Intercellular cell adhesion molecule-1, vascular cell adhesion molecule-1, and regulated on activation normal T cell expressed and secreted are expressed by human breast carcinoma cells and support eosinophil adhesion and activation

Abstract

Eosinophils are usually associated with parasitic and allergic diseases; however, eosinophilia is also observed in several types of human tumors, including breast carcinomas. In this study we examined several human breast carcinoma cell lines for adhesion molecule expression and the ability to bind and activate eosinophils. MDA-MB-435S and MDA-MB-468 cells constitutively expressed both intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and this expression was enhanced by treatment with tumor necrosis factor-alpha (TNF-alpha). BT-20 and SK-BR-3 cells only expressed ICAM-1 or VCAM-1 after stimulation with TNF-alpha. Eosinophils constitutively bound to MDA-MB-435S cells, but not to BT-20 cells. Stimulation with TNF-alpha slightly enhanced eosinophil adhesion to MDA-MB-435S cells and dramatically increased adhesion to BT-20 cells. Greater than 80% of eosinophil adhesion to these cell lines was blocked with an anti-alpha4-integrin monoclonal antibody. Both MDA-MB-435S and BT-20 cells also released eosinophil activator(s). Supernatants from TNF-alpha-treated, but not control-treated, cell lines increased eosinophil adhesion to fibronectin and increased eosinophil transmigration across fibronectin-coated transwell plates. Enzyme-linked immunosorbent assays showed that TNF-alpha-stimulated breast carcinoma cells released the chemokine regulated on activation, T cell expressed and secreted (RANTES). Addition of an anti-RANTES antibody to breast carcinoma cell supernatants partially blocked eosinophil activation suggesting that RANTES in these supernatants was participating in eosinophil activation. These data show that TNF-alpha-stimulated breast carcinoma cells express mediators that can both bind and activate eosinophils, suggesting a mechanism for eosinophil localization to breast carcinoma sites.

Figure 1.

Adhesion molecule expression by breast carcinoma cells. Cell lines were treated with M199/A alone (time, 0) or M199/A containing 20 ng/ml TNF-α for 4 or 24 hours (time, 4, 24). A: After the incubation, cells were lysed in TRIzol and total RNA was isolated. RT-PCR was performed as described in Methods. B: Alternatively, cells were lysed in Triton X-100 containing buffer and the soluble proteins were separated on SDS-PAGE. Western blotting was performed using 2 μg/ml of the specified mAbs directed against P-selectin (G1), E-selectin (ES-1), ICAM-1 (BBA 4), or VCAM-1 (1.G11B1). Experiments were performed at least three times with equivalent data. Representative data are shown.

Figure 2.

VCAM-1 and ICAM-1 are surface expressed on breast carcinoma cells. MDA-MB-435S (A) or BT-20 (B) cells were treated with TNF-α as described in Figure 1 ▶ . Cell surface expression of VCAM-1 or ICAM-1 was determined using a modified ELISA as described in Methods. Data represent the mean ± SEM of at least three experiments. * P < 0.05 as compared to control.

Figure 3.

Eosinophils bind to breast carcinoma cells. Freshly isolated eosinophils (2 × 10⁶/ml) were added to MDA-MB-435S (A) or BT-20 (B) cells treated with buffer alone or TNF-α as described in Figure 1 ▶ . After 15 minutes, nonadherent and loosely adherent cells were removed and eosinophil adhesion was determined as described in Methods. Data represent the mean ± SEM of at least three experiments. * P < 0.05 as compared to control.

Figure 4.

Eosinophil adhesion to MDA-MB-435S and BT-20 cells requires α4-integrins. MDA-MB-435S cells were treated with buffer alone (A) or TNF-α (B) for 24 hours as described in Figure 1 ▶ . Alternatively, BT-20 cells were treated TNF-α for 24 hours as described in Figure 1 ▶ (C). MDA-MB-435S (A and B) or BT-20 cells (C) were pretreated for 15 minutes at 37°C with 5 μg/ml of the specified mAbs directed against ICAM-1 (BBA4) or VCAM-1 (1.G11B1). Alternatively, eosinophils were preincubated with α4-integrin mAb (H2/1) or β2-integrin mAb (7E4) for 15 minutes before addition to the cell monolayers. Eosinophil adhesion was determined in the continued presence of the specified mAbs as described in Methods. Data are mean ± SEM of at least three experiments. * P < 0.05 as compared to control.

Figure 5.

Both MDA-MB-435S and BT-20 cells release eosinophil activator(s). Supernatant from either MDA-MB-435S (A) or BT-20 (B) cells treated with buffer alone treated with TNF-α for 4 or 24 hours were collected. Forty-eight-well plates were coated with either 0.2% gelatin or 25 μg/ml fibronectin (Fn) for 2 hours at 37°C. Plates were washed once with HBSS and then freshly isolated eosinophils (1–2 × 10⁶/ml) were added. Eosinophils were stimulated with a 1:10 dilution of the specified supernatants. After 10 minutes, the nonadherent and loosely adherent eosinophils were removed and adhesion was quantified using eosinophil peroxidase activity as described in Methods. Data are mean ± SEM of at least three experiments. * P < 0.05 as compared to control.

Figure 6.

Breast carcinoma cells release eosinophil chemoattractant(s). Supernatants from MDA-MB-435S or BT-20 cells were collected as described in Figure 5 ▶ . Freshly isolated eosinophils (4 × 10⁶/ml) were added to the upper chamber of a 3-μm pore transwell dish coated with 25 μg/ml of fibronectin. Supernatants were placed in the lower chamber at a 1:10 dilution. M199/A or M199/A with an equivalent amount of TNF-α was used as controls (control). Eosinophils that had migrated into the lower chamber after 90 minutes were counted as described in Methods. Experiments were performed three times with equivalent data. Data represent the mean ± range of a single experiment.

Figure 7.

RANTES is released by TNF-α-stimulated breast carcinoma cells. Supernatants from MDA-MB-435S or BT-20 cells were collected as described in Figure 5 ▶ . RANTES present in the supernatants was assayed by ELISA according to the manufacturer’s instructions. Data represent the mean ± SEM of at least three experiments.