Acute-Phase Concentrations of Lipopolysaccharide (LPS)-Binding Protein Inhibit Innate Immune Cell Activation by Different LPS Chemotypes via
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感染与免疫杂志 2005年第1期
Institute for Microbiology and Hygiene, Charite University Medical Center, Humboldt University Berlin, Berlin
Leibniz Research Center Borstel, Center for Medicine and Bioscience, Borstel
Department of Anesthesiology, University of Lübeck, Lübeck, Germany
ABSTRACT
The chain length of bacterial lipopolysaccharide (LPS) is a crucial factor for host-pathogen interaction during bacterial infection. While rough (R)-type and smooth (S)-type LPSs have been shown to differ in their ability to interact with the bactericidal/permeability-increasing protein, little is known about the differential mode of interaction with the acute-phase reactant LPS-binding protein (LBP). At lower concentrations, LBP catalyzes the binding of LPS to CD14 and enhances LPS-induced cellular activation via Toll-like receptor 4. In humans, however, concentrations of LBP in serum increase during an acute-phase response, and these LBP concentrations exhibit inhibitory effects in terms of cellular activation. The mechanisms of inhibition of LPS effects by LBP are not completely understood. Here, we report that human high-dose LBP (hd-LBP) suppresses binding of both R-type and S-type LPS to CD14 and inhibits LPS-induced nuclear translocation of NF-B, although cellular uptake of R-type LPS was found to be increased by hd-LBP. In contrast, we found that hd-LBP enhanced the binding and uptake of S-type LPS only under serum-free conditions, whereas in the presence of serum, hd-LBP inhibited cellular binding and uptake. This inhibitory effect of serum could be mimicked by the addition of purified high-density lipoprotein (HDL) to serum-free medium, indicating an LBP-mediated transfer of preferentially S-type LPS to plasma lipoproteins such as HDL. A complete understanding of the host's mechanisms to modulate the proinflammatory effects of LPS will most likely help in the understanding of inflammation and infection and may lead to novel therapeutic intervention strategies.
INTRODUCTION
Lipopolysaccharide (LPS) is a prominent cell wall component of gram-negative bacteria and represents one of the most potent activators of the human innate immune system (36). A high sensitivity of the host for detecting LPS is mandatory in order to mount an early and rapid response against invading gram-negative bacteria. Research of the last decade has revealed the chemical composition and the requirements for biologic activity of LPS (2). While a certain set of lipid chains within the lipid A anchor and specific carbohydrates of the core region are absolutely required for activity, the long O chain consisting of up to 80 repeating oligosaccharide units varies greatly between different bacterial strains (35). Numerous reports have shown that the chain length of the carbohydrate unit of LPS that results in either a long (smooth [S]-type) or a short (rough [R]-type) O chain influences the host-pathogen interaction. Studies comparing R- and S-type LPS of Salmonella isolates have shown that colonization of the gut differs (30), as does resistance to complement-mediated killing (20). In addition, interaction with host neutrophils differs in bacteria carrying either R- or S-form LPS (31), which may be due to a distinct sensitivity to the activity of bactericidal/permeability-increasing protein (BPI), a neutrophilic bactericidal protein of the host (5).
Monocytes and macrophages are important regulatory and effector cells in innate immunity, and they express a specific receptor system for the detection of LPS represented by the combined actions of the membrane-bound isoform of CD14 (mCD14) with the central transmembrane signaling unit of Toll-like receptor 4 (TLR-4) and the accessory protein MD-2 (1, 29). Lower concentrations of LPS-binding protein (LBP), a protein closely related to BPI, have been documented to enhance the sensitivity for LPS by 3 to 4 orders of magnitude by catalyzing the binding of LPS to CD14 (14, 37), thus facilitating the release of numerous mediators, among them cytokines such as tumor necrosis factor alpha, interleukin-1, and interleukin-6 (34, 36).
Whereas early LPS recognition is crucial for the host to mount an immune reaction against invading gram-negative bacteria, regulatory mechanisms are important to prevent an overwhelming reaction that may lead to the development of pathological conditions and dysregulation of the immune system, as observed in sepsis and septic shock. Several mechanisms to blunt an immune reaction initiated by LPS have been described, including the induction of cellular and systemic states of LPS tolerance (24, 41, 45), cellular internalization and subsequent endolysosomal deacylation of major endotoxic forms of LPS (28), and the neutralizing transfer of LPS to plasma lipoproteins such as high-density lipoprotein (HDL) followed by intestinal excretion via the liver-bile duct pathway (27). Moreover, several serum and intracellular proteins that facilitate the neutralization of LPS have been described. For example, BPI has been shown to intercalate irreversibly into LPS aggregates to counteract the LBP-driven transport to CD14 (7). Furthermore, soluble CD14 (sCD14) and LBP, both involved in profound enhancement of LPS-induced signaling at low concentrations, have been shown to inhibit cellular activation by LPS at higher concentrations. Although the soluble isoform of CD14 is a prerequisite for the activation of CD14-negative cells by LPS (9, 13) and also participates in the activation of CD14-positive cells by LPS (14, 21), inhibitory effects at higher concentrations of sCD14 have been reported recently. LPS-induced activation of human monocytes and macrophages and LPS stimulation of human whole blood were both inhibited by the addition of sCD14 (17, 42).
LBP has been shown to catalyze the transfer of LPS to mCD14 and sCD14 at lower concentrations (13, 44). Analysis of LBP knockout mice has furthermore shown that LBP plays a pivotal role in immune responses against gram-negative bacteria (19, 26). Normal concentrations of LBP in human serum range between 5 and 10 μg/ml and are strongly increased during an acute-phase response by up to 200 μg/ml (3, 32, 46). It was recently shown that acute-phase concentrations of LBP are protective in a mouse model of bacteremia (25) and inhibit LPS-induced cellular activation of human monocytes (46). The molecular mechanisms for the inhibitory effects of both LPS recognition proteins (CD14 and LBP) are not completely understood. Soluble CD14 has been shown to transport membrane-bound LPS to serum lipoproteins such as HDL (23). In the circulation, LBP has been found to be predominantly associated with serum lipoproteins and to transfer LPS to HDL, low-density lipoprotein, very-low-density lipoprotein, and chylomicrons, resulting in the clearance of LPS from the bloodstream (39, 40, 43). Moreover, neutralization of LPS by LBP-catalyzed transfer to plasma lipoproteins has been shown to be strongly enhanced by the presence of sCD14 (42). In addition, it has recently been shown that high-density LBP (hd-LBP) dissociates cell-bound LPS from mCD14, thereby inhibiting LPS-induced cellular activation and suppressing the transfer of LPS from sCD14 to soluble forms of MD-2 (38). By employing gel permeation chromatography for the analysis of LBP and sCD14 interaction with meningococcal lipooligosaccharide, an increased concentration of LBP was found to be largely ineffective in the enhancement of sCD14-lipooligosaccharide complex formation and in the amplification of proinflammatory activation of human umbilical vein endothelial cells (12).
Here, we analyzed the molecular mechanisms of the anti-inflammatory effects of hd-LBP with respect to different chemical isoforms of LPS, one that contains a large carbohydrate unit (S type) and the other that does not (R type). High-dose LBP suppresses binding of both LPS chemotypes to CD14 and inhibits LPS-induced nuclear translocation of NF-B. Opposite to these common inhibitory effects of hd-LBP, we observed chemotype-specific differences in LBP-mediated major clearance pathways of LPS. The cellular uptake of R-type LPS was successively increased by hd-LBP both in the absence and presence of human serum and, furthermore, in a partially mCD14-independent fashion. In contrast, LBP-mediated cellular uptake of S-type LPS was found to be strictly dependent on mCD14 and decreased in the presence of serum or purified HDL. Our data indicate a preferential transfer of S-type LPS to plasma lipoproteins by acute-phase concentrations of LBP, which may have implications for the understanding of the pathophysiology of gram-negative systemic infections.
MATERIALS AND METHODS
Electrophoretic mobility shift assay (EMSA). CHO/CD14 cells stably transfected with CD14 (15) were seeded into 6-mm-diameter dishes (4 x 105 cells/dish) and allowed to adhere overnight in Ham’s F12 medium (PAA, Linz, Austria) containing 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 μg/ml). For stimulation, medium was replaced by fresh medium supplemented with 2% pooled human serum (Sigma, Munich, Germany). Cells were stimulated with 10 pg of Escherichia coli F515 R-type LPS (kindly provided by H. Brade, Leibniz-Research Center Borstel, Borstel, Germany)/ml or 500 pg of E. coli O111:B4 S-type LPS (Sigma-Aldrich, Deisenhofen, Germany)/ml for 1 h. Human recombinant LBP (kindly provided by Xoma Corp., Berkeley, Calif.) was added at final concentrations of 1, 3.3, and 10 μg/ml just before R- or S-form LPS was added at concentrations of 100 or 500 pg/ml, respectively. Nuclear extracts were prepared as described elsewhere previously (15). Experiments were analyzed with a PhosphorImager (Molecular Dynamics, Krefeld, Germany) and performed at least three times. One representative experiment is shown.
Native PhastGel and SDS electrophoresis. To analyze binding of R-type and S-type LPS to sCD14 and to investigate concentration-dependent effects of LBP on sCD14-LPS complex formation, an automated form of nondenaturing gel electrophoresis was performed by using the PhastSystem apparatus (Amersham Bioscience, Freiburg, Germany). Briefly, R-type LPS from E. coli F515 or S-type LPS from E. coli O111:B4 at a concentration of 0.025 or 0.113 μg/μl, respectively, was incubated in Dulbecco's phosphate-buffered saline (without magnesium or calcium; Life Technologies/GIBCO BRL, Eggenstein, Germany) in the absence or presence of 0.25 μg of recombinant human sCD14 (Biometec, Greifswald, Germany)/μl at 37°C for 10 min. For analysis of LBP effects, recombinant human LBP (Xoma Corp.) was added at final concentrations of 0.0005, 0.0025, 0.025, 0.125, and 0.25 μg/μl. Subsequently, the samples were chilled, and 4x native sample buffer (pH 7.6) was added. Native PhastGel electrophoresis was performed at 4°C by automated application of 1 μl of each sample to PhastGel Homogeneous-20 gels equipped with PhastGel native buffer strips. Electrophoretic separation was performed at a constant voltage of 400 V. Integrity of sCD14 and LBP proteins during the incubation procedure was verified by adjusting native samples to denaturing conditions by the addition of sodium dodecyl sulfate (SDS) to a final concentration of 2%, heating at 95°C for 5 min, and PhastGel SDS electrophoresis. Following electrophoresis, automated silver staining of native and SDS gels was performed according to the manufacturer's protocol.
Cellular surface binding and uptake of LPS. Cellular surface binding and uptake of LPS were assessed by flow cytometry employing BODIPY (Molecular Probes)-labeled R-type LPS from Salmonella enterica serovar Minnesota R595 and Oregon Green-labeled LPS from E. coli O111:B4 (kindly provided by E. Latz, University of Massachusetts, Worcester, Mass.). HDL was purchased from Calbiochem. The cells were harvested with cell dissociation solution (GIBCO BRL), and 5 x 105 cells were incubated with 100 ng of LPS/ml at 37°C in the presence or absence of 2% pooled human serum or in the presence of 300 μg of HDL/ml for 60 min. Recombinant LBP was added immediately before LPS was added to the cells. Cells were washed twice with phosphate-buffered saline and analyzed for LPS uptake by flow cytometry (FACStar; Becton Dickinson, Heidelberg, Germany). Experiments were performed at least twice, and one representative experiment is shown. The percentage of internalized LPS was determined by incubating cells with trypan blue for fluorescence quenching of cell surface-bound LPS (22). The proportion of cell surface-bound LPS was calculated by subtraction of the amount of internalized LPS from the total quantity of cell-associated LPS.
RESULTS
High concentrations of LBP (hd-LBP) inhibit LPS-induced signaling in CHO/CD14 cells. Inhibition of LPS-induced activation of CD14-positive cells by hd-LBP has been shown previously by employing rat alveolar macrophages and murine RAW267 cells (16, 25). Since we chose to use CD14-transfected CHO cells as a model system for the following experiments, we first aimed to confirm the inhibitory effect of hd-LBP towards CHO/CD14 cells by employing both R- and S-type LPS (Fig. 1). As S-type LPS is known to be a weaker activator, cells were stimulated with 10 pg of E. coli F515 R-type LPS/ml and 500 pg of E. coli O111:B4 S-form LPS/ml in medium containing 2% pooled human serum without or with the addition of recombinant human LBP for 60 min and assayed for NF-B translocation by EMSA. A significant NF-B translocation was observed in the absence of additional LBP, most likely due to LBP present in the serum. The addition of 1 ng of LBP/ml resulted in an increase of NF-B translocation after stimulation with both R-type and S-type LPS. Compared to lower LBP concentrations, the addition of 10 μg of LBP/ml, however, resulted in a profound decrease of NF-B translocation in CHO/CD14 cells induced by both LPS chemotypes.
HD LBP inhibits LPS binding to CD14. To investigate whether LPS-CD14 complex formation is influenced by the addition of LBP, we determined the binding of R- and S-type LPS to sCD14 by employing native gel electrophoresis (Fig. 2). In confirmation of previous reports, limited complex formation between R-type LPS and sCD14 in vitro was found to occur in the absence of LBP (Fig. 2A and B, lane 3). The addition of LBP at concentrations corresponding to LBP-to-LPS molar ratios of approximately 1:260 and 1:26, respectively, led to an increase of complex formation (Fig. 2A and B, lanes 5 and 6). In contrast, higher concentrations of LBP corresponding to LBP-to-LPS molar ratios of approximately 1:5 and 1:2.5, respectively, resulted in a profound reduction of LPS-sCD14 complex formation, which was completely absent when the highest concentration (22 pmol [1.32 μg/ml] [Fig. 2A and B, lanes 7 and 8]) of LBP was present. In comparison to binding of R-type LPS, binding of S-type LPS to sCD14 was found to be strictly dependent on the presence of LBP in the in vitro complexation assay (Fig. 2C). The LBP-to-S-type LPS molar ratios used here were approximately 1:1,340, 1:280, and 1:27 (Fig. 2C, lanes 4 to 6). Similar to the results obtained for R-type LPS, higher concentrations of LBP (corresponding to LBP-to-S-type LPS molar ratios of approximately 1:5 and 1:2.5) resulted in a strong reduction of LPS-sCD14 complex formation (Fig. 2C, lanes 7 and 8). Two different migrating complexes are visible in Fig. 2C (lanes 4 to 6). Since many S-form LPS preparations are heterogeneous, consisting of short LPS molecules containing molecules lacking O antigen or containing only one unit as well as long LPS molecules containing 30 to 40 O-antigen units, these two bands may very well reflect this heterogeneity. The integrity of the recombinant sCD14 and LBP proteins in the samples throughout the prior native analytical procedures was confirmed by SDS-polyacrylamide gel electrophoresis (PAGE) analysis performed in parallel (Fig. 2A to C, lower panels).
Cellular binding and internalization of R-type and S-type LPS are differently affected by hd-LBP. To investigate the effects of hd-LBP on CD14-dependent and CD14-independent cellular binding and/or internalization of LPS, we performed flow cytometry experiments by employing CD14-transfected CHO cells or vector control cells and fluorescence-labeled R- and S-form LPS preparations in the presence or absence of 2% human serum (Fig. 3). Cells were incubated with BODIPY-labeled R-type LPS from S. enterica serovar Minnesota R595 or Oregon Green-labeled S-type LPS from E. coli O111:B4 and assayed for LPS binding and internalization by flow cytometry. Signals induced by R-type LPS from S. enterica serovar Minnesota R595 by CHO/CD14 cells as well as by CHO/vector cells were successively elevated by increasing concentrations of LBP independent of the presence or absence of human serum (Fig. 3). Binding and internalization of S-type LPS from E. coli O111:B4, in contrast, were found to be LBP dependent and largely dependent on the presence of mCD14 (Fig. 3A and C). In the presence of 2% human serum, the addition of hd-LBP induced a decrease in signal in CHO/CD14 cells, which is in contrast to the observed behavior of R-type LPS (Fig. 3B). In the absence of CD14, S-type LPS, in contrast to R-type LPS, failed to bind significantly to the cells irrespective of the absence or presence of both LBP and serum (Fig. 3C and D).
It should be noted that the mCD14-expressing CHO cells exhibited higher overall counts representing stronger LPS uptake than vector control cells for both LPS chemotypes. To investigate whether the LBP concentration might affect the proportion of internalized LPS compared to cell surface-bound LPS, we performed quenching experiments by adding trypan blue to the cells before flow cytometry. By this approach, however, we failed to observe any significant difference in the ratios of internalized-to-surface-bound LPS in both CD14-positive and CD14-negative CHO cells (data not shown).
High-density lipoprotein decreases cellular binding and uptake of S-type LPS in the presence of hd-LBP. To investigate whether plasma lipoproteins such as HDL are the major serum acceptors of LPS responsible for the observed decrease of S-form LPS binding to CD14-positive cells at higher LBP concentrations, CHO/CD14 cells were incubated with fluorescence-labeled S-type LPS in the presence of purified HDL and increasing concentrations of LBP under serum-free conditions (Fig. 4). In addition, the LBP dose response profile for S-type LPS binding and uptake by CHO/CD14 cells in the presence of 2% human serum was reiterated. Increasing concentrations of recombinant LBP were found to induce a successive elevation of S-type LPS binding to CHO/CD14 cells in the absence of serum and no supplementation of plasma lipoproteins (Fig. 4). In contrast, in the presence of purified HDL, a comparable increase in cellular S-type LPS binding to CD14/CHO cells was observed only in the range of lower LBP concentrations up to 100 ng/ml, whereas higher concentrations of LBP induced a progressive reduction in S-form LPS binding to the CD14-transfected CHO cells in a manner analogous to that in the presence of 2% human serum (Fig. 4).
DISCUSSION
The pivotal role of low levels of LBP in mounting a rapid immune reaction against gram-negative bacteria has been shown in several prior studies (26, 37), especially by employing LBP knockout mice which are highly susceptible to infections by S. enterica serovar Typhimurium (8, 18, 19). Acute-phase levels of LBP in human sera are strongly elevated, reaching concentrations of up to 200 μg/ml, which is 20-fold higher than normal levels (4, 10, 11). The function of these elevated LBP levels has been associated with an inhibitory effect on LPS-induced cell activation and was thought to be a host mechanism to blunt an otherwise overwhelming immune reaction (25, 38, 46). Currently discussed mechanisms thought to be involved in this inhibitory activity are the ability of LBP to transport LPS into HDL particles (27, 39, 43) and, according to a more recent study, the detachment of LPS from mCD14 and the inhibition of LPS transfer from CD14 to MD-2 (38). In this report, the release of R-type LPS from mCD14 by increased concentrations of LBP has been found to be paralleled by a sustained cellular association of LBP-LPS complexes to membrane domains that are unable to induce signaling (38).
In our present study, we confirm the inhibitory effect of high concentrations of LBP in two different novel assays and present evidence for two distinct pathways for the two distinct chemotypes of LPS analyzed (summarized in Fig. 5). Our results show that in CD14-transfected CHO cells, hd-LBP exhibits an inhibitory effect on proinflammatory signaling induced by both R and S forms of LPS. The results confirm the dose response profiles obtained for rough-type LPS with rat alveolar macrophages and murine RAW267 cells (16, 25). In addition, native gel electrophoresis experiments, while confirming the enhancement of LPS-CD14 complex formation by LBP at lower concentrations, showed that hd-LBP induced a suppression of sCD14-LPS complexation for both R and S types of LPS. Binding of disaggregated LPS to membrane-bound or soluble isoforms of CD14 has been well documented to be a central step in the activation of host cells by minute quantities of LPS (13, 14, 37, 44) (Fig. 5). Our data show that the ability of LBP to catalyze LPS binding to CD14 at low concentrations is completely reversed at high concentrations, suggesting that the inhibitory effects of larger amounts of LBP are due to the inhibition of this central step in LPS signaling. A similar mechanism was postulated recently when it was found that high LBP concentrations are able to remove cell-bound LPS from CD14 (38).
Furthermore, we present evidence that major pathways of LPS clearance are strongly enhanced by hd-LBP. According to our results, distinct mechanisms of LPS clearance are employed by LBP for R- and S-type LPS. In spite of decreased binding of LPS to CD14 and profoundly reduced proinflammatory signaling, cellular surface adhesion and uptake of R-form LPS were found to be elevated in the presence of hd-LBP, partially independent of CD14 (Fig. 5). Therefore, hd-LBP apparently favors an mCD14-independent pathway of cellular uptake of R-type LPS. It should be mentioned, however, that the S-type LPS used here, like many S-type preparations, is most likely a heterogeneous mixture of both short and long LPS chains (as confirmed by our gel shift experiments [Fig. 2C]). Evidence for the existence of at least one additional cellular receptor for LBP-LPS complexes distinct from mCD14 mediating "silent" internalization of LBP-opsonized LPS aggregates has been reported previously (15, 38). Recently, it was postulated that scavenger receptors are involved in LBP-dependent LPS uptake. By employing human coronary endothelial cells stimulated by internalized LPS interacting with cytoplasmatic TLR-4, it was found that internalized LBP-LPS complexes, in contrast to sCD14-LPS complexes, were unable to stimulate cells (6). This result is in line with our finding that increased concentrations of LBP result in inhibition of sCD14 binding to LPS.
In contrast to R-type LPS and to our findings with assays employing S-type LPS under serum-free conditions, the cellular binding and uptake of S-form LPS in the presence of serum were found to be strongly decreased at higher concentrations of LBP. The latter result indicates that hd-LBP facilitates a transfer of S-form LPS to plasma components and reduces the adherence of this LPS chemotype to CD14-positive cells, resulting in the inhibition of signal transduction (Fig. 5). In our assay for cellular binding and uptake of S-form LPS offering both HDL- and CD14-expressing cells as binding sites for LPS, we show for the first time that preferential binding of S-form LPS to HDL compared to cell-exposed mCD14 depends on the concentration of LBP present. In our test system, LBP concentrations of up to 100 ng/ml transferred LPS predominantly to mCD14-expressing cells, whereas concentrations higher than 1 μg/ml preferentially induced LPS binding to HDL. In addition, we found no difference in the ratio between surface-bound and internalized LPS for R-form LPS at different concentrations of LBP, which is in line with recently published results of others (38). It should be mentioned here that other soluble mediators, such as MD-2, may also influence binding, uptake, and signaling of LPS intensively. This needs to be addressed in future studies.
A ternary complex mechanism of catalytic transfer of LPS molecules from aggregate ultrastructures to CD14 by LBP has been postulated (44). Our observation of profound inhibitory effects of higher LBP concentrations in the formation of sCD14-LPS complexes may be explained by an opsonization density specification of the model. At lower concentrations of LBP, the opsonization density of LBP molecules on the surface of LPS aggregates would be low and would facilitate the access of CD14 molecules via their N-terminally located LPS recognition site. In contrast, for higher LBP concentrations, the opsonization density of LBP molecules on the LPS aggregates would be considerably higher, and a sterical hindrance of the access of CD14 molecules to the tightly LBP-coated surface of the LPS aggregate may thus be an explanation for the inhibition of sCD14-LPS complex formation. Another explanation would be that LPS complexed to hd-LBP may bind only transiently to CD14 before being internalized by a CD14-independent pathway, not allowing the assembly of the LPS receptor complex containing CD14, TLR-4, and other associated molecules as described previously by Pfeiffer et al. (33). This would explain the enhancement of silent LPS uptake by mCD14 at hd-LBP concentrations shown in Fig. 3 (Fig. 5).
Taken together, our data confirm and expand previous studies postulating a dual role of LBP and CD14 in the initiation of proinflammatory signaling and clearance or neutralization of LPS. Signal induction is apparently inhibited by hd-LBP due to interference with LPS-CD14 complex formation, whereas major LPS clearance pathways are increasingly favored at higher concentrations of LBP. Moreover, we show for the first time that the beneficial effects of higher LBP concentrations differ with regard to the LPS chemotype, with S-form LPS being predominantly transported to plasma lipoproteins, whereas R-form LPS is taken up mainly by cells via mCD14-dependent and mCD14-independent pathways. Our findings indicate that the elevated concentrations of LBP during the acute-phase response provide a shift in the balance of the LPS-directed dual actions of the protein disfavoring proinflammatory cell activation and favoring clearance of LPS via major cellular and systemic pathways in a chemotype-selective manner.
ACKNOWLEDGMENTS
We acknowledge the excellent technical assistance of Frnzi Creutzburg (Berlin), Nina Grohmann, and Stefanie Adam (Borstel).
This work was supported by the Bundesministerium für Bildung und Forschung (BMBF, CAPNetz, project C5), the Deutsche Forschungsgemeinschaft (DFG, Innate Immunity, project Schr 726, 1-2), Clinique La Prairie Research (to C.A.), DFG STA 609 1-2 (to C.S.), and SFB 633-03, project A7 (to R.R.S.).
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Leibniz Research Center Borstel, Center for Medicine and Bioscience, Borstel
Department of Anesthesiology, University of Lübeck, Lübeck, Germany
ABSTRACT
The chain length of bacterial lipopolysaccharide (LPS) is a crucial factor for host-pathogen interaction during bacterial infection. While rough (R)-type and smooth (S)-type LPSs have been shown to differ in their ability to interact with the bactericidal/permeability-increasing protein, little is known about the differential mode of interaction with the acute-phase reactant LPS-binding protein (LBP). At lower concentrations, LBP catalyzes the binding of LPS to CD14 and enhances LPS-induced cellular activation via Toll-like receptor 4. In humans, however, concentrations of LBP in serum increase during an acute-phase response, and these LBP concentrations exhibit inhibitory effects in terms of cellular activation. The mechanisms of inhibition of LPS effects by LBP are not completely understood. Here, we report that human high-dose LBP (hd-LBP) suppresses binding of both R-type and S-type LPS to CD14 and inhibits LPS-induced nuclear translocation of NF-B, although cellular uptake of R-type LPS was found to be increased by hd-LBP. In contrast, we found that hd-LBP enhanced the binding and uptake of S-type LPS only under serum-free conditions, whereas in the presence of serum, hd-LBP inhibited cellular binding and uptake. This inhibitory effect of serum could be mimicked by the addition of purified high-density lipoprotein (HDL) to serum-free medium, indicating an LBP-mediated transfer of preferentially S-type LPS to plasma lipoproteins such as HDL. A complete understanding of the host's mechanisms to modulate the proinflammatory effects of LPS will most likely help in the understanding of inflammation and infection and may lead to novel therapeutic intervention strategies.
INTRODUCTION
Lipopolysaccharide (LPS) is a prominent cell wall component of gram-negative bacteria and represents one of the most potent activators of the human innate immune system (36). A high sensitivity of the host for detecting LPS is mandatory in order to mount an early and rapid response against invading gram-negative bacteria. Research of the last decade has revealed the chemical composition and the requirements for biologic activity of LPS (2). While a certain set of lipid chains within the lipid A anchor and specific carbohydrates of the core region are absolutely required for activity, the long O chain consisting of up to 80 repeating oligosaccharide units varies greatly between different bacterial strains (35). Numerous reports have shown that the chain length of the carbohydrate unit of LPS that results in either a long (smooth [S]-type) or a short (rough [R]-type) O chain influences the host-pathogen interaction. Studies comparing R- and S-type LPS of Salmonella isolates have shown that colonization of the gut differs (30), as does resistance to complement-mediated killing (20). In addition, interaction with host neutrophils differs in bacteria carrying either R- or S-form LPS (31), which may be due to a distinct sensitivity to the activity of bactericidal/permeability-increasing protein (BPI), a neutrophilic bactericidal protein of the host (5).
Monocytes and macrophages are important regulatory and effector cells in innate immunity, and they express a specific receptor system for the detection of LPS represented by the combined actions of the membrane-bound isoform of CD14 (mCD14) with the central transmembrane signaling unit of Toll-like receptor 4 (TLR-4) and the accessory protein MD-2 (1, 29). Lower concentrations of LPS-binding protein (LBP), a protein closely related to BPI, have been documented to enhance the sensitivity for LPS by 3 to 4 orders of magnitude by catalyzing the binding of LPS to CD14 (14, 37), thus facilitating the release of numerous mediators, among them cytokines such as tumor necrosis factor alpha, interleukin-1, and interleukin-6 (34, 36).
Whereas early LPS recognition is crucial for the host to mount an immune reaction against invading gram-negative bacteria, regulatory mechanisms are important to prevent an overwhelming reaction that may lead to the development of pathological conditions and dysregulation of the immune system, as observed in sepsis and septic shock. Several mechanisms to blunt an immune reaction initiated by LPS have been described, including the induction of cellular and systemic states of LPS tolerance (24, 41, 45), cellular internalization and subsequent endolysosomal deacylation of major endotoxic forms of LPS (28), and the neutralizing transfer of LPS to plasma lipoproteins such as high-density lipoprotein (HDL) followed by intestinal excretion via the liver-bile duct pathway (27). Moreover, several serum and intracellular proteins that facilitate the neutralization of LPS have been described. For example, BPI has been shown to intercalate irreversibly into LPS aggregates to counteract the LBP-driven transport to CD14 (7). Furthermore, soluble CD14 (sCD14) and LBP, both involved in profound enhancement of LPS-induced signaling at low concentrations, have been shown to inhibit cellular activation by LPS at higher concentrations. Although the soluble isoform of CD14 is a prerequisite for the activation of CD14-negative cells by LPS (9, 13) and also participates in the activation of CD14-positive cells by LPS (14, 21), inhibitory effects at higher concentrations of sCD14 have been reported recently. LPS-induced activation of human monocytes and macrophages and LPS stimulation of human whole blood were both inhibited by the addition of sCD14 (17, 42).
LBP has been shown to catalyze the transfer of LPS to mCD14 and sCD14 at lower concentrations (13, 44). Analysis of LBP knockout mice has furthermore shown that LBP plays a pivotal role in immune responses against gram-negative bacteria (19, 26). Normal concentrations of LBP in human serum range between 5 and 10 μg/ml and are strongly increased during an acute-phase response by up to 200 μg/ml (3, 32, 46). It was recently shown that acute-phase concentrations of LBP are protective in a mouse model of bacteremia (25) and inhibit LPS-induced cellular activation of human monocytes (46). The molecular mechanisms for the inhibitory effects of both LPS recognition proteins (CD14 and LBP) are not completely understood. Soluble CD14 has been shown to transport membrane-bound LPS to serum lipoproteins such as HDL (23). In the circulation, LBP has been found to be predominantly associated with serum lipoproteins and to transfer LPS to HDL, low-density lipoprotein, very-low-density lipoprotein, and chylomicrons, resulting in the clearance of LPS from the bloodstream (39, 40, 43). Moreover, neutralization of LPS by LBP-catalyzed transfer to plasma lipoproteins has been shown to be strongly enhanced by the presence of sCD14 (42). In addition, it has recently been shown that high-density LBP (hd-LBP) dissociates cell-bound LPS from mCD14, thereby inhibiting LPS-induced cellular activation and suppressing the transfer of LPS from sCD14 to soluble forms of MD-2 (38). By employing gel permeation chromatography for the analysis of LBP and sCD14 interaction with meningococcal lipooligosaccharide, an increased concentration of LBP was found to be largely ineffective in the enhancement of sCD14-lipooligosaccharide complex formation and in the amplification of proinflammatory activation of human umbilical vein endothelial cells (12).
Here, we analyzed the molecular mechanisms of the anti-inflammatory effects of hd-LBP with respect to different chemical isoforms of LPS, one that contains a large carbohydrate unit (S type) and the other that does not (R type). High-dose LBP suppresses binding of both LPS chemotypes to CD14 and inhibits LPS-induced nuclear translocation of NF-B. Opposite to these common inhibitory effects of hd-LBP, we observed chemotype-specific differences in LBP-mediated major clearance pathways of LPS. The cellular uptake of R-type LPS was successively increased by hd-LBP both in the absence and presence of human serum and, furthermore, in a partially mCD14-independent fashion. In contrast, LBP-mediated cellular uptake of S-type LPS was found to be strictly dependent on mCD14 and decreased in the presence of serum or purified HDL. Our data indicate a preferential transfer of S-type LPS to plasma lipoproteins by acute-phase concentrations of LBP, which may have implications for the understanding of the pathophysiology of gram-negative systemic infections.
MATERIALS AND METHODS
Electrophoretic mobility shift assay (EMSA). CHO/CD14 cells stably transfected with CD14 (15) were seeded into 6-mm-diameter dishes (4 x 105 cells/dish) and allowed to adhere overnight in Ham’s F12 medium (PAA, Linz, Austria) containing 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 μg/ml). For stimulation, medium was replaced by fresh medium supplemented with 2% pooled human serum (Sigma, Munich, Germany). Cells were stimulated with 10 pg of Escherichia coli F515 R-type LPS (kindly provided by H. Brade, Leibniz-Research Center Borstel, Borstel, Germany)/ml or 500 pg of E. coli O111:B4 S-type LPS (Sigma-Aldrich, Deisenhofen, Germany)/ml for 1 h. Human recombinant LBP (kindly provided by Xoma Corp., Berkeley, Calif.) was added at final concentrations of 1, 3.3, and 10 μg/ml just before R- or S-form LPS was added at concentrations of 100 or 500 pg/ml, respectively. Nuclear extracts were prepared as described elsewhere previously (15). Experiments were analyzed with a PhosphorImager (Molecular Dynamics, Krefeld, Germany) and performed at least three times. One representative experiment is shown.
Native PhastGel and SDS electrophoresis. To analyze binding of R-type and S-type LPS to sCD14 and to investigate concentration-dependent effects of LBP on sCD14-LPS complex formation, an automated form of nondenaturing gel electrophoresis was performed by using the PhastSystem apparatus (Amersham Bioscience, Freiburg, Germany). Briefly, R-type LPS from E. coli F515 or S-type LPS from E. coli O111:B4 at a concentration of 0.025 or 0.113 μg/μl, respectively, was incubated in Dulbecco's phosphate-buffered saline (without magnesium or calcium; Life Technologies/GIBCO BRL, Eggenstein, Germany) in the absence or presence of 0.25 μg of recombinant human sCD14 (Biometec, Greifswald, Germany)/μl at 37°C for 10 min. For analysis of LBP effects, recombinant human LBP (Xoma Corp.) was added at final concentrations of 0.0005, 0.0025, 0.025, 0.125, and 0.25 μg/μl. Subsequently, the samples were chilled, and 4x native sample buffer (pH 7.6) was added. Native PhastGel electrophoresis was performed at 4°C by automated application of 1 μl of each sample to PhastGel Homogeneous-20 gels equipped with PhastGel native buffer strips. Electrophoretic separation was performed at a constant voltage of 400 V. Integrity of sCD14 and LBP proteins during the incubation procedure was verified by adjusting native samples to denaturing conditions by the addition of sodium dodecyl sulfate (SDS) to a final concentration of 2%, heating at 95°C for 5 min, and PhastGel SDS electrophoresis. Following electrophoresis, automated silver staining of native and SDS gels was performed according to the manufacturer's protocol.
Cellular surface binding and uptake of LPS. Cellular surface binding and uptake of LPS were assessed by flow cytometry employing BODIPY (Molecular Probes)-labeled R-type LPS from Salmonella enterica serovar Minnesota R595 and Oregon Green-labeled LPS from E. coli O111:B4 (kindly provided by E. Latz, University of Massachusetts, Worcester, Mass.). HDL was purchased from Calbiochem. The cells were harvested with cell dissociation solution (GIBCO BRL), and 5 x 105 cells were incubated with 100 ng of LPS/ml at 37°C in the presence or absence of 2% pooled human serum or in the presence of 300 μg of HDL/ml for 60 min. Recombinant LBP was added immediately before LPS was added to the cells. Cells were washed twice with phosphate-buffered saline and analyzed for LPS uptake by flow cytometry (FACStar; Becton Dickinson, Heidelberg, Germany). Experiments were performed at least twice, and one representative experiment is shown. The percentage of internalized LPS was determined by incubating cells with trypan blue for fluorescence quenching of cell surface-bound LPS (22). The proportion of cell surface-bound LPS was calculated by subtraction of the amount of internalized LPS from the total quantity of cell-associated LPS.
RESULTS
High concentrations of LBP (hd-LBP) inhibit LPS-induced signaling in CHO/CD14 cells. Inhibition of LPS-induced activation of CD14-positive cells by hd-LBP has been shown previously by employing rat alveolar macrophages and murine RAW267 cells (16, 25). Since we chose to use CD14-transfected CHO cells as a model system for the following experiments, we first aimed to confirm the inhibitory effect of hd-LBP towards CHO/CD14 cells by employing both R- and S-type LPS (Fig. 1). As S-type LPS is known to be a weaker activator, cells were stimulated with 10 pg of E. coli F515 R-type LPS/ml and 500 pg of E. coli O111:B4 S-form LPS/ml in medium containing 2% pooled human serum without or with the addition of recombinant human LBP for 60 min and assayed for NF-B translocation by EMSA. A significant NF-B translocation was observed in the absence of additional LBP, most likely due to LBP present in the serum. The addition of 1 ng of LBP/ml resulted in an increase of NF-B translocation after stimulation with both R-type and S-type LPS. Compared to lower LBP concentrations, the addition of 10 μg of LBP/ml, however, resulted in a profound decrease of NF-B translocation in CHO/CD14 cells induced by both LPS chemotypes.
HD LBP inhibits LPS binding to CD14. To investigate whether LPS-CD14 complex formation is influenced by the addition of LBP, we determined the binding of R- and S-type LPS to sCD14 by employing native gel electrophoresis (Fig. 2). In confirmation of previous reports, limited complex formation between R-type LPS and sCD14 in vitro was found to occur in the absence of LBP (Fig. 2A and B, lane 3). The addition of LBP at concentrations corresponding to LBP-to-LPS molar ratios of approximately 1:260 and 1:26, respectively, led to an increase of complex formation (Fig. 2A and B, lanes 5 and 6). In contrast, higher concentrations of LBP corresponding to LBP-to-LPS molar ratios of approximately 1:5 and 1:2.5, respectively, resulted in a profound reduction of LPS-sCD14 complex formation, which was completely absent when the highest concentration (22 pmol [1.32 μg/ml] [Fig. 2A and B, lanes 7 and 8]) of LBP was present. In comparison to binding of R-type LPS, binding of S-type LPS to sCD14 was found to be strictly dependent on the presence of LBP in the in vitro complexation assay (Fig. 2C). The LBP-to-S-type LPS molar ratios used here were approximately 1:1,340, 1:280, and 1:27 (Fig. 2C, lanes 4 to 6). Similar to the results obtained for R-type LPS, higher concentrations of LBP (corresponding to LBP-to-S-type LPS molar ratios of approximately 1:5 and 1:2.5) resulted in a strong reduction of LPS-sCD14 complex formation (Fig. 2C, lanes 7 and 8). Two different migrating complexes are visible in Fig. 2C (lanes 4 to 6). Since many S-form LPS preparations are heterogeneous, consisting of short LPS molecules containing molecules lacking O antigen or containing only one unit as well as long LPS molecules containing 30 to 40 O-antigen units, these two bands may very well reflect this heterogeneity. The integrity of the recombinant sCD14 and LBP proteins in the samples throughout the prior native analytical procedures was confirmed by SDS-polyacrylamide gel electrophoresis (PAGE) analysis performed in parallel (Fig. 2A to C, lower panels).
Cellular binding and internalization of R-type and S-type LPS are differently affected by hd-LBP. To investigate the effects of hd-LBP on CD14-dependent and CD14-independent cellular binding and/or internalization of LPS, we performed flow cytometry experiments by employing CD14-transfected CHO cells or vector control cells and fluorescence-labeled R- and S-form LPS preparations in the presence or absence of 2% human serum (Fig. 3). Cells were incubated with BODIPY-labeled R-type LPS from S. enterica serovar Minnesota R595 or Oregon Green-labeled S-type LPS from E. coli O111:B4 and assayed for LPS binding and internalization by flow cytometry. Signals induced by R-type LPS from S. enterica serovar Minnesota R595 by CHO/CD14 cells as well as by CHO/vector cells were successively elevated by increasing concentrations of LBP independent of the presence or absence of human serum (Fig. 3). Binding and internalization of S-type LPS from E. coli O111:B4, in contrast, were found to be LBP dependent and largely dependent on the presence of mCD14 (Fig. 3A and C). In the presence of 2% human serum, the addition of hd-LBP induced a decrease in signal in CHO/CD14 cells, which is in contrast to the observed behavior of R-type LPS (Fig. 3B). In the absence of CD14, S-type LPS, in contrast to R-type LPS, failed to bind significantly to the cells irrespective of the absence or presence of both LBP and serum (Fig. 3C and D).
It should be noted that the mCD14-expressing CHO cells exhibited higher overall counts representing stronger LPS uptake than vector control cells for both LPS chemotypes. To investigate whether the LBP concentration might affect the proportion of internalized LPS compared to cell surface-bound LPS, we performed quenching experiments by adding trypan blue to the cells before flow cytometry. By this approach, however, we failed to observe any significant difference in the ratios of internalized-to-surface-bound LPS in both CD14-positive and CD14-negative CHO cells (data not shown).
High-density lipoprotein decreases cellular binding and uptake of S-type LPS in the presence of hd-LBP. To investigate whether plasma lipoproteins such as HDL are the major serum acceptors of LPS responsible for the observed decrease of S-form LPS binding to CD14-positive cells at higher LBP concentrations, CHO/CD14 cells were incubated with fluorescence-labeled S-type LPS in the presence of purified HDL and increasing concentrations of LBP under serum-free conditions (Fig. 4). In addition, the LBP dose response profile for S-type LPS binding and uptake by CHO/CD14 cells in the presence of 2% human serum was reiterated. Increasing concentrations of recombinant LBP were found to induce a successive elevation of S-type LPS binding to CHO/CD14 cells in the absence of serum and no supplementation of plasma lipoproteins (Fig. 4). In contrast, in the presence of purified HDL, a comparable increase in cellular S-type LPS binding to CD14/CHO cells was observed only in the range of lower LBP concentrations up to 100 ng/ml, whereas higher concentrations of LBP induced a progressive reduction in S-form LPS binding to the CD14-transfected CHO cells in a manner analogous to that in the presence of 2% human serum (Fig. 4).
DISCUSSION
The pivotal role of low levels of LBP in mounting a rapid immune reaction against gram-negative bacteria has been shown in several prior studies (26, 37), especially by employing LBP knockout mice which are highly susceptible to infections by S. enterica serovar Typhimurium (8, 18, 19). Acute-phase levels of LBP in human sera are strongly elevated, reaching concentrations of up to 200 μg/ml, which is 20-fold higher than normal levels (4, 10, 11). The function of these elevated LBP levels has been associated with an inhibitory effect on LPS-induced cell activation and was thought to be a host mechanism to blunt an otherwise overwhelming immune reaction (25, 38, 46). Currently discussed mechanisms thought to be involved in this inhibitory activity are the ability of LBP to transport LPS into HDL particles (27, 39, 43) and, according to a more recent study, the detachment of LPS from mCD14 and the inhibition of LPS transfer from CD14 to MD-2 (38). In this report, the release of R-type LPS from mCD14 by increased concentrations of LBP has been found to be paralleled by a sustained cellular association of LBP-LPS complexes to membrane domains that are unable to induce signaling (38).
In our present study, we confirm the inhibitory effect of high concentrations of LBP in two different novel assays and present evidence for two distinct pathways for the two distinct chemotypes of LPS analyzed (summarized in Fig. 5). Our results show that in CD14-transfected CHO cells, hd-LBP exhibits an inhibitory effect on proinflammatory signaling induced by both R and S forms of LPS. The results confirm the dose response profiles obtained for rough-type LPS with rat alveolar macrophages and murine RAW267 cells (16, 25). In addition, native gel electrophoresis experiments, while confirming the enhancement of LPS-CD14 complex formation by LBP at lower concentrations, showed that hd-LBP induced a suppression of sCD14-LPS complexation for both R and S types of LPS. Binding of disaggregated LPS to membrane-bound or soluble isoforms of CD14 has been well documented to be a central step in the activation of host cells by minute quantities of LPS (13, 14, 37, 44) (Fig. 5). Our data show that the ability of LBP to catalyze LPS binding to CD14 at low concentrations is completely reversed at high concentrations, suggesting that the inhibitory effects of larger amounts of LBP are due to the inhibition of this central step in LPS signaling. A similar mechanism was postulated recently when it was found that high LBP concentrations are able to remove cell-bound LPS from CD14 (38).
Furthermore, we present evidence that major pathways of LPS clearance are strongly enhanced by hd-LBP. According to our results, distinct mechanisms of LPS clearance are employed by LBP for R- and S-type LPS. In spite of decreased binding of LPS to CD14 and profoundly reduced proinflammatory signaling, cellular surface adhesion and uptake of R-form LPS were found to be elevated in the presence of hd-LBP, partially independent of CD14 (Fig. 5). Therefore, hd-LBP apparently favors an mCD14-independent pathway of cellular uptake of R-type LPS. It should be mentioned, however, that the S-type LPS used here, like many S-type preparations, is most likely a heterogeneous mixture of both short and long LPS chains (as confirmed by our gel shift experiments [Fig. 2C]). Evidence for the existence of at least one additional cellular receptor for LBP-LPS complexes distinct from mCD14 mediating "silent" internalization of LBP-opsonized LPS aggregates has been reported previously (15, 38). Recently, it was postulated that scavenger receptors are involved in LBP-dependent LPS uptake. By employing human coronary endothelial cells stimulated by internalized LPS interacting with cytoplasmatic TLR-4, it was found that internalized LBP-LPS complexes, in contrast to sCD14-LPS complexes, were unable to stimulate cells (6). This result is in line with our finding that increased concentrations of LBP result in inhibition of sCD14 binding to LPS.
In contrast to R-type LPS and to our findings with assays employing S-type LPS under serum-free conditions, the cellular binding and uptake of S-form LPS in the presence of serum were found to be strongly decreased at higher concentrations of LBP. The latter result indicates that hd-LBP facilitates a transfer of S-form LPS to plasma components and reduces the adherence of this LPS chemotype to CD14-positive cells, resulting in the inhibition of signal transduction (Fig. 5). In our assay for cellular binding and uptake of S-form LPS offering both HDL- and CD14-expressing cells as binding sites for LPS, we show for the first time that preferential binding of S-form LPS to HDL compared to cell-exposed mCD14 depends on the concentration of LBP present. In our test system, LBP concentrations of up to 100 ng/ml transferred LPS predominantly to mCD14-expressing cells, whereas concentrations higher than 1 μg/ml preferentially induced LPS binding to HDL. In addition, we found no difference in the ratio between surface-bound and internalized LPS for R-form LPS at different concentrations of LBP, which is in line with recently published results of others (38). It should be mentioned here that other soluble mediators, such as MD-2, may also influence binding, uptake, and signaling of LPS intensively. This needs to be addressed in future studies.
A ternary complex mechanism of catalytic transfer of LPS molecules from aggregate ultrastructures to CD14 by LBP has been postulated (44). Our observation of profound inhibitory effects of higher LBP concentrations in the formation of sCD14-LPS complexes may be explained by an opsonization density specification of the model. At lower concentrations of LBP, the opsonization density of LBP molecules on the surface of LPS aggregates would be low and would facilitate the access of CD14 molecules via their N-terminally located LPS recognition site. In contrast, for higher LBP concentrations, the opsonization density of LBP molecules on the LPS aggregates would be considerably higher, and a sterical hindrance of the access of CD14 molecules to the tightly LBP-coated surface of the LPS aggregate may thus be an explanation for the inhibition of sCD14-LPS complex formation. Another explanation would be that LPS complexed to hd-LBP may bind only transiently to CD14 before being internalized by a CD14-independent pathway, not allowing the assembly of the LPS receptor complex containing CD14, TLR-4, and other associated molecules as described previously by Pfeiffer et al. (33). This would explain the enhancement of silent LPS uptake by mCD14 at hd-LBP concentrations shown in Fig. 3 (Fig. 5).
Taken together, our data confirm and expand previous studies postulating a dual role of LBP and CD14 in the initiation of proinflammatory signaling and clearance or neutralization of LPS. Signal induction is apparently inhibited by hd-LBP due to interference with LPS-CD14 complex formation, whereas major LPS clearance pathways are increasingly favored at higher concentrations of LBP. Moreover, we show for the first time that the beneficial effects of higher LBP concentrations differ with regard to the LPS chemotype, with S-form LPS being predominantly transported to plasma lipoproteins, whereas R-form LPS is taken up mainly by cells via mCD14-dependent and mCD14-independent pathways. Our findings indicate that the elevated concentrations of LBP during the acute-phase response provide a shift in the balance of the LPS-directed dual actions of the protein disfavoring proinflammatory cell activation and favoring clearance of LPS via major cellular and systemic pathways in a chemotype-selective manner.
ACKNOWLEDGMENTS
We acknowledge the excellent technical assistance of Frnzi Creutzburg (Berlin), Nina Grohmann, and Stefanie Adam (Borstel).
This work was supported by the Bundesministerium für Bildung und Forschung (BMBF, CAPNetz, project C5), the Deutsche Forschungsgemeinschaft (DFG, Innate Immunity, project Schr 726, 1-2), Clinique La Prairie Research (to C.A.), DFG STA 609 1-2 (to C.S.), and SFB 633-03, project A7 (to R.R.S.).
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