INTERNATIONAL JOIIRNAL OF LEPROSY^ Volume 61, Number 4
Printed in tlw
Differential Production of Interleukin 1 (IL-1),IL-6, Tumor
Necrosis Factor, and IL-1 Receptor
Antagonist by Human Monocytes Stimulated withMycobacterium
leprae and M. bovis BCG1
Katsuya Suzuki, Yasuo Fukutomi, Masanori Matsuoka,Keiko Torii,
Hidetoshi Hayashi, Takemasa Takii,
Yasukazu Oomoto, and Kikuo Onozaki2
Leprosy and tuberculosis, caused by in-fection with
Mycobacterium leprae and M.tuberculosis, respectively, are chronic
infec-tious diseases from which more than 20 mil-lion patients are
suffering worldwide 24).Although these two mycobacteria are com-mon
in several of their constituents, suchas peptidoglycan and the
arabinogalactan-mycolic acid complex 23), the clinical as-pects of
the two diseases are complicated.Based on the clinical features as
well as im-munologic responses, leprosy patients canbe grouped into
four types or categories:lepromatous (L), tuberculoid (T),
borderline(B) and indeterminatc (I). Patients with tu-berculoid
leprosy manifest a strong cell-me-diated immunity (CM!) response to
M. lep-rae but produce a relatively low levei ofantibody. In
contrast, in lepromatous pa-tients there is a prominent humoral
anti-body response but an anergy in CMI to Al.leprae (I").
Previous studies have suggested that theseimmunological
deviations result from theaccumulation of a particular subset of T
' Received for publication on 22 June 1993; ac-cepted for
publication in revised form on 9 September1993.
= K. Suzuki, M.Sc.; K. Torii, B.S.; H. Hayashi, Ph.D,Assistant
Professor; T. Takii, M.Sc., Assistant Profes-sor; K. Onozaki,
Ph.D., Professor, Department of Hy-gienic Chemistry, Faculty of
Pharmaceutical Sciences,Nagoya City University, Mizuho, Nagoya 467,
Japan.Y. Fukutomi, Ph.D., Senior lnvestigator; M. Matsu-oka,
D.V.Sc., Room Chief of Laboratory 4, NationalInstitute for Leprosy
Rcsearch, Kiyosc, Tokyo 204,Japan. Y. Oomoto, Ph.D., Research
Scientist, De-partment oflmmunology, Otsuka Pharmaceutical
Co.,Ltd., Tokushima, Tokushima 770, Japan.
Reprint requcsts to Dr. Onozaki.
in the lesions, namely, tuberculoid lesionscontam n a
predominance of CD4+ T cells■,vhereas lepromatous lesions contamn
main-ly CD8+ T cells ('")• It also has been re-ported that in
tuberculoid lesions interleu-kin 2 (IL-2) and interferon-gamma
(IFN--y)mRNA, products of the Th 1 -type helper Tcells, were being
expressed while in lepro-matous lesions IL-4, IL-5, and IL-10mRNA,
products of the Th2-type helper Tcells, were exprcssed (1').
Immunologicaldysfunctions of macrophages also have beenreported.
Macrophages of lepromatous pa-tients suppress T-cell responses and
pro-duce minor or only low leveis of IL-1 andtumor necrosis factor
(TNF) (2. 2'. 10). Sincemacrophages function as
antigen-presentingcells to T cells and produce a variety
ofimmunoregulatory cytokincs, it is possiblethat the primary
interaction between mac-rophages and mycobacteria results in
IL-1, IL-6 and TNF are major cytokinesproduced by macrophages,
and these cyto-kines stimulate immunological and inflam-matory
reactions (4). In contrast, IL-1 re-ceptor antagonist (IL-ira),
which is alsoproduced by macrophages, inhibits IL- 1 ac-tivity as
well as IL-1-triggered chain reac-tions by competitively binding to
the IL-1receptors (5). IL-10 also inhibits macro-phage functions
and influences the subse-quent macrophage/T-cell interaction
(7)•Therefore, macrophage function is regulat-ed by both
immunostimulative and im-munosuppressive cytokines.
In this paper, we studied the productionof cytokines, IL-1,
IL-6, TNF and IL-ira,by human monocytes stimulated with liveor
killed Al. lepraeor BCG, and showed that
610^ International.Iournal of Lepras .) ,^1993
M. leprae is a very poor inducer of immu-nostimulatory cytokines
compared to BCG.However, a substantial amount of IL-1 racan be
induced by stimulation with A1. lep-rae even when no other
cytokines are in-duced. In addition, the phagocytosis of A1.leprae
and cytokine production appeared todepend partially on serum
MATERIALS AND METHODSReagent. Human recombinant IL-1a (2
x 10' U/ml) was provided by Dr. M. Ya-mada, Dainippon
Pharmaceutical Co., Osa-ka, Japan, and human rccombinant IL-2
byShionogi Co., Osaka. Concentrated buffycoat from healthy donors
was supplied byAichi Red Cross Blood Center, Aichi, Ja-pan. RPMI
1640 and polymyxin 13 werepurchased from Sigma Chemical Co.,
St.Louis, Missouri, U.S.A., and FBS was ob-tained from Bocknek,
Mycobacteria. A1. leprae strain Thai-53were grown in the foot
pads of nude mice('). Mouse foot pads were aseptically re-moved,
minced with scissors, and homog-enized with 7H12 medium. After
centrifug-ing the homogenate for 10 min at 100 x g,the supernatants
were obtained and againcentrifuged for 20 min at 3500 x g.
Theprecipitates were resuspended with 7H12medium, and the bacillary
number was de-termined by the method of Shepard andMcRae ( 27 ).
The bacillary number was con-sistent with that counted under
microscopywith a hematocytometer. Freeze-diied ^ í.bovis BCG were
obtained from Japan BCGCompany, Tokyo, Japan. The BCG weresuspended
with phosphate buffered saline(PBS). These mycobacteria were
homoge-nized by mild sonication. The bacillarynumber was counted
under microscopy witha hematocytometer. Heat-killed mycobac-teria
were obtained by treating them at 120°Cfor 15 min.
Supernatants of monocytes stimulatedwith mycobacteria. The buffy
coat fromhealthy donors was diluted 1:3 in Hanks'balanced salt
solution (HBBS). Mononucle-ar cens (MNC) were separated over
Ficoll-Hypaque, washed twice in HBBS, and sus-pended in RPMI 1640
medium supple-mented with 100 U/ml of penicillin G, 100µg/m1 of
streptomycin and 15 mM HEPES.The number of monocytes was
estimatedby incubating the cell suspension in a he-
matocytometer at 37°C for 3 min in air con-taining 5% CO, and
then counting thespreading cens. The spreading ccll numberwas
consistent with that of cells adhering tothe tissue culture plate.
One ml of a MNCsuspension containing monocytes (1 x 10 6cens/ml)
was added to each well of a 24-well plate (Falcon, Lincoln, New
Jersey,U.S.A.). After 2 hr of culturing at 37°C inair containing 5%
CO,, the cens were washedtwice with HBBS. More than 90% of
theadherent cclls were monocytes as deter-mined by morphological
criteria withGiemsa staining and the ability to phago-cytose latex
beads. To the adherent mono-cytes, 1 ml of RPMI 1640
supplementedwith 1% fetal bovine serum (FBS) or humanserum (HS),
untreated or heat inactivatedat 56°C for 30 min, containing
mycobacteriawere added, and then the cells were culturedat 37°C.
Although the medium was endo-toxin-free according to the Limulus
ame-bocyte assay (sensitivity limit of 0.1 ng/ml),we usually added
polymyxin 13 (5 lig/mi) tothe culture to inhibit the effect of a
smallamount ofendotoxin. After each culture pe-riod, the
supernatants were obtained by cen-trifugation. These antibiotics
(peniciilin G,streptomycin and polymyxin B) did not af-fect the
viability of A1. leprae or BCG for 1week or 24 hr, respectively, as
determinedby the metabolism of palmitic acid ( 8 ).
Assay for IL-1 activity. 1L-1 activity wasdetermined by a
proliferation assay with anIL-1-dependent mouse T-cell line, Dl
ON4M,which was provided by Dr. S. J. Hopkins,University of
Manchester, Manchester,U.K.(' 2 ). In brief, cens were cultured
inRPMI 1640, 10 mM HEPES, antibiotics, 5x 10 -5 M
2-mercaptoethanol, 10% FBS,concanavalin A (3 ag/mi), IL-2 (40
U/ml)and standard IL-la or test samples. Cells (1x 10 4 ) were
cultured in wells of flat-bottommicrotiter plates at 37°C in 5% CO,
in air.After 3 days of culture, ccll proliferationactivity was
assessed by the 3-[4,5-dimeth-ylthiazol-2-yl]-2,5-diphenyltetrazoli
umbromide or thiazolyl blue (MTT) method.After solubilization of
the formozan with20% SDS and 50% DMF (dimethyl for-mamide) in
water, the absorbance at 595nm was measured on an ELISA
autoreader(Bio-Rad). IL-1 activity was expressed asunir equivalent
to standard recombinant IL-1a.
61, 4^Suzuki, et al.: Monoeyte Cytokincs atter M. leprae and
Assay for 11,-6 activity. The biologicalactivity of 1L-6 was
measureci by its prolif-erative action on the I L-6-depenclent m
u-rine hybridoma clone MI-I60.BSF2 (provid-ed by Dr. T. Hirano,
Osaka University,Osaka, Japan) (15). Proliferation was mea-sured
b■,' the MTT method (2"). One unit ofIL-6 activity was defined as
the reciprocaiof the dilution ofsamples that exhibited 50%of
Assay for TNF activity. The activity ofTNF was determined by a
L929 libroblastcell lytic assay (27). 13riefly, 100 mi of a
sus-pension of TNF-sensitive mouse L929 fi-broblast cells (5 x 109
Mis/mi) was culturedwith serially diluted test samples in wells ofa
flat-bottom microtiter plate at 37°C for 18hr in air containing 5%
CO in the presenceof actinomycin D (1 lig/mi). After culture,the
plates were washed, and cell lysis wascletermined by staining the
plates with crys-tal violei (0.5%) in metlizinol-water (1:25,v/v).
After the dye-stained cells were solu-bilized with 0.1 ml of 0.1%
SDS, the dyeuptake was calculated by an ELISA auto-reader. One unit
of TNF activity was de-fined as the reciprocai of the dilution
ofsamples that lysed 50% of the L929 cens.
Determination of ra. I L- 1ra contentwas determined by an ELISA
using mousemonoclonal antibody (IgG) and rabbit poly-clonal
antibody (IgG) against human re-combinant IL-ira.
Phagocytosis by monocytes of M. lepraeand BCG. Monocytes (5 x
106 cens) werecultured in RPMI 1640 medium supple-mented with I%
untreated or heat-inacti-vated FBS or HS with killed M. leprae
orkilled BCG (1.5 x 10') on coverslips in 24-well culture plates.
After culture for 24 hr,the coverslips were washed and stained
withacid-fast staining. Phagocytosis of the my-cobacteria by
monocytes was determinedunder microscopy.
RESULTSCytokine production by monocytes stim-
ulated with M. leprae and BCG. In miei-to determine the
difference of cytokine pro-duction by monocytes stimulated with
AI.lepra(' and BCG, human monocytes (1 x106cells) were treated with
varying numbersof killed AI. leprae, live or killed BCG. Be-cause
cytokine induction by a humanmonocytic cell line with
requires at least 1% FBS (17), in this studywe first conducted
the experiment in thepresence of 1% heat-inactivated FBS. TNF'ias
determined after 6 hr ofculture and IL-1, IL-6, and IL-ira were
determined after24 hr of culture. As shown in Figure 1, upto 3 x
106 of AI. leprac induced no detect-able IL-1, IL-6 or TNF. At I x
10 theyinduced only very low leveis of these threecytokines. In
contrast, BCG, either live orkilled, induced these cytokines at
more than3 x 104 or 105 edis; as shown 109-106 wasthe optimal. Live
BCG induced largeramounts of cytokines than killed BCG atlower
numbers of bacteria. Thus, BCG ap-peared to be far more potent than
M. leprae.In contrast to IL-1, IL-6 and TNF. IL-irawas induced by
AI. /eprae-stimulatedmonocytes in a dose-dependent manner.BCG also
induced IL- 1 ra, but its produc-tion was inversely related to the
number ofbacteria. Although the data are not shown,104 live or
killed BCG induced more IL-irathan 105 BCG. A control culture with
poly-myxin B alone did not induce detectableleveis of any of the
Kinetics of cytokine production by mono-cytes. The differences
in cytokine produc-tion might have resulted from differentkinetics.
Therefore, monocytes were stim-ulated. with 10' mycobacteria, and
kineticsstudies of cytokine production were con-ducted. IL-1 and
IL-6 production by mono-cytes stimulated with either BCG or Al.
lep-ra(' increased with the duration of theincubation period and
peaked at 18-24 hr.TN F production by BCG-stimulatedmonocytes
peaked ai 12-18 hr. M. lepraecontinuously induced IL-1ra up to 44
hr,but BCG did so only up to 12.5 hr. There-fore, the different
cytokine production bymonocytes stimulated with Al. leprae andBCG
did not appear to result from differentkinetics.
FBS and HS and the effect of heat-inac-tivation. The experiments
in Figures 1 and2 were conducted in the presence of
heat-inactivated FBS. Since it is reported thatphagocytosis of AI.
leprac by human mono-eles depends on complement C3 (25),
wedetermined the effect of heat-inactivationof the serum on
cytokine production bymonocytes stimulated with killed AI. lepracor
BCG. A comparison of FBS and HS wasalso made. As demonstrated in
1993612^ International Journal of Lepro.ti_t'
8 0 00
^ M.Ieprac ( killed )® OCO
^ OCO ( laled )
3x10 13.104^10'^3x10 5^106^3x10'^10' 10 6^3x10 6^10i106^3x10
1 0 5 10 610 ^3x10 ^106^3x10 6^10
Number of mycobacteria / monocytes 10 6 cells
Number of mycobacteria / monocytes 10 cells
L7 M.leprae (killed)
® OCO^ OCO (kled)
FIG. I. Dose response of cytokine production by M. leprae- or
BCG-stimulated monocytes. Human mono-cytes (10' cells) were
cultured in medium supplemented with 1% hcat-inactivatcd fetal
bovine serum with varyingnumbers of killed .t/. leprae, live or
killed BCG. After culture for 24 hr, amounts of IL-1, IL-6, and
IL-Ira inculture supernatants were dctcrmined as described in
Materiais and Methods. Amount of tumor necrosis factor(TNF) was
determined aftcr a 6-hr culture. Mean ± of S.D. of triplicate
cultures is shown. A = IL-I; B = IL-6; C = TNF; D = IL-Ira.
monocytes in untrcated HS produced thehighest leveis of
cytokines in response toeither mycobacteria. Heat-inactivated
FBSexhibited a lower effect. Although the dataare not shown, there
was no difference be-tween untreated and heat-inactivated FBS.The
monocytes in heat-inactivated HS pro-duced the lowest leveis of
cytokine. In thereexperiments, however, BCG was again morepotent
than M. leprae.
Effect of serum on phagocytosis of my-cobacteria by monocytes.
In order to de-termine the effect of serum on the phago-cytosis of
there mycobacteria by monocytes,
the monocytes were cultured in mediumsupplemented with untreated
or heat-inac-tivated FI3S or HS with killed .%í. leprae orkilled
BCG. Monocytes in untreated HSwere most active in the phagocytosis
of AI.leprae. Monocytes in other cultures exhib-ited lower leveis
of phagocytosis. There wasno difference between heat-inactivatcd
HS,untrcated and heat-inactivatcd FBS. Incontrast to AI. leprae,
there was no differ-ence in the phagocytosis of BCG
betweenuntreated and hcat-inactivated HS. Mono-cytes in FBS either
untreated or heat-inac-tivated exhibited lower leveis of
4.5 6.5 12 .5 18 24 44
18 2 42^7.5^6.5^12.5
Incubation Time ( h )
61, 4^Suzuki, et al.: Monoc}ate Ci'tokincs n¡tei M. leprae and
6000 - B
^ M.leprac (killed)
^ OCO (kaled)
Incubation Time (h )
^ M.leprae ( killed )
^ BCG ( killed )
FIG. 2. Kinetics of cytokine production by monocytes. Monocytes
(10" cells) were cultured in mediumsupplemented with 1%
heat-inactivated fetal bovine serum with killed M. leprae, live or
killed BCG (10') forperiods indicated. After culture, amounts of
IL-1, IL-6, tumor necrosis factor (TNF) and IL-Ira in
culturesupernatants were determined. Mcan ± S.D. of triplicate
cultures is shown. A = IL-1; B = IL-6; C = TNF; D= IL- Ira.
tosis (data not shown). However, under thesame experimental
conditions, BCG wereusually phagocytosed more than M. leprae.
Live and killed M. leprae in induction ofcytokines by monocytes.
Untreated HS ap-peared to be the most efficient constituentof the
medium for monocytes to producecytokines in response to
mycobacteria. Themonocytes then were cultured in the pres-ence of
untreated HS, and their ability toproduce cytokines in response to
live orkilled M. leprae and live BCG was deter-mined. As shown in
Figure 3, significant butlow leveis of IL-1 and IL-6 were inducedby
a small number of ALI. leprae. No differ-ence was observed between
live or killed M.leprae. In contrast to IL-1 and IL-6, TNF
was not induced by up to 106 Al. leprae.BCG was more potent than
M. leprae. IL-1 ra was equally produced by 10 5 to 10' M.leprae.
Again, no difference was observedbetween live and killed A. leprae.
In con-trast to the results in FBS, BCG induced IL-1 ra in a
DISCUSSIONIn this study, we demonstrated the dif-
ference between M. leprae and BCG in re-gard to their ability to
induce cytokine pro-duction from human monocytes. The efI'ectsof
live and killed mycobacteria and thoseof heat-inactivation of serum
were also de-termined. BCG has been used as a vaccinefor almost 50
years. Macrophages from mice
6 I 4^ International fournal of Leprosy^ 1993
THE TABLE. Effects of sentiu and heat inactivation on IL - 1 and
IL - 6 production bynionocyles."
Iluman serum Fetal bovine serumSlim ula^t
Untreated Ileat-inactIvated Ileat-inactivated
IL-1 (U/m1)None 9.4 ± 8.9 ND 4.7 ± 1.5
leprae 67.1 ± 8.9 8.8 ± 3.3 48.3 ± 7.2BCG 1669.8 ± 159.4 479.1 ±
38.2 890.7 ± 80.9
IL-6 (Vim!)None 80.6 ± 67.5 ND NDA/. /cprae 713.5 ±^119.5 29.0 ±
2.0 124.7 ± 22.5BCG 2884.3 ± 136.2 1712.0± 195.5 1097.3 ± 141.5
Monocytes (5 x 10" cells) were cultureel in mettium supplemented
with I% untreated or heat-inactivatedhurnan serum or
heat-inactivated fetal bovine semi]) with killed A/. hprae or
killed BCG (1.5 x 10'). Afterculture for 24 hr, the amounts of IL-1
and 1L-6 in the culture supernatants were determined. Mean ± S.D.
oftriplicate cultures is shown. ND = Not detected (< 3
administered BCG became tumoricidal andthosc from a normal mouse
produced IL-6in response to BCG in vitro (14,22,.) There-fore, it
is expected that BCG is a potentinducer of cytokines from animal
and hu-man macrophages. The components of M.tuberculosis muramyl
dipeptide (MDP), li-poarabinomannan (LAM), and proteinshave been
shown to induce cytokine pro-duction by human monocytes or
macro-phages 21' 29)• AI. tuberculosis also is re-ported to induce
IL-1 and TNF by humanmonocytes (=v). However, as far as we knowit
is not clear what kind of cytokines BCGinduce in human monocytcs
and that if theydo, how BCG differs from virulent M. tu-berculosis.
In addition, an immunosuppres-sive cytokine IL-ira has not been
investi-gated in relation to its production bymacrophages/monocytes
stimulated withmycobacteria. In this study BCG, live orkilled,
induced production of IL- 1, TNF,IL-6 and IL-ira by human
monocytes.Therefore, BCG, similar to M. tubercidosis,appeared to be
a potent inducer of cyto-kines. IL-1, IL-6 and TNF were induced ina
dose-dependent manner at small bacterialnumbers and decreased at
higher numbers.However, IL- Ira production was inverselyrelated to
the bacillary number, suggestingthat a very small number ofBCG can
induceIL-ira even when other cytokines are notinduced. It is known
that immunologicaltolerance can be induced by repeated injec-tion
of a very small amount or a very large
amount of antigens (13). It is possible, there-fore, that
IL-1ra, an immunosuppressivecytokine, may play a role in the
low-doseantigen-induced tolerance. IL-1ra produc-tion is induccd by
IL-4, granulocyte mono-cyte-colony stimulating factor, T-cell
growthfactor-f1, and IL-lO (5. =8). However, becauselL-1ra
dccreased with the increase of theother cytokines, there may be a
cytokinecapable of inhibiting IL- ira production.
Studies revealcd that killed tubercu-losis or BCG protected
animais from tu-berculosis oniy weakly, but live BCG did sofor a
long period ())• The same was true witholher bacteria. Mouse
macrophages stim-uiated withwith live Listeria inonocvlo
genesproduced much more IL-1 than those stim-ulated with killed
bacteria (18). In our study,at low bacillary numbers live BCG was
moreeffective than killed BCG in the inductionof IL-6 and TNF, but
not of IL-1 or IL-ira.At high cell numbers, however, there wasno
difference. Therefore, the differentialprotective effect between
live and killed BCGcould not be explained by IL-1 induction.
When compared with BCG, AI. leprae in-duced only small amounts
oU IL-1, IL-6 andTNF. A kinetic study indicated that the
dif-ference had not resulted from the differentkinetics of cytokine
production. It was ofnote, however, that IL-lra was induced ina
dose-dependent manner. The differentdose-dependent responses
between BCG andM. leprae presumably were due to the dif-ferent
efficacies of these mycobacteria. Thus,
J. 1 I C//rI Y/G
5000 - B
n 105 3x10' 106^3x10'^10 7
• M lepra° (kdled)
73210 4̂ 105^3x10
6Number of mycobacteria / monocytes 10 cells
• M.leprae (k7led)
61, 4^Suzuki, et al.: Monocyte Cytokines uiter M. leprae and
6Number of mycobacteria / monocytes 10 cells
FIG. 3. Comparison of live and killcd .tt. Ieprae in induction
ofcytokincs. Monocytes (10' cells) were cultured
in medium supplemented with 1% untreated human serum with live
or killcd ;t1. leprae or live BCG (10'). After
culture for 24 hr, amounts of IL-1, IL-6 and IL-ira in culture
supernatants were dctermined. Amount of tumor
necrosis factor (TNF) was dctermined after a 6-hr culture. Mean
± S.D. of triplicate cultures is shown. A = IL-I; B = IL-6; C =
TNF; D = IL- Ira.
the finding indicates that IL-ira is the mostreadily inducible
cytokine by these myco-bacteria.
It is reported that complement C3 bindsto phenolic glycolipid-I
in AI. leprae, there-by facilitating phagocytosis of M. leprae
bymonocytes through complement receptors( 25). Therefore, we
examined the effect ofheat inactivation of HS and FBS on
phago-cytosis and cytokine production. Indeed,monocytes in fresh HS
phagocytosed M.leprae more than those in heat-inactivatedHS or FBS.
In contrast to M. leprae, therewas no difference in the
phagocytosis of BCGbetween untreated and heat-inactivated HS,
indicating that complement is not involved.Monocytes in FBS,
untreated or heat-in-activated, exhibited the smallest levei
ofphagocytosis. It was of note, however, thatunder any experimental
condition BCG wasphagocytosed more than M. leprae. In par-allel to
the phagocytosis, monocytes in freshHS produced more cytokines than
those inheat-inactivated HS. However, when wecompared HS and FBS
the cytokine pro-duction was not in parallel because mono-cytes in
heat-inactivated HS and FBS pha-gocytosed A1. leprae at the same
levei.Therefore, phagocytosis is not the sole fac-tor responsible
for cytokine induction. It is
616^ International Journal of Leprosy^ 1993
also interesting that HS is more efficient thanheat-inactivated
HS in the case of BCG.Since there was no difference between
thephagocytosis by these monocytes, heat-la-bile serum components
may augment thecapacity of monocytes in cytokine produc-tion.
When we compared live and killed M.leprae there was no
difference. In addition,AL leprae appeared to be a very poor
in-ducer of immunostimulatory cytokines I L-1, IL-6 and TNF, while
BCG stimulatedmuch cytokine production under any ex-perimental
condition. In contrast, IL-ira (animmunosuppressive cytokine) was
inducedby both mycobacteria. Since macrophagefunction is determined
by a balance be-tween stimulatory and suppressive cyto-kines, M.
leprae appeared to confer im-munosuppressive effects rather
thanimmunostimulant ones. BCG induces localinflammation and
systemic immunityagainst Al. tuberculosis ())• However, it isnot
known whether primary infection of M.leprae induces any actue
symptoms. There-fore, the potent cytokine-inducing ability ofBCG
was implicated in their immunostim-ulatory effect. In contrast,
this study sug-gests that A/. leprae escape from host de-fenses by
inducing the least levei ofimmunostimulatory or
proinflammatorycytokine production but inducing substan-tial
amounts of the immunosuppressive cy-tokine IL- 1 ra. Macrophages
from patientswith leprosy are reported to be defective intheir
ability to present M. leprae antigensto sensitized T cells (9' 21).
Since IL-1 andIL-6 play important roles in antigen pre-sentation,
not onl■; the production of lowlevels of IL-1 and IL-6 but also the
pro-duction ofsubstantial amounts of IL- 1 ra areimplicated in the
immunodysfunction. Thelow levei production of TNF further favorsthe
infection and multiplication of M. Lep-rac in monocytes because TNF
enhancesthe production ofreactive nitrogen oxide bymurine
macrophages and inhibits myco-bacterial growth in murine and human
mac-rophages (3.1. In this regard it may be in-teresting to
investigate cytokine productionby patients' monocytes. The cells
may pro-duce more IL-ira and less IL-1, IL-6 andTN F in response to
AL leprae than those ofhealthy individuais. It might also be
inter-esting to investigate the component of M.
leprae which is responsible for IL-1raThrough genetic
engineering the IL-
1 ra-inducing component could be deletedfrom Al. leprae. Such
mutated AL lepra('may be able to induce more cytokines,
thusbecoming a good vaccine.
SUMNIARYHuman blood monocytes cultured in var-
ious serum conditions were stimulated withycobacterium lepmeor
AL bovis BCG and
their cytokine-inducing abilities were com-pared. BCG, either
live or killed, inducedproduction of interleukin 1 (I L-1), IL-6,
tu-mor necrosis factor (TNF), and IL-1 recep-tor antagonist (IL-
Ira). Live BCG at a lowerbacterial number was more potent
thankilled BCG in the induction of IL-6 andTNF. In contrast to BCG,
killed M. lepraeinduced few cytokines except for IL- Ira.Similar
results were obtained when mono-cytes were cultured in the presence
of un-treated or heat-inactivated fetal bovine se-rum (FBS). When
FBS and human serum(HS) were compared and the effect of
heatinactivation was investigated, monocytes inHS produced the most
cytokines, then thosein FBS, irrespective of heat inactivation,
andthose in heat-inactivated HS produced theleast cytokines. There
were no differencesbetween live and killed AL leprae, and BCGwere
far more potent than AL leprae in aliof our experimental
conditions, indicatingthat the poor cytokine (IL- 1, IL-6 and
TNF)-inducing ability ofM. leprae was not due totheir viability.
Cytokine production waspartially in parallel with the phagocytosis
ofthe mycobacteria. These results suggest thatAI. leprae favor
their infection by evokinglittle host reaction through the
induction ofonly low leveis of immunostimulatory orproinflammatory
cytokines but a substan-tial amount of immunosuppressive
RESUMENSe investigó la producción de citocinas por los mo-
nocitos de sangre periférica mantenidos bajo diversascondiciones
de cultivo en respuesta a la estimulacióncon Alycobacterium lepra('
o con ti. bons. El BCG,vivo o muerto, indujo la producción de
interleucina-1(IL-1), IL-6, factor necrosante de tumores (TNF), y
clantagonista del receptor para la IL-1 (IL-Ira). El BCGvivo fue
más potente que cl BCG muerto en la induc-ción de IL-6 y TNF. En
contraste con cl BCG, cl .ti.leprae muerto sói() indujo la
producción de algunas
61, 4^Suzuki, et al.: Alonocptc C't'tokines afiei M. leprae and
citocinas, entre cilas cl IL-ira. Se obtuvieron los mis-mos
resultados cuando los monocitos se cultivaron enpresencia de suero
fetal de bovino (SFB) fresco o inac-tivado por calor. Cuando se
compararon cl SFB y clsuero humano (SH) y se investig• el erecto de
su inac-tivación por calor, se encontró que los monocitos enSH
produjeron más citocinas que las producidas porlos MN en SFB,
independientemente de si cl SFI3 es-tuvo o no inactivado por calor.
Los MN en SH inac-tivado por calor produjeron la menor cantidad de
ci-tocinas. No 'tubo diferencia entre el .il. lepra' vivo ycl M.
leprae muerto por calor; cl BCG fue ms potenteque el ;tl. leprae
bajo todas las condiciones ensayadas,indicando que la pobre
capacidad inductora dc cito-cinas (IL-1, IL-6 y TNF) dcl .t/.
leprae no estuvo re-lacionada con su viabilidad. La producción de
citoci-nas estuvo parcialmente en paralelo con la fagocitosisde
micobacicrias. Estos resultados sugieren que At lep-rae favorece su
implantación por evocar una mínimareacción dei huésped, induciendo
en él la producciónde bajos niveles de citocinas
inmunoestimulatorias oproeinllamatorias y una cantidad substancial
dc cito-cinas inmunosupresoras.
RESUMEDes monocytcs dc sang humain cultivés dans du
serum sous diverses conditions ont été stimuiés par
duA1t'cobacterium leprae ou du BCG de Al. bons et leurcapacité à
induire des cytokines a été comparéc. LcBCG, vivant ou tué,
provoquait ia production d'in-terlcukine 1 (IL-l), IL-6, de facteur
nécrosant des tu-mcurs (FNT) cd d'antagonistc du rcccptcur d'IL-1
(IL-lra). Le BCG vivant en quantité bactérienne moindreétait plus
puissant que le 13C0 tué dans l'inductiond'IL-6 et FNT. En
contraste avec le BCG, le ;t/. lepraetué induisait peu dc
cytokines, excepté I'IL- ira. Dcsrésultats semblablcs ont été
obtenus par des monocytcscultivés en présence dc scrum bovin foctal
(SBF) nontraité ou inactiveé par la chalcur. Quand le SBF et
lescrum humain (SH) ont été comparés et que I'effet
del'inactivation par la chalcur a été examiné, les mono-cytcs
cultivés dans lc scrum humain produisaicnt leplus de cytokines,
suivis par ceux cultivés dans le SBF,indépendamment de
l'inactivation par la chalcur, etccux cultivés dans lc SH inactivé
parla chalcur le moinsde cytokines. II n'y avait pas de différence
entre le AI.leprae vivant et tué, et le BCG était beaucoup
pluspuissant que .tl. leprae dans toutes nos conditions
ex-périmentales, indiquant que la faible capacité de M.Ileprae à
induire des cytokines (iL-1, IL-6, et FNT)n'était pas dele à sa
viabilité. La production de cytokineétait particllement en
paralèlle avec la phagocytose desmycobactéries. Ces résultats
suggèrent que .tl. lepraefavoris son infection en suscitant peu dc
réaction de1'hôte par l'induction de faibles taux de cytokines
sti-mulant l'immunité ou l'inflammation, mais un tauxsubstantial dc
Acknowledgment. We thank Dr. S. Hida for the iL-lra ELISA. This
work was supported in part by a grant
from the U.S.-Japan Cooperative Medical Scicnce Pro-gram. The
authors also wish to gratcfully acknowlcdgcthe Sasakawa Memorial
Health Foundation for theirfinancial support of this study.
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