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Serum interferon-alpha level in first degree relatives of systemic lupus erythematosus patients: Correlation with autoantibodies titers

Serum interferon-alpha level in first degree relatives of systemic lupus erythematosus patients: Correlation with autoantibodies titers
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  ORIGINAL ARTICLE Serum interferon-alpha level in first degree relativesof systemic lupus erythematosus patients: Correlationwith autoantibodies titers Dina Shahin  a, * , Ahmed M. El-Refaey  b , Amany K. El-Hawary  b ,Adel Abdel Salam  a , Sherine Machaly  c , Nashwa Abousamra  d ,Reham M. El-farahaty  d a Rheumatology and Immunology, Internal Medicine Department, Mansoura School of Medicine, Egypt b Pediatrics and Pediatric Nephrology, Mansoura University Children’s Hospital, Egypt c Rheumatology and Rehabilitation Department, Mansoura School of Medicine, Egypt d Haematology Unit, Clinical Pathology Department, Mansoura School of Medicine, Egypt Received 11 May 2011; accepted 12 June 2011Available online 28 July 2011 KEYWORDS Interferon- a ;Systemic lupus erythemato-sus;First degree relatives;Autoantibodies Abstract  Background and objectives:  Interferon- a  (IFN- a ), a cytokine with both antiviral andimmune-regulatoryfunctions,wassuggestedasausefultoolwhichcanevaluatecurrentsystemiclupuserythematosus(SLE)diseaseactivityandidentifypatientswhoareatriskoffuturediseaseflares.Inthecurrent study, serum IFN- a  levels and associated demographic, and serological features in EgyptianSLE patients and their first degree relatives (FDRs) in comparison to unrelated healthy controls(UHCs) were examined, in order to identify individuals at the greatest risk for clinical illness. Abbreviations : IFN- a , interferon- a ; SLE, systemic lupus erythemato-sus; FDRs, first degree relatives; UHCs, unrelated healthy controls;DCs, dendtritic cells; ANA, antinuclear antibodies; ds DNA, antidouble stranded DNA; ACR, American College of Rheumatology;ILE, incomplete lupus erythematosus; ELISA, enzyme linked immunesorbent assay; Hb, hemoglobin; WBCs, white blood cell count; ESR,erythrocyte sedimentation rate; Ig, immunoglobulin.*Corresponding author. Tel.: +2 0145790767.E-mail address: dinashahin68@yahoo.com (D. Shahin).1110-8630    2011 Ain Shams University. Production and hosting byElsevier B.V. All rights reserved.Peer review under responsibility of Ain Shams University.doi:10.1016/j.ejmhg.2011.06.009 Production and hosting by Elsevier The Egyptian Journal of Medical Human Genetics (2011)  12 , 139–146 Ain Shams University The Egyptian Journal of Medical Human Genetics www.ejmhg.eg.netwww.sciencedirect.com  Methods:  In a cross-sectional study, blood samples were drawn from 54 SLE patients, 93 of theirFDRswhoconsentedtoenrollintothestudyand76UHCs.MeasurementofserumIFN- a byamod-ifiedELISAwascarriedout.DatawereanalyzedforassociationsofserumIFN- a levelswithautoan-tibodies titer. Results:  MeanserumIFN- a inFDRswasstatisticallyhigherthantheUHCsandlowerthaninSLEpatients ( P  < 0.0001) and it was correlated with ANA titer ( r  = 0.6,  P  < 0.0001) and anti ds DNAtiter ( r = 0.62, P  < 0.0001). Conclusion:  IFN- a  is a crucial player in the complicated autoimmune changes that occur in SLEandserumIFN- a canbeausefulmarkeridentifyingpersonswhoareatriskoffuturediseasedevelop-ment.   2011 Ain Shams University. Production and hosting by Elsevier B.V. All rights reserved. 1. Introduction Systemic lupus erythematosus (SLE) is a multisystem autoim-munediseasecharacterizedbywidespreadimmunologicabnor-malities and multiple organ involvement [1]. The precisepathogenesis of SLE remains unknown, however, genetic fac-tors in the presence of a permissive environment are involved[2].Oneofthefactorssuggestedtoplayaroleinthepathogenesisof SLE is interferon- a  (IFN- a ), a cytokine with both antiviraland immune-regulatory functions [3], which has been found inincreased levels in SLE patients [4–9]. The potential role of thiscytokineinSLEdevelopmentisfurthersuggestedbythefindingthat SLE patients produce IFN- a  in an abated fashion and thiscytokineinducesmonocytestomatureintodendriticcells(DCs)[10]; a key regulator of antigen presentation.A causative role for IFN- a  in SLE is suggested moredirectly by the observation that IFN- a  therapy in patients withnonautoimmune disorders, induced autoantibody production,including antinuclear antibodies (ANA) and anti doublestranded DNA (ds DNA), and occasionally also a lupus-likesyndrome [11–13] which typically resolved after IFN- a  therapydiscontinuation [13,14]. Furthermore, IFN- a  administrationfrequently lead to other autoimmune disorders such as im-mune mediated thyroid diseases [15], type I diabetes mellitus[16], multiple sclerosis and inflammatory arthritis [17]. SLE is a familial disease and SLE family members havehighersusceptibilitytodevelopSLEandnon-SLEautoimmunedisorders [18,19], and showed more prevalent autoantibodiesthan the unrelated healthy population [20–22]. Abnormallyhigh levels of serum IFN- a  were frequently found in healthyfirst degree relatives (FDRs) of SLE patients [23]. Advancesin genetics, immunology and environmental epidemiology sug-gestthatearlyorevenpreclinical identificationofindividualsathigh risk to evolve into lupus may be feasible [20,24,25].In the current study, we examined serum IFN- a  levels andassociated demographic, immunologic and laboratorial fea-tures in Egyptian SLE patients and their FDRs in comparisonto unrelated healthy controls (UHCs) in order to identify indi-viduals at the greatest risk for clinical illness. 2. Patients and methods 2.1. Study design In a cross-sectional analysis, SLE patients, who were consecu-tively seen in, or referred to the Rheumatology and Immunol-ogy clinics – Mansoura University Hospital and MansouraUniversity Children’s Hospital between September 2009– 2010 were invited to participate if they had at least one siblingwho or their guardians consented to enroll in the study.The study was approved by the Ethical Committee of Mansoura School of Medicine and informed consent wasobtained from every study participant or their guardians. Allthe clinical data were assessed at the time of blood donationand all the demographic, clinical, and serological characteris-tics of the studied subjects were evaluated and recorded by arheumatologist. 2.2. Study population The studied subjects were divided into three categories:(1) SLE patients: All included lupus patients fulfilled the1997 American College of Rheumatology (ACR) revisedcriteria for SLE Classification [26]. No more criteriawere required for patients’ enrollment.(2) First-degree relatives (FDRs): Defined as brothers andsisters of included SLE patients who or their guardiansprovided informed consent. They underwent thoroughclinical examination to screen for autoimmune diseases.FDRs having at least one but fewer than four of the cri-teria for SLE were defined as incomplete lupus erythe-matosus (ILE) [27].(3) Unrelated healthy controls (UHCs): They were recruitedfrom medical students, nursing staff, healthy relatives of non autoimmune disease patients. All the controls weresubjected to athorough history taking and clinical exam-ination toascertain theabsence ofanypersonalorfamilyhistory of autoimmune disease. 2.3. Exclusion criteria Individuals with a personal history of malignancy, recent infec-tion, viral hepatitis or treatment by IFN- a  were not eligible forthe present study. 2.4. Laboratorial analysis From each patient 5 ml venous blood was collected, onemilliliter was withdrawn into plastic tube containing thedipotassium salt of EDTA at a final concentration of 1.2 mg EDTA/ml venous blood which was used for perform-ing CBC using a cell counter (Sysmex K ·  20), 2 ml wasadded into a tube containing trisodium citrate for perform-140 D. Shahin et al.  ing ESR in a ratio of one volume citrate to four volumeblood.Twomlwascollectedintocleandryplastictubeandallowedto clot, the yielded serum was used for performing ANA assayusing ANA serum enzyme immune assay kit (BinDazyme, Bir-mingham, UK), double stranded DNA antibody assay usingenzyme linked immune sorbent assay (ELISA) (Immulisa, Im-mco Diagnostic, NY, USA) and serum level of IFN- a  whichwas measured using an enzyme linked immuno sorbent assay(ELISA) kit (Bender, Medsystems, Vienna, Austria). To neu-tralize heterotrophile antibodies and avoid false-positive levelsof IFN- a , a modified protocol described by Aly et al. [28] byadding5%mouseserumtotheassaybufferwasusedinthecur-rent assay. 2.5. Statistical analysis Data were analyzed using SPSS (version 17). Data were ex-pressed as mean ± SD and frequencies. Differences betweencontinuous variables were analyzed by  t -test and one way AN-OVA followed by Tukey’s post hoc test to determine signifi-cance between groups. Dichotomous variables were analyzedby  2 and Fisher’s exact test whenever applicable.  P  values<0.05 were considered significant. 3. Results 3.1. General characteristics of studied population Demographic features and laboratorial characteristics of theincluded participants were given in Tables 1 and 2. Thestudy population consisted of fifty-four SLE patients,ninety-three FDRs and seventy-six UHCs with mean ageof (by years) 21.2 ± 11.1, 19.5 ± 9.8and 20.9 ± 11.8, respec-tively. Meanwhile, females represented 81.5% of SLEpatients, 67.7% of FDRs and 63.2% of UHCs, adult popu-lation consisted of 51.9% of SLE patients, 48.4% of FDRsand 47.4% of UHCs. Table 1. However, 33.3% of the ILE( n  = 18) subset of FDRs were adults and 66.7% were pedi-atrics and 52% of the asymptomatic FDRs ( n  = 75) wereadults and 48% were pediatrics the difference was not statis-tically significant.The mean hemoglobin levels were comparable in the stud-ied groups. SLE patients and FDRs had significantly lowerWBCs than did the UHCs and only SLE patients showed sig-nificantly higher ESR and lower platelet count as comparedwith FDRs and UHCs. (Table 1). 3.2. Serum INF- a The mean levels of IFN- a  were 65.3 ± 53.4 pg/ml for SLE pa-tients, 19.5 ± 23 pg/ml for FDRs and 5.2 ± 5.2 pg/ml forUHCs as shown in Fig. 1. The difference was statistically sig-nificant by ANOVA ( P  < 0.0001). Tukey’s post hoc test re-vealed that the difference between patients and FDRs wasstatistically significant ( P  < 0.0001 with a 95% CI 33.6–58)and the difference between FDRs and UHCs was found tobe statistically significant ( P  = 0.007 with a CI of 3.3–25.4)(Fig. 1).Within the subset of FDRs, the mean serum IFN- a  inFDRs with ILE (53.8 ± 31.4 pg/ml) was significantly higherthan in asymptomatic FDRs (11.3 ± 8.4 pg/ml) ( P  < 0.0001)and the asymptomatic FDRs had a significant higher meanserum IFN- a  than UHCs ( P  < 0.0001). (Table 2). Table 1  General features of studied population. SLE patients( n  = 54)FDRs( n  = 93)UHCs( n  = 76) P  forANOVA * Scheffe post hoc test P  value patients vs.FDRs P  value FDRs vs.UHCsAge/in years (mean ± SD)Totalparticipants21.2 ± 11.1 19.5 ± 9.8 20.9 ± 11.8 nsAdults 29.5 ± 9.4 28.3 ± 6.1 31.6 ± 8 nsPediatrics 12.2 ± 2.9 11.3 ± 3.5 nsGender  n  (%)Female 44 (81.5%) 63 (67.7%) 48 (63.2%)Male 10 (18.5%) 30 (32.3%) 28 (36.8%)Age category  n  (%)Adults 28 (51.9%) 45 (48.4%) 36 (47.4%)Pediatrics 26 (48.1%) 48 (51.6%) 40 (52.6%)ILE – 18 – Hb 11.3 ± 1.9 13.3 ± 1.6 13.5 ± 1.6 <0.0001 <0.0001 nsWBCs 4.6 ± 1.6 4.9 ± 1.1 6 ± .8 <0.0001 <0.0001 nsPlatelet 262.3 ± 107.3 342.6 ± 96.2 374.3 ± 119.9<0.0001 ns 0.001ESR 71.6 ± 35.2 14.5 ± 3.6 12.9 ± 2.6 <0.0001 <0.0001 ns P  significant <0.05.ns = non significant.FDRs = first degree relatives, ILE = incomplete lupus erythematosus, UHCs = unrelated healthy controls, Hb = hemoglobin,WBCs = white blood cell count, ESR = erythrocyte sedimentation rate, ANA = antinuclear antibody, dsDNA = double stranded DNA. * One way ANOVA. Serum interferon-alpha level in first degree relatives of systemic lupus erythematosus patients: Correlation 141  3.3. Autoantibodies 1)  Antinuclear antibody (ANA) : The overall mean ANAtiter in SLE patients (66.2 ± 26.5 IU/ml) was higherthan in FDRs (12.7 ± 8.3 IU/ml) or the UHCs (6 ±4.9 IU/ml), the difference was significant between thethree groups ( P  < 0.0001) (Fig. 2). Further analysiswas carried out and the difference between patientsand FDRs ( P  < 0.0001, 95% CI 47.7–59.3), on one side,and FDRs and UHCs, on the other side, ( P  = 0.008,95% CI 1.4–11.9) were statistically significant. (Fig. 2).Similarly FDRs with ILE showed higher mean ANAtitre (24.2 ± 2.8 IU/ml) than in asymptomatic FDRs(10 ± 6.7 IU/ml) or the UHCs (6 ± 4.9 IU/ml)( P  < 0.0001) (Table 2).2)  Anti ds DNA antibody (ds DNA) : Although FDRs in thecurrent study showed significantly lower anti ds DNA(21.3 ± 12.6 IU/ml) as compared to SLE patients(107.2 ± 34.2 IU/ml) ( P  < 0.0001, 95% CI 78–93.8),they had higher overall mean levels as compared withUHCs (11.2 ± 9.9) ( P  = 0.003, 95% CI 3–17.3)(Fig. 3). Further analysis of FDRs subgroups andUHCs, a rising anti ds DNA titer was observed; startingby 11.2 ± 9.9 in UHCs to 17.4 ± 10.3 in asymptomaticFDRs to 37.7 ± 6.4 in FDRs with ILE. The differencewas statistically significant,  P  < 0.0001 (Table 2). 3.4. Pearson’s correlations between serum IFN- a  levels and  participants characteristics FDRs revealed, On one hand, a significant negative correla-tions between serum IFN- a  levels and FDRS age ( r  =  0.34, P  = 0.001), mean hemoglobin ( r  =  0.5,  P  < 0.0001), and Table 2  Characteristics of FDRs subsets in comparison to unrelated healthy controls. UHCs( n  = 76)FDRs ( n  = 93)  p  forANOVAScheffe posthoc testAsymptomaticFDRs ( n  = 75)FDRs with ILE( n  = 18)  p 1  p 2 Age (in years) 20.9 ± 11.8 20.3 ± 10 16.2 ± 8.6 ns –  – Hb 13.5 ± 1.6 13.6 ± 1.6 12 ± 1.4 <0.0001 ns<0.0001 WBCs 5.6 ± .8 5.1 ± 1.1 4.3 ± .7 <0.0001<0.0001 <0.0001Platelet 374.3 ± 119.9 343.2 ± 104.2 339.8 ± 52.1 0.04ns nsESR 12.9 ± 2.6 13.5 ± 3 18.5 ± 2.8 <0.0001 ns<0.0001 ANA (mean ± SD) (IU/ml)6 ± 4.9 10 ± 6.7 24.2 ± 2.8<0.0001 <0.0001 <0.0001Anti ds DNA(mean ± SD) (IU/ml)11.2 ± 9.9 17.4 ± 10.3 37.7 ± 6.4 <0.0001 <0.0001<0.0001 IFN- a  (mean ± SD) (pg/ml)5.2 ± 5.2 11.3 ± 8.4 53.8 ± 31.4<0.0001 <0.0001 <0.0001  p  Significant <0.05.  p 1 =  p  Value for asymptomatic first degree relatives vs. unrelated healthy controls.  p 2 =  p  Value for first degree relatives with incomplete lupus vs. asymptomatic first degree relatives.ns = non significant.IFN- a  = interferon- a , FDRs = first degree relatives, ILE = incomplete lupus erythematosus, UHCs = unrelated healthy controls,Hb = hemoglobin, WBCs = white blood cell count, ESR = erythrocyte sedimentation rate, ANA = antinuclear antibody, ds DNA = doublestranded DNA. Figure 1  Mean serum INF-alpha levels in SLE patients, theirfirst degree relatives and healthy unrelated controls (ANOVA P  < 0.0001). 142 D. Shahin et al.  WBCs ( r  =  0.3,  P  = 0.006), and on the other hand, it waspositively correlated with ESR ( r  = 0.5,  P  < 0.0001), ANA ti-ter ( r  = 0.6,  P  < 0.0001), and anti ds DNA ( r  = 0.62, P  < 0.0001) (Table 3). Furthermore, a nearly similar patternof correlations was observed in the total participants (Table 3). 4. Discussion Detecting autoimmune disease in early or preclinical stages isclinically important because the institution of treatment priorto the onset of organ damage has a greater chance to amelio-rate or even cure the disease [29]. Lately, much insight hasbeen gained regarding IFN- a  as a causal factor for SLE,and was suggested as a useful tool which can evaluate currentdisease activity and identify patients who are at risk of futuredisease flares [30]. Even though preclinical identification of individuals at risk of SLE might be feasible [11,20,24,31],the development of quantitative SLE risk profile is still defi-cient [24].Many previous investigators [4–9] observed increased levelsand activity of IFN- a  in SLE patients. This was consistent withthe findings in the present study. More interestingly, FDRs inthe current study had significant higher IFN- a  than UHCs.Given that SLE family members share the same genetic back-ground with their SLE relative patients [19,32] and that serumIFN- a  activity was estimated to have a broad-sense heritabilityof 48% in SLE families [23]; FDRs of SLE patients may prob-ably produce IFN- a  in an abated fashion as in SLE patients[3]. Indeed, SLE blood represented a dendritic cell (a key con-troller of immunity, tolerance, and IFN- a  production) induc-ing environment [10,33]. It was found that the serum of SLEpatients contains an IFN- a  inducing factors that induce theproduction of large quantities of IFN- a  in normal blood leu-kocytes in vitro [34,35].Further investigations of this IFN- a  inducing factor in SLErevealed that immune complexes containing nucleic auto-antibodies were essential for the induction of expression of IFN- a  [14]. In agreement with these observations, auto-antibodies titers and serum IFN- a , in the current study, clearlyshowed stepwise increase in the studied groups going fromUHCs to asymptomatic FDRs to FDRs with ILE to SLE pa-tients. Similar findings were recently reported by Li et al. [24].They showed that IFN- a  genes expression were the specificitiesthat most clearly showed stepwise increase in their studiedgroups starting by low ANA healthy controls, through highANA healthy controls and ending by SLE patients. Data fromearlier studies on auto-antibodies profile of FDRs of SLE pa-tients [20,36] were in accordance to the data from the presentstudy, indicating a familial basis to mount an immuneresponse.Previous investigators suggested at least three phases in thedevelopment of SLE autoimmunity [37,38]; normal immunityprogresses to benign autoimmunity through the influence of genetic composition and environment. Later, benign autoim-munity progresses to pathogenic autoimmunity. Symptomsof clinical illness appear soon after pathogenic autoimmunitydevelops. Our serologic and clinical findings along with theirstrong association with serum IFN- a  levels, in the currentstudy, further supported a crescendo of autoimmunity culmi-nating in clinical illness. First, the UHCs in the present studyrepresented the phase of normal immunity, and the asymptom-atic FDRs represented the phase of benign autoimmunity.Finally, the FDRs with ILE, in the present study, symbolizedthe third pathogenic autoimmunity phase, that is marked bythe presence of more ominous autoantibodies and inflamma-tory markers.In-depth reading of the strong association between IFN- a levels and autoantibodies titer in the current study further indi-cated an essential role for IFN- a  in the development of SLE.Our results could be explained by the hypothesis formulatedby RO ¨ nnblom and Alm [3] in which type I IFN system is cru-cial to the development of SLE in two distinct phases. Initially,autoantibodies against nucleic acids and associated proteinsare generated during infections (exogenous IFN- a  inducers).In the second phase, immune complexes form and act asendogenous IFN- a  inducers, which sustain the autoimmuneprocess by prolonging the production of IFN- a . Furthermore,persistently elevated IFN- a  might promote autoantibody classswitch from immunoglobulin (Ig) M to the more pathogenicIgG class [39]. This proposed mechanism will operate in thegenetically predisposed individuals, such as SLE family mem-bers, to develop SLE. In accordance to the results of the pres-ent work, data from previous studies showed a good Figure 2  Mean serum ANA titer in SLE patients, their firstdegree healthy unrelated controls (ANOVA  P  < 0.0001). Figure 3  Mean serum anti ds DNA titer in SLE patients, theirfirst degree relatives and healthy unrelated controls (ANOVA P  < 0.0001). Serum interferon-alpha level in first degree relatives of systemic lupus erythematosus patients: Correlation 143
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