Abstract
Neutrophils are recognised to play a pivotal role at the interface between innate and acquired immunities following their recruitment to inflamed tissues and lymphoid organs. While neutrophil trafficking through blood vessels has been extensively studied, the molecular mechanisms regulating their migration into the lymphatic system are still poorly understood. Here, we have analysed neutrophil-lymphatic vessel interactions in real time and in vivo using intravital confocal microscopy applied to inflamed cremaster muscles. We show that antigen sensitisation of the tissues induces a rapid but transient entry of tissue-infiltrated neutrophils into lymphatic vessels and subsequent crawling along the luminal side of the lymphatic endothelium. Interestingly, using mice deficient in both TNF receptors p55 and p75, chimeric animals and anti-TNFα antibody blockade we demonstrate that tissue-release of TNFα governs both neutrophil migration through the lymphatic endothelium and luminal crawling. Mechanistically, we show that TNFα primes directly the neutrophils to enter the lymphatic vessels in a strictly CCR7-dependent manner; and induces ICAM-1 up-regulation on lymphatic vessels, allowing neutrophils to crawl along the lumen of the lymphatic endothelium in an ICAM-1/MAC-1-dependent manner. Collectively, our findings demonstrate a new role for TNFα as a key regulator of neutrophil trafficking into and within lymphatic system in vivo.
Highlights
The dynamics of neutrophil migration into the tissue and lymphatic vessels was analysed by intravital confocal microscopy in TNFα-stimulated mouse cremaster muscles. (a) Representative 3D-reconstructed still image (2 μm cross-section) from a LysM-GFP ×αSMA-CherryRFP mouse [exhibiting both endogenous GFP
In vivo fluorescent-immunostaining with non-blocking anti-PECAM-1 and/or a non-inhibitory dose of anti-LYVE-1 mAbs was applied to the tissues to visualise endothelial cells and lymphatic vessels, respectively, to allow the tracking of GFPhigh neutrophils responses into the lymphatic vasculature by intravital confocal microscopy
We observed that following their entry into the lymphatic vessels, intravasated neutrophils were firmly attached to the lymphatic endothelial cells (LEC) and were crawling along the luminal surface of the lymphatic endothelium. (Fig. 1f and Video 3)
Summary
The dynamics of neutrophil migration into the tissue and lymphatic vessels was analysed by intravital confocal microscopy in TNFα-stimulated mouse cremaster muscles. (a) Representative 3D-reconstructed still image (2 μm cross-section) from a LysM-GFP ×αSMA-CherryRFP mouse [exhibiting both endogenous GFP-. Fluorescent neutrophils (green) and RFP-fluorescent pericytes/smooth muscle cells (red) and immunostained with a non-blocking anti-PECAM-1 mAb (blue)] cremaster tissue showing a neutrophil within the lymphatic vessel (yellow arrow) post TNFα-stimulation. (f) Representative 3D-reconstructed still image of a lymphatic vessel from a TNFα-stimulated cremaster tissue of a LysM-GFP mouse and immunostained with an anti-LYVE-1 mAb (red) in vivo. Leukocyte integrins and selectins have been implicated in neutrophil trafficking to LNs28, though their exact contribution to cell migration through afferent lymphatic vessels near sites of inflammation in vivo is still unclear. Despite these seminal but conflicting reports, further investigations are required to fully understand the mechanisms associated with this response. The present findings identify a previously unknown role for TNFαin orchestrating sequential interactions of neutrophils with tissue-associated lymphatic vessels during the acute inflammatory response of antigen sensitisation; and highlight TNFαas a potential target for the manipulation of neutrophil regulation of adaptive immune responses within the lymphatic system
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