An increased level of apoptotic material and B cell activation leading to autoantibody production are hallmarks of systemic lupus erythematoses (SLE). Increased FAS expression, apoptosis, and CD154-mediated signaling, enabling T-B cell interaction are involved in the pathogenesis of SLE. This study addresses the expression profile of CD154 and FAS in the peripheral blood of patients with SLE, rheumatoid arthritis (RA) and normal healthy control donors. Surface markers on peripheral blood T and B cells from patients and healthy control donors were assessed using flow cytometry. The expression of CD154 and FAS were significantly increased in T and B cells of SLE patients as compared to healthy control donors and RA patients. In SLE and RA patients, FAS expression strongly correlated with CD154 expression on T cells, which was not found in healthy control donors. FAS expression was also associated with the occurrence of anti-DNA antibodies. We demonstrate high CD154 and FAS expression as a characteristic feature of SLE. This pattern may reflect simultaneous activation of apoptosis and activation of B-T cell interaction in SL

Dejica, Doru

Herrmann, Martin

Kern, Peter

Manea, Maria

Mueller, Ruediger

Schett, Georg

Sheriff, Ahmed

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Rheumatol Int (2009) 30:181–185DOI 10.1007/s00296-009-0933-4ORIGINAL ARTICLEIncreased expression of CD154 and FAS in SLE patients’ lymphocytesMaria Elena Manea · Ruediger B. Mueller · Doru Dejica · Ahmed SheriV · Georg Schett · Martin Herrmann · Peter Kern Received: 3 January 2009 / Accepted: 28 March 2009 / Published online: 30 April 2009©  Springer-Verlag 2009Abstract An increased level of apoptotic material and Bcell activation leading to autoantibody production are hall-marks of systemic lupus erythematoses (SLE). IncreasedFAS expression, apoptosis, and CD154-mediated signaling,enabling T–B cell interaction are involved in the pathogen-esis of SLE. This study addresses the expression proWle ofCD154 and FAS in the peripheral blood of patients withSLE, rheumatoid arthritis (RA) and normal healthy controldonors. Surface markers on peripheral blood T and B cellsfrom patients and healthy control donors were assessedusing Xow cytometry. The expression of CD154 and FASwere signiWcantly increased in T and B cells of SLEpatients as compared to healthy control donors and RApatients. In SLE and RA patients, FAS expression stronglycorrelated with CD154 expression on T cells, which wasnot found in healthy control donors. FAS expression wasalso associated with the occurrence of anti-DNA antibod-ies. We demonstrate high CD154 and FAS expression as acharacteristic feature of SLE. This pattern may reXectsimultaneous activation of apoptosis and activation of B–Tcell interaction in SLE.IntroductionSystemic lupus erythematoses (SLE) is a systemic autoim-mune disease characterized by plasma B cells secretingnuclear autoantibodies [11], especially the pathognomonicanti-double-stranded DNA autoantibodies (anti-dsDNA). Band T cell interaction is required for the induction ofhumoral immunity. T cells can induce B cell proliferationand diVerentiation via the ligation of CD154 by CD40 as acostimulatory molecule. The interaction of T and B cells inSLE is of major importance for the pathogenesis of the dis-ease. CD154 hyperexpression in patients with active SLEwas shown on T cells, B cells, and monocytes and corre-lated with disease activity [4]. Interestingly, not only mem-brane bound, but also soluble CD154 levels correlate withdisease activity and enhanced titers of anti-dsDNA inpatients with SLE, indicating a central role for the patho-genesis of CD154 [9]. Underlining the importance of theCD154–CD40 interaction in SLE, treatment with a mono-clonal Abs to CD154 signiWcantly decreased disease activ-ity in SLE patients [3]. However, this treatment isassociated with thromboembolic events [13] as a severeside eVect leading to the hypothesis that CD154 cross-link-ing has eVects aside co-stimulation.An accumulation of apoptotic remnants in the tissue isobserved in a subgroup of patients with SLE. FAS (CD95)and FAS–ligand interaction are associated with the induc-tion of apoptosis [7]. The stimulation of autoreactive BM. E. Manea and R. B. Mueller contributed equally to the work.M. E. Manea · D. DejicaDepartment of Immunopathology,“Iuliu Hatieganu” University of Medicine and Pharmacy, Str Croitorilor no 19-21, 3400 Cluj-Napoca, RomaniaR. B. Mueller · A. SheriV · G. Schett · M. HerrmannDepartment for Internal Medicine 3, Institute for Clinical Immunology, University of Erlangen-Nürnberg, Erlangen, GermanyR. B. Mueller (&)Department of Rheumatology, Kantonsspital St. Gallen, Rorschacherstr. 95, 9000 St. Gallen, Switzerlande-mail: ruediger.mueller@kssg.ch; ruediger.Mueller@gmx.orgP. KernFranz von Prümmer Klinik, Bahnhofstraße 16,97769 Bad Brückenau, Germany123182 Rheumatol Int (2009) 30:181–185cells with anti-CD40 leads to an increase in FAS-mediatedapoptosis in vitro [8]. In parallel, elevated plasma levels ofsCD154 in patients with SLE increase CD95 expression onB cells [16]. B cells, stimulated via CD40, are highly sus-ceptible to FAS-mediated apoptosis [8], when exposed toFAS-ligand expressing T cells [14]. Consequently, wehypothesized that the CD154–CD40 mediated interactionand the upregulation of FAS may occur simultaneously inSLE. Therefore, we measured the expression of FAS,CD40, and CD154 on T and B lymphocytes in a cohort of52 patients with SLE, 32 patients with rheumatoid arthritis(RA), and 40 normal healthy control donors. The expres-sion of these surface markers was correlated with eachother and with the clinical course of SLE.Materials and methodsPatient dataThe patient population comprises 52 SLE and 32 RApatients. All patients were treated with disease modifyinganti-rheumatic drugs (DMARDs) and/or corticosteroids. Atthe time of Wrst analysis, all patients had an established dis-ease. Disease activity diVered, and was assessed by serumlevels of C-reactive protein (CRP), erythrocyte sedimenta-tion rates [10], and SLE disease activity index (SLEDAI)score for each patient and the use of medication wasrecorded (Table 1). Forty age and sex-matched healthydonors served as controls. The study was approved by theUniversity of Erlangen–Nuremberg Institutional ReviewBoard, and written informed consent was obtained from allindividuals before entering the study.Immune phenotypingWhole blood was stained for 30 min with saturatingamounts of directly labeled mAbs. After automated lyses ofthe red blood cells (Q-Prep, Beckman Coulter, Cambridge,MA), the leucocytes were Wxed with 1% paraformaldehydeand analyzed using Xow cytometry (EPICS, BeckmanCoulter, Cambridge, MA). The following phycoerythrin(PE), Xuorescein-isothiocyanate (FITC), or CyChrome(Cy5) conjugated monoclonal antibodies were used: anti-CD4 (FITC/PE), anti-CD19 (FITC/PE/Cy5) (PE), anti-CD40 (FITC), anti-CD154 (FITC), and anti-CD95 (PE)(BD Pharmingen, Heidelberg, Germany). Unlabeled cellswere used as controls for determining background andautoXuorescence.Statistical analysisDiVerences in data distribution were analyzed by two-tailedStudent t tests. Correlation analysis was performed follow-ing the Pearson test. Comparison of two correlationanalysis was assessed by the MetaAnalysis program 5.3 ofRalf Schwarzer; http://web.fu-berlin.de/gesund/gesu_engl/meta_e.htm.ResultsExpression of CD154 in SLE patients in comparison to RA and healthy control donorsSince ligation of the costimulatory molecule CD40 on Bcells by its ligand CD154 on T cells plays a central role forB cell activation, we assessed the expression of both mole-cules using Xow cytometry. The individual percentages ofCD154 expressing B cells (CD19pos, Fig. 1a) and T helpercells (CD4pos, Fig. 1b) were signiWcantly increased inpatients with SLE compared with healthy control donorsand patients with RA (B cells) or only compared withhealthy control donors (T helper cells). In detail,0.6 § 0.7% of B cells from healthy control donors werepositive for CD154, which was comparable to RA patients(0.6 § 0.8%). In contrast, CD154 expression was signiW-cantly more frequent (2.1 § 3.2%) in patients suVeringfrom SLE. Similarly, CD154 expression was also increasedin T helper cells of patients with SLE (1.8 § 3.1%) as com-pared to healthy control donors (0.6 § 0.8%). The data sug-gest that expression of the costimulatory molecule CD154is increased in T and B cells of SLE patients. Thus, anincreased expression of CD154 in B cells also discriminatesSLE patients from other autoimmune diseases such as RA[12].Expression of CD40 in SLE patients in comparison to RA and healthy control donorsIn parallel, we assessed CD40, the ligand of CD154 inperipheral blood cells of patients with SLE and RA as wellas healthy control donors. CD40 expression was found inTable 1 Patient dataRA patients mean (range)SLE patientsmean (range)NHD mean (range)Number 32 52 40Age 54.6 (27–69) 45.3 (20–72) 36.3 (22–68)Sex (female/male) 32 f, 9 m 45 f, 7 m 27 f, 13 mC-reactive protein 0.5 (0–1.5) 2.5 (0.1–17) n.d.SLEDAI n.a. 4.3 (0–16) n.a.Anti-DNA n.d. 60.5 (0–1380) n.dANA n.d. 1974 (0–10000) n.d.123Rheumatol Int (2009) 30:181–185 183the majority of B cells although there was a trend to a lowerexpression in SLE patients (71.9 § 22.9%) than in healthycontrol donors (85.4 § 14.1%) and RA patients(79.2 § 18.6%) (Fig. 1c). Thus, in contrast to the Wndingsmade for CD154 expression on SLE patient’s B cells nodiscriminative propensity for CD40 expression could befound among RA patients, healthy donors and SLEpatients.Expression of FAS in SLE patients in comparison to RA and healthy control donorsAn accumulation of apoptotic material is observed in vari-ous tissues of patients with SLE. Since CD154 leads to anaugmented expression of CD95, the expression of this pro-apoptotic molecule was also analyzed. FAS expression wasincreased in B cells (Fig. 2a) and T cells (Fig. 2b) frompatients with SLE in comparison to healthy control donors.In detail, 38 § 20% of B cells and 66 § 14% of T cellsexpressed FAS in SLE patients. In healthy control donorsonly 19 § 7% of B cells and 51 § 9% of T cells expressedFAS. Importantly also RA patients displayed a lowerexpression of FAS in B cells (25 § 14%), whereas itsexpression in T cells (63 § 13%) was comparable to theexpression in T cells of SLE patients.Correlation of CD154 and FAS expressionTo delineate an association between CD154 and CD95expression correlation analysis was performed. There was asigniWcant correlation (r = 0.75; P = 0.001, Fig. 2d)between the percentages of CD154pos and CD95pos T cellsin patients with SLE. To delineate whether this balance issimilar a correlation analysis was also performed in healthycontrol donors. However, the correlation was much lesspronounced (r = 0.35, P = 0.0252, Fig. 2e). In RA patients,the correlation of CD154pos and CD95pos T cells was simi-lar as observed in SLE patients (r = 0.72, P = 0.0001, datanot shown). Importantly, the correlations calculated forSLE and RA patients diVered signiWcantly (P = 0.003) fromthe correlation calculated in healthy control donors. Thus,CD154 and CD95 expression strongly correlate in T cellsof patients with SLE and RA.Since FAS is associated with the induction of apoptosisand an increase in apoptotic material is a hallmark of SLE,we next addressed whether increased FAS expression inT cells is associated with increased SLE disease activity.Indeed, patients with SLE and high FAS expression inT cells also display an increased amount of anti-DNAantibodies (Fig. 2c).DiscussionIn this study, an increased expression of CD154 and FASwas denoted in peripheral blood T and B cells of patientswith SLE as compared to healthy control donors. More-over, the expression of CD154 in B cells and FAS in T andB cells of SLE was higher than the expression of both mol-ecules in lymphocytes of RA patients. Moreover, there wastight correlation observed between CD154 and FAS expres-sion in SLE and RA. These Wndings lead us to the hypothe-sis of an altered balance between CD154 and FAS onT cells in autoimmunity. FAS expression was also associ-ated with the occurrence of anti-DNA antibodies a goodmarker of disease activity in SLE.In coherence with various other publications, we alsofound an increased expression of CD154 on T cells [4] andB cells [6] in patients with SLE. In contrast to the publisheddata [4], the increased expression of CD154 did not corre-late with disease activity as assessed by SLEDAI, but core-lated to the occurrence of anti-DNA antibodies levels,which is a surrogate marker for disease activity in SLEpatients. Thus, we assume that CD154 plays an importantrole in patients with SLE that might lead to an increase inclinical disease activity as shown for the correlation ofFig. 1 CD154 and CD40 expression in SLE: T and B cells wereisolated from full blood by gradient centrifugation. The percentage ofa CD154pos B and b CD4+ T cells and c CD40pos B cells was assessedusing Xow cytometry. All data are represented as dots for every indi-vidual patient and healthy control donor. Percentages of cellsexpressing certain surface markers are demonstrated for patients withRA and SLE represented on the Wrst and second and such found onhealthy control donors (NHD) on the right column, representatively.Statistical diVerences were calculated employing the student’s t test123184 Rheumatol Int (2009) 30:181–185CD95pos with CD154 on T cells on one hand and the occur-rence of anti-DNA antibodies on the other hand.Treatment with neutralizing antibody against CD154[13] leads to the occurrence of thromboembolic events.Because anti-phospholipid antibodies are associated withvascular events in SLE and of monoclonal antibodies toCD40 was linked to the anti-phospholipid antibodies invivo [2], we analyzed whether increased levels of CD40 ofCD154 expressing cells correlate with anti-phospholipidantibodies in our cohort. No association could be found(data not shown).Autoantibodies against FAS–ligand can be found in 30%of the patients with SLE. It has been suggested that thisleads to an inhibition of FAS/FAS-L-mediated apoptosis[15]. In contrast, we demonstrated that the percentage ofFAS expressing cells is increased on lymphocytes ofpatients with SLE. Whether this increase in FAS expressionis a reaction on autoreactive anti-FAS antibodies remainsunclear. However, the level of apoptotic material in patientswith SLE is increased. This may be due to a clearance defectof apoptotic material in patients with SLE [5]. Alternatively,it could also be associated with an increased induction ofapoptosis [12], potentially mediated by increased expressionof FAS. Since the expression level of FAS correlates withCD154 and, in parallel, the disease activity, an increase indisease activity may lead to an increase in apoptosis. Incoherence, an increased expression of CD154 leads to anincreased expression of FAS–ligand and induced, subse-quently, apoptosis in biliary epithelial cells [1].In summary, we showed that the expression of CD154and FAS on SLE patients’ T and B cells was increased andcorrelated with each other in tight correlation. FAS expres-sion was also associated with the occurrence of anti-DNAantibodies a good marker of disease activity in SLE.Acknowledgments This work was supported by “DeutscheForschungsgemeinschaft” SFB 643 (project B5), by the Interdisciplin-ary Centre for Clinical Research (IZKF) (project number A4 and N2)at the University Hospital of the University of Erlangen–Nuremberg,by the European Commissions [E.U. (QLK3-CT-2002-02017_APOCLEAR)], by the Lupus Erythemathodes Selbsthilfegemeinschaft e.V.,by the “Responsif GmbH” Erlangen, by the “Doktor Robert PXeger”Foundation, Bamberg, by the ELAN-Fond of the university of Erlan-gen Nuremberg (Pro.-Nr. 53410026), A grand dedicated to Maria Ele-na Manea and Doru Deica by the Alexander von HumboldtFoundation.Fig. 2 CD95 expression in SLE: T and B cells were isolated from fullblood by gradient centrifugation. Percentages of CD95pos cells amonga CD4pos T and b CD19pos T cells were assessed with Xow cytometry.All data are represented as dots for every individual patient in twocolumns. Percentages of cells expressing CD95 are demonstrated forpatients with RA, SLE, and healthy control donors (NHD). StatisticaldiVerences were calculated employing the Student’s t test. c Patientswith SLE were separated into two groups: negative (left) and positive(right) for anti-DNA antibodies. Percentages of CD95pos T cells aredemonstrated for both groups as mean § standard deviation. StatisticaldiVerences were calculated employing the Student’s t test. Correlationanalysis of CD154 and CD95 expression on T cells of SLE patientsd and healthy control donors e Every dot represents the individualexpressional percentage found within one patient for CD154 and CD95on T cells. The correlation line is depicted as a line with the 95%conWdence interval within the graph123Rheumatol Int (2009) 30:181–185 185References1. AVord SC, Ahmed-Choudhury J, Randhawa S, Russell C,Youster J, Crosby HA, Eliopoulos A, Hubscher SG, Young LS,Adams DH (2001) CD40 activation-induced, Fas-dependentapoptosis and NF-kappaB/AP-1 signaling in human intrahepaticbiliary epithelial cells. FASEB J 15:2345–2354. doi:10.1096/fj.01-0088com2. 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Increased expression of CD154 and FAS in SLE patients' lymphocytes

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Increased expression of CD154 and FAS in SLE patients' lymphocytes

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