Modulation of macrophage function by gamma-irradiation. Acquisition of the primed cell intermediate stage of the macrophage tumoricidal activation pathway

Macrophage activation for tumor cell killing is a multistep pathway in which responsive macrophages interact sequentially with priming and triggering stimuli in the acquisition of full tumoricidal activity. Although this synergistic response of normal macrophages to sequential incubation with activation signals has been well established, characterization of the intermediate stages in this pathway has been difficult, due in large measure to the instability of the intermediate cell phenotypes. We have developed a model system for examination of macrophage-mediated tumor cell lysis, with the use of the murine macrophage tumor cell line RAW 264.7. These cells, like normal macrophages, exhibit a strict requirement for interaction with both interferon-gamma (IFN-gamma, the priming signal) and bacterial lipopolysaccharide (LPS, the triggering signal) in the development of tumor cytolytic activity. In this system, the priming effects of IFN-gamma decay rapidly after withdrawal of this mediator and the cells become unresponsive to LPS triggering. We have recently observed that gamma-irradiation of the RAW 264.7 cells also results in development of a primed activation state for tumor cell killing. The effects of gamma-radiation on the RAW 264.7 cell line are strikingly similar to those resulting from incubation with IFN-gamma, with the exception that the irradiation-induced primed cell intermediate is stable and responsive to LPS triggering for at least 24 hr. Treatment with gamma-radiation also results in increased cell surface expression of major histocompatibility complex-encoded class I antigens; however, class II antigen expression is not induced. Irradiation-induced development of the primed phenotype is not solely the result of cytostatic effects as treatment of the cells with a radiomimetic drug, mitomycin C, results in decreases in [3H]thymidine incorporation that are similar to those observed after irradiation, without concomitant development of cytolytic potential. In addition, priming by gamma-radiation does not appear to be mediated by the release of soluble autoregulatory factors. This alternate pathway for induction of the primed macrophage activation state should serve as a useful tool for identification of molecules important to the functional potential of primed cells, and for elucidation of the biochemical mechanisms of the priming event in tumoricidal activation.     

Expression of a 120,000 dalton protein during tumoricidal activation in murine peritoneal macrophages

Modulation of protein expression during interferon-gamma (IFN-gamma)-lipopolysaccharide (LPS)-mediated macrophage tumoricidal activation has been examined by metabolic radiolabeling of various murine peritoneal macrophage populations with [35S]methionine followed by SDS-PAGE analysis. Although both IFN-gamma and LPS are capable of stimulating the expression of several proteins when used independently, combined treatment induced the enhanced or de novo expression of a 120,000 dalton polypeptide. The expression of this protein was synergistically regulated by both IFN-gamma and LPS in a manner strongly reminiscent of the functional synergism that these two agents exhibit with respect to induction of tumoricidal activity. p120 expression could be seen first at approximately 3 hr after the addition of both agents, reached optimal expression by 6 hr, and maintained elevated synthesis for up to 24 hr. This time course corresponds closely to that seen for the acquisition of tumoricidal competence. Macrophages elicited in the primed state of activity in vivo with methyl vinyl ether co-polymer II (MVE-II) did not express p120, but could be induced to do so when treated with low doses of LPS. Under similar conditions, MVE-II-elicited cells also acquire tumoricidal activity. Macrophages obtained from mice chronically infected with bacillus Calmette-Guerin constitutively expressed both p120 and cytolytic activity. If such macrophages were cultured for 24 hr, the expression of both events decayed and was lost, but could be restored by treatment with low doses of LPS. Thus the data support a strong correlation between the expression by macrophages of a novel 120,000 dalton protein and the expression of tumor cytotoxicity.     

Colony-stimulating factors and regulation of macrophage tumoricidal and microbicidal activities

Conditioned medium from antigen- or mitogen-stimulated spleen cells, lymphokines, contained factors that induced formation of granulocyte and macrophage colonies in cultures of bone marrow cells (CSF). Lymphokines also contained factors that induced macrophage non-specific tumoricidal activity against fibrosarcoma 1023, antibody-dependent tumoricidal activity against lymphoma 18-8, and antimicrobial activities against amastigotes of the protozoan parasite, Leishmania tropica. The factors that regulated macrophage effector functions, however, were different from those that induced colony formation, and could be distinguished from CSF by Sephadex gel chromatography or heat sensitivity. To further analyze a role for CSF in induction of macrophage effector activities, conditioned medium from several nonlymphoid cell sources (L-929, WEHI-3, and endotoxin-treated lung cells) were assayed for CSF activities and capacity to induce tumoricidal and microbicidal activities. Conditioned medium that contained either macrophages CSF (CSF-1) or the factor that induced formation of both macrophage and granulocyte colonies failed to activate macrophages for effector activities against fibrosarcoma 1023, lymphoma 18-8, and L. tropica amastigotes (either resistance to infection or intracellular destruction). These data suggest that CSF has no direct role in activation of macrophages for tumoricidal and microbicidal activities against these targets.     

Activation of C3H/HeJ macrophage tumoricidal activity and cytokine release by R-chemotype lipopolysaccharide preparations. Differential effects of IFN-gamma

We have investigated the relative immunostimulatory activities of S-chemotype LPS and R-chemotype LPS preparations on C3H/HeJ peritoneal macrophages in vitro. As assessed by either secretion of TNF-alpha or IL-1, some of the R-chemotype LPS manifest significant activity on these normally LPS-unresponsive cells. The expression of IL-1 activity by R-LPS-stimulated C3H/HeJ macrophages was unaffected by IFN-gamma; however, this cytokine significantly enhanced TNF-alpha production by the same cells. The R-chemotype LPS preparations alone were not able to activate C3H/HeJ macrophages to become tumoricidal but activity could readily be demonstrated in the presence of IFN-gamma. Of potential importance is the observation that the profile of relative activity of the various R-chemotype LPS preparations for macrophage activation does not parallel that previously obtained by us for the C3H/HeJ B-lymphocyte activation.     

Macrophage activation for microbicidal activity against Leishmania major: inhibition of lymphokine activation by phosphatidylcholine-phosphatidylserine liposomes

Resident peritoneal macrophages from untreated mice develop microbicidal activity against amastigotes of the protozoan parasite Leishmania tropica (current nomenclature = Leishmania major) after in vitro exposure to LK from antigen-stimulated leukocyte culture fluids. This LK-induced macrophage microbicidal activity was completely abrogated by addition of 7:3 phosphatidylcholine: phosphatidylserine liposomes. Liposome inhibition was not due to direct toxic effects against the parasite or macrophage effector cell; factors in LK that induce macrophage microbicidal activity were not adsorbed or destroyed by liposome treatment. Other phagocytic particles, such as latex beads, had no effect on microbicidal activity. Moreover, liposome inhibition of activated macrophage effector function was relatively selective: LK-induced macrophage tumoricidal activity was not affected by liposome treatment. Liposome inhibition was dependent upon liposome dose (5 nmoles/culture) and time of addition of leishmania-infected, LK-treated macrophage cultures. Addition of liposomes through the initial 8 hr of culture completely inhibited LK-induced macrophage microbicidal activity; liposomes added after 16 hr had no effect. Similarly, microbicidal activity by macrophages activated in vivo by BCG or Corynebacterium parvum was not affected by liposome treatment. Liposome treatment also did not affect the increased resistance to infection induced in macrophages by LK. These data suggest that liposomes interfere with one or more early events in the induction of activated macrophages (macrophage-LK interaction) and not with the cytotoxic mechanism itself (parasite-macrophage interaction). These studies add to the growing body of data that implicate cell lipid in regulatory events controlling macrophage effector function.     

Inhibition of macrophage tumoricidal activity by immune complexes and altered erythrocytes

Engagement of the macrophage membrane by biologic particles including insoluble immune complexes inhibited the development of lymphokine-mediated nonspecific tumoricidal activity by murine macrophages. The degree of inhibition was dependent on the dose of particles and the lymphokine concentration. Inhibition was not due to macrophage cell death or to diminution of cell adherence after ingestion of the immune complexes. Soluble immune complexes were not inhibitory, although approximately 10% of the complexes became cell-associated. Monomeric or heat-aggregated IgG was also not inhibitory. IgG-opsonized erythrocytes (EA) were inhibitory and inhibition was dependent on the degree of opsonization. In contrast, nonopsonized erythrocytes (E), which did not bind to macrophages, were not inhibitory. Phagocytosis of glutaraldehyde-treated E or E carrying IgM antibody and complement (EAC) also led to a reduction of tumorilytic activity. Insoluble immune complexes were inhibitory when added either before or after lymphokine. Phagocytosis was neither sufficient nor necessary to cause inhibition because 1) ingestion of polystyrene latex beads did not diminish tumoricidal activity, and 2) macrophages plated on IgG-coated surfaces were inhibited with respect to the tumoricidal function. Inhibition was not affected when indomethacin (10(-6) M) was included in the assay, which indicated that prostaglandins were not involved in the process. Thus, macrophage tumoricidal responsiveness may be compromised by interaction of biologic substances with macrophage plasma membranes. This process may thereby inactivate an important host defense mechanism against neoplastic cells.     

Inhibition of lymphokine-induced macrophage microbicidal activity against Leishmania major by liposomes: characterization of the physicochemical requirements for liposome inhibition

Resident peritoneal macrophages from untreated mice develop potent microbicidal activity against amastigotes of Leishmania major after in vitro treatment with lymphokine (LK) from mitogen-stimulated spleen cells. LK-induced macrophage microbicidal activity was completely and selectively abrogated by treatment with phosphatidylcholine-phosphatidylserine (PC/PS) liposomes. Other macrophage effector functions (phagocytosis, tumoricidal activity) were unaffected, as was cytotoxicity by macrophages activated in vivo or by LK in vitro before liposome treatment. Activation factors in LK were not adsorbed or destroyed by liposomes. Liposome-induced inhibition was unaffected by indomethacin and was fully reversible: macrophages washed free of liposomes developed strong microbicidal activity with subsequent LK treatment. Changes in liposomal lipid composition markedly altered suppressive effects, but inhibition was not dependent on liposome size, cholesterol content, charge, or number of lamellae. Liposomes composed of PC alone or in combination with any of five different phospholipids were not suppressive. In contrast, inhibition was directly dependent on PS concentration within PC/PS liposomes. Phosphoserine was not inhibitory nor was dimyristoyl PS (synthetic saturated PS). However, the lysophospholipid metabolite of PS, lysoPS, was strongly suppressive. These studies suggest that the reversible and selective inhibition of LK-induced macrophage microbicidal activity by PC/PS liposomes is mediated by PS and its lysoPS metabolite.     

Modulation of macrophage Ia-expression by lipopolysaccharide. I. Induction of Ia expression in vivo

Experiments were performed to analyze the modulation of macrophage Ia expression and biosynthesis by Salmonella minnesota-derived lipopolysaccharide (LPS) in vivo. The i.p. injection of LPS into LPS-responder mice caused a dramatic increase in the Ia expression of the peritoneal macrophage population harvested 1 wk after injection. As little as 1 ng of lipid-rich Re595 LPS per mouse caused a significant I-Ak increase, and 1 microgram was optimal; wild-type S. minnesota LPS was less active. No I-Ak induction by LPS was observed in the LPS-nonresponder strain C3H/HeJ. LPS-induced macrophages showed a 6- to 16-fold increase in I-Ak expression by radioimmunoassay (RIA), a 3- to 10-fold increase in the proportion of I-Ak-positive cells, and a 10- to 15-fold increase in I-Ak biosynthetic capacity. The magnitude of this induction by LPS was comparable to increases observed after injection of live Listeria monocytogenes. The kinetics of I-Ak induction by LPS and by L. monocytogenes were different: LPS caused an initial decrease in I-Ak expression 1 day after injection, and I-Ak induction by LPS occurred more slowly and maintained heightened expression longer. Several H-2 gene products (H-2Kk, I-Ak, and I-Ek) were augmented in LPS-induced macrophages. In keeping with increased I-A and I-E expression, LPS-induced macrophages were more effective than normal macrophages in presenting antigen to T lymphocytes. We suggest that the modulation of macrophage Ia expression is one important mechanism contributing to the immunoregulatory activity of LPS.     

Stimulation of macrophage tumoricidal activity by the growth and differentiation factor CSF-1

The effect of the macrophage growth and differentiation factor CSF-1 on the tumoricidal capacity of murine peritoneal exudate macrophages was investigated. Pretreatment of peptone-elicited macrophages 1 day with 300-1200 U/ml CSF-1 induced moderate killing and greatly stimulated lymphokine (LK)-induced killing of [3H]thymidine-labeled TU5 sarcoma cells to levels above that seen with fresh macrophages. Further addition of CSF-1 at Day 1 at the time of the tumor lysis assay promoted moderate increases in spontaneous and LK-induced activity. CSF-1 did not stimulate freshly harvested exudate macrophages to lyse TU5 targets in the presence or absence of lymphokine (LK) activators. Lipopolysaccharide (LPS) at 0.1-1000 ng/ml did not stimulate cytotoxicity, and the low endotoxin content and the use of polymyxin B and C3H/HeJ mice excluded a role for LPS in these experiments. Incubation of the macrophages with IFN and the myeloid growth factors IL-3 and GM-CSF did not stimulate tumoricidal activity. CSF-1 has been proposed as a therapeutic agent to restore myeloid cell numbers in induced (cancer chemotherapy, bone marrow transplantation, etc.) and natural aplastic anemias. These studies show that CSF-1 also may be useful in combination with LK activators to promote resistance to cancer in mature mononuclear cells. CSF-1 may have similar effects in LK-activated macrophages to enhance resistance to infectious diseases.     

Prostaglandin regulation of macrophage function: Effect of endogenous and exogenous prostaglandins

The expression of macrophage antitumor activity and the production of prostaglandins (PG) by operationally defined macrophage populations differed under varying culture conditions. Culture conditions that caused increased PGE₂ production by activated macrophages resulted in an inhibition of their tumoricidal activity. In contrast, production of high levels of PGE₂ by resident and elicited macrophages was associated with an increase in antitumor activity. The activation of resident or elicited cells by lipopolysaccharide (LPS) could be blocked by indomethacin. Treatment of these macrophages with PGE₂ alone also resulted in their activation and subsequent tumor cell destruction. Activation of resident and elicited macrophages by LPS appears to be mediated by PGE₂.     

In vitro modulation of macrophage tumoricidal activity

Summary NaIO₄ treatment of mouse adherent peritoneal cells or lymphocyte-free cloned macrophages enhances their cytotoxic and tumoricidal activity. 5×10^–3 M NaIO₄ treatment of nontumoricidal BCG-activated macrophages renders them completely tumoricidal, whereas the same treatment of stimulated (peptone-normal) macrophages renders them weakly tumoricidal. Addition of LPS in nanogram quantities too low to enhance tumor cell killing by untreated peptone-normal macrophages causes NaIO₄-treated peptone-normal macrophages to be maximally tumoricidal. The activating action of NaIO₄, MAF, or LPS can be potently, but inconsistently, blocked or reversed by the reducing agent NaBH₄ or the aldehyde-reacting agent dimedone. NaIO₄ treatment of lymphocyte-free macrophage colonies does not make them cytotoxic, but NaIO₄-treated colony macrophages are cytotoxic for tumor cells when cultured in 10 ng/ml LPS (an amount of LPS inadequate to render untreated colony macrophages cytotoxic). Supernatants of NaIO₄-treated adherent peritoneal cells contain MAF activity. Thus, the NaIO₄-induced enhancement of peritoneal cell tumoricidal activity may result from both direct NaIO₄ activating effects on macrophages and indirect NaIO₄ effects through NaIO₄-induced MAF production.     

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.     

Expression of macrophage p120 depends on early protein synthesis

We have previously identified a group of early proteins preceding the expression of a 120-kDa protein (p120) which coincides with tumoricidal activation in peritoneal macrophages. In the present report, we have asked whether the in vitro induction of new or enhanced expression of p120 depends on early protein synthesis and RNA synthesis during the treatment period. Expression of p120 was sensitive to pretreatment of the macrophages with either actinomycin D or cycloheximide, indicating that both active protein synthesis and RNA synthesis were required. When poly-adenylated RNA isolated from various macrophage populations was translated in a rabbit reticulocyte in vitro translation system, only mRNA isolated from cells which express p120 was able to direct synthesis of a 120-kDa polypeptide. This product showed identical mobility to p120 induced in intact activated macrophages radiolabeled with [35S]methionine. The presence of translatable p120 mRNA was dependent upon treatment of thioglycollate-elicited macrophages with both IFN-gamma plus LPS at low doses, as is expression of p120 in intact cells. Accumulation of translatable p120 mRNA was blocked by treatment with cycloheximide, indicating that active protein synthesis was required during the induction period. These results suggest that the presence of specific translatable mRNA encoding the p120 polypeptide is dependent upon the expression of early macrophage gene products.     

Inducible expression of murine IP-10 mRNA varies with the state of macrophage inflammatory activity

We have examined the expression of inducible inflammatory genes in murine macrophages from different tissues and at different stages of inflammatory activity. Although i.v. administration of IFN-gamma (10,000 U/mouse) strongly induced expression of IP-10 mRNA in the adherent cell population of the spleen, thioglycollate-elicited peritoneal macrophages were essentially unresponsive at the same dose. In contrast, D3 mRNA was expressed in both cell populations. This differential sensitivity of IP-10 mRNA expression was not restricted to stimulation by IFN-gamma as it was also seen when LPS (25 micrograms/mouse) was administered i.v. Expression of JE and KC mRNA, which encode cytokines related to IP-10, were also differentially expressed in elicited peritoneal macrophages from mice injected with LPS. Differential sensitivity was at least partially related to the state of macrophage activation because IP-10 mRNA was highly inducible in resident but not thioglycollate-elicited peritoneal macrophages. The eliciting agent was also an important determinant because proteose-peptone-elicited peritoneal macrophages were nearly as sensitive as splenic macrophages with respect to expression of IP-10 mRNA. IFN-gamma treatment induced IP-10 and D3 mRNA rapidly and transiently with the same time course in the spleen. IP-10 mRNA was not induced by IFN-gamma in TG-elicited macrophages regardless of the time after treatment. This differential expression of IP-10 was a consequence of different concentration requirements for IFN-gamma in the two cell types; thioglycollate-elicited macrophages required five- to 10-fold more IFN-gamma than did resident cells to achieve comparable IP-10 mRNA levels whether the agent was provided in vitro or in vivo. Thus variable sensitivity for induction of IP-10 mRNA was a characteristic of the macrophage itself and was not mediated by other cellular or molecular elements present in the inflammatory peritoneal cavity. The reduced sensitivity to IFN-gamma or LPS for expression of IP-10, JE, and KC mRNA as compared with TNF-alpha or D3 mRNA suggests that this distinct pattern of regulation may be restricted to members of these two related cytokine gene families that exhibit cell-type specific chemoattractant activity.     

Differential susceptibility of activated macrophage cytotoxic effector reactions to the suppressive effects of transforming growth factor-beta 1

We examined the effects of TGF-beta 1 on induction of several activated macrophage antimicrobial activities against the protozoan parasite Leishmania, and on induction of tumoricidal activity against the fibrosarcoma tumor target 1023. TGF-beta by itself did not affect the viability of either the intracellular or extracellular target in concentrations up to 200 ng/ml. As little as 1 ng/ml TGF-beta, however, suppressed more than 70% of the intracellular killing activity of macrophages treated with lymphokines. In contrast, more than 100 ng/ml TGF-beta was required to suppress intracellular killing by cells activated with an equivalent amount of recombinant IFN-gamma. Addition of TGF-beta for up to 30 min after exposure to activation factors significantly reduced macrophage killing of intracellular parasites. Pretreatment of macrophages with TGF-beta was even more effective: treatment of cells with TGF-beta for 4 h before addition of activation factors abolished all macrophage intracellular killing activity. Regardless of treatment sequence, however, TGF-beta had absolutely no effect, at any concentration tested, on activated macrophage resistance to infection induced by lymphokines or by the cooperative interaction of IFN-gamma and IL-4. Effects of TGF-beta on tumoricidal activity of activated macrophages was intermediate to that of its effects on intracellular killing or resistance to infection. Lymphokine-induced tumor cytotoxicity was marginally (25%) affected by TGF-beta; 200 ng/ml was able to suppress IFN-gamma-induced tumoricidal activity by 40%. Thus, TGF-beta dramatically suppressed certain activated macrophage cytotoxic effector reactions, but was only partially or not at all effective against others, even when the same activation agent (IFN-gamma) was used. The biochemical target for TGF-beta suppressive activity in these reactions may be the pathway for nitric oxide production from L-arginine, because TGF-beta also inhibited the generation of nitric oxide by cytokine-activated macrophages.     

Induction of activated macrophages in C3H/HeJ mice by avirulent Salmonella

A single injection of viable Salmonella typhimurium SL3235, an avirulent organism blocked in the aromatic pathway, induced the generation of activated peritoneal macrophages in three different C3H mouse strains, including macrophage-defective C3H/HeJ mice. Macrophages obtained from immunized mice were cytotoxic for B16 melanoma cells, P815 mastocytoma cells, and TU-5 fibrosarcoma cells and microbicidal in vitro for the obligate, intracellular, protozoan parasite Leishmania major. The capacity of live SL3235 to activate C3H/HeJ macrophages contrasts with the failure of live Bacillus Calmette-Guérin to induce activated macrophages in this mouse strain. Although viable SL3235 were capable of fully activating cells of both normal and defective mice, a dose-dependent difference was observed in the number of organisms necessary for induction of tumoricidal macrophages in C3HeB/FeJ (normal) and C3H/HeJ (defective) animals. As few as 80 viable SL3235 were capable of activating C3HeB/FeJ macrophages whereas 5 X 10(4) organisms were required to activate C3H/HeJ macrophages. Maximal macrophage activation occurred 7 to 10 days after SL3235 inoculation in C3H/HeJ and C3HeB/FeJ mice. Acetone-killed cells of SL3235 had some but not all of the activity of the living Salmonella. A single in vivo injection of the nonviable preparation resulted in the induction of tumoricidal macrophages in C3HeB/FeJ but not in C3H/HeJ mice, even when tested over a wide dosage range. Injection of acetone-killed cells of SL3235 did, however, result in a population of primed macrophages in C3H/HeJ mice, as explanted cells could be induced to express activated macrophage effector activities after additional treatment in vitro with either LPS or IFN-gamma. Thus, in vivo administration of viable SL3235 is, by itself, capable of eliciting the full series of steps required for activation of C3H/HeJ macrophages, whereas killed SL3235 only provides signals sufficient to prime these defective macrophages for further activation in vitro. AI 15613     

Role of protein kinase C and intracellular calcium mobilization in the induction of macrophage tumoricidal activity by interferon-gamma

These studies were designed to test the hypothesis that changes in intracellular Ca2+ levels and activation of the calcium ion- and phospholipid-dependent protein kinase C were required for the induction of macrophage tumoricidal activity by interferon-gamma (IFN-gamma). Phenothiazines and R24571, known antagonists of calcium-binding proteins and therefore nonspecific inhibitors of protein kinase C, blocked in a dose-dependent manner the induction of macrophage cytocidal activity by either natural or recombinant IFN-gamma. Macrophages depleted of intracellular Ca2+ by chelation with Quin 2, were also unresponsive to IFN-gamma. These treatments effected neither the binding of IFN-gamma to its cell surface receptor nor the normal intracellular processing of IFN-gamma. Activators of protein kinase C (such as phorbol esters) and Ca2+ ionophores when added alone did not effect the activation state of the macrophage population. However, macrophages exposed to both drugs in combination were elevated into the primed activation state such that in the presence of a second signal (lipopolysaccharide or heat killed Listeria monocytogenes), the cells were triggered to express full levels of tumoricidal activity. The capacity of phorbol esters to induce cellular activation correlated with their ability to bind and to activate protein kinase C. No synergistic effect was observed between IFN-gamma and protein kinase C activators and/or Ca2+ ionophores, indicating that the drugs could only prime and could not trigger macrophages for tumor cell killing. These results thus support the concept that protein kinase C activation and mobilization of intracellular Ca2+ are essential steps in the pathway of IFN-gamma-dependent induction of non-specific tumoricidal activity in macrophages.     

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.     

Effect of cisplatin, lipopolysaccharide, muramyl dipeptide and recombinant interferon-gamma on murine macrophages in vitro. I. Macrophage-mediated tumor cell lysis

Murine macrophage monolayers treated with cisplatin, lipopolysaccharide (LPS), muramyl dipeptide (MDP) or recombinant interferon-gamma (rIFN gamma) were observed to have significantly increased tumoricidal activity. rIFN gamma had synergistic effects with cisplatin, LPS or MDP in activating macrophages. However, MDP showed much more pronounced synergism with cisplatin and LPS than with rIFN gamma. Supernatants collected from these activated macrophage monolayers also showed increased tumoricidal activity. Tumor cell lysis mediated by cisplatin-treated macrophages did not require priming with rIFN gamma though it may be necessary as a first signal for the increased macrophage activation with LPS and MDP.