Cellular basis for adriamycin-induced augmentation of cell-mediated cytotoxicity in culture.

The cellular basis for the augmented cell-mediated cytotoxic (CMC) response seen when spleen cells from Adriamycin (ADM)-treated mice were stimulated in culture was investigated. Under conditions where mature macrophages were reduced (adherent or silica-sensitive cells removed) at time of alloantigen challenge, the cells from ADM-treated mice developed levels of CMC activity much higher than the low levels which were developed by similar subsets of cells from nontreated control mice. This indicates that ADM treatment enriched a subset of cells in spleen which was nonadherent, silica-insensitive, and nonphagocytic but was capable of providing accessory function. When mature macrophages were not removed, the capability to develop an augmented level of CMC was shown to be associated with a subset of cells from ADM-treated mice which was adherent to either plastic or nylon wool. In recombination experiments, it was found that the removal of Thy 1.2+ cells from the adherent subset from ADM-treated mice had little effect on the response, while their removal from the adherent subset from nontreated mice resulted in elevated levels of response. A similar effect was obtained when Lyt 2.2+ cells were eliminated but not when Lyt 1.2+ cells were removed. This indicates that a Thy 1.2+, Lyt 1-2+ cell, involved in regulation of the response, was missing from or failed to function in the ADM-treated population. Based on these findings, ADM apparently induces modifications in two cell subsets: (a) immature cells of the monocyte/macrophage lineage which can provide accessory function; and (b) adherent, Lyt 2+ T-cells which cooperate in maintaining levels of CMC activity at "normal" levels.

Correlation between adriamycin-induced augmentation of interleukin 2 production and of cell-mediated cytotoxicity in mice.

Based on the observation that spleen cells from Adriamycin-treated mice could develop augmented levels of cytotoxic T-lymphocyte activity in response to heat-treated and/or X-irradiated alloantigens, it was postulated that modulations in soluble mediators could be involved in this phenomenon. In fact, in this study Adriamycin-induced increases in the levels of prostaglandin E2 and interleukin 2 activity have been observed with isolated cells. The "interleukin 2-like" activity was indistinguishable from that of partially purified interleukin 2 in terms of ability to restore responsiveness to experimentally inhibited primary alloantigen response cultures and to maintain long-term cultures of activated T-cells. Furthermore this latter activity was completely ablated by antiinterleukin 2 monoclonal antibody. While the modification in prostaglandin E2 production did not appear to play a role in determining augmentation of cytotoxic T-cell activity, the modification in interleukin 2 production was consistent with the possibility that this is a primary mechanism of Adriamycin-induced augmented cell-mediated cytotoxicity.

Application of the polymerase chain reaction (PCR) to quantify micro-metastasis in an experimental animal.

In vitro cultured r/mHM-SFME-1 cells were injected into the hind foot pads of Balb/c mice. Metastasis was detected in the lungs of tumor-bearing mice by means of both PCR and histological methods. Primers for the PCR were set to amplify a 128 bp exon-1 sequence of the human c-Ha-ras1 gene which had been introduced into the cells. Resulted PCR bands were densitometorically quantified using a bioimage analyzer, and more than 1 x 10(4) tumor cells were detectable in the mouse lung. The number of tumor cells per lung estimated from the amount of PCR products was 1 x 10(5), 15 x 10(5), 1 x 10(5) and 40 x 10(5) on days 7, 14, 21 and 28 respectively after the tumor injection. No metastases were histologically observed on days 7 and 14. Then, the possibility of using this model system for evaluation of a treatment against micro-metastases is discussed.

Comparison of the biological activity of synthetic N-acylated asparagine or serine linked monosaccharide lipid A analogs.

The mitogenicity, lethal toxicity, induction of tumor necrosis factor (TNF), production of nitric oxide (NO) and antitumor activity against Meth A fibrosarcoma by chemically synthesized N-acylated asparagine-linked (A-701, A-702 and A-703) or N-acylated serine-linked (A-607) nonphosphorylated acylglucosamine and 4-0-phosphorylated acylglucosamine (A-103) derived lipid A analogs were determined. compound A-607 (with tetradecanoyl and (R)-3-tetradecanoyloxytetradecanoyl at the C-2 and C-3 positions) induced a significant incorporation of 3H-thymidine into splenocytes of C3H/He mice at concentrations ranging from 3.13 to 50 microM, but the mitogenic activity of A-701 (2-N-acetylglucosamine), A-702 (tetradecanoyl at the C-2), and A-703 (with (R)-tetradecanoyloxytetradecanoyl and tetradecanoyl at the C-2 and C-3) was very weak. The lethality of A-703 and A-103 (with (R)-3-tetradecanoyloxytetradecanoyl at the C-2 and C-3) was weaker than that of A-607 at doses of 300 and 750 nmol/kg in C57BL/6 mice loaded with D-galactosamine. Peritoneal macrophages, stimulated with A-701-A-703, caused production of TNF which induce L929 cell lysis in vitro, and A-703 showed a high production of TNF. The compounds, except for A-607, exhibited little NO production by macrophages, but did induce the NO production in the presence of interferon gamma. Induction of TNF and NO inducible activity by A-703 was lower than that of A-607. A-703, A-607 and A-103 showed antitumor activity against Meth A fibrosarcoma in BALB/c mice. When A-703 or A-103 with muramyl dipeptide was administered, A-703 failed to show combined effects, but A-103 did. We concluded from these findings that the biological potency of asparagine compounds appears to be placed between serine- and amino-free compounds.

Selective imbalances of cellular immune responses by adriamycin.

It has been demonstrated that spleen cells from mice treated with adriamycin not only develop an increased cell-mediated immune response during culture with allogeneic tumor cells, but also have increased phagocytic activity following culture, respond to heat-treated (45 degrees C) alloantigen, develop an increased suppressor cell function, and are less sensitive to a suppressor cell activity. Thus, changes in spleen cell subpopulations occurring in the donor mice consequent to drug treatment result in demonstrable selective imbalances of cellular functions involved in the immune response. One cell type which has been implicated as being necessary in the expression of all these functions is the monocyte-macrophage, and it is suggested that an effect of adriamycin on progenitors of this cell type may lead to the imbalances.

Cell surface changes of in situ macrophages induced by superimposed antigen.

Mice received an injection of sheep erythrocytes (SRBC) into the footpad " prepared" or "not prepared" with a 7-day-prior injection of a protein-bound polysaccharide from Coriolus versicolor (PSK; Krestin), and the ultrastructure of in situ macrophages was studied at various intervals after the injection. A single SRBC injection into the footpad induced linear cell arrangements of several macrophages. The macrophages showed no prominent morphological alterations after SRBC digestion. When PSK-stimulated subcutaneous macrophages were challenged by SRBC, they rapidly sent out numerous long cytoplasmic projections which radiated in all directions. Such projections of neighboring macrophages tended to contact one another. At the following stage, a pronounced sequential alteration was noted, characterized by the interlocking of elongated projections. This provided massive aggregations of "activated" macrophages. These observations suggest the possibility that intercellular communication among "activated" macrophages was elicited, particularly in the subcutaneous region, and maintained through an intensive interaction of cytoplasmic projections. Further, the present results histologically support our previous report which shows that the "PSK-prepared" footpad site but not the "prepared" one supports development of a splenic humoral immune response following injection of superimposed SRBC.

Further cytological observation on 'activation' by superimposed antigen of inflammation-mediated macrophages.

Compact cell aggregates were induced by an injection of a superimposed antigen, sheep erythrocytes (SRBC) into PSK (a protein-bound polysaccharide)-'prepared' mouse footpad. Major cell types in the cell aggregate were macrophages which rapidly digested superimposed SRBC but not PSK substances which were retained over seven days within their phagosomes. Macrophages without PSK like substances-containing phagosomes occurred invariably outside the cell aggregate. The macrophages in the cell aggregate were interlocked via their cytoplasmic projections. Ruthenium red, a specific dye for extracellular proteoglycans, clearly revealed their surface coats and also closely contacted zones of adjoining macrophages. The surface coats were not uniform in distribution and became sporadically thickened deposits at invaginations of the plasma membrane. Ladder-like structures among adjacent cytoplasmic projections also showed a strong affinity to the dye. It is suggested that a primary function of these structures is the maintenance of close contact of aggregating macrophages. Another observation was that binucleate macrophages occurred in the cell aggregates. Careful inspection on sections of well-preserved tissues concludes that such cells were not formed by a cell fusion between mononucleate macrophages in the cell aggregates. Formation of such a binucleate cell from the macrophage in the aggregates is also discussed.

Selective toxicity of glycyrrhetinic acid against tumorigenic r/m HM-SFME-1 cells is potentially attributed to downregulation of glutathione.

Natural products from plants are expected to play significant roles in creating new, safe and improved chemopreventive and therapeutic antitumor agents. Selectivity is also an important issue in cancer prevention and therapy. The present study was designed to extend our previous study on the c-Ha-ras and c-myc-induced tumor cell-selective antiproliferative effects of a licorice component, glycyrrhetinic acid (GA). An in silico ligand-receptor docking simulation revealed that GA acts as an 11ß-hydroxysteroid dehydrogenase type 2 inhibitor. GA disrupted the redox balance in tumor cells through upregulation of reactive oxygen species and downregulation of glutathione (GSH). The GA-induced GSH reduction and cytotoxicity were enhanced by an inhibitor of GSH, l-buthionine-[S,R]-sulfoximine. N-acetyl-l-cysteine, an antioxidant and precursor of GSH, restored the GA-induced GSH reduction and cytotoxicity in tumor cells. Taken together, these data highlighting the downregulation of GSH by GA and the efficacy of GSH in ameliorating GA-mediated cytotoxicity support the notion that GSH is involved in the selective toxicity of GA toward tumor cells.

Effector molecules from antitumor macrophages induced with OK-432 and cyclophosphamide.

The present study was designed to determine whether antitumor activity of macrophages induced with OK-432 and cyclophosphamide was mainly dependent on their ability to produce a soluble factor, that is, L-arginine-dependent nitric oxide as measured by nitrite concentration. Nitrite production by peritoneal macrophages from NIH Swiss mice pretreated with OK-432 (125 KE/kg) i.p. twice at 1-week intervals and with cyclophosphamide (200 mg/kg) i.p. 2 days before the second OK-432 treatment, increased with time for 24 h, and proportionally depended on macrophage numbers. Nitrite production was inhibited by actinomycin D and puromycin but not by mitomycin C. NG-Monomethyl-L-arginine, a specific competitive inhibitor of L-arginine-dependent nitric oxide synthesis, also inhibited production. There was a close correlation between nitrite production and antitumor activity in macrophages from mice pretreated with either OK-432 and cyclophosphamide, OK-432, or thioglycolate broth. OK-432 increased both nitrite production and antitumor activities when added to the macrophage from mice pretreated with OK-432 but not with thioglycolate broth. Both activities of macrophages from mice pretreated with OK-432 and cyclophosphamide were enhanced with increasing concentrations of L-arginine (0.125-1 mM) in the culture medium. D-Arginine, however, did not substitute for L-arginine. Neither activity was affected by contact between the macrophage and the EL4 cell. The macrophage showed antitumor activity through a membrane filter though the activity was greatly reduced. This antitumor activity of macrophages through a membrane was also inhibited by NG-Monomethyl-L-arginine, and increased by OK-432. However, conditioned media, obtained by culturing macrophages induced with OK-432 and cyclophosphamide, inhibited growth of EL4 cells. This activity was carried out by dialysable and non-dialysable factors. One of the dialysable factors was nitrite, an oxidized product of nitric oxide. The antitumor activity of non-dialysable factors was heat-stable and production of factors was increased by NG-Monomethyl-L-arginine and OK-432. Also, non-dialysable factors increased both antitumor and nitrite production activities of OK-432-elicited macrophages, when incubated with factors. Such activity of factors was also heat-stable. The production of factors increased with incubation time of macrophages, and was not inhibited by NG-Monomethyl-L-arginine. These results indicate that in vitro antitumor activity of macrophages induced with OK-432 and cyclophosphamide was mainly dependent on L-arginine-dependent nitric oxide, and that macrophage-associated soluble factors other than nitric oxide were also needed to inhibit fully tumor growth in vitro.

Effects of injection routes of growing tumors and PSK or OK-432 on antiproliferative activity of mouse serum.

The present study examined the effects of various treatments on the antiproliferative activity of mouse serum. Its activity was estimated against the growth of EL4 tumor cells and L929 cells and against splenic blastogenesis in culture. The activity varied among mouse strains tested and among individuals in any strain. However, normal outbred NIH Swiss mice showed the highest activity among the strains and the least variation among individuals. The activity of serum from NIH Swiss mice constantly decreased 7 or 14 days after an injection of 10(6) Ehrlich or sarcoma 180 tumor cells subcutaneously in the right-hind footpad, intradermally in the right side of the chest or into the palm. Other routes, such as intraperitoneal, intravenous in the tail vein, subcutaneous in the right side of the chest and intramuscular in the left thigh, however, hardly affected the activity. The activity also decreased 7 days after an injection into the footpad of a biological response modifier such as PSK or OK-432. The antiproliferative activity of mouse serum seem to depend on macrophages but not natural killer-cell activity, because treatment with silica but not anti-(asialo-GM1) antibody totally reduced the activity. The active fraction was heat-stable (100 degrees C, 30 min) and its molecular mass was 127-140 kDa.

Cyclophosphamide modifies the induction kinetics but not cell types and cytotoxic mechanisms of antitumor cells elicited with OK-432 plus attenuated tumor cells.

The present study was designed to examine whether the antitumor cells induced by treatment with mitomycin-C-treated EL4 cells (EL4MMC) plus OK-432 plus cyclophosphamide differed from those induced by treatment with EL4MMC plus OK-432 in terms of their cell types and antitumor mechanisms. Antitumor activity of peritoneal exudate cells (PEC) from mice receiving either treatment was nonspecific, and inhibition of their target cell growth increased for up to 24 h. Macrophage toxin, silica and trypan blue abrogated the activity in vitro and in vivo, respectively. The activity of the PEC was inhibited with inhibitors of the arachidonic acid cascade, such as dexamethasone, 4-bromophenacyl bromide and nordihydroguaiaretic acid but not esculetin, ibuprofen, indomethacin and BW755C. NG-monomethyl-L-arginine, a specific competitive inhibitor of the L-arginine-dependent nitric oxide synthesis, also inhibited the activity. These results and morphological observations indicated that antitumor cells in the PEC from mice receiving either treatment were macrophages, and that their activity was closely related to the arachidonic acid cascade and to nitric oxide. Antitumor activity of the PEC spontaneously decayed in vitro and this decay was inhibited by the addition of OK-432 or lipopolysaccharide. On the other hand, cyclophosphamide sustained the appearance of antitumor cells in mice treated with EL4MMC plus OK-432. Therefore, cyclophosphamide treatment did not modify cell types and cytotoxic mechanisms of antitumor cells elicited with EL4MMC plus OK-432, but did modify the induction kinetics of such antitumor macrophages.

Enhancement of antileukemic effect in combination of 5-fluorouracil and OK-432.

OK-432, a streptococcal preparation with potent antitumor activity, has been evaluated for its efficacy in experimental and clincal trials. This preparation, however, was ineffective against mouse L1210 leukemia in all treatment schedules. The results of this study indicate that treatments with OK-432 and nucleic acid antimetabolites, such as 5-fluorouracil (5-FU), exert a synergistic effect against L1210 leukemia probably in conjunction with the immunologic defenses of the host. This therapeutic synergism was to some extent dependent on the dose level of 5-FU and was not produced against the 5-FU-resistant L1210 subline. In mice pre-treated with OK-432 prior to the leukemia implantation, there was no synergistic effect as a result of post-treatment with 5-FU. When BDF mice had previously received X-ray irradiation or administration of corticosteroids, the synergism could not be expected either. Comparative combination therapy with BCG or group C streptococcus resulted in failure to produce therapeutic synergism in this system.

Appearance of a helper activity by mouse serum in cultured lymphoid cells.

Manipulations which inhibit the development of non-specific suppressor T cells did not inhibit the development of a helper activity in culture. These manipulations include culturing only those spleen cells nonadherent to Sephadex G-10, CY-treatment of spleen donor mice, or culturing spleen cells in the presence of MS instead of FCS. Further investigation of the helper cell developed in cultures supplemented with MS indicated that both in vivo and in vitro immunizations with SRBC induced the most effective helper activity, and SRBC-ghosts or even sonicated SRBC-ghosts were also effective for in vitro immunization. CM containing IL 2 inhibited the development of a helper activity when added on Day 0 but not on Day 4. rIL 2, however, slightly augmented the development of a helper activity even when added on Day 0. The kinetics of anti-SRBC PFC response was not changed in the presence of the helper cell and augmentation of the response by the helper cell was seen on all the days tested. Augmentation occurred without further SRBC addition to anti-SRBC PFC generation cultures whereas it did not occur without "intact" responder spleen cells. The helper cell was resistant to X-irradiation and to the treatment with anti-Thy 1.2 plus C' but sensitive to the treatment with anti-Lyt 1.2 plus C'. The activity of nonspecific suppressor T cells dominated over that of the helper cell and the helper activity appeared only when the ratio of suppressor cells to helper cells was reduced to less than 1:4.

Augmented induction of antitumor cells in vivo by cyclophosphamide fails to benefit antitumor resistance of the host.

The present study was designed to examine whether cyclophosphamide augmented induction of antitumor cells and antitumor resistance in C57BL/6 mice pretreated with mitomycin-C-treated EL4 cells (EL4MMC) plus OK-432, a streptococcal preparation. C57BL/6 mice were pretreated with EL4MMC (10(7] plus OK-432 (2.5 KE) i.p. twice at 1-week intervals. When the mice received an i.p. injection of cyclophosphamide at 200 mg/kg 2 days before the last treatment, the antitumor activity of their spleen cells and peritoneal exudate cells (PEC) was effectively augmented 7-8 days after the last treatment. Splenic antitumor activity disappeared 15 days after the last treatment whereas augmented antitumor activity of the PEC was detected even 28 days after the last treatment. This cyclophosphamide effect was dose-dependent and 200 mg/kg was the most effective among the doses tested. If the EL4MMC plus OK-432 treatment was injected at a s.c. site, it was also effective in combination with cyclophosphamide. The antitumor activity of the PEC from s.c.-pretreated mice, however, was lower than that from i.p.-pretreated mice. Despite the fact that cyclophosphamide effectively augmented induction of antitumor cells in C57BL/6 mice pretreated with EL4MMC plus OK-432, it diminished rather than augmented, under all conditions tested, the ability of the mice to resist a challenge of live EL4 cells. Reduction of antitumor resistance by cyclophosphamide was also observed in an experimental system of a semi-syngeneic host (BDF1) tumor (EL4). These results indicate that augmentation of in vivo induction of certain kinds of antitumor cells does not necessarily result in a beneficial augmentation of the host's ability to resist tumor growth.

Inhibition of macrophage nitric oxide production by arachidonate-cascade inhibitors.

We examined whether inhibitors of the arachidonic acid cascade inhibited nitric oxide (NO) production, as measured by nitrite concentration, either in macrophages or by their cytosolic fractions. Nitrite production by peritoneal macrophages from mice receiving OK-432 treatment was significantly inhibited by phospholipase A2 inhibitors [dexamethasone and 4-bromophenacyl bromide (4-BPB)], lipoxygenase inhibitors [nordihydroguaiaretic acid (NDGA) and ketoconazole] and a glutathione S-transferase (leukotrienes LTA4-LTC4) inhibitor (ethacrynic acid). However, caffeic acid and esculetin, inhibitors of 5- and 12-lipoxygenase respectively, were not inhibitory. On the other hand, indomethacin, a cyclooxygenase inhibitor, slightly inhibited whereas another inhibitor, ibuprofen, did not. Inhibition of the nitrite production by dexamethasone, 4-BPB, NDGA and ethacrynic acid was also demonstrated when the macrophages were restimulated ex vivo with OK-432 or with lipopolysaccharide. The inhibitory activity of dexamethasone, NDGA and ethacrynic acid was significantly reduced by ex vivo restimulation with OK-432, whereas that of 4-BPB was hardly affected. Furthermore, the inhibitory activity of dexamethasone, NDGA and ethacrynic acid was much higher when the macrophages were continuously exposed to the agents than when they were pulsed. Meanwhile, inhibition by 4-BPB was almost the same with either treatment. In addition, the inhibitory activity of these agents was not blocked with L-arginine, a substrate of NO synthases, or with arachidonate metabolites (LTB4, LTC4 and LTE4). Ethacrynic acid and 4-BPB, but not dexamethasone and NDGA, also inhibited nitrite production by the cytosolic fractions from OK-432-restimulated peritoneal macrophages, and the inhibitory activity of 4-BPB was superior to that of ethacrynic acid. These agents, however, did not inhibit nitrite production from sodium nitroprusside, a spontaneous NO-releasing compound. These results indicate that dexamethasone, 4-BPB, NDGA and ethacrynic acid inhibited the production of NO by macrophages through at least two different mechanisms: one was inhibited by dexamethasone, NDGA and ethacrynic acid and the other by 4-BPB. Furthermore, 4-BPB and ethacrynic acid directly inhibited the activity of the NO synthase in macrophages, suggesting that the agents work by binding to the active site(s) of the enzyme.

Effect of OK-432 on immunization with mitomycin-C-treated L1210 cells.

The pretreatment of B6D2F1 mice with OK-432 alone was not so effective to retard the growth of L1210 leukemia, and the leukemic cell was poorly immunogenic in the mice. However, when OK-432 was given by ip injection in combination with mitomycin-C-treated L1210 cells, the growth of L1210 leukemia was significantly retarded and some mice did not "take" the leukemia. Meanwhile, BCG was also effective in this respect. Differences in properties as an immunopotentiator between OK-432 and BCG were suggested by the experiments of in vitro cytotoxicity test of spleen cells.