K. POPKO1, E. GORSKA1, O. POTAPINSKA1, M. WASIK1, A. STOKLOSA2,
R. PLYWACZEWSKI2, M. WINIARSKA1, D. GORECKA2, P. SLIWINSKI2,
M. POPKO3, T. SZWED3, U. DEMKOW1
FREQUENCY OF DISTRIBUTION OF INFLAMMATORY CYTOKINES
IL-1, IL-6 AND TNF-α GENE POLYMORPHISM IN PATIENTS
WITH OBSTRUCTIVE SLEEPAPNEA
1Department of Laboratory Medicine and Clinical Immunology of Developmental Age,
Warsaw Medical University, Warsaw; Poland; 2Second Clinic of Pulmonary Diseases,
Institute of Tuberculosis and Lung Diseases, Warsaw, Poland; 3Department of Gastroenterology,
Bielanski Hospital, Warsaw, Poland
Obesity is one of the most commonly identified factors for the obstructive sleep
apnea syndrome (OSAS). Adipose tissue is the source of many cytokines, among
them there are IL-6, IL-1, and TNF-α. The level of inflammatory cytokines increases
in people with OSAS and obesity. The aim of this study was to evaluate the
distribution of genotypes in inflammatory cytokine genes in people with obesity-
related OSAS. The examined group consisted of 102 person with obesity related-
OSAS and 77 normal weight person without OSAS. Genotyping of DNA sequence
variation was carried out by restriction enzyme (IL-1: Taq I, IL-6: Lwe I, TNF-α:
Nco I) analysis of PCR amplified DNA. The study revealed a significant correlation
between polymorphism located in the promoter region of inflammatory cytokine
genes and obesity-related OSAS.
Keywords: inflammatory cytokines, IL-6, IL-1, TNF-α, gene polymorphism, obesity, OSAS
Obesity-related disorders became one of the most serious social problems,
reaching a form of civilization-related disease. Obesity is a multifactor disease in
which genetic factors play an important role. These factors account for up to 40%
of causes leading to obesity. There are a great number of genes affecting food
intake and energy expenditure (1, 2).
JOURNAL OF PHYSIOLOGYAND PHARMACOLOGY 2008, 59, Suppl 6, 607-614
One of the consequences of obesity is the obstructive sleep apnea syndrome
(OSAS). OSAS is caused by blockage of the airway, usually when the soft tissue
in the rear of the throat collapses during sleep. With each apnea event, the brain
briefly arouses people with OSAS in order to resume breathing, but,
consequently, sleep is fragmented and of poor quality. Sleep apnea is as common
as is adult diabetes. Risk factors include male gender, overweight (3), and the age
over forty, but sleep apnea can strike anyone at any age, even children (4). Pro-
inflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor
alpha (TNF-α) have been shown to modulate physiological sleep. Plasma IL-1
concentration is highest at the onset of sleep in humans suggesting a sleep wake
cycle variation of this cytokine (5). Vgontzas et al (6) determined the plasma
levels of IL-1, TNF-α, and IL-6 in patients with OSAS, narcolepsy, and
hypersomnia. The concentration of TNF- was significantly elevated in patients
with OSAS and narcolepsy, and it correlated with the intensity of sleepiness
measured as the mean nap sleep latency. IL-6 levels were elevated only in OSAS
and correlated with body mass index (6, 7).
Serious consequences accompanying obesity and leading to the development
of OSAS may be caused by increased level of inflammatory cytokines, such as
IL-1, IL-6, and TNF-α. It is possible that polymorphisms located in cytokines’
genes affect the level of protein expression. It is known that IL-6 plays a role in
lipid metabolism and energy expenditure (8, 9). The polymorphism found in point
174 (G174C) of a promoter region of IL-6 gene affects the level of interleukin 6
expression (10). A similar role in adipose tissue metabolism plays TNF-α. The
level of it depends on the presence of polymorphism G308A in the promoter
region of TNF-α gene. The presence of the Aallele provokes a double increase of
TNF-α gene expression and leads to higher TNF-α production (11, 12). Another
inflammatory cytokine with a great impact on body mass regulation is interleukin
1 (IL-1). Similarly, to IL-6 and TNF- α, the influence of IL-1 on lipid metabolism
and energy expenditure seems connected with the concentration of this cytokine.
The presence of T allele in a homozygotic set causes four-fold increase of IL-1β
production (13, 14).
The purpose of the present study was to evaluate the frequency of IL-1, IL-6,
TNF-α gene polymorphism in OSAS adults with overweight (BMI>25) or
Written informed consent was obtained from all patients and controls during the enrollment
visit. The study protocol was approved by the Warsaw University Medical Ethics Committee in
The study group consisted of 102 patients (74 men and 28 women) (body mass index, BMI>25
kg/m2), aged 21-77, with newly diagnosed OSAS (apnea-hypopnea index, AHI≥5) and 77 non-
apneic controls (AHI <5) aged 18-65 (39 men and 38 women) and BMI-matched. To confirm the
diagnosis, all patients underwent standard polysomnography in the sleep disorder center. All
patients were referred to the Lung Diseases Clinic of the Institute of Tuberculosis and Lung
Diseases in Warsaw, Poland.
Genomic DNA was isolated using Genomic Midi AX isolation kit with ion-exchange
membranes (A&ABiotechnology, Gdynia, Poland). A1 ml blood sample was diluted with saline in
v/v 1:1. After 30 min incubation at 50°C with lysing buffer and proteinase K solution, the mixture
was vortexed, placed in the column, and centrifuged. The column was washed twice with buffer and
transferred into a new tube. The elution buffer was added and centrifugation at 4000 rpm was
applied. This step was repeated twice. The sample was centrifuged for 2 min with isopropanol.
Supernatant was removed and the pellet was washed with ethanol. After centrifugation, supernatant
was removed and the pellet was air-dried for 10 min. Afterward, pure DNAwas dissolved in 200 µl
of sterile water.
Genotyping was done using a PCR-restriction fragment length polymorphism analyses.
Amplification was carried out in a 50 µl volume containing 300 ng genomic DNA, 0.1 µM of each
primer (DNA-Gdansk, Gdansk, Poland):
Interleukin 1-beta (IL-1B+T3954C)
? for 5’- GCT TTT TTG CTG TGA GTC CCG-3’
rev 5’-CTC AGG TGT CCT CGAAGAAAT CAAA-3’
? Interleukin 6 (IL-6 C174G)
for 5’TGA CTT CAG CTT TAC TCT TTG T-3’
rev 5’CTG ATT GGAAAC CTT ATT AAG-3’
? TNF-α (TNF-α A308T)
for 5’- TCC TCC CTG CTC CGA TTC CG-3’
rev 5’- AGG CAA TAG GTT TTG AGG GCC AT-3’,
200 µM of each dNTP (DNA-Gdansk, Gdansk, Poland), 3 mM of magnesium chloride, and 0.2
U of Taq Gold polymerase (Applied Biosystems, Warrington, UK). Thirty five cycles were
conducted in a thermocycler, Mastercycler personal (Eppendorf, Hamburg, Germany). The
amplified PCR products were digested with the addition of appropriate enzymes. The digested
samples were separated by electrophoresis on a 2% agarose gel.
Frequency of distribution analysis was performed with a Chi2square test. Statistical
significance was accepted at P<0.05.
Genotype distribution and G308A allele frequency in controls and OSAS
patients are shown in Table 1. Homozygotic A/A genotype was not found in the
examined population. Statistical analysis did not show any significant difference
in the frequency of distribution of G/G and G/A genotypes in the OSAS patients
compared with the control group. However, 308Aallele was more common in the
OSAS than in control individuals; the difference reached 5-7%.
Genotype distribution and allele frequency of the G174C polymorphism in IL-
6 gene differed between the OSAS patients and control subjects (Table 2).
Genetic analyses revealed the more frequent presence of C allele (in homozygotic
C/C and heterozygotic G/C sets) in the OSAS patients, analyzed as a whole
group, compared with the controls. Significant gender differences were also
observed regarding the C and G alleles of the IL-6 genotype in the group of men,
but not women (Table 2).
No differences were observed in the frequency of IL-1 (C3954T) genotype
distribution. The presence of T and C alleles was similar in the population of
OSAS patients and control subjects studied (Table 3).
OSAS appears to result from inflammatory state with increased levels of IL-1,
IL-6, and TNF-α. Cytokine level may be dependent on polymorphic changes in
related genes. The results of this study support the hypothesis about the
relationship between the IL-6 genotype and OSAS. The C-containing genotypes
(C/C or G/C) were more frequently found in OSAS patients than in control
subjects. The influence of G174C polymorphism on sleep regulation may be
caused by the allele influence on the cytokine expression rate (10). The amount of
IL-6 in circulation is regulated at the level of gene expression. Transcription of IL-
6 DNA is tightly regulated by many factors, such as NFIL-6, NF-κB, Fos/Jun
CRBP, and glucocorticoid receptors (15, 16). It is suggested that part of the
promoter region 180 to 123 plays a key role in transcription induction by viruses,
IL-1, TNF-α, PDGF, and EGF. Activation of the IL-6 promoter involves synergism
between the transcription factors NFIL-6 (158 to 145) and NF-κB (73 to 64).
Additionally, the region 225 to 164 containing the G174C polymorphism has been
reported to demonstrate a negative regulatory effect on gene expression (16, 17).
Studies on the repression of the IL-6 promoter demonstrate binding of the
glucocorticoid receptor to a region around -201. The G174C polymorphism
seems located close enough to this side, may influence binding of the
glucocorticoid receptor, and may affect its ability to repress transcriptional
activation (17). It is also possible that the change of G into C creates the potential
OSAS whole group
Table 1.Frequency of polymorphism G308Ain TNF-αgene in OSAS patients and in the control group.
OSAS whole group
Table 3. Frequency of polymorphism C3954T in IL-1 gene in OSAS patients and in the control group.
OSAS whole group
Table 2. Frequency of polymorphism G174C in IL-6 gene in OSAS patients and in the control group.
binding side for the transcription factor NF-1. This factor has inhibitory effects on
IL-6 gene transcription.
The interaction between steroid hormones and IL-6 expression can explain a
gender-dependent influence of G174C polymorphism on IL-6 expression
observed in the present study in which the C allele was often found in obese
OSAS men, but not women.
The present findings also suggest the relationship between the G308A
polymorphism located in the promoter region of TNF-α gene and the
development of OSAS. However, we could not substantiate any statistical
significance in the frequency of the Aallele occurrence in the population samples
studied. The function of this polymorphism remains unclear. It is located in the
DNA sequence responsible for the binding of AP-2 transcription factor (18). The
role of -308A allele in the positive regulation of gene expression is supported by
many earlier studies (19). This polymorphic variant has been shown to affect the
promoter region of the TNF-α, the gene leading to a higher rate of transcription
compared with -308G allele. The presence of polymorphism in the regulatory
region of a gene may lead to differences in gene expression.
We found no correlation between the C3954T polymorphism in the IL-1 gene
and OSAS. Allele frequencies were very similar in the control group and in the
OSAS population. It is difficult to find a direct relationship between this
polymorphism and OSAS. However, the existence of such a relationship is
confirmed in a study of Um et al (20). These authors have found that the T-allele
carriers (T/T and C/T) have a lower level of total cholesterol, TG, BMI, and WHR
compared with C/C homozygotes.
The mechanism by which the IL-1β gene polymorphism influences obesity-
related OSAS is still unknown. Pociot et al (13, 14) have reported that
homozygosis for the IL-1β -3954T allele is associated with a four-fold increase
in the production of IL-1β compared with the cases homozygous for the C allele.
It is possible that the C3954T polymorphism located in the promoter region of IL-
1β has indirect influence on obesity and OSAS development by affecting adipose
tissue metabolism. IL-1β is secreted by human adipose cells (21). This secretion
is regulated by TNF-α production in obesity. An increase in TNF-α production
may act on adipose tissue to increase the expression and release of IL-1β. The IL-
1β regulates lipid metabolism and synergizes with other effects of TNF-α (22).
The present findings led us to suggest that polymorphism of inflammatory
cytokine genes has an important influence on OSAS development. It is possible
that this influence is exerted via changes in the expression of cytokines.
Shamsuzzaman et al (23) have reported that plasma C-reactive protein is elevated
in patients with OSAS. Teramoto et al (24) have reported that plasma levels of
IL-6 and TNF-α are elevated in patients with OSAS, which is associated with the
level of hsCRP. Those results may explain the relationship between
polymorphism in promoter regions of inflammatory cytokine genes and the
development of OSAS.
We conclude that inflammatory cytokines gene polymorphisms may
contribute to the OSAS pathogenesis. OSAS may have a strong genetic basis due
to the effects from a number of genes including those for inflammatory mediators.
Acknowledgments: Supported by an intramural grant from Warsaw Medical University in
Conflicts of interest: The authors had no conflicts of interest to declare in relation to this article.
1. Wickelgren I. Obesity: how big a problem? Science 1998; 280: 1364-1367.
2. Maes HH, Neale MC, EaVes LJ. Genetic and environmental factors in relative body weight and
human adiposity. Behav Genet 1997; 27: 325-351.
3. Popko K, Gorska E, Wasik M et al. Frequency of distribution of leptin receptor gene polymorphism
in obstructive sleep apnea patients. J Physiol Pharmacol 2007; 58 Suppl 5: 551-561.
4. Hatipo U, Rubinstein I. Inflammation and obstructive sleep apnea syndrome pathogenesis: A
working hypothesis. Respiration 2003; 70: 665-671.
5. Moldofsky H, Lue FA, Eisen J et al. The relationship of interleukin-1 and immune functions to
sleep in humans. Psychosom Med 1986; 48: 309-318.
6. Vgontzas AN, Papanicolaou DA, Bixler EO. Elevation of plasma cytokines in disorders of
excessive daytime sleepiness: Role of sleep disturbance and obesity. J Clin Endocrinol Metab
1997; 82: 1313-1316.
7. Entzian P, Linnemann K, Schlaak M et al. Obstructive sleep apnea and circadian rhythms of
hormones and cytokines. Am J Respir Crit Care Med 1996; 153: 1080-1086.
8. Papanicolaou DA, Petrides JS, Tsigos C et al. Exercise stimulates interleukin-6 secretion:
inhibition by glucocorticoids and correlation with catecholamines. Am J Physiol 1996; 271:
9. Plata-Salaman CR. Central nervous system mechanisms contributing to the cachexia-anorexia
syndrome. Nutrition 2000; 16: 1009-1012.
10. Fishman D, Faulds G, Jeffery R et al. The effect of novel polymorphism in the interleukin-6
(IL-6) gene on IL-6 transcription and an association with systemic-onset juvenile chronic
arthritis. J Clin Invest 1998; 102: 1369-1376.
11. Brandt E, Schorr U, Kunc I et al. Tumor Necrosis Factor-alpha 308 G/Apolymorphism in obese
Caucasians. Int J Obes 2001; 25: 581-585.
12. Hoffstedt J, Eriksson P, Hellstrom L, Rossner S, Ryden M, Arner P. Excessive fat accumulation
is associated with the TNF-alpha- 308G/A promoter polymorphism in women but not in men.
Diabetologia 2000; 43: 117-120.
13. Pociot F, Molvig J, Wogensen L, Worsaae H, Nerup A. TaqI polymorphism in the human
interleukin-1 beta (IL-1 beta) gene correlates with IL-1 beta secretion in vitro. Eur J Clin Invest
1992; 22: 396-402.
14. Pociot F, Ronningen KS, Bergholdt R, Lorencen T, Johannesen J, Ye K. Genetic susceptibility
markers in Danish patients with type 1 (insulin-dependent) diabetes-evidence for polygenicity
in men. Danish Study Group of Diabetes in Childhood. Autoimmunity 1994; 19: 169-178.
15. Matsusaka T, Fujikawa K, Nishio Y et al. Transcription factors NF-IL6 and NF-κB
synergistically active transcription of the inflammatory cytokines, inerleukin-6 and interleukin-
8. Proc Natl Acad Sci USA 1993; 90: 10193-10197.
16. Ray A, Prefontaine KE. Physical association and functional antagonism between the p65 Download full-text
subunit of transcription factor NF-kappa B and the glucocorticoid receptor. Proc Natl Acad Sci
USA 1994; 91: 752-756.
17. Ray A, Prefontaine KE, Ray P. Down-modulation of interleukin-6 gene expression by 17 beta-
estradiol in the absence of high affinity DNA binding by the estrogen receptor. J Biol Chem
1994; 269: 12940-12946.
18. Wilson AG, di Giovine FS, Blakemore AI, Duff GW. Single base polymorphism in the human
tumor necrosis factor alpha (TNF-alpha) gene detectable by NcoI restriction of PCR product.
Hum Mol Genet 1992; 1: 353.
19. Wilson AG, Symons JA, McDowel TL, McDevitt HO, Duff GW. Effects of a polymorphism in
the human tumor necrosis factor alpha promoter on transcriptional activation. Proc Natl Acad
Sci USA 1997; 94: 3195-3199
20. Um JY, Chung HS, Song MY, Shin HD, Kim HM. Association of interleukin-1beta gene
polymorphism with body mass index in women. Clin Chem 2004; 50: 647-650.
21. Fain JN, Cheema PS, Bahouth SW, Lloyd Hiler M. Resting release by human adipose tissue
explants in primary culture. Biochem Biophys Res Commun 2003; 300: 674-678.
22. Zhang HH, Kumar S, Barnett AH, Eggo MC. Dexamethasone inhibits tumor necrosis factor-
alpha-induced apoptosis and interleukin-1 beta release in human subcutaneous adipocytes and
preadipocytes. J Clin Endocrinol Metab 2001: 86: 2817-2825.
23. Shamsuzzaman AS, Winnicki M, Lanfranchi Pet al. Elevated C-reactive protein in patients with
obstructive sleep apnea. Circulation 2002; 105: 2462–2464.
24. Teramoto S,Yamamoto H, Ouchi Y. Increased C-reactive protein and increased plasma
interleukin-6 may synergistically affect the progression of coronary atherosclerosis in
obstructive sleep apnea syndrome. Circulation 2002; 105: 2462-2464.
Received: May 23, 2008.
Accepted: September 23, 2008.
Author’s address: Katarzyna Popko, Department of Laboratory Diagnostics and Clinical
Immunology of the Developmental Age, Warsaw Medical University, Marszalkowska 24 St., 00-
576 Warsaw; phone: +48 22 6296517; e-mail: email@example.com