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

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Subcellular localization of CD66, CD67, and NCA in human neutrophils.

CD66 and CD67 are granulocyte-specific activation antigens; their surface expression is up-regulated when neutrophils are activated. CD66 antibodies r...
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