PMC

The peroxisome proliferator-activated receptor γ (PPARγ) antagonist GW9662 partially reverses the effect of human serum upon dendritic cell (DC) CD1 expression. Fetal bovine serum (FBS)-DCs or human serum (HS)-DCs were generated as previously described in the presence or absence of the PPARγ antagonist GW9662 (10 μm) for 3 days. Cells were then stained with monoclonal antibody (mAb) against CD1a and CD1d and analysed by flow cytometry. (a) The presence of GW9662 had no effect upon FBS-DC CD1a expression but partially reversed the inhibition of CD1a in HS-DCs. (b) GW9662 had no effect upon FBS-DC CD1d expression but reversed the up-regulation of CD1d in HS-DCs. These results are representative of three independent experiments using different DC donors.

Human serum inhibits group 1 CD1 expression by dendritic cells (DCs). CD14⁺ monocytes were cultured for 72 hr in interleukin (IL)-4 and granulocyte–macrophage colony-stimulating factor (GM-CSF) in fetal bovine serum (FBS)-containing medium (FBS-DCs), or similar medium containing additional 10% human pooled AB serum [human serum (HS)-DCs] to derive immature monocyte-derived DCs. Cell surface expression of CD1a, CD1b, CD1c, CD1d, CD14, CD209, and human leucocyte antigen (HLA)-DR were assessed by monoclonal antibody (mAb) staining and flow cytometry. The presence of human serum (HS-DCs) inhibited expression of CD1a, CD1b and CD1c and promoted up-regulation of CD1d. HS-DCs also lacked expression of CD14 and expressed CD209, consistent with a DC phenotype. These results are representative of five independent experiments using different DC donors.

Human serum regulates dendritic cell (DC) activation of CD1-restricted T cells. (a) CD1c-restricted T cells do not proliferate in response to co-culture with human serum (HS)-DCs. CD1c-restricted autoreactive Ye2.3 (left panel) or CD1d-restricted, α-galactosyl ceramide (α-GalCer)-reactive J3N.5 T cells (right panel) were incubated with fetal bovine serum (FBS)-DCs or HS-DC for 72 hr and proliferation was measured. For J3N.5, α-GalCer was used at a final concentration of 100 ng/ml. FBS-DCs, expressing both CD1c and CD1d, produced proliferation of both CD1c-reactive Ye2.3 and CD1d-reactive J3N.5 T cells. Note that HS-DCs expressing lower cell surface levels of CD1c failed to activate CD1c-restricted Ye2.3. (b) CD1c-restricted T cells do not produce interferon (IFN)-γ in response to HS-DCs. CD1-restricted T cells were cultured with DCs as described above. Note that CD1c-restricted Ye2.3 T cells do not make IFN-γ in response to culture with HS-DCs. Results represent triplicate samples ± standard error of the mean (SEM). These data are representative of three independent experiments using different DC donors.

Human serum regulates dendritic cell (DC) expression of CD1 at the level of transcription. (a) Cell surface expression of CD1 is not a result of altered cellular distribution of CD1. Fetal bovine serum (FBS)-DCs (upper panels) or human serum (HS)-DCs (lower panels) were generated as described and stained with monoclonal antibodies (mAbs) against CD1 molecules (green staining) and the lysosomal marker lysosome-associated membrane glycoprotein 1 (LAMP-1) (red staining). Note that, in contrast to FBS-DCs, HS-DCs express no group 1 CD1a, CD1b or CD1c but express increased levels of CD1d. (b) Alteration of DC CD1 expression is regulated at the level of transcription. CD1 mRNA expression by FBS-DCs and HS-DCs was assessed using quantitative real-time polymerase chain reaction. Results are expressed as relative fold change in HS-DCs compared with FBS-DCs. Note that, while HS-DCs have decreased transcription of CD1a, CD1b and CD1c, CD1d is increased when compared with FBS-DCs. (c) Inhibition of DC CD1a expression is reversible. Left panel: FBS-DCs were differentiated as described and at day 3 (red arrow) 10% human serum (red triangles, dashed line) was added to cultures and inhibited further acquisition of CD1a. In contrast, cells continued in culture without the addition of human serum continued to increase cell surface expression of CD1a (squares, solid line). Right panel: HS-DCs were generated over a 3-day culture period and at day 3 (red arrow) human serum was removed from cultures and CD1a cell surface expression increased over the next 48 hr (red triangles, dashed line) while cultures in which human serum remained failed to express CD1a (squares, solid line). These results are representative of three independent experiments using different DC donors. MFI, mean fluorescence intensity.

Human serum dendritic cells (DCs) are refractory to lipopolysaccharide (LPS)-induced maturation. (a) Human serum (HS)-derived DCs fail to express the maturation marker CD83, up-regulate the costimulatory molecule CD86, or produce interleukin (IL)-12p70 in response to LPS. Fetal bovine serum (FBS)-DCs or HS-DCs were generated as described and were further cultured in the presence of LPS (1 μg/ml) for 3 days to induce maturation. DCs were then assessed by monoclonal antibody (mAb) staining and flow cytometry or enzyme-linked immunosorbent assay (ELISA) to assess IL-12p70 production. Note that HS-DCs express less CD83 and CD86 and fail to produce IL-12p70 in response to LPS as a maturation stimulus. Error bars on the IL-12p70 ELISA represent the standard error of the mean (SEM) from triplicate samples. (b) HS-DCs are less effective at supporting an allogeneic mixed lymphocyte reaction (MLR). Immature FBS-DCs or HS-DCs were derived as described and cultured for an additional 72 hr in the presence of medium alone or LPS (100 ng/ml) as a maturation signal. DCs were then irradiated and used to stimulate allogeneic CD4⁺ T cells. Note that maximum allogeneic stimulatory capacity was decreased for immature HS-DCs (right panel; closed squares) when compared with immature FBS-DCs (left panel; closed squares). Additionally, LPS (open squares) had a lesser effect upon increasing the ability of HS-DCs to stimulate allogeneic T cells than upon the ability of FBS-DCs to stimulate these T cells. These results are representative of three independent experiments using different DC donors. Results represent triplicate samples ± SEM. c.p.m., counts per minute; MFI, mean fluorescence intensity.

Polar human serum (HS) lipids inhibit dendritic cell (DC) expression of group 1 CD1 molecules. (a) Inhibition of DC group 1 CD1 expression is mediated by organic soluble molecules. Human serum was separated into aqueous, interphase and organic fractions and added to CD14⁺ cells differentiated to DCs in the presence of interleukin (IL)-4 and granulocyte–macrophage colony-stimulating factor (GM-CSF) in fetal bovine serum (FBS)-containing medium. DCs were also derived in the presence of similar medium containing 10% HS as a positive control. Cells were analysed for CD1a, CD1b and CD1c cell surface expression by flow cytometry. Note that the organic phase of human serum contained the CD1 inhibitory activity. (b) Polar serum lipid fractions inhibit CD1a. Total human serum organic extract was separated using silica column elution with progressively polar solvents (chloroform, followed by acetone, and then methanol). CD14⁺ cells were cultured in medium alone without cytokines (open diamond) or differentiated to DCs in IL-4 and GM-CSF in medium containing FBS (open square), similar medium plus 10% HS (open triangle), total HS organic extract (inverted open triangles), HS chloroform extract (solid circles), HS acetone extract (solid squares), or HS methanol extract (solid triangles). Cells were then assessed for CD1a expression by monoclonal antibody (mAb) staining and flow cytometry. Note that the more polar acetone and methanol fractions inhibited DC expression of CD1a. (c) Lysophosphatidic acid (LPA) and cardiolipin (CL) inhibit CD1a expression. FBS-DCs or HS-DCs were generated as described. Additionally, additional FBS-DCs were cultured in the presence of LPA or CL at concentrations of 1 and 10 μm. DCs were then assessed for CD1a cell surface expression by mAb staining and flow cytometry. Note that LPA and CL inhibited CD1a expression at both 1 and 10 μm. These results are representative of four independent experiments using different DC donors. MFI, mean fluorescence intensity.

Human serum lipids activate peroxisome proliferator-activated receptor γ (PPARγ). (a) Human serum (HS) activates PPARγ. HCT-116 colon cancer cells were transiently transfected with a DR1- PPAR-response element (PPRE)-luciferase reporter construct and cultured for 18 hr in the presence of medium alone or in the presence of increasing concentrations of human serum (1–10%) to assess activation of PPARγ. Cells were then lysed and luciferase activity was measured. Fold induction was calculated by comparing the ratio of activity in human serum-treated cells compared with medium control cells. Note that increasing the percentage of human serum (from 1 to 10%) led to increased PPARγ activity. (b) Human serum polar lipid fractions activate PPARγ. HCT-116 cells transfected with the PPRE-luciferase construct were cultured as described above in the presence of medium (negative control), the non-polar human serum chloroform fraction, more polar human serum acetone and methanol fractions, or the PPARγ agonist troglitazone. Cells were then assayed for luciferase activity. Note that the more polar human serum methanol and acetone eluted fractions caused approximately a twofold induction of luciferase activity while the non-polar chloroform-eluted human serum had no effect upon activation of PPARγ. (c) Lysophosphatidic acid (LPA) and cardiolipin (CL) activate PPARγ. LPA and CL, which inhibit DC expression of group 1 CD1 molecules, were assayed for PPARγ activity as described above. Troglitazone was used as a positive control. Note that both CL and LPA led to increased induction of PPARγ activity. Results represent triplicate samples ± standard error of the mean (SEM). (d) Cotransfection of the PPRE luciferase reporter with plasmids expressing PPARβ/δ, PPARγ, or empty vector. Transfected cells were treated with the indicated compounds for 24 hr prior to luciferase assays. Error bars are the SEM for triplicate samples. A two-way analysis of variance (ANOVA) was performed comparing PPAR-expressing plasmids with the vector controls for each treatment group. The statistical significance is indicated: *, P < 0·05; **, P < 0·01; ***, P < 0·001. (e) Agarose gel showing specific bands amplified from mRNA derived from whole blood monocytes (lane 1) and the same cell preparation after 3 days in culture using standard media containing fetal bovine serum (FBS) with granulocyte–macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 (lane 2). (f, g) Real-time polymerase chain reaction of human DC mRNA using probes for (f) human PPARγ angiopoietin related (PGAR) and (g) human fatty acid binding protein 4 (aP2). For panels (f) and (g), the * symbol indicates P < 0·05 using a one-way analysis of variance (ANOVA) test for each treatment compared to the value for each gene in monocytes. RGA, rosiglitazone.