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Continuous alveolar macrophage (AM) and tumor-infiltrated (TIM) cell lines have been generated from C57B16J mice by in vitro infection with the J2 retrovirus carrying the v-raf and v-myc oncogens. Four cloned AM cell lines (AMJ2-C8, AMJ2-C10, AMJ2-C11, AMJ2-C20) and 3 cloned TIM cell lines (TIMJ2-C4, TIMJ2-C7 and TIMJ2-C15) were expanded for further characterization. Flow cytometry detected the product of the raf gene in the cytoplasm of all these cell lines. Studies on the tumoricidal properties of these AM and TIM cell lines demonstrated differences in their response to a panel of known macrophage activators. Four of these cell lines (AMJ2-C8, AMJ2-C10, TIMJ2-C7 and TIMJ2-C15) were activated following exposure to recombinant murine interferon gamma (rMuIFN-gamma) but not lipopolysaccharide (LPS) or muramyl dipeptide (MDP). AMJ2-C20 was only activated by incubation with rMuIFN-gamma plus LPS. AMJ2-C11 and TIMJ2-C4 are the cell lines that most closely resembled the response pattern of the parental AM and TIM, since they could be activated by either the combination of rMuIFN-gamma plus LPS or rMuIFN-gamma plus MDP. Constitutive expression of MHC-class-II antigens was low on AMJ2-C11 or TIMJ2-C4 but was increased following exposure to rMuIFN-gamma. Neither cell line secreted substantial amounts of IL-1 or TNF but both secreted large amounts of IL-6. Thus these cell lines could be powerful tools to study AM and TIM activation and cytotoxicity.

Murine macrophages from different anatomical sites were compared for their ability to become tumoricidal and to secrete interleukin-1 (IL-1) and tumor necrosis factor (TNF) following stimulation in vitro by several biological response modifiers (BRM). Peritoneal macrophages (PM), alveolar macrophages (AM), and tumor-infiltrating-macrophages (TIM), isolated from B16F10 melanoma colonies in the lung, were incubated overnight with BRM [recombinant murine interferon gamma (rMulFN-gamma), lipopolysaccharide (LPS), muramyl dipeptide (MDP)], either alone or in combination. PM exhibited an increased cytotoxic response following incubation with LPS or rMuIFN-gamma but not with MDP. Both AM and TIM were induced to become tumoricidal following incubation with rMuIFN-gamma plus LPS or rMuIFN-gamma plus MDP but not after stimulation with any BRM alone; the level of cytotoxicity obtained with TIM incubated with rMuIFN-gamma plus LPS was slightly lower than that observed with PM or AM, while with rMuIFN-gamma plus MDP both AM and TIM had lower cytotoxicity than PM. Secretion of IL-I and TNF was observed in PM stimulated with LPS or MDP but not with rMuIFN-gamma. Likewise, secretion of IL-I by AM or TIM was also induced with LPS, although less than that obtained with PM. AM stimulated with LPS secreted larger amounts of TNF than PM while TIM secreted very low amounts of TNF. However, this result may be a consequence of the enzymatic isolation procedure used to obtain TIM since TNF secretion was also impaired in LPS-stimulated normal lung macrophages isolated by a similar enzymatic procedure, or enzyme-treated PM. Our results suggest that TIM obtained from lung metastases share certain functional characteristics with normal AM and respond to BRM in like manner with respect to induction of tumoricidal activity and cytokine secretion.

Inoculation of thioglycollate-elicited peritoneal exudate cells (PEC) into C57BL/6 mice reduced the rate of lung clearance of several intravenously (i.v.) injected murine tumor cells, and increased by up to 100-fold the number of artificially-induced metastatic lung nodules produced by the i.v. injection of B16 melanoma or Lewis lung carcinoma (3LL) tumor cells. Maximum effects were observed when PEC were injected either before, or shortly after, tumor cells. Modulation of lung clearance or metastasis formation was observed only with PEC and not with a variety of other cells, such as splenocytes, thymocytes P815 mastocytoma cells, or several macrophage-like cell lines (PU5-1.8 and IC-21). Lysates of PEC were as efficient in reducing lung clearance and augmenting metastasis formation as were intact viable PEC. Lysates of other cell types, including P815 and the macrophage-like cell lines, were unable to produce these effects. PEC populations, enriched for macrophages by adherence to plastic or by percoll density gradient sedimentation, also increased the number of B16-induced artificial metastasis, implicating the macrophage as the cell responsible for these observations.

The in vivo anti-tumor activity of 2 recombinant cytokines, interleukin-2 (rIL-2) and human hybrid interferon alpha (rHuIFN-alpha A/D), were tested using the murine reticulum cell sarcoma M5076. Experimental hepatic metastases, following i.v. injection of tumor cells, and tumor growth and spontaneous metastases, following s.c. injection of tumor cells, were inhibited to a greater extent in mice treated with a combination of these cytokines than in animals treated with either one alone. When used in conjunction with surgical removal of the s.c. tumor, treatment of mice with both cytokines significantly prolonged survival of tumor-bearing animals. Injection of normal mice with a combination of cytokines, but not with either cytokine alone, resulted in a marked increase in cytotoxic activity of hepatic effector cells. The effector cells in these mice appeared to be NK cells since this enhanced cytotoxicity was markedly reduced in animals treated in vivo with anti-asialo GM1 or in NK-deficient beige mice. Furthermore, no in vivo efficacy was observed in M5076-bearing beige mice treated with these cytokines. Thus, injection of mice with rIL-2 and rHuIFN-alpha A/D results in the induction of an NK-cell-like population in the liver with enhanced cytotoxic activity that correlates with the observed anti-tumor activity in vivo in this murine model. These results suggest that combinations of cytokines, in particular IFN-alpha and IL-2, can be effectively used in combination for the treatment of tumors and/or metastases.

The effect of a human recombinant hybrid alpha interferon (referred to as rHuIFN-alphaA/D) on pulmonary metastases induced by intravenous injection of B16 F10 melanoma cells in C57BL/6 mice was examined; rHuIFN-alphaA/D has been previously shown to have anti-viral, anti-proliferative and immunomodulatory activities in murine cells. Pretreatment of mice with 4 daily intraperitoneal injections of rHuIFN-alphaA/D resulted in a marked decrease in the number of pulmonary metastases. This inhibition was dose-dependent but was not seen when mice were similarly treated with rHuIFN-alphaA, a human recombinant alpha interferon subtype which is inactive on murine cells. Treatment of mice with rHuIFN-alphaA/D following B16 F10 injection resulted in no significant inhibition of pulmonary metastases. Mice given a similar treatment regimen of rHuIFN-alphaA/D had elevated natural killer (NK) cell activity as measured by in vitro cytotoxicity against YAC-I or in vivo pulmonary clearance of B16 F10 cells. Pretreatment of mice with 10 daily injections of rHuIFN-alphaA/D resulted in decreased NK activity and less inhibition of metastases. Therefore, in this model system, rHuIFN-alphaA/D inhibits metastases when given in the appropriate treatment schedule. Furthermore, the data are consistent with the hypothesis that rHuIFN-alphaA/D-induced inhibition is a consequence of the immunomodulation of NK cells, which prevent the establishment of pulmonary metastases.

The ability of recombinant interferons (IFNs) to modulate recombinant interleukin-2 (rIL-2) augmentation of natural killer (NK)-cell activity and to modulate the generation of activated killer (AK) cells was examined. Incubation of murine spleen cells for 18 hr with either human rIL-2 or a human hybrid recombinant IFN alpha, rHuIFN-alpha A/D, which is active on murine cells, resulted in a dose-dependent increase in NK activity; however, recombinant murine IFN gamma, rMuIFN-gamma, had little activity. A more than additive augmentation of cytotoxicity was obtained when spleen cells were incubated with the combination of rIL-2 and rHuIFN-alpha A/D. Incubation of murine spleen cells with rIL-2 for 3 days resulted in a dose-dependent induction of AK cells which were cytotoxic to an NK-resistant tumor target cell. In contrast to the results observed on NK activity, incubation of spleen cells with rHuIFN-alpha A/D and rIL-2 inhibited AK-cell activity. Partially purified murine IFN-alpha had inhibitory activity comparable to that of rHuIFN-alpha A/D. The addition of rHuIFN-alpha A/D at the initiation of culture of spleen cells with rIL-2 (day 0) resulted in maximal inhibition of cytotoxicity; inhibition was reduced or absent if rHuIFN-alpha A/D was added on day 1 or 2 of culture. The proliferation of spleen cells incubated with rIL-2 was also inhibited by rHuIFN-alpha A/D. Addition of rMuIFN-gamma to spleen-cells and rIL-2 increased the cytolytic activity of AK cells and did not inhibit rIL-2-induced proliferation of spleen cells. Similar data were also obtained with human peripheral blood lymphocytes and recombinant cytokines. Incubation of human peripheral blood lymphocytes with rIL-2 and recombinant human IFN-alpha A (rHuIFN-alpha A) or recombinant human IFN-gamma (rHuIFN-gamma) resulted in a more than additive increase in NK activity. Human AK-cell cytotoxicity was inhibited by rHuIFN-alpha A but enhanced by rHuIFN-gamma. Thus recombinant IFNs have differential effects on rIL-2-induced cytotoxic cells, resulting in augmentation or inhibition of activity, which is dependent on both the type of IFN and the cytotoxic activity examined. These results may have important implications for the potential therapeutic use of combinations of these cytokines.

Several murine tumor-cell lines were tested by isotope release assays for their susceptibility to lysis by either activated peritoneal macrophages (apMPh), macrophage-like (MPh-like) cell lines, or natural killer (NK) cells. The qualitative selectivity of tumor-cell lysis by these different effector cells was quite disparate. The rank order of target cell susceptibility to lysis by apMPh in 24 h assay was L5178Y greater than P815 approximately equal to RL male greater than YAC-1 approximately equal to MBL-2. This was seen regardless of whether peritoneal MPh (pMPh) were activated by LPS or poly I:C. Two MPh-like cell lines, PU-5R and FC-1, had a pattern of cytotoxic activity against these target cells that closely paralleled that associated with apMPh, although levels of reactivity differed quantitatively among the effector cells. In contrast, the MPh-like cell line RAW-264 expressed a qualitatively different pattern of target-cell selectivity, preferentially lysing MBL-2, which was relatively refractory to lysis by other MPh-like cell lines or apMPh in the 24 h cytolytic assay. When spontaneous or interferon (IFN)-augmented NK activity was measured against the same panel of target cells, the pattern of selectivity was qualitatively different from that observed for apMPh. The consistent rank order of susceptibility to lysis by NK cells was YAC-1 greater than RL male 1 greater than P815 approximately equal to L5178Y approximately equal to MBL-2. The characteristic patterns of target-cell selectivity for apMPh or NK cells were the same for all of the strains of mice tested. From the different selectivity patterns of apMPh and NK cells, it is concluded that lysis of target cells is not based solely on inherent sensitivity to cytolysis. Instead, selectivity of lysis is probably due to variations in expression of target-cell structures recognized by each type of effector cell, and/or in susceptibility to the lytic mechanism(s) of the various effector populations.

A series of monosaccharides were tested for their ability to inhibit the effector phase of macrophage-mediated cytolysis against two susceptible murine tumor target cells, L5178Y and RL male I. Two monosaccharides, D-mannose and N-acetyl-D-galactosamine, were found to decrease cytotoxicity consistently in a dose-dependent manner. However, D-mannose preferentially inhibited lysis of RL male I target cells with little effect on lysis of L5178Y target cells, while the reverse was found with N-acetyl-D-galactosamine. Neither monosaccharide interfered with the activation of macrophages by polyinosinic:polycytidylic acid. Natural killer cell activity was decreased by a 25 mM concentration of D-mannose but not by N-acetyl-D-galactosamine, although increasing concentrations of N-acetyl-D-galactosamine were inhibitory. Neither monosaccharide affected cytotoxicity by alloimmune T cells. Cytotoxicity of macrophage-like cell lines against tumor target cells was also decreased by monosaccharides but the pattern of inhibition was different from that seen with activated macrophage effector cells. Both D-mannose and N-acetyl-D-galactosamine inhibited glucose oxidation by activated macrophages but only D-mannose significantly decreased protein synthesis of activated macrophages. These results indicate that monosaccharides can inhibit macrophage-mediated cytotoxicity in a selective manner with the pattern dependent on the tumor target cell used in the assay.

Antibodies reactive with effector cells were shown to augment the cytotoxicity of spleen cells from athymic nude and euthymic mice. The addition of alloantibody to the assay or pretreatment of the effector cells with alloantibody resulted in increased cytotoxicity against the human cell K562, a relatively poor target for spontaneous mouse NK activity. When monoclonal antibodies were tested, cytotoxicity was markedly increased by some antibodies, such as anti-H-2, anti-la, and anti-Thy 1.2, while others had no effect. The degree of augmentation of cytotoxicity was dependent on the concentration of antibody added. Nylon-wool-nonadherent nu/nu splenic effector cells mediated the antibody-induced cytotoxicity and anti-asialo GMI plus complement abolished activity, indicating that the cells mediating the cytotoxicity were NK cells and not mature T cells, B cells or macrophages. When spleen cells from mice having different levels of NK activity were evaluated in this system, the magnitude of augmentation by antibody correlated with the level of spontaneous NK activity and no increased cytotoxicity was found with cell populations that had low spontaneous NK activity. Testing a panel of target cells, showed that certain human and mouse cell lines, with low to moderate susceptibility to spontaneous NK activity, were sensitive to antibody-induced NK-cell-mediated cytotoxicity whereas others were completely resistant. Both Fc-IgG receptor-positive and -negative cell lines were susceptible target cells. These results indicate that antibodies reactive with murine NK cells can increase their cytolytic activity.

The in vivo anti-tumor effects of a recombinant human hybrid interferon alpha, rHuIFN-alpha A/D, and recombinant murine interferon gamma (rMuIFN-gamma) were evaluated against experimental hepatic metastases and s.c. tumor growth of the murine reticulum cell sarcoma M5076. The 2 interferons were equally active in preventing experimental hepatic metastases. However, the interferons differed in their relative ability to influence the growth of the same tumor when treatment was initiated following injection of tumor cells. Greater efficacy was obtained in the treatment of metastatic foci in the liver with rHuIFN-alpha A/D, while rMuIFN-gamma was more active in the therapy of an s.c. growing M5076 tumor. These results demonstrate that the same tumor growing at different sites can have different relative sensitivities to IFN-alpha and IFN-gamma.

The in vitro antiproliferative effects of recombinant interferons (IFNs) alpha and gamma against the murine reticulum-cell sarcoma M5076 and the malignant melanoma B16 F10 were evaluated using the human hybrid IFN-alpha A/D (rHuIFN-alpha A/D), which is active on murine cells, and recombinant murine IFN gamma (rMuIFN-gamma). An isobologram analysis was used to evaluate the interactive antiproliferative effects of the recombinant IFNs on these two tumor cell lines. The data, in contrast to prior reports, indicate that rHuIFN-alpha A/D and rMuIFN-gamma interact in an additive rather than a synergistic manner against M5076 cells. When a similar analysis was performed on B16 F10 cells, synergy was obtained. Thus, either a synergistic or an additive antiproliferative effect can be obtained by combining IFN-alpha and IFN-gamma, depending upon the cell line used in the assay.