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Updated: Jun 10, 2025
Isolation of Primary Mouse Lung Endothelial Cells
Published on: November 10, 2021
Amitriptyline Decreases Mouse Lung Endothelial Cell Inflammatory Responses to Packed Red Blood Cell Microparticles
Lindsey Wattley1, Ryan Chae1, Christopher Nguyen1
1Department of Surgery, University of Cincinnati, Cincinnati, Ohio.
Introduction:
Large-volume packed red blood cell (pRBC) transfusion is associated with lung injury and worsened outcomes. Amitriptyline reduces lung injury and inflammation in a murine sepsis model. We hypothesized that red cell microparticles (MP) activate endothelial cells, leading to lung injury and that treatment with amitriptyline would blunt the inflammatory response MPs through inhibition of acid sphingomyelinase (ASM).
Methods:
Murine pRBCs were obtained from C57Bl/6 mice and stored in AS3 for 14 d. The MPs were isolated from pRBCs by serial centrifugation. Mouse lung endothelial cells (MLECs) were pretreated with amitriptyline (0, 2.5, 25, 27 μM, n = 5) for 30 min prior to MP treatment. Chemokine secretion and adhesion molecule shedding was assessed. ASM activity was measured from cell lysates.
Results:
MPs increased the secretion of chemokines and shedding of adhesion molecules in MLECs at both four and 24 h. Amitriptyline treatment of MLECs decreased ASM activity in the setting of MPs. Amitriptyline pretreatment decreased the secretion of chemokines and shedding of adhesion molecules in response to MPs at 4 h but did not decrease adhesion molecule shedding at 24 h CONCLUSIONS: Endothelial cell treatment with MPs induces secretion of chemokines responsible for chemotaxis (keratinocyte chemoattractant, regulated upon activation normal T cell expressed and presumably secreted, and G-granulocyte colony-stimulating factor) as well as many downstream proinflammatory effects (interleukin-6). Additionally, MPs induce adhesion molecule shedding (vascular cell adhesion molecule-1, intracellular adhesion molecule-1, P-selectin, and E-selectin), which has been shown to be associated with endothelial cell activation. Amitriptyline pretreatment decreases MLEC inflammatory response and ASM activity is decreased. These data suggest that ASM inhibition in MLECs is a potential strategy to blunt the inflammatory response to the red blood cell storage lesion.
Insights
Red blood cell microparticles activate lung endothelial cells, causing inflammation. Amitriptyline treatment reduced this response by inhibiting acid sphingomyelinase (ASM), suggesting a therapeutic strategy for transfusion-related lung injury.
Area of Science:
- Immunology
- Cell Biology
- Transfusion Medicine
Background:
- Packed red blood cell (pRBC) transfusion can cause lung injury.
- Red cell microparticles (MPs) may activate endothelial cells, contributing to this injury.
- Amitriptyline has shown anti-inflammatory effects in sepsis models.
Purpose of the Study:
- To investigate if red cell MPs activate lung endothelial cells.
- To determine if amitriptyline can mitigate MP-induced inflammation via acid sphingomyelinase (ASM) inhibition.
Main Methods:
- Isolated MPs from stored murine pRBCs.
- Treated mouse lung endothelial cells (MLECs) with MPs and amitriptyline.
- Assessed chemokine secretion, adhesion molecule shedding, and ASM activity.
Main Results:
- MPs increased chemokine secretion and adhesion molecule shedding in MLECs.
- Amitriptyline decreased ASM activity in MLECs exposed to MPs.
- Amitriptyline reduced MP-induced inflammation at 4 hours but not 24 hours.
Conclusions:
- Endothelial cell activation by MPs involves pro-inflammatory chemokines and adhesion molecules.
- Amitriptyline pretreatment reduces MLEC inflammatory response and ASM activity.
- ASM inhibition is a potential strategy to mitigate inflammation from red blood cell storage lesions.
