Tuberculosis 94 (2014) 123e130

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Tuberculosis
journal homepage: http://intl.elsevierhealth.com/journals/tube

IMMUNOLOGICAL ASPECTS

Pleural fluid mononuclear cells (PFMCs) from tuberculous pleurisy

can migrate in vitro in response to CXCL10
Changyou Wu a, *, Jiangjun Ma a, Yanquan Xu a, Xianlan Zhang b, Suihua Lao b, Binyan Yang a
a
Institute of Immunology, Zhongshan School of Medicine, Key Laboratory of Tropical Disease Control Research of Ministry of Education,
Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou 510080, PR China
b
Chest Hospital of Guangzhou, Guangzhou 510095, PR China

a r t i c l e i n f o s u m m a r y

Article history: We recently reported that pleural fluid mononuclear cells (PFMCs) from tuberculous pleurisy stimulated
Received 1 April 2013 with Bacillus Calmette-Guérin (BCG) or TB antigens produced high levels of cytokines. However, it was
Received in revised form still unclear what mechanism of the PFMCs used to migrate into the pleural fluids in TB infection. In the
21 October 2013
present study, we found that CD3þCD4þ and CD3þCD8þ T cells from PFMCs expressed significantly high
Accepted 27 October 2013
levels of CXCR3 compared to PBMCs. In addition, the levels of CXCL10 (the ligand for CXCR3) in pleural
fluids were significantly higher than those in normal serum and cancerous fluids. After stimulation with
Keywords:
BCG, PFMCs produced high levels of CXCL10. Importantly, the synthesis of CXCL10 was mainly dependent
TB
Migration
on the BCG-induced production of IFNs, because the neutralization of endogenous IFN-a or IFN-g with
Immune response mAbs significantly reduced the production of CXCL10 from BCG-stimulated PFMCs. In addition, the
TB antigen response tubercular pleural fluid (TBPF) or exogenous CXCL10 induced the migration of PFMCs, indicating that
IFN-a or IFN-g modulated the immune response through the expression of CXCL10 to aid the recruitment
and selective homing of activated/effector cells to the site of Mycobacterium tuberculosis (M.tb) infection.
Taken together, the levels of CXCL10 in pleural fluids were high and BCG-stimulated PFMCs expressed
high levels of CXCL10, and CXCL10 induced the migration of PFMCs into the pleural fluids in TB infection.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction divided into four subfamilies according to the N-terminal

Tuberculosis (TB) is a leading cause of morbidity and mor-
tality in the world with 9 million new cases and 2 million deaths
annually [1]. In addition, one-third of all people on the planet are
thought to harbor latent TB, defined as M.tb infection in the
absence of any clinical symptoms. Individuals with latent TB are
not infectious, but they have an about 10% lifetime risk of pro-
gression to active, infectious and potentially lethal disease [2].
Both innate and adaptive immune responses are necessary to
control M.tb, but they are not sufficient to sterilizing immunity.
Mycobacterium bovis bacillus Calmette-Guérin (BCG) is currently
administered as the only available vaccine for the prevention of
tuberculosis in humans [3].
Infection of human cells with M.tb induces the release of
various chemoattractants. The human chemokine system com-
prises more than 50 chemokines and 18 chemokine receptors [4].
Chemokines are identified chemotactic cytokines and can be
* Corresponding author. Tel./fax: þ86 20 87332448.
E-mail address: changyou_wu@yahoo.com (C. Wu).
cysteine positioning in their amino acid sequence: C, CC, CXC and
CX3C or into two categories on the basis of their physiological
features (inducible and constitutive) [4]. Chemokine receptor
expression varies among different leukocyte populations;
therefore, different leukocyte types can be specifically recruited
to the site of inflammation in response to local chemokine milieu
[5]. IFN-g-induced protein 10 (CXCL-10 or CXCL10), which has a
molecular mass of 10 kDa, is a member of the CXC superfamily
[6]. CXCL10 is expressed in predominance in Th1-mediated
pathological inflammatory processes [7e9]. Blockage of
CXCL10 inhibits the recruitment of effector T cells to sites of
inflammation following injury [10,11]. On the other hand, CXCL10
has been shown to inhibit angiogenesis and to have antitumor
properties [12,13].
IFNs are originally identified on the basis of their ability to
induce cellular resistance to viral infections [13]. In most cases, IFNs
have been divided into two classes (types I and II) on the basis of
their structure, function, and cellular origin. Although type I IFNs
(IFN-a/b) can be secreted by virtually all virus-infected cells, type II
IFN (IFN-g) is mainly produced as a result of stimulation of
T "bodlymphocytes and NK cells [14]. Although the protective effect

1472-9792/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tube.2013.10.008
124 C. Wu et al. / Tuberculosis 94 (2014) 123e130
of IFN-g against M.tb has been established, the effects of type I IFNs
are still unclear. Recent studies have highlighted the production of
type I IFNs following bacterial infections [15,16]. It has also been
demonstrated that interferon may induce the expression of certain
chemokine genes, such as those of CXCL9, CXCL10 and CXCL11
[17,18]. However, it was still unclear what mechanism the T cells
used to migrate into the pleural fluids in TB infection. We found that
CD3þCD4þ and CD3þCD8þ T cells from PFMCs expressed signifi-
cantly high levels of CXCR3 compared to PBMCs, and CXCL10
induced the migration of PFMCs migrated into the pleural fluids in
TB infection.
2. Materials and methods
2.1. Subjects and samples
63 patients with tuberculous pleurisy, including 32 men and 31
women aged 19e78 years (mean ¼ 29.26 years) and 12 patients
with lung cancer, consisting of 7 men and 5 women aged 18e82
years (mean ¼ 37.96 years). All participants were enrolled at the
Chest Hospital of Guangzhou, Guangzhou, China. The patients were
diagnosed as pleural fluid (PF) by clinical symptoms, physical
examination and radiographical evidence. PF was collected from
each patient after obtaining written informed consent before the
start of anti-tuberculosis treatment. All patients were seronegative
for HIV, hepatitis B virus (HBV) and hepatitis C virus (HCV) without
a history of autoimmune diseases (AD). Healthy donors were
recruited from blood center in Guangzhou, China and written
informed consent was obtained from all of the healthy donors. This
investigation was approved by the Medical School Review Board at
Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou,
China.
2.2. Antigens and antibodies
Bacillus Calmette-Guérin (BCG) was purchased from Chengdu
Institute of Biological Products, Chengdu, China. Mouse monoclonal
antibody against human IFN-a (all of IFN-a subtypes) was pur-
chased from PBL InterferonSource, and neutralizing antibodies
against IFN-g, isotype control antibody, IgG1 kappa were purchased
from BD Biosciences (BD PharMingen). Purified rhCXCL10, anti-
human CXCL10 and rhIFN-a were purchased from Peprotech
(Rocky Hill, NJ, USA). The following antibodies from BD Biosciences
were used for flow cytometry: CD3-PECF594, CD4-APC, CD8-FITC,
CD14-PEcy7, CD16b-FITC, CXCR3-APC, CCR4-PEcy7, CCR6-APC,
CCR7-PE and CXCL10-PE.
2.3. Mononuclear cell preparation
Peripheral blood mononuclear cells (PBMCs) and pleural fluid
mononuclear cells (PFMCs) were isolated from blood and pleural
fluid by Ficoll-Hypaque (Tianjin Hao Yang Biological Manufac-
ture, Tianjin, China) density gradient centrifugation at 400  g
for 20 min. The cells were then washed with Hanks’ balanced salt
solution (Sigma Chemical Co, St. Louis, Mo) at 400  g for 8 min.
Monocytes (CD14þcells) were positively selected with CD14
microbeads (Miltenyi) with a mean purity of 95% determined by
FACS. PFMC, PFMC and CD14þ cells were finally suspended at a
concentration of 1  106 cells/ml in complete RPMI 1640 medium
(Gibco, Grand Island, NY, USA) supplemented with 10% heat-
inactivated fetal calf serum (FCS; Sijiqing, China), 100 U/ml
penicillin, 100 mg/ml streptomycin, 50 mM 2-mercaptoethanol
and 2 mM L-glutamine (Gibco). The PF supernatants from
tuberculous pleurisy (TP) were cryopreserved at 80  C until the
assay.
2.4. Isolation of neutrophil granulocytes
Granulocytes were isolated from heparinized blood collected
from healthy adult volunteers. After centrifugation for 30 min at
400  g, the upper plasma layer and the interphase were removed.
The neutrophils (thin white cell layer above the red blood cell
pellet) were collected with a pasteur pipette into a conical tube. The
remaining red blood cells were lysed with excess volume of red
blood cell lysis solution for 10 min at room temperature. The
neutrophils were centrifuged at 300  g for 10 min at 20  C. The
cells at 1  10 6 cells/ml in RPMI 1640 were cultured.
2.5. Flow cytometry analysis
For cell surface staining, PFMCs and PBMCs were washed with
PBS buffer containing 0.1% BSA and 0.05% sodium azide and incu-
bated with anti-CD3, anti-CD4, anti-CD8, anti-CXCR3, anti-CCR4,
anti-CCR6 and anti-CCR7 for 30 min at 4  C. The cells were there-
after washed twice and resuspended in PBS buffer. For the detection
of intracellular cytokines, cells were incubated with IFN-g for 12 h in
the presence of brefeldin A (10 mg/ml; SigmaeAldrich, St Louis, MO).
After stimulation, cells were washed twice with PBS buffer con-
taining 0.1% BSA and 0.05% sodium azide. Thereafter, cells were
incubated with anti-CD3, anti-CD14 and anti-CD16b for surface
markers at 4  C in the dark for 30 min. The cells were then washed
twice and fixed in 4% paraformaldehyde, followed by permeabiliza-
tion and stained for anti-CXCL10 for 30 min at 4  C. Flow cytometry
was performed using BD FACSCalibur (Becton Dickinson, San Jose,
CA, USA) or FACSAria II (Becton Dickinson, San Jose, CA, USA) and the
data were analyzed using FlowJo software (TreeStar, San Carlos, CA).
2.6. ELISA
Cell-free culture supernatants were harvested and quantified by
enzyme-linked immunosorbent assay (ELISA) kits for CXCL10 (BD
PharMingen), IFN-g (BD PharMingen) and IFN-a (Mabtech). The
sensitivities of ELISA kits were 7.8 pg/ml for CXCL10, 3.9 pg/ml for
IFN-g and 4.0 pg/ml for IFN-a. All ELISA assays were performed ac-
cording to the manufacturer’s instructions. The ability of cells to
secrete CXCL10 in response to cytokines was determined using a
sandwich ELISA (BD PharMingen). A flat-bottom 96-well microtiter
plate (Greiner) was coated with 100 ml/well of anti-human CXCL10
mAb (2 mg/ml in the mixture of sodium carbonate and sodium bi-
carbonate, pH 9.5) over night at 4  C. The plate was subsequently
washed with PBST, pH 7.0, and 0.05% Tween-20, and blocked with
10% fetal calf serum (Hangzhou sijiqing company). CXCL10 standards
(rHuCXCL10) were made in a solution consisting of phosphate-
buffered saline (PBS), pH 7.0, and 10% fetal calf serum (FCS) using
serial dilutions. Standards or supernatants (100 ml/well) were plated
in triplicate and incubated at ambient temperature for 2 h. After three
washes,100 ml/well of biotinylated anti-human CXCL10 mAb (100 ng/
ml in PBS, pH 7.0, 10% FCS) was added and followed by 100 ml/well of
streptavidineperoxidase conjugate. The chromogen substrate was
used at 100 ml/well and after 30 min,10% H2SO4 was added to stop the
reaction. Plates were read at 450 nm in an automated microplate
reader (Bio-Tek Instruments, Inc., Richmond, CA). IFN-g and IFN-a
were also determined by ELISA using a specific ELISA kit.
2.7. PCR
Cells were stimulated with BCG (10 mg/ml). After stimulation,
total RNA was isolated using RNAeasy mini kit (Qiagen, Valencia,
CA) and residual DNA was removed using RNAse-free DNAse
(Qiagen). Reverse transcription of total RNA to cDNA was performed
at 37  C using Reaction Ready-First Strand cDNA Synthesis kit
 C. Wu et al. / Tuberculosis 94 (2014) 123e130
(Promega). Gene expression was measured using a SYBR Green-
based real-time quantitative PCR in which GAPDH was used as
the housekeeping gene. Amplification of cDNA was conducted in a
real-time PCR system (Applied biosystems step one plus) at the
following conditions: 95  C, 5 min, 95  C, 45s, 65  C, 45 s, for 20e35
cycles. The oligonucleotide primers used were: GAPDH: F-GCA TGG
CCT TCC GTG TCC and R-TGA GTG TGG CAG GGA CTC; IFN-a (all of
IFN-a subtypes): F-TGG CCT TGA CCT TTG CTT TAC and R-TCC TCA
TCT GTG CCA GGA G.
2.8. Chemotaxis Assay
Cell migration was evaluated by using a 24-well millicell
chamber technique with polyethylene terephthalate filters (5-mm
pore size; Millipore, USA). PFMCs migration was quantitated by a
modification of the Boyden chamber technique. Briefly, PFMCs
were suspended at 2  106 cells/ml in migration medium (con-
taining RPMI-1640 plus 1% BSA, 100 U/ml penicillin and 100 mg/ml
streptomycin), and rested at 37  C for 2 h to equilibrate in migration
medium before being subjected to a Chemotaxis Assay. After
incubation period, a total of 200 ml containing 2  105 cells were
added to the upper chamber of 5-mm pore size trans-well inserts.
The lower chamber of each well contained 1.2 ml of migration
medium alone or in the presence of TBPF, CXCL10 (Peprotech) or
anti-human CXCL10 (Peprotech). The chambers were incubated for
2 h, 4 h and 6 h at 37  C in 5% CO2. The numbers of cells migrating to
the lower chamber were counted by microscopy. The assay was
performed in triplicate for each condition.
2.9. Statistical analysis
One-way analysis of variance with the NewmaneeKeuls
multiple-comparison test was used to compare the differences
between multiple groups (GraphPad Prism software; version 5.0).
Wilcoxon matched-pairs test (two-tailed) was used to determine
Figure 1. The expression of chemokine receptors on T cells from PFMCs and PBMCs. The expression of CXCR3, CCR4, CCR6 and CCR7 on CD3þCD4þ and CD3þCD8þ T cells from
PFMCs (A, n ¼ 3) and PBMCs (B, n ¼ 3) were analyzed by flow cytometry. Dark histogram: control; white histogram: cell surface staining. (C). Percentage of chemokine receptors on
CD3þ, CD4þ and CD8þ T cells from PFMCs and PBMCs. Data are expressed as mean  SD, whereas error bars represent of three independent experiments with the similar results.
*P < 0.05, * *P < 0.01, ns: not significant.
125
statistical differences between two groups. Differences were
considered significant at P < 0.05.
3. Results
1. The expression of chemokine receptors on CD3þCD4þ and
CD3þCD8þ T cells from PFMCs and PBMCs
In the first set of experiments, we examined the expression of
chemokine receptor on PFMCs and PBMCs. As indicated in
Figure 1(A)e(C), CXCR3, CCR4 and CCR6 were expressed higher on
CD3þCD4þ T cells from PFMCs than PBMCs. CXCR3 was also
significantly higher on CD3þCD8þ T cells from PFMCs than PBMCs
(*P < 0.05, * *P < 0.01). In contrast, there was no significant dif-
ference in the expression of CCR7 on CD3þCD4þ and CD3þCD8þ
T cells between PFMCs and PBMCs.
2. The levels of CXCL10 in tubercular pleural fluids and the produc-
tion of CXCL10 in PFMCs and PBMCs after stimulation with BCG
To determine whether chemokines were present at the local site
of M.tb infection, we detected the levels of CXCL10 in pleural fluid by
ELISA based on the expression of chemokine receptors. Statistical
results in Figure 2(A) showed that the levels of CXCL10 were
significantly higher in tubercular pleural fluids than cancerous and
normal sera (*P < 0.05, * *P < 0.01). To address whether BCG can
induce CXCL10 production directly, PFMCs and PBMCs were stim-
ulated with BCG for 12 h and the culture supernatants were
collected and used for determination of CXCL10 by ELISA
(Figure 2(B)). The results showed that BCG significantly induced the
production of CXCL10 by PFMCs and PBMCs (*P < 0.05, * *P < 0.01).
3. The correlation between the levels of CXCL10 and IFN-g in the
TBPF, and IFN-g induced the production of CXCL10 by PFMCs
and PBMCs
126 C. Wu et al. / Tuberculosis 94 (2014) 123e130
We first determined the levels of CXCL10 and IFN-g in the TBPF
by ELISA. The results showed that the levels of CXCL10 were posi-
tively correlated with the levels of IFN-g (R [2] ¼ 0.7013, P＜0.0001)
(Figure 3(A)). Because IFN-g has been shown to be able to induce
the expression of certain chemokine genes, we next sought to
determine whether exogenous IFN-g induced CXCL10 secretion in
PFMCs and PBMCs. PFMCs and PBMCs were cultured and stimu-
lated with different concentrations of IFN-g and cell culture su-
pernatants were collected at different time points to determine the
concentration of CXCL10 by ELISA. As shown in Figure 3(B) and (C),
IFN-g induced the expression of CXCL10 by PFMCs and PBMCs in
dose- and time-dependent manner (* *P < 0.01).
4. The expression of CXCL10 on CD3þ, CD14þ and CD16bþ cells
from PFMCs and PBMCs, and exogenous IFN-g induced CXCL10
production by monocytes
To investigate possible sources of CXCL10, CD3þ, CD14þ and
CD16bþ cells from PFMCs and PBMCs were cultured and stimulated
with IFN-g and examined for the production of CXCL10. The results
showed that PFMCs but not PBMCs spontaneously expressed
CXCL10 detected by FACS (Figure 4(A)). As indicated in Figure 4(A),
CXCL10 was higher on CD14þ and CD16bþ cells in PFMCs than
Figure 2. The levels of CXCL10 in PFMCs fluids (TBPF) and the production of CXCL10 in
PFMCs and PBMCs after stimulation with BCG. (A). High levels of CXCL10 protein in
tubercular pleural fluid. Scatter-plot showing CXCL10 levels in patients with tubercu-
lous pleurisy (n ¼ 63), cancerous pleurisy (n ¼ 12) and normal serum (n ¼ 15). Each
symbol represents the value for an individual person and horizontal bars represent the
medians. (B). BCG induced the production of CXCL10 by PFMCs and PBMCs. Levels of
CXCL10 protein in BCG-stimulated PFMCs (n ¼ 13) and PBMCs (n ¼ 13) were measured
by ELISA. *P < 0.05, * *P < 0.01, ns: not significant.
PBMCs, and the expression of CXCL10 was higher on CD14þ than
CD16bþ cells. In contrast, the expression of CXCL10 had no different
on CD3þ T cells by PFMCs and PBMCs.
To investigate the effect of IFN-g on the production of CXCL10,
monocytes or neutrophils were incubated with exogenous IFN-g
and the production of CXCL10 was detected with ELISA. The results
showed that IFN-g significantly enhanced the production of CXCL10
in dose- and time-dependent manner by monocytes and neutro-
phils (Figure 4(B and C)) (* *P < 0.01).
5. Effect of IFN-g on CXCL10 production in BCG-stimulated PFMCs
and PBMCs
To further evaluate the effect of endogenous IFN-g on BCG-
induced CXCL10 production, a neutralizing antibody to IFN-g
(5 mg/ml) or an isotype-matched control antibody (5 mg/ml) was
added to the cultures of BCG-stimulated PFMCs and PBMCs for 12 h
Figure 3. The levels of CXCL10 and IFN-g in the tubercular pleural fluid and IFN-g
induced the production of CXCL10 by PFMCs and PBMCs. A. The correlation between
CXCL10 and IFN-g in the TBPF (n ¼ 36). Statistical significance and correlations were
determined with the spearman test (R2 ¼ 0.7013). P < 0.0001. B and C. IFN-g induced
the production of CXCL10 by PFMCs and PBMCs. Cell culture supernatants were
collected at different time points after stimulation and analyzed for CXCL10 secretion
by ELISA. Dose- and time-dependent expression of CXCL10 by PFMCs (B, n ¼ 5) and
PBMCs (C, n ¼ 5) stimulated with IFN-g. * *P < 0.01.
 C. Wu et al. / Tuberculosis 94 (2014) 123e130
at 37  C. As indicated in Figure 5(A and B), neutralization of IFN-g in
the cell cultures significantly inhibited the production of CXCL10
secretion (*P < 0.05). In contrast, an isotype-matched control
antibody had no effect.
Figure 4. The expression of CXCL10 on CD3þ, CD14þ, CD16bþ cells from PFMCs and
PBMCs and the production of CXCL10 in monocytes and neutrophils after stimulation
with IFN-g. A. The cells were cultured in medium alone or stimulated with IFN-g
(10 ng/ml) for 12 h. After stimulation, cells were harvested and FACS staining was
conducted (A, n ¼ 5) Dark histogram: control, white histogram: intracellular cytokine
staining. B and C. IFN-g induced the production of CXCL10 by monocytes (B, n ¼ 3) and
neutrophils (C, n ¼ 5). Cell culture supernatants were collected at different time points
after stimulation and analyzed for CXCL10 secretion by ELISA. Dose- and time-
dependent expression of CXCL10 by monocytes and neutrophils stimulated with
IFN-g. * *P < 0.01.
127
6. IFN-a induced the production of CXCL10
Stimulation of PFMCs and PBMCs with BCG leads to the pro-
duction of different cytokines including IFN-a. However, we found
that freshly isolated PFMCs but not PBMCs spontaneously
expressed IFN-a detected by RT-PCR (Figure 6(A)), indicating that
PFMCs were activated in vivo. It is important to note that IFN-a
mRNA was significantly expressed at 8 h after stimulation of PFMCs
and PBMCs with BCG (* *P < 0.01). IFN-a mRNA was higher in
PFMCs than PBMCs after stimulation with BCG. To further assess the
expression of IFN-a in different cell subsets, we examined the
expression of IFN-a on neutrophils, monocytes and non-monocytes
from PBMCs (Figure 6(B)). Notably, IFN-a production was found to
be largely confined to the neutrophils and monocytes (* *P < 0.01),
whereas non-monocytes expressed low levels of IFN-a. Moreover,
the levels of IFN-a in the supernatants of cultures of PFMCs that had
been stimulated with BCG were measured. Briefly, PFMCs were
resuspended at 2  106 cells/ml in medium (containing RPMI-1640
plus 1% BSA, 100 U/ml penicillin and 100 mg/ml streptomycin).
When BCG at different concentrations (1.25e20 mg/ml) was added
to the cultures of PFMCs (a total of 200 ml containing 4  105 cells/
well) and incubated for 12 h, 24 h, and 48 h, the levels of IFN-a
protein at 24 h were significantly increased and rapid declines at
48 h (Figure 6(C)).
Since BCG was able to induce the expression of IFN-a, we
analyzed whether IFN-a induced the production of CXCL10 in
PFMCs, PBMCs, neutrophils and monocytes. When IFN-a was added
to the cultures of PFMCs PBMCs, neutrophils or monocytes at
different concentrations (0.1e10 ng/ml) and incubated for 12 h,
24 h, 48 h and 72 h, the levels of CXCL10 protein were significantly
increased in a dose- and time-dependent manner (Figure 6(D)e(G))
* P < 0.05, * *P < 0.01.
Figure 5. Anti-IFN-g antibody inhibited the production of CXCL10 by PFMCs and
PBMCs. Levels of CXCL10 protein in BCG-stimulated PFMCs (A, n ¼ 3) and PBMCs (B,
n ¼ 3) with or without neutralizing anti-IFN-g antibody or an isotype-matched control
antibody for 12 h. CXCL10 in the culture supernatant was measured by ELISA. *P < 0.05,
ns: not significant.
128 C. Wu et al. / Tuberculosis 94 (2014) 123e130

Figure 6. Production of IFN-a and IFN-a induced the production of CXCL10. A. BCG induced the production of IFN-a by PFMCs and PBMCs. PFMCs and PBMCs were incubated with
BCG for 8 h and the levels of for IFN-a were measured by StepOnePlus Real Time PCR system and used SYBR Green qPCR Mastermix. GAPDH served as an internal control. The similar
results were in an additional three experiments. * *P < 0.01. B. BCG induced the expression of IFN-a by neutrophils, monocytes and non-monocytes. Neutrophils, monocytes and
non-monocytes were treated with BCG and the levels of IFN-a mRNA were measured by StepOnePlus Real Time PCR system. The similar results were in an additional three ex-
periments. * *P < 0.01. C. The levels of IFN-a in the supernatants of cultures of PFMCs that had been stimulated with BCG were measured (n ¼ 5). BCG was added to the culture
medium of PFMCs at different concentrations (1.25e20 mg/ml) and cultured for 12 h, 24 h, and 48 h. IFN-a induced the production of CXCL10 by PFMCs (D, n ¼ 5), PBMCs (E, n ¼ 5),
monocytes (F, n ¼ 5) and neutrophils (G, n ¼ 5). Dose- and time-dependent expression of CXCL10 by PFMCs, PBMCs, monocytes and neutrophils stimulated with IFN-a. Cell culture
supernatants were collected at different time points after stimulation and analyzed for CXCL10 secretion by ELISA. Each symbol represents the value for an individual person and
horizontal bars represent the medians. *P < 0.05, * *P < 0.01. Anti-IFN-a antibody inhibited the production of CXCL10 by PFMCs (H, n ¼ 3) and PBMCs (I, n ¼ 6). *P < 0.05, * *P < 0.01,
ns: not significant.

To further understand the effect of IFN-a on BCG-induced
CXCL10 production, a neutralizing antibody to IFN-a (5 mg/ml) or
an isotype-matched control antibody (5 mg/ml) was added to BCG-
stimulated PFMCs and PBMCs for 12 h at 37  C. As indicated in
Figure 6(H) and (I), the production of CXCL10 induced by BCG was
significantly neutralized with 5 mg/ml of anti-IFN-a antibody
(*P < 0.05) but not with an isotype-matched control antibody.
7. PFMCs show enhanced migration in response to CXCL10
To characterize the biological properties of the CXCL10, PFMCs
were assessed for its migration properties. Based on the expression
of CXCR3 on T cells from PFMCs, the chemotactic properties of
CXCL10 on PFMCs were analyzed in a trans-well migration assay. As
demonstrated in Figure 7(A), CXCL10 significantly induced migra-
tion of PFMCs at a different time points with the maximum
migration observed at 6 h. Moreover, we found that neutralization
of CXCL10 in the cultures significantly decreased the number of
migrating cells. In contrast, an isotype-matched control antibody
had no effect (Figure 7(B)) (* *P < 0.01).
4. Discussion
vIn this study, we showed that PFMCs responded to BCG stim-
ulation to produce different inflammatory chemokines which
switched their migratory capacity through the expression of
various chemokine receptors. In addition, we demonstrated that
the BCG induced production of IFN-g and IFN-a regulating the
expression of CXCL10 in an autocrine and paracrine fashion.
Following interaction with BCG, the PFMCs showed a rapid
and robust production of IFN-a and CXCL10. Moreover, the pro-
duction of CXCL10 may regulate the recruitment of different
leukocyte populations expressing CXCR3, such as immature DC,
monocytes, macrophages, activated T lymphocytes and NK cells
to the infected tissues. [19e22] M.tb-induced production of CCL2,
CCL3, CCL4, CCL5, and CXCL8 has been previously observed in
human alveolar macrophages and bronchoalveolar lavage or
pleural fluid from pulmonary TB patients [23,24]. However, the
induction of IFN-a and CXCL10 from PFMCs following M.tb
infection was a novel finding. A particular feature by this che-
mokine was its inducibility by IFNs, therefore we investigated
whether IFN-a and IFN-g released from M.tb-infected PFMCs
could modulate the expression of CXCL10. We found that BCG-
induced CXCL10 expression was significantly dependent on IFN-
a and IFN-g since its neutralization reduced both the mRNA
steady-state level and the protein content secreted into the su-
pernatants of BCG-infected PFMCs cultures. The remaining
expression of CXCL10 observed in the presence of neutralizing
IFN-a and IFN-g Abs could be ascribed to the activation of the NF-
lB pathway induced by the direct interaction of BCG with the
PFMCs. Indeed, the infection of DCs by M.tb-stimulated a rapid
binding of NF-lB to the lB site within the CXCL10 gene promoter
 C. Wu et al. / Tuberculosis 94 (2014) 123e130
Figure 7. CXCL10 induced the migration of PFMCs. PFMCs from TP patients were
incubated in presence or absence of CXCL10, anti-CXCL10 and TBPF for 2 h, 4 h and
6 h in a trans-well migration assay. The number of cells migration to the lower
chamber was determined by counting under microscopy. Data are expressed as
mean  SD, and error bars represent of three independent experiments with the
similar results. * *P < 0.01, ns: not significant.
that may cooperate with other transcription factors in the
regulation of CXCL10 gene transcription [25,26]. In addition, we
also observed that IFN-a and IFN-g could directly caused a sig-
nificant increase in CXCL10 mRNA expression and protein pro-
duction in treated cells. Altogether, these results suggest that the
expression of CXCL10 is regulated in M.tb-infected PFMCs in two
pathways: first, by direct interactions between mycobacterial
components and PFMCs leading to the activation of the NF-lB
complex and, second, in a paracrine and autocrine fashion by the
release of IFN-a and IFN-g from M.tb-infected cells.
The functional impact of chemokines produced by infected
PFMCs on the recruitment of T lymphocytes of CD3þCD4þ and
CD3þCD8þ phenotypes was investigated since these cells would be
recruited to the site of M.tb-infection due to the expression of CCR4,
CCR6 and CXCR3.
In summary, our results suggest the following sequence of
events that may regulate some aspects of the immune response
against M.tb. At early phases of M.tb-infection, before the gen-
eration of Ag-specific T cells, PFMCs derived CXCL10 may recruit
NK cells to the site of infection where they may directly destroy
M.tb-infected cells and produce IFN-g, which stimulates macro-
phage activation. At the later stages of infection when M.tb-
specific Th1 and Tc1 are expanded, their recruitment is mediated
by the expression of inflammatory chemokines from the M.tb-
infected PFMCs. The release of IFN-g sustains the activation of
macrophages required to maintain the inflammatory response
within granuloma through interactions of macrophages with
CD3þCD4þ and CD3þCD8þ T cells. The production of IFN-g may
also reinforce the production of CXCL10 from monocytes, T cells,
and neutrophils to sustain the lymphocyte traffic into the alve-
olar compartment.
The importance of the IFN-a/CXCL10 axis in the regulation of the
immune response against M.tb has to be confirmed in vitro to
define a possible protective role of IFN-a. Indeed, although some
studies demonstrated a clinical improvement in drug-sensitive or
multidrug-resistant TB patients treated with IFN-a, opposite results
were obtained in mice where the induction of type I IFN by
M.tb-infection may be pathogenic [27e30]. A better understanding
of these events may improve and reveal new targets for TB treat-
ment and prevention.
129
Ethical approval: Not required.
Funding: None.
Competing interests: None declared.
Acknowledgments
This work was supported by the Program of China during the
Twelfth Five-Year Plan Period (2013ZX10003007002003).
References
[1] Dye C, Williams BG. The population dynamics and control of tuberculosis.
Science 2010;328:856e61.
[2] WHO global tuberculosis control report 2010. Summary. Cent Eur J Public
Health 2010;18:237.
[3] Matsuo K, Yasutomi Y. Mycobacterium bovis Bacille Calmette-Guerin as a
vaccine vector for global infectious disease control. Tuberc Res Treat
2011;2011:574591.
[4] Zlotnik A, Yoshie O. Chemokines: a new classification system and their role in
immunity. Immunity 2000;12:121e7.
[5] Thelen M. Dancing to the tune of chemokines. Nat Immunol 2001;2:129e34.
[6] Luster AD. Chemokinesechemotactic cytokines that mediate inflammation.
N Engl J Med 1998;338:436e45.
[7] Sorensen TL, Tani M, Jensen J, Pierce V, Lucchinetti C, Folcik VA, Qin S,
Rottman J, Sellebjerg F, Strieter RM, Frederiksen JL, Ransohoff RM. Expression
of specific chemokines and chemokine receptors in the central nervous sys-
tem of multiple sclerosis patients. J Clin Invest 1999;103:807e15.
[8] Zhao DX, Hu Y, Miller GG, Luster AD, Mitchell RN, Libby P. Differential
expression of the IFN-gamma-inducible CXCR3-binding chemokines, IFN-
inducible protein 10, monokine induced by IFN, and IFN-inducible T cell
alpha chemoattractant in human cardiac allografts: association with cardiac
allograft vasculopathy and acute rejection. J Immunol 2002;169:1556e60.
[9] Medoff BD, Wain JC, Seung E, Jackobek R, Means TK, Ginns LC, Farber JM,
Luster AD. CXCR3 and its ligands in a murine model of obliterative bron-
chiolitis: regulation and function. J Immunol 2006;176:7087e95.
[10] Hancock WW, Gao W, Csizmadia V, Faia KL, Shemmeri N, Luster AD. Donor-
derived IP-10 initiates development of acute allograft rejection. J Exp Med
2001;193:975e80.
[11] Luster AD, Greenberg SM, Leder P. The IP-10 chemokine binds to a specific cell
surface heparan sulfate site shared with platelet factor 4 and inhibits endo-
thelial cell proliferation. J Exp Med 1995;182:219e31.
[12] Arenberg DA, Kunkel SL, Polverini PJ, Morris SB, Burdick MD, Glass MC,
Taub DT, Iannettoni MD, Whyte RI, Strieter RM. Interferon-gamma-inducible
protein 10 (IP-10) is an angiostatic factor that inhibits human non-small cell
lung cancer (NSCLC) tumorigenesis and spontaneous metastases. J Exp Med
1996;184:981e92.
[13] Luster AD, Leder P. IP-10, a -C-X-C- chemokine, elicits a potent thymus-
dependent antitumor response in vivo. J Exp Med 1993;178:1057e65.
[14] Goodbourn S, Didcock L, Randall RE. Interferons: cell signalling, immune
modulation, antiviral response and virus countermeasures. J Gen Virol
2000;81:2341e64.
[15] Bogdan C. The function of type I interferons in antimicrobial immunity. Curr
Opin Immunol 2000;12:419e24.
[16] Weiden M, Tanaka N, Qiao Y, Zhao BY, Honda Y, Nakata K, Canova A, Levy DE,
Rom WN, Pine R. Differentiation of monocytes to macrophages switches the
Mycobacterium tuberculosis effect on HIV-1 replication from stimulation to
inhibition: modulation of interferon response and CCAAT/enhancer binding
protein beta expression. J Immunol 2000;165:2028e39.
[17] Moser B, Loetscher P. Lymphocyte traffic control by chemokines. Nat Immunol
2001;2:123e8.
[18] Kawakami K, Qureshi MH, Zhang T, Koguchi Y, Shibuya K, Naoe S, Saito A.
Interferon-gamma (IFN-gamma)-dependent protection and synthesis of che-
moattractants for mononuclear leucocytes caused by IL-12 in the lungs of
mice infected with Cryptococcus neoformans. Clin Exp Immunol 1999;117:
113e22.
[19] Dufour JH, Dziejman M, Liu MT, Leung JH, Lane TE, Luster AD. IFN-gamma-
inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in
effector T cell generation and trafficking. J Immunol 2002;168:3195e204.
[20] Garcia-Lopez MA, Sanchez-Madrid F, Rodriguez-Frade JM, Mellado M,
Acevedo A, Garcia MI, Albar JP, Martinez C, Marazuela M. CXCR3 chemokine
receptor distribution in normal and inflamed tissues: expression on activated
lymphocytes, endothelial cells, and dendritic cells. Lab Invest 2001;81:409e18.
[21] Groom JR, Richmond J, Murooka TT, Sorensen EW, Sung JH, Bankert K, von
Andrian UH, Moon JJ, Mempel TR, Luster AD. CXCR3 chemokine receptor-
ligand interactions in the lymph node optimize CD4þ T helper 1 cell differ-
entiation. Immunity 2012;37:1091e103.
[22] Yokobori N, Schierloh P, Geffner L, Balboa L, Romero M, Musella R,
Castagnino J, De Stefano G, Aleman M, de la Barrera S, Abbate E, Sasiain MC.
CD3 expression distinguishes two gammadeltaT cell receptor subsets with
130 C. Wu et al. / Tuberculosis 94 (2014) 123e130
different phenotype and effector function in tuberculous pleurisy. Clin Exp
Immunol 2009;157:385e94.
[23] Sadek MI, Sada E, Toossi Z, Schwander SK, Rich EA. Chemokines induced by
infection of mononuclear phagocytes with mycobacteria and present in lung
alveoli during active pulmonary tuberculosis. Am J Respir Cell Mol Biol
1998;19:513e21.
[24] Mendez SP, Trejo A, Miranda E. Role of type I interferon in the bacillus
Calmette-Guerin-induced expression of CXCL10 from human monocytes.
Mediators Inflamm 2004;13:343e8.
[25] Lande R, Giacomini E, Grassi T, Remoli ME, Iona E, Miettinen M, Julkunen I,
Coccia EM. IFN-alpha beta released by Mycobacterium tuberculosis-infected
human dendritic cells induces the expression of CXCL10: selective recruit-
ment of NK and activated T cells. J Immunol 2003;170:1174e82.
[26] Ohmori Y, Schreiber RD, Hamilton TA. Synergy between interferon-gamma
and tumor necrosis factor-alpha in transcriptional activation is mediated by
cooperation between signal transducer and activator of transcription 1 and
nuclear factor kappaB. J Biol Chem 1997;272:14899e907.
[27] Giosue S, Casarini M, Ameglio F, Zangrilli P, Palla M, Altieri AM, Bisetti A.
Aerosolized interferon-alpha treatment in patients with multi-drug-resistant
pulmonary tuberculosis. Eur Cytokine Netw 2000;11:99e104.
[28] Manca C, Tsenova L, Bergtold A, Freeman S, Tovey M, Musser JM, Barry CR,
Freedman VH, Kaplan G. Virulence of a Mycobacterium tuberculosis clinical
isolate in mice is determined by failure to induce Th1 type immunity and is
associated with induction of IFN-alpha/beta. Proc Natl Acad Sci U S A 2001;98:
5752e7.
[29] Manca C, Tsenova L, Freeman S, Barczak AK, Tovey M, Murray PJ, Barry C,
Kaplan G, Hypervirulent M. tuberculosis W/Beijing strains upregulate type I
IFNs and increase expression of negative regulators of the Jak-Stat pathway.
J Interferon Cytokine Res 2005;25:694e701.
[30] Mayer-Barber KD, Andrade BB, Barber DL, Hieny S, Feng CG, Caspar P, Oland S,
Gordon S, Sher A. Innate and adaptive interferons suppress IL-1alpha and
IL-1beta production by distinct pulmonary myeloid subsets during Myco-
bacterium tuberculosis infection. Immunity 2011;35:1023e34.