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P-selectin glycoprotein ligand-1 (PSGL-1) on T helper 1 but not on T helper 2 cells binds to P-selectin and supports migration into inflamed skin.

Borges E, Tietz W, Steegmaier M, Moll T, Hallmann R, Hamann A, Vestweber D - J. Exp. Med. (1997)

Bottom Line: PSGL-1 on Th2 cells, although expressed at similar levels as on Th1 cells, did not support binding to P-selectin.Thus, the P-selectin-binding form of PSGL-1 distinguishes Th1 cells from Th2 cells.Furthermore, PSGL-1 is relevant for the entry of Th1 cells into inflamed areas of the skin.

View Article: PubMed Central - PubMed

Affiliation: Institut für Zellbiologie, ZMBE, Universität Münster, Germany.

ABSTRACT
We have shown recently that mouse Th1 cells but not Th2 cells are selectively recruited into inflamed sites of a delayed-type hypersensitivity (DTH) reaction of the skin. This migration was blocked by monoclonal antibodies (mAb) against P- and E-selectin. Here we show that Th1 cells bind to P-selectin via the P-selectin glycoprotein ligand-1 (PSGL-1). This is the only glycoprotein ligand that was detectable by affinity isolation with a P-selectin-Ig fusion protein. Binding of Th1 cells to P-selectin, as analyzed by flow cytometry and in cell adhesion assays, was completely blocked by antibodies against PSGL-1. The same antibodies blocked partially the migration of Th1 cells into cutaneous DTH reactions. This blocking activity, in combination with that of a mAb against E-selectin, was additive. PSGL-1 on Th2 cells, although expressed at similar levels as on Th1 cells, did not support binding to P-selectin. Thus, the P-selectin-binding form of PSGL-1 distinguishes Th1 cells from Th2 cells. Furthermore, PSGL-1 is relevant for the entry of Th1 cells into inflamed areas of the skin. This is the first demonstration for the importance of PSGL-1 for mouse leukocyte recruitment in vivo.

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PSGL-1 from Th1 cells but not from Th2 cells can be affinity  isolated by P-selectin–IgG. Equal numbers of Th1 or Th2 cells were surface biotinylated, and detergent extracts were incubated with either immobilized human Ig as control (Co), P-selectin–IgG (P), or affinity-purified antibodies against PSGL-1 (α). Specifically bound proteins were  either directly electrophoresed (lanes 1–5 and 8) or subjected to a reprecipitation (as indicated) with affinity-purified anti–PSGL-1 rabbit antibodies (lane 6) or immobilized nonimmune rabbit antibodies (lane 7).  Isolated proteins were separated on 6% polyacrylamide gels under reducing conditions. The material in lanes 6 and 7 corresponds to twice as  many cells as used for lanes 1–5 and 8. The front of the gel is marked by an  arrow on the left. Molecular mass markers (in kD) are indicated on the left.
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Figure 1: PSGL-1 from Th1 cells but not from Th2 cells can be affinity isolated by P-selectin–IgG. Equal numbers of Th1 or Th2 cells were surface biotinylated, and detergent extracts were incubated with either immobilized human Ig as control (Co), P-selectin–IgG (P), or affinity-purified antibodies against PSGL-1 (α). Specifically bound proteins were either directly electrophoresed (lanes 1–5 and 8) or subjected to a reprecipitation (as indicated) with affinity-purified anti–PSGL-1 rabbit antibodies (lane 6) or immobilized nonimmune rabbit antibodies (lane 7). Isolated proteins were separated on 6% polyacrylamide gels under reducing conditions. The material in lanes 6 and 7 corresponds to twice as many cells as used for lanes 1–5 and 8. The front of the gel is marked by an arrow on the left. Molecular mass markers (in kD) are indicated on the left.

Mentions: We have recently shown that Th1 cells but not Th2 cells efficiently enter sites of acute inflammation in the skin (19). This migration was blocked by a combination of antibodies against P- and E-selectin (19). Based on these findings, we have now examined which molecules on Th1 cells would function as ligands for P-selectin in this process. We first analyzed which glycoproteins on the surface of Th1 cells bind to P-selectin. To this end, equal numbers of Th1 and Th2 cells were surface biotinylated and subjected to affinity isolation experiments with a P-selectin–Ig fusion protein. As shown in Fig. 1 (lane 5), two proteins of 230 and 130 kD were isolated with P-selectin–Ig that could be reprecipitated with affinity-purified antibodies of the rabbit antiserum 124 against an amino-terminal peptide of mouse PSGL-1 (lane 6). The 230-kD form is the not completely reduced dimeric form of the 130-kD PSGL-1. No selectin ligands could be isolated with the P-selectin probe from Th2 cells (lane 2), although the PSGL-1 protein was detectable with antibodies (lane 3) at similar levels as on Th1 cells (lanes 3 and 4). Interestingly, the 130-kD monomeric form of PSGL-1 on Th2 cells was slightly smaller than the one on Th1 cells (compare lane 3 with 5). Whereas antibodies against PSGL-1 precipitated a broader band from Th1 cells (lane 4), including a slightly smaller molecular species of the size as in Th2 cells, P-selectin–Ig only precipitated the higher molecular weight species. We conclude that only Th1 cells express a modified form of PSGL-1 that is capable of binding to P-selectin. Furthermore, PSGL-1 was the only glycoprotein ligand on Th1 cells that could be detected by this technique.


P-selectin glycoprotein ligand-1 (PSGL-1) on T helper 1 but not on T helper 2 cells binds to P-selectin and supports migration into inflamed skin.

Borges E, Tietz W, Steegmaier M, Moll T, Hallmann R, Hamann A, Vestweber D - J. Exp. Med. (1997)

PSGL-1 from Th1 cells but not from Th2 cells can be affinity  isolated by P-selectin–IgG. Equal numbers of Th1 or Th2 cells were surface biotinylated, and detergent extracts were incubated with either immobilized human Ig as control (Co), P-selectin–IgG (P), or affinity-purified antibodies against PSGL-1 (α). Specifically bound proteins were  either directly electrophoresed (lanes 1–5 and 8) or subjected to a reprecipitation (as indicated) with affinity-purified anti–PSGL-1 rabbit antibodies (lane 6) or immobilized nonimmune rabbit antibodies (lane 7).  Isolated proteins were separated on 6% polyacrylamide gels under reducing conditions. The material in lanes 6 and 7 corresponds to twice as  many cells as used for lanes 1–5 and 8. The front of the gel is marked by an  arrow on the left. Molecular mass markers (in kD) are indicated on the left.
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Related In: Results  -  Collection

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Figure 1: PSGL-1 from Th1 cells but not from Th2 cells can be affinity isolated by P-selectin–IgG. Equal numbers of Th1 or Th2 cells were surface biotinylated, and detergent extracts were incubated with either immobilized human Ig as control (Co), P-selectin–IgG (P), or affinity-purified antibodies against PSGL-1 (α). Specifically bound proteins were either directly electrophoresed (lanes 1–5 and 8) or subjected to a reprecipitation (as indicated) with affinity-purified anti–PSGL-1 rabbit antibodies (lane 6) or immobilized nonimmune rabbit antibodies (lane 7). Isolated proteins were separated on 6% polyacrylamide gels under reducing conditions. The material in lanes 6 and 7 corresponds to twice as many cells as used for lanes 1–5 and 8. The front of the gel is marked by an arrow on the left. Molecular mass markers (in kD) are indicated on the left.
Mentions: We have recently shown that Th1 cells but not Th2 cells efficiently enter sites of acute inflammation in the skin (19). This migration was blocked by a combination of antibodies against P- and E-selectin (19). Based on these findings, we have now examined which molecules on Th1 cells would function as ligands for P-selectin in this process. We first analyzed which glycoproteins on the surface of Th1 cells bind to P-selectin. To this end, equal numbers of Th1 and Th2 cells were surface biotinylated and subjected to affinity isolation experiments with a P-selectin–Ig fusion protein. As shown in Fig. 1 (lane 5), two proteins of 230 and 130 kD were isolated with P-selectin–Ig that could be reprecipitated with affinity-purified antibodies of the rabbit antiserum 124 against an amino-terminal peptide of mouse PSGL-1 (lane 6). The 230-kD form is the not completely reduced dimeric form of the 130-kD PSGL-1. No selectin ligands could be isolated with the P-selectin probe from Th2 cells (lane 2), although the PSGL-1 protein was detectable with antibodies (lane 3) at similar levels as on Th1 cells (lanes 3 and 4). Interestingly, the 130-kD monomeric form of PSGL-1 on Th2 cells was slightly smaller than the one on Th1 cells (compare lane 3 with 5). Whereas antibodies against PSGL-1 precipitated a broader band from Th1 cells (lane 4), including a slightly smaller molecular species of the size as in Th2 cells, P-selectin–Ig only precipitated the higher molecular weight species. We conclude that only Th1 cells express a modified form of PSGL-1 that is capable of binding to P-selectin. Furthermore, PSGL-1 was the only glycoprotein ligand on Th1 cells that could be detected by this technique.

Bottom Line: PSGL-1 on Th2 cells, although expressed at similar levels as on Th1 cells, did not support binding to P-selectin.Thus, the P-selectin-binding form of PSGL-1 distinguishes Th1 cells from Th2 cells.Furthermore, PSGL-1 is relevant for the entry of Th1 cells into inflamed areas of the skin.

View Article: PubMed Central - PubMed

Affiliation: Institut für Zellbiologie, ZMBE, Universität Münster, Germany.

ABSTRACT
We have shown recently that mouse Th1 cells but not Th2 cells are selectively recruited into inflamed sites of a delayed-type hypersensitivity (DTH) reaction of the skin. This migration was blocked by monoclonal antibodies (mAb) against P- and E-selectin. Here we show that Th1 cells bind to P-selectin via the P-selectin glycoprotein ligand-1 (PSGL-1). This is the only glycoprotein ligand that was detectable by affinity isolation with a P-selectin-Ig fusion protein. Binding of Th1 cells to P-selectin, as analyzed by flow cytometry and in cell adhesion assays, was completely blocked by antibodies against PSGL-1. The same antibodies blocked partially the migration of Th1 cells into cutaneous DTH reactions. This blocking activity, in combination with that of a mAb against E-selectin, was additive. PSGL-1 on Th2 cells, although expressed at similar levels as on Th1 cells, did not support binding to P-selectin. Thus, the P-selectin-binding form of PSGL-1 distinguishes Th1 cells from Th2 cells. Furthermore, PSGL-1 is relevant for the entry of Th1 cells into inflamed areas of the skin. This is the first demonstration for the importance of PSGL-1 for mouse leukocyte recruitment in vivo.

Show MeSH
Related in: MedlinePlus