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Regulation of ICAM-1 in cells of the monocyte/macrophage system in microgravity.

Paulsen K, Tauber S, Dumrese C, Bradacs G, Simmet DM, Gölz N, Hauschild S, Raig C, Engeli S, Gutewort A, Hürlimann E, Biskup J, Unverdorben F, Rieder G, Hofmänner D, Mutschler L, Krammer S, Buttron I, Philpot C, Huge A, Lier H, Barz I, Engelmann F, Layer LE, Thiel CS, Ullrich O - Biomed Res Int (2015)

Bottom Line: In contrast, ICAM-1 expression increased in macrophage-like differentiated human U937 cells during the microgravity phase of parabolic flights and in long-term microgravity provided by a 2D clinostat or during the orbital SIMBOX/Shenzhou-8 mission.In nondifferentiated U937 cells, no effect of microgravity on ICAM-1 expression could be observed during parabolic flight experiments.Thus, ICAM-1 can be considered as a rapid-reacting and sustained gravity-regulated molecule in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Anatomy, Faculty of Medicine, University of Zurich, Winterthurerstraß 190, 8057 Zurich, Switzerland.

ABSTRACT
Cells of the immune system are highly sensitive to altered gravity, and the monocyte as well as the macrophage function is proven to be impaired under microgravity conditions. In our study, we investigated the surface expression of ICAM-1 protein and expression of ICAM-1 mRNA in cells of the monocyte/macrophage system in microgravity during clinostat, parabolic flight, sounding rocket, and orbital experiments. In murine BV-2 microglial cells, we detected a downregulation of ICAM-1 expression in clinorotation experiments and a rapid and reversible downregulation in the microgravity phase of parabolic flight experiments. In contrast, ICAM-1 expression increased in macrophage-like differentiated human U937 cells during the microgravity phase of parabolic flights and in long-term microgravity provided by a 2D clinostat or during the orbital SIMBOX/Shenzhou-8 mission. In nondifferentiated U937 cells, no effect of microgravity on ICAM-1 expression could be observed during parabolic flight experiments. We conclude that disturbed immune function in microgravity could be a consequence of ICAM-1 modulation in the monocyte/macrophage system, which in turn could have a strong impact on the interaction with T lymphocytes and cell migration. Thus, ICAM-1 can be considered as a rapid-reacting and sustained gravity-regulated molecule in mammalian cells.

Show MeSH
ICAM-1 surface expression reacts rapidly and reversibly to microgravity. (a) Microscopy of ICAM-1, TUNEL, HCS CellMask, and DAPI including surface calculation for HCS. In order to identify nuclei, cells were stained with DAPI (A). Apoptotic cells were identified by TUNEL reaction (B) and HCS CellMask label (C) which can be retained to a higher extend in nonapoptotic cells. ICAM-1 intensity is depicted in (D). A merge of TUNEL, DAPI, and ICAM-1 (E) shows an apoptotic cell (◄) and a living cell (→). The automated calculation of an iso-surface is exclusively done for living cells using the HCS CellMask channel as shown in the merge with TUNEL, DAPI, and ICAM-1 (F). (b)–(j) BV-2 microglial cells were treated with PMA ((e), (f), and (g)) or TNF-α ((h), (i), and (j)) at the onset of microgravity or during the 1 g in-flight control phase or left untreated ((b), (c), and (d)). Cells were fixed in flight after 20 sec normogravity (1 g) (-●-) or 20 sec microgravity (μg) (-○-). Cells were stained, imaged, and analyzed as described above. The mean intensity of the ICAM-1 signal was binned into mean intensity fluorescence (MIF) categories and the number of cells (frequency) is plotted against these intensity categories ((b), (e), and (h)). ICAM-1 fluorescence intensity of all analyzed cells ((c), (f), and (i)) is depicted for normogravity (triangles) and microgravity (squares). Mean ICAM-1 fluorescence intensity of all analyzed cells ((d), (g), and (j)) was pooled for normogravity (black bar) and microgravity (open bar). For automated imaging, the unified random sampling module was utilized and 63 randomized images of each sample were recorded and at least 500 single cells from 3 independent experiments from 3 different parabolas were analyzed. Mean intensity and SEM are shown and student's t-test showed highly significant difference of the fluorescence values of  ***P < 0.0001, n = 3.
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fig3: ICAM-1 surface expression reacts rapidly and reversibly to microgravity. (a) Microscopy of ICAM-1, TUNEL, HCS CellMask, and DAPI including surface calculation for HCS. In order to identify nuclei, cells were stained with DAPI (A). Apoptotic cells were identified by TUNEL reaction (B) and HCS CellMask label (C) which can be retained to a higher extend in nonapoptotic cells. ICAM-1 intensity is depicted in (D). A merge of TUNEL, DAPI, and ICAM-1 (E) shows an apoptotic cell (◄) and a living cell (→). The automated calculation of an iso-surface is exclusively done for living cells using the HCS CellMask channel as shown in the merge with TUNEL, DAPI, and ICAM-1 (F). (b)–(j) BV-2 microglial cells were treated with PMA ((e), (f), and (g)) or TNF-α ((h), (i), and (j)) at the onset of microgravity or during the 1 g in-flight control phase or left untreated ((b), (c), and (d)). Cells were fixed in flight after 20 sec normogravity (1 g) (-●-) or 20 sec microgravity (μg) (-○-). Cells were stained, imaged, and analyzed as described above. The mean intensity of the ICAM-1 signal was binned into mean intensity fluorescence (MIF) categories and the number of cells (frequency) is plotted against these intensity categories ((b), (e), and (h)). ICAM-1 fluorescence intensity of all analyzed cells ((c), (f), and (i)) is depicted for normogravity (triangles) and microgravity (squares). Mean ICAM-1 fluorescence intensity of all analyzed cells ((d), (g), and (j)) was pooled for normogravity (black bar) and microgravity (open bar). For automated imaging, the unified random sampling module was utilized and 63 randomized images of each sample were recorded and at least 500 single cells from 3 independent experiments from 3 different parabolas were analyzed. Mean intensity and SEM are shown and student's t-test showed highly significant difference of the fluorescence values of  ***P < 0.0001, n = 3.

Mentions: Experiments from different parabolas (1 g and μg, resp.) and different flights were analyzed. The experiments were performed in a sequence of three consecutive μg and 1 g phases. A quadruple fluorescent staining was performed using TUNEL (rhodamine) for detection of apoptotic cells, DAPI for the nuclei, high content screening (HCS) CellMask deep red for the delineation of cells, and FITC-labeled anti ICAM-1 antibody for identification of cell surface expression of ICAM-1. Cells were imaged with a widefield microscope (Leica Microsystems, Wetzlar, Germany) using the uniform random sampling module and identified by an iso-surface calculation (Imaris, Bitplane AG, Zurich, Switzerland). This quadruple staining allowed the exclusion of apoptotic cells in a highly reliable fashion. An example of an apoptotic cell and a living cell is depicted in Figure 3(a). The mean intensity of the ICAM-1 signal was analyzed in nondamaged and nonapoptotic cells only and binned into intensity categories. The relative frequency of these cells was plotted against the fluorescence intensity (Figures 3(b), 3(e), and 3(h)).


Regulation of ICAM-1 in cells of the monocyte/macrophage system in microgravity.

Paulsen K, Tauber S, Dumrese C, Bradacs G, Simmet DM, Gölz N, Hauschild S, Raig C, Engeli S, Gutewort A, Hürlimann E, Biskup J, Unverdorben F, Rieder G, Hofmänner D, Mutschler L, Krammer S, Buttron I, Philpot C, Huge A, Lier H, Barz I, Engelmann F, Layer LE, Thiel CS, Ullrich O - Biomed Res Int (2015)

ICAM-1 surface expression reacts rapidly and reversibly to microgravity. (a) Microscopy of ICAM-1, TUNEL, HCS CellMask, and DAPI including surface calculation for HCS. In order to identify nuclei, cells were stained with DAPI (A). Apoptotic cells were identified by TUNEL reaction (B) and HCS CellMask label (C) which can be retained to a higher extend in nonapoptotic cells. ICAM-1 intensity is depicted in (D). A merge of TUNEL, DAPI, and ICAM-1 (E) shows an apoptotic cell (◄) and a living cell (→). The automated calculation of an iso-surface is exclusively done for living cells using the HCS CellMask channel as shown in the merge with TUNEL, DAPI, and ICAM-1 (F). (b)–(j) BV-2 microglial cells were treated with PMA ((e), (f), and (g)) or TNF-α ((h), (i), and (j)) at the onset of microgravity or during the 1 g in-flight control phase or left untreated ((b), (c), and (d)). Cells were fixed in flight after 20 sec normogravity (1 g) (-●-) or 20 sec microgravity (μg) (-○-). Cells were stained, imaged, and analyzed as described above. The mean intensity of the ICAM-1 signal was binned into mean intensity fluorescence (MIF) categories and the number of cells (frequency) is plotted against these intensity categories ((b), (e), and (h)). ICAM-1 fluorescence intensity of all analyzed cells ((c), (f), and (i)) is depicted for normogravity (triangles) and microgravity (squares). Mean ICAM-1 fluorescence intensity of all analyzed cells ((d), (g), and (j)) was pooled for normogravity (black bar) and microgravity (open bar). For automated imaging, the unified random sampling module was utilized and 63 randomized images of each sample were recorded and at least 500 single cells from 3 independent experiments from 3 different parabolas were analyzed. Mean intensity and SEM are shown and student's t-test showed highly significant difference of the fluorescence values of  ***P < 0.0001, n = 3.
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Related In: Results  -  Collection

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fig3: ICAM-1 surface expression reacts rapidly and reversibly to microgravity. (a) Microscopy of ICAM-1, TUNEL, HCS CellMask, and DAPI including surface calculation for HCS. In order to identify nuclei, cells were stained with DAPI (A). Apoptotic cells were identified by TUNEL reaction (B) and HCS CellMask label (C) which can be retained to a higher extend in nonapoptotic cells. ICAM-1 intensity is depicted in (D). A merge of TUNEL, DAPI, and ICAM-1 (E) shows an apoptotic cell (◄) and a living cell (→). The automated calculation of an iso-surface is exclusively done for living cells using the HCS CellMask channel as shown in the merge with TUNEL, DAPI, and ICAM-1 (F). (b)–(j) BV-2 microglial cells were treated with PMA ((e), (f), and (g)) or TNF-α ((h), (i), and (j)) at the onset of microgravity or during the 1 g in-flight control phase or left untreated ((b), (c), and (d)). Cells were fixed in flight after 20 sec normogravity (1 g) (-●-) or 20 sec microgravity (μg) (-○-). Cells were stained, imaged, and analyzed as described above. The mean intensity of the ICAM-1 signal was binned into mean intensity fluorescence (MIF) categories and the number of cells (frequency) is plotted against these intensity categories ((b), (e), and (h)). ICAM-1 fluorescence intensity of all analyzed cells ((c), (f), and (i)) is depicted for normogravity (triangles) and microgravity (squares). Mean ICAM-1 fluorescence intensity of all analyzed cells ((d), (g), and (j)) was pooled for normogravity (black bar) and microgravity (open bar). For automated imaging, the unified random sampling module was utilized and 63 randomized images of each sample were recorded and at least 500 single cells from 3 independent experiments from 3 different parabolas were analyzed. Mean intensity and SEM are shown and student's t-test showed highly significant difference of the fluorescence values of  ***P < 0.0001, n = 3.
Mentions: Experiments from different parabolas (1 g and μg, resp.) and different flights were analyzed. The experiments were performed in a sequence of three consecutive μg and 1 g phases. A quadruple fluorescent staining was performed using TUNEL (rhodamine) for detection of apoptotic cells, DAPI for the nuclei, high content screening (HCS) CellMask deep red for the delineation of cells, and FITC-labeled anti ICAM-1 antibody for identification of cell surface expression of ICAM-1. Cells were imaged with a widefield microscope (Leica Microsystems, Wetzlar, Germany) using the uniform random sampling module and identified by an iso-surface calculation (Imaris, Bitplane AG, Zurich, Switzerland). This quadruple staining allowed the exclusion of apoptotic cells in a highly reliable fashion. An example of an apoptotic cell and a living cell is depicted in Figure 3(a). The mean intensity of the ICAM-1 signal was analyzed in nondamaged and nonapoptotic cells only and binned into intensity categories. The relative frequency of these cells was plotted against the fluorescence intensity (Figures 3(b), 3(e), and 3(h)).

Bottom Line: In contrast, ICAM-1 expression increased in macrophage-like differentiated human U937 cells during the microgravity phase of parabolic flights and in long-term microgravity provided by a 2D clinostat or during the orbital SIMBOX/Shenzhou-8 mission.In nondifferentiated U937 cells, no effect of microgravity on ICAM-1 expression could be observed during parabolic flight experiments.Thus, ICAM-1 can be considered as a rapid-reacting and sustained gravity-regulated molecule in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Anatomy, Faculty of Medicine, University of Zurich, Winterthurerstraß 190, 8057 Zurich, Switzerland.

ABSTRACT
Cells of the immune system are highly sensitive to altered gravity, and the monocyte as well as the macrophage function is proven to be impaired under microgravity conditions. In our study, we investigated the surface expression of ICAM-1 protein and expression of ICAM-1 mRNA in cells of the monocyte/macrophage system in microgravity during clinostat, parabolic flight, sounding rocket, and orbital experiments. In murine BV-2 microglial cells, we detected a downregulation of ICAM-1 expression in clinorotation experiments and a rapid and reversible downregulation in the microgravity phase of parabolic flight experiments. In contrast, ICAM-1 expression increased in macrophage-like differentiated human U937 cells during the microgravity phase of parabolic flights and in long-term microgravity provided by a 2D clinostat or during the orbital SIMBOX/Shenzhou-8 mission. In nondifferentiated U937 cells, no effect of microgravity on ICAM-1 expression could be observed during parabolic flight experiments. We conclude that disturbed immune function in microgravity could be a consequence of ICAM-1 modulation in the monocyte/macrophage system, which in turn could have a strong impact on the interaction with T lymphocytes and cell migration. Thus, ICAM-1 can be considered as a rapid-reacting and sustained gravity-regulated molecule in mammalian cells.

Show MeSH