Vol. 88, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
June 13, 1979
Pages 854-860
PRO-TEASE INHIBITORS
ANTAGONIZE THE ACTIVATION
OF POLYMORPHONUCLEAR
LEUKOCYTE OXYGEN CONSUMPTION Bernard
D. Goldstein,
Gisela
Witz,
Marie
Amoruso,
and Walter
Departments of Environmental Medicine and Medicine, University Medical Center, New York, New York
Received
April
Troll
New York 10016
27,1979
SUMMARY: We report that the burst of oxygen consumption, as well as the resultant production of O-. and H 0 occurring in activated human polymorphonuclear leukocytes is ? nhibite 8 8'y various compounds which have in common the ability to antagonize the effects of proteolytic enzymes. This effect of protease inhibitors was observed with a variety of stimuli, both phagocytic and non-phagocytic, used to activate 0;. production in human polymorphonuclear leukocytes. Inhibition was also noted in rat polymorphonuclear leukocytes and alveolar macrophages. The results indicate that proteolysis may be involved in activating the burst of oxygen consumption following stimulation of phagocytic cells. INTRODUCTION Phagocytosis accompanied 0;.
of bacteria
by a burst
and H202 (l-2).
The importance
A similar
by the
frequent
disease
whose
cytosis
and degranulation
other
All
PMN lack
However remain
this
the molecular
leukocytes
results
occurs
to leukocyte
burst
in the
in other
formation
phagocytic
antibacterial
in children
(PMN) is
cells
activity
with
of O2 consumption
chronic despite
of
is
(3). indicated
granulomatous normal
phago-
(1,4). phagocytosis
and insoluble
stimuli
that
process
observed
to the
oxidase
of these
event
infections
soluble
nucleotide
of 02 consumption
of this
In addition
by polymorphonuclear
stimuli
responsible are
for
followed
mechanisms
of bacteria which the
lead
burst
by a lag responsible
there
are a number
to activation
of the
of O2 consumption
in
period for
of at least the
activation
20-30
of pyridine
PMN (5-6). seconds.
process
unknown. METHODS
Materials: Cytochrome C, horse heart, Type III; bovine blood, e-aminocaproic acid; F-Met-Leu-Phe, Abbreviation:
PMN, polymorphonuclear
leukocytes.
0006-291X/79/110854-07$01.00/0 Copyright @ I979 by Academic Press, Inc. All rights of reproduction in any form reserved.
854
superoxide dismutase, p-tosyl-L-arginine
type 1, methyl
Vol. 88, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Soybean and lima bean ester and benzamidine were obtained from Sigma. trypsin inhibitor were purchased from Worthington. Phosphoramidon, leupeptin pepstatin, chymostatin, elastatinal and antipain were obtained through the U.S.-Japan Cooperative Cancer Research Program. Phorbol myristate acetate was purchased from P. Borchert, Univ. of Minn. Trasylol and diisopropylfluorophosphate were gifts from T. Finlay. The calcium ionophore A23187 was provided by R.L. Hamill, Lilly Laboratories. Ac-Ala-Ala-Pro-Ala-CH2Cl was a gift from J. Powers. Cell Preparation: Blood was obtained from healthy human volunteers in accordance with the Declaration of Helsinki. After preparation by sedimentation in 1% dextran (m.wt.264,000) at 250C for 45 min., and osmotic shock to remove red cells, PMN were resuspended in balanced salt solution (128mM NaCl, 12mM KCl, 1mM CaCl 2mM MgC12, 2mM glucose, 4mM PO pH 7.4) at a cell concentration of P'07/ml. Rat PMN were isolated in !' similar manner from the pooled aortic blood of four female Sprague Dawley rats. The lavage fluid from the lungs of these ratswas pooled for the preparation of alveolar macrophages by both hypaque-ficoll gradient (7) and by a second procedure in which lavage cells adherent to plastic petri dishes were studied (8). Assays of oxygen activation: 0 consumption was measured as previously described (9) using equipment ? ocated in the laboratory of M.J. Broekman and A.J. Marcus. 0;. was detected by the reduction of ferricytochrome C that was inhibited b superoxide dismutase (1). In some studies, mixtures containing 1.75 x 10i cells/ml and 44 PM cytochrome C were incubated for 10 min. at 37O with activators in the absence and presence of inhibitors. After incubation, the cells were pelleted and the absorbance of the supernatant was recorded from 600-480 nm on a Perkin Elmer 552 spectrophotometer. 02. produc ion, as measured by cytochrome C reduction, was calculated (E=21.1 nM- f cm-l at 550 nm) using a baseline value obtained from a sample containing the activator and superoxide dismustase, 34 ug/ml. In other studies, the reduction of cytochrome C was measured at 37C using a continous assay with samples containing 0.72 x 106 cells/ml, 45 uM cytochrome C, 0.351~9 p horbol myristate acetate/ml with and without added inhibitors. Hydrogen peroxide production was assayed by following the decrease in scopoletin fluorescence (MPF-3 Perkin Elmer fluorescence spectrophotometer) as described by Root et al (2). In each study control samples contained the solvent used to solubilize the inhibitor. RESULTS AND DISCUSSION The addition burst
of phorbol
of O2 consumption
myristate
beginning
In the presence
of the protease
soybean
trypsin
inhibitor,
produces
little
overt
of phosphoramidon results (120
mM) in the
trasylol less
in resting
10% inhibition
(fig. produced
or epsilon-aminocaproic of stimulated
phorbol
PMN oxygen
stimulated
mixture
40 seconds
(figure
myristate
1).
by phorbol 1).
acetate
consumption.
Inclusion 78% inhibition
acid
O2 consumption.
855
in a
diisopropylfluorophosphate,
of O2 consumption
PMN incubation
(80 units/ml) than
inhibitors
or phosphoramidon, change
to human PMN results
in approximately
to PMN previously
in inhibition
acetate
Addition
myristate
acetate
of benzamidine while
(80 mM) resulted
in
Vol. 88, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL
5
RESEARCH COMMUNICATIONS
IO
15
Time (mid Figure 1. Oxygen consumption of stimulated polymorphonuclear leukocytes. After a 5 minute equilibration period with or without protease inhibitors phorbol myristate acetate was added to a final concentration of 118 rig/ml. Curve A, no inhibitor; curve 8, 133 pM soybean trypsin inhibitor; curve C, 17 mM phosphoramidon; curve 0, 2.7 mM diisopropylfluorophosphate; curve E, no inhibitor initially, 13 mM phosphoramidon added at ten minutes. PMN oxygen consumption 'n the absence of inhibitors was 110.4 218.3 (mean + SD) nanomoles/5 x 10 ii cells/l0 min.
Further studies
evidence
of PMN superoxide
reduction
of cytochrome
reduction
in the
ference
effects.
ferricytochrome This
mixture
produced
there
inhibitor system
0;.
while
at a rate
160 PM soybean lo-30%
135 mM benzamidine
e.g.
by adding study
less
having
various
inhibitors
trypsin inhibition
C reducing 77 ug/ml,
dismutase
34 ug/ml
of that
of stimulated
of 3mM diisopropylof cytochrome
inhibitor
C reduction.
and 430 PM lima
of cytochrome
had no effect.
856
in
oxidase
one tenth
In the presence inhibition
superoxide
to a cytochrome
superoxide than
C
be due to an inter-
x 10m3 M, xanthine
as necessary,
was a complete
produced
system,
present 4.6
could
in
by the
in PMN cytochrome
inhibitors
assay
was obtained
as measured
The decrease
was tested
1 nmol/min/ml).
2OmM phosphoramidon,
this
This
of acetaldehyde
PMN (approximately
trypsin
in the
C 75 PM, and,
fluorophosphate
1).
inhibitors
production,
of protease
used in the
consisting
of protease
radical
C (Table
compounds
concentrations
system
anion
presence
of these
dismutase-like the
of an effect
A partial
C reduction interference
bean in
BIOCHEMICAL
Vol. 88, No. 3, 1979
Table
1.
Effect
of
protease
leukocytes
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
inhibitors stimulated
production
on 0;. by phorbol
in
myristate
polymorphonuclear
acetate. Percent
Compound
Concentration
Soybean
Lima
trypsin
bean
inhibitor
trypsin
inhibitor
Pepstatin
of
control
160pM 80pM 16uM 1.6pM
3 53 96 98
430pM
50
750uM
99
Phosphoramidon
20mM 5mM
5 95
135mM 67mM 27mM
15 45 94
80mM
100
Leupeptin
60mM
100
Antipain
60nlM 25mM 3.5mM
44 1;;
3omM
100
Benzamidine
Epsilon-aminocaproic
acid
TAME* Trasylol
100
Elastatinal
units/ml
99
20mM 40mM
64 37
Ac-Ala-Ala-Pro-Ala-CH2C1
4.8mM
89
Chymostatin
2.8mM
62
*
p-Toluenesulfonyl
of soybean
trypsin
tetrazolium that
studies
only
effects
studies,
it
the
as well
as other
of this
noted
proteins,
upon cytochrome serine
was elected
to further
compounds
02 consumption,
that
C reduction.
esterase
using
confirm
of H202 production.
of PMN H202 production
following
Our findings be precluded
of the
importance
toward interpreting inhibition
measure
related
greater
stimulation
857
would
the
A
(10).
In view
inhibitor
a third
in PMN nitro-blue
by Amano et al
use of diisopropylfluorophosphate
by antiprotease
rate
ester
was previously
depending
of the
methyl
inhibitor,
reduction
indicate for
L-arginine
than with
the
of 02 consumption
to activated
PMN
50% decrease
in the
125 rig/ml
phorbol
Vol. 88, No. 3, 1979
myristate
BIOCHEMICAL
acetate
or 10 pg/ml
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
digitonin
was observed
diisopropylfluorophosphate.
9 x 1o-4
in the
A 40% prolongation
presence
of the
lag
of period
before H202 production was observed at a diisopropylfluorophosphate concentration of 1 x 10m4M. The effects of other protease inhibitors
on
H202 production
shown
in Table
were
similar
to those
myristate
acetate
0;./6.2
by 100%.
x lo5
Similarly,
2. These
activation
proteases
suggest process
of human PMN include
inhibitory
enzyme,
effects
cathepsin
trasylol
G or elastase.
G, and a potent
In a recent
duction.
this
is
to a large
derivatives of protease
which
are
inhibitor
are
shown
pinpoint
any single The neutral
and cathepsin granules.
effects
G, a The
a serine
were
be expected
esterase,
observed
to inhibit
has been suggested
known
is
based
is
with cathepsin had little
that
a
of human PMN 0;.
on inhibition
to have nonspecific
effects
in
antagonizing
process.
in the activation extent
The
stimulated
Ac-ala-ala-pro-ala-CH2C1 it
858
is
implicate
would
dishes.
production
intracellular
minor
(12)
involved
petri
clearly
collagenase,
of which
abstract
enzyme
Interpretation
the activation
of PMN elastase
chymotrypsin-like
methylketone
for
Relatively
both
inhibitor
However,
does not
in
160 pM
cells.
effects
located
(8),
of 02. generation
inhibitors
of diisopropylfluorophosphate
or chymostatin,
(11).
all
macrophages
et al
and non-phagocytic,
elastase,
inhibitor,
of alveolar
to plastic
common to phagocytic
responsible
trypsin
PMN 0;.
protease
phorbol
min and 20.7
inhibition
to inhibit
that
with
by Johnston
adherent
phagocytic
of inhibitory
chymotrypsin-like
effect
of 0;.
cells/l0
Soybean
in complete
inhibitor both
as being
x lo6
as described
preparation
trypsin
findings
The pattern
e.g.
studies
stimulated
by 97% and that
resulted
of agents,
PMN protease
production
macrophage
by a variety
a general
O;./l.Z
nmole
min respectively.
inhibitor
of soybean
Table
11.1
when measured
alveolar
ability
suspensions
cells/30 PMN 0;.
trypsin
by the
macrophage
produced
160 uM, inhibited
soybean
in the
1.
Rat PMN and alveolar
nmole
observed
complicated
pro-
by chloroeffects by lack
(13). of
Vol. 88, No. 3, 1979
Table
BIOCHEMICAL
Inhibition
2. activated
in
AND BIOPHYSICAL
by soybean trypsin human
inhibitor
polymorphonuclear
(SBTI)
leukocytes
non-particulate
RESEARCH COMhWNlCATlONS
of 0;.
production
by particulate
and
stimuli.* 0-e production (nmoleG/l.75 x 106 PMN)
Initiator
Control
Zymosan
(10
Latex
(10
Phorbol
particles/cell)
Concanavalin
acetate
A (37
Ionophore NaF
A23187
(10
(0.86
unknown
site
the
granules
membrane
enzyme inhibitable
oxidase
Other
(15).
It
lag
period
types
A direct be completely
in active
0
22
0
19
0
4
1
5
0
site
the
1.099
inhibitor
the cell
membrane,
have also
conceivable
that
more than
and fluidity, effect
there
where
microns
in
0;.
in
to the
between
a cell
and soybean
trypsin
of events
as well
to pyridine
on this
in view proteases
as changes
nucleotide
inhibitors
particularly
is
including
is a cascade
one protease,
of protease
configuration
study
in relation
been reported,
leading
precluded,
NaF
be in PMN granules as following
need not
with
for
latter
of the reported
and other
enzyme
(17). Degranulation
disease
7
by diisopropylfluorophosphate
involving (16)
activation.
similarity
0
of the added
PMN proteases
is also
polarity
enzyme can not
This fuse
(14).
in membrane
25
37OC for 10 minutes except was 20 minutes. serum/ml buffer. Particles
location
of activation.
produced
the
0
pg/ml)
the
inhibitor
1
vM)
concerning
stimulation
during
ug/ml)
us/ml)
Samples were incubated at which the incubation time Sample contained 0.172 ml diameter.
information
1
pg/ml)
(0.6
Digitonin
**
(1.72
(0.02M)
F-Met-Leu-Phe
*
9
particles/cell)**
myristate
SBTI (160 $1)
patients
and enzyme release is
thought
in PMN of chronic
to be normal.
859
However,
the
granulomatous literature
reveals
Vol. 88, No. 3, 1979
no instances est
in which
to do so,
as well
BIOCHEMICAL
neutral
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
proteases
as to test
for
were the
measured.
presence
It would
of a protease
be of interinhibitor.
ACKNOWLEDGEMENTS: We thank Bruce Lippmann for technical assistance and LaVerne for preparing the manuscript. We also thank Drs. I.M. Goldstein, Levitz, T. Finlay, P. Elsbach, and R. Wiesner for helpful discussions, M.J. Broekman for assistance in measuring 02 consumption. The work was ported by NIH grants ES 00673, HL 18163, CA 16060 and center grant ES
Marilyn M. and Dr sup00260.
REFERENCES 1. Curnatte, J.T., Whitten, D.M., and Babior, B.M. (1974) New Eng. J. Med. 290, 593-597. J.A. (1977) J. Clin. Invest. 60: 1266-1279. 2. Root, R.K., and Metcalf, M.L. (1975) J. Exp. Med. 141, 257-264. Drath, D.B., and Karnovsky, S: Baehner, Karnovsky, M.J. and Karnovsky, M.L. (1968) J. Clin. Invest. R.L., 47, 187-192 5. Repine, J.E., White, J.G., Clawson, C.C., and Holmes, B.M. (1974) J. Clin.
Invest.
54, 83-90.
6. Cohen, H.J., and Chovaniec, M.E. (1978) J. Clin. Invest. 61, 1081-1087 7. Goldstein, B.D., Hamburger, S.J., Falk, G.A. and Amoruso, M.A. (1977) Life Sci. 21, 1637-1644 8. Johnston, R.B., Godzik, C.A., and Cohn, Z.A. (1978) J. Exp. Med. 148, 115-127 9. Bressler, N.M., Broekman, M.J., and Marcus, A.J. (1979) Blood 53, 167-178. 10. Amano, D., Kagosaki, Y., and Usui, T. (1975) Biochem. Biophys. Res. Commun. 66, 272-279. 11. Tuhy, P.M., and Powers, J.C. (1975) FEBS Letter 50, 359-361. 12. Simchowitz, L., Mehta, J., and Spilberg, I. (1979) Federation Proc. 38, 1454A. 13. Rossman, T., Norris, C., and Troll, W. (1974) J. Biol. Chem. Chem. 249, 3412-3417. 14. Goldstein, I.M., Cerqueira, M., Lind, S., and Kaplan, H.B. (1977) J. Clin. Invest. 59, 249-254. 15. Wintroub, B.U., Goetzl, E.J., and Austen, K.F. (1974) J. Exp. Med. 140, 812-824. G., and Korchak, H.M. (1978) Proc. Natl. Acad. Sci. USA 16. Weissmann, 75, 3818-3822. 17. Argos, P., Garavito, R.M., Eventoff, W., Rossman, M.G., and Branden, C. I. (1978) J,, Mol. Biol. 126, 141-158.
860
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
June 13, 1979
Pages 854-860
PRO-TEASE INHIBITORS
ANTAGONIZE THE ACTIVATION
OF POLYMORPHONUCLEAR
LEUKOCYTE OXYGEN CONSUMPTION Bernard
D. Goldstein,
Gisela
Witz,
Marie
Amoruso,
and Walter
Departments of Environmental Medicine and Medicine, University Medical Center, New York, New York
Received
April
Troll
New York 10016
27,1979
SUMMARY: We report that the burst of oxygen consumption, as well as the resultant production of O-. and H 0 occurring in activated human polymorphonuclear leukocytes is ? nhibite 8 8'y various compounds which have in common the ability to antagonize the effects of proteolytic enzymes. This effect of protease inhibitors was observed with a variety of stimuli, both phagocytic and non-phagocytic, used to activate 0;. production in human polymorphonuclear leukocytes. Inhibition was also noted in rat polymorphonuclear leukocytes and alveolar macrophages. The results indicate that proteolysis may be involved in activating the burst of oxygen consumption following stimulation of phagocytic cells. INTRODUCTION Phagocytosis accompanied 0;.
of bacteria
by a burst
and H202 (l-2).
The importance
A similar
by the
frequent
disease
whose
cytosis
and degranulation
other
All
PMN lack
However remain
this
the molecular
leukocytes
results
occurs
to leukocyte
burst
in the
in other
formation
phagocytic
antibacterial
in children
(PMN) is
cells
activity
with
of O2 consumption
chronic despite
of
is
(3). indicated
granulomatous normal
phago-
(1,4). phagocytosis
and insoluble
stimuli
that
process
observed
to the
oxidase
of these
event
infections
soluble
nucleotide
of 02 consumption
of this
In addition
by polymorphonuclear
stimuli
responsible are
for
followed
mechanisms
of bacteria which the
lead
burst
by a lag responsible
there
are a number
to activation
of the
of O2 consumption
in
period for
of at least the
activation
20-30
of pyridine
PMN (5-6). seconds.
process
unknown. METHODS
Materials: Cytochrome C, horse heart, Type III; bovine blood, e-aminocaproic acid; F-Met-Leu-Phe, Abbreviation:
PMN, polymorphonuclear
leukocytes.
0006-291X/79/110854-07$01.00/0 Copyright @ I979 by Academic Press, Inc. All rights of reproduction in any form reserved.
854
superoxide dismutase, p-tosyl-L-arginine
type 1, methyl
Vol. 88, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Soybean and lima bean ester and benzamidine were obtained from Sigma. trypsin inhibitor were purchased from Worthington. Phosphoramidon, leupeptin pepstatin, chymostatin, elastatinal and antipain were obtained through the U.S.-Japan Cooperative Cancer Research Program. Phorbol myristate acetate was purchased from P. Borchert, Univ. of Minn. Trasylol and diisopropylfluorophosphate were gifts from T. Finlay. The calcium ionophore A23187 was provided by R.L. Hamill, Lilly Laboratories. Ac-Ala-Ala-Pro-Ala-CH2Cl was a gift from J. Powers. Cell Preparation: Blood was obtained from healthy human volunteers in accordance with the Declaration of Helsinki. After preparation by sedimentation in 1% dextran (m.wt.264,000) at 250C for 45 min., and osmotic shock to remove red cells, PMN were resuspended in balanced salt solution (128mM NaCl, 12mM KCl, 1mM CaCl 2mM MgC12, 2mM glucose, 4mM PO pH 7.4) at a cell concentration of P'07/ml. Rat PMN were isolated in !' similar manner from the pooled aortic blood of four female Sprague Dawley rats. The lavage fluid from the lungs of these ratswas pooled for the preparation of alveolar macrophages by both hypaque-ficoll gradient (7) and by a second procedure in which lavage cells adherent to plastic petri dishes were studied (8). Assays of oxygen activation: 0 consumption was measured as previously described (9) using equipment ? ocated in the laboratory of M.J. Broekman and A.J. Marcus. 0;. was detected by the reduction of ferricytochrome C that was inhibited b superoxide dismutase (1). In some studies, mixtures containing 1.75 x 10i cells/ml and 44 PM cytochrome C were incubated for 10 min. at 37O with activators in the absence and presence of inhibitors. After incubation, the cells were pelleted and the absorbance of the supernatant was recorded from 600-480 nm on a Perkin Elmer 552 spectrophotometer. 02. produc ion, as measured by cytochrome C reduction, was calculated (E=21.1 nM- f cm-l at 550 nm) using a baseline value obtained from a sample containing the activator and superoxide dismustase, 34 ug/ml. In other studies, the reduction of cytochrome C was measured at 37C using a continous assay with samples containing 0.72 x 106 cells/ml, 45 uM cytochrome C, 0.351~9 p horbol myristate acetate/ml with and without added inhibitors. Hydrogen peroxide production was assayed by following the decrease in scopoletin fluorescence (MPF-3 Perkin Elmer fluorescence spectrophotometer) as described by Root et al (2). In each study control samples contained the solvent used to solubilize the inhibitor. RESULTS AND DISCUSSION The addition burst
of phorbol
of O2 consumption
myristate
beginning
In the presence
of the protease
soybean
trypsin
inhibitor,
produces
little
overt
of phosphoramidon results (120
mM) in the
trasylol less
in resting
10% inhibition
(fig. produced
or epsilon-aminocaproic of stimulated
phorbol
PMN oxygen
stimulated
mixture
40 seconds
(figure
myristate
1).
by phorbol 1).
acetate
consumption.
Inclusion 78% inhibition
acid
O2 consumption.
855
in a
diisopropylfluorophosphate,
of O2 consumption
PMN incubation
(80 units/ml) than
inhibitors
or phosphoramidon, change
to human PMN results
in approximately
to PMN previously
in inhibition
acetate
Addition
myristate
acetate
of benzamidine while
(80 mM) resulted
in
Vol. 88, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL
5
RESEARCH COMMUNICATIONS
IO
15
Time (mid Figure 1. Oxygen consumption of stimulated polymorphonuclear leukocytes. After a 5 minute equilibration period with or without protease inhibitors phorbol myristate acetate was added to a final concentration of 118 rig/ml. Curve A, no inhibitor; curve 8, 133 pM soybean trypsin inhibitor; curve C, 17 mM phosphoramidon; curve 0, 2.7 mM diisopropylfluorophosphate; curve E, no inhibitor initially, 13 mM phosphoramidon added at ten minutes. PMN oxygen consumption 'n the absence of inhibitors was 110.4 218.3 (mean + SD) nanomoles/5 x 10 ii cells/l0 min.
Further studies
evidence
of PMN superoxide
reduction
of cytochrome
reduction
in the
ference
effects.
ferricytochrome This
mixture
produced
there
inhibitor system
0;.
while
at a rate
160 PM soybean lo-30%
135 mM benzamidine
e.g.
by adding study
less
having
various
inhibitors
trypsin inhibition
C reducing 77 ug/ml,
dismutase
34 ug/ml
of that
of stimulated
of 3mM diisopropylof cytochrome
inhibitor
C reduction.
and 430 PM lima
of cytochrome
had no effect.
856
in
oxidase
one tenth
In the presence inhibition
superoxide
to a cytochrome
superoxide than
C
be due to an inter-
x 10m3 M, xanthine
as necessary,
was a complete
produced
system,
present 4.6
could
in
by the
in PMN cytochrome
inhibitors
assay
was obtained
as measured
The decrease
was tested
1 nmol/min/ml).
2OmM phosphoramidon,
this
This
of acetaldehyde
PMN (approximately
trypsin
in the
C 75 PM, and,
fluorophosphate
1).
inhibitors
production,
of protease
used in the
consisting
of protease
radical
C (Table
compounds
concentrations
system
anion
presence
of these
dismutase-like the
of an effect
A partial
C reduction interference
bean in
BIOCHEMICAL
Vol. 88, No. 3, 1979
Table
1.
Effect
of
protease
leukocytes
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
inhibitors stimulated
production
on 0;. by phorbol
in
myristate
polymorphonuclear
acetate. Percent
Compound
Concentration
Soybean
Lima
trypsin
bean
inhibitor
trypsin
inhibitor
Pepstatin
of
control
160pM 80pM 16uM 1.6pM
3 53 96 98
430pM
50
750uM
99
Phosphoramidon
20mM 5mM
5 95
135mM 67mM 27mM
15 45 94
80mM
100
Leupeptin
60mM
100
Antipain
60nlM 25mM 3.5mM
44 1;;
3omM
100
Benzamidine
Epsilon-aminocaproic
acid
TAME* Trasylol
100
Elastatinal
units/ml
99
20mM 40mM
64 37
Ac-Ala-Ala-Pro-Ala-CH2C1
4.8mM
89
Chymostatin
2.8mM
62
*
p-Toluenesulfonyl
of soybean
trypsin
tetrazolium that
studies
only
effects
studies,
it
the
as well
as other
of this
noted
proteins,
upon cytochrome serine
was elected
to further
compounds
02 consumption,
that
C reduction.
esterase
using
confirm
of H202 production.
of PMN H202 production
following
Our findings be precluded
of the
importance
toward interpreting inhibition
measure
related
greater
stimulation
857
would
the
A
(10).
In view
inhibitor
a third
in PMN nitro-blue
by Amano et al
use of diisopropylfluorophosphate
by antiprotease
rate
ester
was previously
depending
of the
methyl
inhibitor,
reduction
indicate for
L-arginine
than with
the
of 02 consumption
to activated
PMN
50% decrease
in the
125 rig/ml
phorbol
Vol. 88, No. 3, 1979
myristate
BIOCHEMICAL
acetate
or 10 pg/ml
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
digitonin
was observed
diisopropylfluorophosphate.
9 x 1o-4
in the
A 40% prolongation
presence
of the
lag
of period
before H202 production was observed at a diisopropylfluorophosphate concentration of 1 x 10m4M. The effects of other protease inhibitors
on
H202 production
shown
in Table
were
similar
to those
myristate
acetate
0;./6.2
by 100%.
x lo5
Similarly,
2. These
activation
proteases
suggest process
of human PMN include
inhibitory
enzyme,
effects
cathepsin
trasylol
G or elastase.
G, and a potent
In a recent
duction.
this
is
to a large
derivatives of protease
which
are
inhibitor
are
shown
pinpoint
any single The neutral
and cathepsin granules.
effects
G, a The
a serine
were
be expected
esterase,
observed
to inhibit
has been suggested
known
is
based
is
with cathepsin had little
that
a
of human PMN 0;.
on inhibition
to have nonspecific
effects
in
antagonizing
process.
in the activation extent
The
stimulated
Ac-ala-ala-pro-ala-CH2C1 it
858
is
implicate
would
dishes.
production
intracellular
minor
(12)
involved
petri
clearly
collagenase,
of which
abstract
enzyme
Interpretation
the activation
of PMN elastase
chymotrypsin-like
methylketone
for
Relatively
both
inhibitor
However,
does not
in
160 pM
cells.
effects
located
(8),
of 02. generation
inhibitors
of diisopropylfluorophosphate
or chymostatin,
(11).
all
macrophages
et al
and non-phagocytic,
elastase,
inhibitor,
of alveolar
to plastic
common to phagocytic
responsible
trypsin
PMN 0;.
protease
phorbol
min and 20.7
inhibition
to inhibit
that
with
by Johnston
adherent
phagocytic
of inhibitory
chymotrypsin-like
effect
of 0;.
cells/l0
Soybean
in complete
inhibitor both
as being
x lo6
as described
preparation
trypsin
findings
The pattern
e.g.
studies
stimulated
by 97% and that
resulted
of agents,
PMN protease
production
macrophage
by a variety
a general
O;./l.Z
nmole
min respectively.
inhibitor
of soybean
Table
11.1
when measured
alveolar
ability
suspensions
cells/30 PMN 0;.
trypsin
by the
macrophage
produced
160 uM, inhibited
soybean
in the
1.
Rat PMN and alveolar
nmole
observed
complicated
pro-
by chloroeffects by lack
(13). of
Vol. 88, No. 3, 1979
Table
BIOCHEMICAL
Inhibition
2. activated
in
AND BIOPHYSICAL
by soybean trypsin human
inhibitor
polymorphonuclear
(SBTI)
leukocytes
non-particulate
RESEARCH COMhWNlCATlONS
of 0;.
production
by particulate
and
stimuli.* 0-e production (nmoleG/l.75 x 106 PMN)
Initiator
Control
Zymosan
(10
Latex
(10
Phorbol
particles/cell)
Concanavalin
acetate
A (37
Ionophore NaF
A23187
(10
(0.86
unknown
site
the
granules
membrane
enzyme inhibitable
oxidase
Other
(15).
It
lag
period
types
A direct be completely
in active
0
22
0
19
0
4
1
5
0
site
the
1.099
inhibitor
the cell
membrane,
have also
conceivable
that
more than
and fluidity, effect
there
where
microns
in
0;.
in
to the
between
a cell
and soybean
trypsin
of events
as well
to pyridine
on this
in view proteases
as changes
nucleotide
inhibitors
particularly
is
including
is a cascade
one protease,
of protease
configuration
study
in relation
been reported,
leading
precluded,
NaF
be in PMN granules as following
need not
with
for
latter
of the reported
and other
enzyme
(17). Degranulation
disease
7
by diisopropylfluorophosphate
involving (16)
activation.
similarity
0
of the added
PMN proteases
is also
polarity
enzyme can not
This fuse
(14).
in membrane
25
37OC for 10 minutes except was 20 minutes. serum/ml buffer. Particles
location
of activation.
produced
the
0
pg/ml)
the
inhibitor
1
vM)
concerning
stimulation
during
ug/ml)
us/ml)
Samples were incubated at which the incubation time Sample contained 0.172 ml diameter.
information
1
pg/ml)
(0.6
Digitonin
**
(1.72
(0.02M)
F-Met-Leu-Phe
*
9
particles/cell)**
myristate
SBTI (160 $1)
patients
and enzyme release is
thought
in PMN of chronic
to be normal.
859
However,
the
granulomatous literature
reveals
Vol. 88, No. 3, 1979
no instances est
in which
to do so,
as well
BIOCHEMICAL
neutral
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
proteases
as to test
for
were the
measured.
presence
It would
of a protease
be of interinhibitor.
ACKNOWLEDGEMENTS: We thank Bruce Lippmann for technical assistance and LaVerne for preparing the manuscript. We also thank Drs. I.M. Goldstein, Levitz, T. Finlay, P. Elsbach, and R. Wiesner for helpful discussions, M.J. Broekman for assistance in measuring 02 consumption. The work was ported by NIH grants ES 00673, HL 18163, CA 16060 and center grant ES
Marilyn M. and Dr sup00260.
REFERENCES 1. Curnatte, J.T., Whitten, D.M., and Babior, B.M. (1974) New Eng. J. Med. 290, 593-597. J.A. (1977) J. Clin. Invest. 60: 1266-1279. 2. Root, R.K., and Metcalf, M.L. (1975) J. Exp. Med. 141, 257-264. Drath, D.B., and Karnovsky, S: Baehner, Karnovsky, M.J. and Karnovsky, M.L. (1968) J. Clin. Invest. R.L., 47, 187-192 5. Repine, J.E., White, J.G., Clawson, C.C., and Holmes, B.M. (1974) J. Clin.
Invest.
54, 83-90.
6. Cohen, H.J., and Chovaniec, M.E. (1978) J. Clin. Invest. 61, 1081-1087 7. Goldstein, B.D., Hamburger, S.J., Falk, G.A. and Amoruso, M.A. (1977) Life Sci. 21, 1637-1644 8. Johnston, R.B., Godzik, C.A., and Cohn, Z.A. (1978) J. Exp. Med. 148, 115-127 9. Bressler, N.M., Broekman, M.J., and Marcus, A.J. (1979) Blood 53, 167-178. 10. Amano, D., Kagosaki, Y., and Usui, T. (1975) Biochem. Biophys. Res. Commun. 66, 272-279. 11. Tuhy, P.M., and Powers, J.C. (1975) FEBS Letter 50, 359-361. 12. Simchowitz, L., Mehta, J., and Spilberg, I. (1979) Federation Proc. 38, 1454A. 13. Rossman, T., Norris, C., and Troll, W. (1974) J. Biol. Chem. Chem. 249, 3412-3417. 14. Goldstein, I.M., Cerqueira, M., Lind, S., and Kaplan, H.B. (1977) J. Clin. Invest. 59, 249-254. 15. Wintroub, B.U., Goetzl, E.J., and Austen, K.F. (1974) J. Exp. Med. 140, 812-824. G., and Korchak, H.M. (1978) Proc. Natl. Acad. Sci. USA 16. Weissmann, 75, 3818-3822. 17. Argos, P., Garavito, R.M., Eventoff, W., Rossman, M.G., and Branden, C. I. (1978) J,, Mol. Biol. 126, 141-158.
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