1 Research and 3 Medical Services, Stratton Veterans Affairs Medical Center, and Departments of 2 Medicine and 4 Physiology and Cell Biology, Albany Medical College, Albany, New York 12208
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ABSTRACT |
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Endotoxin selectively induces monocyte Mn superoxide dismutase
(SOD) without affecting levels of Cu,Zn SOD, catalase, or glutathione peroxidase. However, little is known about the structure-activity relationship and the mechanism by which endotoxin induces Mn SOD. In
this study we demonstrated that a mutant Escherichia
coli endotoxin lacking myristoyl fatty acid at the
3' R-3-hydroxymyristate position of the lipid A moiety retained its full capacity to coagulate Limulus amoebocyte lysate compared
with the wild-type E. coli endotoxin
and markedly stimulated the activation of human monocyte nuclear
factor-B and the induction of Mn SOD mRNA and enzyme activity.
However, in contrast to the wild-type endotoxin, it failed to induce
significant production of tumor necrosis factor-
and macrophage
inflammatory protein-1
by monocytes and did not induce the
phosphorylation and nuclear translocation of mitogen-activated protein
kinase. These results suggest that
1) lipid A myristoyl fatty acid,
although it is important for the induction of inflammatory cytokine
production by human monocytes, is not necessary for the induction of Mn
SOD, 2) endotoxin-mediated induction
of Mn SOD and inflammatory cytokines are regulated, at least in part,
through different signal transduction pathways, and
3) failure of the mutant endotoxin
to induce tumor necrosis factor-
production is, at least in part,
due to its inability to activate mitogen-activated protein kinase.
lipopolysaccharide; tumor necrosis factor; nuclear factor-B; mitogen-activated protein kinase
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INTRODUCTION |
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ENDOTOXIN, a lipopolysaccharide (LPS) of the cell wall of gram-negative bacteria, is responsible for a host of toxic effects that occur in patients infected with these microorganisms, including fever, disseminated intravascular coagulation, and hemodynamic changes that may lead to multiple organ failure characteristics of septic shock (19, 24). On the other hand, LPS exhibits immunostimulatory effects (19, 24) and induces the antioxidant enzyme Mn superoxide dismutase (SOD) (1, 27), which are beneficial to the host. Evidence suggests that induction of Mn SOD may be responsible for LPS-induced protection against pulmonary oxygen toxicity (31, 33). However, the serious toxicities of LPS limit any potential clinical use for its beneficial effects.
The endotoxic effects of LPS are caused indirectly through the
activation of monocytes and macrophages, leading to the release of
toxic cytokines, such as tumor necrosis factor (TNF), interleukin (IL)-1, IL-6, and macrophage inflammatory proteins (MIPs) (4, 5, 19,
24). The endotoxic principle of LPS resides in its lipid A component.
For full endotoxic activity in humans, a lipid A structure containing
two (1
6)-linked
D-glucosamine residues, two
phosphoryl groups, and six fatty acids in a defined arrangement as
present in Escherichia coli lipid A is
required (19, 24, 25).
The mechanism and the structural requirement for the induction of Mn
SOD by LPS are not clear. Recently, Somerville et al. (29) reported a
1,000- to 10,000-fold reduction in the ability of a mutant
E. coli LPS lacking the myristoyl
fatty acid moiety at the 3'
R-3-hydroxymyristate position of lipid
A (nonmyristoyl LPS, nmLPS) to stimulate E-selectin expression by human
endothelial cells and TNF- production by adherent monocytes compared
with the wild-type LPS (wtLPS). In the current study we demonstrated that nmLPS at a concentration as high as 1 µg/ml failed to
significantly induce TNF-
and MIP-1
production by human
monocytes, whereas it markedly induced the activation of nuclear
factor-
B (NF-
B) and the induction of Mn SOD mRNA and enzyme
activity. In addition, nmLPS failed to activate mitogen-activated
protein kinase (MAPK), a principal component of the signal transduction
pathway known to be involved in the wtLPS-induced production of TNF-
(4, 11, 12, 23, 30). These results suggest that lipid A myristoyl fatty
acid, although it is important for the induction of inflammatory cytokine production by human monocytes, is not necessary for the induction of Mn SOD. This mutant LPS can be used to distinguish the
intracellular signal transduction pathways for LPS-induced cytokine
production and Mn SOD induction and to study the potential beneficial
effect of Mn SOD induction without the toxic effects of inflammatory
cytokines.
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MATERIALS AND METHODS |
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Materials.
The endotoxins wtLPS (JM83 E. coli
K-12) and nmLPS (BMS67C12 E. coli
K-12) were kindly provided by John E. Somerville (Bristol-Myers Squibb
Pharmaceutical Research Institute, Princeton, NJ). The nmLPS
preparation was free of wtLPS contamination as determined by gas
chromatography and mass spectroscopy. Purified Mn SOD (from E. coli) and Cu,Zn SOD (from bovine
erythrocytes) were obtained from Sigma Chemical (St. Louis, MO). Mn SOD
and Cu,Zn SOD had a specific activity of 4,400 U/mg protein as
determined according to the method described by McCord and Fridovich
(18). Polyclonal goat anti-recombinant human TNF- antibody
[affinity-purified IgG, 1 mg/ml; 50% neutralization dose
0.02-0.04 µg/ml by cell lytic assay using murine L929
fibroblasts] and normal goat IgG were purchased from R & D Systems (Minneapolis, MN). Rabbit polyclonal antiphosphotyrosine
and mouse monoclonal anti-rat MAPK antibodies were obtained from
Transduction Laboratories (Lexington, KY). Enhanced chemiluminescence
Western blotting detection reagents were from Amersham Life Science
(Arlington Heights, IL).
Endotoxin assay. The endotoxin activity of wtLPS and nmLPS was determined with the Limulus amoebocyte lysate (LAL) assay (QCL-1000, BioWhittakker, Walkersville, MD).
Isolation of human monocytes. Human mononuclear cells were isolated from venous blood of normal volunteers (after the nature and possible risks of the studies were explained and informed consent was obtained) using Isolymph (Gallard-Schlesinger Industries, Carle Place, NY). Cells (5 × 106/ml in RPMI 1640 plus antibiotics and 5% autologous serum) were allowed to adhere to tissue culture plates for 2 h. Adherent cells, which consisted of ~90% monocytes as judged by a nonspecific esterase stain, were used for the current studies.
Measurements of TNF- and MIP-1
.
Adherent monocytes were treated with or without LPS (0.01 ng/ml-1.0 µg/ml) for 1, 4, or 18 h at 37°C according to
Somerville et al. (29). In some experiments, goat anti-recombinant
human TNF-
IgG or normal goat IgG (0.16 mg/ml) was added to the
incubation medium. The amounts of TNF-
activity and MIP-1
protein
released into the medium or present in cell lysates (collected by
scraping and sonication) were determined by the anti-TNF-
antibody-inhibitable lysis of murine L929 fibroblasts as described
previously (34) and ELISA (R & D Systems), respectively, carried out
according to the manufacturer's instructions.
Measurements of SOD activity. Adherent monocytes were treated with or without (as control) LPS (1 µg/ml) for 1, 4, or 18 h at 37°C. Cells were then collected by scraping and sonicated, and protein contents were determined using bicinchoninic acid according to Smith et al. (28). Aliquots of cell extracts (50 µg/lane) were then assayed for SOD activity using nondenaturing PAGE (8%) according to the method described by Beauchamp and Fridovich (2), on the basis of the inhibitory effect of SOD on the reduction of tetrazolium by superoxide generated by photochemically reduced riboflavin, as described previously (35). The SOD activity gels were quantified using a computing densitometer (Molecular Dynamics, Sunnyvale, CA). In each assay, purified E. coli Mn SOD and bovine erythrocyte Cu,Zn SOD (25-800 mU) were used to obtain standard curves from which cell extract Mn SOD and Cu,Zn SOD activities, respectively, were derived.
Northern analysis of TNF- and Mn SOD mRNAs.
Northern blot analysis was performed as described previously (38).
Briefly, adherent monocytes were treated with or without 1 µg/ml LPS
for 4 h at 37°C, and the total cellular RNA was isolated by the
single-step method of Chomczynski and Sacchi (7) using Tri Reagent
(Molecular Research Center, Cincinnati, OH). For Northern blots,
denatured RNA samples (20 µg/lane) were electrophoresed in 1.2%
agarose gels, transferred to nylon membrane (Genescreen plus, New
England Nuclear, Boston, MA) by capillary blotting, and stained with
methylene blue to visualize the quality and size of 18S and 28S
ribosomal RNA species. The membrane was then prehybridized as described
previously (38). Hybridization was carried out with 100 µg/ml
denatured salmon testis DNA and human TNF-
or Mn SOD cDNA probes
that had been labeled by random hexanucleotide priming (GIBCO BRL,
Gaithersburg, MD) to a specific activity of >109 cpm/µg DNA. After the
samples were washed, autoradiographs were obtained and radioactive
signals were quantified using a computing densitometer.
Electrophoresis mobility shift assay for NF-B.
Adherent monocytes were treated with or without 1 µg/ml LPS for 1 or
4 h at 37°C, and the nuclear extracts were obtained
according to Osnes et al. (21). For the electrophoresis mobility shift assay (EMSA), 3 µg of nuclear proteins were incubated for 30 min at
room temperature with ~100,000 cpm (5 ng) of an oligonucleotide containing NF-
B consensus sequence (5'-AGT TGA
AGG C-3') that had
been 5'-end labeled with
[
-32P]ATP using T4
polynucleotide kinase (Promega, Madison, WI). Competition was carried
out using a 100-fold excess of the unlabeled oligonucleotide 10 min
before addition of the radiolabeled probe. Samples were then
electrophoresed in a 6% nondenaturing polyacrylamide gel. Autoradiographs were obtained and radioactive signals were quantified using a computing densitometer.
Immunoprecipitation and immunoblotting for MAPK. Immunoprecipitation and immunoblotting for MAPK were performed as described previously (16). Briefly, adherent monocytes were treated with or without 1 µg/ml LPS for 1 h at 37°C. After hypotonic lysis, cytoplasmic and nuclear extracts were prepared according to the method of Wen et al. (37) and immunoprecipitated by overnight incubation with antiphosphotyrosine at 4°C with rocking. Protein A-agarose was added and rocking continued for 1 h at 4°C. Samples were eluted with 2× sample solubilizer, and protein was separated by discontinuous SDS-PAGE (9%), transferred to Immobilon membranes (Millipore, Bedford, MA) by electroblotting, and blocked with 5% milk in Tris-buffered saline containing 0.1% Tween. Membranes were then incubated with mouse monoclonal anti-MAPK antibody (1:1,000) overnight at room temperature, then with horseradish peroxidase-labeled secondary antibody, rabbit anti-mouse IgG (1:1,000, Dako, Carpenteria, CA) for 1 h. Immunoblots were visualized by chemiluminescence using the enhanced chemiluminescence Western blotting detection system, exposed to X-ray film, and quantified using a computing densitometer.
Statistical analysis. Data from two groups were compared by a two-tailed t-test, and those from more than two groups were compared by one-way ANOVA with correction for multiple comparison (10). A difference is considered to be significant at P < 0.05.
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RESULTS |
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Effect of nmLPS on TNF- and MIP-1
production.
To determine whether the reduced capacity of nmLPS to activate human
monocytes was restricted to TNF-
production, we measured the
production of TNF-
and MIP-1
, a member of the chemokine
-family (39), in response to wtLPS and nmLPS. As shown in Fig. 1A,
at 4 h after incubation, wtLPS induced a concentration (0.01 ng/ml-1.0 µg/ml)-dependent release of TNF-
and MIP-1
into
the medium. In contrast, nmLPS even at a concentration as high as 1 µg/ml failed to induce a significant amount of cytokine release. Time-course experiments using 1 µg/ml of LPS (Fig.
1B) demonstrated wtLPS-induced
cytokine release within 1 h, reached a near plateau at 4 h, and lasted
for 18 h. Production of TNF-
and MIP-1
by nmLPS-treated cells was
markedly reduced throughout these time periods. Measurement of TNF-
activity in cell lysates revealed negligible TNF-
in wtLPS- and
nmLPS-treated cells (Fig. 1C), suggesting that reduced TNF-
release by nmLPS-treated monocytes was
in fact due to a decreased production rather than an impaired secretion
of the cytokine.
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Effect of nmLPS on induction of Mn SOD. We then determined whether nmLPS also had a similarly reduced capacity to induce Mn SOD. LPS has been shown to selectively induce Mn SOD without affecting other antioxidant enzymes, including Cu,Zn SOD, catalase, and glutathione peroxidase (1, 27). The results (Fig. 2) revealed that at 18 h, but not at 1 or 4 h, after incubation, wtLPS induced a marked increase (2.5-fold) in monocyte Mn SOD activity, whereas it had no effect on Cu,Zn SOD activity. nmLPS also induced a significant increase (1.5-fold) in the Mn SOD activity at 18 h, although to a lesser degree than wtLPS.
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Effect of nmLPS on TNF- and Mn SOD mRNAs.
Northern analysis of TNF-
and Mn SOD mRNAs (Fig.
5) further supported the above-observed
effects of wtLPS and nmLPS in the production of TNF-
and the
induction of Mn SOD enzyme activity by human monocytes. wtLPS markedly
enhanced the levels of TNF-
(17-fold) and Mn SOD (7-fold) mRNAs
compared with control, nontreated cells. nmLPS also induced a marked
increase (6-fold) in the level of Mn SOD mRNA, whereas it had a
markedly reduced effect on TNF-
mRNA (1-fold increase vs. 17-fold
increase by wtLPS).
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Effect of nmLPS on activation of NF-B.
Considerable evidence suggests that activation of NF-
B is essential
for the induction of monocyte and macrophage TNF-
mRNA by LPS (4,
24, 26). Whether induction of monocyte Mn SOD mRNA by LPS is also
dependent on NF-
B is not clear. We took advantage of the
differential effects of wtLPS and nmLPS observed above to determine the
potential role of NF-
B activation in the LPS-induced induction of
TNF-
and Mn SOD mRNAs using the EMSA. As shown in Fig.
6, wtLPS and nmLPS markedly activated
NF-
B at 1 h after treatment. However, by 4 h the effect was largely
gone (data not shown). There was no difference between wtLPS and nmLPS.
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Effect of nmLPS on activation of MAPK.
The MAPK signal transduction pathway plays an important role in the
LPS-induced production of TNF- (4, 11, 12, 23, 30). We determined
whether failure of nmLPS to induce TNF-
production was due to its
inability to activate MAPK. Cytoplasmic and nuclear fractions were
immunoprecipitated with antiphosphotyrosine, then immunoblot analysis
of phosphorylated MAPK was performed. As shown in Fig.
7, wtLPS markedly increased the nuclear
content of tyrosine-phosphorylated MAPK. In contrast, nmLPS failed to induce tyrosine phosphorylation and nuclear translocation of MAPK. No
tyrosine-phosphorylated MAPK was detectable in the cytoplasmic fractions of wtLPS- or nmLPS-treated monocytes (data not shown).
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DISCUSSION |
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The data presented in this study demonstrated that deletion of
myristoyl fatty acid at the 3'
R-3-hydroxymyristate position of the
E. coli lipid A moeity (nmLPS)
resulted in a markedly reduced ability to induce the production of not
only TNF- (29), but also MIP-1
, by human monocytes, suggesting a
more generalized phenomenon of impaired induction of inflammatory
cytokine production by nmLPS. Furthermore, the impaired induction of
inflammatory cytokine production by nmLPS was associated with an
impaired activation (tyrosine phosphorylation and nuclear
translocation) of MAPK but a normal activation of NF-
B.
The production of TNF- by monocytes is regulated at the
transcriptional and translational levels (4, 14, 15). The exact signal
transduction pathway(s) by which LPS induces TNF-
production has not
been fully established. However, LPS stimulates TNF-
gene
transcription and translation, and it requires the activation of the
protein tyrosine kinase (PTK)/ras/raf-1/MEK/MAPK signal transduction
pathway and NF-
B (4, 11, 12, 14, 15, 23, 30). Thus the inability to
activate MAPK by nmLPS may explain the observed impairment in TNF-
production by nmLPS-treated human monocytes. The exact location and
mechanism by which nmLPS fails to activate MAPK are not clear. It is
likely to be at the level of PTK activation (receptor-PTK interaction)
or ras activation, since other members of the MAPK pathway do not have
access to the plasma membrane at which wtLPS or nmLPS is acting.
Further studies are necessary to clarify this point.
Activation of NF-B, although essential, is not sufficient for the
induction of TNF-
mRNA and production of TNF-
by LPS in
monocytes/macrophages. Peritoneal macrophages from LPS-resistant, C3H/HeJ mice are able to respond to LPS by activating NF-
B normally (9) but are unable to induce TNF-
mRNA and produce TNF-
(3). The
TNF-
synthetic pathway in C3H/HeJ macrophages is intact, since they
are able to produce TNF-
normally on costimulation with
interferon-
and LPS (6). Our observation that nmLPS activated monocyte NF-
B normally but failed to induce TNF-
mRNA and TNF-
production is consistent with this concept. Recent studies suggest that
concerted participation of cis-acting
regulatory elements at the Egr-1 (42), AP-1/CRE-like (20), NF-IL6
(C/EBP
) (22), and
B3 sites is required for optimal induction of
the TNF-
promoter by LPS in human monocytes.
Binding of LPS to a circulating LPS-binding protein, which in turn
binds to the monocyte LPS membrane receptor mCD14, is essential in the
efficient cellular response to low concentrations (<100 ng/ml) of LPS
(4, 30, 32, 41). However, the concentration of LPS used in the current
study (1 µg/ml) is not dependent on mCD14 binding to induce TNF-
production by human monocytes (4, 30). Somerville et al. (29)
demonstrated that the effect of nmLPS can be inhibited by a monoclonal
anti-CD14 antibody, MY4. In addition, at high concentrations, nmLPS
competitively inhibits wtLPS-induced E-selectin induction in human
endothelial cells. It was, therefore, suggested that nmLPS could serve
as an LPS antagonist through competitive binding to mCD14 (29). A
recent study by Cunningham et al. (8) reveals that nmLPS, compared with
wtLPS, binds normally to immobilized soluble CD14 (sCD14) and a panel
of 23 different point-mutated sCD14 molecules. Thus failure of nmLPS to induce TNF-
production by human monocytes is
unlikely due to an impaired binding to CD14.
Little is known about the regulation of Mn SOD gene expression and the
signal transduction pathway(s) responsible for the LPS-mediated
induction of Mn SOD. Our results suggest that inductions of Mn SOD and
TNF- by LPS are mediated through different signal transduction
pathways, since nmLPS was able to induce Mn SOD mRNA and enzyme
activity without inducing TNF-
mRNA and activity. These results are
consistent with previous observations in LPS-resistant C3H/HeJ mice.
Peritoneal macrophages from C3H/HeJ mice are unable to produce TNF-
in response to LPS (3); however, LPS is able to induce Mn SOD normally
in these macrophages compared with macrophages from LPS-sensitive,
C3H/HeOuJ mice (13). Although LPS induction of Mn SOD in human
monocytes, as demonstrated in the current study and in peritoneal
macrophages from C3H/HeJ mice (9, 13), is associated with the
activation of NF-
B, the role of NF-
B activation in the induction
of Mn SOD by LPS cannot be conclusively ascertained.
The reason for the reduced increase of Mn SOD activity in nmLPS-treated
moncytes in the face of an almost comparable increase in Mn SOD mRNA,
compared with wtLPS-treated cells, is not clear. However, we
demonstrated that neutralization of TNF- activity did not reduce Mn
SOD activity of wtLPS-treated cells to the level of nmLPS-treated
monocytes, suggesting that TNF-
produced by wtLPS-treated cells
could not account for the observed difference in the levels of Mn SOD
activity. Because nmLPS is unable to activate MAPK, it is possible that
the MAPK pathway via cross talk contributes to the signal transduction
cascade involved in the induction of Mn SOD by LPS and that the
inability of nmLPS to activate MAPK is responsible for the reduced
increase of Mn SOD activity in nmLPS-treated monocytes. Further studies
are necessary to determine the role of MAPK in the induction of Mn SOD
by LPS. In addition, IL-1, which is known to be produced by
wtLPS-treated monocytes, can also induce Mn SOD (17). It is not clear
whether nmLPS induces monocytes to produce IL-1 and whether IL-1
produced by wtLPS-treated monocytes can account for the observed
difference in the levels of MnSOD activity between wtLPS- and
nmLPS-treated cells.
In summary, we have demonstrated that induction of Mn SOD and TNF-
production by LPS in human monocytes can be dissociated, suggesting
that induction of Mn SOD and TNF-
is regulated by different signal
transduction pathways. The ability of nmLPS to induce Mn SOD without
inflammatory cytokine production provides an opportunity to study the
potential benefits of an LPS-induced increase in Mn SOD without the
associated toxic effects of inflammatory cytokines.
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ACKNOWLEDGEMENTS |
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This work was supported by the Department of Veterans Affairs, Office of Research and Development, Medical Research Service.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: M.-F. Tsan, Research Service (151), Stratton VA Medical Center, 113 Holland Ave., Albany, NY 12208.
Received 13 March 1998; accepted in final form 2 June 1998.
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