A timetable of 24-hour patterns for human lymphocyte subpopulations

Specific lymphocyte cell surface molecules involved in antigen recognition and cell activation present different circadian patterns, with peaks and troughs reflecting a specific time-related compartment of immune cell function. In order to study the dynamics of variation in expression of cytotoxic lymphocyte cell surface molecules that trigger immune responses, several lymphocyte cell surface clusters of differentiation (CD) and antigen receptors, analyses were performed on blood samples collected every 4 h for 24 h from eleven clinically-healthy men. Assays for serum melatonin (peaking at night) and cortisol (peaking near awakening) confirmed 24-h synchronization of the subjects to the light-dark schedule. A significant (p≤0.05) circadian rhythm could be demonstrated for six of the 10 lymphocyte subpopulations, with midday peaks for CD8+dim (T cytotoxic cells, 11:15 h), gammadeltaTCR (gamma-delta T cell receptor-expressing cells, 11:33 h), CD8+ (T suppressor/cytotoxic cells, 12:08 h), and for CD16+ (natural killer cells, 12:59 h), and peaks during the night for CD4+ (T helper/inducer cells, 01:23 h) and CD3+ (total T cells, 02:58 h). A borderline significant rhythm (p = 0.056) was also observed for CD20+ (total B cells), with a peak late in the evening (23:06 h). Acrophases for 3 subsets, CD8+bright (T suppressor cells, 15:22 h), HLA-DR+ (B cells and activated T cells, 23:06 h) and CD25+ (activated T lymphocytes with expression of the alpha chain of IL2 receptor, 23:35 h), where a 24-h rhythm could not be definitively determined, nevertheless provide information on the location of their highest values and possible physiological significance. Thus, specific lymphocyte surface molecules present distinctly-timed profiles of nyctohemeral changes that indicate a temporal (i.e., circadian) organization of cellular immune function, which is most likely of physiological significance in triggering and regulating immune responses. Such a molecular cytotoxic timetable can potentially serve as a guide to sampling during experimental, diagnostic, therapeutic and/or other medical procedures.     

Twenty-Four-Hour Analysis of Lymphocyte Subpopulations and Cytokines in Healthy Subjects

Fourteen healthy male subjects were studied under resting conditions for 24 or 48 h. The 24-h variations of ACTH and Cortisol with peaks in the morning were confirmed. Interleukin-2 release was profoundly reduced at 8:00. A cosine function could be fitted to lymphocyte subpopulations including populations such as T_{helper-memory} cells (CD4+/CDw29) and HLA-DR-bearing cells displaying peak values during the night and minimal values at 9:00. Numbers of natural killer cells (CD57) were not correlated to other cell populations and no rhythm could be detected. Interleukin-1 (3 was detectable in some plasma samples only with an interleukin-1 ELISA kit (Quantakine HS^R), but neither a 24-h rhythm nor reproducible results could be obtained for day 1 and 2 of the study. We conclude that there might be a temporal relation between the parameters analyzed: Higher levels of endogenous Cortisol in the morning hours probably inhibit the cellular interleukin-2 synthesis and are responsible for an enhanced migration of lymphocytes from the blood into the tissues of the reticuloendothelial system. These results might be indicative of a circadian organization of the immune system which may play a role in both physiological and pathological functioning. (Chronobiology International, 13(6), 423-434, 1996)     

Twenty-Four-Hour Analysis of Lymphocyte Subpopulations and Cytokines in Healthy Subjects

Fourteen healthy male subjects were studied under resting conditions for 24 or 48 h. The 24-h variations of ACTH and Cortisol with peaks in the morning were confirmed. Interleukin-2 release was profoundly reduced at 8:00. A cosine function could be fitted to lymphocyte subpopulations including populations such as T_{helper-memory} cells (CD4+/CDw29) and HLA-DR-bearing cells displaying peak values during the night and minimal values at 9:00. Numbers of natural killer cells (CD57) were not correlated to other cell populations and no rhythm could be detected. Interleukin-1 (3 was detectable in some plasma samples only with an interleukin-1 ELISA kit (Quantakine HS^R), but neither a 24-h rhythm nor reproducible results could be obtained for day 1 and 2 of the study. We conclude that there might be a temporal relation between the parameters analyzed: Higher levels of endogenous Cortisol in the morning hours probably inhibit the cellular interleukin-2 synthesis and are responsible for an enhanced migration of lymphocytes from the blood into the tissues of the reticuloendothelial system. These results might be indicative of a circadian organization of the immune system which may play a role in both physiological and pathological functioning. (Chronobiology International, 13(6), 423–434, 1996)     

Temporal structuring of delivery in the absence of a photoperiod: preparturient myometrial activity of the rhesus monkey is related to maternal body temperature and depends on the maternal circadian system.

No convincing evidence exists that the shift from myometrial contractures to contractions, which determines the synchronized 24-h rhythm in the dynamics of the primate uterus, may be attributed to an endogenous circadian rhythm. We therefore wished to ascertain whether a 24-h periodic shift would also occur in the myometrial activity of animals kept under constant conditions. We studied five pregnant rhesus monkeys, kept in continuous darkness from 56-77 days gestational age until delivery at 117-167 days gestational age. During the last week before delivery we determined the individual phase, level, and amplitude of circadian changes in maternal body temperature and 24-h myometrial activity patterns in the form of contractions. In all five monkeys, a rhythm with a period of 24-h characterized the temporal incidence of preparturient contraction activity. A consistent phase lag of 6-7 h from the temperature crest was observed in four out of the five animals. The circadian phase of all individual rhythms was idiosyncratic among animals. We conclude that endogenous rhythms in body temperature and preparturient myometrial activity are truly circadian. In addition, these rhythms are either interdependent or subject to the same maternal timekeeping mechanism, supporting the hypothesis that the exact time of the day at which birth occurs in the rhesus monkey depends on the maternal circadian system.     

Circadian rhythmicity in leukocytes immune responses in the freshwater snake,Natrix piscator

Circadian rhythm is observed in most of the physiological functions including immune response. The use of animal models other than mammals is useful in understanding how the vertebrate circadian system is organized and how this biological clock has changed throughout the vertebrate evolution. The present study was aimed to examine the circadian variability in the innate immune responses of leukocytes in the freshwater snake, Natrix piscator. Leukocytes were isolated and processed for total and differential leukocyte count, leukocyte phagocytosis, NBT reduction, nitrite production, and lymphocyte proliferation. Experiments were conducted for seven time points at 24, 4, 8, 12, 16, 20, and 24 h in three seasons – summer, winter, and spring. Cosinor analysis revealed that among leukocytes, only lymphocyte count showed circadian variation in summer. Percent phagocytosis and phagocytic index had significant rhythm of 24 h in winter and summer season, respectively. The acrophase of NBT reduction and nitrite release were coming during the evening hours in summer and during morning hours in winter and had circadian rhythmicity. A significant phase shift in nitrite release was observed with a trend of delayed phase shift from winter to summer. Circadian rhythm was also observed in lymphocyte proliferation (basal and concanavalin A stimulated). It is evident from the present study that animals synchronize their immune activity according to the time of the day and season. Enhancement of immune function helps the individual cope with seasonal stressors that would otherwise jeopardize the survival of animal.     

Comprehensive Mapping of Regional Expression of the Clock Protein PERIOD2 in Rat Forebrain across the 24-h Day

In mammals, a light-entrainable clock located in the suprachiasmatic nucleus (SCN) regulates circadian rhythms by synchronizing oscillators throughout the brain and body. Notably, the nature of the relation between the SCN clock and subordinate oscillators in the rest of the brain is not well defined. We performed a high temporal resolution analysis of the expression of the circadian clock protein PERIOD2 (PER2) in the rat forebrain to characterize the distribution, amplitude and phase of PER2 rhythms across different regions. Eighty-four LEW/Crl male rats were entrained to a 12-h: 12-h light/dark cycle, and subsequently perfused every 30 min across the 24-h day for a total of 48 time-points. PER2 expression was assessed with immunohistochemistry and analyzed using automated cell counts. We report the presence of PER2 expression in 20 forebrain areas important for a wide range of motivated and appetitive behaviors including the SCN, bed nucleus, and several regions of the amygdala, hippocampus, striatum, and cortex. Eighteen areas displayed significant PER2 rhythms, which peaked at different times of day. Our data demonstrate a previously uncharacterized regional distribution of rhythms of a clock protein expression in the brain that provides a sound basis for future studies of circadian clock function in animal models of disease.     

Alteration of circadian rhythmicity of CD3+CD4+ lymphocyte subpopulation in healthy aging

The CD4+ T helper/inducer and the CD8+ T suppressor/cytotoxic are major lymphocyte subsets that play a key role in cell-mediated immunity. Aging-related changes of immune function have been demonstrated. The purpose of this study is to analyze the dynamics of variation of these specific lymphocyte subsets in the elderly. In our study cortisol and melatonin serum levels were measured and lymphocyte subpopulation analyses were performed on blood samples collected every four hours for 24 hours from fifteen healthy young middle-aged subjects (age range 36-55 years) and fifteen healthy elderly male subjects (age range 67-79 years). A clear circadian rhythm was validated for the time-qualified changes of CD3+ and CD4+ cells with acrophase at night and for the time-qualified changes of CD8+ cells with acrophase at noon in young middle-aged subjects and for the time-qualified changes of CD3+ cells with acrophase at night and for the time-qualified changes of CD8+ cells with acrophase at noon in elderly subjects. No clear circadian rhythm was validated for the time-qualified changes of CD4+ cells in elderly subjects. No statistically significant correlation among lymphocyte subsets was found in elderly subjects. In elderly subjects CD3+ lymphocyte percentage was higher in the photoperiod and in the scotoperiod and cortisol serum level were higher in the scotoperiod in respect to young middle-aged subjects. In the elderly there is an alteration of circadian rhythmicity of T helper/inducer lymphocytes and this phenomenon might contribute to the aging-related changes of immune responses.     

Rhythms in human bone marrow and blood cells

In 24h studies of bone marrow (BM), circadian stage-dependent variations were demonstrated in the proliferative activity of BM cells from subsets of 35 healthy diurnally active men. On an average, the percentage of total BM cells in deoxyribonucleic acid (DNA) synthesis phase was 188% greater at midday than at midnight (circadian rhythm: p = 0.018; acrophase or peak time of 13: 16h). Patients with malignant disease (n = 15) and a normal cortisol circadian rhythm showed higher fractions of BM cells in S-phase at midday. Colony-forming units--granulocyte/macrophage (CFU-GM), an indicator of myeloid progenitor cells, showed the same circadian variation as DNA S-phase (average range of change or ROC = 136%; circadian rhythm: p < 0.001; acrophase of 12:09h). Deoxyribonucleic acid S-phase and CFU-GM in BM both showed a circannual rhythm (p = 0.015 and 0.008) with an identical acrophase of August 12. The daily peak in BM glutathione content, a tripeptide involved in cellular defense against cytotoxic damage, preceded BM proliferative peaks by 4-5 h (ROC = 31-90%; circadian rhythm: p = 0.05; acrophase of 08:30h). Myeloid (ROC = 57%; circadian rhythm: p = 0.056; acrophase at 08:40h) and erythroid (ROC = 26%; circadian rhythm: p = 0.01; acrophase of 13:01h) precursor cells were positively correlated (r = 0.41; p < 0.001), indicating a circadian temporal relationship and equal influence on S-phase of total BM cells. Yield of positive selected CD34+ progenitor stem cells also showed significant circadian variation (ROC = 595%; circadian rhythm: p = 0.02; acrophase of 12:40h). Thus, the temporal synchrony in cell cycling renders BM cells more sensitive at specific times to hematopoietic growth factors and cell cycle-specific cytotoxic drugs. Moreover, proper timing of BM harvesting may improve progenitor cell yield. When using marker rhythms in the blood to allow for individualized timing of BM procedures, the times of low values in white blood corpuscles, neutrophils, and lymphocytes and high values in cortisol were predictive of the times of highest BM erythroid, myeloid, and total S-phase numbers occurring in the following 12 h.     

Hypoxic alterations of cortisol circadian rhythm in man after simulation of a long duration flight

Fatigue is often reported after long duration flights. Mild hypobaric hypoxia caused by pressurisation may be involved in this effect through disruption of circadian rhythms, independently of the number of time zones crossed. In this controlled crossover study, we assessed the effects of two levels of hypoxia equivalent to 8000 and 12,000 ft on the circadian rhythm of plasma cortisol, a marker of the circadian time structure. Sixteen healthy young male volunteers (23-39 years) were exposed in a hypobaric chamber for 8 h (08:00-16:00 h) to 8000 ft, followed 4 weeks later to 12,000 ft. Plasma cortisol was assayed during two 24-h cycles (control and hypoxic exposure) every 2h in all subjects. We found a significant change in the pattern of cortisol secretion during both hypoxic exposures, with an initial fall in cortisol followed by a transient rebound, whereas the phase and the 24-h mean level remained unchanged. The change in cortisol pattern followed the alterations in autonomic balance assessed by heart rate variability (HRV) spectral analysis. The normalised high frequencies and the low-to-high frequencies ratio showed a significant shift toward sympathetic dominance with some differences in time course for both altitudes studied. HRV analysis improved the interpretation of cortisol 24-h profiles. Our data, which strongly suggest that prolonged mild hypoxia alters the expression of cortisol circadian rhythm, should be taken into account to interpret secretory rhythm changes after transmeridian flights.     

Circadian and Ultradian Variations in the Plasma Level of Maturational Gonadotropin (GTH II) in the Indian Catfish,Clarias batrachus

Analysis of changes in the plasma levels of GTH II over a 24-h day/night cycle revealed statistically significant circadian or ultradian variation or both in its circulating level during the gonadally active months of the reproductive cycle. The 24-h average and amplitude of circadian or ultradian rhythm in GTH II increased with the advancement of ovarian development and maturation. In April, a single peak in GTH II level was noticed in the night at 20.6 h. In contrast, in May, June and July a biphasic pattern (ultradian rhythm) was noticed with two characteristic peaks, one in the photophase and the other in the scotophase. In addition, in May a statistically significant circadian rhythm in plasma GTH II was validated with a peak located at 20.1 h. These rhythms seem to have physiological significance. The ultradian rhythm with two peaks during the reproductively active phase appears to provide a suitable physiological milieu for the temporally different yet synchronous population of oocytes for the secretion of steroids. Thus, the observed temporal organization in GTH II may have physiological consequences leading to accomplishment of reproductive process at appropriate time of the year.     

Circadian and Ultradian Variations in the Plasma Level of Maturational Gonadotropin (GTH II) in the Indian Catfish, Clarias batrachus

Analysis of changes in the plasma levels of GTH II over a 24-h day/night cycle revealed statistically significant circadian or ultradian variation or both in its circulating level during the gonadally active months of the reproductive cycle. The 24-h average and amplitude of circadian or ultradian rhythm in GTH II increased with the advancement of ovarian development and maturation. In April, a single peak in GTH II level was noticed in the night at 20.6 h. In contrast, in May, June and July a biphasic pattern (ultradian rhythm) was noticed with two characteristic peaks, one in the photophase and the other in the scotophase. In addition, in May a statistically significant circadian rhythm in plasma GTH II was validated with a peak located at 20.1 h. These rhythms seem to have physiological significance. The ultradian rhythm with two peaks during the reproductively active phase appears to provide a suitable physiological milieu for the temporally different yet synchronous population of oocytes for the secretion of steroids. Thus, the observed temporal organization in GTH II may have physiological consequences leading to accomplishment of reproductive process at appropriate time of the year.     

Circadian variation in cell-adhesion molecule expression by normal human leukocytes

Adhesion molecules located on the surface of blood-borne leukocytes permit adherence of leukocytes to the microvascular endothelium, diapedesis of leukocytes across vessel walls, formation of intimate multicell interactions, and enhanced transmembrane signal transduction. Since some leukocyte-mediated immune functions exhibit nocturnal intensification, the current study was conducted to investigate the hypothesis that expression of selected cell adhesion molecules (CAM) varies with circadian periodicity. Blood was collected from normal human donors over a 24-h period and CAM expression by monocytes, neutrophils, and lymphocytes evaluated by monoclonal antibody binding and flow cytometry. All leukocyte classes exhibited significant circadian-like variation (p < 0.05) in CD62L (L-selectin) expression. Similarly, a diurnal variation (p < 0.05) in monocyte and neutrophil CD54 (ICAM-1) was observed. Finally, neutrophils demonstrated a circadian-like variation (p < 0.05) in CD11a (LFA-1a). The rhythmic alterations in CAM expression may be clinically relevant, since changes in CAM expression have the potential to modulate the leukocyte-induced pathogenesis associated with disease progressions such as nocturnal asthma, the nighttime exacerbations of rheumatoid arthritis, and the high nocturnal incidence of cerebrovascular and cardiovascular crisis.     

Linear demasking techniques are unreliable for estimating the circadian phase of ambulatory temperature data.

Clinical investigators often use ambulatory temperature monitoring to assess the endogenous phase and amplitude of an individual's circadian pacemaker for diagnostic and research purposes. However, an individual's daily schedule includes changes in levels of activity, in posture, and in sleep-wake state, all of which are known to have masking or evoked effects on core body temperature (CBT) data. To compensate for or to correct these masking effects, many investigators have developed "demasking" techniques to extract the underlying circadian phase and amplitude data. However, the validity of these methods is uncertain. Therefore, the authors tested a variety of analytic methods on two different ambulatory data sets from two different studies in which the endogenous circadian pacemaker was not synchronized to the sleep-wake schedule. In both studies, circadian phase estimates calculated from CBT collected when each subject was ambulatory (i.e., free to perform usual daily activities) were compared to those calculated during the same study when the same subject's activities were controlled. In the first study, 24 sighted young and older subjects living on a 28-h scheduled "day" protocol were studied for approximately 21 to 25 cycles of 28-h each. In the second study, a blind man whose endogenous circadian rhythms were not synchronized to the 24-h day despite his maintenance of a regular 24-h sleep-wake schedule was studied for more than 80 consecutive 24-h days. During both studies, the relative phase of the endogenous (circadian) and evoked (scheduled activity-rest) components of the ambulatory temperature data changed progressively and relatively slowly, enabling analysis of the CBT rhythm at nearly all phase relationships between the two components. The analyses of the ambulatory temperature data demonstrate that the masking of the CBT rhythm evoked by changes in activity levels, posture, or sleep-wake state associated with the evoked schedule of activity and rest can significantly obscure the endogenous circadian component of the signal, the object of study. In addition, the masking effect of these evoked responses on temperature depends on the circadian phase at which they occur. These nonlinear interactions between circadian phase and sleep-wake schedule render ambulatory temperature data unreliable for the assessment of endogenous circadian phase. Even when proposed algebraic demasking techniques are used in an attempt to reveal the endogenous temperature rhythm, the phase estimates remain severely compromised.     

Characterization of the interaction between galectin-1 and lymphocyte glycoproteins CD45 and Thy-1

Galectin-1 is a sugar binding protein specific for beta-galactosides and not requiring metal ions for binding activity. It exists as a soluble protein which forms a noncovalent homodimer and is expressed with a broad tissue distribution. Recently, galectin-1 has been shown to play a possible role in the immune system mediating apoptosis of activated T cells with indirect evidence suggesting that galectin-1 interacts with the heavily glycosylated, transmembrane, protein phosphotyrosine phosphatase CD45. The interaction of galectin-1 with purified lymphocyte cell surface proteins was studied using surface plasmon resonance in a BIAcoretrade mark. Galectin-1 was shown to bind CD45 and Thy-1 in a carbohydrate-dependent manner. Several galectin-1 molecules could bind each CD45 molecule. The dissociation constant of dimeric galectin-1 binding to CD45 was measured at approximately 5 microM, indicating the concentration at which cross-linking of cell surface glycoproteins by galectin-1 would occur. A possible role for galectin-1 in the organization of cell surface glycoproteins is discussed.     

Effects of ultradian lighting and feeding schedules on the circadian rhythm of body temperature in monkeys

In estimating, by use of cosinor-test, the 12- and 24-h component parameters of body temperature circadian rhythm in monkeys under ultradian schedules of lighting and feeding (LD 6:6; DL 6:6) we have shown that an intensive 12-h component is registered in both cases. The presence of a 24-h component of circadian rhythm depends on the zeitgeber phase. This component is present in LD 6:6 (lighting hours 07:00-13:00 and 19:00-01:00) and is absent in DL 6:6 (01:00-07:00 and 13:00-19:00). We hold that the most satisfactory explanation of the phenomena observed is that 12-h component is the result of a masking effect induced by the 12-h schedule (exogenous component) whereas the 24-h component reflects the intrinsic pacemaker work (endogenous component). It should be noted that in our case the masking effect in body temperature rhythm is circadian phase-dependent.     

同步遥测分析大鼠棕色脂肪产热与体温昼夜节律变化的时间关系

目的：同步遥测棕色脂肪组织（BAT）产热与体核温度昼夜律变化的时间曲线,分析二者昼夜节律变化的时间关系。方法：实验用成年雄性SD大鼠,在22℃环境温度下,明暗时间各12h,昼光时间为06：00h-18：00h,同步无线遥测体核温度（TC）、BAT温度（T队T）、腋窝温度（Tax）和动物活动的昼夜节律变化。结果：①在昼光中,TBAT较TC低0．67％,而在暗光中二者则相似。大鼠从昼光进入暗光时,TBAT升高的速率较TC升高速率快,开始上升的时间较TC提前8min;而从暗光进入昼光时,TBAT开始下降的时间则较TC提前4min。②Tax的昼夜节律幅度与TC相似,但无论动物在明光期或暗光期中,Tax均低于同步测量的TC。③从昼光期转入暗光期时,动物的行为活动出现增加反应先于TBAT和TC开始上升的时间。结论：实验结果证明,在暗光期中大鼠TC升高与BAT产热增加有关,说明BAT昼夜节律性产热的变化在维持体温昼夜生理节律中有重要的作用。     

Plasticity of the intrinsic period of the human circadian timing system

Human expeditions to Mars will require adaptation to the 24.65-h Martian solar day-night cycle (sol), which is outside the range of entrainment of the human circadian pacemaker under lighting intensities to which astronauts are typically exposed. Failure to entrain the circadian time-keeping system to the desired rest-activity cycle disturbs sleep and impairs cognitive function. Furthermore, differences between the intrinsic circadian period and Earth's 24-h light-dark cycle underlie human circadian rhythm sleep disorders, such as advanced sleep phase disorder and non-24-hour sleep-wake disorders. Therefore, first, we tested whether exposure to a model-based lighting regimen would entrain the human circadian pacemaker at a normal phase angle to the 24.65-h Martian sol and to the 23.5-h day length often required of astronauts during short duration space exploration. Second, we tested here whether such prior entrainment to non-24-h light-dark cycles would lead to subsequent modification of the intrinsic period of the human circadian timing system. Here we show that exposure to moderately bright light ( approximately 450 lux; approximately 1.2 W/m(2)) for the second or first half of the scheduled wake episode is effective for entraining individuals to the 24.65-h Martian sol and a 23.5-h day length, respectively. Estimations of the circadian periods of plasma melatonin, plasma cortisol, and core body temperature rhythms collected under forced desynchrony protocols revealed that the intrinsic circadian period of the human circadian pacemaker was significantly longer following entrainment to the Martian sol as compared to following entrainment to the 23.5-h day. The latter finding of after-effects of entrainment reveals for the first time plasticity of the period of the human circadian timing system. Both findings have important implications for the treatment of circadian rhythm sleep disorders and human space exploration.     

Integration of human sleep-wake regulation and circadian rhythmicity.

The human sleep-wake cycle is generated by a circadian process, originating from the suprachiasmatic nuclei, in interaction with a separate oscillatory process: the sleep homeostat. The sleep-wake cycle is normally timed to occur at a specific phase relative to the external cycle of light-dark exposure. It is also timed at a specific phase relative to internal circadian rhythms, such as the pineal melatonin rhythm, the circadian sleep-wake propensity rhythm, and the rhythm of responsiveness of the circadian pacemaker to light. Variations in these internal and external phase relationships, such as those that occur in blindness, aging, morning and evening, and advanced and delayed sleep-phase syndrome, lead to sleep disruptions and complaints. Changes in ocular circadian photoreception, interindividual variation in the near-24-h intrinsic period of the circadian pacemaker, and sleep homeostasis can contribute to variations in external and internal phase. Recent findings on the physiological and molecular-genetic correlates of circadian sleep disorders suggest that the timing of the sleep-wake cycle and circadian rhythms is closely integrated but is, in part, regulated differentially.