(Received for publication, December 11, 1996, and in revised form, March 14, 1997)
From the Laboratory of Molecular Immunology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1760
Aggregation of the high affinity IgE receptor
(FcRI) in a mast cell line resulted in activation of the p42 and the
stress-activated p38 mitogen-activated protein (MAP) kinases. Selective
inhibition of these respective kinases with PD 098059 and SB 203580 indicated that p42 MAP kinase, but not p38 MAP kinase, contributed to
the production of the cytokine, tumor necrosis factor-
, and the
release of arachidonic acid in these cells. Neither kinase, however,
was essential for Fc
RI-mediated degranulation or constitutive
production of tumor growth factor-
. Studies with SB 203580 and the
p38 MAP kinase activator anisomycin also revealed that p38 MAP kinase negatively regulated activation of p42 MAP kinase and the responses mediated by this kinase.
Stimulation of mast cells by aggregation of membrane IgE receptors
(FcRI), leads to recruitment of the tyrosine kinase Syk and
activation of Syk-dependent signaling cascades (1, 2). These cascades include activation of phospholipase C and sphingosine kinase for mobilization of calcium ions and
PKC1 (3, 4) and the activation of p42 MAP
kinase cascade through Ras (2, 5). These cascades lead ultimately to
secretion of intracellular granules, a response primarily driven by the increase in [Ca2+]i and activation of PKC (6),
and a cPLA2-mediated release of arachidonic acid. The
activation of cPLA2 is dependent on increase of
[Ca2+]i and phosphorylation by MAP kinase (2, 7,
8).
Stimulated mast cells also produce a variety of cytokines that include
interleukins 1, 3, 4, 5, and 6 as well as TNF and granulocyte-macrophage colony-stimulating factor (9, 10). Typically,
increased expression of cytokine mRNA and protein is detectable 30 min to several hours after the addition of stimulant (11). These
cytokines, particularly TNF
, are thought to mediate pathologic
inflammatory reactions (10) and protective responses to bacterial
infection (12). The production and release of TNF
are regulated
through signals transduced by calcium and PKC, although there are
indications that additional Fc
RI-mediated signals may operate for
optimal production of TNF
in cultured RBL-2H3 mast cells. Compared
with antigen, other stimulants are relatively weak inducers of TNF
production when doses of stimulants are matched for maximal stimulation
of degranulation (13). Also, concentrations of Ro31-7549 that block
PKC, secretion of granules, and release of TNF
only partially block
production of TNF
(13).
The present objective was to determine whether stimulation of MAP
kinases induces additional signals for production of TNF. A linkage
between these events has not been established in mast cells.
Antigen-induced stimulation of p42 MAP kinase coincides with the
activation of its upstream regulators, Ras, Raf, and MEK-1 (2, 5), and
persists through the period when production of TNF
would be most
apparent (14). As noted in this paper, however, RBL-2H3 cells also
possess the mammalian homologue of the yeast HOG-1 protein kinase, p38
MAP kinase. We have utilized the MEK-1 inhibitor, PD 098059 (15, 16),
and the p38 MAP kinase inhibitor, SB 203580 (17), to evaluate the role
of these MAP kinases in the production of TNF
and, for comparison,
the release of arachidonic acid, degranulation, and production of
TGF
. Release of arachidonic acid is thought to be dependent on
phosphorylation of cPLA2 by MAP kinase, although the
identity of the MAP kinase is uncertain (18). Degranulation and TGF
production were assumed to be MAP-kinase-independent responses (7, 19).
We show that, while p42 MAP kinase regulated production of both TNF
and arachidonic acid, p38 MAP kinase negatively regulated the
activation of p42 MAP kinase and the responses mediated by this
kinase.
Reagents were obtained from the following sources:
all reagents for cell culture from Life Technologies, Inc.; ATP from
Boehringer Mannheim; adenosine 5-[
-32P]triphosphate
tetra(triethyl-ammonium) salt and [14C]arachidonic acid
from DuPont NEN; phenyl-Sepharose from Pharmacia (Uppsala, Sweden); MAP
kinase substrates (myelin basic protein and a myelin basic peptide,
residues 94-102), polyclonal antibodies against the COOH-terminal
peptide of rat MAP kinase R2 (erk1-CT), anti-phosphotyrosine monoclonal
antibody, 4G10, and p42 MAP kinase glutathione S-transferase
fusion protein from Upstate Biotechnology Inc. (Lake Placid, NY);
polyclonal antibodies against p38 MAP kinase, MEK, and
cPLA2 from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA);
TGF
1 kit and Factor-testTM Mouse TNF-
from Genzyme
Corp. (Cambridge, MA). Ro31-7549 was obtained from LC Laboratories.
The compounds PD 098059 and SB 203580 were synthesized in the Tsukuba
Research Laboratories, Eisai Co., according to the procedures of
Bridges et al. (20) and Adams et al. (21),
respectively, and purified by column chromatography and
recrystallization. These compounds were determined to be >95% pure on
the basis of high performance liquid chromatography and NMR analysis.
The antigen, DNP-BSA, and O-dinitrophenol-specific monoclonal IgE were kindly supplied by Dr. Henry Metzger (NIAMS, National Institutes of Health, Bethesda, MD).
The RBL-2H3 cell line was maintained in complete growth medium (minimum essential medium) supplemented with 15% fetal calf serum, glutamine, antibiotic, and antimycotic agents. Trypsinized cells were plated into 150-mm culture dishes or six-well Costar cluster plates and were incubated overnight in complete growth medium with O-dinitrophenol-specific IgE (0.5 µg/ml) and, for measurement of arachidonic acid release, [14C]arachidonic acid (0.1 µCi/ml).
Cultures were washed the next day and replenished with the required
medium. For the assay of hexosaminidase or
[14C]arachidonic acid, experiments were performed in a
PIPES-buffered medium (25 mM PIPES, pH 7.2, 159 mM NaCl, 5 mM KCl, 0.4 mM
MgCl2, 1.0 mM CaCl2, 5.6 mM glucose, and 0.1% fatty acid-free fraction V bovine
serum albumin). For [32P]phosphorylation of proteins,
cultures were incubated for 90 min with 32P-labeled
orthophosphate in PIPES-buffered medium exactly as described (7). For
all other assays, experiments were performed in complete growth medium
supplemented with 15% fetal calf serum (for measurement of TNF),
5% fetal calf serum (for measurement of TGF
), or 0.1% bovine serum
albumin and 25 mM Hepes, pH 7.2 (for assay of MAP kinases
and separation of proteins by immunoprecipitation and electrophoresis).
The inhibitors were added either 30 min (PD 098059) or 15 min (SB
203580 and indomethacin) before stimulation of cultures with antigen
(DNP-BSA) as described in the figure legends.
Release of the granule marker, hexosaminidase, was
determined by colorimetric assay of medium and cell lysates by
previously described procedures (6). For measurement of release of
arachidonic acid, cells were labeled to equilibrium with
[14C]arachidonic acid before the addition of inhibitors
and antigen as described above. Reactions were terminated by placing
cultures on ice and rapidly removing medium. The medium was briefly
centrifuged (Beckman Microfuge for 30 s) to remove extraneous
cells. Both medium and cell lysates (in 0.1% Triton X-100) were
assayed for hexosaminidase (6) and radiolabel (22). Values were
expressed as the percentage of intracellular hexosaminidase or
radiolabel that was released into the external medium, and they were
corrected for spontaneous release from unstimulated cells. It should be noted that, in RBL-2H3 cells, arachidonic acid is metabolized in part
to leukotriene C4/B4 and prostaglandin
D2 via the 5-lipoxygenase and cyclooxygenase pathways,
respectively (23, 24). Release of radiolabel, as measured in this
paper, was an estimate of total release of
[14C]arachidonic acid and its metabolites. The cytokines
were assayed as described elsewhere (19). Whole cell lysates were
prepared by freezing and thawing the cultures three times. TGF was
assayed with a human TGF
enzyme-linked immunosorbent assay kit,
which utilized a mouse monoclonal anti-human antibody that
cross-reacted with rat TGF
. TNF
was assayed with a murine TNF
enzyme-linked immunosorbent assay kit, which utilized a monoclonal
hamster anti-murine antibody that reacted with mouse or rat TNF
and
-
. The limits of detection for these assays were 25 pg of
TGF
/106 cells and 6 pg of TNF
/106 cells.
Values were corrected for spontaneous release in the absence of
stimulant (
3% for release of hexosaminidase,
2% for release of
arachidonic acid, and undetectable release of TNF
) except for
TGF
, which was produced constitutively in RBL-2H3 cells (19).
After
stimulation of cultures in six-well cluster plates, the cultures were
washed once, and the medium was removed. The plates were then placed on
ice before the addition of 510 µl of a Tris buffer (25 mM
Tris, pH 7.5, 25 mM NaCl, 1 mM
Na3VO4, 2 mM EGTA, 1.5 mM dithiothreitol, 2.5 mM
p-nitrophenyl phosphate, and 20 µg/ml leupeptin and
aprotinin). Cells were disrupted by freezing and thawing three times.
The lysate was centrifuged (15,800 × g for 10 min),
and 450 µl of the supernatant fraction was mixed with 50 µl of
ethylene glycol and 80 µl of washed phenyl-Sepharose. The
phenyl-Sepharose was washed beforehand with 300 µl of the Tris
buffer. The mixture was kept on ice for 5 min for binding of MAP kinase
to the beads. After centrifugation, the phenyl-Sepharose beads were
washed with 1 ml of 10% (v/v) ethylene glycol and then with 30% (v/v)
ethylene glycol. Finally, MAP kinase was eluted by incubating the beads
with 75 µl of 60% ethylene glycol for 5 min on ice. After
centrifugation of the suspension, 15 µl of supernatant was incubated
(15 min, 30 °C) in a solution that contained 50 mM Tris
(pH 7.5), 10 mM MgCl2,
[-32P]ATP (10 Ci/mmol, 37 kBq/tube), and 25 µg of
MAP kinase substrate peptide (peptide 94-102 of bovine myelin basic
protein). The phosphorylated peptide was isolated by centrifugation of
the incubation mixture through phosphocellulose membrane (SpinZyme;
Pierce), which was then washed twice with 500 µl of 75 mM
H3PO4 for the assay of radioactivity.
p42 and p38 MAP kinases were
immunoprecipitated with the appropriate polyclonal antibodies by
procedures described elsewhere (2). Equal amounts of immunoprecipitated
proteins from 5 × 106 cells were incubated in a MOPS
buffer (25 mM -glycerol phosphate, 1 mM
EGTA, 1 mM sodium orthovanadate, 1 mM
dithiothreitol, and 25 mM MOPS, pH 7.2) with
Mg2+-[
-32P]ATP (10 µCi in 150 µM cold ATP, 25 µM MgCl2) and 5 µM myelin basic protein (18 kDa) as substrate in a final
volume of 30 µl. The mixture was incubated at 30 °C for 12 min.
The reaction was terminated by the addition of 30 µl of 2 × SDS
sample buffer. MEK was immunoprecipitated with anti-MEK antibody and
assayed similarly except that p42 MAP kinase glutathione
S-transferase fusion protein (1 µg/assay) was used as
substrate for phosphorylation. Proteins were separated by 12%
SDS-PAGE. Radioactive proteins were detected by autoradiography.
The preparation of cell lysates and immunoprecipitates, analysis of proteins by SDS-PAGE, and transfer to nitrocellulose paper were performed as described elsewhere (2, 7) with the following exception: cPLA2 was separated on NOVEX 10% Tris/glycine gels for 3 h at 35 mA and 4 °C as described by Kramer and co-workers (25). Previously described procedures were used for isolation and detection of [32P]MEK (7). Otherwise, proteins were detected by the immunoblotting technique with antibodies against MEK, p42 MAP kinase, cPLA2, or anti-phosphotyrosine. Secondary antibodies included horseradish peroxidase-conjugated antibody against rabbit IgG or mouse IgG. Finally, proteins were visualized by the ECL System (Amersham Corp.) or by autoradiography.
As shown in
Fig. 1, the MEK inhibitor PD 098059 attenuated
antigen-induced [32P]phosphorylation of MEK (panel
A) and the activation of MEK as determined by in vitro
assay of immunoprecipitated MEK (panel B). Activation of p42
MAP kinase was also attenuated, as indicated by the change in
electrophoretic migration of p42 MAP kinase (panel C) or by
the assay of MAP kinase activity of immunoprecipitated p42 MAP kinase
(panel D). The extent of these inhibitions was dependent on
the concentration of PD 098059. As shown in Fig. 2, the
suppression of MAP kinase activation by PD 098059 (panel A)
was associated with similar dose-dependent suppression of
arachidonic acid release (panel B) and TNF production
(panel C). The suppression of the latter two responses was
highly correlated (r > 0.95). All three responses were
inhibited by ~50% with 10 µM PD 098059. As will be
described later, activation of cPLA2 was also inhibited by
PD 098059. These results suggested that release of arachidonic acid and
production of TNF
were both regulated by p42 MAP kinase.
The Effect of PD 098059 on Degranulation and TGF
To test the selectivity of PD 098059, we next examined
the effects of this compound on antigen-stimulated degranulation and the constitutive production of TGF, which are thought not to be
regulated by MAP kinase (7, 19). PD 098059 had only minimal effects on
stimulated release of the granule constituent, hexosaminidase (Fig.
3A) and the production of TGF
in
unstimulated cells (Fig. 3B). The only significant effect
was partial inhibition (<30%) of degranulation at 50 µM
PD 098059 (Fig. 3A).
The p38 MAP Kinase Inhibitor, SB 203580, Enhances Activation of p42 MAP Kinase, Release of Arachidonic Acid, and Production of TNF
Antigen stimulation also resulted in
increased activity of p38 MAP kinase (Fig.
4A, compare lanes 1 and
2). The p38 kinase inhibitor, SB 203580, inhibited this
activation (Fig. 4A, lanes 3 and 4).
Interestingly, antigen activation of p42 MAP kinase was enhanced
significantly by SB 203580. This enhancement was apparent when cells
were stimulated with 20 or 200 ng/ml antigen (Fig. 4B). The
latter concentration of antigen was known to elicit maximal activation
of p42 MAP kinase.2 These results suggested
that p38 MAP kinase negatively regulates p42 MAP kinase and that this
regulation is alleviated by SB 203580.
The enhanced activation of p42 MAP kinase in the presence of SB 203580 was associated with increased release of arachidonic acid (Fig.
5A) and production of TNF (Fig.
5B). In the experiment shown in Fig. 5B, cells
were stimulated with a low concentration of antigen (6 ng/ml) to
maximize enhancement of the TNF
response (250% increase in Fig.
5B). At optimal doses of antigen enhancement of TNF
production was less (40-80% increase) but still significant (data not
shown).
Because pyridinyl imidazoles that are closely related to SB 203580 have cyclooxygenase-inhibitory activity (25, 26), experiments were conducted to determine whether blockade of cyclooxygenase activity with indomethacin (27) altered accumulation of radiolabel in the medium by suppressing metabolism [14C]arachidonate via this enzyme. Unlike SB 203580, 10 µM indomethacin did not significantly alter release of radiolabel from antigen-stimulated cells (7.8 ± 0.4% release over 15 min versus 7.2 ± 0.2% release in the absence of indomethacin; mean ± S.E. in eight cultures from two experiments). It seemed probable, therefore, that SB 203580 enhanced release rather than the accumulation of [14C]arachidonic acid in the medium.
In contrast to the increased release of arachidonic acid and production
of TNF, SB 203580 had no significant effect on antigen-induced degranulation (Fig. 5C) or constitutive production of TGF
(Fig. 5D). Collectively, these results provided further
evidence for the notion that release of arachidonic acid and TNF
production are both regulated by p42 MAP kinase. In addition, the
results suggested that p38 MAP kinase negatively modulates these
responses through p42 MAP kinase.
The above results suggested that release of arachidonic acid, as well
as production of TNF, was regulated by p42 MAP kinase. As in other
systems (25, 28), the phosphorylation of cPLA2 in
stimulated RBL-2H3 cells leads to decreased electrophoretic mobility of
the enzyme (7). The connection between p42 MAP kinase and the release
of arachidonic acid via cPLA2 was further demonstrated by
the finding that the antigen-induced retardation of electrophoretic
migration of cPLA2 (25) was suppressed by PD 098059 but not
by SB 203580 (Fig. 6).
Activation of p38 MAP Kinase by Anisomycin Partially Suppresses Activation of p42 MAP Kinase and Release of Arachidonic Acid
The
p38 MAP kinase activator, anisomycin, markedly activated this kinase
(Fig. 7A) but much less so p42 MAP kinase
(Fig. 7B). The combination of anisomycin and antigen
revealed inhibitory communication between these two kinase. For
example, the combination of stimulants resulted in less activation of
p38 MAP kinase (Fig. 7C, lane 3) than that
induced by antigen (Fig. 7C, lane 2) or anisomycin (Fig. 7A, lane 2) alone. The
combination also caused less activation of p42 MAP kinase (Fig.
7D, lane 3) than that by antigen alone (Fig.
7D, lane 2). Thus, stimulation of p42 MAP kinase
by antigen appeared to block activation of p38 MAP kinase by
anisomycin, and conversely stimulation of p38 MAP kinase by anisomycin
appeared to partially block activation of p42 MAP kinase by antigen.
Consistent with the latter situation, anisomycin partially suppressed
antigen-induced release of arachidonic acid (25 ± 4% reduction,
mean of three experiments). This reduction corresponded to an
approximately 25% reduction in p42 MAP kinase activation as determined
by densitometric analysis of the blots shown in Fig. 7D and
two other experiments. Anisomycin almost totally blocked (by 83 ± 4%) antigen-induced production of TNF, probably as a consequence,
however, of its known inhibitory actions on protein synthesis at the
translation step (29). Presumably, de novo synthesis of
TNF
would be especially sensitive to inhibitors of protein
synthesis.
Past studies have shown that the responses evoked by antigen in
RBL-2H3 cells were dependent on calcium and signals generated through
PKC or MAP kinase. These studies indicated, for example, that PKC
regulated degranulation (6) as well as the production and secretion of
TNF, although it appeared likely that additional Fc
RI-mediated
signals facilitated TNF
production (13). Activation of p42 MAP
kinase, in contrast, was associated with phosphorylation of
cPLA2 and release of arachidonic acid (2, 7). These
studies, however, did not address the issue of whether other MAP
kinases, such as p38 MAP kinase, regulated cPLA2.
The present results demonstrate that both p38 and p42 MAP kinases are
activated in antigen-stimulated cells. Activation of the latter kinase
appears to be most closely related to release of arachidonic acid and
production of TNF. All three events are inhibited by the MEK
inhibitor, PD 098059 (Fig. 2), and enhanced by the p38 MAP kinase
inhibitor, SB 203580 (Figs. 4 and 5). Both compounds are reported to be
selective inhibitors of MEK (i.e. PD 098059) and p38 MAP
kinase (i.e. SB 203580) when tested against a wide range of
kinases (15-17). In addition, the enhancement of responses in the
presence of SB 203580, in contrast to the attenuation of p42 MAP kinase
activation by the p38 MAP kinase activator, anisomycin (Fig. 7),
suggest that p38 MAP kinase negatively regulates activation of p42 MAP
kinase and its associated responses. Antigen-stimulated degranulation
and the constitutive production of TGF
in RBL-2H3 cells are
minimally affected by the inhibitors (Figs. 3 and 5). Collectively, the
results support the notion that p42 MAP kinase regulates release of
arachidonic acid and promotes an additional signal for stimulating
TNF
production but does not regulate degranulation. Interestingly,
the p38 MAP kinase inhibitor, SB 203580, was first identified as an
inhibitor of cytokine biosynthesis in lipopolysaccharide-stimulated human monocytes (17) and was subsequently shown to suppress TNF
production in lipopolysaccharide-injected mice (30). The compound also
possessed anti-inflammatory activity in mouse models of arthritis
(collagen- and adjuvant-induced), whereas cellular immune responses
measured ex vivo were unaffected (30). It is possible,
therefore, that different MAP kinase pathways are utilized for
activating gene transcription for cytokine synthesis when synthesis is
induced by inflammatory agents or through multimeric immunologic
receptors such as Fc
RI.
The question has been raised whether p38 rather than p42 MAP kinase is responsible for the activation of cPLA2 (18, 25). cPLA2 is phosphorylated by both kinases in thrombin-stimulated platelets, although the phosphorylation by p38 MAP kinase does not appear to activate cPLA2 (25). Our results indicate that p42 MAP kinase regulates phosphorylation of cPLA2 and release of arachidonic acid and suggest, therefore, that this kinase is the activator of cPLA2 at least in RBL-2H3 cells.
Mast cells, including RBL-2H3 cells, also contain a low molecular
weight secreted form (type II) of PLA2 (31) in secretory granules, and this form is presumably released along with other granule
constituents in activated cells (32). A role for secreted PLA2 is unlikely, however, because total suppression of
degranulation by selective inhibitors of PKC, such as Ro31-7549 and
calphostin C, minimally affects release of arachidonic acid in RBL-2H3
cells (Refs. 7 and 33 and see below).3 In
addition to the correlations between activation of the p42 MAP
kinase/cPLA2 pathway and release of arachidonic acid noted here with PD 098059 and SB 203580, similar correlations have been noted
in previous studies with less specific MAP kinase inhibitors. Activation of the entire Raf/MEK/p42 MAP kinase pathway and release of
arachidonic acid were suppressed equally by the glucorticoid, dexamethasone, and the kinase inhibitor, quercetin, while effects on
degranulation were apparent only at relatively high concentrations of
these agents (7, 34). Correlations were noted with the PKC inhibitor,
Ro31-7549. This inhibitor transiently delayed activation of p42 MAP
kinase in antigen-stimulated RBL-2H3 cells. There was a corresponding
transient delay in the release of arachidonic acid, although the
cumulative release eventually equaled that observed in the absence of
Ro31-7549 (14). On the basis of these and other results, we have
suggested that the p42 MAP kinase/cPLA2 pathway, although
transiently activated by PKC, was predominantly activated by Ras
through recruitment of Shc/Grb2/Sos or Vav by FcRI (14). Others have
reported that fatty acids, particularly arachidonic acid, activate p42
and p44 MAP kinases through PKC (35). This scenario is unlikely in
antigen-stimulated RBL-2H3 cells, however, because of the predominance
of the PKC-independent (i.e. Ro31-7549-resistant) pathway
in RBL-2H3 cells.
The present data extend previous findings on the regulation of TNF
synthesis and release. This cytokine is synthesized de novo
and subsequently secreted via Golgi in a PKC- and
calcium-dependent manner (13). The PKC inhibitor,
Ro31-7549, blocks secretion of TNF
but only partially suppresses
synthesis of TNF
(6, 13, 19). Thus, additional signals may be
necessary for optimal stimulation of TNF
synthesis. Antigen is a
particularly potent stimulant of TNF
production when compared with
the combination of calcium ionophore and PKC agonist, phorbol
12-myristate 13-acetate (13, 19). These observations and the present
studies with the MAP kinase inhibitors suggest that optimal production
of TNF
is achieved through activation of both PKC and p42 MAP
kinase.
The present findings may explain why antigen-induced activation of p42
MAP kinase (34), release of arachidonic acid (22, 34) and production of
TNF (36) exhibit similar sensitivity to dexamethasone. All three
responses are totally suppressed in RBL-2H3 cells that have been
treated with 10 nM dexamethasone, whereas
antigen-stimulated hydrolysis of phosphoinositides, increase in
[Ca2+]i, and degranulation (22, 34) are only
partially suppressed by treatment of cells with 100 nM
dexamethasone. Dexamethasone, as noted above, inhibits the entire
Raf/MEK/p42 MAP kinase/cPLA2 pathway at nanomolar
concentrations (34). Therefore, if p42 MAP kinase is the common
regulator of TNF
production and arachidonic acid release, the
similar sensitivities to dexamethasone would be expected.
The connections between the p42 MAP kinase pathway and cytokine
production are unknown for mast cells, but recent reports indicate the
following connections in other types of cells. The overexpression of
Raf1 (37, 38) or p42 MAP kinase (39) results in enhanced expression of
a variety of cytokine genes in T cells and macrophages (37, 39), the
inactivation of IB (38), and the enhanced binding activity of
cytokine transcription factors such as NF-
B and AP-1 (39).
In conclusion, the above results provide the first indication that p42
MAP kinase regulates antigen-mediated production of TNF in a mast
cell line and that p38 MAP kinase may negatively regulate the p42 MAP
kinase/cytokine pathway. We can, for the first time, broadly define the
regulatory pathways for all three functional responses of mast cells to
antigen as follows. Along with elevated [Ca2+]i,
the additional primary signals are as follows: for degranulation (6)
and secretion of newly formed TNF
(13), activation of PKC (6); for
cPLA2-mediated release of arachidonic acid, activation of
p42 MAP kinase (Refs. 2, 7, and 34, and this paper); and for production
of TNF
, the coactivation of PKC and p42 MAP kinase (Ref. 13 and this
paper).