Early apoptotic Jurkat T cells undergo capping of CD43, and its polylactosaminyl saccharide chains serve as ligands for phagocytosis by macrophages. This suggests the presence of a polylactosaminoglycan-binding receptor on macrophages. Here we show that this receptor is nucleolin, a multifunctional shuttling protein present in nucleus, cytoplasm, and on the surface of some types of cells. Nucleolin was detected at the surface of macrophages, and antinucleolin antibody inhibited the binding of the early apoptotic cells to macrophages. Nucleolin-transfected HEK293 cells expressed nucleolin on the cell surface and bound the early apoptotic cells but not phosphatidylserine-exposing late apoptotic cells. This binding was inhibited by anti-nucleolin antibody, by polylactosamine-containing oligosaccharides, and by anti-CD43 antibody. Deletion of the antibody binding region of nucleolin resulted in loss of the apoptotic cell-binding ability. Moreover, truncated recombinant nucleolin in solution containing this region blocked the apoptotic cell binding to macrophages, and the blocking effect was cancelled by the oligosaccharides. These results indicate that nucleolin is a macrophage receptor for apoptotic cells.Macrophages and other phagocytes recognize and ingest apoptotic cells in tissue, preventing their lysis and subsequent release of harmful or immunogenic intracellular components. Therefore, clearance of apoptotic cells by phagocytes is crucial in the maintenance of tissue turnover and homeostasis. Moreover, it has been suggested that the apoptotic cell-ingested phagocytes play a role in suppression or resolution of inflammation (1-5).Cells undergoing apoptosis display a variety of "eat me" signals, namely cell-surface changes to be recognized by phagocytes. These include externalization of phosphatidylserine (PS),3 as yet little identified alterations of carbohydrates, and unidentified alterations of other membrane components such as intercellular adhesion molecule-3 and "thrombospondin binding sites" (1-5). Among these, the most common and the best-characterized change is externalization of PS, although the mechanism of externalization has not been fully understood (6, 7).In contrast to the poor understanding of the nature of eat me signals, various proteins of phagocyte membrane or in extracellular fluid have been reported as receptors or bridging factors for apoptotic cells. For externalized PS on apoptotic cells, CD36 (8), CD68 (9), CLA-1 (10), LOX-1 (11), and PS receptor (12) have been reported as phagocyte receptors; a serum protein ␤ 2 -glycoprotein I (13), complement component C3bi (14), and milk fat globule-epidermal growth factor-factor 8 (15) have been reported to bridge apoptotic cells and phagocytes through exposed PS. For intercellular adhesion molecule-3 on apoptotic cells, CD14 was suggested to be a macrophage receptor (16). Thrombospondin is also known as a bridging protein between unidentified sites on apoptotic cells and CD36 or vitronectin receptor (␣ v ␤ 3 integrin) on phagocytes (17). In addi...

Photodynamic therapy (PDT) induces selective cell death of neoplastic tissue and connecting vasculature by combining photosensitizers with light. Here we clarified the types of cell death induced by PDT in combination with the photosensitizer talaporfin sodium (mono-L-aspartyl chlorine e6, NPe6) in order to evaluate the potential of this therapy as a treatment for glioma. PDT with NPe6 (NPe6-PDT) induces dose-dependent cell death in human glioblastoma T98G cells. Specifically, cell death modalities were observed in NPe6-PDT treated T98G cells, including signs of apoptosis (activation of caspase-3, expression of phosphatidylserine, and DNA fragmentation) and necrosis (stainability of propidium iodide). In addition, high doses of NPe6-PDT decreased the proportion of apoptotic cell death, while increasing necrosis. Closer examination of apoptotic characteristics revealed release of cytochrome-c from mitochondria as well as activation of both caspse-9 and caspase-3 in cells treated with low doses of NPe6-PDT. Benziloxycarbonyl-Leu-Gln(OMe)-His-Asp(OMe)-fluoromethyl-ketone (Z-LEHD-fmk), a caspase-9 specific inhibitor, and benziloxycarbonyl-Asp(OMe)-Gln-Met-Asp(OMe)-fluoromethyl-ketone (Z-DQMD-fmk), a caspase-3 specific inhibitor, showed dose-dependent prevention of cell death in NPe6-PDT treated cells, indicating that mitochondrial apoptotic pathway was a factor in the observed cell death. Further, the cell morphology was observed after PDT. Time- and NPe6-dose dependent necrotic features were increased in NPe6-PDT treated cells. These results suggest that NPe6-PDT could be an effective treatment for glioma if used in mild doses to avoid the increased necrosis that may induce undesirable obstacles.

Photodynamic therapy (PDT) using photosensitizer induces several types of cell death, such as apoptosis, necrosis, and autophagy, depending on the PDT procedure, photosensitizer type, and cell type. We previously demonstrated that PDT using the photosensitizer talaporfin sodium (mono-L-aspartyl chlorine e6, NPe6; NPe6-PDT) induces both mitochondrial apoptotic and necrotic cell death in human glioblastoma T98G cells. However, details regarding the mechanism of necrosis caused by NPe6-PDT are unclear. Here, we investigated whether or not necroptosis, a recently suggested form of programmed necrosis, is involved in the necrotic cell death of NPe6-PDT-treated T98G cells. Leakage of lactate dehydrogenase (LDH) from the cell layer into conditioned medium was significantly increased by NPe6 (25 and 50 μg/ml)-PDT, indicating that NPe6-PDT induces necrosis in these cells. NPe6 (25 μg/ml)-PDT treatment also induced conversion of microtubule-associated protein 1 light-chain 3 (LC3)-I into phosphatidylethanolamine-conjugated LC3-II accompanying autophagosome formation, indicators of autophagy; however, of note, NPe6 (50 μg/ml)-PDT did not induce such autophagic changes. In addition, both necrostatin-1 (a necroptosis inhibitor) and knockdown of necroptotic pathway-related proteins [e.g., receptor interacting serine-threonine kinase (RIP)-1, RIP-3, and mixed lineage kinase domain-like protein (MLKL)] inhibited leakage of LDH caused by NPe6 (25 μg/ml)-PDT. Taken together, the present findings revealed that NPe6-PDT-induced necrotic cell death is mediated in part by the necroptosis pathway in glioblastoma T98G cells.

Oxidative stress is known to cause various damages to cellular components such as nucleic acids, lipids, and proteins. [1][2][3] The damages to the cellular components are induced by free radical reactions including degradation, adduct formation, cross-linking, bond-breaking reactions, and so forth.1-3) Therefore oxidatively stressed cells are thought to undergo cell-membrane damages including membrane protein denaturation and cross-linking by free radicals.We previously studied the responses of macrophages to oxidatively damaged cells such as oxidized erythrocytes, 4,5) neutrophils, 6) and Jurkat T cells, 7) using various oxidants, and found that macrophages recognize and remove these oxidatively damaged cells. [4][5][6][7] The determinants on the oxidized cell surface that macrophages recognize were not chemically modified nor denatured membrane structure, but were preexisting carbohydrate chains containing sialyl residues and polylactosaminoglycan structures, possibly sialylpoly-N-acetyllactosaminyl chains, of membrane glycoproteins.4-7) Then, a question how macrophages discriminate the preexisting cellsurface carbohydrate chains of oxidized cells from those of unoxidized cells arose, and this was explained by the hypothesis that membrane glycoproteins aggregate to form clusters upon cell oxidation, presumably due to free radical-mediated protein cross-linking, and the resultant clusters of their extracellular polylactosaminoglycans provide multivalent and therefore high-affinity ligands for macrophage receptors. [4][5][6][7][8] However, there was no conclusive evidence for the membrane glycoprotein aggregation on oxidatively damaged cells [9][10][11][12] and for the presence of the putative macrophage receptors. [13][14][15] In another series of recent works on apoptotic cells, we found that CD43, a major membrane sialoglycoprotein of hematopoietic cells containing sialyl residues and poly-Nacetyllactosaminyl chains, of Jurkat cells aggregates to form clusters and caps at an early stage of apoptosis, and the early apoptotic Jurkat cells were recognized and taken up by macrophages through the clustered and capped CD43. 16) CD43 is a heavily sialylated membrane glycoprotein, 17) and the determinants on CD43 glycoprotein of early apoptotic cells were suggested to be sialylpoly-N-acetyllactosaminyl sugar chains 16) similarly to the determinants on oxidized cells for the macrophage recognition described above. In addition, we identified the macrophage receptor for sialylpoly-Nacetyllactosaminyl chains as nucleolin, 18) a multifunctional shuttle protein present in nucleus and cytoplasm and on the surface of some types of cells including macrophages, 19,20) and demonstrated that nucleolin expressed on cell surface recognizes early apoptotic cells but not non-apoptotic cells. 18)We also demonstrated that phosphatidylserine (PS), a wellinvestigated cell-surface marker for apoptotic cells, 21-23) is not exposed on the cell surface at the early stage of apoptosis, but is at relatively later stages. 24)Considering the s...

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