Arch Gynecol Obstet (2012) 285:749–755 DOI 10.1007/s00404-011-2049-9

GENERAL GYNECOLOGY

Lymphocyte subpopulations in Chinese women with Turner syndrome Hongye Fan • Dandan Wang • Haiyan Zhu Jie Li • Yali Hu • Yayi Hou

•

Received: 3 April 2011 / Accepted: 1 August 2011 / Published online: 7 September 2011 Ó Springer-Verlag 2011

Abstract Purpose Turner syndrome (TS) is associated with deficiency of cellular and humoral immunity. However, the characteristics of lymphocyte subpopulations in Chinese women with TS have not been reported. In this study, the percentage of lymphocyte subpopulations and the mRNA expression of some transcription factors were determined in patients with TS. The effect of the hormone substitution on lymphocyte subpopulations was also analyzed. Methods Thirteen Chinese TS women and eight age and sex-matched healthy volunteers were studied. The percentage and mean fluorescence intensity (MFI) of lymphocyte subpopulations including CD3?CD4?, CD3? CD8?, CD19-CD138?, CD4?CD25?FoxP3? and CD4?CD8-IL17A? cells were determined by flow cytometry. The mRNA expression of some transcription factors were detected by RT-PCR.

Results Compared to control, the percentage of CD3? CD4? cells was significantly reduced (p \ 0.05), while the percentage of CD19-CD138?, CD4?CD25?FoxP3? and CD4?CD8-IL17A? cells was significantly increased in TS patients. No difference was observed in the percentage of CD3?CD8?, CD19? B cells between TS patients and healthy volunteers, with the similar changes in the mean fluorescence intensity of these cells. The mRNA expression of some transcription factors slightly enhanced in TS patients. Estrogen therapy did not affect the percentage of lymphocyte subpopulations. Conclusion These findings suggested that Turner syndrome might be associated with changes of lymphocyte subpopulations. Keywords Turner syndrome  Lymphocyte subpopulation  Transcription factors

Introduction H. Fan and D. Wang have contributed equally to this manuscript. H. Fan  Y. Hou Immunology and Reproductive Biology Lab of Medical School and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China D. Wang  H. Zhu  J. Li  Y. Hu The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, People’s Republic of China Y. Hu (&)  Y. Hou (&) Jiangsu Key Laboratory of Molecular Medicine, Nanjing University, Nanjing 210093, People’s Republic of China e-mail: [email protected] Y. Hou e-mail: [email protected]

Turner syndrome (TS) is a disorder that characterized by short stature, absent pubertal development and infertility due to ovarian dysgenesis with no estrogen production [1]. TS is caused by the presence of only one normally functioning X-chromosome. The other sex chromosome can be missing (45, X) or abnormal and mosaicism is often present [2]. Immunological disturbances have previously been described in TS, with an association to reduced levels of circulating T- and B-lymphocytes [3, 4]. But the mechanism for the underlying immunodeficiency has not been explored [5, 6]. Some studies reported that TS patients had an increased incidence of autoimmune disease [7, 8] and presume that the maladjustment of immune function may exist in TS patients.

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T and B lymphocytes are the two major types of lymphocyte in adaptive immune responses. Effector T helper (Th) cells derive from progenitor naı¨ve CD4? T cells. Naive Th cells can differentiate into Th1, Th2, Th17 or Regulatory T (Treg) cells under the influence of different cytokines. Th1 cells were long considered to be the major effectors in multiple autoimmune diseases, while Th2 cells have been known to be involved in atopy and asthma. More recently, Th17 cells have been implicated as culprits in a plethora of autoimmune and other inflammatory diseases. Treg cells have a crucial role in suppressing immune responses to self-antigens and in preventing autoimmune diseases. An imbalance between Th17 and Treg cell function may be central in some of these diseases. Most circulating human B cells can express CD19, while plasma cells can be identified by strong expression of CD138 and lost in CD19 expression [9]. CD25 is an activation marker for T cells and is therefore also expressed by effector Th1 and Th2 cells. FOXP3 seems to be the most promising marker of natural Treg cells. Moreover, it was reported that the transcriptional factor RORct directed development of IL-17-secreting T cells, whereas T-bet and GATA3, which are involved in the development of Th1 and Th2 cells, respectively, inhibited differentiation of Th17 cells. To our knowledge, there is no report on lymphocyte subpopulations and their transcription factors in Chinese TS patients. Therefore, in the present study, according to the main markers for different subclasses of lymphocytes, we investigated the percentage of lymphocyte subpopulations in peripheral blood of 13 TS patients. We also detected the mRNA expression levels of some transcription factors such as RORct, T-bet and GATA3. In addition, estrogen therapy is given to TS patients to induce puberty. Since estrogen may be a contributing cause of the higher incidence of autoimmune diseases in women, the effect of hormone substitution on lymphocyte subpopulation in the TS patients was also analyzed.

Materials and methods Patients and control subjects A total of 13 female TS patients, aged from 14 to 33 years, were enrolled in this study, and all the patients underwent karyotype detection and genetically confirmed according to the population census of Jiangsu province. Eight age- and sex-matched healthy volunteers were used as controls. Before sample collection, each patient was given a physical examination, gynecological, cardiac and abdominal ultrasonic tests, and a medical history was attained, especially focusing on autoimmune diseases, tumor, and family history. Seven of thirteen patients had estrogen intake. This

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protocol was approved by the Ethics Committee at The Drum Tower Hospital of Nanjing University Medical School, and informed consent was obtained from each patient and healthy volunteer. All human subjects were in compliance with the Helsinki Declaration. Staining for T cells subsets, B cells, regulatory T cells and Th17 cells 4 mL heparinized peripheral blood samples were collected from each patient in the fasting state. Th17 cells staining 100 ll peripheral blood was stimulated with 10 ll phorbol myristate acetate (50 ng/ml) and 10 ll ionomycin (1 lg/ml) for 5 h. The 100 ll blood was also treated with 5 ll brefeldin A (5 lg/ml, all from Sigma-Aldrich) in the last hour. Cells were incubated with FITC conjugated anti-human CD3, APC conjugated anti-human CD8 at room temperature for 20 min. After fixation and permeabilization, cells were incubated with PE conjugated anti-human IL17A (all from BD Bioscience) for 20 min. Then cells were analyzed and 10,000 events in the CD3? T cell gate were collected for each sample. Staining for T cell subsets, B cells and regulatory T cells Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation and divided into three tubes. Cells in the first tube were incubated with APC conjugated anti-human CD3, FITC conjugated anti-human CD4 and PE conjugated anti-human CD8 (all from BD Bioscience) for detecting CD3?CD4? and CD3?CD8? T cells frequency. Cells in the second tube were incubated with APC conjugated anti-human CD19 and PE conjugated anti-human CD138 (all from BD Bioscience) to examine the frequency of CD19?B cells and the percentage of CD19-CD138? plasma cells. Cells in the third tube were incubated with FITC conjugated antihuman CD4, APC conjugated anti-human CD25 and PE conjugated anti-human Foxp3 (all from eBioscience, USA). Cells were analyzed by flow cytometry, according to the manufacturer’s instructions and flow data were analyzed with CellQuest software (Becton–Dickinson). Reverse transcription polymerase chain reaction (RT-PCR) RORct expression was analyzed with the following primers: forward: 50 -GTAACGCGGCCTACTCCTG-30 reverse: 50 -GTCTTGACCACTGGTTCCTGT-30 (32 cycles). T-bet expression was analyzed with the following primers:

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Forward: 50 -CCACCAGCCACTACAGGATG-30 Reverse: 50 -GGACGCCCCCTTG-TTGTTT-30 (32 cycles). GATA3 expression was analyzed with the following primers: Forward: 50 -GTGCTTTTTAACATCGACGGTC-30 Reverse: 50 -AGGGGCTGAG-ATTCCAGGG-30 (32 cycles). GAPDH expression was analyzed with the following primers: Forward: 50 -ATGGGGAAGGTGAAGGTCG-30 Reverse: 50 -GGGGTCAT-TGATGGCAACAATA-30 (24 cycles). Total RNAs were isolated by Trizol method. A 2 lg total RNA was reverse-transcribed using reverse transcriptionpolymerase chain reaction. Then the first strand complementary was gained in the presence of M-MLV reverse transcriptase and oligo-dT primers. After RT reaction, 1 lL of the incubation mixture was used as the template for the following PCR. 1 lL 10 mmol/L mixture of all four deoxynucleotide triphosphates, 0.2 lL Taq DNA polymerase and 12 lL nuclease-free water were added to adjust the final volume to 25 lL. After an initial incubation at 94°C for 1 min, temperature cycling was initiated with each cycle as following: denaturation at 94°C for 30 s, annealing at 60°C for 20 s and elongation at 72°C for 30 s. The PCR products were then separated on 1.5% agarose gel containing 0.5 lg/ml ethidium bromide. The gel was put on UV-transilluminator and photographed. Serum hormone and autoimmune antibodies Serum concentrations of estradiol (E2), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), thyroglobulin antibodies (TGAb), sex hormone binding

globulin (SHBG), and autoimmune antibodies, including antinuclear antibody (ANA), anti double strand DNA (dsDNA) antibody, rheumatoid factor, were all determined by routine standard methods and compared to age-related reference ranges used at the clinical laboratory, the Affiliated Drum Tower Hospital of Nanjing University Medical School. Statistical analysis Data were shown as mean ± standard deviation (SD) and analyzed using unpaired t test or Mann–Whitney test by GraphPad Prism software (version 5.0). All p values were two-sided. p \ 0.05 and \0.01 were considered significant and notably significant, respectively.

Results Patient demographics The karyotypes and manifestations of gynecological, cardiac and abdominal ultrasonic for all the patients have shown in Table 1. The mean stature of all the patients was 149.3 cm (46.5 * 156.0 cm), and mean bodyweight was 47.5 kg (31.0 * 68.0 kg). Except for one with iritis, other patients were not concomitant with diagnosed autoimmune disease or tumor. The levels of rheumatoid factor (RF) were all within normal scope, and serum ANA and antidsDNA were all negative when analyzed by enzyme linked immunosorbent assay (ELISA).

Table 1 Clinical characteristics for patients with Turner syndrome Age

Karyotype

E2 (pmol/L)

FSH (mIU/ml)

TSH (mIU/L)

TGAb (IU/ml)

SHBG (nmol/L)

Gynecological ultrasound manifestation

Turner syndrome (n = 13) 14

45, XO/46, X, r,(X)(32:7)

126

3.62

1.87

167.6

17

46, XX/45, XO(26:4)

\73.4

79.5

3.08

\10

9

17

45, XO

\73.4

101

2.82

255.9

47

18

45, XO/46, XO?Mar (1:1)

168

68.7

3.89

10.9

20

50

No uterus and ovary Small uterus Naı¨ve uterus, no ovary naı¨ve uterus, no ovary

19

45, XO

\73.4

104

2.44

\10

44

Primordial uterus, no ovary

20

45, XO

195

49.8

1.94

1466

53

21

45, XO

77.5

67.9

6.45

[4000

41

No ovary Naı¨ve uterus

21

45, XO/46, X, i, (Xq) (1:3)

\73.4

56.6

6.61

1743

30

23

45, XO/46, X, i, (Xq) (3:2)

\73.4

103

6.52

87

32

Naı¨ve uterus, ovarian dysgenesis Naı¨ve uterus, no ovary

24 30

45, XO/46, XO?Mar (11:4) 46, XX, inv (X)

174 213

2.78 7.41

3.46 1.66

180.3 \10

32 63

No ovary Polycystic ovary

31

45, XO/46, XX (1:5)

352

20.8

2.54

11.2

43

No left ovary

33

45, XO

266

29.6

4.53

87

74

Ovarian cyst

E2 Estradiol, FSH follicle-stimulating hormone, TSH thyroid-stimulating hormone, TGAb thyroglobulin antibodies, SIIBC sex hormone binding globulin

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Changes of CD4? and CD8? T cell subsets Flow cytometry analysis showed that the frequency of CD3?CD4? T cells decreased significantly in patients with Turner syndrome compared to normal controls [(54.36 ± 9.26%, n = 8) vs. (16.06 ± 9.10%, n = 8, p \ 0.0001)] (Fig. 1a), while the frequency of CD3?CD8? T cells had no difference [(29.44 ± 6.95%, n = 8) vs. (32.54 ± 6.01%, n = 8, p [ 0.05)] (Fig. 1b). Mean fluorescence intensity (MFI) for CD4? T cells decreased, but CD8? T cells increased significantly in TS patients compared to healthy controls (Table 2). Absolute cell numbers for CD4? T cells significantly decreased in TS patients compared to healthy controls, and CD8? T cells had no difference (Table 2). CD19?B cells with no change while CD19CD138?cells increased in TS patients In addition to T cell subsets, we also compared B cells percentage in TS patients with healthy controls, and found that there was no significant change in percentage of peripheral blood CD19? B cells within lymphocytes in

TS patients [(5.89 ± 2.08%, n = 13) vs. (4.95 ± 2.94%, n = 8, p [ 0.05)] (Fig. 2a), while the frequency of CD19-CD138? cells within peripheral blood lymphocytes increased obviously [(0.82 ± 0.37%, n = 13) vs. (0.31 ± 0.11%, n = 8, p \ 0.01 by Mann–Whitney test)] (Fig. 2b). The mean fluorescence intensity also showed that TS patients had much higher CD138 positive cells in peripheral blood (Table 2). CD19-CD138? absolute cell numbers significantly increased in TS patients compared to healthy controls (Table 2). Regulatory T cells and Th17 cells increased in TS patients To further investigate whether peripheral circulating Th17 cells and regulatory T cells participate in the pathogenesis of Turner syndrome, the percentage of these two cell subsets in total peripheral blood lymphocytes were determined by flow cytometry analysis. We found that the frequency of CD3?CD8-IL17A? T cells increased markedly in TS patients compared to healthy controls [(0.056 ± 0.055%, n = 13) vs. (0.025 ± 0.007%, n = 8,

Fig. 1 Changes of CD3?CD4? and CD3?CD8? T cells percentage within lymphocytes population between TS patients and healthy volunteers. a The frequency of CD3?CD4? T cells had notable significance between the two groups (*** means p \ 0.001 vs. control group). b No difference was found in CD3?CD8? T cells population between the two groups

Table 2 Mean fluorescence intensity (MFI) and absolute cell number (ACN) for different lymphocyte subpopulations

CD3?CD4? CD3?CD8? CD19? CD19-CD138? CD4?CD25?Foxp3? CD3?CD8-IL17A?

123

TS patients

control

p value

MFI

17.93 ± 8.18

89.54 ± 29.36

\0.001

ACN (cell/ul)

2364 ± 1375

8282 ± 2264

\0.0001

MFI

163.5 ± 124.6

50.18 ± 49.07

0.031

ACN (cell/ul)

4514 ± 1308

4903 ± 1262

0.555

3.33 ± 0.41

2.97 ± 0.42

0.060

ACN (cell/ul)

MFI

735.7 ± 388.2

914.5 ± 310.1

0.274

MFI ACN (cell/ul)

4.07 ± 0.74 131.7 ± 69.86

3.15 ± 0.22 49.27 ± 23.08

0.003 0.004

MFI

27.99 ± 2.57

25.41 ± 1.65

0.020

ACN (cell/ul)

1006 ± 685.2

305.5 ± 235.7

0.003

MFI

2.59 ± 0.35

2.27 ± 0.38

0.060

ACN (cell/ul)

9.51 ± 10.52

0.30 ± 0.84

0.002

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Fig. 2 TS patients had no change in peripheral blood CD19? B cells percentage compared to controls (a), but the frequency of CD19CD138? cells increased significantly in patients (b), (** means p \ 0.01 vs. control group)

Fig. 3 Both the percentages of CD4?CD25?FoxP3? Tregs (a) and CD3?CD8-IL17A? T cells (b) increased in TS patients compared to control group (* means p \ 0.05 vs. control group)

p \ 0.05 by Mann–Whitney test)] (Fig. 3a). And we also found that the percentage of CD4?CD25?Foxp3? T cells increased significantly in TS patients [(0.65 ± 0.51%, n = 13) vs. (0.21 ± 0.17%, n = 8, p \ 0.05 by Mann– Whitney test)] (Fig. 3b). The mean fluorescence intensity and absolute cell numbers for both Treg and Th17 cells showed the same changes (Table 2). Changes of transcriptional factors Transcriptional factors RORct, T-bet and GATA3 were detected in the present study. The levels of mRNA expression of RORct, T-bet and GATA3 slightly enhanced

in TS patients, but no significantly different from those in healthy volunteers (Fig. 4) Effect of estrogen therapy on the frequency of lymphocyte subpopulations We separated TS patients into two groups according to with or without drug intake, and found that estrogen therapy had no effects on the frequency of CD4? or CD8? T cells, CD19? B cells or CD19-CD138? plasma cells, CD3?CD8-IL17A? T cells, CD4?CD25?FoxP3? T cells, or mRNA expression of transcription factors in those patients (Figs. 1–4, data not shown).

Fig. 4 Changes of transcriptional factors RORct, T-bet and GATA3 between the two groups

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Discussion Compared to healthy volunteers, the percentage of CD3?CD4? cells was significantly reduced (p \ 0.05), while the percentage of CD19-CD138?, CD4?CD25? FoxP3? and CD4?CD8-IL17A? cells was significantly increased (p \ 0.05) in Chinese women with TS. However, no difference was observed in the percentage of CD3?CD8?, CD19? cells. The levels of mRNA expression of some transcription factors slightly enhanced in TS patients. Estrogen therapy did not affect the percentage of lymphocyte subpopulations. Our present results were not entirely in concordance with some earlier studies. A slight but significantly decreased percentage of circulating T and B cells had been reported in twenty patients of Turner’s syndrome [3]. But this study was performed in 1981 and the quality of monoclonal antibody produced in that time may influence the results of detection on levels of T- and B-lymphocytes. On the other hand, some other studies reported lymphocyte subpopulations in 15 girls with TS, aged 5–17 years and showed most girls had normal percentages of lymphocyte subpopulations as compared to the 5–95% percentiles agerelated reference ranges including activated CD4? and CD8? T-cells, but the CD4?/CD8? ratio was in the lower range [10]. However, here authors just compared the difference of lymphocyte subpopulations in young Turner girls with or without otitis. Results showed that no major immunological deficiency was found that could explain the increased incidence of otitis media in the young Turner girls. In our present study, the reduced percentage of CD3?CD4? cells and the normal percentage of CD3?CD8? cells were observed in Chinese women with TS. Although CD4?/CD8? ratio was lower in these patients, but it is due to reduced percentage of CD3?CD4? cells, other than a consequence of a slightly increased CD8? population like early findings [10]. Furthermore, since naı¨ve CD4? T cells can differentiate into Th1, Th2, Th17 or Regulatory T (Treg) cells under the influence of different cytokines, transcriptional factors RORct, T-bet and GATA3 were detected. RORct can direct development of IL-17-secreting T cells, whereas T-bet and GATA3 are involved in the development of Th1 and Th2 cells. The levels of mRNA expression of some transcription factors slightly enhanced in TS patients, but no significantly different from those in healthy volunteers. This suggests that the balance of Th1/Th2 may be retained in TS patients. Furthermore, in our studied TS patient, HIV infection was negative and, there was no evidence of other viral infection, generalized sepsis, acute pyelonephritis, or disseminated fungal infection or history of immunosuppressive therapy. All of these are known to cause a severe reduction in the CD4? population. So the reduced

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percentage of CD3?CD4? cells in peripheral blood of TS patients remains to need clarified. Th17 cells may be involved in the pathogenesis of many human autoimmune diseases [11, 12]. Plasma cells can be identified by strong expression of CD138. We found that the percentage of CD4?CD8-IL17A? and CD19CD138? cells was significantly increased in TS patients, but the detection of autoantibody was limited with the amount of sample from TS patients. So it is very hard for us to suspect that TS patients have an increased incidence of autoimmune disease like those of some studies. Moreover, recent study was conducted in a total of 30 participants (20 TS patients and 10 controls). The results showed that these patients retained regulatory T cell frequency and function despite an increased prevalence of autoimmunity. These findings suggest that the autoimmune predisposition in Turner Syndrome is not due to alterations in regulatory T cells [13]. However, our preset results showed that the percentage of CD4?CD25?FoxP3? cells was significantly increased in TS patients. These differences may be attributed to different range of age in TS patients and the methods of prepared peripheral blood mononuclear cells (PBMCs). Their range of age in TS patients (from 8 to 62) was different from ours (14–33) and frozen PBMCs were used. Surface marker of cells can be influenced by frozen, so frozen PBMCs may be not ideal cells for analyzing lymphocyte subpopulation. CD25 is an activation marker for T cells and is therefore also expressed by effector Th1 and Th2 cells. FOXP3 is the most promising marker of natural Treg cells. Tregs suppress a variety of physiological and pathological immune responses and Treg-mediated suppression is a multi-step process [14, 15]. This suggested that the maintenance of peripheral self-tolerance and immune homeostasis in Chinese women with TS patients might be, at least in part, attributed to higher percentage of Treg cells. Estrogen therapy did not affect the percentage of lymphocyte subpopulations in TS patients. There have been many evidences that the sex hormones influence the immune system and that the lack of estrogens might influence the immune response negatively [16, 17]. Turner syndrome is a disorder that characterized by short stature, absent pubertal development and infertility due to ovarian dysgenesis with no estrogen production [1]. Estrogen therapy is usually used to induce puberty [18]. However, some patients studied were treated with conjugated oral estrogen, the influence of the hormone on lymphocyte subpopulations seemed to be not important. This may ascribe the wider age span and the relatively small size of the study cohort. In conclusion, we found that the percentage of lymphocyte subpopulations in Chinese women with TS was different from that in matched healthy volunteers.

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The treatment with conjugated oral estrogen did not influence the immune system in TS patients. The difference between our results and others may be attributed to the used quality of monoclonal antibody, the methods of prepared samples, and genetics of TS patients. Further studies are needed to elucidate immunological difference between TS patients and matched healthy volunteers. Acknowledgments This work was supported by National Key Basic Research Program of China (2010CB945104), Special Research Grant (XK200709 to YH) for the Key Laboratory from the Department of Health, Jiangsu Province and the Project Foundation of Jiangsu Province Department of Health, China (Grant No. H200754). Conflict of interest

None.

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