©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Lipopolysaccharide (LPS) Signal Transduction and Clearance
DUAL ROLES FOR LPS BINDING PROTEIN AND MEMBRANE CD14 (*)

(Received for publication, October 24, 1994; and in revised form, January 4, 1995)

Julie A. Gegner Richard J. Ulevitch Peter S. Tobias (§)

From the Department of Immunology, The Scripps Research Institute, La Jolla, California 92037

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Under physiological conditions, lipopolysaccharide (LPS) activation of cells involves the LPS binding protein (LBP) and either membrane or soluble CD14. We find LPS forms a ternary complex with LBP and membrane CD14 (mCD14). Subsequent to complex formation and distinct from signal transduction, LBP and LPS internalize. Internalization can be separated from signal transduction with the anti-LBP antibody 18G4 and the anti-CD14 antibody 18E12. 18G4 inhibits LBP binding to mCD14 without blocking signal transduction or LPS transfer to soluble CD14; 18E12 inhibits signal transduction without affecting LPS binding and uptake. These data show that while LPS signal transduction and LPS clearance utilize both LBP and mCD14, the pathways bifurcate after LPS binding to mCD14.


INTRODUCTION

LPS, (^1)released from the outer membrane of Gram-negative bacteria, triggers cells to synthesize and release a cascade of inflammatory mediators that in vivo may progress to septic shock(1) . Two effector molecules involved in the LPS response are LBP and CD14(2) . LBP is a 60-kDa serum glycoprotein that binds to the lipid A moiety of LPS with a dissociation constant near 10M(3) . LBP appears to function as a catalytic transfer protein in delivering LPS to soluble CD14(4) . LBP also enhances the binding of LPS to the membrane form of CD14(5) , although the nature of the LBP interaction with mCD14 has not been evaluated.

Membrane CD14, an LPS receptor, is a 55-kDa glycoprotein attached to the cell surface by a glycosylphosphatidylinositol (GPI) anchor. Because mCD14 does not transverse the membrane, it is not known how the intracellular signal is initiated. Chimera constructs that replaced the GPI anchor with transmembrane domains demonstrate that the anchor is not required for signal transduction(6) . It has been proposed that a yet unidentified transmembrane transducer protein interacts with LPS and mCD14 to transmit the signal(6) . Membrane CD14 is found primarily on myeloid lineage cells and is responsible for the enhanced sensitivity of mCD14-positive cells, including transfected cell lines (7, 8, 9) , to LPS.

In addition to responding to LPS, cells internalize LPS(10, 11) . The scavenger receptor has been implicated in the clearance of LPS by hepatic uptake in mice, RAW 264.7 macrophage cells, and scavenger receptor-transfected CHO cells(12) . Scavenger receptor ligands, such as acetylated low density lipoprotein (AcLDL), were shown to compete in binding and metabolism with lipid IV(A), the bioactive precursor of lipid A. However, the ligands were unable to completely inhibit lipid IV(A) uptake. LBP and mCD14 also augment LPS clearance(11) , although any overlap of LBP- and mCD14-mediated clearance with that of the scavenger receptor has not been evaluated. Luchi and Munford (11) showed that serum, a source of LBP, and CD14 enhance but are not absolutely required for LPS internalization by human neutrophils. It seems likely from these and other studies that LPS clearance is a separate event from LPS signal transduction (9, 10, 11, 12) . Here, we have investigated the surface binding, internalization, and signal transduction properties of LBP and LPS with the model system of CD14-transfected CHO cells to further define the mechanism of LPS-initiated cellular activation.


EXPERIMENTAL PROCEDURES

Reagents

Re595 LPS was isolated from lyophilized Salmonella minnesota Re595 bacteria as described(13) . The ^3H-labeled LPS from Escherichia coli K12 LCD25 was a gift from R. S. Munford (University of Texas, Dallas, TX) or was purchased from List Biological Laboratories (Campbell, CA). The specific activity of the ^3H-labeled LPS typically was 1.45 times 10^6 dpm/µg. AcLDL was a gift from M. Krieger (Massachusetts Institute of Technology, Cambridge, MA). Monoclonal antibodies to LBP (18G4, 2B5) and CD14 (28C5, 18E12) were a gift from A. Moriarty and D. Leturcq (R. W. Johnson Pharmaceutical Research Institute, La Jolla, CA). MY4 was purchased from Coulter Immunology (Hialeah, FL). Reacti-Gel (6times) was purchased from Pierce. Sepharose CL-4B was purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus and Proteinase K were purchased from the Sigma. NF-kappaB probe was purchased from Promega.

Cells

CHO-K1 cells were transfected with pEE14 construct containing the full-length human LBP cDNA (CHO-hLBP) and maintained as described(14) . CHO-K1 cells were transfected with pRc/RSV (CHO-RSV) or a pRcRSV construct containing the DNA coding for the complete human CD14 protein (CHO-hCD14) as described(15) . 70Z/3-RSV, 70Z/3-hCD14, and 70Z/3-hCD14CI cells were transfected and maintained as described(7) . The pRc/RSV-hCD14CI plasmid contained the transmembrane domain and cytoplasmic tail of a murine class I molecule. THP-1 cells were provided by D. Altieri (The Scripps Research Institute, La Jolla, CA). CD14 expression was induced in the THP-1 cells with calcitriol(16) .

S-Labeled LBP

To prepare S-labeled LBP, CHO-hLBP cells were grown to confluence. The medium was replaced with serum-free RPMI 1640 without glutamine and methionine plus Nutridoma-SP from Boehringer Mannheim. L-[S]Methionine was added to a final concentration of 1.2 mCi (1 nmol of methionine)/20 ml. After an approximate overnight incubation of 16 h, the culture supernatant was removed and centrifuged for 5 min at 1500 rpm to remove cells. S-labeled LBP was purified by applying the supernatant to an affinity column made by coupling the anti-LBP monoclonal antibody 18G4 to Reacti-Gel (6times). The column was washed with phosphate-buffered saline (PBS), pH 7.2, plus 2 mM EDTA, 150 mM NaCl, and 0.05% NaN(3). S-Labeled LBP was eluted with a linear glycine pH gradient of pH 5.2 (0.1 M) to 2.5 (40 mM) and neutralized with 1.5 M Tris base. Collection tubes were precoated with 3 mg/ml bovine serum albumin (BSA) for 30 min at 37 °C to minimize adhesion of LBP to the tubes. BSA also was added as a carrier protein at 0.3 mg/ml to the collected fractions. S-Labeled LBP was dialyzed into PBS plus 2 mM EDTA, 150 mM NaCl and stored at 4 °C. The S-labeled LBP was observed as a single band by SDS-polyacrylamide gel electrophoresis and autoradiography. The specific activity of the S-labeled LBP was quantified by ELISA standardized against a known amount of LBP purified from human serum(17) . The LPS binding activity of the S-labeled LBP was tested by sizing chromatography. The column matrix was Sepharose CL-4B. To minimize nonspecific adhesion of S-labeled LBP to the column, the S-labeled LBP sample was mixed with an equal volume of serum. The elution time of S-labeled LBP (50 nM) with and without a stoichiometric excess of Re595 LPS (2.5 µM) was compared. In the absence of LPS, S-labeled LBP eluted in the included volume approximately with BSA; when LPS was added, at least 90% of the S-labeled LBP co-eluted in the void volume with the majority of the LPS.

Binding Assay

LBPbulletLPS complexes were formed by incubating for 2 min at 37 °C. Cells were then added as described in the text. Adherent cells were first detached by addition of PBS plus 2 mM EDTA for 3 min at 20 °C. Cells were pelleted, washed once in binding buffer, and resuspended in binding buffer. Binding buffer contained 20 mM Hepes, pH 7.4, 2 mM EDTA, 150 mM NaCl, and 0.3 mg/ml BSA. When metabolic inhibitors were specified, cells were incubated for 30 min at 37 °C in binding buffer plus 10 mM NaN(3), 2 mM NaF, and 5 mM deoxyglucose (15) prior to addition of LBP and LPS. Sample volume was 100 µl. After complex formation, cells plus any associated LBP and LPS were pelleted by centrifugation for 60 s at 6000 times g. After centrifugation, the supernatant was removed, and the pellet was resuspended in 100 µl of binding buffer. Samples were mixed with 200 µl of 2% SDS and 50 mM EDTA, pH 7.4, and assayed in scintillation fluid for radioactivity. Bound LBP or LPS was calculated from the radioactivity associated with the cell pellet; free ligand was determined from the radioactivity remaining in solution. Quantification of mCD14 expression on CHO-hCD14 cells was done by Scatchard analysis of the binding of I-Fab fragments of anti-CD14 monoclonal antibody 28C5 to CHO-hCD14 cells. The average for CHO-hCD14 cells was 4.4 times 10^5 CD14 molecules/cell. The average molecular mass used for the K12 LCD25 LPS monomer was 4000 Da(10) ; the average molecular mass used for Re595 LPS was 2300 Da. The Re595 LPS molecular weight was calculated from its structure(18) .

Cleavage of CD14

PI-PLC was added to cells in binding buffer at 4 units/1 times 10^6 cells/100 µl for 30 min at 12 °C. Cleavage of CD14 was measured by pelleting the cells and assaying the supernatant for sCD14 by ELISA. Control cells were incubated at 12 °C without PI-PLC.

Chase Experiments

5 times 10^5 CHO-hCD14 cells were incubated with 20 nMS-labeled LBP and 10 nM^3H-labeled LPS for 30 min at 37 °C. Cells were pelleted and resuspended in 30 µl of 1 µM unlabeled purified LBP and incubated for 20 min at 37 °C. Control cells were resuspended in 30 µl of binding buffer. Cells were then diluted with 120 µl of binding buffer, pelleted, and counted. Background binding was determined by adding 30 µl of 1 µM unlabeled LBP to the S-labeled LBP before addition of LPS and cells. AcLDL (100 µg/ml) and 18E12 (10 µg/ml) were added to cells as indicated in the text prior to addition of radiolabeled ligands.

Cell Stimulation

For NF-kappaB activation, CHO-hCD14 cells were grown overnight in 1% human serum. Antibodies were added to a final concentration of 10 µg/ml for 10 min prior to addition of LPS. Re595 LPS (0.1-100 ng/ml) was added for 40 min at 37 °C. Cells were washed twice with cold PBS and removed with PBS plus 2 mM EDTA. Samples were prepared, and the NF-kappaB assay was performed as described(19) . NF-kappaB activation was quantified using the PhosphorImager (Molecular Dynamics). Sample loading was normalized by dividing the NF-kappaB activation bands by the control band. The human TNF whole blood assay ex vivo was performed as described(20, 21) . Briefly, blood from two donors was incubated with or without antibodies (10 µg/ml) for 10 min at 20 °C. Re595 LPS (0.3-10 ng/ml) was added, and the blood was incubated for 4 h at 37 °C. The cells were pelleted, and the supernatant was added to WEHI 164 clone 13 cells(20) . TNF production was quantified by its cytolytic activity(21) .


RESULTS

LBPbulletLPSbulletCD14 Ternary Complex

We first performed studies to evaluate whether binding of LBP to CHO-hCD14 cells required both mCD14 and LPS. Fig. 1shows the results of S-labeled LBP binding to transfected CHO cells in the presence and absence of LPS. Incubation of S-labeled LBP or S-labeled LBPbulletLPS complexes with cells was done at 4 °C. We found that as long as S-labeled LBPbulletLPS complexes were formed at 37 °C, subsequent incubation temperature of either 4 or 37 °C has little effect on the equilibrium binding of S-labeled LBP and LPS to cells (data not shown). In the absence of LPS, direct binding of S-labeled LBP to CHO-hCD14 cells was not observed. Only when LPS was added was S-labeled LBP binding to CHO-hCD14 cells observed. S-Labeled LBP did not adhere to CHO-RSV control cells that lacked CD14, and the anti-CD14 antibody MY4, which blocks LPS binding to CD14(22) , also blocks S-labeled LBP binding. The binding of S-labeled LBP to cells only in the presence of membrane CD14 and LPS can be most easily explained by the formation of an LBPbulletLPSbulletmCD14 ternary complex. LPS-dependent binding of S-labeled LBP was observed in other cell lines that expressed membrane CD14 such as 70Z/3-hCD14, 70Z/3-hCD14CI, calcitriol-induced THP1, and RAW cells but not in the empty vector 70Z/3-RSV control cells or in uninduced THP1 cells (data not shown).


Figure 1: Binding profile of LBP to CHO cells. S-Labeled LBP (40 nM) incubated at 37 °C with or without Re595 LPS (40 nM) was added to cells, and complexes were allowed to form for 40 min at 4 °C. The anti-CD14 antibody MY4 (10 µg/ml) was added to CHO-hCD14 cells for 10 min at 4 °C prior to addition of LBP and LPS. LBP binds to CHO cells only when both mCD14 and LPS are present.



LBP binding to mCD14 is dependent not only on LPS but also on the relative amount of LPS to LBP. As shown in Fig. 2, the optimal LPS concentration for maximum association of S-labeled LBP with cells occurs at approximately 1:1 to 1:2 LBP-LPS stoichiometry. Very high levels of LPS block S-labeled LBP binding to cells. If LPS simply was binding to mCD14 in competition with LBPbulletLPS complexes, then one would expect the amount of mCD14 present to effect the optimal LPS concentration for LBP binding. However, we observed that even though the amount of S-labeled LBP bound increased with increasing mCD14, the LPS concentration that yields maximum S-labeled LBP binding remained the same (see Fig. 2). We evaluated up to a 10-fold range in cell number without observing a shift in the S-labeled LBP binding maximum (data not shown). ^3H-Labeled LPS binding to cells also reaches a maximum at approximately 1:1 to 1:2 LBP-LPS stoichiometry(15) . However, ^3H-labeled LPS binding does not return to background levels when excess LBP is present but rather reaches a plateau(15) . We evaluated a 100-fold molar excess of LBP to ^3H-labeled LPS without observing a reduction in ^3H-labeled LPS binding (data not shown).


Figure 2: Effects of LPS on LBP binding to CHO-hCD14. S-Labeled LBP incubated at 37 °C with increasing amounts of Re595 LPS subsequently was incubated with cells for 40 min at 4 °C. An LBP binding maximum is observed when the LBP to LPS molar ratio is approximately 1:1 to 1:2. The binding maximum is dependent on LBP concentration but not on cell number.



LPS and LBP Form Large Complexes Anchored by mCD14

When the LBP to LPS molar ratio was held constant at 1:1, we were unable to saturate LBPbulletLPS complex binding to mCD14. Fig. 3A shows that binding of S-labeled LBP to CHO-hCD14 cells is linear to a 1000 molar excess of LBP and LPS to mCD14. Similar results were obtained at 4 °C (data not shown). Evidence that the LBPbulletLPS complexes remain associated with mCD14 under conditions that prevent internalization is their release by PI-PLC (see Fig. 5). The inability to saturate binding to mCD14 suggests that LBPbulletLPS complexes self-associate on the cell surface. When either LBP or LPS concentration is held constant, binding saturation occurs. Fig. 3B shows saturation of ^3H-labeled LPS and S-labeled LBP binding to cells when the LBP concentration is held constant at 12 nM; Fig. 3C shows saturation of ^3H-labeled LPS and S-labeled LBP binding when the LPS concentration is held constant at 12 nM. Saturation occurs at an LBP to LPS molar ratio of approximately 1:2. These data support the idea that a ternary LBPbulletLPSbulletmCD14 complex forms since one would not expect LPS binding saturation to occur at limiting LBP if LBP was catalytically delivering LPS to mCD14.


Figure 3: Binding saturation. Panel A evaluates LBP binding to CHO-hCD14 cells as a function of LBP when the LBP to LPS molar ratio is held constant at 1:1. Equal molar amounts of S-labeled LBP and Re595 LPS were incubated with 1.5 times 10^5 CHO-hCD14 cells for 40 min at 37 °C in the presence of inhibitors to internalization. No binding saturation is observed. Panel B shows binding saturation when the LBP concentration was held constant at 12 nM; panel C shows saturation of both S-labeled LBP and ^3H-labeled LPS binding when the LPS concentration was held constant at 12 nM. Complexes in panel A and panel B were formed by incubating S-labeled LBP and ^3H-labeled LPS with 5 times 10^5 CHO-hCD14 cells for 20 min at 37 °C with inhibitors to internalization.




Figure 5: PI-PLC cleavage of mCD14 GPI tail releases bound LBP. 1 times 10^6 cells were incubated with 220 nMS-labeled LBP + 220 nM Re595 LPS for 40 min at either 4 or 37 °C. Cells then were incubated with or without PI-PLC for 20 min at 12 °C. Cells were pelleted, and the supernatant was assayed for CD14 by ELISA. The pellet was counted to determine cell-associated LBP.



LPS and LBP Bind to mCD14 with Similar Kinetics

To further evaluate if LBP transfers LPS to mCD14 or forms a ternary LBPbulletLPSbulletmCD14 complex, we evaluated the binding of S-labeled LBP and ^3H-labeled LPS to mCD14 as a function of time. Fig. 4shows that even when LPS is added in a 5-fold molar excess to LBP, both S-labeled LBP and ^3H-labeled LPS bind to mCD14 with the same association half-life of 1.2 ± 0.1 min. The association kinetics in combination with the previous data (see Fig. 1) imply that LBP and LPS associate with mCD14 as an LBPbulletLPS complex. The observed LBP to LPS stoichiometry under these conditions is approximately 1:4. Variability in the stoichiometry in which LBP and LPS bind mCD14 suggests that multiple LPS molecules bind LBP.


Figure 4: LBP and LPS bind mCD14 with similar kinetics. 10 nMS-labeled LBP and 50 nM^3H-labeled LPS were incubated with 5 times 10^5 cells at 37 °C in binding buffer with inhibitors to internalization. Panel A is a plot of the natural logarithm of the amount of ligand bound at equilibrium (F()) minus ligand bound at time t (F(t)). From the slopes of the curves, LBP and LPS have the same association half-life of 1.2 ± 0.1 min. Panels B and C show that even though the LBP to LPS binding stoichiometry is approximately 1:4, LPS and LBP reach equilibrium together.



LBP and LPS Internalization

Treatment of cells expressing GPI-anchored mCD14 with PI-PLC removes mCD14 and any mCD14-associated LPS on the outer cell membrane(7) . Fig. 5shows that when LBPbulletLPSbulletmCD14 complexes are formed at 4 °C, essentially all of the S-labeled LBP is released by PI-PLC. However, after complex formation at 37 °C, 40% of the S-labeled LBP remains associated with the cells after PI-PLC treatment. Unlike S-labeled LBP binding, the amount of mCD14 released by PI-PLC is not effected by the binding conditions. The resistance of S-labeled LBP binding to release by PI-PLC suggests S-labeled LBP transfers from mCD14 under conditions permissive to internalization, i.e. at 37 °C in the absence of inhibitors.

We next evaluated the resistance of S-labeled LBP and ^3H-labeled LPS binding to dissociation by protease as a function of time at 37 °C. Fig. 6shows that the resistance of both S-labeled LBP and ^3H-labeled LPS binding to release increases at 1%/min. This is similar to a value determined for the rate of LPS internalization by human neutrophils(11) . Internalized S-labeled LBP also should be stable to conditions favoring its dissociation. As shown in Fig. 7, 35% of the S-labeled LBP is resistant to dissociation after dilution into a 300-fold excess of unlabeled LBP. However, when inhibitors to internalization are added, only 10% is resistant to dissociation. The inability to completely dissociate S-labeled LBP by PI-PLC, protease, or unlabeled LBP suggests that LBP is internalized with LPS. Since the amount of mCD14 released by PI-PLC is not affected by incubation conditions, mCD14 either recycles to the cell surface or is not directly involved in LBPbulletLPS complex internalization.


Figure 6: LBP and LPS binding to CHO-hCD14 cells becomes protease resistant after incubation at 37 °C. S-Labeled LBP (25 nM) and ^3H-labeled LPS (25 nM) were incubated with 5 times 10^5 CHO-hCD14 cells for 20 min at 4 °C. Samples were shifted to 37 °C for the indicated time and returned to 4 °C. Proteinase K (0.4 mg/ml) was added at 4 °C for 20 min. Cells were pelleted, washed once in binding buffer, and counted to determine protease-resistant LBP and LPS binding.




Figure 7: Effect of 18E12 and AcLDL on S-labeled LBP resistance to dissociation. 5 times 10^5 cells were incubated with S-labeled LBP (20 nM) and ^3H-labeled LPS (10 nM) for 30 min at 37 °C. 18E12 (10 µg/ml), AcLDL (100 µg/ml), or inhibitors to internalization were added to cells before addition of radiolabeled LBP and LPS. Unlabeled LBP then was added as described under ``Experimental Procedures.'' S-Labeled LBP resistant to displacement by LBP is plotted.



18E12 inhibits LPS-stimulated TNF release as well as other cellular responses with little effect on LPS binding(15) . Fig. 7shows that 18E12 does not inhibit S-labeled LBP internalization. Hampton et al.(12) demonstrated that LPS interacts with scavenger receptor-transfected CHO cells and that this interaction is blocked by AcLDL. To test the possibility that scavenger receptor endogenous to CHO cells is involved in LBPbulletLPS complex internalization, AcLDL was added to the cells prior to LPS and LBP binding. AcLDL had little effect on either the extent of LBPbulletLPS complex binding (data not shown) or on LBP resistance to dissociation (see Fig. 7). Thus, inhibition of LPS-dependent signaling by 18E12 does not inhibit LBP internalization nor does AcLDL, an LPS-competitive ligand for the scavenger receptor.

Cellular Activation by LPS

Anti-CD14 and anti-LBP monoclonal antibodies were used to help differentiate between LPS internalization and signal transduction pathways. How these antibodies effect LPS binding and activation of NF-kappaB in CHO-hCD14 cells is shown in Fig. 8. The anti-CD14 antibodies 18E12 and 28C5 inhibited NF-kappaB activation as previously shown (15) as did the anti-LBP antibody 2B5, whereas the anti-LBP antibody 18G4 had little effect on NF-kappaB activation. Additional experiments showed that the effects of these antibodies on TNF release in whole blood paralleled their effect on NF-kappaB activation in CHO-hCD14 cells; 18G4 was not inhibitory, whereas 18E12, 28C5, and 2B5 caused a diminished response (data not shown). Fig. 8also summarizes the effects of these antibodies on LPS and LBP binding. As previously noted(6) , 18E12 blocks activation without disrupting LBP and LPS binding. We observed 90 ± 3% of LBP and LPS to bind when 18E12 was added. Alternatively, the anti-LBP antibody 18G4 inhibited LBP binding and reduced LPS binding to 10% above background without diminishing activation. As determined by fluorescence spectroscopy, 18G4 does not inhibit transfer of fluorescein isothiocyanate-LPS (22) by LBP to sCD14. (^2)These results corroborate previous studies involving deacylated LPS (10) and the scavenger receptor (12) that demonstrated LPS binding to the cell membrane is not sufficient for signal transduction regardless of whether the initial binding is to mCD14 or the scavenger receptor.


Figure 8: Effect of antibodies on binding and cell activation. The bargraph on the left illustrates the effect of the indicated antibodies (10 µg/ml) on binding of S-labeled LBP (36 nM) and ^3H-labeled LPS (72 nM) to 5 times 10^5 CHO-hCD14 cells (4 nM). Anti-CD14 antibodies were added to cells for 10 min at 4 °C prior to addition of LBPbulletLPS complexes; anti-LBP antibodies were added to LBPbulletLPS complexes for 10 min at 4 °C prior to addition of cells. Complexes were formed for 30 min at 37 °C in binding buffer. 18G4 reduced LBP binding to background levels, whereas LPS binding was observed at 10 ± 2% above background; 18E12 reduced LBP and LPS binding only slightly to 90 ± 3% of the controls. 2B5 and 28C5 reduced binding to background levels. The bargraph on the right illustrates the effect of the indicated antibodies (10 µg/ml) on NF-kappaB activation of CHO-hCD14 cells by Re595 LPS (100 ng/ml). The control antibody and 18G4 did not diminish NF-kappaB activation, whereas 2B5, 18E12, and 28C5 were inhibitory.




DISCUSSION

The binding of S-labeled LBP to cells, only in the presence of mCD14 and LPS, provides direct evidence for the formation of an LBPbulletLPSbulletmCD14 ternary complex. This is congruent with the earlier work of Wright et al.(23) , which showed that LBP binding to macrophages is LPS dependent. The optimal LBP to LPS stoichiometry for LBP binding to mCD14 is approximately 1:1 to 1:2, which agrees with the stoichiometry observed for LBP binding to LPS immobilized on plastic microtiter plates(3) . Stoichiometry estimates are approximate because LPS is heterogeneous in size(24) . However, even when using the same LPS preparation, variable amounts of LPS associate with LBP and mCD14 when the LBP to LPS molar ratio is varied. This implies that in addition to LPS monomers, LPS aggregates bind to LBP and mCD14. An alternative explanation for variability in stoichiometry is that LBP has multiple LPS binding sites. However, inspection of its primary sequence does not reveal any repeated motifs. Further characterization of the LBP structure will aid in distinguishing between these possibilities.

LPS is highly aggregated in aqueous solutions with monomer solubility in the range of 10M(26) . Incubation of LPS with LBP alters its aggregation properties. Size analysis of LBP and LPS by sucrose gradients shows that 1) incubation of LBP with excess LPS increases the apparent size of LBP to co-sediment with the aggregated LPS and 2) incubation of stoichiometric amounts of LBP and LPS reduces the apparent size of LPS to co-sediment with LBP near the apparent size of LBP alone. (^3)When binding to cells is evaluated, LPS concentrations in large excess to LBP inhibit LBP binding to mCD14. While it is possible that LPS directly competes with LBPbulletLPS complexes for mCD14, insensitivity of optimal LBP binding to cell number and hence mCD14 concentration suggests that LPS aggregation of LBP is the primary factor. One phenomenon that is difficult to explain from LBP and LPS size analysis in solution is the inability to saturate LBPbulletLPS complex binding to mCD14 (i.e.Fig. 5A). Whereas equal molar amounts of LBP and LPS are not observed to aggregate in solution, they do accumulate via mCD14 on the cell surface. The flexibility in the LBPbulletLPS complex stoichiometry may enable LBP to accommodate in vivo fluctuations in LPS concentration, while the ability of mCD14 to sequester large numbers of LBPbulletLPS complexes may provide for efficient clearance of LPS, especially where local LPS concentrations are high.

At first glance, the mechanism by which LBP interacts with mCD14 appears distinct from its interaction with sCD14. Whereas LBP catalyzes the transfer of LPS to sCD14(4) , LBP associates with mCD14 as an LBPbulletLPS complex. This leads us to conclude that a membrane component, distinct from mCD14, forms a weak interaction with LBP to stabilize its association with the cell. Stabilization cannot be attributed to LBP interactions with the GPI tail of mCD14 since in studies not shown LBP binding is observed in 70Z/3-hCD14CI cells (6) that express a CD14 transmembrane construct. It is likely that 18G4 binds to the LBP epitope involved in the stabilization. Since 18G4 has no effect on the transfer of LPS to sCD14 or cell activation through mCD14, LBP-facilitated binding of LPS to mCD14 must occur even in the presence of 18G4. Whereas LBP binding is at background levels in the presence of 18G4, 10% of LPS binds, and cell activation ensues. This behavior suggests that transfer of LPS to mCD14 by LBP in the presence of 18G4, which resembles LBP and LPS interactions with sCD14, is sufficient for cell activation.

Internalization of LPS has been characterized in numerous systems, and it now seems evident that both LBP and mCD14 participate in this process. Scavenger receptors have been identified as binding LPS(12) , but our results suggest that internalization initiated by first anchoring to mCD14 occurs independently of the scavenger receptor. In our model, LBP delivers LPS to the cell surface through formation of a ternary LBPbulletLPSbulletmCD14 complex. Internalization and signal transduction pathways diverge at this point. Fig. 9illustrates how the antibodies 18E12 and 18G4 separate the pathways. In signal transduction, LPS and mCD14 interact with a putative transducer to initiate an intracellular response. 18E12 is thought to block the LPS-mCD14 transducer interaction and thus prevent cellular activation by LPS. There is no evidence that LBP is directly involved in the transducer activation. Indeed, the ability to eliminate LBP binding without impeding signaling implies that it is not. In the internalization pathway, LBP stays closely associated with LPS, whereas mCD14 either remains or returns to the cell surface. 18G4 is thought to inhibit LBP and LPS internalization by preventing LBP and LBPbulletLPS complexes from accumulating on the cell surface.


Figure 9: Internalization and signal transduction utilize LBP and mCD14. LBP binds LPS in serum and delivers it to mCD14 on the cell surface. Some complexes initiate cellular activation, perhaps through a transmembrane transducer molecule, whereas other complexes are internalized. Panel A illustrates the effects of 18E12. 18E12 allows binding and internalization of LBPbulletLPS complexes but blocks the epitope on mCD14 involved in signal transduction, presumably the LPS-mCD14 transducer interaction. Panel B illustrates the effects of 18G4. LBP association with mCD14 is stabilized through a weak interaction with an unidentified membrane component. 18G4 recognizes the LBP epitope involved in this interaction and prevents LBP binding and internalization. 18G4 does not affect transfer of LPS to mCD14 and subsequent cellular activation.



Maximal LPS binding occurs when LBP is not limiting. During an acute phase response, LBP serum levels rise from 0.1-5 µg/ml to geq50 µg/ml(23, 25) . It is possible that while there is sufficient LBP present in serum to initiate signaling by LPS and thereby initiate an acute phase response, LBP levels rise to ensure efficient clearance of LPS from circulation. LBP is synthesized by hepatocytes(23, 25) , and LPS is cleared by the liver(12) . Future research may show that hepatic macrophages (Kupfer cells) facilitate clearance of LPS in an LBP- and mCD14-dependent manner. Indeed, the recent identification of mCD14 up-regulation in response to LPS in mouse Kupfer cells (27) supports the hypothesis that LBP and mCD14 are involved in LPS clearance. Any coordinated effect of the scavenger receptor with LBP and mCD14 awaits further investigation. In conclusion, the LPS signal transduction and internalization pathways are optimal when both LBP and mCD14 participate and diverge after the initial LBPbulletLPSbulletmCD14 ternary complex forms.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants AI32021 (to P. S. T.) and AI15136 and GM28985 (to R. J. U.) and National Research Service Award Trainee Grant GM08172 and an American Heart Association Fellowship (to J. A. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Immunology, The Scripps Research Institute, 10666 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-554-8215; Fax: 619-554-3289.

(^1)
The abbreviations used are: LPS, lipopolysaccharide; LBP, LPS binding protein; mCD14, membrane CD14; sCD14, soluble CD14; GPI, glycosylphosphatidylinositol; AcLDL, acetylated low density lipoprotein; TNF, tumor necrosis factor; PI-PLC, phosphatidylinositol-specific phospholipase C; PBS, phosphate-buffered saline; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; CHO, Chinese hamster ovary.

(^2)
J. A. Gegner, R. J. Ulevitch, and P. S. Tobias, unpublished results.

(^3)
P. S. Tobias, unpublished results.


ACKNOWLEDGEMENTS

We thank K. Soldau for quantifying surface CD14, N. Wolfson for performing the TNF assay, and J. Han and J.-D. Lee for providing the transfected cell lines.


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