production in mouse macrophages via MAP kinase activation
The Gonda (Goldschmied) Medical Diagnosis Research Center, Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel
Correspondence: C. Brodie, Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel. E-mail: chaya{at}mail.biu.ac.il
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production.
Macrophages expressed NGF and the NGF receptors TrkA and p75. Treatment
of J744 cells or peritoneal macrophages with NGF induced a large
increase in the production of TNF-. In addition, NGF induced the
secretion of nitric oxide in interferon-
-treated J774 cells or
lipopolysaccharide-treated peritoneal macrophages. The induction of
TNF- production by NGF was blocked by K252a, an inhibitor of the
TrkA receptor. NGF induced phosphorylation and activation of
extracellular signal-regulated kinase, Erk1/Erk2 and c-Jun
amino-terminal kinase, whereas it did not induce phosphorylation of p38
mitogen-activated protein kinase. Inhibition of the MAP kinase-Erk
kinase pathway with PD 098059 decreased the secretion of TNF- by
NGF. Our results suggest that NGF has an important role in the
activation of macrophages during inflammatory responses via activation
of mitogen-activated protein kinases.
Key Words: NGF • NGF receptors • nitric oxide
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Macrophages play important roles in various aspects of inflammatory
responses, immunity, host defense, and tissue repair
[16
]. Macrophages secrete a large number of cytokines,
such as tumor necrosis factor (TNF)- and interleukin (IL)-1, which
mediate some of their effects. Another important factor that plays a
role in the ability of macrophages to kill pathogens and virally
infected and transformed cells is nitric oxide (NO)
[17
]. The expression of inducible NO synthase (iNOS) in
macrophages is induced by a combination of stimuli such as interferon
(IFN)- and bacterial lipopolysaccharide (LPS), IL-1, or TNF-
[18
, 19
]. The signaling pathways involved
in the induction of TNF- and NO include activation of various
members of the mitogen-activated protein (MAP) kinase superfamily,
depending on the types of cells and stimuli [20
21
22
].
In this study, we examined the expression of NGF and its receptors in
mouse macrophages and studied the signals involved in NGF’s effects on
the production of TNF- by mouse macrophages.
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and mouse
TNF- enzyme-linked immunosorbent assay (ELISA) kits were obtained
from Genzyme (Boston, MA). Antiphosphorylated and antinonphosphorylated
p44/42MAPKErk1/Erk2, c-Jun amino-terminal kinase (JNK),
p38MAPK antibodies, and nonradioactive p44/42 MAP kinase
assay kits were purchased from New England Biolabs (Beverly, MA). The
LPS (serotype 0127:B8) and anti-NGF antibody were obtained from Sigma
(St. Louis, MO), and mouse NGF was obtained from Alamone Laboratories
(Jerusalem, Israel). Anti-TrkA was from Santa Cruz Biotechnology (Santa
Cruz, CA), and mouse anti-p75 was from Chemicon (Temecula, CA).
Cell cultures
The mouse monocyte-macrophage cell line J774 was maintained in
medium consisting of Dulbecco’s modified Eagle’s medium containing
10% heat-inactivated fetal calf serum, 2 mM glutamine, penicillin (50
U/mL), and streptomycin (0.05 mg/mL). Cells were split once a week by
short exposure to 0.25% trypsin.
Peritoneal macrophages were prepared from 8-week-old BALB/c mice. Cells were collected by peritoneal lavage 4 days after intraperitoneal injection of 1.5 mL of sterile 3% thioglycolate broth. Cells (106/mL) were plated in six-well tissue culture plates in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, penicillin (100 U/mL), streptomycin (100 µg/mL), and L-glutamine (2 mM). Nonadherent cells were removed by washing the plates 4 h after plating, and the adherent macrophages were cultured in the presence or absence of different stimuli. The cultures consisted of 98% macrophages as determined by staining with anti-CD14 antibody.
Preparation of mRNA, and reverse transcriptase-PCR analysis
Total RNA was extracted from primary cultures of peritoneal
macrophages or J774 cells (7 x 106 cells per
10-cm-diameter plate) with TRI Reagent® (Gibco-BRL, Gaithersburg,
MD). The digestion of the remaining DNA was performed with 2 µL of
RQ1 deoxyribonuclease (1 U/µL) (Promega, Madison, WI). Five
micrograms of total RNA were transcribed into cDNA with ExpandTM
reverse transcriptase (RT; Boehringer Mannheim, Mannheim,
Germany), using 50 pmol of oligo(dt)15. Relative
levels of NGF and the NGF receptors, compared with the mRNA level for
the ribosomal protein S-12, were determined by semiquantitative RT-PCR
[14
]. All reactions were carried out in a total volume
of 50 µL containing 1 µg of cDNA, 1 U of Taq DNA
polymerase (Appligene, Strasbourg, France), 200 µM concentration of
each deoxyribonucleoside triphosphate, and 50 pmol of each primer. The
primer sequences are shown in Table 1
.
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View this table: [in a new window] |
Table 1. Primer Sequences Used for This Study
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Measurement of TNF- and NGF production
Supernatants of untreated and treated macrophages were
collected, and levels of TNF- or NGF were determined using a
commercial ELISA kit. The detection levels of TNF- in this assay
system were 
10–20 pg/mL and 3–5 pg/mL, respectively.
NO secretion
NO secretion was determined by measuring NO2 as
described elsewhere [25
]. Briefly, 100-µL volumes of
culture supernatants were mixed with equal volumes of Griess reagent
(one part 0.1% N-naphthyl ethylenediamine dihydrochloride
in H2O and one part 1% sulfanilamide in 5%
H3PO4) at room temperature for 10 min. Optical
density at 540 nm was measured, and NO2- was
quantified using NaNO2 as the standard.
Preparation of cell homogenates
Cells were washed, and plates were placed on ice, scraped with a
rubber policeman, and centrifuged at 700 g for 10 min. The
supernatants were aspirated, and the cell pellets were resuspended in
100 µL of lysis buffer (25 mM Tris-HCl, pH 7.4, 50 mM NaCl, 0.5%
sodium deoxycholate, 2% Nonidet P-40, 0.2% sodium dodecyl sulfate, 1
mM phenylmethylsulfonyl fluoride, 50 µg/mL of aprotinin, 50 µM
leupeptin, 0.5 mM Na3VO4) on ice for 15 min.
The cell lysates were centrifuged for 15 min at 12,000 g in
an Eppendorf® microcentrifuge (Vaudaux-Eppendorf, Basel,
Switzerland), supernatants were removed, and 2x sample buffer was
added.
Immunoblot analysis
Lysates (30 µ/g of protein) were resolved by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and were transferred to
nitrocellulose membranes. The membranes were blocked with 5% dry milk
in phosphate-buffered saline (PBS) and subsequently stained with the
primary antibody. Specific reactive bands were detected using a goat
anti-rabbit or goat anti-mouse immunoglobulin G conjugated to
horseradish peroxidase (Bio-Rad, Hercules, CA), and the immunoreactive
bands were visualized by using an enhanced chemiluminescence (ECL)
Western blotting detection kit (Amersham, Arlington Heights, IL).
MAP kinase phosphorylation
Phosphorylation of Erk1/Erk2 was analyzed using a rabbit
polyclonal anti-phospho-specific p44/42 MAP kinase antibody directed
against the dually phosphorylated form of Erk MAP kinase enzyme
(Thr-202 and Tyr-204). Phosphorylation of p38MAPK was
analyzed using anti-phospho-p38MAPK (Thr-180 and Tyr-182),
and phosphorylation of JNK was analyzed using anti-phospho-SAPK/JNK
(Thr-183 and Tyr-185). For these assays, cells were serum starved for
1 h and then stimulated with NGF for different periods of time.
Cells were washed with ice-cold PBS and harvested in a lysis buffer,
and cell lysates were subjected to Western blot analysis. Membranes
were stained with the appropriate antibodies followed by anti-rabbit
antibody conjugated to horseradish peroxidase.
Immune complex kinase assay
Erk1/Erk2 activity was measured using a nonradioactive p44/42
MAP kinase assay kit (New England Biolabs) according to the
manufacturer’s instructions. Briefly, cell lysates (50 µg) were
incubated with an antimonoclonal phospho-p44/42 MAP kinase (Thr-202 and
Tyr-204) to selectively immunoprecipitate active Erk1/Erk2. Kinase
assays were performed by incubating immunoprecipitates in 50 µL of
kinase buffer containing 10 µM adenosine triphosphate and 10 µg of
Elk-1 fusion protein for 30 min at room temperature. The reaction was
stopped by the addition of 30 µL of 2x Laemmli sample buffer, and
samples were boiled for 5 min. The phosphorylation of Elk-1 was
measured by Western blotting using anti-phospho-Elk-1 antibody
(Ser-383).
Immunofluorescence staining
Cells were stimulated with LPS for 24 h and were stained
for p75 and TrkA as previously described [7
]. Briefly,
cells (106/mL) were washed with PBS plus 5% bovine serum
albumin and incubated with anti-p75 or anti-TrkA antibodies for 30 min.
The cells were washed three times and incubated with anti-rabbit
antibody conjugated to fluorescein isothiocyanate. After washings,
cells were analyzed with a FACScan® analyzer (Becton Dickenson,
Paramus, NJ), and then the percentage of positive cells and mean
fluorescence intensity were calculated. Nonspecific staining was
determined using a rabbit control antibody.
Statistical analysis
The results are presented as the means ± SE.
All data were analyzed using an analysis of variance to determine the
level of difference between the treatments.
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Figure 1. NGF expression in mouse macrophages. Expression of NGF mRNA in
macrophages is shown (A). RNA of peritoneal macrophages or J774 cells
was extracted, and the samples were processed for RT-PCR. The relative
levels of NGF mRNA were estimated by a semiquantitative RT-PCR.
Reaction mixture with 1 µL of RNA instead of cDNA was used as a
control to exclude any contaminants as a source of amplified fragments
(0). The RT-PCR products were visualized by ethidium bromide staining.
The results of one representative experiment from four similar
experiments are presented. NGF secretion was measured in peritoneal
macrophages (stippled bars)and in J774 cells (striped bars) (B) using
an ELISA. For these experiments, cells were treated with LPS (1
µg/mL) for 24 h.
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Figure 2. Expression of TrkA and p75 in peritoneal macrophages. Macrophages were
plated in the absence or presence of LPS (100 ng/mL) for different
periods. The relative levels of TrkA and p75 mRNA, compared with the
mRNA of the ribosomal protein S-12, were estimated using a
semiquantitative RT-PCR. RNA was extracted, and the samples were
processed for RT-PCR using primers specific for TrkA and p75. mRNA was
detected using Southern blotting followed by hybridization with a
32P-labeled internal primer as described in Materials and
Methods (A). Expression of TrkA (B) and p75 (C) proteins was examined
by immunofluorescence staining using anti-p75 or anti-TrkA antibodies
followed by donkey anti-rabbit antibody conjugated to fluorescein
isothiocyanate and flow cytometry analysis. Cells were treated with LPS
(1 µg/mL) for 24 h. As a control, we used a rabbit control
antibody, which served as a species-matched antibody (NC). Results of a
representative experiment out of four similar experiments are
presented.
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production, J774
cells (Fig. 3A
) and peritoneal macrophages (Fig. 3B)
were incubated with NGF
(100 ng/mL) or LPS (1 µg/mL) for 24 h, and TNF- production
was determined using an ELISA. Untreated cells produced low levels of
TNF-, whereas LPS induced a large increase in TNF- production, as
expected. Treatment of both cell types with NGF (100 ng/mL) induced an
increase in TNF- production. The kinetics of TNF- production by
NGF was slower than that induced by LPS. Thus, LPS had already induced
an increase in TNF- production after 8 h of treatment, whereas
NGF’s effects were first observed after 12 h of treatment (data
not shown). The increase was dose dependent and was blocked by the
anti-NGF antibody, indicating that these effects were specific (data
not shown). The anti-NGF antibody did not block the secretion of
TNF- induced by LPS (data not shown).
 View larger version (31K): [in a new window] |
Figure 3. TNF- secretion by NGF in mouse macrophages. J774 cells (A) or
peritoneal macrophages (B) were treated with NGF (100 ng/mL) or LPS (1
µg/mL) for 24 h. Supernatants were collected, and TNF-
secretion was measured using an ELISA. Results are the means ±
SE of data from four separate experiments. *,
P < 0.002; **, P < 0.001.
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(100 U/mL) alone induced a small
increase in NO secretion. Concomitant treatment of the cells with
IFN- (100 U/mL) and NGF induced an increase in the production of NO
over that observed with NGF alone (Fig. 4A
). Initial effects were observed with 25 ng/mL of NGF, and maximal
effects were obtained with 100 ng/mL of NGF (data not shown). Similar
effects were observed in the induction of the iNOS protein (Fig. 4B)
.
Thus, IFN- induced an increase in the expression of iNOS, and
treatment with NGF further increased this expression. In contrast, NGF
induced only a small increase in the production of NO in J774 cells
treated with LPS (100–1,000 ng/mL) (data not shown).
 View larger version (24K): [in a new window] |
Figure 4. Induction of iNOS and NO by NGF in macrophages. J774 cells (A) were
treated with NGF (100 ng/mL) in the absence or presence of IFN- (50
U/mL) for 24 h, and the production of NO (A) or the expression of
iNOS (B) was determined. Peritoneal macrophages were treated with NGF
in the absence and presence of LPS (100 ng/mL), and the production of
NO (C) or the expression of iNOS (D) was determined. NO was measured in
the culture supernatants using Griess reagent. Results are means ± SE of data from four separate experiments. For iNOS
expression, cells were harvested, and the expression of iNOS was
examined by Western blot analysis. Results of a representative
experiment out of four similar experiments are presented. *,
P < 0.05; **, P; lt 0.005.
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(10–500 U/mL) alone or with IFN- and NGF did not induce NO
secretion (data not shown).
NGF’s effects are mediated via activation of the TrkA receptor
To examine the signaling pathways involved in the induction of
TNF- by NGF in macrophages, we used J774 cells. We found, as
previously described [26
], that J774 cells expressed the
NGF receptor TrkA and that NGF induced tyrosine phosphorylation of TrkA
in these cells (data not shown). To examine the role of TrkA in NGF’s
effects, we used K252a, which inhibits the tyrosine kinase activity of
this receptor [27
]. Pretreatment of the cells with K252a
(50 ng/mL) inhibited the production of TNF- by NGF (Fig. 5A
) and the production of NO in response to NGF plus IFN- (Fig. 5B)
, suggesting the involvement of TrkA activity in these effects.
Treatment of the cells with K252a did not reduce the production of
TNF- induced by LPS (data not shown) or the production of NO induced
by IFN- (Fig. 5B)
.
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Figure 5. K252a inhibits the induction of TNF- and NO by NGF. J774 cells
were treated with K252a (50 ng/mL) for 1 h and then with NGF for
24 h. The secretion of TNF- (A) was measured using an ELISA.
The results are means ± SESE of data from four
separate experiments. *, P < 0.002; **,
P < 0.001. The secretion of NO was measured as
described in Materials and Methods (B).
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has been shown to be mediated by the
activation of different members of the MAP kinase family
[20
21
22
]. We therefore examined whether NGF can induce
activation of the various MAP kinases in macrophages and which of the
pathways is involved in the induction of TNF- by NGF. J774 cells were treated with NGF (100 ng/mL) for different periods of time, and the phosphorylation of Erk1/Erk2 was examined using anti-phospho-specific p44/p42 (Erk1/Erk2) antibodies. As presented in Figure 6A , NGF induced phosphorylation of Erk1/Erk2. The maximal effect was observed after 15 min, and the phosphorylation declined to its basal level after 60 min. Similar results were obtained for Erk1/Erk2 activity using a p44/42MAPK immunoprecipitation kinase assay. For these experiments, J774 cells were stimulated with NGF for various periods of time, and the activity of Erk1/Erk2 was determined by measuring the phosphorylation of the substrate Elk-1. The activity of Erk1/Erk2 reached a maximum after 15 min and declined after 30 min (Fig. 6B) .
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Figure 6. Phosphorylation and activation of Erk1/Erk2 by NGF. J774 cells
were stimulated with NGF for the time periods indicated, and the
phosphorylation (A) or the kinase activity (B) of Erk1/Erk2 was
determined. Erk1/Erk2 phosphorylation was detected by Western blot
analysis using an anti-phospho-Erk1/Erk2 antibody. The membranes were
also blotted with the anti-Erk1/Erk2 antibody. For the kinase activity,
the phosphorylated form of Erk1/Erk2 was immunoprecipitated, and the
kinase activity of Erk1/Erk2 was determined with a nonradioactive
kinase assay kit using the substrate Elk-1 as described in Materials
and Methods.
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Figure 7. Phosphorylation of JNK and p38MAPK by NGF. J774 cells were
stimulated with NGF for the time periods indicated, and the
phosphorylation of JNK (A) or p38MAPK (B) was determined
using anti-phospho-specific JNK or p38 antibodies. The membranes were
also blotted with anti-JNK or anti-p38 antibodies. The results are from
one representative experiment of three similar experiments.
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induced by NGF production, we used the specific MEK inhibitor PD 098059
[28
]. Pretreatment of the cells with PD 098059 blocked
the phosphorylation of Erk1 and Erk2 by NGF (Fig. 8A
). In parallel, pretreatment of the cells with PD 098059 induced a
strong inhibitory effect on the production of TNF- induced by NGF
(Fig. 8B)
.
 View larger version (25K): [in a new window] |
Figure 8. PD 098059 inhibits Erk1/Erk2 phosphorylation and TNF- production by
NGF. J774 cells were treated with PD 098059 (20 µM) for 1 h and
then with NGF for 15 min (A) or for 24 h (B). The phosphorylation
of Erk1/Erk2 was determined by Western blot analysis using
anti-phospho-Erk1/Erk2 antibodies as described for Fig. 6A
, and TNF-
secretion was determined using an ELISA (B). The results are means ± SE of data from four separate experiments. *,
P < 0.001; **, P < 0.002.
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B [32
, 33
]. We also found that macrophages expressed the NGF receptors TrkA and p75. Our findings of TrkA expression are in accordance with various reports regarding the expression of this receptor on immunocompetent cells such as B and T lymphocytes [12 , 34 ], mast cells, eosinophils [35 ], and macrophages [26 , 31 ]. Our results indicate that stimulation of macrophages with LPS induces up-regulation of the TrkA receptor, similar to its effect on the expression of NGF. Although TrkA is expressed on a large number of hematopoietic and lymphoid cells, the expression of p75 has been reported mainly on B lymphocytes [7 ]. In contrast, no expression of p75 has been found on T lymphocytes or mast cells [12 ], and the expression of this receptor on macrophages appears to be controversial. Thus, Susaki et al. [26 ] reported that macrophages do not express p75 protein, and Ehrhard et al. [10 ] reported a lack of p75 mRNA expression on human monocytes. In contrast, Garaci et al. [31 ] reported that human macrophages expressed p75 and that its expression was increased after HIV infection. Our finding that p75 is expressed on mouse macrophages can be attributed either to differences in experimental conditions or to a different degree of macrophage activation or differentiation. Indeed, our findings that activation of macrophages by LPS induced a strong down-regulation of p75 mRNA further support this assumption.
The differential regulation of TrkA and p75 expression by LPS may affect the responsiveness of macrophages to NGF and the ability of NGF to activate specific signaling pathways such as those of Erk1/Erk2 and JNK. Indeed, the relative expression and activity of TrkA and p75 have been reported to affect the ability of NGF to induce cell differentiation and survival in neuronal cells because of differential activation of specific signaling pathways [36 , 37 ].
NGF induced the production of TNF- in mouse macrophages, and this
effect was mediated by activation of TrkA. TNF- is a
well-characterized proinflammatory cytokine involved in host defense
and a wide range of pathological conditions. In addition, NGF also
induced the production of NO in the presence of IFN- or LPS. iNOS
has been reported to be induced in macrophages after stimulation with
LPS and IFN-, TNF- and IFN-, IFN- and phorbol esters, and
other stimuli [17
, 18
, 38
].
Thus, it appears that NGF can provide a complementary signal for
IFN- that can replace the one provided by LPS for the induction of
iNOS in J774 cells. The induction of NO by NGF may be mediated by
proinflammatory cytokines that are secreted by macrophages. Because NGF
induces the production of IL-1 [26
] and TNF- in mouse
macrophages, it is possible that these factors mediate, at least in
part, the effects of NGF on iNOS induction and NO secretion.
Activation of macrophages by bacterial LPS or by inflammatory cytokines initiates a signal transduction cascade that involves activation of various members of the MAP kinase superfamily. The three main members, Erk1/Erk2, p38MAPK, and JNK, are differentially activated in macrophages in response to specific stimuli [20 21 22 ]. We found that in J774 cells NGF induced activation of Erk1/Erk2 and, to a lesser extent, of JNK, whereas it did not affect the phosphorylation of p38MAPK. Activation of these signaling pathways by NGF has been described mainly in PC-12 cells [39 ], whereas there have been only few reports describing the occurrence of NGF signaling in immunocompetent cells [40 ]. The activation of Erk1/Erk2 by NGF in PC-12 cells has been associated with the ability of NGF to induce neuronal cell differentiation [41 ], and activation of JNK was associated with apoptosis of PC-12 cells in response to NGF withdrawal [42 ].
Our results suggest that activation of Erk1/Erk2 is involved in the
production of TNF- in response to NGF, because the MEK inhibitor PD
098059 exerted a marked inhibitory effect on TNF- production.
Because PD 098059 did not block completely the induction of TNF-
production by NGF, we cannot at this point exclude the possibility that
JNK or other signaling pathways are involved in this effect. Different
members of the MAP kinase family have been implicated in the regulation
of TNF- expression. In a recent study, Ajizian et al. reported that
in macrophages the p38 and Erk pathways are involved in the
up-regulation of TNF- production in response to LPS and IFN-
[20
]. In another study, p42MAPK was shown to
be necessary for the synthesis of TNF- induced by FcR
cross-linking [22
]. In peripheral blood mononuclear
cells, inhibition of the protein kinase C and p42MAPK
pathways reduced the production of TNF- [43
].
Collectively, these results suggest that the regulation of TNF-
expression may be mediated by different members of the MAP kinase
family, depending on the cell type and the specific stimuli.
The secretion of NGF by macrophages and its effects on macrophage
activation suggest that in addition to its neurotrophic effects, NGF is
also involved in macrophage function. One possible role of
macrophage-derived NGF is in nerve regeneration. Recently, activated
macrophages have been reported to play a role in nerve regeneration via
secretion of unidentified factors [44
]. Indeed, the
expression and secretion of NGF by activated macrophages implicate NGF
as a possible mediator of this process. NGF also activates macrophages
and induces the secretion of IL-1-ß, TNF-, and NO. Thus, the
interplay between NGF and other cytokines can lead to beneficial or
deleterious effects on the nervous system. For example, induction of
low levels of TNF- can provide a neuroprotective signal, which may
contribute to the effects of NGF on nerve regeneration and repair,
whereas higher concentrations of TNF- can lead to cell death
[45
]. Since macrophages and other immunocompetent cells
express NGF receptors, NGF may act in an autocrine or paracrine manner
in inflammatory responses and in nerve regeneration.
Received June 9, 2000; revised January 18, 2001; accepted January 19, 2001.
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receptor cross-linking activates p42, p38 and JNK/SAPK mitogen-activated protein kinases in murine macrophages. Role for p42MAPK in Fc receptor-stimulated TNF- synthesis J. Immunol. 158,3433-3438[Abstract]B modulates lipopolysaccharide-induced microglial nerve growth factor expression Glia 22,401-407[Medline] and IL-8: engagement of different signal transduction pathways including the p42MAP kinase pathway J. Leukoc. Biol. 64,124-133[Abstract]This article has been cited by other articles:
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