Immunological senescence in mice and its reversal by coenzyme Q10

Mechanisms o f Ageing and Development, 7 (1978) 189-197

189

©Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands

I M M U N O L O G I C A L S E N E S C E N C E IN MICE A N D ITS R E V E R S A L BY C O E N Z Y M E Qlo*

EMILE G. BLIZNAKOV New England Institute, Ridgefield, Connecticut 06877 (U.S.A.)

(Received November 15, 1976; in revised form June 13, 1977)

SUMMARY A pronounced suppression of the humoral, hemolytic, primary immune response in old (22 months) mice was demonstrated as compared with this response in young (10 weeks) mice. The suppression is associated with a lower thymus weight:body weight ratio. In contrast, the ratios spleen weight:body weight and liver weight:body weight in 10 weeks and 22 months old mice remain almost constant. A single administration of coenzyme Ql0 - a non-toxic, non-specific stimulant of the host defense system - partly compensates the age-determined suppression of the humoral, immune response. This suppression probably results from an age-dependent imbalance of T cells: B cells ratio and a decline of their immunological responsiveness which is compensated by the administration of eoenzyme Qlo.

INTRODUCTION Coenzyme Q is now generally recognized as an important component of the mitochondrial electron transport processes of respiration and coupled oxidative phosphorylation, and therefore is of fundamental importance to the intraeellular energy-producing systems. Evidence has been obtained for the existence of coenzyme Q deficiencies in some pathological processes in human cardiac, gingival, and dystrophic tissues, rats with induced hypertension, mice with hereditary muscular dystrophy, Friend virus induced leukemia and others. The therapeutic application and potential of coenzyme Q was reviewed by Folkers [1, 2]. Coenzyme Q is now among the agents being used experimentally and clinically to enhance non-specifically the host resistance. In contrast to other materials, in use for this

*Presented in part at International Symposium on Ooenzyme Q, Biomedical and Clinical Aspects, Lake Yamanaka, Japan, September 16-17, 1976 [4].

190 purpose, extensive toxicological studies, including our own, revealed no significant abnormalities that would contraindicate the use of coenzyme Q in humans. Administration of various members of the coenzyme Q family into experimental animals results in increased resistance to a variety of bacterial and protozoal infections, as well as viral and chemical carcinogeneses. It has been postulated that this enhanced resistance is mediated via stimulation of various parameters of the host defense system, a process which has high cellular energy requirement. The experimental evidence has been reviewed recently by Bliznakov [3, 4]. The host defense system is subject to age-related changes ocurring in both animals and man, although it is only in laboratory animals that the phenomenon has been well studied. These age-related changes are characterized in general as a gradual decline of the activity of many parameters of the host defense system. This decline forms the base for a definitive relationship between senescence and an increased rate of incidence of infectious diseases and cancer as well as mortality due to these causes in both animals and man. Recent reviews adequately cover the extensive literature [5-9]. In the present study we attempted to demonstrate the decline of the humoral immunological responsiveness in aged mice as a representative parameter of the host defense system and the possibility of compensation of this decline by administration of coenzyme Q~o-

MATERIALS AND METHODS Female CF1 mice were used throughout the experiments. They were purchased from Charles River Breeding Laboratories, Inc., Wilmington, Ma. (Carworth Division) and were maintained in air-conditioned rooms (22 -+ I °C) on a 12-hour light and dark cycle in metal cages with free access to food and water. Fresh sterile sheep red blood cells (SRBC, Baltimore Biological Laboratories, Baltimore, Md.) were centrifuged and washed three times with sterile 0.9% sodium chloride solution (saline). Primary immunization was accomplished with SRBC, suspended in 0.2 ml saline, and administered via the tail vein. The day of the antigen administration is designated as day 0. Commercially available pure coenzyme Q10 (2,3-dimethoxy-5-methyl-6-decaprenyl benzoquinone) was used and was administered as an emulsion in sterile 5% glucose solution containing 0.4% of Tween 20 [poly(oxyethylene sorbitol monolaurate)] used as emulsifier. The concentration of coenzyme Qlo in the emulsion was 250/~g/ml. The emulsion (total volume 200 ml) was prepared in a 500 ml Waring blender, kept in a water bath at 60 °C and protected from light. The time of homogenization was 45 s. The particle size of the emulsion was under 5/~m. The method used to prepare the emulsion and the subsequent handling are of critical importance. On day four after the SRBC administration, and 4 h before the first blood collection, six groups of 22 month old mice (25 mice in each group) were treated with six different doses of coenzyme Qlo emulsion injected into the tail vein.

191 The control mice (50 mice, 22 months old and 50 mice, 10 weeks old) were injected with the same mixture, omitting coenzyme Qlo. At suitable intervals after the administration of coenzyme Qlo, blood was collected from each mouse with heparinized capillary tubes by retroorbital venus plexus puncture. Equal volumes of blood from all animals in a group were pooled, the plasma was separated by centrifugation and the samples were stored at - 4 0 °C until hemolysin titers were determined. This determination was carried out using the 50% end point method [10]. Eight to ten plasma dilutions were used. The best fitting regression line between probit percentage hemolysis and the log of the plasma dilution was determined by computer analysis. The experimental points shown on the figures represent the determined values, with standard deviation indicated. Control mice (25 mice 22 months old, and 25 mice 10 weeks old) were sacrificed, weighed and organs (spleen, liver and thymus) were excised and weighed. The data obtained were statistically analyzed by the Student's t test. In a preliminary experiment, the humoral, hemolytic, antibody response in 6, 8, 10, 12, 14 and 16 week old mice (25 mice per age group) was evaluated using the methods described above. All glassware was heated for 5 h at 170 °C. Non-pyrogenic sterile saline and sterile glucose solutions (Travenol Laboratories, Deerfield, I1.), syringes, needles and pipets were used throughout. Possible contamination with bacterial endotoxin (a strong toxic stimulant of the host defense system activity) of the components used for preparation of emulsions was precluded by the exclusive use of only non-pyrogenic materials. Criteria recommended by the U.S. Pharmacopeia were used for evaluation.

RESULTS As shown in Fig. 1, the humoral, hemolytic, primary response of young mice rises steeply until they reach the age of 10 weeks. Thereafter, until the age of 16 weeks, this increase decelerates, thus forming almost a plateau. This is especially apparent on day 5 after the antigen administration (peak response day). For this reason 10 weeks was selected as the age of the control group for the consecutive experiments. The marked suppression of the humoral hemolytic, primary immune response as a function of age of the mice is demonstrated in Fig. 2. Clearly, the hemolytic antibody level in 22 month old mice is less than 50% of the level obtained in 10 week old mice. This reduction is not accompanied by any shifting of the appearance of the peak antibody level day. The profound hemolytic antibody depression in old mice can be partly reversed by a single intravenous administration of coenzyme Qlo on day 4 (Fig. 2). Figure 3 illustrates the dose (coenzyme Qlo)-response (hemolytic antibody level on day 5) relationship in 22 month old mice.

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Fig. 1. Hemolytic, primary, immune response (7 X 107 SRBC per mouse) in 6, 8, 10, 12, 14 and 16 week old female mice determined at various days after SRBCadministration. The experimental points shown represent the determined values for pooled plasma from 25 mice with standard deviations indicated. Fig. 2. Hemolytic, primary, immune response (5 X 1 0 7 SRBC per mouse) in 10 week and 22 month old CF1 female mice, and compensation of the age-dependent suppression of this response by intravenous administration of eoenzyme Ql0 emulsion (125 #g/mouse). The experimental points shown represent the determined values for pooled plasma from 25 mice with standard deviations indicated.

The organs' weight data and the ratios of liver, spleen and thymus weight and body weight are presented in Table I. The results show that the body, liver and spleen weight continue to increase with age, but the ratios between the liver and spleen and the body weight remain practically constant. In contrast, the thymus weight continues to increase with age, but this increase is at a much slower rate than the body weight increase which results in a significantly lower value of the thymus weight:body weight ratio. This ratio in 22 month old mice represents only 69.6% of the same ratio in 10 week old mice.

DISCUSSION In a recent critical commentary, Walford [11] strongly emphasizes that many publications of great immunologic sophistication are shockingly naive gerontologically. He notes that at least one-third of the papers now being published which purport to con. tern immunology and aging do not include, in reality, animals older than young-adult

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BODY, LIVER, SPLEEN AND THYMUS WEIGHT IN 10 WEEKS AND 22 MONTHS OLD CF1 FEMALE MICE

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Fig. 3. Compensation of the suppressed hemolytic, primary, immune response (5 X 10 7 SRBC per

mouse) in 22 month old mice on the peak antibody day (day 5 after the antigen administration) as a function of the dose of eoenzyme Qlo- The experimental points shown represent the determined values for pooled plasma from 25 mice with standard deviations indicated.

or mid-adult. He strongly recommends that only aminals at or beyond the 50% survival point should be used in any experiment concerning aging. The mice used in our experiments are reaching 50% survival rate at age 16 months [ 12] ;therefore, their age is well beyond the requirement established by Walford [ 11 ]. Besides considering mice as "old" when they are not, an additional pitfall in comparative immunogerontologic studies consists of using mice of various ages as controls ("young", "adult" or "young-adults"). It has been established that the age of peak response varies markedly with the immune parameter measured, and also with the strain of mice used in the experiments (reviewed in ref. [11] ). In order to evaluate this parameter in our test system, we compared the immunological response in young mice at age 6, 8, 10, 12, 14 and 16 weeks. Consequently, as a control group in our studies we used mice aged 10 weeks which according to our comparative study, are at a relative plateau formation of their immunological development, after a steep ascendance which occurs earlier. Although bone marrow (B) lymphocytes are directly involved in antibody formation, it is now a well recognized postulate that their collaboration with thymus derived (T) lymphocytes is necessary for the development of a normal humoral immunological response to certain antigens (reviewed in refs. [13 and 14] ). This cognizance resulted in

195 the concept of two distinct groups of antigens, thymus-dependent and thymus-independent, or as Miller prefers to designate them, antigens that can activate T cells and those which cannot [15]. For some antigens the interaction of T cells--B cells is initiated by accessory ceils (A cells, macrophages) which act by processing the antigen or by supplying necessary extracellular factor(s) [6]. Systemic investigations of immunocompetent cell deficiencies in aged mice indicate that both B cells and T cells mediated immune responses decline with advancing age [59]. In contrast, macrophage populations from young and old mice are indistinguishable [6]. This implies that the afferent compartment of the immune system is not engaged in the immunological impairment manifested in old mice. Heidrick and Makinodan [6] reported a 10-fold reduction in the proliferative capacity of T cells and a 5- to 10-fold reduction in B cell proliferative capacity in old mice. As a result, the T cell-B cell ratio is altered. Their studies revealed also that an optimal ratio of T cells--B cells is required to generate a maximal response to SRBC and that the optimal ratio is the same for both young and old ceils. In a more recent study, Teasdale et al. [16] observed that in humans absolute and percentage B-cell counts showed no significant variation with age, while T-cell counts all showed significant and gradual decrease with advancing age. Although B-cell numbers remained stable, B-cell functions were impaired with aging. Surprisingly, there have been few reports on age-related changes in the functional capacity of the thymus. Hirokawa and Makinodan [17] showed that the extent to which T cells can mature is dependent upon the degree of involution the thymic tissue has undergone with age. It was suggested [ 18] that the thymus-dependent T cells are the key factor and their exhaustion is responsible for aging in mammals and probably other vertebrates. It was also suggested that with aging the thymus begins producing not only fewer cells, but less efficient T cells. Pachciarz and Teague [19] emphasized that physiologic thymic function(s) must continue throughout life in order to maintain T-cell effectiveness. Only recently we began to realize the complexities of cell interactions in the immune response. Segre et al. [20] reviewing the present knowledge concluded that the first immunologic lesion of aging in mice is an increase in T-cell suppressor function. This is followed by a decrease in T-cell helper function and finally by a loss of B-cell function. Our results clearly demonstrate the profound suppression of the primary immune response in aged mice to SRBC, a thymus-dependent antigen [21, 22]. This suppression is accompanied by a reduced thymus weight:body weight ratio. In contrast, the ratios spleen weight: body weight and liver weight:body weight in 10 week and in 22 month old mice remain almost constant. A single administration of coenzyme Qlo emulsion, a stimulant of the host defense system, partially but significantly compensates the agedependent suppression of the humoral immune response. This compensation is dependent on the dose of coenzyme Qlo- We have extensively studied the non-linear response of the host defense system upon stimulation. This response forms a W- or M-shaped curve (depending on the selection of parameters used for the representation) and is the property of the host defense system and not of the stimulant used [23, 24].

196 Indirect evidence suggests that coenzyme Q stimulates both the B-cell and T-cell mediated responses [3, 4]. Furthermore, administration of coenzyme Q in experimental animals and humans induces no significant cellular proliferative effect on the host defense system [3, 4]. Thus the stimulating effect is believed to be mediated via a more efficient performance by existing cells rather than by an increased number of cells. This more efficient performance, conferred by coenzyme Qlo, compensates the decline of B-cell and especially T-cell functions in aged mice and probably also restores the functional balance between T cells and B cells required for an optimal response to SRBC. A possible effect via the afferent compartment of the immune system (macrophages) is not considered here because of the delayed administration of coenzyme Qlo (on day 4 after the antigen). An additional, intriguing possibility is suggested by some limited studies reviewed by Wilson [25], implying an impairment of the intracellular process of respiration at the mitochondrial level in senescence. This is also accompanied by changes in the mitochondrial ultrastructure [26]. Similarly, Siliprandi e t al. [27] demonstrated that energylinked processes are partially or completely lost during the aging ofmitochondria but this is, within certain limits, a reversible phenomenon. Further qualitative and quantitative studies at this mitochondrial level should shed light on the relationship between immunological impairment and the resulting increased incidence of cancer and infections in senescence as well as on the compensation of this impairment by coenzyme Q.

ACKNOWLEDGEMENTS This work was derived from research programs supported in part by grants from individuals, corporations and the following foundations: Griffis, Heddens-Good, Ivy Fund, Landegger, Virginia and D. K. Ludwig, J. M. McDonald, Roy R. and Marie S. Neuberger, Anne S. Richardson Fund, Fannie E. Rippel, Rockledge, Scheider, Arnold Schwartz Fund for Health and Education Research, United Order of True Sisters, Gilbert Verney, Wahlstrom, Wallace Genetic, Raymond J. Wean and Whitehall. The assistance and advice of Dr. S. J. Tao for the computer program and statistical analysis is gratefully acknowledged. We thank N. Hastings, C. Kache, G. Katopodis, A. Santini and C. Torcellini for technical assistance.

REFERENCES 1 K. Folkers, Survey on the vitamin aspects of coenzyme Q, Int. J. Vitam. Res., 39 (1969) 334. 2 K. Folkers, The potential of coenzyme Qlo (NSC-140865) in cancer treatment, Cancer Chemoth. Rep., 4 (1974) 19. 3 E. G. Bllznakov, From sharks to coenzyme Qlo, in Sh. M. Reichard, M. R. Escobar and H. Friedman (eds.), The Reticuloendothelial System in Health and Disease." Functions and Charac. teristics, Plenum Press, New York, 1976, p. 441.

197 4 E. G. Bliznakov, Coenzyme Q in experimental infections and neoplasia, in K. Folkers and Y. Yamamura, (eds.), Biomedical and Clinical Aspects of Coenzyme Q, Elsevier/North-Holland Biomedical Press, Amsterdam, 1977, p. 73. 5 T. Makinodan, E. H. Perkins and M. G. Chert, Immunologic activity of the aged, Adv. Gerontol., 3 (1971) 171. 6 M. L. Heidrick and T. Makinodan, Nature of cellular deficiencies in age-related decline of the immune system, Gerontologia, 18 (1972) 305. 7 L. J. Greenberg and E. J. Yunis, Immunologic control of aging: a possible primary event, Gerontologia, 18 (1972) 247. 8 K. E. Cheney and R. L. Walford, Immune function and dysfunction in relation to aging, Life Sci., 14 (1974) 2075. 9 W. H. Adler, Aging and immune function, BioScience, 25 (1975) 652. 10 E. A . Kabat and M. M. Mayer, Experimental Immunochemistry, 2nd Edn., Charles C. Thomas, Springfield, II., 1961. 11 R. L. Walford, When is a mouse "old"?, J. Immunol., 117 (1976) 352. 12 G. C. Cotzias, S. T. Miller, A. R. Nicholson, Jr., W. H. Maston and L. C. Tang, Prolongation of the life-span in mice adapted to large amount of L-dopa, Proc. Nat. Acad. Sci. USA, 71 (1974) 2466. 13 J. F. A. P. Miller, Lymphocyte interactions in antibody responses, Int. Rev. Cytol., 33 (1972) 77. 14 H. N. Claman, "Signal theory" in cellular immunology: collaboration between T- and Bqymphocytes in the immune response,Ann. N. Y. Acad. Sci., 249 (1975) 27. 15 J . F . A . P . Miller, T- cell regulation of immune response, Ann. N. Y. Acad. Sci., 249 (1975) 9. 16 C. Teasdale, J. Thatcher, R. H. Whitehead and L. E. Hughes, Age dependence o f T lymphocytes, Lancet, I (1976) 1410. 17 K. Hirokawa and T. Makinodan, Thymic involution: effect on T cell differentiation, J. Immunol., 114 (1975) 1659. 18 F. M. Burnet, An immunological approach to ageing, Lancet, II (1970) 358. 19 J. A. Pachciarz and P. O. Teague, Age-associated involution of cellular immune function. I. Accelerated decline of mitogen reactivity of spleen cells in adult thymectomized mice, J. Immuno£, 116 (1976) 982. 20 D. Segre and M. Segre, Age-related changes in B and T lymphoeytes and decline of humoral immune responsiveness in aged mice, Mech. Ageing Dev., 6 (1977) 115. 21 H. N. Claman, E. A. Chaperon and R. F. Triplett, Thymus--marrow cell combinations. Synergism in antibody production, Proc. Soc. Exp. Biol. Med., 122 (1966) 1167. 22 J. F. A. P. Miller and G. F. Mitchell, Thymus and antigen-reactive cells, Transplant. Rev., 1 (1969) 3. 23 E. G. Bfiznakov and A. D. Adler, Nonlinear response of the reticuloendothelial system upon stimulation, Path. Microbiol.. 38 (1972) 393. 24 E. G. Bllznakov, lmmunostimulation or immunodepression? Eur. Z Clin. Biol. Res., 26 (1977) 73. 25 P. D. Wilson, Enzyme changes in ageing mammals, Gerontologia, 19 (1973) 79. 26 P. D. Wilson and L. M. Franks, The effect of age on mitochondrlal ultrastrueture, Gerontologia, 21 (1975) 81. 27 D. Siliprandi, N. Siliprandi, G. Scutari and F. Zoccarato, Restoration of some energy lim~ea processes lost during the ageing of rat liver mitochondrla,'Biochem. Biophys. Res. Commun., 55 (1973) 563.