To examine the objection, firstly, the effects of CS on pro inflammatory cytokine secretion including many IL 8 were evaluated. Then, the involvement of TLR2 and TLR4 in IL 8 production was studied and, finally, signaling pathways in human mono cyte derived macrophages after exposure to CS medium were investigated. The findings explain the possible mech anisms behind the initial inflammatory process in lungs. Methods Isolation of PBMC and culture of human monocyte derived macrophages Peripheral blood mononuclear cells were sepa rated by density gradient centrifugation of buffy coats obtained from normal blood donors. Thereafter, neutrophils were pre pared by centrifugation on a Percoll density gradient. The remained cells used for preparation of lymphocyte fraction by centrifugation on a Percoll density gradient.

Human blood monocytes were obtained using RosetteSep according to manufacturers instructions. Briefly, fresh blood was incubated with RosetteSep cocktail at room temperature followed by Ficoll Paque gradient centrifuga tion. The enriched monocytes were collected from the Ficoll plasma interface and purity was assessed by FACS analysis using a FITC labeled anti CD14 mAb. Macrophages were obtained by culturing monocytes for 5 days in medium containing 2. 5 ng ml GM CSF and 25 ng ml M CSF, as described before. CS medium preparation CS medium was prepared as described before. Briefly, a smoking machine was used to direct main and side stream smoke from one cigarette through 5 ml culture medium.

Hereafter, absorbance was measured spectrophotometrically and the media was standardized to a standard curve of CS medium concentration against absorbance at 320 nm. This concentration was serially diluted with untreated media and applied to the cells. Freshly prepared CS medium was used in all experiments. Nontoxic concentrations of CS medium were detected performing different toxicological assays and FACS analysis. Quantification of human cytokines Cells were plated at a density of 5 105 cells ml in 96 well cell culture plates and stimulated with different concen trations of CS medium or LPS for overnight. In defined experiments, cells were pretreated with SB 203580 or curcumin for 30 min before stimulation with CS medium. Hereafter, supernatants were collected and stored at 20 C prior to cytokine quantification. Commer cially available enzyme linked immunosorbent assay kits or Cytometric Anacetrapib Beads Array kits were used to quantify cytokine secretion according to the manufacturers instructions. For CBA, analyses were run on a FACSCali bur. Quadruplicate samples were mixed and used as a sample for the assay.