Chemical Hypoxia Brings to Light Altered Autocrine Sphingosine-1-Phosphate Signalling in Rheumatoid Arthritis Synovial Fibroblasts

Figure 5

Impact of SPL inhibition on CoCl₂-mediated chemokine/cytokine secretion in normal FLS and RAFLS. Human primary FLS from normal (a, c) and RA (b, d, e, f) donors were incubated with 200 μM CoCl₂ in the presence of SPL inhibitor SM4 (3 μM) or the inactive analog SM3 (3 μM) and sphingosine (1 μM) for 24 h. Where indicated, cells were pretreated with S1P₃ antagonist CAY10444 (5 μM) or S1P₂ antagonist JTE-013 (5 μM) for 30 min before stimulation with CoCl₂ in combination with sphingosine (Sph), SM4, or SM3. The data are the means ± SE from three experiments. For statistical comparative analyses, chemokine levels in the samples stimulated with CoCl₂ were compared to that of other samples (a–d) or chemokines produced by cells stimulated with CoCl₂ in combination with Sph and SM4 were compared to those produced by cells incubated with the S1P receptor antagonists prior to cell stimulation (f). ; ; . Cytokine/chemokine secretion in RAFLS supernatants was analyzed using Proteome Profiler Human Cytokine Array panel A (e). Circled pairs of duplicate spots represent one cytokine/chemokine.

(a)

(b)

(c)

(d)

(e)

(f)