ABSTRACT
As a consequence of blood-borne bacterial sepsis, endotoxin or lipopolysaccharide (LPS) from the cell walls of gram-negative bacteria can trigger an acute inflammatory response, leading to a series of pathological events and often resulting in death. To block this inflammatory response to endotoxin, a novel lipid A analogue, E5531, was designed and synthesized as an LPS antagonist, and its biological properties were examined in vitro and in vivo. In murine peritoneal macrophages, E5531 inhibited the release of tumor necrosis factor alpha (TNF-α) by Escherichia coli LPS with a 50% inhibitory concentration (IC50) of 2.2 nM, while E5531 elicited no significant increases in TNF-α on its own. In support of a mechanism consistent with antagonism of binding to a cell surface receptor for LPS, E5531 inhibited equilibrium binding of radioiodinated LPS ([125I]2-(r-azidosalicylamido)-1, 3′-dithiopropionate-LPS) to mouse macrophages with an IC50 of 0.50 μM. E5531 inhibited LPS-induced increases in TNF-α in vivo when it was coinjected with LPS into C57BL/6 mice primed with Mycobacterium bovis bacillus Calmette-Guérin (BCG). In this model, the efficacy of E5531 was inversely correlated to the LPS challenge dose, consistent with a competitive antagonist-like mechanism of action. Blockade of the inflammatory response by E5531 could further be demonstrated in other in vivo models: E5531 protected BCG-primed mice from LPS-induced lethality in a dose-dependent manner and suppressed LPS-induced hepatic injury in Propionibacterium acnes-primed or galactosamine-sensitized mice. These results argue that the novel synthetic lipid A analogue E5531 can antagonize the action of LPS in in vitro and suppress the pathological effects of LPS in vivo in mice.
As a major constituent of the outer membranes of infectious gram-negative bacteria (32), lipopolysaccharide (LPS) triggers many pathophysiological events. Most of the toxic properties of LPS can be attributed to the hydrophobic fatty-acylated disaccharide lipid A portion of the molecule (8). In both in vitro and in vivo systems, LPS-sensitive cells such as monocytes and macrophages can be stimulated by LPS or lipid A to release cytokines such as tumor necrosis factor alpha (TNF-α), interleukin 1 (IL-1), IL-6, and other mediators. In vivo, release of pathophysiological concentrations of these cytokines and cellular mediators can lead to tissue damage. For this reason, a variety of therapeutic interventions have been undertaken to block the action of endotoxin and the sequelae of cellular activation by endotoxin. These approaches include administration of anti-lipid A antibodies, anticytokine antibodies, or soluble neutralizing receptors for cytokines or antagonism of cytokine receptors (1, 29, 35, 36).
For maximal therapeutic effect, it may be necessary to block the action of endotoxin at the primary event of receptor activation in order to block LPS-induced cellular activation and the subsequent release of the broad spectrum of potentially deleterious cytokines and cellular mediators that lead to septic shock. Although the mechanism of cellular activation by LPS has yet to be fully elucidated, several possible cell surface receptors for LPS have been proposed, and their mechanism of activation is being studied (16, 19, 23). Part of our understanding of cellular activation comes from the study of receptor antagonists. It has been reported that the LPSs from two nonenteric, nonpathogenic bacteria, Rhodobacter capsulatusand Rhodobacter sphaeroides, are poor agonists, and the lipid A’s derived from these LPSs can antagonize more pathogenic LPSs (14, 17, 22, 28). These findings imply that certain lipid A derivatives can inhibit the acute inflammatory response to LPS and may be useful for treatment of LPS-induced shock or mortality. Although several reports have demonstrated a role for LPS antagonists, some inconsistencies in extrapolating in vitro studies to in vivo models still exist (5), possibly due to species differences in the animal model used (6) or the confounding presence of weak agonistic activity (24).
E5531 is a chemically synthesized lipid A analog that, while demonstrating potent antagonistic activity against LPS, possesses no intrinsic agonistic activity. We have previously reported that E5531 antagonizes LPS-induced cytokine production in a variety of in vitro assays (12). In this study, we further describe the in vitro and in vivo antagonism of LPS by E5531 in several models of LPS-mediated cellular activation, hepatotoxicity, and lethality.
MATERIALS AND METHODS
Animals.Pathogen-free male C57BL/6 mice were obtained from SLC Inc. (Shizuoka, Japan) and Jackson Laboratories (Bar Harbor, Maine), and pathogen-free male BALB/c mice were obtained from Charles River Japan Inc. (Atsugi, Japan).
The animals were housed at a constant temperature of 23 ± 1°C in 55% ± 5% humidity with a 12-h light and 12-h dark cycle and were provided with pellet food and water ad libitum.
Reagents.E5531, 6-O-{2-deoxy-6-O-methyl-4-O-phosphono-3-O-[(R)-3-Z-dodec-5-enoyloxy decyl]-2-[3-oxo-tetradecanoylamido]-β-d-glucopyranosyl}-2-deoxy-3-O-(R)-3-hydroxydecyl-2-[3-oxo-tetradecanoylamido]-1-O-phosphono-α-d-glucopyranose tetrasodium salt, was synthesized at the Eisai Research Institute of Boston (Andover, Mass.). LPS from Escherichia coli (a phenol extract of serotype O111:B4) was obtained from Sigma Chemical Co. (St. Louis, Mo.) or from List Biological Inc., (Campbell, Calif.). SyntheticE. coli lipid A (serotype O111:B4; catalog no. LA-15-PP) was purchased from Daiichi Chemical Co., (Tokyo, Japan).d-(+)-Galactosamine HCl (GalN), peroxidase (type XII), and bovine serum albumin were purchased from Sigma Chemical Co. Recombinant murine TNF-α, rabbit anti-mouse TNF-α polyclonal antiserum, and a mouse TNF-α enzyme-linked immunosorbent assay (ELISA) kit (Factor-Test mTNF-α; Genzyme Corp., Boston, Mass.) were purchased from Genzyme Corp. (Cambridge, Mass.). N-Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Pierce, Rockford, Ill.), o-phenylenediamine (Wako Pure Chemical Industries Ltd., Osaka, Japan), and other commercially available reagents were used for this study.
[125I]ASD-LPS was prepared by the method of Wollenweber and Morrison (33) with E. coli (O111:B4) LPS (List Biochemicals, Campbell, Calif.) and was stored at −20°C. The LPS concentration of [125I]ASD-LPS was estimated by Endospecy (Seikagaku Kogyo Co., Ltd., Tokyo, Japan), and a specific radioactivity of ∼4.1 μCi/μg was obtained.
For in vitro studies, E5531 and LPS were solubilized in sterile water (LyphoMed Inc., Rosemont, Ill.), and lipid A was solubilized in 0.025% aqueous triethylamine solution. All solutions were sonicated with an ultrasonicator (VW-380; Heat Systems-Ultrasonics Inc., Farmingdale, N.Y.) for 1 to 2 min immediately before each experiment, and serial dilutions were made in Ca2+- and Mg2+-free Hanks balanced salt solution from Sigma Chemical Co. For in vivo studies, E5531 was dissolved in pyrogen-free 5% glucose solution (Otsuka Pharmaceutical Co., Tokushima, Japan) and sonicated just before use. This solution was serially diluted in 5% glucose. LPS was dissolved in pyrogen-free 0.9% saline (Otsuka Pharmaceutical Co.). Live Mycobacterium bovis bacillus Calmette-Guérin (BCG) was obtained from Japan BCG Inc. (Tokyo, Japan). It was suspended in pyrogen-free 0.9% saline just prior to use at a concentration of 10 mg/ml.Propionibacterium acnes ATCC 6918 was suspended in pyrogen-free 0.9% saline at 20 mg/ml just prior to the experiment.
Preparation of murine peritoneal macrophages.Peritoneal macrophages were isolated from mice treated intraperitoneally with 2 mg of a cell wall preparation from heat-killedM. bovis BCG (Ribi Immunochem Research Inc., Hamilton, Mont.) in 200 μl of pyrogen-free 0.9% saline. Three to 6 days later, the animals were killed with CO2 gas and macrophages were isolated by aseptic lavage with ice-cold RPMI 1640 supplemented with penicillin (80 U/ml), streptomycin (100 μg/ml), 1 mM sodium pyruvate (Sigma Chemical Co.), 2% heat-inactivated fetal bovine serum (JRH Biosciences, Lenexa, Kans.), and 10 U of heparin per ml. The harvested cells were centrifuged and the pellet was resuspended with 1 ml of erythrocyte lysing buffer (Sigma Chemical Co.), followed by the addition of 40 ml of serum-free RPMI 1640 containing the antibiotics specified above. After three washes by centrifugation, the final cell pellet was diluted to a concentration of 2 × 106 cells/ml in RPMI 1640 containing 10% fetal bovine serum and was allowed to adhere to 24-well plastic tissue culture plates (Falcon Primaria; Becton Dickinson, Lincoln Park, N.J.). After 3 h at 37°C in 5% CO2, the nonadherent cells were removed from the plate by washing twice with serum-free RPMI 1640.
In vitro analysis of E5531 in mouse peritoneal macrophages.Analysis of inhibition of LPS-induced release of TNF-α by E5531 was performed with cultures of mouse peritoneal macrophages in RPMI 1640 supplemented with 10% fetal bovine serum. E5531 was added to achieve the various concentrations, followed by the addition of LPS at a final concentration of 10 ng/ml. After incubation for 3 h at 37°C, the cultures were clarified by centrifugation (2,000 × gfor 5 min at 4°C), and the resulting supernatants were stored at −80°C until the assay for TNF-α was performed.
Analysis of LPS binding to peritoneal macrophages.Adherent macrophages (2 × 106 cells/well) in 24-well plastic tissue culture plates were washed twice with 1 ml of prewarmed (37°C) serum-free RPMI 1640, followed by the addition of 200 μl of medium supplemented with 2% fetal bovine serum, 25 μl of inhibitor in RPMI 1640 containing 2% fetal bovine serum, and finally, 25 μl of [125I]ASD-LPS (∼60 ng of 4.1 μCi/μg). After incubation for 1 h at 37°C in 5% CO2, the wells were washed three times with 1.0 ml of 50 mM Tris buffer (pH 7.4) containing 150 mM NaCl and 2 mg of bovine serum albumin per ml. After solubilization of the cells with 1 ml of 0.1 N NaOH, 950 μl of each sample was analyzed for 125I with an autogamma counter (Gamma 5500B; Beckman Instruments, Fullerton, Calif.). Nonspecific binding was evaluated by the addition of 1 mg of unlabelled LPS per ml with the [125I]ASD-LPS. Specific binding was calculated by subtracting the nonspecific binding from the total binding. These binding studies used 2% fetal calf serum, which we have found to be optimal for LPS binding to mouse macrophages. At lower serum concentrations, binding is reduced, presumably due to decreased concentrations of LPS binding protein. At higher concentrations (10% or more), some inhibition of binding is seen, likely due to interference with other factors in serum.
Analysis of plasma TNF-α increases and mortality induced by LPS in BCG-primed mice.For BCG priming, 0.2 ml of a 10-mg/ml BCG suspension was intravenously (i.v.) injected into the tail veins of C57BL/6 mice (ages, 5 to 6 weeks), and the primed mice were used for experiments 10 to 14 days later (30). To examine the effect of E5531 on TNF-α production, 0.4 ml of a pyrogen-free 5% glucose solution containing the various amounts of LPS and E5531 was i.v. injected into the tail vein. One hour later, mice were anesthetized with ether and 30 μl of blood was drawn from the retro-orbital vein and placed in a heparinized hematocrit tube. Plasma TNF-α levels were determined by ELISA.
To examine the protective effect of E5531 against LPS-induced mortality in BCG-primed mice, 6 μg of LPS mixed with the indicated amount of E5531 was i.v. injected and mortality was monitored for 44 h.
Protective effect of E5531 on LPS-induced hepatic injury inP. acnes-primed or GalN-sensitized mice.BALB/c mice (age, 8 weeks) were primed 7 days prior to use by i.v. injection of 100 μl of 20 mg of P. acnes per ml in pyrogen-free 0.9% saline into the tail vein. Hepatitis was induced by LPS in primed mice by i.v. injection of 0.4 ml of pyrogen-free 5% glucose solution containing 1 μg of LPS mixed with the various amounts of E5531. Alternatively, mice were sensitized with GalN as reported previously (10, 20). The tail veins of male BALB/c mice were injected with 0.3 ml of pyrogen-free 5% glucose containing 20 mg of GalN (pH 7.0) and 1.0 ng of LPS mixed with 0, 10, 30, 100, or 300 ng of E5531. Additional mice were given GalN alone. Twenty-four hours after injection, mice were anesthetized with ether and approximately 0.5 ml of blood was drawn from the abdominal aorta and placed in tubes containing heparin, plasma was prepared, and l-alanine aminotransferase (ALT; EC 2.6.1.2) activities were determined by standard spectrophotometric methods (11) with an latrozyme TA-LQ Kit (Iatron Laboratories Inc., Tokyo, Japan).
Inhibition of LPS-mediated induction of TNF-α release in primary cultures of mouse peritoneal macrophages. E5531 was added to cultures of mouse peritoneal macrophages at the indicated concentrations, immediately followed by the addition of 10 ng of LPS per ml. After a 3-h incubation at 37°C, supernatants were assayed for TNF-α as described in the Materials and Methods section. Each point and error bar represent the mean and standard error of the mean obtained for three separate cultures in a single experiment and represents three experiments.
Analysis of TNF-α.TNF-α was measured by an ELISA with anti-mouse TNF-α polyclonal antiserum purified by affinity chromatography (protein A-Sepharose [MAPS-II; Bio-Rad, Richmond, Calif.]). Fab′ fragments were generated as described previously (31) and conjugated to peroxidase withN-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate.
Ninety-six-well flat-bottom plates (model no. 3590; Costar, Cambridge, Mass.) were coated overnight at 4°C with 100 μl of 10 μg of anti-mouse TNF-α immunoglobulin G per ml dissolved in 0.1 M sodium carbonate-bicarbonate buffer (pH 9.6). The plates were then washed and blocked with 10 mM phosphate buffer (NaH2PO4-Na2HPO4; pH 7.4) containing 1% bovine serum albumin in 0.15 M NaCl. Plasma samples (100 μl) diluted with blocking buffer containing 0.05% Tween 20 were then added. The plate was allowed to stand overnight at 4°C, washed three times as described above, incubated with 100 μl of anti-mouse TNF-α Fab′ peroxidase at 37°C for 2 h, washed as described above, and then incubated with 100 μl of 0.1 M citrate-Na2HPO4 buffer (pH 5.0) containing 0.017% H2O2 and 0.05%o-phenylenediamine for 30 min at room temperature. After termination by the addition of 50 μl of 2 N H2SO4, the optical density was measured at 490 nm. Immunoreactive TNF-α levels in the test plasma were calculated from a standard curve produced with recombinant mouse TNF-α. In order to detect low levels of TNF-α, a sensitive bioassay with L-P3 cells, a subline of L-929 cells, was also used (13, 34). The detection limit of the assay method was 0.4 ng/ml. Supernatants from in vitro analyses were tested for TNF-α with a mouse TNF-α ELISA kit (Factor-Test mTNF-α; Genzyme Corp.).
Statistical analysis.Results are expressed as the mean ± standard error of the mean. For in vitro studies, the concentration of E5531 that inhibited 50% of the LPS-induced increases in TNF-α concentrations (the 50% inhibitory concentration [IC50]) was calculated by a log-linear interpolation from the two points that span the 50% value. In the studies with animals, statistically significant differences between the control and treated groups were determined by Student’s t test or a one-way analysis of variance with Fisher’s protected least significant difference procedure (25). In lethality studies, the statistically significant differences between the control group and the treated groups were determined by χ2 test. Differences withP values of less than 0.05 were considered statistically significant. The 50% effective doses (ED50s) of E5531 were calculated by a nonlinear least-squares method.
RESULTS
Inhibition of LPS-mediated release of TNF-α in murine peritoneal macrophages.Unstimulated primary cultures of mouse peritoneal macrophages produced no measurable TNF-α. However, 3 h of incubation with LPS (10 ng/ml) triggered the release of 1,973 ± 120 pg of TNF-α per ml (n = 3). As shown in Fig. 1, E5531 inhibited this LPS-triggered TNF-α release at concentrations of 0.1 to 100 nM in a dose-dependent manner (IC50 = 2.2 nM), with complete inhibition observed at concentrations greater than 100 nM. These results indicate that E5531 is a potent antagonist of LPS-mediated activation of murine macrophages.
Antagonistic effect of E5531 on LPS-induced plasma TNF-α increases in mice.In vivo antagonism of LPS by E5531 was determined by analysis of the ability of E5531 to inhibit LPS-induced plasma TNF-α increases in BCG-primed mice. In preliminary experiments, it was determined that peak plasma TNF-α levels were observed 1 h after LPS administration (data not shown). As shown in Table 1, LPS doses of greater than 0.1 μg induced dose-dependent increases in plasma TNF-α levels. E5531 inhibited this response, and the degree of inhibition was inversely correlated to the amount of LPS used as an agonist. Thus, 3 μg of E5531 completely inhibited the LPS response with 0.1 μg of LPS, but inhibition by 3 μg of E5531 was reduced to 50% when mice were challenged with three times as much LPS (0.3 μg). These results indicate that the efficacy of E5531 is related to the challenge dose of LPS used. E5531 alone, even at 100 μg/mouse, did not elicit detectable amounts of TNF-α in plasma (less than 0.4 ng/ml), indicating that it lacks agonistic activity in BCG-primed mice (data not shown).
Effect of E5531 on TNF-α production induced by different doses of LPS in BCG-primed mice
Antagonism of LPS-induced lethality in BCG-primed mice.The i.v. administration of 6 μg of LPS to BCG-primed mice resulted in 90% lethality within 16 h (Fig. 2). No further deaths occurred in this group during a further 20 h of observation. Administration of E5531 along with the LPS challenge protected them from death in a dose-dependent manner, with complete protection achieved with doses of 30 and 100 μg. Compared to the results for the controls, the protection provided by E5531 was statistically significant at doses of greater than 3 μg/animal.
E5531 suppression of LPS-induced mortality in BCG-primed mice. BCG-primed mice (10 animals per group) were i.v. injected with 6 μg of LPS in 0.4 ml of 5% glucose only (•) or along with 3 μg (○), 10 μg (■), 30 μg (□), or 100 μg (▴) of E5531, and mortality was assessed at the indicated times. ∗, P< 0.05; ∗∗, P < 0.01 versus control group (χ2 test; n=10). The control was 5% glucose solution plus LPS.
Effect of E5531 on LPS-induced hepatic injury in P. acnes-primed or GalN-sensitized mice.When P. acnes-primed mice were dosed i.v. with LPS, plasma ALT values increased more than 14-fold compared to those in mice receiving vehicle only, indicating that LPS injured the livers of these animals (Table2).
Inhibitory effect of E5531 on LPS-mediated hepatotoxicitya
E5531 suppressed this LPS-induced hepatic injury in a dose-dependent manner. E5531 (100 μg) inhibited LPS-induced increases in plasma ALT activity by 76%. Furthermore, this protection from hepatic injury was reflected in a trend toward decreased mortality. Animals treated with E5531 only had no measurable liver damage; plasma ALT levels in P. acnes-primed mice treated with 100 μg of E5531 alone were the same as those in P. acnes-primed mice (Table 2).
In another murine hepatic injury model in which GalN is used as a sensitizing agent, simultaneous administration of 1 ng of LPS with 20 mg of GalN induced a 26-fold increase in plasma ALT activity (Table 2). E5531 inhibited this LPS-induced increase in plasma ALT activity in a dose-dependent fashion, with statistically significant suppression by E5531 at doses of 0.03, 0.1, and 0.3 μg/animal. Under these conditions, the ED50 for inhibition of LPS-induced ALT release by E5531 was approximately 15 ng/animal. Plasma ALT activities in animals treated with E5531 and GalN were no higher than those in animals treated with GalN alone, indicating that E5531 did not produce any hepatic injury on its own. Taken together, these two sets of experiments measuring LPS-induced hepatic injury in different sensitized animal models argue that E5531 is capable of inhibiting LPS-mediated hepatotoxicity.
E5531 inhibits [125I]ASD-LPS binding to mouse macrophages in vitro.Analysis of binding of [125I]ASD-LPS to primary cultures of murine macrophages indicates that binding is both specific and saturable. As shown in Fig. 3, E5531 inhibited the specific binding of [125I]ASD-LPS in a dose-dependent manner, with an IC50 of 0.77 μg/ml (0.5 μM), and greater than 80% inhibition was observed with 10 μg of E5531 per ml. LPS and lipid A were also tested for their ability to inhibit [125I]ASD-LPS binding. Specific binding of [125I]ASD-LPS was inhibited >90% by 100 μg ofE. coli LPS per ml, with an IC50 of 0.69 μg/ml. Lipid A inhibited specific LPS binding with an IC50 of 11.2 μg/ml (5.6 μM). These results indicate that cell surface LPS receptors have a greater affinity for E5531 than for lipid A.
Inhibition of [125I]ASD-LPS binding to mouse peritoneal macrophages by E5531. Mouse macrophages were incubated for 1 h with 60 ng of 4.1 μCi of [125I]ASD-LPS per μg and the indicated concentration of E5531 (•), lipid A (■), or LPS (○) in RPMI 1640 containing 2% fetal bovine serum. The cells were washed, followed by the addition of 0.1 N NaOH to solubilize the radioactivity. Aliquots of the mixture were analyzed with a gamma counter. Specific binding of [125I]ASD-LPS was determined by subtracting the value for nonspecific binding from the total binding. Nonspecific binding was evaluated separately by adding 1 mg of unlabelled LPS per ml to the incubation mixture. Each point represents the mean value of specific binding for duplicate cultures, and the results are expressed as counts per minute per culture well.
DISCUSSION
Lipid X, a monosaccharide biosynthetic precursor of lipid A, has been reported to protect mice and sheep against the acute lethal toxicity of LPS (21). However, lipid X has proven to be a poor LPS antagonist in vivo (5), and difficulties in obtaining purely antagonistic preparations of lipid X have yielded confusing results (2, 15). More promisingly, nontoxic LPS or lipid A’s obtained from nonpathogenic bacteria such as R. capsulatus and R. sphaeroides, as well as the disaccharide biosynthetic precursors of LPS such as lipid IVA, have been shown to be more potent inhibitors of the LPS-induced production of TNF-α in murine RAW 264.7 macrophage-like tumor cells in vitro (14, 17, 18) and in mice (17, 22). As reported previously, E5531 is a novel synthetic analogue of the lipid A from R. capsulatus (4, 12). In vitro, E5531 inhibited the LPS-induced release of cytokines such as TNF-α, IL-1, and IL-6 from human monocytes and human whole blood (4). In addition, E5531 potently inhibited the binding of LPS to human monocytes and monocyte-derived macrophages. In this study, similar inhibition of LPS-induced TNF-α release and inhibition of LPS binding was observed in mouse macrophages. The ability of 100 and 1,000 nM E5531 to completely inhibit induction of TNF-α by LPS also implies that E5531 has no intrinsic LPS-like agonistic activity, and in fact, no significant increases in TNF-α concentrations were detected in cultures treated with concentrations of E5531 up to 10 or 100 μM. Thus, unlike lipid IVA (9) and bacterium-derived lipid A from R. sphaeroides (24), E5531 was devoid of agonistic activity in human and mouse systems, even when it was tested at high concentrations.
E5531 inhibits the interaction of [125I]ASD-LPS with the cell surface of mouse macrophages, with an IC50of 0.77 μg/ml (0.50 μM) for ∼240 ng of LPS per ml. This makes E5531 a more potent inhibitor than E. coli lipid A (IC50 = 11.2 μg/ml or 5.6 μM) but a less potent inhibitor than E. coli LPS, which has an estimated IC50 of approximately 23 nM (0.69 μg/ml; using an estimated average molecular mass of ≈30 kDa). By using this estimated molar basis, LPS inhibits binding of [125I]ASD-LPS more potently than lipid A which is the toxicophore and presumably the determinant of LPS binding to cells. This discrepancy in affinity is likely due to a physicochemical characteristic of the more hydrophobic lipid A that limits its solubility or ability to form monomers or small aggregates. At this time, we are unsure if E5531 possesses greater solubility than E. coli lipid A or a higher affinity for the LPS receptor. However, either conclusion supports the idea that E5531 may exert its effect at a cell surface receptor(s) where measurable binding of LPS occurs. Finally, while we have not addressed the role of LPS binding protein (LBP) in these studies, we have found LPS binding to be immeasureable without some serum (2% is optimal in this system) or partially purified LBP from rabbit serum (ion-exchange-purified LBP). Use of partially purified LBP in a similar binding assay with a mouse macrophage cell line (RAW 264.7 cells) yielded results similar to those presented in Fig. 3.
In order to prove the efficacies of LPS antagonists in rodents, a variety of animal models have been developed. In all cases, steps had to be taken to overcome the relative insensitivity of rodents to endotoxin compared to the sensitivity of humans (3, 26, 30). To this end, we have used sensitization or priming with BCG or P. acnes as well as GalN (7, 10, 20, 27, 30). Priming or sensitization of mice with BCG or P. acnes increases their sensitivity to LPS and causes accumulation of activated macrophages in the reticuloendothelial system (30). As described in Table 1, BCG-primed mice generated high levels of TNF-α in response to endotoxin. This response enabled us to examine the in vivo antagonistic activity of E5531 against LPS using the levels of TNF-α in plasma as an index. Consistent with the idea that E5531 is a competitive antagonist of LPS, the effective dose of E5531 was affected by the dose of LPS used; administration of higher doses of LPS required higher doses of antagonist. Because it is well accepted that TNF-α is a key mediator of the pathological action of endotoxin and of the morbidity caused by endotoxin, suppression of LPS-induced TNF-α generation by E5531 in vivo encouraged investigation of the effect of E5531 on LPS-induced lethality and hepatic injury. As expected from results measuring the reduction in the level of TNF-α, E5531 also suppressed lethality caused by LPS in our BCG-primed murine model in a dose-dependent manner (Fig. 2).
Administration of LPS induces hepatic injury in the P. acnes-primed murine model. By using the plasma ALT level as an indicator of hepatotoxicity, E5531 antagonized this LPS-mediated effect with an ED50 of about 10 μg/animal. In this model, as well as in mice sensitized with GalN, hepatotoxicity has been attributed to the generation of TNF-α (10, 20). Therefore, it is likely that the suppressive effect of E5531 on the hepatic injury may be due to attenuation of LPS-induced cytokine generation (including the generation of TNF-α).
In the LPS-induced hepatitis model with mice sensitized with GalN, doses of LPS as low as 1 ng/mouse caused liver injury. In this case, the effective dose of E5531 needed to suppress this injury was as low as 30 ng/mouse, again suggesting that the dose of E5531 required for antagonistic efficacy was related to the LPS dose in vivo.
In conclusion, E5531 inhibits LPS-mediated cellular activation and LPS binding in murine macrophages in vitro. In vivo, E5531 suppresses the LPS response and hepatic injury in mice in an apparently competitive manner. These results suggest that the lipid A derivative suppresses LPS responses through blockade of an LPS receptor both in vitro and in vivo.
FOOTNOTES
- Received 13 April 1998.
- Returned for modification 11 June 1998.
- Accepted 19 August 1998.
- Copyright © 1998 American Society for Microbiology
