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

Although CD14 has been implicated in the immune recognition of bacterial li popolysaccharide (LPS) from Gram-negative bacteria and also peptidoglycan ( PGN) and lipoteichoic acid (LTA) from the outer cell wall of Grampositive b acteria, accumulating evidence has suggested the possible existence of othe r functional receptor(s). In this study, we have used fluorescence recovery after photobleaching (FRAP) in order to get the first dynamic picture of t he innate recognition of bacteria. We have found that the diffusion coeffic ient of CD14 remains unaffected after LPS ligation and that the diffusion c oefficients of FITC-LPS and FITC-LTA bound to cells differ from that of CD1 4. Furthermore, FITC-LPS/LTA rapidly become immobile when bound to cells, s uggesting that FITC-LPS/LTA must briefly associate with CD14 in the initial attachment process and rapidly move on to an immobile receptor or to a com plex of receptors. Further FRAP experiments revealed that heat shock protei n 70 (hsp70) and hsp90 are immobile in cell membranes, and antibodies again st them were found to block the transfer of LPS to the immobile receptor an d to inhibit interleukin 6 production upon LPS stimulation. These experimen ts indicated that LPS transfers from CD14 to hsp70 and hsp90, which may be part of an LPS/LTA multimeric receptor complex. Thus, hsps are implicated a s mediators of the innate activation by bacteria.