During the experimental period, the glial cells were incubated in the absence and presence of recombinant HSPs, including recombinant HSP60 or -70 containing low endotoxin (10 μg/mL; Assay Designs), or H
2O
2 (50 μM; Sigma-Aldrich) for 48 hours. Any effect from possible endotoxin contamination was excluded in the control experiments by using the HSPs pretreated with proteinase K or boiled for 20 minutes before incubation. Positive controls included interferon-γ (IFN-γ; 20 ng/mL; R&D Systems), an inducer of glial antigen presentation,
24 and purified
Escherichia coli LPS (100 ng/mL; Sigma-Aldrich), a TLR4 ligand.
17,18 In the in vitro experiments, we also used specific treatments to inhibit MyD88 or TIRAP (50 μM; Imgenex, San Diego, CA), both of which are adaptor proteins involved in TLR signaling. The MyD88 inhibitor was a cell-permeable peptide containing a sequence from the MyD88 TIR homodimerization domain that blocks MyD88 homodimerization, thereby inhibiting MyD88-dependent TLR signaling.
32,33 The second inhibitor was a cell-permeable peptide that contains a TIRAP sequence and functions as a TIRAP decoy by binding to TIR-interacting domains on specific TLRs, thereby blocking the TIR–TIR domain interaction between TIRAP and the receptor.
34,35 These inhibitor treatments were applied 24 hours before the incubation with HSPs or H
2O
2. Control cultures, prepared from cells of an identical passage, were simultaneously incubated in the absence of these treatments. At the end of the experimental period, glial expression of TLRs and MHC class II was determined by Western blot analysis, and TNF-α levels were measured in the glial conditioned medium by enzyme-linked immunosorbent assay (ELISA). In addition, the antigen-presenting function was determined by coculturing glial cells with T cells, as previously described.
24 At the end of the 48-hour co-incubation period, T-cell proliferation and cytokine secretion were determined. Glial cells were treated with mitomycin C (100 μg/mL; Sigma-Aldrich) for 1 hour, before they were mixed with the T cells, to exclude the possibility that the cytokine production in co-cultures would be from glial cells.
36 Wells without antigen were simultaneously processed as the control. In addition, untreated co-culture wells and control wells containing T cells cultured alone were included in each plate. Cell viability was determined with a live/dead kit containing calcein AM (Molecular Probes) as previously described.
4,16,22,37 All in vitro experiments were performed in triplicate wells and independently repeated three times in separate cell cultures. An integrated value was obtained for treated and untreated cultures after background subtraction using negative controls. Statistical differences between treated and untreated cultures were analyzed by Mann-Whitney rank sum test, using these normalized values.