Recognition and functional activation of the human
IgA receptor (FcαRI) by C-reactive protein
Jinghua Lua,1, Kristopher D. Marjonb,1, Lorraine L. Marnellb,c, Ruipeng Wanga, Carolyn Moldb,d, Terry W. Du Closb,c,d,2,
and Peter Suna,2
aStructural Immunology Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville,
MD 20852; Departments ofdInternal Medicine andbMolecular Genetics and Microbiology, University of New Mexico, Albuquerque, NM 87131; andcVeterans
Affairs Medical Center, Albuquerque, NM 87108
Edited* by Jeffrey V. Ravetch, The Rockfeller University, New York, NY, and approved February 11, 2011 (received for review December 8, 2010)
C-reactive protein (CRP)is animportant biomarker for inflammatory
diseases. However, its role in inflammation beyond complement-
the major IgA receptor, FcαRI, as a ligand for pentraxins. CRP recog-
nized FcαRI both in solution and on cells, and the pentraxin binding
site on the receptor appears distinct from that recognized by IgA.
Further competitive binding and mutational analysis showed that
FcαRI boundto the effector face ofCRPin a region overlapping with
complement C1q and Fcγ receptor (FcγR) binding sites. CRP cross-
linking of FcαRI resulted in extracellular signal-regulated kinase
(ERK) phosphorylation, cytokine production, and degranulation in
expression, phagocytosis, and TNF-α secretion. The ability of CRP to
activate FcαRI defines a function for pentraxins in inflammatory
responses involving neutrophilsandmacrophages. It also highlights
the innate aspect of otherwise humoral immunity-associated anti-
serum amyloid P component|CD89|acute phase protein
marker of infection (1). Interest in the biological activities of CRP
has increased dramatically in recent years because of its associa-
tion with inflammatory diseases such as atherosclerosis and au-
toimmune diseases such as systemic lupus erythematosus. Other
pentraxins include serum amyloid P component (SAP), pentraxin
3 (PTX3), neuronal pentraxin 1 (NPTX1) and neuronal pentraxin
2 (NPTX2). They are innate pattern-recognition molecules tar-
geted to various microbial and self determinants including poly-
saccharides, phosphocholine, and phosphoethanolamine on the
surface of microorganisms, apoptotic or necrotic cells, and nu-
clear autoantigens. CRP and SAP are produced in the liver in
response to inflammatory cytokines such as IL-6 and IL-1. Al-
though the role of CRP in pathogen clearance through comple-
ment activation has been established (2), the participation of
pentraxins in activating cellular immune functions is poorly un-
derstood because of a lack of knowledge of their cellular recep-
tors. CRP and SAP have been shown to bind and activate Fcγ
receptors (FcγR) on monocytes and macrophages (1, 3–6). In
addition, CRP suppressed immune complex-mediated nephro-
toxic nephritis in a mouse model (7). Despite their distinct folds,
both antibody and pentraxins bind FcγR in a 1:1 stoichiometry,
obligating pathogen opsonization or immune complex formation
as the mechanism for receptor clustering and activation (6, 8, 9).
In addition, they share an overlapping binding site on FcγR,
predicting a mutually exclusive FcγR association between anti-
bodies and pentraxins.
Human macrophages and neutrophils express a major receptor
for IgA, Fcα receptor I (FcαRI)/CD89, which activates through
the common Fc receptor (FcR) γ-chain. FcαRI activation by IgA
immune complexes leads to phagocytosis, antigen presentation,
and the release of cytokines, superoxide, and other inflammatory
mediators (10). Despite sharing the common γ-chain for signal-
ing, IgA and IgG antibodies recognize their own receptors and do
not cross-react. The structural recognition of IgA by FcαRI is
-reactive protein (CRP), a member of the pentraxin family, is
a major acute-phase protein in humans and is a clinical
distinct from that of IgG by Fcγ receptors (FcγR) (8, 11). Nev-
ertheless, the ability of pentraxins to bind FcγR with broad spe-
cificities and the functional similarity between FcγR and FcαRI
prompted us to investigate whether pentraxins recognize the re-
ceptor for IgA. Here we identify FcαRI as a receptor for pen-
traxins. The establishment of specific interactions of pentraxins
with FcR provides insight into the mechanism by which these
soluble pattern-recognition molecules activate macrophages and
neutrophils. The finding also reveals a role for FcαRI in the in-
nate immune response.
Pentraxins Recognize FcαRI in Solution. CRP and SAP first were
shown to bind FcγRI-transfected cells and activate phagocytosis
through FcγRI and FcγRIIa (4, 5). More recently, a systematic
solution binding study revealed a broader recognition between
pentraxins and all isoforms of FcγR (6). This broader recognition
between pentraxins and FcγR is supported by their closely related
structures, in that CRP and SAP share identical structural folds
and form similar pentamers. Similarly, FcγR consist of homolo-
gous tandem Ig-like domains with IgG binding sites located in the
two structurally similar membrane proximal domains (8, 12, 13).
The permissive pentraxin–FcγR recognition led us to investigate
further pentraxin recognition of other FcR, including an IgA re-
ceptor, FcαRI, and an IgE receptor, FcεRI. Both FcαRI and
FcεRI consist of two tandem Ig-like domains. Functionally, FcαRI
and FcεRI share a common signaling γ-chain with FcγR and
participate in antibody-mediated inflammation, phagocytosis, and
cytokine release. To examine whether pentraxins interact with
these FcR, recombinant FcαRI and FcεRI were immobilized on
CM5 BIAcore sensorchips together with FcγRIIa (CD32A) as a
control. The binding with various dilutions of CRP, SAP, or PTX3
as the analytes showed that CRP and SAP, but not PTX3, bound
to immobilized FcαRI, with affinities of 2.8 ± 0.2 and 3.2 ± 0.2
μM, respectively (Fig. 1), similar to their binding to FcγR (6). The
kinetic rate constants for CRP and SAP binding to FcαRI are
quite different. Although CRP binding to FcαRI (Ka= 3.1 ±1.4 ×
105M−1·s−1; Kd= 0.35 ± 0.02 s−1) resembles the pentraxin and
IgG binding to the low-affinity FcγR (6), the SAP binding to
FcαRI (Ka= 1.5 ± 0.5 × 104M−1·s−1; Kd= 0.031 ± 0.008 s−1)
displays slower kinetic association and dissociation rates. The
molecular basis for the observed differential rate constants be-
tween CRP and SAP binding to FcαRI is not clear. FcαRI is lo-
cated genetically on human chromosome 19 in a region close to
the leukocyte receptor complex (LRC) that encodes killer cell Ig-
like receptor (KIR), immunoglobulin-like transcript/leukocyte Ig-
Author contributions: J.L., K.D.M., L.L.M., and R.W. performed research; and C.M.,
T.W.D.C., and P.S. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1J.L. and K.D.M. contributed equally to this work.
2To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| March 22, 2011
| vol. 108
| no. 12www.pnas.org/cgi/doi/10.1073/pnas.1018369108
15). FcαRI is most homologous to NKp46 and KIR, sharing
30–35% in sequence identity. Structurally, FcαRI also resembles
KIR and NKp46, with a similar juxtaposition in its two Ig-like
domains, which is opposite those in FcγR and FcεRI (Fig. 1B).
However, CRP failed to bind immobilized FcεRI, KIR, and
NKp46 (Fig. S1), suggesting that the pentraxin recognition of
and FcγR further extends the functional similarity between them.
CRP Recognizes FcαRI on Transfected RBL Cells. To determine
whether the observed solution binding between pentraxins and
FcαRI also occurred on cell surfaces, we investigated CRP
binding to RBL cells, a rat basophilic leukemia cell line, stably
transfected with a Gly-248 variant of FcαRI (referred to as
“G248 cells”) (16). The expression of FcαRI on G248 cells can
be detected readily by an anti-FcαRI antibody, MIP8a, (Fig. 2).
G248 cells and untransfected RBL cells were incubated with
CRP, followed by a FITC-labeled anti-CRP antibody (2C10) for
FACS analysis (Fig. 2C). Greater CRP binding was observed to
G248 cells than to untransfected RBL cells, although significant
binding to the untransfected cells was observed also, probably
because of the binding of CRP to rat FcγR on RBL cells (17, 18).
More importantly, the binding of CRP to G248 cells was reduced
to the level of RBL cells in the presence of MIP8a but not in the
presence of its isotype control. The binding of CRP to G248
cells was dose dependent with an apparent Kdof 0.3 μM (Fig.
2D). These results are in agreement with the data obtained by
surface plasmon resonance (SPR) measurements and showed
that CRP recognizes FcαRI specifically both in solution and on
Pentraxin-Binding Site on FcαRI Is Distinct from That of IgA. The
crystal structure of FcαRI in complex with the Fc region of IgA
showed that IgA recognizes the N-terminal Ig-like domain of
FcαRI (D1) (Fig. 1B) (11). This recognition is very different
from that of IgG binding to FcγR, which involves both the N-
and C-terminal domains (D1 and D2) of the receptor (8, 9). The
stoichiometry of these interactions is also different, with IgG
binding to FcγR at 1:1, and IgA binding to FcαRI at 1:2. The IgG
binding site on FcγR partially overlaps with that of the pen-
traxins, so that pentraxins compete with IgG for FcγR binding
(6). To determine whether pentraxins and IgA share a common
binding site on FcαRI, a solution binding competition experi-
ment between CRP and IgA was carried out using soluble FcαRI
as analyte on an IgA-immobilized sensor chip. If CRP shares a
common binding site with IgA, the addition of CRP to the analyte
would be expected to block the receptor binding to immobilized
IgA. However, the addition of CRP to FcαRI enhanced the re-
ceptor binding to IgA (Fig. 3A). The enhanced binding response
probably is caused by the binding of the higher molecular mass of
the CRP–FcαRI complex to IgA, suggesting that CRP and IgA
bind to distinct regions of FcαRI. Using the FcαRI-transfected
RBL cells (G248 cells), we observed that although MIP8a blocked
both IgA and CRP binding to the transfected RBL cells (Figs. 2B
and 3C), a second anti-FcαRI mAb (A59), which binds to the D2
domain of FcαRI away from the IgA binding site, partially
inhibited CRP but not IgA binding to the FcαRI-transfected cells
(Fig. 3 B and C). Similarly, preincubation with IgA failed to block
CRP binding to FcαRI on the transfected G248 cells (Fig. 3B).
Conversely, unlabeled CRP at a concentration of 1.3 μM failed to
of IgA (Fig. 3C). These results are consistent with CRP and IgA
binding to distinct regions of FcαRI and raise the possibility that
CRP and IgA could interact simultaneously with FcαRI and po-
tentially costimulate cells.
FcαRI and FcγRIIa Bind to Similar Regions on CRP. The pentameric
ring of pentraxins has two faces, a ligand-binding face that rec-
ognizes microbial ligands in a calcium-dependent manner and an
effector face that interacts with complement C1q and FcγR. To
determine whether FcαRI also binds to the effector face of the
pentraxins, competitive CRP binding between C1q and FcαRI
was carried out using BIAcore with immobilized recombinant
FcαRI and FcγRIIa. CRP, when present at 2.7 μM in the analyte,
displayed binding similar to that of immobilized FcαRI and
FcγRIIa (Fig. 4A). In contrast, C1q did not bind either receptor.
Because both C1q and FcγRIIa interact with the effector face of
CRP, their binding to CRP is mutually exclusive. As expected,
the CRP binding to FcγRIIa was partially reduced with the ad-
dition of 0.25 μM of C1q to the CRP-containing analyte and was
eliminated when the concentration of C1q was increased to 1 μM
(Fig. 4A). Similarly, the presence of 1 μM but not 0.25 μM of C1q
blocked the CRP binding to immobilized FcαRI. Because C1q
exists as a hexamer of trimer with each trimeric head capable of
binding to one pentameric CRP (19), 0.25 and 1 μM of C1q are
serial dilutions of CRP, SAP, or PTX3 in micromolar concentrations (u) and
immobilized FcαRI on a CM5 sensor chip. (B) The D1 (green) and D2 (blue)
domains of FcγRIIa (3D5O), FcεRI (1F2Q), FcαRI (1OW0), and KIR2DL2 (2DL2)
are shown in respective D2 orientations. The CRP binding sites on FcγRIIa and
the IgA binding site on FcαRI are shown as surface patches.
CRP and SAP bind to FcαRI in solution. (A) The binding between
(MIP8a) (gray) or isotype control (black) staining of RBL cells (A) or FcαRI-
transfected RBL (G248) cells (B). (C) CRP (150 μg/mL) bound to G248 cells
(gray) better than to RBL cells (black). The CRP binding to G248 cells was
blocked by MIP8a (heavy dashed line) but not by its isotype control (thin
dashed line). Filled areas represent unstained cells. Horizontal axes show
fluorescence intensities. (D) Dose-dependent CRP binding to G248 or RBL
cells detected using FITC-2C10. Data are representative of at least three
CRP binds to FcαRI on transfected RBL cells. (A and B) Anti-FcαRI
Lu et al.PNAS
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expected to titrate a maximum of 1.5 and 6 μM of CRP, re-
spectively. This prediction is consistent with the observed partial
or no inhibition of CRP (2.7 μM) binding to FcR at the lower
concentration of C1q and the complete blockage of CRP binding
at the higher concentration of C1q. The stoichiometric inhibition
of CRP binding to FcαRI by C1q suggests that FcαRI also
interacts with the effector face of CRP. Thus, CRP recognition
of C1q, FcαRI, and FcγR are mutually exclusive.
To determine whether FcγR and FcαRI recognize similar sites
on pentraxins, we examined the receptor binding of CRP mutants
that previously had been identified as defective in FcγR binding.
His-38, Thr-173, and Leu-176 form part of the putative FcR
binding site on CRP, and mutations of each one reduced both
FcγR and C1q binding significantly (6, 20). When these CRP
mutants were assayed for FcαRI binding using BIAcore, H38A
and L176A bound to the receptor similarly to wild-type CRP, but
T173A showed increased FcαRI binding compared with the wild
type (Fig. 4B). These mutational data suggest that although both
FcαRI and FcγR recognize the same face of CRP, the specific
interface residues are likely to differ. This notion is not surprising,
because FcαRI adopts a 3D domain arrangement opposite that of
FcγR (Fig. 1B). The T173A mutant of CRP will provide a useful
reagent to look at differential effects of the two receptor classes.
Based on the assumption that FcαRI and FcγR bind to a
similar site on pentraxins, a docking model for FcαRI binding to
CRP was generated using the crystal structure of the SAP–
FcγRIIa complex (Fig. 4C). Despite its opposite domain orien-
tation, FcαRI could be docked onto CRP based on the FcγRIIa-
complexed SAP structure because of the pentameric symmetry of
pentraxins. The model shows that it is possible for FcαRI to in-
teract with pentraxins in a diagonal orientation similar to FcγRIIa
in the SAP-complexed structure. However, unlike FcγRIIa, which
contacts the A and C subunits of SAP, the opposite D1–D2 hinge
angle of FcαRI results in the receptor contacting the A and D
FcαRI complex shows Thr-173 and Leu-176 but not His-38 as the
immediate receptor-contacting residues (Table S1), consistent
with the mutant binding data showing that the binding of Thr-173
but not His-38 was affected as compared with the wild-type CRP.
CRP Cross-Linking of FcαRI Leads to the Activation of Cellular
Functions. As do FcγR and FcεRI, FcαRI associates with the
common FcR γ-chain and signals through the γ-chain immuno-
receptor tyrosine-based activation motif (ITAM) (10). Cross-
linking of FcαRI leads to activation of several kinases including
spleen tyrosine kinase (Syk) and ERK (21). We have shown
previously that pentraxin recognition of FcγR results in phagocy-
tosis and cytokine secretion by monocytes and macrophages (1, 5,
6, 22, 23). To investigate whether CRP recognition of FcαRI
results in receptor activation, we examined both ERK phosphor-
ylation and degranulation in FcαRI-transfected RBL cells (9.4
cells) upon CRP cross-linking. Because RBL cells express FcεRI,
which associates with and can compete with FcαRI for the FcR
γ-chain, and FcαRI is known to exist in a γ-chain–free form (10),
we obtained an FcαRI-transfected RBL cell line, referred to as
“RBL 9.4,” that expresses a chimeric FcαRI with the cytosolic
domain of the receptor replaced by that of the FcR γ-chain (24).
ERK phosphorylation was readily detectable in these RBL 9.4
binding IgA followed by anti-IgA cross-linking (Fig. 5A). More
importantly, the binding of CRP to FcαRI-expressing RBL 9.4
cells followed by cross-linking with anti-CRP antibody (2C10) in-
duced higher levels of ERK phosphorylation by both Western blot
and FACS analysis than thesame treatment ofuntransfected RBL
cells (Fig. 5 A and B). CRP-induced ERK phosphorylation was
detectable up to 15 min after the cross-linking. RBL cells express
the high-affinity IgE receptor, FcεRI, and the FcR γ-chain, and
cross-linking of FcεRI by IgE leads to potent degranulation as
measured by the release of β-hexosaminidase. This γ-chain–
dependent degranulation also was observed in G248 cells upon
antibody cross-linking of FcαRI (16). Importantly, significant re-
binding response for CRP (2.9 μM), recombinant FcαRI (4.7 μM), or their
combination onto an IgA immobilized CM5 sensor chip. CRP alone did not
bind IgA. (B) The binding of CRP alone (red) in the presence of IgA (1,000 μg/
mL) (green), or mAb A59 (blue) to G248 cells (solid lines) and RBL cells (dashed
lines). Unstained G248 cells (gray line) and RBL cells (shaded gray) are shown.
Horizontal axes of the histograms show fluorescence intensities. (C) Cy3-IgA
binding to G248 cells alone (green) or in the presence of 150 μg/mL CRP (red),
blocking MIP8a (blue shaded), and nonblocking anti-FcαRI mAb A59 (blue
line). Unstained G248 cells (black line) and Cy3-IgA–stained RBL cells (shaded
gray) are shown. (D) Bar graph shows the geometric mean channel fluo-
rescence for Cy3–IgA binding.
CRP and IgA bind at nonidentical sites on FcαRI. (A) The solution
binding between C1q and immobilized FcαRI or FcγRIIa to CRP using BIAcore.
Recombinant FcαRI and FcγRIIa were immobilized individually on CM5 chips.
The analytes were CRP (2.7 μM) in the presence or absence of 0.25 or 1 μM
C1q. C1q alone resulted in close to zero response at 0.25 μM and negative
responses at 1 μM, probably because of higher binding to the dextran sulfate
surface in the reference cell. (B) The equilibrium binding responses of wild-
type and mutant CRP to immobilized FcαRI. (Sensorgrams are shown in Fig.
S2.) (C) The structure of the SAP–FcγRIIa complex (3D5O) and the docked
CRP–FcαRI model in two orthogonal views. The mutation sites used in B are
shown by green sticks. The putative interface residues in the CRP–FcαRI
model are listed in Table S1.
FcαRI recognizes the CRP effector face. (A) Competitive solution
| www.pnas.org/cgi/doi/10.1073/pnas.1018369108Lu et al.
lease of β-hexosaminidase was observed in both RBL 9.4 cells and
G248 cells but not in untransfected cells upon CRP cross-linking
(Fig. 5 C and D). The level of degranulation induced by CRP was
comparable to that induced by IgA. In addition to degranulation,
activated mast cells also produce IL-4 (25, 26). Because RBL is
a mast cell line, we tested the level of IL-4 secretion upon cross-
4 was detected upon CRP cross-linking of G248 cells. Further, the
cytokine production was inhibited by piceatannol, a known Syk
inhibitor of FcR γ-chain signaling in mast cells (27, 28) (Fig. 5E).
These results suggest that CRP cross-linking activates an FcR
γ-chain signaling pathway through FcαRI.
CRP Induces Neutrophil Surface Expression of FcαRI, Phagocytosis,
and TNF-α Production. IgA cross-linking of FcαRI induced the
receptor surface redistribution into lipid raft-like domains in
FcαRI-transfected A20 cells (29). In neutrophils, FcαRI is mo-
bilized rapidly from intracellular granules to the surface by che-
mokines and other mediators (30). We examined the effect of
CRP binding on FcαRI surface expression on neutrophils using
confocal microscopy. Labeled RBC coated with pneumococcal C
polysaccharide (PnC) as CRP ligands were incubated with neu-
trophils, and the expression distribution of FcαRI was measured
with mAb A59 and AF488-labeled secondary antibody. In-
terestingly, FcαRI was diffusely distributed on neutrophils, with a
significant amount residing in the intracellular compartment (Fig.
6A). Binding of CRP-opsonized sheep red blood cells (SRBC)
resulted in a sharp, thin layer of the receptor entirely distributed
on the surface of treated neutrophils with no detectable in-
tracellular localization of the receptor (Fig. 6B). This result sug-
gests that CRP binding induces the surface expression of FcαRI.
We next examined the role of CRP binding to FcαRI in phago-
cytosis of bacteria by neutrophils. Streptococcus pneumoniae se-
rotype 27 (Pn27) was used because it expresses the CRP ligand
phosphocholine in its capsule as well as its cell wall. FITC-
conjugated Pn27 were opsonized with CRP and incubated with
neutrophils. Phagocytosis was determined from the FITC inten-
sity associated with neutrophils after quenching extracellular
fluorescence. Phagocytosis of Pn27 was increased with CRP
opsonization, and preincubation of the neutrophils with the anti-
FcαRI (MIP8a) significantly inhibited the phagocytosis of CRP-
opsonized Pn27 (Fig. 6C). Pretreatment with A59 or an IgG1
isotype control did not inhibit the phagocytosis. Activated neu-
trophils produce type 1 inflammatory cytokines, including TNF-α
(31, 32). We then examined whether CRP activation of FcαRI
induces TNF-α production. The result showed that cross-linking
CRP with an anti-CRP antibody (2C10) induced the neutrophils
to secrete TNF-α, and the TNF-α production upon CRP or IgA
cross-linking was blocked by an Fab fragment of the anti-FcαRI
cretion in FcαRI-transfected RBL cells. (A) FcαRI-transfected 9.4 cells or
untransfected RBL cells were preincubated with CRP or IgA (200 μg/mL), then
incubated with 2C10, anti-IgA, or MIP8a at time point 0. Cells were lysed at
time point 0 and at 5 min, and phospho-ERK was detected by Western blot.
Blots were stripped and reprobed for total ERK. Results are representative of
three experiments. (B) RBL 9.4 cells (open bars) or untransfected RBL cells
(solid bars) were treated as in A, and ERK phosphorylation was determined at
5, 10, and 15 min by flow cytometry. (C) FcαRI-transfected 9.4 cells (solid lines)
or RBL cells (dashed lines) were preincubated with CRP (circle) or IgA (square),
then cross-linked with 2C10 or anti-IgA. No significant β-hexosaminidase
release was detected in 9.4 cells treated with 2C10 alone (diamond). β-
Hexosaminidase release was measured and expressed as the percentage of
total activity. Mean ± SEM of triplicate wells from one experiment are
shown. (D) β-Hexosaminidase release was measured as in C except on FcαRI-
transfected G248 cells with streptavidin cross-linked biotin-CRP. (E) IL-4 se-
cretion in G248 cells (open bars) or untransfected RBL cells (solid bars) treated
with streptavidin cross-linked with CRP or without piceatannol.
CRP induces ERK phosphorylation, degranulation, and cytokine se-
TNF-α secretion. (A and B) Confocal images of neutrophils stained with anti-
FcαRI (A59) and AF488 goat anti-mouse (green). Neutrophils were incubated
for 30 min at room temperature with PKH26 (red)-labeled (A) or CRP-
opsonized (B) PnC-SRBC. (C) Uptake of CRP-opsonized FITC-S. pneumoniae
by neutrophils, expressed as phagocytic index (bacteria per 100 neutrophils),
with or without inhibitors. Data are mean ± SEM of four experiments.
***P < 0.001; **P < 0.01. (D) CRP or IgA (200 μg/mL) cross-linking induced
TNF-α release in human neutrophils. The TNF-α secretion upon either CRP or
IgA treatment was inhibited by the Fab fragment of MIP8a.
CRP induces neutrophil FcαRI surface expression, phagocytosis, and
Lu et al.PNAS
| March 22, 2011
| vol. 108
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antibody (MIP8a), (Fig. 6D). These data suggest that CRP can
activate neutrophils effectively through FcαRI.
That pentraxins recognize both FcαRI and FcγR is counterintu-
itive because the two receptors have opposite D1/D2 domain
structural arrangements. In addition, IgA and IgG bind their
receptors in distinctly different modes, and the two isotypes of
antibodies do not cross-react. The ability of pentraxins to recog-
nize both families of FcR probably results from their pentameric
structure, which makes it possible to contact the same secondary
structure elements from the two receptors with opposite domain
arrangements using symmetrical but different pentraxin subunits.
However, pentraxin recognition of FcαRI is not entirely the result
of its permissive ligand binding, because pentraxins failed to bind
FcεRI despite its closer sequence and structural homology than
FcαRI to FcγR. Likewise, pentraxins did not recognize other
“FcαRI-like” receptors, such as KIR and NKp46. In addition to
binding, we showed that CRP cross-linking of FcαRI led to the
activation of ERK, degranulation, and cytokine production in
FcαRI-transfected cells, as well as to the induction of cell-surface
FcαRI expression, phagocytosis of bacteria, and TNF-α release
FcαRI is expressed primarily on cells of the myeloid lineage, in-
Similar to FcγR, the expression of FcαRI is up-regulated by LPS,
TNF-α, and other proinflammatory stimulators (33) but is down-
regulated by polymeric IgA (34). The regulation of FcR expression
CRP during the acute-phase response, suggesting their potential
involvement in pentraxin-mediated innate immunity, especially
early in infection before effective antibody responses. The expres-
response to chemoattractants, and this increase was shown to be
caused by its release from intracellular storage granules (30).
Similarly, we found CRP treatment induces redistribution of the
receptor to the cell membrane, potentially contributing to the ac-
tivation of the receptor on macrophages and neutrophils during
infection. Because cells expressing FcαRI often express FcγR, it
whether such coengagement activates their functions synergisti-
cally. Alternatively, it is not clear whether the structural difference
between FcαRI and FcγR would result in a different functional
outcome in pentraxin-mediated FcR activation, thus contributing
to cell-type–dependent pathogen responses.
Macrophages and neutrophils are major innate inflammatory
responders to infection. Their effector functions are initiated
primarily through the activation of Toll-like receptors (TLR) by
microbial and endogenous TLR ligands and of FcR by antibody
immune complexes. The recent characterization of pentraxins as
ligands forFcγR and currentlyforFcαRI adds another dimension,
an FcR-mediated innate immune response, as a potential contri-
bution to host defense against pathogens, parallel to the TLR
pathway. TLR- and FcR-mediated innate immune responses have
both similar and contrasting features. TLR and FcR often are
coexpressed on myeloid immune cells. Both TLR and FcR ex-
pression are regulated by inflammation and infection. Although
there are more TLR than FcR, the larger number of TLR pre-
sumably reflects their direct recognition of diverse microbial and
pathogenic ligands, and TLR are activated directly in response to
theincrease in theconcentration of these ligands. In contrast,FcR
recognize a small number of conserved pentraxins and achieve
ligand diversity through the pattern recognition of the pentraxin–
ligand binding. The activation of FcR then would depend on the
increased concentration of pentraxins during infection. It is pos-
sible that both microbial activation of TLR pathways and CRP-
opsonized microbial pathogen activation of FcR pathways occur
concurrently, resulting in synergistic and complementary innate
immune responses, and that together they provide a powerful first
line of host immune defense.
Materials and Methods
Reagents are listed in SI Materials and Methods.
BIAcore Binding Experiments. SPR studies were performed using a BIAcore
3000 (GE Healthcare) with BIAevaluation 4.1 software in 10 mM Hepes (pH
7.4), 0.15 M NaCl, 1.0 mM CaCl2at a flow rate of 50 μL/min. For affinity
analysis, FcγRIIa and FcαRI were immobilized on carboxylated dextran CM5
sensor chips using primary amine coupling. Serial dilutions of SAP, CRP, and
PTX3 from 7.2–0.04 μM were added. For C1q competition binding experi-
ments, the analytes consisted of 2.72 μM CRP with 0.4 mg/mL C1q. To
measure the competition between human IgA and CRP for FcαRI binding,
a CM5 chip was coupled with IgA at levels of 6,000–9,000 resonance units
(RU). The analytes consisted of 2.9 μM of CRP with or without 4.7 μM of
refolded FcαRI. The dissociation constants were obtained by either steady-
state or kinetic curve fittings.
Cell Surface Binding by FACS Analysis. An RBL cell line was stably transfected
with a Gly-248 variant of human FcαRI (16). Human FcαRI exists in two
common alleles (Gly and Ser) as a result of an SNP at amino acid 248 in the
cytoplasmic domain of the receptor gene. The G248 variant of FcαRI pro-
duced a more robust proinflammatory cytokines than did the S248 variant in
transfected cells as well as in human neutrophils. RBL cells and G248 cells
were harvested with trypsin and washed in PBS containing 0.1% BSA and
0.05% sodium azide (PAB). Cells were incubated with CRP or Cy3-IgA for
30 min at 4 °C and washed twice with PAB. CRP binding was detected with
an anti-CRP mAb (FITC-2C10). Data were acquired using a FACScan (BD
Biosciences) or Accuri flow cytometer (AccuriCytometer, Inc.) and analyzed
with FlowJo software (Tree Star, Inc.).
Homology Modeling of the FCαRI-CRP Complex. An initial complex between
FcαRI (1OW0) and CRP (1GNH) was prepared by manual superposition of the
corresponding components onto FcγRIIa and SAP in the SAP–FcγRIIa complex
(3D5O). Docking was performed by tumbling FcαRI over CRP but was largely
constrained to the contact interface in the SAP–FcγRIIa complex using the
shape-only correlation in Hex5 with standard parameters. After clustering,
the three lowest-energy orientations (−371.4 to −392.4 kJ/mol) were se-
lected as the final model.
ERK Phosphorylation Assay. RBL or 9.4 RBL cells were harvested with trypsin
and then washed in Tyrode’s buffer (130 mM NaCl, 5 mM KCl, 2 mM CaCl2,
1 mM MgCl2, 5.6 mM Glucose, 10 mM Hepes, 0.5% BSA, pH 7.4) and resus-
pended at 5.0 × 106cells/mL One hundred-microliter aliquots of cells were
incubated with buffer or CRP (200 μg/mL) for 1 h at 37 °C. Cells were in-
cubated with 2C10 (40 μg/mL) or buffer. For flow cytometry analysis, cells
were fixed with 2% formaldehyde, followed by 90% methanol and washed
with PBS plus 4% FBS. Cells were stained with P-p44/42 MAPK (T202/Y204)
(1:100) rabbit Ab (Cell Signaling Technologies) for 15 min, washed twice, and
then stained with a secondary Alexa Fluor 488 F(ab′)2goat anti-rabbit IgG
(1:500) (Invitrogen) for 15 min. For Western blotting, RBL or RBL 9.4 cells
were seeded at 1.5 × 106cells in 60-mm dishes overnight in complete me-
dium. After treatment, cells were washed with ice-cold HBSS and then lysed
with HBSS containing 1% Triton X-100 with protease and phosphatase
inhibitors (Thermo Scientific). Lysates were incubated for 20 min on ice,
centrifuged at 20,000 × g for 25 min, separated by 10% SDS/PAGE, and
transferred to pvdf membranes. Membranes were probed with P-p44/42
MAPK (T202/Y204) rabbit Ab and then probed with anti-rabbit IgG HRP (Cell
Signaling Technologies). Membranes were stripped with Restore (Thermo
Scientific) and probed for total ERK using p44/42 MAPK.
Degranulation and IL-4 Production Assays. RBL cells or transfected RBL cells
(G248 or 9.4) were cultured overnight in 48-well plates and then washed in
Tyrode’s buffer. Some cells were incubated with 200 μg/mL IgA or CRP for 1 h
at 37 °C. Buffer was removed, and buffer or 40 μg/mL of F(ab′)2anti-IgA or
2C10 was added, and cells were incubated at 37 °C. For G248 cells, 50 μg/mL
of CRP aggregates (AggCRP) were added at time point 0, and activity was
measured over time. Supernatants were collected, and β-hexosaminidase
activity was measured with respect to total release determined by lysis with
1% Triton X-100. Activity was measured by incubation with substrate, 1.4
mg/mL 4-nitrophenyl-N-acetyl β-D-glucosaminide in 75 mM sodium citrate,
pH 4.5, for 1 h at 37 °C. Reactions were stopped by addition of 0.2 M glycine,
pH 10.7, and activity was calculated from the A405 (% release = 100 × su-
pernatant A405/A405 of detergent lysed cells). To assay for IL-4 production,
G248 or untransfected RBL cells were seeded into 96-well plates at a den-
sity of 2 × 104and were preincubated with or without biotin-labeled CRP
| www.pnas.org/cgi/doi/10.1073/pnas.1018369108Lu et al.
(100 μg/mL) and/or piceatannol (25 μg/mL) (Sigma) for 30 min, followed by
streptavidin (20 μg/mL) (Sigma) cross-linking of CRP. After 20 h incubation at
37 °C, the supernatant was assayed for rat IL-4 production using ELISA (R&D
Systems, Inc.) according to manufacturer’s instructions. Data shown are
mean ± SEM of triplicate wells from one representative experiment.
Confocal Microscopy. Human neutrophils were incubated in chamber slides
(Thermo Scientific) for 2 h. PnC-SRBC were incubated with 150 μg/mL of CRP
for 45 min at 37 °C, washed, and added to polymorphonuclear leukocytes
(PMN) at an 8:1 ratio for 10 min. Cells were washed with PBS, fixed with 4%
paraformaldehyde, and permeabilized with 0.2% Triton X-100 for 5 min.
Slides were treated with Image-iT (Invitrogen). Cells were stained with anti-
FcαRI antibody A59, washed, stained with a goat anti-mouse antibody la-
beled with Alexa Fluor 488 (Invitrogen), washed, and mounted in ProLong
Gold Antifade (Invitrogen). Images were acquired using a Zeiss LSM 510
inverted laser scanning microscope.
Neutrophil Phagocytosis and Cytokine Secretion Assays. Neutrophils were
purified by Ficoll-Hypaque centrifugation and resuspended at 2 × 106cells/mL
in RPMI-1640 plus 10% FCS. S. pneumoniae serotype 27 (Pn27) (ATCC) was
grown to log phase, washed in PBS, heat killed, and FITC conjugated. Neu-
trophils were combined with FITC-Pn27 and 100 μg/mL CRP, centrifuged
FITC-fluorescence on gated neutrophils after washing and adding Trypan blue
to quench uningested bacteria and expressed in phagocytic index as number
of Pn27 ingested/100 neutrophils. For cytokine secretion, neutrophils were
treated for 1 h with CRP or IgA (200 μg/mL) with or without the Fab fragment
of MIP8a (15 μg/mL). Medium was removed, 2C10 or anti-IgA (40 μg/mL) was
added, and cells were incubated overnight at 37 °C. Supernatants were ana-
lyzed for TNF-α using an R&D Systems ELISA kit.
ACKNOWLEDGMENTS. We thank Dr. Jeffrey Edberg for providing the FcαRI-
transfected G248 RBL cell line, Dr. Renato Monteiro for providing 9.4 RBL
cells, and Dr. Barbara Bottazzi for providing the recombinant PTX3. Images
were generated in the Cancer Center Fluorescence Microscope Shared Re-
source, University of New Mexico. This work was supported by intramural
research funding from the National Institute of Allergy and Infectious Dis-
eases, by National Research Service Award F31AI080178 (to K.D.M.), by Na-
tional Institutes of Health Grant R21 AI085414, and by a Merit Review Award
from the Department of Veterans Affairs.
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| no. 12