1Department of Biochemistry, Hebrew University-Hadassah Medical School; 2Blood Bank, Hadassah University Hospital; and 3Department of Hematology, Hadassah University Hospital, Jerusalem, Israel 91120
Submitted 22 November 2002 ; accepted in final form 1 May 2003
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ABSTRACT |
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erythrocytes; adherence; acridine orange; band-3; phosphatidylserine
It has been proposed that RBC aging is associated with clustering of band-3 at the RBC surface, and the clustered band-3 binds to IgG antibodies, leading to RBC phagocytosis (10). It has also been proposed that band-3 clustering is responsible for the enhanced adherence of Plasmodium falciparum-infected RBC to endothelial cells (EC) (11). Other studies have shown that RBC recognition by macrophages and adherence to EC are induced also by phosphatidylserine (PS) at the RBC surface, which is known to be translocated to the outer leaflet of the membrane in hemoglobinopathies and other pathological conditions (8). Both band-3 clustering and PS exposure are known to increase with RBC aging (2, 6).
In studying band-3 clustering and its implications to RBC intercellular interactions, a widely used procedure has been the treatment of RBC with acridine orange (AO), because it has been assumed to be a specific inducer of band-3 clustering (1, 10, 12). However, although RBC intercellular interactions are also induced by PS, the effect of AO treatment on RBC membrane phospholipids has not been examined. The present study was undertaken to examine the influence of AO treatment on PS level at the RBC surface.
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METHODS |
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To detect PS at the RBC surface, in the present study we used allophycocyanin-conjugated recombinant human annexin V (rh annexin V-APC; Bender Medsystems, Vienna, Austria) as the PS ligand. APC was used here instead of a fluorescein isothiocyanate (FITC), which is commonly used for PS determination, because the FITC fluorescence spectrum overlaps with that of AO; FITC is excited at 488 and emits at 520 nm, at which AO exhibits high emission, too. In contrast, APC can be excited at 630 and emits at 660 nm, away from the AO fluorescence spectrum. The fluorescence of the AO-treated RBC was compared with that of control RBC, which were incubated for 15 min in AO-free PBS.
For positive control, PS translocation was induced by treatment of RBC with calcium and Ca2+-ionophore. RBC at 16% hematocrit were equilibrated in PBS supplemented with 1 mM CaCl2 for 3 min at 37°C. Subsequently, the Ca-ionophore A-23187 (Sigma Chemical, St. Louis, MO) was added to the RBC suspension to a final concentration of 4 µM and incubated for 1 h at 37°C. To terminate the process, calcium was removed by RBC washing with PBS containing 1 mM EDTA. The cells were then washed three times in Ca2+/Mg2+-free PBS containing 1% bovine serum albumin to remove the ionophore (9).
For annexin V-APC binding, 2 x 106 RBC were suspended in 200 µl of HEPES-buffered saline supplemented with 2.5 mM CaCl2 and incubated with 5 µl annexin V-APC for 15 min at 37°C in the dark. The fluorescently labeled cells were then washed once, suspended in 500 µl of the same buffer, and determined in fluorescence-activated cell sorter (FACS). Data acquisitions were performed on a Becton Dickinson FACScan (Becton Dickinson, San Jose, CA), and analysis was done with CellQuest software (Becton Dickinson). The fluorescence of annexin V-APC-labeled erythrocytes was recorded at the emission 660 nm (channel FL4) with the excitation at 630 nm by a red diode laser. A total of 10,000 events were acquired for each sample. The percentage of annexin V-APC-positive erythrocytes was determined from the fluorescence signal in excess over that obtained with a negative (unlabeled) control RBC for each sample.
To determine the AO incorporation into RBC membrane, 2 x 106 AO-treated RBC (as above) were suspended in 500 µl of HEPES-buffered saline (10 mM HEPES-Na, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) and analyzed in the flow cytometer for AO fluorescence (excitation wavelength 488 nm, emission wavelength 505545 nm). The percentage of AO-labeled erythrocytes was determined from the autofluorescence signal in excess over that obtained with a negative control (not treated with AO) RBC.
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RESULTS AND DISCUSSION |
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Figure 2A shows that treatment of RBC with AO strongly increased the binding of annexin to the cell surface in a dose-dependent manner, up to about 30% annexin V-APC-labeled cells. This effect is not due to possible ATP depletion (which is unlikely to be significant in such short-time treatment), because 1) PS translocation was not detected in negative control RBC, subjected to the same duration of incubation, and 2) it has previously been shown that inhibition of aminophospholipid translocase (flipase), or rapid ATP depletion, does not lead to cell surface exposure of PS in normal RBC, even after overnight incubation (3).
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Determination of AO incorporation (by the AO fluorescence emission of AO-treated RBC, depicted in Fig. 2B) shows that at 1 mM AO 95% of the RBC are already labeled with AO (Fig. 2, BI). However, this figure also shows that the incorporation of AO to RBC was monotonously increased with its buffer concentration, as determined by the mean intensity of AO fluorescence in the cells (Fig 2, BII). Comparing Fig. 2A and Fig. 2BII reveals a clear correlation between the increase in the mean AO fluorescence and the percent of annexin V-APC-labeled RBC, which did not exceed 30%, although all the cells were labeled with AO. This discrepancy can be explained in two ways: 1) the AO incorporation into the cell has to exceed a certain level to induce PS translocation, and 2) the PS at the cell surface has to exceed a certain threshold level to produce a detectable signal of the annexin V-APC fluorescence.
The results of this study demonstrate that treatment of RBC with AO is very effective in inducing translocation of PS to the cell surface. It is thus plausible to conclude that the use of AO as a specific inducer of band-3 clustering for studying RBC intercellular interaction is questionable. Because PS is well known to facilitate RBC adhesion and recognition by other cells, the AO-induced RBC intracellular interaction is not necessarily due to band-3 clustering. It is possible that band-3 clustering and PS translocation are interdependent, as previously suggested (5), but this interrelation has yet to be explored.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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