Induction of CCL18 mRNA and protein in synovial fluid and blood polymorphonuclear neutrophils. (a) Synovial fluid polymorphonuclear neutrophils (SF PMN) of patients with rheumatoid arthritis (RA) and blood PMN from healthy donors and patients with RA were incubated with 20 ng/ml IL-10, 10-7 M vitamin D3 and 10 ng/ml TNF-α for 48 hours. Levels of CCL18 in the supernatant were determined by ELISA. Data represent the geometric mean ± SEM for three independent experiments performed in duplicate. (b) PMN from healthy donors were incubated for 24 hours with various amounts of recombinant TNF-α. Total RNA was amplified by semiquantitative RT-PCR with primers for CCL18 and actin and subjected to agarose gel electrophoresis. This result is representative of three independent experiments with PMN from three different donors. (c) Blood PMN from healthy donors were incubated in the presence or absence of SF from patients with RA for 10 hours, then washed twice and incubated for a further 48 hours with 20 ng/ml IL-10 and 10-7 M vitamin D3. Data represent the geometric mean ± SEM for three independent experiments performed in duplicate.