Viscosity of lymphocyte plasma membrane in aging mice and its possible relation to serum cholesterol

Mechanisms of Ageing and Development, 10 (1979) 71-79 @Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands

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VISCOSITY OF LYMPHOCYTE PLASMA MEMBRANE IN AGING MICE AND ITS POSSIBLE RELATION TO SERUM CHOLESTEROL*

B. RIVNAY**, A. GLOBERSON** and M. SHINITZKY*** Departments of Cell Biology** and Membrane Research***, The Weizmann Institute of Science, Rehovot (Israel) (Received July 25,197 8)

SUMMARY

Lymphocyte membrane fluidity was examined in aged mice and characterized as a qualitative and effective change which takes place in the aging process. Fluorescence polarization of diphenylhexatriene-labelled mouse spleen cells is substantially higher in cells from old mice (20-36 months) than young mice (2-7 months). A similar difference was also observed with isolated plasma membranes from spleen cells of old and young mice. The overall estimate is that the lipid microviscosity in the lymphocyte plasma membrane from old mice is about 20% higher than that of young mice. The cholesterol/ phospholipid ratio determined for the isolated plasma membrane preparations was 0.68 and 0.9, respectively, which is probably the main cause for the difference in membrane viscosity. An elevated cholesterol/phospholipid ratio was also observed in the blood serum of old mice. It is plausible that the source of excess membrane cholesterol in the old mouse lymphocytes originates in the high serum cholesterol.

INTRODUCTION

Many of the immune functions decrease markedly with age [l-8] . A considerable volume of studies focuses on changes in cell population, kinetics, interaction and effect of factors in relation to lymphocyte functions and aging. However, physical changes occurring with age may also affect the behavior of lymphocytes, yet such changes have hardly been considered. It has been reported that serum cholesterol level rises with aging by 20-25%, both in mice and in humans [9, lo]. Since serum lipoproteins exchange efficiently cholesterol with plasma membranes of blood cells [ 1l-161, it is of interest to test whether the plasma membranes of lymphocytes are enriched with cholesterol in the aging animal, and whether the excess cholesterol originates from the serum. *Supported by the Minerva Foundation.

12

The effects of cholesterol in both artificial and biological membranes have been extensively studied and discussed [ 17-261 . Many of the biological functions were found to be modulated by cholesterol. We therefore considered the possibility that elevated membrane cholesterol and viscosity act to reduce lymphocyte functions in addition to the above-cited changes in lymphocyte population which take place in the aging animal. The present study compares intact lymphocytes, their isolated plasma membranes and sera from old and young mice in relation to cholesterol level and membrane microviscosity.

MATERIALSANDMETHODS Mice

(C3H/eb X C57BL/6J)F 1 male mice were obtained from the Animal Breeding Center of the Weizmann Institute. The mice were fed on Purina chow and were given water ad libitum. Cells and membranes

Spleen cells from old (>20 months) and young (2-7 months) mice were used. Cells were teased through a stainless net into RPMI-1640 tissue culture medium, washed once, treated with ACK (0.15 M NH4C1 and 0.01 M KHCOs) for 3-4 min to remove red cells, and resuspended in RPMI. Plasma membrane preparation was carried out as described by Ladoulis et al. [27] and Allan and Crumpton [28] . The membrane preparations (the interface between 30 and 40%sucrose) were kept frozen at -80 “c in small batches in 10 miV Tris-HCl, pH 7.4, and were used immediately or within 24 h after thawing. The purity of the membrane preparation was assessed by the following assays: 5’nucleotidase activity was measured according to the method of Wildnell and Unkeles [29] ; acid phosphatase according to the method of Goldman and Raz [30] ; NADH dehydrogenase according to the method of Wallach and Kamat [31]. Phosphate was determined by the method of Ames and Dubin

1321. Sera

Pooled serum batches were prepared by bleeding from the orbital sinus of about 30 mice from each age group. Animals with any overt pathological manifestation, especially among the aged animals, were discarded. The blood was allowed to clot, spun down and the serum was collected. The sera were heat-inactivated at 56 “c for 30 min to avoid further alteration of lipid composition and were stored at -20 “c in small batches which were used immediately after thawing. Color appearance did not display differences in hemoglobin content. Lipid analysis

Lipids were extracted from sera and membranes according to the method of Renkonen et al. [33] , using three successive extractions. The chloroform phases of these

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extractions were pooled and concentrated before further use. Lipids were kept at -20 “c under nitrogen. Cholesterol was determined calorimetrically by the method of Rude11and Morris [34] . Phospholipids were represented by organic phosphate determination by the method of Ames and Dubin [32]. For the separation of lipid classes, thin-layer chromatography was carried out on 0.25 mm thick silica gel plates (Merck, W. Germany) with petroleum ether:ethyl ether:acetic acid (80:20:1) (all of analytical grade; purchased from Frutarom, Israel), as described by Wood et al. [35]. Spots were revealed with iodine vapors showing Rf values of 0, 0.23-0.30 and 0.95 for phospholipids, free cholesterol and cholesterol esters, respectively, based on known markers. Extraction of the spots was done with chloroform:methanol:water (62:25:4). Membrgne microviscosity The fluorescence polarization method, with 1,6-diphenyl-1,3,5-hexatriene (DPH).as

a probe, was employed for derivation of the apparent membrane microviscosity. The method is comprehensively reviewed in ref. [36]. Intact lymphocytes or isolated plasma membranes were incubated with an equal volume of a dispersion of 2 X 10e6 M DPH in PBS for 1 h; cells were washed once with PBS but membrane suspensions were measured without further washing. Simultaneous measurements of Iii/Z*and Z1were taken at the temperature range of 040 “c. I,, and Z,,are the fluorescence intensities polarized parallel and perpendicular to the polarization of the excitation beam. With these values, the fluorescence anisotropy (r) and the relative quantum yield could be determined [36,37], and the apparent microviscosity ($ could be evaluated. When a single measurement was taken @e/r - I)-’ were used as the best estimations for microviscosity [36], in which r. is the limiting fluorescence anisotropy for DPH. Additional hydrodynamic parameters could be derived from the empirical dependence of Ij on temperature: 17= A~AE/RT

(1)

These parameters include the unit flow volume (A) and the flow activation energy (aE) ]371.

RESULTS

The strain of mice chosen for this study was (C3H/eb X C57BL/6J)F1 hybrids. This is a long-lived strain, which is also resistant to leukemic transformations due to C3H genetic contributions (N. Haran-Ghera, personal communication). The choice of age was based on the stage of responsiveness of the animals. We have observed that at 3-9 months of age the immune response is maximal in several independent assays. Cells from older animals show a decline in activity which reaches a low response plateau at 20 months until 3 years of age [7,8] . We have therefore chosen the age groups of 3-9 months to represent young mice and above 20 months to represent old mice. Fluorescence polarization of DPH-labelled intact spleen lymphocytes from young and old mice had mean Is/Z1values of 1.77 and 1.83, respectively. These values correspond

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to about 20% increase in @e/r - 1)-l. performed

with 60 mice displaying

tribution

of plasma membranes

membranes

Figure 1 presents the sum of several experiments

the above differences. to the fluorescence

In order to determine

polarization

were isolated from spleen cells of both ages. Table I characterizes

parations.

Based on 5’nucleotidase

enriched

7- and 9-fold, respectively.

activity

the con-

of intact cells, plasma these pre-

[31] , the old and young preparations

The slightly higher enrichment

were

of the preparations

from young mouse cells with lysosomes (acid phosphatase) and endoplasmic reticulum (NADH dehydrogenase) is negligible, since the absolute amounts of these contaminants

Fig. 1. Fluorescence polarization (Ztt/Il or (rc/r - 1)-r)’ of DPH as measured in lymphocytes from young and old mice. Measurements were performed on cell suspensions from single spleens (0); or from a pool of n spleens (a).

TABLE I CHARACTERIZATION Marker enzyme

5’~nucleotidase

NADH dehydrgenase

Acid phosphatase

OF PLASMA MEMBRANE PREPARATIONS Cellularfraction

Specific activity (mol (mg prot) -l min -l) * Young

Old

PNS** PM*** Factor of enrichment

34.2 314.6

35.5 255.0

PNS PM Factor of enrichment

144.5 248.4

PNS PM Factor of emichment

*Each is a mean of duplicate or triplicate. **PNS = post-nuclear supernatant. ***PM = plasma membrane.

9.2

1.2 316.7 311.7

1.72 0.0167 0.0493

1.17 0.0911 0.094 1

2.95

1.03

15

are very small. Fluorescence polarization measurements were performed on the intact cells in comparison to isolated plasma membranes. The temperature dependence of the determined microviscosities is given in Fig. 2. It is important to note that the microviscosity of the isolated membranes is substantially higher than that obtained with the corresponding intact cells probably due to contribution from inner cell membranes [38-42] . The marked differences in fluorescence polarization could also be interpreted in terms of monitoring of different distribution of cell subpopulations in the spleens at the various ages, but as far as lymphocytes, monocytes and macrophages are concerned, the distribution is similar [43] . Table II specifies the various hydrodynamic parameters derived from Fig. 2 (see Materials and Methods and ref. [36]). Serum components, mostly lipoproteins, interact with blood cells and exchange lipids. The apparent determinant for cholesterol transfer into lymphocyte plasma membranes is the C/PL ratio of the interacting cholesterol pools [13, 151. Lipid analysis of

25.C

37% 1 32

I 33

I 34

I 35 .

VT x Id (‘K-’ )

Fig. 2. Temperature profiles of microviscosity of intact cells (A, 4) and isolated membranes (0, *I from old (empty symbols) and young (filed symbols) mouse spleens.

TABLE II HYDRODYNAMIC PARAMETERS DERIVED FROM OLD AND YOUNG INTACT LYMPHOCYTES AND ISOLATED MEMBRANES A* (poise)

Intact lymphocytes Isolated plasma membranes

AE (kcaI/mol)

ii (3 7 “C) (poise)

Old

Young

Old

Young

Old

Young

7 x 1o-4 2.5 x lo+

2.1 x 10-4 1.4 x 10-s

4.98 4.85

5.69 5.08

2.5 6.6

2.1 5.2

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the two membrane

preparations

(Table III) demonstrates

an elevated C/PL in old mouse

membrane preparations. It is therefore plausible that the higher C/PL found in lymphocyte membranes from old mice (Table III) originates from a higher C/PL in their serum. Table IV presents the data on lipid analysis of serum from old and young mice. As shown, C/PL in the old mouse serum is indeed higher by about 15%, compared to the young mouse serum, though the total cholesterol/phospholipid

ratio remains approximately

unaltered.

TABLE III CHOLESTEROL

AND PHOSPHOLIPID ANALYSIS OF PLASMA MEMBRANE PREPARATIONS

Age

Young (5 months)

Old (23 months)

Cholesterol @mol/mg protein) Phospholipids** &mol/mg protein) C/PL (molar)

1.095 + 0.066* 1.606 f 0.039 0.68 (0.63-0.74)

1.331 f 0.082 1.477 + 0.153 0.90 (0.77-1.07)

*Mean values of 2-3 samples 5 S.D. **Total phosphate in lipid extract.

TABLE IV LIPID ANALYSIS OF OLD AND YOUNG MOUSE SERA

Serum Total cholesterol (TC) (mol/ml serum) Organic phosphate (mol/ml serum) TC/organic phosphate Lipids isolated on TLC*+ TC/PL* FC/PL* *TLC = thin-layer chromatography; PL = phospholipids. ‘Molar ratios.

Young (5 months)

Old (23 months)

3.18 t 0.14 ( = 1.228 f 0.053 mg/ml) 3.02 + 0.12 1.05 (0.97-1.14)

4.21 + 0.19 (= 1.636 f 0.023 mg/ml) 3.88 * 0.23 1.08 (0.98-1.20)

0.89 (0.68-1.17) 0.200 (0.187-0.214)

0.88 (0.82-0.94) 0.231 (0.220-0.243)

TC = total cholesterol;

FC = free (unesterlfied)

cholesterol;

DISCUSSION

Spleen cells from old and young mice were shown here to differ mostly in the cholesterol/phospholipid (C/PL) ratio of their plasma membrane. This parameter is now considered to be the main determinant of lipid fluidity of both artificial and biological membranes [36, 441. The determined value of C/PL of the membranes from young mice is 0.68 whereas that of old mice is 0.90. This substantial difference in C/PL correlates well with the observed increase in microviscosity in membranes from old mice. An increase in C/PL was shown to increase 1, and A (see eqn. (1)) but to decrease hE in both liposomes

II

and isolated membrane

membranes preparations,

[36].

Similar differences

as well as with the intact

were also observed here between cells (see Table II). The protein

the to

lipid ratio, which can also affect the membrane lipid fluidity, is virtually the same in the cells from old and young mice (see Table III). Other factors which were not determined in this study, the lecithin to sphingomyelin ratio [45] or the degree of unsaturation of the phospholipid acyl chains [19] could in principle also contribute to the observed differences in fluidity. The physiological implications of the observed increase in membrane microviscosity with aging relate primarily to modulation of the membrane protein dynamics. Thus, proteins in membranes from old animals are expected to have smaller rates of diffusion and an overall increase of exposure on both sides of the membrane [Sl, 521. The observed difference in enzymic activities of the various cellular compartments (Table I) could partly originate from the difference in microviscosity. This is supported by the observation that elevation of cholesterol in vivo or in vitro leads to similar differences in the activity of cellular enzymes [46-48] . Beside enzymes, the activity of receptors and antigens is similarly modulated by lipid microviscosity [SO-521 . It is thus probable that due to the increase in microviscosity, aging will be accompanied by reduced cellular functions as a result of impaired

receptor-mediated

processes [26] and in extreme cases by the onset of autoim-

mune activity due to the exposure of naturally

hidden antigenic determinants.

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