Interleukin-18 amplifies M2 polarization of macrophage which leads excessive angiogenesis

[Speaker] Yui Yamazaki:1
[Co-author] Takuro Kobori:1, Atsuko Niwa:1, Takashi Nishinaka:1, Shuji Mori:2, Masahiro Nishibori:3, Hideo Takahashi:1
1:Department of Pharmacology, Faculty of Medicine, Kindai University, Japan, 2:Department of Pharmacology, School of Pharmacy, Shujitsu University, Japan, 3:Department of Pharmacology, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan

M2 macrophage promotes pathologic angiogenesis through a release of pro-angiogenic mediators or the direct cell-cell interaction with endothelium cell in the microenvironment of several chronic inflammatory diseases including rheumatoid arthritis and cancer. In such microenvironment, interleukin-18 (IL-18) is involved in excessive angiogenesis. However, detail mechanism of angiogenesis by M2 macrophage in the microenvironment containing IL-18 remains unsolved. Therefore, we examined the effect of M2 macrophage on angiogenesis in coexistence environment of IL-18. In addition, we focused on osteopontin which is detected at higher levels in the microenvironments of rheumatoid arthritis and cancer. Expression levels of CD163 in the mouse leukemic monocyte macrophage cell line, RAW264.7 were measured using flow cytometry. Matrigel tube formation assay was performed using the mouse endothelial cell line, b.End5 cells. 96-well plates were filled with 50 µL matrigel and allowed to solidify at 37°C for 30 min. Then, b.End5 cells were gently seeded on top of the gel. Subsequently, polarized RAW264.7 cells were co-incubated with b.End5 cells on matrigel for 16 h. The areas and total lengths of the tubes were calculated as the degree of tube formation. IL-18 treatment amplified macrophage polarization toward M2-like phenotype which was indicated increment of CD163 level. Co-culture of b.End5 with RAW264.7 (-) increased the area and length of tubular structures moderately compared with the control group without RAW264.7. Interestingly, IL-10 when combined with IL-18 significantly augmented the tube formation compared to RAW264.7 (IL-10). Additionally, these augmenting effects of increment of CD163 expression and tube formation was significantly suppressed by anti-osteopontin antibody. These results indicated that IL-18 acts synergistically with IL-10 to augment M2 polarization of macrophage with characteristics of increasing CD163 expression. Additionally, macrophage-derived mediators like osteopontin may be involved in this augmenting M2 polarization of macrophage. Furthermore, M2 macrophages induce excessive angiogenesis through the direct cell-cell interaction with endothelial cells, possibly mediated by CD163.
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