Abstract
Integrin-mediated leukocyte adhesion to endothelial cells is a crucial step in immunity against pathogens. Whereas the outside-in signaling pathway in response to the pro-inflammatory cytokine tumour necrosis factor (TNF) has already been studied in detail, little knowledge exists about a supposed TNF-mediated inside-out signaling pathway. In contrast to the outside-in signaling pathway, which relies on the TNF-induced upregulation of surface molecules on endothelium, inside-out signaling should also be present in an endothelium-free environment. Using single-cell force spectroscopy, we show here that stimulating Jurkat cells with TNF significantly reinforces their adhesion to fibronectin in a biomimetic in vitro assay for cell-surface contact times of about 1.5 seconds, whereas for larger contact times the effect disappears. Analysis of single-molecule ruptures further demonstrates that TNF strengthens sub-cellular single rupture events at short cell-surface contact times. Hence, our results provide quantitative evidence for the significant impact of TNF-induced inside-out signaling in the T-lymphocyte initial adhesion machinery.
Highlights
Immunity against pathogens critically depends on the ability of leukocytes to rapidly transmigrate from the bloodstream into inflamed tissue and to the site of infection
To explore whether tumour necrosis factor (TNF) influences the adhesion of lymphocytes in the absence of an in vivo endothelial cell layer, we carried out in vitro adhesion experiments on surfaces coated with fibronectin
For quantifying the interaction of Jurkat cells with fibronectin as a function of TNF, we carried out Single-cell force spectroscopy (SCFS) experiments: a single living cell is attached to a tipless cantilever and pressed onto a fibronectin-functionalized surface using an atomic force microscope (Fig. 1a)
Summary
Immunity against pathogens critically depends on the ability of leukocytes to rapidly transmigrate from the bloodstream into inflamed tissue and to the site of infection. Loss of FAN causes a defective recruitment of leukocytes to sites of infection[11], RACK1 and EED have been described as direct interaction partners of the cytoplasmic domains of integrins[12,13,14], and nSMase[2] has been implicated in the adhesion, migration and confluence-dependent contact inhibition of cells[15] These findings led us to speculate that TNF may control the inflammatory recruitment and the binding of T cells to the endothelium by directly impacting on the adhesiveness of T cell integrins via inside-out signaling, in addition to its well-established effects on the outside-in signaling via endothelial cells. This result provides quantitative evidence for the existence of an inside-out signaling mechanism involved in the TNF-mediated activation of lymphocyte adhesion
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