Immunomodulatory effects of extracorporealphotochemotherapy in systemic sclerosisGabor Papp a, Ildiko Fanny Horvath a, Sandor Barath a, Edit Gyimesi a,Judit Vegh a, Peter Szodoray b, Margit Zeher a,⁎
a Division of Clinical Immunology, 3rd Department of Medicine, Medical and Health Science Center, University of Debrecen,Debrecen, Hungaryb Institute of Immunology, Rikshospitalet, University of Oslo, Oslo, Norway
Received 18 July 2011; accepted with revision 30 September 2011Available online 7 October 2011
Abstract The aim of this study was to evaluate the clinical and immunomodulatory effects ofextracorporeal photochemotherapy (ECP) in systemic sclerosis (SSc). We enrolled 16 patientswith diffuse cutaneous SSc, who received 12 ECP treatments in total. After ECP treatments,the dermal thickness reduced and the mobility of joints improved. Internal organ involvementdid not deteriorate. The percentages and numbers of peripheral Th17 cells decreased, thevalues of Tr1 and Treg cells increased, and the suppressor capacity of Treg cells improved. Inter-estingly, we found a positive correlation between the reduction of IL-17 levels and skin thicknessmeasured by ultrasound. Moreover, levels of CCL2 and TGF-beta decreased, while the concen-
Systemic sclerosis (SSc) is a chronic systemic autoimmune dis-ease characterized by pathogenic immune activation and abnor-mal collagen deposition resulting in fibrosis of the skin andinternal organs, such as heart, lungs, or kidneys. There are twodifferent clinical subsets of SSc: the diffuse cutaneous form ischaracterized by rapidly progressive fibrosis of the skin and vis-ceral organs, while in the limited cutaneous form the skin andorgan fibrosis is generally limited and the progression is slow [1].
Despite the intensive research, there is still no proventreatment in halting fibrosis and preventing progression ofthe disease. Since therapeutic options are limited mainly tothe management of the complications, the diffuse cutaneousform has the highest mortality among connective tissue dis-eases, with overall 55% survival at 10 years [2]. Besides bio-logics, extracorporeal photochemotherapy (ECP), also knownas photopheresis, is one of the promising therapeutic strate-gies in the diffuse cutaneous form of the disease.
ECP is a special immunomodulatory therapy, which is basedon apheresis technology. The separation of leukocyte richplasma from the red blood cell fraction is followed by its exvivo exposition to 8-methoxypsoralen (8-MOP) and ultraviolet(UV) A light, and finally, the re-infusion of the treated cells.The majority of treated cells die within 72 h, since due tothe 8-MOP and UV-A irradiation, cells become incapable ofproliferation and undergo apoptosis [3]. The advantage ofphotopheresis is its minimal toxicity and side effects, com-pared to other immunosuppressive treatments. In a random-ized, double-blind, placebo-controlled trial, ECP was shownto induce significant improvement of skin and joint involve-ment in patients with recent disease onset [4]. Anotherstudy demonstrated that after ECP, the dermal echo intensityincreased, while the dermal thickness reduced, which sug-gests that ECP is more likely to improve dermal edema, thenfibrosis [5]. Behind the clinical amelioration, multiple mecha-nisms are assumed, involving (I) apoptosis induction in treatedlymphocytes, (II) modulation of antigen-presenting cells, (III)reinforcement of immunoregulatory activity, and (IV) stimula-tion of anti-inflammatory cytokines [6]. However, the exactmechanism of its action is not fully understood yet.
In order to assess these possible beneficial effects of ECPtherapy, we performed a wide-spectrum of analyses, rangingfrom clinical evaluation to serological markers and immune-competent cell analyses. We assessed the skin, joint andorgan involvements, and determined a wide spectrum of circu-lating cytokines and peripheral immune-competent cell typeswith regulatory and effector properties, reflecting overall dis-turbances in immune homeostasis, characteristic to patientswith diffuse cutaneous SSc. In order to determine the changesin suppressor capability of CD4+CD25+ T regulative (Treg)cells, we carried out an in vitro functional test after each pro-cedure. We also estimated how ECPmay affect the parametersof serum levels of complements and autoantibodies.
2. Materials and methods
2.1. Patients
Sixteen patients suffering from diffuse cutaneous SSc (14 fe-male and 2 male; mean age 46.5±13.2 years) were enrolled
in the present study, recruited from the outpatient clinic forsystemic autoimmune diseases at the Division of Clinical Im-munology, 3rd Department of Medicine, Medical and HealthScience Center, University of Debrecen, where they receivedregular follow-up and treatment. The diagnosis of SSc wasestablished according to the corresponding diagnostic criteria[7]. The mean disease duration was 3.9 years (range 0.5–7 years). For 1 year before the beginning of photopheresistherapy, patients received only 2×400 mg pentoxyphyllinand 2.5 mg amlodipine treatment. Sixteen sex- and age-matched healthy individuals (14 female and 2 male; meanage 43.9±12.8 years) served as controls for the laboratory re-sults. No patients, or controls enrolled in this study had ongo-ing or previous infections during the study. Informed writtenconsent was obtained from the subjects, and the study hasbeen approved by the ethics committee of the University ofDebrecen. All experiments carried out were in compliancewith the Declaration of Helsinki.
2.2. Method of extracorporeal photochemotherapy
We performed ECP procedures by using THERAKOS UVAR XTSPhotopheresis System (Therakos Inc., Raritan, NJ, USA). Inthe first step, whole blood was drawn from the patient intothe centrifuge bowl, in which the buffy coat was separated,than accumulated into a recirculation bag. After approxi-mately 250–300 ml of buffy coat was collected, the hemato-crit sensor stopped the collection. A photosensitizing agent,8-MOP was automatically added to the cell concentrate inthe treatment chamber; the final concentration of 8-MOPwas 334 ng/ml. Subsequently, peripheral blood mononuclearcells (PBMCs) were irradiated by UV-A with the irradiationdose of 1.5–2 J/cm2, and finally re-infused to the patient.
The patients were treated using the standard protocol inwhich ECP cycles were carried out once in every 6 weeks.Each ECP cycle consisted of two procedures on consecutivedays. Patients received 6 cycles in total during the wholetherapy period.
2.3. Skin and joint assessment
Assessment of the skin involvement was performed at baseline,and after 6 weeks of each treatment. It has been earlier dem-onstrated that the skin on the back of the hand is thicker is SSc,and its echographic values significantly differ from those ofnormal skin on the back of the hand [5]. According to these for-mer observations, echographic measurements of dermal thick-ness were carried out at 4 different places on the extensorsurface of the upper limbs (upper arm, forearm, back of thehand, base of the 3 rd finger) in this study. The echographused was HP SONOS 5500 ultrasound system (Hewlett Packard,Andover, MA, USA), which works with 15–6L (15 MHz) trans-ducer. The investigation depth was 10 mm, and at every exam-ination we did three measurements and took the average ofthe data. The procedure was done by the same licensed spe-cialist before and after each treatment. Skin involvementwas also determined by modified Rodnan skin score (MRSS),which is a standard outcome measure for skin disease in SScand calculated by the summation of skin thickness in 17 differ-ent regions of the body [8]. Moreover, we assessed the changesin the tightness of oral aperture as well.
152 G. Papp et al.
Each patient was examined for changes in joint move-ment at baseline and after the last ECP treatment. We ex-amined the mobility of the upper limbs (shoulders, elbows,wrists) and lower limbs (hips, knees, ankles) on both side,registered and measured the changes in degrees.
2.4. Determination of the visceral organ involvement
Prior to the first and after the last treatment, patientsunderwent diagnostic procedures, including chest X-ray,high-resolution computed tomography, spirometry/diffusioncapacity test, Doppler echocardiography, abdominal ultraso-nography, esophagus passage radiography and routine labo-ratory tests.
2.5. Blood samples for laboratory investigations
Blood samples were obtained from patients prior to the begin-ning of the therapy and 6 weeks after each cycle, in order toassess the effects of ECP. Laboratory samples were alsoobtained from the sixteen healthy controls. Serum sampleswere stored at −70 °C until further analysis, while cell sampleswere processed immediately.
2.6. Determination of lymphocyte subpopulationand activated T cells
To identify lymphocyte subpopulations, we used monoclonalantibodies against cluster of differentiation (CD)3, CD4, CD8,CD19 and CD56 (BD Biosciences, San Diego, CA, USA and Immu-notech, Marseille, France). The expression of T-lymphocyteactivation markers such CD69 and human leukocyte antigen(HLA)-DR was also determined on CD3+ cells (BD Biosciences).Samples were processed according to the Coulter Q-PREP pro-tocol and system (Beckman Coulter Inc., Miami, FL, USA), asdescribed previously [9]. Measurements were performed on aCoulter FC500 flow cytometer (Beckman Coulter Inc.). Thefollowing peripheral immune-competent cell types were in-vestigated: T cells (CD3+), T-helper cells (CD4+), cytotoxic T(Tc) cells (CD8+), B cells (CD19+), early-activated T lympho-cytes (CD3+ CD69+), late-activated T lymphocytes (CD3+ HLA-DR+), natural killer (NK) cells (CD56+) and NKT cells (CD3+CD56+). The B, T, T-helper, activated T, NK and NKT cellswere quantified as their percentage in the entire lymphocytepopulation.
2.7. Identification of CD4+ T cell subsets byintracytoplasmic cytokine assessment
The method for intracellular staining of CD4+ T cell subsets hasbeen performed, as described previously [10]. The followingmonoclonal antibodies were used: fluorescein isothiocyanate(FITC)-labeled anti-interferon (IFN)-gamma, phycoerythrin(PE)-labeled anti-interleukin (IL)-4, PE-conjugated anti-IL-10(all from BD Biosciences), or PE-labeled anti-IL-17 (R&D Sys-tems, Minneapolis, MN, USA). Measurements were performedand data were collected on a Coulter FC500 flow cytometer(Beckman Coulter Inc.). Based on intracytoplasmic staining,the phenotypes within CD4+ cells were determined as follows:T helper (Th)1 cells: CD4+ IFN-gammam IL-4-; Th2 cells: CD4+
IFN-gamma− IL-4+; T regulative type 1 (Tr1) cells: CD4+ IL10+;Th17 cells: CD4+ IL17+. Cells were quantified as their percent-age in the CD4+ lymphocyte population.
2.8. Determination of CD4+CD25+bright FoxP3+Treg cells
Cell surface (CD4, CD25) staining and intracellular [forkheadbox P3 (FoxP3)] staining were carried out on freshly isolatedPBMCs from heparinized blood as described previously [10].The following reagents were used: Ficoll (Sigma Aldrich, StLouis, MO, USA), CD4-FITC monoclonal antibody (Sigma Al-drich), CD25-phycoerythrin-Cy5 (PC5) (Immunotech, Marseille,France), FoxP3-PE, clone: PCH101 (eBioscience, San Diego, CA,USA) and intracellular staining kit (eBioscience).
2.9. Suppression functional assay of CD4+CD25+Treg cells
Suppression functional assay was used, as described by us previ-ously [10]. PBMCs were isolated from heparinized whole bloodby density gradient centrifugation over Ficoll (Sigma Aldrich).CD4+CD25+ T cells were isolated from PBMCs by using MiltenyiRegulatory T Cell Isolation Kit (Miltenyi Biotech, Bergisch Glad-bach, Germany), according to the manufacturer's instructions.Briefly, non-CD4+ T cells were depleted by indirectmagnetic la-beling with Biotin-Antibody Cocktail and Anti Biotin Microbe-ads. LD Columns (Miltenyi Biotech) were used to deplete non-CD4+ cells. CD25+ T cells were purified from the pre-enrichedCD4+ T cell fraction by positive selection process. In thesesteps, CD4+ T cells were directly labeled with anti-CD25microbeads and CD4+CD25+ T cells were eluted from the MSColumn (Miltenyi Biotech). Magnetically isolated 1×105 CD4+CD25+ and CD4+CD25− T cells were cultured in 200 μl ofcRPMI 1640 in 96-well U bottom plates for 72 h separately,also together, in co-cultures. CD4+CD25+ and CD4+CD25− cellswere cultured in 1:1 ratio in the mixed lymphocyte reaction.For polyclonal stimulation, cells were stimulated with anti-CD3/CD28 T cell expander microbeads (Dynal, Oslo, Norway)in 1 bead/cell concentration. Proliferation was investigatedby using a terazolium-based assay (EZ4U Proliferation Kit, Bio-Medica Inc, San Diego, CA, USA). The substrate was added inthe last 2.5 h to the culture and finally optical density (OD)values were detected at 450 nm by ELISA reader. OD values ofmixed lymphocyte culture were corrected by OD values ofCD4+CD25+ T cells cultured alone. Suppression activity was de-termined, as the ratio of OD values of the CD4+CD25− T cell cul-tures and mixed lymphocyte reactions.
2.10. Evaluation of circulating cytokines by multiplexcytokine assay and ELISA
Serum levels of cytokines and chemokines, including tumor ne-crosis factor (TNF)-alpha, IFN-gamma, IL-1-alpha, IL-1-beta, IL-1RA, IL-2, IL-4, IL-6, IL-8, IL-10, IL-13, IL-17, chemokine (C-Cmotif) ligand 2 (CCL2) also known as monocyte chemoattrac-tant protein-1 (MCP-1)/(MCAF), fibroblast growth factor(FGF), vascular endothelial growth factor (VEGF), epidermalgrowth factor (EGF) and hepatocyte growth factor (HGF)were measured at the Tissue Engineering Laboratory of the
Table 1 Mean±standard deviation values of the dermalthickness measured at the extensor surface of 4 differentregions, before the first and after the last ECP treatment.Differences were considered statistically significant atpb0.05.
Before ECPtreatment
After the 6thcycle of ECP
Upper arm 0.98±0.28 mm 0.84±0.22 mm p=0.016Forearm 1.01±0.21 mm 0.90±0.19 mm p=0.017Hand 1.12±0.23 mm 0.99±0.24 mm p=0.013Finger 1.25±0.24 mm 1.06±0.25 mm pb0.001
ECP: extracorporeal photochemotherapy.
153Photopheresis in systemic sclerosis
Institute of Human Physiology and Clinical Experimental Re-search, Semmelweis University. Measurements were carriedout by using Fluorokine Multianalyte Profiling (MAP) Kits(R&D Systems, Minneapolis, MN, USA), according to the manu-facturer's instruction. Briefly, a sandwich immunoassay-basedprotein array system, which contains dyed microspheres con-jugated with a monoclonal antibody specific for a target pro-tein was used in this assay. Antibody-coupled beads wereincubated with the serum sample (antigen) after which theywere incubated with biotinylated detection antibody beforefinally being incubated with streptavidin–phycoerythrin.These captured bead complexes were then read by a multi-analyte bioassay detection system (Luminex 200 System,Luminex, Austin, TX, USA). Acquisition and preliminary analysiswere carried out using Applied Cytometry System STarStation3.0 software (Applied Cytometry, Dinnington, UK).
Transforming growth factor (TGF)-beta levels were mea-sured by BD OptEIA enzyme-linked immunosorbent assay(ELISA) kits (BD Biosciences) according to the manufacturer'sinstructions.
2.11. Determination of complement levels, assess-ment of anti-ENA and anti-Scl-70
As part of routine diagnostic evaluation, levels of comple-ment C3 and C4 were determined by nephelometry, whileanti-extractable nuclear antigen (ENA) and anti-Scl-70 auto-antibodies were determined by ELISA technique.
2.12. Statistical analyses
The SPSS ver. 12.0 (SPSS Inc., Chicago, IL, UDA) was used forstatistical analysis. To assess the distribution of the dataKolmogorov–Smirnov test was used. In cases of normal distri-bution, we determined mean±standard deviation (SD) valuesand used two-sample t test for statistical comparison of the ex-perimental data. In cases of distributions different from that ofnormal, median, minimum and maximum values were calcu-lated, and Mann–Whitney test was used. The general linearmodel-repeated measures ANOVA analysis was used to evalu-ate the significance of changes in parameters over time.When the strength of the linear relationship between two vari-ables was evaluated, Pearson's correlation coefficient wasused, while in cases of non-normal distribution, Spearman'scorrelation coefficient was applied. Differences were consid-ered statistically significant at pb0.05.
3. Results
3.1. Skin and joint involvements
When changes in skin involvement were analyzed in compar-ison to baseline values, a significant improvement was ob-served. Based on echographic measurements, the dermalthickness progressively reduced during the whole therapyperiod. We found significant difference between baselinevalues and those measured after the last cycle at each inves-tigated places (Table 1). The modified Rodnan skin scorealso decreased as a result of the treatment. Significant re-duction was observed already after the first ECP cycle,
compared to the baseline (29.81±3.56 vs. 32.69±4.36, re-spectively, p=0.049), and values decreased continuouslyafter each treatment (MRSS after the last cycle: 20.17±3.76) (Fig. 1). We measured significant increase in the oralapertures after the last treatment, compared to the base-line values (3.84±0.32 cm vs. 2.96±0.53 cm, respectively,pb0.001).
Analysis of changes in joint mobility revealed clear im-provement. ECP therapy increased the ranges of motions ofboth upper and lower limbs (Table 2).
3.2. Visceral organ involvement
Pulmonary fibrosis was the most common organ involvement,and 9 of the patients had low DLCO score (lower than 75% ofthe expected value). None of our patients showed more than5% change during the follow-up period, which indicated noprogression of the pulmonary fibrosis. The distribution oforgan involvement was as follows: pulmonary fibrosis n=9;esophageal dysmotility n=4; pulmonary arterial hypertensionn=3; serositis n=1. Although organ involvements did notshow improvement through the treatments, at least theywere stabile and did not deteriorate during the ECP therapy.
3.3. Peripheral T cells, T-cell subsets and B cells
We found no significant differences in peripheral blood CD3+,CD4+ and CD8+ T-cell and B-cell numbers and percentagesbetween patients and controls. Interestingly, patientshave significantly increased percentages of late-activated T(CD3+HLA-DR+) cell, compared to control values (3.72±1.59%vs. 2.24±1.43%, respectively, p=0.018). ECP treatment didnot change significantly the number of these cell types.
3.4. Peripheral NK and NKT cells
Both numbers and percentages of peripheral NK cells were sig-nificantly higher in patients, compared to healthy individuals(NK numbers: 0.199±0.102 G/l vs. 0.131±0.038 G/l, respec-tively, p=0.043; NK percentages: 13.16±6.65% vs. 9.01±3.77%, respectively, p=0.038). On the other hand, values ofNKT cells were significantly lower, compared to controls (NKTnumbers: 0.0194±0.0177 G/l vs. 0.0399±0.0284 G/l, respec-tively, p=0.041; NKT percentages: 1.18±1.04% vs. 2.16±
Figure 1 Effects of consecutive cycles of ECP on the modified Rodnan skin scores (MRSS). Bars show the mean and the standard de-viation (SD). Statistically significant changes in values are indicated by (*).
154 G. Papp et al.
1.59%, respectively, p=0.047). According to our results, ECPtreatments had no effect on NK and NKT cell counts.
3.5. Peripheral Th1, Th2 and Th17 cells
Th1 cell numbers and percentages were significantly lowerin patients before treatments, compared to control values(Th1 numbers: 0.119±0.067 G/l vs. 0.167±0.045 G/l, re-spectively, p=0.034; Th1 percentages: 13.72±5.23% vs.18.25±4.71%, respectively, p=0.017), and these values didnot changes during the ECP therapy. Values of Th2 cellswere similar in patients and controls, and did not changedue to treatments. Regarding Th17 cells, both numbers andpercentages were significantly elevated in patients, com-pared to healthy individuals (Th17 numbers: 0.0129±0.0062 G/l vs. 0.0073±0.0034 G/l, respectively, p=0.007;Th17 percentages: 1.61±0.74% vs. 1.09±0.48%, respective-ly, p=0.025), and interestingly, already after the secondECP cycle, these values significantly decreased (Th17 num-bers: 0.0129±0.0062 G/l vs. 0.0085±0.0032 G/l, respective-ly, p=0.021; Th17 percentages: 1.61±0.74% vs. 1.14±0.37%,respectively, p=0.032) and became similar to control values(Fig. 2A).
Table 2 Significant differences between the mobility of certaintreatment. Differences were considered statistically significant at
Absolute numbers and percentages of both Tr1 and CD4+CD25+Treg cells were significantly lower in patients prior to receivingthe first ECP treatment, compared to control values (Tr1 num-bers: 0.00347±0.00126 G/l vs. 0.00691±0.00254 G/l, respec-tively, pb0.001; Tr1 percentages: 0.45±0.17% vs. 0.84±0.33%, respectively, pb0.001; CD4+CD25+ Treg numbers:0.0359±0.0094 G/l vs. 0.0479±0.0075 G/l, respectively,p=0.001; CD4+CD25+ Treg percentages: 4.88±1.17% vs. 6.14±0.93%, respectively, p=0.002). During the 9 months of therapy,values of both of these regulatory cell types significantly elevat-ed after the second ECP cycle (Tr1 numbers: 0.00347±0.00126 G/l vs. 0.00442±0.00108 G/l, respectively, p=0.041;Tr1 percentages: 0.45±0.17% vs. 0.59±0.19%, respectively,p=0.043; CD4+CD25+ Treg numbers: 0.0359±0.0094 G/l vs.0.0454±0.0138 G/l, respectively, p=0.048; CD4+CD25+ Tregpercentages: 4.88±1.17% vs. 6.02±1.48%, respectively,p=0.021) (Fig. 2B and C). As a result of the changes mentionedearlier, the initially elevated Th17/Treg ratio significantly de-creased after the second treatment (0.35±0.17 vs. 0.21±0.11,respectively, p=0.042), and became similar to control values(0.22±0.21).
joints measured before the first and after the last ECPpb0.05.
Figure 2 Effects of consecutive cycles of ECP on the absolute numbers and percentages of peripheral Th17 (A), Tr1 (B) and CD4+CD25+ Treg (C) cells. Control values are gained from healthy individuals (n=16). Cells were quantified as their percentage in theCD4+ lymphocyte population. Bars show the mean and the standard deviation (SD). Statistically significant changes are indicatedby (*).
The in vitro suppressor capability of CD4+CD25+ Treg cells wasreduced in patients, compared to that found in healthy individ-uals (1.66±0.30 vs. 2.21±0.75, respectively, p=0.014), how-ever, already after the first cycle, it improved significantly(1.66±0.30 vs. 2.29±0.46, respectively, pb0.001) and becamesimilar to control values (Fig. 3).
3.8. Levels of circulating cytokines
Measured biomarkerswere cataloged in five functional subsets:cytokines that drive, albeit not exclusively, pro-inflammatoryeffects (IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-6, IL-17, TNF-alpha, IFN-gamma), cytokines with mainly anti-inflammatoryeffects (IL-1Ra, IL-10, IL-13), cytokines with pro-fibroticeffects (TGF-beta, FGF, VEGF, EGF), cytokinewith anti-fibroticeffects (HGF) and chemokines (CCL2, IL-8). According to our re-sults, levels of cytokines withmainly pro-inflammatory effects,showed no significant changes over the time of therapy. Con-cerning anti-inflammatory cytokines, the concentration ofcirculating IL-10 significantly elevated already after thesecond cycle of ECP treatment, compared to baseline(5.18±4.38 pg/ml vs. 2.15±2.01 pg/ml, respectively, p=0.022) (Fig. 4A). Additionally, levels of IL-1Ra showed a statisti-cally significant increasing trend over time (F=2.919,p=0.028)(Fig. 4B). The pro-fibrotic cytokine TGF-beta decreased signifi-cantly already after the first treatment, compared to baselinevalues (1.28±0.46 pg/ml vs. 1.77±0.84 pg/ml, respectively,p=0.049) (Fig. 4C), moreover, levels of HGF showed a statisti-cally significant upward trend over time (F=2.687, p=0.041)(Fig. 4D). Interestingly, CCL2 chemokine levels significantly de-creased already after the second treatment, compared to base-line values (377.99±83.62 pg/ml vs. 463.11±115.01 pg/ml,respectively, p=0.037) (Fig. 4E).
Figure 3 Effects of consecutive cycles of ECP on the in vitro supprewas determined, as the ratio of optical density (OD) values of the CD4values are gained from healthy individuals (n=16). Bars show thechanges are indicated by (*).
3.9. Levels of autoantibodies and complements
Among patients, we found 15 both anti-ENA and anti-Scl-70antibody-positive individuals. No changes in levels of auto-antibodies and complements were detected during the in-vestigated period.
3.10. Association between clinical amelioration andchanges in peripheral immune parameters
We performed a broad spectrum of correlation analyses toexplore any possible association between the immunomodu-latory and the clinical effects of photopheresis. Significantcorrelations was observed between the reduction of abso-lute numbers and percentages of peripheral Th17 cells andthe reduction of the skin thickness measured by ultrasoundscanner at the base of 3rd finger (Th17 numbers: R=0.784,p=0.001; Th17 percentages: R=0.649, p=0.009) and fore-arm (Th17 numbers: R=0.532, p=0.043; Th17 percentages:R=0.518, p=0.048).
4. Discussion
Several studies confirmed good clinical response to photo-pheresis treatments in many diseases, such as graft-versus-host disease (GVHD), cutan T-cell lymphoma and a few auto-immune conditions. Concerning SSc, only two trials werereported in the last decade, and both of them underlinedthe positive effect of ECP on clinical symptoms of SSC. Oneof them, in which 13 patients were studied, found that ECPmay improve dermal edema, not fibrosis [5]. In the otherstudy, which was a double-blind, placebo-controlled clinicaltrial with 64 patients, clear improvement was observed inboth skin severity scores and joint involvement [4]. Our re-sults reinforce these earlier observations, since our patients
ssion capability of CD4+CD25+ Treg cells. Suppression capability+CD25− T cell cultures and mixed lymphocyte reactions. Controlmean and the standard deviation (SD). Statistically significant
Figure 4 Effects of consecutive cycles of ECP on the serumlevels of IL-10 (A), IL-1Ra (B), TGF-beta (C) HGF (D) and CCL2(E). Bars show the mean and the standard deviation (SD). Statis-tically significant changes in values are indicated by (*).
157Photopheresis in systemic sclerosis
showed significant amelioration of symptoms during ECP thera-py. MRSS scores decreased, joint mobility improved, oral aper-ture progressively increased, and mimical function becamebetter. Before our present investigations, the immunobiologicalmechanisms of ECP did not studied in scleroderma in details.
Clinical and pathological evidence support the conceptthat SSc is primarily a vascular disease that is mediated byautoimmune processes and results in tissue fibrosis. Recentstudies as well as our investigations highlighted the keyroles of the innate and adaptive immune system in autoim-mune processes characteristic to SSc. Besides the abnormal-ities of NK and NKT cells [11,12], alterations in Th17, Tr1 andCD4+CD25+ Treg cell subsets were also demonstrated in SSc[13–16]. In accordance with the literature and our previousresults, we found increased NK and Th17 cell numbers andpercentages, while values of NKT, Th1, Tr1 and CD4+CD25+Treg cells were decreased in SSc patients, compared tothose found in controls. These results reflect that the al-tered Th17 and regulatory T cells ratio may play a pathogen-ic role by tipping the fine balance toward enhanced immunereactivity. ECP treatment seems to have a strong effect onmost of the T-cell subtypes, but not on NK and NKT cellcounts. Based on studies focusing on immunological effectsof ECP in GVDH, photopheresis, besides the direct elimina-tion of autoreactive cells, may increase the proportions ofperipheral CD4+CD25+ Treg cells, contributing to the resto-ration of disproportional autoimmune responses [17–19].We also observed that effect on CD4+CD25+ Treg subsets,however, changes in proportions of T cell subsets seem tobe more complex. While ECP increases the numbers and per-centages of IL-10-producing Tr1 cells, values of Th17 cellsdecrease following the therapy. According to our results, thechanges in the parameters of Th17 cells seem to be muchmore pronounced in the patients with greater improvementin skin thickness during the therapy. Additionally, we observedimprovement in the suppressor effect of CD4+CD25+ Tregcells, and elevation in the concentration of the circulatinganti-inflammatory cytokine, IL-10. These findings indicatethat not only quantitative changes of T-cell subsets, but alsoqualitative changes may be responsible for the good clinicalresponse to ECP treatments. Along with IL-10 levels, concen-tration of IL-1Ra showed significant increase during the thera-py. The cytokine IL-1Ra, by inhibiting competitively thebinding of IL-1 to cell surface receptors, prevents the pro-in-flammatory effects of IL-1, thus functions as a major naturallyoccurring anti-inflammatory protein [20]. Since the elevatedIL-1 beta levels impair the suppression ability of CD4+CD25+Treg cells and contribute to the generation of Th17 cells[21], the increase in levels of IL-1Ra, along with the increasedIL-10 levels, may also contribute to the deceleration of the en-hanced autoimmune responses.
Regarding fibrogenesis, growing evidence indicates thecritical involvement of infiltrating activated macrophagesand T-cells in the production of a variety of pro-fibrotic cy-tokines such as TGF-beta, platelet-derived growth factor(PDGF), CCL2, IL-2, IL-4, IL-6 and IL-17, all of which induceor promote fibrosis and fibroproliferation [16,22,23]. Addi-tionally, local tissue response to the vascular injury involvesactivation of matrix metalloproteinases leading to extracellu-lar breakdown; and release of angiogenic growth factors suchas VEGF or FGF [24], which also contribute to pathologic tissueremodeling processes.We found that ECP significantly reduces
TGF-beta and CCL2 levels in SSc, leading to attenuated pro-fibrotic activity, which at least partly explain the clinicalamelioration observed due to the therapy. Interestingly, arecent study reported decreased levels of HGF, which is apro-angiogenic but anti-fibrotic factor, in the disease [25]. Anin vitro study revealed that high concentration of HGF inhibitscollagen production in cultured fibroblasts derived from pa-tients with SSc [26]. Based on these findings, a deficiency or re-duction of HGF may prevent vascular repair and increase tissuefibrosis in the disease. According to our results, ECP treatmentsincrease the levels of HGF, which, together with the decreasein TGF-beta and CCL2 levels, may be an effective moleculartherapeutic response resulting in attenuated fibrosis.
Although photopheresis may not improve the previouslydeveloped fibrosis in SSc, the therapy contributes to the de-celeration of disease progression. Additionally, the fact thatour patients did not show any adverse reaction to ECP, un-derlines the minimal toxicity of ECP, which is a clear advan-tage compared with other immunomodulatory therapies.
The longer follow-up of patients is necessary to assess,how persistent the clinical improvements and changes in lab-oratory parameters are, and estimate how often should ECPtreatment be repeated to achieve the best results in SSc. Webelieve that further investigation of the complex mecha-nisms of ECP will open new avenues to control derailed im-mune machinery, thus this method can be a potentiallypowerful element of the modern therapeutic arsenal in SSc.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgment
This work was supported by the TÁMOP 4.2.1./B-09/1/KONV-2010-0007 project. The project is co-financed by theEuropean Union and the European Social Fund.
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