Soluble CD14 Discriminates Slight Structural Differences between Lipid As That Lead to Distinct Host Cell Activation

MS/MS spectra at m/z 1948 of F. nucleatum lipid A (A) and two corresponding proposed structural patterns (B and C).

TLR4/MD-2-specific responsiveness to F. nucleatum lipid A. A, Peritoneal macrophages from C3H/HeN (○) and C3H/HeJ (•) mice were stimulated with the indicated doses of test specimens for 24 h in the presence of 5% FBS. After incubation, the supernatants were collected and IL-6 production was determined by ELISA. Experiments were done at least three times, with representative results presented. Each assay was done in triplicate and the data are expressed as the mean ± SD. The mean values were significantly different from medium alone. ∗, p < 0.01. B, Ba/κB, Ba/mTLR2, and Ba/mTLR4/mMD-2 cells were stimulated with 10 ng/ml of the test specimens for 4 h in the presence of 5% FBS, after which luciferase activities were measured. Results are shown as relative luciferase activity, which was determined as the ratio of stimulated to nonstimulated activity. C, Ba/κB, Ba/mTLR2, and Ba/mTLR4/mMD-2 cells were stimulated with the indicated doses of the LPL- or nontreated test specimens for 4 h in the presence of 5% FBS, after which luciferase activities were measured.

LAL activity of F. nucleatum lipid A. The indicated doses of F. nucleatum lipid A (•) and compound 506 (○) were mixed with LAL reagent, after which LAL activity was determined using a quantitative chromogenic assay. Representative results from three independent experiments are presented.

Contribution of FBS to lipid A-induced cytokine production. Cell surface expressions of mCD14 on human monocytes (A) and HGF (D) were determined with a specific Ab (solid line) or its isotype control (dotted line), as described in Materials and Methods. Human monocytes (B and C) and HGF (E and F) were stimulated with the indicated doses of F. nucleatum lipid A (•) or compound 506 (○) for 24 h in the absence (B and E) or presence (C and F) of 5% FBS. After incubation, the supernatants were collected, and TNF-α (B and C) and IL-8 (E and F) were determined by ELISA. Each assay was done in triplicate and the data are expressed as the mean ± SD. The mean values were significantly different from F. nucleatum lipid A at each dose. ∗, p < 0.01.

Contribution of FBS to lipid A-induced up-regulation of costimulatory molecule. Murine peritoneal exudate cells were stimulated with the indicated doses of F. nucleatum lipid A (circle) or compound 506 (square) for 24 h in the absence (open symbol) or presence (closed symbol) of 5% FBS. After incubation, the cells were stained with Abs specific for CD80 and CD86, or an isotype-matched control. Values in the histograms represent the MFI of cells stained with CD80 (A) and CD86 (C), and the graph is representative of results from three experiments. Representative results for the expressions of CD80 (B) and CD86 (D) on cells stimulated with (bold line) or without (thin line) 10 ng/ml of the test specimens are shown. Dotted lines indicate cells stained with the isotype control Ab. Values indicated in the histograms represent the MFI of cells stained with each Ab.

Contribution of FBS to lipid A-induced TRIF/TICAM-1-dependent cell activation. A, IFN-β mRNA expression in murine peritoneal macrophages stimulated with the indicated doses of F. nucleatum lipid A (FnLA) or compound 506 (506) for 3 h in the absence or presence of 5% FBS. B and C, Peritoneal macrophages were stimulated with the indicated doses of FnLA (•), compound 506 (○), or Pam₃CSK₄ (▵) for 24 h in the absence (B) or presence (C) of 5% FBS. After incubation, the supernatants were collected and IP-10/CXCL10 production was determined by ELISA. Experiments were done at least three times, with representative results presented. Each assay was done in triplicate and the data are expressed as the mean ± SD. The mean values were significantly different from FnLA at each dose. ∗, p < 0.01.

Contribution of sCD14 to lipid A-elicited NF-κB activation via human TLR4. Ba/hTLR4/hMD-2 cells were stimulated with the indicated doses of F. nucleatum lipid A (•) or compound 506 (○) for 4 h under the following culture conditions: medium alone (A), 1% FBS (B), 500 ng/ml CD14 (C), and 500 ng/ml CD14 together with 50 ng/ml LBP (D). NF-κB activation was measured using a luciferase assay. Results are shown as relative luciferase activity, which was determined as the ratio of stimulated to nonstimulated activity.