Protein Tyrosine Phosphatase Gamma (PTPgamma) is a Novel Leukocyte Marker Highly Expressed by CD34 Precursors

Abstract

Protein Tyrosine Phosphatase gamma (PTPgamma) is a receptor-like transmembrane protein belonging to the family of classical protein tyrosine phosphatases. PTPgamma is known to regulate haematopoietic differentiation in a murine embryonic stem cells model. We have recently demonstrated that PTPgamma mRNA is expressed in monocytes, tissue-localized myeloid dendritic cells and in both myeloid and plasmacytoid dendritic cells in peripheral blood. We now developed a PTPgamma specific antibody that recognizes the protein by flow cytometry. PTPgamma expression was detected in monocytes and both myeloid and plasmacytoid dendritic cells, while PMN showed a low but consistent staining in contrast with previous mRNA data. B cells were found to express the phosphatase while T cells were negative. In keeping with RNA data, PTPgamma was detected in monocyte-derived dendritic cells and its expression rose upon LPS stimulation. Finally, we discovered that CD34(+) haematopoietic precursors express high PTPgamma level that drops during in vitro expansion induced by IL-3 and SCF growth factors. We therefore propose PTPgamma as a new functionally regulated leukocyte marker whose role in normal and pathological context deserve further investigation.

Keywords: Egg yolk immunoglobulin (IgY); fluorescence activated cell sorting (FACS); haematopoietic progenitors; human protein tyrosine phosphatase gamma (PTPγ).

Figure 1

A.) Western blot analysis of conditioned media with chPTPγ antibody: Nctr = medium from 293 cells transfected with empty vector, PTPγ = medium from 293 cells transfected with PTPγ extra cellular domain cDNA, producing and secreting a soluble 120 KD protein. B) Northern blot analysis of haemopoietic cell lines for PTPγ; PTPζ is the other member of subtype V receptor type tyrosine phosphatase and is used as a negative control; β-actin is showed for total RNA estimation. As a positive control for PTPζ hybridization we utilized human brain RNA on the same blot (data not shown). C) FACS analysis of K562, K562 transfected with PTPγ full-length cDNA (K562 γ1) and U937 cell lines for PTPγ using chPTPγ antibody. Isotype control staining is showed in gray.

Figure 2

FACS analysis of peripheral blood PMN (CD15⁺; CD14⁻), T lymphocytes (CD3⁺), B lymphocytes (CD19⁺), monocytes (CD14⁺), myeloid (CD1c⁺/CD19⁻) and plasmacytoid (CD303⁺) DC using chPTPγ antibody. Isotype control is shown in gray, one representative experiment of eight from individual donors is shown.

Figure 3

A) FACS analysis of immature (upper) or LPS-matured (lower) monocyte-derived DC using chPTPγ antibody; isotype control is shown in gray. B) double staining of purified peripheral blood precursors (upper) at the time of separation and after three days of culture in the presence of SCF and IL-3 (lower). In red: cells treated with anti-CD34 and chPTPγ; in black: cells treated with anti-CD34 and chicken isotype IgY. One representative experiment of four is shown. Each experiment involved purification, differentiation (for DC) and staining of cells from individual donors.