Expression of the inflammatory chemokines CCL2, CCL5 and CXCL2 and the receptors CCR1-3 and CXCR2 in T lymphocytes from mammary tumor-bearing mice

Abstract

Chemokines and their receptors have been studied in several solid tumor models as mediators of inflammation. In turn, inflammation has been implicated in the promotion and progression of tumors, and as such, chemokines have been proposed as novel molecular targets for chemotherapy. While the expression of these molecules has been described in tumor cells, endothelial cells, macrophages and neutrophils, less attention has been paid to the expression profile of these molecules by T lymphocytes in the periphery or infiltrating the tumor. Using the D1-DMBA-3 murine mammary adenocarcinoma model, we aimed to better characterize the differential expression of chemokines and/or their receptors in the host and in the tumor microenvironment, and specifically, in the T cells of tumor-bearing mice compared to normal control animals. We found that T lymphocytes from tumor-bearing mice express the pro-inflammatory chemokines, CCL2, CCL5 and CXCL2, as well as the chemokine receptors, CCR1, CCR2, CCR3 and CXCR2.

Fig. 1. Expression of proinflammatory chemokines in mammary tumor-bearing mice

A) DA-3 mammary tumor cells express CCL5 and CXCL2, but not CCL2 mRNA. B) Production of CCL2 and CXCL2 protein in the tumor cystic fluid of 3-week tumor bearers. C) CCL2 protein levels are increased in the circulation of 3-week mammary tumor-bearing mice. These data are representative of at least three independent experiments with at least six mice per group. ^*Significant difference (p<0.005) between control and tumor bearers’ serum levels of CCL2.

Fig. 2. Increased expression of chemokines by T lymphocytes of tumor-bearing mice

A) Increased expression of chemokine mRNA by T lymphocytes from tumor-bearing mice. B) Increased protein levels of CCL5. C) Increased protein levels of CCL2. D) Increased CXCL2 protein secretion in tumor-bearing mice. For mRNA expression studies of chemokines, purified splenic T cells from normal (N) and 4-week tumor-bearing mice (T) were cultured for 4 h +/− 10 μg/ml Con A. Total RNA was isolated and 20 μg was subjected to a multi-probe RNase protection assay. These data are representative of at least three independent experiments with six mice per group. For protein secretion studies, purified splenic T cells from normal and 4-week tumor-bearing mice were cultured overnight +/− 10 μg/ml Con A. Cell-free supernatants were collected and assayed by specific ELISAs. These data are representative of at least four independent experiments with at least five mice per group. ^*Significant difference (p<0.005) between control and tumor bearers’ T lymphocytes treated with Con A. No significant differences were noted for unstimulated T cells between control and tumor-bearing mice for CCL2, CCL5, and CXCL2.

Fig. 3

The expression of CC and CXC chemokine receptors is altered in splenic T cells of tumor-bearing mice. A) RNase protection assay of CC chemokine receptors. B) Western Blot of CC receptors. C) RNase protection assay of CXC receptors. For RNase protection studies, splenic T cells from normal and tumor-bearing mice were cultured for 2 h +/− 10 μg/ml Con A. Total RNA was isolated and 20 μg was subjected to a multi-probe CC or CXC chemokine protection assay. For Western Blotting studies, splenic T cells from normal and tumor-bearing animals were cultured for 3 h +/− 10 μg/ml Con A. Total protein was extracted and 50 μg from each sample was run. These data are representative of at least three independent experiments with six mice per group.

Fig. 4

The expression of CC and CXC chemokines is altered in splenic CD4⁺ and CD8⁺ T cell subsets of tumor-bearing mice. A) RNase protection of CC and CXC chemokines, and protein expression of: B) CCL5, C) CCL2, and D) CXCL2. Splenic CD4⁺ and C8⁺ T cells from normal and tumor-bearing mice were isolated by Miltneyi magnetic bead technique and then cultured for 2 h +/− 10 μg/ml Con A for RNA and overnight for protein. Total RNA was isolated and 20 μg was subjected to a multi-probe protection assay. For protein expression studies, cell-free supernatants were collected and assayed by specific ELISAs. These data are representative of at least three independent experiments with at least six mice per group. ^*Significant difference (p<0.005) between control and tumor bearers’ T lymphocytes treated with Con A.

Fig. 5

The expression of CC and CXC chemokine receptors is altered in splenic CD4⁺ and CD8⁺ T cell subsets of tumor-bearing mice. Total RNA was isolated and 20 μg was subjected to a multi-probe protection assay. These data are representative of at least three independent experiments with at least six mice per group.

Fig. 6

The effect of CCL2 on chemokine (A) and chemokine receptor (B) expression in T cells from mammary tumor-bearing mice. Splenic T cells from mammary tumor-bearing mice were cultured in the presence of 10 ng/ml of recombinant murine CCL2 (rmCCL2) for 4 h. Total RNA was isolated and 20 μg was subjected to a multi-probe protection assay for chemokines and chemokine receptors. These data are representative of at least three independent experiments with six mice per group.

Fig. 7

Expression of chemokines and chemokine receptors by T cells. A–C. Immunostaining of tumor sections at 3-weeks of growth showing co-localization of CD3-immunoreactivity (red in A) with CCR1-immunoreactivity (green in B) in tumor-infiltrating T cells. Double-labeled cells in the merged image are indicated by open arrows (C). D–I. Immunolabeling of T-cells with CD3 antibody (brown reaction product) combined with in situ hybridization (silver grains) using ³⁵S-labeled cRNAs for detection of CCR3 (D–E) and CCL5 mRNAs (F–G). Grain density indicative of CCR3 expression is concentrated over T cells located within periarteriole lymphoid sheaths in the spleens of normal mice (D), and is increased in the spleens of tumor-bearing mice (E). T cells within the sheaths are located to the left and the dashed line in D indicates the sheath margin. The arrow in D indicates a cluster of labeled T cells outside the sheath. The arrows in E indicate labeled cells within the sheath. F–G. Double labeling of tumor-infiltrating T cells at 3-weeks post tumor implantation for CCL5 and CD3. H-I. Merged confocal microscope images of CD8 (red) and CCL5 (green) immunostained tumor section showing CD8⁺ T cells that express CCL5 (open arrows in H). These cells are shown in higher maginification in I. Bar in A = 35 μm in A–C and I, 17 μm in D–E, 7 μm in F–G and 93 μm in H.