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A hallmark feature of inflammation is the orchestrated recruitment of neutrophils from the bloodstream into inflamed tissue. Although selectins and integrins mediate recruitment in many tissues, they have a minimal role in the lungs and liver. Exploiting an unbiased in vivo functional screen, we identified a lung and liver homing peptide that functionally abrogates neutrophil recruitment to these organs. Using biochemical, genetic, and confocal intravital imaging approaches, we identified dipeptidase-1 (DPEP1) as the target and established its role as a physical adhesion receptor for neutrophil sequestration independent of its enzymatic activity. Importantly, genetic ablation or functional peptide blocking of DPEP1 significantly reduced neutrophil recruitment to the lungs and liver and provided improved survival in models of endotoxemia. Our data establish DPEP1 as a major adhesion receptor on the lung and liver endothelium and identify a therapeutic target for neutrophil-driven inflammatory diseases of the lungs.
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Dipeptidasas/metabolismo , Neutrófilos/fisiología , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Animales , Cilastatina/farmacología , Cilastatina/uso terapéutico , Dipeptidasas/antagonistas & inhibidores , Dipeptidasas/genética , Modelos Animales de Enfermedad , Endotoxemia/mortalidad , Endotoxemia/patología , Endotoxemia/prevención & control , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Humanos , Lipopolisacáridos/farmacología , Hígado/efectos de los fármacos , Hígado/inmunología , Hígado/metabolismo , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Infiltración Neutrófila/efectos de los fármacos , Péptidos/síntesis química , Péptidos/química , Péptidos/farmacología , Tasa de SupervivenciaRESUMEN
The Canadian Society for Immunology (CSI) established a formal Equity, Diversity, and Inclusion (EDI) Committee with the goal of providing EDI advocacy and leadership within the CSI, as well as in the broader scientific community. A first task of this committee was to review the publicly available historical data on gender representation within the CSI's membership, leadership, award recipients, and conference chairs/presenters as a step in establishing a baseline reference point and monitoring the trajectory of future success in achieving true inclusion. We found that, except for overall membership and a specific subset of awards, all categories showed a historical bias toward men, particularly prior to 2010. Bias persists in various categories, evident even in recent years. However, we note an encouraging trend toward greater gender parity, particularly in the roles of President, symposium presenters, and workshop chairs, especially from 2017 onward. We present these findings as well as our recommendations to enhance inclusivity. These include a more comprehensive collection and secure storage of self-identification data, emphasis on EDI as an essential component of all annual meeting activities, and innovative measures of outreach, collaboration, and leadership with the aim of making the CSI a model for improving EDI in other professional research societies.
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Distinciones y Premios , Liderazgo , Femenino , Humanos , Masculino , Canadá , Estudios Retrospectivos , Sociedades MédicasRESUMEN
Reported herein is the synthesis of sialyl LewisX analogues bearing a trans-bicyclo[4.4.0] dioxadecane-modified 3- O,4- C-fused galactopyranoside scaffold that locks the carboxylate pharmacophore in either the axial or equatorial position. This novel series of bicyclic galactopyranosides are prepared through a stereocontrolled intramolecular cyclization reaction that has been evaluated both experimentally and by density functional theory calculations. The cyclization precursors are obtained from ß-d-galactose pentaacetate in a nine-step sequence featuring a highly diastereoselective equatorial alkynylation and Cu(I) catalyzed formation of the acetylenic α-ketoester moiety. Preliminary biological evaluations indicate improved activity as P-selectin antagonists for the axially configured analogues as compared to their equatorial counterparts.
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Compuestos Bicíclicos Heterocíclicos con Puentes/química , Galactosa/química , Antígeno Sialil Lewis X/química , Estructura MolecularRESUMEN
Focal adhesion kinase (FAK)-related nonkinase (PTK2 isoform 6 in humans, hereafter referred to as FRNK) is a cytoskeletal regulatory protein that has recently been shown to dampen lung fibrosis, yet its role in inflammation is unknown. Here, we show for the first time that expression of FRNK negatively regulates IL-4-mediated inflammation in a human model of eosinophil recruitment. Mechanistically, FRNK blocks eosinophil accumulation, firm adhesion and transmigration by preventing transcription and protein expression of VCAM-1 and CCL26. IL-4 activates STAT6 to induce VCAM-1 and CCL26 transcription. We now show that IL-4 also increases GATA6 to induce VCAM-1 expression. FRNK blocks IL-4-induced GATA6 transcription but has little effect on GATA6 protein expression and no effect on STAT6 activation. FRNK can block FAK or Pyk2 signaling and we, thus, downregulated these proteins using siRNA to determine whether signaling from either protein is involved in the regulation of VCAM-1 and CCL26. Knockdown of FAK, Pyk2 or both had no effect on VCAM-1 or CCL26 expression, which suggests that FRNK acts independently of FAK and Pyk2 signaling. Finally, we found that IL-4 induces the late expression of endogenous FRNK. In summary, FRNK represents a novel mechanism to negatively regulate IL-4-mediated inflammation.
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Mediadores de Inflamación/metabolismo , Inflamación/inmunología , Interleucina-4/inmunología , Proteínas Tirosina Quinasas/metabolismo , Adhesión Celular/inmunología , Movimiento Celular/inmunología , Células Cultivadas , Quimiocina CCL26 , Quimiocinas CC/biosíntesis , Activación Enzimática , Eosinófilos/inmunología , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Quinasa 2 de Adhesión Focal/genética , Quinasa 2 de Adhesión Focal/metabolismo , Factor de Transcripción GATA6/biosíntesis , Factor de Transcripción GATA6/genética , Expresión Génica/genética , Células Endoteliales de la Vena Umbilical Humana , Humanos , Proteínas Tirosina Quinasas/genética , Interferencia de ARN , ARN Interferente Pequeño , Factor de Transcripción STAT6/metabolismo , Transducción de Señal/inmunología , Transcripción Genética/genética , Molécula 1 de Adhesión Celular Vascular/biosíntesisRESUMEN
It has been known for many years that neutrophils and platelets participate in the pathogenesis of severe sepsis, but the inter-relationship between these players is completely unknown. We report several cellular events that led to enhanced trapping of bacteria in blood vessels: platelet TLR4 detected TLR4 ligands in blood and induced platelet binding to adherent neutrophils. This led to robust neutrophil activation and formation of neutrophil extracellular traps (NETs). Plasma from severely septic humans also induced TLR4-dependent platelet-neutrophil interactions, leading to the production of NETs. The NETs retained their integrity under flow conditions and ensnared bacteria within the vasculature. The entire event occurred primarily in the liver sinusoids and pulmonary capillaries, where NETs have the greatest capacity for bacterial trapping. We propose that platelet TLR4 is a threshold switch for this new bacterial trapping mechanism in severe sepsis.
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Bacterias/inmunología , Plaquetas/inmunología , Neutrófilos/inmunología , Sepsis/microbiología , Sepsis/fisiopatología , Receptor Toll-Like 4/metabolismo , Alanina Transaminasa/sangre , Animales , Epitelio/patología , Humanos , Lipopolisacáridos/metabolismo , Hígado/metabolismo , Ratones , Neutrófilos/enzimología , Sepsis/inmunologíaRESUMEN
[This corrects the article DOI: 10.3389/fimmu.2022.1020056.].
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Although microtubules have long been implicated in cell locomotion, the mechanism of their involvement remains controversial. Most studies have concluded that microtubules play a positive role by regulating actin polymerization, transporting membrane vesicles to the leading edge, and/or facilitating the turnover of adhesion plaques. Here we used wild-type and mutant CHO cell lines with alterations in tubulin to demonstrate that microtubules can also act to restrain cell motility. Tubulin mutations or low concentrations of drugs that suppress microtubule dynamics without affecting the amount of microtubule polymer inhibited the rate of migration by preventing microtubule reorganization in the trailing portion of the cells where the more dynamic microtubules are normally found. Under these conditions, cells along the edge of a wound still extended lamellipodia and elongated toward the wound but were inhibited in their ability to retract their tails, thus retarding forward progress. The idea that microtubules normally act to restrain cell locomotion was confirmed by treating cells with high concentrations of nocodazole to depolymerize the microtubule network. In the absence of microtubules, wild-type CHO and HeLa cells could still move at near normal speeds, but the movement became more random. We conclude that microtubules act both to restrain cell movement and to establish directionality.
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Movimiento Celular/fisiología , Microtúbulos/metabolismo , Seudópodos/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Células CHO , Movimiento Celular/efectos de los fármacos , Cricetinae , Cricetulus , Microtúbulos/genética , Nocodazol/farmacología , Seudópodos/genética , Tubulina (Proteína)/genética , Moduladores de Tubulina/farmacologíaRESUMEN
During an inflammatory response, endothelial cells undergo morphological changes to allow for the passage of neutrophils from the blood vessel to the site of injury or infection. Although endothelial cell junctions and the cytoskeleton undergo reorganization during inflammation, little is known about another class of cellular structures, the focal adhesions. In this study, we examined several focal adhesion proteins during an inflammatory response. We found that there was selective loss of paxillin and focal adhesion kinase (FAK) from focal adhesions in proximity to transmigrating neutrophils; in contrast the levels of the focal adhesion proteins ß1-integrin and vinculin were unaffected. Paxillin was lost from focal adhesions during neutrophil transmigration both under static and flow conditions. Down-regulating endothelial paxillin with siRNA blocked neutrophil transmigration while having no effect on rolling or adhesion. As paxillin dynamics are regulated partly by FAK, the role of FAK in neutrophil transmigration was examined using two complementary methods. siRNA was used to down-regulate total FAK protein while dominant-negative, kinase-deficient FAK was expressed to block FAK signaling. Disruption of the FAK protein or FAK signaling decreased neutrophil transmigration. Collectively, these findings reveal a novel role for endothelial focal adhesion proteins paxillin and FAK in regulating neutrophil transmigration.
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Endotelio/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Neutrófilos/metabolismo , Paxillin/metabolismo , Migración Transendotelial y Transepitelial/inmunología , Adhesión Celular/genética , Células Cultivadas , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Endotelio/inmunología , Endotelio/patología , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/inmunología , Adhesiones Focales/patología , Humanos , Inflamación , Rodamiento de Leucocito/genética , Mutación/genética , Neutrófilos/inmunología , Neutrófilos/patología , Paxillin/genética , Paxillin/inmunología , ARN Interferente Pequeño/genética , Migración Transendotelial y Transepitelial/genética , Transgenes/genéticaRESUMEN
The endothelium actively participates in neutrophil migration out of the vasculature via dynamic, cytoskeleton-dependent rearrangements leading to the formation of transmigratory cups in vitro, and to domes that completely surround the leukocyte in vivo. Leukocyte-specific protein 1 (LSP1), an F-actin-binding protein recently shown to be in the endothelium, is critical for effective transmigration, although the mechanism has remained elusive. Herein we show that endothelial LSP1 is expressed in the nucleus and cytosol of resting endothelial cells and associates with the cytoskeleton upon endothelial activation. Two-photon microscopy revealed that endothelial LSP1 was crucial for the formation of endothelial domes in vivo in response to neutrophil chemokine keratinocyte-derived chemokine (KC) as well as in response to endogenously produced chemokines stimulated by cytokines (tumor necrosis factor α [TNFα] or interleukin-1ß [IL-1ß]). Endothelial domes were significantly reduced in Lsp1(-/-) compared with wild-type (WT) mice. Lsp1(-/-) animals not only showed impaired neutrophil emigration after KC and TNFα stimulation, but also had disproportionate increases in vascular permeability. We demonstrate that endothelial LSP1 is recruited to the cytoskeleton in inflammation and plays an important role in forming endothelial domes thereby regulating neutrophil transendothelial migration. The permeability data may underscore the physiologic relevance of domes and the role for LSP1 in endothelial barrier integrity.
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Proteínas de Unión al Calcio/fisiología , Permeabilidad Capilar/fisiología , Endotelio/metabolismo , Neutrófilos/fisiología , Migración Transendotelial y Transepitelial , Animales , Western Blotting , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Permeabilidad Capilar/efectos de los fármacos , Células Cultivadas , Citoesqueleto/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Endotelio/citología , Endotelio/efectos de los fármacos , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos , Microscopía Confocal/instrumentación , Microscopía Electrónica de Transmisión , Microscopía de Fluorescencia por Excitación Multifotónica/instrumentación , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/efectos de los fármacos , Neutrófilos/citología , Neutrófilos/ultraestructura , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) and other respiratory pathologies in vivo is difficult due to the limited accessibility and inherent motion artifacts of the lungs. Nonetheless, various approaches have been developed to overcome these challenges. This protocol describes a method for intravital fluorescence microscopy to study real-time leukocyte-endothelial interactions in the pulmonary microcirculation in an experimental model of ALI. An in vivo lung imaging system and 3-D printed intravital microscopy platform are used to secure the anesthetized mouse and stabilize the lung while minimizing confounding lung injury. Following preparation, widefield fluorescence microscopy is used to study leukocyte adhesion, leukocyte rolling, and capillary function. While the protocol presented here focuses on imaging in an acute model of inflammatory lung disease, it may also be adapted to study other pathological and physiological processes in the lung.
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Lesión Pulmonar Aguda , Síndrome de Dificultad Respiratoria , Lesión Pulmonar Aguda/patología , Animales , Pulmón/patología , Ratones , Microcirculación/fisiología , Microscopía Fluorescente , Síndrome de Dificultad Respiratoria/diagnóstico por imagen , VacioRESUMEN
Introduction: Intestinal roundworms cause chronic debilitating disease in animals, including humans. Traditional experimental models of these types of infection use a large single-dose infection. However, in natural settings, hosts are exposed to parasites on a regular basis and when mice are exposed to frequent, smaller doses of Heligmosomoides polygyrus, the parasites are cleared more quickly. Whether this more effective host response has any negative consequences for the host is not known. Results: Using a trickle model of infection, we found that worm clearance was associated with known resistance-related host responses: increased granuloma and tuft cell numbers, increased levels of granuloma IgG and decreased intestinal transit time, as well as higher serum IgE levels. However, we found that the improved worm clearance was also associated with an inflammatory phenotype in and around the granuloma, increased smooth muscle hypertrophy/hyperplasia, and elevated levels of Adamts gene expression. Discussion: To our knowledge, we are the first to identify the involvement of this protein family of matrix metalloproteinases (MMPs) in host responses to helminth infections. Our results highlight the delicate balance between parasite clearance and host tissue damage, which both contribute to host pathology. When continually exposed to parasitic worms, improved clearance comes at a cost.
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Nematospiroides dubius , Humanos , Ratones , Animales , Cicatriz , Inmunidad , Granuloma , InflamaciónRESUMEN
Leukocyte transmigration can be affected by shear stress; however, the mechanisms by which shear stress modulates transmigration are unknown. We found that adhesion of eosinophils or an eosinophilic cell line to intereukin 4-stimulated endothelial cells led to a shear-dependent increase in endothelial cell intracellular calcium and increased phosphorylation of extracellular signal-regulated kinase (ERK) 2, but not c-Jun NH2-terminal kinase or p38 mitogen-activated protein kinase. Latex beads coated with antibodies were used to characterize the role of specific endothelial cell surface molecules in initiating signaling under shear conditions. We found that ligation of either vascular cell adhesion molecule-1 or E-selectin, but not major histocompatibility complex class I, induced a shear-dependent increase in ERK2 phosphorylation in cytokine-stimulated endothelial cells. Disassembly of the actin cytoskeleton with latrunculin A prevented ERK2 phosphorylation after adhesion under flow conditions, supporting a role for the cytoskeleton in mechano-sensing. Rapid phosphorylation of focal adhesion kinase and paxillin occurred under identical conditions, suggesting that focal adhesions were also involved in mechanotransduction. Finally, we found that Rho-associated protein kinase and calpain were both critical in the subsequent transendothelial migration of eosinophils under flow conditions. These data suggest that ligation of leukocyte adhesion molecules under flow conditions leads to mechanotransduction in endothelial cells, which can regulate subsequent leukocyte trafficking.
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Movimiento Celular/inmunología , Células Endoteliales/fisiología , Eosinófilos/fisiología , Mecanotransducción Celular/inmunología , Actinas/metabolismo , Adulto , Calcio/fisiología , Calpaína/fisiología , Adhesión Celular/inmunología , Línea Celular , Movimiento Celular/fisiología , Citoesqueleto/metabolismo , Selectina E/metabolismo , Células Endoteliales/enzimología , Endotelio Vascular/citología , Endotelio Vascular/enzimología , Endotelio Vascular/inmunología , Humanos , Interleucina-4/fisiología , Péptidos y Proteínas de Señalización Intracelular , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Tirosina Quinasas/fisiología , Molécula 1 de Adhesión Celular Vascular/metabolismo , Quinasas Asociadas a rhoRESUMEN
Eotaxin-3/CCL26 is an agonist for chemokine receptor 3 (CCR3) and a natural antagonist for CCR1, CCR2 and CCR5. CCL26 expression by non-haematopoietic cells has been well documented; however, no studies to date have demonstrated CCL26 expression by leucocytes. In this study, we investigated the ability of human monocytic cells to produce CCL26 in response to cytokines. We found that interleukin-4 (IL-4) increased the expression of CCL26 messenger RNA (mRNA) and protein in U937 cells, in human monocytes and in human monocyte-derived macrophages. Tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) alone did not induce CCL26 expression, yet these pro-inflammatory cytokines synergized with IL-4 to increase CCL26 protein expression. Signal transducer and activator of transcription 6 (STAT6) was not affected by costimulation with TNF-alpha, suggesting that the synergy between IL-4 and TNF-alpha occurs at a step downstream of STAT6 activation. Co-incubation of interferon-gamma (IFN-gamma) with IL-4 had no effect on CCL26 protein release. By contrast, pretreatment with IFN-gamma decreased total STAT6 protein, blocked IL-4-mediated STAT6 phosphorylation and decreased IL-4-mediated CCL26 mRNA expression and protein release. These data show that IL-4 and pro-inflammatory cytokines such as TNF-alpha, IL-1beta and IFN-gamma regulate CCL26 synthesis in human monocytic cells, which may be important in regulating monocyte inflammatory responses.
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Quimiocinas CC/biosíntesis , Monocitos/metabolismo , Transducción de Señal/inmunología , Western Blotting , Quimiocina CCL26 , Quimiocinas CC/inmunología , Ensayo de Inmunoadsorción Enzimática , Expresión Génica , Regulación de la Expresión Génica , Humanos , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucina-1beta/inmunología , Interleucina-1beta/metabolismo , Interleucina-4/inmunología , Interleucina-4/metabolismo , Monocitos/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Células U937RESUMEN
Eosinophils are traditionally associated with allergic and parasitic inflammation. More recently, eosinophils have also been shown to have roles in diverse processes including development, intestinal health, thymic selection, and B-cell survival with the majority of these insights being derived from murine models and in vitro assays. Despite this, tools to measure the dynamic activity of eosinophils in situ have been lacking. Intravital microscopy is a powerful tool that enables direct visualization of leukocytes and their dynamic behavior in real-time in a wide range of processes in both health and disease. Until recently eosinophil researchers have not been able to take full advantage of this technology due to a lack of tools such as genetically encoded reporter mice. This mini-review examines the history of intravital microscopy with a focus on eosinophils. The development and use of eosinophil-specific Cre (EoCre) mice to create GFP and tdTomato fluorescent reporter animals is also described. Genetically encoded eosinophil reporter mice combined with intravital microscopy provide a powerful tool to add to the toolbox of technologies that will help us unravel the mysteries still surrounding this cell.
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Eosinófilos/citología , Microscopía Intravital , Animales , Ciego/citología , Colorantes Fluorescentes/metabolismo , Genes Reporteros , Intestino Delgado/citología , Pulmón/citología , Ganglios Linfáticos/citología , Ratones Endogámicos C57BL , Músculos/citologíaRESUMEN
The COVID-19 pandemic is shining a spotlight on the field of immunology like never before. To appreciate the diverse ways in which immunologists have contributed, Nature Reviews Immunology invited the president of the International Union of Immunological Societies and the presidents of 15 other national immunology societies to discuss how they and their members responded following the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
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COVID-19/epidemiología , Infecciones por Coronavirus/epidemiología , Cooperación Internacional , Pandemias , Neumonía Viral/epidemiología , Síndrome Respiratorio Agudo Grave/epidemiología , Sociedades Científicas/organización & administración , Antivirales/síntesis química , Antivirales/uso terapéutico , Betacoronavirus/efectos de los fármacos , Betacoronavirus/inmunología , Betacoronavirus/patogenicidad , COVID-19/inmunología , COVID-19/prevención & control , COVID-19/terapia , Vacunas contra la COVID-19 , Relaciones Comunidad-Institución , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/terapia , Salud Global/tendencias , Humanos , Educación del Paciente como Asunto/organización & administración , Equipo de Protección Personal/provisión & distribución , Neumonía Viral/inmunología , Neumonía Viral/terapia , SARS-CoV-2 , Síndrome Respiratorio Agudo Grave/inmunología , Síndrome Respiratorio Agudo Grave/terapia , Vacunas Virales/biosíntesisRESUMEN
The endocannabinoid (EC) system mediates protection against intestinal inflammation. In this study, we investigated the effects of blocking EC degradation or cellular reuptake in experimental colitis in mice. Mice were treated with trinitrobenzene-sulfonic acid in presence and absence of the fatty acid amide hydrolase (FAAH) blocker URB597, the EC membrane transport inhibitor VDM11, and combinations of both. Inflammation was significantly reduced in the presence of URB597, VDM11, or both as evaluated by macroscopic damage score, myeloperoxidase levels, and colon length. These effects were abolished in CB(1)- and CB(2)-receptor-gene-deficient mice. Quantitative reverse transcription polymerase chain reaction after induction of experimental colitis by different pathways showed that expression of FAAH messenger RNA (mRNA) is significantly reduced in different models of inflammation early in the expression of colitis, and these return to control levels as the disease progresses. Genomic DNA from 202 patients with Crohn's disease (CD) and 206 healthy controls was analyzed for the C385A polymorphism in the FAAH gene to address a possible role in humans. In our groups, the C385A polymorphism was equally distributed in patients with CD and healthy controls. In conclusion, drugs targeting EC degradation offer therapeutic potential in the treatment of inflammatory bowel diseases. Furthermore, reduction of FAAH mRNA expression is involved in the pathophysiological response to colitis.
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Moduladores de Receptores de Cannabinoides/metabolismo , Colitis/tratamiento farmacológico , Endocannabinoides , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/metabolismo , Amidohidrolasas/antagonistas & inhibidores , Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Animales , Ácidos Araquidónicos/uso terapéutico , Benzamidas/uso terapéutico , Carbamatos/uso terapéutico , Colitis/metabolismo , Enfermedad de Crohn/genética , Enfermedad de Crohn/metabolismo , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , Polimorfismo Genético , ARN Mensajero/metabolismo , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB2/genética , Ácido Trinitrobencenosulfónico/uso terapéuticoRESUMEN
Eosinophils are core components of the immune system, yet tools are lacking to directly observe eosinophils in action in vivo. To better understand the role of tissue resident eosinophils, we used eosinophil-specific CRE (eoCRE) mice to create GFP and tdTomato reporters. We then employed intravital microscopy to examine the dynamic behaviour of eosinophils in the healthy GI tract, mesentery, liver, lymph node, skin and lung. Given the role of eosinophils in allergic airway diseases, we also examined eosinophils in the lung following ovalbumin sensitization and challenge. We were able to monitor and quantify eosinophilic behaviours including patrolling, crawling, clustering, tissue distribution and interactions with other leukocytes. Thus, these reporter mice allow eosinophils to be examined in real-time in living animals, paving the way to further understanding the roles eosinophils play in both health and disease.
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Eosinófilos/inmunología , Animales , Eosinófilos/citología , Eosinófilos/fisiología , Femenino , Tracto Gastrointestinal/citología , Tracto Gastrointestinal/inmunología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Intravital/métodos , Pulmón/citología , Pulmón/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Especificidad de Órganos , Ovalbúmina/inmunología , Neumonía/inmunología , Neumonía/patologíaRESUMEN
Leukocyte recruitment plays a critical role during both normal inflammation and chronic inflammatory diseases, and ongoing studies endeavor to better understand the complexities of this process. Focal adhesion kinase (FAK) is well known for its role in cancer, yet it also has been shown to regulate aspects of neutrophil and B16 melanoma cell recruitment by rapidly influencing endothelial cell focal adhesion dynamics and junctional opening. Recently, we found that FAK related non-kinase (FRNK), a protein that is often used as a FAK dominant negative, blocked eosinophil transmigration by preventing the transcription of vascular cell adhesion molecule-1 (VCAM-1) and eotaxin-3 (CCL26). Surprisingly, the blocking occurred even in the absence of endogenous FAK. To better understand the role of FAK in leukocyte recruitment, we used a FAK-specific inhibitor (PF-573228) and determined the effect on IL-4 induced eosinophil recruitment in vitro and in vivo. PF-573228 prevented the expression of VCAM-1 and CCL26 expression in IL-4-stimulated human endothelial cells in vitro. As a result, eosinophil adhesion and transmigration were blocked. PF-572338 also prevented IL-4-induced VCAM-1 expression in vivo. Using brightfield intravital microscopy, we found that PF-573228 decreased leukocyte rolling flux, adhesion, and emigration. We specifically examined eosinophil recruitment in vivo by using an eosinophil-GFP reporter mouse and found PF-573228 attenuated eosinophil emigration. This study reveals that a FAK inhibitor influences inflammation through its action on eosinophil recruitment.
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Quimiotaxis de Leucocito/fisiología , Eosinófilos/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/antagonistas & inhibidores , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Animales , Células Cultivadas , Quimiotaxis de Leucocito/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Quinolonas/farmacología , Sulfonas/farmacologíaRESUMEN
Matrix metalloproteinase-9 (MMP-9) is present in the tertiary granules of neutrophils and is rapidly released following stimulation. We examined the pathways that regulate tumor necrosis factor (TNF)-mediated MMP-9 release and found this to be dependent on the TNF receptor I. TNF rapidly activated extracellular signal-regulated kinase and p38 mitogen-activated protein kinases, but neither of these pathways was critical for MMP-9 release. Many neutrophil responses to TNF require beta2-integrin-dependent signaling and subsequent Src family kinase activation. In contrast, we found that MMP-9 release from tertiary granules was only partially affected by blocking beta2-integrin-mediated adhesion. Similarly, blocking Src family kinases with the inhibitor PP2 only attenuated TNF-induced MMP-9 release. Blocking beta2-integrin-mediated adhesion and Src family kinases did not result in additive inhibition of MMP-9 release. In contrast, inhibiting protein kinase C (PKC) with a pan-specific inhibitor blocked greater than 85% of MMP-9 release. Inhibitors against specific PKC isoforms suggested a role for PKC alpha and PKC delta in maximal MMP-9 release. These data suggest that MMP-9 release from tertiary granules uses beta2-integrin-independent signaling pathways. Furthermore, PKC isoforms play a critical role in regulating tertiary granule release.