Phosphatidylserine as an anchor for plasminogen and its plasminogen receptor, histone H2B, to the macrophage surface

Abstract

Background: Plasminogen (Plg) binding to cell surface Plg receptors (Plg-Rs) on the surface of macrophages facilitates Plg activation and migration of these cells. Histone H2B (H2B) acts as a Plg-R and its cell surface expression is up-regulated when monocytes are differentiated to macrophages via a pathway dependent on L-type Ca(2+) channels and intracellular Ca(2+).

Objectives: We sought to investigate the mechanism by which H2B, a protein without a transmembrane domain, is retained on the macrophage surface.

Methods: THP-1 monocytoid cells were induced to differentiate with interferon gamma + Vitamin D3 or to undergo apoptosis by treatment with camptothecin. Flow cytometry and cell surface biotinylation followed by Western blotting were used to measure the interrelationship between Plg binding, cell surface expression of H2B and outer membrane exposure of phosphatidylserine (PS).

Results: H2B interacted directly with PS via an electrostatic interaction. Anti-PS or PS binding proteins, annexin V and protein S, diminished H2B interaction with PS on the surface of differentiated or apoptotic cells and these same reagents inhibited Plg binding to these cells. L-type Ca(2+) channels played a significant role in PS exposure, H2B surface expression and Plg binding induced either by differentiation or apoptosis.

Conclusions: These data suggest that H2B tethers to the surface of cells by interacting with PS on differentiated or apoptotic monocytoid cells. L-type Ca(2+) channels regulate PS exposure on the surface of these cells. The exposed PS interacts directly with H2B and hence provides sites for Plg to bind to.

Fig. 1

Binding of human H2B to phosphatidylserine (PS). (A) Recombinant H2B was allowed to bind to microtiter wells coated with BSA, PS, PC or DNA. Recombinant H2B (0.4–5 μg mL⁻¹) was added to the wells for 2 min at 22 °C. After washing thoroughly with PBS, anti-H2B, followed by Alexa-488 anti-rabbit IgG, was added to detect bound H2B. H2B bound to DNA (positive control) and PS but not to BSA or PC. *P<0.001 for PS and DNA vs. BSA as analyzed by a one-way ANOVA test. (B) Binding of H2B (5 μg mL⁻¹) to immobilized PS was inhibited by KCl, indicative of an electrostatic interaction. Data are the means ± SD from triplicates. (C) H2B or α-enolase was added to BSA or PS-coated wells, and anti-H2B or anti-enolase was used for detection. Binding anti-enolase to enolase-coated wells is a control to validate the detection system. *P = 0.01 for PS vs. BSA. #P = 0.02 for enolase vs. BSA as analyzed by Student’s t-test.

Fig. 2

H2B binds to the macrophage surface via PS and this interaction contributes to plasmin generation. (A) Binding of Alexa-488 Plg as detected by FACS to THP-1 cells induced to differentiated by IFNγ+ VD3 (48 h). The cells were either untreated or treated with KCl for 5 min and washed before addition of the labeled Plg.KCl reduced Plg binding to differentiated THP-1 cells compared with unwashed cells (*P ≤ 0.001, by a one-way ANOVA). (B) Effect of KCl washes (5 min) on H2B surface expression. After washing, the cells were biotinylated, and the biotinylated proteins were subjected to Western blotting (left panels). Intensities of the Western blot bands were measured by densitometry and expressed as the fold-increase in H2B, α-enolase and CD14 relative to their levels on non-stimulated THP-1 cells (right panel). Data are the means ± SD from triplicate blots. H2B, striated bars; α-enolase, gray bars; CD14, open bars. *P ≤0.002 vs. cell surface H2B on IFNγ+ VD3-treated cells as analyzed by Student’s t-test. KCl treatment eluted surface H2B but did not alter surface expression of α-enolase and CD14. (C) THP-1 cells were differentiated with IFNγ+ VD3 in the absence or presence of either anti-PS (50 μg mL⁻¹) or its isotype control for 48 h. Cells were subjected to FACS staining by anti-H2B followed by Alexa-488 anti-rabbit IgG. Specific mean fluorescence intensity (MFI) values were quantified by subtracting the binding obtained with secondary antibody alone. Data are means ± SD from triplicate experiments. Anti-PS reduced H2B surface expression on the IFNγ+ VD3-treated cells compared with IFNγ+ VD3 and isotype control-treated cells (*P < 0.001 by ANOVA). The isotype control has no inhibitory effect on IFNγ+ VD3-mediated H2B surface expression. (D) THP-1 cells were treated with IFNγ+ VD3 in the absence or presence of anti-PS antibody (50 μg mL⁻¹) and its isotype control (IgM). Cells were incubated with Plg (200 nM) for 1 h and then uPA(3 nM) was added with the chromogenic substrate S-2251. Plasmin generation was measured at 405 nm over 2 h. Data are representative of three experiments. Note the 60% reduction of plasmin generation at 120 min in anti-PS-treated cells compared with isotype control-treated cells.

Fig. 3

Inhibition of cell surface H2B expression and Plg binding by annexin V and protein S. (A) THP-1 cells were differentiated with IFNγ+ VD3 in the absence or presence of various concentrations of annexin V or protein S for 48 h. Cell surface biotinylation followed by Western blot of biotinylated proteins with anti-H2B indicated a dose-dependent inhibition of H2B localization on the cell surface. Annexin V and protein S did not alter differentiation-induced α-enolase and CD14 cell surface localization. Intensities of the Western blot bands of H2B, α-enolase and CD14 are expressed as the fold increase compared with non-stimulated THP-1 cells (lower panel). Bar represents means ± SD from three independent Western blots; H2B, striated bars; α-enolase, gray bars; CD14, open bars. (B) THP-1 cells were treated with IFNγ+ VD3 in the presence or absence of annexin V or protein S, and Alexa-488 Plg binding was analyzed by FACS. Data are presented as means ± SD of four independent experiments. Differentiation-induced Plg binding was inhibited by annexin V and protein S.*P < 0.001 for IFNγ+ VD3 vs. untreated cells, **P < 0.001 for annexin V vs. IFNγ+ VD3 alone treated cells and ***P ≤ 0.001 for protein S vs. IFNγ+ VD3 alone treated cells as analyzed by ANOVA.

Fig. 4

PS exposure, Plg binding and H2B surface expression induced by differentiation and apoptosis. THP-1 cells were pretreated with zVAD-fmk, amlodipine and verapamil for 1 h or were untreated. The cells were either differentiated with IFNγ+ VD3 for 48 h or induced to apoptosis with camptothecin for 24 h and then labeled with either FITC-annexin V to detect PS exposure (A), Alexa-488 Plg (B) or anti-H2B followed by Alexa-488 anti-rabbit IgG (C) and analyzed by FACS. Data are presented as fold change over untreated THP-1 cells from three independent experiments. *P < 0.001 vs. untreated cell; #P ≤0.001 vs. camptothecin-treated cells as analyzed by a one-way ANOVA test.

Fig. 5

Effect of the L-type calcium channel blockers, amlodipine and verapamil, on PS exposure, Plg binding and H2B surface expression. THP-1 cells were either pretreated with amlodipine or verapamil for 1 h or were untreated. The cells were then differentiated with IFNγ + VD3 for 48 h or induced to apoptosis with camptothecin for 24 h. Cells were labeled with FITC-annexin V (A), Alexa-488 Plg (B) or anti-H2B followed by Alexa-488 anti-rabbit IgG (C) and analyzed by FACS. Data are presented as fold change over untreated THP-1 cells from three independent experiments. *P ≤0.001 vs. untreated cells; #P ≤ 0.001 vs. IFNγ+ VD3 alone treated cells; **P ≤ 0.001 vs. camptothecin alone treated cells as analyzed by ANOVA test.

Fig. 6

Phosphatidylserine exposure, Plg binding and H2B surface expression on human peripheral blood monocytes induced to differentiate or to undergo apoptosis. Isolated human blood monocytes (open bar) were either subjected to FACS analysis or cultured. The cultured cells were either induced to differentiate (striated bar) by continued culture for 5 days or induced to undergo apoptosis with camptothecin (black bar) for 24 h. The cells were then harvested and labeled with either FITC-annexin V to detect PS exposure (A), Alexa-488 Plg (B) or anti-H2B followed by Alexa-488 anti-rabbit IgG (C) and analyzed by FACS. Data are presented as fold change compared with freshly isolated monocytes from three independent experiments. *P < 0.001 vs. untreated cells as analyzed by one-way ANOVA test.

Fig. 7

Role of PS in apoptosis-induced Plg binding and H2B surface expression. IFNγ+ VD3 and camptothecin-treated (24 h) cells were either untreated or pretreated with anti-PS or its isotype control IgM. Cells were washed and allowed to bind exogenously added recombinant H2B (5 μg mL⁻¹). Cells were then washed and subjected to FACS staining with either anti-H2B (A) or Alexa-488 labeled Plg (B). Specific mean fluorescence intensities are presented. Data are the means ± SD from triplicate experiments. *P < 0.001 vs. untreated; #P < 0.001 vs. IFNγ+ VD3 treated; ##P < 0.001 vs. IFNγ+ VD3 + ExoH2B treated and IFNγ+ VD3 + ExoH2B+isotype IgM treated; §P < 0.001 vs. camptothecin treated; §§P < 0.001 vs. camptothecin + ExoH2B treated and camptothecin + ExoH2B + isotype IgM treated, as analyzed by one-way ANOVA test.