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2 Is Essential for Specific Functions of Fc
R and FcR1
* The Blood Research Institute, The Blood Center of Southeastern Wisconsin, Milwaukee, WI 53226;
Department of Pediatrics, National Taiwan University Hospital, Taipei, Taiwan;
Institute of Molecular Medicine, Nanjing University, Nanjing, Peoples Republic of China;
Department of Pharmacology, Byk Gulden, Konstanz, Germany; and
¶ Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI 53226
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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2 (PLC2) plays a critical role in the
functions of the B cell receptor in B cells and of the FcR
chain-containing collagen receptor in platelets. Here we report that
PLC2 is also expressed in mast cells and monocytes/macrophages and
is activated by cross-linking of Fc
R and FcR. Although
PLC2-deficient mice have normal development and numbers of mast
cells and monocytes/macrophages, we demonstrate that PLC2 is
essential for specific functions of FcR and FcR. While
PLC2-deficient mast cells have normal mitogen-activated protein
kinase activation and cytokine production at mRNA levels, the mutant
cells have impaired FcR-mediated Ca2+ flux and inositol
1,4,5-trisphosphate production, degranulation, and cytokine secretion.
As a physiological consequence of the effect of PLC2 deficiency, the
mutant mice are resistant to IgE-mediated cutaneous inflammatory skin
reaction. Macrophages from PLC2-deficient mice have no detectable
FcR-mediated Ca2+ flux; however, the mutant cells have
normal FcR-mediated phagocytosis. Moreover, PLC2 plays a
nonredundant role in FcR-mediated inflammatory skin
reaction. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R, which binds IgG; Fc
R, which binds IgA; and
FcR, which binds IgE. High affinity FcRs are referred to as FcRI,
and low affinity FcRs are referred to as FcRII/III. High affinity FcRs
can bind noncomplexed, monomeric Igs, while low affinity FcR bind
aggregated Igs or Abs complexed to multivalent Ags
(1, 2, 3).
FcRs are widely distributed in hemopoietic cells and mediate a wide
array of biological functions (1, 2). FcRI is expressed
primarily by mast cells and basophils (3). Cross-linking
of the FcRI on mast cells induces the release of granules containing
histamine, serotonin, 
-hexosaminidase, and mast cell-specific
proteases, accompanied by transcription and secretion of multiple
cytokines, including IL-1 through -6, IL-9, IL-10, IL-13, IL-16,
TNF-, TGF-, and GM-CSF, ultimately leading to an allergic
reaction (4, 5). FcRs, including FcRI, FcRII, and
FcRIII, are widely expressed in most hemopoietic cells, including
monocytes/macrophages, neutrophils, NK cells, and platelets
(6, 7, 8). These receptors also mediate a wide array of
biological functions, including respiratory burst, release of
inflammatory mediators and cytokines, endocytosis, Ab-dependent cell
cytotoxicity (ADCC), and anaphylaxis (6, 7, 9, 10).
FcRs with immunoreceptor tyrosine-based activation motifs are capable
of triggering cell activation. Ab-induced FcR aggregation activates
several cytoplasmic protein tyrosine kinases, including Src family
kinases, Syk family kinases, and Tec family kinases
(11, 12, 13, 14, 15, 16, 17, 18). In turn, the activated tyrosine kinases rapidly
phosphorylate many intracellular substrates, leading to activation of
multiple signaling pathways, which include phospholipase C (PLC).
PLC hydrolyzes phosphatidylinositols to generate diacylglycerol and
inositol phosphates, including inositol 1,4,5-trisphosphate
(IP3) (19). The diacylglycerol
activates protein kinase C (PKC), while IP3
mediates the mobilization of Ca2+ from internal
stores, resulting in a transient intracellular
Ca2+ ([Ca2+]i) flux
(19). PLC is activated by FcR in mast cells
(20), by FcRIIIA in NK cells (21, 22, 23), and
by FcRI or FcRIIA in neutrophils (24) and monocytes
(18, 25). The PLC/Ca2+/PKC
pathway has been shown to be involved in the activation of all types of
mitogen-activated protein kinases (MAPK; ERKs, c-Jun N-terminal kinases
(JNKs), and p38 MAPKs) (26, 27, 28, 29, 30), although the
PKC-independent Grb2/Sos/Raf1 pathway plays a primary role in the
activation of MAPKs (27, 31, 32, 33, 34). Activated PKC can
promote the activation of ERK1 and ERK2 (26, 27, 28). Calcium
and PKC also participate in JNK activation (29, 30). In
addition, PKC is required for the maximum activation of p38 MAPK
(29, 30, 35). The PLC/Ca2+/PKC
pathway leads to the activation of transcription factors, including
NFAT and Elk-1 (36, 37, 38). Ultimately, activation of
the FcR induces phagocytosis, ADCC, respiratory burst, degranulation,
production of inflammatory cytokines, and enhanced Ag presentation
(1).
Two isoforms of PLC, PLC1 and PLC2, have been identified. The
PLC1 isoform is ubiquitously expressed, whereas PLC2 is
predominantly expressed in hemopoietic cells (39). The
potential physiological role of PLC1 has been approached by the
derivation of mice that lack the enzyme. Disruption of the PLC1 gene
resulted in embryonic lethality during early to midgestation on
approximately embryonic day 9 (40). This early embryonic
lethality precludes in vivo analysis of the role of PLC1 in Ig
superfamily receptor signaling. However, studies of PLC1-deficient
fibroblasts have shown that epidermal growth factor failed to mobilize
intracellular Ca2+ in the mutant cells, while
epidermal growth factor -induced DNA synthesis, cell growth, and cell
migration were normal (40). Unlike PLC1, disruption of
the PLC2 gene does not lead to embryonic lethality.
PLC2-deficient B cells have impaired BCR-mediated signaling,
resulting in xid-like immunodeficiency with defective B cell
development and function (41). PLC2 is also an
essential component of FcR-chain-containing collagen receptor
signaling pathways, wherein PLC2 deficiency causes platelet
dysfunction and fetal hemorrhage (41). The first
characterization of PLC2-deficient mice has shown that PLC2
deficiency correlates with defective FcR-mediated ADCC activity in
NK cells and decreased FcR-induced degranulation in mast cells
(41). Here we reveal that both PLC1 and PLC2 are
involved in FcR signaling in mast cells, and that PLC2 is
essential for specific FcR-mediated functions. PLC2 deficiency
results in partially impaired FcR-induced Ca2+
flux and IP3 production; decreased
FcR-mediated release of serotonin, arachidonic acid, and histamine;
and resistance to IgE-mediated inflammatory skin reaction. In addition,
we demonstrate that PLC2 plays a nonredundant role in FcR-induced
Ca2+ flux in monocytes/macrophages and in
FcR-mediated inflammatory skin reaction in vivo. Interestingly,
PLC2 deficiency did not affect FcR-mediated phagocytosis in
monocytes/macrophages.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Wild-type and PLC2-deficient mast cells (2.5 x
106/ml) were preloaded with mouse anti-TNP
IgE (4 µg/ml; C38-2; BD PharMingen, San Diego, CA) overnight and
subsequently stimulated with rabbit anti-mouse IgE Ab (10 µg/ml;
R35-72; BD PharMingen). After stimulation, cells were lysed on ice and
centrifuged to remove debris as previously described (42).
The supernatant was incubated with Abs against PLC1 (Santa Cruz
Biotechnology, Santa Cruz, CA) or PLC2 (Santa Cruz Biotechnology).
Immune complexes were precipitated with protein A-Sepharose, washed
three times in lysis buffer, eluted with sample buffer for SDS-PAGE,
and transferred to nitrocellulose. Membranes were probed with
anti-phosphotyrosine (4G10; Upstate Biotechnology, Lake Placid, NY)
or the designated Abs and visualized with an ECL detection system
(Amersham Pharmacia Biotech, Arlington Heights, IL).
Calcium fluorometry
Bone marrow-derived mast cells (2 x 106/ml) were incubated with IgE anti-DNP mAb (10 mg/ml; SPE-7; Sigma-Aldrich, St. Louis, MO) in DMEM with 10% FBS on ice for 1 h. Then, Indo-1 (10 µg/ml; Molecular Probes, Eugene, OR) was added to the cells, followed by further incubation at room temperature for 30 min. The cells were washed and stimulated with DNP-human serum albumin (HSA; 100 ng/ml; Sigma). The calcium concentration was determined by flow cytometry.
For macrophages/monocytes, leukocytes were purified from mouse
peripheral blood with Polymorphprep (NYCOMED PHARMA, Oslo, Norway)
according to the manufacturer. The cells were loaded with Indo-1 (10
µg/ml) at room temperature for 30 min, followed by incubation with
anti-FcII/III monoclonal IgG (10 µg/ml; 2.4G2; BD PharMingen)
and PE-conjugated anti-Mac-1 Abs (BD PharMingen) on ice for 30 min.
The cells were washed and warmed to room temperature, then stimulated
with rabbit anti-rat IgG (10 µg/ml; Southern Biotechnology
Associates, Birmingham, AL). The calcium concentration was determined
by flow cytometry in Mac-1-positive cells.
[Ca2+]i was determined as previously described (43). The maximum ratio (Rmax) was determined by adding ionomycin (10 µg/ml) in cells, and the minimum ratio (Rmin) was determined following depletion of external Ca2+ by 5 mM EGTA. The [Ca2+]i was calculated according to the equation [Ca2+]i = kDa (R - Rmin)/(Rmax - R)(Sf2/Sb2), where Kd is 230 nM, and Sf2 and Sb2 are the fluorescence intensities at 490 nm of the Ca2+-free and Ca2+-saturated indicators, respectively.
Measurement of IP3
FcR-mediated IP3 induction was measured with the IP3 radioreceptor assay kit (PerkinElmer, Palo Alto, CA) according to manufacturer’s instructions. Briefly, the bone marrow-derived mast cells were suspended at 1 x 106/ml and incubated with mouse IgE mAb (4 µg/ml) for 12 h. The cells were extensively washed in medium, stimulated with anti-mouse IgE mAb (10 µg/ml) for 0, 2, 5, and 10 min, then collected and subjected to IP3 radioreceptor assay.
MAPK assay
To examine the activation of ERK1/2, p38, and JNK1/2,
wild-type and PLC2-deficient mast cells (3 x
106) were preloaded with mouse anti-TNP IgE
overnight and subsequently stimulated with rabbit anti-mouse IgE Ab
(10 µg/ml) for 0, 10, and 30 min. Cells were lysed, and cell lysate
was immunoprecipitated with anti-ERK1/2, anti-p38, and
anti-JNK1/2 (all from Santa Cruz Biotechnology) Abs, and in vitro
kinase assays were employed to measure the activation of three MAPKs.
Autophosphorylation of ERK1 and ERK2 was measured. The myelin basic
protein (MBP) peptide was used as substrate for p38, while the
GST-c-Jun peptide was used as substrate for JNK.
Mast cells and degranulation assay
Bone marrow cells from wild-type or mutant mice were cultured in RPMI 1640 medium containing 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 25 U/ml rIL-3 (R&D Systems, Minneapolis, MN), and 10% heat-inactivated FBS (HyClone, Logan, UT) for 4–8 wk, with medium replacement every 3–4 days.
For the serotonin release assay, bone marrow-derived mast cells were suspended at 1 x 106/ml and incubated with mouse IgE mAb (4 µg/ml) and 5 µCi/ml [3H]serotonin (NEN, Boston, MA) for 16 h. The cells were extensively washed in medium and then stimulated with anti-mouse IgE mAb (10 µg/ml) for 60 min. [3H]Serotonin released into the supernatant and remaining in the cell pellet was quantitated.
For the arachidonic acid release assay, bone marrow-derived mast cells were suspended at 1 x 106/ml and incubated with mouse IgE mAb (4 µg/ml) for 12 h, followed by incubation with 2.5 µCi/ml [3H]arachidonic acid (PerkinElmer) for 2 h. The cells were extensively washed in medium and then stimulated with anti-mouse IgE mAb (10 µg/ml) for 60 min. [3H]Arachidonic acid released into the supernatant and remaining in the cell pellet was quantitated.
For histamine release, 8- to 10-wk-old PLC2-deficient and wild-type
mice were injected i.v. with 2 µg of monoclonal mouse anti-DNP
IgE (Sigma-Aldrich) diluted in 200 µl of DMEM. Twenty-four hours
later the mice were injected i.v. with 500 µg of DNP-HSA
(Sigma-Aldrich) for 2 min. The mice were euthanized, and blood was
immediately collected. Serum histamine concentration was determined
using a competitive histamine immunoassay kit (Immunotech,
Westbrook, ME).
RT-PCR
Mouse anti-TNP IgE-sensitized wild-type and
PLC2-deficient mast cells (5 x 105
cells) were stimulated with anti-mouse IgE mAb (10 µg/ml). Total
RNA was prepared from cells by RNAzol B (Tel-Test, Inc., Friendswood,
TX), and first-strand cDNA was synthesized from total RNA with the
GeneAmp RT-PCR kit (PerkinElmer). The specific primers for each
individual cytokine and actin are as follows: IL-2 forward,
TGGAGCAGCTGTTGATGGACCTAC; IL-2 reverse,
AGATGATGCTTTGACAGAAGGCTATC; IL-3 forward,
GTGGCCGGGATACCCACCGTTTAAC; IL-3 reverse, TGGCAGCGCAGAGTCATTCGCAGAT;
IL-4 forward, GAGATCATCGGCATTTTGAAC; IL-4 reverse,
CTTGGACTCATTCATGGTGCA; IL-6 forward, ATGAAGTTCCTCTCTGCAAGAGAC;
IL-6 reverse, GTAGCATCCATCATTTCTTTGTAT; TGF- forward,
AGACGGAATACAGGGCTTTCGATTC; TGF- reverse,
CTTGGGCTTGCGACCCACGTAGTA; IFN- forward,
CTTCTTCAGCAACAGCAAGGCGAAA; IFN- reverse,
CCCCCAGATACAACCCCGCAATCA; actin forward, ACTCCTATGTGGGTGACGAG; and
actin reverse, CAGGTCCAGACGCAGGATGGC. Actin was used as the RNA
control.
IgE-mediated inflammatory skin reaction
Eight- to 10-week-old wild-type and PLC2-deficient mice were
lightly anesthetized and injected intradermally on the basolateral side
with 20 ng/ml of monoclonal mouse anti-DNP IgE (Sigma-Aldrich)
diluted in 20 µl of DMEM. DMEM alone was used as the negative
control. Twenty-four hours later the mice were injected i.v. with 100
µg of DNP-HSA (Sigma-Aldrich) in 100 µl of PBS with 1% Evans blue
dye (Sigma-Aldrich). Sixty minutes later, extravazation was visualized
by blue staining of the injection sites at the reverse side of skin
sections as an indication for a positive inflammatory skin
reaction.
FcR-mediated inflammatory skin reaction
Wild-type and PLC2-deficient mice were injected intradermally
on the basolateral side with rabbit IgG anti-OVA or saline in 25
µl. Two hours later, 500 µg of OVA with 1% Evans blue in 100 µl
of DMEM was injected i.v. About 30 min later extravazation was
visualized by blue staining of the injection sites at the reverse side
of skin sections as an indication of a positive inflammatory skin
reaction.
Macrophages
Macrophages were obtained through two methods. First, bone
marrow cells from wild-type and PLC2-deficient mice were cultured in
DMEM containing 2 mM glutamine, 100 U/ml of penicillin, 100 µg/ml of
streptomycin, 10% FBS, and 10% L929 cell-conditioned medium as a
source of M-CSF. Nonadherent cells were removed 1–2 days later and
transferred to a fresh 150-mm plate. Nonadherent cells were removed
from this secondary culture 4 days later and discarded. The adherent
cultures were used 5–7 days after initial harvest from the mice, when
confluence was achieved. Cultures generated via this protocol were
almost pure macrophages. Second, macrophages were obtained from
thioglycolate-elicited peritoneum. Wild-type and PLC2-deficient mice
were injected i.p. with 1 ml of 5% thioglycolate, and peritoneal
exudate cells were harvested 3 days later. The cells were suspended in
-MEM supplemented with 10% heat-inactivated FCS at 1 x
106 cell/ml. They were plated in 24-well culture
plates at 1 ml/well and incubated for 6 h at 37°C in 5%
CO2. The adherent macrophages were obtained after
the nonadherent cells were rinsed away with PBS.
FcR-mediated phagocytosis
Thioglycollate-elicited peritoneal wild-type and
PLC2-deficient macrophages were cultured on slides and incubated
with anti-SRBC IgG2a- or IgG2b-opsonized SRBCs. Nonopsonized SRBCs
were used as negative controls. After 1-h culture at 37°C, the slides
were washed with water, fixed in 0.25% glutaraldehyde, and
photographed. Phagocytosis was quantified by calculating the percentage
of phagocytosis (percentage of macrophages containing at least one
ingested SRBC) and the phagocytic index (the percentage of phagocytosis
times the mean number of phagocytosed SRBC per macrophage) after
counting 30–40 macrophages.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-deficient mast cells have impaired FcR-induced
Ca2+ flux, but normal activation of MAPKs
Cross-linking of FcRI on mast cells activates protein tyrosine
kinases and induces elevation of
[Ca2+]i. Activation of both PLC
members, PLC1 and PLC2, is regulated through tyrosine kinases. We
first examined the expression pattern of PLC1 and PLC2 in mast
cells derived from wild-type mouse bone marrow. PLC1 and PLC2
were immunoprecipitated from mast cell lysates and identified in
Western blots by PLC1- or PLC2-specific Abs, respectively. As
shown in Fig. 1
A, both PLC1
and PLC2 were expressed in wild-type mast cells. Next, we examined
whether PLC1 and PLC2 could be activated, indicated by tyrosine
phosphorylation, following FcR engagement in the mast cells. The
bone marrow-derived mast cells were coated with monoclonal murine IgE
and subsequently activated with rabbit anti-mouse IgE Ab. As shown
in Fig. 1B, not only were both PLC1 and PLC2 activated
by engagement of FcR in wild-type mast cells, but the time courses
of activation of the two PLCs were comparable. In addition, PLC2
deficiency had no effect on the expression and activation of PLC1 in
PLC2-deficient mast cells (Fig. 1C).
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R on mast cells led to an initial spike in
[Ca2+]i, followed by a sustained
plateau of intermediate Ca2+ concentration that
slowly decayed to basal levels. As shown in Fig. 1
D,
although PLC2-deficient mast cells exhibited an increase in
intracellular Ca2+ concentration following FcR
cross-linking, the amplitude of Ca2+ elevation
was decreased compared with that in wild-type cells. These results were
in contrast to the effects of PLC2 deficiency on BCR signaling,
where cross-linking of the BCR failed to induce any increase in
[Ca2+]i in PLC2-deficient B
cells (41). Consistent with the decreased
Ca2+ flux, FcR-mediated
IP3 induction was also reduced (Fig. 1E). These decreases in Ca2+ flux and
IP3 production were not due to decreased
expression of FcR on PLC2-deficient mast cells, as the level of
FcR expression on PLC2-deficient mast cells was comparable to
that on wild-type mast cells (Fig. 1F).
We next sought to determine whether the reduced magnitude of the
Ca2+ signal in PLC2-deficient mast cells
affected the activation of other signaling events and cellular
function. Engagement of the FcR leads to activation of the PLC
pathway, which, in turn, can promote and/or enhance activation of all
three types of MAPKs, including ERKs, JNKs, and p38 MAPKs (29, 30, 35, 44, 45). Therefore, we evaluated the extent of ERK, JNK,
and p38 MAPK activation in PLC2-deficient, relative to wild-type,
mast cells in response to FcR cross-linking. As shown in Fig. 1G, PLC2 deficiency had no effect on the activation of
any of these three types of MAPKs.
PLC2-deficient mast cells have impaired degranulation, but
normal cytokine transcription, after FcR engagement
Engagement of the FcR induces the release of granules and the
secretion of multiple cytokines, including IL-1 through -6, IL-9,
IL-10, IL-13, IL-16, TNF-, TGF-, GM-CSF, and IFN-, leading to
allergic reactions. We examined the ability of PLC2-deficient mast
cells to release inflammatory mediators and cytokines after FcR
ligation. As illustrated in Fig. 2A, degranulation, as measured
by the release of [3H]serotonin, was reduced in
PLC2-deficient mast cells. In addition, the FcR-mediated release
of arachidonic acid was reduced in PLC2-deficient mast cells (Fig. 2B). However, ionomycin, but not PMA, was able to restore
the ability of PLC2-deficient mast cells to release arachidonic acid
to the same extent as that of wild-type mast cells (Fig. 2B). Next, we examined the extent of histamine release by
mast cells following engagement of FcR in wild-type and
PLC2-deficient mice. Wild-type mice showed an increase in the serum
concentration of histamine, whereas increases in the serum histamine
concentration in PLC2-deficient mice were decreased relative to
those in wild-type animals (Fig. 2C). These data
demonstrated that Ca2+ flux amplitude (Fig. 1D) correlated with the extent of degranulation.
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R induces the
production of multiple cytokines. It has been shown that
FcR-mediated elevation of
[Ca2+]i is required for cytokine
production at transcriptional levels (46, 47). An increase
in intracellular Ca2+ concentration activates
protein phosphatase calcineurin, leading to nuclear translocation of
the transcription factor NFAT (48, 49). FcR-mediated
activation of PKC is involved in the activation of transcriptional
factors Jun/Fos (50, 51), components of the NFAT complex
(49). NFAT regulates the transcription of several cytokine
genes (52, 53). In addition, the
PKC/Ca2+-independent Ras/Raf-1/ERK cascade has
been shown to be involved in FcR-mediated induction of the
transcription factors Elk-1 and NFAT (54). Moreover, the
promoters for controlling the expression of different cytokines are
different (55, 56, 57, 58). To determine whether the expression of
different cytokines depends on the PKC/Ca2+
pathway and requires different amplitudes and durations of
Ca2+ elevation, we examined the effect of
impaired FcR-mediated elevation of intracellular
Ca2+ in PLC2-deficient mast cells on the
production of different cytokines. Quantitative RT-PCR was conducted to
determine the induction of different cytokine mRNA transcripts in bone
marrow-derived wild-type and PLC2-deficient mast cells following
engagement of the FcR. Cross-linking of FcR induced comparable
levels of cytokine mRNA in PLC2-deficient and wild-type mast cells
(Fig. 2D). Nonetheless, FcR-induced secretion of cytokine
proteins, exemplified by the secretion of IL-6, was decreased in
PLC2-deficient mast cells (Fig. 2E). Interestingly,
ionomycin, but not PMA, was able to restore the ability of
PLC2-deficient mast cells to secrete IL-6 (Fig. 2F).
Therefore, although the production of cytokines at the transcription
level is not affected by the decreased amplitude of the
Ca2+ signal, the secretion of cytokines is
sensitive to the amplitude of the Ca2+
flux.
PLC2-deficient mice are resistant to IgE-mediated inflammatory
skin reaction
The ability of PLC2-deficient mice to develop an inflammatory
skin reaction in response to IgE-mediated activation was examined. In
passive inflammatory skin reaction, local extravasation, fibrin
deposition, and tissue swelling are induced by local injection of
Ag-specific IgE, followed by i.v. antigenic challenge. As shown in Fig. 3, IgE-mediated passive cutaneous
anaphylaxis in PLC2-deficient mice was decreased compared with that
in wild-type controls. These data demonstrate that PLC2 plays an
essential role in the FcR-mediated inflammatory skin reaction.
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R-induced Ca2+ flux in macrophages is dependent
upon the presence of PLC2
There are three classes of Fc receptors for IgG in the mouse: the
high affinity receptor FcRI, which is capable of binding monomeric
IgG, and the two low affinity receptors, FcRII and FcRIII, which
bind polymeric IgG. All three classes of FcR are expressed on
monocytes/macrophages. We first examined the expression pattern of
PLC1 and PLC2 in macrophages. In contrast to mast cells, which
express both PLC1 and PLC2, only PLC2 was detected in
wild-type, bone marrow-derived macrophages, and neither PLC isoform
was detectable in PLC2-deficient, bone marrow-derived macrophages
(Fig. 4A). This expression
pattern indicates that PLC2 may be the sole PLC isoform available
for FcR signaling in macrophages. Engagement of FcRII/III in
Mac1+ macrophages/monocytes induced only a
transient increase in [Ca2+]i,
which quickly returned to basal levels (Fig. 4B). As
expected, cross-linking of FcRII/III failed to induce any increase
in [Ca2+]i in PLC2-deficient
Mac1+ cells (Fig. 4B). These data
demonstrate that Ca2+ flux in response to
engagement of the FcR in macrophages/monocytes is dependent upon
PLC2.
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R-regulated phagocytosis is normal in PLC2-deficient
macrophages
One of the important biological responses mediated by the FcR
is phagocytosis. FcRI- and FcRII/III-mediated phagocytoses were
compared in wild-type and PLC2-deficient macrophages.
FcR-mediated phagocytosis was assessed by the ability of
thioglycolate-elicited peritoneal macrophages to internalize SRBCs
opsonized with IgG. IgG2a-opsonized SRBCs are bound and internalized by
the high affinity FcRI, while IgG2b-opsonized SRBCs are bound and
internalized by the low affinity FcRII/III. As shown in Fig. 5, macrophages from PLC2-deficient
mice internalized either IgG2a-opsonized (percentage of phagocytosis,
91.7 ± 2.9%; phagocytic index, 9.5 ± 1.7) or
IgG2b-opsonized (percentage of phagocytosis, 72.5 ± 3.5%;
phagocytic index, 2.0 ± 0.3) particles as efficiently as did
wild-type macrophages (IgG2a: percentage of phagocytosis, 93.7 ±
3.0%; phagocytic index, 9.7 ± 0.8; IgG2b: percentage of
phagocytosis, 80.0 ± 7.0%; phagocytic index, 2.7 ± 0.4).
Since PLC2-deficient macrophages fail to express either isoform of
PLC (Fig. 4A), we conclude that the PLC pathway is
dispensable for FcRI- and FcRII/III-mediated phagocytosis in
macrophages.
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2 is essential for FcR-mediated passive inflammatory skin
reaction
IgG immune complexes can trigger anaphylaxis via engagement of the
FcR on mast cells (59). To assess the role of PLC2
in the FcR-mediated passive inflammatory skin reaction in vivo,
wild-type and PLC2-deficient mice were injected intradermally at the
basolateral side with rabbit anti-OVA IgG Abs. The extent of
inflammatory skin reaction was determined by assessing the degree of
extravazation of blue dye on the reverse side of skin sections removed
from sites of intradermal injection. As shown in Fig. 6, the FcR-mediated inflammatory skin
reaction in PLC2-deficient mice was decreased compared with that in
wild-type controls. These data demonstrate that PLC2 plays an
essential role in the FcR-mediated inflammatory skin reaction.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R and FcR. However, the relative contributions of PLC1 and
PLC2 to generation of Ca2+ and activation of
PKC following engagement of FcR or FcR are not clear. In
addition, no data have been reported describing the physiological roles
of these signals in the functions of FcR and FcR. In the present
studies we demonstrate that both PLC1 and PLC2 are expressed and
activated by FcR in mast cells, while only PLC2 is expressed and
activated by FcR in monocytes/macrophages. Consistent with its
expression and activation patterns, PLC2 deficiency partially
impaired FcR-induced Ca2+ flux in mast cells
and totally abrogated FcR-induced Ca2+ flux in
monocytes/macrophages. The impaired signals in PLC2-deficient mast
cells upon FcR engagement had no effect on the activation of all
three types of MAPKs or on the transcription of cytokines, whereas they
markedly reduced FcR-mediated serotonin, arachidonic acid, and
histamine release, leading to resistance to IgE-mediated inflammatory
reaction. Surprisingly, although abrogation of
Ca2+ flux in PLC2-deficient macrophages
dramatically altered the FcR-mediated inflammatory reaction, it did
not affect FcR-mediated phagocytosis in monocytes/macrophages.
Although engagement of the FcR activates both PLC1 and PLC2,
previous studies have suggested that PLC1, but not PLC2, is the
primary contributor to IP3 production and
Ca2+ flux in a mast cell line, RBL-2H3
(60). Contrary to current belief, our results clearly
demonstrate the important role of PLC2 in FcR signaling and
function in primary mast cells. Nonetheless, it is highly possible that
PLC1 also plays an essential role in FcR function. One simple
model is that an initial spike in
[Ca2+]i is required for the release
of Ca2+ stores from the endoplasmic reticulum via
IP3-gated channels (61). Emptying of
these stores then causes the opening of plasma membrane
Ca2+ channels (store-operated channel (SOC)),
allowing ions from outside the cell to maintain an elevated
[Ca2+]i (61, 62). A
relatively high level of IP3 is required to empty
internal stores sufficiently to open the SOC and generate a sustained
flux. Lower levels of IP3 produce a smaller
release phase that generally decays rapidly (61).
Therefore, cross-linking of the FcR activates both PLC1 and
PLC2, leading to an initial
[Ca2+]i spike high enough to open
the SOC and generate a sustained flux. The absence of PLC2 reduces
the level of IP3 production by FcR engagement
(Fig. 1E), resulting in decreases in both the initial spike
and the sustained plateau of
[Ca2+]i. Consistent with this
model, there is no detectable difference in the activation kinetics of
PLC1 and PLC2 by FcR engagement (Fig. 1B). However,
it is also possible that PLC1 and PLC2 play unique roles in
FcR function. Previous studies have shown that phosphoinositol
3-kinase inhibitor, wortmannin, specifically blocks the activation of
PLC1, but not PLC2 (60). The different activation
mechanisms of PLC1 and PLC2 indicate that their relative
contributions to FcR function may differ. Further studies of
PLC1-deficient and PLC1/PLC2 double-deficient mast cells will
help to address this issue.
The different effects of PLC2 deficiency on FcR signaling in mast
cells and on FcR signaling in macrophages could be due to the
different mechanisms by which these two receptors signal. Engagement of
the FcR activates both PLC1 and PLC2 as well as sphingosine
kinase (63). Sphingosine kinase phosphorylates sphingosine
into sphingosine-1-phosphate, which alone is able to trigger a
Ca2+ response (63). The absence of
PLC2 reduces the level of IP3 production by
FcR engagement, leading to decreases in both the initial spike and
the sustained plateau of [Ca2+]i.
The residual Ca2+ induction by FcR engagement
in PLC2-deficient mast cells could be due to the activation of
PLC1 and/or sphingosine kinase. In contrast, engagement of FcR in
monocytes/macrophages only activates PLC2 to produce
IP3. Therefore, PLC2 deficiency totally
abrogates FcR-induced Ca2+ flux in
monocytes/macrophages.
Engagement of FcR activates Src family kinases, Syk family kinases,
and Tec family kinases (11, 13, 14, 15, 16, 17, 18). Through adapter
proteins linker for activation of T cells (LAT), Src homology 2
domain-containing leukocyte protein-76, and p95Vav,
activated tyrosine kinases, in turn, initiate downstream pathways,
including PLC/PKC/Ca2+ and MAPKs (ERK, p38,
and JNK) (64, 65, 66). It is known that the Grb2/Sos/Raf1
pathway is a potent pathway activating MAPKs (27, 31, 32, 33, 34).
Upon receptor ligation, through the adaptor protein Grb2, a Ras guanine
nucleotide exchange factor, Sos, is recruited to the membrane, where it
activates Ras (67, 68). Activated Ras associates with and
activates Raf-1 Ser/Thr kinase, leading to a cascade of kinase
activation, which finally activates ERK1 and ERK2 kinases (27, 32, 54). Nonetheless, a number of studies have shown that the
PLC/PKC/Ca2+ pathway also contributes to the
activation of all three MAPKs (ERK, p38, and JNK) (26, 27, 28, 29, 30, 33, 35, 45). However, PLC2 deficiency had no effect on the
activation of any of the three MAPKs, suggesting that activation of
MAPKs is either independent of PKC/Ca2+ signals
or insensitive to the decreased amplitude of the
PKC/Ca2+ signals. Our previous studies, which
showed that BCR ligation in PLC2-deficient B cells generates no
PKC/Ca2+ signals, but activates all three MAPKs
(41), support the idea that MAPK activation is independent
of PKC/Ca2+ signals.
The signals that lead to induction of cytokines are not fully
understood. The elevated levels of
[Ca2+]i initiate association of
Ca2+ with calmodulin, which activates the
Ca2+/calmodulin-dependent phosphatase,
calcineurin (38). Calcineurin, in turn, activates the NFAT
family of transcription factors (69, 70), which interact
with the AP-1 transcription activation complex and cooperatively bind
to the composite NFAT/AP-1 site to regulate the expression of multiple
genes, including IL-2 (38). PKC also plays an essential
role in the induction of cytokines, in that PKC is essential for
activation of NF-
B (71, 72) and production of IL-2
(72). Whereas the promoters that control the expression of
various cytokines are different (55, 56, 57, 58) and may be
differentially affected by the amplitude and duration of
PKC/Ca2+ signals, the transcription of all the
cytokines that we examined was not affected by the impaired amplitude
of FcR-mediated Ca2+ flux observed in
PLC2-deficient mast cells. Therefore, the transcription of these
cytokines may either be independent of or insensitive to the amplitude
and duration of the PKC/Ca2+ signal generated
upon cross-linking of the FcR in mast cells. To discriminate between
these possibilities, it will be necessary to examine FcR-mediated
cytokine expression in mast cells that are deficient in both PLC1
and PLC2.
Intracellular signals generated upon cross-linking of FcRs, similar
to those initiated by engagement of the FcR, involve activation of
Src family and Syk family tyrosine kinases as well as adapter proteins
LAT, Src homology 2 domain-containing leukocyte protein-76, and B cell
linker protein, and lead to activation of the PLC pathway
(15, 25, 73, 74, 75, 76). Among these signal transduction pathway
components, Src family kinases, Syk kinase and the adapter protein
LAT have all been shown to play critical roles in
FcR-regulated phagocytosis (75, 77, 78, 79). In addition,
FcR-mediated phagocytosis is via Ca2+-dependent
and Ca2+-independent pathways
(80, 81, 82, 83) Moreover, PKC, which is activated by PLC, has
been reported to play a role in regulating phagocytosis (83, 84). It was surprising, therefore, that PLC2-deficient
macrophages phagocytosed IgG2a (FcRI-dependent) or IgG2b
(FcRII/III-dependent) immune complexes normally compared with
wild-type macrophages, especially in light of the observation that
these mutant macrophages did not express the alternative PLC1
isoform and failed to mobilize Ca2+ in response
to FcR cross-linking. These data support the idea that the
PLC/PKC/Ca2+ pathway is not required for
FcR-mediated phagocytosis. Nevertheless, our previous studies
demonstrated that PLC2 is essential for FcR-containing collagen
receptor-mediated release of ATP/ADP and thromboxin
A2 in platelets (41) and is
essential for FcR-mediated ADCC activity in NK cells
(41). Therefore, PLC2 is essential for certain
functions of FcR, but it is dispensable for other functions of these
receptors.
Following engagement of FcR and FcR in mast cells, inflammatory
mediators, including histamine, serotonin, and -hexosaminidase, are
released from granules, leading to allergic reactions
(61). Previous studies have demonstrated that activation
of PKC and elevation of intracellular Ca2+ are
sufficient and necessary for the release of these inflammatory
mediators from granules (85, 86, 87). By contrast, studies
have shown that the MAPKs are not required for the release of these
inflammatory mediators, but are involved in the production of
arachidonic acid and cytokines (27, 33, 88). Here we
demonstrate that PLC2 deficiency partially impaired
Ca2+ signals following engagement of the FcR
in mast cells, resulting in a reduced FcR-mediated release of
serotonin, histamine, and arachidonic acid, but had no effect on
cytokine transcription. These results support the idea that
PKC/Ca2+ signals are essential for mast cell
degranulation, whereas MAPKs are involved in cytokine production.
However, these results also reveal that mast cell degranulation,
secretion of cytokines, and resulting allergic reactions are dependent
upon and sensitive to the amplitude of PKC/Ca2+
signals. Moreover, PLC2 deficiency affects the release of
arachidonic acid. One possible explanation is that although the release
of arachidonic acid is regulated primarily through MAPK, PKC may
transiently influence this release (33).
PLC2-deficient mice were resistant to FcR-mediated cutaneous
inflammatory skin reaction. Similarly, PLC2-deficient mice were
resistant to FcR-mediated cutaneous inflammatory skin reaction,
demonstrating that the PLC/PKC/Ca2+ pathway
also plays an essential role in the FcR-mediated inflammatory skin
reaction. These results identify PLC2 as a possible new therapeutic
target for the prevention or treatment of allergic reactions.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Demin Wang, The Blood Research Institute, The Blood Center of Southeastern Wisconsin, Milwaukee, WI 53226. E-mail address: dwang{at}bcsew.edu
3 Abbreviations used in this paper: BCR, B cell receptor; ADCC, Ab-dependent cell cytotoxicity; [Ca2+]i, intracellular Ca2+; HSA, human serum albumin; IP3, inositol 1,4,5-trisphosphate; JNK, c-Jun N-terminal kinase; LAT, linker for activation of T cells; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; PKC, protein kinase C; PLC2, phospholipase C2; SLP76, SH2-domain-containing leukocyte protein of 76 kDa; SOC, store-operated channel; LAT, linker for activation of T cell.
Received for publication February 19, 2002. Accepted for publication October 11, 2002.
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