Lipopolysaccharide Rapidly Traffics to and from the Golgi Apparatus with the Toll-like Receptor 4-MD-2-CD14 Complex in a Process That Is Distinct from the Initiation of Signal Transduction
Mammalian responses to LPS require the expression of Toll-like receptor 4 (TLR4), CD14, and MD-2. We expressed fluorescent TLR4 in cell lines and found that TLR4 densely localized to the surface and the Golgi. Similar distributions were observed in human monocytes. Confocal imaging revealed rapid recycling of TLR4-CD14-MD-2 complexes between the Golgi and the plasma membrane. Fluorescent LPS followed these trafficking pathways in CD14-positive cells. The TLR4- adapter protein, MyD88, translocated to the cell surface upon LPS exposure, and cross-linking of surface TLR4 with antibody induced signaling. Golgi-associated TLR4 expression was disrupted by brefeldin A, yet LPS signaling was preserved. We conclude that LPS signaling may be initiated by surface aggregation of TLR4 and is not dependent upon LPS trafficking to the Golgi. Mammalian responses to LPS require the expression of Toll-like receptor 4 (TLR4), CD14, and MD-2. We expressed fluorescent TLR4 in cell lines and found that TLR4 densely localized to the surface and the Golgi. Similar distributions were observed in human monocytes. Confocal imaging revealed rapid recycling of TLR4-CD14-MD-2 complexes between the Golgi and the plasma membrane. Fluorescent LPS followed these trafficking pathways in CD14-positive cells. The TLR4- adapter protein, MyD88, translocated to the cell surface upon LPS exposure, and cross-linking of surface TLR4 with antibody induced signaling. Golgi-associated TLR4 expression was disrupted by brefeldin A, yet LPS signaling was preserved. We conclude that LPS signaling may be initiated by surface aggregation of TLR4 and is not dependent upon LPS trafficking to the Golgi. The first line defense of multicellular organisms against bacterial pathogens relies upon germ line-encoded receptors that recognize a variety of conserved molecular structures on microorganisms (1Medzhitov R. Janeway Jr., C.A. Cell. 1997; 91: 295-298Google Scholar, 2Fearon D.T. Locksley R.M. Science. 1996; 272: 50-53Google Scholar, 3Aderem A. Ulevitch R.J. Nature. 2000; 406: 782-787Google Scholar). One family of such pattern recognition receptors is the type I transmembrane signaling receptors known as Toll-like receptors (TLRs). 1The abbreviations used are: TLR, Toll-like receptor; IL, interleukin; LPS, lipopolysaccharide; GFP, green fluorescent protein; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; HEK, human embryonic kidney; MALP-2, M. fermentans-derived membrane lipopeptide macrophage-activating lipopeptide of 2 kDa; mAb, monoclonal antibody; FRAP, fluorescent recovery after photobleaching; TNF, tumor necrosis factor; HRP, horseradish peroxidase; FACS, fluorescence-activated cell sorting; PBS, phosphate-buffered saline; TNFR, tumor necrosis factor receptor; ER, endoplasmic reticulum; PBMC, peripheral blood mononuclear cell; GM1, monosialo ganglioside GM1; APC, allophycocyanin; BODIPY, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-5-indacene; rLBP, recombinant lipopolysaccharide binding protein 1The abbreviations used are: TLR, Toll-like receptor; IL, interleukin; LPS, lipopolysaccharide; GFP, green fluorescent protein; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; HEK, human embryonic kidney; MALP-2, M. fermentans-derived membrane lipopeptide macrophage-activating lipopeptide of 2 kDa; mAb, monoclonal antibody; FRAP, fluorescent recovery after photobleaching; TNF, tumor necrosis factor; HRP, horseradish peroxidase; FACS, fluorescence-activated cell sorting; PBS, phosphate-buffered saline; TNFR, tumor necrosis factor receptor; ER, endoplasmic reticulum; PBMC, peripheral blood mononuclear cell; GM1, monosialo ganglioside GM1; APC, allophycocyanin; BODIPY, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-5-indacene; rLBP, recombinant lipopolysaccharide binding protein These receptors are all characterized by an intracellular signaling domain that is homologous to that of the IL-1 receptor and an extracellular domain with leucine-rich repeats (4Rock F.L. Hardiman G. Timans J.C. Kastelein R.A. Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 588-593Google Scholar, 5Du X. Poltorak A. Wei Y. Beutler B. Eur. Cytokine Netw. 2000; 11: 362-371Google Scholar). The most extensively studied microbial product known to engage TLRs is lipopolysaccharide (LPS; endotoxin), a complex glycolipid that comprises the major portion of the outer leaflet of the outer membrane of Gram-negative bacteria (6Raetz C.R. Annu. Rev. Biochem. 1990; 59: 129-170Google Scholar). A potent immune response is orchestrated upon the recognition of LPS by mammalian cells, including the production and release of cytokines, activation of complement, and various other effects that result in the killing and clearance of the pathogen. Uncontrolled hyperinflammatory host responses to LPS may lead to life-threatening complications such as septic shock, multiorgan failure, and death (7Bone R.C. Ann. Intern. Med. 1991; 115: 457-469Google Scholar). Toll-like receptor 4 is the signaling receptor for LPS and requires the small glycosylated protein MD-2 for optimal signaling (8Poltorak A. He X. Smirnova I. Liu M.Y. Huffel C.V. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Google Scholar, 9Hoshino K. Takeuchi O. Kawai T. Sanjo H. Ogawa T. Takeda Y. Takeda K. Akira S. J. Immunol. 1999; 162: 3749-3752Google Scholar, 10Shimazu R. Akashi S. Ogata H. Nagai Y. Fukudome K. Miyake K. Kimoto M. J. Exp. Med. 1999; 189: 1777-1782Google Scholar, 11Schromm A.B. Lien E. Henneke P. Chow J.C. Yoshimura A. Heine H. Latz E. Monks B.G. Schwartz D.A. Miyake K. Golenbock D.T. J. Exp. Med. 2001; 194: 79-88Google Scholar, 12Visintin A. Mazzoni A. Spitzer J.A. Segal D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12156-12161Google Scholar, 13Nagai Y. Akashi S. Nagafuku M. Ogata M. Iwakura Y. Akira S. Kitamura T. Kosugi A. Kimoto M. Miyake K. Nat. Immunol. 2002; 3: 667-672Google Scholar). We have recently observed that a mutant form of MD-2 (C95Y) completely abrogated LPS responses and that wild-type MD-2 was able to confer LPS responsiveness in TLR4-positive cells lacking MD-2 expression (11Schromm A.B. Lien E. Henneke P. Chow J.C. Yoshimura A. Heine H. Latz E. Monks B.G. Schwartz D.A. Miyake K. Golenbock D.T. J. Exp. Med. 2001; 194: 79-88Google Scholar, 12Visintin A. Mazzoni A. Spitzer J.A. Segal D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12156-12161Google Scholar). Whereas there is widespread agreement that CD14, TLR4, and MD-2 expression are necessary for optimal responses to LPS, the mechanism of cellular activation remains in doubt. Wright and co-workers (14Thieblemont N. Thieringer R. Wright S.D. Immunity. 1998; 8: 771-777Google Scholar,25Thieblemont N. Wright S.D. J. Exp. Med. 1999; 190: 523-534Google Scholar) observed that LPS is internalized and trafficked to the Golgi apparatus. Indeed, they reported that in the absence of internalization and movement of LPS to the Golgi, LPS did not activate mammalian cells (14Thieblemont N. Thieringer R. Wright S.D. Immunity. 1998; 8: 771-777Google Scholar). Wright proposed these events to be the critical initiators of signal transduction. TLR4 has now been reported to be localized to the Golgi in certain epithelial cells (15Hornef M.W. Frisan T. Vandewalle A. Normark S. Richter-Dahlfors A. J. Exp. Med. 2002; 195: 559-570Google Scholar). Together, these data suggested that LPS stimulates innate immune responses by activating an internal receptor, TLR4, that normally resides in the Golgi apparatus. Since the discovery and development of green fluorescent protein (GFP), the subcellular localization, trafficking, and fate of proteins be studied in cells the fluorescent protein as a Annu. Rev. Biochem. 1998; Scholar, J. 2001; Scholar, J. E. A. Nat. Rev. Cell. 2001; Scholar). of GFP, such as yellow fluorescent protein and cyan fluorescent protein have been These be for of We have a protein with in to the of and TLR4 as a to the receptor These were expressed in human embryonic cells. cells TLR4 and expression of the fluorescent receptors these cells to to the We the subcellular localization, trafficking, and of TLR4, CD14, and LPS in cells by of and and cells as as fluorescent We that LPS to the Golgi and found that TLR4 is expressed in the Golgi of cells as as monocytes. TLR4 is LPS to between the cell surface and the Golgi signal not TLR4 expression in the Golgi was not necessary for cells to to antibody cross-linking of surface TLR4 was able to signaling in cells and human monocytes. These that LPS signaling is initiated on the plasma membrane. of TLR4 in an Golgi and the movement of LPS to internal of TLR4 is necessary for signaling to were PBS, and were was was a LPS was and by as M. Y. J. Immunol. 2000; Scholar). and were The fermentans-derived membrane macrophage-activating lipopeptide of 2 was G. The for human TLR4 was in the by C. Janeway and R. The expression was C. of The was to as in with a these were by and M. R.M. Science. 2000; Scholar). The R.M. Science. 2000; Scholar). of and TLR4 was on and on in to fluorescent The and for were and The and for TLR4 were and The were with and and in and The mammalian expression and was a of K. Miyake of The Golgi of the of human to was The fluorescent were by of in by H. the and for and The were and and in cell lines of cells the fluorescent protein were by C. H. Cell. of of cells in the of and by fluorescence-activated cell cell lines were by The fluorescent cell lines were in with and in a A cell line TLR4 and MD-2 was by of as with a human MD-2 A. Mazzoni A. Spitzer J.A. Segal D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12156-12161Google Scholar). were by and as A. Mazzoni A. Spitzer J.A. Segal D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12156-12161Google Scholar). cells were in and in and surface proteins were 4 for in of a of in were and in for and in of and a of and and were for in of protein and with 2 of a antibody and of protein for 4 were in by and to These were in and with the The were with and by to cells that were a of 2 The cells were with the to an of an of a of the was with a The the cells were as cells were with in the of MD-2 in with the for MD-2 A. Mazzoni A. Spitzer J.A. Segal D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12156-12161Google Scholar). that have that all of these for MD-2 responses to Latz and A. of the cells were in and was a the and for all the data of and are as the S.D. of Confocal was with a with an A and a was were on proteins were the for and GFP, the line of a was were with a were with a were to the between the of the as of the signal was to be were with and the Fluorescent recovery after were by an of and the line of a recovery was observed as in the were after with in for 2 cell and imaging were a for was monoclonal CD14, and as were with antibody were by the for of and the to these of the was of cells observed with was on cells in to the Golgi by was the MD-2 was for by of as a antibody and as the antibody was by antibody were by of in and in with human for they were in and in in for on The cells were in with they were with for followed by a with binding was by with on for The cells were for with with a with with the cells were in the with a expression of TLR4 and in was by were with an antibody with after in human for were with and CD14-positive cells to and were for E. LPS was to a to and with the the was LPS by a The of the LPS was with LPS was in human human and human for to cells were in and with LPS for the in were with PBS, and in by in PBS, and by an of were to protein binding by 4 in with PBS, 4 cells were in and for were for by embryonic cells are in all of the known of the LPS signaling receptor, including TLR4, and cells are normally to cells not and are to the known to activate these We and have observed that cells with TLR4 LPS responsiveness they are with MD-2 in the of in recombinant MD-2 (11Schromm A.B. Lien E. Henneke P. Chow J.C. Yoshimura A. Heine H. Latz E. Monks B.G. Schwartz D.A. Miyake K. Golenbock D.T. J. Exp. Med. 2001; 194: 79-88Google Scholar, 12Visintin A. Mazzoni A. Spitzer J.A. Segal D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12156-12161Google Scholar, H. Chow J.C. J. 2000; Scholar). cells that were The cell lines were with a in that is the of the factor and with the microbial cell LPS and the fermentans-derived membrane lipopeptide LPS responsiveness to cells cells The responses observed were responses observed in cells that not an not cellular responses to TLR4 by the were by a of and of as by and imaging of the various cell lines were and were to and data not The responsiveness was in of of GFP, was with expression of these proteins the did not result in activation not proteins of TLR4 and with fluorescent the are signaling receptors that confer and recognition of in cells. The of the and as a protein protein and to be the of the The were that the subcellular of the receptors by signal by a signal in the as is were on and observed was expressed in subcellular We observed plasma membrane expression and in a for TLR4 a monoclonal antibody revealed a surface expression of the protein of the fluorescent protein used human expressed of TLR4 and and with followed by with revealed that the glycosylated of TLR4 and MD-2 are expressed on the cell We to MD-2 TLR4 to of these protein not A. Mazzoni A. Spitzer J.A. Segal D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12156-12161Google Scholar). These that TLR4 and MD-2 the and on the cell surface as a protein We to the intracellular that is in A fluorescent subcellular for the Golgi complex of the of human to with the for TLR4 2 fluorescent is known to in Golgi Science. with 2 These the in TLR4 as the Golgi apparatus. the pattern of TLR4 was an of and the of TLR4 in cells, human with a monoclonal of revealed TLR4 on the surface of these cells of that TLR4 is expressed in a with the that TLR4 resides on the cell surface and in the Golgi. The Golgi complex is an intracellular that is for proteins and are the by the Golgi complex and are in for to as plasma to be to the to these and the Golgi complex plasma membrane that are by J. 2001; Scholar, R. K. J. J. 2001; Scholar). The of the to be used in a known as are with these are a that the FRAP, a small of in the cell is by with a the movement of the be recovery in the of the We to to the Golgi complex after of Golgi-associated of with to the Golgi completely the the the cells were of recovery revealed a and recovery of Golgi of the Golgi-associated was 4 after rapid movement of between cell We the recovery of Golgi after is to protein the recycling of the membrane to the Golgi apparatus. cells were with the protein for 2 to The recovery of Golgi observed in the cells was to that in cells, that rapid of TLR4 the plasma membrane TLR4 in the Golgi These data that TLR4 is a protein that and between major cellular the Golgi complex and the plasma membrane. of TLR4 by LPS requires the of a glycosylated protein with a signal MD-2 has been suggested to with TLR4 in the intracellular We have recently reported that MD-2 be cells to TLR4 responses to LPS (11Schromm A.B. Lien E. Henneke P. Chow J.C. Yoshimura A. Heine H. Latz E. Monks B.G. Schwartz D.A. Miyake K. Golenbock D.T. J. Exp. Med. 2001; 194: 79-88Google Scholar, 12Visintin A. Mazzoni A. Spitzer J.A. Segal D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12156-12161Google Scholar). MD-2 LPS responsiveness to TLR4-positive cells, a to LPS responses (11Schromm A.B. Lien E. Henneke P. Chow J.C. Yoshimura A. Heine H. Latz E. Monks B.G. Schwartz D.A. Miyake K. Golenbock D.T. J. Exp. Med. 2001; 194: 79-88Google Scholar). We expressed a of MD-2 in cells that expressed and MD-2 by in to the and trafficking of MD-2. of MD-2 by antibody surface not that MD-2 a and complex with TLR4 on the cell membrane 4 A and data not We MD-2 between the membrane and the Golgi of cells that were with MD-2 were for MD-2 and observed by We observed TLR4, MD-2 between the cell membrane and the Golgi and that MD-2 with TLR4 all 4 data not to followed the pattern of movement the cell membrane to the Golgi, used to to on the cell surface and was found to be internalized to the Golgi These lead to the that the LPS signaling receptor of TLR4, and CD14, is on the plasma membrane and that protein complex is between the plasma membrane and the Golgi. A of is that surface CD14, TLR4, and MD-2 in and that is by the of These between the cell membrane and the Golgi, all of including receptors in the absence of signaling. A, LPS binding and in was in cells with CD14, MD-2 and CD14, were with for various and binding and were by cells were with the CD14, MD-2 and CD14, with The cells were and activation was by after of are to the and are of cells were with and with in for Confocal of cells are and cells were for and with were by and a of a is for cell and 2 in the These the trafficking of and the of and LPS, the surface of cells to the the of MD-2 and in LPS binding and cells with MD-2 and binding and internalization of binding and of not not was observed in cells with and of with MD-2 did not binding in with cells with of MD-2 on the of the of LPS in the absence of signaling. of CD14-positive cells with LPS did not result in the activation of the release of not the of these cells to of MD-2 expression LPS responsiveness the absence of membrane these LPS binding and were after of a that is after the of that the in A not between cellular LPS binding and LPS was The that LPS binding and internalization in cells not We with MD-2 and these cells to the cell surface of cells, LPS binding was in cells, the by CD14-positive cells internalized of and of LPS were after cells were to LPS type of of LPS to of these an that was not in cells We imaging in cells to the of the LPS internalization and to the of TLR4 in intracellular LPS We in recombinant MD-2 to signaling in to the effects of upon We that recombinant MD-2 TLR4 signaling in response to found that MD-2 to LPS responses in cells with not cells were with on the after the of LPS, membrane of LPS and was and were observed to between the plasma membrane and an intracellular The movement the cell membrane to the of the cell was to require between 4 and a that to a movement of The of movement and the fate of LPS with to were between cells and that expressed in with TLR4 that in the absence of MD-2 TLR4 expression there is that cells be by the intracellular of LPS not to be to result in cellular Whereas is that the movement of LPS to the Golgi is not to activate signal have that LPS internalization and trafficking to the Golgi are necessary for signaling to (15Hornef M.W. Frisan T. Vandewalle A. Normark S. Richter-Dahlfors A. J. Exp. Med. 2002; 195: 559-570Google Scholar, N. Wright S.D. J. Exp. Med. 1999; 190: 523-534Google Scholar). We the subcellular of LPS signaling by the expression of a of in and cells in to to the intracellular signaling domain of TLRs to the signaling R. P. E. A. C. S. Janeway Jr., C.A. Cell. 1998; Scholar) and is for responses to LPS, such as the production and release of cells to LPS, the was expressed in LPS a portion of the intracellular was observed to to the of the plasma membrane is with the that is to the domain of surface TLR4, signal is A the small to of the Golgi the J. 2001; Scholar, S. K. J. J. 2001; Scholar). We disrupted the Golgi by brefeldin A to cells in to the of these the release of such as was not Confocal imaging of brefeldin cells that the was in the the in the subcellular of TLR4, signaling in brefeldin cells was not result that signaling not require Golgi to TLR4 was of a signal be with a of that TLR4 not be to protein binding with monoclonal to TLR4 The were extensively with PBS, and cells were to the surface proteins by the monoclonal We observed that cross-linking of surface TLR4 was to activate not to release in cells. to have that LPS of the antibody for the of the cells are in the absence of MD-2 Similar to the in cell be by against including and not by an mAb, Gram-negative the complex a potent immune response to of LPS that are The of TLR4 as the LPS signal is by the that are to Gram-negative bacterial K. Takeuchi O. Kawai T. Sanjo H. Ogawa T. Takeda Y. Takeda K. Akira S. J. Immunol. 1999; 162: 3749-3752Google Scholar, I. J. Immunol. Scholar). The of TLR4 not a in the response to LPS may be by the host immune by the production of these the to may to septic and multiorgan The molecular mechanism of TLR4 is of in the of and for the in of the of J. R. Med. 1997; Scholar, C. J. P. A. J.C. G. B. Med. Scholar, for Med. 1990; Scholar). has been that bacterial lipopolysaccharide has the to mammalian and activate cells. The discovery of as an LPS binding receptor that movement as a result of the of to and to the that after LPS is the a portion of the LPS the Golgi apparatus. the with movement the membrane to Golgi be in not as was has been the mechanism of cellular activation was in to the movement of LPS, a variety of on The discovery of receptor 4 as the LPS signal for to the of LPS immune cells. all of the TLR4 has to the IL-1 receptor is known to signal by complexes with the receptor; complex has a for the MyD88, and is to the domain of the signaling the of TLRs to the has yet to be TLRs in the The complex (15Hornef M.W. Frisan T. Vandewalle A. Normark S. Richter-Dahlfors A. J. Exp. Med. 2002; 195: 559-570Google Scholar) reported that TLR4 is localized in the Golgi in epithelial cells and of LPS with TLR4 in was that signaling in the Golgi and that the receptor was an intracellular We proteins of yellow and cyan fluorescent protein with TLRs in to be able to the subcellular of TLRs the to cells for antibody has to not the of TLR4 that was in to the of These are receptors expression in cells in the of immune responses to bacterial that cells that be to be the Golgi of cells, epithelial cells, is on the cell is that the expression of TLR4 in and human be in the that the Golgi, of has surface expression of TLR4 in be to the of the mammalian outer membrane that cells are with a We that the Golgi of TLR4 normally as a of TLR4 protein and that cells may the surface expression of TLR4 by the of TLR4 in the Golgi A. Mazzoni A. Spitzer Segal D.M. J. Immunol. 2001; Scholar, M. N. D. A. J. 2000; Scholar). We that of the epithelial cell outer membrane result in the discovery of surface TLR4 in these cells, there is to that the recycling of membrane the surface to the Golgi and is to cells. that TLR4 requires MD-2 in to LPS signal transduction. that MD-2 to the extracellular domain of TLR4 R. Akashi S. Ogata H. Nagai Y. Fukudome K. Miyake K. Kimoto M. J. Exp. Med. 1999; 189: 1777-1782Google Scholar, Silva J. K. U. Ulevitch R.J. J. 2001; Scholar) and that binding has MD-2 is a protein that LPS responsiveness in cells that TLR4 MD-2 (11Schromm A.B. Lien E. Henneke P. Chow J.C. Yoshimura A. Heine H. Latz E. Monks B.G. Schwartz D.A. Miyake K. Golenbock D.T. J. Exp. Med. 2001; 194: 79-88Google Scholar, 12Visintin A. Mazzoni A. Spitzer J.A. Segal D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12156-12161Google Scholar). observed that the receptor complex was the plasma membrane and that MD-2 with TLR4 between the plasma membrane and the Golgi in the absence of to the cell surface to the Golgi, to the T. E. Thieringer R. J. Exp. Med. 1999; 190: Scholar). data that the in TLR4, CD14, and MD-2 all are to is and the surface to the Golgi R. K. J. J. 2001; Scholar, A. A. E. M. P. A. I. G. K. M. S. T. G. G. H. Heine H. C. G. J. S. G. Eur. J. Immunol. 2001; Scholar, M. Miyake K. Golenbock D.T. K. J. Sci. 2002; 115: Scholar). have found that cells were with the to in the the plasma membrane to the Golgi CD14, TLR4, and LPS not Whereas the that LPS is internalized cells and to the Golgi cell signaling not is with the that cellular activation on the cell these data be to be The of cells to by LPS after brefeldin A that Golgi of TLR4 is not necessary to be that these the of the Golgi to the We found that after LPS an intracellular to the plasma membrane in cells, to the that signaling on the plasma membrane. most surface TLR4 is of a signal to cells, the antibody cross-linking in and monoclonal that are of with TLR4 be to be the of TLR4 that first with LPS as bacteria are by that the responses to LPS the cell surface may have the to other the including the Golgi and other be to LPS and to LPS activate cells. such are to and Whereas LPS and have found that they with other of cellular C. M. J. J. 1998; Scholar, S. C. Jr., 2001; Scholar). an to the of used to bacterial internalization in cells by these cells with receptors that were of these receptors that with the these the response was by the of internalization Golenbock D.T. J. Immunol. 1997; Scholar). of the protein family have been suggested to to LPS that the by pathogens that have the to mammalian cells. of receptors in be intracellular signaling that a form of host defense against certain intracellular pathogens R. M. X. J. M. 2001; Scholar, N. Y. A. G. J. 2001; Scholar). of the of the of is that the of responses to LPS not intracellular the The data that the movement of LPS the cell and the Golgi of TLR4 is necessary for signal to We that LPS is internalized by the of and is to the Golgi with the movement of LPS has data the that signaling with the cross-linking and of surface have observed to be to signal transduction. The of and is with