Clin Exp Immunol 2000; 120:307316

Frequent reversible membrane damage in peripheral blood B cells in human T cell

lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic
paraparesis (HAM/TSP)
Y. FURUKAWA, C. R. M. BANGHAM, G. P. TAYLOR*, J. N. WEBER* & M. OSAME Department of Immunology
and *Department of GU Medicine and Communicable Disease, Imperial College School of Medicine at St Mary's, London, UK, and
Third Department of Internal Medicine, Faculty of Medicine, Kagoshima University, Kagoshima, Japan
(Accepted for publication 14 January 2000)

SUMMARY
Apoptosis in peripheral blood lymphocyte populations in HTLV-I-infected people in vivo was
examined, to study the lymphocyte dynamics in HTLV-I infection. Freshly isolated lymphocytes from
10 non-infected healthy people, eight asymptomatic HTLV-I carriers and 15 patients with HAM/TSP
were stained with FITC-labelled annexin V to detect phosphatidylserine (PS) residue exposure at the
outer plasma membrane leaflet as an early marker of apoptosis. There was no significant difference in
annexin V positivity in CD41 and CD81 lymphocytes between non-infected subjects, asymptomatic
carriers and HAM/TSP patients, but there was a greatly increased exposure of PS on CD191
lymphocytes (B cells) detected by FITCannexin V in 12 out of 15 (80%) HAM/TSP patients, while
only two out of eight (25%) asymptomatic carriers and none of the non-infected healthy people showed
this aberrant PS exposure on B cells. The intensity of annexin V staining of B cells in HAM/TSP was
intermediate, as distinct from the high annexin V staining on advanced apoptotic cells. However,
annexin V positivity was decreased when the cells were stained after 24 h of culture, suggesting that the
intermediate PS exposure on the B cell in HAM/TSP is not a consequence of an apoptotic process, but
rather reflects reversible membrane damage. B cells with PS exposure in vivo might provide a site for
coagulation and inflammation, and so contribute to the pathogenesis of HAM/TSP and its complications.
Keywords

HAM/TSP B lymphocyte

annexin V

INTRODUCTION
HTLV-I is the aetiologic agent of HAM/TSP [1,2] and adult T cell
leukaemia (ATL) [3,4]. One of the characteristic features of
HAM/TSP is a high proviral load [5], and most people infected
with HTLV-I have a strong cytotoxic T lymphocyte (CTL)
response to the Tax antigen [6,7] of the virus. If the CTL are
active in vivo, we reasoned that it may be possible to detect
apoptosis of the target cells infected with HTLV-I, which are
CD41 lymphocytes. We also thought that the CTL themselves
might be undergoing apoptosis, because of chronic antigenic
stimulation by the virus [8]. In HTLV-I infection there are reports
that FasL can induce apoptosis of ATL cells in vitro [9].
Furthermore, Tax can either induce [10,11] or inhibit [12,13]
apoptosis of different transfected or HTLV-I-infected cell lines.
However, these results were obtained in vitro, and may not reflect
the lymphocyte turnover in vivo. In HIV infection there are
Correspondence: C. R. M. Bangham, Department of Immunology,
Imperial College School of Medicine at St Mary's, Norfolk Place, London
W2 1PG, UK.
E-mail: c.bangham@ic.ac.uk
q 2000 Blackwell Science

membrane damage

apoptosis

reports that not only CD41 and CD81 T cells [14], but also B cells
are undergoing apoptosis [15,16]. Based on these findings, we
sought early apoptotic changes of lymphocytes in different
populations from freshly isolated peripheral blood mononuclear
cells (PBMC).
The problem with detecting apoptotic cells in freshly isolated
peripheral blood is that late apoptotic cells detectable by the
conventional electrophoresis method (DNA ladder) or terminal
deoxynucleotidyl transferase-(TdT) mediated dUTP nick end
labelling (TUNEL) are cleared rapidly by the mononuclear
phagocyte system, so that apoptotic cells cannot be detected
in vivo. To overcome this problem, we detected a change
normally associated with early apoptosis: the exposure of
phosphatidylserine (PS) residue on the outer plasma membrane
leaflet detected by FITC-conjugated annexin V [17]. Vital cells
maintain plasma membrane asymmetry and PS is kept on the inner
plasma membrane. Apoptotic lymphocytes expose PS at the outer
membrane leaflet early after onset of the execution effector phase
of apoptosis, even though the integrity of the membrane has not
been compromised at this stage [18]. Annexin V was shown to
interact strongly and specifically with PS and can be used to detect

307

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Y. Furukawa et al:

apoptosis [19]. We used flow cytometry with annexin V to detect

PS on the outer membrane leaflet. In this study, we could not
detect a significant increase of apoptosis in CD41 and CD81
lymphocytes in HAM/TSP. However, surprisingly there was a
very high degree of abnormal exposure of PS residues at the B cell
outer plasma membrane leaflet mainly in HAM/TSP patients.
However, this annexin V positivity was reversible after short-term
culture, and the intensity of staining did not correlate with the
degree of advanced apoptotic change after culture. The results
show that there is widespread reversible membrane damage in
peripheral blood B cells in HAM/TSP. We also measured anticardiolipin and anti-b 2-glycoprotein I (b 2-GPI) antibodies,
because such antibodies are often found in patients with evidence
of membrane damage, and they are of pathogenic significance

because they are associated with a thrombotic tendency [20]. We

discuss the possible involvement of membrane-damaged B cells in
the pathogenesis of HAM/TSP.
PATIENTS AND METHODS
Patients and lymphocyte preparation
Cell samples were derived from 10 uninfected healthy adult
controls, eight HTLV-I-infected asymptomatic carriers and 15
HAM/TSP patients. The HTLV-I-infected individuals were
predominantly Afro-Caribbean in origin. Peripheral venous blood
was collected and heparinized and kept on ice until cell
preparation and analysis. PBMC were prepared using Histopaque
(Sigma, Poole, UK).

Asymptomatic
carrier

Non-infected

HAM/TSP

0.5

0.3
(0.6)

3.5

0.6
(1.5)

27.9

0.8
(2.5)

43.5

55.7

56.1

39.7

40.4

30.9

Annexin V

CD4
0.7

0.2
(0.7)

3.7

1
(2.4)

27

0.7
(2.7)

76.7

22.5

56.1

39.2

46.6

25.7

CD8

0.5

0.1
(1.7)

3.9

0.9
(9.5)

1.8

26.1
(87.5)

90.8

8.5

86.5

8.7

68.4

3.7

CD19
Fig. 1. FITCannexin V positivity in lymphocyte populations. Two-colour staining was carried out for the simultaneous determination of surface
phenotype and annexin V positivity. Cells from a representative healthy non-infected person, an asymptomatic HTLV-I carrier and a HAM/TSP patient
were simultaneously labelled with FITCannexin V and PE-conjugated anti-CD4 or anti-CD8 or anti-CD19 antibodies. Numbers refer to the percentage of
cells in each quadrant. Numbers in parentheses refer to the percentage of annexin V1 cells among the CD41 or CD81 or CD191 populations. Frequent
annexin V1 B (CD191) lymphocytes were observed in the HAM/TSP patient.
q 2000 Blackwell Science Ltd, Clinical and Experimental Immunology, 120:307316

Detection of apoptotic cells

Detection of apoptotic cells was by TUNEL with B cell surface
marker. PBMC (106) were washed with PBS and centrifuged at
300 g for 5 min. The pellet was resuspended in 100 m l of
PBS22% fetal calf serum (FCS) and 20 m l of PE-conjugated antihCD19 were added and incubated for 30 min on ice. Cells were
washed twice with ice-cold PBS, and then fixed with 1 ml of fresh
2% paraformaldehyde (prewarmed to 378C) and incubated for
30 min at room temperature on a horizontal shaker (150 rev/min).
Cells were then permeabilized with 100 m l 01% Triton X-100 in
01% sodium citrate for 2 min on ice and then washed twice with
PBS. Cells were resuspended in TUNEL buffer with TdT
(Boehringer Mannheim), and incubated for 60 min at 378C in
the incubator and then washed twice with PBS and analysed by
flow cytometry.
Measurement of HTLV-I proviral load
HTLV-I proviral load was quantified using a HTLV-I-specific
polymerase chain reaction (PCR) in serial dilutions of DNA
extracted from PBMC [21]. The proviral load was calculated from

309

100

26.0
35.4

53.9
30.0

80

60

40

FITC-labelled annexin V and surface marker antibody staining

Annexin V staining was performed according to the manufacturer's manual (Boehringer Mannheim, Lewes, UK) with minor
modifications. Cells (5  105) were washed with PBS and
resuspended in 100 m l of HEPES buffer (10 mm HEPES/NaOH,
pH 74, 150 mm NaCl, 5 mm KCl, 1 mm MgCl2, 18 mm CaCl2)
containing FITCannexin V (2 m g/ml final concentration), then
incubated for 30 min on ice with 20 m l of PE-conjugated
antibodies to surface markers (anti-hCD4 (13B8.2), anti-hCD8
(B9.11), anti-hCD19 (J4.119); Immunotech, Luton, UK). In some
experiments, propidium iodide (PI) dye (1 m g/ml final concentration; Sigma) was added to discriminate necrotic cells from
apoptotic cells. After staining, cells were washed with HEPES
buffer and resuspended in 05 ml of 1% paraformaldehyde and
analysed on an Epics Profile II flow cytometer (Coulter, High
Wycombe, UK). The threshold for annexin V was made by
staining cells without CaCl2, and the threshold for surface markers
was made with isotypic control antibody. Cells were gated on (i)
the major population of normal-sized lymphoid cells, according to
the forward and side scatters, and (ii) a wider gate to include the
advanced apoptotic cells. Annexin V staining was classified as
negative (under the threshold), highly positive (as high as PI1
cells), and intermediately positive (between negative and highly
positive). Cells were also simultaneously labelled with FITC
annexin V, PE-conjugated anti-hCD21 (LB21; Serotec, Oxford,
UK) and PE covalently linked to cyanin 5.1 (PC5)-conjugated antihCD19 (J4.119; Immunotech) antibody and analysed as described
above. In certain experiments, cells were simultaneously labelled
with FITC-conjugated anti-hCD95 (UB2; Immunotech) or FITCconjugated anti-hIgD (HJ9; Sigma) or FITC-conjugated antihCD40 (EA-5; Ancell, Nottingham, UK) with PE-conjugated
anti-hCD19 (J4.119) antibody and analysed as described above.
In other experiments, cells were simultaneously labelled with
FITC-conjugated anti-FasL (H11; Alexis Corp., Nottingham, UK)
with PE-conjugated antibodies to surface markers (anti-hCD4
(13B8.2), anti-hCD8 (B9.11), anti-hCD19 (J4.119)) and analysed
as described above. In certain experiments, cells were labelled
with FITC-conjugated anti-hCD154 (CD40 ligand; 2431;
Ancell) with PE-conjugated anti-hCD19 (J4.119) and analysed.

Percent annexin V cells in lymphocyte populations

Reversible B cell membrane damage in HAM/TSP

20

4 .1
3 .7
.
.
3.4 2 3 2 0
.
1 4

8.3
.
5.6 6 8
4.9
.
6.0 5 9

9 .0
4.0

Co. A.C. HAM

CD8

Co. A.C. HAM

CD19

0
Co. A.C. HAM
CD4

P < 0.001

Fig. 2. FITCannexin V positivity in lymphocyte populations in 10 noninfected control people (Co.), eight asymptomatic carriers (A.C.) and 15
HAM/TSP patients (HAM). Each point represents the mean value of one
person from two to five different experiments. Numbers indicate the mean
values ^ s.d. Bars mark mean values ^ s.d. Annexin V positivity in
CD191 cells (B cells) was significantly higher in HAM/TSP patients than
in non-infected people (P , 0001).

the Poisson distribution of PCR negatives at the endpoint dilution.

Viral load was expressed as number of copies per 100 PBMC.
B cell isolation using magnetic cell sorting
In some experiments B cells were negatively isolated using
MACS magnetic microbeads (Miltenyi Biotec, Bisley, UK). PBMC
were washed with ice-cold PBS25% FCS and stained with
magnetic microbead-conjugated anti-hCD16 for 30 min and then
further stained with magnetic microbead-conjugated anti-hCD14,
Table 1. Surface marker expression in non-infected people, asymptomatic
carriers and HAM/TSP patients (%)

CD4
CD8
CD19
CD95 (Fas)

CD951/CD41
CD951/CD81

CD951/CD191

q 2000 Blackwell Science Ltd, Clinical and Experimental Immunology, 120:307316

Non-infected

Asymptomatic carrier

502 ^ 80
(n 10)
300 ^ 64
(n 10)
97 ^ 44
(n 10)
263 ^ 98
(n 10)

518 ^ 93
472 ^ 73
(n 8)
(n 13)
279 ^ 62
300 ^ 113
(n 8)
(n 13)
115 ^ 49
127 ^ 64
(n 8)
(n 14)
301 ^ 51
449 ^ 132
(n 7)
(n 13)
001 , P , 002
0001 , P , 001
437 ^ 134
569 ^ 127
(n 4)
(n 10)
327 ^ 132
564 ^ 177
(n 4)
(n 9)
001 , P , 002
0001 , P , 001
53 ^ 28
72 ^ 54
(n 6)
(n 13)

393 ^ 215
(n 4)
232 ^ 159
(n 5)
43 ^ 24
(n 10)

HAM/TSP

310

Y. Furukawa et al:
0h

Forward scatter

(a)

24 h

1
2

1
2

(b)
* HAM 1

Side scatter
* HAM 2
HAM 3

Normal gate (gate 1)

Annexin V

1.6

0.1
(0.2)

2.7

25.9
(87.4)
68.7

4.2
(10.4)
56.6

3.7

0.9
(2.2)

35.6

HAM 4
HAM 6
* HAM 7
* HAM 8
* HAM 9
* HAM 10
* HAM 11
HAM 12

CD19

* A.C. 1
* A.C. 2

Wide gate (gate 2)

Annexin V

2.6

0.6
(2.0)

4.8

24.3
(85.4)
69

3.6

1.9
(5.4)

20

40

60

80

100

Percent annexin V positivity

3.7
(10.5)
60.2

29.4

CD19
Fig. 3. Time course study of annexin V1 B cells in HAM/TSP. (a) Freshly isolated peripheral blood mononuclear cells (PBMC) (left column) and 24 h
cultured PBMC (right column) from HAM/TSP patient (case 1 in Fig. 2) were labelled with FITCannexin V and PE-conjugated anti-CD19 antibody and
analysed in the normal PBL gate (gate 1) and in the wide gate (gate 2). Annexin V1 B cells were further divided into regions of high and intermediate
annexin V staining. Numbers refer to the percentage of cells in each region. Numbers in parentheses refer to the percentage of annexin V1 cells among the
CD191 population. (b) Time course study of 11 HAM/TSP patients and two asymptomatic carriers with remarkable annexin V positivity in B cells. B,
Percentage annexin V intermediately positive cells in fresh B cell population; A, percentage annexin V intermediately positive cells in B cell after 24 h
culture. Hatched bars represent percentage annexin V highly positive cells after 24 h culture. Case numbers in HAM patients are the same as in Fig. 2.
*Cases in which the annexin V positivity (high 1 intermediate) decreased after 24 h culture.

anti-hCD56 and anti-hCD3 for 15 min. Cells were then passed

through the column matrix in the magnetic field, and the eluted
cells were collected. The purity of negatively selected B cells was
9598% CD191 by flow cytometry.
Time course study of B cells in HAM/TSP
PBMC (106/ml) were cultured in RPMI with l-glutamine
supplemented with 10% FCS, penicillin, and streptomycin.
Freshly isolated PBMC and cultured PBMC or B cells were

labelled with FITCannexin V and PE-conjugated anti-CD19

antibody and analysed in the normal PBL gate and in the wide
gate. Cells were also analysed by the TUNEL assay. PBMC were
also cultured in RPMI with 10% FCS in the presence of 20 m g/ml
soluble fusion proteins (Fas-Fc, TNF-R1-Fc, LARD-Fc, DR4-Fc,
provided by Drs G. R. Screaton and X.-N. Xu, Oxford, UK)
comprising the extracellular domains of the receptors fused to the
constant Fc domain of the IgG1 heavy chain. In certain
experiments, PBMC and negatively isolated B cells were cultured

q 2000 Blackwell Science Ltd, Clinical and Experimental Immunology, 120:307316

35

Percent annexin V high positive

cells after culture

20.0

30
25
20

annexin V (intermediate)
annexin V (high)
TUNEL

15
10
5
0
24 h

48 h

CD4

15.0

10.0

r = 0.50
0.001 < P < 0.01

5.0

0.0 .
00

2.0

4 .0

6.0

8.0

10.0

Percent annexin V intermediate positive

cells in fresh PBMC

35

Wide gate

(b)

30

Percent annexin V high positive

cells after culture

Percent positivity in B lymphocyte

0h

311

(a)

Normal gate

25
20
15
10
5
0
0h

24 h

48 h

Fig. 4. Comparison between annexin V intermediate or high positive B

cells and terminal deoxynucleotidyl transferase-(TdT) mediated dUTP
nick end labelling (TUNEL)-positive B cells in fresh and after culture from
a non-infected person. Freshly isolated peripheral blood mononuclear cells
(PBMC) and cultured PBMC for 24 h to 48 h were labelled with FITC
annexin V and PE-conjugated anti-CD19 antibody and analysed in normal
lymphocyte gate (upper panel) and in wide gate (lower panel). Cells were
also analysed by the TUNEL assay.

with RPMI containing 10% patients' own serum or normal human

AB serum for 24 h and labelled with FITCannexin V and PEconjugated anti-CD19 antibody and analysed in the normal PBL
gate and in the wide gate.
Isolation of annexin V binding cells
Annexin V binding cells were isolated using an apoptotic cell
isolation kit (Miltenyi Biotec). Ten million PBMC were
suspended in 80 m l binding buffer, and 20 m l of annexin Vcoupled microbeads were added to this solution and incubated at
68C for 15 min. Cells were then washed with 2 ml of binding
buffer and resuspended in 500 m l of binding buffer. The cells
were passed through the RS1 column in a magnetic field, and the
annexin V1 cells were collected.
Detection of caspase 3 activation in B cells
Activation of caspase 3 was detected at the single-cell level, using
the profluorescent caspase 3 substrate PhiPhiLux-G1D2
(OncoImmune Inc., Gaithersburg, MD) in FL1 in combination
with PE-conjugated anti-CD19 antibody staining in FL2 by flow

20.0

CD8

15.0

10.0

r = 0.49
.
0 001 < P < 0.01
5.0

0.0 .
00

5.0

10.0

15.0

20.0

25.0

Percent annexin V intermediate positive

cells in fresh PBMC
(c)
40.0
Percent annexin V high positive
cells after culture

Percent positivity in B lymphocyte

Reversible B cell membrane damage in HAM/TSP

CD19

35.0
30.0
25.0
20.0
15.0

r = 0.15
0.1 < P < 0.5

10.0
5.0
0.0
0.0

20.0

40.0

60.0

80.0

100.0

Percent annexin V intermediate positive

cells in fresh PBMC
Fig. 5. Test for correlation between the annexin V positivity (intermediately positive cells) in the normal gate before culture, and annexin V
highly stained cells in the wide gate after 24 h culture in the mixed
peripheral blood mononuclear cells (PBMC) in each lymphocyte
population. (a) In CD41 lymphocyte population. (b) In CD81 lymphocyte
population. (c) In CD191 lymphocyte population. W, Non-infected people;
O, asymptomatic carriers; X, HAM/TSP patients.

cytometry. One million PBMC were centrifuged at 200 g for

5 min and 50 m 1 of 10 m m substrate solution (PhiPhiLux-G1D2)
were added to the pellet and incubated in a 5% CO2 incubator at
378C for 60 min. Cells were washed with ice-cold flow cytometry

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312

Y. Furukawa et al:

buffer and centrifuged at 200 g for 5 min. The cell pellet was
resuspended in 100 m l flow cytometry buffer, 20 m 1 PEconjugated anti-hCD19 (J4.119) were added and incubated on
ice for 30 min. The cells were then washed with flow cytometry
buffer, resuspended in 1 ml of cytometry buffer and analysed.

HAM/TSP
0 day

Uninfected
0 day

(a)

(0.7%)

0.1%

1.4%

Uninfected
3 day
(97.5%)

15.9%

8.5%

103

103

10
Annexin V

Detection of anti-cardiolipin antibody and anti-b 2-GPI antibody

by ELISA
Both antibodies were measured using commercial kits (Shield
Diagnostics, Dundee, UK) according to the manufacturer's
manual. Anti-cardiolipin (CL) antibody was called negative when

10

10

10

101

101

101

101

10

102

8.0%

81.0%

10.8%

(42.5%)

0.4%

13.5%
0

10

0.2%

103

10

10

10

10

10

100
100

101

102

103

CD19

Caspase 3

10

(7.1%)

0.4%

0.1%
3

102

(3.9%)

0.5%

0.1%
10

103

102

102

5.2%

9.2%
10

10

10

101

102

103

100
100

101

102

(36.5%)

5.3%

12.3%
1

100 0
10

0.5%

100
100

103

101

102

103

CD19
(b)
Annexin V beads positively selected PBMC
HAM/TSP
0 day

Caspase 3

(3.3%)

3.1%

0.3%
10

Uninfected
3 day

10

102

102
3.3%

21.4%

93.3%

101

100
0
10

(89.5%)

69.0%

1.5%

8.1%

101

101

102

100
0
10

103

101

102

103

CD19
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Reversible B cell membrane damage in HAM/TSP

lower than 16 PL units/ml, borderline at 1621 PL units/ml, and
positive at . 21 PL units/ml. Anti-b 2-GPI antibody was called
negative when , 10 U/ml, borderline at 1015 U/ml, and positive
at . 15 U/ml, according to the manufacturer's manual.
Statistical analysis
Fluorescent staining intensities are given as mean ^ s.d. percent
positive lymphocytes. The means of different populations were
compared using Student's t-test (two-tailed).
RESULTS
Annexin V staining and surface marker staining
A typical result of annexin V staining with surface marker
antibodies is shown in Fig. 1. The frequencies of annexin V1
lymphocytes were not remarkable in the CD41 or CD81
populations, but there was remarkably high FITCannexin V
staining of CD191 lymphocytes in the HAM/TSP patient.
Annexin V1 CD191 B lymphocytes were widespread in both
clinically progressing and stable HAM/TSP. Twelve out of 15
HAM/TSP patients (80%) showed a large proportion of annexin
V1 B cells (539 ^ 300%, n 15) in contrast to uninfected
people (90 ^ 40%, n 10), and the difference between the
group means was significant (P , 0001). Asymptomatic carriers
showed a more variable range of annexin V1 B cells (260 ^
354%, n 8). Two out of eight asymptomatic carriers showed a
high proportion of annexin V1 B cells, similar to HAM/TSP, but
the remaining six asymptomatic carriers showed lower positivity,
similar to uninfected people (Fig. 2). The B cell frequency (as
percentage of PBMC) did not differ between the three groups
(Table 1). CD95 (Fas) expression was significantly higher in
HAM/TSP patients than in non-infected people (0001 ,
P , 001), and was also higher than in asymptomatic carriers
(001 , P , 002). However, this difference was found mainly in
the CD81 population and not in the CD191 population (Table 1).
Annexin V positivity in the B cell population did not correlate
with the percentage of either total B cells or surface IgD1 B cells.
Annexin V1 B cells were found in both CD21-positive and
-negative cells (data not shown). FasL was not detected on the
surface of either CD41, CD81 or CD191 cells in uninfected
people, asymptomatic carriers or HAM/TSP patients (data not
shown).
HTLV-I viral load and annexin V positivity in B cell population
Mean HTLV-I proviral load in HAM/TSP patients (102 copies/
100 PBMC) was significantly higher than in asymptomatic
carriers (006 copies/100 PBMC; 0001 , P , 001). There was
no correlation between the HTLV-I viral load and annexin V

313

positivity in the B cell population within any subject group. We

also tested for correlation between viral load and CD95 expression
or between viral load and annexin V positivity in each
subpopulation in HAM/TSP. However, there was no significant
correlation (data not shown).
Time course study of annexin V1 CD191 lymphocytes
A typical result of annexin V staining before culture and after 24 h
culture is shown in Fig. 3a. In this case, the majority of CD191 B
lymphocytes were stained with annexin V before culture, but this
decreased after 24 h culture. In many cases, the total of annexin
V1 (intermediate 1 high) B cells after 24 h culture was less than
the annexin V1 B cells before culture (Fig. 3b), and the total
number of B cells increased only slightly after 24 h culture (final
cell number 115 ^ 21% of starting cell number; n 11;
002 , P , 005). These results suggest that not all of the
annexin V1 cells were undergoing apoptosis. When we also
analysed cells present in the wide gate as well as in the normal
lymphocyte gate, there were annexin V highly positive cells in the
wide gate; the number of these cells increased after culture
(Fig. 3a). PI-stained cells were seen in this annexin V highly
stained CD191 cell population and not in the annexin V
intermediate cells, which suggests that the annexin V highly
stained cells were more advanced apoptotic or secondary necrotic
cells (data not shown). The proportion of CD191 cells in the wide
gate that were positive in the TUNEL assay reached a similar
proportion to the annexin V highly positive CD191 cells in the
wide gate (Fig. 4). We also compared annexin V positivity
(intermediately positive cells) in the normal gate before culture,
and annexin V highly stained cells in the wide gate after 24 h
culture in the PBMC in each lymphocyte population. There was a
positive correlation between the annexin V positivity (intermediately positive cells) in the normal gate before culture and annexin
V highly stained cells in the wide gate after 24 h culture in the
mixed PBMC in both CD41 and CD81 cells. However, no such
correlation was seen in CD191 cells (Fig. 5). We also blocked the
known death receptor ligands, using soluble form of these receptors
(FAS-Fc, TNFR1-Fc, LARD-Fc, DR4-Fc) to test whether there was
a contribution of these pathways in PS exposure. However, there
was no difference in annexin V positivity after 24 h culture with
or without these blocking reagents (data not shown).
Detection of caspase 3 activation in B cells
PBMC from a HAM/TSP patient with high annexin VFITC
positivity in B cells were examined for caspase activity present in
the B cell population. While 975% of the B cells were positive for
annexin V, caspase 3 was activated in only 39% of the B cells
(Fig. 6a, middle column). Similar results were obtained from the

Fig. 6. Flow cytometric analysis of caspase 3 activation and annexin V positivity in B cells from a HAM/TSP patient. (a) Upper row, FITCannexin V and
PE-conjugated anti-CD19 antibody was simultaneously labelled. Numbers refer to the percentage of cells in each quadrant. Numbers in parentheses refer to
the percentage of annexin V1 cells among the CD191 population. Lower row, caspase 3-activated cells are shown on the ordinate and CD191 cells on the
abscissa. Numbers refer to the percentage of cells in each quadrant. Numbers in parentheses refer to the percentage of caspase 3-activated cells among the
CD191 population. Left column, fresh peripheral blood mononuclear cells (PBMC) from an uninfected person. Middle column, fresh PBMC from a HAM/
TSP patient. Right column, PBMC after 3 days culture of uninfected person. In the HAM/TSP patient, 975% of the fresh B cells were intermediately
FITCannexin V1 however, caspase 3 was activated only in 39% of the B cells (middle column). After 3 days culture of uninfected PBMC, late apoptotic
B cells were highly stained with annexin V and caspase 3 was activated (right column). High threshold for caspase 3 activation in ordinate. Caspase 3
activation in B cells is shown as a positive control. (b) Flow cytometric analysis of caspase 3 activation in cells positively selected with annexin V beads in
fresh PBMC from a HAM/TSP patient (left column) and after 3 days' culture from an uninfected person (right column). Caspase 3 was not activated in
HAM/TSP patients. Caspase 3 was activated in B cells after 3 days culture from an uninfected person.
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Y. Furukawa et al:

flow cytometric analysis of caspase 3 activation and annexin V

staining in peripheral blood B cells from a second HAM/TSP
patient (data not shown). The threshold for caspase 3 activation
was made using fresh PBMC from an HTLV-I uninfected person
(Fig. 6a, left column). The threshold for caspase 3 was higher than
that for other surface markers because the fluorescence of the
precleaved profluorescent protease is not completely quenched, as
described in the manufacturer's manual. As a positive control, we
used PBMC cultured for 3 days (Fig. 6a, right column). Of the
cells positively selected with annexin V-coupled microbeads,
964% were B cells, and caspase 3 was activated in only 33% of
this B cell population (Fig. 6b, left column).
Annexin V positivity under culture with patient's own serum
We cultured the PBMC and negatively isolated B cells from
HAM/TSP patients who harboured a high proportion of annexin
V1 B cells with RPMI containing 10% FCS or patients' own
serum or normal human AB serum, to test whether a serum factor
contributes to B cell membrane damage. Similarly, we cultured
PBMC and negatively isolated B cells from non-infected healthy
controls with RPMI containing 10% FCS or patient's serum or
normal human AB serum, to test whether a serum factor can
initiate B cell membrane damage. In some patients, the annexin V
positivity of B cells remained higher in culture with 10% patient's
own serum than with 10% FCS or normal AB serum. However,
we could not induce membrane damage in uninfected people's B
cells, using patient's serum under the culture conditions used (data
not shown).
Anti-CL antibody and anti-b 2-GPI antibody
Three out of 14 HAM/TSP patients showed a moderately high
level of anti-CL antibody, and two out of 14 had borderline titres.
On the other hand, in asymptomatic carriers only one out of seven
showed a borderline level of anti-CL antibody, but the anti-CL
antibody titre did not correlate with annexin V positivity on the B
cells, and anti-b 2-GPI antibody was negative in all HAM/TSP
patients and in asymptomatic carriers.
DISCUSSION
There is a strong and chronically activated CTL response to the
Tax antigen in many HTLV-I-infected people [6,7]. The original
purpose of this study was to detect apoptotic cells in the CD41
lymphocyte population, which are the host cells for HTLV-I, and
also in the CD81 cells, which include CTL in the freshly isolated
PBMC. In CD41 lymphocytes, we could not detect a significant
difference in the extent of apoptosis between non-infected,
asymptomatic carriers and HAM/TSP patients, using FITC
annexin V (Fig. 1 and Fig. 2). One possible explanation for this
result is that the target cells that express Tax antigen constitute a
very small population among the HTLV-I-infected cells at any
one time in the circulating PBMC in vivo [22], so that even if
CTL are killing infected target cells, the low death rate cannot be
detected. It is also possible that apoptosis of HTLV-I-infected
lymphocytes occurs only in tissues such as lymph nodes, spleen or
lung, so that apoptosis is not detected in the peripheral blood. The
other possibility is that Tax-expressing cells are protected from
apoptosis by Tax [12,13], although there are contradictory reports
that Tax can promote apoptosis [10,11].
In the CD191 population, there was a markedly increased
exposure of PS residues on the B cell outer plasma membrane

leaflet, mainly in HAM/TSP patients. Although the mean value of

annexin V positivity of CD191 cells in non-infected people (90 ^
40%) was higher than in CD41 cells (37 ^ 34%; 0001 ,
P , 001) or in CD81 cells (56 ^ 60%; 01 , P , 05), the
annexin V positivity of CD191 cells was much higher in HAM/TSP
patients (539 ^ 30%) (Fig. 2). To test whether annexin V positivity
was confined to a particular subclass of B cells, we stained the cells
with antibodies to IgD and CD21. IgD1 B cells represent recirculating B cells and CD211 B cells represent memory B cells
[23]. However, the proportion of annexin V1 B cells did not
correlate with the proportion of surface IgD1 B cells, and CD211
B cells were found among both annexin V-positive and -negative
B cells. This suggests that annexin V positivity is not restricted to
memory B cells, and therefore that the membrane damage (i.e.
abnormal PS exposure) is not confined to B cells that are specific
to HTLV-I antigens.
Because the proportion of annexin V1 B cells in freshly
isolated PBMC was so high in HAM/TSP patients, and the B cell
number did not decrease in these patients, we doubted whether all
of the annexin V1 cells were really undergoing apoptosis. We
therefore analysed freshly isolated PBMC and cultured PBMC in
the normal lymphocyte gate as well as in the wider gate to include
late apoptotic cells, because late apoptotic cells have an altered
forward scatter and side scatter [15]. When PBMC were cultured
for 24 h and analysed in the wider gate, annexin V highly stained
CD191 cells appeared (Fig. 3a). PI-stained CD191 cells were in
this annexin V highly stained CD191 cell population, and not in
the annexin V intermediately stained cell population, and the
proportion of annexin V highly stained cells correlated strongly
with the proportion of TUNEL1 cells (Fig. 4), suggesting that
annexin V highly stained cells are late apoptotic. It is usually
thought that early apoptotic cells bind intermediate amounts of
annexin V, whereas late apoptotic cells bind more annexin V, and
eventually, apoptotic cells also become PI1, because of secondary
necrosis [24]. However, in our study the majority of intermediately stained CD191 cells which were detected in the freshly
isolated PBMC became annexin V2 (Figs 3a,b). One may argue
that the annexin V2 cells proliferated, but this is unlikely, because
the percentage of annexin V2 CD191 lymphocytes before culture
was so small and the increase in the number of B cells in PBMC
after 24 h culture was small (115 ^ 21% after culture, n 11;
002 , P , 005). Thus cell proliferation cannot account for the
observed increase in annexin V low cells. Moreover, when we
compared the annexin V positivity of freshly isolated PBMC in
the normal gate with the annexin V highly stained advanced
apoptotic cells in the wide gate after 24 h of culture, there was a
positive correlation in the CD41 and CD81 populations in HAM/
TSP, but there was no correlation in CD191 cells (Fig. 5).
Although B cells from normal people have a greater tendency to
undergo apoptosis than T cells during in vitro culture [25], and
50% of the B cells will die in 24 days [26], there was no
difference in annexin V highly stained (advanced apoptotic)
CD191 lymphocytes between HAM/TSP patients and uninfected
people after 24 h culture. We concluded that annexin V
intermediate positive staining in the CD41 and CD81 populations
reflected early apoptotic change, and the cells became annexin V
highly stained after 24 h of culture, whereas in the CD191
population in HAM/TSP patients, although some of the annexin V
intermediately stained cells became annexin V high, most became
negative after 24 h culture. PS is actively maintained at the inside
of the plasma membrane by aminophospholipid translocase [27],

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Reversible B cell membrane damage in HAM/TSP

and appears on the outside after the disruption of the transmitochondrial potential, which is an irreversible point of the
apoptotic process [28]. Our finding that PS residue exposure on
the B cell outer plasma membrane leaflet was reversible in HAM/
TSP implies that intermediate PS exposure is not always a
consequence of the apoptotic process. As a further test of whether
the annexin V1 B cells in HAM/TSP patients are really apoptotic,
we examined caspase 3 activation in B cells. Although 975% of
the B cells were positive for annexin VFITC, caspase 3 was
activated in only 33% of the B cells (Fig. 6a, middle column). To
confirm this result further, we isolated annexin V binding cells
using annexin V-coupled microbeads. Again, 964% of the
annexin V positively selected cells were B cells, and caspase 3
was activated in only 33% of these B cells (Fig. 6b, left column).
However, we could not detect caspase 3 activation in the B cells
of HAM/TSP patients with very high frequencies of annexin V1 B
cells. These results confirm that this PS exposure on B cells in
HAM/TSP patients is not a result of apoptosis.
One possible explanation for the aberrant exposure of PS on B
cells in HAM/TSP is that the B cell membrane damage is caused
from outside the cell. Because annexin V intermediate staining
became negative in the culture, it is not likely that non-B cells in
peripheral blood were causing the B cell membrane damage. In
blocking experiments using a soluble protein of known death
signal-transducing TNFR family receptors [29], annexin V
staining of the B cells did not differ from the control culture
(data not shown), and the CD95 (Fas) expression on B cells was
low both in non-infected and asymptomatic carriers and HAM/
TSP patients (Table 1). FasL expression was not detected on
CD41, CD81 or CD191 cells (data not shown). We also
examined CD40 and CD154 (CD40 ligand) expression in noninfected people, asymptomatic carriers and HAM/TSP patients.
CD40 was expressed on most of the B cells in all cases, and there
was no difference among the three subject groups. CD40 ligand
was not expressed, but again there was no significant difference
among the three subject groups (data not shown). These findings
did not give further clues to the mechanism of increased annexin
V staining of B cells, but suggested that this increased staining
was not a result of apoptosis. Although we cannot conclude that
this abnormal PS exposure on B cells is specific to HAM/TSP, it
has been reported that in HIV infection there is not a high level of
annexin V positivity, although there are significant levels of
mitochondrial depolarization in fresh PBMC [14].
It has been suggested that the cell membrane damage and PS
residue exposure are caused by anti-b 2-GPI antibody in the antiphospholipid antibody syndrome [30]. In this model, anti-b 2-GPI
antibodies induce formation of multiple complexes of b 2-GPI
molecules on the cell surface because of their bivalent or
multivalent property, and injure the plasma membrane of these
cells directly or perhaps by binding the cellular Fc receptor,
resulting in aberrant PS exposure. In our study, three of 14 HAM/
TSP patients showed a moderately high level of anti-CL antibody,
and two out of 14 had borderline levels; on the other hand, in
asymptomatic carriers only one out of seven showed a borderline
level of anti-CL antibody. However, anti-CL antibody titre did not
correlate with the annexin V positivity on the B cells, and we
could not detect anti-b 2-GPI antibody either in HAM/TSP
patients or in asymptomatic carriers. Although we could not
detect anti-b 2-GPI antibody, it remains possible that some
cofactor other than anti-b 2-GPI binds to the B cell membrane
with its antibody, and causes membrane damage. We also tested if

315

some soluble factor other than anti-CL antibody was causing B

cell membrane damage, by culturing isolated B cells with the
patients' own serum. In some patients, intermediate staining of
annexin V in the B cells persisted after 24 h culture when cultured
with the patient's own serum, but most of the others did not. In
addition, patients' sera did not induce annexin V positivity in noninfected subjects' B cells after 24 h culture (data not shown).
Thus, some other factor or condition may be required to induce B
cell membrane damage.
Cells that expose PS on the surface, such as those in late
apoptosis, are usually recognized and scavenged from the
circulation by phagocytes [31]. One possible explanation why
PS-exposing B cells are not removed in HAM/TSP is the degree of
PS exposure. Distinct from the advanced stage of apoptosis, the
degree of PS exposure on fresh B cells of HAM/TSP patients
is intermediate, and may therefore not be recognized by the
mononuclear phagocytic system. Another possibility is that
phagocytic clearance needs additional molecules to be exposed
on the apoptotic cells, such as the ligand for a Vb 3 vitronectin
receptor integrin receptors [32], the ligand for class B scavenger
receptor CD36 [33] and the ligand for the antigen recognized by
the MoAb 61D3 [34].
With regard to the possible significance of PS exposure, the
PS which becomes surface-exposed on apoptotic cells catalyses
reactions of the coagulation cascade [20,35,36], and may elicit
diverse humoral and cellular responses of the inflammatory and
haemostatic systems [37]. It has been reported that nearly 70% of
HAM/TSP patients show MRI imaging abnormalities in the brain
which could be ischaemic or inflammatory lesions [38]. The
abnormal exposure of PS on B cells in HAM/TSP may disturb the
microcirculation and contribute to these complications and
therefore could contribute to the spinal cord lesions seen in
HAM/TSP.
Note added in proof. Since this manuscript was submitted, a
paper has been published in The Journal of Immunology
(164:132232) by S.R. Dillon et al., in which the authors show
that annexin V binding to normal murine B cells does not
necessarily reflect early apoptosis, but appears to pay a role in
receptor-mediated signalling.
REFERENCES
1 Gessain A, Barin F, Vernant JC, Gout O, Maurs L, Calender A, de The
G. Antibodies to human T-lymphotropic virus type-I in patients with
tropical spastic paraparesis. Lancet 1985; 2:40710.
2 Osame M, Usuku K, Izumo S, Ijichi N, Amitani H, Igata A, Matsumoto
M, Tara M. HTLV-I associated myelopathy, a new clinical entity.
Lancet 1986; 1:10312.
3 Poiesz BJ, Ruscetti FW, Gazdar AF, Bunn PA, Minna JD, Gallo RC.
Detection and isolation of type C retrovirus particles from fresh and
cultured lymphocytes of a patient with cutaneous T-cell lymphoma.
Proc Natl Acad Sci USA 1980; 77:74159.
4 Yoshida M, Miyoshi I, Hinuma Y. Isolation and characterization of
retrovirus from cell lines of human adult T-cell leukemia and its
implication in the disease. Proc Natl Acad Sci USA 1982; 79:20315.
5 Yoshida M, Osame M, Kawai H et al. Increased replication of HTLV-I
in HTLV-I-associated myelopathy. Ann Neurol 1989; 26:3315.
6 Jacobson S, Shida H, McFarlin DE, Fauci AS, Koenig S. Circulating
CD81 cytotoxic T lymphocytes specific for HTLV- I pX in patients
with HTLV-I associated neurological disease. Nature 1990; 348:245
8.
7 Parker CE, Daenke S, Nightingale S, Bangham CR. Activated, HTLV-

q 2000 Blackwell Science Ltd, Clinical and Experimental Immunology, 120:307316

316

8
9
10
11

12
13
14
15

16

17

18

19

20

21

Y. Furukawa et al:

1-specific cytotoxic T-lymphocytes are found in healthy seropositives

as well as in patients with tropical spastic paraparesis. Virology 1992;
188:62836.
Van Parijs L, Abbas AK. Homeostasis and self-tolerance in the
immune system: turning lymphocytes off. Science 1998; 280:2438.
Debatin KM, Goldman CK, Waldmann TA, Krammer PH. APO-1induced apoptosis of leukemia cells from patients with adult T-cell
leukemia. Blood 1993; 81:29727.
Yamada T, Yamaoka S, Goto T, Nakai M, Tsujimoto Y, Hatanaka M.
The human T-cell leukemia virus type I Tax protein induces apoptosis
which is blocked by the Bcl-2 protein. J Virol 1994; 68:33749.
Chlichlia K, Moldenhauer G, Daniel PT, Busslinger M, Gazzolo L,
Schirrmacher V, Khazaie K. Immediate effects of reversible HTLV-1
tax function: T-cell activation and apoptosis. Oncogene 1995; 10:269
77.
Copeland KF, Haaksma AG, Goudsmit J, Krammer PH, Heeney JL.
Inhibition of apoptosis in T cells expressing human T cell leukemia
virus type I Tax. AIDS Res Hum Retrovir 1994; 10:125968.
Brauweiler A, Garrus JE, Reed JC, Nyborg JK. Repression of bax gene
expression by the HTLV-1 Tax protein: implications for suppression of
apoptosis in virally infected cells. Virology 1997; 231:13540.
Macho A, Castedo M, Marchetti P et al. Mitochondrial dysfunctions in
circulating T lymphocytes from human immunodeficiency virus-1
carriers. Blood 1995; 86:24817.
Carbonari M, Cibati M, Cherchi M, Sbarigia D, Pesce AM, DellAnna
L, Modica A, Fiorilli M. Detection and characterization of apoptotic
peripheral blood lymphocytes in human immunodeficiency virus
infection and cancer chemotherapy by a novel flow immunocytometric
method. Blood 1994; 83:126877.
Samuelsson A, Sonnerborg A, Heuts N, Coster J, Chiodi F. Progressive
B cell apoptosis and expression of Fas ligand during human
immunodeficiency virus type 1 infection. AIDS Res Hum Retrovir
1997; 13:10318.
Martin SJ, Reutelingsperger CP, McGahon AJ, Rader JA, van Schie
RC, LaFace DM, Green DR. Early redistribution of plasma membrane
phosphatidylserine is a general feature of apoptosis regardless of the
initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J
Exp Med 1995; 182:154556.
van Engeland M, Nieland LJ, Ramaekers FC, Schutte B, Reutelingsperger CP. Annexin V-affinity assay: a review on an apoptosis
detection system based on phosphatidylserine exposure. Cytometry
1998; 31:19.
Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST,
van Oers MH. Annexin V for flow cytometric detection of
phosphatidylserine expression on B cells undergoing apoptosis. Blood
1994; 84:141520.
Casciola Rosen L, Rosen A, Petri M, Schlissel M. Surface blebs on
apoptotic cells are sites of enhanced procoagulant activity: implications for coagulation events and antigenic spread in systemic lupus
erythematosus. Proc Natl Acad Sci USA 1996; 93:16249.
Tosswill JHC, Taylor GP, Clewley JP, Weber JN. Quantification of
proviral DNA load in HTLV-I infections. J Virol Methods 1998;
75:2126.

22 Beilke MA, In DR, Gravell M et al. In situ hybridization detection of

HTLV-I RNA in peripheral blood mononuclear cells of TSP/HAM
patients and their spouses. J Med Virol 1991; 33:6471.
23 Chapple MR, MacLennan IC, Johnson GD. A phenotypic study of B
lymphocyte subpopulations in human bone marrow. Clin Exp Immunol
1990; 81:16672.
24 Lens SM, Tesselaar K, den Drijver BF, van Oers MH, van Lier RA. A
dual role for both CD40-ligand and TNF-alpha in controlling human B
cell death. J Immunol 1996; 156:50714.
25 Vermes I, Haanen C, Richel DJ, Schaafsma MR, Kalsbeek Batenburg
E, Reutelingsperger CP. Apoptosis and secondary necrosis of
lymphocytes in culture. Acta Haematol 1997; 98:813.
26 Ashman RF, Peckham D, Stunz LL. Regulation of B cell apoptosis.
Adv Exp Med Biol 1996; 406:14554.
27 Bratton DL, Fadok VA, Richter DA, Kailey JM, Guthrie LA, Henson
PM. Appearance of phosphatidylserine on apoptotic cells requires
calcium-mediated nonspecific flip-flop and is enhanced by loss of the
aminophospholipid translocase. J Biol Chem 1997; 272:2615965.
28 Castedo M, Hirsch T, Susin SA, Zamzami N, Marchetti P, Macho A,
Kroemer G. Sequential acquisition of mitochondrial and plasma
membrane alterations during early lymphocyte apoptosis. J Immunol
1996; 157:51221.
29 Golstein P. Cell death: TRAIL and its receptors. Curr Biol 1997;
7:7503.
30 Takeya H, Mori T, Gabazza EC et al. Anti-beta2-glycoprotein I
(beta2GPI) monoclonal antibodies with lupus anticoagulant-like
activity enhance the beta2GPI binding to phospholipids. J Clin Invest
1997; 99:22608.
31 Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson
PM. Exposure of phosphatidylserine on the surface of apoptotic
lymphocytes triggers specific recognition and removal by macrophages. J Immunol 1992; 148:220716.
32 Savill J, Dransfield I, Hogg N, Haslett C. Vitronectin receptormediated phagocytosis of cells undergoing apoptosis. Nature 1990;
343:1703.
33 Ren Y, Silverstein RL, Allen J, Savill J. CD36 gene transfer confers
capacity for phagocytosis of cells undergoing apoptosis. J Exp Med
1995; 181:185762.
34 Flora PK, Gregory CD. Recognition of apoptotic cells by human
macrophages: inhibition by a monocyte/macrophage-specific monoclonal
antibody. Eur J Immunol 1994; 24:262532.
35 Bombeli T, Karsan A, Tait JF, Harlan JM. Apoptotic vascular
endothelial cells become procoagulant. Blood 1997; 89:242942.
36 Flynn PD, Byrne CD, Baglin TP, Weissberg PL, Bennett MR.
Thrombin generation by apoptotic vascular smooth muscle cells. Blood
1997; 89:437884.
37 Reutelingsperger CP, van Heerde WL. Annexin V, the regulator of
phosphatidylserine-catalyzed inflammation and coagulation during
apoptosis. Cell Mol Life Sci 1997; 53:52732.
38 Nakagawa M, Izumo S, Ijichi S, Kubota H, Arimura K, Kawabata M,
Osame M. HTLV-I-associated myelopathy: analysis of 213 patients
based on clinical features and laboratory findings. J Neurovirol 1995;
1:5061.

q 2000 Blackwell Science Ltd, Clinical and Experimental Immunology, 120:307316