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IMMUNOBIOLOGY
Phosphatidylserine exposure in B lymphocytes: a role for lipid packing
James I. Elliott, Alessandro Sardini, Joanne C. Cooper, Denis R. Alexander, Suzel Davanture, Giovanna Chimini, and Christopher F. Higgins
Plasma membrane lipids are usually distributed asymmetrically, with phosphatidylserine (PS) confined to the inner leaflet. PS exposure at the outer leaflet occurs
early in apoptosis, but it is also constitutive on some nonapoptotic cell populations where it plays a role in cell signaling. How PS is transported (“flopped”) to
the cell surface is unknown. Contrary to
previous reports that normal murine B
lymphocytes lack lipid asymmetry, we
show that PS is normally restricted to the
inner leaflet of these cells. PS exposure
on normal B cells did, however, occur
spontaneously ex vivo. Consistent with
the hypothesis that loss of PS asymmetry
is regulated by CD45, PS is constitutively
exposed on viable, CD45-deficient B cells.
We show that calcium-stimulated PS exposure in B cells is strain variable, ABCA1
independent, and both preceded by and
dependent on a decrease in lipid packing.
This decrease in lipid packing is concomitant with cell shrinkage and consequent
membrane distortion, both of which are
potently inhibited by blockers of volume-
regulatory Kⴙ and Clⴚ ion channels. Thus,
changes in plasma membrane organization precede PS translocation. The data
suggest a model in which PS redistribution may occur by a translocaseindependent mechanism at energetically
favorable sites of membrane perturbation
where lipid packing is decreased. (Blood.
2006;108:1611-1617)
© 2006 by The American Society of Hematology
Introduction
The anionic phospholipid phosphatidylserine (PS) is largely confined to the inner leaflet of the plasma membrane of healthy cells,
and its flopping to the outer leaflet and exposure to the extracellular
face of the membrane is an early event in apoptosis, the safe
removal of cellular debris, and initiation of the clotting cascade.1
The mechanisms by which PS distribution is altered are poorly
understood but are generally assumed to involve proteins facilitating either inward or outward PS transport (“flippases” or “floppases,” respectively), and/or scramblases mediating bidirectional,
headgroup-nonspecific phospholipid transport.2,3 The rate of PS
translocation has, for example, been found to be sensitive to the
altered expression of the ATP-binding cassette transporter ABCA1,
suggesting that this protein might either directly transport PS or
regulate another transporter.4 PS exposure in apoptosis is also
accompanied by a decrease in the normal packing of plasma
membrane phospholipids, which can be detected by increased
binding of the lipophilic dye MC540.5 While it is not known
whether the 2 phenomena are mechanistically related, exposure of
PS (detected by binding of annexin V [AV]) and decreased lipid
packing have been reported to identify equivalent populations of
early apoptotic cells.6,7
Loss of PS asymmetry can also occur in the absence of
apoptosis, for example, in T cells stimulated via the ATP receptor
P2X7,8 and in a population of nonapoptotic CD4⫹CD45RBlo
activated/memory T cells.9 PS has also been reported to be
constitutively exposed on most or all viable murine B cells,10,11
with a level of surface PS of 10 to 100 times that of T cells that does
not increase upon stimulation, indicating complete loss of lipid
asymmetry.10,11 In contrast to these reports, we show that PS
distribution on murine B lymphocytes, as for most cells, is
asymmetric, although PS is rapidly exposed ex vivo in the absence
of exogenous signal. PS exposure was accelerated by stimulation
with a calcium ionophore and was preceded by and dependent on a
decrease in plasma membrane lipid packing. This change in lipid
packing could not be dissociated from cell shrinkage and membrane deformity, suggesting that loss of PS asymmetry may occur
at sites of membrane-bending at which lipid packing is decreased in
one leaflet.
From the Medical Research Council (MRC) Clinical Sciences Centre,
Faculty of Medicine, Imperial College, Hammersmith Hospital Campus,
London, United Kingdom; the Laboratory of Lymphocyte Signalling and
Development, The Babraham Institute, Babraham, Cambridge, United
Kingdom; and the Centre d’Immunologie de Marseille-Luminy, Parc
Scientifique et Technologique de Luminy, Marseille, France.
Scientifique (CNRS) (G.C. and S.D.), and specific funding from the European
Union (G.C. and S.D.).
Submitted November 15, 2005; accepted April 24, 2006. Prepublished online
as Blood First Edition Paper, May 9, 2006; DOI 10.1182/blood-200511-012328.
Supported by the Medical Research Council of Great Britain (J.I.E., A.S., and
C.F.H.), institutional funding from Institut National de la Santé et de la
Recherche Médicale (INSERM) and Centre National de la Recherche
BLOOD, 1 SEPTEMBER 2006 䡠 VOLUME 108, NUMBER 5
Materials and methods
Mice
Friend virus B-type (FVB/n) and nonobese diabetic (NOD) mice were bred
at the Biological Services Unit at Imperial College, Hammersmith Campus,
London, United Kingdom. Mice lacking CD45 have been described
elsewhere12 and were compared with age-matched nontransgenic mice from
the same breeding program at The Babraham Institute, Babraham, Cambridge, United Kingdom. Mice lacking ABCA1 have been described
elsewhere4 and were maintained on a DBA/1J background in a pathogenfree facility at Charles River Laboratories in Lyon, France. Where studied,
The online version of this article contains a data supplement.
Reprints: James I. Elliott, MRC Clinical Sciences Centre, Faculty of Medicine,
Imperial College, Hammersmith Hospital Campus, Du Cane Rd, London, W12
0NN, United Kingdom; e-mail: [email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2006 by The American Society of Hematology
1611
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1612
ELLIOTT et al
BLOOD, 1 SEPTEMBER 2006 䡠 VOLUME 108, NUMBER 5
used as a measure of the volume of spherical cells,13 its sensitivity being
greatest when light is collected over an angle of less than 10 degrees14 as in
the FACScalibur. Distortion of the plasma membrane was measured by an
increase in side light scatter (SSC), which has previously been shown to be
concomitant with cell shrinkage (decrease in FSC) early in apoptosis.14-16
DIDS (Sigma) was dissolved in water. Tamoxifen (Sigma) was dissolved in ethanol. Calcium ionophore, glybenclamide (Sigma), and the
Ca2⫹ indicator Fluo-4AM (used at 0.25 ␮M; Molecular Probes, Leiden, The
Netherlands) were dissolved in DMSO. All results are representative of at
least 3 independent experiments.
Note on calcium ionophore concentrations
Because calcium ionophore (A23 187) is highly lipophilic, the concentration of ionophore required to induce PS flop (or MC540 uptake) is, in our
experience, more dependent on the amount of cell membrane in the tube
(and this is likely to include red cell contamination/debris/dead cells gated
out during analysis) than on volume of the reaction mix. For this reason,
responses of cells from different sources were compared in a single tube (to
ensure identical ionophore to membrane concentrations in a given experiment). Changing ionophore and cell concentrations in a reaction mix have
equivalent (but opposite) effects on responses.
Confocal imaging of murine lymphocytes stained
with MC540 and AV
Murine mesenteric lymph node cells, freshly dissected, were adhered to
glass cover slips coated with poly-L-lysine (Sigma). Cells were exposed for
1 minute to 10 ␮M calcimycin in the presence or absence of 1 ␮M MC540,
Figure 1. PS is localized in the inner membrane leaflet of murine B cells and
exposed on stimulation. (A) Lymphocytes from C57BL/10 mice were labeled with
anti-CD4APC (T-cell–specific) and anti-CD19PERCP (B-cell–specific) antibodies and
preincubated with AVFITC. (Ai) Density plots of the rate of PS exposure (binding of AV)
by T cells (CD4⫹; left panel) and B cells (CD19⫹; right panel) following stimulation
with 5 ␮M calcimycin (arrows). (Aii) PS exposure by T- and B-cell populations
following stimulation. Data are from the experiment shown in panel Ai, plotted as
histograms to compare AV binding by cell populations gated at t ⫽ 0 seconds (black
line) and t ⫽ 500 seconds (gray line) in T cells (left panel) and B cells (right panel). (B)
Lymphocytes from C57BL/10 mice were labeled with anti-CD19PERCP (B-cell–
specific) antibodies, preincubated with AVFITC and stimulated with calcimycin at the
doses shown. Density plots illustrate the rate and degree of PS exposure (AV
binding). (C) Spontaneous PS exposure and cell death in B-lymphocyte populations
ex vivo. Lymphocytes from C57BL/10 mice were labeled with anti-CD4CYCHROME and
anti-CD19APC antibodies, to distinguish T and B cells, and incubated with AVFITC. (Ci)
Histograms show PS exposure (AV binding) by T cells (CD4⫹) and B cells (CD19⫹) 30
minutes (black lines) and 2 hours (gray lines) after animals were killed. (Cii) Line
graph shows the proportion (⫾ SD) of cells stained with anti-CD19 antibody (B cells)
within “live cell” gates as determined by forward and side light scatter at times shown
following the time the animals were killed.
ABCA1-deficient mice were compared with wild-type mice from the same
breeding colony. All other strains were from Harlan-Olac (Bicester,
United Kingdom). All home office and local ethical guidelines for the
care of laboratory animals were followed.
Real-time flow cytometry
Mesenteric lymph node cells were prepared from adult mice. Cell suspensions in phenol red–free Dulbecco modified Eagle medium (DMEM;
Sigma, Poole, United Kingdom) were stained with CD19PE, CD19PERCP,
CD19APC, CD4APC, or CD4CYCHROME (Becton Dickinson, San Jose, CA)
antibodies, as indicated. The use of differently labeled antibodies enabled B
cells from 2 mouse strains to be detected differentially, and hence studied
simultaneously in the same tube. Cells were washed and resuspended in
DMEM and, where indicated, equilibrated with AVFITC or AVCY5 (AV;
Becton Dickinson), or with 0.05 ␮M merocyanine 540 (MC540; Sigma) for
3 minutes. Where cells were stimulated, baseline fluorescence was established for 30 seconds to 1 minute prior to addition of 2 ␮M to 5 ␮M calcium
ionophore (calcimycin, A23 187; Sigma). Cells were monitored for PS
exposure continuously in real time by flow cytometry on a FACScalibur
machine and analyzed using CellQuest (Becton Dickinson) or FlowJo
(Treestar.com, Ashland, OR) software. Forward light scatter (FSC) was
Figure 2. PS exposure by B cells is strain dependent. (A) Lymphocytes from NZW
and DBA/2 mice were labeled with anti-CD19APC and anti-CD19PERCP antibodies,
respectively (to allow B cells from each strain to be easily distinguished), mixed, and
preincubated with AVFITC. Panels show density plots of the rate of PS exposure
(binding of AV) by B cells from NZW (left panel) and from DBA/2 mice (center panel)
measured in the same tube following stimulation with 3 ␮M calcimycin (arrows). The
right panel shows an alternative representation of the same data, plotted as the
percentage of cells with exposed PS as a function of time. (B) An equivalent
experiment to that in panel A but with lymphocytes derived from NOD and C57BL/10
mice. (C) Cells were stained as in panel A to identify B-cell populations after mixing.
Bars show the percentage (mean ⫾ SD, n ⫽ 4) of B cells from DBA/2 and NZW (left
panel), or NOD and C57BL/10 mice (right panel), with exposed PS approximately 7
minutes after stimulation with 3 ␮M calcimycin. (D) Cells from NZW and NOD mice
were stained as in panel A to distinguish B-cell populations. Panels show density plots
of the rate of PS exposure (binding of AV) by B cells from NZW (left panel) and NOD
mice (center panel) following stimulation with 3 ␮M calcimycin (arrows). The rate of
PS exposure was equivalent in the 2 strains. In similar experiments, the rate of PS
exposure by B-cell populations from NOD and/or NZW mice was faster than that that
by cells from 129, C3H, CBA, FVB/n, and NIH mice (not shown).
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BLOOD, 1 SEPTEMBER 2006 䡠 VOLUME 108, NUMBER 5
PHOSPHATIDYLSERINE EXPOSURE IN B CELLS
1613
ionophore resulted in marked PS exposure in both lymphocyte
populations. Thus, as for most cell types and in contrast to previous
reports,10,11 unstimulated B lymphocytes (like T lymphocytes)
possess an asymmetric lipid bilayer with PS largely restricted to the
inner leaflet. Stimulation resulted in PS “flopping” to the outer
leaflet: the time at which PS exposure was detectable and the peak
AV-binding per cell depended on the concentration of calcimycin
added (Figure 1B).
To assess the possibility that mouse strain–dependent differences in B-cell distribution of PS account for differences between
our data and those published previously, we studied lymphocytes
from a variety of normal and autoimmune strains. To ensure direct
comparison of responses, B cells from strains being studied were
stained with differently labeled anti-CD19 antibodies (to allow
subsequent discrimination during analysis) and mixed before
stimulation. Responses were compared by real-time flow cytometry in a single tube. PS was restricted to the inner leaflet of B cells
from all mice, although significant strain-dependent differences in
Figure 3. Calcium ionophore–stimulated PS exposure in murine B cells is
ABCA1 independent. Lymphocytes from ABCA1-deficient mice and age-matched
controls from the same colony were labeled with anti-CD19APC and anti-CD19PERCP
antibodies, respectively, mixed and preincubated with AVFITC or MC540. Histograms
show (A) the percentage of CD19⫹ B cells having exposed PS (indicated by binding
of AV) following stimulation with 5 ␮M calcimycin (arrow), or (B) MC540 uptake by
CD19⫹ B cells following stimulation with 2.5 ␮M calcimycin (arrow). ABCA1⫺/⫺, red
line; parental, blue line.
successively fixed in a solution of 4% formaldehyde, 4% sucrose in
phosphate-buffered saline (PBS), and, where indicated, stained with
AV-Alexa Fluor 568 (Molecular Probes). All cells were also stained with
4⬘,6-diamidino-2-phenylindole dihydrochloride (DAPI; 15 mg/mL; 1:10 000
dilution; Molecular Probes, Eugene, OR) for nuclear DNA. Cells were
imaged with a Leica SP confocal microscope equipped with a 100⫻/1.4
PlanApoChromat oil-immersion objective lens (Leica, Wetzlar, Germany).
Merocyanine 540 was excited with the 488-nm line of an argon laser and
AV–Alexa Fluor 568 with the 561-nm line of a solid state laser; the emitted
fluorescence collected through a triple-dichroic mirror (488/568/663) with a
498-nm to 700-nm bandpass filter and 575-nm to 700-nm bandpass filter,
respectively. DAPI staining of nuclear DNA was excited with the 351-nm
line of an ultraviolet (UV) laser, and emission fluorescence collected with a
396-nm to 508-nm bandpass filter. Stacks of confocal sections separated by
0.3-␮m increments were taken and images analyzed with Metamorph 5.0v1
software (Universal Imaging, Downington, PA). Figures were prepared
with Adobe Photoshop 6.0 software (Adobe Systems, San Jose, CA).
Results
PS exposure on unstimulated and stimulated B cells
It has been reported that PS is exposed at high levels on
unstimulated murine B cells and that further exposure does not
occur on stimulation.10,11 We assessed levels of cell-surface PS on
rapidly isolated lymphocytes from C57BL/10 mice by real-time
flow cytometry in the continuous presence of AVFITC. After basal
binding of AVFITC was established, cells were treated with calcium
ionophore (calcimycin, A23 187) to stimulate nonspecific uptake of
Ca2⫹ and consequent PS exposure (Figure 1). Unstimulated B and
T cells studied in the same tube exhibited comparably low-level
binding of AV, indicating restriction of PS to the inner leaflet of the
plasma membrane in both cell types. Stimulation with calcium
Figure 4. A decrease in lipid packing precedes PS exposure in B cells. (A)
Lymphocytes from C57BL/10 mice were labeled with anti-CD19APC antibody and
preincubated with either 0.05 ␮M MC540 or AVFITC. Histograms show (mean
fluorescence units) the rate of (Ai) MC540 binding or (Aii) PS exposure (binding of AV)
by CD19⫹ B cells following stimulation with 3 ␮M (red lines), 2 ␮M (blue lines), or
1 ␮M (green lines) calcimycin. (Bi) Lymphocytes from C57BL/10 and NOD mice were
labeled with anti-CD19APC and anti-CD19PERCP antibodies, respectively, mixed, and
preincubated with 0.05 ␮M MC540. Panels show density plots of the rate of decrease
in lipid packing (increased uptake of MC540) by B cells from C57BL/10 (left panel)
and from NOD mice (center panel) in the same tube following stimulation with 2 ␮M
calcimycin (arrows). The right panel shows an alternative plot of the same data
plotted as the percentage of cells binding high levels of MC540 as a function of time.
Red lines, NOD B cells; blue lines, C57BL/10 B cells. (Bii) Histograms of MC540
binding by the B-cell populations shown in panel Bi, gated at t ⫽ 0 seconds and
t ⫽ 400 seconds. Red lines, NOD B cells; blue lines, C57BL/10 B cells. (C) Results of
3 independent experiments equivalent to that in panel Bi but with lymph node cells
derived from NOD (red lines) and DBA/2 (green lines) mice. (D) Calcium uptake in
stimulated B cells. (Di) Lymphocytes from the C57BL/10 (red line) and NZW (blue
line) mice were labeled with anti-CD19APC and anti-CD19PERCP antibodies respectively, mixed, incubated with 0.25 ␮M of the calcium-sensitive indicator Fluo-4AM for
10 minutes, washed, and stimulated with 0.2 ␮M calcimycin (arrow). Plots show Ca2⫹
uptake as a function of time. (Dii) Shows an equivalent experiment to that in panel Di
but with lymph node cells derived from DBA/2 (red line) and NZW (blue line) mice.
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1614
ELLIOTT et al
BLOOD, 1 SEPTEMBER 2006 䡠 VOLUME 108, NUMBER 5
Figure 5. Decreased lipid packing is concomitant with and dependent on cell shrinkage. (A) Lymphocytes from NOD mice were stained with anti-CD19 antibody, preincubated with
0.05 ␮M MC540, and stimulated with 2 ␮M calcimycin (arrows) alone (red lines) or together with 10 ␮M clotrimazole (blue lines). Plots show (Ai) cell shrinkage (decrease in forward light
scatter [FSC]), (Aii) percentage of MC540hi cells, and (Aiii) membrane buckling (side light scatter, [SSC]) plotted as a function of time. (B) Lymphocytes from NOD mice were stained with
anti-CD19 antibody. (Bi) Cells were preincubated with AVFITC alone (left panel) or together with 1 ␮M tamoxifen (right panel) and stimulated with 2 ␮M calcimycin (arrows). Density plots
show the change in AV binding as a function of time. (Bii) Cells were preincubated with 0.05 ␮M MC540 and stimulated with 2 ␮M calcimycin (arrows) alone (red lines) or in the presence of
1 ␮M tamoxifen (added immediately before baseline recording; blue lines). Plots show cell shrinkage (left panel; decrease in forward light scatter [FSC]); mean MC540 fluorescence units
(center panel), membrane buckling (right panel; side light scatter [SSC]); all plotted as a function of time.
sensitivity to stimulation were apparent. For example, responses of
B cells from the autoimmune-prone New Zealand white (NZW)
and NOD strains of mice were comparable (not shown), but
responded more rapidly than those from C57BL/10, DBA/2
(Figure 2), and 6 other nonautoimmune prone strains (129/Ola,
BALB/c, C3H, CBA/Ca, FVB/n, NIH; not shown). That responses
were more pronounced in cells from autoimmune-prone mouse
strains is consistent with our recent observation that calcium
ionophore–stimulated lymphocytes from patients with systemic
lupus erythematosus (SLE) and rheumatoid arthritis (RA) externalize PS more readily than do control cells.17
We suggest that the apparent discrepancy with previous reports
arises from the fact that, in contrast to T cells, PS in B cells
becomes exposed to the surface ex vivo within 2 hours even in the
absence of exogenous stimulation (Figure 1C). Moreover, this
exposure of PS on B cells was associated with their relatively rapid
death ex vivo, as evidenced by a steadily falling proportion of
CD19⫹ cells within the live gate population (Figure 1C). The
disproportionate death of CD19⫹ B cells ex vivo is routinely
observed in our hands, although the rate appears to be affected by
factors such as temperature and serum concentration (data not
shown). In previous studies,10,11 B lymphocytes appear to have
been maintained for a relatively long period ex vivo as this
population was enriched over magnetic bead columns prior to
analysis. In our experiments, by contrast, B cells were not
physically isolated, but instead gated electronically on a flow
cytometer on the basis of fluorescent antibody binding.
PS translocation by B cells is independent of ABCA1
The rate of calcium ionophore–stimulated PS exposure has previously been reported to be decreased in erythrocytes and fibroblasts
lacking the ABC transporter ABCA1, and increased in HeLa cells
overexpressing ABCA1 following transfection,4 leading to the
suggestion that ABCA1 might transport PS to the cell surface or
regulate a heterologous PS transporter. However, we showed that
the rates of PS exposure by ionophore-stimulated B lymphocytes
from parental and from ABCA1⫺/⫺ mice could not be distinguished
(Figure 3), indicating that ABCA1 is not involved in calcium
ionophore–stimulated PS transport in murine B cells.
Figure 6. PS exposure on B cells from CD45ⴚ/ⴚ and parental mice. (A) Lymphocytes
from CD45⫺/⫺ (solid line) or parental (CD45⫹/⫹) mice (dotted line) were labeled with
anti-CD19APC and anti-CD19PE antibodies, respectively, to discriminate between CD19⫹ B
cells from each mouse after mixing. Cells were mixed and preincubated with AVFITC to
assess PS exposure. (B) To compare the rate of cell death among spleen cells from
parental (C57BL/6) and CD45⫺/⫺ mice, the sub-G1 DNA content of lymphocytes at t ⫽ 0
and 24 hours was assessed through uptake of propidium iodide (PI) analyzed by flow
cytometry. Percentages of cells containing sub-G1 levels of DNA are shown. (C) Lymphocytes from CD45⫺/⫺ (gray line) or parental (CD45⫹/⫹) mice (black line) were labeled with
anti-CD19APC and anti-CD19FITC antibodies, respectively, to discriminate between CD19⫹
B cells from each mouse, mixed, and incubated with MC540 to assess lipid packing.
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BLOOD, 1 SEPTEMBER 2006 䡠 VOLUME 108, NUMBER 5
Cell shrinkage and MC540 uptake by murine B cells
MC540 is a fluorescent, lipophilic dye that binds preferentially to
the outer leaflet of plasma membrane bilayers with relatively
loosely packed lipids,5,18-20 including those of apoptotic cells.6,7 We
compared the kinetic relationship between MC540-binding (to
measure lipid packing) and AV-binding (to measure PS exposure).
Following calcium ionophore–stimulation in the presence of
MC540, dye uptake exhibited a rapid increase (Figure 4A).
Increased MC540 uptake preceded loss of PS asymmetry and,
indeed, occurred at concentrations of ionophore below that required to stimulate detectable PS exposure (Figure 4A). Hitherto,
the only event (other than calcium uptake) shown to precede PS
translocation following calcimycin-stimulation is cell shrinkage
due to K⫹ efflux via KCa3.1 (formerly IKCa1).21 Cell shrinkage and
consequent buckling of the cell membrane can be measured in flow
cytometry by a decrease in forward light scatter (FSC) and
concomitant increase in side light scatter (SSC).14-16 The rates of
MC540 uptake (Figure 4B-C and Figure S1, which is available on
the Blood website; see the Supplemental Figures link at the top of
the online article) and cell shrinkage (Figure S1), as for PS
externalization (AV-binding), were strain dependent with B cells
from NOD and NZW mice exhibiting a relatively rapid response,
suggesting that strain-dependent variation in rates of PS translocation reflects upstream differences in rates of cell shrinkage and
decrease in lipid packing. Strain-dependent variations did not,
however, appear to reflect differences in calcium uptake (Figure
4D). Decreased B-cell lipid packing (onset of MC540 uptake),
shrinkage, and membrane buckling were concomitant (Figure
5A-B). Both membrane distortion and decreased lipid packing
depended on cell shrinkage, as evidenced by their inhibition by
clotrimazole, which block KCa3.1-dependent K⫹ efflux, and also by
tamoxifen, which inhibits volume-regulated chloride channels.22,23
DIDS and glybenclamide, which are thought to inhibit loss of lipid
asymmetry downstream of cell shrinkage, had no effect on MC540
uptake (not shown). Together, these data strongly suggest that
PHOSPHATIDYLSERINE EXPOSURE IN B CELLS
1615
decreased lipid packing requires cell shrinkage and membrane
buckling, and that all precede PS exposure.
PS is constitutively exposed on CD45-deficient B lymphocytes
We have recently shown that PS translocation in T cells is
negatively regulated by the tyrosine phosphatase CD45.9 Consistent with this finding (and in contrast to parental B cells) we found
PS to be constitutively exposed on CD45⫺/⫺ B cells (Figure 6). One
possible explanation for the elevated exposure of PS on B cells
from CD45⫺/⫺ mice is that these cells might lack normal signaling
pathways mediating survival, thereby leading to increased apoptosis. However, we have found no evidence for increased apoptosis in
peripheral T or B cells from CD45⫺/⫺ mice, assessed using
4 different assays (Figure 6, and Figure S2). It therefore seems
unlikely that the high PS expression on more than 90% CD45⫺/⫺
B cells could be significantly explained by apoptosis, particularly
as B-cell numbers are higher in CD45⫺/⫺ mice than the parentals.24
Thus, B cells from CD45⫺/⫺ mice are viable but have exposed PS.
Importantly, unstimulated CD45⫺/⫺ B cells exhibit higher SSC and
MC540 binding than do parental B cells stained in the same tube
(Figure 6), consistent with the hypothesis that membrane curvature
and decreased lipid packing promote PS exposure.
MC540 binding and PS exposure occur at sites of
membrane blebbing
The finding that PS exposure depends on prior cell shrinkage, an
increase in membrane buckling, and decreased lipid packing
suggests a model whereby calcium ionophore–stimulated PS
exposure might occur at energetically favorable sites on developing
membrane blebs (see “Discussion”). We therefore analyzed MC540
binding and PS exposure by calcium ionophore–stimulated lymphocytes by confocal microscopy. As we have found MC540 to reduce
the binding of AV per cell, the 2 stains were analyzed separately.
Figure 7A shows a stack of confocal sections through calcium
ionophore–stimulated lymphocytes stained with AV. Blebs are
Figure 7. Merocyanine 540 and annexin V preferentially bind to membrane blebs on calcium ionophore–stimulated lymphocytes. Confocal sections of murine
mesenteric lymph node cells are shown. Lymphocytes from C57BL/10 mice were stimulated with 10 ␮M calcimycin and stained with 1 ␮M MC540 (green) or annexin V
AlexaFluor 568 (red). DAPI staining of nuclear DNA is in blue. Scale bars indicate 10 ␮m. (A) Stack of confocal sections separated by 0.3 ␮m increments showing membrane
blebs. (B) Single confocal sections.
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1616
ELLIOTT et al
visible as small protrusions at the cell surface. Consistent with the
hypothesis, binding of both AV and MC540 occur preferentially at
sites of membrane blebbing (Figure 7). Bleb localization is
somewhat more apparent for AV than MC540, consistent with the
fact that MC540 exhibits greater background binding to unstimulated cells.
Discussion
In most cells, phospholipids are distributed asymmetrically in the
plasma membrane, with the great majority of PS in the inner leaflet.
PS translocation to the outer leaflet is required for the immunologically silent removal of apoptotic cells.25 PS exposure can also occur
on nonapoptotic cells, for example on murine T lymphocytes
following stimulation of the ATP receptor P2X7 (note: murine B
cells do not express P2X7).8 Moreover, PS is constitutively exposed
at the surface of many cells of the CD45RBlo subpopulation of
viable CD4⫹ activated/memory lymphocytes. Importantly, PS
distribution between inner and outer leaflets modulates the activity
of several membrane proteins.9
Reports that viable murine B cells, in contrast to T cells, do not
exhibit lipid asymmetry have been suggested to reflect a key aspect
of their biology.10,11 Here, we show that, in contrast to published
findings,10,16 PS in B cells, as in T cells, is in fact largely confined to
the inner leaflet of the plasma membrane and surface exposure can
readily be induced following stimulation with a calcium ionophore.
A likely explanation for the previous report that PS distribution is
not asymmetric is the observation that B cells spontaneously
translocate PS to the cell surface relatively rapidly ex vivo. In
contrast to previous studies, we did not physically isolate B cells,
but instead gated cells electronically during analysis of flow
cytometric data, thereby reducing time spent by cells ex vivo.
The rate of PS translocation in B cells, as measured by the
proportion of responding cells at a given time, was found to be
strain dependent. These strain-dependent events also correlated
with differences in rates of MC540 uptake, supporting the hypothesis that decreased lipid packing is required for PS translocation.
Significantly, in view of previous findings that lymphocytes from
patients with rheumatoid arthritis and systemic lupus erythematosus have relatively high rates of PS translocation,17 the rate of PS
exposure was highest in B cells from the autoimmune-prone strains
NZW and NOD. Both NZW and NOD strains are also associated
with susceptibility to lupuslike disease.26-28 In principle, a tendency
toward PS exposure might promote susceptibility to SLE through
increases in shedding of proinflammatory microvesicles,29,30 presentation of PS itself as an autoantigen,31 or subsequent increased
release of other autoantigens in the form of apoptotic debris. As the
increased rate of PS translocation in NOD and NZW lymphocytes
is downstream of an increased rate of cell shrinkage and concomitant decrease in lipid packing, the data suggest that aberrant volume
regulation may contribute toward susceptibility to lupuslike disease.
We have shown previously that ionophore-stimulated PS exposure is subsequent to and dependent on cell shrinkage caused by K⫹
efflux via KCa3.1.21 We show here that cell shrinkage results in a
decrease in lipid packing, as evidenced by increased uptake of the
dye MC540, presumably a consequence of membrane buckling.
Thus, significant changes in the organization of the lipid bilayer
appear to precede PS translocation. Uptake of MC540 and decreased lipid packing occur simultaneously and are both blocked
BLOOD, 1 SEPTEMBER 2006 䡠 VOLUME 108, NUMBER 5
by inhibitors of volume regulatory ion channels (along with cell
shrinkage). Hence, PS translocation appears to depend on upstream
cell shrinkage and concomitant decrease in lipid packing. Tamoxifen, a potent inhibitor of volume-regulated chloride channels,
blocked PS exposure at concentrations as low as 1 ␮M, making it,
to our knowledge, the most potent inhibitor of lipid scrambling
described to date.
It is an apparent paradox that lipid packing decreases upon cell
shrinkage, whereas an increase in packing might be anticipated.
Nevertheless, as has been observed previously,14-16 distortion of the
plasma membrane (measured by increase in SSC) is concomitant
with cell shrinkage early in apoptosis. We suggest, therefore, that
buckling of the plasma membrane as cells shrink distorts the lipid
bilayer locally such that MC540 inserts into the plasma membrane
at sites of decreased lipid binding at the apex of blebs (Figure 8).
PS⫹ blebs/microvesicles are known to form and be shed following
Figure 8. Model for calcium-stimulated PS translocation. (A) In the absence of
stimulation, the plasma membrane of most healthy cells is asymmetric, with PS and
PE restricted to the inner leaflet and PC largely restricted to the outer leaflet. (B)
Following stimulation with a calcium ionophore, K⫹ and Cl⫺ ions leave the cell, water
follows, and the cell shrinks.21 As a consequence of cell shrinkage, the plasma
membrane buckles, prior to the shedding of microvesicles/blebs. At the apex of
microvesicles/blebs the packing of phospholipids is relatively loose, reducing the
energetic barrier to outward movement of PS and PE. A decrease in lipid packing in
the outer leaflet allows the insertion of MC540. In the inner leaflet, PS and PE are
tightly packed, increasing the energetic favorability of outward phospholipid “flop.” At
the base of microvesicles/blebs, PC is tightly packed in the outer leaflet and PS/PE
loosely packed in the inner leaflet, favoring inward phospholipid flip. (C) PS and PE
flop out at the apex, and PC flips in at the base. Thus, averaged across the
microvesicle/bleb, transport of phospholipids appears headgroup-nonspecific and
bidirectional (“scrambling”). (D) As it is no longer tethered to cytoplasmic/cytoskeletal
proteins, PS in the outer leaflet is relatively free to move laterally outside of the bleb
(unfilled arrow). (E) Microvesicles/blebs are shed. Phospholipid balance is partially
restored, with most PS/PE distributed in inner and outer leaflets. Consistent with the
model above, both we32 and others33 have found that shed particles, but not cell
remnants, exhibit high levels of exposed PS, though some movement of PS out of the
bleb is likely and has been found on cell remnants in other systems.34
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
BLOOD, 1 SEPTEMBER 2006 䡠 VOLUME 108, NUMBER 5
stimulation with calcium ionophore.2,32,33,35 Our observation that
cell shrinkage, which is required for PS exposure, is strongly
associated with decreased lipid packing provides a model for
headgroup-nonspecific loss of lipid asymmetry (“scrambling”;
Figure 8). In this model, bending of the membrane during cell
shrinkage creates domains at the base and apex of blebs in which
phospholipids in the outer leaflet are in relative excess or deficit,
respectively, creating regions favorable for phospholipid flip-flop.
Movement of inner leaflet phospholipids such as PS to the outer
leaflet would be favored at the apex of blebs and that of PC to the
inner leaflet favored at the base. This model predicts that phospholipid movement is bidirectional and headgroup nonspecific (consistent with the phenomenon of phospholipid “scrambling”), and that
PS exposure is predominantly found on shed particles and is less
apparent on the cell bodies that remain following shedding.32,33
Consistent with our hypothesis, MC540 binding and PS exposure
on calcium ionophore–stimulated lymphocytes both localized to
sites of membrane blebbing (Figure 7). The model also implies that
a dedicated protein may not be required to mediate PS transloca-
PHOSPHATIDYLSERINE EXPOSURE IN B CELLS
1617
tion. The search for a specific protein that mediates bidirectional
transport has been a notable failure.36,37 While it has been shown
that the rate of PS exposure is sensitive to altered expression of the
ABC transporter, ABCA1, in human B cells,38 murine erythrocytes,4 and following ABCA1 transfection,4,38 the rate of PS
exposure by murine ABCA1⫺/⫺ and parental B cells could not be
distinguished (Figure 3). Hence, whereas ABCA1 can modulate
rates of PS exposure, it does not appear to be required. If ABCA1 is
a floppase, therefore, its loss can be compensated for by another
enzyme in genetically modified murine B lymphocytes. Alternatively, and perhaps more likely, ABCA1 is not itself a floppase but
can modulate the rate of PS translocation indirectly.
Based on studies of bacterial membranes, it has recently been
suggested that phospholipid flip-flop may be promoted by a wide
range of ␣-helical membrane-spanning proteins that provide energetically favorable sites for charged headgroups to traverse the
hydrophobic interior of bilayer.39 This model, which is complementary to our own, removes the need to posit (but does not disprove)
the existence of a specific bidirectional phospholipid transporter.
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2006 108: 1611-1617
doi:10.1182/blood-2005-11-012328 originally published
online May 9, 2006
Phosphatidylserine exposure in B lymphocytes: a role for lipid packing
James I. Elliott, Alessandro Sardini, Joanne C. Cooper, Denis R. Alexander, Suzel Davanture,
Giovanna Chimini and Christopher F. Higgins
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