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Prepublished online as a Blood First Edition Paper on April 17, 2002; DOI 10.1182/blood-2002-01-0027.
IMMUNOBIOLOGY
From the Division of Haematology, Hanson Institute,
Institute of Medical and Veterinary Science (IMVS), Adelaide, South
Australia.
Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is an
immunoglobulin-immunoreceptor tyrosine-based inhibitory motif
(Ig-ITIM) superfamily member that recruits and activates protein-tyrosine phosphatases, SHP-1 and SHP-2, through its intrinsic ITIMs. PECAM-1-deficient (PECAM-1 The development and maintenance of a diverse B-cell
repertoire is governed by signals through the B-cell receptor (BCR)
antigen complex, modulated by regulatory B-cell coreceptors. This
process is dependent on both the strength and duration of signaling
thresholds transduced by the BCR complex on B cells. Developing and
activated B cells undergo selective mechanisms during positive and
negative selection, maturation, and activation that determine their
survival and functional capacity. Upon exposure to self-antigen, B
cells can be physically eliminated by deletion in the bone marrow,
undergo self-editing, be functionally impaired by anergy, or become
activated leading to autoimmunity.1 Several factors
influence the fate of developing and activated B cells, including their
response to self-antigen, the microenvironment, and mechanisms of
B-cell coreceptor ligation.
Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a
130-kd glycoprotein and a member of the immunoglobulin (Ig) superfamily. In human beings, it is expressed at the lateral junctions of endothelial cells and on the surface of hematopoietic cells, including monocytes, neutrophils, natural killer cells, platelets, and
naive T and B cells.2 There is emerging evidence to
suggest that PECAM-1 may be an important regulator of antigen-induced cell activation in the context of lymphocytes. Inhibitory effects have
been observed in response to PECAM-1 cross-linking. Specifically, induction of immune stimulation of the T-cell antigen receptor (TCR)
complex on Jurkat T cells leads to tyrosine phosphorylation of PECAM-1
and recruitment of protein-tyrosine phosphatases (PTPs) SHP-2 and
possibly SHP-1.3 In addition, coligation of the TCR complex with immunoreceptor tyrosine-based inhibitory motif
(ITIM)-bearing PECAM-1 on Jurkat T cells leads to a transient block in
calcium mobilization.4 At present, there has been little
information available on the role of PECAM-1 in B-cell physiology. In
human beings, PECAM-1 is expressed on naive follicular mantle zone B cells and plasma cells but not on germinal center-specific memory B
cells.5 Signaling studies of PECAM-1 have suggested that PECAM-1 may function as an inhibitory receptor in vivo. Its cytoplasmic domain contains 2 ITIMs that upon phosphorylation of the putative tyrosine residues recruit and activate PTPs SHP-1 and SHP-2 under physiologically relevant conditions.6-8 Thus, PECAM-1
might negatively regulate BCR signaling by recruiting SHP-1 and/or
SHP-2. Consistent with this hypothesis, functional analysis of chimeric
receptors containing the extracellular and transmembrane domains of
Fc The important regulatory role of B-cell coreceptors has been
demonstrated by targeted disruption of coinhibitory receptor genes in
mice, where the absence of an inhibitory receptor leads to uncoupling
of signaling pathways responsible for control of B-cell tolerance and
activation.11-14 Immunologic defects observed include
alterations in B-cell development pathways, B-lymphocyte antigen
receptor signal transduction and, in some cases, predisposition to
development of high-affinity autoantibodies and spontaneous autoimmune
disease. Because the primary function of PECAM-1 was originally thought to be in cell adhesion, preliminary characterization of PECAM-1-deficient mice focused on a functional evaluation of PECAM-1 in the context of inflammatory adhesion-dependent cascades and
not in the context of an inhibitory coreceptor in immune
cells.15 To elucidate the function of PECAM-1 during
B-cell development and activation in vivo, we initially examined in
vitro and in vivo functional B-cell responses of B6 PECAM-1-deficient
mice compared with B6 wild-type PECAM-1+/+ mice.
Subsequently, we examined whether B6 PECAM-1-deficient mice showed any
evidence of spontaneous autoimmune disease with age.
Mice
Flow cytometric analysis
Cell proliferation assay Splenocytes from wild-type and PECAM-1 /
6-week-old mice were depleted of T cells by incubating with Thy1.2
monoclonal antibodies and Low-Tox M rabbit complement (Cedarlane,
Hornby, ON) for 1 hour at 37°C. Resting B cells were separated from
nonlymphoid cells, dead T cells, and red blood cells by a discontinuous
50%/75% Percoll gradient (Amersham Pharmacia Biotech, Castle Hill,
Australia). A total of 105 purified B cells (> 98%
B220+) were plated into 96-well flat-bottom trays in
complete RPMI 160 media containing 10% fetal calf serum. B cells were
stimulated in triplicate with indicated concentrations of
lipopolysaccharide (LPS) (Sigma Chemical, St Louis, MO),
affinity-purified F(ab')2 goat antimouse IgM (Jackson
ImmunoResearch Laboratories), affinity-purified whole Ig goat antimouse
IgM (Jackson ImmunoResearch Laboratories), and hampster antimouse CD40
(clone HM40-3) (PharMingen) with and without murine recombinant
interleukin-4 (IL-4) (Peprotech, Rocky Hill, NJ) for 72 hours.
Proliferation was assessed by the incorporation of a cell titer
96-assay reagent (Promega, Madison, WI) added during the last 2 hours
of culture, followed by measuring OD values at 490 nm.
Apoptosis assays To determine apoptosis levels in wild-type and PECAM-1/ B cells, 5 × 105 purified B
cells were cultured in complete RPMI in the presence or absence of 50 µg/mL whole anti-IgM. At times 0 and 17 hours, cells were washed
twice in phosphate-buffered saline (PBS) and stained at 0°C with
propidium iodide and FITC-conjugated annexin V (Immunotech) as per the
manufacturer's instructions and analyzed on an EPICS XL-MLC flow cytometer.
Differential CFSE labeling to compare survival of
PECAM-1+/+ and PECAM-1 / mice using
Percoll gradient separation as described above. Lymphocytes from
PECAM-1+/+ and PECAM-1/ mice were labeled
with different intensities of the fluorescein-based vital dye 5 and 6 carboxy fluorescein diacetate succinimidylester (CFSE) using previously
described methodology.16 This allows 2 different cell
populations to be tracked independently and their survival in the
recipient animals to be directly compared. Briefly, cells were
suspended at 5 × 107 mL in PBS/0.1% bovine serum
albumin (BSA), and CFSE (Molecular Probes, Eugene, OR) from a 5 mM
stock dissolved in dimethyl sulfoxide was added to give a final
concentration of 10 µM and 2.5 µM for the PECAM-1+/+
and PECAM-1/ lymphocytes, respectively, to give
"fully" and a "quarter" labeled populations, respectively.
After 10 minutes' incubation at 37°C, cells were washed in ice-cold
PBS/0.1% BSA 3 times to remove unbound CFSE. Equal numbers of
"fully" labeled PECAM-1+/+ and "quarter" labeled
PECAM-1/ lymphocytes were mixed, and a total of
3 × 107 per mouse was injected via the lateral tail vein
of PECAM-1+/+ recipients. At days 7 and 30, 3 mice were
killed, spleens removed, and splenic lymphocytes isolated. Single-cell
suspensions were stained with antibodies directed against biotinylated
CD5 (Ly-1) (T-cell marker) (PharMingen) and PECAM-1 (390, a gift from
Dr Steve Albelda), with streptavidin-PE as a secondary label, and with
CD45R-PE (B220) (B-cell marker) (PharMingen). Analysis was restricted
to lymphocyte populations as defined by forward- and side-scatter
characteristics. Data were collected on an EPICS XL-MCL and analyzed
using WinMDI version 2.8 software. These data allowed the relative
survival of PECAM-1/ versus PECAM-1+/+ T
and B lymphocytes to be compared by direct ratio. Because this approach
internally controls for variation in injection volume, it is more
accurate than injecting individual populations of cells into
separate recipients.
Calcium mobilization assays Wild-type and PECAM-1/ purified splenic B cells
(5 × 106/mL) were loaded with 3 µM Fura-2/AM
(Molecular Probes) in medium at room temperature for 45 minutes. Cells
were washed twice with PBS and resuspended in the same cell
concentration in PBS supplemented with 1 mM CaCl2 and 1 mM
MgCl2. A total of 2.0 mL of cell suspension was added to a
cuvette with a small stirrer. Calcium mobilization profiles were
recorded at 510-nm emission wavelength excited by 340 nm and 380 nm
using a Luminescence Spectrophotometer LS-50B (Perkin Elmer, Bucks,
England). After establishing a baseline, 1 to 10 µg/mL
affinity-purified F(ab')2 goat antimouse IgM (Jackson ImmunoResearch Laboratories) (cross-linking BCR alone) was added to
Fura-2-loaded murine PECAM-1+/+ and
PECAM-1/ splenic B cells. Cumulative calcium
mobilization was evaluated by integration for 4 minutes of calcium
mobilization over the baseline given before stimulation.
Immunizations and serum immunoglobulin assays Eight-week-old wild-type and PECAM-1/ mice were
bled for isolation of serum for Ig determination. These mice were then
immunized intraperitoneally with either 100 µg alum-precipitated
dinitrophenol-keyhole limpet hemocyanin (DNP-KLH)
(Calbiochem-Novabiochem, San Diego, CA) or 20 µg alum-precipitated
DNP-Ficoll (Biosearch Technologies, Novato, CA). Mice were bled at days
7, 14, and 21 after primary immunization. At day 21, mice were
rechallenged with DNP-KLH and bled at day 35 to assess the secondary
response. Sera were collected, and the anti-DNP antibodies were
determined by DNP- and isotype-specific enzyme-linked immunosorbent
assay (ELISA). In DNP-KLH experiments, sera isolated from
PECAM-1+/+ and PECAM-1/ mice at days 7, 14, and 21 were diluted from 1:1000, while at day 35 sera were diluted from
1:10 000. Briefly, Nunc Maxisorb ELISA plates were coated with DNP-BSA
(5 µg/mL) (Sigma Chemical), blocked with 1% skim milk and 0.05%
Tween 20, and incubated with sera serially diluted in PBS-Tween 20 plus 0.5% skim milk for 1 hour at 37°C. Bound antibody was detected
with secondary horseradish peroxidase-conjugated rabbit antimouse
isotype-specific antisera (Zymed Laboratories, San Francisco, CA) using
3,3',5,5'-tetramyl benzidine (TMB) as a substrate. Isotype-specific Ig
levels in unimmunized animals were determined using a mouse MonoAB ID
kit from Zymed Laboratories.
Assays for autoantibodies Sera were collected monthly for 16 months from wild-type and PECAM-1/ mice and assayed for the presence of
antinuclear antibodies (ANAs). Briefly, sera were diluted from an
initial 1:50 dilution in PBS and applied to HEp-2000 slides
(Immunoconcepts, Sacramento, CA) for 30 minutes at room temperature.
Following 2 15-minute washes in PBS, bound antibodies were detected
with fluorescein-conjugated antimouse IgG (Fab'2) 1:100
(Silenus Laboratories, Hawthorn, Australia) for 30 minutes and examined
using an immunofluorescence microscope. Anti-double-stranded DNA
(dsDNA) autoantibodies were determined using a commercial ELISA assay
(Alpha Diagnostic International, San Antonio, TX) as per the
manufacturer's instructions. Based upon the manufacturer's
recommendations, a definitive positive ELISA reading was defined as an
OD 450 nm reading of 0.2 above the negative control. Therefore, any
ELISA readings above 0.48 were considered positive.
Pathology Kidney, lungs, foot joints, and other organs were removed from aged PECAM-1+/+ (n = 8) and PECAM-1/
(n = 8) mice and then were fixed in 10% formalin in PBS and embedded in paraffin. Sections were then cut and stained for hematoxylin and
eosin and periodic acid-Schiff (PAS) reaction. Kidney portions were
also embedded in Tissue-Tek OCT compound: (Sakura Finetek USA Inc.,
Torrance, CA) and snap-frozen. Frozen sections (4 µm) were cut and
air-dried, fixed in cold acetone for 30 minutes, blocked in 10% normal
sheep serum, washed in PBS, and stained with fluorescein-conjugated
sheep antimouse IgG (Fab'2) 1:100 dilution (Silenus
Laboratories). Proteinuria was detected in long-term PECAM-1+/+ (n = 8) and PECAM-1/ mice
(n = 8) using Multistix 9 Reagent Strips for Urinalysis (Bayer
Australia, Pymble, Australia). Urinary protein levels of more than 1 g/L (100 mg/dL) were considered as positive evidence of proteinuria.
PECAM-1 expression of wild-type and PECAM-1 / mice has been
previously reported.15 These mice have been subsequently
backcrossed onto pure B6 background. To check the level of PECAM-1
expression on the surface of primary cells derived from
PECAM-1+/+ and PECAM-1/ mice, we routinely
performed flow cytometry studies to confirm homozygous mutants of
PECAM-1 and wild-type controls. Cell surface staining of mouse spleen
cells derived from PECAM-1+/+ and PECAM-1/
mice confirmed lack of expression in homozygous mutants of PECAM-1 compared with wild-type controls (Figure
1A). In these studies, we observed that
more than 95% of the splenic lymphocytes from wild-type mice are
positive for surface expression of PECAM-1. This is in contrast to
humans, where PECAM-1 expression is restricted to naive B and T cells
and not memory B and T cells.5,17
Impaired B-cell development in the bone marrow of
PECAM-1 / mice compared with wild-type
(PECAM-1+/+) controls by flow
cytometry.13,18-19 Evaluation of the total numbers of bone
marrow cells derived from age- and sex-matched 5-week-old
PECAM-1+/+ and PECAM-1/ mice revealed
comparable cell numbers (10.4 × 106 per femur ± 3.8 × 106 vs 10.9 × 106 per femur ± 3.4 × 106) (n = 17). Assessment of early pro-B cells in bone
marrow defined by dual positivity for
CD43+B220+ revealed comparable percentages of
pro-B cells in 5-week-old PECAM-1+/+ and
PECAM-1/ mice (Figure 1B, Table
1).
However, assessment of the later stages of B-cell development revealed
a reduction in mature IgM+IgDhi B cells but not
in immature IgM+IgDlo B cells in 5-week-old
PECAM-1 Transient block in B-cell maturation and increased B-1a cells in
peripheral B-cell pools of PECAM-1 / mice show
decreased body and spleen weight and decreased numbers of cells in the
spleen (8.5 × 107 vs 13.9 × 107) and
peritoneum (0.52 × 106 vs 1.11 × 106)
compared with wild-type PECAM-1+/+ control mice (Table
2). Single staining experiments for the expression of IgM and IgD demonstrate that the decrease in spleen cellularity is mainly due to a reduction in mature IgD-expressing cells
(44.7% vs 52.1%) (Table 1).
Dual staining with B220 and IgD/IgM supports this interpretation with
the PECAM-1
In preliminary experiments, we identified B-1a cells in the peritoneal
lavage of PECAM-1+/+ mice by using dual staining of
CD5/B220, CD5/IgM, CD5/Mac-1, and CD5/CD3 markers. In each case, a
distinct population of cells was identified (15.3%-16.6%) as the B-1a
cell population, defined as
CD5+B220lowIgM+Mac-1+CD3
One possible explanation for the transitional defect from immature to
mature B cells in PECAM-1
An alternative explanation for the deficiency of mature recirculating B
cells in the periphery of PECAM-1 Hyperproliferation of PECAM-1 / B cells in response to B-cell antigen receptor
stimulation by anti-IgM cross-linking, mitogen stimulation by LPS, and
CD40 stimulation using an anti-CD40 antibody in the presence and
absence of IL-4. In these experiments, T-cell-depleted splenic B cells
from wild-type and PECAM-1/ mice were obtained by
T-cell depletion with anti-Thy1.2 and complement lysis and purified
through Percoll gradient centrifugation. Resting B cells were obtained
from the 50% to 75% interface of the Percoll gradient and used for
proliferation assays. These resting B cells from
PECAM-1/ mice showed increased proliferation to all
doses of F(ab')2 anti-IgM and to stimulation with LPS
compared with wild-type controls (Figure 5A). In contrast, the resting B cells
from PECAM-1/ mice did not show increased proliferation
to varying doses of anti-CD40 with or without IL-4 compared with
wild-type controls (Figure 5B). These results are consistent with the
concept that PECAM-1 is a negative regulator of activation-induced
B-cell proliferation involving a BCR-dependent pathway and not the CD40
receptor pathway. To test if PECAM-1 expression is important for
negative signaling through Fc receptors, we examined B-cell
proliferation in response to varying doses of intact anti-IgM to
determine effects on BCR-Fc RIIB1 cross-linking. As shown in Figure
5A, resting B cells from PECAM-1/ mice showed increased
proliferation to all doses of intact anti-IgM stimulation. However, a
reduction in proliferation was observed by intact anti-IgM stimulation
compared with F(ab')2 anti-IgM stimulation. These results
are suggestive that PECAM-1 expression is not required for
FcRIIB1-mediated inhibition of B-cell proliferation.
Enhanced kinetics in calcium responses of
PECAM-1 / 13,18,21 and CD72/ B
cells.19 Because PECAM-1 can recruit and activate PTPs,
principally involving SHP-2 and to a lesser extent SHP-1, we wanted to
test the ability of wild-type and PECAM-1/ B cells to
increase their intracellular Ca++ levels after anti-IgM
F(ab')2 cross-linking. As shown in Figure 6, PECAM-1/ B cells
showed increased levels of free Ca++ following varying
doses of anti-IgM stimulation compared with wild-type controls,
particularly at subthreshold concentrations. This increase in free
Ca++ levels was observed over all concentrations of
anti-IgM tested. These results indicate that PECAM-1 negatively
regulates BCR-mediated activation pathways upstream of
Ca++ release.
Humoral immune responses of PECAM-1 / mice using an
isotype-specific capture ELISA to assess any functional defect caused
by loss of PECAM-1 expression in B-cell function. At 8 weeks of age,
PECAM-1/ mice showed a significant increase
(Student t test; P < .001; n = 8) in the
level of serum IgM compared with wild-type control mice, while all
other Ig isotypes were comparable (Figure
7A). To test the ability of
PECAM-1/ mice to mount normal humoral immune responses
to T-cell-dependent and T-cell-independent antigens, 8-week-old
wild-type and PECAM-1/ mice were immunized with DNP-KLH
or DNP-Ficoll. Mice immunized with DNP-Ficoll were bled at days 7, 14, and 21 and sera tested for DNP-specific antibodies using an
isotype-specific ELISA. As shown in Figure 7B, PECAM-1/
mice showed elevated IgG2a, IgG2b,
IgG3, and IgA responses to DNP-Ficoll at day 7 and to a
lesser extent at day 14 compared with wild-type control mice
(Student t test; P < .001; n = 8). By day
21, only the IgG2b response was still elevated. This
alteration in response to T-cell-independent antigen is of potential
significance, particularly because all of the mice had been housed in a
clean barrier-protected environment.
Mice immunized with DNP-KLH were bled at days 7, 14, and 21 and were
rechallenged with DNP-KLH at day 21 and bled at day 35 to determine
their ability to mount a secondary antibody response. As shown in
Figure 7C, PECAM-1 Autoantibody production in PECAM-1 RIIB1 22 and CD22 23 have rendered mice
susceptible to induction of autoimmune disease, we monitored sequential
serum samples from aging wild-type control and PECAM-1/
mice for the presence of ANAs. As shown in Figure
8A, sera from 9-month-old
PECAM-1/ mice showed positive homogenous nuclear
staining of HEp-2 epithelial cells, while sera from age-matched
wild-type control mice showed no reactivity. Fifty percent of
PECAM-1/ mice showed evidence of ANA positivity, with
titers reaching levels of 3200 at 9 months of age. By 17 months of age,
63% of PECAM-1/ mice showed evidence of ANAs with
titers up to 3200 (Figure 8B). Because the homogenous nuclear staining
pattern of HEp-2 cells is consistent with autoantibody specificity
against dsDNA and dsDNA complexed to histone 2A/2B or histone 1, we
next determined IgG titers against dsDNA by ELISA. As shown in Figure
8C, the sera from 9-month-old PECAM-1/ mice that
stained positive for ANAs (Figure 8A) were confirmed to have
specificity of antibody recognition for dsDNA with higher readings than
age- and sex-matched wild-type control sera.
Immune complex-mediated glomerulonephritis has been strongly
associated with the deposition of specific isotypes of autoantibodies and/or cationicity to produce pathogenicity in animal models of autoimmune disease and in human systemic lupus
erythematosus.24,25 Previous studies on the CD22-deficient
mice revealed that these mice developed high-titer anti-dsDNA
antibodies of IgG2a isotype but showed no evidence of immune
complex-mediated glomerulonephritis with age.23 Seminal
studies of Fc
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Abstract
Introduction
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