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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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Presence of soluble P-selectin fragment in the plasma. Plasma samples  were collected from wild-type (WT), ΔCT, and P-selectin–deficient (P-KO) mice.  (a) Soluble P-selectin levels were determined using a sandwich ELISA. After subtracting the value of P-KO plasma, an average of fivefold increase in plasma  P-selectin was detected in ΔCT mice compared with the wild-type. (b) Western  blot analysis of plasma P-selectin. 10 μl of plasma or lysates from 5 × 107 platelets  was loaded in each lane. The blot was probed with a polyclonal antibody against  P-selectin. (c) Increased levels of soluble P-selectin in the plasma of wild-type  mice stimulated with LPS or TNF-α. Wild-type mice were injected intraperitoneally with LPS or murine recombinant TNF-α (both at 20 μg/g body weight) and  plasma samples were collected 4 h later. Plasma from P-KO mice was used to determine the background for the ELISA. The OD values shown were obtained after subtracting the value of P-KO plasma. *, P < 0.002 ; **, P = 0.02; n = 3 or 4.
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Figure 10: Presence of soluble P-selectin fragment in the plasma. Plasma samples were collected from wild-type (WT), ΔCT, and P-selectin–deficient (P-KO) mice. (a) Soluble P-selectin levels were determined using a sandwich ELISA. After subtracting the value of P-KO plasma, an average of fivefold increase in plasma P-selectin was detected in ΔCT mice compared with the wild-type. (b) Western blot analysis of plasma P-selectin. 10 μl of plasma or lysates from 5 × 107 platelets was loaded in each lane. The blot was probed with a polyclonal antibody against P-selectin. (c) Increased levels of soluble P-selectin in the plasma of wild-type mice stimulated with LPS or TNF-α. Wild-type mice were injected intraperitoneally with LPS or murine recombinant TNF-α (both at 20 μg/g body weight) and plasma samples were collected 4 h later. Plasma from P-KO mice was used to determine the background for the ELISA. The OD values shown were obtained after subtracting the value of P-KO plasma. *, P < 0.002 ; **, P = 0.02; n = 3 or 4.

Mentions: The fact that the baseline leukocyte rolling was not unusually high in ΔCT mice, combined with the weak fluorescence staining of the ΔCT vessels, suggests that the ΔCT– P-selectin delivered to the surface of endothelial cells might be removed from the plasma membrane in vivo. Indeed, using a sandwich ELISA, we have detected an over fivefold increase in the level of soluble P-selectin in the plasma of the mutant mice (Fig. 10 a). Consistent with the result of the ELISA, Western blot analysis of the plasma using a polyclonal antibody against P-selectin revealed a fragment at ∼100 kD whose level was significantly increased in ΔCT mice compared with wild-type mice (Fig. 10 b). Since the capture antibody used in the ELISA is a monoclonal antibody (RB40.34) against the lectin domain of P-selectin (Bosse and Vestweber, 1994), and because of the estimated molecular weight, we believe that the P-selectin fragment in the plasma detected by Western blot contains the NH2-terminal 100-kD extracellular portion of P-selectin. Consistent with this possibility, the soluble P-selectin fragment in the plasma was not recognized by the anti-CT antibody in Western blot analysis (data not shown). In addition, treatment of wild-type mice with LPS or TNF-α induced a fourfold increase in the levels of soluble P-selectin in the plasma (Fig. 10 c), indicating that shedding of P-selectin into the plasma may represent a general mechanism of P-selectin downregulation after inflammatory responses.


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)

Presence of soluble P-selectin fragment in the plasma. Plasma samples  were collected from wild-type (WT), ΔCT, and P-selectin–deficient (P-KO) mice.  (a) Soluble P-selectin levels were determined using a sandwich ELISA. After subtracting the value of P-KO plasma, an average of fivefold increase in plasma  P-selectin was detected in ΔCT mice compared with the wild-type. (b) Western  blot analysis of plasma P-selectin. 10 μl of plasma or lysates from 5 × 107 platelets  was loaded in each lane. The blot was probed with a polyclonal antibody against  P-selectin. (c) Increased levels of soluble P-selectin in the plasma of wild-type  mice stimulated with LPS or TNF-α. Wild-type mice were injected intraperitoneally with LPS or murine recombinant TNF-α (both at 20 μg/g body weight) and  plasma samples were collected 4 h later. Plasma from P-KO mice was used to determine the background for the ELISA. The OD values shown were obtained after subtracting the value of P-KO plasma. *, P < 0.002 ; **, P = 0.02; n = 3 or 4.
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Figure 10: Presence of soluble P-selectin fragment in the plasma. Plasma samples were collected from wild-type (WT), ΔCT, and P-selectin–deficient (P-KO) mice. (a) Soluble P-selectin levels were determined using a sandwich ELISA. After subtracting the value of P-KO plasma, an average of fivefold increase in plasma P-selectin was detected in ΔCT mice compared with the wild-type. (b) Western blot analysis of plasma P-selectin. 10 μl of plasma or lysates from 5 × 107 platelets was loaded in each lane. The blot was probed with a polyclonal antibody against P-selectin. (c) Increased levels of soluble P-selectin in the plasma of wild-type mice stimulated with LPS or TNF-α. Wild-type mice were injected intraperitoneally with LPS or murine recombinant TNF-α (both at 20 μg/g body weight) and plasma samples were collected 4 h later. Plasma from P-KO mice was used to determine the background for the ELISA. The OD values shown were obtained after subtracting the value of P-KO plasma. *, P < 0.002 ; **, P = 0.02; n = 3 or 4.
Mentions: The fact that the baseline leukocyte rolling was not unusually high in ΔCT mice, combined with the weak fluorescence staining of the ΔCT vessels, suggests that the ΔCT– P-selectin delivered to the surface of endothelial cells might be removed from the plasma membrane in vivo. Indeed, using a sandwich ELISA, we have detected an over fivefold increase in the level of soluble P-selectin in the plasma of the mutant mice (Fig. 10 a). Consistent with the result of the ELISA, Western blot analysis of the plasma using a polyclonal antibody against P-selectin revealed a fragment at ∼100 kD whose level was significantly increased in ΔCT mice compared with wild-type mice (Fig. 10 b). Since the capture antibody used in the ELISA is a monoclonal antibody (RB40.34) against the lectin domain of P-selectin (Bosse and Vestweber, 1994), and because of the estimated molecular weight, we believe that the P-selectin fragment in the plasma detected by Western blot contains the NH2-terminal 100-kD extracellular portion of P-selectin. Consistent with this possibility, the soluble P-selectin fragment in the plasma was not recognized by the anti-CT antibody in Western blot analysis (data not shown). In addition, treatment of wild-type mice with LPS or TNF-α induced a fourfold increase in the levels of soluble P-selectin in the plasma (Fig. 10 c), indicating that shedding of P-selectin into the plasma may represent a general mechanism of P-selectin downregulation after inflammatory responses.

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