Haworth C, Brennan FM, Chantry D, Turner M, Maini RN, Feldmann M. As confirmed by tradition supernatant transfer experiments, maximal inhibition of T cell activation was also dependent on the direct contact between the T cells and GM-CSF-treated monocytes during antigen demonstration. Collectively, these results imply that GM-CSF can either inhibit or enhance the re-stimulation of primed T cells by antigen-presenting monocytes or MoDC, respectively. (Dpt) was purchased from ARTU Biologicals (Lelystad, The Netherlands). The content of the major allergen Der p1 in the lot used was 185 g/mg of Dpt protein draw out. Indomethacin was from Sigma (St Louis, MO). Isolation and tradition of cells The human being T helper cell clone CFTS 4:3.1 (TCC) was isolated from a skin punch biopsy of a patient with atopic dermatitis as described previously [19,20]. It specifically recognizes the major allergen of house dust mite, Der p1, in association with the MHC class II restriction molecule HLA-DPw4 (P. Baselmans = 8), the proliferative response induced by monocytes when used at a 1:1 percentage (4 104 APC/well) was comparable to the degree of T cell proliferation stimulated by MoDC at a 1:10 percentage (4 103 APC/well). Consequently, these numbers of APC/well were used in subsequent experiments to allow for a assessment between the relative stimulatory capacities Pectolinarigenin of MoDC and monocytes. Open in a separate windowpane Fig. 1 Monocyte-derived dendritic cells (MoDC) are more potent stimulators of T cell clones (TCC) than monocytes or autologous EpsteinCBarr disease (EBV)-B cells. Different numbers of antigen-pulsed antigen-presenting cells (APC), autologous EBV-transformed B cells (?) or allogeneic MoDC (?) and monocytes (?) from HLA-DPw4 matched donors, were co-cultured with TCC (4 104 cells/well). The proliferative response Pectolinarigenin was identified at day time 3. The results represent the mean ct/min ( s.e.m.) of three different experiments. Proliferation ideals of non-(Dpt)) for 20 h in presence or absence of GM-CSF (300 U/ml) or with GM-CSF and IL-4 (300 U/ml and 120 U/ml, respectively). Data symbolize the imply ct/min ( s.e.m.) of at least four self-employed experiments, except for GM-CSF-treated MoDC, where two experiments were performed. Proliferation ideals of non-Dpt-stimulated TCC have been subtracted. GM-CSF-treated monocytes secrete PGE2 and a variety of immunomodulatory cytokines GM-CSF has been reported to induce the secretion of prostaglandins by monocytes [5,22,23]. Consequently, supernatants harvested after 20 h from monocyte cultures comprising GM-CSF or the combination of GM-CSF and IL-4 were assayed for the presence of PGE2 by specific ELISA. As demonstrated in Table 1, high levels of PGE2 were found in the supernatants of GM-CSF-treated monocytes. The production of PGE2 was almost completely suppressed when Pectolinarigenin indomethacin was present during the incubation of monocytes with GM-CSF. IL-4 also inhibited GM-CSF-induced PGE2 production to about 90%. Table 1 Effect of GM-CSF, IL-4, and indomethacin on cytokine production by monocytes* in the absence or presence of either GM-CSF or GM-CSF and indomethacin (indo) or IL-4. Cell-free supernatant was collected 24 h after specific T cell activation. The percent of TCC response ( s.e.m.) is definitely shown relative to that induced by antigen-pulsed non-treated monocytes. The levels of IL-10 and PGE2 are indicated as the mean ( s.e.m.) of at least three self-employed experiments. TCC proliferation is definitely partially restored by indomethacin, anti-IL-10 antibody or exogenous IL-2 Since earlier reports shown IL-10 to inhibit antigen demonstration by mononuclear cells, we tested whether the addition of neutralizing anti-IL-10 antibody only or in combination with indomethacin would restore the ability of monocytes to fully activate the TCC. As demonstrated in Fig. 4, antigen pulsing of monocytes in the presence of GM-CSF and anti-IL-10 antibody slightly relieved inhibition of YAP1 TCC proliferation. This effect was comparable to that achieved by obstructing PGE2 through the addition of indomethacin. However, by combining anti-IL-10 antibody with indomethacin at the time of monocyte incubation with antigen and GM-CSF, T cell proliferation was restored to about two-thirds of the level that was stimulated by monocytes pulsed in the absence of GM-CSF. Moreover, if indomethacin was supplemented to the co-culture of monocytes and T cells in order to block ongoing PGE2 synthesis, T cell growth was still inhibited by about 35% compared with control levels of TCC.