The mechanisms of excessive migration of activated neutrophils into inflamed lungs, credited with tissue damage, are not fully understood. We explored the hitherto unknown expression of leukocyte-specific protein 1 (LSP1) in human and mouse lungs and neutrophils and examined its role in neutrophil migration in acute lung inflammation. Autopsied septic human lungs showed increased LSP1 labeling in epithelium, endothelium, and leukocytes, including in their nuclei compared with normal lungs. We induced acute lung inflammation through intranasal administration of E. coli lipopolysaccharide (LPS) (80 μg) in LSP1-deficient (Lsp1(-/-)) and wild-type (WT) 129/SvJ mice. Immunocytochemistry and Western blots showed increased expression of LSP1 and phosphorylated LSP1 in lungs of LPS-treated WT mice. Histology showed more congestion, inflammation, and Gr-1(+) neutrophils in lung of WT mice than Lsp1(-/-) mice. LPS-treated WT mice had significantly more neutrophils in bronchoalveolar lavage (BAL) and myeloperoxidase levels in lungs compared with Lsp1(-/-) mice. However, there were no differences in lung tissue and BAL concentrations of keratinocyte-derived chemokine, monocyte chemoattractant protein-1, macrophage inflammatory protein-1α and -1β, vascular permeability, and phosphorylated p38 MAPK between LPS-treated WT and Lsp1(-/-) mice, whereas TNF-α concentration was higher in BAL fluid from LPS-treated WT. Immunoelectron microscopy showed increased LSP1 in the nuclei of LPS-treated neutrophils. We also found increased levels of phosphorylated LSP1 associated with plasma membrane, nucleus, and cytosol at various times after LPS treatment of murine bone marrow-derived neutrophils, suggesting its role in modulation of neutrophil cytoskeleton and the membrane. These data collectively show increased expression of LSP1 in inflamed mouse and human lungs and its role in neutrophil recruitment and lung inflammation.

Background Asthma is a major cause of morbidity and mortality in humans. The mechanisms of asthma are still not fully understood. Leukocyte-specific protein-1 (LSP-1) regulates neutrophil migration during acute lung inflammation. However, its role in asthma remains unknown. Methods An OVA-induced mouse asthma model in LSP1-deficient (Lsp1−/−) and wild-type (WT) 129/SvJ mice were used to test the hypothesis that the absence of LSP1 would inhibit airway hyperresponsiveness and lung inflammation. Results Light and electron microscopic immunocytochemistry and Western blotting showed that, compared with normal healthy lungs, the levels of LSP1 were increased in lungs of OVA-asthmatic mice. Compared to Lsp1−/− OVA mice, WT OVA mice had higher levels of leukocytes in broncho-alveolar lavage fluid and in the lung tissues (P < 0.05). The levels of OVA-specific IgE but not IgA and IgG1 in the serum of WT OVA mice was higher than that of Lsp1−/− OVA mice (P < 0.05). Deficiency of LSP1 significantly reduced the levels of IL-4, IL-5, IL-6, IL-13, and CXCL1 (P < 0.05) but not total proteins in broncho-alveolar lavage fluid in asthmatic mice. The airway hyper-responsiveness to methacholine in Lsp1−/− OVA mice was improved compared to WT OVA mice (P < 0.05). Histology revealed more inflammation (inflammatory cells, and airway and blood vessel wall thickening) in the lungs of WT OVA mice than in those of Lsp1−/− OVA mice. Finally, immunohistology showed localization of LSP1 protein in normal and asthmatic human lungs especially associated with the vascular endothelium and neutrophils. Conclusion These data show that LSP1 deficiency reduces airway hyper-responsiveness and lung inflammation, including leukocyte recruitment and cytokine expression, in a mouse model of asthma.

Asthma is one of the most serious forms of respiratory disease, causing economic losses, high mortality, and morbidity globally. However, its etiology is still ambiguous. Many inflammatory cells, including T lymphocytes, eosinophils, mast cells, macrophages, and neutrophils, are recruited into the airway of asthma lung patients. In the previous study, we reported that LSP1 deficiency reduces neutrophil migration and acute lung inflammation, but its role in asthma remains unknown. Therefore, in this study, we developed an asthma‐like disease in LSP1‐deficient (Lsp1−/−) and wild‐type (WT) 129/SvJ mice (n=6 each treatment group) through two intraperitoneal (IP) injections of 2μg OVA/2mg alum two weeks apart, followed by three aerosol challenges with 1% OVA two days apart after two weeks of the last IP injection. We hypothesized that the absence of LSP1 inhibited airway inflammation in this OVA‐induced asthma mouse model due to inhibition of immune cell migration.Immunohistochemistry staining and western blot data showed that the level of LSP1 increased in asthma mouse lungs, compared with normal healthy lungs. Results from immuno‐gold as well as immunofluorescent staining illustrated that LSP1 was expressed on endothelium, bronchiolar epithelium, neutrophils, eosinophils, lymphocytes, alveolar macrophages, and pulmonary intravascular macrophages. There was no significant difference in peripheral leukocyte numbers between normal and asthmatic WT and Lsp1−/− mice. However, compared to Lsp1−/− asthma mice, WT asthma mice had higher levels leukocytes in broncho‐alveolar lavage fluid, higher myeloperoxidase, a marker of neutrophils, and eosiperoxidase levels in lungs (P<0.05). The level of OVA‐specific IgE, but not IgA and IgG1, in the serum of WT asthma mice was higher than that of Lsp1−/− asthma mice (P<0.05). Other research reported that IL‐4, IL‐5, IL‐6, IL‐13, and KC are associated with eosinophilia and neutrophil migration. We found that the absence of the LSP1 gene significantly reduced the levels of IL‐4, IL‐5, IL‐6, IL‐13, and KC (P<0.05) but not the levels of IL‐17, eotaxin, IFN‐gamma, and total protein in broncho‐alveolar lavage fluid. The airway hyper‐responsiveness response to methacholine in Lsp1−/− asthma mice was ameliorated compared to WT asthma mice (P<0.05). Histopathology evaluation findings showed more recruited inflammatory cells, as well as airway and blood vessel wall thickening, in the lungs of WT mice than in those of Lsp1−/− mice. These data indicate that LSP1 promotes airway inflammation associated with asthma in this mouse model. LSP1 may be a potential target for therapeutic intervention in allergic lung disease or asthma in humans.Support or Funding InformationWe thank to the Natural Science and Engineering Research Council (NSERC), Graduate Student Scholarship from Integrated Training Program in Infectious Disease, Food Safety and Public Policy (ITraP), Devolved Graduate Scholarship from Department of Veterinary Biomedical Sciences, and Graduate Student Scholarship from Western College of Veterinary Medicine, University of Saskatchewan, Canada for supporting this research. We thank Ms. LaRhonda Sobchishin for the technical support provided for the electron microscope technique and Ms. Eiko Kawamura for technical support in using a confocal microscope.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.