©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Enzymatically Deacylated Lipopolysaccharide (LPS) Can Antagonize LPS at Multiple Sites in the LPS Recognition Pathway (*)

Richard L. Kitchens , Robert S. Munford (§)

From the (1) Departments of Microbiology and Internal Medicine and the Immunology Graduate Program, The University of Texas Southwestern Medical Center, Dallas, Texas 75235

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Like other tetraacyl partial structures of lipopolysaccharide (LPS) and lipid A, LPS that has been partially deacylated by acyloxyacyl hydrolase can inhibit LPS-induced responses in human cells. To identify the site(s) of inhibition in the LPS recognition pathway, we analyzed the apparent binding affinities and interactions of H-labeled enzymatically deacylated LPS (dLPS) and [H]LPS with CD14, the LPS receptor, on THP-1 cells. Using (i) incubation conditions that prevented ligand internalization and (ii) defined concentrations of LPS binding protein (LBP), which facilitates LPS and dLPS binding to CD14, we found that dLPS can antagonize LPS in at least three ways. 1) When the concentration of LBP in the medium was suboptimal for promoting LPS-CD14 binding, low concentrations of dLPS were able to compete with LPS for binding CD14, suggesting competition between LPS and dLPS for engaging LBP. 2) When LBP was present in excess, dLPS could compete with LPS for binding CD14, but only at dLPS concentrations that were at or above its Kfor binding CD14 (100 ng/ml). 3) In contrast, substoichiometric concentrations of dLPS (1 ng/ml) inhibited LPS-induced (3 ng/ml) interleukin-8 release without blocking LPS binding to CD14. Functional antagonism was possible without competition for cell-surface binding because both LPS-induced interleukin-8 release and dLPS inhibition occurred at concentrations that were far below their respective CD14 binding Kvalues. In addition to its expected ability to compete with LPS for binding LBP and CD14, dLPS thus potently antagonizes LPS at an undiscovered site that is distal to LPS-CD14 binding in the LPS recognition pathway.


INTRODUCTION

Lipopolysaccharide (LPS() or endotoxin) is a bioactive glycolipid constituent of the outer membranes of Gram-negative bacteria. Responses to LPS in human cells show stringent requirements for certain structural features of lipid A, the bioactive center of LPS, including a diglucosamine backbone that bears both 1 and 4` phosphates and acyloxyacyl-linked (secondary) fatty acids (1) . For example, a synthetic tetraacyl lipid A partial structure that lacks secondary fatty acids (compound 406 or LA-14-PP) is virtually inactive in in vitro assays using human cells (1) . LPS molecules bearing tetraacyl lipid A structures may be generated by a leukocyte enzyme, acyloxyacyl hydrolase (2) , which removes secondary fatty acyl chains from a variety of bacterial LPSs (3) . Enzymatically deacylated LPS (dLPS) has greatly reduced stimulatory activity in human cells. Moreover, Pohlman et al. (4) showed that dLPS inhibits the ability of LPS, but not other agonists, to stimulate adhesion of neutrophils to human endothelial cells. Subsequently, others showed that dLPS (5, 6, 7, 8) and tetraacyl lipid A partial structures (derived either by purification of biosynthetic precursors (7, 9, 10) or by chemical synthesis) (11, 12) can inhibit a variety of LPS-induced responses in human monocyte/macrophages, neutrophils, endothelial cells, and whole blood.

The basis for the LPS-specific inhibitory action shown by these analogs is controversial. As initially suggested by Pohlman et al. (4) , the analogs may compete with LPS for binding certain critical molecules in the LPS recognition pathway. A likely target would be CD14, an important LPS receptor that, in the presence of LPS binding protein (LBP), mediates cellular responses to low concentrations of LPS (13, 14, 15, 16) . In keeping with this notion, other investigators (17, 18, 19, 20) have found that high concentrations of synthetic tetraacyl lipid A analogs ( e.g. LA-14-PP and PE-4) can inhibit LPS binding to monocytes, presumably by competing with LPS for binding CD14. In contrast, we found that dLPS and LA-14-PP can inhibit responses to LPS without inhibiting CD14-mediated LPS uptake by cells of the human THP-1 monocytic cell line (8) . To attempt to resolve this issue, we performed quantitative analyses of the binding of [H]LPS and [H]dLPS to THP-1 cells under conditions that prevent ligand internalization. Our results indicate that, depending upon the experimental conditions, dLPS can prevent the LPS-CD14 interaction by competing with LPS for binding either LBP or CD14. In addition, dLPS can also antagonize the stimulatory activity of LPS without inhibiting the binding of LPS to CD14. Remarkably, although dLPS binds to CD14 with substantially lower affinity than does LPS, 50% inhibition of the response to LPS can occur with dLPS:LPS ratios of 0.2 or less. Our results indicate that LPS antagonism by lipid A analogs may occur at three distinct sites in the LPS recognition pathway, and they point to the existence of an undiscovered inhibitory mechanism that occurs after the interaction of LPS with CD14.


MATERIALS AND METHODS

Cells

Cells of the THP-1 human monocyte cell line (21) were obtained from D. Altieri (Scripps Research Institute, La Jolla, CA) and cultured as described previously (8) . To induce elevated expression of CD14, the cells were exposed to 0.05 µM 1,25-dihydroxyvitamin Dfor 48 h.

Reagents

Purified LBP from acute phase rabbit serum was generously provided by Dr. Peter S. Tobias (Scripps Research Institute, La Jolla, CA), and each lot was tested to determine the ratio of LBP to LPS that produced maximal binding to CD14. Unless otherwise stated, a molar and functional excess of LBP was used in the binding assays. On a weight basis, LBP was typically present at 15-80-fold higher concentrations than LPS. Proteinase K, phosphatidylinositol-specific phospholipase C (PI-PLC from Bacillus cereus), Nonidet P-40, sodium deoxycholate, SDS, iodoacetamide, phenylmethylsulfonyl fluoride, aprotinin, leupeptin, TPCK, and ZPCK were obtained from Sigma. Octylglucoside (octyl--D-glucopyranoside) was from Calbiochem (San Diego, CA). Anti-CD14 mAbs 60bca and 26ic in mouse ascites fluid were provided by Dr. Robert F. Todd (University of Michigan, Ann Arbor).

LPS Preparations

[H]LPS was biosynthetically labeled using Escherichia coli LCD25 (22) to a specific activity of 6 10dpm/µg of LPS. A molecular weight of 4,000 was estimated for the LPS monomer (8) . Unlabeled LPS was derived from the same strain, LCD25. dLPS was produced from these preparations using acyloxyacyl hydrolase as described previously (5) . The specific activity of [H]dLPS was 4.2 10dpm/µg. LPS from Salmonella minnesota R5 was repurified using deoxycholate (23) , derivatized with SASD and radioiodinated using Iodogen (Pierce) as described elsewhere (24) . This preparation did not contain protein impurities (<0.1% by weight) by silver staining or by fluorography of SDS-PAGE gels.

Preparation of Fab Fragments of mAb 60bca

60bca was purified on -bind G (Pierce) before preparation of antibody fragments. Fab fragments were purified from papain digests using protein A-Sepharose (unbound fraction) followed by gel filtration chromatography on Sephacryl S-100-HR (Sigma). Preparations were pure as assessed by Coomassie Blue-stained SDS-PAGE gels.

Measurement of CD14 Expression

CD14 expression was measured by analyzing the binding of mAb 60bca (Fab) as described previously (25, 26) . Briefly, cells were incubated with increasing concentrations of radioiodinated antibody fragments for 1 h on ice. Cell-bound and free antibody fragments were separated by centrifugation over a 10% sucrose cushion. Nonspecific binding was determined in the presence of unlabeled 60bca (whole IgG) at a 150-fold excess over the highest concentration of labeled antibody used. The data were analyzed by hyperbolic curve fitting or Scatchard analysis (below). The number of molecules/cell was derived from specific radioactivities determined by measuring labeled Fab concentrations spectrophotometrically and assuming a molecular weight of 50,000.

LPS Binding Assay

Equilibrium binding of [H]LPS to the cells was performed by the method of Kirkland et al. (26) with minor modifications. THP-1 cells were washed in ice-cold HNE buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA), preincubated for 30 min at 37 °C in SEBDAF buffer (20 mM HEPES (pH = 7.4), 150 mM NaCl, 1 mM EDTA, 300 µg/ml BSA, 10 mM NaN, 2 mM NaF, 5 mM deoxyglucose) to prevent ligand internalization. The cells were then centrifuged, resuspended in either SEBDAF buffer or RPMI-B (RPMI 1640 containing 300 µg/ml BSA, 10 mM NaN, 2 mM NaF, and 10 mM deoxyglucose), and counted using a hemocytometer. Cells used for determination of nonspecific binding were preincubated for at least 15 min on ice with mAb 60bca (usually 0.7 µl of ascites fluid in 50-100 µl of binding buffer) before adding LPS. The LPS was sonicated (8) either in SEBDAF buffer or RPMI-B, and purified LBP was added to the LPS at room temperature 10 min before mixing with the cells. In some experiments the LPS and LBP were added to the cells at the same time, with similar results. The LPS (in 10-20 µl) was added to the cells (4 10VD3-induced cells in 50 µl, final volume) and allowed to bind with frequent mixing for 30 min at 37 or 10 °C. 450 µl of ice-cold binding buffer (without inhibitors) were then added, and the mixtures were centrifuged at 6,000 rpm (2,800 g) for 15 s. The H contained in 200 µl of supernatant was counted to determine the amount of unbound LPS, the remaining supernatant was aspirated, and the cells were washed with 500 µl of binding buffer (without inhibitors). The cells were resuspended in 100 µl of buffer, and the cell-associated H was counted after adding 0.2 ml of 1% SDS, 10 mM EDTA and 3 ml of scintillation fluid. The time required to wash the cells resulted in minimal dissociation since the t for ligand dissociation was greater than 1 h (not shown). The previously described (26) wash step (30 min in 50 µl of binding buffer) was omitted in most experiments since this step had little or no effect on the results of the assay. Fewer than 10% of the cells were permeable to trypan blue following incubation in SEBDAF or RPMI-B.

Data Analysis

Kand Bvalues were determined by curve fitting to the hyperbolic function ( B [free LPS]/( K+ [free LPS]) using GraphPad InPlot Software (Graph Pad Software Inc., San Diego, CA) or by Scatchard analysis. The K, or equilibrium dissociation constant, is defined as the concentration of free ligand required to saturate half of the available CD14 binding sites. Brefers to the maximum number of ligand binding sites under saturating conditions.

Stripping Cell Surface-bound Ligands

Labeled LPS or dLPS were bound to the cells and washed as described above. The surface-bound ligands were then stripped by two methods. (i) The cells were incubated on ice for 45 min in 500 µl of 0.02% proteinase K in phosphate-buffered saline (27) , 500 µl of buffer containing 20% fetal calf serum were added, the cells were recovered by centrifugation, and the radioactivity in an aliquot of supernatant was counted. (ii) Stripping with PI-PLC was performed as previously described (26) using 0.5 unit of PI-PLC with the washed cells in a 50-µl final volume for 30 min at 10 °C. The proportions of tritiated ligands in the cells and culture supernatants were determined by scintillation counting. In some experiments, acid stripping of the ligands was attempted by resuspending the washed cells in ice-cold phosphate-buffered saline adjusted to pH 3 with acetic acid for 5 min, followed by centrifugation to recover the cells and supernatants.

Cross-linking LPS to CD14

VD3-induced THP-1 cells (5 10cells in 10 ml of RPMI 1640 + 1% fetal calf serum) were incubated in the dark for 10 min at 37 °C with 20 ng/ml I-ASD-LPS in the presence or absence of the same concentrations of unlabeled dLPS added 1 min before the LPS. The cell suspension was transferred to a 10-cm dish on ice, and the cross-linker was activated for 2 min with a hand-held 254-nm UV light (UVP, San Gabriel, CA). The cells were washed in cold phosphate-buffered saline and lysed for 30 min on ice in 200 µl of lysis buffer (20 mM Tris(Cl), pH 7.5, 250 mM NaCl, 2 mM EDTA, 20 mM octylglucoside, 1% (w/v) Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 5 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 0.1 mM TPCK, and 0.1 mM ZPCK). The lysate was centrifuged for 15 min at 12,000 g, and 200 µl (4 mg protein) of lysate supernatant were passed over a 50-µl column of protein A-Sepharose containing prebound CD14 mAb 26ic. The column was washed with 1 ml of lysis buffer followed by 200 µl of lysis buffer diluted 1:10 in 100 mM Tris, pH 6.8, 2 mM EDTA, and 10% glycerol. The CD14 was recovered by adding 50 µl of 2 SDS sample buffer to the protein A-Sepharose, heating in boiling water for 5 min, and removing the beads by centrifugation. The samples were run on a 16-cm, 10% polyacrylamide SDS-PAGE gel, and the CD14 bands were detected by autoradiography.

Cell Stimulation and Interleukin-8 (IL-8) Assays

VD3-induced THP-1 cells were washed in cold HNE buffer and resuspended in RPMI 1640 + 300 µg/ml BSA at 0.8 10cells/100 µl, final volume, in microcentrifuge tubes. For comparison with cross-linking assays, the cells were resuspended in RPMI 1640 + 1% fetal calf serum at 5 10cells/ml in six-well culture plates. LPS or dLPS were prepared in RPMI + 300 µg/ml BSA by sonication (see above), and LBP or 1% fetal calf serum was added at room temperature 10 min before adding to the cells. The cells were incubated for the specific times at 37 °C, pelleted by brief centrifugation, and IL-8 was measured in the culture supernatants by enzyme-linked immunosorbent assay (R & D Systems, Minneapolis, MN).


RESULTS

THP-1 Cells Express CD14

THP-1 cells cultured in the presence of VD3 express elevated levels of cell-surface CD14 (8, 28) as determined by fluorescence-activated cell sorting analysis. Quantitative analysis of CD14 expression was performed by binding I-labeled Fab fragments of an CD14 mAb to cells that had been exposed to VD3 for 48 h. This analysis revealed an average of 5.2 (± 2.3 S.D., n = 5) 10binding sites/cell ( K= 10 M), in good agreement with previous estimates (3 10and 6 10sites/cell) (25, 26) made under similar conditions using a different CD14 mAb. Uninduced cells expressed low levels of CD14 that were undetectable by fluorescence-activated cell sorting (1.3 (± 0.6 S.D.) 10binding sites/cell, n = 7)), also in agreement with a previous report (25) .

Pretreatment of Cells in SEBDAF Buffer Inhibits Internalization of LPS and dLPS at 37 °C

To study binding of LPS or dLPS to the cell surface in the absence of ligand internalization, THP-1 cells were preincubated with azide, deoxyglucose, and fluoride (SEBDAF buffer) to deplete cellular ATP. Preincubation for 30 min at 37 °C in SEBDAF buffer did not result in significant cell loss or permeability to trypan blue. Subsequent binding incubations were done in SEBDAF buffer or in RPMI 1640 that contained the same inhibitors (RPMI-B). Ligand binding to SEBDAF-pretreated cells was performed at 10 or 37 °C. Two methods were used to strip the surface-bound ligands from the cells. (i) PI-PLC cleaves CD14 from its lipid anchor (26) and (ii) proteinase K digests unprotected proteins on the extracellular surface of the plasma membrane (27) . As shown in , the percentages of cell-bound ligands released by either method were only modestly decreased following binding at 37 °C compared to binding at 10 °C. This indicates that internalization was minimal at 37 °C. Treatment of the cells with PI-PLC removed most of the bound LPS and dLPS, providing evidence that the ligands remained bound to CD14. When the metabolic inhibitors were not added, most of the bound LPS or dLPS became internalized ( i.e. could not be stripped) (). In separate experiments (not shown), proteinase K treatment did not significantly reduce the cell number or increase cellular permeability to trypan blue. However, this treatment destroyed many of the cells that were already trypan blue-permeable (10% or less) before proteinase K treatment. In experiments not shown, LPS and dLPS were not removed by a conventional stripping protocol for protein ligands (acetate-buffered saline at pH 3) even though this treatment dramatically increased cell permeability to trypan blue. The rate of spontaneous dissociation during the stripping procedures was consistent with the slow, monophasic dissociation rates for LPS and dLPS ( tgreater than 1 h) (data not shown), suggesting that these ligands dissociate from CD14. Taken together, the data in indicate that, under the conditions of the binding assay, LPS and dLPS bind to CD14 with minimal internalization and that most of each ligand remains associated with CD14 after 30 min at 37 or 10 °C.

Specific Binding of LPS and dLPS to THP-1 Cells Is CD14-dependent

An incubation temperature of 37 °C was used for most experiments since internalization was not significantly increased at this temperature compared to 10 °C. As shown in Fig. 1 , most of the cellular binding of [H]LPS or [H]dLPS was inhibited by a large excess of unlabeled LPS or dLPS. The absence of LBP or the presence of anti-CD14 mAb 60bca also inhibited specific binding, indicating that LBP and CD14 mediate essentially all measurable specific binding in these cells. As shown previously (8) , a small but measurable amount of LPS binding to CD14 occurred in the absence of LBP. Similar results were obtained when ligands were bound to the cells in the presence of SEBDAF buffer (not shown). Analysis of [H]LPS and [H]dLPS Binding to CD14-The specific binding of LPS and dLPS to CD14 was saturable when studied in SEBDAF buffer (Fig. 2, A and B). In the presence of excess LBP, the apparent equilibrium dissociation constant ( K) for LPS was 33 ng/ml (± 1, n = 2) or 8.3 nM. This value is similar to those reported by Kirkland et al., who used human serum as a source of LBP at 10° or 37 °C ( K= 27-49 nM). Analysis of dLPS binding (Fig. 2 B) showed a lower binding affinity, as reflected in a higher K(101 ng/ml (± 14, n = 2) or 28 nM). The results obtained by Scatchard analysis were similar (not shown). dLPS binding in RPMI-B (not shown) yielded results that were similar to those obtained in SEBDAF buffer. In RPMI-B, LPS binding was more erratic, presumably due to increased LPS aggregate structure in the presence of divalent cations, and could not be analyzed with confidence. The analysis of LPS and dLPS binding affinities in uninduced THP-1 cells (not shown) yielded similar results to those obtained in VD3-induced cells.


Figure 1: Binding of [H]LPS and [H]dLPS to THP-1 cells. VD3-induced THP-1 cells were preincubated in SEBDAF buffer, and [H]LPS or [H]dLPS (25 ng/ml) was bound to the cells (4 10cells in 50 µl RPMI-B) with a molar excess of purified LBP (375 ng/ml) at 37 °C as described under ``Materials and Methods.'' Specific binding was inhibited by mixing a 100-fold excess of unlabeled competitor (100 LPS or 100 dLPS) with the labeled ligand, preincubating for 10 min at room temperature with LBP, and adding this mixture to the cells. Binding was also measured in the absence of LBP ( No LBP), and binding to CD14 was inhibited by preincubating the cells with mAb 60bca for 15 min before adding the labeled ligands ( CD14 mAb). Standard deviations (not shown) were within 10% of the means of triplicate determinations.




Figure 2: Analysis of LPS and dLPS binding. VD3-induced THP-1 cells were pretreated in SEBDAF buffer, and the binding of increasing concentrations of [H]LPS ( A) or [H]dLPS ( B) was measured in the presence of excess LBP. Assays were performed in triplicate with 4 10cells/50 µl of SEBDAF buffer at 37 °C. Specific binding is shown by filled circles and error bars which denote the mean ± S.D. of triplicate determinations. Nonspecific binding ( dashed lines) was determined in the presence of CD14 mAb 60bca.



Bvalues (maximal binding at saturation) for LPS and dLPS (Fig. 2, A and B) were somewhat variable but always within the same order of magnitude as Bvalues obtained from the binding of I-Fab fragments of CD14 mAb 60bca (above). This suggests that the binding stoichiometry of LPS (or dLPS) to CD14 is near 1:1, in contrast to previously reported values of 8:1 and 20:1 (26) .

When LBP Is Limiting, Low Concentrations of dLPS Can Inhibit LPS Binding to CD14

When the LBP concentration is suboptimal, concentrations of dLPS that are far below its Kcan inhibit binding of LPS to CD14. As shown in , the binding of 2 nM [H]LPS (8 ng/ml) was submaximal in the presence of 0.1 nM or lower concentrations of LBP. Under these conditions, adding an equal concentration of dLPS inhibited LPS binding to CD14 by approximately 50%, whereas this amount of dLPS had very little inhibitory effect at LBP concentrations (1 nM) that produced maximal LPS binding. The inhibition of LPS binding by dLPS presumably occurs by competition for binding to LBP.

In the Presence of Excess LBP, High Concentrations of dLPS Can Inhibit LPS Binding to CD14

As shown in Fig. 3, 50% inhibition of the binding of [H]LPS (3 ng/ml) occurred in the presence of a concentration of dLPS (approximately 100 ng/ml) that was equal to the binding Kfor dLPS. This result was expected since the Kof a ligand is defined as the concentration required to occupy half of the available receptor binding sites at equilibrium. Low concentrations of dLPS (below 10 ng/ml) did not significantly inhibit LPS binding, since these concentrations are far below the Kfor dLPS. Our data do not rule out the possibility that dLPS might inhibit the ability of LPS to bind to CD14 by sequestering the LPS in aggregate structures. This explanation seems unlikely, however, since sucrose gradient analyses indicated that both LPS and dLPS were highly disaggregated in the presence of LBP and SEBDAF buffer (not shown), and the inhibitory ability of dLPS was similar whether the binding was performed in the presence (RPMI-B) or absence (SEBDAF) of divalent cations (Fig. 3).


Figure 3: Inhibition of [H]LPS binding to CD14 by high concentrations of dLPS in the presence of excess LBP. VD3-induced THP-1 cells were pretreated in SEBDAF buffer, and [H]LPS (3 ng/ml) was bound to the cells (4 10cells in 50 µl) in the presence of increasing concentrations of unlabeled dLPS at 37 °C. Binding was performed in SEBDAF buffer ( closed circles) or RPMI-B ( open triangles). Error bars denote standard deviations from the means of triplicate determinations.



Low Concentrations of dLPS, in the Presence of Excess LBP, Inhibit LPS-induced IL-8 Production without Inhibiting LPS Binding to CD14

To compare binding with function, we studied the effects of LPS and dLPS on the production of IL-8 under similar conditions used for the binding assay without the metabolic inhibitors. As shown in Fig. 4 , the IL-8 response was maximal (EC) at 1-3 ng/ml LPS and half-maximal (EC) at approximately 0.2 ng/ml. (The assay measures the release of IL-8 into the culture supernatant after a 2-h incubation. In experiments not shown, the cells continued to release IL-8 at a slower rate after 2 h but also with the maximal response at a dose of approximately 1 ng/ml.) The EC(0.2 ng/ml or 5 10 M) and EC(1-3 ng/ml) are far below the Kfor LPS binding (33 ng/ml or 8 10 M), indicating a large spare receptor effect (29) with respect to CD14 binding.


Figure 4: LPS dose response for IL-8 induction. VD3-induced THP-1 cells (8 10in 100 µl, final volume, of RPMI 1640 + 300 µg/ml BSA) were incubated at 37 °C with increasing concentrations of LPS in the presence of excess LBP for 2 h at 37 °C. IL-8 in the culture medium was measured as described under ``Materials and Methods.'' Closed circles and bars denote means and ranges of duplicate determinations.



dLPS did not stimulate IL-8 release and strongly inhibited this response to LPS. As shown in Fig. 5, a brief preincubation with dLPS almost completely suppressed the response to an equal amount of LPS. Substoichiometric concentrations of dLPS (with respect to LPS) were also very inhibitory even when added simultaneously with LPS (not shown), in keeping with the previously observed effects of dLPS on LPS-induced NF-B, IL-1 (8) , and protein tyrosine phosphorylation (data not shown). The effects of varying concentrations of dLPS on the LPS dose response (Fig. 5) are difficult to analyze since they show characteristics of noncompetitive inhibition ( i.e. maximal control levels of activity are not attained with increasing LPS concentrations in the presence of dLPS) and competitive inhibition (a lower but approximately equal plateau of activity occurs with increasing LPS concentrations in the presence of each concentration of dLPS). Most importantly, dLPS inhibits maximal stimulatory concentrations of LPS at dLPS concentrations that are far below its binding Kwithout significantly inhibiting LPS binding to CD14.


Figure 5: Inhibition of LPS-induced IL-8 by dLPS. Incubations were performed as described in Fig. 4 except that dLPS (with excess LBP) was added 10 min before the addition of LPS. No dLPS, closed circles; 0.1 ng/ml dLPS, open circles; 0.4 ng/ml dLPS, open triangles; and 1 ng/ml dLPS, open squares. Data points and error bars denote means and ranges of duplicate determinations.



dLPS Inhibits LPS Function without Inhibiting Cross-linking of LPS to CD14

To obtain biochemical evidence that the direct interaction between LPS and CD14 was not inhibited by low concentrations of dLPS that inhibit LPS responses, we observed cross-linking to CD14 (25) using LPS that was derivatized with the photoactivatable cross-linker, SASD. We photoactivated I-ASD-LPS to CD14 after it had incubated with the cells for 10 min. Immunoprecipitation of CD14 from detergent lysates showed that the LPS cross-linked to CD14 (Fig. 6, inset, lane 1). Cross-linking was not significantly inhibited in the presence of an equal amount of unlabeled dLPS ( lane 2), but it was abolished when the cells were preincubated with mAb 60bca ( lane 3). A functional comparison to this experiment was made by measuring IL-8 release for 90 min under the same conditions. As also shown in Fig. 6, the addition of dLPS strongly inhibited the IL-8 response under conditions that did not prevent cross-linking LPS to CD14 on the cells. We conclude that low concentrations of dLPS that do not block binding of LPS to CD14 can nevertheless antagonize LPS, presumably at a site that is distal to CD14 binding.


Figure 6: dLPS inhibits CD14-mediated IL-8 induction by LPS without inhibiting cross-linking of I-ASD-LPS to CD14. I-ASD-LPS was cross-linked to VD3-induced THP-1 cells after binding for 10 min in the presence or absence of the same amount of unlabeled dLPS. IL-8 release into the culture supernatant was measured under identical conditions except that the incubations were continued for 90 min at 37 °C. Data points and error bars denote means and ranges of duplicate determinations. Inset, autoradiographs of CD14 immunoprecipitates, analyzed using SDS-PAGE. Lane 1, I-ASD-LPS alone; lane 2, 20 ng/ml unlabeled dLPS + I-ASD-LPS; lane 3, pretreatment with CD14 mAb 60bca before binding with I-ASD-LPS. The arrow indicates the migration position of CD14.




DISCUSSION

A quantitative dissection of potential inhibitory mechanisms has not been reported previously for any of the lipid A analogs that are known to act as LPS antagonists. Two measurements were critical for such an analysis. We first compared the binding affinity of the antagonistic LPS analog (dLPS) to that of LPS. Competition between LPS and dLPS for binding CD14 would be expected to occur at or near their respective binding Kvalues. The second measurement was the ECfor LPS stimulation of IL-8, which we found to be almost 100-fold lower than the LPS binding K, indicating that most of the LPS molecules that can bind to these cells do not necessarily contribute to signaling. This phenomenon, called the ``spare receptor effect,'' has been observed in many known receptor systems (29) . Large numbers of spare receptors serve to increase the dose sensitivity of cells to agonists by binding more molecules at low agonist concentrations. Antagonism that occurs at low concentrations of agonist and antagonist ( i.e. well below their respective binding Kvalues) should involve mechanisms other than competition for receptor binding.

We found that dLPS (and presumably other lipid A analogs) can block LPS signaling in at least three ways as follows.

Competing with LPS for Binding LBP

When the concentration of LBP is suboptimal for facilitating LPS-CD14 binding, low concentrations of antagonist can inhibit LPS binding to cell-surface CD14 (). Competition for binding LBP is the likely explanation, as Tobias et al. (30) reported that LPS binds to LBP with a stoichiometry of 1:1, and that the tetraacyl lipid A structures, lipid IVA and dLPS, inhibit binding of LBP to immobilized LPS. Competition for limiting amounts of LBP could be an important inhibitory mechanism in extravascular sites where serum components are scarce. Competition for LBP may also be an important factor when in vitro LPS stimulation assays are done with a limiting concentration of serum in the incubation medium (11, 12, 31) . Even in the absence of added serum, the sensitivity of cells to LPS, although mediated largely by CD14, may depend in part upon residual LBP that remains bound to the cells after washing (32) .

Competing with LPS for Binding CD14

When LBP concentrations are optimal for promoting LPS-CD14 binding, only high concentrations of antagonist can compete with LPS for binding CD14 (Fig. 3). Approximately 50% inhibition of LPS (3 ng/ml) binding was observed in the presence of 100 ng/ml dLPS; this is at the dLPS binding K, or the concentration at which half of the available binding sites are engaged by dLPS, so inhibition of LPS binding would be expected at this dLPS concentration. (The ability of an antagonist to inhibit binding is determined by its binding Krather than by the ratio of antagonist to agonist (29) .) This mechanism probably accounts for the ability of high concentrations of lipid A analogs to inhibit LPS binding to CD14-expressing cells. For example, 300 ng/ml tetraacyl lipid A analog (PE-4) was required to block the binding of labeled LPS (30 ng/ml) to human monocytes in the presence of serum (18) . Competition for CD14 binding may be the inhibitory mechanism in most cases in which high analog concentrations are required to block LPS stimulation.

Competing with LPS at a Site That Is Distal to CD14 in the Signaling Pathway

As noted above, large numbers of spare CD14 receptors facilitate maximal cellular responses at ligand concentrations that are far below the binding Kof either the antagonist or agonist. Competition for binding to CD14 does not occur at these low concentrations (Fig. 3) because the ability of the antagonist to inhibit agonist binding competitively is determined by their respective binding Kvalues. It has been common practice to determine binding Kvalues for receptor antagonists by measuring the ability of the antagonist to inhibit functional responses induced by the appropriate agonist (29) . Our data show that this method is not appropriate for dLPS, since functional responses to LPS can be inhibited by dLPS concentrations that are 100-fold below the Kfor dLPS binding. Moreover, these low concentrations of dLPS can inhibit LPS signaling without blocking LPS binding to CD14 (Fig. 6). We believe that dLPS is not a CD14 receptor antagonist under these circumstances. Rather, it may inhibit the interaction of LPS with another molecule that is distal to CD14 in the signal pathway. Particularly intriguing is the observation that, despite the lower binding affinity of dLPS for CD14, substoichiometric concentrations of dLPS can block LPS stimulation.

There are several models that could explain the third mechanism of dLPS inhibition. The idea that dLPS might block the binding of LPS to a high affinity subclass of CD14 cannot be formally excluded. Under certain conditions, we have observed high affinity binding of LPS to CD14, with an apparent dissociation constant ( K) of approximately 2 ng/ml or 500 pM (data not shown). However, this finding probably does not reflect a saturable subclass of CD14 or point to the site of dLPS inhibition, since dLPS binds with lower affinity than LPS and does not significantly inhibit high affinity LPS binding. Another possibility is that antagonist and agonist molecules bind to multiple binding sites on the same CD14 molecule (possibly involving the multiple L XXL XL X motifs on CD14) (33) . In this model, the binding of dLPS might produce an allosteric effect that could inhibit receptor function without inhibiting the binding of LPS to another site. Data regarding the existence of multiple LPS binding sites on CD14 are conflicting (26, 34) , and our data suggest that the binding stoichiometry between LPS (or dLPS) and CD14 is approximately 1:1. Moreover, the low number of LPS and dLPS molecules that interact functionally are not statistically likely to bind to the same molecule of CD14 since such concentrations are so far below the Kvalues for both ligands.

A more likely model, shown in Fig. 7, postulates the existence of a low abundance receptor/effector molecule that binds to LPS or dLPS either in the context of CD14 (LPSCD14 complex) or after transfer of the ligands from CD14 to this molecule. Thus, dLPS antagonizes LPS in this model by interfering with the ability of LPS to interact with the receptor/effector. This model would also be consistent with the spare-receptor effect (Fig. 4), as well as with scenarios proposed to explain how LPS and soluble CD14 (sCD14) stimulate responses in CD14-negative cells (35, 36) . The ability of the proposed molecule to bind the ligands directly (without CD14) could account for the ability of high concentrations of LPS to stimulate cells in the presence of antibodies that block CD14 binding (8, 13, 14, 32) , as well as cells that express little or no CD14 (27, 37) .


Figure 7: dLPS can antagonize LPS at three sites in the LPS recognition pathway: ( 1) by competing for binding LBP, ( 2) by competing for binding CD14, and ( 3) by interacting at a site distal to CD14.



Alternatively, dLPS might antagonize LPS responses by inducing a negative signal that somehow rapidly inhibits LPS recognition. Our attempts to detect the signal-inducing ability of dLPS in THP-1 cells have not been revealing. Some investigators have reported, however, that tetraacyl or other lipid A partial structures induce priming effects that increase the sensitivity of cells to LPS (12, 38, 39) . Others have reported that low concentrations of a synthetic tetraacyl lipid A stimulate the migration of human neutrophils (40, 41) . These data support the notion that tetraacyl antagonists can deliver signals to cells, offering an interesting alternative interpretation of their inhibitory mechanism.

  
Table: Release of LPS or dLPS bound to THP-1 cells

[H]LPS or [H]dLPS (usually 25 ng/ml), in the presence of a 2-fold molar excess of purified LBP in RPMI-B, was allowed to bind at the indicated temperatures to VD3-induced THP-1 cells that had been pretreated in SEBDAF buffer. The cells were washed, and the bound ligands were stripped as described under ``Materials and Methods.'' The amount of ligand released is expressed as the percentage of radiolabeled ligand initially bound to the cells. The data are shown as mean ± S.D. of triplicates (number of experiments).


  
Table: Low concentrations of dLPS inhibit LPS binding to CD14 when LBP is limiting

Low concentrations of dLPS inhibit LPS binding to CD14 when LBP is limiting. VD3-induced THP-1 cells (4 10in 50 µl of RPMI-B) were pretreated in SEBDAF buffer and 2 nM [H]LPS (8 ng/ml) were then added, alone or in the presence of 2 nM unlabeled dLPS. The fraction inhibited by dLPS is expressed as the percent inhibition of LPS bound in the absence of dLPS. Values are mean ± S.D. of triplicate determinations.



FOOTNOTES

*
This work was funded by National Institutes of Health, NIAID Grant AI18188. 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 Internal Medicine, UT-Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9113. Tel.: 214-648-3480; Fax: 214-648-9478.

The abbreviations used are: LPS, lipopolysaccharide; dLPS, enzymatically deacylated LPS that lacks secondary fatty acyl chains; LBP, LPS-binding protein; VD3, 1,25-dihydroxyvitamin D; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone; ZPCK, N-CBZ-L-phenylalanine chloromethyl ketone; BSA, bovine serum albumin; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; PI-PLC, phosphatidylinositol-specific phospholipase C; SASD, sulfosuccinimidyl 2-( p-azidosalicylamido)ethyl-1,3`-dithiopropionate.


ACKNOWLEDGEMENTS

We thank Leon Eidels for critical review of the manuscript.


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