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Salmonella-Type Heptaacylated Lipid A Is
Inactive and Acts as an Antagonist of
Lipopolysaccharide Action on Human Line
Cells
Ken-ichi Tanamoto and Satoko Azumi
J Immunol 2000; 164:3149-3156; ;
doi: 10.4049/jimmunol.164.6.3149
http://www.jimmunol.org/content/164/6/3149
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References
Salmonella-Type Heptaacylated Lipid A Is Inactive and Acts as
an Antagonist of Lipopolysaccharide Action on Human Line
Cells
Ken-ichi Tanamoto2 and Satoko Azumi1
T
he biological activity of lipid A depends on its chemical
structure, and extraordinary progress has been made on
the relationship between chemical structure and biological activity of lipid A by using chemically synthesized lipid A
analogues (1). Recently, several nontoxic lipid As that closely
chemically resemble active lipid A but are completely devoid of
endotoxic activity have been reported (2– 4). Many of these preparations have also been found to act as antagonists (3, 5, 6), and
simple chemical modifications have also been found to change
biologically active lipid A to completely nontoxic derivatives (7,
8). These findings indicate that the biological activity of lipid A is
controlled by fine structural variations that are still ill-defined. In
addition, cells of different species have been discovered to respond
in different ways to certain lipid A derivatives, such as lipid A
precursor structure, which is an agonist in mouse cells but an antagonist in human cells (9 –11), indicating that the cells (or receptor molecules) discriminate fine differences in the chemical structure of lipid A. These findings indicate that understanding the
biological activity of lipid A is not a simple task and that the
mechanism of action of lipid A is different in humans and mice.
Escherichia coli- and Salmonella-type lipid A have the most
typical lipid A structures so far defined, and both have been synthesized chemically (12–14) and well characterized biologically by
many investigators (15–20). The chemical structure of these lipid
As consists of a hexa- and heptaacylated diglucosamine bearing
Division of Microbiology, National Institute of Health Sciences, Tokyo, Japan
Received for publication August 9, 1999. Accepted for publication January 3, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
S.A. was supported by a grant from the Japan Science and Technology Corporation.
2
Address correspondence and reprint requests to Dr. Ken-ichi Tanamoto, Division of
Microbiology, National Institute of Health, Sciences, 1-18-1 Kamiyoga, Setagayaku,
Tokyo 158-8501, Japan. E-mail address: [email protected]
Copyright © 2000 by The American Association of Immunologists
six and seven fatty acids, respectively, as shown in Fig. 1. The only
structural difference between them is the hexadecanoyl acid attached to the hydroxyl residue of 3-hydroxy tetradecanoic acid
bound to the 2 position of reducing glucosamine.
Previous studies on structure-activity relationships performed
by using chemically synthesized analogues have revealed that Salmonella-type lipid A is one of the active lipid As and exhibited
activity in all of the test systems employed at that time, although
its activity was a little less than that of E. coli-type synthetic lipid
A, which has, therefore, been subsequently recognized as representative of the structure of active lipid A.
In view of the species-specific actions of endotoxin, it is necessary to clarify the actual action of lipid A molecules on human
cells to better understand endotoxicosis, and thus the activity of
endotoxin must be considered based on its actions on human cells.
In this study, we show that one of the typical lipid As from
Salmonella is not an activator on human cells.
Materials and Methods
Materials
Recombinant TNF-␣ standards and rabbit polyclonal antisera against murine TNF-␣ were obtained from Asahi Kasei Kogyo (Tokyo, Japan). Rabbit
IgG was obtained from Zymed Laboratories (South San Francisco, CA).
Anti-human and -mouse I␬B-␣ antisera (1309 and 751, respectively) were
gifts from Dr. Nancy Rice (Advanced BioScience Laboratories-Basic Research Program, National Cancer Institute-Frederick Cancer Research and
Development Center). Synthetic lipid A analogues 506 and 516 were a gift
of Daiichi Kagaku (Tokyo, Japan). RPMI 1640 medium with glutamine
and IMDM were the products of Life Technologies (Grand Island, NY).
Quantitative Limulus assay reagent (Endospecy) was obtained from Seikagaku Kogyo (Tokyo, Japan). Pyrogen-free water was a product of Otsuka
Seiyaku (Tokyo, Japan). LPS was extracted from E. coli 03K2a2b:H2,
Salmonella minnesota (S type), Salmonella typhimurium LT2, and Salmonella abortus equi by the aqueous phenol method (21). Lipid A was obtained as an insoluble substance after 1% acetic acid treatment of LPS at
100°C for 90 min (22). The polysaccharide portion was obtained from the
supernatant of the reaction mixture by evaporating to dryness and washing
with chloroform three times. No residual lipid A portion was detected by
0022-1767/00/$02.00
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The stimulation of both THP-1 and U937 human-derived cells by Salmonella lipid A preparations from various strains, as assessed
by TNF-␣ induction and NF-␬B activation, was found to be very low (almost inactive) compared with Escherichia coli lipid A, but
all of the lipid As exerted strong activity on mouse cells and on Limulus gelation activity. Experiments using chemically synthesized
E. coli-type hexaacylated lipid A (506) and Salmonella-type heptaacylated lipid A (516) yielded clearer results. Both lipid A
preparations strongly induced TNF-␣ release and activated NF-␬B in mouse peritoneal macrophages and mouse macrophage-like
cell line J774-1 and induced Limulus gelation activity, although the activity of the latter was slightly weaker than that of the
former. However, 516 was completely inactive on both THP-1 and U937 cells in terms of both induction of TNF-␣ and NF-␬B
activation, whereas 506 displayed strong activity on both cells, the same as natural E. coli LPS. In contrast to the action of the lipid
A preparations, all the Salmonella LPSs also exhibited full activity on human cells. However, the polysaccharide portion of the LPS
neither exhibited TNF-␣ induction activity on the cells when administered alone or together with lipid A nor inhibited the activity
of the LPS. These results suggest that the mechanism of activation by LPS or the recognition of lipid A structure by human and
mouse cells may differ. In addition, both 516 and lipid A from Salmonella were found to antagonize the 506 and E. coli LPS action
that induced TNF-␣ release and NF-␬B activation in THP-1 cells. The Journal of Immunology, 2000, 164: 3149 –3156.
3150
Salmonella-TYPE LIPID A IS INACTIVE ON HUMAN CELLS
FIGURE 1. Structure of E. coli-type
hexaacylated and Salmonella-type heptaacylated lipid A molecules synthesized chemically (506 and 516, respectively).
either fatty acid analysis by Gas chromatography or by Limulus gelation
assay.
Gas-liquid chromatography analysis was performed on a model GC-14A
(Shimadzu, Kyoto, Japan) with a HiCap-CBM5 fused silica capillary column (25 m ⫻ 0.25 mm; GL Science, Toyko, Japan) and temperature programs 120°C for 3 min to 250°C at 3°C/min. Nitrogen was used as
carrier gas.
Mass spectrometry
Liquid secondary-ion mass spectrometry was performed on a VG ZAB2SEQ spectrometer (VG Analytical, Manchester, U.K.) operated at 8 kV in
a negative mode. The cesium gun was operated at 30 kV. Current-controlled scans were acquired at a rate of 30 s per decade. A mixture of
ethanolamine and m-nitrobenzyl alcohol (1:1) was used as the matrix.
Induction of TNF-␣ release from mouse peritoneal
macrophages, J774-1, THP-1, and U937 cells
Mouse peritoneal macrophages were obtained by washing the peritoneal
cavity of female BALB/c mice (6 –10 wk old, Japan SLC, Hamamatsu,
Japan) with 5 ml of serum-free IMDM (23). The cell number was adjusted
to 2 ⫻ 106 cells/ml. After adhesion, the cells were incubated with the
stimulant for 6 h. J774-1, THP-1, and U937 cells were grown in RPMI
1640 medium supplemented with 10% (v/v) heat-inactivated FCS, 50 ␮M
2-ME, 5 mM HEPES, penicillin (100 U/ml), and streptomycin (100 ␮g/ml)
in a 5% CO2 atmosphere at 37°C. J774-1 cells were harvested by scraping
with cell scraper (Costar, Cambridge, MA) and suspended in a fresh medium. The cells (1 ⫻ 106 cells/ml/well of 24-well dishes) were allowed to
adhere to plastic for 3 h at 37°C, washed twice with medium, and incubated
an additional 4 h for TNF-␣ induction with the stimulant. THP-1 cells (2 ⫻
105 cells/ml/well of 24-well dishes) were prepared for the experiments by
adding 100 ng/ml of PMA (Sigma, St. Louis, MO) and 0.1 ␮M 1,25dihydroxy vitamin D3 (Wako Pure Chemical Industries, Tokyo, Japan) to
cell suspensions in RPMI 1640 medium with 10% FCS (11, 24). The cell
suspensions were allowed to differentiate and adhere to plastic for 72 h at
37°C. After washing, the cells were incubated an additional 24 h with the
stimulant. U937 cells (1 ⫻ 106 cells/ml/well of 24-well dishes) were prepared for the experiments by adding 100 ng/ml of PMA to cell suspensions
in RPMI 1640 medium with 10% FCS (25). The cell suspensions were
allowed to differentiate and adhere to plastic for 48 h at 37°C. After washing, the cells were incubated an additional 8 h with the stimulant. The
supernatant of the test samples was stored at ⫺80°C until used to determine
TNF-␣.
TNF-␣ assay
The TNF-␣ produced was measured by cytotoxicity assay against L929
murine fibroblast cells. L929 cells were grown in tissue culture flasks in
RPMI 1640 medium (Life Technologies) supplemented with 10% FCS, 50
␮M 2-ME, 5 mM HEPES, penicillin (100 U/ml), and streptomycin (100
␮g/ml). Cells were detached with trypsin, washed, resuspended in medium
at 4 ⫻ 105 cells/ml, and 100 ␮l aliquots were plated in 96-well flat-bottom
plates (catalog no. 25860-96; Corning Glass, Corning, NY). After incubation for 3–5 h at 37°C in 5% CO2, 50 ␮l of actinomycin D (4 ␮g/ml) in
RPMI 1640 medium was added to each well, and 50 ␮l of test sample was
Activation of NF-␬B
Endotoxin-mediated NF-␬B activation was assessed by analyzing the degradation of I␬B-␣ protein in the cytosol fraction by Western blotting
(26 –28).
Crude cytosol fractions of LPS-treated and -untreated cells were prepared as follows (29). J774-1 cells (2 ⫻ 106/2.5 ml/well of 6-well dishes)
or THP-1 cells (1.25 ⫻ 106/well) pretreated with 20 ␮g/ml of cycloheximide for 30 min were treated with the test sample for 15 and 60 min,
respectively, and washed once at 4°C with 5 ml of PBS and were immediately frozen. The frozen cells were lysed on ice for 10 min with 100 ␮l
of lysis buffer (10 mM HEPES-KOH, 5 mM EDTA, and 10 mM KCl, pH
7.9) containing 0.5% (for J774-1) or 0.1% (for THP-1) Nonidet P-40, 1
mM DTT, and a protease inhibitor mixture (Boehringer Mannheim, Mannheim, Germany). The lysate was centrifuged for 5 min at 15,000 rpm at
4°C, and the resultant supernatant was used as the crude cytosol fraction.
The crude cytosol fractions (50 ␮g of protein) were subjected to 10%
SDS-PAGE, and I␬B-␣ was detected as follows. Protein was transferred
for 1 h onto a polyvinylidene difluoride membrane (Millipore, Bedford,
MA) with a semidry blotting apparatus. The membrane was then probed
with human (or mouse) I␬B-␣ antisera (1:1000 dilution) followed by a
peroxide-labeled goat anti-rabbit IgG Ab (Boehringer Mannheim; diluted
1:10000). Proteins were detected by using an enhanced chemiluminescence
system (Amersham, Arlington Heights, IL).
Limulus amoebocyte gelation activity
Activation of the proclotting enzyme of the horseshoe crab was tested by
a quantitative Limulus assay reagent (Endospecy). Pyrogen-free water was
used to dilute the test samples. A 50-␮l amount of each sample was incubated with the same volume of lysate containing chromogenic substrate in
96-well flat plates (Costar) for 30 min at 37°C. The reaction was stopped
by adding 200 ␮l of 0.6 M acetic acid. The chromogen ( p-nitroaniline) was
measured at 405 nm using a microplate reader (Molecular Devices, Menlo
Park, CA).
Inhibition assay of endotoxin-mediated TNF-␣ induction and
NF-␬B activation in THP-1 cells
Inhibition of endotoxin-mediated TNF-␣ induction and NF-␬B activation
in THP-1 cells were performed by adding the indicated concentration of the
inhibitor to the assay system followed immediately by the addition of agonist, and TNF-␣ production or the degradation of I␬B-␣ was compared
with a control containing agonist alone. Inhibition of TNF-␣ induction is
expressed as percent TNF-␣ production with production in the presence of
agonist alone equal to 100%.
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Gas-liquid chromatography conditions
then added to the wells (final volume; 200 ␮l/well). The results are expressed as means of triplicate determinations. To determine whether the
cytotoxic activity against L929 cells was due to TNF-␣, aliquots of supernatants from macrophage culture were incubated for 12 h with polyclonal
rabbit antiserum to TNF-␣ using nonspecific IgG as the control. The polyclonal Ab to TNF-␣ completely abolished the cytotoxicity of the supernatants in all the samples tested.
The Journal of Immunology
3151
Table I. Fatty acid composition of lipid A from various Salmonella and E. coli LPSa
Amount of Fatty Acid (mol/4 mol ␤-OH C14:0)
Lipid A From
C12:0
C14:0
␤-OH C14:0
C16:0
S. minnesota
S. abortus equi
S. typhimurium LT2
E. coli
1.32
1.18
0.89
0.86
1.13
0.97
1.24
1.18
4
4
4
4
0.68
0.90
0.62
0
a
Fatty acid composition was analyzed by gas-liquid chromatography. Molar ratios of the fatty acids are expressed by
assuming the number of 3-hydroxytetradecanoic acid molecules in each LPS to be 4.
Results
Fatty acid composition and liquid secondary-ion mass
spectrometry analysis of lipid As from Salmonella and
E. coli LPS
FIGURE 2. Salmonella LPS and lipid A induction of TNF-␣ release by
mouse cells. A, Murine peritoneal macrophages. B, J774-1 cells. Cells (A,
2 ⫻ 106 cells/ml/well of 24-well dishes in IMDM; B, 1 ⫻ 106 cells/ml/well
in RPMI 1640 medium supplemented with 10% (v/v) FCS) were cultured
with test sample. After 6 h (A) or 4 h (B) of incubation at 37°C, the
supernatants were examined for TNF-␣. The results are expressed as
means ⫾ SD for triplicate wells from one of three experiments with similar
results.
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To examine the fatty acid composition of LPS and lipid A from the
Salmonella species used in the present study, i.e., S. abortus equi,
S. minnesota (S type), and S. typhimurium LT2 lipid As were hydrolyzed, and the methyl-fatty acids liberated were analyzed by
gas-liquid chromatography. The results are shown in Table I,
where the molar ratios of the fatty acids are expressed by assuming
the number of 3-hydroxytetradecanoic acid molecules in each LPS
to be 4. The composition of all the lipid As was very similar, with
the major component of all these lipid A preparations being heptaacylated diglucosamine lipid A, as also confirmed by the results
of mass-spectrometry (data not shown).
FIGURE 3. Salmonella LPS and lipid A induction of TNF-␣ release by
human line cells. A, Human THP-1 cells. Cells (2 ⫻ 105 cells/ml/well)
were incubated with 100 ng/ml of PMA and 0.1 ␮M 1,25-dihydroxyvitamin D3 for 3 days in RPMI 1640 medium containing 10% FCS at 37°C. B,
Human U937 cells. Cells (1 ⫻ 106 cells/ml/well) were incubated with 100
ng/ml of PMA for 2 days in RPMI 1640 medium containing 10% FCS at
37°C. After an additional 24 h (A, THP-1) or 8 h (B, U937) of incubation in
medium containing 10% FCS with 10 ␮l of test sample, the supernatants were
assayed for TNF-␣. Values represent the mean concentration of TNF-␣ SD for
triplicate wells from one of three experiments with similar results.
3152
Salmonella-TYPE LIPID A IS INACTIVE ON HUMAN CELLS
Salmonella LPS and lipid A induction of TNF-␣ release by
mouse peritoneal macrophages and mouse macrophage-like
J774-1 cells
of each lipid A was 103 times less or more than that of their
parent LPS.
TNF-␣ release into the medium was estimated by cytotoxicity
against actinomycin-D-sensitized L929 murine fibroblasts. Murine
peritoneal macrophages were found to react to stimulation by all
Salmonella LPSs from the three different species, S. abortus equi,
S. minnesota, and S. typhimurium. As shown in Fig. 2A, the cells
started to secrete TNF-␣ at a LPS concentration of 0.1 ng/ml, and
TNF-␣ production by the macrophages increased dose-dependently. Although the activity of the lipid A preparations was less
than that of their respective parent LPS, they stimulated cells at a
concentration of 1–10 ng/ml.
A similar response to Salmonella LPS and lipid A was observed
in mouse macrophage-like J774-1 cells. Although the activity of
each of the lipid As was about 10 times less than that of its parent
LPS, the same as in mouse peritoneal macrophages with regard to
the minimum stimulatory dose, significant TNF-␣ production was
recognized at a dose of 1 ng/ml (Fig. 2B).
E. coli- and Salmonella-type synthetic lipid A induction of TNF␣ release by mouse peritoneal macrophages and mouse
macrophage-like J774-1 cells
Salmonella LPS and lipid A induction of TNF-␣ release by
THP-1 and U937 line cells
Human THP-1 and U937 line cells also reacted to stimulation with
all three Salmonella LPSs, and both lines started to secrete TNF-␣
at a concentration of 10 ng/ml. The LPS-induced production of
TNF-␣ increased dose-dependently (Fig. 3). However, the cells did
not produce TNF-␣ when exposed to any of the lipid A preparations, and significant amounts of TNF-␣ were first detected at a
concentration of 10 ␮g/ml in THP-1 cells, showing that the activity
Lipid A preparations obtained from natural LPS are usually heterogeneous, and all of the lipid A preparations from Salmonella
LPS used in the present study partially included a significant
amount of E. coli-type lipid A and may also have included contaminating parent LPS, both of which stimulate human cells. Because the data obtained by using these preparations are, therefore,
not reliable, the same experiments described above were performed with chemically synthesized Salmonella-type lipid A (516;
Fig. 1) using E. coli-type synthetic lipid A (506; Fig. 1) as a control. The results in mouse cells are shown in Fig. 4, A and B.
Murine peritoneal macrophages reacted to stimulation with 516 in
a dose-dependent manner and started to secrete TNF-␣ at a concentration of 10 ng/ml (Fig. 4A). A similar response to 516 was
also observed in mouse macrophage-like J774-1 cells (Fig. 4B).
The activity of 506 in inducing TNF-␣ was greater than that of
516, and the activity was expressed even at 1 ng/ml in both mouse
peritoneal and J774-1 line cells, showing that its activity is about
ten times more powerful than that of 516.
Synthetic lipid A, 506 and 516, induction of TNF-␣ release by
THP-1 and U937 line cells
Human THP-1 and U937 line cells also reacted to stimulation with
E. coli-type hexaacylated lipid A and started to secrete significant
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FIGURE 4. E. coli- and Salmonella-type synthetic lipid A induction of TNF-␣ release by
mouse and human cells. A, Murine peritoneal
macrophages. B, J774-1 cells. C, Human THP-1
cells. D, Human U937 cells. Experiments were
performed as described in Figs. 2 and 3. The results are expressed as means ⫾ SD of triplicate
wells from one of two experiments with similar
results.
The Journal of Immunology
amounts of TNF-␣ at a concentration of 1 ng/ml. The LPS-induced
production of TNF-␣ by both cells increased dose-dependently
(Fig. 4, C and D). In contrast, neither human THP-1 or U937 cells
of produced TNF-␣ at all when stimulated with 516, even at a
concentration of 10 ␮g/ml (Fig. 4, C and D).
Activation of NF-␬B in mouse macrophage-like J774-1 cells by
Salmonella LPS and lipid A, and E. coli- and Salmonella-type
synthetic lipid As
To examine endotoxin-mediated NF-␬B activation, J774-1 cells
were treated with LPS and lipid A from S. abortus equi, and cell
extracts were analyzed for degradation of I␬B-␣ protein by immunoblotting. The results are shown in Fig. 5A. Stimulation of the
cells with both S. abortus equi LPS and lipid A resulted in a rapid
decrease and disappearance of I␬B-␣ protein at 1 ng/ml ml and 10
ng/ml, respectively. Both synthetic lipid A preparations 506 and 516
also expressed strong I␬B-␣ degrading activity in J774-1 cells (Fig.
FIGURE 7. Limulus amoebocyte gelation activity of Salmonella abortus equi LPS and lipid A, and synthetic lipid As. Quantitative Limulus
assay reagent was incubated with test samples for 30 min, and the chromogen released was measured. Œ, Salmonella abortus equi LPS; ‚, lipid
A; F, 506; and f, 516.
5B). However, the activity of 506 was about ten times stronger than
that of 516, consistent with the results of TNF-␣ induction.
Activation of NF-␬B in THP-1 line cells by Salmonella LPS and
lipid A, and synthetic lipid As
Human THP-1 line cells also reacted to stimulation with Salmonella LPS and E. coli-type hexaacylated lipid A, and I␬B-␣ protein
degradation started at a concentration of 1 ng/ml (Fig. 6, A and B).
In contrast, Salmonella lipid A exhibited much less activity than
the parent LPS (104 times less), and Salmonella type synthetic
lipid A did not induce degradation of I␬B-␣ at all, even at a concentration of 1 ␮g/ml (Fig. 6).
Limulus amoebocyte gelation activity of Salmonella LPS and
lipid A, and synthetic lipid As
Activation of the clotting system cascade of the horseshoe crab
was used to assess differences in the activity of Salmonella LPS
and lipid A, 506 and 516. The results are shown in Fig. 7. Although the activity of 516 was slightly lower than that of 506, all
of the preparations exhibited strong Limulus gelation activity.
Inhibition of the TNF-␣ induction activity of E. coli LPS and
506 by Salmonella lipid A and 516
FIGURE 6. Activation of NF-␬B in human cells by Salmonella LPS and
lipid A, and synthetic lipid As. THP-1 cells (1.25 ⫻ 106/well) were treated
with cycloheximide (20 ␮g/ml) for 30 min and then with indicated concentrations of test sample for 60 min. Cytosol extracts were prepared and
the I␬B-␣ levels were analyzed by Western blotting (50 ␮g protein/lane).
Because the lipid A from Salmonella and synthetic Salmonellatype lipid A failed to stimulate TNF-␣ production by human
THP-1 cells, their antagonistic activity on the response of cells to
active LPS was tested by adding them to the cell cultures together
with the agonist. As shown in Fig. 8, both Salmonella lipid A and
synthetic Salmonella-type lipid A displayed inhibitory activity on
TNF-␣ production stimulated by E. coli LPS. Suppression by synthetic Salmonella-type lipid A was observed in a dose-dependent
manner without regard to the stimulatory dose of the agonist (10
and 100 ng/ml). Significant inhibitory effects were observed at an
agonist to antagonist ratio of 1:10 (w/w), and almost complete
inhibition was possible at a 100-fold excess of both antagonists to
stimulant.
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FIGURE 5. Activation of NF-␬B in mouse cells by Salmonella LPS and
lipid A, and synthetic lipid As. Endotoxin-mediated NF-␬B activation was
examined by analyzing for degradation of I␬B-␣. J774-1 cells (2 ⫻ 106/2.5
ml/well) were treated with indicated concentrations of test sample for 15
min. Cytosol extracts were prepared and the I␬B-␣ levels were analyzed by
Western blotting (50 ␮g protein/lane).
3153
3154
Salmonella-TYPE LIPID A IS INACTIVE ON HUMAN CELLS
FIGURE 9. Inhibitory activity of 516 on the NF-␬B activation of E. coli
LPS in human cells. Various concentrations of 516 in 10 ␮l of water were
added to THP-1 cells prepared as described in Fig. 6, followed immediately
by the addition of 10 ␮l of 506. After 60 min of incubation at 37°C, cytosol
extracts were prepared and the I␬B-␣ levels were analyzed by Western
blotting (50 ␮g protein/lane).
Discussion
FIGURE 8. Inhibitory activity of Salmonella lipid A and 516 on the
TNF-␣ induction activity of E. coli LPS in human cells. Various concentrations of antagonist (A, Salmonella lipid A; B, 516) in 10 ␮l of water were
added to THP-1 cells prepared as described in Fig. 3, followed immediately
by the addition of 10 ␮l of E. coli LPS. After 24 h of incubation at 37°C,
the supernatants were assayed for TNF-␣. Inhibition is expressed as percent TNF-␣ production, with TNF-␣ production in the presence of agonist
alone equal to 100%. Values represent the mean % inhibition ⫾ SD for
triplicate wells. Concentrations of E. coli LPS: E, 10 ng/ml; F, 100 ng/ml.
Inhibition by 516 of NF-␬B activation induced by E. coli LPS in
THP-1 cells
The antagonistic activity of synthetic Salmonella-type lipid A on
the response of human THP-1 line cells to I␬B-␣-degrading activity of 506 was tested. As shown in Fig. 9, synthetic Salmonellatype lipid A (516) displayed inhibitory activity on the degradation
of I␬B-␣ in response to 506. An almost total inhibitory effect was
observed at an agonist to antagonist ratio of 1:100 (w/w).
Activity of the polysaccharide portion of Salmonella LPS on
induction of TNF-␣ and inhibition of TNF-␣ induction by parent
LPS in THP-1 line cells
Because the activity of LPS and lipid A from Salmonella on human cells was completely different, the action of the polysaccharide portion from S. abortus equi was examined in regard to
TNF-␣ induction activity and the inhibitory activity on TNF-␣
induction of active 506. No TNF-␣ induction by polysaccharide
alone was recognized, even at 10 ␮g/ml, and no LPS activity was
recovered when the polysaccharide portion and lipid A from S.
In the present study, we found that all lipid A preparations isolated
from several Salmonella strains exhibited far lower activity on
human cells in inducing TNF-␣ and NF-␬B activation than E. coli
lipid A. According to the fatty acid analysis by gas-liquid chromatography and mass spectrometry (data not shown), the major
lipid A structure of all of the lipid A preparations from the Salmonella strains used in the present study was heptaacylated diglucosamine, which is referred to as Salmonella-type lipid A, because
it is the typical structure present in Salmonella, as reported previously (30 –32). In addition to the Salmonella-type lipid A, they
also contained a significant amount of E. coli-type hexaacylated
diglucosamine lipid A structure.
Because natural lipid A usually consists of a mixture of various
structures originated from the biosynthesis and extraction procedure, as was also the case in the present study, and its biological
activity depends on the chemical structure, experiments using natural lipid A may give rise to misunderstandings regarding structure-activity relationships.
To confirm the phenomenon observed when natural Salmonella
lipid A was used, and to obtain more definitive results, experiments have been performed with synthetic E. coli- and Salmonellatype lipid A (506 and 516, respectively). The results showed that
Salmonella-type lipid A is completely inactive on human cells, but
is a strong agonist in mice and active in inducting Limulus gelation
activity as well. In contrast, E. coli-type lipid A exhibited full
activity in human cells as well.
Both of these lipid As, the Salmonella-type heptaacylated and E.
coli-type hexaacylated forms, had been successfully synthesized
chemically in the past (12–14), and their biological properties had
been carefully tested by many investigators (15–20). The Salmonella-type lipid A had been demonstrated to be very active in mice
and rabbits and subsequently had been thought to be a typical lipid
A (18 –20) along with that from E. coli. However, Salmonella-type
lipid A (516) was not tested for activity on human systems, and
nobody at that time expected that it would be discriminated by
human and mouse cells.
Although natural Salmonella lipid A contains a significant
amount of the E. coli-type hexaacylated diglucosamine structure,
as recognized by mass spectrometry, its activity on human cells
was mach lower than expected. In the present study, biologically
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abortus equi were added to the cells together (data not shown).
Furthermore, the polysaccharide showed no inhibitory activity on
the TNF-␣ induction activity of its parent LPS, even when a 103
times excess of polysaccharide to lipid A (w/w) was added to the
cells (10 ng/ml of LPS plus 10 ␮g/ml of polysaccharide) (data not
shown).
The Journal of Immunology
Based on the results of the present and previous studies, it seems
that higher animals have higher specificity for the recognition of
lipid A structures as observed in the case of lipid A precursor and
chemically modified lipid A analogues (39). This seems to suggest
that the active endotoxin is effectively detoxified in higher animals.
Experiments concerning the structure-activity relationships of
lipid As have mainly been performed in animal systems thus far,
and thus an important finding in the present study had been overlooked. Accordingly, the study should be performed again from
the viewpoint of human endotoxicosis by using a human system
and chemically pure materials.
Acknowledgments
We thank Dr. Nancy Rice for the use of anti-human and -mouse I␬B-␣
antisera (1309 and 751, respectively).
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