Neutrophil‐Derived Microvesicle Uptake under Flow Conditions in an In Vitro Model of Pulmonary Vascular Inflammation

Abstract

BackgroundDespite elevation of circulating microvesicles (MVs) during systemic inflammation, little is known of their cell/tissue‐specific interactions and uptake under these conditions. We recently found that during subclinical endotoxaemia in mice, circulating MV uptake increases in the pulmonary circulation, primarily through enhanced uptake by lung marginated monocytes. To further understand MV‐cell interactions within the pulmonary circulation, we developed an in vitro parallel flow chamber‐based model of MV uptake by human lung microvascular endothelial cells (HLMECs) and monocytes.MethodsDynamic fluid flow conditions at variable shear stresses (0.56–10.95 dyn/cm2) were produced using the ibidi pump system, with primary HLMECs adhered to μ‐Slides I Luer, with 0.4, 0.6, 0.8 mm chamber heights. HLMECs were adapted to flow for 18 h prior to MV uptake experiments. MVs were produced by N‐formylmethionyl‐leucyl‐phenylalanine (fMLP, 1 μM) stimulation of neutrophils, labelled with the fluorescent lipophilic dye, DiD, and administrated to the flow chamber system. Uptake of labelled MVs was assessed after 1 h by flow cytometric measurement of cell‐associated DiD (mean fluorescence intensity: MFI). For MV uptake under inflammatory conditions, HLMECs were stimulated with TNFα (10 ng/mL, 18 h) and then incubated with MVs under flow, with or without adherent monocytes.ResultsAt a shear stress of 5.00 dyn/cm2, i.e. an upper estimate for lung capillaries, neutrophil‐MV uptake by HLMECs was reduced as compared to uptake in a tissue culture well (flow: 221±52 vs well: 554±210 MFI; p<0.01). Increasing shear stresses over the range of 0.56–10.95 dyn/cm2 reduced MV uptake by HLMECs. TNFα pre‐treatment of HLMECs increased MV uptake 2‐fold under flow conditions (5 dyn/cm2) (untreated:221±52 vs TNFα: 435±117 MFI; p<0.01) and, in contrast to untreated HLMECs, increasing shear stress enhanced MV uptake by TNFα‐treated HLMECs (interaction p<0.0001). In monocyte‐HLMEC co‐culture, MV uptake was similar between cell types in tissue culture wells (monocytes: 149±62, HLMECs: 212±94 MFI), but 3‐fold higher by monocytes than HLMECs under flow conditions (5 dyn/cm2) (monocytes: 1573±166, HLMECs: 491±186 MFI; p<0.01).ConclusionUnder physiological flow conditions, marked differences in neutrophil‐MV uptake by HLMECs were observed compared to tissue culture well incubation. Uptake by HLMECs decreased with increasing shear stress under flow, but this effect was reversed by TNFα pre‐stimulation. In the case of monocytes (adhered to TNFα‐treated HLMECs), MV uptake was enhanced with flow and substantively exceeded that of HLMECs, consistent with our previous in vivo findings in endotoxaemic mice. Our results provide a strong caution for using standard tissue well cultures for MV uptake studies and suggest that this approach utilising more physiologically relevant flow conditions would be essential to define in vivo interplay between circulating MVs and their target cells within the pulmonary vasculature.Support or Funding InformationBritish Journal of Anaesthesia/Royal College of Anaesthetists and Chelsea & Westminster Health Charity