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Wissel, H*; Rupp, J; Burkhardt, W*; Tschirch, E; Wauer, R R.*; Rüdiger, M*†

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Chlamydia pneumonoae (Cpn) cause pulmonary infections. Cpn alter the expression of toll like receptor (TLR) 4 in alveolar type II (ATII)-cells. Subsequently, nuclear factor kappaB (NF-kappaB) is activated and tumor necrosis factor α (TNFα) and macrophage inflammatory protein 2 (MIP-2) are produced. Intratracheal application of perfluorocarbons (PFC) has been shown beneficial in animals with bacterial pneumonia. PFC do not only improve gas exchange, but also reduce pulmonary production of TNFα. The mechanism, however, is still poorly understood. The purpose of this study was to investigate whether PFC-incubation of ATII-cells prevents Cpn-induced increase of TNFα. Furthermore, the underlying pathway had to be elucidated. ATII-cells were isolated from rat lungs, incubated with Cpn (strain TW 183) and PF5080. TNFα and MIP-2 concentration in supernatant was analysed by ELISA. Western blot analysis was performed on nuclear and cytoplasmatic extracts and membrane fractions; protein and receptor expression was analysed by immunocytochemistry and confocal laser scan microscopy. PF5080 pre-incubation prevented the Cpn-induced secretion of TNFα (30 ±10 vs. 700 ±50 pg/ml) and MIP-2 (100 ±20 vs. 4440 ±40 pg/ml). Cpn incubation caused a 2.2 fold increase of TNFα-receptor 1 expression when compared with control cells. The rise was prevented by PF5080 pre-incubation. TNFα production is mainly regulated by NF-κB activation. Cpn caused a decrease in cytoplasmatic IκBα (5 ±1 vs. 13 ±3) and an increase in nuclear p65 (30 ±10 vs. 6 ±1). The NF-κB activation was prevented by PF5080 pre-incubation. The ATII-cell response is mediated by an interaction of Cpn with toll like receptor (TLR) 4 that was 1.5 fold higher after Cpn-incubation, compared to control. No similar increase was found in PF5080 pre-incubated cells. After 24 h of Cpn incubation in 50% of cells Cpn were adherent to cell surface and in 88 ±6% Cpn were found in the perinuclear region. In PF5080 pre-incubated ATII-cells only 34 ±4% showed Cpn on the cell surface and 5 ±1% intracellular Cpn. PFC pre-treatment of isolated ATII-cells did prevent Cpn-induced increase in TNFα. The most likely explanation is a reduced contact of Cpn with the ATII-cells that prevents intracellular uptake and activation of the cellular cascade that induces TNFα production. Since labeled PF5080 was found in the membranes of ATII-cells, the effect could be explained by a stabilization of the cellular membrane.

This study was supported by Medizinischer Forschungsfonds Tirol

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