Regulation of Nitric Oxide Production by Murine Peritoneal Macrophages Treated in Vitro with Chemokine Monocyte Chemoattractant Protein 1

Monocyte chemoattractant protein 1 (MCP-1) is an important mediator of monocyte/macrophage recruitment and activation at the sites of chronic inflammation and neoplasia. In the current study, the role of nitrogen monoxide (NO) in the activation of murine peritoneal macrophages to the tumoricidal state in response to in vitro MCP-1 treatment and the regulatory mechanisms involved therein were investigated. Murine peritoneal macrophages upon activation with MCP-1 showed a dose- and time-dependent production of NO together with increased tumoricidal activity against P815 mastocytoma cells. N-monomethyl- -arginine (L-NMMA), a specific inhibitor of the -arginine pathway, inhibited the MCP-1-induced NO secretion and generation of macrophage-mediated tumoricidal activity against P815 (NO-sensitive, TNF-resistant) cells but not the L929 (TNF-sensitive, NO-resistant) cells. These results indicated -arginine-dependent production of NO to be one of the effector mechanisms contributing to the tumoricidal activity of MCP-1-treated macrophages. Supporting this fact, expression of iNOS mRNA was also detected in the murine peritoneal macrophages upon treatment with MCP-1. Investigating the signal transduction pathway responsible for the NO production by the MCP-1-activated murine peritoneal macrophages, it was observed that the pharmacological inhibitors wortmannin, H-7 (1-(5-isoquinoline sulfonyl)-2-methyl piperazine dihydrochloride), and PD98059 blocked the MCP-1-induced NO production, suggesting the probable involvement of phosphoinositol-3-kinase, protein kinase C, and p42/44 MAPkinases in the above process. Various modulators of calcium and calmodulin (CaM) such as EGTA, nifedipine, TMB-8 (3,4,5-trimethoxybenzoic acid-8-(diethylamino)octyl ester), A23187, and W-7 (N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide) were also found to modulate the in vitro macrophage NO release in response to MCP-1. This observation indicated the regulatory role of calcium/CaM in the process of MCP-1-induced macrophage NO production. Similarly, the role of serine/threonine and protein tyrosine phosphatases in the above pathway was suggested using the specific inhibitors of these phosphatases, okadaic acid and sodium orthovanadate.     

Tumor necrosis factor-alpha-dependent production of reactive nitrogen intermediates mediates IFN-gamma plus IL-2-induced murine macrophage tumoricidal activity.

We have previously established that IFN-gamma plus IL-2 induces murine macrophage tumoricidal activity. The purpose of this study was to identify the effector molecules that account for the IFN-gamma plus IL-2-induced macrophage cytotoxicity against P815 mastocytoma cells. ANA-1 macrophages and normal thioglycollate-elicited mouse peritoneal macrophages produced little or no detectable nitrite (NO2-) after incubation with IFN-gamma alone or IL-2 alone; however, IL-2 synergized with IFN-gamma for the production of NO2-. IFN-gamma plus IL-2 did not induce NO2- production or tumoricidal activity in ANA-1 macrophages that were cultured in medium devoid of L-arginine or in ANA-1 macrophages that were incubated with NG-monomethyl-L-arginine. As observed previously with ANA-1 macrophage tumoricidal activity, IL-4 inhibited IFN-gamma plus IL-2-induced, but not IFN-gamma plus LPS-induced, NO2- production. IL-4 also selectively decreased the ability of IFN-gamma and/or IL-2 to augment TNF-alpha mRNA expression in ANA-1 macrophages. Lastly, incubation of ANA-1 macrophages with anti-TNF mAb selectively inhibited the ability of IFN-gamma plus IL-2 to induce NO2- production and tumoricidal activity. These results indicate that IFN-gamma plus IL-2-induced tumoricidal activity is dependent upon the metabolism of L-arginine to reactive nitrogen intermediates, and they establish a role for TNF-alpha as a required intermediate for IL-2-dependent NO2- production and tumoricidal activity.     

Acidic polysaccharide isolated from Phellinus linteus induces nitric oxide-mediated tumoricidal activity of macrophages through protein tyrosine kinase and protein kinase C

Mushroom polysaccharides are increasingly being utilized to treat a wide variety of diseases. Aqueous extracts from the Phellinus linteus have been reported to have anti-tumor and immunomodulatory properties. In particular, acidic polysaccharide (PL) isolated from P. linteus induced a secretory and cellular macrophage response. However, the exact mechanism by which PL regulates the macrophage functions remains unclear. PL-treated murine peritoneal macrophages in vitro and in vivo dramatically induced the production of NO. PL enhanced the lytic death of B16 cells through the production of NO. The present study examined signal molecules that may participate in PL-elicited responses by macrophages. The data demonstrated that a protein kinase C (PKC) inhibitor, staurosporine, and a protein tyrosine kinase (PTK) inhibitor, genistein, inhibited the tumoricidal activity of macrophages induced by PL. In addition, these inhibitors blocked the production of NO and the expression of surface molecules in PL-stimulated macrophages. Furthermore, CD11b/CD18 possibly mediates PL-induced cell activation. These results suggest that PL stimulates NO production for tumoricidal activity and induces cell-mediated immunity by increasing surface molecules, and the process may be a mechanism by which PL produces its therapeutic effects.     

IL-4 inhibits the costimulatory activity of IL-2 or picolinic acid but not of lipopolysaccharide on IFN-gamma-treated macrophages

We reported previously that IL-2 induces tumoricidal activity in IFN-gamma-treated murine macrophages. The present study was performed to investigate the regulation of IL-2-dependent tumoricidal activity in murine macrophage cell lines. The v-raf/v-myc-immortalized murine macrophage cell lines ANA-1, GG2EE, and HEN-CV did not express constitutive levels of cytotoxic activity against P815 mastocytoma cells. Moreover, these macrophage cell lines did not become tumoricidal after exposure to IL-4, IFN-gamma, IL-2 or LPS. However, these macrophages developed cytotoxic capabilities after incubation with either IFN-gamma plus IL-2 or IFN-gamma plus LPS. IL-4 inhibited IFN-gamma plus IL-2- but not IFN-gamma plus LPS-induced tumoricidal activity. This effect of IL-4 was not restricted to v-raf/v-myc-immortalized macrophage cell lines because similar results were obtained by using a macrophage cell line that was established from a spontaneous histiocytic sarcoma. The suppressive activity of IL-4 on the ANA-1 macrophage cell line was dose-dependent (approximately 12-200 U/ml) and was neutralized by the addition of anti-IL-4 mAb. IL-4 decreased the IFN-gamma-induced expression of mRNA for the p55 (alpha) subunit of the IL-2R in ANA-1 macrophages. Therefore, at least one mechanism by which IL-4 may have inhibited IFN-gamma plus IL-2-induced tumoricidal activity was by reducing macrophage IL-2R alpha mRNA expression. We have previously reported that picolinic acid, a tryptophan metabolite, is a costimulator of macrophage tumoricidal activity. We now report that IL-4 also inhibited IFN-gamma plus picolinic acid-induced cytotoxicity in ANA-1 macrophages. We propose that IL-2 and picolinic acid may have a common mechanism of action that is susceptible to IL-4 suppression.     

Role of bacterial DNA in macrophage activation by group B streptococci

Bacterial DNA (CpG DNA) induces macrophage activation and the production of inflammatory mediators, including tumor necrosis factor (TNF) and nitric oxide (NO) by these cells. However, the role of bacterial DNA in the macrophage response to whole bacteria is unknown. We used overlapping strategies to estimate the relative contribution of bacterial DNA to the upregulation of TNF and NO production in macrophages stimulated with antibiotic-treated group B streptococci (GBS). Selective inhibitors of the bacterial DNA/TLR9 pathway (chloroquine, an inhibitory oligonucleotide, and DNase I) consistently inhibited GBS-induced TNF secretion by 35-50% in RAW 264.7 macrophages and murine splenic macrophages, but had no effect on inducible nitric oxide synthase (iNOS) accumulation or NO secretion. Similarly, splenic and peritoneal macrophages from mice lacking TLR9 expression secreted 40% less TNF than macrophages from control mice after GBS challenge but accumulated comparable amounts of iNOS protein. Finally, studies in both RAW 264.7 cells and macrophages from TLR9-/- mice implicated GBS DNA in the upregulation of interleukins 6 (IL-6) and 12 (IL-12) but not interferon-beta (IFNbeta), a key intermediary in macrophage production of iNOS/NO. Our data suggest that the bacterial DNA/TLR9 pathway plays an important role in stimulating TNF rather than NO production in macrophages exposed to antibiotic-treated GBS, and that TLR9-independent upregulation of IFNbeta production by whole GBS may account for this difference.     

Activation of macrophages with N-formyl-methionyl-leucyl-phenylalanine: involvement of protein kinase C and tyrosine kinase

N-formyl-methionyl-leucyl-phenylalanine (fMLP) a potent chemotactic peptide stimulates immune responses by activating macrophages and other cells of the immune system. The present study reports inhibition of fMLP-induced activation of murine peritoneal and P388D-1 macrophage cell line by protein kinase C (PKC) inhibitors, H-7 and chelerythrine chloride. Similarly, tumoricidal activity was also downregulated by protein tyrosine kinase (PTK) inhibitors genestein and lavendustin A. Further, fMLP increased tyrosine phosphorylation of several proteins in murine macrophages, which were inhibited in presence of genestein and lavendustin A. These findings suggest the involvement of PKC and PTK in the activation of murine macrophages with fMLP.     

Binding of activated macrophages to tumor cells through a macrophage lectin and its role in macrophage tumoricidal activity

A 45-60 kDa Gal/GalNAc-specific macrophage lectin was found to participate in the interaction between tumor cells and tumoricidal macrophages activated by an antitumor streptococcal preparation, OK-432, and in the tumoricidal activity of the activated macrophages. The binding between OK-432-elicited activated macrophages and murine mastocytoma P-815 cells was inhibited on preincubation of the macrophages with a neoglycoprotein (Gal-BSA) or a complex-type glycopeptide (unit B) which was a specific inhibitor of the macrophage lectin. This binding of the macrophages to P-815 cells was also inhibited on the addition of anti-macrophage lectin antiserum. Contrary to the case of OK-432-elicited macrophages, the binding of thioglycolate-elicited (responsive) macrophages to P-815 cells was inhibited only a little by Gal-BSA and unit B, and not inhibited by the antiserum. Furthermore, the tumoricidal activity of the activated macrophages was inhibited by the addition of the anti-macrophage lectin antiserum. These results suggest that the binding of activated macrophages to tumor cells through the Gal/GalNAc-specific macrophage lectin is an important part of the tumor cell killing mechanism.     

Augmentation of an antitumor CTL response In vivo by inhibition of suppressor macrophage nitric oxide

Evidence is provided that inhibition of macrophage NO production can augment in vivo CTL responses. Specifically, administration of NG-monomethyl-l -arginine (NGMMA) via osmotic pumps increases the tumor-specific CTL response against the P815 mastocytoma in the peritoneal cavity of preimmunized mice. Both the magnitude and duration of the CTL response were increased. That the augmented CTL response resulted from inhibition of the NO synthase pathway is supported by the finding that macrophage NO production from NGMMA-treated mice was reduced. Also, in vitro inhibition of NO production by peritoneal exudate cells from P815 tumor-challenged mice augmented the secondary CTL response observed. Cell proliferation was augmented by NGMMA in these cultures, suggesting that macrophage NO may suppress CTL by inhibiting clonal expansion. NO-mediated inhibition was observed in vivo in this experimental system, even though the CTL response is not suppressed, in that tumor rejection occurs. Therefore, the present results are consistent with the conclusion that macrophage NO-mediated inhibition of the CTL response is a side effect of activating macrophages rather than resulting from the action of a distinct subset of what have long been termed suppressor macrophages. Most important, the results indicate that NO-mediated suppressor macrophage activity can be an important CTL immunoregulatory element in vivo.     

Regulation of the RON receptor tyrosine kinase expression in macrophages: blocking the RON gene transcription by endotoxin-induced nitric oxide

Previous studies have shown that activation of the RON receptor tyrosine kinase inhibits inducible NO production in murine peritoneal macrophages. The purpose of this study is to determine whether inflammatory mediators such as LPS, IFN-gamma, and TNF-alpha regulate RON expression. Western blot analysis showed that RON expression is reduced in peritoneal macrophages collected from mice injected with a low dose of LPS. The inhibition was seen as early as 8 h after LPS challenge. Experiments in vitro also demonstrated that the levels of the RON mRNA and protein are diminished in cultured peritoneal macrophages following LPS stimulation. TNF-alpha plus IFN-gamma abrogated macrophage RON expression, although individual cytokines had no significant effect. Because LPS and TNF-alpha plus IFN-gamma induce NO production, we reasoned that NO might be involved in the RON inhibition. Two NO donors, S-nitroglutathione (GSNO) and (+/-)-S-nitroso-N-acetylpenicillamine (SNAP), directly inhibited macrophage RON expression when added to the cell cultures. Blocking NO production by NO inhibitors like TGF-beta prevented the LPS-mediated inhibitory effect. In Raw264.7 cells transiently transfected with a report vector, GSNO or SNAP inhibited the luciferase activities driven by the RON gene promoter. Moreover, GSNO or SNAP inhibited the macrophage-stimulating protein-induced RON phosphorylation and macrophage migration. We concluded from these data that RON expression in macrophages is regulated during inflammation. LPS and TNF-alpha plus IFN-gamma are capable of down-regulating RON expression through induction of NO production. The inhibitory effect of NO is mediated by suppression of the RON gene promoter activities.     

Cellular models of macrophage tumoricidal effector mechanisms in vitro. Characterization of cytolytic responses to tumor necrosis factor and nitric oxide pathways in vitro

The recently described L-arginine-dependent nitric oxide (NO) pathway has been proposed to interact synergistically with the TNF pathway in murine macrophage-mediated tumor cytotoxicity in vitro. We have employed an experimental construct in which these two pathways were independently expressed by two different effector cell populations. The TNF-dependent pathway was committed by murine 3T3 cells transfected with the cDNA encoding human pro-TNF. The NO pathway was executed by the murine EMT-6 mammary adenocarcinoma cell line treated with murine rIFN-gamma and LPS. Controls for the TNF pathway committed by the transfectant included lysis of the TNF-sensitive murine L929 cell in coculture, secretion of TNF, and absence of nitrite synthesis. For the NO pathway controls included lysis of the murine P815 mastocytoma cocultured with activated EMT-6 cells that had been pretreated with murine rIFN-gamma and LPS, production of nitrite by this activated effector cell, and an absence of TNF secretion. The target cell panel included L929, EMT-6, P815, and murine B16 melanoma and TU-5 sarcoma cell lines. All targets on this panel were susceptible to lysis by LPS-triggered murine bacillus Calmette-Guérin-activated macrophages. The 3T3 transfectant caused significant lysis of cocultured L929 and TU-5 targets. The EMT-6 effector cell only caused significant lysis of the P815 target. When both effector cells were cocultured with these target cells, lysis of the P815 target was observed to be additive or superadditive; however, for all the other targets, cytotoxicity was comparable with or subadditive compared with that seen with the 3T3 transfectant effector cell alone. Thus, these two pathways do not appear to account for the broad, potent tumoricidal activity observed for activated macrophages in vitro.     

Monocyte chemoattractant protein-1/CC chemokine ligand 2 enhances apoptotic cell removal by macrophages through Rac1 activation

Apoptotic cell removal (efferocytosis) is an essential process in the regulation of inflammation and tissue repair. We have shown that monocyte chemoattractant protein-1/CC chemokine ligand 2 (MCP-1/CCL2) enhances efferocytosis by alveolar macrophages in murine bacterial pneumonia. However, the mechanism by which MCP-1 exerts this effect remains to be determined. Here we explored that hypothesis that MCP-1 enhances efferocytosis through a Rac1/phosphatidylinositol 3-kinase (PI3-kinase)-dependent mechanism.We assessed phagocytosis of apoptotic cells by MCP-1 treated murine macrophages in vitro and in vivo. Rac activity in macrophages was measured using a Rac pull down assay and an ELISA based assay (GLISA). The downstream Rac1 activation pathway was studied using a specific Rac1 inhibitor and PI3-kinase inhibitor in in vitro assays.MCP-1 enhanced efferocytosis of apoptotic cells by murine alveolar macrophages (AMs), peritoneal macrophages (PMs), the J774 macrophage cell line (J774s) in vitro, and murine AMs in vivo. Rac1 activation was demonstrated in these cell lines. The effect of MCP-1 on efferocytosis was completely negated by the Rac1 inhibitor and PI3-kinase inhibitor.We demonstrated that MCP-1 enhances efferocytosis in a Rac1-PI3 kinase-dependent manner. Therefore, MCP-1-Rac1-PI3K interaction plays a critical role in resolution of acute lung inflammation.     

B cell stimulatory factor-1 (interleukin 4) activates macrophages for increased tumoricidal activity and expression of Ia antigens

Macrophages are activated by lymphokines (LK) to kill tumor cell and microbial targets. Interferon-gamma (IFN) is the major LK activity in conventional, antigen or mitogen-stimulated spleen cell culture fluids for induction of these macrophage effector functions. In view of the recent demonstration that murine macrophage-like cell lines have receptors for B cell stimulatory factor-1/interleukin 4 (BSF-1), a possible role for BSF-1 in regulation of macrophage function was considered. In this communication, thioglycollate-elicited murine peritoneal macrophages were shown to express about 2300 high affinity (Ka approximately 2 X 10(10) M-1) BSF-1 receptors/cell. Peritoneal macrophages treated with purified, T cell-derived BSF-1 developed potent tumoricidal activity against fibrosarcoma target cells. The concentration of BSF-1 that induced 50% of maximal tumor cytotoxicity was 38 +/- 4 U/ml for seven experiments; similar dose-responses were observed with recombinant BSF-1. That BSF-1 dose-responses for induction of macrophage-mediated tumor cytotoxicity were not affected by 5 micrograms/ml polymyxin B suggested that contaminant endotoxins played little or no role in cytotoxic activity. BSF-1 alone (less than or equal to 500 U/ml) was not directly toxic to tumor cells or macrophages. Macrophage tumoricidal activity induced by BSF-1 but not by IFN was inhibited greater than or equal to 90% with monoclonal anti-BSF-1 antibody. BSF-1 induced Ia antigen expression on peritoneal macrophages and increased (twofold to threefold) FcR(II)-dependent binding of murine IgG immune complexes to bone marrow-derived macrophages (greater than 98% macrophages). Based on these findings, it was concluded that BSF-1 is a potent macrophage activation factor.     

In vitro and in vivo induction of nitric oxide by murine macrophages stimulated with Bordetella pertussis

Abstract Nitric oxide (NO) exhibits potent antimicrobial activity in vitro. The function of NO in host defenses in vivo, however, is presently unclear. Experiments were undertaken to determine the production of NO in vitro from murine peritoneal and alveolar macrophages, and murine macrophage cell line (J774A.1) stimulated with Bordetella pertussis or pertussis toxin (PT). In addition, we determined circulating levels of NO in the sera and bronchoalveolar lavage (BAL) fluids of mice infected intranasally with B. pertussis . The results of this study showed that in vitro murine peritoneal macrophages induce production of NO in response to B. pertussis and PT. In addition, murine macrophage cell line, J774A.1 also induces NO production after stimulation with B. pertussis . NO production was also detected in alveolar macrophages from mice infected intranasally with B. pertussis . Finally, a significant increment of circulating levels of NO was noted, in the sera but not in the BAL fluids, of mice infected intranasally with B. pertussis .     

Inhibition of macrophage tumoricidal activity by glucocorticoids

In this study, the effect of corticosteroids on the activation of macrophages to a fully tumoricidal state was examined. Thioglycolate-elicited peritoneal exudate macrophages from C3H/HeJ mice were rendered cytolytic for P815 mastocytoma cells in a two-signal tumoricidal assay that used recombinant interferon-gamma (rIFN-gamma; 1 to 10 U/ml) as a "priming" signal and butanol-extracted lipopolysaccharide (But-LPS; 0.1 to 5 micrograms/ml) as a "trigger" signal. Treatment of macrophages with either rIFN-gamma alone or But-LPS alone failed to result in significant cytolytic ability. Tumoricidal activity was markedly inhibited in a dose-dependent fashion when glucocorticoids were added simultaneously to the cultures with rIFN-gamma and But-LPS at concentrations ranging from 1 X 10(-10) to 1 X 10(-5) M. Nonglucocorticoid sex hormones failed to inhibit tumoricidal activity in this system under identical culture conditions. Inhibition was most effective if the glucocorticoids were added simultaneously with the priming and triggering signals (rIFN-gamma and But-LPS); however, if the glucocorticoids were added 24 hr after the signals were provided to the cultures, suboptimal inhibition was observed. Experiments that dissociated the priming phase of activation from the triggering phase showed that glucocorticoids inhibited both the rIFN-gamma-induced priming stage as well as the But-LPS-induced triggering stage of activation. These observations provide evidence that glucocorticoids, but not other steroid hormones, inhibit the activation of macrophages to a fully tumoricidal state by interfering with either the priming or triggering signals in this two-signal model of macrophage activation.     

Cytolytic mechanisms of activated macrophages. Tumor necrosis factor and L-arginine-dependent mechanisms act synergistically as the major cytolytic mechanisms of activated macrophages

We examined the cytolytic mechanisms of activated macrophages by using proteose peptone- or thioglycollate broth-induced mouse peritoneal macrophages or mouse macrophage hybridomas as effector cells, L.P3 cells, a clone of L929 cells, and P815 cells as target cells, and IFN-gamma and LPS as activators. It was determined that TNF is the main cytolytic molecule against L.P3 cells from the following results: 1) activated macrophages can produce TNF; 2) TNF shows cytotoxic activity against L.P3 cells; 3) the addition of anti-TNF antibody inhibited most of the cytolytic activity of activated macrophages against L.P3 cells. On the other hand, it was concluded that the main cytolytic mechanism against P815 cells is the production of NO2-/NO3- from L-arginine, from the following results: 1) activated macrophages can produce NO2-; 2) NaNO2 shows high cytotoxic activity against P815 cells; 3) the depletion of L-arginine from the medium inhibited most of the cytolytic activity of activated macrophages against P815 cells and NO2- production by activated macrophages. In this study, however, cytostatic effects of L-arginine-dependent effector mechanism were not studied. Thus, these results show that activated macrophages can express at least two cytolytic mechanisms independently, namely, the one that appears to be mediated by the L-arginine-dependent effector mechanism and the second that appears to be mediated directly by TNF. Furthermore, it was demonstrated that TNF and L-arginine-dependent NO2- production act synergistically as killing mechanisms of activated macrophages. These mechanisms can explain the cytolytic activity of activated macrophages against a variety of target cells.     

Granules of human CD3+ large granular lymphocytes contain a macrophage regulating factor(s) that induces macrophage H2O2 production and tumoricidal activity but decreases cell surface Ia antigen expression.

CTL (cytotoxic T lymphocytes) and LGL (large granular lymphocytes) exocytose cytoplasmic granules on activation after recognition of their target, releasing granule-associated molecules. We have previously suggested that this process could release immunoregulatory molecules. In this study we investigated whether normal human LGL granules contained a factor regulating different macrophage activity. Human CD3+ LGL cells were generated by activating peripheral blood lymphocytes (PBL) for 10-12 days with recombinant human IL-2 (rhIL-2), and granules were isolated from disrupted cell homogenate by Percoll gradient fractionation. Solubilized granules were tested for macrophage-activating factor (MAF) activity in three different macrophage assays. When M-CSF-differentiated murine bone marrow-derived macrophages were incubated 9 hr with human LGL granules, they were fully activated to lyse the TNF-resistant P815 tumor cells. The granule-MAF showed a synergistic effect with rhIL-1 beta, rmTNF-alpha, and rmIFN-tau in the cytolytic assay. In addition, proteose-peptone-elicited murine peritoneal macrophages profoundly increased H2O2 production after activation with human LGL granules. However, unlike IFN-tau, no increase in peritoneal macrophage Ia antigen expression was detected after incubation with granules. Moreover, granule-MAF suppressed Ia induction by IFN-tau. These results confirm that human CD3+ LGL granules contain a molecule(s) capable of regulating macrophage function.     

Alteration of in vitro murine peritoneal macrophage function by dietary enrichment with eicosapentaenoic and docosahexaenoic acids in menhaden fish oil

The effects of diets containing menhaden fish oil (MFO), compared with those of diets containing safflower oil (SAF) or an essential fatty acid deficient hydrogenated coconut oil (HCO), on in vitro activation of tumoricidal capacity by murine macrophages were assessed. Mice fed the experimental diets for 4 weeks were injected intraperitoneally with sterile thioglycollate broth 3 days before use. There was no difference between any of the groups with respect to total peritoneal exudate cells or the percentage of macrophages, although the fatty acid profile of purified adherent macrophages closely paralleled that of the diets. Macrophages from mice fed MFO killed fewer P815 mastocytoma cells upon activation with recombinant interferon gamma (IFN gamma) and lipopolysaccharide. Macrophages from all diets were equally competent for tumoricidal capacity when activated pharmacologically with calcium ionophore, phorbol 12-myristate 13-acetate, and lipopolysaccharide (LPS), suggesting that MFO diet macrophages were hyporesponsive to IFN gamma. Priming with higher concentrations of IFN gamma restored the partial defect in activation of MFO macrophages. When activated for 24 hr with high levels of LPS, macrophages from mice fed SAF displayed little cytolytic capacity; addition of indomethacin. (1 microM) resulted in enhanced levels of P815 kill. In contrast, MFO and HCO diet macrophages were highly cytolytic with similar LPS treatment with or without indomethacin. Macrophages from mice fed SAF produced threefold more prostaglandin E in response to LPS than did MFO and HCO diet macrophages. These results suggest that dietary manipulation of fatty acids can alter activation of tumoricidal capacity of macrophages, possibly both dependent and independent of changes in eicosanoid synthesis.