IVIg inhibits the constitutive maturation of monocyte-derived DCs.
(A) DCs were generated as described in “Materials and methods” in the presence of GM-CSF and IL-4. At day 5, immature DCs were cultured in the presence of IVIg (0.15 mM; middle row) or the absence of IVIg (Ctl, top row) for an additional 48 hours. Equimolar concentration of human serum albumin (HSA; bottom row) was used as control. At day 7, cells were harvested and analyzed for the expression of surface molecules using flow cytometry with labeled antibodies. Percentages of cells positive for the indicated markers are depicted in upper right corners. Results shown are representative of 4 independent experiments from different donors. (B) The inhibitory effect of IVIg on DC is mediated by IgG molecules. IgG molecules were affinity purified from Sandoglobulin on Protein G column followed by gel filtration. Five-day-old DCs were incubated with affinity-purified IgG (0.15mM) for 48 hours. Cells treated with medium alone (control) are denoted by thick lines, and thin lines represent IgG-treated cells. (C) DCs obtained by culturing monocytes for 5 days were incubated independently in the presence of equimolar concentrations (0.15 mM) of IVIg (thin lines) or F(ab′)2 fragments of IVIg (dashed lines) or Fc fragments (thick lines) for 48 hours (bottom row). Top row shows results of control experiment. Cells were analyzed for the expression of surface molecules using flow cytometry, and data were processed using CellQuest software.
