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Figure 5 | Arthritis Research & Therapy

Figure 5

From: Expression and regulation of CCL18 in synovial fluid neutrophils of patients with rheumatoid arthritis

Figure 5

CCL18 induction of polymorphonuclear neutrophils co-cultured with endothelial cells requires cell–cell contact and TNF-α. (a) CCL18 levels were measured by ELISA in culture supernatants of 5 × 105 peripheral blood mononuclear cells, 5 × 106 polymorphonuclear neutrophils (PMN) or 5 × 105 EA-hy.926 cells alone or after co-culture with EA-hy.926 cells after incubation for 48 hours. Direct cell–cell contact of PMN and EA-hy.926 cells was prevented by Boyden chambers (bc) as indicated. Data represent the geometric mean ± SEM of CCL18 measured in four independent experiments performed in duplicate. (b) CCL18 levels were measured in culture supernatants of PMN and EA-hy.926 cells alone or after co-culture of both cell types after incubation for 48 hours. Cultures were supplemented with anti-TNF-α antibodies (infliximab; 50 μg/ml) or 10 ng/ml TNF-α as indicated. Data represent the geometric mean ± SEM of three independent experiments performed in duplicate. (c) RNA was prepared from PMN and EA-hy.926 cells alone or after co-culture of both cell types after incubation for 24 hours. Cultures were supplemented with TNF-α (10 ng/ml) as indicated. RNA samples were amplified by semiquantitative RT-PCR with primers for CCL18 and actin and subjected to gel electrophoresis. This result is representative of three independent experiments with PMN from three different donors. (d) TNF-α levels were measured by ELISA in culture supernatants of PMN and EA-hy.926 cells alone and in co-cultures of PMN and EA-hy.926 cells before or after γ-irradiation (40 Gy) of one of these cell types. The irradiated cell type is marked with an asterisk. Data represent the geometric mean ± SEM for three independent experiments performed in duplicate.

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