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Originally published In Press as doi:10.1074/jbc.M206404200 on October 7, 2002

J. Biol. Chem., Vol. 277, Issue 49, 47818-47825, December 6, 2002
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An Essential Role for Albumin in the Interaction of Endotoxin with Lipopolysaccharide-binding Protein and sCD14 and Resultant Cell Activation*

Theresa L. GioanniniDagger §, DeSheng Zhang§, Athmane Teghanemt§, and Jerrold P. Weiss§||

From the Departments of Dagger  Biochemistry, || Microbiology, and § Internal Medicine, Inflammation Program, Division of Infectious Diseases, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, and Veterans Affairs Medical Center, Iowa City, Iowa 52242

Received for publication, June 27, 2002, and in revised form, October 2, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Experiments utilizing endotoxin aggregates, lipooligosaccharides (LOS) isolated from metabolically labeled Neisseria meningitidis serotype group B, demonstrate that albumin is an essential component of lipopolysaccharide binding protein- (LBP) and sCD14-dependent 1) disaggregation of LOS and 2) LOS activation of human umbilical vein endothelial cells (HUVEC). Aggregates of LOS (LOSagg) with an apparent Mr >=  2 × 107 were isolated by gel sieving on Sephacryl HR S500 in buffered balanced salts solution plus albumin. Incubation of LOSagg with LBP and sCD14 promoted LOSagg disaggregation in an albumin-dependent fashion to complexes that contain LOS and sCD14, but no LBP, with an apparent Mr ~ 60,000 (LOS:sCD14) as determined by Sephacryl S200 chromatography. Isolation by gel filtration of LOSagg:protein aggregates formed by the interaction of LOSagg with either LBP or sCD14 alone revealed that the sequence of LOS-protein interactions as well as the step(s) at which albumin is necessary for the production of bioactive LOS:sCD14 were specific. Efficient generation of LOS:sCD14 required 1) interaction of LOSagg with LBP before interaction with CD14 and 2) the presence of albumin during the interaction of LBP with LOSagg. Activation of HUVEC by LOSagg, as measured by IL-8 production, required both LBP and sCD14 and was thirty times more potent in the presence of albumin. In contrast, LOS:sCD14 did not require additional LBP, sCD14, or albumin to activate HUVEC but depended on the presence of albumin for optimal solubility/stability once formed. The albumin effect is apparently specific, because neither ovalbumin nor gelatin substituted for albumin in facilitating LBP:sCD14-dependent disaggregation of LOSagg or activation of endothelial cells. These results indicate that albumin is an essential facilitator of LBP/sCD14-induced LOS disaggregation that is required for activation of endothelial cells by LOSagg.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Host cell responses to Gram-negative bacteria are orchestrated by immediate recognition of and response to minute quantities of bacteria and/or bacterial products that mobilize defense mechanisms. Although normally protective, similar responses to higher concentrations of these products may provide the basis for much of the pathologic sequelae in certain invasive infections. For example, the pathogenicity and severity of infection provoked by Neisseria meningitidis (NMB)1 has been correlated with high circulating levels of meningococcal endotoxin, lipooligosaccharide (LOS) (1). LOS is a unique amphiphathic glycolipid found in the outer leaflet of the outer membrane of NMB and related Gram-negative bacteria (2, 3). LOS, like lipopolysaccharide (LPS), contains the bioactive lipid A moiety composed of the disaccharide backbone of beta (1'right-arrow6)-linked D-glucosamine modified by O-phosphorylethanolamine and substituted with symmetrically arranged amide- and ester-linked 3-hydroxy-substituted fatty acids (4-6).

Host cell response to LOS, like other endotoxins, is mediated through specific interactions with several host proteins including the lipopolysaccharide binding protein (LBP) and CD14 in either a membrane-bound (mCD14) or soluble form (sCD14) to activate cells, such as macrophages, neutrophils, and endothelial cells, with the resultant release of a broad array of pro-inflammatory mediators (7-13). The cell protein(s) thought to be primarily responsible for triggering cell responses to endotoxin includes members of the Toll-like receptor protein family, most notably, Toll-like receptor 4 complexed to accessory proteins such as MD-2 (14-20). In addition to these protein interactions, it has been suggested by a number of earlier studies that serum albumin may be a factor in the host defense system by facilitating interactions of endotoxin with protein components responsible for cell activation and clearance (21-27). In particular, interactions between the lipid A portion of endotoxins and albumin have been noted to be important in delivery of endotoxin to cell acceptors (21, 24, 26).

We have recently reported the generation of an acetate auxotroph of NMB that permits radiolabeling of LOS to high specific activity (28). The physical state of the highly radioactive LOS has been examined by gel filtration chromatography under conditions that produce material that can be used directly in bioassays at concentrations that are pathophysiologically relevant (28). These studies, as well as recent studies with lipopolysaccharide isolated from Escherichia coli K12 (29), have shown that 1) activation of endothelial cells, as measured by production of IL-8, or 2) activation of blood leukocytes, as measured by lucigenin-enhanced chemiluminescence, can be correlated with changes in the physical and/or biochemical mode of presentation of LOS or LPS (28).

This gel filtration system has been utilized to isolate apparently homogeneous populations of LOS aggregates (LOSagg) and protein:LOS aggregates/complexes (28, 29). Subsequently, the bioactive form of LOS delivered to and responsible for response in mCD14-deficient endothelial cells consists of a complex of LOS with sCD14 that is significantly smaller in molecular size than is LOSagg. Generation of this bioactive LOS containing complex is greatly facilitated by interaction with LBP and involves changes in physical state from an aggregate with an apparent Mr 2 × 107 to an active complex with an apparent Mr <=  100,000. Thus, cellular activation requires the transfer of the amphipathic glycolipid between complex hydrophobic environments, from endotoxin-rich aggregates or monolayers in the bacterial membrane to CD14 and subsequently to Toll-like receptor 4/MD-2 in a host cell membrane. When this transfer of endotoxin occurs across and within aqueous spaces, as it does with sCD14, a role for a lipid carrier might be anticipated. Previous studies have suggested that LBP can play a direct lipid transfer as well as LPS-binding role (30-32). We now show that efficient LBP/sCD14-dependent disaggregation of LOS and activation of endothelial cells additionally require the presence of albumin. In summary, these findings indicate that albumin is essential for efficient fluid-phase interaction of endotoxin-binding proteins with LOS and subsequent generation of the bioactive species responsible for activation of cells deficient in mCD14. Such a role for albumin is consistent with its prominence in body fluids as a lipid carrier and its ability to bind endotoxin weakly.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Dr. Stephen Carroll, Xoma Corporation (Berkeley, CA) generously provided LBP, sCD14, and rabbit polyclonal anti-LBP antibodies. Sephacryl HR S500 was obtained from Amersham Biosciences. Human serum albumin (HSA) was endotoxin-free, 25% stock solution prepared by Baxter Healthcare Corp., Glendale, CA. Bovine serum albumin was purchased from Calbiochem (San Diego, CA). Electrophoresis-grade gelatin, fatty acid-free human serum albumin, and ovalbumin were purchased from Sigma. Polyclonal rabbit anti-CD14 antibodies (M305) directed against CD14 were purchased from Santa Cruz Biotechnology, Santa Cruz, CA. Monoclonal murine antibodies to CD14 (MEM-18 and 18E12) were respectively purchased from Accurate Biochemicals and a gift from Johnson & Johnson Corp., New Brunswick, NJ. HUVEC, endothelial cell basal medium, fetal bovine serum, bovine brain extract, human endothelial growth factor, hydrocortisone, and gentamicin were from Clonetics (San Francisco, CA). Bovine type 1 collagen was obtained from Collaborative Research Products. 96-well Optiplates were from Anthos Labtec Instruments (New Castle, DE). [1,2-14C]Acetic acid sodium salt (110 mCi/mmol) was purchased from Moraveck Biochemicals, Inc. (Brea, CA). 3H-LPS, purchased from List Biological Laboratories, Campbell, CA, was dissolved in endotoxin-free water to a final concentration of 100 µg/ml and sonicated twice on ice for 10 min. Aliquots were stored frozen at -70 °C until needed.

Preparation of 14C-LOS from Neisseria meningitidis Serogroup B-- [14C]LOS from the mutant strain of encapsulated N. meningitidis serogroup B (NMB ACE-1) was metabolically labeled and isolated as previously described (28). Briefly, the acetate auxotroph strain NMB ACE-1 was generated by allelic exchange of the putative meningocococcal gene encoding the PDH E1 component (pdhA) with a plasmid containing a copy of pdhA disrupted by insertion of a Kmr cassette (28). The bacteria were cultured at 37 °C in 5% CO2/95% atmosphere on Difco GC medium-based agar supplemented with 1× isovitalex. Radiolabeling was carried out in Morse's defined broth medium (33) supplemented with 1× isovitalex, 10 mM sodium bicarbonate, and 1.5 mM sodium acetate. To obtain [14C]LOS of high specific radioactivity, 160 µCi/ml of [1,2-14C]acetate was added to the medium during growth of meningococci to late log phase.

[14C]LOS was purified from radiolabeled bacteria by a modification of the hot phenol-water method (28). After ethanol precipitation, the [14C]LOS-containing pellet was dried and resuspended in cold distilled water to an estimated concentration of 100 µg of LOS/ml and then sonicated at room temperature for 15 min in a water bath sonicator. The composition of [14C]LOS was routinely analyzed for 14C-labeled fatty acid content by thin layer chromatography, and quantitation was performed by image analysis as described previously (28). The specific radioactivity of [14C]LOS was calculated from image and GC-MS analyses as described by Giardina et al. (28).

Chromatography-- Columns of Sephacryl HR S500 (1.5 cm × 18 cm) were pre-equilibrated in Hepes-buffered (10 mM, pH 7.4) Hanks' balanced salts solution with divalent cations (HBSS+) ± 0.1% HSA. Aliquots of resuspended and sonicated [14C]LOS were diluted with or without HSA and incubated at 37 °C for 15 min before gel filtration chromatography. Chromatography on Sephacryl HR S500 of [14C]LOS, purified as described above, provides a population of [14C]LOS aggregates that elute in the void volume peak fractions, i.e. LOSagg, apparent Mr >=  2 × 107. These fractions served as a source of [14C]LOSagg in subsequent experiments.

Chromatographed samples contained from 4 ng to 8 µg LOSagg in 0.2-0.5 ml of column buffer with or without 0.1% HSA. Fractions (1 ml) were collected at a flow rate of 0.5 ml/min at room temperature. To prepare protein:LOS aggregates, samples were incubated with the indicated proteins for 15 min at 37 °C before application to the column. Samples collected in the absence of albumin were used immediately to prevent loss of material caused by nonspecific sticking. Aliquots of the collected [14C]LOS fractions were analyzed by liquid scintillation spectroscopy using a Beckman LS liquid scintillation counter. Recoveries of the radiolabeled LOSagg + HSA ranged from 70% to 90%; however, recoveries of protein:LOS aggregates in the absence of albumin were lower (in the range of 50-80%). To preclude contamination of purified LOS preparations, all solutions were pyrogen-free and sterile-filtered. The glass columns and connecting tubing were either autoclaved or washed extensively with 70% ethanol. After chromatography, fractions selected for use in bioassays were pooled and passed through sterile syringe filters (0.22 µm pore size) with greater than 90% recovery of labeled material in the sterile filtrate. Fractions were stored under sterile conditions at 4 °C for more than 3 months with no detectable changes in chromatographic or functional properties.

The column was calibrated using [14C]oleate-labeled E. coli PL-2 and [14C]acetate as markers of the void (10-11 ml) and inclusion volumes (25-26 ml), respectively, in column buffer with or without 0.1% albumin. Additional standards included blue dextran (Mr approx  2,000,000), thyroglobulin (650 and 1300 kDa, monomers and dimers, respectively), ferritin (440 kDa), catalase (232 kDa), and aldolase (158 kDa). Elution profiles of bacteria, blue dextran, and acetate were unaffected by the presence of albumin in the column buffer.

Sephacryl S200 chromatography was performed on an AKTA FPLC system using a 1.6 cm × 30 cm column with a flow rate 0.5 ml/min in Hepes-buffered HBSS+; 1-ml fractions were collected and evaluated for the presence of [14C]LOS by liquid scintillation spectroscopy. The column was equilibrated with Bio-Rad gel filtration standards that include thyroglobulin (Vo), gamma -globulin, ovalbumin, myoglobin, and vitamin B12 (Vi).

Immunoblotting and Immunocapture-- To demonstrate the presence of LBP or CD14 in isolated LOSagg:protein aggregates, samples of LOSagg plus LBP or CD14 were incubated as described with or without HSA, and the aggregates were isolated in the void volume of Sephacryl HR S500 columns equilibrated in Hepes-buffered HBSS+ (1.5 cm x 6 cm). Shorter columns were used to reduce losses of LOSagg during chromatography without albumin and allow efficient isolation of the large aggregates. Void volume fractions containing LOSagg were precipitated with trichloroacetic acid; the precipitated material was washed and then resuspended in SDS-PAGE sample buffer. Control samples containing only LBP or sCD14 with no LOS were also chromatographed and treated in a similar manner. In addition to these samples, control samples of LBP and sCD14 of varying concentrations were electrophoresed using an Amersham Biosciences PhastGel system through either 12.5% or 10-15% acrylamide gels and transferred to nitrocellulose by semi-dry transfer using the same system. To block nonspecific background on the immunoblots, the nitrocellulose was washed with phosphate-buffered saline (PBS) containing 0.05% Tween-20 and then blocked with 3% BSA in the same buffer for 1 h at 25 °C. After washing, the blots were treated with the appropriate primary antibody (1:1000 rabbit anti-LBP or 1:500 rabbit anti-CD14 serum) diluted in 1% BSA/PBS/0.05% Tween-20 overnight at 25 °C. After washing with PBS/0.05% Tween-20, the blots were incubated with secondary antibody conjugated to horseradish peroxidase (donkey anti-rabbit IgG) for 1 h at 25 °C and washed with PBS/0.05% Tween-20. The blots were then developed using the Pierce SuperSignal substrate system.

To capture protein-containing aggregates/complexes of LOS, appropriate antibodies and controls (1 µg/well) were diluted in Hepes-buffered HBSS+/1% HSA and applied to Nunc-Immuno Maxisorp plates in pH 9.6 bicarbonate buffer overnight at 25 °C. The wells were rinsed with HBSS+/Hepes/1% HSA three times and then incubated for 2 h with each well filled with blocking buffer (Hepes/HBSS+/1% HSA). The ligand to be captured by the antibody was then incubated overnight with shaking at 25 °C. The supernatant was removed and an aliquot reserved for evaluation of the unbound LOS radioactivity. The wells were washed three times with HBSS+/Hepes/1% HSA. Captured material was eluted with 2% SDS by incubation for 15 min at 37 °C. The supernatant was removed and the recovery was evaluated by liquid scintillation spectroscopy.

Human Cells-- HUVEC were routinely cultured on collagen-coated plates (Costar, Cambridge, MA) at 37 °C, 5% CO2, and 95% relative humidity in endothelial cell basal medium supplemented with 5% fetal bovine serum, 12 µg/ml bovine brain extract, 10 ng/ml human endothelial growth factor, 1 µg/ml hydrocortisone, and 50 µg/ml gentamicin. Cells were subcultured and grown to confluence (~4-5 days). Cell monolayers were then washed twice with warm HBSS+ to remove traces of serum before adding experimental media. Experiments were done with cells between passages 2-6.

HUVEC Activation Assays-- Cells in 48-well tissue culture plates were incubated for at least 6 h at 37 °C, 5% CO2, and 95% humidity in Dulbecco's minimal essential medium and 0.1% albumin or, where indicated, without albumin or with 0.1% ovalbumin or gelatin with various concentrations of [14C]LOS with or without LBP (0.2 µg/ml) and sCD14 (0.5 µg/ml). Activation of HUVEC was assessed by measuring accumulation of extracellular IL-8 by ELISA (34). Equivalent responses were observed with 0.1-0.5 µg/ml of LBP and 0.25-1.0 µg/ml sCD14.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

LOSagg Isolated in the Presence and Absence of Albumin-- We have previously described the purification of an apparently homogeneous population of LOS aggregates from NMB ACE1 with an Mr >=  2 × 107 as determined by gel filtration (Sephacryl S500) in Hepes-buffered HBSS+ supplemented with 0.1% HSA (28). To examine the possible role of albumin in LBP/sCD14-dependent disaggregation of LOSagg and activation of HUVEC, we purified LOSagg in the absence of albumin. Neither the chromatographic profile of LOS nor the overall recovery of LOS was affected by the absence of albumin (Fig. 1). The predominant population of LOS aggregates (LOSagg) formed in the absence or presence of albumin migrated with similar apparent Mr (>=  2 × 107).


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  		Fig. 1.   Isolation of [14C]LOS aggregates by Sephacryl S500 HR chromatography with and without albumin. [14C]LOS was isolated from NMB ACE-1 by hot phenol-water extraction and ethanol precipitation as previously described (28). Aliquots of the purified [14C]LOS were incubated for 15 min at 37 °C in Hepes-buffered HBSS+ ± 1% HSA before chromatography through Sephacryl S500 HR using the same buffer as described under "Experimental Procedures." The void volume peak fraction with albumin (LOSaggHSA) or without albumin (LOSaggNo HSA) was utilized as a source of a relatively homogeneous population of LOS (LOSagg). Data are expressed as percent of total [14C]LOS recovered. Total recovery of [14C]LOS was 70-90%.

Albumin Is Required for LBP/sCD14-dependent Disaggregation of LOSagg and Activation of HUVEC-- The isolation of LOSagg with or without albumin provided a source of LOSagg to examine the effect of albumin on the physical state of LOS-containing aggregates formed during the interaction of LOSagg with endotoxin-binding proteins and the role of albumin in cell activation by endotoxin. As we have previously shown (28), LOSagg formed and isolated in the presence of albumin (LOSaggHSA) was quantitatively disaggregated when incubated with LBP, sCD14, and albumin (Fig. 2). In contrast, LOSagg formed and isolated in the absence of albumin (LOSaggNo HSA) and incubated with LBP and sCD14 without albumin was not significantly disaggregated (Fig. 2A).


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  		Fig. 2.   LBP- and sCD14-dependent disaggregation of [14C]LOS and endothelial cell activation by [14C]LOS with or without albumin. A, LOSaggHSA or LOSaggNo HSA (100 ng of LOS/ml, ca. 20 nM) was recovered after Sephacryl S500 chromatography (fraction 11, Fig. 1) and incubated for 15 min at 37 °C with LBP (500 ng/ml, 10 nM) and sCD14 (5 µg/ml, 100 nM) in Hepes-buffered HBSS+ ± 1% HSA and then analyzed by gel filtration chromatography in HBSS+ ± 1% HSA as described under "Experimental Procedures." Results shown are representative of > 3 experiments. Data are expressed as percent of total [14C]LOS recovered. Total recovery of [14C]LOS was 70-90% with albumin and 50-70% without albumin. B, cell activation of HUVEC by LOSaggHSA or LOSaggNo HSA was measured as described under "Experimental Procedures." All samples contained LBP (100 ng/ml, 2 nM) and sCD14 (250 ng/ml, 5 nM). LOS aggregates were incubated for 20 h with the proteins either in the presence (black-down-triangle , black-square) or absence (down-triangle, ) of albumin. The amount of LOS added was calculated from the experimentally determined specific activity of LOS. Data shown represent the means ± S.E. of data from three or more experiments.

The presence of albumin exerted similar influences on the activation of endothelial cells by LOSagg in the presence of LBP and sCD14. LOSaggHSA isolated and incubated with LBP and sCD14 in the presence of albumin potently activated HUVEC, whereas LOSaggNo HSA with LBP and sCD14 incubated in the absence of albumin did not (Fig. 2B). If albumin was added either only during the formation/isolation of LOSagg or introduced only during the incubation of cells with LBP and sCD14, substantial but not optimal LOS-dependent cell activation was observed (data not shown). Different commercial sources of human albumin including fatty acid-free albumin, bovine serum albumin, and human serum albumin purified by gel filtration chromatography all had similar effects (data not shown).

The requirement for albumin is apparently specific: neither gelatin nor ovalbumin supported LOS-dependent cell activation (Fig. 3B) or LBP/sCD14-dependent disaggregation of LOSagg (Fig. 3A). Although the chromatographic profile of NMB ACE-1 LOS was similar in 0.1% ovalbumin (LOSaggOVAL) and 0.1% HSA (compares Fig. 1 and 3A), the recovered LOSaggOVAL was not significantly disaggregated during incubation with LBP and sCD14 in the presence of ovalbumin (Fig. 3A). Overall recoveries of [14C]LOS either from the chromatographic step or from cell cultures were not appreciably different under the various experimental conditions. These findings strongly suggest that albumin is an essential cofactor for LBP/sCD14-dependent disaggregation of LOSagg and activation of endothelial cells.


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  		Fig. 3.   Determination of the specificity of albumin in endothelial cell activation and in LBP and sCD14-dependent disaggregation of LPSagg. A, [14C]LOSagg were isolated by gel filtration chromatography (Sephacryl S500) as described under "Experimental Procedures" in the presence of 0.1% ovalbumin and then a sample (LOSaggOVAL, 100 ng/ml) was either re-chromatographed to check for homogeneity or incubated with 10 nM LBP and sCD14 (5 µg/ml, ca. 100 nM) in Hepes-buffered HBSS+/0.1% ovalbumin for 15 min at 37 °C before characterization by gel filtration chromatography in the same buffer. Results shown are representative of at least two experiments. Data are expressed as percent of total [14C]LOS recovered. Total recovery of [14C]LOS was > 70%. B, cell activation of HUVEC by LOSagg was measured as described under "Experimental Procedures." LOSagg was isolated by Sephacryl S500 chromatography in Hepes-buffered HBSS+. To samples containing LOSagg, LBP (100 ng/ml, 2 nM) and sCD14 (250 ng/ml, 5 nM) and 1% of the indicated proteins were added and incubated for 20 h. The amount of LOS added was calculated from the experimentally determined specific activity of LOS. Data shown represent the means ± S.E. of data from three or more experiments.

Fig. 4 illustrates that albumin is also needed for optimal LBP/sCD14-dependent activation of HUVEC by E. coli K12 (LCD25) lipopolysaccharide (LPS). Incubation of LPSagg plus LBP, sCD14, and albumin produces disaggregation of LPSagg (29). However, recoveries of LPSagg and LPSagg:protein complexes during Sephacryl S500 chromatography in the absence of albumin were low (< 30%), precluding a direct assessment of the role of albumin in LPB/sCD14-dependent disaggregation of LPS.


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  		Fig. 4.   Specific potentiation of the LBP, sCD14-dependent activation of endothelial cells by LPS by serum albumin. Cell activation of HUVEC by LPS was determined by measurement of the accumulation of IL-8 as described under "Experimental Procedures." To samples containing LPS, LBP (100 ng/ml, 2 nM) and sCD14 (250 ng/ml, 5 nM) and 1% of the either human serum albumin or ovalbumin was added and incubated for 20 h. The accumulation of IL-8 product represents the product generated from 0.3-1 ng/ml LPS. Control is IL-8 accumulation in the presence of albumin. Data shown represent the means ± S.E. of the data.

LBP//sCD14-dependent Disaggregation of LOSagg Requires Ordered Interaction of LBP and sCD14 with LOSagg-- To better define where and when albumin is needed to facilitate LBP/sCD14-dependent disaggregation of LOSagg, we first examined whether the combined action of LBP and sCD14 in the presence of albumin on LOSaggHSA required the simultaneous presence of these proteins or ordered sequential interactions with LOSaggHSA. For this purpose, gel chromatography was employed to recover LOSagg exposed to either LBP or sCD14 alone. The aggregates formed with LOSaggHSA were separated from any free protein that was not physically associated with LOSaggHSA. In a manner similar to the recently described interactions of LPSagg with LBP (29), incubation of LOSaggHSA with LBP alone had no direct disaggregating effects at the concentration utilized in these assays. However, LOSagg recovered after exposure to LBP (i.e. (LOSagg:LBP)HSA; Fig. 5A) was associated with LBP as determined by immunoblot (Fig. 5 insert) and was disaggregated by subsequent treatment with sCD14 to an extent comparable to the disaggregation observed by simultaneous treatment of LOSaggHSA with LBP, sCD14, and albumin (for comparison see Figs. 2A and 5A).


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  		Fig. 5.   LBP/sCD14-dependent aggregation of [14C]LOSagg requires specific sequential exposure [14C]LOSagg to LBP, sCD14, and albumin. A, isolated LOSaggHSA (Fig. 1; 1 µg/ml, ca. 200 nM) was incubated with 100 nM LBP in Hepes-buffered HBSS+/1% HSA for 15 min at 37 °C before characterization by gel filtration chromatography. LBP:LOSaggHSA (100 ng LOS/ml, ca. 20 nM) was recovered after Sephacryl S500 chromatography (fraction 11) and then incubated for 15 min at 37 °C + sCD14 (5 µg/ml; ca. 100 nM) in HBSS+/1% HSA and then again analyzed by gel filtration chromatography. Results shown are representative of >=  3 experiments. Data are expressed as percent of total [14C]LOS recovered. Total recovery of [14C]LOS (from both columns) was >70%. Immunoblot analysis (insert) of isolated aggregates recovered after incubation of LOSaggHSA + LBP indicate that the recovered void volume fraction contains LBP. Lane 1 represents 5 ng of LBP; lane 2 represents LBP:LOSaggHSA; lane 3 is LBP:LOSaggNo HSA (see below, C). No LBP was detected in the corresponding void volume fractions during chromatography of purified LBP without LOSagg nor was a signal detected in the void volume when LOSagg was chromatographed alone. B, purified LOSaggHSA (100 ng/ml, ca. 20 nM) was incubated with sCD14 (5 µg/ml, ca. 100 nM) in Hepes-buffered HBSS+/0.1% HSA and then analyzed by gel filtration chromatography. The peak fraction of "sCD14:LOSaggHSA (100 ng LOS/ml) recovered after Sephacryl S500 chromatography (fraction 11) was then incubated for 15 min at 37 °C with 10 nM LBP in Hepes-buffered HBSS+/1% HSA and then again analyzed by gel filtration chromatography. Results shown are representative of >=  3 experiments. Data are expressed as percent of total [14C]LOS recovered. Total recovery of [14C]LOS was > 70%. Immunoblot analysis of the peak fraction demonstrated that less than 1% of the added sCD14 was associated with the LOSaggHSA after column chromatography under these conditions (data not shown). C, isolated LOSaggNo HSA (Fig. 1; 1 µg/ml, ca. 200 nM) was incubated with 100 nM LBP in Hepes-buffered HBSS+ (no HSA) for 15 min at 37 °C before characterization by gel filtration chromatography. (LBP:LOSagg)No HSA (100 ng LOS/ml, ca. 20 nM) was recovered after Sephacryl S500 chromatography (fraction 11) and then incubated for 15 min at 37 °C + sCD14 (5 µg/ml, ca. 100 nM) in Hepes-buffered HBSS+/1% HSA and then again analyzed by gel filtration chromatography. Results shown are representative of >=  3 experiments. Data are expressed as percent of total [14C]LOS recovered. Total recovery of [14C]LOS (from both columns) was > 70%. Immunoblot analysis (insert above; lane 3) of isolated aggregates recovered after incubation of LOSaggNo HSA + LBP indicate that the recovered void volume fraction contains LBP.

Treatment of LOSaggHSA directly with sCD14 in albumin resulted in little or no disaggregation (Fig. 5B) under the conditions and concentrations indicated. The addition of LBP (+ albumin) to the recovered LOSaggHSA treated with sCD14 (LOSagg:sCD14)HSA also did not produce disaggregation of LOSagg (Fig. 5B). The lack of disaggregation is consistent with the results of immunoblots of the sCD14-treated LOSagg that indicated no significant association of sCD14 with recovered LOSagg. Therefore, efficient LBP/sCD14-dependent disaggregation of LOSagg requires prior interaction of LOSagg with LBP in the presence of albumin before exposure to sCD14.

Albumin Is Required During the Interaction of LOSagg with LBP for Subsequent sCD14-dependent Disaggregation-- Because extraction of LOS from LOSagg by sCD14 occurred only after formation of LOSagg:LBP, we expected that this would be the step in which the presence of albumin would be most essential. However, as shown in Fig. 5C, the absence of albumin during the exposure of LOSagg to LBP precluded subsequent sCD14 disaggregation of LOSagg even when albumin was introduced with sCD14. Immunoblots of LOSagg recovered after incubation with LBP with or without albumin indicated similar association of LBP with LOSagg in the presence or absence of albumin (compare blots in Fig. 5A inserts). Apparently, the requirement for albumin during the interaction of LOSagg with LBP is not to permit LBP-LOSagg interactions, but rather to promote a presentation of LOS in LOSagg:LBP that facilitates sCD14-dependent disaggregation.

Albumin Is Needed as a Cofactor for LOS:sCD14 Complex-- The known lipid-, including endotoxin-, binding properties of albumin introduced the possibility that albumin might be an essential constituent of the bioactive disaggregated complex. To test this possibility, we generated preparative amounts of the bioactive disaggregated complex (Mr ~ 100,000) from the Sephacryl S500 chromatography for analysis on Sephacryl S200, a matrix with resolution more appropriate to the smaller size of the complex. The latter chromatography was carried out in Hepes-buffered HBSS+ without added albumin to permit simultaneous monitoring of the elution of [14C]LOS and the bulk albumin carried with the complex from the prior (S500) chromatography step. The [14C]LOS eluted as a very sharp and symmetric peak just after albumin (Fig. 6A), suggesting that [14C]LOS was part of a complex with Mr ~ 60,000 and not physically associated with albumin. [14C]LOS in this complex was immunoprecipitated by a monoclonal antibody directed toward an epitope outside the endotoxin-binding site of CD14 (18E12; Fig. 6B). Neither an antibody whose epitope overlaps the endotoxin-binding site of CD14 (MEM18) (35, 36) nor an affinity-purified polyclonal antibody to LBP was able to capture the [14C]LOS-containing complex. These findings suggest that the 60-kDa complex contains one molecule of LOS (4.8 kDa) and one molecule of sCD14 (ca. 50 kDa), i.e. LOS:sCD14 complex.


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  		Fig. 6.   Characterization of the bioactive LOS complex formed by LBP and sCD14-dependent disaggregation of LOSagg. A, the product formed by disaggregation of LOSagg after treatment with LBP and sCD14 was isolated from Sephacryl S500 chromatography as described under "Experimental Procedures," concentrated using Amicon Centricon-10, and re-chromatographed on Sephacryl S200 (1.5 cm × 30 cm column) on Amersham Biosciences AKTA FPLC in Hepes buffered HBSS+. The solid line represents [14C]LOS cpm recovered per fraction and the dashed line represents protein absorbance at 280 nm, i.e. elution of albumin remaining from the S500 chromatography column buffer. B, the disaggregated LOS complex isolated from S500 was incubated in wells containing 1 µg of the absorbed antibodies as indicated (see "Experimental Procedures"). The amount of [14C]LOS that was captured by interaction with the antibodies and remained associated after washing was determined as described under "Experimental Procedures" and is expressed as percent of total [14C]LOS added to the sample. Only monoclonal antibody 18E12, an antibody that binds CD14 outside the endotoxin binding region and that does not capture aggregates of LOSagg:LBP, captures the disaggregated LOS complex. The data shown represent the mean ± S.E. of three experiments. C, the effect of anti-CD14 antibodies and anti-LBP antibody on the ability of LOS:sCD14 to activate endothelial cells was examined by pre-incubation of the LOS:sCD14 complex with the indicated antibodies for 30 min at 37 °C before addition to the cells. The effect of the absence of albumin added during the pre-incubation and activation of the cells was also examined. Results are expressed as percent of control, i.e. production of IL8 formed by activation of cells with LOS:sCD14. Data represent the mean ± S.E.

The peak [14C]LOS-containing fraction (LOS:sCD14) recovered from Sephacryl S200 chromatography potently activated HUVEC without the further addition of LBP and/or sCD14 in contrast to LOSagg (Fig. 6C). Cell activation by this fraction was blocked by 18E12, but not by MEM18 or anti-LBP antibodies, confirming that it is the LOS:sCD14 complex that is responsible for cell activation. Although albumin is not physically associated with the bioactive LOS complex, addition of albumin to this complex appears to be needed for solubility/stability of the complex and, hence, maximal cell activation (Fig. 6C).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have previously demonstrated that LOS isolated from an acetate auxotroph of N. meningitidis can be resolved by gel sieving on Sephacryl S500 in the presence of 0.1% albumin to yield a population of LOS aggregates that can be disaggregated efficiently by interaction with LBP and sCD14 in a buffer system compatible with direct bioassay of isolated species at pathophysiologically relevant concentrations (28). The disaggregated LOS-containing complex is responsible for the maximal cell activation by LOS as measured by the production of IL-8 in HUVEC (28). In this study, we provide evidence that albumin is an essential component in the formation of the active LOS species. The chromatographic profile of LOS in the absence or presence of albumin is identical and generates a major species with Mr > 2 × 107 (Fig. 1). However, results presented here indicate that albumin is necessary for the disaggregation of LOSagg promoted by interaction with LBP and sCD14 (Fig. 2A) and for maximal cellular response to LOS in HUVEC as measured by IL-8 production (Fig. 2B).

Albumin has unique structural properties that include six hydrophobic sites responsible for its ability to bind fatty acids, hydrophobic drugs, bile acids, and steroid hormones (37-40). The ability of albumin to bind and transport long chain fatty acids accounts for the unexpected solubility of these compounds in plasma and permits their transport between different tissues and organs. Associations between endotoxin and albumin have been demonstrated in many studies and in diverse biological settings including whole serum (24, 41-44). These interactions involve primarily the lipid A region, most notably by increasing the solubility of isolated lipid A (23, 25, 26, 41, 45). Reduced recoveries observed during chromatography with LPS in the absence of albumin would be consistent with these previous findings and support a role for albumin to facilitate the "solubility" of LPS in aqueous solutions. An activating role of albumin on endotoxin activity in vitro and in vivo has been previously shown (23, 43, 46). Complexes of albumin with isolated lipid A or intact LPS are bioactive, especially when presented in disaggregated form (27, 42, 47-50). Our findings also show a striking parallel between albumin-dependent disaggregation of endotoxin and endotoxin-dependent cell activation (Figs. 2-4). However, we have shown by high resolution gel sieving chromatography on Sephacryl S200 and immunoassays that the bioactive complex is smaller than albumin (ca. 60 kDa) and likely contains one molecule each of sCD14 and LOS ("LOS:sCD14") (Fig. 6). Although albumin is not associated with the bioactive complex in a high affinity interaction, albumin apparently is nonetheless needed to maintain the solubility/stability of the bioactive species thereby facilitating its delivery to Toll-Like Receptor 4-dependent cell membrane acceptor systems2. The formation of bioactive LPS-albumin complexes in earlier studies was accomplished by using high concentrations of the reactants, long incubations times, and EDTA, and were generated in the absence of LBP and/or sCD14 (11, 27, 32, 51-53). In contrast, under conditions more closely resembling physiological levels of endotoxin, LBP, sCD14, and albumin (0.1-1%), we observed virtually quantitative conversion of LOSagg to LOS:sCD14 within 15 min at 37 °C. These findings strongly suggest that endotoxin:sCD14, not endotoxin-albumin, complexes are physiologically relevant. We suggest that albumin, instead, may function mainly to stabilize otherwise labile intermediates formed as endotoxin is extracted and transferred from a complex hydrophobic environment to specific protein acceptors across aqueous space.

More insight into the function of albumin has emerged by introducing albumin at various stages of LOS:protein interactions and isolating the intermediate species. From this experimental approach, we have obtained evidence that disaggregation of LOSagg required an ordered interaction with LBP, sCD14, and albumin. LBP, not sCD14, engaged LOSagg in a significant manner both in the presence or absence of albumin. But disaggregation to a bioactive form of LOS by interaction of sCD14 with the LBP:LOSagg occurred only when the association of LBP with LOSagg included exposure to albumin. Interactions of LOSagg with LBP must precede exposure to sCD14 since, even in the presence of albumin, sCD14 did not interact with LOSagg to form a stable intermediate that can be subsequently disaggregated. Therefore, LBP alone associated with aggregates of LOS in a way that permitted the transfer of individual endotoxin molecules to sCD14. This finding is consistent with the postulated catalytic role of LBP in that a single LBP: LOSagg provided a vehicle for generation of numerous LOS:sCD14 complexes. The fact that LBP efficiently catalyzed transfer of individual molecules of LOS to sCD14 only when LOSagg:LBP was formed in the presence of albumin suggests an essential role for albumin in maintaining a physical presentation of LOSagg:LBP needed for efficient extraction and transfer of individual molecules of LOS to sCD14. Given the highly amphipathic structure of LOS, it is very likely that lipophilic groups in lipid A within LOSagg are sequestered away from the external aqueous environment and are involved in intermolecular hydrophobic interactions that stabilize the large aggregates. Presumably LBP binding destabilizes endotoxin aggregates, at least in part, by inducing topological rearrangements of endotoxin molecules that modify intermolecular interactions by exchanging attractive forces between individual LOS molecules to include interactions between LOS molecules and LBP. The fact that albumin was needed at this stage to facilitate subsequent extraction and transfer of individual molecules of LOS to sCD14 suggests that the "active" configuration of LOSagg:LBP is one in which lipophilic groups within lipid A are more exposed (Fig. 7). In the absence of albumin, such a physical presentation would probably be unstable and lead to further rearrangements that may then render LOSagg:LBP refractory to disaggregation with sCD14 even when albumin is subsequently added (Figs. 5C and 7).


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  		Fig. 7.   Model for the mode of interaction of albumin in LBP/sCD14-dependent disaggregation of LOSagg and cell activation. Albumin is essential during the interaction of LBP with LOSagg to produce a LBP:LOSagg aggregate that presents LOS in such a way as to permit efficient transfer of LOS from the aggregate to a molecule of sCD14. The complex of LOS:sCD14 formed, which is stabilized by the presence of albumin, is responsible for cell activation as monitored by the production of IL-8 by endothelial cells. The participation of albumin is as a transiently or loosely associated cofactor that permits efficient transfer/protection of the amphipathic LOS molecule between the endotoxin-binding proteins across an aqueous milieu.

Therefore, although albumin may directly form bioactive complexes with LPS (lipid A) under highly artificial conditions, we believe its more physiological role in endotoxin-dependent cell activation reflects its ability to associate, however transiently, with exposed lipid A as endotoxin moves across aqueous spaces from a complex hydrophobic environment to specific protein acceptors. The requirement for albumin is apparently specific because neither ovalbumin nor gelatin was able to substitute for albumin in promoting disaggregation or cell activation. It is possible that this function could be fulfilled by other "lipid carriers." However, it is also possible the weak endotoxin binding properties of albumin previously described (27, 43, 53) are ideally adapted and suited for this function. In either case, this is likely a natural function of albumin in body fluids given its abundance at these sites. Whether albumin is also needed to facilitate transfer to acceptors that are themselves within complex hydrophobic environments (e.g. mCD14, lipoproteins) remains to be determined. It is worth noting that, even in extensively washed cells, albumin is still present, suggesting a tight association of albumin with the cell membrane and so implying the availability of albumin for participation in an exchange between LBP and mCD14 if necessary (43, 54).

Although all endotoxins have several highly conserved structural characteristics, there are many structural variations among the broad array of Gram-negative bacterial endotoxin species present in nature (5, 2). These include differences in the number, structure, and site of linkage of fatty acids within lipid A, the presence of other sub-stochiometric substituents with lipid A and inner carbohydrate core regions as well as the overall composition and length of the oligo- or polysaccharide chain. Such structural variations may affect the solubility of endotoxin aggregates and/or protein-endotoxin complexes in aqueous environments and, hence, the need for albumin to maintain these aggregates in a dispersed form potentially reactive with downstream host acceptors. A similar role for albumin in promoting LBP/sCD14-dependent transformation of both E. coli K12 LPS and NMB LOS was observed (see Figs. 2 and 4), but the structural differences between these two endotoxin species may be too limited to adequately test this hypothesis. It should be noted that the ability of albumin to facilitate transfer of hydrophobic molecules in aqueous environments between proteins has been observed previously. For example, albumin promotes a bidirectional transfer of cholesterol between cells and extracellular proteins that contributes significantly to cholesterol efflux (55, 56). This occurs despite (because of?) the low affinity of cholesterol for albumin. This effect is also albumin-specific and not replicated by either ovalbumin or gelatin. Therefore, it seems likely that the role described in this study will apply not only to NMB LOS and E. coli K12 LPS, but to a much broader array of endotoxin species.

In summary, the results presented in this paper indicate that albumin is an important cofactor in the formation and delivery of bioactive endotoxin-containing complexes to endotoxin-responsive cells. We propose that this role of albumin is conferred by its ability to transiently shield the hydrophobic portion of the lipid A moiety from a hydrophilic environment and thereby permit more effective extraction and transfer of monomers of endotoxin to specific host targets. We have shown an essential role for albumin in the interaction of endotoxin with LBP and sCD14. Additional studies are needed to see if this role extends to other key acceptors in endotoxin-dependent cell activation (e.g. mCD14, Toll-like receptor 4, MD-2) or in the clearance and inactivation of endotoxin.

    ACKNOWLEDGEMENTS

We thank Dr. William Nauseef for careful reading of the manuscript and Dr. Steve Carroll, Xoma Corp. (Berkeley, CA), for providing recombinant LBP, sCD14, and anti-LBP antibodies.

    FOOTNOTES

* This work was supported by United States Public Health Service Grants DK05472 and PO144642 (to J. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Dept. of Internal Medicine, GH 34W, Iowa City, IA 52242. Tel.: 319-338-0581 (ext. 7534); Fax: 319-339-7162; E-mail: theresa-gioannini@uiowa.edu.

Published, JBC Papers in Press, October 7, 2002, DOI 10.1074/jbc.M206404200

2 A. Teghanemt, T. L. Gioannini, K. A. Zarember, and J. P. Weiss, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: NMB, Neisseria meningitidis; BSA, bovine serum albumin; EDTA, ethylenediaminetetracetic acid; HBSS, Hanks' balanced salt solution; Hepes, 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid; HSA, human serum albumin; HUVEC, human umbilical vein endothelial cells; LBP, lipopolysaccharide binding protein; LOS, lipooligosaccharide; LPS, lipopolysaccharide; LOSagg, lipooligosaccharide aggregates; NMB ACE-1, Neisseria meningitidis serotype B acetate auxotroph; PBS, phosphate buffered saline; sCD14, soluble CD14..

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P. Prohinar, F. Re, R. Widstrom, D. Zhang, A. Teghanemt, J. P. Weiss, and T. L. Gioannini
Specific High Affinity Interactions of Monomeric Endotoxin{middle dot}Protein Complexes with Toll-like Receptor 4 Ectodomain
J. Biol. Chem., January 12, 2007; 282(2): 1010 - 1017.
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 Innate ImmunityHome page
J. Andra, T. Gutsmann, P. Garidel, and K. Brandenburg
Invited review: Mechanisms of endotoxin neutralization by synthetic cationic compounds
Innate Immunity, October 1, 2006; 12(5): 261 - 277.
[Abstract] [PDF]

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D. M. B. Post, D. Zhang, J. S. Eastvold, A. Teghanemt, B. W. Gibson, and J. P. Weiss
Biochemical and Functional Characterization of Membrane Blebs Purified from Neisseria meningitidis Serogroup B
J. Biol. Chem., November 18, 2005; 280(46): 38383 - 38394.
[Abstract] [Full Text] [PDF]

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A. Teghanemt, D. Zhang, E. N. Levis, J. P. Weiss, and T. L. Gioannini
Molecular Basis of Reduced Potency of Underacylated Endotoxins
J. Immunol., October 1, 2005; 175(7): 4669 - 4676.
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 Innate ImmunityHome page
T.L. Gioannini, A. Teghanemt, DeS. Zhang, E.N. Levis, and J.P. Weiss
Monomeric endotoxin:protein complexes are essential for TLR4-dependent cell activation
Innate Immunity, April 1, 2005; 11(2): 117 - 123.
[Abstract] [PDF]

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 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
H. P. Jia, J. N. Kline, A. Penisten, M. A. Apicella, T. L. Gioannini, J. Weiss, and P. B. McCray Jr.
Endotoxin responsiveness of human airway epithelia is limited by low expression of MD-2
Am J Physiol Lung Cell Mol Physiol, August 1, 2004; 287(2): L428 - L437.
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T. L. Gioannini, A. Teghanemt, D. Zhang, N. P. Coussens, W. Dockstader, S. Ramaswamy, and J. P. Weiss
Isolation of an endotoxin-MD-2 complex that produces Toll-like receptor 4-dependent cell activation at picomolar concentrations
PNAS, March 23, 2004; 101(12): 4186 - 4191.
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 Innate ImmunityHome page
T. L. Gioannini, A. Teghanemt, K. A. Zarember, and J. P. Weiss
Regulation of interactions of endotoxin with host cells
Innate Immunity, December 1, 2003; 9(6): 401 - 408.
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S. H. Han, J. H. Kim, M. Martin, S. M. Michalek, and M. H. Nahm
Pneumococcal Lipoteichoic Acid (LTA) Is Not as Potent as Staphylococcal LTA in Stimulating Toll-Like Receptor 2
Infect. Immun., October 1, 2003; 71(10): 5541 - 5548.
[Abstract] [Full Text] [PDF]

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