Post-treatment changes of six cytokines in active pulmonary tuberculosis: differences between patients with stable or increased fibrosis.

Abstract

Little information is available regarding the relationship between the fibrotic evolution of pulmonary tuberculosis (PTB) and cytokine levels in human bronchoalveolar lavage fluid (BALF) and serum.
To evaluate correlations between profibrotic cytokine levels and post-treatment lung fibrotic evolution.
BALF and serum amounts of pro- or anti-inflammatory cytokines were obtained by ELISA before and 6 months after the start of anti-tuberculosis chemotherapy in 13 subjects with PTB. BALF levels were recalculated as ELF (epithelial lining fluid) levels by the urea method. High resolution computed tomography (HRCT) of both lungs was performed at the same time.
When comparing pre- and post-treatment radiological data, the scores for 2-10 mm nodules, consolidation and fibrosis presented significant differences (P < 0.05). Concomitantly, pre-treatment vs. 6 month concentrations of ELF IFN-gamma and TNF-alpha were decreased (P < 0.05), while those of IL-4 and IL-10 were increased (P < 0.012). At serum level, IFN-gamma decreased, as did TNF-alpha, TGF-beta1 and PDGF-BB (P < 0.05). When the patients were subdivided into two groups, 1) stable or 2) increasing HRCT fibrosis score, significant increases in the second group were observed for ELF/ serum values of TGF-beta1 and ELF PDGF-BB (P < 0.05) at 6 months post-treatment. Only serum TGF-beta1 values were significantly associated with the same group before treatment.

Full text

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INT J TUBERC LUNG DIS 9(1):98–104
© 2005 IUATLD
Post-treatment changes of six cytokines in active
pulmonary tuberculosis: differences between
patients with stable or increased fibrosis
F. Ameglio,* M. Casarini,† E. Capoluongo,‡ P. Mattia,§ G. Puglisi,¶ S. Giosuè†
*Department of Clinical Pathology, General Hospital S Giovanni Calibita, †Department of Cardiovascular and
Respiratory Sciences, La Sapienza University, ‡Department of Biochemistry, Catholic University of the Sacred Heart,
§Department of Radiology and ¶Chest Clinic, Carlo Forlanini Hospital, Roma, Italy
S U M M A R Y
SETTING: Little information is available regarding the
relationship between the fibrotic evolution of pulmo-
nary tuberculosis (PTB) and cytokine levels in human
bronchoalveolar lavage fluid (BALF) and serum.
OBJECTIVE: To evaluate correlations between profi-
brotic cytokine levels and post-treatment lung fibrotic
evolution.
DESIGN: BALF and serum amounts of pro- or anti-
inflammatory cytokines were obtained by ELISA before
and 6 months after the start of anti-tuberculosis chemo-
therapy in 13 subjects with PTB. BALF levels were recal-
culated as ELF (epithelial lining fluid) levels by the urea
method. High resolution computed tomography (HRCT)
of both lungs was performed at the same time.
RESULTS: When comparing pre- and post-treatment ra-
diological data, the scores for 2–10 mm nodules, consol-
idation and fibrosis presented significant differences
(P ? 0.05). Concomitantly, pre-treatment vs. 6 month
concentrations of ELF IFN-? and TNF-? were de-
creased (P ? 0.05), while those of IL-4 and IL-10 were
increased (P ? 0.012). At serum level, IFN-? decreased,
as did TNF-?, TGF-?1 and PDGF-BB (P ? 0.05). When
the patients were subdivided into two groups, 1) stable
or 2) increasing HRCT fibrosis score, significant in-
creases in the second group were observed for ELF/
serum values of TGF-?1 and ELF PDGF-BB (P ? 0.05)
at 6 months post-treatment. Only serum TGF-?1 values
were significantly associated with the same group before
treatment.
KEY WORDS: fibrogenic cytokines; pro-inflammatory
cytokines; pulmonary tuberculosis; HRCT; fibrosis;
treatment
VARIOUS STUDIES have highlighted the function of
different cytokines or their producing cells in the field of
resistance to Mycobacterium tuberculosis infection1,2–10
or have described the differential cytokine patterns in
various clinical phases of pulmonary tuberculosis
(PTB).5,9,11–18 Less information exists regarding pos-
sible activities exerted by specific cytokines on pecu-
liar aspects of the disease evolution, such as lung fi-
brotic outcome.15,19
Recent studies have shown that some cytokines,
including interleukin-4 (IL-4), IL-10, transforming
growth factor-?1 (TGF-?1), platelet derived growth
factor-BB (PDGF-BB), fibroblast growth factor (FGF)
and vascular endothelium growth factor (VEGF), af-
fect fibroblast proliferation or activation and collagen
deposition. In particular, Th-1 cytokines, including
interferon-gamma (IFN-?) and IL-2, exert an inhibi-
tory activity, while Th-2 cytokines and the above-
mentioned growth factors exhibit an enhancing ac-
tion on both fibroblast proliferation and other func-
tions promoting fibrogenic processes.6–8,20–22
It is widely accepted that the inflammatory re-
sponse, including all the regulatory mediators of in-
flammation, both enhancers and inhibitors, is respon-
sible for the evolution of the disease, i.e., its resolution
or its progression to fibrosis.2,7,22–24 In addition, ani-
mal models have suggested that a beneficial response
to M. tuberculosis may be induced by Th-1 biological
regulators, while TGF-?1 and other anti-inflamma-
tory cytokines may worsen the clinical outcome, in-
ducing fibrotic processes. One could thus speculate
that each individual may present rapid resolution of
the disease or, alternatively, slow evolution towards
fibrosis, depending on a genetically-determined abil-
ity to drive the cytokine network.20,25–28
From a general point of view, it is assumed that the
Correspondence to: Dr Franco Ameglio, Clinical Pathology, General Hospital S Giovanni Calibita, Isola Tiberina 39, Rome,
00186 Italy. Tel: (?39) 06 6837367. Fax: (?39) 06 6879345. e-mail: francoameglio@hotmail.com, francoameglio@
virgilio.it
Article submitted 1 December 2003. Final version accepted 5 April 2004.

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Fibrogenic cytokines in pulmonary tuberculosis
99
first response to M. tuberculosis infection is of Th-1
type. When this response is not able to eliminate the
microorganism, there is a shift towards a Th-2 re-
sponse, even if effective treatment is rapidly followed
by a new Th-1 shift and subsequent M. tuberculosis
eradication. Excessive persistence of Th-2 response
may lead to lung fibrosis.26,28–31
Our group has previously evaluated the behaviour
of various cytokines in the ELF of PTB patients treated
with different regimens.14,15 In the present study, we
analysed serum and ELF concentrations of six cytok-
ines, IFN-?, TNF-?, IL-4, IL-10, TGF-?1 and PDGF-
BB,32,33 in 13 patients affected with active PTB tuber-
culosis prior to anti-tuberculosis chemotherapy and 6
months after start of combined standard treatment.
To obtain ELF concentrations, and eliminate any
bias due to different individual bronchoalveolar lav-
age (BAL) dilutions,15,34 the urea correction method
was applied to the bronchoalveolar lavage fluid (BALF)
concentrations determined by enzyme-linked immuno-
sorbent assay (ELISA).
Patients also underwent high resolution computed
tomography (HRCT) of the chest to establish changes
in certain evolutive indices.29 Analysis of these images
produced different scores for bronchial modifications,
fibrosis, cavitation or consolidation and nodules (both
miliary and those 2–10 mm).
The aim of this study was to evaluate the varia-
tions in both the above-mentioned cytokines moni-
tored over time in the ELF and serum of patients and
the radiological scores. Correlations of the cytokine
levels and radiological scores were also explored to
verify whether some of the structural changes corre-
sponded to biochemical modifications. Finally, subdi-
viding the patients into two groups, depending on
whether their HRCT fibrotic scores (pre-treatment vs.
6 months after start of treatment) were stable or in-
creasing, the differences between the ELF and serum
cytokine levels were compared.
MATERIALS AND METHODS
Patients
During the period January 2002–June 2003, 74 hu-
man immunodeficiency virus negative adult patients
with PTB were observed at the Carlo Forlanini Hos-
pital in Rome. Of these, 13 white patients who shared
the following eligibility criteria were enrolled in the
study: sputum positive for acid-fast bacilli;30 M. tuber-
culosis positivity confirmed by conventional sputum
culture;16 fever; weight loss of ?5% over the previous
6 months; no evidence of other concurrent diseases.
There were 10 males and three females, with an aver-
age age of 34 years (range 19–54). None of the patients
had previously received anti-tuberculosis treatment.
All patients were submitted to the same chemo-
therapy regimen, consisting of isoniazid (H, 5 mg/kg
daily), ethambutol (E, 15–25 mg/kg daily), rifampicin
(R, 10 mg/kg daily), and pyrazinamide (Z, 15–30 mg/
kg daily) for 2 months, followed by RH for 4 months:
2EHRZ/4RH. No corticosteroids or immunosuppres-
sive agents were administered. At the end of the study,
eight patients were cured after 6 months of treatment,
and the other five continued until complete recovery.
All patients gave informed consent and the project
was approved by the ethics committee.
HRCT and arbitrary score generation
Chest HRCT was performed in the supine position
and at end inspiration using Tomoscan LX (Philips
Medical Systems, Eindhoven, The Netherlands). The
HRCT scan results were assessed for the presence,
distribution and extent of the following parameters:
1) parenchymal consolidation (dense increased opac-
ity obliterating vascular structures); 2) ground glass
pattern (increased attenuation without obscuration
of pulmonary vessels); 3) lung nodules: miliary nod-
ules (interstitial 1–2 mm, uniform sizes, well defined
nodules, random distribution); nodules 2–10 mm (al-
veolar, centrilobular, adjacent to vessel or bronchus,
subpleural and interstitial nodules); 4) bronchial le-
sions: bronchial wall thickening, bronchiectasis, bron-
chial distortion and bronchial occlusion; 5) cavita-
tion: thin- and thick-walled, regularity or irregularity
of borders, air-fluid level; and 6) fibrosis: parenchy-
mal bands, irregular linear opacities with parenchy-
mal distortion and secondary traction bronchiectasis.
The extent of involvement was assessed as previ-
ously reported.15 HRCT findings were analysed retro-
spectively by two independent chest radiologists
blinded to the patients’ clinical data and who had not
taken part in the case selection. A final decision on the
findings was reached by consensus.
Bronchoalveolar lavage
Fiberoptic bronchoscopy was performed in all cases
before and 6 months after the start of chemotherapy
using the previously described method31 to evaluate
disease evolution over a period of time. BAL was also
performed in the most involved pulmonary site, as de-
tected by HRCT. BALF was centrifuged and frozen at
?80?C. Different biochemical variables were deter-
mined on BALF samples. Their values were subse-
quently corrected by the urea method34 to obtain the
ELF concentrations. This method consists of deter-
mining urea concentrations in the serum and BALF of
each patient, and calculating a ratio (serum urea/
BALF urea) with the data obtained. This ratio repre-
sents the dilution factor of each BALF. BALF values
determined for the different variables were multiplied
by the urea ratio to calculate the ELF concentrations.
Cytokine concentrations
To determine the urea content of ELF and serum sam-
ples, a commercially available kit was purchased
(Sigma 65 UV, Sigma, Milan, Italy). Cytokine concen-

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100
The International Journal of Tuberculosis and Lung Disease
trations were measured in serum and ELF. Determi-
nations of human IL-4, IL-10, IFN-?, TNF-?, TGF-?1
and PDGF-BB were measured by ELISA (R&D Sys-
tems, Minneapolis, MN, USA). Kit sensitivities applied
to BALF before urea correction and to serum were the
following: IL-4 ? 1 pg/ml; IL-10 ? 0.5 pg/ml; IFN-? ?
8 pg/ml; TNF-? ? 4.4 pg/ml; TGF-?1 ? 7 pg/ml; and
PDGF-BB ? 8 pg/ml. When the cytokine concentra-
tions of BALF were under the detection limits, ELF
concentrations were also considered under the detec-
tion limits and urea correction was not applied.
Statistical analysis
Only non-parametric tests were used, due to the lim-
ited number of patients examined, the lack of knowl-
edge of the data distribution and the use of semiquan-
titative scores. Data are therefore expressed as medians
and ranges. For data comparisons, Kruskall Wallis,
Friedman or Wilcoxon’s paired tests were used. Cor-
relations were analysed using Spearman’s rank test.
RESULTS
On the basis of the scores on chest HRCT scan indi-
cated by the two expert radiologists, an evaluation of
10 variables was obtained for each subject before and
after 6 months of treatment at the site from which the
BAL was obtained. The median scores and ranges of
each individual parameter are presented in Table 1.
There is a weak significance for consolidation, while a
clear significance can be seen for the 2–10 mm nodule
score and for the increased fibrosis score.
The ground glass score is reported in this Table,
but as the scores were always negative, no further
evaluations were made. The HRCT results were taken
as the reference for evaluating clinical changes.
The values for IL-4, IL-10, IFN-?, PDGF-BB, TNF-?
and TGF-?1, in both ELF and serum, obtained for the
same patients at different times, were analysed (Table
2). Only IL-4 and IL-10 showed significant increases
over time in ELF, while IFN-? and TNF-? had an in-
verse behaviour. Changes observed for the other cyto-
kines were not significant. Serum concentrations of
IFN-?, PDGF-BB, TGF-?1 and TNF-? fell consis-
tently over time. Significant positive correlations be-
tween the ELF and serum cytokine levels were only
observed for TGF-?1 and PDGF-BB (R ? 0.55, P ?
0.001 and R ? 0.38, P ? 0.02, respectively).
To analyse whether the different cytokines were in-
terrelated, all possible correlations were extensively
analysed for both ELF and serum (data not shown).
Interestingly, at both 0 and 6 months, the following
cytokines, all of them Th-2 or fibrogenic mediators,
were significantly correlated in ELF: IL-4, IL-10,
TGF-?-1 and PDGF-BB.
For serum, a significant correlation was observed
before and after treatment between PDGF-BB and
TGF-?1, together with highly significant correlations
between pre-treatment IFN-? and TNF-?. Few signif-
icant correlations were recorded between ELF cyto-
Table 1
ranges) observed for different variables in lung tuberculosis
patients before and 6 months after start of treatment
Differences between the HRCT scores (medians and
HRCT extent score
HRCT variablesT0 T6
P
Miliary nodules ?2 mm
Nodules 2–10 mm
Consolidation
Cavity
Bronchial wall thickening
Bronchiectasis
Bronchial distortion
Bronchial occlusion
Fibrosis
0 (0–3)
2 (1–3)
2 (0–3)
0 (0–3)
2 (0–3)
1 (0–3)
2 (0–3)
2 (0–3)
2 (0–4)
0 (0–1)
1 (0–1)
0 (0–3)
0 (0–2)
1 (0–3)
1 (0–4)
1 (0–4)
0 (0–3)
3 (1–4)
0.16
0.0004*
0.05*
0.07
0.21
0.85
0.83
0.09
0.02*
*Significant difference.
HRCT ? high resolution computed tomography; T0 ? pre-treatment evalua-
tion; T6 ? evaluation after 6 months of treatment; P ? significance of differ-
ences between T0 and T6.
Table 2
before and 6 months after start of treatment
Medians and ranges of the levels of six cytokines observed in patients’ ELF or serum
CytokinesSample T0T6
P
IFN-?
ELF (pg/mL)
Serum (pg/mL)
ELF (pg/mL)
Serum (pg/mL)
ELF (pg/mL)
Serum (pg/mL)
ELF (pg/mL)
Serum (pg/mL)
ELF (pg/mL)
Serum (ng/mL)
ELF (pg/mL)
Serum (pg/mL)
451 (103–2600)
39 (10–63)
46 (UDL-220)
3.1 (UDL-13)
16.3 (UDL-130)
1.2 (UDL-1.7)
8.4 (UDL-43.1)
0.9 (UDL-1.8)
1299 (550–9360)
69.1 (45.2–93.7)
138 (42–3797)
3357 (2548–7584)
334 (142–1365)
15 (3–39)
19.3 (UDL-65)
1.0 (UDL-5.6)
63.4 (13–170)
UDL (UDL-2.2)
25.2 (5.2–172.4)
UDL (UDL-1.1)
1820 (504–5312)
45.2 (19.8–82.8)
233 (48–8256)
2303 (1386–5412)
0.05*
0.02*
0.02*
0.03*
0.007*
0.40
0.01*
0.23
0.40
0.05*
0.24
0.04*
TNF-?
IL-4
IL-10
TGF-?1
PDGF-BB
*Significant difference.
ELF ? epithelial lining fluid; T0 ? pre-treatment evaluation; T6 ? evaluation after 6 months of treatment; P ? significance
of differences between T0 and T6; UDL ? under detection limit.

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Fibrogenic cytokines in pulmonary tuberculosis
101
kine concentrations and HRCT scores evaluated at
local level. IFN-? was inversely correlated with bron-
chial occlusion (R ? ?0.55, P ? 0.05), while IL-10
was inversely related to bronchial distortion after
treatment (R ? ?0.57, P ? 0.05). As previously re-
ported, TNF-? was significantly correlated with the
cavitation score (P ? 0.05). Fibrosis score was not
significantly correlated with the cytokine levels, but
when the patients were subdivided into two groups,
in relation to whether their fibrosis score was stable or
increased over time (Table 3), the following signifi-
cant increases were observed in cytokine levels: serum
TGF-?1 at time 0, ELF PDGF-BB at time 6 months,
and serum and ELF TGF-?1 at time 6 months.
DISCUSSION
In a previous study, patients with PTB were observed
before and after standard 2-month treatment.14 No sig-
nificant differences were found for the same HRCT
scores14 analysed in this report. In the present study, in-
stead, significant changes were observed when 2–10 mm
nodules and consolidation were considered, suggesting
that a longer treatment period is necessary to obtain
significant variations. A similar variation was registered
in another, previous study, when 2-month standard
treatment was associated with inhalatory administra-
tion of IFN-?, suggesting that this cytokine synergised
with anti-tuberculosis treatment.14,35,36 Another change
was found for the fibrosis score, which was increased
between pre- and post-treatment. ELF cytokine levels
were also consistent with those calculated previously.14–16
These decreases between pre- and post-treatment sug-
gest that both levels are higher when the microorgan-
isms are numerous,4,19,24 and indicate a subsequent
reduction associated with M. tuberculosis eradica-
tion.14,16 New findings were observed for IL-4 and IL-10,
which increased over time; these changes were in keep-
ing with the models reported in the literature, when the
bacterial load is adequately reduced and IL-4 and IL-10
are expected to increase to minimise inflammatory tis-
sue damage.1 These two mediators presented similar
behaviour, in keeping with previous results showing
that the untreated active phase of PTB was character-
ised by a Th-1 immunological response.5,7,11 An ap-
parently contrasting study, performed on PTB tissues
by immunohistochemistry, only detected TGF-?1,
while the other mediators were negative, probably due
to the lower sensitivity of this technique as compared
with ELISA and sampling type.37
When the serum concentrations were analysed, sig-
nificant reductions were observed for IFN-?, TNF-?,
TGF-?1 and PDGF-BB.17,38 These new findings indi-
cate a reduction in inflammatory cytokines induced by
effective treatment and a systemic reduction in pro-
fibrotic cytokines. In addition, TGF-?1 and TNF-? con-
centrations determined in serum and ELF were signif-
icantly correlated, suggesting that the serum values may
be dependent on the cytokine amounts released in the
lung area.
Another important point is the correlation be-
tween the levels of different cytokines observed at dif-
ferent times. As expected, IL-4, IL-10 and fibrogenic
PDGF-BB and TGF-?1 were highly correlated.3,12 In
the serum, at the same observation time, a significant
correlation was registered between the two fibrogenic
cytokines TGF-?1 and PDGF-BB and between IFN-?
and TNF-?. The latter represent two biological medi-
ators known to be correlated in various other models,
such as psoriasis.39 TNF-? has also been found to cor-
relate with M. tuberculosis virulence.40 As seen for
ELF, serum presented a significant correlation be-
tween TGF-?1 and PDGF-BB post treatment, indicat-
ing a strong relationship between their syntheses.
An extensive analysis of correlations was also made
concerning HRCT scores and cytokine amounts. The
Table 3
or increased (n ? 7) fibrosis scores over time, analysed before and 6 months after start of treatment
Medians and ranges of the levels of six cytokines observed in patients’ ELF or serum, subdivided into stable (n ? 6)
Cytokines
(units)
T0T6
T0
P
T6
P
Sample StableIncreasedStableIncreased
IFN-?
ELF (pg/mL)
Serum (pg/mL)
ELF (pg/mL)
Serum (pg/mL)
ELF (pg/mL)
Serum (pg/mL)
ELF (pg/mL)
Serum (pg/mL)
ELF (pg/mL)
Serum (ng/mL)
ELF (pg/mL)
Serum (pg/mL)
301 (103–2600)
30.5 (10–235)
72.5 (UDL-172)
4.2 (0.4–13)
11.5 (UDL-130)
0.6 (UDL-1.2)
3.5 (2–39)
UDL (UDL-1)
1257.5 (550–9360)
64.5 (45.2–69.0)
127 (UDL-650)
3164 (2548–4761)
490 (115–1601)
41 (23–63)
46 (UDL-220)
2.5 (1–10)
18 (UDL-53)
1 (UDL-1.7)
12 (3–43)
UDL (UDL-1.8)
1580 (784–9360)
82.1 (63.8–93.7)
308 (72–3797)
5048 (3065–7584)
206 (126–1032)
17.5 (10–33)
19.5 (UDL-65)
1.7 (0.6–2.6)
45.5 (13–170)
0.8 (UDL-2.2)
20.5 (5–85)
UDL (UDL-1.1)
1165 (504–2507)
32.5 (19.8–38.6)
172.5 (UDL-325)
1603 (1412–2303)
423 (283–1520)
15 (UDL–25)
19 (UDL-65)
1.5 (0.4–5.6)
64 (44–160)
UDL (UDL-1.5)
34 (16–172)
UDL (UDL-0.8)
2637 (1678–5312)
67.5 (45.3–82.8)
884 (157–8256)
3304 (1386–5412)
0.48
0.46
0.67
0.62
0.77
0.45
0.07
0.47
0.28
0.05*
0.48
0.14
0.12
0.54
0.61
0.71
0.25
0.42
0.31
0.25
0.02*
0.01*
0.03*
0.14
TNF-?
IL-4
IL-10
TGF-?1
PDGF-BB
*Significant difference.
ELF ? epithelial lining fluid; T0? pre-treatment evaluation; T6 ? evaluation after 6 months of treatment; P ? significance of differences between stable and
increased fibrotic scores at T0 and T6; UDL? under detection limit.

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The International Journal of Tuberculosis and Lung Disease
data have not been reported because of frequent non
significant correlations, with the exception of TNF-?
with the cavity score,15 IFN-? with bronchial occlu-
sion and IL-10 with bronchial distortion. No data are
available regarding a correlation between fibrosis and
cytokine levels in PTB.
Although no direct correlation could be seen be-
tween fibrosis scores and pro-fibrogenic cytokines,
the separation of patients into stable or increased fi-
brosis score over time showed significant differences
in TGF-?1 and/or PDGF-BB in ELF or serum. Stable
fibrosis score was clearly associated with lower TGF-
?1 values, while the opposite was observed for in-
creased fibrosis scores. In particular, the TGF-?1 and
PDGF-BB levels were significantly different at 6 months,
while only TGF-?1 showed significant changes before
treatment, suggesting that the serum values of this mol-
ecule might be further analysed as a prognostic index
for fibrotic evolution in PTB. These data highlight the
role of TGF-?1 and its expression by producing cells
in the clinical outcome of this disease, which may lead
to impaired respiratory function. It can be postulated
that targeting the active TGF-?1 molecule or its activa-
tion pathway or subsequent signal transmission might
be useful to reduce fibrotic evolution in patients start-
ing with high TGF-?1 levels, with the aim of attenu-
ating possible lung function impairment or disability.
In conclusion, our work shows that, at ELF or
serum level, on diagnosis, patients with pulmonary
tuberculosis are characterised by a cytokine pattern
not exclusive of a Th-1 or Th-2 response. After effec-
tive treatment, there is a serum/ELF decrease of IFN-?
and TNF-?, and serum TGF-?1 and PDGF-BB. In
contrast, ELF values of IL-4 and IL-10 were signifi-
cantly increased, suggesting a local synthesis of anti-
inflammatory cytokines, a possible indication of an
incompletely resolved process, possibly leading to the
induction of fibrotic mechanisms. As reported for
cavitation and TNF-?, fibrosis may also be related to
TGF-?1 and PDGF-BB values.
Acknowledgements
The authors gratefully acknowledge the collaboration of Angelo Di
Genesio MD, and Giuseppe Moro MD in providing urea determi-
nation and Susan Watson for the preparation of the manuscript.
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