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Fluorescence recovery after photobleaching reveals that LPS rapidly transfers from CD14 to hsp70 and hsp90 on the cell membrane

Kathy Triantafilou^1,,, Martha Triantafilou¹, Shab Ladha², Alan Mackie², Russell L. Dedrick³, Nelson Fernandez¹ and Richard Cherry¹

¹ University of Essex, Department of Biological Sciences, Central Campus, Wivenhoe Park, Colchester, CO4 3SQ, UK
² Department of Food Biophysics, Institute of Food Research, Norwich Research Park, Colney Lane, Norwich, NR4 7UA, UK
³ Molecular Immunology, XOMA (US) LLC, 2910 Seventh Street, Berkeley, CA 94710, USA
Present address: School of Biological Sciences, University of Portsmouth, King Henry Building, King Henry I Street, Portsmouth, PO1 2DY, UK

View larger version (16K): [in a new window]   Fig. 1. Characterization of 26ic PE-Fab probe. PE-Fab probe against CD14 was characterized using HPLC (A) and flow cytometry (B-E). HPLC size exclusion chromatography separated the 1:1 labelled PE-Fab (1), from free PE (2) and from free Fab (3). The arrow indicates the fractions collected for further analysis. For testing the binding of the PE-Fab conjugate, CHO-CD14 cells were incubated with no probe (B), 40 nM of PE-labelled Fab (C), 40nM unconjugated PE (D) or 40 nM of PE-Fab in the presence of 10 ng ml-1 of unlabelled LPS (E). 10,000 cells were analysed for fluorescence on a FACalibur flow cytometer. The histograms display relative cell numbers (y axis) as a function of relative fluorescence intensities (x axis).

View larger version (23K): [in a new window]   Fig. 2. Lateral diffusion of CD14 before and after LPS/LTA stimulation. FRAP curve of 26ic PE-Fab bound to CHO-CD14 (A) and MonoMac 6 cells (B) and measured at 22°C. FRAP curve of 26ic PE-Fab bound to CHO-CD14 (C) and MonoMac 6 cells (D) after stimulation with 250 ng ml-1 of LPS and measured at 22°C. FRAP curve of 26ic PE-Fab bound to CHO-CD14 (E) and MonoMac 6 cells (F) after stimulation with 250 ng ml-1 of LTA and measured at 22°C. The best fit to the experimental data is shown. The diffusion coefficient of CD14 was the mean and standard deviation from several (10) individual curves.

View larger version (10K): [in a new window]   Fig. 3. Characterization of FITC-LPS probe. CHO-CD14 cells were incubated with no FITC-LPS probe (A), 10 ng ml-1 of FITC-LPS probe (B), or with 10 ng ml-1 of FITC-LPS in the presence of 100x excess of unlabelled LPS (C). 10,000 cells were analysed for fluorescence on a FACalibur flow cytometer. The histograms display relative cell numbers (y axis) as a function of relative fluorescence intensities (x axis).

View larger version (23K): [in a new window]   Fig. 4. Lateral diffusion of FITC-LPS bound to cells. FRAP curve of FITC-LPS bound to CHO-CD14 cells (A,B) and measured at 22°C (A) or at 37°C (B). FRAP curve of FITC-LPS bound to MonoMac 6 cells (C,D) and measured at 22°C (C), or at 37°C (D). FRAP curve of FITC-LPS bound to ECV-304 (E,F) cells and measured 22°C (E), or at 37°C (F). The best fit to the experimental data is shown. The diffusion coefficient of FITC-LPS bound to cells was the mean and standard deviation from several (10) individual curves.

View larger version (17K): [in a new window]   Fig. 5. Characterization and FRAP measurements of FITC-LTA probe. CHO-CD14 cells were incubated with no FITC-LTA probe (A), 10 ng ml-1 of FITC-LTA probe (B) or 10 ng ml-1 of FITC-LTA in the presence of 100x excess of unlabelled LTA (C). 10,000 cells were analysed for fluorescence on a FACalibur flow cytometer. The histograms display relative cell numbers (y axis) as a function of relative fluorescence intensities (x axis). FRAP curves of FITC-LTA bound to CHO-CD14 (D) ECV-304 cells (E) and Mono-Mac 6 cells (F), measured at 22°C. The best fit to the experimental data is shown. The diffusion coefficient of FITC-LPS bound to cells was the mean and standard deviation from several (10) individual curves.

View larger version (17K): [in a new window]   Fig. 6. FRAP measurements of heat shock proteins. FRAP curve of hsp70 OG-Fab bound to MonoMac 6 (A) and ECV-304 cells (B) and measured at 22°C. FRAP curve of hsp90 OG-Fab bound to MonoMac 6 (C) and ECV-304 cells (D) and measured at 22°C. The best fit to the experimental data is shown. The diffusion coefficients of hsp70 and hsp90 were the mean and standard deviation from several (10) individual curves.

View larger version (9K): [in a new window]   Fig. 7. IL-6 inhibition experiments. MonoMac 6 cells (A) and ECV-304 cells (B) were pre-incubated with MY4, anti-hsp70 or anti-hsp90 polyclonal sera, or a combination of these antibodies prior to stimulation with 1 ng ml-1 of native LPS, in 5% HPS. Control experiments with irrelevant polyclonal sera were also performed. The biological activity of LPS was tested by measuring induction of IL-6 using an ELISA. Each data point represents several independent experiments.

View larger version (8K): [in a new window]   Fig. 8. Schematic representation of LPS transfer from CD14. LPS transfers from CD14 and is released in the lipid bilayer of the membrane before it reaches the signal transduction complex that contains hsp70 and hsp90.

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