Figure 6.
DCs derived from TLR-4–deficient mice are not sensitive to sHA stimulation. (A) Bone marrow–derived DCs were obtained from either C3H/HeN (wild-type) and C3H/HeJ (TLR-4 mutant) or C57BL/10ScSn (wild-type) and C57BL/10ScCr (TLR-4 mutant). On day 6 of culture the DCs were either incubated with 100 ng/ml of bacterial cell wall lysates containing LPS and other substances such as lipoprotein, or 100 ng/ml highly purified LPS from Salmonella abortus equi or Salmonella minessota strain R595 as positive and negative controls. Alternatively, 10 or 50 μg/ml sHA or 100 μg/ml HMW-HA were added. After 48 h of treatment the cell free supernatants were screened for their TNF-α content by ELISA. Data represent the mean TNF-α release of triplicate wells; pg/mg total protein ± SD. (B) DCs were treated for 48 h with the same substances described in A, washed, and coincubated for 4 d with 105 alloreactive T cells at a DC/TC ratio of 1:20. T cell proliferation was determined on day 5 by addition of 1 μCi of 3[H]thymidine for the final 18 h. Results are shown in counts per minute (CPM) ± SD of triplicate wells. (C) Day 6 bone marrow–derived DCs of either C57BL/10Sn (black bars) or C57BL/10Cr (white bars) were incubated for 24 h with 30 μg/ml sHA or 100 ng/ml LPS and/or pretreated with 10 mg/ml polymyxin B (polyB) 1 h before stimulation. After 24 h of incubation DCs were stained for Iab, B7–1, and B7–2 expression and analyzed by flow cytometry. Results are shown a mean fluorescence intensities (MFI).