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The Journal of Immunology, 2005, 174: 6013-6022.
Copyright © 2005 by The American Association of Immunologists

CD40 Ligation Activates Murine Macrophages via an IFN-{gamma}-Dependent Mechanism Resulting in Tumor Cell Destruction In Vitro 1

Ilia N. Buhtoiarov*, Hillary Lum*, Gideon Berke§, Donna M. Paulnock{dagger}, Paul M. Sondel*,{ddagger} and Alexander L. Rakhmilevich2,*,{ddagger}

Departments of* Human Oncology and{dagger} Medical Microbiology and Immunology and{ddagger} UW Comprehensive Cancer Center, University of Wisconsin, Madison, WI 53792; and§ Department of Immunology, Weizmann Institute of Science, Rehovot, Israel


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
We have shown previously that agonistic anti-CD40 mAb induced T cell-independent antitumor effects in vivo. In this study, we investigated mechanisms of macrophage activation with anti-CD40 mAb treatment, assessed by the antitumor action of macrophages in vitro. Intraperitoneal injection of anti-CD40 mAb into C57BL/6 mice resulted in activation of peritoneal macrophages capable of suppressing B16 melanoma cell proliferation in vitro, an effect that was greatly enhanced by LPS and observed against several murine and human tumor cell lines. Anti-CD40 mAb also primed macrophages in vitro to mediate cytostatic effects in the presence of LPS. The tumoristatic effect of CD40 ligation-activated macrophages was associated with apoptosis and killing of tumor cells. Activation of macrophages by anti-CD40 mAb required endogenous IFN-{gamma} because priming of macrophages by anti-CD40 mAb was abrogated in the presence of anti-IFN-{gamma} mAb, as well as in IFN-{gamma}-knockout mice. Macrophages obtained either from C57BL/6 mice depleted of T and NK cells by Ab treatment, or from scid/beige mice, were still activated by anti-CD40 mAb to mediate cytostatic activity. These results argued against the role of NK and T cells as the sole source of exogenous IFN-{gamma} for macrophage activation and suggested that anti-CD40 mAb-activated macrophages could produce IFN-{gamma}. We confirmed this hypothesis by detecting intracytoplasmic IFN-{gamma} in macrophages activated with anti-CD40 mAb in vivo or in vitro. IFN-{gamma} production by macrophages was dependent on IL-12. Taken together, the results show that murine macrophages are activated directly by anti-CD40 mAb to secrete IFN-{gamma} and mediate tumor cell destruction.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The interaction between the CD40 molecule, expressed on APC, such as dendritic cells and macrophages, and CD40L, expressed on T cells, plays a pivotal role in activation of adaptive immunity (1, 2, 3). This pathway for T cell activation has been the focus of strategies to activate antitumor immune responses against "weak" tumor Ags. When stimulated by CD40-CD40L interaction, APC produce IL-12 and costimulatory molecules B7-1 and B7-2, which are necessary for activating effector CD8+ T cells. Such activation of tumor-specific T cells by certain vaccines via CD40-CD40L interaction (4) prompted attempts to activate antitumor immune T cells in tumor-bearing hosts by means of CD40 ligation. Indeed, it has been shown that direct activation of APC, either by CD40L (5, 6) or agonistic anti-CD40 mAb (7, 8, 9), resulted in CD8+ T cell-dependent antitumor effects in some murine tumor models. Despite the importance of these observations, many aspects of CD40 biology, which may have therapeutic implications, remain to be elucidated. Thus, we have shown recently that CD40 ligation can induce the activation of NK cells, which can mediate antitumor effects even in the absence of T cells (10). However, depletion of NK cells reduced but did not fully abrogate the antitumor effect of anti-CD40 mAb in some tumor models (10), suggesting that cells other than T cells and NK cells may contribute to the antitumor effects of anti-CD40 mAb.

Although it is clear that APC such as macrophages can be activated by direct CD40 ligation, as we (10) and others (11, 12) have shown, the mechanisms of activation of antitumor macrophages via CD40 ligation have not been characterized. In this study, we show that macrophages can be directly activated by anti-CD40 mAb to produce IFN-{gamma} and mediate destruction of tumor targets in vitro, especially in the presence of a second signal such as LPS.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and cell lines

Female C57BL/6 mice 7–10 wk old were obtained from Harlan Spargue Dawley or The Jackson Laboratory. C57BL/6 IFN-{gamma}-knockout (KO) 3 mice, FcR-KO mice, CD40-KO mice, and C3H/HeJ mice were purchased from The Jackson Laboratory. CB-17 scid/beige mice were obtained from Taconic Farms. Housing, care, and use of mice were conducted in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals. The murine cell lines (B16 melanoma, L5178Y lymphoma, and Renca carcinoma) and human cell lines (M21 melanoma, NIH:OVCAR-3 ovarian carcinoma, and Jurkat lymphoma) were grown in RPMI 1640 complete medium (except the OVCAR cell line, which was grown in DMEM) containing 10% FBS (Sigma-Aldrich), 2 mM L-glutamine, and 100 U/ml penicillin/streptomycin (both from Invitrogen Life Technologies) at 37°C in a humidified 5% CO2 atmosphere.

Abs and reagents

The FGK 45.5 hybridoma cells producing a stimulatory anti-CD40 mAb (13) were a gift from Dr. F. Melchers (Basel Institute for Immunology, Basel, Switzerland). The Ab was obtained from ascites of nude mice injected with the hybridoma cells and enriched for IgG by ammonium sulfate precipitation. For in vivo studies, mice were injected i.p. with 0.5 mg of either anti-CD40 mAb or rat IgG control Ab (Sigma-Aldrich) in 0.5 ml of PBS. Rat anti-mouse IFN-{gamma} mAb (clone R4-6A2; American Type Culture Collection) was obtained from ascites of nude mice injected with the hybridoma cells and enriched for IgG by ammonium sulfate precipitation. Rat anti-mouse IL-12 mAb (clone C17.8) was obtained from Genzyme. Recombinant murine IFN-{gamma} was obtained from Roche.

In vivo effector cell depletion

Mice were depleted of T cells with a mixture of anti-CD4 mAb, GK1.5 hybridoma (300 µg) and anti-CD8 mAb, and 2.43 hybridoma (300 µg) i.p. (on days –2 and 2) relative to i.p. injection of anti-CD40 mAb (day 0). Control mice were given i.p. 600 µg of rat IgG. NK cells were depleted according to the same schedule by i.p. injection with 250 µg of anti-NK1.1 mAb, PK136 hybridoma. All these hybridomas were obtained from American Type Culture Collection, and the ascites were enriched for IgG by ammonium sulfate precipitation. Successful cell subset depletion was confirmed either by flow cytometry (CD4+ and CD8+ T cells) or in the 4-h cytotoxic test against NK-sensitive YAC-1 target cells (NK cells).

Macrophage purification and activation

Peritoneal exudate cells (PEC) were obtained by a peritoneal cavity lavage with 5 ml of the cold RPMI 1640 complete medium. Collected PEC were placed into 96-well flat-bottom cell culture clusters (Corning Glass) at a concentration of 1–1.5 x 106 cells/ml, 0.2 ml/well. The peritoneal macrophage population was enriched by adhesion on plastic for 1.5–2 h, followed by washing and aspiration of nonadherent cells. Flow cytometry revealed that 95% of adherent cells were macrophages based on their expression of the F4/80 macrophage marker. To activate macrophages in vitro, adherent PEC from naive C57BL/6 mice were incubated for 48 h in complete medium containing 25 µg/ml of either anti-CD40 mAb or rat IgG, unless otherwise indicated.

Macrophage-induced tumor cytostasis assay

Antitumor cytostatic activity of macrophages was determined by the inhibition of DNA synthesis in target tumor cells (10). Briefly, adherent PEC (prepared from 2 to 3 x 105 PEC/well) obtained from mice 5 days after i.p. injection of anti-CD40 mAb or rat IgG or obtained 48 h after in vitro stimulation were cocultured with B16 tumor cells (1 x 104/well) in medium alone or in the presence of LPS (Sigma-Aldrich) for 48 h. To estimate DNA synthesis, cells were pulsed with [3H]thymidine (1 µCi/well) during the last 6 h of incubation. [3H]Thymidine incorporation was determined by beta scintillation of total cells harvested from the wells onto glass fiber filters (Packard Instrument), using the Packard Matrix 9600 Direct Beta Counter (Packard Instrument). Unless stated otherwise, results are expressed as counts per 5 min for triplicate wells ± SEM.

NO production

Peritoneal macrophages (prepared from 2 to 3 x 105 PEC/well) from mice subjected to various treatments were incubated for 48 h. Nitrite accumulation in macrophage supernatants was determined using Griess reagent (Sigma-Aldrich). Equal volumes of supernatants and Griess reagent were mixed for 10 min, and the A540 nm was recorded and compared with a standard nitrite curve ranging from 0 to 120 µM.

Flow cytometric analysis

Peritoneal cells (0.5–1 x 106) from C57BL/6 mice were stained for 40 min at 4°C with FITC-conjugated rat anti-mouse Ab against the macrophage-specific F4/80 Ag (Serotec), FITC-conjugated rat IgG2b Ab (BD Pharmingen) as an isotype control, PE-conjugated anti-CD40 mAb, clone IC10 (eBioscience), and PE-conjugated rat IgG2a Ab (BD Pharmingen) as an isotype control. Propidium iodide was added to stain dead cells that subsequently were excluded from the analysis. Stained cells were analyzed using a FACScan cytofluorometer (BD Biosciences), and data were collected for 10,000 events/sample. For cell sorting, peritoneal cells from naive mice were stained with FITC-conjugated anti-F4/80 mAb, and F4/80+ cells were selected for using a FACSVantage SE with DiVa upgrade (BD Biosciences).

Detection of intracellular IFN-{gamma} by flow cytometry

PEC from mice injected i.p. with 0.5 mg of either anti-CD40 mAb or rat IgG i.p. were obtained 1–5 days after treatment. PEC were seeded into 6-well cell culture clusters (Costar; Corning Glass) at a concentration of 1 x 106 cells/ml, 5 ml/well, and enriched for macrophages by adherence to plastic during 1.5 h before staining. In other experiments, PEC from naive C57BL/6 mice were stimulated in vitro with anti-CD40 mAb or rat IgG for 24 h before staining. To enable accumulation of cytoplasmic IFN-{gamma} in the endoplasmic reticulum, cells were incubated in medium containing monensin (1 µl/1 ml; eBioscience) for 4 h. Cells were harvested by gentle scrapping with a rubber policeman and assayed for intracellular IFN-{gamma} as described elsewhere (14, 15) and according to the eBioscience 2004 Catalog and Reference Manual. Briefly, macrophages were resuspended in PBS with 2% FBS (flow buffer) at a concentration of 1 x 106/ml and stained for the murine mature macrophage-specific F4/80 surface Ag with anti-F4/80-FITC mAb (Serotec), 5 µg/1 x 106 cells, at 4°C for 40 min. As an isotype control, rat IgG2b-FITC mAb (BD Biosciences) was used. Cells were centrifuged, the cell pellet was resuspended in 0.1 ml of the flow buffer, and 0.1 ml of IC Fixation Buffer (eBioscience) was added for 20 min. Before performing the intracellular staining for cytoplasmic IFN-{gamma}, cell membranes were permeabilized with permeabilization buffer (eBioscience) for 5 min and washed in the same buffer two additional times. After the final cell wash, the pellet was resuspended in 0.1 ml of permeabilization buffer, and cells were stained for cytoplasmic IFN-{gamma} with PE-conjugated rat anti-mouse IFN-{gamma} mAb (XMG1.2; eBioscience), 2 µg/1 x 106 cells, at 4°C for 40 min. As a reference and isotype Ab control, rat IgG1-PE mAb (eBioscience) was used. Finally, cells were resuspended in 0.3 ml of flow buffer and analyzed using a FACScan cytofluorometer (BD Biosciences). Analysis of data collected for 10,000 events/sample was performed using the CellQuest software (BD Biosciences).

Assay for tumor cell apoptosis

Peritoneal cells (3 x 106/ml) obtained from mice 5 days after anti-CD40 mAb treatment were allowed to adhere to 12-well plates and cocultured with L5178Y tumor cells (1 x 105/ml) with or without LPS (10 ng/ml). After 24 or 48 h, cells were harvested and adjusted to 1 x 106 cells/ml flow buffer. To distinguish macrophages from tumor cells, macrophages were labeled with anti-F4/80-APC mAb (eBioscience) for 45 min at 4°C. To detect tumor cells undergoing apoptosis, 10 µl of annexin V-FITC mAb (BD Pharmingen) were added to the cells for 20 min at room temperature. Before flow cytometric analysis, 7-aminoactinomycin D (7-AAD) (eBioscience) was added to cells to determine induced changes in cell membrane integrity and permeability. Analysis was based on gating F4/80-APC-negative cells (L5178Y cells) and monitoring annexin V and 7-AAD double staining.

Statistical analysis

A two-tailed Student’s t test was used to determine significance of differences between experimental and relevant control values.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD40 ligation activates macrophages to mediate tumoristatic effects in vitro

In the first series of experiments, we determined if the in vivo injection of anti-CD40 mAb induced activation of macrophages capable of mediating an antitumor effect in vitro. As anti-CD40 mAb was to be administered i.p., we first determined the proportion of naive peritoneal macrophages, which expressed the CD40 molecule. Fig. 1A shows that all F4/80+ cells (macrophages) were positive for CD40 expression. Separate analyses determined that the remaining cells in the left upper quadrant in Fig. 1A were primarily CD40+, F4/80–, and B220+ B cells (data not shown).



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  		FIGURE 1. Anti-CD40 mAb induces macrophage activation in vivo. A, CD40 expression on peritoneal macrophages. Peritoneal cells were obtained from naive C57BL/6 mice and stained with FITC-conjugated anti-F4/80 mAb and PE-conjugated anti-CD40 mAb. RBC were excluded from the analysis. The results are shown as a dot plot of viable peritoneal cells. B and C, PEC were harvested from C57BL/6 mice (four mice per group) 5 days after a single i.p. injection of either anti-CD40 mAb or rat IgG (0.5 mg of each). PEC were placed in 96-flat microwell cell culture plates in triplicates, and adherent cells were cocultured with B16 melanoma tumor cells for 48 h in medium containing various concentrations of LPS. [3H]Thymidine was added to the wells 6 h before harvesting. B, The results are presented as mean ± SE of [3H]thymidine incorporation into B16 tumor cells. Asterisks indicate proliferation <1 x 103 counts. C, Cell culture supernatants were collected from the same triplicate wells after 42 h and tested for nitrite concentration (mean ± SE) using the Griess reagent. Asterisks indicate nitrite levels below the detection limits.

 
C57BL/6 mice were injected i.p. with anti-CD40 mAb, and PEC were collected 5 days postinjection. Fig. 1B shows that adherent PEC from anti-CD40 mAb-treated mice were able to suppress proliferation of B16 cells compared with PEC from rat IgG-treated mice. Proliferation of B16 cells alone was generally similar to that of B16 cells in the presence of PEC from rat IgG-treated mice (data not shown). PEC alone, from control or anti-CD40 mAb-treated mice, incorporated <1000 counts of [3H]thymidine (data not shown).

The observed tumor inhibition effect of anti-CD40 mAb-activated macrophages was increased greatly in the presence of LPS in a dose-dependent fashion (Fig. 1B). In the absence of PEC, LPS did not substantially affect proliferation of B16 cells (data not shown). The observed in vitro tumor inhibitory effect of macrophages from anti-CD40 mAb-treated mice was associated with the ability of these same macrophages to produce NO in vitro (Fig. 1C). This activating effect of anti-CD40 mAb on peritoneal macrophages was reproduced in more than a dozen experiments that showed varying degrees of direct in vitro antitumor effect by macrophages from anti-CD40 mAb-treated mice and consistent anti-CD40 mAb-induced priming of PEC to LPS.

To determine the target specificity of this macrophage-mediated antitumor effect, we tested the sensitivity of different murine (B16 melanoma, L5178Y lymphoma, and Renca carcinoma) and human (M21 melanoma, Jurkat lymphoma, and OVCAR carcinoma) cell lines to the cytostatic effect of macrophages activated with anti-CD40 mAb +/– LPS. We found comparable (to B16) inhibition of proliferation of all cell lines tested (Fig. 2). As in the experiment depicted in Fig. 1, enhanced antitumor activity of the activated macrophages was associated with increased production of NO (data not shown).



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  		FIGURE 2. CD40 ligation-activated macrophages are cytostatic for various murine and human tumor targets. PEC were harvested from four to six C57BL/6 mice 5 days after a single i.p. injection of 0.5 mg of anti-CD40 mAb. Adherent peritoneal cells from anti-CD40 mAb-treated mice were cocultured with the murine (B16 melanoma, L5178Y lymphoma, and Renca carcinoma) or human (M21 melanoma, OVCAR carcinoma, and Jurkat lymphoma) tumor cells for 48 h in medium with or without 10 ng/ml LPS. Control wells contained tumor cells without macrophages. [3H]Thymidine was added to the wells 6 h before harvesting. The results are presented as mean ± SE of triplicate wells of [3H]thymidine incorporation into tumor cells. A combined graph of three separate experiments is shown.

 
Next, we wanted to ascertain that activation of macrophages with anti-CD40 mAb was due to specific interaction with the CD40 molecule on the surface of the macrophage rather than due to nonspecific interaction with the FcR. We used two approaches to address this issue. First, we tested anti-CD40 mAb in FcR-KO mice. The results in Fig. 3A show that anti-CD40 mAb induced a similar degree of antitumor activity by macrophages in control C57BL/6 and FcR-KO mice. This result indicates that interaction of anti-CD40 mAb and FcRs on macrophages (or other cells) was not the mechanism of the observed macrophage activation. In the second approach, anti-CD40 mAb was injected into CD40-KO mice. Fig. 3B shows that all tumoristatic activity of macrophages induced by anti-CD40 mAb, and most of that induced by anti-CD40 mAb + LPS in control mice, was abrogated in CD40-KO mice, thus confirming that macrophages were activated via CD40 ligation.



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  		FIGURE 3. Anti-CD40 mAb induces macrophage activation via CD40 ligation in vivo. Control C57BL/6 mice and FcR-KO mice (A), CD40-KO mice (B), or C3H/HeJ mice (C) were injected i.p. with 0.5 mg of anti-CD40 mAb (or the same dose of rat IgG (A and C) or PBS (B) as controls). Adherent peritoneal cells were cocultured with B16 cells for 48 h in medium with or without 10 ng/ml LPS. The results are presented as mean ± SE of triplicate wells of [3H]thymidine incorporation into B16 tumor cells. *, Proliferation < 1 x 103 counts.

 
Because macrophages from anti-CD40 mAb-treated mice were very sensitive to low doses of LPS (Fig. 1, B and C), the possibility remained that endotoxin contamination of our media and/or anti-CD40 mAb was responsible for macrophage activation induced by the anti-CD40 mAb given alone (16, 17). To exclude endotoxin contamination as a cause of responsiveness to anti-CD40 mAb, we used LPS-resistant C3H/HeJ mice. The results showed that macrophages from C3H/HeJ mice injected with anti-CD40 mAb retained their tumoritoxicity in vitro (Fig. 3C). As expected, LPS failed to further augment this antitumor effect in C3H/HeJ mice (Fig. 3C). These results prove that endotoxin contamination was not responsible for macrophage activation by anti-CD40 mAb in vivo.

We determined next whether the activation of macrophages with anti-CD40 mAb was direct or indirect. Adherent PEC were obtained from naive C57BL/6 mice and stimulated in vitro with various doses of anti-CD40 mAb (5–50 µg/ml). After 48 h of stimulation, the cells were washed and incubated with B16 target cells in the presence or absence of LPS for an additional 48 h. The results in Fig. 4A show that anti-CD40 mAb induced a dose-dependent activation of antitumor macrophages that was most readily detected in the presence of 0.1–10 ng/ml LPS. The priming effect of anti-CD40 mAb on macrophages was similar in magnitude to that of IFN-{gamma}, as shown in the experiment presented in Fig. 4B. In a separate experiment, we confirmed that anti-CD40 mAb can directly activate macrophages for antitumor activities by using in vitro activation of peritoneal macrophages positively sorted by flow cytometry. Naive PEC from untreated mice were sorted for F4/80 expression. The selected population consisted of 97% F4/80+ macrophages. These cells were stimulated in vitro as described in the legend for Fig. 4 with anti-CD40 mAb (25 µg/ml) + LPS (10 ng/ml). These anti-CD40 mAb-activated, LPS-triggered macrophages induced a statistically significant (p < 0.05) suppression of B16 tumor cell proliferation (57,414 ± 903), as compared with sorted macrophages incubated either with medium,anti-CD40 mAb alone, or LPS alone (110,139 ± 8,405, 110,000 ± 1,154, and 73,261 ± 4,557, respectively).



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  		FIGURE 4. Anti-CD40 mAb induces tumoristatic activity of macrophages in vitro. PEC from four naive C57BL/6 mice were harvested and enriched for macrophages by adherence to plastic. A, Anti-CD40 mAb primes macrophages to subsequent challenge with LPS. Macrophages were cultured in medium containing various concentrations of anti-CD40 mAb, as shown in the figure, for 48 h. Subsequently, the cells were washed of anti-CD40 mAb and coincubated with B16 tumor cells in the presence of various doses of LPS for the next 48 h. [3H]Thymidine was added to the wells 6 h before harvesting. Tumoristatic effect mediated by activated macrophages was assessed by inhibition of [3H]thymidine incorporation into B16 cells. B, Anti-CD40 mAb primes macrophages to LPS similar to exogenous IFN-{gamma}. Adherent PEC from four naive C57BL/6 mice were cultured in the presence of either anti-CD40 mAb (25 µg/ml) or IFN-{gamma} (100 IU/ml) for 48 h. After repeated cell washes, macrophages were cocultured with B16 melanoma cells in the presence of LPS (10 ng/ml) for the next 48 h. Tumoristatic activity mediated by activated macrophages was assessed in the [3H]thymidine incorporation assay.

 
Anti-CD40 mAb-induced activation of macrophages is IFN-{gamma} dependent

The results in Fig. 4B suggested that anti-CD40 mAb and IFN-{gamma} may have similar mechanisms of macrophage activation. Therefore, we determined the role of IFN-{gamma} in activation of macrophages by anti-CD40 mAb. In the first approach, we determined whether anti-CD40 mAb treatment, in vivo and in vitro, would activate macrophages in IFN-{gamma}-KO mice. The results show that the antitumor effect of anti-CD40 mAb observed in control mice in the absence of LPS was abolished in IFN-{gamma}-KO mice, both in vivo (Fig. 5A) and in vitro (Fig. 5B). Similarly, the enhanced antitumor effects of CD40 ligation-activated macrophages in the presence of LPS were virtually abrogated in IFN-{gamma}-KO mice (Fig. 5, A and B). These results demonstrate that endogenous IFN-{gamma} is involved in the activation of antitumor macrophages by anti-CD40 mAb.



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  		FIGURE 5. Anti-CD40 mAb activation of macrophages is IFN-{gamma} dependent. A, PEC from IFN-{gamma}-KO mice or conventional C57BL/6 mice injected i.p. with anti-CD40 mAb or rat IgG were harvested 3 days later. Adherent cells were coincubated with B16 melanoma cells in medium +/– LPS (10 ng/ml) for 48 h. Tumor cell proliferation was measured by [3H]thymidine incorporation in B16 cells. B, Adherent PEC from naive IFN-{gamma}-KO or conventional C57BL/6 mice were stimulated in vitro with anti-CD40 mAb (25 µg/ml) for 48 h. After cell washes, macrophages were stimulated with LPS (10 ng/ml) in the presence of B16 melanoma cells for another 48 h. C, PEC from C57BL/6 mice injected i.p. with 0.5 mg of anti-CD40 mAb or rat IgG were harvested 5 days later. Adherent cells were coincubated with B16 melanoma cells in medium +/– LPS (10 ng/ml), with or without anti-IFN-{gamma} mAb (50 µg/ml), for 48 h. Tumor cell proliferation was measured by [3H]thymidine incorporation in B16 cells. D, PEC from naive C57BL/6 mice were enriched for macrophages and stimulated in vitro with anti-CD40 mAb (25 µg/ml) for 48 h and then with LPS (10 ng/ml) for another 48 h. Blocking anti-IFN-{gamma} mAb (50 µg/ml) and anti-IL-12 mAb (50 µg/ml) were added to cultures during both steps as indicated. Tumoristatic effect by activated macrophages was assessed by inhibition of DNA synthesis in B16 cells coincubated with activated macrophages for the last 48 h and measured by [3H]thymidine incorporation into B16 cells. The results of all assays are presented as means ± SE of [3H]thymidine incorporation in B16 cells in triplicates, four mice per group. Asterisks indicate proliferation < 1 x 103 counts.

 
In a second approach, we attempted to block CD40 ligation-induced macrophage activation by the addition to the culture of anti-IFN-{gamma} mAb. When the macrophages were first activated in vivo with anti-CD40 mAb and then further stimulated in vitro with LPS, addition of anti-IFN-{gamma} mAb caused partial inhibition of tumoritoxicity induced by anti-CD40 mAb + LPS but not by the in vivo anti-CD40 mAb alone (Fig. 5C). When macrophages were first activated in vitro with anti-CD40 mAb and then with LPS, anti-IFN-{gamma} mAb substantially reduced the antitumor effect of anti-CD40 mAb-primed, LPS-triggered macrophages (Fig. 5D). Anti-IL-12 mAb mediated a similar inhibitory effect on macrophage activation by anti-CD40 mAb + LPS (Fig. 5D), suggesting that both IFN-{gamma} and IL-12 play an important role in CD40 ligation-induced activation of macrophages.

The role of T cells and NK cells in CD40-induced activation of antitumor macrophages

As NK cells and T cells can produce IFN-{gamma} in response to different stimuli, we thought to determine whether these cells play a role in anti-CD40 mAb-induced macrophage activation in vivo. In a first approach, C57BL/6 mice were depleted of T cells, NK cells, or both T cells and NK cells, with appropriate mAbs. The depleting efficacy of anti-CD4, anti-CD8, and anti-NK1.1 mAbs was confirmed in separate experiments. Cell subset-depleted and control mice were injected with anti-CD40 mAb, and the activity of macrophages was tested 5 days later in a 48-h cytostatic assay. The results in Fig. 6A show that peritoneal macrophages from T cell-depleted, NK cell-depleted, and T/NK cell-depleted mice retained their ability to be primed with anti-CD40 mAb to mediate an enhanced antitumor effect in the presence of low-dose LPS.



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  		FIGURE 6. Anti-CD40 mAb activation of macrophages does not require the presence of NK or T cells. A, C57BL/6 mice were depleted of either T cells, NK cells, or both T and NK cells (T/NK) as described in Materials and Methods. These mice were injected with a single dose of anti-CD40 mAb (0.5 mg). Control mice without cell depletion (no depl.) were injected with anti-CD40 mAb or rat IgG (0.5 mg). Five days after the treatment, PEC were harvested, enriched for macrophages, and stimulated with LPS (10 ng/ml) in the presence of B16 target cells for 48 h. Tumoristatic effects mediated by activated macrophages were assessed by reduction of B16 melanoma cells proliferation in the [3H]thymidine incorporation assay (mean ± SE of triplicates, four mice per group). The tumoristasis of PEC in the presence of LPS for all four groups of mice that received anti-CD40 mAb was greater (p < 0.05) than the tumoristasis induced by LPS treatment of the control PEC (from rat IgG-treated mice). B, Scid/beige mice received a single injection of either anti-CD40 mAb (0.5 mg) or rat IgG (0.5 mg). Five days after the treatment, adherent PEC were stimulated with LPS (10 ng/ml) in the presence of B16 tumor cells for 48 h. Tumoristatic effects mediated by activated macrophages were assessed by [3H]thymidine incorporation assay. The results are presented as means ± SE of triplicates, four mice per group. An asterisk indicates that the tumoristatic effect seen for anti-CD40 mAb + LPS treatment was greater (p < 0.05) than that for rat IgG + LPS treatment.

 
These findings were confirmed in another approach using T cell- and NK cell-deficient scid/beige mice (Fig. 6B). PEC from anti-CD40 mAb-treated scid/beige mice exhibited a statistically significant (p < 0.05) suppression of B16 cell proliferation in the presence of LPS compared with PEC from rat IgG-treated mice. Taken together, these findings demonstrate that T cells and NK cells are not required for anti-CD40 mAb-induced macrophage activation.

Macrophages activated by anti-CD40 mAb contain cytoplasmic IFN-{gamma}

As our experiments showed that the antitumor activity of macrophages was induced by CD40 ligation in the absence of T and NK cells but required IFN-{gamma}, we hypothesized that macrophages could secrete IFN-{gamma} upon stimulation with anti-CD40 mAb. To test this hypothesis, we measured the percentage of F4/80+ PEC that had detectable cytoplasmic IFN-{gamma} following stimulation with anti-CD40 mAb in vivo and in vitro. Twenty-four hours after i.p. injection of anti-CD40 mAb, peritoneal macrophages showed a substantially enhanced expression of intracellular IFN-{gamma} compared with macrophages from rat IgG-treated mice. IFN-{gamma} expression in macrophages diminished by day 2 following anti-CD40 mAb injection and remained low through day 5 (Fig. 7A). These results were confirmed in vitro by showing that peritoneal macrophages stimulated in culture with anti-CD40 mAb for 24 h expressed a large percentage of IFN-{gamma}+ cells (30% of total cells or 75.8% of F4/80+ cells) compared with 1.2% of IFN-{gamma}+ cells among unstimulated macrophages (Fig. 7B). The results in Fig. 7B also show that IFN-{gamma} production by anti-CD40 mAb-activated macrophages was dependent on IL-12 because the percentage of IFN-{gamma}+,F4/80+ cells after anti-CD40 mAb treatment decreased from 75.8 to 3.3% when anti-IL-12 mAb was added at the same time as the anti-CD40 mAb (Fig. 7, Bd).



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  		FIGURE 7. Anti-CD40 mAb induces IL-12-dependent production of IFN-{gamma} by macrophages. A, Macrophages stimulated in vivo with anti-CD40 mAb contain cytoplasmic IFN-{gamma}. PEC from C57BL/6 mice treated with 0.5 mg of either rat IgG or anti-CD40 mAb were collected on day 1 (rat IgG and anti-CD40 mAb) and days 2, 3, 4, and 5 (anti-CD40 mAb) after the treatment and tested for intracellular IFN-{gamma} as described in Materials and Methods. FITC-conjugated rat-anti-mouse F4/80 mAb was used to gate macrophages. Data show histograms of gated macrophages pooled from three mice per group. Shaded areas represent F4/80+ cells staining positively for isotype mAb, and open areas represent F4/80+ cells staining positively for intracellular IFN-{gamma}. Mean fluorescence intensity values for the level of IFN-{gamma} expression are shown in each histogram. F4/80+ peritoneal cells from naive mice did not express IFN-{gamma} (data not shown), similar to data shown here for PEC from animals treated with rat IgG and stained on day 1 (control). B, Anti-IL-12 mAb abrogates detection of cytoplasmic IFN-{gamma} in macrophages stimulated in vitro with anti-CD40 mAb. Peritoneal macrophages from naive C57BL/6 mice were stimulated in vitro with 50 µg/ml of either rat IgG (a and b) or anti-CD40 mAb (c and d) for 24 h. Anti-CD40 mAb-stimulated cells were also incubated in the presence of 50 µg/ml blocking anti-IL-12 mAb (d) or rat IgG (c). Adherent cells were stained with FITC-conjugated rat anti-mouse F4/80 mAb and PE-conjugated anti-IFN-{gamma} mAb (b–d) or PE-conjugated isotype mAb (a). Data show dot plots of macrophages pooled from three mice. The numbers noted in the right upper quadrant of each dot plot are the percentages of F4/80+ adherent peritoneal cells staining positively for control IgG (a) or for IFN-{gamma} (b–d). The percentage of F4/80+ cells from anti-CD40 mAb-stimulated PEC staining positively for the isotype control mAb was similar to that from control mice (data not shown).

 
CD40 ligation-activated macrophages induce apoptosis and killing of tumor cells

We next focused on why tumor cells cultured with activated macrophages show decreased incorporation of [3H]TdR. Activated macrophages (from anti-CD40 mAb-treated mice) were cultured with B16 tumor cells in the presence of LPS for 42 h, [3H]TdR was added for 6 h, and radioactivity was determined. Control tumor cells incorporated 270,000 counts, whereas tumor cells cocultured with the activated macrophages incorporated only 740 counts. When supernatants from replicate cocultures of activated macrophages and B16 cells were harvested at 42 h and used to replace the medium from 42-h cultures of B16 cells before the 6 h of incubation with [3H]TdR, the [3H]TdR incorporation remained at 240,000 counts. This proves that the decrease in [3H]TdR incorporation by tumor cells cultured with activated macrophages cannot be accounted for merely by macrophage secretion of cold thymidine, which competes with the uptake of [3H]TdR by proliferating tumor cells.

In related studies, B16 tumor cells were cultured for 48 h with anti-CD40-activated macrophages and LPS under standard culture conditions or in parallel wells where the medium was replaced twice a day. We found no difference in the level of B16 tumoristatic activity between standard cultures and cultures that received replacement with fresh medium (data not shown). This indicated that the inhibition of [3H]TdR incorporation by tumor cells cultured with activated macrophages is not likely the result of viable tumor cells showing inhibited proliferation due to consumption of nutrients required for growth by the activated macrophages.

We next explored whether the macrophage-induced tumor cytostasis in our system was associated with apoptosis and killing of tumor cells. This was tested by culturing L5178Y lymphoma cells with activated macrophages and then staining with annexin V and 7-AAD for flow cytometric analysis of apoptosis and cell death, respectively. L5178Y cells were sensitive to anti-CD40 mAb-induced tumoristasis (Fig. 2) and grew in suspension rather than as an adherent monolayer. Thus, they can be harvested for flow cytometric assessment of apoptotic cells, without requiring trypsinization of adherent cells, as needed for B16 cells. During the first 24 h of culture in the absence of LPS (Fig. 8A, left column), macrophages from anti-CD40 mAb-treated mice induced more apoptosis (48% of the cells are annexin V+ in the two right quadrants in Fig. 8A) than macrophages from mice injected with rat IgG (23% annexin V+). The anti-CD40 mAb-activated macrophages have induced an "early apoptotic" state in the tumor cells as 30% of them were found in the right lower quadrant in Fig. 8A (annexin V+ but 7-AAD–). After 48 h of culture in the absence of LPS (the left column of Fig. 8B), anti-CD40 mAb-activated macrophages induced apoptotic death (annexin V+ and 7-AAD+) in the majority of the tumor cells (80%). In contrast, only 13% of tumor cells were annexin V+ and 7-AAD+ after 48 h of culture with macrophages from rat IgG-treated mice. Apoptotic cell death increased when macrophages were further activated by culturing with LPS. After 24 h (Fig. 8A, right column), 67% of tumor cells were annexin V+ when cultured with anti-CD40-treated macrophages with LPS vs 26% for tumor cells cultured with LPS and control macrophages. By 48 h (Fig. 8B, right column), 94% of the tumor cells cultured with the activated macrophages had undergone apoptotic death (annexin V+ and 7-AAD+) vs only 28% in the control culture.



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  		FIGURE 8. CD40 ligation-activated macrophages can induce apoptotic death in tumor cells. L5178Y tumor cells were cultured in medium (no M{phi}) or in the presence of macrophages obtained from mice 5 days after the injection of either rat IgG or anti-CD40 mAb. The cells were cultured either in medium alone or in the presence of 10 ng/ml LPS for 24 h (A) or 48 h (B). After the indicated period of coincubation, cells were stained with APC-conjugated anti-F4/80 mAb, and positive cells (macrophages) were excluded from additional analysis. L5178Y cells were assayed for apoptotic transformation as revealed by expression of phosphatidylserine detected by annexin V-FITC and for altered membrane permeability, as detected by the DNA-binding dye 7-AAD. Numbers indicate the percentage of L5178Y cells that are viable (left lower quadrant: annexin V–, 7-AAD–), early apoptotic (right lower quadrant: annexin V+, 7-AAD–), or dead due to apoptosis (right upper quadrant: annexin V+, 7-AAD+).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
CD40-CD40L interactions are involved in generation of immune responses (1, 2, 3). APC (mainly dendritic cells and macrophages) present a MHC class II-restricted peptide Ag to CD4+ T cells, which recognize the Ag via their T cell Ag receptors, and induce an activation process in the CD4+ T cells. The CD40L expressed on activated CD4+ T cells, then interacts with the CD40 molecule expressed on the membrane of the APC. APC activated through the CD40-CD40L interaction produce IL-12 and express costimulatory molecules B7-1 and B7-2, which results in activation of CD8+ effector T cells (1, 18). The importance of B7-1 and B7-2 induced on APC by CD40 ligation has been demonstrated in the generation of systemic antitumor CD8+ T cell responses (6). Activation of tumor-specific immune responses resulting in reduction of tumor growth was achieved either by using CD40L (5, 6, 11, 12) or agonistic anti-CD40 mAb (7, 8, 9, 19, 20, 21). In these studies, the protective therapeutic effect has been shown to involve CD8+ T cells.

While evaluating antitumor immune mechanisms in poorly immunogenic murine tumor models to potentially simulate the clinical setting, we have recently identified an antitumor pathway for CD40 ligation that does not appear to work through the activation of systemic CD8+ T cell immunity. By using weakly immunogenic mouse B16, NXS2, and CT26-Ep21.6 tumors, we have shown that in vivo treatment of tumor-bearing mice with anti-CD40 mAb results in the in vivo activation of NK cells and causes T cell-independent, NK-dependent antitumor and antimetastatic effects (10). In addition, we have shown that peritoneal macrophages can be activated by CD40 ligation to mediate antitumor effects in vitro (10), implying that macrophages may contribute to the antitumor effects of anti-CD40 mAb in vivo. Indeed, our preliminary data suggest that the antitumor effect of anti-CD40 mAb in vivo is reduced in mice depleted of macrophages (data not shown).

In the present study, we evaluated the mechanisms of activation of macrophages by anti-CD40 mAb. Our results show that treatment with anti-CD40 mAb, in vivo and in vitro, induced peritoneal macrophages to mediate tumoristatic effects. These results are in agreement with the findings of Alderson et al. (22), showing that CD40L-transfected cells activated human monocytes in vitro to become tumoricidal. In addition, our results show that CD40 ligation had a direct effect on macrophages, as it was observed in vitro with both macrophages enriched by adherence to plastic and also with macrophages isolated by positive sorting. Furthermore, activation of cytostatic macrophages by anti-CD40 mAb was demonstrated in mice in the absence of T and NK cells. These data do not preclude the participation of T and NK cells in CD40 ligation-induced macrophage activation in conventional mice but demonstrate that T and NK cells were not required. In contrast, our results show that IFN-{gamma} was involved in the activation of macrophages by anti-CD40 mAb, both in vivo and in vitro. These results are in keeping with our previous findings showing an important role of IFN-{gamma} in antimetastatic effects induced by anti-CD40 mAb (10).

IFN-{gamma} is a well-known activator of macrophages (23, 24). In our studies, anti-CD40 mAb and IFN-{gamma} had a comparable effect on priming macrophages to LPS in vitro. Our data show that in the absence of IFN-{gamma}, anti-CD40 mAb loses its ability to fully activate macrophages. This result suggests that IFN-{gamma} induced by CD40 ligation may act alone as the primary agent responsible for macrophage activation. Alternatively, IFN-{gamma} induced by anti-CD40 mAb may collaborate with anti-CD40 mAb or with other cytokines or downstream molecules induced by CD40 ligation, which alone are ineffective in the absence of IFN-{gamma}. In favor of the second possibility, it has been shown that CD40L and IFN-{gamma} could be synergistic in activation of tumoricidal macrophages (12) or induction of chemokines (25). Similarly, the synergy of IFN-{gamma} and TNF-{alpha}, another cytokine induced by CD40 ligation (26), has been documented (27, 28). The molecular mechanisms that play a role in our experimental system require additional characterization.

Somewhat unexpectedly, our findings show that anti-CD40 mAb activates macrophages to produce IFN-{gamma}. Recent studies have challenged the paradigm that only lymphoid cells produce IFN-{gamma} by showing that under certain conditions macrophages were able to produce IFN-{gamma} (29). Stimulation of peritoneal macrophages with Corynebacterium parvum (30), IL-12 (31), or a combination of IL-12 and IL-18 (32, 33) results in release of IFN-{gamma}. Our results confirm and extend these observations by showing that macrophages produced IFN-{gamma} in response to anti-CD40 mAb via an IL-12-dependent mechanism because anti-IL-12 mAb neutralized CD40 ligation-induced expression of IFN-{gamma} in macrophages (Fig. 7B). Taken together, our data suggest the following sequence of events in response to CD40 ligation: anti-CD40 mAb interacts with the CD40 receptor on macrophages; these activated macrophages produce IL-12 (6); IL-12, in turn, induces production of IFN-{gamma} by macrophages; and IFN-{gamma} activates macrophages to mediate antitumor effects, which are enhanced additionally by stimulation with LPS.

Although the inhibition of tumor cell [3H]TdR incorporation by CD40-activated macrophages is IFN-{gamma} dependent, a few mechanisms could account for inhibition of tumor cell [3H]TdR incorporation. First, we have ruled out the possibility that cold thymidine secreted by activated macrophages competed with "hot" thymidine to decrease the detectable [3H]TdR incorporation into proliferating B16 tumor cells. Second, we have shown that activated macrophages do not slow the proliferation of viable tumor cells merely by consuming nutrients in the medium. Fig. 8 shows that the macrophage-induced cytostasis was associated with early apoptosis after 24 h (staining with annexin V but not 7-AAD), which progresses to apoptosis (staining with annexin V) and killing (bright staining with 7-AAD) of tumor cells after 48 h. In addition, using the crystal violet assay and eosin dye exclusion, we have confirmed that fewer tumor cells were present in the wells with anti-CD40 mAb + LPS-activated macrophages compared with control cultures (data not shown). These experiments prove that the mechanism for the inhibition of [3H]TdR incorporation by tumor cells following 48 h of incubation with activated macrophages in these studies is macrophage-mediated apoptotic death of the tumor cells. In addition, preliminary studies suggest that the apoptotic effect of CD40 ligation-activated macrophages on tumor cells is caspase independent because this effect was not reduced in the presence of Z-VAD-fmk (34), a pan-caspase inhibitor (data not shown). Finally, it will be important to determine whether the cytostatic and cytotoxic functions of CD40 ligation-activated macrophages are tumor specific or may be directed against other targets, including infected or normal cells.

At present, it is not clear if CD40 ligation-activated macrophages can mediate their antitumor effects via soluble factors. Although IFN-{gamma} had a direct tumoristatic effect against B16 tumor cells in vitro, inducing ~50% inhibition of tumor cell proliferation at the dose of 1 ng/ml (data not shown), this cytostatic effect of recombinant murine IFN-{gamma} was much weaker than that induced by anti-CD40 mAb-activated macrophages in the presence of LPS (Figs. 1 and 4). Moreover, only relatively low levels of IFN-{gamma} (12–23 pg/ml) could be detected by ELISA in 48-h supernatants of macrophages stimulated with anti-CD40 mAb + LPS (data not shown). These findings indicate that soluble IFN-{gamma} (whether released by macrophages or by the small number of NK cells that may be contaminating this macrophage population) is not a crucial effector molecule that is directly responsible for the tumor stasis observed in this system. Additionally, indomethacin did not affect the tumoristatic effect of CD40 ligation-activated macrophages (data not shown), suggesting that PG that could be secreted by activated macrophages do not play a significant role in these antitumor effects. It is possible that NO or TNF-{alpha}, both known to mediate macrophage cytotoxic functions (24), are involved in the antitumor effects induced by anti-CD40 mAb. In agreement with the studies of others (12, 35), we show here that anti-CD40 mAb-activated macrophages secrete NO (Fig. 1B). TNF-{alpha} can be also secreted by macrophages in response to CD40 ligation (12, 36). Studies addressing the roles of NO and TNF-{alpha} as effector molecules in the antitumor effects of anti-CD40 mAb-activated macrophages observed in the experiments shown here are underway in our laboratory.


   Acknowledgments
 
We thank Drs. Jacqueline Hank and Joseph Connor, as well as Mildred Felder, for providing cell lines and helpful discussions, Kathy Schell for flow cytometric assistance, and Brian Schmidt for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant CA87025 and a grant from the Midwest Athletes Against Childhood Cancer Fund. Back

2 Address correspondence and reprint requests to Dr. Alexander L. Rakhmilevich, University of Wisconsin, K4/413 Clinical Science Center, Department of Human Oncology, 600 Highland Avenue, Madison, WI 53792. E-mail address: rakhmil{at}humonc.wisc.edu Back

3 Abbreviations used in this paper: KO, knockout; PEC, peritoneal exudate cell; 7-AAD, 7-aminoactinomycin D. Back

Received for publication May 17, 2004. Accepted for publication March 3, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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