RESUMEN
Immunoglobulin A (IgA) is generally considered as a non-inflammatory regulator of mucosal immunity, and its importance in diversifying the gut microbiota is increasingly appreciated. IgA autoantibodies have been found in several autoimmune or chronic inflammatory diseases, but their role in pathophysiology is ill-understood. IgA can interact with the Fc receptor FcαRI on immune cells. We now established a novel IgA autoimmune blistering model, which closely resembles the human disease linear IgA bullous disease (LABD) by using genetically modified mice that produce human IgA and express human FcαRI. Intravital microscopy demonstrated that presence of IgA anti-collagen XVII, - the auto-antigen in LABD-, resulted in neutrophil activation and extravasation from blood vessels into skin tissue. Continued exposure to anti-collagen XVII IgA led to massive neutrophil accumulation, severe tissue damage and blister formation. Importantly, treatment with anti-FcαRI monoclonal antibodies not only prevented disease, but was also able to resolve existing inflammation and tissue damage. Collectively, our data reveal a novel role of neutrophil FcαRI in IgA autoantibody-mediated disease and identify FcαRI as promising new therapeutic target to resolve chronic inflammation and tissue damage.
Asunto(s)
Inmunoglobulina A , Receptores Fc , Animales , Anticuerpos Monoclonales/uso terapéutico , Autoanticuerpos , Inflamación/tratamiento farmacológico , RatonesRESUMEN
In epithelial cancers, the epidermal growth factor receptor (EGFR) and integrin α6ß4 are frequently overexpressed and found to synergistically activate intracellular signaling pathways that promote cell proliferation and migration. In cancer cells, the ß4 subunit is phosphorylated at tyrosine residues not normally recognized as kinase substrates; however, the function of these phosphotyrosine residues in cancer cells is a subject of much debate. In EGFR-overexpressing carcinoma cells, we found that the Src family kinase (SFK) inhibitor PP2 reduces ß4 tyrosine phosphorylation following the activation of EGFR. However, siRNA mediated knockdown of the SFKs Src, Fyn, Yes and Lyn, individually or in combination, did not affect the EGF-induced phosphorylation of ß4. Using phospho-peptide affinity chromatography and mass spectrometry, we found that PLCγ1 binds ß4 at the phosphorylated residues Y1422/Y1440, but were unable to verify this interaction in A431 carcinoma cells that overexpress the EGFR. Furthermore, using A431 cells devoid of ß4 or reconstituted with phenylalanine specific mutants of ß4, the activation of several downstream signaling pathways, including PLCγ/PKC, MAPK and PI3K/Akt, were not substantially affected. We conclude that tyrosine-phosphorylated ß4 does not enhance EGFR-mediated signaling in EGFR-overexpressing cells, despite the fact that this integrin subunit is highly tyrosine phosphorylated in these cells.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/genética , Integrina beta4/metabolismo , Neoplasias Cutáneas/metabolismo , Tirosina/metabolismo , Animales , Línea Celular Tumoral , Humanos , Integrina beta4/fisiología , Espectrometría de Masas , Fosforilación , Fosfotirosina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Neoplasias Cutáneas/genéticaRESUMEN
Most clinically used anticancer mAbs are of the IgG isotype, which can eliminate tumor cells through NK cell-mediated antibody-dependent cellular cytotoxicity and macrophage-mediated antibody-dependent phagocytosis. IgG, however, ineffectively recruits neutrophils as effector cells. IgA mAbs induce migration and activation of neutrophils through the IgA Fc receptor (FcαRI) but are unable to activate NK cells and have poorer half-life. Here, we combined the agonistic activity of IgG mAbs and FcαRI targeting in a therapeutic bispecific antibody format. The resulting TrisomAb molecules recruited NK cells, macrophages, and neutrophils as effector cells for eradication of tumor cells in vitro and in vivo. Moreover, TrisomAb had long in vivo half-life and strongly decreased B16F10gp75 tumor outgrowth in mice. Importantly, neutrophils of colorectal cancer patients effectively eliminated tumor cells in the presence of anti-EGFR TrisomAb but were less efficient in mediating killing in the presence of IgG anti-EGFR mAb (cetuximab). The clinical application of TrisomAb may provide potential alternatives for cancer patients who do not benefit from current IgG mAb therapy.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antineoplásicos Inmunológicos/farmacología , Neutrófilos/inmunología , Animales , Anticuerpos Biespecíficos/farmacología , Anticuerpos Monoclonales/farmacología , Antígenos CD/inmunología , Línea Celular Tumoral , Cetuximab/farmacología , Femenino , Células HCT116 , Humanos , Inmunoglobulina G/inmunología , Células Asesinas Naturales/inmunología , Macrófagos/inmunología , Masculino , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Melanoma Experimental/terapia , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Inmunológicos , Receptores Fc/inmunologíaRESUMEN
Vascular abnormalities in tumors have a major impact on the immune microenvironment in tumors. The consequences of abnormal vasculature include increased hypoxia, acidosis, high intra-tumoral fluid pressure, and angiogenesis. This introduces an immunosuppressive microenvironment that alters immune cell maturation, activation, and trafficking, which supports tumor immune evasion and dissemination of tumor cells. Increasing data suggests that cancer endothelium is a major barrier for traveling leukocytes, ranging from a partial blockade resulting in a selective endothelial barrier, to a complete immune infiltration blockade associated with immune exclusion and immune desert cancer phenotypes. Failed immune cell trafficking as well as immunosuppression within the tumor microenvironment limits the efficacy of immunotherapeutic approaches. As such, targeting proteins with key roles in angiogenesis may potentially reduce immunosuppression and might restore infiltration of anti-tumor immune cells, creating a therapeutic window for successful immunotherapy. In this review, we provide a comprehensive overview of established as well as more controversial endothelial pathways that govern selective immune cell trafficking across cancer endothelium. Additionally, we discuss recent insights and strategies that target tumor vasculature in order to increase infiltration of cytotoxic immune cells during the therapeutic window of vascular normalization hereby improving the efficacy of immunotherapy.
Asunto(s)
Endotelio/inmunología , Neoplasias/inmunología , Animales , Humanos , Tolerancia Inmunológica/inmunología , Inmunoterapia/métodos , Transducción de Señal/inmunología , Microambiente Tumoral/inmunologíaRESUMEN
Surgical resection of the primary tumor provides the best chance of cure for patients with colorectal carcinoma (CRC). However, bacterial translocation during intestinal surgery has been correlated with poor long-term oncological outcome. Therefore, we investigated the influence of bacterial contamination during colon surgery on CRC liver metastases development. Blood and liver samples of patients undergoing resection of primary CRC or liver metastases were collected. Cell numbers, activation markers and inflammatory mediators were determined. Tumor cell adhesion and outgrowth after sham- or colectomy operations were determined in a rat model, in which tumor cells had been injected into the portal vein. White blood cells and granulocytes were increased in per- and post-operative patient blood samples. IL-6 was also increased post-operatively compared to the preoperative level. Expression of NOX-2, NOX-4 and polymorphonuclear cells (PMNs) numbers were elevated in post-operative human liver samples. In vitro stimulation of macrophages with plasma of rats after colectomy resulted in production of reactive oxygen species (ROS). Colectomy in rats increased D-lactate levels in plasma, supporting bacterial translocation. Decreased expression of tight junction molecules and increased tumor cell adhesion and outgrowth was observed. Treatment with a selective decontamination of the digestive tract (SDD) cocktail decreased tumor cell adherence after colectomy. In conclusion, postoperative bacterial translocation may activate liver macrophages and PMNs, resulting in ROS production. As we previously showed that ROS release led to liver vasculature damage, circulating tumor cells may adhere to exposed extracellular matrix and grow out into liver metastases. This knowledge is pivotal for development of therapeutic strategies to prevent surgery-induced liver metastases development.
RESUMEN
Neutrophils represent the most abundant population of circulating cytotoxic effector cells. Moreover, their number can be easily increased by treatment with granulocyte-colony stimulating factor or granulocyte macrophage-colony stimulating factor, without the need for ex vivo expansion. Because neutrophils express Fc receptors, they have the potential to act as effector cells during monoclonal antibody therapy of cancer. Additionally, as neutrophils play a role in the regulation of adaptive immune responses, exploiting neutrophils in mAb therapy may result in long-term antitumour immunity. There is limited evidence that neutrophils play a prominent role in current immunoglobulin G-based immunotherapy. However, as IgA induces neutrophil recruitment, novel therapeutic strategies that aim to target the IgA Fc receptor FcαRI may fully unleash the potential of enlisting neutrophils as cytotoxic effector cells in antibody therapy of cancer.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/fisiología , Neoplasias/inmunología , Neutrófilos/fisiología , Inmunidad Adaptativa/fisiología , Predicción , Humanos , Inmunoterapia/métodos , Neoplasias/terapia , Células Tumorales CultivadasRESUMEN
Endothelial cells line the lumen of the vessel wall and are exposed to flow. In linear parts of the vessel, the endothelial cells experience laminar flow, resulting in endothelial cell alignment in the direction of flow, thereby protecting the vessel wall from inflammation and permeability. In order for endothelial cells to align, they undergo rapid remodeling of the actin cytoskeleton by local activation of the small GTPase Rac1. However, it is not clear whether sustained and local activation of Rac1 is required for long-term flow-induced cell alignment. Using a FRET-based DORA Rac1 biosensor, we show that local Rac1 activity remains for 12 h upon long-term flow. Silencing studies show that the RhoGEF Trio is crucial for keeping active Rac1 at the downstream side of the cell and, as a result, for long-term flow-induced cell alignment. Surprisingly, Trio appears to be not involved in flow-induced activation of Rac1. Our data show that flow induces Rac1 activity at the downstream side of the cell in a Trio-dependent manner and that Trio functions as a scaffold protein rather than a functional GEF under long-term flow conditions.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Células Cultivadas , Células Endoteliales/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Hidrodinámica , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Inflammation is part of the complex biological response of body tissues to harmful stimuli, such as pathogens. It serves as a protective response that involves leukocytes, blood vessels and molecular mediators with the purpose to eliminate the initial cause of cell injury and to initiate tissue repair. Inflammation is tightly regulated by the body and is associated with transient crossing of leukocytes through the blood vessel wall, a process called transendothelial migration (TEM) or diapedesis. TEM is a close collaboration between leukocytes on one hand and the endothelium on the other. Limiting vascular leakage during TEM but also when the leukocyte has crossed the endothelium is essential for maintaining vascular homeostasis. Although many details have been uncovered during the recent years, the molecular mechanisms from the vascular part that drive TEM still shows significant gaps in our understanding. This review will focus on the local signals that are induced in the endothelium that regulate leukocyte TEM and simultaneous preservation of endothelial barrier function.
Asunto(s)
Endotelio/metabolismo , Leucocitos/citología , Migración Transendotelial y Transepitelial , Humanos , Transducción de SeñalRESUMEN
ICAM-1 is required for firm adhesion of leukocytes to the endothelium. However, how the spatial organization of endothelial ICAM-1 regulates leukocyte adhesion is not well understood. In this study, we identified the calcium-effector protein annexin A2 as a novel binding partner for ICAM-1. ICAM-1 clustering promotes the ICAM-1-annexin A2 interaction and induces translocation of ICAM-1 into caveolin-1-rich membrane domains. Depletion of endothelial annexin A2 using RNA interference enhances ICAM-1 membrane mobility and prevents the translocation of ICAM-1 into caveolin-1-rich membrane domains. Surprisingly, this results in increased neutrophil adhesion and transendothelial migration under flow conditions and reduced crawling time, velocity, and lateral migration distance of neutrophils on the endothelium. In conclusion, our data show that annexin A2 limits neutrophil transendothelial migration by organizing the spatial distribution of ICAM-1.
Asunto(s)
Anexina A2/metabolismo , Células Endoteliales/inmunología , Molécula 1 de Adhesión Intercelular/metabolismo , Leucocitos/inmunología , Neutrófilos/inmunología , Caveolina 1/metabolismo , Adhesión Celular , Movimiento Celular , Células Endoteliales de la Vena Umbilical Humana , Humanos , Unión Proteica , Transporte de Proteínas , Agregación de Receptores , Migración Transendotelial y TransepitelialRESUMEN
During immune surveillance and inflammation, leukocytes exit the vasculature through transient openings in the endothelium without causing plasma leakage. However, the exact mechanisms behind this intriguing phenomenon are still unknown. Here we report that maintenance of endothelial barrier integrity during leukocyte diapedesis requires local endothelial RhoA cycling. Endothelial RhoA depletion in vitro or Rho inhibition in vivo provokes neutrophil-induced vascular leakage that manifests during the physical movement of neutrophils through the endothelial layer. Local RhoA activation initiates the formation of contractile F-actin structures that surround emigrating neutrophils. These structures that surround neutrophil-induced endothelial pores prevent plasma leakage through actomyosin-based pore confinement. Mechanistically, we found that the initiation of RhoA activity involves ICAM-1 and the Rho GEFs Ect2 and LARG. In addition, regulation of actomyosin-based endothelial pore confinement involves ROCK2b, but not ROCK1. Thus, endothelial cells assemble RhoA-controlled contractile F-actin structures around endothelial pores that prevent vascular leakage during leukocyte extravasation.
Asunto(s)
Actinas/metabolismo , Endotelio Vascular/metabolismo , Células Endoteliales de la Vena Umbilical Humana/citología , Leucocitos/citología , Transducción de Señal , Migración Transendotelial y Transepitelial , Proteína de Unión al GTP rhoA/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinas/genética , Animales , Permeabilidad Capilar , Células Cultivadas , Endotelio Vascular/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular , Leucocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Neutrófilos/citología , Neutrófilos/metabolismo , Proteína de Unión al GTP rhoA/genéticaRESUMEN
The Rho family of small GTPases forms a 20-member family within the Ras superfamily of GTP-dependent enzymes that are activated by a variety of extracellular signals. The most well known Rho family members are RhoA (Ras homolog gene family, member A), Cdc42 (cell division control protein 42), and Rac1 (Ras-related C3 botulinum toxin substrate 1), which affect intracellular signaling pathways that regulate a plethora of critical cellular functions, such as oxidative stress, cellular contacts, migration, and proliferation. In this review, we describe the current knowledge on the role of GTPase Rac1 in the vasculature. Whereas most recent reviews focus on the role of vascular Rac1 in endothelial cells, in the present review we also highlight the functional involvement of Rac1 in other vascular cells types, namely, smooth muscle cells present in the media and fibroblasts located in the adventitia of the vessel wall. Collectively, this overview shows that Rac1 activity is involved in various functions within one cell type at distinct locations within the cell, and that there are overlapping but also cell type-specific functions in the vasculature. Chronically enhanced Rac1 activity seems to contribute to vascular pathology; however, Rac1 is essential to vascular homeostasis, which makes Rac1 inhibition as a therapeutic option a delicate balancing act.
Asunto(s)
Endotelio Vascular/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Músculo Liso Vascular/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Vasos Sanguíneos/citología , Vasos Sanguíneos/metabolismo , Movimiento Celular/fisiología , Endotelio Vascular/citología , Fibroblastos/metabolismo , Humanos , Músculo Liso Vascular/citología , Estrés Oxidativo/fisiologíaRESUMEN
Endothelial cell-cell junctions maintain a restrictive barrier that is tightly regulated to allow dynamic responses to permeability-inducing angiogenic factors, as well as to inflammatory agents and adherent leukocytes. The ability of these stimuli to transiently remodel adherens junctions depends on Rho-GTPase-controlled cytoskeletal rearrangements. How the activity of Rho-GTPases is spatio-temporally controlled at endothelial adherens junctions by guanine-nucleotide exchange factors (GEFs) is incompletely understood. Here, we identify a crucial role for the Rho-GEF Trio in stabilizing junctions based around vascular endothelial (VE)-cadherin (also known as CDH5). Trio interacts with VE-cadherin and locally activates Rac1 at adherens junctions during the formation of nascent contacts, as assessed using a novel FRET-based Rac1 biosensor and biochemical assays. The Rac-GEF domain of Trio is responsible for the remodeling of junctional actin from radial into cortical actin bundles, a crucial step for junction stabilization. This promotes the formation of linear adherens junctions and increases endothelial monolayer resistance. Collectively, our data show the importance of spatio-temporal regulation of the actin cytoskeleton through Trio and Rac1 at VE-cadherin-based cell-cell junctions in the maintenance of the endothelial barrier.
Asunto(s)
Antígenos CD/metabolismo , Cadherinas/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Uniones Intercelulares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Antígenos CD/genética , Cadherinas/genética , Permeabilidad Capilar/genética , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , GTP Fosfohidrolasas/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Células Endoteliales de la Vena Umbilical Humana , Humanos , Uniones Intercelulares/genética , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/genética , Proteína de Unión al GTP rac1/genéticaRESUMEN
Leukocyte transendothelial migration (TEM) is one of the crucial steps during inflammation. A better understanding of the key molecules that regulate leukocyte extravasation aids to the development of novel therapeutics for treatment of inflammation-based diseases, such as atherosclerosis and rheumatoid arthritis. The adhesion molecules ICAM-1 and VCAM-1 are known as central mediators of TEM. Clustering of these molecules by their leukocytic integrins initiates the activation of several signaling pathways within the endothelium, including a rise in intracellular Ca (2+), activation of several kinase cascades, and the activation of Rho-GTPases. Activation of Rho-GTPases has been shown to control adhesion molecule clustering and the formation of apical membrane protrusions that embrace adherent leukocytes during TEM. Here, we discuss the potential regulatory mechanisms of leukocyte extravasation from an endothelial point of view, with specific focus on the role of the Rho-GTPases.