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. 2005;2005(1):37-43.
doi: 10.1155/JBB.2005.37.

Impact of Tumor-Derived CCL2 on Macrophage Effector Function

M S Brault, R A Kurt

Impact of Tumor-Derived CCL2 on Macrophage Effector Function

M S Brault et al. J Biomed Biotechnol. 2005.

Abstract

Monocyte chemoattractant protein-1 (MCP-1, CCL2) is produced by many different types of cells. In the current investigation, the effect of tumor-derived CCL2 on macrophages was evaluated to determine the extent to which this chemokine influenced the innate immune response to cancer. To do this, we used the 4T1 murine mammary carcinoma cell line that constitutively expresses CCL2 and generated 4T1 expressing an antisense CCL2 transcript. The antisense-CCL2-expressing 4T1 produced no detectable CCL2. Macrophages from female BALB/c mice were exposed to supernatants from these tumor cells. The results showed that tumor-derived CCL2 was capable of modulating cytokine gene expression but not protein production in resting, activated, and tumor-associated macrophages. In addition, tumor-derived CCL2 did not affect phagocytic activity, nitric oxide production, or cytolytic activity of the macrophages. Overall, these data suggest that tumor-derived CCL2 does not directly influence macrophage-mediated antitumor activity.

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Figures

Figure 1
Figure 1
Tumor-derived CCL2 production. Supernatants were taken from A4 and G7 tumor cells at different time points and evaluated for CCL2 production by ELISA. The data are representative of three separate experiments with standard deviation shown.
Figure 2
Figure 2
Cytokine analysis by RT-PCR. Resting (a) and activated (b) macrophages were examined for IL-12, IL-18, and TNF-α expression. GAPDH was used as a positive control. The data represent one of three separate experiments. For the densitometric analysis, the optical densities were calculated by comparison to the positive control (GAPDH).
Figure 2
Figure 2
Cytokine analysis by RT-PCR. Resting (a) and activated (b) macrophages were examined for IL-12, IL-18, and TNF-α expression. GAPDH was used as a positive control. The data represent one of three separate experiments. For the densitometric analysis, the optical densities were calculated by comparison to the positive control (GAPDH).
Figure 2
Figure 2
Cytokine analysis by RT-PCR. Resting (a) and activated (b) macrophages were examined for IL-12, IL-18, and TNF-α expression. GAPDH was used as a positive control. The data represent one of three separate experiments. For the densitometric analysis, the optical densities were calculated by comparison to the positive control (GAPDH).
Figure 2
Figure 2
Cytokine analysis by RT-PCR. Resting (a) and activated (b) macrophages were examined for IL-12, IL-18, and TNF-α expression. GAPDH was used as a positive control. The data represent one of three separate experiments. For the densitometric analysis, the optical densities were calculated by comparison to the positive control (GAPDH).
Figure 3
Figure 3
Cytokine production by ELISA. (a) Resting and activated macrophages were examined for cytokine production after exposure to A4 and G7 supernatants. The data are representative of three separate experiments. The error bars represent the standard deviation of the mean of duplicate wells analyzed by ELISA. (b) Following exposure of macrophages to rCCL2 ( μg/mL), supernatants were assayed for the same cytokines. (c) TAM harvested from four-week A4 and G7 tumors were analyzed for cytokine production. The data shown are representative of three separate experiments, with error bars denoting the standard deviation from the mean.
Figure 3
Figure 3
Cytokine production by ELISA. (a) Resting and activated macrophages were examined for cytokine production after exposure to A4 and G7 supernatants. The data are representative of three separate experiments. The error bars represent the standard deviation of the mean of duplicate wells analyzed by ELISA. (b) Following exposure of macrophages to rCCL2 ( μg/mL), supernatants were assayed for the same cytokines. (c) TAM harvested from four-week A4 and G7 tumors were analyzed for cytokine production. The data shown are representative of three separate experiments, with error bars denoting the standard deviation from the mean.
Figure 3
Figure 3
Cytokine production by ELISA. (a) Resting and activated macrophages were examined for cytokine production after exposure to A4 and G7 supernatants. The data are representative of three separate experiments. The error bars represent the standard deviation of the mean of duplicate wells analyzed by ELISA. (b) Following exposure of macrophages to rCCL2 ( μg/mL), supernatants were assayed for the same cytokines. (c) TAM harvested from four-week A4 and G7 tumors were analyzed for cytokine production. The data shown are representative of three separate experiments, with error bars denoting the standard deviation from the mean.
Figure 4
Figure 4
Macrophage effector function. (a) Macrophages exposed to A4 (▪) and G7 (□) supernatants were evaluated for total NO production by measuring the concentration of nitrite. The amount of nitrite was measured in three separate experiments from duplicate wells by ELISA. Error bars represent the standard deviation of the mean. (b) Macrophages were exposed to A4 and G7 supernatants for 72 hours to test their ability to modulate cytolytic activity. The figure represents macrophage-mediated cytolysis of the tumor cells. The data are representative of three separate cell counts/experiment, with error bars showing standard deviation from the mean. (c) The ability of macrophages exposed to A4 (▪) and G7 (□) supernatants to phagocytose E coli was determined through confocal microscopy. Using both light and florescence settings simultaneously, it was possible to distinguish phagocytosed bacteria. The data are representative of two separate experiments where cell counts were determined from three separate fields of view/slide. Error bars represent standard deviation from the mean.
Figure 4
Figure 4
Macrophage effector function. (a) Macrophages exposed to A4 (▪) and G7 (□) supernatants were evaluated for total NO production by measuring the concentration of nitrite. The amount of nitrite was measured in three separate experiments from duplicate wells by ELISA. Error bars represent the standard deviation of the mean. (b) Macrophages were exposed to A4 and G7 supernatants for 72 hours to test their ability to modulate cytolytic activity. The figure represents macrophage-mediated cytolysis of the tumor cells. The data are representative of three separate cell counts/experiment, with error bars showing standard deviation from the mean. (c) The ability of macrophages exposed to A4 (▪) and G7 (□) supernatants to phagocytose E coli was determined through confocal microscopy. Using both light and florescence settings simultaneously, it was possible to distinguish phagocytosed bacteria. The data are representative of two separate experiments where cell counts were determined from three separate fields of view/slide. Error bars represent standard deviation from the mean.
Figure 4
Figure 4
Macrophage effector function. (a) Macrophages exposed to A4 (▪) and G7 (□) supernatants were evaluated for total NO production by measuring the concentration of nitrite. The amount of nitrite was measured in three separate experiments from duplicate wells by ELISA. Error bars represent the standard deviation of the mean. (b) Macrophages were exposed to A4 and G7 supernatants for 72 hours to test their ability to modulate cytolytic activity. The figure represents macrophage-mediated cytolysis of the tumor cells. The data are representative of three separate cell counts/experiment, with error bars showing standard deviation from the mean. (c) The ability of macrophages exposed to A4 (▪) and G7 (□) supernatants to phagocytose E coli was determined through confocal microscopy. Using both light and florescence settings simultaneously, it was possible to distinguish phagocytosed bacteria. The data are representative of two separate experiments where cell counts were determined from three separate fields of view/slide. Error bars represent standard deviation from the mean.
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References

    1. Martinet N, Beck G, Bernard V, et al. Mechanism for the recruitment of macrophages to cancer site. In vivo concentration gradient of monocyte chemotactic activity. Cancer. 1992;70(4):854–860. - PubMed
    1. Vaddi K, Newton R.C. Regulation of monocyte integrin expression by beta-family chemokines. J Immunol. 1994;153(10):4721–4732. - PubMed
    1. Carr M.W, Roth S.J, Luther E, Rose S.S, Springer T.A. Monocyte chemoattractant protein 1 acts as a T-lymphocyte chemoattractant. Proc Natl Acad Sci USA. 1994;91(9):3652–3656. - PMC - PubMed
    1. Allavena P, Bianchi G, Zhou D, et al. Induction of natural killer cell migration by monocyte chemotactic protein-1, -2 and -3. Eur J Immunol. 1994;24(12):3233–3236. - PubMed
    1. Loetscher P, Seitz M, Clark-Lewis I, Baggiolini M, Moser B. Activation of NK cells by CC chemokines. Chemotaxis, Ca2+ mobilization, and enzyme release. J Immunol. 1996;156(1):322–327. - PubMed