Association of immunological cell profiles with specific clinical phenotypes of scleroderma disease

Research Article Association of Immunological Cell Profiles with Specific Clinical Phenotypes of Scleroderma Disease José Manuel López-Cacho,1Soledad Gallardo,1Manuel Posada,2 Miriam Aguerri,1David Calzada,1Teodoro Mayayo,3María Luisa González-Rodríguez,4 Antonio María Rabasco,4Carlos Lahoz,1,5 and Blanca Cárdaba1,5 1Department of Immunology, IIS-Jim´ enez D´ ıaz Foundation, Reyes Cat´ olicos Avenue 2, 28040 Madrid, Spain 2Institute of Rare Diseases Research, Carlos III Institute of Health, EuroBioBank, and CIBERER, 28029 Madrid, Spain 3Sani-Red S.L, Barcelona Scientific Park, 08013 Barcelona, Spain 4Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Seville, 41012 Seville, Spain 5CIBER of Respiratory Diseases (CIBERES), 28029 Madrid, Spain Correspondence should be addressed to Blanca C´ ardaba; [email protected] Received 7 January 2014; Revised 17 February 2014; Accepted 16 March 2014; Published 10 April 2014 Academic Editor: Richard Gomer Copyright © 2014 Jos´ eManuelL ´ opez-Cacho et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This study aimed to search the correlation among immunological profiles and clinical phenotypes of scleroderma in wellcharacterized groups of scleroderma patients, comparing forty-nine scleroderma patients stratified according to specific clinical phenotypes with forty-nine healthy controls. Five immunological cell subpopulations (B, CD4+and CD8+T-cells, NK, and monocytes) and their respective stages of apoptosis and activation were analyzed by flow cytometry, in samples of peripheral blood mononuclear cells (PBMCs). Analyses of results were stratified according to disease stage, time since the diagnosis, and visceral damage (pulmonary fibrosis, pulmonary hypertension, and cardiac affliction) and by time of treatment with corticosteroids. An increase in the percentages of monocytes and a decrease in the B cells were mainly related to the disease progression. A general apoptosis decrease was found in all phenotypes studied, except in localized scleroderma. An increase of B and NK cells activation was found in patients diagnosed more than 10 years ago. Specific cell populations like monocytes, NK, and B cells were associated with the type of affected organ. This study shows how, in a heterogeneous disease, proper patient’s stratification according to clinical phenotypes allows finding specific cellular profiles. Our data may lead to improvements in the knowledge of prognosis factors and to aid in the analysis of future specific therapies. 1. Introduction Scleroderma is a rare autoimmune disease of unknown etiology which affects thousands of people around the world. Itsprevalenceisestimatedtobebetween15and35cases per 100000 inhabitants [1–3]. Its first symptoms can be seen around the third and fourth decade of the life but, in some cases, symptoms can exist for several years without a correct diagnosis. Scleroderma is three times more common in womenthanmen.Thediseaseisnotlinkedinanyconsistent way to race, season, geography, occupation, or socioeconomic status. Environmental etiologies are nonetheless possible [1]. Scleroderma is a complex autoimmune disease characterized by fibrosis in all the organs, although its name is derived fromthefibrosisofskincausedbythedisease.Damageinthe endothelium seems to be the initial lesion responsible for the cascade of events that results in the disease [4,5]leadingthree main types of alterations: vascular occlusion, immune system alterations, and connective tissue proliferation. Fibrosis of the internal organs leads to respiratory problems, dysphagia, bowel alterations, and kidney and cardiac dysfunctions; these complications lead to marked disability, loss of quality of life, and high rates of mortality [1]. There are two main types of scleroderma, localized and systemic. Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 148293, 8 pages http://dx.doi.org/10.1155/2014/148293 2BioMed Research International Localized scleroderma affects mainly the skin, while systemic scleroderma may affect many parts of the body. Localized scleroderma (LSc) can be classified as morphea, linear, or linear “en coup de sabre.” Systemic scleroderma (SSc) can be divided into three major subtypes, limited cutaneous systemic sclerosis (lcSSc), diffuse cutaneous systemic sclerosis (dcSSc) (sclerosis of proximal extremities, trunk, and face), and systemic sclerosis sine scleroderma (organ fibrosis only; no skin thickening). Differences have been described in immunological cell subpopulations in patients with different types of complications and visceral involvement [6–8]. The results of studies in scleroderma patients which have investigated the number and the percentages of lymphocytes [9–12], their activation [13,14], and apoptosis states [14,15]have shown some discrepancies. Reasons for these discrepancies are not clearly apparent but could be related to differences in subtype, stage, and activity of the disease, demographic characteristics of the patients, and methodological issues in obtaining and analyzing patient samples [15]. To clarify these discrepancies, we have studied the relationship between clinical scleroderma phenotypes and the different cellular subpopulations in well-characterized groups of scleroderma patients, comparing their results with healthy controls and stratifying them according to scleroderma subtypes, time since the diagnosis of the disease, and presence of complications (pulmonary fibrosis, pulmonary hypertension, and cardiac affliction) and by time with corticosteroids treatment. Clinical characteristics like gastroesophageal reflux, digital ulcers, joint involvement, scleroderma renal crisis, or Rodnan total skin [16] score were not considered phenotypic strata in this study. 2. Materials and Methods 2.1. Subjects. Scleroderma patients and controls were ascertained from Spanish Association of Scleroderma (AEE), with the collaboration of the Institute of Rare Diseases Research (IIER), Instituto de Salud Carlos III (ISCIII), and the Spanish Federation of Rare Diseases (FEDER). As all scleroderma patients were prevalent cases, we validated their diagnosis checking that they met the classification criteria for SSc established by the ARA in 1980 [17] instead of the new one established in 2013 by the ACR/EULAR [18]. The identification of the different types of scleroderma as well as the organ damages was based on clinical features extracted from the clinical records which were made by rheumatologists and/or internal medicines specialists at hospitals of Spain. Time elapsed since the onset of the first symptoms to the diagnosis of the disease was estimated through telephone surveys, with the cases who accepted to participate in this study. Clinical medical records were revised to confirm some dates, when necessary criteria for pulmonary fibrosis, heart lesions, and pulmonary hypertension were obtained from echocardiography and/or pulmonary function tests stated in clinical records. Controls were selected according to the age and gender of each patient recruited who was responsible for providing Table 1: Participant distribution by subsets. Comparison Group Patients Controls CONT 49 Patients PAT 49 Type of scleroderma LSc 6 dcSSc 32 lcSSc 11 Time since the diagnosis of the disease (years) 0–3 13 4–10 15 >11 21 Treatment with corticoids (years) No 21 0–3 17 >311 Pulmonary fibrosis No 26 Yes 23 Cardiac affliction No 42 Yes 7 Pulmonary hypertension No 31 Yes 18 his/her own controls among his/her friends and/or neighborhoods. Controls demonstrated no clinical findings suggestive of infections, allergic manifestations, or immunological disorders. Forty-nine scleroderma patients (43 females and 6 males) and 49 healthy subjects (26 females and 23 males) were included in the study. Although there was a higher presence of females in scleroderma patients than in control group (87.7% versus 53.06%), the analysis according to sex did not show any statistically significant association with cellular population studied. The mean age ±standard deviation in the total group of patients was 48.0 ± 10.9 years and 48.4 ± 13.1 years for controls (Table 1). During the assays, thirteen patients were receiving corticosteroid treatment (11 with prednisone, 1 with methylprednisolone, and 1 with deflazacort), 4 patients were undergoing treatment with other immunosuppressive drugs, and 11 patients were treated with a combination of an immunosuppressive with a corticosteroid. None of the patients took the medication in the 12 hours before the samples were collected. All participants signed an informed consent in which they agreed to participate in the project. The project was also approved by the Ethics Committee of both participating institutions (Instituto de Salud Carlos III and IIS-Fundaci´ on Jim´ enez D´ ıaz). 2.2. Isolation of Peripheral Blood Mononuclear Cells (PBMCs). PBMCs from donors were isolated from venous blood by density gradient sedimentation on Lymphoprep (Comercial Rafer, Zaragoza, Spain) following the manufacturer’s instructions and resuspended in freezing medium containing 90% FBS(Lonza,Verviers,Belgium)and10%DMSO(SigmaAldrich, St. Louis, USA). The cells were aliquoted into cryogenicvialsandstoredinliquidnitrogenuntiluse. PBMCswerethawedinRPMI1640supplementedwith BioMed Research International 3 FL4 log: FL4 log logarithmic FL3 log: FL3 log logarithmic FL4 log: FL4 log logarithmic FL4 log: FL4 log logarithmic FL1 log: FL1 log logarithmic FL3 log: FL3 log logarithmic FL2 log: FL2 log logarithmic FL5 log: FL5 log logarithmic FL3 log: FL3 log logarithmic 11E1 1E2 1E3 1E4 1 1E1 1E2 1E3 1E4 11E1 1E2 1E3 1E4 1 1E1 1E2 1E3 1E4 ANEX-V+CD25+ 11E1 1E2 1E3 SS lin: SS lin Exp-SSC low 0 100 200 300 500 11E1 1E2 1E3 1 1E1 1E2 11E1 1E2 1E3 1 1E1 1E2 1E3 CD3+CD8+ CD3+CD4+CD8+ Monocytes CD4+SSC B cells (CD19+) NK cells (CD56+) CD3+CD4+ CD3+cells Figure 1: Characterization of cellular subpopulations analyzed by flow cytometry. FL1: Annexin-V+cells; FL2: CD3+or/and CD56+cells; FL3: CD4+cells; FL4: CD19+or CD8+cells; FL5: CD25+cells; SSC: side-scatter detector. 5% FBS inactivated, 1% L-glutamine, 1% peni-streptomycin, and 1 mM sodium pyruvate (Flow Laboratories, Irvine, UK) and after two washed, the PBMCs were resuspended in saline buffer and stained according recommendations of the specific commercial antibody. Quantification and viability were determined by trypan blue exclusion. Before doing apoptosis and activation analysis, controls with freeze PBMCs samples were carried out for testing quality of assays. Samples included for analysis had a viability of at least 90%. All biological samples were collected from the Institute of Rare Diseases Research Biobank, belonging to European Rare Diseases Biobank-EuroBioBank. 2.3. Immunophenotyping. Cellular phenotype was analyzed by flow cytometry using a single PBMCs sample labeled with a five-color combination of monoclonal antibodies for Annexin-V-FITC (FL-1) (Ref: ANXVKF-100T, Inmunostep, Salamanca, Spain), CD3-PE (Ref: CYT-3PE5) and CD56-PE conjugated (FL-2) (Ref: CYT-56PE) (Vitro-Cytognos, Salamanca, Spain), CD4-ECD (FL-3) (Ref: 6604727, Beckman Coulter, Izasa, Barcelona, Spain), CD8-PC5 (Ref: CYT-8C2) and CD19-PC5 conjugated (FL-4) (Ref: CYT-19C2) (VitroCytognos, Salamanca, Spain), and CD25-PC7 (FL-5) conjugated (Ref: PN A52882, Beckman Coulter, Izasa, Barcelona, Spain) by using Cytomics FC500 and CXP software (Beckman Coulter, Brea, USA). Unlike other studies, the different signal intensities of this combination of fluorochromes allowed us to analyze several clusters of differentiation at the same time using only one determination (Figure 1). At least 5,000 events were acquired in a list mode and all the data were analyzed by cell quest software (CXP Beckman Coulter). Five-color flow cytometry analysis was performed as follows. Briefly, 2⋅105PBMCs were labeled with 20 𝜇L of each monoclonal antibody (CD3-PE, CD56-PE, CD4-ECD, CD8PC5, CD19-PC5, and CD25-PC7 conjugated) and placed in an ice-water bath for 20 minutes. AfterwashingwithPBS,20𝜇L of Annexin-V-FITC was delivered and the tubes were placed in an ice-water bath for 15 minutes in darkness. Then, the cells were resuspended in 200 𝜇L of binding buffer (PBS) for immediate flow cytometry analysis. Live PBMCs were electronically gated by forwardand right-angle scatters; different types of cells were then gated and the percentage and intensity of expression were evaluated. When the gated cells were uncertain or very few, the data were excluded. Negative controls included a human isotype-matched nonrelevant immunoglobulin: Mouse IgG2a-PE (CYT-IC006PE, Cytognos) and IgG2bPE (CYT-IC007PE, Cytognos), IgG1-PECy5 Mouse Isotypic Control (CYT-IC005C, Cytognos), Mouse IgG2aPC7 (PN 4BioMed Research International CONT PAT LSc lcSSc dcSSc NO PF PF NO PH PH 10 20 30 40 B lymphocytes Cells (%) ∗ ∗ ∗ ∗ ‡ Figure 2: Box-plot of B lymphocytes. ∗Statistically significant relative to controls. ‡Statistically significant relative to patients without pulmonary hypertension. A12692, Beckman Coulter), and Mouse IgG1-ECD Isotypic Control (A07797, Beckman Coulter). Figure 1 (upper panel) summarizes the selection of cell populations studied: percentages of T lymphocytes CD3+CD4+and CD3+CD8+were evaluatedforthetotalnumberofCD3 +cells. Percentages of B cells (CD3−CD19+), NK cells (CD3−CD56+), and monocytes were evaluated regarding total number of cells. Monocytes were gated based on CD3−CD4+expression and high sidescatter (SSC). After the determination of cellular subpopulations, the percentages of apoptosis (Annexin-V+) and activation (CD25+expression) of each subpopulation were evaluated regarding their respective overall number of cells (lower panel of Figure 1). 2.4. Statistical Analysis. Global analysis was performed by Kruskal-Wallis test and comparisons between groups were performed using a nonparametric two-tailed Mann-Whitney test for independent variables. A 𝑃value of <0.05 was used. WeusedtheRprogramforthestatisticalanalysisandtheboxplots design. 3. Results Table 2 showsthemediansofthepercentagesofeachtypeof cellular subpopulations studied, as well as their activation or apoptosis stages, grouped by clinical phenotype. Significant differences were detected in scleroderma patients when we compare their results with the control group. We found a decrease in the percentage of B lymphocytes and higher values of monocytes. We did not find any difference in activation states, although for apoptosis values we observed a decrease in all the subsets of cells compared to controls. According to scleroderma phenotypes, we found a progressive reduction in the percentages of B lymphocytes in the most severe phenotypes of the disease (Figure 2); this decrease in the diffuse systemic variant (𝑃 < 0.01)compared CONT PAT LSc lcSSc dcSSc NO PF PF NO PH PH 20 40 60 80 Overall apoptosis Apoptosis (%) ∗ ∗ ∗∗∗ ∗ ∗ † Figure 3: Box-plot of overall apoptosis regarding scleroderma type and presence of PH and PF. ∗Statistically significant relative to controls. †Statistically significant relative to patients without pulmonary hypertension. CONT PAT NO PF PF NO PH PH 0 10 20 30 40 50 60 Activated NK cells Cells (%) ∗ ∗ Figure 4: Box-plot of activated NK cells. ∗Statistically significant relative to controls. to controls was statistically significant. A statistically significant increase in monocytes was shown relative to controls (3.35%) only in patients affected by systemic sclerodermas (7.41% in limited form and 7.91% in diffuse form). No difference in cell activation was found among scleroderma phenotypes. As for overall apoptosis, we found a decrease in systemic but not in localized sclerodermas (Figure 3). Apoptosisvaluesbycellularpopulation(Table 2) were decreased in several cellular subsets in systemic sclerodermas. The reduction was in all the subsets analyzed in the diffuse form and B cells and in monocytes in the limited form. Localized sclerodermas showed an apoptotic decrease in monocytes. In patients with pulmonary fibrosis (PF), we found a reduction in the percentages of B lymphocytes (Figure 2)and an increase of activated NK cells (Figure 4). Patients with and without fibrosis showed increased monocyte percentages (Table 2) and similar apoptotic profiles. We obtained reduced percentages in all cell subpopulations except in CD4 T-cells in patients with PF. BioMed Research International 5 Table 2: Cell subpopulation percentages (medians) stratified by subsets. Cell population Overall Type Pulmonary fibrosis Pulmonary hypertension Cardiac affliction Time since the diagnosis of the disease (years) Corticosteroids treatment (years) Controls 𝑛=49 Patients 𝑛=49 LSc 𝑛=6 lcSSc 𝑛=11 dcSSc 𝑛=32 No 𝑛=26 Yes 𝑛=23 No 𝑛=31 Yes 𝑛=18 No 𝑛=42 Yes 𝑛=7 0–3 𝑛=13 4–10 𝑛=15 >10 𝑛=21 No 𝑛=21 0to3 𝑛=17 >3 𝑛=11 General CD4+T-cells 65.04 62.46 62.36 66.17 63.84 62.36 63.84 65.66 62.19 62.46 68.35 64.16 63.84 61.91 62.31 62.46 65.46 CD8+T-cells 29.86 27.16 28.45 25.77 25.14 27.66 25.65 26.15 27.5 27.66 25.14 28.81 24.44 29.13 25.26 29.23 25.14 Bcells 15.62 11.14 16.19 12.13 10.56 14.02 9.97 16.87 5.98‡12.39 6.19 12.51 12.13 11.01 18.50 10.56 9.97 NK cells 11.09 11.73 10.09 9.74 11.77 9.89 12.4 10.51 12.7 11.65 12.34 9.43 11.65 12.34 14.81 9.89 10.39 Monocytes 3.35 7.41 4.65 7. 4 1 7. 9 1 7. 4 1 7. 2 5 7. 6 6 3.78 7. 2 5 1 2 . 81 6.87 7. 5 3 4.43 6.87 7.53 4.43 Activation Overall 7.32 8.07 9.37 8.68 6.46 6.46 8.63 6.87 8.67 8.20 6.91 8.38 5.14 9.52∗8.67 8.68 6.46 CD4+T-cells 5.05 6.88 7.81 8.47 5.78 5.78 7.21 5.54 7.08 6.69 7.99 8.17 4.67 7.44 5.23 7.69 6.69 CD8+T-cells 4.34 5.66 7.00 5.29 4.49 5.29 8.25 5.08 8.28 6.09 4.17 6.61 3.87 7.09 7.56 6.90 4.17 B cells 9.79 11.61 13.11 5.41 11.98 11.87 11.48 11.33 15.69†11.87 10.00 12.19 9.03 13.48 8.54 13.48 14.71 NK cells 14.93 19.57 24.57 22.41 15.68 18.41 21.39 18.99 22.44 19.57 21.30 23.27 11.76 23.48 17.05 23.09 17.71 Monocytes 70.80 70.10 81.75 66.67 61.90 77.36 59.42 74.88 61.52 74.75 58.60 62.93 84.68 61.90 84.45 70.66 50.00 Apoptosis Overall 29.00 19.16 31.23 18.51 15.41 19.67 19.16 14.67 25.32†19.16 19.61 16.77 15.41 25.22 20.21 19.61 12.88 CD4+T-cells 22.56 17.48 22.61 13.97 11.05 17.66 17.48 11.10 21.88†17.48 21.36 13.23 11.05 22.79 18.44 18.22 11.05 CD8+T-cells 21.42 16.75 28.57 21.05 10.63 16.67 21.05 10.49 24.8 16.75 22.00 21.07 10.35 27.08 16.71 21.69 9.84 B cells 30.97 19.42 38.59 15.52 18.92 19.12 20.92 15.26 28.13†20.92 18.92 18.85 19.12 22.35 20.89 24.68 18.92 NK cells 45.68 35.43 68.02 37.93 30.77 37.93 32.67 33.10 45.32 36.69 32.67 37.31 27.99 40.81 31.18 47.54 33.33 Monocytes 80.00 62.90 59.67 65.97 62.90 58.58 66.67 58.32 67.48 62.90 62.04 57.44 62.04 66.26 62.93 66.41 55.88 Bold letters indicate statistically significant results relative to controls. ‡Statistically significant compared to controls and without pulmonary hypertension. †Statistically significant relative to patients without pulmonary hypertension. ∗Indicate those data statistically significant compared to four and ten years from the diagnosis of the disease. 6BioMed Research International In the group of patients with pulmonary hypertension (PH), we found the lowest percentages of B cells (Figure 2). This group was the only one without an increase of monocytes (Table 2). We also found significant increases of activated B, NK, and CD8+cells. The increase of CD8+cellsisaltered like in pulmonary fibrosis. Regarding apoptosis, we found decreases in all the subpopulations of patients without pulmonary hypertension when comparing these subpopulations to controls. In addition, we found statistically significant differences when comparing absence and presence of PH, withlowerapoptosisfoundintheabsenceofpulmonary hypertension (Table 2). We found few significant differences in patients affected by cardiac affliction (CA) probably due to the low number of patients tested (𝑛=7);weonlyfoundanincreaseof monocytes (Table 2). Regarding time since the diagnosis of the disease, we only found an increase of monocytes in patients diagnosed from 4 to 10 years (Table 2). Patients diagnosed more than 10 years previously showed an increase in the percentages ofactivatedBandNKcells;theseresultswereverysimilar to those obtained in patients diagnosed from 0 to 3 years (Table 2). We also found the lowest percentages of apoptosis in the group of patients diagnosed from 4 to 10 years ago (Table 2), but a progressive recovery is produced with time, except for B cells and monocytes. Regarding to the treatment time with corticosteroids, thetreatment decreases the percentages of B cells and, in patients treated during more than 3 years, a reduction of the percentages of monocytes and their activation state are produced (Table 2). In this group we also obtained the smallest percentages of apoptosis (Table 2). In addition, the group of patients diagnosed from zero to three years had higher percentages of activated NK cells. 4. Discussion There have been conflicting results concerning changes in the immune cell subpopulations involved in scleroderma [11,19– 21] due to the heterogeneity of the disease and differences in study methods. In order to shed light on this issue, we have studied a well-defined clinical population, analyzing both the cell subpopulations and their activation and apoptosis stages and also correlating the data with different clinical phenotypes. We obtained normal values of T lymphocytes, as has been described by other authors [11,20–22]. However, we found a reduction of B-cells in the worse-prognosis situations of thediseasebuthigherpercentagesofmonocytesinalmost all SSc subsets. These results can be also correlated with the conditions of visceral involvement that triggers the worst prognosis (Figure 2 and Table 2). The monocyte fraction of peripheral blood contains precursors to potential regulators of fibrosis [23]suchas fibrocytes and macrophages [18,24,25]. Several studies obtained functional abnormalities and increased numbers of these cells in scleroderma patients [23,26–29]. Our results are consistent with many data that indicate the association between elevated levels of peripheral blood fibrocytes and monocytes and diverse forms of organ remodeling such as renal fibrosis, cirrhosis, or different types of pulmonary fibrosis [29]. An abnormal remodeling in scleroderma is usually more common in systemic sclerodermas [30]. However, our monocytes results must be considered with caution because monocytes were not detected directly through a CD14 marker and, instead, were selected indirectly, as CD3−CD4+,and some monocytes cannot express CD4. The action of B cells in scleroderma could be related to their functions in the regulation of the immune response [31] and antibody production and abnormalities in them can induce or develop autoimmune diseases like scleroderma. Analyzing the activation states, we obtained a higher overall activation in patients in whom the time elapsed since diagnosiswasgreaterorinpatientswithvisceralinvolvement (especially in B and NK cells). Some authors determine that survival rates of scleroderma patients begin their decline in theperiodoftimeoffourtotenyearssincetheygotothe hospital [32,33]. As detected in our study, the development of serious visceral involvement portends early mortality in patients with scleroderma [32,34,35] and is concomitant with elevated activation states of immune cells [28,36,37]. The role played by NK cells in autoimmune diseases is both a diseasecontrolling and a disease-promoting role and NK cells may be implicated in the onset, maintenance, or progression of these diseases [38], thus suggesting their involvement in the scleroderma [39]. Activation markers like CD25 play a role in cell contact interactions between T-cells and other cells, including fibroblasts. However, the analysis of other lymphocytes activation markersasCD69orMHCClassIIcouldbeinteresting, especially for determining monocyte activation. Inclusion of cytokine production from these cell populations could also enhance information about their states of activation. Apoptosis values may be difficult to evaluate due to the method for conserving the cells. In this study, as frozen cells were used, the freezing effect was checked and was homogenous in all samples. Annexin V staining precedes the loss of membrane integrity which accompanies the later stages of cell death resulting from either apoptotic or necrotic processes, for that was selected as cellular death marker. We found a decrease in different subsets in the disease, possibly because of apoptosis resistance in the cells of scleroderma patients. Apoptosis resistance is the primary mechanism leading to the development of fibrotic lesions and could be one of the reasons why some types of sclerodermas have more visceral fibrosis and worse prognosis, especially in the case of diffuse forms, where the apoptosis rates are the lowest. Some authors [40] have described that, during later phases of systemic sclerosis, inflammatory events become less intense and the immune response acts as a low-grade amplifier of fibrogenesis and microangiopathy, presenting a significant therapeutic challenge. This could explain the differences that we have found between the patients analyzed according to timesincediagnosis:adiminutioninthebeginningofthe disease, which could coincide with the endothelial damage; after four to ten years, the disease could coincide with the development of fibrosis in the internal organs, where we have BioMed Research International 7 obtained apoptosis diminutions; and, finally, until about ten years following diagnosis, the apoptosis tends to increase and thefibrosistendstoslowdown. In patients diagnosed more than ten years previously, the reduction in the apoptosis of B cells and monocytes is parallel to an increase in the overall activation, especially in the activation of B and NK cells. This coincides with decreases in the survival rates of the scleroderma patients [32,33]and organ affliction tends to appear in the late-onset stages of the disease [30]. The detected alterations in these subpopulations couldhavesomeinfluenceonsurvivalratesandcouldbe useful in the prognosis of the disease. The usual treatment of scleroderma—especially of organ fibrosis—[30] is based on immunosuppressive drugs and corticosteroids that increase the apoptosis of the inflammatory cells [41]. In our results, the treatment seems to increase theapoptosisinthegrouptreatedwithcorticosteroidsfrom zero to three years but, in patients with more than three years treated, the situation reverts to diminished percentages. This could be one of the reasons that make treatment with corticosteroids inefficient in patients who have had the disease for many years. In addition to this, two things must be taken into account: both kinds of drugs can interfere with the action of B cells, although they usually do not reduce their numbers and corticosteroids produce an inhibitory effect in NK cells [42] but these cells have a lower sensitivity to these kinds of drugs [43]. These data coincide with our results where more time is needed to reduce their activation. However, corticosteroids are useful in monocytes, producing a reduction in the patients with more time with the disease. 5. Conclusions In conclusion, an overall decrease in apoptosis and in the numbers of B cells together with an increase of monocytes and activated B and NK cells is directly correlated with the severity of the disease. These results suggest that these cells contribute to the immunological abnormalities observed in sclerodermaandcouldannouncetheappearanceofcomplications or a worsening in the disease. Conflict of Interests The authors declare that they have no financial relationships related to any products involved in this study that might lead to a conflict of interests. Acknowledgments The authors would like to thank the Spanish Federation of Rare Diseases (FEDER, Federaci´ on Espa˜ nola de Enfermedades Raras) and the Spanish Association of Scleroderma (Asociaci´ on Espa˜ nola de Esclerodermia, AEE) for their assistance in recruiting volunteers. They also thank the Spanish Rare Diseases Biobank, EuroBioBank, partner for their support in the biological samples management. They also would like to acknowledge the help of the IIS-Fundaci´ on Jim´ enez D´ ıaz and especially Ignacio Mahillo from the Epidemiology Department for assistance with statistical analysis. They also wouldliketothanktheUniversityofSeville.Thisworkwas supported by the enterprise By Biotech & Science by Grants from Corporaci´ on Tecnol´ ogicaAndaluzaandAgenciaIDEA (no. exp. 34323). Miriam Aguerri and David Calzada were supported by Fundaci´ on Conchita R´ abago, Madrid, Spain. References [1] J. Barnes and M. D. 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