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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 2 (January 15), 1998:
pp. 656-662
A Naturally Occurring Mutation in FC RIIA: A Q to
K127 Change Confers Unique IgG Binding Properties to the
R131 Allelic Form of the Receptor
By
Cynthia F. Norris,
Luminita Pricop,
Sean S. Millard,
Scott
M. Taylor,
Saul Surrey,
Elias Schwartz,
Jane E. Salmon, and
Steven
E. McKenzie
From the Department of Pediatrics, Children's Hospital of
Philadelphia, Philadelphia, PA; the Department of Medicine, The
Hospital for Special Surgery and Graduate Program in Immunology,
Cornell University Medical College, New York, NY; the Departments of
Pediatrics and Research, duPont Hospital for Children, Wilmington, DE;
and the Department of Pediatrics, Jefferson Medical College,
Philadelphia, PA.
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ABSTRACT |
Fc RIIa is widely expressed on hematopoietic cells. There are two
known allelic polymorphic forms of FcRIIa,
FcRIIa-R131 and FcRIIa-H131, which differ
in the amino acid at position 131 in the second Ig-like domain. In
contrast to FcRIIa-R131, FcRIIa-H131
binds hIgG2 but not mIgG1, and this
differential binding has clinical implications for host defense,
autoimmune disease, immunohematologic disease, and response to
therapeutic monoclonal antibodies. We identified a novel FcRIIA
genotype in a healthy individual homozygous for FcRIIA
R/R131 in whom a C to A substitution at codon 127 changes
glutamine (Q) to lysine (K) in one of the two FcRIIA genes. This
individual's homozygosity for FcRIIA-R/R131 leads to
the prediction that the receptors on her cells would not bind
hIgG2. Monocyte and neutrophil phagocytosis of
hIgG2-opsonized erythrocytes was significantly higher
(P < .05) for cells from this K/Q127,
R/R131 individual than for Q/Q127, R/R
131 donors. Platelet aggregation stimulated by an
mIgG1 anti-CD9 antibody in this individual was
significantly different (P < .05) from Q/Q127,
H/R131 and Q/Q127, H/H131 donors
and similar to Q/Q127, R/R131. Our data show
that the K127/R131 receptors have a unique
phenotype, binding both hIgG2 and mIgG1. Further functionally significant mutations in human Fc receptors and
possible novel mechanisms for inherited differences in disease susceptibility should be sought with unbiased screening methods.
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INTRODUCTION |
Fc RECEPTOR IIA for IgG (FcRIIa,
CD32), the most widely distributed Fc receptor, is expressed on
neutrophils, monocyte/macrophages, and platelets. Unlike the group of
multisubunit immune recognition receptors to which other Fc
receptors belong, FcRIIa as a single unit possesses both ligand
binding and signal transducing activities. Allelic polymorphism of
FcRIIa influences receptor function. There are two known
codominantly expressed alleles of FcRIIA that differ in the amino
acid at position 131 in the second Ig-like extracellular domain. The
two forms are FcRIIA-R131 (Arginine, codon CGT) and
FcRIIA-H131 (Histidine, codon CAT). The relative
frequency of the R131 and H131 allotypes varies
in different ethnic groups.1-3 A second polymorphism in
FcRIIa at position 27 (glutamine or tryptophan) is not linked to the
polymorphism at position 131 and does not affect receptor function.4 In contrast to FcRIIA-R131 and
all of the other Fc receptors, FcRIIA-H131 is unique
in its efficient binding of the human (h) IgG2
subclass.4-7 The clinical consequences of the differential
binding of IgG subclasses are profound. Individuals homozygous for
R131 are at higher risk for serious infection with
encapsulated organisms,8-10 which are cleared via a
predominant IgG2 response and for the consequences of
impaired immune complex removal associated with renal involvement in
systemic lupus erythematosus.11,12 Conversely, individuals
homozygous for the H131 allotype may be at higher risk for
autoimmune disorders mediated by IgG2, such as
heparin-induced thrombocytopenia.13,14 The FcRIIA
allotypes also differ in the binding of specific murine and rat IgG
subclasses.15,16 In particular, FcRIIA-R131
binds murine IgG1 (mIgG1) well, but
FcRIIA-H131 does so minimally. Clinically, this
differential binding has been noted to affect the therapeutic utility
of certain murine monoclonal antibodies (MoAbs).17
In the course of screening an African-American population with a
polymerase chain reaction single-stranded conformational polymorphism
(PCR-SSCP) method to determine the
FcRIIA-H/R131 genotype distribution,18 we
identified a healthy individual with a novel FcRIIA genotype. She
has no history of infectious or immune complex-mediated diseases. In
this individual homozygous for FcRIIA-R/R131, a C to A
substitution at codon 127 changes wild-type glutamine (Q) to lysine (K)
in one of the two FcRIIA genes. Cells from this individual would be
predicted not to bind hIgG2 via FcRIIa. We examined the
functional consequences of this mutation in neutrophils, monocytes and
platelets from this individual compared with those of
R/R131, H/R131, and H/H131
individuals, all of whom were wild-type (Q/Q) at position 127. We show
that the K127 substitution imparts to an
FcRIIA-R131 molecule the ability to interact with the
hIgG2 subclass and enhance monocyte and neutrophil
phagocytosis in comparison with that of wild-type homozygous
Q/Q127, R/R131. In addition, we show that the
mutant receptor retains the ability to bind mIgG1 and thus
has a unique phenotype.
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MATERIALS AND METHODS |
Cell preparation and PCR amplification.
Peripheral blood (10 mL) was collected in a heparinized tube.
Collection of blood samples from donors was performed with informed consent after obtaining the approval of the Institutional Review Board.
The erythrocytes were selectively lysed and genomic DNA was isolated
from the resulting white blood cell pellet with an automated nucleic
acid extractor according to manufacturer's instructions (Applied
Biosystems, Inc, Foster City, CA). Genomic DNA was reconstituted in
sterile water (1 mL) and the concentration was determined by optical
density at 260 nm on a spectrophotometer.
Oligonucleotide primers were chosen that selectively amplify the
FcRIIA gene and not the highly homologous FcRIIB and FcRIIC genes. One of two sense primers (PCR1 or PCR3) from the second extracellular domain was used. The antisense primer (4INM) was in the
intron immediately downstream of the second extracellular domain where
the sequence for FcRIIA, FcRIIB, and FcRIIC diverge. The
resulting PCR product was either 322 bp (using PCR1) or 277 bp (using
PCR3). Both products contained the distal portion of the second
extracellular FcRIIA exon (which contained the polymorphism at codon
131 and the mutation at codon 127), the splice junction, and the
proximal portion of the downstream intron. The primers are as follows:
PCR1, 5 GGA GAA ACC ATC ATG CTG AG 3; PCR3, 5 CTG
GTC AAG GTC ACA TTC TTC 3; and 4INM, 5 CAA TTT TGC TGC TAT GGG C 3.
PCR reactions were performed in 100 µL containing buffer (50 mmol/L
KCl, 10 mmol/L Tris-HCl, pH 8.3, 0.001% [wt/vol] gelatin, 1.5 mmol/L
MgCl2), 200 ng of sense and 200 ng of antisense primer, 130 to 860 ng of genomic DNA, and 400 µmol/L each of dATP, dCTP, dGTP,
and TTP. After 5 minutes of incubation at 95°C, 2.5 U of AmpliTaq
DNA polymerase (Perkin-Elmer/Cetus, Norwalk, CT) was added. The mixture
was amplified for 30 to 35 cycles using a GeneAmp PCR System 9600 (Perkin-Elmer/Cetus). Each cycle consisted of a denaturing step
(94°C for 30 seconds), an annealing step (50°C for 30 seconds),
and an elongation step (72°C for 30 seconds), in that order. PCR
amplification was performed on five separate occasions for the
individual with the codon 127 substitution (4 from an initial blood
draw and 1 from a second blood draw).
SSCP analysis.
We previously described an SSCP protocol for identification of the
FcRIIA-H/R131 genotype.2 Briefly, a 0.65 ng
(typically 5.4 to 6.3 µL) sample of the above-described PCR product
and 10 µL of loading buffer (95% [vol/vol] formamide, 0.05%
[wt/vol] xylene cyanol, 20 mmol/L EDTA) were heated to 100°C for
10 minutes and then placed immediately on wet ice. All subsequent steps
were performed in a cold room at 4°C. Samples were loaded onto a
nondenaturing 8% (wt/vol) polyacrylamide-TBE (92 mmol/L Tris, 95 mmol/L borate, 2.5 mmol/L EDTA) gel (18 × 24 cm; SE 600; Hoefer
Scientific Instruments, San Francisco, CA) with a 37.5:1 ratio of
acrylamide to bisacrylamide. The gel apparatus was further cooled by
the Hoefer SE 6160 heat exchanger with a continuous flow of cold water
surrounding the chamber. Electrophoresis was performed in a
discontinuous buffer (25 mmol/L Tris, 192 mmol/L glycine) at 200 V for
6 hours. Gels were silver stained according to the manufacturer's
instructions (silver stain kit; Bio-Rad, Melville, NY).
Automated DNA sequence analysis.
PCR products were purified with Magic PCR Preps DNA Purification System
(Promega, Madison, WI), and then automated DNA sequence analysis with
dye-labeled dideoxynucleotide chain terminators was performed following
the manufacturer's instructions (Taq dideoxy terminator cycle
sequencing; Applied Biosystems, Inc). The reaction products were
analyzed on a laser-based, fluorescence emission 373A DNA sequencer
(Applied Biosystems, Inc). All DNA sequences were determined in both
directions using sense (PCR1 or PCR3) and antisense (4INM) primers.
Subcloning and analysis of FcRIIA PCR products.
The FcRIIA PCR product containing the substitution at codon 127 was
obtained as previously described using PCR1 and 4INM primers. The
product was purified with Magic PCR Preps DNA Purification System
(Promega) and subcloned directly into the pT7Blue T-Vector using the
pT7Blue T-Vector Kit (Novagen, Madison, WI) following the
manufacturer's instructions. Individual colonies were isolated and
grown at 37°C, and the subcloned FcRIIA DNA was amplified directly from individual colonies using PCR and vector-based primers that flank the cloning site. The sequence of each PCR product was
determined in both directions by automated DNA sequence analysis as
described above.
Quantitation of FcR expression by flow cytometry.
Leukocytes from fresh anticoagulated blood were prepared as previously
reported.12,19 Briefly, isolated monocytes or PMNs were
incubated with saturating concentrations of the following MoAbs: murine
IgG1 or IgG2b (controls), IV.3
(mIgG2b specific for FcRII; Medarex, Inc, Annandale,
NJ), 41H16 (specific for FcRIIA-R131; generously
provided by Dr Theodore Zipf, University of Texas Cancer Center,
Houston, TX20), CLB Gran 1 (specific for
FcRIIIB12; Research Diagnostics Inc, Flanders, NJ), and
22 (specific for FcRI12; Medarex, Inc). This was
followed by incubation with phycoerythrin-conjugated goat antimouse IgG
F(ab)2. After washing with phosphate-buffered saline
and fixation with 1% (vol/vol) paraformaldehyde, specific cell-associated immunofluorescence was quantitated on a FACScan (Becton
Dickinson, San Jose, CA) as previously described.5
Assay of neutrophil (PMN) FcR-mediated phagocytosis.
Fresh human peripheral blood was collected in a heparinized syringe and
separated by centrifugation through a discontinuous two-step
Ficoll-Hypaque gradient.5 Neutrophils (PMNs) were isolated
from the lower interface and washed with Hanks' balanced salt
solution. Bovine erythrocytes were coupled to IV.3 Fab (anti-FcRII CD32 MoAb), hIgG1 (human IgG1-myeloma protein),
hIgG2 (human IgG2-myeloma protein), or
mIgG1 (murine IgG1-myeloma protein) by a
biotin-avidin technique.5 The resulting E-IV.3,
E-hIgG1, E-hIgG2, and E-mIgG1 were
used as probes of FcR-mediated internalization. The density of
opsonization was determined by flow cytometry as described previously
and corresponded to low opsonization to maximize the capacity to detect
differences among the FcRIIA genotypes.12 Erythrocyte
phagocytosis by PMNs and monocytes was quantitated as reported
previously.5 Briefly, phagocytes were combined with E-IV.3,
E-hIgG2, E-hIgG1, or
E-mIgG1, centrifuged at 44g for 3 minutes, and then incubated at 37°C for 15 minutes to allow for
maximum internalization. After hypotonic lysis of noninternalized E,
phagocytosis was quantitated by light microscopy. At least 400 cells
per slide were counted in duplicate without knowledge of the donor
FcRIIA genotype. The data are expressed as the phagocytic index (PI;
number of ingested erythrocytes per 100 PMN). To enable simultaneous
quantitation of FcRIIa function in multiple donors with a range of
different erythrocyte probes, a flow cytometric assay was used.
Erythrocytes coupled to IgG or Fab were labeled with lipophilic red dye
PKH-26 and then fluorescence was determined.21 The
phagocytosis assay was performed as described above and, after lysis of
noninternalized erythrocytes, phagocyte-associated PKH-26 fluorescence
was quantitated by flow cytometry.21a To
compare individuals of different FcRIIA genotypes and to control for interexperiment variability using both assays of phagocytosis, data are
expressed as the percentage of PI of the R/R131 homozygote
studied in each experiment (%PI = [PI-K127 or
H131/PI-R131] × 100).
Platelet aggregation assay.
Peripheral blood (20 mL) was collected in a polypropylene tube
(Sarstedt, Nuembrecht, Germany) on two occasions from the individual with the K/Q127, R/R131 FcRIIA genotype for
platelet aggregation analysis. This analysis was also performed for
individuals from each of the known FcRIIA genotypes (n = 5 to 7 per
genotype). Platelet-rich plasma and platelet-poor plasma were obtained
by differential centrifugation (800 rpm [132g] for 15 minutes, followed by 3,000 rpm [1,862g] for 15 minutes), and
the platelet count of the platelet-rich plasma was adjusted to
300,000/µL with the platelet-poor plasma. Aggregation studies were
performed in one of two aggregometers (PAP 4; Biodata, Hatboro, PA or
Chronolog, Havertown, PA) calibrated to each other by parallel analysis
of the same samples. All samples were determined to be free of
spontaneous aggregation and to have normal aggregation in response to
standard agonists (thrombin and collagen). Alb-6, an mIgG1
MoAb also directed against CD9 (AMAC, Inc, Westbrook, ME), was diluted
to 200 ng/µL and added to 0.5 mL of platelet-rich plasma at a final
concentration of 15 µg/mL, and aggregation was monitored. Lag time
(the time from the addition of the antibody agonist until the onset of
aggregation as manifested by the start of the marked deflection in the
light transmission recording) and the final percentage of aggregation
were determined. The final antibody concentration chosen, 15 µg/mL,
is a potent stimulus, as shown by platelets from Q/Q127,
H/R131 heterozygotes, which aggregate but demonstrate a
prolonged lag time.
Statistical analysis.
Results of phagocytosis assays for monocytes and neutrophils from
donors with different FcRIIA genotypes were compared using the
paired Student's t-test (two-sided,  < .05). Likewise,
platelet aggregation lag time data for platelets from donors with
different FcRIIA genotypes were compared using the paired Student's
t-test (two-sided, < .05).
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RESULTS |
Detection of the Q to K127 change in FcRIIA.
To determine the FcRIIA-H/R131 genotype distribution in
healthy individuals to compare with that in disease populations, we analyzed PCR products from genomic DNA of 50 African-Americans by
SSCP.2 This FcRIIA-specific PCR product includes the
known H/R131 polymorphism and extends from the middle of
the exon encoding the second Ig-like extracellular domain through the
downstream intron. PCR product from one African-American individual had
a unique SSCP pattern (Fig 1A) that, when
subjected to automated DNA sequence analysis, showed homozygosity for
R131 and heterozygosity for a C to A mutation in codon 127 (Fig 1B). This substitution changes the codon from CAG (glutamine, Q)
to AAG (lysine, K). To ensure that this was not an artifactual
PCR-induced mutation, several steps were taken. Sequence analysis of an
independent PCR reaction from the same genomic DNA preparation was
confirmatory, as were the results from a second independent preparation
of genomic DNA from the same individual. Because codon 127 in the
highly homologous FcRIIB and FcRIIC genes is AAG (K), we ruled
out amplification of the homologous genes by sequence analysis of subcloned PCR products. At every position in the PCR product where FcRIIA diverges from FcRIIB/C, the sequence agreed exactly with FcRIIA. Individually sequenced subclones showed the C to A
substitution in a distribution consistent with a heterozygote (6 of 15, A; 9 of 15, C). Thus, we established that this individual has a novel C
to A mutation at FcRIIA codon 127.
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| Fig 1.
Detection of the FcRIIA K 127 mutation. (A) SSCP
analysis of the FcRIIA PCR product from genomic DNA is shown. In
lane 4, a unique SSCP pattern (arrowheads, top band) is seen in
comparison with H/R131 (lanes 1 through 3),
R/R131 (lanes 5 and 6), and H/H131 (lanes 7 and
8) samples. (B) DNA sequence analysis of the PCR product with the
unique SSCP pattern shows heterozygosity for C (blue) and A (green) at
the first nucleotide of codon 127. The sequence also indicates
R/R131 homozygosity (G, black, at the second nucleotide of
codon 131).
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The K127 mutation occurs within a 14-amino acid stretch
implicated as important in the binding of IgG ligands by the FcRIIa receptor (Fig 2).22,23 To
examine the functional consequences of this change, we performed
analysis of cells from the individual with the variant using
well-defined antibody reagents that distinguish FcRIIA-H131 from R131.
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| Fig 2.
Sequence comparison of the amino acids from position 124 to 137 for FcRIIA wild-type (Q127, green), the described
mutation (K127, red diamond), and FcRIIB/C. Note that
NFS (blue, underlined) at 135-137 is a site for N-linked glycosylation
in FcRIIB/C. There is a conservative L (IIA) to S (IIB/C) change at
position 132.
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Cell surface expression of FcRIIA K127 mutant is
equivalent to wild-type.
To examine the effect of the K127 mutation on phagocyte
function, we first assessed receptor expression. Flow cytometry was
performed concurrently on monocytes and PMNs from disease-free controls with Q/Q127, H/H131 and Q/Q127,
R/R131 genotypes and the individual with the
K/Q127, R/R131. Using anti-FcRII MoAb IV.3,
there was similar fluorescence on monocytes (mean channel fluorescence
intensity in arbitrary units [MFI]: 526, 600, and 509, respectively)
and PMN (MFI, 468, 538, and 547, respectively). IV.3 is a ligand
binding site MoAb that recognizes both the
Q127/R131 and Q127/H131
allotypes of FcRIIa. Experiments with MoAb 41H16, which specifically recognizes FcRIIa-R131, supported similar expression of
FcRIIa on K/Q127, R/R131 and
Q/Q127, R/R131 PMNs (MFI, 550 v 506).
Previous studies using these MoAbs have shown similar expression of
FcRIIa in populations of disease-free individuals of each known
genotype.5,20 Although we cannot rule out the possibility
of differential binding of the anti-FcRIIa MoAbs to the
K127/R131 variant, our results suggest that the
mutant receptor is present at equivalent levels to wild-type receptors.
Additionally, recognition by ligand binding site MoAbs supports the
possibility that it is a functional receptor.
The Q to K127 change enhances the phagocytosis of
erythrocytes coupled to hIgG2 in an R131
homozygote.
FcRIIa is a major phagocytic receptor on monocytes and PMNs, and
internalization of E-hIgG2 is dependent on FcRIIA
genotype.5,12 PMN and monocytes from H/H131
individuals efficiently bind and ingest E-hIgG2, whereas
this is minimal or absent in R/R131 homozygotes.
H/R131 heterozygotes have an intermediate capacity to
recognize hIgG2.12 In contrast,
mIgG1 is efficiently bound by R/R131
homozygotes and minimally recognized by H/H131 individuals.
To examine the effect of the K127 variant on
hIgG2 and mIgG1 handling, we simultaneously
quantitated E-hIgG2 and E-mIgG1 phagocytosis by
monocytes from the individual with the K127 variant and
compared those results with results from H/H131 and
R/R131 donors. Separate matched triplet experiments were
performed, each with a pair of homozygote donors and the variant.
Phagocytosis of E-IV.3, an FcRIIa-specific probe that is not
selective for allotypes, was assessed in each experiment.
E-hIgG1, which is recognized by both alleles of FcRIIa
and by FcRI and FcRIIIa, was a control for general phagocytic
potential of donor monocytes. As shown in
Fig 3A, phagocytosis of E-hIgG2
was higher for the K/Q127, R/R131 variant than
for the group of Q/Q127, R/R131 homozygotes
(P < .05, n = 3), whereas there was no difference in the
capacity to internalize E-IV.3 and E-hIgG1. Although the K/Q127 heterozygote showed enhanced recognition of
hIgG2, there was no detectable change in the association
with mIgG1. We cannot exclude the possibility that, with a
wide range of E-mIgG1 opsonization densities, a difference
would be evident, but availability of blood from the variant donor was
limited. Formal proof that there is a change in hIgG2
binding in the absence of a reciprocal change in mIgG1
binding will require phagocytosis experiments using cells transfected
with the FcRIIA-K127/R131.
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| Fig 3.
Monocyte (A) and PMN (B) internalization of erythrocytes
coupled with specific human IgG myeloma proteins (E-hIgG1
and E-hIgG2), murine IgG1 myeloma protein
(E-mIgG1), or anti-FcRII MoAb IV.3 (E-IV.3). The
phagocytic index shown for each erythrocyte probe reflects simultaneous
experiments with phagocytes from individuals of each of three FcRIIA
genotypes: Q/Q127, H/H131; Q/Q127,
R/R131; and K/Q127, R/R131. The % Phagocytic Index = (PIdonor/ PIQ/Q127,
R/R131) × 100. Values represent the mean ± SD of
two to five experiments comparing the K127 variant with
different H/H131 and R/R131 homozygotes. % PI
were compared using the paired Student's t-test. *P < .05, K/Q127, R/R131 v
Q/Q127, R/R131; **P < .01, K/Q127, R/R131 v Q/Q127,
H/H131; ***P = .051, K/Q127,
R/R131 v Q/Q127, H/H131.
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In the experiments with PMN, evidence for differential binding capacity
of K/Q127, R/R131 was underscored.
Internalization of E-hIgG2 for the heterozygote variant was
two to three times greater than that of simultaneously studied
R/R131 donors (P < .005, paired t-test, n = 5), whereas handling of E-IV.3 by PMN was comparable for all
genotypes in the matched triplet experiments (Fig 3B). Equivalent
expression of FcRIIIb (CD16; MoAb CLB Gran 1) and the absence of
FcRI (CD64; MoAb 22) in the individuals with each of the three
genotypes (data not shown) minimizes the possibility of these receptors
confounding the analysis of the impact of the Q to K127
mutation on PMN function. For the level of opsonization of
E-hIgG2 used in these studies, we have previously shown
phagocytosis measured by microscopy in a population of wild-type
R/R131 donors to be 1.2 ± 2.4 erythrocytes/PMN (range,
0 to 6).12 In the current studies, assessment by microscopy
showed R/R131 donors to internalize 4 ± 1 erythrocytes/PMN compared with 14 ± 1 in the K/Q127,
R/R131 variant. This difference was confirmed with the flow
cytometric assay of phagocytosis, which provided the opportunity to
measure the function of a greater number of phagocytes and multiple
probes. Given that the individual with the variant is heterozygous for the K127 substitution and thus only half of the FcRIIa
receptor molecules on the PMN or monocyte are the
K127/R131 form, our data show enhanced binding
of hIgG2 by the variant receptor over the wild-type
Q127/R131.
Platelet aggregation mediated by mIgG1 antiplatelet
antibody.
The phagocyte data showed clearly that the
K127/R131 receptor interacted well with
hIgG2. The data also suggested that interaction of
K127/R131 receptor proteins with
mIgG1 was comparable to that of
Q127/R131 and distinct from
Q127/H131. We performed experiments with an
mIgG1 antiplatelet CD9 antibody to extend these findings in
cells (platelets) expressing FcRIIa as the sole Fc receptor for
IgG.24-26 There is a differential ability of platelets to
be activated by murine antiplatelet antibodies of the mIgG1
subclass depending on the FcRIIA-H/R131 genotype:
R/R131 > H/R131 >> H/H131, as
manifested by increases in lag time.2,27,28 We quantitated platelet aggregation from known wild-type FcRIIA-H/H131,
R/R131 and H/R131 donors and the individual
with the Q to K127 mutation after stimulation with Alb-6,
an mIgG1 anti-CD9 antiplatelet antibody (15 µg/mL). As
predicted, platelets from all donors, except Q/Q127,
H/H131, underwent aggregation in response to Alb-6 and
proceeded to an equivalent final extent. Platelets from all donors also
had negligible spontaneous aggregation and equivalent aggregation to
standard agonists, including thrombin and collagen. As shown in
Fig 4, when stimulated with Alb-6, the lag
time for the variant K/Q127, R/R131 platelets
was no different from that of Q/Q127, R/R131,
but was significantly shorter than that of platelets from both Q/Q127, H/R131 and Q/Q127,
H/H131 donors (P < .05). The comparison with
Q/Q127, H/R131 is particularly important,
because on those platelets 50% of the receptors are of the
Q127/H131 form, whereas 50% are
Q127/R131. If K127/R131
receptors did not interact well with mIgG1, then the
platelets from the donor with the variant K/Q127,
R/R131 genotype should have behaved like the
Q/Q127, H/R131 platelets. Instead, they were
significantly different from Q/Q127, H/R131
platelets and indistinguishable from Q/Q127,
R/R131. These platelet activation results indicate that the
FcRIIA K127/R131 receptors retain
significant interaction with mIgG1.
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| Fig 4.
Platelet aggregation triggered by mIgG1
antiplatelet CD9 antibody Alb-6. Lag time in minutes (mean ± SEM) is
shown for platelets from the variant individual and the three wild-type
FcRIIA genotypes. *Lag time is significantly shorter (P < .05) for K/Q127, R/R131 and Q/Q127,
R/R131 than for Q/Q127, H/R131 and
Q/Q127, H/H131.
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| |
DISCUSSION |
We identified an individual with a novel FcRIIA genotype in which a
C to A nucleotide substitution leads to a Q to K127 amino
acid change in the setting of homozygosity for R/R131. We
showed increased phagocytosis of hIgG2-opsonized
erythrocytes by this individual's monocytes and neutrophils in
comparison with those of wild-type Q/Q127,
R/R131 individuals. On platelets, there was a significant
difference in the interaction with mIgG1 in comparison with
platelets from Q/Q127, H/R131 and
Q/Q127, H/H131 donors, but no difference from
Q/Q127, R/R131. Neither surface expression as
assessed by MoAb IV.3 nor interaction with hIgG1 were
affected by the K127 mutation. These results indicate that
this novel mutation converts a receptor with minimal interaction with
the hIgG2 subclass (wild-type Q127/R131) into one with clearly detectable
interaction (variant K127/R131). In addition,
interaction with mIgG1 is preserved. In our studies of
phagocytes, we were careful to establish equivalent expression of the
Fc receptors at physiologic surface densities on cells with the
normal phagocytic machinery. Previous experiments have identified
critical regions for IgG binding within the extracellular domains of
FcRIIa. The present study confirms their importance in unmanipulated
human phagocytes and demonstrates the impact even in the context of
other Fc receptors (IIIb on PMN and Ia on monocytes). These features
of our comparative studies may be an advantage over studies of
transfected Fc receptor genes in heterologous cells.
The K127/R131 FcRIIa molecule we describe
has a unique phenotype it recognizes hIgG2-like
Q127/H131 and mIgG1-like
Q127/R131. The structural basis for ligand
binding specificity by this variant and the previously described
FcRIIA allelic isoforms is unknown. The unique phenotype of the
FcRIIA-K127/R131 molecule implies that
interactions between amino acids in the ligand binding pocket, such as
at 127 and 131, with each other and with specific IgG residues
contribute to the differential IgG binding by the receptor isoforms.
Chimeric molecules with other Fc and Fc receptors as well as a
large number of site-directed mutants have been used in transfected
cells in vitro to study FcRIIA ligand binding, but the 127 position
has not been explored by any of these investigators because there was
no prior evidence to implicate its
importance.23,29-31 Once the FcRIIa crystal structure has been determined, structure/function studies with wild-type and the mutant K127/R131 receptors
will shed light in the future on the nature of differential receptor
interactions with various IgG subclasses.32-34
This is the first example of a naturally occurring mutation in a human
Fc receptor that alters receptor function. Further examples are
anticipated as useful genetic screening methods and precise functional
studies are more widely applied. Growing evidence supports the view
that genetic variation in the Fc receptors has functional
significance and may be associated with susceptibility to human
disease.35-44 Polymorphisms of FcRIIa and FcRIIIb and their association with disease underscore their roles as specific genetic risk factors. Rapid determination of the
FcRIIA-H/R131 genotype continues to be important in
studies that link human immune, infectious, and inflammatory disorders
to this polymorphism.3,9,12,45 Because the K127
mutation is not resolved at the level of reactivity with available anti-FcRII MoAb or with allele-specific genotyping, the current work
shows the importance of unbiased genetic screening methods such as SSCP
(which we used), DGGE, or direct automated sequence analysis in
examining genetic variations in FcRIIa and other Fc
receptors.2,46 We have not seen the Q to K127
mutation in any other of the approximately 200 healthy individuals screened to date. However, we and others have noted that cells from
individuals of known H/R131 genotypes occasionally have
anomalous binding, platelet aggregation, or phagocytosis of IgG ligands
mediated by FcRIIa. Thus, in the future, it will be of great
interest to more fully examine the prevalence of this or related
FcRIIA mutations in such individuals.
| |
FOOTNOTES |
Submitted January 13, 1997;
accepted September 11, 1997.
Supported in part by the following grants from the Public Health
Service: NIH T32 HL07150 (C.F.N.), R01 DK16691 (E.S., S.S., S.McK.),
P30 HD28815 (S.McK.), P01 HL40387 (S.McK.), and R01 AR38889 (J.E.S.).
The flow cytometry Core Facility at the Hospital for Special Surgery is
supported in part by the Cornell Multipurpose Arthritis and
Musculoskeletal Disease Center (P60 AR 30692).
Address reprint requests to Steven E. McKenzie, MD, PhD, duPont
Hospital for Children, 1600 Rockland Rd, Wilmington, DE 19899.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Donna Patterson for her assistance and Diana Cassel
and Margaret Keller for their advice and encouragement.
| |
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Abstract
Introduction
Abstract