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Isolation and Characterization of Complement Receptor Type 1 (CR1)
Storage Vesicles From Human Neutrophils Using Antibodies to the
Cytoplasmic Tail of CR1
By Anoopa Kumar, Erica Wetzler, and Melvin Berger
Neutrophil (PMN) activation is associated with increased
surface expression of several membrane proteins that are
translocated from intracellular pools. Indirect evidence suggests that the intracellular storage pools of complement receptor type 1 (CR1) in resting PMN are distinct from traditional granules and may be the secretory vesicles in which
albumin is also stored, but it is not known if this compartment is homogeneous or heterogeneous. To isolate and
characterize the CR1-containing vesicles, we used antibodies against unique sequences in the cytoplasmic tail of CR1.
Affinity-purified IgG was used to adsorb CR1 storage vesicles
from the light membrane fraction (g-band) of nitrogen cavitates of resting PMN. The immunoadsorbent could quantitatively remove the CR1-containing vesicles, whereas control
adsorbents with nonimmune IgG showed no specific binding of CR1. Immunoblots of specifically isolated vesicles also
showed enrichment of albumin, decay-accelerating factor,
FcgRIII, and CR3; whereas HLA class I was not detectable in
these vesicles. Enzyme assay of specifically isolated vesicles
after treatment with Triton X-100 showed that these vesicles
contained most of the cells’ latent alkaline phosphatase. An
additional population of vesicles containing albumin, but not
CR1, and that did not bind to anti-CR1 adsorbent was also
identified. Immunoelectron microscopy showed that the
specifically isolated vesicles had mean diameters of 0.086 to
0.1 mm and stained positive for CR1 and albumin. These
results indicate that CR1 storage vesicles can be isolated
with antibodies against the cytoplasmic tail of CR1 and show
that these vesicles also contain albumin as well as glycosylphosphatidyl inositol-anchored proteins. These results are
most compatible with the hypothesis that CR1-containing
vesicles have arisen by endocytic retrieval of proteins that
had been on the plasma membrane.
q 1997 by The American Society of Hematology.
H
its cytoplasmic tail protruding outwards and available for
binding to immobilized antibody. Magnetic beads were
coated with antibodies generated against synthetic peptides
corresponding to unique sequences in the tail of CR1. These
antibodies bound to native CR1 in detergent lysates of PMN
and were used to isolate the CR1-containing vesicles. This
immunoisolation technique has allowed direct characterization of the specifically isolated vesicles by immunochemical
and enzymatic methods and direct visualization by electron
microscopy. The results of these analyses have important
implications for hypotheses about the formation of this
unique neutrophil secretory compartment and may allow in
vitro studies of the mechanisms of its selective translocation
during PMN activation.
UMAN NEUTROPHILS (PMN) acquire increased
functional capabilities upon exposure to chemoattractants.1,2 This is primarily due to a marked increase in the
plasma membrane expression of several functionally important proteins, which are rapidly translocated from preformed intracellular storage pools during PMN activation.3-10
These intracellular storage compartments represent a readily
mobilizable pool of proteins that are needed early in PMN
activation and that appear on the plasma membrane before
significant degranulation occurs. Proteins that are translocated to the surface with similar rapid kinetics belong to
several different families and have diverse structures, including traditional membrane-spanning proteins as well as phosphatidyl inositol-anchored proteins. In addition, secretion of
soluble proteins also occurs.11-14 The number and identity of
the structures in which these proteins are stored remains
unknown and they have not been completely characterized.
Electron microscopic studies suggest that complement receptor type 1 (CR1; the complement C3b/C4b receptor,
CD35) is stored in resting PMN in intracellular vesicles that
are distinct from primary and specific granules.8 In initial
attempts at subcellular fractionation of resting neutrophils
using sucrose gradients, CR1 was found in fractions separate
from the traditional granules, but these fractions also contained the plasma membranes.15 In more recent studies using
Percoll density gradients and free-flow electrophoresis, CR1
was found in fractions enriched in secretory proteins that
were distinct from the fractions containing markers for the
plasma membrane or traditional granules.11 However, these
putative secretory vesicles have only been partially purified,
and it is thus far still not clear whether all of the different
proteins present in the secretory vesicle fraction are actually
stored in the same structures or whether different types of
proteins are stored in different structures having similar densities. We therefore sought to purify CR1 storage vesicles
by immunoisolation techniques using antibodies against the
cytoplasmic tail of CR1, because the receptor should be
stored with its extracellular domain inside these vesicles and
MATERIALS AND METHODS
Antibodies to cytoplasmic tail of CR1. The predicted amino acid
sequence of CR1 has been previously reported by Klickstein et al.16
The sequence of the cytoplasmic tail [(1495) KHRKGNNAHENPKEVAIHLHSQGGSSVHPRTLQTNEENSRVLP (1537, C-terminal)] contains two stretches of unique sequence (underlined) that
were selected to maximize the specificity for CR1. The first peptide
is a 19-mer corresponding to residues 1500-1518, and the second is
an 11-mer beginning after a region homologous to the epidermal
growth factor receptor phosphorylation site (residue 1527) and ex-
From the Department of Pediatrics, Case Western Reserve University School of Medicine, Cleveland, OH.
Submitted August 2, 1996; accepted January 29, 1997.
Supported by National Institutes of Health Grant No. AI22687.
Address reprint requests to Melvin Berger, MD, PhD, Department
of Pediatrics, Rainbow Babies and Children’s Hospital, 2101 Adelbert Rd, Cleveland, OH 44106.
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.
q 1997 by The American Society of Hematology.
0006-4971/97/8912-0040$3.00/0
Blood, Vol 89, No 12 (June 15), 1997: pp 4555-4565
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KUMAR, WETZLER, AND BERGER
tending to include the C-terminus (residue 1537). The peptides were
custom synthesized by Multiple Peptide Systems (San Diego, CA),
with cysteines added to the C-terminus of the 19-mer and to the Nterminus of the 11-mer to facilitate conjugation. The peptides were
coupled through the terminal cysteine thiol to keyhole limpet hemocyanin (KLH) with the heterobifunctional cross-linking agent maleimidobenzoyl-N-hydroxysuccinimide ester, in a ratio of 1 part peptide to 1 part KLH (wt/wt).
For development of polyclonal antisera, the peptide-KLH conjugates were suspended in Dulbecco’s phosphate-buffered saline (PBS)
at 3.1 mg/mL and emulsified by mixing with an equal volume of
complete Freund’s adjuvant. A total volume of 0.6 mL containing
1 mg conjugate was injected into five to six subcutaneous sites in
the back of NZW rabbits for the initial immunization. Subsequent
boosters consisted of similar doses of conjugate and were administered with complete Freund’s adjuvant on day 21 after the initial
immunization and on days 42 and 63 using incomplete Freund’s
adjuvant. Rabbits were bled on days 52, 73, and 80; and sera were
separated and frozen at 0807C. The antibody titers were determined
against the original immunizing conjugate as well as unconjugated
peptides using an enzyme-linked immunosorbent assay (ELISA).
Affinity purification of peptide-specific antibodies. Rabbit antisera were immunoaffinity purified using immobilized peptide columns. Ten milligrams of purified synthetic peptide was coupled to
2 mL of cyanogen bromide-activated agarose gel (Multiple Peptide
Systems). After deactivation of the remaining activated sites, the
gel was equilibrated in 0.1 mol/L MOPS (3-[N-Morpholino]propanesulfonic acid; Sigma Chemical Co, St Louis, MO) buffer
(pH 7.5) at room temperature and poured into a column. The rabbit
serum was diluted with an equal volume of MOPS buffer and passed
through the column. The column was then washed with an excess
of MOPS buffer and the bound antibody was eluted with a pH 3.0
elution buffer (Bio-Rad Laboratories, Richmond, CA). The eluates
were extensively dialyzed against PBS (pH 7.4) and protein concentrations were measured using the micro BCA method (Pierce, Rockford, IL).
Preparation of PMN lysates. Polymorphonuclear leukocytes
(PMN) were isolated from heparinized peripheral blood drawn from
healthy adult donors using Percoll density gradients as previously
described.4,8 Contaminating erythrocytes were removed by lysis with
hypotonic saline, and PMN were washed in Hanks’ Balanced Salt
Solution without Ca2/ or Mg2/ and held at 07C in this solution until
beginning the lysis or the N2 cavitation process. Cells prepared in
this way are routinely greater than 95% pure by microscopic examination and greater than 95% viable as determined by trypan blue
dye exclusion. Lysates were prepared from 1 1 108 PMN/mL by
suspension in a lysis buffer consisting of PBS (pH 7.2) with 1%
NP-40, 10 mmol/L EDTA, 0.2% sodium dodecyl sulfate (SDS), and
the following protease inhibitors: 10 mmol/L benzamidine, 1 mmol/
L phenylmethylsulfonyl fluoride (PMSF), 100 mmol/L Na-p-tosylL-lysine chloromethyl ketone, 100 mmol/L N-tosyl-L-phenylalanine
chloromethyl ketone, 100 mmol/L leupeptin, and 1 mmol/L pepstatin.11 The mixture was held on ice for 1 hour with occasional
vortexing, the intact cells and cell debris were separated with a
microfuge, and the supernatant was collected and frozen at 0807C
until further analysis.
ELISA to detect binding of antipeptide sera to CR1 in PMN lysates. In initial experiments, the previously described capture
ELISA for the detection of CR1 in PMN lysates11 was modified for
testing the reactivity of antisera raised against synthetic CR1 peptides. Intact CR1 in detergent lysates of PMN was captured on
F(ab*)2 of a mouse monoclonal antibody (MoAb; 3D9) to the extracellular domain of CR1 that was coated onto the plate. After washing,
rabbit antisera to the cytoplasmic tail peptides were added, incubated,
and washed. Binding of rabbit antibody was then detected with
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peroxidase-conjugated goat antirabbit IgG. In later experiments, purified rabbit antipeptide IgG was used as the coating antibody at a
concentration of 10 mg/mL. After the wells were blocked with 5%
nonfat dry milk in PBS-Tween (PBST; 0.5% Tween-20; PBSTmilk), they were incubated with or without PMN lysates and the
captured CR1 was detected with biotinylated MoAb to its extracellular domain, followed by the addition of streptavidin-peroxidase conjugate. After additional washing, the reaction was developed with
o-phenylene diamine substrate and plates were read in an automated
ELISA reader. Normal rabbit IgG was included as a control in all
plates.
Isolation of CR1 storage vesicles from nitrogen cavitates of resting
PMN using antipeptide IgG conjugated to magnetic beads. As a
first approach to immunoisolation of CR1-containing vesicles, we
used a modification of the above-described assay. Instead of PMN
lysates prepared with detergent, nitrogen cavitates, in which the
vesicles should remain intact, were used and Tween-20 was omitted
from all washing buffers. Resting PMN were held at 07C to prevent
the spontaneous upregulation of plasma membrane CR1 expression
that may accompany rewarming of isolated PMN.4 The cells were
suspended in relaxation buffer5 [100 mmol/L KCl, 3 mmol/L NaCl,
1 mmol/L ATP(Na)2 , 3.5 mmol/L MgCl2 , 10 mmol/L PIPES; pH
7.3] containing protease inhibitors as described above and were subjected to nitrogen cavitation at 47C as described by Borregaard et
al.5 Nuclei and unbroken cells were removed from the crude PMN
cavitate by centrifugation at 500g for 10 minutes at 47C. The resulting postnuclear supernatant (PNS) was layered over a 20-mL
Percoll gradient (density Å 1.065 g/mL), with a 1-mL Percoll cushion (1.12 g/mL) at the bottom.11 When the gradient was centrifuged
for 30 minutes at 37,000g, a light membrane fraction containing the
secretory vesicles (g-band) was found as a distinct band in the
upper part of the gradient, whereas the traditional granules formed
a broader band at the bottom of the gradient tube. The g-band and
the material just above the band containing the granules was aspirated. The Percoll from the g-band was pelletted by ultracentrifugation at 180,000g for 1 hour.5 The supernatant was collected and used
for the isolation of vesicles containing CR1.
Magnetic beads with covalently bound sheep antirabbit IgG (Dynabeads M-280; Dynal, Inc, Lake Success, NY) were saturated with
purified immune or control rabbit IgG at 5 to 10 mg/mg beads at
47C overnight and then washed with PBS as described earlier.17,18
The beads were then incubated with PBS containing 5% nonfat dry
milk and 1% bovine serum albumin (BSA) for 1 hour at 47C and
washed with PBS. Based on results in ELISA assays and other
systems, the following steps were used to prevent nonspecific binding. The g-band prepared as above was first precleared two times
in wells of Immulon IV microtiter plates (Dynatech Laboratories,
Chantilly, VA) coated with 10 mg/mL of nonimmune rabbit IgG,
each time by incubating for 45 minutes at 47C. The g-band from
these wells was then further precleared by incubation with magnetic
beads saturated with nonimmune rabbit IgG for 45 minutes at 47C.
The beads were removed from the g-band by applying a magnet to
the wall of the test tube for about 5 minutes at 47C. This precleared
g-band was then incubated with beads saturated with purified immune or control nonimmune rabbit IgG overnight at 47C. After
washing with the relaxation buffer containing the protease inhibitors
described above, beads were either directly processed for electron
microscopic studies or were resuspended in PBS with 1% Triton X100 or hot 2% SDS-polyacrylamide gel electrophoresis (SDSPAGE) sample buffer,19 both containing protease inhibitors, for analysis by enzyme assay or SDS-PAGE and Western blots, respectively.
To analyze the different proteins present in the bound vesicles
using both immunoblots and ELISAs, in preliminary experiments
we studied the use of SDS sample buffer versus PBS-Triton X-100
for solubilizing the proteins. The results from these experiments
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CR1 STORAGE VESICLES FROM PMN
4557
indicated that, whereas SDS in the sample buffer readily solubilized
CR1, an integral membrane protein found in the membranes of the
vesicles, this concentration of SDS interfered with subsequent
ELISA assays. On the other hand, Triton X-100 failed to release all
of the CR1 present in the vesicles bound to the adsorbent. However,
the soluble protein albumin was readily released from these vesicles
by Triton X-100 and could then be quantitated by ELISA. This
detergent was also satisfactory for assay of latent alkaline phosphatase, which is not accessible in the absence of detergent.14 Therefore,
we used aliquots solubilized with SDS sample buffer for immunoblot
analysis of CR1, albumin, FcgRIII, decay-accelerating factor (DAF),
a-chain of CR3, and HLA class I; whereas we used samples solubilized in PBS–Triton X-100 for analysis of the total contents of
albumin by ELISA and alkaline phosphatase by enzyme assay.
Immunoblotting. Protein samples were run on either 7% or 10%
SDS-PAGE under nonreducing conditions in a Mini Protean II Dual
Slab Cell (Bio-Rad). In 7% gels, the following prestained molecular
weight (MW) markers (Bio-Rad) were used: myosin (205 kD), bgalactosidase (117 kD), BSA (89 kD), and ovalbumin (47 kD). In
10% gels, phosphorylase B (101 kD), BSA (83 kD), ovalbumin (50.6
kD), carbonic anhydrase (35 kD), soybean trypsin inhibitor (29.1
kD), and lysozyme (20.9 kD) were used. Separated proteins on SDS
gels from the vesicle isolations were then transferred onto nitrocellulose membranes in 192 mmol/L glycine, 25 mmol/L Tris (pH 8.3),
and 20% (vol/vol) methanol, using a Mini transblot electrophoretic
transfer cell (Bio-Rad). The membrane was blocked with PBSTmilk for 1 hour at room temperature and then reacted with MoAb
against CR1, human albumin (Accurate Chemical and Scientific
Corp, Westbury, NY), DAF (CD55; Wako Chemicals USA, Inc,
Richmond, VA), FcgRIII (CD16b; clone 3G8), HLA class I (clone
W6/32), or a-chain of CR3 (CD11b, Dako-C3bi-R; Dako A/S,
Glostrup, Denmark) at 1 mg/mL in PBST-milk and incubated for 2
hours at room temperature. This was followed by incubation with
1:1,000 dilution of F(ab*)2 of goat antimouse IgG conjugated to
alkaline phosphatase (Organon Teknika Corp, West Chester, PA)
for 1 hour and then by three additional washes with PBST. The
reaction was developed with 5-bromo-4-chloro-3-indolylphosphatetoluidine and nitroblue tetrazolium chloride (Sigma Chemical Co)
and was stopped by washing extensively with distilled water.
ELISA to detect human albumin. Albumin was determined in
1% Triton X-100 extracts of isolated vesicles using an ELISA assay.
To determine whether albumin was free and could be detected in
the absence of detergent, isolated vesicles and unbound fractions
were also resuspended in PBS without Triton X-100 but containing
protease inhibitors. All washes in these steps were made with PBS
containing protease inhibitors but without any detergent to prevent
the premature lysis of intact vesicles. An IgG fraction from rabbit
polyclonal antihuman albumin (Boehringer Mannheim Biochemicals, Indianapolis, IN) was used as the capture antibody at 1:5,000
dilution. The unreacted sites in the ELISA plate wells were blocked
with PBST buffer containing 5% nonfat dry milk and 1% BSA.
After incubation with the samples and washes as before, the plates
were incubated with 1:1,000 dilution of mouse antihuman albumin
MoAb (Accurate Chemical and Scientific Corp) and washed. The
binding of this antibody was detected with peroxidase-conjugated
goat antimouse IgG (Sigma Chemical Co). After additional washing,
the reaction was developed and read as described above.
Assay for alkaline phosphatase. Latent alkaline phosphatase activity was measured in samples of isolated vesicles after they were
permeabilized with PBS containing Triton X-100 as described by
Borregaard et al.14 The samples were diluted in sodium-barbital
buffer, pH 10.5,20 and were incubated with PNP substrate (1 mg/
mL; Sigma Chemical Co) for 90 minutes at 377C. At the end of the
incubation the reaction was stopped with ice-cold sodium barbital
buffer and the optical density was immediately measured at 405 nm
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using a Thermomax reader (Molecular Devices, Sunnyvale, CA).
Human placental alkaline phosphatase (P-3895; Sigma Chemical
Co) was used as a standard to assure linearity of the assays in the
microplate reader. Enzyme activity in isolated fractions was expressed as conventional units: 1 mmol/L substrate converted per
minute using 18.6 mmol/L01 cm01 as the extinction coefficient for
p-nitrophenol.21 Alkaline phosphatase activity was also determined
in vesicles resuspended in PBS without Triton X-100 but containing
protease inhibitors. As with assays for albumin release, all washes in
these assays were made with PBS containing the protease inhibitors
described above, but without any detergent to prevent the premature
lysis of intact vesicles.
Immunoelectron microscopy. For localization of CR1 or albumin, vesicles isolated on magnetic beads were fixed by the addition
of 4% paraformaldehyde in 0.1 mol/L phosphate buffer, pH 7.4, for
30 minutes at room temperature followed by 8% paraformaldehyde
also for 30 minutes at room temperature. Beads were embedded in
10% gelatin, which was then infused overnight at 47C with polyvinyl
pyrrolidone-sucrose.22 The blocks were then mounted on specimen
holders and stored in liquid N2 . Ultrathin cryosections were cut on
a Reichert Ultracut FC E (Reichert Optische Werke AG, Vienna,
Austria) at 01307C, placed on Formvar-coated copper grids (Electron Microscopy Sciences, Fort Washington, PA), and then immunostained as previously described.22 Briefly, sections were blocked using 1% ovalbumin in Tris-buffered saline (TBS; pH 7.6), containing
1004 mol/L PMSF for 60 minutes. Sections were stained with antiCR1 MoAbs 3D9 and C543 or anti-albumin MoAb (Accurate) or
with nonimmune mouse IgG1 (MOPC 21; Sigma Chemical Co) in
TBS containing 1% ovalbumin, 0.2% cold water fish gelatin (Sigma
Chemical Co), and 1004 mol/L PMSF for 90 minutes and were
washed four times in TBS-PMSF. Sections were then stained with
5-nm gold-conjugated goat antimouse IgG for 90 minutes, washed
again with TBS-PMSF, and washed four additional times with distilled water. The sections were postembedded in a 9:1 mixture of
methylcellulose (Sigma Chemical Co) and 3% uranyl acetate and
were examined with a JEOL 100 CX transmission electron microscope (JEOL Ltd, Tokyo, Japan).
RESULTS
Reactivity of antipeptide antibodies with intact CR1. To
specifically isolate the vesicles in which CR1 is stored in
resting PMN, we desired to use antibodies against the cytoplasmic tail of CR1, which should be exposed on the outside
of these vesicles. We therefore developed antibodies against
KLH conjugates of synthetic peptides corresponding to
unique sequences within the tail of CR1 and confirmed their
reactivity with these peptides. Because reactivity of antisera
with synthetic peptides does not necessarily indicate that
these antibodies do in fact recognize their determinants in
the native molecule, we tested these antibodies against CR1
in a capture ELISA system. The results indicated that the
antisera to both peptides reacted with native CR1 in detergent
lysates of PMN (Fig 1). We then affinity-purified the specific
IgG from the antisera using peptides bound to agarose. We
used a sandwich ELISA in which the purified IgG preparations were applied to ELISA plates to test their ability to
capture intact CR1 from PMN lysates. Captured CR1 was
then detected with biotinylated MoAb to its extracellular
domain and peroxidase-streptavidin conjugates. The purified
IgG preparations against both peptides clearly bound CR1.
In contrast, purified normal rabbit IgG showed no reactivity
with CR1. This confirmed that the antipeptide rabbit antibod-
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4558
KUMAR, WETZLER, AND BERGER
Fig 1. Reactivity of rabbit antisera against the 19-mer and 11-mer
peptides of cytoplasmic tail of CR1. Rabbit sera were tested at a
dilution of 1:100 in an ELISA system in which native CR1 was captured from detergent neutrophil extracts (NE) onto wells precoated
with F(ab*)2 of mouse MoAb to the extracellular domain of CR1. Binding of rabbit IgG to the captured CR1 was detected with peroxidaseconjugated goat antirabbit antibody and o-phenylene diamine. The
sera were tested in parallel in wells that received neutrophil extract
containing CR1 ("NE) but nonimmune rabbit IgG and in wells that
did not receive CR1 (ÏNE). The reactivity in wells with immune sera
that had not received NE was equal to or less than the values obtained for nonimmune sera with NE. The results represent the mean
of duplicate determinations from two separate experiments.
ies bound specifically to the cytoplasmic tail of native CR1.
We then attempted to use these antibodies to isolate CR1
storage vesicles directly on magnetic beads.
Isolation of CR1 storage vesicles using magnetic beads
conjugated with antipeptide antibodies. For isolation of intact CR1 storage vesicles, we initially used the whole PNS
from the N2 cavitate in preliminary experiments. Because the
results from these experiments showed excessive nonspecific
binding, we further fractionated the PNS using Percoll density gradients.11 We next prepared the light membrane fraction or g-band11 from the PNS of nitrogen cavitates of resting
PMN. Tween and other detergents were omitted from all
washing buffers so that the vesicles would remain intact and
could be isolated without lysis. To preserve the integrity of
CR1, six different protease inhibitors were incorporated in
solutions used in every step of the process. To minimize
nonspecific binding, unreacted sites on the beads were
blocked with 5% nonfat dry milk and 1% BSA and preclearing steps in which the g-band was incubated in ELISA wells
coated with normal rabbit IgG and then on magnetic beads
conjugated with normal rabbit IgG were included (see the
Materials and Methods for details). The amount of CR1
present in PNS was 37.0 { 10.08 ng/mL (mean { SEM,
n Å 3) as determined by the indirect ELISA using soluble
CR1 (T-cell Sciences, Cambridge, MA) as the standard. The
amount of CR1 present in g-band from the Percoll gradients
was more than 85% of that found in PNS, in agreement with
previous results,11 and during the preclearing steps there was
a less than 5% loss of CR1.
Affinity-purified antibodies against the 11 and 19 residue
peptides were combined and bound to sheep antirabbit IgG
that was covalently attached to magnetic beads, which then
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served as an adsorbent to capture CR1 vesicles from the
precleared g-band. The material unbound to the beads after
the overnight incubation was collected to determine the efficiency of capturing the CR1-containing vesicles present in
the g-band. After washing the beads with relaxation buffer
containing protease inhibitors, the vesicles bound to the
beads were either lysed by the addition of hot SDS-PAGE
sample buffer for analysis on SDS-PAGE and Western blots
or were resuspended in PBS containing Triton X-100 for
analysis by ELISA.
Western blots for CR1 and other proteins. The proteins
present in the SDS eluates of the beads were separated on
gels that were subsequently transferred onto nitrocellulose
membranes and were used to determine the presence of CR1
and other proteins of specific interest in the isolated vesicular
structures. When probed with MoAb 3D9 against the extracellular domain of CR1, these blots showed enrichment of
the Ç205-kD band corresponding to CR1 in the specifically
isolated vesicles (Fig 2). We used two different amounts of
beads to optimize recovery of CR1 vesicles while minimizing nonspecific binding. As shown in Fig 2, although a large
proportion of the CR1 was bound to 25 mL of beads, a
considerable amount of the CR1 remained unbound. When
100 mL of beads were used, the CR1 was totally adsorbed
from the g-band. In contrast, neither 25 nor 100 mL of control
beads bearing nonimmune rabbit IgG showed any appreciable binding of CR1. These results indicate that the immunoadsorbent with antibodies against the cytoplasmic tail
could quantitatively capture CR1-containing vesicles present
in the g-band.
Identical blots probed with antialbumin MoAb showed a
distinct band at 67 kD (Fig 2). However, the results of the
blots in material unbound to 100 mL of immune beads suggest that an equal amount of albumin was still present in the
unbound material, whereas the CR1 had been completely
removed from this fraction. The proportion of albumin bound
to both 25 and 100 mL of anti-CR1 (immune) beads is much
greater than the very small amounts bound to equivalent
aliquots of nonimmune beads. Taken together, these results
suggest that the CR1 vesicles contain albumin, but that there
is also albumin in structures not bearing CR1. This conclusion was studied in more detail using ELISAs, as described
below.
Additional blots that were run in parallel were reacted
with antibodies against FcgRIII, DAF, CD11b (the a-chain
of CR3), and HLA class I (Fig 2). A comparison of the
amount of each protein present in PNS in relation to g-band
indicates that most of the FcgRIII and HLA class I present
in the whole PNS seems to be present in the g-band, whereas
only some of the CR3 and DAF are found in the g-band.
Previous studies have shown that a substantial amount of
the FcgRIII in PMN is present in intracellular vesicles that
morphologically resemble those in which CR1 is stored.23
In density gradients of cavitates of resting neutrophils, most
of the CR3 localizes in fractions containing specific granules
and gelatinase granules, whereas only a small fraction of the
CR3 sediments with the light membranes11 and/or the plasma
membrane-enriched fractions.24-26 Thus, the amount of CR3
in the g-band that we used for isolation of these vesicles is
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CR1 STORAGE VESICLES FROM PMN
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Fig 2. Western blot of PNS, gband, and fractions from vesicle
isolation experiments using immune or nonimmune beads. For
the detection of CR1 (MW !
Ò205 kD) and a-chain of CR3
(MW ! 165 kD), samples were
run on a 7% SDS-PAGE and
transferred onto nitrocellulose
filters. CR1 and CR3 in the figure
are shown on portion of blot
above 117 kD, as determined by
the marker b-galactosidase. For
the detection of FcgRIII (MW !
50 to 70 kD), albumin (HSA; MW
! 67 kD), DAF (MW ! 70 kD), and
HLA class I (MW ! 46 kD), samples were run on a 10% SDSPAGE and transferred onto nitrocelluose filters (refer to the
Materials and Methods for details). FcgRIII, albumin, and DAF
in the figure represent the portion of the blot between 101 kD
and 35 kD, as determined by the
markers phosphorylase B and
carbonic anhydrase. HLA class I
in the figure represents the portion of the blot between 50 kD
and 35 kD, as determined by the
markers ovalbumin and carbonic
anhydrase. The volume of each
sample was adjusted to equal
the volume of g-band applied to
allow direct comparisons.
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KUMAR, WETZLER, AND BERGER
only a small fraction of the cells’ total content of this protein.
Apart from kinetic studies of its mobilization,7 there are few
data regarding the localization of DAF in PMN. The results
of the blots for FcgRIII, a-chain of CR3, and DAF in material bound to 100 mL immune beads show that, of the amount
present in the g-band, most of these proteins are contained
in structures adsorbed by the anti-CR1 bearing beads. In
contrast, control beads bearing nonimmune rabbit IgG
showed very little or no binding of these proteins. These
results indicate that the efficiency of isolation of these proteins was similar to that for CR1, suggesting that they are
packaged together. Subcellular fractionation studies by Bjerrum and Borregaard27 have shown that a majority of HLA
class I is localized on the plasma membrane and therefore
it has been used as a plasma membrane marker in human
neutrophils. The results of the blots prepared from the vesicles isolated by anti-CR1 bearing beads show that HLA class
I is not present in these vesicles (Fig 2). This shows that the
binding of vesicles to beads bearing antibodies to the tail of
CR1 is specific and that there is no contamination with
plasma membrane fragments.
ELISA for albumin and assay for alkaline phosphatase.
To better quantitate the albumin in the immunoisolated vesicles, they were permeabilized with PBS containing 1% Triton X-100 and their content of human albumin was quantitated by ELISA. A comparison of the material bound and
Fig 3. Fractionation of human albumin during adsorption of CR1containing vesicles. Bound vesicles on beads and unbound supernatants were resuspended in PBS containing 1% Triton X-100 and ELISA
was performed as described in the Materials and Methods. ( ) The
material bound to either 25 mL or 100 mL beads bearing anti-CR1
peptide IgG (Immune) or to 25 mL or 100 mL beads bearing nonimmune rabbit IgG (Non-Imm). g-Band and material unbound (j) to
beads were also included to compare the efficiency of isolation. The
volume of each sample was adjusted back to the same volume as
the initially applied g-band to allow direct comparison. The results
shown are the mean Ô SEM of duplicate determinations from each
of three separate experiments.
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Table 1. Recovery of Freely Accessible and Vesicle-Enclosed
(Latent) Albumin From 100 mL of Anti-CR1 Beads
Nanograms applied
Total amount Å 104.5 { 26.4 ng
(75.7% latent)
Nanograms recovered
Unbound
47.9 { 15.9 ng
(59.5% latent)
Bound
62.9 { 12.5 ng
(96.5% latent)
Release of albumin from g-band and fractions bound and unbound
to anti-CR1 beads by treatment with PBS with or without Triton X100. Bound vesicles, g-band, or unbound supernatants were resuspended in PBS without detergent or PBS containing 1% Triton X-100,
and ELISA was performed as described in the Materials and Methods.
Results for recovery represent the total albumin released in the presence of Triton X-100 in all cases. Results for the proportion of albumin
in each fraction that was vesicle-enclosed or latent (ie, accessible
only in the presence of Triton X-100) are shown in parentheses and
were calculated using the equation:
% Latent Å
Total (/ Triton) 0 Freely Accessible (0 Triton)
1 100.
Total (/ Triton)
The results are the mean { SEM of duplicate determinations from
each of three separate experiments similar to those in Fig 3.
unbound to 100 mL of immune beads indicates that about
45% of the applied albumin did not bind to anti-CR1 bearing
beads (Fig 3, middle set of bars). These results are comparable to those obtained by immunoblotting (Fig 2), which
showed that there was a considerable amount of albumin in
the unbound fraction, even though there was no CR1 detectable in this fraction of material not bound to 100 mL of
immune beads. There was minimal binding of albumin to
beads bearing nonimmune IgG (Fig 3, right most set of bars).
These results imply that, although the CR1 vesicles contain
albumin, there is also a substantial fraction of albumin present in a pool distinct from CR1-containing vesicles.
To determine whether the albumin in the various fractions
was free or contained within vesicles, release of albumin
from the isolated CR1 vesicles and other fractions was tested
in the absence and presence of Triton X-100 (Table 1). Of
the total amount of albumin detected in the g-band in the
presence of Triton, only Ç25% was freely accessible in the
absence of Triton, thus indicating that Ç75% was contained
within intact vesicles (latent). The results also show that only
about 3.5% of the albumin present in the vesicles bound to
anti-CR1 bearing beads was detectable in the absence of
Triton X-100, indicating that 96.5% was latent. In the fraction that was unbound to anti-CR1 bearing beads, about 40%
of the albumin was freely accessible, whereas approximately
60% was detectable only in the presence of Triton X-100.
Nonimmune beads showed very little binding of albumin
(11.1 { 9.4 ng; °10% of total applied).
Thus, in the fraction unbound to 100 mL of immune beads,
a significant amount of the albumin is still stored inside
vesicles, because 60% of it can only be detected in the presence of detergent. However, because the immunoblot for
CR1 (Fig 2) shows that this fraction contains little or no
CR1, this must be a subset of vesicles that do not contain
CR1. This suggests that there is heterogeneity in the secre-
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CR1 STORAGE VESICLES FROM PMN
4561
tory vesicle compartment. Because the majority of the albumin contained in the unbound fractions as well as in the
bound fractions is only accessible in the presence of the
detergent and hence is contained in intact vesicles, there
seems to have been relatively little damage to or breakage of
vesicles during the isolation procedures. These observations
strongly suggest that the vesicles adsorbed to the beads are
intact and that the isolation technique we have used is specific. Hence, the release of bound albumin might be adaptable as a useful tool for vesicle-membrane fusion studies.
The total recovery of alkaline phosphatase activity in the
vesicles was determined in samples that were permeabilized
with PBS containing Triton X-100, as described earlier by
Borregaard et al.14 The results in Fig 4 show that alkaline
phosphatase is specifically enriched in CR1 containing vesicles because the amount of enzyme bound to 100 mL of
immune beads (Fig 4, stippled bar, middle pair) is much
greater than the minimal amount bound to nonimmune beads
(Fig 4, stippled bar, right most pair). It should be noted that
the latter did not contain any CR1 (Fig 2). Furthermore, Fig
4 also shows that about °40% of the alkaline phosphatase
applied to 100 mL of immune beads failed to bind (solid bar,
middle pair). This contrasts with Fig 2, which showed that
this fraction did not contain any CR1.
Determination of whether the alkaline phosphatase in the
various fractions was freely accessible or enclosed in vesicles (latent) was determined by comparing samples not con-
Fig 4. Fractionation of alkaline phosphatase during adsoption of
CR1-containing vesicles. Bound vesicles and unbound supernatants
were resuspended in PBS containing 1% Triton X-100 and assayed
for alkaline phosphatase using PNP substrate in sodium-barbital
buffer, pH 10.5, as described in the Materials and Methods. ( ) The
material bound to either 25 mL or 100 mL beads conjugated to antiCR1 peptide IgG (Immune) or to 25 mL or 100 mL beads conjugated
to nonimmune rabbit IgG (Non-Imm). (j) g-Band and unbound material. All volumes were adjusted to equal the volume of the g-band
originally applied to allow direct comparisons. The results shown are
the mean Ô SEM of duplicate determinations from each of three
separate experiments.
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Table 2. Recovery of Freely Accessible and Latent Alkaline
Phosphatase From 100 mL of Anti-CR1 Beads
Milliunits applied
Milliunits recovered
Total activity Å 1470 { 60 mU
(61.9% latent)
Unbound
515 { 25 mU
(21.4% latent)
Bound
720 { 135 mU
(88.2% latent)
Accessibility of alkaline phosphatase in g-band and fractions bound
and unbound to anti-CR1 beads in the presence and absence of Triton
X-100. Bound vesicles, g-band, or unbound supernatants were resuspended in PBS without detergent or PBS containing 1% Triton X-100,
and alkaline phosphatase activity was assayed as described in the
Materials and Methods. Results for total activity recovered represent
the alkaline phosphatase activity detected in the presence of Triton
X-100. Results for the proportion of alkaline phosphatase that was
latent (ie, accessible only in the presence of Triton X-100) were calculated using the equation:
% Latent Å
Total (/ Triton) 0 Freely Accessible (0 Triton)
1 100.
Total (/ Triton)
The results are the mean { SEM of duplicate determinations from
each of three separate experiments similar to those in Fig 4.
taining detergent with samples permeabilized with PBS containing Triton X-100, respectively.14 The results in Table 2
show that more than 60% of the alkaline phosphatase present
in the g-band applied to the immunoadsorbent was latent
(detectable only in the presence of Triton X-100), whereas
less than 40% was readily accessible (nonlatent). In the vesicles that were bound to immune beads, more than 88% of
the detectable alkaline phosphatase activity was latent, thus
suggesting that latent alkaline phosphatase is stored inside
CR1 containing vesicles. In the fraction that was unbound
to immune beads, about 21% of the alkaline phosphatase
activity was latent, whereas 79% was freely accessible.
These results suggest that most of the alkaline phosphatase
detected in the unbound fraction represents fragments of or
vesicles derived from the plasma membrane, in which the
active site of the enzyme is exposed externally. Because only
21% of the alkaline phosphatase activity in the fraction not
bound to immune beads was latent and this fraction contained Ç40% of the total alkaline phosphatase recovered,
we conclude that less than 10% of the latent alkaline phosphatase in the cell is present in vesicles that do not contain
CR1.
Nonimmune beads bound very little of either latent or
freely accessible alkaline phosphatase activity. Of the small
amount of alkaline phosphatase bound to the nonimmune
beads, there was no enrichment of vesicle-enclosed enzyme,
because the proportion that was latent (Ç50%, not shown)
was no higher than that in the material applied (Table 2,
total activity).
The lack of binding of HLA class I to anti-CR1 beads as
indicated in Fig 2, the very small amount of freely accessible
alkaline phosphatase bound to the anti-CR1 bearing beads
(Fig 4 and Table 2), and the minimal nonspecific binding of
latent alkaline phosphatase to nonimmune beads all suggest
that there is very little nonspecific binding of intracellular
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4562
KUMAR, WETZLER, AND BERGER
Fig 5. Immunoelectron microscopy of vesicles isolated on
magnetic beads coated with antibodies to CR1 peptides. (A)
Vesicles stained with a mixture
of MoAbs 3D9 and C543 to the
extracellular domains of CR1. (B)
Vesicles stained with antihuman
albumin MoAb. (C) Vesicles
stained with control nonimmune
antibody MOPC 21. Arrows
show vesicles specifically bound
to beads bearing antibodies to
CR1 tail peptides. (A) and (B)
show staining with 5-nm goldconjugated goat antimouse IgG.
(C) fails to show staining with
the same gold-conjugated goat
antimouse IgG. Bars ! 0.1 mm.
or plasma membrane vesicles onto the anti-CR1 beads and,
thus, that the technique we have adopted for the isolation of
CR1 vesicles is very specific.
Electron microscopy for CR1 and albumin. An advantage of the use of magnetic beads as the immunoadsorbent
is that these particles can be processed directly for EM studies,18,28 thus obviating any losses or alterations in the vesicles
that might occur in trying to elute them from other adsorbents. Cryosections of vesicles bound to magnetic beads
bearing antibodies against the CR1 tail peptides were probed
with a mixture of MoAbs 3D9 and C543 against the extracellular domain of CR1, and the latter antibodies were identified
with gold-conjugated goat antimouse IgG. The vesicles isolated on magnetic beads conjugated with antipeptide rabbit
IgG stained positively for CR1 (Fig 5A). These structures
appear as small vesicles with irregular borders with mean
diameters of 0.086 to 0.1 mm, consistent with the size and
shape of the CR1-containing structures in sections of intact
resting cells.8 The somewhat variable appearance of the vesicles bound to the beads is compatible with results in other
systems in which immunomagnetic beads have been used to
isolate small secretory or synaptic vesicles.29 These vesicles
also stained positively with MoAb against albumin (Fig 5B).
In contrast, cryosections of vesicles probed with nonimmune
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mouse IgG (MOPC 21) did not bind any gold particles (Fig
5C). Cryosections from beads conjugated with nonimmune
IgG did not show any bound vesicles, and none of the beads
showed any typical appearing primary or secondary granules.
DISCUSSION
In this study we used antibodies against synthetic peptides
from the cytoplasmic tail of CR1 to isolate CR1-containing
storage vesicles from resting PMN. Previous studies from
our laboratory showed that the sites of intracellular storage
of CR1 in resting neutrophils are structurally unique and
morphologically distinguishable from traditional neutrophil
granules.8 More recent density gradient and free-flow electrophoresis studies have suggested that CR1 may be stored
in secretory vesicles that also contain albumin and latent
alkaline phosphatase.11 Several lines of indirect evidence,
including kinetic and pharmacological studies, suggest that
DAF may also be stored in these vesicles, because the kinetics of upregulation of DAF and CR1 expression in response
to different stimuli are similar and also resemble those for
translocation of alkaline phosphatase to the plasma membrane and for secretion of albumin.7,11,12,14 CR1, alkaline
phosphatase, and some but not all of the cell’s CR3 are
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CR1 STORAGE VESICLES FROM PMN
4563
considered readily mobilizable because they can clearly be
translocated to the PMN surface without exocytosis of traditional granule constituents such as myeloperoxidase, lactoferrin, or B12 binding protein.11-14,30 Also, immunochemistry
and double-labeling immunoelectron microscopy studies
with antibodies against cytochrome b558 and CR3 has shown
that these proteins colocalized with albumin in the same
vesicles.24 However, the movement of an additional portion
of the CR3 can be differentiated from that of CR1 pharmacologically and kinetically11,31 and its storage pool is physically
distinct.11,15 It thus seems clear that CR1 is stored separately
from the traditional granules and most of the CR3. However,
despite these results, there has been no direct evidence on
whether the different readily mobilizable proteins (CR1, alkaline phosphatase, DAF, and albumin) are stored together
in a single type of secretory vesicle or whether they are
stored in distinct structures that share some physical properties.
Earlier studies attempting to identify the distinct intracellular pools of CR1 and CR3 by density gradient centrifugation failed to resolve the CR1 storage compartment from
the plasma membrane-containing fractions.15 Although highvoltage free-flow electrophoresis did result in separation of
secretory vesicles from neutrophil plasma membrane fragments,11 it was not clear that this technique resulted in the
isolation of a single population of biochemically homogeneous vesicles.
To approach this problem of heterogeneity of vesicular
structures in PMN, we used a specific immunoaffinity technique. Similar immunoisolation techniques using antibodies
to exposed cytoplasmic tails of other receptors and transporters have been successfully used in the isolation and characterization of a variety of vesicular structures from other types
of cells.29,32-39 The specific enrichment we achieved using
antibodies to the cytoplasmic tail of CR1 is indicated in the
Western blot of vesicles isolated using immune rabbit IgG
as opposed to normal IgG (Fig 2). The lack of binding of
CR1-containing vesicles to beads bearing nonimmune IgG
and the lack of binding of the plasma membrane marker HLA
class I to the beads bearing anti-CR1 show the specificity of
this immuno-affinity technique. Electron micrographs of the
isolated CR1 containing structures bound to their adsorbent
showed somewhat irregularly shaped vesicles that correspond in size to the CR1-containing vesicles demonstrated
earlier in fixed, intact PMN.8 These vesicles stained positively with MoAb to the extracellular domain of CR1 as well
as with MoAb to albumin.
Normal human neutrophils have been shown to contain
intracellular albumin in small, membrane-limited structures
that have been referred to as secretory vesicles because albumin is readily secreted after stimulation by chemoattractants.11,13 Previous studies from our laboratory have shown
that, in fMLP-stimulated PMN, albumin was endocytosed
into multivesicular bodies and that the membranes of these
structures contained CR1 that originated from the plasma
membrane and that was initially reinternalized in small endocytic vesicles along with the endocytosed albumin tracer.40
Our present study provides positive confirmation that CR1containing vesicles in resting cells also contain albumin by
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both the immunoblotting studies as well as the immunoelectron microscopy of specifically isolated vesicles. Because
earlier observations indicate that PMN do not synthesize
albumin,13 its presence in CR1 vesicles is consistent with
previous results, suggesting it gains access to these vesicles
as they form during the endocytic process.13,40 The presence
of endocytosed plasma proteins in the CR1 storage vesicles
suggests that they may be analogous to the a-granules in
which endocytosed plasma proteins are stored in platelets.41
It has been previously shown that secretory vesicles can
be separated from plasma membrane vesicles by using freeflow electrophoresis and that their markers appear on the
plasma membrane after mild PMN stimulation.13,42 Our results show that the CR1-containing vesicles also contain the
GPI-linked proteins DAF, FcgRIII, and alkaline phosphatase
in addition to some CR3 and about half of the albumin,
which support earlier kinetic studies and other indirect observations.7,9,11,23-26,31 Previous electron microscopic observations have indicated that some FcgRIII in human neutrophils
is present in small vesicles23 and that stimulation by chemoattractants causes a rapid translocation of intracellular
FcgRIII to the PMN surface.9 Those results are thus also
consistent with our current studies, which suggest that a
major fraction of the intracellular FcgRIII is stored in the
CR1 vesicles. Our observations that the GPI-linked proteins
DAF, alkaline phosphatase, and FcgRIII are stored in the
CR1 vesicles suggest that the cytoplasmic tail of CR1, or
the lack of any cytoplasmic tail at all by the GPI-linked
proteins, may not be the major determinant of the intracellular packaging of these proteins. However, it is not known
how these structurally different proteins are sorted into the
same storage compartment. We speculate that mixing of proteins initially packaged in different compartments may take
place on the plasma membrane, perhaps after gradual exocytosis of different types of vesicles and granules in resting
cells as they circulate or after initial constitutive plasma
membrane expression. Reinternalization of these membrane
proteins along with endocytosed soluble plasma proteins13
probably accounts for the content of diverse types of membrane proteins and soluble constituents in the CR1 vesicles.
However, there must be some specificity of membrane internalization or recycling, because HLA class I antigens are
not included in the same structures as CR1 and these other
proteins, consistent with the previous separation of secretory
vesicles from plasma membrane fragments.11,27
Our immunoblotting studies indicate that CR1 was completely removed from the g-band by the immunoadsorbent.
However, our results also show that a significant amount of
albumin is found separately from the CR1 vesicles. The fact
that the majority of the albumin in the fractions that did not
bind to the anti-CR1 adsorbent was detectable only in the
presence of Triton X-100 indicates that these fractions also
contain intact vesicles. These data thus indicate that there
are populations of albumin-containing vesicles that are distinct from CR1 vesicles, suggesting that they may have
arisen by endocytosis of membrane domains free from CR1.
Although about 40% of the alkaline phosphatase did not
bind to the anti-CR1 beads, only Ç21% of this activity was
latent, as compared with 88% latency of the activity bound
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4564
KUMAR, WETZLER, AND BERGER
to the anti-CR1 containing beads. Thus, greater than 90%
of the latent alkaline phosphatase activity in the cell is stored
in the CR1 vesicles, in agreement with previous results from
Borregaard’s laboratory.11 Most of the alkaline phosphatase
not bound to the anti-CR1 adsorbent is thus likely to be in
plasma membrane fragments or right-side out vesicles.
Our results suggest that CR1, DAF, alkaline phosphatase,
and FcgRIII are stored in the same structures. These vesicles
also contain albumin and a small amount of the cell’s CR3,
but they are distinct from and not contaminated with plasma
membranes. Using these isolated CR1 storage vesicles and
the knowledge that they contain releasable soluble products
(albumin) as well as latent enzyme activity, we may now be
able to study the mechanisms of their fusion with plasma
membranes in vitro. This should allow us to determine how
the exocytosis of readily mobilizable compartments such as
the CR1 storage vesicles differs from that of the traditional
primary and secondary granules and should greatly advance
our understanding of the sequence of events during early
neutrophil activation.
ACKNOWLEDGMENT
We are grateful to Drs Alan Tartakoff and Carolina Jost for advice
and helpful discussions.
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1997 89: 4555-4565
Isolation and Characterization of Complement Receptor Type 1 (CR1)
Storage Vesicles From Human Neutrophils Using Antibodies to the
Cytoplasmic Tail of CR1
Anoopa Kumar, Erica Wetzler and Melvin Berger
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