MATERIALS
AND
In vitro
METHODS
Male New Zealand white rabbits (R. and R. Rabbitry, Stanwood, WA), 1.5-2 kg, were housed in the University of Washington Vivarium and provided food and water ad libiturn. Experirnental protocols involving animals were reviewed and accepted by the University of Washington Animal Care Committee and meet the standards set by the National Institutes of Health for animal care and use.
Culture After
Materials rabbit
IFN-y
(lot
number
6951-11)
design
Alveolar macrophages isolated by BAL were centrifuged at 800 rpm for 7 mm and resuspended in RPM! 1640 at a concentration of 5 x 10 cells/mi. Aliquots (1 ml) of the cell suspension were pipetted into 12-well tissue culture plates. Macrophages were then treated with IFN-y (0.1-1000 units/mi), LPS (0.1-1000 ng/ml), or both IFN--y (0.1-1000 units/ml) and LPS (10 ng/ml). IFN-’y was added to cultures 1 h before LPS. Each experimental condition was performed in duplicate.
Animals
Recombinant
experimental
plates incubation,
were
incubated at 37#{176}Cin 5% CO2 cell supernatants were aspirated,
was
ruption.
In vivo New (Fig. LPS
experimental
design
Zealand white rabbits 1). Animals received after
pretreatment
were divided into either IFN-’y alone,
with
IFN-’y.
by adsorption using washed rabbit red blood cells to remove naturally occurring goat hemagglutinating antibodies. When mixed and incubated for 30 mm at 37#{176}Cwith an equal volume of conditioned medium from endotoxin (LPS)stimulated rabbit macrophages (TNF source), 1/400 diluted antiserum neutralized 2000 units TNF/ml. The anti-HrTNF contained 40 mg total protein/mi.
TNF
assay
TNF natants
was of
assayed by L929 the macrophage
cytotoxicity cultures
A81od
580
Journal
-24
dehydrogenase
of Leukocyte
[14]. were
Sempling
-0.50
were
ob-
Cell free superserially diluted
0.51
1.52
P 2.5
3
4
5
6
7
bows
harvest
Immediately after lethal intravenous injection with pentobarbital, bronchoalveolar lavage (BAL) was done under low pressure with six 40-mi aliquots of normal saline solution at 4#{176}C.Lavage was performed using an in situ technique by surgically isolating the trachea and positioning a sterile large bore IV catheter in the trachea. A differential cell count of each lavage preparation was done using Diff-Quik reagents (American Scientific Products, McGaw Park, IL). Preparations with greater than 5% polymorphonuclear cells or red blood cells (RBCs) were excluded from the study. Previous studies have shown by morphologic evidence, as indicated by an eccentric nucleus and membrane ruffling, as well as functional evidence, as indicated by sheep RBC rosetting, nonspecific esterase staining, and the ability to phagocytose latex beads, that this method consistently yields a greater than 95% alveolar macrophage population [13]. Macrophage viability, assessed by exclusion of the vital dye trypan lactate
samples
IFN Alone
TTr 4$
blue and at 24 h.
Blood
three groups LPS alone, or
tained before administration of LPS and at selected intervals for 7 h after LPS administration. Blood samples of animals receiving IFN#y alone were obtained after the final dose of IFN-y. Group I received subcutaneous injections of IFN-7, 25,000 units/kg (5 pig/kg), on 3 consecutive days. Group II received a single intravenous infusion of LPS, 1 &g/kg, over 30 mm. Group III received subcutaneous injections of IFNy, 25,000 units/kg (5 pg/kg) on 3 consecutive days before intravenous infusion of LPS, 1 /Lg/kg, over 30 mm. Serum TNF was measured in the three groups. Because of its membrane-bound nature, PCA was not measured in vivo.
(St. Louis, MO). The lyophilized powder was suspended in pyrogen-free, distilled H20 with 0.1% triethylamine (Sigma) at a final concentration of I mg/mi, dispersed by sonication at 20#{176}C, and stored at - 70#{176}C. LPS aliquots were resuspended to working concentration in RPM! 1640 media (GIBCO, Grand Island, NY) and sonicated before each experiment. Culture medium, RPM! 1640, was obtained from GIBCO. Goat polyclonal anti-human recombinant TNF was provided by John Mathison of the Scripps Clinical and Research Foundation, La Jolla, CA. This antibody was prepared by immunizing goats with human recombinant TNF. The globulin fraction was prepared by ammonium sulfate precipitation, diisopropyl fluorophosphate (DFP) treatment, and dialysis against 10 mM Hepes saline (pH 7.4), followed
macrophage
24 h. centri-
fuged to remove any cellular debris, and stored at - 70#{176}Cfor TNF assay. Cell lysates for PCA determinations were prepared from the macrophage monolayer by one freeze (70#{176}C)-thaw (37#{176}C) cycle, followed by ultrasonic dis-
provided by Genentech, Inc., South San Francisco, CA. Desired concentrations of IFN-’y were prepared from a stock solution of 1.8 x 106 units/ml (5 x 106 units/mg) immediately prior to each use. The IFN-y preparation was free of endotoxin contamination as determined by Limulus amebocyte lysate assay (minimal detectable endotoxin level 0.125 Eu/ml or < 25 pg/ml). Escherichia coil 0111:B4 LPS, extracted by hot phenol-water technique, was obtained from Sigma
Alveolar
for
release,
Biology
is greater
Volume
than
52,
T -0.5
0
0.5
1
1.5
2
2.5
3
4
5
6
7
hows
TT 4$
IFN
-24
-0.50
0.5
1
1.5
and
2
2.5
LPS
3
4
5
6
hours
Fig. 1. In vivo protocol for rabbits receiving IFN-y alone (5 sg/kg, SC, single dose on 3 consecutive days), LPS alone (1 zg/kg, IV over 30 mm), or IFN-1 (5 &g/kg, SC, single dose on 3 consecutive days) plus LPS (1 &g/kg, IV over 30 mm after final IFN-y injection). Blood samples were obtained 30 mm prior to final IFN--y or LPS injection and for 7 h thereafter.
90%
December
LPS Alone
1992
7
from
1:2 to
1:1024
in 96-well
culture
plates.
All
sample
5
dilu-
tions were performed in duplicate. Transformed murine fibroblasts (NCTC clone L929 American Type Culture Collection, Rockville, MD) at a concentration of 5 x 10 cells/well in RPMI 1640 media supplemented with 5% horse serum (Hyclone Laboratories, Logan, UT) were added to each well containing the sample dilutions. L929 cells were pretreated with actinomycin D (Sigma Chemical Co., St. Louis, MO), 1 g/ml, for 15 mm before addition to the sample dilutions. Culture plates were then incubated at 37#{176}Cin 5% CO2. After 24 h the cells were washed with PBS and fixed and stained in 0.1% crystal violet in 20% methanol.
4 *
3
V.) C
V
2
0
U. U. 1
2 I-
Dried monolayers were solubilized in 0.1 M sodium citrate in 50% ethanol and the absorbance read at 550 nm in a Vmax microplate reader (Molecular Devices, Menlo Park, CA). One unit of TNF activity was defined as that activity that produced 50% cytolysis of the L929 monolayer. A linear regression was performed to determine the point between serial curred.
dilutions LPS
of the (0.1-1000
TNF samples ng/ml) and
exhibited no direct cytotoxicity Verification that L929 cytotoxicity was confirmed by incubating antibody. L929 cytotoxicity dependent the presence
PCA
fashion when of anti-TNF.
where 50% IFN-y (0.1-1000
cytolysis ocunits/ml)
against the L929 cell line. was due to TNF activity selected samples with TNF was inhibited in a dose-
sample
supernatants
were
tested
clotting 0.1 ml
assay was performed on macrophage of pooled citrated plasma and 0.1 ml
in
of
25 mM CaC12 as previously described [15]. Briefly, the time required for clot formation, which reflects the amount of macrophage PCA present in the sample, was converted to units of activity by interpolation from a standard curve. The standard curve was generated by serially diluted rabbit brain thromboplastin clotting times. Neat rabbit brain thromboplastin was assigned an activity of 100,000 mU. Each sample was assayed in duplicate.
Statistical
analysis
Data are expressed as means ± SEM. increase were calculated as (treatment
Data expressed value-control
0.1
as fold value)/
1
10
Interferon 3. Effect
Fig. pressed
assay
A one-stage lysates using
0
of IFN--1
as fold
Gamma
on alveolar
increase
100
from
Dose
macrophage
unstimulated
(units/mI)
TNF
control
1000
production.
levels.
*p
TNF
ex-
0.05,
AND
In vitro
METHODS
Male New Zealand white rabbits (R. and R. Rabbitry, Stanwood, WA), 1.5-2 kg, were housed in the University of Washington Vivarium and provided food and water ad libiturn. Experirnental protocols involving animals were reviewed and accepted by the University of Washington Animal Care Committee and meet the standards set by the National Institutes of Health for animal care and use.
Culture After
Materials rabbit
IFN-y
(lot
number
6951-11)
design
Alveolar macrophages isolated by BAL were centrifuged at 800 rpm for 7 mm and resuspended in RPM! 1640 at a concentration of 5 x 10 cells/mi. Aliquots (1 ml) of the cell suspension were pipetted into 12-well tissue culture plates. Macrophages were then treated with IFN-y (0.1-1000 units/mi), LPS (0.1-1000 ng/ml), or both IFN--y (0.1-1000 units/ml) and LPS (10 ng/ml). IFN-’y was added to cultures 1 h before LPS. Each experimental condition was performed in duplicate.
Animals
Recombinant
experimental
plates incubation,
were
incubated at 37#{176}Cin 5% CO2 cell supernatants were aspirated,
was
ruption.
In vivo New (Fig. LPS
experimental
design
Zealand white rabbits 1). Animals received after
pretreatment
were divided into either IFN-’y alone,
with
IFN-’y.
by adsorption using washed rabbit red blood cells to remove naturally occurring goat hemagglutinating antibodies. When mixed and incubated for 30 mm at 37#{176}Cwith an equal volume of conditioned medium from endotoxin (LPS)stimulated rabbit macrophages (TNF source), 1/400 diluted antiserum neutralized 2000 units TNF/ml. The anti-HrTNF contained 40 mg total protein/mi.
TNF
assay
TNF natants
was of
assayed by L929 the macrophage
cytotoxicity cultures
A81od
580
Journal
-24
dehydrogenase
of Leukocyte
[14]. were
Sempling
-0.50
were
ob-
Cell free superserially diluted
0.51
1.52
P 2.5
3
4
5
6
7
bows
harvest
Immediately after lethal intravenous injection with pentobarbital, bronchoalveolar lavage (BAL) was done under low pressure with six 40-mi aliquots of normal saline solution at 4#{176}C.Lavage was performed using an in situ technique by surgically isolating the trachea and positioning a sterile large bore IV catheter in the trachea. A differential cell count of each lavage preparation was done using Diff-Quik reagents (American Scientific Products, McGaw Park, IL). Preparations with greater than 5% polymorphonuclear cells or red blood cells (RBCs) were excluded from the study. Previous studies have shown by morphologic evidence, as indicated by an eccentric nucleus and membrane ruffling, as well as functional evidence, as indicated by sheep RBC rosetting, nonspecific esterase staining, and the ability to phagocytose latex beads, that this method consistently yields a greater than 95% alveolar macrophage population [13]. Macrophage viability, assessed by exclusion of the vital dye trypan lactate
samples
IFN Alone
TTr 4$
blue and at 24 h.
Blood
three groups LPS alone, or
tained before administration of LPS and at selected intervals for 7 h after LPS administration. Blood samples of animals receiving IFN#y alone were obtained after the final dose of IFN-y. Group I received subcutaneous injections of IFN-7, 25,000 units/kg (5 pig/kg), on 3 consecutive days. Group II received a single intravenous infusion of LPS, 1 &g/kg, over 30 mm. Group III received subcutaneous injections of IFNy, 25,000 units/kg (5 pg/kg) on 3 consecutive days before intravenous infusion of LPS, 1 /Lg/kg, over 30 mm. Serum TNF was measured in the three groups. Because of its membrane-bound nature, PCA was not measured in vivo.
(St. Louis, MO). The lyophilized powder was suspended in pyrogen-free, distilled H20 with 0.1% triethylamine (Sigma) at a final concentration of I mg/mi, dispersed by sonication at 20#{176}C, and stored at - 70#{176}C. LPS aliquots were resuspended to working concentration in RPM! 1640 media (GIBCO, Grand Island, NY) and sonicated before each experiment. Culture medium, RPM! 1640, was obtained from GIBCO. Goat polyclonal anti-human recombinant TNF was provided by John Mathison of the Scripps Clinical and Research Foundation, La Jolla, CA. This antibody was prepared by immunizing goats with human recombinant TNF. The globulin fraction was prepared by ammonium sulfate precipitation, diisopropyl fluorophosphate (DFP) treatment, and dialysis against 10 mM Hepes saline (pH 7.4), followed
macrophage
24 h. centri-
fuged to remove any cellular debris, and stored at - 70#{176}Cfor TNF assay. Cell lysates for PCA determinations were prepared from the macrophage monolayer by one freeze (70#{176}C)-thaw (37#{176}C) cycle, followed by ultrasonic dis-
provided by Genentech, Inc., South San Francisco, CA. Desired concentrations of IFN-’y were prepared from a stock solution of 1.8 x 106 units/ml (5 x 106 units/mg) immediately prior to each use. The IFN-y preparation was free of endotoxin contamination as determined by Limulus amebocyte lysate assay (minimal detectable endotoxin level 0.125 Eu/ml or < 25 pg/ml). Escherichia coil 0111:B4 LPS, extracted by hot phenol-water technique, was obtained from Sigma
Alveolar
for
release,
Biology
is greater
Volume
than
52,
T -0.5
0
0.5
1
1.5
2
2.5
3
4
5
6
7
hows
TT 4$
IFN
-24
-0.50
0.5
1
1.5
and
2
2.5
LPS
3
4
5
6
hours
Fig. 1. In vivo protocol for rabbits receiving IFN-y alone (5 sg/kg, SC, single dose on 3 consecutive days), LPS alone (1 zg/kg, IV over 30 mm), or IFN-1 (5 &g/kg, SC, single dose on 3 consecutive days) plus LPS (1 &g/kg, IV over 30 mm after final IFN-y injection). Blood samples were obtained 30 mm prior to final IFN--y or LPS injection and for 7 h thereafter.
90%
December
LPS Alone
1992
7
from
1:2 to
1:1024
in 96-well
culture
plates.
All
sample
5
dilu-
tions were performed in duplicate. Transformed murine fibroblasts (NCTC clone L929 American Type Culture Collection, Rockville, MD) at a concentration of 5 x 10 cells/well in RPMI 1640 media supplemented with 5% horse serum (Hyclone Laboratories, Logan, UT) were added to each well containing the sample dilutions. L929 cells were pretreated with actinomycin D (Sigma Chemical Co., St. Louis, MO), 1 g/ml, for 15 mm before addition to the sample dilutions. Culture plates were then incubated at 37#{176}Cin 5% CO2. After 24 h the cells were washed with PBS and fixed and stained in 0.1% crystal violet in 20% methanol.
4 *
3
V.) C
V
2
0
U. U. 1
2 I-
Dried monolayers were solubilized in 0.1 M sodium citrate in 50% ethanol and the absorbance read at 550 nm in a Vmax microplate reader (Molecular Devices, Menlo Park, CA). One unit of TNF activity was defined as that activity that produced 50% cytolysis of the L929 monolayer. A linear regression was performed to determine the point between serial curred.
dilutions LPS
of the (0.1-1000
TNF samples ng/ml) and
exhibited no direct cytotoxicity Verification that L929 cytotoxicity was confirmed by incubating antibody. L929 cytotoxicity dependent the presence
PCA
fashion when of anti-TNF.
where 50% IFN-y (0.1-1000
cytolysis ocunits/ml)
against the L929 cell line. was due to TNF activity selected samples with TNF was inhibited in a dose-
sample
supernatants
were
tested
clotting 0.1 ml
assay was performed on macrophage of pooled citrated plasma and 0.1 ml
in
of
25 mM CaC12 as previously described [15]. Briefly, the time required for clot formation, which reflects the amount of macrophage PCA present in the sample, was converted to units of activity by interpolation from a standard curve. The standard curve was generated by serially diluted rabbit brain thromboplastin clotting times. Neat rabbit brain thromboplastin was assigned an activity of 100,000 mU. Each sample was assayed in duplicate.
Statistical
analysis
Data are expressed as means ± SEM. increase were calculated as (treatment
Data expressed value-control
0.1
as fold value)/
1
10
Interferon 3. Effect
Fig. pressed
assay
A one-stage lysates using
0
of IFN--1
as fold
Gamma
on alveolar
increase
100
from
Dose
macrophage
unstimulated
(units/mI)
TNF
control
1000
production.
levels.
*p
TNF
ex-
0.05,
