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The Journal of Immunology, 2006, 177: 4880-4887.
Copyright © 2006 by The American Association of Immunologists, Inc.

Native High-Density Lipoprotein Augments Monocyte Responses to Lipopolysaccharide (LPS) by Suppressing the Inhibitory Activity of LPS-Binding Protein1

Patricia A. Thompson and Richard L. Kitchens2

Department of Internal Medicine, Division of Infectious Diseases, University of Texas Southwestern Medical Center, Dallas, TX 75390


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
High-density lipoprotein (HDL) is an abundant plasma lipoprotein that is generally thought to be anti-inflammatory in both health and infectious disease. It binds and neutralizes the bioactivity of the potent bacterial lipids, LPS and lipoteichoic acid, that stimulate host innate immune responses. LPS-binding protein (LBP) plays an important role in augmenting leukocyte responses to LPS, whereas high concentrations of LBP, in the range of those found in plasma, can be inhibitory. We found that native HDL (nHDL) augmented human monocyte responses to LPS in the presence of inhibitory concentrations of LBP as measured by production of TNF and other cytokines. HDL did not stimulate cells in the absence of LPS, and it did not augment responses that were stimulated by IL-1beta or lipoteichoic acid. This activity of HDL was inhibited by trypsin treatment, suggesting that one or more protein constituents of HDL are required. In contrast to nHDL, low-density lipoprotein, and reconstituted HDL did not possess this activity. The total lipoprotein fraction of normal plasma had activity that was similar to that of nHDL, whereas lipoproteins from septic patients with reduced HDL levels had a reduced ability to augment responses to LPS; this activity was restored by adding normal HDL to the patient lipoproteins. Our results demonstrate a novel proinflammatory activity of HDL that may help maintain sensitive host responses to LPS by suppressing the inhibitory activity of LBP. Our findings also raise the possibility that the decline of HDL during sepsis may help control the response to LPS.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Lipopolysaccharide (LPS or endotoxin) is an important determinant of the innate immune response to Gram-negative bacteria due in part to its abundance in the outer bacterial membrane and to the sensitivity of the host mechanisms that recognize it. Most common forms of LPS are recognized by TLR4 (1) in cooperation with other LPS-binding proteins (LBP),3 which include MD-2, an essential coreceptor (2), CD14, an important LPS-binding receptor (3), and LBP, a secreted protein that transfers LPS to CD14 (4). These proteins create a sensitive recognition system that allows the host to detect trace amounts of LPS. The ability to sense the presence of LPS contributes to resistance to infection, whereas in advanced infection, it contributes to the pathophysiology of severe sepsis, which leads to organ failure, shock, and death. To prevent exaggerated responses to LPS, the host has evolved numerous LPS control mechanisms that exert their effects by intracellular (e.g., tolerance and anti-inflammatory cytokines) or by extracellular (e.g., plasma lipoproteins and inhibitory LPS-binding proteins) processes.

LBP can either potentiate or inhibit responses to LPS by mechanisms that depend primarily upon the LBP concentration (5). Its ability to potentiate LPS recognition may explain why LBP-deficient mice have impaired resistance to i.p. infection by Salmonella typhimurium (4, 6) and Escherichia coli (7) and to pulmonary infection by Klebsiella pneumoniae (8, 9). LBP can extract LPS from bacterial membranes (10) and promote its rapid binding to CD14 (11). Whereas low concentrations of LBP favor cell stimulation, higher LBP concentrations are inhibitory (5). In the serum of patients with severe sepsis, high acute phase concentrations of LBP can markedly inhibit monocyte responses to LPS (12). When administered i.p., LBP can rescue animals from the toxic effects of LPS or Gram-negative bacteria (5). Thus, when LBP levels rise both in the plasma (12) and extravascular fluids (7, 13, 14, 15) during Gram-negative sepsis, it may help to control responses to LPS. Although three LPS inhibitory mechanisms have been reported for LBP (16), factors that modulate the balance between the stimulatory and inhibitory activities of LBP have not been described.

High-density lipoprotein (HDL) particles are multifunctional lipoprotein complexes that transport lipids and have several anti-inflammatory properties that are thought to be important for protecting against atherosclerosis and other inflammatory diseases (17, 18). These include lipid transport and antioxidant activities that remove or inactivate inflammatory lipids and the ability of HDL to inhibit various inflammatory responses in endothelial cells. HDL and other plasma lipoproteins can also bind and neutralize the bioactivity of Gram-negative bacterial LPS (19) and Gram-positive bacterial lipoteichoic acid (LTA) (20), both of which can elicit strong proinflammatory responses. Although it is well-established that plasma lipoproteins provide an important host mechanism for controlling responses to LPS (21), there is no compelling evidence that the role of HDL is uniquely important compared with those of other lipoprotein classes. Van Lenten et al. (22) reported that HDL becomes proinflammatory during the acute phase response by augmenting monocyte chemotaxis induced by oxidized low-density lipoprotein (LDL), whereas normal HDL inhibits LDL-induced chemotaxis. During severe and prolonged inflammation such as that induced by bacterial sepsis, circulating HDL levels decline dramatically (23, 24, 25, 26, 27), and it is unclear how the loss of HDL impacts inflammation and immunity in the infected host. It is generally believed that declining HDL levels impair the host’s ability to neutralize LPS (23). To the contrary, we found no reduction in the rate or extent of LPS binding to lipoproteins in the serum of septic patients with extremely low HDL levels, despite the fact that HDL is the major lipoprotein carrier of LPS in normal serum (24). In these patients, the LPS-binding function of HDL was replaced by that of non-HDL lipoproteins in proportion to their phospholipid content. Thus, it is possible that the loss of HDL in septic patients serves to reduce its proinflammatory activity.

Most in vitro assays that have assessed the effects of HDL on LPS responses have been performed in mixtures (e.g., diluted serum) that already contain significant concentrations of native HDL (nHDL) with low concentrations of LBP and other serum proteins. To test the effects of lipoproteins on the inhibitory activity of LBP, we performed our assays in serum-free medium and found that nHDL, but not LDL, augmented LPS responses by decreasing the ability of LBP to inhibit LPS bioactivity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

LPS from E. coli 014 (Ra chemotype) was provided by Dr. R. S. Munford (University of Texas Southwestern Medical Center, Dallas, TX) and was free of contaminating protein on silver-stained SDS-PAGE gels. Outer membrane fragments ("blebs") were isolated from culture supernatants of S. typhimurium as described (10). The Salmonella express wild-type S-form LPS that contains O-Ag polysaccharide chains. The purified LPS or blebs were diluted in HEPES-buffered saline that contained 0.1 mM EDTA and 0.3 mg/ml BSA. Purified LPS was sonicated to disperse large aggregates. Recombinant human LBP and soluble CD14 (sCD14) were produced by baculovirus expression (16, 28). Recombinant human IL-1beta was from R&D Systems, and LTA isolated from S. aureus was obtained from Sigma-Aldrich. DuoSet ELISA reagents for human cytokines were from BD Pharmingen. All other reagents were from Sigma-Aldrich unless stated otherwise.

Plasma lipoproteins

Plasma lipoprotein fractions were isolated by ultracentrifugal flotation in KBr in the presence of protease inhibitors as described (24, 29). The total lipoprotein fraction was prepared in a single step at density (d) < 1.21 g/ml. Individual fractions were isolated sequentially from plasma to obtain very low-density lipoprotein (VLDL) (d < 1.019; also contained intermediate-density lipoprotein and chylomicrons), then LDL (1.063 > d > 1.019), and finally HDL (1.21 > d > 1.063). Total cholesterol and phospholipids were measured colorimetrically as previously described (24), and total protein was measured by the bicinchoninic acid method (Pierce) using BSA as a standard. Partial trypsinization of nHDL was performed by incubating nHDL for 1 h at 37°C with trypsin (25 µg/mg nHDL) or buffer ("mock-treated nHDL") as described (30). The nHDL was separated from the trypsin and proteolytic fragments by chromatography on Superose 6HR 10/30 (Amersham Biosciences). In cell activation experiments, the trypsinized nHDL concentration was equalized to that of mock-treated nHDL according to the phospholipid concentration, which is not affected by trypsin treatment. Reconstituted HDL (R-HDL) (31), prepared from purified human apoA-I and egg phosphatidylcholine, was provided by Dr. P. Lerch (Swiss Red Cross Blood Transfusion Service, Bern, Switzerland). ELISAs for apolipoprotein (apo)E, apoB, and serum amyloid A (SAA) were performed as previously described (24). The capture Abs for apoA-I, apoA-II, and apoC-I ELISAs were affinity purified polyclonal goat anti-human IgG from Academy Biomedical, and the detection Abs were biotin derivatives of the same Abs. Bound Abs were detected by streptavidin-HRP (Jackson ImmunoResearch Laboratories) and 3,3',5,5' tetramethylbenzidine/H2O2 substrate (BD Pharmingen). Normal human serum was used to construct standard curves in ELISAs for apoA-I, A-II, E, and B, and the results were normalized to published plasma concentrations of each protein (32). The apoC-I ELISA used purified human apoC-I (BioDesign International) as a standard.

Lipoproteins from critically ill patients

Total lipoprotein fractions were derived from frozen (–70°C) serum samples selected from those of a study that was previously described in detail (24). The samples were from 13 critically ill patients who had sustained blunt trauma or severe burn injury and four healthy volunteers. All patients met the criteria for systemic inflammatory response syndrome, and eight of the patients had confirmed bacterial infections; two were classified as sepsis and six were classified as severe sepsis. The serum was prepared from clotted venous blood that was obtained by informed consent with the approval of the Institutional Review Board of University of Texas Southwestern Medical Center.

The HDL content of the total lipoprotein fractions (determined as percentage of total phospholipids or cholesterol and expressed as milligrams per deciliter) was determined by fractionating the lipoproteins by size on a Superose 6 column (24). Phospholipid recovery from the column ranged from 93 to 109%; the phospholipid distribution in the HDL, LDL, and VLDL fractions of each sample agreed closely with our previous fractionations of the same samples (24), indicating that the lipoprotein particles had not significantly deteriorated during storage. Similar recoveries were obtained for total cholesterol. For cell stimulation experiments, individual lipoprotein fractions (HDL, LDL, and VLDL) were prepared from pooled serum by sequential ultracentrifugal flotation as described above.

Cells

THP-1 cells (a human monocyte cell line) cultured in 1,25 dihydroxyvitamin D3 (VD3) for 4 days to induce CD14 expression are referred to as THP-1 (VD3) cells (16). In LPS stimulation experiments, the cells (5 x 105 cells/50 µl) were incubated in suspension at 37°C for 2–4 h in serum-free medium (RPMI 1640 containing 20 mM HEPES buffer (pH 7.4) and 0.1 mg/ml BSA) in the presence of 20 pg/ml 014 LPS or 100 pg/ml bleb LPS, added simultaneously (without preincubation) with LBP and lipoproteins. The cells were maintained in suspension by brief oscillation (3 s) at 2-min intervals using an Eppendorf Thermomixer (Brinkmann Instruments). Cytokines were measured in cell-free supernatants by ELISA sets from BD Biosciences. The LPS and bleb concentrations were chosen from LPS dose-response experiments as concentrations that produced near maximal cytokine responses. In pilot experiments, all reagents were checked for effects on cell viability by the WST-1 assay (Roche) and for possible LPS contamination by induction of cytokines in the presence of LBP. PBMC were isolated from normal heparinized venous blood by separation on Histopaque 1077 (Sigma-Aldrich) (28). The PBMC (5 x 105/50 µl) were stimulated in suspension with E. coli 014 LPS in serum-free medium (RPMI 1640 containing final concentrations of 0.4 mM Mg2+, 1.27 mM Ca2+, 20 mM HEPES buffer (pH 7.4) and 0.1 mg/ml BSA) in the presence or absence of nHDL as described above.

Statistics

All statistical analyses were performed using Prism 4.03 (GraphPad Software). These included mean, SD, SEM, the one-sample t test to determine the difference of the mean of any group from a theoretical value of 1.0, ANOVA using Bonferroni’s multiple comparison test to determine significant differences between multiple groups, and the Mann-Whitney U test to determine differences between two groups. Linear regression analysis with Pearson correlation coefficient (r) and significance (p) were used for x-y plots.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
nHDL suppresses the inhibitory activity of LBP

In serum-free medium, low concentrations of LBP promoted maximal stimulation of cell responses by LPS, whereas LBP was inhibitory at higher concentrations (Fig. 1A). These LBP concentrations are within the normal physiologic range; normal human plasma LBP levels were 3.5 ± 1.3 µg/ml (mean ± SD, n = 9) in our assays, and LBP concentrations in extravascular fluids are generally expected to be lower. However, during acute inflammation and infection, LBP concentrations become elevated (28 ± 17 µg/ml, n = 36; Ref. 24). In the presence of the higher LBP concentrations (0.03–3.0 µg/ml), nHDL enhanced the production of TNF by 2- to 9-fold, whereas at lower LBP concentrations, nHDL either had no effect or was inhibitory (Fig. 1A and Table I). In the presence of 25 µg/ml LBP, nHDL also augmented the cell response to LPS in two experiments (3.5- and 20.8-fold, respectively; data not shown). Similar results were obtained when IL-8 and IL-1beta were used as response readouts in selected experiments (data not shown). PBMC responses to LPS were also augmented and inhibited by low and high concentrations of LBP, respectively. The LBP concentration was extended to 10 µg/ml, which generally was more strongly inhibitory than 3 µg/ml (data not shown). As shown in Table II, nHDL augmented the PBMC response to LPS 2.3- and 3.2-fold in the presence of 3 and 10 µg/ml LBP, respectively, whereas nHDL had no significant effect at 0.003 µg/ml LBP.


Figure 1
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  		FIGURE 1. nHDL augments LPS-induced TNF in monocytes in the presence of inhibitory concentrations of LBP. A, THP-1 (VD3) cells were incubated in suspension for 4 h at 37°C in the absence (•) or presence ({blacktriangleup}) of nHDL (200 µg of protein/ml) and increasing concentrations of LBP (0–3 µg/ml) in serum-free medium containing purified E. coli (Ra) LPS (20 pg/ml). TNF was measured in the culture supernatants by ELISA. Annotations above the symbols denote the fold increase in TNF production in the presence of nHDL over that produced in the absence of nHDL. The graph shows the results of a single representative experiment; a summary of similar experiments is shown in Table I. B, Cells were incubated as described in A with S. typhimurium outer membrane fragments containing 100 pg of LPS/ml. The data are shown as mean and range of duplicate determinations obtained in a single experiment, which was repeated with similar results.

 

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  		Table I. nHDL augments LPS-induced TNF in monocytes in the presence of inhibitory concentrations of LBPa

 

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  		Table II. nHDL augments LPS-induced TNF in normal PBMC in the presence of inhibitory concentrations of LBPa

 
nHDL also augmented responses of cells that were stimulated with outer membrane fragments (blebs) from Gram-negative bacteria (Fig. 1B). LBP concentration curves (Fig. 1) showed that nHDL augmented the cell response only when the LBP concentrations were at or above levels that yielded maximal stimulation, suggesting that nHDL interferes with the inhibitory activity of LBP. In contrast, nHDL did not augment the cell response at low stimulatory concentrations of LBP.

nHDL did not augment cell responses in the absence of LPS. nHDL alone had no stimulatory activity in the presence or absence of LBP in THP-1 cells (Fig. 2A) or PBMC (data not shown), indicating that nHDL was not contaminated with LPS or other stimulatory molecules. When the cells were stimulated with rIL-1beta, LBP and nHDL had no effect (Fig. 2B). Whereas increasing concentrations of LBP potentiated and inhibited the cell response to LTA, nHDL was strongly inhibitory at all LBP concentrations in keeping with a previous report (20). nHDL did not augment the ability of sCD14 to promote cell responses to LPS in the absence of LBP; in an experiment in which the cells were stimulated with preformed LPS-sCD14 complexes, we observed that 20 pg of LPS/ml induced 1.03 ± 0.01 ng of TNF/ml in the absence of nHDL and 0.92 ± 0.11 ng of TNF/ml in the presence of nHDL (100 µg/ml). Although LBP inhibited responses to cell bound LPS (i.e., when LBP was added after LPS monomers had already bound to cell surface CD14) as previously described (16), nHDL had no effect on this inhibitory activity of LBP in two experiments (data not shown).


Figure 2
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  		FIGURE 2. nHDL has no stimulatory activity in the absence of LPS. A, THP-1 (VD3) cells were incubated as described in Fig. 1 in the presence or absence of LPS, nHDL (500 µg protein/ml), and LBP as noted in the legends. Mean ± range of duplicate determinations in a representative experiment are shown. Each isolated HDL or other lipoprotein preparation was tested in the absence of LPS with similar negative results (data not shown). B, THP-1 (VD3) cells were stimulated with rIL-1beta (10 ng/ml) or LTA (200 ng/ml) in the presence or absence of nHDL (200 µg/ml), and LBP as noted. TNF was measured in the culture supernatants by ELISA. The data are shown as mean ± SD of four determinations in two experiments.

 
The effect of nHDL was concentration-dependent. As shown in Fig. 3A, the greatest response enhancement was seen at high nHDL concentrations. In contrast, LDL had no enhancing effect in the presence of LBP, and at the highest LDL concentration, the LBP-stimulated cell responses were inhibited, presumably by the ability of LDL to bind and neutralize LPS (Fig. 3B). For comparison to normal HDL and LDL concentrations in human plasma, based on lipid content of the preparations, we calculated that 1400 µg/ml nHDL protein and 564 µg/ml LDL protein are equivalent to normal plasma levels in our assays. Also in contrast to nHDL, R-HDL was strongly inhibitory (Fig. 3C). R-HDL contains only apoA-I and phosphatidylcholine in a formulation that is optimized for LPS binding and neutralization (31).


Figure 3
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  		FIGURE 3. nHDL (not LDL or R-HDL) augments the response to LPS in a concentration-dependent manner. THP-1 (VD3) cells were incubated for 2 h as described in Fig. 1 in the presence of LPS, LBP (0, 0.03, and 0.3 µg/ml), and (A) increasing concentrations of nHDL as noted in (micrograms of protein per milliliter), or (B) increasing concentrations of LDL, or (C) R-HDL or nHDL. TNF was measured in the culture supernatants by ELISA. Mean ± range of duplicate determinations in a representative experiment are shown. The experiment was repeated with similar results.

 
The active nHDL component is a protein

Mild trypsinization can partially remove protein components from the surface of nHDL particles without altering the lipid composition or size of the particles (30). Under these conditions, we found that after trypsin treatment, the phospholipid and cholesterol contents of nHDL were unaffected, whereas the total protein content of the particles was decreased by 24 ± 4% in two experiments. Trypsinization did not alter the mobility of nHDL during Superose 6 chromatography (data not shown). As shown in Fig. 4, trypsinized nHDL had a reduced ability to enhance cell responses in the presence of an inhibitory concentration of LBP, suggesting that the active nHDL constituent is a protein or possibly more than one protein. The trypsinized nHDL preparation did not contain an inhibitor as evidenced by the ability of a mixture of control and trypsinized nHDL to enhance cell responses.


Figure 4
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  		FIGURE 4. The active component of nHDL is destroyed by trypsin. THP-1 (VD3) cells were incubated for 4 h as described in Fig. 1 in the presence of LPS (20 pg/ml) and 0.3 µg/ml LBP in the absence ({blacksquare}) or presence of 50 µg/ml mock-treated nHDL (Figure 4), trypsin-treated nHDL ({square}), or 50 µg/ml each of mock-treated and trypsin-treated nHDL (Figure 4). TNF was measured in the culture supernatants by ELISA and shown as percentage of control (absence of nHDL). The data are expressed as mean ± SD of seven determinations in three experiments using two HDL preparations. *, Significant differences (p < 0.001) between groups in the ANOVA/Bonferroni test. The trypsin-HDL group was not significantly different from the no HDL control group (p > 0.05).

 
Lipoproteins from septic patients with low HDL levels have decreased ability to augment LPS responses in the presence of LBP

We previously found that circulating HDL cholesterol (HDL-C) and phospholipid levels decline dramatically in septic patients. In the serum of patients with severe sepsis, we measured levels as low as 1 and 10% of normal HDL-C and phospholipid levels, respectively (24). To begin to determine the functional impact that such a loss of HDL might have, we isolated the total lipoprotein fractions from the serum of 13 critically ill patients and compared their ability to augment LPS responses in the presence of inhibitory concentrations of LBP to those of control lipoproteins isolated from four healthy volunteers. As shown in Table III, the patients had extremely low serum HDL-C and HDL phospholipid (HDL-PL) levels; levels of certain apos that are normally HDL-specific (i.e., apoA-I, apoA-II, and apoC-I) were also dramatically reduced. As expected, we found that the patient serum had elevated levels of SAA, a major acute phase reactant, which usually replaces apoA-I as the most abundant HDL apo during the acute phase response (33). Most of the patients had normal or elevated levels of apoB, the major apo of LDL and VLDL, and apoE, which occurs in triglyceride-rich lipoproteins and in a minor subclass of HDL (24, 26) (Table III).


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  		Table III. Serum lipoprotein and apo levels in normal subjects and critically ill patientsa

 
To assess the effects of the lipoproteins on cell responses to LPS in the presence of LBP, we first added the lipoproteins to the cells on the basis of equal amounts of protein. As shown in Fig. 5A, lipoproteins from healthy volunteers augmented the monocyte response to LPS in the presence of an inhibitory concentration of LBP, whereas the lipoproteins derived from six patients generally had decreased activity. The ability to augment the response correlated significantly with the HDL-C content of the lipoproteins as shown in Fig. 5A and with HDL-PL as shown in Table IV. In a second set of experiments, which included lipoproteins from six additional patients and two additional controls, we assessed the effects of lipoproteins added on the basis of both equal protein (Fig. 5B) and equal phospholipids (Fig. 5C). Because phospholipids are the major determinants of lipoprotein binding (24) and neutralization of LPS (34), we reasoned that adding equal amounts of phospholipid would minimize bias when comparing samples that contained different phospholipid concentrations. Our results again showed that the normal lipoproteins augmented the response to LPS to a greater degree than did lipoproteins of septic patients and that the response to LPS correlated positively with the HDL content (Fig. 5, B and C, and Table IV). Four patient and two control samples from Fig. 5, B and C, also showed a similar correlation in the presence of 0.3 µg/ml LBP (data not shown), whereas the fold-increases in TNF were slightly lower than those obtained with 3 µg/ml LBP. Although the TNF fold increases in Fig. 5A are somewhat higher in both patients and controls than those in the other panels, probably due to day-to-day differences in the assay, the relative differences between patients and controls in each experiment are similar. As shown in Table IV, the LPS-augmenting activities of the lipoprotein preparations also showed significant positive correlations to their content of HDL apo (apoA-I, A-II, and C-I), whereas there was no correlation with the major protein components of LDL and VLDL (apoB and apoE) or with the acute phase HDL component, SAA. The activity of the normal lipoproteins was not due to contamination; they had no ability to stimulate the cells in the absence of LPS in the presence or absence of LBP (data not shown). The low activity of the patient lipoproteins was not due to suppression of nHDL activity or by LPS neutralization by other lipoproteins. Fig. 5D shows that the addition of normal nHDL to lipoproteins pooled from four patients restored the ability to augment the cell response to LPS.


Figure 5
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  		FIGURE 5. Total lipoprotein fractions from critically ill patients with low HDL levels have decreased ability to augment LPS-induced TNF. THP-1 (VD3) cells were incubated for 4 h as described in Fig. 1 in the presence of LPS (20 pg/ml), 0.3 µg/ml LBP (A), or 3 µg/ml LBP (B and C), and total lipoprotein fractions of serum from four healthy volunteers (•) and 13 critically ill patients ({blacktriangleup}) at a final concentration of 80 µg protein/ml (A and B) or 80 µg of phospholipid/ml (C). TNF was measured in the culture supernatants by ELISA and expressed as the fold increase above that obtained in the absence of lipoproteins (y-axis) and plotted against the final concentration of HDL (milligrams of cholesterol per deciliter) in each assay (x-axis). Each symbol represents one subject, and error bars denote mean and range of duplicate determinations. The same subjects were analyzed in B and C, whereas additional subjects were analyzed in A. Linear regression analyses are shown with Pearson correlation coefficients (r) and significance (p). D, Pooled total lipoprotein fractions (patient Lp) (80 µg of phospholipid/ml) from four patients were assayed in the presence of 3 µg/ml LBP either alone or in combination with normal nHDL (100 µg/ml) as described in C. D shows the mean ± SD of four determinations in two experiments. Asterisks denote significant differences between the groups (*, p < 0.05; **, p < 0.01; and ***, p < 0.001) by the ANOVA/Bonferroni test.

 

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  		Table IV. HDL and HDL-specific apo levels correlate positively with the ability to augment LPS-induced TNF by total lipoprotein fractions from normal and critically ill subjectsa

 
Because the compositions of lipoproteins can change dramatically during acute inflammation and infection, we next compared the activities of normal and acute phase HDL. We isolated the HDL fraction of pooled serum samples from three normal subjects and compared its activity to that of HDL isolated from pooled serum from three patients whose total lipoprotein fractions had low HDL content and possessed low ability to augment the response to LPS (Fig. 5). As shown in Fig. 6, the pooled patient HDL had no ability to augment TNF in the presence of an inhibitory concentration of LBP, whereas the normal nHDL augmented the TNF response as expected. It should be noted that the higher patient HDL concentration tested (200 µg of PL/ml) was equivalent to the HDL-PL concentration in the pooled patient serum (208 µg of HDL-PL/ml or 20.8 mg/dL). The results indicate that the decline in the response-enhancing activity of the total lipoprotein fractions of patients is due not only to a decline in HDL concentration but also to a decline in the activity of the HDL itself. Although our analysis of isolated patient HDL should be considered preliminary since it does not include a large cross section of patients, it shows that acute phase HDL can completely lose its ability to suppress the inhibitory activity of LBP. In summary, our results indicate that the lipoproteins of septic patients have a decreased ability to augment cell responses to LPS in the presence of inhibitory LBP concentrations as a result of decreased HDL levels and/or decreased levels of one or more normal HDL-associated proteins.


Figure 6
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  		FIGURE 6. Loss of LPS-enhancing activity in acute phase nHDL. THP-1 (VD3) cells were incubated for 4 h as described in Fig. 1 in the presence of LPS (20 pg/ml), 3 µg/ml LBP, and nHDL (100 or 200 µg phospholipid/ml) derived from pooled serum of three healthy volunteers and from three patients that showed low LPS enhancing responses in Fig. 5. TNF production in the presence of nHDL is shown as the fold increase above that obtained in the absence of nHDL. The mean ± SD of four determinations in two experiments are shown. *, Fold-changes significantly >1.0 by the one-sample t test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our findings reveal a novel proinflammatory activity of HDL that may appear to contradict the widely accepted view that HDL is an LPS inhibitor. This apparent discrepancy can be explained by the ability of nHDL to differentially affect early and late (or prolonged) responses to LPS. LPS binds to CD14 on monocyte surfaces more rapidly than it binds to nHDL and other native lipoproteins (29, 35). Thus, under conditions in which the cells compete with lipoproteins for LPS, early responses to LPS may be unimpeded. In contrast, nHDL and other lipoproteins can inhibit late or prolonged responses to LPS by binding and neutralizing LPS bioactivity (19) and by removing LPS from cell surfaces (28, 29). In keeping with this, we found that preincubating LPS, LBP, and nHDL together for 30 min before adding to THP-1 monocytes, ablated the ability of nHDL to augment the cell response, whereas preincubating only LBP and nHDL did not (data from two experiments; data not shown). Thus, differences in timing of these opposing effects of nHDL provide a rationale for our hypothesis that nHDL can augment beneficial early responses to LPS, whereas it helps to control exaggerated or prolonged responses to LPS that may become detrimental to the host.

In contrast to nHDL, artificial R-HDL preparations that are optimized for LPS binding, compete more effectively for LPS, resulting in significant inhibition of cell responses at early time points (29, 31, 34, 36). It has also been suggested that R-HDL causes rapid down-regulation of monocyte CD14 expression (36), however, we found that R-HDL did not decrease the density of membrane-bound CD14 in THP-1 cells (data not shown). Maximal or prolonged monocyte responses to LPS require prolonged contact of LPS with the cell surface (16, 28, 29, 37). Therefore, the enhanced ability of R-HDL to remove LPS that has already bound to cell surface CD14 increases R-HDL’s inhibitory potency (29). When LPS is prebound to monocytes, R-HDL inhibits early cell responses (e.g., those occurring at 4 h or less), whereas inhibition of responses by nHDL requires extended incubation (16–24 h) (29, 38). LPS inhibition by nHDL becomes more apparent under noncompetitive conditions in which LPS and nHDL are preincubated before adding to cells or administering to live animals. Some studies suggest that under these conditions nHDL has the least inhibitory potency compared with other lipoprotein classes (39, 40, 41). Even when LPS and nHDL were added simultaneously to whole blood, all other lipoprotein classes outperformed nHDL as LPS inhibitors (34), and significant LPS inhibition required the addition of several thousand micrograms per milliliter of nHDL, concentrations that are above the physiologic range. The ability of transgenic overexpression of human apoA-I to inhibit LPS responses in mice has been interpreted as an inhibitory effect of nHDL (42). The degree of inhibition was surprising in view of an increase in HDL of only 2-fold in the transgenic mice. The inhibition may have been due to the formation of an inhibitory HDL subclass. For example, the apoA-I transgene promotes the formation of a significant amount of pre-beta HDL, which has a discoidal structure similar to that of R-HDL. Conversely, in experiments in which lipoprotein levels were pharmacologically reduced, the resulting hyperresponsiveness to LPS was normalized by administering the total lipoprotein fraction of human plasma (21), whereas the effect of the nHDL fraction alone was not reported. In summary, although R-HDL is a potent and rapid LPS inhibitor, the inhibitory effect of nHDL is slow, and the studies cited above raise the possibility that normal nHDL is not the most potent LPS inhibitor of the native lipoprotein classes. Indeed, nHDL probably plays little or no role in LPS neutralization during severe sepsis, since we found that when HDL levels decline in these patients, LPS is bound preferentially by LDL and VLDL (24).

The ability of nHDL to augment the cellular response to LPS appears to be due entirely to its ability to suppress the inhibitory activity of LBP. nHDL did not augment the cell response to IL-1beta at any LBP concentration or to LPS in the presence of very low concentrations of LBP. The experiments in Fig. 1 and Table I reveal that in the low range of LBP concentrations, nHDL either had no effect or was inhibitory. The inhibitory effect of nHDL occurred only when the LBP concentration was exceedingly low; this may be due to the ability of nHDL to absorb LPS under conditions in which LBP was only marginally stimulatory, or it may have resulted from a direct inhibitory effect of nHDL on LBP activity. If the interaction of LBP with HDL merely reduces the effective concentration of LBP throughout the LBP dose curve, it is difficult to explain how this interaction would increase maximal cell activation at high LBP concentrations. HDL may either differentially affect inhibitory vs stimulatory activities of LBP, or HDL may have an additional effect on LPS recognition by the cells.

The mechanism of suppression of LBP inhibitory activity by nHDL is unclear. LBP can inhibit LPS bioactivity by three known mechanisms (16): 1) by transferring LPS to lipoproteins, 2) by forming LPS-LBP aggregates that bind CD14 and are internalized but do not signal, and 3) by interfering with the transfer of CD14-bound LPS to the MD-2/TLR4 signaling receptor. Mechanism 1) can be excluded because the LPS was not neutralized by nHDL. Inhibitory mechanism 3) does not appear to be influenced by nHDL, since we found that nHDL did not reverse the ability of LBP to inhibit responses to cell-bound LPS monomers (16). This suggests that nHDL interferes with LBP inhibitory activity at a step before the binding of LPS to membrane-bound CD14. Thus, nHDL may interfere with mechanism 2) or another inhibitory mechanism that has not yet been described. The activity of nHDL requires one or more HDL-specific proteins (Figs. 3 and 4), and one possible mechanism could involve specific binding of LBP to the HDL-associated protein(s). Studies of the association of LBP with lipoproteins are problematic and have yielded contradictory results. Wright and coworkers (43) reported that LBP associates exclusively with HDL, whereas Vreugdenhil et al. (44) reported that LBP associates exclusively with non-HDL lipoproteins that contain apoB. Because LBP can bind to both immobilized HDL (45) or LDL (44), it seems unlikely that our HDL-specific protein is essential for LBP binding to lipoprotein particles. Thus, it is more likely that the mechanism involves a specific interaction between LBP and the HDL-specific protein(s) either after LBP binds to the HDL particle or after the protein dissociates from the HDL particle. Alternatively, the active HDL protein may interact with LPS in a way that alters LPS-LBP complex formation. Another possibility is that the HDL protein binds an LBP-associated lipid and promotes its exchange with LPS (46). Studies are now in progress to identify the active HDL protein.

Based on the results of the current study, it would be reasonable to hypothesize that the high concentration of HDL in the circulation would suppress the inhibitory activity of plasma LBP in vivo. This hypothesis is supported by the findings of Schumann and coworkers (5), who first showed that LBP can strongly inhibit in vivo responses to LPS and Gram-negative bacteria and can rescue mice from LPS toxicity. They noted that LBP was inhibitory when administered i.p., whereas i.v. administration was ineffective. Indeed, we and others have observed sensitive responses to LPS in assays of whole blood, which contains microgram per milliliter concentrations of LBP. In contrast, we and others have found that the same concentrations of LBP in serum-free medium can be strongly inhibitory (5), and we show here that this inhibitory effect is significantly reversed by the presence of nHDL (Fig. 1 and Table I). Thus, normal HDL may preserve the ability of circulating leukocytes to respond sensitively to LPS, whereas during severe inflammation and sepsis, the decline of HDL coupled with the increase of LBP may work together to help control the inflammatory response to LPS, and non-HDL lipoproteins may assume the major role in binding, neutralizing, and clearing LPS (24). It is unclear whether the ability of normal HDL to augment the LPS response should be expected to be confined to the blood; we have not yet determined the effects of nHDL on the responses of macrophages of various types and differentiation states.

Our findings raise the possibility that HDL may play a novel and unexpected role in promoting the early inflammatory response to Gram-negative bacteria. This property of normal HDL described in our experiments stands in contrast to previous reports that HDL becomes proinflammatory only when modified by the acute phase response (22). These studies reveal that acute phase HDL enhances LDL-induced monocyte chemotactic activity, whereas normal HDL is inhibitory. This proinflammatory activity of acute phase HDL is thought to be due to modifications such as the loss of paraoxonase, which can destroy chemotactic lipid hydroperoxides that are carried by LDL (reviewed in Ref. 47). In contrast, we found that acute phase lipoproteins isolated from critically ill patients had decreased proinflammatory activity as measured by their decreased ability to augment LPS bioactivity in the presence of inhibitory LBP concentrations. Although our data suggest that this is due to a reduction in the HDL content of the acute phase lipoproteins, our results also show that acute phase HDL itself can lose this activity (Fig. 6). Additional experiments will be required to determine whether the loss of this activity in acute phase HDL precedes or follows the decline in HDL levels in the plasma during disease progression in the host. Our results suggest that the alteration and marked decline of circulating HDL that occurs during severe inflammation and infection (24) may help to control inflammation by reducing the proinflammatory activities of HDL.


   Acknowledgments
 
We thank Dr. Robert Munford for providing LPS and for helpful discussions and critical reading of the manuscript. We also thank Drs. Borna Mehrad, Patrick Rensen, and Jimmy Berbée for critically reading the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant AI45896 from the National Institute of Allergy and Infectious Diseases. Back

2 Address correspondence and reprint requests to Dr. Richard L. Kitchens, Department of Internal Medicine, Division of Infectious Diseases, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9113. E-mail address: richard.kitchens{at}UTSouthwestern.edu Back

3 Abbreviations used in this paper: LBP, LPS-binding protein; HDL, high-density lipoprotein; LTA, lipoteichoic acid; nHDL, native HDL; sCD14, soluble CD14; LDL, low-density lipoprotein; VLDL, very low-density lipoprotein; R-HDL, reconstituted HDL; apo, apolipoprotein; SAA, serum amyloid A; HDL-C, HDL cholesterol; HDL-PL, HDL-phospholipid; VD3, 1,25 dihydroxyvitamin D3. Back

Received for publication December 12, 2005. Accepted for publication July 24, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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