Vol. 185, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
June 30, 1992
RESEARCH COMMUNICATIONS Pages 1115-1121
ZINC HYDROXIDESTIMULATESSUPBROXIDEPRODUCTIONBY RAT ALVEOLARMACROPHAGES Keiki
Ogino*,
Yukie Izumi, Hironobu Ishiyama, Tomoko Murata, Haruo Kobayashi,and Tatsuya Houbara
Department of Public Health, Yamaguchi University School of Medicine,1144 Kogushi, Ube 755,Japan Received May 21, 1992 SUMMARY:The effect of zinc hydroxide on superoxide (0;) production by rat alveolar macrophages was determined by chemiluminescence and by cytochrome c reduction. Zinc ions had no effect on the chemiluminescence of unstimulated alveolar macrophages. By contrast, zinc hydroxide (ZnOH,), a neutralized form of zinc ions, increased the chemiluminescence level and 0, release. Increased 0, release was inhibited by pertussis toxin, isoquinoline sulfonamide and pretreatment with EGTA. These findings indicate that zinc hydroxide formation from zinc compounds can stimula e the 0; production by alveolar macrophages by receptor-mediated and Ca2f B 1992Academic Press,Inc. dependent process.
During phagocytosis polymorphonuclear
leukocytes,
(02) and H202 (1,Z). during
or chemical
phagocytosis
activation,
produce substantial
These oxidants
are utilized
; they are also thought
e.g.,
ischemic
heart
tinal
disorders
(5),
failure
(3),
respiratory
renal
disease
(6),
Zinc has been shown to inhibit (8) through
its
the free
radical
macrophages (11). industrial accompanying
setting
causes fever,
leukocytosis
+To whom correspondence
within
of superoxide
to be involved disorders
killing
in tissue
(4),
gastrointes(7).
by stimulated
(9) or its
damage,
macrophages
inhibitory
effect
High doses of zinc might eventually
affect
By contrast,
quantities
and atherosclerosis
effect
damage induced
the same time would adversely
as well as
for microbial
0; production
membrane stabilizing
on NADPHoxidase and ATPase (10). minimize
macrophages,
by activated
the bacteriocidal
the inhalation chills,
macrophages, capacity
but at
of the
of zinc oxide fumes in an
and respiratory
4 to 12 hr after
symptoms with
inhalation
(12).
There
should be addressed. 0006-291X/92
1115
$4.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
BIOCHEMICAL
185, No. 3, 1992
may be a close
relationship
AND BIOPHYSICAL
between zinc and phagocytosis.
zinc hydroxide,
a neutralized
by rat alveolar
macrophages and discuss
relation
to signal
RESEARCH COMMUNICATIONS
zinc compound form,
transduction
stimulated
We found that 02 production
the mechanism of activation
in
pathway.
MATERIALS AND METHODS Materials: Bovine serum albumin, horse heart ferricytochrome c (type VI), phorbol myristate acetate, pertussis toxin, EGTA and superoxide dismutase (bovine erythrocytes, 3000 lJ/mg protein) were obtained from Sigma Chemical. Catalase (65,000 U/mg protein) was obtained from Boehringer Mannheim GmbH. ZnSO4 of 99.9% purity was purchased from Wako Pure Chemicals. Mouse sodium metrizoate ficoll was obtained from Japan Immuno Research Laboratory and 2-methyl-6-phenyl-3,7-dihydroimidazo[l,2]pyrazin-3-one was purchased from Tokyo Kasei. 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride was obtained from Seikagaku Kogyo. Preparation of zinc hydroxide: Zinc sulfate was dissolved in double distilled water and was then adjusted to pH 7.0 with NaOH. A white opaque liquid was obtained. The concentration of zinc hydroxide was expressed as that of zinc sulfate. Preparation of alveolar macrophages: Alveolar macrophages were collected from adult Wistar rats bv bronchoalveolar lavage with calcium and magnesium-free Dulbecco's phosphate-buffered saline-(13). After centrifugation at 70 x g for 5 min and hypotonic lysis of contaminating erythrocytes, cells were suspended in Hanks' balanced salt solution (HBSS) containing 0.1% bovine serum albumin and layered on mouse sodium metrizoate ficoll (d=1.090), following which they were centrifuged at 1000 x g for 30 min. Macrophages, recovered from the upper band, were washed twice with HBSS by centrifugation at 70 x g for 5 min. Cells were resuspended in HBSS. Chemiluminescence: Chemiluminescence was measured using a Cypridina luciferin'analog, 2-methyl-6-phenyl-3,7-dihydroimidazo[l,2]pyrazin-3-one (CLA) as a probe with an Aloka Co. luminescence reader at 37OC (14). Reaction mixtures contained 1.0 x lo6 macrophages, 1.0 pM CLA, and O-l mM zinc sulfate or zinc hydroxide in 1 ml of continuously stirred HBSS. Intensity of light emission was expressed by subtracting initial non-specific luminescence from maximal luminescence. CLA was dissolved double distilled water and the concentration of CLA was determined by E = 8900 M-' cm-'. nm 410
in
Superoxide release: Superoxide was measured, following the superoxide dismutase (SOD) inhibitable reduction of ferricvtochrome c at 550 nm in a single spectrophotometer, by a modification of the procedure of Babior et al. (15). Reaction mixtures contained 1.0 x lo6 macrophages, 0.1 mM ferricytochrome c, and 1 mM zinc hydroxide or 1 pM phorbol myristate acetate (PMA) in 1 ml of HBSS. Before incubation, 10 pg/ml of SOD was added as reference. After incubation for 10 min at 37'C, reaction mixtures were chilled, clarified by centrifugation, and used to measure ferroc tochrome c at 550 nm. To investigate the role of GTP-binding protein, Ca' Y and protein kinase C on zinc hydroxide-induced superoxide production, pertussis toxin (16), I-(5-isoquinolinesulfonyl)-2-methylpiperazine, an inhibitor of protein kinase C (17), EGTA (18) were added. 1116
Vol.
185,
No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
RESULTS Zinc
hydroxide-induced
CL&dependent
CLA-dependent of
zinc
chemiluminescence
hydroxide.
single
peak
at
2.8
min,
chemiluminescence
inhibited
by catalase
nescence.
The pH of
sulfate
was 6.7.
reaction
mixture
1).
time
Zinc
reaction zinc
pH. The level (Fig.
of 0.7
mixtures
min
the 0.1
of
hydoxide-induced
hydroxide-
was not
on chemilumi-
addition
of
1 mM zinc
mM had no effect
on the
chemiluminescence
was a significant zinc
showed
Zinc
by SOD, but
below
logarithmic
1).
had no effect after
addition
hydroxide
(Fig.
sulfate zinc
on the
by zinc
inhibited ions
2) and there
between
dramatically
induced
was completely
the
p < 0.01)
correlation
hydroxide
concentration
values
intensity.
release
Superoxide
nmol/106
a lag
(Table
and chemiluminescence
reduction.
with
However,
was dose-dependent
Superoxide
increased
The chemiluminescence
induced
(R=0.977,
chemiluminescence
release Superoxide
cell
per
was determined released
10 min.
Zinc
from
by SOD-inhibitable resting
hydroxide
macrophages
cytrochrome was 0.9
c + 0.4
(1 mM) and PMA (1 PM) augmented
II--
&
INCUBATION TIME
Fig.
1
MIN
1. CLA-dependent chemiluminscence due to zinc hydroxide. The reaction mixtures contained 1 x lo6 cells, 1 ,uM CLA, 1 mM zinc hydroxide, and HBSS in a total volume of 1.0 ml (system 1). Zinc hydroxide was omitted from system 1 for control (system 2). System 3 contained 15 units of SOD, in addition to the ingredients contained in system 1. Results are expressed as a single luminescence measurement in one representative experiment. 1117
a
Vol.
185,
No.
3,
BIOCHEMICAL
1992
Table
1.
Effect
of of
AND
BIOPHYSICAL
some chemicals on CLA-dependent alveolar macrophages
Chemicals
35240 hydroxide
1 mM
Zinc hydroxide + SOD 30 units
725240
sulfate
Zinc
sulfate
Results indicate
O2
release
(500
14700
(5)
* 62143
(5)
f. 6844
(5)
754980
f. 48037
(5)
41950
I? 4618
(4)
mM
36975
+I 4228
(4)
are expressed the number
of
as the mean experiments.
and 22.4-fold,
f
SD.
The
respectively
number
in
(Table
parentheses
2).
1-(5-isoquinolinesulfonyl)-2-methylpiperazine
EGTA (10 mM) inhibited 62%,
5172
1 mM 0.1
6.6-fold
rig/ml),
intensity
1 mM
Zinc hydroxide 1 mM + catalase 1300 units Zinc
f
respectively
COMMUNICATIONS
chemiluminescence
Maximal light (counts/min)
Control Zinc
RESEARCH
zinc
(Table
hydroxide-induced
02 release
Pertussis
toxin
(100
and
by 61%,
PM), 43%,
and
3).
DISCUSSION The present oxide
release
process.
study from
rat
The respiratory
demonstrated alveolar burst
.Ol
that
macrophages of
2.
hydroxide by the
phagocytes
is
Concentration
can
induce
super-
receptor-mediated
triggered
1
1
Zinc Hydroxide
Fig.
zinc
by phagocytosable
10
(mM)
CLA-dependent chemiluminescence of macrophages at various zinc hydroxide concentrations. The chemiluminescence was determined in the presence of 1 pM CLA, 1 x lo6 cells, and zinc hydroxide as described in Methods. CLA light emission was expressed as counts per min. The results are mean + SD of three determinations.
1118
Vol.
185, No. 3, 1992 Table
BIOCHEMICAL
Effect
2.
AND BIOPHYSICAL
of zinc hydroxide and phorbol 0; release by macrophages
RESEARCH COMMUNICATIONS
myristate
02 production (nmol/106 cell/l0 Control Zinc
hydroxide
PMA
(bacteria,
0.9 f 0.4
(5)
1 mM
5.9 f 0.3
(5)
1 PM
20.2 f 0.4
(5)
by a number complement acids,
soluble
factors,
factors
phorbol
and endotoxin IgG and/or
respiratory Fc part system pertussis
burst
on the
esters,
immunoglobulins surface
toxin
Table
which
3.
of
phagocytes
blocks
Effect
a The cells expressed
of the
the
avidly
activity
23).
toxin
when coated
and triggers
affinity
C3b component
(22,
activating
ingested
high
receptors of
the
for
action
GTP-binding
protein
on zinc hydroxide-induced macrophages
0;
% inhibition min)
1 mM
5.6 f 0.7
500 rig/ml
2.2 2 0.7
61
3.2 f 0.8
43
100 pM lmMa
5.3 f 0.6
10 mM
2.0 f 0.8
were preincubated for 60 min at 37°C. Results as the mean f SD of five experiments. 1119
the
complement
The inhibitory
of the
and
fatty
material,
0; production (nmol/106 cell/l0
+ 1-(5-isoquinolinesulfonyl) -2-methylpiperazine
+ EGTA
is
etc.)
lectins,
platelet
particulate
of some chemicals release by alveolar
Zinc hydroxide
Zinc hydroxide
, cytochalasins,
and the
Chemicals
+ pertussis
leukotrienes diacylglycerol,
because
debris,
peptides,
Non-bacterial
21),
cell
the number
chemotactic
to complement,
(20,
indicate
materials,
,e.g.
(19).
exposed
of the
aggregate
Ca 2+ ionophores,
detergents,
factor, with
of
viruses,
on
min)
Values are mean f SD. The numbers in paretheses of experiments.
particles
acetate
62
are
of (16)
Vol.
on zinc the
hydroxide-induced
receptor
before
pathway.
Isoquinoline
inhibits
the
our It
BIOCHEMICAL
185, No. 3, 1992
difficult
have
calcium
which
exogeneous
Ca
2t
phosphodiesteric of
have
been
of
zinc
hydroxide
C,
is
we still
phagocytosis.
depletes
with
reports
previous
protein-linked
do
Pre-
extracellular 02 release
the
receptor. recognition
since
induces
on
Therefore,
hydroxide-induced
ionized was by the
activation
of
4,5-biphosphate
with
and protein
oxide
and itself
is
the
capable
macrophages.
Therefore,
investigations
of the
ACKNOWLEDGMENT: of
This
However,
phagocytosis in
by neutralization
an amphoteric summary,
suppress
02 production
Zn(OH)2
Ministry
(17).
immunoglobulin
60 min
stimulated
and form
the
kinase
of receptor-mediated
phosphatidylinositol
to
(8).
a weak acid
hydroxide
zinc
acts
transduction
not
and Ca 2t mobilization,
shown
by phagocytes form
In
hydroxide
(19).
ions
production
for
zinc
inositolphosphates
phosphorylation Zinc
37°C
of
of
hydroxide
a GTP-binding
hydrolysis
signal
action
with
zinc
coincided
induce
in the
or does
mechanism
that
that
dependent
of
concerned
proof
which
formation
site the
not
Our finding
stimulators
ylated
is
zinc
of protein
by FMLP (24)
the
EGTA (10 mM) at
(18).
protein
RESEARCH COMMUNICATIONS
that
an inhibitor
burst
to speculate
of
indicated
GTP-binding
that
substantial
treatment
release
sulfonamide,
suggest
of macrophages not
the
respiratory
findings is
0;
AND BIOPHYSICAL
present of
consists findings
stimulating these
work
Education,
study,
by phagocytes. with insoluble
provide
direct
superoxide
underlying
in part,
and Culture,
the Zinc
ions
give
hydroxide
particles
evidence
that
fume
02
hydrox-
white
is (25).
zinc
by alveolar
an important
zinc
inhibit
NaOH. Zinc
production
may play
was supported, Science
present
of
results
mechanisms
the
and to
role
in the
fever.
by a Grant-in-Aid
from
Japan.
REFERENCES 1. 2. 3.
Johnston, R.B.jr, Godzik, C.A., and Cohn, Z.A. (1978) 148, 115-127. and Cohn, Z.A. (1980) J. Exp. Med. 152, Murray, H.Y., Romson, J.L., Hook, B.G., Kunkel, S.L., Abrams, G.D., and Lucchesi, B.R. (1983) Circulation 67, 1016-1023.
1120
J.
Exp.
Med.
1590-1608. Schork, M.A.,
Vol.
185, No. 3, 1992
4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Weiss, E.B., and Bellino, J.R. (1986) Chest 89, 709-716. Ogino, K., Hobara, T., Ishiyama, H., Yamasaki, K., Izumi, Y., and Oka, S. (1992) Eur. J. Pharmacol. 212, 9-13. Adachi, T., Fukuta, M., Ito, Y., Hirano, K., Sugiura, M., and Sugiura, K. (1986) Biochem. Pharmacol. 35, 341-345. Quinn, M.T., Parthasarathy, S., Fong, L.G., and Seinberg, D. (1987) Pro. Natl. Acad. Sci. U.S.A. 84, 2995-2998. Chvapil, M., Stankova, L., Bernhard, D.S., Weldy, P.L., Carlson, J.B. (1977) Infec. Immun. 16, 367-373. E.C., and Campbell, Bettger, W.J., and O'Dell, B.Y. (1981) Life Sciences 28, 1425-1438. Mustafa, M.G., Cross, C.E., Munn, R.J., and Hardie, J.A. (1971) J. Lab. Clin. Med. 77, 563-571. Badway, J.A., and Karnovsky, M.L. (1980) Ann. Rev. Biochem. 49, 695726. Noel, N.E., and Ruthman, J.C. (1988) Am. J. Emerg. Med. 6, 609-610. Cardozo, C., Edelman, J., Jagirdar, J., and Lesser, M. (1991) Am. Rev. Respir. Dis. 144, 173-178. Nakano, M., Sugioka, K., Ushijima, Y., and Goto, T. (1986) Anal. Biochem. 159, 363-369. Babior, B.M., Kipness, R.S., and Curnutte, J.T. (1973) J. Clin. Invest. 52, 741-744. Heyworth, P.G. and Segal, A.W. (1986) Biochem. J. 239, 723-731. Gerard, C., McPhail, L.C., Marfat, A., Stimler-Gerard, N.P., and McCall, C.E. (1986) J. Clin. Invest. 77, 61-65. Bass, D.A., Scully, S.P., Segel, G.B., and Lichtman, A. (1986) J. Clin. Invest. 77, 1349-1356. Rossi, F. (1986) Biochim. Biophys. Acta 853, 65-89. Allen, R.C., Stjerholm, R.L., and Steele, R.H. (1972) Biochem. Biophys. Res. Commmun. 47, 679-684. Johnston, R.B., Jr., Lehmeyer, J.E., and Guthrie, L.A. (1976) J. Exp. Med. 143, 1551-1556. Muller-Eberhard, H.J., and Schreiber, R.D. (1980) Adv. Immunol. 29, l-53. Whaley, K., and Ferguson, A. (1981) Mol. Aspects. Med. 4, 209-273. Fujita, I., Takeshige, K., and Minakami, S. (1986) Biochem. Pharmacol. 35, 4555-4562. Cotton, F.A. and Wilkinson, G.(1976) Basic inorganic chemistry: The Chemistry of Anions, p. 108. John Wiley & Sons, Inc., New York.
1121
BIOCHEMICAL
AND BIOPHYSICAL
June 30, 1992
RESEARCH COMMUNICATIONS Pages 1115-1121
ZINC HYDROXIDESTIMULATESSUPBROXIDEPRODUCTIONBY RAT ALVEOLARMACROPHAGES Keiki
Ogino*,
Yukie Izumi, Hironobu Ishiyama, Tomoko Murata, Haruo Kobayashi,and Tatsuya Houbara
Department of Public Health, Yamaguchi University School of Medicine,1144 Kogushi, Ube 755,Japan Received May 21, 1992 SUMMARY:The effect of zinc hydroxide on superoxide (0;) production by rat alveolar macrophages was determined by chemiluminescence and by cytochrome c reduction. Zinc ions had no effect on the chemiluminescence of unstimulated alveolar macrophages. By contrast, zinc hydroxide (ZnOH,), a neutralized form of zinc ions, increased the chemiluminescence level and 0, release. Increased 0, release was inhibited by pertussis toxin, isoquinoline sulfonamide and pretreatment with EGTA. These findings indicate that zinc hydroxide formation from zinc compounds can stimula e the 0; production by alveolar macrophages by receptor-mediated and Ca2f B 1992Academic Press,Inc. dependent process.
During phagocytosis polymorphonuclear
leukocytes,
(02) and H202 (1,Z). during
or chemical
phagocytosis
activation,
produce substantial
These oxidants
are utilized
; they are also thought
e.g.,
ischemic
heart
tinal
disorders
(5),
failure
(3),
respiratory
renal
disease
(6),
Zinc has been shown to inhibit (8) through
its
the free
radical
macrophages (11). industrial accompanying
setting
causes fever,
leukocytosis
+To whom correspondence
within
of superoxide
to be involved disorders
killing
in tissue
(4),
gastrointes(7).
by stimulated
(9) or its
damage,
macrophages
inhibitory
effect
High doses of zinc might eventually
affect
By contrast,
quantities
and atherosclerosis
effect
damage induced
the same time would adversely
as well as
for microbial
0; production
membrane stabilizing
on NADPHoxidase and ATPase (10). minimize
macrophages,
by activated
the bacteriocidal
the inhalation chills,
macrophages, capacity
but at
of the
of zinc oxide fumes in an
and respiratory
4 to 12 hr after
symptoms with
inhalation
(12).
There
should be addressed. 0006-291X/92
1115
$4.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
BIOCHEMICAL
185, No. 3, 1992
may be a close
relationship
AND BIOPHYSICAL
between zinc and phagocytosis.
zinc hydroxide,
a neutralized
by rat alveolar
macrophages and discuss
relation
to signal
RESEARCH COMMUNICATIONS
zinc compound form,
transduction
stimulated
We found that 02 production
the mechanism of activation
in
pathway.
MATERIALS AND METHODS Materials: Bovine serum albumin, horse heart ferricytochrome c (type VI), phorbol myristate acetate, pertussis toxin, EGTA and superoxide dismutase (bovine erythrocytes, 3000 lJ/mg protein) were obtained from Sigma Chemical. Catalase (65,000 U/mg protein) was obtained from Boehringer Mannheim GmbH. ZnSO4 of 99.9% purity was purchased from Wako Pure Chemicals. Mouse sodium metrizoate ficoll was obtained from Japan Immuno Research Laboratory and 2-methyl-6-phenyl-3,7-dihydroimidazo[l,2]pyrazin-3-one was purchased from Tokyo Kasei. 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride was obtained from Seikagaku Kogyo. Preparation of zinc hydroxide: Zinc sulfate was dissolved in double distilled water and was then adjusted to pH 7.0 with NaOH. A white opaque liquid was obtained. The concentration of zinc hydroxide was expressed as that of zinc sulfate. Preparation of alveolar macrophages: Alveolar macrophages were collected from adult Wistar rats bv bronchoalveolar lavage with calcium and magnesium-free Dulbecco's phosphate-buffered saline-(13). After centrifugation at 70 x g for 5 min and hypotonic lysis of contaminating erythrocytes, cells were suspended in Hanks' balanced salt solution (HBSS) containing 0.1% bovine serum albumin and layered on mouse sodium metrizoate ficoll (d=1.090), following which they were centrifuged at 1000 x g for 30 min. Macrophages, recovered from the upper band, were washed twice with HBSS by centrifugation at 70 x g for 5 min. Cells were resuspended in HBSS. Chemiluminescence: Chemiluminescence was measured using a Cypridina luciferin'analog, 2-methyl-6-phenyl-3,7-dihydroimidazo[l,2]pyrazin-3-one (CLA) as a probe with an Aloka Co. luminescence reader at 37OC (14). Reaction mixtures contained 1.0 x lo6 macrophages, 1.0 pM CLA, and O-l mM zinc sulfate or zinc hydroxide in 1 ml of continuously stirred HBSS. Intensity of light emission was expressed by subtracting initial non-specific luminescence from maximal luminescence. CLA was dissolved double distilled water and the concentration of CLA was determined by E = 8900 M-' cm-'. nm 410
in
Superoxide release: Superoxide was measured, following the superoxide dismutase (SOD) inhibitable reduction of ferricvtochrome c at 550 nm in a single spectrophotometer, by a modification of the procedure of Babior et al. (15). Reaction mixtures contained 1.0 x lo6 macrophages, 0.1 mM ferricytochrome c, and 1 mM zinc hydroxide or 1 pM phorbol myristate acetate (PMA) in 1 ml of HBSS. Before incubation, 10 pg/ml of SOD was added as reference. After incubation for 10 min at 37'C, reaction mixtures were chilled, clarified by centrifugation, and used to measure ferroc tochrome c at 550 nm. To investigate the role of GTP-binding protein, Ca' Y and protein kinase C on zinc hydroxide-induced superoxide production, pertussis toxin (16), I-(5-isoquinolinesulfonyl)-2-methylpiperazine, an inhibitor of protein kinase C (17), EGTA (18) were added. 1116
Vol.
185,
No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
RESULTS Zinc
hydroxide-induced
CL&dependent
CLA-dependent of
zinc
chemiluminescence
hydroxide.
single
peak
at
2.8
min,
chemiluminescence
inhibited
by catalase
nescence.
The pH of
sulfate
was 6.7.
reaction
mixture
1).
time
Zinc
reaction zinc
pH. The level (Fig.
of 0.7
mixtures
min
the 0.1
of
hydoxide-induced
hydroxide-
was not
on chemilumi-
addition
of
1 mM zinc
mM had no effect
on the
chemiluminescence
was a significant zinc
showed
Zinc
by SOD, but
below
logarithmic
1).
had no effect after
addition
hydroxide
(Fig.
sulfate zinc
on the
by zinc
inhibited ions
2) and there
between
dramatically
induced
was completely
the
p < 0.01)
correlation
hydroxide
concentration
values
intensity.
release
Superoxide
nmol/106
a lag
(Table
and chemiluminescence
reduction.
with
However,
was dose-dependent
Superoxide
increased
The chemiluminescence
induced
(R=0.977,
chemiluminescence
release Superoxide
cell
per
was determined released
10 min.
Zinc
from
by SOD-inhibitable resting
hydroxide
macrophages
cytrochrome was 0.9
c + 0.4
(1 mM) and PMA (1 PM) augmented
II--
&
INCUBATION TIME
Fig.
1
MIN
1. CLA-dependent chemiluminscence due to zinc hydroxide. The reaction mixtures contained 1 x lo6 cells, 1 ,uM CLA, 1 mM zinc hydroxide, and HBSS in a total volume of 1.0 ml (system 1). Zinc hydroxide was omitted from system 1 for control (system 2). System 3 contained 15 units of SOD, in addition to the ingredients contained in system 1. Results are expressed as a single luminescence measurement in one representative experiment. 1117
a
Vol.
185,
No.
3,
BIOCHEMICAL
1992
Table
1.
Effect
of of
AND
BIOPHYSICAL
some chemicals on CLA-dependent alveolar macrophages
Chemicals
35240 hydroxide
1 mM
Zinc hydroxide + SOD 30 units
725240
sulfate
Zinc
sulfate
Results indicate
O2
release
(500
14700
(5)
* 62143
(5)
f. 6844
(5)
754980
f. 48037
(5)
41950
I? 4618
(4)
mM
36975
+I 4228
(4)
are expressed the number
of
as the mean experiments.
and 22.4-fold,
f
SD.
The
respectively
number
in
(Table
parentheses
2).
1-(5-isoquinolinesulfonyl)-2-methylpiperazine
EGTA (10 mM) inhibited 62%,
5172
1 mM 0.1
6.6-fold
rig/ml),
intensity
1 mM
Zinc hydroxide 1 mM + catalase 1300 units Zinc
f
respectively
COMMUNICATIONS
chemiluminescence
Maximal light (counts/min)
Control Zinc
RESEARCH
zinc
(Table
hydroxide-induced
02 release
Pertussis
toxin
(100
and
by 61%,
PM), 43%,
and
3).
DISCUSSION The present oxide
release
process.
study from
rat
The respiratory
demonstrated alveolar burst
.Ol
that
macrophages of
2.
hydroxide by the
phagocytes
is
Concentration
can
induce
super-
receptor-mediated
triggered
1
1
Zinc Hydroxide
Fig.
zinc
by phagocytosable
10
(mM)
CLA-dependent chemiluminescence of macrophages at various zinc hydroxide concentrations. The chemiluminescence was determined in the presence of 1 pM CLA, 1 x lo6 cells, and zinc hydroxide as described in Methods. CLA light emission was expressed as counts per min. The results are mean + SD of three determinations.
1118
Vol.
185, No. 3, 1992 Table
BIOCHEMICAL
Effect
2.
AND BIOPHYSICAL
of zinc hydroxide and phorbol 0; release by macrophages
RESEARCH COMMUNICATIONS
myristate
02 production (nmol/106 cell/l0 Control Zinc
hydroxide
PMA
(bacteria,
0.9 f 0.4
(5)
1 mM
5.9 f 0.3
(5)
1 PM
20.2 f 0.4
(5)
by a number complement acids,
soluble
factors,
factors
phorbol
and endotoxin IgG and/or
respiratory Fc part system pertussis
burst
on the
esters,
immunoglobulins surface
toxin
Table
which
3.
of
phagocytes
blocks
Effect
a The cells expressed
of the
the
avidly
activity
23).
toxin
when coated
and triggers
affinity
C3b component
(22,
activating
ingested
high
receptors of
the
for
action
GTP-binding
protein
on zinc hydroxide-induced macrophages
0;
% inhibition min)
1 mM
5.6 f 0.7
500 rig/ml
2.2 2 0.7
61
3.2 f 0.8
43
100 pM lmMa
5.3 f 0.6
10 mM
2.0 f 0.8
were preincubated for 60 min at 37°C. Results as the mean f SD of five experiments. 1119
the
complement
The inhibitory
of the
and
fatty
material,
0; production (nmol/106 cell/l0
+ 1-(5-isoquinolinesulfonyl) -2-methylpiperazine
+ EGTA
is
etc.)
lectins,
platelet
particulate
of some chemicals release by alveolar
Zinc hydroxide
Zinc hydroxide
, cytochalasins,
and the
Chemicals
+ pertussis
leukotrienes diacylglycerol,
because
debris,
peptides,
Non-bacterial
21),
cell
the number
chemotactic
to complement,
(20,
indicate
materials,
,e.g.
(19).
exposed
of the
aggregate
Ca 2+ ionophores,
detergents,
factor, with
of
viruses,
on
min)
Values are mean f SD. The numbers in paretheses of experiments.
particles
acetate
62
are
of (16)
Vol.
on zinc the
hydroxide-induced
receptor
before
pathway.
Isoquinoline
inhibits
the
our It
BIOCHEMICAL
185, No. 3, 1992
difficult
have
calcium
which
exogeneous
Ca
2t
phosphodiesteric of
have
been
of
zinc
hydroxide
C,
is
we still
phagocytosis.
depletes
with
reports
previous
protein-linked
do
Pre-
extracellular 02 release
the
receptor. recognition
since
induces
on
Therefore,
hydroxide-induced
ionized was by the
activation
of
4,5-biphosphate
with
and protein
oxide
and itself
is
the
capable
macrophages.
Therefore,
investigations
of the
ACKNOWLEDGMENT: of
This
However,
phagocytosis in
by neutralization
an amphoteric summary,
suppress
02 production
Zn(OH)2
Ministry
(17).
immunoglobulin
60 min
stimulated
and form
the
kinase
of receptor-mediated
phosphatidylinositol
to
(8).
a weak acid
hydroxide
zinc
acts
transduction
not
and Ca 2t mobilization,
shown
by phagocytes form
In
hydroxide
(19).
ions
production
for
zinc
inositolphosphates
phosphorylation Zinc
37°C
of
of
hydroxide
a GTP-binding
hydrolysis
signal
action
with
zinc
coincided
induce
in the
or does
mechanism
that
that
dependent
of
concerned
proof
which
formation
site the
not
Our finding
stimulators
ylated
is
zinc
of protein
by FMLP (24)
the
EGTA (10 mM) at
(18).
protein
RESEARCH COMMUNICATIONS
that
an inhibitor
burst
to speculate
of
indicated
GTP-binding
that
substantial
treatment
release
sulfonamide,
suggest
of macrophages not
the
respiratory
findings is
0;
AND BIOPHYSICAL
present of
consists findings
stimulating these
work
Education,
study,
by phagocytes. with insoluble
provide
direct
superoxide
underlying
in part,
and Culture,
the Zinc
ions
give
hydroxide
particles
evidence
that
fume
02
hydrox-
white
is (25).
zinc
by alveolar
an important
zinc
inhibit
NaOH. Zinc
production
may play
was supported, Science
present
of
results
mechanisms
the
and to
role
in the
fever.
by a Grant-in-Aid
from
Japan.
REFERENCES 1. 2. 3.
Johnston, R.B.jr, Godzik, C.A., and Cohn, Z.A. (1978) 148, 115-127. and Cohn, Z.A. (1980) J. Exp. Med. 152, Murray, H.Y., Romson, J.L., Hook, B.G., Kunkel, S.L., Abrams, G.D., and Lucchesi, B.R. (1983) Circulation 67, 1016-1023.
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4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
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RESEARCH COMMUNICATIONS
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