Tumor-mediated immunosuppression: Prevention by inhibitors of prostaglandin synthesis

The addition of MC16 tumor cells (a prostaglandin E₂-producing cell line induced in C57B1/6J mice by methylcholanthrene) to cultures of normal syngeneic spleen cells inhibits the antibody response of these cells to sheep red blood cells. This inhibition can be blocked by adding to the cultures prostaglandin synthetase inhibitors, such as indomethacin, flufenamic acid and aspirin. These MC16 tumor cells are also immunosuppressive . Mice bearing the syngeneic MC16 tumor become unresponsive to sheep red blood cells as the tumor grows. As in the test system, inhibitors of prostaglandin synthetases seem to block the immunosuppressive activity of MC16 cells since tumor-bearing mice, treated therapeutically with indomethacin, responded normally in their production of antibody to sheep red blood cells.     

Prostaglandin-mediated suppression of delayed-type hypersensitivity to infected erythrocytes during Babesia microti infection in mice

The mechanism of suppression of delayed-type hypersensitivity (DTH) to intraerythrocytic Babesia microti which occurs during infection in mice was examined. The suppression was not specific for anti-parasite DTH; infected mice immunized and challenged with sheep red blood cells had a similar depression of anti-sheep red blood cell DTH. Sublethal or lethal irradiation did not significantly alter the suppression of the DTH response, and cyclophosphamide pretreatment of infected mice also had no effect on suppression. Multiple passive transfer experiments using serum or regional lymph node cells from immunized or infected and immunized (suppressed) donor animals failed to demonstrate any ability to transfer suppression of DTH. Adherent cells from the spleens or peritoneal exudates of suppressed mice, however, did significantly depress the ability of immunized mice to express a DTH response. The cells responsible for this suppression were Thy 1- and nonspecific esterase+. Treatment of suppressive cell populations with 10 micrograms/ml indomethacin for 24 hr in vitro abrogated their suppressive ability, and in vivo administration of indomethacin to suppressed mice also restored DTH to normal levels. By examining levels of prostaglandin E2 (PGE2) in supernates of cultured peritoneal exudate cells from immune or suppressed mice, it was shown that infected mice had peritoneal exudate cells which produced significantly more PGE2 than similar cells from immune mice. These data suggest that B. microti infection elicits synthesis of PGE2 by macrophage-like cells which results in suppression of DTH to parasite as well as heterologous antigens.     

The effect of indomethacin on the activation and effector function of suppressor cells from tumor-bearing mice

Summary The spleens of mice with large M-1 fibrosarcomas contain two populations of suppressor cells with the properties of macrophages and T cells. In this study, we tested the effect of indomethacin on suppressor cell activation and effector function. Neither the activation nor the effector function of the suppressor macrophages was inhibited by indomethacin, and the activity of suppressor macrophages correlated with the tumor size. In contrast, the treatment of tumor-bearing mice with indomethacin from the day of injection of tumor cells completely blocked the in vivo activation of suppressor T cells. Indomethacin did not, however, depress suppressor T cell activity if mice were treated only during the third week of tumor growth. The effector function of the suppressor T cells, as assessed in mixing assays, was partially blocked by indomethacin, while selective suppression by low-molecular-weight factors was completely blocked if indomethacin was present in the cultures. Furthermore, the in vitro activation of suppressor cells by soluble factors secreted by tumor-bearer spleen cells was completely blocked by indomethacin, and this inhibition was reversed by prostaglandin E₁. These data are consistent with the hypothesis that prostaglandins are involved in the activation, but not the effector function, of tumor-activated suppressor T cells.     

Immunosuppressive activity of human chorionic gonadotrophin preparations in vivo: evidence for gonadal dependence

Human chorionic gonadotrophin preparations (hCG), when injected ip daily for 4 days, suppress the delayed-type hypersensitivity (DTH) response of mice to sheep red blood cells. Preparations of crude hCG, purified hCG subunits, and hCG that was formed by recombining the purified subunits showed immunosuppressive activity in accord with their gonadotrophic activity. The immunosuppressive effects in male and female mice were comparable. However, removal of the gonads completely abrogated the immunosuppressive activity of hCG in both males and females, suggesting that the effect of hCG is mediated by a factor released from the gonads. We conclude that the hCG molecule itself exhibits immunosuppressive activity in vivo in both male and female mice and that the gonads are required for the expression of this activity.     

Defects in tumor cell immunogenicity: lymphokine signals in in vitro cytolytic responses to tumor alloantigens

The B16 melanoma of C57BL/6 mice illustrates a deficiency in immunostimulation which may be important in some host-tumor relationships. B16 immunizes very poorly, even against its own major histocompatibility complex (MHC) antigens. We have compared the anti-MHC cytolytic response induced in vitro by B16 and by other tumors of both lymphoid and nonlymphoid origin. We have also studied the role of indomethacin and exogenous lymphokines in facilitating these responses and examined the relationship of specific and nonspecific effector cells induced. In contrast to normal lymphoid cells and two lymphoid tumor cells (EL4 and WEHI-265), the three nonlymphoid tumors, B16, Lewis lung tumor (3LL), and MC-2 fibrosarcoma, failed to induce primary cytolytic responses by themselves. MC-2 and B16 represented two different defects in immunogenicity. MC-2, which we have shown previously to induce an in vivo cytolytic response, could also immunize in vitro provided that prostaglandin production was blocked with indomethacin. In contrast B16, which is poorly immunogenic in vivo, immunized in vitro only if a concanavalin A-induced lymphokine supernatant (CS) was added as an exogenous source of "signal 2." High concentrations of the interleukin 2-containing Con A-induced spleen cell culture supernatant-induced non-H-2b-specific lymphokine-activated killer (LAK) cells in the absence of B16 stimulator cells. However, lymphokine concentrations too low to induce LAK cells enabled the otherwise nonimmunogenic B16 cells to induce specific cytolytic activity.     

Impairment of T-helper function by a Plasmodium berghei-derived immunosuppressive factor

Mice injected with an immunosuppressive factor (ISF) extracted from Plasmodium berghei-infected rat erythrocytes have a reduced antibody response to unrelated antigens. T-cells from ISF-treated mice failed to provide adequate help to naive, syngeneic B-cells in the primary IgM response in vitro to sheep red blood cells and to dinitrophenylated keyhole limpet hemocyanin. The same T-cells, however, were able to cooperate with memory B-cells in the secondary IgG response. No other cellular deficit was detected in ISF-treated mice; B-cells and macrophages behaved normally, and there was no detectable excess of suppressor cells. The T-cell impairment was not reflected in decreased production of interleukin 2, but was also shown by the diminished delayed type hypersensitivity reaction to sheep red blood cells of ISF-treated mice.     

Impairment of T-Helper Function by a Plasmodium berghei-Derived Immunosuppressive Factor1

Mice injected with an immunosuppressive factor (ISF) extracted from Plasmodium berghei-infected rat erythrocytes have a reduced antibody response to unrelated antigens. T-cells from ISF-treated mice failed to provide adequate help to naive, syngeneic B-cells in the primary IgM response in vitro to sheep red blood cells and to dinitrophenylated keyhole limpet hemocyanin. The same T-cells, however, were able to cooperate with memory B-cells in the secondary IgG response. No other cellular deficit was delected in ISF-treated mice; B-cells and macrophages behaved normally, and there was no detectable excess of suppressor cells. The T-cell impairment was not reflected in decreased production of interleukin 2, but was also shown by the diminished delayed type hyperaensitivity reaction to sheep red blood cells of ISF-treated mice.     

Immunosuppression by spleen cells from Moloney leukemia. III. Evidence for a suppressor cell that is not the leukemic, virus-producing cell.

Spleens of mice bearing MuLV (Moloney)-induced leukemia contain cells that inhibit the antibody response of normal syngeneic lymphocytes to sheep RBC in Marbrook cultures. In order to determine whether these immunosuppressive cells are virus-infected tumor cells or normal cells we pretreated leukemic spleen cell suspensions with syngeneic mouse antiserum to Moloney leukemia antigen(s) (plus complement) and with rat anti-Moloney serum (plus complement). The cytotoxic treatment killed approximately 20% to 30% and 60% to 70% of the cells, respectively. The remaining viable cell population was tested for MuLV production (in an infectious center assay on S+L-fibroblasts), for lethal effect on newborn mice, and for immunosuppressive activity. After the treatment with anti-Moloney sera the number of MuLV-releasing cells decreased 10-fold and the leukemogenic potential in vivo decreased 100-fold as compared to leukemic spleen cells pretreated with nonimmune mouse and rat sera (plus complement). In contrast, the ability of the antisera-treated cells to inhibit anti-SRBC response remained undiminished. This indicates that, in part, the immunosuppressive cells in the leukemic spleen are normal, noninfected cells, involved, perhaps, in immune regulation.     

Trichinella spiralis: inhibition of sheep hemagglutinins in mice

One hundred and twenty-four mice were injected intraperitoneally with sheep red blood cells. The mice had been previously either orally inoculated with T. spiralis (16 mice), or injected intraperitoneally during 7 consecutive days with normal saline (12 mice), normal mouse serum (6 mice), or infected mouse serum (6 mice), normal rabbit serum (6 mice), sera from lightly (36 mice) or heavily infected rabbits (36 mice), and rabbit anti-lymphocyte serum (6 mice). The homologous serum clearly demonstrated an immunosuppressive effect on the production of sheep hemagglutinins; however, it was impossible to conclude that heterologous serum has such an activity since the normal rabbit serum used as control demonstrated the same activity. The inhibition of hemagglutinin production has also been observed in mice infected with T. spiralis. The presence of a suppressive agent released by the parasite or antigenic competition is discussed as the possible mediator of immunological unresponsiveness.     

Induction of antitumor immunity by indomethacin

Irradiated tumor cells given, together with indomethacin, to syngeneic mice induced an antitumor response and conferred protection against a challenge of a lethal dose of murine mammary (4T1) and lung (3LL) carcinoma cells. Continuous administration of indomethacin was crucial throughout the entire period of immunization and challenge, as no protection was achieved when the drug was given during only one of these procedures. Antitumor immunity was long-lasting and, when tested in the 4T1 model, 48% of mice were resistant to a second challenge of lethal tumor cells. Tumor-free immune mice that were given indomethacin for more than 300 days remained healthy with normal white blood cell counts and normal spleen size. Cells isolated from immune mice were able to kill tumor cells in culture after in vitro activation by interleukin-2, in a manner similar to cells from naive normal control mice. In addition, the mitogenic response of their T cells was as high as that of the control naive mice. While indomethacin was able to induce antitumor immunity to 4T1 and 3LL murine carcinoma cells, both of which contain a high concentration of endogenic prostaglandin E₂ (PGE2), no such immunity was achieved to murine tumor cells with a low concentration of endogenic PGE2. These results suggest a correlation between PGE2 concentration and the ability of indomethacin to induce antitumor immunity. We therefore suggest that an immunotherapy protocol with long-term dispensation of a tolerable dose of an immunomodulator, given together with irradiated autologous tumor cells, may stimulate antitumor responses to tumors containing high concentrations of endogenic PGE2. Received: 12 August 1999 / Accepted: 21 September 1999     

Ala-1: murine alloantigen of activated lymphocytes. II. T and B effector cells express ala-1.

Ala-1 (activated lymphocyte antigen-1) is a murine alloantigen expressed only on activated peripheral T and B lymphocytes. The presence or absence of Ala-1 on specific functional lymphocyte subsets was determined by treating the relevant cell population with anti-Ala-1 and complement, and assaying for residual functional activity. By this method, Ala-1 was shown to be on in vivo primed killer T cells cytotoxic for allogeneic tumor cells. It was also found on helper T cells generated in vivo to sheep red blood cells, and on IgM and IgG plaque-forming cells (PFC) to sheep red blood cells. In contrast, splenic precursors of helper cells and of IgM PFC to sheep red blood cells were completely resistant to treatment with anti-Ala-1 and complement. These findings indicate that effector cells can be distinguished from their nonactivated precursors by their expression of Ala-1.     

Effect of an orally applied herbal immunomodulator on cytokine induction and antibody response in normal and immunosuppressed mice

The influence of the oral administration of a herbal immunomodulator, consisting of an aqueous-ethanolic extract of the mixed herbal drugs Thujae summitates, Baptisiae tinctoriae radix, Echinaceae purpureae radix and Echinaceae pallidae radix, on cytokine induction and antibody response against sheep red blood cells was investigated in mice. The treatment of the animals with the extract caused no enhancement of the cytokine titers in the serum. Spleen cells isolated from the treated mice, however, produced higher amounts of IL-2, IFNgamma and GM-CSF ex vivo in comparison to spleen cells isolated from control animals, especially after additional stimulation by lipopolysaccharides or concanavalin A. The application of the extract also triggered the production of IL-1 and TNFalpha by peritoneal macrophages ex vivo. The influence of the herbal extract on the antibody response was examined by the plaque forming cell assay. The administration of the extract caused a significant enhancement of the antibody response against sheep red blood cells, inducing an increase in the numbers of splenic plaque forming cells and the titers of specific antibodies in the sera of the treated animals. In mice, immunosuppressed by old age or additional treatment with hydrocortisone, the therapy with the extract resulted in a normalization of the antibody response against sheep red blood cells.     

Novel sulfated polysaccharide derived from red-tide microalga Gyrodinium impudicum strain KG03 with immunostimulating activity in vivo

The high-sulfate-containing exopolysaccharide p-KG03 is produced by the red-tide microalga Gyrodinium impudicum strain KG03. The immunostimulatory effects of this sulfated exopolysaccharide were investigated by isolating peritoneal macrophages from mice 10 or 20 days after they had received a single dose of p-KG03 (100 or 200 mg/kg body weight). The cytotoxicity of the isolated macrophages for B16 tumor cells was tested, as B16 tumor cells are sensitive to tumor necrosis factor α (TNF-α) and nitric oxide. The activities of natural killer cells from the p-KG03-treated mice against YAC-1 mouse lymphoma cells were also tested. The nonspecific immune functions mediated by natural killer cells and macrophages were increased by treatment with p-KG03 in vivo. These results suggest that p-KG03 has immunostimulatory effects and enhances the tumoricidal activities of macrophages and NK cells in vivo. In addition, p-KG03 treatment increased the plaque-forming cell response to sheep red blood cells, as well as the levels of IgM and IgG Exposure to p-KG03 also increased the production by macrophages of cytokines, such as interleukins -1β and -6, and TNF-α. This is the first report of a marine microalgal sulfated polysaccharide having immunostimulatory activities. The p-KG03 polysaccharide may be useful for the development of biotechnological and pharmaceutical products that incorporate bioactive marine exopolysaccharides.     

The antimetastatic effect of macrophages restored by indomethacin: concomitant tumor immunity model

The role of macrophages acting as immunologic antitumor effectors and promoters of tumor growth are poorly understood as yet. We investigated the role of macrophage in model of concomitant immunity (CI), a phenomenon of secondary tumor rejection during the primary tumor growth. It has been shown that the period of CI weakening can coincide with appearance of tumor metastases. We used mammary carcinoma (MC) artificial lung metastases to evaluate the influence of macrophages from various period of CI on the development of metastases in mice. Our results indicated that macrophages are responsible for the late period of CI weakening and suppression. To investigate weather prostaglandins can mediate suppressive effect of macrophages we used experiments with indomethacin and we found that inhibition of prostaglandin E2 synthesis by indomethacin restored antimetastatic effect of concomitant immune macrophages.     

Mechanisms of action of synthetic polyelectrolytes and polyampholytes on the immune response]

We investigated the effect of polyacrilic acid (PAA) on the immune response in mice of various strains on sheep red blood cells and also the influence of poly-2-methyl-5-vinyl-pyridine (PMVY), PAA and their statistical copolymers on antibody-forming cells (AFC) production in cultures of T- and B-lymphocytes in vivo. PAA was seen to increase accumulation of AFC in the spleen of mice depending on their genotypes. PMVP and PAA were found to intensify the cooperating interaction of T- and B-lymphocytes, whereas their copolymers exert quite an opposite effect. The injection of copolymers to the recipients of cooperating T- and B-lymphocytes practically results in the complete elimination of the cooperation effect between T- and B-lymphocytes in the immune response to sheep erythrocytes without cytostatic action of cell proliferation.     

Induction of impaired antitumor immunity by fusion of MHC class II-deficient dendritic cells with tumor cells

To dissect the role of Ag presentation through MHC class I and/or II pathways by dendritic cell (DC)-tumor fusion cells, we have created various types of DC-tumor fusion cells by alternating fusion cell partners. Fusions of MC38/MUC1 carcinoma cells with DC from wild-type (WT-DC), MHC class I knockout (IKO-DC), class II knockout (IIKO-DC), or class I and II knockout (I/IIKO-DC) mice created WTDC-fusion cells (FC), IKO-FC, IIKO-FC, and I/IIKO-FC, respectively. MHC class II- and MUC1-positive fusion cells were constructed by fusion of B16/MUC1 melanoma cells with IKO-DC (IKO/B16-FC). Immunization of MUC1 transgenic mice with 5 x 10(5) WTDC-FC, IKO-FC, IIKO-FC, or I/IIKO-FC provided 100, 91.7, 61.5, and 15.4% protection, respectively, against tumor challenge with MC38/MUC1 cells. In contrast, all mice immunized with irradiated MC38/MUC1 tumor cells or WT-DC developed tumors. One group of mice was immunized with 5 x 10(5) IKO/B16-FC and then challenged with B16/Ia(+)/MUC1 on one flank and MC38/MUC1 on the other flank. Immunization of these mice with IKO/B16-FC resulted in 100 and 78.6% protection against B16/Ia(+)/MUC1 and MC38/MUC1 tumor challenge, respectively. The antitumor immunity induced by immunization with IKO/B16-FC was able to inhibit the growth of MHC class II-negative tumor. In addition, in vivo results correlated with the induction of Ag-specific CTL. Collectively, the data indicate that MHC class II Ag presentation targeting activation of CD4 T cells is indispensable for antitumor immunity.     

Resistance to tumor growth mediated by Listeria monocytogenes. Destruction of experimental malignant melanoma by LM-activated peritoneal and lymphoid cells.

A murine experimental model of nonspecific tumor destruction mediated by cells activated by Listeria monocytogenes (LM) is described. B16 melanoma growth is prevented or suppressed in the syngeneic host when tumor cells are inoculated in contact with viable LM. In vitro, cultured B16 cells are destroyed by LM immune peritoneal or splenic cells in the presence of the bacterial antigen(s). Activation of LM immune cells in vitro is immunologically specific. Replacement of LM by sheep red blood cells or bovine serum albumin in the in vitro cultures aborts the cytotoxic effect. Further, no tumor cell killing is obtained when thioglycollate-induced or normal peritoneal cells are substituted for LM immune cells in the in vitro cultures. Normal spleen cells in the presence of LM are weakly cytotoxic for B16 cells. Normal peritoneal cells plus LM or LM alone are not. Elimination of thymus derived "T" cells by anti-theta C3H or rabbit anti-mouse brain serum (RAMB) abrogated the cytotoxic effect. Therefore, LM-induced tumor destruction probably occurs through nonspecific mechanism(s) consequent to activation of host "T" cells by specific immune reactivity to LM antigen(s).     

Mechanism of the suppressive effect of interferon on antibody synthesis in vivo.

Mouse interferon preparations significantly suppress the in vivo antibody response to sheep red blood cells (SRBC), a thymus-dependent antigen, and to Salmonella typhimurium lipopolysaccharide (LPS), a thymus-independent antigen. It is also possible to effect the late responses of antigen sensitive "memory" cells observed during secondary immunization by administration of interferon prior to primary immunization. The immunosuppressive activity of interferon was time- and dose-dependent. Maximum suppression was produced when animals were given 1.5 times 10-5 units of interferon between 4 and 48 hr before antigenic stimulation. These findings suggested that interferon affects some early event(s) in the process of antibody synthesis which might be related to the general inhibitory effect of interferon on rapidly dividing cells and viral m-RNA translation. In addition, the use of nonadherent spleen cell cultures from interferon-treated mice, immunized in vitro with a thymus-independent antigen, indicated that in this situation the inhibitory effect of interferon was due to an action on B lymphocytes. A variety of soluble "suppressive" factors are secreted by T cells as a consequence of activation by mitogens or specific antigens in vitro. Since T cells are recognized as one of the sources of interferon, it is suggested that interferon should be investigated as a suppressor T cell-produced lymphokine which can regulate B cell expression.     

Discordant effect of aspirin and indomethacin on intestinal tumor burden in Apc(Min/+)mice

Epidemiologic and animal studies indicate that sustained use of non-steroidal anti-inflammatory drugs (NSAIDs) have a chemopreventive effect against the incidence of colorectal neoplasia and subsequent mortality. We previously demonstrated that sulindac significantly reduces intestinal tumor load in Apc(Min/+)mice and the tumor regression was not necessarily correlated with prostaglandin biosynthesis. In the present study, we further investigate the relationship of NSAID treatment and tumorigenesis in the Apc(Min/+)mouse model. We demonstrate that indomethacin (9 ppm) is a very potent chemopreventive agent, reducing tumor load by 85% and significantly inhibiting basal and ex vivo prostaglandin formation (P< 0.006 and P< 0.0001, respectively). Aspirin (400 ppm) has a similar impact on reducing prostaglandin levels, but in contrast to indomethacin, is uneffective in reducing the tumor load. The data indicate a discordance between the impact of different NSAIDs on tumorigenesis in Apc(Min/+)mice.     

The interactions between indomethacin and cytotoxic drugs in mice bearing B-16 melanomas

We have compared the effects of indomethacin alone (100 microgram/mouse/day) with those of indomethacin plus adriamycin, 5-FU, nitrogen mustard, thioTEPA, and vincristine on B-16 tumor cell proliferation in vivo. As we have previously described, after four days of treatment with indomethacin, subcutaneous tumors were slightly smaller and lighter in weight, but contained more melanoma cells. Addition of indomethacin to cytotoxic regimens resulted in either no change or a decrease in the effectiveness of the chemotherapy. In previous studies we demonstrated that treatment of tumor-bearing mice with a long-acting synthetic analogue of PGE2 (di-M-PGE2) stimulated the synthesis of endogenous prostaglandins. In order to evaluate if these endogenously synthesized prostaglandins were responsible for the inhibition of B-16 growth in vivo, mice were treated with di-M-PGE2 or di-M-PGE2 plus indomethacin. Addition of indomethacin did not alter the tumor inhibitory effects of di-M-PGE2.