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Research Center Borstel, Center for Medicine and Biosciences, Borstel, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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, IL-1
, and IL-6.
Chemically, LPS consist of a hydrophilic heteropolysaccharide that is
covalently linked to a hydrophobic lipid portion, called lipid A, which
anchors the molecule to the membrane. It has been shown that lipid A
harbors the "endotoxic principle" of LPS (2) and that
a peculiar molecular conformation ("endotoxic conformation") of
lipid A is a prerequisiste for its endotoxic activity (3).
The primary chemical structure of lipid A defines its molecular
conformation, which may, in its extremes, be either conical, with the
cross-section of the hydrophilic backbone being smaller than that of
the hydrophobic moiety, or cylindrical with identical cross-sections.
Thus, lipid A of the endotoxically most active LPS from
Escherichia coli, which consists of a 1,6-linked
D-glucosamine disaccharide carrying six saturated
fatty acids and two negatively charged phosphates at defined locations
(2, 4), has a conical conformation (3).
Variations of this structural arrangement, such as a reduction in the
number of charges or the number of acyl chains, a change in their
distribution, or degree of saturation, results in a dramatic reduction
in biological activity (5) and a transition from a conical
to a cylindrical conformation.
Binding of LPS to membrane-bound CD14 (mCD14),3 an LPS receptor, is one of the first steps in the activation of monocytes/macrophages (6). However, for two reasons mCD14 cannot be the protein initiating the intracellular signaling: 1) mCD14 lacks a transmembrane domain and 2) at higher LPS concentrations activation can be achieved also after blockade of mCD14 by anti-CD14 Abs or in the absence of CD14 (7).
From previous studies it is known that the LPS-binding protein LBP, an acute-phase serum protein, forms complexes with the LPS molecules and transports these directly into the host cell membrane (8) or to mCD14 (6), which is known to be a coreceptor for the signaling protein. Because it has been shown that the soluble form of CD14 (sCD14) transports endotoxin molecules directly into phospholipid membranes (9), it is reasonable to assume that mCD14 operates in a similar manner.
From our findings of a correlation between the molecular conformation of lipid A and its ability to induce cytokine production in mononuclear cells, some characteristics of the signaling protein can be defined: 1) it should be accessible to modulations via its outermost transmembrane domain (binding site in the membrane) and 2) it should be sensitive to mechanical deformation (i.e., by the endotoxically active conformation of lipid A). These characteristics are fulfilled by ion channels, in particular by a mechanosensitive channel as found by Martin et al. in macrophages (10).
It has previously been shown that K+ channels are
involved in the LPS-induced activation of monocytes, macrophages
(11, 12, 13, 14, 15), and endothelial cells (16). Most of
these authors (11, 12, 14), using nonselective
K+ channel blockers, have provided evidence for a
regulatory activity of K+ channels for
posttransscriptional processes. In particular, Walev et al.
(12) have demonstrated in comprehensive experiments using
K+-rich and K+-depleted
media as well as K+ channel blockers that
K+ efflux plays an important role in
LPS-stimulated IL-1 synthesis in monocytes. As the authors
claim, this was the first example for the control of a proteolytic
process by the major intracellular ion. Maruyama et al.
(13) and Hoang et al. (16) have already
suggested the involvement of Ca2+-sensitive
K+ channels in LPS signaling. McKinney and Gallin
(15), by measuring the whole-cell inwardly rectifying
K+ current in murine macrophage J774.1 cells,
have shown that LPS treatment changes the density of inwardly
rectifying K+ channels.
From these findings and considering that a most important requirement for signaling, the involvement of energy, would obviously be fulfilled by an ion channel, we have proposed that the modulation of a K+ channel by the lipid A moiety of LPS, depending on its shape, is one initial transmembrane step in LPS signaling.
In this paper, using excised outside-out membrane patches, we provide strong experimental evidence that a high-conductance Ca2+- and voltage-dependent K+ channel is involved in transmembrane signal transduction in macrophages as an early step and that the modulation of the channel by endotoxin is strongly sensitive to the conformation of lipid A.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Quinine, lidocaine, tetraethylammonium (TEA), charybdotoxin, iberiotoxin, and paxilline were obtained from Sigma (Deisenhofen, Germany); chlorpromazine (CPZ) was obtained from Fluka (Deisenhofen, Germany).
The anti-CD14 Ab MEM18 (IgG 1) was a kind gift of V. Horejshi (Academy of Sciences of the Czech Republic, Prague, Czech Republic).
Endotoxins
S-form LPS from the wild-type strain Salmonella enterica sv. Minnesota, which expresses the complete core sugar and O-chain polysaccharide, was obtained from phenol-killed bacteria by phenol/water extraction. Re LPS from the deep rough mutant of S. enterica sv. Minnesota strain R595, which is the LPS with only two sugars attached to lipid A, was extracted according to the phenol/chloroform/petrolether procedure. The LPS preparations were lyophilized and used in the natural salt form. Lipid A was isolated from deep rough mutant LPS from E. coli strain F515 by acetate buffer treatment (0.1 M, 100°C for 1–3 h). This lipid A contains two major subspecies, a hexaacyl and a pentaacyl fraction. These were separated by preparative TLC (kindly performed by U. Zähringer, Research Center Borstel, Germany), purified, and subsequently converted to the triethylammonium salt forms. Synthetic tetraacyl lipid A (compound) 406 was a kind gift of Shoichi Kusumoto (University of Osaka, Japan).
Electrophysiology
The patch-clamp experiments were performed at room temperature
in the outside-out excised-patch configuration (17). Patch
pipettes were made from borosilicate glass (Hilgenberg, Malsfeld,
Germany) and had resistances of (5.6 ± 1.1) M
(n = 32). The bathing solution in the patch-clamp
experiments was HBSS ([K+] = 5.8 mM,
[Ca2+] = 1 mM) (Biochrom, Berlin, Germany). LPS
was dissolved in pyrogen-free H2O in a stock
solution (1 mg/ml) and diluted in HBSS. In all experiments, LPS was
added to the bathing solution at least 5 min before measurement. The
pipette solution contained 140 mM KCl, 1.2 mM
MgCl2, 10 mM HEPES, 0.97 mM
CaCl2, 1.92 mM EGTA; pH was adjusted with KOH to
7.2. For the determination of the Ca2+-dependence
of the channel, the concentration of free Ca2+
was adjusted with different concentrations of
CaCl2 and EGTA and controlled photometrically
using the Ca2+-sensitive dye fura-2.
K+ current was amplified with a patch-clamp
amplifier (LM-PC/A; List-Electronic, Darmstadt, Germany), filtered with
an 8-pole Bessel-filter (Frequency Devices, Haverhill, MA) (10/20/50
kHz), and digitized with a 12-bit analog digital converter (Dalanco
Spry Model 250; Dalanco, Rochester, NY); sample frequency was 100 kHz,
and recording time was 6 or 12 s. The software applied for
recording and further evaluation of the time series was "sample250"
and "day+night" (Division of Biophysics, University of Kiel, Kiel,
Germany). The open probability, defined as the probability to find one
specific channel in an open state at a certain time t, was
calculated from the ratios p(k) of the total
occupancies of the several levels k to the recorded time
(18):
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Open probability is normalized to the maximum number of channels visible in one patch under any of the applied experimental conditions. Experiments were done at least in triplicate, and in the respective figures the results are depicted from one representative experiment.
Cell culture
Monocytes were isolated from human peripheral blood by the
Hypaque-Ficoll gradient method and stored in RPMI 1640 medium
(endotoxin 
0.01 EU/ml; Biochrom, Berlin, Germany) plus 2%
penicillin/streptomycin/glutamine plus 4% human serum (blood group AB,
heat inactivated) at 37°C and 6% CO2. For
dividing the lymphocytes from the monocytes, all nonadherent cells were
removed after 2 h. M-CSF (2 ng/ml) was added to the medium for 7
days to differentiate the monocytes to macrophages (19).
The cells were used for electrophysiological experiments on days 6–9.
To determine the cytokine-inducing capacity of LPS and its inhibition
by various channel blockers, human mononuclear cells and macrophages
were stimulated by adding LPS and the blockers in the concentrations
mentioned below and subsequently incubated for 4 h. TNF-
production was determined in the supernatant using the ELISA technique;
cytokine-mRNA production was determined by the RT-PCR technique.
Viability was found to be >96% applying the trypan blue method.
TNF- ELISA
Supernatants were collected after centrifugation of the culture
plates for 10 min at 400 x g and stored at -20°C
until determination of cytokine content. TNF- in the cell
supernatant was determined in a sandwich ELISA as described elsewhere
(20). Ninety-six-well plates (Greiner, Solingen, Germany)
were coated with a mAb against TNF- (clone 6b from Intex AG,
Muttent, Switzerland). Cell culture supernatants and the standard
(rTNF-; Intex) were diluted with buffer. After exposure to
appropriately diluted test samples and serial dilutions of standard
rTNF-, the plates were exposed to peroxidate-conjugated rabbit
anti-rTNF- Ab. The plates were shaken 16–24 h at 4°C. For
removal of free Ab, the plates were washed six times in distilled
water. Subsequently, the color reaction was started by addition of
tetramethylbenzidine/H2O2
in alcoholic solution and after 5–15 min stopped by the addition of 1
M sulfuric acid. In the color reaction, the substrate is cleaved
enzymatically, and the product can be measured photometrically. This
was done on an ELISA reader (Rainbow; Tecan, Crailsheim, Germany) at a
wavelength of 450 nm, and the values were related to the standard.
TNF- was determined in duplicate at two different dilutions, and the
values were averaged.
PCR experiments
After incubation, cells were washed in PBS,
centrifuged (400 x g, 5 min), and the pellets were
frozen at -70°C until further analysis. The mRNA of 5 x
105 cells/assay was isolated using
oligo(dT)-coated magnetic beads (Dynal, Hamburg, Germany) according to
the manufacturer. Reverse transcription was performed in a reaction mix
containing 1 mM oligo(dT)20 primers, 10 mM dNTP
(Pharmacia, Freiburg, Germany), 10 mM DTT (Life Technologies,
Karlsruhe, Germany), 10 U RNAGuard (Pharmacia), and 200 U Superscript
(Life Technologies) at 37°C for 1 h. PCR was conducted using the
following gene-specific intron-spanning primers at the specified
annealing temperatures: -actin (sense, 5'-AGC GGG AAA TCG TGC GTG;
antisense, 5'-CAG GGT ACA TGG TGG TGC C; 55°C), IL-6 (sense, 5'-CTT
TTG GAG TTT GAG GTA TAC CTA G; antisense, 5'-GCT GCG CAG AAT GAG ATG
AGT TGT C; 52°C), TNF- (sense, 5'-GAG TGA CAA GCC TGT AGC;
antisense, 5'-CCC TTC TCC AGC TGG AAG; 55°C). The reaction mixture
contained 1.5 ml of the cDNA preparation, 20 mM sense and antisense
primers, 10 mM dNTPs (Pharmacia), and 1.25 U Taq Polymerase
(Life Technologies) in a final volume of 50 ml. DNA fragments were
analyzed in a 2% agarose gel electrophoresis.
Small-angle x-ray diffraction
Small-angle x-ray diffraction measurements for the determination of the aggregate structure of pentaacyl lipid A in the absence and presence of chlorpromazine were performed at the European Molecular Biology Laboratory outstation at the synchrotron radiation facility HASYLAB (c/o Deutsches Elektronen Synchrotron, Hamburg, Germany). The lipid A samples were prepared in 85% HEPES buffer (20 mM), incubated at 50°C, vortexed, and recooled to 4°C. This temperature cycle was repeated twice, and the samples were stored at 4°C 24 h before measurement. From the aggregate structures, the conformation of the individual molecules was inferred: cylindrical in the case of lamellar structures (the cross-sections of the hydrophilic and hydrophobic moieties are identical) and conical/concave in the case of inverted cubic and HII structures (the cross-section of the hydrophobic is larger than that of the hydrophilic portion) (3).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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1 µm2, n
= 10/day) on days 1–3 to 
2.5 on day 5. We focused our experiments
on the high-conductance channel (210 pS in symmetrical 140 mM
K+). It was characterized to be calcium- and
voltage-dependent, which can be taken from the left shift of the open
probability with increasing Ca2+ concentration
and from its increase with increasing voltage, respectively (Fig. 1
). It was further characterized to be
sensitive to quinine (100 µM) and paxilline (5 µM). These
properties are indicative of a MaxiK channel (21, 22)
Surprisingly, however, the channel was only sensitive to very high
concentrations of charybdotoxin and iberiotoxin (50 nM). This
behavior may be explained by the type of subunit present in the
MaxiK channel in macrophages, which can modulate the affinity of these
compounds to the MaxiK channel (23, 24) The complete
characterization of the subunit will be subject of further work.
Here, we used paxilline as a channel blocker, which is known to be
selective for MaxiK channels independent of the subunit
(25).
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, a and b), and
the value of the membrane potential, at which first events could be
observed, was decreased. From these results it can be concluded that
endotoxin leads to a considerable increase of channel activity at a
given membrane potential. However, this increase of channel activity
does not affect the single-channel conductance, but can be quantified
by calculating the open probability of the channel. Thus, the open
probability showed a dose-dependent increase upon the addition of Re
LPS to the outside of the membrane in physiological concentrations
(2–20 ng/ml) (Fig. 3a).
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c).
Furthermore, the increase of the open probability following the
addition of Re LPS (10 ng/ml) could be inhibited by compound 406 (Fig. 2d), showing the direct inhibition of the LPS-induced
activation of the signal transducer by an endotoxin antagonist.
Compound 406 was applied here in a 10-fold higher concentration merely
to make its antagonizing activity more obvious (27).
The distinction between agonists and antagonists has to take place at
the signaling protein, because Delude et al. (28) have
already ruled out CD14 for this function. To investigate a possible
CD14 dependence of channel activation, CD14 was blocked by a monoclonal
anti-CD14 Ab (MEM18) inhibiting binding of LPS to CD14 at a
concentration of 10 µg/ml (29). This led to inhibition
of channel activation by Re LPS (20–40 ng/ml) (Fig. 3b),
clearly demonstrating the upstream involvement of CD14 in channel
activation in accordance to the proposed role of CD14 in the activation
process by endotoxin (6, 30).
Based on results from the literature showing that potassium channel
blockers can inhibit macrophage activation (12, 13), we
verified the physiological relevance of our findings by incubating HMDM
with various specific or nonspecific channel blockers and subsequently
determining LPS-induced TNF- and IL-6 production. We found a clear
correlation between the sensitivity of the channel to the various
agents and their ability to inhibit LPS-induced TNF- and IL-6
production. In particular, the MaxiK-selective blocker paxilline
inhibited cytokine production, whereas apamin as a blocker of
small-conductance Ca2+-activated
K+ channels (31) neither blocked the
channel nor inhibited cytokine production (Fig. 4). The TNF- release was almost
completely suppressed by the nonselective channel blockers quinine (100
µM) and TEA (10 mM) in freshly isolated monocytes and macrophages
(data not shown). Paxilline (5 µM) blocked TNF- release in
macrophages, but had no significant effect on monocytes (Fig. 5). These results are consistent with the
expression pattern of MaxiK channels in monocytes/macrophages and
indicate that MaxiK channels are the essential targets of paxilline in
the blockade of cytokine production.
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, a and b). For
this, it is assumed that lipid A and CPZ molecules form stable
complexes, which are preserved when extracted from the aggregate, e.g.,
by the action of LBP.
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c). In a number of independent control experiments it
was shown that in this concentration range CPZ alone had no effect on
the open probability. This observation excludes the possibility that
CPZ in this low concentration range and in the absence of lipid A
causes membrane alterations that would affect channel gating. At the
endotoxin concentration applied, CPZ concentrations above 1 µM led to
channel blocking as was observed for CPZ alone at comparable
concentrations (data not shown). This decreased channel activity at
higher CPZ concentrations is consistent with the decrease in the
IL-6-inducing capacity described by Thiéblemont et al.
(32). With these experiments we have, for the first time,
shown a direct relation between the capacity of endotoxin to activate
the immune system, i.e., to induce cytokine production in macrophages,
and the structure and conformation of the endotoxin molecules.
First evidence for an involvement of K+
channels in LPS signal transduction has been provided by Maruyama et
al. (13), who described the inhibition of LPS-induced
cytokine production by the nonspecific K+ channel
blocker quinine, and by Walev et al. (12), who have
provided a first example for the control of a proteolytic process by
the major intracellular ion by showing that K+
efflux plays an important role in LPS-stimulated IL-1 synthesis in
monocytes. McKinney and Gallin (15) have shown that LPS
treatment changes the density of inwardly rectifying
K+ channels, and Hoang et al. (16)
and Maruyama et al. (13) have described the activation of
high-conductance Ca2+-activated
K+ channels by LPS in artery smooth muscle cells
and in human alveolar macrophages. And most interestingly, in a very
recent publication Chen et al. (34) have reported on the
expression of activated large conductance
Ca2+-activated K+ channels
in vascular smooth muscle in rats with endotoxic shock. The authors
suggest that the channels are activated by an LPS-induced
overproduction of NO, i.e., as a secondary or even tertiary step of LPS
signaling, and that they contribute to endotoxin-mediated vascular
hyporeactivity.
In our experiments, we applied LPS in physiological concentrations (a few nanograms) to the outside of outside-out membrane patches. Hereby, we have shown that the interaction between LPS and the channel in the cytoplasmic membrane of macrophages behaves in a manner consistent with known characteristics of LPS signaling, regarding both biological data as well as the physicochemical parameters of endotoxin, pointing toward an LPS signaling mechanism via a MaxiK channel as a primary step.
Kirber et al. (35) reported on the activation of a MaxiK channel in vascular smooth muscle cells by free negatively charged fatty acids, in particular myristic acid (14:0). In our experiments, the underlying mechanism of channel activation is not the interaction of fatty acids per se, because the agonistic hexaacyl lipid A activates the channel, whereas the antagonistic tetraacyl lipid A part structure, compound 406, does not. Obviously, channel activation by free fatty acids and by lipids underlie different mechanisms. For the latter, the molecular conformation is a most important parameter that is governed by the number of fatty acids in relation to the size of the backbone.
It has previously been shown that the sensitivity of monocytes to
activation by LPS goes along with their adherence (15) and
that the number of K+ channels expressed on
freshly isolated monocytes is very low and is increased by adherence
(36), long-term stimulation with LPS (37), or
mechanical stress (10). Furthermore, Martin et al.
(10) have shown that, during the process of adherence of
monocytes to vascular endothelium, stimulation of the merging
macrophages takes place, leading to the activation of potassium
channels. These effects are consistent with the observed increase in
the number of high-conductance Ca2+-dependent
K+ channels expressed during the differentiation
of monocytes to macrophages and the different effects of paxilline on
LPS-induced TNF- release from monocytes and macrophages (see
above).
In this paper, we have provided strong experimental evidence that
modulation of an ion channel is a very early step in LPS-induced
transmembrane signaling in macrophages. We could define the channel to
be a high-conductance Ca2+-sensitive and
voltage-dependent K+ channel. Channel modulation
is strongly dependent on the molecular conformation of the lipid A
moiety and involves the membrane-bound LPS receptor mCD14, as shown by
the anti-CD14 Ab-mediated blockade of the channel activation. The
involvement of further membrane-bound or transmembrane proteins remains
to be elucidated. It has been shown in the literature that members of
the Toll-like receptor (TLR) family play an important role in LPS
signaling (38, 39, 40, 41). Thus, TNF- release could be
significantly reduced by an Ab recognizing TLR4/MD-2 complexes
(41) (it has been previously shown that TLR4-mediated
signaling requires the associated protein MD-2 (42)).
However, from our channel-blocking experiments with paxilline, it
follows that the TLR pathway is not independent from the described
K+ channel. Furthermore, our data together with
earlier findings in the literature (12, 15) reveal a
switch between the mechanisms of LPS-induced activation of monocytes
and macrophages. Nevertheless, it seems very likely that in both cell
types K+ channels are involved in LPS-induced
transmembrane signaling, because induction of cytokine production in
macrophages could be inhibited by the selective channel blocker
paxilline as well as by the nonselective blockers quinine and TEA and
that in monocytes only by the latter. This implies that the channels
involved in activation of monocytes and macrophages differ in their
structure and function. Also, from our earlier work on the correlation
between molecular conformation and cytokine-inducing capacity of lipid
A using PBMC and the present results on macrophages it seems evident
that this correlation is valid independent of the particular signaling
mechanisms in the different cell types or of the state of
differentiation.
Details of the signaling cascade, but also of the interaction mechanisms of LPS with the channel, remain to be elucidated. This includes also the involvement of further membrane-bound and serum proteins and a deeper understanding of the role of agonistic and antagonistic compounds such as lipid A precursors or part structures and phospholipids (43).
To our knowledge, our data show for the first time the direct involvement of a transmembrane protein in the very early steps of LPS signaling.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ulrich Seydel, Division of Biophysics, Department of Immunochemistry and Biochemical Microbiology, Research Center Borstel, Center for Medicine and Biosciences, Parkallee 10, D-23845 Borstel, Germany.
3 Abbreviations used in this paper: mCD14, membrane-bound CD14; sCD14, soluble CD14; CPZ, chlorpromazine; HMDM, human macrophages derived from peripheral blood monocytes; LBP, LPS-binding protein; RsLPS, LPS from Rhodobacter sphaeroides; RsLA, lipid A from Rhodobacter sphaeroides; TEA, tetraethylammonium; TLR, Toll-like receptor.
Received for publication June 23, 2000. Accepted for publication October 18, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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in human monocytes. EMBO J. 14:1607.[Medline]
from human alveolar macrophages. Am. J. Respir. Cell Mol. Biol. 10:514.[Abstract]
-subunit homolog. Proc. Natl. Acad. Sci. USA 96:4137. subunit of the high-conductance calcium-activated potassium channel contributes to the high-affinity receptor for charybdotoxin. Proc. Natl. Acad. Sci. USA 94:2853. subunit (KCNMB4) makes the large conductance, voltage- and Ca2+-activated K+ channel resistant to charybdotoxin and iberiotoxin. Proc. Natl. Acad. Sci. USA 97:5562.This article has been cited by other articles:
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 C. Stamme, M. Muller, L. Hamann, T. Gutsmann, and U. Seydel Surfactant Protein A Inhibits Lipopolysaccharide-Induced Immune Cell Activation by Preventing the Interaction of Lipopolysaccharide with Lipopolysaccharide-Binding Protein Am. J. Respir. Cell Mol. Biol., September 1, 2002; 27(3): 353 - 360. [Abstract] [Full Text] [PDF]
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T. Gutsmann, M. Muller, S. F. Carroll, R. C. MacKenzie, A. Wiese, and U. Seydel Dual Role of Lipopolysaccharide (LPS)-Binding Protein in Neutralization of LPS and Enhancement of LPS-Induced Activation of Mononuclear Cells Infect. Immun., November 1, 2001; 69(11): 6942 - 6950. [Abstract] [Full Text] [PDF]
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2, 5.12 nm =
aQ/
, Control; •, plus 5
nM (
,
plus coincubated pentaacyl lipid A and CPZ (5 nM/100 nM). Curves
represent the results from one representative of at least three
independent experiments.