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Significance of serum interleukin-8 levels in patients with Behcet's disease: high levels may indicate vascular involvement

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Background  Interleukin-8 (IL-8) has been shown previously to associate with different individual clinical manifestations and activity of Behcet's disease (BD), but its association with vascular involvement has not been established.Methods 
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   © 2009 The International Society of DermatologyInternational Journal of Dermatology   2009, 48  , 259–264  259  Abstract  Background  Interleukin-8 (IL-8) has been shown previously to associate with different individual clinical manifestations and activity of Behcet’s disease (BD), but its association with vascular involvement has not been established.  Methods  Forty-five untreated patients with BD and 29 healthy individuals were included in the study. The activity of patients was based on the existence of two or more symptoms and a statistically significantly high Behcet’s Disease Activity Index (BDAI) at the time of the study. IL-8, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) results were evaluated with respect to activity, vascular involvement, and other specific individual clinical manifestations of the disease.  Results  IL-8 levels were found to be significantly elevated in active BD compared with inactive BD (  P   = 0.006) and healthy controls (  P   = 0.000), with median values of 267 (53–2000), 137 (52–290), and 58 pg/mL (53–160 pg/mL), respectively. Unlike ESR and CRP, IL-8 levels showed a high correlation with BDAI scores (  r   = 0.743, P   = 0.00) and the number of active clinical manifestations (  r   = 0.646, P   = 0.00). Serum levels of IL-8 were increased in patients with oral ulcers, genital ulcers, eye lesions, and vascular lesions, with median values and significance levels of 254.5 (53–2000), P   = 0.05; 254.5 (52–1400), P   = 0.03; 254.5 (72–2000), P   = 0.029; and 593 pg/mL (110–2000 pg/mL), P   = 0.001, respectively. In addition, IL-8 levels in the active patient group with vascular involvement were significantly higher than the levels in those without vascular involvement.  Conclusion  Serum IL-8 levels are increased in the active phase of BD. This marker may be useful in the early detection of vascular involvement.  BlackwellPublishingLtdOxford,UKIJDInternationalJournalofDermatology0011-90591365-4632©2008TheInternationalSocietyofDermatologyXXX  Report  IL-8levelsinBehcet’sdisease  KartalDurmazlar   etal.Report  Significance of serum interleukin-8 levels in patients with Behcet’s disease: high levels may indicate vascular involvement  Selda Pelin Kartal Durmazlar, MD  , Gul Bahar Ulkar, MD  , Fatma Eskioglu, MD  , Semih Tatlican, MD  , Ali Mert, MD  , and Ahmet Akgul, MD  From the Departments of Dermatology and Microbiology, Ankara Yıldırım Beyazıt Training and Research Hospital, and Department of Cardiovascular Surgery, Ministry of Health Ankara Yuksek Ihtisas Education and Research Hospital, Ankara, Turkey  Correspondence  Selda Pelin Kartal Durmazlar, MD  Tunali Hilmi Cad. 83/4 Kavaklidere Ankara Turkey E-mail: pelin@drcom  Introduction  Behcet’s disease (BD) is manifested by a triad of relapsingiritis, aphthous stomatitis, and genital ulcers.   1  The diagnosisis established by clinical criteria only.   2  It is now recognized as amultisystemic, immunoinflammatory disorder involvingvessels of all sizes.   3  The disease is most prevalent in Mediterra-nean countries, the Middle East, and Japan, but has aworldwide distribution. Vasculo-Behcet’s disease (VBD),which involves the arterial and venous system, is found in 15–38% of patients with BD.   4  Three major manifestations of VBD have been identified: venous occlusion, arterial occlusion,and aneurysm formation, with a clear preponderance of venous lesions (88%) compared with arterial involvement(12%).   5  The coexistence of arterial and venous involvementis not frequent and is one of the major causes of morbidityand mortality.   6  Venous involvement, including superficialthrombophlebitis and deep venous thrombosis, is a characteristicmanifestation. The occlusion of major veins, arteries, andaneurysms often causes bleeding, infarction, organ failure,and restricted movement of the extremities. The rupture of suchaneurysms may be fatal. Vascular lesions in the lung, includingthrombosis, aneurysm, and arteriobronchial fistula, causerecurrent episodes of dyspnea, cough, chest pain, and hemo-ptysis.   7  Cardiac manifestations, such as coronary and valvulardisease, occur in some patients.   8,9  Computed tomography,magnetic resonance imaging, angiography, and ventilation–perfusion scintigraphy are useful for the detection of vascularlesions in suspected patients.   10  Surgery should be avoided on acute exacerbation of BDbecause of progressive graft thrombosis and the formation of new aneurysms at the anastomosis;   4  therefore, the activity of the disease is very important for patients who are candidatesfor elective vascular operations.Although the etiopathogenesis of BD has not yet beenclarified, different immunologic abnormalities have beenreported, with increased spontaneous secretion of tumornecrosis factor-  α  (TNF-  α  ), interleukin-6 (IL-6), and interleukin-8   International Journal of Dermatology   2009, 48  , 259–264© 2009 The International Society of Dermatology   260Report  IL-8 levels in Behcet’s disease  Kartal Durmazlar   et al.  (IL-8) in monocyte cultures obtained from BD patients.   11  Among the pro-inflammatory chemokines, IL-8 plays a roleof particular importance in BD, as elevated serum levels havebeen reported by many investigators.   12–18  Activated peripheralpolymorphonuclear leukocytes (PMNLs) and the infiltrationof PMNLs into lesions are characteristic findings of BD. Theincrease in the level of IL-8 up-regulates neutrophil chemo-taxis, as IL-8 mRNA expression has been reported to be moreprominent in patients with active BD than in patients withinactive disease.   19  IL-8, a major chemokine known as neutrophilactivating factor, attracts and activates leukocytes and hasbeen assumed to represent a notable link between immunesystem activation and endothelial alterations in BD. Vascularinvolvement in BD is a life-threatening complication of thedisease, and so early diagnosis is very important.Although IL-8 has been shown previously to be associatedwith different individual clinical manifestations   18  and activityof BD, its association with vascular involvement has not beenestablished. Therefore, the present study was conducted toevaluate IL-8 as a serologic marker for the assessment of theactivity of BD with vascular involvement and other individualclinical manifestations. Serum levels of IL-8 were comparedwith values of C-reactive protein (CRP), an acute phasereactant, and the erythrocyte sedimentation rate (ESR). Theresults were controlled with healthy individuals.  Materials and Methods   Subjects  Forty-five patients with BD who attended the Department of Dermatology (20 men and 25 women; mean age, 31.62 ± 6.82 years) and were diagnosed according to the International Study Group criteria,   2  and 29 sex- and age-matched healthy control subjects (14 men and 15 women; mean age, 28.67 ± 5.52 years), were included in the study. The control group consisted of healthy volunteers working in our hospital. There was no potential bias when obtaining the control group. Patients who had other illnesses, such as autoimmune disease, that might affect the outcome and patients taking any medication affecting the immune system were excluded from the study. Informed consent was obtained from all subjects. Patients were grouped according to their active signs and vascular involvement. A pathergy test was performed and the number of active manifestations was recorded for each patient (ranging between one and five).   Determination of disease activity  As there is no accepted specific clinical activity scoring system or laboratory screening profile for BD, patients were considered to have active disease if they showed the existence of two or more symptoms, with worsening of clinical symptoms and a lack of well-being, at the time of the study. The Behcet’s Disease Activity Index (BDAI) was also measured in our patients, according to the method presented by Bhakta et al   .   20  and described in detail by Lawton et al   .   21  Overall disease activity scores were derived from the addition of the scores according to the evaluation of the duration of each clinical feature (0–4) and the patients’ and clinicians’ impression of the disease activity. As it has been shown that the inclusion of ESR and CRP measurements does not add significantly to the overall measurement of disease activity,   21  and they were not included as criteria in BDAI,   20,21  ESR and CRP were not used in the determination of the activity of the disease. Patients who had no BD symptoms over a 4-week period, or less than two symptoms with healing and an overall status of wellbeing, were grouped as inactive BD.   Vascular examination  All patients and healthy volunteers were examined carefully for vascular involvement. Venous and arterial systems were examined using Doppler ultrasonography and computed tomography. All clinical and laboratory findings indicative of vascular involvement were noted, and the Department of Cardiovascular Surgery was consulted.   Detection of IL-8 by enzyme-linked immunosorbent assay (ELISA)  The IL-8 level was measured using ELISA with an IL-8/NAP1 ELISA kit (Bender Medsystems, Vienna, Austria). Venous blood samples (5 cm   3  ) were drawn from patients and healthy volunteers. The serum was separated by centrifugation at g   value: 274 for 10 min and stored at –70 °  C until use.   Statistical analysis  The results were analyzed using SPSS for Windows®, Version 11.5 (SPSS Inc., Chicago, IL, USA). Each variant was evaluated by one-sample Kolmogorov–Smirnov test for compatibility with a normal distribution. As the data did not fit a normal distribution, the nonparametric Kruskal–Wallis test was used for intergroup comparison and the Mann–Whitney U   -test or Bonferroni-corrected Mann–Whitney U   -test for between-group comparison, as indicated. The relationship between the variables was evaluated by Spearman correlation. The level of significance was set at 0.05, but, in the Bonferroni-corrected test, the significance threshold of 0.05 yielded to a value of 0.0167. The data are presented as median values and their individual ranges (in parentheses).  Results  The numbers of patients suffering from individual activeclinical manifestations of the disease to varying degrees wereas follows: 30 with oral ulceration (66.7%), 32 with genitalulceration (71.1%), 26 with ocular involvement (57.8%), 13with vascular involvement (28.8%), and 24 with cutaneouslesions (53.3%), such as erythema nodosum (EN) andpapulopustular eruption (PPE). Twenty-eight patients (62.2%)were found to have a positive pathergy test. According to ourevaluation scale, 33 patients were considered to have active   © 2009 The International Society of DermatologyInternational Journal of Dermatology   2009, 48  , 259–264  261  Kartal Durmazlar   et al.IL-8 levels in Behcet’s disease  Report  and 12 patients to have inactive disease. Consistent with this,the active group had a statistically significantly higher BDAIscore (median, 14; range, 8–25) than the inactive group (median,3; range, 1–5) (  P  = 0.00) (Table 1). According to the vascularexamination, venous involvement was found in eight patientsin the active group and in five patients in the inactive group;other types of vascular involvement, such as arterial occlusionand aneurysm, were not found. Five of the eight patients withactive BD had deep venous thrombosis and three had superficialthrombophlebitis. All of the patients with venous involvementin the inactive group had superficial thrombophlebitis.In the intergroup comparison of active BD, inactive BD andhealthy controls according to the serum levels of IL-8 andCRP, and ESR, a significant difference was detected for eachparameter (  P  = 0.00) (Table 1). To determine which group/ groups caused the difference, between-group comparisonswere performed. IL-8 levels were found to be elevated inactive BD compared with inactive BD and healthy controls.IL-8 levels were also found to be elevated in inactive BDcompared with healthy controls. IL-8 levels showed a positiveassociation with disease activity. ESR was found to be ele-vated in active and inactive disease when compared withhealthy controls, but no difference was detected betweenactive and inactive disease groups. CRP was not found to beelevated in active disease compared with inactive disease andhealthy controls (Table 1). Unlike ESR and CRP, IL-8 levelswere found to show a high correlation with BDAI scores(  r  = 0.743, P  = 0.00) and the number of active clinicalmanifestations (  r  = 0.646, P  = 0.00) (Table 2).The evaluation of the association between specific individ-ual clinical manifestations and serum levels revealedincreased IL-8 levels in patients with oral ulcers (  P  = 0.05),genital ulcers (  P  = 0.033), eye lesions (  P  = 0.029), and vascu-lar lesions (  P  = 0.001) (Table 3, Fig. 1). No association wasfound between IL-8 levels and EN/PPE or the presence of apositive pathergy test.The serum levels of IL-8 in active BD patients with vascularinvolvement were found to be increased four- to five-fold,with a median of 974 pg/mL (range, 396–2000 pg/mL),compared with those in active BD patients without vascularinvolvement (200 pg/mL; range, 53–724 pg/mL) (  P  = 0.00).In addition, the serum levels of IL-8 in inactive BD patientswith vascular involvement were found to be increased two-fold, with a median of 162.50 pg/mL (range, 52–148 pg/mL),compared with those in inactive BD patients without vascularinvolvement (median, 98 pg/mL; range, 52–148 pg/mL)(  P  = 0.15) (Table 4). CRP and ESR showed no significantassociation with patients with vascular involvement.  Discussion  The main pathological processes in BD include an inflammatoryprocess of the small arteries and veins and thrombosis as aresult of vasculitis of the vasa vasorum.   17  Histopathologic Table 1 Comparison of the serum interleukin-8 (IL-8), C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR) values, and Behcet’s Disease Activity Index (BDAI) scores, between the groups Median (range)IL-8 (pg/mL)CRP (mg/L)ESR (mm/h)BDAI score Group I (active BD) ( n   = 33)267 (53–2000)14.8 (3.1–99.9)24 (2–95)14 (8–25)Group II (inactive BD) ( n   = 12)137 (52–290)6.0 (3.1–33.6)27 (8–49)3 (1–5)Group III (healthy control) ( n   = 29)58 (53–160)3.1 (3.1–9.9)6 (2–18)Significance for groupsI–II–III* P   = 0.00* P   = 0.00* P   = 0.00I–II† P   = 0.006† P   = 0.035† P   = 0.849‡ P   = 0.00I–III† P   = 0.000† P   = 0.027† P   = 0.000II–III† P   = 0.001† P   = 0.000† P   = 0.001 * P  significantly different by Kruskal–Wallis test ( P  < 0.05).† P  significantly different by Bonferroni-corrected Mann–Whitney U  -test ( P  < 0.0167).‡ P  significantly different by Mann–Whitney U  -test ( P  < 0.05).Data are presented as the median values with their individual ranges in parentheses. Table 2 Relationship between the variables VariableSpearman correlation coefficient ( r  )  P   value IL-8–BDAI0.7430.00*ESR–BDAI–0.2050.177CRP–BDAI0.2190.148IL-8–Total+0.5550.00* BDAI, Behcet’s Disease Activity Index; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; IL-8, interleukin-8; Total+, number of active clinical manifestations for each patient.* P  < 0.05.   International Journal of Dermatology   2009, 48  , 259–264© 2009 The International Society of Dermatology   262Report  IL-8 levels in Behcet’s disease  Kartal Durmazlar   et al.  studies have revealed cellular infiltrations, consisting of lymphocytes, plasmocytes, monocytes, and PMNLs to varyingdegrees, depending on the stage of the BD lesion. As cytokinesare involved in the regulation of the functions of lymphocytesand phagocytes, they play an important role in the pathogenesisof the disease.   22  Zouboulis et al   .   23  reported that endothelialcells could be responsible, at least in part, for the enhanced IL-8 secretion in the active stage of the disease, but Mantas et al   .   24  reported the cellular source of IL-8 to be diverse in BD, witha possible major contribution by lymphocytes.The observations of Kobayashi et al   .   25  in six patients withaneurysm as a result of BD suggest that VBD should beclassified as a neutrophilic vasculitis targeting the vasa vasorum.Aneurysm formation may be related to the degeneration of the arterial wall caused by inflammation of the vasa vasorum.The chemotactic and phagocytic activities of neutrophils in Table 3 Association of interleukin-8 (IL-8) levels (pg/mL) with individual clinical manifestations and pathergy reaction ManifestationNo. of patientsIL-8 level in patients with manifestationIL-8 level in patients without manifestation P   valueMedian(Range)Median(Range) Oral ulcer30254.5(53–2000)148(52–1400)0.05*Genital ulcer32254.5(53–2000)148(52–1100)0.033*Ocular lesion26254.5(72–2000)148(52–1400)0.029*Vascular lesion13593(110–2000)160(52–724)0.001*EN/PPE24209(53–2000)180(56–1100)0.856Pathergy-positive28209(53–2000)160(52–1400)0.266 EN/PPE, erythema nodosum/papulopustular eruption.* P  < 0.05 by Mann–Whitney U  -test.Data are presented as the median values with their individual ranges in parentheses. Figure 1 Median interleukin-8 (IL-8) levels (pg/mL) as a function of the absence and presence of individual clinical manifestations and positive pathergy reaction. EN/PPE, erythema nodosum/ papulopustular eruption IL-8 levelMedianRange  P   value Active BD ( n   = 3)With vascular involvement ( n   = 8)974396–20000.00*Without vascular involvement ( n   = 25)20053–724Inactive BD ( n   = 12)With vascular involvement ( n   = 5)162.5110–2900.15*Without vascular involvement ( n   = 7)9852–148 * P  < 0.05 by Mann–Whitney U  -test.Data are presented as the median values with their individual ranges. Table 4 Comparison of interleukin-8 (IL-8) levels (pg/mL) in active and inactive Behcet’s disease (BD) patients in the presence or absence of vascular involvement  © 2009 The International Society of DermatologyInternational Journal of Dermatology   2009, 48 , 259–264 263 Kartal Durmazlar   et al.IL-8 levels in Behcet’s disease Report patients with BD have been reported to be high, 26  and Sahin et al  . 27  have demonstrated an enhanced interaction of neutrophils with cultured endothelial cells after pretreatmentwith serum from patients with BD. IL-8 secretion on incuba-tion of human dermal microvascular endothelial cells withserum from BD patients indicates that chemotaxis is an initialprocess of inflammation, and suggests that serum factors,such as circulating anti-endothelial antibodies, 28  may provokea rapid tissue response in BD. These antibodies do not producea cytotoxic effect, but activate endothelial cells to producecytokines; therefore, these cytokines are elevated in the sera of patients with active BD. 29  IL-8 up-regulates neutrophil chemo-taxis, as mRNA expression has been reported to be moreprominent in patients with active BD than in patients withinactive disease. 19  Katsantonis et al  . 30  have reported IL-8 to bea reliable serum marker for the assessment of the activity of BD in the follow-up of clinical and therapeutic studies.Gur-Toy et al  . 18  have reported that serum IL-8 levels displaya positive association with a number of active manifestations.According to individual comparisons of the clinical manifes-tations, IL-8 levels were found to have a significantly highassociation with oral ulcers, skin lesions, and ocular involve-ment; however, no significant association was detected betweenIL-8 levels and vascular involvement. The main limitation of this study was that none of the patients were untreated.Medications, such as glucocorticosteroids and immunu-suppressives, 31  reduce transcription of the pro-inflammatorycytokines (IL-8, TNF- α , IL-1, etc.). Colchicine is generallyused for the treatment of BD, and it has been shown to reducethe elevated levels of IL-8 in patients with familial Mediterraneanfever. 32  Therefore, studies including patients taking suchdrugs cannot reflect the true levels of cytokines. A theory canbe proposed to explain this situation: immunosuppressivetreatment may affect the levels of cytokines, which, in turn,may affect the outcome of the study.In the current study, serum levels of IL-8 were found toshow a positive correlation with BDAI scores and the totalnumber of active clinical manifestations. Significantly higherserum IL-8 levels were found in the active BD group than inthe inactive BD and control groups. These results are consist-ent with the study of Katsantonis et al  ., 30  which indicated thatIL-8 was a reliable marker for the activity of disease. The evalu-ation of the association between specific individual clinicalmanifestations and serum levels revealed increased IL-8 levelsin patients with oral ulcers, genital ulcers, eye lesions, and vascularlesions. No significant association was detected between IL-8levels and EN/PPE or positive pathergy reaction. ESR and CRPshowed no significant correlation with disease activity, butESR was independently associated with BD, as elevated levelswere detected in patients relative to healthy controls.In addition, extremely high serum levels of IL-8 were foundin active BD patients with vascular involvement, supportingthe theory 23  that endothelial cells may be responsible, in part,for the secretion of IL-8 in the active phase of BD. Endothelialinjury is a characteristic finding in BD, and can be observedeven in patients with no clinical vascular involvement. 33–35 According to our results, four- to five-fold increased levels of IL-8 in the active phase may indicate additional endothelialinjury in any part of the body. This marker may be useful forthe early detection of vascular involvement and activity of BDin patients who are candidates for elective vascular operations.Treatment with glucocorticosteroids, colchicine andimmunosuppressives in BD affects the level of cytokines. Thisis why we chose a study group of untreated BD patients. Thisselection criterion decreased the number of patients in ourstudy population and also decreased the number of patientswith vascular involvement, a possible study limitation. Conclusion The vascular findings in 45 untreated BD patients haveshown that serum IL-8 levels increase in the active phase of the disease, and four- to five-fold increased levels of IL-8 inactive BD may indicate endothelial injury in any part of thebody. This marker may be useful for the early detection of venous involvement, and further investigation is mandatoryin patients with arterial involvement. High serum IL-8 levelsare a reliable marker for the detection of the activity of disease.Our study may provide additional benefit for the subtyping of active and inactive BD. Acknowledgments The authors are grateful to Banu Tabanli (Hacettepe UniversityFaculty of Medicine, Department of Biostatistics, Ankara,Turkey) for statistical editing and Dr Murat Ozeren (MersinUniversity Faculty of Medicine, Department of CardiovascularSurgery, Mersin, Turkey) for editorial assistance. References 1Behcet H. Über rezidivierende, aphtöse, durch ein virus verursachte Geshwüre am Mund am Auge und an den Genitalien. Dermatol Wochenschr  1937; 105 : 1152–1157.2International Study Group for Behcet’s disease. Criteria for diagnosis of Behcet’s disease. Lancet   1990; 335 : 1078–1080.3Rizzi B, Bruno S, Dammacco R. Behcet’s disease: an immune-mediated vasculitis involving vessels of all sizes. Int J Clin Lab Res  1997; 27 : 225–232.4Ozeren M, Mavioglu I, Dogan OV, et al  . Reoperation results of arterial involvement in Behcet’s disease. Eur J Vasc Endovasc Surg   2000; 20 : 512–516.5Koc Y, Güllü I, Akpek G, et al  . Vascular involvement in Behcet’s disease.  J Rheumatol   1992; 19 : 402–410.6Yazici H, Basaran G, Hamuryudan V, et al  . The ten-year mortality in Behcet’s syndrome. Br J Rheumatol   1996; 35 : 139–141.
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