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1.
Nat Immunol ; 24(12): 2021-2031, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37903858

RESUMO

S100A8/S100A9 is a proinflammatory mediator released by myeloid cells during many acute and chronic inflammatory disorders. However, the precise mechanism of its release from the cytosolic compartment of neutrophils is unclear. Here, we show that E-selectin-induced rapid S100A8/S100A9 release during inflammation occurs in an NLRP3 inflammasome-dependent fashion. Mechanistically, E-selectin engagement triggers Bruton's tyrosine kinase-dependent tyrosine phosphorylation of NLRP3. Concomitant potassium efflux via the voltage-gated potassium channel KV1.3 mediates ASC oligomerization. This is followed by caspase 1 cleavage and downstream activation of pore-forming gasdermin D, enabling cytosolic release of S100A8/S100A9. Strikingly, E-selectin-mediated gasdermin D pore formation does not result in cell death but is a transient process involving activation of the ESCRT III membrane repair machinery. These data clarify molecular mechanisms of controlled S100A8/S100A9 release from neutrophils and identify the NLRP3/gasdermin D axis as a rapid and reversible activation system in neutrophils during inflammation.


Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Humanos , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Gasderminas , Neutrófilos/metabolismo , Selectina E/metabolismo , Calgranulina A/metabolismo , Calgranulina B/metabolismo , Inflamação/metabolismo
2.
Proc Natl Acad Sci U S A ; 121(19): e2319057121, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38687790

RESUMO

Eosinophil recruitment is a pathological hallmark of many allergic and helminthic diseases. Here, we investigated chemokine receptor CCR3-induced eosinophil recruitment in sialyltransferase St3gal4-/- mice. We found a marked decrease in eosinophil extravasation into CCL11-stimulated cremaster muscles and into the inflamed peritoneal cavity of St3gal4-/- mice. Ex vivo flow chamber assays uncovered reduced adhesion of St3gal4-/- compared to wild type eosinophils. Using flow cytometry, we show reduced binding of CCL11 to St3gal4-/- eosinophils. Further, we noted reduced binding of CCL11 to its chemokine receptor CCR3 isolated from St3gal4-/- eosinophils. This was accompanied by almost absent CCR3 internalization of CCL11-stimulated St3gal4-/- eosinophils. Applying an ovalbumin-induced allergic airway disease model, we found a dramatic reduction in eosinophil numbers in bronchoalveolar lavage fluid following intratracheal challenge with ovalbumin in St3gal4-deficient mice. Finally, we also investigated tissue-resident eosinophils under homeostatic conditions and found reduced resident eosinophil numbers in the thymus and adipose tissue in the absence of ST3Gal-IV. Taken together, our results demonstrate an important role of ST3Gal-IV in CCR3-induced eosinophil recruitment in vivo rendering this enzyme an attractive target in reducing unwanted eosinophil infiltration in various disorders including allergic diseases.


Assuntos
Eosinófilos , Camundongos Knockout , Receptores CCR3 , Sialiltransferases , beta-Galactosídeo alfa-2,3-Sialiltransferase , Animais , Receptores CCR3/metabolismo , Receptores CCR3/genética , Sialiltransferases/metabolismo , Sialiltransferases/genética , Eosinófilos/metabolismo , Eosinófilos/imunologia , Camundongos , Quimiocina CCL11/metabolismo , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Líquido da Lavagem Broncoalveolar
3.
Blood ; 139(23): 3402-3417, 2022 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-35303071

RESUMO

Neutrophils are key players during host defense and sterile inflammation. Neutrophil dysfunction is a characteristic feature of the acquired immunodeficiency during kidney disease. We speculated that the impaired renal clearance of the intrinsic purine metabolite soluble uric acid (sUA) may account for neutrophil dysfunction. Indeed, hyperuricemia (HU, serum UA of 9-12 mg/dL) related or unrelated to kidney dysfunction significantly diminished neutrophil adhesion and extravasation in mice with crystal- and coronavirus-related sterile inflammation using intravital microscopy and an air pouch model. This impaired neutrophil recruitment was partially reversible by depleting UA with rasburicase. We validated these findings in vitro using either neutrophils or serum from patients with kidney dysfunction-related HU with or without UA depletion, which partially normalized the defective migration of neutrophils. Mechanistically, sUA impaired ß2 integrin activity and internalization/recycling by regulating intracellular pH and cytoskeletal dynamics, physiological processes that are known to alter the migratory and phagocytic capability of neutrophils. This effect was fully reversible by blocking intracellular uptake of sUA via urate transporters. In contrast, sUA had no effect on neutrophil extracellular trap formation in neutrophils from healthy subjects or patients with kidney dysfunction. Our results identify an unexpected immunoregulatory role of the intrinsic purine metabolite sUA, which contrasts the well-known immunostimulatory effects of crystalline UA. Specifically targeting UA may help to overcome certain forms of immunodeficiency, for example in kidney dysfunction, but may enhance sterile forms of inflammation.


Assuntos
Antígenos CD18 , Ácido Úrico , Animais , Antígenos CD18/metabolismo , Humanos , Imunidade Inata , Inflamação , Camundongos , Infiltração de Neutrófilos , Neutrófilos , Ácido Úrico/farmacologia , Ácido Úrico/urina
4.
Nat Immunol ; 10(1): 101-8, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19060902

RESUMO

The Duffy antigen receptor for chemokines (DARC) belongs to a family of 'silent' heptahelical chemokine receptors that do not couple to G proteins and fail to transmit measurable intracellular signals. DARC binds most inflammatory chemokines and is prominently expressed on venular endothelial cells, where its function has remained contentious. Here we show that DARC, like other silent receptors, internalized chemokines but did not effectively scavenge them. Instead, DARC mediated chemokine transcytosis, which led to apical retention of intact chemokines and more leukocyte migration across monolayers expressing DARC. Mice overexpressing DARC on blood vessel endothelium had enhanced chemokine-induced leukocyte extravasation and contact-hypersensitivity reactions. Thus, interactions of chemokines with DARC support their activity on apposing leukocytes in vitro and in vivo.


Assuntos
Movimento Celular , Quimiocinas/metabolismo , Sistema do Grupo Sanguíneo Duffy/metabolismo , Leucócitos/imunologia , Receptores de Superfície Celular/metabolismo , Animais , Células Cultivadas , Cães , Sistema do Grupo Sanguíneo Duffy/genética , Endotélio Vascular/imunologia , Endotélio Vascular/metabolismo , Humanos , Camundongos , Transporte Proteico , Receptores de Superfície Celular/genética
5.
Haematologica ; 105(7): 1845-1856, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-31699792

RESUMO

Leukocyte recruitment into inflamed tissue is highly dependent on the activation and binding of integrins to their respective ligands, followed by the induction of various signaling events within the cell referred to as outside-in signaling. Src family kinases (SFK) are the central players in the outside-in signaling process, assigning them a critical role for proper immune cell function. Our study investigated the role of SFK on neutrophil recruitment in vivo using Hck-/- Fgr-/- Lyn-/- mice, which lack SFK expressed in neutrophils. We show that loss of SFK strongly reduces neutrophil adhesion and post-arrest modifications in a shear force dependent manner. Additionally, we found that in the absence of SFK, neutrophils display impaired Rab27a-dependent surface mobilization of neutrophil elastase, VLA3 and VLA6 containing vesicles. This results in a defect in neutrophil vascular basement membrane penetration and thus strongly impaired extravasation. Taken together, we demonstrate that SFK play a role in neutrophil post-arrest modifications and extravasation during acute inflammation. These findings may support the current efforts to use SFK-inhibitors in inflammatory diseases with unwanted neutrophil recruitment.


Assuntos
Neutrófilos , Quinases da Família src , Animais , Membrana Basal , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas , Quinases da Família src/genética
6.
Arterioscler Thromb Vasc Biol ; 37(6): 1076-1086, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28428216

RESUMO

OBJECTIVE: Platelet function has been intensively studied in the adult organism. However, little is known about the function and hemostatic capacity of platelets in the developing fetus as suitable in vivo models are lacking. APPROACH AND RESULTS: To examine fetal platelet function in vivo, we generated a fetal thrombosis model and investigated light/dye-induced thrombus formation by intravital microscopy throughout gestation. We observed that significantly less and unstable thrombi were formed at embryonic day (E) 13.5 compared with E17.5. Flow cytometry revealed significantly lower platelet counts in E13.5 versus E17.5 fetuses versus adult controls. In addition, fetal platelets demonstrated changed activation responses of surface adhesion molecules and reduced P-selectin content and mobilization. Interestingly, we also measured reduced levels of the integrin-activating proteins Kindlin-3, Talin-1, and Rap1 during fetal development. Consistently, fetal platelets demonstrated diminished spreading capacity compared with adults. Transfusion of adult platelets into the fetal circulation led to rapid platelet aggregate formation even in young fetuses. Yet, retrospective data analysis of a neonatal cohort demonstrated no correlation of platelet transfusion with closure of a persistent ductus arteriosus, a process reported to be platelet dependent. CONCLUSIONS: Taken together, we demonstrate an ontogenetic regulation of platelet function in vivo with physiologically low platelet numbers and hyporeactivity early during fetal development shedding new light on hemostatic function during fetal life.


Assuntos
Plaquetas/metabolismo , Hemostasia , Ativação Plaquetária , Trombose/sangue , Animais , Moléculas de Adesão Celular/sangue , Bases de Dados Factuais , Modelos Animais de Doenças , Permeabilidade do Canal Arterial/sangue , Feminino , Idade Gestacional , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Recém-Nascido de muito Baixo Peso , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Adesividade Plaquetária , Transfusão de Plaquetas , Nascimento Prematuro/sangue , Estudos Retrospectivos , Transdução de Sinais , Trombocitopenia/sangue
7.
Blood ; 123(12): 1887-96, 2014 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-24458438

RESUMO

Emerging evidence suggests a role of the cytokine midkine (MK) in inflammation. In this study, its functional relevance for recruitment of polymorphonuclear neutrophils (PMNs) during acute inflammation was investigated. Intravital microscopy and histologic analysis of tumor necrosis factor-α-stimulated cremaster muscle venules revealed severely compromised leukocyte adhesion and extravasation in MK(-/-) mice compared with MK(+/+) animals. Systemic administration of recombinant MK completely rescued the adhesion defect in MK(-/-) mice. In a hind limb ischemia model, leukocyte accumulation in MK(-/-) mice was significantly diminished compared with MK(+/+) animals. However, MK did not lead to an inflammatory activation of PMNs or endothelial cells suggesting that it does not serve as classical proinflammatory cytokine. Unexpectedly, immobilized MK mediated PMN adhesion under static and flow conditions, whereas PMN-derived MK was dispensable for the induction of adhesion. Furthermore, adhesion strengthening remained unaffected by MK. Flow cytometry revealed that immobilized, but not soluble MK, significantly promoted the high affinity conformation of ß2 integrins of PMNs. Blocking studies of low-density lipoprotein receptor-related protein 1 (LRP1) suggested that LRP1 may act as a receptor for MK on PMNs. Thus, MK seems to support PMN adhesion by promoting the high affinity conformation of ß2 integrins, thereby facilitating PMN trafficking during acute inflammation.


Assuntos
Antígenos CD18/fisiologia , Inflamação/fisiopatologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Neutrófilos/fisiologia , Animais , Antígenos CD11/fisiologia , Antígenos CD18/genética , Adesão Celular/imunologia , Adesão Celular/fisiologia , Citocinas/imunologia , Citocinas/fisiologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/imunologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/fisiologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Midkina , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/imunologia , Fatores de Crescimento Neural/fisiologia , Neutrófilos/imunologia , Neutrófilos/patologia , Receptores de LDL/imunologia , Receptores de LDL/fisiologia , Proteínas Supressoras de Tumor/imunologia , Proteínas Supressoras de Tumor/fisiologia
8.
J Immunol ; 192(5): 2366-73, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24470498

RESUMO

ChemR23 is a chemotactic receptor expressed by APCs, such as dendritic cells, macrophages, and NK cells. Chemerin, the ChemR23 ligand, was detected by immunohistochemistry, to be associated with inflamed endothelial cells in autoimmune diseases, such as lupus erythematosus, psoriasis, and rheumatoid arthritis. This study reports that blood and lymphatic murine endothelial cells produce chemerin following retinoic acid stimulation. Conversely, proinflammatory cytokines, such as TNF-α, IFN-γ, and LPS, or calcitriol, are not effective. Retinoic acid-stimulated endothelial cells promoted dendritic cell adhesion under shear stress conditions and transmigration in a ChemR23-dependent manner. Activated endothelial cells upregulated the expression of the atypical chemotactic receptor CCRL2/ACKR5, a nonsignaling receptor able to bind and present chemerin to ChemR23(+) dendritic cells. Accordingly, activated endothelial cells expressed chemerin on the plasma membrane and promoted in a more efficient manner chemerin-dependent transmigration of dendritic cells. Finally, chemerin stimulation of myeloid dendritic cells induced the high-affinity binding of VCAM-1/CD106 Fc chimeric protein and promoted VCAM-1-dependent arrest to immobilized ligands under shear stress conditions. In conclusion, this study reports that retinoic acid-activated endothelial cells can promote myeloid and plasmacytoid dendritic cell transmigration across endothelial cell monolayers through the endogenous production of chemerin, the upregulation of CCRL2, and the activation of dendritic cell ß1 integrin affinity.


Assuntos
Fatores Quimiotáticos/imunologia , Células Dendríticas/imunologia , Células Endoteliais/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Migração Transendotelial e Transepitelial/imunologia , Animais , Antineoplásicos/farmacologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Adesão Celular/imunologia , Linhagem Celular , Quimiocinas , Fatores Quimiotáticos/genética , Células Dendríticas/citologia , Células Endoteliais/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Knockout , Receptores CCR , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/imunologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/imunologia , Migração Transendotelial e Transepitelial/efeitos dos fármacos , Migração Transendotelial e Transepitelial/genética , Tretinoína/farmacologia , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/imunologia
9.
J Infect Dis ; 212(2): 247-57, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-25605866

RESUMO

BACKGROUND: Neutrophilic inflammation often persists for days despite effective antibiotic treatment and contributes to brain damage in bacterial meningitis. We propose here that myeloid-related protein 14 (MRP14), an abundant cytosolic protein in myeloid cells, acts as an endogenous danger signal, driving inflammation and aggravating tissue injury. METHODS: The release pattern of MRP14 was analyzed in human and murine cerebrospinal fluid (CSF), as well as in isolated neutrophils. Its functional role was assessed in a mouse meningitis model, using MRP14-deficient mice. RESULTS: We detected large quantities of MRP14 in CSF specimens from patients and mice with pneumococcal meningitis. Immunohistochemical analyses and a cell-depletion approach indicated neutrophils as the major source of MRP14. In a meningitis model, MRP14-deficient mice showed a better resolution of inflammation during antibiotic therapy, which was accompanied by reduced disease severity. Intrathecal administration of MRP14 before infection reverted the phenotype of MRP14-deficient mice back to wild type. Moreover, intrathecal injection of MRP14 alone was sufficient to induce meningitis in a Toll-like receptor 4 (TLR4)-CXCL2-dependent manner. Finally, treatment with the MRP14 antagonist paquinimod reduced inflammation and disease severity significantly, reaching levels comparable to those achieved after genetic depletion of MRP14. CONCLUSIONS: The present study implicates MRP14 as an essential propagator of inflammation and potential therapeutic target in pneumococcal meningitis.


Assuntos
Calgranulina B/líquido cefalorraquidiano , Meningite Pneumocócica/líquido cefalorraquidiano , Transportadores de Cassetes de Ligação de ATP/líquido cefalorraquidiano , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Estudos de Casos e Controles , Ceftriaxona/farmacologia , Ceftriaxona/uso terapêutico , Quimiocina CXCL2/biossíntese , Humanos , Masculino , Meningite Pneumocócica/tratamento farmacológico , Meningite Pneumocócica/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout
10.
Blood ; 121(21): e118-28, 2013 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-23525796

RESUMO

In adult mammals, leukocyte recruitment follows a well-defined cascade of adhesion events enabling leukocytes to leave the circulatory system and transmigrate into tissue. Currently, it is unclear whether leukocyte recruitment proceeds in a similar fashion during fetal development. Considering the fact that the incidence of neonatal sepsis increases dramatically with decreasing gestational age in humans, we hypothesized that leukocyte recruitment may be acquired only late during fetal ontogeny. To test this, we developed a fetal intravital microscopy model in pregnant mice and, using LysEGFP (neutrophil reporter) mice, investigated leukocyte recruitment during fetal development. We show that fetal blood neutrophils acquire the ability to roll and adhere on inflamed yolk sac vessels during late fetal development, whereas at earlier embryonic stages (before day E15), rolling and adhesion were essentially absent. Accordingly, flow chamber experiments showed that fetal EGFP(+) blood cells underwent efficient adhesion only when they were harvested on or after E15. Fluorescence-activated cell sorter analysis on EGFP(+) fetal blood cells revealed that surface expression of CXCR2 and less pronounced P-selectin glycoprotein ligand-1 (PSGL-1) begin to increase only late in fetal life. Taken together, our findings demonstrate that inflammation-induced leukocyte recruitment is ontogenetically regulated and enables efficient neutrophil trafficking only during late fetal life.


Assuntos
Movimento Celular/imunologia , Sistema Imunitário/embriologia , Leucócitos/citologia , Microvasos/embriologia , Saco Vitelino/embriologia , Animais , Adesão Celular/imunologia , Eritroblastos/citologia , Feminino , Sangue Fetal/citologia , Proteínas de Fluorescência Verde/metabolismo , Sistema Imunitário/citologia , Migração e Rolagem de Leucócitos/imunologia , Leucócitos/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Microvasos/citologia , Microvasos/imunologia , Neutrófilos/citologia , Neutrófilos/metabolismo , Selectina-P/metabolismo , Gravidez , Receptores de Interleucina-8B/metabolismo , Saco Vitelino/irrigação sanguínea , Saco Vitelino/citologia
11.
J Immunol ; 190(12): 6450-6456, 2013 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-23670187

RESUMO

Chemokines, acting on their cognate receptors on infiltrating leukocytes, drive the inflammatory response. We have been interested in determining roles and potential mechanisms for the atypical chemokine-scavenging receptor D6 in the regulation of inflammation. In this study, we show that a psoriasis-like pathology that arises in inflamed skins of D6-deficient mice is characterized by a massive and aberrant localization of neutrophils to the dermal/epidermal junction, which is associated with development of the pathology. Such misplacement of neutrophils is also seen with D6-deficient mice in other inflammatory models, suggesting a role for D6 in the spatial positioning of neutrophils within inflamed sites. We further show that D6 functions cell autonomously in this context and that D6, expressed by neutrophils, limits their migrational responses to CCR1 ligands such as CCL3. Our data therefore indicate that D6 is able to play a cell-autonomous role as a migratory rheostat restricting migration of D6-expressing cells such as neutrophils toward ligands for coexpressed inflammatory chemokine receptors. These data have important implications for our understanding of the roles for D6 in regulating inflammation and for our understanding of the control of spatial positioning of leukocytes at inflamed sites.


Assuntos
Doenças do Sistema Imunitário/imunologia , Transtornos Leucocíticos/imunologia , Psoríase/imunologia , Receptores de Quimiocinas/imunologia , Animais , Modelos Animais de Doenças , Inflamação/imunologia , Camundongos , Camundongos Knockout , Microscopia Confocal , Psoríase/patologia , Pele/imunologia , Pele/patologia
12.
J Leukoc Biol ; 2024 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-39312228

RESUMO

Newborns are at high risk to develop sepsis. This is linked to innate immune responses at birth which are not completely adapted to postnatal life. Neutrophils are key players of innate immunity and exhibit a marked ontogenetic regulation of their functionality. Here, we studied the NLRP3 inflammasome in neonatal neutrophils and found lower baseline expression of NLRP3, pro-caspase-1 and the K+-channel KV1.3 compared to adult neutrophils. Following stimulation with LPS/Nigericin, ASC oligomerization, caspase-1 activation and IL-1ß release were significantly reduced in neonatal compared to adult neutrophils. Similarly, stimulation of neonatal neutrophils with E-selectin led to reduced NLRP3 inflammasome activation accompanied by diminished release of the alarmin S100A8/A9. Taken together, our results strongly indicate diminished NLRP3 inflammasome activation in neonatal neutrophils leading to a significant reduction of released IL-1ß and S100A8/A9. These findings identify reduced neutrophil NLRP3 inflammasome activation as critical component contributing to the inherent susceptibility to infections in neonates.

13.
Blood ; 116(5): 841-9, 2010 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-20407037

RESUMO

The receptor for advanced glycation end products (RAGE) contributes to the inflammatory response in many acute and chronic diseases. In this context, RAGE has been identified as a ligand for the beta(2)-integrin Mac-1 under static in vitro conditions. Because intercellular adhesion molecule (ICAM)-1 also binds beta(2)-integrins, we studied RAGE(-/-), Icam1(-/-), and RAGE(-/-) Icam1(-/-) mice to define the relative contribution of each ligand for leukocyte adhesion in vivo. We show that trauma-induced leukocyte adhesion in cremaster muscle venules is strongly dependent on RAGE and ICAM-1 acting together in an overlapping fashion. Additional in vivo experiments in chimeric mice lacking endothelium-expressed RAGE and ICAM-1 located the adhesion defect to the endothelial compartment. Using microflow chambers coated with P-selectin, CXCL1, and soluble RAGE (sRAGE) demonstrated that sRAGE supports leukocyte adhesion under flow conditions in a Mac-1- but not LFA-1-dependent fashion. A static adhesion assay revealed that wild-type and RAGE(-/-) neutrophil adhesion and spreading were similar on immobilized sRAGE or fibrinogen. These observations indicate a crucial role of endothelium-expressed RAGE as Mac-1 ligand and uncover RAGE and ICAM-1 as a new set of functionally linked adhesion molecules, which closely cooperate in mediating leukocyte adhesion during the acute trauma-induced inflammatory response in vivo.


Assuntos
Quimiotaxia de Leucócito/fisiologia , Endotélio Vascular/metabolismo , Molécula 1 de Adesão Intercelular/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Músculo Esquelético/irrigação sanguínea , Vasculite/imunologia , Doença Aguda , Animais , Adesão Celular , Forma Celular , Quimiotaxia de Leucócito/efeitos dos fármacos , Humanos , Leucotrieno B4/farmacologia , Ligantes , Antígeno de Macrófago 1/metabolismo , Camundongos , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/deficiência , Músculo Esquelético/lesões , Músculo Esquelético/patologia , Neutrófilos/patologia , Quimera por Radiação , Proteínas Recombinantes de Fusão/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Vasculite/etiologia , Vênulas/patologia
14.
Cardiovasc Res ; 118(5): 1289-1302, 2022 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-33881519

RESUMO

AIMS: Neutrophil trafficking within the vasculature strongly relies on intracellular calcium signalling. Sustained Ca2+ influx into the cell requires a compensatory efflux of potassium to maintain membrane potential. Here, we aimed to investigate whether the voltage-gated potassium channel KV1.3 regulates neutrophil function during the acute inflammatory process by affecting sustained Ca2+ signalling. METHODS AND RESULTS: Using in vitro assays and electrophysiological techniques, we show that KV1.3 is functionally expressed in human neutrophils regulating sustained store-operated Ca2+ entry through membrane potential stabilizing K+ efflux. Inhibition of KV1.3 on neutrophils by the specific inhibitor 5-(4-Phenoxybutoxy)psoralen (PAP-1) impaired intracellular Ca2+ signalling, thereby preventing cellular spreading, adhesion strengthening, and appropriate crawling under flow conditions in vitro. Using intravital microscopy, we show that pharmacological blockade or genetic deletion of KV1.3 in mice decreased neutrophil adhesion in a blood flow dependent fashion in inflamed cremaster muscle venules. Furthermore, we identified KV1.3 as a critical component for neutrophil extravasation into the inflamed peritoneal cavity. Finally, we also revealed impaired phagocytosis of Escherichia coli particles by neutrophils in the absence of KV1.3. CONCLUSION: We show that the voltage-gated potassium channel KV1.3 is critical for Ca2+ signalling and neutrophil trafficking during acute inflammatory processes. Our findings do not only provide evidence for a role of KV1.3 for sustained calcium signalling in neutrophils affecting key functions of these cells, they also open up new therapeutic approaches to treat inflammatory disorders characterized by overwhelming neutrophil infiltration.


Assuntos
Canais de Potássio de Abertura Dependente da Tensão da Membrana , Animais , Cálcio/metabolismo , Inflamação , Canal de Potássio Kv1.5 , Potenciais da Membrana/fisiologia , Camundongos , Infiltração de Neutrófilos
15.
J Immunol ; 182(4): 2074-83, 2009 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-19201860

RESUMO

Activation of vitamin D receptor (VDR) by 1,25-dihydroxyvitamin D(3) (1,25-vitD) reprograms dendritic cells (DC) to become tolerogenic. Previous studies suggested that 1,25-vitD could inhibit the changes brought about by differentiation and maturation of DCs. Underpinning the described phenotypic and functional alterations, there must be 1,25-vitD-coordinated transcriptional events. However, this transcriptional program has not been systematically investigated, particularly not in a developmental context. Hence, it has not been explored how 1,25-vitD-regulated genes, particularly the ones bringing about the tolerogenic phenotype, are connected to differentiation. We conducted global gene expression analysis followed by comprehensive quantitative PCR validation to clarify the interrelationship between 1,25-vitD and differentiation-driven gene expression patterns in developing human monocyte-derived and blood myeloid DCs. In this study we show that 1,25-vitD regulates a large set of genes that are not affected by differentiation. Interestingly, several genes, impacted both by the ligand and by differentiation, appear to be regulated by 1,25-vitD independently of the developmental context. We have also characterized the kinetics of generation of 1,25-vitD by using three early and robustly regulated genes, the chemokine CCL22, the inhibitory receptors CD300LF and CYP24A1. We found that monocyte-derived DCs are able to turn on 1,25-vitD sensitive genes in early phases of differentiation if the precursor is present. Our data collectively suggest that exogenous or endogenously generated 1,25-vitD regulates a large set of its targets autonomously and not via inhibition of differentiation and maturation, leading to the previously characterized tolerogenic state.


Assuntos
Calcitriol/imunologia , Células Dendríticas/imunologia , Tolerância Imunológica/genética , Transcrição Gênica/imunologia , Vitaminas/imunologia , Western Blotting , Calcitriol/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Quimiocina CCL22/imunologia , Quimiocina CCL22/metabolismo , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Tolerância Imunológica/imunologia , Imuno-Histoquímica , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Receptores de Calcitriol/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica/genética , Vitaminas/metabolismo
16.
J Am Soc Nephrol ; 21(1): 42-52, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19917782

RESUMO

The homing of dendritic cells and T cells to secondary lymphoid organs requires chemokine receptor 7 (CCR7) expression on these cells. T cells mediate the pathogenesis of experimental accelerated nephrotoxic serum nephritis (NTS), including its suppression by regulatory T cells (Tregs), but the contribution of CCR7 to this disease is unknown. Here, we compared the development of NTS in CCR7-knockout (KO) and wild-type (WT) mice. Compared with WT mice, CCR7KO mice developed more severe disease with significantly more inflammatory cells infiltrating the kidney. These cells included FoxP3(+) Tregs, which were virtually absent from WT kidneys. The adoptive transfer of WT Tregs into CCR7KO mice at the time of immunization protected the recipients from disease; these cells homed to secondary lymphoid organs but not to kidneys. Conversely, adoptive transfer of CCR7KO Tregs into WT mice did not inhibit development of NTS. These data suggest that NTS can develop without CCR7 expression, but Treg-mediated disease suppression, which seems to occur in secondary lymphoid organs, requires CCR7.


Assuntos
Movimento Celular/fisiologia , Nefrite/metabolismo , Nefrite/patologia , Receptores CCR7/metabolismo , Linfócitos T Reguladores/patologia , Linfócitos T Reguladores/fisiologia , Doença Aguda , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Regulação para Baixo/fisiologia , Sistema Imunitário/fisiopatologia , Rim/metabolismo , Rim/patologia , Linfonodos/metabolismo , Linfonodos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Nefrite/fisiopatologia , Receptores CCR7/genética , Índice de Gravidade de Doença
17.
Am J Pathol ; 175(1): 119-31, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19498001

RESUMO

The Duffy antigen/receptor for chemokines (DARC) is a chemokine-binding protein that is expressed on erythrocytes and renal endothelial cells. DARC-mediated endothelial transcytosis of chemokines may facilitate the renal recruitment of macrophages and T cells, as has been suggested for neutrophils. We studied the role of Darc in two mouse models of prolonged renal inflammation, one that primarily involves the tubulointerstitium (unilateral ureteral obstruction), and one that requires an adaptive immune response that leads to glomerulonephritis (accelerated nephrotoxic nephritis). Renal expression of Darc and its ligands was increased in both models. Leukocytes effectively infiltrated obstructed kidneys in Darc-deficient mice with pronounced T-cell infiltration at early time points. Development of interstitial fibrosis was comparable in both genotypes. Nephrotoxic nephritis was inducible in Darc-deficient mice, with both an increased humoral immune response and functional impairment during the early phase of disease. Leukocytes efficiently infiltrated kidneys of Darc-deficient mice, with increased cell numbers at early but not late time points. Taken together, renal inflammation developed more rapidly in DARC-deficient mice, without affecting the extent of renal injury at later time points. Thus, genetic elimination of Darc in mice does not prevent the development of renal infiltrates and may even enhance such development during the early phases of interstitial and glomerular diseases in mouse models of prolonged renal inflammation.


Assuntos
Quimiotaxia de Leucócito , Sistema do Grupo Sanguíneo Duffy , Glomerulonefrite , Macrófagos , Receptores de Superfície Celular , Linfócitos T , Animais , Camundongos , Quimiocinas/imunologia , Quimiotaxia de Leucócito/imunologia , Sistema do Grupo Sanguíneo Duffy/genética , Sistema do Grupo Sanguíneo Duffy/imunologia , Ensaio de Imunoadsorção Enzimática , Fibrose , Citometria de Fluxo , Glomerulonefrite/imunologia , Glomerulonefrite/patologia , Processamento de Imagem Assistida por Computador , Imunoglobulina G/sangue , Imuno-Histoquímica , Macrófagos/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologia
18.
J Immunol ; 181(5): 3353-63, 2008 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-18714007

RESUMO

D6 scavenges inflammatory chemokines and is essential for the regulation of inflammatory and immune responses. Mechanisms explaining the cellular basis for D6 function have been based on D6 expression by lymphatic endothelial cells. In this study, we demonstrate that functional D6 is also expressed by murine and human hemopoietic cells and that this expression can be regulated by pro- and anti-inflammatory agents. D6 expression was highest in B cells and dendritic cells (DCs). In myeloid cells, LPS down-regulated expression, while TGF-beta up-regulated expression. Activation of T cells with anti-CD3 and soluble CD28 up-regulated mRNA expression 20-fold, while maturation of human macrophage and megakaryocyte precursors also up-regulated D6 expression. Competition assays demonstrated that chemokine uptake was D6 dependent in human leukocytes, whereas mouse D6-null cells failed to uptake and clear inflammatory chemokines. Furthermore, we present evidence indicating that D6 expression is GATA1 dependent, thus explaining D6 expression in myeloid progenitor cells, mast cells, megakaryocytes, and DCs. We propose a model for D6 function in which leukocytes, within inflamed sites, activate D6 expression and thus trigger resolution of inflammatory responses. Our data on D6 expression by circulating DCs and B cells also suggest alternative roles for D6, perhaps in the coordination of innate and adaptive immune responses. These data therefore alter our models of in vivo D6 function and suggest possible discrete, and novel, roles for D6 on lymphatic endothelial cells and leukocytes.


Assuntos
Fator de Transcrição GATA1/fisiologia , Regulação da Expressão Gênica , Leucócitos/metabolismo , Receptores CCR10/genética , Animais , Células Cultivadas , Células Dendríticas , Células Endoteliais , Humanos , Inflamação/imunologia , Camundongos , Receptor D6 de Quimiocina
19.
J Immunol ; 181(11): 8171-81, 2008 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19039854

RESUMO

D6 scavenges inflammatory chemokines and is essential for the regulation of inflammatory and immune responses. Mechanisms explaining the cellular basis for D6 function have been based on D6 expression by lymphatic endothelial cells. In this study, we demonstrate that functional D6 is also expressed by murine and human hemopoietic cells and that this expression can be regulated by pro- and anti-inflammatory agents. D6 expression was highest in B cells and dendritic cells (DCs). In myeloid cells, LPS down-regulated expression, while TGF-beta up-regulated expression. Activation of T cells with anti-CD3 and soluble CD28 up-regulated mRNA expression 20-fold, while maturation of human macrophage and megakaryocyte precursors also up-regulated D6 expression. Competition assays demonstrated that chemokine uptake was D6 dependent in human leukocytes, whereas mouse D6-null cells failed to uptake and clear inflammatory chemokines. Furthermore, we present evidence indicating that D6 expression is GATA1 dependent, thus explaining D6 expression in myeloid progenitor cells, mast cells, megakaryocytes, and DCs. We propose a model for D6 function in which leukocytes, within inflamed sites, activate D6 expression and thus trigger resolution of inflammatory responses. Our data on D6 expression by circulating DCs and B cells also suggest alternative roles for D6, perhaps in the coordination of innate and adaptive immune responses. These data therefore alter our models of in vivo D6 function and suggest possible discrete, and novel, roles for D6 on lymphatic endothelial cells and leukocytes.


Assuntos
Fator de Transcrição GATA1/imunologia , Regulação da Expressão Gênica/imunologia , Células-Tronco Hematopoéticas/imunologia , Leucócitos/imunologia , Modelos Biológicos , Receptores CCR10/imunologia , Animais , Quimiocinas/genética , Quimiocinas/imunologia , Quimiocinas/metabolismo , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Regulação da Expressão Gênica/genética , Células-Tronco Hematopoéticas/citologia , Humanos , Leucócitos/metabolismo , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Camundongos , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Receptores CCR10/biossíntese , Receptores CCR10/genética , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/farmacologia , Receptor D6 de Quimiocina
20.
Front Immunol ; 11: 588245, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33414784

RESUMO

Uromodulin (UMOD) is produced and secreted by tubular epithelial cells. Secreted UMOD polymerizes (pUMOD) in the tubular lumen, where it regulates salt transport and protects the kidney from bacteria and stone formation. Under various pathological conditions, pUMOD accumulates within the tubular lumen and reaches extratubular sites where it may interact with renal interstitial cells. Here, we investigated the potential of extratubular pUMOD to act as a damage associated molecular pattern (DAMP) molecule thereby creating local inflammation. We found that intrascrotal and intraperitoneal injection of pUMOD induced leukocyte recruitment in vivo and led to TNF-α secretion by F4/80 positive macrophages. Additionally, pUMOD directly affected vascular permeability and increased neutrophil extravasation independent of macrophage-released TNF-α. Interestingly, pUMOD displayed no chemotactic properties on neutrophils, did not directly activate ß2 integrins and did not upregulate adhesion molecules on endothelial cells. In obstructed neonatal murine kidneys, we observed extratubular UMOD accumulation in the renal interstitium with tubular atrophy and leukocyte infiltrates. Finally, we found extratubular UMOD deposits associated with peritubular leukocyte infiltration in kidneys from patients with inflammatory kidney diseases. Taken together, we identified extratubular pUMOD as a strong inducer of leukocyte recruitment, underlining its critical role in mounting an inflammatory response in various kidneys pathologies.


Assuntos
Inflamação/imunologia , Leucócitos/imunologia , Uromodulina/imunologia , Músculos Abdominais/imunologia , Animais , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Nefropatias/imunologia , Masculino , Camundongos Endogâmicos C57BL , Polimerização
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