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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 10 (November 15), 1999:
pp. 3448-3455
Enhanced Liver Uptake of Opsonized Red Blood Cells After In Vivo
Transfer of Fc RIIA cDNA to the Liver
By
Petr Bezdicek,
Stefan Worgall,
Imre Kovesdi,
Moo-Kyung Kim,
Jong-Gu Park,
Theresa Vincent,
Philip L. Leopold,
Alan D. Schreiber, and
Ronald G. Crystal
From the Division of Pulmonary and Critical Care Medicine, Weill
Medical College of Cornell University, New York, NY; GenVec, Inc,
Rockville, MD; InKine Pharmaceutical Co, Inc, Blue Bell, PA; and The
University of Pennsylvania School of Medicine, Philadelphia, PA.
 |
ABSTRACT |
Fc receptors convey to phagocytic cells the ability to recognize,
bind, and internalize IgG-coated cells and microorganisms. The present
study demonstrates the use of adenovirus (Ad)-mediated gene transfer of
human Fc receptor IIA cDNA to convert normally nonphagocytic cells
(hepatocytes) into functional equivalents of phagocytic cells. Ad
vector in vitro transfer and expression of FcRIIA cDNA in primary
rat hepatocytes was confirmed by flow cytometry anti-FcRIIA
immunodetection, and the function of the receptor was demonstrated by
enhanced binding and phagocytosis of 51Cr-labeled
IgG-opsonized erythrocytes. After in vivo gene transfer to rats,
expression of FcRIIA cDNA in hepatocytes was confirmed by Northern
analysis and immunohistochemistry. Rats infected with the Ad vector
carrying the FcRIIA cDNA demonstrated enhanced clearance of
opsonized erythrocytes, but not nonopsonized erythrocytes, from the
circulation with increased sequestration within the liver. Together,
these data demonstrate that Ad-mediated FcRIIA gene transfer can
convert normally IgG-nonphagocytic cells into phagocytic cells capable
of recognizing, binding, and ingesting an opsonized particulate
antigen, suggesting that gene transfer strategies might be used to
transiently augment host defense by enhancing the clearance of immune complexes.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
PHAGOCYTOSIS OF IgG-coated cells and
microorganisms, an important component of host defense, is mediated by
receptors for the Fc portion of IgG (Fc receptors) expressed on
monocytes/macrophages and neutrophils.1-4 The 3 classes of
Fc receptors (FcRI, FcRII, and FcRIII) differ by size,
structure, ligand binding specificity, and cellular
distribution.1-3 The importance of Fc receptors in host
defense is highlighted by disorders such as systemic lupus erythematosus and chronic renal and liver failure, in which enhanced susceptibility to infection is associated with a downregulation of
Fc receptors.5-7 Likewise, when there is a deficiency in the number and/or function of mononuclear and/or polymononuclear phagocytes, such as occurs in association with chemotherapy and prolonged administration of corticosteroids, the adaptive immune system
cannot effectively use IgG opsonization as a strategy to aid in host
defense.8-11
Based on these concepts, we hypothesized that in vivo clearance of
IgG-coated cells might be enhanced by inducing Fc receptor expression in normally nonphagocytic cells in the liver, an organ that
normally plays a major role in the clearance of IgG-coated complexes.12-14 To evaluate this hypothesis, we used an
adenovirus (Ad) gene transfer vector to express normal human FcRIIA,
a FcRII allotype with affinity for IgG2,2,15 cDNA in rat
liver cells in vitro and in vivo, and evaluated the ability of the
modified liver cells to phagocytize opsonized red blood cells. The in
vitro data demonstrate that FcRIIA cDNA genetic modification of
primary rat hepatocytes enhances the ability of these cells to bind and phagocytize IgG opsonized cells. The in vivo data demonstrate that
FcRIIA cDNA transfer to hepatocytes enhances the clearance of IgG
opsonized cells by the liver from the circulation.
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MATERIALS AND METHODS |
Adenovirus vectors.
The replication-deficient recombinant Ad vectors AdCMVFcRIIA and
AdCMVNull are both E1a , partial
E1b, partial E3 vectors based on
adenovirus type 5 (Ad5), in which an expression cassette containing a
promoter driving the expression of a recombinant gene is inserted at
the site of the E1 deletion.16,17 AdCMVFcRIIA contains
an expression cassette of the cytomegalovirus (CMV) early/intermediate promoter/enhancer followed by the human FcRIIA cDNA isoform H/R 13118 and a SV 40 stop/poly (A) signal. AdCMVNull is
identical, except that it lacks the gene in the expression
cassette.19 The Ad vectors were propagated, purified, and
stored at  70°C, as previously described.16,17
Titers of viral preparations were determined by plaque assay using 293 cells.20 All preparations were free of replication
competent Ad.21
Primary hepatocyte cultures.
Primary hepatocyte cultures22 were established from 250 to
300 g female Sprague-Dawley rats (Taconic, Germantown, NY). Animals were anesthetized by intramuscular injection of ketamine (60 mg/kg; Fort Dodge Lab, Inc, Fort Dodge, IA) and xylazine (5 mg/kg; Butler Co,
Columbus, OH). The portal vein was cannulated and the liver was
perfused and digested with collagenase solution (GIBCO BRL, Gaithersburg, MD). Hepatocytes were grown in 1:1 mixture of Dulbecco's Modified Eagle Medium (DMEM; Biofluids, Rockville, MD) and Waymouth Medium (Biofluids) containing dexamethasone (20 ng/mL; Sigma, St Louis,
MO), insulin-transferrin-sodium-selenite media supplement (1 µL/mL;
Sigma), and gentamicin (10 µg/mL; Sigma), penicillin G (50 U/mL;
GIBCO BRL), and streptomycin (50 µL/mL; GIBCO BRL) on fibronectin
(Sigma)-coated 6-well tissue culture plates (Becton Dickinson Labware,
Franklin Lakes, NJ).
Expression and function of FcRIIA in primary rat
hepatocytes.
To assess expression of FcRIIA by immunodetection flow cytometry
analysis (EPICS XL; Coulter Corp, Miami, FL), primary rat hepatocyte
cultures were infected with AdCMVFcRIIA at multiplicity of infection
(moi) 10, using noninfected cells and cells infected with
AdCMVNull as controls. The cells were incubated with phosphate-buffered saline solution, pH 7.4 (PBS; Biofluids) containing 2% goat serum on
ice for 30 minutes followed by mouse anti-FcRIIA monoclonal antibody
IV.3 (20 µg/mL; Medarex, Annandale, NJ) for 30 minutes on ice. The
cells were then washed in PBS, incubated with fluorescein isothiocyanate (FITC)-conjugated goat antimouse [F(ab)2]
fragments (Boehringer Mannheim, Indianapolis, IN) for 30 minutes,
washed in PBS, and analyzed by flow cytometry. Isotype-matched mouse monoclonal antibodies (IgG2b; Sigma) were analyzed as negative controls.
The function of the FcRIIA protein expressed by the rat hepatocytes
was assessed by quantifying binding and phagocytosis of Ig-opsonized
51Cr-labeled sheep red blood cells (SRBC)23 by
the hepatocytes. Primary rat hepatocytes (5 × 105/well) were infected with AdCMVFcRIIA (moi 10), using
noninfected cells and cells infected with AdCMVNull as controls. After
48 hours, the hepatocytes were incubated for 60 minutes with IgG rabbit
antisheep red blood cell antibody (Accurate Chemical & Scientific Corp,
Westbury, NY) -coated, 51Cr-labeled (DuPont NEN, Boston,
MA) SRBC (Accurate). Nonopsonized 51Cr-labeled SRBC were
used as controls. To evaluate the binding of IgG-opsonized
51Cr-SRBC to the FcRIIA cDNA-modified hepatocytes, the
hepatocytes were washed 3 times in PBS and then lysed by
incubation in 0.5% sodium dodecyl sulphate (SDS) solution (Sigma) for
10 minutes. To evaluate phagocytosis, hepatocytes were incubated with
SRBC for 60 minutes, washed 3 times in PBS, incubated for
60 seconds in hypotonic SRBC lysing buffer (31 mmol/L ammonia chloride,
2 mmol/L potassium bicarbonate, 20 µmol/L ethylenediaminetetraacetic acid; all from Sigma) to lyse bound noninternalized SRBC, washed, and
then lysed in 0.5% SDS for 10 minutes. To evaluate phagocytosis at
different time points, hepatocytes were incubated with SRBC for 10, 30, and 60 minutes. The hepatocyte to SRBC ratio for all incubations was
1:500. The radioactivity of lysates was measured using a gamma-counter.
Expression and function of FcRIIA in vivo.
To evaluate the expression of FcRIIA cDNA and the function of
FcRIIA protein in vivo, female Sprague-Dawley rats (250 to 300 g)
were anesthetized by intramuscular injection of ketamine (60 mg/kg) and
xylazine (5 mg/kg). Based on the knowledge that greater than 90% of an
intravenously administered Ad goes to the liver,24,25 the
Ad vectors (AdCMVFcRIIA, AdCMVNull) were administered to the liver
via the external jugular vein (109 plaque-forming units
[pfu] in 100 µL 0.9% NaCl). Naive animals were used
as controls.
Northern analysis was used to demonstrate in vivo FcRIIA transcripts
in the liver. After 48 hours, animals were killed (pentobarbital overdose intraperitoneally), total RNA was extracted, and expression of
FcRIIA mRNA was assessed by Northern analysis. Total RNA was extracted (RNA Extraction Kit; Clontech, Palo Alto, CA) and transferred (10 µg/lane) to nylon membranes after electrophoretic separation through a 1% agarose gel. The membranes were assessed using a human
FcRIIA cDNA probe labeled with 32P-deoxycytidine
triphosphate (dCTP; Random Primer Labeling Kit; Stratagene, La Jolla, CA) for 2 hours using standard
methods.26 32P-labeled human -actin cDNA was
used as a positive control.22 To analyze the expression and
distribution of the FcRIIA in the liver, livers were harvested 48 hours after administration of AdFcRIIA, AdNull or from uninfected
animals. The organs were fixed with 4% paraformaldehyde for 24 hours
and then transferred to 70% ethanol before embedding.
Immunohistochemical staining for FcRIIA was performed on 5-µm,
paraffin-embedded sections using the anti-FcRII antibody IV.3. An
isotype (IgG2b) -matched antibody was used as a control. The antibodies
were incubated for 16 hours at 4°C and then washed and incubated
with biotinylated rabbit antimouse [F(ab')2]
(Boehringer Mannheim) for 30 minutes, followed by streptavidin-FITC
(Boehringer Mannheim) for 30 minutes. A rabbit anti-FITC alkaline
phosphatase-labeled antibody (Boehringer Mannheim) was then incubated
for 30 minutes, followed by detection with 4-nitro blue tetrazolium
chloride (NBT)-5-bromo-4-chloro-3-indoyl-phosphate (BCIP)
substrate (Boehringer Mannheim). The samples were
counterstained with nuclear fast red (DIGENE, Beltsville, MD) and
analyzed by light microscopy.
To determine if in vivo transfer of the FcRIIA to the liver was
associated with enhanced clearance of IgG opsonized RBC from the
circulation, rats were anesthetized and Ad vectors were administered as
described above. Forty-eight hours later, 51Cr-labeled rat
red blood cells (RRBC) were opsonized with IgG rabbit anti-RRBC
antibodies (Accurate). The IgG-coated, 51Cr-labeled RRBC
were administered intravenously via the femoral vein (3.4 × 108 RRBC in 500 µL 0.9% NaCl). Nonopsonized
51Cr-labeled RRBC were used as controls. At 5, 15, 30, 60, 90, and 120 minutes after injection of the 51Cr-labeled
RRBC, blood samples were collected (100 µL) from the external jugular
vein and radioactivity of the samples was measured using a gamma
counter. Clearance of RBC from the circulation was expressed as a
percentage of RBC survival in the peripheral blood.27 The
value obtained at 5 minutes after injection was considered 100%.
Animals were killed after 120 minutes by pentobarbital overdose.
To determine the uptake of the IgG-opsonized 51Cr-labeled
RRBC in different organs, rats were infected with Ad vectors, and then
opsonized and nonopsonized RRBC were administered as described above.
Animals were killed 120 minutes after administration of the RRBC.
Liver, lung, and spleen were removed, weighed, and homogenized in 20 mL
of H2O. Total radioactivity of the organ (dpm) was
determined using a gamma-counter and expressed as a percentage of the
total radioactivity injected intravenously.
To further analyze the uptake of IgG coated particles in vivo,
fluorescent-labeled IgG-coated microspheres were injected intravenously in rats that had been infected with AdFcRIIA or AdNull 48 hours previously. The fluorescent microspheres were prepared by mixing Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL) with human IgG (Sigma) for
10 minutes at 4°C, pH 8.0, followed by the addition of glycine (1 mg/mL) to stop the reaction and dialysis in 10 mmol/L HEPES, 140 mmol/L
KCl, pH 7.2, for 2 hours to remove unbound biotin. The biotinylated IgG
was then bound to 40 nm Neutr-Avidin-labeled red fluorescent
microspheres (Molecular Probes, Eugene, OR). The number of microspheres
injected was 6 × 1012 particles/animal. One hour
after the injection of the fluorescent-coated microspheres, the animals
were killed and frozen sections of the livers were prepared by
immediately freezing the tissue in TBS tissue freezing medium (Electron
Microscopy Sciences, Fort Washington, PA). Frozen sections were fixed
in 4% paraformaldehyde for 30 minutes and evaluated by fluorescence microscopy.
Statistical analysis.
The results are expressed as the mean ± standard error of the mean.
Statistical comparisons were made using the unpaired 2-tailed Student's t-test.
| |
RESULTS |
AdFcRIIA transfer to rat hepatocytes in vitro.
To demonstrate the ability of an Ad vector expressing the AdFcRIIA
cDNA (AdFcRIIA) to transfer and express human FcRIIA in vitro in
rat hepatocytes, primary rat hepatocyte cultures were infected with
AdFcRIIA (moi, 10). Quantification of expression of FcRIIA on the
cell surface, determined by flow cytometry, demonstrated positive
FcRIIA staining in 46% of cells after AdFcRIIA infection
(Fig 1). No expression of FcRIIA was
demonstrated in noninfected or AdNull-infected primary rat hepatocytes.
These results suggest that Ad-mediated FcRIIA cDNA transfer results in expression of the transgene in primary rat hepatocyte tissue cultures.
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| Fig 1.
Expression of FcRIIA on the surface of primary rat
hepatocyte cultures after Ad vector-mediated gene transfer of FcRIIA
cDNA. Primary hepatocytes were infected with AdFcRIIA and AdNull at
10 moi. After 48 hours, cells were incubated with anti-FcRIIA
monoclonal antibody for 30 minutes, washed, and labeled with
FITC-conjugated goat antimouse F(ab')2 IgG for 30 minutes, washed, and fixed in 1% paraformaldehyde. Isotype-matched
controls were used for all reactions. Shown is flow cytometry of (A)
naive controls, (B) AdNull-infected cells, and (C) AdFcRIIA-infected
cells.
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Function of FcRIIA transferred in vitro to
rat hepatocytes.
Function of the FcRIIA protein expressed on the primary rat
hepatocytes after in vitro Ad-mediated gene transfer was assessed using
a phagocytic assay using radiolabeled SRBC. Cells infected with
AdFcRIIA (moi, 10) showed increased binding of IgG-coated SRBC
(Fig 2A; P < .001, for 30 and 60 minutes compared with values for noninfected and AdNull-infected cells)
and phagocytosis of IgG-coated SRBC (Fig 2B; P < .006, for 30 and 60 minutes compared with values for noninfected and AdNull-infected
cells). Increased binding and phagocytosis by hepatocytes expressing
FcRIIA was observed after 10 minutes of incubation with opsonized
erythrocytes with significant increase and plateau after 30 and 60 minutes. Noninfected and AdNull-infected rat hepatocytes showed a
minimal amount of nonspecific binding and internalization of both
nonopsonized and IgG-opsonized SRBC. These observations provide
evidence that Ad-mediated transfer of FcRIIA cDNA results in
expression of a functional phagocytic receptor on primary hepatocytes,
enabling these cells to recognize, bind, and internalize opsonized
particles.
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| Fig 2.
Binding and phagocytosis of opsonized SRBC by primary rat
hepatocyte cultures at different time points after Ad-mediated
FcRIIA cDNA gene transfer. Primary hepatocytes were infected with
AdNull and AdFcRIIA at 10 moi. After 48 hours, cells were incubated
with IgG-coated 51Cr-labeled SRBC for 10, 30, and 60 minutes. As a control, the cells were incubated with nonopsonized SRBC
for 60 minutes. To evaluate binding of IgG-coated SRBC to primary
hepatocytes, cells were washed with PBS and then lysed by incubation
with 0.5% SDS for 10 minutes. To evaluate SRBC phagocytosis by the
primary hepatocytes, cells were washed with PBS and the SRBC bound to
the cell surface were lysed by incubation with hypotonic lysis buffer
for 1 minute. The cells were lysed by incubation with 0.5% SDS for 10 minutes and the radioactivity of lysate was quantified. (A) Binding of
opsonized SRBC at 10, 30, and 60 minutes to hepatocytes that were not
infected (control;  ), infected with AdNull ( ), or infected with
AdFcRIIA ( ). Also indicated as controls are parallel cultures of
cells incubated with nonopsonized SRBC for 60 minutes with uninfected
hepatocytes ( ), AdNull-infected hepatocytes ( ), and
AdFcRIIA-infected hepatocytes ( ). (B) Phagocytosis of opsonized
RBC at 10, 30, and 60 minutes. The symbols are identical to that in
(A). For all data, shown are the means of activity (dpm)/well from 3 measurements ± standard error.
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AdFcRIIA transfer to rat hepatocytes in vivo.
To analyze if FcRIIA can be expressed in vivo in rat hepatocytes
after intravenous administration of the AdFcRIIA vector (109 pfu), total RNA was extracted from rat liver and
assessed by Northern analysis. This experiment demonstrated that
intravenous administration of AdFcRIIA resulted in expression of
FcRIIA mRNA transcripts in the liver of the experimental animals. No FcRIIA mRNA transcripts were observed in the liver of noninfected animals or animals infected with AdNull, although control -actin mRNA transcripts were similar in all samples (not shown). Analysis of
liver sections of animals infected with Ad FcRIIA or AdNull by
immunohistochemistry showed positive staining for FcRIIA in liver
parenchymal cells (hepatocytes) of the animals infected with Ad
FcRIIA
(Fig 3C and
E), but not in control naive (Fig 3A and D) or AdNull-infected animals
(Fig 3B and E). These observations confirm that in vivo Ad-mediated
transfer of FcRIIA results in expression of the FcRIIA transgene
in the liver.
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| Fig 3.
Expression of FcRIIA in rat liver in vivo after
Ad-mediated FcRIIA cDNA gene transfer. Sprague Dawley rats received
Ad FcRIIA or AdNull intravenously (109 pfu). After 48 hours, the livers were removed and fixed, and paraffin-embedded
sections were analyzed for FcRIIA expression by immunohistochemistry
using anti-FcRIIA antibody IV.3. (A and D) Naive control; (B and E)
AdNull; (C and F) Ad FcRIIA-infected. (A, B, and C, bar = 50 µm;
E, D, and F [high power representative of A, B, and C], bar = 50 µm.)
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AdFcRIIA induced enhanced clearance of opsonized RBC
in vivo.
The function of FcRIIA expressed after intravenous administration of
AdFcRIIA (109 pfu) was assessed by the clearance of
radiolabeled IgG-opsonized and nonopsonized RRBC from the peripheral
blood of experimental animals. Nonopsonized RRBC were not effectively
cleared in noninfected animals or in AdNull and AdFcRIIA-infected
animals (Fig 4A; P > .2, all
comparisons, all time points). In contrast, IgG-coated (opsonized) RRBC
were rapidly cleared from the blood in the animals receiving
AdFcRIIA. One hour after administration of RRBC in noninfected
animals, 54% ± 3% RRBC remained in the blood compared with 33% ± 4% in AdNull and 9% ± 1% in FcRIIA-infected animals (Fig 4B; P < .001, comparing values for
FcRIIA-infected animals with AdNull and noninfected animals at all
time points after intravenous injection of radiolabeled RRBC).
Interestingly, infection with AdNull also increased clearance of
IgG-coated RRBC compared with noninfected animals, but much less than
that observed with AdFcRIIA (P < .01, all comparisons).
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| Fig 4.
Clearance of 51Cr-labeled SRBC from the
circulation after in vivo Ad-mediated FcRIIA cDNA gene transfer.
Sprague-Dawley rats were administered AdFcRIIA or AdNull
intravenously (109 pfu). After 48 hours,
51Cr-labeled opsonized (IgG-coated) RRBC or nonopsonized
RRBC as controls were injected intravenously. At 5 to 120 minutes,
blood samples were collected and 51Cr-radioactivity
measured using a gamma scintillation counter. The level of
51Cr-RRBC in the blood was expressed as a percentage of the
value obtained at 5 minutes (100%) after injection. (A) Clearance of
nonopsonized RRBC. Control, no vector administered (); AdNull ();
AdFcRIIA (). Each point represents the mean and standard error
from 3 animals in each group. (B) Clearance of opsonized RRBC. The
symbols are identical to those used in (A). Each point represents mean
and standard error from 14 animals in control group, 12 animals in
group infected with AdNull, and 17 animals infected with AdFcRIIA.
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To determine the uptake of RRBC in different organs (liver, lung, and
spleen), animals were killed 120 minutes after administration of
radiolabeled RRBC. No significant difference was observed in uptake of
nonopsonized RRBC in noninfected and AdNull and AdFcRIIA-infected animals (Fig 5A; P > .3, all
comparisons). In contrast, AdFcRIIA-infected animals showed
significant increase in the liver uptake of IgG-coated RRBC (69% ± 6%) compared with AdNull infected (41% ± 4%) and noninfected animals (27% ± 2%; Fig 5B; P < .04, AdFcRIIA-infected animals with AdNull and noninfected animals). An
increased number of IgG-coated fluorescent microspheres was present in
the liver sections of the animals infected with Ad FcRIIA (Fig 6C)
compared with naive controls (Fig 6A) or animals infected
with AdNull (Fig 5B). The presence of fluorescent microspheres in the
control and Ad Null group most likely reflects the uptake by resident
liver macrophages. Taken together, these observations provide evidence
that Ad-mediated transfer of FcRIIA cDNA leads to the expression of
a functional phagocytic receptor in hepatocytes and transforms these
normally nonphagocytic cells into cells that are able to internalize
opsonized particles and enhance their clearance from the peripheral
blood.
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| Fig 5.
Organ sequestration of radiolabeled RRBC after in vivo
Ad-mediated FcRIIA cDNA gene transfer. Sprague-Dawley rats were
administered AdFcRIIA or AdNull intravenously (109 pfu).
After 48 hours, 51Cr-labeled opsonized (IgG-coated) RRBC or
nonopsonized RRBC as controls were injected intravenously. After 120 minutes, the liver, lung, and spleen were removed and homogenized.
Total radioactivity of the organs, measured in a gamma scintillation
counter, is presented as a percentage of the total dose of injected
radioactivity (dpm). (A) Organ sequestration after administration of
nonopsonized RRBC. (B) Organ sequestration after administration of
opsonized RRBC. Shown are mean percentages with standard errors from 4 animals in the control group, 6 animals in the AdNull-infected group,
and 4 animals in the AdFcRIIA-infected group.
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| Fig 6.
Localization of IgG fluorescent microspheres in vivo
after Ad-mediated FcRIIA cDNA gene transfer. Sprague Dawley rats
received Ad FcRIIA or AdNull intravenously (109 pfu).
After 48 hours, IgG fluorescent microspheres (6 × 1012
particles) were injected intravenously, and 1 hour later the livers
were removed and frozen section were prepared and analyzed by
fluorescence microscopy. (A) Naive control; (B) AdNull-infected; and
(C) Ad FcRIIA-infected. Bar = 50 µm.
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DISCUSSION |
This study demonstrates Ad-mediated transfer of FcRIIA receptor cDNA
can convert nonphagocytic cells to phagocytic cells, capable of
accelerating the clearance of IgG opsonized particulates in vivo.
First, in vitro infection of primary rat hepatocytes with AdFcRIIA
resulted in expression of the FcRIIA on the cell surface. Second,
the transfected receptor was functional, enabling normally
nonphagocytic cells to recognize, bind, and phagocytose opsonized RBC.
Third, intravenous administration of AdFcRIIA resulted in expression
of FcRIIA in hepatocytes, resulting in increased clearance of
opsonized RRBC from the circulation of the experimental animals, with
enhanced sequestration of RRBC in the liver.
FcRIIA receptor.
Fc receptors are receptors on the surface of phagocytic cells that
recognize and bind the Fc portion of the IgG molecule.1-4 In humans, there are 3 major Fc receptors: FcRI (CD64), FcRII (CD32), and FcRIII (CD16). They all exhibit a disulfide loop structure in their extracellular domain that is characteristic for all
Ig gene superfamily members. Additional heterogeneity of the Fc
receptors results in the occurrence of different allelic forms.28,29 The FcRIIA allotype is a 40-kD
protein with wide cellular distribution and is present on the surface
of monocytes, macrophages, granulocytes, platelets, Langerhans' cells,
B and T lymphocytes, and some mesangial cells.2 FcRIIA
is the only allotype of the Fc receptor that can directly mediate a
phagocytic signal in the absence of an accessory chain and in the
absence of other Fc receptors.2,3,15 It is the only
human Fc receptor, which recognizes IgG2
efficiently,2,3,15 a component of the host defense system
that plays an important role in defense against infection with
encapsulated bacteria, such as, Streptococcus pneumoniae,
Hemophilus influenzae, and Neisseria
meningitis.30,31
Transfer of FcRIIA to nonphagocytic cells.
Using transfection with recombinant plasmids, various Fc receptors
have been expressed on nonphagocytic cell lines (COS-1, Jurkat T cells,
and fibroblasts).15,18,32 These in vitro experiments resulted in the expression of the functional Fc receptors on the
cell surface assessed by the phagocytosis of opsonized RRBC. Tyrosine
phosphorylation of multiple proteins (eg, FcRIIA, ZAP-70, p72SYK, and phospholipase Cl subunit) and an increase in
intracellular Ca2+ concentration was observed after
cross-linking of FcRIIA with anti-FcRIIA monoclonal
antibody,15 and incubation with inhibitors of tyrosine
kinase reduced phagocytic function of the transfected cells.33
In the present study, expression of FcRIIA was observed in liver
parenchymal cells. Furthermore, increased clearance of IgG opsonized
RBC with increased sequestration of RBC in the liver was observed in
experimental animals infected with AdFcRIIA, when compared with
noninfected animals and animals infected with AdNull. Some degree of
enhancement of the clearance of opsonized RRBC was also observed after
infection with AdNull compared with noninfected animals. This is likely
due to activation of resident phagocytic cells by administration of the
Ad vector per se, because there was no expression of the human
FcRIIA visible in the AdNull- infected animals. Infection with the
Ad vectors (AdFcRIIA, AdNull) did not affect the clearance of
nonopsonized RBC. The increased localization of IgG-coated fluorescent
microspheres after Ad FcRIIA transfer in vivo in the liver provide
additional evidence for increased uptake of IgG opsonized particles by
liver cells of these animals.
Possible clinical significance.
One application of this approach may be in in vivo enhancement of host
defense in clinical situations in which phagocytosis of IgG-targeted
complexes is impaired due to the downregulation of the Fc receptors,
eg, in systemic lupus erythematosus, chronic liver disease, and chronic
kidney disease.5-7 In regard to defects that alter the
binding of IgG-coated targets due to genetic polymorphisms (and
resulting dysfunction of FcRIIA), an arginine (R131)/histidine change at amino acid position 131 in the second Ig-like domain of the
FcRIIA receptor is common.28,29 This mutation is
clinically significant because FcRIIA on leukocytes of individuals
homozygous for R131/R131 (~25% of Caucassians) are unable to
efficiently bind IgG2-opsonized bacteria. These individuals have a
higher incidence of infections with encapsulated bacteria and increased mortality.30,31 The R131/R131 genotype is also a risk
factor for development of lupus nephritis in patients with systemic
lupus erythematosus probably due to its lower affinity for certain
immune complexes.34 Production of reactive oxygen
intermediates by neutrophils in response to antineutrophil cytoplasmic
antibodies is also significantly increased in individuals with
Wegener's granulomatosis who express the R131/R131
genotype.35,36
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ACKNOWLEDGMENT |
The authors thank H. Carpenter and B. Ferris for technical assistance
and N. Mohamed for help preparing this manuscript.
| |
FOOTNOTES |
Submitted December 14, 1998; accepted July 10, 1999.
Supported in part by Grants No. P01 HL51746, P01 HL59312, and AI 22193 from the National Institutes of Health; the Cystic Fibrosis Foundation
(Bethesda, MD); the Will Rogers Memorial Fund (White Plains, NY);
GenVec, Inc (Rockville, MD); and InKine Pharmaceutical Co, Inc (Blue
Bell, PA).
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.
Address reprint requests to Ronald G. Crystal, MD,
Division of Pulmonary and Critical Care Medicine, The New York
Hospital-Cornell Medical Center, 520 E 70th St, ST 505, New York, NY
10021; e-mail: geneticmedicine{at}mail.med.cornell.edu.
| |
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