interleukin-8 and arginyl-glycyl-aspartyl-serine

Apoptotic cells are phagocytosed as soon as they appear in vivo. In this study, we first determined precisely at what stage apoptotic cells are phagocytosed by macrophages, and then examined the subsequent cytokine production. Phagocytosis was confirmed by flow cytometry and confocal laser microscopy, whereas the subsequent response was examined by ELISA and RT-PCR for quantitative and semiquantitative measurement of the protein and mRNA levels of cytokines, respectively. Even the cell populations containing very early apoptotic cells, such as IL-2-dependent CTLL-2 cells cultured in the absence of IL-2 for 4 h and a murine leukemic cell line, P388 cells, treated with etoposide for 5 h, were phagocytosed by macrophages. Although the cell populations containing the very early apoptotic cells used in this study were FITC-Annexin V-negative and did not show a decrease in cell size as compared with untreated cells, they showed a very small increase in phosphatidylserine on the cell surface, as detected with Cy3-Annexin V, and a decrease in mitochondrial membrane potential, indicating that the cell populations had already started the apoptotic process. Phagocytosis of such populations containing very early apoptotic cells was inhibited by phospho-L-serine much more significantly than Arg-Gly-Asp-Ser. In addition, macrophages hardly produced either proinflammatory or anti-inflammatory cytokines after phagocytosis, thus being an almost null response. These results are contrary to the generally accepted concept that the phagocytosis of apoptotic cells leads to the production of anti-inflammatory cytokines, suggesting instead that cells starting to undergo apoptosis are quickly phagocytosed by macrophages without any inflammation in vivo.

Topics: Animals; Apoptosis; Cell Division; Cell Line; Cell Line, Tumor; Coculture Techniques; Cytokines; Humans; Interleukin-8; Kupffer Cells; Leukemia P388; Macrophages; Macrophages, Alveolar; Macrophages, Peritoneal; Mice; Monocytes; Oligopeptides; Phagocytosis; Phosphoserine; RNA, Messenger; Time Factors

Chemotherapy and radiotherapy are performed for cancer patients with the hope that dying cancer cells are safely scavenged by phagocytic cells such as macrophages. In this study, we examined cytokine production by macrophages during and after the phagocytosis of etoposide-treated P388 cells in vitro and in vivo. Etoposide caused apoptosis as early as 5 h after treatment, as assessed as to the exposure of phosphatidylserine, increase in membrane permeability and DNA ladder formation. Phagocytosis by phorbol myristate acetate (PMA)-treated THP-1 cells occurred marginally when P388 cells were treated with etoposide for 10 h, while it occurred significantly with P388 cells treated for 24 h, as evidenced by flow cytometry and confocal microscopy. PMA-treated THP-1 cells produced pro-inflammatory cytokines, such as interleukin (IL)-1alpha, IL-8 and macrophage migration inhibitory factor (MIF), but not anti-inflammatory cytokines among those tested at the mRNA level during and after the phagocytosis of apoptotic cells. IL-8 and MIF were also produced at the protein level, and the IL-8 production was dependent on cell-to-cell contact when the plasma membranes of apoptotic cells were intact enough not to leak one of the cytoplasmic enzymes, lactate dehydrogenase. In addition, etoposide-treated P388 cells induced neutrophil infiltration as well as MIP-2 production upon injection into the peritoneal cavity of either normal mice or mice with sterile peritonitis. When macrophages ingesting and/or binding apoptotic P388 cells were isolated from the mice with sterile peritonitis using a cell sorter, they were found to produce MIP-2 upon culture.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Chemokine CXCL2; Chemokines; Coculture Techniques; Cytokines; Etoposide; Flow Cytometry; Humans; Interleukin-8; Leukemia P388; Macrophages; Male; Mice; Microscopy, Confocal; Neoplasm Transplantation; Oligopeptides; Phagocytosis; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Because it is generally believed that apoptosis is not associated with inflammation, we hypothesized that the interaction of phagocytes with apoptotic cells provides a negative or null signal for inflammation. However, we recently found that the interaction led to the production of proinflammatory cytokines but not antiinflammatory cytokines, although the apoptotic cell membranes appeared to be intact. In this study, we examined in detail the relationship among the kinetics of apoptosis, phagocytosis and production of cytokines by macrophages. Among the time points examined, murine CTLL-2 cells became apoptotic in terms of cell size and exposure of phosphatidylserine after 12 h of culture in the absence of IL-2, and at the same time they began to be phagocytosed and lead to proinflammatory cytokine production by PMA-treated THP-1 cells (human macrophages). The phagocytosis of apoptotic cells by macrophages was also confirmed by confocal laser microscopy. The coculturing of human macrophages with murine apoptotic cells led to the production of human proinflammatory cytokines, notably IL-8, at both the mRNA level and the protein level. The coculturing of monocyte-derived macrophages with the apoptotic cells also led to the production of IL-8 protein. Both the phagocytosis and production of the cytokines were suppressed by either phospho-L-serine or RGDS (Arg-Gly-Asp-Ser), but not by RGES (Arg-Gly-Glu-Ser). Thus, the production of proinflammatory cytokines and phagocytosis of apoptotic CTLL-2 cells appear to be closely interrelated.

Topics: Animals; Apoptosis; Cell Differentiation; Cell Line; Cytokines; Humans; Inflammation; Interleukin-8; Macrophages; Mice; Monocytes; Oligopeptides; Phagocytosis; Phosphoserine; T-Lymphocytes, Cytotoxic; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured