Mycobacterium tuberculosis H37Rv induces monocytic release of interleukin-6 via activation of mitogen-activated protein kinases: inhibition by N -acetyl-L-cysteine

Mycobacteriumtuberculosis H37Rv inducesmonocytic release ofinterleukin-6via activationofmitogen-activated protein kinases:inhibition byN -acetyl-L-cysteinePalaniappan Natarajan & Sujatha Narayanan

Department of Immunology, Tuberculosis Research Centre, Chetput, Chennai, India

Correspondence: Sujatha Narayanan,

Department of Immunology, Tuberculosis

Research Centre, Mayor V.R. Ramanathan

Road, Chetput, Chennai 600 031, India. Tel.:

191 (0) 44 2836 9627; fax: 191 (0) 44 2836

2528; e-mail: [email protected]

Received 30 October 2006; revised 20 February

2007; accepted 22 February 2007.

First published online 24 May 2007.

DOI:10.1111/j.1574-695X.2007.00256.x

Editor: Willem van Eden

Keywords

mitogen-activated protein kinases; N -acetyl-L-

cysteine (NAC); Mycobacterium tuberculosis .

Abstract

The release of proinflammatory cytokines after mycobacterial infection is a host

immune response that may be propitious or deleterious to the host. Elevated levels

of interleukin (IL)-6 are present in plasma of patients with active tuberculosis

infection. The aim of this study was to investigate the role of mitogen-activated

protein kinases in the secretion of interleukin-6 in THP-1 cells and human primary

monocytes that were infected with Mycobacterium tuberculosis H37Rv, and its

regulation by N-acetyl-L-cysteine, a potential antimycobacterial agent. Exposure of

THP-1 human monocytes to M. tuberculosis H37Rv induced rapidly, in a time-

dependent manner, the phosphorylation of mitogen-activated protein kinase

kinase 3/6 and p38 mitogen-activated protein kinase, accompanied by an

upregulation of interleukin-6. Using highly specific inhibitors of mitogen-acti-

vated protein kinase kinase-1, p38 mitogen-activated protein kinase and nuclear

factor-kB, we found that extracellular-signal regulated kinase 1/2, p38 mitogen-

activated protein kinase and nuclear factor-kB were essential for M. tuberculosis

H37Rv-induced interleukin-6 production in human primary monocytes. Pretreat-

ment with N-acetyl-L-cysteine reduced, in a dose-dependent manner, M. tubercu-

losis H37Rv-induced activation of mitogen-activated protein kinase kinase 3/6

and interleukin-6 production in THP-1 cells.

Introduction

The outcome of a mycobacterial infection is determined by

an interplay between the innate and the acquired arms of the

immune response. Mycobacterium-infected macrophages or

monocytes secrete proinflammatory cytokines including

tumor necrosis factor-a (TNF-a), interleukin (IL)-1, IL-6,

and IL-12 as well as anti-inflammatory cytokines including

IL-4 and IL-10 (Orme & Cooper, 1999; Van Crevel et al.,

2002). These cytokines play critical roles in the recruitment

of monocytes and lymphocytes from the bloodstream to the

infected area, in the control of inflammatory response, in

subsequent granuloma formation and the outcome of

mycobacterial infections. Significant quantities of IL-6 are

produced by human and murine macrophages in response

to M. tuberculosis infection in vitro, and elevated concentra-

tions of IL-6 are also present in plasma of patients with

tuberculosis (TB) (Ogawa et al., 1991; el-Ahmady et al.,

1997). Nagabhushanam et al. (2003) have reported that IL-6

produced by M. tuberculosis-infected macrophages selectively

inhibited macrophage responses to interferon (IFN)-g.

This might ultimately lead to prolonged intracellular survi-

val of M. tuberculosis and establishment of a chronic

infectious state. Hence, IL-6 production by immune cells

during infection with M. tuberculosis is of interest.

Of late, many studies have targeted the signaling cascades

that are induced by mycobacterial strains in monocytes/

macrophages, namely, phosphatidylinositol (PI) 3-kinase,

protein kinase C and mitogen-activated protein kinase

(MAPK) cascades. MAPK activation in macrophages

appears to play an important role in the production of

various effector molecules (cytokines, chemokines and

reactive nitrogen intermediates) following a mycobacterial

infection (Schorey & Cooper, 2003; Koul et al., 2004).

MAPKs represent highly conserved serine-threonine kinases

that are activated by upstream MAPK kinases (MKK or

MAPKK or MEK) through a Th-XXX-Tyr phosphorylation

motif and are critical for cell proliferation, differentiation

and death, as well as for inflammatory responses. The

MAPKs comprise three distinct subfamilies with multiple

FEMS Immunol Med Microbiol 50 (2007) 309–318 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

subisoforms: p38 MAPK with a, b, g and d isoforms; c-jun

NH2 terminal kinases or stress-activated protein kinase,

with p46 and p54 as the main isoforms, and extracellular-

signal regulated kinase (ERK), which has p44 (ERK1) and

p42 (ERK2) isoforms. The c-jun NH2 terminal kinase and

p38 MAPK are primarily induced in response to cellular

stress, osmolarity, heat shock, UV irradiation and also to

inflammatory cytokines. ERK is mainly activated by growth

factors and phorbol esters (Cowan & Storey, 2003; Song

et al., 2003).

Using highly specific, cell-permeable inhibitors of MAPK

activity, several studies have reported the involvement of

the ERK and p38 MAPK pathways in cytokine release from

monocytes/macrophages that are incubated with mycobac-

teria or mycobacterial antigens (Chan et al., 2001; Reiling

et al., 2001; Ameixa & Friedland, 2002; Bhattacharyya et al.,

2002; Bluementhal et al., 2002; Roach & Schorey, 2002; Tse

et al., 2002; Song et al., 2003; Mendez-Samperio et al., 2004;

Jung et al., 2006). Song et al. (2003) demonstrated that both

ERK and p38 MAPKs were essential for M. tuberculosis

H37Rv-induced TNF-a production, whereas activation of

the p38 MAPK pathway alone was essential for M. tubercu-

losis H37Rv-induced IL-10 production. But information

regarding the involvement of MAPKs in IL-6 release from

monocytes/macrophages that are infected with mycobacter-

ia is scarce.

Among the MAPKs, p38 MAPK has been implicated in

regulating inflammatory cytokine biosynthesis and tran-

scription (Lee et al., 1994). One of the best studied activators

of p38 MAPK is the MKK 3/6, which lies directly upstream

of p38 MAPK. Among ERK, c-jun NH2 terminal kinase and

p38 MAPK, MKK3/6 activates only p38 MAPK (Yamaguchi

et al., 1995; Moruguchi et al., 1996). In cardiac myocytes, the

stimulation of p38 MAPK by MKK6 activates the transcrip-

tion factor nuclear factor (NF)-kB, to induce IL-6 gene

expression and release (Craig et al., 2000). Activation of

both NF-kB and p38 MAPK is also found in monocytes

following infection with M. tuberculosis (Ghosh, 1999;

Mendez-Samperio et al., 2001, 2004). But whether the

involvement of MKK3/6, p38 MAPK and NF-kB results in

the induction of IL-6 in M. tuberculosis H37Rv-infected

monocytes is not known.

Furthermore, NAC, an inhibitor of NF-kB in monocyte

cell lines (Tsuji et al., 1999), has been shown to inhibit IL-6

induction in M. tuberculosis H37Rv-infected human mono-

cyte-derived macrophages (Venkataraman et al., 2006).

Haddad (2001) have reported that inhibition of lipopoly-

saccharide-induced IL-6 formation in rat alveolar epithelial

cells by NAC is p38 MAPK dependent. But whether NAC

utilizes MAPK pathway to inhibit IL-6 production in

M. tuberculosis H37Rv-infected cells is not yet known. Given

this background, the focus of the current study was to

elucidate the role of MKK3/6, p38 MAPK and NF-kB in the

induction of IL-6, in M. tuberculosis H37Rv-infected THP-1

cell line and human primary monocytes, and the effect of

NAC upon this.

Materials and methods

Reagents

Antibodies against total and phosphorylated forms of

MKK3/6 and p38 MAPKs were purchased from Cell Signal-

ing Technology (Beverly, MA). Lipopolysaccharide derived

from Escherichia coli, serotype 055:B5, PD98059, SB203580

and Bay 11-7082 were purchased from Calbiochem Bios-

ciences (San Diego, CA). Horseradish peroxidase-linked

secondary antibodies, polyvinylidene difluoride membrane

and enhanced chemiluminescence kit (ECL) were from

Amersham Biosciences (Piscataway, NJ). Histopaque-1077,

NAC and dimethylsulfoxide (DMSO) were from Sigma

Chemicals (St Louis, MO). Middlebrook 7H9 medium was

from Difco laboratories (Sparks, MD). Endotoxin-free fetal

calf serum (FCS), RPMI 1640 (with glutamine and HEPES),

albumin–dextrose–catalase supplement, antibiotics and

phosphate-buffered saline (PBS) pH 7.2 were from Invitro-

gen Corporation (Carlsbad, CA). The IL-6 enzyme-linked

immunosorbent assay (ELISA) kit was from BD Biosciences

(San Jose, CA).

Cell culture

THP-1 cells (from National Center for Cell Sciences, Pune,

India) were maintained in endotoxin-free RPMI-1640 med-

ium containing 10% heat-inactivated fetal bovine serum

(FBS), 100 units mL�1 penicillin, 100 mg mL�1 streptomycin,

2 mM glutamine, and 20 mM sodium bicarbonate at 37 1C

in a humidified, 5% CO2 atmosphere. For experimental

purposes, cells were washed twice with RPMI-1640 medium

containing 10% FCS to remove the antibiotics, and sus-

pended in endotoxin-free RPMI-1640 medium containing

10% FCS.

Isolation and culture of human primarymonocytes

Whole blood was obtained from healthy volunteers. All

healthy controls gave their informed consent before being

enrolled, and the study was approved by the TB Research

Centre (TRC) – Institutional Ethics Committee review

board. They had no previous history of clinical TB, all were

negative for HIV and had received the Mycobacterium bovis

bacillus Calmette–Guerin (BCG) vaccinations as children.

Venous blood was drawn into sterile collection tubes, and

peripheral blood mononuclear cells were isolated by density

sedimentation over Histopaque-1077. Cells were incubated

for 1 h at 37 1C, and nonadherent cells were removed

FEMS Immunol Med Microbiol 50 (2007) 309–318c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

310 P. Natarajan & S. Narayanan

by pipetting off the supernatant. Adherent monocytes

recovered were 4 95% CD141 cells. The cells were then

incubated at 37 1C in a humidified, 5% CO2 atmosphere

until used in experiments. To show that the stimulatory

capacity of the H37Rv was not the result of contamination

with lipopolysaccharide, experiments were performed with

the addition of the specific lipopolysaccharide-inhibiting

oligopeptide polymyxin B (10.0mg mL�1) before mycobac-

terial stimulation.

Processing of H37Rv for infection

Mycobacterium tuberculosis H37Rv was grown in 7H9 med-

ium with albumin–dextrose complex and mid-log phase

cultures were used for infecting THP-1 human monocytes.

The bacterial suspension was washed and resuspended in

RPMI containing 10% FCS. Bacterial clumps were disaggre-

gated by vortexing five times (each cycle �2 min) with

3-mm sterile glass beads. The bacterial suspension was

passed through 27 gauge needle several times to disaggregate

any remaining clumps. The total number of bacilli per

milliliter of suspension was ascertained by counting in a

Thoma counting chamber.

Infection and preparation of cell lysates

THP-1 cells were seeded in 24-well tissue culture plates at a

density of 0.5� 106 cells per well. For serial kinetic studies of

phosphorylated forms of MAPKs, the cells were cultured

with medium alone (control), or infected with M. tubercu-

losis H37Rv at a multiplicity of infection (MOI) bacteria:

monocyte ratio of 10:1 (or 1 mg mL�1 lipopolysaccharide as

the positive control) for various periods. In inhibition

experiments, the culture wells were first pretreated with 1,

10, or 30 mM PD98059 (MEK1 inhibitor), or SB203580 (p38

MAPK inhibitor), or 1, 5, or 10 mM Bay 11-7082 (NF-kB

inhibitor) for 60 min before infection and, once infected, the

cultures were left for 4 h to allow phagocytosis of the bacilli.

After 4 h, the cells were washed thrice with PBS and then

resuspended in RPMI-1640 medium supplemented with

10% FCS (no inhibitors were added following the initial 4 h

infection period) (Tse et al., 2002). To serve as control, the

volume of the diluent DMSO (0.1% v/v) contained in 30 mM

PD98059 or SB203580 and in 10 mM Bay 11-7082 was added

to the cell culture. The viability of the infected monolayers

vs. an uninfected control was monitored by the trypan blue

dye exclusion method and found to be 4 98% in all of the

experiments described. After incubation with M. tuberculosis

H37Rv at various time points, 0.5 million cells were lysed

with 100 mL of 2� sample buffer [125 mM Tris (pH 6.8),

4% SDS, 20% glycerol, 100 mM DTT, and 0.05% bromo-

phenol blue], and denatured at 95 1C for 5 min. The lysates

were centrifuged at 1500 g for 15 min and the supernatants

were stored at � 80 1C for future use (Song et al., 2003).

Determination of MAP kinase phosphorylationthrough Western immunoblotting

An 80-mL sample was loaded onto a 12.5% sodium

dodecyl sulfate polyacrylamide gel electrophoresis (SDS-

PAGE) gel, and run at 100 V. The proteins were transferred

electrophoretically onto a polyvinylidene difluoride

membrane for 1 h 15 min at 95 V, at 4 1C by wet blot (Bio-

Rad), in transfer buffer comprising 25 mM Tris-HCl,

192 mM glycine, and 20% methanol, pH 8.3. The membrane

was washed in Tris buffered saline (TBS, pH 7.6) for 5 min.

Then the membrane was blocked with 5% nonfat dry

milk in TBS containing 0.1% Tween 20 (0.1% TBST)

for 1 h at room temperature. After three washes with

0.05% TBST, the membrane was incubated overnight at

4 1C with rabbit polyclonal antihuman phospho and non-

phospho MKK3/6, p38 Abs (1:1000) in 0.1% TBST

containing 5% bovine serum albumin (BSA), with gentle

shaking. After three washes with 0.05% TBST, the

membrane was incubated with donkey antirabbit polyclonal

Ab conjugated to horseradish peroxidase (1:300) in 0.1%

TBST containing 5% nonfat dry milk for 1 h at room

temperature. After three washes with 0.05% TBST, the blots

were developed using ECL. Blots were analyzed using GS 700

Imaging Densitometer (Bio-Rad Laboratories, Hercules,

CA).

NAC treatment of THP-1 cells and MAP kinaseactivation

We determined the effects of NAC on M. tuberculosis

H37Rv-induced activation of MKK3/6 and secretion of IL-6

in THP-1 cells. THP-1 cells were pretreated with different

concentrations of NAC (1, 10, 50 mM) for 2 h and washed

twice with RPMI containing 10% FCS to remove extracel-

lular NAC (Haddad, 2001). Then the cells were infected with

M. tuberculosis H37Rv at an MOI of 10:1 (or 1 mg mL�1

lipopolysaccharide as the control). At defined time points

(45 min for H37Rv and 30 min for lipopolysaccharide-

induced cultures) the cells were lysed as described before

and the lysates were stored at � 80 1C for analysis of

the phosphorylation status of MKK3/6. Certain wells were

incubated for 24 h and the culture supernatants were

collected after centrifugation at 1500 g for 15 min at 4 1C.

The resulting supernatants were stored at � 80 1C for

estimation of IL-6.

IL-6 measurement in culture supernatants

THP-1 cells were either left untreated or were treated with

vehicle (DMSO), PD98059, SB203580, Bay 11-7082 or NAC

followed by incubation with M. tuberculosis H37Rv or

lipopolysaccharide for 24 h. Alternatively, human mono-

cytes (at a concentration of 1 million mL�1) were either left


311NAC and MAPK in tuberculosis

untreated or were treated with vehicle (DMSO), 30mM

PD98059 or SB203580, or 5 mM Bay 11-7082 followed by

incubation with M. tuberculosis H37Rv for 24 h. The cell-free

supernatants were removed and assayed for IL-6 by ELISA

using the human BD OPTEIA IL-6 assay kit according to

the manufacturer’s protocol. The lower limit of detection

was 4.7 pg mL�1. The viability of the NAC-treated mono-

layers vs. an untreated control was monitored by the

Trypan blue dye exclusion method and found to be 4 97%

in all of the experiments described. Also the viability of the

infected human primary monocytes was not found to be

affected.

Statistical analysis and data presentation

The data from independent experiments are presented as

mean� SD. Statistical evaluation of the difference in mean

separation was performed by one-way ANOVA, followed by

post hoc Tukey’s test, and the a priori level of significance at

95% confidence level was considered at Po 0.05.

Fig. 1. p38 and MKK3/6 MAPKs are activated in Mycobacterium tuberculosis H37Rv-infected THP-1 human monocytes. THP-1 human monocytes were

left untreated (‘0’ min) or treated with Mycobacterium tuberculosis H37Rv (bacteria: host cell, 10:1) or lipopolysaccharide (1 mg mL�1) for various

lengths of time (indicated in minutes in a, b, c and d). Cells were then lysed and aliquots of total cell lysates were separated by 12.5% SDS-PAGE and

immunoblotted as described. Blots were incubated overnight with specific antiphospho-MKK3/6 (p-MKK3/MKK6) or antiphospho-p38 (p-p38), as well

as specific control Abs for the unphosphorylated form of the kinases, anti-MKK3 (MKK3) or anti-p38; followed by appropriate peroxidase-coupled

secondary antibodies and were visualized by ECL. The unphosphorylated forms were used to ensure that the total MKK3 or p38 protein was present in

equal amounts in all the lanes (a–d, bottom). The graphs to the right of a, b, c and d show the corresponding densitometric analyses of blots probed with

antiphospho-MAPK antibodies. Data shown are the mean� SD of three independent experiments performed in triplicate.



Results

Mycobacterium tuberculosis H37Rv infectionleads to the activation of MKK3/6 and p38MAPKs in THP-1 human monocytes

The activation of some MAPK family members by

M. tuberculosis H37Rv in human monocytes has already

been reported (Song et al., 2003). To study the phosphoryla-

tion profile of the MKK3/6, an upstream activator of p38

MAPK, we challenged THP-1 human monocytes with

M. tuberculosis H37Rv at an MOI of 10:1. Time-dependent

phosphorylation of both MKK3/6 and p38 MAPK was

observed. Mycobacterium tuberculosis H37Rv induced strong

phosphorylation of MKK3/6, 15 min postinfection, and the

level of phosphorylation decreased at 30 min. The peak

activation occurred again at 45 min and then it remained

�threefold higher than the basal level till 2 h (Fig. 1c).

A similar kinetics was observed for p38 MAPK except that

there was a slight decline at 60 min postinfection (Fig. 1d).

In contrast to M. tuberculosis H37Rv, the peak activation of

both MAPKs in the case of induction by lipopolysaccharide,

occurred at the 30-min time point and then declined close to

baseline at 2 h (Fig. 1a and b). Total MKK3 and p38 levels

remained consistent throughout the infections, indicating

that phosphorylation was specific to the external stimuli by

the mycobacteria (Fig. 1a–d, bottom).

p38 MAPK and NF-jB pathways are critical forM. tuberculosis H37Rv-induced IL-6 formationin THP-1 human monocytes

The p38 MAPK and NF-kB signaling pathways have been

implicated in mediating the expression of various cytokines

in cells stimulated with lipopolysaccharide (Ghosh, 1999;

Haddad, 2001). To further explore the essential role of p38

Fig. 2. Mycobacterium tuberculosis H37Rv sti-

mulates IL-6 secretion in THP-1 human mono-

cytes through p38 MAPK and NF-kB pathways.

THP-1 human monocytes were preincubated

with medium containing 0.1% DMSO as solvent

control, 1, 10, or 30mm PD98059 or SB203580,

or 1, 5, or 10 mm Bay 11-7082 for 60 min before

incubation with lipopolysaccharide – 1 mg mL�1

(a, b and c) or with Mycobacterium tuberculosis

H37Rv (d, e and f) at an MOI of 10:1 for 24 h.

Supernatants were harvested after 24 h and IL-6

formation was measured by ELISA. Data shown

are the mean� SD of three independent experi-

ments performed in triplicate; bars represent the

mean� SD. �Po 0.05.



MAPK and NF-kB in the secretion of IL-6 from THP-1

human monocytes by M. tuberculosis H37Rv, we assayed

IL-6 cytokine formation in the presence of highly specific

inhibitors of MEK1, p38 MAPK, NF-kB. The cells were

preincubated with PD98059 or SB203580 or Bay 11-7082

in increasing doses for 60 min before incubation with

M. tuberculosis H37Rv or lipopolysaccharide. IL-6 produc-

tion by THP-1 cells in response to M. tuberculosis H37Rv

was significantly reduced by SB203580 at all the three doses

used – the reduction was 13.9%, 39.7% and 82.8% for 1, 10

and 30 mM SB203580, respectively (Fig. 2e). In the case of

Bay 11-7082, the reduction was significant only with 5 and

10mM doses (35.4% and 60.7% inhibition; Fig. 2f). Even

though the MEK1 inhibitor PD98059, brought about a

slight increase with 1mM concentration, the 10 and 30 mM

doses did not have any effect on the IL-6 formation (Fig.

2d). Interestingly, lipopolysaccharide-induced IL-6 produc-

tion was significantly reduced by all the three inhibitors used

(Fig. 2a–c) – 1, 10 and 30 mM PD98059 inhibited 31.4%,

45.9% and 63.6%, respectively; 10 and 30 mM SB203580

caused inhibition of 36.2% and 74.8%, respectively; 1, 5 and

10mm Bay 11-7082 inhibited 22.3%, 55.6% and 68.2%,

respectively. The observed inhibition was not due to DMSO,

as DMSO alone did not exhibit any inhibitory effects at this

concentration (0.1%). These results show that p38 MAPK

and NF-kB are involved in the signaling of IL-6 production

during mycobacterial infection in THP-1 cells.

ERK1/2, p38 MAPK and NF-jB pathways arecritical for M . tuberculosis H37Rv-induced IL-6formation in human primary monocytes

ERK1/2 and p38 MAPKs have been shown to be pivotal in

mycobacterial 38-kDa protein-induced IL-6 formation in

human monocytes. To dissect the signaling mechanism

behind stimulation with whole bacilli, human primary

monocytes were preincubated with pathway inhibitors for

60 min before incubation with M. tuberculosis H37Rv for

24 h, and cell-free supernatants were assayed for IL-6

formation. IL-6 production was significantly reduced by all

the three inhibitors used (Fig. 3) – 30mM PD98059, 30 mM

SB203580 and 5 mM Bay 11-7082 inhibited 61.6%, 70.39%

and 90%, respectively. The observed inhibition was not due

to DMSO, as DMSO alone did not exhibit any inhibitory

effects at this concentration (0.1%). These results show that

ERK1/2, p38 MAPK and NF-kB are involved in the signaling

of IL-6 production during mycobacterial infection of

human primary monocytes.

The role of NAC in mediating M. tuberculosisH37Rv-induced activation of MKK3/6

From the serial kinetic studies of MKK3/6 in THP-1 cells

incubated with M. tuberculosis H37Rv or lipopolysacchar-

ide, we observed that the peak activation of MKK3/6 occurs

at 45 or 30 min, respectively (Fig. 1). We chose these time

points to determine the effect of NAC over the activation of

MKK3/6. Cells were pretreated with NAC for 2 h before

stimulation by M. tuberculosis H37Rv for 45 min or lipopo-

lysaccharide for 30 min. Western blot analysis of cell lysates

using specific Abs revealed that with NAC pretreatment the

amount of both M. tuberculosis H37Rv-induced (Fig. 4b)

and lipopolysaccharide-induced (Fig. 4a) phosphorylation

of MKK3/6 was reduced in a dose-dependent manner.

Scanning densitometric analyses of the blots probed with

antiphospho-MKK3/6 showed that only 10 and 50 mM

doses of NAC significantly reduced lipopolysaccharide-

induced phosphorylation, but M. tuberculosis H37Rv-

induced phosphorylation was significantly reduced in all

the three doses of NAC with maximum effect at 50 mM

(right panel of Fig. 4). The steady, phosphorylation-inde-

pendent state of MKK3/6 was mostly not affected by any of

the aforementioned treatments (Fig. 4a and b, bottom).

NAC down-regulates IL-6 formation in THP-1 cellsincubated with M. tuberculosis H37Rv orlipopolysaccharide

Our studies demonstrated that MKK3/6 and p38 MAPK

follow an almost identical phosphorylation profile upon

infection with M. tuberculosis H37Rv, and p38 MAPK was

involved in mediating M. tuberculosis H37Rv-induced for-

mation of IL-6 in THP-1 cells (Figs 1 and 2). As MKK3/6 is

known to activate p38 MAPK and our results also show that

M. tuberculosis H37Rv-induced phosphorylation of MKK3/

6 is reduced by NAC in THP-1 cells, we were interested in

Fig. 3. Effects of MEK or p38 MAPK or NF-kB inhibitors on Mycobacter-

ium tuberculosis H37Rv-mediated IL-6 secretion in human primary

monocytes. Thirty micromolar PD98059 or 30mM SB203580 or 5mM

Bay 11-7082 or 0.1% DMSO were added to monocytes for 60 min

before stimulation with Mycobacterium tuberculosis H37Rv. The super-

natants were harvested after 24 h for IL-6 cytokine assessment using

ELISA. Data shown are the mean� SD of nine independent experiments

performed in duplicate. �Po 0.05, as compared with Mycobacterium

tuberculosis H37Rv (MOI 10:1).



looking at the effect of NAC on M. tuberculosis H37Rv-

induced IL-6 formation in THP-1-cells. Cells were pre-

treated with NAC for 2 h before stimulation by M. tubercu-

losis H37Rv or lipopolysaccharide for 24 h and IL-6 was

estimated in the culture supernatants. It was found that

NAC significantly suppressed, in a dose-dependent manner,

both M. tuberculosis H37Rv (Fig. 5b) and lipopolysacchar-

ide-induced (Fig. 5a) IL-6 production in THP-1-cells. The

suppression was significant from 1 mM dose (10.5% inhibi-

tion) in the case of lipopolysaccharide, but only from

10 mM dose (34% inhibition) for M. tuberculosis H37Rv

infected cultures. In both cases the maximum suppression

happened with the 50 mM dose (90% – lipopolysaccharide;

80.5% – M. tuberculosis H37Rv).

Discussion

Although it has been demonstrated that MAPKs are

important in mediating the secretion of several effector

molecules in monocytes/macrophages infected with

mycobacteria, little is known about the role of MAPK

activation during the secretion of IL-6 cytokine by

Fig. 4. The attenuating effect of NAC on MKK3/6 phosphorylation. THP-1 human monocytes were cultured with medium alone (control), preincubated

in the presence of NAC (50 mm) for 2 h before exposure to medium alone for 15 min, exposed to lipopolysaccharide (1 mg mL�1) for 30 min or

Mycobacterium tuberculosis H37Rv (MOI 10:1) for 45 min, or preincubated in the presence of NAC (1–50 mm) for 2 h followed by exposure to

lipopolysaccharide (1 mg mL�1) for 30 min (a) or Mycobacterium tuberculosis H37Rv (MOI 10:1) for 45 min (b). Cells were then lysed, and aliquots of total

cell lysates were separated by SDS-PAGE and immunoblotted as described. Blots were incubated overnight with specific antiphospho-MKK3/6, as well

as specific control Abs for the unphosphorylated form of the kinase, anti-MKK3; followed by appropriate peroxidase-coupled secondary antibodies and

were visualized by ECL. The MKK3 Ab was used to ensure that the total MKK3 protein was present in equal amounts in all lanes (a–b, bottom). The

graphs to the right of a and b are the corresponding densitometric analyses of the blots probed with antiphospho-MKK3/6 antibodies (data from three

independent experiments performed in triplicate; bars represent the mean� SD). �Po 0.05, as compared with lipopolysaccharide (1 mg mL�1) or

Mycobacterium tuberculosis H37Rv (MOI 10:1).



monocytes after infection with M. tuberculosis H37Rv. In the

current study, we have demonstrated the following:

(1) Mycobacterium tuberculosis H37Rv induces phosphory-

lation of MKK3/6 in THP-1 cells,

(2) the phosphorylation profiles of MKK3/6 and p38

MAPKs are different in THP-1 human monocytes induced

with lipopolysaccharide and M. tuberculosis H37Rv,

(3) p38 MAPK and NF-kB are involved in mediating both

lipopolysaccharide and M. tuberculosis H37Rv-induced IL-6

formation in THP-1cells,

(4) ERK1/2, p38 MAPK and NF-kB are involved in mediat-

ing M. tuberculosis H37Rv-induced IL-6 formation in hu-

man primary monocytes,

(5) NAC, an antioxidant, reduced in a dose-dependent

manner the phosphorylation of MKK3/6, as well as IL-6

formation in both lipopolysaccharide and M. tuberculosis

H37Rv-induced THP-1 cells.

In recent years, THP-1 cells have been utilized extensively

as a faithful model in the study of infection, host cell signaling

and intracellular survival of mycobacteria (Carter et al., 1999;

Maiti et al., 2001; Riendeau & Kornfeld, 2003). Upon induc-

tion by lipopolysaccharide and M. tuberculosis H37Rv, THP-1

cells showed distinct, rapid activation of MKK3/6 and p38

MAPK in a time-dependent manner. This is the first report

that MAPKs, MKK3/6 and p38 are rapidly phosphorylated by

M. tuberculosis H37Rv in THP-1 cells. The activation induced

by the bacilli reduced drastically in both kinases at 30 min and

then increased to the peak level at 45-min time point. This

fall and rise of the p38 MAPK activation observed in our

study is consistent with the data of Song et al. (2003) on

infection of M. tuberculosis H37Rv in human peripheral

blood monocytes. Moreover, the activation of both MAPKs

by lipopolysaccharide slowly reduced to baseline at 2 h, but

the bacilli-induced activation continued to stay twofold

higher than the basal activation even at the 2-h time point.

This prolonged activation might help the bacilli to sustain

reasonable levels of IL-6 during infection.

Recently, it was shown that Bacillus Calmette–Guerin

induces TNF-a, IL-6, and IL-10 mRNA expression in a

NF-kB-dependent manner, and that M. tuberculosis H37Rv

also induces TNF-a production in human blood monocytes,

through activation of ERK1/2 and p38 MAPKs (Song et al.

2003; Cheung et al., 2005). While dissecting the signaling

mechanisms underlying IL-6 induction by lipopolysacch-

aride and of M. tuberculosis H37Rv in THP-1 cells using

specific cell permeable inhibitors, we observed that lipopo-

lysaccharide-induced IL-6 formation was sensitive to phar-

macological inhibition of ERK1/2, p38 MAPK and NF-kB,

but M. tuberculosis H37Rv-induced IL-6 formation was

sensitive only to inhibition of p38 MAPK and NF-kB.

However, the involvement of ERK1/2 pathway in bacilli-

induced IL-6 cannot be completely ruled out, unless a second

specific inhibitor is used and studies involving cross-talk

between the pathways are conducted in relation to IL-6

secretion. Inhibition by SB203580 led to the highest reduction

of IL-6 formation induced by both lipopolysaccharide and

M. tuberculosis H37Rv, followed by Bay 11-7082. This implies

that p38 MAPK plays a greater role in the regulation of IL-6

formation by both lipopolysaccharide and M. tuberculosis

H37Rv. These data reinforce the previous finding that activa-

tion of the IL-6 gene by M. tuberculosis LAM or lipopolysac-

charide is mediated by NF-kB (Zhang et al., 1994).

While this work was in progress, Jung et al. (2006)

demonstrated that both ERK1/2 and p38 MAPK are in-

volved in the induction of IL-6 by 38 kDa antigen of

M. tuberculosis in human monocytes derived from health

volunteers and TB patients. When we performed inhibition

experiments in human primary monocytes infected with

M. tuberculosis H37Rv, we confirmed the involvement of

ERK1/2, p38 MAPK and NF-kB pathways in IL-6 secretion.

The discrepancy seen in our data obtained with THP-1cells

could be due to the use of intact mycobacteria in our study

compared with the use of an individual antigen (Jung et al.,

2006). Another possibility could be the differences between

human primary monocytes and the THP-1 cell line, as

Fig. 5. The effect of NAC on lipopolysaccharide/

Mycobacterium tuberculosis H37Rv-induced IL-6

formation of THP-1 human monocytes. THP-1

human monocytes were preincubated with NAC

for 2 h followed by exposure to lipopolysacchar-

ide 1 mg mL�1 (a) or with Mycobacterium tuber-

culosis H37Rv (MOI 10:1) (b) for 24 h.

Supernatants were harvested after 24 h and IL-6

formation was measured by ELISA. �Po 0.05, as

compared with lipopolysaccharide (1 mg mL�1) or

Mycobacterium tuberculosis H37Rv (MOI 10:1).

Data shown are the mean� SD of three inde-

pendent experiments performed in triplicate;

bars represent the mean� SD.



differences in MAPK activation between macrophage cell

lines and primary macrophages have been noted previously

(Rao, 2001).

IL-6 has been shown to promote the growth of mycobac-

teria in peripheral blood monocytes. Newman et al. (1991)

reported that increased survival of Mycobacterium avium

intracellulare in isolated macrophages is correlated with the

efficiency with which TNF-a and IL-6 are produced in

response to M. avium intracellulare infection. A study by

Tse et al. (2002) showed that ERK and p38 MAPKs are

involved in controlling the growth of M. avium morpho-

types. Furthermore, NAC, an antioxidant has been proved to

reduce IL-6 production from M. tuberculosis H37Rv-

infected human monocyte-derived macrophages and also

to cause growth inhibition of M. tuberculosis H37Rv in

blood cultures (Venkataraman et al., 2006). Based on these

data, we hypothesized that NAC might act on MAPK to

bring about changes in IL-6 production in monocytes, and

our findings proved this to be true. NAC reduced, in a dose-

dependent manner, the phosphorylation of MKK3/6 and

the formation of IL-6 in both lipopolysaccharide and

M. tuberculosis H37Rv treated THP-1 cells. This is the first

ever report linking NAC, MKK3/6 and IL-6 formation in

M. tuberculosis H37Rv infection. Interestingly, as M. tuber-

culosis H37Rv-induced IL-6 was shown to inhibit macro-

phage responses to IFN-g, NAC can serve as a potential

therapeutic agent in improving macrophage responses in TB

(Nagabhushanam et al., 2003).

From all these findings, it is reasonable to speculate that

immediately after infection of human primary monocytes,

M. tuberculosis H37Rv phosphorylates ERK1/2 and p38

MAPKs; these in turn might interact with NF-kB and other

transcription factors, finally resulting in the induction of

IL-6 formation after 24 h (Carter et al., 1999). On the other

hand, NAC, in addition to inhibiting MKK3/6 phosphoryla-

tion, might also interact with p38 and NF-kB in the process

of reducing IL-6 formation in THP-1 cells, because NAC is

known to inhibit NF-kB activation and cytokine expression

in THP-1 cells (Tsuji et al., 1999; Haddad, 2001). Future

studies are therefore necessary to delineate the biochemical

cross-talk between the MAPK and NF-kB pathways involved

in the M. tuberculosis H37Rv-induced formation of IL-6 in

THP-1 cells. As several reports have pointed out that the

biological behavior of pathogenic mycobacteria is different

from that of the nonpathogenic strains, experiments with

different strains of mycobacteria will give a clear insight into

the regulation of IL-6 formation in monocytes and macro-

phages.

Acknowledgements

We thank Mrs Fathima Rehman for her help in statistical

analysis. Palaniappan Natarajan is grateful to Council of

Scientific and Industrial Research (CSIR), India, for a Senior

Research Fellowship.

References

Ameixa C & Friedland JS (2002) Interleukin-8 secretion from

Mycobacterium tuberculosis-infected monocytes is regulated by

protein tyrosine kinases but not by ERK1/2 or p38 mitogen-

activated protein kinases. Infect Immun 70: 4743–4746.

Bhattacharyya A, Pathak S, Kundu M & Basu J (2002) Mitogen-

activated protein kinases regulate Mycobacterium avium-

induced tumor necrosis factor-alpha release from

macrophages. FEMS Immunol Med Microbiol 34: 73–80.

Bluementhal A, Ehlers S, Ernst M, Flad HD & Reiling N (2002)

Control of mycobacterial replication in human macrophages:

roles of extra-cellular signal-regulated kinases and p38

mitogen-activated protein kinase pathways. Infect Immun 70:

4961–4967.

Carter BA, Knudtson LK, Monick MM & Hunninghake WG

(1999) The p38 mitogen-activated protein kinase is required

for NF-kB-dependent gene expression. The role of TATA-

binding protein (TBP). J Biol chem 274: 30858–30863.

Chan DE, Morris RK, Belisle TJ, Preston H, Remigio KL, Brennan

JP & Riches HWD (2001) Induction of inducible nitric oxide

synthase-NO � by lipoarabinomannan of Mycobacterium

tuberculosis is mediated by MEK-ERK, MKK7-JNK, and NF-

kB signaling pathways. Infect Immun 69: 2001–2010.

Cheung KWB, Lee CWD, Li CBJ, Lau YL & Lau SYA (2005) A role

for double-stranded RNA-activated protein kinase PKR in

Mycobacterium-induced cytokine expression. J Immunol 175:

7218–722.

Cowan JK & Storey BK (2003) Mitogen-activated protein kinases:

new signaling pathways functioning in cellular responses to

environmental stress. J Exp Biol 206: 1107–1115.

Craig R, Larkin A, Mingo AM, Thuerauf JD, Andrews C,

McDonough MP & Glembotski CC (2000) p38 MAPK and

NF-kB collaborate to induce interleukin-6 gene expression

and release. Evidence for a cytoprotective autocrine signaling

pathway in a cardiac myocyte model system. J Biol Chem 275:

23814–23824.

el-Ahmady O, Mansour M, Zoeir H & Mansour O (1997)

Elevated concentrations of interleukins and leukotriene in

response to Mycobacterium tuberculosis infection. Ann Clin

Biochem 34: 160–164.

Ghosh S (1999) Regulation of inducible gene expression by the

transcription factor NF-kB. Immunol Res 19: 183–189.

Haddad JJ (2001) Reduction-oxidation signaling mediating p38

MAPK-dependent regulation of pro-inflammatory cytokine

biosynthesis: on the mechanism of glutathione as a novel

immunoregulatory antioxidant thiol. Int Arch Biosci

1001–1013.

Jung SB, Yang CS, Lee JS et al. (2006) The mycobacterial 38-

kilodalton glycolipoprotein antigen activates the mitogen-

activated protein kinase pathway and release of



proinflammatory cytokines through Toll-like receptors 2 and 4

in human monocytes. Infect Immun 74: 2686–2696.

Koul A, Herget T, Klebl TB & Ullrich A (2004) Interplay between

mycobacteria and host signaling pathways. Nat Rev Microbiol

2: 189–202.

Lee JC, Laydon JT, McDonnell PC et al. (1994) A protein kinase

involved in the regulation of inflammatory cytokine

biosynthesis. Nature 372: 739–746.

Maiti D, Bhattacharyya A & Basu J (2001) Lipoarabinomannan

from Mycobacterium tuberculosis promotes macrophage

survival by phosphorylating Bad through a

phosphatidylinositol 3-kinase/Akt pathway. J Biol Chem 276:

329–333.

Mendez-Samperio P, Palma J & Vazquez A (2001) Roles of

intracellular calcium and NF-kB in the Bacillus

Calmette–Guerin-induced secretion of interleukin-8 from

human monocytes. Cell Immunol 211: 113–122.

Mendez-Samperio P, Ayala H, Trejo A & Ramirez FA (2004)

Differential induction of TNF-a and NOS2 by mitogen-

activated protein kinase signaling pathways during

Mycobacterium bovis infection. J Infect 48: 66–73.

Moruguchi T, Kuroyanagi N, Yamaguchi K et al. (1996) A novel

kinase cascade mediated by mitogen-activated protein kinase

kinase 6 and MKK3. J Biol Chem 271: 13675–13679.

Nagabhushanam V, Solache A, Ting L, Escaron CJ, Zhan JY &

Ernst JD (2003) Innate inhibition of adaptive immunity:

Mycobacterium tuberculosis-induced IL-6 inhibits macrophage

responses to IFN-g. J Immunol 171: 4750–4757.

Newman GW, Gan HX, McCarthy PL Jr. & Remold HG (1991)

Survival of human macrophages infected with Mycobacterium

avium intracellulare correlates with increased production of

tumor necrosis factor-alpha and IL-6. J Immunol 147:

3942–3948.

Ogawa T, Uchida H, Kusumoto Y, Mori Y, Yamamura Y &

Hamada S (1991) Increase in tumor necrosis factor alpha- and

interleukin-6-secreting cells in peripheral blood mononuclear

cells from subjects infected with Mycobacterium tuberculosis.

Infect Immun 59: 3021–3025.

Orme IM & Cooper AM (1999) Cytokine/chemokine cascades in

immunity to tuberculosis. Immunol Today 20: 307–312.

Rao KM (2001) MAP kinase activation in macrophages. J Leukoc

Biol 69: 3–10.

Reiling M, Bluementhal A, Flad HD, Ernst M & Ehlers S (2001)

Mycobacteria-induced TNF-alpha and IL-10 formation by

human macrophages is differentially regulated at the level of

mitogen-activated protein kinase activity. J Immunol 167:

3339–3345.

Riendeau JC & Kornfeld H (2003) THP-1 cell apoptosis in

response to mycobacterial infection. Infect Immun 71:

254–259.

Roach K & Schorey JS (2002) Differential regulation of

the mitogen-activated protein kinases by pathogenic

and nonpathogenic mycobacteria. Infect Immun 70:

3040–3052.

Schorey JS & Cooper AM (2003) Macrophage signaling upon

mycobacterial infection: the MAP kinases lead the way. Cell

Microbiol 5: 133–142.

Song C-H, Lee J-S, Lim K, Kim H-J, Park J-K, Paik T-H & Jo E-K

(2003) Role of mitogen-activated protein kinase pathways in

the production of tumour necrosis factor-a, IL-10, and

monocyte chemotactic protein-1 by Mycobacterium

tuberculosis H37Rv-infected human monocytes. J Clin

Immunol 23: 194–201.

Tse HM, Josephy SL, Chan ED, Fouts D & Cooper AM (2002)

Activation of the mitogen-activated protein kinases signaling

pathway is instrumental in determining the ability of

Mycobacterium avium to grow in murine macrophages.

J Immunol 168: 825–833.

Tsuji F, Miyake Y, Aono H, Kawashima Y & Mita S (1999) Effects

of bucillamine and N-acetyl-L-cysteine on cytokine

production and collagen-induced arthritis. Clin Exp Immunol

115: 26–31.

Van Crevel R, Ottenhoff TH & Van der Meer JW (2002) Innate

immunity to Mycobacterium tuberculosis. Clin Microbio Rev 15:

294–309.

Venkataraman V, Rodgers T, Linares R, Reilly N, Swaminathan S,

Hom D, Millman A, Wallis R & Connell N (2006) Tuberculosis

immunity in healthy and HIV infected subjects. AIDS Res Ther

3: 5–16.

Yamaguchi K, Shirakabe K, Shibuya H, Irie K, Oishi I, Ueno N,

Taniguchi T, Nishida E & Matsumoto K (1995) Identification

of a member of the MAPKKK family as a potential

mediator of TGF-beta signal transduction. Science 270:

2008–2011.

Zhang Y, Broser M & Rom WN (1994) Activation of the

interleukin 6 gene by Mycobacterium tuberculosis or

lipopolysaccharide is mediated by nuclear factors NF-IL6 and

NF-kB. Proc Natl Acad Sci USA 15: 2225–2222.



Documents

Mycobacterium tuberculosis H37Rv induces monocytic release of interleukin-6 via activation of mitogen-activated protein kinases: inhibition by N -acetyl-L-cysteine