Circadian rhythms in surface molecules of rat blood lymphocytes

Carme Pelegrí1, Jordi Vilaplana2, Cristina Castellote1, Manel Rabanal1, Àngels Franch1, and Margarida Castell1

1 Grup d'Autoimmunitat i Tolerància, 2 Grup de Cronobiologia, Departament de Fisiologia- Divisió IV, Facultat de Farmàcia, Universitat de Barcelona, 08028 Barcelona, Spain


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present article examines whether the expression of certain surface molecules that trigger immune responses shows a circadian rhythm. We also analyzed the rhythms in the number and percentage of lymphocyte subpopulations, in the leukocyte differential counts, and in the total red and white blood cell counts. Blood samples obtained from rats at 2-h intervals for 24 h were stained with several mouse monoclonal antibodies directed against lymphocyte surface molecules and processed by flow cytometry. The number of B, total T, Tgamma delta , Th, and Ts/c cells followed a 24-h rhythm with a peak in the first half of the resting period. The expression of CD45, CD5, CD3, and CD4 followed a circadian rhythm. Their acrophases suggested temporal association between CD45 and CD5 at the end of the active phase and between CD4 and CD3 at the beginning of this phase. This temporal organization could have an important role for immune cell function.

leukocytes; CD3; CD4, CD5; CD45


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

RECENT RESEARCH HAS FOCUSED on the physiological interactions between the nervous, endocrine, and immune systems. It is now clear that the three systems share some mediator molecules and receptors and contribute jointly to the maintenance of homeostasis (5). Secretion of glucocorticoid hormones from the hypothalamic-pituitary-adrenal axis shows circadian variation, as do those systems influenced by glucocorticoids. Although the interactions between the neuroendocrine and the immune elements are complex (5), the immune system is influenced by cortisol levels and shows daily fluctuations in some aspects (1, 2, 41). Thus the number of circulating leukocytes and the percentage of each leukocyte type show daily rhythmic variations (reviewed in Refs. 15 and 16). In humans, total white blood cell counts peak in the evening/night. Among them, neutrophil and NK cell numbers reach a maximum in the afternoon, monocytes and lymphocytes at night, T cells at midnight, and B cells in the early morning (33, 36). In nocturnal animals like rat and mouse, the number of total white blood cells, lymphocytes, and Th and B cells peaks during the resting period (12, 14, 25, 29). In mice, the percentages of lymphocyte subsets also show circadian rhythms and a significant increase in total T and Th cell percentages occurs during the activity period (23).

Immune functions are also subjected to circadian rhythmicity. Thus contact hypersensitivity responses vary according to the time of antigen application (35). Circadian periodicity has also been observed in T cell responses to phytohemagglutinin (40), tetanus toxoid (17), lipopolysaccharide, and concanavalin A (13). Moreover, cytokine production in human whole blood shows a circadian variation (6, 32). Circadian variations can also be observed in symptoms of immunoinflammatory disorders. In rheumatoid arthritis, joint inflammation is most severe in the early morning (2, 30), whereas asthma exacerbations commonly occur at night (27). Therefore, circadian rhythms condition not only cell counts but also lymphocyte reactivity and function. In response to foreign antigens, T helper cell activation is triggered by specific interaction of the T cell receptor with foreign antigens presented by specialized cells. This triggering requires the participation of antigen-independent adhesion and costimulatory molecules (9, 43). Lymphocyte activation changes the expression of surface molecules by clustering some of them or down- or upregulating others (21). The absence of costimulatory signals blocks the activation of T cells (19). Moreover, a recent report shows that several T cell responses are directly related to the number of engaged receptors (37). Here, we examine whether the expression of surface molecules involved in antigen recognition and cell activation follows a circadian rhythm. To this end, several mouse monoclonal antibodies (MAb) directed against rat lymphocyte surface molecules, double staining techniques, and flow cytometry were applied to blood samples obtained from rats at 2-h intervals for 24 h.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animal maintenance. Seventy-two male Wistar rats (Charles River, France), 5 wk old on arrival, were kept in an isolated sound room in standard conditions of temperature (22 ± 2°C) and 12:12-h light-dark cycles (300 lux/0 lux). They were housed in individual cages, fed food and water ad libitum, and given 10 days for adaptation.

Experimental design and blood sampling. Rats were randomly assigned to 12 groups of 6 rats each. Each group was bled only once, to avoid effects due to stress, at a determined Zeitgeber time (ZT). The ZT was used as the reference to detect rhythmicity of the studied variables. Considering that light on corresponds to ZT0 h and light off to ZT12 h, blood samples were taken at ZT1 until ZT23 every 2 h. Because rats are nocturnal rodents, ZT0 corresponds approximately to the beginning of their resting phase, whereas ZT12 corresponds to the beginning of the active phase. Rats were anaesthetized with ether and bled by retro-orbital puncture. Two milliliters of blood were collected in EDTA tubes (Sardstedt, Canovelles, Spain).

Blood cell counts and lymphocyte subsets analysis. Total red and white blood cell counts, hematocrit and hemoglobin, and leukocyte differential counts were determined automatically by means of a Coulter Counter JT hemocytometer (Hialeah).

Lymphocyte subsets were determined after double staining with a panel of anti-rat lymphocyte antibodies, summarized in Table 1, by flow cytometry. The phenotypes characteristic of lymphocyte subsets were chosen according to the presence or absence of specific clusters of differentiation (CD) and antigen receptors at the cell surface: CD45+ cells as total lymphocytes, Ig+/RT-1B+ cells or Ig+/CD45RABC+ cells as B lymphocytes, CD5+/TCRalpha beta + or CD3+ as total T lymphocytes, CD4+ as Th lymphocytes, CD8+/NKR-P1- cells as Ts/c lymphocytes, CD8+/TCRgamma delta + cells as Tgamma delta lymphocytes, and CD8+/NKR-P1+ cells as NK cells. Before lymphocyte staining, erythrocytes were eliminated by osmotic lysis (31). Cells (106) were then reacted with primary nonlabeled mouse MAb for 20 min at 4°C and subsequently washed with phosphate-buffered saline (PBS) containing 2% fetal calf serum (FCS) and 0.1% NaN3. Cells were incubated for 20 min with phycoerythrin-conjugated goat anti-mouse IgG antibody (Southern Biotechnology Associates, Birmingham, AL) diluted in PBS and containing 2% of rat serum to avoid cross reactions. After being washed again, cells were reacted with normal mouse immunoglobulins (15 min at 4°C) and fluorescein isothiocyanate-conjugated antibodies (20 min at 4°C; Table 1). Finally, cells were washed with PBS, fixed with 1% paraformaldehyde, and stored at 4°C in the dark until analysis. For each animal, a negative control staining was included using an isotype-matched MAb.

                              
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Table 1.   Antibodies used to label rat blood lymphocytes

Before the experiment, appropriate dilutions of each primary and secondary antibody were established to use saturating concentrations of immunoreagents for 106 mononuclear cells. During the experiment, the same batch and antibody dilution were used to eliminate variations in fluorescence emissions due to conjugated antibodies.

All analyses were performed with an Epics XL flow cytometer (Coulter). All samples were analyzed considering the same gate (Fig. 1). The gate was set to include 100% of cells labeled with MAb against immunoglobulins, CD4, and CD8. The number of fluorescent cells was expressed as the percentage of total gated lymphocytes. For each staining antibody, the mean fluorescence intensity (MFI) of the positive cells obtained from each sample was used to measure the expression of surface molecules. For each molecule studied, all samples collected over the course of 24 h were analyzed correlatively at the cytometer, i.e., with the same conditions of voltage.


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Fig. 1.   A: forward (FS)/side scatter (SS) dot plot of a representative rat blood sample showing the selected gate for lymphocytes. B: representative histogram of frequency distribution of fluorescence intensity obtained after staining with FITC-anti-rat CD5 and phycoerythrin (PE)-anti-rat TCRalpha beta monoclonal antibody (MAb). C and D: separate fluorescence intensity distribution for each staining in B.

Statistical analysis. The rhythmicity of each variable was studied by Cosinor analysis (4), and this study was then complemented with conventional analysis of variance (ANOVA).

Cosinor analysis, by using the least squares method, approximates the following sinusoidal function to the experimental data
<IT>Y<SUB>t</SUB>=M+A×</IT>cos [(2 × &pgr;/&tgr;) × <IT>t</IT> − &phgr;]
where Yt is the value of the cosine function at time t (ZT in our case), M is the mean level of the oscillation or mesor (acronym for midline estimating statistic over rhythm), A is the amplitude (the extent of oscillation from the mesor or half of the total oscillation), pi  is the pi number, tau  is the period (24 h in our case), and phi  is the acrophase (the time at which the cosine function reaches the maximum value). Therefore, Cosinor analysis determines the best-fitting sinusoidal wave by estimating three parameters: mesor, amplitude, and acrophase.

By Cosinor analysis, we determined the fiduciary limits of the mesor, the amplitude, and the acrophase at 95% probability level. When the range determined by the fiduciary limits of the amplitude contains the 0 value, it cannot be excluded that amplitude is 0, and, therefore, the existence of a rhythm is not statistically significant. In other words, to test the statistical significance of the presence of the rhythm, we determined whether the null hypothesis of zero amplitude is or is not rejected at 0.05 of alpha level.

On the other hand, the fiduciary limits of the acrophase allow determining whether there are significant differences between the acrophases of different variables. When the range determined by the fiduciary limits of the acrophase of one variable has some point of coincidence with that of another one, the possibility that both acrophases are equal cannot be discarded. In this study, this statement has been applied to establish whether two variables differ in their acrophases.

Cosinor analysis has been complemented by ANOVA. For each dependent variable that has a significant circadian rhythm according to the Cosinor analysis, an ANOVA was performed to confirm differences in the variable according to the independent (grouping) variable ZT. Moreover, when ZT has a significant effect on the dependent variable, a post hoc comparison (LSD test) was performed comparing the group with the ZT nearest the acrophase with the group with the ZT nearest the bathyphase (i.e., acrophase + 12 h).

In the MFI data, the percentage of variation (PV) was calculated. PV was defined as the difference between maximum and minimum values of the fitted function (i.e., twice amplitude) divided by mesor and expressed as percentage [PV = 100 × (2 × amplitude)/mesor].


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Erythrocyte counts, hemoglobin, hematocrit, and total and differential leukocyte counts. Results concerning red blood cells are summarized in Table 2. Erythrocyte counts, hematocrit, and hemoglobin concentration displayed a circadian rhythm with all acrophases placed in the middle of the resting period.

                              
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Table 2.   Circadian rhythm parameters (and fiduciary limits) obtained for erythrocyte count, hematocrit, and hemoglobin and total and differential leukocyte counts and percentages

The number of total circulating white blood cells showed a significant circadian rhythm (Table 2), and the maximum value was found at the beginning of the resting period (Fig. 2). The number of the main leukocyte populations, i.e., lymphocytes, granulocytes, and monocytes also showed a circadian variation, with maximal counts at the beginning of the resting period in the three cases (Table 2 and Fig. 2). The contribution of each population to the increase in total leukocyte count can be estimated from the amplitude of each particular rhythm. Thus granulocytes, lymphocytes, and monocytes contribute to the amplitude of the leukocyte count with about 21% for granulocytes, 72% for lymphocytes, and only 7% for monocytes.


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Fig. 2.   Acrophases corresponding to the number of total leukocytes, leukocyte types, and lymphocyte subpopulations in peripheral blood from Wistar rats. ZT, Zeitgeber time.

The proportion of each leukocyte population displayed a daily variation and presented different acrophases over the course of the day (Table 2). The percentage of lymphocytes was maximal at the beginning of the activity period, when the total count of white blood cells was minimal, i.e., almost 12 h after the leukocyte acrophase.

Percentage and absolute number of lymphocyte subsets. B cell percentage presented a mesor of 28.7 (27.4-29.5) when determined as RT-1B+Ig+ cells or a mesor of 26.3 (25.1-27.5) when determined as CD45RABC+Ig+ cells with respect to circulating blood lymphocytes. These proportions did not show a circadian rhythm. To establish the T cell population, two stainings were also applied: TCRalpha beta /CD5 and CD3. TCRalpha beta +CD5+ cells displayed a significant circadian rhythm with a mesor of 58.8% (56.8-60.8) and an amplitude of 4.2% (0.8-7.6), this proportion being maximal at the beginning of the active period with a ZT of 13.3 h (9.8-17.2). The CD3+ cell percentage presented a mesor of 56.9% (55.3-58.5), but the study of the circadian rhythmicity showed only borderline statistical results. However, the best estimation of the acrophase (ZT 10.7 h) was similar to that of TCRalpha beta +CD5+ cells.

The percentage of T helper cells or CD4+ lymphocytes had a mesor of 48.0% (46.5-49.5) and showed a significant daily variation with an amplitude of 3.3% (0.7-5.9) and an acrophase at the beginning of the active period, at ZT 14.4 h (10.8-17.9), close to that obtained for the T cell population. The mesor of percentage of CD8+NKR-P1- cells, which mainly include the Ts/c subpopulation, was about 14.3% (13.8-14.8), but the proportion of these cells did not present a significant rhythm over the course of the day.

About 1.2% (1.1-1.3) of blood lymphocytes presented the TCRgamma delta +CD8+ cell phenotype, but this small population did not show a significant daily rhythmicity. By means of the double positive labeling NKR-P1+CD8+, NK cells were identified. The mesor indicated that about 4.8% (4.4-5.3) of blood lymphocytes were NK cells and that the daily variation of the percentage of this population was not significant.

From the total lymphocyte count and the percentages of each subpopulation, the absolute number of each lymphocyte subpopulation was calculated. Results of circadian variation analysis are shown in Table 3 and in Figs. 2 and 3. The number of all lymphocyte subsets displayed a significant circadian rhythm by the Cosinor analysis. These results were confirmed by ANOVA with the exception of Tgamma delta + cells. However, this population followed a clear sinusoidal oscillation as shown in Fig. 3. The acrophases of B, total T lymphocytes, and the different T cell subsets were placed between ZT 3.5 and 6.2 h. This was similar to the ZT of the total lymphocyte count, which corresponds to the first half of the resting period. On the other hand, the NK cell count showed a 24-h period rhythm with maximal values at the end of the active phase, i.e., about 7 h before the acrophase of the other blood lymphocyte subpopulations.

                              
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Table 3.   Circadian rhythm parameters (and fiduciary limits) calculated from absolute number of blood circulating lymphocyte subpopulations



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Fig. 3.   Sinusoidal function obtained by Cosinor analysis of absolute number of lymphocyte subsets (P < 0.05 in all cases). For each ZT group, mean and SE are shown. Graphs have been duplicated on the x axis to facilitate rhythm visualization. Light/dark schedule is indicated.

Expression of surface molecules on lymphocytes. The circadian rhythmicity found in the expression of 11 glycoproteins expressed on the lymphocyte surface is summarized in Table 4 and Fig. 4. The sinusoidal function of the expression of those molecules that showed significant rhythmicity can be visualized in Fig. 5.

                              
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Table 4.   Circadian rhythm parameters (and fiduciary limits) calculated from the expression of lymphocyte surface molecules (MFI)



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Fig. 4.   Acrophases corresponding to the expression of lymphocyte surface molecules from Wistar rats. The points with fiduciary limits indicate a significant circadian rhythm, whereas those points without fiduciary limits represent the best acrophase estimation. Molecules with related acrophases have been grouped in pointed squares.



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Fig. 5.   Sinusoidal function obtained by Cosinor analysis of the expression of some surface molecules (P < 0.05 in all cases). For each ZT group, mean and SE are shown. Graphs have been duplicated on the x axis to facilitate rhythm visualization. Light/dark schedule is indicated. MFI, mean fluorescence intensity.

The CD45 molecule, expressed on every lymphocyte, exhibited a circadian rhythm with a variation of almost 8% and an acrophase at the end of the activity period. Considering the B cell population, no significant 24-h rhythm was detected in immunoglobulin, RT-1B, or CD45RABC surface expression. Concerning T cells, the expression of alpha beta or gamma delta T cell receptors did not show a significant circadian rhythm. In contrast, CD5 molecule expression in TCRalpha beta + cells displayed a 24-h rhythm with a daily variation of about 8% and the acrophase at the end of the active period.

CD3 is a molecule that is expressed on every T cell. A high CD3 daily variation expression (of about 20%) was detected. This variation showed a circadian rhythm in all T cells, with maximal expression at the end of the resting period. In regard to CD3 expression only on Th cells, a significant rhythm was also detected with an acrophase close to that of all T cells. Th cells also showed a variation of about 17% in CD4 expression, and there was a 24-h period rhythmicity with the acrophase at the beginning of the active phase.

Neither the CD8 surface molecule present on Ts/c cells, TCRgamma delta + cells, and NK cells nor the NKR-P1 receptor, present in the NK cell surface, presented significant circadian variations.

Concerning T cell surface molecules, the closeness between the acrophases for CD45 and CD5 molecules and between the acrophases for CD3 and CD4 molecules can be highlighted. CD45 acrophase, at ZT 22.8 (19.7-1.8), did not differ statistically from that of CD5, found at ZT 21.9 (17.4-2.3). That is, both acrophases were placed at the end of the active phase. On the other hand, CD3 acrophase was found at ZT 10.4 (8.6-12.2), whereas that of CD4 was at ZT 13.2 (10.2-16.3), and there were not statistical differences between them. Significant differences were detected between acrophases of CD45-CD5 and those of CD3-CD4 molecules. The study carried out over the course of the day showed that they were sited in antiphase positions (ZT of about 22 h vs. ZT of about 11-12 h).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The main finding in this study is the presence of circadian rhythms in the expression of certain lymphocyte surface molecules in healthy male Wistar rats. Moreover, circadian rhythms in the number and proportion of blood circulating erythrocytes, leukocytes, and lymphocyte subsets are confirmed.

Circadian rhythms in the number of blood cells have been previously reported (revised in Refs. 15 and 16). In the present study, we confirmed that the number of red and white cells in the peripheral blood of Wistar rats followed a circadian rhythm with maximal values during the resting period. The acrophase of the leukocyte number occurred around the beginning of this period, as already described for Lewis and Sprague-Dawley rats (12, 14) and similar to what has been reported previously in mice (25). Daily changes in blood leukocyte counts have been attributed to a rhythmic cell distribution between the circulating and the marginal compartments, between blood and other tissues, and to a rhythmic influx of new cells (36). Although all leukocyte populations contribute to the circadian rhythm, our results showed that lymphocytes were the main contributor to that oscillation.

The circadian rhythm in blood lymphocytes presented the acrophase at the beginning of the resting period, and, thus, minimal values were found at the beginning of the activity period. This distribution may reflect the immune system physiology. Because antigen entry probably occurs during the active phase, the immune system places its cells in strategic locations of the body, like the spleen and the lymph nodes, where the environment makes more effective the antigen-cell interaction. Therefore, lymphocytes migrate from blood to lymphoid tissues when the entering of antigen is most likely to occur. Accordingly, during the resting period, the number of cells in rat submaxillary lymph nodes has minimal values (13).

In addition to the study of rhythmicities in red and white blood cell counts, we investigated the presence of circadian rhythms in lymphocyte population cell number and, more importantly, in the expression of their surface molecules. The blood counts of the major and minor lymphocyte subsets followed a 24-h circadian variation. Acrophases for B, Talpha beta , and Tgamma delta lymphocytes and for CD4 and CD8 T cell subsets were placed in the first half of the rat resting period, whereas the peak for blood NK cells was detected during the second half of the active phase. These results are in accord with those of McNulty et al. (29), who reported an increase in B and Ts/c lymphocytes during the rat early resting period. The results are also in accord with those of Griffin and Whitacre (14) and Deprés-Brummer et al. (12), who found minimal numbers of rat blood T CD4+ cells and CD8+ cells during the active phase. The maximal number of NK cells detected here in the rat active phase is in accord with results described in humans (7), where the circulating NK count peaks in the beginning of the active phase and is low at rest phase.

In addition to identifying lymphocyte phenotype, the use of fluorescein-conjugated MAbs and flow cytometry allows the experimenter, under certain conditions, to establish proportionality between fluorescence intensity and the density of surface molecules bound to the monoclonal antibodies (3, 34). Although calibration beads can be applied to establish linearity in fluorescence and to enable the transformation of arbitrary units of mean fluorescence intensity into absolute units, it is also plausible to use the mean fluorescence intensity as a measure of the quantity of antibody molecules bound to cell membranes, which is proportional to the number of receptors on cell surface (34). To use mean fluorescence intensity as a direct measure of the number of surface molecules, some conditions must be taken into account. These conditions include, first, the use of the same batch of FITC-MAb (because conjugation can vary from time to time). Second, the antibodies used must be in saturating concentrations (to bind all possible sites on the cell). Finally, cells must be analyzed in the same conditions of cytometer voltage (to avoid variations due to cytometer setup). All these conditions were met in the present work: saturating concentrations of antibodies were established previously, and the same batch (and even the same dilution) of each antibody was used throughout the 24-h study. Moreover, in the cytometer, each sample was analyzed considering the same gate and for each molecule studied. Finally, all samples collected throughout the 24-h period were analyzed in a correlative matter to assure the same conditions of voltage and to eliminate other possible variations due to the cytometer.

We analyzed the daily variability in the expression of the lymphocyte surface molecules involved in the first steps of the immune response, i.e., antigen recognition and cell activation. In response to foreign antigens, T helper cell activation is triggered by the specific interaction of the T cell receptor (TCRalpha beta ) with a specific antigen bound to self-major histocompatibility complex (MHC) molecules on the surface of a presenting cell (43). The TCR-antigen interaction does not suffice to activate T cells; the complete activation requires the participation of adhesion and costimulatory molecules from both the T cell and the antigen-presenting cell. These interactions lead to the establishment of an immunologic synapse (21). Among others, the CD3 complex transmits activation signals to the cell when the TCR binds antigen (42), and CD4 and CD8 surface molecules, in separate subsets of blood T cells, act as coreceptors to enhance the TCR-CD3 signaling. CD45 or leukocyte common antigen (LCA) is a protein phosphatase with a critical role in signal transduction and T cell activation (20). CD5 on T cells is also considered an accessory molecule for T cell activation. Crosslinking of T cell CD5 with its ligand CD72 on antigen-presenting B cells may enhance T cell activation (26). On the other hand, lymphocyte activation produces changes in the expression of surface molecules, clustering some of them or producing the down- or upregulation of others (21). The role of surface molecules is such that the absence of costimulatory signals produced a failure in the activation of T cells (19). Moreover, a recent report shows that several T cell responses are directly related to the number of engaged receptors (37).

The study of CD45, CD5, CD3, and CD4 molecules on T cells revealed that their surface expression followed a circadian rhythm presenting a variation from 8 to 21% throughout the day. According to their acrophases, two groups can be defined: one for CD3 and CD4 and another for CD45 and CD5. Acrophases of CD3 and CD4 were found maximally at the beginning of the active phase, near the moment at which the number of Th lymphocytes in peripheral blood was minimal. On the other hand, acrophases of CD5 and CD45 molecules coincided at the end of the active period, 4 h before the maximal blood circulating lymphocyte count. Although the association between these maximal expressions was merely statistical, a physiological significance could be postulated. CD3 and CD4 acrophases may reflect preparation for the encounter with antigen in lymphoid tissues during the active phase, whereas CD5 and CD45, which do not interact directly with antigen but behave as transducting signals essential to cell activation, increase their expression after antigen interaction. This supports the hypothesis that although the immune activities involved in the initial encounter with antigen should peak while an animal is active, the resolution stages of such responses tend to peak during the resting period because immune responses are energetically expensive (33).

The study of CD8 molecule expression in Ts/c cells did not show a significant 24-h rhythm but showed the best estimation of the acrophase at the same time as CD4 molecule for Th cells. This also suggests the preparation of Ts/c cells for the encounter of the antigen and could indicate that the expression of these coreceptors was similarly regulated in Th and Ts/c cells in terms of time.

Antigen recognition is also needed to activate B cells. In this case, the antigen is bound to a specific immunoglobulin receptor on B cells. The signal transduction events in B cells that result from antigen interaction are quite similar to those of T cells. CD45 and its B-restricted isoform CD45RABC transduct signals to the cytoplasmic compartment (43). RT-1B molecule is the MHC class II molecule in rat that is essential whenever B cells behave as antigen-presenting cells. No circadian rhythm was found in the expression of either immunoglobulins, CD45RABC, or RT-1B on B cells. The best estimation of their acrophases appeared between the second half of the resting period and the beginning of the active phase. During this period, B cells may prepare for antigen contact during the active phase.

Finally, we studied NKR-P1 and CD8 molecules on NK cells, which participate in innate immunity. The biological functions of NKR-P1, a glycoprotein of the C-type lectin superfamily, are unknown, although a role in target cell recognition and killing has been suggested (24). No significant circadian rhythm was found in either NKR-P1 or CD8 expression on NK cells, although the best estimation of their acrophases was sited in both cases at the end of the resting period. These peaks coincided with minimal numbers of NK cells in blood (12 h later than acrophase of NK cell counts). NK cells may prepare their receptors for an effective encounter with an unspecific antigen that, in this case, would take place in a fast manner and during the active phase, because NK cells contribute to innate immunity and do not require previous sensitization.

In summary, we detected circadian rhythmicity in the expression of certain lymphocyte surface molecules essential in triggering immune responses. The temporal relationship between the expression of some molecules has been demonstrated, and the physiological role for this temporal organization is discussed. These results may shed some light on the circadian rhythmicity of certain lymphocyte functions.


    ACKNOWLEDGEMENTS

We thank Dr. Antoni Díez-Noguera for providing the software to perform the Cosinor analysis.


    FOOTNOTES

M.R. is a fellowship holder from the University of Barcelona. This study was supported by the Generalitat de Catalunya (1998SGR-033).

Address for reprint requests and other correspondence: C. Pelegrí. Departament de Fisiologia-Divisió IV, Facultat de Farmàcia, Universitat de Barcelona, Av. Joan XXIII s/n, E-08028 Barcelona, Spain (E-mail: cpelegri{at}farmacia.far.ub.es).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published September 11, 2002;10.1152/ajpcell.00084.2002

Received 25 February 2002; accepted in final form 4 September 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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