Food restriction in female Wistar rats. VII. Mitochondrial parameters in resting and proliferating splenic lymphocytes

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Archives of Gerontology and Geriatrics 19 (1994) 31-42

ARCHIVES O F GERONTOLOGY AND G E R I A T R I C S

Food restriction in female Wistar rats. VII. Mitochondrial parameters in resting and proliferating splenic lymphocytes Carlo Pieri*, Rina Recchioni, Fausto Moroni, Fiorella Marcheselli, Maurizio Marra Cytology Center, Gerontological Research Department of LN.R.C.A., Via Birarelli, 8, 60121 Ancona, Italy Received 28 February 1994; revision received 29 April 1994; accepted 2 May 1994 Abstract

The effect of food restriction on the mitochondria of resting and proliferating rat splenocytes was examined, measuring the membrane potential and mass of these organelles, by means of the specific fluorescent probes Rhodamine-123 and Nonyl Acridine Orange, respectively. Food restriction was applied on an every-other-day schedule (EOD) starting at the age of 3.5 months. The ad libitum fed (AL) animals were killed when they were 4, 11 and 24 months old, whereas the EOD rats were killed at 11 and 26 months. Resting lymphocytes from AL rats showed an age-dependent increase of both membrane potential and mass of their mitoehondria. However, the mitochondrial mass increased to a larger extent when compared with the membrane potential resulting in a decrease of the respiratory quotient (RQ), i.e. of the respiratory activity per unit of mitochondrial mass. In EOD animals, the mitochondrial membrane potential was lower and the mitochondrial mass was higher than in the corresponding age-matched controls, resulting in a further decrease of RQ. Following mitogenic stimulation, most of the cells from young and adult AL rat showed an increase of membrane potential and mass of their mitochondria. In contrast about 50% of cells from old AL rats had depolarized organelles after 72 h from the stimulation. Food restriction was able to prevent these alterations allowing the majority of cells, including those from old animals, to maintain the hyperpolarization of their mitochondria during the 3-day culture. In light of the well known sensitivity of mitochondrial membrane potential to peroxidative stress, present data suggest that the increase of respiration occurring during mitogenesis may increase free radical production, which is better tolerated by cells from EOD animals than by those from AL animals. Keywords: Food restriction; Lymphocytes; Mitochondria * Corresponding author. 0167-4943/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0167-4943(94)00556-M

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1. Introduction

It has long been recognized that the responsiveness of lymphocytes to mitogenic stimulation is impaired during aging (Pisciotta et al., 1967; Hallgreen et al., 1973; Weksler and Hutteroth, 1974; Gershon et al., 1979). Various methods have been developed to experimentally modify this decline. Vitamin E, reduced glutathione and oligoelements supplementation (Corwin and Shloss, 1980; Meydani et al., 1986; Fabris et al., 1990; Furukawa et al., 1990) as well as feeding the animals with a calorie-restricted diet (Gerbase De-Lima, 1975; Weindruch et al., 1979; Fernandes et al., 1990; Pieri et al., 1990a) were all able to prevent the loss of immune function in old animals. During the last few years, several attempts were made to explain the mechanism through which diet restriction preserves the lymphocyte response and some interesting information was obtained. Diet restriction augments Ca 2+ level, IL-2 synthesis and high affinity IL-2 receptor expression in response to mitogens (Fernandes et al., 1990). In addition, diet restriction increases membrane fluidity of splenocytes (Fernandes et al., 1990; Pieri et al., 1990a), probably because it modifies their fatty acid composition (Fernandes et al., 1990). Although these parameters may account for the improvement of proliferative response by diet restriction, other parameters may equally play a role in regulating such a response. It has been proposed that the inhibition of thymidine incorporation into aged splenocytes may in part be coupled to a defect in responsiveness of mitochondria and/or mitochondrial ATP production (Verity et al., 1983). In a previous work from our laboratory (Pieri et al., 1993a) we demonstrated that the mitochondrial functionality of proliferating rat splenocytes was impaired during aging. By means of flow cytometry, we measured the membrane potential and mass of these organdies, using the specific fluorescent probes Rhodamine-123 (Rh-123) and Nonyl-Acridine Orange (NAO), respectively (Johnson et al., 1980; Retinaud et al., 1988). The main results were that old resting cells had higher mitochondrial mass and lower membrane potential when compared with the young ones. Upon mitogenic stimulation, most of the lymphocytes from young rats increased their membrane potential and mass. In contrast, about half of the cells from old animals showed depolarized organelles after 72 h stimulation (Pieri et al., 1993a). This impairment at mitochondrial level may contribute to reducing the proliferative response of rat splenocytes during aging. In the present paper we extend our analysis to the lymphocytes from adult and old food-restricted animals. It is well known that dietary restriction prolongs the lifespan of the experimental animals and exerts numerous beneficial effects on some age-dependent alterations at cellular level (for review see Yu, 1990; Masoro et al., 1991). It is relevant to this present work that both mitochondrial structure and function were better preserved in some organs of food-restricted animals than in animals fed ad libitum (Weindruch et al., 1980; Rumsey et al., 1987; Iwasaki et al., 1988; Laganiere and Yu, 1989a). However, there are no data concerning the lymphocyte mitochondria.

C. Pieri et aL /Arch. Gerontol. Geriatr. 19 (1994) 31-42

33

:2. Material and methods

2.1. Animals Female Wistar rats from our own breed were used. By the age of 3.5 months one group of animals was food restricted by feeding on an every-other-day schedule (EOD) with the same commercial chow given ad libitum (AL) to the other groups of young, adult and old rats. As reported in detail previously (Pieri et al., 1990a), food restriction applied in this way was able to prolong the mean, median and maximum lifespan of the animals. The EOD rats were killed at 11 and 26 months of age, after 24 h feeding. The young, adult and old AL groups were killed when they were 4, 11 and 24 months old. Each experimental group consisted of 5 animals. 2.2. Cell preparation and culture Splenic lymphocytes were prepared by Ficol-Hypaque gradient centrifugation (Boyum, 1968). After repeated washes in Hanks' solution, they were resuspended (2 x 106cells/ml) in RPMI-1640 supplemented with 2 mM glutamine, 10% fetal calf serum, 100 units/ml penicillin and 100/~g/ml streptomycin. The cells were stimulated with Con A (5/~g/ml) and incubated at 37°C and 5% CO2 atmosphere for various durations, before staining for mitochondrial parameters determination. Microcultures were performed on round bottomed plates at a final concentration of 3 x 105 cells in 0.2-ml volumes of complete medium. Cultures were stimulated with 5 t~g/ml Con A, incubated as previously described and pulsed with 2/~Ci/well [3Hlthymidine (6.7 Ci/mmol, Amersham, UK) for the last 6 h of a 72-h incubation period. Cell suspensions were collected on glass fiber filters (Labtek) by means of a cell harvester (Skatron, AS). Filters were dried and radioactivity was measured in a liquid scintillation counter (Packard-Tricarb). 2.3. Flow cytometric analysis The splenocytes were stained for mitochondrial membrane potential determination according to Darzynkiewicz et al. (1981) as previously described (Pieri et al., 1992a). At different time points the cells were stained by 25/zM Rh-123 (Molecular Probes) at room temperature for 20 min in the dark, washed twice with phosphate buffered saline (pH 7.4) and suspended in RPMI-1640. The same procedure was followed for the staining with 5 /zM NAO (Polyscience) for 15 min according to Retinaud et al. (1988). Before analysis, 10/zg/ml ethidium bromide (EB) was added to each sample to monitor the dead cells, which were excluded from the analysis of Rh-123 and NAO fluorescence by gating for red fluorescence. Measurements of the fluorescence, on a cell-by-cell basis, were carried out in a Coulter Epics V flow cytometer (Coulter, Hialeah, FL). The argon ion laser was tuned to 488 nm. The green fluorescence of Rh-123- or NAO-stained cells was detected between 500 and 540 nm, whereas the red fluorescence emitted by EB, which penetrated only dead cells, was measured at wavelengths higher than 615 nm. Ten thousand events were collected from each sample and data were analysed by program packages provided

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C. Pieri et al./Arch. Gerontol. Geriatr. 19 (1994) 31-42

by the manufacturer. Values were assessed for significance by two-way (diet and age) analysis of variance (ANOVA) test, with significant differences identified by a P value <0.05. 3. Results

The age and time-dependent changes of the proliferative response of splenic lymphocytes are reported in Table 1. As was expected, the amount of labeled thymidine incorporated by the cells decreased with aging. Food restriction enhanced the proliferative activity of splenocytes from both adult and old animals. It has to be noted, however, that an age-dependent decrease of isotope incorporation was also present in cells from the EOD groups. Figs. 1 and 2 report examples of histograms showing the distribution of Rh-123 and NAO fluorescences, respectively, over the cell population from both old AL and EOD animals, obtained with unstimulated control and Con A-stimulated cells at 72 h. All the data reported in the present paper were calculated from the integral of probe fluorescence. To emphasize, however, the difference between the two experimental groups shown in the figures, the logarithm of the fluorescence was reported in the abscissa. Examining the control cultures, lower Rh-123 (Fig. 1) and higher NAO (Fig. 2) uptake were observed in cells from EOD old animals as compared with those from AL ones. This resulted in different values of mean fluorescence (if) of both probes, and these data are reported in Table 2. In agreement with our previous paper (Pieri et al., 1993a) an increase o f f f of both fluorochromes was observed during aging. However, NAO/Y increased to a higher extent than ff of Rh-123, resulting in a progressive age-dependent decrease of the respiratory quotient (RQ), i.e. the respiratory efficiency per unit of mitochondrial mass (Leprat et al., 1990). Considering the resting splenocytes from EOD rats, ff of Rh-123 was maintained at a low level, whereas that of NAO increased with the age of the animals and reached values higher than those found in old AL animals.

Table 1 Effect of age and food restriction on proliferative responses of spleen cells to Con A (5 #g/ml) Animals

[3H]Thymidine incorporation (counts/min × 10 -3)

Young A L Adult A L Old AL Adult E O D Old EOD

100.5 67.3 17.5 86.7 51.1

4- 8.4 -¢- 5.2 a -4- 3.0 a'b 4- 8.1 c 4- 6.4 a,c,d

Values are the means ± S.E.M. of 5 animals per each group. Statistically different vs: ayoung AL, badult AL, Cold AL and dadult EOD.

C. Pieri et al./Arch. Gerontol. Geriatr. 19 (1994) 31-42

35

Rh-123 400

300'

fl Z

200 -

100-

I

I

|

I

50

100

150

200

400 u

300

EOD

200

100

50

100

150

200

Log of fluorescence intensity (channel number) Fig. 1. Histograms of Rh-123 distribution of control (D) and Con A-stimulated (Q) splenic lymphocytes as measured at 72 h from the application of the mitogen (5 #.g/ml). The cells included in channels 0-75, 75-150 and > 150 were considered to have depolarized, normally polarized ad hyperpolarized mitochondria, respectively.

This resulted in a significant decrease of RQ in adult and old EOD rats, when compared with the age-matched controls. Mitogenic stimulation induced profound changes in the uptake of both probes, which were also reflected in/T value modifications, but they can be better appreciated considering the distribution of the fluorescence within the cell populations. Indeed, as also shown in the histograms of Fig. 1, three distinctly different fluorescent populations could be recognized after Rh-123 staining. The first population (channels 0-75) bound low amount of dye, representing cells with partially or totally depolarized mitochondria. The second cell population (channels 75-150) was likely

C Pieri et aL/Arch. Gerontol. Geriatr. 19 (1994) 31-42

36

NAO 400 -1

i i

AL

300 "

,,a

200 -

Z:

100-

50

100

150

200

400

m

EOD

300

200 Z

100

I

i

l

i

50

100

150

200

Log of fluorescence intensity (channel number) Fig. 2. Histograms of NAO fluorescence distribution of control (El) and Con A-stimulated (O) splenic lymphocytes as measured at 72 h from the application of the mitogen (5/~g/ml). The cells included in channels 0-100, 100-155 ad > 155 were considered to have low, normal and high mitochondrial mass, respectively.

Table 2 Age and diet-dependent changes of Rh-123 and NAO mean fluorescence as well as of RQ in resting splenocytes Animals

Rh- 123

Young AL Adult AL Old AL Adult EOD Old EOD

38.1 40.4 43.1 38.9 38.1

± .4.4.4.4-

1.6 1.4 1.2 a 1.1 c 1.3 c

NAO

RQ

38.2 ± 1.8 42.1 ± 1.2 47.0.4- 1.3 a,b 44.7 .4- 1.5 a 52.8 .4- 2.0 a'b'c'd

0.998 .4- 0.009 0.959 .4- 0.008 a 0.917.4- 0.011 a,b 0.870 .4- 0.010 a'b'c 0.722 4- 0.007 a,b,c,d

Values are the means ± S.E.M. of 5 animals per each group. Statistically different vs. ayoung AL, badult AL, Cold AL and dadult EOD.

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C Pieri et al./Arch. Gerontol. Geriatr. 19 (1994) 31-42

to include the lymphocytes which had normally polarized mitochondria and the third, highly fluorescent (channel > 150) population arose from hyperpolarized organelles. The same distribution of the fluorescence was observed when NAO fluorescence was taken into account (Fig. 2). However, in this case the first population was included between channels 0 and 100, the second between channels 100 and 155, and the third contained the cells with fluorescence higher than channel 155. As shown in Figs. 1 and 2 mitogenic stimulation resulted in an increase of the first and third populations at the expense of the second one, for both probes. Figs. 3 and 4 summarize the results, i.e. the changes in cell populations showing low, normal and high Rh-123 and NAO uptake, respectively. After 24 h with Con A stimulation about 50-55% of the cells from AL animals were able to increase their uptake of Rh-123. This percentage increased with culture time in young and adult rats, but it decreased by 48 and 72 h in old animals. In contrast, the number of cells showing depolarized mitochondria passed from 21% by 24 h to 45% and 48% at the other two time points in old AL animals. The number of cells with low Rh-123 uptake did not overcome 17 and 26% in young and adult AL rats, respectively, by 72 h. Food restriction deeply influenced the distribution into the three different cell populations. Indeed, it increased the number of splenocytes with high Rh-123 uptake, even in the adult animals, but, furthermore, it allowed an increasing number of cells from old animals to maintain the hyperpolarization of their mitochondria

Rh-123

uptake

100

high

norma I

low 8O

60_

40.

i

i

i

¢

24

48

72

24

!

48

i

l

i

72

24

48

72

hours

Fig. 3. Time-dependent changes of cell populations showing low, normal and high Rh-123 fluorescence during lymphocytes proliferation. The symbols represent cells from young (A), adult (O), and old AL ([:l) rats, and adult (0) and old (1) EOD rats.

38

C. Pieri et al./Arch. Gerontol. Geriatr. 19 (1994) 31-42 NAO

uptake

100.

normal

low

high

80.

o

60. 0 -

40.

20.

0

J

24

v

i

i

v

i

1

i

i

48

72

24

48

72

24

48

72

hours

Fig. 4. Time-dependent changes of cell populations showing low, normal ad high NAO fluorescence during lymphocytes proliferation. Symbols as in Fig. 3.

during the 3-day culture. It is of interest that in EOD animals the increase of the number of highly fluorescent cells occurred mainly at the expense of those with low Rh-123 uptake. The same age- and diet-dependent effect could be seen when NAO fluorescence distribution was taken into account (Fig. 4). However, it is worth noting that the number of cells showing loss of mitochondrial mass was lower than that showing mitochondrial depolarization, as is particularly evident in cells from old AL animals. 4. Discussion

The age-dependent changes of mitochondria parameters of splenocytes have already been investigated in our laboratory (Pieri et al., 1993a). In the present work, we repeated the analysis of the different age groups together with food-restricted animals, in order to avoid artifacts in fluorescence measurements (expecially F ) due to different settings of the flow cytometer. Thus, on the same day, one animal of each group was killed, the splenocytes were prepared, stained and analysed strictly under the same conditions. The time and the age-dependent patterns of mitochondrial membrane potential and mass of splenic lymphocytes were similar to the ones described in our previous paper (Pieri et al., 1993a). Resting cells from AL animals had a higher mitochondrial mass than in young ones; however, the RQ was lower in the former group than in the latter.

C. Pieri et al./Arch. Gerontol. Geriatr. 19 (1994) 31-42

39

These data are in agreement with those reported by Weindruch et al. (1980) showing a significant age-dependent decrease of the respiratory control index in mitochondria isolated from mouse spleens. Compared with the age-matched AL animals, cells from EOD rats showed a higher mitochondrial mass and a lower membrane potential resulting in a very low RQ which was already evident in the adult group (Table 2). The physiological significance of these differences between AL and EOD animals remains to be disclosed together with the possibility that the increase of NAO fluorescence seen during aging in both experimental models is due to an increase in the size and/or in the number of mitochondria per cell. Food restriction markedly influences the distribution of the cells selected on the basis of their Rh-123 fluorescence when they are stimulated to proliferate. The number of cells with depolarized mitochondria decreases and that with hyperpolarized organdies increases in EOD as compared with AL age-matched rats. These differences cannot be explained by a lower number of stimulated cells in samples from AL rats compared with those from EOD ones. The number of cells able to modify the membrane potential of their mitochondria in both directions, i.e. depolarization or hyperpolarization, was at least 85% in both models, irrespective of the age and the diet given to the animals. This means that 85% of the cells received the stimulus necessary to activate their mitochondria. The mechanism(s) regulating mitochondrial activation is not completely disclosed, although the activation does not seem to be connected with blast transformation (Pieri et al., 1992a). Moreover, the differences in the mitochondrial response between cells from AL and EOD animals cannot be explained by a different sensitivity to the culture conditions. Three-day culture of unstimulated cells resulted in only a 5-8% increase of the number of cells with low Rh-123 uptake (not shown). Thus, the depolarization of mitochondria seems to be closely related to events occuring during the proliferative process, and a likely candidate is the peroxidative stress. Support for this possibility stems from experiments on isolated mitochondria showing that the activation of lipid peroxidation, regardless of the nature of prooxidant, causes efflux of Ca 2+ and other cations from mitochondria, fall of transmembrane potential and swelling (Marshansky et al., 1983; Masini et al., 1985). It is well known that mitogenic stimulation causes free radicals production which seems to be involved in the activation of lymphocytes as positive mediators (Wrogemann et al., 1978; Chaudhri et al., 1986; Fidelus, 1988). However, their level must be kept low by the antioxidant defense system to avoid damage to the cells. We found marginal differences of Rh-123 and NAO uptake after 24 h of mitogenic stimulation (Fig. 3), thus the early production (within 2 h) (Whitacre and Cathcart, 1992) of free radicals in mitogen-stimulated cells can hardly account for the observed modifications occurring to a larger extent at 48 and 72 h. The respiratory activity of mitochondria increases during proliferation, increasing the risk of superoxide radical production (Nohl et al., 1978), especially in old cells (Nohl, 1986; Hansford, 1987). Thus, the observed time- and age-dependent damages of mitochondria, as well as the preservation of their functional integrity elicited by food restriction, may be linked either to the amount of free radical produced during

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the cycling of the cell or to the level of antioxidants which may differ in the two dietary models, or both. This hypothesis is also supported by some recent data from our laboratory. The addition of the antioxidant reduced glutathione (GSH) into the culture medium eliminated the differences in the proliferative response of lymphocytes from EOD and AL animals (Pieri et al., 1993b) suggesting that the antioxidant defense system plays a key role in determinating these differences. Moreover, GSH was able to improve the mitochondrial parameters of proliferating splenoeytes from young animals (Pieri et al., 1992a), supporting the view either that a peroxidative stress, although low, also occurred in stimulated young cells or that mitochondria are very sensitive to this stress. More recently, we confirmed the extremely high sensitivity of mitochondria to the peroxidative stress induced by the proliferative stimulation, analysing cells from vitamin E deficient animals. In the 11-month-old deficient rats 60% of the cells showed depolarization and 40% loss of mitochondrial mass after 72 h from the stimulation (Pieri et al., 1993c). This mitochondrial impairment due to the lack of the antioxidant vitamin E was fully prevented by the addition of another antioxidant, GSH, into the culture medium (Pieri et al., 1993d). In conclusion, data of the present work show that mitochondrial function of splenic lymphocytes is better preserved in old EOD animals than in the AL ones. This might be due to a better preservation of the antioxidant defense system in lymphocytes from food-restricted animals as it occurs in other types of cells (Koizumi et al., 1987; Laganiere and Yu, 1987, 1989b; Pieri et al., 1990b, 1992b). The improvement of mitochondrial function may play a role in the improvement of proliferative response elicited by the dietary intervention.

Acknowledgements The authors thank Mr G. Mazzarini for editing the text and Mrs M. Glebocki for reading the manuscript.

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