LPS-binding protein circulates in association with apoB-containing lipoproteins and enhances endotoxin-LDL/VLDL interaction

Evidence for a physiological role for LBP in inflammation is supported by studies that demonstrate enhanced mortality and uncontrolled multiplication and spread of bacteria in LBP knockout mice compared with wild-type mice after intraperitoneal administration of bacteria (11). The results of these studies indicate that LBP is required to induce a rapid inflammatory response, which is essential for the resistance to bacteria. However, LBP has the paradoxical dual function of sensitizing the immune system to endotoxin and, on the other hand, enhancing detoxification of endotoxin. LBP catalyzes the transfer of LPS into lipoproteins, thereby enhancing LPS detoxification (12). Likewise, LBP catalyzes the lipoprotein neutralization of lipoteichoic acid, a component of the cell membrane of gram-positive bacteria (13). Lamping et al. demonstrated in a murine model that high levels of LBP in the circulation, as seen during an acute-phase response, inhibit LPS effects and prevent mortality induced by endotoxemia (14). The latter observation strongly supports a physiological role for LBP-dependent detoxification of LPS in the host defense.

Endotoxemia induces an acute-phase response characterized by multiple physiological adaptations. This response appears to play a role in host defense mechanisms, although its physiological relevance needs further elucidation. One aspect of the acute-phase response is a dramatic rise in circulating levels of LBP (15). Concomitantly, large changes in serum lipid and lipoprotein concentrations occur. Circulating levels of total cholesterol, LDL cholesterol, and HDL cholesterol decrease, whereas serum triglyceride and VLDL levels increase (16). In addition, alterations in apolipoprotein levels are observed (16, 17). ApoA-I concentrations drop, and HDL becomes depleted in apoA-I (16, 18). In contrast, apoB levels are not affected by either viral or bacterial infection (16).

Others found evidence for an association of LBP with apoA-I–containing lipoproteins in plasma from healthy persons (12). We consider that the physical association of LBP with these lipoproteins may be important for the cooperation of LBP and lipoproteins in the detoxification of endotoxin. However, the strong reduction of apoA-I and HDL levels that coincides with the dramatic raise in LBP levels during endotoxemia seems in contrast with this cooperative function. Because it is firmly established that LDL and VLDL are critical in the survival of infection with gram-negative bacteria (19) and that circulating levels of these lipoproteins are relatively high during inflammation compared with HDL levels, the present study was undertaken to investigate whether LBP associates with LDL and VLDL. To this end, the distribution of LBP among lipoproteins was studied in serum of healthy and septic persons. Subsequently, we investigated the effect of the association of LBP with lipoproteins and apolipoproteins on the LPS-binding capacity.

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Reagents.

LPS from Escherichia coli, serotype 055:B5, was purchased from Sigma Chemical Co. (St. Louis, Missouri, USA). Purified apoA-I and apoB were derived from Calbiochem (La Jolla, California, USA) and ICN Radiochemicals Inc. (Irvine, California, USA), respectively. Polyclonal antibodies to human LBP were obtained by immunizing rabbits with purified human LBP. Protein A–purified anti-LBP IgG was biotinylated following standard procedures. Anti-human LBP mAb HM14 was obtained by immunizing mice with LBP following classical procedures. Horseradish peroxidase–labeled mAb against apoA-I and apoB were gifts from L. Sorell (Center for Genetic Engineering and Biotechnology, Havana, Cuba).

Blood samples.

Blood was collected from healthy donors and from septic patients. Informed consent was obtained from healthy donors and relatives of septic patients. To prepare serum, blood was allowed to clot for 2 hours at room temperature. Serum was separated by centrifugation and stored at –80°C until use. Fresh serum was used for isolation of lipoprotein fractions.

Lipid electrophoresis and Western blot analysis.

Agarose electrophoresis was performed in barbital buffer (pH 8.6) using the Paragon Lipoprotein Electrophoresis kit P/N (Beckman Instruments, Brea, California, USA) according to the manufacturer’s instructions. Agarose gel electrophoresis was followed by electrophoretic transfer onto an Immobilon-P membrane (Millipore Corp., Bedford, Massachusetts, USA) in blotting buffer (25 mM TRIS, 192 mM Glycine, 10% methanol) using the Phast system (Pharmacia Biotech A/B, Uppsala, Sweden). After transfer, the membranes were blocked with 1% BSA in PBS for 1 hour at 37°C and washed with 0.1% BSA 0.5% Tween in PBS. For detection of LBP, membranes were incubated with biotin-labeled polyclonal antibody specific for human LBP, washed, and incubated with peroxidase-labeled streptavidin. After detection of LBP, apoA-I was probed on the same blot after elution of the LBP antibodies by incubation with 0.1 M Glycine-HCl buffer (pH 2.5) for 15 minutes at 37°C. Localization of apoA-I was followed by detection of apoB in a similar protocol. For immunodetection of apoA-I and apoB, peroxidase-labeled mAb’s were used. Interactions of LPS with serum lipoproteins were studied by preincubation of biotin-labeled LPS with human serum overnight at 37°C. After preincubation, serum was subjected to electrophoresis and blotted and membranes were incubated with peroxidase-labeled streptavidin. To study LPS binding to isolated (apo)lipoproteins, human serum was first subjected to electrophoresis and blotted and membranes were incubated with biotinylated LPS, followed by incubation with peroxidase-labeled streptavidin (Zymed Laboratories Inc., South San Francisco, California, USA). Peroxidase activity was detected by chemiluminescent substrates (SuperSignal Substrate, Pierce Chemical Co., Rockford, Illinois, USA) according to the manufacturer’s recommendation.

Lipoprotein fractionation.

Lipoproteins were isolated from pooled fresh normal human serum (six healthy donors) by a 22-hour single spin density gradient ultracentrifugation at 200,000g and 17°C according to Terpstra et al. (20), using a Beckman XL-80 ultra centrifuge with a SW40 rotor and ultraclear centrifuge tubes (Beckman Instruments). A step gradient was constructed from 2 ml serum adjusted to density 1.250 g/ml with KBr and sucrose; two NaCl/KBr solutions with density 1.225 and 1.100 g/ml, respectively; and 0.998 g/ml endotoxin-free water. All solutions contained 0.1 mg/ml EDTA. The lipoprotein fractions VLDL, LDL, HDL2, and HDL3 were collected by aspiration, dialyzed extensively against PBS at 4°C, and stored at –80°C until use. Cholesterol concentrations in the isolated lipoprotein subfractions were determined using an enzymatic colorimetric test from F. Hoffman LaRoche, AG (Basel, Switzerland). The lipoprotein fractions used contained no detectable LBP as assessed by LBP-specific ELISA (15).

Purification of LBP and production of LBP-depleted human serum.

LBP was isolated from human plasma by selective-affinity immunosorption as described previously (21). Plasma from healthy volunteers was kindly provided by the local blood bank. In short, anti-hLBP mAb HM-14 was cross-linked to CNBr-activated Sepharose (Pharmacia Biotech A/S) according to the manufacturer’s instructions. Human plasma was applied to the anti-LBP column, and unbound proteins were washed out with 0.5 M MgCl. Bound LBP was eluted with 0.1 M Glycine-HCl buffer (pH 2.5). LBP-depleted serum was produced by passing serum over the anti-LBP column. LBP-depleted serum contained less LBP than 0.05% of normal serum as assessed by LBP-ELISA.

Preparation of biotin-labeled LBP and LPS.   And another 3 pages.

The structure and function of LBP are extensively studied; however, knowledge concerning the in vivo forms or associations with other serum components and the effect of these associations on the function of LBP is limited. In the present study, we obtained evidence that LBP circulates in association with apoB-containing lipoproteins in healthy persons and in septic patients. This association is functional, as LBP bound to LDL and VLDL was observed to enhance the LPS-binding capacity of these lipoproteins, a process known to result in protection from the deleterious effects of LPS toxicity.

In search of the factors involved in the association of LBP with apoB-containing lipoproteins in serum, we found that this interaction is at least in part mediated by an interaction of LBP with apoB. Although LBP was not associated with apoA-I or HDL in serum, our experiments demonstrate that LBP does bind to purified apoA-I. Evidence for a specific interaction of LBP with apoA-I is supported by studies by Massamiri et al., who demonstrated that binding of LBP to reconstituted HDL is partially blocked by antibodies against apoA-I (23). In the present study, we observed a tenfold higher affinity of LBP for apoB compared with apoA-I (Figure ​(Figure8c).8c). The amount of apoA-I molecules in serum, however, is about 30 times higher than the amount of apoB under healthy conditions, therefore, it is not possible to subscribe the predominant interaction of LBP with LDL and VLDL to the higher affinity of LBP for apoB. It is possible that LBP interacts with domains of apoA-I that are masked by incorporation in native HDL. Furthermore, other constituents than apoB may contribute to the high affinity of LBP for LDL and VLDL and consequently increase the competition for binding of LBP to HDL.

The LBP-transporting lipoproteins LDL and VLDL were also found to be the predominant LPS-binding lipoproteins in normal human serum, an observation which is supported by others (5, 24). Studies performed in rodents report the preferential binding of LPS to HDL (4, 25–27). Relatively high HDL levels in rodents seem to account for the predominant binding of LPS to HDL, as high LDL levels in Watanabe heritable hyperlipidemic rabbits and high VLDL levels in apoE knockout mice result in the predominant binding of LPS to LDL (5, 28) and to VLDL (J. Kuiper personal communication), respectively. In contrast to rodents, the HDL:LDL/VLDL ratio is low in humans, which most likely causes the differences observed for the distribution of LPS among lipoproteins in humans and rodents.

Our data further demonstrate that LBP associated with LDL and VLDL strongly enhances binding of LPS to these lipoproteins in a dose-dependent fashion. However, presence of LBP is not required for the interaction of LPS with LDL and VLDL. ApoB, present in LDL and VLDL, was demonstrated to have high affinity for LPS and therefore may account for LPS binding under LBP-free conditions. This interaction of LPS with apoB is inhibited by presence of LBP, implicating competition of LBP and LPS for apoB. It can be hypothesized from these data that apoB functions as an anchor for LBP on LDL and VLDL. This bound LBP may catalyze the binding of LPS to other lipoprotein components and thereby enhance the total LPS-binding capacity. Given that others found that a lipid-lipid interaction accounts for the association of LPS with lipoproteins (5) and have demonstrated that LBP enhances binding of LPS to phospholipid membranes (29, 30), it is likely that LBP linked to apoB enhances intercalation of LPS into the phospholipid membrane of the lipoproteins. Other proteins present in LDL and VLDL may, however, also contribute to the LPS-binding capacity of these lipoproteins. ApoE, present in VLDL, for instance, was demonstrated to bind LPS (31). Most interestingly, apoE-deficient mice display increased susceptibility to endotoxemia (32), the latter supporting a possible role for apoE in the scavenging of LPS.

During infection, lipoproteins are proven to be fundamental for the survival of the host (1, 2, 9, 10, 19). Under these conditions, lipoprotein levels and composition are known to be altered profoundly (33). These changes prompted us to evaluate the interaction of lipoproteins with LBP and endotoxins in septic serum. The changes in lipoprotein composition and the more than tenfold enhanced LBP levels did not significantly affect the distribution profile of LBP. In serum of septic patients, LBP is also colocated predominantly with apoB (Figure ​(Figure10).10). These observations indicate that during sepsis, approximately tenfold more LBP is associated with LDL and VLDL. This is of particular interest, as we observed that the LBP-induced upregulation of LPS binding to these lipoproteins is dose-dependent. Only a small fraction of the total LBP in septic sera migrates as free LBP. Whether there is a difference in biologic activity between lipoprotein-associated and non–lipoprotein-associated LBP in the host response to endotoxin is currently under our investigation. The predominant incorporation of LPS in LDL and VLDL is also observed in septic serum and does not seem to be influenced by the alterations in lipoprotein composition and the presence of free LBP. Our results, which indicate that LPS and LBP both predominantly bind to LDL and VLDL under septic conditions and that apoB forms a binding site for LBP and LPS, are in line with the findings of others (16) that in contrast to apoA-I, apoB levels stay high during infection. The remaining high concentration of apoB in LDL and VLDL seems functional, as it may contribute to the enhanced binding of LBP and, as a consequence LPS, to these lipoproteins during infection.

It is firmly established that binding of LPS to lipoproteins reduces LPS toxicity (3). In accordance, hypolipidemia results in enhanced LPS-induced lethality in animals (2), and in humans, low levels of cholesterol predict an increased risk of death from infection (34), which emphasizes the significance of lipoproteins in protection from bacteria and their toxins. The protective function of lipoproteins is considered to be due to an increase in the clearance of LPS by formation of LPS-lipoprotein complexes and to prevention of its binding to cells. In addition, it was recently demonstrated that lipoproteins, including LDL, also promote the release of cell-associated LPS, which was proved to be dependent on LBP (35). This inhibition of endotoxin binding reduces activation of monocytes and thereby the secretion of cytokines (3,35, 36). This protective property of lipoproteins is not only described for gram-negative bacteria, but also the toxic effects of fragments of gram-positive bacteria are inhibited by LDL, a process catalyzed by the presence of LBP (13). The beneficial role for LDL in the host defense against bacteria is supported by a study that demonstrates that LDL-receptor–deficient mice with elevated circulating LDL concentrations are protected against lethal endotoxemia and severe infections with gram-negative micro-organisms (19).

The uptake of LPS into LDL, which we consider beneficial during acute infection, should be considered as potentially harmful during chronic inflammation. Since we studied LPS binding to LDL and VLDL, and not toxicity, we cannot exclude that LPS binds to LDL and VLDL in a manner by which it retains some toxic activity. In the long term, these complexes may play a pathogenic role in the development of atherosclerosis. Although LDL protects endothelial cells from acute LPS toxicity by formation of LPS-LDL complexes, these complexes migrate across the endothelium and, via unknown pathways, increase the secretion of monocyte chemotactic activity by endothelial cells (28). [In other words, infections in the intima media of the artery walls and the LDL immune response contributes to the formation of atheroma. Thus atheroma is like the formation of a boil from the presences of bacteria, and the leakage of this boil like atheroma results in ischemic events.}  As a consequence, transport of LDL-LPS complexes into the artery wall may initiate an inflammatory response and provoke an atherosclerotic reaction.

In conclusion, we found strong evidence for an association of LBP with apoB-containing lipoproteins in the circulation of healthy persons. Also in septic patients with extremely high circulating LBP concentrations, LBP is predominantly bound to apoB-containing lipoproteins. Most interestingly, LBP associated with LDL and VLDL enhances the LPS-binding capacity of these lipoproteins in a dose-dependent manner. Accordingly, in serum from healthy persons and from septic patients, LDL and VLDL are the predominant LPS-binding lipoproteins. Overall, the data of this study suggest that LBP is a cofactor of circulating LDL and VLDL, which facilitates the uptake of endotoxin by these lipoproteins. This implies an important role for LBP/LDL and VLDL complexes in the defense against bacteria and endotoxin.

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We thank M. Poeze for the assistance in obtaining the serum of septic patients. This work was supported by a grant of the Dutch Digestive Diseases Foundation, The Netherlands