Technical Reference #1696

1696.

The LPS Receptor Generates Inflammatory Signals from the Cell Surface Eicke Latz; Alberto Visintin; Egil Lien; Kate A. Fitzgerald; Terje Espevik; Douglas T. Golenbock, University of Massachusetts, Journal of Endotoxin Research, 9(1696), (2003)
Link To Paper

Abstract:
Bacterial lipopolysaccharides (LPSs) are recognized in mammals by a receptor complex composed of CD14 Toll-like receptor 4 (TLR4) and MD-2. The mechanism of TLR4 function remains to be elucidated. We constructed chimeric TLR molecules C-terminally fused to fluorescent proteins and stably expressed these chimeric constructs in cells. Confocal microscopy revealed TLR4 to be expressed on the plasma membrane and the Golgi apparatus. Time-lapse confocal imaging showed rapid recycling of TLR4/CD14/MD-2 complexes between the Golgi and the plasma membrane. Membrane TLR4 engagement by antibody was sufficient to induce signaling and pharmacological disruption of the Golgi did not affect cellular responses to LPS. Thus LPS signaling commences after LPS recognition by surface-expressed TLR4 independent of LPS trafficking to the Golgi.

Materials & Methods:
For confocal microscopy cells were seeded on 35-mm glass-bottomed gamma-irradiated tissue culture dishes (MatTek Corp. Ashland MA USA) and imaged using a Zeiss Axiovert 100-M inverted microscope equipped with a LSM 510 laser scanning unit. Yellow fluorescent protein was excited with the 514 nm or 488 nm line of a 25-mW argon laser. Red fluorophores were excited with a helium/neon laser emitting at 543 nm or a helium/neon laser emitting at 633 nm as appropriate for the fluorophore used. Channel separation was granted by choosing band-pass or long-pass filters that optimally separate the fluorescence emissions between the different photomultipliers. Furthermore if crossover of the fluorescence signal was detected tracks were scanned separately. For fluorescence recovery after photobleaching (FRAP) experiments an area of interest was readily photobleached by intense laser illumination and fluorescence recovery was observed over time using low laser power. To block release of newly synthesized protein cells were pretreated with 200 mg/ml cycloheximide for 2 h before FRAP experiments. Fluorescence loss in photobleaching (FLIP) was performed by repetitive photobleaching of a selected cell area. Single confocal scans were taken intermittently to monitor the effect of the photobleaching on total cellular fluorescence. Live-cell images and confocal time-lapse fluorescence imaging were performed at 37°C using a warm-stage apparatus (Zeiss). Indirect immunofluorescence staining for FACS was done using purified mouse monoclonal antibodies (TLR4: HTA125) and isotype matched control antibodies (Sigma) as primary antibodies. After counterstaining with an APC-conjugated goat anti-mouse secondary antibody (Caltag) the cells were analyzed by flow cytometry (Becton Dickinson LSR) using a helium/neon laser emitting at 633 nm to excite APC. Membrane CD14 was visualized by a Tricolor-conjugated anti- CD14 antibody (Caltag Burlingame CA USA).

Microscopic Technique
Fluorescence Microscopy, Immunofluorescence, Confocal Microscopy

Cell Type(s)
HEK-293