Theme : Dreams

Effects of 48 Hours Sleep Deprivation on Human Immune Profile

Levent Öztürk 1, Zerrin Pelin 2, Derya Karadeniz 2, Hakan Kaynak 2, Lütfi Çakar 1, Erbil Gözükirmizi 2
1Department of Physiology and 2 Sleep Disorders Unit, Department of Neurology, Cerrahpasa Medical School, Istanbul 34303, Turkey
Abstract
It is a common belief that sleep deprivation increases the susceptibility to diseases. In order to evaluate the effects of sleep deprivation on immune profile in humans, peripheral venous blood was obtained from sixteen healthy young male volunteers. Ten of the volunteers underwent 48 hours of sleep deprivation and the other six maintained their regular sleep schedule and acted as controls. The first blood samples were taken at the end of the first polysomnographic recording at 8:00 a.m. After this sampling, ten subjects were sleep deprived for 48 hours in sedentary conditions. The second and third blood samples were taken at the 24th and 48th hours. The subjects were recorded again to verify rebound effects of sleep deprivation after the third blood sampling. In this second polysomnographic recording, all sleep-deprived subjects showed slow wave and REM sleep rebound. The last blood samples were taken at the 72nd hour of study at 8:00 a.m. CD4, CD8, CD5, CD16, CD19 surface antigen positive lymphocyte subsets, serum IgG, IgM, and cortisol levels were assessed in all samples. Our results showed that the proportion of NK cells were decreased during sleep deprivation and returned to normal values after recovery sleep. In the control group, we did not observe any changes in the same direction as the sleep-deprived group.

Current Claim: Sleep deprivation causes decrease in natural killer cell proportions and may increase susceptibility to illness.

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The number of people exposed to sleep deprivation is increasing in modern society. It has been believed that sleep is essential to recovery from illness and; conversely, lack of sleep impairs host defense and increases susceptibility to infections. In the last two decades, many researchers interested in the relationship between sleep deprivation and immunity along with an increasing body of literature has suggested that sleep is important for the proper functioning of host defense systems. However, available data is far from elucidating the way in which sleep affects the immune system.
In animals, sustained sleep loss lasting about sixteen days has a lethal outcome due to systemic infection and afebrile septicemia with opportunistic microorganisms (Everson, 1993, 1995). In a following study, the breakdown of gut wall by the bacterial invasion has been reported as a failed defense in the late phases of total sleep deprivation (Bergmann et al., 1996). Isolated REM sleep deprivation in rats resulted in a reduced primary antibody response to sheep erythrocytes and decreased antigen uptake in the spleen and liver (Moldofsky, 1994). Conversely, Benca et al. (1989), found that spleen cell numbers, spleen cell mitogen responses and in vitro and in vivo splenic antibody-secreting cell responses appeared to be unaffected in rats by sleep deprivation.

Deprivation studies involving humans are fewer than animal studies, but they do not have the disadvantage of stressor-induced forced wakefulness required in animal experiments, which makes it difficult to distinguish stress reactions from the effects of sleep deprivation per se. Humans are the only species that know cognitively why they are being kept from sleeping. Hence, in human studies of total sleep deprivation (TSD), plasma levels of glucocorticoids and urinary catecholamines have generally been found unchanged unless the deprivation procedure also involves intensive physical (Opstad and Aakvaag, 1983) and/or mental activity (Radomski et al., 1992). As reviewed by Dinges et al. (1995), there is also no compelling evidence to suggest that sleep deprivation in humans is invariably associated with stress reactions. One of the first systematic studies on the effects of TSD on the human immune system reported that there were no statistically significant changes in the number of circulating polymorphonuclear leukocytes, monocytes or B lymphocytes during or after 77 hours TSD, but decreased leukocyte phagocytic activity relative to baseline levels during TSD (Palmblad et al., 1976). Without any defining alteration in stress hormones, Moldofsky et al. (1989a) described an increase in NK activity during the early evening hours and delayed nocturnal rise in lymphocyte proliferation to Poke Weed Mitogen (PWM) in 6 men deprived for 64h. Soon after this study, the same group reported that 40h of sleep deprivation led to significant increases in nocturnal rise of lymphocyte proliferation to PWM, but they did not observe any change in in vitro NK activity as reported before (Moldofsky et al., 1989b). A wide range of immune parameters were evaluated by Dinges et al. (1994), before, during and after 64 hours of total sleep deprivation in twenty healthy young adults and their results indicated that 64h sleep-deprived subjects had increased number and activities of CD56(+) and CD57(+) NK cells and decreased counts of CD4(+) and CD16(+) cells. Finally, Irwin et al., (1994, 1996) reported that modest loss of sleep in humans (that is, early-night and late-night partial sleep deprivation) resulted in a significant decrease in activity and number of NK cells, which returned to baseline levels after one night of recovery sleep.

As a result of getting different results in various sleep-deprivation protocols, we aimed to assess the alterations of immune parameters by using flow cytometric analysis in 48h sleep-deprived subjects and also in controls who maintained their regular sleep schedule during the study protocol.

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Subjects
Seventeen healthy male subjects with an age range of 19-21 years enrolled in the study as volunteers. Two physicians completed their physical and psychiatric evaluations, including complete blood counts. They were all non-smokers and restricted from the use of alcohol, caffeine and other medications during the study protocol. Sixteen men (ten sleep deprived, six controls) completed the study protocol; while one subject was dropped at the end of first night polysomnography because he had increased sleep latency due to restless leg syndrome.
Sleep deprivation protocol
Before the deprivation protocol, subjects were recorded with classical polysomnography (PSG) to exclude any sleep disorders, such as sleep-related breathing disorders, periodic limb movements during sleep, etc. Following PSG, ten subjects underwent continuous awakening (deprivation group) beginning at 8:00 a.m. for 48 hours, while the other six subjects (control group) maintained their regular sleep schedule. Subjects were kept under sedentary and constant environmental conditions for 48 hours duration and were supervised by two physicians in order to keep them awake. Meals were provided ad libitum. At the end of a 48 hour awake period, sleep-deprived subjects were allowed to sleep. Recovery sleep was also monitored with classical polysomnography to demonstrate rebound effects of sleep deprivation. This study protocol was approved by The Ethical Committee of the University of Istanbul, Cerrahpasa Medical School.

Blood samples
Venous blood samples were collected at the end of baseline sleep and drawn at 24-hour intervals at 8:00 a.m. every morning during four consecutive days: baseline, beginning of deprivation protocol after eight hours of sleep; at the 24th hour (the middle of deprivation); 48th hour (end of deprivation); and 72nd hour. At each blood drawing, a 20 ml blood sample was taken in an anticoagulant-free syringe. Ten ml of each sample was immediately taken to the laboratory for the measurement of lymphocyte subsets by flow cytometry. The remaining 10 ml of each sample was centrifuged to separate serum and stored at -20°C until IgG, IgM and cortisol measurements were performed.

a)Complete blood counts: White blood cell, red blood cell and platelet counts, hemoglobin concentrations, hematocrit levels, mean corpuscular volume (MCH), mean corpuscular hemoglobin content (MCHC), and red cell distribution width (RDW) measurements were performed by using a standard method (Medonic CA-570 Counter) in the initial evaluation of subjects and at the end of the sleep deprivation period.

b)Lymphocyte subpopulations: Lymphocyte enumeration was performed by using monoclonal antibodies (Table 1) in flow cytometer (EPICS Profile II, Coulter Corp). B-cells (CD19), T-helpers (CD4), T-suppressors (CD8), mature T-cells (CD5), and (CD16+) NK cells were assessed.

c)Immunoglobulin measurements: Serum IgG and IgM levels were measured by using nephelometry which is based on the light scattering properties of antigen-antibody complexes in solution. In conjunction with nephelometer, commercially available antibody packs (QM300, Sanofi Diagnostics Pasteur, Inc.) were used.

d) Cortisol measurement: Cortisol levels were assayed together by using a radioimmunoassay kit (DSL-2100 ACTIVE, Diagnostic Systems Laboratories Inc., Texas). Minimum detectable level was 0,3 µg/dl.

Statistical analysis
Comparisons between the results of consecutive days in each group (both deprivation and control) were performed by Wilcoxon Matched Pairs Signed Ranks Test. The student's t-test was used to compare sleep parameters between baseline and recovery sleep.

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Figure 1
Table 2 shows comparison of sleep parameters which were scored according to standard criteria (Rechtschaffen and Kales, 1968) during baseline and recovery sleep. Compared with baseline sleep, recovery sleep has a reduced number of awakenings and wake after sleep onset (WASO) time. Amounts of delta sleep were increased during recovery at the expense of Stage 2 .
Complete blood counts did not show any significant difference between control and deprivation groups at the beginning of the study. Furthermore, during deprivation procedure, no noteworthy changes in blood counts were obtained.

(CD16+) NK cells decreased in the first 24 hours of sleep deprivation. This decline was about 37% (p<0.02) and remained at the same level at the 48th hour. After recovery period, (CD16+) cells returned to baseline values. Unlike NK cells, other lymphocyte subpopulations did not show any changes. T-helper cells (CD4) showed a significant (p<0.05) increase with 24-hour sleep deprivation and returned to baseline levels at the end of the 48th hour, although deprivation was at its maximum. B-lymphocytes (CD19) did not change during deprivation period but they decreased significantly after recovery (p<0.01). CD5 and CD8 subgroups did not show any statistically significant change in deprivation or control groups (Table 3). Control group results showed the same variability in CD4 and CD19 subsets, but the results of (CD16+) NK cells were entirely different from the deprivation group and were not statistically significant (Table 4). NK cell changes in both sleep-deprived and control groups are shown in Figure 1.

Serum IgG and IgM levels of sleep deprived and control groups did not show any changes. Serum cortisol levels of both groups maintained their normal values during the study period.

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As an important part of immune response, NK cells are efficient, nonspecific effector cells that are not restricted by the major histocompatibility complex. They are rapidly activated and capable of protecting the host until more specific immune competent cells are activated. The NK cells are an important line of defense in acute and chronic viral infections and prevention of the development and progression of cancer. These cells are highly influenced by sleep, sleep deprivation and a variety of different stresses. Changes in NK cell counts and activity in sleep deprivation studies vary from one another. In the present study, NK cells decreased during 48 hours sleep deprivation and returned to baseline levels after recovery. Compared to baseline values, the reduction of NK cells was 37% and 31% at the 24th and 48th hours of deprivation, respectively. Sustained decrement of NK cells during 48h vigil was contrary to the findings of Dinges et al. (1994). They found an increase in NK cells during 64 hours of sleep deprivation, but this increment began after an initial decrease obtained at the 24th hour. They hypothesized that this decrement may be a transient effect of sleep loss and real deprivation effect may manifest itself when the deprivation period is lengthened; however, our results did not support this consideration. Moldofsky et al. (1989b), reported continuous decrement in NK cell activity during 40 hours of sleep deprivation and they obtained the lowest value during recovery sleep. As the decrement during recovery sleep continued, they could not interpret these results as a deprivation effect; however, in our study the corrective effects of recovery sleep on NK cells and not getting the same undulation in the control group as in the sleep-deprived group were indications of the sleep deprivation effect on NK cells. These findings were also consistent with other results that showed immunosupressive effects of sleep deprivation (Irwin et al., 1994, 1996).
The advantage of our study was to include a separate control group which preserved sleeping habit. As there are not established normal values of lymphocyte subgroups, we consider that determination of difference from controls may reflect real variability. The only robust finding between two groups was obtained from NK cells. The other parameters changing significantly during deprivation, like CD4 increase at the 24th hour and CD19 decline after recovery sleep, showed the same variability direction as in controls.

In humans, sleep deprivation studies have been considered to be limited by stress reactions. But there is little indication of stress-mediated immunologic changes during experimentally-induced sleep loss in humans. Previous studies failed to find elevated urinary catecholamine levels (Palmblad et al., 1976, 1979) or plasma cortisol levels (Palmbald et al., 1976; Moldofsky et al., 1989a; Dinges et al., 1994) during TSD. Apart from biological markers, psychosocial measures of distress during TSD showed that emotional distress does not account for these changes (Dinges et al., 1994). In our study, serum cortisol measurements of the sleep-deprived group were parallel with the control group. Furthermore, comparisons with the control group enabled us to isolate the effects of sleep deprivation from other effects of experimental conditions.

In some studies, NK cell activity was found to be decreased, especially during Stage 4 sleep (Shahal et al., 1992; Moldofsky et al., 1986, 1994). With these results, it becomes difficult to interpret the decline in NK cells when sleep drive reached its maximum, as in our study at the 48th hour. Single blood sampling may lead to limitation in understanding these changes of immune cells, but we tried to eliminate this inconvenience by drawing blood always at a precise time of the day, in the morning, from both sleep-deprived and normal sleeping control subjects. We thought that the comparison of day-to-day changes in each group would reduce difficulties concerning single blood sampling.

The time selected for sampling was 8:00 a.m., because this was the first hour after baseline sleep for all subjects. Perhaps it would have been better to have taken samples during the night, but this approach also has the risk of disrupting the subjects' sleep and also has the disadvantage of taking blood during different sleep stages for each subject. We considered, in the light of previous data which showed NK cells were influenced by different sleep stages (Shahal et al., 1993; Moldofsky, 1995), that sampling immediately after sleep would be beneficial. Another missing point was the evaluation of lymphocytes as percentile values. Global variability in lymphocyte subgroups can be shown by this way. Assessment of the absolute number of NK cells would be beneficial.

The practical implication of 37% reduction of NK cells is not known. As a part of nonspecific immune response, NK cells have a role in protection of primary herpes virus infection and lymphokine induced killer cytotoxicity and these functions may have a prognostic role in identifying patients at risk for recurrence or progression of malignant disease (Biron et al., 1989; Fawzy et al., 1993). For this reason, our data implied that sleep becomes more important in chronically ill patients than healthy subjects.

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The authors do not wish to include any acknowledgments.

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