Rhythmic variations of different hemopoietic cell lines and maturation stages in aging mice

Mechanisms o f Ageing and Development, 42 (1988)91--104

91

ElsevierScientificPublishersIrelandLtd.

RIIYTHMIC VARIATIONS OF DIFFERENT HEMOPOIETIC CELL LINES AND MATURATION STAGES IN AGING MICE

OLAV SLETVOLI~, OLE DIDRIK LAERUM" and TROND RIISE b •The Gade Institute, Department of Pathology, University of Bergen, N-5021 Haukeland Hospital and bSectionfor Medical lt~formatics and Statistics, University of Bergen (Norway)

(ReceivedJune30th, 1987) (RevisionreceivedSeptember4th, 1987) SUMMARY Non-proliferative and proliferative myeloid, and lymphoid and erythroid bone marrow cells were studied in aging female C3H mice. A chronobiological approach was used and mice aged 16, 21 and 26 months were examined vs. 3 month-old mice every 3 h during the 24-h period in three different experiments. Significant circadian fluctuations were observed in most of the cell populations, even in the oldest mice. The rhythmicity patterns might be different at different times of the year, and in young mice seasonal fluctuations in the 24-h mean values were observed. The absolute numbers of the 24-h means seemed to be highest at 21 months of age in all cell lines and maturation stages. Sinus function fitting indicated a decline of the amplitudes in aging mice. Minor age-related phase-differences were indicated in some populations. However, the fitting of original data to single sinus functions was variable and often obscured important features in the cell number variations. The present investigation illustrates the importance of performing time-sequence studies in hematology.

K e y words: Aging; Hemopoiesis; Cell lines; Maturation stages; Circadian rhythms;

Seasonal differences; Sinus function fitting INTRODUCTION Reports on quantitative alterations of hemopoiesis in aging mice have mainly shown inconsistent results [1--5]. However, in recent studies [6--8] we concluded that neglect of the strong rhythmic variability in hemopoiesis (see reviews by Haus et al. [9], Laerum and Aardal [10]) may lead to conflicting results. When applying a chronobiological approach, we observed an age-related shift of the proliferation kinetics in the murine bone marrow, indicating a decline of the hemopoietic reserve 0047-6374/88/$03.50 Printedand Publishedin Ireland

© 1988ElsevierScientificPublishersIrelandLtd

92 capacity [6]. In addition, we found age-related increased numbers of blood granulocytes showing strong circadian variations even at advanced age of the mice

[7]. As an extension of previous studies on how aging affects white blood cell physiology in mice [6--8], the present investigation deals with the cell numbers in different cell lines and morphological maturation stages in the bone marrow. Thus, animals of different ages were studied at different times of the 24-h period and also at different times of the year. In addition, the data have been fitted to single sinus functions, a commonly used method when describing rhythms in biology. MATERIALSAND METHODS

Experimental conditions Female (virgins) C3H mice (SPF quality, Gamle Bomholdtgaard, Denmark) were purchased 12 months old, and animals from this stock supplied " o l d " mice throughout the whole study. Young female control mice of the same strain were purchased 2 months old from the same colony and were synchronized to the same laboratory conditions as the older mice for at least 3--4 weeks before each experiment. Pathogen testing before the onset of the experiments showed that the mice were free of Bacillus piliformis, Pasteurella multocida, ectoparasites, tape worm, Reo 3 virus, PMV, LCM, but contaminated with MHV and Pasteurella pneumotropica. Post-recovery titers against Sendai virus were also observed. Fenbendazole was given against Syphacia ob Velata. The original number of aging mice was limited. Therefore, the maximal life-span was not examined in our colony. A minor number of deaths was observed before 24 months of age, while 26 months represented the steeply declining part of the survival curve. These findings were in line with previous reports. Five mice were kept in each cage on a 12 : 12 h artificial light-dark regimen with lights on at 0800 h (Mean European Time = MET). In the dark period no use of light was allowed in the animal room. In the animal room there was constant temperature (22°C _ 0.5) and humidity (54070 _ 1). The mice had free access to food (Altromin, Ringsted, Denmark) and water. Mice aged 16, 21 and 26 months were examined vs. 3-month-old controls every 3 h over a 24-h period in February, June and November, respectively. At each timepoint four animals were killed by exsanguination after carbon dioxide narcosis and the femurs were removed and cleaned under sterile conditions. One femur was flushed with 5 ml 0.907o NaCl - - 2.5 ml from each end of the bone. The concentration of nucleated bone marrow cells was measured on a Coulter Counter following red cell lysis. Each mouse was carefully dissected. In addition, we examined both the total and the differential leukocyte counts in peripheral blood. Different bone marrow stem cells were measured, as well. Flow cytometric DNA analysis of bone marrow, thymus and spleen was also performed. Any pathology led to elimination from the study.

93

Differential counts The bone marrow cells were washed once in saline and therafter centrifuged at 400 g. After removal of the supernatant one drop of inactivated horse serum was added, and the cells were thoroughly resuspended Two smears were made from each mouse. The smears were rapidly air-dried and fixed in methanol after at least 6 h. Following May-Grfinwald Giemsa staining, 300 nucleated bone marrow cells were counted per slide on coded specimens. Differential counts were made for nonproliferative myeloid cells (metamyelocytes and granulocytes) and proliferative myeloid cells (myelocytes and earlier stages), erythroid cells and lymphocytes. The discrimination of cells with ring-formed or clover-leaf nuclei as proliferative or not was done according to the results of a pilot study with differential counting of autoradiographic smears of bone marrow ceils after a short-time incubation with tritiated thymidine as described by Lundmark [11]. The differential counts were used for calculation of the total number of cells within each compartment by multiplying with the total bone marrow cellularity.

Statistical analysis Student's t-test was used to evaluate the original data. In addition, the data were analyzed by use of the non-linear regression program BMDP3R described by Dixon and Brown [12]. The estimation of the parameters was done by an iterative procedure. This consists of a non-linear fitting to single sinus functions, which is complementary to the single cosinor analysis described by Halberg et al. [13]. RESULTS Each cell line and maturation stage will be described separately and circadian rhythmicity and phasing will be related to season and age. The 24-h mean values in aging mice will be expressed as percent of means in young mice, while the 24-h mean values in young mice will be related to season, as a control. In addition, the calculated data from the sinus function fitting will be presented. The amplitudes in the different groups of aging mice will be expressed as percent of the respective mesor (mesor = time-adjusted mean) and as percent of the amplitudes in young mice.

Non-proliferative myeloid bone marrow cells Circadian rhythmicity andphasing (Fig. 1): In young mice there were significant peak-trough differences in all three experiments (P < 0.05). In addition, young mice had maximum values at different times of the 24-h period, indicating a seasonal pattern of the peak phasing. Significant circadian variations were found in mice aged 16 and 26 months ( P < 0.05), as well, but not in 21-month-old mice (P > 0.05). However, aging mice seemed to have maximum values at least 3 h later than young mice in every experiment. The peak values in young mice mainly coincided with the trough values in the aging mice.

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95 The differences in circadian pattern and phasing had important implications. When comparing 16-month-old vs. young mice, the oldest mice had a significantly higher cell number than young mice exclusively at 1000 h. In the rest of the 24-h period there were insignificant peak-trough difference (P > 0.05). When comparing 21-month-old vs. young mice, the older mice had significantly higher values at 1300 h and 2200 h, and at 0700 h in the end of the experiment. In most of the remaining 24-h period the 21-month-old mice had insignficantly higher values. Twenty-sixmonth-old mice had insignificantly higher values than young mice at six different time-points. However, at 0400 h the young mice had a significantly higher value! Thus, the conclusions were highly dependent on the time of the day. 24-H mean: While the differences were insignificant when comparing mice examined in June and February (P > 0.05), the June value was significantly lower than the November value (P < 0.05), indicating seasonal differences. Both 16- and 26-month-old mice had insignficantly higher values than young mice (P > 0.05), while the 21-month-old mice had significantly higher numbers than young mice (P < 0.05), indicating age-related differences (Fig. 6). Proliferative myeloid bone marrow cells Circadian rhythmicity and phasing (Fig. 2): In young mice there were significant peak-trough differences in June and November (P < 0.05), but not in February (P > 0.05). There were also seasonal differences in the peak phasing. The peak-trough differences in aging mice were insignificant (P > 0.05). When comparing young and old mice at single time-points, there was insignificant difference between the 16-month-old mice and young controls. Twenty-one-month-old mice had values consistently above the curve for young mice with significant differences at 2200 h and between 0400 h and 0700 h in the end of the experiment (P < 0.05). The overlapping between the 26-month-old and the young mice was variable, but young mice had a significantly higher cell number at 0400 h (P< 0.05). 24-H mean: Young mice examined in June had significantly lower cell numbers than in November (P < 0.05), while the difference between June and February was insignficant (P > 0.05), indicating seasonal variations (Fig. 5). Both 16: and 21month-old mice had cell numbers significantly higher than young mice (P < 0.05, P < 0.01) (Fig. 6). There was no significant difference between 26-month-old and young mice (P > 0.05), even though the old mice had the highest value observed in all the three experiments. These results indicate a maximal number of myeloid proliferative cells in 21-month-old mice and a levelling off by 26 months. L ymphocytes Circadian rhythmicity and phasing (Fig. 3): Young mice had prominent circadian variations with significant peak-trough differences in June and November (P < 0.05,

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P < 0.01), but not in February (P > 0.05). In different months the maximum values were found at different times of the day, indicating seasonal differences of the peak phasing. There were insignificant fluctuations in aging mice (P > 0.05). Sixteen-month-old mice had significantly lower lymphocyte numbers than young mice at 1000 h and 2200 h, while the differences were insignificantly different during the rest of the 24-h period. There were insignificant differences between 21-month old and young mice at any single time-point. Twenty-six-month-old mice had a significantly higher value than young mice at 0700 h, while at 0400 h and 1600 h the young mice had values considerably but not significantly above those in 26-monthold mice. Twenty-four mean: There were insignificant differences between the lymphocyte numbers in young mice examined at different times of the year (P > 0.05) (Fig. 5). Sixteeen-month-old mice had significantly lower numbers than young mice (P < 0.05), while the differences between young mice and 21- and 26-month-old mice were insignificant (P > 0.05) (Fig. 6).

Erythroid bone marrow ceils Circadian rhythmicity and phasing (Fig. 4): Young mice examined in November had significant peak-trough difference (P < 0.05), but not those examined in February or June or any of the aging groups. Twenty-four mean: The number of erythroid cells in young mice examined in February was insignificantly higher than in November (P > 0.05), but significantly higher than in June (P < 0.01), indicating seasonal variations (Fig. 5). Only 21month-old mice had significantly higher values than young mice (P < 0.05), indicating a maximal cell number at this age (Fig. 6). Sin us function fitting Non-proliferative myeloid cells: Although sinusoidal rhythms were indicated for some of the groups (Table I), there was no age-related differences when the amplitudes were expressed as percent of the mesor value in each group (Fig. 7), while the amplitudes in aging mice as percent of amplitudes in young mice were maximal at 16 months and thereafter declined (Fig. 8). However, important details in the original data, especially age-related phase-relationships were obscured by sinus function fitting. Proliferative myeloid cells: Sinusoidal rhythms were indicated in most of the groups (Table I). The amplitudes in aging mice expressed as percent of amplitudes in young mice were lowest in 21- and 26-month-old mice indicating an age-related decline of the amplitude (Fig. 8). The changes in the amplitudes of aging mice expressed as percent of the respective mesor (Fig. 7) were inconsistent. Although the fit might be statistically significant (Table I), important features in the original data were hidden. Lymphoid cells: The fitting varied considerably (Table I). The amplitudes

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Fig. 5. The 24-h m e a n numbers _+ S.E.M. o f different hemopoietic cell lines and maturation stages in young mice examined at three different times of the year. The significance of seasonal difference is indicated. Fig. 6. The 24-h m e a n o f different hemopoietic cell lines and maturation stages in different groups of aging mice expressed as percent of m e a n in young mice. The significance of the differences between young and old mice is indicated.

TABLE I T H E SIGNIFICANCE OF F I T T I N G T H E C I R C A D I A N V A R I A T I O N S OF C E L L N U M B E R S IN D I F F E R E N T BONE M A R R O W C E L L LINES A N D M A T U R A T I O N STAGES TO SINGLE SINUS FUNCTIONS Mice of different ages were examined in three different experiments.

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Fig. 7. The amplitudes from the sinus function fitting of cell numbers in different cell lines and maturation stages expressed as percent of mesor values in different groups of aging mice. Fig. 8. The amplitudes from the sinus function fitting of cell numbers in different cell lines and maturation stages in aging mice expressed as percent of amplitudes in young mice.

expressed as percent of mesors were unchanged during aging (Fig. 7), while the amplitude expressed as percent of amplitude in young mice ratio was highest in 16month-old mice (Fig. 8). Erythroid cells: Considerable and significant sinusoidal changes were found in a single experiment (Table I, Fig. 7), while there were minor age-related changes of the amplitudes (Fig. 8). DISCUSSION

The present study showed that the number of cells in the different cell lines and maturation stages in the mouse bone marrow varied significantly during the 24-h period. Consistent co-variations of different cell lines were not seen. This variability in hemopoiesis was in accordance with previous reports (see reviews by Haus et ai. [9], Laerum and Aardal [10]). Even at high ages of the mice there were significant fluctuations in some of the cell lines. However, the extent of the circadian variations sometimes seemed to decline with increasing age, and the phasing of the rhythms could be slightly different when comparing young and old mice. These age-related effects on the 24-h periodicity were consistent with recent reports on blood cell physiology in aging mice [6--8], However, we cannot exclude that ultradian

102

fluctuations might be hidden in the present curves. Thus, Ryabykh et al. [14] have reported periods of few hours in rhythms of the different cell populations in the bone marrow. The concept of seasonal rhythms in mice may be debated. Thus, one population of 3-month-old mice experiences not more than one season. In addition, such studies are complicated by the fact that the mean life span of the C3H strain only comprises 7m8 seasons. Therefore, seasonal variations may represent seasonal differences rather than defined seasonal rhythms. In addition, any experiment on mice which considers age-related changes is challenged by potential seasonal variations. When studying age-related changes in mice from the same original stock, experiments at different times of the year are required. Therefore, the results may be influenced by seasonal differences. However, the problem is not solved by analyzing all the different age groups at one specific time of the year. Thus, it is not known whether specific age-related differences may be revealed at one season, but not at another. Therefore, it seems just as relevant to examine different groups of aging mice during 24-h periods at different times of the year as to examine the different age-groups at only one specific time-point of the day, which is most commonly done. In the present study we also estimated the 24-h mean values of absolute numbers in each compartment. Thus, we found an age-related increase of both nonproliferative and proliferative myeloid cells in the oldest age groups. These results were in line with Boggs et al. [15] who described an age-related increased number of peroxidase-positive cells in the bone marrow of mice. In addition, the findings were consistent with our recent report on the proliferative activity in the bone marrow measured by flow cytometry [7]. Thus, we observed that the total number of cells in the different phases, but especially the G 1 + G o phase cells increased with aging. The fraction of S- and G 2 phase cells decreased, indicating a decline of the total proliferative reserve capacity. However, when estimating the total bone marrow proliferative activity, several different cell lines are included, as well. Due to such differences and widely differing methods, the results are not fully comparable. Our results showing an increase of the granulocyte pool may also be due to a compensation phenomenon to maintain a higher blood granulocyte number in steady state, alternatively an inefficient myelopoiesis. Thus, Boggs et al. [15] found that although the absolute increase of granulocyte numbers in peripheral blood after endotoxin stimulation was equal in young and old mice, the relative increase seemed to decline in old mice, due to higher steady state numbers. In line with Kay et al. [2] the age-related changes in the absolute lymphocyte numbers were inconsistent, although the relative numbers seemed to decline (data not shown). However, there are reports on age-related shifts between different B cells as well as between helper/suppressor T cell effects in the bone marrow [16,17|. In the present study the maximal number of erythroid cells were found in 21-

103 month-old mice, while there were no differences between control mice and the oldest age-group. Thus, our results were somewhat different when compared with recent reports indicating an age-related reduction of the erythropoictic capacity, affecting maturing erythroid precursors [18,19]. However, the methods were not comparable and the data were expressed differently. Generally, the bone marrow differential count and related quantitative estimates have been focused insufficiently when examining the hemopoiesis in aging mice. This neglect may be related to the fact that microscopic examination of bone marrow smears is a rather tedious and time-consuming procedure with a high degree of variance, even when counting large numbers of cells, In mice there are various "uncommon" myelomonocytic forms, as well [20], which contribute to an additional uncertainty. However, we tried to solve this problem by classifying myeloid cells according to proliferative capacity, relating morphology to the ability of myeloid cells to incorporate thymidin into DNA (proliferative/non-proliferative myeloid cells). The method of fitting original data in biology to sinus or related functions is widely used [12]. However, it is not reasonable to expect any biological variation strictly to follow such functions. Although the fit may be statistically significant, important features in the original data may be hidden. Thus, in the present study visual evaluation combined with Student's t-test seemed to be just as appropriate as sinus function fitting. However, the amplitudes in aging mice expressed as percent of ~hnplitudes in young mice were declining with increasing age, indicating a reduced non-random variability with aging, although the significance was variable. In conclusion, significant fluctuations were found within the various cell lines and maturation stages in the murine bone marrow during the 24-h period. The pattern of the circadian variations might be different at different times of the year and seasonal fluctuations in the 24-h mean values were observed in young mice. There was an age-related decline of the amplitudes and sometimes the rhythmicity patterns were different at different ages. The absolute numbers of the 24-h means in all the cell lines and maturation stages seemed to be maximal at 21 months of age. While the relative numbers of myeloid cells increased, that of lymphoid cells declined with increasing age. The relative numbers of erythroid cells were unchanged. The present study illustrates the importance of performing experiments at various times of the day. Fitting of hematological data to single sinus functions may obscure important features of the variations. ACKNOWLEDGMENTS This work was supported by The Norwegian Research Council for Science and Humanities and the Norwegian Cancer Society. We thank Ms. Astri Helgesen, Ms. Gro Olderoty and Ms. Dagny Ann Sandnes for expert technical assistance.

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