Expression profiles of cytokines/chemokines and cytotoxic factors in DP macrophages. Infiltrating macrophages were isolated from the cardiac tissues by collagenase digestion followed by adhesion to the plastic dish. DP macrophages were separated from other macrophages by MACS based on the presence or absence of CD8. Prior to the MACS sorting, we confirmed by light microscopy that macrophages detached from the plastic dish were in a single-cell suspension (data not shown). (A) Macrophages collected from the cardiac tissues were reacted with FITC-conjugated anti-CD4 (OX-35) and PE-conjugated anti-CD8 (OX-8) Abs. MACS was conducted using anti-PE microbeads. The cells selected positively and negatively are shown in the top and bottom panels, respectively. Experiments were repeated at least twice, and representative results are shown. (B) The expression of cytokines/chemokines (IL-18, IFN-γ, RANTES, and MCP-1) in DP macrophages was analyzed by quantitative real-time RT-PCR. The data were compared with those of CD8^- macrophages. Results are represented as a fold (mean ± SD of repeated experiments done in triplicate) against control macrophages. (C) The expression patterns of cytotoxic factors (Fas L, perforin, and granzyme B) in DP macrophages (right columns) were compared with those in CD8^- macrophages (left columns). Data are represented as mean ± SD of repeated experiments done in triplicate. (D) Immunofluorescent double staining for CD68 (ED-1, green) and Fas L (N-20, red), or CD68 (ED-1, green) and granzyme B (N-19, red) in the cardiac tissue section (left panels). Infiltrating macrophages isolated from the tissues were stained for CD68 (ED-1, green) and granzyme B (N-19, red; right panel). Total magnification: × 80. ^*P < .05.