Role of various cytokines have been implicated in the development and perpetuation
of Sjögren\u27s syndrome (SS), but no specific cytokine could be determined as a major contributor
to the SS. Salivary and serum interleukin 6 (IL-6) levels have been studied previously
in patients with SS, but data upon salivary and serum basic fibroblast growth
factor (bFGF) in SS are lacking. The aim of this study was to evaluate levels of salivary
and serum IL-6 and bFGF in 18 patients with SS, age range 32–79, mean 54.05 years.
Control group consisted of 23 healthy participants, mean age 25 years. Serum IL-6 and
bFGF levels were not significantly different between patients with SS and healthy controls.
Elevated levels of salivary IL-6 and bFGF in patients with SS when compared to
the healthy controls were found (p<0.001). We might speculate that higher levels of salivary
IL-6 and bFGF in patients with SS might originate from local production probably
having source in the salivary glands

Ana Cekić-Arambašin

Andrija Bošnjak

Josip Lukač

Mišo Virag

Nada Čikeš

Vanja Vučićević Boras

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Coll. Antropol. 28 Suppl. 2 (2004) 305–311UCD 616.72-002.77: 577.27Original scientific paperThe Significance of Salivary andSerum Interleukin 6 and BasicFibroblast Growth Factor Levels inPatients with Sjögren's SyndromeVanja Vu~i}evi} Boras1, Nada ^ike{2, Josip Luka~3, Ana Ceki}-Aramba{in1,Mi{o Virag4 and Andrija Bo{njak51 Department of Oral Medicine, School of Dental Medicine, University of Zagreb, Zagreb,Croatia2 Department for Clinical Immunology and Rheumatology, University Hospital Center»Zagreb«, Zagreb, Croatia3 Department for Nuclear Medicine and Oncology, University Hospital »Sestre milosrdnice«,Zagreb, Croatia4 Department for Maxillofacial Surgery, University Hospital »Dubrava«, Zagreb, Croatia5 Department of Periodontology, School of Dental Medicine, University of Zagreb, Zagreb,CroatiaA B S T R A C TRole of various cytokines have been implicated in the development and perpetuationof Sjögren's syndrome (SS), but no specific cytokine could be determined as a major con-tributor to the SS. Salivary and serum interleukin 6 (IL-6) levels have been studied pre-viously in patients with SS, but data upon salivary and serum basic fibroblast growthfactor (bFGF) in SS are lacking. The aim of this study was to evaluate levels of salivaryand serum IL-6 and bFGF in 18 patients with SS, age range 32–79, mean 54.05 years.Control group consisted of 23 healthy participants, mean age 25 years. Serum IL-6 andbFGF levels were not significantly different between patients with SS and healthy con-trols. Elevated levels of salivary IL-6 and bFGF in patients with SS when compared tothe healthy controls were found (p<0.001). We might speculate that higher levels of sali-vary IL-6 and bFGF in patients with SSmight originate from local production probablyhaving source in the salivary glands.Key words: salivary cytokines, serum cytokines, Sjögren's syndrome305Received for publication October 13, 2003IntroductionSjögren's syndrome (SS) is a chronicinflammatory disorder characterized bylymphocytic infiltration of lacrimal andsalivary glands, which results in dry eyesand mouth1. Despite the extensive world-wide literature, pathogenesis of SS re-mains poorly understood. It has been pos-tulated that a combination of immuno-logic, genetic and environmental factorsare involved in the development of SS 2.Interesting data on cytokine productionfrom the salivary glands of SS patientshave been reported in the past few years.From reviewing the published literatureit is difficult to obtain a clear picture ofthe cytokine profile in SS. This is largelydue to the major differences in techniquesand specimens used in these studies. Allthe available data clearly suggest an insitu immune response in the exocrineglands of SS patients, with the epithelialcell playing the role of antigen presentingcell. As a consequence T-cells are attra-cted and become activated. Produced cy-tokines are responsible for inflammatoryperpetuation and stimulation of B-lym-phocytes3.The proinflammatory cytokine IL-6play an important role in the inflamma-tory, autoimmune and malignant pro-cesses. IL-6 is generated by lymphocytes,endothelial cells, fibroblasts and epithe-lial cells. It is well known that collagendiseases and other autoimmune diseasesare associated with elevated levels of se-rum IL-64. Some authors5–7 found ele-vated levels of salivary IL-6 in patientswith SS, but data upon serum IL-6 in SSare scarce.The role of basic fibroblast growth fac-tor (bFGF) has been implicated in the nu-merous processes of normal and neoplas-tic tissues of the human and animalbodies. BFGF has a pleiotrophic effectsand can act as a growth or differentiationfactor depending on the cell type and lo-cal environment8. Various tissues fromectodermal, endodermal and neural ori-gin have been shown to produce bFGFand serum bFGF levels are elevated inpatients with wide spectrum of tumors9–11.There are few reports in the literaturewhich evaluate serum and salivary IL-6levels in patients with SS, but no dataupon bFGF either salivary or serum inpatients with SS could be found in theworldwide literature. The aim of thisstudy was to evaluate significance of sali-vary and serum IL-6 and bFGF levels inpatients with SS.Materials and MethodsPrior to investigation informed con-sent according to FDA12 was obtainedfrom each participant. The patient groupconsisted of 18 participants, all women,with confirmed SS according to the Vitaliet al.13, revised version of the Europeancriteria set for classification. The patientswith SS were age range 32–79 years,mean 54,05 years. Control group consist-ed of 23 participants, 15 women and 8men, mean age 25 who were healthy anddid not take any medications 1 monthprior to investigation.Whole unstimulated saliva specimenswere collected from each participantwhile they were sitting, into calibratedtubes for five minutes according to WuWang et al.14. Afterwards the saliva spec-imens were stored at –70 °C and kept un-til analysis. A 5 ml of fasting blood fromcubital vein was taken from each partici-pant between 7 and 9 A.M., frozen andstored at –70 °C until analysis.For determination of salivary and se-rum bFGF levels ELISA (Quantikine, HSFGF basic, R&D Systems, Minneapolis,USA) was performed. Concentrations ofsalivary and serum IL-6 were determinedusing an ELISA (Milenia Biotec, Bad Nau-heim, Germany). Sensitivity for bFGFwas 0.22 pg/mL and for IL-6 was 1.2pg/mL.306V. Vu~i}evi} Boras et al.: Cytokines in Sjögren's Syndrome, Coll. Antropol. 28 Suppl. 2 (2004) 305–311Statistical analysis was performed us-ing Mann Whitney U-test and valueslower than 0.001 were considered as sta-tistically significant (p<0.001). The inves-tigated parameters that did not show anysignificance were then analyzed by use ofMann Whitney U-test corrected for ties,i.e. all the values that were zero were ex-cluded from the analysis.ResultsNo significant differences in serumIL-6 values between patients with SS andcontrols could be found.No significant differences in serumbFGF between patients with SS and con-trols could be found using Mann-WhitneyU-test (Table 1).Significant difference in salivary IL-6between patients with SS and controlgroup was found by use of Mann-WhitneyU-test (p<0.001) (Table 2).Significant difference in salivary bFGFvalues between patients with SS and con-trol group was found when Mann-Whit-ney U-test corrected for ties was perfor-med (p<0.001) (Table 3).DiscussionFox et al.15 evaluated IL-6 in labialsalivary glands in patients with SS with307V. Vu~i}evi} Boras et al.: Cytokines in Sjögren's Syndrome, Coll. Antropol. 28 Suppl. 2 (2004) 305–311TABLE 1DIFFERENCE OF SERUM BASIC FIBROBLAST GROWTH FACTOR (bFGF) BETWEENPATIENTS WITH SJÖGREN'S SYNDROME (SS) AND CONTROL GROUPDG NMeanrankSumof ranksMann-WhitneyU-testpbFGF(serum)SS 18 17.78 320.00Controls 17 18.24 310.00 149.000 0.895Total 35TABLE 2DIFFERENCE OF SALIVARY INTERLEUKIN 6 (IL-6) BETWEEN PATIENTS WITHSJÖGREN'S SYNDROME (SS) AND CONTROL GROUPDG NMeanrankSumof ranksMann-WhitneyU-testpIL-6(saliva)SS 18 27.61 497.00Controls 23 15.83 364.00 88.000 0.001Total 41TABLE 3DIFFERENCE OF SALIVARY BASIC FIBROBLAST GROWTH FACTOR (bFGF) BETWEEN PATIENTSWITH SJÖGREN'S SYNDROME (SS) AND CONTROL GROUP (MANN-WHITNEY U-TEST CORRECTEDFOR TIES)DG NMeanrankSumof ranksMann-WhitneyU-testpbFGF(saliva)SS 8 14.44 115.50 8.500 0.001Controls 11 6.77 74.50Total 19oligonucleotide probes and in situ hybrid-ization technique which demonstratedthat IL-6 mRNA was found both in infil-trating lymphocytes and the epithelialcells. Furthermore, Fox et al.5 reportedthat IL-1 and IL-6 mRNA were found insalivary gland epithelial cells using re-verse transcriptase and polymerase chainreaction. Oxholm et al.16 found greaterstaining of duct cells with IL-6, IFN-,and TNF- in labial salivary glands of SSpatients when compared to the normalglands. Grisisus et al.7 found that sali-vary IL-6 concentration was elevated inpatients with SS when compared to thehealthy subjects. As seen in our study,the same authors found that higher IL-6concentrations were associated with in-creased disease activity. Also, salivarylevels were higher than serum levels inpatients with SS, and these findings sup-port the contention that salivary IL-6 isproduced locally. Tischler et al.6 reportedthat salivary IL-6 levels in the wholemixed saliva were significantly elevatedin patients with primary SS when com-pared to the patients with dry mouth, butno significant differences in serum IL-6were noticed between two groups. Sameauthors also stated that elevated salivaryIL-6 levels are result of a local produc-tion. Study of Fox et al.15 confirmed thatsalivary IL-6 levels were significantly ele-vated in the parotid saliva of patientswith SS when compared to the healthycontrols and patients with primary bi-liary cirrhosis (PBC). On the contrary, se-rum IL-6 were elevated in both SS andPBC patients, although those levels wereless than those found in the saliva of thesame patients. Sun et al.17 stated that in-creased gene expression of IL-6 wasfound in patients with primary SS, butthat the IL-6 was also found in minor sal-ivary glands in control subjects. Authors5–7stated that increased salivary IL-6 levelscould accurately monitor salivary glandinvolvement in SS patients. We have alsotried to evaluate the role of either sali-vary and/or serum bFGF levels. The re-sults of this study show that salivarybFGF levels in patients with SS are ele-vated when compared to the controlgroup. It is interesting to note that in pa-tients with elevated salivary bFGF a fin-ding of more active disease together withadditional systemic diseases was noticed.Five patients with more active SS andsignificantly elevated salivary bFGFshowed clinical and/or laboratory signs ofdisease such as elevated erythrocyte sedi-mentation rate, elevated levels of C-reac-tive protein, decreased hemoglobin levels.Patient GZ had also recurrent herpeszoster infection together with SS. PatientSD had also brain hemangioblastomaand skin melanoma. Patient BA had ele-vated serum IgM together with skin tu-berculosis and liver cirrhosis. PatientsPM and CD had more active disease withpersistent SS-A levels more than 100 RU.Serum bFGF levels were not signifi-cantly different in patients with SS whencompared to the control group. These re-sults would implicate a local productionof bFGF in patients with SS, but exactplace of bFGF origin remains to be eluci-dated. In addition, elevated salivarybFGF might be a result of a chronic in-flammation, which might play a role inthe fibrosis seen in the later stages ofgland involvement. Furthermore, to ourknowledge no literature data upon sali-vary and serum bFGF exist and our re-sults are difficult to compare. Furtherstudies involving parotid and whole sa-liva in patients with SS with regard toIL-6 and bFGF levels are needed.AcknowledgmentThe authors wish to thank Mrs. La-dika-Davidovi} for excellence in perform-ing ELISA and dr. Iva Alajbeg for herhelp in statistical analysis.308V. Vu~i}evi} Boras et al.: Cytokines in Sjögren's Syndrome, Coll. Antropol. 28 Suppl. 2 (2004) 305–311R E F E R E N C E S1. FOX, R. I., H. KANG: Rheumatology. (W.B.Saunders, Philadelphia, 1993). — 2. TZIOUFAS, A.G., H. M. MOUTSOPOULOS: Rheumatology. (Mosby,London, 1998). — 3. FOX, P. C., P. M. SPEIGHT, Crit.Rev. Oral Biol. Med., 7 (1996) 144. — 4. KISHIMOTO,T., Blood, 74 (1989) 1. — 5. GRISIUS, M. M., D. K.BERMUDEZ, P. C. FOX, J. Rheumatol., 24 (1997)1089. — 6. TISCHLER, M., I. YARON, I. SHIRAZI, Y.YOSIPOV, M. YARON, Rheumatol. Int., 18 (1999)125. — 7. FOX, P. C., M. M. GRISIUS, D. BERMU-DEZ, D. SUN: (Plenum Press, New York, 1998). — 8.PARTRIDGE, M., D. CHANTRY, M. TURNER, M.FELDMANN, J. Invest. Dermatol., 96 (1991) 771. —9. CHODAK, G., V. HOSPELHORN, S. JUDGE, R.MAYFORTH, H. KOEPPEN, J. SASSE, Cancer Res.,48 (1988) 2083. — 10. WATANABE, H., A. HORI, M.SENO, Biochem. Biophys. Res. Commun., 175 (1991)229. — 11. BLOTNICK, S., G. E. PEOPLES, M. R.FREEMAN, T. J. EBERLEIN, M. KLAGSBURN,Proc. Natl. Acad. Sci. USA, 91 (1994) 2890. — 12.FDA: Guidance for institutional Review Boards andclinical investigators. (FDA, 1998). — 13. VITALI, C.,S. BOMBARDIERI, R. JONSSON, Ann. Rheum. Dis.,61 (2002) 554. — 14. WU WANG, C. Y., M. PATEL, J.FENG, M. MILLES, S. L. WANG, Arch. Oral Biol., 40(1995) 1093. — 15. FOX, R. I., H. KANG, D. ANDO, J.ABRAMS, E. PISA, J. Immunol., 152 (1994) 5532. —16. OXHOLM, P., T. E. DANIELS, K. BENDTZEN,Autoimmunity, 12 (1992) 185. — 17. SUN, D., M. R.EMMERT-BUCK, P. C. FOX, Autoimmunity, 28(1998) 125.V. Vu~i}evi} BorasDepartment of Oral Medicine, School of Dental Medicine, University of Zagreb,Gunduli}eva 5, 10000 Zagrebe-mail: vvboras@hotmail.comZNA^AJ SALIVARNOG I SERUMSKOG INTERLEUKINA 6 I BAZI^NOGFAKTORA RASTA FIBROBLASTA U BOLESNIKA SA SJÖGRENOVIMSINDROMOMS A @ E T A KPretpostavlja se da uloga citokina mo`e biti uklju~ena u nastanak i razvoj Sjögre-nova sindroma (SS), ali do sada niti jedan specifi~ni citokin nije naveden kao glavnifaktor koji doprinosi SS. Salivarni i serumski interleukin 6 (IL-6) su ve} prije ispitivaniu oboljelih od SS, dok podataka o salivarnom i serumskom bazi~nom faktoru rasta fi-broblasta (bFGF) u oboljelih od SS nema. Cilj ovog ispitivanja je bio ustanoviti vri-jednosti salivarnog i serumskog IL-6 i bFGF u 18 bolesnika sa SS, starosti 32–79, pro-sje~ne starosti 54.05 godina. Kontrolna skupina se sastojala od 23 zdrava ispitanika,srednje dobi 25 godina. Serumske vrijednosti IL-6 i bFGF se nisu zna~ajno razlikovaleu oboljelih od SS u odnosu na kontrolnu skupinu. Povi{ene vrijednosti salivarnog IL-6 ibFGF su ustanovljene u oboljelih od SS u odnosu na kontrolnu skupinu (p<0.001).Mo`emo zaklju~iti da povi{ene vrijednosti salivarnog IL-6 i bFGF u oboljelih od SS-amogu biti posljedica lokalne proizvodnje, vjerojatno unutar `lijezda slinovnica.309V. Vu~i}evi} Boras et al.: Cytokines in Sjögren's Syndrome, Coll. Antropol. 28 Suppl. 2 (2004) 305–311

The Significance of Salivary and Serum Interleukin 6 and Basic Fibroblast Growth Factor Levels in Patients with Sjögren\u27s Syndrome

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