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Tyrosine sulfation of native mouse Psgl-1 is required for optimal leukocyte rolling on P-selectin in vivo.

Westmuckett AD, Thacker KM, Moore KL - PLoS ONE (2011)

Bottom Line: We observed that rolling flux fractions were lower and leukocyte rolling velocities were higher in Tpst DKO → B6 venules compared to WT → B6 venules.Similar results were observed on immobilized P-selectin in vitro.Finally, Tpst DKO leukocytes bound less P-selectin than wild type leukocytes despite equivalent surface expression of Psgl-1.

View Article: PubMed Central - PubMed

Affiliation: Oklahoma Medical Research Foundation, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America. Andrew-Westmuckett@omrf.org

ABSTRACT

Background: We recently demonstrated that tyrosine sulfation is an important contributor to monocyte recruitment and retention in a mouse model of atherosclerosis. P-selectin glycoprotein ligand-1 (Psgl-1) is tyrosine-sulfated in mouse monocyte/macrophages and its interaction with P-selectin is important in monocyte recruitment in atherosclerosis. However, whether tyrosine sulfation is required for the P-selectin binding function of mouse Psgl-1 is unknown. Here we test the function of native Psgl-1 expressed in leukocytes lacking endogenous tyrosylprotein sulfotransferase (TPST) activity.

Methodology/principal findings: Psgl-1 function was assessed by examining P-selectin dependent leukocyte rolling in post-capillary venules of C57BL6 mice transplanted with hematopoietic progenitors from wild type (WT → B6) or Tpst1;Tpst2 double knockout mice (Tpst DKO → B6) which lack TPST activity. We observed that rolling flux fractions were lower and leukocyte rolling velocities were higher in Tpst DKO → B6 venules compared to WT → B6 venules. Similar results were observed on immobilized P-selectin in vitro. Finally, Tpst DKO leukocytes bound less P-selectin than wild type leukocytes despite equivalent surface expression of Psgl-1.

Conclusions/significance: These findings provide direct and convincing evidence that tyrosine sulfation is required for optimal function of mouse Psgl-1 in vivo and suggests that tyrosine sulfation of Psgl-1 contributes to the development of atherosclerosis.

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Related in: MedlinePlus

P-selectin dependent leukocyte rolling in vitro.Bone marrow leukocytes from wild type (n = 3) and Tpst DKO→B6 (n = 3) mice were isolated and their rolling on immobilized mouse P-selectin/IgM (site density  = 100 sites/µm2) was observed at 1 dyn/cm2. (A) For each animal, the number of rolling cells in 4 fields of view were averaged. (B) Rolling velocities of 10 leukocytes were determined in the same 4 fields of view as the number of rolling cells. Values are reported as mean ± S.E.M.
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pone-0020406-g003: P-selectin dependent leukocyte rolling in vitro.Bone marrow leukocytes from wild type (n = 3) and Tpst DKO→B6 (n = 3) mice were isolated and their rolling on immobilized mouse P-selectin/IgM (site density  = 100 sites/µm2) was observed at 1 dyn/cm2. (A) For each animal, the number of rolling cells in 4 fields of view were averaged. (B) Rolling velocities of 10 leukocytes were determined in the same 4 fields of view as the number of rolling cells. Values are reported as mean ± S.E.M.

Mentions: For wild type leukocytes, we observed 176±15 rolling cells/mm2, whereas for Tpst DKO→B6 leukocytes we observed only 97±6 rolling cells/mm2 (n = 12 fields from 3 independent paired experiments) (Fig. 3A). Statistical analysis showed that the number of rolling cells in the Tpst DKO→B6 group was significantly lower than the WT group (p<0.0001, d = 2.1).


Tyrosine sulfation of native mouse Psgl-1 is required for optimal leukocyte rolling on P-selectin in vivo.

Westmuckett AD, Thacker KM, Moore KL - PLoS ONE (2011)

P-selectin dependent leukocyte rolling in vitro.Bone marrow leukocytes from wild type (n = 3) and Tpst DKO→B6 (n = 3) mice were isolated and their rolling on immobilized mouse P-selectin/IgM (site density  = 100 sites/µm2) was observed at 1 dyn/cm2. (A) For each animal, the number of rolling cells in 4 fields of view were averaged. (B) Rolling velocities of 10 leukocytes were determined in the same 4 fields of view as the number of rolling cells. Values are reported as mean ± S.E.M.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3102115&req=5

pone-0020406-g003: P-selectin dependent leukocyte rolling in vitro.Bone marrow leukocytes from wild type (n = 3) and Tpst DKO→B6 (n = 3) mice were isolated and their rolling on immobilized mouse P-selectin/IgM (site density  = 100 sites/µm2) was observed at 1 dyn/cm2. (A) For each animal, the number of rolling cells in 4 fields of view were averaged. (B) Rolling velocities of 10 leukocytes were determined in the same 4 fields of view as the number of rolling cells. Values are reported as mean ± S.E.M.
Mentions: For wild type leukocytes, we observed 176±15 rolling cells/mm2, whereas for Tpst DKO→B6 leukocytes we observed only 97±6 rolling cells/mm2 (n = 12 fields from 3 independent paired experiments) (Fig. 3A). Statistical analysis showed that the number of rolling cells in the Tpst DKO→B6 group was significantly lower than the WT group (p<0.0001, d = 2.1).

Bottom Line: We observed that rolling flux fractions were lower and leukocyte rolling velocities were higher in Tpst DKO → B6 venules compared to WT → B6 venules.Similar results were observed on immobilized P-selectin in vitro.Finally, Tpst DKO leukocytes bound less P-selectin than wild type leukocytes despite equivalent surface expression of Psgl-1.

View Article: PubMed Central - PubMed

Affiliation: Oklahoma Medical Research Foundation, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America. Andrew-Westmuckett@omrf.org

ABSTRACT

Background: We recently demonstrated that tyrosine sulfation is an important contributor to monocyte recruitment and retention in a mouse model of atherosclerosis. P-selectin glycoprotein ligand-1 (Psgl-1) is tyrosine-sulfated in mouse monocyte/macrophages and its interaction with P-selectin is important in monocyte recruitment in atherosclerosis. However, whether tyrosine sulfation is required for the P-selectin binding function of mouse Psgl-1 is unknown. Here we test the function of native Psgl-1 expressed in leukocytes lacking endogenous tyrosylprotein sulfotransferase (TPST) activity.

Methodology/principal findings: Psgl-1 function was assessed by examining P-selectin dependent leukocyte rolling in post-capillary venules of C57BL6 mice transplanted with hematopoietic progenitors from wild type (WT → B6) or Tpst1;Tpst2 double knockout mice (Tpst DKO → B6) which lack TPST activity. We observed that rolling flux fractions were lower and leukocyte rolling velocities were higher in Tpst DKO → B6 venules compared to WT → B6 venules. Similar results were observed on immobilized P-selectin in vitro. Finally, Tpst DKO leukocytes bound less P-selectin than wild type leukocytes despite equivalent surface expression of Psgl-1.

Conclusions/significance: These findings provide direct and convincing evidence that tyrosine sulfation is required for optimal function of mouse Psgl-1 in vivo and suggests that tyrosine sulfation of Psgl-1 contributes to the development of atherosclerosis.

Show MeSH
Related in: MedlinePlus