The cytokines were measured in the cell culture supernatants; TNF-α after 4 h incubation at 37°C and IL-6, IL-10 and IL-12 after 18 h incubation at 37°C using commercial ELISA kits (Milenyi Biotec Thermo Scientific). The cytokine responses were measured without (spontaneous) and after stimulation and the values of the spontaneous secretion were deducted from the challenge values to allow for comparison with other, similar studies (e.g. [20]). Counting of white blood cells showed only minor differences in the monocyte/lymphocyte ratio between controls

and subjects with sarcoidosis. The participants GSK458 were supplied with a pump and filter holders preloaded with cellulose acetate filters (Mixed Cellulose Esters, 25 mm PCM Casettes, 0·8 µm pore size; Zeflon International Inc., Ocala, FL, USA). The subjects turned on the pump and sampling was performed for about 4 h. The exact volume sampled was read from a volume meter attached to the pump and was usually

MLN0128 about 2·5 m3. The filters were analysed for their content of N-acetylhexosaminidase (NAHA) using an enzyme technique [10,21]. One ml of a fluorogenic enzyme substrate (4-methylumbelliferyl N-acetyl-β-D-glucosaminide; Mycometer A/S, Copenhagen, Denmark) was added to the filter, followed by 2 ml of a developer after an incubation period of around 30 min, determined by the room temperature. The liquid was sucked out through the filter and placed in a cuvette. The fluorescence in the cuvette was read in a fluorometer (Picofluor; Turner Designs, Sunnyvale, CA, USA). To decrease the variance induced by methodological variations in the analysis technique, the fluorescence values were divided by 10 and rounded off to the nearest whole number to express NAHA activity in units Erastin (NAHA U/m3). Values in the different groups were calculated using spss version 17–W7 and expressed as mean and standard error of the mean. Differences between groups were evaluated using the Mann–Whitney

test and the intercorrelations assessed using Spearman’s-test. A P-value of < 0·05 was considered statistically significant. The spontaneous secretion of different cytokines from PBMC is reported in Table 1. The spontaneous secretion of all cytokines was higher from PBMC from subjects with sarcoidosis with significant differences for TNF-α and IL-12. Table 2 reports the secretion of cytokines after incubation with different FCWA and LPS. After stimulation with S-glucan the secretion of TNF-α was significantly higher among subjects with sarcoidosis, but there were no differences for the other cytokines. Stimulation with P-glucan caused a high secretion of all cytokines, which was significantly higher among subjects with sarcoidosis. Chitin was a comparatively weak inducer of cytokines in both groups except for IL-6, and no differences were found between controls and sarcoidosis.