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Roberta Martinelli,
Masataka Kamei, Peter T Sage,
Ramiro Massol,
Laya Varghese,
Tracey Sciuto,
Mourad Toporsian,
Ann M Dvorak,
Tomas Kirchhausen,
Timothy A Springer,
Christopher V Carman
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ABSTRACT: Basic mechanisms by which cellular barriers sense and respond to integrity disruptions remain poorly understood. Despite its tenuous structure and constitutive exposure to disruptive strains, the vascular endothelium exhibits robust barrier function. We show that in response to micrometer-scale disruptions induced by transmigrating leukocytes, endothelial cells generate unique ventral lamellipodia that propagate via integrins toward and across these "micro-wounds" to close them. This novel actin remodeling activity progressively healed multiple micro-wounds in succession and changed direction during this process. Mechanical probe-induced micro-wounding of both endothelia and epithelia suggests that ventral lamellipodia formed as a response to force imbalance and specifically loss of isometric tension. Ventral lamellipodia were enriched in the Rac1 effectors cortactin, IQGAP, and p47Phox and exhibited localized production of hydrogen peroxide. Together with Apr2/3, these were functionally required for effective micro-wound healing. We propose that barrier disruptions are detected as local release of isometric tension/force unloading, which is directly coupled to reactive oxygen species-dependent self-restorative actin remodeling dynamics.
The Journal of Cell Biology 04/2013; 201(3):449-65. · 10.26 Impact Factor
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ABSTRACT: CD4(+)CXCR5(+)Foxp3(+) follicular regulatory T cells (T(FR) cells) inhibit humoral immunity mediated by CD4(+)CXCR5(+)Foxp3(-) follicular helper T cells (T(FH) cells). Although the inhibitory receptor PD-1 is expressed by both cell types, its role in the differentiation of T(FR) cells is unknown. Here we found that mice deficient in PD-1 and its ligand PD-L1 had a greater abundance of T(FR) cells in the lymph nodes and that those T(FR) cells had enhanced suppressive ability. We also found substantial populations of T(FR) cells in mouse blood and demonstrated that T(FR) cells in the blood homed to lymph nodes and potently inhibited T(FH) cells in vivo. T(FR) cells in the blood required signaling via the costimulatory receptors CD28 and ICOS but were inhibited by PD-1 and PD-L1. Our findings demonstrate mechanisms by which the PD-1 pathway regulates antibody production and help reconcile inconsistencies surrounding the role of this pathway in humoral immunity.
Nature Immunology 12/2012; · 26.01 Impact Factor
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ABSTRACT: Adaptive immunity requires that T cells efficiently scan diverse cell surfaces to identify cognate Ag. However, the basic cellular mechanisms remain unclear. In this study, we investigated this process using vascular endothelial cells, APCs that possess a unique and extremely advantageous, planar morphology. High-resolution imaging revealed that CD4 memory/effector T cells dynamically probe the endothelium by extending submicron-scale, actin-rich "invadosome/podosome-like protrusions" (ILPs). The intimate intercellular contacts enforced by ILPs consistently preceded and supported T cell activation in response to endothelial MHC class II/Ag. The resulting calcium flux stabilized dense arrays of ILPs (each enriched in TCR, protein kinase C-θ, ZAP70, phosphotyrosine, and HS1), forming what we term a podo-synapse. Similar findings were made using CD8 CTLs on endothelium. Furthermore, careful re-examination of both traditional APC models and professional APCs suggests broad relevance for ILPs in facilitating Ag recognition. Together, our results indicate that ILPs function as sensory organelles that serve as actuators of immune surveillance.
The Journal of Immunology 03/2012; 188(8):3686-99. · 5.79 Impact Factor
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ABSTRACT: Programmed death ligand-1 (PD-L1) plays a critical role in T-cell regulatory function. Here, we report a newly discovered effect of PD-L1 on angiogenesis. We demonstrate that PD-L1 and its receptor CD80, but not PD-1, are expressed by primary murine lung and heart vascular endothelial cells and the miscrovascular endothelial cell line (MS1) at both the mRNA and protein levels in vitro. The inhibition of PD-L1 or CD80 expression in MS1 cells, by small-interfering RNA transfection, led to a significant up-regulation of vascular endothelial growth factor receptor 2 expression and cell proliferation levels in MS1 cells. Furthermore, MS1 cells were found to have a significantly lower proliferation and vascular endothelial growth factor receptor 2 expression levels when they were co-cultured with PD-L1-expressing normal corneal epithelial cells, as compared to MS1 cells co-cultured with PD-L1(-/-) corneal epithelial cells. In a suture-induced corneal angiogenesis model, we observed a significantly higher level of angiogenic response in PD-L1(-/-) knockout mice as compared to wild-type mice, although there was no significant difference in the expression of inflammatory cytokines (interleukin-1α, interleukin-1β, or tumor necrosis factor-α) or the infiltration of innate immune cells (neutrophils and macrophages) between the two groups. We conclude that the expression of PD-L1 in both vascular endothelial cells and corneal epithelial cells regulates corneal angiogenesis.
American Journal Of Pathology 04/2011; 178(4):1922-9. · 4.89 Impact Factor
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ABSTRACT: Regulatory T cells (Tregs) and the PD-1: PD-ligand (PD-L) pathway are both critical to terminating immune responses. Elimination of either can result in the breakdown of tolerance and the development of autoimmunity. The PD-1: PD-L pathway can thwart self-reactive T cells and protect against autoimmunity in many ways. In this review, we highlight how PD-1 and its ligands defend against potentially pathogenic self-reactive effector T cells by simultaneously harnessing two mechanisms of peripheral tolerance: (i) the promotion of Treg development and function and (ii) the direct inhibition of potentially pathogenic self-reactive T cells that have escaped into the periphery. Treg cells induced by the PD-1 pathway may also assist in maintaining immune homeostasis, keeping the threshold for T-cell activation high enough to safeguard against autoimmunity. PD-L1 expression on non-hematopoietic cells as well as hematopoietic cells endows PD-L1 with the capacity to promote Treg development and enhance Treg function in lymphoid organs and tissues that are targets of autoimmune attack. At sites where transforming growth factor-beta is present (e.g. sites of immune privilege or inflammation), PD-L1 may promote the de novo generation of Tregs. When considering the consequences of uncontrolled immunity, it would be therapeutically advantageous to manipulate Treg development and sustain Treg function. Thus, this review also discusses how the PD-1 pathway regulates a number of autoimmune diseases and the therapeutic potential of PD-1: PD-L modulation.
Immunological Reviews 07/2010; 236:219-42. · 11.15 Impact Factor
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Immunological Reviews - IMMUNOL REV. 01/2010; 236(1):219-242.
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ABSTRACT: Immune system functions require blood leukocytes to continuously traffic throughout the body and repeatedly cross endothelial barriers (i.e., diapedese) as they enter (intravasation) and exit (extravasation) the circulation. The earliest studies to directly characterize diapedesis in vivo suggested co-existence of two distinct migratory pathways: between (para-cellular) and through (trans-cellular) individual endothelial cells. However, in the absence of conclusive in vitro observations, the latter pathway remained poorly accepted. The recent emergence of unambiguous in vitro reports of trans-cellular diapedesis has begun to illuminate mechanisms for this pathway and has renewed interest in its physiological roles. A thorough reevaluation of the existing literature reveals a large number of studies documenting significant use of the trans-cellular pathway in diverse in vivo settings. These include constitutive trafficking in bone marrow and lymphoid organs as well as upregulated extravasation in peripheral tissues during inflammation. Here we collectively summarize these in vivo observations alongside the emerging in vitro data in order to provide a framework for understanding the settings, mechanisms and roles for the trans-cellular route of diapedesis.
Frontiers in Bioscience 02/2009; 14:5066-83. · 3.52 Impact Factor
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ABSTRACT: The real-time observation of protein dynamics in living cells and organisms is of fundamental importance for understanding biological processes. Most approaches to labeling proteins exploit noncovalent interactions, unsuitable to long-term studies, or genetic fusion to naturally occurring fluorescent proteins that often have unsatisfactory optical properties. Here we used the fungal enzyme cutinase and its suicide substrate p-nitrophenyl phosphonate to covalently attach a variety of labels to the integrin lymphocyte function-associated antigen-1 (LFA-1) on the surface of living cells. Cutinase was embedded in the extracellular domain of LFA-1 with no appreciable influence on integrin function and conformational regulation. p-nitrophenyl phosphonate-conjugated fluorochromes, including the very bright and stable quantum dots, bound efficiently and specifically to LFA-1/cutinase. The availability of a genetically encoded tag that binds covalently to quantum dots could foster the development of new experimental strategies for the study of protein dynamics in vivo.
Proceedings of the National Academy of Sciences 10/2007; 104(37):14753-8. · 9.68 Impact Factor
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ABSTRACT: Diapedesis is critical for immune system function and inflammatory responses. This occurs by migration of blood leukocytes either directly through individual microvascular endothelial cells (the "transcellular" route) or between them (the "paracellular" route). Mechanisms for transcellular pore formation in endothelium remain unknown. Here we demonstrate that lymphocytes used podosomes and extended "invasive podosomes" to palpate the surface of, and ultimately form transcellular pores through, the endothelium. In lymphocytes, these structures were dependent on Src kinase and the actin regulatory protein WASP; inhibition of podosome formation selectively blocked the transcellular route of diapedesis. In endothelium, membrane fusion events dependent on the SNARE-containing membrane fusion complex and intracellular calcium were required for efficient transcellular pore formation in response to podosomes. These findings provide insights into basic mechanisms for leukocyte trafficking and the functions of podosomes.
Immunity 07/2007; 26(6):784-97. · 21.64 Impact Factor
