gms | German Medical Science

Objectives: Low serum levels or deficiency of 1,2-dihydroxyvitamine D3 (VD3) are associated with a higher mortality in trauma patients with sepsis or ARDS, although the molecular mechanisms are not yet understood. VD3 is known to stimulate lung maturity, alveolar type II cell differentiation and pulmonary surfactant synthesis. In addition, VD3 is actively involved in the innate immune system and the defense of epithelial cells in case of infection. In this study we investigated the impact VD3 on the alveolar-capillary barrier on the cell and molecular level in a defined co-culture model of alveolar epithelial cells and microvascular endothelial cells.

Methods: NCI-H441 cells from an ATII-like epithelial cell line were seeded apically on semi-permeable transwell filters, representing the epithelial lining of the alveoli. From basolateral side outgrowth-endothelial cells as a source of endothelial cells isolated from the peripheral blood were seeded and co-cultures were cultured to form a confluent cell layer. With an EVOM we measured the epithelial resistance TEER indicating the epithelial barrier properties. In addition, stimulation with different concentrations of LPS was used to mimic bacterial infection. Gene expression of tight junctional molecules, inflammatory cytokines and antimicrobial peptides was analyzed by real time PCR, while corresponding proteins were evaluated by ELISA or and immune-fluorescence staining or Westernblots, respectively. Statistical evaluation was performed by Annova for at least 3 independent sets of experiments.

Results and Conclusion: VD3 effectively protected the alveolar capillary barrier after LPS treatment indicated by TEER measurement as a functional parameter. Noticeably, it inhibited the IL-6 secretion in both H441 cells and OECs in the co-culture system treated with high LPS concentration and also in the mono-culture of OECs. In addition, VD3 could significantly suppress the alveolar surfactant molecule SP-A expression, which was highly expressed in the co-culture system after LPS infection. VD3 induced high levels of the antimicrobial peptide LL-37 with the ability to inactivate LPS which seems to be one of the key factors in the protective function of VD3.