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The Journal of Immunology, 2000, 164: 2728-2737.
Copyright © 2000 by The American Association of Immunologists

Induction of Chemokine Secretion and Enhancement of Contact-Dependent Macrophage Cytotoxicity by Engineered Expression of Granulocyte-Macrophage Colony-Stimulating Factor in Human Colon Cancer Cells1

Hisashi Shinohara, Seiji Yano, Corazon D. Bucana and Isaiah J. Fidler2

Department of Cancer Biology, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated the role of tumor cell-derived GM-CSF in recruitment and tumoricidal activation of tissue macrophages. Transfection of the murine GM-CSF gene into KM12SM human colon cancer cells decreased the tumorigenicity of transfected cells and nontransfected bystander colon cancer cells in nude mice. Sequential tissue sections from sites injected with high GM-CSF-producing tumor cells (but not from nontransfected or low GM-CSF-producing cells) demonstrated a dense infiltration of polymorphonuclear cells (PMN), followed by infiltration of macrophages, which correlated with expression of the macrophage-inflammatory protein-1{alpha} and the monocyte chemoattractant protein-1 (MCP-1) in mouse PMN and macrophages. GM-CSF-producing KM12SM cells were highly sensitive to lysis by mouse macrophages and also increased macrophage-mediated lysis of bystander nontransfected KM12SM cells. The incubation of macrophages with GM-CSF induced expression of the CD11b surface adhesion molecule, which was associated with increased attachment to tumor cells. All KM12SM cells were sensitive to macrophage-mediated lysis in the presence of rGM-CSF and recombinant MCP-1. Collectively, the results demonstrate that tumor cell-derived GM-CSF stimulates PMN and macrophages to secrete macrophage-inflammatory protein-1{alpha} and MCP-1, which triggers recruitment of mononuclear cells, induces expression of adhesion molecules on macrophages, and enhances contact-dependent cytolysis of tumor cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tumor-associated macrophages play a pivotal role in host resistance against cancer (1, 2). Successful antitumor response by macrophages requires their recruitment to the tumor site and induction of tumoricidal properties (3). Once activated to the tumoricidal state, macrophages can lyse tumor cells directly or initiate specific antitumor immune responses (4). Essential to generation of specific immunity is GM-CSF, a pleiotropic cytokine that induces the differentiation and proliferation of myeloid precursor cells (5, 6, 7, 8, 9). GM-CSF activates APC, which in turn primes CD4+/CD8+ T lymphocytes (10, 11). These discoveries have led to efforts to modulate GM-CSF activity for therapeutic purposes. The implantation of GM-CSF-transduced cells has been shown to induce a dense inflammatory infiltrate composed of polymorphonuclear (PMN)3 cells, tissue macrophages, and dendritic cells (12, 13, 14, 15), suggesting that tumor-derived GM-CSF could lead to recruitment of immune cells to initiate antitumor response. The composition of the leukocyte population recruited into inflammatory sites is largely controlled by such chemokines as macrophage-inflammatory protein-1{alpha} (MIP-1{alpha}) and monocyte chemoattractant protein-1 (MCP-1) (16, 17, 18, 19). Whether GM-CSF induces recruitment of leukocytes by this pathway is unknown.

To produce lysis of target cells, tumor-associated macrophages must bind to the target cells and secrete cytotoxic molecules (4). These tumoricidal activities of macrophages were originally shown to be induced by two sequential signals, IFN-{gamma} and LPS (20), and our laboratory has demonstrated that systemic activation of macrophages to the tumoricidal state can also be accomplished by administration of synthetic bacterial cell wall analogues (21, 22). Engineered expression of chemokines, e.g., MCP-1 (23, 24, 25) and GM-CSF (15, 26, 27), can also induce tumoricidal properties in macrophages, but whether the expression of these diverse molecules is linked to tumor cell destruction by tumoricidal macrophages is unknown.

In the present study, we determined whether tumor cell-derived GM-CSF can lead to recruitment and tumoricidal activation of tissue macrophages. We report that transfection of the murine GM-CSF gene into KM12SM human colon cancer cells leads to the recruitment of PMN and macrophages into the tumor cell inoculation site via GM-CSF-regulated secretion of MIP-1{alpha} and MCP-1. This sequence induces contact-dependent macrophage-mediated tumor cell lysis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Athymic Ncr-nu/nu male mice were purchased from the Animal Production Area, National Cancer Institute-Frederick Cancer Research Facility (Frederick, MD). Mice were maintained according to institutional guidelines in facilities approved by The American Association for Accreditation of Laboratory Animal Care in accordance with U.S. Department of Agriculture, Department of Health and Human Services, and National Institutes of Health regulations and standards.

Reagents

Eagle’s MEM, Ca2+-, Mg2+-free HBSS, and FBS were purchased from M. A. Bioproducts (Walkersville, MD). Murine rGM-CSF and rat anti-mouse GM-CSF mAb were purchased from Genzyme (Cambridge, MA). Murine rMCP-1 was purchased from R&D Systems (Minneapolis, MN). Hamster anti-mouse MCP-1 mAb and murine rIFN-{gamma} (sp. act., 107 U/mg) were purchased from PharMingen (San Diego, CA). Salmonella LPS, normal rat IgG, and NG-methyl-L-arginine (NMA) were purchased from Sigma (St. Louis, MO). All reagents used in tissue culture (except LPS) were free of endotoxin, as determined by the Limulus amebocyte lysate assay (Associates of Cape Cod, Woods Hole, MA).

Tumor cells and transfection

KM12SM human colon carcinoma cells and HT-29 human colon carcinoma cells (28) were grown as adherent monolayer cultures in MEM supplemented with 5% FBS, vitamins, sodium pyruvate, L-glutamine, and nonessential amino acids at 37°C in a humidified atmosphere containing 5% CO2. All cultures were free of mycoplasma, reovirus type 3, pneumonia virus of mice, K virus, encephalitis virus, lymphocyte choriomeningitis virus, ectromelia virus, and lactate dehydrogenase virus (assayed by M. A. Bioproducts). KM12SM cells plated at a density of 2 x 105/100-mm plates were transfected with the plasmid pcDNA3/GM-CSF encoding the murine GM-CSF gene under control of the CMV promoter using a mammalian transfection kit (Stratagene, La Jolla, CA) (15). After 3 wk, neomycin (800 µg/ml)-resistant clones were selected. DNA extracted from each clone was processed for detection of murine GM-CSF <2;tj;2>gene by PCR using 5'-GAATTCAAGCTTGATGTGGCTG-3' and 5'-ATTCAGAGCTGGCCTGGGCTT-3' primers. Expression of murine GM-CSF mRNA and protein was analyzed by Northern blots and ELISA, respectively, as described below.

Tumor cell-conditioned medium

Tumor cells (1 x 106) were plated onto 30-mm plates in medium containing 5% FBS. After 12 h, the cultures were washed with serum-free medium and refed with 2 ml of medium containing 5% FBS. The supernatants were collected 48 h later, centrifuged at 3000 x g at 4°C for 15 min, divided into aliquots, and stored at -80°C until use.

In vivo studies

Cultures of parental and GM-CSF-transfected KM12SM tumor cells in their exponential growth phase were harvested by a brief exposure to a 0.25% trypsin-0.1% EDTA solution. The cell suspension was pipetted to produce a single cell suspension, washed, and resuspended in HBSS. Cell viability was determined by trypan blue exclusion, and only single cell suspensions of greater than 90% viability were used. Different numbers of cells (1 x 105 to 5 x 106 in 0.2 ml HBSS) were injected into the subcutis (s.c.) of the right flank of nude mice. To determine tumorigenicity in orthotopic site, tumor cells (1 x 106 to 5 x 106 in 0.05 ml HBSS) were injected into the cecal wall of nude mice following laparotomy under methoxyflurane anesthesia (22). The incision was closed in one layer with wound clips (22). Tumor volume (TV) was estimated by the formula: TV = L (mm) x W2 (mm2)/2, where L and W represent the length and the width of the tumor mass, respectively.

Immunohistochemistry

Mice were injected s.c. with tumor cells (2 x 106/0.2 ml HBSS). The injection site was excised 1, 3, or 7 days after inoculation. The tissues were placed in OCT compound (Miles Laboratories, Elkhart, IN) and snap frozen in liquid nitrogen. Immunohistochemical staining was performed by the immunoperoxidase technique (29). Briefly, frozen sections (10 µm) were fixed with cold acetone, rinsed with PBS, and treated with 3% hydrogen peroxide in methanol for 10 min. Nonspecific reactions were blocked by incubating the sections in a solution containing 5% normal horse serum and 1% normal goat serum. The sections were incubated overnight at 4°C with 1/500 dilution of a rat anti-Ly-6G mAb specific for PMN (PharMingen) and with 1/70 dilution of rat anti-macrophage scavenger receptor (Scav-R) mAb (Serotic, Oxford, U.K.). The samples were then rinsed three times with PBS and incubated for 60 min with a peroxidase-labeled anti-rat IgG Ab (Jackson ImmunoResearch, West Grove, PA) at 1/200 dilution at room temperature, followed by incubation with diaminobenzidine substrate (Research Genetics, Huntsville, AL) for 5 min. The sections were rinsed with distilled water and counterstained with Gill’s hematoxylin (22, 29).

Collection and cultivation of mouse PMN and macrophages

PMN and peritoneal exudate macrophages (PEM) were collected by peritoneal lavage of mice given an i.p. injection of 1.5 ml of thioglycolate broth (Baltimore Biological Laboratories, Cockeysville, MD) 1 day and 4 days before harvest, respectively (22). The cells were washed with HBSS and plated onto a plastic surface for 1 h in serum-free MEM. For PMN cultures, the nonadherent cells were then collected and processed for further analysis. For PEM cultures, the nonadherent cells were removed by washing with medium. At that time, more than 98% of adherent cell populations were macrophages according to morphology and phagocytic criteria (30).

ELISA for GM-CSF and MCP-1

PEM (2 x 105) were incubated in 24-well plates for 24 h with medium alone, medium containing 10 ng/ml rGM-CSF, or tumor cell-conditioned medium. The cultures were then washed and incubated for an additional 72 h in medium containing 5% FBS. Cell-free supernatants were prepared and used for ELISA. The level of murine GM-CSF in tumor cell-conditioned medium was determined using an ELISA kit purchased from R&D Systems; the detection limit was 10 pg/ml. The assessment of murine MCP-1 in PEM supernatants was performed using an ELISA kit purchased from Biosource International (Camarillo, CA); the detection limit was 39 pg/ml.

Immunostaining of macrophages with anti-CD11b Ab

PEM (3 x 104) were plated into four-well chamber slides (Nunc, Naperville, IL) for 1 h, and nonadherent cells were removed by washing. The adherent cells were incubated for an additional 72 h in medium (control), medium containing rGM-CSF (10 ng/ml), or tumor cell-conditioned medium. The PEM were then fixed in ice-cold acetone for 5 min and rinsed twice in PBS. Immunostaining was performed by the technique described above. The Abs used were a rat anti-mouse CD11b mAb (PharMingen, San Diego, CA) at 1/500 dilution and a peroxidase-labeled anti-rat IgG Ab (Jackson ImmunoResearch) at 1/200 dilution.

Scanning electron microscopy

KM12 cells admixed with PEM (1:10 ratio) were plated onto glass coverslips. After 48 h, the cells were fixed and treated as described in detail previously (31). The specimens were mounted directly on double-stick carbon tabs and transferred to aluminum specimen mounts. The samples were coated with platinum/palladium alloy and examined in a Hitachi S520 scanning electron microscope at an accelerating voltage of 5 kV.

Macrophage-mediated cytotoxicity

Macrophage-mediated tumor cytotoxicity was assessed by a radioactive release assay as described in detail previously (22, 30). PEM (1 x 105/38-mm2 well) were incubated in 96-well plates for 20 h with medium alone or with medium containing rIFN-{gamma} (10 U/ml) plus LPS (10 ng/ml). Tumor target cells in their exponential growth phase were incubated for 24 h in medium containing 0.2 µCi/ml [3H]thymidine (>2500 Ci/mmol; ICN Biomedicals, Costa Mesa, CA). The tumor cells were washed three times with HBSS to remove unbound radioisotope, harvested by a brief trypsinization, and resuspended in medium. The target cells were plated at a density of 1 x 104 cells/38-mm2 well containing 1 x 105 control or test macrophages to obtain an initial macrophage:target cell ratio of 10:1. Radiolabeled target cells were also plated alone as a negative control. After 72 h of coincubation, the cultures were washed twice with PBS, and adherent viable cells were lysed with 0.1 ml of 0.1 N KOH. The lysates were harvested with a Harvester 96 (Tomtec, Orange, CT), and the radioactivity was monitored in a liquid scintillation counter. Cytotoxicity was calculated using the formula: cytotoxicity (%) = ((A - B)/A) x 100, where A is cpm in target cells cultured alone, and B is the cpm in test cultures.

Northern blot analyses

Poly(A)+ mRNA was extracted from tissues or cell cultures using the FastTrack kit (Invitrogen, San Diego, CA). mRNA (2 µg) was electrophoresed in 1% denaturing formaldehyde/agarose gels, transferred to GeneScreen nylon membrane (DuPont, Boston, MA), and UV cross-linked with 120,000 µJ/cm2 using a UV Stratalinker 1800 (Stratagene). Hybridizations were performed as described previously (32). Nylon filters were washed three times at 55–60°C with 30 mM NaCl, 3 mM sodium citrate (pH 7.2), and 0.1% SDS (w/v). Signals were developed on Hyperfilm-MP (Amersham, Buckinghamshire, U.K.). mRNA expression was quantified on an LKB Ultrascan XL laser densitometer (Pharmacia LKB Biotechnology, Uppsala, Sweden); each sample measurement was calculated from the ratio of the average areas between the specific mRNA transcripts and the 1.3-kb GAPDH mRNA transcript in the linear range of the film.

A 180-bp partial murine MIP-1{alpha} cDNA probe was synthesized by RT-PCR with a set of primers (5'-TATGGAGCTGACACCCCGAC-3' and 5'-GATGTATTCTTGGACCCAGGT-3') (33). Additional cDNA probes used in the analysis were a 1.3-kb PstI cDNA fragment corresponding to rat GAPDH (34), a 1-kb PstI cDNA fragment of murine GM-CSF (15), a 0.8-kb XhoI/NotI cDNA fragment of murine MCP-1 (a gift from Dr. Mukaida, Kanazawa, Japan) (35), and a 1.7-kb BamHI cDNA fragment of murine macrophage metalloelastase (MME) (36). The cDNA fragments were purified by agarose gel electrophoresis, recovered using GeneClean (BIO 101, La Jolla, CA), and radiolabeled by the random primer technique using [{alpha}-32P]dNTP (Amersham).

Statistical analysis

The significance of the in vitro data was analyzed by the unpaired Student’s t test (two-tailed). The significance of the in vivo data was analyzed by the Mann-Whitney U test. p values less than 0.05 were regarded as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of KM12SM cell lines engineered to produce murine GM-CSF

Multiple neomycin-resistant clones were isolated from cultures of KM12SM cells transfected with the murine GM-CSF gene. Three clones producing >5 ng GM-CSF/106 cells/24 h were combined, and the line was designated KM-H (high). Three clones producing 0.01–0.1 ng GM-CSF/106 cells/24 h were combined to yield the KM-L (low) line. PCR analysis confirmed that both KM-L and KM-H lines expressed the murine GM-CSF gene (Fig. 1GoA). However, the KM-H cells expressed nearly 33 times the level of murine GM-CSF mRNA as the KM-L cells (Fig. 1GoB). ELISA demonstrated that KM-L cells produced 0.05 ng GM-CSF/106 cells/24 h, whereas KM-H cells produced 9.92 ng GM-CSF/106 cells/24 h. Transfection of the murine GM-CSF gene did not alter the in vitro growth of KM12SM cells (data not shown). cDNA probes designed to analyze murine MIP-1{alpha} and MCP-1 mRNA cross-reacted with the human counterpart in mononuclear cells. Nevertheless, neither transfectant nor nontransfectant cells cultured with rGM-CSF expressed these chemokine genes (Fig. 1GoB).



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  		FIGURE 1. Characterization of KM12SM cell lines engineered to produce murine GM-CSF. KM12SM cells were transfected with pcDNA3/GM-CSF plasmid. Clones producing >5 ng GM-CSF/106 cells/24 h (KM-H) and clones producing 0.01–0.1 ng GM-CSF/106 cells/24 h (KM-l) were isolated. A, Detection of murine GM-CSF gene. Cellular DNA extracted from nontransfected (KM-P), KM-L, and KM-H cells was amplified by PCR, and the products were resolved on agarose gel. Southern blots probed by 32P-labeled murine GM-CSF cDNA are shown. B, Gene expression of murine GM-CSF and chemokines in KM12SM cells. Samples of mRNA (2 µg) extracted from the three lines of KM12SM cells, KM-P cells treated for 72 h with 10 ng/ml of rGM-CSF, and human mononuclear cells (HMNC) were resolved on agarose gel and processed for Northern blots using 32P-labeled murine GM-CSF, murine MIP-1{alpha}, and murine MCP-1 cDNA probes. Relative densitometric intensity of the transcripts (corrected for GAPDH) is recorded for each case. ND, not detected.

 
Inhibition of tumorigenicity of murine GM-CSF-transfected KM12SM cells

In the next set of experiments, we correlated the expression of murine GM-CSF with tumorigenicity of the KM12SM cells. Tumor cells were injected s.c. (ectopic site) or into the cecal wall (orthotopic site) of nude mice. Mice injected with 1 x 106 nontransfectant KM12SM (KM-P) or KM-L cells had progressively growing tumors that by 5 wk reached mean tumor volumes of about 2000 mm3 in the subcutis and 60–80 mm3 in the cecal wall. In contrast, none of the mice injected with as many as 5 x 106 KM-H cells had detectable tumors (Table IGo).


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  		Table I. Tumorigenicity of KM-P, KM-L, and KM-H cells in nude mice1

 
Inhibition of tumorigenicity of bystander cells by GM-CSF-transfected KM12SM cells

Next, we investigated whether tumor cells engineered to express murine GM-CSF can inhibit the outgrowth of bystander parental cells or different human colon cancer cells. Nude mice were injected s.c. with 1 x 106 KM-P cells or 1 x 105 HT-29 cells either alone, or admixed with 5 x 106 KM-H cells. Both KM-P and HT-29 cells produced rapidly growing tumors in the injected mice (Fig. 2Go). In contrast, none of the mice injected with KM-H and KM-P cells developed measurable tumors. The coinjection of KM-H cells and HT-29 cells also resulted in significant inhibition of HT-29 cell growth.



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  		FIGURE 2. Regression of tumorigenicity of bystander human colon cancer cells by GM-CSF-producing KM12SM cells. Nude mice (five per group) were injected s.c. with 1 x 106 parental KM12SM cells, or 1 x 105 HT-29 cells either alone or mixed with 5 x 106 KM-H cells. Values are median of tumor volume estimated as described in Materials and Methods. *, p < 0.05 as compared with appropriate controls. This is one representative experiment of two.

 
Infiltration of PMN and macrophages into the sites of tumor cell inoculation

Mice were injected s.c. with 1 x 106 KM-P, KM-L, or KM-H cells. The sites of tumor cell inoculation were excised on days 1, 3, and 7. The tissues were processed for immunohistochemical staining using mAb against Ly-6G, specific for PMN, and Scav-R, specific for macrophage. The results shown in Fig. 3Go demonstrate that regardless of cell type, on day 1, the sites of tumor cell inoculation had few PMN and macrophages. Between days 3 and 7, the PMN infiltrate diminished at the site of KM-P and KM-L injection. The number of macrophages was slightly increased on the tumor periphery, and injected cells developed discrete tumor nodules by day 7. In contrast, the site of KM-H inoculation exhibited numerous PMNs on day 3 and was densely infiltrated by macrophages on day 7. Only a few KM-H cells survived among the infiltrating macrophages.



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  		FIGURE 3. Infiltration of PMN and macrophages into site of tumor cell inoculation. Nude mice were injected s.c. with 1 x 106 KM-P, KM-L, or KM-H cells. The sites of inoculation were excised on days 1, 3, and 7. Immunohistochemical staining was performed using mAb against Ly-6G, specific for PMN, and Scav-R, specific for macrophages. Localization of PMN and macrophages was identified by incubating samples with diaminobenzidine. Bar = 100 µm.

 
Increased gene expression of chemokines at sites of tumor cell inoculation

To determine whether tumor cell-derived GM-CSF triggered recruitment of PMNs and macrophages, the s.c. sites of inoculated tumor cells (1.5 x 1.5 cm) were excised on days 1, 3, and 7 for analysis of chemokine gene expression. The results measured by Northern blot analysis are shown in Fig. 4Go. On day 1, the site of inoculated KM-H cells, but not KM-P or KM-L cells, demonstrated a high level of murine GM-CSF mRNA. This expression was decreased by day 7, most likely due to a decrease in number of tumor cells. We next determined expression levels of mRNA encoding murine MIP-1{alpha} and MCP-1. Sites of KM-P or KM-L inoculation expressed low levels of MIP-1{alpha} and MCP-1 mRNA, whereas the KM-H site expressed nearly a 20-fold increase in MIP-1{alpha} and 5-fold increase in MCP-1 mRNA expression (Fig. 4Go). The levels of MIP-1{alpha} and MCP-1 mRNA expression were also decreased by day 7.



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  		FIGURE 4. Expression of GM-CSF and chemokine genes at the site of tumor cell inoculation. Nude mice were injected s.c. with 1 x 106 KM-P, KM-L, or KM-H cells, and tissue samples were collected from the inoculation site on days 1, 3, and 7. Samples of mRNA (2 µg) extracted from each tissue were resolved on agarose gel and processed for Northern blot analysis using 32P-labeled murine GM-CSF, murine MIP-1{alpha}, and murine MCP-1 cDNA probes. The relative densitometric intensity of the transcripts (corrected for GAPDH) was recorded for each case. ND, not detected.

 
In vitro induction of MIP-1{alpha} and MCP-1 gene expression in leukocytes by GM-CSF

To prove that GM-CSF can induce secretion of MIP-1{alpha} and MCP-1 in leukocytes, mouse PMN and PEM were collected and treated for up to 8 h with 10 ng/ml rGM-CSF, equivalent to that in KM-H tumor cell-conditioned medium; the expression of mRNA encoding MIP-1{alpha} and MCP-1 was then measured by Northern analysis. The data are shown in Fig. 5Go. Treatment of PMN for 4 h with rGM-CSF resulted in a nearly 7-fold increase in MIP-1{alpha} and a nearly 10-fold increase in MCP-1 mRNA, which persisted through 8 h of exposure. Treatment of PEM for 4 h with rGM-CSF also demonstrated a nearly 17-fold increase of MIP-1{alpha} and a nearly 30-fold increase of MCP-1 mRNA, which were further increased by 8 h up to 40-fold increase over control levels. Enhanced expression of MME mRNA in PEM treated with rGM-CSF confirmed the correct fractionation of leukocytes in this set of experiments (15).



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  		FIGURE 5. Induction of chemokine gene expression in leukocytes by GM-CSF. Mouse PMN and PEM were collected and treated for up to 8 h with 10 ng/ml rGM-CSF. mRNA (2 µg) extracted from each sample was resolved on agarose gel and processed for Northern blot analysis using 32P-labeled murine MIP-1{alpha}, murine MCP-1, and murine MME cDNA probes. The relative densitometric intensity of the transcripts (corrected by GAPDH) was recorded for each case. ND, not detected.

 
Correlation between macrophage secretion of MCP-1 and expression of CD11b

To determine whether expression of MCP-1 correlates with macrophage-mediated tumor cell lysis, we incubated PEM (2 x 105) with medium alone, medium containing 10 ng/ml rGM-CSF, or tumor cell-conditioned medium. After 72 h, culture supernatants were analyzed by ELISA for levels of MCP-1 protein. As shown in Fig. 6GoA, PEM incubated in medium alone or in medium conditioned by parental KM-P cells did not secrete detectable levels of MCP-1 protein. PEM cultured in medium conditioned by KM-L cells secreted low levels of MCP-1 protein. PEM cultured in medium conditioned by KM-H cells had a 15-fold increase in MCP-1 protein, a level comparable with that in PEM exposed to an equivalent concentration of rGM-CSF, suggesting that GM-CSF induced secretion of MCP-1 by macrophages.



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  		FIGURE 6. A, Regulation of macrophage secretion of MCP-1 by GM-CSF. PEM (2 x 105) were incubated with medium alone, medium containing 10 ng/ml rGM-CSF, or medium conditioned by KM-P, KM-L, or KM-H cells. After 72 h, culture supernatants were analyzed for the presence of MCP-1 protein using a specific ELISA. {dagger}, Undetectable levels. B, Expression of CD11b on macrophages. PEM plated into chamber slides were incubated with medium alone, medium containing 10 ng/ml rGM-CSF, or medium conditioned by KM-P or KM-H cells. Expression of CD11b protein was detected by indirect immunoperoxidase assay using an anti-murine CD11b mAb. Bar = 50 µm. C, Ultrastructural interaction of macrophages with target KM12SM tumor cells. KM-P or KM-H cells were plated onto PEM (on coverslips) to obtain an initial macrophage:target cell ratio of 10:1. After 48 h, the cells were fixed and processed for scanning electron microscopy. T, tumor cell; M, macrophage. Bar = 10 µm.

 
We next determined whether treatment of macrophages with GM-CSF regulated the expression of the CD11b surface adhesion molecule, a leukocyte ß2-integrin whose expression is known to be induced by MCP-1 (37, 38). PEM plated into chamber slides were incubated with medium alone, medium containing 10 ng/ml rGM-CSF, or tumor cell-conditioned medium, and analyzed by immunohistochemical staining with anti-CD11b Ab. As shown in Fig. 6GoB, PEM cultured in medium conditioned by KM-H cells as well as medium containing an equivalent concentration of rGM-CSF stained positive for the CD11b protein, whereas control PEM or PEM cultured with medium conditioned by parental KM-P tumor cells did not. CD11b is expressed on macrophages and mature dendritic cells (11, 39, 40). Mature dendritic cells are characterized by expression of MHC class I and II as well as CD40, CD83, and CD86 (39, 40, 41). Mature dendritic cells, however, do not express macrophage markers such as CD14, FC{gamma} receptor, and F4/80 (11, 40). The cells shown in Fig. 6GoB stained positive for F4/80, suggesting that they are not mature dendritic cells. The addition of 1 µg/ml anti-GM-CSF Ab inhibited the expression of CD11b (data not shown). Scanning electron microscopy revealed that overexpression of CD11b on macrophages was associated with their increased attachment to tumor cells (Fig. 6GoC).

Macrophage-mediated tumor cell lysis

We next examined whether the production of murine GM-CSF from KM12SM cells increased their sensitivity to mouse PEM-mediated in vitro lysis (Fig. 7Go). PEM activated by incubation with IFN-{gamma} and LPS lysed all target cells (KM-P, KM-L, KM-H). The lysis of KM-P and KM-L cells could be blocked by the addition of NMA, a specific inhibitor of inducible NO synthase (42). The addition of NMA partially blocked lysis of KM-H cells (Fig. 7Go). KM-H cells were sensitive to lysis mediated by control PEM, whereas KM-P and KM-L were not (Fig. 7Go). The addition of anti-GM-CSF and anti-MCP-1 Abs (at different concentration) partially inhibited cytotoxicity against KM-H cells by 45% and 33%, respectively (Fig. 7Go). All tumor cells incubated with rGM-CSF became sensitive to lysis by PEM, which was blocked by the addition of anti-GM-CSF or anti-MCP-1 Abs. Furthermore, all tumor cells incubated with rMCP-1 became sensitive to lysis by PEM, which was blocked by addition of anti-MCP-1 Abs.



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  		FIGURE 7. Macrophage-mediated cytotoxicity against parental and GM-CSF-transfected KM12SM cells. A, PEM (1 x 105) were incubated for 24 h with medium alone or medium containing rIFN-{gamma} (10 U/ml) plus LPS (0.1 ng/ml). The PEM were washed; [3H]thymidine-labeled KM-P (P), KM-L (L), or KM-H (H) cells (1 x 104) were added, and the cultures were incubated for 72 h with medium alone or medium containing 3 mM of NMA. B, C, and D, PEM were incubated with medium containing 1 µg/ml of normal rat IgG (IgG), rat anti-immune GM-CSF Ab (anti-GM-CSF), or hamster anti-murine MCP-1 Ab (anti-MCP-1) in the absence (B) or presence of 1 ng/ml murine rGM-CSF (C) or murine rMCP-1 (D). Radiolabeled target cells were also plated alone as an additional control. The values are the mean ± SD of triplicate cultures. *, p < 0.05; **, p < 0.005 as compared with the cytolysis of KM-P. {dagger}, p < 0.05; {ddagger}, p < 0.005 as compared with the cytolysis in cultures of target cells treated with medium (A) or normal rat IgG (B, C, and D). This is one representative experiment of three.

 
Increased macrophage-mediated lysis of parental KM12SM cells by cocultivation with GM-CSF-producing cells

In the final set of experiments, we examined whether tumor cells expressing GM-CSF could induce macrophage-mediated lysis against non-GM-CSF-producing parental cells. PEM (1 x 105) were incubated with tumor cells (1 x 104) at different ratios of radiolabeled KM-P cells and nonradiolabeled KM-H cells. As shown in Fig. 8Go, incubation of increasing numbers of target KM-P cells with KM-H cells increased cytotoxicity, reaching 70% at the 1:4 ratio.



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  		FIGURE 8. Increase in macrophage-mediated cytotoxicity against parental KM12SM cells by cocultivation with GM-CSF-producing KM-H cells. PEM (1 x 105) were incubated with a mixture of tumor cells (1 x 104) at different ratios of [3H]thymidine-labeled KM-P cells (target cells) and unlabeled KM-H cells. Radiolabeled target cells were also plated alone as an additional control. Cytotoxicity was determined after 72 h of cocultivation. The values are the mean ± SD of triplicate cultures. *, p < 0.0001 as compared with the cytolysis in the absence of KM-H cells. This is one representative experiment of three.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our study demonstrates that transfection of the murine GM-CSF gene into KM12SM human colon cancer cells inhibits their tumorigenicity in nude mice. Transfection likewise inhibited the tumorigenicity of bystander parental KM12SM and HT-29 human colon cancer cells. The mechanisms for the suppression of tumorigenicity involved GM-CSF-regulated secretion of MIP-1{alpha} and MCP-1, recruitment of PMN and macrophages into the site of tumor cell inoculation, and induction of contact-dependent macrophage cytotoxicity, which resulted from up-regulation of adhesion molecules.

Previous studies have demonstrated that in addition to stimulating the proliferation and differentiation of myeloid progenitor cells, GM-CSF also regulates inflammatory responses (5, 6). Earlier studies indicate that the sites of GM-CSF-producing tumor cells contain a dense inflammatory infiltrate composed of granulocytes, tissue macrophages, dendritic cells, and CD4+/CD8+ lymphocytes (12, 13, 14, 15). The mechanism for the recruitment of inflammatory cells in response to GM-CSF has been unknown (6). The present nude mouse model using GM-CSF-transfected human colon cancer cells shows that maximal infiltration of PMN occurred 3 days after tumor cell inoculation, and recruitment of macrophages occurred by day 7. Tissue expression levels of the MIP-1{alpha} and MCP-1 genes peaked within 24 h, immediately before the appearance of infiltrating leukocytes, and the rise and fall of these chemokines correlated with the expression of tumor cell-derived GM-CSF. Data generated in vitro demonstrate that GM-CSF can induce expression of MIP-1{alpha} and MCP-1 mRNA in both PMN and macrophages, suggesting that the infiltrating myeloid cells per se may be the source of these chemokines. Tumor-derived cytokines are known to stimulate tumor-proximal resting macrophages (43). During infiltration into the tumors, macrophages are exposed to increasing concentration of signals such as disrupted extracellular matrix proteins (44). The secretion of monocyte chemotactic factors, e.g., MCP-1 and MIP-1{alpha}, is required for macrophage infiltration and activation (45). Our results show that the expression of MCP-1 and MIP-1{alpha} in the KM-H tumors was maximal by day 1 after injection when the tumors contained few PMN and macrophages (Fig. 3Go). The increase in infiltrating cells was associated with a decline in MCP-1 and MIP-1{alpha} message. These data suggest the possibility that host lymphoid cells in the subcutis are more responsive to tumor-derived GM-CSF than lymphoid cells that infiltrate the tumor a few days later. In short, it appears that the early inflammatory cells may be involved in GM-CSF-mediated secretion of chemokines.

MIP-1{alpha} is known to attract PMN, monocytes, and dendritic cells (17, 18). MCP-1 is a similarly potent attractant for monocytes and activated CD4+/CD8+ T lymphocytes (17, 19, 46). The present results demonstrate that these two critical chemoattractants mediate the recruitment of inflammatory leukocytes into tumors expressing GM-CSF. While a recent report demonstrated that GM-CSF enhances EBV-induced synthesis of MIP-1{alpha} in human PMN (47), the current study is the first to establish the role of GM-CSF in regulating synthesis of MIP-1{alpha} and MCP-1 in murine PMN and macrophages. In the mouse, two CXC chemokines, keratinocytes (KC) and MIP-2, have been shown to induce neutrophil recruitment (48). Whether GM-CSF regulates their secretion is to be determined. Recent data also suggest that KC and MIP-2 can increase expression of CD11b on PMNs (49, 50). Whether these chemokines enhance contact-dependent tumor cell lysis by PMNs is unclear.

There is a considerable body of evidence that GM-CSF plays a crucial role in CD4+/CD8+ T cell-mediated immune response by activating dendritic APC (10, 13, 14). Specific immunity, however, cannot explain the present results showing that engineered expression of GM-CSF in human KM12SM cells inhibits tumorigenicity not only in the transfected cells, but also in bystander parental and different human colon cancer cells implanted into nude mice. Tumor-associated macrophages activated in response to appropriate stimuli play a key role in nonspecific antitumor immunity (1, 4), and the present in vitro generated data prove that the variants of KM12SM cells are susceptible to lysis by macrophages activated with rIFN-{gamma} plus LPS, which are themselves potent inducers of macrophage tumoricidal activity (20). GM-CSF has been shown to activate monocytes to become cytotoxic against human melanoma cells in vitro (26), and the present results demonstrate that GM-CSF-producing KM12SM cells were highly sensitive to lysis by control macrophages. In the presence of rGM-CSF, all lines of KM12SM cells became sensitive to lysis mediated by PEM, an event blocked by anti-GM-CSF Abs. Furthermore, GM-CSF-producing KM cells increased macrophage-mediated lysis of bystander parental cells, providing strong evidence that tumor-derived GM-CSF is an exogenous regulator of macrophage tumoricidal activity, i.e., GM-CSF attracts macrophages into the tumor site and activates them to become tumoricidal.

The precise mechanism that regulates GM-CSF-mediated tumor cell lysis by macrophages is still unclear (39). There is compelling evidence that MCP-1 can stimulate macrophage-mediated inhibition of tumor growth in vitro and in vivo (19, 23), and we have previously reported that the in vitro treatment of macrophages with MCP-1 enhances LPS-induced cytotoxic properties (24, 25). The present data show that the lysis of GM-CSF-producing KM12SM cells by control macrophages can be decreased by the addition of anti-MCP-1 Abs and that all variant KM cells became sensitive to lysis by macrophages in the presence of rMCP-1, suggesting that macrophage-produced MCP-1 may mediate the antitumor properties of GM-CSF.

For macrophage-mediated tumor cytotoxicity, the macrophages must bind avidly to target cells and the inhibitory molecules must be secreted (4). Our present results suggest that MCP-1 enhances binding of macrophages to tumor target cells. This molecule can regulate the expression of ß2 integrins, which play a key role in chemotaxis, phagocytosis, and other adhesion-dependent functions of myeloid cells (51). In our study, medium conditioned by GM-CSF-producing KM12SM cells, as well as medium containing an equivalent concentration of rGM-CSF, induced expression of the CD11b surface adhesion molecule on PEM, and the expression of CD11b directly correlated with macrophage secretion of MCP-1, as measured by ELISA and attachment of PEM to tumor cells. These data suggest that MCP-1 may have a role in contact-dependent macrophage cytotoxicity, one involving induction of adhesion molecules.

Fig. 9Go summarizes our data and illustrates our working model for the mechanism of nonspecific antitumor immune response against GM-CSF-transfected cancer cells. Tumor cell-derived GM-CSF stimulates PMN and macrophages to secrete MIP-1{alpha} and MCP-1, which in turn can trigger recruitment of myeloid cells into the inoculation site. MCP-1 also induces expression of the CD11b adhesion molecules on macrophages, which allows for increased attachment to target tumor cells and their lysis. Data from our laboratory also show that GM-CSF induces expression of metalloelastase in PEM (52), which correlates with cleavage of plasminogen to angiostatin (53), suggesting that tumor-derived GM-CSF can lead to inhibition of neoplastic angiogenesis (15). Collectively, these data recommend the therapeutic use of the GM-CSF gene for treatment of human cancer.



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  		FIGURE 9. A working model. Tumor cell-derived GM-CSF stimulates PMN and macrophages (M{phi}) to secrete MIP-1{alpha} and MCP-1 (1 ). These chemokines trigger chemotaxis of myeloid cells into the tumor inoculation site (2 ). MCP-1 can also induce expression of the CD11b adhesion molecules on macrophages (3 ), which allows for an increase in contact-dependent cytolysis against tumor cells (4 ).

 


   Acknowledgments
 
We thank Walter Pagel for his critical editorial review, and Lola López for expert assistance in the preparation of this manuscript.


   Footnotes
 
1 This work was supported by Cancer Center Support Core Grant CA16672 and Grant R35-CA42107 from the National Cancer Institute, National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Isaiah J. Fidler, Department of Cancer Biology, Box 173, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. E-mail address: Back

3 Abbreviations used in this paper: PMN, polymorphonuclear; MCP-1, monocyte chemoattractant protein-1; MIP, macrophage-inflammatory protein; MME, murine macrophage metalloelastase; NMA, NG-methyl-L-arginine; PEM, peritoneal exudate macrophage. Back

Received for publication September 10, 1999. Accepted for publication December 15, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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