Subcellular neutrophils
localization
of CD66,
Thomas
P. Ducker
and
Department
of Medicine,
University
Keith
M. Skubitz of Minnesota
Medical
Abstract: CD66 and CD67 are granulocyte-specific activation antigens; their surface expression is up-regulated when neutrophils are activated. CD66 antibodies recognize an 180-kd neutrophil surface protein that is also recognized by anti-carcinoembryonic antigen (CEA) antibodies and is therefore a nonspecific cross-reacting antigen (NCA). CD67 antibodies recognize an 100-kd neutrophil surface protein that is attached to the membrane via a glycosyl-phosphatidylinositol anchor. To identify an intracellular pool from which CD66 and CD67 could be up-regulated, the subcellular distribution of proteins recognized by CD66 and CD67 monoclonal antibodies and polyclonal anti-CEA was studied. Neutrophil plasma membranes, granules, and cytoplasm were prepared by nitrogen cavitation and differential centrifugation and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Most of the 180-kd protein recognized by CD66 antibodies and the 100-kd protein recognized by CD67 antibodies were located in the secondary granule fraction, with lesser amounts detectable in the plasma membrane fraction. Several NCA species ranging from 40 to 200 kd were identified, and the distribution of these NCAs was different in the primary granules, secondary granules, and plasma membrane fractions. The major NCAs in the plasma membrane fraction were of 95 to 100 and 180 to 200 kd; the secondary granule fraction contained major NCAs of 42, 85, 95 to 100, and 180 to 200 kd. NCAs were also detected in the primary granule fraction, the most prominent being of 90-100 kd; no NCA of 180 to 200 kd was detected in the primary granules. The presence ofCD66, CD67, and NCAs in the secondary granules suggests secondary granules as a likely source from which surface with the
primary
11-16; Key -
these antigens activation. granules
is
The
could be potential
recruited role
J.
unknown.
to for
Leukoc.
the NCAs
cell in
Biol.
52:
1992. Words:
carcinoembryonic
antigen
activation
antigen
granulocyte
INTRODUCTiON The recently granulocyte-specific
defined
CD66 “activation
and antigens”
CD67, and NCA in human
CD67 antigens in that they
tected in low density on resting neutrophils regulated on the surface of stimulated neutrophils modulation of the surface expression of CD66 with cell activation suggests that they may play
are
are de-
are up[1, 2]. The and CD67 a role in the
School,
and
the Masonic
Center
of the CD66 antigen cytoplasts, suggesting
could that
Center, Minneapolis,
Minnesota
not be detected in neutrophil the protein is mobilized to the
surface from intracellular stores [3, 4]. Immunoprecipitation studies demonstrated that the CD66 antigen was present on a protein of approximately 180 kd [1, 3] that also appears to be one of the major endogenous substrates for neutrophil ecto-protein kinase activity [5-7]. Phosphoamino acid analysis of this protein revealed that it contained predominantly phosphotyrosine, with a lower level of phosphoserine. Preclearing studies demonstrated that this protein is also recognized by CD1S monoclonal antibodies [6-8]. The CD67 antigen is present on a lOO-kd neutrophil surface protein, and its expression on the neutrophil surface is up-regulated by stimulation with fMLP and TPA and C5a [2]. The expression of the CD67 antigen is decreased when neutrophils are treated with phosphatidylinositol-specific phospholipase C, demonstrating that it is linked to the membrane via a glycosyl-phosphatidylinositol anchor [2J. Early work trying to identify tumor-specific antigens led to the description of carcinoembryonic antigen (CEA) [9, 10]. CEA was first described as a l80-kd tumor-associated cell surface glycoprotein on colon cancer cells. Since the discovcry of CEA [9, 10], a number of closely related, crossreacting antigens have been found in normal cells [11, 12]. Of these cross-reacting antigens, the classical “nonspecific crossreacting antigen” (NCA) is found on granulocytes and macrophages [11-13]. The CEA gene is one ofa family ofat least 17 closely related genes that belong to the immunoglobulin gene superfamily [11, 12]. In neutrophils, four species of NCA of 160, 95, 90, and 55 kd have been identified using surface labeling with i25I [11, 14-16]. The 180-kd neutrophil phosphoprotein recognized by CD66 monoclonal antibodies is also recognized by polyclonal anti-CEA [6] and thus may represent the 160-kd form of the classical NCA on human neutrophils or a new NCA. Furthermore, immunoblotting studies have revealed that CD66 antibodies react with purified CEA, NCA, and biliary glycoprotein (BGP) [6]. Similarly, van der Schoot et al. [17] have found that the neutrophil protein recognized by CD66 monoclonal antibodies can be adsorbed by polyclonal anti-CEA, and Watt et al. [18] have found that CD66 antibodies react with CEA-related molecules. In addition, polyclonal anti-CEA can preclear the 100-kd neutrophil protein recognized by CD67 monoclonal antibodies [17]. Although studies of CEA, NCA, and BGP suggest a potential function in both homotypic and hetero-
but
functional responses of neutrophils to stimulation. The CD66 antigen is up-regulated by stimulation with the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP), the calcium ionophore A23187, and 12-0tetradecanoylphorbol-13-acetate (TPA) [1, 3, 4]. Up-regulation
Abbreviations: lin
BGP,
fMLP,
gen; M;
PBS,
biliary
NCA,
nonspecific
Reprint neapolis,
Received
Journal
CEA,
carcinoembryonic
gel MN
saline;
antigen; SDS-PAGE,
electrophoresis;
requests:
1gM,
cross-reacting
phosphate-buffered
acrylamide
glycoprotein;
N-formylmethionyl-leucyl-phenylalanine;
TPA,
Keith
M.
NMS, sodium
normal dodecyl
anti-
immunoglobumouse
serum;
sulfate-poly-
12-O-tetradecanoylphorbol-13-acetate.
Skubitz,
Box
325,
University
Hospital,
Min-
55455.
November
27,
of Leukocyte
1991;
accepted
Biology
February
Volume
11, 1992.
52, July
1992
11
typic adhesion [19-21], the roles of CD66, CD67 and NCA in neutrophil function are unknown; their appearance on stimulated cells suggests a potentially important role in neutrophil function. Because many neutrophil activation antigens are upregulated from intracellular stores [22-29], we examined the subcellular distribution of proteins recognized by CD66 and CD67 monoclonal antibodies and also polyclonal anti-CEA. Neutrophil plasma membranes and granules were prepared by nitrogen then analyzed
cavitation by sodium
and differential centrifugation dodecyl sulfate-polyacrylamide
and gel
electrophoresis (SDS-PAGE) and immunoblotting. The data suggest that most of the neutrophil proteins recognized by CD66 and CD67 monoclonal antibodies are located in the secondary granule fraction, with lesser amounts detectable in the plasma membrane fraction. NCA species with different electrophoretic patterns were detected in the primary granule, secondary granule, and plasma membrane fractions.
MATERIALS
AND METHODS
Cell Preparation Normal peripheral blood neutrophils were prepared from heparinized (2 U/ml) blood by a modification of the method of Boyum as previously described [30] and were suspended at the indicated concentrations in 145 mM NaCl and 20 mM HEPES (Gibco, Grand Island, NY), pH 7.3 (NaCl-HEPES), or phosphate-buffered saline (PBS), pH 7.3. Differential cell counts on Wright-stained cells routinely revealed more than 95% neutrophils. Viability as assessed by trypan blue dye exclusion was greater than 98%.
Antibodies
and Reagents
The CD15 monoclonal antibodies AHN-1 (immunoglobulin M, 1gM) and AHN-1.1 (1gM) [31] and the CD45 antibody AHN-12 [32] have been previously described. The CD66 antibody gran 10 [3, 4] and the CD16 antibody CLB FcR-gran 1 were gifts ofDrs. E. van der Schoot and A. von dem Borne, CLB, Amsterdam, The Netherlands. Monoclonal antibody PMN7C3 (IgG3) [33] was a gift ofDr. H. Malech, National Institutes of Health, Bethesda, MD. The CD67 antibody G1OF5 [2, 3] was a gift of Dr. J. Thompson, VA Medical Center, Lexington, KY. Rabbit anti-CEA was obtained from Accurate Chemical Corp., Westbury, NY. Monoclonal antibody T84.1, which reacts with CEA [34], was a gift of Dr. R. Paxton, City of Hope, Duarte, CA. CEA, purified from human colon cancer cells metastatic to the liver, BGP, purified from human bile, and NCA, purified from colon cancer metastatic to liver [34-36], were gifts from Dr. R. Paxton.
Subceliular
Fractionation
of Human
Neutrophils
Neutrophils from 240 ml of whole blood were isolated described above and suspended at 5 x 107/ml in PBS. isopropylfluorophosphate (Sigma) was added to 10 mM the cells were incubated for 15 mm at 0#{176}C.Neutrophil cellular fractionation was performed as described by regaard et al. [37]. Neutrophils (4 x 108)
as Diand subBorwere
resuspended in 15 ml of ice-cold relaxation KC1, 3 mM NaCl, 1 mM adenosine (Sigma), 3.5 mM MgCl2, 10 N,N-bis(2-ethanesulfonic acid) (PIPES),
buffer [100 mM triphosphate (Na)2 mM piperazinepH 7.4 (Sigma)].
This
20
12
solution
Journal
was
then
equilibriated
of Leukocyte
Biology
for
Volume
mm
at 4#{176}C with
52, July
1992
nitrogen at 350 psi with constant slow stirring ruption bomb (minibomb, Parr Instrument IL). The cavitate was then collected dropwise 62.5 mM EGTA (Sigma), pH 7.4, in relaxation
in a cell disCo., Moline, in 300 jl o buffer, pH
7.4. Nuclei and unbroken cells were removed by centrifugation at 500g for 20 mm at 4#{176}C.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 (final concentration 1 x relaxation buffer, pH 6.8, with 1.25 mM EGTA). The 1.120 density Percoll (14 ml) was laid under 1.050 density Percoll (14 ml) in 50-ml polycarbonate tubes, and the supernatant was loaded onto the Percoll gradients, which had been precooled to 4#{176}C.The gradient was then centrifuged at 4#{176}Cfor 15 mm at 48,000g. The cytoplasmic fraction and the three bands representing the azurophiic granules (a fraction), specific granules (f3 fraction), and plasma membranes (y fraction) were then collected, and the Percoll was removed from the samples by centrifugation at l8O,000g for 90 mm at 4#{176}C.The pelleted membranes and granules were washed with relaxation buffer and centrifuged again at l8O,000g for 90 mm at 4#{176}C.The samples were then either resuspended in reducing Laemmli sample buffer [38], incubated at 100#{176}C for 2 mm, and stored at - 70#{176}C, or resuspended in nonreducing Laemmli sample buffer, incubated at 23#{176}Cfor 30 mm, and stored at - 70#{176}Cuntil use. Protein concentrations of the fractions were determined by the bicinchoninic acid protein assay (Pierce).
32p Labeling, Immunoprecipitation, Gel Electrophoresis of Neutrophii
and Poiyacrylamide Proteins
Two 32P-labeling techniques were utilized. Metabolic labeling with H332PO4 was performed exactly as previously described [6, 8]. 32 labeling of cell proteins by incubation of intact viable cells with [y-32P]ATP, a technique that presumably utilizes a neutrophil ecto-protein kinase activity, was performed as previously described [7, 8]. Immunoprecipitation, SDS-PAGE, and autoradiography were performed as previously described [8].
Immunobiotting Samples were suspended Tris-HC1, pH 6.8, 2% bromphenol blue with [38] and incubated for trophil protein or 1 jg applied to each lane of
in Laemmli sample buffer (62 mM SDS, 10% glycerol, and 0.001% or without 5% 2-mercaptoethanol) 2 mm at 100#{176}C, and 15 g of neuof purified CEA, BGP, or NCA was a 10% polyacrylamide gel and dcc-
trophoresis was performed in the Laemmli buffer system. Molecular weight standards were purchased from Sigma. Neutrophil extract was prepared by suspending 1 x 108 cells, pretreated with diisopropylfluorophosphate, in 1 ml of lysis buffer (20 mM Tris-HC1, pH 7.6, 150 mM NaC1, 0.5% Nonidet P-40, 0.02% NaN3, and 2 mM phenylmethylsulfonyl fluoride), incubating on ice for 15 mm, and centrifuging at l3,700g for 15 mm at 4#{176}C.The supernatant was mixed with twice-concentrated Laemmli sample buffer as above. The gels were then equilibriated in transfer buffer (4 parts 60 mM Tris-HC1, pH 9.2, 49 mM glycine, and 1 part methanol) for 30 mm and electroblotted onto Immobilon paper (Millipore Corp., Bedford, MA) at 200 mA for 1 h at 23#{176}C.After transfer of proteins onto the Immobilon paper, the Immobion strips were washed twice with 20 mM TrisHC1, pH 7.5, 0.5 M NaCl containing 0.3% Tween-20 (Sigma) (TBS-Tween). The strips were then blocked with TBS-Tween containing 5% normal goat serum (Gibco) overnight. After two washes with incubated for 2 h in rabbit
TBS-Tween, antibody
or
the Immobilon was mouse monoclonal
ABCD
ABCD
ABCD
ABCD
200-. 116_
97.4-
cv)
6645-
29-
Fig.
1. Immunoblots
granules
(a
of neutrophil
fraction)
polyacrylamide or coli
gel
normal
shown.
(lanes
mouse
Fifteen
under serum
cytoplasm Neutrophils
reducing (NMS)
as
described
of protein was 116,000; phosphorylase
diluted
1:1000
(lanes were
conditions,
micrograms
j3-galactosidase,
antibodies
D).
in
A),
plasma
transferred in
membranes
fractionated the
by
TBS-Tween
(y
centrifugation
to Immobilon-P,
text.
This
with
goat serum. After three washes in TBS-Tween, incubated for 1 h at 23#{176}Cwith biotinylated IgG or biotinylated goat antimouse IgG and Cooper Biomedical, Malvern, PA) diluted Tween with 5% normal goat serum. The washed three times in TBS-Tween without
was
Proteins
used
serum
albumin,
5%
normal
the blots were goat antirabbit 1gM (Cappel, 1:1000 in TBSImmobilon was goat serum and
RESULTS Localization
Some neutrophil membrane sion can be up-regulated granules [22-29]. Therefore,
of CD66 and CD67 Antigens are
proteins whose also located the presence
surface expresin intracellular of the activation
antigens CD66 and CD67 in primary granules, secondary granules, and plasma membranes was studied by immunoblotting. Percoll gradients were used to purify a plasma membrane-rich fraction (‘y fraction), primary granules (a fraction), and secondary granules (j3 fraction), as well as cytosol as described in Methods. The CD66 monoclonal antibody gran 10 detected an 180-kd protein in both the plasma membrane (Fig. 1, lane B) and secondary granule (lane C) fractions but not in cytosol (lane A) or primary granules (lane D). Similarly, the CD67 antibody G1OF5 identified an 100-kd protein in both the plasma membrane (lane B) and secondary granule (lane C) fractions but not in cytosol (lane A) or primary granules (lane D). As expected, the CD15 antibody
AHN-1.1
identified
multiple
proteins
primarily
Ducker
(lanes
in the and Skubitz
B),
secondary
discontinuous
Percoll
immunoblotted performed
as molecular
then incubated for 30 mm with avidin-conjugated alkaline phosphatase (Cappel) diluted 1:5000 in TBS-Tween with 5% normal goat serum. After washing twice in TBS-Tween and twice in water, proteins were visualized by incubation in 0.3 mg/ml nitroblue tetrazolium (Sigma), 0.2 mg/mI 5-bromo-4-chloro-3-indoylphosphate (Sigma), 100 mM NaCl, 50 mM MgCl2, and 100 mM Tris-HC1, pH 9.5, for 20 mm at 23#{176}C[39]. The reaction was stopped by washing in distilled water.
Subceiiular
fraction) over
and
experiment
loaded in each lane. a, 97,400; bovine
NMS
CD15
CD67
CD66
66,000;
with with
five
weight ovalbumin,
gran different
standards 45,000;
granules
(/3 fraction)
gradients, 10 (CD66), donors
were and
(lanes
separated
by
G1OF5 and
myosin
(CD67),
representative
heavy
carbonic
C),
and
primary
SDS-PAGE
in a
AHN-l.l immunoblots
chain,
anhydrase,
200,000;
10%
(CD15), are
Escherichia
29,000.
plasma membrane and secondary granule fractions but normal mouse serum (NMS) did not (Fig. 1). In addition, as previously reported for other CD45 antibodies [23], the CD45 antibody AHN-12 identified an 180-kd protein in the /3 and -y fractions but not in the a fraction or cytosol (not shown). The type III Fc-y receptor, like most CEA species, is anchored to the membrane by a glycosyl-phosphatidylinositol linkage [40-42]. In contrast to CD67, the CD16 antibody CLB FcR-gran 1 detected the type III Fc-’y receptor only in the membrane fraction, but not in the a, 13, or cytoplasm fraction (data not shown).
Immunoprecipitation of 32P-Labeled CD67 Monoclonal Antibodies
Neutrophil
Proteins
with
To further characterize the antigen recognized by CD67 antibodies, we attempted to determine if the protein was phosphorylated. Two radiolabeling techniques were utilized. In one, viable neutrophils were incubated with H332PO4 to metabolically radiolabel phosphoproteins. In the second radiolabeling technique, viable neutrophils were incubated with [y-32P]ATP utilizing a reaction in which presumably phosphorylation occurs via a neutrophil ecto-protein kinase activity [5, 7, 8]. No detectable 32P-labeled proteins were immunoprecipitated by G1OF5 from extracts of cells radiolabeled by either technique (data not shown). These experiments were performed with cells from four different donors. As expected [3], G1OF5 did immunoprecipitate an 125I labeled protein from surface-labeled neutrophils (data not shown). Also as expected [6, 8], CD15 and CD66 antibodies immunoprecipitated a 180-kd ‘2P-labeled protein from extracts
of cells
Reactivity
of
labeled
with
G1OF5
with
y..32P
CEA,
(data
not
shown).
BGP,
and NCA
Because the CD67 antibody B13.9 [17] has been shown to react with purified CEA, the reactivity of G1OF5 with purified CEA and the related proteins BGP and NCA was tested by immunoblotting. As expected, monoclonal antibody G1OF5 detected the 100-kd protein in the neutrophil
Subcellular
localization
of CD66,
CD67,
and
NCA
13
extract (Fig. 2, lane A). However, no reactivity of G1OFS with purified CEA (lane B), BGP (lane C), or NCA (lane D) was detected, although these proteins were readily detected by immunoblotting with the anti-CEA antibody T84.1 (ref. 6,
and
not
ABCD
shown).
Subcellular
200
Localization
of NCAS in Human fractions
subcellular
munoblotting NCAs (Fig.
with polyclonal rabbit anti-CEA to detect 3). Anti-CEA detected a complex pattern of promolecular fraction
also
studied
by
im-
0
45-
size from 40 to 200 (Fig. 3, lane C). In con-
NCAs were also detected in the primary granule fraction (lane D). The most prominent NCA in the primary granule fraction was of 80-120 kd; much less of the NCA of 180 to 200 kd was observed in the a fraction than in the y and
anti-CEA Fig.
3.
Immunoblots
()(
fraction)
primary
of 85 and 42 kd (lane C) fraction compartment.
were and
prominent largely res-
experiments
Neutrophil pression probably regulated
activation is associated of a number of molecules play a role in neutrophil molecules may modulate
specific response. regulated intracellular
effector molecules Several molecules after stimulation pools as well
with increased surface ex[22-29]. These molecules function, and the upneutrophil function or be
involved in the neutrophil whose surface expression is uphave been shown to be located in as on the surface, and the in-
ABCD
weight
granules
B), (a
using standards
different were
D).
anti-CEA
Representative donors
the
(/3
same
as
A),
plasma
fraction)
Neutrophils
rabbit
text.
(lanes
granules
(lanes
polyclonal in the five
cytoplasm
secondary
fraction)
with
as described
creased surface tion from these plc, CD11 and granules regulation
NRS
of neutrophil
(lanes
immunoblotted (NRS), lar
DISCUSSION
-
29
trast, few anti-CEA reactive proteins were detected in the cytoplasm fraction (lane A); in some experiments no reactivity with cytosol was detected (not shown). The most prominent NCA species identified in the plasma membrane fraction were of 95 to 100 kd and 180 to 200 kd (lane B).
i3 fractions. NCA species in the secondary granule tricted to this subcellular
-
C,)
Neutrophil
teins ranging in apparent kd in the secondary granule
were
Neutrophils
ABCD
were or
membranes
(lanes
normal
immunoblots
C),
rabbit of five
are
shown.
Proteins
in
Figure
1.
and
fractionated
used
and serum separate
as molecu-
expression is thought to be due to translocaintracellular pools to the surface. For examCD18 have been found in the secondary
and also is associated
[22, 23, 25]. Similarly, secondary and tertiary here demonstrate that present in the secondary
the
tertiary granules, with degranulation
and their upof these granules
CD45 has also been found in the granules [23]. The studies reported the CD66 and CD67 antigens are also granule fraction as well as on the
membrane. Thus, secondary granules are a likely source from which up-regulation of these antigens with stimulation occurs. Although the subcellular fractionation technique used in this study resolved cytosol and the a, /3, and ‘y fractions, heterogeneity within these fractions is likely. For examplc, evidence for the existence of subpopulations of granules among the “specific granules” has been presented [43]. In addition, CD66 and CD67 may be present in the “tertiary granules” that contain gelatinase or other granule populations that are distributed largely in the /3 fraction obtained by the fractionation technique employed in this study [44, 45]. CD67 has been shown to be anchored to the membrane via a glycosyl-phosphatidylinositol linkage [2, 46]. CD16, the type III FcR-’y receptor, is also attached to the membrane via a glycosyl-phosphatidylinositol linkage [40-42]. Depending on the conditions of stimulation, CD16 expression has been reported to increase or decrease on the neutrophil surface after stimulation [4, 47, 48]. Interestingly, CD16 could not be detected in the study. Jost et al.
Fig.
2.
Purified resolved
Immunoblotting
CEA, by
periments purified neutrophil
14
and in
of
purified
NCA,
and
SDS-PAGE
Immobilon-P, described
BGP,
the
CEA,
BGP,
proteins;
Journal
under
immunoblotted
text.
is shown.
CEA,
Fifteen lane
the
CD67
immunoblot
micrograms
NCA
was
loaded
B,
CEA;
lane
of Leukocyte
and
of in C,
Biology
NCA
neutrophil conditions,
with
A representative or
BGP,
solubilized reducing
with
antibody from
lane.
BGP;
lane
Volume
D,
to
separate
protein Lane
were
G1OF5
three
neutrophil each
G1OF5.
proteins transferred
or A,
exlg
solubilized
NCA.
52,
July
as
1992
of
secondary [40] have
granule fraction also reported that
in the present CD16 could not
be detected in the secondary granule fraction, suggesting that the intracellular pool from which CD16 can be upregulated must reside in a different granule fraction. However, one cannot exclude the possibility that CD16 normally resides in a highly mobilizable granule fraction that is distributed in the secondary granule fraction obtained by this fractionation technique but is mobilized to the cell surface during cell preparation. The 180-kd protein recognized by CD66 antibodies is also recognized by anti-CEA and is thus an NCA of neutrophils. Others have demonstrated that CD67 antibodies react with
CEA due CEA
[17]; our results did not confirm this, but this may be to the limits of the sensitivity of the assay we employed. and NCA have been shown to undergo homophilic and
heterophilic adhesion CD67 may also perform CD67, multiple NCA
[19-21]. This suggests that CD66 adhesive functions. Like CD66 species recognized by anti-CEA
and and were
found in the secondary granules, and the pattern of NCA molecules observed in the secondary granules was similar to that observed in the membranes. In contrast to CD66 and CD67, however, multiple NCA species were also detected in the a-granule fraction. However, the NCAs detected in the a fraction differed in molecular weight from those in the secondary granule and membrane fractions. The NCA species in the secondary granule fraction might serve as an intracellular pool from which to augment surface expression of these molecules after appropriate stimulation. The potential role of NCA molecules in the primary granule fraction is less clear.
11. Thompson, J., and Zimmerman, W. The carcinoembryonic antigen gene family: structure, expression and evolution. Tumor BioL 9, 63, 1988. 12. Zimmerman, W., and Thompson, J. 1990. Recent developments concerning the carcinoembryonic antigen gene family and their clinical implications. Tumor Biol. 11, 1, 1990. 13. Wahren, B., Gadler, F., Gahrton, G., Hammarstr#{246}m, S., Hareland, Y., Hyd#{233}n,N., Ljungdahl, E., M.hl#{233}n, A., Rud#{233}n, U., and Wikiund, M. NCA: A differentiation antigen of myelopoietic cells in humans and hominoid monkeys. Ann. NY. Acad. Sci. 417, 344, 1983. 14. Kuroki, M., Matsuo, Y., Kuroki, M., and Matsuoka, Y. Nonspecific
643,
We thank Drs. E. van der Schoot, A. von dem Borne, J. Thompson, and H. Malech for providing antisera; Dr. R. Paxton for providing purified CEA, BGP, and NCA, and antibody T84.1; Dr. A. Skubitz for helpful discussions and a critical review of the manuscript; and C. Stocke for manuscript preparation. Supported in part by National Institutes
of Health the
Masonic
grant
CA36248,
Memorial
Hospital
the
Leukemia Fund,
Task
Force,
Inc.
M.,
identifies weight
(NCA)
expressed
munol. 17.
van
24,
der
1. Majdic, 0. Cluster report: CD66. In Leucocyte Typing IV (Knapp, W., et al, Eds.) New York: Oxford University Press, p. 838, 1989. 2. Stockinger, H. Cluster report: CD67. In Leucocyte Typing IV (Knapp, W. et al). New York: Oxford University Press, p. 840, 1989. 3. van der Schoot, CE., Daams, M., Kr. von dem Borne, A.E.G., Skubitz, KM., Skubitz, A.P.N., Kniep, B., Agthoven, A.V., Brailly, H., Romagne, F., Lanini, S., Civin, C.!., Fackler, M.J., Chorvath, B., Duraj, J., Horesji, V., Basil, V., Hildreth, J., Hyman, J. , and Tetteroo, PAT. Biochemical analysis of the myeloid antibody panel. In Leucocyte Typing IV (Knapp, W. et al., Eds.) New York: Oxford University Press, p. 868, 1989. 4. Tetteroo, PAT., Bos, M.J.E., Visser, F.J., and Kr. von dem Borne, A.E.G. Neutrophil activation detected by monoclonal antibodies. j ImmunoL 136, 3427, 1986. 5. Dusenbery, K.E., Mendiola, JR., and Skubitz, KM. Evidence for ecto-protein kinase activity on the surface of human neutrophils. Bioc/zein. Biophys. Res. Commun. 153, 7, 1988. 6. Skubitz, K.M., Ducker, T.P., and Goueli, S.A. CD66 monoclonal antibodies recognize a phosphotyrosine-containing protein bearing a carcinoembryonic antigen cross-reacting antigen on the surface of human neutrophils. j ImmunoL 148, 852 (1992). 7. Skubitz, K.M., Ehresmann, D.D., and Ducker, T.P. Characterization of human neutrophil ecto-protein kinase activity released by kinase substrates. j ImmunoL 147, 638, 1991. 8. Skubitz, K.M., Mendiola, J.R., and Collett, MS. CDI5 monoclonal antibodies react with a phosphotyrosine-containing protein on the surface of human neutrophils. j Immunol. 141, 4318, 1988. 9. Gold, P., and Freedman, S.0. Demonstration of tumor-specific antigens in human colonic carcinomata by immunological tolerance and absorption techniques. j Exp. Med. 121, 439, 1965. 10. von Kleist, S., and Burtin, P. Misc en evidence dans les tumeurs coliques humaines de l’adulte normal. C. R. Hebd Seances Acad. Sci. 253, 1543, 1966.
and Skubitz
new forms of crossreacting and 160,000 in normal
1177,
by
human
and
Mach, J. -P. antigen (CEA)
antigens granulocytes.
of
molecular
Mol.
Im-
1987.
Schoot,
CE.,
Kuijpers,
T.W.,
Daams,
M.,
and
von
dem
Borne, belong
A.E.G.K. The granulocyte CD66 and CD67 antigens to the CEA family and therefore to the Ig-supergene family. Blood 76, 196a, 1990. 18. Watt, SM., Sala-Newby, G., Hoang, T., Gilmore, D.J., Grunert, F., Nagel, G., Murdoch, S.J., Tchilian, E., Lennox, ES., and Waldmann, H. CD66 identifies a neutrophil-specific within
members
REFERENCES
Buchegger, F., Schreyer, M., antibody against carcinoembryonic
two 90,000
epitope
Ducker
antigen
1984.
16. Audette, Monoclonal
ACKNOWLEDGMENTS
and
cross-reacting
granulocytes: six species with different peptide sizes and membrane anchoring forms. Biochem. Biophys. Res. Commun. 166, 701, 1990. 15. Buchegger, F., Schreyer, M., Carrel, S., and Mach, J. -P. Monoclonal antibodies identify a CEA crossreacting antigen of 95 kD (NCA-95) distinct in antigenicity and tissue distribution from the previously described NCA of 55 kD. Int. j Cancer 33,
the
of
molecules. 19. Oikawa, G. , and
the
antigen
(CEA)
expressed
ophilic
adhesion.
Benchimol,
(NCA)
Fuks,
Rojas,
M.,
mily,
functions
sion
molecule. R.F.,
III,
Peters,
WA.,
large
subunit
Lacal,
of
N.,
with
1990.
Bilary
supergene intercellular
1, 527,
Crowley,
CA.,
localization
formerly
neutrophil
P.,
Pulido,
R.,
location j BioL
Berger,
M.,
ta.koff,
AM.
gp
of
110),
adhesion.
Sanchez-Madrid,
in
1991.
Wetzler,
EM.,
Miller,
L.J.,
human
F.,
ofT200 and Mol Chem. 263, 9946,
Intracellular
3019,
j
the
a surface Clin. Invest.
Welter,
sites neutrophils.
Bainton,
D.F.,
and
Mollinedo,
glycoproteins 1988.
E.,
Turner,
JR.
for storage and recycling Proc. Nail. Acad. Sci.
Borregarrd,
N.,
and
specific
C3bi/fibrinogen,
granules fibronectin,
containing
, and Tarof C3b USA 88,
Springer,
and
receptors vitronectin
for in
F.
in human
Stimulated mobilization of monocyte Mac-l and p150,95 sion proteins from an intracellular vesicular compartment cell surface. j Clin. Invest. 80, 535, 1987. 26. Singer, 1.1., Scott, S., Kawka, D.W., and Kazazis, D.M. somes:
faadhe-
1990. RE.,
Subcellular
(MOla;
a human molecule.
C.P.
Rosin,
B.M.
Shirota,
antigen, adhesion
1984.
receptors
25.
Commun.
Beauchemin,
Stanners,
A.,
Mol
and heter164, 39, 1989.
immunoglobulin
Dffer
Babior,
antigen
homophilic
a Ca2-dependent
Growth
associated
1280,
Intracellular neutrophils. 24.
as
Arnaout,
and
glycoprotein 23.
and
of the
vitro
Cell
Todd,
74,
A.,
in
S.,
by
adhesion
Matsuoka, Y., Kosaki, activity of non-specific
Carcinoembryonic as an intercellular
a member
of
carcinoembryonic Res.
Jothy,
is expressed
family
surface:
Biophys.
A.,
Fuks,
and
cell
Biochem.
S.,
glycoprotein,
22.
that
antigen
on CHO
K. , and Stanners, C.P. tumor marker, functions Cell 57, 327, 1989. 21.
system
Blood 78, 63, 1991. S., Inuzuka, C., Kuroki, M., Nakazato, H. Cell adhesion
cross-reacting
20.
hematopoietic
carcinoembryonic
human
TA.
adheto the Adhelaminin, poly-
morphonuclear leukocytes and monocytes. J. Cell Biol. 109, 3169, 1989. 27. Yoon, P.S., Boxer, L.A., Mayo, L.A., Yang, A.Y., and Wicha, MS. Human neutrophil laminin receptors: activationdependent receptor expression. j ImmunoL 138, 259, 1987. 28. Heiple, J.M., and Ossowskf, L. Human neutrophil plasmino-
Subcellular
localization
of CD66,
CD67,
and
NCA
15
gen activator is localized in specific granules and is translocated to the cell surface by exocytosis. j Exp. Med. 164, 826, 1986. 29. O’Shea, J.J., Brown, E.J., Seligmann, BE., Metcalf, J.A., Frank, MM., and Gallin, J.I. Evidence for distinct intracellular pools of receptors for C3b and C3bi in human neutrophils. j Immunol. 164, 2580, 1985. 30. Craddock, P.R., Hammerschmidt, D.E., White, J.G., Dalmasso, A.P., and Jacob, H.S. Complement (C5a)-induced granulocyte aggregation in vitro: a possible mechanism of complementmediated leukostasis and leukopenia. j Clin. Invest. 60, 260, 1977.
1987. Harvath,
L.,
Balke,
J.A.,
Christiansen,
NP.,
Russel,
A.A.,
and
Skubitz, KM. Selected antibodies to leukocyte common antigen (CD45) inhibit human neutrophil chemotaxis. j ImmunoL 146, 949, 1991. 33. Melnick, D.A., Nauseef, W.M., Markowitz, S.D., Gardner, J.P., and Malech, H.L. Biochemical analysis and subcellular localization of a neutrophil-specific antigen, PMN-7, involved in the respiratory burst. j ImmunoL 134, 3346, 1985. 34. Hefta, S.A., Paxton, R.J., and Shively, J.E. Sequence and glycosylation site identity of two distinct glycoforms of nonspecific cross-reacting antigen as demonstrated by sequence analysis and fast atom bombardment mass spectrometry. j BioL Chem. 265, 8618, 1990. 35. Paxton, R.J., Hefta, S.A., Hefta, L.J.F., Hinoda, Y., Lee, T.D., and Shively, J.E. 1989. Structural studies of the carcinoembryonic antigen gene family: sequence analysis and posttranslational modifications. In The Carcinoembryonic Antigen Gene Family (Yachi, A., and Shively, J.E., Eds.) Amsterdam: Elsevier Science Publishers, p. 23, 1989. 36. Shively, J.E., Hinoda, Y., Hefta, L.J.F., Neumaier, M., Hefta, S.A., Shively, L., Paxton, R.J., and Riggs, AD. 1989. Molecular cloning of members of the carcinoembryonic antigen gene family. In The Carcinoembryonic Antigen Gene Family (Yachi, A., and Shively, J.E., Eds.) Amsterdam: Elsevier Science Publishers, p. 97, 1989. 37. Borregaard, N., Heiple, J.M., Simons, E.R., and Clark, R.A. Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: translocation during
16
Journal
Blood
75,
744,
of Leukocyte
Biology
Volume
52, July
1992
during the as227, 680, 1970.
J.J.
Biochem-
antigen
NB1.
1990.
40. Jost, CR., Huizinga, TW.J., de Goede, R., Fransen, JAM., Tetteroo, P.A.T., Daha, MR., and Ginsel, L.A. Intracellular localization and de novo synthesis of FcRIII in human neutrophil granulocytes. Blood 75, 144, 1990. 41. Huizinga,
31. Skubitz, KM. and Snook, R.W., II. Monoclonal antibodies that recognize lacto-N-fucopentaose III (CD15) react with the adhesion-promoting glycoprotein family (LFA-1/HMAC-1/GP 150,95) and CR1 on human neutrophils.j ImmunoL 139, 1631, 32.
J. Cell BioL 97, 52, 1983. U.K. Cleavage of structural proteins sembly of the head of bacteriophage T4. Nature 39. Stroncek, D.F., Skubitz, KM., and McCullough, ical characterization of the neutrophil-specific activation.
38. Laemmli,
Kleijer,
T.W.J., M.,
Kr.
van von
der
dem
Schoot, Borne,
CE., A.E.G.,
PAT. The P1-linked receptor FcRIII of neutrophils. Nature 333, 667, 1988. 42.
Selvaraj,
P.,
Rosse,
W.F.,
Silber,
R.,
Jost,
C., Klaassen, R., D. , and Tetteroo, is released on stimulation Roos,
and
Springer,
TA.
major Fc receptor in blood has a phosphatidylinositol and is deficient in paroxysmal nocturnal haemoglobinuria. ture 333, 565, 1988. 43. Perez, H.D., Marder, S., Elfman, F., and Ives, HE. neutrophils contain subpopulations of specific granules ing
different
Biochem.
sensitivities
to
Res.
Commun.
Biophys.
changes
in
145,
cytosolic
976,
free
The
anchor Na-
Human exhibitcalcium.
1987.
44. Dewald, B., Bretz, U., and Baggiolini, M. Release of gelatinase from a novel secretory compartment of human neutrophils. j Clin. Invest. 70, 518, 1982. 45. Borregaard, N., Christensen, L., Bjerrum, OW., Birgens, H.S., and Clemmensen, I. Identification of a highly mobilizable subset of human neutrophil intracellular vesicles that contains tetranectin and latent alkaline phosphatase. j Clin. Invest. 85, 408, 1990. 46. van der Schoot, CE., Huizinga, T.W.J., van’t E.T., Wijmans, R., Pinkster, J., and Kr. von dem Deficiency of glycosyl-phosphatidylinositol-linked glycoproteins
of
hemoglobinuria, nc assay. Blood
description 76, 1853,
leukocytes
in
of a new 1990.
Veer-Korthof,
Borne, A.E.G. membrane
paroxysmal
diagnostic
nocturnal
cytofluoromet-
Huizinga, T.W.J., de Haas, M., Kleijer, M., Nuijens, J.H., Roos, D., and Kr. von dem Borne, A.E.G. Soluble Fc-y receptor III in human plasma originates from release by neutrophils. j Clin. Invest. 86, 416, 1990. 48. Skubitz, K.M., Stroncek, D.F., and Sun, B. Neutrophil-specific 47.
antigen
linkage.
j
NB1 is Leukoc.
anchored BioL
via
49,
163,
a
glycosyl-phosphatidylinositol 1991.