Characterization of TNF receptor subtype expression and signaling on pulmonary endothelial cells in mice

Abstract

TNF plays a crucial role in the pathogenesis of acute lung injury. However, the expression profile of its two receptors, p55 and p75, on pulmonary endothelium and their influence on TNF signaling during lung microvascular inflammation remain uncertain. Using flow cytometry, we characterized the expression profile of TNF receptors on the surface of freshly harvested pulmonary endothelial cells (PECs) from mice and found expression of both receptors with dominance of p55. To investigate the impact of stimulating individual TNF receptors, we treated wild-type and TNF receptor knockout mice with intravenous TNF and determined surface expression of adhesion molecules (E-selectin, VCAM-1, ICAM-1) on PECs by flow cytometry. TNF-induced upregulation of all adhesion molecules was substantially attenuated by absence of p55, whereas lack of p75 had a similar but smaller effect that varied between adhesion molecules. Selective blockade of individual TNF receptors by specific antibodies in wild-type primary PEC culture confirmed that the in vivo findings were due to direct effects of TNF receptor inhibition on endothelium and not other cells (e.g., circulating leukocytes). Finally, we found that PEC surface expression of p55 dramatically decreased in the early stages of endotoxemia following intravenous LPS, while no change in p75 expression was detected. These data demonstrate a crucial in vivo role of p55 and an auxiliary role of p75 in TNF-mediated adhesion molecule upregulation on PECs. It is possible that the importance of the individual receptors varies at different stages of pulmonary microvascular inflammation following changes in their relative expression.

Expression, Regulation, and Function of Atypical Chemerin Receptor CCRL2 on Endothelial Cells

“…Consistent with previous reports, LPS injection upregulated VCAM-1 on liver and lung endothelial cells in both genotypes (Fig. 7, 8) (24, 26–29). …”

“…Primary human endothelial cells (HUVEC and HDMEC) treated in vitro with pro-inflammatory stimuli upregulated CCRL2 and bound Fc-Chemerin, indicating conserved regulation in primary EC across species. Liver and lung endothelial cells from LPS-dosed mice of both genotypes upregulated VCAM- 1, which is consistent with previous reports (24, 26, 39). It is not yet clear why CCRL2 is expressed endogenously at higher levels in mouse lung ECs compared to liver ECs, although it is well documented that ECs isolated from anatomically different vascular beds are phenotypically and functionally distinct in leukocyte adhesion and trafficking mechanisms (20, 40, 41).…”

Alveolar macrophage-derived microvesicles mediate acute lung injury

“…We first investigated MV-associated content of TNF, IL-1β and IL-6 as indices of their proinflammatory activity, because these cytokines are important mediators of ALI18 28–30 and are known to be expressed within the alveoli in the early phase of ALI. MVs were isolated from BALF samples taken from mice at 1 hour after i.t.…”

Novel concepts of acute lung injury and alveolar-capillary barrier dysfunction

“…Despite their important role in pathogen containment, excessive alveolar neutrophil recruitment has been associated with injury to the alveolar-capillary barrier in ALI (136). Neutrophils are marginated in the alveolar capillaries in infectious or noninfectious ALI, and this process involves engagement of chemokine-receptor interactions (267) and of different families of endothelial and epithelial adhesion molecules, such as JAMs (junctional adhesion molecules), ICAM-1 (intercellular adhesion molecule-1), PECAM-1 (platelet endothelial cell adhesion molecule-1), and VCAM-1 (vascular adhesion molecule-1), which are upregulated upon release of inflammatory mediators like TNF-␣ (16,93,131). In addition, leukocyte-expressed adenosine receptor A(2b) and eicosanoid expression are attributed a role in pulmonary neutrophil recruitment in LPS-induced ALI or after Pseudomonas aeruginosa infection in mice (102,124).…”