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.     

Macrophage activation: synergism between hybridoma MAF and poly(I). Poly(C) delivered by liposomes

High concentrations of a murine T cell hybridoma culture supernatant containing macrophage-activating factor (MAF) rendered resident mouse peritoneal macrophages cytotoxic for P815 mastocytoma cells. The capacity of the hybridoma-derived MAF (MAFH) to induce tumoricidal activity increased 10(3) to 10(4)-fold when the lymphokine was encapsulated into liposomes. Combinations of MAFH and poly(I) X poly(C) acted synergistically to render macrophages potently cytotoxic. Subthreshold (nonactivating) concentrations of free or liposome-encapsulated MAFH increased the potency of free poly(I) X poly(C) and liposome encapsulated poly(I) X poly(C). Either as free agent or encapsulated in liposomes, single-stranded poly(I) or poly(C) did not activate macrophages in the presence or absence of MAFH. Double-stranded poly(I) X poly(C) was thus required for macrophage activation and synergism with MAFH.     

Enhancement of human monocyte tumoricidal activity by recombinant M-CSF

Activated monocytes are an important component of immunologic defense against neoplastic disease. A variety of agents capable of inducing tumoricidal activity have been described, including bacterial LPS, IFN-gamma, IL-1, IL-2, TNF, and GM-CSF. We now show that pretreatment of monocytes with recombinant human macrophage-specific colony stimulating factor (M-CSF) augments the tumoricidal activity of human peripheral blood monocytes induced by other activating agents. Monocytes were preincubated for three days with M-CSF at 10(3) U/ml, washed, and treated for an additional two days with secondary activators. Tumoricidal activity was measured in a 6-h 51Cr-release assay using NK-resistant WEHI 164 cells that had been treated with actinomycin D. Pretreatment of monocytes with M-CSF significantly increased tumoricidal activity induced by LPS, IFN gamma, LPS plus IFN gamma, and LPS plus PMA. Pretreatment with IL-1, IL-2, IL-3, IL-4, or GM-CSF was not as effective as M-CSF in increasing tumoricidal activity. Enhanced tumoricidal activity was directly correlated to the increased TNF production resulting from M-CSF pretreatment. TNF antiserum completely blocked tumoricidal activity, demonstrating that TNF was responsible for the M-CSF-mediated increase in tumor cell lysis. M-CSF pretreatment also enhanced non-TNF mediated tumoricidal activity by monocytes, as seen by increased killing of the TNF-resistant target P815. This study demonstrated that in addition to the role of M-CSF in the proliferation and differentiation of monocyte/macrophage precursors, M-CSF also augments an effector function of mature blood monocytes.     

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.     

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.     

Characterization of IL-2 receptor expression and function on murine macrophages

This study was designed to examine the expression and function of IL-2R on murine macrophages. We used a model system of murine macrophage cell lines (ANA-1 and GG2EE) that was established by infecting normal murine bone marrow-derived cells with the J2 (v-raf/v-myc) recombinant murine retrovirus. ANA-1 macrophages did not constitutively express detectable levels of mRNA for the p55, IL-2R alpha. However, a brief exposure to IFN-gamma was sufficient to induce IL-2R alpha mRNA in ANA-1 macrophages. Flow cytometric analysis indicated that ANA-1 macrophages expressed low constitutive levels of IL-2R alpha on their cell surface that were augmented after treatment of the cells with IFN-gamma. Affinity binding and cross-linking of [125I]IL-2 to ANA-1 macrophages demonstrated that IL-2R alpha and the p70-75, IL-2R beta were both present on ANA-1 macrophages constitutively. IFN-gamma increased the expression of IL-2R alpha on ANA-1 macrophages but did not increase the expression of IL-2R beta on these macrophages. Although IL-2 alone did not induce the tumoricidal activity of ANA-1 macrophages, IL-2 acted synergistically with IFN-gamma to induce macrophage tumoricidal activity. These data demonstrate the expression of IL-2R on murine macrophage cell lines and establish the role of IL-2 as a costimulator of macrophage-mediated tumoricidal activity.     

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.     

Macrophage-activating factor produced by a T cell hybridoma: physiochemical and biosynthetic resemblance to gamma-interferon

Biochemical and biosynthetic evidence has been obtained which indicates that the macrophage activating factor (MAF) produced by the murine T cell hybridoma clone 24/G1, which primes macrophages for nonspecific tumoricidal activity, is a form of gamma-interferon (IFN gamma). MAF and antiviral activities were generated by the clone in proportional amounts under a variety of culture conditions. Production of both activities showed identical dependence on cell density even when production precipitously ceased at cell concentrations greater than 1.2 X 10(6) cells/ml. MAF and antiviral activities displayed identical sensitivities to pH and temperature and were indistinguishable on the basis of binding to insolubilized polynucleotides. Dye-ligand chromatography of a stimulated hybridoma supernatant on a column of Matrex Gel Red A resulted in the 1500-fold purification and 100% recovery of the MAF activity. A qualitatively identical elution profile was also obtained for the antiviral activity; however, only 32% of the original activity was recovered. When subjected to gel filtration on a high performance liquid chromatography system (HPLC), the MAF and antiviral activities present in the Matrex Red pool displayed identical elution profiles and exhibited an apparent m.w. of 50,000. This technique resulted in another 10-fold purification and 50% recovery of the two activities. On HPLC chromatofocusing the MAF activity in the Matrex Red pool could be resolved into seven chromatographically distinct species yet could not be resolved from the antiviral activity on either a qualitative or quantitative basis. These results thus provide quantitative molecular evidence to support the concept that IFN gamma can act as a MAF.     

Macrophage activation factor from EL-4, a murine T-cell line: antigenic characterization by hamster monoclonal antibodies to murine interferon-gamma

A cloned variant of the EL-4 murine T-cell line treated with phorbol myristate acetate (PMA) releases a factor that activates macrophages for nonspecific tumor cytotoxicity. This macrophage activation factor (MAF) is both physicochemically (Mr 25,000; pH 2 stable) and biologically different from interferon-gamma (IFN-gamma). However, EL-4 MAF may represent a breakdown product or otherwise altered fragment of IFN-gamma. We examined this possibility with a unique pair of hamster monoclonal antibodies against different epitopes of murine IFN-gamma. Both antibodies inhibited IFN-gamma-induced fibroblast antiviral activity; H21 but not H1 antibody also inhibited lymphokine (LK)-induced macrophage-mediated tumor cytotoxicity. Neither antibody, however, had any effect on the EL-4 MAF throughout a broad dose response. Moreover, passage through a H21 immunoaffinity chromatography column or addition of staphylococcal protein A and antibody completely inhibited LK-induced macrophage tumoricidal activity but did not affect the activity in EL-4 MAF. Identical effects in both fluid and solid phase were observed with polyclonal rabbit antisera to murine IFN-gamma. Results with all of these antibodies strongly suggest that the EL-4 MAF and murine IFN-gamma are antigenically distinct.     

Role of endogenously produced interleukin-6 as a second signal in murine thymocyte proliferation induced by multiple cytokines: regulatory effects of transforming growth factor-beta

Previous studies have demonstrated that murine thymocytes proliferate in the presence of submitogenic concentrations of phytohemagglutinin-P (PHA-P) and various cytokines such as interleukin-1 (IL-1), interleukin-4 (IL-4), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6). We report that C3H/HeJ thymocytes stimulated with PHA-P and IL-1, IL-4, or TNF-alpha secrete significant levels of IL-6 as determined on B9 hybridoma cells. The possibility that thymocyte proliferation induced by these cytokines was mediated through IL-6 was investigated utilizing a neutralizing monoclonal antibody against murine IL-6, MP5 20F3.1. The results demonstrate that MP5 20F3.1 inhibited the proliferative response of thymocytes and B9 hybridoma cells to recombinant MuIL-6 (but not HuIL-6) and neutralized the endogenous IL-6 produced in the thymocyte cultures, but did not have any measurable effects on the proliferative responses induced by IL-1, IL-4, or TNF-alpha. Although the level of endogeneously produced IL-6 did not play a measurable role in the proliferative response induced by TNF-alpha, the addition of higher concentrations of IL-6 augmented the proliferation of murine thymocytes induced by rMu TNF-alpha. In addition, recombinant human transforming growth factor-beta 1 (rHu TGF-beta 1) significantly inhibited thymocyte proliferation induced by HuIL-1, rMuIL-4, rMuIL-6, and rMuTNF-alpha. The studies suggest that IL-1, IL-4, or TNF-alpha mediate a proliferative signal on murine thymocytes independent of IL-6 and that the proliferative signals provided by these cytokines as well as IL-6 are inhibitable by rHu TGF-beta 1.     

Interleukin-4 downregulates interleukin-6 production by human alveolar macrophages at protein and mRNA levels.

Effects of interleukin-4 (IL-4) on IL-6 production by human alveolar macrophages (AM) obtained by bronchoalveolar lavage from healthy donors was examined at the protein and gene levels. IL-6 production was quantitated by enzyme immunoassay (EIA) and bioassay using the IL-6 dependent murine hybridoma cell line MH60.BSF2. Results showed that when activated with LPS, AM released significantly more biologically active IL-6 than blood monocytes. Human rIL-4 significantly suppressed IL-6 production by AM and monocytes stimulated with LPS. Northern blot analysis revealed that IL-4 reduced the expression of IL-6 mRNA in LPS-stimulated AM and monocytes. The inhibitory effect was most pronounced when IL-4 was added with LPS or within the first 4 hr after LPS to AM or monocytes. The suppressive effect of IL-4 was completely neutralized by pretreatment with anti-IL-4 antibody. IL-4 also showed a suppressive effect on IL-6 production by macrophages generated in vitro by maturation of blood monocytes with granulocyte-macrophage colony stimulating factor (GM-CSF). These observations suggest that IL-4 may play a critical role in in situ regulation of immune responses through suppression of IL-6 production.     

Nitric oxide-mediated tumoricidal activity of murine microglial cells

Experimental metastases in the brain of mice are infiltrated by microglia, and parabiosis experiments of green fluorescent protein (GFP(+)) and GFP(-) mice revealed that these microglia are derived from circulating monocytes (GFP(+), F4/80(+), and CD68(+)). These findings raised the question as to whether microglia (specialized macrophages) possess tumoricidal activity. C8-B4 murine microglia cells were incubated in vitro in medium (control) or in medium containing both lipopolysaccharide and interferon-γ. Control microglia were not tumoricidal against a number of murine and human tumor cells, whereas lipopolysaccharide/interferon-γ-activated microglia lysed murine and human tumor cells by release of nitric oxide. Parallel experiments with murine peritoneal macrophages produced identical results. Neither activated microglia nor activated macrophages lysed nontumorigenic murine or human cells. Collectively, these data demonstrate that brain metastasis-associated microglia are derived from circulating mononuclear cells and exhibit selective and specific tumoricidal activity.     

IFN-beta 2/IL-6 augments the activity of human natural killer cells

MHC nonrestricted cytotoxic cells play an important role in the killing of tumor cells in vitro and potentially in vivo. The activity of these cells is regulated by several cytokines such as IL-2 and IFN. In the present study we provide first evidence that IL-6 significantly augments the cytotoxic activity of human NK cells. IL-6 is produced by many different cells and is also known as IFN-beta 2, B cell stimulatory factor 2, hybridoma growth factor, hepatocyte-stimulating factor, and 26 kDa protein. IL-6 stimulates the activity of human CD3- NK cells but not that of CD3+ non-MHC-restricted cytotoxic T lymphocytes. As is the case with IL-2, the IL-6-mediated augmented cytotoxicity was a result of a more efficient lysis, but was not caused by an increased effector to target cell binding. Moreover, the effect of IL-6 on NK cell activity was blocked by a mAb directed against IL-2, and IL-6 itself was found to be a potent inducer of IL-2 production in cultured human PBMC. Thus it may be concluded that IL-6 enhances the cytotoxic activity of NK cells via IL-2. This newly recognized property of IL-6, which is produced by almost any cell, may be of importance in host defense against microbes and malignancies and therefore could contribute to improve the adoptive immunotherapy by using lymphokine-activated killer cells.     

Downregulation of tumor necrosis factor receptors of macrophages by interferons and interleukin-1. Role of protein kinase C activation

Freshly harvested murine peritoneal macrophages and a line of transformed murine macrophages (RAW) were used in experiments designed to investigate the effect of different interferons (IFN) and interleukin-1 (IL-1) on tumor necrosis factor (TNF) receptors. Low concentrations of IFN-gamma or somewhat higher concentrations of IFN-alpha drastically downregulated the TNF receptors of RAW cells. A similar, but less pronounced, downregulation of TNF receptors was observed in peritoneal macrophages treated with these IFNs. This downregulation could not be accounted for by an induction of TNF secretion. Furthermore, IFN-alpha and gamma interacted synergistically in downregulating TNF receptors of RAW cells. IL-1 also downregulated TNF receptors. When RAW cells were treated with inhibitors of protein kinase C, the downregulation of TNF receptors by IFNs or IL-1 was reversed, and TNF binding increased up to 2-fold over that of untreated cells. Such increase was also observed in RAW cells treated only with the inhibitor of protein kinase C, staurosporine. However, TNF receptors decreased in peritoneal macrophages treated with staurosporine. This finding was explained by activation of macrophages by staurosporine, which induced secretion of TNF. These findings indicate that protein kinase C activity regulates TNF receptors in macrophages.     

Murine eosinophils and IL-1: alpha IL-1 mRNA detection by in situ hybridization. Production and release of IL-1 from peritoneal eosinophils

The presence of IL-1 mRNA in eosinophils from mice infected with larvae of the parasite Mesocestoides corti was investigated by in situ hybridization technique. S35 labeled cDNA probe for alpha IL-1, hybridized with mRNA in murine eosinophils and macrophages. After 6 h of LPS stimulation eosinophils were able to express mRNA in their cytoplasm. This expression was highly increased by the addition of indomethacine. The IL-1 mRNA expression in murine macrophages was higher than in eosinophils in LPS-stimulated cells. This difference was statistically significant, p less than 0.001. To test if eosinophils may produce and release IL-1 in the culture medium, we isolated these cells in a Percoll gradient. Cell preparations with a purity exceeding 94% were cultured with various stimuli and their supernatants were tested for IL-1 activity. Eosinophils produced 169.65 +/- 73 U/ml when stimulated with LPS (n = 14). A dose-dependent response was obtained when the eosinophils were in the presence of the calcium ionophore A23187. Controls were performed to rule out the contribution of the contaminating population on the thymocyte proliferating activity. They were also used to detect other possible causes of interference in the assay, such as leukotrienes or TNF. IL-1 in supernatants was also detected using a conversion assay such as EL-4 thymoma cells. IL-1 activity was first detected in culture supernatants 18 h later, maximal production being in the first 24 h. In accordance with our hybridization results, an increase in IL-1 activity was obtained when eosinophils were stimulated with LPS and treated with indomethacine. The factor had a molecular mass between 16 to 20 kDa that corresponded to the described for murine IL-1. Inasmuch as IL-1 is an important mediator of inflammatory reactions this IL may enhance the proinflammatory action of eosinophils.     

Stimulatory effects of purified macrophage colony-stimulating factor on murine resident peritoneal macrophages

A purified preparation of macrophage colony-stimulating factor (M-CSF) free of interferon and endotoxin activity was studied for its effects on resident murine peritoneal macrophages. M-CSF was found to induce profound morphologic alterations in resident macrophages. These changes included a marked increase in cell size, membrane ruffling, and cytoplasmic vacuolization. Further, after 72 hr of incubation with 1000 U/ml of M-CSF, there were significant increases in macrophage DNA synthesis as measured by autoradiography (P less than 0.001), and in macrophage monolayer protein content (P less than 0.01). None of these changes was seen in control macrophages or those exposed to recombinant interferon-gamma (IFN). Low activity levels of the ectoenzymes 5'-nucleotidase (5'NTD) and alkaline phosphodiesterase I (APD) have been associated with certain macrophage functions, particularly the expression of tumor cytotoxicity. Macrophage monolayers exposed to M-CSF demonstrated an unaltered level of 5'NTD activity from controls and a significantly increased level of APD activity (P less than 0.01) and did not demonstrate an increased ability to kill tumor cells, as measured by the 51Cr-release assay. On the other hand, IFN caused significant decreases in both 5'NTD (P less than 0.05) and APD (P less than 0.01) and also induced marked tumoricidal activity in macrophage monolayers. These results indicate that purified M-CSF induces highly specific alterations in the functional activity and morphologic appearance of resident macrophages and these changes are distinct from those induced by IFN.