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Involvement of JAK2 and MAPK on type II nitric oxide
synthase expression in skin-derived dendritic cells
M. T. CRUZ,
1,2
C. B. DUARTE,
2
M. GONC¸ALO,
3
A. P. CARVALHO,
2
AND M. C. LOPES
1,2
1
Faculdade de Farma´cia e
3
Faculdade de Medicina (Servico de Dermatologia),
2
Centro de
Neurocieˆncias, Universidade de Coimbra, 3000 Coimbra, Portugal
Cruz, M. T., C. B. Duarte, M. Gonc¸alo, A. P. Carvalho,
and M. C. Lopes. InvolvementofJAK2andMAPKontypeII
nitric oxide synthase expression in skin-derived dendritic
cells. Am. J. Physiol. 277 (Cell Physiol. 46): C1050–C1057,
1999.—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-a (TNF-a) and granulocyte-
macrophage colony-stimulating factor (GM-CSF)]. Expres-
sion of the inducible isoform of NO synthase (iNOS) was
confirmed by immunofluorescence with an antibody against
iNOS.ThetyrosinekinaseinhibitorgenisteindecreasedLPS-
and GM-CSF-induced nitrite (NO
2
2
) production. The effect of
LPS and cytokines on NO
2
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
2
production evoked by LPS,
TNF-a, 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-a or GM-CSF on NO
2
2
production.
Immunocytochemistry studies revealed that the transcrip-
tion factor nuclear factor-kB 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 majorrole inthe induction of iNOS in FSDC.
mitogen-activated protein kinase; Janus kinase 2; nuclear
factor-kB
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 constitu-
tively expressed in cells of neural and endothelial
origin, respectively, and they are regulated by physi-
ological changes in the intracellular calcium concentra-
tion. 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 consis-
tent 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 character-
ized 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), afamily of structur-
allyrelated kinasesthat areinvolved incellular events,
such as growth, differentiation, and stress responses
(25). In mammalian cells, three subgroups of MAPK
havebeendetectedandinclude 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 genetranscription (13, 25).
The signaling events required for NO production in
DC have not been identified yet. Studies on LC (and
DC)havebeen hampered bythedifficultiesin 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-1b (IL-1b), and tumor necrosis
factor-a (TNF-a)]on theproduction of NO by FSDCand
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
cytokinesontheintracellulardistributionofthenuclear
transcription factor nuclear factor-kB (NF-kB) and
whether JAK2 is involvedin NF-kB activation.
MATERIALS AND METHODS
Materials. The rabbit anti-mouse iNOS polyclonal anti-
bodywas purchased from TransductionLaboratories (Lexing-
ton, KY), the rabbit anti-human NF-kB p65 was from Serotec
(Oxford, UK), and the fluorescein isothiocyanate (FITC)-
conjugatedswineanti-rabbitimmunoglobulin wasfromDAKO
(Copenhagen, Denmark). The Prolong Antifade Kit was ob-
tained from Molecular Probes Europe (Leiden, The Nether-
lands). LPS from Escherichia coli (serotype 026:B6) was
obtained from Sigma Chemical (St. Louis, MO), the mouse
TNF-a receptor was from Boehringer Mannheim (Carnaxide,
Portugal), and mouse IL-1b receptor was purchased from
Pharmingen (San Diego, CA). The murine GM-CSF receptor
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. Section 1734
solely to indicate this fact.
0363-6143/99 $5.00 Copyright
r
1999 the American Physiological SocietyC1050 http://www.ajpcell.org
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 fromSigma 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 env
AKR
-
myc
MH2
fusion gene. FSDC show characteristics of immature
DC and express low levels of major hisotcompatibility com-
plex II molecules (I-A
d,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 cellprecursors (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-tetrazoliumbromide(MTT;
see Ref. 24). After removal of cell-free supernatants for the
nitrite (NO
2
2
) 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. Superna-
tants 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 forma-
zan. 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 620nm.
NO
2
2
measurement. The production of NO was assessed as
the accumulation of NO
2
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% H
3
PO
4
], during 10 min. The absor-
bance at 550 nm was measured in an automated plate reader
(SLT). The NO
2
2
concentration was determined from a sodium
nitrite standard curve.
Immunofluorescence microscopy. For immunofluorescence
analysis,FSDC were grownon glass coverslipsand werefixed
and permeabilized by immersing the coverslips in 220°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
temperaturewitharabbit polyclonalantibodydirectedagainst
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 experi-
ments consisted of processing the same preparations as
described, except for the omission of the primary antibody,
and resulted inno specificstaining.
Data analysis. Results are presented as means 6 SE of the
indicated number of experiments. Mean values were com-
pared using one-way ANOVA and the Bonferroni multiple
comparison test. Thesignificance levelwas 0.05.
RESULTS
NO
2
2
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-a, and IL-1b) for 48 h, and the culture superna-
tants were collected for the NO
2
2
measurement (Fig. 1).
Cell-free supernatants, from cells incubated in the
presence of LPS (5 µg/ml), showed an increase in NO
2
2
concentration (from 0.63 6 0.06 to 24.1 6 2.92 µM),
reflecting an increase in NO production. NO
2
2
produc-
tion was also stimulated upon incubation of FSDC with
100U/ml TNF-a (from 0.63 6 0.06 to4.76 6 0.82 µM)or
200 ng/ml GM-CSF (from 0.63 6 0.06 to 6.32 6 1.1µM).
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 pro-
duction was observed in FSDC cultures exposed to
IL-1b (Fig. 1).
Fig. 1. Nitrite concentration in super-
natantsofnonstimulatedfetal skinden-
dritic cells (FSDC) maintained in cul-
ture medium (control) or in cells
stimulated for 48 h with 5 µg/ml lipo-
polysaccharide (LPS) or cytokines [100
U/ml tumor necrosis factor-a (TNF-a),
10 ng/ml interleukin-1b (IL-1b), or 200
ng/ml granulocyte-macrophage colony-
stimulating factor (GM-CSF)]. Cells
were also incubated with aminoguani-
dine (AG; 1 mM) during the period of
stimulation with the cytokines or LPS.
Each value represents the mean 6 SE
from 3–24 experiments, performed in
duplicate.*P, 0.05,**P, 0.01, ***P,
0.001, and ns, not significant.
C1051NITRIC OXIDE SYNTHASE IN A SKIN DENDRITIC CELL LINE
To confirm whether NO
2
2
was formed via the induc-
tion 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 inhib-
ited NO
2
2
accumulation in the culture supernatant
induced either by LPS or by the cytokines TNF-a and
GM-CSF (Fig. 1).
Because TNF-a may be involved in the activation of
cytotoxic events (34) and considering that stimulation
of macrophages with LPS plus interferon-g (IFN-g)
produces cytotoxic amounts of NO
2
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). Approxi-
mately 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-a-treated
cellsand in thecells maintained inculture 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-a.
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
NO
2
2
production upon stimulation of the cells with LPS,
TNF-a, or GM-CSF. The genistein (30 µM) slightly
reduced NO
2
2
production induced by TNF-a (100 U/ml)
to 92.8 6 6.6%, whereas NO
2
2
production induced by
LPS (5 µg/ml) and byGM-CSF (200 ng/ml) was reduced
to 24.6 6 5.5 and 56.1 6 13.3% of the control, respec-
tively (Table 2). Genistin, the inactive analog of genis-
tein (1), did not affect NO
2
2
production evoked by LPS,
TNF-a, 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.
Wenextinvestigatedtherole of MAPK kinase (MEK),
p38 MAPK, and JAK2 in the activation of NO
2
2
produc-
tion 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 concentra-
tions 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 NO
2
2
production, which indicates that
the MEK signaling cascade was not involved in the
LPS-induced NO production in this cell line. In con-
trast, NO
2
2
production induced by either GM-CSF or
TNF-a was slightly reduced, to 89.5 6 6.1 and 83.2 6
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 treat-
ment with 20 µM SB-203580. The p38 MAPK inhibitor
reduced LPS-, GM-CSF-, and TNF-a-induced NO
2
2
pro-
duction to 78.8 6 3.6, 72.4 6 7.0 and 85.3 6 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 6 2.9% of the control (P , 0.05; data
not shown). Under these experimental conditions, no
morphological evidence of celldeath was observed.
Tyrphostin B42 was a potent inhibitor of LPS-,
GM-CSF- and TNF-a-induced NO production in FSDC
(Table 2), which indicates that the JAK pathway plays
an important role in the regulation of iNOSexpression.
This compound was the most effective protein kinase
inhibitorinpreventingthe cytokine-induced NO produc-
tion; at 20 µM, tyrphostin B42 inhibited NO
2
2
produc-
tion induced by TNF-a and by GM-CSF to 48.6 6 13.1
and 38.2 6 7.5% of the control, respectively. The JAK2
inhibitor reduced NO
2
2
formation induced by LPS to
41.9 6 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
2
formation evoked by LPS, TNF-a, and
GM-CSF to 76.3 6 5.8, 62.6 6 1.05 and 69.5 6 3.2% of
the control, respectively (Table 2).
Translocation of NF-
k
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
Table 1. Effect of LPS and cytokines on the MTT
reduction by FSDC
MTT Reduction,
% Control
GM-CSF (200 ng/ml) 99.165.7*
(n519)
LPS (5 µg/ml) 88.362.6*
(n520)
TNF-a (100 U/ml) 95.163.2*
(n519)
Values are means 6 SE; no. of experiments performed in duplicate
in parentheses. Fetal skin dendritic cells (FSDC) were incubated
with lipopolysaccharide (LPS), tumor necrosis factor-a (TNF-a), or
granulocyte-macrophage colony-stimulating factor (GM-CSF) at the
indicatedconcentrationfor48h. The 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-tetrazolium bromide (MTT) assay was performed as de-
scribed in
MATERIALS AND METHODS. Results are expressed as a
percentage of MTT reduction by control cells maintained in culture
medium. *Not significant.
C1052 NITRIC OXIDE SYNTHASE IN A SKIN DENDRITIC CELL LINE
Fig. 2. Detection of inducible nitric oxide synthase
(iNOS) protein expression in the FSDC line maintained
for 48 h in culture medium, in the absence (A)orinthe
presence of 5 µg/ml LPS (B) or 200 ng/ml GM-CSF (C).
Immunostaining was performed as described in
MATERI-
ALS AND METHODS (magnification 3250). Cells present in
8 fields in 2 preparations were counted.
C1053NITRIC OXIDE SYNTHASE IN A SKIN DENDRITIC CELL LINE
nuclear transcription factor NF-kB and whether the
observed effects could beattributed to JAK2.
The effect of LPS, GM-CSF, and TNF-a on the
intracellular distribution of NF-kB in FSDC was exam-
ined immunocytochemically, using a specific antibody
against the p65 subunit. Before LPS stimulation, p65
was distributed throughout the cytoplasm (Fig. 3A).
WhenFSDC 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-a for 15, 30,
or 60 min, no accumulation of p65 in the nuclei of the
cells was observed (datanot shown).
DISCUSSION
In the present work, we show that LPS, TNF-a, 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 activa-
tion of JAK2 is crucial for the translocation ofNF-kBto
the nucleus upon activationwith LPS.
We found that LPS was more potent than TNF-a or
GM-CSF in activating NO
2
2
production due to stimula-
tion of iNOS expression. In contrast to the significant
effect of LPS, TNF-a, and GM-CSF on the NO
2
2
produc-
tion by FSDC, no effect of IL-1b 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-g in
bone marrow DC andLC (20, 31), in contrast to the lack
of effect of IFN-g 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
expressiNOSevenin the presence ofLPSandGM-CSF.
However, our results are in agreement with those
obtainedin highly purified bone marrowDC stimulated
with IFN-g 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 iNOSappears to be regulated
in a cell-specific manner(14, 23, 28–30).
We observed that genistein inhibited the LPS- and
GM-CSF-induced NO
2
2
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
celltypesshowingthatgenisteinsuppresses the expres-
sion of iNOS activity induced by cytokines (23, 29). In
contrast, genistein had no effect on NO
2
2
production
induced by TNF-a (Table 2).
The JAK2 inhibitor tyrphostin B42 was a potent
antagonist of the GM-CSF-induced NO
2
2
production,
although SB-203580 also inhibited the NO
2
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
bc 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 evi-
dence 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-a in FSDC. Other
authors reported the involvement of the p38 MAPK in
Table 2. Effect of different kinase inhibitors and of genistin on LPS
and cytokine-induced NO
2
2
production by FSDC
Nitrite Production, %
Genistein
(30 µM)
Genistin
(30 µM)
PD-098059
(30 µM)
SB-203580
(10 µM)
SB-203580
(20 µM)
SB-203580
(10 µM)
1
PD-098059
(30 µM)
Tyrphostin
B42
(20 µM)
LPS (5 µg/ml) 24.665.5‡ 91.261.1§ 103.4613.5§ 84.762.3† 78.863.6‡ 76.365.8‡ 41.967.9‡
(5) (3) (4) (5) (5) (3) (4)
TNF-a (100 U/ml) 92.866.6§ 103.768.1§ 83.264.4† 89.360.9§ 85.363.4† 62.661.1‡ 48.6613.1*
(4) (4) (3) (4) (4) (2) (4)
GM-CSF (200 ng/ml) 56.1613.3* 128.4613.1§ 89.566.1§ 90.663.9§ 72.467.0† 69.563.2* 38.267.5‡
(5) (2) (5) (5) (4) (2) (3)
Values are means 6 SE;no.ofexperimentsperformedinduplicateinparentheses.FSDCwereincubatedwiththeinhibitorsattheindicated
concentrations in the presence of LPS, TNF-a, or GM-CSF for 48 h. NO
2
2
levels in the culture supernatants were analyzed by the Griess
reaction, as described in
MATERIALS AND METHODS. Results are expressed as percentage of maximal NO
2
2
production by cells maintained in
culture medium in the presence of LPS or cytokines and in the absenceof inhibitors. *P , 0.05, †P , 0.01, ‡P , 0.001, and §not significant.
C1054 NITRIC OXIDE SYNTHASE IN A SKIN DENDRITIC CELL LINE
Fig. 3. Translocation of p65 into the nuclei of
FSDC in response to LPS. FSDC were main-
tained in culture medium (A), treated with 5
µg/ml LPS for 30 min (B), or preincubated
with 50 µM tyrphostin B42 for 2 h before
stimulation with 5 µg/ml LPS for 30 min (C).
Cells were then fixed and incubated with
antibody against p65 followed by FITC-conju-
gated anti-rabbit immunoglobulins as de-
scribed in
MATERIALS AND METHODS. Intracellu-
lar localization of FITC-labeled p65 was
observed using a confocal fluorescent micro-
scope (magnification 3300).
C1055NITRIC OXIDE SYNTHASE IN A SKIN DENDRITIC CELL LINE
the signaling pathways of TNF-a. The TNF-a-stimu-
lated phosphorylation and activation of cytosolic phos-
pholipase A
2
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-a and IL-1a in astrocytes (6), but, in serum-
starved mesangial cells, the inhibition of p38 MAPK
promoted IL-1b-induced iNOS expression and subse-
quent 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
productioninducedbyTNF-a in FSDC (Table 2).TNF-a
increased ERK1 and ERK2 phosphorylation in IEC-6
cells, and PD-098059 inhibited TNF-a-induced IEC-6
cell growth (7). In contrast, in mouse astrocytes, iNOS
expression induced by TNF-a and IL-1a was only
partially affected by PD-098059 (6). However, our
results suggest that both GM-CSF and TNF-a induce
NO
2
2
production mainly through the JAK signaling
pathway (Table 2). Accordingly, the activation of iNOS
expression in DLD-1 cells seems to require JAK activ-
ity, especially the IFN-g-activated JAK2 (15).
The lack of effect of PD-098059 on NO
2
2
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-g/LPS has been reported to
require tyrosine kinase activity, specifically JAK2 (27),
and the Ras/MAPK signaling pathway does not appear
tobe involved in the IFN-g/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
2
production in FSDC (Table 2). The p38
MAPK is also involved in the NO
2
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 andsignal
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
(Table2). It isalso possible thattheMAPK andtheJAK
signaling pathways cooperate in FSDC to trigger the
NO production induced by LPS, GM-CSF, and TNF-a.
In fact, in HCD-57 cells, a significant contribution of
thecytosolictyrosinekinaseJAK2to the erythropoietin-
induced activation of the Ras/MEK cascade was ob-
served (3).
Activation of the NF-kB was shown to represent a
crucial step in the induction of iNOS (38). In FSDC we
demonstrated that LPS, but not TNF-a or GM-CSF,
induced the translocation of NF-kB 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-kB after
LPS stimulation. To our knowledge, this is the first
report showing the involvement of JAK2 in the NF-kB
activation. Taken together, our results indicate that,
althoughJAK2 participates in the induction of iNOSby
LPS and cytokines in FSDC, this kinase is coupled to
the activation of NF-kB 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 trans-
locationofNF-kBtothenucleusinthe same experimen-
tal conditions, suggesting that the effect of p38 MAPK
in the expression of iNOS is not mediated via NF-kB
activation.
It is clear that JAKs serve to phosphorylate the
signaltransducerandactivatoroftranscription(STATs)
when the cytokine receptorlacks intrinsic kinase activ-
ity. 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-kB is not the only transcription factor
involved in the induction of iNOS by LPS and cyto-
kines.
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
lymphocyteproliferationbyinhibitingorinducingapop-
tosis (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.
We thank Dr. G. Girolomoni (Laboratory of Immunology, Istituto
Dermopatico dell’Immacolata, Istituto diRicovero 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 assistanceinthe
utilization of the fluorescent confocal microscope.
This work was supported by Praxis/P/SAU/126/96.
Address for reprint requests and other correspondence: M. C.
Lopes, Faculdade de Farma´cia da Universidade de Coimbra, Rua do
Norte, 3000 Coimbra Codex, Portugal.
Received 21 December 1998; accepted in final form 9August 1999.
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