| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Departments of Medicine, Molecular Biology and Genetics, and Oncology, Division of Rheumatology, Johns Hopkins University School of Medicine, Baltimore, MD 21205
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Transgenic (Tg) mouse model systems have clearly demonstrated that B cells that generate a BCR capable of recognizing self Ag with high affinity may edit that receptor by replacing the IgH and/or IgL chain gene segments through further V(D)J recombination (11, 12, 13). Self-reactive immature B cells that fail to edit their receptors successfully are negatively selected at an immature stage of development (14, 15) and die by apoptosis in situ, analogous to thymocytes expressing an autoreactive TCR (16, 17). B cells that survive this stringent selection process are exported to the peripheral lymphoid organs to participate in immune surveillance.
The plasma membrane of a healthy cell typically exhibits an asymmetric distribution of its major phospholipids, maintained by the activity of aminophospholipid translocase (18). Virtually all of the phosphatidylserine (PS) and most of the phosphatidylethanolamine (PtE) and phosphatidylinositol (PI) molecules reside on the inner leaflet of the plasma membrane, with sphingomyelin (SM) largely confined to the outer leaflet, and PC distributed equally between the leaflets (19). During the early stages of apoptosis, cells lose their membrane phospholipid asymmetry and expose PS on the outer leaflet of the plasma membrane, triggering their phagocytosis by macrophages that bear PS receptors (20). Rapid phagocytosis prevents secondary necrosis and inflammation within the surrounding tissue (reviewed in Refs. 21 and 22).
Annexin V (AnV) is a member of a large family of Ca2+ and phospholipid binding proteins (23). In the presence of physiological concentrations of Ca2+, AnV has high affinity for negatively charged phospholipids, especially PS. Based on its affinity for PS, fluorescently labeled AnV is often used in flow cytometric assays to detect cells undergoing apoptosis (24, 25). The original intent of the studies described below was to use recombinant AnV (rAnV) to study apoptosis at various stages during murine B cell development. Contrary to our expectations, we found that rAnV binds to the outer leaflet of the plasma membrane in healthy B cells, associates with the BCR in membrane microdomains in anti-IgM-activated B cells, and affects the signaling activity of the BCR on splenic B cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The Tg mice expressing the Ig H+L chain genes from the 3-83 BCR that confers reactivity to the mouse MHC class I Ags H-2Kb and -Kk (26) were provided by Dr. David Scott (American Red Cross, Rockville, MD) and maintained on an H-2d background by continuous backcrosses to BALB/c mice (National Cancer Institute, Frederick, MD). 3-83 Tg mice were also bred to H-2b ß2m-/- mice (27), which were the gift of Dr. Mark Soloski (Johns Hopkins University). Mice expressing Ig H+L transgenes encoding a BCR specific for HEL and anti-HEL/soluble HEL double Tg mice (28) were obtained from Dr. Tim Behrens (University of Minnesota, Minneapolis, MN) or from The Jackson Laboratory (Bar Harbor, ME). VH12 Tg mice (29), which carry a rearranged Ig H chain gene from a PC-specific B-1a clone, were provided by Dr. Stephen Clarke (University of North Carolina, Chapel Hill). Both anti-HEL Tg and VH12 Tg mice were maintained as heterozygotes by continuous backcrosses to C57BL/6 mice. Control non-Tg mice were offspring from these same matings. Mice were maintained in specific pathogen-free conditions in our facility and unless otherwise noted were used in experiments at 4–20 wk of age.
Reagents
Recombinant human AnV was cloned by PCR, expressed in
Escherichia coli, then purified and conjugated to FITC or
biotin as previously described (30). Our reagent gave
results identical with those using commercially prepared versions of
FITC-rAnV (Clontech, Palo Alto, CA) or biotin-rAnV (PharMingen, San
Diego, CA). TUNEL assays were performed with a kit (Roche,
Indianapolis, IN) according to the manufacturer’s instructions.
4',6-Diamidino-2-phenylindole and DiOC6 were
obtained from Molecular Probes (Eugene, OR); 7AAD, MC540, Quantum Red
(QR)-SA, and FITC- cholera toxin (CTx; ß subunit) were purchased from
Sigma (St. Louis, MO). Unlabeled SA used in cross-linking experiments
was purchased from ICN (Irvine, CA). Anti-B220-PE (clone RA3-6B2),
anti-CD3
-biotin (clone 2C11), and anti-CD19-biotin (clone
1D3) mAbs were obtained from PharMingen. Unlabeled and PE-labeled goat
anti-mouse IgM, PE-goat anti-mouse IgG2a, and goat
anti-mouse IgD-biotin were purchased from Southern Biotechnology
Associates (Birmingham, AL). Both FITC- and biotin-conjugated versions
of 54.1, the mAb that recognizes the 3-83 Id, were gifts from Terri
Grdina (American Red Cross). The anti-mouse MHC class I
(H-2Kb, Ld) mAb 28-14-8, a
mouse IgG2a mAb, was provided by Dr. Mark Soloski (Johns Hopkins
University, Baltimore, MD). The mouse anti-
-fodrin mAb 1622 was
purchased from Chemicon International (Temecula, CA), and
HRP-conjugated sheep anti-mouse Ig was purchased from Amersham
(Arlington Heights, IL). 4G10, a mouse anti-phosphotyrosine mAb,
was obtained from Upstate Biotechnology (Lake Placid, NY). 60.815 is a
human anti-human poly(A)DP-ribose polymerase (PARP) antisera that
cross-reacts on mouse PARP (31); HRP-conjugated goat
anti-human Ig was purchased from Southern Biotechnology
Associates.
Apoptosis control cell line
103-bcl2 cells (32) were obtained from Dr. Naomi Rosenberg (Tufts University, Boston, MA) and were grown in RPMI 1640 supplemented with 10% FBS, 4 mM glutamine, 10 mM HEPES, 50 µM 2-ME, and antibiotics at 33°C in a 5% CO2 incubator. For induction of apoptosis, the cells were shifted to 39°C for 12–16 h.
Cell preparation and purification
BM cells were isolated from femurs and tibias by careful disruption in PBS using a mortar and pestle. Cells were resuspended, depleted of bone fragments by passive sedimentation, and pelleted at 1000 x g. Splenocytes were obtained by crushing spleens between glass slides, then resuspending and pelleting the cells as described for BM. To avoid the potential loss or gain of rAnV+ cells, BM and spleen samples were not depleted of RBC by Ficoll treatment or hypotonic lysis; instead, RBC and dead cells were gated out electronically after flow cytometric analysis. All cells were resuspended in FACS wash buffer (FACS WB; HBSS, 1% BSA, and 10 mM HEPES, pH 7.4) at a concentration of 20–30 x 106 c/ml before staining. To enrich for B cells, BM or spleen preparations were stained with anti-CD19-biotin followed by a wash by SA-conjugated magnetic beads, then passed over a MiniMacs separation column according to the manufacturer’s instructions (Miltenyi Biotec, Sunnyvale, CA).
Pronase digestion of splenocytes
Splenocytes were purified by Ficoll-gradient centrifugation and resuspended in HBSS and 10 mM HEPES, pH 7.5, before incubation for 5 min at 37°C in the presence or the absence of 50 µg/ml pronase (Roche). Cells were transferred to an ice bucket, and an excess of ice-cold HBSS and 10% newborn calf serum was added. Cells were then recovered by centrifugation and washed two additional times in HBSS and 10% newborn calf serum. Finally, cells were resuspended in FACS WB and analyzed by flow cytometry as described below.
Cell staining and flow cytometry
For staining with FITC- or biotin-rAnV, 1–1.5 x
106 cells were transferred to 5-ml tubes and
washed with 1 ml of FACS WB and 2 mM CaCl2 or WB
and 2 mM EGTA, then pelleted at 1000 x g. The
interaction between AnV and PS is calcium dependent and as such should
be abolished in the presence of EGTA. Cells were then incubated on ice
for 20 min in the presence of saturating amounts of the appropriate
FITC-, PE-, and/or biotin-conjugated mAbs and FITC- or biotin-rAnV in a
total volume of 100 µl of FACS WB with Ca2+ or
EGTA. Cells were washed with 1.5 ml of WB and
Ca2+ or EGTA, pelleted, then resuspended in 100
µl WB and Ca2+ or EGTA containing a saturating
amount of QR-SA. After another 20-min incubation on ice, the cells were
washed and pelleted as before, then resuspended in 0.5 ml of WB and
Ca2+ or EGTA and analyzed on a FACScan using
CellQuest software (Becton Dickinson, Mountain View, CA), except for
those analyses shown in Fig. 3
B, which were performed on a
Coulter EPICS-EL cytometer (Coulter, Hialeah, FL) and analyzed with
FloJo software (Tree Star, San Carlos, CA). MC540 was added just before
FACS analysis at a final concentration of 0.1 µg/ml. Cells were
stained with DiOC6 (40 nM, final concentration)
for 15 min at 37°C and were washed before staining with other
reagents. Detectors for forward and side light scatter were set on a
linear scale, whereas logarithmic detectors were used for all three
fluorescence channels (FL-1, FL-2, and FL-3). Compensation for spectral
overlap between FL channels was performed for each experiment using
single-color-stained cell populations. Wherever possible, instrument
settings were saved to disk and used again with slight modifications if
necessary in related experiments. All cells were collected ungated to
disk, and data were analyzed using CellQuest software. Unless otherwise
noted, RBC and dead cells were excluded by electronically gating data
on the basis of FSC vs SSC profiles; a minimum of
104 cells of interest were analyzed further.
|
Liposomes, generated essentially as previously described (33), were composed of 70% PC and 30% of each of the phospholipids PS, PI, phosphatidic acid, SM, and PtE, or 100% PC. Pure preparations of lipids (Sigma) were added to glass tubes in appropriate combinations, mixed gently, evaporated to dryness under N2 gas, resuspended in HEPES buffer to a final concentration of 1 mM, then sonicated on ice for 5 min. Liposomes were used within 2 h of preparation. Before staining cells with FITC-AnV, liposomes were added to the FITC-rAnV solution at a predetermined optimal concentration (5 µM), followed by incubation for 10 min at room temperature. Cells were stained with the AnV/liposome mixture in the usual manner, washed thoroughly, stained with B220-PE followed by 7AAD, and then analyzed on a FACScan.
Western immunoblotting
To analyze the status of -fodrin cleavage in various cell
populations, cells were washed twice with PBS, pelleted, and
resuspended in an equal volume of PBS and 2x SDS gel loading buffer
(17% glycerol, 5% SDS, 0.05% bromophenol blue, 1.4 M 2-ME, and 0.45
M Tris, pH 6.8), and then boiled 10 min. Cellular debris was removed by
centrifugation for 5 min at 14,000 rpm, and supernatants were
transferred to fresh tubes. Extracts corresponding to 1.2–2 x
106 cell equivalents were loaded into each lane
of an 8.5% SDS-PAGE gel and blotted after electrophoresis to
Hybond-ECL nitrocellulose membrane (0.45 µm pore size; Amersham,
Arlington Heights, IL) at 55 V for 5 h in transfer buffer (25 mM
Tris, 192 mM glycine, and 15% methanol). Nonspecific binding was
blocked by overnight incubation in BLOTTO (5% nonfat milk powder and
0.1% Tween 20 in PBS) at 4°C. Blots were probed with the
anti--fodrin mAb 1622 at a dilution of 1/2,500 in BLOTTO for
1 h at room temperature. Filters were washed three times for 5 min
each time with PBS/0.1% Tween 20 (PBS-T), then incubated for 30 min at
room temp with BLOTTO containing a 1/2,500 dilution of HRP-conjugated
sheep anti-mouse Ig (Amersham). Filters were washed extensively
(once for 15 min, then twice for 5 min each time) with PBS-T, and the
specific protein complexes were identified using the ECL-Western
blotting detection system (Amersham). For subsequent analysis of PARP
cleavage, blots were stripped for 30 min at 50°C in strip buffer (0.1
M 2-ME, 2% SDS, and 62.5 mM Tris-HCl, pH 6.7), washed extensively with
PBS-T, incubated in BLOTTO O/N at 4°C, then probed as described above
with the anti-human PARP mAb 60.815 at a dilution of 1/5,000. After
washing, the blot was incubated with goat anti-human Ig-HRP at a
1/15,000 dilution, then developed by ECL. For anti-phosphotyrosine
immunoblotting, cells were stimulated as noted in the text, washed
twice in protein-free HBSS containing 1.5 mM
CaCl2 and 1 mM
Na3VO4, lysed for 10 min on
ice in TNT lysis buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tris-Cl
(pH 7.6), 1 mM Na3VO4, 1 mM
PMSF, 1 µg/ml aprotinin, and 1 µg/ml leupeptin), then centrifuged
to remove debris. Supernatants (from 106
cells/lane) were run on 11% acrylamide gels and transferred to
nitrocellulose. Filters were blocked overnight in TBS/BSA (10 mM
Tris-Cl (pH 8.0), 150 mM NaCl, and 2% BSA), then incubated at room
temperature for 1 h with 1 µg/ml 4G10 diluted in TBS/BSA. The
blot was washed three times (10 min each time) with PBS-T, incubated
with sheep anti-mouse-Ig-HRP (1/6,000) for 30 min at room
temperature, then washed three times with PBS-T (10 min each) and
developed by ECL.
Immunofluorescence microscopy
Cells were stained (or not) with mAbs as indicated in the text, fixed for 30 min at room temperature in fresh 2% paraformaldehyde solution, then centrifuged and washed twice. For GM1 staining, fixed cells were incubated with FITC-CTx (8 µg/ml) for 30 min, then washed before analysis. Confocal microscopy was performed on a scanning confocal microscope system (model LSM 410, Carl Zeiss, Thornwood, NY).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We prepared rAnV and conjugated it to either FITC or biotin for
use in flow cytometry (30). To confirm the ability of rAnV
to detect cells undergoing apoptosis, we stained 103-bcl2, a
temperature-sensitive Abelson virus-transformed pro-B cell line
(32). This cell line can be induced to undergo apoptosis
by shifting cells from the permissive temperature (33°C) to a
nonpermissive temperature (39°C) (32). As predicted from
previous studies (30), when FITC-rAnV is used in
combination with the DNA-binding dye 7-aminoactinomycin D (7AAD;
excluded from viable cells with an intact plasma membrane) to stain
103-bcl2 cells cultured at 39°C overnight, we can readily detect both
apoptotic (rAnV+7AAD-) and
dead (rAnV+7AAD+) cells
(Fig. 1). The light scatter
characteristics of the latter cells correlate well with their rAnV/7AAD
profiles, as most of these are
FSClowSSCint/high (data not
shown). Only sorted
rAnV-7AAD- (viable) cells
and not rAnV+7AAD-
(apoptotic) cells incorporated [3H]thymidine
upon subsequent culture for 72 h at the permissive temperature
(data not shown). The
rAnV+7AAD- cells also
exhibited membrane blebbing and nuclear condensation, and their DNA
displayed internucleosomal fragmentation, all characteristic features
of apoptosis (30) (data not shown).
|
Given that rAnV so clearly demarcates apoptotic 103-bcl2 cells in vitro, we proceeded to use this reagent to stain primary B cells directly ex vivo. We isolated BM cells and splenocytes from a variety of mice, including normal nontransgenic (non-Tg) mice and mice expressing transgenes encoding both the H and L chains of an Ab specific for the MHC class I molecule H-2K (clone 3-83) (26). The 3-83 Ab, isolated from a B-2 clone, binds to H-2Kk with high affinity and to H-2Kb with moderate affinity, but does not bind with appreciable affinity to H-2Kd (34, 35). Thus, in H-2d 3-83 Tg mice or in H-2b ß2m-/- 3-83 Tg mice (whose cells lack surface expression of MHC class I molecules) (27), nearly all the immature BM B cells and mature peripheral B cells express the Tg BCR and stain with the 3-83 Id-specific mAb 54.1 (35, 36) (data not shown). However, in H-2b or H-2k 3-83 Tg mice, B cell development is arrested at an immature stage, and those Tg B cells remaining are IgMlowIgD- (14).
We used anti-B220-PE and FITC-rAnV to stain BM and splenic cells
isolated from 3-83 Tg mice on both a negatively selecting background
(H-2b
ß2m+/-) and a background
permissive for the survival of cells expressing the Tg BCR
(H-2b
ß2m-/-). Contrary to
expectation, we found that most of the live-gated
B220+ cells in the
ß2m-/- Tg BM and spleen
bind rAnV despite the absence of a self Ag that might trigger apoptosis
(Fig. 2). These Tg B cells bind rAnV at
intermediate levels (rAnVint): significantly
higher than background staining in the absence of
Ca2+, but lower than the levels found on dead or
dying cells (Fig. 2 and see below). Furthermore, a substantial
proportion (
40–50%) of B cells in both non-Tg and
H-2b
ß2m+/- Tg BM and spleen
also bind rAnV at intermediate levels (Fig. 2).
|
). We
also stained cells from anti-HEL/soluble HEL (sHEL) double-Tg mice,
where the presence of HEL as a self Ag induces anergy in the B cells
expressing the anti-HEL BCR (28). Again, a large
fraction of the anergic Tg B cells in these mice bound FITC-rAnV
(Fig. 2).
Finally, we examined VH12 Tg mice, which bear an
IgH transgene isolated from a B-1 cell clone specific for PC. In
VH12 Tg mice, the rearranged IgH transgene
efficiently mediates positive selection of developing B cells into the
B-1 cell subset (29). When we stained BM and spleen cells
from these mice with anti-B220-PE and FITC-rAnV, we again found
that a large fraction of Tg BM cells bound rAnV, as did nearly all the
splenic B-1 cells (Fig. 2 and our manuscript in preparation).
Interestingly, we did not observe similar rAnV binding on splenic T
cells. In general, <10% of CD3+ splenic T cells
(Fig. 2) or thymocytes (data not shown) in any of the mice we analyzed
(including TCR Tg mice; our manuscript in preparation), bind rAnV above
background levels. Thus, PS exposure on nonapoptotic cells is not a
general characteristic of all lymphocytes, but is instead a feature
that is relatively specific to the B cell lineage.
We performed two experiments to address the binding specificity of
rAnV. First, FITC-rAnV was preincubated with liposomes prepared from a
panel of phospholipids (33) before staining BM cells. The
rAnV binding to viable (7AAD-) non-Tg or
H-2d 3-83 Tg BM B cells was strongly inhibited by
5 µM PS-containing liposomes and partially inhibited by PI (another
negatively charged phospholipid), but was virtually unaffected by PC,
PtE, phosphatidic acid, or SM (Fig. 3A). Thus, rAnV is binding in
a specific fashion to negatively charged phospholipids, especially PS,
exposed on the surface of BM B cells. Second, we sought to determine
whether rAnV was binding to a B cell-specific lipoprotein rather than
to the lipid membrane itself. Splenocytes were incubated with pronase
before staining with anti-CD19-PE and rAnV-FITC. As shown in Fig. 3B, pronase treatment completely eliminated anti-CD19
staining, but only reduced rAnV staining 2-fold. Thus, rAnV is almost
certainly binding to PS in the plasma membrane itself.
Most rAnV-binding B cells are not apoptotic
Our initial experiments suggested that most of the B cells that
bind rAnV in Ig Tg and non-Tg BM are not undergoing apoptosis. To
confirm this observation, we examined rAnV-binding BM B cells using a
wide variety of independent assays to detect apoptosis. One of the
hallmarks of the later stages of apoptosis is internucleosomal
DNA cleavage (37). This form of DNA degradation can
be detected either by agarose gel electrophoresis or by the TUNEL assay
(38). Cellular DNA isolated from FACS-sorted
rAnVintB220+7AAD-
H-2d 3-83 Tg BM cells did not exhibit
laddering on agarose gels (data not shown). Likewise, when we subjected
non-Tg or H-2d 3-83 Tg BM cells to TUNEL
analysis, we found that only a small proportion of rAnV-binding cells
were also TUNEL+, and that these cells stained
brightly with rAnV (rAnVhigh; Fig. 4A). Virtually none of the
dominant rAnVint B cell population is
TUNEL+. Similar results were obtained using
splenocytes from VH12 Tg mice, again ruling out
potential idiosyncrasies with the 3-83 Tg mice (data not shown). These
results are in striking contrast to the staining patterns observed for
induced 103-bcl2 cells, in which nearly all the rAnV-binding cells are
also TUNEL+ (Fig. 4A). We also
examined sorted rAnV+7AAD-
3-83 Tg BM B cells by fluorescence microscopy after staining the cells
with the DNA-binding dye 4',6-diamidino-2-phenylindole to mark nuclei.
These cells displayed a normal nuclear staining pattern, and their
general morphology did not reveal the membrane blebbing that generally
accompanies apoptosis (data not shown).
|
m), an event that
generally occurs concomitant with or just before surface exposure of PS
(39). To detect a possible loss of
m in rAnV+ B cells,
we stained non-Tg and 3-83 Ig Tg BM cells with the lipophilic dye
DiOC6 (40). Cells that have lost
their m are unable to retain
DiOC6, as we observed in control experiments with
induced 103-bcl2 cells (Fig. 4B). In distinction to
apoptotic 103-bcl2 cells, the majority of both non-Tg and 3-83 Tg
rAnVint BM B cells retain
DiOC6 at high levels (Fig. 4B).
Merocyanine 540 (MC540) is a dye that intercalates into loosely packed
lipid membranes (41). Cells at the later stages of
apoptosis begin to lose the integrity of their lipid arrangement and
will bind MC540 (42). As shown in Fig. 4C,
apoptotic 103-bcl2 cells that bind the highest levels of rAnV also
stain with MC540. Similarly, in 3-83 Tg or non-Tg BM, the cells binding
the highest amounts of rAnV also bind MC540. The dominant population of
rAnVint Tg B cells falls into both
MC540+ and MC540- subsets
(Fig. 4C). These results indicate that although the majority
of these B cells maintain their overall membrane integrity, there are
alterations in the lipid packing on those cells binding the highest
levels of rAnV (see Discussion).
Because most cytometric assays for apoptosis measure events that occur
after the exposure of PS, we next turned to biochemical analyses of
rAnV-binding B cells. One of the very earliest events in cells
undergoing apoptosis is the cleavage of various protein substrates by
caspases (reviewed in Ref. 43). The DNA repair enzyme PARP
is cleaved almost immediately after the onset of apoptosis
(44). A second target protein is -fodrin (nonerythroid
spectrin), a ubiquitous membrane-associated cytoskeletal protein whose
cleavage during apoptosis is thought to play a role in membrane
blebbing (45). To determine whether these proteins have
been cleaved in rAnV-binding BM B cells, we prepared whole cell lysates
from immunoaffinity-purified
CD19+rAnV+
H-2d 3-83 Tg BM B cells, non-Tg
CD19+ BM cells, or induced 103-bcl2 cells and
subjected them to immunoblot analysis with Abs specific for -fodrin
or PARP. Although extensive cleavage of -fodrin (240 kDa) into its
150- and 120-kDa products is clearly evident in the induced 103-bcl2
sample, none of the 150-kDa product and only a small amount of the
120-kDa product were found in the BM B cell samples (Fig. 5A). The presence of the
150-kDa product is most indicative of active apoptosis, because it is
only transiently formed (46). The 120-kDa band is more
ubiquitous, as it is generated even in the largely
rAnV- splenocyte population (Fig. 5A)
and in purified splenic T cells that are essentially
rAnV- (data not shown). Strikingly, no
degradation of PARP is detectable among rAnV+ BM
B cells, in clear contrast to the induced 103-bcl2 cells, which exhibit
extensive PARP cleavage (Fig. 5B). Thus, by multiple
independent criteria, these rAnV-binding B cells are not apoptotic.
|
To examine the distribution of PS on the surface of B cells, we
used fluorescence microscopy to analyze both Ig Tg and non-Tg
splenocytes costained with FITC-rAnV and goat anti-mouse-IgM-PE.
Cells kept on ice and examined while still cold exhibited a homogeneous
staining pattern for both PS and IgM (Fig. 6A). However, as the cells
warmed to room temperature and the IgM molecules began to cap at one
pole of the cell (46), the PS clearly traveled with the
IgM into the cap (Fig. 6B). This co-capping phenomenon did
not depend upon cross-linking PS with rAnV, because we also found PS in
caps of IgM formed before we stained with rAnV (Fig. 6C).
The co-capping we observed is specific; if we first cross-linked IgM on
splenic B cells, then fixed the cells and stained with FITC-rAnV and an
Ab to MHC class I, the PS entered the IgM cap while the MHC molecules
remained homogeneously distributed (Fig. 6D). We also found
that B220 (CD45R) was generally absent from PS/IgM caps in
anti-IgM-treated cells (data not shown; see Discussion).
Thus, to our surprise, a lipid probe (rAnV) and a protein probe
(anti-IgM Ab) simultaneously altered their membrane distribution in
capped B cells.
|
One of the components of lipid rafts is the GM1 glycosphingolipid,
which binds the ß subunit of CTx (50). FITC-labeled CTx
can therefore be used to stain fixed cells and directly visualize these
raft domains (51, 52). To test whether GM1 is present in
IgM/PS caps, we stimulated splenocytes with unlabeled anti-IgM,
stained them with rAnV-biotin followed by Texas Red-streptavidin
(TR-SA), then fixed the cells and stained them with FITC-CTx. As shown
in Fig. 6E, PS and GM1 colocalize in the
anti-IgM-induced caps. Interestingly, we found that PS, IgM, and
GM1 also colocalize on anti-IgM-treated anergic B cells from an
anti-HEL/sHEL double-Tg mouse (Fig. 6F)
(28). This indicates that anergic B cells are still
capable of assembling IgM into rAnV- and CTx-binding domains, and
implies that their functional deficiencies lie downstream of this early
event (53, 54).
PS, IgM, and GM1 are frequently co-capped in vivo on B-1 cells and anergic B-2 cells
When we assessed the phenotypes of splenocytes directly ex vivo by
fixing the cells immediately after isolation and staining them with
FITC-CTx and anti-IgM-PE, we frequently found cells in the
anti-HEL Ig/sHEL double-Tg mice that had already capped their
surface IgM in vivo, presumably mediated by their interaction(s) with
Tg sHEL protein (Fig. 6H). This in vivo capping was never
observed in the anti-HEL Ig Tg B cells, which developed in the
absence of the sHEL Ag (Fig. 6G). Pre-capping in vivo was
also evident in many of the splenic B-1 cells from the
VH12 Ig Tg mice (data not shown), whose BCR
recognizes PC, a ubiquitous lipid species (Fig. 2). This constitutive
capping of the BCR in B-1 cells and in anergic B-2 cells may have
implications for the signaling activities of the BCR under these
physiologic circumstances (see Discussion).
Disruption of PS/IgM co-capping interferes with BCR signaling events
Having found that PS associates with IgM undergoing capping in
anti-Ig-treated B cells, we investigated whether this association
has any influence on signaling by the BCR. We stained splenic B cells
with rAnV-biotin and then treated the washed cells with SA for 20 min
at 37°C. Confocal microscopy revealed that this treatment resulted in
clustering of PS on the B cell surface without significantly affecting
the distribution of surface IgM (Fig. 7A). We then incubated rAnV-
plus SA-treated or untreated splenocytes with various amounts of a
cross-linking anti-IgM Ab for 5 min at 37°C. Lysates from these
cells were prepared and analyzed for changes in the pattern of
tyrosine-phosphorylated proteins by Western blot (Fig. 7B).
As expected, B cells treated with as little as 0.6 µg/ml anti-IgM
showed a striking increase in the levels of many phosphorylated species
compared with the pattern seen in lysates from control unstimulated
cells (Fig. 7B, lanes 5, 7, 9, and
11). Notably, one phosphoprotein with an approximate
molecular mass of 16 kDa present in lysates of unstimulated cells lost
its phosphotyrosine(s) after anti-IgM treatment (Fig. 7B, compare lanes 1 and 5).
Pretreatment of the B cells with rAnV-biotin and SA resulted in at
least one striking change in this pattern; phosphorylation of the
16-kDa protein was maintained regardless of surface IgM cross-linking,
implying that the preclustering of PS disrupts the dephosphorylation of
this protein after BCR stimulation. If we co-cross-link PS and IgM by
adding SA to cells prestained with rAnV-biotin and anti-IgM-biotin,
the 16-kDa species is almost completely dephosphorylated (Fig. 7B, lane 14), as it is with anti-IgM-biotin
and SA alone (lane 13). Pretreatment of B cells with
rAnV-biotin and SA resulted in several additional changes in the
phosphorylation pattern, including the accelerated disappearance (i.e.,
from 10 to 4 µg/ml anti-IgM) of a phosphoprotein of 150 kDa
(Fig. 7B, compare lanes 7 and 8). This
result indicates that sequestration of PS by rAnV cross-linking also
interferes with kinase activity in anti-IgM-treated B cells. Thus,
we conclude that the presence of PS or of membrane alterations
associated with rAnV binding can influence the nature of the BCR
signal.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The exposure of PS on the surface of apoptotic cells is a
well-documented phenomenon (reviewed in Ref. 21). We
report here our surprising finding that viable developing and mature B
cells bearing a BCR that can mediate positive selection, and thus
inclusion of a B cell into the peripheral repertoire, display an
altered membrane structure that allows rAnV binding to the plasma
membrane. Furthermore, we have shown that rAnVint
B cells do not display any of the other well-known biochemical or
morphologic characteristics associated with apoptosis, including
internucleosomal DNA fragmentation, loss of mitochondrial
m, nuclear chromatin condensation, or the
cleavage of proteins such as -fodrin and PARP (Figs. 4 and 5 and
data not shown). Therefore, the exposure of PS can be unlinked from the
apoptosis pathway in lymphocytes. Our data do not imply, however, that
rAnV cannot be used to detect apoptotic B cells. In addition to its
clear utility in identifying apoptotic B cells in vitro
(25) (Fig. 1), this reagent does detect small populations
of rAnVhigh TUNEL+
DiOC6- BM B cells that
apparently are undergoing apoptosis (Fig. 4, A and
B).
How do PS+ B cells escape engulfment by BM macrophages?
It is believed that morphological changes on the surface of cells
dying in vivo, including the exposure of PS, provide key signals to
neighboring phagocytes that induce these scavengers to engulf and
digest them (reviewed in Ref. 55). This specialized
process of cellular housekeeping prevents the induction of inflammation
and tissue damage that can arise when cells undergo a necrotic death.
If TUNEL+ self-reactive thymocytes can be found
within thymic macrophages (17), how can B cells with
exposed PS escape phagocytosis by BM macrophages? One possibility is
that the PS receptors expressed by macrophages may be dependent upon
the overall density of PS on the target cell, allowing
rAnVint B cells to survive in an environment
where rAnVhigh cells are targets for destruction.
However, it seems more likely that rAnVint B
cells lack additional surface changes present on apoptotic cells that
are required to trigger phagocytosis (55). At least five
macrophage surface molecules, including the vitronectin receptor
vß3 integrin, CD36;
the class A scavenger receptor, CD14; and the ATP binding cassette
transporter, ABC-1, have been shown to be involved in the removal of
apoptotic cells, in some cases through adhesive bridging interactions
with extracellular matrix proteins on the apoptotic target (21, 55). In addition to PS redistribution on apoptotic cells, some
carbohydrate changes at the cell surface may provide important ligands
for phagocytes (56). It is therefore probable that the
viable PS+ BM B cells we have identified (Fig. 2)
lack the full spectrum of requisite ligands for the surveying
macrophages (21, 55, 57).
The role of PS associated with the BCR
Mature B cells triggered through their Ag receptors initiate a
signaling cascade characterized by the phosphorylation and
dephosphorylation of multiple cellular proteins. To achieve this signal
amplification, IgM is associated with several accessory molecules,
including Ig (CD79) and Igß (CD79ß), which trigger Src and
Syk family tyrosine kinases, the phosphatases CD45 (B220) and
SH2-containing phosphatase-1 (SHP-1) (via its association with
CD22), and the costimulatory molecules CD40, CD19, and CD21
(54, 58, 59). Our results suggest that PS exposed on the
surface of viable B cells may play a role as a component of membrane
microdomains (Fig. 6 and see below). Upon cross-linking with the
appropriate Abs, externalized PS co-caps with several proteins
expressed on B cells, including IgM, CD19, and MHC class I, although PS
seems to co-cap most efficiently with IgM (data not shown and Fig. 6).
Importantly, disruption of the activation-induced cosegregation of PS
and IgM (by pretreating B cells with rAnV-biotin and SA) led to at
least two notable changes in the phosphotyrosine patterns in lysates
from cells subsequently stimulated with anti-IgM (Fig. 7). Thus, we
postulate that PS is an important component of the membrane remodeling
machinery in B lymphocytes required to achieve optimal association of
signaling molecules with their intracellular targets.
Our results parallel several recent studies aimed at understanding the three-dimensional structure of the synapse between T cells and APC (52, 60, 61, 62, 63, 64). Discrete membrane microdomains, often referred to as lipid rafts, have been associated with signal transducing molecules in both nonlymphoid and lymphoid cells. Using FITC-CTx to detect GM1 in sphingolipid-cholesterol-rich rafts, one group demonstrated that these rafts aggregate in the zone of contact between T cells and anti-CD3-coated beads (52). Furthermore, these studies showed that CD28 exerts its costimulatory effects on T cells by recruiting rafts into the TCR/APC contact site, rather than by integrating CD28 and TCR signals in the nucleus, to affect gene expression (52, 62). The membrane compartmentalization between rafts and nonrafts is required for efficient T cell activation, probably by recruiting intracellular kinases to the synapse while maintaining segregation of phosphorylated substrates from phosphatases (60, 64). Interestingly, in line with a report that the phosphatase CD45 is excluded from lipid rafts in T cells (65), we found that B220 (CD45R) is generally absent from PS/IgM caps in anti-IgM-treated cells (data not shown).
It remains unclear whether the association of IgM and PS in the B cell membrane precedes signaling through the BCR or whether this association is induced by BCR signaling. Experiments to address this question, using fluorescence resonance energy transfer, are currently in progress. It will also be of interest to elucidate further the mechanism by which PS externalization is controlled in healthy cells, perhaps by regulated expression of the aminophospholipid translocase or scramblase enzymes known to affect lipid movement in the plasma membrane (66).
Although very few resting mature T cells or thymocytes bind rAnV ex
vivo (Fig. 2 and data not shown), T cells activated with Con A in vitro
show a uniform increase in rAnV-FITC binding (data not shown).
Interestingly, the PS molecules exposed on Con A blasts or on the small
subset of splenic T cells that bind rAnV (Fig. 2) also migrate into
anti-CD3- or anti-Thy-1-induced caps (data not shown). The
infrequency of resting T cells compared with resting B cells that bind
rAnV in vivo (Fig. 2) might reflect developmentally regulated
differences in the signaling pathways within B and T cells.
Endocytosis of IgM molecules bound to their cognate Ag leads to Ag processing and presentation to T cells and is therefore an integral part of the immune response. In addition to their role in concentrating appropriate signaling molecules, rafts in membranes are thought to function in sorting and trafficking transmembrane proteins through the endocytic pathway (67). Our confocal experiments have revealed that PS tagged with FITC-rAnV is not only capped, but is also internalized with the BCR on anti-IgM-treated B cells (data not shown). This suggests that PS may play a unique and significant functional role in B cell Ag presentation and might further explain its presence on a greater fraction of B cells vs. T cells.
What is the nature of the altered membrane structure on rAnV-binding B cells?
Although the liposome binding inhibition experiments
(33) indicated that FITC-rAnV binding to B cells is
strongly inhibited by preincubation of this reagent with PS-containing
liposomes (Fig. 3A), it remains possible that rAnV binding
reflects something other than PS externalization on these cells. It is
unlikely that rAnV is binding to a lipid-modified glycoprotein, because
binding was not affected by pretreatment of cells with pronase (Fig. 3B). rAnV may bind an additional, as yet unidentified, lipid
bearing a binding site identical or overlapping with that on PS.
Alternatively, rAnV may be detecting a specialized arrangement of
lipids in the membrane, perhaps a shift from a typical lamellar bilayer
to hexagonal phases, for instance (reviewed in Ref. 68).
This hypothesis is supported by our observation that a significant
fraction (generally 20–25%) of rAnV-binding B cells also stain with
MC540 (Fig. 4C), a dye that is sensitive to the molecular
packing of membrane lipids and that intercalates into loosely packed
lipid membranes (69). An altered lipid arrangement in
localized regions of contact with Ag or with other cells may increase
the efficiency of delivering appropriate signals to the B cell (Fig. 7B), as has been shown for T cells (52, 62). As
discussed above, it seems reasonable to postulate that the changes in
membrane structure revealed by rAnV and CTx on anti-IgM-stimulated
cells (Fig. 6) may be analogous to those observed in T cells receiving
costimulatory signals (52, 62).
Caps containing IgM, PS, and GM1 exist on B-1 cells and anergic B-2 cells ex vivo
Both B-1 and anergic B-2 cells are thought to receive continuous
stimulation through their Ag receptors. In the B-1 lineage, this
stimulus is thought to come from a low affinity interaction of the BCR
with self Ag, which may play a role in the initial selection of these
cells into the B-1 lineage (29). The BCR signaling differs
between B-1 and B-2 cells, especially with respect to synergy between
IgM and CD19 (70). Anergic B-2 cells also display
signaling defects specific to the BCR as opposed to other accessory
molecules, such as CD40 and cytokine receptors (71, 72).
Our surprising finding that IgM, PS, and GM1 are cocapped in B-1 and
anergic B-2 cells examined immediately ex vivo (Fig. 6) may offer an
insight as to the mechanism of these signaling differences. The BCR may
be sequestered from interaction with other regulatory surface receptors
due to its highly focal distribution in the membrane, leading to
alterations in B cell signaling behavior. Further experimentation will
be required to determine which other signaling molecules are
specifically excluded from or included within these BCR domains.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Current address: ZymoGenetics, 1201 Eastlake Avenue East, Seattle, WA 98102.
3 Address correspondence and reprint requests to Dr. Mark Schlissel at the current address: Department of Molecular and Cell Biology, 439 LSA, University of California, Berkeley, CA 94720-3200. E-mail address:
4 Abbreviations used in this paper: BCR, B cell receptor; BM, bone marrow; CTx, cholera toxin; MC540, merocyanine 540; m, mitochondrial transmembrane potential; PARP, poly(A)DP-ribose polymerase; PC, phosphatidylcholine; PI, phosphatidylinositol; PS, phosphatidylserine; PtE, phosphatidylethanolamine; QR, Quantum Red; rAnV, recombinant annexin V; SA, streptavidin; 7AAD, 7-amino-actinomycin D; (s)HEL, (soluble) hen egg lysozyme; SM, sphingomyelin; Tg, transgenic; ß2m, ß2-macroglobulin; WB, wash buffer.
Received for publication September 8, 1999. Accepted for publication November 10, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  S. R. Stowell, S. Karmakar, C. M. Arthur, T. Ju, L. C. Rodrigues, T. B. Riul, M. Dias-Baruffi, J. Miner, R. P. McEver, and R. D. Cummings Galectin-1 Induces Reversible Phosphatidylserine Exposure at the Plasma Membrane Mol. Biol. Cell, March 1, 2009; 20(5): 1408 - 1418. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Jendholm, M. Samuelsson, L.-O. Cardell, A. Forsgren, and K. Riesbeck Moraxella catarrhalis-dependent tonsillar B cell activation does not lead to apoptosis but to vigorous proliferation resulting in nonspecific IgM production J. Leukoc. Biol., June 1, 2008; 83(6): 1370 - 1378. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Smrz, P. Lebduska, L. Draberova, J. Korb, and P. Draber Engagement of Phospholipid Scramblase 1 in Activated Cells: IMPLICATION FOR PHOSPHATIDYLSERINE EXTERNALIZATION AND EXOCYTOSIS J. Biol. Chem., April 18, 2008; 283(16): 10904 - 10918. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Jackson, N. Harp, D. Patel, J. Zhang, S. Willson, Y. J. Kim, C. Clanton, and J. D. Capra CD45RO enriches for activated, highly mutated human germinal center B cells Blood, December 1, 2007; 110(12): 3917 - 3925. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. R. Barker, J. G. Parvani, D. Meyer, A. S. Hey, K. Skak, and N. L. Letvin IL-21 Induces Apoptosis of Antigen-Specific CD8+ T Lymphocytes J. Immunol., September 15, 2007; 179(6): 3596 - 3603. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Wirenfeldt, L. Dissing-Olesen, A. Anne Babcock, M. Nielsen, M. Meldgaard, J. Zimmer, I. Azcoitia, R. G. Q. Leslie, F. Dagnaes-Hansen, and B. Finsen Population Control of Resident and Immigrant Microglia by Mitosis and Apoptosis Am. J. Pathol., August 1, 2007; 171(2): 617 - 631. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Smrz, L. Draberova, and P. Draber Non-apoptotic Phosphatidylserine Externalization Induced by Engagement of Glycosylphosphatidylinositol-anchored Proteins J. Biol. Chem., April 6, 2007; 282(14): 10487 - 10497. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. R. Stowell, S. Karmakar, C. J. Stowell, M. Dias-Baruffi, R. P. McEver, and R. D. Cummings Human galectin-1, -2, and -4 induce surface exposure of phosphatidylserine in activated human neutrophils but not in activated T cells Blood, January 1, 2007; 109(1): 219 - 227. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Fischer, S. Voelkl, J. Berger, R. Andreesen, T. Pomorski, and A. Mackensen Antigen recognition induces phosphatidylserine exposure on the cell surface of human CD8+ T cells Blood, December 15, 2006; 108(13): 4094 - 4101. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. A. Luster, J. He, X. Huang, S. N. Maiti, A. J. Schroit, P. G. de Groot, and P. E. Thorpe Plasma Protein beta-2-Glycoprotein 1 Mediates Interaction between the Anti-tumor Monoclonal Antibody 3G4 and Anionic Phospholipids on Endothelial Cells J. Biol. Chem., October 6, 2006; 281(40): 29863 - 29871. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. I. Elliott, A. Sardini, J. C. Cooper, D. R. Alexander, S. Davanture, G. Chimini, and C. F. Higgins Phosphatidylserine exposure in B lymphocytes: a role for lipid packing Blood, September 1, 2006; 108(5): 1611 - 1617. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Watanabe, S. Kondo, K. Mizuno, W. Yano, H. Nakao, Y. Hattori, K. Kimura, and T. Nishida Promotion of Corneal Epithelial Wound Healing In Vitro and In Vivo by Annexin A5 Invest. Ophthalmol. Vis. Sci., May 1, 2006; 47(5): 1862 - 1868. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. A. Shah, H. Peng, and S. Carbonetto Role of non-raft cholesterol in lymphocytic choriomeningitis virus infection via {alpha}-dystroglycan. J. Gen. Virol., March 1, 2006; 87(Pt 3): 673 - 678. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Li, M. Wang, L. Tan, C. Wang, J. Ma, N. Li, Y. Li, G. Xu, and J. Li Docosahexaenoic acid changes lipid composition and interleukin-2 receptor signaling in membrane rafts J. Lipid Res., September 1, 2005; 46(9): 1904 - 1913. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Massullo, L. J. Druhan, B. A. Bunnell, M. G. Hunter, J. M. Robinson, C. B. Marsh, and B. R. Avalos Aberrant subcellular targeting of the G185R neutrophil elastase mutant associated with severe congenital neutropenia induces premature apoptosis of differentiating promyelocytes Blood, May 1, 2005; 105(9): 3397 - 3404. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Jackson and J. D. Capra IgH V-Region Sequence Does Not Predict the Survival Fate of Human Germinal Center B Cells J. Immunol., March 1, 2005; 174(5): 2805 - 2813. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. D. Arditti, A. Rabinkov, T. Miron, Y. Reisner, A. Berrebi, M. Wilchek, and D. Mirelman Apoptotic killing of B-chronic lymphocytic leukemia tumor cells by allicin generated in situ using a rituximab-alliinase conjugate Mol. Cancer Ther., February 1, 2005; 4(2): 325 - 332. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Z. Wang, M. A. Brehm, and R. M. Welsh Preapoptotic Phenotype of Viral Epitope-Specific CD8 T Cells Precludes Memory Development and Is an Intrinsic Property of the Epitope J. Immunol., October 15, 2004; 173(8): 5138 - 5147. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. A. Maianski, D. Roos, and T. W. Kuijpers Bid Truncation, Bid/Bax Targeting to the Mitochondria, and Caspase Activation Associated with Neutrophil Apoptosis Are Inhibited by Granulocyte Colony-Stimulating Factor J. Immunol., June 1, 2004; 172(11): 7024 - 7030. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. W. Kuijpers, N. A. Maianski, A. T. J. Tool, K. Becker, B. Plecko, F. Valianpour, R. J. A. Wanders, R. Pereira, J. Van Hove, A. J. Verhoeven, et al. Neutrophils in Barth syndrome (BTHS) avidly bind annexin-V in the absence of apoptosis Blood, May 15, 2004; 103(10): 3915 - 3923. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. C. Frasch, P. M. Henson, K. Nagaosa, M. B. Fessler, N. Borregaard, and D. L. Bratton Phospholipid Flip-Flop and Phospholipid Scramblase 1 (PLSCR1) Co-localize to Uropod Rafts in Formylated Met-Leu-Phe-stimulated Neutrophils J. Biol. Chem., April 23, 2004; 279(17): 17625 - 17633. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Dietzen, K. L. Page, and T. A. Tetzloff Lipid rafts are necessary for tonic inhibition of cellular tissue factor procoagulant activity Blood, April 15, 2004; 103(8): 3038 - 3044. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. P. Mone, P. Huang, H. Pelicano, C. M. Cheney, J. M. Green, J. Y. Tso, A. J. Johnson, S. Jefferson, T. S. Lin, and J. C. Byrd Hu1D10 induces apoptosis concurrent with activation of the AKT survival pathway in human chronic lymphocytic leukemia cells Blood, March 1, 2004; 103(5): 1846 - 1854. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. K. Callahan, M. S. Halleck, S. Krahling, A. J. Henderson, P. Williamson, and R. A. Schlegel Phosphatidylserine expression and phagocytosis of apoptotic thymocytes during differentiation of monocytic cells J. Leukoc. Biol., November 1, 2003; 74(5): 846 - 856. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Dias-Baruffi, H. Zhu, M. Cho, S. Karmakar, R. P. McEver, and R. D. Cummings Dimeric Galectin-1 Induces Surface Exposure of Phosphatidylserine and Phagocytic Recognition of Leukocytes without Inducing Apoptosis J. Biol. Chem., October 17, 2003; 278(42): 41282 - 41293. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. E. Kagan, G. G. Borisenko, B. F. Serinkan, Y. Y. Tyurina, V. A. Tyurin, J. Jiang, S. X. Liu, A. A. Shvedova, J. P. Fabisiak, W. Uthaisang, et al. Appetizing rancidity of apoptotic cells for macrophages: oxidation, externalization, and recognition of phosphatidylserine Am J Physiol Lung Cell Mol Physiol, July 1, 2003; 285(1): L1 - L17. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. K. Callahan, P. M. Popernack, S. Tsutsui, L. Truong, R. A. Schlegel, and A. J. Henderson Phosphatidylserine on HIV Envelope Is a Cofactor for Infection of Monocytic Cells J. Immunol., May 1, 2003; 170(9): 4840 - 4845. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Meffre and M. C. Nussenzweig Deletion of immunoglobulin beta in developing B cells leads to cell death PNAS, August 20, 2002; 99(17): 11334 - 11339. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Y. H. Mak, J. J. Brosens, M. Christian, F. A. Hills, L. Chamley, L. Regan, and J. O. White Regulated Expression of Signal Transducer and Activator of Transcription, Stat5, and its Enhancement of PRL Expression in Human Endometrial Stromal Cells in Vitro J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2581 - 2588. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Kunzelmann-Marche, J.-M. Freyssinet, and M. C. Martinez Loss of Plasma Membrane Phospholipid Asymmetry Requires Raft Integrity. ROLE OF TRANSIENT RECEPTOR POTENTIAL CHANNELS AND ERK PATHWAY J. Biol. Chem., May 24, 2002; 277(22): 19876 - 19881. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. van den Eijnde, M. J. B. van den Hoff, C. P. M. Reutelingsperger, W. L. van Heerde, M. E. R. Henfling, C. Vermeij-Keers, B. Schutte, M. Borgers, and F. C. S. Ramaekers Transient expression of phosphatidylserine at cell-cell contact areas is required for myotube formation J. Cell Sci., March 12, 2002; 114(20): 3631 - 3642. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Cipriani, G. Borsellino, H. Knowles, D. Tramonti, F. Cavaliere, G. Bernardi, L. Battistini, and C. F. Brosnan Curcumin Inhibits Activation of V{gamma}9V{delta}2 T Cells by Phosphoantigens and Induces Apoptosis Involving Apoptosis-Inducing Factor and Large Scale DNA Fragmentation J. Immunol., September 15, 2001; 167(6): 3454 - 3462. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. S. Berenson, R. H. Rasp, J.-T. Gau, J. L. Ryan, and H. C. Yohe Differences in splenic B-lymphocyte ganglioside expression and accessibility in normal and endotoxin-hyporesponsive mice J. Leukoc. Biol., June 1, 2001; 69(6): 969 - 976. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. R. Dillon, A. Constantinescu, and M. S. Schlissel Annexin V Binds to Positively Selected B Cells J. Immunol., January 1, 2001; 166(1): 58 - 71. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Reichlin, Y. Hu, E. Meffre, H. Nagaoka, S. Gong, M. Kraus, K. Rajewsky, and M. C. Nussenzweig B Cell Development Is Arrested at the Immature B Cell Stage in Mice Carrying a Mutation in the Cytoplasmic Domain of Immunoglobulin {beta} J. Exp. Med., December 27, 2000; 193(1): 13 - 24. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Cuervo, A. V. Gomes, J. A. Barnes, and J. F. Dice Selective Degradation of Annexins by Chaperone-mediated Autophagy J. Biol. Chem., October 20, 2000; 275(43): 33329 - 33335. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
