Journal of Gerontology: MEDICAL SCIENCES
1999, Vol. 54A, No. 12, M60l-M606
Copyright 1999 by The Gerontological Society (if America
Effect ofAge on Plasma Membrane Asymmetry
and Membrane Fluidity in Human Leukocytes and Platelets
1. M. Noble,l T. H. Thomas,' and G. A. Ford2
Departments of 'Medicine and 2Clinical Pharmacology, University of NewcastleUponTyne,United Kingdom.
Background. We determined whether ageing changes in plasma membrane phospholipid asymmetry were related to
changes in membrane physical characteristics.
Methods. Plasma membrane asymmetry was determined in polymorphonuclear leukocytes (PMN), lymphocytes, and
platelets from 45 healthy young (mean 29 years, 26 male) and 28 healthy elderly (mean 70 years, 15 male) subjects by flow
cytometric measurement of annex in V binding to cell surface phosphatidylserine. Membrane fluidity in lymphocytes and
platelets from young and elderly subjects was determined by fluorescence polarization of 1,6-diphenyl-l,3,5-hexatriene (DPH)
and (4-trimethylammonium)-DPH (1MA).
Results. In elderly subjects, a higher proportion of lymphocytes had specific annexin V binding to phosphatidylserine (PS)
than in young subjects (young: median percentage of cells with specific annexin V binding to PS 5.3 [second to fourth quintiles
range 3.8-8.7]; elderly: 8.5 [5.2-17.2]; p = .028). No ageing change in annexin V binding to PMN was observed (young:
35.0% [21.8-53.5]; elderly: 39.6% [27.4-69.8]; p = .42). Platelets had no specific annexin V binding (young: median
molecules of annexin V specific binding 3.8 [0.4-11.3]; elderly: -1.4 [-4.8-1.7]; p = .23). Superficial membrane fluidity was
increased in lymphocytes (TMA anisotropy, young: 0.271 [0.259-0.289]; elderly: 0.262 [0.242-0.279]; p = .004), but not in
platelets (young: 0.273 [0.259-0.293]; elderly: 0.269 [0.248-0.284];p =.12). Lymphocyte annexin V binding correlated with TMA
(r =-.65, p =.022), but not DPH anisotropy (r =-.39, p =.18).
Conclusions. Plasma membrane asymmetry is decreased with age in human lymphocytes, but not in human PMN or
platelets. The increased proportion of lymphocytes with loss of plasma membrane asymmetry corresponds to the ageing
changes in superficial membrane fluidity observed in lymphocytes. Such alterations in lymphocyte plasma membrane structure
with age could account for changes in membrane-bound receptor function described with ageing, and may contribute to
alterations in immune responsiveness and vascular thrombosis seen in older humans.
P
LASMA membrane asymmetry is crucial in the maintenance of membrane and cell function. In eukaryotic cells the
aminophospholipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE), are localized to the inner membrane
leaflet, whereas phosphatidylcholine (PC) and sphingomyelin
are situated in the outer membrane leaflet (1). Loss of plasma
membrane asymmetry, and the resultant appearance of PS on
the outer plasma membrane leaflet, increase the thrombogenic
potential of circulating erythrocytes, polymorphonuclear leukocytes (PMN), and platelets (2), and are also associated with disease exacerbations in sickle cell anemia (3). Increased PS exposure also occurs in apoptosis, and are a trigger for the recognition
and removal of damaged and apoptotic cells by circulating
macrophages (4). The appearance of PS on the outer cell surface has been shown to be a good marker for loss of plasma
membrane asymmetry (1) and can be assessed in vitro by measuring the binding of annexin V, a 35-kDa protein which binds
with high affinity to PS but does not penetrate the cell plasma
membrane (5).
Plasma membrane fluidity is largely determined by the constituent phospholipids in the membrane (6). Although membrane
fluidity in deep hydrophobic regions of the plasma membrane is
probably determined by the amount of cholesterol and the degree
of unsaturation of phospholipid fatty acid side chains (6), superficial membrane fluidity is determined by phospholipid head
groups at the inner or outer plasma membrane surface. In most
cells, the inner membrane leaflet, which contains most of the
PS within the plasma membrane, is more fluid than the outer
(7). If membrane asymmetry is altered, and PS is redistributed
between the inner and outer membrane leaflets, one might expect a corresponding change in plasma membrane fluidity in
the superficial outer layers 'of the membrane (8). Alterations in
membrane fluidity have been shown to alter the function of
many integral membrane receptors (9). Thus, alterations in
membrane asymmetry could alter membrane fluidity and the
function of integral membrane proteins.
Ageing is associated with alterations in membrane lipid composition (10), membrane physical properties (11), receptor
function (12), and antigen expression (13). No previous work
has examined whether phospholipid asymmetry is altered in
cells of ageing humans, although ageing changes in cholesterol
distribution within the plasma membranes of mice synaptic
plasma membranes have been reported (14). A loss of membrane asymmetry with age, leading to changes in membrane
fluidity (8), could account for many of the alterations in membrane function that are observed with increasing age.
Ageing changes in membrane function have been described
in human polymorphonuclear cells (PMN) (15), lymphocytes
(12), and platelets (16). We determined whether ageing changes
in phospholipid asymmetry occurred with age in human PMN,
lymphocytes, and platelets using flow cytometric determination
of fluorescein-conjugated annexin V binding to PS. We also exM601
M602
NOBLEETAL.
Annexin V Binding
ture with 1.5 mL 6% dextran to sediment red blood cells. For
platelet studies, platelet-rich plasma was obtained by centrifugation of sodium citrate-anticoagulated venous blood ( 120g, 10
minutes) from six healthy young and six healthy elderly subjects. Platelet aggregation was prevented by incubation with an
equal volume of the tetrapeptide, gly-pro-arg-pro (2.5 mM,
Sigma, Poole, UK), which binds to fibrinogen and inhibits fibrin
production (18). Five microliters leukocyte-rich plasma or 6 ul,
platelet-rich plasma plus gly-pro-arg-pro was incubated on ice
for 10 minutes with 2.5 ul. fluorescein isothiocyanate (FITC)conjugated annexin V (lmmunotech, supplied by Coulter
Electronics, Luton, UK) and 8 ul. binding buffer (Hepes 10 mM,
NaCl 140 mM, cao, 3 mM). The binding of annexin V to PS is
calcium dependent, requiring an external calcium concentration
of greater than 1 mM (5). To determine nonspecific binding of
annexin V, experiments were repeated with 5 mM EDTA and no
calcium in the binding buffer. Two microliters PE-conjugated
anti-CD45 monoclonal antibody (Dako, High Wycombe, UK)
was added to leukocyte incubations to allow determination of
lymphocyte and PMN populations from the leukocyte suspension (Figure 1). Immediately prior to measurement of annexin
binding on the flow cytometer, samples were diluted with 200
ul, of the appropriate ice-cold binding buffer.
Cell preparation.-To investigate leukocyte annexin V binding, 10 mL heparinized venous blood from 20 healthy young
and 19 healthy elderly subjects was incubated at room tempera-
Flow cytometric analysis.-Samples were analyzed by flow
cytometry (FACStar, Becton Dickinson Immunocytometry
Systems, Mountain View, CA) immediately after incubation
amined ageing changes in deep and superficial membrane fluidity in these cell types using fluorescence polarization, and the
relationship between alterations in membrane asymmetry and
membrane fluidity.
MATERIALS AND METHODS
Subjects
Forty-five healthy young (mean 29 years, range 21-35, 26
male) and 28 healthy elderly (mean 70 years, range 63-81, 15
male) subjects were studied, all of whom met the Senieur
Criteria for immunogerontological studies (17). Young subjects
were recruited from clinical and laboratory staff, and elderly
subjects were independent community dwellers. Ethical approval was obtained from the Newcastle Health Authority/
Newcastle University Joint Ethics Committee and all subjects
gave written informed consent prior to the study. Venous blood
was collected into glass tubes containing appropriate anticoagulant and kept at room temperature prior to use in experiments.
The time from venesection to the start of incubations did not
exceed 15 minutes.
-r
~-r-----------------------------------'
Side Scatter
N
o
o
o
~.4-_---'-_~~""---'-'-'r-T'T---.------r--,r--r"""-rT""1.....-----r--'--'-""T"""'1rtrT"1---::---,--.--r--.-r.-rTi
Figure I. Flow cytometric scatter plot of leukocyte suspension from one young subject, showing identification of lymphocyte and PMN regions. Plot of side
scatter (loglO fluorescence units) against PE-conjugated anti-CD45 binding (logIO fluorescence units) showing gated PMN and lymphocyte regions.
PLASMA MEMBRANE ASYMMETRYAND MEMBRANE FLUIDITY
with annexin V. PMN and lymphocytes were identified from
dot plots of anti-CD45 staining and side scatter (Figure 1). Dot
plots of 10,000 leukocytes or 20,000 platelets were obtained for
each subject. Data were analyzed using WinMDI version 2.7
(Joseph Trotter, Scripps Institute, San Diego, CA) software for
PC (1995). 256-channel frequency histograms for fluorescein
fluorescence (annexin V binding) were obtained for each cell
type. The proportion of PMN or lymphocytes within each cell
population with specific binding of annexin V to PS was determined in young and elderly subjects by placing a marker at the
distal end of the nonspecific binding curve, and measuring the
number of cells distal to this marker (19) (Figure 2). Fluorescence values were converted to median number of molecules of
FITC per cell using quantitative fluorescence bead standards
(Dako Fluorospheres, High Wycombe, UK).
Membrane Fluidity
Cell preparation.-To obtain mixed mononuclear leukocytes (MMC) which comprise >95% lymphocytes, heparinized
blood from 18 healthy young and 19 healthy elderly subjects
o
or--,.......,........,.~..,...,.,....--,--"'r--++~r,..u.:Io..,4-l,mI,l4o\1r~..u.;;'\1
............_
10°
101
102
103
104
Annexin V Binding (Fluorescence Units)
Figure 2. Annexin V binding in human lymphocytes. Frequency histogram
of Fl'I'Csconjugated annexin V fluorescence (FL l-Height) for one representative young and elderly subject. Nonspecific binding (incubation with annexin
and 5 mM EDTA in absence of calcium, young subject): - - ; annexin V
binding (young subject): _ _ ; annexin V binding (elderly subject): _ .
M603
was diluted 1:1 in Dulbecco's phosphate-buffered saline (PBS,
Sigma), and centrifuged on a density gradient (histopaque1077, Sigma) (400g, 25 minutes) (20). The MMC suspension
was washed once with PBS (250g, 15 minutes) and counted
with a hemocytometer.
Platelet-rich plasma was obtained from EDTA-anticoagulated blood from 16 healthy young and 17 healthy elderly subjects by centrifugation (l20g, 10 minutes), washed once, and
resuspended in PBS. Cells were counted using a laboratory
Coulter counter.
PMN were isolated from heparinized blood by centrifugation on histopaque-l 077. The red cell and PMN pellet was resuspended in PBS and subjected to dextran sedimentation. The
PMN pellet obtained from the supernatant was subjected to one
cycle of hypotonic lysis (distilled water 15 seconds, followed
by double-strength PBS), to remove contaminating red blood
cells, and one wash prior to counting with a hemocytometer
(20,21).
Fluidity determinaiion-s-Ccst suspensions (MMC: 106 cells
in 2 mL PBS with 5 mM glutamine; platelets: 107 cells in 2 mL
PBS with 5 mM glucose; PMN 106 cells in 2 mL PBS with
5 mM glucose) were prepared for assessment of membrane fluidity. Membrane fluidity was assessed by measuring the steadystate fluorescence polarization of 1,6-diphenyI-l ,3,5-hexatriene
(DPH), which localizes deep within the plasma membrane bilayer, and its hydrophilic derivative, 1-(4-trimethylammonium)-6-diphenyl-1,3,5-hexatriene (TMA), which is situated
more superficially on the outer plasma membrane leaflet (6).
Two milliliters of cell suspension was incubated with 2 ~M
DPH or TMA at 37°C in an optical glass cuvette. Fluorescence
anisotropy readings were obtained using a computer-controlled
spectrofluorimeter (Perkin Elmer LS50B). Excitation and
emission wavelengths were 360 and 430 nm respectively.
Anisotropy was calculated as previously described (22): (IvvG X Ivh) / (Ivv + 2G X Ivh) where Ivv and Ivh are the intensities detected with the excitation in the vertical (v) and the analyzer in the vertical (v) or horizontal (h) positions, and G is the
correction factor for the optical system. Final dye concentrations
were 2 X 1~ M, and 30-minute preincubation of DPH-containing samples was required to allow dye uptake into cells. For each
sample, between five and eight readings were obtained over 5
minutes, and the mean taken. The short time course ensured that
TMA was located solely in the outer plasma membrane leaflet.
The fluorescence anisotropy of a membrane probe is the inverse
of the fluidity of the lipid region in which it is situated.
Statistical Analysis
Data for each group were found to be distributed nonparametrically, and the Mann-Whitney U test was used for statistical
analysis. Data for annexin binding for all cell types were expressed as ranges from second to fourth quintiles inclusive, due
to the logarithmic distribution of data leading to large upper
ranges in both young and elderly groups. Spearman regression
analysis, to compare annexin V binding with superficial and
deep membrane fluidity, was performed for lymphocyte samples from eight elderly subjects and four young subjects in
whom both parameters had been measured.
NOBLEETAL.
M604
Table 2. DPH and TMA Anisotropy in Healthy Young
and Healthy Elderly Subjects*
Table I. Specific Annexin Binding to PS
in Human Lymphocytes, PMN, and Platelets
Young
Elderly
Percent of cells with specific annexin V binding
8.5% (5.2-17.2)
Lymphocytes
5.3% (3.8-8.7)
39.6% (27.4-69.8)
PMN
35.0% (21.8-53.5)
Specific annexin V binding (molecules)
Lymphocytes
4.7 X 103 (3.8--6.3)
PMN
15.6 X lQ3 (9.0-24.3)
Platelets
3.8 (0.4-11.3)
4.6 X 10-' (3.9--6.6)
13.8 X 103 (10.0-20.2)
-1.4 (-4.8-1.7)
Cell Type
Young
Elderly
p
DPH
MMC
Platelet
0.173 (0.152--0.186)
0.181 (0.164-0.196)
0.161 (0.126--0.182)
0.160 (0.144-0.194)
.017
.007
TMA
MMC
Platelet
0.271 (0.259--0.289)
0.273 (0.259--0.293)
0.262 (0.242--0.279)
0.269 (0.248--0.284)
.12
p
.028
.42
.94
.62
.23
Specific annexin binding to PS was determined by incubating leukocyte or
platelet suspensions with annexin in the presence of calcium (1.7 mM) to measure total binding, or EDTA (5 mM) to measure nonspecific binding. The percent of lymphocytes or PMN distal to nonspecific binding on the annexin V
256-channel frequency histogram and median fluorescence of these cells were
calculated. Median fluorescence values were converted to molecules of annexin
V bound by calibration with fluorescent bead standards. n =20 young and 19
elderly subjects for lymphocyte and PMN data; 6 young and 6 elderly subjects
for platelet data. Data are all second to fourth quintile ranges. Mann- Whitney U
test was used for statistical significance.
RESULTS
Annexin V Binding in Lymphocytes
Following incubation with FITC-conjugated annexin V, all
samples had increased fluorescences compared to control samples. However, a large proportion of this increase was due to
nonspecific annexin V binding as it occurred in the absence of
calcium. Most lymphocytes exhibited only nonspecific binding,
but a small proportion had specific annexin V binding to PS, indicating that these cells had decreased membrane asymmetry.
The proportion of lymphocytes with specific annexin V binding
was significantly increased in elderly subjects compared to
young (p = .028). There was no difference between young and
elderly groups in the number of molecules of annexin binding
specifically to PS (p = .94) in cells with reduced membrane
asymmetry (Table 1).
AnnexinV Binding in PMN
In contrast to lymphocytes, most PMN had some specific annexin V binding; in some subjects, more than one population of
cells with differing amounts of annexin V binding were present.
Specific annexin V binding to PS in PMN was unchanged with
increasing age, both in the proportion of cells lying outside the
range for nonspecific binding (p = .42), and in the number of
molecules of annexin V specifically bound on these cells (p =
.62) (Table 1).
Annexin V Binding in Platelets
There was no specific binding of annexin V to PS on resting
platelets from either young or elderly subjects (Table 1).
Fluorescence Polarization Measurements
DPH anisotropy was decreased in the elderly subjects for
both human platelets (p = .007) and MMC (p = .017), indicating that the fluidity at the core of the membrane in both platelets
and MMC was increased in healthy elderly subjects compared
to young. TMA anisotropy was reduced in MMC from elderly
subjects (p = .004). In contrast, platelet TMA anisotropy was
unchanged [95% confidence interval for differences between
.004-
*Membrane fluidity of MMC and platelets was determined by measuring
the steady-state fluorescence polarization of DPH and TMA in healthy young
and healthy elderly subjects. Data are expressed as median (range) for each
group. n = 18 young and 19 elderly subjects for MMC data; 16 young and 17
elderly subjects for platelet data. Mann-Whitney U test was used for statistical
significance.
medians: (-0.003,0.160) p = .12] (Table 2). Therefore membrane fluidity in the superficial layers of the outer leaflet of the
plasma membrane was significantly increased in MMC, but not
platelets, from elderly subjects.
Measurements of PMN membrane fluidity were unreliable
due to high intersubject variability.This was related to the hypotonic lysis stage ofPMN preparation used to remove contaminating red blood cells. Unlike PMN, MMC can be prepared on a
density gradient, without the need to remove red blood cells.
When isolated MMC were subjected to hypotonic lysis, large
changes in anisotropy were observed for both TMA (young [n =
2]: control mean 0.277, with hypotonic lysis 0.267; elderly [n =
2]: control 0.284, with hypotonic lysis 0.270) and DPH (young:
control 0.180, with hypotonic lysis 0.187; elderly: control
0.200, with hypotonic lysis 0.187), which were greater than expected intrasubject variability for lymphocyte anisotropy (coefficient of variation: TMA 1.87%; DPH 2.47%). Thus hypotonic
lysis could disrupt membrane properties in both MMC and
PMN.
Regression analysis was performed in eight elderly subjects
and four young subjects, in whom both specific annexin V binding and membrane fluidity measurements in lymphocytes were
obtained. Lymphocyte annexin V binding correlated with TMA
anisotropy (R =-.65, p = .022) (Figure 3), but not with DPH
anisotropy (R =-.39,p = .18).
DISCUSSION
These results demonstrate an increase in the proportion of
lymphocytes with specific binding of annexin V to PS in elderly subjects compared to young, indicating that an increased
proportion of lymphocytes in elderly individuals has lost membrane asymmetry. No ageing change in the amount ofPS present on the outer surface of PMN was found, suggesting that
plasma membrane asymmetry in PMN is preserved with ageing.
The finding that resting platelets have no external PS expression
is not surprising, as the appearance of PS on the surface of
platelets would make them highly thrombogenic, and thus likely
to be rapidly removed from the circulation (2). It is possible that
the increased PS expression on lymphocytes from older subjects
could be related to alterations in lymphocyte subsets with ageing (23), and further studies are required to investigate this possibility. The ageing change in lymphocyte plasma membrane
asymmetry may be related to the previously reported ageingrelated increase in lymphocyte apoptosis (24) and could have
PLASMA MEMBRANE ASYMMETRY AND MEMBRANE FLUIDITY
.29
M605
0
.28
~
00
b
0
0
.27
00
...
(5
0
• ...-1
~
<r:
<r:
~
...
.26
...
~
.25
...
.24
0
10
5
15
20
25
Annexin V Binding - % Of Cells With Specific Binding
Figure 3. Relationship between PS expression and superficial membrane fluidity in human lymphocytes. PS expression was measured by annexin V binding
(expressed as median number of molecules per cell for each subject) and membrane fluidity by TMA anisotropy in 12 healthy subjects (8 elderly., 4 young 0). PS
expression was inversely correlated with TMA anisotropy (r -.65, p .022), indicating increased superficial membrane fluidity associated with increased PS
expression on the outer cell membrane surface.
=
=
important implications for the function of membrane bound
receptors, thrombogenesis, and cell survival.
Plasma membrane asymmetry is thought to be regulated by
three membrane enzymes: an ATP-requiring floppase which
moves all phospholipids indiscriminately from the inner to the
outer plasma membrane leaflet; an ATP-requiring aminophospholipid translocase, which transports aminophospholipids from
the outer to the inner membrane leaflet; and a Ca 2+-dependent
scramblase, which moves phospholipids rapidly in both directions across the membrane, leading to a loss of asymmetry (1).
Zhao and colleagues have shown that the amount of scramblase
in different cell types is related to the proportion of cells with
loss of membrane asymmetry (19). Our finding that ageing is
associated with an increased proportion of lymphocytes with
loss of membrane asymmetry could therefore be explained by
alterations in the amount or activity of scramblase with increased age.
Techniques which have been used to demonstrate phospholipid asymmetry include the use of chemical reagents or phospholipases to alter the phospholipid composition of the outer
membrane leaflet, strategies to monitor the redistribution of exogenously added labelled phospholipids across the bilayer, and
the activation of hemostatic processes such as the PS-mediated
activation of prothrombin to thrombin by prothrombinase
(5,25). These have demonstrated that the appearance of PS on
the outer plasma membrane leaflet is a good marker for loss of
plasma membrane asymmetry. Annexin V binds to aminophospholipids, but cannot cross the plasma membrane. Binding to
PS occurs with much higher affinity and at lower calcium concentrations than binding to PE (5), and is therefore an effective
method of measuring the appearance of PS on the outer leaflet
of the plasma membrane. The use of annexin V to detect cell
surface PS has been validated against the prothrombinase assay
in red blood cells (5).
Plasma membrane fluidity is related to the phospholipid and
cholesterol content of the plasma membrane. As membrane
asymmetry is lost, and PS redistributed to the outer membrane
leaflet, one might expect a corresponding increase in superficial
plasma membrane fluidity. The correlation between the proportion of lymphocytes with specific annexin V binding to PS and
TMA, but not DPH, anisotropy supports this hypothesis. No
loss of plasma membrane asymmetry or corresponding increase
in superficial membrane fluidity with age was observed in
platelets. None of the subjects in this study had extremes of
daily diet that could have influenced the membrane fluidity results. PMN membrane fluidity could not be measured in this
study due to the preparation methods normally used (21), causing high inter- and intrasubject variability in results.
M606
NOBLE ETAL.
In conclusion, these data demonstrate an age-associated increase in PS expression and thereby reduced membrane asymmetry in lymphocytes, but not PMN or platelets. The increase
in lymphocyte PS expression correlates with an increase in
superficial membrane fluidity observed in older subjects.
Reductions in lymphocyte plasma membrane asymmetry are
likely to be associated with alterations in membrane bound enzyme and ion channel function which could account for the
changes in cell membrane receptor-mediated function described with ageing. The reduction in plasma membrane function and alterations in superficial membrane fluidity associated
with loss of plasma membrane asymmetry in human lymphocytes could contribute to the decline in cell-mediated immunity
with age which leads to increased rates of infection (26) and
malignant disease (27) in older people.
ACKNOWlEOOMENT
JMN is a Medical Research Council Clinical Training Fellow. TIlT is supported by the Northern Counties Kidney Research Fund. Address correspondence to Dr. G.A. Ford, Wolfson Unit of Clinical Pharmacology, University of
Newcastle Upon Tyne, Claremont Place, Newcastle Upon Tyne NE2 4HH UK.
E-mail: [email protected]
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ReceivedJuly 20,1998
AcceptedApril 15, 1999