RESUMEN
BACKGROUND: Scleroderma renal crisis (SRC) is a life-threatening complication of systemic sclerosis (SSc). Autoantibodies (Abs) against endothelial cell antigens have been implicated in SSc and SRC. However, their detailed roles remain poorly defined. Pro-inflammatory cytokine interleukin-6 (IL-6) has been found to be increased in SSc, but its role in SRC is unclear. Here, we aimed to determine how the autoantibodies from patients with SSc and SRC affect IL-6 secretion by micro-vascular endothelial cells (HMECs). METHODS: Serum IgG fractions were isolated from either SSc patients with SRC (n = 4) or healthy individuals (n = 4) and then each experiment with HMECs was performed with SSc-IgG from a separate patient or separate healthy control. IL-6 expression and release by HMECs was assessed by quantitative reverse transcription and quantitative PCR (RT-qPCR) and immunoassays, respectively. The mechanisms underlying the production of IL-6 were analyzed by transient HMEC transfections with IL-6 promoter constructs, electrophoretic mobility shift assays, Western blots and flow cytometry. RESULTS: Exposure of HMECs to IgG from SSc patients, but not from healthy controls, resulted in a time- and dose-dependent increase in IL-6 secretion, which was associated with increased AKT, p70S6K, and ERK1/2 signalling, as well as increased c-FOS/AP-1 transcriptional activity. All these effects could be reduced by the blockade of the endothelial PAR-1 receptor and/or c-FOS/AP-1silencing. CONCLUSIONS: Autoantibodies against PAR-1 found in patients with SSc and SRC induce IL-6 production by endothelial cells through signalling pathways controlled by the AP-1 transcription factor. These observations offer a greater understanding of adverse endothelial cell responses to autoantibodies present in patients with SRC.
Asunto(s)
Autoanticuerpos/inmunología , Células Endoteliales/inmunología , Interleucina-6/inmunología , Enfermedades Renales/inmunología , Sistema de Señalización de MAP Quinasas/inmunología , Receptor PAR-1/inmunología , Esclerodermia Sistémica/inmunología , Adulto , Línea Celular , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Extravillous trophoblast (EVT) migration and invasion is the crucial step for normal placental development. IL-11 is a cytokine regulating cell migration and invasion in cells and is a critical factor for successful implantation of an embryo. Higher expression of thrombin receptor PAR-1 was reported in early pregnancy. The precise role of thrombin in trophoblast functions is not well understood. In this study, we asked whether thrombin can induce IL-11 secretion in trophoblasts if yes, which physiological cell functions are possibly affected? In this study, HTR-8/SVneo cells, which were originally derived from first-trimester villous explants of early pregnancy were used as the extravillous trophoblast (EVT) model. BeWo cells were used as the cytotrophoblast model. For gene silencing, qPCR and ELISA, each experiment was performed in triplicates for minimum three times. Here, we found that thrombin stimulates IL-11 gene expression and protein secretion in HTR-8/SVneo cells but not in BeWo cells. PAR-1 was the only receptor which was highly expressed in HTR-8/SVneo cells. Thrombin-mediated expression and secretion of IL-11 were mainly activated via PAR-1 receptor. Rac1, but not Rho-kinase activation is required for thrombin-induced IL-11 secretion. We also found that thrombin stimulation significantly enhanced cell migration that was inhibited after silencing the IL-11 gene. In conclusion, this study demonstrates the role of thrombin in regulating human EVT migration via IL-11 secretion. We propose that thrombin might regulate EVT migration through the decidua and spiral artery remodeling. Failure of thrombin-dependent EVT migration results in pregnancy disorder, such as preeclampsia.
Asunto(s)
Interleucina-11/metabolismo , Placentación/inmunología , Receptor PAR-1/metabolismo , Trombina/metabolismo , Trofoblastos/inmunología , Línea Celular , Movimiento Celular/inmunología , Femenino , Humanos , Interleucina-11/inmunología , Embarazo , Primer Trimestre del Embarazo , Receptor PAR-1/inmunología , Trombina/inmunologíaRESUMEN
Protease activity of allergens has been suggested to be involved in the pathogenesis of allergic diseases. The major allergen Der f 3 from Dermatophagoides farinae harbors serine protease activity, but its immunopathogenesis remains unclear. This study aims to explore the effect of Der f 3 on the airway epithelial barrier and on the molecular pathways by which Der f 3 induces inflammation. RNA-seq was performed to identify differentially expressed genes in bronchial airway epithelial cells (AEC) between native Der f 3 and heat-inactivated (H) Der f 3, coupled with real-time PCR (RT-PCR) and ELISA for validation. Unlike other protease allergens such as that induce Th2-promoting alarmins (IL-25, IL-33, TSLP) in AECs, Der f 3 induced pro-inflammatory cytokines and chemokines including IL-6, IL-8 and GM-CSF, which are known to promote Th17 response. These pro-inflammatory mediators were induced by Der f 3 via the MAPK and NF-κB pathways as well as the store-operated calcium signaling. Gene silencing with small interfering RNA in A549 and BEAS-2B cells indicated that activation of AECs by Der f 3 was mainly dependent on protease-activated receptor 2 (PAR-2), while PAR-1 was also required for the full activation of AECs. Double knock-down of PAR-1 and PAR-2 largely impaired Der f 3-inducecd IL-8 production and subsequent signaling pathways. Our data suggest that Der f 3 induces pro-inflammatory mediators in human epithelial cell lines via the PARs-MAPK-NF-κB axis. Our results provide a molecular mechanism by which Der f 3 may trigger the Th17-skewed allergic response toward house dust mites.
Asunto(s)
Alérgenos/inmunología , Proteínas de Artrópodos/inmunología , Células Epiteliales/inmunología , Pyroglyphidae/inmunología , Receptor PAR-1/inmunología , Receptor PAR-2/inmunología , Mucosa Respiratoria/inmunología , Serina Endopeptidasas/inmunología , Células A549 , Alérgenos/farmacología , Animales , Proteínas de Artrópodos/farmacología , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/genética , Señalización del Calcio/inmunología , Citocinas/genética , Citocinas/inmunología , Células Epiteliales/patología , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Técnicas de Silenciamiento del Gen , Humanos , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Mediadores de Inflamación/inmunología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Sistema de Señalización de MAP Quinasas/inmunología , FN-kappa B/genética , FN-kappa B/inmunología , Receptor PAR-1/genética , Receptor PAR-2/genética , Serina Endopeptidasas/farmacología , Células Th17/inmunología , Células Th17/patología , Células Th2/inmunología , Células Th2/patologíaRESUMEN
We previously demonstrated that Cry j1, the major pollen allergen of Cryptomeria japonica (Japanese cedar), transiently increases protease activity and intracellular Ca2+ concentration in cultured human keratinocytes, and delays recovery after stratum corneum barrier disruption in human skin ex vivo. Topical application of tranexamic acid or trypsin-type serine protease inhibitors accelerates barrier recovery. We hypothesized that tranexamic acid might prevent the transient protease activity increase and the barrier recovery delay induced by Cry j1. Here, we tested this hypothesis and examined the mechanism involved. In cultured human keratinocytes, knock-down of protease-activated receptor 1 (PAR-1) reduced the transient increase of calcium induced by Cry j1, whereas knock-down of PAR-2 did not. Knock-down of thrombin significantly reduced the transient increases of calcium concentration and protease activity. Tranexamic acid, soybean trypsin inhibitor, or bivalirudin (a thrombin inhibitor) also reduced the calcium elevation induced by Cry j1 and/or thrombin. Co-application of tranexamic acid or bivalirudin with Cry j1 to human skin ex vivo blocked the delay of barrier recovery. These results suggest that thrombin and PAR-1 or PAR-1-like receptor might mediate the adverse effects of Cry j1 on human epidermal keratinocytes, and could open up a new strategy for treating inflammatory skin diseases.
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Antígenos de Plantas/inmunología , Cryptomeria/inmunología , Queratinocitos/efectos de los fármacos , Proteínas de Plantas/inmunología , Trombina/inmunología , Ácido Tranexámico/farmacología , Calcio/inmunología , Línea Celular , Epidermis/efectos de los fármacos , Epidermis/inmunología , Humanos , Queratinocitos/inmunología , Permeabilidad/efectos de los fármacos , Receptor PAR-1/inmunologíaRESUMEN
In antineutrophil cytoplasmic antibody-associated vasculitis (AAV), Toll-like receptors (TLRs) may be engaged by infection-associated patterns and by endogenous danger signals, linking infection and innate inflammation with this autoimmune disease. This study examined intrarenal TLR2, TLR4, and TLR9 expression and renal injury in AAV, testing the hypothesis that increased TLR expression correlates with renal injury. Patients with AAV exhibited both glomerular and tubulointerstitial expression of TLR2, TLR4, and TLR9, with TLR4 being the most prominent in both compartments. Glomerular TLR4 expression correlated with glomerular segmental necrosis and cellular crescents, with TLR2 expression correlating with glomerular segmental necrosis. The extent and intensity of glomerular and tubulointerstitial TLR4 expression and the intensity of glomerular TLR2 expression inversely correlated with the presenting estimated glomerular filtration rate. Although myeloid cells within the kidney expressed TLR2, TLR4, and TLR9, TLR2 and TLR4 colocalized with endothelial cells and podocytes, whereas TLR9 was expressed predominantly by podocytes. The functional relevance of intrarenal TLR expression was further supported by the colocalization of TLRs with their endogenous ligands high-mobility group box 1 and fibrinogen. Therefore, in AAV, the extent of intrarenal TLR4 and TLR2 expression and their correlation with renal injury indicates that TLR4, and to a lesser degree TLR2, may be potential therapeutic targets in this disease.
Asunto(s)
Vasculitis Asociada a Anticuerpos Citoplasmáticos Antineutrófilos/inmunología , Glomerulonefritis/inmunología , Glomérulos Renales/inmunología , Receptor Toll-Like 2/análisis , Receptor Toll-Like 4/análisis , Anciano , Vasculitis Asociada a Anticuerpos Citoplasmáticos Antineutrófilos/patología , Vasculitis Asociada a Anticuerpos Citoplasmáticos Antineutrófilos/fisiopatología , Anticuerpos Anticitoplasma de Neutrófilos/inmunología , Femenino , Fibrinógeno/análisis , Tasa de Filtración Glomerular , Glomerulonefritis/patología , Glomerulonefritis/fisiopatología , Proteína HMGB1/análisis , Humanos , Glomérulos Renales/patología , Glomérulos Renales/fisiopatología , Masculino , Persona de Mediana Edad , Peroxidasa/inmunología , Receptor PAR-1/inmunología , Índice de Severidad de la Enfermedad , Receptor Toll-Like 9/análisisRESUMEN
AIM: This study aimed to analyze the predictive, prognostic and diagnostic value of autoantibodies to coagulation factor II thrombin receptor (F2R; protease-activated receptor 1, PAR1) (PAR1-AB) in patients with primary epithelial ovarian cancer (EOC). MATERIALS AND METHODS: A total of 197 patients with primary EOC and 200 healthy female blood donors were included in the study. Enzyme-linked immunosorbent assay was applied to determine PAR1-AB levels in blood sera taken preoperatively. Correlation of PAR1-AB with clinicopathological outcome, progression-free (PFS) and overall (OS) survival was analyzed and patients were compared with controls. RESULTS: PAR1-AB was significantly negatively correlated with histological grading (p=0.008) and was significantly lower in the patient group compared to healthy controls (p<0.001). There was no significant correlation of PAR1-AB level with PFS or OS. CONCLUSION: This study showed PAR1-AB to significantly decrease in patients with primary EOC and with histological high-grade carcinoma. The relevance of PAR1-AB in early detection of ovarian cancer and follow-up for EOC should be further investigated.
Asunto(s)
Autoanticuerpos/sangre , Neoplasias Glandulares y Epiteliales/sangre , Neoplasias Ováricas/sangre , Receptor PAR-1/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Supervivencia sin Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Persona de Mediana Edad , Neoplasias Glandulares y Epiteliales/diagnóstico , Neoplasias Ováricas/diagnóstico , Pronóstico , Receptor PAR-1/genéticaRESUMEN
γδ T cells are non-conventional, innate-like T cells, characterized by a restricted T-cell receptor repertoire. They participate in protective immunity responses against extracellular and intracellular pathogens, tumour surveillance, modulation of innate and adaptive immune responses, tissue healing, epithelial cell maintenance and regulation of physiological organ function. In this study, we investigated the role of neutrophils during the activation of human blood γδ T cells through CD3 molecules. We found that the up-regulation of CD69 expression, and the production of interferon-γ and tumour necrosis factor-α induced by anti-CD3 antibodies was potentiated by neutrophils. We found that inhibition of caspase-1 and neutralization of interleukin-18 did not affect neutrophil-mediated modulation. By contrast, the treatment with serine protease inhibitors prevented the potentiation of γδ T-cell activation induced by neutrophils. Moreover, the addition of elastase to γδ T-cell culture increased their stimulation, and the treatment of neutrophils with elastase inhibitor prevented the effect of neutrophils on γδ T-cell activation. Furthermore, we demonstrated that the effect of elastase on γδ T cells was mediated through the protease-activated receptor, PAR1, because the inhibition of this receptor with a specific antagonist, RWJ56110, abrogated the effect of neutrophils on γδ T-cell activation.
Asunto(s)
Elastasa de Leucocito/inmunología , Activación de Linfocitos , Activación Neutrófila/inmunología , Neutrófilos/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Linfocitos T/inmunología , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Complejo CD3/inmunología , Humanos , Interferón gamma/inmunología , Lectinas Tipo C/inmunología , Neutrófilos/citología , Receptor PAR-1/inmunología , Linfocitos T/citología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
BACKGROUND: In this study, we investigated the mechanism of platelet activation in patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), as well as the activation of the alternative complement pathway by platelets in AAV. METHODS: CD62P and platelet-leukocyte aggregates in AAV patients were tested by flow cytometry. Platelets were stimulated by plasma from active AAV patients. The effect of the thrombin-protease-activated receptors (PARs) pathway was evaluated by blocking thrombin or PAR1 antagonists. After platelets were activated by plasma from AAV patients, Ca/Mg-Tyrode's buffer and Mg-EGTA buffer were used to measure complement activation in liquid phase and on the surface of platelets. RESULTS: The levels of CD62P-expressing platelets and platelet-leukocyte aggregates were significantly higher in active AAV patients than those in remission and normal controls. Platelets were activated by plasma from active AAV patients (percentage of CD62P-expressing platelets, 97.7 ± 3% vs. 1 ± 0.2%, p < 0.0001, compared with those incubated with healthy donor plasma), and this was inhibited by thrombin or PAR1 antagonists (percentage of CD62P-expressing platelets, 97.7 ± 3% vs. 2.7 ± 1%, p < 0.0001, 97.7 ± 3% vs. 5 ± 1.4%, p < 0.0001, respectively). Platelets activated by plasma from AAV patients could trigger complement activation via the alternative pathway, as demonstrated by significant elevation of C3a, C5a, and sC5b-9 and significantly more C3c and C5b-9 deposition on the surface of platelets. CONCLUSIONS: Platelets were activated in AAV patients, and such activation was at least partially attributed to the thrombin-PARs pathway. Activated platelets triggered the alternative complement pathway in AAV.
Asunto(s)
Vasculitis Asociada a Anticuerpos Citoplasmáticos Antineutrófilos/inmunología , Vía Alternativa del Complemento/inmunología , Activación Plaquetaria/inmunología , Receptor PAR-1/inmunología , Trombina/inmunología , Anciano , Plaquetas/inmunología , Plaquetas/metabolismo , Proteínas del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Selectina-P/inmunología , Selectina-P/metabolismo , Activación Plaquetaria/efectos de los fármacos , Receptor PAR-1/antagonistas & inhibidores , Receptor PAR-1/metabolismo , Transducción de Señal/inmunología , Trombina/metabolismoRESUMEN
BACKGROUND: Proteolytic cleavage of protease-activated receptor 1 (PAR1) can result in potent downstream regulatory effects on inflammation. Although PAR1 is expressed throughout the gastrointestinal tract and activating proteases are increased in inflammatory bowel disease, the effect of PAR1 activation on colitis remains poorly understood, and has not previously been studied in pediatric disease. METHODS: Expression of PAR1 and inflammatory cytokines in colonic biopsies from pediatric patients with Crohn's disease exhibiting active moderate to severe colitis was measured by quantitative PCR. The functional relevance of these clinical data was further studied in a mouse model of Citrobacter rodentium-induced colitis. RESULTS: PAR1 expression was significantly upregulated in the inflamed colons of pediatric patients with Crohn's disease, with expression levels directly correlating to disease severity. In patients with severe colitis, PAR1 expression uniquely correlated with Th17-related (IL17A, IL22, and IL23A) cytokines. Infection of PAR1-deficient (PAR1) and wildtype mice with colitogenic C. rodentium revealed that disease severity and colonic pathology were strongly attenuated in mice lacking PAR1. Furthermore, Th17-type immune response was completely abolished in the colons of infected PAR1 but not wildtype mice. Finally, PAR1 was shown to be essential for secretion of the Th17-driving cytokine IL-23 by C. rodentium-stimulated macrophages. CONCLUSIONS: This study demonstrates a strong link between PAR1 expression, Th17-type immunity, and disease severity in both pediatric patients with Crohn's disease and C. rodentium-induced colitis in mice. The data presented suggest PAR1 exerts a proinflammatory role in colitis in both humans and mice by promoting a Th17-type immune response, potentially by supporting the production of IL-23.
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Colitis/inmunología , Enfermedad de Crohn/inmunología , Citocinas/inmunología , Receptor PAR-1/metabolismo , Células Th17/inmunología , Adolescente , Animales , Niño , Citrobacter rodentium , Colitis/inducido químicamente , Colitis/genética , Colon/inmunología , Colon/patología , Enfermedad de Crohn/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Interleucina-17/inmunología , Subunidad p19 de la Interleucina-23/inmunología , Interleucinas/inmunología , Masculino , Ratones , Receptor PAR-1/inmunología , Índice de Severidad de la Enfermedad , Interleucina-22RESUMEN
Microvascular endothelial cells maintain a tight barrier to prevent passage of plasma and circulating immune cells into the extravascular tissue compartment, yet endothelial cells respond rapidly to vasoactive substances, including thrombin, allowing transient paracellular permeability. This response is a cornerstone of acute inflammation, but the mechanisms responsible are still incompletely understood. Here, we demonstrate that thrombin triggers MALT1 to proteolytically cleave cylindromatosis (CYLD). Fragmentation of CYLD results in microtubule disruption and a cascade of events leading to endothelial cell retraction and an acute permeability response. This finding reveals an unexpected role for the MALT1 protease, which previously has been viewed mostly as a driver of pro-inflammatory NF-κB signaling in lymphocytes. Thus, MALT1 not only promotes immune cell activation but also acutely regulates endothelial cell biology, actions that together facilitate tissue inflammation. Pharmacologic inhibition of MALT1 may therefore have synergistic impact by targeting multiple disparate steps in the overall inflammatory response.
Asunto(s)
Caspasas/inmunología , Cisteína Endopeptidasas/inmunología , Células Endoteliales/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Proteínas de Neoplasias/inmunología , Trombina/farmacología , Animales , Transporte Biológico , Proteínas Adaptadoras de Señalización CARD/genética , Proteínas Adaptadoras de Señalización CARD/inmunología , Caspasas/genética , Línea Celular , Cisteína Endopeptidasas/genética , Enzima Desubiquitinante CYLD , Células Endoteliales/citología , Células Endoteliales/inmunología , Regulación de la Expresión Génica , Quinasa I-kappa B/genética , Quinasa I-kappa B/inmunología , Ratones , Ratones Transgénicos , Microtúbulos/ultraestructura , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , FN-kappa B/genética , FN-kappa B/inmunología , Proteínas de Neoplasias/genética , Permeabilidad/efectos de los fármacos , Cultivo Primario de Células , Receptor PAR-1/genética , Receptor PAR-1/inmunología , Transducción de Señal , Trombina/metabolismoRESUMEN
INTRODUCTION: In addition to its central role in coagulation, thrombin is involved in non-hemostatic activities such as inflammation. Direct inhibition of thrombin activity (e.g. with dabigatran) or reducing its generation by inhibition of Factor Xa (e.g. with rivaroxaban) may therefore have anti-inflammatory effects. MATERIALS AND METHODS: Microarray experiments were performed to identify transcriptome-wide changes in mRNA expression levels induced by thrombin in the presence and absence of the PAR-1 antagonist vorapaxar in primary human umbilical vein endothelial cells (HUVECs). On this basis, HUVECs were incubated with recalcified plasma, with or without rivaroxaban (0.3-3000nM), dabigatran (0.3-10,000nM), or vorapaxar (0.3-10nM). Expression levels of preselected pro-inflammatory genes were quantified by real-time PCR. RESULTS: Vorapaxar abolished 67 of the 69 transcripts altered by more than twofold on addition of thrombin to HUVECs. ELAM-1, VCAM-1, ICAM-1, MCP-1, IL-8, CXCL1, and CXCL2 were among the genes most strongly induced by thrombin. Inflammatory gene expression after stimulation of thrombin generation was concentration-dependently suppressed by vorapaxar, dabigatran, and rivaroxaban. However, dabigatran at low concentrations (3-300nM) increased significantly the expression levels of CXCL1, CXCL2, IL-8, ELAM-1, MCP-1, and tissue factor. CONCLUSION: In HUVECs, plasma-induced transcriptional changes are mediated by thrombin-induced PAR-1 activation. Rivaroxaban downregulated the expression of pro-inflammatory markers and tissue factor to a similar extent to dabigatran.
Asunto(s)
Antitrombinas/farmacología , Dabigatrán/farmacología , Células Endoteliales/efectos de los fármacos , Inhibidores del Factor Xa/farmacología , Rivaroxabán/farmacología , Trombina/inmunología , Transcriptoma/efectos de los fármacos , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/inmunología , Lactonas/farmacología , Piridinas/farmacología , Receptor PAR-1/antagonistas & inhibidores , Receptor PAR-1/inmunologíaRESUMEN
PROBLEM: Risk factors for preterm birth include placental abruption, giving rise to excessive decidual thrombin, and intrauterine bacterial infection. Human endometrial endothelial cells (HEECs) express Toll-like receptors (TLRs), and infection-derived agonists trigger HEECs to generate specific inflammatory responses. As thrombin, in addition to inducing coagulation, can contribute to inflammation, its effect on HEEC inflammatory responses to the TLR4 agonist, bacterial lipopolysaccharide (LPS), was investigated. METHOD OF STUDY: HEECs were pre-treated with or without thrombin or specific protease-activated receptor (PAR) agonists, followed by treatment with or without LPS. Supernatants were measured for cytokines and chemokines by ELISA and multiplex analysis. RESULTS: Thrombin significantly and synergistically augmented LPS-induced HEEC secretion of interleukin (IL)-6, IL-8, granulocyte colony-stimulating factor (G-CSF), and growth-regulated oncogene-alpha (GRO-α), and significantly augmented monocyte chemotactic protein (MCP)-1, tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF) secretion additively. Similar to thrombin, a PAR1 agonist synergistically augmented the LPS-induced HEEC secretion of inflammatory IL-6, IL-8, G-CSF, and GRO-α. CONCLUSION: Thrombin, via PAR1 activation, synergistically augments LPS-induced HEEC production of chemokines involved in immune cell recruitment and survival, suggesting a mechanism by which intrauterine abruption and bacterial infection may together be associated with an aggravated uterine inflammatory response.
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Endometrio/inmunología , Células Endoteliales/inmunología , Lipopolisacáridos/toxicidad , Receptor PAR-1/inmunología , Trombina/inmunología , Citocinas/inmunología , Femenino , HumanosRESUMEN
BACKGROUND: The inflammatory response plays a key role at different stages of cancer development. Allelic variants of the interleukin 1A (IL1A), interleukin 4 (IL4), nuclear factor kappa B1 (NFKB1) and protease-activated receptor 1 (PAR1) genes may influence not only the inflammatory response but also susceptibility to cancer development. Among major ethnic or continental groups, these polymorphic variants present different allelic frequencies. In admixed populations, such as the Brazilian population, data on distribution of these polymorphisms are limited. Here, we collected samples of cancer-free individuals from the north, northeast, midwest, south and southeast regions of Brazil and from the three main groups that gave rise to the Brazilian population: Native Americans from the Brazilian Amazon, Africans and Europeans. We describe the allelic distributions of four IL1A (rs3783553), IL4 (rs79071878), NFKB1 (rs28362491) and PAR1 (rs11267092) gene polymorphisms, which the literature describes as polymorphisms with a risk of cancer or worse prognosis for cancer. RESULTS: The genotypic distribution of the four polymorphisms was statistically distinct between Native Americans, Africans and Europeans. For the allelic frequency of these polymorphisms, the Native American population was the most distinct among the three parental populations, and it included the greatest number of alleles with a risk of cancer or worse prognosis for cancer. The PAR1 gene polymorphism allelic distribution was similar among all Brazilian regions. For the other three markers, the northern region population was statistically distinct from other Brazilian region populations. CONCLUSION: The IL1A, IL4, NFKB1 and PAR1 gene polymorphism allelic distributions are homogeneous among the regional Brazilian populations, except for the northern region, which significantly differs from the other four Brazilian regions. Among the parental populations, the Native American population exhibited a higher incidence of alleles with risk of cancer or worse prognosis for cancer, which can indicate greater susceptibility to this disease. These genetic data may be useful for future studies on the association between these polymorphisms and cancer in the investigated populations.
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Predisposición Genética a la Enfermedad , Interleucina-1alfa/genética , Interleucina-4/genética , Subunidad p50 de NF-kappa B/genética , Neoplasias/etnología , Neoplasias/genética , Receptor PAR-1/genética , Alelos , Población Negra , Brasil , Expresión Génica , Frecuencia de los Genes , Genética de Población , Humanos , Indígenas Sudamericanos , Interleucina-1alfa/inmunología , Interleucina-4/inmunología , Subunidad p50 de NF-kappa B/inmunología , Neoplasias/diagnóstico , Neoplasias/inmunología , Polimorfismo de Nucleótido Simple , Pronóstico , Receptor PAR-1/inmunología , Riesgo , Población BlancaAsunto(s)
Vacunas contra el Cáncer/uso terapéutico , Neoplasias Hematológicas/tratamiento farmacológico , Oligopéptidos/uso terapéutico , Vacunas contra el Cáncer/efectos adversos , Vacunas contra el Cáncer/inmunología , Ensayos Clínicos Fase I como Asunto , Ensayos Clínicos Fase II como Asunto , Neoplasias Hematológicas/inmunología , Neoplasias Hematológicas/patología , Humanos , Elastasa de Leucocito/inmunología , Oligopéptidos/efectos adversos , Oligopéptidos/inmunología , Receptor PAR-1/inmunología , Resultado del TratamientoRESUMEN
Development of acute experimental autoimmune encephalomyelitis (EAE) depends on Th17 cells expressing the nuclear factor NR4A2. However, in mice lacking NR4A2 in T cells, a late-onset disease is still inducible, despite a great reduction in acute inflammation. We here reveal that development of this late onset disease depends on cytotoxic T-cell-like CD4(+) T cells expressing the T-box transcription factor Eomesodermin (Eomes). T-cell-specific deletion of the Eomes gene remarkably ameliorates the late-onset EAE. Strikingly, similar Eomes(+) CD4(+) T cells are increased in the peripheral blood and cerebrospinal fluid from patients in a progressive state of multiple sclerosis. Collective data indicate an involvement of granzyme B and protease-activated receptor-1 in the neuroinflammation mediated by Eomes(+) CD4(+) T cells.
Asunto(s)
Encefalomielitis Autoinmune Experimental/inmunología , Granzimas/inmunología , Esclerosis Múltiple Crónica Progresiva/inmunología , Receptor PAR-1/inmunología , Proteínas de Dominio T Box/inmunología , Células Th17/inmunología , Adulto , Animales , Encefalomielitis Autoinmune Experimental/genética , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Esclerosis Múltiple Recurrente-Remitente/inmunología , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/inmunología , Proteínas de Dominio T Box/genética , Linfocitos T Colaboradores-Inductores/inmunología , Adulto JovenRESUMEN
In breast cancer, the prognosis of human epidermal growth factor receptor 2 (HER2)-positive patients (20-25%) has been dramatically improved by the clinical application of the anti-HER2 antibody drugs trastuzumab and pertuzumab. However, the clinical outcomes of HER2-negative cases with a poor prognosis have not improved, and novel therapeutic antibody drugs or diagnostic molecular markers of prognosis are urgently needed. Here, we targeted protease-activated receptor 1 (PAR1) as a new biomarker for HER2-negative patients. The developed anti-PAR1 antibody inhibited PAR1 activation by matrix metalloprotease 1 and thereby prevented cancer-cell migration and invasion. To estimate PAR1 expression levels in HER2-negative patient tissues using the antibody, user-friendly immunohistochemistry with fluorescence nanoparticles or quantum dots (QDs) was developed. Previously, immunohistochemistry with QDs was affected by tissue autofluorescence, making quantitative measurement extremely difficult. We significantly improved the quantitative sensitivity of immunohistochemistry with QDs by using an autofluorescence-subtracted image and single-QD imaging. The immunohistochemistry showed that PAR1 expression was strongly correlated with relapse-free survival time in HER2-negative breast cancer patients. Therefore, the developed anti-PAR1 antibody is a strong candidate for use as an anticancer drug and a prognostic biomarker for HER2-negative patients.
Asunto(s)
Neoplasias de la Mama/diagnóstico , Diagnóstico por Imagen/métodos , Recurrencia Local de Neoplasia/diagnóstico , Puntos Cuánticos , Adulto , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/cirugía , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Femenino , Humanos , Inmunohistoquímica , Metaloproteinasa 1 de la Matriz/metabolismo , Persona de Mediana Edad , Estadificación de Neoplasias , Cuidados Posoperatorios , Pronóstico , Receptor ErbB-2/metabolismo , Receptor PAR-1/antagonistas & inhibidores , Receptor PAR-1/inmunologíaAsunto(s)
Antiinflamatorios/farmacocinética , Células Mesangiales/efectos de los fármacos , Células Mesangiales/inmunología , Pirazoles/farmacología , Piridonas/farmacología , Receptor PAR-1/inmunología , Trombina/antagonistas & inhibidores , Trombina/inmunología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Inhibidores del Factor Xa/farmacología , HumanosRESUMEN
The plasminogen (Plg)/plasmin (Pla) system is associated with a variety of biological activities beyond the classical dissolution of fibrin clots, including cell migration, tissue repair, and inflammation. Although the capacity of Plg/Pla to induce cell migration is well defined, the mechanism underlying this process in vivo is elusive. In this study, we show that Pla induces in vitro migration of murine fibroblasts and macrophages (RAW 264.7) dependent on the MEK/ERK pathway and by requiring its proteolytic activity and lysine binding sites. Plasmin injection into the pleural cavity of BALB/c mice induced a time-dependent influx of mononuclear cells that was associated with augmented ERK1/2 and IκB-α phosphorylation and increased levels of CCL2 and IL-6 in pleural exudates. The inhibition of protease activity by using a serine protease inhibitor leupeptin or two structurally different protease-activated receptor-1 antagonists (SCH79797 and RWJ56110) abolished Pla-induced mononuclear recruitment and ERK1/2 and IκB-α phosphorylation. Interestingly, inhibition of the MEK/ERK pathway abolished Pla-induced CCL2 upregulation and mononuclear cell influx. In agreement with a requirement for the CCL2/CCR2 axis to Pla-induced cell migration, the use of a CCR2 antagonist (RS504393) prevented the Plg/Pla-induced recruitment of mononuclear cells to the pleural cavity and migration of macrophages at transwell plates. Therefore, Pla-induced mononuclear cell recruitment in vivo was dependent on protease-activated receptor-1 activation of the MEK/ERK/NF-κB pathway, which led to the release of CCL2 and activation of CCR2.
Asunto(s)
Movimiento Celular/inmunología , Quinasas MAP Reguladas por Señal Extracelular/inmunología , Fibrinolisina/inmunología , Quinasas Quinasa Quinasa PAM/inmunología , Sistema de Señalización de MAP Quinasas/inmunología , Monocitos/inmunología , Receptor PAR-1/inmunología , Receptores CCR2/inmunología , Animales , Benzoxazinas/farmacología , Movimiento Celular/efectos de los fármacos , Quimiocina CCL2/inmunología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Macrófagos Peritoneales/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , FN-kappa B/inmunología , Cavidad Pleural/inmunología , Receptores CCR2/antagonistas & inhibidores , Compuestos de Espiro/farmacología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/inmunologíaRESUMEN
Viral myocarditis is estimated to cause ~20% of sudden death in people under the age of 40. A variety of viruses have been found to cause myocarditis including coxsackievirus B3 (CVB3). Many studies have been performed with CVB3 because there is a mouse model of CVB3-induced myocarditis. Studies have shown that the TLR3-IFNß pathway plays a central role in the innate immune response to CVB3 infection. Our laboratory studies the role of protease activated receptors (PAR) in different biological responses including viral infection. We examined the effect of a deficiency in either PAR1 or PAR2 on CVB3-induced myocarditis. Interestingly, we found that PAR1 knockout mice had increased cardiac injury whereas PAR2 knockout mice had decreased cardiac injury. Our studies support the notion that PARs modulate the innate immune response and can have both positive and negative effects on TLR-dependent responses.
Asunto(s)
Infecciones por Coxsackievirus/complicaciones , Inmunidad Innata , Miocarditis/inmunología , Miocarditis/virología , Receptor PAR-1/inmunología , Receptor PAR-2/inmunología , Animales , Infecciones por Coxsackievirus/inmunología , Modelos Animales de Enfermedad , Enterovirus/inmunología , Enterovirus/fisiología , Corazón/virología , Interacciones Huésped-Patógeno , Humanos , Ratones , Ratones Noqueados , Miocarditis/genética , Receptor PAR-1/genética , Receptor PAR-2/genética , Receptores Toll-Like/inmunologíaRESUMEN
Protease-activated receptors (PARs) are a subfamily of four G-protein-coupled receptors mediating multiple functions. PARs expression was studied in subpopulations of human lymphocytes. Our results indicate that natural killer cells expressed mRNA for PAR1, PAR2 and PAR3, CD4+ T cells expressed PAR1 and PAR2, while γδ and CD8+ T cells only expressed PAR1. PAR4 was absent at mRNA level and B cells did not express any PAR. Analyses of the cell surface PARs expression by flow cytometry were consistent with the mRNA data and also between different donors. PAR1 is the most abundant member of the PAR family present in lymphocytes.