Figure 1
Figure 1. Production of macrophage chemotactic factors by apoptotic BL cells. (A) Chemotaxis of HMDM to EBV-positive (Mutu-BL) and EBV-negative (BL2) lines undergoing spontaneous (■) or UV-induced apoptosis (). Bcl-2 transfectants that are protected from apoptosis () are shown for comparison. Chemotaxis is shown as fold increase above that of background (medium alone), which was set to 1. CCL5 (100 ng/mL) is included as a positive control. Levels of apoptosis (assessed using annexin V) for Mutu-BL: 23% (BL), 15% (+Bcl-2), 89% (+UV); for BL2: 44% (BL), 18% (+Bcl-2), 99% (+UV). Data shown are means plus or minus SEM of replicate high-power fields. Experiment shown is representative of 3. Student t test (background vs BL cells): **P < .005, ***P < .001. (B) Chemotaxis of human monocyte/macrophage cell line MonoMac6 to Mutu-BL cells undergoing spontaneous or UV-induced apoptosis. Student t test (background vs BL cells): ***P < .001. (C) Kinetics of macrophage chemoattractant release from BL cells undergoing apoptosis. Mutu-BL cells were induced into apoptosis by UV irradiation and assayed at the indicated times. Chemotaxis of HMDM toward apoptotic BL cells (■, mean ± SD) and apoptosis (assessed using annexin V, ) were assessed in parallel. Experiment is representative of 2 identical. (D) Presence of macrophage chemoattractant activity in cell-free supernatants of BL cells undergoing apoptosis. Chemotaxis of HMDM to UV-induced Mutu-BL cells (■) and to cell-free supernatant (S/N, ▩) from the same cells. Cells were 94% apoptotic (assessed using annexin V) in this experiment. Student t test (cells vs S/N): P = .2 (ns indicates not significant). (E) Blockade of macrophage chemotaxis to apoptotic BL cells by PTX. Chemotaxis to UV-induced Mutu-BL cells of HMDM (▩) or HMDM pretreated with PTX (100 ng/mL) for 12 hours before chemotaxis assay (■). Student t test (control vs PTX-treated HMDM): ***P < .001. Levels of apoptosis (assessed using annexin V) were 67% (Mutu), 85% (Mutu + UV). Note there was no loss in viability of macrophages after PTX treatment (91% viable PTX-treated macrophages at the end of the chemotaxis assay vs 90% for control macrophages in the experiment shown). (F) Inhibition of macrophage chemotaxis to apoptotic BL cells by the viral chemokine antagonist, vMIPII. Chemotaxis of HMDM to UV-induced Mutu-BL cells in the absence (▩) or presence (■) of vMIPII (60 ng/mL). Student t test (control vs vMIPII-treated HMDM): ***P < .001. Levels of apoptosis (assessed using annexin V) were 51% (Mutu), 88% (Mutu + UV). Macrophage viability (> 90%) was unaffected by vMIPII.

Production of macrophage chemotactic factors by apoptotic BL cells. (A) Chemotaxis of HMDM to EBV-positive (Mutu-BL) and EBV-negative (BL2) lines undergoing spontaneous (■) or UV-induced apoptosis (). Bcl-2 transfectants that are protected from apoptosis () are shown for comparison. Chemotaxis is shown as fold increase above that of background (medium alone), which was set to 1. CCL5 (100 ng/mL) is included as a positive control. Levels of apoptosis (assessed using annexin V) for Mutu-BL: 23% (BL), 15% (+Bcl-2), 89% (+UV); for BL2: 44% (BL), 18% (+Bcl-2), 99% (+UV). Data shown are means plus or minus SEM of replicate high-power fields. Experiment shown is representative of 3. Student t test (background vs BL cells): **P < .005, ***P < .001. (B) Chemotaxis of human monocyte/macrophage cell line MonoMac6 to Mutu-BL cells undergoing spontaneous or UV-induced apoptosis. Student t test (background vs BL cells): ***P < .001. (C) Kinetics of macrophage chemoattractant release from BL cells undergoing apoptosis. Mutu-BL cells were induced into apoptosis by UV irradiation and assayed at the indicated times. Chemotaxis of HMDM toward apoptotic BL cells (■, mean ± SD) and apoptosis (assessed using annexin V, ) were assessed in parallel. Experiment is representative of 2 identical. (D) Presence of macrophage chemoattractant activity in cell-free supernatants of BL cells undergoing apoptosis. Chemotaxis of HMDM to UV-induced Mutu-BL cells (■) and to cell-free supernatant (S/N, ▩) from the same cells. Cells were 94% apoptotic (assessed using annexin V) in this experiment. Student t test (cells vs S/N): P = .2 (ns indicates not significant). (E) Blockade of macrophage chemotaxis to apoptotic BL cells by PTX. Chemotaxis to UV-induced Mutu-BL cells of HMDM (▩) or HMDM pretreated with PTX (100 ng/mL) for 12 hours before chemotaxis assay (■). Student t test (control vs PTX-treated HMDM): ***P < .001. Levels of apoptosis (assessed using annexin V) were 67% (Mutu), 85% (Mutu + UV). Note there was no loss in viability of macrophages after PTX treatment (91% viable PTX-treated macrophages at the end of the chemotaxis assay vs 90% for control macrophages in the experiment shown). (F) Inhibition of macrophage chemotaxis to apoptotic BL cells by the viral chemokine antagonist, vMIPII. Chemotaxis of HMDM to UV-induced Mutu-BL cells in the absence (▩) or presence (■) of vMIPII (60 ng/mL). Student t test (control vs vMIPII-treated HMDM): ***P < .001. Levels of apoptosis (assessed using annexin V) were 51% (Mutu), 88% (Mutu + UV). Macrophage viability (> 90%) was unaffected by vMIPII.

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