Differential regulation and function of CD73, a glycosyl-phosphatidylinositol-linked 70-kD adhesion molecule, on lymphocytes and endothelial cells

Abstract

CD73, otherwise known as ecto-5'-nucleotidase, is a glycosyl-phosphatidylinositol-linked 70-kD molecule expressed on different cell types, including vascular endothelial cells (EC) and certain subtypes of lymphocytes. There is strong evidence for lymphocyte CD73 having a role in several immunological phenomena such as lymphocyte activation, proliferation, and adhesion to endothelium, but the physiological role of CD73 in other cell types is less clear. To compare the biological characteristics of CD73 in different cell types, we have studied the structure, function, and surface modulation of CD73 on lymphocytes and EC. CD73 molecules on lymphocytes are shed from the cell surface as a consequence of triggering with an anti-CD73 mAb, mimicking ligand binding. In contrast, triggering of endothelial CD73 does not have any effect on its expression. Lymphocyte CD73 is susceptible to phosphatidylinositol phospholipase, whereas only a small portion of CD73 on EC could be removed by this enzyme. Furthermore, CD73 on EC was unable to deliver a tyrosine phosphorylation inducing signal upon mAb triggering, whereas triggering of lymphocyte CD73 can induce tyrosine phosphorylation. Despite the functional differences, CD73 molecules on lymphocytes and EC were practically identical structurally, when studied at the protein, mRNA, and cDNA level. Thus, CD73 is an interesting example of a molecule which lacks structural variants but yet has a wide diversity of biological functions. We suggest that the ligand-induced shedding of lymphocyte CD73 represents an important and novel means of controlling lymphocyte-EC interactions.

Figure 1

1E9 and 4G4 mAbs recognize distinct epitopes of the CD73 molecule. 4G4 is a mouse IgG₁, and 1E9 is a mouse IgG₃ mAb. (a) FACS^® staining of HEC cells using 1E9 and FITC anti– mouse IgG₁ (anti–mouse IgG₁ faintly cross-reacts with IgG₃ causing the low positivity). (b) FACS^® staining of HEC cells using 4G4 and FITC anti–mouse IgG₁. (c) HEC cells were sequentially treated with 1E9 mAb, 4G4 mAb, and FITC anti–mouse IgG₁. (d) FACS^® staining of HEC cells using 4G4 mAb and FITC anti– mouse IgG₃. (e) FACS^® staining of HEC cells using 1E9 mAb and FITC anti–mouse IgG₃. (f) HEC cells were sequentially treated with 4G4 mAb, 1E9 mAb, and FITC anti–mouse IgG₃. Percentage of positive cells and the mean fluorescence intensity are indicated in the upper right hand corner of each panel.

Figure 2

Triggering of CD73 at 37°C results in CD73 downregulation in lymphocytes but not in EC. (A) Lymphocytes, HECs, and HUVECs were incubated for 2 h in presence of an antiCD73 mAb 4G4, anti-CD73 mAb 1E9, or a negative control mAb 3G6 at 37°C, and the expression of CD73 on the cell surface was analyzed by IF and FACS^®. The arrows in the middle and bottom panels point at the CD73 positive subpopulation of PBL. (B) Downregulation of CD73 on lymphocyte surface is temperature dependent. CD73 expression was analyzed by IF staining and FACS^® after incubating lymphocytes and HECs in presence of an anti-CD73 (◆̶ , continuous line) or in presence of a negative control mAb (○̶ , dashed line) for various time periods at 37° or 4°C. Percentage of positive cells is shown in the graphs on the left, and the MFI of positive cells is shown in the graphs on the right.

Figure 2

Triggering of CD73 at 37°C results in CD73 downregulation in lymphocytes but not in EC. (A) Lymphocytes, HECs, and HUVECs were incubated for 2 h in presence of an antiCD73 mAb 4G4, anti-CD73 mAb 1E9, or a negative control mAb 3G6 at 37°C, and the expression of CD73 on the cell surface was analyzed by IF and FACS^®. The arrows in the middle and bottom panels point at the CD73 positive subpopulation of PBL. (B) Downregulation of CD73 on lymphocyte surface is temperature dependent. CD73 expression was analyzed by IF staining and FACS^® after incubating lymphocytes and HECs in presence of an anti-CD73 (◆̶ , continuous line) or in presence of a negative control mAb (○̶ , dashed line) for various time periods at 37° or 4°C. Percentage of positive cells is shown in the graphs on the left, and the MFI of positive cells is shown in the graphs on the right.

Figure 3

Downregulated CD73 is rapidly replaced by new CD73 molecules. (A) Lymphocytes, HECs, and HUVECs in suspension were preincubated with negative control mAb, anti-CD73 mAb 4G4, or anti-CD73 mAb 1E9, at 4°C; washed twice and incubated in complete medium at 37°C for 15 min; and then stained with either anti-CD73 mAb and a second stage FITC-conjugated Ab, or only with a second stage Ab. The arrows in the middle and bottom panels point at the CD73 positive subpopulation of PBL. (B) FACScan^® analysis of lymphocytes and HECs after a saturating preincubation on ice with an anti-CD73 mAb and a subsequent incubation in complete medium at 37°C for various periods of time. After incubation, cells were either labeled with both anti-CD73 mAb and a second stage, FITC-conjugated Ab (◆̶ , continuous line) or only with a second stage, FITC-conjugated Ab (○̶ , dashed line). MFI is measured for all cells. (C) CD73 can be detected from the lymphocyte supernatant but not from the EC supernatant. Lymphocytes, HECs, and HUVECs were incubated with the anti-CD73 mAb as described above, and the cell lysate and the incubation supernatant were analyzed for presence of CD73 using either both a primary and a secondary Ab or only a secondary Ab in a dot blot assay, as described in Materials and Methods. The three spots in each panel represent different numbers of cells. For lymphocytes: left spot, 1 × 10⁶ cells, middle spot, 0.5 × 10⁶ cells, and right spot, 0.25 × 10⁶ cells except that the right spot of the cell lysate detected with both first and second stage Abs has 1.5 × 10⁶ cells. For EC: left spot, 0.9 × 10⁶ cells, middle spot, 0.6 × 10⁶ cells, and right spot, 0.3 × 10⁶ cells.

Figure 3

Downregulated CD73 is rapidly replaced by new CD73 molecules. (A) Lymphocytes, HECs, and HUVECs in suspension were preincubated with negative control mAb, anti-CD73 mAb 4G4, or anti-CD73 mAb 1E9, at 4°C; washed twice and incubated in complete medium at 37°C for 15 min; and then stained with either anti-CD73 mAb and a second stage FITC-conjugated Ab, or only with a second stage Ab. The arrows in the middle and bottom panels point at the CD73 positive subpopulation of PBL. (B) FACScan^® analysis of lymphocytes and HECs after a saturating preincubation on ice with an anti-CD73 mAb and a subsequent incubation in complete medium at 37°C for various periods of time. After incubation, cells were either labeled with both anti-CD73 mAb and a second stage, FITC-conjugated Ab (◆̶ , continuous line) or only with a second stage, FITC-conjugated Ab (○̶ , dashed line). MFI is measured for all cells. (C) CD73 can be detected from the lymphocyte supernatant but not from the EC supernatant. Lymphocytes, HECs, and HUVECs were incubated with the anti-CD73 mAb as described above, and the cell lysate and the incubation supernatant were analyzed for presence of CD73 using either both a primary and a secondary Ab or only a secondary Ab in a dot blot assay, as described in Materials and Methods. The three spots in each panel represent different numbers of cells. For lymphocytes: left spot, 1 × 10⁶ cells, middle spot, 0.5 × 10⁶ cells, and right spot, 0.25 × 10⁶ cells except that the right spot of the cell lysate detected with both first and second stage Abs has 1.5 × 10⁶ cells. For EC: left spot, 0.9 × 10⁶ cells, middle spot, 0.6 × 10⁶ cells, and right spot, 0.3 × 10⁶ cells.

Figure 3

Downregulated CD73 is rapidly replaced by new CD73 molecules. (A) Lymphocytes, HECs, and HUVECs in suspension were preincubated with negative control mAb, anti-CD73 mAb 4G4, or anti-CD73 mAb 1E9, at 4°C; washed twice and incubated in complete medium at 37°C for 15 min; and then stained with either anti-CD73 mAb and a second stage FITC-conjugated Ab, or only with a second stage Ab. The arrows in the middle and bottom panels point at the CD73 positive subpopulation of PBL. (B) FACScan^® analysis of lymphocytes and HECs after a saturating preincubation on ice with an anti-CD73 mAb and a subsequent incubation in complete medium at 37°C for various periods of time. After incubation, cells were either labeled with both anti-CD73 mAb and a second stage, FITC-conjugated Ab (◆̶ , continuous line) or only with a second stage, FITC-conjugated Ab (○̶ , dashed line). MFI is measured for all cells. (C) CD73 can be detected from the lymphocyte supernatant but not from the EC supernatant. Lymphocytes, HECs, and HUVECs were incubated with the anti-CD73 mAb as described above, and the cell lysate and the incubation supernatant were analyzed for presence of CD73 using either both a primary and a secondary Ab or only a secondary Ab in a dot blot assay, as described in Materials and Methods. The three spots in each panel represent different numbers of cells. For lymphocytes: left spot, 1 × 10⁶ cells, middle spot, 0.5 × 10⁶ cells, and right spot, 0.25 × 10⁶ cells except that the right spot of the cell lysate detected with both first and second stage Abs has 1.5 × 10⁶ cells. For EC: left spot, 0.9 × 10⁶ cells, middle spot, 0.6 × 10⁶ cells, and right spot, 0.3 × 10⁶ cells.

Figure 4

CD73 is not detectable in permeabilized lymphocytes after mAb 4G4 treatment at 37°C. FACS^® analysis was performed with permeabilized and non-permeabilized lymphocytes after 2 h incubation at 37°C in presence of either anti-CD73 mAb 4G4 or negative control mAb. The arrows in the middle panel point at the CD73-positive subpopulation of PBL.

Figure 5

Cross-linking of CD73 results in different staining patterns on lymphocyte and endothelial surfaces. Lymphocytes and EC were stained with mAb 4G4 and a second stage FITC-conjugated Ab, as described in Materials and Methods. (a) Patching of CD73 on lymphocyte surface after cross-linking at 4°C. (b) At 37°C, the majority of lymphocytes becomes CD73 negative due to CD73 cross-linking, and the remaining CD73-reactivity is found in large patches (arrow). (c) Capping of CD73 on EC after cross-linking at 4°C (arrow). (d) Capping of CD73 on EC after cross-linking at 37°C (arrows). (e) Punctate staining pattern of adherent HEC cells stained with antiCD73 mAb at 4°C. (f) Punctate staining pattern of adherent HEC cells stained with anti-CD73 mAb at 37°C. (g) Adherent HEC cells stained with a negative control mAb 3G6 at 37°C. In a, b, c and d, cells were stained in suspension and analyzed microscopically on cytospin preparations. Bar, 10 μm.

Figure 6

Molecular mass of CD73 from lymphocytes and EC. (A) ¹²⁵I-labeled HECs were lysed, and immunoprecipitations were performed with a negative control mAb (lane 1), mAb 4G4 (lane 2, arrow), and mAb Hermes-3, which recognizes the CD44 molecule (lane 3) as described in Materials and Methods. (B) Lysates from freshly isolated ¹²⁵I-labeled PBL were immunoprecipitated with a negative control mAb (lane 1), mAb 4G4 (lane 2, arrow), and mAb Hermes-3. No significant size differences were observed between CD73 molecules from lymphocytes and EC.

Figure 7

Northern blot analysis of lymphocyte and endothelial CD73 mRNA. PolyA⁺ RNA (10 μg/lane), prepared from human PBL and from cultured HECs, was denatured and separated by electrophoresis on a 1.1% agarose gel. The RNA was blotted onto a Hybond N membrane and hybridized with a ³²P-labeled CD73 cDNA probe. Posthybridization wash conditions were 0.1× SSC, 0.1% SDS at 65°C for 1 h. SSC is 0.15 M NaCl, 0.015 M Na citrate. The signal detected from lymphocyte RNA was several times stronger than that from HEC RNA, and thus, the exposure time for the PBL blot is 12 h and the exposure time for the HEC blot is two weeks.

Figure 8

PI-PLC treatment of HEC cells. HEC cells in suspension were treated with PI-PLC and analyzed by immunofluorescence staining and FACS^® for the expression of CD73. Negative control staining was performed using a class-matched, nonbinding mAb as the first stage antibody.

Figure 9

Characterization of function of endothelial CD73 molecule. (A) Endothelial CD73 has an intact ecto-5′-NT activity that is inhibitable by anti-CD73 Ab but not with a negative control mAb. Conversion of [¹⁴C]AMP to [¹⁴C]adenosine and [¹⁴C]inosine by endothelial ecto-5′-NT was quantitated, as described in Materials and Methods. The results shown are the mean of duplicates from two independent experiments ± SEM. (B) mAb triggering of CD73 on lymphocyte surface causes tyrosine phosphorylation of certain protein substrates. PBL enriched for CD73⁺ cells were treated with a negative control, 1E9 and 4G4 mAb's for different periods of time, and lysed. Tyrosine phosphorylation was detected from the lysates by tyrosine blotting, as described in the Materials and Methods. Arrows point at the 26- and 28-kD tyrosine-phosphorylated products induced by anti-CD73 triggering. (C) mAb triggering of CD73 on the endothelial surface does not cause tyrosine phosphorylation. Adherent HECs were triggered with either an anti-CD73 mAb or a negative control mAb for 15, 30, or 45 min, cells were lysed, and tyrosine phosphorylation of intracellular proteins was detected from the lysates by tyrosine blotting, as described in Materials and Methods.

Figure 9

Characterization of function of endothelial CD73 molecule. (A) Endothelial CD73 has an intact ecto-5′-NT activity that is inhibitable by anti-CD73 Ab but not with a negative control mAb. Conversion of [¹⁴C]AMP to [¹⁴C]adenosine and [¹⁴C]inosine by endothelial ecto-5′-NT was quantitated, as described in Materials and Methods. The results shown are the mean of duplicates from two independent experiments ± SEM. (B) mAb triggering of CD73 on lymphocyte surface causes tyrosine phosphorylation of certain protein substrates. PBL enriched for CD73⁺ cells were treated with a negative control, 1E9 and 4G4 mAb's for different periods of time, and lysed. Tyrosine phosphorylation was detected from the lysates by tyrosine blotting, as described in the Materials and Methods. Arrows point at the 26- and 28-kD tyrosine-phosphorylated products induced by anti-CD73 triggering. (C) mAb triggering of CD73 on the endothelial surface does not cause tyrosine phosphorylation. Adherent HECs were triggered with either an anti-CD73 mAb or a negative control mAb for 15, 30, or 45 min, cells were lysed, and tyrosine phosphorylation of intracellular proteins was detected from the lysates by tyrosine blotting, as described in Materials and Methods.

Figure 9

Characterization of function of endothelial CD73 molecule. (A) Endothelial CD73 has an intact ecto-5′-NT activity that is inhibitable by anti-CD73 Ab but not with a negative control mAb. Conversion of [¹⁴C]AMP to [¹⁴C]adenosine and [¹⁴C]inosine by endothelial ecto-5′-NT was quantitated, as described in Materials and Methods. The results shown are the mean of duplicates from two independent experiments ± SEM. (B) mAb triggering of CD73 on lymphocyte surface causes tyrosine phosphorylation of certain protein substrates. PBL enriched for CD73⁺ cells were treated with a negative control, 1E9 and 4G4 mAb's for different periods of time, and lysed. Tyrosine phosphorylation was detected from the lysates by tyrosine blotting, as described in the Materials and Methods. Arrows point at the 26- and 28-kD tyrosine-phosphorylated products induced by anti-CD73 triggering. (C) mAb triggering of CD73 on the endothelial surface does not cause tyrosine phosphorylation. Adherent HECs were triggered with either an anti-CD73 mAb or a negative control mAb for 15, 30, or 45 min, cells were lysed, and tyrosine phosphorylation of intracellular proteins was detected from the lysates by tyrosine blotting, as described in Materials and Methods.