Mac ophage Con ol o Mycobac e ial G ow h Induced by
Picolinic Acid Is Dependen on Hos Cell Apop osis
1
Te esa F. Pais and Rui Appelbe g
2
The e ec s o picolinic acid (PA) on he in amac ophagic g ow h o Mycobac e ium a ium we e s udied. PA educed M. a ium
g ow h inside mouse mac ophages and led o a comple e con ol o mycobac e ial g ow h when added oge he wi h IFN-
g
. The
mechanism in ol ed did no equi e TNF-
a
, NO, o he espi a o y bu s , and was no dependen on ei he i on o zinc wi h-
holding. The mycobac e ios a ic ac i i y o he mac ophages was associa ed wi h he induc ion o mo phological changes ha
culmina ed in apop osis a day 4 o ea men . PA alone induced apop osis in mac ophages, and his e ec was inc eased by IFN-
g
ea men . Apop osis a day 4 o in ec ion was educed by inhibi ing mac ophage ac i a ion wi h he p os aglandin 15 deoxy-
p os aglandin J
2
o by ea ing he cells wi h he an ioxidan N-ace ylcys eine. Mycobac e ial g ow h was pa ially es o ed in
mac ophages ea ed wi h PA and IFN-
g
when 15 deoxy-p os aglandin J
2
was added, concomi an wi h a delay in apop osis.
N-Ace ylcys eine o glu a hione could also comple ely e e he mycobac e ios a ic e ec s o PA o PA plus IFN-
g
.The Jou nal
o Immunology, 2000, 164: 389–397.
Mycobac e ium a ium is an in acellula acul a i e bac-
e ium ha p oli e a es inside hos mac ophages (1).
Upon ac i a ion by cy okines such as IFN-
g
and/o
TNF-
a
, mouse mac ophages can cause a limi ed es ic ion o he
g ow h o M. a ium h ough a mechanism ha is nei he dependen
on NO (2) o on eac i e oxygen in e media es (3, 4), bu which
may in ol e i on dep i a ion (5, 6). In con as , many esea che s
ha e ailed o ind such a p o ec i e e ec o IFN-
g
when s udying
M. a ium in ec ions in cul u ed human monocy es (7, 8), despi e
clinical e idence o a majo ole o IL-12/IFN-
g
pa hways in he
con ol o in ec ions by mycobac e ia in humans (9). We ha e
p e iously epo ed ha , in addi ion o IFN-
g
/TNF-
a
ac i a ion,
he e may be addi ional signals equi ed o lead o comple e con-
ol o M. a ium in ec ion in mice (10). I is he e o e impo an o
s udy he ole o o he po en ial induce s o mac ophage an imy-
cobac e ial mechanisms.
Se e al s udies ha e shown an in ol emen o yp ophan ca-
abolism in he an imic obial mechanisms o he mac ophage. Du -
ing he in i o ac i a ion o human ib oblas and monocy es wi h
IFN-
g
, he deple ion o yp ophan due o he induc ion o 2,3-
indoleamine dioxygenase is pa ially esponsible o he inhibi ion
o in acellula g ow h o Toxoplasma gondii (11–15), Leishmania
dono ani (12), Legionella pneumophila (16), Chlamydia spp. (12–
14, 17), o CMV (18). Deple ion o yp ophan should no a ec
he in acellula g ow h o M. a ium because mycobac e ia a e able
o syn hesize all o he amino acids equi ed o g ow h (19). The
induc ion o yp ophan ca abolism has been epo ed in pa ien s
wi h cance (20), in mice in ec ed wi h in luenza i us (21), in
AIDS pa ien s (22), and in humans in ec ed wi h Mycobac e ium
ube culosis and Salmonella (23). Howe e , he possible ole o
he ca aboli es o yp ophan me abolism as pa o he an imic o-
bial esponses has ne e been se iously conside ed in hose s udies.
Picolinic acid (PA)
3
is one o he na u ally occu ing deg ada ion
p oduc s o yp ophan. This molecule is able o o m s able com-
plexes wi h ansi ion me al ions (24), and i s chela ing p ope ies
seem o acili a e he abso p ion o zinc om he in es ine (25) and
explain he inhibi o y e ec o PA in he axenic g ow h o Esch-
e ichia coli (26) as well as in he g ow h o no mal kidney a cells
(27, 28). PA is also umo icidal in i o (29), an e ec ha is ela ed
o he induc ion o mac ophage-media ed cy os a ic ac i i y (30).
The idea ha PA plays a ole in immune mechanisms is s eng h-
ened by he obse a ion ha mice ea ed wi h PA ha e enhanced
le els o mRNA o IL-1 and TNF-
a
and highe esis ance o Can-
dida albicans (31). In i o s udies wi h mac ophages con i med
ha PA is a modula o o mac ophage unc ions because i co-
s imula es mac ophage ac i a ion and enhances he an i umo ac-
i i y o pe i oneal mac ophages ea ed wi h IFN-
g
(32). In his
wo k, we ha e assessed he abili y o PA o modula e an i-M.
a ium mechanisms in cul u ed mouse mac ophages. We show ha
adminis a ion o PA o mac ophages in ec ed wi h M. a ium in-
duced mycobac e ial g ow h inhibi ion s ongly syne gizing wi h
IFN-
g
in he induc ion o comple e mycobac e ios asis.
Ma e ials and Me hods
Mycobac e ia
A smoo h anspa en (SmT) a ian o M. a ium s ain 25291 was ob ained
om he Ame ican Type Cul u e Collec ion (Manassas, VA). S ain 2-151
SmT and GIR10 SmT we e gi en by J. Belisle (Colo ado S a e Uni e si y,
Fo Collins, CO) and B. Heym (Ins i u Pas eu , Pa is, F ance), espec-
i ely. The mycobac e ia we e g own in Middleb ook 7H9 b o h (Di co,
De oi , MI) con aining 10% albumin/dex ose/ca alase (ADC) supplemen
(Di co) and 0.04% Tween 80 (Sigma, S . Louis, MO) a 37°C un il mid-log
phase. The bac e ia we e collec ed by cen i uga ion, washed in saline con-
aining 0.04% Tween 80, and hen esuspended in a small olume o he
same solu ion. The suspension was b ie ly sonica ed un il no bac e ial
Labo a o y o Mic obiology and Immunology o In ec ion, Ins i u e o Molecula and
Cell Biology, Uni e si y o Po o, Po o, Po ugal
Recei ed o publica ion July 12, 1999. Accep ed o publica ion Oc obe 14, 1999.
The cos s o publica ion o his a icle we e de ayed in pa by he paymen o page
cha ges. This a icle mus he e o e be he eby ma ked ad e isemen in acco dance
wi h 18 U.S.C. Sec ion 1734 solely o indica e his ac .
1
This wo k was suppo ed by Con ac P/SAU58/96 om he PRAXIS XXI P o-
g amme (Lisbon). T.F.P. ecei ed a ellowship om he PRAXIS XXI P og amme.
2
Add ess co espondence and ep in eques s o D . Rui Appelbe g, Labo a o y o
Mic obiology and Immunology o In ec ion, Ins i u e o Molecula and Cell Biology,
Rua do Campo Aleg e 823, 4150-171 Po o, Po ugal. E-mail add ess: appelb@
ibmc.up.p
3
Abb e ia ions used in his pape : PA, picolinic acid; 15d-PGJ
2
, 15 deoxy-p os a-
glandin J
2
; iNOS, inducible NO syn hase; NAC, N-ace ylcys eine; PPAR-
g
, pe oxi-
some p oli e a o -ac i a ed ecep o -
g
; SmT, smoo h anspa en ; SOD, supe oxide
dismu ase.
Copy igh © 2000 by The Ame ican Associa ion o Immunologis s 0022-1767/00/$02.00
clumps we e isualized by op ical mic oscopy. The p epa a ion was dilu ed
and ozen in aliquo s a 270°C. The aliquo s we e hawed a 37°C and
dilu ed o he desi ed concen a ion be o e inocula ion, acco ding o a p e-
ious quan i ica ion. Fo de e mining numbe s o iable mycobac e ia, se-
ial dilu ions o he suspensions we e pla ed on solid Middleb ook 7H10
aga medium (Di co) supplemen ed wi h 10% OADC (5% BSA, 0.06%
oleic acid, and 2% dex ose).
Cell cul u es
Bone ma ow-de i ed mac ophages we e ob ained by cul i a ing bone
ma ow cells om BALB/c o C57BL/6 mice, ob ained by lushing he
emu s wi h HBSS (Li e Technologies, Paisley, U.K.). In some expe i-
men s, mice gene ically de icien in he genes o he inducible NO syn-
hase (iNOS
2/2
, in a C57BL/6 backg ound), he TNF ecep o p55
(p55
2/2
, in a C57BL/6 backg ound), o he p47
phox
p o ein (p47
phox2/2
,in
a mixed 129 3C57BL/6 backg ound) we e used. To emo e ib oblas s,
he cells we e cul u ed o e nigh on cell cul u e dishes wi h DMEM (Li e
Technologies) supplemen ed wi h 10 mM HEPES, 1 mM sodium py u a e,
10 mM glu amine, 10% o hea -inac i a ed Myoclone cal se um (Li e
Technologies), and 10% o L929 cell-condi ioned medium. The nonadhe -
en cells we e collec ed wi h wa m HBSS medium and cul u e o 9 days
wi h L929 cell-condi ioned medium un il hey we e comple ely di e en-
ia ed, as p e iously desc ibed (3). Pe i oneal mac ophages we e ob ained
om C57BL/6 mice by injec ing hem i.p. wi h 1 ml o 4% o hioglyco-
la e. The pe i oneal exuda e was collec ed 4 days la e by washing he
pe i oneal ca i y wi h 10 ml o HBSS. The cells we e esuspended a he
concen a ion o 2–3 310
6
cells/ml in comple e DMEM and cul u ed o
2 h o cells o adhe e. Then he cells we e washed wi h wa m HBSS, and
he emaining adhe en cells we e in ec ed. When necessa y, he numbe o
mac ophages was de e mined by lysing he cells wi h 3% ce imide (Sig-
ma) and 0.25% P onase (Sigma) and coun ing he nuclei in a
hemocy ome e .
S udies in axenic medium
A o al o 5 310
5
CFU/ml o M. a ium s ains 25291 SmT, GIR10 SmT,
and 2-151 SmT was inocula ed in DMEM o in DMEM plus PA (2, 4, 8,
16, 32 mM). The mycobac e ia we e cul u ed in 24-well pla es a 37°C in
aCO
2
incuba o . Saponin was added o he wells a day 6 o a inal con-
cen a ion o 0.1%, and he cul u es we e b ie ly sonica ed o dis up he
clumps be o e se ial dilu ion and pla ing on Middleb ook 7H10 medium.
In ec ion o mac ophages and inhibi ion s udies
Each well con aining ;0.5 310
6
mac ophages was in ec ed wi h 0.2 ml
o DMEM con aining 1–5 310
6
CFU o M. a ium o 4 h. A e in ec ion,
he cul u es we e washed wi h HBSS o emo e unphagocy osed bac e ia.
Mac ophages om iplica e wells ( ime 0) we e immedia ely lysed in
0.1% saponin, and se ial dilu ions we e pla ed in 7H10 solid medium. To
es he an i-M. a ium ac i i y o PA (Sigma), bone ma ow-de i ed mac-
ophages we e ea ed daily a e in ec ion wi h 2 mM PA, 100 U/ml IFN-
g
(R&D Sys ems, Minneapolis, MN) o PA (2 mM) plus IFN-
g
(100 U/ml)
un il day 3 o in ec ion. Fe ic ci a e, i on sul a e, zinc sul a e (Me ck,
Da ms ad , Ge many), apo ans e in (Sigma), IL-4 (R&D), and an i-TNF
mAbs (clone MP6-XT22; DNAX, Palo Al o, CA) we e added a e in ec-
ion, whe e indica ed. Ca alase (bo ine o e y h ocy e; Sigma o Calbio-
chem, San Diego, CA) o supe oxide dismu ase (SOD; Sigma o Calbio-
chem) was added du ing phagocy osis and a e washing he ex acellula
bac e ia in he indica ed expe imen s. N-Ace ylcys eine (NAC; Sigma) o
glu a hione (Sigma) was added o mac ophages du ing in ec ion (10 mM),
1 h a e in ec ion a a inal concen a ion o 50 mM, washed away, and
hen added a days 0 and 1 a a inal concen a ion o 5 mM, whe e indi-
ca ed. In he expe imen s wi h pe i oneal mac ophages, IFN-
g
(100 U/ml)
was added a days 0 and 1, and PA (2 mM) was added un il day 3 o
in ec ion. The p os aglandin 15d-PGJ
2
(Cayman Chemical, Ann A bo ,
MI) was added a e in ec ion a a inal concen a ion o 6
m
M. The my-
cobac e ial g ow h was moni o ed by lysing mac ophages in iplica e
wells up o day 6 o in ec ion. The esul s a e exp essed as CFU 61SD
o he means.
Elec on mic oscopy
Mac ophages in ec ed wi h M. a ium s ain 25291 SmT we e p ocessed o
elec on mic oscopy a day 4 o in ec ion. Mac ophages we e ixed o 1 h
a oom empe a u e wi h 2.5% glu a aldehyde in 0.1 M cacodyla e bu e
con aining 0.1 M suc ose, 5 mM Ca
21
,and5mMMg
21
, pH 7.2. The cells
we e sc aped o he cul u e dishes wi h a ubbe policeman and ea ed
wi h 1% osmium e oxide. Pos ixa ion was done in 1% u anyl ace a e.
A e dehyd a ion in e hanol, he samples we e embedded in Epon. Sec-
ions we e s ained wi h u anyl ace a e and lead ci a e.
Analysis and quan i ica ion o apop osis
Apop osis was s udied wi h di e en assays. Fo he use o he annexin
V-FITC s aining, mac ophages we e cul u ed on pe i dishes and in ec ed
wi h M. a ium s ain 25291 SmT. A day 4 o in ec ion, nonadhe en cells
we e collec ed by aspi a ion o he medium and adhe en mac ophages
we e de ached by incuba ing he dishes wi h cold PBS con aining 0.5 mM
EDTA du ing 1 h. Cells we e pooled and washed once wi h binding bu e
(10 mM HEPES bu e con aining 0.14 mM NaCl and 2.5 mM CaCl
2,
pH
7.4). A o al o 2–5 310
5
cells was incuba ed in 100
m
l o binding bu e
con aining 2.5
m
l o annexin V-FITC (Pha Mingen, San Diego, CA) o 20
min in ice. P opidium iodide (1
m
g/ml) was added be o e analysis on a
FACSo e low cy ome e (Bec on Dickinson, Moun ain View, CA) o
exclude mechanically damaged cells. To e alua e apop osis by s aining
DNA wi h p opidium iodide, mac ophages we e ob ained as desc ibed
abo e and we e pe mea ed wi h 70% e hanol o 10 min. A e cen i u-
ga ion, he cells we e s ained o 20 min a 37°C wi h 50
m
g/ml p opidium
iodide in PBS con aining 100
m
g/ml RNase (Boeh inge Mannheim, O -
weile , Ge many). Cells we e analyzed o p opidium iodide luo escence
(DNA con en ) by FACS. To pe o m DNA labeling wi h luo escein
dUTP, mac ophages we e s ained wi h he APO-DIRECT ki , acco ding o
he manu ac u e ’s ins uc ions (Phoenix Flow Sys ems, San Diego, CA).
Finally, cellula iabili y was also s udied by looking a he mi ochond ial-
dependen educ ion o MTT o o mazan, as desc ibed be o e (33). B ie ly,
mac ophages we e incuba ed wi h MTT (0.25 mg/ml) o 1ha 37°C, and
he o mazan was dissol ed wi h DMSO (Me ck). The ex en o educ ion
o MTT o o mazan was de e mined by measu ing he OD a 550 nm.
Resul s
PA inhibi s he in amac ophagic g ow h o M. a ium and
syne gizes wi h IFN-
g
in con olling mycobac e ial p oli e a ion
We es ed he abili y o PA o enhance he an i-M. a ium ac i i y
o IFN-
g
in bone ma ow-de i ed mac ophages om BALB/c
mice. Mac ophages we e in ec ed wi h he i ulen M. a ium s ain
25291SmT and ea ed a e phagocy osis wi h PA (2 mM), IFN-
g
(100 U/ml), o PA plus IFN-
g
daily du ing he i s 3 days o
in ec ion. The addi ion o PA alone o he in ec ed mac ophages
inhibi ed he in amac ophagic g ow h o M. a ium measu ed by
CFU (Fig. 1). This dec ease was al eady signi ican a day 4 o
in ec ion (p,0.05). When PA was added oge he wi h IFN-
g
,
mycobac e ial p oli e a ion was comple ely con olled, as no sig-
ni ican inc ease (p.0.05) in bac e ial numbe s was obse ed
du ing he 6 days o in ec ion. The same bac e ios a ic e ec was
seen in eshly explan ed pe i oneal mac ophages elici ed wi h ei-
he casein o hioglycola e ( esul s no shown). The induc ion o
mac ophage an imycobac e ial ac i i y by PA was also obse ed
wi h wo addi ional s ains o M. a ium (Fig. 1). The e ec s o PA
on he in amac ophagic g ow h we e independen o he sensi i -
i y o he di e en s ains o PA in axenic medium. Thus, al hough
M. a ium s ain Gi 10 g ew 1 log in axenic medium con aining 32
mM PA and was 4 imes mo e esis an o PA in axenic medium
(IC
50
54 mM) han s ains 25291 SmT o 2-151 SmT (IC
50
51
mM), he in amac ophagic g ow h a day 4 o s ain Gi 10 was
educed by bo h PA ( om 0.37 log o 0.11 log) o PA plus IFN-
g
( om 0.37 log o 20.04 log) o simila ex en s as obse ed wi h
he wo o he s ains: om 0.52 log o 0.20 log wi h PA and om
0.52 log o 0.05 log wi h PA plus IFN-
g
o s ain 2-151 SmT, and
om 0.35 log o 0.24 log wi h PA and om 0.35 log o 0.07 log
wi h PA plus IFN-
g
o s ain 25291 SmT, in he same expe imen .
The e ec o PA in he in amac ophagic g ow h o M. a ium is
no dependen on commonly desc ibed bac e icidal mechanisms
The a es o no mal a kidney cell g ow h by PA (27, 28) is
abolished by he addi ion o ei he Zn
21
o Fe
31
(28) because PA
in e e es wi h he in acellula le els o hese me al ions (27). On
he o he hand, i has been shown ha i on is c ucial o M. a ium
g ow h (5, 6). The e o e, we es ed whe he he e ec s o PA o PA
390 MYCOBACTERIOSTASIS INDUCED BY PA
plus IFN-
g
could be blocked by supplemen a ion o he cul u e
media wi h excess amoun s o hese ions. As shown in Fig. 2, he
an i-M. a ium mechanism induced by PA was no due o he che-
la ion o Fe o Zn
21
ions, as he addi ion o ei he zinc sul a e o
i on in wo di e en ionic o ms (II o III) did no a ec he g ow h
inhibi ion induced by PA. I is also unlikely ha PA ac ed o p e-
en binding o i on o ans e in, as an excess o apo ans e in
did no e e PA g ow h inhibi ion (Fig. 2A). Because he an i u-
mo cos imula o y e ec o PA is associa ed wi h he enhancemen
o iNOS ac i i y and wi h inc eased le els o TNF-
a
mRNA ex-
p ession (34), we in es iga ed he ole o NO and o TNF-
a
in he
PA an imycobac e ial mechanism. We ound ha he e was no
di e ence in M. a ium g ow h inhibi ion when bone ma ow-de-
i ed mac ophages om iNOS gene-dis up ed mice o TNF ecep-
o p55 gene-dis up ed mice we e used in he expe imen s and
compa ed wi h he espec i e con ol mac ophages de i ed om
mice wi h simila backg ound (C57BL/6, Fig. 3). In addi ion, he
neu aliza ion o TNF-
a
wi h a mAb did no a ec he in amac-
ophagic g ow h inhibi ion in mac ophages ea ed wi h PA o PA
plus IFN-
g
(Fig. 2). T ea men wi h IL-4, which inhibi s he co-
s imula o y umo icidal ac i i y o PA (35), did no e e i s bac-
e ios a ic e ec (Fig. 2). Finally, he addi ion o ca alase o SOD
plus ca alase did no in e e e wi h PA bac e ios a ic ac i i y (Fig.
2), showing ha unc ion o he NADPH oxidase is p obably no
equi ed o he e ec s o PA. This esul was con i med by using
bone ma ow-de i ed mac ophages om p47
phox2/2
mice (Fig. 3),
which showed he same abili y o wild- ype mac ophages ( om
ei he he C57BL/6 o he 129 s ains, al hough he la e da a a e
no shown) o es ic g ow h o M. a ium a e s imula ion wi h
PA 6IFN-
g
.
PA an imycobac e ial ac i i y occu s concomi an ly wi h he
induc ion o apop osis in ea ed mac ophages
We obse ed ha a e 3 days o in ec ion, he numbe o adhe en
mac ophages, measu ed by he P onase-ce imide me hod as well
as by he educ ion o MTT, dec eased when he cul u es we e
ea ed wi h PA plus IFN-
g
( esul s no shown). PA also induced
changes in cell mo phology. A day 4 o in ec ion, PA- ea ed
mac ophages showed nume ous small elec on- anspa en acu-
oles in he cy oplasm (Fig. 4B) when compa ed wi h non ea ed
cells (Fig. 4A), and a day 6 o in ec ion he e was ex ensi e bleb-
bing o he ea ed cells, which became agmen ed in o small es-
icles (Fig. 4D), whe eas non ea ed in ec ed cells had a no mal
appea ance (Fig. 4C). As hese changes we e mo phologically
compa ible wi h apop o ic modi ica ions, we measu ed by FACS
analysis he numbe o apop o ic cells by assessing, as a ma ke o
an ea ly s age o apop osis (36), he exposu e o phospha idylse -
ine a he ou e lea le o he plasma memb ane a day 4 (Fig. 5A)
and, as a ma ke o a la e s age, he agmen a ion o DNA by
p opidium iodide s aining (37) a day 6 (Fig. 5B). Mac ophages
ea ed wi h PA plus IFN-
g
showed clea signs o apop osis al-
eady a day 4 wi h an inc eased exposu e o phospha idylse ine
(29.9% 66 o posi i e cells) when compa ed wi h IFN-
g
- ea ed
(4.8% 60.8), PA- ea ed (6.9% 63.6), o non ea ed cells (1.18%
60.2). A day 6, he highes pe cen age o cells wi h agmen ed
DNA was seen in mac ophages ea ed wi h PA plus IFN-
g
(58%).
PA alone was also able o induce DNA agmen a ion, wi h 18%
o he cells showing hypoploid DNA when compa ed wi h IFN-
g
- ea ed o non ea ed cells. These esul s we e u he con i med
by labeling DNA agmen s wi h luo escein dUTP ( esul s no
shown).
The inhibi ion o apop osis inc eases bac e ial numbe s in PA-
ea ed mac ophages
Da a p esen ed o da e sugges ha he e ec s o PA on M. a ium
we e media ed h ough he mac ophage, by modula ing i s unc-
ion. Howe e , hey canno o mally exclude a di ec e ec o PA
on he mycobac e ia. We he e o e es ed he e ec s o ei he a
deac i a o o he mac ophage, 15d-PGJ
2
(38, 39), o an inhibi o
o apop osis, NAC (39–42). Pe i oneal mac ophages elici ed wi h
hioglycola e, known o exp ess he pe oxisome p oli e a o -ac i-
a ed ecep o -
g
(PPAR-
g
) and o espond o 15d-PGJ
2
(39), we e
in ec ed wi h M. a ium 25291 SmT and s imula ed wi h IFN-
g
(100 U/ml, a days 0 and 1), wi h PA (2 mM daily), o wi h PA
plus IFN-
g
. As an indica o o mac ophage ac i a ion, we analyzed
he le els o ni i es in he supe na an s om he mac ophage cul-
u es. When cells we e ea ed wi h IFN-
g
o PA plus IFN-
g
in he
p esence o 6
m
M 15d-PGJ
2
, he e was no ac i a ion o mac o-
phages because ni i es we e no de ec able in he cul u es ( esul s
no shown). Mac ophages ea ed wi h PA plus IFN-
g
in he p es-
ence o 15d-PGJ
2
did no show he mo phological changes seen in
mac ophages ea ed wi h PA plus IFN-
g
in he absence o he
FIGURE 1. Comple e es ic ion o g ow h o h ee isola es o M. a ium is induced by a combined s imula ion o BALB/c mac ophages wi h IFN-
g
and
PA. A e in ec ion, mac ophages we e ei he no ea ed (M) o ea ed daily wi h IFN-
g
(100 U/ml) ( ), PA (2 mM) (E), PA plus IFN-
g
(F) un il day
3 o in ec ion. Each poin ep esen s he mean o iplica e de e mina ions 6SD. S a is ically signi ican g ow h inhibi ion is labeled p o p,0.05,
pp o p,0.01, and ppp o p50, acco ding o S uden ’s es .
391The Jou nal o Immunology
p os aglandin ( esul s no shown). 15d-PGJ
2
ea men also p e-
en ed he induc ion o apop osis in cells ea ed wi h PA plus
IFN-
g
, as e alua ed by he main enance o he abili y o educe
MTT by he mi ochond ial espi a o y chain (Fig. 6A)o by he
educ ion in he exposu e o phospha idylse ine (Fig. 6B). The
educ ion o apop osis by 15d-PGJ
2
in mac ophages ea ed wi h
FIGURE 2. Exclusion o se e al molecules as
media o s o he mycobac e ios a ic ac i i y o
PA 6IFN-
g
. Bone ma ow-de i ed mac ophages
om BALB/c mice we e in ec ed wi h M. a ium
s ain 25291 SmT and incuba ed as desc ibed in he
p e ious igu e in medium (open ba s) o wi h
IFN-
g
(ligh s ippled ba s), PA (hea y s ippled
ba s), and PA plus IFN-
g
( illed ba s), in he p es-
ence o i on ci a e (5 and 10
m
g/ml), apo ans e in
(T , 0.5 and 1 mg/ml), and IL-4 (200 and 400 U/ml)
(A), o , in o he expe imen s (Band C), i on sul a e
(5
m
g/ml), i on ci a e (10
m
g/ml), zinc sul a e (10
m
g/ml), ca alase (0.1 mg/ml), SOD (1500 U/ml), o
an i-TNF (10
m
g/ml). In he expe imen depic ed in
Band C, he i on and zinc sal s we e added daily, as
we e PA and IFN-
g
. Ca alase (0.01 and 0.1 mg/ml)
and SOD (1500 U/ml) we e added du ing and a e
phagocy osis. The e ec s o he di e en com-
pounds we e compa ed wi hin each incuba ion
g oup (con ols, IFN-
g
- ea ed, PA- ea ed, o PA 6
IFN-
g
- ea ed mac ophages) wi h he espec i e
con ol gi en no compound. S a is ically signi ican
di e ences a e labeled p(p,0.05) o pp (p,
0.01), acco ding o S uden ’s es .
FIGURE 3. Mycobac e ios asis induced by IFN-
g
plus PA is independen o NO, TNF-
a
signaling h ough p55, o he NADPH oxidase. Bone ma ow-
de i ed mac ophages o s ains o mice gene ically de icien o iNOS, TNFRp55, and NADPH oxidase we e es ed o an i-M. a ium ac i i y induced by
PA 6IFN-
g
. Mac ophages om C57BL/6 mice we e used as con ols o all expe imen s, and simila da a we e ob ained wi h mac ophages om s ain
129 (no shown). S a is ics as in Fig. 1.
392 MYCOBACTERIOSTASIS INDUCED BY PA
PA plus IFN-
g
was associa ed wi h a signi ican inc ease in he
numbe o iable bac e ia (Fig. 6C). The p os aglandin did no
induce any signi ican changes in he numbe o in acellula my-
cobac e ia in mac ophages ea ed wi h PA o IFN-
g
, o in non-
ea ed mac ophages.
Reac i e oxygen in e media e p oduc ion may lead o p o-
g ammed cell dea h, which can be p e en ed by he an ioxidan
NAC (40–43). We he e o e es ed he e ec s o his compound on
bo h apop osis and mycobac e ios asis induced by PA. We ound
ha by adding NAC du ing phagocy osis (10 mM) and a e
phagocy osis (50 mM) du ing 1 h be o e ea men wi h PA o
bone ma ow-de i ed mac ophages, we could educe apop osis as
measu ed by exposu e o phospha idylse ine a day 4 o ea men
wi h PA plus IFN-
g
(Fig. 7A). Fu he mo e, we comple ely e-
e ed he e ec o PA and PA plus IFN-
g
in he inhibi ion o M.
a ium g ow h inside he mac ophages wi h he NAC ea men
(Fig. 7B). Addi ion o glu a hione was also e ec i e in e e ing
he mycobac e ios a ic e ec (Fig. 7C).
Discussion
We ha e shown ha PA, a e minal ca aboli e o yp ophan,
induced an i-M. a ium ac i i y in mu ine mac ophages and
syne gized wi h IFN-
g
, leading o comple e mycobac e ios asis.
FIGURE 4. Mo phological changes induced by PA a e sugges i e o apop osis. Mac ophages in ec ed wi h M. a ium 25291 SmT only (Aand C)o
ea ed wi h PA (Band D) we e p ocessed o elec on mic oscopy a day 4 o in ec ion (Aand B) o mic opho og aphed in phase con as a day 6 (Cand
D). Magni ica ions: A,39,550; B,37,580; C,3160; D,3325.
393The Jou nal o Immunology

All h ee s ains o M. a ium es ed o da e had he same deg ee o
suscep ibili y o he mycobac e ios a ic e ec o PA and PA plus
IFN-
g
in mac ophages, independen ly o he suscep ibili y o PA in
axenic medium. Also, he g ow h o he Gi 10 s ain was no a -
es ed in axenic media wi h concen a ions o PA up o 32 mM,
whe eas his s ain was as easily con olled by ac i a ed mac o-
phages ea ed wi h PA as he o he wo s ains o M. a ium. These
da a s ongly a gue agains a di ec e ec o PA on mycobac e ia,
bu sugges an e ec media ed h ough he mac ophage.
PA is able o chela e me al ions. Howe e , i on o zinc wi h-
holding was unlikely he cause o es ic ion o M. a ium g ow h
inside mac ophages ea ed wi h PA o PA plus IFN-
g
. No only
was he addi ion o he ion me als ine ec i e in e e ing he bac-
e ios asis, bu also he inhibi ion o he g ow h o M. a ium in-
duced by PA plus IFN-
g
was highe han he one ob ained wi h
o he i on chela o s (4). As p e iously shown by Va esio and co-
wo ke s (34), PA is also a mac ophage agonis leading o highe
sec e ion o cy okines such as TNF-
a
and o NO. In he sys em
desc ibed in his wo k, howe e , TNF-
a
was no in ol ed in mod-
ula ing mycobac e ial g ow h inhibi ion ollowing PA s imula ion,
as ei he he neu aliza ion o he cy okine wi h speci ic mAbs o
he use o bone ma ow-de i ed mac ophages om TNF ecep o
p55
2/2
mice ailed o ha e an e ec in he mac ophages’ an i-M.
a ium ac i i y induced by PA o PA plus IFN-
g
. Finally, nei he
oxygen- eac i e species gene a ed by he NADPH oxidase no NO
was media ing g ow h inhibi ion a e s imula ion o mac ophages
wi h PA o PA plus IFN-
g
. Ins ead, we ha e ound a new an i-M.
a ium mac ophage e ec o mechanism ha is concomi an wi h
he induc ion o apop osis and ha could only be e e ed by
blocking p og ammed cell dea h. O he s ha e shown ha human
monocy es in ec ed wi h M. a ium and ea ed wi h hyd ogen pe -
oxide en e apop osis a e 6 h wi h concomi an bac e ial killing
(44). Fu he mo e, bacillus Calme e-Gue´ in and M. ube culosis
we e also killed in human monocy es a ew hou s a e he apo-
p o ic s imulus gi en by ATP (45, 46) o Fas-L (47). The induc ion
o apop osis and mycobac e ios asis in mu ine mac ophages by PA
plus IFN-
g
had di e en ea u es om he p e iously desc ibed.
We did no obse e bac e ial killing, bu a he an inabili y o M.
FIGURE 5. Ex ensi e apop osis is induced by IFN-
g
plus PA o e an
ex ended pe iod o days. In ec ed mac ophages we e s ained wi h annexin
V-FITC a day 4 o in ec ion (A) o ixed wi h 70% e hanol and s ained
wi h p opidium iodide a day 6 (B) and analyzed by FACS. Mac ophages
ea ed wi h hyd ogen pe oxide (10 mM) o killed a 56°C we e used as
apop o ic o nec o ic con ols, espec i ely.
FIGURE 6. The mac ophage deac i a o 15d-PGJ
2
inhibi s bo h apop osis and mycobac e ios asis induced by IFN-
g
plus PA. In ec ed pe i oneal
mac ophages ea ed wi h PA (days 0, 1, 2, and 3), IFN-
g
(days 0 and 1), o PA plus IFN-
g
in he p esence o 6
m
M 15d-PGJ
2
( ) o medium (M) we e
assayed o MTT educ ion a day 4 o in ec ion (A) o o in acellula iable bac e ia (C). The exposu e o phospha idylse ine in mac ophages ea ed wi h
PA plus IFN-
g
in medium alone (black line) o in he p esence o 6
m
M 15d-PGJ
2
(g ay line) was compa ed by s aining wi h annexin V-FITC (B). The
his og ams co espond o cells ga ed o nega i e p opidium iodide s aining. The illed his og am ep esen s non ea ed mac ophages. S a is ics as in Fig.
1.
394 MYCOBACTERIOSTASIS INDUCED BY PA
a ium o g ow in ea ed mac ophages. This is mo e consis en
wi h wha is gene ally obse ed in i o, in which killing o M.
a ium is seldom seen and when i occu s i akes place h oughou
a p o ac ed pe iod o weeks a he han hou s.
While he es ic ion o M. a ium g ow h was obse ed om he
ea ly ime poin s o ea men wi h PA o PA plus IFN-
g
, apop o ic
changes o he phagocy es we e only de ec ed la e , s a ing o
become p ominen a day 4 o in ec ion. This sugges s ha he
mechanism leading o mycobac e ios asis occu s in pa allel o is
he same ha will esul in he p og ammed cell dea h pa hway,
and ha mycobac e ios asis is no a consequence o he la e cel-
lula apop o ic changes.
A inal a gumen agains a di ec ole o PA on he inhibi ion o
M. a ium g ow h inside mac ophages comes om he abili y o
abolish i s e ec s by p e en ing apop osis o he mac ophages.
Al hough he h ee agen s used o ha pu pose, 15d-PGJ
2
, NAC,
and glu a hione, ha e pleio opic e ec s, hei ac ions a e conco -
dan ega ding he inhibi ion o apop osis, making i less likely ha
he e ec s obse ed we e due o o he biological e ec s sha ed by
hese di e en molecules. This s ongly sugges s a link be ween
he mycobac e ios a ic mechanism and ha o p og ammed cell
dea h. In addi ion, he bac e ios a ic and apop o ic e ec s o PA
plus IFN-
g
we e closely dependen on he ac i a ion o mac o-
phages by IFN-
g
because he mechanism was e e ed by he p os-
aglandin 15d-PGJ
2
in hioglycola e-elici ed mac ophages. By
binding o PPAR-
g
, 15d-PGJ
2
has been shown o block exp ession
o genes media ed by ansc ip ion ac o s associa ed o in lamma-
o y changes such as AP-1, NF-
k
B, and S a 1 (38). Al hough he
known consequence o he ac i a ion o PPAR-
g
in mac ophages
is he inhibi ion o he exp ession o genes such as hose coding o
gela inase B, TNF-
a
, and iNOS (38, 39), we obse ed ha ano he
e ec was he inhibi ion o apop osis. As a ole o TNF-
a
and NO
in he exp ession o he PA ea men had been excluded, we eel
ha he inhibi ion o apop osis is he cause o he abolished an i-M.
a ium ac i i y. In addi ion, whene e we ailed o e e he my-
cobac e ios a ic e ec induced by PA plus IFN-
g
, e.g., by supply-
ing me al ions, eac i e oxygen sca enge s, o cy okine modula-
o s, o by using he mac ophages om he gene-dis up ed mouse
s ains, we also ailed o de ec any di e ences in he mo pholog-
ical al e a ions desc ibed in his wo k.
The mechanism whe eby PA igge s apop osis is no cla i ied,
bu i could in ol e he gene a ion o eac i e oxygen species o an
al e a ion o he cy oplasmic edox po en ial, as sugges ed by he
esul s ob ained wi h NAC and glu a hione (48, 49). NAC is a hiol
compound ha can ac ei he as a hyd oxyl adical sca enge o by
inc easing he in acellula le els o glu a hione, which p o ec s he
cell agains oxida i e damage. Al hough he addi ion o ca alase o
ca alase plus SOD did no e e he bac e ios a ic e ec o PA in
ou s udies, NAC blocked he mycobac e ios asis induced by PA
and PA plus IFN-
g
p obably because o i s an iapop o ic e ec . A
ole o NAC in blocking oxygen- eac i e species gene a ed by he
NADPH oxidase wi h he consequen inhibi ion o mycobac e i-
os asis does no seem plausible because no di e ences we e ound
when p47
phox
-de icien mice we e compa ed wi h wild- ype con-
ols. Also, no e idence exis s o sugges ha oxygen- eac i e spe-
cies gene a ed ou side he phagosome (e.g., he cy osol o he mi-
ochond ia) may lead o mycobac e ial g ow h es ic ion inside
hei acuola loca ion. I has been epo ed ha i on (III) picoli-
na e ca alyzes he p oduc ion o hyd oxyl adical in he p esence o
supe oxide adical and hyd ogen pe oxide (50). E en hough he
addi ion o ca alase o SOD plus ca alase did no e e PA o PA
plus IFN-
g
e ec , i is possible ha he enzymes did no each he
si e in which O
2
2
and H
2
O
2
a e o med, p esumably in acellula .
A localized p oduc ion o he hyd oxyl adical could ini ia e lipid
pe oxida ion and cell damage wi h subsequen apop osis (46). On
he o he hand, eac i e oxygen species also ac as second mes-
senge s in he ac i a ion o NF-
k
B (51), a anc ip ion ac o ha
is associa ed wi h ansduc ion o p oapop o ic signals in di e en
cells (52, 53). As 15d-PGJ
2
and NAC can inhibi ac i a ion o
NF-
k
B (38, 51, 54), we pos ula e ha ac i a ion o NF-
k
B in mac-
ophages ea ed wi h PA and PA plus IFN-
g
is cen al o bo h
apop osis and induc ion o mycobac e ios asis.
IFN-
g
clea ly enhanced he apop o ic and bac e ios a ic ac i i y
o PA in in ec ed mac ophages. Tha e ec is no a combina ion o
wo sepa a e an i-M. a ium ac i i ies because in hioglycola e-elic-
i ed pe i oneal mac ophages, he ac i i y o PA plus IFN-
g
was
e e ed by 15d-PGJ
2
wi hou dis u bing he ac i i ies o PA alone.
In e es ingly, IFN-
g
is a cy okine ha may inhibi cell g ow h and
FIGURE 7. The inhibi o o apop osis, NAC, p e en s he induc ion o
mycobac e ios asis by IFN-
g
plus PA. A, Exposu e o phospha idylse ine
was analyzed by s aining he cells wi h annexin V-FITC. His og ams ep-
esen mac ophages ea ed wi h PA plus IFN-
g
in medium (black line) o
mac ophages ha had been wi h NAC be o e and du ing ea men wi h PA
plus IFN-
g
(g ay line) a e ga ing on cells excluding p opidium iodide. B,
E ec s o PA o PA plus IFN-
g
on he g ow h o M. a ium s ain 25291
we e compa ed be ween mac ophages cul u ed in medium (le ) and mac-
ophages ea ed wi h NAC ( igh ). C, E ec s o wo di e en an ioxidan s
in he inhibi ion o bac e ios a ic ac i i y o PA. M. a ium CFU we e de-
e mined a e 6 days o cul u e wi h he indica ed s imuli in he absence
(M) o p esence o NAC ( ) o glu a hione (p). S a is ics as in Fig. 1.
395The Jou nal o Immunology
induce apop osis (55, 56). Fu he mo e, a de ec i e S a 1 ansc ip-
ion ac o in U3 cells inac i a ed IFN-
g
signaling and dec eased
he cons i u i e exp ession o caspases (57). Ac i a ion o mac o-
phages wi h IFN-
g
could p ime he cell o he apop o ic e ec o
PA. The dissec ion o he ansduc ion signals in ol ed in his
mechanism could b ing impo an in o ma ion o he unde s and-
ing o he an imycobac e ial mechanisms.
In summa y, we desc ibe a no el pa hway o mycobac e ial
g ow h con ol ha is igge ed by PA in IFN-
g
-ac i a ed mac o-
phages and ha is ela ed o changes occu ing du ing he p o-
g ammed cell dea h o he hos cell. This pa hway does no in ol e
p e iously desc ibed e ec o mechanisms such as he espi a o y
bu s o NO.
Acknowledgmen s
We hank D s. J. Mudge , C. Na han, and J. MacMicking o supplying
b eede s o iNOS
2/2
mice; D . Tak Mak o supplying b eede s o
p55
2/2
mice; D s. S e en Holland and B ahm H. Segal o he gi o
p47
phox2/2
mice; DNAX o he an i-TNF hyb idoma; and D . M. T.
Sil a, Ms. Paula Macedo, and Ms. I ene Ba os o help wi h he
elec on mic oscopic analysis.
Re e ences
1. F ehel, C., C. de Chas ellie , C. O edo, and P. Be che. 1991. In amac ophagic
g ow h o Mycobac e ium a ium du ing in ec ion o mice. In ec . Immun.
59:2207.
2. Gomes, M. S., M. Flo´ ido, T. F. Pais, and R. Appelbe g. 1999. Imp o ed clea -
ance o Mycobac e ium a ium upon dis up ion o he inducible ni ic oxide syn-
hase gene. J. Immunol. 162:6734.
3. Appelbe g, R., and I. M. O me. 1993. E ec o mechanisms in ol ed in cy okine-
media ed bac e ios asis o Mycobac e ium a ium in ec ions in mu ine mac o-
phages. Immunology 80:352.
4. Segal, B. H., T. M. Dohe y, T. A. Wynn, A. W. Chee e , A. She , and
S. M. Holland. 1999. The p47
phox2/2
mouse model o ch onic g anuloma ous
disease has no mal g anuloma o ma ion and cy okine esponses o Mycobac e-
ium a ium and Schis osoma mansoni eggs. In ec . Immun. 67:1659.
5. Gomes, M. S., and R. Appelbe g. 1998. E idence o a link be ween i on me-
abolism and N amp1 gene unc ion in inna e esis ance agains Mycobac e ium
a ium.Immunology 95:165.
6. Gomes, M. S., G. Dom, J. Ped osa, J. R. Boelae , and R. Appelbe g. 1999.
E ec s o i on dep i a ion on Mycobac e ium a ium g ow h. Tube cle Lung Dis.
79:321.
7. Dou as, G. S., D. L. Looke , A. E. Va e , and A. J. C owle. 1985.
g
In e e on
ac i a es human mac ophages o become umo icidal and leishmanicidal bu en-
hances eplica ion o mac ophage-associa ed mycobac e ia. In ec . Immun. 50:1.
8. Shi a suchi, H., J. L. Johnson, H. Toba, and J. J. Ellne . 1990. S ain- and dono -
ela ed di e ences in he in e ac ion o Mycobac e ium a ium wi h human mono-
cy es and i s modula ion by in e e on-
g
.J. In ec . Dis. 162:932.
9. O enho , T. H. M., D. Kuma a a ne, and J.-L. Casano a. 1998. No el human
immunode iciencies e eal he essen ial ole o ype-I cy okines in immuni y o
in acellula in ec ions. Immunol. Today 19:491.
10. Flo´ ido, M., A. S. Gonc¸al es, R. A. Sil a, S. Ehle s, A. M. Coope , and
R. Appelbe g. 1999. Resis ance o i ulen Mycobac e ium a ium o in e e on-
g
-media ed an imic obial ac i i y sugges s addi ional signals o induc ion o
mycobac e ios asis. In ec . Immun. 67:3610.
11. P e e ko n, E. R. 1984. In e e on
g
blocks he g ow h o Toxoplasma gondii in
human ib oblas s by inducing he hos cells o deg ade yp ophan. P oc. Na l.
Acad. Sci. USA 81:908.
12. Mu ay, H. W., A. Szu o-Sudol, D. Wellne , M. J. Oca, A. M. G ange ,
D. M. Libby, C. D. Ro he mel, and B. Y. Rubin. 1989. Role o yp ophan deg-
ada ion in espi a o y bu s -independen an imic obial ac i i y o
g
in e e on-
s imula ed human mac ophages. In ec . Immun. 57:845.
13. Thomas, S. M., L. F. Ga i y, C. R. B and , C. S. Schobe , G.-S. Feng,
M. W. Taylo , J. M. Ca lin, and G. I. By ne. 1993. IFN-
g
-media ed an imic obial
esponse: indoleamine 2,3-dioxygenase-de icien mu an hos cells no longe in-
hibi in acellula Chlamydia spp. o Toxoplasma g ow h. J. Immunol. 150:5529.
14. Gup a, S. L., J. M. Ca lin, P. Pya i, W. Dai, E. R. P e e ko n, and M. J. Mu phy,
J . 1994. An ipa asi ic and an ip oli e a i e e ec s o indoleamine 2,3-dioxyge-
nase enzyme exp ession in human ib oblas s. In ec . Immun. 62:2277.
15. Naginemi, C. N., K. Pa dhasa adhi, M. C. Ma ins, B. De ick, and J. J. Hooks.
1996. Mechanisms o in e e on-induced inhibi ion o Toxoplasma gondii epli-
ca ion in human e inal pigmen epi helial cells. In ec . Immun. 64:4188.
16. Geb an, S. J., Y. Yamamo o, C. New on, T. W. Klein, and H. F iedman. 1994.
Inhibi ion o Legionella pneumophila g ow h by
g
in e e on in pe missi e A/J
mouse mac ophages: ole o eac i e oxygen species, ni ic oxide, yp ophan,
and i on (III). In ec . Immun. 62:3197.
17. Meh a, S. J., R. D. Mille , J. A. Rami ez, and J. T. Summe sgill. 1998. Inhibi ion
o Chlamydia pneumoniae eplica ion in Hep-2 cells by in e e on-
g
: ole o
yp ophan ca abolism. J. In ec . Dis. 177:1326.
18. Bodaghi, B., O. Gou eau, D. Zipe o, L. Lau en , J.-L. Vi elizie , and
S. Michelson. 1999. Role o IFN-
g
-induced indoleamine 2,3 dioxygenase and
inducible ni ic oxide syn hase in he eplica ion o human cy omegalo i us in
e inal pigmen epi helial cells. J. Immunol. 162:957.
19. Ra ledge, C. 1982. Nu i ion, g ow h and me abolism. In The Biology o he
Mycobac e ia, Vol. 1: Physiology, Iden i ica ion and Classi ica ion.
C. Ra ledge and J. S an o d, eds. Academic P ess, London, pp.183–271.
20. Yasiu, H., K. Takai, R. Yoshida, and O. Hayaishi. 1986. In e e on enhances
yp ophan me abolism by inducing pulmona y indoleamine 2,3 dioxygenase: i s
possible occu ence in cance pa ien s. P oc. Na l. Acad. Sci. USA 83:6622.
21. Yoshida, R., Y. U ade, M. Tokuda, and O. Hayaishi. 1979. Induc ion o in-
doleamine 2,3-dioxygenase in mouse lung du ing i us in ec ion. P oc. Na l.
Acad. Sci. USA 76:4084.
22. Fuchs, D., A. Fo sman, L. Hagbe g, M. La sson, G. No k ans, G. Reibnegge ,
E. R. We ne , and H. Wach e . 1990. Immune ac i a ion and dec eased yp o-
phan in pa ien s wi h HIV-1 in ec ion. J. In e e on Res. 10:599.
23. Wannemache , R. W. 1977. Key ole o a ious indi idual amino acids in hos
esponse o in ec ion. Am. J. Clin. Nu . 30:1269.
24. Baila , J. C., J ., ed. 1956. The Chemis y o he Coo dina ion Compounds. Re-
inhold Publishing Co p., New Yo k.
25. E ans, G. W., and P. E. Johnson. 1980. Cha ac e iza ion and quan i a ion o a
zinc-binding ligand in human milk. Pedia . Res. 14:876.
26. Collins, J. J., C. R. Adle , J. A. Fe nandez-Pol, D. Cou , and G. S. Johnson. 1979.
T ansien g ow h inhibi ion o Esche ichia coli K-12 by ion chela o s: in i o
inhibi ion o ibonucleic acid syn hesis. J. Bac e iol. 138:923.
27. Fe nandez-Pol, J. A. 1977. I on: possible cause o he G
1
a es induced in NRK
cells by picolinic acid. Biochem. Biophys. Res. Commun. 78:136.
28. Fe nandez-Pol, J. A. 1977. T ansi ion me al ions induce cell g ow h in NRK cells
synch onized in G
1
by picolinic acid. Biochem. Biophys. Res. Commun. 76:413.
29. Leu hause , S. W. C., L. W. Obe ley, and T. D. Obe ley. 1982. An i umo ac i i y
o picolinic acid in CBA/J mice. J. Na l. Cance Ins . 68:123.
30. Ru mann, R., R. D. Welke , T. Sai o, M. A. Chi igos, and L. Va esio. 1984. In
i o ac i a ion o mac ophages bu no na u al kille cells by picolinic acid
(PLA). J. Immunopha macol. 6:291.
31. Blasi, E., R. Mazzolla, L. Pi zu a, R. Ba luzzi, and F. Bis oni. 1993. P o ec i e
e ec o picolinic acid on mice in ace eb ally in ec ed wi h le hal doses o Can-
dida albicans.An imic ob. Agen s Chemo he . 37:2422.
32. Va esio, L., M. Clay on, E. Blasi, R. Ru man, and D. Raadzioch. 1990. Picolinic
acid, a ca aboli e o yp ophan, as he second signal in he ac i a ion o IFN
g
-
p imed mac ophages. J. Immunol. 145:4265.
33. Zinga elli, B., M. O’Conno , H. Wong, A. L. Salzman, and C. Szabo´. 1996.
Pe oxyni i e-media ed DNA s and b eakage ac i a es poly-adenosine diphos-
pha e ibosyl syn he ase and causes cellula ene gy deple ion in mac ophages
s imula ed wi h bac e ial lipopolysaccha ide. J. Immunol. 156:350.
34. Melillo, G., G. W. Cox, D. Radzioch, and L. Va esio. 1993. Picolinic acid, a
ca aboli e o L- yp ophan, is a cos imulus o he induc ion o eac i e ni ogen
in e media e p oduc ion in mu ine mac ophages. J. Immunol. 150:4031.
35. Cox, G. W., U. Cha opadhyay, J. J. Oppenheim, and L. Va esio. 1991. IL-4
inhibi s he cos imula o y ac i i y o IL-2 o picolinic acid bu no o lipopoly-
saccha ide on IFN-
g
- ea ed mac ophages. J. Immunol. 147:3809.
36. Ve mes, I., C. Haanen, H. S e ens-Nakken, and C. Reu elingspe ge . 1995. A
no el assay o apop osis: low cy ome ic de ec ion o phospha idylse ine ex-
p ession on ea ly apop o ic cells using luo escein labeled annexin V. J. Immunol.
Me hods 184:39.
37. Tel o d, W. G., L. E. King, and P. J. F ake . 1994. Rapid quan i a ion o apop osis
in pu e and he e ogeneous cell popula ions using low cy ome y. J. Immunol.
Me hods 172:1.
38. Rico e, M., A. C. Li, T. M. Wilson, C. J. Kelly, and C. K. Glass. 1998. The
pe oxisome p oli e a o -ac i a ed ecep o -
g
is a nega i e egula o o mac o-
phage ac i a ion. Na u e 391:79.
39. Jiang, C., A. T. Ting, and B. Seed. 1998. PPAR-
g
agonis s inhibi p oduc ion o
monocy e in lamma o y cy okines. Na u e 391:82.
40. Hockenbe y, D. M., Z. N. Ol ai, X.-M. Yin, C. L. Milliman, and
S. J. Ko smeye . 1993. Bcl-2 unc ions in an an ioxidan pa hway o p e en
apop osis. Cell 75:241.
41. Maye , M., and M. Noble. 1994. N-ace yl-L-cys eine is a plu ipo en p o ec o
agains cell dea h and enhance o ophic ac o -media ed cell su i al in i o.
P oc. Na l. Acad. Sci. USA 91:7496.
42. Fe a i, G., C. Y. I. Yan, and L. A. G eene. 1995. N-Ace ylcys eine (D- and
L-s e eoisome s) p e en s apop o ic dea h o neu onal cells. J. Neu osci.
15:2857.
43. Um, H.-D., J. M. O ens ein, and S. M. Wahl. 1996. Fas media es apop osis in
human monocy es by a eac i e oxygen in e media e dependen pa hway. J. Im-
munol. 156:3469.
44. Laochum oon o apong, P., S. Paul, K. B. Elkon, and G. Kaplan. 1996. H
2
O
2
induces monocy e apop osis and educes iabili y o Mycobac e ium a ium-M.
in acellula e wi hin cul u ed human monocy es. In ec . Immun. 64:452.
45. Molloy, A., P. Laochum oon o apong, and G. Kaplan. 1994. Apop osis, bu no
nec osis, o in ec ed monocy es is coupled wi h killing o in acellula bacillus
Calme e-Gue´ in. J. Exp. Med. 180:1499.
46. Lammas, D. A., C. S obe , C. J. Ha ey, N. Kend ick, S. Panchalingam, and
D. S. Kuma a a ne. 1997. ATP-induced killing o mycobac e ia by human mac-
ophages is media ed by pu ine gic P2Z(P2 37) ecep o s. Immuni y 7:433.
47. Oddo, M., T. Renno, A. A inge , T. Bakke , H. R. MacDonald, and
P. R. A. Meylan. 1998. Fas ligand-induced apop osis o in ec ed human mac o-
phages educes he iabili y o in acellula Mycobac e ium ube culosis. J. Im-
munol. 160:5448.
396 MYCOBACTERIOSTASIS INDUCED BY PA
48. Ueda, S., H. Nakamu a, H. Masu ani, T. Sasada, S. Yoneha a, A. Takabayashi,
Y. Yamaoka, and J. Yodoi. 1998. Redox egula ion o caspase-3(-like) p o ease
ac i i y: egula o y oles o hio edoxin and cy och ome c.J. Immunol. 161:6689.
49. Kane, D. J., T. A. Sa a ian, R. An on, H. Hahn, E. B. G alla, J. S. Valen ine,
T. O d, and D. E. B edesen. 1993. Bcl-2 inhibi ion o neu al dea h: dec eased
gene a ion o eac i e oxygen species. Science 262:1274.
50. Bannis e , W. H., J. V. Bannis e , A. J. F. Sea le, and P. J. Tho nalley. 1983. The
eac ion o supe oxide adicals wi h me al picolina e complexes. Ino g. Chim.
Ac a 78:139.
51. Sch ek, R., P. Riebe , and P. A. Baue le. 1991. Reac i e oxygen in e media es as
appa en ly widely used messenge s in he ac i a ion o he NF-
k
B ansc ip ion
ac o and HIV-1. EMBO J. 10:2247.
52. Lin, K.-I., S.-H. Lee, R. Na ayanan, J. M. Ba aban, J. M. Ha dwick, and
R. R. Ra an. 1995. Thiol agen s and Bcl-2 iden i y an alpha i us-induced apo-
p o ic pa hway ha equi es ac i a ion o he ansc ip ion ac o NF-
k
B. J. Cell
Biol. 131:1149.
53. G imm, S., M. K. A. Baue , P. A. Baue le, and K. Schulze-Os ho . 1996. Bcl-2
down- egula es he ac i i y o ansc ip ion ac o NF-
k
B induced upon apop o-
sis. J. Cell Biol. 134:13.
54. S aal, F. J. T., M. Roede e , L. He zenbe g, and L. He zenbe g. 1990. In acel-
lula hiols egula e ac i a ion o nuclea ac o
k
B and ansc ip ion o human
immunode iciency i us. P oc. Na l. Acad. Sci. USA 87:9943.
55. No elli, F., F. di Pie o, P. F. di Celle, S. Be ini, P. A aca i, G. Ga o a, and
G. Fo ni. 1994. En i onmen al signals in luencing exp ession o he IFN-
g
e-
cep o on human T cells con ol whe he IFN-
g
p omo es p oli e a ion o apo-
p osis. J. Immunol. 152:496.
56. Deiss, L. P., E. Feins ein, H. Be issi, O. Cohen, and A. Kimchi. 1995. Iden i i-
ca ion o a no el se ine/ h eonine kinase and a no el 15-kD p o ein as po en ial
media o s o he
g
in e e on-induced cell dea h. Genes De . 9:15.
57. Kuma , A., M. Commane, T. W. Flickinge , C. M. Ho a h, and G. R. S a k.
1997. De ec i e TNF-
a
-induced apop osis in STAT1-null cells due o low con-
s i u i e le els o caspases. Science 278:1630.
397The Jou nal o Immunology