Mechanisms o f Ageing and Development, 59 ( 1991 ) 27--35 Elsevier Scientific Publishers Ireland Ltd.
27
SOLUBLE INTERLEUKIN-2 RECEPTOR RELEASE DEFECT IN VITRO IN
ELDERLY SUBJECTS
CALOGERO CARUSO a, GABRIELE DI LORENZO b, MARIA ASSUNTA MODICA a, GIUSEPPINA CANDORE a, MARIA RITA PORTELLI a, GRAZIA CRESCIMANNO b, ANTONINO INGRASSIA b, GIUSEPPE BARBAGALLO SANGIORGI b and ALFREDO SALERNO a ~aIstituto di Patologia generale dell'Universith di Palermo and t'Istituto di Medicina lnterna e Geriatria dell'Universitb di Palermo (Italy) (Received July 3rd, 1990) (Revision received November 2nd, 1990)
SUMMARY
The evidence from several studies indicates that as individuals age, they may display immune dysfunctions, mostly T cell dysfunctions. Recently, a soluble form of the receptor for interleukin-2 (IL-2) (slL-2R) has been demonstrated in human sera and in vitro stimulated culture supernatants from human T lymphocytes. In the present paper, we report in vitro slL-2R production from peripheral blood mononuclear cells in elderly subjects. The results show that no difference exists for unstimulated cultures, whereas after mitogen stimulation the elderly subjects showed the lowest values compared with young ones. These findings suggest that slL-2R may provide a new tool for the study of T lymphocyte dysfunctions in old age.
Key words: T cells; Elderly; Soluble form of receptor for IL-2 (slL-2R) INTRODUCTION
The study of ageing humans and experimental animals has documented many alterations in immune function, the most widely accepted of which is the dysfunction of T lymphocytes. Depression of T-dependent immunity is manifested by an impaired capacity to develop delayed hypersensivity, as well as by reduced lymphocyte response to T-dependent antigens and mitogens [ 1,2].
Correspondence to: Calogero Caruso, Istituto di Patologia generale, Corso Tukory 211, 90134 Palermo, Italy. 0047-6374/91/$03.50 Printed and Published in Ireland
© 1991 Elsevier Scientific Publishers Ireland Ltd.
28
Current theories of T cell activation recognize that upon reacting with a mitogen, T cells synthesize and secrete interteukin-2 (IL-2) and express IL-2 receptor (IL-2R). The acquisition of IL-2R on activated T cells requires the prescence of IL-2 for optimal receptor expression. Subsequent to the interaction with antigen or mitogen the progress of a T cell through the phases of the cell cycle is critically determined by a cascade ofintracellular signals generated by the binding of IL-2 to a IL-2R. Hence, both T cell and monocyte-derived interleukins regulate the T cell proliferative response [3 71. Recent studies have shown that activated T cells shed IL-2R (slL-2R) into the cell culture supernatant and that this soluble IL-2R is capable, under specific experimental conditions, of binding IL-2. While there is no clear evidence that slL-2R have a physiologic role, it might function to downregulate I L-2 driven immunoproliferation [8--11]. Previously, it has been demonstrated that IL-2 secretion by T lymphocytes of both experimental animals and humans decreased with age [ 1,2,12]. In the present study, we have examined elderly and young individuals for in vitro slL-2R production. The results show that the in vitro production of slL-2R is decreased in aged subjects. MATERIALS AND METHODS
Sample population Fifty-three subjects were studied; 21 were old subjects (8 females and 13 males, range 66--87 years). None of the selected subjects was affected by neoplastic, infectious, or autoimmune disease, nor received any drug influencing immune functions at the time of the study. The remainders consisted of 32 healthy adults (11 females and 21 males, range 2 1 ~ 8 years). None of these young subjects had a history of prolonged disease and none was ill or taking any drug at the time of the study.
Cell cultures [13
16]
Peripheral blood mononuclear cells (MNC) were isolated from fresh heparinized venous blood. The MNC were separated on Ficoll-Hypaque density gradient (Nycomed As Diagnostica, Oslo, Norway) and washed twice in complete medium RPMI 1640 (Gibco Grand Island, NY, U.S.A.) supplemented with 10% heat-inactivated fetal calf serum (Flow Laboratories, Irvine, Scotland, U.K.), gentamycin, streptomycin and Hepes (Boehringer, Mannheim GmbH, Germany). A cell suspension of 5 x 106/ml was incubated v/v, in 13 x 100 mm plastic cultures tubes (No., 2027, Falcon Plastics, Oxnard, California, U.S.A.), with 50 gg of phytohaemoagglutinin (PHA) (CalbiochemBehring Corp., San Diego, CA, U.S.A.) or as a control with complete medium alone. The 50 gg/ml dose of PHA was used because previous experiments showed it yielded the optimal IL-2 production [13,14,16]. The cell cultures were performed for 24 h at 37°C in a humidified incubator with 5% CO2. The cultures were harvested and centrifuged (600 rev./min for 10 min) to
29
separate the cells and supernatants; harvested supernatants were stored at 70°C until assay. In some experiments (N = 15) old or young MNC were divided in two aliquotes. The first one was processed as above discussed; the second one was stimulated for 48 h with PHA and proliferation assayed, as previously described [ 17 ]. Briefly, MNC (0.1 ml) were cultured in triplicate, in 96 fiat-bottom microtitre plates (Flow Laboratories); 0.1 ml of PHA was added at 50/~g/ml and the cultures maintained for 2 days in a humidified atmosphere at 37°C. Four hours before harvesting, each well was pulsed with 0.5/~Ci of [methyl-3H]thymidine (spec. act. 5.0 Ci/mmol: the Radiochemical Centre, Amersham, U.K.), and cultures were processed with an automated sample harvester for liquid scintillation counting. Radioactivity was measured in a counter and expressed as disintegrations per minute (dpm). The results were expressed as mean Adpm which is equal to the dpm of the cells in mitogencontaining media minus the dpm of the cells in medium without mitogen.
Quantification of slL-2R [ 14,15,18 ] An enzyme immunoassay test kit was used for the quantitative determination of slL-2R (Cellfree, T Cell Sciences, Inc., Cambridge, MA, U.S.A.). An anti-lL-2R monoclonal coating antibody was first adsorbed onto polystyrene microtiter wells. Soluble IL-2R present in the sample or standard binds to antibody on the coated well; unreacted sample components were removed by washing. An enzyme conjugated anti-IL-2R monoclonal antibody directed against a second epitope on the IL-2R molecule was then added and bound to the IL-2R captured by the first antibody. Unbound enzyme conjugated anti-IL-2R was removed during a wash step and substrate solution was added to the wells. A colored product was formed in proportion to the amount of slL-2R present in the sample. The reaction was terminated by addition to stop solution and absorbance at 490 nm was measured with a Titertek enzyme-linked immuno-sorbent assay (ELISA) reader (Flow, Rockville, MD, U.S.A.). The values of slL-2R concentrations were calculated by conversion to a numerical value of the absorbance of the test wells compared with the standard curve. The detection limit in our laboratory was 50 units/ml slL-2R.
Statistical analysis Data were expressed as mean ~ S.D. The values for the different groups were compared with Student's t-test. Correlations were calculated by linear regression. Statistical elaboration was performed by using Statgraphics, a statistical graphics system (STSC, Inc. and statistical graphics corporation). RESULTS
Concerning the production of slL-2R in the supernatant from unstimulated 24 h cultures (medium alone), in most subjects we observed very low production, i.e. under
31
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Fig. 2. Mean slL2-R production by PHA-stimulated MNC cultures in elderly (N = 21) and young (N = 32) individuals. The horizontalbars indicate the mean values ( ± S.D.) of slL2-R (units/2.5 x 106)which are significantly (P = 0.045) higher for young group (333.8 ± 171.6 versus 233.7 ± 193.4).
The exact role played by released IL-2R in T cell proliferation remains to be clarified. Thus, there is no solid evidence for the time being to show that sIL-2R participates in T cell responses. Therefore, the age-associated alteration in the production of sIL-2R observed in this study could not be a contributable factor for the age-associated immune function impairment. To determine whether slL-2R concentration accumulated by an individual's M N C after 24 h of stimulation with PHA is associated with the lymphocyte proliferative response, we used regression analysis to compare sIL-2R levels and proliferative responses in 15 subjects, both young and old ones. Figure 3 shows that there is a significant correlation between levels of sIL-2R at 24 h and proliferation to PHA at 48 h (r = 0.61; P = 0.015). DISCUSSION These experiments show that after 24 h in vitro stimulation with PHA cultures from elderly individuals are characterized by a decreased release of slL-2R compared with
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Fig. 3. Correlation between sIL-2R level measured at 24 h of culture and proliferative response to PHA determined at 48 h of culture (r = 0.61: P = 0.015).
cultures from young subjects. Recently, it has been demonstrated that age associated immune defect shows a wide range of impairment since a considerable proportion of elderly subjects displays responses comparable with those of adult controls [ 19]. Accordingly we can see that several old subjects display slL-2R values comparable with those of young subjects. However, healthy aged subjects taken as a whole display reduced SIL-2R release. It is well known that lymphocytes are activated by IL-2 through the interaction of IL-2 with a specific IL-2 receptor. This receptor is composed by two different chains of 75 k D a and 55 k D a respectively. The 55 k D a subunit is also known as Tac or CD25 and can be recognized by specific antibodies. Lymphocytes increase their expression of CD25 upon in vitro activation and release the CD25 molecule in the supernatant or in sera when the process occurs in vivo [3,4,6,8--12,20]. The exact function of released IL-2P. remains to be clarified. If slL-2R could clearly downregulate the IL-2-driven proliferation, logically, one would expect to observe an increase with age in slL-2R production because slL-2R, presumably, competes for binding of IL-2 and makes less IL-2 molecules available for the cell, dependent on IL-2 to proliferate. Although it has been suggested that the role of the released IL-2R is to downregulate IL-2-dependent cell-mediated responses, other possibilities for the function of this protein should be considered. The rapid increase in soluble IL-2R after cellular activation has occurred may represent the means, by a proteolytic cleavage mechanism, for activated cells to reduce surface receptor density below a critical threshold level, thus preventing an ongoing, perhaps undesirable, response. Alternatively, the pro-
33 duction of released IL-2R may locally confine the effects of IL-2, hence keeping immune responses local. Another possibility would be that the soluble IL-2R could serve as a binding protein, effectively prolonging the half-life of IL-2 [10,11]. However, our results (Fig. 3) demonstrate a high correlation between production of slL-2R and T cells proliferation of both young and old individuals suggesting that the age-associated alteration in the production of slL-2R observed in this study should be a contributable factor for the decreased T cell proliferation manifested by many groups [1,2] in the ageing population. Several aberrations of the IL-2/IL-2R system have been observed in elderly subjects, including decreased IL-2 production, impaired responsiveness to exogenous IL-2, altered cellular expression of IL-2R, increased serum levels of slL-2R and decreased slL-2R in vitro production [1, 2, 12, 21--24 and present results]. Concerning the low synthesis of IL-2 in both aged humans and age experimental animals, the most significant element controlling the quantity of IL-2 produced by aged lymphocytes appears to be the decreased number of precursors of T helper lymphocytes which seem the major cellular source of IL-2 [25]. Expression oflI-2 receptor is also deficient in aged animals and humans [2,22,23]. Since II-2 modulated the expression of the IL-2R, the decreased production of slL-2R can be due to diminished IL-2 synthesis [26]. In a recent report, levels of slL-2R values were studied in 13 young and 15 old subjects. Seventy-two-hour culture supernatants of cells from elderly donors contained significantly less IL-2 and slL-2R in the culture media compared to cultures of cells from young donors. Obtained results suggested, then, that decreased expression of both IL-2 and IL-2R mRNA contributed to the low synthesis of IL-2 and IL-2R, respectively [ 23 ]. As a matter of fact, high affinity IL-2R are similarly expressed on PHA-stimulated lymphoblasts from young and elderly individuals, but there are markedly fewer lymphoblasts in 72 h cultures of cells from elderly as compared to young individuals [24]. Recently, in order to analyze the first steps of T cell activation in elderly subjects, we studied in vitro interleukin production by peripheral mononuclear cells after 24 h PHA stimulation [27]. There was a significant decrease in IL-2 and gammainterferon (7-IFN) production by mononuclear cells of the elderly subjects with respect to young ones. No significant difference was instead observed between the old subjects and young ones as regards interleukin-4 (IL-4) production. These preliminary results suggest that in elderly age there is an impairment of Th l-like cells which produce IL-2 and 3~-IFN, but not of TH2-1ike cells that produce IL-4 [28--30]. Concerning the mechanisms of these impairments, one possibility can be a relative loss of T helper cells. However, it is not possible to show consistent changes in the percentage of T cells and CD4 ÷ or CD8 ÷ subsets in elderly individuals [31]. Although rough abnormalities in T cell subsets have not been found, more subtle differences remain a possibility. In fact, we have recently observed by using limiting dilution microculture techniques that the fraction of mitogen-responsive T cell precursors is reduced in old subjects [32,33].
34
The evidence from experimental studies indicates that age-associated changes in immune functions are contributing significantly to the increase in susceptibility to certain diseases like the autoimmune ones [ 1 ]. It is intriguing that the same aberrations of IL2/IL2-R system have been described in normal young subjects with HLAB8,DR3 phenotype and in patients with autoimmune diseases linked to this phenotype [13--16,18,34 37]. ACKNOWLEDGEMENT
This work was supported by grants from Ministero della Pubblica Istruzione, Rome. REFERENCES
1 2 3 4 5 6 7 8 9
10
11 12 13
14
15
16 17
T. Makinodan, J. Lubinski and T.C. Fong, Cellular, biochemical, and molecular basis of T-cell senescence. Arch. Pathol. Lab. Med., 111 (1987) 910--914. M.L. T h o m a n and W.O. Weigle, The cellular and subcellular bases of immunosenescence. Adv. lmmunol., 46 (1989) 221--261. G. Moiler (ed.), Interleukins and lymphocyte activation, lmmunol. Rev., 63 (1982) 5--209. G. Moiler (ed.), IL-2: receptors and genes. ImmunoL Rev., 92 (1986) 1--156. G. Moiler (ed.), G a m m a interferon, lmmunoL Rev., 97 (1987) 5 ~ 5 . W.C. Greene and W.J. Leonard, The h u m a n interleukin-2 receptor. Annu. Rev. lmmunol., 4 (1986) 69--95. W.E. Paul and J. Ohara, B-cell stimulatory l:actor-l/interleukin-4. Annu. Rev. Immttntd.. 5 (1987) 429 459. W.C. Greene and W.J. Leonard, The h u m a n interleukin-2-receptor. Annu. Rev. lmmunoL, 4 (1986) 69--95. L.A. Rubin, C.C. Kurman, W.E. Biddison, N.D. Goldman and D.L. Nelson, A monoclonal antibody, 7G7/B6, binds to an epitope of h u m a n interleukin-2 (1L-2) receptor that is distinct from that recognized by IL-2 or anti-TAC. Hybridoma, 4 (1985) 91--102. L.A. Rubin, C. Kurman, M.E. Fritz, W.E. Biddison, B. Boutin, R. Yarchoan and D.L. Nelson, Soluble interleukin-2 receptors are released from activated h u m a n lymphoid cells in vitro. J. ImmunoL, 135 (1985) 3172--3177. L.A. Rubin, G. Jay and D.L. Nelson, The released interleukin-2 receptor binds interleukin-2 efficiently. J. lmmunoL, 137 (1986) 3841--3844. S.R.C. Gottesman, Changes in T cell mediated immunity with age - - an update. Rev. Biol. Res. Aging, 3 (1987) 95--127. C. Giordano, C. Caruso, F. Pant/), M.A. Modica, A.M. Zambito, M.P. Amato and A. Galluzzo, Dissociated production of interleukin-2 and immune gamma-interferon by phytohemoagglutinin stimulated peripheral mononuclear cells in type I (insulin-dependent) diabetes. J. C/in. Lab. hTmnmo/., 27 (1988) 73--76. C. Giordano, F. Pant/), C. Caruso, M.A. Modica, A.M. Zambilo, N. Sapienza. M.P. Amato and A. Galluzzo, Interleukin-2 and soluble interleukin 2-receptor secretion dcfect in vitro in newly diagnosed type 1 diabetic patients. Diahetes, 38 (1989) 310 315. M.A. Modica, G. Di Lorenzo, A. Galluzzo, C. Giordano, M.R. Portelli, G. Candore and C. Caruso. Soluble interleukin-2 receptor secretion defect in vitro in HLA-B8, DR3 positive subjects. Autoimmunity, 7 (1990) 87 96. M.A. Modica, A.M. Zambito, G. Candore and C. Caruso, Markers o f t lymphocyte activation in HLA-BS, DR3 positive subjects, hmnunobioh~g.v. 181 (1990) 257 266. M.A. Modica, G. C a m m a r a t a and C. Caruso, HLA-BS,DR3 phenotype and lymphocytc responses to phytohaemoagglutinin. J. hJmumogenet., 17 (1990) 101 It)7.
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36 37
C. Giordano, A. Galluzzo, A. Marco, F. Pant6, M.P. Amato, C. Caruso and G.D. Bompiani, Increased soluble interleukin-2 receptor levels in the sera of type 1 diabetic patients. Diabetes Res.. 8 (1988) 135--138. W. Barcellini, M.O. Borghi, C. Sguotti, R. Palmieri, D. Frasca, P.L. Meroni, G. Doria and C. Zanussi, Heterogeneity of immune responsiveness in healthy elderly subjects. Clin. lmmunol, hnmunoputhol.. 47 (1988) 142--151. G. Pizzolo, The soluble interleukin-2 receptor as a new biological marker in diseases, lmmunol. Clin., 7 (1988) 13--21. C. Saadeh, C. Auzenne, D. Nelson and F. Orson, Sera from the aged contain higher levels of the IL-2 receptor compared to young adults. Fed. Proc.. 45 (1986) 378. S. Negoro, H. Hara, S. Miyata, O. Saiki, T. Tanaka, K. Yoshizaki, T. lgarashi and S. Kishimoto, Mechanisms of age-related decline in antigen-specific T cell proliferative response: IL-2 receptor expression and recombinant IL-2 induced proliferative response of purified Tac-positive T cells. Mech. Ageing Dev.. 36 (1986) 223--241. J.E. Nagel, R.K. Chopra, F.J. Chrest, M.T. McCoy, E.L. Schneider, N.J. Holbrook and W.H. Adler, Decreased proliferation, interleukin 2 synthesis, and interleukin 2 receptor expression are accompanied by decreased mRNA expression in phytohemagglutinin-stimulatedcells from elderly donors. J. Clin. Invest., 81 (1988) 1096--1102. J.E. Nagel, R.K. Chopra, D.C. Powers and W.H. Adler, Effect of age on the human high affinity interleukin 2 receptor of phytohaemagglutinin stimulated peripheral blood lymphocytes. Clin. Exp. lmmunol., 75 (1989) 286--291. R.A. Miller, Age-associated decline in precursor frequency for different T cell-mediated reactions with preservation of helper of cytotoxic effect per precursor cell. J. Immunol., 132 (1984) 6 3 ~ 8 . K.A. Smith and D.A. Cantrell, Interleukin 2 regulates its own receptors. Proe. Natl. Acad. Sci. U.S.A. 82 (1985) 864--868. C. Caruso, G. Candore, M.A. Modica, M.R. Portelli, M. Durante and G. Di Lorenzo, Dissociated production of interleukin-4 and gamma-interferon by PHA stimulated mononuclear cells in elderly subjects. European Federation o['hnmunological Societies, 10th Meeting Edinburgh, (1990) abstract 38- I. B.A. Tortes, W.L..Farrar and H.M. Johnson, Interleukin-2 regulates immune interferon (IFN-',/) production by normal and suppressor cell cultures. J. Immunol., 128 (1982) 2217--2219. T.R. Mosmann and R.L. Coffman, Different patterns oflymphokine secretion lead to different functional properties. Annu. Rev. Immunol., 7 (1989) 145--173. T.R. Mosmann and R.L. Coffman, Heterogeneity of cytokine secretio patterns and functions of helper T cells. Adv. ImmunoL, 46 (1989) 111--147. D. Matour, M. Melnicoff, D. Kaye and D.M. Murasko, The role o f t cell phenotypes in decreased lymphoproliferation of the elderly. Clin. hmnunol, hmJlunopathol.. 50 (19891 82--99. C. Caruso, M.A. Modica, G. Candore, G. Di Lorenzo, M. Durante and C. Di Giulio, In vivo thymopentin modulation of mitogen responsive T cell precursor frequency, hu. J. hmnunother.. 5 (1989) 133--137. C. Caruso, G. Candore, M.A. Modica, C. Di Giulio, A. lngrassia and G. Di Lorenzo, In vitro thymopentin modulation of mitogen responsive T cell precursor frequency. Thymus, (1990) in press. M. Malkovski, P.M. Sondel, W. Strober and A.G. Dalgleish, The interleukins in acquired disease. Clin. Exp. lmmunol., 74 (1988) 151--161. M.N. Manoussakis, G.K. Papadopoulos, A.A. Drosos and H.M. Moutsopoulos, Soluble interleukin 2 receptor molecules in the serum of patients with autoimmune diseases. Clin. Immunol. Immunopathol., 50 (1989) 321--332. C. Caruso, G. Candore and M.A. Modica, Non response to hepatitis B vaccine. New. Engl. J. Med., 322 (1990) 550~551 (letter). C. Caruso, G. Candore and M.A. Modica, HLA-DR3 and immunoresponsiveness. Lancet, 336 (1990) 50~-507 (letter).
27
SOLUBLE INTERLEUKIN-2 RECEPTOR RELEASE DEFECT IN VITRO IN
ELDERLY SUBJECTS
CALOGERO CARUSO a, GABRIELE DI LORENZO b, MARIA ASSUNTA MODICA a, GIUSEPPINA CANDORE a, MARIA RITA PORTELLI a, GRAZIA CRESCIMANNO b, ANTONINO INGRASSIA b, GIUSEPPE BARBAGALLO SANGIORGI b and ALFREDO SALERNO a ~aIstituto di Patologia generale dell'Universith di Palermo and t'Istituto di Medicina lnterna e Geriatria dell'Universitb di Palermo (Italy) (Received July 3rd, 1990) (Revision received November 2nd, 1990)
SUMMARY
The evidence from several studies indicates that as individuals age, they may display immune dysfunctions, mostly T cell dysfunctions. Recently, a soluble form of the receptor for interleukin-2 (IL-2) (slL-2R) has been demonstrated in human sera and in vitro stimulated culture supernatants from human T lymphocytes. In the present paper, we report in vitro slL-2R production from peripheral blood mononuclear cells in elderly subjects. The results show that no difference exists for unstimulated cultures, whereas after mitogen stimulation the elderly subjects showed the lowest values compared with young ones. These findings suggest that slL-2R may provide a new tool for the study of T lymphocyte dysfunctions in old age.
Key words: T cells; Elderly; Soluble form of receptor for IL-2 (slL-2R) INTRODUCTION
The study of ageing humans and experimental animals has documented many alterations in immune function, the most widely accepted of which is the dysfunction of T lymphocytes. Depression of T-dependent immunity is manifested by an impaired capacity to develop delayed hypersensivity, as well as by reduced lymphocyte response to T-dependent antigens and mitogens [ 1,2].
Correspondence to: Calogero Caruso, Istituto di Patologia generale, Corso Tukory 211, 90134 Palermo, Italy. 0047-6374/91/$03.50 Printed and Published in Ireland
© 1991 Elsevier Scientific Publishers Ireland Ltd.
28
Current theories of T cell activation recognize that upon reacting with a mitogen, T cells synthesize and secrete interteukin-2 (IL-2) and express IL-2 receptor (IL-2R). The acquisition of IL-2R on activated T cells requires the prescence of IL-2 for optimal receptor expression. Subsequent to the interaction with antigen or mitogen the progress of a T cell through the phases of the cell cycle is critically determined by a cascade ofintracellular signals generated by the binding of IL-2 to a IL-2R. Hence, both T cell and monocyte-derived interleukins regulate the T cell proliferative response [3 71. Recent studies have shown that activated T cells shed IL-2R (slL-2R) into the cell culture supernatant and that this soluble IL-2R is capable, under specific experimental conditions, of binding IL-2. While there is no clear evidence that slL-2R have a physiologic role, it might function to downregulate I L-2 driven immunoproliferation [8--11]. Previously, it has been demonstrated that IL-2 secretion by T lymphocytes of both experimental animals and humans decreased with age [ 1,2,12]. In the present study, we have examined elderly and young individuals for in vitro slL-2R production. The results show that the in vitro production of slL-2R is decreased in aged subjects. MATERIALS AND METHODS
Sample population Fifty-three subjects were studied; 21 were old subjects (8 females and 13 males, range 66--87 years). None of the selected subjects was affected by neoplastic, infectious, or autoimmune disease, nor received any drug influencing immune functions at the time of the study. The remainders consisted of 32 healthy adults (11 females and 21 males, range 2 1 ~ 8 years). None of these young subjects had a history of prolonged disease and none was ill or taking any drug at the time of the study.
Cell cultures [13
16]
Peripheral blood mononuclear cells (MNC) were isolated from fresh heparinized venous blood. The MNC were separated on Ficoll-Hypaque density gradient (Nycomed As Diagnostica, Oslo, Norway) and washed twice in complete medium RPMI 1640 (Gibco Grand Island, NY, U.S.A.) supplemented with 10% heat-inactivated fetal calf serum (Flow Laboratories, Irvine, Scotland, U.K.), gentamycin, streptomycin and Hepes (Boehringer, Mannheim GmbH, Germany). A cell suspension of 5 x 106/ml was incubated v/v, in 13 x 100 mm plastic cultures tubes (No., 2027, Falcon Plastics, Oxnard, California, U.S.A.), with 50 gg of phytohaemoagglutinin (PHA) (CalbiochemBehring Corp., San Diego, CA, U.S.A.) or as a control with complete medium alone. The 50 gg/ml dose of PHA was used because previous experiments showed it yielded the optimal IL-2 production [13,14,16]. The cell cultures were performed for 24 h at 37°C in a humidified incubator with 5% CO2. The cultures were harvested and centrifuged (600 rev./min for 10 min) to
29
separate the cells and supernatants; harvested supernatants were stored at 70°C until assay. In some experiments (N = 15) old or young MNC were divided in two aliquotes. The first one was processed as above discussed; the second one was stimulated for 48 h with PHA and proliferation assayed, as previously described [ 17 ]. Briefly, MNC (0.1 ml) were cultured in triplicate, in 96 fiat-bottom microtitre plates (Flow Laboratories); 0.1 ml of PHA was added at 50/~g/ml and the cultures maintained for 2 days in a humidified atmosphere at 37°C. Four hours before harvesting, each well was pulsed with 0.5/~Ci of [methyl-3H]thymidine (spec. act. 5.0 Ci/mmol: the Radiochemical Centre, Amersham, U.K.), and cultures were processed with an automated sample harvester for liquid scintillation counting. Radioactivity was measured in a counter and expressed as disintegrations per minute (dpm). The results were expressed as mean Adpm which is equal to the dpm of the cells in mitogencontaining media minus the dpm of the cells in medium without mitogen.
Quantification of slL-2R [ 14,15,18 ] An enzyme immunoassay test kit was used for the quantitative determination of slL-2R (Cellfree, T Cell Sciences, Inc., Cambridge, MA, U.S.A.). An anti-lL-2R monoclonal coating antibody was first adsorbed onto polystyrene microtiter wells. Soluble IL-2R present in the sample or standard binds to antibody on the coated well; unreacted sample components were removed by washing. An enzyme conjugated anti-IL-2R monoclonal antibody directed against a second epitope on the IL-2R molecule was then added and bound to the IL-2R captured by the first antibody. Unbound enzyme conjugated anti-IL-2R was removed during a wash step and substrate solution was added to the wells. A colored product was formed in proportion to the amount of slL-2R present in the sample. The reaction was terminated by addition to stop solution and absorbance at 490 nm was measured with a Titertek enzyme-linked immuno-sorbent assay (ELISA) reader (Flow, Rockville, MD, U.S.A.). The values of slL-2R concentrations were calculated by conversion to a numerical value of the absorbance of the test wells compared with the standard curve. The detection limit in our laboratory was 50 units/ml slL-2R.
Statistical analysis Data were expressed as mean ~ S.D. The values for the different groups were compared with Student's t-test. Correlations were calculated by linear regression. Statistical elaboration was performed by using Statgraphics, a statistical graphics system (STSC, Inc. and statistical graphics corporation). RESULTS
Concerning the production of slL-2R in the supernatant from unstimulated 24 h cultures (medium alone), in most subjects we observed very low production, i.e. under
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Fig. 2. Mean slL2-R production by PHA-stimulated MNC cultures in elderly (N = 21) and young (N = 32) individuals. The horizontalbars indicate the mean values ( ± S.D.) of slL2-R (units/2.5 x 106)which are significantly (P = 0.045) higher for young group (333.8 ± 171.6 versus 233.7 ± 193.4).
The exact role played by released IL-2R in T cell proliferation remains to be clarified. Thus, there is no solid evidence for the time being to show that sIL-2R participates in T cell responses. Therefore, the age-associated alteration in the production of sIL-2R observed in this study could not be a contributable factor for the age-associated immune function impairment. To determine whether slL-2R concentration accumulated by an individual's M N C after 24 h of stimulation with PHA is associated with the lymphocyte proliferative response, we used regression analysis to compare sIL-2R levels and proliferative responses in 15 subjects, both young and old ones. Figure 3 shows that there is a significant correlation between levels of sIL-2R at 24 h and proliferation to PHA at 48 h (r = 0.61; P = 0.015). DISCUSSION These experiments show that after 24 h in vitro stimulation with PHA cultures from elderly individuals are characterized by a decreased release of slL-2R compared with
32 700~ 600:-
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L
54
60
_
:
7
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J
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D P M x 1000
Fig. 3. Correlation between sIL-2R level measured at 24 h of culture and proliferative response to PHA determined at 48 h of culture (r = 0.61: P = 0.015).
cultures from young subjects. Recently, it has been demonstrated that age associated immune defect shows a wide range of impairment since a considerable proportion of elderly subjects displays responses comparable with those of adult controls [ 19]. Accordingly we can see that several old subjects display slL-2R values comparable with those of young subjects. However, healthy aged subjects taken as a whole display reduced SIL-2R release. It is well known that lymphocytes are activated by IL-2 through the interaction of IL-2 with a specific IL-2 receptor. This receptor is composed by two different chains of 75 k D a and 55 k D a respectively. The 55 k D a subunit is also known as Tac or CD25 and can be recognized by specific antibodies. Lymphocytes increase their expression of CD25 upon in vitro activation and release the CD25 molecule in the supernatant or in sera when the process occurs in vivo [3,4,6,8--12,20]. The exact function of released IL-2P. remains to be clarified. If slL-2R could clearly downregulate the IL-2-driven proliferation, logically, one would expect to observe an increase with age in slL-2R production because slL-2R, presumably, competes for binding of IL-2 and makes less IL-2 molecules available for the cell, dependent on IL-2 to proliferate. Although it has been suggested that the role of the released IL-2R is to downregulate IL-2-dependent cell-mediated responses, other possibilities for the function of this protein should be considered. The rapid increase in soluble IL-2R after cellular activation has occurred may represent the means, by a proteolytic cleavage mechanism, for activated cells to reduce surface receptor density below a critical threshold level, thus preventing an ongoing, perhaps undesirable, response. Alternatively, the pro-
33 duction of released IL-2R may locally confine the effects of IL-2, hence keeping immune responses local. Another possibility would be that the soluble IL-2R could serve as a binding protein, effectively prolonging the half-life of IL-2 [10,11]. However, our results (Fig. 3) demonstrate a high correlation between production of slL-2R and T cells proliferation of both young and old individuals suggesting that the age-associated alteration in the production of slL-2R observed in this study should be a contributable factor for the decreased T cell proliferation manifested by many groups [1,2] in the ageing population. Several aberrations of the IL-2/IL-2R system have been observed in elderly subjects, including decreased IL-2 production, impaired responsiveness to exogenous IL-2, altered cellular expression of IL-2R, increased serum levels of slL-2R and decreased slL-2R in vitro production [1, 2, 12, 21--24 and present results]. Concerning the low synthesis of IL-2 in both aged humans and age experimental animals, the most significant element controlling the quantity of IL-2 produced by aged lymphocytes appears to be the decreased number of precursors of T helper lymphocytes which seem the major cellular source of IL-2 [25]. Expression oflI-2 receptor is also deficient in aged animals and humans [2,22,23]. Since II-2 modulated the expression of the IL-2R, the decreased production of slL-2R can be due to diminished IL-2 synthesis [26]. In a recent report, levels of slL-2R values were studied in 13 young and 15 old subjects. Seventy-two-hour culture supernatants of cells from elderly donors contained significantly less IL-2 and slL-2R in the culture media compared to cultures of cells from young donors. Obtained results suggested, then, that decreased expression of both IL-2 and IL-2R mRNA contributed to the low synthesis of IL-2 and IL-2R, respectively [ 23 ]. As a matter of fact, high affinity IL-2R are similarly expressed on PHA-stimulated lymphoblasts from young and elderly individuals, but there are markedly fewer lymphoblasts in 72 h cultures of cells from elderly as compared to young individuals [24]. Recently, in order to analyze the first steps of T cell activation in elderly subjects, we studied in vitro interleukin production by peripheral mononuclear cells after 24 h PHA stimulation [27]. There was a significant decrease in IL-2 and gammainterferon (7-IFN) production by mononuclear cells of the elderly subjects with respect to young ones. No significant difference was instead observed between the old subjects and young ones as regards interleukin-4 (IL-4) production. These preliminary results suggest that in elderly age there is an impairment of Th l-like cells which produce IL-2 and 3~-IFN, but not of TH2-1ike cells that produce IL-4 [28--30]. Concerning the mechanisms of these impairments, one possibility can be a relative loss of T helper cells. However, it is not possible to show consistent changes in the percentage of T cells and CD4 ÷ or CD8 ÷ subsets in elderly individuals [31]. Although rough abnormalities in T cell subsets have not been found, more subtle differences remain a possibility. In fact, we have recently observed by using limiting dilution microculture techniques that the fraction of mitogen-responsive T cell precursors is reduced in old subjects [32,33].
34
The evidence from experimental studies indicates that age-associated changes in immune functions are contributing significantly to the increase in susceptibility to certain diseases like the autoimmune ones [ 1 ]. It is intriguing that the same aberrations of IL2/IL2-R system have been described in normal young subjects with HLAB8,DR3 phenotype and in patients with autoimmune diseases linked to this phenotype [13--16,18,34 37]. ACKNOWLEDGEMENT
This work was supported by grants from Ministero della Pubblica Istruzione, Rome. REFERENCES
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