Phagocytosis of LM-Mel-45 Melanoma and Jurkat Cells by THP-1 Macrophages

Abstract

Exposing macrophages to IFN-g or IL-4 polarizes them toward M1-like inflammatory or M2-like tissue repair phenotypes, respectively. It is uncertain whether the macrophage phenotypes are equally effective at phagocytosing melanoma and non-melanoma bait cells. We hypothesize that phenotypic polarization of macrophages will improve their phagocytic ability and that phagocytic vulnerability will depend on bait cell type. This study compares phenotypic polarization of macrophages on phagocytosis of LM-Mel-45 (LM45) melanoma cells and non-melanoma (Jurkat) cells at 2 h or 16 h. Macrophage differentiation to an M0 phenotype was induced by exposing THP-1 monocytes to 50 ng/ml PMA for two days, followed by 4 days rest in RPMI 1640, 10% FBS. M1-like and M2-like phenotypes were induced by 24 h exposure to 20 ng/ml IFN-γ and 20 ng/mL IL-4, respectively. Phenotypic state was measured by surface expression of CCR7, CD209, and CD172. Bait cells (LM45 or Jurkat) were treated with 0.5 mM staurosporine for 24 h to induce apoptosis and stained with CellTrace® Violet. Macrophages were stained with CellTracker® Orange CMRA. Bait cells were added to adherent macrophages to measure phagocytosis (2 or 16 h). To qualitatively monitor the phagocytic process, time-lapse imaging every 15 min for 16 h was done on a Zeiss Axiovert 200M microscope with a 10x objective. To quantify phagocytosis, adhered and loose cells were harvested and prepared for flow cytometry. Cells were analyzed for violet, orange, or both orange and violet staining. Positive phagocytosis was expressed as the percentage of orange macrophages with violet staining above the violet cutoff. M0 macrophages are positive for CD172. Exposure to IFN-γ increases levels of CD209 and CCR7, and CD172 remains present. Exposure to IL-4 increases levels of CD209 with little change in CCR7 and CD172 remains present. Time-lapse imaging shows phagocytosis of LM45 cells via a “nipping” behavior rather than engulfment. When Jurkat cells serve as bait, multiple bait cells will attach to a single macrophage. Blebbing in bait cells is seen by 8 h. Phagocytic rate toward LM45 cells over 2 h averaged 6.7 ± 1.8% for M0 macrophages and 5.6 ± 2.3% for IFN-γ treated macrophages. Phagocytic rate toward LM45 cells over 16 h averaged 13.9 ± 1.3% for M0 macrophages and 14.9 ± 1.8% for IFN-γ treated macrophages. The differences were not significant. Phagocytic rate toward Jurkat cells averaged 22.4 ± 3.5% for M0 macrophages and 26.9 ± 4.0% for IFN-γ treated macrophages. This difference was not significant. Phagocytosis was significantly greater for Jurkat cells than LM45 cells (P<0.001). In summary, IFN-γ treatment does not increase macrophage phagocytic ability, refuting our hypothesis. However, macrophages phagocytose Jurkat cells more effectively than LM45 melanoma, suggesting that chemotherapy designed to increase phagocytic vulnerability may have potential in melanoma treatment. KCOM Graduate Program Grant #851-068 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.