Prostaglandins and asthma: The use of blood components for metabolic studies Tadao
Okazaki,
James
B. lee,
M.D., M.D.,
and
Daniel
Vervloet,
Carl
E. Arbesman,
M.D., M.D.
Ahmad Buffalo,
Attallah, N.
Ph.D.,
II.
Experiments were carried out to study a possible role of prostuglandi.ns (PGs) in the pathogenesis of human bronchial asthma. Leukocyte, plasma, and ,serum of ambulatory asthmatic as well as nonasthmatic indinidwcls were userl. PGF teas measured by competitive radioimmunoassay with anti-PGF,, antibody. It teas found that PGF was spontaneously released from the leukocytes of both asthmatic and nonasthmatic individuals. The PGF release as studied with the cells of asthmatic individuals was enhanced by the additions of arachidonic acid and/or dibutyryl cyclic AHP. Antigenic challenge of sensitive cells failed to show any genernl effect on the amount of PGF released. The amount of spontaneously released PGP had no correlation with the amount of maximal antigenic hi,stamine release. Preincubation of the cells with arachidonic acid haa no effect on subsequent antigenic kistaminc release. Plasma PGF levels in nonasthmatic and in asthmatic i,ndiciduals Cere not significantly different and both v:ere considerably lower than the serum levels. The mean serum PGF level in the asthmatic group was higher than in the nonnsthmatic, indicating probably a greater release of PGF from blood cells.
The prostaglandins (PGs) are a class of naturally occurring acidic lipids of highly potent biologic activities.l Of the three biologically important PGs, PGFs and PGEs are actively metabolized in the lung2 whereas PGAs escape degradation by the lung. PGF,, is the major PG found in the lung of several species, including man.3 PGF,, is a potent bronchoconstrictor in contrast to PGE’s which relax tracheobranchial smooth muscle.4-7 Asthmatic individuals have been shown to possess much higher sensitivity to the bronchoconstrictor action of PGF,, than nonasthmatic subjects. +I0 In addition, PGF,, has been shown to be released from sensitized guinea pig lung following antigenic challenge, where antigenic release of PGE has not been detected by specific radioimmunoassay.” Immunoglobulin E-mediated histamine release in vitro from human leukocytes and lung fragments has been shown to be modified by PGE’s and PGF’s.~~~ l3 From the Allergy Research Laboratory of the Buffalo General Hospital and the Departments of Medicine and Microbiology of the State University of New York at Buffalo. Supported in part by United States Public Health Service Research Grant 5ROl-AI-01303 and in part by United States Public Health Service Allergic Clinical Centers Grant 3-P15-AI-10397 of the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md. Presented in part at the Thirtieth Annual Meeting of The American Academy of Allergy, Jan. 21, 1974, Bal Harbour, Fla. Received for publication Oct. 8, 1974. Accepted for publication Feb. 12, 1975. Reprint requests to: Dr. Carl E.. Arbesman, Clinical Professor of Medicine, The Buffalo General Hospital, 100 High St., Buffalo, N. Y. 14203. Vol.
57, No.
8, pp.
164-133
VOLUME NUMBER
Prostaglandins
57 2
0I
0
:
I
5
10
and
I
I
20
40
asthma
125
TIME, MIN.
FIG. 1. Time bars represent of 4 asthmatic
course of spontaneous the standard errors individuals.
PGF of
release mean
from from
human leukocytes at 37” 4 experiments using the
C. Vertical leukocytes
These observations suggest that PG’s may have an important role in hypersensitivity phenomena. It has been proposed that an increased ratio of PGF/PGE may be of importance in the pathogenesis of human bronchial asthma.l In this paper we report the results of preliminary experiments for the study of a possible role of PG’s in the hypersensitivity phenomena. MATERIALS
AND
METHODS
Leukocytes were suspended in Tris buffer (2.5 x 10-Z M Tris [hydroxymethyl] amino1.2 x 10-i M sodium chloride; 5.0 x 10-3 M methane, Sigma Chemical Co., St. Louis, Missouri; potassium chloride; 6.0 x 10-k M calcium chloride; 1.0 x 10-a M magnesium chloride; 0.03% human serum albumin) and antigenic release and fluorometric assay of histamine were performed according to a method slightly modified from the one described by May.14 The final leukocyte counts ranged from 3 to 4 x lOa/ml. The blood used for the assay of plasma and serum PGF was cooled in ice mater immediately after being drawn from the central cubital vein. The plasma was obtained by centrifuging heparinized blood at 1,200 g at a temperature between 0 and 4” C for 10 min. Serum was separated from the cells by allowing the nonheparinized blood to coagulate, at 37” C for 30 min. All the samples were stored in silieonized glass or polystyrene tubes below -20’ C prior to the assay. The group of patients studied consisted of equal number of males and females 14 years of age or older. They had mild to no wheezing, no aspirin hypersensitivity, and positive skin tests to environmental allergens. They intermittently had sympathomimetic or xanthine drugs, but had no medication for 48 hr and no aspirin, indomethacin, or steroids for 1 wk before the morning of blood collection. Healthy males and females approximately in equal numbers, over 15 years of age without a history of allergy, were the sources for the normal blood. PGF was measured by radioimmunoassay as described by Caldwell and associates.15 Anti-PGF,, antibody was obtained commercially (Clinical Assays, Inc., Cambridge, Mass.) as well as by immunizing rabbits with PGF,, (Donated by Dr. John
126
Okazaki
J. ALLERGY
et al.
CLIN. IMMUNOL. FEBRUARY 1976
10 --
2 --
o-r-
0 ARACHIDONATE,
2.5
5
)rG/ML
FIG. 2. The effect of arachidonic acid on total and cellular PGF levels measured after 40 min of incubation at 37” C. Representative experiment. Mean and duplicate values from one patient are shown.
Pike, Upjohn Co., Kalamazoo, Mich.) conjugated with crystalline bovine serum albumin (Sigma Chemical Co., St. Louis, MO.). The recovery of radioactive PGF,, (Prostaglandin F,,-9-H3, Amersham-Searle Co., Des Plaines, Ill.) routinely measured as internal standards for every specimen was i5%-90%. The specificity of the assay for PGF,, was such that 3 to 6 times as much PGF,, (donated by Dr. John Pike, Upjohn Co., Kalamazoo, Mich.) as PGF,, was required for the 50% inhibition of the binding between the antibody and tritium-labeled PGF,,. Arachidonic acid (Sigma Chemical Co., St. Louis, MO.) in the concentrations used did not influence the assays of either PGF?, or histamine. Furthermore, neither the pH of the media nor spontaneous histamine release was affected by additibn of arachidonic acid to the incubation media. Crude extracts of short ragweed pollen (Center Laboratories, Inc., Port Washington, N. Y.) were dialyzed against normal saline before use as the challenging antigen. This antigen did not influence the assay of PGFI,. PGE, (Donated by Dr. John Pike, Upjohn Co., Kalamazoo, Mich.), PGA (Donated by Dr. John Pike, Upjohn Co., Kalamazoo, Mich.), and N6, 02-dibutyryl cyclic AMP, sodium salt (Sigma Chemical Co., St. Louis, MO.) wore obtained from the manufacturers. RESULTS Spontaneous
PGF
release
The cell suspensions were incubated at 37O C for up to 40 min. The suspensions were cooled in ice water after varying periods of incubation. PGF released during the incubation was measured with the supernatants obtained by centrifuging the suspension at 150 g for 8 min at a temperature between 0 and 4’ C. The cells of both nonasthmatic and asthmatic individuals were found to release PGF
VOLUME NUMBER
Prostaglandins
57 2
t I
1.0 2 x 10-q
0
and
asthma
127
II 5 x 10
-3
CYCLIC AMPI f110-3 FIG. 3. Effect of dibutyryl cyclic AMP of incubation ot 37” C. Representative
on leukocyte experiment.
PGF release measured after 40 min Mean and duplicate values are shown.
spontaneously (Fig. 1). The small amount of release at time 0 seems to be an experimental error representing the amount released during the handling of the cells. The incubation period of 40 min was used for all subsequent experiments. The precursor of PGF,,, arachidonic acid (
the difference between 0.1, paired t test).
in the amount of PGF released between control without antigen and the tests with antigen. No typical antigen dose-responsecurve was obtained for the release of the PGF: The amount released did not significantly change by the varying concentration of challenging antigen. Spontaneous PGF release (without added antigen) is shown in Fig. 5 in comparison with the amount of maximal antigenic histamine release from other aliquots of the same cell suspension used for the histamine release. The cells were incubated for 40 min at 37O C for both spontaneous PGF release and antigenic histamine release. No correlation was observed between the amounts of two compounds released.
VOLUME NUMBER
Prostaglandins
57 2
.
4,O
6.0
PROSTAGLANDIN F, FIG. 5. Maximal antigenic after 40 min of incubation and PG releases (Spearman’s
acid
on leukocyte
histamine
Antigenic
histamine
8.0
Control
measured histamine
release release
(%)
Arachidonate No.
l
(&ml)
2.5
5.0
10
:
::
:i
2
5:
i
:i
2”:
;:
ii
Mean 21 33t 31t *Each value represents the mean of two to four measurements. tNot significantly different from control (p > 0.1, paired t test).
Effect of arachidonic
acid on antigenic
histamine
129
NG/ML
histamine release and spontaneous PGF release at 37” C. No correlation was found between the rank order correlation coefftcient = 0).
TABLE II. Effect of arachidonic
Exp.
asthma
.
.
2,o
and
31t
release
The cells were incubated with arachidonic acid ( 210 pg/ml) for 40 min at 37’ C prior to antigenic challenge (Table II). Although statistically significant enhancement (p < 0.01) of antigenie histamine release was noted in Experiment 3 at 2.5 pg/ml of arachidonic acid, generally no significant effect was seen by the addition of PG precursor. Plasma
and
serum
PGF levels
As shown in Fig. 6, mean serum levels of PGF were significantly higher than the levels obtained for plasma both in normal and in asthmatic individuals. The
130
Okazaki
et
J. ALLERGY
al.
T NORMAL
20
T
PLASM ASTHMA
1
SERWl NOWAL
CLIN. IMMUNOL. FEBRUARY 1976
ASTHNA . . . . . . . . .
10
: . .
5.0
. .
z g 2.0 LL E B 2 1.0 5 F -50
. . . . .
.
.
.
. . .
.
+ : .
. .
l .*
.
-05
.
:.; .
I10
.. .
.
+
.20
.. .. .. .
. .
.
L
FIG. 6. coagulate values. significant
PGF for The (p
levels in 30 min difference
Okazaki,
James
B. lee,
M.D., M.D.,
and
Daniel
Vervloet,
Carl
E. Arbesman,
M.D., M.D.
Ahmad Buffalo,
Attallah, N.
Ph.D.,
II.
Experiments were carried out to study a possible role of prostuglandi.ns (PGs) in the pathogenesis of human bronchial asthma. Leukocyte, plasma, and ,serum of ambulatory asthmatic as well as nonasthmatic indinidwcls were userl. PGF teas measured by competitive radioimmunoassay with anti-PGF,, antibody. It teas found that PGF was spontaneously released from the leukocytes of both asthmatic and nonasthmatic individuals. The PGF release as studied with the cells of asthmatic individuals was enhanced by the additions of arachidonic acid and/or dibutyryl cyclic AHP. Antigenic challenge of sensitive cells failed to show any genernl effect on the amount of PGF released. The amount of spontaneously released PGP had no correlation with the amount of maximal antigenic hi,stamine release. Preincubation of the cells with arachidonic acid haa no effect on subsequent antigenic kistaminc release. Plasma PGF levels in nonasthmatic and in asthmatic i,ndiciduals Cere not significantly different and both v:ere considerably lower than the serum levels. The mean serum PGF level in the asthmatic group was higher than in the nonnsthmatic, indicating probably a greater release of PGF from blood cells.
The prostaglandins (PGs) are a class of naturally occurring acidic lipids of highly potent biologic activities.l Of the three biologically important PGs, PGFs and PGEs are actively metabolized in the lung2 whereas PGAs escape degradation by the lung. PGF,, is the major PG found in the lung of several species, including man.3 PGF,, is a potent bronchoconstrictor in contrast to PGE’s which relax tracheobranchial smooth muscle.4-7 Asthmatic individuals have been shown to possess much higher sensitivity to the bronchoconstrictor action of PGF,, than nonasthmatic subjects. +I0 In addition, PGF,, has been shown to be released from sensitized guinea pig lung following antigenic challenge, where antigenic release of PGE has not been detected by specific radioimmunoassay.” Immunoglobulin E-mediated histamine release in vitro from human leukocytes and lung fragments has been shown to be modified by PGE’s and PGF’s.~~~ l3 From the Allergy Research Laboratory of the Buffalo General Hospital and the Departments of Medicine and Microbiology of the State University of New York at Buffalo. Supported in part by United States Public Health Service Research Grant 5ROl-AI-01303 and in part by United States Public Health Service Allergic Clinical Centers Grant 3-P15-AI-10397 of the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md. Presented in part at the Thirtieth Annual Meeting of The American Academy of Allergy, Jan. 21, 1974, Bal Harbour, Fla. Received for publication Oct. 8, 1974. Accepted for publication Feb. 12, 1975. Reprint requests to: Dr. Carl E.. Arbesman, Clinical Professor of Medicine, The Buffalo General Hospital, 100 High St., Buffalo, N. Y. 14203. Vol.
57, No.
8, pp.
164-133
VOLUME NUMBER
Prostaglandins
57 2
0I
0
:
I
5
10
and
I
I
20
40
asthma
125
TIME, MIN.
FIG. 1. Time bars represent of 4 asthmatic
course of spontaneous the standard errors individuals.
PGF of
release mean
from from
human leukocytes at 37” 4 experiments using the
C. Vertical leukocytes
These observations suggest that PG’s may have an important role in hypersensitivity phenomena. It has been proposed that an increased ratio of PGF/PGE may be of importance in the pathogenesis of human bronchial asthma.l In this paper we report the results of preliminary experiments for the study of a possible role of PG’s in the hypersensitivity phenomena. MATERIALS
AND
METHODS
Leukocytes were suspended in Tris buffer (2.5 x 10-Z M Tris [hydroxymethyl] amino1.2 x 10-i M sodium chloride; 5.0 x 10-3 M methane, Sigma Chemical Co., St. Louis, Missouri; potassium chloride; 6.0 x 10-k M calcium chloride; 1.0 x 10-a M magnesium chloride; 0.03% human serum albumin) and antigenic release and fluorometric assay of histamine were performed according to a method slightly modified from the one described by May.14 The final leukocyte counts ranged from 3 to 4 x lOa/ml. The blood used for the assay of plasma and serum PGF was cooled in ice mater immediately after being drawn from the central cubital vein. The plasma was obtained by centrifuging heparinized blood at 1,200 g at a temperature between 0 and 4” C for 10 min. Serum was separated from the cells by allowing the nonheparinized blood to coagulate, at 37” C for 30 min. All the samples were stored in silieonized glass or polystyrene tubes below -20’ C prior to the assay. The group of patients studied consisted of equal number of males and females 14 years of age or older. They had mild to no wheezing, no aspirin hypersensitivity, and positive skin tests to environmental allergens. They intermittently had sympathomimetic or xanthine drugs, but had no medication for 48 hr and no aspirin, indomethacin, or steroids for 1 wk before the morning of blood collection. Healthy males and females approximately in equal numbers, over 15 years of age without a history of allergy, were the sources for the normal blood. PGF was measured by radioimmunoassay as described by Caldwell and associates.15 Anti-PGF,, antibody was obtained commercially (Clinical Assays, Inc., Cambridge, Mass.) as well as by immunizing rabbits with PGF,, (Donated by Dr. John
126
Okazaki
J. ALLERGY
et al.
CLIN. IMMUNOL. FEBRUARY 1976
10 --
2 --
o-r-
0 ARACHIDONATE,
2.5
5
)rG/ML
FIG. 2. The effect of arachidonic acid on total and cellular PGF levels measured after 40 min of incubation at 37” C. Representative experiment. Mean and duplicate values from one patient are shown.
Pike, Upjohn Co., Kalamazoo, Mich.) conjugated with crystalline bovine serum albumin (Sigma Chemical Co., St. Louis, MO.). The recovery of radioactive PGF,, (Prostaglandin F,,-9-H3, Amersham-Searle Co., Des Plaines, Ill.) routinely measured as internal standards for every specimen was i5%-90%. The specificity of the assay for PGF,, was such that 3 to 6 times as much PGF,, (donated by Dr. John Pike, Upjohn Co., Kalamazoo, Mich.) as PGF,, was required for the 50% inhibition of the binding between the antibody and tritium-labeled PGF,,. Arachidonic acid (Sigma Chemical Co., St. Louis, MO.) in the concentrations used did not influence the assays of either PGF?, or histamine. Furthermore, neither the pH of the media nor spontaneous histamine release was affected by additibn of arachidonic acid to the incubation media. Crude extracts of short ragweed pollen (Center Laboratories, Inc., Port Washington, N. Y.) were dialyzed against normal saline before use as the challenging antigen. This antigen did not influence the assay of PGFI,. PGE, (Donated by Dr. John Pike, Upjohn Co., Kalamazoo, Mich.), PGA (Donated by Dr. John Pike, Upjohn Co., Kalamazoo, Mich.), and N6, 02-dibutyryl cyclic AMP, sodium salt (Sigma Chemical Co., St. Louis, MO.) wore obtained from the manufacturers. RESULTS Spontaneous
PGF
release
The cell suspensions were incubated at 37O C for up to 40 min. The suspensions were cooled in ice water after varying periods of incubation. PGF released during the incubation was measured with the supernatants obtained by centrifuging the suspension at 150 g for 8 min at a temperature between 0 and 4’ C. The cells of both nonasthmatic and asthmatic individuals were found to release PGF
VOLUME NUMBER
Prostaglandins
57 2
t I
1.0 2 x 10-q
0
and
asthma
127
II 5 x 10
-3
CYCLIC AMPI f110-3 FIG. 3. Effect of dibutyryl cyclic AMP of incubation ot 37” C. Representative
on leukocyte experiment.
PGF release measured after 40 min Mean and duplicate values are shown.
spontaneously (Fig. 1). The small amount of release at time 0 seems to be an experimental error representing the amount released during the handling of the cells. The incubation period of 40 min was used for all subsequent experiments. The precursor of PGF,,, arachidonic acid (
the difference between 0.1, paired t test).
in the amount of PGF released between control without antigen and the tests with antigen. No typical antigen dose-responsecurve was obtained for the release of the PGF: The amount released did not significantly change by the varying concentration of challenging antigen. Spontaneous PGF release (without added antigen) is shown in Fig. 5 in comparison with the amount of maximal antigenic histamine release from other aliquots of the same cell suspension used for the histamine release. The cells were incubated for 40 min at 37O C for both spontaneous PGF release and antigenic histamine release. No correlation was observed between the amounts of two compounds released.
VOLUME NUMBER
Prostaglandins
57 2
.
4,O
6.0
PROSTAGLANDIN F, FIG. 5. Maximal antigenic after 40 min of incubation and PG releases (Spearman’s
acid
on leukocyte
histamine
Antigenic
histamine
8.0
Control
measured histamine
release release
(%)
Arachidonate No.
l
(&ml)
2.5
5.0
10
:
::
:i
2
5:
i
:i
2”:
;:
ii
Mean 21 33t 31t *Each value represents the mean of two to four measurements. tNot significantly different from control (p > 0.1, paired t test).
Effect of arachidonic
acid on antigenic
histamine
129
NG/ML
histamine release and spontaneous PGF release at 37” C. No correlation was found between the rank order correlation coefftcient = 0).
TABLE II. Effect of arachidonic
Exp.
asthma
.
.
2,o
and
31t
release
The cells were incubated with arachidonic acid ( 210 pg/ml) for 40 min at 37’ C prior to antigenic challenge (Table II). Although statistically significant enhancement (p < 0.01) of antigenie histamine release was noted in Experiment 3 at 2.5 pg/ml of arachidonic acid, generally no significant effect was seen by the addition of PG precursor. Plasma
and
serum
PGF levels
As shown in Fig. 6, mean serum levels of PGF were significantly higher than the levels obtained for plasma both in normal and in asthmatic individuals. The
130
Okazaki
et
J. ALLERGY
al.
T NORMAL
20
T
PLASM ASTHMA
1
SERWl NOWAL
CLIN. IMMUNOL. FEBRUARY 1976
ASTHNA . . . . . . . . .
10
: . .
5.0
. .
z g 2.0 LL E B 2 1.0 5 F -50
. . . . .
.
.
.
. . .
.
+ : .
. .
l .*
.
-05
.
:.; .
I10
.. .
.
+
.20
.. .. .. .
. .
.
L
FIG. 6. coagulate values. significant
PGF for The (p
levels in 30 min difference
