Microbes and Infection 8 (2006) 2492e2500

www.elsevier.com/locate/micinf

Original article

Functional and phenotypic changes in monocytes from

patients with tuberculosis are reversed with treatment
Marı́a D. Sánchez1, Yoenis Garcı́a1, Carlos Montes, Sara C. Parı́s,
Mauricio Rojas, Luis F. Barrera, Mauricio A. Arias2, Luis F. Garcı́a*
Grupo de Inmunologı́a Celular e Inmunogenética, Facultad de Medicina, Sede de Investigación Universitaria,
Universidad de Antioquia, Cra 53 No. 61-30, Lab. 410, Medellı́n, Colombia

Received 14 March 2006; accepted 23 June 2006

Available online 13 July 2006

Abstract

Alterations of monocyte/macrophages have been reported in patients with tuberculosis (TB), but their significance is poorly understood.
Blood mononuclear cells from patients with different clinical forms of TB, at various times of anti-TB treatment, and healthy tuberculin positive
individuals, were double-stained for CD14 plus CD206, TLR-2, IFN-gR1, CD40, HLA-DR, CD36 and CD163, and analyzed by flow cytometry.
Monocytes were infected with Mycobacterium tuberculosis H37Rv and 24 h later the phenotype, induction of necrosis and apoptosis and pro-
duction of tumor necrosis factor TNFa, interleukin (IL)-10 and IL-12p40 were determined. TB patients presented higher percentage of CD14þ
cells but lower percentage of CD14þDRþ and CD14þCD36þ cells. Expression of CD14, HLA-DR and CD36 was decreased in TB patients.
Normal percentages and expression were restored during anti-TB treatment. Monocytes from TB patients underwent necrosis and apoptosis after
M. tuberculosis infection, whereas monocytes from healthy controls exhibited only apoptosis. Anti-TB treatment reverted necrosis. There were
no differences between the various clinical forms of TB. In vitro M. tuberculosis infection decreased expression of the membrane molecules
studied. HLA-DR and CD36 inhibition correlated with induction of apoptosis. Restoration of monocyte alterations during anti-TB treatment
suggests that such alterations may be caused by the high M. tuberculosis load present during active disease.
Ó 2006 Elsevier SAS. All rights reserved.

Keywords: Tuberculosis; Monocytes; Cytokines; Apoptosis; Necrosis

1. Introduction is an ineffective anti-mycobacterial immune response with

The establishment of active tuberculosis (TB) and its clin-
ical manifestations are closely linked to the host’s immune
response, but the mechanisms underlying the loss of the pro-
tective response in patients with TB are still poorly understood
[1]. The spectrum of immune response ranges from a protective
response in latent TB, to the absence of a response and dissem-
ination of mycobacteria in miliary TB. In pulmonary TB, there
* Corresponding author. Tel.: þ57 4 210 6446; fax: þ57 4 210 6455.
E-mail address: lfgarcia@udea.edu.co (L.F. Garcı́a).
1
These authors contributed equally to this work.
2
Present address: Department of Cellular and Molecular Medicine,
St. George’s, University of London, London SW17 0RE, UK.
a clinically progressive disease, while in pleural TB there is
a localized response that can, in some cases, lead to resolution
of the disease even without treatment [2].
Monocytes/macrophages play a determinant role in the
anti-mycobacterial defense, the inflammatory granulomatous
reaction, and the tissue damage that occurs in TB [3]. These
cells are the primary target for Mycobacterium tuberculosis
infection, and their innate capacity to deal with mycobacteria
defines the early progression of the infection. Accumulation of
macrophages and their precursors, circulating monocytes, at
the infection foci is an important feature of the host response
to mycobacteria and granuloma formation [4]. However, in-
fected phagocytes within the granuloma eventually die either
by apoptosis or necrosis [5], and the type of cell death may

1286-4579/$ - see front matter Ó 2006 Elsevier SAS. All rights reserved.
doi:10.1016/j.micinf.2006.06.005
 M.D. Sánchez et al. / Microbes and Infection 8 (2006) 2492e2500
differentially condition the progression of the infection. There
is evidence that apoptotic macrophages contain the mycobac-
teria [6] and the derived apoptotic bodies can be phagocytosed
by dendritic cells as a source for antigen presentation to T cells
[7]. Contrariwise, necrotic macrophages may release myco-
bacteria, able to infect incoming phagocytes, and inflamma-
tory components that contribute to tissue damage [8].
Thus alterations in monocyte/macrophage function could
favor the establishment of active TB. These changes may
alter: (1) the expression of membrane molecules able to bind
M. tuberculosis or to transmit activating signals, such as
complement receptors, mannose receptor (CD206), scavenger
receptors (ScR), Toll-like receptors (TLR), or Interferon-
gamma receptor (IFNgR); (2) the pattern of cytokine
production in response to mycobacterial stimuli, particularly
interleukin (IL)-12, tumor necrosis factor (TNF) a and
IL-10; (3) their antigen presenting capacity because dimin-
ished expression of major histocompatibility complex antigens
and/or co-stimulatory molecules; (4) their capacity to become
properly activated by T-cell derived cytokines; (5) the produc-
tion of the anti-mycobacterial effector molecules; and (6) the
pattern of cell death by infected macrophages leading to
necrosis, rather than apoptosis.
Therefore, a more detailed characterization of the monocyte
phenotype and function in patients with TB is needed to better
understand the level of immune dysfunction and possibly
provide some prognostic information that would help to estab-
lish a more effective treatment. It is also important to determine
the effect of anti-mycobacterial treatment on these alterations.
In this report we show that monocytes from TB patients have
reduced expression of CD14, HLA-DR and CD36. This re-
duction correlated with M. tuberculosis-induced monocyte
death. Importantly, M. tuberculosis infection induces necrosis
in monocytes from TB patients. The abnormalities in pheno-
type and monocyte function were reversed with the anti-TB
treatment.
2. Materials and methods
2.1. Study population
Patients with a diagnosis of TB were recruited from differ-
ent health units in the metropolitan area of Medellı́n, Colom-
bia. These patients included 127 with pulmonary TB, 45 with
pleural TB, 19 with miliary TB, and 14 with other forms of TB
including meningitis, joint, renal, and ganglionary. TB was
diagnosed by sputum smear staining for acid fast bacilli and
mycobacteria culturing. Clinical and epidemiological criteria,
X-ray, biopsy, and adenosine deaminase levels in pleural ef-
flux, were considered when direct visualization of mycobacte-
ria was not possible. One hundred and seventy patients were
studied at time of diagnosis (0 months of treatment), and 76
of them (cohort group) were followed during the 6 months
of anti-TB treatment. There were also 131 patients who
were studied independently at 0, 3 or 6 months of anti-TB
treatment. HIV-positive individuals, and patients with other
immunodeficiencies, cancer, autoimmune diseases, as well as
2493
pregnant women, were excluded. As controls, 113 tuberculin
positive healthy individuals were recruited from medical and
laboratory personnel from the Facultad de Medicina, Universi-
dad de Antioquia, and other institutions where the patients
were recruited. The tuberculin skin test consists of the intra-
dermal injection of 0.1 ml (2 U) of purified protein derivative
(RT 23, SSI Copenhagen, Denmark). Indurations 10 mm,
measured after 72 h, were considered positive. Written in-
formed consent was obtained from all subjects studied. The
study was approved by the Ethics Committee of the Facultad
de Medicina, Universidad de Antioquia.
2.2. Reagents and antibodies
RPMI-1640 and phosphate-buffered saline (PBS) were
purchased from Gibco-BRL (Grand Island, NY); lymphocyte
separation medium, pooled human serum (PHS) from Bio-
Whittaker (Walkersview, MD); fetal calf serum (FCS) from
HyClone (South Logan, UT); trypan blue, NaN3, and bovine
serum albumin (BSA) from SigmaeAldrich (St. Louis,
MO); ethidium bromide (EtBr) from BE ICN (Cleveland,
OH); terminal deoxynucleotidyl transferase (TdT) dUTP bio-
tin Nick End Labeling (TUNEL) from Mebstain (Medical
and Biological Laboratories, Nagoya, Japan); DIOC6 (3,3 0 -
dihexyloxacarbocyanine iodide) from Molecular Probes
(Eugene, OR); paraformaldehyde from Fisher Scientific (Pitts-
burgh, PA); Middlebrook 7H9 and 7H10 media from Becton
Dickinson (Cockeysville, MD); and glycerol from Mallin-
ckrodt Baker (Xalostoc, Mexico). Antibodies used in flow cy-
tometry are shown in Table 1.
2.3. Culture and maintenance of M. tuberculosis H37Rv
Mycobacterium tuberculosis H37Rv was obtained from the
Instituto Nacional de Salud (Bogotá, Colombia), and grown in
7H9 liquid medium. Cultures were harvested at exponential
growing phase. To disaggregate clumps, mycobacteria were
suspended in DMEM, and sonicated at 2.5 W output for
3 min at 4  C (Sonics Vibra Cell, model CV33, Newtown,
CT), centrifuged for 5 min at 400  g, the supernatant diluted
in PBS at 0.1 OD and frozen at 70  C in DMEM containing
30% glycerine and 10% FCS. The number of colony forming
units was determined as described [9].
2.4. Phenotyping of circulating monocytes
Mononuclear cells (PBMCs) were separated from 35 ml of
defibrinated blood on Lymphocyte separation medium, washed
and resuspended in RPMI-1640 supplemented with 0.5% PHS
plus 2 mM L-glutamine and 10 mM HEPES. Cell viability was
always 95% as tested by trypan blue exclusion. Five hundred
thousand PBMC were washed and resuspended in 50 ml of
staining buffer (PBS plus 1% PHS and 0.1% NaN3), contain-
ing anti-CD14-PE plus antibodies against either HLA-DR or
CD40, involved in antigen presentation [10], CD36 and
CD163, two scavenger receptors associated with removal of
apoptotic cells [11], or mannose receptor (CD206), TLR-2
2494 M.D. Sánchez et al. / Microbes and Infection 8 (2006) 2492e2500

Table 1
Antibodies
Antibodies to: Clone Isotype Source
CD3-Cy UCHT1 IgG1* BD PharMingen, San Diego, CA
CD14-PE M5E2 IgG2a BD PharMingen
CD36-FITC CB38 (NL07) IgM BD PharMingen
CD40-FITC 5C3 IgG1 BD PharMingen
HLA-DR-FITC TU36 IgG2b Caltag Laboratories, Burlingame, CA
Mannose receptor-Cy-Chrome (CD206) 19.2 IgG1 BD PharMingen
TLR2-FITC TL2.1 IgG2a BD Biosciences
IFNg-R1-FITC 2E2 Hamster IgG Santa Cruz Biotechnology, Santa Cruz, CA
CD163-PE GHI/61 IgG1 BD PharMingen
Mouse IgG-FITC/PE/Cy-Chrome MOPC-21 IgG1 BD PharMingen
Mouse IgG- FITC/PE G155e178 IgG2a BD PharMingen
Mouse IgG-FITC 27e35 IgG2b BD PharMingen
Mouse IgM-FITC G155e228 IgM BD PharMingen
Mouse IgG-FITC BPC-4 IgG2b Ancell, Bayport, MN
Hamster IgG-FITC IgG Santa Cruz Biotechnology

and IFN-gRa, associated with phagocytosis and macrophage
activation [12], labeled as described in Table 1. Isotype control
antibodies were used as control for non-specific binding. Cells
were incubated on ice for 20 min, washed, resuspended in PBS
and fixed with 2% paraformaldehyde. Ten thousand events
were acquired in an EPICS XL flow cytometer (Coulter, Hia-
leah, FL), and the percentage of positive cells and the mean
fluorescence intensity (MFI) was analyzed using the software
Windows Multiple Document Interface 2.8 (WinMDI, Scripts
Research Institute, La Jolla, CA.).
2.5. Culture of monocytes and in vitro
infection with M. tuberculosis
PBMC (5  105 CD14þ cells) were cultured in 1 ml of
medium in 24-well plates (BD Falcon, Bedford, MA) for 2 h
at 37  C. After incubation, the wells were extensively washed
with warm PBS containing 0.5% PHS. Adherent cells were
cultured in RPMI-1640 supplemented with 10% PHS plus
2 mM L-glutamine and 10 mM HEPES for 24 h. The percent-
age of contaminating CD3þ cells was always 5%. The
number of adherent cells was determined in replicate well
by counting the nuclei [13]. The cells were infected with
M. tuberculosis at a multiplicity of infection (MOI) of 5 for
24 h. After incubation, culture supernatants were collected
and frozen at 70  C, and the cells fixed with 2% paraformal-
dehyde. Thereafter, the cells were harvested using rubber
scrapers (Sarstedt, Newton, NC) and stained for determination
of surface markers and cell death by flow cytometry.
2.7. Determination of cell death of
M. tuberculosis-infected monocytes
Apoptosis and necrosis were determined by simultaneous
staining with DIOC6 and EtBr [14]. M. tuberculosis-infected
monocytes (5  105) were resuspended in 2 ml PBS and in-
cubated with 25 ml (1.3 mg/ml) of EtBr and 10 ml of 7 mM
DIOC6 for 20 min at room temperature in darkness, followed
by fixation with 2% paraformaldehyde. The cells were
washed and resuspended in PBS for flow cytometry analysis.
Cells with high fluorescence intensity for DIOC6 and ne-
gative for EtBr were considered alive. Cells with low fluores-
cence intensity for DIOC6 and EtBr negative were considered
apoptotic, and cells positive for EtBr were considered
necrotic [14].
The TUNEL technique was used together with FITC-
labeled anti-CD36 and anti-HLA-DR to determine the ex-
pression of HLA-DR and CD36 on apoptotic cells. For this
purpose, 1  106 M. tuberculosis-infected monocytes were
fixed with 2% paraformaldehyde, washed and permeabilized
with PBS containing 0.5% Tween-20 and 0.2% BSA. The
TdT reagent (TdT buffer, dUTPs-PE, and TdT enzyme)
was added in 18:1:1 ratio. After incubating for 1 h at
37  C, the cells were washed and resuspended in PBS con-
taining 0.1% NaN3. Anti-CD36-FITC, anti-HLA-DR-FITC,
or the respective isotype controls, were added at a final
concentration of 5 mg/ml. The cells were washed and re-
suspended in deionized water plus 0.2% BSA before flow
cytometry analysis. Cells in absence of TdT enzyme were
used as negative control.

2.6. Cytokine measurement 2.8. Statistical analysis

TNFa, IL-10 and IL-12p40 levels in culture supernatants
from M. tuberculosis-infected and non-infected monocytes
were determined by ELISA using commercial kits (Duoset,
R&D, Minneapolis, MN) following the manufacturer’s
instructions.
Comparisons between groups were done by one- and two-
way ANOVA. Comparison between infected and non-infected
cultures was done by two-tailed paired t-test. Results were
considered significant when P < 0.05. All analyses were
done using Prism 4 software (Graphpad, San Diego, CA).
 M.D. Sánchez et al. / Microbes and Infection 8 (2006) 2492e2500 2495

3. Results pleural TB (P < 0.01), but not in patients with other forms
of TB; whereas CD14 MFI (Fig. 1B) was lower in all forms
3.1. Demographic description of the subjects studied of TB (P < 0.001), as compared to healthy controls. The per-
centage of CD14þHLA-DRþ cells (P < 0.01; Fig. 1C) and
A total of 205 patients with different forms of TB were CD14þCD36þ (P < 0.001; Fig. 1E) cells, as well as the
recruited, of whom 106 were male and 101 female. The expression of HLA-DR (P < 0.001; Fig. 1D) and CD36
mean age of males was 38.1  12.8 years and of females (P < 0.001; Fig. 1F) on CD14þ monocytes was lower
34.0  11.8 years (P ¼ 0.02). The mean age of controls was (P < 0.001) in TB patients, irrespective of the form of the
41.0  10.3 years. Tuberculin skin test was done in only 81 disease, except for patients with other forms of TB where no
TB patients due to refusal to be tested and non-compliance difference was found in the percentage of HLA-DRþ cells.
at the time of measuring the skin response. Fourteen patients Expression of CD40, MR, TLR-2, CD163 and IFN-gR1 in
(17%) were tuberculin negative with 0 mm of induration in TB patients was not different compared to healthy controls
13 of them, whereas 67 were positive (83%) with an indura- (data not shown).
tion of 15.8 mm (range 10e26). All patients responded to
anti-TB treatment. 3.3. Effect of anti-TB treatment on the expression
of CD14, HLA-DR and CD36
3.2. Expression of CD14 and cell surface molecules
associated with monocyte function Expression of CD14, and HLA-DR on CD14þ cells steadily
increased over time in the cohort group to reach those levels
The percentage of circulating CD14þ monocytes (Fig. 1A) observed in controls (Fig. 2A and B). Recovery of CD36
was increased in patients with pulmonary (P < 0.001) and MFI on CD14þ cells was only observed at 6 months of

A ** B ***
*** 800
***
40 ***
30 600
% CD14+cells

MFI CD14

20 400

10 200

0
0
Controls Pulmonary Pleural Miliary + Others Controls Pulmonary Pleural Miliary + Others

C ** D ***
*** 400 ***
100
***
MFI DR (CD14+ cells)
% DR+ (CD14+) cells

75 300

50 200

25 100

0 0
Controls Pulmonary Pleural Miliary + Others Controls Pulmonary Pleural Miliary + Others

E *** F ***
*** ***
*** *** 800
***
MFI CD36 (CD14+ cells)

100
% CD36+ (CD14+) cells

80 600

60 400
40
200
20

0 0
Controls Pulmonary Pleural Miliary + Others Controls Pulmonary Pleural Miliary + Others

Fig. 1. Expression of CD14, HLA-DR and CD36 by monocytes from healthy control and patients with different clinical forms of TB. Fresh PBMC (5  105) were
obtained from healthy tuberculin positive controls (n ¼ 58), patients with pulmonary (n ¼ 45), pleural (n ¼ 16) and miliary or other forms (n ¼ 11) of TB, and
immunostained with specific monoclonal antibodies as described in Section 2. Ten thousand events were analyzed by flow cytometry and the results shown as
percentage of positive PBMC (A, C, D) or the mean fluorescence intensity (B, D, E) of CD14 (A, B), HLA-DR on CD14þ cells (C, D) and CD36 on CD14þ
cells (E, F). Comparison between groups was done by one-way ANOVA. **P < 0.01; ***P < 0.001.
2496 M.D. Sánchez et al. / Microbes and Infection 8 (2006) 2492e2500

A B C
*** ** ***

HLA-DR MFI on CD14+ cells

**
** *** ***

CD36 MFI on CD14+cells

800 400 500

600 400
300
CD14 MFI
300
400 200
200
200 100 100

0 0 0

0 3 6 0 3 6 0 3 6
Months of anti-TB treatment Months of anti-TB treatment Months of anti-TB treatment

Fig. 2. Expression of CD14, HLA-DR and CD36 by monocytes from TB patients at different times of anti-TB treatment. Fresh PBMC (5  105) were obtained
from a cohort of TB patients at 0, 3 and 6 months of anti-TB treatment, and immunostained with specific monoclonal antibodies as described in Section 2. Ten
thousand events were analyzed by flow cytometry and the results shown as mean fluorescence intensity of CD14 (A) (n ¼ 30), HLA-DR on CD14þ cells (B)
(n ¼ 20) and CD36 on CD14þ cells (C) (n ¼ 20). Comparison between times of treatment was done by one-way ANOVA. **P < 0.01; ***P < 0.001.

treatment (Fig. 2C). The effect of anti-TB treatment was also Since no significant differences were observed between pa-
observed in the cross-sectional group of TB patients (data not tients with different forms of TB, they are shown as a single
shown). The percentage of CD14þ, CD14þDRþ and group. M. tuberculosis infection of monocytes from controls
CD14þCD36þ cells also returned to normal during anti-TB (Fig. 3A and C) and TB patients (Fig. 3B and D) reduced
treatment (data not shown). Expression of CD40, MR, TLR- the percentage of positive cells (Fig. 3A and B) and the
2, IFN-gRa and CD163 was not affected by anti-TB treatment expression (Fig. 3C and D) of the markers studied, with the
(data not shown). exception of the percentage CD163 in healthy controls; and
the expression of CD36 on cells from TB patients which
3.4. Effect of M. tuberculosis infection on the expression were not affected.
of monocyte surface molecules
3.5. Effect of anti-TB treatment on
To determine whether the changes observed ex vivo may be M. tuberculosis-induced cell death
induced by in vitro infection with M. tuberculosis, monocytes
from controls and TB patients, at the beginning of treatment, Monocytes from TB patients upon in vitro exposure to PPD
were infected or not with M. tuberculosis for 24 h (MOI: 5). or infection with M. tuberculosis exhibit a mixed pattern of

A B
HEALTHY CONTROLS TB PATIENTS
% Positive cells ± 95% CI
% Positive cells ± 95% CI

80 80
70 70
60 60
50 50
40 40
30 30
20 20
10 10
0
*** *** ns *** *** *** ***
0
*** *** * *** *** *** ***
Mtb - + - + - + - + - + - + - + Mtb - + - + - + - + - + - + - +
DR CD36 CD163 MR IFNγR TLR2 CD40 DR CD36 CD163 MR IFNγR TLR2 CD40

C 250
225 D 100
80
200 60
50 50
MFI ± 95% CI
MFI ± 95%CI

40 40
30 30
20 20
10 10
0
*** *** *** *** *** * *** 0
*** ns *** *** *** * ***
Mtb - + - + - + - + - + - + - + Mtb - + - + - + - + - + - + - +
DR CD36 CD163 MR IFNγR TLR2 CD40 DR CD36 CD163 MR IFNγR TLR2 CD40

Fig. 3. Effect of in vitro infection with Mycobacterium tuberculosis H37Rv on the expression of monocyte surface molecules. Monocyte monolayers obtained from
healthy controls (A and C) and TB patients (B and D) were infected with M. tuberculosis (5:1) for 24 h. Thereafter, the cells were stained with anti-CD14-FITC
plus PE-labeled monoclonal antibodies against monocyte surface molecules as indicated in the Fig. The percentage of double positive cells (A and B) and the mean
fluorescence intensity (MFI) (C and D) were determined by flow cytometry. Comparisons for each surface molecule on infected and non-infected cells were done
by two-tail paired Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.
 M.D. Sánchez et al. / Microbes and Infection 8 (2006) 2492e2500 2497

necrosis and apoptosis, whereas cells from healthy controls
only show apoptosis [14]. Here, we tested whether the type
of monocyte death varies in the different clinical forms of
TB, and if the type of death changes during anti-TB treatment.
After 24 h of infection, nearly half of M. tuberculosis-infected
monocytes from healthy controls died from apoptosis
(Fig. 4A), whereas fewer than 10% died from necrosis. In TB
patients, irrespective of the form of the disease, about one third
of the cells died from necrosis, and a similar percentage of cells
showed apoptosis. This death pattern was reverted by anti-TB
treatment (Fig. 4B), with percentages of necrosis and apoptosis
similar to healthy controls at the third month of treatment.
3.6. Effect of apoptosis on HLA-DR and CD36
expression by M. tuberculosis-infected monocytes
during M. tuberculosis infection favoring a Th1 response [16].
Thus, we measured TNFa, IL-10 and IL-12p40 in supernatants
of monocyte cultures infected with M. tuberculosis from
healthy controls and TB patients with the clinical forms studied
(Fig. 6). M. tuberculosis infection induced production of TNFa
by healthy controls and TB patients, with no differences be-
tween the various forms of disease (Fig. 6A). M. tuberculosis in-
duced IL-10 production by the different groups (Fig. 6B), with
the exception of patients with other forms of TB in whom the
difference between infected and non-infected cultures was not
significant. Increased IL-12p40 production was observed in
healthy controls and patients with pulmonary and other forms
of TB, but not in patients with pleural TB (Fig. 6C).
4. Discussion

We next examined whether the down-regulation of expres-
sion of the molecules studied induced by M. tuberculosis infec-
tion could be associated with induction of apoptosis. Monocytes
from healthy controls were infected or not with M. tuberculosis,
and double stained with TUNEL and anti-HLA-DR or anti-
CD36. A high percentage of monocytes expressed HLA-DR
(Fig. 5A) and CD36 (Fig. 5D) prior to infection. After 24 h,
non-infected cells showed a marked decrease in the expression
of both molecules, although the cells remain viable, as shown by
the negligible number of TUNELþ cells (Fig. 5B and E). M. tu-
berculosis infection further inhibited HLA-DR and CD36 ex-
pression (Fig. 5C and F), but this inhibition was accompanied
by great increase of TUNELþ-CD36 and -HLA-DR cells,
suggesting that inhibition of the expression of monocyte surface
molecules after in vitro M. tuberculosis infection may be due to
cell death induced by the infection.
3.7. Cytokine production by M. tuberculosis-infected
monocytes
Induction of apoptosis and necrosis is modulated by the
TNFa/IL-10 ratio and M. tuberculosis induces production of
both cytokines [14,15]. IL-12 also plays an important role
Our results demonstrate that active TB, irrespective of the
clinical form of the disease, results in changes of the exp-
ression, as well as in the percentage of monocytes expressing
surface molecules known to play important functions in the
anti-TB response. The results also confirm previous observa-
tions [14] that TB patients have an altered pattern of monocyte
death upon in vitro infection with M. tuberculosis.
Patients with pulmonary and pleural TB had increased per-
centage of circulating CD14þ cells, but CD14 expression was
significantly decreased in all clinical forms of TB. However,
the expression levels and the percentage of CD14þ monocytes
recovered to normal values with anti-TB treatment. We [17]
and others [18] have reported that TB patients have augmented
serum levels of soluble CD14 that return to normal values dur-
ing anti-TB treatment. It is possible that during active TB,
CD14 is released from the monocyte membrane into circula-
tion [19], explaining its low expression on monocyte mem-
brane, and the increased levels of serum sCD14. Successful
anti-TB treatment may result in retention of mCD14 on the
cell membrane with subsequent decrease of sCD14. Impor-
tantly, sCD14 has immunomodulatory effects, such as inhibi-
tion of IL-2 and IFNg production [20], which have been
reported in patients with TB [21].

A 100
B 100
% cells ± 95% CI

80 80
% cells ± 95% CI

Necrotic
60 Apoptotic 60
Live
40 40

20 20

0 0

Control Pulmonar Pleural Miliary 0 3 6

Time of treatment

Fig. 4. Effect of in vitro infection with M. tuberculosis and anti-tuberculosis treatment on monocyte death. (A) Monocytes obtained from healthy individuals
(n ¼ 18), and TB patients with pulmonary (n ¼ 13), pleural (n ¼ 4) and other forms of TB (n ¼ 2) were cultured at 5  105 cells/well and infected for 24
with M. tuberculosis at a MOI of 5. Then the cells were washed and incubated in the presence of EtBr and DIOC6, and 5000 events were acquired by flow cy-
tometry. The cells with high fluorescence intensity for DIOC6 and negative for EtBr were considered alive. The cells with low fluorescence intensity for DIOC6 and
EtBr negative were considered apoptotic, and the cells positive for EtBr were considered necrotic. (B) Monocyte monolayers from TB patients at 0 (n ¼ 19), 3
(n ¼ 19) and 6 (n ¼ 16) months of anti-TB treatment were infected and the percentage of live, apoptotic and necrotic cells detected as described above.
2498 M.D. Sánchez et al. / Microbes and Infection 8 (2006) 2492e2500

A 2 h non-infected B 24 h non-infected C 24 h M.tb-infected

79% 22% 2% 5%

80%
DR

D E F

60% 15% 3% 1%
CD36

82%

TUNEL

Fig. 5. Effect of M. tuberculosis infection and cell adherence on CD36 and HLA-DR expression by blood-derived monocytes. Monocytes were allowed to adhere
onto plastic wells for 2 h (A and D) and then infected (C and F) or not (B and E) with M. tuberculosis at a MOI of 5 and cultured for 24 h. After culture, the cells
were tested for expression of HLA-DR (A, B, C) and CD36 (D, E, F) by staining with specific monoclonal antibodies, and analyzed by flow cytometry.
Figure shows one representative experiment of three.

Our group has previously reported that monocytes from TB
patients have decreased ability to phagocytose M. tuberculosis
[22], and here we show that such patients have diminished per-
centage of CD14þDRþ circulating monocytes and low expres-
sion of HLA-DR on CD14þ cells. This evidence suggests that
during active TB, monocytes, and presumably their descen-
dent macrophages and myeloid DC, have impaired capacity
to engulf, process and present mycobacterial antigens to T
cells, as shown by other authors [23]. These abnormalities
may be partially responsible for the depressed specific T-cell
responses observed in TB patients.
CD36 was also found to be severely impaired, both in the
percentage of positive cells and its expression on CD14þ cells.
CD36 is a member of the scavenger receptors (SR) type B fam-
ily and its expression is up-regulated during monocyte to mac-
rophage maturation [11]. CD36 binds oxLDL and HDL and
participates in removal of apoptotic cells. There is evidence
that transforming growth factor (TGF)-b down-regulates
CD36 expression [24], suggesting that the high serum levels
of TGF-b present in TB patients [25] may be responsible for
the low expression and decreased percentage of CD36þ mono-
cytes. The reduction of CD36þ positive monocytes and its de-
creased expression suggest that monocytes/macrophages from
TB patients may have an altered ability to recognize and
remove apoptotic cells, allowing these cells to become late
necrotic and favoring bacterial dissemination.
The induction of apoptosis and necrosis of monocytes by in
vitro infection with M. tuberculosis confirms our previous re-
ports [14]. Monocytes from individuals with latent TB under-
went apoptosis upon infection with M. tuberculosis, whereas
monocytes from TB patients presented both apoptosis and ne-
crosis, irrespective of the type of the disease. Anti-TB treat-
ment reversed the induction of necrosis. These results
further suggest that apoptosis may be a protective mechanism
in M. tuberculosis infection whereas monocyte necrosis is an
important event in the deregulated inflammatory response
that leads to the tissue damage and mycobacterial spread
that occurs in active TB.
One important question that arises from our results relates
to the cause(s) of monocyte alterations in TB patients. The dif-
ferences between healthy tuberculin-positive individuals and
TB patients and the reversibility of the observed alterations
with anti-TB treatment provide strong evidence that changes
in monocytes are caused by active infection. Results also sug-
gest that systemic factors, from bacterial or host origin, may
be responsible for such effects. Circulating mycobacterial
products, such as ManLAM [26], may participate in the mono-
cyte abnormalities found in TB patients. On the host side, sys-
temic mediators (cytokines, hormones, sCD14, among others)
may be responsible for monocyte alterations in TB patients.
They may affect the generation of new monocytes required
at the granulomatous inflammation. They may also change
 M.D. Sánchez et al. / Microbes and Infection 8 (2006) 2492e2500 2499

TNF-α HLA-DR and CD36 expression on apoptotic cells was more

pg/ml (Geometric mean ± 95%CI)
10000 *** *** ** * striking, suggesting that it may be a consequence of the M. tu-
-
+ berculosis-induced monocyte death. However, more detailed
1000 experiments are required to differentiate the effects mediated
by cell death and those mediated by M. tuberculosis infection
100 independently of cell death.
Finally, we cannot exclude that the absence of significant
10 differences between different clinical forms of TB may be
due to either the high variability within the groups of patients,
1 or to the small number of patients in some groups (i.e. mili-
Controls Pulmonary Pleural Miliary + Others
ary). However, results suggest that monocyte phenotypes are
TB not predictive for TB susceptibility, and that monocytes are
IL-10 not responsible for the spectrum of immune responses found
pg/ml (Geometric mean ± 95%CI)

10000 in the various forms of TB. Rather, these differences may be

*** *** * ns
due to variable alterations of the T-cell dependent acquired
1000 immune response.

100
Acknowledgements
-
10
+
This work was supported by Colciencias (Bogotá, Colom-
bia), grant 1115-04-11957. The authors acknowledge the
1 patients and control individuals that participated in this study,
Controls Pulmonary Pleural Miliary + Others
the collaboration of the personnel of the institutions where
TB
the patients were recruited, and the personnel of the Flow
IL-12p40 Cytometry Unit.
pg/ml (Geometric mean ± 95%CI)

1000 ns
* **
10
10
1 of macrophages and monocytes, Infect. Immun. 64 (1996) 683e690.
Controls Pulmonary Pleural Miliary + Others
TB
Fig. 6. Cytokine production by monocytes from patients with different clinical
forms of TB, infected in vitro with M. tuberculosis. Monocyte monolayers
from healthy controls (n ¼ 23), and patients with pulmonary (n ¼ 33), pleural
(n ¼ 8) and miliary plus other TB forms (n ¼ 5) were infected or not with
M. tuberculosis at a MOI of 5 during 24 h. Thereafter, the culture supernatants
were collected and assayed for TNFa (A), IL-10 (B) and IL-12p40 (C) by
ELISA. Differences between infected and non-infected cultures in the different
groups were analyzed by two-way ANOVA. *P < 0.05; **P < 0.01;
***P < 0.001; ns, not significant.
the subsets of circulating monocyte by affecting their state of
maturation [27], or inducing an alternative type of activation
[28]. Chemokines [29] and adhesion molecules [30] induced
during active TB may also define the migration of monocyte
subsets from circulation into affected tissues.
Although down-regulation of the membrane molecules after
in vitro infection with M. tuberculosis cannot explain the ex
vivo findings, it may be relevant at the granuloma level. The
down-regulation of HLA-DR and CD36 expression by non-
infected monocytes, with no evidence of diminished cell viabil-
ity, is in agreement with our own previous observation [14].
However, in M. tuberculosis-infected cultures the decrease in
**
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