Biochemical Characterization of the Neutrophil

Biochemical Characterization of the Neutrophil-Specific Antigen NB1
By David F. Stroncek, Keith M. Skubitz, and J. Jeffrey McCullough
Neutrophil-specific alloantibodies and the antigens they
recognize are important in clinical medicine, but little is
known about the structure of these antigens. Alloimmunization t o the antigen N B l is a clinically important cause of
neonatal neutropenia and febrile transfusion reactions. To
study the immunochemistry of the NB1 antigen, we prepared neutrophil plasma membranes and granules by nitrogen cavitation and differential centrifugation and then
analyzed them by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) and immunoblotting with
alloantibodies t o several neutrophil-specific antigens. Two
different antisera t o the neutrophil-specific antigen NB1
identified an approximately 55-Kd protein by immunoblotting on neutrophil membranes from four NB1-positive
donors but not on neutrophil membranes from five NB1negative donors. Four anti-NB1 antisera immunoprecipitated a 58- t o 64-Kd protein from extracts of NB1-positive
neutrophils surface-labeled with ”‘1 using lactoperoxidase,
but not from similarly treated NB1-negative neutrophils.
Normal human serum did not immunoprecipitate or immunoblot any proteins from these same neutrophil preparations. The NB1 antigen was detected by immunoblotting in
secondary granules but was not found in primary granules.
The electrophoretic mobility of the antigen was decreased
slightly by reduction, suggesting that intrachain disulfide
bonds were present. After reduction, the antigen could no
longer be recognized by anti-NB1 antisera, but treatment
of the antigen with periodate had no effect on the ability of
anti-NB1 antisera t o recognize the antigen, suggesting that
it is not a carbohydrate. The data suggest that the
neutrophil-specific antigen NB1 is present on a 58- t o
64-Kd surface glycoprotein that is also present in secondary granules, and that the NB1 epitope is not a carbohydrate but probably resides in the tertiary structure of the
protein backbone.
0 1990 by The American Society of Hematology.
A
family of three structurally and functionally related leukocyte adhesion glycoproteins (GP), LFA- 1, HMac- 1, and GP
150,95.6 Many neutrophil-specific MoAbs recognize the
oligosaccharide lacto-N-fucopentaose 111 (CDl5) and react
with the neutrophil membrane receptors C R 3 and CRl.’.’
MoAbs have also been used to identify two types of neutrophil Fc-y-receptors: the Fc-y-receptor 111 (CDl6) a 50- to
70-Kd protein’.’’ and the Fc-y-receptor I1 (CDw32) a 40-Kd
protein.” There is evidence that the neutrophil-specific
antigen NA1 is on the neutrophil Fc-y-receptor 111. Several
CD16 MoAbs that react with the Fc-y-receptor 111 react
with NAl-positive but not NAl-negative cell^.'^-'^ Direct
identification of the N A l and NA2 antigens using alloantisera havz not been reported, however.
We used immunoblotting and cell surface ‘251-labelingand
immunoprecipitation to identify proteins bearing the neutrophil-specific antigen N B l and studied its distribution in
neutrophil granules. Alloantisera to NB1 identified the N B l
antigen on a 58- to 64-Kd GP located on neutrophil plasma
membranes and secondary granules.
VARIETY OF neutrophil-specific antigens have been
described in the past 20 years.’.2 Alloantibodies to
these antigens are important in some clinical situations,
including alloimmune neonatal neutropenia, primary autoimmune neutropenia of childhood, secondary autoimmune
neutropenia, drug-induced neutropenia, and leukocytemediated transfusion reactions.’ Antigens NB1 and NB2
constitute the neutrophil-specific biallelic N B antigen
~ y s t e m . ~These
.~
antigens are present in whites with a
phenotypic frequency of 97% and 32%, respectively.2 Antibodies to N B l are common in alloimmune neonatal neutropenia.
In antigen-negative mothers, antibodies to this neutrophil
antigen develop during pregnancy with an antigen-positive
child. The children are often born neutropenic, but the
neutropenia revolves spontaneously in 2 weeks to 6 months as
the antibody is catabolized.’
Several neutrophil antigens have been identified and
characterized using murine monoclonal antibodies (MoAbs):
but little is known about the antigens defined by neutrophilspecific alloantisera. MoAbs have been used to identify a
From the Departments of Laboratory Medicine and Pathology,
and Medicine, University of Minnesota Medical School, the Masonic Cancer Center, Minneapolis; and the American Red Cross St
Paul Regional Blood Service, MN.
Submitted February 21. 1989; accepted October 9. 1989.
Supported in part by National Institutes of Health Grant
No.CA36248, The American Red Cross, The Leukemia Task Force,
and the Masonic Memorial Hospital Fund, Inc.
Presented at the annual meeting of the Central Society for
Clinical Research, November 11, 1988. Chicago, Illinois. and
published in abstract form in Clin Res 368774,1988.
Address reprint requests to David F. Stroncek. MD, Department
of Laboratory Medicine and Pathology, Box 198 UMHC, Harvard
St at E River Rd, Minneapolis. MN 55455.
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 I734 solely to
indicate this fact.
0 1990 by The American Society of Hematology.
0006-4971/90/7503-003483.00/0
744
MATERIALS AND METHODS
Preparation of neutrophils. Heparinized (2 U/mL) peripheral
blood (PB) was obtained from healthy donors of known neutrophil
antigen types. Neutrophils were isolated with Ficoll-Hypaque by a
modification of the method of Boyum as previously de~cribed”.’~
and
were suspended at the indicated concentrations in phosphatebuffered saline (PBS), pH 7.4. Differential cell counts on Wright’sstained cells routinely revealed more than 95% neutrophils.
Isolation of neutrophil membranes. Neutrophils from 240 mL
whole blood (approximately 4 x 10’ cells) were suspended at 2 x
10n/mL in PBS containing 5 mmol/L diisopropylfluorophosphate
(DFP) (Sigma Chemical, St Louis, MO), incubated for 10 minutes
at 0°C. and washed twice with 40 mL PBS at 4°C.” All reactions
with DFP were performed in a fume hood, and all articles containing
DFP were washed with 5 mmol/L NaOH before being removed
from the hood. Plasma membranes were then prepared by nitrogen
cavitation and differential centrifugation as described.” Neutrophils
were suspended in 20 mL Hanks’ balanced salt solution (HBSS)
without calcium, pH 7.4 (GIBCO, Grand Island, NY) containing
2.5 mmol/L MgCI, at 4OC. The cell suspension was placed in a cell
Blood, Vol75, No 3 (February 1). 1990: pp 744-755
BIOCHEMICAL CHARACTERIZATION OF NB1
disruption bomb (Parr Instrument, Moline, IL, model no. 4639) and
equilibrated with nitrogen at 350 psi for 20 minutes at 4OC. The
suspension was then released dropwise into 2.0 mL 25 mmol/L
EDTA, pH 7.2 (Sigma) and was centrifuged at 500 g for 10 minutes
at 4OC. The supernatant was then centrifuged at 20,000 g for 20
minutes at 4OC to remove the lysosomes and mitochondria, and the
remaining supernatant was then centrifuged at 100,000 g for 60
minutes at 4OC. The resulting pellet was resuspended in 10 mmol/L
N-2-Hydroxyethyl piperazine-N'-2-ethanesulfonic acid (HEPES)
(Sigma), pH 7.2, centrifuged at 100,000 g for 60 minutes at 4OC,
resuspended in 1 mmol/L HEPES, pH 7.2, and centrifuged at
100,000 g for 60 minutes at 4OC, and the membrane-rich pellet was
then resuspended in 0.5 mol/L Tris-HC1, pH 7, (BioRad Laboratories, Richmond, CA) with 1% sodium dodecyl sulfate (SDS)(Sigma)
and stored at -8OOC.
Isolation of primary and secondary neutrophil granules.
Neutrophils from 240 mL whole blood were isolated and treated
with DFP as described. Neutrophil subcellular fractionation was
performed as described by Borregaard et al.19 Neutrophils were
resuspended in 20 mL ice-cold relaxation buffer [ 100 mmol/L KCl,
3 mmol/L NaCl, 1 mmol/L adenosine triphosphate (Na), (Sigma),
3.5 mmol/L MgCl,, 10 mmol/L piperazine-N, N'-bis (2-ethanesulfonic acid) (PIPES), pH 7.3 (Sigma)]. This solution was then
equilibrated for 20 minutes at 4OC with nitrogen at 350 psi with
constant slow stirring in the cell disruption bomb. The suspension
was then collected dropwise into 2.2 mL 12.5 mmol/L EDTA, pH
7.4 (Sigma) in relaxation buffer. Nuclei and unbroken cells were
removed by centrifugation at 500 g for 20 minutes at 4OC. Percoll
(Pharmacia Fine Chemicals, Piscataway, NJ) was adjusted to a
density of 1.120 and 1.050 g/mL with relaxation buffer 10 times
concentrated. The 1.050 density Percoll was laid over 1.120 density
Percoll, and the supernate was loaded onto the Percoll gradients,
which had been precooled to 4OC. The gradient was then centrifuged
at 4OC for 15 minutes at 48,000 g. The three bands representing the
azurophilic granules, specific granules, and plasma membranes were
then collected, and the Percoll was removed from the samples by
centrifugation at 100,000 g for 90 minutes at 4OC. The pelleted
membranes and granules were washed with relaxation buffer,
centrifuged again at 100,000 g for 90 minutes at 4OC and then
resuspended in 0.5 mol/L Tris, pH 7 with 1% SDS, and stored at
-8OOC. Protein concentrations of the fractions were determined by
the bicinchoninic acid (BCA) protein assay (Pierce).
Separation of the soluble and membrane components of secondary granules. Secondary granules from 2 x 10' neutrophils were
prepared by centrifugation over discontinuous Percoll gradients as
described. Isolated secondary granules were suspended in 20 mmol/
L Tris-HCI, pH 7, frozen and thawed twice, sonicated for 2 minutes
at OOC, and then centrifuged at 120,000 g for 90 minutes at 4OC. The
supernatant was removed and stored at -8OOC until use, and the
pellet was resuspended in 500 mmol/L NaCl with 20 mmol/L
Tris-HCI, pH 7, sonicated again for 2 minutes at OOC, frozen and
thawed, and again centrifuged at 120,000 g a t 4OC. The supernatant
was removed and stored at -8OoCuntil use. The resulting membranerich pellet was resuspended in 0.5 mmol/L Tris-HCI, pH 7, with 1%
SDS and stored at -8OOC until use.
Alloantisera and MoAbs. Human alloantisera were a gift from
the American Red Cross Neutrophil Serology Reference Laboratory, St Paul, MN. All four anti-NB1 antisera were from women
alloimmunized during pregnancy. Antisera NIH-50-3-04-01-01 and
NIH-FD-251-601-C are reference anti-NB1 antisera from the
National Institute of Health, Bethesda, MD, and their titers in
granulocyte immunofluorescencewere 1:256 and 1:512, respectively.
Anti-NB1 17.009 and 17.003 are reference antisera from the
American Red Cross Neutrophil Serology Reference Laboratory.
The titers in granulocyte immunofluorescence of 17.009 and 17.013
745
were 1:2056 and 1:1024, respectively. MoAbs AHN-1.1 (CD15),8
AHN-10 (antineutrophil-elastase),20 and AHN-9 (anti-human
lactoferrin):' have been described previously.
Immunoblotting. Samples were suspended in Laemmli sample
buffer [62 mmol/L Tris-HC1, pH 6.8, 2% SDS, 10% glycerol, and
0.001% bromphenol blue with or without 5% 2-mercaptoethanol
(ME)], incubated for 2 minutes at 100°C, and 15 pg protein was
applied to each lane of a 10% polyacrylamide gel and electrophoresis
was performed in the Laemmli buffer system.,, The gels were then
equilibrated in transfer buffer [25 mmol/L Tris-HCI, pH 8.3, 192
mmol/L glycine (BioRad), 20% methanol] for 30 minutes and
electroblotted onto nitrocellulose (BioRad) at 30 V for 16 hours at
-5OC, and then 150 V for 1 hour at -5OC.,' Alternatively,
electroblotting was performed at 30 V for 1 hour at -5OC with 0.1%
SDS added to the transfer buffer as indicated.24
After transfer of proteins onto nitrocellulose, the nitrocellulose
strips were blocked with 20 mmol/L Tris-HCI, pH 7.5,500 mmol/L
NaC1, containing 0.3% Tween-20 (Sigma) (TBS-Tween) with 20%
normal goat serum (GIBCO) for 4 hours with constant rocking.
After two washes with TBS-Tween, the nitrocellulose was incubated
overnight in human alloantibody or mouse MoAb diluted in TBSTween with 10% normal goat serum. After two washes in TBSTween, the blots were incubated for 1 hour at 23OC with biotinylated
goat anti-human IgG or biotinylated goat anti-mouse IgG and IgM
(Cappel, Cooper Biomedical, Malvern, PA) diluted 1:1,000 in
TBS-Tween with 10% normal goat serum. The nitrocellulose was
washed twice in TBS-Tween without goat serum and then incubated
for 30 minutes with avidin-conjugated alkaline phosphatase (Cappel) diluted 1:5,000 in TBS-Tween with 10% normal goat serum.25
After two TBS-Tween washes, proteins were visualized by incubation in 0.3 mg/mL nitroblue tetrazolium (NBT) (Sigma), 0.2
mg/mL 5-bromo-4-chloro-3-indolylphosphate
(BCIP) (Sigma), 100
mmol/L NaCI, 50 mmol/L MgCI, and 100 mmol/L Tris-HC1, pH
9.5 for 10 minutes at 23OCZ6The reaction was stopped by washing in
5 mmol/L EDTA, 20 mmol/L Tris-HC1, pH 6.8, at 23OC.
Modifications of transferred proteins. In some cases, as indicated, membrane preparations were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose paper as described; the nitrocellulose was then incubated in
1% SDS, 0.5 mol/L Tris-HCI, pH 7, with or without 5% ME for 30
minutes at 60OC. The nitrocellulose was then washed three times
with TBS-Tween, blocked, incubated with antisera, and developed
as described. In other cases, as indicated, samples were transferred
to nitrocellulose and the nitrocellulose was washed once in 50
mmol/L sodium acetate buffer (Sigma), pH 4.5, incubated in 50
mmol/L sodium acetate buffer, pH 4.5, with and without 40
mmol/L sodium periodate (Sigma) for 1 hour in the dark at 23OC,
and washed with 50 mmol/L sodium acetate buffer, ph 4.5, three
times.,' The nitrocellulose was then blocked, treated with antibody,
and developed.
'Z51-labelingof neutrophils. Neutrophils were surface-labeled
with
by using lactoperoxidase as previously described with minor
modification2': 2 x lo7cells were suspended in 1 mL PBS at O°C and
5 U lactoperoxidase (Sigma), 1 mCi of NalZ51(carrier-free, Amersham, Arlington Heights, IL), and 10 pL 0.06% H,O, was added.
After 5 minutes, an additional 10 pL 0.06% H202was added, and the
teaction was terminated 5 minutes later by adding 9 mL PBS and
DFP to 5 mmol/L. The cells were incubated on ice for 10 minutes,
recovered by centrifugation at 400 g for 8 minutes, and then washed
with 10 mL PBS. All steps were performed at Oo to 4OC.
Immunoprecipitation and PAGE. Immunoprecipitation was performed as previously described with minor modifications2': 2 x 10'
radiolabeled cells were suspended in 1 mL cell solubilization buffer
[20 mmol/L Tris-HC1, pH 7.6, containing 150 mmol/L NaCI, 0.5%
Nonidet P-40 (NP-40) (Sigma), 0.02% NaN,, and 2 mmol/L
746
phenylmethylsulfonylfluoride (PMSF) (Sigma)], and incubated on
ice for 1 hour. The suspensions were then centrifuged at 9,800 g for
30 minutes at 4°C. The resulting supernatants (750 pL) were mixed
with 60 pL normal human serum (NHS) in a final volume of 1.25
mL containing 20 mmol/L Tris-HCI, pH 8.2, 100 mmol/L NaCI,
0.5% NP-40, 1 mmol/L EDTA, 0.125 mg/mL gelatin, 0.02%NaN,,
and 2 mmol/L PMSF. The mixture was incubated 1 hour at 4"C,
then 600 pL 10% Staphylococcus aureus suspension (Pansorbin A,
Calbiochem, LaJolla, CA), was added. The mixture was incubated
for 20 minutes at 4°C. and then centrifuged at 2,000 gfor 20 minutes
at 4°C. The resulting supernatant was cleared once by adding 300
pL Pansorbin, incubating 20 minutes at 4OC, and centrifuging as
above. This treatment with Pansorbin was repeated, and the resulting supernatant was used for immunoprecipitation or analyzed by
SDS-PAGE.
Radiolabeled cell proteins were immunoprecipitated in reaction
mixtures containing cell extract, antibody (4 pL), 20 mmol/L Tris
HCI, pH 8.2, 100 mmol/L NaCI, 0.5% NP-40, 1 mmol/L EDTA,
0.125 mg/mL gelatin, and 2 mmol/L PMSF in a total volume of
approximately 300 pL in 10 x 75-mm glass tubes. After the
suspension was incubated overnight at O°C, 50 pL 10%Staphylococcus aureus was added. After 15 minutes at OOC, the mixture was
washed twice by adding 1 mL wash buffer (20 mmol/L Tris-HC1,
pH 8.2, 1 mmol/L EDTA, 100 mmol/L NaC1, 0.5% NP-40, 2.5
mol/L KC1, and 0.25 mg/mL gelatin) and centrifuging at 2,000 g
for 20 minutes at 4°C. The pellet was then suspended in 1 mL buffer
containing 20 mmol/L Tris-HCI, pH 8.2, transferred to an Eppendorf tube, and centrifuged at 13,700 g for 10 minutes at 4°C. The
precipitate was suspended in reducing Laemmli sample buffer (62
mmol/L Tris-HCI, pH 6.8, 2% SDS, 10% glycerol, 5% ME, and
0.001% bromphenol blue), incubated at 100°C for 2 minutes and
analyzed by SDS-PAGE." Molecular weight (mol wt) standards
were purchased from Sigma. Gel slabs were stained, dried, and
examined by autoradiography with Kodak X-Omat XAR-5 film.
Solid-phase antibody-binding assays. Antibodies were assayed
for cell-binding activity by a solid-phase assay, as described?8 50 pL
PBS containing 0.1 or 0.5 pg, as indicated, of each subcellular
fraction was applied to wells of a Linbro 96-well microtiter plate and
incubated at 23°C for 6 hours. Two hundred microliters PBS
containing 50 mg/mL BSA and 0.02%NaN, was then added to each
well and incubated for 8 hours at 23OC. This solution was then
removed, and 50 pL supernatant containing the indicated antibody
to be tested was added and incubated for 1 hour at 23°C. Excess
antibody was removed by washing three times with 200 pL ice-cold
PBS containing 1 mg/mL BSA and 0.1% Triton X-100(BioRad);
'251-labeledsheep anti-mouse immunoglobulin (100,000 cpm, specific activity 10-50 pCi/pg, Amersham) was then added in 50 pL
buffer containing 20 mmol/L Tris-HC1, pH 7.6, 1 mmol/L EDTA,
100 mmol/L NaCI, and 20 mg/mL BSA, and incubated for 1 hour
at 23°C. After washing as above, 100 pL 2 mol/L NaOH was added;
after 30 minutes at 23"C, the solution was transferred to glass tubes,
and the radioactivity was counted.
'zP-Labeling of neutrophil proteins. Neutrophil proteins were
labeled with 32Punder conditions designed to detect ecto-protein
kinase activity as previously describedz9:10 pCi of [-pSZP]ATP
(specificactivity 4,500 Ci/mmol, ICN, Costa Mesa, CA) was added
to 1 x lo6neutrophils in 60 pL buffer containing 140 mmol/L NaCI,
1.8 mmol/L Caz+,0.8 mmol/L Mg2+, 1 mmol/L Mn2+,and 20
mmol/L HEPES, pH 7.4, and was incubated at 37OC for 10 minutes.
DFP to a final concentration 10 mmol/L was then added, and the
mixture was incubated for 2 minutes more at 23°C. The mixture was
then centrifuged at 13,000 g for 2 minutes at 23°C. and the cells
were solubilized in cell solubilization buffer containing 200 pmol/L
Na,VO, and 200 pmol/L Na,MoO,. Immunoprecipitation and
STRONCEK, SKUBITZ, AND McCULLOUGH
analysis by SDS-PAGE and autoradiography were performed as
previously described.,'
Typing neutrophils for alloantigens. Neutrophils were typed
with American Red Cross or National Institute of Health reference
antisera by using a granulocyte agglutination assay and a granulocyte immunofluorescence assay with 1% paraformaldehyde-fixed
neutrophils as previously de~cribed.'.~'Neutrophils from all NBlpositive donors were typed positive, and all NB1-negative neutrophils were typed negative in both assays.
Antibody adsorption. Alloantisera were adsorbed by incubating
granulocytes with test serum at a ratio of packed granulocytes to
serum of 1:2. After incubation at 37°C for 1 hour with occasional
mixing, the mixture was centrifuged at 6,000 g for 5 minutes. The
serum was removed and centrifuged again at 6,000 g for 5 minutes.
The serum was then removed and frozen at -80°C until use.
Alloantisera were also adsorbed by using pooled outdated platelets
as previously described.2
RESULTS
Identification of NBl antigen by immunoblotting. N B l positive neutrophil plasma membranes prepared by differential centrifugation were analyzed by immunoblotting with
two antisera to NBI, 17.009 (Fig 1, lanes A through D) and
17.013 (Fig 1, lanes E through H). Both antisera reacted
with a protein of approximately 55 Kd; however, anti-NB1,
17.009, had a higher titer. N H S (lane I) did not react with
any proteins. Based on these results, plasma membranes
prepared by differential centrifugation from nine donors
were analyzed by immunoblotting with anti-NB1 antisera
17.009 and 17.013 a t a dilution of 1:lOO (Fig 2). Both
anti-NB1 antisera reacted with a protein of approximately
55-Kd on all four NB1-positive membrane preparations (Fig
2, lanes A through D and J through M). No protein of this
size was detected on any of the five NB1-negative plasma
membrane preparations (Fig 2, lanes E through I and N
through R). A 60-Kd protein was visible in immunoblots of
three of four NB1-positive membrane preparations probed
with anti-NB1 17.009 but was also apparent with two
NB1-negative membrane preparations (Fig 2, lanes A and B
and D through F), and more weakly apparent on one
NB1-positive and two NB 1-negative membrane preparations
(Fig 2, lanes C, G, and I). Anti-NBl 17.013 did not react
with this protein on any of the nine membrane preparations
tested (Fig 2, lanes J through R). NHS did not recognize any
protein from the plasma membrane preparations of five of
the nine donors tested. A representative blot with NHS is
shown (lane S). NHS occasionally reacted very weakly with
a 60-Kd protein with two NB1-positive and two N B l negative membrane preparations (data not shown).
One of the membrane preparations was prepared from
cells obtained from the person who produced the anti-NB1
17.009. Immunoblots of these membranes with anti-NB1
17.013 showed no reaction (lane R), and immunoblots with
the autologous serum, anti-NB1 17.009, showed no reaction
with the 55-Kd protein but weakly stained the 60-Kd protein
(lane I). Sera from the other eight neutrophil donors did not
immunoblot any proteins on autologous neutrophil membrane preparations. Three anti-NA1 antisera, three antiN A 2 antisera, two anti-NB2 antisera, two anti-Mart antisera, and polyspecific anti-HLA antiserum did not detect
747
BIOCHEMICAL CHARACTERIZATION OF NB1
Fig 1. Immunoblots of neutrophil plasma membranescontaining NE1 antigen probed with
two different anti-NB1 antisera
(17.009 and 17.013) at various
dilutions. Membranes prepared
by differential centrifugation
were separated by SDS-PAGE
in a 10% gel under nonreducing
conditions, transferred onto nitrocellulose, and probed with
anti-NB1 antisera 17.009 (lanes
A through Dl or 17.013 (lanes E
through HI. The anti-NB1 dilutions used were 1:30 (lanes A
and E), 1:lOO (lanes B and F),
1:3OO (lanes C and GI. and 1:
1,000 (lanes D and HI. Lane I
was probed with a dilution of
1:30 of NHS. Fifteen micrograms of protein was applied t o
each lane. Molecular weight
standards were phosphorylase
a. 97.400: BSA, 68.000: ovalbumin.45.000: and carbonic anhvdrase, 29,000.
ANTI-NBl 17.009
A
97.4-
B
E
m
m
I
2
0.L
-
G
9
H
I
--
-
l
29-
T l O N 1:
30 100 300 .ooO
ANTI-NB 1 1 7 . 0 0 9
ABCD
F
NHS
L .
proteins on NAI. NA2, NBI, NB2, and Mart-positive
neutrophil membranes in this assay (not shown).
Coomassie blue staining of gels of plasma membrane
proteins of the four NBI-positive donors and five NBInegative donors electrophoresed under reducing or nonreducing conditions did not detect differences among any proteins
(data not shown).
Eflect of reduction on NBI antigen. When plasma
membrane proteins were separated by SDS-PAGE under
reducing conditions before immunoblotting with anti-NBI
17.009. the 55-Kd protein was no longer detected, but the
60-Kd protein was still stained (Fig 3). Under these conditions, antiserum 17.009 detected a 60-Kd protein on all four
NBI-positive membrane preparations and four of five NBl-
29
D
C
- l a p
ANTI-NB1 17.013
E F G H I
30 100 300 lo00
30
negative membrane preparations (Fig 3, lanes A through D
and F through I). No protein was identified on one NBInegative membrane preparation (Fig 3, lane E). The inability to detect the 55-Kd protein by immunoblotting when
SDS-PAGE was performed under reducing conditions could
have been the result of failure of the anti-NBI antiserum to
recognize reduced antigen.
To determine if anti-NBI antiserum could recognize
reduced NBI antigen, NBI-positive neutrophil membranes
were analyzed by SDS-PAGE under nonreducing conditions
and were then transferred onto nitrocellulose. The nitrocellulose was then incubated in 0.5 mol/L Tris-HCI, pH 7.0.
containing 1% SDS with or without 5% ME for 30 minutes at
6OOC. After three washes with TBS-Tween. the nitrocelluANTI-NB 1 17.0 1 3
J K L M
NOPOR
NHS
S
-
NB1
+
NB1
-
NB1
+
NB1
-
Fig 2. Immunoblots of neutrophil plasma membranes from nine different donors probed with anti-NB1 antisera. Plasma membrane
preparations prepared by differential centrifugation from four NBl-positiie donors (lanes A through D and J through M) and five
NBl-negative donors (lanes E through I and N through R) were separated by SDS-PAGE in a 10% gel under nonreducing condition8 and
immunoblotted with anti-NB1 17.009 (lanes A through I)and anti-NB1 17.013 (lanes J through R) at a dilution of 1:lOO. A representative
immunoblot of an NB1-positive membrane preparation with NHS at a dilution of 1:lOO is shown in lane S. Fifteen micrograms of protein
was applied t o each lane. Molecular weight standards were as described in the legend t o Fig 1.
STRONCEK, SKUBIlZ. AND MCCUUOUGH
748
ABCD
J
" I
E F G H I
.PT I
~
<.rrJ.-.
'f.
---7
K
J
-
-
97.4
66-
45-
29 -
NB1
+
NB1
-
Fig 3. Immunoblots with anti-NE1 of neutrophil plasma membrane preparations separated under reducing conditions before
immunoblotting. Membranes of the name nine donors described in the legend t o Fig 2 were prepared by differential centrifugation and
were separated by SDS-PAGE in a 10% gel under reducing conditions and immunoblotted with anti-NE1 17.009 (dilution of 1:lOO).
Immunoblots of the four NE1-positive membrane preparations are shown in lanes A through D, and immunoblots of the five NE1-negative
membrane preparations are shown in lanes E through 1. A representative blot with NHS at a dilution of 1:lo0 on an NE1-positive membrane
preparation is shown in lane J. A representative blot with anti-NE1 17.009 of plasma membranes separated under nonreducing conditions
is shown in lane K. Fifteen micrograms of protein was applied t o each lane. Molecular weight standards were as described in the legend t o
Fig 1.
lose was then probed with anti-NBI 17.009 (dilution of
1:lOO) and developed as described. Anti-NBI reacted with
the 55-Kd protein on the nitrocellulose incubated in buffer
alone, (Fig 4. lane A) and buffer containing SDS (lane B).
but not on nitrocellulose incubated in buffer containing SDS
and M E (lane C). This same treatment did not decrease the
ability of AHN-1.1 to recognize its carbohydrate determi-
ANTI-N61
A
m
r'
B
C
nant (lanes D through F). The proteins recognized by
AHN-1.1 were not as well visualized when samples were
electrophoresed under nonreducing as compared with reducing conditions before immunoblotting. Similar results were
obtained when nitrocellulose blots were incubated with SDS
and M E at 37OC for 1 hour or at IOOOC for 2 minutes (data
not shown).
NHS
AHN-1.1
D
E
F
G
H
I
66-
29-
Fig 4. Immunoblots of neutrophil plasma membrane proteins reduced eftor transfer onto nitroceJllulose paper. NE1-positive plasma
membranes were separated by SDS-PAGE in a 10% gel under nonreducing conditions and transferred onto nitrocellulose. The
nitrocellulose strip. were then incubated in Tris buffer (control) Ilsnes A. D, and G) or in buffer containing 1% SDS without (lanes E, E, and
H) or with (lanes C, F. and II 8% ME for 30 minutes at 6 O X before immunoblotting with anti-NE1 17.009 (lanes A through C). AHN-1.1 (lanes
D through F), or NHS (lanes G through I)at dilutions of 1:lOO. 1:200, and 1:loo, respectively. Fifteen micrograms of protein was applied t o
each lane. Molecular weight standards were as described in the legend t o Fig 1.
BIOCHEMIW CHARACTERIZATION OF NB 1
749
A
97.4
(3
B
D
C
-
66- o
I7
O
NHS
AHN-1.1
ANTI- NB1
E
F
G
H
I
r& Ii ,
r b
45-
x
Fig 5. Immunoblots of neutrophil plauru membrane protoim treated with periodate. NE1-positive mombranos were sopmated by
SDS-PAGE in a 10% gel under reducing (Ian08 D through FI or nonroducing conditions llanos A through C and G through I1 and wore
transforred Onto nitrocellulose. N i t r o c ~ l l u l o strips
s~
were incubeted in .odium acetate buffer with IIanes C, F. end I)or without (lanes E, E.
and HI 40 m d / L sodium periodate, or were not further treated (lanes A. 0. and GI before immunoblotting with anti-NB1 llanos A through
C ) , AHN-1.1 llanos D through FI, or NHS llanos G through I).Fifteen micrograms of protein was applied t o each lane. Molecular weight
standards were as described in the legend t o Fig 1.
detect the 55-Kd protein (lane C). Similarly. treatment of
transferred membrane proteins with 50 mmol/L sodium
pcriodatc in PRS. pH 7.4, for 16 hours at 23OC did not affect
the epitopc (diita not shown). Lectin chromatography''
showed that the SSKd protein bound to concanavalin A
indicating that the protein is glycosylated.
Identification of NRI antiKen by immrinoprecipitation.
All four anti-NRI antisera tested immunoprecipitated a 58to 64-Kd "'I-labeled protein from extracts of surface-labeled
NRI-positive neutrophils (Fig 6. lanes R through E and G
through J). No radiolabeled proteins were immunoprecipitated by NHS (lanes I.: and K). In contrast. no 58- to 64-Kd
Efect of periodate treatment on N R l antigen. To detcrmine the role of carbohydrates in the NRI cpitopc. plasma
membrane proteins from an NRI-positive donor were transferred onto nitrocellulose and incubated for I hour with 40
mmol/L sodium pcriodate in 50 mmol/L sodium acetate
MoAb AWN-1.1. which
buffer. pH 4.5. As
recognizes the oligosaccharide lacto-.V-fucopcntaosc 111. no
longer reacted with pcriodatc-oxidired neutrophil membrane
proteins (Fig 5. lane F) but did react with membranes
incubated with sodium acetate (lane E). In contrast. trcatment of the transferred membrane proteins with sodium
pcriodatc did not affect the ability of anti-NRI antisera to
ABCOEF
G H I J K
97.4c)
LMNOP
ORSTU
. -
a
66-
-
'$
29-
Y
NB1 +
NBl
+
NB1
-
Ne1
-
Fig 6. Immunoprocipitmtion and SDS-PAGE undr r.dudng conditkm of '%&dod
nwtrophll s u r f " protdm. Nwtrophlh were
sutfaco lobeled with '
9 using Iactoporoxidase. solubilized. p r e c l r r e d with NHS. and then immunoprecipitated with the indicated antisera
as dewibed in the text. Lane A, cell extract from donor A; lanes B through U: immunoprecipitates of cells from: donor A (NB1-positive1
(lanes E through FI; donor E lNB1-positive1 llanos G through KI: donor C (NB1-negative) (lane8 L through PI; and donor D (NB1-negative1
(h~108
Q through VI. Immunoprecipitating antibodies: 17.009 (anti-NE1I llanos E. G. L. and 01: NIH-60-3-04-01-01 (anti-NE1I llanos C. H, M,
and RI; 17.013 (anti-NE1I ( h e 8 0 , I. N, and SI:NIH-FD-261-601-C (anti-NE1I (lanes E. J. 0, and TI; and NHS (lanos F. K. P. and Ul. Proteins
used as molecular weight standard# were as described in the legend t o Fig 1 with the addition of myosin heavy chain, 200.000; Escherichia
coli E-~lactosidare.
116.000.
750
SlRONCEK. SKUBTTZ. AND McCULLOUGH
proteins were detected in immunoprecipitates from N R I negative cells (lanes L through U). The patterns of radiolabeled proteins present in the cell extracts of all four donors
were similar; a representativeextract is shown in lane A for
comparison. Three of the four antibodies to N R I tested also
immunoprecipitated an 80-Kd protein from extracts of
N R I - p s i t i v e and NRI-negative cells (Fig 6).lmmunoprecip
itation of the 80-Kd protein by only three of the four
anti-NRI antisera. as well as immunoprecipitation of this
protein from NRI-negative cells. suggested that these three
antisera may also detect another undescribed neutrophil
antigen in addition to N R I . Both the 58- 1064-Kd and 80-Kd
proteins had slightly decreased electrophoretic mobilities
under reducing conditions as compared with nonreducing
conditions (Fig 7). IgG purified from anti-NRI antiserum
17.009 also immunoprecipitated the 58- to 64-Kd and 80-Kd
proteins (data not shown).
So radiolabeled proteins could be detected in immunoprecipitates using antisera to N A I , NA2. NR2. and Mart from
extracts of radiolabeled cells bearing these antigens as
determined by immunofluorescence. A representativeexperiment using these antisera is shown in Fig 8 . Similarly, no
"'I-labeled proteins were immunoprecipitated from any of
the neutrophil extracts tested by anti-Kell, anti-PLA'. or
anti-DulTy (data not shown).
116
97.4
-
-
c3
' 0
F
X
r'
45
-
29
-.
Fig 7.
proteins by anti-NE1 antisera and SDS-PAGE under rsducing end
nonreducing conditions. Neutrophils were r u r h c e labeled with '?
using lactoperoxidase, solubilired. prwleared with NHS, immunoprecipitated with anti-NE1 17.009. and then analyred by SDSPAGE under reducing (lane A) and nonreducing (lane E) conditions.
Molecular weight standards were as described in the legend t o
Fig 6.
ABCDEFGH I J K L
-
200
97.4 66
--
<3
%
45-
X
5
'4
29
'C
Fig 8. Immunoprscipitntion and SDS-PAGE undr reducing
conditions of '%labeled neutrophil w r f a r x protdns. Neutrophils
from a donor whose neutrophils were positive for NA2 and Mart
and negative for NA1 and NE1 were surface labeled with '? using
lactoperoxidow. solubilized. preclwred w i t h NHS, and then immunoprecipitated with the indicated alloantisera as described in the
text. Lane A. cell extract: lanes E through L, immunopreciphtes
of colls. Immunoprecipitating antibodies: NHS (lane E). 17.009
(anti-NE1) (lane C), 17.013 (anti-NE1 (lane D), NIH-50-3--1-01
(anti-NE1) (lane El. 17.019 (anti-NE2) (lane FI. 17.002 (anti-Mart)
(lane GI. 17.014 (anti-Mart) (lane HI. 17.012 (anti-NA2) (lane I).
17.007 (anti-NA2) (lane J), 17.006 (anti-NAl) (lane K). 17.016
(anti-NAl) (lane L). Molecular weight standards were as described
in the legend to Fig 6.
To explore further the relationship between the two
proteins immunoprecipitated by anti-NRI antiserum. immunoprecipitationstudies were performed with anti-NRI 17.009
that had been adsorbed with NRI-positive cells. N R I negative cells. or poled platelets. After adsorption of antiN R I antiserum with NRI-positive neutrophils, the antiserum no longer reacted with NR I -positive cells in neutrophil
agglutination and immunofluorescence assays. Anti-NRI
antiserum adsorbed with N R I -negative cells and platelets
continued to react with NRI-positive neutrophils in these
assays, however. and had only a slight decrease in titer
( I :2056 to 1:1023 in the granulocyte immunofluorescence
assay). Anti-NRI antiserum ad.sorbed with XRI-positive
cells no longer immunoprecipitated the 58- to 64-Kd protein
from N R I -positive neutrophils; however. the 80-Kd protein
was still immunoprecipitated (Fig 9. lanes C. E. H.J. M. and
0).Anti-NRI antiserum adsorbed with NRI-negative neutrophils (lanes R. G . and 1.) and anti-NRI antiserum
adsorbed with poled platelets (lanes D. I.and N) continued
to immunoprecipitate both proteins from N R I -positive ncutrophils. Immunoprecipitation studies were also performed
with NRI-negative neutrophils and. as expected. anti-NRI
antiserum (lane P) and anti-NRI antiserum adsorbed with
NR1-negative neutrophils (lane 0).
NRI-positive neutro-
BIOCHEMICAL CHARACTERIZATION OF NB1
ABCDE
F G H I J
--200 97.4
66-
-
*'c
-
KLMNO
P Q R S
- -
I
I
I
c
X
)I--
5
75 1
.
45-
29
NBl +
NB1 +
NB1 +
NBl
-
Fig 9. Immunoprecipitation with anti-NE1 antisorum proodsorbed with neutrophils 01 platdots and SDS-PAGE u n d r reducing
conditions of '?-labeled neutrophil surface proteins. Neutrophils were surface labeled with '
9 using lactoperoxidase. solubilized,
precleared with NHS. and thon immunoprecipitated with the indicnted antisore. Immunoprecipitates of extracts from donor E
lNE1-positive1 (bnes A through E): donor F (NE1-positive1 (lanes F through J): donor G (NB1-positive) (lanes K through 0 ) :donor of
anti-NE1 sora 17.009 (NEl-negative) (lanos P through S). Immunoprecipitating antibodies: 17.009 (anti-NE1 1 (lanes A. F, K. and PI: 17.009
adsorbed with NE1-n.geticn neutrophils (lanos B, G. L. and 0 ) :17.009 adsorbed with NB1-positive neutrophils from donor 0 (lanes C. H. M.
and Rl: 17.009 adsorbed with pooled platelets (lanos D. 1. N. and SI:and 17.009 adsorbed with NB1-positive neutrophils from donor H (lanes
E, J. and 01. Molecular weight standards ware as described in the legend to Fig 6.
phils (lane R). or pooled platelets (lane S) immunoprecipitated the 80-Kd protein but not the 58- to 64-Kd protein.
Anti-NRI antiserum adsorbed with NRI-positive neutrophils no longer immunoblotted the 55-Kd protein and antiNRI antiserum adsorbed with NRI-negative neutrophils
continued to immunoblot the 55-Kd protein (data not shown).
To confirm further that the N R I antigen i s present on the
58- to 64-Kd protein and not on the 80-Kd protein. neutrophils from the person who donated anti-NRI antiserum
17.009 were immunoprecipitated with autologous serum
(Fig 9. lane P) and three other anti-NRI antisera (data not
shown). This donor's neutrophils were NRI-negative. and
she developed anti-NRI while pregnant with a child whose
neutrophils were NRI-positive. The 80-Kd protein was
immunoprecipitated by hcr own serum ( I 7.009) (Fig 9. lane
P) and with twoof three other antibodies to N R I tested. but
the 58- to 64-Kd protein was not immunoprecipitated by any
of the four antibodies (data not shown). NHS did not
immunoprecipitateeither protein. The presence of the 80-Kd
protein on N R I -negative neutrophils (including neutrophils
obtained from a person who produced alloantibodies to N R I )
provida further evidence that this 80-Kd protein does not
contain the N R I antigen.
Immunoprecipitation studies were also performed with
anti-NRI antiserum using extracts of neutrophils labeled
with "P by the neutrophil ecto-kinase."' Under these conditions. several proteins were radiolabeled as expected but no
"P-labeled proteins were immunoprecipitated by the antiN R I antiserum (data not shown).
Suhcelliilar localization of NRI antigen. Recause some
neutrophil membrane structures arc also located in ncutrophi1 granules. primary and secondary granules were probed
with anti-NRI antisera by immunoblotting. A mixture of
primary and secondary neutrophil granules was prcparcd by
nitrogen cavitation and dilfcrcntial centrifugation. Thcse
granules were then analyzed by immunoblotting with antiNRI antiserum 17.009. Granules prepared from all four
N R I -positive neutrophils contained a protein of approximately 55 Kd (Fig 10. lanes A through D). whercas no
protein could be detected in granules prepared from NRInegative neutrophils (lanes E through I). N H S did not detect
proteins in these granule preparations (lane J).
To localize the N R I antigen more precisely. Percoll
gradients were used to purify neutrophil plasma membranes.
primary granules. and secondary granules. As expected,
anti-NRI antiserum immunoblotted a 55-Kd protein on the
plasma membranes prepared from two NRI-positive donors
(Fig I I . !ana A and R). In addition. this antiserum detected
a 55-Kd protein on secondary granules from both donors
(lanes C and D). A faint 55-Kd protein was sometimes
identified with this antiserum in the primary granule fraction
(lanes E and F). N H S did not react with proteins in any of
the three fractions (not shown).
To confirm the nature of the plasma membrane. primary
granule. and secondary granule fractions. these preparations
wcrealso probed by immunoblotting with AHN-1.l.a CD15
MoAb known to detect an oligosaccharide present on proteins and lipids in neutrophil membranes and secondary
granules.'." AHN-9. a MoAb that reacts with the secondary
granule protein lactoferrin. and MoAb AHN-IO. which
reacts with the primary granule protein elastase. As
expected." AHN- I . I detected proteins in both plasma membrane and secondary granule fractions (Fig I I . lanes G
through J ) but not in the primary granule fractions (lanes K
and I-). Also as expected. AHN-9 reacted only with the
STRONCEK. SKUBITt. AND McCULLOUGH
752
J
97.4X
r'
45-
29-
NB1
+
NB1
-
secondary granule fraction (not shown) and A H N - I 0 reacted strongly with a 30-Kd protein in primary granules only
weakly with secondary granules and not at all with the
plasma membrane fractions (not shown). I n addition. a
solid-phase radioimmunoasay was performed using MoAbs
to cathespin G (AWN-I I ) . ;I second antilactoferrin antibody
(AH%-9.1). and the CD15 antibody AHN-1.1 (Fig 12). As
expected. cathespin G was greatly concentrated in the
primary granulcs and lactoferrin was mostly in the secondary
granules. confirming the separation of the subcellular fractions as detected by immunoblotting.
To determine i f NRI was located in the secondary granule
membranes or in the soluble granule contents. secondary
granules were sonicated and frec7e-thawcd twice as dc-
Fig 11. lmmunoblots of neutrophil plasma
membranes. primary granules. and secondary granules prepared from two different NBl-positive
donors by centrifugatkn over discontinuous Percdl
gradients. separated by SDS-PAGE in a 10% gel
under nonreducing conditions (lanes A through F)
or reducing conditions (lanes G through L ) and
immunoblotted with anti-NE1 17.009 (dilution of
1:lOO) (lanes A through F) or AHN-1.1 (lanes G
through L). as described in the text. Cell fractions:
Plasma membranes (lanes A. B, G, and HI, secondary granules (lanes C, D, 1, and J). and primary
granules (lanes E. F. K. and I.) Fifteen micrograms
of protein was loaded in u c h lane. Molecular
weight standards were as described in the legend
t o Fig 8.
97.4'0
(3
66-
v
x
5
4529-
Fig 10. Immunoblots of neutrophil granules
with anti-NE1 antisera. A mixture of primary and
secondary granules was prepared by differential
centrifugation and separated by SDS-PAGE in a
10% gel under nonreducing conditions. and immunoblotted with anti-NE1 17.009 (dilution of 1:lOO)
as described in the fext. The grenules were prepared from NB1-positive neutrophils (lanes A
through D) and NBl-nogmiva neutrophils (lanes E
through I) from the same nine donors described in
the legends to Figs 2 and 3. A representative
immunoblot of NE1-positive granules probed with
NHS is shown in lane J. Fifteen micrograms of
protein was added to each lane. Molecular weight
standards ware os described in the legend to Fig 6.
scribed above and the resulting supernatants and granule
membranes were separated by SDS-PAGE. transfcrrcd to
nitrocellulose. and probed with antibodies. Almost a l l the
NRI antigen was in the pclletcd granule membrane fraction
(Fig 13. lane C):only a small amount of N R I antigen was
within the soluble contents of the granule or looscly associated with the granule membrane (Fig 13. lane A). As
expected, probing with A H S - 9 demonstrated lactoferrin
primarily in the soluble contents of the secondary granules
(lanes G and H). Also as expected." AHN-1.1 identified
proteins in the soluble granule contents (lanes D and E) as
well as a higher mol-wt protein observed primarily in the
membrane fraction (lane F). When a greater amount of
protein from the membrane fraction was applied to the gel.
ANTI- NB1
AHN-1.1
ABCDEF
G H I J K L
BIOCHEMICAL CHARACTERIZATION OF NE1
753
both the highcr and lowcr mol-wt proteins wcrc visualized by
probinpwith A H N - I . I (not shown).
n
Ea
0
DISCUSSION
-6ooo
U
c
3
mo4OOo
%
U
0
-
E =
c
U
0
AHN. 1 1
AHN.9.1
AHN. 1 . 1
Antibody
Fig 12. Reaction of MoAbs with subcellular fractions as
determined by radioimmunoassay. Aliquot. of each subcellular
fraction as indicated (0.1 pg for each test antibody except
AHN-1.1. for which 0.6 pg of each fraction was used) was
adsorbed t o microtiter wells: the wells were then blocked with
BSA. and antibody binding was determined as described in the
text. Primary granule fraction (solid bars). secondary granule
fraction (horizontal hatched bars). membrane fraction (dotted
bars), cytoplesm (diagonal hatched bars). Specific binding (cpm
bound using culture supernatant of the test hybridoma as the test
antibody
cpm bound using culture media as the test supernatant) is shown as the mean of two separate determinntions. In
each case. the negative control culture media yielded a signal of <:
1lOCpm.
ANTI-NB1
ABC
(3
I oF
97.466-
X
r'
Neutrophil-specific antibodies arc often idcntificd in autoimmunc neutropenia of childhood. neonatal alloimmunc
ncutropcnia, and transfusion-rclatcd acute lung injury.'
Most frcqucntly. these antibodics arc directed against the
ncutrophil antigen systcms S A and NR."." The ncutrophilspecific XR antigen systcm is composed of two alleles, S R I
and S R 2 . ' Although monoclonal antiscra havc been used to
identify a varicty of Icukocytc surface antigens. littlc is
known about the structurcs dcfincd by neutrophil-spccific
alloantisera. Our study shows that the ncutrophil-specific
antigcn S R I is prescnt on a 58- to 64-Kd protein located on
ncutrophil plasma mcmbrancs and on mcmbrancs of sccondary grmulcs. All anti-NRI antibodics tested dctcctcd a
55-Kd protein by immunoblotting on plasma membrane
prcparations from YRI-positivc individuals but did not react
with this protcin on plasma membrane prcparations from
YRI-ncpativc individuals. Antibodies to VRI immunoprecip
itatcd a 5 8 - to 64-Kd protein from NRI-positive neutrophils.
but did not immunoprecipitate any protein in this mol-wt
rangc from N R I -negative neutrophils. Adsorption studies
provided furthcr cvidcncc that the 58- to 64-Kd glycoprotcin
bears thc S R I antigen. The ability of anti-NRI antisera to
immunoprccipitatc thc 58- to 64-Kd G P was lost whcn
antiscra wcrc adsorbed with NRI-positive neutrophils but
AHN-1.1
AHN-9
NHS
-
GHI
JKL
DEF
1
c
4529-
Fig 13. Immunoblots of soluble and membrane componms of 8uondary granulos. Secondary granules from NE1-poskive noutrophlls
were wsponded in 20 mmol/L Tris-HCI, pH 7.0. freoze-thawed, sonicated, and centrifuged as described in the Material and Mothods
section. The supernatant ("firn supernatant") W.S saved, and the pellet was resuspended in Mx) mmol/L NaCI. 20 mmol/L Tris-HCI. pH
7.0. freoze-thawed. sonicated, and centrifuged again. The t w o supernatants and the resulting membranes were analyzed by SDS-PAGE in a
10% gel under nonreducing conditions (lanes A through C) or reducing conditions (lanes D through L). and immunobloned with anti-NB1
117.0091 (lanes A through C). AHN-1.1 (lanes D through F). ANN-9 (antilactoferrin) (lanes c) through I).and NHS (lanes J through L). First
supernatant (lanes A. D. (3, and J ) , second supernatant (lanes B. E. H. and K):pelleted membranes (Ian- C. F. 1. and L). Fitteon micrograms
of protein was added t o each lane. Molecular weight standards were as described in the legend to Fig 1.
STRONCEK, SKUBITZ, AND McCULLOUGH
754
was not lost when antisera were adsorbed with NB1-negative
neutrophils. These results were consistent with the preliminary results reported by M ~ l d e r . ~ ~
The 58- to 64-Kd G P immunoprecipitated by anti-NB1
had a slightly decreased electrophoretic mobility under
reducing as compared with nonreducing conditions. The
epitope on the 58- to 64-Kd protein recognized by anti-NB1
antisera was destroyed by reducing agents but not by
treatment with periodate. The 58- to 64-Kd protein bearing
the N B l antigen was present not only on neutrophil plasma
membranes but also in secondary granules. Most of the N B l
antigen in secondary granules was located in the granule
membrane.
Three of four anti-NB1 antisera reacted with an 80-Kd
G P in addition to the 58- to 64-Kd GP. Reference anti-NB1
antiserum NIH-FD-25 1-601-C never reacted with this protein, however. The presence of this protein on neutrophil
membranes did not correlate with the presence of N B l
antigens on intact cells as measured by granulocyte agglutination and immunofluorescence assays. Further evidence
that NB1 was not present on the 80-Kd protein was the
ability of anti-NB1 17.009 to immunoprecipitate the 80-Kd
protein from autologous neutrophils. The antibody to NB1
from this donor was an alloantibody detected when her child
developed neonatal alloimmune neutropenia. Because the
donor made antibodies to the NB1 antigen and because she
had no history of autoimmune neutropenia, her neutrophils
should not contain the NB1 antigen; thus, the 80-Kd protein
detected on her cells should not contain the NB1 antigen.
However, the ability of three of four anti-NB1 antisera to
immunoprecipitate the 80-Kd gp whereas four antisera to
N A l , three antisera to NA2, one antiserum to NB2, and two
antisera to Mart did not precipitate the 80-Kd protein
suggests, however, a possible relationship between this protein and development of anti-NB1 antibodies. Serologic
attempts to identify an undescribed neutrophil antigen with
these antisera that could represent the 80-Kd protein have
not identified such an antigen system.
We were unable to identify a protein bearing the NA1,
NA2, NB2, or Mart antigens. Other investigators reported
that some CD16 MoAbs react with NAl-positive cells but
not with NAl-negative cells and that anti-NA1 alloantibodies block the ability of these CD16 MoAbs to bind N A I positive ne~trophils.’~.’~
Although these reports showed that
the CD16 MoAbs immunoprecipitate a 50- to 60-Kd protein,
no immunoprecipitation studies have been reported with
anti-NA1 alloantibodies.
Thus, anti-NB1 antisera identified a 58- to 64-Kd G P on
the surface of NB1-positive human neutrophils that was also
present in the secondary granule membranes. The electrophoretic mobility of this protein was altered slightly by
reducing agents, consistent with the presence of intrachain
disulfide bonds, and the epitope was sensitive to reducing
agents but not treatment with periodate. These results
suggest the NB1 epitope is not a carbohydrate but probably
resides in the tertiary structure of the protein backbone. The
role of this protein in neutrophil function remains undefined.
ACKNOWLEDGMENT
We thank Raji Shankar and John Mediolia for excellent technical
assistance, Drs A. Dalmasso and A. Skubitz for helpful discussion
and a critical review of the manuscript, and Bobbie Gibson for
manuscript preparation.
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