http://informahealthcare.com/mor ISSN 1439-7595 (print), 1439-7609 (online) Mod Rheumatol, 2014; 24(1): 97–105 © 2013 Japan College of Rheumatology DOI 10.3109/14397595.2013.854052
ORIGINAL ARTICLE
Peripheral blood mononuclear cell microchimerism in Turkish female patients with systemic sclerosis
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¨ zkan • Nuran Tu¨rkc¸apar • Orhan Ku¨c¸u¨ks¸ ahin • Ali S¸ ahin • Tu¨lin O ¨ zturk • S¸ u¨kran Erten • Asuman Sungurog˘lu Elif Berna Ko¨ksoy • Gu¨ls¸ ah O • Murat Turgay Gu¨lay Kınıklı
•
Received: 7 December 2012 / Accepted: 15 February 2013 Ó Japan College of Rheumatology 2013
Abstract Objectives To investigate microchimerism (Mc) in peripheral blood mononuclear cells (PBMC) taken from female patients with systemic sclerosis (SSc) and healthy females. We also intended to research the association between Mc and the clinical subsets. Methods This study included 50 females with lcSSc, 30 females with dcSSc and 40 healthy females. The Y-chromosome sequences were studied by RT-PCR in DNA obtained from PBMC. Results Mc was found in 28 (35 %) patients and 8 (20 %) healthy controls as well as in 6 dcSSc patients with son(s) (27.3 %), 10 lcSSc patients with son(s) (32.3 %) and 7 control females with son(s) (18.9 %) (p [ 0.05). Mc was detected in 6 nulliparous lcSSc patients (31.6 %) and in 1 nulliparous dcSSc patient (11.1 %) (p [ 0.05). The mean time elapsed between the first pregnancy and the diagnosis of SSc was 3.5 (0–49) years in the Mc-positive patients and 14 (0–55) years in the negative patients (p = 0.020). The mean A. S¸ ahin (&) Division of Rheumatology, Sanliurfa Education and Research Hospital, 63100 Sanliurfa, Turkey e-mail: [email protected] ¨ zkan G. O ¨ zturk A. Sungurog˘lu T. O Department of Medical Biology, Ibni Sina Hospital, Ankara University School of Medicine, Ankara, Turkey N. Tu¨rkc¸apar O. Ku¨c¸u¨ks¸ ahin E. B. Ko¨ksoy M. Turgay G. Kınıklı Department of Internal Medicine, Rheumatology, Ibni Sina Hospital, Ankara University School of Medicine, Ankara, Turkey S¸ . Erten Division of Rheumatology, Atatu¨rk Education and Research Hospital, Ankara, Turkey Published online: 14 March 2013
modified Rodnan skin scores (ModRSS) of the patients with and without Mc was 10 (4–24) and 13 (4–26), respectively (p = 0.038). The relationship between Mc and the system involvement, disease severity, autoantibody profile, number of children and age of children was not found. Conclusions Various etiological factors rather than just one play a role in the development of scleroderma. Mc is thought to be one factor that shortens the elapsed time of disease development in SSc. Mc is inversely related to the ModRSS, and no association was detected between Mc and autoantibodies or the clinical subsets. Keywords Female Lymphocyte Microchimerism Systemic sclerosis Turkish
Introduction Systemic sclerosis (SSc) is an autoimmune disease that involves the skin, lung, heart, kidney and gastrointestinal system. The etiology of the disease, however, remains unknown. Microvascular changes, excessive extracellular matrix production and immune system dysregulation are responsible for SSc pathogenesis [1]. Fibrosis in the internal organs and skin triggered by complex interactions in the autoimmune system accounts for the clinical manifestation of the disease. SSc is mainly classified into two subtypes: limited cutaneous systemic sclerosis (lcSSc) and diffuse cutaneous systemic sclerosis (dcSSc) [1]. LcSSc involves distal parts of the body, such as the hands and feet, whereas dcSSc involves the skin and many internal organs and has a higher mortality and morbidity. The etiologic factors that cause the clinical differences observed in the patients are unknown [2]. In graft-versus-host disease (GVHD), which develops as a chronic complication in allogeneic stem cell transplant
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patients, the development of fibrosis in organs and tissues similar to scleroderma and even being positive for autoantibodies suggests that microchimerism might have a role in the pathogenesis of scleroderma [3]. Microchimerism (Mc) is the presence of a small amount of DNA or cells from one individual in another individual. The most common and natural resource of microchimerism is pregnancy. SSc, of which GVHD is the iatrogenic cause, is observed more in women than in men and occurs mostly in young women after childbirth. During pregnancy, hemopoietic cells travel to both the mother and the fetus through the placenta, and these microchimeric cells have been shown to maintain their presence for years. The cells belonging to the mother who has given birth to a baby and those of the baby received by the mother can remain in circulation for years. This hypothesis suggests that disease development is not only present in women who have children but also in men and in women who did not bear a child. The presence of Mc is thought to play a role in not only scleroderma but also in other autoimmune diseases and in non-autoimmune diseases (such as malignancies) [4–11]. In this study, the Mc presence in the peripheral blood lymphocytes of patients with diffuse and limited type scleroderma and its relationship with child-bearing and the gender of both the children and the patient was investigated. Moreover, another aim was to investigate the relationship of these data to the pathogenesis and clinical course of the disease.
Materials and methods The investigation was based on the SSc patients who were followed in the inpatient and outpatient clinics of Ankara University Faculty of the Medicine Department of Rheumatology and healthy age- and gender-matched volunteers without any known diseases and medication use. According to the diagnostic criteria of the American College of Rheumatology (ACR) [12], two groups were formed: the group of 80 female patients diagnosed with SSc and the group of 40 healthy female controls. The patient group was divided into two subgroups: patients with dcSSc (n = 30) and patients with lcSSc (n = 50). For this research, the Ankara University Faculty of Medicine Ethics Committee approval, dated 06.28.2010 and with decision number 13-265, was obtained. This study was performed in accordance with the Helsinki declaration 2008. Inclusion criteria 1.
All patients with newly diagnosed SSc according to the ACR diagnostic criteria [12] or those patients who received treatment and follow-up.
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2.
3.
Female patients with a history of birth (especially those who have male children), including those with a history of abortion. Young unmarried patients without a history of birth or abortion.
Exclusion criteria 1.
2.
3. 4. 5.
Patients from the control group who have different additional diseases (such as malignancy and autoimmune diseases) and have previously identified/known autoantibodies (such as RF, ANA and ANCA). Patients with severe psychiatric disorders that could affect cooperation (especially the treatment of psychosis and depression within the last 30 days). Patients with a history of blood transfusion. Patients with a history of peripheral blood stem cell transplantation (allogeneic transplant). Patients with a history of drug use in the control group (such as oral contraceptives and antidepressants).
Observation and laboratory techniques All patients and healthy controls were included in a detailed anamnesis and physical examination. Recent and older data from the patient files were examined. Each patient’s duration of illness, examination, prevalence of skin involvement, telangiectasia and calcinosis and other findings were recorded. Skin scores were calculated for all patients, and the patients were divided into two groups according to the skin involvement as either patients with dcSSc or lcSSc. Raynaud’s phenomenon was evaluated according to its presence at the time of the diagnosis or its emergence during follow-up. Lung involvement was monitored using laboratory and imaging methods, such as the pulmonary functional test (PFT), diffusion lung of carbon monoxide (DLCO), and high resolution computerized tomography (HRCT), 6-min walk test (6MWT), New York Heart Association (NYHA) functional class (I–IV) to decide whether the patient had or needed treatment for any of these issues. Pulmonary arterial hypertension (PAH) was diagnosed as a mean pulmonary arterial systolic pressure C40 mmHg on echocardiography (echo) or C25 mmHg on right heart catheterization [13]. Interstitial lung involvement/disease (ILD) was confirmed as previously proposed recommendations [14]. Heart involvement was detected using various methods, such as electrocardiography (ECG) and echo. Arrhythmia, conduction defects, measurement of left ventricular ejection fraction, history or presence of pericarditis, echo findings, and the need for treatment were decided accordingly.
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Gastrointestinal tract involvement, mouth appearance, dry mouth, dysphagia, reflux symptoms, malabsorption, weight loss, diarrhea and constipation, and previous findings, such as those of esophagogastroduodenoscopy/colonoscopy, esophageal manometry were assessed according to the results. Cardiac/heart involvement and gastrointestinal involvement was defined as recent literature [15, 16] Renal involvement was determined as proteinuria, hematuria, and the signs and symptoms of scleroderma renal crisis (SRC) [17]. The pregnancy and abortion histories of the patients with children (not necessarily alive) were recorded. The patients with children were also questioned about the gender of their children (with a preference for those with male children), the age of their children and the time period between their birth and the emergence of the disease symptoms. In the control group, the patients’ age and the time period between the most recent birth and blood collection were recorded. The ModRSS and ‘‘Medsger’s disease severity scale’’ were calculated [18]. ANA, anti-Scl-70, anti-centromere, anti-SS-A, anti-SS-B, anti-U1-RNP and other existing immunoblotting data were obtained from the patient files. The aim of this study was explained to the participants, and written informed consent forms were obtained from all subjects included in the study. DNA extraction and real-time PCR Genomic DNA was extracted from the peripheral blood samples of patients and healthy subjects using a PureLinkTM Genomic DNA Mini Kit (InvitrogenTM, catalog No. K182002), according to the manufacturer’s instructions. The DNA was stored at -20 °C until PCR amplification. The DNA integrity and quantity were assessed by agarose gel electrophoresis and spectrophotometry, respectively. For this purpose, a 1 % agarose gel was used, and the 260 and 280 nm optical density ratio values (OD260/ OD280) were determined. The SRY levels in the peripheral blood were analyzed using a Light Cycler System (Roche, Mannheim, Germany). As a standard template, DNA derived from a normal adult male was used. The primers were SRY-F (forward primer) 50 -TGGCGATTAAGTCAAATTCGC-30 and SRY-R (reverse primer) 50 -CCCCCTAGTACCCTGACAATGTATT-30 [19]. Simultaneously, the same amount of target DNA was tested for the GAPDH gene to confirm the quality (ability to amplify) of the extracted DNA. The primers were GAPDH-F 50 -AAC AGCGACACCCACTCCTC-30 and GAPDH-R 50 -CATAC CAGGAAATGAGCTTGACAA-30 [20]. For the PCR assay, 50 ng of extracted DNA and the LightCyclerÒ 480 SYBR Green I Master Mix (Roche) were used. The PCR reaction was started with denaturation at 95 °C for 5 min, followed by forty cycles at 95 °C for 30 s, 62 °C
for 30 s, and 72 °C for 30 s and then 72 °C for 5 min. Figure 1 below shows agarose gel electrophoresis of the PCR-amplified SRY regions. The Light Cycler System software was used for analyzing the amplified data. In all groups, the presence of microchimerism was verified with the Light CyclerÒ (with RT-PCR), and DNA amplification curves were obtained and screened. The DNA isolation and PCR were performed with particular care to avoid any contamination. Evaluation of data and statistical analysis For the registration and statistical analysis of the data, SPSS (Statistical Package for Social Sciences, Chicago, IL, USA) 16.0 software package was used. Descriptive statistical data were presented as the mean ± standard deviation (median or minimum–maximum). The suitability of the data to a normal distribution was checked using the Kolmogorov–Smirnov and Shapiro–Wilk normality tests. Parametric tests were used for the evaluation of those data complying with a normal distribution, and non-parametric tests were used for the evaluation of those data not complying with a normal distribution. A comparison of continuous variables between the two groups was conducted using Student’s t test for parametric variables and the Mann–Whitney U test for non-normal variables. To evaluate the correlation between two continuous variables, we used the Pearson correlation coefficients test for parametric variables. The Kruskal–Wallis non-parametric analysis of variance was used for the comparison of three groups. Values of p \ 0.05 were considered statistically significant.
Results In this study, 80 female patients with SSc were evaluated, with 50 (62.5 %) lcSSc patients and 30 (37.5 %) dcSSc patients. The mean age of the lcSSc patients was 46.20 ± 14.6 (44, 18–80) years, and the mean age of the dcSSc patients was 47.23 ± 14.1 (44, 27–76) years. In the control group, 40 healthy women were evaluated. The mean age of the control women was 42.55 ± 11.4 (40, 28–73) years. There was no age difference between the patient and control groups (p = 0.375). In the control group, 37 women (92.5 %) had sons, 1 (2.5 %) had not given birth (nulliparous), and 2 (5 %) had a daughter. A total of 53 (66.25 %) patients had a son. In the dcSSc patient group, 22 women (73.3 %) had sons, 4 (13.3 %) had daughters, and 4 (13.3 %) had not given birth. In the lcSSc patient group, 31 female patients (62 %) had sons, 7 (14 %) had daughters, 10 (20 %) had not given birth, and 2 (4 %) had miscarriages (Table 1).
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Fig. 1 Microchimerism according to SRY on agarose gel electrophoresis. MPP microchimerism-positive patient, MNP microchimerismnegative patient, NT non-templated, negative control with water, SRY sex-determining region of Y (137 bp) Table 1 Characteristic features and Mc statuses of the groups dcSSc
lcSSc
Control 40
p
N (%)
30 (37.5)
50 (62.5)
Age (years)
47.23 ± 14.17 (44)
46.20 ± 14.69 (44)
42.55 ± 11.40 (40)
0.375
Age at first birth (years)
24.92 ± 5.1
24.11 ± 3.8
26.21 ± 3.8
0.095
Age at diagnosis (years)
36.10 ± 11.89 (33.5)
40.14 ± 14.23 (37.5)
0.188
Time between birth to diagnosis (years)
10.2 ± 9.4 (7.5)
14.32 ± 14 (12)
0.390
Disease duration (years)
11.47 ± 8.9 (9)
6.20 ± 6.5 (4)
Number of children
2.69 ± 1.3 (1–6)
2.76 ± 1.2 (1–6)
1.74 ± 0.9 (1–5)
Mothers who had boys, n (%)
22 (73.3)
31 (62)
37 (92.5)
Mothers who had girls, n (%) Nulliparous patients, n (%)
4 (13.3) 4 (13.3)
7 (14) 10 (20)
2 (5) 1 (2.5)
0.001* \0.05*
Pregnancy history
Patients who had abortions, n (%)
0.004*
2 (4)
Microchimerism n (%) Mothers who had boys, n (%)
6 (66.7)
10 (52.6)
Mothers who had girls, n (%)
2 (22.2)
2 (10.5)
Nulliparous patients, n (%)
1 (11.1)
Patients who had abortions, n (%)
6 (31.6)
7 (87.5)
[0.05
1 (12.5)
1 (5.3)
Data expressed as the mean ± SD (median) dcSSc diffuse cutaneous systemic sclerosis, lcSSc limited cutaneous systemic sclerosis * Statistically significant
The mean number of children was 2.69 ± 1.37 (1–6) in the dcSSc patient group and 2.76 ± 1.21 (1–6) in the lcSSc patient group. The mean number of children in the control group was 1.74 ± 0.91 (1–5). The difference between the number of children in the patient and control groups was significant (p \ 0.05). The median duration of illness was 4 (1–28) years in the lcSSc group and 9 (1–35) years in the dcSSc group. The
difference in the duration of illness between these two groups was significant (Mann–Whitney test, p = 0.001) (Table 1). The mean age of the women at their first birth was 26.21 ± 3.8 (17–34) years in the control group, 24.92 ± 5.1 (17–37) years in the dcSSc patient group and 24.11 ± 3.8 (18–33) years in the lcSSc patient group. There was no significant difference with respect to the age
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Table 2 Systemic involvement and autoantibody profiles of SSc patients according to their clinical subsets
dcSSc n (%)
lcSSc n (%)
Gastrointestinal involvement
23 (76.7)
20 (40)
Raynaud’s phenomenon
28 (93.3)
47 (94)
[0.05
5 (16.7)
3 (6)
[0.05
Cardiac involvement dcSSc diffuse cutaneous systemic sclerosis, lcSSc limited cutaneous systemic sclerosis, SRC scleroderma renal crisis, PAP pulmonary arterial pressure a
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Statistically significant difference between 2 groups according to the PAP level (p = 0.001) * Statistically significant
Renal involvement (SRC)
–
p
OR (95 %CI) 0.001*
4.92 (1.78–13.70)
1 (2)
13 (43.3)
2 (4)
\0.05*
6 (20)
3 (6)
[0.05
ANA positivity
29 (96.7)
47 (94)
[0.05
Anti-Scl-70 positivity
23 (76.7)
13 (26)
\0.05*
9.34 (3.26–27.02)
Anti-centromere positivity
2 (6.7)
20 (40)
\0.05*
9.33 (1.997–43.627)
Anti-SS-A positivity
5 (16.7)
13 (26)
[0.05
Anti-SS-B positivity
1 (3.3)
6 (12)
[0.05
5 (10)
[0.05
Interstitial lung involvement Pulmonary hypertensiona
Anti-U1-RNP positivity
at first birth (one-way ANOVA, p = 0.095). The elapsed time between the diagnosis and birth was a mean of 10.2 (median 7.5, 0–30) years in the dcSSc patients and 14.32 (median: 12, 0–55) years in the lcSSc patients. This difference was not significant (p = 0.390). The mean age at diagnosis in the patients with dcSSc and lcSSc was 36.10 ± 11.8 (33.5, 18–56) and 40.14 ± 14.2 (37.5, 15–77) years, respectively. Again, there was no significant difference (p = 0.188) (Table 1). The ModRSS was a median of 22 (12–26) in the dcSSc patients and 10 (4–24) in the lcSSc patients, which was a significant difference (p \ 0.05). The median Medsger’s disease severity scale was 2 (1–3) in the dcSSc patients and 1 (1–2) in the lcSSc patients, which was also a significant difference (p \ 0.05). The median ModRSS was 22 (12–26) in the dcSSc patients who had son(s) and 24 (14–24) in the dcSSc patients who did not have sons (p = 0.872). The median ModRSS was 8 (4–14) in the lcSSc patients who had son(s) and 10 (4–24) in lcSSc patients who did not have sons (p = 0.577). The median Medsger’s disease severity scale was 2 (1–3) in the dcSSc patients who had son(s) and 2 (1–2) in the dcSSc patients who did not have sons (p = 0.447). The median Medsger’s disease severity scale was 1 (1–2) in the lcSSc patients who had sons and 1 (1–2) in the lcSSc patients who did not have sons (p = 0.296). Moreover, the Raynaud phenomenon was detected in 28 (93.3 %) patients with dcSSc and in 47 (94 %) patients with lcSSc (p [ 0.05) (Table 2). The median pulmonary arterial pressure (PAP) was 28 (20–50) mmHg in the dcSSc patients and 25 (20–45) mmHg in the lcSSc patients (p = 0.001) (Table 2). Cardiac involvement was found in 5 (16.7 %) patients with dcSSc and in 3 (6 %) patients with lcSSc. Pulmonary hypertension was found in 6 (20 %) dcSSc patients and in 3 (6 %) lcSSc patients (p [ 0.05) (Table 2).
–
18.52 (3.74–90.91)
Gastrointestinal involvement was observed in 23 (76.7 %) patients with dcSSc and in 20 (40 %) patients with lcSSc (Pearson Chi-square, p = 0.001) (Table 2). Gastrointestinal involvement was 4.92 times higher in the dcSSc patients than in the lcSSc patients (OR 4.92, 95 % CI 1.78–13.70), and 1 (2 %) patient with lcSSc had renal involvement (Table 2). Interstitial lung disease was identified in 13 (43.3 %) patients with dcSSc and in 2 (4 %) patients with lcSSc (Pearson Chi-square, p \ 0.05). Similarly, interstitial lung disease was 18.52 times higher in dcSSc patients than in lcSSc patients (OR 18.52, 95 % CI 3.74–90.91) (Table 2). Overall, 29 (96.7 %) patients with dcSSc and 47 (94 %) patients with lcSSc had ANA positivity (p [ 0.05) (Table 2). Anti-Scl-70 was positive in 23 (76.7 %) patients with dcSSc and in 13 (26 %) patients with lcSSc. Anti-Scl70 positivity was 9.34 times greater in the dcSSc group than in the lcSSc group (OR 9.34, 95 % CI 3.26–27.02) (Table 2). The anti-centromere antibody was positive in 2 (6.7 %) patients with dcSSc and in 20 (40 %) patients with lcSSc. Anti-centromere antibody positivity was 9.33 times more likely in lcSSc than in dcSSc (OR 9.33, 95 % CI 1.997–43.627) (Table 2). Five (10 %) patients with lcSSc were anti-U1-RNP- positive, and 1 (2 %) patient with lcSSc was anti-Ku positive (Table 2). Anti-SS-A was positive in 5 (16.7 %) patients with dcSSc and in 13 (26 %) patients with lcSSc (p [ 0.05) (Table 2). Anti-SS-B was positive in 1 (3.3 %) patient with dcSSc and in 6 (12 %) patients with lcSSc (p [ 0.05) (Table 2). Two (6.6 %) patients with dcSSc had ankylosing spondylitis (HLA-B27-positive). Diabetes mellitus (DM) was observed in 4 (8 %) patients with lcSSc and in 1 (3.3 %) patient with dcSSc. Familial Mediterranean fever (FMF) was detected in 1 (2 %) patient with lcSSc, and Meniere disease was detected in 1 (2 %) patient with lcSSc. Kikuchi Fujimato disease was present in 1 (2 %) patient with lcSSc, and
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chronic hepatitis C (HCV) was present in 1 (3.3 %) patient with dcSSc. One (2 %) patient with lcSSc had primary biliary cirrhosis, 1 (2 %) patient with lcSSc had otosclerosis, and three (6 %) patients with lcSSc had hypothyroidism. Mc was detected in 8 (within group 20 %, between groups 22.2 %) women from the control group, 9 (within group 30 %, between groups 25 %) women from the dcSSc group, and 19 (within group 38 %, between groups 52.8 %) women from the lcSSc group (p = 0.180). The child status of the Mc-positive patients diffuse cutaneous SSc was as follows: 6 (66.7 %) had son(s), 2 (22.2 %) had daughter(s), and 1 (11.1 %) was nulliparous-nulligravid. The child status of the Mc-positive patients limited cutaneous SSc was as follows: 10 (52.6 %) had son(s), 2 (10.5 %) had daughter(s), 6 (31.6 %) were nulliparous-nulligravid, and 1 (5.3 %) had a miscarriage. In the control group, 7 (87.5 %) Mc-positive women had son(s) and 1 (12.5 %) Mc-positive woman was nulliparous-nulligravid (Table 1). There were no significant differences between Mc and lung involvement (p = 0.555), renal involvement (p [ 0.05), Raynaud’s phenomenon (p = 0.337), cardiac involvement (p = 0.706), pulmonary hypertension (p = 0.483), or interstitial lung disease (p = 0.177). The median PAP of the patients with Mc was 25 (20–40) mmHg, and the median PAP of the patients without Mc was 26 (20–50) (p = 0.151). In addition, there were no significant differences between Mc and ANA positivity (p = 0.609), anti-Scl-70 positivity (p = 0.777), anti-centromere positivity (p = 0.372), anti-U1-RNP positivity (p = 0.653), anti-SS-A positivity (p = 0.694), or anti-SS-B positivity (p = 0.232). The median ModRSS was 10 (4–24) in the Mc-positive patients and 13 (4–26) in the Mc-negative patients (Mann– Whitney test, p = 0.038). The median of the Medsger’s disease severity scale was 1 (1–2) in the Mc-positive patients and 1 (1–3) (p = 0.118) in the Mc-negative patients. Of the 28 microchimerism-positive patients (75 %), 21 had a Medsger’s disease severity scale of 1, and 7 (25 %) had a score of 2. In the patients with Mc, the median number and age of their children were 2 (1–5) and 20 (1–5) years, respectively. No significant difference was detected between the presence of Mc and the number of children or the age of the children (p = 0,881 and p = 0,865, respectively). The mean age of the women was 44.25 ± 13.95 (42.5, 18–76) in those with Mc and 45.67 ± 13.47 (41.5, 27–80) in those without Mc. Additionally, the mean ages of the dcSSc patients with Mc, lcSSc patients with Mc and control women were 45.11 ± 13.71 (43, 30–76), 42.79 ± 15.28 (38, 18–70) and 46.75 ± 11.99 (46.5, 31–65) years, respectively. The elapsed time between the first birth and the development of the SSc symptoms (diagnosis) was a median of 3.5 (0–49) years in the Mc-positive patients and a median
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of 14 (0–55) years in the Mc-negative patients, which was a significant difference (Mann–Whitney test, p = 0.020). Additionally, Mc was detected in 2 (4 %) patients with lcSSc associated with DM and in 1 (2 %) patient with lcSSc associated with FMF.
Discussion The main trend that we identified was that SSc was more prevalent in women who had given birth and even more common in those who had male children. This observation can be explained by fetal cells. Fetal cells that pass through the placenta can be observed in the maternal blood as early as 4–5 weeks of pregnancy [21]. This passage is not just one way, as the maternal cells can also pass through the fetal circulation. For the formation and continuation of pregnancy, a certain amount of fetal cells above the threshold level is required in maternal blood circulation. Any passage below the threshold value may result in miscarriage, whereas excess passage can result in autoimmune disease susceptibility and complications in pregnancy. Various mouse model experiments have shown that peripheral antigen-specific tolerance against the fetus can develop by ‘‘clonal deletion’’ in the maternal immune system and provide for the continuation of pregnancy [22–24]. Fetal Mc may play a role in the pathogenesis of SSc cells and/or is thought to be a marker of inflammation. Although these fetal cells carry microchimeric CD3 and CD4 T cell markers, a reaction may not develop due to the development of immune tolerance during pregnancy. The role of these cells in the pathogenesis of SSc was first discussed by Scott Pereira [25]. Similarly, various other authors have reported that scleroderma, evolving as a result of the transplacental passage of cells between the mother and fetus, may be a chronic form of GVHD [25]. Nonautologous microchimeric cells that carry Y chromosome DNA sequences of female patients with male children can trigger a reaction similar to GVHD and play a role in the pathogenesis of this disease [26–29]. However, showing the presence of these cells is not only important but very difficult. Thirty women with dcSSc, seventeen with lcSSc and twenty-two healthy women in a study of CD4? and CD8? T cells containing the Y chromosome DNA sequences were investigated by quantitative PCR [30]. In this study, 82.9 % (39/47 patients) had SSc, and 63.6 % (14/22 controls) of the control group had Mc. This difference was not significant (p = 0.138). Mc was detected in 26/30 (86.7 %) of the diffuse cutaneous group and in 14/17 (82.4 %) of the limited cutaneous group, showing that the two patient groups did not differ with respect to the existence of Mc (p = 1.0). These T cells
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consisted mostly of CD4? T cells. CD4? and CD8? microchimeric cells express CD25 (IL-2 receptor) and undergo oligo-clonal expansion when they are activated. In this study, however, the prevalence of skin involvement and disease severity were not taken into account [30]. In our study, Mc was detected in 35 % (28/80) of the SSc group and in 20 % (8/40) of the control group using an RT-PCR technique. This difference was insignificant (p = 0.180). Mc was detected in 9/30 (30 %) of the diffuse cutaneous group and 19/50 (38 %) of the limited cutaneous group, which was an insignificant difference (p [ 0.05). Sawaya et al. [31] observed that microchimeric cells could be detected in skin biopsies approximately 50 years after the last birth, which demonstrates that in the early stage of the disease, microchimeric cells are detected in high concentrations in the non-involved skin areas but that the microchimeric cell ratio decreases after disease progression and inflammatory cell migration as a result of a bacterial, viral or chemical triggers. The longevity of these microchimeric cells can explain the development of the disease in nulliparous or male patients with SSc. SSc has been proposed to develop due to the settling of autologous inflammatory cells in the inflamed area, followed by the release of pro-inflammatory cytokines and fibroproliferative and vascular events as a result of the migration of microchimeric cells, which are activated through certain environmental causes (such as viral or other infections and chemical or other factors) in the ‘‘target microenvironment’’ (for example, in SSc, the target can be skin, lungs, heart or other organs) [31]. In our study, the median of the ModRSS was 10 (4–24) in the patients with Mc and 13 (4–26) in the non-microchimeric patients. The difference between these two groups was significant (Mann–Whitney test, p = 0.038). This situation demonstrates that the chance of detecting microchimeric cells in the peripheral blood circulation can be decreased in patients with high skin involvement as these cells migrate from the circulation to the skin (the target tissue). However, no significant relationship was detected among Mc and the Medsger’s disease severity score, other organ involvement or the autoantibody profile. Evidence has shown that these microchimeric cells play a protective role in pregnancy, transplantation, tissue damage repair, many autoimmune diseases, including type 1 diabetes mellitus, breast cancer and some other malignancies. However, these cells can also be detected in healthy women. An understanding of how these microchimeric cells are activated to cause SSc is still not yet clarified [31]. In our study, the mean disease duration was calculated as 11.47 ± 8.9 (9, 1–35) years in the diffuse cutaneous group and as 6.20 ± 6.5 (4, 1–28) years in the limited cutaneous group. The duration between the first birth and
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the onset of the symptoms of SSc (diagnosis) was a median of 3.5 (0–49) years in the Mc-positive group and 14 (0–55) years in the Mc-negative group. The difference between these two groups was significant (Mann–Whitney test, p = 0.020). This situation suggests that Mc shortens the onset of the disease and thus could be a triggering factor in the development of the disease. We found that the presence of Mc in the peripheral blood mononuclear cells was detected in a nulliparousnulligravid 76-year-old female patient with dcSSc, demonstrating that microchimeric cells can live in the blood circulation for many years. Furthermore, Mc was detected in a 48-year-old healthy woman with no pregnancy history (nulligravid) in the control group. In the limited cutaneous group, the nulliparous patients with detected microchimerism were 18, 20, 29, 34, 38 and 60 years old. In other words, our oldest nulliparous patients with detected Mc were 60 and 76 years old, revealing the presence of a lifelong continuous microchimeric situation. In addition, the median age of the children of the patients with Mc was 20 (1–50), and thus we demonstrated the presence of Mc in the blood mononuclear cells even 50 years after giving birth. Microchimeric T lymphocytes have been shown to be detected in the maternal circulation even 40 years after the last pregnancy [32]. A study conducted in Italy researched the presence of Mc in the peripheral blood mononuclear cells of female patients with SSc by nested-PCR and RT-PCR techniques and its relationship with the clinical and serological findings. Y chromosome-specific sequences were detected in 41 of 54 female patients with SSc (76 %) by the nestedPCR technique but only in 8 patients (15 %) by the RT-PCR technique [33]. Of the 19 patients who never gave birth to a male child and did not have a history of miscarriage, Mc was detected in 2 (10 %) by RT-PCR and in 13 (68 %) by nested-PCR. The nested-PCR technique was found to be of high sensitivity but low specificity. The relationship between Mc and the sub-types of SSc (diffuse and limited cutaneous), disease activity and organ involvement could not be shown [33]. However, in patients with systemic lupus erythematosus (SLE), Mc was found to be related to disease activity by the nested-PCR technique [34]. The beneficial and/or harmful effects of continuous Mc and its biological importance are not yet understood. Mc is believed to potentially play a role in growth and development, tissue repair, and the diversity of responses to infection, immune surveillance and autoimmunity [34]. As microchimeric cells can show ‘‘fluctuation’’ with respect to time and disease activity, the time point of taking the blood sample is also important. In our study, Mc was not detected in blood samples taken at different times in one microchimeric patient with dcSSc. This situation confirms that microchimeric cells can ‘‘fluctuate’’ and
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suggests that the timing is just as important as the technique. However, to prove the role of timing, the procedure should be performed at different times with more patients and controls. This situation could affect the detection of Mc (limitation) in our study. In one study, the presence of maternal Mc was shown in the peripheral blood mononuclear cells of 22 % of the healthy females [35]. We also detected Mc in the peripheral blood mononuclear cells of 20 % of the healthy females in our study. Mc can be a factor in the development of autoimmunity but cannot be the cause of autoimmunity itself [36]. In other words, as a result of an environmental exposure or a trigger, the fetal microchimeric cells will divide and proliferate, causing autoimmunity to then develop [36]. Although a life-long microchimeric condition can induce the development of autoimmune diseases, such as SSc, it is an indispensable condition for the continuation of life. In one study, the presence of Y chromosome DNA sequences in the peripheral blood mononuclear cells, i.e., Mc, was shown in 11 out of 35 healthy women with male children (31 %). In this study, Mc was detected in 12 out of 20 SSc patients (60 %) by the PCR method. Additionally in this study, Mc was shown in 8 women who had previous miscarriages and/or transfusions. Y chromosome DNA could show the presence of persistent male fetal cells in a woman who gave birth to a male child. In this study, the mean time between the birth of the last male child in the patients with SSc and the performance of this study was calculated to be 21 years [37]. In our study, we showed the presence of Mc in 7 out of 37 (18.9 %) healthy women who gave birth to a male child. In another study, Mc was investigated in 43 female patients with SSc who did not receive immunosuppressive treatment (such as steroids, methotrexate, and cyclophosphamide) in the last 30 days. The ages of these patients ranged between 20 and 87 years, and they all had given birth to at least one male child. Their age of diagnosis was between 20 and 68 years, and the ages of their male children were between 2 and 31 years. In this study, the sexdetermining region of Y (SRY) was detected in only 6.9 % of the patients. The disease involvement in these patients was not included in this study [38]. In our study, the median age of the children of the microchimeric patients was 20 (1–50) years, and the median number of children was 2 (1–5). In addition, the age of diagnosis of the microchimeric patients with male children was 18–69 years. In our study, the medication usage of the patients was discarded, but women who did not use any medications were investigated in the control group. As we have emphasized previously, in addition to ‘‘fluctuation’’, another factor that affects the detection of Mc is the use of ‘‘medication’’, especially ‘‘immunosuppressive medications’’. This situation could also affect the
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detection of Mc (limitation) in our study. In the future, if patients who have been diagnosed recently and did not receive any treatment are investigated, then different results may be found. In a study conducted in Spain, the presence of Mc was investigated in 47 female SSc patients (30 limited cutaneous and 17 diffuse cutaneous) and 40 healthy female individuals, with 19 lcSSc patients, 10 dcSSc patients and 26 control women having male children [39]. In this study, the presence of Mc was detected in 4 patients (13.7 %), 2 each in both sub-groups, and in 2 healthy women (7.6 %), but this difference was not significant [39]. In summary, we determined in this study that the Mc ratio in patients with dcSSc and lcSSc was not different than that in the control group. This ratio was also not related to the number of births and the sex of the children. We also could not show a significant difference associated with the clinical course of the disease. Scleroderma is a heterogeneous disease, and many factors play a role, rather than a specific etiological reason, in the development of scleroderma. However, we have shown that the presence of Mc shortens the time of onset of the disease. Acknowledgments We would like to thank to ‘‘Ankara Tıplılar Vakfı’’ for funding this project. We also thank Mrs. Zeynep BIYIKLI for the statistical analyses. In addition, the summary of this research was accepted as a poster presentation at the 5th Turkish-Greek Rheumatology Days, October 14–16, 2011. Conflict of interest
None.
References 1. LeRoy EC, Black C, Fleischmajer R, Jablonska S, Krieg T, Medsger TA Jr, et al. Scleroderma (systemic sclerosis): classification, subsets and pathogenesis. J Rheumatol. 1988;15(2):202–5. 2. Gabrielli A, Avvedimento EV, Krieg T. Scleroderma. N Engl J Med. 2009;360(19):1989–2003. 3. Ruzek MC, Jha S, Ledbetter S, Richards SM, Garman RD. A modified model of graft-versus-host-induced systemic sclerosis (scleroderma) exhibits all major aspects of the human disease. Arthritis Rheum. 2004;50(4):1319–31. 4. Jimenez SA. Artlett CM Microchimerism and systemic sclerosis. Curr Opin Rheumatol. 2005;17(1):86–90. 5. Lee ES, Bou-Gharios G, Seppanen E, Khosrotehrani K, Fisk NM. Fetal stem cell microchimerism: natural-born healers or killers? Mol Hum Reprod. 2010;16(11):869–78. 6. Sarkar K, Miller FW. Possible roles and determinants of microchimerism in autoimmune and other disorders. Autoimmun Rev. 2004;3(6):454–63. 7. Bianchi DW, Zickwolf GK, Weil GJ, Sylvester S, DeMaria MA. Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum. Proc Natl Acad Sci USA. 1996; 93(2):705–8. 8. Lo YM, Patel P, Sampietro M, Gillmer MD, Fleming KA, Wainscoat JS. Detection of single-copy fetal DNA sequence from maternal blood. Lancet. 1990;335(8703):1463–4.
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9. Nelson JL. Maternal-fetal immunology and autoimmune disease: is some autoimmune disease auto-alloimmune or allo-autoimmune? Arthritis Rheum. 1996;39(2):191–4. 10. Artlett CM. Microchimerism in health and disease. Curr Mol Med. 2002;2(6):525–35. 11. Lambert NC, Lo YM, Erickson TD, Tylee TS, Guthrie KA, Furst DE, et al. Male microchimerism in healthy women and women with scleroderma: cells or circulating DNA? A quantitative answer. Blood. 2002;100(8):2845–51. 12. Subcommittee for Scleroderma Criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee. Preliminary criteria for the classification of systemic sclerosis (scleroderma). Arthritis Rheum. 1980;23(5):581–90. 13. McLaughlin VV, Archer SL, Badesch DB, Barst RJ, Farber HW, Lindner JR, et al. ACCF/AHA 2009 Expert Consensus Document on Pulmonary Hypertension. A report of the American College of Cardiology Foundation Task Force on Expert Consensus Document and the American Heart Association. Circulation. 2009; 119:2250–94. 14. Khanna D, Brown KK, Clements PJ, Elashoff R, Furst DE, Goldin J, Seibold JR, Silver RM, Tashkin DP, Wells AU. Systemic sclerosis-associated interstitial lung disease proposed recommendations for future randomized clinical trials. Clin Exp Rheumatol. 2010;28(Suppl. 58):S55–62. 15. Boueiz A, Mathai SC, Hummers LK, Hassoun PM. Cardiac complications of systemic sclerosis: recent progress in diagnosis. Curr Opin Rheumatol. 2010;22:696–703. 16. Clements PJ, Becvar R, Drosos AA, Ghattas L, Gabrielli A. Assessment of gastrointestinal involvement. Clin Exp Rheumatol. 2003;21(3 Suppl 29):S15–8. 17. Denton CP, Lapadula G, Mouhon L, Muller-Ladner U. Renal complications and scleroderma renal crisis. Rheumatology (Oxford). 2009;48(Suppl 3):iii32–5. 18. Medsger TA Jr, Bombardieri S, Czirjak L, Scorza R, Della Rossa A, Bencivelli W. Assessment of disease severity and prognosis. Clin Exp Rheumatol. 2003;21(Suppl 29):S42–6. 19. Shimada K, Murakami K, Shozu M, Segawa T, Sumitani H, Inoue M. Sex-determining region Y levels in maternal plasma: evaluation in abnormal pregnancy. J Obstet Gynaecol Res. 2004;30(2):148–54. 20. Ponchel F, Toomes C, Bransfield K, Leong FT, Douglas SH, Field SL, et al. Real-time PCR based on SYBR—green I fluorescence: an alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions. BMC Biotechnol. 2003;3:18. 21. Thomas MR, Williamson R, Craft I, Yazdani N, Rodeck CH. Y chromosome sequence DNA amplified from peripheral blood of women in early pregnancy. Lancet. 1994;343(8894):413–4. 22. Schroder J, Tilikianen A, De la Chapelle A. Fetal leukocytes in the maternal circulation after delivery. Transplantation. 1974;17(4):346–54. 23. Schroder J. Transplacental passage of blood cells. J Med Genet. 1975;12(3):230–42. 24. Jiang S-P, Vacchio MS. Multiple mechanisms of peripheral T-cell tolerance to the fetal ‘‘allograft’’. J Immunol. 1998; 160(7):3086–90.
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25. Black CM, Stevens WM. Scleroderma. Rheum Dis Clin North Am. 1989;15(2):193–212. 26. Artlett CM, Welsh KI, Black CM, Jimenez SA. Fetal-maternal HLA compatibility confers susceptibility to systemic sclerosis. Immunogenetics. 1997;47(1):17–22. 27. Nelson JL, Furst DE, Maloney S, et al. Microchimerism and HLA-compatible relationships of pregnancy in scleroderma. Lancet. 1998;351(9102):559–62. 28. Artlett CM. Pathophysiology of fetal microchimeric cells. Clin Chim Acta. 2005;360(1–2):1–8. 29. Gammill SH, Nelson JL. Naturally acquired microchimerism. Int J Dev Biol. 2010;54(2–3):531–43. 30. Artlett CM, Cox LA, Ramos RC, Dennis TN, Fortunato RA, Hummers LK, et al. Increased microchimeric CD4? T-lymphocytes in peripheral blood from women with systemic sclerosis. Clin Immunol. 2002;103(3 Pt 1):303–8. 31. Sawaya HH, Jimenez SA, Artlett CM. Quantification of fetal microchimeric cells in clinically affected and unaffected skin of patients with systemic sclerosis. Rheumatology (Oxford). 2004;43(8):965–8. 32. Artlett CM, Dito CG, Christner PJ. Methodology for detecting trace amounts of microchimeric DNA from peripheral murine white blood cells by real-time PCR. Biol Proced Online. 2003;5:103–7. 33. Mosca M, Giuliano T, Curcio M, Doveri M, De Feo F, Tani C, et al. Comparison of real-time PCR and nested PCR for the detection of Y chromosome sequences in the peripheral blood mononuclear cells of patients with systemic sclerosis. Ann Rheum Dis. 2009;68(1):155–6. 34. Mosca M, Curcio M, Lapi S, Valentini G, D’Angelo S, Rizzo G, et al. Correlations of Y chromosome microchimerism with disease activity in patients with SLE: analysis of preliminary data. Ann Rheum Dis. 2003;62(7):651–4. 35. Lambert NC, Erickson TD, Yan Z, Pang JM, Guthrie KA, Furst DE, et al. Quantification of maternal microchimerism by HLAspecific real-time polymerase chain reaction: studies of healthy women and women with scleroderma. Arthritis Rheum. 2004;50(3):906–14. 36. Christner PJ, Artlett CM, Conway RF, Jimenez SA. Increased numbers of microchimeric cells of fetal origin are associated with dermal fibrosis in mice following injection of vinyl chloride. Arthritis Rheum. 2000;43(11):2598–605. 37. Evans PC, Lambert N, Maloney S, Furst DE, Moore JM, Nelson JL. Long-term fetal microchimerism in peripheral blood mononuclear cell subsets in healthy women and women with scleroderma. Blood. 1999;93(6):2033–7. 38. Burastero SE, Galbiati S, Vassallo A, Sabbadini MG, Bellone M, Marchionni L, et al. Cellular microchimerism as a lifelong physiologic status in parous women. An immunologic basis for its amplification in patients with systemic sclerosis. Arthritis Rheum. 2003;48(4):1109–16. 39. Selva-O’Callaghan A, Mijares-Boeckh-Behrens T, Prades EB, Solans-Laque R, Simeon-Aznar CP, Fonollosa-Pla V, et al. Lack of evidence of foetal microchimerism in female Spanish patients with systemic sclerosis. Lupus. 2003;12(1):15–20.
ORIGINAL ARTICLE
Peripheral blood mononuclear cell microchimerism in Turkish female patients with systemic sclerosis
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¨ zkan • Nuran Tu¨rkc¸apar • Orhan Ku¨c¸u¨ks¸ ahin • Ali S¸ ahin • Tu¨lin O ¨ zturk • S¸ u¨kran Erten • Asuman Sungurog˘lu Elif Berna Ko¨ksoy • Gu¨ls¸ ah O • Murat Turgay Gu¨lay Kınıklı
•
Received: 7 December 2012 / Accepted: 15 February 2013 Ó Japan College of Rheumatology 2013
Abstract Objectives To investigate microchimerism (Mc) in peripheral blood mononuclear cells (PBMC) taken from female patients with systemic sclerosis (SSc) and healthy females. We also intended to research the association between Mc and the clinical subsets. Methods This study included 50 females with lcSSc, 30 females with dcSSc and 40 healthy females. The Y-chromosome sequences were studied by RT-PCR in DNA obtained from PBMC. Results Mc was found in 28 (35 %) patients and 8 (20 %) healthy controls as well as in 6 dcSSc patients with son(s) (27.3 %), 10 lcSSc patients with son(s) (32.3 %) and 7 control females with son(s) (18.9 %) (p [ 0.05). Mc was detected in 6 nulliparous lcSSc patients (31.6 %) and in 1 nulliparous dcSSc patient (11.1 %) (p [ 0.05). The mean time elapsed between the first pregnancy and the diagnosis of SSc was 3.5 (0–49) years in the Mc-positive patients and 14 (0–55) years in the negative patients (p = 0.020). The mean A. S¸ ahin (&) Division of Rheumatology, Sanliurfa Education and Research Hospital, 63100 Sanliurfa, Turkey e-mail: [email protected] ¨ zkan G. O ¨ zturk A. Sungurog˘lu T. O Department of Medical Biology, Ibni Sina Hospital, Ankara University School of Medicine, Ankara, Turkey N. Tu¨rkc¸apar O. Ku¨c¸u¨ks¸ ahin E. B. Ko¨ksoy M. Turgay G. Kınıklı Department of Internal Medicine, Rheumatology, Ibni Sina Hospital, Ankara University School of Medicine, Ankara, Turkey S¸ . Erten Division of Rheumatology, Atatu¨rk Education and Research Hospital, Ankara, Turkey Published online: 14 March 2013
modified Rodnan skin scores (ModRSS) of the patients with and without Mc was 10 (4–24) and 13 (4–26), respectively (p = 0.038). The relationship between Mc and the system involvement, disease severity, autoantibody profile, number of children and age of children was not found. Conclusions Various etiological factors rather than just one play a role in the development of scleroderma. Mc is thought to be one factor that shortens the elapsed time of disease development in SSc. Mc is inversely related to the ModRSS, and no association was detected between Mc and autoantibodies or the clinical subsets. Keywords Female Lymphocyte Microchimerism Systemic sclerosis Turkish
Introduction Systemic sclerosis (SSc) is an autoimmune disease that involves the skin, lung, heart, kidney and gastrointestinal system. The etiology of the disease, however, remains unknown. Microvascular changes, excessive extracellular matrix production and immune system dysregulation are responsible for SSc pathogenesis [1]. Fibrosis in the internal organs and skin triggered by complex interactions in the autoimmune system accounts for the clinical manifestation of the disease. SSc is mainly classified into two subtypes: limited cutaneous systemic sclerosis (lcSSc) and diffuse cutaneous systemic sclerosis (dcSSc) [1]. LcSSc involves distal parts of the body, such as the hands and feet, whereas dcSSc involves the skin and many internal organs and has a higher mortality and morbidity. The etiologic factors that cause the clinical differences observed in the patients are unknown [2]. In graft-versus-host disease (GVHD), which develops as a chronic complication in allogeneic stem cell transplant
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patients, the development of fibrosis in organs and tissues similar to scleroderma and even being positive for autoantibodies suggests that microchimerism might have a role in the pathogenesis of scleroderma [3]. Microchimerism (Mc) is the presence of a small amount of DNA or cells from one individual in another individual. The most common and natural resource of microchimerism is pregnancy. SSc, of which GVHD is the iatrogenic cause, is observed more in women than in men and occurs mostly in young women after childbirth. During pregnancy, hemopoietic cells travel to both the mother and the fetus through the placenta, and these microchimeric cells have been shown to maintain their presence for years. The cells belonging to the mother who has given birth to a baby and those of the baby received by the mother can remain in circulation for years. This hypothesis suggests that disease development is not only present in women who have children but also in men and in women who did not bear a child. The presence of Mc is thought to play a role in not only scleroderma but also in other autoimmune diseases and in non-autoimmune diseases (such as malignancies) [4–11]. In this study, the Mc presence in the peripheral blood lymphocytes of patients with diffuse and limited type scleroderma and its relationship with child-bearing and the gender of both the children and the patient was investigated. Moreover, another aim was to investigate the relationship of these data to the pathogenesis and clinical course of the disease.
Materials and methods The investigation was based on the SSc patients who were followed in the inpatient and outpatient clinics of Ankara University Faculty of the Medicine Department of Rheumatology and healthy age- and gender-matched volunteers without any known diseases and medication use. According to the diagnostic criteria of the American College of Rheumatology (ACR) [12], two groups were formed: the group of 80 female patients diagnosed with SSc and the group of 40 healthy female controls. The patient group was divided into two subgroups: patients with dcSSc (n = 30) and patients with lcSSc (n = 50). For this research, the Ankara University Faculty of Medicine Ethics Committee approval, dated 06.28.2010 and with decision number 13-265, was obtained. This study was performed in accordance with the Helsinki declaration 2008. Inclusion criteria 1.
All patients with newly diagnosed SSc according to the ACR diagnostic criteria [12] or those patients who received treatment and follow-up.
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2.
3.
Female patients with a history of birth (especially those who have male children), including those with a history of abortion. Young unmarried patients without a history of birth or abortion.
Exclusion criteria 1.
2.
3. 4. 5.
Patients from the control group who have different additional diseases (such as malignancy and autoimmune diseases) and have previously identified/known autoantibodies (such as RF, ANA and ANCA). Patients with severe psychiatric disorders that could affect cooperation (especially the treatment of psychosis and depression within the last 30 days). Patients with a history of blood transfusion. Patients with a history of peripheral blood stem cell transplantation (allogeneic transplant). Patients with a history of drug use in the control group (such as oral contraceptives and antidepressants).
Observation and laboratory techniques All patients and healthy controls were included in a detailed anamnesis and physical examination. Recent and older data from the patient files were examined. Each patient’s duration of illness, examination, prevalence of skin involvement, telangiectasia and calcinosis and other findings were recorded. Skin scores were calculated for all patients, and the patients were divided into two groups according to the skin involvement as either patients with dcSSc or lcSSc. Raynaud’s phenomenon was evaluated according to its presence at the time of the diagnosis or its emergence during follow-up. Lung involvement was monitored using laboratory and imaging methods, such as the pulmonary functional test (PFT), diffusion lung of carbon monoxide (DLCO), and high resolution computerized tomography (HRCT), 6-min walk test (6MWT), New York Heart Association (NYHA) functional class (I–IV) to decide whether the patient had or needed treatment for any of these issues. Pulmonary arterial hypertension (PAH) was diagnosed as a mean pulmonary arterial systolic pressure C40 mmHg on echocardiography (echo) or C25 mmHg on right heart catheterization [13]. Interstitial lung involvement/disease (ILD) was confirmed as previously proposed recommendations [14]. Heart involvement was detected using various methods, such as electrocardiography (ECG) and echo. Arrhythmia, conduction defects, measurement of left ventricular ejection fraction, history or presence of pericarditis, echo findings, and the need for treatment were decided accordingly.
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Gastrointestinal tract involvement, mouth appearance, dry mouth, dysphagia, reflux symptoms, malabsorption, weight loss, diarrhea and constipation, and previous findings, such as those of esophagogastroduodenoscopy/colonoscopy, esophageal manometry were assessed according to the results. Cardiac/heart involvement and gastrointestinal involvement was defined as recent literature [15, 16] Renal involvement was determined as proteinuria, hematuria, and the signs and symptoms of scleroderma renal crisis (SRC) [17]. The pregnancy and abortion histories of the patients with children (not necessarily alive) were recorded. The patients with children were also questioned about the gender of their children (with a preference for those with male children), the age of their children and the time period between their birth and the emergence of the disease symptoms. In the control group, the patients’ age and the time period between the most recent birth and blood collection were recorded. The ModRSS and ‘‘Medsger’s disease severity scale’’ were calculated [18]. ANA, anti-Scl-70, anti-centromere, anti-SS-A, anti-SS-B, anti-U1-RNP and other existing immunoblotting data were obtained from the patient files. The aim of this study was explained to the participants, and written informed consent forms were obtained from all subjects included in the study. DNA extraction and real-time PCR Genomic DNA was extracted from the peripheral blood samples of patients and healthy subjects using a PureLinkTM Genomic DNA Mini Kit (InvitrogenTM, catalog No. K182002), according to the manufacturer’s instructions. The DNA was stored at -20 °C until PCR amplification. The DNA integrity and quantity were assessed by agarose gel electrophoresis and spectrophotometry, respectively. For this purpose, a 1 % agarose gel was used, and the 260 and 280 nm optical density ratio values (OD260/ OD280) were determined. The SRY levels in the peripheral blood were analyzed using a Light Cycler System (Roche, Mannheim, Germany). As a standard template, DNA derived from a normal adult male was used. The primers were SRY-F (forward primer) 50 -TGGCGATTAAGTCAAATTCGC-30 and SRY-R (reverse primer) 50 -CCCCCTAGTACCCTGACAATGTATT-30 [19]. Simultaneously, the same amount of target DNA was tested for the GAPDH gene to confirm the quality (ability to amplify) of the extracted DNA. The primers were GAPDH-F 50 -AAC AGCGACACCCACTCCTC-30 and GAPDH-R 50 -CATAC CAGGAAATGAGCTTGACAA-30 [20]. For the PCR assay, 50 ng of extracted DNA and the LightCyclerÒ 480 SYBR Green I Master Mix (Roche) were used. The PCR reaction was started with denaturation at 95 °C for 5 min, followed by forty cycles at 95 °C for 30 s, 62 °C
for 30 s, and 72 °C for 30 s and then 72 °C for 5 min. Figure 1 below shows agarose gel electrophoresis of the PCR-amplified SRY regions. The Light Cycler System software was used for analyzing the amplified data. In all groups, the presence of microchimerism was verified with the Light CyclerÒ (with RT-PCR), and DNA amplification curves were obtained and screened. The DNA isolation and PCR were performed with particular care to avoid any contamination. Evaluation of data and statistical analysis For the registration and statistical analysis of the data, SPSS (Statistical Package for Social Sciences, Chicago, IL, USA) 16.0 software package was used. Descriptive statistical data were presented as the mean ± standard deviation (median or minimum–maximum). The suitability of the data to a normal distribution was checked using the Kolmogorov–Smirnov and Shapiro–Wilk normality tests. Parametric tests were used for the evaluation of those data complying with a normal distribution, and non-parametric tests were used for the evaluation of those data not complying with a normal distribution. A comparison of continuous variables between the two groups was conducted using Student’s t test for parametric variables and the Mann–Whitney U test for non-normal variables. To evaluate the correlation between two continuous variables, we used the Pearson correlation coefficients test for parametric variables. The Kruskal–Wallis non-parametric analysis of variance was used for the comparison of three groups. Values of p \ 0.05 were considered statistically significant.
Results In this study, 80 female patients with SSc were evaluated, with 50 (62.5 %) lcSSc patients and 30 (37.5 %) dcSSc patients. The mean age of the lcSSc patients was 46.20 ± 14.6 (44, 18–80) years, and the mean age of the dcSSc patients was 47.23 ± 14.1 (44, 27–76) years. In the control group, 40 healthy women were evaluated. The mean age of the control women was 42.55 ± 11.4 (40, 28–73) years. There was no age difference between the patient and control groups (p = 0.375). In the control group, 37 women (92.5 %) had sons, 1 (2.5 %) had not given birth (nulliparous), and 2 (5 %) had a daughter. A total of 53 (66.25 %) patients had a son. In the dcSSc patient group, 22 women (73.3 %) had sons, 4 (13.3 %) had daughters, and 4 (13.3 %) had not given birth. In the lcSSc patient group, 31 female patients (62 %) had sons, 7 (14 %) had daughters, 10 (20 %) had not given birth, and 2 (4 %) had miscarriages (Table 1).
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Fig. 1 Microchimerism according to SRY on agarose gel electrophoresis. MPP microchimerism-positive patient, MNP microchimerismnegative patient, NT non-templated, negative control with water, SRY sex-determining region of Y (137 bp) Table 1 Characteristic features and Mc statuses of the groups dcSSc
lcSSc
Control 40
p
N (%)
30 (37.5)
50 (62.5)
Age (years)
47.23 ± 14.17 (44)
46.20 ± 14.69 (44)
42.55 ± 11.40 (40)
0.375
Age at first birth (years)
24.92 ± 5.1
24.11 ± 3.8
26.21 ± 3.8
0.095
Age at diagnosis (years)
36.10 ± 11.89 (33.5)
40.14 ± 14.23 (37.5)
0.188
Time between birth to diagnosis (years)
10.2 ± 9.4 (7.5)
14.32 ± 14 (12)
0.390
Disease duration (years)
11.47 ± 8.9 (9)
6.20 ± 6.5 (4)
Number of children
2.69 ± 1.3 (1–6)
2.76 ± 1.2 (1–6)
1.74 ± 0.9 (1–5)
Mothers who had boys, n (%)
22 (73.3)
31 (62)
37 (92.5)
Mothers who had girls, n (%) Nulliparous patients, n (%)
4 (13.3) 4 (13.3)
7 (14) 10 (20)
2 (5) 1 (2.5)
0.001* \0.05*
Pregnancy history
Patients who had abortions, n (%)
0.004*
2 (4)
Microchimerism n (%) Mothers who had boys, n (%)
6 (66.7)
10 (52.6)
Mothers who had girls, n (%)
2 (22.2)
2 (10.5)
Nulliparous patients, n (%)
1 (11.1)
Patients who had abortions, n (%)
6 (31.6)
7 (87.5)
[0.05
1 (12.5)
1 (5.3)
Data expressed as the mean ± SD (median) dcSSc diffuse cutaneous systemic sclerosis, lcSSc limited cutaneous systemic sclerosis * Statistically significant
The mean number of children was 2.69 ± 1.37 (1–6) in the dcSSc patient group and 2.76 ± 1.21 (1–6) in the lcSSc patient group. The mean number of children in the control group was 1.74 ± 0.91 (1–5). The difference between the number of children in the patient and control groups was significant (p \ 0.05). The median duration of illness was 4 (1–28) years in the lcSSc group and 9 (1–35) years in the dcSSc group. The
difference in the duration of illness between these two groups was significant (Mann–Whitney test, p = 0.001) (Table 1). The mean age of the women at their first birth was 26.21 ± 3.8 (17–34) years in the control group, 24.92 ± 5.1 (17–37) years in the dcSSc patient group and 24.11 ± 3.8 (18–33) years in the lcSSc patient group. There was no significant difference with respect to the age
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Table 2 Systemic involvement and autoantibody profiles of SSc patients according to their clinical subsets
dcSSc n (%)
lcSSc n (%)
Gastrointestinal involvement
23 (76.7)
20 (40)
Raynaud’s phenomenon
28 (93.3)
47 (94)
[0.05
5 (16.7)
3 (6)
[0.05
Cardiac involvement dcSSc diffuse cutaneous systemic sclerosis, lcSSc limited cutaneous systemic sclerosis, SRC scleroderma renal crisis, PAP pulmonary arterial pressure a
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Statistically significant difference between 2 groups according to the PAP level (p = 0.001) * Statistically significant
Renal involvement (SRC)
–
p
OR (95 %CI) 0.001*
4.92 (1.78–13.70)
1 (2)
13 (43.3)
2 (4)
\0.05*
6 (20)
3 (6)
[0.05
ANA positivity
29 (96.7)
47 (94)
[0.05
Anti-Scl-70 positivity
23 (76.7)
13 (26)
\0.05*
9.34 (3.26–27.02)
Anti-centromere positivity
2 (6.7)
20 (40)
\0.05*
9.33 (1.997–43.627)
Anti-SS-A positivity
5 (16.7)
13 (26)
[0.05
Anti-SS-B positivity
1 (3.3)
6 (12)
[0.05
5 (10)
[0.05
Interstitial lung involvement Pulmonary hypertensiona
Anti-U1-RNP positivity
at first birth (one-way ANOVA, p = 0.095). The elapsed time between the diagnosis and birth was a mean of 10.2 (median 7.5, 0–30) years in the dcSSc patients and 14.32 (median: 12, 0–55) years in the lcSSc patients. This difference was not significant (p = 0.390). The mean age at diagnosis in the patients with dcSSc and lcSSc was 36.10 ± 11.8 (33.5, 18–56) and 40.14 ± 14.2 (37.5, 15–77) years, respectively. Again, there was no significant difference (p = 0.188) (Table 1). The ModRSS was a median of 22 (12–26) in the dcSSc patients and 10 (4–24) in the lcSSc patients, which was a significant difference (p \ 0.05). The median Medsger’s disease severity scale was 2 (1–3) in the dcSSc patients and 1 (1–2) in the lcSSc patients, which was also a significant difference (p \ 0.05). The median ModRSS was 22 (12–26) in the dcSSc patients who had son(s) and 24 (14–24) in the dcSSc patients who did not have sons (p = 0.872). The median ModRSS was 8 (4–14) in the lcSSc patients who had son(s) and 10 (4–24) in lcSSc patients who did not have sons (p = 0.577). The median Medsger’s disease severity scale was 2 (1–3) in the dcSSc patients who had son(s) and 2 (1–2) in the dcSSc patients who did not have sons (p = 0.447). The median Medsger’s disease severity scale was 1 (1–2) in the lcSSc patients who had sons and 1 (1–2) in the lcSSc patients who did not have sons (p = 0.296). Moreover, the Raynaud phenomenon was detected in 28 (93.3 %) patients with dcSSc and in 47 (94 %) patients with lcSSc (p [ 0.05) (Table 2). The median pulmonary arterial pressure (PAP) was 28 (20–50) mmHg in the dcSSc patients and 25 (20–45) mmHg in the lcSSc patients (p = 0.001) (Table 2). Cardiac involvement was found in 5 (16.7 %) patients with dcSSc and in 3 (6 %) patients with lcSSc. Pulmonary hypertension was found in 6 (20 %) dcSSc patients and in 3 (6 %) lcSSc patients (p [ 0.05) (Table 2).
–
18.52 (3.74–90.91)
Gastrointestinal involvement was observed in 23 (76.7 %) patients with dcSSc and in 20 (40 %) patients with lcSSc (Pearson Chi-square, p = 0.001) (Table 2). Gastrointestinal involvement was 4.92 times higher in the dcSSc patients than in the lcSSc patients (OR 4.92, 95 % CI 1.78–13.70), and 1 (2 %) patient with lcSSc had renal involvement (Table 2). Interstitial lung disease was identified in 13 (43.3 %) patients with dcSSc and in 2 (4 %) patients with lcSSc (Pearson Chi-square, p \ 0.05). Similarly, interstitial lung disease was 18.52 times higher in dcSSc patients than in lcSSc patients (OR 18.52, 95 % CI 3.74–90.91) (Table 2). Overall, 29 (96.7 %) patients with dcSSc and 47 (94 %) patients with lcSSc had ANA positivity (p [ 0.05) (Table 2). Anti-Scl-70 was positive in 23 (76.7 %) patients with dcSSc and in 13 (26 %) patients with lcSSc. Anti-Scl70 positivity was 9.34 times greater in the dcSSc group than in the lcSSc group (OR 9.34, 95 % CI 3.26–27.02) (Table 2). The anti-centromere antibody was positive in 2 (6.7 %) patients with dcSSc and in 20 (40 %) patients with lcSSc. Anti-centromere antibody positivity was 9.33 times more likely in lcSSc than in dcSSc (OR 9.33, 95 % CI 1.997–43.627) (Table 2). Five (10 %) patients with lcSSc were anti-U1-RNP- positive, and 1 (2 %) patient with lcSSc was anti-Ku positive (Table 2). Anti-SS-A was positive in 5 (16.7 %) patients with dcSSc and in 13 (26 %) patients with lcSSc (p [ 0.05) (Table 2). Anti-SS-B was positive in 1 (3.3 %) patient with dcSSc and in 6 (12 %) patients with lcSSc (p [ 0.05) (Table 2). Two (6.6 %) patients with dcSSc had ankylosing spondylitis (HLA-B27-positive). Diabetes mellitus (DM) was observed in 4 (8 %) patients with lcSSc and in 1 (3.3 %) patient with dcSSc. Familial Mediterranean fever (FMF) was detected in 1 (2 %) patient with lcSSc, and Meniere disease was detected in 1 (2 %) patient with lcSSc. Kikuchi Fujimato disease was present in 1 (2 %) patient with lcSSc, and
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chronic hepatitis C (HCV) was present in 1 (3.3 %) patient with dcSSc. One (2 %) patient with lcSSc had primary biliary cirrhosis, 1 (2 %) patient with lcSSc had otosclerosis, and three (6 %) patients with lcSSc had hypothyroidism. Mc was detected in 8 (within group 20 %, between groups 22.2 %) women from the control group, 9 (within group 30 %, between groups 25 %) women from the dcSSc group, and 19 (within group 38 %, between groups 52.8 %) women from the lcSSc group (p = 0.180). The child status of the Mc-positive patients diffuse cutaneous SSc was as follows: 6 (66.7 %) had son(s), 2 (22.2 %) had daughter(s), and 1 (11.1 %) was nulliparous-nulligravid. The child status of the Mc-positive patients limited cutaneous SSc was as follows: 10 (52.6 %) had son(s), 2 (10.5 %) had daughter(s), 6 (31.6 %) were nulliparous-nulligravid, and 1 (5.3 %) had a miscarriage. In the control group, 7 (87.5 %) Mc-positive women had son(s) and 1 (12.5 %) Mc-positive woman was nulliparous-nulligravid (Table 1). There were no significant differences between Mc and lung involvement (p = 0.555), renal involvement (p [ 0.05), Raynaud’s phenomenon (p = 0.337), cardiac involvement (p = 0.706), pulmonary hypertension (p = 0.483), or interstitial lung disease (p = 0.177). The median PAP of the patients with Mc was 25 (20–40) mmHg, and the median PAP of the patients without Mc was 26 (20–50) (p = 0.151). In addition, there were no significant differences between Mc and ANA positivity (p = 0.609), anti-Scl-70 positivity (p = 0.777), anti-centromere positivity (p = 0.372), anti-U1-RNP positivity (p = 0.653), anti-SS-A positivity (p = 0.694), or anti-SS-B positivity (p = 0.232). The median ModRSS was 10 (4–24) in the Mc-positive patients and 13 (4–26) in the Mc-negative patients (Mann– Whitney test, p = 0.038). The median of the Medsger’s disease severity scale was 1 (1–2) in the Mc-positive patients and 1 (1–3) (p = 0.118) in the Mc-negative patients. Of the 28 microchimerism-positive patients (75 %), 21 had a Medsger’s disease severity scale of 1, and 7 (25 %) had a score of 2. In the patients with Mc, the median number and age of their children were 2 (1–5) and 20 (1–5) years, respectively. No significant difference was detected between the presence of Mc and the number of children or the age of the children (p = 0,881 and p = 0,865, respectively). The mean age of the women was 44.25 ± 13.95 (42.5, 18–76) in those with Mc and 45.67 ± 13.47 (41.5, 27–80) in those without Mc. Additionally, the mean ages of the dcSSc patients with Mc, lcSSc patients with Mc and control women were 45.11 ± 13.71 (43, 30–76), 42.79 ± 15.28 (38, 18–70) and 46.75 ± 11.99 (46.5, 31–65) years, respectively. The elapsed time between the first birth and the development of the SSc symptoms (diagnosis) was a median of 3.5 (0–49) years in the Mc-positive patients and a median
Mod Rheumatol, 2014; 24(1): 97–105
of 14 (0–55) years in the Mc-negative patients, which was a significant difference (Mann–Whitney test, p = 0.020). Additionally, Mc was detected in 2 (4 %) patients with lcSSc associated with DM and in 1 (2 %) patient with lcSSc associated with FMF.
Discussion The main trend that we identified was that SSc was more prevalent in women who had given birth and even more common in those who had male children. This observation can be explained by fetal cells. Fetal cells that pass through the placenta can be observed in the maternal blood as early as 4–5 weeks of pregnancy [21]. This passage is not just one way, as the maternal cells can also pass through the fetal circulation. For the formation and continuation of pregnancy, a certain amount of fetal cells above the threshold level is required in maternal blood circulation. Any passage below the threshold value may result in miscarriage, whereas excess passage can result in autoimmune disease susceptibility and complications in pregnancy. Various mouse model experiments have shown that peripheral antigen-specific tolerance against the fetus can develop by ‘‘clonal deletion’’ in the maternal immune system and provide for the continuation of pregnancy [22–24]. Fetal Mc may play a role in the pathogenesis of SSc cells and/or is thought to be a marker of inflammation. Although these fetal cells carry microchimeric CD3 and CD4 T cell markers, a reaction may not develop due to the development of immune tolerance during pregnancy. The role of these cells in the pathogenesis of SSc was first discussed by Scott Pereira [25]. Similarly, various other authors have reported that scleroderma, evolving as a result of the transplacental passage of cells between the mother and fetus, may be a chronic form of GVHD [25]. Nonautologous microchimeric cells that carry Y chromosome DNA sequences of female patients with male children can trigger a reaction similar to GVHD and play a role in the pathogenesis of this disease [26–29]. However, showing the presence of these cells is not only important but very difficult. Thirty women with dcSSc, seventeen with lcSSc and twenty-two healthy women in a study of CD4? and CD8? T cells containing the Y chromosome DNA sequences were investigated by quantitative PCR [30]. In this study, 82.9 % (39/47 patients) had SSc, and 63.6 % (14/22 controls) of the control group had Mc. This difference was not significant (p = 0.138). Mc was detected in 26/30 (86.7 %) of the diffuse cutaneous group and in 14/17 (82.4 %) of the limited cutaneous group, showing that the two patient groups did not differ with respect to the existence of Mc (p = 1.0). These T cells
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DOI 10.3109/14397595.2013.854052
consisted mostly of CD4? T cells. CD4? and CD8? microchimeric cells express CD25 (IL-2 receptor) and undergo oligo-clonal expansion when they are activated. In this study, however, the prevalence of skin involvement and disease severity were not taken into account [30]. In our study, Mc was detected in 35 % (28/80) of the SSc group and in 20 % (8/40) of the control group using an RT-PCR technique. This difference was insignificant (p = 0.180). Mc was detected in 9/30 (30 %) of the diffuse cutaneous group and 19/50 (38 %) of the limited cutaneous group, which was an insignificant difference (p [ 0.05). Sawaya et al. [31] observed that microchimeric cells could be detected in skin biopsies approximately 50 years after the last birth, which demonstrates that in the early stage of the disease, microchimeric cells are detected in high concentrations in the non-involved skin areas but that the microchimeric cell ratio decreases after disease progression and inflammatory cell migration as a result of a bacterial, viral or chemical triggers. The longevity of these microchimeric cells can explain the development of the disease in nulliparous or male patients with SSc. SSc has been proposed to develop due to the settling of autologous inflammatory cells in the inflamed area, followed by the release of pro-inflammatory cytokines and fibroproliferative and vascular events as a result of the migration of microchimeric cells, which are activated through certain environmental causes (such as viral or other infections and chemical or other factors) in the ‘‘target microenvironment’’ (for example, in SSc, the target can be skin, lungs, heart or other organs) [31]. In our study, the median of the ModRSS was 10 (4–24) in the patients with Mc and 13 (4–26) in the non-microchimeric patients. The difference between these two groups was significant (Mann–Whitney test, p = 0.038). This situation demonstrates that the chance of detecting microchimeric cells in the peripheral blood circulation can be decreased in patients with high skin involvement as these cells migrate from the circulation to the skin (the target tissue). However, no significant relationship was detected among Mc and the Medsger’s disease severity score, other organ involvement or the autoantibody profile. Evidence has shown that these microchimeric cells play a protective role in pregnancy, transplantation, tissue damage repair, many autoimmune diseases, including type 1 diabetes mellitus, breast cancer and some other malignancies. However, these cells can also be detected in healthy women. An understanding of how these microchimeric cells are activated to cause SSc is still not yet clarified [31]. In our study, the mean disease duration was calculated as 11.47 ± 8.9 (9, 1–35) years in the diffuse cutaneous group and as 6.20 ± 6.5 (4, 1–28) years in the limited cutaneous group. The duration between the first birth and
Modern Rheumatology 103
the onset of the symptoms of SSc (diagnosis) was a median of 3.5 (0–49) years in the Mc-positive group and 14 (0–55) years in the Mc-negative group. The difference between these two groups was significant (Mann–Whitney test, p = 0.020). This situation suggests that Mc shortens the onset of the disease and thus could be a triggering factor in the development of the disease. We found that the presence of Mc in the peripheral blood mononuclear cells was detected in a nulliparousnulligravid 76-year-old female patient with dcSSc, demonstrating that microchimeric cells can live in the blood circulation for many years. Furthermore, Mc was detected in a 48-year-old healthy woman with no pregnancy history (nulligravid) in the control group. In the limited cutaneous group, the nulliparous patients with detected microchimerism were 18, 20, 29, 34, 38 and 60 years old. In other words, our oldest nulliparous patients with detected Mc were 60 and 76 years old, revealing the presence of a lifelong continuous microchimeric situation. In addition, the median age of the children of the patients with Mc was 20 (1–50), and thus we demonstrated the presence of Mc in the blood mononuclear cells even 50 years after giving birth. Microchimeric T lymphocytes have been shown to be detected in the maternal circulation even 40 years after the last pregnancy [32]. A study conducted in Italy researched the presence of Mc in the peripheral blood mononuclear cells of female patients with SSc by nested-PCR and RT-PCR techniques and its relationship with the clinical and serological findings. Y chromosome-specific sequences were detected in 41 of 54 female patients with SSc (76 %) by the nestedPCR technique but only in 8 patients (15 %) by the RT-PCR technique [33]. Of the 19 patients who never gave birth to a male child and did not have a history of miscarriage, Mc was detected in 2 (10 %) by RT-PCR and in 13 (68 %) by nested-PCR. The nested-PCR technique was found to be of high sensitivity but low specificity. The relationship between Mc and the sub-types of SSc (diffuse and limited cutaneous), disease activity and organ involvement could not be shown [33]. However, in patients with systemic lupus erythematosus (SLE), Mc was found to be related to disease activity by the nested-PCR technique [34]. The beneficial and/or harmful effects of continuous Mc and its biological importance are not yet understood. Mc is believed to potentially play a role in growth and development, tissue repair, and the diversity of responses to infection, immune surveillance and autoimmunity [34]. As microchimeric cells can show ‘‘fluctuation’’ with respect to time and disease activity, the time point of taking the blood sample is also important. In our study, Mc was not detected in blood samples taken at different times in one microchimeric patient with dcSSc. This situation confirms that microchimeric cells can ‘‘fluctuate’’ and
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suggests that the timing is just as important as the technique. However, to prove the role of timing, the procedure should be performed at different times with more patients and controls. This situation could affect the detection of Mc (limitation) in our study. In one study, the presence of maternal Mc was shown in the peripheral blood mononuclear cells of 22 % of the healthy females [35]. We also detected Mc in the peripheral blood mononuclear cells of 20 % of the healthy females in our study. Mc can be a factor in the development of autoimmunity but cannot be the cause of autoimmunity itself [36]. In other words, as a result of an environmental exposure or a trigger, the fetal microchimeric cells will divide and proliferate, causing autoimmunity to then develop [36]. Although a life-long microchimeric condition can induce the development of autoimmune diseases, such as SSc, it is an indispensable condition for the continuation of life. In one study, the presence of Y chromosome DNA sequences in the peripheral blood mononuclear cells, i.e., Mc, was shown in 11 out of 35 healthy women with male children (31 %). In this study, Mc was detected in 12 out of 20 SSc patients (60 %) by the PCR method. Additionally in this study, Mc was shown in 8 women who had previous miscarriages and/or transfusions. Y chromosome DNA could show the presence of persistent male fetal cells in a woman who gave birth to a male child. In this study, the mean time between the birth of the last male child in the patients with SSc and the performance of this study was calculated to be 21 years [37]. In our study, we showed the presence of Mc in 7 out of 37 (18.9 %) healthy women who gave birth to a male child. In another study, Mc was investigated in 43 female patients with SSc who did not receive immunosuppressive treatment (such as steroids, methotrexate, and cyclophosphamide) in the last 30 days. The ages of these patients ranged between 20 and 87 years, and they all had given birth to at least one male child. Their age of diagnosis was between 20 and 68 years, and the ages of their male children were between 2 and 31 years. In this study, the sexdetermining region of Y (SRY) was detected in only 6.9 % of the patients. The disease involvement in these patients was not included in this study [38]. In our study, the median age of the children of the microchimeric patients was 20 (1–50) years, and the median number of children was 2 (1–5). In addition, the age of diagnosis of the microchimeric patients with male children was 18–69 years. In our study, the medication usage of the patients was discarded, but women who did not use any medications were investigated in the control group. As we have emphasized previously, in addition to ‘‘fluctuation’’, another factor that affects the detection of Mc is the use of ‘‘medication’’, especially ‘‘immunosuppressive medications’’. This situation could also affect the
Mod Rheumatol, 2014; 24(1): 97–105
detection of Mc (limitation) in our study. In the future, if patients who have been diagnosed recently and did not receive any treatment are investigated, then different results may be found. In a study conducted in Spain, the presence of Mc was investigated in 47 female SSc patients (30 limited cutaneous and 17 diffuse cutaneous) and 40 healthy female individuals, with 19 lcSSc patients, 10 dcSSc patients and 26 control women having male children [39]. In this study, the presence of Mc was detected in 4 patients (13.7 %), 2 each in both sub-groups, and in 2 healthy women (7.6 %), but this difference was not significant [39]. In summary, we determined in this study that the Mc ratio in patients with dcSSc and lcSSc was not different than that in the control group. This ratio was also not related to the number of births and the sex of the children. We also could not show a significant difference associated with the clinical course of the disease. Scleroderma is a heterogeneous disease, and many factors play a role, rather than a specific etiological reason, in the development of scleroderma. However, we have shown that the presence of Mc shortens the time of onset of the disease. Acknowledgments We would like to thank to ‘‘Ankara Tıplılar Vakfı’’ for funding this project. We also thank Mrs. Zeynep BIYIKLI for the statistical analyses. In addition, the summary of this research was accepted as a poster presentation at the 5th Turkish-Greek Rheumatology Days, October 14–16, 2011. Conflict of interest
None.
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