* Institut für Molekulare Medizin und Zellforschung, AG Tumorimmunologie und Vakzineforschung, and
AG Biophysik und Strahlenbiologie, Medizinische Fakultät der Universität Freiburg, 79104 Freiburg, and
Jomol Pharma GmbH, Regensburg, Germany
Correspondence: Silke Pfannes, AG Tumorimmunologie/Vakzine, Institut für Molekulare Medizin und Zellforschung, Stefan-Meier-Strasse 8, D-79104 Freiburg i.Br., Germany. E-mail: pfannes{at}uni-freiburg.de
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(TNF-) by bone
marrow-derived macrophages (BMDMs) of different mouse inbred strains
after they were stimulated with the lipopeptide
P3CSK4, a water-soluble synthetic analogue of
the lipidated N terminus of bacterial lipoprotein. The lipopeptide was
able to induce a strong, long lasting release of oxygen radicals in
BALB/c mouse macrophages. Furthermore, it induced nitric oxide release
from BMDMs of several mouse strains (BALB/c, C57Bl/6, C57Bl/10ScSn,
Sv129, NMRI, and LPS-nonresponder C57Bl/10ScCr). Stimulation with
P3CSK4 also resulted in comparable production
of TNF- in LPS-responder and nonresponder BMDMs from C57Bl/10ScSn
mice and C57Bl/10ScCr mice, respectively. All three antitumoral
mediators reached functional levels or concentrations as shown by the
strong cytostatic/cytotoxic activity of lipopeptide-activated
macrophages for the cell lines Abelson 8-1, M12.5/P815, and L929, which
are sensitive to ROIs, nitric oxide, and TNF-, respectively. We
found that synthetic lipopeptides can induce the secretion of effective
levels of soluble tumor-cytotoxic/cytostatic mediators in
BMDMs of LPS-responsive and, of particular interest, also of
LPS-unresponsive mice. This result could indicate that the highly
effective bacterial-macrophage activators
P3CSK4 and LPS use different receptors and/or
different intracellular signal transduction pathways.
Key Words: nitric oxide • oxidative burst • tumor necrosis factor- • lipopeptide
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(TNF-) and interleukin (IL) 1ß [4
,
5
], as well as reactive oxygen intermediates and reactive
nitrogen intermediates (ROIs and RNIs, respectively), e.g., superoxide,
hydrogen peroxide, and nitric oxide [6
]. Bacterial
compounds such as lipopolysaccharides (LPSs) and muramyl-dipeptides
(MDPs) are potent activators of macrophages and have been investigated
extensively as inducers of antitumoral activities [7
,
8
]. We and others [9
10
11
] recently have
shown a similar immunostimulating potential for lipopeptides and
lipoproteins from the outer cell walls of gram-negative bacteria. Thus,
we could also show that synthetic lipopeptides, which represent
analogues of the lipidated N-termini of native lipoproteins, stimulate
macrophages for the enhanced production of cytokines such as TNF-,
IL-1, and IL-6 and the release of nitrogen radicals [12
,
13
]. Furthermore, we could demonstrate that
lipopeptide-stimulated macrophages are cytostatic/cytotoxic for tumor
cells in vitro [14
]. Mice with mutations in the LPS gene
locus on chromosome 4 are highly resistant to LPS effects
[15
, 16
]. In contrast, no mouse
nonresponder strain has been described so far for lipopeptides or
lipoproteins [17
]. Furthermore, lipoproteins and
lipopeptides can induce similar mitogenic and immunogenic responses in
cells from both LPS-responder and LPS-nonresponder mice
[17
18
19
20
21
]. Here we show that the water-soluble, synthetic
lipopeptide
N-palmitoyl-S-[2,3-bis(palmitoyloxy)-propyl]-(R)-cysteinyl-(lysyl)3-lysine
(P3CSK4) induced the release of oxygen and
nitrogen radicals as well as the production of TNF- in bone
marrow-derived macrophages from various mouse inbred strains.
Macrophages from C57Bl/10ScSn (LPS-responder) and C57Bl/10ScCr
(LPS-nonresponder) mice were activated to a comparable extent, and this
stimulation could be enhanced by simultaneous addition of interferon
(IFN-). Stimulation of ROI, RNI, and TNF- production by
P3CSK4 reached functional levels, as was shown
by strong cytostasis/cytotoxicity of the lipopeptide-activated
macrophages on Abelson 8-1, M12.5/P815, and L929 tumor cells,
respectively. |
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was purchased from
Pharmingen/Becton Dickinson, Hamburg, Germany. LPS from
Salmonella abortus equi was a kind gift from C. Galanos,
Max-Planck-Institut für Immunbiologie, Freiburg, Germany, and
S. abortus equi S1301 kindly donated by B. Kleine, Institut
für Molekulare Medizin und Zellforschung, AG Tumorimmunologie und
Vakzineforschung, Freiburg, Germany.
NG-Monomethyl-L-arginine
(L-NMMA) and N-acetyl-L-cysteine
(NAC) were purchased from Sigma (Deisenhofen, Germany).
Mice
Female and male strains BALB/c, C57Bl/6, C57Bl/10, and 129 SV
mice, 6 to 10 weeks old, were obtained from the breeding facilities at
the Max-Planck-Institut für Immunbiologie. For some experiments,
strains C57Bl/6 and C57Bl/10 mice were purchased from Bomholtgård, Ry,
Denmark.
Tumor cells
The murine fibroblast cell line L929, the murine mastocytoma
cell line P815, and the murine B-cell lymphoma cell line M12.4 were
kept in RPMI-1640 medium (Gibco BRL, Eggenstein, Germany), supplemented
with 10% heat-inactivated fetal calf serum (FCS), 1% nonessential
amino acids, 2 mM L-glutamine, 100 U/mL of penicillin, and
100 µg/mL of streptomycin (cRPMI) (all from Seromed Biochrom KG,
Berlin, Germany). The murine B-cell lymphoma cell line Abelson 8-1 was
kept in Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L of
glucose (Seromed Biochrom KG), supplemented with 10% FCS, 2 mM
L-glutamine, 1% nonessential amino acids, 100 U/mL of
penicillin, and 100 µg/mL of streptomycin (cDMEM).
Murine bone marrow-derived macrophages
Murine bone marrow-derived macrophages (BMDMs) were
differentiated in vitro from bone marrow precursor cells as described
in detail by Hoffmann et al. [14
]. Briefly, bone marrow
cells were flushed from femurs and tibias of 6- to 10-week-old mice,
washed twice in RPMI 1640, and grown for 10 days in liquid cultures in
Teflon film bags (SLG, Gauting, Germany) at 37°C and 5%
CO2. The culture medium consisted of RPMI 1640 supplemented
with 15% L-cell-conditioned medium as a source of macrophage
colony-stimulating factor, 10% heat-inactivated FCS, 5%
heat-inactivated horse serum, 1 mM sodium pyruvate (both from Seromed
Biochrom KG), 50 U/mL of penicillin, 50 µg/mL of streptomycin, and
5 x 10-5 M 2-mercaptoethanol. Cultures were set up
with 6 x 106 cells/50 mL. After harvesting, the
macrophages were washed once, counted, and resuspended at 2 x
106 cells/mL in test medium. For the preparation of
L-cell-conditioned medium, 105 L929 cells/mL were cultured
in 100-mL batches in cell culture flasks (Falcon; Becton Dickinson,
Heidelberg, Germany) in cRPMI 1640 at 37°C and 5% CO2.
After 7 days, the culture supernatants were harvested, cleared from
cell debris by centrifugation (1,500 x g, 4°C, 15
min), and stored at -20°C.
Induction and determination of nitric oxide release in murine BMDMs
Mature BMDMs were harvested, washed once, and resuspended in
cRPMI medium. Cells (105/well) were seeded into the wells
of 96-well flat-bottom microtiter plates (Falcon) and stimulated with
various concentrations of P3CSK4 or LPS in the
presence or absence of the costimulus IFN- or the nitric oxide
synthase (NOS) inhibitor L-NMMA in a total volume of 150
µL. Culture supernatants were harvested after 42 h (and for the
kinetics studies also after 19 and 72 h). All assays were
performed in triplicate. Production of nitric oxide was determined by
measuring nitrite, a stable metabolite of nitric oxide, in the culture
supernatants using the Griess reaction [6
]: One hundred
microliters of culture supernatant were mixed with 100 µL of Griess
reagent [1% sulfanilamide and 0.1%
N-(1-naphthyl)ethylendiamine in 2.5% phosphoric acid], and
the absorbance at 550 nm was monitored with a Dynatech MRX
enzyme-linked immunosorbent assay plate reader (Denkendorf, Germany).
Nitrite concentrations were calculated by using sodium nitrite as a
standard.
Macrophage-mediated growth inhibition of Abelson 8-1 tumor
cells
Macrophage-mediated tumor-cytostatic/cytotoxic activity against
Abelson 8-1 tumor cells was determined by applying the alkaline
phosphatase assay, as described by Modolell et al. [23
].
Briefly, cocultures of 5 x 103 Abelson 8-1 tumor
cells together with 1 x 105 BMDMs were set up in
cDMEM in flat-bottom microtiter plates in a total volume of 200 µL
and incubated in the presence of various stimuli at 37°C and 10%
CO2. After 3 days, the plates were centrifuged at 660 x g for 2 min, and the supernatants were decanted. To each
well, 100 µL of buffer (pH 10.2) containing diethanolamine (200 mM),
MgCl2 (2 mM), Triton X-100 (1%), and
p-nitrophenylphosphate (10 mM) were added, and the plates
were incubated for 60 min at room temperature in the dark on a
horizontal shaker. The enzyme reaction was stopped by adding 100
µL/well of 0.5 M NaOH. Absorbance was measured at 405 and 490 nm in
an automated enzyme-linked immunosorbent assay reader (MRX Dynatech,
Denkendorf, Germany). Optical density values of cultures containing
tumor cells and unstimulated effecter cells were set to 100%.
Macrophage-mediated cytostasis of P815 and M12.4 tumor cells
Target cells were seeded into flat-bottom microtiter plates
(104/100 µL/well in cRPMI) and incubated at 37°C and
5% CO2 for 24 h. Mature BMDMs were harvested the
following day, cell density was adjusted to 2 x
106/mL, and 50 µL were added to each well. The effecter
cells were stimulated with either P3CSK4 or LPS
from S. abortus equi S1301 in a final volume of 200 µL for
20 h. Then cells were pulsed for 4 h by the addition of
23.125 kBq [3H]TdR/well (925 kBq/mL; Amersham,
Braunschweig, Germany). After freezing and thawing, labeled culture DNA
was transferred to glass fiber filters with an automatic cell harvester
(LKB1295-001; Pharmacia Upjohn, Freiburg, Germany). The incorporated
radioactivity was measured in a liquid scintillation counter (LKB
Betaplate 1205; Pharmacia Upjohn). Cytostasis was expressed as
percentage of inhibition of [3H]TdR incorporation
compared with control cultures (cocultures of unstimulated BMDMs and
tumor cells).
Macrophage-mediated tumor cell cytotoxicity
The killing of labeled tumor cells was measured by a
[3H]thymidine ([3H]TdR) release assay.
Briefly, tumor cells were cultured in cRPMI and were labeled with
13.875 kBq of [3H]TdR/mL for 20 h at 37°C and 5%
CO2. Four hours before use, the cells were washed twice
with RPMI 1640 and maintained in cRPMI without radioactivity. The
assays were performed in flat-bottom microtiter plates (Becton
Dickinson) at an effector-to-target cell ratio of 10:1. BMDMs
(105/100 µL) were added to the wells and stimulated with
either P3CSK4 or LPS (from Escherichia
coli strain O55:B5 [Sigma]) in a final volume of 200 µL for
2 h. Thereafter, the macrophages were washed twice, and
104 labeled tumor cells were added to each well in a final
volume of 200 µL. The cocultures were incubated for 48 h at
37°C and 5% CO2. At the end of the coculture period, the
radioactivity released into the supernatant was determined in an
aliquot of 100 µL/well by liquid scintillation counting. Results are
expressed as the percentages of specific lysis, as calculated by the
following formula: percent specific lysis =
([cpmexp -
cpmspont]/[cpmtotal -
cpmspont]) x 100. Spontaneous release
(cpmspont) was determined in microwells containing only
labeled tumor cells. Total release (cpmtotal) was
determined by lysis of the tumor cells with 0.5% sodium dodecyl
sulfate.
Detection of ROIs by chemiluminescence
BMDMs were suspended in BM 86 medium (Boehringer Mannheim,
Mannheim, Germany) supplemented with 2% FCS and placed into
chemiluminescence (CL) tubes (5 x 105 BMDMs/100
µL). Four hundred microliters of Hanks’ balanced saline solution
without phenol red (Seromed Biochrom KG) containing the stimulus were
added, and the macrophages were stimulated for different periods at
37°C. The CL reaction was started by the addition of 10 µL of
lucigenin (final concentration, 0.1 mM; Sigma), and the baseline was
recorded for 5 min at 37°C. Thereafter, macrophages were incubated
with a known inducer of CL (40 µL/tube of zymosan; final
concentration, 500 µg/mL; Sigma), and the CL response was measured
with an LB 9505 C luminometer (Berthold, Wildbad, Germany).
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 View larger version (46K): [in a new window] |
Figure 1. Dose-dependent induction of NO release by the lipopeptide
P3CSK4. Murine BMDMs from the four different
inbred mouse strains C57BI/6 (a), C57BI/10ScSn (b), BALB/c (c), and
129Sv (d) were seeded into the wells of 96-well microtiter plates and
stimulated with P3CSK4 or LPS. NO release was
determined by measuring the nitrite concentration in the culture
supernatants using the Griess reaction. Values represent means ±
SD of triplicate cultures.
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 View larger version (38K): [in a new window] |
Figure 2. Inhibition of iNOS activity by L-NMMA. Macrophages from
BALB/c, C57BI/10ScSn, or C57BI/10ScCr mice were stimulated with
P3CSK4 without or with the iNOS inhibitor
L-NMMA or in medium alone (control). Nitrite concentrations
in the culture supernatants were determined using the Griess reaction.
Values represent means ± SD of triplicate cultures.
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-mediated tumor cytotoxicity of BMDMs
stimulated with P3CSK4|
View this table: [in a new window] |
Table 1. Induction of Tumor Cytotoxicity in BALB/c BMDMs by
P3CSK4
|
. As shown in Table 1
, by means of lysis of the
TNF--sensitive tumor cell line L929, P3CSK4
stimulation of BMDMs for only 2 h was sufficient to produce
effective quantities of this cytokine, resulting in up to 85% lysis
during the subsequent coculture. In contrast, stimulation of
macrophages with LPS under the same conditions was less efficient,
leading to the lysis of 47% of the tumor cells. Addition of
L-NMMA to these cultures resulted in only partial
inhibition of the cytotoxic effect, indicating that RNIs were most
probably also involved in the process, however not as the main cause of
tumor cell death.
Induction of the oxidative burst and of ROI-mediated
antitumoral activity in BMDMs by
P3CSK4
As demonstrated in the preceding experiments,
lipopeptide-stimulated BMDMs are able to produce TNF- and RNIs to
attack different sensitive target cells. Other members of the family of
cytostatic/cytotoxic factors released by activated macrophages are
ROIs. We therefore investigated the induction of the oxidative burst in
BMDMs by determination of the lucigenin-enhanced CL. For this approach,
5 x 105 BMDMs were stimulated with
P3CSK4 or LPS from E. coli O111:B5
at various concentrations for 1 h at 37°C. Recording of the CL
reaction was started after the addition of lucigenin and zymosan as the
triggering substances. As can be seen in Figure 3a
, both P3CSK4 and LPS primed BMDMs for the
zymosan-mediated oxidative burst. In comparison to unstimulated BMDMs
(i.e., CL activity set to 100%), P3CSK4- as
well as LPS-stimulated macrophages showed maximum production of ROIs
after an incubation period of 60 min. However, whereas the oxidative
burst of LPS-primed BMDMs increased only twofold (200% CL), maximum CL
of BMDMs stimulated with 10 and 50 µg/mL of
P3CSK4 was over 400% and nearly 800% that of
comparable control cells, respectively.
 View larger version (32K): [in a new window] |
Figure 3. Induction of oxidative burst in BMDMs. (a) Murine BMDMs were stimulated
with P3CSK4 or LPS from E. coli
O111:B4. Lucigenin was added to the samples, and a baseline of
lucigenin-enhanced CL was recorded. The priming effect of
P3CSK4 or LPS on the production of ROIs was
determined after the addition of zymosan as triggering substance. (b)
The time course of ROI production of murine
P3CSK4 or LPS from E. coli
O111:B4-primed BMDMs after restimulation with P815 tumor cells was
recorded. (c) The CL response of cocultured BMDMs was integrated, and
enhancement of ROI production was calculated by setting the CL response
of cocultured, unstimulated BMDMs (Control) to 100%.
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To further substantiate these findings, BALB/c macrophages stimulated
with various concentrations of P3CSK4 were
cultured together with cells of the murine B-cell lymphoma Abelson 8-1
at an effector-to-target cell ratio of 20:1. Macrophage-mediated tumor
cell growth inhibition (TCGI) was determined by measuring the alkaline
phosphatase activity of the remaining viable tumor cells after 3 days.
As can be seen in Figure 4
, P3CSK4-activated macrophages showed a
strong, dose-dependent TCGI, resulting in up to 90% growth
reduction at agent concentrations of 10, 1, and 0.1 µg/mL.
Macrophages activated with 0.01 µg/mL of
P3CSK4 were not able to bring about growth
reduction. The activity of P3CSK4 could not be
inhibited by the addition of L-NMMA at a concentration
leading to complete blocking of nitric oxide release (1 mM) nor by
addition of anti-TNF- antibodies at appropriate concentrations (data
not shown). In contrast, when preincubated for 1 h in medium
containing the oxygen radical scavenger NAC, the macrophages showed a
markedly reduced TCGI capacity (approximately 50% reduction at an NAC
concentration of 83 µM and a P3CSK4
concentration of 0.1 µg/mL). This strongly suggests that
P3CSK4 is capable not only of inducing
effective quantities of nitric oxide but also of generating ROIs at
concentrations sufficient for antitumoral effects.
 View larger version (52K): [in a new window] |
Figure 4. P3CSK4 induced macrophage-mediated TCGI against
Abelson 8-1 tumor cells. For determination of tumor cell growth
inhibition, murine BMDMs from BALB/c mice and Abelson 8-1 tumor cells
were cocultured for 3 days in flat-bottom microtiter plates in the
presence of P3CSK4 either alone or together
with NAC. Tumor cell numbers at the end of the incubation time were
determined by alkaline phosphatase assay as described in Materials and
Methods. The growth of tumor cells in the presence of unstimulated
macrophages served as controls (0% inhibition). Values represent
means ± SD of triplicate determinations.
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 View larger version (30K): [in a new window] |
Figure 5. Comparison of NO release induced by P3CSK4 and
LPS in BMDMs from LPS-responder C57Bl/10ScSn (A) and -nonresponder
C57Bl/10ScCr (B) mice. Murine BMDMs were stimulated with
P3CSK4 or LPS (inserts). Nitrite concentrations
in the culture supernatants were determined using the Griess reaction.
Values represent means ± SD of triplicate cultures.
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 View larger version (31K): [in a new window] |
Figure 6. Time course of NO release induced by
P3CSK4 and LPS. Murine BMDMs from LPS-responder
C57Bl/10ScSn or LPS-nonresponder C57Bl/10ScCr mice were stimulated with
P3CSK4 and LPS for the times indicated. Culture
supernatants were harvested at the time points indicated and tested for
nitrite contents using the Griess reaction. Values represent means ± SD of triplicate cultures.
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is known to enhance the release of nitric oxide from murine
macrophages activated by other stimuli, e.g. LPS or TNF-. In the
next set of experiments, we therefore examined whether the
P3CSK4-induced release of nitric oxide could
also be enhanced by IFN- in an additive or even synergistic way and,
if so, whether LPS responder and nonresponder cells would respond in a
similar way. BMDMs from C57Bl/10ScSn and C57Bl/10ScCr mice were
stimulated with different concentrations of
P3CSK4 (0.01–1 µg/mL) in the absence or
presence of 0.5–12.5 U/mL of IFN- for 42 h. As can be seen in
Figure 7
, all IFN- concentrations tested showed a marked synergistic
activity especially with 0.1 and 1 µg/mL of
P3CSK4. Similarly to LPS responder animals, LPS
nonresponder macrophages produced nearly comparable amounts of
NO2- after combined stimulation with the
lipopeptide and IFN-.
 View larger version (50K): [in a new window] |
Figure 7. Enhancement of the P3CSK4-induced NO release
from murine BMDMs by IFN-. Murine BMDMs from C57Bl/10ScSn and
C57Bl/10ScCr mice were stimulated with P3CSK4
either alone or together with IFN-. Nitrite concentrations in
supernatants were determined using the Griess reaction. Values
represent means ± SD of triplicate cultures.
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-sensitive cell line L929 as well as against the
nitric oxide-sensitive cell line P815 in BMDMs from both LPS-responder
and -nonresponder animals. As expected, only BMDMs from LPS-responder
mice showed a comparable cytostatic activity against the two tumor cell
lines after stimulation with LPS.
 View larger version (40K): [in a new window] |
Figure 8. Cytostatic activity of BMDMs against L929 or P815 tumor cells. Tumor
cells were incubated in microtiter plates. BMDMs were added to each
well and stimulated with P3CSK4 or LPS from
S. abortus equi S1301. Proliferating tumor cells were pulsed
with [3H]TdR. Cytostasis was expressed as percentage of
inhibition of [3H]TdR incorporation into target cell DNA
compared to control cultures.
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Animals of the C57Bl/10ScCr strain, like those of the LPS-nonresponder
strain C3H/HeJ, bear a mutation in the LPS gene on chromosome 4
[15
]. However, whereas the C3H/HeJ allele
(Lpsd) is codominant, that of C57Bl/10ScCr
animals is strictly recessive [15
]. In addition, the two
strains differ in their IFN- response to microbial stimuli; whereas
C3H/HeJ (as well as C57Bl10/ScSn) splenocytes readily produce IFN-
after stimulation with various bacteria, C57Bl/10ScCr mice are unable
to exhibit such an IFN- response [27
]. Yaegashi et
al. [28
] showed that this is due to the inability of the
respective macrophages to produce IFN-ß after microbial stimulation
and thereby provide the necessary help for the IFN--producing T and
natural killer cells. On the other hand, Munder et al.
[29
] reported that murine BMDMs themselves are able to
produce large amounts of IFN- if stimulated properly (in their case
with a combination of IL-12 and IL-18). Especially for the induction of
nitric oxide release, IFN- is known to be an extremely potent
costimulus [30
], either directly or by inducing TNF-
[31
].
Whether murine BMDMs respond to lipopeptide stimulation by
releasing IFN- is presently under investigation. However, our
results in which C57Bl/10ScCr cells showed a slightly lower secretion
of nitric oxide after lipopeptide stimulation as compared with
macrophages of the congenic C57Bl/10ScSn animals could reflect a less
pronounced reaction of the former and in consequence a lower endogenous
production of IFN- after lipopeptide stimulation. In agreement with
this assumption are the results we obtained after adding exogenous
IFN- to the two cell populations: In C57Bl/10ScCr macrophages, the
synergistic effect was much more pronounced and showed a more stringent
dose dependency than in C57Bl/10ScSn cells, resulting finally in the
release of comparable quantities of nitric oxide from both cell
populations.
Cox et al. [32
] showed that NO must reach a threshold
level to be active in tumor cell destruction. The same is likely to be
true for other soluble mediators of tumor cytotoxicity, e.g., TNF-
and oxygen radicals. We previously showed that production of TNF- is
induced in murine BMDMs after stimulation with synthetic lipopeptides
[12
]. Here we demonstrated for the first time a release
of ROIs by lipopeptide-stimulated BMDMs that exceeded by far the
oxidative burst induced in these cells by LPS. Furthermore, we showed
that all three of the soluble mediators reached functional, i.e.,
tumoricidal, levels after stimulation of BMDMs with
P3CSK4, because we obtained substantial killing
or growth arrest of tumor cells sensitive to either TNF- (L929),
RNIs (P815 and M12.4), or ROIs (Abelson 8-1) after cocultivation with
lipopeptide-activated macrophages.
Well-known targets for regulation by ROIs within the cells are
several proteins known to be involved in LPS signaling, namely the
mitogen-activated protein kinases (MAPKs) extracellular regulated
kinases 1 (ERK1) and 2 (ERK2), as well as the nuclear transcription
factor NF-
B [33
, 34
]. After stimulation
of murine macrophages (BMDMs as well as cells of the macrophage cell
line RAW 264.7) with the lipopeptide P3CSK4, we
could detect both a dose- and time-dependent activation of the MAPKs
ERK1 and ERK2, as well as a translocation of the activated NF-B
[M. R. Müller, S. D. C. Pfannes, M. Ayoub, P.
Hoffmann, W. G. Bessler, and K. Mittenbühler, unpublished
results]. Hence, intracellular signaling by LPS and lipopeptides seems
to follow similar routes as far as these two steps are concerned.
Earlier within the signal transduction chain, however, there seem to be
differences between LPS- and lipopeptide-driven stimuli: Both molecules
bind to cells via CD14, but whereas the presence of this molecule is
essential for LPS signaling, it is not for lipopeptides, but only
enhances its effect [35, 36; M. R. Müller et al.,
unpublished results]. Several recent publications identify different
members of the Toll receptor family as being responsible for the
transmembrane signaling step after binding of LPS to CD14. Kirschning
et al. [37
] showed transfer of LPS responsiveness to
otherwise LPS-unresponsive cells by transfection with human Toll-like
receptor (TLR)-2. In contrast, Poltorak et al. [38
]
demonstrated that the LPS gene (see above) corresponds to the gene of
TLR-4 and that the LPS-nonresponder mice used here (C57Bl/10ScCr) bear
a null mutation in this gene. Because we could show a comparable
reactivity to lipopeptides for macrophages from both C57Bl/10ScSn
LPS-responder and C57Bl/10ScCr LPS-nonresponder animals, which was
reflected in a similar release of nitric oxide as well as similar
cytostatic and cytotoxic activity against various tumor target cell
lines, participation of TLR-4 in lipopeptide signaling seems highly
unlikely. Preliminary results indicated that the release of ROIs is
also similar in these LPS-responder and -nonresponder macrophages after
lipopeptide stimulation [P. Hoffmann et al., unpublished results].
With the same line of evidence, Takeuchi et al.
[39
] demonstrated that macrophage-activating
lipopeptide-2-induced cytokine and NO synthesis was mediated by TLR-2.
Whether other members of the TLR family also play a role in lipopeptide
signaling must be elucidated in future studies.
The authors appreciate the excellent technical assistance of Angelika Haber and Marianne Eckert.
Received May 30, 2000; revised November 27, 2000; accepted November 29, 2000.
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, a cofactor in the interferon production induced by gram-negative bacteria in mice J. Exp. Med. 181,953-960 upon combined stimulation with interleukin (IL)-12 and IL-18: a novel pathway of autocrine macrophage activation J. Exp. Med. 187,1-6-induced TNF- is a prerequisite for in vitro production of nitric oxide generated in murine peritoneal macrophages by IFN- Eur. J. Immunol. 28,838-843[Medline]-dependent production of reactive nitrogen intermediates mediates IFN- plus IL-2-induced murine macrophage tumoricidal activity J. Immunol. 149,3290-3296[Abstract]
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