RESUMEN
OBJECTIVE: To investigate the role of proteinase-activated receptor 4 (PAR-4) in mediating joint inflammation and pain in mice. METHODS: Knee joint blood flow, edema, and pain sensitivity (as induced by thermal and mechanical stimuli) were assessed in C57BL/6 mice following intraarticular injection of either the selective PAR-4 agonist AYPGKF-NH(2) or the inactive control peptide YAPGKF-NH(2). The mechanism of action of AYPGKF-NH(2) was examined by pretreatment of each mouse with either the PAR-4 antagonist pepducin P4pal-10 or the bradykinin antagonist HOE 140. Finally, the role of PAR-4 in mediating joint inflammation was tested by pretreating mice with acutely inflamed knees with pepducin P4pal-10. RESULTS: PAR-4 activation caused a long-lasting increase in joint blood flow and edema formation, which was not seen following injection of the control peptide. The PAR-4-activating peptide was also found to be pronociceptive in the joint, where it enhanced sensitivity to a noxious thermal stimulus and caused mechanical allodynia and hyperalgesia. The proinflammatory and pronociceptive effects of AYPGKF-NH(2) could be inhibited by pepducin P4pal-10 and HOE 140. Finally, pepducin P4pal-10 ameliorated the clinical and physiologic signs of acute joint inflammation. CONCLUSION: This study demonstrates that local activation of PAR-4 leads to proinflammatory changes in the knee joint that are dependent on the kallikrein-kinin system. We also show for the first time that PARs are involved in the modulation of joint pain, with PAR-4 being pronociceptive in this tissue. Thus, blockade of articular PAR-4 may be a useful means of controlling joint inflammation and pain.
Asunto(s)
Artralgia/metabolismo , Artritis/etiología , Artritis/metabolismo , Receptores Proteinasa-Activados/metabolismo , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/farmacología , Bradiquinina/administración & dosificación , Bradiquinina/análogos & derivados , Bradiquinina/farmacología , Antagonistas del Receptor de Bradiquinina B2 , Modelos Animales de Enfermedad , Edema/metabolismo , Inyecciones Intraarticulares , Ratones , Ratones Endogámicos C57BL , Oligopéptidos/administración & dosificación , Oligopéptidos/farmacología , Receptor de Bradiquinina B2/metabolismo , Receptores Proteinasa-Activados/agonistas , Flujo Sanguíneo Regional/efectos de los fármacos , Flujo Sanguíneo Regional/fisiologíaRESUMEN
Proteinase-activated receptors (PARs) are a family of G-protein-coupled receptors that are activated by endogenous serine proteinases that cleave the N-terminal domain of the receptor unmasking a "tethered ligand" sequence. Trypsin and other agonists at PAR(2) act on peripheral nerves to augment the transfer of nociceptive information. We tested whether PAR(2) agonists also exert a spinal pronociceptive effect by i.t. administering the selective ligand, Ser-Leu-Ile-Gly-Arg-Leu-NH(2) (SLI-GRL). This produced thermal and mechanical hyperalgesia in rats and mice and augmented mechanical and thermal hyperalgesia seen in the formalin inflammatory pain test. Effects of SLIGRL were abrogated in PAR(2)-deficient mice and were not seen with the inactive control peptide, Leu-Arg-Gly-Ile-Leu-Ser-NH(2). Surprisingly, electrophysiological studies, using whole-cell recording from rat substantia gelatinosa neurons, failed to demonstrate an increase in excitatory transmission or neuronal excitability following treatment with SLIGRL or trypsin. In fact, the actions of trypsin were consistent with a decrease in dorsal horn excitability. SLIGRL and trypsin did, however, depolarize and increase the excitability of large, medium and small primary afferent, dorsal root ganglion neurons. The effects were associated with an increase in conductance at hyperpolarized potentials and a decrease in conductance at depolarized potentials. PAR(2)-like immunoreactivity was found in DRG but not in spinal dorsal horn. These results suggest that activation of DRG neuron cell bodies may account for the pronociceptive actions of i.t. applied PAR(2) agonists. They also imply that pathophysiological release of PAR(2)-activating proteases in the vicinity of DRG neurons may produce profound effects on nociceptive processing in vivo.
Asunto(s)
Ganglios Espinales/citología , Hiperalgesia/fisiopatología , Neuronas Aferentes/efectos de los fármacos , Oligopéptidos/farmacología , Receptor PAR-2/agonistas , Animales , Formaldehído , Ganglios Espinales/fisiología , Calor , Hiperalgesia/inducido químicamente , Inyecciones Espinales , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Noqueados , Neuronas Aferentes/fisiología , Dolor/inducido químicamente , Dolor/fisiopatología , Ratas , Ratas Wistar , Receptor PAR-2/deficiencia , Receptor PAR-2/genética , Médula Espinal/efectos de los fármacos , Médula Espinal/fisiopatología , Tripsina/farmacologíaRESUMEN
Attaching-effacing bacteria are major causes of infectious diarrhea in humans worldwide. Citrobacter rodentium is an attaching-effacing enteric pathogen that causes transmissible murine colonic mucosal hyperplasia. We characterized colonic inflammation and ion transport at 3, 7, 10, 30, and 60 d after infection of C57Bl/6 mice with C. rodentium. Macroscopic damage score was significantly increased 7 and 10 d after infection. Colonic wall thickness was increased at 7, 10, 30, and 60 d. Myeloperoxidase (MPO) activity was significantly increased at 3, 7, and 10 d and returned to control levels by days 30 and 60. The expressions of inducible nitric oxide synthase and cyclooxygenase-2 were increased by C. rodentium infection. Significant reductions in the epithelial secretory response to carbachol, but not to electrical field stimulation or forskolin, were observed at 3 and 10 d of infection. Translocation of enteric bacteria into the mesenteric lymph nodes was observed 10 d following infection. There was no difference in response to infection between animals deficient in inducible nitric oxide synthase and wild-type controls. The COX-2 inhibitor rofecoxib caused decreased wall thickness and MPO activity at day 10. However, COX-2 inhibition did not alter infection-induced changes in ion transport. Citrobacter rodentium infection causes colonic inflammation, mucosal hyperplasia, and nitric-oxide-independent epithelial dysfunction in association with increased permeability to luminal bacteria.
Asunto(s)
Citrobacter rodentium , Colitis/metabolismo , Colon/metabolismo , Infecciones por Enterobacteriaceae/metabolismo , Mucosa Intestinal/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Animales , Traslocación Bacteriana , Carbacol/farmacología , Permeabilidad de la Membrana Celular , Agonistas Colinérgicos/farmacología , Colitis/microbiología , Colitis/patología , Colitis/fisiopatología , Colon/efectos de los fármacos , Colon/microbiología , Colon/patología , Colon/fisiopatología , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa 2/farmacología , Infecciones por Enterobacteriaceae/complicaciones , Infecciones por Enterobacteriaceae/microbiología , Infecciones por Enterobacteriaceae/patología , Infecciones por Enterobacteriaceae/fisiopatología , Hiperplasia , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/microbiología , Mucosa Intestinal/fisiopatología , Secreciones Intestinales/metabolismo , Transporte Iónico , Lactonas/farmacología , Ganglios Linfáticos/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo II/deficiencia , Óxido Nítrico Sintasa de Tipo II/genética , Peroxidasa/metabolismo , Sulfonas/farmacología , Factores de TiempoRESUMEN
BACKGROUND: Activation of colonic proteinase activated receptor-1 (PAR1) provokes colonic inflammation and increases mucosal permeability in mice. The mechanism of inflammation is not neurogenic like in the paw of rats but depends on PAR1-mediated activation monocytic cells. PAR1 activation in the colon increases the release of lymphocyte T helper-1 (TH1) cytokines. Moreover, PAR1 expression is increased in biopsies from patients with inflammatory bowel disease, and its activation during TH1-mediated colitis in mice increases all of the hallmarks of inflammation. METHODS: This study aimed to characterize the effects of PAR1 activation in oxazolone-mediated colitis, involving a TH2 cytokine profile. RESULTS: Intracolonic administration of oxazolone increased myeloperoxidase activity, damage score, and interleukin (IL)-4, IL-10, tumor necrosis factor alpha, and IL-1beta mRNA expression but lowered interferon-gamma mRNA expression, indicating colonic inflammation of a TH2 profile. The concurrent intracolonic administration of a PAR1 agonist in oxazolone-treated mice inhibited colitis, resulting in a reduction of myeloperoxidase activity, damage score, and inflammatory cytokine mRNA expression. Using PAR1-deficient mice, we confirmed that the anti-inflammatory effects of PAR1 agonists were mediated by PAR1. Moreover, in PAR1-deficient mice or in mice treated with a PAR1 antagonist, oxazolone-induced colitis was exacerbated, showing an endogenous modulatory role for PAR1 in this TH2 cytokine profile of colitis. CONCLUSIONS: Thus, as opposed to a previously shown proinflammatory role for PAR1 in a TH1 cytokine-mediated colitis, our new data show anti-inflammatory role for PAR1 activation in the setting of TH2 cytokine colitis model.
Asunto(s)
Colitis/inmunología , Colitis/fisiopatología , Receptor PAR-1/fisiología , Animales , Colitis/veterinaria , Citocinas/inmunología , Inflamación/fisiopatología , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos C57BL , Oxazolona , ARN Mensajero/biosíntesis , Receptor PAR-1/antagonistas & inhibidores , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células TH1/inmunologíaRESUMEN
Citrobacter rodentium is a bacterial pathogen that causes a murine infectious colitis equivalent to enterohemorrhagic Escherichia coli infection in humans. Colonic luminal fluid from C. rodentium-infected mice, but not from sham-infected mice, contains active serine proteinases that can activate proteinase-activated receptor-2 (PAR2). We have identified granzyme A and murine trypsins to be present in C. rodentium-infected luminal fluid, as determined by mass spectrometry and Western blot analysis. Inflammatory indices (colonic mucosa macroscopic damage score, increased intestinal wall thickness, granulocyte infiltration, and bacterial translocation from the colonic lumen to peritoneal organs) were all increased in C. rodentium-infected mice, compared with sham-infected mice. Soybean trypsin inhibitor-treated wild-type mice and untreated PAR2-deficient (PAR2-/-) mice (compared with their wild-type littermates) both had substantially reduced levels of C. rodentium-induced inflammation. These data point to an important role for both pathogen-induced host serine proteinases and PAR2 in the setting of infectious colitis.
Asunto(s)
Citrobacter rodentium/fisiología , Colitis/metabolismo , Colitis/microbiología , Endopeptidasas/metabolismo , Receptor PAR-2/metabolismo , Animales , Colitis/enzimología , Colitis/patología , Granzimas , Ratones , Serina Endopeptidasas/metabolismo , Tripsina/metabolismoRESUMEN
Proteinase-activated receptor-1 (PAR1), a G protein-coupled receptor activated by thrombin, is highly expressed in different cell types of the gastrointestinal tract. The activity of thrombin and of other proteinases is significantly increased in the colon of inflammatory bowel disease (IBD) patients. Since PAR1 activation in tissues other than the gut provoked inflammation, we hypothesized that PAR1 activation in the colon is involved in the pathogenesis of IBD. Here, we demonstrate that PAR1 is overexpressed in the colon of IBD patients. In mice, intracolonic administration of PAR1 agonists led to an inflammatory reaction characterized by edema and granulocyte infiltration. This PAR1 activation-induced inflammation was dependent on B and T lymphocytes. Moreover, PAR1 activation exacerbated and prolonged inflammation in a mouse model of IBD induced by the intracolonic administration of trinitrobenzene sulfonic acid (TNBS), while PAR1 antagonism significantly decreased the mortality and severity of colonic inflammation induced by TNBS and dextran sodium sulfate. In these 2 models, colitis development was strongly attenuated by PAR1 deficiency. Taken together, these results imply an important role for PAR1 in the pathogenesis of experimental colitis, supporting the notion that PAR1 inhibition may be beneficial in the context of IBD and possibly in other chronic intestinal inflammatory disorders.