Caveolae-dependent and -independent uptake of albumin in cultured rodent pulmonary endothelial cells

Although a critical role for caveolae-mediated albumin transcytosis in pulmonary endothelium is well established, considerably less is known about caveolae-independent pathways. In this current study, we confirmed that cultured rat pulmonary microvascular (RPMEC) and pulmonary artery (RPAEC) endothelium endocytosed Alexa488-labeled albumin in a saturable, temperature-sensitive mode and internalization resulted in co-localization by fluorescence microscopy with cholera B toxin and caveolin-1. Although siRNA to caveolin-1 (cav-1) in RPAEC significantly inhibited albumin uptake, a remnant portion of albumin uptake was cav-1-independent, suggesting alternative pathways for albumin uptake. Thus, we isolated and cultured mouse lung endothelial cells (MLEC) from wild type and cav-1-/- mice and noted that ∼ 65% of albumin uptake, as determined by confocal imaging or live cell total internal reflectance fluorescence microscopy (TIRF), persisted in total absence of cav-1. Uptake of colloidal gold labeled albumin was evaluated by electron microscopy and demonstrated that albumin uptake in MLEC from cav-1-/- mice was through caveolae-independent pathway(s) including clathrin-coated pits that resulted in endosomal accumulation of albumin. Finally, we noted that albumin uptake in RPMEC was in part sensitive to pharmacological agents (amiloride [sodium transport inhibitor], Gö6976 [protein kinase C inhibitor], and cytochalasin D [inhibitor of actin polymerization]) consistent with a macropinocytosis-like process. The amiloride sensitivity accounting for macropinocytosis also exists in albumin uptake by both wild type and cav-1 -/- MLEC. We conclude from these studies that in addition to the well described caveolar-dependent pulmonary endothelial cell endocytosis of albumin, a portion of overall uptake in pulmonary endothelial cells is cav-1 insensitive and appears to involve clathrin-mediated endocytosis and macropinocytosis-like process. © 2013 Li et al

Cyclic stretch induced IL-33 production through HMGB1/TLR-4 signaling pathway in murine respiratory epithelial cells.

Interleukin 33 (IL-33), an inflammatory and mechanically responsive cytokine, is an important component of a TLR4-dependent innate immune process in mucosal epithelium. Although TLR4 also plays a role in sensing biomechanical stretch, a pathway of stretch-induced TLR4-dependent IL-33 biosynthesis has not been revealed. In the current study, we show that short term (6 h) cyclic stretch (CS) of cultured murine respiratory epithelial cells (MLE-12) increased intracellular IL-33 expression in a TLR4 dependent fashion. There was no detectable IL-33 in conditioned media in this interval. CS, however, increased release of the notable alarmin, HMGB1, and a neutralizing antibody (2G7) to HMGB1 completely abolished the CS mediated increase in IL-33. rHMGB1 increased IL-33 synthesis and this was partially abrogated by silencing TLR4 suggesting additional receptors for HMGB1 are involved in its regulation of IL-33. Collectively, these data reveal a HMGB1/TLR4/IL-33 pathway in the response of respiratory epithelium to mechanical stretch

Activation of TLR4 by LPS or cyclic stretch increases intracellular IL-33.

<p>MLE-12 cells were transfected with 50 nM of TLR4-specific siRNA (TLR4siRNA) or nonspecific siRNA control (consiRNA) using Lipofectamine 2000 for 48h before stretch. (A) Transfected MLE-12 cells were further treated with 100 ng/ml LPS or control solution for 24h and IL-33 production were measured in cell lysate by ELISA. (B) IL-33 production after 6h cyclic stretch in transfected cells treated with or without TLR4-specific siRNA. (C) Total TLR4 expression in whole cell lysate and cytoplasm in MLE-12 cells with (cyclic S) or without (con) stretching were measured by Western blot and normalized by β-actin. ***P<0.001 when compared between groups denoted by horizontal lines.</p

Cyclic, but not static, stretch induced increase in IL-33.

<p>Mouse IL-33 (mIL-33) expression in whole cell lysate after static (static S ~18% elongation) or cyclic stretch (cyctic S ~18% elongation, 1HZ) was detected by western blot at different time point (4h, 6h, 8h) and normalized by β-actin (right graph). ***P<0.001 compared with control (con). Each stretching group collected three wells for a single experiment, the bar graphs illustrate data representative of three independent experiments and western blot at the different time point was run separately.</p

Cyclic stretch activates TLR4-dependent signaling (NF-κB translocation) and pro-inflammatory state (IL-6 release into medium).

<p>(A) MLE-12 cells were transfected with 50nM TLR-4 specific siRNA or negative control siRNA using Lipofectamine 2000 for 48 h. TLR-4 expression in control group (con), negative control siRNA group (con siRNA) and TLR-4 specific siRNA group (TLR4 siRNA) were detected by western blot and normalized by β-actin (upper graph). (B) Stretch induced NF-κB nucleus translocation were TLR-4 dependent. Total and nuclear NF-κB were measured by Western blot and its nucleus/total NF-κB ratio was analyzed after 6h cyclic stretch in transfected cells. (C) IL-6 secretion in transfected cell media after 6h cyclic stretch was measured by ELISA and was TLR-4 dependent. Each group collected from three wells in a single experiment and the data were presented as mean ± SD from three separate experiments. **P<0.01, ***P<0.001 when compared between groups denoted by horizontal lines.</p

Cyclic stretch increases HMGB1 expression in a TLR4-dependent fashion.

<p>(A) Cell culture media with or without cyclic stretch were concentrated and HMGB1 contents were detected by Western blot. (B) HMGB1 in cell culture media with or without stretch was directly measured by ELISA. (C) Media of the cells which transfected with nonspecific siRNA control (consiRNA) or TLR-4-specific siRNA (TLR4 siRNA) with or without stretch (con vs. cyclic S) were concentrated and HMGB1 contents were measured by Western blot. (D) HMGB1 in media of the transfected cells following 6h cyclic stretch (cyclic S) was measured by ELISA. **P<0.01, ***P<0.001 compared with control. Each stretching group collected from three wells and represented a single experiment, the bar graphs illustrate data representative of three independent experiments.</p

Cyclic stretch induced increase in cytosolic and nuclear levels of IL-33.

<p>Nuclear and cytoplasm protein fractions of MLE-12 cells with (cyclic S) or without (con) 6h stretch were isolated, and mIL-33 in nucleus and cytoplasm were measured by Western blot (A) or ELISA (B). Lamin B and β-actin (A) served as a loading control for nucleus and cytoplasmic protein, respectively. **P<0.01, ***P<0.001 compared with control (con). Each stretching group collected from three wells for a single experiment, the bar graphs illustrate data representative of three independent experiments.</p