School of Biotechnology, Banaras Hindu University, Varanasi, India
Correspondence: Prof. Ajit Sodhi, School of Biotechnology, Banaras Hindu University, Varanasi 221005, India. E-mail: ajit.sodhi{at}lycos.com
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B, and G-proteins.
The expression of phospho-p42/44 MAPK and phospho-I
B was also
observed. The role of protein phosphatases in the above pathway has
been suggested using the specific inhibitors of these phosphatases,
i.e., okadaic acid and sodium orthovanadate.
Key Words: chemoattractant iNOS signal transduction kinases phosphatases
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Among the numerous chemoattractants characterized so far, fMLP was one of the first to be identified and possess potent chemotactic properties for phagocytic leukocytes [13 14 15 ]. Polymorphoneutrophils and macrophages express membrane receptors for fMLP: a high-affinity formyl peptide receptor (FPR) and a low-affinity variant, FPR-like 1 (FPR1) [1 , 16 17 18 19 20 21 ], which allow them to respond to chemotactic gradients and cause their migration into sites of infection/inflammation. Binding of fMLP to its receptor also activates these cells to elaborate effector functions necessary to fight infection or malignancies [19 , 22 , 23 ]. In vivo and in vitro studies have shown fMLP to activate macrophages to induce lysozyme production, superoxide anion formation, and release of proinflammatory cytokines, resulting in the attainment of tumoricidal properties [19 , 22 23 24 25 26 ]. However, in spite of these evidences, the molecular mechanism(s) by which fMLP triggers macrophage activation and how these activated macrophages cause cytolysis of tumor cells in a target-specific manner remain to be fully understood.
The tumoricidal properties of activated macrophages are attributed, among other causes, to the secretion of cytolytic-effector molecules that induce apoptotic killing of tumor cells [27 , 28 ]. The production of nitric oxide (NO) represents one of the principle characteristics of activated murine macrophages [28 ]. In the macrophage system, NO is formed enzymatically from the terminal guanidino-nitrogen of L-arginine by the Ca-independent, inducible NO synthase (iNOS), which yields L-citrulline as a coproduct [29 ]. Various studies have established the secretion of NO by activated macrophages to be a vital effector mechanism and indicated it as a potent inducer of tumor-cell cytotoxicity [27 , 30 31 32 ]. In addition, other studies have also shown NO to exert antimetastatic effect toward tumor growth [33 ].
In view of these facts, in the present study we demonstrate fMLP-induced NO production in murine peritoneal macrophages and its contribution toward macrophage tumoricidal activity. The role of various protein kinases and phosphatases in the fMLP-induced signal transduction pathway leading to NO production in the peritoneal macrophages was also investigated.
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Cell cultures and reagents
P815 (murine mastocytoma cell line) was obtained from the
National Tissue Culture Facility (Pune, India). All cell cultures were
maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal
calf serum (FCS), penicillin (100 U/ml), streptomycin (100 µg/ml),
and gentamycin (20 µg/ml). Medium RPMI 1640, sodium orthovanadate,
okadaic acid, NG-monomethyl-L-arginine (L-NMMA),
wortmannin, recombinant macrophage chemotactic activating factor (MCAF)
or monocyte chemoattractant protein 1 (MCP-1), and tosyl phenylalanine
chloromethyl ketone (TPCK) were purchased from Sigma Chemical Co. (St.
Louis, MO). FCS was purchased from Biological Industries (Israel).
3H-thymidine was obtained from Bhabha Atomic Research
Centre (Mumbai, India). Genistein, phorbol myristate acetate (PMA), and
H-7 [1-(5-isoquinoline sulphonyl)-2-methyl-piperazine
dihydrochloride] were purchased from LC Laboratories (Woburn,
MA). PD98059 and phospho-p42/44 mitogen-activated protein kinase (MAPK)
antibodies were purchased from New England Biolabs (Beverly, MA).
Polyclonal antibodies against iNOS, horseradish peroxidase
(HRP)-conjugated rabbit immunoglobulin G (IgG), and chemiluminescence
Western blotting kit were obtained from Santa Cruz Biotechnology (Santa
Cruz, CA).
Isolation and activation of macrophages
Macrophage monolayers were prepared as described previously
[34
]. Peritoneal exudate cells (PEC) were harvested
using chilled, serum-free RPMI 1640 culture medium. PEC was added to
each well of a 24-well tissue-culture plate (A/S Nunc, Denmark). After
2 h, nonadherent cells were removed by vigorous washing. More than
95% of the adherent cells were macrophages, as determined by
morphology and nonspecific esterase staining. Approximately 2 x
106 macrophages adhered to each well of a 24-well
tissue-culture plate (A/S Nunc) and were subsequently cultured in a
final volume of 500 µl complete medium for 1824 h to form a
well-spread monolayer.
Macrophage monolayers (2x106 cells/well) grown in 24-well tissue-culture plates (A/S Nunc) were incubated in a final volume of 500 µl medium or the same volume of medium containing different doses of fMLP or lipopolysaccharides (LPS; 10 µg/ml) for various time intervals as indicated in Results. Thereafter, the cell-free culture supernatants were collected by centrifugation at 250 g for 10 min. The supernatants were assayed for the release of nitrite, and the cell lysates were prepared as described below and proceeded for immunoblotting using iNOS antibodies.
Coculture of macrophages and tumor cells
Macrophage monolayers (2x106 cells/well) grown in
24-well tissue-culture plates (A/S Nunc) were cultured in medium alone
or medium containing fMLP (10 µg/ml) or LPS (10 µg/ml). After
12 h incubation at 37°C in CO2 incubator, the cells
were washed thoroughly. Tumor target cells (2x105/well)
were added in the prewetted transwell inserts and placed over the
macrophage monolayers in each well of the 24-well tissue-culture
plates. Prior to the addition of the tumor cells, the inhibitor L-NMMA
(500 µM) was added in a few wells as indicated in Results. The
cocultures were incubated for 24 h, and thereafter, the tumor
cells were collected for cell viability assay and the cell-free culture
supernatants for nitrite assay.
Nitrite assay for estimation of NO production
The concentration of stable nitrite, the end product from NO
generation by effector macrophages, was determined by the method of
Ding et al. [35
] based on Griess reaction. Briefly, 100
µl culture supernatants were incubated with an equal volume of Griess
reagent (one part 1% sulphanilamide in 2.5%
H3PO4 plus one part of 0.1%
napthyl-ethylene-diamine dihydrochloride in distilled water:two parts
mixed together within 12 h of use and kept chilled) at room
temperature for 10 min in a 96-well plate. The absorbance at 540 nm was
measured with an Emax microplate reader (Molecular Devices, Palo Alto,
CA). Nitrite content (µmoles/106 cells) was quantified by
extrapolation from a sodium nitrite standard curve in each experiment.
Macrophage-mediated cytotoxicity
Cytotoxicity was measured by the release of radioactivity as
described previously [34
]. Target cells (P815) in
exponential growth phase were incubated in medium containing 0.5
µCi/ml 3H-thymidine. After 24 h of incubation, the
cells were washed and used as targets in the cytotoxicity assay.
Labeled tumor target cells were plated into wells containing treated or
untreated macrophages at an effector:target cell ratio of 10:1. After
24 h of coincubation, cell-free culture supernatants were
collected, and an aliquot was counted for radioactivity in a liquid
scintillation counter (LKB). The percentage of macrophage-mediated
cytotoxicity was calculated as follows: % Cytotoxicity = 100 x experimental cpm - spontaneous cpm/total cpm -
spontaneous cpm, where "experimental cpm" represents the amount of
radioactivity released in culture wells containing effector +
target cells; "spontanous cpm" represents spontaneous release,
i.e., radioactivity release in the cultures of target cells alone; and
"total cpm" represents the total release, i.e., radioactivity
released from the target cells lysed with 1 N NaOH. Spontaneous release
was consistently less than 10% of the total release.
Percentage viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) assay
Percent viability of tumor cells was determined by MTT assay as
described earlier [36
]. Briefly, the tumor cells
(2x105 cells/well), harvested after 24 h of
coincubation with macrophages, were placed in each well of a 96-well
tissue-culture plate (A/S Nunc). MTT (10 µl of 5 mg/ml) was added,
and the cells were incubated for 4 h. A purple formazan product
formed was solubilized by the addition of 100 µl acidic isopropanol
(0.04 N HCl in isopropanol). The absorbance of each well was measured
with a microplate enzyme-linked immunosorbent assay (ELISA) reader
using a wavelength of 570 nm. The relative cell viability was
calculated according to the formula: Relative cell viability =
absorbance experimental/absorbance control x 100, where
"absorbance control" represents tumor cells incubated in medium
alone, and "absorbance experimental" represents tumor cells
coincubated with untreated or fMLP/LPS-treated macrophages.
Preparation of cell lysates and immunoblotting
After stimulation for the specified time intervals, macrophages
were washed with ice-cold phosphate-buffered saline containing 1 mM
Na3VO4 and then lysed in 50 µl lysis buffer
[20 mM Tris-HCl, pH 8, 137 mM NaCl, 10% glycerol (v/v), 1% Triton
X-100 (v/v), 1 mM Na3VO4, 2 mM
ethylenediaminetetraacetate (EDTA), 1 mM phenylmethylsulfonyl fluoride
(PMSF), 20 µM leupeptin, and 0.15 U/ml aprotonin] for 20 min at
4°C. The lysates were centrifuged at 13,000 g for 15 min,
and the supernatants (containing Triton X-100-soluble proteins) were
separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels at
20 mA. The separated proteins were transferred to nitrocellulose (8 h
at 125 mA), immunoblotted with the respective primary antibodies,
HRP-tagged secondary antibody, and visualized by the
chemiluminescence.
RNA isolation, reverse transcription (RT), and polymerase chain
reaction (PCR)
Total RNA was isolated from the murine peritoneal macrophages by
TRIzol reagent (Gibco BRL, Grand Island, NY) in accordance with the
suppliers instructions. The RNA was reverse-transcribed using a
ThermoScript kit (Gibco BRL) and amplified by PCR using the specific
primers indicated below. The thermocycle conditions were 40 cycles of
94°C for 1 min, 55°C for 1 min, and 72°C for 2 min, after which
an additional extension step at 72°C for 5 min was included.
Electrophoresis of amplified DNAs was carried out on 1.5% agarose gel
and stained with ethidium bromide. The primer sequences are as follows:
mouse iNOS forward primer, 5'-CAAAGTCAAATCCTACCAAAGTGACCTG-3'; iNOS
reverse primer, 5'-TGCTACAGTTCCGAGCGTCAAAGACCTG-3'; mouse ß-globin
forward, 5'-CCTGCAGTGTCTGATATTGTTG-3'; ß-globin reverse,
5'-AACACACCATTGCGATGAA-3'. The possible contamination of any PCR
component was excluded by performing a PCR reaction with these
components in the absence of RT product in each set of experiments
(negative control).
Statistical analysis
The statistical significance of difference between the test
groups was analyzed by Students t-test (two-tailed).
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View this table: [in a new window] |
Table 1. Production of NO by fMLP-Treated Murine Peritoneal Macrophages
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![]() View larger version (14K): [in a new window] |
Figure 1. Kinetics of NO production by murine peritoneal macrophages treated with
fMLP. Macrophages (106 cells/well) were cultured for
24 h in medium alone or with fMLP (10 µg/ml) or LPS (10 µg/ml)
for different time durations. The cell-free culture supernatants were
collected thereafter and assayed for nitrite as described in Materials
and Methods. Values are mean ± SD and are
representative of three independent experiments done in triplicate.
P < 0.05 versus values for untreated macrophages.
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![]() View larger version (24K): [in a new window] |
Figure 2. Expression of iNOS in murine peritoneal macrophages treated with fMLP.
Macrophages (106 cells/well) were cultured for 24 h in
medium alone or medium containing 10 µg/ml LPS or fMLP in the
presence or absence of TPCK. Thereafter, the cells were lysed and used
for immunoblotting against iNOS antibodies as described in Materials
and Methods. The figure is one of the representatives of three
independent experiments having similar results. Lane 1: Macrophage + LPS; lane 2: macrophage + fMLP; lane 3: untreated macrophage;
lane 4: macrophage + 50 µM TPCK; lane 5: macrophage + 50
µM TPCK + LPS; lane 6: macrophage + 100 µM TPCK +
fMLP.
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![]() View larger version (57K): [in a new window] |
Figure 3. RT-PCR analysis of iNOS mRNA expression in fMLP-treated murine
peritoneal macrophages. Macrophages (106 cells/well) were
incubated in medium alone or medium containing the indicated doses of
fMLP for 12 h. Thereafter, total RNA was extracted, and RT-PCR was
carried as described in Materials and Methods. The PCR products were
resolved on 1.5% agarose gel, stained with ethidium bromide, and
visualized on a UV transluminator. The lower panel represents the
expression of ß-globin mRNA in the untreated and fMLP-treated
macrophages. Densitometric analysis of the iNOS RT-PCR blot is
indicated by the Raw volume represented in the bar graph. Lane 1:
Untreated macrophages; lane 2: macrophages treated with 10 µg/ml
fMLP; lane 3: macrophages treated with 5 µg/ml fMLP. The figure is
representative of three independent experiments with similar results.
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View this table: [in a new window] |
Table 2. Effect of L-N-Monomethyl Arginine (L-NMMA) on the
Tumoricidal Activity of fMLP-Treated Macrophages
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Role of protein kinases in the induction of NO production in
fMLP-activated peritoneal macrophages
The regulation of NO production by protein kinase C (PKC),
phosphoinositol-3-kinase (PI-3K), serine/threonine MAPK, and tyrosine
kinases was examined in the fMLP-treated murine peritoneal macrophages
preincubated with specific inhibitors and/or activators for these
kinases (Fig. 4
). Phorbol ester, PMA (200 nM) that activates PKC, increased NO
production in untreated macrophages and displayed no significant effect
on fMLP-treated macrophages. Conversely, a specific inhibitor of PKC,
H7 (10 µM), caused a fourfold decrease in fMLP-induced nitrite
release as compared with those incubated with fMLP alone. Similar
inhibition was also observed with the PI-3K inhibitor, wortmannin (200
nM), and the p42/44 MAPK inhibitor, PD98059 (25 µM). Protein tyrosine
kinase inhibitor genistein (10 µg/ml) also significantly inhibited
nitrite release by macrophages activated with fMLP. Pertussis toxin
(100 ng/ml) also caused inhibiton of fMLP-induced NO production in the
murine peritoneal macrophages. In all of the above experiments,
macrophages incubated with the inhibitors alone exhibited minimal
nitrite release as in the untreated macrophages (Fig. 4)
.
![]() View larger version (15K): [in a new window] |
Figure 4. Effect of protein kinase inhibitors on NO production by fMLP- treated
murine peritoneal macrophages. Macrophages (106 cells/well)
were incubated for 24 h in medium alone or medium containing 10
µM H7, 200 nM PMA, 200 nM wortmannin, 25 µM PD98059, 10 µg/ml
genistein, or 100 ng/ml pertussis toxin in the absence or presence of
10 µg/ml fMLP. The cell-free culture supernatants were then collected
and assayed for nitrite as described in Materials and Methods. Values
are mean ± SD and are representative of three
independent experiments done in triplicate.
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View larger version (11K): [in a new window] |
Figure 5. Expression of phospho-p42/44 MAPK in murine peritoneal macrophages
treated with fMLP. Macrophages (106 cells/well) were
incubated in medium alone or medium containing 10 µg/ml fMLP or LPS
for 15 min. Thereafter, the cells were lysed and used for
immunoblotting against phospho-p42/44 MAPK antibodies as described in
Materials and Methods. Lane 1: Untreated macrophage; lane 2:
macrophages incubated with fMLP; lane 3: macrophages incubated with
LPS. The figure is one representative of three independent experiments
having similar results.
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B in murine
peritoneal macrophages
B
expression. In response to fMLP treatment, the peritoneal macrophages
exhibited a time-dependent increase in the expression of phospho-I
B
with maximal expression between 30 and 60 min of stimulation. No
phospho-I
B expression was observed in the untreated macrophages
(Fig. 6
). |
View larger version (13K): [in a new window] |
Figure 6. Expression of phospho-I B in murine-peritoneal macrophages treated
with fMLP. Macrophages (106 cells/well) were incubated in
medium alone or medium containing 10 µg/ml fMLP for the indicated
time intervals. Thereafter, the cells were lysed and used for
immunoblotting against phospho-I B antibodies as described in
Materials and Methods. The figure is one representative of three
independent experiments having similar results. Lane 1: Untreated
macrophage; lane 2: macrophages incubated with fMLP for 5 min; lane 3:
macrophages incubated with fMLP for 15 min; lane 4: macrophages
incubated with fMLP for 30 min; lane 5: macrophages incubated with fMLP
for 60 min.
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Figure 7. Effect of protein phosphatase inhibitors on NO production by
fMLP-treated murine peritoneal macrophages. Macrophages
(106 cells/well) were incubated for 24 h in medium
alone (MEDIUM) or medium containing 1 nM okadaic acid [OKA (low)], 10
µM okadaic acid [OKA (high)] and 100 µM sodium orthovanadate in
the absence or presence of 10 µg/ml fMLP. The cell-free culture
supernatants were then collected for nitrite assay as described in
Materials and Methods. Values are mean ± SD and are
representative of three independent experiments done in triplicate.
P < 0.05 versus values for macrophages incubated with
medium alone.
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(macrophage-inflammatory
protein-1
) contribute to trypanocidal activity in human macrophages
through NO production [41
]. The possibility that the
enhanced NO production by the murine macrophages in response to fMLP
could possibly be a result of endotoxin contamination is ruled out
because all the reagents were free of endotoxin as demonstrated by the
amoebocyte lysate assay. NO is considered to be one of the principal effectors of macrophage-mediated cytotoxicity [27 , 28 , 30 , 31 ]. The contribution of NO in the fMLP-induced macrophage tumoricidal activity was investigated. The results from the macrophage-tumor cell cocultures correlated the increased nitrite levels in the culture supernatants to the increase in the cytotoxicity toward the NO-sensitive P815 cells, indicating the role of NO in macrophage-mediated cytotoxicity in response to fMLP. The ability of L-NMMA to abrogate the fMLP-induced macrophage tumoricidal activity against P815 cells and its reversal by excess L-arginine in macrophage-tumor cell coculture further support the involvement of NO in macrophage-mediated cytotoxicity. Previous studies from our laboratory along with a number of others have shown that NO produced by activated macrophages is responsible for cytostatic or cytolytic activity for tumor cells in vivo and in vitro [27 , 30 , 31 , 42 ]. The present findings are of relevance, in view of the studies on the role of chemoattractants in macrophage infiltration to neoplastic tissues and the expression of iNOS in tumor-infiltrating mononuclear cells in colon adenoma [43 ].
The signal transduction mechanism for the fMLP-induced NO production in murine peritoneal macrophages was investigated. The results of the studies with the inhibitors demonstrated that macrophage NO production in response to fMLP was blocked significantly by the tyrosine kinase inhibitor genistein, PI3K inhibitor wortmannin, and PKC inhibitor H7, suggesting the involvement of the respective kinases in the above process. Additionally, the augmentation of NO production by PKC activator PMA confirmed the participation of PKC. In agreement with our observations, the involvement of PKC and tyrosine kinases in the production of NO by macrophages has been demonstrated earlier [44 45 46 ]. These studies have proposed that the signaling cascade leading to macrophage activation and effector functions involves an initial wave of tyrosine phosphorylation, generation of membrane-inositides, and activation of a variety of serine/threonine kinases such as MAPKs, which transmit the signal to the nucleus triggering the expression of proinflammatory genes [47 48 49 50 51 ]. In support of this, the expression of phospho-p42/44 MAPK and the inhibition of fMLP-induced NO production by MAPK inhibitor PD98059 in the murine peritoneal macrophages are shown presently. The NO production in response to fMLP was also found to be sensitive to pertussis toxin, suggesting a role for the Gi-proteins. The fact that the functional response to chemokines as well as classical chemoattractants is mediated through 7-membrane spanning, G-protein-coupled receptors (GPCR) [52 , 53 ] supports our observation. For fMLP, two GPCR receptors, FPR and FPRL1, have been cloned [17 18 19 20 21 ]. Together, these findings suggest the fMLP-induced signal transduction for NO production to involve the activation of Gi-protein-coupled receptors, PI3K, PKC, and p42/44 MAPK. The involvement of these signal intermediates has been demonstrated earlier for chemoattractants in multiple cell types [54 55 56 57 ].
The iNOS gene contains regulatory sites in its promoter region for the
transcription factor nuclear factor-
B (NF-
B) [58
].
NF-
B exists in the cytoplasm in its inactive form along with the
inhibitory protein I
-B [59
]. Activation of signaling
cascade in response to external stimuli leads to phosphorylation of
I
B and its dissociation from the NF-
B complex; proteolytic
degradation of I
B by I
B protease; and translocation of the
activated NF-
B and its binding to the relevant gene promoter. The
present data on the phosphorylation of I
B in fMLP-treated murine
peritoneal macrophages and the inhibition of iNOS expression by TPCK,
which blocks NF-
B activation by inhibiting the I
B protease
[60
, 61
], implicate the involvement of
NF-
B in the fMLP-induced signal transduction for macrophage NO
production.
Finally, the role of protein phosphatases was also studied, considering
their importance in modulating reversible phosphorylation events that
are integral to the signal-transduction pathway of macrophage
activation [62
63
64
]. Using the inhibitors of
serine/threonine protein phosphatases, okadaic acid, it is observed
that the production of NO in macrophages treated with fMLP requires
serine/threonine phosphatase activity (Fig. 3)
. The inhibition of
serine/threonine PP2A up-regulated the NO production of fMLP-treated
macrophages, whereas the inhibition of PP1 down-regulated the same.
Such an opposing regulatory role of PP1 and PP2A on NO production and
the differential sensitivity of the same toward different
concentrations of okadaic acid are supported by earlier studies in
intact mammalian cells [65
]. The enhanced expression of
NO release observed in the presence of 1 nM okadaic acid was because of
the fact that at doses less than 1 nM, okadaic acid preferentially
inhibits PP2A, and higher doses that inhibit PP1 in addition to PP2A
completely block NO production [65
, 66
].
These findings correlated to similar observations at the level of iNOS
expression in the fMLP-activated macrophages (unpublished results) and
are in agreement with earlier studies on the requirement of PP1A and
PP2A in the activation of iNOS gene in macrophages [67
].
However, the role of tyrosine phosphatases in this process could not be
resolved, because the tyrosine phosphatase inhibitor, sodium
orthovanadate (10 µM), could not cause any significant augment in the
production of NO by the fMLP-treated murine peritoneal
macrophages. In contrast, the involvement of protein tyrosine
phosphatases in the production of NO is shown for macrophages
treated with interferon-
(IFN-
) [68
].
In summary, considering the ability of fMLP in the recruitment
and activation of phagocytic leukocytes under inflammatory settings and
its pathophysiological consequences, our present data identify NO to be
one of the major effector molecules responsible for the tumoricidal
activity of murine peritoneal macrophages in response to fMLP. Further,
this study also suggests that the signal transduction cascade, leading
to the expression of this effector molecule in macrophages, involves
the participation of protein kinases, phosphatases, and the
transcription factor NF-
B in response to fMLP treatment in vitro. A
schematic diagram of the possible fMLP-induced signal-transduction
pathway for NO production in murine peritoneal macrophages, as
suggested, is represented in Figure 8
.
![]() View larger version (50K): [in a new window] |
Figure 8. A schematic representation of the possible signal transduction pathway
leading to NO production in murine peritoneal macrophages in response
to in vitro fMLP treatment. The specific inhibitors/activators used in
dissecting the signaling pathway in the present study are indicated in
uppercase italics, whereas the inhibition or activation of the
individual events in the signal cascade is shown by the symbols
provided in the diagram key. The poorly characterized signaling events
are indicated by "?".
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Received May 15, 2001; revised August 12, 2001; accepted August 12, 2001.
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