LPS induced changes in the MHC class I antigen presentation pathway in
DCs. Immature CD34-derived LCs (A) and monocyte-derived
DCs (B) were activated by addition of 0.5 μg of LPS.
At time 0, 6, and 48 h post-LPS, cells were harvested and fixed,
and the expression of DC activation marker CD83, MHC class II, and MHC
class I was monitored by flow cytometry. (C) LPS
mediated a coordinate induction of MHC class I components. CD34-derived
LCs were induced with LPS for 0–48 h, after which the cells were
extracted in 1% Triton X-100, and membranes from separated lysates
were probed with antibodies specific for the indicated proteins. MCP20
is a constitutive, noninducible component of the human proteasome and
serves as a loading control for each time point. (D) LPS
increases TAP-mediated peptide transport in CD34-derived LCs.
Streptolysin O-permeabilized immature (□) and LPS-matured
(▵) CD34-derived LCs were incubated with the iodinated
RRYQNSTEL peptide at 37°C for the indicated time periods.
Translocation was assessed by binding of the hot, glycosylated reporter
peptide to Con A-Sepharose beads.