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Auris Nasus Larynx

journal homepage: www.elsevier.com/locate/anl

The differences in the expression of fractalkine and its receptor in

conditions of tonsillar hypertrophy and chronic tonsillitis
Elif Koclu Hetemoglu a,*, Seda Turkoglu Babakurban a, Yunus Kasım Terzi b,
Feride Iffet Sahin b, Selim Sermed Erbek a
a
Başkent University Department of Otorhinolaryngology, Fevzi Cakmak Avenue 5th Street No: 48 06490 Bahcelievler, Ankara, Turkey
b
Başkent University Department of Medical Genetics, Sehit H. Temel Kuguoglu Avenue No:34 06490 Bahcelievler, Ankara, Turkey

A R T I C L E I N F O A B S T R A C T

Article history: Objective: Fractalkine, member of chemokine family, is involved in many inflammatory processes
Received 2 December 2017 in the human body. The aim of this study is to compare expression levels of fractalkine ligand and its
Accepted 2 December 2018 receptor in chronic tonsillitis and hypertrophic tonsil samples.
Available online xxx
Methods: The study was conducted at Baskent University Departments of Otorhinolaryngology
and Medical Genetics. It is designed as a prospective, non-randomized, controlled clinical study.
Keywords:
Total 97 samples, obtained from adenotonsillectomy due to chronic tonsillitis or tonsillar
Chronic tonsillitis
Hypertrophic tonsil
hypertrophy, were participated in the study. Fractalkine and its receptor expression levels were
Fractalkine expression determined and comparison was made between the tissue groups. c.839C > T (T280 M)
polymorphism of fractalkine receptor was analyzed, then relationship between polymorphism
and the expression level of fractalkine receptor was investigated.
Results: Fractalkine receptor expression was significantly higher in the hypertrophic tonsil group
than chronic tonsillitis group (p < 0.05).
Conclusion: Fractalkine, member of chemokine family, and its receptor may play role in preventing
chronic-recurrent tonsillitis.
© 2018 Published by Elsevier B.V.

1. Introduction Chronic tonsillitis is persistent and repetitive inflammation of

Palatine tonsils are components of the secondary lymphoid
system. They constitute one of the body’s first lines of defense
against pathogenic microorganisms. Inflammation due to
pathogen proliferation and activation on tonsil tissue causes
tonsillitis. Especially in chronic cases, surgical excision is the
most appropriate therapeutic approach [1].
* Corresponding author.
E-mail addresses: kocluelif@gmail.com (E. Koclu Hetemoglu),
turkoglu_seda@yahoo.com (S. Turkoglu Babakurban), ykterzi@gmail.com (Y.
K. Terzi), feridesahin@hotmail.com (F.I. Sahin), selimerbek@gmail.com (S.S.
Erbek).
https://doi.org/10.1016/j.anl.2018.12.001
0385-8146/© 2018 Published by Elsevier B.V.
palatine tonsils. Tonsillar hypertrophy is a process characterized by
the palatine tonsil enlargement, which is not always accompanied
by the infective process. Although this hypertrophy may be
associated with chronic tonsillitis, presence of hypertrophic tonsil
patients without history of chronic tonsillitis shows us that these
two diseases are emerging at the same tissues with different
pathophysiological mechanisms. Hypertrophic tonsils and chronic
tonsillitis have been compared in terms of histological features,
oxidant-antioxidant amounts, variable inflammation markers and
statistical differences have been found [2–4].
Chemokines are the strategic molecules play role in
transporting leukocytes in homeostatic and inflammatory
conditions such as adhesion, proliferation, cell maturation

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and proinflammatory gene expression [5,6]. Although there are
similarities between amino-acid sequences, chemokines are
grouped by position of the cysteine residues in the N terminal
ends. These subgroups are C–, CC–, CXC–, CX3C–. After
being stimulated by chemokines, intracellular functions are
regulated by G protein binded signal pathways.
Fractalkine is the only member of CX3C– subgroup. Its
genetic code located on 16th chromosome and consists of
373 amino acids [7]. Fractalkine expression was first described
in endothelial cells, which are activated by interleukin 1 (IL1)
and tumor necrosis factor a (TNFa). Further studies showed
that fractalkine had wide mRNA distribution in human body [8–
11]. Fractalkine and its receptor (CX3CL1, CX3CR1 respec-
tively) expressions have been shown in B cells of germinal
center of palatine tonsils [12]. Fractalkine genotype distribution
has also been investigated in tonsil tissue and c.839C > T
(T280 M) single nucleotide polymorphism was found related
with decreased risk of chronic tonsillitis [13]. Chronic tonsillitis
is an infective, inflammatory disease, therefore altered
fractalkine and its receptor levels may play role on this
process. The aim of this study was to determine differences
infractalkine and its receptor expression levels between chronic
tonsillitis and tonsillar hypertrophy. We also investigated the
presence of fractalkine ligand and its receptor in adenoid tissues
and the correlation between expression level and polymorphism
of fractalkine receptor in these tissues.
2. Materials and methods
This study was conducted at Baskent University Departments
of Otorhinolaryngology and Medical Genetics. It was designed as
a prospective, non-randomized, controlled clinical study. Local
ethics committee approved the study and informed consents were
obtained from all of the participants and their family.
2.1. Collecting samples
Total 97 samples from patients under 18 years old were used in
the study. These samples were obtained from tonsillectomy
(
adenoidectomy) surgeries due to chronic tonsillitis or tonsillar
hypertrophy, between January–June 2015. Paradise criteria were
used to decide tonsillectomy [14]. A questionnaire was applied for
searching frequency of acute tonsillitis. Patients with more than
seven episodes in the previous year, five episodes in each of the
previous two years or three episodes in each of the previous three
years grouped as chronic tonsillitis. Patients with snoring
examined for tonsillary and adenoid hypertrophy. Brodsky scale
was used to diagnose tonsillar hypertrophy, grade 3–4 patients
went to surgery. After medical treatment, patient with snoring and
Table 1
The enzyme cut pattern to detect c.839C > T (T280 M) single nucleotide polymorphism.
Region Enzyme
c.839C > T (T280 M) BsmB1
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>%80 obstructive adenoid tissue in choana went to adenoidect-
omy. Patients with immunodeficiency, malignancy, ciliary
dysfunction, cystic fibrosis and congenital syndrome were
excluded. 3 groups were formed from obtained tissues:
adenoidectomy materials (n:33), tonsillectomy materials due to
chronic tonsillitis without tonsillar hypertrophy (n:32) and
tonsillar hypertrophy without chronic tonsillitis (n:32).
2.2. Genotyping
Tissues were sent to DNA extraction with ‘tissue DNA isolation
kit’ (NucleoSpin Tissue DNA Isolation Kit, Macherey-Nagel
GmbH&Co., Düren, Germany). Target areas of fractalkine gene
were amplified with forward (50 -AGAATCATCCA-
GACGCTGTTTTCC-30 ) and reverse (50 -CACAGGACAGC-
CAGGCATTTCC-30 ) primers. Length of sequence known DNA
fragments obtained from PCR reaction were truncated by site
specific restriction endonuclease enzyme. 311 base pairs (bp)
amplicon was digested with restriction enzyme BsmB1 (R0580L,
_
New England BioLabsInc, Ipswich, U.K.) to detect c.839C > T
(T280 M) single nucleotide polymorphism (SNP). The enzyme
cut pattern is shown in the Table 1. After restriction enzyme
digestion, samples were run on % 2 agarose gel. There are cutting
patterns of randomly chosen 19 patients in Fig. 1. Accorging to the
cutting areas genotypes were determined.
2.3. Fractalkine and its receptor expressions
Tissues obtained from the surgeries were sent to Department of
Medical Genetics on the same day as surgeries. From these
tissues, RNA isolation was performed using guanidiumisothio-
cyanate and phenol-containing tripure (Roche Diagnostics
GmbH, Mannheim, Germany). These RNAs were transformed
into complementary DNA (cDNA) with Transcriptor High
Fidelity cDNA Synthesis Kit (Roche Diagnostics GmbH,
Mannheim, Germany). Expression levels of fractalkine
(CX3CL1) (Assay ID: 102721), fractalkine receptor (CX3CR1)
and GAPDH (housekeeping gene) (Assay ID: 141139) were
determined via Real Time Polymerase Chain Reaction (RT-PCR)
technique. Semi quantitative RT-PCR was performed with the
LightCycler1 480 II System: after first denaturation at 95  C for
10 min, 45 cycles; 95  C for 10 min, 60  C for 30 s, at 72  C 1 s.
Expression levels were investigated using 2DDCt technique and
comparisons were made between the tissue groups.
2.4. Statistical analysis
SPSS software (Statistical Package for the Social Sciences,
version 20.0, SSPS Inc, Chicago, IL) was used for statistical
Length of PCR sequence Cutting pattern
Wild type (CC) = 118 + 118 + 75
311 bp Heterozygous (CT) = 194 + 118 + 75
Homozygous mutant (TT) = 194 + 118
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Fig. 1. Gel electrophoresis image of the amplification products of the fractalkine gene region, length of sequence known DNA fragments were truncated by site
specific restriction endonuclease enzyme BsmB1. Patient no 1 has 194 + 118 (Homozygous TT), patient no 2,4,5 have 118 + 118 + 75 (wild CC), patinet no 3 has
194 + 118 + 75 (heterozygous CT) cutting pattern.
assessments. Chi-Square test, Student-t test, Mann–Whitney U
test, Kruskal–Wallis test were used for comparing the findings.
3. Results
Total 97 tissues were included in the study. 56 of them were
from males, 41 were from females. Mean age was
5.94
2.95. There were no statistically significant difference
in terms of age and gender. Allele frequencies of three groups
were compared and there was no significant difference (Table 2).
Expressions of fractalkine and its receptor were investigated
in chronic tonsillitis and tonsillar hypertrophy group. Although
there was no significant difference in fractalkine expression,
fractalkine receptor expression was significantly higher in
tonsillar hypertrophy group than chronic tonsillitis group
(p = 0.005) (Fig. 2).
Chronic tonsillitis and tonsillar hypertrophy groups were
combined to create tonsil group to analyze if there was genotype
differences between tonsil and adenoid tissues. There was no
significant difference between genotypes of tonsil and adenoid
groups (p = 0.342).
The expression levels of fractalkine receptor and ligand did
not differ according to the fractalkine receptor genotype
distribution (p > 0.05)
4. Discussion
As a chemokine, fractalkine is expressed by variable tissues. It
is also expressed in tonsil tissue, which is a part of the immune
system [12]. Although fractalkine expression has been shown on
tonsil tissue before, there is no study investigating expression
Table 2
Demographic data and allele frequencies of the groups.
Chronic tonsillitis group
Age in years (mean + SD) 6.44
3.02
Gender (Male/Female) 16/16
Alleles (CC/CT/TT) 22/9/1
Allele frequency C/T (%) 53/11 (82,8/17,2)
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3
differences of fractalkine and its receptor between chronic
tonsillitis and tonsillar hypertrophy conditions. Due to our results,
fractalkine receptor expression is significantly higher in tonsillar
hypertrophy group than chronic tonsillitis group. This result gives
us rise to thought that elevated levels of fractalkine receptor may
play role in preventing chronic-recurrent tonsillitis. There are
some studies that support anti-inflammatory effects of fractalkine
receptor. Ahadzadeh et al. [15] showed that CX3CR1 reduces
renal injury in mice with angiotensin 2 induced hypertension by
modulating the invasion of inflammatory cells. Kim et al. [16]
reported that CX3CR1+ macrophages and CD8+ T cells induced
protective IgA production by B cells independently of mesenteric
lymph nodes and Peyer patches. Castro-Sanchez et al. [17]
showed that CX3CR1- mices have more neuroinflammation and
neurodegeneration by overproduction of proinflammatory
markers in Parkinson disease. Another idea, undiscovered
chemokines may stimulate the same receptor and induce many
other inflammatory processes. These processes may be revealed
with new studies.
The mechanism of tonsillar hypertrophy without chronic-
recurrent infection has not been explained yet clearly. Another
hypothesis for elevated levels of fractalkine receptor expression
in tonsillar hypertrophy can be associated with effect of
fractalkine on hypertrophy mechanisms of tonsil tissue.
We could not find correlation between fractalkine receptor
genotype distribution and expression level. Because of this we
suggest that nucleotide substitution is not on a specific area that
can change expression level.
We considered tonsils as a whole tissue and did not separate
into cell groups. Previous immunohistochemical studies have
shown CX3CL1 and CX3CR1 expression on germinal centers
Tonsillar hypertrophy group Adenoid group p Value
5.34
2.4 6.03
3.33 0.720
20/12 20/13 0.630
22/10/0 19/12/2 0.609
54/10 (84,3/15,7) 50/16 (75,7/24,3) 0.402
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Fig. 2. Means and +/ standart errors of the expression levels of CX3CL1 and CX3CR1 in chronic tonsillitis and tonsillary hypertrophy groups.
and memory B cells of secondary lymphoid system
[11,12]. CX3CR1 expression change in conditions of tonsillary
hypertrophy and chronic tonsillitis may be due to memory B
cells in germinal centers. Further immunohistochemical studies
can support this hypothesis.
There are lots of studies that show us that fractalkine is
playing important roles in acute and chronic inflammation in
almost every system. Hosokawa et al. [18] showed that in acute
inflammation of periodontal tissue fractalkine and its receptor
expression was elevated and this elevation was up-regulated
with lipopolysaccharide of Porphyromonasgingivalis. In
another study Ikejima et al. [19] reported that elevated
fractalkine expression was detected in ruptured plaques in
unstable angina pectoris. Ruth et al. [10] reported that soluble
fractalkine level in rheumatoid arthritis is significantly higher
than in osteoarthritis. Raychaudhuri et al. [20] showed elevated
levels of fractalkine and its receptor on inflammed skin tissue in
psoriasis disease. These two studies show us that fractalkine is
playing role in rheumatologic diseases. Fractalkine may also
play role as tumor suppressor. Ohta et al. [21] reported that high
level of fractalkine expression is a good prognostic factor of
colorectal carcinomas.
Expression of fractalkine and its receptor in germinal centers
of tonsil tissue has been reported [12]. Fractalkine genotype
distribution has also been investigated in tonsil tissue and
c.839C > T (T280 M) single nucleotide polymorphism was
found related with decreased risk of chronic tonsillitis [13]. We
studied expression levels of fractalkine and its receptor and
found higher receptor expression in hypertrophic tonsils.
Further studies with larger groups are required to establish
the role of the fractalkine and its receptor in tonsil tissue and its
clinical consequences.
5. Conclusion
This is the first study investigating expressions of fractalkine
and its receptor in different situations of tonsil tissue. Elevated
levels of fractalkine receptor on hypertrophic tonsil tissue may
be associated with preventive mechanisms against chronic
tonsillitis or playing role on hypertrophy of tonsil tissue. New
Please cite this article in press as: Koclu Hetemoglu E, et al. Auris Nasus Larynx (2018), https://doi.org/10.1016/j.anl.2018.12.001
studies searching these mechanisms may guide us to new
treatment modalities.
Acknowledgments
This research did not receive any specific grant from funding
agencies in the public, commercial or not-for-profit sectors.
References
[1] Schwaab M, Gurr A, Hansen S, Minovi AM, Thomas JP, Sudhoff H,
et al. Human beta-Defensins in different states of diseases of the
tonsillapalatina. Eur Arch Otorhinolaryngol 2010;267(5):821–30.
[2] Kutluhan A, Ugras S, Kiris M, Cankaya H, Kiroglu AF, Yurttas V.
Differences in clinical and histopathologic features between chronic
adenotonsillitisand chronic adenotonsillar hypertrophy. Kulak Burun
Bogaz Ihtis Derg 2003;10(2):61–7.
[3] Kiroglu AF, Noyan T, Oger M, Kara T. Oxidants and antioxidants in
tonsillar and adenoidal tissue in chronic adenotonsillitis and adenoton-
sillar hypertrophy in children. Int J Pediatr Otorhinolaryngol
2006;70:35–8.
[4] Zagor M, Minarowska A, Knas M, Krajewska K, Niemcunowicz-Janica
A, Marciniak J, et al. N-acetyl-b-hexosaminidase in chronic tonsillitis
and tonsillar hypertrophy. Otolaryngol Pol 2013;67:204–8.
[5] Dorgham K, Ghadiri A, Hermand P, Rodero M, Poupel L, Iga M, et al.
An engineered CX3CR1 antagonist endowed with anti-inflammatory
activity. J Leukoc Biol 2009;86:903–11.
[6] Gerard C, Rollins BJ. Chemokines and disease. Nat Immunol
2001;2:108–15.
[7] Nomiyama H, Imai T, Kusuda J, Miura R, Callen DF, Yoshie O. Human
chemokines fractalkine (SCYD1), MDC (SCYA22) and TARC
(SCYA17) are clustered on chromosome 16q13. Cytogenet Cell Genet
1998;81(1):10–1.
[8] Bazan JF, Bacon KB, Hardiman G, Wang W, Soo K, Rossi D, et al. A
new class of membrane-bound chemokine with a CX3C motif. Nature
1997;385:640–4.
[9] Umehara H, Bloom ET, Okazaki T, Nagano Y, Yoshie O, Imai T.
Fractalkine in vascular biology: from basic research to clinical disease.
Arterioscler Thromb Vasc Biol 2004;24:34–40.
[10] Ruth JH, Volin MV, Haines GK, Woodruff DC, Katschke KJ, Woods
JM, et al. Fractalkine, a novel chemokine in rheumatoid arthritis and in
rat adjuvant-induced arthritis. Arthritis Rheum 2001;44:1568–81.
[11] Foussat A, Bouchet-Delbos L, Berrebi D, Durand-Gasselin I, Cou-
lomb-L’Hermine A, Krzysiek R, et al. Deregulation of the expression
of the fractalkine/fractalkine receptor complex in HIV-1-infected
patients. Blood 2001;98:1678–86.
G Model
ANL-2551; No. of Pages 5
E. Koclu Hetemoglu et al. / Auris Nasus Larynx xxx (2018) xxx–xxx
[12] Corcione A, Ferretti E, Bertolotto M, Fais F, Raffaghello L, Gregorio
A, et al. CX3CR1 is expressed by human B lymphocytes and mediates
CX3CL1 driven chemotaxis of tonsil centrocytes. PLoS One 2009;4
(December (12)):e8485.
[13] Babakurban ST, Erbek SS, Terzi YK, Arslan F, Sahin FI. Fractalkine
receptor polymorphism and chronic tonsillitis. Eur Arch Otorhinolar-
yngol 2014;271(7):2045–8.
[14] Paradise JL, Bluestone CD, Bachman RZ, Colborn DK, Bernard BS,
Taylor FH, et al. Efficacy of tonsillectomy for recurrent throat infection
in severely affected children: results of parallel randomized and
nonrandomized clinical trials. N Engl J Med 1984;310:674–83.
[15] Ahadzadeh E, Rosendahl A, Czesla D, Steffens P, Prüner L, Meyer-
Schwesinger C, et al. The chemokine receptor CX3CR1 reduces renal
injury in mice with angiotensin 2 induced hypertension. Am J Physiol
Renal Physiol 2018;1152(10).
[16] Kim YI, Song JH, Ko HJ, Kweon MN, Kang CY, Reinecker HC, et al.
CX3CR1+ macrophages and CD8+ T cells control intestinal IgA
production. J Immunol 2018;201(August (4)):1287–94.
Please cite this article in press as: Koclu Hetemoglu E, et al. Auris Nasus Larynx (2018), https://doi.org/10.1016/j.anl.2018.12.001
5
[17] Castro-Sanchez S, Garcia-Yagüe AJ, Lopez Royo T, Casarejos M,
Lanciego JL, Lastres-Becker I. Cx3cr1-deficiency exacerbates alpha-
synuclein-A53T induced neuroinflammation and neurodegeneration in
a mouse model of Parkinson’s disease. Glia 2018;00:1–11.
[18] Hosokawa Y, Nakanishi T, Yamaguchi D, Nakae H, Matsuo T. Expres-
sion of fractalkine (CX3CL1) and its receptor, CX3CR1, in periodontal
diseased tissue. Clin Exp Immunol 2005;139(3):506–12.
[19] Ikejima H, Imanishi T, Tsujioka H, Kashiwagi M, Kuroi A, Tanimoto
T, et al. Upregulation of fractalkine and its receptor, CX3CR1, is
associated with coronary plaque rupture in patients with unstable
angina pectoris. Circ J 2010;74:337–45.
[20] Raychaudhuri SP, Jiang WY, Farber EM. Cellular localization of
fractalkine at sites of inflammation: antigen-presenting cells in psoria-
sis express high levels of fractalkine. Br J Dermatol 2001;144:1105–13.
[21] Ohta M, Tanaka F, Yamaguchi H, Sadanaga N, Inoue H, Mori M. The
high expression of fractalkine results in a better prognosis for colorectal
cancer patients. Int J Oncol 2005;26:41–7.