostaglandin F1α (6-keto-PGF1α) and of prostaglandin E2 (PGE2) were measured in the dorsal horn with the use of mass spectrometry after peripheral inflammation. Expression of the prostanoid receptors was determined by immunohistology. Effects of prostacyclin receptor (IP) activation on spinal neurons were investigated with biochemical assays (cyclic adenosine monophosphate-, glutamate release-measurement, Western blot analysis) in embryonic cultures and adult spinal cord. The specific IP antagonist Cay10441 was applied intrathecally after zymosan-induced mechanical hyperalgesia in vivo. RESULTS: Peripheral inflammation caused a significant increase of the stable PGI2 metabolite 6-keto-PGF1α in the dorsal horn of wild-type mice (n = 5). IP was located on spinal neurons and did not colocalize with the prostaglandin E2 receptors EP2 or EP4. The selective IP-agonist cicaprost increased cyclic adenosine monophosphate synthesis in spinal cultures from wild-type but not from IP-deficient mice (n = 5-10). The combination of fluorescence-resonance-energy transfer-based cyclic adenosine monophosphate imaging and calcium imaging showed a cicaprost-induced cyclic adenosine monophosphate synthesis in spinal cord neurons (n = 5-6). Fittingly, IP activation increased glutamate release from acute spinal cord sections of adult mice (n = 13-58). Cicaprost, but not agonists for EP2 and EP4, induced protein kinase A-dependent phosphorylation of the GluR1 subunit and its translocation to the membrane. Accordingly, intrathecal administration of the IP receptor antagonist Cay10441 had an antinociceptive effect (n = 8-11). CONCLUSION: Spinal prostacyclin synthesis during early inflammation causes the recruitment of GluR1 receptors to membrane fractions, thereby augmenting the onset of central sensitization.
Assuntos
AMP Cíclico/fisiologia , Nociceptividade/fisiologia , Prostaglandinas I/fisiologia , Receptores de AMPA/metabolismo , Medula Espinal/fisiologia , Animais , Comportamento Animal/efeitos dos fármacos , Western Blotting , Cálcio/metabolismo , Cromatografia Líquida de Alta Pressão , Epitopos , Feminino , Transferência Ressonante de Energia de Fluorescência , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/fisiologia , Dor/psicologia , Gravidez , Prostaglandinas I/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Medula Espinal/citologia , Medula Espinal/metabolismo , Espectrometria de Massas em Tandem , Translocação GenéticaRESUMO
Sphingosine-1-phosphate (S1P) is generated through phosphorylation of sphingosine by two sphingosine kinases (SPHK-1 and -2). As extra- and intracellular messenger S1P fulfils multiple roles in inflammation such as mediating proinflammatory inputs or acting as chemoattractant. In addition, S1P induces cyclooxygenase-2 (COX-2) expression and the synthesis of proinflammatory prostanoids in several cell types. Here, we analysed in vivo the regulation of S1P level as well as potential interactions between S1P and COX-dependent prostaglandin synthesis during zymosan-induced inflammation. S1P and prostanoid levels were determined in the blood and at the site of inflammation under basal conditions and during zymosan-induced inflammation using wild type and SPHK-1 and -2 knockout mice. We found that alterations in S1P levels did not correlate with changes in plasma- or tissue-concentrations of the prostanoids as well as COX-2 expression. In the inflamed tissue S1P and prostanoid concentrations were reciprocally regulated. Prostaglandin levels increased over 6h, while S1P and sphingosine level decreased during the same time, which makes an induction of prostanoid synthesis by S1P in zymosan-induced inflammation unlikely. Additionally, despite altered S1P levels wild type and SPHK knockout mice showed similar behavioural nociceptive responses and oedema sizes suggesting minor functions of S1P in this inflammatory model.
Assuntos
Inflamação/fisiopatologia , Lisofosfolipídeos/biossíntese , Prostaglandinas/biossíntese , Esfingosina/análogos & derivados , Animais , Ciclo-Oxigenase 2/biossíntese , Edema/fisiopatologia , Inflamação/induzido quimicamente , Camundongos , Camundongos Knockout , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Esfingosina/biossíntese , ZimosanRESUMO
Multi-Epitope-Ligand-Carthography (MELC) allows consecutive immunohistochemical visualization of up to 100 proteins on the same tissue sample. Subsequent biomathematical analysis of these images allows a quantitative description of changes in protein networks. We used the MELC technology to study the effect of the nonopioid analgesic drug dipyrone on protein network profiles associated with arachidonic acid-dependent signaling pathways. MELC analysis with 31 different fluorescence-labeled tags was used to compare the effect of dipyrone on protein networks in spinal cords of mice with zymosan-induced hyperalgesia, a common model for inflammatory pain. We found that the number of motifs which describe the colocalization of 5-lipoxygenase (5-LO) or 12-LO with other proteins increased disproportionally after dipyrone treatment. Activation of 5-LO and 12-LO induces their translocation to membrane compartments which was also reflected by MELC results. Although no changes in 5-LO or 12-LO expression were seen by Western blot analysis or by immunohistochemistry in spinal cords of dipyrone-treated mice, the activation of both enzymes was verified by determining LO-products. Spinal amounts of 5(S)-hydroxyeicosatetraenoic acid (HETE) and 12(S)-HETE, which are generated by 5-LO and 12-LO, respectively, were significantly increased in spinal cords of dipyrone-treated animals. In primary spinal cord neurons, dipyrone selectively and dose-dependently increased 5(S)-(HETE) and 12(S)-HETE synthesis. Thus, we show for the first time that monitoring protein network profiles by topological proteomic analysis is a useful tool to identify mechanisms of drug actions.
Assuntos
Ácido Araquidônico/metabolismo , Dipirona/farmacologia , Nociceptores/metabolismo , Proteômica/métodos , Transdução de Sinais/efeitos dos fármacos , Animais , Araquidonato 12-Lipoxigenase/metabolismo , Araquidonato 5-Lipoxigenase/metabolismo , Imuno-Histoquímica/métodos , Camundongos , Prostaglandina-Endoperóxido Sintases/metabolismo , Proteoma/efeitos dos fármacos , Medula Espinal/citologia , Medula Espinal/ultraestrutura , Estatísticas não ParamétricasRESUMO
Macrophages (Mφ) undergo activation to pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes in response to pathophysiologic stimuli and dysregulation of the M1-M2 balance is often associated with diseases. Therefore, studying mechanisms of macrophage polarization may reveal new drug targets. Human Mφ polarization is generally studied in primary monocyte-derived Mφ (PBMC Mφ) and THP-1-derived Mφ (THP-1 Mφ). We compared the polarization profile of THP-1 Mφ with that of PBMC Mφ to assess the alternative use of THP-1 for polarization studies. Cellular morphology, the expression profiles of 18 genes and 4 cell surface proteins, and phagocytosis capacity for apoptotic cells and S. aureus bioparticles were compared between these Mφ, activated towards M1, M2a, or M2c subsets by stimulation with LPS/IFNγ, IL-4, or IL-10, respectively, for 6h, 24h and 48h. The Mφ types are unique in morphology and basal expression of polarization marker genes, particularly CCL22, in a pre-polarized state, and were differentially sensitive to polarization stimuli. Generally, M1 markers were instantly induced and gradually decreased, while M2 markers were markedly expressed at a later time. Expression profiles of M1 markers were similar between the polarized Mφ types, but M2a cell surface markers demonstrated an IL-4-dependent upregulation only in PBMC Mφ. Polarized THP-1 Mφ but not PBMC Mφ showed distinctive phagocytic capacity for apoptotic cells and bacterial antigens, respectively. In conclusion, our data suggest that THP-1 may be useful for performing studies involving phagocytosis and M1 polarization, rather than M2 polarization.
Assuntos
Polaridade Celular/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Fagocitose/imunologia , Staphylococcus aureus/imunologia , Biomarcadores/análise , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Linhagem Celular , Humanos , Interferon gama/farmacologia , Interleucina-10/farmacologia , Interleucina-4/farmacologia , Lipopolissacarídeos/farmacologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismoRESUMO
Platelets are well known for their role in hemostasis but are also increasingly recognized for their supporting role in innate immune responses. Here, we studied the role of platelets in the development of peripheral inflammation and found that platelets colocalize with macrophages in the inflamed tissue outside of blood vessels in different animal models for cutaneous inflammation. Collagen-treatment of macrophages isolated from paws during zymosan-induced inflammation induced thromboxane synthesis through the platelet-expressed collagen receptor glycoprotein VI. Deletion of glycoprotein VI or its downstream effector thromboxane A2 receptor (TP) reduced zymosan-induced mechanical allodynia without altering macrophage recruitment or formation of macrophage/platelet complexes. Instead, macrophages in inflamed paws of glycoprotein VI- and TP-deficient mice exhibited an increased expression of anti-inflammatory markers and synthesized less proinflammatory mediators (prostaglandin E2 and IL6). TP expression on platelets was necessary to mediate increased prostaglandin E2 and IL6 synthesis, whereas TP expression on macrophages was sufficient to decrease the expression of the anti-inflammatory macrophage marker CD206, showing that TP activation on platelets and macrophages regulates different aspects of macrophage activation.
Assuntos
Macrófagos/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores de Tromboxano A2 e Prostaglandina H2/metabolismo , Pele/patologia , Animais , Plaquetas/metabolismo , Colágeno/química , Feminino , Deleção de Genes , Inflamação , Lectinas Tipo C/metabolismo , Masculino , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Receptores de Superfície Celular/metabolismo , Tromboxano A2/metabolismoRESUMO
Inflammatory pain is based on stimulation and sensitization of peripheral endings of sensory neurons (nociceptors) by pronociceptive mediators. These mediators can be released by resident cells, as well as invading immune cells. Although neutrophils are known to release various mediators, which can stimulate or sensitize nociceptors, the extent of their contribution to nociceptive responses is unclear. Here, we studied the contribution of neutrophils to zymosan-induced inflammatory pain, which is characterized by an early recruitment of high numbers of neutrophils. Surprisingly, antibody-mediated neutrophil depletion caused a complete loss of edema formation but had no effect on mechanical pain thresholds. Blockage of the interaction between neutrophils and platelets or endothelial cells using antibodies directed against CD11b and CD162 reduced neutrophil recruitment to the site of inflammation. Again, the treatment decreased zymosan-induced edemas without altering mechanical pain thresholds. Also, HLB-219 mice, which have five to 10 times less platelets than WT mice, showed reduced neutrophil recruitment to the site of inflammation and decreased edema sizes, whereas, again, mechanical thresholds were unaltered. The effects observed in HLB-219 mice were relatively small and not reproduced in vWF-deficient mice or after antibody-mediated blockage of GPIbα. Flow chamber and transmigration assays showed that platelets were not necessary for neutrophil adhesion to endothelial cells but increased their transmigration. Taken together, zymosan-induced mechanical allodynia is, in contrast to edema formation, independent of neutrophils, and recruitment of neutrophils is only partly influenced by interactions with platelets.
Assuntos
Movimento Celular/efeitos dos fármacos , Edema/induzido quimicamente , Hiperalgesia/induzido quimicamente , Neutrófilos/metabolismo , Zimosan/toxicidade , Animais , Antígeno CD11b/biossíntese , Antígeno CD11b/genética , Antígeno CD11b/imunologia , Movimento Celular/genética , Movimento Celular/imunologia , Edema/genética , Edema/imunologia , Edema/metabolismo , Edema/patologia , Hiperalgesia/genética , Hiperalgesia/imunologia , Hiperalgesia/metabolismo , Hiperalgesia/patologia , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Mutantes , Neutrófilos/imunologia , Neutrófilos/patologia , Dor/induzido quimicamente , Dor/genética , Dor/imunologia , Dor/metabolismo , Dor/patologiaRESUMO
Sphingosine 1-Phosphate (S1P) modulates various cellular functions such as apoptosis, cell differentiation, and migration. Although S1P is an abundant signaling molecule in the central nervous system, very little is known about its influence on neuronal functions. We found that S1P concentrations were selectively decreased in the cerebrospinal fluid of adult rats in an acute and an inflammatory pain model. Pharmacological inhibition of sphingosine kinases (SPHK) decreased basal pain thresholds and SphK2 knock-out mice, but not SphK1 knock-out mice, had a significant decrease in withdrawal latency. Intrathecal application of S1P or sphinganine 1-phosphate (dihydro-S1P) reduced the pain-related (nociceptive) behavior in the formalin assay. S1P and dihydro-S1P inhibited cyclic AMP (cAMP) synthesis, a key second messenger of spinal nociceptive processing, in spinal cord neurons. By combining fluorescence resonance energy transfer (FRET)-based cAMP measurements with Multi Epitope Ligand Cartography (MELC), we showed that S1P decreased cAMP synthesis in excitatory dorsal horn neurons. Accordingly, intrathecal application of dihydro-S1P abolished the cAMP-dependent phosphorylation of NMDA receptors in the outer laminae of the spinal cord. Taken together, the data show that S1P modulates spinal nociceptive processing through inhibition of neuronal cAMP synthesis.