Correlation of serum tumor necrosis factor-alpha, interleukin-4 and soluble interleukin-2 receptor levels with radiologic and clinical manifestations in active pulmonary tuberculosis.

Abstract

The precise clinical manifestations of tuberculosis are likely to result from a complex interaction between the host and the pathogen. We took serum samples from a group of patients with a variety of clinical and radiological stages of pulmonary tuberculosis in order to characterize tumor necrosis factor-alpha (TNF-alpha), interleukin-4 (IL-4) and soluble interleukin-2 receptor (sIL-2R) response. We further evaluated whether the levels of TNF-alpha, IL-4 and soluble IL-2R are related with each other, and also evaluated the levels of TNF-alpha, IL-4 and sIL-2R after anti-tuberculosis therapy and relation with radiologic scores. Forty-three inpatients with active pulmonary tuberculosis and 19 healthy controls participated in the study. Patients were divided into four categories radiologically on chest X-ray (minimal, moderate-advanced, far-advanced and with miliary infiltration). Concentrations of TNF-alpha (20.9+/-10/15.4+/-8 pg/ml) and sIL-2R (2569+/-842/1444+/-514 pg/ml) were statistically different between patients and controls (p=0.02 and p=0.0001, respectively). Before chemotherapy there was a positive correlation between TNF-alpha and sIL-2R (r=0.34), but there was no correlation between IL-4 and TNF-alpha, and between IL-4 and sIL-2R (r=-0.23 and r=-0.22). The TNF-alpha level was not statistically different in four groups before and after chemotherapy. Results of this study provided some evidence confirming the previously reported role of TNF-alpha, IL-4 and sIL 2R in the control of tuberculosis, but these cytokines were not found related with disease severity.

Full text

Page 1

THE precise clinical manifestations of tuberculosis are
likely to result from a complex interaction between
the host and the pathogen. We took serum samples
from a group of patients with a variety of clinical and
radiological stages of pulmonary tuberculosis in
order to characterize tumor necrosis factor-a (TNF-
a), interleukin-4 (IL-4) and soluble interleukin-2
receptor (sIL-2R) response. We further evaluated
whether the levels of TNF-a, IL-4 and soluble IL-2R
are related with each other, and also evaluated the
levels of TNF-a, IL-4 and sIL-2R after anti-tuberculosis
therapy and relation with radiologic scores. Forty-
three inpatients with active pulmonary tuberculosis
and 19 healthy controls participated in the study.
Patients were divided into four categories radiologi-
cally on chest X-ray (minimal, moderate-advanced,
far-advanced and with miliary infiltration). Concen-
trations of TNF-a (20.99 /10/15.49 /8 pg/ml) and sIL-2R
(25699 /842/14449 /514 pg/ml) were statistically dif-
ferent between patients and controls (p?/0.02 and
p?/0.0001, respectively). Before chemotherapy there
was a positive correlation between TNF-a and sIL-2R
(r?/0.34), but there was no correlation between IL-4
and TNF-a, and between IL-4 and sIL-2R (r?/? /0.23
and r?/? /0.22). The TNF-a level was not statistically
different in four groups before and after chemother-
apy. Results of this study provided some evidence
confirming the previously reported role of TNF-a, IL-
4 and sIL 2R in the control of tuberculosis, but these
cytokines were not found related with disease sever-
ity.
Key
Cytokine
words: Tuberculosis,Radiologicmanifestation
Mediators of Inflammation, 12, 9?/14 (2003)
Correlation of serum tumor
necrosis factor-a, interleukin-4
and soluble interleukin-2 receptor
levels with radiologic and clinical
manifestations in active
pulmonary tuberculosis
Levent Kart1,CA, Hakan Buyukoglan2, Ishak
O. Tekin3, Remzi Altin1, Zuhal Senturk2,
Inci Gulmez2, Ramazan Demir2and
Mustafa Ozesmi2
1Department of Pulmonary Medicine and
3Department of Immunology, Faculty of Medicine,
Zonguldak Karaelmas University, 67600 Zonguldak,
Turkey; and2Department of Pulmonary Medicine,
Faculty of Medicine, Erciyes University, Turkey
CACorresponding Author
Tel: ? /90 372 2610169
Fax: ? /90 372 2610155
E-mail: kartlevent@yahoo.co.uk,
leventkart@karaelmas.edu.tr
Introduction
Cytokines are primarily involved in host responses to
disease or infection and any involvement with
homeostatic mechanism has been less than dramatic.
Many cytokines are produced during tuberculosis
(TB),1,2with a predominance of Th1 cytokines during
the early stage3,4and Th2 cytokines in the later stages
of the infection.5These cytokines exert important
roles to limit or exacerbate the disease depending on
their balance and combinations. An understanding of
the basis of these associations and correlations during
TB could be useful in elucidating protection/patho-
genesis.
Some cytokines promote inflammation and are
called proinflammatory cytokines [tumor necrosis
factor-a
(TNF-a), interleukin
whereas other cytokines suppress the activity of
proinflammatory cytokines and are called anti-in-
flammatory cytokines (IL-4, IL-10, IL-13).6TNF-a has
harmful effects, such as acute-phase pathophysiolo-
gic events including fever and tissue necrosis. It also
plays a protective role against mycobacterial infec-
(IL)-1,IL-6, IL-8],
tion.7,8Soluble interleukin 2 receptor (sIL-2R) is a
surrogate marker of T-lymphocyte activation and
proliferation. A soluble fraction of the IL-2 receptor,
released from the cell membrane, is detectable in
serum and its concentration is known to be elevated
in TB.9
In the literature, most reports on cytokines during
TB are from studies on in vitro-stimulated lymphoid
cells with few reports on in vivo plasma levels. We
consider immunity of an individual to TB to some-
times be reflected in the plasma levels of some
cytokines. In addition, the plasma is easily accessible,
thus requiring simple procedures and equipment to
process it. In the present study we therefore exam-
ined the levels of TNF-a, IL-4 and sIL-2R in the serum
of pulmonary TB patients. To understand systemic T-
cell response in pulmonary tuberculosis to some
degree, we analyzed TNF-a, IL-4 and sIL-2R in serum.
Also, to evaluate the hypothesis that different clinical
and radiological manifestations of active pulmonary
tuberculosis are associated with different patterns of
cellular immune response systemically, we took
serum samples from a group of patients with a
Research Communication
ISSN 0962-9351 print/ISSN 1466-1861 online/03/010009-06 – 2003 Taylor & Francis Ltd
DOI: 10.1080/0962935031000096926
9

Page 2

variety of clinical and radiological stages of pulmon-
ary tuberculosis. We further evaluated whether the
levels of TNF-a, IL-4 and sIL-2R are related with each
other and also evaluated the levels of TNFa, IL-4 and
sIL-2R before and after anti-TB therapy.
Materials and methods
Subjects
Forty-three inpatients (20 male and 23 female) with
active pulmonary TB and 19 healthy controls (eight
male and 11 female) participated in the study. All
patients were recruited from the Tuberculosis Hospi-
tal and Erciyes University Medical Faculty, Clinic of
Pulmonology in Kayseri, which is located in the
central region of Turkey. Their mean age was 36.49 /
15.5 years (range 16?/67 years). On entry, all patients
had positive smear for acid-fast bacilli in sputum or
bronchial lavage and subsequent cultures of these
specimens yielded tubercle bacilli. None of the
patients had any evidence of concomitant bacterial
or viral infections as indicated by sputum and blood
cultures and viral serologic study including HIV. Five
patients had diabetes mellitus. All patients were
administered anti-TB therapy in which isoniazid,
rifampicin, pyrazinamide and streptomycin or etham-
butol were used.
We evaluated patients with physical examinations,
chest radiographs and routine laboratory tests. All
patients had pulmonary TB symptoms such as cough,
fever, and hemoptysis. To assess the presence, form,
spread and size of the TB cavities and infiltrations in
the lungs, all 43 patients received plain postero-
anterior and lateral chest radiographs. To avoid
observer bias, three pulmonary physicians initially
assessed the radiographs independently before the
laboratory studies. Patients were divided radiologi-
cally into four categories; minimal, moderate-ad-
vanced, far-advanced, and with miliary infiltration.
In minimal TB, minimal lesions include those of slight
to moderate density but that do not contain demon-
strable cavitations. They may involve a small part of
one or both lungs, but the total extent, regardless of
distribution, should not exceed the volume of the
lung on one side that occupies the space above the
second condrosternal junction and the spine of the
fourth thoracic vertebra or the body of the fifth
thoracic vertebra. Moderate-advanced TB lesions
include disseminated lesions of slight to moderate
density that may extend throughout the total volume
of one lung or the equivalent in both lungs; dense or
confluent lesions limited in extent to one-third of the
volume of one lung; and total diameter of cavitations,
if present, must be less than 4 cm. In far-advanced
TB, total cavity diameters are more than 4 cm and
Mediators of Inflammation? Vol 12? 2003
lesions are more extensive than moderately ad-
vanced.10,11
Additionally, appetite and weakness were asked
and assessed as present or not. Weight loss was
measured as a difference between weight of patients
before disease and on the time of applying to our
clinic. Body mass index, erythrocyte sedimentation
rate, and body temperature were measured initially
and after chemotherapy. Body mass index was
measured as body weight (kg)/square of height
(m2). Tuberculin skin tests with PPD (purified protein
derivative) were conducted in 13 patients, reactions
were calculated 72 h after by the same technician
(mean 14.69 /6.6 mm). A control group of 19 healthy
volunteer subjects (eight women and 11 men) with a
mean age of 34.99 /12.7 years (range 22?/65 years)
was also studied. We also evaluated normal volun-
teers with chest radiographs, routine laboratory tests
and physical examinations. None of the control
groups had any evidence of infections and systemic
disorders. All patients and volunteers gave informed
consent and the Erciyes University Medical Faculty,
Department of Pulmonology, approved the protocol.
Immunoassays
All serum samples were determined from the patients
and the control group of peripheral venous blood
samples prior to the initiation and the sixth month of
anti-TB chemotherapy, and samples were preserved
at ? /708C. All samples were assigned code numbers
and processed by an investigator. For the assay,
frozen serum samples were transferred by cold chain
rules (with CO2 ice) to the Zonguldak Karaelmas
University Medical Faculty Laboratory of Immunol-
ogy on the same day. All measurement was per-
formed in 24 h. IL-4, sIL-2R and TNF-a enzyme-linked
immunosorbent assay (ELISA) kits were purchased
by Biosource International Inc. (Camarillo, California,
USA) and used according to the recommendations of
the manufacturer. The minimum detectable dose of
TNF-a is 1.7 pg/ml, of sIL-2R is 12 pg/ml and that of
IL-4 is 0.1 pg/ml, and there is no cross-reactivity with
other cytokines. All samples were assayed in dupli-
cate. These parameters were measured in patients
after 6 months of anti-TB treatment.
Statistical analysis
The Kolmogorov?/Smirnov test was used for con-
firmation of parameters. All figures are presented as
mean9 /SD unless otherwise stated. Non-parametric
Mann?/Witney U, Wilcoxon and Kruskal?/Wallis tests
were used for comparisons between the groups of
subjects. Spearman rank correlation was used to
examine the relationship between the expression
individual cytokines and clinical parameters. All pB/
0.05 were considered significant.
L. Kart et al.
10

Page 3

Results
Patient demographics
Demographic data of the 43 patients recruited from
Erciyes University and Tuberculosis Hospital are
presented in Table 1.
Immunoassay findings
We studied the total amount of IL-4, sIL-2R and TNF-a
in serum. The mean concentration of IL-4, TNF-a and
sIL-2R levels are presented in Table 2. Concentrations
of TNF-a and sIL-2R were statistically different (p?/
0.02 and p?/0.0001, respectively), but IL-4 levels
were not significantly different between controls and
patients (p?/0.40) (Table 2). Before chemotherapy
there was a positive correlation between TNF-a and
sIL-2R (r?/0.34, p?/0.02), but there was no correla-
tion between IL-4 and TNF-a, and between IL-4 and
sIL-2R (r?/? /0.23 and r?/? /0.22). After chemother-
apy, IL-4, TNF-a and sIL-2R levels were not correlated
with each other (r?/0.16, r?/0.10, and r?/? /0.01).
Also, TNF-a, IL-4 and sIL-2R levels were not corre-
lated with the PPD reaction in 13 tuberculosis
patients.
The concentrations of TNF-a, IL-4 and sIL-2R are
presented in Table 3 according to chest X-ray scores
in patients. To analyze the differences between
cytokine concentrations of the affected lungs, the
Wilcoxon rank-paired test was employed, as well as
for the X-ray scores. Patients with TB showed
correlation between sIL-2R and TNF-a levels accord-
ing to chest X-ray lesions in moderate-advanced (r?/
0.33), far-advanced (r?/0.27) and miliary (r?/0.91)
TB. Nevertheless, IL-4 and sIL-2R levels were not
correlated in minimal, moderate-advanced and far-
advanced cases. In minimal and far advanced cases,
IL-4 and TNF-a levels showed negative correlation
(r?/? /0.57 and r?/? /0.34). There is no correlation
between the concentrations of IL-4, sIL-2R and TNF-a
in moderate-advanced and miliary TB. After che-
motherapy, the concentration of sIL-2R and TNF-a
levels was correlated in far-advanced cases (r?/0.32).
TNF-a, IL-4 and sIL-2R levels of four groups were not
different statistically before and after chemotherapy
as well (p ?/0.05). In four groups of patients there
were statistically differences for serum sIL-2R levels
before and after chemotherapy, but TNF-a and IL-4
levels were not statistically different before and after
chemotherapy. In patients with TB, there was no
correlation for TNF-a, IL-4 and sIL-2R levels with
body mass index, age, weight loss, fever and
erythrocyte sedimentation rate (p?/0.05). There
was no statistically difference between widespread
of lesions and age (p?/0.11). Considering TNF-a, IL-4
and sIL-2R levels, there were no statistical differences
between male and female, with and without cavity,
diabetic and non-diabetic, with and without sequelae
(p?/0.05) (Table 4).
Discussion
The pleiotropic cytokine TNF-a has been shown to
be associated with both protection and pathogenesis
in mycobacterial infections.12In mice experimentally
infected with Micobacterium bovis bacillus Calmette-
Gue ´rin (BCG), in vivo administration of anti-TNF-a
antibodies inhibited the development of granulomas
in host organs resulting in the dissemination of the
Table 1. Demographics and clinical characteristics of TB
patients
Total patients43
Sex
Age, mean9 /SD
20 men; 23 women
369 /15 (range 16?/67)
Chest X-ray
Minimal (%)
Moderate-advanced (%)
Far-advanced (%)
Milier (%)
Cavity (%)
9 (20.9%)
19 (44.2%)
11 (25.6%)
4 (9.3%)
12(27.9%)
Symptoms and findings
Weight loss
Weight loss, mean9 /SD (kg)
Boy mass index9 /SD (kg/m2)
Appetite
Weakness
Fever?/378C
Body temperature, mean9 /SD (8C)
28 (65.1%)
3.249 /3
19.69 /3
25 (58.1%)
23 (53.4%)
25 (58.1%)
37.99 /0.9
Table 2. Age, weight, body mass index (BMI), TNF-a and s IL 2R in controls and patients before and after chemotherapy
ParameterCase X9 /SD
(n?/43)
Case9 /SD*
(n?/43)
Control9 /SD
(n?/19)
p1
p2
Age
BMI
Weight
TNF-a (pg/ml)
sIL-2R (pg/ml)
IL-4 (pg/ml)
369 /15
199 /2
579 /8
219 /10
25699 /842
0.59 /1
?/
349 /12
249 /3
689 /12
159 /8
14459 /515
0.39 /0.3
0.95
0.00
0.01
0.02
0.00
0.40
?/
239 /3
669 /11
229 /26
14579 /457
0.59 /0.6
?/0.05
?/0.05
0.26
0.81
0.15
Data presented as mean9 /SD. p1, Comparison of case and control groups before chemotherapy; p2, comparison of case and control groups
after chemotherapy.
*Levels of cytokine after anti-tuberculosis chemotherapy.
Cytokine and radiologic correlation in tuberculosis patients
Mediators of Inflammation? Vol 12? 200311

Page 4

bacilli.13,14An increased production of TNF-a was
reported following stimulation of monocytes by
lipopolysaccharides or BCG in newly diagnosed TB
patients versus monocytes from chronic cases.15Our
findings agree with the aforementioned reports that
elevated amounts of TNF-a are detectable during the
early stages of TB, and extended it to plasma after
chemotherapy of pulmonary TB patients.
Virulentandavirulent
strains were shown to induce TNF-a in murine
macrophages, but the virulent strain induced TNF-a
later than the avirulent one.16These studies suggest
that TNF-a may be required early to limit mycobac-
terial multiplication. It is therefore apparent that the
rapid induction of TNF-a is a crucial factor in the
elimination of TB in the host. However, the level of
TNF-a in our study was variable from patient to
patient. The variation could be owing to many factors
including host and bacterial factors, but not disease
severity and other accompanied diseases such as
diabetes mellitus (Table 4).
Previous reports have suggested that an association
between TNF-a with pathology in TB accompanied
by fever, weight loss, shock and tissue necrosis.17,18
Human infection with Micobacterium tuberculosis
can lead to a range of clinical outcomes, from
asymptomatic lifelong infection to progressive pri-
mary TB to what has been traditionally regarded as
typical reactivation disease with pulmonary infiltrates
and often parechymal lung destruction.19Cellular
immune response phenotypic CD4?T-cell differ-
ences seem to be related to different clinical pre-
sentations of illness. A preponderance of CD4?T
cells at the site of infection is associated with recovery
following anti-TB therapy.20
The macrophage-activating molecule, TNF, partici-
pates in the development of microbicidal granulo-
mas13and may be critical to the immune response
directed at M. tuberculosis in situ.21The activation
and recruitment of cells into an area of inflammation
is a crucial step in the development of delayed-type
hypersensitivity (DTH) responses. DTH is immuno-
logically a process similar to cell-mediated immunity,
involving T cells and cytokines. However, DTH leads
to pathologic responses, such as granulomatous
inflammation, calcification, caseation necrosis, and
cavity formation. Granulomas usually form as a result
of the persistence of a non-degradable product or as
the result of DTH responses.22The formation of
tuberculous granulomas in humans is associated with
the expression of characteristic cytokine profiles and
indicates that the expression of certain cytokines is
associated with the development of specific patholo-
gic features resulting in granulomas.23In our study,
TNF-a levels were no different before and after
chemotherapy as high level. This indicates that
TNF-a may play a significant role in DTH and
granulomatous reaction in TB. However, TNF-a
Micobacteriumavium
Table 3. Concentrations of TNF-a, SIL-2R and IL-4 according to chest X-ray findings before and after chemotherapy
TNF-a (pg/ml)
p
SIL-2R (pg/ml)
p
IL-4 (pg/ml)
p
Before chemotherapy After chemotherapy
Before chemotherapy After chemotherapy
Before chemotherapy After chemotherapy
Total (n?/43)
219 /10
229 /27
0.75
25709 /842
14579 /457
0.00
0.59 /1
0.59 /0.6
0.86
Minimal (n?/9)
219 /8
199 /10
0.61
24759 /1063
13299 /395
0.003
0.19 /0.0
0.19 /0.0
0.34
Moderate-advanced (n?/19)
199 /8
289 /39
0.38
24469 /639
15879 /402
0.000
0.39 /0.5
0.79 /0.7
0.10
Far-advanced (n?/11)
239 /15
169 /6
0.20
26359 /705
12829 /424
0.000
1.29 /2.0
0.69 /0.7
0.39
Miliary (n?/4)
239 /9
229 /10
0.93
31859 /1465
16039 /800
0.028
0.19 /0.0
0.59 /0.7
0.39
p*
0.76
0.79
0.68
0.32
0.28
0.08
Data presented as mean9 /SD.
*Comparison between four groups of cytokine levels.
L. Kart et al.
12Mediators of Inflammation? Vol 12? 2003

Page 5

levels were not correlated to sequela and cavity
formation in our cases.
Th1-type cells secrete high levels of IL-2, TNF-a
and interferon-g. This activates macrophages and
promotes cell-mediated immune responses against
invasive intracellular pathogens. IL-2 receptor may
exist on the cell membrane as a signal transducing
unit or in a soluble form in the extra cellular fluid.
Similar to the literature, our results showed that sIL-
2R levels were higher in TB patients in the early
stages of disease with correlating TNF-a levels. sIL-2R
levels were statistically reduced in four groups after
chemotherapy independently of TNF-a level. In a
recent study, TNF-a was readily detected in sputum
from TB patients at baseline and responded to anti-
TB therapy.24The elevated TNF-a level even after
chemotherapy may be due to the systemic response
being different to the local response.
In TB patients, clinical and radiological manifesta-
tion varieties may be related to anti-inflammatory
cytokine response variability rather than proinflam-
matory cytokines. In active pulmonary TB, certain
cytokines have been postulated to relate to cavity
formation, although the detailed mechanism of cavity
formation is not yet known. Abe and coworkers
showed that increased serum vascular endothelial
growth factor levels subdue cavity formation in active
pulmonary TB. In their study, serum levels of IL-8
and TNF-a were not related to cavity formation.25To
parallel this study, we observed that sIL-2R, IL-4 and
TNF-a levels were not significantly different between
groups with and without cavities.
Cytokines such as IL-1b, IL-6, IL-8, IL-12, inter-
feron-g and TNF-a have been detected at higher
concentrations in more affected lungs,26suggesting
that these mediators are associated with the degree of
TB disease and may be related to the intensity of
inflammation. In contrast to this knowledge, our
results showed that TNF-a and sIL-2R levels were
not related to chest X-ray scores of tuberculosis.
Radiological abnormalities were quantified by means
of four different scores evaluated by three physicians
inreadingthe conventional
images. Significant differences between the four
groups were not observed for sIL-2R, IL-4 and TNF-
a, suggesting that these mediators were not asso-
chestradiographs
ciated with the severity of tuberculous disease. In
contrast to our findings, Casarini et al. found that
significant difference was observed for TNF-a, sug-
gesting that this mediator was associated with the
severity of tuberculous disease as evaluated by high
resolution computerized tomography.27In a different
study, IL-2 concentrations were also inversely corre-
lated with radiological score, unlike our results.28
Several of the immune theories focus on the central
role of Th1/Th2 cross-regulation. In particular, it has
been hypothesized that there is a switch from a Th1
to a Th2-dominant cell-mediated immune response
leading to active disease, as is seen in other infec-
tions. However, attempts at isolating Th2 cells and
Th2-type cytokines from the infection has not always
been successful. IL 4 expression in samples from
infected individuals was shown to be lower than in
those from uninfected controls.29This would seem to
indicate that the role played by Th1/Th2 immunity
might not be a simple one. A second hypothesis is
that a true switch from a Th1 to a Th2 response does
not necessarily occur, but instead that the relative
strength of the Th1 response determines latency or
active disease in TB.30Presence of the correlation
between TNF-a and sIL-2R levels in our study
showed the cross-regulation of cytokines in active
pulmonary TB. These findings are in concordance
with the literature. When the severity of the disease is
increasing, the correlation of these two cytokines
seems more significant. But IL-4 response was not
correlated with TNF-a and sIL-2R levels, similar to
literature.
Evidence from the murine model of TB has linked
major histocompatibility complex class I restricted
CD8?T cells with protective immunity against M.
tuberculosis infection.31Further evidence suggests
that these CD8?cells are more important in control-
ling pulmonary infection during the chronic phase of
disease rather than in the early stages of infection.32
CD8?cells are capable of producing macrophage-
activating cytokines and display cytolytic activity
against infected macrophages.33Previous studies
have shown increased IL-4 cytokine production in
TB patients compared with healthy normal subjects
or contacts,34,35although others have demonstrated
reduced type 1 cytokine production in the absence of
Mediators of Inflammation? Vol 12? 2003
Table 4. Concentrations of TNF-a, SIL-2R and IL-4 according to gender and clinical conditions before and after chemotherapy
TNF-a (pg/ml)SIL-2R (pg/ml) IL-4 (pg/ml)
NBefore
chemotherapy
After
chemotherapy
Before
chemotherapy
After
chemotherapy
Before
chemotherapy
After
chemotherapy
Male/female
Sequela, ? //? /
Cavitary, ? //? /
Diabetes, ? //? /
20/23
26/17
12/31
5/38
19/22
21/21
20/21
18/21
29/16
19/27
14/25
16/23
2540/2594
2743/2305
2410/2631
2945/2520
1498/1421
1467/1442
1391/1483
1540/1446
0.4/0.6
0.6/0.4
1.2/0.2*
0.1/0.5*
0.7/0.4
0.4/0.9
0.9/0.4
0.5/0.5
*pB/0.05.
Cytokine and radiologic correlation in tuberculosis patients
13