Figure 3.

Expression and function of GPR84 in human THP-1 monocytes. THP-1 monocytes were treated with or without LPS (100 ng.ml⁻¹) for 24 h. After membrane preparation the specific binding of either varying concentrations of the GPR84 antagonist [³H]9543 was measured in membranes from LPS-treated THP-1 cells to allow estimation of both K_d and Bmax (a) or the specific binding of a single concentration of [³H]9543 that was close to 10 times the estimated K_d was measured in membranes from both vehicle and LPS- treated cells (b(i)). Data in a are from a representative experiment of four separate studies in which B_max = 293 ± 55 and K_d = 0.18 ± 0.02 nM, whilst in b(i) they are means ± S.E.M. (n = 7). LPS-treated cells expressed significantly higher amounts of the receptor ***p < 0.001 (two-tail t-test). Equivalent assessments of levels of GPR84 mRNA were conducted (b(ii)). Data are means ± S.E.M. (n = 6) *p < 0.05 (two-tail t-test). (c) LPS treatment was associated with increased levels of basal binding of [³⁵S]GTPγS (n = 4) ***p < 0.001 (two-tail t-test). The ability of varying concentration of 2-HTP (d,e) and PSB-16671 (f,g) to promote binding of [³⁵S]GTPγS was measured in membranes of both untreated (vehicle) and LPS-treated cells. In (d,f) data are shown from representative experiments and illustrates the enhanced efficacy of the ligand following LPS treatment, whilst in e and g data pooled from multiple experiments were normalised and illustrate that the potency of the ligands was not affected by the upregulation of GPR84.