Update Participation of Lipopolysaccharide-binding Protein in Lipopolysaccharide-dependent Macrophage Activation P. S. Tobias, J. Mathison, D. Mintz, J.-D. Lee, V. Kravchenko, K. Kato, J. Pugin, and R. J. Ulevitch The Scripps Research Institute, La Jolla, California
Only recently has the mechanism for lipopolysaccharide (LPS) recognition by macrophages been elucidated. In contrast to many ligand receptor interactions, the interaction of LPS with its receptor, CDI4, on myeloid cells is greatly enhanced by prior complexation of LPS with LPS-binding protein (LBP), a recently discovered plasma glycoprotein. LBP is found in normal serum or plasma in the 5 to 10 ~g/ml range. In plasma, it reacts rapidly but transiently with LPS. LPS-LBP complexes then react with CDl4 bearing cells. Blocking CDl4 with monoclonal antibodies or removal ofLBP from plasma blocks the ability of the cells to react with LPS-LBP complexes and also blocks release of cytokines and other mediators from the cells. In the normal lung, bronchoalveolar lavage fluid contains low levels of LBP. However, during acute lung injury, LBP levels may rise by transudation and enhance activation of alveolar macrophages to release injurious mediators. Description of this pathway for LPS recognition by macrophages and other leukocytes offers the possibility of developing new reagents to block LPS recognition and prevent the development of endotoxemia.
When we senselipopolysaccharide, we are likely to turn on everydefense at our disposal; we willbomb,defoliate, blockade, seal off, and destroy all tissue in the area ... Pyrogen is released from leukocytes, adding fever to hemorrhage, necrosis, and shock. It is a shambles ... All this seemsunnecessary, panic-driven. There is nothing intrinsically poisonous about endotoxin, but it must look awful, or feel awful, when sensed by cells. -Lewis Thomas in The Lives of a Cell (1974) The consequences of endotoxemia include fever, hypotension, diffuse intravascular coagulation, acute respiratory distress, multi-organ failure, and ultimately death. While the mechanisms of these pathologic conditions may not yet be (Received in original form February 28, 1992 and in revised form March 24, 1992) Address correspondence to: P. S. Tobias, Ph.D., IMM-12, Department of Immunology, The Scripps Research Institute, 10666 N. Torrey Pines Rd., La Jolla, CA 92037. Abbreviations: adult respiratory distress syndrome, ARDS; 2-{p-azidosalicylamido)ethyl-l,3'-dithiopropionyl, ASD; bronchoalveolar lavage fluid, BAL; bactericidal/permeability increasing protein, BPI; Chinese hamster ovary, CHO; lipopolysaccharide-coated erythrocytes, E-LPS; fluoresceinlabeled LPS, F-LPS; interleukin, IL; lipopolysaccharide-binding protein, LBP; lipopolysaccharide, LPS; monoclonal antibodies, mAb; monocytes/ macrophages, Met>; peritoneal elicited macrophages, PEM; phosphatidylinositol, PI; tumor necrosis factor, TNF. Am. J. Respir. Cell Mol. BioI. Vol. 7. pp. 239-245, 1992
clear, it is certain that the endotoxin or lipopolysaccharides (LPS) from gram-negative bacteria activate monocytes/macrophages (Met» to secrete a variety of products that ultimately lead to the clinical features of endotoxemia. Because LPS are not themselves toxic, it is the inappropriate Met> response that initiates endotoxic injury. Not all responses to LPS are inappropriate, although the difference between an appropriate and an inappropriate response is unclear. The appropriate response probably includes tumor necrosis factor (TNF) and interleukin-l (IL-l) since these cytokines have been implicated in resistance to infection (1-6). The same mediators have been implicated in pathologic gram-negative septic shock (7-9). Thus, a central problem is to understand how Met> recognize LPS. Clarifying how responses are initiated may help to clarify how an appropriate response differs from an inappropriate response and may lead to development of methods to forestall the pathophysiologic changes that comprise gram-negative septic shock. This review will focus on recent progress in understanding Met> recognition of LPS.
LPS It is useful to briefly review LPS structure (Figure 1) (10-12). The lipid A moiety is the most highly conserved part of the structure, typically with two glucosamines, two phosphate esters, and five or more fatty acids. Studies of synthetic lipid A reveal that all the endotoxic activity of an LPS is derived from the lipid A moiety (11); however, not all lipid /Ji.s are
240
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 7 1992
Chain
~ l..l-----~I----- . .l l - - . f Lipid A ~
endotoxically active (13). The core region contains several sugars, among them 3-deoxY-D-manno-2-octulosonate (KDO), a sugar unique to LPS, and is typically conserved within a given genus of bacteria. The core region may also contain phosphate groups as well as ethanolamine moieties, often in substoichiometric amounts. The most variable part of the LPS structure is the a-antigen, responsible for the individual antigenic signatures of individual strains of gram-negative bacteria. Strains with an intact a-antigen are termed "smooth" strains, whereas those lacking the a-antigen and retaining variable amounts of the core sugars are termed "rough" mutants. These terms derive from the appearance of bacterial colonies. Isolates of LPS are rarely homogeneous, probably reflecting an inhomogeneous composition of the bacterial outer membrane (14). Different isolates from even the same strain of bacteria can show considerable variation in composition (15). Considerable experimental evidence supports the contention that LPS acts via a specific plasma membrane receptor (16-25). Recent studies from our laboratory have identified a novel mechanism for LPS recognition that differs from the standard ligand-receptor model in that it involves a plasma protein, LPS-binding protein (LBP) , which forms highaffinity complexes with LPS and a plasma membrane glycoprotein, CD14, which acts as a receptor for the LPS-LBP complexes (16). This review will focus on the properties of these two proteins and the evidence that they form a highaffinity recognition system for LPS. Our current understanding of the role of LBP in the LPS-dependent activation of macrophages, and possibly certain other cells, is illustrated in Figure 2. LPS shed from the surfaces of gram-negative bacteria binds to LBP. The complexes subsequently interact with CD14, leading to M¢ activation and cytokine secretion. The fact that there are other cells, such as B cells, which have no CD14 but nevertheless respond to LPS, suggests that there is an alternative to the CD14 pathway. This alternative pathway may be used even by CD14 bearing cells when they are in environments, such as the extravascular spaces, where LBP is low. The precise mechanism by which LPS-LBP engagement of CD14 leads to M¢ activation is by no means clear; our current working hypothesis will be described.
Figure 1. General chemicalstructure of bacterial lipopolysaccharides. The three parts of LPS are not drawn to scale. Depending on the value of n, molecular weights may be as large as 20,000. The O-specific chain is typically the immunodominant structure and is highly variable among bacterial strains. Mutants lacking the O-specific chain and more or less of the core region are "rough" mutants. Re mutants contain only lipid A and the two Kdo residues. The lipid A fragment has all the endotoxic activity of the intact molecule.
LBP LBP was first discovered as an acute-phase reactant with affinity for LPS (26), and we suggested that it might be an LPS inactivator by analogy with the protective effects of some other acute-phase reactants (27). This hypothesis could hardly have been more wrong. Our recent data show that the LBP in normal plasma is sufficient to dramatically enhance the ability of LPS to activate macrophages, and the reason for the rise in LBP during an acute-phase response remains unknown. One conjecture is that increasing LBP in the vascular compartment serves to enhance the availability of LBP in the extravascular compartment, especially at sites of inflammation. As deduced from cD NA clones, the amino acid sequences of human and rabbit LBP are shown in Figure 3. Both mature secreted proteins are composed of 456 amino acids, of which 69% are identical. Human LBP has five potential sites for N-linked glycosylation, three of which are also found in rabbit LBP. The protein isolated from serum is glycosylated, but
LBP LPS
Bacteria ---+ I::Zl
~ ------tv
1
1 \?
/'
~
~?
_ _ -+-t
IL-1 IL·6 IL·8 TNF-a
Figure 2. The involvement of LBP in LPS-dependent Mel> activation. See text for details.
Update
Figure 3. Comparison of the eDNA-derived amino acid sequences of LBP and BPI. Only one pair of cysteines is fully conserved at positions 135 and 175. Rabbit and human LBP have conserved glycosylation sites at Asn residues 276, 327, and 363. Native rabbit LBP has a single plasmin (or trypsin) cleavage site at Arg(99)-Leu(100). Cleavage at this position destroys LPS-binding activity. The LPS-binding activity of BPI resides in the N-terminal half of the molecule (30).
241
Bovine BPI Human BPI Human LBP Rabbit LBP Consensus
1 tNPGiVaRIT vNPGvVvRIs aNPG1VaRIT tNPGIItRIT -NPG-V-RIT
qKGLDYAcQq qKGLDYAsQq dKGLqYAaQe dKGLEYAare -KGLDYA-Q-
GvLtLQkELe GtaALQkELk G1LALQsELl GILALQrkLl G-LALQ-EL-
kITiPnFsGn rIkiPDYsds rITIPDFtGd eVTIPDsdGd -IT-PDF-G-
50 FkIKyLGkGq FkIKHLGkGh LrIpHvGrGr FrIKHFGraq F-IKHLG-G-
Bovine BPI Human BPI Human LBP Rabbit LBP Consensus
51 YsFFSMvIqg YsFYSMdIre YeFhSLnIhs YkFYSLkIpr Y-FYS--I--
FnLpnSqiRP FqLpsSqism ceLlhSalRP FeLlrgtlRP F-L--S--RP
IPdkGLdLSI vPnvGLkFSI vPgqGLsLSI IPgqGLsLdI -P--GL-LSI
rDAsIkIrGk SnAnIkIsGk SDssIrVqGr SDAyIhVrGs SDA-I---G-
100 WKaRKnFiKL WKaqKrFLKM WKvRKsFFKL WKvRKaFLrL WK-RK-FLKL
Bovine BPI Human BPI Human LBP Rabbit LBP Consensus
101 gGnFDLSVeG sGnFDLSIeG qGsFDvSVkG knsFDLyVkG -G-FDLSV-G
iSIlagLnLG mSISadLkLG iSISvnLILG ItISvhLvLG -SIS--L-LG
yDpaSGhsTV SnptSGkPTI SEs.SGrPTg SEs.SGrPTV SE--SG-PTV
TCSSCSSgIn TCSSCSShIn yC1SCSSdIa TtSSCSSdIq TCSSCSS-I-
150 tVrIhIsgss sVhVhIsksk dVeVdm.sgd nVeldI.egd -V-V-I----
Bovine BPI Human BPI Human LBP Rabbit LBP Consensus
151 IGWLiqLFrk vGWLiqLFhk sGWLlnLFhn leeL1nLLqs -GWL--LF--
rIESILqksM kIESaLrnkM qIESkFqkvL qIDarLrevL -IES-L----
trkICEvVts nSqVCEkVtn eSrICEmIqk eSkICrqIee -S-ICE----
tVSSkLQPYF sVSSkLQPYF sVSSdLQPYL aVtahLQPYL -VSS-LQPY-
200 QTLPVTTklD QTLPVmTkID QTLPVTTeID QTLPVTTqID QTLPVTT-ID
Bovine BPI Human BPI Human LBP Rabbit LBP Consensus
201 kvAGVDYSLV SvAGInYgLV SfAdIDYSLV SfAGIDYSLm S-AGIDYSLV
apPRATAnnL apPatTAetL eaPRATAqmL eaPRATAgmL --PRATA--L
DwLLKGEfFs DVqMKGEfYs EVMFKGEiFh DVMFKGEiFp DVMFKGE-F-
laHRSPppFa enHhnPppFa rnHRSPvtLl IdHRSPvdFl --HRSP--F-
250 pPaLaFPsdH pPvMeFPaaH aavMsLPeeH aPaMnLPeaH -P-M--P--H
Bovine BPI Human BPI Human LBP Rabbit LBP Consensus
251 dRMVYLgISE dRMVYLg1SD nkMVYFaISD sRMVYFsISD -RMVY--ISD
YfFNTAgFVY YfFNTAgLVY YvFNTAsLVY YvFNTAsLaY Y-FNTA-LVY
qkaGaLNLtl qeaGvLkMtl heeGyLNFsi hksGywNFsi ---G-LNF--
rDDMIPkESk rDDMIPkESk tDDMIPpDSn tDaMVPaDln -DDMIP--S-
300 fRLTTKfFgi fRLTTKfFgt iRLTTKsFrp iRrTTKsFrp -RLTTK-F--
Bovine BPI Human BPI Human LBP Rabbit LBP Consensus
301 LIPqvAkMFP FlPevAkkFP FVPrlArLYP FVPllAnLYP FVP--A-L-P
dMqMqLfiwa NMkiqihvsa NMnLeLqgsv NMnLeLqgtv NM-L-L----
slpPkLtMkP stpPhLsvqP psaPILnFsP nseqlvnLst ---P-L---P
ssLdlifvLD tgLtfyPavD gnLsvdPyME enLleePeMD --L---P-MD
350 tqAFaILPnS VqAFaVLPnS IdAFvlLPsS IeALvVLPsS I-AF-VLP-S
Bovine BPI Human BPI Human LBP Rabbit LBP Consensus
351 SlDPIFILem Slas1Fligm SkEPvFrLsv arEPvFrLgv S-EP-F-L--
n1NlSvvvga hTtgSmevsa aTNvSatltf aTNvSat1tl -TN-S-----
ksdrliGeLr esnrlvGeLK ntskitGfLK ntrkitGfLK ------G-LK
IdkLllELKh IdrLllELKh pgkvkvELKe pgrLqvELKe ---L--ELK-
400 SdIGpFsVEs SnIGpFPVEL SkVGIFnaEL SkVGgFnVEL S--G-F-VEL
Bovine BPI Human BPI Human LBP Rabbit LBP Consensus
401 LqsviNYvmp LqdiMNYiVp Lea1LNYyIl LealLNYyIl L---LNY-I-
tivlPvINkK ilvlPrVNEK nt1yPkfNDK nnlyPkVNEK ----P-VNEK
LqkGFPLPLp LqkGFPLPtp LaeGFPLPLl LahrFPLPLl L--GFPLPL-
ayIeLFnLtL arVQLYnvvL krVQLYdLgL rhIQLYdLIL ---QLY-L-L
450 QpyqdFLLFG QpHqnFLLFG QiHkdFLFLG QtHenFLLvG Q-H--FLLFG
Bovine BPI Human BPI Human LBP Rabbit LBP Consensus
451 AdVQYs AdVvYk AnVQYmrv AnIQYrrv A-VQY---
the extent of glycosylation is unknown. Secretion signal sequences of 26 and 25 residues, respectively, for the rabbit and human proteins are observed in the cDNA sequences. One other protein has sufficient sequence and functional similarity to suggest that it is a homologous protein; this is the bactericidal/permeability increasing protein (BPI) of neutrophil granules (28, 29). The sequences of human LBP and BPI are45 % identical and the two proteins share the ability to bind LPS and also have some antigenic cross-reactivity (28). A 25-kD N-terminal fragment of BPI is known which contains all, the LPS-binding and -neutralizing activity of complete BPI (30). However, BPI has not yet been demon-
strated to haveany function in endotoxemia. Its physiologic roleis probably to enhance bacterial killingbytheneutrophil (29). Hydropathy plots (31) and other predictors of protein secondary structure do not clearlyreveal any structural features of LBP or BPI that might form an LPS-binding site. Several otherproteins appear to have some sequence similarity (32, 33); however, theirfunctions probably do not include binding LPS, and thus the significance of the similarity is hard to assess. Biosynthesis of LBP appears to be restricted to the liver, most probably to the hepatocyte. Liver is the only tissue in which mRNA for LBP has been found (R. Schumann, per-
242
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULARBIOLOGY VOL. 7 1992
sonal communication). While Kupffer cell mRNA has not been studied, alveolar macrophages do not contain mRNA for LBP (T. Martin, personal communication). Pulse-chase labeling studies with explanted rabbit hepatocytes show an initial 50-kD intracellular LBP that is later found extracellularly as a 58- and 60-kD doublet (34). Culture in the presence of tunicamycin leads to intracellular accumulation of the 50-kD LBP. Similar data have more recently been obtained with human or rabbit LBP transfected into Chinese hamster ovary (CRG) cells. These results suggest the intracellular synthesis of an unglycosylated form ofLBP, followed by post-translational processing and secretion of the doublet isolable from serum. Levels of LBP in normal serum are in the 5 to 10 /Lglml range, with levels in acute-phase serum varying widely to above 200 /Lglml. As yet, the relation of serum LBP concentrations to outcomes in sepsis patients is unknown, but of considerable interest. The ability of LBP to bind to LPS has been shown to involve interaction with the lipid A moiety of LPS (35). This conclusion is derived from the observations that a variety of smooth LPS all bind to LBP similarly, the sugars from Salmonella minnesota Re595 LPS do not block LBP binding to LPS, and the binding of LBP to lipid A decreases as the structure of lipid A is dissected away. The apparent dissociation constant for S. minnesota Re595 LPS-LBP complexes is near 10- 9 M. In serum or plasma, the association of LPS with LBP is transient (26). LPS shed from the surfaces of gram-negative bacteria partitions among several potential ligands, among them lipoproteins (36) and transferrin (37) as well as LBP. LPS in complex with LBP also partitions, some going on to associate with Met> and some transferring to lipoproteins. The kinetics of these reactions, especially in vivo, have not been seriously studied.
LBP Involvement in LPS-dependent M Activation Rabbit peritoneal elicited macrophages (PEM) , elicited with mineral oil, are useful in studying LPS-dependent activation because they are obtainable in large quantity in a quiescent state and produce TNF in readily measurable amounts upon activation. While PEM respond to LPS in the absence of LBP if sufficient LPS is used, the presence of LBP dramatically enhances TNF responses to rough and smooth LPS as well as lipid A, especially at lower doses of LPS (Table 1). Similar effectsare seen on the production of other mediators, such as IL-l and IL-8. Substances that lack lipid A, such as heat-killed Staphylococcus aureus, peptidoglycan, or phorbol ester, do not show any effect of LBP. The increased levels of secreted TNF derive from induction of higher levels of TNF mRNA as well as a longer mRNA half-life. Immunodepletion of LBP from whole rabbit blood leads to a diminution of the ability of peripheral monocytes in whole blood to secrete TNF in response to LPS, although the response to heat-killed S. aureus is not diminished (38).
CD14 CD14 was first identified and cloned as a myeloid marker of unknown function (39), although it has since been found on a few other cells (40-42). The sequences of the human (43), rabbit, and murine (44) CD14s are shown in Figure 4. The mature surface protein apparently has no transmembrane
TABLE 1
LBP enhances LPS-induced TNF production in PEM [LBP] Stimulus
ng/ml
100 ng/ml
0
TNF (U/ml) E. coli 0111 :B4
1 0.1
6
Only recently has the mechanism for lipopolysaccharide (LPS) recognition by macrophages been elucidated. In contrast to many ligand receptor interactions, the interaction of LPS with its receptor, CDI4, on myeloid cells is greatly enhanced by prior complexation of LPS with LPS-binding protein (LBP), a recently discovered plasma glycoprotein. LBP is found in normal serum or plasma in the 5 to 10 ~g/ml range. In plasma, it reacts rapidly but transiently with LPS. LPS-LBP complexes then react with CDl4 bearing cells. Blocking CDl4 with monoclonal antibodies or removal ofLBP from plasma blocks the ability of the cells to react with LPS-LBP complexes and also blocks release of cytokines and other mediators from the cells. In the normal lung, bronchoalveolar lavage fluid contains low levels of LBP. However, during acute lung injury, LBP levels may rise by transudation and enhance activation of alveolar macrophages to release injurious mediators. Description of this pathway for LPS recognition by macrophages and other leukocytes offers the possibility of developing new reagents to block LPS recognition and prevent the development of endotoxemia.
When we senselipopolysaccharide, we are likely to turn on everydefense at our disposal; we willbomb,defoliate, blockade, seal off, and destroy all tissue in the area ... Pyrogen is released from leukocytes, adding fever to hemorrhage, necrosis, and shock. It is a shambles ... All this seemsunnecessary, panic-driven. There is nothing intrinsically poisonous about endotoxin, but it must look awful, or feel awful, when sensed by cells. -Lewis Thomas in The Lives of a Cell (1974) The consequences of endotoxemia include fever, hypotension, diffuse intravascular coagulation, acute respiratory distress, multi-organ failure, and ultimately death. While the mechanisms of these pathologic conditions may not yet be (Received in original form February 28, 1992 and in revised form March 24, 1992) Address correspondence to: P. S. Tobias, Ph.D., IMM-12, Department of Immunology, The Scripps Research Institute, 10666 N. Torrey Pines Rd., La Jolla, CA 92037. Abbreviations: adult respiratory distress syndrome, ARDS; 2-{p-azidosalicylamido)ethyl-l,3'-dithiopropionyl, ASD; bronchoalveolar lavage fluid, BAL; bactericidal/permeability increasing protein, BPI; Chinese hamster ovary, CHO; lipopolysaccharide-coated erythrocytes, E-LPS; fluoresceinlabeled LPS, F-LPS; interleukin, IL; lipopolysaccharide-binding protein, LBP; lipopolysaccharide, LPS; monoclonal antibodies, mAb; monocytes/ macrophages, Met>; peritoneal elicited macrophages, PEM; phosphatidylinositol, PI; tumor necrosis factor, TNF. Am. J. Respir. Cell Mol. BioI. Vol. 7. pp. 239-245, 1992
clear, it is certain that the endotoxin or lipopolysaccharides (LPS) from gram-negative bacteria activate monocytes/macrophages (Met» to secrete a variety of products that ultimately lead to the clinical features of endotoxemia. Because LPS are not themselves toxic, it is the inappropriate Met> response that initiates endotoxic injury. Not all responses to LPS are inappropriate, although the difference between an appropriate and an inappropriate response is unclear. The appropriate response probably includes tumor necrosis factor (TNF) and interleukin-l (IL-l) since these cytokines have been implicated in resistance to infection (1-6). The same mediators have been implicated in pathologic gram-negative septic shock (7-9). Thus, a central problem is to understand how Met> recognize LPS. Clarifying how responses are initiated may help to clarify how an appropriate response differs from an inappropriate response and may lead to development of methods to forestall the pathophysiologic changes that comprise gram-negative septic shock. This review will focus on recent progress in understanding Met> recognition of LPS.
LPS It is useful to briefly review LPS structure (Figure 1) (10-12). The lipid A moiety is the most highly conserved part of the structure, typically with two glucosamines, two phosphate esters, and five or more fatty acids. Studies of synthetic lipid A reveal that all the endotoxic activity of an LPS is derived from the lipid A moiety (11); however, not all lipid /Ji.s are
240
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 7 1992
Chain
~ l..l-----~I----- . .l l - - . f Lipid A ~
endotoxically active (13). The core region contains several sugars, among them 3-deoxY-D-manno-2-octulosonate (KDO), a sugar unique to LPS, and is typically conserved within a given genus of bacteria. The core region may also contain phosphate groups as well as ethanolamine moieties, often in substoichiometric amounts. The most variable part of the LPS structure is the a-antigen, responsible for the individual antigenic signatures of individual strains of gram-negative bacteria. Strains with an intact a-antigen are termed "smooth" strains, whereas those lacking the a-antigen and retaining variable amounts of the core sugars are termed "rough" mutants. These terms derive from the appearance of bacterial colonies. Isolates of LPS are rarely homogeneous, probably reflecting an inhomogeneous composition of the bacterial outer membrane (14). Different isolates from even the same strain of bacteria can show considerable variation in composition (15). Considerable experimental evidence supports the contention that LPS acts via a specific plasma membrane receptor (16-25). Recent studies from our laboratory have identified a novel mechanism for LPS recognition that differs from the standard ligand-receptor model in that it involves a plasma protein, LPS-binding protein (LBP) , which forms highaffinity complexes with LPS and a plasma membrane glycoprotein, CD14, which acts as a receptor for the LPS-LBP complexes (16). This review will focus on the properties of these two proteins and the evidence that they form a highaffinity recognition system for LPS. Our current understanding of the role of LBP in the LPS-dependent activation of macrophages, and possibly certain other cells, is illustrated in Figure 2. LPS shed from the surfaces of gram-negative bacteria binds to LBP. The complexes subsequently interact with CD14, leading to M¢ activation and cytokine secretion. The fact that there are other cells, such as B cells, which have no CD14 but nevertheless respond to LPS, suggests that there is an alternative to the CD14 pathway. This alternative pathway may be used even by CD14 bearing cells when they are in environments, such as the extravascular spaces, where LBP is low. The precise mechanism by which LPS-LBP engagement of CD14 leads to M¢ activation is by no means clear; our current working hypothesis will be described.
Figure 1. General chemicalstructure of bacterial lipopolysaccharides. The three parts of LPS are not drawn to scale. Depending on the value of n, molecular weights may be as large as 20,000. The O-specific chain is typically the immunodominant structure and is highly variable among bacterial strains. Mutants lacking the O-specific chain and more or less of the core region are "rough" mutants. Re mutants contain only lipid A and the two Kdo residues. The lipid A fragment has all the endotoxic activity of the intact molecule.
LBP LBP was first discovered as an acute-phase reactant with affinity for LPS (26), and we suggested that it might be an LPS inactivator by analogy with the protective effects of some other acute-phase reactants (27). This hypothesis could hardly have been more wrong. Our recent data show that the LBP in normal plasma is sufficient to dramatically enhance the ability of LPS to activate macrophages, and the reason for the rise in LBP during an acute-phase response remains unknown. One conjecture is that increasing LBP in the vascular compartment serves to enhance the availability of LBP in the extravascular compartment, especially at sites of inflammation. As deduced from cD NA clones, the amino acid sequences of human and rabbit LBP are shown in Figure 3. Both mature secreted proteins are composed of 456 amino acids, of which 69% are identical. Human LBP has five potential sites for N-linked glycosylation, three of which are also found in rabbit LBP. The protein isolated from serum is glycosylated, but
LBP LPS
Bacteria ---+ I::Zl
~ ------tv
1
1 \?
/'
~
~?
_ _ -+-t
IL-1 IL·6 IL·8 TNF-a
Figure 2. The involvement of LBP in LPS-dependent Mel> activation. See text for details.
Update
Figure 3. Comparison of the eDNA-derived amino acid sequences of LBP and BPI. Only one pair of cysteines is fully conserved at positions 135 and 175. Rabbit and human LBP have conserved glycosylation sites at Asn residues 276, 327, and 363. Native rabbit LBP has a single plasmin (or trypsin) cleavage site at Arg(99)-Leu(100). Cleavage at this position destroys LPS-binding activity. The LPS-binding activity of BPI resides in the N-terminal half of the molecule (30).
241
Bovine BPI Human BPI Human LBP Rabbit LBP Consensus
1 tNPGiVaRIT vNPGvVvRIs aNPG1VaRIT tNPGIItRIT -NPG-V-RIT
qKGLDYAcQq qKGLDYAsQq dKGLqYAaQe dKGLEYAare -KGLDYA-Q-
GvLtLQkELe GtaALQkELk G1LALQsELl GILALQrkLl G-LALQ-EL-
kITiPnFsGn rIkiPDYsds rITIPDFtGd eVTIPDsdGd -IT-PDF-G-
50 FkIKyLGkGq FkIKHLGkGh LrIpHvGrGr FrIKHFGraq F-IKHLG-G-
Bovine BPI Human BPI Human LBP Rabbit LBP Consensus
51 YsFFSMvIqg YsFYSMdIre YeFhSLnIhs YkFYSLkIpr Y-FYS--I--
FnLpnSqiRP FqLpsSqism ceLlhSalRP FeLlrgtlRP F-L--S--RP
IPdkGLdLSI vPnvGLkFSI vPgqGLsLSI IPgqGLsLdI -P--GL-LSI
rDAsIkIrGk SnAnIkIsGk SDssIrVqGr SDAyIhVrGs SDA-I---G-
100 WKaRKnFiKL WKaqKrFLKM WKvRKsFFKL WKvRKaFLrL WK-RK-FLKL
Bovine BPI Human BPI Human LBP Rabbit LBP Consensus
101 gGnFDLSVeG sGnFDLSIeG qGsFDvSVkG knsFDLyVkG -G-FDLSV-G
iSIlagLnLG mSISadLkLG iSISvnLILG ItISvhLvLG -SIS--L-LG
yDpaSGhsTV SnptSGkPTI SEs.SGrPTg SEs.SGrPTV SE--SG-PTV
TCSSCSSgIn TCSSCSShIn yC1SCSSdIa TtSSCSSdIq TCSSCSS-I-
150 tVrIhIsgss sVhVhIsksk dVeVdm.sgd nVeldI.egd -V-V-I----
Bovine BPI Human BPI Human LBP Rabbit LBP Consensus
151 IGWLiqLFrk vGWLiqLFhk sGWLlnLFhn leeL1nLLqs -GWL--LF--
rIESILqksM kIESaLrnkM qIESkFqkvL qIDarLrevL -IES-L----
trkICEvVts nSqVCEkVtn eSrICEmIqk eSkICrqIee -S-ICE----
tVSSkLQPYF sVSSkLQPYF sVSSdLQPYL aVtahLQPYL -VSS-LQPY-
200 QTLPVTTklD QTLPVmTkID QTLPVTTeID QTLPVTTqID QTLPVTT-ID
Bovine BPI Human BPI Human LBP Rabbit LBP Consensus
201 kvAGVDYSLV SvAGInYgLV SfAdIDYSLV SfAGIDYSLm S-AGIDYSLV
apPRATAnnL apPatTAetL eaPRATAqmL eaPRATAgmL --PRATA--L
DwLLKGEfFs DVqMKGEfYs EVMFKGEiFh DVMFKGEiFp DVMFKGE-F-
laHRSPppFa enHhnPppFa rnHRSPvtLl IdHRSPvdFl --HRSP--F-
250 pPaLaFPsdH pPvMeFPaaH aavMsLPeeH aPaMnLPeaH -P-M--P--H
Bovine BPI Human BPI Human LBP Rabbit LBP Consensus
251 dRMVYLgISE dRMVYLg1SD nkMVYFaISD sRMVYFsISD -RMVY--ISD
YfFNTAgFVY YfFNTAgLVY YvFNTAsLVY YvFNTAsLaY Y-FNTA-LVY
qkaGaLNLtl qeaGvLkMtl heeGyLNFsi hksGywNFsi ---G-LNF--
rDDMIPkESk rDDMIPkESk tDDMIPpDSn tDaMVPaDln -DDMIP--S-
300 fRLTTKfFgi fRLTTKfFgt iRLTTKsFrp iRrTTKsFrp -RLTTK-F--
Bovine BPI Human BPI Human LBP Rabbit LBP Consensus
301 LIPqvAkMFP FlPevAkkFP FVPrlArLYP FVPllAnLYP FVP--A-L-P
dMqMqLfiwa NMkiqihvsa NMnLeLqgsv NMnLeLqgtv NM-L-L----
slpPkLtMkP stpPhLsvqP psaPILnFsP nseqlvnLst ---P-L---P
ssLdlifvLD tgLtfyPavD gnLsvdPyME enLleePeMD --L---P-MD
350 tqAFaILPnS VqAFaVLPnS IdAFvlLPsS IeALvVLPsS I-AF-VLP-S
Bovine BPI Human BPI Human LBP Rabbit LBP Consensus
351 SlDPIFILem Slas1Fligm SkEPvFrLsv arEPvFrLgv S-EP-F-L--
n1NlSvvvga hTtgSmevsa aTNvSatltf aTNvSat1tl -TN-S-----
ksdrliGeLr esnrlvGeLK ntskitGfLK ntrkitGfLK ------G-LK
IdkLllELKh IdrLllELKh pgkvkvELKe pgrLqvELKe ---L--ELK-
400 SdIGpFsVEs SnIGpFPVEL SkVGIFnaEL SkVGgFnVEL S--G-F-VEL
Bovine BPI Human BPI Human LBP Rabbit LBP Consensus
401 LqsviNYvmp LqdiMNYiVp Lea1LNYyIl LealLNYyIl L---LNY-I-
tivlPvINkK ilvlPrVNEK nt1yPkfNDK nnlyPkVNEK ----P-VNEK
LqkGFPLPLp LqkGFPLPtp LaeGFPLPLl LahrFPLPLl L--GFPLPL-
ayIeLFnLtL arVQLYnvvL krVQLYdLgL rhIQLYdLIL ---QLY-L-L
450 QpyqdFLLFG QpHqnFLLFG QiHkdFLFLG QtHenFLLvG Q-H--FLLFG
Bovine BPI Human BPI Human LBP Rabbit LBP Consensus
451 AdVQYs AdVvYk AnVQYmrv AnIQYrrv A-VQY---
the extent of glycosylation is unknown. Secretion signal sequences of 26 and 25 residues, respectively, for the rabbit and human proteins are observed in the cDNA sequences. One other protein has sufficient sequence and functional similarity to suggest that it is a homologous protein; this is the bactericidal/permeability increasing protein (BPI) of neutrophil granules (28, 29). The sequences of human LBP and BPI are45 % identical and the two proteins share the ability to bind LPS and also have some antigenic cross-reactivity (28). A 25-kD N-terminal fragment of BPI is known which contains all, the LPS-binding and -neutralizing activity of complete BPI (30). However, BPI has not yet been demon-
strated to haveany function in endotoxemia. Its physiologic roleis probably to enhance bacterial killingbytheneutrophil (29). Hydropathy plots (31) and other predictors of protein secondary structure do not clearlyreveal any structural features of LBP or BPI that might form an LPS-binding site. Several otherproteins appear to have some sequence similarity (32, 33); however, theirfunctions probably do not include binding LPS, and thus the significance of the similarity is hard to assess. Biosynthesis of LBP appears to be restricted to the liver, most probably to the hepatocyte. Liver is the only tissue in which mRNA for LBP has been found (R. Schumann, per-
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sonal communication). While Kupffer cell mRNA has not been studied, alveolar macrophages do not contain mRNA for LBP (T. Martin, personal communication). Pulse-chase labeling studies with explanted rabbit hepatocytes show an initial 50-kD intracellular LBP that is later found extracellularly as a 58- and 60-kD doublet (34). Culture in the presence of tunicamycin leads to intracellular accumulation of the 50-kD LBP. Similar data have more recently been obtained with human or rabbit LBP transfected into Chinese hamster ovary (CRG) cells. These results suggest the intracellular synthesis of an unglycosylated form ofLBP, followed by post-translational processing and secretion of the doublet isolable from serum. Levels of LBP in normal serum are in the 5 to 10 /Lglml range, with levels in acute-phase serum varying widely to above 200 /Lglml. As yet, the relation of serum LBP concentrations to outcomes in sepsis patients is unknown, but of considerable interest. The ability of LBP to bind to LPS has been shown to involve interaction with the lipid A moiety of LPS (35). This conclusion is derived from the observations that a variety of smooth LPS all bind to LBP similarly, the sugars from Salmonella minnesota Re595 LPS do not block LBP binding to LPS, and the binding of LBP to lipid A decreases as the structure of lipid A is dissected away. The apparent dissociation constant for S. minnesota Re595 LPS-LBP complexes is near 10- 9 M. In serum or plasma, the association of LPS with LBP is transient (26). LPS shed from the surfaces of gram-negative bacteria partitions among several potential ligands, among them lipoproteins (36) and transferrin (37) as well as LBP. LPS in complex with LBP also partitions, some going on to associate with Met> and some transferring to lipoproteins. The kinetics of these reactions, especially in vivo, have not been seriously studied.
LBP Involvement in LPS-dependent M Activation Rabbit peritoneal elicited macrophages (PEM) , elicited with mineral oil, are useful in studying LPS-dependent activation because they are obtainable in large quantity in a quiescent state and produce TNF in readily measurable amounts upon activation. While PEM respond to LPS in the absence of LBP if sufficient LPS is used, the presence of LBP dramatically enhances TNF responses to rough and smooth LPS as well as lipid A, especially at lower doses of LPS (Table 1). Similar effectsare seen on the production of other mediators, such as IL-l and IL-8. Substances that lack lipid A, such as heat-killed Staphylococcus aureus, peptidoglycan, or phorbol ester, do not show any effect of LBP. The increased levels of secreted TNF derive from induction of higher levels of TNF mRNA as well as a longer mRNA half-life. Immunodepletion of LBP from whole rabbit blood leads to a diminution of the ability of peripheral monocytes in whole blood to secrete TNF in response to LPS, although the response to heat-killed S. aureus is not diminished (38).
CD14 CD14 was first identified and cloned as a myeloid marker of unknown function (39), although it has since been found on a few other cells (40-42). The sequences of the human (43), rabbit, and murine (44) CD14s are shown in Figure 4. The mature surface protein apparently has no transmembrane
TABLE 1
LBP enhances LPS-induced TNF production in PEM [LBP] Stimulus
ng/ml
100 ng/ml
0
TNF (U/ml) E. coli 0111 :B4
1 0.1
6
