Depression of cell-mediated immunity in plasmacytoma-bearing mice.

Previous reports have documented that mice bearing plasmacytomas (PC) are suppressed in their ability to mount a primary antibody response, whereas cellular immunity appears to be normal. Studies presented here provide evidence that T cell responsiveness is also depressed in BALB/c mice bearing the Lieberman plasmacytoma (Lpc-1). For instance, splenocytes from mice bearing large tumours were impaired in their in vitro ability to respond to the T cell mitogen, mount an appropriate alloreactive cytolytic T lymphocyte response, and produce interleukin-2 (IL-2). A population of suppressor cells was detected in the spleens 7 days after tumour implantation as evidenced by their ability to prevent normal splenocytes not only from responding to antigens in mixed lymphocyte culture, but also from producing IL-2. A similar inhibitory effect was observed with culture supernatants of these cells, indicating the existence of a soluble suppressive factor. Therefore, the present study demonstrates that cellular immune responses are impaired in mice bearing Lpc-1 tumours and that this effect may be due to the generation of suppressor cells and/or a suppressive factor.

The mechanism of action of a synthetic immunomodulator, 3,6-bis(2-piperidinoethoxy)acridine trihydrochloride (CL 246,738), in natural killer cell activation in animals.

CL 246,738 is a low molecular weight, synthetic immunomodulator. The present study was done to determine the interaction among interferon (IFN), macrophages, and natural killer (NK) cells in mice following oral administration of CL 246,738. Splenic NK activity as evidenced by lysis of YAC-1 lymphoma cells in vitro was found to be augmented by the compound not only in normal mice, but also in immunodeficient beige and nude mice. Lytic activity remained elevated from one to seven days after a single treatment and the peak activation varied depending on the source of NK cells. NK cell activity associated with the peritoneal exudate cell population peaked at day 1 and returned to normal by day 2, whereas NK cell activity of peripheral blood lymphocytes peaked at day 3 and remained significantly elevated until day 7. Liver associated NK activity peaked at day 4 and remained significantly elevated at day 7 after treatment with CL 246,738. Lung associated NK activity was elevated by day 1 after treatment, peaked at day 4 and returned to normal by day 7 after drug administration. The drug was also effective in inducing IFN in all mouse strains tested. When these drug-treated mice were given antibody to IFN-(alpha + beta) but not to IFN-(beta), both IFN levels and NK cell activity decreased, suggesting the importance of IFN-(alpha) in this system. Furthermore, mice that had received carrageenan prior to, but not after CL 246,738 administration showed reduced serum IFN titers as well as decreased NK cell activity, indicating that macrophages played an intermediate role in immune enhancement by the drug.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of 3,6-bis(2-piperidinoethoxy) acridine trihydrochloride (CL 246, 738) on acute graft vs host reactions in mice.

3,6-Bis (piperidinoethoxy) acridine trihydrochloride (CL 246, 738) prevented the development of graft vs host (GVH) disease in normal BDF1 mice injected with C57BL/6 parental spleen cells. A single oral dose (50 mg/kg) given on day 0 or day -1 of GVH induction prevented the day 10 GVH-associated suppression of mitogen responsiveness and IL-2 production. The drug was ineffective if given later (days 3-7) in the reaction. The protective effect of CL 246,738 was neutralized by injecting drug-treated GVH mice with antibody to asialo GM-1 (ASGM-1). This suggested that the protective mechanism was not due to a direct effect of the drug on donor cells but rather was achieved indirectly through the activation of host ASGM-1+ cells which then rejected donor lymphocytes. This hypothesis was supported by immunofluorescence which showed that the donor-host chimerism seen in control GVH mice was not found in drug-treated GVH mice. Direct verification of this hypothesis was provided by data which showed that the transfer of CL 246, 738-activated large granular lymphocytes from normal F1 mice can prevent donor-induced immunosuppression in GVH mice. The results suggest that CL 246,738 is a potent immunostimulant which can boost natural resistance of normal unirradiated mice.

Reconstitution of cytolytic alloreactivity with N-[4-[(4-fluorophenyl) sulfonyl]phenyl]acetamide (CL 259,763) in animals immunocompromised by cyclophosphamide.

A novel synthetic immunopotentiator, i.e. N-[4-[(4-fluorophenyl)sulfonyl]phenyl]acetamide (CL 259,763), was investigated for its potential in reconstituting the cell-mediated immune response of animals whose immunologic system had been severely depressed by cytoreductive agents. It was demonstrated that lymphocytes from mice which had received 300 mg/kg of cyclophosphamide (CY) immediately following antigen sensitization had a reduced capability of responding to alloantigens in mixed lymphocyte culture and failed to generate effective cytolytic T-lymphocytes (CTL) capable of destroying appropriate tumor target cells in a cytotoxicity assay. However, treatment of these immunocompromised animals with CL 259,763 produced a significant restoration of alloreactivity, as evidenced by an enhancement of the CTL response. Although effective doses of CL 259,763 ranged from 20 to 300 mg/kg, the optimal effect was observed at 75 mg/kg. Findings from a time course study indicated that the maximum restoration occurred when CL 259,763 was given to mice 2-5 days after, but not before or simultaneously with, CY treatment. Both the immunoimpairment by CY and its reversal by CL 259,763 appeared not to be antigen specific. The lessened immunoreactivity of CY-treated mice was explicable by the presence of suppressor cells in their spleens. These suppressors were able to adhere to plastic and resisted treatment with anti-Thy 1.2 antibody, indicating a macrophage characteristic. Flow cytometric analysis indicated a quantitative depletion of all T-lymphocytes, including Thy-1.2(+), Lyt-1(+), Lyt-2(+) and L3T4(+) subsets in the spleens of CY-treated mice; however, a population of Mac-1(+) cells was markedly expanded.(ABSTRACT TRUNCATED AT 250 WORDS)

Restoration of cytolytic T-lymphocyte response with a new immunopotentiator, N-(4-[(4-fluorophenyl)sulfonyl]phenyl)acetamide (CL 259, 763), in mice.

The immunorestorative characteristics of a novel synthetic immunomodulator, N-(4-[(4-fluorophenyl)sulfonyl]phenyl)acetamide (CL 259, 763), has been investigated in several experimental models. In one situation, the compound was shown to enhance the induction of a cytolytic T-lymphocyte response to the murine MBL-2 leukemia implanted in its syngeneic host in which only a minimal reactivity to the tumor is normally displayed. In a Vaccinia virus model, the compound similarly augmented the lytic activity of cytolytic T-lymphocyte to virus-infected targets in not only viral antigen-primed but also cyclosporin A-impaired mice. Likewise, the alloreactive cytolytic T-lymphocyte activity was recovered in animals immunocompromised by inoculation with murine plasmacytomas or cytoreductive anticancer drugs, such as cyclophosphamide and 5-fluorouracil. Thus, the present findings suggest that CL 259,763 is effective in potentiating the immune response to weak antigens as well as in restoring alloreactivity by sparing the immunotoxicity associated with the administration of cytotoxic drugs and the growth of neoplasms.

Low molecular weight immunopotentiators.

It has long been recognized that modulation of the immune system by various agents may have potential for the management of certain infectious and neoplastic diseases. Both natural products as well as chemically synthesized compounds have been investigated for immunotherapeutic potential. Over the years, conflicting reports on the clinical efficacy of these agents have left the early promise of immunotherapy unfulfilled. However, the manipulation of the immune system to generate a desired effect is becoming feasible as the mechanisms which regulate the immune network are better understood. Much of the early work on immunotherapy concentrated on the development of immunopotentiators, agents which enhance the host's own immune system against cancer cells or infectious pathogens. Furthermore, with the development of subunit and/or synthetic vaccines, which are often weakly immunogenic, the importance of developing agents capable of acting as adjuvants became apparent. As a result, the utility of immunopotentiators has now extended to the area of vaccines. There are a number of reviews available on immunomodulators [see Fenichel, R. L. and Chirigos, M. A. (eds) (1984), Immune Modulation Agents and Their Mechanisms, Marcel Dekker, New York]. The purpose of this article is to provide an update on low molecular weight agents capable of potentiating the immunological network. Attention will be given to those agents which have undergone significant clinical development in the areas of cancer, infectious diseases and vaccination over the past several years. These agents will be categorized as to whether they are naturally occurring or chemically synthesized.

Effect of hyperthermia on the sensitivity of human colon carcinoma cells to mitoxantrone.

The effect of temperature on the cytotoxicity of mitoxantrone (MX) was investigated by exposing human WIDR colon carcinoma cells to elevated temperatures in the presence of drug. The survival of treated cells was determined by their ability to proliferate and to form colonies in vitro. It was demonstrated that incubation of WIDR cells with MX at 42 degrees C produced not only a higher toxicity but also a more rapid action than when cells were exposed to drug at 37 degrees C. By measuring the amounts of 3H-MX incorporated by treated tumor cells, it was shown that cells at 42 degrees C induced an approximately threefold increase in drug uptake when compared to cells at 37 degrees C. The effect seemed long-lasting but preheating did not induce tolerance to a subsequent exposure to MX hyperthermia. Therefore, it is clear that the cytotoxic potency of MX can be augmented by elevated temperature possibly by enhancing the uptake of drug. The present findings may prove useful for designing an effective clinical regimen of MX thermochemotherapy.

Internalization and re-expression of antigens of human melanoma cells following exposure to monoclonal antibody.

Modulation of the surface membrane of human Sk-Mel-28 melanoma cells by monoclonal antibody (MoAb) 96.5 recognizing p97 determinants was examined using direct radioimmunoassay and indirect fluorescent antibody-staining techniques. It was determined that the majority of 111In-labeled antibody that remained associated with cells after a 24-hr incubation at 37 degrees C had been internalized because MoAb 96.5 was no longer visible on the cell surface. A second treatment of these cells with the same antibody 24 hr later not only increased the cell-associated radioactivity, reflecting an increase of total antibody bound, but also rendered these cells membrane immunofluorescent again, indicating the re-expression of surface antigens. Autoradiographs of the electrophoretically analyzed membrane components of Sk-Mel-28 cells further demonstrated the appearance of newly synthesized 97-kDa proteins that were immunoprecipitable with MoAb 96.5. Taken together, the present findings suggest that p97 antigens undergo endocytosis in Sk-Mel-28 cells following exposure to MoAb 96.5. However, the same antigens were regenerated and expressed on the cell surface within a period of 24 hr. The re-expression of tumor cell surface antigen following initial internalization of the MoAb-antigen complex may have implications for diagnosis and therapy.

Relationship of chemical structures of anthraquinones with their effects on the suppression of immune responses.

A series of 37 anthraquinones were evaluated for their ability to inhibit the induction of cytolytic T-lymphocytes in a mixed lymphocyte culture system, useful as a preliminary screen for immunosuppressive agents. These compounds were also tested for their ability to prevent the production of antibody in mice. It was demonstrated that 1,4-bis [(2-aminoethyl)amino]-5, 8-dihydroxy-9,10-anthracenedione dihydrochloride (AEAD, 2) derived from mitoxantrone (MX, 1) by removing hydroxyethyl groups from both side chains was extremely active in depressing immune responses in vitro and in vivo. Four additional anthraquinones related to AEAD were also identified to share similar suppressive activity. They include a Schiff base, 1,4-dihydroxy-5,8-bis[[2-[(3-pyridinylmethylene)amino]ethyl]amino] -9,10-anthracenedione; a dimer with N-terminals methylated, 1,1-[ethylenebis (iminoethyleneimino)]-bis [5,8-dihydroxy-4-[(2-methylamino-ethyl)amino] anthraquinone tetrahydrochloride; an oxazolidine, 1,4-dihydroxy-5,8-bis [[2-(2-propyl-3-oxazolidinyl)ethyl]amino] anthraquinone; and its polymeric oxazolidine, poly [5,8-dihydroxy-1,4-anthraquinonyleneiminoethylene-3,2-oxazolidine- diyltrimethylene-2,3-oxazolidinediylethyleneimino]. These compounds may warrant further consideration as candidates for the treatment of refractory autoimmune diseases and in organ transplantation.

The mechanism of action of 3,6-bis(2-piperidinoethoxy)acridine trihydrochloride (CL 246,738) in the potentiation of natural killer cells.

An effort has been made to determine the mechanism by which the immunomodulator 3,6-bis(2-piperidinoethoxy)acridine trihydrochloride (CL 246,738) enhances the cytotoxic activity of natural killer (NK) cells. Orally administered CL 246,738 produced augmentation of NK cell activity in mice in a dose-related fashion over a dose range of 10 to 160 mg/kg, with a peak stimulation occurring at 40 mg/kg. The stimulatory effect was short-lived and only persisted for 3 days after a single oral dose of the drug. However, it could be boosted by a subsequent treatment. With anti-asialo GM-1 (anti-ASGM-1) antibody used as an NK cell marker, it was determined that the compound increased the number of ASGM-1-positive cells in mice, as indicated by radioimmunoassay and immunofluorescence staining. NK cells of beige mice were also activated by CL 246,738. Furthermore, the compound at concentrations of 0.02 to 0.2 microgram/ml induced NK cell activity in vitro, with a minimum 3-day incubation being required for optimal activation. This effect was dependent on the presence of macrophages and was inhibited by anti-IFN-alpha + beta but not anti-IFN-beta antibody. Taken together, it is postulated that the compound functions by stimulating macrophages to release IFN-alpha, which subsequently activates NK cells. As an effective stimulator of IFN and NK cells, CL 246,738 may prove clinically useful in the immunotherapy of certain types of malignancy.

Inhibition of the induction of alloreactivity with mitoxantrone.

A clinically active anticancer agent, mitoxantrone (MX): 1, 4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]-9, 10-anthracenedione dihydrochloride, was studied for its potential inhibitory effect on alloreactivity induction. Addition of MX to mixed lymphocyte cultures (MLC) not only inhibited the proliferative response of lymphocytes to alloantigens but also prevented the generation of cytolytic T lymphocytes (CTL). MX showed a long-lasting effect in vitro and acted at the inductive rather than the effector phase of the CTL response as indicated by its failure to alter the activity of those CTL already generated in MLC. MX also inhibited CTL induction in mice. However, the precursors of CTL appeared to be spared in these animals as supported by limiting dilution analysis and also because CTL could be reactivated by exposure of splenocytes to the same or different alloantigens in MLC. The present findings demonstrate that MX is a potent immunosuppressive agent and as such might prove to be clinically useful in the treatment of autoimmune diseases or find utility in the organ transplantation field.

Modulation of the immune response to tumors by a novel synthetic compound, N-[4-[(4-fluorophenyl)sulfonyl]phenyl] acetamide (CL 259,763).

CL 259,763, N-[4-[(4-fluorophenyl)sulfonyl]phenyl] acetamide, is an orally active compound capable of modifying the reactivity of certain lymphoid cell populations affected by the growth of a tumor. The compound augmented the response of lymphocytes from tumor-primed animals to syngeneic tumor cells, resulting in a marked increase in tumor cell destruction. Likewise, it enhanced macrophage inhibitory effects on the growth of tumor cells in vitro. These "activated" macrophages were detectable in peritoneal exudates of treated mice 4 to 12 days after receiving a single oral dose of CL 259,763, with peak activity being demonstrable by day 7. The compound also restored the alloreactivity of lymphocytes from immunodepressed mice bearing the Lieberman plasma cell tumor, possibly by interfering with suppressor cells. Macrophages and lymphocytes from treated mice released significantly more IL-1 and IL-2-like factors in culture than did the control counterparts. Sera from treated mice also possessed more colony stimulating factor than those from normal mice. Immunoadjuvant effects were evident when the compound was administered with an inactivated L1210 leukemia vaccine and it enhanced the effectiveness of cytotoxic chemotherapy when given to mice challenged with P388 murine leukemia. These immunomodulating effects of CL 259,763 may hopefully be exploited in efforts to augment the immune response of the host to a progressively growing tumor.

Induction of tumor-inhibitory macrophages with a novel synthetic immunomodulator, 3,6-bis(2-piperidinoethoxy)acridine trihydrochloride (CL 246,738).

3,6-bis(2-piperidinoethoxy)acridine trihydrochloride (CL 246,738) has been investigated for its immunomodulatory effect on murine macrophages. Incubation of macrophages harvested from the peritoneal cavities of normal mice with the compound for 48 to 72 hr rendered these cells inhibitory to the growth of tumor cells in vitro. Activation of tumor-inhibitory macrophages occurred over a range of concentrations (0.025 to 0.1 micrograms/ml) producing no direct inhibitory effects on tumor cells. Treatment of effector cells with carrageenan abrogated the effect, whereas treatment with anti-Thy-1.2 antibody and C did not, suggesting that the primary effectors were macrophages rather than T lymphocytes. These activated macrophages also manifested in vitro tumor cytolysis. In vivo studies indicated that peritoneal macrophages from mice treated with single oral doses of 100 to 400 mg/kg of the compound were also inhibitory to tumor cell growth in vitro. Effector macrophages became demonstrable in mice as early as 1 day after drug administration, reached peak activity at day 12, and disappeared by day 31, indicating a rapid onset but long-persisting effect. The tumor cytostatic activity of these macrophages was augmented by endotoxin at the dose of endotoxin that, in itself, had no effect. The addition of protease inhibitors, N-alpha-p-tosyl-L-lysine chloromethyl ketone and aprotinin, to cultures markedly diminished the cytostatic effect, suggesting that the release of neutral protease(s) could account for the inhibitory effects of the macrophages. On the other hand, hydrogen peroxide and arginase seemed excluded as the mechanism of action because the effect was not sensitive to treatment with catalase and exogenous arginine. The present findings indicate that CL 246,738 is an orally active immunopotentiator capable of inducing tumor-inhibitory macrophages both in vitro and in vivo.

Activation of tumor-cytostatic macrophages with the antitumor agent 9,10-anthracenedicarboxaldehyde bis[(4,5-dihydro-1H-imidazole-2-yl)hydrazone] dihydrochloride (bisantrene).

An investigation was carried out to determine the potential for activating tumor-inhibitory macrophages with the cytotoxic antitumor agent, bisantrene. Macrophages prepared from peritoneal exudates of mice treated i.p. with bisantrene were extremely active in inhibiting the growth of tumor cells. The minimal effective in vivo dose of this drug appeared to be 25 mg/kg, with peak activation being achieved at doses of 50 to 100 mg/kg. Effector cells became detectable 2 days after treatment and persisted for at least 4 weeks. Incubation of effector and target cells for 48 hr seemed necessary to achieve the maximum inhibitory effect. Treatment with carrageenan in vitro and in vivo abolished tumor cytostasis, whereas treatment with anti-T-cell antibody plus complement had no effect, suggesting that macrophages rather than T-lymphocytes were responsible for the observed results. Culture supernatants of activated macrophages were found to have antiproliferative effects on tumor cells, indicating that a cytostatic factor(s) was produced by these macrophages. Hydrogen peroxide and neutral proteases apparently did not function as cytostatic mediators since activated macrophages were resistant to treatment with catalase, N-alpha-p-tosyl-L-lysine chloromethyl ketone, and aprotinin. The present findings suggest that, in addition to direct toxicity to tumor cells, bisantrene may act as a macrophage-activating immunopotentiator. This observation may be of potential clinical usefulness in the design of immunochemotherapeutic trials for certain types of cancer.

Induction of alloreactive immunosuppression by 1,4-bis [( 2-aminoethyl)amino]-5,8-dihydroxy-9,10-anthracenedione dihydrochloride (CL 232,468).

1,4-bis[2-aminoethyl)amino]-5,8-dihydroxy-9,10-anthracenedione (AEAD) has been investigated for its potential immunosuppressive effect on cell-mediated immune responses. Addition of the compound to mixed lymphocyte cultures (MLC) not only significantly inhibited these cells from responding to alloantigens but also prevented the induction of cytolytic T lymphocytes (CTL). A structurally related compound, mitoxantrone, was also found to be active in inhibiting CTL induction. AEAD had to be present during the first 3 days of a 5-day MLC in order to produce a significant effect and it had no effect on those CTL already generated, suggesting that it acted upon induction of CTL rather than the effector phase. Lymphocytes from mice treated with the compound were incapable of responding to alloantigens in vitro and the effect was dose- and time-dependent. Furthermore, lymphocytes from treated mice were found to inhibit CTL generation from normal mouse lymphocytes, indicating that a suppressor cell population might be induced in the spleens of animals treated with the compound. The present findings clearly demonstrate that AEAD is a compound with potent immunosuppressive activity on alloreactive immune responses.