Role of CD34 in LPS induced acute lung inflammation

CD34, a pan-selectin binding protein when glycosylated, has been shown to be involved in leukocyte migration to the site of inflammation. However, only one report is available on the expression and role of CD34 in neutrophil recruitment during acute lung inflammation. Hence, we proceeded to study the expression of CD34 in lung tissue and its role in migration of neutrophils into the lungs using a mouse model of endotoxin induced acute lung inflammation. Acute lung inflammation was induced via intranasal E. coli O55:B5 LPS (50 μg) instillation in CD34 KO and WT C57BL/6 mice. Mice (male and female) from each phenotype (i.e. WT and CD34 KO) were divided into five sub-groups comprising of 0, 3, 9, 15 and 24-hour LPS treatment time-points. Each time-point had five-seven WT mice and five-seven CD34 KO mice. Experiments were carried out to look at several factors associated with an inflammatory response in the lungs of mice, including peripheral blood cell counts, total and differential leukocyte cell counts in BALF, adherent neutrophils left in the lung after lavage (MPO levels, Gr-1 IF staining), lung vascular permeability (BAL total protein), lung inflammation scoring (H&E staining), and cytokine and chemokine levels in BALF. Our results demonstrate that intranasal LPS challenge induces time-dependent inflammatory responses in WT mice (N = 5-7/time-point) and CD34 KO mice (N = 5-7/time-point). While there was no difference in the total or differential leukocyte counts, lung MPO content in CD34 KO was lower than WT group at 3 h time-point (p=0.0308). The MPO levels in CD34 KO mice begin to rise at 9 h (p=0.0021), as opposed to an early 3 h rise in WT mice (p=0.0001), indicating that CD34 KO mice display delays in lung neutrophil recruitment kinetics. CD34 KO mice do not display loss of lung vascular barrier properties as suggested by lower total protein in BAL supernatant in CD34 KO mice at 3 h (p=0.0452) and 24 h (p=0.0113) time-points. In addition, immunofluorescence lung staining shows distinct alveolar staining with Gr-1, a marker for polymorphonuclear leukocytes and a subset of inflammatory monocytes/macrophages. Lung immunofluorescent staining in WT mice at baseline (0 h No LPS) reveals CD34 expression in the bronchiolar epithelium, in addition to alveolar septa. Several pro-inflammatory cytokines and chemokines (TNF-α, IL-1β, KC, MIP-1α, IL-6, IL-10 and IL-12 p40 sub-unit; p