RESUMO
The migration of neutrophils from the blood circulation to sites of infection or injury is a key immune response and requires the breaching of endothelial cells (ECs) that line the inner aspect of blood vessels. Unregulated neutrophil transendothelial cell migration (TEM) is pathogenic, but the molecular basis of its physiological termination remains unknown. Here, we demonstrated that ECs of venules in inflamed tissues exhibited a robust autophagic response that was aligned temporally with the peak of neutrophil trafficking and was strictly localized to EC contacts. Genetic ablation of EC autophagy led to excessive neutrophil TEM and uncontrolled leukocyte migration in murine inflammatory models, while pharmacological induction of autophagy suppressed neutrophil infiltration into tissues. Mechanistically, autophagy regulated the remodeling of EC junctions and expression of key EC adhesion molecules, facilitating their intracellular trafficking and degradation. Collectively, we have identified autophagy as a modulator of EC leukocyte trafficking machinery aimed at terminating physiological inflammation.
Assuntos
Autofagia/fisiologia , Células Endoteliais/fisiologia , Infiltração de Neutrófilos/fisiologia , Migração Transendotelial e Transepitelial/fisiologia , Animais , Quimiotaxia de Leucócito/fisiologia , Células Endoteliais/patologia , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Junções Intercelulares/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/fisiologiaRESUMO
Aging is associated with dysregulated immune functions. Here, we investigated the impact of age on neutrophil diapedesis. Using confocal intravital microscopy, we found that in aged mice, neutrophils adhered to vascular endothelium in inflamed tissues but exhibited a high frequency of reverse transendothelial migration (rTEM). This retrograde breaching of the endothelium by neutrophils was governed by enhanced production of the chemokine CXCL1 from mast cells that localized at endothelial cell (EC) junctions. Increased EC expression of the atypical chemokine receptor 1 (ACKR1) supported this pro-inflammatory milieu in aged venules. Accumulation of CXCL1 caused desensitization of the chemokine receptor CXCR2 on neutrophils and loss of neutrophil directional motility within EC junctions. Fluorescent tracking revealed that in aged mice, neutrophils undergoing rTEM re-entered the circulation and disseminated to the lungs where they caused vascular leakage. Thus, neutrophils stemming from a local inflammatory site contribute to remote organ damage, with implication to the dysregulated systemic inflammation associated with aging.
Assuntos
Envelhecimento/imunologia , Transporte Biológico/imunologia , Inflamação/imunologia , Neutrófilos/imunologia , Animais , Quimiocina CXCL1/imunologia , Células Endoteliais/imunologia , Endotélio Vascular/imunologia , Feminino , Junções Intercelulares/imunologia , Pulmão/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Interleucina-8B/imunologia , Vênulas/imunologiaRESUMO
Neutrophils require directional cues to navigate through the complex structure of venular walls and into inflamed tissues. Here we applied confocal intravital microscopy to analyze neutrophil emigration in cytokine-stimulated mouse cremaster muscles. We identified differential and non-redundant roles for the chemokines CXCL1 and CXCL2, governed by their distinct cellular sources. CXCL1 was produced mainly by TNF-stimulated endothelial cells (ECs) and pericytes and supported luminal and sub-EC neutrophil crawling. Conversely, neutrophils were the main producers of CXCL2, and this chemokine was critical for correct breaching of endothelial junctions. This pro-migratory activity of CXCL2 depended on the atypical chemokine receptor 1 (ACKR1), which is enriched within endothelial junctions. Transmigrating neutrophils promoted a self-guided migration response through EC junctions, creating a junctional chemokine "depot" in the form of ACKR1-presented CXCL2 that enabled efficient unidirectional luminal-to-abluminal migration. Thus, CXCL1 and CXCL2 act in a sequential manner to guide neutrophils through venular walls as governed by their distinct cellular sources.
Assuntos
Quimiocina CXCL1 , Quimiocina CXCL2 , Sistema do Grupo Sanguíneo Duffy , Neutrófilos , Receptores de Superfície Celular , Migração Transendotelial e Transepitelial , Animais , Músculos Abdominais/efeitos dos fármacos , Músculos Abdominais/imunologia , Músculos Abdominais/metabolismo , Quimiocina CXCL1/genética , Quimiocina CXCL1/imunologia , Quimiocina CXCL1/metabolismo , Quimiocina CXCL2/genética , Quimiocina CXCL2/imunologia , Quimiocina CXCL2/metabolismo , Sistema do Grupo Sanguíneo Duffy/genética , Sistema do Grupo Sanguíneo Duffy/imunologia , Sistema do Grupo Sanguíneo Duffy/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Junções Intercelulares/efeitos dos fármacos , Junções Intercelulares/imunologia , Junções Intercelulares/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neutrófilos/citologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Migração Transendotelial e Transepitelial/efeitos dos fármacos , Migração Transendotelial e Transepitelial/genética , Migração Transendotelial e Transepitelial/imunologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Cellular senescence is a hallmark of advanced age and a major instigator of numerous inflammatory pathologies. While endothelial cell (EC) senescence is aligned with defective vascular functionality, its impact on fundamental inflammatory responses in vivo at single-cell level remain unclear. To directly investigate the role of EC senescence on dynamics of neutrophil-venular wall interactions, we applied high resolution confocal intravital microscopy to inflamed tissues of an EC-specific progeroid mouse model, characterized by profound indicators of EC senescence. Progerin-expressing ECs supported prolonged neutrophil adhesion and crawling in a cell autonomous manner that additionally mediated neutrophil-dependent microvascular leakage. Transcriptomic and immunofluorescence analysis of inflamed tissues identified elevated levels of EC CXCL1 on progerin-expressing ECs and functional blockade of CXCL1 suppressed the dysregulated neutrophil responses elicited by senescent ECs. Similarly, cultured progerin-expressing human ECs exhibited a senescent phenotype, were pro-inflammatory and prompted increased neutrophil attachment and activation. Collectively, our findings support the concept that senescent ECs drive excessive inflammation and provide new insights into the mode, dynamics, and mechanisms of this response at single-cell level.
RESUMO
Fibromyalgia is a debilitating widespread chronic pain syndrome that occurs in 2 to 4% of the population. The prevailing view that fibromyalgia results from central nervous system dysfunction has recently been challenged with data showing changes in peripheral nervous system activity. Using a mouse model of chronic widespread pain through hyperalgesic priming of muscle, we show that neutrophils invade sensory ganglia and confer mechanical hypersensitivity on recipient mice, while adoptive transfer of immunoglobulin, serum, lymphocytes, or monocytes has no effect on pain behavior. Neutrophil depletion abolishes the establishment of chronic widespread pain in mice. Neutrophils from patients with fibromyalgia also confer pain on mice. A link between neutrophil-derived mediators and peripheral nerve sensitization is already established. Our observations suggest approaches for targeting fibromyalgia pain via mechanisms that cause altered neutrophil activity and interactions with sensory neurons.
Assuntos
Dor Crônica , Fibromialgia , Humanos , Neutrófilos , Hiperalgesia , Gânglios SensitivosRESUMO
The migration of neutrophils into inflamed tissues is a fundamental component of innate immunity. A decisive step in this process is the polarized migration of blood neutrophils through endothelial cells (ECs) lining the venular lumen (transendothelial migration (TEM)) in a luminal-to-abluminal direction. By real-time confocal imaging, we found that neutrophils had disrupted polarized TEM ('hesitant' and 'reverse') in vivo. We noted these events in inflammation after ischemia-reperfusion injury, characterized by lower expression of junctional adhesion molecule C (JAM-C) at EC junctions, and they were enhanced by blockade or genetic deletion of JAM-C in ECs. Our results identify JAM-C as a key regulator of polarized neutrophil TEM in vivo and suggest that reverse TEM of neutrophils can contribute to the dissemination of systemic inflammation.
Assuntos
Moléculas de Adesão Celular/imunologia , Endotélio Vascular/imunologia , Imunoglobulinas/imunologia , Inflamação/imunologia , Neutrófilos/imunologia , Migração Transendotelial e Transepitelial/imunologia , Animais , Endotélio Vascular/citologia , Endotélio Vascular/patologia , Processamento de Imagem Assistida por Computador , Inflamação/patologia , Camundongos , Microscopia Confocal , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/patologiaRESUMO
Neutrophil trafficking is a key component of the inflammatory response. Here, we have investigated the role of the immunomodulatory lectin Galectin-9 (Gal-9) on neutrophil recruitment. Our data indicate that Gal-9 is upregulated in the inflamed vasculature of RA synovial biopsies and report the release of Gal-9 into the extracellular environment following endothelial cell activation. siRNA knockdown of endothelial Gal-9 resulted in reduced neutrophil adhesion and neutrophil recruitment was significantly reduced in Gal-9 knockout mice in a model of zymosan-induced peritonitis. We also provide evidence for Gal-9 binding sites on human neutrophils; Gal-9 binding induced neutrophil activation (increased expression of ß2 integrins and reduced expression of CD62L). Intra-vital microscopy confirmed a pro-recruitment role for Gal-9, with increased numbers of transmigrated neutrophils following Gal-9 administration. We studied the role of both soluble and immobilized Gal-9 on human neutrophil recruitment. Soluble Gal-9 significantly strengthened the interaction between neutrophils and the endothelium and inhibited neutrophil crawling on ICAM-1. When immobilized, Gal-9 functioned as an adhesion molecule and captured neutrophils from the flow. Neutrophils adherent to Gal-9 exhibited a spread/activated phenotype that was inhibited by CD18 and CD44 neutralizing antibodies, suggesting a role for these molecules in the pro-adhesive effects of Gal-9. Our data indicate that Gal-9 is expressed and released by the activated endothelium and functions both in soluble form and when immobilized as a neutrophil adhesion molecule. This study paves the way for further investigation of the role of Gal-9 in leukocyte recruitment in different inflammatory settings.
Assuntos
Antígenos CD18/metabolismo , Galectinas/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Receptores de Hialuronatos/metabolismo , Neutrófilos/metabolismo , Migração Transendotelial e Transepitelial , Animais , Adesão Celular , Humanos , CamundongosRESUMO
Recent advances have provided evidence for the involvement of neutrophils in both innate and adaptive immunity, robustly challenging the old dogma that neutrophils are short-lived prototypical innate immune cells solely involved in acute responses to microbes and exerting collateral tissue damage. There is now ample evidence showing that neutrophils can migrate into different compartments of the lymphoid system where they contribute to the orchestration of the activation and/or suppression of lymphocyte effector functions in homeostasis and during chronic inflammation, such as autoimmune disorders and cancer. In support of this notion, neutrophils can generate a wide range of cytokines and other mediators capable of regulating the survival, proliferation and functions of both T and B cells. In addition, neutrophils can directly engage with lymphocytes and promote antigen presentation. Furthermore, there is emerging evidence of the existence of distinct and diverse neutrophil phenotypes with immunomodulatory functions that characterise different pathological conditions, including chronic and autoimmune inflammatory conditions. The aim of this review is to discuss the mechanisms implicated in neutrophil trafficking into the lymphoid system and to provide an overview of the immuno-regulatory functions of neutrophils in health and disease in the context of adaptive immunity. Copyright © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Assuntos
Linfonodos/fisiologia , Neoplasias/imunologia , Neutrófilos/fisiologia , Animais , Doenças Autoimunes/imunologia , Modelos Animais de Doenças , Humanos , FenótipoRESUMO
Ischemia/reperfusion (I/R) injury is a severe inflammatory insult associated with numerous pathologies, such as myocardial infarction, stroke and acute kidney injury. I/R injury is characterized by a rapid influx of activated neutrophils secreting toxic free radical species and degrading enzymes that can irreversibly damage the tissue, thus impairing organ functions. Significant efforts have been invested in identifying therapeutic targets to suppress neutrophil recruitment and activation post-I/R injury. In this context, pharmacological targeting of neutrophil elastase (NE) has shown promising anti-inflammatory efficacy in a number of experimental and clinical settings of I/R injury and is considered a plausible clinical strategy for organ care. However, the mechanisms of action of NE, and hence its inhibitors, in this process are not fully understood. Here we conducted a comprehensive analysis of the impact of NE genetic deletion on neutrophil infiltration in four murine models of I/R injury as induced in the heart, kidneys, intestine and cremaster muscle. In all models, neutrophil migration into ischemic regions was significantly suppressed in NE-/- mice as compared with wild-type controls. Analysis of inflamed cremaster muscle and mesenteric microvessels by intravital and confocal microscopy revealed a selective entrapment of neutrophils within venular walls, most notably at the level of the venular basement membrane (BM) following NE deletion/pharmacological blockade. This effect was associated with the suppression of NE-mediated remodeling of the low matrix protein expressing regions within the venular BM used by transmigrating neutrophils as exit portals. Furthermore, whilst NE deficiency led to reduced neutrophil activation and vascular leakage, levels of monocytes and prohealing M2 macrophages were reduced in tissues of NE-/- mice subjected to I/R. Collectively our results identify a vital and non-redundant role for NE in supporting neutrophil breaching of the venular BM post-I/R injury but also suggest a protective role for NE in promoting tissue repair. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Assuntos
Elastase de Leucócito/fisiologia , Neutrófilos/fisiologia , Traumatismo por Reperfusão/enzimologia , Migração Transendotelial e Transepitelial/fisiologia , Remodelação Vascular/fisiologia , Animais , Membrana Basal/enzimologia , Membrana Basal/patologia , Membrana Basal/fisiopatologia , Modelos Animais de Doenças , Deleção de Genes , Rim/irrigação sanguínea , Rim/patologia , Elastase de Leucócito/deficiência , Elastase de Leucócito/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Infiltração de Neutrófilos/fisiologia , Neutrófilos/enzimologia , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Vênulas/enzimologia , Vênulas/patologia , Vênulas/fisiopatologiaRESUMO
Intracellular adhesion molecule-1 (ICAM-1) is a transmembrane glycoprotein expressed on the cell surface of numerous cell types such as endothelial and epithelial cells, vascular smooth muscle cells, and certain leukocyte subsets. With respect to the latter, ICAM-1 has been detected on neutrophils in several clinical and experimental settings, but little is known about the regulation of expression or function of neutrophil ICAM-1. In this study, we report on the de novo induction of ICAM-1 on the cell surface of murine neutrophils by lipopolysaccharide (LPS), tumor necrosis factor, and zymosan particles in vitro. The induction of neutrophil ICAM-1 was associated with enhanced phagocytosis of zymosan particles and reactive oxygen species (ROS) generation. Conversely, neutrophils from ICAM-1-deficient mice were defective in these effector functions. Mechanistically, ICAM-1-mediated intracellular signaling appeared to support neutrophil ROS generation and phagocytosis. In vivo, LPS-induced inflammation in the mouse cremaster muscle and peritoneal cavity led to ICAM-1 expression on intravascular and locally transmigrated neutrophils. The use of chimeric mice deficient in ICAM-1 on myeloid cells demonstrated that neutrophil ICAM-1 was not required for local neutrophil transmigration, but supported optimal intravascular and extravascular phagocytosis of zymosan particles. Collectively, the present results shed light on regulation of expression and function of ICAM-1 on neutrophils and identify it as an additional regulator of neutrophil effector responses in host defense.
Assuntos
Endotoxemia/induzido quimicamente , Endotoxemia/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Molécula 1 de Adesão Intercelular/biossíntese , Lipopolissacarídeos/toxicidade , Neutrófilos/metabolismo , Animais , Modelos Animais de Doenças , Endotoxemia/genética , Endotoxemia/patologia , Molécula 1 de Adesão Intercelular/genética , Camundongos , Camundongos Knockout , Neutrófilos/patologia , Fagocitose/efeitos dos fármacos , Fagocitose/genética , Espécies Reativas de Oxigênio/metabolismo , Migração Transendotelial e Transepitelial/efeitos dos fármacos , Migração Transendotelial e Transepitelial/genéticaRESUMO
The stereochemistry of all four stereoisomers of brivaracetam was determined using vibrational circular dichroism (VCD) spectroscopy. By comparing experimentally obtained VCD spectra and computationally simulated ones, the absolute configurations can be confidently assigned without prior knowledge of their relative stereochemistry. Neither the corrected mean absolute errors analysis of the nuclear magnetic resonance (NMR) data, nor the matching of experimental and calculated infrared spectra allowed the diastereoisomers to be distinguished. VCD spectroscopy itself suffices to establish the absolute configurations of all diastereoisomers. The relative stereochemistry could also be statistically confirmed by matching experimental and computed NMR spectra using the CP3 algorithm. The combination of VCD and NMR is recommended for molecules bearing more than one chiral center, as the relative configurations obtained from NMR serve as an independent check for those established with VCD. Analysis of the calculated VCD spectra reveals that the localized NH2 scissoring mode at around 1600 cm(-1) is characteristic for intramolecular hydrogen bonding, while the orientation of the ethyl group is reflected by the delocalized modes between 1150 and 1050 cm(-1).
Assuntos
Etano/química , Pirrolidinonas/química , Dicroísmo Circular , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Teoria Quântica , EstereoisomerismoRESUMO
Leukocyte extravasation is one of the essential and first steps during the initiation of inflammation. Therefore, a better understanding of the key molecules that regulate this process may help to develop novel therapeutics for treatment of inflammation-based diseases such as atherosclerosis or rheumatoid arthritis. The endothelial adhesion molecules ICAM-1 and VCAM-1 are known as the central mediators of leukocyte adhesion to and transmigration across the endothelium. Engagement of these molecules by their leukocyte integrin receptors initiates the activation of several signaling pathways within both leukocytes and endothelium. Several of such events have been described to occur during transendothelial migration of all leukocyte subsets, whereas other mechanisms are known only for a single leukocyte subset. Here, we summarize current knowledge on regulatory mechanisms of leukocyte extravasation from a leukocyte and endothelial point of view, respectively. Specifically, we will focus on highlighting common and unique mechanisms that specific leukocyte subsets exploit to succeed in crossing endothelial monolayers.
Assuntos
Leucócitos/fisiologia , Migração Transendotelial e Transepitelial/fisiologia , Animais , Adesão Celular/fisiologia , Moléculas de Adesão Celular/fisiologia , Humanos , Inflamação/etiologia , Inflamação/patologia , Inflamação/fisiopatologia , Molécula 1 de Adesão Intercelular/fisiologia , Leucócitos/classificação , Camundongos , Modelos Biológicos , Molécula 1 de Adesão de Célula Vascular/fisiologiaRESUMO
The dimethyl sulfoxide (DMSO) solubility data from Enamine and two UCB pharma compound collections were analyzed using 8 different machine learning methods and 12 descriptor sets. The analyzed data sets were highly imbalanced with 1.7-5.8% nonsoluble compounds. The libraries' enrichment by soluble molecules from the set of 10% of the most reliable predictions was used to compare prediction performances of the methods. The highest accuracies were calculated using a C4.5 decision classification tree, random forest, and associative neural networks. The performances of the methods developed were estimated on individual data sets and their combinations. The developed models provided on average a 2-fold decrease of the number of nonsoluble compounds amid all compounds predicted as soluble in DMSO. However, a 4-9-fold enrichment was observed if only 10% of the most reliable predictions were considered. The structural features influencing compounds to be soluble or nonsoluble in DMSO were also determined. The best models developed with the publicly available Enamine data set are freely available online at http://ochem.eu/article/33409 .
Assuntos
Inteligência Artificial , Bases de Dados de Produtos Farmacêuticos , Dimetil Sulfóxido/química , Informática/métodos , Modelos Lineares , Redes Neurais de Computação , Reprodutibilidade dos Testes , Solubilidade , Máquina de Vetores de SuporteRESUMO
The mechanism of leukocyte migration through venular walls in vivo is largely unknown. By using immunofluorescence staining and confocal microscopy, the present study demonstrates the existence of regions within the walls of unstimulated murine cremasteric venules where expression of key vascular basement membrane (BM) constituents, laminin 10, collagen IV, and nidogen-2 (but not perlecan) are considerably lower (<60%) than the average expression detected in the same vessel. These sites were closely associated with gaps between pericytes and were preferentially used by migrating neutrophils during their passage through cytokine-stimulated venules. Although neutrophil transmigration did not alter the number/unit area of extracellular matrix protein low expression sites, the size of these regions was enlarged and their protein content was reduced in interleukin-1beta-stimulated venules. These effects were entirely dependent on the presence of neutrophils and appeared to involve neutrophil-derived serine proteases. Furthermore, evidence was obtained indicating that transmigrating neutrophils carry laminins on their cell surface in vivo. Collectively, through identification of regions of low extracellular matrix protein localization that define the preferred route for transmigrating neutrophils, we have identified a plausible mechanism by which neutrophils penetrate the vascular BM without causing a gross disruption to its intricate structure.
Assuntos
Membrana Basal/fisiologia , Proteínas da Matriz Extracelular/genética , Neutrófilos/fisiologia , Vênulas/fisiologia , Animais , Movimento Celular , Primers do DNA , Regulação da Expressão Gênica , Leucócitos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/irrigação sanguínea , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
A multi-disciplinary approach was used to identify the first pharmacophore model for KCC2 blockers: several physico-chemical studies such as XRD and NMR were combined to molecular modelling techniques, SAR analysis and synthesis of constrained analogues in order to determine a minimal conformational space regrouping few potential bioactive conformations. These conformations were further compared to the conformational space of a different series of KCC2 blockers in order to identify the common pharmacophoric features. The synthesis of more potent analogues in this second series confirmed the usefulness of this KCC2 blocker pharmacophore model.
Assuntos
Anticonvulsivantes/síntese química , Bloqueadores dos Canais de Potássio/síntese química , Prolina/análogos & derivados , Prolina/síntese química , Simportadores/antagonistas & inibidores , Animais , Anticonvulsivantes/farmacologia , Linhagem Celular Tumoral , Furosemida/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Transporte de Íons/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Bloqueadores dos Canais de Potássio/farmacologia , Prolina/farmacologia , Ratos , Relação Estrutura-Atividade , Simportadores/metabolismo , Difração de Raios X , Cotransportadores de K e Cl-RESUMO
This paper describes the specific control strategy of the commercial manufacturing process of an immediate release tablet formulation based on continuous twin-screw wet granulation. This control strategy has been defined by a multidisciplinary team using an enhanced approach, in alignment with the quality by design principles. During process development, experiments have been performed according to multivariate designs first to identify critical material attributes and critical process parameters and then, to define process conditions generating a product having the required quality. Hence, controls have been applied on critical quality attributes and on related critical process parameters and critical material attributes. Due to the specificity of the process that combines batch and continuous unit operations, a specific control strategy has been designed to ensure intermediate and end product quality. Therefore, controls including soft sensor model and in process controls have been developed to continuously monitor granules residual moisture content, assay and dissolution as granules and tablets critical attributes. In addition, process analytical technology implementation enabled increased process understanding and provided support for the development of the control strategy. This study is therefore considered as a real industrial case study of control strategy definition and implementation for an intended commercial continuous manufacturing process.
Assuntos
Tecnologia Farmacêutica , Composição de Medicamentos , Tamanho da Partícula , ComprimidosRESUMO
Neutrophil diapedesis is an immediate step following infections and injury and is driven by complex interactions between leukocytes and various components of the blood vessel wall. Here, we show that perivascular mast cells (MC) are key regulators of neutrophil behaviour within the sub-endothelial space of inflamed venules. Using confocal intravital microscopy, we observe directed abluminal neutrophil motility along pericyte processes towards perivascular MCs, a response that created neutrophil extravasation hotspots. Conversely, MC-deficiency and pharmacological or genetic blockade of IL-17A leads to impaired neutrophil sub-endothelial migration and breaching of the pericyte layer. Mechanistically, identifying MCs as a significant cellular source of IL-17A, we establish that MC-derived IL-17A regulates the enrichment of key effector molecules ICAM-1 and CXCL1 in nearby pericytes. Collectively, we identify a novel MC-IL-17A-pericyte axis as modulator of the final steps of neutrophil diapedesis, with potential translational implications for inflammatory disorders driven by increased neutrophil diapedesis.
Assuntos
Neutrófilos , Migração Transendotelial e Transepitelial , Neutrófilos/fisiologia , Pericitos , Interleucina-17 , MastócitosRESUMO
The venular basement membrane plays a critical role in maintaining the integrity of blood vessels and through its dense and highly organized network of matrix proteins also acts as a formidable barrier to macromolecules and emigrating leukocytes. Leukocytes can however penetrate the venular basement membrane at sites of inflammation, though the associated in vivo mechanisms are poorly understood. Using whole mount immunostained tissues and confocal microscopy, we demonstrate that the venular basement membrane of multiple organs expresses regions of low matrix protein (laminin-511 and type IV collagen) deposition that have been termed low-expression regions (LERs). In the multiple tissues analyzed (eg, cremaster muscle, skin, mesenteric tissue), LERs were directly aligned with gaps between adjacent pericytes and were more prevalent in small venules. As predicted by their permissive nature, LERs acted as "gates" for transmigrating neutrophils in all inflammatory reactions investigated (elicited by leukotriene B(4) [LTB(4)], CXCL1, tumor necrosis factor [TNF]alpha, endotoxin, and ischemia/reperfusion [I/R] injury), and this response was associated with an enhancement of the size of laminin-511 and type IV collagen LERs. Transmigrated neutrophils stained positively for laminins but not type IV collagen, suggesting that different mechanisms exist in remodeling of different basement membrane networks. Collectively the findings provide further insight into characteristics of specialized regions within venular basement membranes that are preferentially used and remodeled by transmigrating neutrophils.
Assuntos
Membrana Basal/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Especificidade de Órgãos , Vênulas/metabolismo , Animais , Membrana Basal/efeitos dos fármacos , Capilares/efeitos dos fármacos , Capilares/metabolismo , Capilares/patologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Colágeno Tipo IV/metabolismo , Perfilação da Expressão Gênica , Laminina/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Modelos Biológicos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Especificidade de Órgãos/efeitos dos fármacos , Fatores de Tempo , Vênulas/efeitos dos fármacos , Vênulas/patologiaRESUMO
Leukocyte transmigration is mediated by endothelial cell (EC) junctional molecules, but the associated mechanisms remain unclear. Here we investigate how intercellular adhesion molecule-2 (ICAM-2), junctional adhesion molecule-A (JAM-A), and platelet endothelial cell adhesion molecule (PECAM-1) mediate neutrophil transmigration in a stimulus-dependent manner (eg, as induced by interleukin-1beta [IL-1beta] but not tumor necrosis factor-alpha [TNF-alpha]), and demonstrate their ability to act in sequence. Using a cell-transfer technique, transmigration responses of wild-type and TNF-alpha p55/p75 receptor-deficient leukocytes (TNFR(-/-)) through mouse cremasteric venules were quantified by fluorescence intravital microscopy. Whereas wild-type leukocytes showed a normal transmigration response to TNF-alpha in ICAM-2(-/-), JAM-A(-/-), and PECAM-1(-/-) recipient mice, TNFR(-/-) leukocytes exhibited a reduced transmigration response. Hence, when the ability of TNF-alpha to directly stimulate neutrophils is blocked, TNF-alpha-induced neutrophil transmigration is rendered dependent on ICAM-2, JAM-A, and PECAM-1, suggesting that the stimulus-dependent role of these molecules is governed by the target cell being activated. Furthermore, analysis of the site of arrest of neutrophils in inflamed tissues from ICAM-2(-/-), JAM-A(-/-), and PECAM-1(-/-) mice demonstrated that these molecules act sequentially to mediate transmigration. Collectively, the findings provide novel insights into the mechanisms of action of key molecules implicated in leukocyte transmigration.