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In Vitro erythrocidal activity of activated spleen cells from young and old mice
Experimental Gerontology 35 (2000) 409 – 416
In Vitro erythrocidal activity of activated spleen cells from young and old mice Rajiv K. Saxenaa,b,*, William H. Adlera,b School of Life Sciences, Jawaharlal Nehru University, New Delhi, India Immunology Section, GRC, NIA, National Institutes of Health, Baltimore, MD, USA Received 7 January 2000; received in revised form 22 February 2000; accepted 22 February 2000
Abstract We have recently reported that activated mouse spleen mononuclear cells (MNCs) efficiently lyse autologous erythrocytes in vitro (Saxena and Chandrasekhar, 2000). In the present study, we have investigated erythrocyte-depleting ability (EDA) of spleen MNCs from young and old mice. Time kinetics of survival of erythrocytes in mitogen-activated spleen cell cultures indicated that the erythrocyte depletion was significantly faster in young spleen cell cultures than in the old. Poorer EDA of old MNCs was in spite of the fact that the susceptibility to lysis actually increased in erythrocytes from old mice. Erythrocytes opsonized by a hamster anti mouse Fas monoclonal antibody, were destroyed with a much greater efficiency by young MNCs, whereas the corresponding effect of opsonization was only moderate for old MNCs. Depletion of macrophages from MNC preparations by plastic adherence as well as carbonyl-iron and magnet treatment had a marginal if any effect on EDA of young and old mouse MNCs, indicating that a lower lymphocyte-associated erythrocidal activity as one of the factors responsible for overall lower EDA associated with spleen derived MNCs of old mice. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Erythrocytes; Aging; Fas; Cytotoxicity; Phagocytosis
1. Introduction Many parameters of T-cell activity are known to decline in the aged. These include T-cell proliferation in response to stimulation through T cell receptor and co-stimulators (Lerner et al., 1988; Song et al., 1993; Engwerda et al., 1994), production and response to growth promoting cytokines (Saxena et al., 1988; Nagelkerken et al., 1991; Ernst et al., 1993) and activation of cytotoxic lymphocytes (Hirano and Nordin, 1976; Saxena et al., 1984; Powers and Belshe, 1993; Mbawuike et al., 1997). We have previously character* Corresponding author. Tel.: ⫹1-304-285-6314; fax: ⫹1-304-285-6126. E-mail address: [email protected] (R.K. Saxena). 0531-5565/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 5 3 1 - 5 5 6 5 ( 0 0 ) 0 0 0 9 1 - 7
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ized mitogen-induced cytotoxic cells in spleen cells from young and old mice and reported differences in their cytolytic activities (Saxena et al., 1981, 1988). Cytotoxic cells generated in young mouse spleen cells activated with Con-A efficiently lysed Fasexpressing cell lines like Hut77 and Jurkat, whereas effector cells generated in old mouse spleen cells had poor lytic activity against these targets (Saxena and Adler, 1999). Con-A activated old spleen cells were, however, markedly more cytotoxic against low Fasexpressing cell lines like P815 (Saxena et al., 1988, Saxena and Adler, 1999). It was proposed that killing through Fas-FasL interaction might be relatively less efficient for activated spleen cells from old mice (Saxena and Adler, 1999). Recently, we have studied the destruction of autologous erythrocytes by Con-A activated mouse spleen mononuclear cells (MNCs) in vitro, and have reported that the erythrocyte counts fall rapidly when co-cultured with MNCs (Saxena and Chandrasekhar, 2000). Phagocytosis of erythrocytes by macrophages in MNC preparations as well as a direct cell-mediated lysis of erythrocytes, contributed to the erythrocyte destruction in vitro (Saxena and Chandrasekhar, 2000). In addition, a role for Fas-FasL and analogous TNF-TNFR interactions in recognition and killing of erythrocytes by spleen MNCs was also suggested (Saxena, 2000; Saxena and Chandrasekhar, 2000). Because FasL-mediated cytotoxic activity appears to be deficient in mitogen-activated old mouse spleen cells, as discussed above, it was of interest to see if young and old MNCs differed in their efficiencies to destroy autologous erythrocytes. In the present study, we have shown that MNCs from old mouse spleen cells had significantly lower erythrocidal activity than the MNCs from young mice. Differences in the erythrocidal activity of old and young MNCs persisted even if macrophages were depleted from the MNC preparations or erythrocytes were opsonized by anti-Fas antibodies.
2. Materials and methods 2.1. Effector and Target cells Spleen effector cells used in this study were derived from young or old C57Bl/6 mice. Young male C57Bl/6 mice (12 to 16 weeks) were obtained from Jackson Laboratories. Old male C57Bl/6 mice (age 24 to 26 months) were from the aging mouse colonies maintained at the National Institute on Aging, National Institutes of Health, Baltimore, MD. Spleen cell suspensions were prepared by teasing the spleens in cold complete culture medium (CM, RPMI 1640 medium supplemented with Gentamicin (50 g/ml), 2-mercaptoethenol (2 ⫻ 10⫺5 M), and 10% fetal calf serum). To get erythrocyte-free spleen cells, erythrocytes were removed by osmotic shock. Spleen cells were pelleted (1800 rpm ⫻ 5 min), 500 l of sterile water was added to the pellet drop by drop, and the tube was vortexed for 15 s, followed immediately with the addition of equal volume of 2 ⫻ phosphate-buffered saline (PBS). Spleen cell preparations were depleted of adherent and phagocytic cells by plastic adherence and carbonyl iron and magnet treatment, or enriched for T cells by passage through Nylon Wool Column, as described before (Saxena et al., 1981). Mouse blood was collected aseptically by eye bleed. The blood was collected into a tube containing sterile PBS. The cells were washed thrice with sterile PBS. The erythrocyte pellet was then suspended in CM. These cell preparations were ⬎ 99.9% erythrocytes, and were used as such.
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Fig. 1. Numbers of mononuclear cells and erythrocytes in Con-A-activated cultures of spleen cells from young and old mice. Spleen cells derived from young and old mice were adjusted to 6 ⫻ 106 mononuclear cells /ml and cultured in 24-well micro culture plates in the presence of Con-A (5 g/ml). At different time points, mononuclear cells and erythrocytes surviving in the culture were determined by counting in a hemocytometer. Panels A and B show the numbers of mononuclear cells and erythrocytes, respectively, in the culture at different time points. Each point is a mean ⫾ SD of three individual experiments. Paired t test indicated that there were highly significant differences between the percentage erythrocyte survivals in old and young spleen cell cultures.
2.2. Reagents and other supplies Special reagents used in this study and their sources are as follows. Carbonyl iron, Concanavalin A (Con A) (Sigma), purified hamster anti mouse Fas monoclonal antibody (PharMingen, CA), hamster IgG (Organon Teknika Corp, West Chester), all plastic disposable culture ware (Costar, USA). 3. Results 3.1. Kinetics of survival of mononuclear cells (MNCs) and erythrocytes in Con-A activated cultures of spleen cells from young and old mice The number of erythrocytes present in spleen cells declined progressively in culture and the rate of decline was significantly enhanced if Con A was added to the cultures (Saxena and Chandrasekhar, 2000). The aim of the present study was to compare the erythrolytic activities associated with spleen cells derived from young and old mice. For this purpose, spleen cells from young and old mice were cultured for 3 days in presence of Con A. The time course of changes in the numbers of mononuclear cells (MNCs, lymphocytes, and macrophages) and erythrocytes in old and young spleen cell coulters are given in Fig. 1. These results show that the changes in numbers of MNCs were essentially similar for young and old mouse spleen cells (Fig. 1A). Fig. 1B depicts changes in the
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Fig. 2. Efficiency of spleen mononuclear cells of young and old mouse spleens to destroy blood-derived erythrocytes from young and old mice. Erythrocyte-free mononuclear cells (5 ⫻ 106/ml) from young and old mouse spleens were freed of erythrocytes by water lysis as described in Section 2, and cultured with blood erythrocytes (10 ⫻ 106/ml) derived from young or old mice, in presence of Con-A (5 g/ml). After 3 days of culture, numbers of residual erythrocytes were determined in different cultures. Each bar represents a mean ⫾ SD of three individual experiments. Difference between O/Y and O/O sets was highly significant (P ⬍ 0.01).
erythrocyte counts in cultured spleen cells. In fresh spleen cell preparations (zero day time point in Fig. 1B), spleen cells from old mice had a greater numbers of erythrocytes than the young mouse spleen cells. Erythrocyte count in young spleen cell cultures declined steeply after the first day of culture. At the end of 3 days of culture, few erythrocytes remained in young mouse spleen cell cultures activated with Con-A. In contrast, in old mouse spleen cell cultures, survival of erythrocytes was significantly better. In absolute terms, erythrocyte count in young spleen cell cultures on the third day was less than 10% of that in old mouse spleen cell cultures. These results indicate that erythrocytes are destroyed at a faster rate in young spleen cell cultures than in the old. Lysis of erythrocytes was not due to soluble cytotoxic factors released by young or the old spleen cells because culture supernatants of Con-A activated young or old spleen cells had no cytotoxic effect on erythrocytes (data not shown). 3.2. Susceptibility of old and young erythrocytes to depletion by spleen MNCs Slower destruction of erythrocytes in old spleen cell cultures could be due to inherent ability of erythrocytes in old spleen cell preparations to resist destruction, or due to decreased capability of old mouse spleen MNCs to destroy erythrocytes, or both. To study these possibilities, we prepared erythrocyte-free spleen cell preparations from old or young mouse spleens, and cultured them with blood-derived erythrocytes from old and young mice. Surviving erythrocyte counts in co-cultures of MNCs and erythrocytes on the third day of culture are shown in Fig. 2. Young MNCs efficiently destroyed erythrocytes from old as well as young mice. Survival of old as well as young erythrocytes co-cultured with old MNCs, was markedly higher, indicating the old MNCs were less efficient in destroying erythrocytes. In addition, significantly fewer old mouse-derived erythrocytes
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Fig. 3. Efficiency of spleen mononuclear cells from old and young mice to destroy blood-derived erythrocytes opsonized with anti-Fas antibody. Erythrocyte-free spleen mononuclear cells (5 ⫻ 106/ml) were cultured with erythrocytes (5 ⫻ 106/ml) from the same mouse with or without prior treatment with an anti-Fas antibody as described in Section 2. After 1 day in culture, numbers of surviving erythrocytes were determined. Results of two experiments each with young (F) or old (E) mice are shown.
survived in co-cultures with old MNCs as compared to young mouse erythrocytes (Fig. 2), indicating that old erythrocytes may be more susceptible to destruction by MNCs. In these experiments, erythrocyte-free effector cell preparations were obtained by lysing the contaminating erythrocytes by osmotic shock as described in Section 2. Similar results were, however, obtained if effector cell preparations were depleted of erythrocytes by using Ficoll density gradient centrifugation. When cultured in absence of MNCs, young as well as old mouse erythrocytes survived equally well (above 80% survival on the third day), indicating that there were no inherent differences in culture survivability of erythrocytes from old or young mice (results not shown). 3.3 Ability of young and old mouse MNCs to deplete opsonized erythrocytes We have previously shown that anti-Fas antibody can be used to opsonize mouse erythrocytes for a more efficient clearance by spleen MNCs in culture (Saxena and Chandrasekhar, 2000). The ability of old and young mouse spleen cell MNCs to destroy opsonized erythrocytes was examined. For this purpose, erythrocyte-free spleen MNCs from young and old mice were cultured with control or opsonized erythrocytes derived from respective MNC donor mice. In this experiment, culture duration was one day only so that increased EDA due to opsonization of erythrocytes could be assessed. Results in Fig. 3 show that opsonization of erythrocytes augmented their destruction by young as well as old MNCs. Although the mean survival of control and opsonized erythrocytes after
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Table 1 Survival of erythrocytes co-cultured with control and macrophage-depleted MNC preparations from young and old mice Source of MNCs Young spleen Young spleen Old spleen Old spleen
Macrophage depletion No Yes No Yes No Yes No Yes
Residual erythrocytes (⫻ 10⫺6/ml)
% Survival of erythrocytes
0.12 (4.23) 0.51 (6.40) 0.15 (3.87) 0.45 (5.96) 2.11 (5.67) 2.65 (6.78) 2.59 (6.32) 2.95 (7.88)
2.83 7.96 3.87 7.55 37.21 39.08 41.00 37.43
Control and macrophage-depleted spleen cells from old or young mice were cultured (5 ⫻ 106 MNCs/ml) with Con-A (5 g/ml) for 3 days. Residual erythrocyte counts were determined after 3 days of culture. Values in parentheses denote the erythrocyte count in the respective cell preparations at the beginning of the culture.
one day of culture was 70% and 14% respectively for young MNCs, the corresponding figures for old MNCs were 83% and 62%. Thus, the opsonization of erythrocytes considerably augmented their destruction by young MNCs, whereas the effect of opsonization was relatively moderate for old MNCs. 3.4. Effect of depletion of macrophages on the EDA of young and old MNCs Our previous attempts to characterize the effector cell have shown that various subpopulations contributed to the overall EDA of spleen (Saxena and Chandrasekhar, 2000). To assess the contribution of macrophages in erythrocyte destruction by young and old MNCs, survival of erythrocytes in Con-A activated cultures of control and macrophagedepleted spleen cell, preparations from young and old mice were studied. Results of these experiments indicate that the erythrocyte-destroying activity in young as well as old spleen cells remained essentially unaltered as a result of macrophage depletion, even though a small decline in EDA of young macrophage depleted spleen cells was observed (Table 1).
4. Discussion Even though phagocytosis with or without antibody as a facilitating agent is well established as a mechanism of destruction of erythrocytes in vivo, alternative mechanisms of erythrocyte degradation may also operate. This may be especially true for certain pathological conditions associated with anemia. In a recent study, enhanced in vivo clearance rate for deformed erythrocytes could not be ascribed to an increased phagocytic activity (Baerlocher, 1997). In certain forms of hemolytic anemia, anti-erythrocyte autoantibodies may be involved in the mechanism of erythrocyte loss (Smith, 1999). TNF released in large amounts may cause anemia by acting at the level of erythropoiesis or by increasing erythrocyte destruction (Taverne et al., 1994). We recently provided in vitro evidence for the possibility of direct cell mediated lysis as a novel mechanism of erythrocyte destruction (Saxena and Chandrasekhar, 2000). In
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the present communication, we have further extended this study and demonstrated that the in vitro erythrolytic activity of spleen cells is significantly lower in old mice as compared to the young. As there is no evidence for the release of soluble cytotoxic mediators by the spleen cells from young or old mice, in vitro erythrocidal activity is likely to be cell-mediated. Lower EDA in spleen cells from old mice could be due to an inefficient recognition of erythrocytes by old MNCs. This, however, seems unlikely because the facilitation of recognition by using opsonized erythrocytes only marginally improved the EDA for old MNCs while boosting it to a much greater extent in the case of young MNCs. Decline in the ability of old MNCs to destroy erythrocytes may therefore be due to an inherent decline in erythrocidal mechanisms. Relative contribution of macrophage-mediated phagocytosis to overall EDA in young and old MNCs was assessed by studying the effect of depleting macrophages on the EDA of these MNC preparations. Our results indicated that depletion of macrophage had little effect on the destruction of erythrocytes in old or young MNCs. These results indicate that the decline in non-phagocytic mechanisms of erythrocyte destruction may contribute to the overall decline of EDA in old mouse spleen cells. Although a direct erythrolytic effect of MNCs in vitro is clear from our results, the question remains if this mode of erythrocyte destruction actually contributes to erythrocyte turnover in vivo and whether the phenomenon has a role in regulation of erythrocyte turnover as a function of age. It has been reported that the proportion of younger erythrocytes increase in old mice (Gershon and Sheiban, 1991), which is an indication of faster depletion of erythrocytes in old mice. This might appear to argue against a role for direct lysis in erythrocyte destruction in old mice because this parameter declines in old mice. Yet we have also found that the susceptibility to destruction increases in erythrocytes from old mice, which may counter-balance sluggish EDA in old mice. In addition, it is possible that alternative mechanisms for erythrocyte destruction in old mice may compensate for decline in direct erythrocidal efficiency. Accordingly, Shimizu and Hokano (1988) have reported that old or worn red blood cells are eliminated more by mechanical stress than by phagocytosis of macrophage in the senile spleen. Overall homeostasis of erythrocyte numbers in vivo must depend upon a variety of factors that modulate the generation and destruction of erythrocytes. Further studies are needed to define the role of MNC-mediated lysis as a erythrocyte destruction mechanism in erythrocyte turnover in young and old mice.
Acknowledgments Research funding from DST is gratefully acknowledged.
References Baerlocher, G. M., Schlappritzi, E., Tobler, A., & Reinhart, W. H. (1997). The influence of tumour necrosis factor-alpha on phagocytosis of human erythrocytes by autologous monocytes/macrophages. Eur J Haematol 58, 357–359. Bocci, V. (1981). Determinants of erythrocyte aging: a reappraisal. Brit J Haematol 48, 515–522. Bratosin, D., Mazurier, J., Tissier, J. P., Estaquier, J., Huart, J. J., Ameisen, J. C., Aminoff, D., & Montreuil, J. (1998). Cellular and molecular mechanisms of senescent erythrocyte phagocytosis by macrophages. A review. Biochimie 80, 173–95.
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Engwerda, C. R., Handwerger, B. S., & Fox, B. S. (1994). Aged T cells are hyporesponsive to costimulation mediated by CD28. J Immunol 152, 3740 –3747. Ernst D. N., Wigle, W. O., Noonan, D. N., McQuitty, D. N., & Hobbs, M. V. (1993). The age-associated increase in IFN-gamma synthesis by mouse CD8⫹ T cells correlates with shifts in the frequencies of cell subsets defined by membrane CD44, CD45RB, 3G11, and MEL-14 expression. J Immunol 151, 575–587. Gershon, H. & Sheiben, E. (1991). In vitro sequestration of erythrocytes from hosts of various ages. Adv Exp Med Biol 307, 339 –350. Hirano, T. & Nordin, A. A. (1976). Age-associated decline in the in vitro development of cytotoxic lymphocytes in NZB mice. J Immunol 117, 1093–1098. Landaw, S. A.(1988) Factors that accelerate or retard red blood cell senescence. Blood cells 14, 47– 67. Lerner, A., Yamada, T., & Miller, R. A. (1989). Pgp-1hi T lymphocytes accumulate with age in mice and respond poorly to concanavalin A. Eur J Immunol 19, 977–982. Mbawuike, I. N., Acuna, C. L., Walz, K. C., Atmar, R. L., Greenberg, S. B., & Couch, R. B. (1997). Cytokines and impaired CD8⫹ CTL activity among elderly persons and the enhancing effect of IL-12. Mech Ageing Dev 94, 25–39. Nagelkerken, L., Hertogh–Huijbregts, A., Dobber, R., & Drager, A. (1991). Age-related changes in lymphokine production related to a decreased number of CD45RBhi CD4⫹ T cells. Eur J Immunol 21, 273–281. Powers, D. C. & Belshe, R. B. (1993). Effect of age on cytotoxic T lymphocyte memory as well as serum and local antibody responses elicited by inactivated influenza virus vaccine. J Infect Dis 167, 584 –592. Saxena, R. K. (2000). Should erythrocyte destruction in vivo be through phagocytosis alone? J Biosciences. In press. Saxena, R. K. & Adler, W. H. (1999). Cytolytic activity of mitogen activated old and young mouse spleen cells against tumor target cells expressing high or low levels of Fas antigen. Exp Mol Med 31, 137– 41. Saxena, R. K., Adler, W. H., & Nordin, A. A. (1981). Modulation of natural cytotoxicity by alloantibodies. IV. A comparative study of the activation of mouse spleen cell cytotoxicity by anti H-2 antisera, interferon, and mitogens. Cellular Immunol 63, 28 – 40. Saxena, R. K. & Chandrasekhar, B. (2000). A novel non-phagocytic mechanism of erythrocyte destruction involving direct cell mediated cytotoxicity. Int J Hematology. In press. Saxena, R. K., Saxena, Q. B., & Adler, W. H. (1984). Interleukin-2-induced activation of natural killer activity in spleen cells from old and young mice. Immunology 51, 719 –26. Saxena, R. K., Saxena, Q. B., & Adler, W. H. (1988). Lectin-induced cytotoxic activity in spleen cells from young and old mice. Age-related changes in types of effector cells, lymphokine production and response. Immunology 64, 457– 461. Shimizu, K. & Hokano, M. (1988). Elimination of old or worn red blood cells in the senile murine spleen. Acta Histochem 83, 65–70. Smith, L. A. (1999). Autoimmune hemolytic anemias: characteristics and classification. Clin Lab Sci 12, 110 – 4. Song, L., Kim, Y. H., Chopra, J. J., Proust, J. J., Nagel, J. E., Nordin, A. A., & Adler W. H. (1993). Age-related effects in T cell activation and proliferation. Exp Gerontol 28, 313–321. Taverne, J., Sheikh, N., de Souza, J. B., Playfair, J. H., Probert, L., & Kollias, G. (1994). Anemia and resistance to malaria in transgeneic mice expressing human tumor Necrosis Factor. Immunology 82, 397– 403.
In Vitro erythrocidal activity of activated spleen cells from young and old mice Rajiv K. Saxenaa,b,*, William H. Adlera,b School of Life Sciences, Jawaharlal Nehru University, New Delhi, India Immunology Section, GRC, NIA, National Institutes of Health, Baltimore, MD, USA Received 7 January 2000; received in revised form 22 February 2000; accepted 22 February 2000
Abstract We have recently reported that activated mouse spleen mononuclear cells (MNCs) efficiently lyse autologous erythrocytes in vitro (Saxena and Chandrasekhar, 2000). In the present study, we have investigated erythrocyte-depleting ability (EDA) of spleen MNCs from young and old mice. Time kinetics of survival of erythrocytes in mitogen-activated spleen cell cultures indicated that the erythrocyte depletion was significantly faster in young spleen cell cultures than in the old. Poorer EDA of old MNCs was in spite of the fact that the susceptibility to lysis actually increased in erythrocytes from old mice. Erythrocytes opsonized by a hamster anti mouse Fas monoclonal antibody, were destroyed with a much greater efficiency by young MNCs, whereas the corresponding effect of opsonization was only moderate for old MNCs. Depletion of macrophages from MNC preparations by plastic adherence as well as carbonyl-iron and magnet treatment had a marginal if any effect on EDA of young and old mouse MNCs, indicating that a lower lymphocyte-associated erythrocidal activity as one of the factors responsible for overall lower EDA associated with spleen derived MNCs of old mice. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Erythrocytes; Aging; Fas; Cytotoxicity; Phagocytosis
1. Introduction Many parameters of T-cell activity are known to decline in the aged. These include T-cell proliferation in response to stimulation through T cell receptor and co-stimulators (Lerner et al., 1988; Song et al., 1993; Engwerda et al., 1994), production and response to growth promoting cytokines (Saxena et al., 1988; Nagelkerken et al., 1991; Ernst et al., 1993) and activation of cytotoxic lymphocytes (Hirano and Nordin, 1976; Saxena et al., 1984; Powers and Belshe, 1993; Mbawuike et al., 1997). We have previously character* Corresponding author. Tel.: ⫹1-304-285-6314; fax: ⫹1-304-285-6126. E-mail address: [email protected] (R.K. Saxena). 0531-5565/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 5 3 1 - 5 5 6 5 ( 0 0 ) 0 0 0 9 1 - 7
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ized mitogen-induced cytotoxic cells in spleen cells from young and old mice and reported differences in their cytolytic activities (Saxena et al., 1981, 1988). Cytotoxic cells generated in young mouse spleen cells activated with Con-A efficiently lysed Fasexpressing cell lines like Hut77 and Jurkat, whereas effector cells generated in old mouse spleen cells had poor lytic activity against these targets (Saxena and Adler, 1999). Con-A activated old spleen cells were, however, markedly more cytotoxic against low Fasexpressing cell lines like P815 (Saxena et al., 1988, Saxena and Adler, 1999). It was proposed that killing through Fas-FasL interaction might be relatively less efficient for activated spleen cells from old mice (Saxena and Adler, 1999). Recently, we have studied the destruction of autologous erythrocytes by Con-A activated mouse spleen mononuclear cells (MNCs) in vitro, and have reported that the erythrocyte counts fall rapidly when co-cultured with MNCs (Saxena and Chandrasekhar, 2000). Phagocytosis of erythrocytes by macrophages in MNC preparations as well as a direct cell-mediated lysis of erythrocytes, contributed to the erythrocyte destruction in vitro (Saxena and Chandrasekhar, 2000). In addition, a role for Fas-FasL and analogous TNF-TNFR interactions in recognition and killing of erythrocytes by spleen MNCs was also suggested (Saxena, 2000; Saxena and Chandrasekhar, 2000). Because FasL-mediated cytotoxic activity appears to be deficient in mitogen-activated old mouse spleen cells, as discussed above, it was of interest to see if young and old MNCs differed in their efficiencies to destroy autologous erythrocytes. In the present study, we have shown that MNCs from old mouse spleen cells had significantly lower erythrocidal activity than the MNCs from young mice. Differences in the erythrocidal activity of old and young MNCs persisted even if macrophages were depleted from the MNC preparations or erythrocytes were opsonized by anti-Fas antibodies.
2. Materials and methods 2.1. Effector and Target cells Spleen effector cells used in this study were derived from young or old C57Bl/6 mice. Young male C57Bl/6 mice (12 to 16 weeks) were obtained from Jackson Laboratories. Old male C57Bl/6 mice (age 24 to 26 months) were from the aging mouse colonies maintained at the National Institute on Aging, National Institutes of Health, Baltimore, MD. Spleen cell suspensions were prepared by teasing the spleens in cold complete culture medium (CM, RPMI 1640 medium supplemented with Gentamicin (50 g/ml), 2-mercaptoethenol (2 ⫻ 10⫺5 M), and 10% fetal calf serum). To get erythrocyte-free spleen cells, erythrocytes were removed by osmotic shock. Spleen cells were pelleted (1800 rpm ⫻ 5 min), 500 l of sterile water was added to the pellet drop by drop, and the tube was vortexed for 15 s, followed immediately with the addition of equal volume of 2 ⫻ phosphate-buffered saline (PBS). Spleen cell preparations were depleted of adherent and phagocytic cells by plastic adherence and carbonyl iron and magnet treatment, or enriched for T cells by passage through Nylon Wool Column, as described before (Saxena et al., 1981). Mouse blood was collected aseptically by eye bleed. The blood was collected into a tube containing sterile PBS. The cells were washed thrice with sterile PBS. The erythrocyte pellet was then suspended in CM. These cell preparations were ⬎ 99.9% erythrocytes, and were used as such.
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Fig. 1. Numbers of mononuclear cells and erythrocytes in Con-A-activated cultures of spleen cells from young and old mice. Spleen cells derived from young and old mice were adjusted to 6 ⫻ 106 mononuclear cells /ml and cultured in 24-well micro culture plates in the presence of Con-A (5 g/ml). At different time points, mononuclear cells and erythrocytes surviving in the culture were determined by counting in a hemocytometer. Panels A and B show the numbers of mononuclear cells and erythrocytes, respectively, in the culture at different time points. Each point is a mean ⫾ SD of three individual experiments. Paired t test indicated that there were highly significant differences between the percentage erythrocyte survivals in old and young spleen cell cultures.
2.2. Reagents and other supplies Special reagents used in this study and their sources are as follows. Carbonyl iron, Concanavalin A (Con A) (Sigma), purified hamster anti mouse Fas monoclonal antibody (PharMingen, CA), hamster IgG (Organon Teknika Corp, West Chester), all plastic disposable culture ware (Costar, USA). 3. Results 3.1. Kinetics of survival of mononuclear cells (MNCs) and erythrocytes in Con-A activated cultures of spleen cells from young and old mice The number of erythrocytes present in spleen cells declined progressively in culture and the rate of decline was significantly enhanced if Con A was added to the cultures (Saxena and Chandrasekhar, 2000). The aim of the present study was to compare the erythrolytic activities associated with spleen cells derived from young and old mice. For this purpose, spleen cells from young and old mice were cultured for 3 days in presence of Con A. The time course of changes in the numbers of mononuclear cells (MNCs, lymphocytes, and macrophages) and erythrocytes in old and young spleen cell coulters are given in Fig. 1. These results show that the changes in numbers of MNCs were essentially similar for young and old mouse spleen cells (Fig. 1A). Fig. 1B depicts changes in the
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Fig. 2. Efficiency of spleen mononuclear cells of young and old mouse spleens to destroy blood-derived erythrocytes from young and old mice. Erythrocyte-free mononuclear cells (5 ⫻ 106/ml) from young and old mouse spleens were freed of erythrocytes by water lysis as described in Section 2, and cultured with blood erythrocytes (10 ⫻ 106/ml) derived from young or old mice, in presence of Con-A (5 g/ml). After 3 days of culture, numbers of residual erythrocytes were determined in different cultures. Each bar represents a mean ⫾ SD of three individual experiments. Difference between O/Y and O/O sets was highly significant (P ⬍ 0.01).
erythrocyte counts in cultured spleen cells. In fresh spleen cell preparations (zero day time point in Fig. 1B), spleen cells from old mice had a greater numbers of erythrocytes than the young mouse spleen cells. Erythrocyte count in young spleen cell cultures declined steeply after the first day of culture. At the end of 3 days of culture, few erythrocytes remained in young mouse spleen cell cultures activated with Con-A. In contrast, in old mouse spleen cell cultures, survival of erythrocytes was significantly better. In absolute terms, erythrocyte count in young spleen cell cultures on the third day was less than 10% of that in old mouse spleen cell cultures. These results indicate that erythrocytes are destroyed at a faster rate in young spleen cell cultures than in the old. Lysis of erythrocytes was not due to soluble cytotoxic factors released by young or the old spleen cells because culture supernatants of Con-A activated young or old spleen cells had no cytotoxic effect on erythrocytes (data not shown). 3.2. Susceptibility of old and young erythrocytes to depletion by spleen MNCs Slower destruction of erythrocytes in old spleen cell cultures could be due to inherent ability of erythrocytes in old spleen cell preparations to resist destruction, or due to decreased capability of old mouse spleen MNCs to destroy erythrocytes, or both. To study these possibilities, we prepared erythrocyte-free spleen cell preparations from old or young mouse spleens, and cultured them with blood-derived erythrocytes from old and young mice. Surviving erythrocyte counts in co-cultures of MNCs and erythrocytes on the third day of culture are shown in Fig. 2. Young MNCs efficiently destroyed erythrocytes from old as well as young mice. Survival of old as well as young erythrocytes co-cultured with old MNCs, was markedly higher, indicating the old MNCs were less efficient in destroying erythrocytes. In addition, significantly fewer old mouse-derived erythrocytes
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Fig. 3. Efficiency of spleen mononuclear cells from old and young mice to destroy blood-derived erythrocytes opsonized with anti-Fas antibody. Erythrocyte-free spleen mononuclear cells (5 ⫻ 106/ml) were cultured with erythrocytes (5 ⫻ 106/ml) from the same mouse with or without prior treatment with an anti-Fas antibody as described in Section 2. After 1 day in culture, numbers of surviving erythrocytes were determined. Results of two experiments each with young (F) or old (E) mice are shown.
survived in co-cultures with old MNCs as compared to young mouse erythrocytes (Fig. 2), indicating that old erythrocytes may be more susceptible to destruction by MNCs. In these experiments, erythrocyte-free effector cell preparations were obtained by lysing the contaminating erythrocytes by osmotic shock as described in Section 2. Similar results were, however, obtained if effector cell preparations were depleted of erythrocytes by using Ficoll density gradient centrifugation. When cultured in absence of MNCs, young as well as old mouse erythrocytes survived equally well (above 80% survival on the third day), indicating that there were no inherent differences in culture survivability of erythrocytes from old or young mice (results not shown). 3.3 Ability of young and old mouse MNCs to deplete opsonized erythrocytes We have previously shown that anti-Fas antibody can be used to opsonize mouse erythrocytes for a more efficient clearance by spleen MNCs in culture (Saxena and Chandrasekhar, 2000). The ability of old and young mouse spleen cell MNCs to destroy opsonized erythrocytes was examined. For this purpose, erythrocyte-free spleen MNCs from young and old mice were cultured with control or opsonized erythrocytes derived from respective MNC donor mice. In this experiment, culture duration was one day only so that increased EDA due to opsonization of erythrocytes could be assessed. Results in Fig. 3 show that opsonization of erythrocytes augmented their destruction by young as well as old MNCs. Although the mean survival of control and opsonized erythrocytes after
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Table 1 Survival of erythrocytes co-cultured with control and macrophage-depleted MNC preparations from young and old mice Source of MNCs Young spleen Young spleen Old spleen Old spleen
Macrophage depletion No Yes No Yes No Yes No Yes
Residual erythrocytes (⫻ 10⫺6/ml)
% Survival of erythrocytes
0.12 (4.23) 0.51 (6.40) 0.15 (3.87) 0.45 (5.96) 2.11 (5.67) 2.65 (6.78) 2.59 (6.32) 2.95 (7.88)
2.83 7.96 3.87 7.55 37.21 39.08 41.00 37.43
Control and macrophage-depleted spleen cells from old or young mice were cultured (5 ⫻ 106 MNCs/ml) with Con-A (5 g/ml) for 3 days. Residual erythrocyte counts were determined after 3 days of culture. Values in parentheses denote the erythrocyte count in the respective cell preparations at the beginning of the culture.
one day of culture was 70% and 14% respectively for young MNCs, the corresponding figures for old MNCs were 83% and 62%. Thus, the opsonization of erythrocytes considerably augmented their destruction by young MNCs, whereas the effect of opsonization was relatively moderate for old MNCs. 3.4. Effect of depletion of macrophages on the EDA of young and old MNCs Our previous attempts to characterize the effector cell have shown that various subpopulations contributed to the overall EDA of spleen (Saxena and Chandrasekhar, 2000). To assess the contribution of macrophages in erythrocyte destruction by young and old MNCs, survival of erythrocytes in Con-A activated cultures of control and macrophagedepleted spleen cell, preparations from young and old mice were studied. Results of these experiments indicate that the erythrocyte-destroying activity in young as well as old spleen cells remained essentially unaltered as a result of macrophage depletion, even though a small decline in EDA of young macrophage depleted spleen cells was observed (Table 1).
4. Discussion Even though phagocytosis with or without antibody as a facilitating agent is well established as a mechanism of destruction of erythrocytes in vivo, alternative mechanisms of erythrocyte degradation may also operate. This may be especially true for certain pathological conditions associated with anemia. In a recent study, enhanced in vivo clearance rate for deformed erythrocytes could not be ascribed to an increased phagocytic activity (Baerlocher, 1997). In certain forms of hemolytic anemia, anti-erythrocyte autoantibodies may be involved in the mechanism of erythrocyte loss (Smith, 1999). TNF released in large amounts may cause anemia by acting at the level of erythropoiesis or by increasing erythrocyte destruction (Taverne et al., 1994). We recently provided in vitro evidence for the possibility of direct cell mediated lysis as a novel mechanism of erythrocyte destruction (Saxena and Chandrasekhar, 2000). In
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the present communication, we have further extended this study and demonstrated that the in vitro erythrolytic activity of spleen cells is significantly lower in old mice as compared to the young. As there is no evidence for the release of soluble cytotoxic mediators by the spleen cells from young or old mice, in vitro erythrocidal activity is likely to be cell-mediated. Lower EDA in spleen cells from old mice could be due to an inefficient recognition of erythrocytes by old MNCs. This, however, seems unlikely because the facilitation of recognition by using opsonized erythrocytes only marginally improved the EDA for old MNCs while boosting it to a much greater extent in the case of young MNCs. Decline in the ability of old MNCs to destroy erythrocytes may therefore be due to an inherent decline in erythrocidal mechanisms. Relative contribution of macrophage-mediated phagocytosis to overall EDA in young and old MNCs was assessed by studying the effect of depleting macrophages on the EDA of these MNC preparations. Our results indicated that depletion of macrophage had little effect on the destruction of erythrocytes in old or young MNCs. These results indicate that the decline in non-phagocytic mechanisms of erythrocyte destruction may contribute to the overall decline of EDA in old mouse spleen cells. Although a direct erythrolytic effect of MNCs in vitro is clear from our results, the question remains if this mode of erythrocyte destruction actually contributes to erythrocyte turnover in vivo and whether the phenomenon has a role in regulation of erythrocyte turnover as a function of age. It has been reported that the proportion of younger erythrocytes increase in old mice (Gershon and Sheiban, 1991), which is an indication of faster depletion of erythrocytes in old mice. This might appear to argue against a role for direct lysis in erythrocyte destruction in old mice because this parameter declines in old mice. Yet we have also found that the susceptibility to destruction increases in erythrocytes from old mice, which may counter-balance sluggish EDA in old mice. In addition, it is possible that alternative mechanisms for erythrocyte destruction in old mice may compensate for decline in direct erythrocidal efficiency. Accordingly, Shimizu and Hokano (1988) have reported that old or worn red blood cells are eliminated more by mechanical stress than by phagocytosis of macrophage in the senile spleen. Overall homeostasis of erythrocyte numbers in vivo must depend upon a variety of factors that modulate the generation and destruction of erythrocytes. Further studies are needed to define the role of MNC-mediated lysis as a erythrocyte destruction mechanism in erythrocyte turnover in young and old mice.
Acknowledgments Research funding from DST is gratefully acknowledged.
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