Carol J. Burger

Interleukin-12 Overcomes Paclitaxel-Mediated Suppression of T-Cell Proliferation

The antineoplastic agent paclitaxel (TAXOL) is a potent inhibitor of tumor cell division and a useful chemotherapeutic for the treatment of refractory ovarian and breast carcinoma. Multiple immune system actions have been ascribed to paclitaxel, including the capacity to induce macrophage antitumor cytotoxic molecule production. However, T-cells are susceptible to paclitaxels cytostatic functions, and no studies have investigated the effects of direct paclitaxel administration on lymphocyte function in the tumor-bearing host (TBH). Because paclitaxel is currently used as an antitumor chemotherapeutic agent and tumor growth alters leukocyte functions, we assessed T-cell function following chemotherapeutic-type paclitaxel treatment. Paclitaxel administration significantly compromised the proliferative capacity of both normal host and TBH lymphocytes in vitro. Although tumor growth impaired T-cell interferon-gamma (IFN-gamma) production, paclitaxel treatment did not alter IFN-gamma. We speculate that the immunostimulatory cytokine interleukin-12 (IL-12), which promoted T-cell activation and proliferation, was capable of reversing paclitaxel-mediated immunosuppression. Exogenous IL-12 fully reconstituted proliferative reactivity and enhanced IFN-gamma production by both normal host and TBH lymphocytes in vitro. Collectively, these data suggest that chemotherapeutic paclitaxel regimens impart significant but reversible inhibition of lymphocyte populations, and IL-12 may be a useful ancillary immunotherapeutic to overcome paclitaxel-induced modulation of lymphocyte activities.

Taxol-mediated changes in fibrosarcoma-induced immune cell function: Modulation of antitumor activities

The anticancer drug taxol (paclitaxel) inhibits tumors through multiple cytotoxic and cytostatic mechanisms. Independently of these mechanisms, taxol induces distinct immunological efficacy when it acts as a second signal for activation of tumoricidal activity by interferon-γ(IFNγ)-primed murine normal host macrophages. We reported that tumor-distal macrophages, which mediate immunosuppression through dysregulated nitric oxide (NO) and tumor necrosis factor α (TNFα) production, are differentially regulated by taxol. Because taxol influences tumor cell growth dynamics and activates immune cell populations, we assessed the ex vivo immunosuppressive and antitumor activities of taxol-treated normal host and tumor-bearing host (TBH) macrophages. Pretreatment of such cells with taxol partly reconstituted T cell alloantigen reactivity, suggesting that taxol mediates a limited reversal of TBH macrophage immunosuppressive activity. Taxol-treated TBH macrophages significantly suppressed the growth of fibrosarcoma cells (Meth-KDE) through soluble effector molecules and promoted direct cell-mediated cytotoxicity, indicating that taxol enhanced tumor-induced macrophage antitumor activities. Tumor-induced helper T cells, however, showed a higher sensitivity to direct taxol-induced suppression. These data demonstrate that taxol exerts pleiotropic effects on antitumor immune responses with the capacity to abate the immunosuppressive activities of macrophages and promote macrophage-mediated anti-tumor activities simultaneously, but also directly modulating T cell reactivity. Collectively, these studies suggest that the antineoplastic drug taxol may impart antitumor activity through an immunotherapeutic capacity.

Taxol, a microtubule-stabilizing antineoplastic agent, differentially regulates normal and tumor-bearing host macrophage nitric oxide production

Taxol, a potent antitumor chemotherapeutic, promotes in vitro cytotoxic antitumor activities by normal host macrophage (M phi s). Because tumor growth induces functional changes among M phi populations, we determined whether fibrosarcoma growth (Meth-KDE) modified M phi responsiveness to the activating agent taxol. Tumors induce tumor-distal M phi populations to become immune suppressor cells, partially through overproduction of the cytotoxic and proinflammatory molecules nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha). Beneficial to the tumor-bearing host (TBH) when released by tumor-proximal M phi s, NO and TNF-alpha suppress lymphoproliferation and fail to impart antitumor activity when expressed in tumor-distal compartments. We report that taxol differentially regulated normal host and TBH M phi production of the immunosuppressive molecule NO by tumor-distal M phi populations. In response to IFN-gamma-priming and taxol triggering, TBH M phi s increase their production of NO as compared to resting M phi s; however, unlike normal host M phi s, taxol-induced TBH M phi NO production was significantly suboptimal. Modulation of TBH M phi NO production in tumor-distal compartments may alleviate M phi-mediated suppression of T-cell proliferative responses, yet promote sufficient NO production by tumor-associated M phi s to affect cytotoxicity. Collectively, these data leave implications for immunotherapeutic activities by the anticancer drug taxol.

Interleukin 2 (IL-2) activity during tumor growth: IL-2 production kinetics, absorption of and responses to exogenous IL-2

A temporal study assessed the relationship between fibrosarcoma growth and immunologic encumbrance due to the inability of BALB/c mouse splenocytes to elaborate the lymphokine Interleukin 2 (IL-2). Nylon-wool fractionation and antiserum treatments suggested the existence of a mildly nylon-wool-adherent, anti-Lyt 2-sensitive tumor-induced suppressor T (Ts) cell which significantly decreased IL-2 activity. Absorption investigations indicated that ligand-activated tumor-bearing host (TBH) spleen cells were less receptive to IL-2 than their normal counterparts. When splenocytes were antiserum treated before absorption, removal of Lyt 2+ (suppressor T) cells resulted in greater IL-2 absorption by the remaining cells. Purified IL-2 only partially restored suppressed TBH spleen cell mitogen- or alloantigen-induced blastogenesis; whereas, normal host reactivity was significantly augmented. The collective data suggest that TBH spleen cells were capable of producing IL-2 and of responding to the IL-2 amplification signal when tumor-induced Ts cells were depleted.

Tumour Growth Causes Suppression of Autoreactive T-Cell Proliferation by Disrupting Macrophage Responsiveness to Interferon-γ

Normal immune homeostasis is regulated partly by a small population of CD4+ T cells that react to autologous major histocompatibility complex class‐II molecules on self‐cells. Decreased autoreactive T‐cell responses are associated with cancer. Tumour growth causes syngeneic macrophages (Mø) to suppress autoreactive T‐cell proliferation by decreasing Mø class‐II expression and increasing Mø production of the suppressor molecule prostaglandin E2 (PGE2). Because interferon‐γ (IFN‐γ) is a potent Mø activation molecule which regulates both Mø PGE2 and class‐II expression, the effects of IFN‐γ on tumour‐induced suppression of autoreactive T‐cell proliferation were investigated. Exogenous IFN‐γ increased normal host (NH) CD4+ autoreactive T‐cell proliferation stimulated by syngeneic NHMø but decreased proliferation stimulated by tumour‐bearing host (TBH) Mø. Antibody (Ab) neutralization of endogenous IFN‐γ activity reduced TBH Mø‐mediated suppression. Kinetic studies showed that endogenous IFN‐γ suppressor activity was not exclusive during T‐cell activation. Indomelhacin treatment blocked IFN‐γ‐induced suppression in TBH Mø‐T cell cultures. TBH Mø‐T cell cultures contained significantly more PGE2 than those containing NH Mø. Exogenous IFN‐γ increased early PGE2 production in TBH Mø cultures but decreased production in NHMø cultures. The Ab‐mediated neutralization of endogenous transforming growth factor‐β or tumour necrosis factor‐x reduced TBH Mμ‐mediated suppression and blocked IFN‐γ‐induced suppression. Short‐term treatment of Mμ with IFN‐γ before their addition to T cells caused TBH Mμ to stimulate T‐cell proliferation, which suggests that early suppressor molecule production by TBHMø inhibits synthesis or activity of IFN‐γ‐induced stimulatory monokines. These results show that tumour growth causes Mø to suppress autoreactive T‐cell responses by allowing IFN‐γ to induce Mø suppressor molecules, which block production or activity of stimulatory monokines.

Fibrosarcoma‐induced increase in macrophage tumor necrosis factor α synthesis suppresses T cell responses

Tumors down‐regulate T cell responses partly by increasing macrophage (mφ) production of the suppressive molecule prostaglandin E2 (PGE2). Because tumor growth increases mφ tumor necrosis factor a (TNF‐α) production and TNF‐α stimulates mφ PGE2 synthesis, we examined the contribution of TNF‐α to fibrosarcoma‐induced mφ ‐mediated suppression of allo‐ reactive CD4+ T cell proliferation. We showed that tumorbearing host (TBH) mφs express high levels of TNF‐α mRNA, which leads to increased lipopolysaccharide‐ induced TNF‐α production. Timor cells were directly involved in mφ TNF‐α synthesis because fibrosarcoma cells induced normal host (NH) mφ s to produce TNF‐α. Addition of TBH mφ s to allogeneic mixed lymphocyte reaction (MLR) cultures suppressed CD4+ T cell proliferation more than NH mφ s. The neutralization of endogenous TNF‐α activity with anti‐TNF‐α antibody (Ab) treatment reversed TBH, but not NH, mφ ‐mediated suppression. Conversely, exogenous TNF‐α increased NH or TBH mφ‐mediated suppression but stimulated T cell proliferation without mφ s. Kinetic treatment of MLR cultures with anti‐TNF‐α Ab or TNF‐α showed that TNF‐ αproduction and activity occurred at the beginning of T cell proliferation. When arachidonic acid metabolite synthesis was inhibited, TNF‐α‐induced suppression was blocked in NH mφ ‐containing cultures and completely reversed in TBH mφ‐containing cultures. A PGErspecific enzyme‐linked immunosorbent assay showed that TNF‐a addition increased PGE2 production in NH mφ ‐ containing cultures to that of TBH mφ ‐containing cultures. Exogenous PGE2 did not affect the TNF‐α enhancement of T cell proliferation without mφ s. Therefore, suppression induced by TNF‐α was caused by increased mφ PGE2 production and not by TNF‐α in concert with PGE2. Even though TNF‐a is known to enhance lymphocyte proliferation, we show that in the presence of mφs, the main TNF‐α producers, TNF‐α suppresses T cell proliferation. Perhaps increased TNF‐α production during pathological states, such as cancer, triggers the initial stages of suppression.

Tumor modulation of autoreactivity: Decreased macrophage and autoreactive T cell interactions☆

The autologous mixed lymphocyte reaction (AMLR) is an in vitro measure of autoreactivity, a key mechanism in immune homeostasis. In this system, macrophages (M phi) act as accessory cells to autoreactive L3T4+ T cells by presenting self-Ia and releasing soluble modulators. During tumor growth, changes occur in M phi and T cells. Tumor-bearing host (TBH) M phi have a reduced ability to act as accessory cells. In fact, TBH M phi suppressed autoreactivity by 60-70%. The decrease in TBH M phi or T-cell abilities was not due to differences in cell numbers or incubation time. Because tumor growth causes increased prostaglandin E2 (PGE2) production by M phi, indomethacin was used to assess the contribution of prostaglandins. Normal and TBH T-cell reactivity increased nearly 50% when stimulated by normal host M phi, while normal and TBH T-cell reactivity increased nearly 100% when stimulated by TBH M phi. Thus increased prostaglandin production is partly responsible for the increased TBH suppressor M phi activity and in the normal host, suppressor M phi may be responsible for maintaining immune regulation. To assess the direct role of prostaglandins in T-cell hyporesponsiveness, PGE2 was titrated into the cultures. PGE2 suppressed normal and TBH T-cell responsiveness in a dose-dependent manner. Normal host T cells were suppressed to a greater extent than TBH T cells by PGE2 (66% versus 42% suppression, respectively). Reduced Ia expression and active suppressor mechanisms are not the only mechanisms mediating hypoautoreactivity during tumor growth. TBH autoreactive L3T4+ T cells were less responsive to self-Ia; they were only 60-80% as reactive as their normal counterparts. To address whether the helper T (TH)-cell defect involved cytokines, T cells were treated with interleukin (IL)-1, IL-2, and IL-4. In all cases, the TBH T-cell response to the factors was decreased (only 60-75% as reactive as normal T cells). Because TBH M phi-mediated suppression can override the addition of IL-1, IL-2, and IL-4, indomethacin was also added with the exogenous interleukins. This coaddition significantly enhanced normal host autoreactivity above control levels while TBH autoreactivity (the combination of TBH T cells and TBH M phi) only returned to normal host unstimulated levels. Tumor growth modulates the immune response at least by (i) decreasing the accessory cell abilities of TBH M phi through decreased Ia expression and increased production of suppressive molecules such as prostaglandins; and (ii) decreasing the responsiveness to immune enhancing factors by TH cells.

Tumor growth modulates macrophage nitric oxide production following paclitaxel administration.

The antineoplastic agent paclitaxel (Taxol) mimics bacterial lipopolysaccharide (LPS) in normal host macrophages (Mphis), enhancing antitumor cytotoxicity in vitro. Because paclitaxel is used as an antitumor chemotherapeutic agent and tumor growth alters Mphi phenotype and function, we assessed effector molecule production and cytotoxic activity by normal host and tumor-bearing host (TBH) Mphis following paclitaxel administration. Paclitaxel treatment, duplicating human chemotherapeutic regimens, primed normal host splenic Mphis for enhanced production of the cytotoxic mediator nitric oxide (NO); in contrast, paclitaxels NO-inducing activity was significantly suppressed in TBHs. In contrast to NO regulation, Mphi capacity for tumor necrosis factor-alpha (TNF-alpha) production in both normal hosts and TBHs was enhanced by paclitaxel administration. Although tumor growth modulated paclitaxel-induced Mphi NO production, paclitaxel administration enhanced both normal host and TBH Mphi cytotoxic antitumor activity. Blocking NO with a competitive inhibitor abrogated Mphi cytotoxicity, suggesting paclitaxel-induced TBH Mphi NO production, although suboptimal, remains sufficient to mediate antitumor activity. These data demonstrate that paclitaxels in vivo immune activities are differentially regulated during tumor burden and suggest that paclitaxels immunotherapeutic functions may contribute to its success as an anticancer agent.

Tumor-induced modulation of macrophage class II MHC molecule mRNA expression

Class II MHC protein expression in macrophages (M phi) is reduced during tumor growth. Because regulation of class II MHC proteins occurs during transcription, tumor growth may suppress class II MHC protein expression by suppressing mRNA. The decrease in class II mRNA may result from (i) a decrease in M phi responsiveness to an inducing agent, such as interferon-gamma (IFN-gamma), or (ii) an increase in M phi sensitivity to suppressing agents, such as prostaglandin E2 (PGE2). To determine how tumors induce suppression of class II mRNA, M phi were cultured in the presence of IFN-gamma with or without other factors, and Northern blot analyses were performed. Unstimulated normal host (NH) or tumor-bearing host (TBH) M phi do not express detectable class II mRNA. The addition of IFN-gamma induces class II mRNA expression in NH and TBH M phi, but class II mRNA expression is significantly lower in TBH M phi. Kinetic studies suggested that NH M phi class II mRNA is induced faster and in greater amounts than TBH M phi class II mRNA. There is a decrease in M phi class II mRNA stability during tumor growth that may account for the decreased induction by IFN-gamma. Lipopolysaccharide (LPS) suppresses class II mRNA induction in both NH and TBH IFN-gamma-treated M phi, but TBH M phi are more sensitive to its suppression. PGE2 and tumor-necrosis factor-alpha (TNF-alpha), two factors produced by LPS-stimulated M phi, were tested for their ability to modulate class II mRNA expression in NH and TBH IFN-gamma-treated M phi. PGE2 suppressed class II mRNA expression in both NH and TBH M phi. The addition of TNF-alpha to IFN-gamma-treated M phi suppressed class II mRNA in NH M phi but, surprisingly, had an additive effect on IFN-gamma-induced class II mRNA expression. TNF-alpha did not induce class II mRNA expression in TBH M phi in the absence of IFN-gamma. The cause of the reduced class II mRNA expression during tumor growth is a decreased response to IFN-gamma and an increased sensitivity to PGE2. This change may cause the observed suppression mediated by TBH M phi.

Interferon-γ reduces tumor-induced Ia− macrophage-mediated suppression: role of prostaglandin E2, Ia, and tumor necrosis factor-α

Tumor growth enhances macrophage (M phi) suppressor activity by causing M phi to increase synthesis of inhibitory molecules such as prostaglandin E2 (PGE2) or decreasing their expression of up-regulatory molecules such as the class II MHC protein Ia. Although these tumor-induced changes are correlated, it is unknown whether tumor-bearing host (TBH) Ia- M phi become more suppressive by increasing their PGE2 synthesis. To assess the role of PGE2 in tumor-induced Ia- M phi-mediated suppression of CD4+ T-cell alloreactivity, unseparated (Ia(+)-enriched) or Ia(+)-depleted (Ia-) populations of murine normal host (NH) or TBH splenic M phi were added to mixed lymphocyte reaction (MLR) cultures. NH or TBH Ia- M phi were significantly more suppressive than their respective unseparated populations, and TBH Ia- M phi were more suppressive than their NH counterparts. When PGE2 production was blocked with indomethacin, TBH Ia- M phi-mediated suppression was reduced more than suppression mediated by all other M phi populations. A PGE2-specific ELISA showed more PGE2 in Ia- M phi-containing cultures than in those with whole M phi and more in cultures containing TBH Ia- M phi than in their NH counterparts. Because interferon-gamma (IFN-gamma) is a potent M phi activation molecule that regulates both Ia expression and PGE2 production, the effects of IFN-gamma on tumor-induced Ia- M phi-mediated suppression were investigated. Exogenous IFN-gamma reduced suppression mediated by all M phi populations except NH unseparated M phi. IFN-gamma suppressed alloreactivity without M phi or with NH unseparated M phi. Suppression mediated by NH or TBH Ia-, and TBH unseparated M phi was also reduced when M phi were pre-incubated with IFN-gamma before their addition to MLR cultures. IFN-gamma addition did not block Ia- M phi-mediated suppression by decreasing M phi PGE2 production. In fact, IFN-gamma addition increased PGE2 production two-fold in MLR cultures. However, IFN-gamma partly reduced suppression mediated by exogenous PGE2 added to M phi-depleted cultures. Cytofluorometric analysis showed that IFN-gamma increased the percentage of Ia+ M phi in NH and TBH Ia- M phi populations. Blocking TNF-alpha activity with anti-TNF-alpha antibodies caused IFN-gamma to suppress alloreactivity in all M phi-added cultures. Collectively, these data show that tumor-induced suppression mediated by Ia- M phi is caused by increased PGE2 synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)