Introduction We have identified bronchoalveolar lavage NGAL (neutrophil gelatinase associated lipocalin, also known as lipocalin-2) as a potential biomarker in acute lung injury (ALI). Rising NGAL concentrations over the first 72 h of illness, and higher absolute NGAL levels at day 3 predicted ICU survival. The biological mechanism by which NGAL is associated with improved survival is not known. Lipocalin-2 modulates the process of epithelial to mesenchymal transition (EMT) in breast cancer. We have demonstrated the typical morphological and proteomic changes and the invasive phenotype of EMT in alveolar epithelial cells in vitro in response to either ARDS BAL fluid or TNFalpha + TGFbeta, cytokines present in the lavage fluid of patients with ALI. We hypothesised that NGAL could alter EMT in vitro in alveolar epithelium.

Methods A549 cells were stimulated with TNFalpha + TGFbeta in the presence or absence of NGAL (0.1–10 ng/ml). Cells were lysed at 72 h and probed by western blot for E-cadherin, Zo-1, fibronectin and vimentin. Supernatants were analysed by gelatin zymography for MMP-2. The invasive capacity of the cells was assessed using BD Biocoat Matrigel Invasion Assay Chambers.

Results As previously described TNFalpha + TGFbeta caused loss of E-cadherin, Zo-1 and gain of vimentin and fibronectin in A549 cells, with an accompanying morphological change to spindle shaped cells, that secreted MMP-2 and invaded matrigel. Treatment with NGAL did not affect E-cadherin, Zo-1, fibronectin nor vimentin. However, treatment with NGAL abrogated the change in morphology, maintaining an epithelial-like cobblestone appearance (Abstract S48 Figure 1a). Additionally, NGAL inhibited the production of MMP-2 by these cells (from 1.8±0.3 to 0.9±0.2 relative gelatinolytic units, p=0.04) and reduced the capacity of the cells to migrate through basement membrane-type matrix (Abstract S48 Figure 1b).

• Download figure
• Open in new tab
• Download powerpoint

Abstract S48 Figure 1

Conclusion NGAL inhibited some but not all of typical changes of EMT in this in vitro model of ALI. The protective effect of NGAL in patients with ALI may be mediated by maintaining alveolar epithelial morphology, reducing their production of the basement membrane-destructive enzyme MMP-2, and reducing their capacity to migrate to the sub-epithelial interstitial space. Further work is required to assess this mechanism in vivo and to examine the effects of supplementary NGAL in ALI.