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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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Analysis of leukocyte rolling in mesenteric venules.  Baseline rolling was recorded during the first 10 min after exteriorization of the mesentery. The number of rolling leukocytes was  quantified by counting the cells passing through a perpendicular  plane in 1 min. To examine leukocyte rolling on activated endothelium, A23187 was added to the exposed mesentery and leukocyte rolling was recorded for an additional 10 min. The data represent the mean of seven mice of each genotype at 4 wk of age. *,  P < 0.05; **, P = 0.71. A previous study (Mayadas, 1993) has  shown that the number of rolling leukocytes per minute in  P-selectin–deficient mice was minimal: <0.05 and 0.10 ± 0.06 for  resting and activated venules, respectively.
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Figure 8: Analysis of leukocyte rolling in mesenteric venules. Baseline rolling was recorded during the first 10 min after exteriorization of the mesentery. The number of rolling leukocytes was quantified by counting the cells passing through a perpendicular plane in 1 min. To examine leukocyte rolling on activated endothelium, A23187 was added to the exposed mesentery and leukocyte rolling was recorded for an additional 10 min. The data represent the mean of seven mice of each genotype at 4 wk of age. *, P < 0.05; **, P = 0.71. A previous study (Mayadas, 1993) has shown that the number of rolling leukocytes per minute in P-selectin–deficient mice was minimal: <0.05 and 0.10 ± 0.06 for resting and activated venules, respectively.

Mentions: The ability of endothelial ΔCT–P-selectin to mediate leukocyte rolling was assessed by intravital microscopy. Upon exteriorization of the mesentery, the baseline leukocyte rolling, a process shown previously to be dependent on endothelial P-selectin (Mayadas et al., 1993), was still observed in the ΔCT mice, and its frequency was comparable to that of the wild type (Fig. 8). This observation suggests that ΔCT–P-selectin expressed by endothelial cells is capable of mediating functional binding to P-selectin ligands on leukocytes in vivo. Subsequently, the venules were superfused with the calcium ionophore A23187 to induce the degranulation of Weibel-Palade bodies (Sporn et al., 1986; Mayadas et al., 1993). In wild-type mice, a twofold increase in number of rolling leukocytes was observed within 2–3 min of stimulation. In contrast, this increase did not occur in the mutant mice, suggesting the absence of a significant releasable storage pool of P-selectin in the mesenteric endothelial cells of the ΔCT mice (Fig. 8).


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)

Analysis of leukocyte rolling in mesenteric venules.  Baseline rolling was recorded during the first 10 min after exteriorization of the mesentery. The number of rolling leukocytes was  quantified by counting the cells passing through a perpendicular  plane in 1 min. To examine leukocyte rolling on activated endothelium, A23187 was added to the exposed mesentery and leukocyte rolling was recorded for an additional 10 min. The data represent the mean of seven mice of each genotype at 4 wk of age. *,  P < 0.05; **, P = 0.71. A previous study (Mayadas, 1993) has  shown that the number of rolling leukocytes per minute in  P-selectin–deficient mice was minimal: <0.05 and 0.10 ± 0.06 for  resting and activated venules, respectively.
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Related In: Results  -  Collection

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Figure 8: Analysis of leukocyte rolling in mesenteric venules. Baseline rolling was recorded during the first 10 min after exteriorization of the mesentery. The number of rolling leukocytes was quantified by counting the cells passing through a perpendicular plane in 1 min. To examine leukocyte rolling on activated endothelium, A23187 was added to the exposed mesentery and leukocyte rolling was recorded for an additional 10 min. The data represent the mean of seven mice of each genotype at 4 wk of age. *, P < 0.05; **, P = 0.71. A previous study (Mayadas, 1993) has shown that the number of rolling leukocytes per minute in P-selectin–deficient mice was minimal: <0.05 and 0.10 ± 0.06 for resting and activated venules, respectively.
Mentions: The ability of endothelial ΔCT–P-selectin to mediate leukocyte rolling was assessed by intravital microscopy. Upon exteriorization of the mesentery, the baseline leukocyte rolling, a process shown previously to be dependent on endothelial P-selectin (Mayadas et al., 1993), was still observed in the ΔCT mice, and its frequency was comparable to that of the wild type (Fig. 8). This observation suggests that ΔCT–P-selectin expressed by endothelial cells is capable of mediating functional binding to P-selectin ligands on leukocytes in vivo. Subsequently, the venules were superfused with the calcium ionophore A23187 to induce the degranulation of Weibel-Palade bodies (Sporn et al., 1986; Mayadas et al., 1993). In wild-type mice, a twofold increase in number of rolling leukocytes was observed within 2–3 min of stimulation. In contrast, this increase did not occur in the mutant mice, suggesting the absence of a significant releasable storage pool of P-selectin in the mesenteric endothelial cells of the ΔCT mice (Fig. 8).

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