Paracytosis of Haemophilus influenzae through cell layers of NCI-H292 lung epithelial cells.

Haemophilus influenzae penetrates the respiratory epithelium during carriage and invasive disease, including respiratory tract infections. We developed an in vitro model system consisting of lung epithelial NCI-H292 cells on permeable supports to study the passage of H. influenzae through lung epithelial cell layers. The NCI-H292 cells formed tight layers with a Ca(2+)-dependent transepithelial resistance of around 40 omega.cm2. H. influenzae passed through the cell layers without affecting the viability of the cells and [3H]inulin penetration. The passage time was independent of the inoculum of H. influenzae in the apical compartment and was not influenced by the presence of capsule or fimbriae on H. influenzae or by the ability of the bacteria to adhere to the epithelial cells. However, highly adherent strains showed greater paracytosis. Different strains passed through the cell layer independently. The passage time was shorter for rapidly growing strains than for slowly growing strains (10 to 18 h and 30 h, respectively). Microscopic examination revealed the presence of clusters of H. influenzae bacteria between the epithelial cells, indicating that bacterial passage was due to paracytosis. After the addition of chloramphenicol, no bacteria were cultured from the basolateral side, and no bacterial clusters between the epithelial cells were seen, suggesting that de novo bacterial protein synthesis was needed for the bacteria to reach the intercellular space. We conclude that H. influenzae passes through viable cell layers of the human lung epithelial cell line NCI-H292 by paracytosis, requiring bacterial protein synthesis.