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Lipoteichoic Acid Synergizes with Glycosphingolipids To Potently Stimulate Secretion of Interleukin-6 from Human Blood Cells

ABSTRACT In the present study, we found that lipoteichoic acid (LTA) synergizes with glycosphingolipids to stimulate human blood cells to secrete cytokines. We employed globoside, kerasin, and lactosylceramide as representative neutral glycosphingolipids and mixed gangliosides GM2 and GM3 as representative acidic glycosphingolipids. LTA and the glycosphingolipids enhanced cytokine secretion by human whole blood, peripheral blood mononuclear cells, and purified monocytes in a dose-dependent manner. The level of synergy ranged up to ∼10-fold greater than the additive stimulation caused by LTA and glycosphingolipid alone. The greatest synergy was observed with GM3. We also found that LTA synergizes with the synthetic bacterial lipopeptide mimic Pam3CysK4. In contrast, the glycosphingolipids suppressed the stimulation caused by Pam3CysK4. The stimulation of human cells requires the simultaneous presence of LTA and the glycosphingolipids and probably requires their physical interactions, as shown by dot blotting and nondenaturing polyacrylamide gel electrophoresis experiments. We hypothesize that the enhanced stimulation is due to heterooligomers that form between LTA and glycosphingolipids at the subcritical micelle concentrations used in these experiments. Previous studies showed that LTA also synergizes with hemoglobin. The data taken together suggest that LTA may be a pathogen-associated molecular pattern, although its full activity requires the presence of a synergistic partner(s).

It has been established that septic shock caused by gramnegative pathogens is primarily due to the ability of lipopolysaccharide (LPS) to trigger the excessive secretion of proinflammatory cytokines by macrophages (1,10). In contrast, the bacterial component(s) responsible for septic shock caused by gram-positive bacteria has not been confirmed. As a result, a number of constituents of gram-positive bacteria have been studied for their abilities to cause or contribute to shock. These include proteinaceous pyrogenic toxins, which act in vitro as superantigens; peptidoglycan oligomers (28); and lipoteichoic acid (LTA), a bacterial cell membrane component that has structural similarities to LPS. Over the last several years, highly purified LTA free of LPS contamination has been shown to induce monocyte and macrophage secretion of proinflammatory cytokines (7, 14, 18-20, 30, 31), and it shares many other functional features with LPS (12,32). However, a major difference between LPS and LTA is that the injection of purified LPS alone can result in shock in an animal model, whereas the injection of purified LTA alone has never been shown to induce shock. Even when one examines the levels at which LPS and LTA induce cytokine release from macrophages/monocytes in vitro, it is clear that LPS is a more potent immunostimulant than purified LTA.
Recently, our laboratories and others have begun to define molecules of both human and bacterial origin that synergize with LTA and greatly enhance its ability to stimulate cells of the immune system. Muramyl dipeptide, the minimal structural unit of peptidoglycan that retains immunostimulating activity, synergizes with LTA to enhance the induction of inflammatory cytokines in human monocytes (34). In addition, we have shown that hemoglobin synergizes with both weakly active phenol-extracted LTA and the more highly active butanolextracted LTA to markedly enhance cytokine secretion by both human blood monocytes and mouse macrophages (7,14). Synergism with hemoglobin in activating macrophages was noted for LTAs from a variety of different gram-positive bacteria (14) and also for LPS (4,25,26). The mechanisms for the synergy between LTA and these other molecules have not yet been defined at the molecular level. However, we have shown that LTA and hemoglobin physically interact, so one possible hypothesis is that LTA forms a complex with hemoglobin in a way that facilitates the presentation of LTA to the macrophage Toll-like receptors (TLRs). One of the reasons for the notable difference in potency between LTA and LPS may be related to the structural differences between their lipid moieties and their interactions with specific TLRs (TLR2 and TLR4, respectively) (3,11). Even when one examines only the interaction between different species of LPS and the LPS receptor, TLR4, it is clear that differences in the lipid moiety (e.g., the numbers of fatty acid chains) can result in markedly different levels of cytokine secretion. LPS containing less than six fatty acids (e.g., penta-or tetra-acylated LPS) exhibited less activity or was even nonactive, and in one case, signaling was even through a different receptor (1,6). Interestingly, it has been shown that the mixing of the poorly active penta-and tetra-acylated LPS molecules at concentrations below their critical micelle concentrations (CMCs) before addition of the mixture to cells results in increased cytokine secretion (5). This result raises the possibility that the heterooligomers that formed between these two LPS structures were better able to interact with their receptor, subsequently resulting in enhanced cytokine release.
We show here that when LTA is combined with various glycosphingolipids and incubated with either human whole blood or isolated cells, cytokine release is markedly enhanced. We also show that LTA and some of the glycolipids physically interact and that enhanced cytokine release requires that both components be incubated simultaneously with the blood cells. On the basis of these observations, we hypothesize that mixtures of LTA and various glycosphingolipids at concentrations below their CMCs may form heterooligomers that are better able to interact with their receptors.
(The work performed by Shiri Meron-Sudai is in partial fulfillment of a Ph.D. degree from the Sackler Faculty of Medicine, Tel-Aviv University.)

MATERIALS AND METHODS
Reagents. Butanol-extracted Streptococcus pyogenes LTA was purified as described previously (14). The chain length of polyglycerolphosphate and the percent D-alanine content were analyzed by nuclear magnetic resonance (NMR) and were found to be 23 and 65%, respectively (14). The maximum level of LPS contamination of the LTA preparations was Յ0.004% (e.g., 400 pg/ml in 10 g/ml LTA, as determined by the Limulus amoebocyte lysate assay) (14). Ten g/ml polymyxin B (Sigma-Aldrich Co., St. Louis, MO) was added to all reaction mixtures to exclude stimulatory effects due to any residual LPS. The glycosphingolipids used in this study were purchased from Matreya LLC. Synthetic tripalmitoylated bacterial lipopeptide Pam3CysK4 was purchased from InvivoGen (San Diego, CA).
Preparation of human PBMCs and monocytes. Peripheral blood mononuclear cells (PBMCs) were obtained from heparinized blood with Ficoll-Hypaque (Amersham Biosciences), as described previously (17). Briefly, Ficoll-Hypaque was added to whole blood diluted 1:2 in Dulbecco's phosphate-buffered saline (PBS), and the mixture was centrifuged at 700 ϫ g for 30 min at room temperature (RT). The PBMC layer was collected, and the cells were washed three times with cold Dulbecco's PBS. Monocytes were further isolated by allowing them to adhere to plastic. The PBMCs were resuspended in RPMI 1640 (supplemented with penicillin at 100 g/ml and streptomycin at 100 U/ml) with 2% fetal calf serum (FCS) at a concentration of 5 ϫ 10 6 cells/ml. The cells were incubated for 1 h at 37°C in tissue culture flasks to allow the monocytes to adhere to the flasks. Nonadherent cells were removed by washing the flasks three times with Earle's balanced salt solution. RPMI 1640 supplemented with 10% FCS was added to the adherent monocytes, and the cells were incubated overnight at 37°C, during which time the monocytes detached (17). Monocytes purified in this way were collected, washed, and counted. PBMCs or monocytes, at 1.5 ϫ 10 5 to 2 ϫ 10 5 cells/200 l in polypropylene tubes, were diluted in RPMI 1640 supplemented with 5% FCS or donor plasma and exposed to stimuli by overnight incubation.
Whole-blood assays. Heparinized blood obtained from volunteers was diluted 1:5 in saline, as described by Morath et al. (19,20). Diluted blood was incubated with LTA, glycosphingolipids, or mixtures of both components that had been preincubated at 37°C for 1 h. After overnight incubation (i.e., 19 to 22 h), the supernatants were removed and assayed for the levels of interleukin-6 (IL-6) that had been secreted.
IL-6 measurements. Medium from the samples was collected and was assayed for the amount of IL-6 secreted by enzyme-linked immunosorbent assay, according to the manufacturer's protocol (Pierce Endogen; Pierce Biotechnology, Inc.). This method is sensitive down to 300 pg/ml IL-6.
Nondenaturing Western blot analysis. LTA was diluted to 10 g/ml in PBS and incubated in the presence or absence of 2 or 20 g/ml globoside or mixed gangliosides for 30 min at RT. Samples were resolved on a 12% native polyacrylamide gel and transferred to nitrocellulose membranes. The membrane was blocked, washed, and incubated with a 1/5,000 dilution of anti-LTA polyclonal antibody, as described previously (14). The membrane was then washed and incubated with a 1:10,000 dilution of peroxidase-conjugated goat anti-rabbit immunoglobulin G (ICN/Cappel). After the membrane was washed, immunoreactive species were detected with enhanced chemiluminescence Western blotting detection reagents (Amersham Biosciences).
Dot blot assay. Glycosphingolipids (10 to 15 g/0.5 l) and polymyxin B (15 g/0.5 l), as a negative control, were applied to aluminum-packed silica gel plates (high-performance thin-layer chromatography plates; Merck, Darmstadt, Germany) and dried at 50°C for 20 min. The plates were blocked with a blocking solution containing 1% (wt/vol) bovine serum albumin and 0.1% (vol/vol) Tween 20 in PBS for 1 h at RT with gentle agitation. The plates were washed twice with PBS and incubated either with LTA at 40 g/ml in PBS or with PBS for 1.5 h at RT with gentle agitation. The plates were washed with PBS four times for 1 min each time and were then incubated with anti-LTA rabbit serum diluted 1:10,000 in blocking solution for 1 h at RT with agitation. The plates were washed as described above and incubated with goat anti-rabbit horseradish peroxidase diluted 1:40,000 in blocking solution (Jackson ImmunoResearch Laboratories, Inc.) for 45 min at RT with agitation. The plates were washed again, and the immunoreactive species were detected on the dot blots with the enhanced chemiluminescence detection reagent (Amersham).
Statistical analysis. The results are presented as the means Ϯ standard deviations for triplicate or quadruplicate cultures in a single representative experiment of n experiments performed. Statistical significance was assessed by using a one-tailed Student's t test. A P value of Ͻ0.05 was considered significant.

RESULTS
The ceramide-containing glycolipids (i.e., sphingolipids) are grouped according to the nature of the polar head group at position 1 of the ceramide backbone. One group, the glycosphingolipids, are further divided into two groups on the basis of the sugar composition of the head group. The neutral glycosphingolipids contain uncharged sugar residues, while the acidic glycosphingolipids, also known as gangliosides, contain charged sugar residues, such as sialic acid. In the present study, we employed galactosylceramide and lactosylceramide, which are the most predominant of the neutral glycosphingolipids, and globoside. As representatives of the acidic glycosphingolipids, we employed ganglioside GM 2 or GM 3 individually or as a mixture. The gangliosides vary by virtue of the overall size of their oligosaccharide chains, but they all contain only one sialic acid residue per molecule.
The addition of 15 M of various neutral or acidic eukaryotic glycolipids markedly enhanced the level of IL-6 secretion by PBMCs in response to 0.062 M LTA (Fig. 1). It should be noted that the magnitude of stimulation differed among the various sphingolipids, with the greatest level of synergy being found when LTA and GM 3 were incubated with the PBMCs. This is of particular interest, since GM 3 is the most prominent glycosphingolipid found in normal human serum (8,22). By comparison, the level of IL-6 secretion in response to 0.062 M LTA plus GM 3 was equivalent to the level of IL-6 secretion in response to 50 ng LPS (114.53 Ϯ 22 ng/ml and 149.23 Ϯ 8.78 ng/ml, respectively). Results similar to those obtained with PBMCs were obtained in a separate set of experiments with LTA, GM 3 , and purified monocytes (Fig. 2). Human monocytes were also incubated with globoside mixed with LTA at concentrations ranging from 0 to 0.496 M (which correspond to 0 to 2 g/ml). This resulted in molar ratios of LTA to globoside that ranged from 1:480 to 1:60 (Fig. 3). The percent increases caused by the mixtures were the greatest at the lower ratios of LTA to globoside, and at a molar ratio of 1:60, there was no statistically significant increase in the level of stimulation.

Interactions of LTA and sphingolipids.
Having determined the ability of glycosphingolipids to potentiate the activity of LTA, we next sought to examine whether this potentiation was the result of the separate activity of each component on the cells or was a shared activity. For this purpose, either the monocytes were incubated for 1 h with 0.062 M LTA, fol-lowed by washing and overnight incubation with 15 M globoside, or they were incubated for 1 h with 15 M globoside, followed by washing and overnight incubation with 0.062 M LTA. As a positive control, the monocytes were also incubated overnight with a mixture of both components (Fig. 4). The synergistic effects were achieved only when both components were simultaneously incubated with the cells, suggesting the possibility that a physical interaction between the two components may be required in order for synergy to occur.
To determine whether the synergistic effect of the sphingolipid-LTA mixtures could be due to physical interactions between the two molecules, we performed two types of tests for interactions, nondenatured polyacrylamide gel electrophoresis followed by Western blotting and dot blot assays. Although LTA migrates on a sodium dodecyl sulfate-polyacrylamide gel at ϳ8 to 10 kDa, it migrates much more slowly and, because of its microheterogeneous nature, in a much more diffuse manner on a nondenaturing gel (Fig. 5). This migration pattern may be explained by the ability of LTA to form micelles in aqueous solution (33). When LTA is incubated with two different ceramide-containing glycolipids before it is run on a nondenaturing acrylamide gel, the migration of LTA is affected in different ways, depending upon whether the glycolipid is globoside or mixed gangliosides. At roughly equimolar ratios, neither of the glycolipids had a dramatic effect on LTA migration. However, when the ratio of the host glycolipid was increased ϳ10-fold, the use of globoside resulted in LTA migration that was slower and less diffuse, while the use of mixed gangliosides resulted in LTA migration that was more rapid (Fig. 5). The interaction with hemoglobin has also been shown to change the migration of LTA in a nondenaturing gel (14). LTA was also incubated with gelatin in the previous study to control for the nonspecific effect of the protein on LTA migration, and no alteration of migration was observed. In the dot blot assay, we spotted three glycosphingolipids on silica gel thin-layer chromatography plates, incubated them with LTA, and detected LTA binding by using an anti-LTA antiserum (Fig. 6). LTA bound strongly to lactosylceramide and kerasin, less so to GM 2 , and not at all to the negative control, polymyxin B. The negative control plate that was not incubated with LTA showed no binding of primary or secondary antibodies to kerasin, GM 2 , or polymyxin B. There appeared to be a weak cross-reaction with lactosylceramide, but this may have been due to the low titers of antibody against ceramides that can be found in normal mammalian serum (15,21). Nevertheless, there was such a marked difference between the two blots that we are convinced that it successfully makes the point that LTA interacts with lactosylceramide.
Synergy of LTA preparations and glycosphingolipids is not due to lipoprotein contamination. Previous studies have suggested that the contamination of LTA preparations with small amounts of lipoproteins might be responsible for the ability of such LTA preparations to stimulate macrophages to secrete cytokines (13). Although NMR and amino acid analysis did not reveal any protein contamination in our LTA preparations (14), we examined the possibility that a trace amount of a lipoprotein or a lipopeptide other than LTA could be responsible for synergizing with glycolipids and enhancing monocytestimulating activity. We first examined whether the addition of very low concentrations of Pam3CysK4 to LTA would result in enhanced cytokine release (Fig. 7). The results showed that, indeed, low concentrations of Pam3CysK4 do synergize with LTA. However, when the same concentrations of Pam3CysK4 were added to the various glycosphingolipids, it was not able to synergize with any of the glycosphingolipids. Combinations of glycolipids with low concentrations of Pam3CysK4 give only an additive and not a synergistic effect, and this occurred only with globoside (Fig. 8). Furthermore, at higher concentrations of Pam3CysK4, glycolipids inhibited the secretion of IL-6 by the PBMCs. Taken together, these results suggest that the potentiation of stimulation by our LTA preparation and glycosphingolipids is not due to a contaminating Pam3CysK4-like lipopeptide.
Potentiation of LTA activity by glycosphingolipids in human whole blood. We next wished to compare the abilities of different glycolipids to synergize with LTA to stimulate monocytes in whole blood. In these experiments, we used LTA at 0.062 M (0.5 g/ml), because at this concentration LTA alone stimulated the cells only weakly. The concentrations of glycosphingolipids that we added to the cells ranged from the lowest to the highest of the known glycosphingolipid concentrations in normal human serum (8,22), and the molar ratio between LTA and glycosphingolipids was kept at ϳ1:120. Several neutral and acidic glycosphingolipids in a human whole-blood system were examined, and all of them showed an increase in IL-6 secretion when they were mixed with LTA ( Fig. 9). There were no differences in the levels of stimulation when 7.5 M glycosphingolipid (the low concentration of glycosphingolipids in normal human serum) or 15 M glycosphingolipid (the high concentration level of glycosphingolipids in normal human serum) was added. Similar results were obtained when dealanylated LTA was used (data not shown). Pam3CysK4 did not synergize with glycosphingolipids or LTA when whole blood from any of the four different human blood donors was used (data not shown).
In a separate set of experiments, we also assayed the culture supernatants for IL-1␤ secretion. In these experiments, similar synergies were seen when LTA was combined with ceramide containing glycolipids, with GM 3 again giving the highest level of enhancement, in which the level of IL-1␤ secreted was roughly 10-fold higher than the level secreted in response to 50 ng/ml LPS (Fig. 10). In future experiments, it will be of interest to more completely define the profiles of the cytokines secreted and to assess their effects in vivo.

DISCUSSION
LTA is one of the cell wall components of gram-positive bacteria long considered to be a possible contributor to septic shock caused by gram-positive organisms, but its activity in stimulating cells of the immune system to secrete proinflam- VOL. 15,2008 SECRETION OF IL-6 FROM HUMAN BLOOD CELLS matory cytokines has frequently been found to be much less potent than that of LPS. However, previous work from our laboratories showed that butanol-extracted LTA can form complexes and synergize with hemoglobin to potently increase the amount of IL-6 and tumor necrosis factor alpha secreted by macrophages and monocytes in a whole-blood preparation (14). Thus, we have hypothesized that LTA may be a pathogen-associated molecular pattern. However, because the full activity of LTA requires the presence of synergistic partners, it may not be accurate to call it a bona fide pathogen-associated molecular pattern. We investigated the effects that combinations of glycosphingolipids and LTA might have on the stimulation of immune cells of the blood system for several reasons. Many ceramidecontaining molecules, including the glycosphingolipids tested in this study, are normally present in the serum of uninfected individuals at micromolar concentrations (8,22). Sepsis has been shown to result in increased concentrations of several different ceramide species in serum (9), and interestingly, ␣-galactosylceramide (i.e., kerasin) was recently shown to sensitize mice to the development of lethal endotoxic shock (16).
We found that the use of mixtures of LTA and eukaryotic amphiphiles (i.e., ceramide-containing glycolipids, or glycosphingolipids) resulted in a much stronger cytokine response by monocytes than the use of LTA or glycolipid alone. This synergism apparently requires the use of the two amphiphiles at an optimal molar ratio (Fig. 3). The mechanism underlying this synergism is unclear at present. One possible hypothesis is that oligomerization with certain amphiphiles, as shown here for glycosphingolipids, may result in the formation of heterooligomers (e.g., dimers and trimers) between LTA and other amphiphilic molecules that more efficiently bind to a TLR, which may result in an enhanced response. This hypothesis is based on the following results. First, synergy requires the simultaneous presence of both amphiphiles. Second, the two amphiphiles physically interact with each other, as shown by their interaction on dot blots and by nondenaturing polyacrylamide gel electrophoresis followed by Western blotting. Furthermore, all of these experiments were carried out at concentrations below their CMCs. At the CMC, amphiphiles tend to form dimers, tetramers, and oligomers until at the CMC they form micelles (23,24,27). It has been shown that when mixtures of different lipids are used, the oligomers and micelles always contain mixed lipids, with the degree of oligomerization depending upon the concentration and the nature of the amphiphiles (23). This phenomenon may explain the recent observation in which two different LPS species expressed by Burkholderia mallei that are individually inactive due to suboptimal numbers of acyl chains (i.e., tetra-and penta-acylated molecules) became active when they were mixed together (5). It should be noted, however, that the specific effects of different glycosphingolipids on LTA migration in native gels do not necessarily correlate directly with macrophage activation, nor do they necessarily indicate that the mechanisms are the same or different.
At present, there are basically two schools of thought with regard to the proinflammatory properties of LTA: (i) LTA is a gram-positive bacterial component that activates cells of the immune system (e.g., monocytes and macrophages) to secrete tumor necrosis factor alpha, IL-6, and other proinflammatory molecules; or (ii) a copurifying lipoprotein is the component of LTA preparations that stimulates cytokine secretion by monocytes and macrophages and LTA is essentially inactive (13). Since we had shown that our highly purified LTA preparations could synergize with glycolipids to enhance cytokine secretion from monocytes, we wanted to determine if the stimulation of monocytes with the lipopeptide Pam3CysK4 and glycolipids would also result in synergy. To the contrary, we found not only that was there no synergy but also that glycosphingolipids could actually inhibit the activity of Pam3CysK4. Furthermore, we showed that in a similar range of molar ratios, Pam3CysK4 did synergize with LTA and resulted in enhanced cytokine secretion. This result may also stem from the formation of heterooligomers between these two amphiphiles.
In summary, we currently favor the concept that LTA will exhibit potent stimulatory activity under certain conditions, such as when LTA is released from the pathogen (2) in the presence of hemoglobin (which is released from erythrocytes by a hemolytic toxin, such as streptolysin O), muramyl dipeptide (which is released from bacterial cell walls by lysozyme and amidases as part of the host response [29]), or glycosphingolipids (whose levels are known to be increased during sepsis [9]). It is also quite possible that there are other bacterial or host macromolecules that could synergize with LTA to increase proinflammatory cytokine secretion. Future experiments should focus on the synergistic effects of mixtures of LTA and other compounds.