Inhibition of TNFalpha in vivo prevents hyperoxia-mediated activation of caspase 3 in type II cells

BACKGROUND: The mechanisms during the initial phase of oxygen toxicity leading to pulmonary tissue damage are incompletely known. Increase of tumour necrosis factor alpha (TNFalpha) represents one of the first pulmonary responses to hyperoxia. We hypothesised that, in the initial phase of hyperoxia, TNFalpha activates the caspase cascade in type II pneumocytes (TIIcells). METHODS: Lung sections or freshly isolated TIIcells of control and hyperoxic treated rats (48 hrs) were used for the determination of TNFalpha (ELISA), TNF-receptor 1 (Western blot) and activity of caspases 8, 3, and 9 (colorimetrically). NF-kappaB activation was determined by EMSA, by increase of the p65 subunit in the nuclear fraction, and by immunocytochemistry using a monoclonal anti-NF-kappaB-antibody which selectively stained the activated, nuclear form of NF-kappa B. Apoptotic markers in lung tissue sections (TUNEL) and in TIIcells (cell death detection ELISA, Bax, Bcl-2, mitochondrial membrane potential, and late and early apoptotic cells) were measured using commercially available kits. RESULTS: In vivo, hyperoxia activated NF-kappaB and increased the expression of TNFalpha, TNF-receptor 1 and the activity of caspase 8 and 3 in freshly isolated TIIcells. Intratracheal application of anti-TNFalpha antibodies prevented the increase of TNFRI and of caspase 3 activity. Under hyperoxia, there was neither a significant change of cytosolic cytochrome C or of caspase 9 activity, nor an increase in apoptosis of TIIcells. Hyperoxia-induced activation of caspase 3 gradually decreased over two days of normoxia without increasing apoptosis. Therefore, activation of caspase 3 is a temporary effect in sublethal hyperoxia and did not mark the "point of no return" in TIIcells. CONCLUSION: In the initiation phase of pulmonary oxygen toxicity, an increase of TNFalpha and its receptor TNFR1 leads to the activation of caspase 8 and 3 in TIIcells. Together with the hyperoxic induced increase of Bax and the decrease of the mitochondrial membrane potential, activation of caspase 3 can be seen as sensitisation for apoptosis. Eliminating the TNFalpha effect in vivo by anti-TNFalpha antibodies prevents the pro-apoptotic sensitisation of TIIcells

Meson screening masses from lattice QCD with two light and the strange quark

We present results for screening masses of mesons built from light and strange quarks in the temperature range of approximately between 140 MeV to 800 MeV. The lattice computations were performed with 2+1 dynamical light and strange flavors of improved (p4) staggered fermions along a line of constant physics defined by a pion mass of about 220 MeV and a kaon mass of 500 MeV. The lattices had temporal extents Nt = 4, 6 and 8 and aspect ratios of Ns / Nt \geq 4. At least up to a temperature of 140 MeV the pseudo-scalar screening mass remains almost equal to the corresponding zero temperature pseudo-scalar (pole) mass. At temperatures around 3Tc (Tc being the transition temperature) the continuum extrapolated pseudo-scalar screening mass approaches very close to the free continuum result of 2 \pi T from below. On the other hand, at high temperatures the vector screening mass turns out to be larger than the free continuum value of 2 \pi T. The pseudo-scalar and the vector screening masses do not become degenerate even for a temperature as high as 4Tc. Using these mesonic spatial correlation functions we have also investigated the restoration of chiral symmetry and the effective restoration of the axial symmetry. We have found that the vector and the axial-vector screening correlators become degenerate, indicating chiral symmetry restoration, at a temperature which is consistent with the QCD transition temperature obtained in previous studies. On the other hand, the pseudo-scalar and the scalar screening correlators become degenerate only at temperatures larger than 1.3Tc, indicating that the effective restoration of the axial symmetry takes place at a temperature larger than the QCD transition temperature.Comment: Published versio

Chlamydophila pneumoniae induces expression of Toll-like Receptor 4 and release of TNF-α and MIP-2 via an NF-κB pathway in rat type II pneumocytes

Abstract Background The role of alveolar type II cells in the regulation of innate and adaptive immunity is unclear. Toll-like receptors (TLRs) have been implicated in host defense. The purpose of the present study was to investigate whether Chlamydophila pneumoniae (I) alters the expression of TLR2 and/orTLR4 in type II cells in a (II) Rho-GTPase- and (III) NF-κB-dependent pathway, subsequently (IV) leading to the production of (IV) pro-inflammatory TNF-α and MIP-2. Methods Isolated rat type II pneumocytes were incubated with C. pneumoniae after pre-treatment with calcium chelator BAPTA-AM, inhibitors of NF-κB (parthenolide, SN50) or with a specific inhibitor of the Rho-GTPase (mevastatin). TLR2 and TLR4 mRNA expressions were analyzed by PCR. Activation of TLR4, Rac1, RhoA protein and NF-κB was determined by Western blotting and confocal laser scan microscopy (CLSM) and TNF-α and MIP-2 release by ELISA. Results Type II cells constitutively expressed TLR4 and TLR2 mRNA. A prominent induction of TLR4 but not TLR2 mRNA was detected after 2 hours of incubation with C. pneumoniae. The TLR4 protein expression reached a peak at 30 min, began to decrease within 1–2 hours and peaked again at 3 hours. Incubation of cells with heat-inactivated bacteria (56°C for 30 min) significantly reduced the TLR4 expression. Treated bacteria with polymyxin B (2 μg/ml) did not alter TLR4 expression. C. pneumoniae-induced NF-κB activity was blocked by TLR4 blocking antibodies. TLR4 mRNA and protein expression were inhibited in the presence of BAPTA-AM, SN50 or parthenolide. TNF-α and MIP-2 release was increased in type II cells in response to C. pneumoniae, whereas BAPTA-AM, SN50 or parthenolide decreased the C. pneumoniae-induced TNF-α and MIP-2 release. Mevastatin inhibited C. pneumoniae-mediated Rac1, RhoA and TLR4 expression. Conclusion The TLR4 protein expression in rat type II cells is likely to be mediated by a heat-sensitive C. pneumoniae protein that induces a fast Ca2+-mediated NF-κB activity, necessary for maintenance of TLR4 expression and TNF-α and MIP-2 release through possibly Rac and Rho protein-dependent mechanism. These results indicate that type II pneumocytes play an important role in the innate pulmonary immune system and in inflammatory response mechanism of the alveolus.</p