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Division of Hematology, Hanson Institute, Institute of Medical and Veterinary Science, Adelaide, South Australia, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RI cross-linking in vitro. These
results suggest that PECAM-1 acts as a counterregulator in allergic
disease susceptibility and severity and negatively modulates mast cell
activation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI binding domain of FcRI (recombinant human Ab E25) in
atopic individuals has shown promising results. This mAb functions by
decreasing serum IgE to almost undetectable levels and reduces the
early phase (mast cell-mediated) and late phase (inflammation-mediated)
responses to inhaled allergen (2). An alternative
therapeutic option being explored is targeting the molecular
mechanisms that function as negative regulators of the early phase of
mast cell activation. These include the inhibitory coreceptors that
contain intracytoplasmic immunoreceptor tyrosine-based inhibitory
motifs (ITIMs).3 Mast
cells contain several Ig-ITIM-bearing receptors, including the
low-affinity IgG receptor Fc
RIIB, transmembrane glycoprotein
receptor gp49B1, signal regulatory protein (SIRP)
, mast cell
function-associated Ag, and platelet endothelial cell adhesion
molecule-1 (PECAM-1)/CD31 (3, 4, 5, 6, 7). These inhibitory
coreceptors can inhibit mast cell activation by ITIM-mediated
recruitment and activation of distinct Src homology 2 domain-containing
protein-tyrosine phosphatase (SHP)-1 and/or SHP-2 or Src homology 2
domain-containing inositol polyphosphate 5'-phosphatase (SHIP)1 and/or
SHIP2 (5). Adoption of the active configuration of
phosphatases exposes their catalytic active site necessary for
dephosphorylation of the 
- and -chains of FcRI and other
substrates to block the activation signals generated via cross-linking
the FcRIs when allergen is bound to cell surface IgE. During an
allergic response, stimulation of the FcRI pathway induces release
of vasoactive substances from dense granules, including histamine and
serotonin, and synthesis and release of cytokines. A major
problem underlying FcRI-mediated signaling in mast cells is
to define the mechanisms that control direct feedback counterregulatory
pathways that dampen the early phase of mast cell activation pathway
following FcRI engagement.
Generation of mouse models in which mast cell coreceptors have been
deleted by gene targeting strategies has provided new insights into the
regulation of FcRI-mediated mast cell activation in vivo and in
vitro. Mice deficient in mast cell coreceptors, FcRIIB, and gp49B1
exhibit increased vascular permeability and systemic and local
IgE-dependent anaphylactic responses (8, 9).
FcRIIB-deficient mice also display increased vascular
permeability in passive cutaneous IgG-dependent anaphylaxis
(10). This increase in severity of anaphylactic responses
in FcRIIB and gp49B1 mice reflects the fact that these mast cell
coreceptors are essential to provide a direct negative feedback
regulation of FcRI signaling. FcRIIB has a single ITIM and
selectively uses SHIP upon coaggregation with immune complexes to
delivery inhibitory signaling events (11). In contrast,
gp49B1 has two ITIMs and preferentially recruits SHP-1 following
Ab-mediated coligation of gp49B1 to FcRI, inhibiting IgE-induced
mast cell activation in vitro (12).
PECAM-1 is a newly assigned member of the Ig superfamily with six extracellular Ig domains and two cytosolic ITIMs (13, 14, 15, 16). PECAM-1 is highly expressed at the lateral junctions of endothelial cells and at a lower density on the surfaces of neutrophils, monocytes, platelets, NK cells, T and B cell subsets, and mast cells. PECAM-1 has been demonstrated to be involved in neutrophil recruitment in vivo, neutrophil and monocyte chemotaxis, and transendothelial migration of monocytes and neutrophils in vitro (17, 18, 19). Anti-domain 1 PECAM-1 mAbs and soluble PECAM-IgG chimeric molecules have been shown to inhibit transmigration of leukocytes through endothelium and in vivo neutrophil recruitment (17, 18, 20, 21, 22).
Studies in rat mucosal mast cells (RBL-2H3 cell line) have shown that
PECAM-1 becomes tyrosine phosphorylated and preferentially
recruits SHP-2 following FcRI engagement (7, 23).
However, no functional data are available on the ability of PECAM-1 to
modulate mast cell activation either in vitro or in vivo. SHP-2 has
been implicated in both positive and negative signaling pathways,
depending upon the cell type and signaling complexes formed
(24). Analysis of chimeric receptors expressing the
extracellular portion of FcRIIB and intracellular portion of SIRP
(a known SHP-2-binding receptor) in RBL-2H3 cell line has revealed that
the cytoplasmic domain of SIRP is sufficient to inhibit IgE-induced
mediator secretion and cytokine synthesis in mast cells in vitro
(5). Additional studies will be required to precisely
define the functional role of PECAM-1 and SHP-2 in mast cell
function.
Because no naturally occurring deficiency of PECAM-1 has been reported in humans, the availability of PECAM-1-deficient mice provides us with the opportunity to directly test the functional importance of PECAM-1 in local and systemic IgE-dependent anaphylaxis in vivo and mast cell secretory responses of primary bone marrow mast cells (BMMC) in vitro. PECAM-1-/- mice are born at the expected mendelian frequency and remain viable and healthy throughout life (25). To test the hypothesis that PECAM-1 acts as a counterregulator in allergic disease susceptibility and severity, we examined the local cutaneous and passive systemic IgE-dependent anaphylaxis responses of PECAM-1-deficient mice compared with wild-type mice. To complement these studies, we tested whether primary cultured BMMC from wild-type and PECAM-1-/- mice showed any functional differences in mast cell mediator secretion responses in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The construction of the PECAM-1-deficient mice has been previously described in detail (25). These PECAM-1-deficient mice have been further backcrossed six generations onto C57BL/6 background. All mice were housed under pathogen-free conditions in a specific pathogen-free barrier-protected animal house facility at Adelaide University (Adelaide, South Australia). The phenotype of wild-type and PECAM-1-deficient mice was confirmed by flow cytometry analysis of peripheral blood elements using a monoclonal rat anti-mouse PECAM-1 390 Ab directed against Ig domain 2 (a gift from Dr. S. Albelda, University of Pennsylvania Medical Center, Philadelphia, PA) (26).
Derivation of primary BMMC lines
Bone marrow cells were derived from 4- to 8-wk-old femurs of PECAM-1+/+ and PECAM-1-/- mice and were cultured in IL-3-containing BMMC complete medium containing DMEM, 10% FCS, 50% WEHI-3DB- conditioned medium, 2 mM L-glutamine, 100 µM nonessential amino acids, 1 mM sodium pyruvate, and 10-5 M 2-ME as described (12). After 4–5 wk, >97% of suspension cells in the cultures were confirmed as immature mast cells by toluidine blue staining and flow cytometry analysis with specific mast cell markers, including IgE receptor and c-Kit staining.
Isolation of peritoneal mast cells
Peritoneal mast cells were isolated from 10 wild-type mice by
washing with Tyrode’s buffer containing gelatin and purification
through sequential 22% (w/v) metrizamide gradients (Sigma-Aldrich, St.
Louis, MO) (27). Using this procedure, purified peritoneal
mast cells were obtained at >95% purity as assessed by FcRI
expression.
Flow cytometric analysis
Single cell suspensions (1 x 106 cells/ml) of BMMC were single stained with Abs directed against a range of Ags including IgE receptor (anti-DNP mouse IgE, clone SPE-7; Sigma-Aldrich), c-Kit (Ack2; a gift from L. Ashman, Hanson Institute, Adelaide, South Australia, Australia) (28) and PECAM-1 (390). Abs were biotinylated and detected with streptavidin-R-PE (Southern Biotechnology Associates, Birmingham, AL) or detected with labeled secondary Abs, anti-rat R-PE (Southern Biotechnology Associates). Data were collected on an Epics XL-MCL (Beckman Coulter, Fullerton, CA) and analyzed using WinMDI version 2.8 software (copyright 1993–1998, J. Trotter; http://facs.scripps.edu/software.html).
Preparation of monomeric IgE
Commercial preparations of anti-DNP mouse IgE mAb (clone SPE-7; Sigma-Aldrich) were subjected to ultracentrifugation and the supernatants were further purified by Sepharose gel filtration column chromatography before use. Freshly prepared monomeric IgE was used in all our studies.
Skin mast cell determination
Sections of skin were removed from the ears and back of PECAM-1+/+ and PECAM-1-/- mice. These tissue samples were fixed in 10% formalin and embedded in paraffin. Five-micrometer sections were cut and stained with Giemsa stain. The number of tissue mast cells was counted in both skin sections derived from the ears and back of PECAM-1+/+ and PECAM-1-/- mice at x40 magnification across six high-power fields on an Olympus BH-2 microscope (Olympus, Phillips, Australia).
Active systemic anaphylaxis
PECAM-1+/+ and PECAM-1-/- mice were i.v. injected with 3 µg anti-DNP mouse IgE (Sigma-Aldrich). Twenty-four hours later, mice were challenged with i.v. administration of 500 µg of DNP-BSA (Sigma-Aldrich) or PBS. After 1.5 min, mice were sacrificed by CO2 inhalation and blood was immediately collected by cardiac puncture. Serum was isolated from blood samples and tested for serum histamine concentration by competitive histamine ELISA (Immunotech, Marseille, France).
Local cutaneous anaphylaxis
The left and right ears of 5-wk-old PECAM-1+/+ and PECAM-1-/- mice were intradermally injected with either 20 µl of PBS or 10 ng anti-DNP mouse IgE (clone SPE-7; Sigma-Aldrich). Twenty hours later, the mice were challenged with an i.v. injection of 500 µg DNP-BSA (Sigma-Aldrich) or PBS. Ear thickness measurements were recorded at 0.5, 1, 2, 4, and 8 h using a dial thickness gauge (Mitutoyo, Tokyo, Japan) with a minimum sensitivity of 1 µm. Net ear thickness was determined as the difference between the thicknesses of the right and left ears of each mouse.
Histology
In some local cutaneous anaphylaxis experiments, PECAM-1+/+ and PECAM-1-/- mice were sacrificed 30 min after administration with DNP-BSA. Left and right ears were removed, fixed in 10% formalin, and embedded in paraffin. Five-micrometer sections were cut and then stained with Giemsa stain. Intact and degranulated tissue mast cells were counted by examining at x40 magnification using an Olympus BH-2 microscope. A degranulated mast cell was defined as a cell showing release of >10% cell granules.
Electron microscopy
For conventional electron microscopy, BMMCs were fixed with a mixture of 2% paraformaldehyde and 1% glutaraldehyde in 0.2 M cacodylate buffer (pH 7.4), postfixed with 1% OsO4 supplemented with 15% ferrocyanure, dehydrated in ethanol, and embedded in Epon. Ultrathin sections were viewed with a TEM CM120 Philips electron microscope (Philips Electronic Systems, Mahwah, NJ) after counterstaining with uranyl acetate and lead citrate.
Degranulation
The degree of dense granule secretion was determined by measuring the release of serotonin. PECAM-1+/+ and PECAM-1-/- BMMC (1 x 107 cells/ml) were preloaded with 1 µCi 5-hydroxy [3H]tryptamine trifluoroacetate (without IL-3; Amersham Pharmacia Biotech, Little Chalfont, U.K.) overnight in a CO2 incubator at 37°C. Cells were washed once in Tyrode’s buffer (112 mM NaCl, 2.7 mM KCl, 0.4 mM NaH2PO4, 1.6 mM CaCl2, 1 mM MgCl2, 10 mM HEPES (pH 7.5), 0.05% (w/v) gelatin, 0.1% (w/v) glucose) and resuspended in 200 µl of Tyrode’s buffer. For stimulating with IgE alone, 10 µg/ml anti-DNP mouse IgE (without IL-3; Sigma-Aldrich) was incubated with cells at 37°C for 15 min. For stimulation with IgE plus polyvalent Ag, DNP-BSA, cells were incubated with 10 µg/ml anti-DNP mouse IgE for 2 h at 37°C. Washed cells were stimulated with varying doses of DNP-BSA (0–100 ng/ml) and incubated for 15 min at 37°C. As a positive control, cells were incubated with 10 µM ionomycin/20 ng/ml PMA for 15 min at 37°C as a check on the integrity of the cell signaling machinery. Samples were centrifuged at 4000 rpm for 5 min, supernatants were separated, and cell pellets were solubilized in 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO). The release of serotonin was measured in triplicate by liquid scintillation in both cell supernatants and pellets and calculated as a percentage of degranulation. The percentage of release values were calculated using the formula [(S/S + P)] x 100, where S and P are the serotonin contents of the supernatants and pellet, respectively, from each sample.
Statistical analysis
Numerical values were expressed as the mean ± SD. Experimental results were evaluated by two-way ANOVA to allow comparison of data obtained from PECAM-1+/+ and PECAM-1-/- mice at different time points. In some cases, the Student t test was used to analyze experimental data at the same time point in the time course.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The generation and preliminary characterization of
PECAM-1-/- mice have been previously reported
(25). Functional assessment of in vitro platelet-collagen
responses has revealed that the PECAM-1-/- mice
have phenotypic features of an inhibitory receptor knockout
(29). In this study, we sought to test the possibility
that PECAM-1 may serve an essential role as a counterregulator of
allergic disease susceptibility and severity. In initial studies, we
demonstrated by flow cytometry that PECAM-1 is highly expressed on the
surface of immature BMMC derived from bone marrow of
PECAM-1+/+ mice, but not in
PECAM-1-/- BMMC (Fig. 1
). We also showed that PECAM-1 was
expressed at a lower density on the surface of mature peritoneal mast
cells compared with immature BMMC (Fig. 2).
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, there was no significant difference (Student’s t test;
p > 0.05; n = 6) in the number of
tissue skin mast cells derived from the ears and back of
PECAM-1+/+ and PECAM-1-/-
mice. Collectively, these data suggest that PECAM-1 is expressed in
mast cells and is not required for mast cell development and populating
tissues.
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Passive systemic IgE-dependent anaphylaxis is dependent upon
passive transfer of anti-DNP mouse IgE Ab followed by i.v.
administration of the cross-linking polyvalent Ag, DNP-BSA. Mice
deficient in FcRI, Src homology 2 domain-containing leukocyte
phosphoprotein-76, linker for activation of T cells, Gab2, and
mast cell-deficient WBB6F1-W/Wv all lead to
diminished or virtually absent systemic IgE-dependent anaphylactic
responses (30, 31, 32, 33, 34, 35), while mice deficient in FcRIIB and
gp49B1 show enhanced systemic IgE-dependent anaphylactic responses
(8, 9). These studies in gene-targeted mice reveal the
importance of components of the FcRI pathway together with the
presence of mast cells and inhibitory receptors in modulating
susceptibility to IgE-dependent anaphylaxis.
To test the in vivo function of PECAM-1 in passive systemic
IgE-dependent anaphylaxis, we challenged
PECAM-1+/+ and
PECAM-1-/- mice with an i.v.
administration of 3 µg monoclonal anti-DNP mouse IgE, then (24 h
later) with 500 µg DNP-BSA or PBS. After 1.5 min mice were
sacrificed, blood was collected, and serum was isolated for
determination of histamine concentration. As shown in Fig. 4, IgE stimulation alone did not induce
an increase in serum histamine concentration in
PECAM-1-/- mice compared with
PECAM-1+/+ mice. In contrast,
PECAM-1+/+ mice showed an increase in serum
histamine concentration upon FcRI cross-linking, while
PECAM-1-/- mice showed a significant elevation
in serum histamine concentration (6,565 ± 2,363 nM vs 12,985
± 3,569 nM; Student’s t test; p < 0.001;
n = 13). These results are consistent with our
hypothesis that the absence of PECAM-1 as an inhibitory receptor leads
to more severe passive systemic IgE-dependent anaphylaxis.
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To determine whether PECAM-1 serves as a negative regulator of
FcRI mast cell activation in local cutaneous anaphylaxis, the left
and right ears of PECAM-1+/+ and
PECAM-1-/- mice were injected intradermally
with 20 µl PBS or 10 ng anti-DNP mouse IgE. Twenty hours later,
mice were challenged with 500 µg DNP-BSA or PBS by i.v. tail vein
injection. Left and right ear tissue swelling was monitored at various
time points from t = 0.5, 1, 2, 4, and 8 h with a
dial thickness gauge. As shown in Fig. 5, the net ear thickness for PECAM-1-/- mice was
significantly increased compared with PECAM-1+/+
mice following FcRI mast cell activation induced by IgE anti-DNP
and cross-linking with DNP-BSA (Student’s t test;
p < 0.001; n = 6). The net ear
swelling peaked at 1 h and slowly subsided by 2 h. Following
induction of Ag-dependent IgE-mediated local anaphylaxis, a 10-fold
increase in net ear thickness was observed for
PECAM-1-/- mice (Fig. 5B) when
compared with Ag-independent IgE/PBS control at peak swelling of 1
h (Fig. 5A). In contrast, PECAM-1+/+
mice showed a 4-fold increase in net ear thickness compared with
Ag-independent IgE/PBS control at peak swelling of 1 h.
Importantly, we observed that the net ear thickness was higher in
PECAM-1-/- mice at all times compared with
PECAM-1+/+ mice. To examine the numbers of
intact vs degranulated tissue mast cells in
PECAM-1+/+ and
PECAM-1-/- mice during local cutaneous
IgE-dependent anaphylaxis, the left and right ears of
PECAM-1+/+ and PECAM-1-/-
mice were injected intradermally with 20 µl PBS or 10 ng
anti-DNP mouse IgE. Twenty hours later, mice were challenged with
500 µg DNP-BSA by i.v. tail vein injection. In these experiments,
mice were sacrificed at t = 0.5 h, ears were
removed, and 5-µm Giemsa-stained sections were examined and
quantitated for intact tissue mast cell content by light
microscopy. The right ears of PECAM-1-/-
mice showed 4-fold fewer intact tissue mast cells 0.5 h
after challenge compared with PECAM-1+/+ mice, as
demonstrated in histological sections (Fig. 6A) and by quantitation of
intact tissue mast cells (Student’s t test;
p < 0.005; n = 5) (Fig. 6B). Importantly, tissue mast cells in skin tissue sections
of PECAM-1-/- mice displayed quantitatively
more degranulation than comparative samples from wild-type mice
(Student’s t test; p < 0.005;
n = 5) (Fig. 6C). These results suggest
that PECAM-1 serves as a negative regulator in dampening local
cutaneous anaphylaxis, which is a hallmark feature of bee stings,
insect bites, and latex hypersensitivity.
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RI
cross-linking in vitro
To investigate the underlying mechanism responsible for the
increased severity of PECAM-1-/- mice to local
and systemic IgE-mediated anaphylaxis due to defective
FcRI-mediated degranulation of mast cells, we next examined the
capacity of wild-type and PECAM-1-/- BMMC to
degranulate by examining their response to anti-DNP IgE
stimulation alone and anti-DNP IgE stimulation with varying doses
of DNP-BSA and PMA/ionomycin. After 4–5 wk in culture, the various
BMMC lines expressed >95% FcRI expression (Fig. 1
) and showed
comparable staining of metachromatic granules with toluidine blue or
alcian blue-safranin (data not shown). Electron microscopy studies of
the various BMMC lines revealed similar cell morphology, membrane
projections, presence of cytoplasmic morphologically distinct granules
including internal vesicles (type I), granules with an electron-dense
core surrounded by membrane vesicles (type II), and electron-dense
granules (type III) (Fig. 7A)
(36). Examination of the ultrastructural components of
various BMMC phenotypes by transmission electron microscopy enabled
quantitation of type III electron-dense granule content in primary
murine BMMC derived from PECAM-1+/+ and
PECAM-1-/- mice. Quantitation of dense granules
from each of the BMMC lines revealed comparable numbers of type III
electron-dense granules per BMMC (Student’s t test;
p > 0.05; n = 15) (Fig. 7B). As the dense granule content was comparable between the
primary murine BMMC lines, we then wanted to correlate the in vivo
studies of local and systemic anaphylaxis with mast cell degranulation
in vitro. As shown in Fig. 7C, FcRI-evoked degranulation
(as indicated by serotonin release) was markedly enhanced in
PECAM-1-/- BMMC. The
PECAM-1+/+ BMMC consistently released
42% of their total granule contents, whereas the
PECAM-1-/- BMMC released 65% of their total
granule contents following stimulation with anti-DNP IgE and
FcRI cross-linking with DNP-BSA. Dose-dependent curves revealed a
peak of degranulation at concentrations of 10 ng/ml DNP-BSA for all
cell lines tested. In contrast, PMA plus ionomycin-induced
degranulation was comparable in wild-type and
PECAM-1-/- BMMC. Therefore, our results suggest
that the secretion machinery in PECAM-1-/- BMMC
is intact but the absence of PECAM-1 leads to defective hyperresponsive
FcRI-mediated degranulation. Our observation that FcRI
cross-linking induces enhanced degranulation in
PECAM-1-/- BMMC suggests that the presence of
PECAM-1 in mast cells plays an important role in preventing
inappropriate mast cell degranulation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
PECAM-1-/- mice displayed a hyperresponsive
phenotype in Ag-dependent IgE-mediated passive systemic anaphylaxis
(Fig. 4) and local cutaneous anaphylaxis (Figs. 5 and 6), which is
suggestive of an inhibitory receptor knockout phenotype.
Systemic anaphylaxis is a health condition whereby the individual
succumbs to a severe allergic reaction to a specific allergen. Because
it is regarded as a reflection of extensive mast cell activation
(32), we decided to assess the severity of this allergic
condition in wild-type and PECAM-1-/- mice. We
chose to evaluate a passive rather than active method of sensitization
to exclude the participation of other Ab classes, which may complicate
the comparison and interpretation of the anaphylactic responses.
Sensitization of mice for active anaphylaxis induces not only an IgE
response but also production of Ag-specific IgG1 Abs (37),
which can participate (with IgE Abs) in both local cutaneous
anaphylaxis (8) and passive systemic anaphylactic
responses (38). Passive sensitization ensures sole
activation of FcRI-containing mast cells and basophils that have
been primed with anti-DNP IgE and receptor cross-linked with
DNP-BSA. As previously mentioned, there was a marked enhancement of
systemic anaphylaxis in PECAM-1-/-
mice when compared with their wild-type counterparts, which was
quantitatively assessed by a sensitive serum histamine assay. This
hyperresponsive phenotype is consistent with the in vivo findings
obtained from FcRIIB-/- mice
(8). In gp49B1-/- mice, systemic
anaphylaxis was measured by enhanced mast cell degranulation in the
trachea and also increased mortality (68 vs 27%) in a shorter time
frame (26 vs 45 min) when compared with their wild-type counterparts
(9). In contrast, mice that were deficient in FcRI,
linker for activation of T cells, Src homology 2
domain-containing leukocyte phosphoprotein-76, Vav, and Gab2 have shown
decreased serum histamine concentrations when compared with normal
serum histamine levels (32, 33, 34, 35). Under these
circumstances, the results are indicative of mast cell insufficiency to
degranulate, thus attenuating effector function. Thus, these findings
indicate that the activatory components of the FcRI signaling
pathway are crucial in IgE-mediated allergic responses. Conversely, as
shown by PECAM-1-/-,
FcRIIB-/-, and
gp49B1-/- mice, abrogation of the inhibitory
receptor leads to enhanced systemic anaphylaxis where there is
extensive mast cell degranulation and death.
The medical condition of local IgE-dependent reactions is highly prevalent in the developed world. It is characterized by typical local allergic responses to insect stings, latex-induced hypersensitivity, and other allergenic substances, such as pollen. A local allergic response is commonly reflected by localized swelling at particular sites of tissue within the body. Taking this important indicator of local anaphylaxis into account, we devised experiments in which to assess the effect of PECAM-1 in regulation of local cutaneous anaphylaxis. Upon induction of local cutaneous anaphylaxis, we found that PECAM-1-/- mice exhibited a 2- to 3-fold greater ear swelling at 1 h following Ag-dependent challenge with IgE and DNP-BSA compared with their wild-type counterparts. In both genotypes of mice, a peak net ear swelling was observed after 1 h of Ag challenge which eventually subsided and returned to basal levels by 8 h. The observations in the PECAM-1-/- mice share features comparable with gp49B1-/- mice, which displayed a 2- to 3-fold greater net swelling at 0.5–2 h after Ag challenge when compared with their wild-type counterparts (9). To investigate whether there was a correlation between these observational findings and mast cell degranulation, ear tissue from wild-type and PECAM-1-/- mice were removed 30 min after the induction of IgE-mediated local cutaneous anaphylaxis and were examined histologically. As defined by tissue histology, the number of morphologically intact mast cells was significantly reduced 3-fold in the Ag-challenged PECAM-1-/- mice at 0.5 h. This finding was also comparable to gp49B1-/- mice, which displayed a 2-fold decrease in intact mast cells at both 0.5- and 4-h time points (9). To further substantiate these suggestions of extensive mast cell degranulation, intact and degranulated mast cells within the skin of Ag-challenged PECAM-1-/- mice were quantitated and were demonstrated to contain a higher percentage of degranulated mast cells when compared with wild-type mice. Hence, these findings are indicative of an enhanced susceptibility to IgE-mediated mast cell degranulation in inhibitory receptor knockout animals.
Previous studies in rat mucosal mast cells, RBL-2H3 cells, have
revealed that PECAM-1 becomes tyrosine phosphorylated upon FcRI
aggregation with anti-FcRI receptor Abs and recruits the
protein-tyrosine phosphatase, SHP-2 (7, 23). At this time,
no studies were reported that examined the functional importance of
PECAM-1 in regulating FcRI-evoked mast cell responses.
Functional studies were also impeded by the lack of naturally occurring
PECAM-1-deficient human individuals, thus prompting us to use
genetically altered PECAM-1-deficient mice to directly assess the
functional importance of PECAM-1 in mast cell activation in vivo and in
vitro. Importantly, because the FcRI is intrinsic to multiple cell
types, including mast cells and basophils, it was imperative to conduct
in vitro studies on IL-3-dependent primary BMMC populations derived
from wild-type and PECAM-1-/- mice to determine
whether PECAM-1 plays an essential role in regulating FcRI-mediated
signaling pathways. Our in vitro studies revealed that PECAM-1 is
highly expressed on the surface of immature murine bone marrow-derived
mast cell progenitors but at a lower density on the surface of mature
peritoneal mast cells (Figs. 1 and 2).
PECAM-1-/- primary BMMC exhibited normal
morphology, equivalent type III dense granule content, and similar
kinetics of growth (data not shown) compared with wild-type BMMC (Fig. 7). PECAM-1-/- primary BMMC showed
hyperresponsive features of Ag-dependent IgE-mediated mast cell
activation, as indicated by elevated FcRI-evoked secretion of dense
granule serotonin following IgE stimulation and DNP-BSA cross-linking
compared with wild-type BMMC (Fig. 7).
This Ag-dependent FcRI-mediated hyperresponsive mast cell phenotype
observed in PECAM-1-/- BMMC was not observed
in other inhibitory receptor knockouts, including
FcRIIB-/- BMMCs and
gp49B1-/- BMMC (10, 39). Loss of
FcRIIB rendered the cells sensitive to IgG-mediated triggering but
did not have a substantial effect on FcRI-mediated activation
(10). In contrast, loss of gp49B1 in BMMC did not result
in any difference in hypersensitivity in Ag-dependent IgE-mediated mast
cell activation (40). These results appear to conflict
with the in vivo enhanced systemic and local cutaneous anaphylaxis
observed in the gp49B1-/- mice
(9). The reason for this discrepancy between the in vivo
and in vitro data is not clear and further studies are required to
resolve this issue.
In summary, immune inhibitory mechanisms are equally as important as
immune system activation. Classical symptoms of cellular
hyperresponsiveness, including autoimmunity and anaphylaxis, are
evident in mice that possess targeted disruptions of their inhibitory
receptors. In this study, we have defined PECAM-1 as a new mast cell
coreceptor that provides a direct feedback mechanism to negatively
attenuate Ag-dependent IgE/FcRI-mediated signal transduction. By
studying the mechanism by which we can manipulate IgE-FcR interactions,
we can identify potential novel and more-effective avenues of
prophylactic treatment for allergy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Denise E. Jackson, Division of Hematology, Hanson Institute, Institute of Medical and Veterinary Science, Frome Road, Adelaide, South Australia 5000, Australia. E-mail address: Denise.Jackson{at}imvs.sa.gov.au
3 Abbreviations used in this paper: ITIM, immunoreceptor tyrosine-based inhibitory motif; PECAM-1, platelet endothelial cell adhesion molecule-1; BMMC, bone marrow mast cell; SIRP, signal regulatory protein; SHP, Src homology 2 domain-containing protein-tyrosine phosphatase; SHIP, Src homology 2 domain-containing inositol polyphosphate 5'-phosphatase.
Received for publication February 25, 2002. Accepted for publication April 17, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain gene. Cell 75:969.[Medline]
RI-mediated mast cell activation. Immunity 12:525.[Medline]
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 R. Goel, B. Boylan, L. Gruman, P. J. Newman, P. E. North, and D. K. Newman The proinflammatory phenotype of PECAM-1-deficient mice results in atherogenic diet-induced steatohepatitis Am J Physiol Gastrointest Liver Physiol, December 1, 2007; 293(6): G1205 - G1214. [Abstract] [Full Text] [PDF]
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 A. Woodfin, M.-B. Voisin, and S. Nourshargh PECAM-1: A Multi-Functional Molecule in Inflammation and Vascular Biology Arterioscler. Thromb. Vasc. Biol., December 1, 2007; 27(12): 2514 - 2523. [Abstract] [Full Text] [PDF]
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 T. S. Dhanjal, C. Pendaries, E. A. Ross, M. K. Larson, M. B. Protty, C. D. Buckley, and S. P. Watson A novel role for PECAM-1 in megakaryocytokinesis and recovery of platelet counts in thrombocytopenic mice Blood, May 15, 2007; 109(10): 4237 - 4244. [Abstract] [Full Text] [PDF]
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 C. M. Udell, L. A. Samayawardhena, Y. Kawakami, T. Kawakami, and A. W. B. Craig Fer and Fps/Fes Participate in a Lyn-dependent Pathway from Fc{epsilon}RI to Platelet-Endothelial Cell Adhesion Molecule 1 to Limit Mast Cell Activation J. Biol. Chem., July 28, 2006; 281(30): 20949 - 20957. [Abstract] [Full Text] [PDF]
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 S. M. Albelda, K. C. Lau, P. Chien, Z.-Y. Huang, E. Arguiris, A. Bohen, J. Sun, J. A. Billet, M. Christofidou-Solomidou, Z. K. Indik, et al. Role for Platelet-Endothelial Cell Adhesion Molecule-1 in Macrophage Fc{gamma} Receptor Function Am. J. Respir. Cell Mol. Biol., August 1, 2004; 31(2): 246 - 255. [Abstract] [Full Text] [PDF]
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V. Rathore, M. A. Stapleton, C. A. Hillery, R. R. Montgomery, T. C. Nichols, E. P. Merricks, D. K. Newman, and P. J. Newman PECAM-1 negatively regulates GPIb/V/IX signaling in murine platelets Blood, November 15, 2003; 102(10): 3658 - 3664. [Abstract] [Full Text] [PDF]
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P. J. Newman and D. K. Newman Signal Transduction Pathways Mediated by PECAM-1: New Roles for an Old Molecule in Platelet and Vascular Cell Biology Arterioscler. Thromb. Vasc. Biol., June 1, 2003; 23(6): 953 - 964. [Abstract] [Full Text] [PDF]
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; n = 13) and
PECAM-1-/- (
; n = 13) mice were
sensitized with 3 µg anti-DNP mouse IgE by i.v. injection.
Twenty-four hours later, mice were challenged with 500 µg DNP-BSA or
PBS; after 1.5 min, mice were sacrificed and blood was collected for
determination of serum histamine concentration. Results represent serum
histamine concentrations of PECAM-1+/+ and
PECAM-1-/- mice with and without Ag stimulation. The mean
values of each group are indicated.
