Mechanical forces induced by the transendothelial migration of human neutrophils.
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ABSTRACT:
SUBMITTER: Rabodzey A
PROVIDER: S-EPMC2479581 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Lymphatic filariasis is a major tropical disease caused by the parasite Brugia malayi. Microfilariae (Mf) circulate in the peripheral blood for 2-3 hours in synchronisation with maximal feeding of the mosquito vector. When absent from the peripheral blood, Mf sequester in the capillaries of the lungs. Mf are therefore in close contact with vascular endothelial cells (EC) and may induce EC immune function and/or wound repair mechanisms such as angiogenesis. In this study, Mf were co-cultured with human umbilical vein EC (HUVEC) or human lung microvascular EC (HLMVEC) and the transendothelial migration of leukocyte subsets was analysed. In addition, the protein and/or mRNA expression of chemokine, cytokine and angiogenic mediators in endothelial cells in the presence of live microfilariae were measured by a combination of cDNA arrays, protein arrays, ELISA and fluorescence antibody tests.Surprisingly, our findings indicate that Mf presence partially blocked transendothelial migration of monocytes and neutrophils, but not lymphocytes. However, Mf exposure did not result in altered vascular EC expression of key mediators of the tethering stage of extravasation, such as ICAM-1, VCAM-1 and various chemokines. To further analyse the immunological function of vascular EC in the presence of Mf, we measured the mRNA and/or protein expression of a number of pro-inflammatory mediators. We found that expression levels of the mediators tested were predominantly unaltered upon B. malayi Mf exposure. In addition, a comparison of angiogenic mediators induced by intact Mf and Wolbachia-depleted Mf revealed that even intact Mf induce the expression of remarkably few angiogenic mediators in vascular EC. Our study suggests that live microfilariae are remarkably inert in their induction and/or activation of vascular cells in their immediate local environment. Overall, this work presents important insights into the immunological function of the vascular endothelium during an infection with B. malayi.
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Project description:The migration of circulating neutrophils towards damaged or infected tissue is absolutely critical to the inflammatory response. L-selectin is a cell adhesion molecule abundantly expressed on circulating neutrophils. For over two decades, neutrophil L-selectin has been assigned the exclusive role of supporting tethering and rolling - the initial stages of the multi-step adhesion cascade. Here, we provide direct evidence for L-selectin contributing to neutrophil transendothelial migration (TEM). We show that L-selectin co-clusters with PECAM-1 - a well-characterised cell adhesion molecule involved in regulating neutrophil TEM. This co-clustering behaviour occurs specifically during TEM, which serves to augment ectodomain shedding of L-selectin and expedite the time taken for TEM (TTT) to complete. Blocking PECAM-1 signalling (through mutation of its cytoplasmic tail), PECAM-1-dependent adhesion or L-selectin shedding, leads to a significant delay in the TTT. Finally, we show that co-clustering of L-selectin with PECAM-1 occurs specifically across TNF- but not IL-1β-activated endothelial monolayers - implying unique adhesion interactomes forming in a cytokine-specific manner. To our knowledge, this is the first report to implicate a non-canonical role for L-selectin in regulating neutrophil TEM.
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Project description: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.
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Project description:Human neutrophils are mediators of innate immunity and undergo dramatic shape changes at all stages of their functional life cycle. In this work, we quantified the forces associated with a neutrophil's morphological transition from a nonadherent, quiescent sphere to its adherent and spread state. We did this by tracking, with high spatial and temporal resolution, the cell's mechanical behavior during spreading on microfabricated post-array detectors printed with the extracellular matrix protein fibronectin. Two dominant mechanical regimes were observed: transient protrusion and steady-state contraction. During spreading, a wave of protrusive force (75 ± 8 pN/post) propagates radially outward from the cell center at a speed of 206 ± 28 nm/s. Once completed, the cells enter a sustained contractile state. Although post engagement during contraction was continuously varying, posts within the core of the contact zone were less contractile (-20 ± 10 pN/post) than those residing at the geometric perimeter (-106 ± 10 pN/post). The magnitude of the protrusive force was found to be unchanged in response to cytoskeletal inhibitors of lamellipodium formation and myosin II-mediated contractility. However, cytochalasin B, known to reduce cortical tension in neutrophils, slowed spreading velocity (61 ± 37 nm/s) without significantly reducing protrusive force. Relaxation of the actin cortical shell was a prerequisite for spreading on post arrays as demonstrated by stiffening in response to jasplakinolide and the abrogation of spreading. ROCK and myosin II inhibition reduced long-term contractility. Function blocking antibody studies revealed haptokinetic spreading was induced by β2 integrin ligation. Neutrophils were found to moderately invaginate the post arrays to a depth of ∼1 μm as measured from spinning disk confocal microscopy. Our work suggests a competition of adhesion energy, cortical tension, and the relaxation of cortical tension is at play at the onset of neutrophil spreading.
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Project description:p38 mitogen-activated protein kinase (MAPK) signalling is critical in the pathophysiology of a variety of inflammatory processes. Leukocyte recruitment to the site of inflammation is a multistep process governed by specific signalling cascades. After adhesion in the lumen, many leukocytes crawl to optimal sites at endothelial junctions and transmigrate to extravascular tissue in a Mac-1-dependent manner. The signalling mechanisms that regulate postadhesion steps of intraluminal crawling, transmigration, and chemotaxis in tissue remain incompletely understood. The present study explored the effect of p38 MAPK inhibitor SB203580 on various parameters of neutrophil recruitment triggered by chemokine KC (CXCL1) gradient. Neutrophil-endothelial interactions in microvasculature of murine cremaster muscle were determined using intravital microscopy and time-lapsed video analysis. SB203580 (100 nM) did not change leukocyte rolling but significantly attenuated neutrophil adhesion, emigration, and transmigration and impaired the initiation of neutrophil crawling and transmigration. In response to KC chemotactic gradient, SB203580 significantly reduced the velocity of migration and chemotaxis index of neutrophils in tissue. The upregulation of Mac-1 expression in neutrophils stimulated by KC was significantly blunted by SB203580 in vitro. Collectively, our findings demonstrate that pharmacological suppression of p38 MAPK significantly impairs multiple steps of neutrophil recruitment in vivo.
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Project description:Neutrophil migration requires β2 integrins and chemoattractant receptor signaling for motility and directionality. G protein subunit Gα13 can facilitate cell migration by mediating RhoA activation induced by G protein-coupled receptors. However, the possible role of Gα13-integrin interaction in migration is unclear. In this study, we show that Gα13 -/- neutrophils are deficient in transendothelial migration and migration on β2 integrin ligand ICAM-1. However, unlike G protein-coupled receptors and integrin inside-out signaling pathways, Gα13 is important in migration velocity and neutrophil spreading but not in directionality nor cell adhesion. Importantly, neutrophil recruitment in vivo was also inhibited in Gα13 -/- mice, suggesting the importance of Gα13 in transendothelial migration of neutrophils in vitro and in vivo. Furthermore, a synthetic peptide (MB2mP6) derived from the Gα13 binding site of β2 inhibited Gα13-β2 interaction and Gα13-mediated transient RhoA inhibition in neutrophils, suggesting that this peptide inhibited integrin outside-in signaling. MB2mP6 inhibited migration of control neutrophils through endothelial cell monolayers or ICAM-1-coated filters, but was without further effect on Gα13 -/- neutrophils. It also inhibited integrin-dependent neutrophil migration velocity without affecting directionality. In vivo, MB2mP6 markedly inhibited neutrophil infiltration into the cardiac tissues induced by ischemia/reperfusion injury. Thus, Gα13-dependent outside-in signaling enables integrin-dependent neutrophil motility without affecting directionality and may be a new therapeutic target for inhibiting neutrophil trafficking but not adhesion.
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Project description:Circulating human T effector memory cell (TEM) recognition of nonself MHC (major histocompatibility complex) molecules on allograft endothelial cells can initiate graft rejection despite elimination of professional antigen-presenting cells necessary for naive T-cell activation. Our previous studies of CD4 TEM have established that engagement of the T-cell receptor not only activates T cells but also triggers transendothelial migration (TEM) by a process that is distinct from that induced by activating chemokine receptors on T cells, being slower, requiring microtubule-organizing center-directed cytolytic granule polarization to and release from the leading edge of the T cell, and requiring engagement of proteins of the endothelial cell lateral border recycling compartment. Although CD4 TEM may contribute to acute allograft rejection, the primary effectors are alloreactive CD8 TEM. Whether and how T-cell receptor engagement affects TEM of human CD8 TEM is unknown.We modeled TEM of CD8 TEM across cultured human microvascular endothelial cells engineered to present superantigen under conditions of venular shear stress in vitro in a flow chamber. Here, we report that T-cell receptor engagement can also induce TEM of this population that similarly differs from chemokine receptor-driven TEM with regard to kinetics, morphological manifestations, and microtubule-organizing center dynamics as with CD4 TEM. However, CD8 TEM do not require either cytolytic granule release or interactions with proteins of the lateral border recycling compartment.These results imply that therapeutic strategies designed to inhibit T-cell receptor-driven recruitment based on targeting granule release or components of the lateral border recycling compartment will not affect CD8 TEM and are unlikely to block acute rejection in the clinic.
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Project description:B cells, like T cells, can infiltrate sites of inflammation, but the processes and B cell subsets involved are poorly understood. Using human cells and in vitro assays, we find only a very small number of B cells will adhere to TNF-activated (but not to resting) human microvascular endothelial cells (ECs) under conditions of venular flow and do so by binding to ICAM-1 and VCAM-1. CXCL13 and to a lesser extent CXCL10 bound to the ECs can increase adhesion and induce transendothelial migration (TEM) of adherent naïve and memory B cells in 10-15 minutes through a process involving cell spreading, translocation of the microtubule organizing center (MTOC) into a trailing uropod and interacting with EC ALCAM. Engagement of the BCR by EC-bound anti-κ light chain antibody also increases adhesion and TEM of κ+ but not λ+ B cells. BCR-induced TEM takes 30-60 minutes, requires Syk activation, is initiated by B cell rounding up and translocation of the MTOC to the region of the B cell adjacent to the EC and also utilizes EC ALCAM for TEM. BCR engagement reduces the number of B cells responding to chemokines and preferentially stimulates TEM of CD27+ B cells that co-express IgD, with or without IgM, as well as CD43. RNA Seq analysis suggests peripheral blood CD19+CD27+CD43+IgD+ cells have increased expression of genes that support BCR activation as well as innate immune properties in comparison to total peripheral blood CD19+ cells.
2023-07-13 | GSE237044 | GEO
Project description: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.
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Project description:ObjectivesRegulatory T (Treg) cells regulate immunity in autoimmune diseases and cancers. However, immunotherapies that target tumor-infiltrating Treg cells often induce unwanted immune responses and tissue inflammation. Our research focussed on exploring the expression pattern of CD177 in tumor-infiltrating Treg cells with the aim of identifying a potential target that can enhance immunotherapy effectiveness.MethodsSingle-cell RNA sequencing (scRNA-seq) data and survival data were obtained from public databases. Twenty-one colorectal cancer patient samples, including fresh tumor tissues, peritumoral tissues and peripheral blood mononuclear cells (PBMCs), were analysed using flow cytometry. The transendothelial activity of CD177+ Treg cells was substantiated using in vitro experiments.ResultsScRNA-seq and flow cytometry results indicated that CD177 was exclusively expressed in intratumoral Treg cells. CD177+ Treg cells exhibited greater activation status and expressed elevated Treg cell canonical markers and immune checkpoint molecules than CD177- Treg cells. We further discovered that both intratumoral CD177+ Treg cells and CD177-overexpressing induced Treg (iTreg) cells had lower levels of PD-1 than their CD177- counterparts. Moreover, CD177 overexpression significantly enhanced the transendothelial migration of Treg cells in vitro.ConclusionsThese results demonstrated that Treg cells with higher CD177 levels exhibited an enhanced activation status and transendothelial migration capacity. Our findings suggest that CD177 may serve as an immunotherapeutic target and that overexpression of CD177 may improve the efficacy of chimeric antigen receptor T (CAR-T) cell therapy.
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