Klaus D. Elgert

Tumor‐induced immune dysfunction: the macrophage connection

Although macrophages (Mφs) mediate tumor cytotoxicity, display tumor‐associated antigens, and stimulate antitumor lymphocytes, cancer cells routinely circumvent these host‐mediated immune activities, rendering the host incapable of mounting a successful antitumor immune response. Evidence supporting a direct causal relationship between cancer and immune dysfunction suggests that the presence of neoplastic tissue leads to immunologic degeneration. Furthermore, substantial data demonstrate that tumor growth adversely alters Mφ function and phenotype. Thus, although Mφs can serve as both positive and negative mediators of the immune system, the importance of Mφs in tumor‐induced immune suppression remains controversial. This review focuses on the evidence that tumor‐derived molecules redirect Mφ activities to promote tumor development. Tumors produce cytokines, growth factors, chemotactic molecules, and proteases that influence Mφ functions. Many tumor‐derived molecules, such as IL‐4, IL‐6, IL‐10, MDF, TGF‐β1, PGE2, and M‐CSF, deactivate or suppress the cytotoxic activity of activated Mφs. Evidence that tumor‐derived molecules modulate Mφ cytotoxicity and induce Mφ suppressor activity is presented. This information further suggests that Mφs in different in vivo compartments may be differentially regulated by tumor‐derived molecules, which may deactivate tumor‐proximal (in situ) Mφ populations while concurrently activating tumordistal Mφs, imparting a twofold insult to the hosts antitumor immune response. J. Leukoc. Biol. 64: 275–290; 1998.

Induction of macrophage suppressor activity by fibrosarcoma-derived transforming growth factor-beta 1: contrasting effects on resting and activated macrophages.

Tumor‐derived transforming growth factor‐β1 (TGF‐β1) suppresses several immune responses. Because tumor growth induces macrophage (mφ) suppressor activity, we determined whether murine fibrosarcoma‐derived TGF‐β1 contributed to mφ‐mediated suppression of autoantigen‐ and alloantigen‐stimulated T cell proliferation. The murine fibrosarcoma Meth‐KDE cell line constitutively produced TGF‐β1. Meth‐KDE tumor‐bearing host (TBH) syngeneic splenic mφs suppressed autoantigen‐ and alloantigen‐stimulated normal host (NH) CD4+ T cell proliferation. Pretreatment with Meth‐KDE supernatants induced NH mφs to suppress T cell proliferation as much as TBH mφs. Anti‐TGF‐β1 antibody treatment reversed Meth‐KDE‐induced NH mφ‐mediated suppression. Recombinant TGF‐β1‐induced mφ‐mediated suppression was not blocked during inhibition of prostaglandin E2 (PGE2), nitric oxide (NO), or TGF‐β1 production. However, Meth‐KDE‐induced mφ‐mediated suppression was partly reduced when PGE2 production was inhibited. Pretreatment with tumor cell‐derived TGF‐β1, but not recombinant TGF‐β1, increased activated mφ PGE2 production. These results show that additional tumor‐derived molecules aid in TGF‐β1‐enhanced PGE2 production. Also, TGF‐β1 alone up‐regulates mφ synthesis of suppressor molecules that are different from PGE2, NO, and TGF‐β1. Although TGF‐β1 has direct suppressor activity on lymphocytes, these results show that release of tumor cell TGF‐β1 also induces mφ suppressor activity. J. Leukoc. Biol. 57: 919–928; 1995.

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.

Macrophage regulation of the T-cell allogeneic response during tumor growth

Abstract One way mixed lymphocyte reactions (MLR) were used to determine how in vivo tumor growth affects in vitro macrophage-T-cell interactions. When normal T-cells were used, reactivity to allogeneic cells required macrophages. In contrast, the nonadherent spleen cell population from week old tumor-bearing mice responded without additional macrophages. At all concentrations, addition of exogenous macrophages enhanced the MLR. Macrophages from normal as well as tumor-bearing mice (TBM) when treated with anti-theta serum and complement exhibited no inhibitory activity towards normal nylon wool nonadherent (NA) T-cells, at concentrations ranging up to 24%. Macrophage enhancement was not contact dependent, though the degree of enhanced activity was dependent upon the host stage of tumor development. During initial phases of tumor growth, T-cell reactivity was maximally enhanced by the presence of normal or TBM macrophages. In the late stages of tumor development, T-cells, when exposed to macrophages from normal or TBM, exhibited a subnormal level of reactivity. Though no in vitro macrophage inhibition of T-cell MLR activity could be detected, experiments are currently being conducted to determine the extent of macrophage involvement in the degeneration of the T-cell immune response during late stages of tumor growth.

Shifts in macrophage (Mφ) surface phenotypes during tumor growth: association of Mac-2+ and Mac-3+ Mφ with immunosuppressive activity

Rat anti-mouse monoclonal antibodies (mAb), anti-Mac-1, -2, and -3, directed against macrophage (M phi) glycoprotein surface antigens, were used to demonstrate a tumor-induced shift in peritoneal M phi subpopulations. This study of the tumor-induced shift was approached in two steps. First, to show that separate phenotypic M phi subpopulations existed and second, to show that a shift in these populations was involved in immunosuppression of the host during tumor growth. Endogenous peroxidase activity was examined among normal and tumor-bearing host (TBH) M phi. A significant increase in the number of peroxidase-positive M phi occurred during tumor growth. Indirect immunofluorescence showed a decrease in Mac-2+ cells and an increase in Mac-3+ cells in TBH M phi populations. When the mAb, anti-Mac-1,-2, and -3 were used in the presence of complement (C), they were cytotoxic for M phi and showed differential depletion of normal and TBH M phi. Peroxidase-positive TBH M phi were susceptible to C-mediated lysis by anti-Mac-1 and -3 but not by anti-Mac-2, whereas no direct relationship was observed among normal host M phi. To demonstrate differences between normal and TBH M phi subpopulations, soluble inhibitory factors were examined from mAb plus C-modified M phi populations. Anti-Mac plus C-treated normal and TBH M phi produced supernatants with different regulatory capabilities as assessed in the mixed-lymphocyte reaction (MLR). Anti-Mac-2 plus C treatment significantly reduced the ability of TBH M phi to produce a soluble suppressor(s) but did not alter normal host M phi-derived suppressor production. In contrast, anti-Mac-1 and -3 plus C treatment of normal host M phi significantly reduced suppressor production. In the TBH, however, anti-Mac-1 plus C had no effect, while anti-Mac-3 plus C had only a limited reduction as compared to the normal host. Determination of levels of prostaglandin E2 (PGE2) in M phi supernatants showed that normal host Mac-1+ M phi were involved in down regulation of PGE2 production. This control was missing in the TBH M phi. Mac-2+ M phi were the apparent producers of PGE2 which accounts for the factor-mediated MLR suppression attributed to TBH Mac-2+ M phi. Collectively, these data suggest that tumor-induced aberrations in immunoregulation can in part be attributed to differences in anti-Mac mAb-defined M phi subpopulations.

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.

Changes in Macrophage Populations: Phenotypic Differences between Normal and Tumor-Bearing Host Macro phages

As a tumor grows, changes occur in the function of macrophages (M phi). This is concomitant with changes in their phenotype. Flow cytometric analysis of monoclonal antibody (mAb)-labeled thioglycollate-elicited peritoneal, and resident splenic, M phi showed a tumor-induced shift of Mac-1, -2, -3, and Ia antigen expression. During tumor growth, the percentage of peritoneal Mac-2+, -3+, and Ia+ M phi decreased significantly (22%, 14%, and 58%, respectively), while Mac-1+ M phi remained unchanged. By analyzing the data on two-dimensional histograms and comparing the sizes of M phi to cell-surface antigen expression, we identified distinct subpopulations of peritoneal M phi. Three distinct size versus antigen expression M phi subpopulations were detected by flow cytometry and consisted of 10-16, 17-22, and 23-27 microns for the small-, medium-, and large-sized populations, respectively. Large-sized Mac-1+ and -2+ M phi decreased (37% and 38%), while large-sized Mac-3+ M phi did not decrease during tumor growth. Medium-sized Mac-3+ M phi decreased 33% during tumor growth, while no differences could be seen in medium-sized Mac-1+ or -2+ M phi. Concomitant with the decrease in large-sized Mac-1+ M phi was an increase in small-sized Mac-1+ M phi. Peritoneal Ia+ M phi were mostly small-sized (4-7-fold increase over the medium-sized and none in the large-sized population). M phi Ia antigen expression was nearly absent in the 21-day tumor-bearing host, with less than 4% of the cells labeling positive (a 73% drop from normal host M phi). In splenic M phi, the percentage of Mac-1+ M phi significantly increased (90%) during tumor growth, while Mac-2+ and -3+ M phi showed a smaller, but still significant, increase (48% and 40%, respectively). Additionally, splenic Ia+ M phi significantly decreased (29%) during tumor growth. More important than the decreased cell numbers was the significant decrease in Ia antigen expression per cell. Unlike the peritoneal M phi, the splenic M phi did not show distinct size versus antigen expression subpopulations, although there was an overall difference in M phi size between normal and TBH. These data suggested that M phi from different anatomical sites are phenotypically different and tumor growth mediates phenotypic alterations in peritoneal and splenic M phi populations. This may be the source of tumor-induced dysfunction of M phi-mediated immune activity.

Prostaglandin E2 production by Mac-2+ macrophages: tumor-induced population shift.

Tumor growth induced a shift in the phenotype of macrophages (MΦ) responsible for factor‐mediated suppression of allogeneic mixed lymphocyte reactions (MLR), and the suppression by tumor‐bearing host (TBH) Mac‐2+ MΦ was in part due to production of prostaglandin E2 (PGE2). Thioglycollate‐elicited peritoneal MΦ from normal and TBH BALB/c mice were modulated with anti‐Mac‐1, ‐2, or ‐3 monoclonal antibodies (mAb) or depleted with mAb plus complement and cultured in the presence or absence of indomethacin. Culture supernatants derived from mAb plus complement‐depleted Mo were added to the MLR at time of initiation and showed that the suppressor phenotype shifted from Mac‐3+ in the normal host to Mac‐2+ in the TBH. Mac‐1+ MΦ also appeared to be involved in suppression by normal host, but not TBH, MΦ. Loss of MLR suppression (increase in MLR reactivity) correlated with an increase in protein content of the culture supernatants. In an effort to explain both this relationship and the mechanism of MLR suppression, PGE2 levels of culture supernatants were determined by radioimmunoassay. Mac‐1+ MΦ were involved in the regulation of PGE2 production in normal hosts, as both activation and depletion caused an increase in PGE2 production. Depletion caused a more dramatic increase in PGE2 production than did activation, suggesting that Mac‐1+ MΦ had a dampening effect on PGE2 production. In contrast, no Mac‐1+ MΦ‐mediated regulatory function occurred in the TBH. Mac‐3+ MΦ were involved in the regulation of PGE2 production in both normal and TBH. Mac‐2+ MΦ were the primary producers of PGE2 in the TBH, but not in the normal host, as their depletion in the TBH caused a significant loss of PGE2 production. Thus, immunosuppression in the TBH was at least partly due to the inability of Mac‐1+ and/or Mac‐3+ MΦ to control production of PGE2 by Mac‐2+ MΦ.

Variations in macrophage antigen phenotype: a correlation between Ia antigen reduction and immune dysfunction during tumor growth.

Variable la antigen expression by macrophages (Mφ) was examined during tumor growth by measuring: i) la antigen masking and immunofluorescence by anti‐la antibody, ii) accessory cell function in concanavalin A (Con A) and mixed lymphocyte reaction (MLR)‐induced T cell proliferation, and iii) Mφ stimulatory function in the MLR. Tumor‐induced progressive loss of la antigen expression was shown by immunofluorescence and corroborated by anti‐la blockade of MLR stimulatory activity of normal but not tumor‐bearing hosts (TBH) splenic Mφ. The TBH splenic Mφ supported Con A‐induced proliferation of syngeneic T cells (la antigen‐independent) but did not support syngeneic T cell proliferation in the MLR (la antigen‐dependent). Irrespective of tissue source, normal and TBH Mφ differed in their MLR stimulatory capabilities. In general, splenic Mφ preparations were better stimulators of allogeneic T cell blastogenesis in the MLR than thioglycollate‐elicited peritoneal Mφ. Kinetic studies with TBH Mφ showed a significant progressive loss in MLR stimulatory activity, which was especially pronounced with peritoneal Mφ. Expression of la antigens by normal but not TBH Mφ were diminished by 24‐h in vitro plating of the peritoneal Mφ. Indomethacin treatment showed Prostaglandin E2 was not a direct in vitro factor in la antigen‐mediated reduction of splenic Mφ MLR stimulatory activity. Taken together, these data delineate a loss of Mφ la antigen expression, resulting in a decrease in la antigen‐mediated functional activities during tumor growth.