Increased Cytokine Production by Peripheral Blood Mononuclear Cells from Healthy Elderly People“ U. FAGIOL0,b A. COSSARIZZA,C S. SANTACATERINA: C. ORTOLAN1,d D. MONT1,C R. PAGANELLIF AND C. FRANCESCHY bInstitute of Internal Medicine University of Padua Padua, ltaly cInstitutc of General Pathology University of Modena Modena, Italy dSS. Giovanni e Paolo Hospital USLI6 Venice, Italy eDepartment ofAlletgology and Clinical Immunology University “La Sapienza Rome, Italy ”
Mammalian aging is associated with a complex derangement of the immune system. In particular, an impaired capability of T lymphocytes to proliferate when stimulated with a variety of mitogens has been reported. This defect was ascribed to a defective production and utilization of a lymphokirie that is crucial for T-cell proliferation, that is, interleukin-2 (IL-2).I However, immune responses are modulated and regulated by a variety of cytokines other than IL-2, whose production and utilization have not been properly studied during immunosenescence, particularly in humans. For this reason, we thought it worthwhile to study the production of other cytokines in supernatants from peripheral blood mononuclear cell cultures from old subjects. As the production of these cytokines can be deeply affected by the presence of clinical or subclinical pathological conditions, particular attention was paid to the selection of the subjects admitted to the study. Our results suggest that physiological aging is associated with a complex derangement of cytokine production. The production of some of them is increased, whereas that of some others is unaffected.
RThedata reported in this review article were obtained during experiments supported by C.N.R. (Progetto Finalizzato “Invecchiamento,” paper no. 92-1-209) and M.U.R.S.T. (40% and 60%) grants to U. Fagiolo and C. Franceschi. 490
FAGIOLO el al.: CYTOKINE PRODUCTION
49 1
MATERL4LS AND METHODS A total of 26 healthy subjects (13 young donors, 26.8 ? 0.8 years old; 13 aged subjects, 80.8 2.1 years) were studied. The aged population was selected according to the strict criteria of SENIEUR Protocol2among an initial population of more than 300 citizens. All of them gave their informed consent. Blood collection, separation of peripheral blood mononuclear cells by density gradient centrifugation, and cell cultures were performed as previously de~cribed.”~ Cytokines were measured in the supernatants of mitogen-stimulated cells. Phytohemagglutinin (PHA-P Difco, Detroit, MI; final concentration 1 pl/ml) or PHA-P plus 12-0-tetradecanoylphorbol 13-acetate (TPA, Sigma Chem. Co., St. Louis, MO; final concentration 1 ng/ml) were used. For each subject and time point, four replicates were prepared. Cultures were incubated a: 37°C in a humidified atmosphere of 5% COz and 95% air, and supernatants were collected after 24, 48, or 72 hours. They were stored at -80°C until use. Tumor necrosis factor-a (TNF-a)was also measured in the serum.
*
TABLE1. Cytokine Production by Peripheral Blood Mononuclear Cells from Aged Healthy Donors Cytokine IL-6 (spontaneous) IL-6 (TPA + PHA induced) TNF-a (spontaneous) TNF-a(TPA + PHA induced) IFN-.I (spontaneous) IFN-.I (PHA induced) IL-1p (spontaneous) IL-lp (TPA-induced)
Production“ Normal Increased Normal Increased Normal Normal Increased Increased
(1) (2)
(3) (4)
“In comparison with young healthy controls: (1) statistically significant after 48 and 72 hours of culture; (2) statistically significant after 24, 48, and 72 hours of culture; (3) statistically significant after 24 hours of culture; and (4) statistically significant after 24,48, and 72 hours of culture.
Cytokines were measured by the following ELISA kits: HU-IFN y ELISA kit from Hbt (Amsterdam, The Netherlands), IL-6 ELISA Kit, and TNF-a ELISA kit (both from Genzyme Corp., Boston, MA). IL-1p was measured by competitive radioimmunoassay purchased from Genzyme. Each supernatant aliquot was tested in duplicate and thawed only once. Measurements were taken according to the manufacturers’ instructions, and results were interpolated from the standard reference curve provided with each kit. RESULTS AND DISCUSSlON The results of this study are summarized in TABLE 1. A significant increase in IL-6 production was detected in cultures from aged donors from 43 hours onwards. A similar phenomenon was observed for TNF-aproduction. In this case, a significant difference was present also after 24 hours. This increased in vitro production of TNF-a after mitogen stimulation was not paralleled by an increased concentration in
492
ANNALS NEW YORK ACADEMY OF SCIENCES
the serum. Indeed, no significant difference in the TNF-a serum level was found between young and aged donors. IL-6 and TNF-a are produced by both monocytes and lymphocytes, and the stimulation with PHA+TPA represents an optimal signal for their production by both cell types. Thus, monocytes and lymphocytes could be responsible for this increased production of cytokines. To assess whether other cytokines, particularly those mainly produced by monocytes or T cells, were also produced at higher levels by cells from aged donors, the production of IL-lp and interferon-y (IFN-y) was also measured. IL-1 is mainly produced by adherent cells. A significant difference in IL-1p production was present at all the time points considered (24, 48, and 72 hours) between young and old subjects. However, the same percentage of monocytes was present in the cultures from the two groups. IFN-y, mainly produced by T lymphocytes, was undetectable in unstimulated cultures. At variance with IL-6 and TNF-a, no difference was found between cultures from young and aged donors at all the times considered. On the whole, our data suggest that a decreased production of cytokines, as reported for IL-2, is not a general phenomenon and that the production of different cytokines is likely regulated in an independent manner. Other investigators have reported an increased production of IL-4 and IFN-y in aged mice in which the . ~ age-associated increase in the producproduction of IL-2 is severely i m ~ a i r e dAn tion of IL-6 has also been reported in aged MRL/lpr mice: probably due to abnormal transcriptional control. Thus, an increased production of this cytokine may be a characteristic of physiological aging, not only in humans. The increase in IL-6 and TNF-a production was mainly present in stimulated cultures. The spontaneous production of all of the cytokines we checked did not differ between young and old subjects. Such a situation is mirrored by the serum concentration of at least one of them, that is, TNF-a, whose levels were similar in young and old subjects. The similar number of monocytes present in the blood of young and aged donors suggests that the increased production of some cytokines, particularly IL-1 p, cannot be ascribed to an increased number of producing cells and that monocytes from aged subjects are functionally intact. In conclusion, both monocytes and T lymphocytes are probably responsible for the increased production of cytokines occurring during the aging process. Dysregulation of cytokine production is probably a most important characteristic of aging, and this phenomenon may be responsible, at least in part, for age-related immune and nonimmune diseases. REFERENCES 1 . COSSARIZZA A,, D. MONTI,M. CANTINI, R. PAGANELLI, G. MONTAGNANI, R. CADOSSI, F. BERSANI & C. FRANCESCHI. 1989. Extremely low frequency pulsed electromagnetic fields increase interleukin-2 (IL-2) utilization and IL-2 receptor expression in lymphocytes from old subjects. FEBS Lett. 2 4 8 141-144. 2. LIGTHART, G. J., J. X. CORBERAND, C. FOURNIER, P. GARANAUD, W. HUMANS,B. KENNES, H. K. MULLER-HERMELINK & G. G. STEINMA”.1984. Admission criteria for immunogerontological studies in man: The SENIEUR protocol. Meih. Ageing Dev. 2 8 47-55. 3. COSSARIZZA, A., D. MONTI,G. MONTAGNANI, C. ORTOLANI, M. MASI,M. ZANNOTTI & C. 1990. Precocious aging of the immune system in Down’s syndrome: FRANCESCHI. Alterations of B-lymphocytes,T-lymphocyte subsets and of cells with NK markers in DS children. Am. J. Med. Genet. 7(suppl): 213-218.
FAGIOLO et al.: CYTOKINE PRODUCTION
493
4. COSSARIZZA, A., c . ORTOLANI, E. FORTI,G . MONTAGNANI, R. PAGANELLI, M. ZANNOTH, M. MARINI, D. MONTl & C. FRANCESCHI. 1991. Age-related expansion of functionally inefficient cells with markers of NK activity in Down's syndrome. Blood 77: 263-1270. 5 . NAGELKERKEN, L., A. HERTOGH-HUIJBREGTS, R. DOBBER & A. DRAGER.1991. Age-related changes in lymphokine production related to a decreased number of CD45RBh'CD4+ T cells. Eur. J. Immunol. 21: 273-281. 6. TANG,B., T. MATSUDA, S. AKIRA,N. NAGATA, S. IKEHARA, T. HIRANO & T. KISHIMOTO. 1991. Age-associated increase in interleukin 6 in MRL/lpr mice. Int. Immunol. 3: 273278.
Mammalian aging is associated with a complex derangement of the immune system. In particular, an impaired capability of T lymphocytes to proliferate when stimulated with a variety of mitogens has been reported. This defect was ascribed to a defective production and utilization of a lymphokirie that is crucial for T-cell proliferation, that is, interleukin-2 (IL-2).I However, immune responses are modulated and regulated by a variety of cytokines other than IL-2, whose production and utilization have not been properly studied during immunosenescence, particularly in humans. For this reason, we thought it worthwhile to study the production of other cytokines in supernatants from peripheral blood mononuclear cell cultures from old subjects. As the production of these cytokines can be deeply affected by the presence of clinical or subclinical pathological conditions, particular attention was paid to the selection of the subjects admitted to the study. Our results suggest that physiological aging is associated with a complex derangement of cytokine production. The production of some of them is increased, whereas that of some others is unaffected.
RThedata reported in this review article were obtained during experiments supported by C.N.R. (Progetto Finalizzato “Invecchiamento,” paper no. 92-1-209) and M.U.R.S.T. (40% and 60%) grants to U. Fagiolo and C. Franceschi. 490
FAGIOLO el al.: CYTOKINE PRODUCTION
49 1
MATERL4LS AND METHODS A total of 26 healthy subjects (13 young donors, 26.8 ? 0.8 years old; 13 aged subjects, 80.8 2.1 years) were studied. The aged population was selected according to the strict criteria of SENIEUR Protocol2among an initial population of more than 300 citizens. All of them gave their informed consent. Blood collection, separation of peripheral blood mononuclear cells by density gradient centrifugation, and cell cultures were performed as previously de~cribed.”~ Cytokines were measured in the supernatants of mitogen-stimulated cells. Phytohemagglutinin (PHA-P Difco, Detroit, MI; final concentration 1 pl/ml) or PHA-P plus 12-0-tetradecanoylphorbol 13-acetate (TPA, Sigma Chem. Co., St. Louis, MO; final concentration 1 ng/ml) were used. For each subject and time point, four replicates were prepared. Cultures were incubated a: 37°C in a humidified atmosphere of 5% COz and 95% air, and supernatants were collected after 24, 48, or 72 hours. They were stored at -80°C until use. Tumor necrosis factor-a (TNF-a)was also measured in the serum.
*
TABLE1. Cytokine Production by Peripheral Blood Mononuclear Cells from Aged Healthy Donors Cytokine IL-6 (spontaneous) IL-6 (TPA + PHA induced) TNF-a (spontaneous) TNF-a(TPA + PHA induced) IFN-.I (spontaneous) IFN-.I (PHA induced) IL-1p (spontaneous) IL-lp (TPA-induced)
Production“ Normal Increased Normal Increased Normal Normal Increased Increased
(1) (2)
(3) (4)
“In comparison with young healthy controls: (1) statistically significant after 48 and 72 hours of culture; (2) statistically significant after 24, 48, and 72 hours of culture; (3) statistically significant after 24 hours of culture; and (4) statistically significant after 24,48, and 72 hours of culture.
Cytokines were measured by the following ELISA kits: HU-IFN y ELISA kit from Hbt (Amsterdam, The Netherlands), IL-6 ELISA Kit, and TNF-a ELISA kit (both from Genzyme Corp., Boston, MA). IL-1p was measured by competitive radioimmunoassay purchased from Genzyme. Each supernatant aliquot was tested in duplicate and thawed only once. Measurements were taken according to the manufacturers’ instructions, and results were interpolated from the standard reference curve provided with each kit. RESULTS AND DISCUSSlON The results of this study are summarized in TABLE 1. A significant increase in IL-6 production was detected in cultures from aged donors from 43 hours onwards. A similar phenomenon was observed for TNF-aproduction. In this case, a significant difference was present also after 24 hours. This increased in vitro production of TNF-a after mitogen stimulation was not paralleled by an increased concentration in
492
ANNALS NEW YORK ACADEMY OF SCIENCES
the serum. Indeed, no significant difference in the TNF-a serum level was found between young and aged donors. IL-6 and TNF-a are produced by both monocytes and lymphocytes, and the stimulation with PHA+TPA represents an optimal signal for their production by both cell types. Thus, monocytes and lymphocytes could be responsible for this increased production of cytokines. To assess whether other cytokines, particularly those mainly produced by monocytes or T cells, were also produced at higher levels by cells from aged donors, the production of IL-lp and interferon-y (IFN-y) was also measured. IL-1 is mainly produced by adherent cells. A significant difference in IL-1p production was present at all the time points considered (24, 48, and 72 hours) between young and old subjects. However, the same percentage of monocytes was present in the cultures from the two groups. IFN-y, mainly produced by T lymphocytes, was undetectable in unstimulated cultures. At variance with IL-6 and TNF-a, no difference was found between cultures from young and aged donors at all the times considered. On the whole, our data suggest that a decreased production of cytokines, as reported for IL-2, is not a general phenomenon and that the production of different cytokines is likely regulated in an independent manner. Other investigators have reported an increased production of IL-4 and IFN-y in aged mice in which the . ~ age-associated increase in the producproduction of IL-2 is severely i m ~ a i r e dAn tion of IL-6 has also been reported in aged MRL/lpr mice: probably due to abnormal transcriptional control. Thus, an increased production of this cytokine may be a characteristic of physiological aging, not only in humans. The increase in IL-6 and TNF-a production was mainly present in stimulated cultures. The spontaneous production of all of the cytokines we checked did not differ between young and old subjects. Such a situation is mirrored by the serum concentration of at least one of them, that is, TNF-a, whose levels were similar in young and old subjects. The similar number of monocytes present in the blood of young and aged donors suggests that the increased production of some cytokines, particularly IL-1 p, cannot be ascribed to an increased number of producing cells and that monocytes from aged subjects are functionally intact. In conclusion, both monocytes and T lymphocytes are probably responsible for the increased production of cytokines occurring during the aging process. Dysregulation of cytokine production is probably a most important characteristic of aging, and this phenomenon may be responsible, at least in part, for age-related immune and nonimmune diseases. REFERENCES 1 . COSSARIZZA A,, D. MONTI,M. CANTINI, R. PAGANELLI, G. MONTAGNANI, R. CADOSSI, F. BERSANI & C. FRANCESCHI. 1989. Extremely low frequency pulsed electromagnetic fields increase interleukin-2 (IL-2) utilization and IL-2 receptor expression in lymphocytes from old subjects. FEBS Lett. 2 4 8 141-144. 2. LIGTHART, G. J., J. X. CORBERAND, C. FOURNIER, P. GARANAUD, W. HUMANS,B. KENNES, H. K. MULLER-HERMELINK & G. G. STEINMA”.1984. Admission criteria for immunogerontological studies in man: The SENIEUR protocol. Meih. Ageing Dev. 2 8 47-55. 3. COSSARIZZA, A., D. MONTI,G. MONTAGNANI, C. ORTOLANI, M. MASI,M. ZANNOTTI & C. 1990. Precocious aging of the immune system in Down’s syndrome: FRANCESCHI. Alterations of B-lymphocytes,T-lymphocyte subsets and of cells with NK markers in DS children. Am. J. Med. Genet. 7(suppl): 213-218.
FAGIOLO et al.: CYTOKINE PRODUCTION
493
4. COSSARIZZA, A., c . ORTOLANI, E. FORTI,G . MONTAGNANI, R. PAGANELLI, M. ZANNOTH, M. MARINI, D. MONTl & C. FRANCESCHI. 1991. Age-related expansion of functionally inefficient cells with markers of NK activity in Down's syndrome. Blood 77: 263-1270. 5 . NAGELKERKEN, L., A. HERTOGH-HUIJBREGTS, R. DOBBER & A. DRAGER.1991. Age-related changes in lymphokine production related to a decreased number of CD45RBh'CD4+ T cells. Eur. J. Immunol. 21: 273-281. 6. TANG,B., T. MATSUDA, S. AKIRA,N. NAGATA, S. IKEHARA, T. HIRANO & T. KISHIMOTO. 1991. Age-associated increase in interleukin 6 in MRL/lpr mice. Int. Immunol. 3: 273278.
