Recombined CC chemokine ligand 2 into B16 cells induces production of Th2-dominanted cytokines and inhibits melanoma metastasis

Immunology Letters 113 (2007) 19–28

Recombined CC chemokine ligand 2 into B16 cells induces production of Th2-dominanted cytokines and inhibits melanoma metastasis夽 Kaimeng Hu, Jun Xiong, Kaihong Ji, Hongyu Sun, Jing Wang, Houqi Liu ∗ Research Center of Developmental Biology & Department of Histology and Embryology, Second Military Medical University, Shanghai 200433, PR China Received 28 March 2007; received in revised form 24 June 2007; accepted 1 July 2007 Available online 21 August 2007

Abstract This study is aimed to verify whether CCL2 can induce Th2 polarization in vivo and subsequently inhibit tumor metastasis. B16 cells (a murine melanoma cell line) highly expressing CCL2 (CCL2-B16 cells) were obtained by transfection with recombinant plasmid CCL2-pcDNA3. Primary thymocytes were co-cultured with CCL2-B16 cells and STAT-6-mediated Th2 polarization was noticed after co-culture. Caudal vein injection of CCL2-B16 cells effectively inhibited pulmonary metastasis in C57BL/6 mice, but not in nude mice, indicating that T cells play a role in CCL2-induced inhibition of tumor metastasis. We found that high level of CCL2 up-regulated the expression of Th2-related cytokine (IL-4) in tumor microenvironment and increased CD4+, CD8+, and CD45RB+ cells in the peripheral blood and tumor tissues. We also demonstrated that inoculation of mice with CCL2-B16 cells prolonged mice survival time when they were reinjected with wildtype B16 cells, implying that CCL2 can activate immuno-memory in mice. It is concluded that high expression of CCL2 can induce Th2 polarization in tumor microenvironment and can effectively inhibit tumor metastasis, which casts new lights on the role of chemokines in reconstruction of immune surveillance in patients suffering from tumors. © 2007 Elsevier B.V. All rights reserved. Keywords: Th2 cells; Chemokine; Tumor microenvironment; Memory T cells; Cell differentiation

1. Introduction The process of carcinogenesis always involves the immune response of the body. Under immune surveillance the immunologic agents can effectively guard against and even kill cancer cells, with chemokines and cytokines as the initiators of the both innate and adaptive immune responses. Unfortunately, immune escape occurs at the initial stage of carcinogenesis, in which the T cells fail to recognize tumor cells and the tumors grow continuously [1–3]. Once malignant transformation happens and there are metastases, it becomes too late for immune agents to handle the tumor. Therefore, one of the problems for tumor immunotherapy is how to reactivity the chemokines function in tumor immunology [2,4].

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This work was supported by the National Natural Science Foundation of China (No. 30428001) and Major Program of “The Eleventh Five-Year Plan” of PLA Medical Science Research (No. 06G62). ∗ Corresponding author. E-mail address: [email protected] (H. Liu). 0165-2478/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2007.07.004

The presence of certain chemokines can effectively slow down the growth of tumor and inhibit its metastasis [5,6]. CC chemokine ligand 2 (CCL2), also named as monocyte chemoattractant protein (MCP-1), is a member of the CC chemokine family. It can recruit leucocytes harboring CC chemokine receptor 2 (CCR2), including monocytes, some T cells, eosinophils, and so on. CCL2 can also gather macrophages that phagocytize tumor cells; especially when a great amount of macrophages were present in the microenvironment of tumor, they can directly or indirectly induce death of tumor cells [7–9]. However, the role of macrophages for tumors remains unclear because some cytokines secreted by macrophages (such as VEGF) can promote the vascularization in tumor tissues and subsequently promote the growth and metastasis of tumors [5,10]. CCL2 is virtually a chemoattractant for most subtypes of leucocytes, including T lymphocyte. Studies have found that endogenous CCL2 can act on T cells and subsequently trigger Th2-type and Th1-type immune responses [11]. CD4 T-cellmediated immune responses can be divided into Th2-type and Th1-type according to the cytokines they secrete. Th1-type response mainly involves IL-2, IFN-␥ and activation of T-bet;

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Th2-type response mainly involves IL-4, IL-5 and activation of GATA-3 and STAT-6. CCL2 participates in the differentiation of T cells; although it depends on the specific receptor and local microenvironment, in most cases CCL2 is involved in Th2-type response [12,13]. Traditionally, the polarization of Th1 cells is thought to contribute to cellular immune response and result in the increase of CD8+ cytotoxic T lymphocytes (CTLs); Th2 cell is associated with hormonal immune response and the increase of CD4+ helper T cells (Th) and memory T cells. Th and memory T cells play very important roles in maintaining the activity of CTLs during anti-tumor immune surveillance [14]. It has been reported that injection of Th2 cells into mice effectively repressed the tumor metastasis, increased T cells sensitivity to tumor antigens, and induced effective anti-tumor immune response [15,16]. So far, to our knowledge, there has been no report on in vivo induction of Th2-dominant differentiation for inhibiting tumor metastasis. The aim of our study is to verify whether high concentration of CCL2 can induce Th2-type immune response and subsequently inhibit tumor metastasis. We introduced CCL2 in eukaryotic cell B16 to obtain B16 cells highly expressing CCL2; and animal experiment demonstrated that CCL2 induced the polarization of Th2 cells in the tumor microenvironment and inhibited pulmonary tumor metastasis. 2. Materials and methods 2.1. Animal Male C57BL/6 mice and nude mice, aged 6–8 weeks, were purchased from Shanghai Slac Laboratory Animal Co., Ltd. and kept in specific-pathogen-free condition. 2.2. Tumor cells and transfection with CCL2 Mouse melanoma B16 cells (Shanghai Institute of Cellular Biology, Academia Sinica) were cultured in complete DMEM (Dulbecco’s modified eagle’s medium, GIBCO, Carlsbad, California) supplemented with 10% (v/v) FBS, 100 U/ml penicillin, and 100 ␮g/ml streptomycin sulfate (GIBCO) at 37 ◦ C in 5% CO2 . The forward primer (5 ccc aag ctt agc caa ctc tca ctg aag cca g) and the reverse primer (5 cgg gat ccg cat cac agt ccg agt cac act ag) for PCR amplification were designed by the computer software Generunner according to the full-length cDNA of mouse CCL2 (GenBank accession No. NM 011333) and were synthesized by Shanghai Sangon Biotechnology Co., Ltd. The total mRNA of activated celiac macrophages was extracted with RNA extraction kit (Bioasia Biotechnologies, Inc., Shanghai). The reverse transcriptase-polymerase chain reaction (RT-PCR) was performed by employing the following protocol: total RNA and OligdT; 70 ◦ C for 10 min, adding AMV (25U, Promaga), 5 ×buffer, 10 mmol/L dNTPs, 42 ◦ C for 1 h, 99 ◦ C for 5 min, 4 ◦ C for 5 min; adding ExTaq, the upper and lower primers, one cycle at 99 ◦ C for 5 min, 30 cycles at 99 ◦ C for 30 s, 58 ◦ C for 30 s, 72 ◦ C for 30 s, followed by 72 ◦ C at 10 min. PCR product (485 bp) was digested with BamH I and Hind III and was inserted

into the correspondingly digested pcDNA3.0 vector (Invitrogen, Carlsbad, California); the recombinant vector pcDNA3.0-CCL2 was then transformed into E. coli DH5 alpha. Positive clones were harvested 24 h later and cultured. B16 cells (1 × 105 ) were seeded in 6-well plates (35 mm) and when cell growth reached 60% confluence, pcDNA3.0-CCL2 and blank pcDNA3.0 vector (as negative control) were used to transfect the cells with liposome Fungene-6 (Roche, Indianapolis, IN). The transfected cells were maintained in a selection medium containing 400 ␮g/ml G418 (Merck, Germany) until the appearance of positive clones. Stable clones were subjected to two rounds of serial dilution in 24- and 96-well plates. Four CCL2-transfected clones (CCL2B16: A4, A5, A6, B1) and two blank vector-transfected clones (mock-B16: H6, I5) were selected for further study. RT-PCR was used to detect the expression of CCL2 mRNA in transfected B16 cells. Four clones of CCL2-B16 (A4, A5, A6, B1), mock-B 16 (H6, I5), and wildtype B16 cells (WT) (1 × 106 ) were cultured for 2 days and the total mRNA was extracted for RT-PCR: 100 ng/␮l template RNA, CCL2 primer 1: cgg gat ccg ctc tct ctt cct cca cca c; primer 2: ccc aag ctt ccg agt cac act agt tca ctg, internal control G3PDH, primer 1: tga agg tcg gtg tga acg gat ttg gc; primer 2: cat gta ggc cat gag gtc cac cac. The reaction was run for 30 cycles. CCL2 levels in the cell suspension were determined by enzyme-linked immunosorbent assay (ELISA): the culture supernatants were harvested and examined with rabbit anti-mouse CCL2 ELISA kit (SIGMA). The standard curves were plotted and the contents of CCL2 were calculated for each cell clone. The mean values and the standard difference were also calculated (n = 5). The chemotactic activity of recombinant CCL2 was tested by the following procedures. Equal volumes of DMEM containing 10% FBS and 2% agarose gel were mixed for perforation of six groups of holes (2.4 mm in diameter) in glass dishes, with three holes located on a line at an equal distance of 2.4 mm in each group. The upper, middle, and lower holes in each group were filled with 10 ␮l suspensions of CCL2-B16 cells, macrophages, and wildtype B16 cells, respectively. The system was cultured at 37 ◦ C for 4 h, fixed with 2% glutaral for 30 min, and observed under microscope after Giemsa staining. The ocular micrometer was used to measure the migrating distance of macrophages to the upper (A, mm) and the lower (B, mm) holes, and the chemotaxis index was calculated as A/B. 2.3. In vitro co-culture system Thymus glands obtained from new born mice were cut into small pieces with scissors, digested with 0.05% trypsin-EDTA at 37 ◦ C for 30 min (GIBCO, Carlsbad, California), and finally made into cell suspensions. Wildtype B16, CCL2-B16, and mock-B16 cells (2 × 105 ) were separately cultured with primary thymocytes (2 × 107 ) in 6-well plates with a final volume of 2 ml. Cultured thymocytes were taken as the control. Two hours later B16 cells adhered to the bottom of plate and the thymocytes still suspended in the medium. Two hours later the thymocytes were harvested from cell suspension. After centrifugation and lysis, the lytic products were labeled with rabbit anti-mouse STAT-6, rabbit anti-mouse

K. Hu et al. / Immunology Letters 113 (2007) 19–28

GATA-3, and goat anti-mouse T-bet (Boster). ␤-Actin was taken as internal control. Four hours after co-culture, the supernatants were collected and the thymocytes were harvested by centrifugation for total RNA extraction. RT-PCR was used to detected the expression cytokine: (1) IL-2: p1: tcc act tca agc tct aca g, p2: gag tca aat cca gaa gat gcc; (2) IFN-␥: p1: tta act caa gtg gca tag atg, p2: gaa gaa ggt agt aat cag gtg (Th1 dominant); (3) IL-4: p1: cga aga aca cca cag aga ctg agc, p2: gac tca ttc atg gtg cag ctt atc g; (4) IL-5: p1: atg act gtc cct ctg tgc ctg gag c, p2: ctg ttt ttc ctg gag taa act ggg g (Th2 dominant). G3PDH served as control and shared the same primer as above. The co-culture experiments were repeated five times and the gray scale ratios of chemokines to G3PDH band were calculated; the mean values and the standard difference were also calculated (n = 5).

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PBS; lavage solutions were collected to prepare cell suspension by centrifugation. Cell smears were prepared for immunofluorescence staining. The red color (Cy3) and the green color (FITC) were used to label CD3 and CCL2, respectively. The immunofluorescence procedure was carried out as following: the smears was immobilized with −10 ◦ C acetone for 7 min and incubated with primary antibody overnight at 4 ◦ C, then subjected to further incubation with fluoresce in isothyocyanate (FITC)-conjugated goat anti-rat IgG (for CCL2) or Cy3 goat anti-rabbit IgG (for T cells) for 30 min at 37 ◦ C, mounted with Fluorescent Mountin Medium (DAKO, Cambridge shire, CA) to prevent bleaching, finally observed under fluorescent microscope. Meanwhile, the cell suspension was marked with immuno-labeled mAbs: goat anti-mouse-FITC CD4+ and goat anti-mouse-PE CD8+, and was analyzed with flow cytometry.

2.4. Establishment of pulmonary metastasis model In order to make clear the anti-tumor effect of CCL2 is through T cells or macrophages, 15 nude mice were injected with 2 × 105 wildtype B16, CCL2-B16, or mock-B16 cells via the caudal veins to establish the pulmonary metastasis models (5 mice in each group); 1 week later the mice were sacrificed for observation of the lung. Meanwhile, fifteen C57BL/6 mice were injected with 2 × 105 wildtype B16, CCL2-B16, and mock-B16 cells via the caudal veins to establish pulmonary metastasis models (5 mice in each group). The animals were sacrificed 10 days later, the lungs were harvested and kept in 10% formalin for 24 h, then imbedded with paraffin and made into 4 ␮m thick sections for H-E staining. Immunohistochemical staining was employed to detect the expression of CCL2, IL-4, and IFN-␥: the sections were de-paraffined, incubated in 3% hydrogen peroxide/methanol at room temperature for 10 min, then cultured with rabbit antimouse antibodies of CCL2, IL-4, and IFN-␥, kept overnight at 4 ◦ C, followed by incubation with HRP goat anti-rabbit IgG; the labeling was visualized using a DAB kit (Maixin, PRC). Peripheral blood samples were obtained from the orbita of the pulmonary metastasis models and were centrifuged with percoll (ρ = 1.088) at 5000 rpm for 30 min. The mononuclear leucocytes were harvested from the single-cell suspension and were labeled with FITC CD4, PE CD8, and FITC-CD45RB. After washed with PBS for three times the mononuclear leucocytes were analyzed by FACScan flow cytometer (BD, cellquest). Meanwhile, the spleens of mice were removed and incubated with 0.25% trypsin-EDTA and the erythrocytes were destroyed by 0.83% NH4 Cl. The remaining spleen cells were collected and made into cell suspension with 0.05 M PBS, then the cell suspension (400 ␮l) was labeled with FITC-CD45RB and analyzed by FACScan flow cytometer (BD, cellquest). 2.5. Establishment of local tumor model The local tumor models were produced by injecting 2 × 105 wildtype B16, CCL2-B16, and mock-B16 cells into the left hind legs of 15 C57BL/6 mice (5 mice in each group). The mice were sacrificed 2 weeks later and the tumor masses and the corresponding adjacent tissues were harvested, washed with

2.6. Establishment of cross-inoculation model In order to observe the effect of CCL2 on long-effect immunity, 40 C57BL/6 mice were evenly randomized into four groups, namely, CCL2-WT, CCL2-CCL2, WT-CCL2, and WTWT group. Mice in CCL2-WT and CCL2-CCL2 groups were firstly injected with 1 × 104 CCL2-B16 cells via caudal veins and, 5 days later, they were re-injected with 1 × 104 wildtype B16 cells and 1 × 104 CCL2-B16 cells, respectively; those in WT-CCL2 and WT-WT were firstly injected with wildtype B16 cells and re-injected with CCL2-B16 and wildtype B16 cells, respectively. The death time of animals was recorded in each group and the survival curves were plotted for statistical analysis. 2.7. Statistical analysis The data were analyzed with SAS 6.12 software. The expression of CCL2 (2.2), the ratios of gray scales of electrophoresis bands (2.3), and the rates of positive cells (2.4, 2.5) were analyzed using ONE-WAY ANOVA analysis. Dunnett’s test was used for the comparison between the CCL2-B16 group and the other groups; Log-Rank test was used for the survival curves of the cross-inoculation mice models. A P value less than 0.05 was taken as statistically significant. 3. Results 3.1. Selection of CCL2-B16 cells with chemotactic activity pcDNA3.0-CCL2 was successfully constructed and introduced into B16 cells. RT-PCR and ELISA showed that A5 clone (CCL2-B16 cells) had the highest expression of CCL2 ([0.57 ± 0.35] ng/ml) (Fig. 1), which was significantly higher than that of control cells (all P < 0.05). A5 clone was chosen for studying the chemotactic activity of recombinant CCL2 protein. We found that A5 cells induced a (5.1 ± 0.2) mm migrating distance of the macrophages and wildtype B16 cells induced a distance of (0.7 ± 0.01) mm, with the chemotaxis index calculated as 5.1 (an index over 2 indicates obvious chemotaxis

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Fig. 1. Expression of CCL2 in each transfected cell clone. (A) The levels of CCL2 mRNA in different cell clones as determined by RT-PCR; (B) the contents of CCL2 proteins in different cell supernatants as determined by ELISA, calculated as the mean ± S.D. (n = 5). A6, A5, and A4 were CCL2-B16 cells; Mock cells were transfected with blank vectors; WT indicated wildtype B16 cells. The level of CCL2 in CCL2-B16 cells was significantly higher than those in the other two groups (*P < 0.05).

Fig. 2. Expression of cytokines mRNA in thymocytes after co-cultured with three types of B16 cells. The total RNA was extracted from the thymocytes and the RT-PCR results were showed in (A). The gray scale ratios of the cytokines to G3PDH were (mean ± S.D., n = 5) shown in (B). WT, MOCK, and CCL2 were the three co-culture groups and the CON group only contained thymocytes (negative control). The expression of IL-4 and IL-5 in CCL2 group was higher than those in the other two groups (both P < 0.05); there was no significant difference in the expression of IL-2 and IFN-␥ between all the groups.

activity), indicating that the recombinant CCL2 protein produced by CCL2-B16 cells had chemotactic activity. 3.2. Th2 polarization induced by high expression of recombinant CCL2 protein in co-culture system Four hours after culture B16 cells adhered to the plate while the thymocytes suspended in the medium. The thymocytes were harvested by centrifugation and the expression of IL-4, IL-5, IL2, and IFN-␥ mRNA was examined by RT-PCR. The expression of IL-2 and IFN-␥ in thymocytes was similar in all the groups (P = 0.076). The expression of IL-4 and IL-5 mRNA in CCL2B16 group was significantly higher than those of other groups (all P < 0.05), suggesting that high concentration of CCL2 can induce Th2 polarization (Fig. 2). Immunobloting results showed that the expression of STAT-6 and GATA-3 (Th2 dominant) was increased in the thymocytes 2 h after co-cultured with CCL2B16, while T-bet (Th1 dominant) was negative in all groups (Fig. 3). The findings indicated that CCL2 may induce Th2 polarization through activating STAT-6. 3.3. High expression of CCL2 inhibits tumor metastasis in mice through T cells Normal C57BL/6 mice were injected with WT-B16, mockB16, or CCL2-B16 cells via the caudal veins to establish pulmonary metastasis model. Ten days later the lungs of mice

in WT-B16 and mock-B16 groups were full of black melanoma colonies (Fig. 4), but almost no melanoma colony was found in the lungs of mice in CCL2-B16 group, indicating CCL2 inhibited the tumor metastasis. However, when the same experiment was performed with nude mice, black melanoma colonies were found all over the lungs of mice in all the three groups 1 week after injection (Fig. 5), which indicated that CCL2 exerted its inhibitory effect via T cells.

Fig. 3. Examination of STAT-6, GATA-3, and T-bet expression in thymocytes after co-cultured with three types of B16 cells by Western blotting. WT, MOCK, and CCL2 were the three co-culture groups and the CON group only contained thymocytes (negative control). The expression of STAT-6 and GATA-3 was increased in CCL2 group and the expression of T-bet was at a low level in all four groups.

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Fig. 4. Inhibition of pulmonary metastasis in C57BL/6 mice model. The lungs of mice were harvested 10 days after they were inoculated with 2 × 105 CCL2-B16, mock-B16, and wildtype-B16 cells. (A) The gross lung tissue and lung sections of wildtype-B16 group (HE staining, 100×); (B) the gross lung tissue and lung sections of CCL2-B16 group (HE staining, 100×). The tumor metastasis was greatly inhibited in CCL2-B 16 group.

Immunohistochemical staining of the pulmonary metastasis tumor samples showed obvious expression of CCL2 and IL-4 in CCL2-B16 group, but not IFN-␥. There were only weak signals in the samples of the other two groups (Fig. 6). Analysis of peripheral blood T cell differentiation showed that the proportions of CD8+, CD4+, and CD45RB+ cells in CCL2-B16 group (n = 5) were significantly higher than those in wildtype B-16 group and mock-B16 group (CD8+ cells: 25.95 ± 0.2 versus 13.72 ± 0.3, 13.22 ± 0.1, both P < 0.01; CD4+ cells: 38.14 ± 0.2 versus 21.74 ± 0.1, 17.09 ± 0.1, both P < 0.05; CD45RB+ cells: 48.14 ± 0.1 versus 17.51 ± 0.3, 10.67 ± 0.4, both P < 0.05). We also found that the positive rate of CD45RB+ spleen cells CCL2-B16 group was higher than those of the other two groups (87.26 ± 0.3 versus 66.9 ± 0.4, 71.68 ± 0.2, P < 0.05) (Fig. 7). The above findings indicated that high expression of CCL2 induced the increase of effector T cells. 3.4. Expression of CCL2 in tumor microenvironment and its chemotactic effect on T cells In the local tumor models, we found that the numbers of CD4+ and CD8+ cells in CCL2-B16 group were sig-

nificantly more than those in wildtype B-16 and mock-B16 cells (CD4+ cells: 30.10 ± 0.2 versus 0.79 ± 0.03, 0.22 ± 0.1; P < 0.01. CD8+ cells: 8.82 ± 0.5 versus 3.72 ± 0.3, 0.17 ± 0.08; P < 0.01). Immunofluoresence results demonstrated strong positive signals of CCL2 and CD3 in CCL2-B16 group, but only weak positive signals of CD3 in the other two groups (Fig. 8). It indicates that high level of CCL2 in tumor microenvironment could help to gather T cells around tumors.

3.5. High expression of CCL2 prolonged survival of mice in cross-inoculation model The survival curves drawn from the cross-inoculation models showed that the survival periods of mice in CCL2-WT and CCL2-CCL2 groups were obviously prolonged compared with those in WT-CCL2 and WT-WT groups. The survival rate of mice in CCL2-WT group was significantly higher than that in WT-CCL2 group (Chi-square = 55.3491, P < 0.05), which received the same types and same amount of cells, but only with different orders (Fig. 9).

Fig. 5. Lung tissues of nude mice after injecting different kinds of B16 cells via caudal veins. (A) Wildtype-B16 group; (B) CCL2-B16 group. There was no obvious difference in the tumor metastasis (in terms of number and size) between the two groups.

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Fig. 6. Immunohistochemical determination of CCL2 and cytokines (IL-4 and IFN-␥) expression in pulmonary metastasis tumors. (A) CCL2-B16 group. The expression of CCL2 and IL-4 was strong and that of IFN-␥ was weaker; (B) wildtype-B16 group. The expression of CCL2, IL-4 and IFN-␥ was weak in lung tumor tissues.

4. Discussion In this study we aimed to study the influence of high CCL2 expression on T cell differentiation in tumor microenvironment in vivo and its inhibitory effect on tumor metastasis. We introduced CCL2 into B16 cells via a eukaryotic vector and found that CCL2 could induce Th2 polarization in vitro. We also established three animal models and they suggested that high concentration of CCL2 can induce Th2 cells polarization and the increase of effector T cells in local microenvironment, and obviously inhibit tumor metastasis. Previous studies only suggested an unclear role of CCL2 in anti-tumor immune response. Although CCL2 can mobilize monocytes to tumor sites, it demonstrates a limited anti-tumor

immune response to breast and ovarian tumors, which we also confirmed with nude mice pulmonary metastasis models [17]. Nude mice have mononuclear-macrophage system, but have no T cells. We found injection of CCL2-B16 cells into nude mice did not reduce the pulmonary metastasis; whereas injection of CCL2-B16 cells into normal mice effectively inhibited tumor metastasis, indicating that macrophages play no important role in inhibiting of tumor metastasis and CCL2 may exert its antitumor effect through other pathways [18]. Van Deventer et al. showed that MIP-1␣ (CCL3) could mobilize a great number of T cells to the tumor site, inducing effective immune surveillance and tumor inhibition. Therefore, it can be deduced that CCL2 may exert its anti-metastasis effect through activating certain functions of T cells [19,20].

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Fig. 7. Proportions of CD4+, CD8+, and CD45RB+ in pulmonary metastasis mice model by flow cytometry. (A) The proportions of CD4+ and C8+ in peripheral monocytes; (B) the proportions of CD8+ and CD45RB+ in peripheral monocytes; (C) the proportion of CD45RB+ in spleen suspension. C, M, and W indicated CCL2-B16, Mock-B16, and wildtype B16 cell groups, respectively. It was found that the proportions of CD4+, CD8+, and CD45RB+ cells were all increased in CCL2-B16 group (*P < 0.05).

The effect of CCL2 on T cells is mainly manifested by its influence on the differentiation of Th cells. CCL2 can induce Th2-dominant immune responses in infections and rheumatic diseases. Traynor et al. found in a human pulmonary Cryptococcus neoformans infection model that CCL2 could induce T2-dominant immune response in a time- and concentrationdependent manner. A number of tumor antigens and chemokines (including CCL2, CCL17 and CCL11) can be detected in the tumor microenvironment of patients with Hodgkin’s disease, which was thought to be related to the differentiation and functioning of T cells [21–23]. In the present study we also found that high concentration of CCL2 in tumor microenvironment can greatly increase Th2-associated cytokines (IL-4 and IL-5) by activating intracellular signal molecules STAT-6 and GATA3, resulting in transformation of Th0 to Th2. In our pulmonary metastasis model, the high expression of CCL2 in CCL2-B16 group was accompanied by high expression of IL-4, implying local high expression of CCL2 can induce transformation of Th cells into Th2 cells. Th1- and Th2-dominant immune responses are the result of Th0 cell differentiation. Mattes and colleagues found injection of Th1 cells into mice did not effectively inhibit the pulmonary

metastasis of melanoma, while Th2 cells demonstrated a better inhibitory effect. It is implied that Th1 cells function only in a short time and Th2 cells activate humoral immune response; the latter can maintain the activity of memory T cells and subsequently prolong the action of CTLs [15,24–26]. Our study found that high concentration of CCL2 did not exert its antitumor activity through Th1 cell polarization. In mice models with decreased pulmonary metastasis, the pulmonary expression of IFN-␥ was weaker, indicating Th1-oriented differentiation was not the dominant [27,28]. In our local tumor model, we found that high expression of CCL2 gathered large number of T cells (CD3+) in the adjacent tumor tissues and the proportions of CD4+ and CD8+ cells were also increased. In our pulmonary metastasis model we found CD4+ and CD8+ cells were increased in CCL2-B16 group. The increase of these cells might be a result of Th2 cell differentiation [29,30]. Previous studies have demonstrated that Th2 cells possessed effective immune memory function, which was also verified by the cross-inoculation model in the present study. The better survival of mice in CCL2-WT group indicated that an initial injection of CCL2-B16 cells could enhance the immune response against the antigens of B16 cells, prolonging the mice

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Fig. 8. Examination of lavage liquids after washing tumor tissues harvested from local tumor models. (A) The proportions of CD4+ and CD8+ as determined by flow cytometry. C, M, and W indicated wildtype B16, mock-B16, and CCL2-B16 groups, respectively. The proportions of CD4+ and CD8+ in CCL2-B16 group were significantly higher than those of the other two groups (* P < 0.05). (B) The immunofluorescence results of cell smears. 1, indicated view under light microscope in CCL2-B16 group, 2, 3, and 4, respectively indicated the colors of green (FITC-CCL2), red (Cy3-CD3), and double labeling result in CCL2-B16 group, implying that CCL2 gathered T cells around the tumor cells. 5, 6 showed the negative results under light microscope and double labeling in wildtype group (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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Fig. 9. Survival curves of mice after cross-inoculation. Mice firstly injected with wildtype B16 cells had a lower survival curve than those firstly injected with CCL2-B16 cells, indicating mice firstly injected with wildtype B16 cells died faster. Log-Rank test showed that the survival curve of CCL2-WT group was significantly higher than that of WT-CCL2 group (P < 0.05).

survival after re-injected with wildtype B16 cells [31,32]. As a marker of memory T cells, CD45RB+ cells were found increased in the peripheral blood and spleen in the present study, which further demonstrates CCL2 functions via Th2 pathway [33,34]. In the present study we investigated the influence of endogenous chemokines on the tumor microenvironment and Th2 polarization, which has not been given much attention by other researchers previously [35]. Our experiment makes it easy to influence Th cells differentiation in vivo and improves the immune response of animals to the existing antigens. We noticed that CCL2 had a limited influence on the growth of local (primary) tumors, but it was effective in inhibiting tumor metastasis, which, however, indicates that CCL2 s role as a treatment for tumor is limited [15,36,37]. In conclusion, high expression of CCL2 can recruit immune cells and inhibit tumor metastasis. CCL2 can mobilize T lymphocytes, activate STAT-6 signal pathway, up-regulate Th2associted cytokines (IL-4, IL-5), and increase the numbers of peripheral CD4+, C8+, and CD45RB+, thus inhibits metastasis and recurrence of tumors. High level of CCL2 might be employed for building a balance between immune surveillance and immune escape, contributing to inhibition of tumor metastasis. References [1] Smyth MJ, Godfrey DI, Trapani JA. A fresh look at tumor immunosurveillance and immunotherapy. Nat Immunol 2001;2(4):293–9. [2] Pardoll D. Does the immune system see tumors as foreign or self? Annu Rev Immunol 2003;21:807–39. [3] Finn OJ. Tumor immunology at the service of cancer immunotherapy. Curr Opin Immunol 2004;16(2):127–9. [4] Ahmad M, Rees RC, Ali SA. Escape from immunotherapy: possible mechanisms that influence tumor regression/progression. Cancer Immunol Immunother 2004;53(10):844–54. [5] Balkwill F. Chemokine biology in cancer. Semin Immunol 2003;15:49–55. [6] Murphy PM. Chemokines and molecular basis of cancer metastasis. New Engl J Med 2001;354:833–5.

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[7] Conti I, Rollins BJ. CCL2 (monocyte chemoattractant protein-1) and cancer. Semin Cancer Biol 2004;14:149–54. [8] Goswami S, Sahai E, Wyckoff JB, Cammer M, Cox D, Pixley FJ, et al. Macrophages promote the invasion of breast carcinoma cells via a colonystimulating factor-1/epidermal growth factor paracrine loop. Cancer Res 2005;65(12):5278–83. [9] Monti P, Leone BE, Marchesi F, Balzano G, Zerbi A, Scaltrini F, et al. The CC chemokine MCP-1/CCL2 in pancreatic cancer progression: regulation of expression and potential mechanisms of ant malignant activity. Cancer Res 2003;63:7451–61. [10] Hiratsuka S, Watanabe A, Aburatani H, Maru Y. Tumour-mediated upregulation of chemoattractants and recruitment of myeloid cells predetermines lung metastasis. Nat Cell Biol 2006;8(12):1369–75. [11] Gu L, Tseng S, Horner RM, Tam C, Loda M, Rollins BJ. Control of TH2 polarization by the chemokine monocyte chemoattractant protein-1. Nature 2000;404(6776):407–11. [12] Le Berre L, Herve C, Buzelin F, Usal C, Soulillou JP, Dantal J. Renal macrophage activation and Th2 polarization precedes the development of nephrotic syndrome in Buffalo/Mna rats. Kidney Int 2005;68(5): 2079–90. [13] Matsukawa A, Lukacs NW, Standiford TJ, Chensue SW, Kunkel SL. Adenoviral-mediated overexpression of monocyte chemoattractant protein-1 differentially alters the development of Th1 and Th2 type responses in vivo. J Immunol 2000;164:1699–704. [14] Yuan J, Latouche JB, Hodges J, Houghton AN, Heller G, Sadelain M, et al. Langerhans-type dendritic cells genetically modified to express full-length antigen optimally stimulate CTLs in a CD4-dependent manner. J Immunol 2006;176(4):2357–65. [15] Mattes J, Hulett M, Xie W, Hogan S, Rothenberg ME, Foster P, et al. Immunotherapy of cytotoxic T cell-resistant tumors by T helper 2 cells: an eotaxin and STAT6-dependent process. J Exp Med 2003;197:387–93. [16] Yao D, Zhang X, Wei H, Tian Z. CCR4 participation in Th type 1 (mycobacterial) and Th type 2 (schistosomal) anamnestic pulmonary granulomatous responses. Cell Mol Immunol 2005;2(3):189–96. [17] Scotton C, Milliken D, Wilson J, Raju S, Balkwill F. Analysis of CC chemokine and chemokine receptor expression in solid ovarian tumours. Br J Cancer 2001;85:891–7. [18] Jassar AS, Suzuki E, Kapoor V, Sun J, Silverberg MB, Cheung L, et al. Activation of tumor-associated macrophages by the vascular disrupting agent 5,6-dimethylxanthenone-4-acetic acid induces an effective CD8+ Tcell-mediated antitumor immune response in murine models of lung cancer and mesothelioma. Cancer Res 2005;65(24):11752–61. [19] Van Deventer HW, Serody JS, McKinnon KP, Clements C, Brickey WJ, Ting JP. Transfection of macrophage inflammatory protein 1 alpha into B16 F10 melanoma cells inhibits growth of pulmonary metastases but not subcutaneous tumors. J Immunol 2002;169:1634–9. [20] Daly C, Rollins BJ. Monocyte chemoattractant protein-1 (CCL2) in inflammatory disease and adaptive immunity: therapeutic opportunities and controversies. Microcirculation 2003;10(3–4):247–57. [21] Owen JL, Lopez DM, Grosso JF, Guthrie KM, Herbert LM, Torroella-Kouri M, et al. The expression of CCL2 by T lymphocytes of mammary tumor bearers: role of tumor-derived factors. Cell Immunol 2005;235(2):122–35. [22] Traynor TR, Herring AC, Dorf ME, Kuziel WA, Toews GB, Huffnagle GB. Differential roles of CC chemokine ligand 2/monocyte chemotactic protein-1 and CCR2 in the development of T1 immunity. J Immunol 2002;168:4659–66. [23] Tsuchiya T, Ohshima K, Karube K, Yamaguchi T, Suefuji H, Hamasaki M, et al. Th1, Th2, and activated T-cell marker and clinical prognosis in peripheral T-cell lymphoma, unspecified: comparison with AILD, ALCL, lymphoblastic lymphoma, and ATLL. Blood 2004;103:236–41. [24] Mendoza L. A network model for the control of the differentiation process in Th cells. Biosystems 2006;84(2):101–14. [25] Iezzi G, Boni A, Degl’Innocenti E, Grioni M, Bertilaccio MT, Bellone M. Type 2 cytotoxic T lymphocytes modulate the activity of dendritic cells toward type 2 immune responses. J Immunol 2006;177(4):2131–7. [26] Mattes J, Foster PS. Regulation of eosinophil migration and Th2 cell function by IL-5 and eotaxin. Curr Drug Targets Inflamm Allergy 2003;2(2):169–74.

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K. Hu et al. / Immunology Letters 113 (2007) 19–28

[27] Yao D, Zhang X, Wei H, Tian Z. Antisense-induced blockade of GATA-3 expression could inhibit Th2 excursion of tumor cells in vitro and in vivo. Cell Mol Immunol 2005;2(3):189–96. [28] Knutson KL, Disis ML. Tumor antigen-specific T helper cells in cancer immunity and immunotherapy. Cancer Immunol Immunother 2005;4(8):721–8. [29] Sallusto F, Lenig D, Mackay CR, Lanzavecchia A. Flexible programs of chemokine receptor expression on human polarized T helper 1 and 2 lymphocytes. J Exp Med 1998;187(6):875–83. [30] Chamoto K, Kosaka A, Tsuji T, Matsuzaki J, Sato T, Takeshima T, et al. Critical role of the Th1/Tc1 circuit for the generation of tumor-specific CTL during tumor eradication in vivo by Th1-cell therapy. Cancer Sci 2003;94:924–8. [31] Nishimura T, Iwakabe K, Sekimoto M, Ohmi Y, Yahata T, Nakui M, et al. Distinct role of antigen-specific T helper type 1 (Th1) and Th2 cells in tumor eradication in vivo. J Exp Med 1999;190: 617–27.

[32] Dobrzanski MJ, Reome JB, Dutton RW. Type 1 and type 2 CD8+ effector T cell subpopulations promote long-term tumor immunity and protection to progressively growing tumor. J Immunol 2000;164(2):916–25. [33] Liu Y, Poon RT, Hughes J, Feng X, Yu WC, Fan ST. Chemokine receptors support infiltration of lymphocyte subpopulations in human hepatocellular carcinoma. Clin Immunol 2005;114(2):174–82. [34] Eriksson K, Nordstrom I, Czerkinsky C, Holmgren J. Differential effect of cholera toxin on CD45RA+ and CD45RO+ T cells: specific inhibition of cytokine production but not proliferation of human naive T cells. Clin Exp Immunol 2000;121(2):283–8. [35] Giles R, Loberg RD. Can we target the chemokine network for cancer therapeutics? Curr Cancer Drug Targets 2006;6(8):659–70. [36] Xu W, Hou W, Yao G, Ji Y, Yeh M, Sun B. Inhibition of Th1- and enhancement of Th2-initiating cytokines and chemokines in trichosanthin-treated macrophages. Biochem Biophys Res Commun 2001;284(1):168–72. [37] Brault MS, Kurt RA. Impact of tumor-derived CCL2 on macrophage effector function. J Biomed Biotechnol 2005;2005:37–43.