Soluble vimentin binds to P‐selectin and attenuates leukocytic infiltration into the lungs in murine endotoxin induced acute lung injury

Abstract

BackgroundAcute lung injury (ALI) is a devastating condition that culminates as inflammation of the lungs mediated by leukocytic infiltration. In animal models, platelets and platelet P‐selectin are well described mediators of ALI and other forms of inflammation through enhancing capture and transmigration of neutrophils across vascular endothelium. Vimentin is an intermediate filament protein mainly thought to be involved in maintaining the cytoskeleton. Recently, there has been increasing evidence of vimentin's role during inflammation, although its precise role is unclear. We had recently observed that recombinant soluble vimentin (sVim) blocks neutrophil adhesion to platelets, endothelial cells, and P‐selectin monolayers; whether these observations translate into attenuation of ALI is unknown. Therefore, we hypothesize that soluble vimentin sVim blocks P‐selectin‐PSGL‐1 interactions and will decrease leukocytic infiltration into the lungs of endotoxin‐exposed mice.MethodsAll experiments were approved by the Institutional Animal Care and Use Committee at Baylor College of Medicine and the Michael E. DeBakey Veterans Affairs Medical Center. Recombinant sVim was expressed in E. coli and His‐purified using affinity chromatography. Binding kinetics of sVim to P‐selectin was determined using both enzyme‐linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). ELISA was performed by detecting sVim binding to immobilized P‐selectin/Fc or PSGL‐1/Fc protein. In SPR (BIACore), sVim was perfused over immobilized P‐selectin/Fc or IgG protein to determine the equilibrium dissociation constant KD. To determine whether sVim inhibits leukocytic infiltration into lungs after endotoxin exposure, C57BL/6 mice were treated with intraperitoneal injections of sVim (3 mg/kg) or vehicle control followed by endotoxin (O111:B4; 15 mg/kg) 1 hour later and then euthanized at 96 hours. Mice were evaluated by blinded investigators for both physical activity and degree of lung inflammation. Activity was determined using the mouse clinical assessment score for sepsis (M‐CASS) and lung inflammation was determined by alveolar septal thickening and leukocytic infiltration on hematoxylin and eosin stained sections.ResultssVim preferentially binds to P‐selectin/Fc and but not PSGL‐1/Fc in ELISA. Using SPR, sVim binds to P‐selectin/Fc with a KD of 125 ± 1.2 nM and not to IgG control. Endotoxin‐exposed mice treated with sVim were more active as determined by the M‐CASS (1.9 ± 0.2 vs 2.5 ± 0.2; 4‐point scale where lower is better; n=10/grp, p<0.05). Mice receiving sVim also had less alveolar septal thickening and leukocytic infiltration into the lungs after exposure to endotoxin.ConclusionSoluble vimentin binds to P‐selectin and not P‐selectin glycoprotein ligand‐1 and decreases leukocytic infiltration into the lungs of endotoxin‐exposed mice. Further studies need to take place to determine whether vimentin also improves gas exchange in the lungs. Finally, dose‐response studies need to be performed to determine the optimal dose and timing of sVim to improve outcomes in acute lung injury.Support or Funding InformationNIH/NHLBI HL‐116524, NIH/NIGMS GM‐112806