Figure 2.
Figure 2. Phagocytosis of apoptotic monocytes induces immune paralysis in iDCs. (A) Apoptotic monocytes interacting with iDCs. Two DiI-stained apoptotic monocytes are surrounded by iDC pseudopods, and engulfed apoptotic fragments are seen within the dendritic cell (fluorescence microscopy, magnification × 100; further described under “Generation of monocyte-derived dendritic cells (DCs) and interaction with apoptotic monocytes”). For the micrograph in panel A, DiI-stained apoptotic cells were dried on a glass slide with Fluoromount-G (Southern Biotech), and then visualized under a Zeiss Axiovert 200 microscope equipped with a Plan-APOCHROMAT 100×/1.40 objective lens (Zeiss, Oberkochen, Germany). The image was captured with a SensiCam (PCO, Kelheim, Germany) and acquired via ImagePro Plus 4.5 software (Media Cybernetics, Silver Spring, MD). Final processing was performed with Adobe Photoshop 8.0 software (Adobe Systems, San Jose, CA). (B) Apoptotic monocyte engulfment down-regulates the expression of maturation-related molecules on DCs. Apoptotic monocytes were offered, or not offered, to iDCs at a ratio of 4:1. Five hours later, iDCs were exposed to 5 ng/mL LPS. HLA-DR-FITC and CD86 FITC expression was measured by flow cytometry. DR and CD86 are expressed at baseline levels in iDCs (mean fluorescence: 918 and 640, respectively) and are up-regulated following exposure to LPS (mean fluorescence: 1256 and 1159, respectively). Up-regulation by LPS is inhibited by exposure to apoptotic monocytes for both DR (mean fluorescence: 886, P < .001) and CD86 (mean fluorescence: 756, P < .01).Values represent the mean ± SEM of 3 experiments. (C) Apoptotic monocyte engulfment decreases IL-12 p40 production by DCs exposed to LPS. Immature DCs secrete IL-12 in response to LPS. Following interaction with apoptotic cells, down-regulation of IL-12 secretion is observed. Data represent the mean ± SD of 3 experiments. (D) Apoptotic monocytes prevent appearance of mature DC morphology in response to LPS. iDCs change morphology, becoming elongated and more “dendritic,” as they transform into mDCs in response to LPS. Following interaction with apoptotic cells, iDC morphology is retained despite exposure to LPS (light microscopy magnification, × 10). For the micrograph in panel D, cells were mounted in RPMI culture medium for visualization under a Nikon Eclipse TS100 microscope equipped with a Nikon Ph2 ADL 10×/0.25 objective lens (Nikon, Melville, NY). The image was then captured with a Nikon Coolpix 995 camera and processed with Adobe Photoshop 8.0 software.

Phagocytosis of apoptotic monocytes induces immune paralysis in iDCs. (A) Apoptotic monocytes interacting with iDCs. Two DiI-stained apoptotic monocytes are surrounded by iDC pseudopods, and engulfed apoptotic fragments are seen within the dendritic cell (fluorescence microscopy, magnification × 100; further described under “Generation of monocyte-derived dendritic cells (DCs) and interaction with apoptotic monocytes”). For the micrograph in panel A, DiI-stained apoptotic cells were dried on a glass slide with Fluoromount-G (Southern Biotech), and then visualized under a Zeiss Axiovert 200 microscope equipped with a Plan-APOCHROMAT 100×/1.40 objective lens (Zeiss, Oberkochen, Germany). The image was captured with a SensiCam (PCO, Kelheim, Germany) and acquired via ImagePro Plus 4.5 software (Media Cybernetics, Silver Spring, MD). Final processing was performed with Adobe Photoshop 8.0 software (Adobe Systems, San Jose, CA). (B) Apoptotic monocyte engulfment down-regulates the expression of maturation-related molecules on DCs. Apoptotic monocytes were offered, or not offered, to iDCs at a ratio of 4:1. Five hours later, iDCs were exposed to 5 ng/mL LPS. HLA-DR-FITC and CD86 FITC expression was measured by flow cytometry. DR and CD86 are expressed at baseline levels in iDCs (mean fluorescence: 918 and 640, respectively) and are up-regulated following exposure to LPS (mean fluorescence: 1256 and 1159, respectively). Up-regulation by LPS is inhibited by exposure to apoptotic monocytes for both DR (mean fluorescence: 886, P < .001) and CD86 (mean fluorescence: 756, P < .01).Values represent the mean ± SEM of 3 experiments. (C) Apoptotic monocyte engulfment decreases IL-12 p40 production by DCs exposed to LPS. Immature DCs secrete IL-12 in response to LPS. Following interaction with apoptotic cells, down-regulation of IL-12 secretion is observed. Data represent the mean ± SD of 3 experiments. (D) Apoptotic monocytes prevent appearance of mature DC morphology in response to LPS. iDCs change morphology, becoming elongated and more “dendritic,” as they transform into mDCs in response to LPS. Following interaction with apoptotic cells, iDC morphology is retained despite exposure to LPS (light microscopy magnification, × 10). For the micrograph in panel D, cells were mounted in RPMI culture medium for visualization under a Nikon Eclipse TS100 microscope equipped with a Nikon Ph2 ADL 10×/0.25 objective lens (Nikon, Melville, NY). The image was then captured with a Nikon Coolpix 995 camera and processed with Adobe Photoshop 8.0 software.

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