Effect of Hormone Replacement Therapy on CD4⁺ and CD8⁺ Numbers, CD4⁺/CD8⁺ Ratio, and Immunoglobulin Levels in Hemodialysis Patients

Abstract

Uremia induces a suppression of the immune status. A large clinical literature suggests that estradiol (E₂) plays a critical role in immune function. A large proportion of women hemodialysis patients faced early menopause and inadequate estrogen levels. The aim of the present study is to evaluate the effect of hormone replacement therapy on immune function in terms of CD4⁺ numbers (inducer/helper T cells), CD8⁺ numbers (cytotoxic/suppressor T cells), CD4⁺/CD8⁺ ratio, and IgG, IgM, IgA levels in woman hemodialysis patients. In our study, 15 female hemodialysis patients (median age 32.6 range 24–45) were treated with triphasic estrogen/progesterone preparation (estradiol 2 mg for 10 days, and afterwards estradiol 2 mg + norethisterone 1 mg for another 10 days, and at the end estradiol 1 mg for 6 days) for 6 months. CD4⁺ numbers, CD8⁺ numbers, and IgG, IgA, and IgM levels were determined before and after HRT. The “paired-samples T” test was used for statistical analysis of pretreatment and posttreatment values. A significant increase was observed for CD4⁺ numbers (582 ± 435 versus 637 ± 445, p = 0.04) and CD4⁺/CD8⁺ ratio (1.4 ± 0.16 to 2.4 ± 0.3, p < 0.01) after hormone replacement therapy (HRT). Serum immunoglobulin levels were not changed significantly. In conclusion, in postmenopausal hemodialysis patients, HRT significantly increased CD4⁺ numbers and CD4⁺/CD8⁺ ratio, but no effect was observed in IgM, IgG, and IgA levels. Long-term clinical effects of HRT on immune system should be investigated in dialysis patients with further studies.

• hemodialysis
• hormone replacement therapy
• CD4⁺
• CD8⁺
• CD4⁺/CD8⁺
• immunoglobulin

Introduction

A large proportion of women hemodialysis patients faced early menopause and inadequate estrogen levels and only a small proportion of these patients are being offered hormone replacement therapy (HRT). Zingraf et al. showed a decrease in estradiol (E₂) levels in 30% of women undergoing dialysis.[1] Mantouvalus et al. reported decreased estrogen concentrations in half of the women having dialysis treatment.[2]

It is well known that end-stage renal disease (ESRD) patients have an immunodeficiency state that involves both B- and T-cell functions. The impairment of the immune system leads to an increased incidence of infections and malignancies, to anergy in cutaneous delayed hypersensitivity tests, and to inadequate responses to antibodies. Estrogen deficiency may be among the many factors that may play a role in the development of this immunodeficient state in ESRD patients. Uremia induces a suppression of the immune status.[3] A large clinical literature suggests that E₂ plays a critical role in immune function.[4]

The aim of the present study is to evaluate the effect of HRT on immune function in terms of CD4⁺ numbers (inducer/helper T cells), CD8⁺ numbers (cytotoxic/suppressor T cells), CD4⁺/CD8⁺ ratio, and IgG, IgM, and IgA levels in woman hemodialysis patients. To the best of our knowledge, this is the first study to determine the effect of HRT on CD4⁺, CD8⁺, CD4⁺/CD8⁺ ratio, serum IgG, M, and A levels in hemodialysis patients.

Materials and Methods

Among a total of 70 patients under treatment in the dialysis unit of the Hospital of Yüzüncü Yıl University, 15 women were eligible for the study. Inclusion criteria were as follows: postmenopausal status (last menses 12 months prior to enrollment); age under 45 years; and lack of prior or current HRT use.

Patient groups were questioned about their gynecologic and obstetrical histories, habits and present complaints before treatment. Each case was given detailed information about possible complications of hormone replacement therapy, and informed consent was taken.

After obtaining detailed history from all patients, their breast, pelvic, and gynecologic examinations, vaginal smear sample, and mammography were obtained in addition to physical examination. Patient history, physical examination, and laboratory results were reviewed.

The patient group was given a triphasic estrogen/progesterone preparation, Trisequens® film-coated tablets, Novo Nordisk (12 blue tbs: estradiol 2 mg, 10 white tbs: estradiol 2 mg + norethisterone 1 mg, 6 red tb: estradiol 1 mg) for 6 months. Venous blood samples after 12 h of fasting and before dialysis were obtained before HRT and after HRT at the end of 6 months after 12 h of fasting. The serum immunoglobulins (Ig) G, A, and M were measured with nephelometry. Complete blood counts were performed by using an automatic analyzer (STKS Coulter), and manual differential counts were performed on Wright-stained PB smears. The numbers of CD4⁺ (inducer/helper T cells) and CD8⁺ (cytotoxic/suppressor T cells) were determined in anticoagulated peripheral venous blood samples. The ratio of CD4⁺ cells and CD8⁺ cells in peripheral blood was determined using anti-CD4⁺ (IgG1-FITC) monoclonal antibody and anti-CD8⁺ (IgG1-PE) monoclonal antibody (Immunotech Kid No:PN IM0747) via flow cytometry (Coulter Epics XL flow cytometer, France). The final results were expressed as mean ± SD.

Statistical analysis was done with the data obtained. “Paired-samples T” test was used for statistical analysis of pretreatment and posttreatment values. Values were given as mean ± standard deviation. The statistical significance limit was set at p < 0.05.

Results

The mean age of our 15 dialysis patients was 33.2 ± 8.7 (range 24–45) years. All patients were on HD treatment twice (n = 2) or thrice (n = 13) weekly. Duration of dialysis before HRT was 30 ± 17 (12–84) months. The mean estradiol level was significantly increased after HRT (23.4 ± 6.0 pg/mL versus 73.1 ± 43.8 pg/mL, p < 0.001).

After 6 months of HRT with the sequential combined drug Novo Nordisk, a significant increase was observed for CD4⁺ number (582 ± 435 versus 637 ± 445, p = 0.04) and CD4⁺/CD8⁺ ratio (1.4 ± 0.16 to 2.4 ± 0.3, p < 0.01) . Laboratory results were shown in Table 1. After HRT, serum immunoglobulins levels were not changed significantly.

Table 1 Laboratory results

	Unit	Before HRT	After HRT	p
CD4^+	Number	582 ± 435	637 ± 445	p = 0.04
CD8^+	Number	391 ± 177	249 ± 56	p = 0.06
CD4^+/CD8^+	Ratio	1.4 ± 0.6	2.4 ± 1.4	p < 0.01
IgG	g/L	12 ± 4	10 ± 4	p = 0.2
IgM	g/L	1.37 ± 0.7	1.33 ± 0.7	p = 0.8
IgA	g/L	2.5 ± 1.9	1.9 ± 1.0	p = 0.8

No thromboembolic complication or side effect attributable to HRT was observed during the study period.

Discussion

It is well known that ESRD patients have an immunodeficiency state that involves both B- and T-cell functions. A reason for the decreased cellular immunity might be a defect in the costimulatory function of antigen-presenting cells, and a persistent inflammatory state of monocytes, which is caused by uremia and by the dialysis treatment. Other factors that might contribute to the decreased immunity are malnutrition, Calcitrol, and hyperparathyroidism.[5&6] A bidirectional interaction between the endocrine and immune systems has been well documented.[7-9] There is evidence that fertile women are more prone to the onset of autoimmune diseases than men, but this increased susceptibility disappears after menopause. Hormonal changes are likely to be responsible for these events.[10] A variety of mechanisms for this phenomenon have been postulated, including E₂'s ability to regulate cytokine production[11] and inhibit apoptosis of peripheral blood mononuclear cells. However, these studies are often inconsistent due to differences in subject selection (e.g., differences in menopausal status or time since onset of menopause) and environmental factors (e.g., nutrition or alcohol intake, etc.).[12] Cutolo et al. suggests that HRT for sex hormone deficiency in postmenopausal women may have pleiotropic effects on immune function.[4] Cellular and humoral immune responses have been found to be higher in hormone replacement therapy users than in nonusers.[13] E₂'s ability to modulate immune cell function was reported for macrophages, thymocytes, and NK activity. Because E₂ is immunomodulatory, E₂ deficiency can modulate immune function.[14-16]

Yang et al. investigated the effect of HRT with continuous use of E2 valerate 2 mg/d and medroxyprogesterone acetate 5 mg/d and reported that women after menopause are prone to impaired responses, but some of the impairment can be restored after HRT. They found no difference in terms of CD4⁺ numbers, CD8⁺ numbers, and CD4⁺/CD8⁺ ratio after HRT.[17] However, Holl et al. reported that cytotoxic T cells and the CD⁺/CD8⁺ ratio were significantly increased after HRT with valerate and 0.15 mg levonorgestrel.[18] These studies were done in otherwise healthy postmenopausal women. In our study, after 6 months of HRT with the sequential combined drug Novo Nordisk, a significant increase was observed for CD4⁺ level (p < 0.04) and CD4⁺/CD8⁺ ratio (p < 0.01) in hemodialysis patients. Yılmazer et al. reported that HRT with transdermal 17B estradiol 50 µg/day and continuous medroxyprogesterone acetate 2.5 mg/d or oral conjugated equine estrogen 0.625 mg/day and continuous medroxyprogesterone acetate 2.5 mg/d have no effect on IgG and M levels in healthy postmenopausal women.[19] We found similar findings for hemodialysis patients.

In conclusion in postmenopausal hemodialysis patients, HRT significantly increased CD4⁺ numbers and the CD4⁺/CD8⁺ ratio, but no effect was observed in IgM, IgG, and IgA levels. The mechanism and long-term clinical effects of HRT on immune and cardiovascular systems should be investigated in dialysis patients with further studies.