The maximal IL 8 amounts can only be gen erated if the resulting mRNA, after NF B translocation, is rapidly stabilized by the p38 MAPK pathway. Block ing the p38 MAPK pathway by SB203580 decreased the amount of IL 8 generation suggesting that p38 MAPK pathway involves in the maximal amounts of IL 8 produc tion after CS exposure. More studies are needed to demon strate that whether Zotarolimus(ABT-578)? the role of p38 MAPK pathway is to stabilize IL 8 mRNA after CS stimulation or the pathway at least partially activates NF B activation. It has been demonstrated that SB203580 attenuates lysophosphatidic acid dependent phosphorylation of I B, NF B and NF B transcription in human bronchial epithelial cells. These findings suggest that SB203580 by itself might be involved in NF B activation.

The increases in phosphor ylated I B levels as well as the dramatic decline in amount of IL 8 secretion by NF B inhibitor showed that NF B activation is involved in the pathways of CS stimulation of human macrophages. The five members of the mammalian NF B family, p65, RelB, c Rel, p50 p105, and p52 p100, exist in unstimulated cells as homo or het erodimers bound to I B family proteins. NF B activa tion leads to the translocation of thetranscription factors from the cytoplasm to the nucleus. We studied the translocation of NF B subunit p65 to the nucleus fol lowing CS activation. We detected an increase in p65 level in nuclear protein extracts following CS exposure. Furthermore, no p65 was detected in the nuclear pro tein extracts where the cells were pre treated with anti TLR4 prior to CS medium stimulation.

These findings confirm that CS induces NF B translocation to the nucleus and that this can be inhibited by blockade of TLR4. In conclusion, the results presented here show that the mechanism underlying IL 8 production by human macro phages after CS medium exposure involves activation of TLR4 specific signaling pathways. It has to be stressed at this point that a secreted TLR2 agonist from different bac terial LPS, induced distinct patterns of cytokine produc tion by macrophages. Therefore, we can not rule out the possibility of the ligation of TLR4 by different LPS or bacterial endotoxins present in CS medium. However, these compounds are part of CS extract and must be con sidered as one of the inflammatory stimuli of CS consti tutes which might triggers initial lung inflammation in COPD.

In our study no analysis done to characterize the chemical nature of the activity present in CS medium. For instance, our CS medium may contain reactive oxygen species which activates NF B and may regulate immune signaling through TLR4. Further studies are needed to Entinostat address firstly the presence of ROS in our CS medium preparations and secondly the possible ROS interaction with TLR4 signaling. During the preparation of the manuscript, Droemann et al.