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(Journal of Leukocyte Biology. 2001;69:129-137.)
© 2001 by Society for Leukocyte Biology

Tumor cell-derived TGF-ß and IL-10 dysregulate paclitaxel-induced macrophage activation

David W. Mullins, Ryan S. Martins, Carol J. Burger and Klaus D. Elgert

Department of Biology, Microbiology and Immunology Section, Virginia Polytechnic Institute and State University, Blacksburg, Virginia

Correspondence: Dr. Klaus D. Elgert, Department of Biology, Microbiology and Immunology Section, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0406. E-mail: kdelgert{at}vt.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Paclitaxel (TAXOLTM) activates in vitro macrophage (Mø) expression of proinflammatory and cytotoxic mediators, including IL-12, tumor necrosis factor {alpha} (TNF-{alpha}), and nitric oxide (NO). However, tumors dysregulate Mø through soluble suppressor molecules, and it is possible that tumors evade paclitaxel-mediated immune effector function through the production of immunomodulatory molecules and inhibition of Mø function in situ. Because Mø activation in the tumor microenvironment is a desirable goal of anti-tumor immunotherapy, we evaluated whether tumor-derived immunomodulatory factors dysregulate paclitaxel-mediated Mø activation. Tumor cell-derived supernatant suppressed paclitaxel’s capacity to induce IL-12, TNF-{alpha}, and NO production by RAW264.7 Mø. Tumor factors also dysregulated paclitaxel-induced expression of a HIV-LTR, promoter-driven luciferase construct in RAW264.7 Mø, suggesting that tumors may inhibit a broad range of Mø functionality. Depletion studies revealed that IL-10 and transforming growth factor-ß1 (TGF-ß1), but not prostaglandin E2 (PGE2), impaired paclitaxel-mediated activation, suggesting that abrogation of these factors in situ might restore paclitaxel’s activating capacity and enhance anti-tumor efficacy.

Key Words: immunosuppression • tumor necrosis factor {alpha} • interleukin-12 • prostaglandin E2 • nitric oxide


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Paclitaxel (registered trade name TAXOLTM) (reviewed in [1 2 ]) is a potent chemotherapeutic agent originally extracted from the bark and leaves of the Western yew Taxus brevifolia. Paclitaxel’s primary mechanism of antineoplastic activity derives from its capacity to halt cell-cycle progression by inhibiting dynamic depolymerization of {alpha}/ß tubulin polymers, leading to rapid neoplastic cell death and inhibition of tumor progression [3 ]. Paclitaxel has been approved to treat breast, ovarian, and lung cancers as well as AIDS-related Kaposi’s sarcoma [4 ].

In addition to its antineoplastic activities, paclitaxel imparts cell cycle-independent effects on immune cell populations, including natural killer cells [5 ], T cells [6 7 ], and macrophages (Mø) [8 9 10 11 12 ]. In murine Mø, paclitaxel stimulates in vitro responses similar to those induced by bacterial lipopolysaccharide (LPS) [13 14 ]. Paclitaxel enhances production of the cytotoxic mediators nitric oxide (NO) and tumor necrosis factor {alpha} (TNF-{alpha}) [8 15 ] and expression of the immunostimulatory cytokine interleukin (IL)-12 [12 ] by Mø from tumor-distal compartments (including the spleen and peritoneal cavity). Furthermore, paclitaxel acts as a second signal for activation of in vitro tumoricidal activity by interferon-{gamma} (IFN-{gamma})-primed, tumor-bearing host (TBH), murine Mø [8 9 ].

To take full advantage of paclitaxel’s immunostimulatory action, we must first understand the impediments to Taxol-mediated activation in context of the tumor microenvironment. Tumors evade anti-tumor immune effector function, in part, through the production and release of factors that down-regulate tumor-associated Mø (TAM) cytotoxic and effector-molecule production [15 16 ]. Tumors maintain elevated levels of prostaglandin E2 (PGE2), transforming growth factor-ß1 (TGF-ß1), and IL-10 [15 ] in situ, rendering tumor-associated Mø incapable of IFN-{gamma} and LPS-induced NO production [17 ] or IL-12 expression [18 ]. Paclitaxel’s efficacy as a Mø-activating agent in the tumor microenvironment remains uncertain.

In the present study, we assessed paclitaxel’s ability to activate Mø effector functions in the presence of tumor-derived cytokines. We used the RAW264.7 Mø cell line transfected with a luciferase reporter gene under the transcriptional control of a nuclear factor-{kappa}B (NF-{kappa}B)-responsive HIV-long-terminal repeat (LTR) promoter. This Mø cell line (referred to as a4) facilitates the characterization of paclitaxel-mediated Mø activation and factor production in the absence or presence of tumor-derived factors. Using this as a model of TAM, we show that tumor-derived factors dysregulate paclitaxel-mediated Mø activation, limiting Mø production of effector molecules and immunostimulatory cytokines. Combined with our previous studies, these data suggest that paclitaxel has immune-activating properties that could provide immunotherapeutic activity but that are dysregulated by tumor-derived suppressor molecules. Through an enhanced understanding of paclitaxel’s immunopharacologic efficacy, novel therapeutic maneuvers may restore paclitaxel’s activating capacity in situ.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tumor cell culture and supernatant preparation
The BALB/c-derived fibrosarcoma designated methylcholanthrene-induced nonmetastatic murine fibrosarcoma (Meth-KDE) [15 ] was used to generate tumor-derived supernatants. The parental tumor is a nonmetastatic fibrosarcoma originally induced by methylcholanthrene [19 ] and maintained in hosts by transplantation. For in vitro studies, the Meth-KDE cell line was established from homogenized tumor mass. Following 2 h of culture (37°C, 5% CO2) in 60 mm glass dishes, nonadherent cells were collected and treated with anti-CD4 [American Type Culture Collection (ATCC); clone GK1.5], anti-CD8 (ATCC; clone 3.155), anti-IAd (ATCC; clone MK-D6), and B cell and anti-immature T cell (ATCC; clone J11.D) antibody (Ab) plus Low-Tox-M rabbit complement (Accurate Chemical Company, Westbury, NY). The remaining tumor cells were cultured for at least 30 days. Meth-KDE cells were maintained by diluting 1:10 in fresh medium every fifth day. Flow cytometric analyses revealed no detectable T cells, B cells, or macrophages, as assessed by staining with phycoerythrin (PE)-conjugated antibodies for CD3, CD45R, and CD14 (Pharmingen, La Jolla, CA), respectively.

Tumor supernatant was obtained by culturing the purified Meth-KDE tumor (4x106 cells/ml at 37°C, 5% CO2) in 24-well culture plates (Corning Cell Wells, Corning, NY) in a total vol of 1.0 ml complete RPMI medium. Cell-free supernatants were collected by centrifugation (400 g, 5 min) at 72 h (optimal time) and passed through a 0.4 micron filter. Fresh Meth-KDE supernatants were depleted of IL-10 or TGF-ß1 as described [15 ]. Briefly, tumor supernatant (1.0 ml) was incubated (2 h at 37°C) in 24-well plates that were coated with cytokine-specific Abs. Prior to addition of the tumor supernatants, plates were coated with anti-IL-10 or anti-TGF-ß1 by adding concentrated Ab preparations to wells, incubating overnight at 4°C, and washing three times with phosphate-buffered saline (PBS). Wells were blocked with sterile PBS containing 5% fetal bovine serum (FBS) for 2 h at room temperature and washed three times with PBS. Meth-KDE supernatants (500 µl/well) were incubated in coated plates or control plates (non-Ab-coated, serum-treated wells) for 2 h at 37°C and then immediately transferred into assays. PGE2-depleted tumor supernatants were prepared by incubating Meth-KDE cells, as described, with 10-7 M indomethacin (Sigma Chemical Co., St. Louis, MO), an arachidonic acid pathway inhibitor. Resulting supernatants did not contain detectable PGE2, as measured by specific enzyme-linked immunosorbent assay (ELISA; Advanced Magnetics, Boston, MA).

Macrophage cell lines
The RAW264.7 Mø cell line (TIB 71), originally derived from the ascites of an Abelson leukemia virus, tumor-induced male mouse [20 ], was acquired from ATCC. The RAW264.7 Mø cell line transfected with a luciferase reporter gene downstream from the NF-{kappa}B-responsive, viral, HIV-1-LTR promoter (a4 Mø) [21 ] was generously provided by Dr. Matthew Sweet (Centre for Molecular and Cellular Biology, University of Queensland, Brisbane, Australia). Briefly, the cell line was established by electroporation of RAW264.7 Mø with the pMC1NeoPolyA neomycin phosphotransferase-expression plasmid (Stratagene, La Jolla, CA) containing HIV-chloramphenicol acetyltransferase (CAT) promoter driving the pGL-2Basic luciferase reporter gene (Promega, Madison, WI). Stable transfectants, subsequently referred to as a4 cells, were selected by incubation with 200 µg/ml G418 (Sigma) for 15 days. RAW264.7 Mø and a4 Mø were maintained by diluting 1:10 in fresh medium every fifth day.

Media and reagents
RAW264.7 Mø and a4 Mø were cultured in RPMI-1640 medium with 2 mM L-glutamine (Sigma). All media contained 50 µg/ml gentamicin sulfate (Tri-Bio Laboratories, State College, PA), 25 mM sodium bicarbonate (NaHCO3), and 25 mM HEPES buffer (Sigma). RPMI-1640 medium was endotoxin-free (<10 pg/ml) as assessed by the Limulus amebocyte lysate assay (Sigma). Cell cultures were supplemented with complete RPMI medium containing 5% FBS (Atlanta Biologicals, Norcross, GA). Paclitaxel (CalBiochem, La Jolla, CA) was dissolved in 100% dimethyl sulfoxide (DMSO; Mallinckrodt Chemical, Paris, KY) to 4 mM stock solution and stored at -80°C. Paclitaxel was diluted to assay concentrations in RPMI-1640 medium immediately before use. The final concentration of DMSO in cultures was <1%. Bacterial lipopolysaccharide (LPS; Escherichia coli serotype 026:B6) was purchased from Sigma. IFN-{gamma} (specific activity 2.98x106 U/ml; endotoxin content <10 pg/ml) was purchased from Genzyme, Inc. (Cambridge, MA). Recombinant TGF-ß1 (100 µg/ml) and rabbit anti-TGFß1 polyclonal Ab (6.5 mg/ml; endotoxin content <10 pg/ml) were generous gifts from Genentech, Inc. (South San Francisco, CA). IL-10 (specific activity 7000 U/ml) was acquired from DNAX (Palo Alto, CA), and anti-murine IL-10 mAbs were obtained from SXC-1 (ATCC; clone HB 10739) hybridoma supernatants. PGE2 and indomethacin were obtained from Sigma.

Luciferase assays
To assess reporter gene expression, a4 cell luciferase activity was measured. First, a4 cells (1x106 cells/well) were cultured overnight in 24-well, tissue-culture plates in a total vol of 1.0 ml complete medium. Cells were washed with fresh RPMI-1640 and recultured in a total vol of 1.0 ml with indicated treatment reagents in complete medium or Meth-KDE tumor supernatants. Culture was continued for 2 h, then cells were lysed with 20 µl cell lysis solution (Promega). Cellular debris were removed by centrifugation at 12,000 g for 5 sec, and cell extracts were assayed for total protein by the Lowry method using Sigma reagents.

Luciferase activity in cell extracts was determined using luciferin (Sigma) substrate in a MgCl2 buffer supplemented with 0.1 M adenosine 5'-triphosphate (ATP). Luciferin substrate mix (100 µL) was added to 10 µL room-temperature cell extract, and luciferase activity was recorded as relative light units using a Berthold (Bundoora, Australia) automated luminometer. According to the manufacturer’s recommendation, luciferase activity was assessed for 10 sec, beginning 10 sec after mixing the substrate and cell extract; luciferase-mediated light production was stable and optimal during this period (unpublished results). Activity was standardized to the concentration of protein in the cell extract and shown as relative light units (RLU)/µg total protein.

Mø nitrite production
RAW264.7 Mø (2x105 cells) were cultured in 96-well, flat-bottom, tissue-culture plates (Corning). Each well contained a total vol of 200 µL, serum-free, RPMI-1640 medium with indicated treatment reagents added at the start of culture. Supernatants for nitrite assessment were collected at 24 h (optimal time) following centrifugation (400 g, 5 min). Mø viability remained >95% throughout the culture periods as assessed by the MTT assay [22 ], and paclitaxel doses up to 35 µM did not decrease Mø viability significantly, as assessed by the Alamar blue (CalBiochem) viability assay [9 ] (unpublished results).

Nitrite levels in culture supernatants were measured using the Griess reagent [23 ]. Briefly, 100 µL Mø supernatants were added to 100 µL Griess reagent (0.1% naphthylenediamine dihydrochloride, 1.0% sulfanilamide, 2.5% H3PO4; Sigma), incubated at room temperature for 10 min, and absorbance read at 570 nm (MR 600 microplate absorbance reader; Dynatech Laboratories, Alexandria, VA).

Cytokine quantification
Cytokine production was induced by culturing Mø, as described for NO production, for 4 h (TNF-{alpha}) or 18 h (IL-12, optimal culture time). Supernatants were collected and immediately assayed for cytokine using specific ELISA. IL-12 content was determined using a p70-specific ELISA (IL-12 DuoSet®; R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol. The ELISA consisted of anti-mouse IL-12 p70 capture antibody adhered to high-affinity protein-binding plates (Nunc MaxiSorp® ELISA plates), biotinylated secondary antibody, and horseradish peroxidase (HRP)-conjugated, strepavidin-detection reagent. The limit of detection for IL-12 was approximately 10 pg/ml. TNF-{alpha} content was determined using a murine TNF-{alpha}-specific ELISA (Quantikine M, R&D Systems) following the manufacturer’s protocol. The limit of detection for TNF-{alpha} was 5 pg/ml. ELISA were developed with a Sigma tetramethylbenzidine liquid substrate system, and absorbance was determined at 450 nm using an MR-600 microplate reader (Dynex, Alexandria, VA).

Western analysis
For Western analysis of IFN consensus sequence-binding protein (ICSBP), Mø were cultured, and cellular proteins were prepared for 12 h. Cells were lysed in 50 mM Tris-Cl, pH 7.6, containing 10 µg/ml leupeptin and aprotinin (Sigma) and 300 mM NaCl. Membranes were pelleted by centrifugation (12,000 g), and protein was determined using the Lowry microtiter method. Protein (15 µg) was denatured by boiling in 2-mercaptoethanol (2-ME; 5%) and separated by sodium dodecyl sulfate-polyacrylamide chain reaction (SDS-PAGE) using a 10% ProtoGel (National Diagnostics, Atlanta, GA) vertical gel, transferred to nitrocellulose, and blocked using 5% nonfat milk. A polyclonal rabbit anti-mouse ICSBP Ab (Zymed Laboratories, Inc., South San Francisco, CA) was used at 2 µg/ml, according to the manufacturer’s recommendation. HRP-conjugated, goat anti-rabbit immunoglobulin G (IgG) secondary Ab (Transduction Laboratories, Lexington, KY) was used at 1:2000 dilution. Bound ICSBP was detected with Luminol reagent (Santa Cruz Biotechnology, Santa Cruz, CA).

Statistics and calculations
Triplicate cultures were tested for nitrite, TNF-{alpha}, IL-12, and luciferase activity. Data are averages of triplicate determinations in a single experiment, and error bars represent standard error of the mean (SE). All experiments were repeated at least three times, and representative experiments are shown. Comparisons were tested for significance by the Student’s t-test, and comparisons are significant at the p < 0.05 level, unless otherwise stated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Tumor cell-derived factors down-regulate paclitaxel-mediated Mø cytokine and NO production
Paclitaxel’s LPS-mimetic capacity to activate murine Mø has been characterized extensively; paclitaxel stimulates in vitro Mø production of cytokines and effector molecules, including TNF-{alpha} [8 ], IL-12 [12 ], and NO [9 ]. Production of these proinflammatory and cytotoxic factors by Mø in the tumor microenvironment could impart distinct immunologic anti-tumor activity. However, most studies have evaluated Mø directly ex vivo from normal, healthy hosts. We showed that recombinant TGF-ß1, IL-10, or PGE2 can inhibit paclitaxel-induced activation of primary (splenic and peritoneal exudate) murine Mø [15 ] and that tumor growth retards Mø IL-12 production ex vivo [12 ]. Thus, tumors may evade the immunotherapeutic effects of paclitaxel by suppressing Mø activation and cytotoxic effector function. Therefore, we evaluated the effect of tumor-derived factors on Mø cytokine and NO production in response to paclitaxel activation.

To evaluate the impact of tumor-derived factors on paclitaxel-mediated Mø activation, we cultured RAW264.7 Mø, without or with 72-h Meth-KDE-derived, cell-free supernatants (1:2 dilution). Previously, we showed that these fibrosarcoma cells produce substantial quantities of TGF-ß1, IL-10, and PGE2 in culture [15 ]. Cytokine production was determined by analyzing spent medium for IL-12 (Fig. 1A ) and TNF-{alpha} (Fig. 1B) using cytokine-specific ELISA. Paclitaxel (10 µM, optimal dose in our hands) induced TNF-{alpha} and IL-12 production. IFN-{gamma} priming enhanced paclitaxel-mediated activation, which correlates with our ex vivo data from normal mice [8 12 ]. Tumor cell-derived supernatant inhibited IL-12 and TNF-{alpha} production, also in parallel with our ex vivo data showing inhibited IL-12 production by TBH Mø [12 ]. Notably, tumor-derived supernatant factors significantly decreased paclitaxel’s capacity significantly to induce IL-12 production by IFN-{gamma}-primed Mø to only 25.5% of the level achieved in the absence of tumor-derived immunomodulatory factors. Although paclitaxel doses as low as 0.1 µM enhanced Mø IL-12 and TNF-{alpha} production, doses in excess of 10 µM did not enhance cytokine production further in the presence of tumor cell-derived supernatants (unpublished results).



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  		Figure 1. Tumor cell-derived factors down-regulate in vitro Mø cytokine and effector-factor production. RAW264.7 Mø (2x105 cells) were cultured without or with 72-h Meth-KDE-derived supernatants (1:2 dilution). Some cultures were primed with IFN-{gamma} (10 U/ml) and activated with paclitaxel (10 µM, optimal dose). Spent culture medium was collected after 4, 18, or 48 h and tested for IL-12 (A), TNF-{alpha} (B), and NO (C), respectively. IL-12 and TNF-{alpha} were evaluated by cytokine-specific ELISA. IL-12 ELISA were p70-specific. Parallel RAW264.7 Mø (2x105 cells) were cultured without or with 72 h Meth-KDE supernatants (1:2 dilution). NO production was measured by colorimetric Griess reaction. Data are averages and SE of triplicate, independent determinations from one of three similar experiments. For ICSBP evaluation (D), RAW264.7 Mø were cultured in fresh medium (lane 1) or primed with IFN-{gamma} (10–100 U/ml, lanes 2–4) for 12 h. Representative data from one of three similar experiments are shown.

 
Priming and activation with IFN-{gamma} (10 U/ml) and paclitaxel (10 µM) for 24 h enhanced RAW264.7 Mø NO production. Tumor-derived supernatant suppressed NO production regardless of priming and activation signals (Fig. 1C) , and Western analyses revealed that tumor cell-derived factors inhibited inducible NO synthase (iNOS) expression (unpublished results). Even in the presence of optimal paclitaxel doses (10.0 µM) and IFN-{gamma}, tumor-derived supernatant decreased Mø NO production by >29%. In conjunction with IFN-{gamma} priming, paclitaxel is capable of partially overcoming tumor-induced suppression of ex vivo Mø effector function [8 ]; the current data suggest that paclitaxel, in combination with IFN-{gamma} priming, may induce modest NO production. However, kinetic analyses revealed that paclitaxel-mediated NO production remained suppressed throughout the course of a 72 h in vitro co-culture with tumor supernatant (unpublished results), suggesting that prolonging paclitaxel exposure may not enhance Mø NO production further in the tumor microenvironment. These data complement ex vivo results demonstrating that TAM are functionally deactivated for NO production in the tumor microenvironment [17 ]. Collectively, these results suggest that paclitaxel may overcome the tumor-induced inhibition partly and stimulate low levels of Mø NO production.

Because tumor-derived factors can inhibit Mø production of IFN-{gamma}-inducible factors, such as IL-12, we assessed the effect of tumor-derived factors on expression of the recently described signal-transduction molecule ICSBP. Mø were primed with IFN-{gamma} (10–100 U/ml) for 24 h. In the absence of tumor-derived factors, RAW264.7 Mø ICSBP expression was increased in a dose-dependent manner by IFN-{gamma} (Fig. 1D) . Addition of 72-h Meth-KDE supernatants (1:2 dilution) suppressed IFN-{gamma}-induced ICSBP expression significantly, even when Mø were primed with high doses of IFN-{gamma} (100 U/ml). These data provide evidence that tumors affect Mø function by dysregulating IFN-{gamma} responsiveness through inhibition of the ICSBP signal-transduction pathway.

Tumor cell-derived factors inhibit paclitaxel-induced activation of a4 Mø reporter gene expression
A hallmark of Mø activation for anti-tumor effector function is the rapid induction of NF-{kappa}B-mediated signal pathways. Because we needed a system that would allow for rapid assessment of Mø response to activating agents in the context of tumor-derived factors, we acquired the a4 Mø cell line. This line was developed by transfecting RAW264.7 Mø with the HIV-1-LTR promoter-driving expression of a luciferase reporter gene construct [21 ]. Using 10 µM paclitaxel [or 50 ng/ml concanavalin A (Con A) as a negative activation control], we established that optimal luciferase expression (a measure of reporter gene expression) occurs following 2 h of incubation with Mø-activating reagents (Fig. 2A ), which agrees with the results of Sweet and Hume [21 ]. To verify that a4 Mø reporter gene expression is paclitaxel-inducible in a dose-dependent manner, a4 cells (1.0x106 cells) were cultured in 1.0 ml complete medium with 0.1–10 µg/ml LPS (serving as a positive control) or physiologically relevant doses (0.1–10 µM) of paclitaxel for 2 h (optimal time). Luciferase activity increased in a dose-dependent manner in response to LPS activation. In a profile mimicking LPS-mediated activation, paclitaxel increased a4 Mø luciferase activity in a dose-dependent manner (Fig. 2B) . The dose-dependent response of the reporter gene in this model resembles LPS closely and paclitaxel-mediated NO, TNF-{alpha}, and IL-12 production profiles shown in primary murine Mø [12 15 ].



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  		Figure 2. Paclitaxel induces reporter gene expression in a4 Mø. A4 Mø (1.0x106 cells) were cultured in 1.0 ml complete medium with Con-A (8 µg/ml) or paclitaxel (10 µM) for 24 h. Lysates were prepared for luciferase analysis at 1, 2, 4, 6, 12, and 24 h (A). To establish that a4 Mø activation is LPS- or paclitaxel-inducible, cells were cultured for 2 h with a range of doses (B). Data are averages and SE of triplicate, independent determinations from one of three similar experiments.

 
Because Meth-KDE fibrosarcoma cells produce soluble factors that modulate Mø anti-tumor effector activity (see Fig. 1 ), we assessed the effect of tumor cell-derived factors on LPS- and paclitaxel-mediated Mø activation using the a4 Mø cell line (Fig. 3 ). The a4 Mø were cultured, as described, without or with tumor cell-derived supernatants (1:2 dilution) from 72-h cultures of Meth-KDE cells (4x106 cells) and activated with LPS (10 µg/ml) or paclitaxel (10 µM) for 2 h. Tumor supernatant was strongly inhibitory of a4 cell activation, suppressing LPS-induced luciferase activity by 32–36% and paclitaxel-induced luciferase activity by 51–52%, respectively (Fig. 3) . At optimal paclitaxel dose (10 µM), luciferase activity increased twofold; in contrast, tumor cell-derived supernatant (1:2 dilution) modulated paclitaxel’s capacity to activate a4 Mø, restricting luciferase activity to a modest 7% increase compared with unactivated cells. Tumor supernatant suppressed paclitaxel-mediated activation of a4 Mø at concentrations as low as 15% of total culture medium, although the inhibitory effect titrated with increasing dilution of the tumor cell-derived supernatant (unpublished results). Increasing concentrations of paclitaxel (up to 35 µM, maximal dose without significant cell death) failed to overcome inhibition (unpublished results).



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  		Figure 3. Tumor cell-derived factors inhibit paclitaxel-induced a4 Mø activation. a4 Mø (1.0x106) were cultured without or with 72-h Meth-KDE, tumor cell-derived supernatants (1:2 dilution). Some cultures were activated with LPS (1.0 or 10 µg/ml) or paclitaxel (1.0 or 10 µM) for 2 h. Lysates were prepared and assayed for luciferase activity. Data are averages and SE of triplicate, independent determinations from one of three similar experiments. *p < 0.05 compared with similarly activated cells in supernatant-free media. Stippled line represents the level of reporter gene expression of unactivated cells, standardized to a value of 1.0 RLU/µg total protein.

 
Tumor cell-derived TGF-ß1 and IL-10 inhibit paclitaxel-induced Mø activation
The Meth-KDE fibrosarcoma produces several immunomodulatory factors, including TGF-ß1, IL-10, and PGE2 [15 16 ], all of which could potentially modulate paclitaxel-mediated activation of Mø. To determine the role of TGF-ß1 (Fig. 4 ) and IL-10 (Fig. 5 ) in the tumor-induced modulation of paclitaxel-mediated Mø activation, Meth-KDE supernatants were depleted of these cytokines by Ab absorption using anti-TGF-ß1 and anti-IL-10 (as described in [15 ]). The role of PGE2 was investigated using the arachidonic acid inhibitor indomethacin (10-7 M) to treat Meth-KDE cells, thus producing PGE2-free supernatants. Next, a4 Mø (1.0x106) were cultured without or with paclitaxel (10 µM) and supplemented with fresh or factor-depleted Meth-KDE supernatant (1:2 dilution). Similarly, activated a4 Mø cultures were treated with exogenous rTGF-ß1, rIL-10, or PGE2 at levels approximately equal to those measured in 72-h Meth-KDE cell supernatants [15 ]. After 2 h, lysates were prepared and assayed for luciferase activity. Parallel cultures were tested for NO production at 24 h, as described.



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  		Figure 4. Tumor cell-derived TGF-ß1 inhibits paclitaxel-induced Mø activation and NO production. a4 Mø (1.0x106) were cultured without or with LPS (10 µg/ml) or paclitaxel (10 µM). Some cultures were supplemented with fresh or TGF-ß1-depleted, Meth-KDE, tumor cell-derived supernatant (1:2 dilution); parallel cultures were treated with rTGF-ß1 (10 ng/ml). After 2 h, lysates were prepared and assayed for luciferase activity (A). NO production was determined at 24 h in parallel cultures (B). Data are averages and SE of triplicate, independent determinations from one of three similar experiments.

 


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  		Figure 5. Tumor cell-derived IL-10 inhibits paclitaxel-induced Mø activation and NO production. a4 Mø (1.0x106) were cultured without or with LPS (10 µg/ml) or paclitaxel (10 µM). Some cultures were supplemented with fresh or IL-10-depleted, Meth-KDE, tumor cell-derived supernatant (1:2 dilution); parallel cultures were treated with IL-10 (3.0 U/ml). After 2 h, lysates were prepared and assayed for luciferase activity (A). NO production was determined at 24 h in parallel cultures (B). Data are averages and SE of triplicate, independent determinations from one of three similar experiments.

 
Meth-KDE, tumor cell-derived supernatant down-regulated paclitaxel-induced, a4 Mø luciferase activity in all cultures. Depletion of TGF-ß1 (Fig. 4A) from tumor supernatant partially reversed suppression in paclitaxel-activated cultures. These data suggest that paclitaxel-induced activation pathways may be highly sensitive to TGF-ß1-mediated inhibition. Addition of physiologic levels [15 ] of recombinant TGF-ß1 (10.0 ng/ml) suppressed reporter gene expression following activation with paclitaxel (Fig. 4A) or 10 µg/ml LPS (unpublished results). Although depletion of TGF-ß1 reconstituted paclitaxel-mediated activation partially, it fully reversed tumor supernatant-mediated suppression of NO production (Fig. 4B) . The overall paucity of paclitaxel-induced NO production in this system emphasizes further the requirement for IFN-{gamma} priming of Mø to achieve a potent cytotoxic effector response.

In a manner similar to TGF-ß1, IL-10 modulated paclitaxel-mediated Mø activation. Depletion of IL-10 (Fig. 5A) from the tumor cell-derived supernatant reversed suppression significantly in LPS- (unpublished results) and paclitaxel-activated cultures. A physiologically relevant dose of recombinant IL-10 (3.0 U/ml) [15 ] suppressed paclitaxel-activated reporter gene expression (Fig. 5A) and NO production (Fig. 5B) . Depletion of IL-10 from supernatants also reconstituted LPS- (unpublished results) and paclitaxel-mediated NO production significantly (Fig. 5B) .

In contrast to TGF-ß1 and IL-10, tumor-derived PGE2 may not play a significant role in tumor-induced suppression of Mø activation. Supernatant from indomethacin (10-7 M)-treated Meth-KDE cells inhibited a4 Mø activation (Fig. 6A ), and exogenous PGE2 (25.0 ng/ml) was only moderately suppressive of paclitaxel-mediated Mø activation compared with TGF-ß1 or IL-10. This is interesting because exogenous PGE2 does inhibit paclitaxel-mediated Mø NO production (Fig. 6B) , and tumor-cell supernatant from indomethacin-treated cultures fails to inhibit NO production (Fig. 6B) . The presence of TGF-ß1 and IL-10 in supernatant could account for the failure of paclitaxel to activate Mø in this culture, but the ability to activate these cells in the presence of exogenous PGE2 demonstrates that PGE2 is not a major suppressor factor in this model. Collectively, these data suggest that Meth-KDE-derived TGF-ß1 (Fig. 4) and IL-10 (Fig. 5) are the primary mediators of tumor-induced suppression of paclitaxel-mediated Mø activation but not NO induction.



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  		Figure 6. Tumor-derived PGE2 inhibits paclitaxel-induced Mø NO production but not paclitaxel-induced Mø activation. a4 Mø (1.0x106) were cultured without or with LPS (10 µg/ml) or paclitaxel (10 µM). Some cultures were supplemented with fresh, Meth-KDE, tumor cell-derived supernatant (1:2 dilution). Parallel cultures were supplemented with supernatant from indomethacin (10-7 M)-treated Meth-KDE cells (1:2 dilution) or exogenous PGE2 (25.0 ng/ml). After 2 h, lysates were prepared and assayed for luciferase activity (A). NO production was determined at 24 h in parallel cultures (B). Indomethacin alone did not induce Mø activation or NO production (unpublished results). Data are averages and SE of triplicate, independent determinations from one of three similar experiments.

 
Pretreatment with tumor cell-derived factors primes Mø for enhanced responsiveness to paclitaxel-induced activation
Studies from our group have shown that tumors prime resident Mø in tumor-distal compartments (spleen, peritoneal cavity) for enhanced effector function on activation with LPS or paclitaxel [8 9 15 ], and others have demonstrated that tumors suppress the activation capacity of in situ Mø [17 18 ]. This apparent inconsistency is explained by the variation in Mø exposure to tumor-derived factors in different in vivo compartments. We asked whether temporal variations in exposure to tumor-derived factors could regulate Mø response to activation differentially. a4 Mø were co-treated or pretreated with activating agents and tumor cell-derived supernatant (Fig. 7 ). For pretreatment, a4 Mø (1x106) were cultured with tumor cell-derived supernatant for 4 h, washed twice in fresh medium, and recultured in fresh medium without or with LPS (10 µg/ml) or paclitaxel (10 µM) for an additional 2 h. For co-treatment cultures, supernatants and activating agents were added simultaneously at the start of the 2-h culture period. In contrast to co-treated cultures, in which tumor cell-derived factors suppressed LPS- and paclitaxel-induced luciferase activity, pretreatment primed the a4 Mø for enhanced response to activation, as indicated by increased luciferase activity (Fig. 7) . These data suggest that tumors may regulate paclitaxel-mediated activation differentially, depending on the in vivo compartment, and these in vitro data closely model previous ex vivo results for tumor-distal (transient treatment) and tumor-proximal (continuous treatment) Mø.



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  		Figure 7. Pretreatment with tumor-derived factors primes Mø for enhanced responsiveness to activation. A4 Mø (1.0x106) were cultured without or with 72-h Meth-KDE, tumor cell-derived supernatant (1:2 dilution). For pretreatment cultures, a4 Mø were cultured with tumor supernatant for 4 h, washed twice in fresh medium, and recultured in fresh medium without or with LPS (10 µg/ml) or paclitaxel (10 µM) for an additional 2 h. For co-treatment cultures, tumor supernatants and activating agents were added simultaneously at the start of the 2 h culture period. Data are averages and SE of triplicate, independent determinations from one of three similar experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Tumor growth alters Mø phenotype and function significantly. Extensive research has investigated how tumors induce Mø production of immunomodulatory signals (reviewed in [16 ]), demonstrating that these factors are important mediators of immunosuppression during cancer. Tumors promote Mø production of cytotoxic molecules, including TNF-{alpha} [24 ], NO [25 ], H2O2 [26 ], reactive oxygen intermediates [27 ], and IL-1 [28 ] in tumor-distal compartments where they may lack anti-tumor efficacy but impart immunosuppression. Simultaneously, tumors maintain high in situ concentrations of suppressor factors that dysregulate TAM IL-12 [17 18 ] and NO production (unpublished results). Therefore, the differential activation properties of Mø in the TBH must come into play when designing optimal anticancer therapies—especially maneuvers that make use of reagents with immune-activating properties such as paclitaxel.

Because tumors influence immune cell responses to paclitaxel through the production of soluble effector molecules, we assessed paclitaxel’s ability to activate Mø cytotoxic and effector functions in the presence of tumor-derived cytokines. Throughout this study, we used a Mø cell line (RAW264.7) with a luciferase reporter gene (a4 Mø) under the transcriptional control of the HIV-1-LTR promoter (a NF-{kappa}B-responsive element). The a4 Mø cell line allows for the characterization of Mø activation and factor production in the absence or presence of tumor cell-derived factors. This system facilitated an analysis of Mø response to paclitaxel in the absence or presence of tumor-derived immunomodulatory factors. Activity of the HIV-1 LTR was shown to be almost completely dependent on LPS or LPS-like stimuli [21 ], making the a4 cells useful for the study of LPS-mimetic molecules on Mø activation. The luciferase reporter gene provides a simple and rapid readout system for activation, and luciferase activity in a4 Mø lysates increased in a dose-dependent manner in response to LPS or paclitaxel activation (Fig. 2) .

Our previous studies [8 9 12 29 ] demonstrated that paclitaxel induces distinct immunologic efficacy when it acts as a second signal for activation of tumoricidal activity by IFN-{gamma}-primed murine Mø. Therefore, we assessed the effects of tumor-derived factors on the NF-{kappa}B-responsive molecules TNF-{alpha}, IL-12, and iNOS using a4 Mø. (In parallel studies with a nontransfected parental line of the a4 cells, RAW264.7, the presence of the reporter construct made no difference in factor production on activation; unpublished results.) Paclitaxel and LPS induced TNF-{alpha} and IL-12 production, in agreement with our previous ex vivo data [8 12 ], although tumor cell-derived supernatant inhibited Mø production of IL-12 (Fig. 1A) and TNF-{alpha} (Fig. 1B) . Priming with IFN-{gamma} (10 U/ml) and activation with paclitaxel (10 µM) for 24 h enhanced NO production (Fig. 1C) . Addition of tumor cell-derived factors down-regulated NO production, even when activated with the optimal dose of LPS. IFN-{gamma} priming modestly increased a4 Mø NO production, supporting the concept that IFN-{gamma} may be critical for mediating Mø priming and activation for cytotoxic anti-tumor responses in situ. Given that tumors modulate IFN-{gamma} production [18 30 ], these data may explain why tumors appear to evade Mø-mediated anti-tumor cytotoxicity in vivo. Loss of IFN-{gamma} production may result from, or lead to, compromised IL-12 production, and tumor cell-derived factors dysregulate Mø production of IL-12 (see Fig. 1B ). The dramatic inhibition of Mø IL-12 production following exposure to tumor supernatants suggests that IL-12 production in situ may be strongly inhibited, effectively breaking the link between Mø-mediated innate immunity and T-cell-mediated acquired immunity. Because specific T-cell anti-tumor cytotoxic responses are necessary to achieve significant regression of neoplasia, tumor-induced dysregulation of IL-12 may account for systemic immunosuppression in the TBH. Another possible mechanism by which tumors modulate Mø activities may involve the control of IFN-{gamma} responsiveness. Because tumor growth modulates IFN-{gamma}-induced Mø production of IL-12, and ICSBP has been implicated in the control of IL-12 p40 expression [31 ], we assessed whether tumor cell-derived factors dysregulate the IFN-{gamma} signaling pathway through differential regulation of ICSBP (Fig. 1D) . Human myeloid cells down-regulate ICSBP expression spontaneously [32 ], and ICSBP knockout mice display a defect in resistance to infection that derives from a failure to induce IL-12 expression [33 ]. We show that tumor cell-derived factors inhibit IFN-{gamma}-induced Mø expression of ICSBP in vitro, suggesting a possible lesion in the immune response during tumor growth. Although the data may suggest that restoration of IFN-{gamma} production in vivo could enhance the activity of Mø-activating agents such as paclitaxel, tumor-mediated modulation of ICSBP expression (Fig. 1D) demonstrates an additional lesion in IFN-{gamma}-mediated signaling in the tumor-bearing host. Because paclitaxel induces optimal activation as a second signal following IFN-{gamma} priming [11 ], tumor-induced dysregulation of ICSBP may explain the paucity of paclitaxel-induced NO production in situ or in the presence of immunomodulatory cytokines. Further studies should investigate the role of ICSBP in tumor-induced Mø dysfunction.

Using a4 Mø reporter gene expression and factor production as readouts, we assessed the effects of the identified tumor-derived suppressor molecules (TGF-ß1, IL-10, and PGE2) produced by the Meth-KDE fibrosarcoma [15 ] on Mø activation. Because we [8 ] and others [11 ] demonstrated that paclitaxel differentially regulates tumor-induced Mø populations [8 9 29 ], we used the a4 Mø cell line to characterize paclitaxel-mediated Mø activation and factor production in the absence or presence of tumor cell-derived factors. The a4 Mø cell line has the specificity and appropriate functionality for these experiments because the cells respond to paclitaxel-mediated activation in a dose-dependent manner (Fig. 2) . When treated with Meth-KDE-derived supernatant, LPS- or paclitaxel-activated a4 Mø cell response is suppressed by approximately 30%–50%, respectively. Notably, paclitaxel failed to induce significant activation of a4 Mø in the presence of tumor supernatants, even when used at the optimal dose level (10 µM). Meth-KDE tumor cell-derived supernatant down-regulated a4 Mø activation in response to LPS or paclitaxel, and depletion of TGF-ß1 (Fig. 4) or IL-10 (Fig. 5) reversed the inhibitory effects of the tumor cell-derived factors partly. In corollary experiments, physiologically relevant levels [15 ] of recombinant TGF-ß1 or IL-10 were added to a4 cells; TGF-ß1 and IL-10 down-regulated a4 Mø luciferase activity (Figs. 4A and 5A) , suggesting that tumor-derived TGF-ß1 and IL-10 may down-regulate Mø activation significantly in situ. Supernatants from indomethacin-treated Meth-KDE cells, which are free of PGE2, inhibited a4 Mø luciferase activity (Fig. 6A) , and exogenous PGE2 only moderately suppressed a4 Mø activation. Collectively, these data suggest that the tumor cell-derived factors TGF-ß1 or IL-10 are the primary mediators of tumor-induced suppression of paclitaxel-mediated Mø activation and that PGE2 plays only a minor role in this model system.

In vivo, Mø exist in tumor-proximal (in situ) and tumor-distal compartments, and these populations encounter different types and levels of inhibitory molecules produced by tumors. Unlike in situ Mø populations, which are constantly exposed to high levels of tumor-derived immunomodulatory factors, Mø in tumor-distal compartments may experience only low-level or transient exposure to these factors. We demonstrated [8 9 15 ] that tumor-distal Mø produced elevated levels of NO and TNF-{alpha} upon activation with LPS or paclitaxel, suggesting that these populations are primed by tumor growth for enhanced cytotoxic and effector-molecule production [8 9 ]. These data contrast with our in vitro results using a4 cells, in which tumor cell-derived factors modulate activation in response to LPS or paclitaxel. We determined whether a4 Mø responses to LPS and paclitaxel are differentially regulated by continuous versus transient exposure to tumor-derived factors. Pretreatment (4 h) of a4 Mø with tumor supernatant led to a priming effect, and these cells demonstrated substantial levels of reporter gene expression on activation (Fig. 7) . These data suggest that transient exposure to tumor cell-derived factors may enhance Mø response to activation, and this scenario may reflect the in vivo response of tumor-distal immune cells. Cells continuously treated with tumor-derived factors demonstrated suppressed LPS- and paclitaxel-induced activation, suggesting that continuous exposure to these factors prevents a4 Mø activation regardless of the stimulus. These data complement our work in primary Mø, suggesting that tumor growth primes distal populations for enhanced cytotoxic and immunosuppressive molecule production and concurrently suppresses in situ Mø anti-tumor activity. Continuous stimulation with certain tumor cell-derived factors may suppress Mø activation, and transient treatment induces a priming effect. Alternatively, tumors may produce distinct priming and suppressing factors; in this scenario, pretreatment (or transient, low-level exposure in tumor-distal compartments) leads to priming. Simultaneously, continuous culture (or chronic high-level exposure in situ) overpowers the priming signals and prevents Mø activation, and our in vitro data [8 9 15 16 29 ] support this model.

Tumor-bearing host immunosuppression remains a major obstacle in cancer therapy. Successful anti-tumor immunity is dependent on the cytokine repertoire present at the tumor site. Tumor cells counteract this repertoire by producing immunosuppressive cytokines, which lead to dysfunctional macrophage killing activity. By defining tumor-induced immunosuppression, measures may be taken to counteract the defect and restore macrophage tumoricidal activity, thus eliminating the tumor. Collectively, these data enhance our understanding of Mø activation in the context of the tumor. These observations suggest that therapies, which disrupt or abrogate tumor production of immunomodulatory factors such as anti-sense-based targeting of TGF-ß1 and IL-10, may improve the efficacy of current chemotherapeutic strategies substantially.


   ACKNOWLEDGEMENTS
 
These studies were supported, in part, by grants from the Virginia Academy of Science (D. W. M.) and the Horsley Cancer Research Fund (K. D. E.). The authors thank Dr. Matthew Sweet (Centre for Molecular and Cellular Biology, University of Queensland, Brisbane, Australia) for providing the a4 Mø cell line. We thank Genentech, Inc. (South San Francisco, CA) for providing rTGF-ß1 and anti-TGF-ß1 Ab and DNAX (Palo Alto, CA) for providing IL-10. We appreciate the technical assistance of Dr. Charles Rutherford (Virginia Tech, Blacksburg, VA).


   FOOTNOTES
 
Current address of David W. Mullins: Carter Immunology Center, University of Virginia, Box 801386, Charlottesville, VA 22908-1386. E-mail: dmullins{at}virginia.edu

Received August 10, 1999; revised August 28, 2000; accepted August 29, 2000.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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