PLC treatment. (0.16-1.25. U!mL at 37#{176}Cfor 1 h) removed only the. GPI-anchored. CD14, resulting in reduced. CDI4 expression so that the amount of CDI4.
Effect of specific
A
inhibitors
on CD1 4-dependent
phagocytosis
150 IL
Phagocytosis
120
>>
U) (0)
90
0
of killed
presence was
U
of 10%
inhibited
.,
C)
30
LPS
to CD!4,
receptor 00
30
60
90
120
mAbs
(Fig.
1 i0
of
B 1
5).
There
and
kinase 90
‘a ‘a
60
250
250
(Fig.
V
30
-
6).
was
0.
9
60
90
CD14
Effect
cci P1-PLC
transfectants
and
LB P-opEc..
120
150
ability
P1-PLC
After
were
seen
and
with
mAb
63D3
tion,
whereas
cells (a P1 3-
with
expressioce
0.16 U/mL;
and
1.25 tJ/
PBS.
CL)14
measured.
rc’Con-
phagtecytosis
illustrates
was
one represen-
of two.
We
used
of
jiM
not
mAb
inhibit
CDI4-dependent
18E12
does
[9,
10,
the
two
transfected
amounts
of CD 14,
pression
by treating
PLC
treatment
only
the
we
cell
cell
activa-
a flow
human
LB P-opEc
devised
(0.16-1.25
U!mL
GPI-anchored so
CD14,
that
THPI-wtCDl4
the
cells cells.
The
Hl-PHS-
or
CD14
expression
Cl)14
anchorage both of C 1)14
resulting of
killed BODIPY,
LB P-opEc (r
fore
the
(Fig. the supported
4).
When
equal
levels
1 h) removed
of
the
was
kinase
seen
with
at
inof
protein
0.02-2
assay
THP1-
expression
cells
70Z/3
edly
enhances
1gM
expression other
jiM
to study
sta-
cell
with
nature
of the
expression
was
At
normally In
lines
expressing
activation
present
and
We
no has
show
Thus
all
functions, facilitate
that
initiate(s)
unique identified.
here
that
LBP
Schffet
inhibition
a!.
demonstrated murine
pre-B
CI)i4)
mark-
including
surface
shown
in
(WI-anchored
forms
of
well were
[13].
In
of the data
primary
this
has of
CD14-dependent
to
form
sup-
of CD14 that
for
of C 1)14 with
(an)
is to addi-
signaling.
the an
study,
able
transmemhrane
as
or support
suggest role
interaction of
studies
CD14
equally
available
also
phagocytosis
express
supstudies
either
function
been
and
in
regardless the
then
CDI4-dependent dose-response
cells
of E. co/i,
protein(s)
lines
it was
equally
THP-l
cell
cell
to LPS,
both
in-
of CDI4 Previous
CDI4
addition,
acidic associated
Gram-negative
well.
not
responses
[12].
anchor.
mCDI4
do
There-
endocytosis an
was forms
equally
that
and
opsonized
of
pH.
into
integral
GPI-anchored
C[)14
fluorescence
at acid
expression
of and
cellular
ligand
The
internalized
C 1)14
a or
BODIPY-conjugated
of phagocytosis be
cells,
a (WI-anchored
CD 14-transfected
(which
THP-i
quenched
of opEc of
LPS-induced tional
correlate
studies may
phagocytosis
bind
is not
GPI-linked
using
that
from
substrate.
phagocytosis
both
membrane by
phagocytosis
of phagocytosis.
for
phagocytosis
port
CDI4
transmembrane
FITC,
iransrnembrane
expressed
to
and
tyrosine
CDI4.
as the
compartment.
CD14-transfected
of the CD14
GPI-anchored
I-Il-
6). iNo inhibition
expressing
of human
substrate
LPS-induced
P1-PLC.
in reduced
found
regardless
of respectively,
(Fig.
derived
line,
used
enhanced
with
different
was
it is useful
ex-
to perform
was
= 0.9)
phagocytosis
phagocytosis
cell
unlike
P1-
that
of cells
at 25
shown).
lines
form
E. co/i
to normalize
CDI4
approximated
of
23 (a protein
cytometric
transmembrane
with
at 37#{176}C for
amount
ability
expressed
cells
71%
respectively
by THP1-wtCI)14,
not
cell
performed
a strategy
THP1-wtCDl4
and
wortmannin,
concentration
monocytic
ported
201.
lines
by 61
inhibition
tyrphostin
transfected
with does
inhibited
DISCUSSION
bly
Phagocytosis of opEc is correlated only with the amount of CD14 and not with the form of membrane anchorage
forms
= 2)
(n
of wortmannin
of
54%
(data
bacteria;
equivalent,
inhibition
THPI-
P1-PLC
were then
Each panel
0.
P1-PLC
twice
to
p
of
inhibitory
THP1-wtCDl4
by THP1-wtCDl4 cells inhibitor bisindolyl-maleimide
tracellular
tmCDl4
74% by
Phagocytosis
was
LBP-opEc
at 100
where
expression
anti-Fry
not
1.25
P1-PLC
U/mL;
0.62
(MEl)
C 1)14
0.16
P1-PLC
V, THP1-wtCi)14.
washed
regression.
linear
by
from
A, THPI-tmCDl4,
phagoc.yteesis
Icetweeme
experircueret
Because
and
were
and (B)
ranging
#{149}, THPI-wtCl)14,
cells
and
cceefficic’ret
determined tative
reblock
at 37#{176}C f(er 60
THPI-RSV,
,
PI-PLC
no P1-PLC;
incubating,
inculcated
were
mireP1-PLC;
0.31 U/mL;
expression
nc’latiore
84%, not
Icy THP-I
H l-PHS-opEc
(Sigma)
THPI-wtCI)14,
A, THPI-tmCl)l4.
(A)
P1-PLC;
no
#{149}. THPI-wtCl)14, U.
cells
cci P1-PLC
THPI-RSV,
0.
expression
ore C1)14
tee ingest
THP-I
ceencentraticens
U/mL
mnL.
and
concentrations
and
phagocytosis kinase C
(MFI)
treatment
their
Trarusfected
1.25 U/mI.
c:eptcer
63
concentrations
cells
concentrations
wtCI)14.
60.3,
197)
respectively.
A 67
hibitor)
I
30
0
1.25
a mean
nM
nM
PHS-opEc
0)
U/mL:
and
does
mAb
and
of HI-PHS-opEc
inhibitor),
and
0
with
lV.3,
was
by THP1-wtCDl4
.
0
mite
by 90
which
plasma
.
0
4.
in the
H1-EDTA
28C5
63D3,
anti-CI)18
(3G8,
phagocytosis
at 25
U-
Fig.
mAb
monocytes
10%
-
0.
0
normal and
14 mAb
Anti-CDI4
binding
by
plasma
by anti-CD
spectively.
0
E. co/i
EDTA
GPI-anchor
of
important
illustrated
role by
phagocytosis
phagocytosis
an with
in LBP
anti-
791
A 80 0>
--
00
20 I’
(-)
Ab:
Ag:
28C5
63D3
60.3
3G8
CD14
CD14
CD18
Fc?RllI
IV.3
Fc’yRII
197
Fig.
FciflI
5.
cytes
Pleagocytosis
in
the
Pleagocyteesis
cef monocytes
II
only
Icy nermal
inhibited
hey
as detennuined
refpositive cells where
B
K. cob
10% E DTA
of
plasnea (B) was
Hl-EE)TA 28C5.
eef killed
presence
coneupared was
monce-
plasnea (A) and anti-CDI4 neAb
by thee percentage
witle a contrcel ceensisting
mc’asured
after
1 h inculcation.
30 20
o
0.
10 0 Ab:
Ag:
LBP
CD14
antibody
viously
fragments.
that
plexes
that
coating
[4].
Of
nm,
binding
cytes
by
human
may
stem
from
note,
in
not
cytes
the
different
with
our
cell
CD14-dependent
cell
anti-CD14
that
18E12
inhibits
and
nuclear
results,
on
bulk
of
the
inhibits vation
and
tumor
dependent
binding
CD 14-dependent for
vides
the
792
Journal
10],
not
factor results
results:
release but
been
that
another
serves
machinery
Leukocyte
Biology
protein, for
Volume
plexes,
thus
pathway,
blocking
we
cytosis
of opEc
from
similar
extent
18G4
sCD14.
Furthermore,
inhibited
bound
but
inhibited
does
further
dependent
pro-
cation-chelating
1997
that
block
LPS
was
of
LBP-opEc
an
antibody
binding
it still
Phagoblocked
to
fragments was
occurred
it is
due
when
not binds
Finally, was
to Ig
LBP
was
serum. et
al.
occurs
have in
asserted
evidence phagocytosis
CD14-dependent
agent.
that
monocytes
via
CDI4-dependent a separate
phagocytosis to support
this
assertion.
is inhibited We,
along
phagocytosis
by with
pathway
[11]. EDTA,
occurs
We
pro-
Complement-
others,
a divalent have
in the
a
and
that
[20].
phagocytosis
because
comopsonic
F(ab’)2
63D3,
complement-dependent
mol-
signal-
cells
phagocytosis
satu-
immune
of experiments.
CD14-dependent for
to
of phagoto the
receptor-dependent
anti-CD14
14 mAb
not
FryRlI, CD14
inhibition secondary
THP1-wtCDl4 both
to bacteria
phagocytosis from
by
that
Grunwald re-
by
Fc
the
a number
by anti-CD
substituted
CD14-
an
performed
on
showed
3).
able
18G4
December
with
CD14/anti-CD14
previously
62,
FryRl,
with
KB acti-
transmembrane
the
we
of CDI8,
of Fc receptors
mAb
then
(Fig.
Based
mAbs,
cooperate
ration
unlikely
which
phagocytosis
to
used
CDI4
as a ligand-focusing
appear
that
effect
18E12
portions not
occurred
factor
Our
using
mAb
little
[10].
do
phagocytosis.
studies
possibility
shown
factor
and
inhibition
extramembrane
mediate
vide
transmembrane
necessary
of
CD14
FryRlIl
has
the not
the
and
necrosis
whereas
nuclear
parallel
18G4
mAb
it has but
that
attachment, of
by anti-CDI4
phagocytosis.
We postulate ecule
[9, but
necrosis
separated
tumor
cytoskeletal results
To disprove
by mono-
anti-LBP
KB activation
binding
phagocytosis LPS
with Previously
binding
substrates
be
ing, the
cytosis
results
et al. were
can
FcyRI
erythroOur
and
opso-
[11].
18E12.
LPS
as an
of E. co/i
LPS-stimulated
LBP-LPS
LBP
Grunwald
lines
factor
com-
macrophages
reported.
types
activation
mAb
release
ported
cell
by
to
of LPS-coupled was
pre-
opsonin
LPS-LBP
of
phagocytosis
CD14-dependent and
with
phagocytosis
monocytic
an
erythrocytes
phagocytosis
Fc’yRII
et al. have
as
description
CDI4-dependent and
acts
macrophages
Consistent
to show
of
FcyRlll
by Wright
LBP
initial
the
but
CD18
of erythrocytes
binding
enhances
[4].
Studies
demonstrated
showing
CD14
shown absence
cifically
A
inhibit
[24,
25].
P1
Recent
mannin
inhibits
actin-rich
and
inhibited
B
of Fry
phagocytic kinase
inhibitor
ment cc’lls
with
were’ incubated
was
then
LBP-opEc
for
30
In
(MEl)
[23]
and
our
system
did
not
23 kinases out
This
cations
by human
monocytes
l-II-EDTA
plasma
dent
phagocytosis
mAb
28C5
tor
mAbs
THPI-wtCDI4
not
60.3.
on
study
cations;
it was
13-chain
of CR3
and
of complement
phagocytosis.
fect
was
well
as
to
that
Indik
et
al.
P1
3-kinase
in
observed for
Fc’yRIIIA/’y mannin dose-dependent
at for
phagocytosis [22,
inhibited
have
were
is not
de-
This
work
in diva-
in the
clearance
a path
CR3,
CR4,
and
may
intercept by
can-
targets.
via
utilized
Grants
number
immunology was
10884-1MM
of The
supported
A115136
(P. 5. T.),
that with
Fe
is
Fcy
rethe
receptors
at
and
from
Scripps
by National
(R.
J. U.),
A101442
(D.
the
l)epart-
Research
Institute.
Institutes
GM28485
(R.
of Health
J. U.),
A132021
S.).
CD18, the
ab-
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by
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does
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protein
recep-
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at
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ACKNOWLEDGMENTS
by anti-CI)14
inhibited
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25,
pathway pathway
in and
phagocytosis 3-kinase
by phagocytes
phagocytic signaling
as yet
phagocytosis
P1
receptors
protein
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or anti-Fcy
phagocytosis
when
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mannin
also
therefore
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system
not
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an
role
of
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inhibitors the
hisindolyl-
activity
Although
[24,
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the
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bacteria
similar fash-
of
C 1)14-dependent
establishes
that
by
C1)14-dependent
of these
with
shown
FryR-mediated
specifically
of Gram-negative
manner have
in a dose-dependent
shown). to
FryR-
cation-indepen-
inhibited
18 mAb
complement
lent
cellular
of EDTA
CDI4-dependent
the
phagocytosis
divalent
significantly
of EDTA
LBP
This
anti-CD
the with
presence
5).
cells
presence
sence
We observed
5). Phagocytosis
Thus,
pendent the
was not
(Fig.
and
in the (Fig.
but
[11].
not
ri
decreases
inhibited
inhibit
inhibit
effects
intracellular divalent
23
de-
[26-28].
We was
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known
not
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be
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concentrations
inhibited
tyrosine rule
not
(data
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cseeasuned.
re-
inhibitors
23
[26-28].
completely do
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mneice, Phagocytosis
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phosphory-
a dose-dependent
p.M
contrast,
that C [29]
to tyrosine
kinase
phagocytosis
6).
tyrphostin
hibited
25
of tyrphostin
(Fig.
kinase
Pluagocytosis of LBF’-opEc Icy TH P1 -wtCI) 14 cells was inwith wortniannin (A) and tyrpleecstime 23 (B) .Aften Ienc.treatef cells with inleibiteer as descrilced in Materials amid Methccds,
into
that would
Furthermore,
tyrphostin in
at
maleimide
6.
of
closure
phagocytosis
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ion
23 (concentratIon)
tyrosine
rosine
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Tyrphostln
with
cells
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leads [23].
receptor-mediated
0 0 0) ‘a
6)
wort-
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their
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Fry
0
0.
(Fig. that
by inhibiting
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CD 14-dependent
U)
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suggests
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it is not
creases
50%
0)
Fig.
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Activation
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clistrihutiomu
Volume
ccl a flmiorescent
62, December
1997
eel hunean
CI)14
requirc’d
3-kinase
M.
NI., Calle’gees.
A., Alenaleam,
R.,
I..
W. F., l)eemlge’. and selective
Crinde’y. C., Viahees. inleileiteen of phospleati-
in tIre’ corrtpletiort
J.
J. 296.
res1eccmeses. Biochenr.
NI. ‘I’.. Swanseeme,
I. A.
(1996)
297-301.
A role’
of maenopineecytcesis
leer pleosphoand
phagocytee-
135. 1249-1260. Park. 1.-C.. Murray. R. K.. Chien. P.. Danlcy, C.. Sclereiheer.A. 1).(1993) Cecreserved cytoplasmie tyrosine re’sidues of t1ecgamnua suheunit are re’quirm.d for a phagocytic signal mediated hey Fc’yltllIA. J. Chic. Incest. 92, Ce!!
Rio!.
2073-2079.
28.
29.
Indik. Z. K.. Pan. X. 9.. Iluaceg. M.-M.. McKenzie’. S. K.. l.c’vinson. A. I.. Schre’iber. A. 1). (1994) Icisertion of ey#{252}eplasmi. tyrosine. se.quences into thee’ nonphagocytic re’cepteer Fe’yRIIB establishes pheageecytie function. Bh,oc! 83, 2072-2080. Borenema, J. L)., Karnitz, I.. NI., Schrcecccs,H. A., Aleraheamee, H. ‘I’., Leihscen, P. 1. (1994) ceatural killer
quenchR. P.
H., Bc’nggnere.
in neutropleil
sis by macropheages. 27.
A region
her
Cancer Re’s. 54, 2419-2423. NI. P. (1993) Wortmarenin is a pote’metpheeespleatiimehileiteer: the neele eel 1ehueesphaticlylinmcsitol 3.4.5-
Jolensoru.
ule’ release
49. 315-32:3. J. H.. Hcebenison, eef a lluore’scent
using
Anaki, iraesitide
Pleagocyte’ C3-me’diate’cl attachrnecet
341-348. 18.
tris1ehcesphate 26.
I.ee,
Bjerknes.
43894399. Powis, C.. Bonjceukliace.
dylinositol-3-kinase. 25.Arearce. A.. Wymann. elylinosirol 3-kinase
prce-
J.-l).,Knavchcnkee, V.. Kirkland, T. N.. Han, I., Mackman. N.. Moriarty. A.. l.eturcq. D., Tecleias, P. 5., Ulevitch, Ri. (1993) CPI-anchorc’d cer intc.gralme’mleracee’fcenmnscr1Cl) 14 cnediate ideretiralcellular responses teeendotcexin. Proc. 1V0t1. lead. Sri. lISA 90. 9930-9934. Theofame, C.. He,rwitz. A. H., Williams. H. E.. Liu, I’.. Chace, 1..Birr, C.. Carnell, S. F., Meszanos. K.. Parent. I. B., Kasler, H., Abenle, S., Trccwn, P. V.., Cazzano-Santcerce. H. (1994) An amino-terminal fragment of hucnan lipeepolysaecharide-IeindicegFenote’inre’tains lipid A binding hut not Ci)14-suimulateery activity. J. Imncunol. 152. 3623-3629. V/ace. C. P.. Park, C. S.. l,au. B. H. 5. (1993) A rapid and sineple. reeicroflucerccmetric’phagceiytcesis assay. J. Incnmunoi. Meth. 162. 1-7.
‘U N. (1995)
anchcer
leerlipcepolysacchanidc’heinehing. J. Rica!. Chem. 270, :361-:368. Jack. R. S..Crunwald, U.. Stelter,F..Workalemahu, C., Schutt, C. (1995) Bothe membrane-bound aced soluble forrirs eel (:1)14 heind tee Cramnegative bacteria. Eon. J. Inmnmuno!. 25. 1436-1441. Ninoneiya, N., Hazeki, K., Fukui. Y.. Se’ya. T.. Okada. ‘I’.. Hazeki. 0.. Ui. M. (1994) Involvement ccl phosphatidylineesitol 3-kinasc’ ice Fr gamma receptor signaling. J. Rio!. Cimenr. 269. 22732-22737. Inehik, Z. K.. Park, 1.-C.. Hunter, S., Sclereiher. A. I). (1996) The molecular dissection of Fe’ ganemna rc’ceptccr mediated eIeagoeytcesis. Blood 86.
Ashe’ndel, C., Zalkow. I... Matter, C. 1. (1994) Wonemanceiru, a pot..nt
bacteria
19-4125.
Katie. K., Tobias. P. S.. Kirkland, 1’. N., Ulevitch, of C1J14 icetce 707./3 cells dramatically enhances
J. Lirp. Mcd.
and 17.
21.
deacyla-
S.. Kirkland.
a versatile’
30.
Teeullec,
Fe receptor sticeeulaiimen cells is associated with and
I).,
ce’ll-mm’diated
Pianetti.
kamue, NI., Baude’n,
P.. Coste’, V.. Becissin,
of ehospIuatim1ylicrc.sitol 3-kinase in preedeiru kicease’ C-irede’peredent graru-
eytotcexicity. H..
J. Exp. Mec!. 180. 1427-1435.
Bellevergue..
P., Beeursier,
P.. Crand-Pe’rnet, 1.. AjaE., l,eenieelle.
F., Duhamel.
L,
Charoce. D.. Kirilovsky, 1. (1991) The hisinclolylmeealeinnide CF l09203X is a potent and selective inhibitor eel proteime kinase’ C. J. Rio!. C/cern. 266, 1577 1-15779.
Effector molecules in expression of the antimicrobial activity of macrophages against Mycobacterium avium complex: roles of reactive nitrogen intermediates, reactive oxygen intermediates, and free fatty acids Tatsuya Satoshi Department
*
Izumo,
Abstract:
tive (FFA),
microbicidal
Immunology
reactive
oxygen avium (m’)
of
free
inhibitors
and
phospholipase
intermediates
(RO!)
A2,
of
nitric
indicating
oxide
roles
synthase
of
RO!,
or
RN!,
and
The
anti-MAC
either
RN!
Listeria
get
or
FFA
activity.
the
combinations
H202-halogenation
+
was
of gave
generated
system
was
by
murine
the
combined
activity
MAC
contact
and ter
ceased MAC
xanthine with
was
m4s
with
MAC
anti-Lm
+
but
ROl
observed
of
m-mediated phase after 804; !997.
university,
as a tarFFA
and
effect,
functions RN!
with
of m4s, FFA
killing
MAC
of
and
not
MAC
stimulation.
in
antiby
J. Leukoc.
Biol.
in early
62:
the
functions
nitrogen
important
roles
RNI
including in the
795-
below
2]. ef-
to
what
myco-
is
known
of antimycobacte-
the
oxide
although
strain [9]. killed that
ROI
found
production
MAC the
RNI
of impor-
against sensitivity
found
that
a
(H202-halogenation
and
was
also
previously
ability
M’cobacterjon,
known [3-7].
activity of
we system
of ms
MAC,
are
dependence
halogenation also
radicals
microbicidal
Second,
effectively
are functions
tuberculosis
(NO)
of m
reported
(RNI)
antimicrobial
Mycobacterium
nitric
H202-mediated
duce
[1,
resistance
intermediates
expression
[8],
We
devised
as antimicrobial
molecules
in
against
m4s
system)
re-
therapeutic
of ms.
play
been
describe
effector
and
of drug
been
role
of host
We
major
reactive
MAC
have
infec-
patients
effective
a central
expression
pathogens.
Abbneviaticnus:
collaborating
relatively
in the
tant
stimulation)
seen 1-24 h afwas initiated
participates the
cells
play
(MAC)
AIDS because
although regimens
(ms)
First,
ii].
af-
the
temporarily
fector
complex in
to treat
organisms,
as prophylactic
Macrophages
has
production
(MAC
organisms
as well
avium.
intractable
of MAC
murine
from 3 h and increased during the first 36 h and continued at least for 4 days. These findings suggest that RN! and FFA rather than RO! are important effectors action
Kajitani,*
Medical
encountered
somewhat
sistance
to
immediately
within 2 h. FFA release was stimulation. RN! production
of anti-MAC
Slcimane
frequently
are
rial
showed an antago!n addition, when
(3)
was
RN!
they
com-
oxidase-acetaldehyde
RN!,
potentiated.
peritoneal
ter
Hiroko
Mycobacterium
are
concerning
antagonism. used
a synergistic
whereas FFA + H202-halogenation nism in exerting bactericidal activity. ROl
an
(Lm)
tions
bacterial
a synergistic effect. system in combina-
showed
monocytogenes
organism,
RN!
bactericidal
of RN! with FFA showed the H202-halogenation
with
of Dermatology,
Disseminated
in murine for ROt
a significant
When
Sano,*
INTRODUCTION
complex (MAC) and the production of these
NaNO2acids), exhibited
tion
Chiaki
acids
FFA in m anti-MAC functions. (2) Acidified derived RN!, FFA (linolenic and arachidonic and the H202-mediated halogenation system bination However,
Shimizu,*
tDepartrneni
reac-
fatty
(1) Intracellular growth of MAC m4s was accelerated by scavengers
RN!
and
activities
(RN!),
Mycobacterium of macrophage
effectors. peritoneal or
studied
intermediates
and
and
of Microbiology
Sato,* Toshiaki Tomioka*
Japan
We
nitrogen
against mode
Akaki,*t, Katsumasa Dekio, and Haruaki
M.
tuberculosis
of MAC inversely
odium
[10,
strains
to
correlated
cone1elc’x;
inwith
ne,
macre)-
RNI.
reactive nitrogen intermediates; NO. nitric oxide: ROl. reactive cexygen interneediates; Lm, Lisieria rnonorytogenes; TNF-a, tuneon necrosis factor a; FFA. free fatty acids; PEA9, phospheolipase A9; N NI MA, BSA, hcovine serurci alhtcnein; SOD, su1eereexide clismutase; FBS, fe’tal beevirie’ senunu; XOA. xanthiree oxidase-acetaldehyde: CFU, colcerey-feerming units; FEC. penitoneal exudate cell; HBSS, Hanks’ balanced salt solution; CL, chemileumireescenee; NOS, nitric oxide synthase; iNOS, ireducible ruiphage;
oxide
tnic
symethase;
etharuesulforeie’ acid. Cornesporedence: and
Immuneclogy.
H EPES.
Hanuaki
N-2-hydncexyethyl1eipenazine-N’-2-
Tomioka. Medical
Shineane
Departme’nt
University,
cef Microbiology
Izumo.
Shimane
693,
1997:
accepted
June
Japan.
Key
Words:
11202-mediated
cidal
activity
. phosphoiipa.se
halogenation A2
.
nitric
oxide
system. . microbisyntha.se
Received
February
18.
1997;
revised
June,
9.
9, 1997.
Journal
of Leukocyte
Biology
Volume 62, December
1997
795
their
virulence
It is also
[12].
reported
that
H202
plays
portant roles in the expression of the bacteriostatic of murine peritoneal ms against a low-virulence M. avium pears
[13].
Therefore,
to participate
provided
that
plied
to ms.
11202-halogenation
in m-mediated
sufficient Third,
hibit
potent
antimycobacterial
found
that
were
more
virulent relationship
to
colonial
and that
FFA
liberated
due
to the
(PLA2)
play
important
cobacteriostatic study,
of RNI,
and
effective
linolenic MAC
because
membrane the tested
potent
[16].
tion/release m4s
when
in vitro
addition, RNI,
ROl,
we
were
stimulated
but
was
Moreover,
for
their
[19]
(HEPES),
ciii,
100
fetal
bovine
serum
used
for
culture.
units/mL cell
Bacterial
24
by
with
MAC
acetate
exhibit FFA
of produc-
mouse
peritoneal
organisms
tture
(1.0
METHODS
probe
te,st.
Tleey
Laboratories,
(serovar
with
were
was
mL)
infection
in
N-285
and
Middlebreecek
Listeria
MI).
Scey broth
100
CFU
for
and
MAC
tor
(serovar
16) were
identified 7H9
by DNA
medium
EGIJ
(Lm)
Inonorytogenes
(I)ifcce was
(l)ifco).
Mice Six-
to
Clea
Ccc., Osaka,
niree-week-old
tece-week-celd
female’
feneale
Kanagawa,
Japan
Special
agents
Linolenie
acid,
arud Zyneosan MO.
Chemicals
xanthine
acid
796
Chemical
Journal
Japan
eight-
Charles
tee
River,
Inc.,
St. Louis,
MO.
(SOD)
and
Canboxy-PTIO
Industries,
of Leukocyte
Sigma
purchased Othe’n
we’re Osaka,
Nc_
catalase’,
bovine
from
was
tetna-reiethylimidazoline-3-oxide-l-ecxyl)
Pure
from
eexidase.
quinacnine,
A were obtained
[tHiarachidonic
dismutase
from
expenimnelets,
obtained
5.5)
at 37#{176}Cfor
1-2
by treating
p.M
cef 20
FeSO.4,
serum
albecmin
Chemical from
American
agents,
including
Co., St.
p.M
cceunted
we,re
7H11
den
cells
62,
oxielase,
10
mixace-
mM
ethyle’nediaminetetnaacetic 5.5)
at 37#{176}C for 2 h. After residual numb ens of
agan anel tryptic
PECs
(3
into
X
soy agar
was poured
The
MAC-infected
reeL)
with
plates
plus
catalase,
CO2
imecuhator
dium
dodecyl
NMMA,
the
temperaturc
After
the
frcem
December
Zymosan
incubawith
0.1 -mL
portion
(1.5
2% cef
10#{176} CFU/
X
cultivaticen, 80
ccentaimling
restrltant
agents,
mn
including
on quinacrimec’ m
lysate’
was
20% lysate’
p.L eef 0.23% kept
mixed BSA.
was
S 01)
at 37#{176}C in a
cultures,
with Thee
see-
at room 120
p.L
number
counted
(en
of 7H
11
10 mL
of
FBS-RPMI
ter;
Falcon)
PECs (2 X 10 cells) were in a FBS-ceeated plastic culture at 37#{176}Cfor 2 h in a CO2 incubator. adluerent
scraped
cccllee,te,d
off
into
Icy subsequent
preparation of
by ms
A-induce’el
5%
gently
1997
a CO2
ielate’s.
su-
Wako
a
Falcon
washing
N-260
cef test
thee m
saline
in
(H BSS),
to the
added
feer 10 miii, and CFU
were
96-well;
culture, incubated at 37#{176}C washed again with 2% FBS-HBSS. then cultivated in tlee medium (0.2
addition
for 5 days.
ccf 1ehospleate-buffe.reel
FBS-RPMI
2 he. After
carleecxy-PTIO,
sulfate was
cf 5%
in-
han-
cell
neonolayer
were
rtes
give’n
hefcere
at 37#{176}Cin
fcer
iniracellulare
for 2 Ic,and
or withccut
nd,
incubated
salt solution M.
fromee reeice
(0 at-heetteer,eeel
air)
onto thee m
incubator
bacterial
and
humidified
containing
irea CO9
ire 0.1
cells)
NY)
ccelleeted
(1 mg) 4 days
A
wells
Ceerning,
CO9-95%e
the medium mL)
l0
neicrceeulture
Works, (5%
were
(PECs)
of Zyneosan
injection
balanced
ment
Volume
by the
activity
exudate
The
FBS-HBSS,
Japan.
Biology
killing in a reaction
Lmn, nespe’ctively.
anti-MAC
Ha-
(2-phenyl-4,4,5,5purchased
Ic. Bacterial
xarethune’
100
cef 10-20
cnM sodium
in 100
FeSOt
organisrees
g/mL
and
ecensisting
(1.0 mL) pM
measured
FBS-Hanks’
agar
acid,
de-
effectors. i’o
with other
1-10
RNI, FFA, and ROl production (NMMA),
Louis,
peroxide
mice
arachidonic
(BSA),
purchased
In some
also used.
monomethyl-L-arginine
diolahe’led
used.
CI3AIJN
were
mice
BALl3/e
were
Japan,
comhinc,d
and
Nal,
consisting
bacterial
ve,st.
16), and
MAC
grown
L)etroit,
in Tryptic
cultured
N-260
was
in an
M. arium N-254 (serecvar 9), N-265 (serovar; ncentypeable). N-339 (senovan 8), and N-445 (senovar 1) and Mycobacierium intracellulare 14),
MI))
effectors
acid in 100 mM sodiunu acetate’ buffer (pH individual tneattnecet of test cteicroorganisms,
Glass
patients
M (pH
system
taldehyde,
Organisms
from
ccf streptomy-
heat-inactivated
(vlv)
the H:O2-lealcegereatiecre system,
in a reactiemn cnixture
10-20
buffer
XOA
of
and
imectcbated
seeded
(serovar
p.g/mL
5%
Ccc,, Walkersville,
FFA,
Fe cer feer2 he when
Peniteenc’al
N-256
100
and
BioWhittaker
(FBS;
activities eef RNI,
trapenitoneal
isolated
C,
the FFA-mediated antimicrobial activity, tlue organisms (- 10#{176} CFU) were treated with 2.5 tee 25 p.g/mL eef FFA (linoleruic or anacluideenic acid) dissolved on finely e’mulsifie’d (Icy sonication) in 1.0 mL of 100 mM sodium acetate bteffer (pH 5.5) at 37#{176}C for 1-2 h. The antimicrobial activity ef a h12O2-Fe2-rnediated halogenation system was measured as fecllows. TIue test organisms (. l0’ CFU)
M
AND
glutamine.
killing by antimicrobial
1iM H202,
system.
MATERIALS
2 mM of penicillin
supple-
and the xanthine oxidase-acetaldehyde (XOA) system were measured as pne7viously described [10, 181 with some, neodifications. For measurement of the antimicrobial activity of RNI against MAC and Lrn, test organisms [- 10#{176} colony-forming units (CFU)] we,re treated with RNI generated in a neacticen mixture (1.0 mnL) containing 1.25 to 10 mg/mL of NaNO2 in 100mM sodium acetate buffer (pH 5.5) at
were
anti-
various
profiles
FFA
not
signifi-
18].
among
examined and
The
components
ms
activity
of they
[10, major
of murine
In
system
tested
the
activ-
a ROl-mediated alone
102)
were
are
anti-MAC
culture
as this
OH,
they
phospholipids
most
used
acids
acid
sulforcic
37#{176}Cfor
system.
killing
arachidonic
activity
A2
antimicrobial
because
11202,
1640 (Nissui Pharmaceutical Ccc.. Tokyo, Japan) with 25 mM N-2-hydnoxye’thylpiperazine-iV’-2-ethane-
terneine
was
in mycobacterial
and
pro-
mycobactericidal/my-
H202-halogenation
system
(02,
of MAC been
of phagocytic
anti-MAC
the
effector
molecules
a close
of phospholipase
m4
RPM! mented
Antimicncebial
[17].
we assessed system
microbicidal cantly
in
were
indicates resistance
Medium
previ-
of MAC
it has
action
ex-
than
phospholipids
roles
H202-halogenation ROl
from
We
killing
of FFA
hydrolytic
FFA,
15].
This
sup-
(FFA)
variants
Moreover,
mechanisms
In this ities
degree
are
acids
[14,
[16].
virulence.
vesicles
fatty
FFA-mediated
the
their
Fe2+
colonial
variants
between
organisms posed
SmD
susceptible SmT
free activity
avirulent
apkilling,
of catalytic
unsaturated
ously
reaction
mycobacterial
amounts
im-
activity strain of
of the m
nes
pncdeceaion
cells 20%
cenesisting FBS-RPMI
centniftcgatiore without
a rest
c)F antimicrobial
cultured
in
elisle (80-mm
diame-
After
nirusireg with
ccf more
than
90%
with
rubbe’n
peclicemen
and
g fcen 5
mm. The
fresh
at 250 period
were
effecteens.
used
ms
for
2%
were
measure-
RN!
production
of ms
RNI
productiore
of
latiecn
in
slight
meedifications.
1.0
mL
mL)
culture
of
in
wasluing tured
5%
previously
(5
coentaining
for and
nitrite,
In
the
were
alone lation
of
that
to
UV-killed
NO2-
was MAC
ire
16-mm
with ire 10/
X
Gniess
the
which
the
medium,
no
36
h of
during
supernatants
increased
and
the
at
absorptien MAC
organisms
significant
indicat-
the
under
ccenelitoere.
release
FFA
Measureme,nt
m4s
of
Test
FFA
nele,ase, (6
mns
pcrfoermed
was
as described
106 ce,lls) were
X
incubated
FBS-RPMI
3.0
of
mL
cells) ing
were
then
tJV-killed
CO2
incubateer
fluid
was
4
containing
cultured MAC
ire 0.2
(1 X
feer up toe 24 h. The
mc’astcne,d
eten
(Packarol
M4
chemilunrine.scence
M
production
by using
Instrument
c’ach
mL
of 10%
FBS-RPMI
10#{176}/mL) in rueicnocultune, radioactivity
a Tni-Carb
Co.,
to some scavengers),
itor]
[23],
Carboxy-PTIO
caused
contain-
(data to play
tivity
of murine
(pH
tc,st
To
examine
the
ROl
in m
7.4,
plee’nel
with 0.1 ciuL sieen of killed mixture
sured md,,
(5
and
a
in
was
l0
X
the
antimicrobial
red-free)
cef 1 mM
MAC
me,asuned
2, acidified
s1eectrom-
and
H202-halogenation
with
killing
containing luminol.
(1
phcetoe’missieen Iumi1eheetccneeter
X
of CL as felleews
ire terceis
The,n. l05/neL) dunireg
ire 0.8 mL
10 mM
HEPES
0.1
of bacterial
was
neL
added
inculcation
(Leemicceureter
of
[22]. HBSS
aced added stmspen-
to the incubation at 37#{176}C was mea-
ATP-237,
Toyo
the
or
these
the
basis
of
5-day
cul-
and
FFA
RNI,
of anti-MAC
Kagaker
not
ac-
shown).
fixed
MAC
was
observed
2B).
This
means
were
findings
mt race!-
M.
separate
experiments,
anti-MAC
activ-
H202-halogenation when
whereas
that
in were
Similar with
organisms
when
acid,
effective
efficacies
the
at 10 M,
effect
5.5
significant
Notably,
in Fig-
linolenic
tested In
of the
As shown [26],
manner.
displayed
and
profiles against
microbicidal
not
killed
was
effectors
were
FFA,
the
system.
Their
shown).
effectively
tericidal
these
at pH
they
also
of RN!, examined
RN!
when
(data
acid
centration
of
system N-265.
N-260 (data
we
a dose-dependent
arachidonic system
of participation
of a cell-free
obtained
no
Fe2+
added
was
sufficient
Fe2
con-
significant
bac-
at 1
amounts
or
5
of cata-
_____________F
None
Fig.
1.
Effects
gr(ewth +
on
after
ROl,
NaNO2-derived
in also
lulare ity
use
M. avium
1.tM (Fig.
iokyo).
O time
SOD
catainhib-
[24],
ms
expression
activity, activity
MAC
IL).
wo,ne’ suspended
modes
ure
were
cells)
ms.
anti-MAC
culture,
(CL) ef ROl
ms
peritoneal
plus
experiment,
m4s
Therefore, in the
of was
(NOS)
scavenger) on
of
ms
of SOD
In this
effects
roles
in
synthase
of cultured
shown).
some
effects
functions
Microbicidal activity of RNI, FFA, and ROl against MAC organisms
increased
Briefly.
not
seem
addition [NO
[25].
cytotoxic
the
growth
(NO-specific
morphology
into
scintillation
Creevc,
by the
on
anti-MAC
bacterial
inhibitor)
no
and
the
NMMA
(PLA2
recovery
The
extent
(ROI
at 37#{176}C in a
liberated
liquid
Dcewners
we,lls
ms.
lases
result
on
by Neve
in
p.Ci/mL cef [tulanachideenic acid (100 Ci/mmol) in a polyprdepylene tube (15 X 90 mm) at 37#{176}Cin a CO2 incubator lc)r 24 Fe. The resultant ms loaded with radioactive anachidonic acid were, thoroughly washe,d with 2% FB S-HBSS to remevc’ uceincoenpecrated [3H]arachidonic acid. The ms (2.5 X 10 10%
representative
inhibitors
peritoneal
tivation from
al. [21].
murine
agents cul-
the
metabolic
quinacrine
accumu-
cultivation, NO2
1 shows
Figure some
reagent,
measuring in
by
Roles of ROI, RNI, and FFA in the expression of m4 anti-MAC activity
there culat 37#{176}C in a
were wells
did not produce
c)rganisms
(2.5
culture
witlu
Icy
echeserved
ms
intervals,
react
thee same
MAC
resultant
experiment
in
were
cells)
accumu-
[201 incubated
at 37#{176}C for 3 h, followed
At
to
quantitated
corutnceh
cultured
the
4 days.
alleewed
was
ccentent nm.
up
of NO2 described
10
X
culture wells (Falcon) with 2% FBS-HBSS. The, in 1.0 mL of 5% FBS-RPMI
removed
et
as
FBS-RPMI
in terms
measured
16-mm
incuhatccr
ture
was
Te,st ms
were,
ing
ms
supennatant
CO2
550
RESULTS
fected
**
Catalase
of
with
thc, me,dium
(1,000 (0.5
NMMA
MAC
ccf seeme,
in
umeits/mL)
plus
=
3).
N-260
catalase
*,**
cometnol ms
(*P
