(Received for publication, July 27, 1995)
From the
The reactive nitrogen species, nitric oxide (NO), plays an
important role in the pathogenesis of neurodegenerative diseases. The
suppression of NO production may be fundamental for survival of
neurons. Here, we report that pretreatment of human ramified microglial
cells with nearly physiological levels of exogenous NO prevents
lipopolysaccharide (LPS)/tumor necrosis factor
(TNF
)-inducible NO synthesis, because by affecting NF-
B
activation it inhibits inducible Ca
-independent NO
synthase isoform (iNOS) mRNA expression. Using reverse transcriptase
polymerase chain reaction, we have found that both NO donor sodium
nitroprusside (SNP) and authentic NO solution are able to inhibit
LPS/TNF
-inducible iNOS gene expression; this effect was reversed
by reduced hemoglobin, a trapping agent for NO. The early presence of
SNP during LPS/TNF
induction is essential for inhibition of iNOS
mRNA expression. Furthermore, SNP is capable of inhibiting
LPS/TNF
-inducible nitrite release, as determined by Griess
reaction. Finally, using electrophoretic mobility shift assay, we have
shown that SNP inhibits LPS/TNF
-elicited NF-
B activation.
This suggests that inhibition of iNOS gene expression by exogenous NO
may be ascribed to a decreased NF-
B availability.
Nitric oxide (NO) ()is a major messenger molecule
playing key roles in many physiological and pathological
processes(1) . NO production is catalyzed by at least two major
forms of the NO synthase (NOS) enzyme: a constitutive
Ca
-dependent NOS isoform (cNOS) and an inducible
Ca
-independent NOS isoform (iNOS), which is expressed
after stimulation with Escherichia coli lipopolysaccharide
(LPS) and cytokines. Recently, we have demonstrated that LPS and/or
TNF
are able to induce iNOS in human ramified microglia leading to
a high NO output(2) . On the other hand, NO release from mouse
microglia is thought to play an important role in neuronal cell
death(3, 4, 5) . In a recent work, Meda et al.(6) suggested a possible involvement of NO
produced by rat microglia after activation with
-amyloid protein
and IFN-
in the pathogenesis of neuronal degradation occurring
with age and in Alzheimer's disease.
Preserving iNOS gene from
its undesirable induction may be important for neuronal survival.
Down-regulation of iNOS expression was reported to be achieved by some
factors such as dexamethasone, interleukin-4, transforming growth
factor-, and basic fibroblast growth factor (7, 8, 9) . Recently, Griscavage et al.(10) have indicated that NO may function as a negative
feedback modulation of inducible NO synthase by interacting with
enzyme-bound heme in activated rat macrophage cell line. Park et
al.(11) have recently observed that glial cells in rat
endogenously produced NO following cytokine stimulation down-regulated
iNOS expression.
However, little is known about the regulatory
effects on the mechanism by the variable low concentrations of the
available NO before iNOS induction. Recently, we have observed that
sodium nitroprusside (SNP), a well known NO donor, elicited inhibition
of LPS-induced iNOS expression in rat neutrophils, suggesting a
possible suppressive effect on iNOS gene expression by exogenous
NO(12) . The promoter region of human iNOS gene in vascular
smooth muscle cells was shown to contain the consensus sequences for
the binding of NFB, a nuclear transcriptional factor(13) ,
and iNOS transcription has been recently observed as being regulated by
NF
B activation(14) .
Here, we used human ramified
microglial cells as a model for a better understanding of the molecular
mechanism by which NO regulates its own production. To this respect, we
hypothesize that a nearly physiological level of exogenous NO may
prevent LPS/TNF-inducible iNOS mRNA expression by affecting
NF
B activation.
As described previously, human ramified microglial cells were
found to release high NO amounts(2) . In fact, we observed that
stimulation of 5 10
microglial cells with a mixture
containing LPS (1 µg/ml) + TNF
(500 units/ml) for 24 h
caused a marked production of nitrite
(NO
), the breakdown product of NO (from
3.52 ± 0.26 to 15.54 ± 0.66 nmol ml
; Fig. 1), as measured by Griess reaction. This increase was
abolished by NOS inhibitor N
-nitro-L-arginine methyl ester (500
µM) (from 15.54 ± 0.66 to 3.07 ± 0.24 nmol
ml
) (Fig. 1). A 0.5-h pretreatment of human
ramified microglial cells with SNP (10 µM) decreased NO
production induced by LPS/TNF
(from 15.54 ± 0.66 to 4.07
± 0.72 nmol ml
) (Fig. 1). However,
this effect was not accounted for by direct modification of iNOS
enzymatic activity, as determined using
[
H]citrulline generation from L-[
H]arginine. In fact, we have observed
that human microglial cells treated with LPS (1 µg/ml) +
TNF
(500 units/ml) for 24 h expressed a
Ca
-independent NOS activity (680 pmol/min/mg of
protein). When SNP (10 µM) were added directly into the
assay mixture or preincubated for 2 h in the enzyme preparations, no
significant variations of iNOS activity were observed (data not shown).
Figure 1:
Effect of SNP on nitrite production in
human ramified microglial cells. Nitrite levels were measured using
Griess reaction in supernatants of human ramified microglial cells
treated with LPS (1 µg/ml) + TNF (500 units/ml) for 24 h.
A 0.5-h SNP (10 µM) pretreatment of LPS/TNF
-treated
cells significantly (*, p
0.01) decreased nitrite levels.
Data are expressed as nmol ml
. Each bar represents the mean ± S.E. of four experiments. LNAME, N
-nitro-L-arginine
methyl ester.
The following results provide evidence that NO was able to reduce
its own production in human ramified microglial cells by affecting iNOS
gene expression. As previously observed(2) , using RT-PCR we
found that an 8-h treatment of microglial cells with LPS/TNF
strongly enhanced iNOS mRNA levels (Fig. 2). When
LPS/TNF
-treated microglial cells were preincubated with NO donor
SNP (10 µM) iNOS gene expression was strongly inhibited (Fig. 2). This effect was significantly reversed by reduced
hemoglobin (oxyhemoglobin; 1 µM), a trapping agent for NO.
To obtain further evidence that the effect of SNP was mediated by its
NO release, SNP was pre-exposed to room light for 2 weeks to prerelease
NO, as verified by determination of nitrite levels (data not shown).
The resulting cyanide, Fe
, and nitrite did not
interfere with iNOS mRNA induction (Fig. 2). Furthermore, 1% of
authentic NO solution was capable of inhibiting iNOS mRNA expression (Fig. 2). Recently, Park et al.(11) have
observed that NO limited iNOS mRNA expression in rat glial cells when
it was produced following stimulation with cytokines. However, we have
observed that the early presence (0.5 h) of SNP during cell stimulation
with LPS/TNF
was essential for iNOS mRNA inhibition. In fact, when
SNP was added 4 h after iNOS up-regulation, no inhibition of iNOS mRNA
synthesis was observed. Our data indicate that nearly physiological
levels of pre-existing exogenous NO may prevent the induction of iNOS
gene expression at least in some cell lines such as human microglial
cells.
Figure 2:
Effect of NO on iNOS gene expression in
human ramified microglial cells. 1.5% agarose gel electrophoresis of
the RT-PCR products for iNOS mRNA (450 bp) and for GAPDH mRNA (195 bp)
after ethidium bromide staining is shown. Lane 1, untreated
cells; lane 2, an 8-h treatment of 5 10
microglial cells with LPS/TNF
strongly enhanced iNOS mRNA
levels; lane 3, pretreatment of cells with NO donor SNP (10
µM) inhibited LPS/TNF
-induced mRNA expression; lane 4, this effect was reversed by oxyhemoglobin; lane
5, sample 2 pretreated with NO-deprived SNP solution; lane
6, sample 2 plus SNP added 4 h after iNOS up-regulation; lane
7, sample 2 pretreated with 1% of authentic NO solution. PCR
products for the GAPDH gene were taken as the reference cellular
transcript. Molecular weight is the 100-bp DNA ladder (Life
Technologies, Inc.).
By using electrophoretic mobility shift assay, we found that
treatment with LPS/TNF induced the activation of NF-
B, which
peaked after a 0.5-h stimulation (Fig. 3), this effect being
observed elsewhere for an established mouse macrophage cell
line(14) . A 0.5-h pretreatment of human microglial cells with
SNP (10 µM) was able to inhibit LPS/TNF
-elicited
NF-
B activation (Fig. 3), suggesting that exogenous NO
inhibition of iNOS expression may be due to a decreased NF-
B
activation. On the other hand, it has been confirmed recently that in
human vascular endothelial cells NO inhibited NF-
B activation,
this effect being mediated by induction and stabilization of
I-
B(20) .
Figure 3:
Effect of SNP on DNA binding activity of
NF-B transcription factor in human ramified microglial cells.
Nuclear extracts of cells were incubated with
P-labeled
double-stranded oligonucleotide containing the consensus NF-
B DNA
binding site in a reaction mixture. Lane 1, untreated cells; lanes 2-5, cells treated with LPS/TNF
for 0.5, 1,
2, and 4 h, respectively; lane 6, the same samples as for lane 2 incubated with 100
cold NF-
B as specific
competitor; lane 7, the same sample as for lane 2 incubated with 100
nonspecific competitor; lane
8, control cells pretreated with SNP (10 µM) for 0.5
h; lanes 9-12, LPS/TNF
-treated cells preincubated
(0.5 h) with SNP for 0.5, 1, 2, and 4 h,
respectively.
Uncontrolled and massive iNOS-induced NO
production was suggested to be potentially detrimental to the tissue
integrity(21) . The present data indicate that nearly
physiological concentrations of NO may keep the iNOS expression
suppressed by preventing NF-B activation. This implies that
induction of iNOS expression could be regulated, at least in part, by
the endogenous NO level likely to be produced by cNOS enzyme, although
no data concerning this matter are available so far. When the basal
level of NO is at a value such as to suppress iNOS expression, the
endogenous NO may contribute to avoid an undesirable and potentially
harmful induction of iNOS expression. If the NO level goes down below
threshold value, iNOS expression may become easily achievable. In this
respect, it seems worthwhile noting that LPS/IFN
, which induces
iNOS gene, is also able to simultaneously decrease cNOS mRNA expression
in human monocyte/macrophage cells (22) . Furthermore, in
endothelial cells TNF
has been shown to regulate cNOS expression
by shortening its mRNA half-life(23) .
Finally, NO donors could be considered potential drugs for the control of microglial iNOS expression induction. Since the latter seems to play an important role in the pathogenesis of some neurodegenerative diseases, exogenous NO donor could be as potentially active for the prevention of those pathologies.