Systemic activation of tumoricidal properties in mouse macrophages and inhibition of melanoma metastases by the oral administration of MTP-PE, a lipophilic muramyl dipeptide

The purpose of these studies was to determine whether the oral administration of a lipophilic analog of muramyl dipeptide, MTP-PE, can produce in situ activation of tumoricidal properties in mouse macrophages. MTP-PE was dissolved in a phosphate-buffered saline to produce micelles. Single or multiple oral administrations of MTP-PE produced tumoricidal activation in both lung and peritoneal macrophages. This was in direct contrast to the i.v. or i.p. administrations of MTP-PE incorporated in liposomes, which produced activation in only lung or only peritoneal macrophages, respectively. The distribution and fate of [3H]-labeled MTP-PE subsequent to oral administration revealed that MTP-PE was found in various organs independent of reticuloendothelial activity. Finally, the repeated twice-weekly oral administrations of MTP-PE inhibited lung and lymph node metastasis in C57BL/6 mice by syngeneic B16 melanoma cells. The oral administration of MTP-PE, however, was not effective in eradicating well-established melanoma metastases. We conclude that the oral administration of a lipophilic muramyl dipeptide produces systemic activation of macrophages. The feasibility of enhancing host defense against infections and cancer by the oral administration of an immunomodulator has obvious clinical advantages.     

Synergistic activation by recombinant mouse interferon-gamma and muramyl dipeptide of tumoricidal properties in mouse macrophages

Mouse peritoneal macrophages were activated to become cytotoxic against B16-BL6 melanoma cells by the combination of subthreshold amounts of murine interferon-gamma (IFN-gamma; 0.1 to 10 U/ml) and N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP; 0.001 to 10 micrograms/ml), but not by the combination of pH 2-treated IFN-gamma and MDP, heat-treated IFN-gamma and MDP, or IFN-gamma and the inactive stereoisomer of MDP, N-acetyl-muramyl-D-alanyl-D-isoglutamine (MDP-D). The encapsulation of intact IFN-gamma and MDP within the same liposome preparation was synergistic for macrophage activation. In contrast, the presentation of identical concentrations of IFN-gamma and MDP in separate liposome preparations did not activate macrophages. These data allow us to conclude that the encapsulation of genetically engineered IFN-gamma and synthetically produced MDP within the same liposome is highly efficient in producing synergistic activation of tumoricidal properties in mouse macrophages.     

Correlation of immunomodulatory and therapeutic activities of interferon and interferon inducers in metastatic disease

The mechanism of therapeutic activity of recombinant murine interferon-gamma (rMu IFN-gamma) and the IFN inducer polyinosinic-polycytidylic acid solubilized with poly-L-lysine in carboxy methyl cellulose (pICLC) in treating metastatic disease was investigated by comparing effector cell augmentation with therapeutic activity in mice bearing experimental lung metastases (B16-BL6 melanoma). Effector cell functions in spleen, peripheral blood, and lung (the organ with tumor) were tested after 1 and 3 weeks of rMu IFN-gamma or pICLC administration (intravenous, three times a week). In these studies, natural killer (NK), lymphokine-activated killer (LAK), cytolytic T lymphocytes (CTL) (against specific and nonspecific targets), and macrophage tumoricidal and tumoristatic activities were measured. rM IFN-gamma and pICLC had therapeutic activity and immunomodulatory activity in most assays of immune function examined. Specific CTL activity of pulmonary parenchymal mononuclear cells (PPMC), but not in splenocytes or peripheral blood lymphocytes (PBL), during week 3 and not during week 1, correlated with the therapeutic activity of rMu IFN-gamma and of pICLC. Macrophage tumoricidal activity in PPMC, but not in alveolar macrophages, also correlated with the therapeutic activity of rMu IFN-gamma, but the opposite was true for the therapeutic activity of pICLC. NK activity of PPMC, but not of splenocytes or PBL, during week 1 correlated with the therapeutic activity of pICLC; in contrast, NK activity at any site did not correlate with the therapeutic activity of rMu IFN-gamma. LAK activity at any site did not correlate with the therapeutic activity of either agent.     

In situ activation of mouse macrophages and therapy of spontaneous renal cell cancer metastasis by liposomes containing the lipopeptide CGP 31362

Summary We determined whether the intravenous administration of multilamellar vesicle liposomes (MLV) containing a lipopeptide analogue of a fragment from the cell wall of gram-negative bacteria (CGP 31 362) can render BALB/c mouse alveolar macrophages tumoricidal in situ and reduce the incidence of spontaneous lung metastasis of syngeneic renal carcinoma (RENCA) cells. Alveolar macrophages (a) incubated in vitro with MLV containing CGP 31 362 (MLV-31 362) and (b) harvested from mice injected i.v. with MLV-31 362 were rendered cytotoxic against the RENCA cells. Maximum cytotoxic activity of the macrophages was induced by injecting 5 µmol MLV consisting of 250 mg phospholipids and 0.5 mg CGP 31 362. The single i.v. injection of 5 µmol MLV-31 362 produced activation of macrophages that lasted for up to 4 days. Repeated i. v. injections of MLV-31 362 produced a continuous antitumor activity in alveolar macrophages. To study the lipopeptide's effects on metastasis, we injected the left kidneys of BALB/c mice with RENCA cells. The kidney with growing tumor was resected 10 days later and, after a further 2 days, groups of mice were injected i.v. with MLV-31 362 or with MLV-HBSS (twice weekly for 3 weeks). Treatment with MLV-31 362 significantly decreased the median number of spontaneous lung metastases. These data demonstrate that the systemic administration of MLV-31 362 can activate murine lung macrophages in situ and reduce the incidence of spontaneous RENCA lung metastases.     

Inhibition of spontaneous pulmonary metastases of Lewis lung carcinoma by oral treatment with Respivax and Broncho-Vaxom

Summary The antimetastatic activity of orally administered polybacterial vaccines, Broncho-Vaxom (BV) and Respivax (RV) was examined in C57BL/6 mice, bearing implants of Lewis lung carcinoma (3LL) in the footpad. The oral administration of BV or RV for 10 consecutive days before or after surgery caused significant reduction of the number and volume of lung metastases. In addition, the therapeutic potential of BV and RV was examined in combination with chemotherapy to determine if there is additive activity. In animals bearing pulmonary micrometastases, treatment with a combination of cyclophosphamide at 50–150 mg/kg with BV or RV was found to be more effective than each of these treatments alone. In immune function studies it was established that the oral administration of BV and RV induced an increase in the number of cells, recovered by broncho-alveolar lavage, and alveolar macrophages were dominant in these cell populations. Furthermore, oral treatment of mice with these vaccines rendered their alveolar macrophages tumoricidal for syngeneic metastatic 3LL cells in vitro. These results show that pulmonary macrophages induced by oral administration of BV and RV played a key role in the inhibition of metastasis in 3LL-bearing mice.     

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.     

In situ activation of tumoricidal properties in rat alveolar macrophages and rejection of experimental lung metastases by intravenous injections of Nocardia rubra cell wall skeleton

Summary The IV injection of squalene-treated cell wall skeleton of Nocardia rubra (N-CWS) into F344 rats rendered their alveolar macrophages (AM) tumoricidal. Maximum tumoricidal activity developed in AM by 24 h after the IV, but not IP or SC, injection of 300 g N-CWS. Tumoricidal activity of AM was maintained for 48–72 h after one IV injection of N-CWS. Experimental lung metastases were produced in female F344 rats by the IV injection of viable syngeneic mammary adenocarcinoma cells. Treatments twice weekly with Hank's balanced salt solution, N-CWS placebo or N-CWS began 3, 7, or 10 days later and were continued for 3 or 4 weeks for a total of six or eight treatments. Practically all the rats (>90%) treated with N-CWS beginning on either day 3 or day 7 after tumor cell challenge survived until day 210, when the experiment was terminated. In contrast, 90% of the rats treated with balanced salt solution or N-CWS placebo died by day 70 of the experiment. Therapy with N-CWS preparation was not successful when the first injection was administered 10 days after tumor cell challenge, suggesting that this therapeutic regimen is effective only against minimal tumor burden. We conclude that in this animal tumor model, the IV injection of N-CWS preparations can render AM tumoricidal and aid in the eradication of pulmonary micrometastases.     

Prostaglandin E2 does not inhibit tumoricidal activity of mouse macrophages against adherent tumor cells

We determined whether endogenously produced PGE2 can down-regulate the tumoricidal properties of macrophages by a negative feedback mechanism. Peritoneal exudate macrophages or resident peritoneal macrophages of mice were incubated in medium (control) or in medium containing IFN-gamma and LPS. Activated macrophages were highly tumoricidal against syngeneic melanoma cells and secreted high levels of PGE2. Treatment with indomethacin or diclofenac sodium (voltaren) completely inhibited the production and secretion of PGE2 but not the tumoricidal activity of activated macrophages measured either immediately after activation or 1 to 3 days thereafter. Finally, the addition of exogenous PGE2 did not alter the ability of peritoneal exudate macrophages to respond to IFN-gamma or of LPS to produce high levels of tumor cell lysis. Collectively, these results show that PGE2 produced by activated macrophages is not a down-regulator of their tumoricidal activity against adherent tumor cells.     

Antimetastatic effect of endogenous tumor necrosis factor induced by the treatment of recombinant interferon γ followed by an analogue (GLA-60) to synthetic lipid A subunit

Summary We have investigated the effect of endogenous production of tumor necrosis factor (TNF) induced by the combination of recombinant interferon (rIFN) as a primer followed by GLA-60 as a trigger (rIFN/GLA-60) on murine lung metastases caused by B16-BL6 melanoma. In order to examine the therapeutic effect of endogenous TNF on tumor metastasis, the ability of multiple administrations of rIFN/GLA-60 to induce TNF production was also tested. The multiple administrations of rIFN/GLA-60 at intervals of 2 days were effective for the induction of endogenous TNF in mice but continuous multiple administrations of them for 2–4 days were not. In tumor-bearing mice, the production of endogenous TNF by rIFN/GLA-60 was less than that of normal mice, but treatment 3 days after the surgical excision of primary tumors showed the endogenous TNF production to be similar to that in normal mice. In the experimental lung metastasis model, intravenous administration of rIFN followed by intravenous or intranasal administration of GLA-60 showed potent inhibition of lung metastases of B16-BL6 melanoma, whereas the reverse sequence of administration (GLA-60/rIFN) or administration of a mixture of rIFN and GLA-60, which cannot induce the production of TNF, caused no inhibition of lung metastases. These results indicated that the regression of tumor metastases by rIFN/GLA-60 was mediated by the production of endogenous TNF in addition to the direct effects of both immunostimulants. Furthermore, the administration of rIFN and GLA-60 significantly inhibited the tumor metastases in spontaneous lung metastasis model. These results may provide a promising approach for the treatment of cancer metastasis as a result of its ability to induce endogenous TNF.     

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.     

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.     

Antitumor activity of Corynebacterium parvum administered into the pleural cavity of mice

Summary We investigated the efficacy of intrapleurally (IPl) injected 0.25 mg Corynebacterium parvum (CP) against pulmonary tumor deposits of four syngeneic murine tumors, C3Hf/Bu or CBA fibrosarcoma (FSa) or mammary carcinoma (MCa), and compared its efficacy with that of intravenous (IV) CP. A mode of action of CP given by IPl administration was also studied. When given to mice prior to the IV inoculation of tumor cells, IPl and IV CP reduced the number of metastases of C3Hf/Bu-FSa and CBA-MCa equally well. Intrapleural injection of CP within a week after tumor cell inoculation was more effective against metastases in the lung than IV CP. A combination of IPl and IV administration of CP was more effective in the therapy of lung metastases of CBA-FSa and CBA-MCa than either single treatment alone. Intrapleural and IV injections of CP augmented concomitant immunity to artificially produced lung metastases of C3Hf/Bu-FSa. Intrapleural CP was less effective than IV CP in inducing complete regression of the SC growing C3Hf/Bu-FSa.In contrast to IV CP, IPl CP did not markedly influence the spleen weight and cellularity. It was also less effective in increasing the liver weight. However, CP by either route of injection led to a significant increase in the lung weight. CP injected IPl, but not IV, caused a significant increase in the number of nucleated cells in the pleural cavity of mice. More than 90% of these cells were macrophages, and they were found to be cytotoxic for in vitro cultures of CBA-MCa cells. Activated macrophages also mediated in vivo antitumor resistance, as shown by the abolition of this resistance by treatment of the mice with carrageenan.     

Activation of natural resistance against lung metastasis of an adenocarcinoma in T-cell depressed spontaneously hypertensive rats by infection with Listeria monocytogenes

Summary We report here our study of the role of natural host defense mechanisms mediated by macrophages and natural killer (NK) cells in an experimental model of spontaneous pulmonary metastases of a mammary adenocarcinoma SST-2 in spontaneously hypertensive rats (SHR) with congenital T-cell depression. To activate macrophages and NK cells, Listeria monocytogenes (LM) was injected IV into SHR which had received a transplantation of SST-2. To assess the antimetastatic responses induced by LM, the number of lung nodules and the lung weight in SHR were evaluated 30 days after tumor inoculation. The growth of lung metastases, though not of primary tumors, was significantly reduced if 10⁷ LM were injected IV into SHR 2, 10 and 20 days after the SC transplantation of 5×10⁴ or 5×10⁵ SST-2. An inhibitory effect of LM on pulmonary metastases was also observed in tumor-excised rats, in which the number of lung metastases and the lung weight were enhanced as compared with those in tumor-bearing rats which had not undergone surgery. Peritoneal resident cells which were harvested from rats injected with LM showed a significant augmentation of tumoricidal activity against SST-2 cells as measured by in vitro cytotoxicity. Similarly, the NK activity of spleen cells of SHR injected with LM increased significantly when compared with untreated SHR. These data suggest that the inhibition of metastatic growth, though not of pirmary tumor growth, was accomplished by the, possibly T-cell independent, activation of macrophages and NK cells.     

Direct activation of tumoricidal properties in rat alveolar macrophages by Nocardia rubra cell wall skeleton

Summary Alveolar macrophages (AM) lavaged from lungs of normal F344 rats were rendered tumoricidal following their direct interaction with squalene-treated Nocardia rubra cell wall skeleton (N-CWS) present in the culture medium. Maximum tumoricidal activity was obtained by incubating AM with 1 g N-CWS/ml for a 24-h period. These AM were cytolytic to syngeneic, allogeneic, and xenogeneic tumorigenic cells. Tumoricidal activity following interaction with N-CWS decreased gradually and was lost completely by 96 h. A second in vitro exposure to N-CWS reactivated AM to their full tumoricidal potential. The present studies suggest that N-CWS can directly activate AM to render them tumoricidal.     

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.     

Augmentation of impaired tumoricidal function in alveolar macrophages from lung cancer patients by cocultivation with allogeneic, but not autologous lymphocytes

 It has been reported that the in vitro development of tumoricidal function in alveolar macrophages from lung cancer patients is reduced significantly when compared to that in peripheral blood monocytes from the same patients or alveolar macrophages from control patients. In the present investigation, a method for potentiating the development of tumoricidal function in alveolar macrophages from lung cancer patients is described. This method, which relies on priming the macrophages with purified, allogeneic peripheral blood lymphocytes from normal donors, could not be demonstrated when autologous lymphocytes from lung cancer patients were used in the priming coculture. The augmentation of tumoricidal function appears to be mediated by one or more soluble factors, since supernatants from cocultures of alveolar macrophages and allogeneic peripheral blood lymphocytes could enhance the cytotoxic function of freshly obtained alveolar macrophages. Furthermore, it appears that NK cells are necessary for this effect, since depletion of CD56⁺/CD57⁺ cells from allogeneic lymphocytes eliminated their capacity to enhance alveolar macrophage cytotoxic function. The augmentation of cytotoxic function elicited in alveolar macrophages by this method was not associated with changes in the secretion of tumor necrosis factor α, or interleukin 1β. Received: 15 March 1997 / Accepted: 11 June 1997     

Effect of L-arginine on the retention of macrophage tumoricidal activity

It has been reported that the tumoricidal activity of macrophages (M phi) depends on L-arginine and that L-arginine metabolites such as reactive nitrogen intermediates alter M phi physical capacities. The aim of this report is to investigate the dose-related effect of L-arginine on the expression and retention of M phi tumoricidal activity. Cytotoxicity of M phi activated by IFN-gamma plus LPS was detected in the presence of about 0.1 mM or more of L-arginine. This paralleled the NO2- production in the presence, but not in the absence, of L-arginine. On the other hand, activated M phi were destined to die and lost their tumoricidal activity with time in the presence of 0.3 mM or more L-arginine. They retained, however, considerable activity in the absence or presence of 0.15 mM L-arginine. This retention of M phi cytotoxicity was longer when M phi were preactivated by 100 ng/ml than 10 ng/ml of LPS in combination with IFN-gamma. Addition of indomethacin, an inhibitor of prostaglandin production, did not prevent the decay of M phi cytotoxicity but rather facilitated it even in the absence of L-arginine. Regardless of indomethacin, consecutive stimulation with LPS or LPS plus IFN-gamma during culture was effective in maintaining the tumoricidal activity at a high level. In addition, we found that M phi which had lost tumoricidal activity during culture in L-arginine deficient medium could be reactivated by LPS to attack tumor target cells.     

Phorbol esters and calcium ionophore can prime murine peritoneal macrophages for tumor cell destruction

Murine macrophages from sites of inflammation develop toward tumoricidal competence by exposure to a macrophage-activating factor such as interferon-gamma (IFN-gamma). To explore the biochemical transductional events initiated by IFN-gamma, peritoneal macrophages from C57BL/6J mice elicited by various sterile irritants were treated in vitro with two pharmacologic agents that mimic the action of certain second messengers. Phorbol myristate acetate (PMA) and the ionophore A23187 cooperatively reproduced the ability of IFN-gamma to prime macrophages for tumoricidal function. Neither agent alone was able to prime macrophages. The two agents acted on the macrophages, and target susceptibility to kill was not altered by PMA and A23187. Only active phorbol esters, which are known to bind and stimulate protein kinase C, were able to cooperate with A23187 to induce priming. A cell-permeable synthetic diacylglycerol (sn-1,2-dioctanoyl glycerol) could also prime for cytolysis. In the presence of PMA, A23187, and EGTA, addition of Ca++ was sufficient for priming, whereas the addition of Mg++ was much less efficient. Priming by IFN-gamma, however, was not blocked by EGTA. Efflux of 45Ca++ from preloaded cells was significantly increased by A23187 and by IFN-gamma. Quin-2/AM, an intracellular chelator of Ca++, blocked priming by IFN-gamma. In summary, the data suggest that priming of macrophages for tumoricidal function by IFN-gamma involves, at least in part, alterations in protein kinase C and in levels of intracellular Ca++.     

Activation of peritoneal macrophages by orally administered ether analogues of lysophospholipids.

Oral administration of dodecylglycerol, inflammatory product of cancerous tissues, and the alkyl lysophospholipid derivative, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3-choline), greatly activated mouse peritoneal macrophages. The activation was dose related and was assessed as increased Fc-mediated ingestion of red blood cells, superoxide production, chemiluminescence activity, and incorporation of radioactive thymidine and leucine. Furthermore, the data show that dodecylglycerol or ET-18-OCH3-choline was capable of inducing equally high levels of macrophage activation and cytotoxic action on tumor cells, just as occurs with intraperitoneal administration. Dodecylglycerol appeared to activate the macrophages at a relatively lower dose (5 micrograms/mouse) than ET-18-OCH3-choline (15 micrograms/mouse). The optimal oral doses required to activate macrophages for ingestion and cytotoxic activities were relatively higher than previously observed when these agents were administered intraperitoneally. Thus, the dose difference provided crucial information for correlating oral dosages with in vivo concentration of these agents as bioassayed by macrophage activation. These observations have extended and further support our earlier findings that these agents are effective immunopotentiators and thus could therapeutically be used to activate macrophages for cytotoxic effects on tumor cells via the oral route.     

Identification and characterization of monoclonal antibodies specific for macrophages at intermediate stages in the 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. A number of mediators have been identified which have activating capability, including in particular IFN-gamma and bacterial LPS. Although the synergistic functional response of normal macrophages to sequential incubation with these activation signals has been well-established, characterization of the intermediate stages in the activation pathway has been difficult. We have developed a model system for examination of various aspects of macrophage activation, through 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 IFN-gamma and LPS in the development of tumor cytolytic activity. In addition, these cells can be stably primed by the administration of gamma-radiation. In the studies reported here, we have used RAW 264.7 cells treated with IFN-gamma alone or with IFN-gamma plus LPS to stimulate the production of rat mAb probes recognizing cell surface changes occurring during the activation process. In this way we have identified three Ag associated with intermediate stages of the activation process. One Ag, TM-1, is expressed on RAW 264.7 cells primed by IFN-gamma or gamma-radiation. This surface Ag thus identifies cells at the primed cell intermediate stage of the tumoricidal activation pathway regardless of the mechanism of activation. A second Ag, TM-2, is expressed on IFN-treated RAW 264.7 cells but not on RAW 264.7 cells primed with gamma-radiation alone. Expression of this Ag can be induced by treatment of irradiated cells with IFN-gamma, but is not induced by IFN-gamma treatment of a noncytolytic cell line, WEHI-3. This Ag thus appears to be an IFN-inducible cell surface protein associated specifically with macrophage activation for tumoricidal activity. Finally, Ag TM-3 is detectable on RAW 264.7 cells primed by either IFN-gamma or gamma-radiation, after subsequent triggering of the primed cells with LPS. The addition of the mAb recognizing this antigen to the function assay of tumor cell killing can inhibit they lytic activity of both triggered cells. Thus, this Ag may play a role in the antitumor effector functions of activated macrophages. Overall, the results suggest that these mAb can serve as useful tools for identification of molecules associated with the process of macrophage activation for tumor cell killing.