RESUMO
Transendothelial migration of neutrophils in postcapillary venules is a key event in the inflammatory response against pathogens and tissue damage. The precise regulation of this process is incompletely understood. We report that perivascular macrophages are critical for neutrophil migration into skin infected with the pathogen Staphylococcus aureus. Using multiphoton intravital microscopy we showed that neutrophils extravasate from inflamed dermal venules in close proximity to perivascular macrophages, which are a major source of neutrophil chemoattractants. The virulence factor α-hemolysin produced by S. aureus lyses perivascular macrophages, which leads to decreased neutrophil transmigration. Our data illustrate a previously unrecognized role for perivascular macrophages in neutrophil recruitment to inflamed skin and indicate that S. aureus uses hemolysin-dependent killing of these cells as an immune evasion strategy.
Assuntos
Macrófagos/imunologia , Neutrófilos/imunologia , Pele/imunologia , Infecções Estafilocócicas/imunologia , Animais , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/metabolismo , Vasos Sanguíneos/imunologia , Vasos Sanguíneos/metabolismo , Citometria de Fluxo , Expressão Gênica/imunologia , Proteínas Hemolisinas/imunologia , Proteínas Hemolisinas/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Microscopia de Fluorescência por Excitação Multifotônica , Infiltração de Neutrófilos/imunologia , Neutrófilos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/irrigação sanguínea , Pele/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/imunologia , Staphylococcus aureus/metabolismo , Imagem com Lapso de Tempo/métodos , Migração Transendotelial e Transepitelial/imunologia , Vênulas/imunologia , Vênulas/metabolismoRESUMO
Monocyte patrolling of the vasculature has been ascribed primarily to the non-classical monocyte subset. However, a recent study of the glomerular microvasculature provided evidence that both classical and non-classical monocytes undergo periods of intravascular retention and migration. Despite this, whether these subsets contribute differentially to acute glomerular inflammation is unknown. This study used glomerular multiphoton intravital microscopy to investigate the capacity of classical and non-classical monocytes to patrol the glomerular microvasculature and promote acute, neutrophil-dependent glomerular inflammation. In imaging experiments in monocyte reporter Cx3cr1gfp/+ mice, co-staining with anti-Ly6B or anti-Ly6C revealed that both non-classical monocytes [CX3C chemokine receptor 1-green fluorescent protein positive (CX3CR1-GFP+)] and classical monocytes (CX3CR1-GFP+ and Ly6B+ or Ly6C+) underwent prolonged (>10 minutes) retention and migration in the glomerular microvasculature. On induction of acute glomerulonephritis, in these behaviors were increased in classical but not non-classical monocytes. Using non-classical monocyte-deficient Csf1rCreNr4a1fl/fl mice, or anti-CCR2 to deplete classical monocytes, the removal of either subset reduced neutrophil retention and activation in acutely inflamed glomeruli, while the depletion of both subsets, via anti-CCR2 treatment in Csf1rCreNr4a1fl/fl mice, led to further reductions in neutrophil activity. In contrast, in a model of CD4+ T cell-dependent glomerulonephritis, the depletion of either monocyte subset failed to alter neutrophil responses. These findings indicate that both classical and non-classical monocytes patrol the glomerular microvasculature and promote neutrophil responses in acutely inflamed glomeruli.
RESUMO
Dermal regulatory T cells (Tregs) are essential for maintenance of skin homeostasis and control of skin inflammatory responses. In mice, Tregs in the skin are characterized by high expression of CD103, the αE integrin. Evidence indicates that CD103 promotes Treg retention within the skin, although the mechanism underlying this effect is unknown. The main ligand of CD103, E-cadherin, is predominantly expressed by cells in the epidermis. However, because Tregs are predominantly located within the dermis, the nature of the interactions between E-cadherin and CD103-expressing Tregs is unclear. In this study, we used multiphoton intravital microscopy to examine the contribution of CD103 to Treg behavior in resting and inflamed skin of mice undergoing oxazolone-induced contact hypersensitivity. Inhibition of CD103 in uninflamed skin did not alter Treg behavior, whereas 48 h after inducing contact hypersensitivity by oxazolone challenge, CD103 inhibition increased Treg migration. This coincided with E-cadherin upregulation on infiltrating myeloid leukocytes in the dermis. Using CD11c-enhanced yellow fluorescent protein (EYFP) × Foxp3-GFP dual-reporter mice, inhibition of CD103 was found to reduce Treg interactions with dermal dendritic cells. CD103 inhibition also resulted in increased recruitment of effector CD4+ T cells and IFN-γ expression in challenged skin and resulted in reduced glucocorticoid-induced TNFR-related protein expression on Tregs. These results demonstrate that CD103 controls intradermal Treg migration, but only at later stages in the inflammatory response, when E-cadherin expression in the dermis is increased, and provide evidence that CD103-mediated interactions between Tregs and dermal dendritic cells support regulation of skin inflammation.
Assuntos
Dermatite de Contato , Linfócitos T Reguladores , Animais , Camundongos , Caderinas/metabolismo , Dermatite de Contato/metabolismo , Inflamação/metabolismo , Cadeias alfa de Integrinas/metabolismo , Oxazolona/metabolismo , Linfócitos T Reguladores/metabolismoRESUMO
In stroke patients, infection is a significant contributor to morbidity and mortality. Moreover, older stroke patients show an increased risk of developing stroke-associated infection, although the mechanisms underlying this increased susceptibility to infection are unknown. In this study, using an experimental mouse model of ischemic stroke, we showed that older (12-15 mo of age) mice had elevated lung bacterial infection and inflammatory damage after stroke when compared with young (8-10 wk of age) counterparts, despite undergoing the same degree of brain injury. Intravital microscopy of the lung microvasculature revealed that in younger mice, stroke promoted neutrophil arrest in pulmonary microvessels, but this response was not seen in older poststroke mice. In addition, bacterial phagocytosis by neutrophils in the lung microvasculature was reduced by both aging and stroke, such that neutrophils in aged poststroke mice showed the greatest impairment in this function. Analysis of neutrophil migration in vitro and in the cremaster muscle demonstrated that stroke alone did not negatively impact neutrophil migration, but that the combination of increased age and stroke led to reduced effectiveness of neutrophil chemotaxis. Transcriptomic analysis of pulmonary neutrophils using RNA sequencing identified 79 genes that were selectively altered in the context of combined aging and stroke, and they were associated with pathways that control neutrophil chemotaxis. Taken together, the findings of this study show that stroke in older animals results in worsening of neutrophil antibacterial responses and changes in neutrophil gene expression that have the potential to underpin elevated risk of stroke-associated infection in the context of increased age.
Assuntos
Pneumonia , Acidente Vascular Cerebral , Idoso , Envelhecimento , Animais , Humanos , Pulmão , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Fagocitose , Pneumonia/metabolismo , Acidente Vascular Cerebral/metabolismoRESUMO
T-cell receptor+ CD4- CD8- double-negative (DN) T cells are a population of T cells present in low abundance in blood and lymphoid organs, but enriched in various organs including the kidney. Despite burgeoning interest in these cells, studies examining their abundance in the kidney have reported conflicting results. Here we developed a flow cytometry strategy to clearly segregate DN T cells from other immune cells in the mouse kidney and used it to characterize their phenotype and response in renal ischemia-reperfusion injury (IRI). These experiments revealed that in the healthy kidney, most DN T cells are located within the renal parenchyma and exhibit an effector memory phenotype. In response to IRI, the number of renal DN T cells is unaltered after 24 h, but significantly increased by 72 h. This increase is not related to alterations in proliferation or apoptosis. By contrast, adoptive transfer studies indicate that circulating DN T cells undergo preferential recruitment to the postischemic kidney. Furthermore, DN T cells show the capacity to upregulate CD8, both in vivo following adoptive transfer and in response to ex vivo activation. Together, these findings provide novel insights regarding the phenotype of DN T cells in the kidney, including their predominant extravascular location, and show that increases in their abundance in the kidney following IRI occur in part as a result of increased recruitment from the circulation. Furthermore, the observation that DN T cells can upregulate CD8 in vivo has important implications for detection and characterization of DN T cells in future studies.
Assuntos
Traumatismo por Reperfusão , Linfócitos T , Camundongos , Animais , Rim , Receptores de Antígenos de Linfócitos T alfa-beta , Receptores de Antígenos de Linfócitos TRESUMO
OBJECTIVE: The endothelial surface layer (ESL), a layer of macromolecules on the surface of endothelial cells, can both impede and facilitate leukocyte recruitment. However, its role in monocyte and neutrophil recruitment in glomerular capillaries is unknown. METHODS: We used multiphoton intravital microscopy to examine monocyte and neutrophil behavior in the glomerulus following ESL disruption with hyaluronidase. RESULTS: Constitutive retention and migration of monocytes and neutrophils within the glomerular microvasculature was unaltered by hyaluronidase. Consistent with this, inhibition of the hyaluronan-binding molecule CD44 also failed to modulate glomerular trafficking of these immune cells. To investigate the contribution of the ESL during acute inflammation, we induced glomerulonephritis via in situ immune complex deposition. This resulted in increases in glomerular retention of monocytes and neutrophils but did not induce marked reduction in the glomerular ESL. Furthermore, hyaluronidase treatment did not modify the prolonged retention of monocytes and neutrophils in the acutely inflamed glomerular microvasculature. CONCLUSIONS: These observations indicate that, despite evidence that the ESL has the capacity to inhibit leukocyte-endothelial cell interactions while also containing adhesive ligands for immune cells, neither of these functions modulate trafficking of monocytes and neutrophils in steady-state or acutely-inflamed glomeruli.
Assuntos
Monócitos , Neutrófilos , Hialuronoglucosaminidase , Células Endoteliais , EndotélioRESUMO
The importance of tetraspanin proteins in regulating migration has been demonstrated in many diverse cellular systems. However, the function of the leukocyte-restricted tetraspanin CD53 remains obscure. We therefore hypothesized that CD53 plays a role in regulating leukocyte recruitment and tested this hypothesis by examining responses of CD53-deficient mice to a range of inflammatory stimuli. Deletion of CD53 significantly reduced neutrophil recruitment to the acutely inflamed peritoneal cavity. Intravital microscopy revealed that in response to several inflammatory and chemotactic stimuli, absence of CD53 had only minor effects on leukocyte rolling and adhesion in postcapillary venules. In contrast, Cd53-/- mice showed a defect in leukocyte transmigration induced by TNF, CXCL1 and CCL2, and a reduced capacity for leukocyte retention on the endothelial surface under shear flow. Comparison of adhesion molecule expression in wild-type and Cd53-/- neutrophils revealed no alteration in expression of ß2 integrins, whereas L-selectin was almost completely absent from Cd53-/- neutrophils. In addition, Cd53-/- neutrophils showed defects in activation-induced cytoskeletal remodeling and translocation to the cell periphery, responses necessary for efficient transendothelial migration, as well as increased α3 integrin expression. These alterations were associated with effects on inflammation, so that in Cd53-/- mice, the onset of neutrophil-dependent serum-induced arthritis was delayed. Together, these findings demonstrate a role for tetraspanin CD53 in promotion of neutrophil transendothelial migration and inflammation, associated with CD53-mediated regulation of L-selectin expression, attachment to the endothelial surface, integrin expression and trafficking, and cytoskeletal function.
Assuntos
Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Citoesqueleto/metabolismo , Integrina alfa3/metabolismo , Selectina L/metabolismo , Neutrófilos/fisiologia , Tetraspanina 25/metabolismo , Animais , Quimiocina CCL2/metabolismo , Quimiocina CXCL1/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Migração Transendotelial e TransepitelialRESUMO
Ischemia-reperfusion injury (IRI) is a major risk factor for chronic renal failure. Caspase-3, an effector responsible for apoptosis execution, is activated within the peritubular capillary (PTC) in the early stage of IRI-induced acute kidney injury (AKI). Recently, we showed that caspase-3-dependent microvascular rarefaction plays a key role in fibrosis development after mild renal IRI. Here, we further characterized the role of caspase-3 in microvascular dysfunction and progressive renal failure in both mild and severe AKI, by performing unilateral renal artery clamping for 30/60 min with contralateral nephrectomy in wild-type (C57BL/6) or caspase-3-/- mice. In both forms of AKI, caspase-3-/- mice showed better long-term outcomes despite worse initial tubular injury. After 3 wk, they showed reduced PTC injury, decreased PTC collagen deposition and α-smooth muscle actin expression, and lower tubular injury scores compared with wild-type animals. Caspase-3-/- mice with severe IRI also showed better preservation of long-term renal function. Intravital imaging and microcomputed tomography revealed preserved PTC permeability and better terminal capillary density in caspase-3-/- mice. Collectively, these results demonstrate the pivotal importance of caspase-3 in regulating long-term renal function after IRI and establish the predominant role of PTC dysfunction as a major contributor to progressive renal dysfunction.NEW & NOTEWORTHY Our findings demonstrate the pivotal importance of caspase-3 in regulating renal microvascular dysfunction, fibrogenesis, and long-term renal impairment after acute kidney injury induced by ischemia-reperfusion injury. Furthermore, this study establishes the predominant role of peritubular capillary integrity as a major contributor to progressive renal dysfunction after ischemia-reperfusion injury.
Assuntos
Injúria Renal Aguda/metabolismo , Caspase 3/metabolismo , Insuficiência Renal Crônica/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Apoptose/fisiologia , Capilares/metabolismo , Feminino , Rim/metabolismo , Camundongos Endogâmicos C57BL , Rarefação Microvascular/patologia , Traumatismo por Reperfusão/patologiaRESUMO
Enterohaemorrhagic E. coli cause major epidemics worldwide with significant organ damage and very high percentages of death. Due to the ability of enterohaemorrhagic E. coli to produce shiga toxin these bacteria damage the kidney leading to the hemolytic uremic syndrome. A therapy against this serious kidney disease has not been developed yet and the impact and mechanism of leukocyte activation and recruitment are unclear. Tissue-resident macrophages represent the main leukocyte population in the healthy kidney, but the role of this important cell population in shiga toxin-producing E. coli-hemolytic uremic syndrome is incompletely understood. Using state of the art microscopy and mass spectrometry imaging, our preclinical study demonstrated a phenotypic and functional switch of tissue-resident macrophages after disease induction in mice. Kidney macrophages produced the inflammatory molecule TNFα and depletion of tissue-resident macrophages via the CSF1 receptor abolished TNFα levels in the kidney and significantly diminished disease severity. Furthermore, macrophage depletion did not only attenuate endothelial damage and thrombocytopenia, but also activation of thrombocytes and neutrophils. Moreover, we observed that neutrophils infiltrated the kidney cortex and depletion of macrophages significantly reduced the recruitment of neutrophils and expression of the neutrophil-attracting chemokines CXCL1 and CXCL2. Intravital microscopy revealed that inhibition of CXCR2, the receptor for CXCL1 and CXCL2, significantly reduced the infiltration of neutrophils and reduced kidney injury. Thus, our study shows activation of tissue-resident macrophages during shiga toxin-producing E. coli-hemolytic uremic syndrome leading to the production of disease-promoting TNFα and CXCR2-dependent recruitment of neutrophils.
Assuntos
Síndrome Hemolítico-Urêmica , Toxina Shiga , Animais , Escherichia coli , Rim , Macrófagos , Camundongos , Infiltração de NeutrófilosRESUMO
The leukocyte-restricted tetraspanin CD53 has been shown to promote lymphocyte homing to lymph nodes (LNs) and myeloid cell recruitment to acutely inflamed peripheral organs, and accelerate the onset of immune-mediated disease. However, its contribution in the setting of chronic systemic autoimmunity has not been investigated. We made use of the Lyn-/- autoimmune model, generating Cd53-/- Lyn-/- mice, and compared trafficking of immune cells into secondary lymphoid organs and systemic autoimmune disease development with mice lacking either gene alone. Consistent with previous observations, absence of CD53 led to reduced LN cellularity via reductions in both B and T cells, a phenotype also observed in Cd53-/- Lyn-/- mice. In some settings, Cd53-/- Lyn-/- lymphocytes showed greater loss of surface L-selectin and CD69 upregulation above that imparted by Lyn deficiency alone, indicating that absence of these two proteins can mediate additive effects in the immune system. Conversely, prototypical effects of Lyn deficiency including splenomegaly, plasma cell expansion, elevated serum immunoglobulin M and anti-nuclear antibodies were unaffected by CD53 deficiency. Furthermore, while Lyn-/- mice developed glomerular injury and showed elevated glomerular neutrophil retention above than that in wild-type mice, absence of CD53 in Lyn-/- mice did not alter these responses. Together, these findings demonstrate that while tetraspanin CD53 promotes lymphocyte trafficking into LNs independent of Lyn, it does not make an important contribution to development of autoimmunity, plasma cell dysfunction or glomerular injury in the Lyn-/- model of systemic autoimmunity.
Assuntos
Autoimunidade , Ativação Linfocitária , Tetraspanina 25/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T , Quinases da Família src/genéticaRESUMO
Regulatory T cells (Tregs) play important roles in limiting inflammatory responses in the periphery. During these responses, Treg abundance in affected organs increases and interfering with their recruitment results in exacerbation of inflammation. However, the mechanisms whereby Tregs enter the skin remain poorly understood. The aim of this study was to use intravital microscopy to investigate adhesion and transmigration of Tregs in the dermal microvasculature in a two-challenge model of contact sensitivity. Using intravital confocal microscopy of Foxp3-GFP mice, we visualized endogenous Tregs and assessed their interactions in the dermal microvasculature. Four hours after hapten challenge, Tregs underwent adhesion with â¼25% of these cells proceeding to transmigration, a process dependent on CCR4. At 24 h, Tregs adhered but no longer underwent transmigration, instead remaining in prolonged contact with the endothelium, migrating over the endothelial surface. Four hours after a second challenge, Treg transmigration was restored, although in this case transmigration was CCR4 independent, instead involving the CCR6/CCL20 pathway. Notably, at 24 h but not 4 h after challenge, endothelial cells expressed MHC class II (MHC II). Moreover, at this time of peak MHC II expression, inhibition of MHC II reduced Treg adhesion, demonstrating an unexpected role for MHC II in Treg attachment to the endothelium. Together these data show that Treg adhesion and transmigration can be driven by different molecular mechanisms at different stages of an Ag-driven inflammatory response. In addition, Tregs can undergo prolonged migration on the inflamed endothelium.
Assuntos
Movimento Celular , Inflamação , Linfócitos T Reguladores/citologia , Animais , Inflamação/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores/imunologiaRESUMO
In addition to their roles in hemostasis and thrombosis, platelets are now recognized as making important contributions to a wide variety of inflammatory responses. This function primarily occurs as a result of intravascular interactions of platelets with leukocytes undergoing recruitment to the site of inflammation. As these interactions occur under the shear forces of flowing blood, they are typically rapid and highly dynamic. As such, the use of rapid frame-rate forms of in vivo microscopy, such as spinning-disk confocal intravital microscopy, have emerged as the optimal approaches for investigating these interactions and delineating their molecular basis and contribution to the inflammatory response. In this review, we provide an overview of the different methodologies employed to image platelet-leukocyte interactions in vivo, and examine the contributions of these interactions to inflammation that have been uncovered by intravital imaging.
Assuntos
Plaquetas/metabolismo , Microscopia Intravital/métodos , Leucócitos/metabolismo , Plaquetas/citologia , Humanos , Leucócitos/citologiaRESUMO
Platelet-leukocyte interactions promote acute glomerulonephritis. However, neither the nature of the interactions between platelets and immune cells nor the capacity of platelets to promote leukocyte activation has been characterized in this condition. We used confocal intravital microscopy to define the interactions of platelets with neutrophils, monocytes, and endothelial cells in glomerular capillaries in mice. In the absence of inflammation, platelets underwent rapid on/off interactions with immune cells. During glomerulonephritis induced by in situ immune complex formation, platelets that interacted with neutrophils or monocytes, but not with other intraglomerular cells, were retained in the glomerulus for prolonged durations. Depletion of platelets inhibited both neutrophil recruitment and activation. Inhibition of platelet activating factor reduced neutrophil recruitment without impacting reactive oxygen species generation, while blocking CXC chemokine ligand 7 (CXCL7) reduced both responses. In contrast, inhibition of the adenosine diphosphate and thromboxane A2 pathways inhibited neutrophil reactive oxygen species generation without affecting neutrophil adhesion. Thus, platelet retention in glomerular capillaries following immune complex deposition stems from prolongation of platelet interactions with immune cells but not other substrates. Pro-inflammatory mediators play divergent roles in promoting neutrophil retention and activation in glomerular capillaries.
Assuntos
Plaquetas/imunologia , Comunicação Celular/imunologia , Glomerulonefrite/imunologia , Glomérulos Renais/citologia , Neutrófilos/imunologia , Animais , Plaquetas/metabolismo , Capilares/citologia , Capilares/diagnóstico por imagem , Capilares/imunologia , Modelos Animais de Doenças , Glomerulonefrite/diagnóstico por imagem , Glomerulonefrite/patologia , Humanos , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Microscopia Intravital , Glomérulos Renais/irrigação sanguínea , Glomérulos Renais/imunologia , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Monócitos/imunologia , Infiltração de Neutrófilos/imunologia , Neutrófilos/metabolismoRESUMO
Memory regulatory T cells (mTregs) have been demonstrated to persist long-term in hosts after the resolution of primary influenza A virus (IAV) infection. However, whether such IAV infection-experienced (IAV-experienced) mTregs differentiate into a phenotypically and functionally distinct Treg subset and what function they play at the infection site remains poorly defined. In this study, we characterized the phenotype, examined the responsiveness and assessed the suppressive function of IAV-experienced memory Tregs (mTregs). In comparison with inexperienced naïve Tregs (nTregs), mTregs exhibited elevated expression of CD39, CD69, CD103, cytotoxic T lymphocyte-associated antigen-4, leukocyte function-associated antigen-1 and programmed cell death-1 and could be activated in an antigen-specific manner in vitro and in vivo. When mTregs and nTregs were adoptively cotransferred into recipient mice, mTregs had a competitive advantage in migrating to the IAV-infected lungs. mTregs were more capable of controlling in vitro proliferation of CD4+ and CD8+ T cells and suppressed CD40 and CD86 upregulation on bone marrow-derived dendritic cells. Adoptively transferred mTregs, but not adoptively transferred nTregs, significantly attenuated body weight loss, lung pathology and immune cell infiltration into the infected lungs after IAV infection. These results suggest that mTregs generated after IAV infection differentiate into a phenotypically distinct and functionally enhanced Treg subset that can be activated in an antigen-specific manner to exert immunosuppression. We propose vaccination to induce such mTregs as a potential novel strategy to protect against severe IAV infection.
Assuntos
Memória Imunológica , Vírus da Influenza A/imunologia , Pulmão/imunologia , Pulmão/virologia , Infecções por Orthomyxoviridae/imunologia , Linfócitos T Reguladores/imunologia , Transferência Adotiva , Animais , Movimento Celular/imunologia , Proliferação de Células , Células Dendríticas/imunologia , Regulação para Baixo , Feminino , Pulmão/patologia , Ativação Linfocitária/imunologia , Camundongos Endogâmicos C57BL , Monócitos/patologia , Infiltração de Neutrófilos , Infecções por Orthomyxoviridae/virologia , Fenótipo , Redução de PesoRESUMO
Nonclassical monocytes undergo intravascular patrolling in blood vessels, positioning them ideally to coordinate responses to inflammatory stimuli. Under some circumstances, the actions of monocytes have been shown to involve promotion of neutrophil recruitment. However, the mechanisms whereby patrolling monocytes control the actions of neutrophils in the circulation are unclear. Here, we examined the contributions of monocytes to antibody- and neutrophil-dependent inflammation in a model of in situ immune complex-mediated glomerulonephritis. Multiphoton and spinning disk confocal intravital microscopy revealed that monocytes patrol both uninflamed and inflamed glomeruli using ß2 and α4 integrins and CX3CR1. Monocyte depletion reduced glomerular injury, demonstrating that these cells promote inappropriate inflammation in this setting. Monocyte depletion also resulted in reductions in neutrophil recruitment and dwell time in glomerular capillaries and in reactive oxygen species (ROS) generation by neutrophils, suggesting a role for cross-talk between monocytes and neutrophils in induction of glomerulonephritis. Consistent with this hypothesis, patrolling monocytes and neutrophils underwent prolonged interactions in glomerular capillaries, with the duration of these interactions increasing during inflammation. Moreover, neutrophils that interacted with monocytes showed increased retention and a greater propensity for ROS generation in the glomerulus. Also, renal patrolling monocytes, but not neutrophils, produced TNF during inflammation, and TNF inhibition reduced neutrophil dwell time and ROS production, as well as renal injury. These findings show that monocytes and neutrophils undergo interactions within the glomerular microvasculature. Moreover, evidence indicates that, in response to an inflammatory stimulus, these interactions allow monocytes to promote neutrophil recruitment and activation within the glomerular microvasculature, leading to neutrophil-dependent tissue injury.
Assuntos
Glomerulonefrite/imunologia , Glomérulos Renais/imunologia , Monócitos/imunologia , Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Animais , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/imunologia , Receptor 1 de Quimiocina CX3C/metabolismo , Capilares/imunologia , Comunicação Celular/imunologia , Glomerulonefrite/metabolismo , Integrinas/imunologia , Integrinas/metabolismo , Glomérulos Renais/irrigação sanguínea , Glomérulos Renais/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Neutrófilos/metabolismo , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismoAssuntos
Insuficiência Renal Crônica , Trombopoetina , Humanos , Plaquetas , Contagem de Plaquetas , RimRESUMO
A medicinal chemistry program to develop potent and selective LABA compounds required the synthesis of both carbon-14 and stable-isotope labelled materials. Carbon-14 labelled AZD7307 was successfully synthesised in 6 steps from [14C]chloroacetyl chloride in an overall radiochemical yield of 10%. In addition, the synthetic route of a stable labelled isotopomer of AZD7307 is also described and synthesised in four linear steps from [13C6]cyclohexylamine hydrochloride in an overall yield of 12%.
Assuntos
Agonistas Adrenérgicos beta/química , Agonistas Adrenérgicos beta/síntese química , Radioisótopos de Carbono/química , Receptores Adrenérgicos beta 2/metabolismo , Técnicas de Química Sintética , Marcação por Isótopo , RadioquímicaRESUMO
Di-docosahexaenoyl (C22:6)-bis(monoacylglycerol) phosphate (BMP) has been identified as a promising biomarker for drug-induced phospholipidosis (DIPL). Both unlabelled and stable isotope labelled versions of BMP were desired for use as internal standards. Isopropylideneglycerol was converted to 4-methoxyphenyldiphenylmethyl-3-PMB-glycerol in three steps. Initially, the 2-postion of the glycerol was protected as a t-butyldiphenylsilyl ether, which proved to be a mistake; deprotection of the ether resulted in the decomposition of the compound. A switch to a t-butyldimethylsilyl ether protecting group resulted in an intermediate that could be deprotected to the alcohol to give the target compound after salt exchange. The same procedure was used to prepare [13 C6 ]BMP from [13 C3 ]glycerol.
Assuntos
Isótopos de Carbono/química , Doenças por Armazenamento dos Lisossomos/induzido quimicamente , Doenças por Armazenamento dos Lisossomos/metabolismo , Monoglicerídeos/química , Fosfatos/química , Fosfatos/síntese química , Fosfolipídeos/metabolismo , Técnicas de Química Sintética , Marcação por Isótopo , RadioquímicaRESUMO
The prospects for complement-targeted therapy in ANCA-associated vasculitis have been enhanced by a recent clinical trial in which C5a receptor 1 (C5aR1) inhibition safely replaced glucocorticoids in induction treatment. C5aR1 primes neutrophils for activation by anti-neutrophil cytoplasmic antibody (ANCA) and is therefore required in models of glomerulonephritis induced by anti-myeloperoxidase antibody. Although humoral and cellular autoimmunity play essential roles in ANCA-associated vasculitis, a role for C5aR1 in these responses has not been described. Here, we use murine models to dissect the role of C5aR1 in the generation of anti-myeloperoxidase autoimmunity and the effector responses resulting in renal injury. The genetic absence or pharmacological inhibition of C5aR1 results in reduced autoimmunity to myeloperoxidase with an attenuated Th1 response, increased Foxp3+ regulatory T cells and reduction in generation of myeloperoxidase-ANCA. These changes are mediated by C5aR1 on dendritic cells, which promotes activation, and thus myeloperoxidase autoimmunity and glomerulonephritis. We also use renal intravital microscopy to determine the effect of C5aR1 inhibition on ANCA induced neutrophil dysfunction. We found that myeloperoxidase-ANCA induce neutrophil retention and reactive oxygen species burst within glomerular capillaries. These pathological behaviors are abrogated by C5aR1 inhibition. Thus, C5aR1 inhibition ameliorates both autoimmunity and intra-renal neutrophil activation in ANCA-associated vasculitis.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Autoimunidade , Glomerulonefrite/imunologia , Glomérulos Renais/imunologia , Ativação de Neutrófilo , Neutrófilos/imunologia , Peroxidase/imunologia , Receptor da Anafilatoxina C5a/imunologia , Animais , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Glomerulonefrite/genética , Glomerulonefrite/metabolismo , Glomerulonefrite/patologia , Imunidade Celular , Imunidade Humoral , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/metabolismo , Peroxidase/genética , Peroxidase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/metabolismo , Explosão Respiratória , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Células Th1/imunologia , Células Th1/metabolismoRESUMO
Neutrophil extracellular traps (NETs) have been documented in glomeruli of patients with glomerulonephritis. However, the dynamics of NET formation in the glomerulus and their functional contribution to acute glomerular injury are poorly understood. Herein, we used in vivo multiphoton microscopy to investigate NET formation in the acutely inflamed glomerulus. Glomerular inflammation was induced using an antibody against the glomerular basement membrane. After induction of inflammation, multiphoton microscopy revealed that approximately 20% of glomeruli contained structures composed of extracellular DNA within the capillaries. These structures were not seen in mice depleted of neutrophils, consistent with them being NETs. Most contained myeloperoxidase, as seen in NETs in other tissues, whereas intraglomerular NETs did not contain significant levels of the histone H2Ax or neutrophil elastase. In vivo imaging revealed that intraglomerular NETs were present only transiently, suggesting that NETs were susceptible to disruption under the high shear conditions in glomerular capillaries. Investigation of NETs under flow conditions in vitro supported this concept. Dissolution of NETs via DNase I did not alter anti-glomerular basement membrane antibody-induced glomerular injury, as assessed via albuminuria, although the degree of microscopic hematuria was reduced by this intervention. These data indicate that in this model of acute, neutrophil-dependent glomerulonephritis, NETs are generated in the glomerular capillaries, where they are short lived and make a modest contribution to glomerular injury.