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(Journal of Leukocyte Biology. 2002;71:262-270.)
© 2002 by Society for Leukocyte Biology

fMLP-induced in vitro nitric oxide production and its regulation in murine peritoneal macrophages

Ajit Sodhi and Subhra K. Biswas

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial N-formyl peptides such as N-formyl-methionyl-leucyl-phenylalanine (fMLP) are important mediators of monocyte/macrophage recruitment and activation at the sites of inflammation. In the current study, the role of nitric oxide (NO) in the activation of murine peritoneal macrophages to tumoricidal state in response to in vitro fMLP treatment has been investigated. Murine peritoneal macrophages on treatment with fMLP showed a dose- and time-dependent production of NO together with increased tumoricidal activity against P815 mastocytoma cells. L-NMMA, a specific inhibitor of L-arginine pathway, inhibited the fMLP-induced NO secretion and macrophage-mediated tumoricidal activity against P815 cells. These results indicate the L-arginine-dependent production of NO to be one of the effector mechanisms contributing to the tumoricidal activity of fMLP-treated macrophages. The expression of iNOS protein and iNOS mRNA is also observed. The pharmacological inhibitors genistein, wortmannin, H7, PD98059, TPCK, and pertussis toxin (PTX) blocked the fMLP-induced NO production, suggesting the involvement of tyrosine kinases, PI3K, PKC, p42/44 MAPkinase, NF-{kappa}B, and G-proteins. The expression of phospho-p42/44 MAPK and phospho-I{kappa}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


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The recruitment of phagocytic leukocytes to sites of inflammation and malignancy is essential for surmounting a successful host-defense reaction. Studies in this aspect have shown phagocytic leukocytes to respond to a large number of chemoattractants with directional cell movements, activation of integrins, generation of superoxide anions, and the release of granule contents, all of which contribute to the first line of host defense [1 ]. Over the past two decades, the importance of classical chemoattractants, such as bacterial chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP), activated complement component 5, leukotriene B4, platelet-activating factor, and chemotactic cytokines (chemokines), over other biological response modifiers (BRMs) has been recognized in view of their ability to selectively attract and activate leukocyte subpopulation in a variety of inflammatory settings [2 3 4 5 6 ]. In addition to their role in acute inflammation, there is evidence for the role of chemoattractants in lymphocyte trafficking [6 , 7 ], haematopoiesis [8 ], allergy [9 ], atherogenesis [10 ], angiogenesis [11 ], and malignancies [12 ]. These studies have attributed much interest in chemoattractants and their receptors from a therapeutic viewpoint.

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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Inbred strains of Balb/c mice of either sex at 8–10 weeks of age were used for obtaining peritoneal macrophages.

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 18–24 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 supplier’s 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 Student’s t-test (two-tailed).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
fMLP induces NO production in murine peritoneal macrophages
Murine peritoneal macrophages treated in vitro with fMLP showed significant NO production (measured by the nitrite concentration) as compared with the untreated macrophages. The production of NO occurred in a dose- and time-dependent manner with maximal response at 24 h after macrophage stimulation with 10 µg/ml fMLP (Table 1 and Fig. 1 ). The fMLP-induced NO production is inhibited in the presence of the L-arginine analog and NOS inhibitor, L-NMMA (500 µM), suggesting that the enhanced nitrite release was a result of NOS-mediated L-arginine metabolism. This was supported by the reversal of L-NMMA-induced inhibition of NO production by the addition of excess L-arginine (2 mM) to the fMLP-activated macrophage cultures. LPS-treated macrophages were taken as the positive control in the above experiment.


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  		Table 1. Production of NO by fMLP-Treated Murine Peritoneal Macrophages

 


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  		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.

 
Expression of iNOS in response to fMLP treatment by murine peritoneal macrophages
The production of NO from L-arginine in macrophages is catalyzed by the enzyme iNOS [29 ]. Treatment of murine peritoneal macrophages with the optimum dose of fMLP (10 µg/ml) or LPS (10 µg/ml) for 24 h induced enhanced iNOS expression in the cell lysates (Fig. 2 ). Peritoneal macrophages pretreated with 50 µM TPCK (cysteine, serine protease inhibitor) inhibited the fMLP- and LPS-induced NO production. iNOS expression could not be detected in the cell lysates of the untreated macrophages.



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  		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.

 
Expression of iNOS mRNA in murine peritoneal macrophages treated with fMLP
Murine peritoneal macrophages were cultured in medium alone or medium containing 5–10 µg/ml fMLP for 12 h, and RT-PCR was carried out using the specific primers for mouse iNOS following total RNA extraction, as mentioned in Materials and Methods. Figure 3 shows that fMLP (5–10 µg/ml) treatment of murine peritoneal macrophages resulted in a significantly enhanced expression of iNOS mRNA compared with the untreated macrophages. The use of an equal RNA template for each reaction was determined by equal expression of ß-globin mRNA.



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  		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.

 
Effect of L-NMMA and involvement of L-arginine-mediated NO production in the tumoricidal activity of fMLP-treated macrophages
The involvement of NO production by L-arginine metabolism in the tumoricidal activity of fMLP-activated macrophages was investigated by studying the cytotoxic effect of fMLP-treated peritoneal macrophages toward the NO-sensitive tumor cell line, P815, in the presence or absence of the NOS inhibitor, L-NMMA. Murine peritoneal macrophages treated with the optimum concentrations of fMLP (10 µg/ml) or LPS (10 µg/ml) for 24 h were coincubated with labeled P815 cells in the presence or absence of L-NMMA (500 µM) and tested for their tumoricidal activity, as indicated in Materials and Methods. fMLP-treated macrophages showed significant cytolytic activity toward the P815 cells as compared with the untreated macrophages (Table 2 ). The presence of L-NMMA in the cocultures inhibited the fMLP- and LPS-induced cytolytic activity of the murine peritoneal macrophages toward P815 cells. The L-NMMA inhibition was reversed by the presence of excess L-arginine (2 mM), confirming the involvement of L-arginine-mediated NO production in the fMLP-induced tumoricidal activity of peritoneal macrophages. Medium-containing fMLP incubated alone for 24 h had no effect on tumor-cell killing (unpublished results).


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  		Table 2. Effect of L-N-Monomethyl Arginine (L-NMMA) on the Tumoricidal Activity of fMLP-Treated Macrophages

 
In a parallel set of experiments under the same conditions, the activated macrophages were coincubated with unlabeled P815 cells in the presence or absence of L-NMMA (500 µM), and the nitrite content in the culture supernatants was checked on completion of the experiment. Correlating with the macrophage-mediated cytotoxicity data, the culture supernatants from fMLP-treated macrophage-tumor cell cocultures showed significantly enhanced NO production, which was inhibited in the presence of L-NMMA (500 µM; Table 2 ). However, the inhibitory action of L-NMMA on fMLP-induced NO release was restored in the presence of excess L-arginine (2 mM) in the cocultures. Untreated macrophage-tumor cell cocultures did not show any significant NO production. Similarly, P815 tumor cells incubated alone in medium containing 10 µg/ml fMLP did not show any significant NO release.

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) .



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  		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.

 
fMLP induces the expression of phospho-p42/44 MAPK in murine peritoneal macrophages
Murine peritoneal macrophages were incubated in medium alone or in medium containing 10 µg/ml fMLP for 15 min, and the cell lysates were assayed for expression of phosphorylated p42/44 MAPK. The macrophages treated with fMLP showed enhanced expression of phospho-42/44 MAPK, and no expression was observed in the untreated macrophages (Fig. 5 ). Significant phospho-p42/44 MAPK expression is also observed in LPS (10 µg/ml)-treated peritoneal macrophages.



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  		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.

 
fMLP induces the expression for phospho-I{kappa}B in murine peritoneal macrophages
Murine peritoneal macrophages were incubated in medium alone or in medium containing 10 µg/ml fMLP for the indicated time intervals, and the cell lysates were used for the detection of phosphorylated I{kappa}B expression. In response to fMLP treatment, the peritoneal macrophages exhibited a time-dependent increase in the expression of phospho-I{kappa}B with maximal expression between 30 and 60 min of stimulation. No phospho-I{kappa}B expression was observed in the untreated macrophages (Fig. 6 ).



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  		Figure 6. Expression of phospho-I{kappa}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{kappa}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.

 
Role of protein phophatases in the induction of NO production by fMLP-treated peritoneal macrophages
The inhibitors for serine/threonine phosphatases, okadaic acid (10 µM and 1 nM), and tyrosine phosphatases, sodium orthovanadate (10 µM), were used to study their effect on the NO production in the fMLP-treated macrophages (Fig. 7 ). High doses of okadaic acid (10 µM), known to inhibit serine/threonine phosphatases PP1A and 2A, inhibited fMLP-induced NO production, and concentrations of okadaic acid <1 nM that specifically inhibits only PP2A moderately enhanced the NO release as compared with the macrophages treated with fMLP. However, treatment of macrophages with sodium orthovanadate (10 µM) did not cause any augmentation of the NO production by the fMLP-activated macrophages.



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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.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Successful host defense against invading microorganisms, parasites, and cancer depends on the migration, accumulation, and activation of phagocytic leukocytes at the relevant sites [22 ]. Formylated peptides of bacterial origin and their synthetic counterpart fMLP have been shown to induce chemotaxis and activation of polymorphonuclear cells, macrophages in vivo [26 ] and in vitro [19 , 22 23 24 25 ]. Investigating the mechanism(s) responsible for the macrophage tumoricidal activity, the present study demonstrates the ability of fMLP to induce the production of NO in vitro by murine peritoneal macrophages. The NO production by the macrophages in response to fMLP was found to be dose- and time-dependent with maximal NO production at 24 h of incubation following treatment with 10 µg/ml fMLP. The involvement of L-arginine metabolism in the generation of NO by fMLP-treated macrophages was evident from the inhibitory effect of NOS inhibitor, L-NMMA, on the production of NO and its reversal in the presence of excess L-arginine. The existence of inducible pathways for NO production from L-arginine has been well-documented in monocytes and macrophages in response to cytokines, bacterial lipopolysaccharides, and anticancer drugs [37 38 39 40 ] but none for chemoattractants. Although fMLP has been found to trigger superoxide anion production and release of lysozyme production and hydrogen peroxide in macrophages [22 23 24 25 ], there is no study regarding the induction of NO by this agent. The fMLP-induced NO production correlated with the enhanced expression of iNOS protein and iNOS mRNA in the fMLP-treated murine peritoneal macrophages. Supporting these observations is a recent study that shows the chemokines RANTES (regulated on activation, normal T expressed and secreted) and MIP-1{alpha} (macrophage-inflammatory protein-1{alpha}) 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-{kappa}B (NF-{kappa}B) [58 ]. NF-{kappa}B exists in the cytoplasm in its inactive form along with the inhibitory protein I{kappa}-B [59 ]. Activation of signaling cascade in response to external stimuli leads to phosphorylation of I{kappa}B and its dissociation from the NF-{kappa}B complex; proteolytic degradation of I{kappa}B by I{kappa}B protease; and translocation of the activated NF-{kappa}B and its binding to the relevant gene promoter. The present data on the phosphorylation of I{kappa}B in fMLP-treated murine peritoneal macrophages and the inhibition of iNOS expression by TPCK, which blocks NF-{kappa}B activation by inhibiting the I{kappa}B protease [60 , 61 ], implicate the involvement of NF-{kappa}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-{gamma} (IFN-{gamma}) [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-{kappa}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 .



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  		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 "?".

 


   ACKNOWLEDGEMENTS
 
We are grateful to Dr. R. K. Singh (University of Nebraska Medical Center, Omaha) for the kind gift of the murine RT-PCR primers.

Received May 15, 2001; revised August 12, 2001; accepted August 12, 2001.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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