Extracellular histones are present in the airways because of cell death occurring during inflammation. They promote inflammation and cause tissue damage due to their cationic nature. The anionic phosphoglycoprotein osteopontin (OPN) is expressed at high levels during airway inflammation and has been ascribed both pro- and anti-inflammatory roles. In this study, it was hypothesized that OPN may neutralize the harmful activities of extracellular histones at the airway mucosal surface. In a model of histone-induced acute lung injury, OPN mice showed increased inflammation and tissue injury, and succumbed within 24 h, whereas wild-type mice showed lower degrees of inflammation and no mortality. In lipopolysaccharide-induced acute lung injury, wild-type mice showed less inflammation and tissue injury than OPN mice. In bronchoalveolar lavage fluid from ARDS patients, high levels of OPN and also histone-OPN complexes were detected. In addition, OPN bound to histones with high affinity in vitro, resulting in less cytotoxicity and reduced formation of tissue-damaging neutrophil extracellular traps (NETs). The interaction between OPN and histones was dependent on posttranslational modification of OPN, i.e., phosphorylation. The findings demonstrate a novel role for OPN, modulating the pro-inflammatory and cytotoxic properties of free histones.

Osteopontin (OPN) plays a role in inflammation via recruitment of neutrophils and tissue remodeling. In this study, we investigated the distribution of OPN-expressing cells in the airway epithelium of normal lung tissue and that from patients with chronic obstructive pulmonary disease (COPD). OPN was detected on the epithelial cell surface of small airways and in scattered cells within the epithelial cell layer. Staining revealed higher OPN concentrations in tissue showing moderate to severe COPD compared to that in controls. In addition, OPN expression was confined to goblet and club cells, and was absent from ciliated and basal cells as detected via immunohistochemistry. However, OPN expression was up-regulated in submerged basal cells cultures exposed to cigarette smoke (CS) extract. Cell fractioning of air-liquid interface cultures revealed increased OPN production from basal compartment cells compared to that in luminal fraction cells. Furthermore, both constitutive and CS-induced expression of OPN decreased during differentiation. In contrast, cultures stimulated with interleukin (IL)-13 to promote goblet cell hyperplasia showed increased OPN production in response to CS exposure. These results indicate that the cellular composition of the airway epithelium plays an important role in OPN expression and that these levels may reflect disease endotypes in COPD.

Phenotypic characterization of submerged human bronchial epithelial cells (HBECs) and OPN production in response to cigarette smoke extract (CSE). (A) HBECs were grown to near confluence and stained to detect markers of basal cells (p63) and goblet cells (MUC5AC). 4′,6-Diamidino-2-phenylindole (DAPI) was used to stain the DNA of nuclei. Micrographs from one representative experiment out of three. Scale bars = 50 µm. (B) HBECs were incubated with CSE (0-5%) for 24 h. OPN levels in cell culture media were determined via enzyme-linked immunosorbent assay (ELISA). Results represent means and standard deviations (SD) of three independent experiments. Statistical analyses were performed using one-way ANOVA with Dunnett's post-hoc test. (C) Submerged HBECs were cultured in the absence and presence of CSE (5%) for 24 h. The mRNA expression of OPN, MUC5AC, UTG, and p63 (fold-change compared to control cells cultured in medium alone) is depicted here. The results represent means and SD of three to five independent experiments. Statistical analyses were performed using a Mann-Whitney U test. (D) Intracellular OPN content and content in the media of cells cultured in the absence and presence of CSE (5%) for 24 h. The results represent means and SD of three independent experiments. Statistical analyses were performed using one-way ANOVA with Tukey's post-hoc test. *P < 0.05, **P < 0.01. " should read:"Phenotypic characterization of submerged human bronchial epithelial cells (HBECs) and OPN production in response to cigarette smoke extract (CSE). (A) HBECs were grown to near confluence and stained to detect markers of basal cells (p63) and goblet cells (MUC5AC). 4′,6-Diamidino-2-phenylindole (DAPI) was used to stain the DNA of nuclei. Micrographs from one representative experiment out of three. Scale bars = 50 µm. (B) HBECs were incubated with CSE (0-5%) for 24 h. OPN levels in cell culture media were determined using ELISA. OPN-release from unstimulated cells (0% CSE) were set to 1, and the other conditions were calculated as fold change. Results represent means and standard deviations (SD) of three independent experiments. Statistical analyses were performed using one-way ANOVA with Dunnett's post-hoc test. (C) Submerged HBECs were cultured in the absence and presence of CSE (5%) for 24 h. The mRNA expression of OPN, MUC5AC, UTG, and p63 (fold-change compared to control cells cultured in medium alone) is depicted here. The results represent means and SD of three to five independent experiments. Statistical analyses were performed using a Mann-Whitney U test. (D) Intracellular OPN content and content in the media of cells cultured in the absence and presence of CSE (5%) for 24 h. The results represent means and SD of three independent experiments. Statistical analyses were performed using one-way ANOVA with Tukey's post-hoc test. *P < 0.05, **P < 0.01. " Published: xx xx xxxx open