1 Faculdade de Farmácia e 3 Faculdade de Medicina (Servico de Dermatologia), 2 Centro de Neurociências, Universidade de Coimbra, 3000 Coimbra, Portugal
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this report, we demonstrate that a fetal
mouse skin-derived dendritic cell line produces nitric oxide (NO) in
response to the endotoxin [lipopolysaccharide (LPS)] and to
cytokines [tumor necrosis factor- (TNF-
) and
granulocyte-macrophage colony-stimulating factor (GM-CSF)].
Expression of the inducible isoform of NO synthase (iNOS) was confirmed
by immunofluorescence with an antibody against iNOS. The tyrosine
kinase inhibitor genistein decreased LPS- and GM-CSF-induced nitrite
(NO
2) production. The effect of LPS
and cytokines on NO
2 production was
inhibited by the Janus kinase 2 (JAK2) inhibitor tyrphostin B42. The
p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB-203580
also reduced the NO
2 production evoked by LPS, TNF-
, or GM-CSF, but it was not as effective as tyrphostin B42. Inhibition of MAPK kinase with PD-098059 also slightly reduced the
effect of TNF-
or GM-CSF on NO
2
production. Immunocytochemistry studies revealed that the transcription
factor nuclear factor-
B was translocated from the cytoplasm into the nuclei of fetal skin-derived dendritic cells (FSDC) stimulated with
LPS, and this translocation was inhibited by tyrphostin B42. Our
results show that JAK2 plays a major role in the induction of iNOS in FSDC.
mitogen-activated protein kinase; Janus kinase 2; nuclear
factor-B
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
NITRIC OXIDE (NO) is generated by the enzyme nitric oxide synthase (NOS), of which three related, but functionally distinct, isoforms have been identified in mammalian cells. Type I and type III NOS are constitutively expressed in cells of neural and endothelial origin, respectively, and they are regulated by physiological changes in the intracellular calcium concentration. In contrast, type II NOS (or iNOS) is expressed in cells with immunoregulatory functions in response to a wide array of proinflammatory cytokines and bacterial cell wall products (16).
Because skin is the first defense against a hostile environment, NO produced by Langerhans cells (LC), keratinocytes, and/or dermal dendritic cells (DC) may have an important contribution to host defense against skin pathogens. In addition, several reports are consistent with NO being involved in skin inflammatory diseases (5) and in the modulation (enhancement or suppression) of antigen presentation (20).
The cellular and molecular mechanisms involved in the control of NO synthesis are a subject of the current investigation. The intracellular signals that regulate the expression of iNOS have been studied in different cell types, and, although it has not been fully characterized yet, iNOS expression appears to be regulated in a cell-specific manner. Protein kinase C (PKC), protein tyrosine kinases (PTKs), and cAMP-dependent protein kinase have been found to be involved in the regulation of iNOS expression (14, 17, 19, 23, 28-30). Activation of some of these kinases may stimulate the mitogen-activated protein kinases (MAPK), a family of structurally related kinases that are involved in cellular events, such as growth, differentiation, and stress responses (25). In mammalian cells, three subgroups of MAPK have been detected and include the extracellular signal-regulated kinases (ERKs, p42/p44), the c-Jun amino-terminal kinases (JNKs), and the p38 MAPKs (25). Cytokines activate members of the MAPK and of the Janus kinase (JAK) families of PTKs which, in turn, activate by phosphorylation one or more transcription factors that are translocated from the cytoplasm to the nucleus to induce gene transcription (13, 25).
The signaling events required for NO production in DC have not been
identified yet. Studies on LC (and DC) have been hampered by the
difficulties in obtaining large amounts of LC devoid of contaminating
cells. In our study, we circumvented this problem by using a mouse
fetal skin dendritic cell line (FSDC) that is representative of early
DC precursors (10). We studied the effect of lipopolysaccharide (LPS)
and cytokines [granulocyte-macrophage colony-stimulating factor
(GM-CSF), interleukin-1 (IL-1
), and tumor necrosis factor-
(TNF-
)] on the production of NO by FSDC and the role played by
genistein-sensitive tyrosine kinases and by the MAPK and JAK pathways
in the process. Furthermore, we investigated the effect of LPS and
cytokines on the intracellular distribution of the nuclear
transcription factor nuclear factor-
B (NF-
B) and whether JAK2 is
involved in NF-
B activation.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials. The rabbit anti-mouse iNOS
polyclonal antibody was purchased from Transduction Laboratories
(Lexington, KY), the rabbit anti-human NF-B p65 was from Serotec
(Oxford, UK), and the fluorescein isothiocyanate (FITC)-conjugated
swine anti-rabbit immunoglobulin was from DAKO (Copenhagen, Denmark).
The Prolong Antifade Kit was obtained from Molecular Probes Europe
(Leiden, The Netherlands). LPS from Escherichia
coli (serotype 026:B6) was obtained from Sigma Chemical
(St. Louis, MO), the mouse TNF-
receptor was from Boehringer
Mannheim (Carnaxide, Portugal), and mouse IL-1
receptor was
purchased from Pharmingen (San Diego, CA). The murine GM-CSF receptor
was from Serotec; SB-203580 was a kind gift of Dr. J. L. Adams from
SmithKline Beecham Pharmaceuticals (King of Prussia, PA). Tyrphostin
B42 and PD-098059 were obtained from RBI (Natick, MA), FCS was from
Biochrom (Berlin, Germany), and trypsin was from GIBCO (Paisley, UK).
Genistein and genistin were from Sigma Chemical. All other reagents
were from Sigma Chemical.
Cell culture. The fetal mouse skin dendritic cell line FSDC was kindly supplied by Dr. G. Girolomoni (10). This cell line was generated from fetal mouse skin by infecting a cell suspension with a retroviral vector carrying an envAKR-mycMH2 fusion gene. FSDC show characteristics of immature DC and express low levels of major hisotcompatibility complex II molecules (I-Ad,b), and their proliferation in serum-free medium occurs in the presence of GM-CSF, but not macrophage colony-stimulating factor, indicating that they are dendritic cell precursors (10).
The cells were cultured in Iscove's medium supplemented with 10% FCS, 1% glutamine, 100 µg/ml streptomycin, and 100 U/ml penicillin.
Cell viability. Assessment of cell
viability was made in all experimental conditions by a colorimetric
assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT; see Ref. 24). After removal of cell-free supernatants for
the nitrite (NO2) assay, 400 µl of
culture medium and 40 µl of MTT solution (5 mg/ml in PBS) were added
to each well. The microplates were further incubated at 37°C for 1 h. Supernatants were then discarded, and 300 µl of acidified isopropanol (0.04 N HCl in isopropanol) were added to the cultures and
mixed thoroughly to dissolve the dark blue crystals of formazan. The
blank assay (no cells) was subtracted from the other readings. Formazan
quantification was performed using an automatic plate reader (SLT) at
570 nm, with a reference wavelength of 620 nm.
NO2
measurement. The production of NO was assessed as
the accumulation of NO
2 in the culture
supernatants, using a colorimetric reaction with the Griess reagent
(11). Briefly, after stimulation for 48 h, the culture supernatants
were collected and mixed with equal volumes of the Griess reagent
[0.1%
N-(1-naphthyl)ethylenediamine
dihydrochloride, 1% sulfanilamide, and 5%
H3PO4],
during 10 min. The absorbance at 550 nm was measured in an automated
plate reader (SLT). The NO
2
concentration was determined from a sodium nitrite standard curve.
Immunofluorescence microscopy. For
immunofluorescence analysis, FSDC were grown on glass coverslips and
were fixed and permeabilized by immersing the coverslips in
20°C methanol-acetone (1:1) for 10 min. Nonspecific binding
was blocked by incubation in PBS supplemented with normal swine serum
(1:20) and 0.5% BSA for 45 min at room temperature. Cells were then
incubated for 90 min at room temperature with a rabbit polyclonal
antibody directed against mouse iNOS (5 µg/ml) or for 2 h with the
rabbit polyclonal antibody directed against human p65 (1:200). After
being rinsed with PBS, the cells were incubated with FITC-conjugated
swine anti-rabbit immunoglobulin (1:40 dilution) in 0.5% BSA-PBS for
45 min. The coverslips were rinsed again as before and were mounted
with a Prolong Antifade Kit on a slide. Cells labeled with
FITC-anti-iNOS were photographed on a Nikon Diaphot-TMD microscope. The
intracellular localization of FITC-labeled p65 was observed using the
488-nm line of a krypton/argon laser on a Bio-Rad MRC 600 fluorescent
confocal microscope. Control experiments consisted of processing the
same preparations as described, except for the omission of the primary
antibody, and resulted in no specific staining.
Data analysis. Results are presented as means ± SE of the indicated number of experiments. Mean values were compared using one-way ANOVA and the Bonferroni multiple comparison test. The significance level was 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
NO2
production by FSDC response to LPS and cytokines.
To determine whether NO production was inducible in FSDC, the mouse
dendritic cell line in culture was treated with LPS or cytokines
(GM-CSF, TNF-
, and IL-1
) for 48 h, and the culture supernatants
were collected for the NO
2 measurement
(Fig. 1). Cell-free supernatants, from
cells incubated in the presence of LPS (5 µg/ml), showed an increase
in NO
2 concentration (from 0.63 ± 0.06 to 24.1 ± 2.92 µM), reflecting an increase in NO production. NO
2 production was also stimulated
upon incubation of FSDC with 100 U/ml TNF-
(from 0.63 ± 0.06 to
4.76 ± 0.82 µM) or 200 ng/ml GM-CSF (from 0.63 ± 0.06 to 6.32 ± 1.1 µM). At a concentration of 200 ng/ml, GM-CSF also caused a
significant increase in the ability of FSDC to stimulate the allogeneic
or syngeneic T cells in the primary mixed-leukocyte reaction (10). No
significant NO production was observed in FSDC cultures exposed to
IL-1
(Fig. 1).
|
To confirm whether NO2 was formed via
the induction of iNOS, the cells were incubated for 48 h in the
presence of the NOS inhibitor aminoguanidine (1 mM; see Ref. 37), which was added simultaneously with the cytokines or LPS. Aminoguanidine completely inhibited NO
2 accumulation
in the culture supernatant induced either by LPS or by the cytokines TNF-
and GM-CSF (Fig. 1).
Because TNF- may be involved in the activation of cytotoxic events
(34) and considering that stimulation of macrophages with LPS plus
interferon-
(IFN-
) produces
cytotoxic amounts of NO
2 (21), which
cause apoptosis (32), we evaluated the effects of LPS and cytokines on
the viability of FSDC, using the MTT assay (Table
1). The results show that neither LPS nor
cytokines significantly affected the FSDC viability (Table 1).
|
Identification of iNOS expression in FSDC line by
immunocytochemistry. Immunofluorescent labeling of FSDC
with the anti-iNOS polyclonal antibody was markedly increased in cells
stimulated with LPS (Fig.
2B) and
GM-CSF (Fig. 2C) compared with the
cells maintained in culture medium (Fig.
2A). Approximately 15-18% and 10-13% of the LPS- and GM-CSF-stimulated cells, respectively, were iNOS positive. The number of cells expressing iNOS in the TNF--treated cells and in the cells maintained in culture medium was
5-8 and 3-5%, respectively (data not shown). These results indicated that FSDC expressed iNOS, which was inducible by LPS, GM-CSF,
and TNF-
.
|
Effect of protein kinase inhibitors on NO production
by stimulated FSDC. To investigate the role of tyrosine
kinases on the expression of iNOS induced by LPS and cytokines in FSDC,
we studied the effect of genistein, a broad-spectrum inhibitor of
tyrosine kinases (1), on the NO2
production upon stimulation of the cells with LPS, TNF-
, or GM-CSF.
The genistein (30 µM) slightly reduced
NO
2 production induced by TNF-
(100 U/ml) to 92.8 ± 6.6%, whereas NO
2
production induced by LPS (5 µg/ml) and by GM-CSF (200 ng/ml) was
reduced to 24.6 ± 5.5 and 56.1 ± 13.3% of the control,
respectively (Table 2). Genistin, the
inactive analog of genistein (1), did not affect
NO
2 production evoked by LPS, TNF-
, or by GM-CSF in FSDC (Table 2). Therefore, the significant effect of
genistein on NO production induced by LPS and GM-CSF can be attributed
to its ability to inhibit PTKs rather than to nonspecific effects.
|
We next investigated the role of MAPK kinase (MEK), p38 MAPK, and JAK2
in the activation of NO2 production by
LPS and cytokines in FSDC. The PD-098059 was used to inhibit the MEK
activation (8), and the p38 MAPK and JAK2 were inhibited with SB-203580
(36) and tyrphostin B42 (22), respectively. The concentrations of
genistein (30 µM), PD-098059 (30 µM), SB-203580 (10 and 20 µM),
and tyrphostin B42 (20 µM) were chosen based on the previously
published studies (8, 14, 22, 36), and the assay of cellular MTT
reduction in the presence of the indicated concentrations of the
compounds revealed the lack of a significant toxic effect (data not shown).
The inhibitor PD-098059 was without effect on LPS-stimulated
NO2 production, which indicates that
the MEK signaling cascade was not involved in the LPS-induced NO
production in this cell line. In contrast,
NO
2 production induced by either
GM-CSF or TNF-
was slightly reduced, to 89.5 ± 6.1 and 83.2 ± 4.4% of the control, respectively (Table 2), in the presence of
30 µM PD-098059, a concentration of the antagonist that fully
inactivates the MEK pathway (8).
The NO production, 48 h after LPS and cytokine stimulation, was only
partially affected by the treatment with 20 µM SB-203580. The p38
MAPK inhibitor reduced LPS-, GM-CSF-, and TNF--induced NO
2 production to 78.8 ± 3.6, 72.4 ± 7.0 and 85.3 ± 3.4% of the control, respectively. However,
at the concentration of 20 µM, SB-203580 inhibited the MTT reduction
by FSDC incubated simultaneously with this inhibitor and LPS by ~86.3 ± 2.9% of the control (P < 0.05; data not shown). Under these experimental conditions, no
morphological evidence of cell death was observed.
Tyrphostin B42 was a potent inhibitor of LPS-, GM-CSF- and
TNF--induced NO production in FSDC (Table 2), which indicates that
the JAK pathway plays an important role in the regulation of iNOS
expression. This compound was the most effective protein kinase
inhibitor in preventing the cytokine-induced NO production; at 20 µM,
tyrphostin B42 inhibited NO
2
production induced by TNF-
and by GM-CSF to 48.6 ± 13.1 and 38.2 ± 7.5% of the control, respectively. The JAK2 inhibitor reduced
NO
2 formation induced by LPS to 41.9 ± 7.9% of the control. Although the JAK pathway plays an important
role in the regulation of iNOS induction by LPS and cytokines, a role
for p38 MAPK and MEK must also be considered in the control of iNOS
expression in FSDC, since the simultaneous utilization of the
inhibitors SB-203580 and PD-098059 reduced
NO
2 formation evoked by LPS, TNF-
,
and GM-CSF to 76.3 ± 5.8, 62.6 ± 1.05 and 69.5 ± 3.2% of
the control, respectively (Table 2).
Translocation of NF-B in nuclei of the
FSDC. In this set of experiments, we investigated the
effect of LPS and cytokines on the intracellular distribution of the
nuclear transcription factor NF-
B and whether the observed effects
could be attributed to JAK2.
The effect of LPS, GM-CSF, and TNF- on the intracellular
distribution of NF-
B in FSDC was examined immunocytochemically, using a specific antibody against the p65 subunit. Before LPS stimulation, p65 was distributed throughout the cytoplasm (Fig. 3A).
When FSDC were treated with LPS for 30 min, p65 was detected in most of
the nuclei of FSDC (Fig. 3B), and
this translocation was inhibited by inhibition of JAK2 with 50 µM
tyrphostin B42 (Fig. 3C). However,
when FSDC were treated with GM-CSF or TNF-
for 15, 30, or 60 min, no
accumulation of p65 in the nuclei of the cells was observed (data not
shown).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the present work, we show that LPS, TNF-, and GM-CSF increase the
expression of iNOS in a skin-derived dendritic cell line (FSDC) by a
mechanism involving the activation of JAK2, thereby leading to the
production of NO. Moreover, in these cells, the activation of JAK2 is
crucial for the translocation of NF-
B to the nucleus upon activation
with LPS.
We found that LPS was more potent than TNF- or GM-CSF in activating
NO
2 production due to stimulation of iNOS expression. In contrast to the significant effect of LPS, TNF-
,
and GM-CSF on the NO
2 production by
FSDC, no effect of IL-1
was observed (Fig. 1) despite the fact that these cells express IL-1 receptors (unpublished observations). LPS was
also recently shown to stimulate the production of cytokines by human
peripheral blood DC (33). Therefore, LPS may cause NO production both
directly and indirectly via the synthesis of cytokines that also
stimulate NO production. Accordingly, other authors have shown that
iNOS expression can be induced by LPS and
IFN-
in bone marrow DC and LC (20,
31), in contrast to the lack of effect of
IFN-
plus LPS on the expression of
iNOS mRNA in mouse epidermal LC (4). We observed that a subpopulation of the FSDC did not show increased expression of iNOS upon stimulation with LPS or GM-CSF, as determined by immunocytochemistry (Fig. 2). We
do not know why a low percentage of cells express iNOS even in the
presence of LPS and GM-CSF. However, our results are in agreement with
those obtained in highly purified bone marrow DC stimulated with
IFN-
plus LPS (20).
The intracellular signaling events involved in iNOS expression are not well understood, and the knowledge of the mechanisms involved in the control of NO synthesis by different cell types is a subject of current interest. It was shown that PKC, PTKs, and cAMP are important regulators of iNOS gene expression (23, 28, 30), but the expression of iNOS appears to be regulated in a cell-specific manner (14, 23, 28-30).
We observed that genistein inhibited the LPS- and GM-CSF-induced
NO2 production, which indicates that
activation of tyrosine kinase pathways is involved in the regulation of
iNOS induction in FSDC. These findings are in agreement with
observations in other cell types showing that genistein suppresses the
expression of iNOS activity induced by cytokines (23, 29). In contrast, genistein had no effect on NO
2
production induced by TNF-
(Table 2).
The JAK2 inhibitor tyrphostin B42 was a potent antagonist of the
GM-CSF-induced NO2 production,
although SB-203580 also inhibited the
NO
2 production induced by GM-CSF
(Table 2). Recent studies indicate that GM-CSF induces the activation
of the MAPK pathway (2), JNK (18), and JAK2 (35, 39). However, in
agreement with our results, JAK2 seems also to be a primary kinase
regulating all of the known GM-CSF signals, as previously reported in
BA/F3 cells (35). Moreover, the JAK2 protein kinase is necessary for
binding and phosphorylation of the GM-CSF receptor
c chain in CV-1
cells (39). These observations can explain the potent effect of
tyrphostin B42 in the GM-CSF-induced NO production by FSDC. To our
knowledge, the present study provides the first evidence for the
signaling pathways involved in the NO production induced by GM-CSF.
Our results also indicate that the p38 MAPK is involved in iNOS
expression by TNF- in FSDC. Other authors reported the involvement of the p38 MAPK in the signaling pathways of TNF-
. The
TNF-
-stimulated phosphorylation and activation of cytosolic
phospholipase A2 are completely
abolished in neutrophils treated with SB-203580 (36). The p38 MAPK
activity is also required for the transcriptional induction of iNOS by
TNF-
and IL-1
in astrocytes (6), but, in serum-starved mesangial
cells, the inhibition of p38 MAPK promoted IL-1
-induced iNOS
expression and subsequent NO production (12). The most likely
explanation for these seemingly inconsistent results is that the
complex regulation of iNOS expression is tissue-specific.
The MEK inhibitor PD-098059 also inhibited NO production induced by
TNF- in FSDC (Table 2). TNF-
increased ERK1 and ERK2 phosphorylation in IEC-6 cells, and PD-098059 inhibited TNF-
-induced IEC-6 cell growth (7). In contrast, in mouse astrocytes, iNOS expression induced by TNF-
and IL-1
was only partially affected by PD-098059 (6). However, our results suggest that both GM-CSF and
TNF-
induce NO
2 production mainly
through the JAK signaling pathway (Table 2). Accordingly, the
activation of iNOS expression in DLD-1 cells seems to require JAK
activity, especially the
IFN-
-activated JAK2 (15).
The lack of effect of PD-098059 on NO2
production induced by LPS rendered the involvement of the p42/p44 MAPK
pathway in iNOS production induced by LPS unlikely. Accordingly, in
glial cells, expression of iNOS stimulated by
IFN-
/LPS has been reported to
require tyrosine kinase activity, specifically JAK2 (27), and the
Ras/MAPK signaling pathway does not appear to be involved
in the IFN-
/LPS-evoked iNOS induction (26). Our results also
indicated that the JAK2 is involved in the NO production induced by
LPS, since tyrphostin B42 was a potent antagonist of the LPS-induced
NO
2 production in FSDC (Table 2). The
p38 MAPK is also involved in the NO
2 production induced by LPS in these cells, although to a much lower extent.
The other subgroup of MAPK, the JNK, may also play a role in the signaling pathway leading to an increase in NO production in FSDC, but this possibility was not investigated in this work, since there are no specific inhibitors available.
Another important aspect is the cross talk and signal integration among
MAPK pathways and among MAPK pathways and other signaling pathways. Our
results demonstrated that p38 MAPK and MEK cooperate in the NO
production induced by LPS and cytokines (Table 2). It is also possible
that the MAPK and the JAK signaling pathways cooperate in FSDC to
trigger the NO production induced by LPS, GM-CSF, and TNF-. In fact,
in HCD-57 cells, a significant contribution of the cytosolic tyrosine
kinase JAK2 to the erythropoietin-induced activation of the Ras/MEK
cascade was observed (3).
Activation of the NF-B was shown to represent a crucial step in the
induction of iNOS (38). In FSDC we demonstrated that LPS, but not
TNF-
or GM-CSF, induced the translocation of NF-
B into the
nucleus (Fig. 3) by a mechanism that involves the JAK2, since the
specific inhibitor of this kinase, tyrphostin B42, completely prevented
the translocation of NF-
B after LPS stimulation. To our knowledge,
this is the first report showing the involvement of JAK2 in the NF-
B
activation. Taken together, our results indicate that, although JAK2
participates in the induction of iNOS by LPS and cytokines in FSDC,
this kinase is coupled to the activation of NF-
B only in response to
stimulation with LPS. Although the p38 MAPK played a minor role in the
stimulation of NO production evoked by LPS, the antagonist SB-203580
was without effect on the translocation of NF-
B to the nucleus in
the same experimental conditions, suggesting that the effect of p38
MAPK in the expression of iNOS is not mediated via NF-
B activation.
It is clear that JAKs serve to phosphorylate the signal transducer and
activator of transcription (STATs) when the cytokine receptor lacks
intrinsic kinase activity. Activated STATs form dimers, translocate to
the nucleus, and bind to response elements to induce transcription
(13). Therefore, it is possible that, in FSDC, the NF-B is not the
only transcription factor involved in the induction of iNOS by LPS and cytokines.
There are potentially significant physiological and physiopathological aspects of iNOS expression and NO production by DC. NO appears to be involved in skin physiology, growth, and remodeling (5). Because it is diffusible across cells, NO produced by LC regulates lymphocyte proliferation by inhibiting or inducing apoptosis (9, 20). Therefore, elucidation of the molecular mechanisms by which endotoxin and cytokine induce NO production by DC is of major importance and may have implications for the design and execution of immunotherapeutic strategies.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. G. Girolomoni (Laboratory of Immunology, Istituto Dermopatico dell'Immacolata, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy) for the kind gift of the fetal skin-derived dendritic cell line. We thank Dr. J. Reis for technical assistance in the utilization of the fluorescent confocal microscope.
![]() |
FOOTNOTES |
---|
This work was supported by Praxis/P/SAU/126/96.
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 and other correspondence: M. C. Lopes, Faculdade de Farmácia da Universidade de Coimbra, Rua do Norte, 3000 Coimbra Codex, Portugal.
Received 21 December 1998; accepted in final form 9 August 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Akiyama, T.,
and
H. Ogawara.
Use and specificity of genistein as inhibitor of protein tyrosine kinases.
Methods Enzymol.
201:
362-370,
1991[Medline].
2.
Bittorf, T.,
R. Jaster,
and
J. Brock.
Rapid activation of the MAP kinase pathway in hematopoietic cells by erythropoietin, granulocyte-macrophage colony-stimulating factor and interleukin-3.
Cell. Signal.
6:
305-311,
1994[Medline].
3.
Bittorf, T.,
R. Jaster,
B. Lüdtke,
B. Kamper,
and
J. Brock.
Requirement for JAK2 in erythropoietin-induced signaling pathways.
Cell. Signal.
9:
85-89,
1997[Medline].
4.
Blank, C.,
C. Bogdan,
C. Bauer,
K. Erb,
and
H. Moll.
Murine epidermal Langerhans cells do not express inducible nitric oxide synthase.
Eur. J. Immunol.
26:
792-796,
1996[Medline].
5.
Bruch-Gerharz, D.,
T. Ruzicka,
and
V. Kolb-Bachofen.
Nitric oxide in human skin: current status and future prospects.
J. Invest. Dermatol.
110:
1-7,
1998[Abstract].
6.
Da Silva, J.,
B. Pierrat,
J. L. Mary,
and
W. Lesslauer.
Blockade of p38 mitogen-activated protein kinase pathway inhibits inducible nitric-oxide synthase expression in mouse astrocytes.
J. Biol. Chem.
272:
28373-28380,
1997
7.
Dionne, S.,
I. D. D'Agata,
F. M. Ruemmele,
E. Levy,
J. St.-Louis,
A. K. Srivastava,
D. Levesque,
and
E. G. Seidman.
Tyrosine kinase and MAPK inhibition of TNF- and EGF-stimulated IEC-6 cell growth.
Biochem. Biophys. Res. Commun.
242:
146-150,
1998[Medline].
8.
Dudley, D. T.,
L. Pang,
S. J. Decker,
A. J. Bridges,
and
A. R. Saltiel.
A synthetic inhibitor of the mitogen-activated protein kinase cascade.
Proc. Natl. Acad. Sci. USA
92:
7686-7689,
1995[Abstract].
9.
Genaro, A. M.,
S. Hortelano,
A. Alvarez,
C. Martínez-A,
and
L. Boscá.
Splenic B lymphocyte programmed cell death is prevented by nitric oxide release through mechanisms involving sustained Bcl-2 levels.
J. Clin. Invest.
95:
1884-1890,
1995[Medline].
10.
Girolomoni, G.,
M. B. Lutz,
S. Pastore,
C. U. Abmann,
A. Cavani,
and
P. Ricciardi-Castagnoli.
Establishment of a cell line with features of early dendritic cell precursors from fetal mouse skin.
Eur. J. Immunol.
25:
2163-2169,
1995[Medline].
11.
Green, L. C.,
D. A. Wagner,
J. Glogowski,
P. L. Skipper,
J. S. Wishnok,
and
S. R. Tannenbaum.
Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids.
Anal. Biochem.
126:
131-138,
1982[Medline].
12.
Guan, Z.,
L. D. Baier,
and
A. R. Morrison.
p38 Mitogen-activated protein kinase down-regulates nitric oxide and up-regulates prostaglandin E2 biosynthesis stimulated by interleukin-1.
J. Biol. Chem.
272:
8083-8089,
1997
13.
Horvath, C. M.,
and
J. E. Darnell, Jr.
The state of the STATs: recent developments in the study of signal transduction to the nucleus.
Curr. Opin. Cell Biol.
9:
233-239,
1997[Medline].
14.
Joly, G. A.,
M. Ayres,
and
R. G. Kilbourn.
Potent inhibition of inducible nitric oxide synthase by geldanamycin, a tyrosine kinase inhibitor, in endothelial, smooth muscle cells, and in rat aorta.
FEBS Lett.
403:
40-44,
1997[Medline].
15.
Kleinert, H.,
T. Waallerath,
G. Fritz,
I. Ihrig-Biedert,
F. Rodriguez-Pascual,
D. A. Geller,
and
U. Förstermann.
Cytokine induction of NO synthase II in human DLD-1 cells: roles of the JAK-STAT, AP-1 and NF-B signaling pathways.
Br. J. Pharmacol.
125:
193-201,
1998[Abstract].
16.
Knowles, R. G.,
and
S. Moncada.
Nitric oxide synthase in mammals.
Biochem. J.
298:
249-258,
1994[Medline].
17.
Kunz, D.,
H. Mühl,
W. Gaby,
and
J. Pfeilschifter.
Two distinct signalling pathways trigger the expression of inducible nitric oxide synthase in rat mesangial cells.
Proc. Natl. Acad. Sci. USA
91:
5387-5391,
1994[Abstract].
18.
Liu, R.,
T. Itoh,
K. Arai,
and
S. Watanabe.
Activation of c-Jun N-terminal kinase by human granulocyte macrophage-colony stimulating factor in BA/F3 cells.
Biochem. Biophys. Res. Commun.
234:
611-615,
1997[Medline].
19.
Lockhart, B. P.,
K. C. Cressey,
and
J. M. Lepagnol.
Supression of nitric oxide formation by tyrosine kinases inhibitors in murine N9 microglia.
Br. J. Pharmacol.
123:
879-889,
1998[Abstract].
20.
Lu, L.,
C. A. Bonham,
F. G. Chambers,
S. C. Watkins,
R. A. Hoffman,
R. L. Simmons,
and
A. W. Thomson.
Induction of nitric oxide synthase in mouse dendritic cells by IFN-, endotoxin, and interaction with allogeneic T cells. Nitric oxide production is associated with dendritic cell apoptosis.
J. Immunol.
157:
3577-3586,
1996[Abstract].
21.
Memer, U. K.,
and
B. Brüne.
Modification of macrophage glyceraldehyde-3-phosphatedehydrogenase in response to nitric oxide.
Eur. J. Pharmacol.
302:
171-182,
1996[Medline].
22.
Meydan, N.,
T. Grunberger,
H. Dadi,
M. Shahar,
E. Arpaia,
Z. Lapidot,
J. S. Leeder,
M. Freedman,
A. Cohen,
A. Gazit,
A. Levitzki,
and
C. M. Roifman.
Inhibition of acute lymphoblastic leukaemia by a JAK2 inhibitor.
Nature
379:
645-648,
1996[Medline].
23.
Miller, D. R.,
J. M. Collier,
and
R. E. Billings.
Protein tyrosine kinase activity regulates nitric oxide synthase induction in rat hepatocytes.
Am. J. Physiol.
272 (Gastrointest. Liver Physiol. 35):
G207-G214,
1997
24.
Mosmann, T.
Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.
J. Immunol. Methods
65:
55-63,
1983[Medline].
25.
Neary, J. T.
MAPK cascades in cell growth and death.
News Physiol. Sci.
12:
286-293,
1997
26.
Nishiya, T.,
T. Uehara,
H. Edamatsu,
Y. Kaziro,
H. Itoh,
and
Y. Nomura.
Activation of STAT1 and subsequent transcription of inducible nitric oxide synthase gene in C6 glioma cells is independent of interferon--induced MAPK activation that is mediated by p21ras.
FEBS Lett.
408:
33-38,
1997[Medline].
27.
Nishiya, T.,
T. Uehara,
and
Y. Nomura.
Herbimycin A suppresses NF-B activation and tyrosine phosphorylation of JAK2 and the subsequent induction of nitric oxide synthase in C6 glioma cells.
FEBS Lett.
371:
333-336,
1995[Medline].
28.
Oddis, C. V.,
R. L. Simmons,
B. G. Hattler,
and
M. S. Finkel.
Protein kinase A activation is required for IL-1-induced nitric oxide production by cardiac myocytes.
Am. J. Physiol.
271 (Cell Physiol. 40):
C429-C434,
1996
29.
Okuda, S.,
F. Kanda,
Y. Kawahara,
and
K. Chihara.
Regulation of inducible nitric oxide synthase expression in L6 rat skeletal muscle cells.
Am. J. Physiol.
272 (Cell Physiol. 41):
C35-C40,
1997
30.
Paul, A.,
K. Doherty,
and
R. Plevin.
Differential regulation by protein kinase C isoforms of nitric oxide synthase induction in RAW 264.7 macrophages and rat aortic smooth muscle cells.
Br. J. Pharmacol.
120:
940-946,
1997[Abstract].
31.
Qureshi, A. A.,
J. Hosoi,
S. Xu,
A. Takashima,
R. D. Granstein,
and
E. A. Lerner.
Langerhans cells express inducible nitric oxide synthase and produce nitric oxide.
J. Invest. Dermatol.
107:
815-821,
1996[Abstract].
32.
Sarih, M.,
V. Souvannavong,
and
A. Adam.
Nitric oxide synthase induces macrophage death by apoptosis.
Biochem. Biophys. Res. Commun.
191:
503-508,
1993[Medline].
33.
Verhasselt, V.,
C. Buelens,
F. Willems,
D. D. Groote,
N. Haeffner-Cavaillon,
and
M. Goldman.
Bacterial lipopolysaccharide stimulates the production of cytokines and the expression of costimulatory molecules by human peripheral blood dendritic cells. Evidence for a soluble CD14-dependent pathway.
J. Immunol.
158:
2919-2925,
1997[Abstract].
34.
Wallach, D.
Cell death induction by TNF: a matter of self control.
Trends Biochem. Sci.
22:
107-109,
1997[Medline].
35.
Watanabe, S.,
T. Itoh,
and
K. Arai.
Roles of JAK kinases in human GM-CSF receptor signal transduction.
J. Allergy Clin. Immunol.
98:
S183-S191,
1996[Medline].
36.
Waterman, W. H.,
T. F. P. Molski,
C. Huang,
J. L. Adams,
and
R. I. Sha'afi.
Tumor necrosis factor--induced phosphorylation and activation of cytosolic phospholipase A2 are abrogated by an inhibitor of the p38 mitogen-activated protein kinase cascade in human neutrophils.
Biochem. J.
319:
17-20,
1996[Medline].
37.
Wolf, D. J.,
and
A. Lubeskie.
Aminoguanidine is an isoform-selective, mechanism-based inactivator of nitric oxide synthase.
Arch. Biochem. Biophys.
316:
290-301,
1995[Medline].
38.
Xie, Q.,
Y. Kashiwabara,
and
C. Nathan.
Role of transcription factor NF-B/ReI in induction of nitric oxide synthase.
J. Biol. Chem.
269:
4705-4708,
1994
39.
Zhao, Y.,
F. Wagner,
S. J. Frank,
and
A. S. Kraft.
The amino-terminal portion of the JAK2 protein kinase is necessary for binding and phosphorylation of the granulocyte-macrophage colony-stimulating factor receptor c chain.
J. Biol. Chem.
270:
13814-13818,
1995