Apoprotein E is synthesized and secreted by resident and thioglycollate-elicited macrophages but not by pyran copolymer- or bacillus Calmette-Guerin-activated macrophages.

Macrophages are active secretory cells that display functionally distinct phenotypes that are regulated by inflammation. We have found that apoprotein E (ApoE), a component of plasma lipoproteins, was synthesized and secreted by resident and nonspecifically stimulated macrophages elicited with thioglycollate broth, but not by activated macrophages obtained from mice treated with bacillus Calmette-Guerin, pyran copolymer, whole Corynebacterium parvum, or bacterial endotoxin. ApoE represented approximately 1% of the newly synthesized protein and approximately 10% of secreted protein of resident and thioglycollate-elicited macrophages. ApoE from thioglycollate-elicited macrophages was indistinguishable from ApoE in mouse plasma lipoproteins, as determined by immunoreactivity, peptide mapping, and molecular weight. When specific antibodies were used to localize cell-associated ApoE, strong immunofluorescence was seen in the Golgi region of resident and thioglycollate-elicited macrophages immediately after removal from the peritoneal cavity, as well as after culture for up to 7 d. In contrast, activated macrophages did not synthesize or secrete ApoE to an appreciable extent and had no immunocytochemically detectable intracellular ApoE. When activated macrophages were cultured in medium containing serum, their activated state, as judged by production of H2O2, declined within 48-72 h in parallel with the induction of synthesis and secretion of ApoE and detection of intracellular ApoE by immunofluorescence. During prolonged culture the rate of synthesis and secretion of ApoE increased in both resident and activated macrophages. Therefore, the synthesis and secretion of ApoE may serve as markers for the functional state of macrophages.

Augmentation of organ-associated natural killer activity by biological response modifiers. Isolation and characterization of large granular lymphocytes from the liver.

Natural killer (NK) activity in the rat and human has been attributed to cells having the morphology of large granular lymphocytes (LGL). However, this association has been less clear in the mouse, largely because of difficulties in obtaining highly enriched populations of LGL from normal spleen and blood. We have previously observed that the administration of the biological response modifier (BRM) maleic anhydride divinyl ether (MVE-2) strongly augmented NK activity in lung and liver, and the augmented NK activity coincided with increased resistance to the formation of experimental metastases in these organs. The degree of NK augmentation was most striking in the liver, an unexpected and previously unreported observation. In the present study, both MVE-2 or Corynebacterium parvum induced a dramatic augmentation of liver NK activity, which reached maximum levels 3-5 d after treatment. This augmentation of NK activity in the liver coincided with a large increase in the number of lymphoid cells with the morphological characteristics of LGL that could be isolated from enzymatically digested suspensions of perfused liver. The yield of LGL per liver following BRM treatment corresponded to a 10-50-fold increase as compared to normal mice. LGL were purified from these enzymatically digested suspensions of perfused liver by depletion of adherent cells on nylon wool columns and subsequent enrichment for low-density lymphoid cells by fractionation on Percoll density gradients. The enrichment of LGL correlated with greatly increased NK activity against YAC-1. Conversely, the higher-density fractions were depleted of both LGL and NK activity. This increase in NK activity in the liver was suppressed by in vivo treatment with anti-asialo GM1 (asGM1) serum. This treatment also resulted in a corresponding reduction in both the total number and percentage of LGL. By flow cytometry analysis, the phenotype of the majority of these highly cytolytic LGL isolated from the livers of BRM-treated mice were asGM1+, Thy-1+, Ly-5+, Qa-5+, Mac-1+, and Gma-1+, whereas these LGL were Ly-1-, Lyt-2-, L3T4-, and surface Ig-. We conclude that the livers of BRM-treated mice can provide a rich source of highly active mouse LGL that could be used for further characterization of this lymphocyte subset. Further, these studies imply a potential for BRM therapy of neoplastic or viral diseases through augmentation of organ-associated immune responses.

In vitro inhibitory effects of polymer-linked methotrexate derivatives on tetrahydrofolate dehydrogenase and murine L5178Y cells.

In vitro studies were made on four synthetic polymeric derivatives of the antitumor agent methotrexate (MTX): 1) divinylether-maleic anhydride-MTX (DIVEMA-MTX), 2) poly-L-lysine-MTX (PL-MTX), 3) polyethyleneimine-MTX (PEI-MTX), and 4) carboxymethyl cellulose-MTX (CMC-MTX). They were tested for their ability to inhibit tetrahydrofolate dehydrogenase (dihydrofolate reductase). Their growth inhibition of murine L5178Y leukemia cells was also studied. 1wo of these polymers, DIVEMA-MTX and PEI-MTX, had similar or only slightly reduced activity compared to equivalent concentrations of MTX, whereas PL-MTX and CMC-MTX had significantly higher (1--3 logs) minimal inhibitory concentrations.

Effect of immunomodulators on effector cells involved in antibody-dependent cellular cytotoxicity: Brief communication.

The effect of four immunomodulators (BCG, Corynebacterium parvum, pyran copolymer, and levamisole) on the cellular arm of antibody-dependent cellular cytotoxicity (ADCC) was investigated in mice with 51Cr-labeled chicken erythrocytes employed as targets. All these drugs, except levamisole, stimulated the effector cells of ADCC in the spleen, but the kinetics of their effect differed. Stimulation of the effector cells of ADCC peaked on day 15 after injection of BCG and C. parvum and on day 7 after injection of pyran, which was less efficient in this respect than the two bacterial immunostimulants. The increase in ADCC activity caused by BCG and C. parvum was eliminated by treatment with carbonyl iron of the splenocyte suspensions.

Role of divalent ion complex formation in pyran--inhibition of nucleic acid biosynthesis.

The degree of inhibition of mammalian DNA-dependent RNA polymerases I and II and Moloney leukemia virus RNA-dependent DNA polymerase by pyran copolymer was dependent on the concentration of the divalent cation cofactor in the reaction mixture. Inhibition was completely blocked by an excess of divalent cations. It was concluded that pyran inhibited these enzymes by complexing with the essential divalent cation cofactor.

Specific potentiation of L1210 vaccine by pyran copolymer.

Pyran copolymer (NSC 46015) was found to potentiate strongly the immune response of C57BL/6J X DBA/2 F1 mice to 10(4) live L1210 tumor cells following suboptimal vaccination with 10(7) radiation-inactivated L1210 cells. Optimal immunity to challenge was produced by concomitant i.p administration of pyran and L1210 vaccine, and activity was dependent upon both pyran and vaccine dosages. In addition, this immunopotentiation seemed to be related to the intrinsic viscosity of different pyran preparations tested, although all the pyran compounds had significant activity. Furthermore, the increased immunity of subsequent live tumor challenge appeared to be specific for the vaccinating cell type.

Chemical and biological adjuvants capable of potentiating tumor cell vaccine.

With an L1210 tumor vaccine model, three biological and two chemical agents were tested for their ability to act as adjuvants. Adjuvant was administered with irradiated L1210 cells to immunize mice against this poorly immunogenic tumor. Two chemicals, pyran copolymer and glucan, and one biological, Brucella abortus strain 456 ether extract, were shown to be strong stimulators of antitumor immunity. Vaccination with irradiated tumor cells or adjuvant alone did not produce host resistance. Optimal immunity to challenge was produced by concomitant administration of either pyran copolymer, glucan, or B. abortus strain 456 ether extract with L1210 vaccine. Antitumor immunity was maximally expressed when vaccine and adjuvant were administered i.p. Evidence for systemic immunity was demonstrated when challenge was at a distal s.c. site. Mice immune to challenge were found to be refractory to a later rechallenge.

Maleic vinyl ether activation of murine macrophages against lung-metastasizing tumors.

A 16,500 molecular weight fraction of maleic vinyl ether (MVE-2) induced tumoristatic and tumoricidal activity in peritoneal macrophages of BALB/c and C57BL/6 mice following i.p. administration. Growth of B16 melanoma cells in vitro was inhibited up to 85% by MVE-2-activated, but not resident, peritoneal macrophages. In a tritiated thymidine release assay, B16 melanoma cells, and to a lesser extent Madison 109 lung carcinoma cells, were also sensitive to the cytolytic action of MVE-2-activated peritoneal macrophages. Administration i.v. of MVE-2 resulted in tumoristatic and tumoricidal activity in alveolar macrophages against radiolabeled B16 and Madison 109 lung carcinoma target cells. MVE-2-activated alveolar macrophages significantly inhibited L5178Y lymphoma colony formation following a 48-hr macrophage-tumor cell coincubation. BALB/c mice bearing the lung-metastasizing Madison 109 lung carcinoma footpad tumor were given MVE-2 i.v., using the same dosing regimen that induced alveolar macrophages to be tumoricidal in vitro. Significant increases in life span were observed, suggesting that the antitumor activity of MVE-2 in this tumor system may be mediated by the activation of alveolar macrophages, with a resulting decrease in metastatic growth in the lung.

Effect of pyran copolymer on activation of murine macrophages: evidence for incomplete activation by use of functional markers.

The degree of activation of peritoneal macrophages elicited by pyran copolymer (MVE-2) was studied in C57BL/6J mice. When cytotoxicity was examined under endotoxin-free culture conditions, the pyran-elicited macrophages could not complete cytolysis of tumor target cells. The macrophages, however, completed cytolysis when pulsed with endotoxin. These results were obtained when either the interval between injection of the pyran copolymer and harvest of the macrophage or the dose of pyran was varied. The pyran-elicited macrophages expressed five markers considered to be typical of inflammatory macrophages, and bound tumor cells to an augmented degree. The pyran-elicited macrophages were capable of secreting a potent cytolytic proteinase when pulsed with endotoxin, but did not secrete cytolytic proteinase spontaneously. The pyran-elicited macrophages, in contrast to inflammatory macrophages, could effect cytostasis of tumor cells; their cytostatic potential was also augmented by addition of endotoxin. Taken together, the results indicated that pyran copolymer elicits primed but not fully activated murine macrophages.

Effects of adriamycin and cyclophosphamide treatment on induction of macrophage cytotoxic function in mice.

The effects of i.p. and s.c. Adriamycin and cyclophosphamide treatment of BALB/c x DBA/2F1 mice were studied alone and in combination with immunotherapeutic agents, pyran copolymer and Bacillus Calmette-Guérin, on macrophage cytotoxic ability, As assessed by direct viable cell counts of MBL-2 leukemia cells, both Adriamycin and cyclophosphamide produced growth-inhibitory macrophages. This function after s.c. cytostatic treatment peaked at Day 1 and decreased progressively, attaining normal control values by Day 6. When adjuvants, such as pyran and B. Calmette-Guérin, were administered i.p. simultaneously with s.c. Adriamycin or cyclophosphamide, adjuvant-induced cytotoxic function was not markedly affected. A better knowledge of the influence of cytostatic agents alone or combined with immunoadjuvants on macrophage cytotoxic ability may be useful in designing more effective chemoimmunotherapy protocols.

Association of macrophage activation with antitumor activity by synthetic and biological agents.

Treatment of normal BALB/c mice i.p. with a number of adjuvants, including pyran copolymer, the copolymer of polyinosinic and polycytidylic acids, Bacillus Calmette-Guérin, glucan, and dextran sulfate, rendered macrophages nonspecifically cytostatic for syngeneic tumor cells. Macrophage activation was highly dose dependent. The validity of the inhibition of DNA synthesis assay for measuring macrophage-induced cytostasis of target cells was proven by demonstrating a concurrent decrease in RNA synthesis and a reduction in viable tumor cell number. Moreover, conditioned supernatants from pyran-activated macrophages did not significantly decrease [3H]thymidine incorporation by freshly added leukemia cells. Biological or synthetic agents that activated macrophages were generally effective systemic antitumor agents against the M109 lung carcinoma. Drugs that did not activate macrophages, such as typhoid vaccine, tilorone, levamisole, WY-13876, and thymosin, were ineffective in prolonging the life of tumor-bearing mice. Pyran treatment i.p. was the most effective antitumor adjuvant in two separate tumor models, and suppression of tumor growth appeared to be related not only to an increase in macrophage tumoricidal function, but also to a larger influx of macrophages responding at the tumor site.

Enhancing activity of various immunoaugmenting agents on the delayed-type hypersensitivity response in mice.

Six immunoaugmenting agents were tested in the delayed-type hypersensitivity reaction (DTH) in normal BALB/c X DBA/2 mice. The agents tested, levan, lentinan, mannozym, maleic anhydride divinyl ether, polyriboinosinic-polycytidylic acid-poly-L-lysine, and highly purified L-cell interferon, gave significant increases in the DTH response above the sheep red blood cell control. The schedule of doses for each agent corresponded with previous experiments from this laboratory of the maximum natural killer cell activity, macrophage activation, and interferon induction. Highly purified L-cell interferon was capable of eliciting a significant DTH response when given 4 hr after the initial challenge with sheep erythrocytes. In addition, lambda-carrageenan, a macrophage-cytotoxic agent which can render the macrophage inactive, was found to suppress the DTH response to levels slightly above phosphate-buffered saline controls. The carrageenan-induced suppression of the DTH response could be abrogated by coadministration with immunoaugmenting agents to levels attained with the immunoaugmenting agents alone.

Immune response of BALB/c X DBA/2F1 mice to a tumor allograft during pyran copolymer-induced tumor enhancement.

The immune response of BALB/c x DBA/2 F1 mice to a transplantable Moloney leukemia virus-induced tumor allograft (MBL-2) was studied to determine the mechanism of pyran copolymer-induced tumor enhancement. The relative levels of humoral, lymphocyte, and macrophage response were followed chronologically by in vitro cytotoxic microassays using 51Cr-labeled target cells. Although pyran increased the titer of humoral cytotoxic antibody, levels of humoral factors capable of abrogating lymphocytoxicity were not enhanced. Furthermore, splenic lymphocyte-mediated cytotoxicity, although slightly diminished in pyran-treated mice, was not significantly affected. Macrophages harvested from allograft-bearing animals exhibited marked tumoricidal activity, which was augmented by pyran treatment. This macrophage-associated activity was specific for MBL-2 cells and not attributable to cytotoxins elaborated into the culture medium. Pyran slightly activated macrophages from nonsensitized mice to become cytotoxic for MBL-2 cells; activation was not T-cell dependent. However, strikingly fewer macrophages infiltrated the allograft in pyran-treated animals as judged by both histopathology and direct measurement. The defect in the migration or deposit of macrophages at the allograft site may have contributed to tumor enhancement.

Cellular and serum involvement in protection against Friend leukemia virus.

Treatment of mice with the immunomodulator pyran copolymer inhibited leukemogenesis produced by Friend leukemia virus (FLV) complex, as evidenced by inhibition of the spleen focus-forming virus and lymphatic leukemia virus, as well as by a significant decrease in splenomegaly. In this report we present data suggesting that the protective effect of pyran is mediated by macrophages. Protection was conferred on normal recipient mice when peritoneal exudate cells from pyran-treated mice were transferred to recipient mice infected 24 hr later with FLV. Animals receiving pyran-activated peritoneal cells had a significant reduction of splenomegaly and of titers of spleen focus-forming virus and lymphatic leukemia virus than did control animals. In contrast, when glycogen-elicited peritoneal exudate cells were transferred, the mice were not protected. Pyran-activated peritoneal cells, but not normal peritoneal cells, also inhibited FLV growth in vitro. Serum from pyran-treated, but not glycogen-treated, mice also transferred resistance to FLV-infected mice.

In vivo modulation of myelopoiesis and immune functions by maleic anhydride divinyl ether copolymer (MVE-2) in tumor-free and MBL-2 tumor-bearing mice treated with cyclophosphamide.

Treatment of normal or MBL-2 tumor-bearing mice with cyclophosphamide (CY) caused severe suppression of myelopoiesis and macrophage (M phi) functions, both of which may limit further use of chemotherapy. Additional treatment with the chemically defined biological response modifier maleic anhydride divinyl ether copolymer (MVE-2) was able to ameliorate the myelosuppressive effects of CY and to restore normal bone marrow cellularity. The stimulatory effects on myelopoiesis, however, could only be obtained by administering MVE-2 at greater than or equal to 3 days after CY, which correlated with an MVE-2-induced simultaneous increase in granulocyte and/or macrophage colony-stimulating factor secretion by bone marrow cells or M phi. Injection of MBL-2 tumor-bearing mice with MVE-2, at 3 days after Cy treatment, caused a decrease in tumor burden and a significant increase in median survival time as compared to treatment with CY alone. At the same time, MVE-2 induced an increase in the number of cytotoxic M phi and a complete restoration within the myelopoietic lineage, which might prevent delayed side effects of CY, such as secondary infections, and might permit more intensive chemotherapeutic treatment. Treatment of MBL-2 tumor-bearing mice with MVE-2, at 6 days after CY, induced a significant increase in M phi cytotoxicity but did not prolong median survival time, probably due to a rapid regrowth of tumor after treatment with CY. Our studies thus show that successful combined therapy with the primary cytotoxic agent CY and the biological response modifier MVE-2 depends on precise timing of the drug regimen and is influenced by the extent and reversibility of CY-induced immunosuppression, as well as by the kinetics of recruitment of new effector cells from bone marrow and by the tumor burden present at the time of treatment.

Macrophage involvement in the protective effect of pyran copolymer against the Madison lung carcinoma (M109).

Pyran copolymer (NSC 46015) therapy markedly enhanced host resistance to a murine lung carcinoma (M109) implanted s.c. Multiple dose schedules were not significantly better than single doses at increasing lifespan. Although tumor necrosis was much more extensive in the lesions of pyran-treated mice, pyran copolymer was not directly toxic to M109 cells in vitro. A comparative histopathological study revealed an intense histiocytic reaction in the connective tissue surrounding the primary tumor in mice receiving pyran as compared to 0.9% NaCl solution-treated controls. Macrophages were often associated with necrobiotic tumor cells. Morphologically activated macrophages were recovered from pyran-treated animals which potently inhibited DNA synthesis of M109 tumor cells in vitro. This response peaked 6 days after drug treatment and was to a large extent specific for neoplastic cells. Our results from both in vivo and in vitro studies support the concept that pyran enhances host resistance to neoplasia by mobilization and activation of the reticuloendothelial elements of the host's defense.

Presence of DNA polymerase in lymphosarcoma in northern pike (Esox lucius).

Northern poke lymphosarcoma DNA polymerase was partially purified from particulate fractions banding at 1.15 to 1.16 g/ml from homogenates prepared from frozen necropsies of tumor-bearing pike. The enzyme behaves as a typical reverse transcriptase, in that it prefers ribotemplates to deoxytemplates. The isoelectric point (pl 5.5) is similar to that of avian myeloblastosis virus polymerase. The pike enzyme elutes from a phosphocellulose column at 0.22 M potassium phosphate, the same as avian myeloblastosis virus DNA polymerase. The enzyme activity is inhibited by pyran, a specific inhibitor of viral DNA polymerases. The most striking difference between the pike lymphoma polymerase and the other viral DNA polymerases tested is the low maximum temperature of 20 degrees, compared to 30 degrees for Rauscher leukemia virus polymerase and 38 degrees for avian myeloblastosis virus and Rous sarcoma virus.

Inability of anti-asialo-GM1 and 2-chloroadenosine to abrogate maleic anhydride-divinyl ether-induced resistance against experimental murine lung carcinoma metastases.

Both macrophages and natural killer cells have been implicated in the antimetastatic activity of maleic anhydride-divinyl ether (MVE-5). In the present study, we attempted to utilize anti-asialo-GM1 antibody and 2-chloroadenosine, agents that kill natural killer (NK) cells and macrophages, respectively, to determine the relative contribution of each effector cell type to the overall host defense. These agents were tested in the M109 lung metastasis model in syngeneic BALB/c mice, and the cytotoxic activities of both peritoneal macrophages and splenic NK cells were followed. The most profound antitumor effect was observed when MVE-5 was given before rather than after i.v. tumor inoculation. Treatment i.p. with MVE-5 at 20 mg/kg produced greater than 98% inhibition of subsequent lung metastases when given 2 days prior to tumor. Anti-asialo-GM1 antibody (25 mg/kg, i.p.) and 2-chloroadenosine (50 mg/kg, i.p.) were administered concurrently with MVE-5. Although each agent exhibited greater selectivity for its respective target, the early (Day 2) inhibitory response was nonspecific. By Day 5 after MVE-5 treatment, 2-chloroadenosine only inhibited macrophage tumoricidal activity, and conversely, anti-asialo-GM1 antibody only inhibited NK reactivity. Despite the ability of these agents to increase survival of metastases in control animals, they only slightly abrogated the antimetastatic activity of MVE-5. Our data suggest that caution should be exercised in using these agents to discriminate macrophage and NK responses.

Role of macrophages in the immunotherapy of Lewis lung peritoneal carcinomatosis.

The underlying cellular mechanisms for the antitumor effects of biological response modifiers (BRMs) have not been clearly resolved. We have investigated this issue in the Lewis lung (3LL) peritoneal carcinomatosis model in which treatment with the BRM MVE-2 slows tumor growth and enhances survival. MVE-2 is a potent inducer of cytotoxic macrophages (m phi s); however, in the vivo tumoricidal properties of these m phi s remain to be firmly established. To directly establish that m phi s were at least in part responsible for the in vivo efficacy of MVE-2, a novel method of obtaining highly enriched m phi suspensions was developed which gave high purity, satisfactory yield, and excellent viability without affecting antitumor activity. Using the 3LL peritoneal carcinomatosis model and adoptive transfer techniques, we directly demonstrate that the majority of antitumor activity was associated with the adherent cell fraction enriched for m phi s. Histological observations supported this conclusion, indicating that MVE-2 treatment initially activated cells associated with nonspecific immunity, retarding tumor growth in the ascites long enough for a multifaceted immune response to develop.

Selective depletion of liver and splenic macrophages using liposomes encapsulating the drug dichloromethylene diphosphonate: effects on antimicrobial resistance.

The current results provide direct evidence for a role of tissue macrophages (M phi) in natural immunity and support the use of immunomodulators to enhance antiviral resistance in immunocompromised individuals. In this study, macrophages (M phi) in the spleen and liver were eliminated by intravenous (i.v.) injection of the drug dichloromethylene diphosphonate (DMDP) encapsulated in liposomes. The effect of this depletion system on peritoneal M phi, peripheral blood leukocytes, splenic natural killer (NK) activity, and natural and immunomodulator-induced host resistance was then assessed. Barrier-maintained CD-1 female mice were inoculated i.v. either with DMDP liposomes, free liposomes (containing no DMDP), or saline on day -2 or on days -3 and -1 before cell population analysis or infection. Single or double treatment with DMDP liposomes had no effect on peritoneal M phi as indicated by no changes in total number, differential counts, or ectoenzyme patterns. Double treatment with DMDP liposomes caused a marked leukocytosis in blood, primarily of lymphocytes and polymorphonuclear leukocytes (PMN), and a transient depression of spontaneous and interferon-inducible splenic NK activity. The effects on host resistance to i.v. infection with Listeria monocytogenes or herpes simplex virus type 2 (HSV-2) indicated that i.v. treatment with DMDP liposomes significantly reduced natural resistance to these microorganisms as evidenced by increased mortality and decreased median survival time. When DMDP liposomes-treated mice were given the immunomodulator maleic anhydride divinyl ether copolymer (MVE-2) intraperitoneally the day before infection with HSV-2, the immunosuppressive effect of DMDP liposomes treatment was significantly reversed.