Article Figures & Data
Figures
- Fig. 1.
Binding of FITC-labeled B. subtilis to human CD14 expressed on CHO cells. Cells were incubated with labeled bacteria in the presence or absence of 1 μg of recombinant LBP per ml. The marginal binding in the presence of LBP is abolished by inclusion of 10 μg of anti CD14 MAb My4 per ml in the incubation mixture but not by the same amount of an isotype-matched control. The ratio of cells to bacteria was 1:20.
- Fig. 2.
(A) Binding of serum-pretreated FITC-labeled B. subtilis to human CD14 expressed on CHO cells. Cells were incubated with labeled bacteria in the presence or absence of 1 μg of recombinant LBP per ml. Binding in the presence of LBP is abolished by inclusion of (i) a 20-fold excess of serum-pretreated unlabeledB. subtilis cells, (ii) Salmonella minnesota Re 595 LPS at a final concentration of 20 μg/ml, or (iii) 10 μg of anti CD14 MAb My4 per ml (but not by the same amount of an isotype-matched control). The ratio of cells to bacteria was 1:20. (B) Binding of plasma-pretreated FITC-labeled B. subtilis to human CD14 expressed on CHO cells. B. subtilis organisms were not pretreated (upper), pretreated with serum (middle), or pretreated with plasma (lower). Binding to CHO cells expressing human CD14 was determined in the presence of 1 μg of recombinant LBP per ml. (C) B. subtilis does not bind to the mutant CD14(39–41, 43–44A), which lacks the binding sites for LPS and E. coli.
- Fig. 3.
Serum treatment of B. subtilis opens up the bacterial cell wall. Mid-log-phase cells were examined by transmission electron microscopy either directly (A), after heat inactivation (B), or after heat inactivation and exposure to 10% NMS for 2 h at 37°C (C).
- Fig. 4.
LTA derived from the cell wall of B. subtilisforms complexes with CD14 (A) and LBP (C) but not with CD14(39–41, 43–44A) (B). Soluble CD14 and LBP were incubated overnight with LTA in the amounts indicated and separated by native polyacrylamide gel electrophoresis. CD14 and LBP were detected by Western blotting. CD14 binding at limiting ligand concentrations results in complexes which migrate faster than the native protein, while CD14 binding at excess ligand concentrations results in complexes that migrate more slowly. (D) The binding to CD14 was repeated with a second highly purified preparation of LTA. s, soluble.
- Fig. 5.
Serum-pretreated B. subtilis organisms are phagocytosed by vitamin D3-induced U937 cells in a CD14- and LBP-dependent fashion. (A) Phagocytosis was performed at 37 or 4°C in the presence or absence of 2.5 μg of murine recombinant LBP per ml. Internalization of the labeled bacteria is inhibitable with Fab fragments of the anti-CD14 MAb biG 14 or with a 20-fold excess of unlabeled bacteria (added where indicated). The ratio of cells to labeled bacteria was 1:50. (B) The same experiment as described for panel A was carried out with uninduced U937 cells, which do not express CD14.
