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January - March 2009: 
Volume 22, Issue 1

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Lymphocyte subsets and early apoptosis in the peripheral blood of patients with Obstructive Sleep Apnoea Syndrome (Preliminary Results)
Abstract
SUMMARY. BACKGROUND: Several lines of evidence suggest immune system derangement in patients with Obstructive Sleep Apnoea Syndrome (OSAS), expressed by elevation in the circulating levels of proinflammatory markers and increase in the total number of neutrophils, but there is less information on possible alterations in lymphocyte expression. OBJECTIVES: The aim of this study was to explore the differences between OSAS patients without comorbidities and healthy control subjects in lymphocyte subsets and in apoptosis. PATIENTS AND METHODS: In 12 otherwise healthy OSAS patients (AHI≥5/h+symptoms) and 12 age- and body mass index (BMI)- matched, healthy control subjects (AHI<5/h) layers of mononuclear cells from were isolated from blood samples following a density gradient centrifugation with Ficoll-Histopaque. The quantification of lymphocyte subsets was carried out by whole blood multicolour flow cytometry. Spontaneous early apoptosis was also assessed by flow cytometry after simultaneous staining with Annexin V and various markers for lymphocytic subsets. Annexin V expression is a characteristic of the early apoptosis of whole blood leucocytes. RESULTS: The absolute number of Large Granular Lymphocytes (T-LGLs) was significantly lower in OSAS patients than in the healthy control subjects. Conversely, the percentage and the absolute numbers of the other lymphocyte subsets tested (CD4+, CD8+, CD19+, ΝΚ and γδ cells) showed no significant difference between patients with OSAS and healthy subjects. No difference was observed in early apoptosis of the above lymphocyte subsets between OSAS patients and control subjects. CONCLUSIONS: The spontaneous apoptosis of peripheral blood lymphocytes is not altered in OSAS patients compared to non-apnoeic individuals. However, the significant reduction of T-LGLs in the peripheral blood of OSAS patients supports the concept of immune derangement in OSAS, as such a decrease has been observed only in a few autoimmune diseases. Pneumon 2009; 22(1):–
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Introduction

Obstructive Sleep Apnoea Syndrome (OSAS) is a common disorder that affects 4% and 2% of middle-aged men and women respectively1. OSAS is characterized by periodic upper airway obstruction and intermittent hypoxia and hypercapnoea, resulting in sleep fragmentation, usually manifested as excessive daytime sleepiness1.

Intermitted hypoxia and hypercapnoea have been considered to be responsible for the activation of endothelial cells and T lymphocytes, either directly or mediated by inflammatory cytokines, and also their programmed cell death, which may lead to immune derangement and cardiovascular disorders2,3. T-lymphocyte activation observed in OSAS4,5 and the regulation between activation and apoptosis in T cell lineages6 may imply a difference in lymphocyte subset counts between OSAS patients and subjects without apnoea. The current literature provides no data on the absolute count and percentages of lymphocyte subsets in OSAS patients. Only one study is reported on the reversible increase of CD4+ lymphocytes in OSAS patients which normalises after CPAP application7, but the apoptosis of lymphocyte subsets of these patients was not examined.

The aim of this study was to examine possible differences between OSAS patients and age- and body mass index (BMI)- matched control subjects in the expression of lymphocyte subsets and in the relative percentages of automatic lymphocyte apoptosis.

Methods

Study population

Subjects (n=12) with polysomnographically confirmed OSAS (AHI≥5/hour+ symptoms), but who were otherwise healthy were initially recruited. All of them had visited the Sleep Laboratory of the Pneumonology Department of the Democritus University of Thrace Medical School for investigation of breathing disturbances during sleep. The control subjects were 12 BMI- and age-matched individuals in whom a diagnosis of OSAS was excluded by polysomnography (AHI<5/hour). None of the participants reported infection, injury, surgical operation, any known collagen or haematological disease or malignancy, during the 3 months prior to the examination. All participants signed a form of voluntary participation and informed consent, and the study was approved by the ethics committee of the institution.

Definitions

Apnoea was defined as complete cessation of airflow for ≥10 sec; hypopnoea as a 30% reduction in airflow for ≥10 sec, accompanied by a ≥4% desaturation or by an electroencephalogram (EEG) recorded arousal. The Apnoea Hypopnoea Index (AHI) was defined as the total number of apnoeas and hypopnoeas per hour of electroencephalographic sleep, and Oxygen Desaturation Index (ODI) as the total number of oxygen desaturation (≥3%) events per hour of encephalographic sleep. OSAS patients were considered to be those who reported symptoms and had an AHI of ≥5/hour of sleep.

Polysomnographic sleep study

All participants received a full polysomnographic sleep study (Alice 4, Respironics, Murraysville, Pennsylvania, USA) in the Sleep Laboratory of the Pneumonology Department of the Democritus University of Thrace Medical School, conducted between 10 pm and 6 am. The parameters tested were EEG, electrooculogram, electromyogram of the submental and tibialis anterior muscles, electrocardiogram (ECG) (for the assessment of cardiac function and arterial pulse), snoring (by a microphone placed at the height of the jugular), airflow (with an oronasal thermistor), oxyhaemoglobin saturation (with pulse oxymetry). Thoracic cage and abdominal motion were recorded by inductive plethysmography. The EEG recordings were scored manually according to standard criteria8.

Flow cytometry and study of apoptosis

Whole venous blood samples were incubated with the following antibodies after lysis with ammonium chloride: CD4-FITC, CD8 PE, CD5-FITC, CD19 PE, CD45 PerCP, CD3-FITC, CD16+CD56 and appropriate isotopic controls (BD Pharmingen). Data acquisition and analysis was performed on a FACSCalibur, flow cytometer, using the CellQuest Pro software (BD biosciences). The study on apoptosis was conducted by staining with Annexin V (BD Pharmingen) (Figure 1).

Statistical analysis

The data analysis was performed with the use of SPSS for Windows, version 15.00 (SPSS Inc, Chicago, Illinois). Quantitative variables were expressed as mean ± standard deviation. Student's t-test was used to compare different characteristics between the two groups. A value of p <0.05 was considered statistically significant.

Results

The comparison of the anthropometric and sleep characteristics of OSAS patients and the control subjects is shown in Table 1.

Lymphocyte subsets

The group of OSAS patients had statistically significantly lower T-LGL counts than the control group (p< 0.041) (Table 2). No differences were observed between the patients and control subjects in the absolute counts and the percentages of CD4+, CD8+, CD19+, NK, and γδ cell populations.

Table 1. Comparison between OSAS patients and control subjects of anthropometric indices, sleep characteristics, and daytime somnolence.

 

Patients

(n=12)

Control Subjects

(n=12)

p

 

AGE (years)

48.25±13.33

47.2±11.27

0.843

ΒΜΙ (kg/m2)

31.78±6.17

31.33±4.35

0.847

ESS

11.63±5.78

6.45±4.02

0.000

ΑΗΙ (/hour)

31.17±18.48

2±1.63

0.000

Average SpO2, %

90.16±3.62

93.93±1.16

0.000

Minimum SpO2, %

82.09±10.17

90.85±1.84

0.000

t<90, %TST

12.19±27.05

0.68±0.99

0.000

 

Apoptosis

No significant difference was observed in the percentage of apoptosis of the lymphocyte subsets between the OSAS patients and the control subjects (Table 3).

Discussion

This study investigated, with the use of flow cytometry, the absolute numbers and percentages of lymphocyte subsets, along with early apoptosis in OSAS patients and age- and BMI-matched control subject.

The main finding was the significantly lower value of the absolute number of T-LGLs of OSAS patients, in comparison with that of control subjects. No significant difference was found, however, between the two groups regarding the percentages or the absolute numbers of the other lymphocyte subsets, namely: CD4+, CD8+, CD19+, NK and γδ+ cells.

Table 2. The absolute count of lymphocyte subsets in OSAS patients and control subjects

 

Patients

(n=12)

Control
Subjects

(n=12)

p

Total
lymphocytes
(/mm3)

3082.92±782.61

3423±564.33

0.625

CD 4+ (/mm3)

1425.48±390.38

1555.47±449.07

0.497

CD 8+ (/mm3)

579.20±274.68

661.80±224.21

0.478

CD 19+ (/mm3)

501.18±240.57

586.98±255.32

0.427

NK (/mm3)

2137.63±614.29

2396.67±525.73

0.306

T-LGL (/mm3)

89.60±84.46

190.39±118.05

0.041

γδ (/mm3)

109.30±76.62

109.29±62.46

0.999

Table 3. Comparison between OSAS patients and control subjects in apoptosis of different lymphocyte subsets, expressed as%.

 

Patients

(n=12)

Controls

(n=12)

p

 

CD 4+ (%)

4.67±1.32

4.43±1.64

0.728

CD 8+ (%)

13.22±7.70

11.15±5.41

0.468

CD 19+ (%)

16.83±7.31

22.18±10.80

0.228

ΝΚ (%)

8.06±1.75

8.13±4.35

0.966

T-LGL (%)

9.65±2.85

8.14±6.09

0.517

 

The decreased number of T-LGLs in the peripheral blood is a known characteristic of various autoimmune diseases such as rheumatoid arthritis9, psoriasis10, multiple sclerosis11 and diabetes mellitus12. The decreased expression of T-LGLs is possibly generated by kinase P13 dysfunction, which influences a wide range of cellular functions, including energy metabolism, cell cycle progression and apoptosis13. The pathway of P13-AKT is important for balanced regulation between survival and apoptosis, and alterations in this pathway have been described in many neoplasms13. A recent report has revealed that T-LGLs exhibit constitutive production of the proinflammatory cytokine Regulated on Activation, Normal T Expressed and Secreted (RANTES), macrophage inflammatory protein 1β (MIP-1β), and interleukin-18 (IL-18). Notably, all three cytokines have been shown to induce the phosphatidylinositol-3 kinase (P13K)14-17. Elevated P13K activity in T-LGLs is thought likely to play an important role in the ability of the pathological cells to avoid homeostatic apoptosis, since inhibition of this pathway leads to apoptosis in the population of cells harbouring the pathological clone18. In this study, however, no increased apoptosis was observed; therefore the decrease exhibited in the T-LGL population cannot be attributed to the defective function of P13 kinase.

One limitation of the present study is that, being in a preliminary stage, only a small number of participants were investigated, rendering the findings inconclusive. In addition, other than flow cytometry no functional methods that could elucidate further the cell population of each subset were used. Moreover, for the apoptosis percentage assessment a sole antibody was used for every lymphocyte subset to evaluate the early apoptosis, while antibodies such as 7-AAD and PI, specific for late apoptosis detection, would have been helpful.

In conclusion, this study indicates a degree of immune derangement in OSAS patients expressed as a decrease in circulating T-LGLs. However, further research is necessary, preferably with the evaluation of differential expression of in OSAS patients before and after CPAP treatment, in order to establish more strongly the interaction between OSAS and immunity.

References

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18.  Schade AE, Powers JJ, Wlodarski MW, et al. Phosphatidylinositol-3-phosphate kinase pathway activation protects leukemic large granular lymphocytes from undergoing homeostatic apoptosis. Blood 2006; 107:4834-4840

References