Eosinophil
granule
Randa Hirohito
in peripheral
blood
I. Abu-Ghazaleh Sandra L. Dunnette,t David Kita,1 Larry L. Thomas,S and Gerald J. GleichtI
*Departmt
of Biochemistry
Immunology,
Mayo
Rush
proteins
Medical
and
Clinic
College,
and
Molecular Mayo
Biology,
Foundation,
Rush-Presbyterian-St.
tAllergic
Diseases
Rochester,
Luke’s
Center,
Loegering,1
Research
Minnesota,
Medical
A.
and
James
Laboratory,
and
Department
Chicago,
1
granulocytes L.
1Department
Checkel,1 of
of Immunology/Microbiology,
Illinois
Abstract: Eosinophils contain four principal cationic proteins, major basic protein (MBP), eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), and eosinophil peroxidase (EPO). To determine the
ties [18, 19]. One of the terms of its helminthotoxicity ECP possesses nibonuclease tivities. In addition, ECP
quantities of these proteins in granulocytes and whether they are specific to eosinophils, their concentrations in lysates of human granulocytes were measured using specific radioimmunoassays. The effect of different methods for eosinophil lysis on the recovery of the proteins was also studied. Maximal recovery occurred at pH 2 for MBP
homologies among EDN, ECP, human, and rat pancreatic ribonucleases, bovine seminal ribonuclease, and angiogenin indicate that they belong to a ribonuclease multigene family [21-24]. Finally, EPO by itself is a potent helminthotoxin [18]. When armed with H2O2 plus a halide, its potency as a helminthotoxin increases dramatically [18, 25]. EPO is also
and pH 5.6 for the other granule proteins. The proteins cosedimented with eosinophils and their concentrations (X± SEM) in ng/106 eosinophils (and in nM/106 eosinophils) were: MBP, 8,982 ± 611 (641.6); EDN, 3,283 ± 116 (178.4); ECP, 5,269 ± 283 (250.9); and EPO, 12,174 ± 859 (171.5). Basophils from a normal person contained (in ng/106 cells) MBP, 2,374; EDN, 214; ECP, 77; and EPO, 17. Highly purified neutrophils contamed (in ng/106 cells) MBP, 3 ± 0.5; EDN, 72 ± 9; and ECP, 50 ± 12. Therefore we conclude that these proteins are mainly expressed in eosinophils, but that certain ones are present in basophils and neutrophils. J. Leukoc. Biol.
able
52: 611-618; Key phil .
Words: cationic basophils
to damage respiratory epithelium Previous studies have suggested predominant protein in the guinea
major protein
basic protein . eosinophil peroxidase . eosinoeosinophil-derived neurotoxin . neutrophils
[2]. that MBP pig eosinophil
is the granule
eosinophil-denived core of the EDN, ECP, [4-6]. These
neunotoxin (EDN) [2]. The crystalloid eosinophil granule consists of MBP whereas and EPO are localized to the granule matrix proteins play an important role in the effector
function of eosinophils. For example, MBP is present at high levels in sputa [3] and is deposited at sites of respiratory epithelial damage in patients with bronchial asthma [2, 3]. In addition, MBP is cytotoxic to mammalian cells [2, 3], helminths [2], protozoa [7], and bacteria [8]. MBP is capable of inducing hyperreactivity of bronchial smooth muscle both 10] and in vivo [11], and of activating also triggers the degranulation of
[14], well.
and mast cells It is a powerful and
[15]. EDN neurotoxin
causes paralysis intrathecally [2, [17], and possesses
has
complement platelets [13], multiple in rabbits
in experimental 16]. EDN is also weak helminthotoxic
funcand
animals a potent activi-
MATERIALS
AND
METHODS
Purification
of the
Eosinophil
Granule
Proteins
The four eosinophil granule proteins MBP, EDN, ECP, and EPO were purified from eosinophils of patients with eosinophilia as described earlier [16, 20]. Briefly, eosinophils from patients with marked eosinophilia were lysed with sucrose and hepanin, and the granules were collected by centnifugation. Granules were lysed by exposure to 0.01 M HCI, pH 2, and by sonication. Insoluble materials were removed by centnifugation and the lysate was applied to a Sephadex G-50 column (Pharmacia, Inc., Piscataway, NJ), equilibrated with 0.025 M sodium acetate buffer, with 0.15 M NaCl, pH 4.2. The void volume peak fractions from the
Eosinophils are believed to play an important role in the body’s immune response to parasites [1, 2] and in the pathogenesis of certain inflammatory diseases, especially asthma [2, 3]. The human eosinophil granule contains four principal proteins: the major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil penoxidase (EPO), and the
guinea pigs when injected ribonuclease
proteins in Like EDN, [20] ac[8]. The
[26] and that ECP accounts for about 30% of the weight of the human eosinophil granule [27]. The distribution of these proteins in peripheral blood granulocytes has not been thoroughly studied. We have measured the quantities of the eosinophil granule proteins in human peripheral blood granulocytes. In the process of these measurements we invesligated the conditions for eosinophil lysis that yield maximal granule protein recovery. Finally, the levels of those proteins in normal plasmas were measured.
INTRODUCTION
basophils tions as
potent eosinophil is ECP [18, 19]. [17, 20] and neurotoxic has bactericidal properties
1992.
#{149}
in vitro [9, [12]. MBP
more
Sephadex G-50 column were ion exchange chromatography to obtain EDN and
EPO ECP
[28]. [17]
The were
pooled and further purified on carboxymethyl-Sephanose
second pooled
peak fractions and dialyzed
by
containing against half-
Abbreviations: BAR , burro antirabbit ; CTAB, cetyltrimethyl ammonium bromide; ECP, eosinophil cationic protein; EDN, eosinophil-derived neurotoxin; EPO, eosinophil peroxidase; MBP, major basic protein; NP-40, nonidet P-40; P1, protease inhibitors; PPACK, D-phenylalanyl-L-prolyl-Larginine chloromethyl ketone; PPF-E, protamine, phosphate, FCS and EDTA buffer; PBS, phosphate-buffered saline; RIA, radioimmunoassay. Reprint requests: Randa I. Abu-Ghazaleh, Department of Biochemistry
and
Molecular
Biology,
Mayo
Clinic
and
Mayo
Foundation,
Rochester,
MN
55905. Received
Journal
May
of Leukocyte
18,
1992;
Biology
accepted
July
Volume
16,
52,
1992.
December
1992
611
strength Dulbecco’s phosphate-buffered saline (PBS) for 4 h, applied to 1.2 x 50 cm hepanin-Sephanose column (Phanmacia), and eluted with a linear gradient of 0.075-1.5 M NaC1 in 0.005 M phosphate buffer, pH 7. The fractions containing EDN eluted
eluted at 0.15-0.2 at 0.35-0.65 M
tions
were
pooled
as
NaC1 and those NaCl [17, 20]. The M
MBP
containing third peak
4
0
2
ECP frac0
[29].
C, Production Rabbits tradermal purified
of Antibodies
0
-2
were immunized against the specific proteins by ininjection of 1 ml of an emulsion of 100 g of the protein in 0.5 ml PBS and 0.5 ml CFA (Sigma
-4 0.1
Chemical Co., St. Louis, MO, No. F-4258). This was followed by booster immunizations 2 weeks to 2 months later with 100 tg protein emulsion in 0.5 ml PBS and 0.5 ml IFA (Sigma, No. F-5506). Animals producing antibodies were subsequently injected subcutaneously with 100 g protein on day 1, followed by 100 tg of protein intravenously on days 2, 3, and 4. Antinabbit IgG used in the double-antibody nadioimmunoassay (RIA) was produced in burros by immunization with an emulsion of 100 g of rabbit IgG (Cappel-Onganon Teknika Corporation, Malvern, PA) in 0.5 ml PBS and 0.5 ml CFA, followed 1 and 2 months later ,Lg of rabbit IgG in 0.5 ml PBS and 0.5 exsanguinated to obtain the antirabbit tiserum.
by injections ml IFA. Burros IgG (BAR-IgG)
of
100
0
C, 0 -I
100 were an-
ECP, ng/tub. Radioimmunoassays All of the eosinophil granule proteins were measured by double-antibody RIA. Briefly, purified standards or unknowns were diluted in PPF-E buffer [0.1% protamine sulfate (Sigma), 0.1 M phosphate buffer (pH 7.5), 0.5% newborn or FCS (Pel-Fneez, Rogers, AR), 0.1% NaN3, and 0.01 M EDTA (Sigma)], pH 7.5. The samples were mixed with specific rabbit antisera and nadiolabeled protein (0.5 ng/tube). Antiserum dilutions were as follows: anti-MBP, 1:5,000; anti-EDN, 1:10,000; anti-ECP, 1:20,000; and anti-
4 0
2 C,
0
0 1
-2
EPO,
1:35,000. After an overnight incubation at 4#{176}C,BARand normal rabbit serum (diluted 20-fold, Pel-Fneez) were added. The tubes were mixed, incubated at room temperatune for 3 h, and centrifuged. Supernatant fluids were decanted and radioactivity in precipitates was measured using a gamma scintillation counter. A set of control tubes to determine intenassay variability and nonspecific and total binding was included. In the MBP assay, samples were reduced and alkylated in Tris-EDTA buffer (0.15 M NaCl, 0.01 M EDTA, and 0.33 M Tnis, pH 8.0) by treating each
IgG
sample (Sigma),
and internal control final concentration
at 20#{176}C with dithiothreitol 0.0075 M, for 1 h followed
0.1
612
Journal
Company,
of Leukocyte
Palo
Biology
Alto,
CA)
with
Volume
52,
RIA
December
100
10
EPO, ng/tube Fig. 1. Double (c). B0 is the protein.
antibody competition bound radiolabeled
B is the
concentrations
slopes
=
respectively.
ECP,
and
bound of cold
-3.146, P EPO
radiolabeled and
0.0001 RIAs
for are
0.5,
for: EDN (a); in the absence
protein
inhibitory
-2.63,
granule
Randa Hirohito
in peripheral
blood
I. Abu-Ghazaleh Sandra L. Dunnette,t David Kita,1 Larry L. Thomas,S and Gerald J. GleichtI
*Departmt
of Biochemistry
Immunology,
Mayo
Rush
proteins
Medical
and
Clinic
College,
and
Molecular Mayo
Biology,
Foundation,
Rush-Presbyterian-St.
tAllergic
Diseases
Rochester,
Luke’s
Center,
Loegering,1
Research
Minnesota,
Medical
A.
and
James
Laboratory,
and
Department
Chicago,
1
granulocytes L.
1Department
Checkel,1 of
of Immunology/Microbiology,
Illinois
Abstract: Eosinophils contain four principal cationic proteins, major basic protein (MBP), eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), and eosinophil peroxidase (EPO). To determine the
ties [18, 19]. One of the terms of its helminthotoxicity ECP possesses nibonuclease tivities. In addition, ECP
quantities of these proteins in granulocytes and whether they are specific to eosinophils, their concentrations in lysates of human granulocytes were measured using specific radioimmunoassays. The effect of different methods for eosinophil lysis on the recovery of the proteins was also studied. Maximal recovery occurred at pH 2 for MBP
homologies among EDN, ECP, human, and rat pancreatic ribonucleases, bovine seminal ribonuclease, and angiogenin indicate that they belong to a ribonuclease multigene family [21-24]. Finally, EPO by itself is a potent helminthotoxin [18]. When armed with H2O2 plus a halide, its potency as a helminthotoxin increases dramatically [18, 25]. EPO is also
and pH 5.6 for the other granule proteins. The proteins cosedimented with eosinophils and their concentrations (X± SEM) in ng/106 eosinophils (and in nM/106 eosinophils) were: MBP, 8,982 ± 611 (641.6); EDN, 3,283 ± 116 (178.4); ECP, 5,269 ± 283 (250.9); and EPO, 12,174 ± 859 (171.5). Basophils from a normal person contained (in ng/106 cells) MBP, 2,374; EDN, 214; ECP, 77; and EPO, 17. Highly purified neutrophils contamed (in ng/106 cells) MBP, 3 ± 0.5; EDN, 72 ± 9; and ECP, 50 ± 12. Therefore we conclude that these proteins are mainly expressed in eosinophils, but that certain ones are present in basophils and neutrophils. J. Leukoc. Biol.
able
52: 611-618; Key phil .
Words: cationic basophils
to damage respiratory epithelium Previous studies have suggested predominant protein in the guinea
major protein
basic protein . eosinophil peroxidase . eosinoeosinophil-derived neurotoxin . neutrophils
[2]. that MBP pig eosinophil
is the granule
eosinophil-denived core of the EDN, ECP, [4-6]. These
neunotoxin (EDN) [2]. The crystalloid eosinophil granule consists of MBP whereas and EPO are localized to the granule matrix proteins play an important role in the effector
function of eosinophils. For example, MBP is present at high levels in sputa [3] and is deposited at sites of respiratory epithelial damage in patients with bronchial asthma [2, 3]. In addition, MBP is cytotoxic to mammalian cells [2, 3], helminths [2], protozoa [7], and bacteria [8]. MBP is capable of inducing hyperreactivity of bronchial smooth muscle both 10] and in vivo [11], and of activating also triggers the degranulation of
[14], well.
and mast cells It is a powerful and
[15]. EDN neurotoxin
causes paralysis intrathecally [2, [17], and possesses
has
complement platelets [13], multiple in rabbits
in experimental 16]. EDN is also weak helminthotoxic
funcand
animals a potent activi-
MATERIALS
AND
METHODS
Purification
of the
Eosinophil
Granule
Proteins
The four eosinophil granule proteins MBP, EDN, ECP, and EPO were purified from eosinophils of patients with eosinophilia as described earlier [16, 20]. Briefly, eosinophils from patients with marked eosinophilia were lysed with sucrose and hepanin, and the granules were collected by centnifugation. Granules were lysed by exposure to 0.01 M HCI, pH 2, and by sonication. Insoluble materials were removed by centnifugation and the lysate was applied to a Sephadex G-50 column (Pharmacia, Inc., Piscataway, NJ), equilibrated with 0.025 M sodium acetate buffer, with 0.15 M NaCl, pH 4.2. The void volume peak fractions from the
Eosinophils are believed to play an important role in the body’s immune response to parasites [1, 2] and in the pathogenesis of certain inflammatory diseases, especially asthma [2, 3]. The human eosinophil granule contains four principal proteins: the major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil penoxidase (EPO), and the
guinea pigs when injected ribonuclease
proteins in Like EDN, [20] ac[8]. The
[26] and that ECP accounts for about 30% of the weight of the human eosinophil granule [27]. The distribution of these proteins in peripheral blood granulocytes has not been thoroughly studied. We have measured the quantities of the eosinophil granule proteins in human peripheral blood granulocytes. In the process of these measurements we invesligated the conditions for eosinophil lysis that yield maximal granule protein recovery. Finally, the levels of those proteins in normal plasmas were measured.
INTRODUCTION
basophils tions as
potent eosinophil is ECP [18, 19]. [17, 20] and neurotoxic has bactericidal properties
1992.
#{149}
in vitro [9, [12]. MBP
more
Sephadex G-50 column were ion exchange chromatography to obtain EDN and
EPO ECP
[28]. [17]
The were
pooled and further purified on carboxymethyl-Sephanose
second pooled
peak fractions and dialyzed
by
containing against half-
Abbreviations: BAR , burro antirabbit ; CTAB, cetyltrimethyl ammonium bromide; ECP, eosinophil cationic protein; EDN, eosinophil-derived neurotoxin; EPO, eosinophil peroxidase; MBP, major basic protein; NP-40, nonidet P-40; P1, protease inhibitors; PPACK, D-phenylalanyl-L-prolyl-Larginine chloromethyl ketone; PPF-E, protamine, phosphate, FCS and EDTA buffer; PBS, phosphate-buffered saline; RIA, radioimmunoassay. Reprint requests: Randa I. Abu-Ghazaleh, Department of Biochemistry
and
Molecular
Biology,
Mayo
Clinic
and
Mayo
Foundation,
Rochester,
MN
55905. Received
Journal
May
of Leukocyte
18,
1992;
Biology
accepted
July
Volume
16,
52,
1992.
December
1992
611
strength Dulbecco’s phosphate-buffered saline (PBS) for 4 h, applied to 1.2 x 50 cm hepanin-Sephanose column (Phanmacia), and eluted with a linear gradient of 0.075-1.5 M NaC1 in 0.005 M phosphate buffer, pH 7. The fractions containing EDN eluted
eluted at 0.15-0.2 at 0.35-0.65 M
tions
were
pooled
as
NaC1 and those NaCl [17, 20]. The M
MBP
containing third peak
4
0
2
ECP frac0
[29].
C, Production Rabbits tradermal purified
of Antibodies
0
-2
were immunized against the specific proteins by ininjection of 1 ml of an emulsion of 100 g of the protein in 0.5 ml PBS and 0.5 ml CFA (Sigma
-4 0.1
Chemical Co., St. Louis, MO, No. F-4258). This was followed by booster immunizations 2 weeks to 2 months later with 100 tg protein emulsion in 0.5 ml PBS and 0.5 ml IFA (Sigma, No. F-5506). Animals producing antibodies were subsequently injected subcutaneously with 100 g protein on day 1, followed by 100 tg of protein intravenously on days 2, 3, and 4. Antinabbit IgG used in the double-antibody nadioimmunoassay (RIA) was produced in burros by immunization with an emulsion of 100 g of rabbit IgG (Cappel-Onganon Teknika Corporation, Malvern, PA) in 0.5 ml PBS and 0.5 ml CFA, followed 1 and 2 months later ,Lg of rabbit IgG in 0.5 ml PBS and 0.5 exsanguinated to obtain the antirabbit tiserum.
by injections ml IFA. Burros IgG (BAR-IgG)
of
100
0
C, 0 -I
100 were an-
ECP, ng/tub. Radioimmunoassays All of the eosinophil granule proteins were measured by double-antibody RIA. Briefly, purified standards or unknowns were diluted in PPF-E buffer [0.1% protamine sulfate (Sigma), 0.1 M phosphate buffer (pH 7.5), 0.5% newborn or FCS (Pel-Fneez, Rogers, AR), 0.1% NaN3, and 0.01 M EDTA (Sigma)], pH 7.5. The samples were mixed with specific rabbit antisera and nadiolabeled protein (0.5 ng/tube). Antiserum dilutions were as follows: anti-MBP, 1:5,000; anti-EDN, 1:10,000; anti-ECP, 1:20,000; and anti-
4 0
2 C,
0
0 1
-2
EPO,
1:35,000. After an overnight incubation at 4#{176}C,BARand normal rabbit serum (diluted 20-fold, Pel-Fneez) were added. The tubes were mixed, incubated at room temperatune for 3 h, and centrifuged. Supernatant fluids were decanted and radioactivity in precipitates was measured using a gamma scintillation counter. A set of control tubes to determine intenassay variability and nonspecific and total binding was included. In the MBP assay, samples were reduced and alkylated in Tris-EDTA buffer (0.15 M NaCl, 0.01 M EDTA, and 0.33 M Tnis, pH 8.0) by treating each
IgG
sample (Sigma),
and internal control final concentration
at 20#{176}C with dithiothreitol 0.0075 M, for 1 h followed
0.1
612
Journal
Company,
of Leukocyte
Palo
Biology
Alto,
CA)
with
Volume
52,
RIA
December
100
10
EPO, ng/tube Fig. 1. Double (c). B0 is the protein.
antibody competition bound radiolabeled
B is the
concentrations
slopes
=
respectively.
ECP,
and
bound of cold
-3.146, P EPO
radiolabeled and
0.0001 RIAs
for are
0.5,
for: EDN (a); in the absence
protein
inhibitory
-2.63,
