(Received for publication, July 13, 1994; and in revised form, January 16, 1995)
From the
Stimulation of resident peritoneal macrophages with S-[2,3-bis(pamitoyloxy)-(2R,2S)-propyl]-N-palmytoyl-(R)-CysSerLys
or S-[2,3-bis(pamitoyloxy)-(2R,2S)-propyl]-N-palmytoyl-(R)-CysAlaLys
,
two synthetic bacterial lipopeptides, promoted the expression of the
inducible form of nitric oxide synthase, exhibiting a temporal pattern
of nitric oxide release that was delayed with respect to the induction
elicited by bacterial lipopolysaccharide. Treatment of macrophages with
genistein blocked the nitric oxide synthesis triggered by the
lipopeptides or lipopolysaccharide. Simultaneous incubation with
lipopolysaccharide and lipopeptide resulted in an antagonistic effect
on nitric oxide synthase mRNA levels and on nitrite plus nitrate
release to the medium.
Triggering with bacterial lipopeptides induced macrophage programmed cell death. In macrophages activated with lipopeptide, apoptosis was observed even in the absence of nitric oxide synthesis, therefore indicating the existence of alternative pathways in the control of programmed cell death in these cells.
The effect of lipopolysaccharide (LPS) ()by itself or
in synergism with other cytokines on the induction of NOS in several
tissues is now well
documented(1, 2, 3, 4) . In M
,
IFN-
and TNF-
synergize with LPS in the release of NO which
is involved in the antimicrobial action of activated
M
(4) . However, in addition to LPS, other components of
the bacterial cell wall such as membrane lipoproteins are able to
modulate immunological responses in M
and B and T lymphocytes,
although their mechanism of action is less
known(5, 6) . Regarding bacterial lipoproteins,
previous work showed that their biological activity can also be
reproduced by synthetic lipopeptides (TPP) that mimic the fatty acid
esterification of these molecules(7) . Indeed, these
lipopeptides have some technical advantages for the use in in vivo experiments since they do not produce necrosis upon injection and
lack toxic and pyrogenic effects(7) . The activation elicited
by these synthetic lipopeptides in M
involves an early tyrosine
phosphorylation of substrates and uses an upstream signaling pathway
partially different from that triggered via LPS(8) .
Activation of M usually leads to programmed cell death and the
release of NO by itself has been reported as one of the signals that
mediates apoptosis at least in peritoneal M
(9) . However,
it is possible that in addition to this molecule other reactive
intermediates or cytokines produced in the course of M
activation
may be involved in the apoptotic process characteristic of activated
cells(9, 10) . Here we show that triggering of
resident peritoneal rat M
with TPP promotes NO synthesis with a
kinetics different from that elicited by LPS, and the presence of both
bacterial products results in a significant blockage of NOS expression
and NO release. Moreover, in TPP-activated cells apoptosis may be
obtained in the absence of NO synthesis, suggesting the existence of
alternative pathways in cell death induction by TPP.
Figure 1:
Bacterial lipopeptides induce NO
synthesis in M. Cultured resident peritoneal M
(3
10
) were incubated with 5 µg/ml of TPP (
), 5
µg/ml of TPP-Ala (
), or 5 µg/ml of LPS (
), and at
the indicated times the concentration of nitrite plus nitrate released
to the medium was measured. Alternatively, cells were incubated for 5
min with 100 µM of genistein prior to stimulation with 5
µg/ml of TPP (solid bars) or 5 µg/ml of LPS (open
bars), and the nitrite and nitrate concentration were measured (panel A). When genistein was added at different times after
stimulation with TPP (
) or LPS (
) the NO synthesis was
measured after 24 h of stimulation (panel B). The
dose-dependent curve for the inhibition of NO release by genistein
(added 5 min prior to M
triggering) was measured after 24 h of
stimulation with TPP or LPS (inset). Results show the mean
± S.E. of three experiments.
Figure 2:
Nitrite and nitrate release in M
activated with lipopeptides. M
were incubated for 36 h with TPP
(
) or TPP-Ala (
), as described in Fig. 1. The
release of nitrate (dotted line) and nitrite (continuous
line) were measured. Results show the mean ± S.E. of three
experiments.
Since the amount of nitrate released represented 27% of the
nitrite concentration, these results suggested a moderate oxidation of
nitrites in the course of TPP stimulation. Indeed, in addition to NO,
TPP also promoted the release of HO
when
stimulated M
cultures were triggered for 1 h with
PDBu(13, 14) . This H
O
production was near additive to the effect elicited by TNF-
and IFN-
(Fig. 3). These results prompted us to study the
role of these cytokines on the TPP-dependent NO release. As Table 1shows, incubation of M
with IFN-
, TNF-
, or
with both molecules inhibited the NO release induced by TPP. Opposite
to this situation, a clear synergism between IFN-
and TNF-
was observed in LPS stimulated M
, suggesting that TPP and LPS use
different pathways in their mechanism of NOS induction. However, an
unexpected antagonism between TPP and LPS in NO release was observed.
Figure 3:
TPP promotes PDBu-dependent
HO
release in M
. Coverslip-attached cells
were stimulated for 24 h with 2 ng/ml of IFN-
or TNF-
in the
absence (open bars) or presence (dashed bars) of 5
µg/ml of TPP. After this incubation period, the cell layers were
extensively washed and triggered with 100 ng/ml of PDBu for 1 h to
release H
O
that was determined fluorometrically
by the oxidation of scopoletin in the presence of horseradish
peroxidase. Results show the mean ± S.E. for triplicates from
one of three independent experiments.
Figure 4:
TPP and LPS antagonize in promoting iNOS
expression. M were incubated for the indicated periods of time in
the absence (lane 1) or with 5 µg/ml of TPP (lanes 2 and 4) and 1 µg/ml of LPS (lanes 3 and 4), respectively. RNA was extracted and analyzed by Northern
blot using a specific probe for iNOS. After normalization for
-actin content, the relative amount of iNOS mRNA is shown in
arbitrary units (a.u.). The figure shows 1 representative
experiment out of three.
Figure 5:
TPP promotes apoptosis in the absence of
NO synthesis. M (2
10
) were incubated for 36
h in the absence (lanes 1 and 3) or presence of 5
µg/ml of TPP (lanes 2 and 4) and 1 mM NMA (lanes 3 and 4). The DNA laddering and
nitrite plus nitrate concentration in the medium were assayed (panel A). The time course of NO release in the absence
(
,
) or presence of TPP (
,
) (panel
B), and the relative content in apoptotic cells (panel C)
were determined. Filled symbols correspond to assays in the
presence of 1 mM NMA. To have a positive control of
NO-dependent apoptosis, cells were incubated with 50 µM of
3-morpholinosydnonimine (
), a NO donor (panel C). An A
value of 0.06 O.D. units corresponding to
control cells was subtracted from each sample. Results show 1
representative experiment out of three.
Figure 6:
TPP induces apoptosis in arginine-free
medium and promotes iNOS expression. M were incubated for 36 h in
arginine-free medium (lanes 1 and 2) or in its
presence (lanes 3-5) and were stimulated with 5
µg/ml of TPP (lanes 2 and 4), or TPP plus 1
µg/ml of LPS (lane 5). The extent of apoptosis (open
bars) or NO release (dashed bars) were measured. An A
value of 0.05 O.D. units corresponding to
control cells was subtracted from each sample. At the time of sampling,
cells were homogenized and analyzed by Western blot using an iNOS
mAb.
Figure 7:
Dose
dependence curve of apoptosis in M stimulated with TPP in
arginine-free medium. M
were incubated for 36 h with the
indicated concentrations of TPP and the extent of apoptosis (
) and
NO release (
) were measured. Results show a representative
experiment out of three cell preparations.
Several bacterial cell wall products, among which are LPS,
lipoproteins, murein, and membrane proteins, share in common the
ability to stimulate various types of immune cells(18) .
However, since the chemical structure of these molecules exhibits great
differences between them, it is conceivable that the signaling pathways
activated after cell triggering with bacterial products might show a
certain degree of specificity. For example, lipopeptides specifically
activate G proteins in human neutrophils(19) .
Additionally, tyrosine phosphorylation of MAP kinases 1 and 2 has been
reported after TPP activation of murine M
from LPS-responsive and
-nonresponsive strains; however, for LPS MAP activation is observed
only in LPS-responsive strains, and the lack of activation in the
LPS-nonresponsive counterparts has been situated at a post-receptor
step but prior to MAP kinase activation(8) . As a result of
M
activation with cytokines and bacterial products, these cells
release various secretory molecules (10) exhibiting a high
chemical activity (NO, H
O
, and
O
). Regarding NO release by TPP-activated
M
, it has been shown that tyrosine kinase inhibitors, such as
genistein, effectively cancelled NO production, revealing the necessity
of tyrosine phosphorylation in the pathway that involves NOS
expression, and a similar conclusion applies for LPS(20) .
However, except for a prolonged sensitivity to genistein when added at
various times after LPS triggering, no significant differences have
been observed between LPS and TPP regarding the involvement of protein
tyrosine kinase activation following M
activation.
The results
reported in this work add new information regarding cell activation and
apoptotic death induction of M by bacterial lipopeptides, which
seem to exhibit some specific characteristics when compared with the
effects elicited by LPS. The release of NO after TPP stimulation is
delayed and quantitatively lower with respect to the response elicited
after LPS challenge. This is opposite to the temporal pattern of MAP
kinase phosphorylation that, at least in murine M
, is more
rapidly activated by lipopeptides(8) . Moreover, simultaneous
treatment of the cells with both TPP and LPS results in a blockage of
the response, as reflected by the decrease in iNOS mRNA levels and
protein, and NO release to the medium, which suggests that some signals
are released in the course of the dual stimulation resulting in a
partially antagonistic response. This result was unexpected since
cooperation would be observed in view of the use of some common
transduction pathways (i.e. early protein tyrosine kinase
stimulation and MAP kinase activation).
In rat M TPP in
addition to NO synthesis promotes the release of
H
O
, clearly observed after triggering with
phorbol esters, and therefore contributing to cell activation with
various oxygen reactive intermediates; however, the study of the
modulation by IFN-
and TNF-
of the TPP response revealed that
these cytokines antagonize the production of NO (a behavior opposite to
their cooperative action in LPS stimulated macrophages, refs. 13, 14),
but result additive (and therefore independent) with TPP in the
production of H
O
.
An additional difference
between TPP and LPS in M activation concerns the commitment for
apoptosis after stimulation. In the absence of NO synthesis LPS fails
to trigger an effective apoptosis(9, 17) , whereas
under these conditions TPP retains the ability to induce cell death of
activated M
, which suggests that an alternative NO-independent
apoptotic pathway is operative in TPP-stimulated cells. Our results
show that when NO is produced, the relative amount of DNA fragmentation
is moderately increased (
30%), suggesting that NO is not the main
apoptotic inducer and that a cooperation exists between pathways that
trigger apoptosis. However, it remains to be determined whether NO may
influence the fraction of the cell population exhibiting DNA cleavage.
Regarding the mechanism of action of TPP in promoting apoptosis,
several possibilities could be envisaged. The contribution of NO has
been attributed either to a blockage in the energetic metabolism (via
aconitase inhibition) or to a direct alteration in the DNA structure as
result of nucleotide deamination directly due to NO, and both cases
have been discussed previously(9, 17) . In addition to
these mechanisms, there is a possible occurrence in TPP-activated
M of an enhanced susceptibility to H
O
or
other oxidant species released in the course of the activation. An
example of this situation has been reported in retrovirus infected T
cells that exhibit an extreme sensitivity to H
O
for apoptosis(21) . Indeed, the occurrence of
physiological pathways leading to apoptosis of antigen-presenting
M
following CD4
T cell activation has been
proposed to be genetically programmed into the repertoire of M
functions(22, 23) .
Finally, and in keeping with
the view that M are programmed for cell death upon
activation(23) , it is possible that the apoptosis observed in
TPP-activated M
lies on the release of several reactive oxygen
intermediates (NO, H
O
,
O
) which may trigger the expression of
genes or modulate the activity of transcriptional factors such as NF-kB
or AP-1 that would be responsible for this process(24) .