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Role of P-selectin cytoplasmic domain in granular targeting in vivo and in early inflammatory responses.

Hartwell DW, Mayadas TN, Berger G, Frenette PS, Rayburn H, Hynes RO, Wagner DD - J. Cell Biol. (1998)

Bottom Line: The deletion did not affect the sorting of P-selectin into alpha-granules of platelets but severely compromised the storage of P-selectin in endothelial cells.Unstored P-selectin was proteolytically shed from the plasma membrane, resulting in increased levels of soluble P-selectin in the plasma.Our results suggest that different sorting mechanisms for P-selectin are used in platelets and endothelial cells and that the storage pool of P-selectin in endothelial cells is functionally important during early stages of inflammation.

View Article: PubMed Central - PubMed

Affiliation: Center for Blood Research, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
P-selectin is an adhesion receptor for leukocytes expressed on activated platelets and endothelial cells. The cytoplasmic domain of P-selectin was shown in vitro to contain signals required for both the sorting of this protein into storage granules and its internalization from the plasma membrane. To evaluate in vivo the role of the regulated secretion of P-selectin, we have generated a mouse that expresses P-selectin lacking the cytoplasmic domain (DeltaCT mice). The deletion did not affect the sorting of P-selectin into alpha-granules of platelets but severely compromised the storage of P-selectin in endothelial cells. Unstored P-selectin was proteolytically shed from the plasma membrane, resulting in increased levels of soluble P-selectin in the plasma. The DeltaCT-P-selectin appeared capable of mediating cell adhesion as it supported leukocyte rolling in the mutant mice. However, a secretagogue failed to upregulate leukocyte rolling in the DeltaCT mice, indicating an absence of a releasable storage pool of P-selectin in the endothelium. Furthermore, the neutrophil influx into the inflamed peritoneum was only 30% of the wild-type level 2 h after stimulation. Our results suggest that different sorting mechanisms for P-selectin are used in platelets and endothelial cells and that the storage pool of P-selectin in endothelial cells is functionally important during early stages of inflammation.

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Immunofluorescence localization of  P-selectin in cultured endothelial cells. (a) Cultured lung microvascular endothelial cells were  double stained for P-selectin and vWF. Identical  fields are shown for P-selectin and vWF staining  in each genotype. In wild-type endothelial cells,  P-selectin colocalized with vWF in a granular  pattern typical of Weibel-Palade bodies. In ΔCT  endothelial cells, strong membrane staining for  P-selectin was observed. vWF staining showed  normal granular localization in the mutant cells.  (b) Cultured brain microvascular endothelial  cells from ΔCT mice. Left, vWF localization in  Weibel-Palade bodies. Middle, the identical field  stained for P-selectin. Colocalization of P-selectin with vWF in Weibel-Palade bodies was observed. Right, a ΔCT brain endothelial cell  with strong membrane staining for P-selectin.  Bars, 3 μm.
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Figure 7: Immunofluorescence localization of P-selectin in cultured endothelial cells. (a) Cultured lung microvascular endothelial cells were double stained for P-selectin and vWF. Identical fields are shown for P-selectin and vWF staining in each genotype. In wild-type endothelial cells, P-selectin colocalized with vWF in a granular pattern typical of Weibel-Palade bodies. In ΔCT endothelial cells, strong membrane staining for P-selectin was observed. vWF staining showed normal granular localization in the mutant cells. (b) Cultured brain microvascular endothelial cells from ΔCT mice. Left, vWF localization in Weibel-Palade bodies. Middle, the identical field stained for P-selectin. Colocalization of P-selectin with vWF in Weibel-Palade bodies was observed. Right, a ΔCT brain endothelial cell with strong membrane staining for P-selectin. Bars, 3 μm.

Mentions: The expression of ΔCT–P-selectin by endothelial cells in vivo was evaluated by fluorescence staining of P-selectin on frozen sections of the lung and heart. Compared with that of the wild type, the positive staining for P-selectin appeared less intense in the ΔCT vessels (data not shown). However, the limited resolution of the method does not permit differentiation of P-selectin localized in the storage granules or on the cell surface. To determine whether the P-selectin molecules without the CT were stored in Weibel-Palade bodies, lung microvascular endothelial cells were isolated and cultured in vitro. The cultured endothelial cells were double stained for P-selectin and vWF, a major component of Weibel-Palade bodies (Wagner et al., 1982). In wild-type endothelial cells, the distribution of P-selectin was colocalized with that of vWF to the Weibel-Palade bodies (Fig. 7 a). Meanwhile, the granular staining for P-selectin was much reduced or absent in ΔCT endothelial cells. Instead, strong membrane staining of P-selectin was observed in these cells, with vWF remaining in the Weibel-Palade bodies (Fig. 7 a). Similar predominant membrane staining of ΔCT–P-selectin was observed in four separate preparations of lung endothelial cell cultures. Therefore, it appears that, unlike the situation in platelets, the cytoplasmic domain plays a critical role in sorting of P-selectin into Weibel-Palade bodies in lung endothelial cells. To our surprise, some brain microvascular endothelial cells isolated from the mutant mice were capable of sorting ΔCT–P-selectin into granules. Although surface staining of ΔCT–P-selectin was still frequently observed in the brain microvascular endothelial cells (Fig. 7 b, right), the ΔCT–P-selectin was also localized in Weibel-Palade bodies in numerous cultured brain endothelial cells (Fig. 7 b, middle).


Role of P-selectin cytoplasmic domain in granular targeting in vivo and in early inflammatory responses.

Hartwell DW, Mayadas TN, Berger G, Frenette PS, Rayburn H, Hynes RO, Wagner DD - J. Cell Biol. (1998)

Immunofluorescence localization of  P-selectin in cultured endothelial cells. (a) Cultured lung microvascular endothelial cells were  double stained for P-selectin and vWF. Identical  fields are shown for P-selectin and vWF staining  in each genotype. In wild-type endothelial cells,  P-selectin colocalized with vWF in a granular  pattern typical of Weibel-Palade bodies. In ΔCT  endothelial cells, strong membrane staining for  P-selectin was observed. vWF staining showed  normal granular localization in the mutant cells.  (b) Cultured brain microvascular endothelial  cells from ΔCT mice. Left, vWF localization in  Weibel-Palade bodies. Middle, the identical field  stained for P-selectin. Colocalization of P-selectin with vWF in Weibel-Palade bodies was observed. Right, a ΔCT brain endothelial cell  with strong membrane staining for P-selectin.  Bars, 3 μm.
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Figure 7: Immunofluorescence localization of P-selectin in cultured endothelial cells. (a) Cultured lung microvascular endothelial cells were double stained for P-selectin and vWF. Identical fields are shown for P-selectin and vWF staining in each genotype. In wild-type endothelial cells, P-selectin colocalized with vWF in a granular pattern typical of Weibel-Palade bodies. In ΔCT endothelial cells, strong membrane staining for P-selectin was observed. vWF staining showed normal granular localization in the mutant cells. (b) Cultured brain microvascular endothelial cells from ΔCT mice. Left, vWF localization in Weibel-Palade bodies. Middle, the identical field stained for P-selectin. Colocalization of P-selectin with vWF in Weibel-Palade bodies was observed. Right, a ΔCT brain endothelial cell with strong membrane staining for P-selectin. Bars, 3 μm.
Mentions: The expression of ΔCT–P-selectin by endothelial cells in vivo was evaluated by fluorescence staining of P-selectin on frozen sections of the lung and heart. Compared with that of the wild type, the positive staining for P-selectin appeared less intense in the ΔCT vessels (data not shown). However, the limited resolution of the method does not permit differentiation of P-selectin localized in the storage granules or on the cell surface. To determine whether the P-selectin molecules without the CT were stored in Weibel-Palade bodies, lung microvascular endothelial cells were isolated and cultured in vitro. The cultured endothelial cells were double stained for P-selectin and vWF, a major component of Weibel-Palade bodies (Wagner et al., 1982). In wild-type endothelial cells, the distribution of P-selectin was colocalized with that of vWF to the Weibel-Palade bodies (Fig. 7 a). Meanwhile, the granular staining for P-selectin was much reduced or absent in ΔCT endothelial cells. Instead, strong membrane staining of P-selectin was observed in these cells, with vWF remaining in the Weibel-Palade bodies (Fig. 7 a). Similar predominant membrane staining of ΔCT–P-selectin was observed in four separate preparations of lung endothelial cell cultures. Therefore, it appears that, unlike the situation in platelets, the cytoplasmic domain plays a critical role in sorting of P-selectin into Weibel-Palade bodies in lung endothelial cells. To our surprise, some brain microvascular endothelial cells isolated from the mutant mice were capable of sorting ΔCT–P-selectin into granules. Although surface staining of ΔCT–P-selectin was still frequently observed in the brain microvascular endothelial cells (Fig. 7 b, right), the ΔCT–P-selectin was also localized in Weibel-Palade bodies in numerous cultured brain endothelial cells (Fig. 7 b, middle).

Bottom Line: The deletion did not affect the sorting of P-selectin into alpha-granules of platelets but severely compromised the storage of P-selectin in endothelial cells.Unstored P-selectin was proteolytically shed from the plasma membrane, resulting in increased levels of soluble P-selectin in the plasma.Our results suggest that different sorting mechanisms for P-selectin are used in platelets and endothelial cells and that the storage pool of P-selectin in endothelial cells is functionally important during early stages of inflammation.

View Article: PubMed Central - PubMed

Affiliation: Center for Blood Research, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
P-selectin is an adhesion receptor for leukocytes expressed on activated platelets and endothelial cells. The cytoplasmic domain of P-selectin was shown in vitro to contain signals required for both the sorting of this protein into storage granules and its internalization from the plasma membrane. To evaluate in vivo the role of the regulated secretion of P-selectin, we have generated a mouse that expresses P-selectin lacking the cytoplasmic domain (DeltaCT mice). The deletion did not affect the sorting of P-selectin into alpha-granules of platelets but severely compromised the storage of P-selectin in endothelial cells. Unstored P-selectin was proteolytically shed from the plasma membrane, resulting in increased levels of soluble P-selectin in the plasma. The DeltaCT-P-selectin appeared capable of mediating cell adhesion as it supported leukocyte rolling in the mutant mice. However, a secretagogue failed to upregulate leukocyte rolling in the DeltaCT mice, indicating an absence of a releasable storage pool of P-selectin in the endothelium. Furthermore, the neutrophil influx into the inflamed peritoneum was only 30% of the wild-type level 2 h after stimulation. Our results suggest that different sorting mechanisms for P-selectin are used in platelets and endothelial cells and that the storage pool of P-selectin in endothelial cells is functionally important during early stages of inflammation.

Show MeSH
Related in: MedlinePlus