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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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Neutrophil recruitment in thioglycollate-induced peritonitis. Peritoneal lavages were collected at indicated time intervals after thioglycollate injection. Total cells in the lavages were  determined using a Coulter counter. Neutrophil numbers were  differentially counted on Wright-Giemsa–stained cytospin preparations. Eight to ten mice of each genotype, 8–10 wk of age, were  used for each time point.
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Figure 9: Neutrophil recruitment in thioglycollate-induced peritonitis. Peritoneal lavages were collected at indicated time intervals after thioglycollate injection. Total cells in the lavages were determined using a Coulter counter. Neutrophil numbers were differentially counted on Wright-Giemsa–stained cytospin preparations. Eight to ten mice of each genotype, 8–10 wk of age, were used for each time point.

Mentions: P-selectin has been shown to play an important role in neutrophil influx during early stages of thioglycollate-induced peritonitis (Mayadas et al., 1993). The effect of the absence of P-selectin storage pool in the mesentery of the ΔCT mice was evaluated in this chemically induced inflammatory model. Thioglycollate was injected into the peritoneal cavities of mutant and wild-type mice and peritoneal lavages were collected after various time intervals. At the 2-h time point, the neutrophil influx in the ΔCT mice was only 30% of that of wild-type mice (Fig. 9). However, this defect in neutrophil emigration in the ΔCT mice was corrected by 4 h after thioglycollate injection. The number of neutrophils recruited to the peritoneal cavity remained comparable between the two genotypes at 24 and 48 h after the induction of peritonitis (Fig. 9). These results indicate that the absence of a stored pool of P-selectin affects only the early stages of inflammation.


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)

Neutrophil recruitment in thioglycollate-induced peritonitis. Peritoneal lavages were collected at indicated time intervals after thioglycollate injection. Total cells in the lavages were  determined using a Coulter counter. Neutrophil numbers were  differentially counted on Wright-Giemsa–stained cytospin preparations. Eight to ten mice of each genotype, 8–10 wk of age, were  used for each time point.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132959&req=5

Figure 9: Neutrophil recruitment in thioglycollate-induced peritonitis. Peritoneal lavages were collected at indicated time intervals after thioglycollate injection. Total cells in the lavages were determined using a Coulter counter. Neutrophil numbers were differentially counted on Wright-Giemsa–stained cytospin preparations. Eight to ten mice of each genotype, 8–10 wk of age, were used for each time point.
Mentions: P-selectin has been shown to play an important role in neutrophil influx during early stages of thioglycollate-induced peritonitis (Mayadas et al., 1993). The effect of the absence of P-selectin storage pool in the mesentery of the ΔCT mice was evaluated in this chemically induced inflammatory model. Thioglycollate was injected into the peritoneal cavities of mutant and wild-type mice and peritoneal lavages were collected after various time intervals. At the 2-h time point, the neutrophil influx in the ΔCT mice was only 30% of that of wild-type mice (Fig. 9). However, this defect in neutrophil emigration in the ΔCT mice was corrected by 4 h after thioglycollate injection. The number of neutrophils recruited to the peritoneal cavity remained comparable between the two genotypes at 24 and 48 h after the induction of peritonitis (Fig. 9). These results indicate that the absence of a stored pool of P-selectin affects only the early stages of inflammation.

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