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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 3 (August 1), 1999:
pp. 1012-1020
Surface Molecule Loss and Bleb Formation by Human Germinal Center B
Cells Undergoing Apoptosis: Role of Apoptotic Blebs in Monocyte
Chemotaxis
By
Carmen Segundo,
Francisco Medina,
Carmen Rodríguez,
Rosalía Martínez-Palencia,
Francisco Leyva-Cobián, and
José A. Brieva
From Servicio de Inmunología, Hospital Universitario Puerta
del Mar, Cádiz; and Servicio de Inmunología, Hospital
Universitario Marqués de Valdecilla, Santander, Spain.
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ABSTRACT |
Human tonsil germinal center (GC) B cells rapidly undergo apoptosis
in culture. Annexin-V binding shows an early event in this process. In
the present study, this method has been used to label apoptotic GC B
cells and to analyze additional surface molecules. The expression of
all of the molecules studied was reduced in apoptotic
(annexin-V+) GC B cells, and the reduction was more
marked for CD11a, CD21, CD22, CD49d, and CD54, molecules that
participate in survival interaction for GC B cells. The analysis of
CD54, one of the molecules that was more drastically reduced, showed
that GC, but not mantle zone, B cells actively secrete CD54 to the
culture supernatant (SN). The secreted CD54 was partly released from
the GC B cells in a particulate form as demonstrated by centrifugation.
Further experiments using filtration, fluorescence microscopy, electron microscopy, and flow cytometry analysis showed that GC B
cells released to the culture SN a population of spherical membranous vesicles of about 0.18 µm in size, similar to the blebs described in
other apoptosis systems. Bleb formation depended on active metabolism,
Ca2+, and, in part, on microfilament integrity. GC
B-cell-derived blebs were clearly associated with apoptosis, as
antiapoptotic stimuli prevented their formation. In addition, GC
B-cell-derived blebs contained the adhesion molecules previously
studied. Consequently, bleb formation might contribute to the surface
molecule loss occurring in apoptotic GC B cells. Finally, a chemotaxis
assay showed that GC B-cell blebs were chemotactic for human monocytes,
suggesting that this mechanism might operate in vivo.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
PROGRAMMED CELL DEATH (PCD) or apoptosis
is a physiological process leading to the elimination of useless and
harmful cells, which is very important for preserving tissue
homeostasis in multicellular organisms.1 Recent reports
indicate that apoptosis is regulated by the complex interaction of
several families of proteins that have been conserved throughout
evolution.2,3 One of these protein families consists of
cysteine-containing, aspartate-specific proteases termed caspases, and
appears to play a key role in the effector phase of
apoptosis.4 PCD causes characteristic morphological changes
in the cells, which include the release of small vesicles derived from
the cytoplasmic membrane (the phenomenon known as blebbing), shrinkage
of the cell and the detachment from the surrounding structures,
chromatin condensation, and nuclear and cellular
fragmentation.5 The process ends with the phagocytosis of
dead cells and apoptotic bodies either by neighboring cells or by
specialized phagocytes.6
Germinal centers (GC) are globular microstructures that consist mainly
of B lymphocytes, which arise within the follicles of secondary
lymphoid tissues on the entrance of T-dependent and some T-independent
Ag.7-9 B cells undergoing cell death by apoptosis are a
main feature of functional GC. Elimination of apoptotic GC B
lymphocytes is carried out by cells of the monocyte/macrophage lineage
that, in this location, are called tingible body
macrophage.10 Increasing evidence supports the view that
the apoptotic pathway is involved in several aspects of the B-cell
physiology within the GC. Thus, a basal GC B-cell propensity to
apoptosis11,12 allows the rescue of those cells that, after
accumulating somatic mutations in their Ig V genes, bear an antibody
(Ab) with high affinity for the Ag exposed on the
follicular dendritic cell (FDC) surface, a process that leads to memory
B-cell selection.13-16 Together with the process of Ig V
gene somatic hypermutation, it has recently been documented that a
fraction of GC B cells goes through a new phase of V(D)J Ig gene
recombination and B-cell receptor (BCR) editing of as yet
unknown significance.17-19 It is thought that PCD also
operates to assure the deletion of B cells bearing defective or
autoreactive Ab generated by these two genetic processes. In this
regard, it has been shown that an important proportion of GC B cells
undergoes apoptosis by the specific recognition of Ag in soluble
form.20-22 This finding has been related to a possible
mechanism for maintaining self-tolerance by the elimination of
self-reactive B-cell clones generated by the Ig diversification
processes occurring during the GC response.20-22 The
propensity of GC B lymphocytes to die is further evidenced by the
observation that they rapidly undergo massive apoptosis in
culture.13,23 In addition, human GC founder B cells have been shown to trigger the apoptosis program even before the onset of
somatic mutations.24 Therefore, GC are sites where B cells exhibit an enhanced tendency to PCD, which is critical for the action
of appropriate selective mechanisms.
Several surface molecules have been demonstrated to participate in the
rescue of GC B cells from apoptosis. Besides the above-mentioned role
for the BCR, the ligation of CD40 appears to deliver survival signals
to these cells.13,25 Moreover, the adhesion molecules CD11a, CD29-CD49d, and CD54 have been shown to contribute to the FDC-GC
B-cell interaction, which appears to be required as well for
maintaining the latter cells alive.26-29 In addition, CD21, the complement receptor 2 (CR2), which is expressed by GC B cells and
which can interact with C3d and CD23 expressed on the FDC surface, also
plays a relevant role in GC B-cell survival.30,31
The initial aim of this study was to examine the fate of several human
GC B-cell surface molecules during their spontaneous apoptosis in
vitro. To this end, use was made of an early event occurring in
apoptotic cells, including GC B cells; this event is the translocation
of phosphatydilserine residues from the inner to the outer leaflet of
the cell membrane, a phenomenon that can be shown by binding with
labeled annexin-V.32 The results show that apoptotic GC B
cells rapidly lose a variety of surface molecules, mainly those
involved in their adhesion to FDC and surrounding estractures. Further
experiments showed that, at least in part, these molecules were
released from the cell surface as membranous vesicles similar to those
described as apoptotic blebs in many cell systems. Finally, these GC
B-cell-derived blebs exhibited chemoattractive activity on monocytes,
suggesting that this mechanism might be operative in vivo.
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MATERIALS AND METHODS |
Materials.
Cycloheximide (Cx), Cytochalasin B (CkB), phorbol 12-myristate
13-acetate (PMA), zymosan, and PKH26-GL were purchased from Sigma (St
Louis, MO). Z-Val-Ala-DL-Asp-fluoromethylketone (VAD-fmk) was
obtained from Bachem Feinchemikalien AG (Budendorf, Switzerland). EDTA
was purchased from Pharmacia Biotech (Uppsala, Sweden). Agar was
provided by Difco Laboratories (Detroit, MI). Interleukin-4 (IL-4) was
provided by Peprotech Inc (Rocky Hill, NJ). The 24-well flat-bottomed
plate used for cell culture was from Nunc (Roskilde, Denmark).
Anti-CD40 mouse monoclonal antibody (MoAb) (MAB 89), fluorescein
isothiocyanate (FITC)-labeled mouse MoAb against CD21 and CD54 and
phycoerythrin (PE)-labeled mouse MoAb against CD11a, and CD40 were
obtained from Immunotech (Luminy, France). FITC-labeled mouse MoAb
against CD20, CD22, HLA DR, and CD11a, and PE-labeled mouse MoAb
against CD20, CD21, CD22, HLA DR, CD38, CD45, CD49d, CD54, and CD58,
and control FITC-labeled and PE-labeled MoAb of appropriate isotypes
were provided by Becton Dickinson (San Jose, CA). FITC-labeled
annexin-V was obtained from Bender Medsystems (Vienna, Austria). Human
CD54 enzyme-linked immunosorbent assay (ELISA) kit was purchased from
R&D System Inc (Minneapolis, MN). Isopore filter (0.1-µm pore) used
to filter cell culture supernatants was provided by Millipore Corp
(Bedford, MA). Polyvinylpyrrolidone-free polycarbonate membrane disc
(5-µm pore) used in chemotaxis assays was obtained from BioRad
Laboratories (Richmond, CA).
Tonsil cell and blood monocyte preparation.
Tonsillar tissue was obtained from subjects undergoing tonsillectomy
for chronic tonsillitis. Single-cell suspensions were prepared and the
cells were separated into T- and non-T (B)-cell populations by a
previously reported rosette technique. B cells were fractionated
further into GC and mantle zone (MZ) B cells on a discontinuous Percoll
gradient as detailed elsewhere.33 Purity of GC B cells was
determined by immunofluorescence and flow cytometry, as
CD38+ CD20high cells, and exhibited a viability
higher than 95%, as determined by trypan blue exclusion test.
Monocytes for chemotaxis assays were obtained from healthy volunteers'
blood. Briefly, blood samples were drawn in sterile tubes, containing
14.3 IU of sodium heparin/mL. Mononuclear cells were isolated by
bouyant density centrifugation on Lymphoprep (Nyergaard, Oslo, Norway)
at 450g for 35 minutes. The cells were recovered from the
interface, washed twice, and resuspended at 1 × 106
monocytes/mL in RPMI 1640 medium supplemented with L-glutamine (10 mmol/L). Monocytes were distinguished by differential cell count
performed on smears using a nonspecific esterase stain as previously
described.34 The monocyte proportion in 13 cell
preparations ranged from 15.5% to 24% (17.6% ± 2.5%; mean ± standard error of mean [SEM]).
Cell culture.
GC and MZ B cells and T cells (106 cells/mL) were incubated
at 37°C with 5% CO2 in a culture medium consisting of
RPMI 1640 supplemented with 10% fetal calf serum (FCS),
L-glutamine (10 mmol/L), and gentamycin (0.05 mg/mL) for 2 hours unless indicated otherwise. GC B cells were also cultured in the
presence of anti-CD40 MoAb (1 µg/mL) + IL-4 (2 ng/mL) and the peptide
VAD-fmk (200 µmol/L) for the indicated times. In some experiments,
cells were stained with PKH26-GL alone or combined with FITC-conjugated
MoAb before the culture.
Preparation of cell-free supernatant (SN) and subcellular particles
derived from cultured GC B and T cells.
Tonsil GC and MZ B cells and T cells (106/mL) were
incubated for 2 hours or the indicated times in medium, and then the
culture SN was centrifuged at 103g for 10 minutes at 4°C to remove cells and large cellular
fragments (cell-free SN). Cell-free SN obtained from cultured GC B
cells was then centrifuged at 105g for 18 hours at
4°C, and the soluble (GC SN SOL) and the precipitated (GC SN PREC)
fractions were recovered, and adjusted to the initial volume in medium.
In some experiments, cell-free SN obtained from GC B-cell cultures was
filtered on a 0.1-µm pore membrane to remove subcellular particles
from the cell-free SN (GC SN FILT).
Cell and subcellular membranous particle staining.
Two-color staining of GC B cells with fluorochrome-conjugated MoAbs and
annexin-V binding assay was performed as previously described.35 In brief, GC B cells (106/mL)
cultured for 2 hours, or the indicated time, were incubated with
FITC-conjugated annexin-V, either alone or in combination with optimal
concentrations of PE-labeled MoAbs against CD11a, CD20, CD21, CD22,
CD38, CD40, CD45, CD49d, CD54, CD58, and HLA DR in 1 mL of
Ca2+ HEPES buffer (10 mmol/L HEPES, 1.8 mmol/L
CaCl2, 150 mmol/L NaCl) for 30 minutes in the dark at
4°C. After two washes with the same buffer, the cells were analyzed
by flow cytometry and by fluorescence microscopy. To explore the
presence of membranous cellular particles in culture SN, tonsillar GC B
and T cells (106 cells/mL) were stained with the lipid
intercalating dye PKH26-GL (4 µmol/L) for 5 minutes at
room temperature. Staining reaction was stopped by adding a similar
volume of FCS, and the cells were washed twice in culture medium.
Stained cells were cultured (106 cells/mL) for 2 hours,
unless indicated otherwise. At the end of the culture period, the
cell-free SN was obtained and the presence of red fluorescent cellular
particles was analyzed by flow cytometry. To investigate the
requirements for the generation of membranous particles, GC B cells
were stained with PKH26-GL as above and cultured for 12 hours in the
presence and in the absence of cycloheximide (10 µg/mL), cytokalasin
B (5 µg/mL), anti-CD40 MoAb (1 µg/mL) + IL-4 (2 ng/mL), EDTA (1 mmol/L), PMA (1 ng/mL), and the peptide VAD-fmk (200 µmol/L). At the
end of the culture period, cell-free SN was recovered and analyzed by
flow cytometry. In some experiments, cell-free SN obtained from
cultured GC B cells was passed through a 0.1-µm pore filter, and the
presence of stained particles in the elutant was analyzed as above. To
test the presence of certain GC B-cell surface molecules in the
membranous particle, PKH26-GL-stained GC B cells were additionally
labeled with appropriate FITC-conjugated MoAbs, washed, and cultured
for 2 hours at 106 cells/mL in culture medium. The
cell-free SN was analyzed by flow cytometry, assessing the expression
of these molecules on the PKH26-GL+ particle fraction.
Flow cytometry.
Fluorescence-activated cell sorting (FACS) analysis was performed on a
FACScalibur instrument (Becton Dickinson) equipped with an air-cooled
argon ion laser emitting 15 mW at 488 nm. The instrument was equipped
with three fluorescence detector photomultiplier tubes, with green
fluorescence (FITC, FL1) being collected through a 585/42-nm bandpass,
orange/red (PE, FL2) through a 585/42-nm bandpass, and red (PerCP, FL3)
through a 650-nm longpass filter. Two-color cell analysis was performed
as previously reported.35 In the analysis of
cellular-derived vesicles contained in the cell-free SN, light scatter
and fluorescence signals were recorded in logarithmic mode. In
experiments to test requirements for cell-derived vesicle generation,
cell-free SN was acquired for 2 minutes at medium flow rate, and the
event numbers were recorded. Negative controls were established as
previously described.36
Electron microscopy (EM).
GC B cells (2 × 106 cells/mL) were cultured for 2 hours either in a semisolid medium consisting of agar (0.5% wt/vol in
culture medium) or on a 0.1-µm Isopore membrane placed at the bottom
of a well of a 24-well microculture plate, and these preparations were
used for transmission and scanning EM studies,
respectively. In addition, cell-free SN obtained from these cultures
was passed through a 0.1-µm pore membrane, and the presence of
subcellular vesicles deposited on these membranes was also investigated
by both transmission and scanning EM. The different samples were treated with glutaraldehyde (2.5%) overnight at 4°C, with osmium tetroxide (1%) for 30 minutes and then dehydrated.
Samples for scanning EM were critical-point dried, mounted in specimen
stubs, gold coated, and analyzed.
Chemotaxis assay.
Monocyte chemotaxis was evaluated by a modified Boyden chamber
technique as previously described.37 The chambers were
separated by polyvinylpyrrolidone-free polycarbonate membrane discs and incubated at 37°C in 5% CO2 in air for 90 minutes.
Different preparations of the SN of cultured GC B and T cells were
used. Culture medium and zymosan activated serum (ZAS) were used as
negative and positive controls, respectively. Two independent observers
counted the migrated cells within a square reticle in 10 high-power
immersion oil fields (hpf). Each experiment was performed in triplicate chambers and the mean calculated.
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RESULTS |
Loss of surface molecules by CG B cells undergoing apoptosis.
It is well known that GC B cells rapidly undergo spontaneous apoptosis
in culture. Annexin-V binding has been described as a good marker for
early stages of apoptosis.32 Accordingly, highly purified
GC B cells were obtained from human tonsils
(Fig 1A.1), and their capacity to bind
FITC-annexin-V was tested after different periods of culture. As can be
seen in Fig 1A.2, the proportion of annexin-V+ GC B cells
increased with time and, after 12 hours, most of the cells (88% ± 5%; mean ± standard error of mean [SEM]; n = 6) showed the annexin-V+ phenotype. This phenomenon could be partly
reversed by adding to the culture either anti-CD40 MoAb + IL-4, a well-established antiapoptotic stimulus in this cell
system,13 or the broad-spectrum caspase-inhibitor
VAD-fmk.38 These results indicate that annexin-V binding
can be used as a marker of GC B-cell apoptosis. The method allowed the
analysis by flow cytometry of additional surface molecules in apoptotic
(annexin-V+) and nonapoptotic
(annexin-V ) GC B cells. GC B cells cultured for 2 hours were used in this analysis because this short period of time was
sufficient for a substantial proportion of the cells to reach the
apoptotic phenotype (from 10% ± 1.5%, at 0 hour, to 33% ± 3%, at 2 hours; mean ± SEM; n = 15). Figure 1B.1 shows an example
of this study and Fig 1B.2 summarizes data obtained in five different
experiments, representing the percentage of remaining molecule
expression, determined as the mean fluorescence intensity (MFI), in
apoptotic GC B cells in comparison to live GC B cells. As can be seen,
annexin-V+ GC B cells showed reduced expression of all the
surface molecules explored. The reduction was not equal for all of the
molecules. Thus, CD20 was the least affected; CD38, CD40, CD45, and HLA
DR exhibited an intermediate level of reduction; and CD11a, CD21, CD22,
CD49d, CD54, and CD58 showed the lowest level of expression. Survival
stimulus (CD40 + IL-4 and VAD-fmk) delayed the entrance of GC B cells
into the annexin-V+ stage, but apoptotic cells emerging in
these cultures showed a low expression of surface molecule similar to
that exhibited by untreated cells (data not shown). During the first 6 hours of culture, the surface molecule loss shown by apoptotic GC B cells was not associated with a decrease in cellular size, as detected
by changes in the forward scatter values. The level of surface molecule
expression by annexin-V+ GC B cells determined as the MFI
for each molecule was similar in cells cultured from 0 to 12 hours
(data not shown).
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| Fig 1.
Human GC B-cell purification (A.1), annexin-V binding
expression kinetics (A.2), and surface molecule expression (B). (A.1)
Unfractionated and GC B cells were obtained from human tonsils and the
expression of CD20 and CD38 on their surface was monitored by
immunofluorescence and flow cytometry. Dot plots corresponding to a
representative experiment are shown. (A.2) GC B cells (106
cells/mL) were cultured for indicated times in the absence and in the
presence of anti-CD40 MoAb (1 µg/mL) + IL-4 (2 ng/mL) or VAD-fmk
(200 µmol/L) and the cells were then labeled with FITC-annexin-V and
analyzed by flow cytometry. Histograms of annexin-V expression in one
representative experiment are shown. (B.1) GC B cells were cultured for
2 hours and simultaneously labeled with FITC-annexin-V and additional
MoAb directed to the indicated molecules. Dot plots of one
representative experiment are shown. All of the surface molecules
examined were positive in more than 90% of the freshly isolated GC B
cells, except for CD49d, which was positive in only 39% ± 5% (mean ± SEM) of the cells. Axis scales of dot plots are logarithmic. (B.2)
Expression of surface molecules in annexin-V+ GC B cells.
The values were obtained as the percentage of the MFI shown for each
surface molecule studied on the annexin-V+ cells, with
respect to that observed on annexin-V cells, which was
considered the control expression. Results represent the mean ± SEM
of five experiments.
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Presence of CD54 in the supernatant of GC B-cell culture.
The surface molecule loss observed in apoptotic GC B cells could be
explained by either endocytosis or external release. To determine which
of these two possible mechanisms operates, the presence of CD54, one of
the molecules that was more drastically lost, was determined in the
supernatant of GC B-cell cultures by ELISA.
Figure 2 shows that CD54 was readily
detected in the SN of GC B-cell cultures, and its quantity increased
over the time of culture. This phenomenon appeared to be restricted to GC B cells, because purified tonsillar MZ B cells, which express similar levels of CD54 on their surface, did not release detectable quantities of this molecule into the 24-hour culture supernatant (Fig
2). To investigate further this phenomenon, cell-free SN obtained after
2 hours of GC B-cell culture was ultracentrifuged, and the recovery of
CD54 was additionally determined in both the precipitate and the
ultrasupernatant fraction. The results showed that 42.2% ± 9.6%
(mean ± SEM; n = 3) of the total CD54 contained in the cell-free
supernatant (3.09 ng/mL ± 0.36; n = 3) was recovered in the
precipitate fraction. This finding suggested that, at least in part,
CD54 was released by the GC B cells in a particulate form.
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| Fig 2.
CD54 detection in culture supernatant. Tonsillar GC and
MZ B cells were cultured, and the cell-free SN was recovered after the
indicated times and tested for their CD54 content by an ELISA
technique. Results (ng/mL) represent the mean ± SEM of eight
experiments.
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Identification of membranous particles released into the GC B-cell
supernatant.
Previous reports have established that apoptotic cells produce plasma
membrane rounded vesicles called blebs.5 Therefore, experiments were designed to test whether the GC B-cell SN contained particles that could be identified as blebs. Cell-free SN obtained from
2-hour GC B-cell cultures was passed through a 0.1-µm pore filter,
and the filter was examined by EM. Figure
3A shows the presence of spherical vesicles retained on the filter as
detected by either scanning or transmission EM (Fig 3A.1 and A.2,
respectively). These particles appeared as rounded plasma membrane
vesicles, with an average diameter of 0.180 µm ± 0.05 (mean ± standard deviation [SD]). Later in the culture, particles of larger
size were also isolated from the SN of GC B-cell cultures, some of them
containing chromatin-condensed material, corresponding to apoptotic
bodies (data not shown). In additional experiments, GC B cells were
incubated for 2 hours in semisolid cultures to observe bleb formation.
Figure 3B shows the process of blebbing by apoptotic GC B cells as
detected by fluorescence microscopy (Fig 3B.1) and by transmission and scanning EM (Fig 3B.2 and B.3, respectively).
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| Fig 3.
Microscopy analysis of released vesicles (A) and their
cellular generation (B). (A) Cell-free SN obtained from GC B cells
cultured for 2 hours was passed through a filter (0.1-µm pore), and
the filter was processed for scanning (A.1) and transmission (A.2) EM.
Filter pores are seen as black holes in A.1. (B) GC B cells were
stained with PKH26-GL and, after 2 hours, were analyzed under
fluorescence microscopy (B.1). GC B cells cultured for 2 hours were
studied by transmission EM (B.2) and scanning EM (B.3).
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Flow cytometry analysis of apoptotic GC B-cell-derived blebs.
PKH26-GL is a fluorescent probe specific for the cell membrane that
can be used as a stable and nontoxic staining of viable cells39 and cellular membrane particles.36 In
an attempt to track apoptotic cell-derived blebs, GC B cells, after
labeling with PKH26-GL, were cultured for 2 hours and the presence of
fluorescent particles was examined in the cell-free SN by flow
cytometry. Figure 4A shows that the forward
versus side scatter dot plot analysis of this cell-free SN did not show
a clear distinction of the blebs, as their size was close to the
detection limit of the cytometer. Nevertheless, the use of the
fluorochrome allowed identification of the presence of a population of
membranous particles (Fig 4B), which disappeared when the GC B-cell
supernatant was filtered through a 0.1 µm pore filter (Fig 4C). In
addition, these particles were not present in the supernatant of
PKH26-GL-labeled tonsillar T lymphocytes cultured for 2 hours (Fig
4D). These results indicate that the method could be useful for
detecting GC B-cell-derived blebs and, accordingly, it was used to
investigate the kinetics of and the requirements for bleb formation in
the present culture system. Figure 5A shows
that the kinetics of bleb accumulation in the SN of GC B-cell cultures
was linear during the first 12 hours. The generation of blebs markedly
decreased when cells were cultured at 4°C and when cells were
treated with EDTA (1 mmol/L) and with blockers of GC B-cell apoptosis
such as anti-CD40 MoAb + IL-4 and VAD-fmk (Fig 5B). Treatment with
cytochalasin B, which disrupts microfilaments, provoked only a partial
decrease (P < .03). The inclusion of cycloheximide (an
inhibitor of protein synthesis) and the PKC-activator, PMA, did not
significantly modify GC B-cell bleb generation (Fig 5B).
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| Fig 4.
Analysis of GC B-cell-derived vesicles by flow
cytometry. Tonsillar GC B cells and T cells were stained with the cell
membrane-specific probe, PKH26-GL, and then cultured for 2 hours. After
this period, cell-free SN was collected and the presence of membranous
vesicles was monitored by flow cytometry. (A) Dot plot representation
of the forward scatter (FS) versus side scatter (SS) values of the SN
containing GC B-cell-derived particles is shown. FL2 histogram
obtained from the analysis of the same sample as in (A), either before
(B) or after (C) filtering through a 0.1-µm pore membrane and of SN
obtained from T-cell culture (D).
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| Fig 5.
Kinetics of and requirements for the generation of
vesicles derived from cultured GC B cells. GC B cells were stained with
PKH26-GL, cultured for 2 hours, and the cell-free SN was analyzed by
flow cytometry. (A) A dot plot of SS versus FL2 parameters was used to
define GC B-cell-derived membranous particles. (B) The count of
PKH26-GL+ particles detected in the SN obtained for
indicated times was recorded. Results of one experiment representative
of three are shown. (C) PKH26-GL-stained GC B cells were cultured for
12 hours at 4°C and at 37°C in the absence (control culture)
and in the presence of cycloheximide (Cx, 10 µg/mL), cytochalasin B
(CkB, 5 µg/mL), EDTA (1 nmol/L), anti-CD40 MoAb (1 µg/mL) + IL-4
(2 ng/mL), PMA (10 ng/mL), and VAD-fmk (200 µmol/L). Values of the
counts of PKH26-GL+ particles were recorded and expressed
as percentages of the control figures. Results represent the mean ± SEM of four experiments.
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Presence of surface molecules in the blebs derived from apoptotic GC
B cells.
The labeling of GC B-cell membrane with PKH26-GL neither modified
annexin-V binding nor affected the detection of surface molecules by
immunofluorescence and flow cytometry (data not shown). Therefore, this
method was used to investigate the presence of the previously studied
surface molecules in the GC B-cell-derived blebs. As can be seen in
Fig 6A, PKH26-GL-stained vesicles, at least in part, expressed CD11a, CD20, CD21, CD22, HLA DR, and CD54.
Figure 6B shows that, apart from CD11a, all of the molecules studied
were detected on most of the PHH26-GL+ particles.
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| Fig 6.
Expression of surface molecules by vesicles derived from
cultured GC B cells. GC B cells were stained with PKH26-GL and
FITC-conjugated MoAb against several surface molecules present on GC B
cells and against an irrelevant antigen (control). After 2 hours,
cell-free SN was obtained and analyzed by flow cytometry. (A) Contour
plots of FL2 (PKH26-GL staining) versus FL1 (indicated molecule) from
one representative example are shown. (B) Values were expressed as the
percentage of particles positive for each molecule. Results represent
the mean ± SEM of three experiments.
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Role of the blebs derived from apoptotic GC B cells as monocyte
chemoattractant.
In the final set of experiments, the possibility that blebs derived
from apoptotic GC B cells could be chemotactic for monocytes was
tested. To this end, the activity of cell-free SN obtained from 2-hour
GC B-cell cultures was determined in a chemotaxis assay that used
normal blood monocytes. Figure 7A shows
that GC B-cell SN exhibited detectable chemotactic activity that
increased in a concentration-dependent manner. This activity was not a
nonspecific phenomenon, as similar SN obtained from cultured T
lymphocytes isolated from the same tonsils did not produce any effect
in the assay (Fig 7B). The monocyte-chemotactic activity shown by GC B-cell SN was lost when it was passed through a 0.1-µm pore filter (Fig 7C), suggesting that the activity was associated with cellular particles released into the SN. In addition, Fig 7D shows that the
monocyte-chemotactic activity shown by the GC B-cell culture SN was
almost exclusively restricted to the membranous precipitate (GC SN
PREC), but not in the soluble (GC SN SOL), fraction obtained by
ultracentrifugation.
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| Fig 7.
Chemoattractant activity on human monocytes by vesicles
derived from GC B cells cultured for 2 hours. (A) Monocyte chemotaxis
was assessed using as stimulus culture medium (negative control), ZAS
(positive control), and different dilutions of GC B-cell culture SN (GC
SN). (B) The chemoattractant activity of GC SN was compared with that
of a similar supernatant obtained from T-cell cultures (T SN). (C)
Comparison of the chemoattractant effect of GC SN before and after
being passed through a 0.1-µm pore membrane (GC SN FILT). (D) GC SN
was centrifuged at 105g, and the precipitated
material (GC SN PREC) and the soluble fraction (GC SN SOL) were
obtained and tested in the chemotaxis assay. The values were expressed
as the mean count of migrated monocytes per high-power field. Results
represent the mean ± SEM of four experiments.
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| |
DISCUSSION |
It is well established that GC B cells are prone to undergo PCD in
vivo, as well as in vitro, and this capacity appears to be essential
for the selection of memory B cells and probably for the deletion of
defective and autoreactive B-cell clones generated during the GC
response.40,41 In the present study, annexin-V binding,
which shows the phosphatidylserine translocation to the membrane
external leaflet, was used for detecting spontaneous apoptosis by
cultured GC B cells isolated from human tonsils. This method has been
demonstrated to define an early stage of apoptosis in many cell
systems, including human tonsil GC B cells, where annexin-V
positiveness coincides with the appearance of initial DNA condensation
and fragmentation.32 Present results reinforce this
assumption, as annexin-V binding by GC B cells was clearly delayed by
two well-established antiapoptotic signals, such as the ligation of
CD40 plus IL-413 and the treatment with VAD-fmk, a peptide
that blocks the caspase cascade at several levels.38 The
use of annexin-V binding allowed the observation of additional surface
molecules on apoptotic GC B cells. Present data indicate that GC B
cells undergoing apoptosis exhibit a markedly reduced expression of
several surface molecules. The finding that MFI values of surface
molecule expression by apoptotic (annexin-V+) GC B cells
remained unaltered during at least 12 hours, even in the presence of
antiapoptotic stimuli, suggested that the surface molecule loss was a
stable phenomenon, apparently occurring during the transition from
annexin-V to annexin-V+ stages. Moreover,
both annexin-V binding and surface molecule loss were clearly related
to the apoptotic status of the GC B cells, as these phenomena were
equally delayed by the antiapoptotic treatments. Therefore, loss of
surface molecules seemed to be a part of the GC B-cell apoptosis program.
Several mechanisms can be invoked to explain the reduced expression of
apoptotic GC B-cell surface molecules. The detection and analysis in
the culture SN of CD54, one of the molecules more drastically lost in
the present system, showed that GC B, but not MZ B, cells released
considerable quantities of this molecule to the culture medium. In
addition, a significant proportion of these molecules was released from
the GC B cells in a particulate form, as they could be precipitated by
ultracentrifugation. This finding was more relevant in light of the
fact that CD54 is a well-known example of a potentially soluble
molecule.42 Cellular blebbing has been described as the
formation and release of small membranous vesicles that occurs as an
initial event of PCD.43 Consequently, the possible
existence of this process during the apoptosis of GC B cells was
examined. Present data indicate that apoptotic GC B cells released
small spherical membranous vesicles of about 0.2 µm in diameter, as
demonstrated by EM and flow cytometry. This latter method revealed that
the molecules that showed low expression on apoptotic GC B cells could
be detected in most of the membranous vesicles derived from these
cells, and allowed a more detailed study of the kinetics of release and
the metabolic requirement for the generation of these vesicles. Thus,
the formation of vesicles derived from apoptotic GC B cells depended on
active metabolism and Ca2+ presence and was largely
independent on protein synthesis, microfilament integrity, and PKC
activation. These characteristics are compatible with those described
for the generation of cellular blebs in several models of apoptotic
systems.44-48 Further experiments indicated that apoptotic
GC B cells also developed surface blebbing, as determined by
fluorescence microscopy and EM. Therefore, it is reasonable to think
that membranous vesicles found in the SN of GC B-cell cultures are
similar to the blebs described during PCD. This idea was also supported
by the finding that bleb formation was prevented by stimuli that
delayed the appearance of apoptosis in these cells. Taken together,
these observations suggest that, at least in part, the blebbing process
can contribute to the explanation of the reduction of surface molecule
expression by apoptotic GC B cells.
Reduced expression of surface proteins by apoptotic GC B cells was not
similar for all of the molecules studied, the adhesion molecules
(CD11a, CD21, CD22, CD49d, CD54, and CD58) being those most affected.
Many of these molecules are involved in the adhesion of GC B cells to
FDC, and possibly also to other surrounding structures, a mechanism
that contributes to preserving GC B-cell survival.26-31 Loss of contact and detachment is a common feature of apoptotic cells.5,43 In fact, disruption of intercellular contact
through integrins and other adhesion molecules causes apoptosis in many cell systems.49,50 Thus, early in the apoptotic program, GC B cells appeared to lose those molecules that allowed their attachment, which probably resulted in the irreversibility of the process.
Apoptotic cells are rapidly eliminated by phagocytosis, which is
important to prevent inflammatory reactions. This process is carried
out either by neighbor or by "professional" phagocytes depending
on the tissue. In the GC, cells of the monocyte/macrophage lineage are
specialized in this task, and apoptotic GC B cells are commonly
observed to be engulfed by cells of this kind, named for this reason
tingible body macrophages.10 Some of the molecular events
involved in the recognition and phagocytosis of apoptotic cells by
macrophages have been clarified.6,51-53 However, little is
known about the mechanisms that locally recruit the macrophage into the
apoptotic focus. Monocytes migrate toward particular sites by following
the gradient of certain chemotactic factors. Therefore, the possibility
that SN derived from cultured GC B cells is chemoattractant for
monocytes was assessed. The data demonstrate that SN from cultured GC
B, but not T, cells exhibited detectable chemotactic activity for
monocytes. In addition, this activity was present in the particulate
fraction of the GC B cell SN, as it was eliminated either by filtering
the SN on membranes with 0.1 µm exclusion pore, or by centrifugation
at 105g. These findings indicate that blebs
released by GC B cells undergoing PCD are capable of attracting
monocytes in vitro. Accordingly, it can be hypothesized that a gradient
of apoptotic blebs released around dying GC B cells attracts local
macrophages in vivo. As membrane blebbing is a rather common event in
apoptosis, it could also operate to attract macrophages into the
apoptotic focus in other cellular systems.
| |
AKNOWLEDGMENT |
The authors thank O. Aliseda and J.M. Geraldía (EM Division,
UCA) and M. Beltrán (Servicio de Anatomía
Patológica, Hospital Puerta del Mar) for technical assistance in
sample preparation and EM analysis.
| |
FOOTNOTES |
Submitted December 3, 1998; accepted March 31, 1999.
Supported by Grants No. 96/2116 and 97/1119 from the Fondo de
Investigaciones Sanitarias of Spain.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to José A. Brieva, MD,
Servicio de Inmunología, Hospital Universitario Puerta del Mar,
Avenida Ana de Viya 21, 11009 Cádiz, Spain; e-mail:
jabrieva{at}mar.hpm.sas.cica.es.
| |
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ABSTRACT
INTRODUCTION