For all those stimulator to T-cell ratios, the CD1a+ cells were the most potent T-cell stimulators

For all those stimulator to T-cell ratios, the CD1a+ cells were the most potent T-cell stimulators. in experimental animals. This appears to play a role in a predominant Th2 cell stimulating potential of DC from the lung environment. DEX exposure of CD1a+ LAF MF498 cells prevented the upregulation of even this low-level expression of CD80 and CD86. The veiled/dendritic morphology and the expression of other relevant cell surface markers and adhesion molecules was not affected by DEX exposure. It is concluded that DEX hampers the maturation of CD1a+ LAF cells into active lung DCs. function and marker expression of DCs isolated from the human lung. Recently, we described [18] the isolation, via FACS-sorting, of low Flt1 autofluorescent (LAF) cells from human bronchoalveolar lavage (BAL). These LAF cells matured, after being cultured overnight, into veiled/dendritic cells with the MF498 morphology and the immunophenotype of immature DCs. The cells showed a strong potency to stimulate naive T-cells, and both contained MF498 and released biologically active IL-1 and IL-6. We also described the marked differences between the CD1a+ and CD1a? subsets of LAF cells [19]. The CD1a+ subset exhibited a higher accessory capability than the CD1a? subset. CD1a+ LAF cells were very poor suppliers of IL-1, IL-6 and TNF-, whereas CD1a? LAF cells were potent producers of these cytokines. CD1a+ LAF cells were (after the overnight maturation period) both positive for and suppliers of S100; CD1a? LAF cells were unfavorable in this respect. We therefore concluded that the CD1a+ LAF cells could be regarded as examples of common DCs from the lung environment, because they can rapidly assume all the characteristics of Langerhans cell-like immature DCs [19]. The above-described method of obtaining common DCs from human BAL enabled us to study, = 24, smoking 17 7 (mean SD) smokes per day], because the yield of cells in smoking individuals is much larger and more workable than in nonsmoking individuals. We also studied the BAL cells of a few subjects who had never smoked (= 3). The mean age of the lavaged subjects was 36 years (range 18C53 years). The procedure was approved by the Medical Ethics Committee of the Erasmus University and University Hospital Dijkzigt. Isolation and purification of DC BAL cells were kept at 4C, washed twice in phosphate buffered saline (PBS) made up of 05% bovine serum albumin and 045% glucose and, subsequently, filtered through a 55 m and a 30 m gauze. BAL cells were sorted on a FACS-Vantage (Becton Dickinson, Erembodegem, Belgium) with a 488-nm laser. Sort windows were generated on autofluorescence (FL1, 530 nm) to create a populace of cells with low autofluorescence (LAF) in order to exclude alveolar macrophages (AM), on forward scatter (FSC) to exclude small MF498 cells (lymphocytes) and debris and on sideward scatter (SSC) to exclude cells with a high SSC (granulocytes and AMs). As described previously, this procedure yields a populace of LAF cells, comprised of DCs and their precursors, with small contaminations of AMs ( 10%) and lymphocytes ( 5%) [18]. A further purification was performed in experiments by sequential labelling of LAF cells with OKT6 (CD1a) conjugated with phycoerythrin and an additional sort windows on fluorescence channel 2 (FL2, 585 nm). Flow cytometry results were reported previously [19]. This method yields CD1a+ DCs with a contamination of AMs of 2%. Blood monocytes were purified according to techniques described in detail elsewhere [20]. In short, monocytes were isolated from heparin blood or buffy coats by subsequent FicollCPaque (1079 g/ml;.