Keywords chronic tonsillitis - tonsillar hypertrophy - adenotonsillectomy - cytokines
Introduction
The tonsils form part of the mucosa-associated lymphoid tissue (MALT), in particular
the nasopharynx-associated lymphoid tissue (NALT), and are rich in B and T lymphocytes,
which are core components of the immune system.[1 ]
[2 ]
[3 ]
[4 ] The tonsil's histological structure is, therefore, closely related to its immunological
role. In children, the density of lymphocytes in the tonsils is greater than in adults,[5 ] with T-cells (helper and cytotoxic) and B cells being greatly increased when the
tonsils are affected by pathological processes.[6 ]
There are two main clinical manifestations of an affected tonsil: tonsillar hypertrophy
and recurrent tonsillitis.[7 ] In cases of hypertrophy, the tonsils increase in size, with no signs of inflammation,
the main symptom being obstruction of the upper airways like in obstructive sleep
apnea syndrome (OSAS), characterized by repetitive upper airway occlusion that causes
intermittent hypoxia. Conversely, tonsillitis presents as an infectious/inflammatory
process, often caused by viral or bacterial agents. These infections trigger local
and systemic reactions that are often mediated by cytokines.[8 ]
Cytokines are produced in a cascade by various types of cells at the site of the lesion,
that is, a cytokine may stimulate its target cells to produce more cytokines. These
substances bind to specific receptors, activating intracellular messengers that regulate
gene transcription. They affect the activity, differentiation, proliferation, and
survival of different cells, including the cells of the immune system, and also regulate
the production and activity of other cytokines. Some cytokines may exert proinflammatory
(Th1 profile) or antiinflammatory activities (Th2 profile) according to the microenvironment
in which they are located. Those considered proinflammatory include IL-6 and TNF-α,
while those considered antiinflammatory include IL-10.[9 ]
[10 ]
Under antigenic stimulation, the intraepithelial T lymphocytes present in the tonsillar
crypts produce Th1 proinflammatory and Th2 antiinflammatory responses. The proinflammatory
Th1 cytokines that are then released include interleukin-2 (IL-2), interferon-γ (IFN-γ),
and tumor necrosis factor-α (TNF-α). The antiinflammatory Th2 cytokines include IL-4,
IL-5, IL-6, and IL-13. All of these cytokines can be measured in peripheral blood.[11 ] The production of Th1 cytokines is dominant in immunocompetent patients. In acute
tonsillitis, either viral or bacterial, the production of cytokines begins with type
Th1, including IFN-γ and TNF-α, and later progresses to the secretion of Th2 cytokines.
This allows a balance to be reached in the defense against external agents, with subsequent
control of the immunological response to ensure that the inflammatory process does
not perpetuate.[12 ]
[13 ]
A significant increase in the levels of the inflammatory cytokines IL-6 and TNF-α
was found in patients with chronic tonsillitis compared with a healthy control group.[14 ] Tumor necrosis factor α and IL-6 are considered potent inflammatory cytokines, since
they play an important role in the acute phase of the inflammation. The quantification
of these cytokines allows the extent of the inflammation to be calculated.[13 ]
Therefore, the objective of the present study was to evaluate possible differences
in the plasma levels of TNF-α, IL-6, and IL-10 in patients submitted to adenotonsillectomy
with clinical conditions of tonsillar hypertrophy and recurrent tonsillitis, either
alone or in combination.
Methods
Ethical Aspects and the Study Population
The institute's internal review board approved the protocol of this prospective, longitudinal
study under reference CAAE 28210014.1.0000.5078. The parents or guardians were given
information on the study, and those willing to allow their children to participate
read and signed the informed consent form. The children who were able to read signed
an assent form.
Children receiving care at the otorhinolaryngology outpatient clinic at Hospital das
Clínicas/Universidade Federal de Goiás, between January 2015 and January 2017, who
were to undergo tonsillectomy or adenotonsillectomy due to tonsillar hypertrophy and
were complaining of obstruction of the upper airways with rhonchi and/or breathing
through the mouth or because of recurrent infection of the tonsils, were admitted
to the study. Children with any other comorbidity were excluded. The patients were
distributed into three different groups according to their clinical symptoms, medical
history of tonsil infections, and the size of the tonsils.[11 ]
Group 1 - Recurrent tonsillitis (RT): Tonsil size grade I or II in children with a history of recurrent tonsillitis and
at least 3 episodes/year for 3 consecutive years, or at least 5 episodes/year for
2 consecutive years, or more than 6 episodes of tonsillitis within a single year.
Group 2 - Recurrent tonsillitis with hypertrophy (RTTH): Hypertrophied tonsils grade III or IV according to the Brodsky grading scale, associated
with recurrent infections.
Group 3 - Tonsillar hypertrophy (TH): Tonsil grade III or IV with no history of infection of the tonsils.
Group 4 - Control: No history of upper respiratory tract infection or adenotonsillar hypertrophy.
Peripheral Blood Sample Collection
A 3-ml sample of peripheral blood was collected into ethylenediaminetetraacetic acid
(EDTA) vacuum tubes for both patients and controls. In the case of the patients, that
sample was taken immediately prior to the surgical procedure and a second sample was
collected 4 to 6 months after the surgery. The blood samples were sent to the immunoregulation
laboratory of the Institute of Tropical Pathology and Public Health to measure cytokine
levels.
Preparation of the Samples
After centrifuging the blood samples at 2,000 rpm for 7 minutes, the separated EDTA
plasma was frozen at -80°C until measurement of the cytokine levels.
Cytokine Level Measurement
The levels of IL-6 and IL-10 were measured in plasma using the immunoenzymatic method
called enzyme-linked immunosorbent assay (ELISA) (BD Bioscience, San Diego, CA, USA)
in accordance with the manufacturer's instructions. Readings were performed using
a Multiskan microplate spectrophotometer (Thermo LabSystems, Milford, MA, USA) at
a 450 nm wavelength. The range of detection of the IL-6 kit used was 4.68 to 300 pg/mL,
while the range of the IL-10 kit was from 7.8 to 500 pg/mL.
The TNF-α cytokine was measured using the Human TNF-α Standard ABTS ELISA Development
Kit (Peprotech, Cranbury, NJ, USA) in accordance with the manufacturer's instructions.
After 25 minutes, optical density was read using the Multiskan microplate spectrophotometer
(Thermo Labsystems, USA) at 405 nm, using the 620 nm filter as reference wavelength.
The detection range of the test was 62.5 to 4,000 pg/mL.
Statistical Analysis
The data were analyzed using the GraphPad Prism statistical program, version 5.0.
(GraphPad Software, San Diego, CA, USA). Medians were calculated and presented as
a horizontal line. The groups were compared using the nonparametric Mann-Whitney U-test.
Statistical significance was defined as p < 0.05.
Results
Sociodemographic Characteristics of the Study Groups
The overall study sample consisted of 35 children of 2 to 14 years of age (mean 7.04
years). Fourteen of the children were female and 21 were male. The RT group consisted
of 7 patients, the RTTH group of 8, and the TH group of 10. Another 10 children formed
the healthy control group ([Table 1 ]).
Table 1
Characteristics of the patients according to study group
Study group
Females
Males
Age (mean ± SD)
Total number of individuals
RT
3
4
7.8 ± 2.5
7
RTTH
3
5
6.5 ± 1.5
8
TH
4
6
6.9 ± 3.1
10
Control
4
6
10.0 ± 3.0
10
Abbreviations: RT, recurrent tonsillitis; RTTH, Recurrent tonsillitis with hypertrophy;
SD, standard deviation; TH, tonsillar hypertrophy.
Cytokine Profile in the Plasma of the Individuals Included in the Study
No statistically significant difference was found in the plasma levels of TNF-α between
any of the study groups in relation to the control group ([Fig. 1A ]). The plasma levels of IL-6 were higher in the RT (p = 0.0394) and TH (p = 0.0009) groups compared with the control group. The levels of IL-6 were also higher
in the TH group compared with the RTTH group (p = 0.0394) ([Fig. 1B ]). No statistically significant difference was found in the plasma levels of IL-10
between any of the study groups in relation to the control group ([Fig. 1C ]).
Fig. 1 Plasma levels of TNF-α (A), IL-6 (B) and IL-10 (C) to each group in the present study
(preoperatively). Plasma levels of TNF-α, IL-6 and IL-10 were quantified in the recurrent
tonsillitis (RT), tonsillar hypertrophy (TH), recurrent tonsillitis with hypertrophy
(RTTH) and controls (DS) groups by the ELISA method. The horizontal bars indicate
medians. The Mann-Whitney statistical method was used and values less than 0.05 were
considered significant.
There was no statistically significant difference in the TNF-α/IL-10 ratio between
any of the groups in the present study ([Fig. 2A ]). The IL-6/IL-10 ratio was greater in the RT (p = 0.0293) and TH groups (p = 0.0005) compared with the control group ([Fig. 2B ]). Between the RT and RTTH groups, the IL-6/IL-10 ratio was greater in the RT group
(p = 0.0091) ([Fig. 2B ]).
Fig. 2 Ratio between plasma levels of TNF-α (A) and IL-6 (B) in relation to IL-10. Plasma
levels of TNF-α, IL-6 and IL-10 were quantified in the recurrent tonsillitis (RT),
tonsillar hypertrophy (TH), recurrent hypertrophy tonsillitis (RTTH) and healthy controls
(DS) groups by the ELISA method. The ratios of pro-inflammatory and anti-inflammatory
cytokine concentrations were calculated for each individual in each group. The horizontal
bars indicate the medians. The Mann-Whitney statistical method was used and values
less than 0.05 were considered significant.
In the patients to be submitted to adenotonsillectomy, the samples taken preoperatively
did not differ from those collected 4 to 6 months after the surgical procedure (late
postoperative period) with respect to the plasma levels of TNF-α ([Fig. 3A ]) (p = 0.2799), IL-6 ([Fig. 3B ]) (p = 0.9684), or IL-10 ([Fig. 3C ]) (p = 1.000).
Fig. 3 Plasma levels of TNF-a (A), IL-6 (B) and IL-10 (C), in preoperatively (1st collection)
and postoperatively (2nd collection) between 4 and 6 months after the surgery. No
significant difference. The horizontal bars indicate the medians. The Mann-Whitney
statistical method was used and values less than 0.05 were considered significant.
Discussion
The present prospective study sought to evaluate the plasma levels of the TNF-α, IL-6,
and IL-10 cytokines in patients submitted to adenotonsillectomy due to tonsillar hypertrophy
and/or recurrent tonsillitis, both preoperatively and postoperatively.
The tonsils and adenoids are the first line of defense against pathogenic agents in
the host. Located in the upper aerodigestive tract, these structures act through both
humoral and cell immunity. Clinically, there are two situations in which the surgical
removal of the tonsils may be necessary: hypertrophy and recurrent tonsillitis.[15 ] Both conditions exert a negative effect on patients' quality of life and health
and, when indicated, surgery to remove the tonsils represents a good solution for
improving the child's quality of life.[15 ]
[16 ]
The present results showed higher IL-6 levels in the RT and TH groups. There was a
significant increase in inflammatory cytokine IL-6 levels in the patients with chronic
tonsillitis compared with the healthy control group. Together with TNF-α, IL-6 is
considered to be one of the most potent antiinflammatory cytokines, since it exerts
an important role in the acute phase of inflammation, such as in the differentiation
of lymphocytes, cell proliferation, and survival, and in potentiating apoptotic signaling.
The measurement of these cytokine levels allows the extent of the inflammation to
be estimated.[15 ]
Interleukin-6 is secreted by many different types of cells including macrophages,
monocytes, eosinophils, hepatocytes, and glial cells, with TNF-α and IL-1 being potent
inducers of this cytokine. Interleukin-6 causes fever and activates the hypothalamic-pituitary-adrenal
axis through its α-receptors (IL-6Rα) and the gp130 subunit (glycoprotein 130, member
of the class I cytokine receptor family).[17 ] As a proinflammatory cytokine, IL-6 promotes neutrophil maturation and activation,
macrophage maturation, and differentiation/maintenance of cytotoxic T lymphocytes
and natural killer (NK) cells. In addition, it activates astrocytes and microglia
and regulates the expression of neuropeptides following neural lesion, contributing
to regeneration.[18 ]
In the RT group, the increase in IL-6 levels could be the result of repeated stimulation
by pathogenic agents, with this increase occurring 24 hours after the contact of these
agents with the host.[19 ]
[20 ]
In the HT group, the increase in IL-6 levels could be the result of OSAS, a pathological
condition that can elevate the serum levels of inflammatory cytokines, including IL-6.
The intermittent hypoxia increases oxidative stress and generates reactive oxygen
species (ROS), which activates oxidant-sensitive transcription factors.[21 ] One of them is nuclear factor kB (NF-kB), which is known to induce many proinflammatory
cytokines, including IL-6.[22 ]
In the present study, the ratios of the serum levels of the inflammatory/antiinflammatory
cytokines were determined. The IL-6/IL-10 ratio was higher in the RT and TH groups
compared with the control group, which is justifiable, since there is an increase
in proinflammatory activity in relation to anti-inflammatory activity, increasing
the ratio between the interleukins. There was a significant increase in the levels
of inflammatory cytokines IL-6 in the patients with recurrent tonsillitis compared
with the healthy control group. The higher IL-6/IL-10 ratio in the RT and TH groups
represents the predominant inflammatory profile in the plasma of these patients.
Both hypertrophy and the presence of recurrent infections in the tonsils are probably
due to immune system dysfunction. An abnormal regulatory mechanism in lymphocyte activation
must cause these deviations in the function of the tonsils, which then go on to harm
the individual, leading to the need for their surgical removal.[17 ]
[18 ]
No statistically significant differences were found in the TNF-α levels between the
study groups, as previously shown in some studies.[14 ]
[23 ] Tumor necrosis factor-α, also known as cachetin, is a proinflammatory cytokine produced
mainly by monocytes, macrophages, and T lymphocytes. Following a surgical procedure,
trauma or during infections, TNF-α is one of the earliest and most potent mediators
of inflammatory response. It triggers important metabolic and hemodynamic changes
and is able to activate different cells to produce other cytokines.[7 ]
[9 ] Some studies have reported serum TNF-α values 10 to 20 times higher than normal
in patients with chronic tonsillitis, with TNF-α production being higher, particularly
in patients with recurrent tonsillitis.[24 ] In the present study, blood sampling was not performed during the acute phase of
tonsillitis, either preoperatively or in the late postoperative period. This fact
could explain the absence of any statistically significant difference between the
groups in relation to TNF-α levels. Nonetheless, another explanation may lie in the
small sample size.
Conclusion
Patients with a history of chronic tonsillitis, in particular those allocated here
to the RT and TH groups, showed higher levels of IL-6 plasma when compared with healthy
controls, highlighting a more inflammatory profile in these patients.
