Impairment of lymphocyte activities in depressed aged subjects

Mechanisms of Ageing and Development, 60 ( 1991) 13-24

13

Elsevier ScientificPublishers Ireland Ltd.

IMPAIRMENT OF LYMPHOCYTE ACTIVITIES IN DEPRESSED AGED SUBJECTS

LUISA GUIDI a, CARLO BARTOLONI a, DANIELA FRASCA f, LEONARDO ANTICO b, ROBERTO PILI a, FLAVIO CURSI a, E. TEMPESTA c, CARLO RUMI d, EDOARDO MENINI e, PIERUGO CARBONIN b, GINO DORIA f and GINO GAMBASSI a Institutes of aClinica Medica Generale and bCattedra di Geriatria. "Psichiatria. aSemeiotica Medica and eChimica Biologica, Universitd Cattolica de/Sacro Cuore, Facoltd di Medicina. Roma and JLaboratorv of Pathology, ENEA-CRE, Casaccia. Roma (Italy)

(Received December4th, 1990) SUMMARY Lymphocyte activities were determined in a population of 26 institutionalized aged subjects, selected as healthy according to the SENIEUR protocol and previously reported to display immunological and endocrinological abnormalities correlated with depressive disorders. The lymphocyte mitotic response to PHA, which was reduced in aged as compared to adult subjects, was found to be significantly lower and negatively correlated with the depression score in the elderly subjects. In supernatants of PHA-stimulated lymphocyte culture from aged subjects, IL-2, IL-4 and 7-IFN levels were very low and more severely affected in the depressed aged group. Each cytokine production was negatively correlated with age and depression score. NK activity was lower in the aged and it could be augmented by the addition of IL-2 or t~-IFN, even though to a lesser extent than in the adult subjects. The nondepressed aged displayed higher levels of IL-2 inducible NK activity than the depressed aged subjects. IL-2 and t~-IFN stimulated NK activities were negatively correlated with depression score. The present work indicates that the psychological status could affect lymphocyte reactivity in the aged. Given the relatively high frequency of affective disorders in these subjects, the psychological status should be considered in studies of immune senescence. Key words: Aging; Psychoneuroendocrinoimmunology; Lymphocyte blastogenesis; Interleukins; Interferon; NK activity Correspondence to: Carlo Bartoloni,M.D., Istitutodi Clinica Medica,UniversitfiCattolicaS. Cuore, L.go A. Gemelli, 8 !-00168 Roma, Italy.

0047-6374/91/$03.50 Printed and Published in Ireland

© 1991 Elsevier ScientificPublishers Ireland Ltd.

14

INTRODUCTION Immunogerontological studies are often influenced by the selection of the healthy aged population. A valid approach to this problem could be the use of a standardized screening procedure, such as the SENIEUR protocol [1], that excludes all subjects suffering from organic diseases and under medical care by drug therapy. In recent years, the influence exerted by affective disorders and chronic stress on immune functions through neuroendocrine pathways has been emphasized [2--5]. Our study is aimed at evaluating possible correlations between depressive disorders and immunosenescence. In a previous study, we examined cutaneous delayed hypersensitivity and lymphocyte subsets in a population of institutionalized elderly subjects, selected according to the SENIEUR protocol. The aged subjects were evaluated for affective disorders and the correlations between depression score and lymphocyte subsets or plasmatic and urinary cortisol levels were described. We report here the results of functional studies performed on cultured lymphocytes from these subjects and adult controls, such as lymphocyte mitotic response to PHA, cytokine (IL-2, IL-4, 3/-IFN) production and basal or cytokinestimulated natural killer (NK) cell activity. MATERIALSAND METHODS

Subjects Twenty-six elderly subjects were selected, according to the SENIEUR protocol [1], out of a population of 130 elderly subjects (> 65 years) all residents in a public institution for retired people (Casa di Riposo Roma III). They were 9 males and 17 females, mean age 81.1 ± 6.4, range 66--95. In some of the analyses they were randomly subdivided in the following age groups: 10 (66--77), 9 (78--85), 7 (86--95). Six adult subjects (laboratory personnel) were studied as controls.

Psychological evaluation The aged subjects underwent a psychodiagnostic examination made by a neuropsychiatrist, according to the criteria of DSM III R (Diagnostic and Statistical Manual of Mental Disorders, Third Edition, Revised, American Psychiatric Association, Washington, DC, 1987). Subsequently, they were examined on the basis of the Hamilton Rating Scale for Depression (HRSD, 6). Nine were classified as depressed, scoring > 15 and 17 as non-depressed. This classification was confirmed also by the Beck Depression Inventory (BDI, 7). The presence of dementia was excluded by means of the Mini Mental State (MMS). The adult controls were submitted to the BDI and found non-depressed.

15 Cell culture

Peripheral blood mononuclear cells (PBM) were obtained from fresh heparinized venous blood by density gradient centrifugation on Ficoll-Isopaque (Lymphoprep, Nycomed As., Norway), as described by Boyum [8]. PBM were cultured in microtiter culture plates (Falcon Plastics 3040, Oxnard, CA) in RPMI 1640 (GIBCO, Grand Island, N.Y.), supplemented with 10% fetal calf serum (FCS, Seromed, West Germany), 10 /~g/ml gentamycin (Eurobio, France), 2 mM L-glutamine (Flow Laboratories, Scotland, U.K.). Mitogenic stimulus was provided by phytohemagglutinin (PHA, GIBCO) at the final concentrations of 1.2%, 0.6% and 0.3%. Cultures were maintained in a 5% CO2 humidified incubator for 72 h. Four hours before harvest, cultures received 0.5 /~Ci of tritiated thymidine (spec. act. 74 GBq/mmol, Amersham International, Amersham, U.K.), in 20 ttl. Cells were harvested with an automated cell device (Skatron, Lier, Norway) and passed onto filter disks that were then transferred to vials containing liquid scintillation fluid (Filter Count, Packard, Downers Grove, IL). Radioactivity was measured in a Minaxi Tri-Carb 4000 scintillation counter (Packard) and expressed as net counts per min (cpm, experimental minus control without PHA). T cell growth factor (TCGF) production was induced by stimulating PBM at the concentration of 2 x 106/ml in 12 x 75-mm round-bottom tubes (Falcon 2054) with 5% PHA, for 48 h in a 5% CO2 humidified incubator. Culture supernatants were collected by centrifugation at 2500 rev./min for 5 min, filtered through 0.2/~m Millex filters (Millipore, Molsheim, France) and stored until use at -20°C. PHA concentration was optimal for maximal TCGF production. TCGF titration

The assay was based on the method of Gillis et al. [9], but involved statistical validation of titration data according to the principles of the biological assay by parallel lines as applied to probit analysis of quantitative responses, as previously described [10]. Briefly, IL-2-dependent CTLL cells (1 x 104/well) were cultured in triplicate with serial dilutions of TCGF-containing supernatants for 20 h, then pulsed 4 h with 0.5 t~Ci of tritiated thymidine (spec. act. 74 GBq/mmol) and harvested by an automated cell harvester, as described above. Net cpm (experimental minus control without TCGF) were expressed as percent of the maximal cpm obtained in the assay. In each titration assay, probit transformation was applied to standard dilutions of TCGF produced by the gibbon line MLA 144, arbitrarily defined to contain 100 units of IL-2/ml and to experimental dilutions of culture supernatants. Parallelism between the standard regression and each experimental regression was verified by analysis of variance. Potency of each experimental supernatant relative to the standard supernatant was calculated as the anti-log2 of the horizontal distance between the experimental and standard lines. A factor by which the titer should be multiplied or divided to obtain the variation due to one standard error was also calculated.

16 T C G F titrations were performed in parallel in the presence of a large excess of mAb I l B l l (anti-murine IL-4, kindly provided by Drs. J. Ohara and W.E. Paul, through the courtesy of Dr. A. Lanzavecchia), in order to determine the possible contribution of IL-4 to the C T L L cell proliferation. It was found that approximately the 30---40% of the T C G F activity present in the culture supernatants could be attributed to the presence of IL-4. Thus, the T C G F activity detectable in stimulated cell supernatants in the experiments reported herein can be ascribed to a mixture of IL-2 and IL-4. I F N assay 3'-IFN assay was performed by a plaque reduction assay in microtiter plates using vesicular stomatitis virus and L cells, according to Landolfo et al. [11]. Titers are expressed in laboratory units. Natural killer cell activity N K cell function was measured after filtration of PBM on sterile nylon wool columns [121, by a modification of the 51Cr release assay [13], employing K 562 cells as targets. Briefly, 1 x 106 target cells were labelled with 200 #Ci Na2CrO4in saline (New England Nuclear, NJ) for 45 min at 37"C. After washings, 10 4 K562 target cells were mixed with effector lymphocytes in RPMI 1640 medium at different ratios (from 50/1 to 6.25/1) of effectors to target cells in round bottom 96 well plates (Nunclon Delta Si, 1-67008, Nunc, Denmark) in the presence of 10% FCS. The plates were centrifuged for 2 min at 250 x g and then incubated at 37°C in 5%, CO2, 95% air, for 4 h. Target cells were incubated with 100 #1 of 10% SDS for maximum release (MR) or with medium for spontaneous release (SR). The assay was terminated by centrifugation at 500 x g for 10 min and radioactivity in 100/~1 of supernatant fluids was counted. The percent specific cytotoxicity was calculated from the formula: (Experimental cpm - SR cpm)/(MR cpm - SR cpm) x 100. All experiments were performed intriplicate. N K activity was also evaluated after 1 h incubation (37"C, 5% CO2) of effectors cells with 10 units/ml of recombinant human IL-2 (Biogen, Geneva, Switzerland) or 10 000 units/ml of recombinant human a - I F N (Roferon A, Roche). Cells were then washed twice and tested for N K activity. Statistical evaluation The frequency distribution o f the various data sets has been analyzed by means of the Kolmogorov-Smirnov test for goodness of fit to a normal distribution. Mean values were compared by two tailed Student's t-test for unpaired data. Correlations were evaluated by linear regression or by non parametric tests (Kendali's ranks coefficient test). For the analysis of correlations the peak responses were used (e.g. PHA 1.2°, N K 50/1).

17

TABL E 1 L Y M P H O C Y T E MITOTIC RESPONSE TO PHA (NET CPM) (MEAN 4- S.D., n)

Mitogen concentration (%) 1.2

Aged All Depressed Non-depressed Age 6 ~ - 7 7 Age 78--85 Age 8 ~ - 9 5 Adults

0.6

0.3

25209# 4- 30616 (26)

21868# 4- 26370

16247# 4- 21565

7156" 34767 28084 28977 16257 80104

6497* 30006 22932 25976 15062 80035

3912" 22777 15752 18861 13592 44592

4± ± 444-

5832 34167 39366 28938 18330 11274

(9) (17) (10) (9) (7) (6)

444444-

5776 29437 30013 30213 15742 15981

444444-

3315 24295 22223 23863 20463 13373

#P < 0.005 vs. adults. *P < 0.05 vs. non-depressed.

RESULTS

The lymphocyte mitotic response to PHA (Table I) was greatly diminished in aged subjects, when compared to the adult controls and was negatively correlated with age (Fig. 1). However, within the aged population, a significant difference could be detected between the depressed elderly and the non-depressed ones, the latter showing the higher levels (Fig. 2). In the aged subjects, the PHA response was negatively correlated with the HRSD score (r = -0.42, P = 0.029). 120,000

90,000 "

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Age ( y e a r s ) Fig. I. Linear regression analysis of mitotic response to PHA (net cpm) on age.

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The cytokine production was greatly reduced in aged as compared to adult subjects (Table II, Fig. 3). The production of IL-2, IL-4 and 3,-IFN was negatively correlated with age (Kendall's tau respectively = -0.34, P = 0.0017; -0.21, P = 0.022; -0.28, P = 0.036). The production of IL-2, IL-4 and 3,-IFN was negatively correlated with the depression score (Kendall's tau = -0.34, P = 0.0025; and -0.3, P = 0.021; and -0.36, P = 0.011, respectively). The production of the different cytokines was positively correlated each other and with the mitotic response to PHA in the overall population, regardless of aging and depression (data not shown)• T A B L E !1 CYTOKINE S.D., n)

PRODUCTION

A F T E R IN V I T R O S T I M U L A T I O N

IL-2

1L-4

W I T H P H A ( M E A N (units/ml) 4-

,y-IFN

Aged All

3.7* + 6.6 (26)

1.6" 4- 3.4

2 . 1 " 4- 5.9

Depressed Non depressed Age 66~77 Age 78--85

2.3 4.3 6.3 3.1

1.1 1.9 2.8 1.5

0.01 3.2 4.5 1.0

Age 86--95 Adults

0.4 4- 1.0 (7) 82.7 4- 24.3 (6)

* P < 0.0001 vs. adults.

+ + -¢4-

6.2 6.8 8.7 5.6

(9) (17) (10) (9)

444-4-

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0.01 54.1 4- 20.4

0.01 81.00

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The results obtained in the N K assays are shown in Tables III, IV and V. A negative correlation was found between N K activity and age for unstimulated N K cells and after stimulation with IL-2 or ct-IFN (Fig. 4). Basal N K activity was lower in aged subjects than in adult controls (Fig. 5). However, no difference could be detected between the depressed and non-depressed aged. A similar behaviour was displayed after stimulation with oe-IFN (Fig. 6). When N K cells were IL-2-stimulated, a difference between the depressed aged and the non-

TABLE III BASAL NK ACTIVITY (% 5tCr RELEASE) (MEAN 4- S.D., n)

Effector/target cell ratio 50/1 Aged All Depressed Non-depressed Age 66--77 Age 78--85 Age 86--95 Adults

39.1" 38.4 39.5 35.8 44.2 37.4 61.7

*P < 0.02 vs. adults.

25/1

44444± ±

17.2 17.5 17.6 22.1 15.1 11.7 24.3

(26) (9) (17) (10) (9) (7) (6)

38.9 38.9 38.8 34.7 41.6 41.3 52.3

12. 5/1

44± 444±

21.3 21.1 22.1 21.4 18.1 26.9 18.7

23.6* 18.6 26.3 21 27.7 22.2 38.8

6.25/1

4444± 4±

13.2 7.2 15.0 16 12.5 10.0 10.8

12.8 9.7 14.4 14.7 13.2 9.5 21.3

4± ± ± 4± ±

10.8 10.7 10.8 15.3 6.6 7.7 10.1

20

TABLE IV c t - I F N - - S T 1 M U L A T E D NK ACTIVITY (% 5JCr R E L E A S E ) ( M E A N 4- S.D., n)

Effector/target cell ratio 50/1 Aged All Depressed Non-depressed Age 6 6 ~ 7 7 Age 78--85 Age 86--95 Adults

59.4#* 48.4 66.3t 53.2 72.1 53 88

25/1

44± 44± ±

28.9 (19) 27.4 (7) 28.7 (12) 30 (9) 30.1 (7) 24.7 (5) 20.7 (6)

51.2" 48.1 53.1 53.7 52.2 45.4 84.4t

12.5/1

44± ± 4± ±

22.4 31.6 15.6 30.2 17.2 13.4 22.7

42.2# 33 47.8t 51 34.2 37.6 60.9t

6.25/1

± ± ± 4± 4~

30.1 20.7 34.2 43.3 15.9 10.1 18.9

26.9#* 21.6 30.3t 32.1 23.9 21.9 47.7t

44± + 44±

18.7 16.1 19.9 25.4 11.8 11.8 16

*P < 0.05 vs. adults. #P < 0.005 vs. NK activity unstimulated. t P < 0.05 vs. NK activity unstimulated.

depressed ones was detected (Fig. 7). Furthermore, in the elderly subjects, the NK activity after stimulation with IL-2 and t~-IFN was negatively correlated with the H R S D score (r = -0.65, P = 0.002 and r = -0.43, P = 0.046, respectively). DISCUSSION

Numerous reports indicate that the lymphocyte response to mitogens is significantly reduced in the aged individuals [9,14--19].

TABLE V IL-2-STIMULATED NK ACTIVITY (% 51Cr R E L E A S E ) ( M E A N ± S.D., n)

Effector/target cell ratio 50/1 Aged All Depressed Non-depressed Age 6 6 ~ 7 7 Age 78--85 Age 86--95 Adults

46.4* 34.6# 53.2 45.9 52.8 36.1 71.1

25/1

+ + ± + ± ± 4-

19.7 11.1 20.7 26.3 14.7 6.9 17.1

(19) (7) (12) (8) (7) (4) (6)

*P < 0.05 vs. adults. #P < 0.05 vs. non-depressed. t P < 0.05 vs. NK activity unstimulated.

43.1 34.4 48.2 41.6 50.3 33.8 59.1

12. 5/1

+ ± 44. ± ± 4-

19.2 16.6 19.4 25.2 14.8 7.7 16.9

31.9" 20.1# 38.8t 33.3 35.2 23.4 48.5

6.25/1

± 44± ± ± :t:

16.6 6.2 17 22.9 11.2 6.9 13.8

19 8.6# 25.1t 20 19.4 16.4 31

± ± + 4± ± 4-

12.9 7.2 11.6 16.1 12.1 9.6 10.9

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22 In some studies on psychiatric patients unselected for age [20,21], the presence of depressive illness was correlated with impairment in the proliferative capacity of lymphocytes. In the present study we demonstrate that depressive disorders in the elderly are correlated with a reduction in lymphocyte response to PHA. A negative correlation of lymphocyte proliferation with depression score was detected. One of the postulated reasons for impairment of lymphocyte proliferation in the aged is a reduction in cytokine production. A reduced production of IL-2 has been demonstrated in PHA-stimulated cultures of lymphocytes from aged humans (9,15,19,22,23). At variance, similar levels of IL-2 were found in supernatants of PHA-stimulated cells from aged and adult subjects [14]. In aged humans the production of y-IFN has been described as normal [23] as well as decreased [24]. In cultures of lymphocytes from aged donors, co-stimulated with phorbol myristate acetate (PMA) and calcium ionophore A23187, Chopra et al. [25] obtained similar levels of IL-2 and higher levels of y-IFN, as compared to young controls, suggesting that the defects observed in PHA-stimulated lymphocytes from the aged subjects could be due to alterations in the transmission of signals from the cell membrane to the nucleus. We found very low levels of IL-2, IL-4 and y-IFN in culture supernatants of PHAstimulated lymphocytes and a negative correlation of the levels of these cytokines with age was detected. Furthermore, cytokine production was negatively influenced by the presence of depression as a negative correlation with the H R S D score was detected. There is very little agreement among investigators on the modifications of NK activity during aging in humans, as it was found reduced [19,26], augmented [27], or unchanged [18,28,29]. Lighthart et al. [28] described a correlation between NK activity and number of CDI6+ cells in the peripheral blood, which were not diminished in the aged. In a SENIEUR population, on the contrary, Facchini et al. [29] detected increased numbers of circulating CDI6+ cells, but, normal NK activity in unfractionated cells. However, NK activity of purified CDI6+ cells was found decreased in aged subjects. These findings may be explained by an increase of CD16+/Leu7+ cells in the elderly, a subset of C D l 6 + cells exhibiting lower NK activity [30]. As far as the psychological aspects are concerned, a reduced NK activity was described in depressed adults [21,31] and in bereaved women [32]. In a previous study, we analyzed lymphocyte subsets and found similar levels of CDI6+ cells in the aged as compared to adults. Leu7+ cells were highly increased in the aged, more in the non-depressed aged than in the depressed ones. CD16+Leu7+ cells, however, were positively correlated with age and were increased in depressed aged (paper submitted). In the present study, we found that N K activity is lower in the aged and can be augmented by addition of IL-2 and ~-IFN, even though to a lesser extent than in adults. Higher levels of IL-2-induced NK activity were detectable in non-depressed

23 aged as c o m p a r e d to the depressed ones. IL-2- a n d I F N - s t i m u l a t e d N K activities were negatively correlated with the H R S D score. I n conclusion, the i m p a i r m e n t of lymphocyte activities was f o u n d m u c h more pron o u n c e d in depressed as c o m p a r e d to non-depressed aged subjects. The high frequency of depression in the elderly (35% o f o u r examined p o p u l a t i o n ) could a c c o u n t for the heterogeneity o f i m m u n e responsiveness observed in aging [14]. Since the depression score was previously f o u n d to be correlated with plasmatic a n d u r i n a r y cortisol levels, it appears that i m m u n o l o g i c a l studies of aging should carefully consider modifications of n e u r o e n d o c r i n e parameters. ACKNOWLEDGEMENTS This research was partly supported by the Italian D e p a r t m e n t o f E d u c a t i o n (Ministero P u b b l i c a Istruzione). We t h a n k the Social Service Officers from the M u n i c i p a l i t y of R o m e (Assessorato ai Servizi Sociali, C o m u n e di R o m a ) a n d Dr. L. De Gasperis, director o f the Casa di Riposo R o m a III, for their kind cooperation. REFERENCES 1 G.J. Ligthart, J.X. Corberand, C. Fournier, P. Galanaud, W. Hijmans, B. Kennes, H.K. MiillerHermelink and G.G. Steinmann, Admission criteria for immunogerontologicalstudies in man: the Senieur protocol. Mech. Ageing Dev., 28 (1984) 47--55. 2 G.H.B. Baker, Psychological factors and immunity. J. Psychosom. Res., 31 (1987) 1--10. 3 G.F. Solomon, Psychoneuroimmunology:interactions between central nervous system and immune system. J. Neurosci. Res., 18 (1987) 1--9. 4 M. Stein, S.E. Keller and S.J. Schleifer, Stress and immunomodulation: the role of depression and neuroendocrine function. J. lmmunol.. 135 (1985) 827s-829s. 5 B.A. Schindler, Stress, affective disorders and immune functions. Med. Clin. N. Am., 69 (1985) 585--597. 6 M. Hamilton, Developmentof a rating scale for primary depressive illness. Br. J. Soc. Clin. Psychol., 6 (1967) 278--296. 7 A.T. Beck and A. Beamesderfer, Assessment of depression: the depression inventory. In P. Pichot (ed.), Psychological Measurements in Psychopharmacology, Vol. 7. Karger, Basel, 1974, pp. 151--169. 8 A. Boyum, Isolation of mononuclear cells and granulocytes from human blood. Isolation of mononuclear cells by one centrifugation and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand. J. Clin. Lab. Invest., $97 (1968) 77--89. 9 S. Gillis, R. Kozak, M. Durante and M.E. Weksler, Immunologicalstudies of aging. Decreased production of and response to T cell growth factor by lymphocyte from aged humans../. Clin. Invest., 67 (1981) 937--942. 10 A. Sette, L. Adorini, E. Marubini and G. Doria, A microcomputer program for probit analysis of IL-2 titration data. J. lmmunol. Methods, 86 (1986) 265--269. 11 S. Landolfo, H. Kirchner and M.M. Simon, Production of immune interferon is regulated by more then one T cell subset: Lyt 1,2,3 and Qat-5 phenotypes of murine T lymphocytes involved in IFN gamma production in primary and secondary MLR. Eur. J. lmmunol.. 12 (1982) 295--299. 12 M.H. Julius, E. Simpson and L.A. Herzenberg, A rapid method for the isolation of functional thymus-derived murine lymphocytes. Eur. J. lmmunol., 3 (1973) 645--649.

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18 19 20 21 22

23 24 25

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