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Apoptosis induction by macrophage-derived reactive oxygen species in myelin-specific T cells requires cell–cell contact
Journal of Neuroimmunology 95 Ž1999. 152–156
Apoptosis induction by macrophage-derived reactive oxygen species in myelin-specific T cells requires cell–cell contact Eilhard Mix a , Uwe K. Zettl b
a,b
, Jurgen Zielasek b, Hans-Peter Hartung ¨
b,1
, Ralf Gold
b,)
a Department of Neurology, UniÕersity of Rostock, D-18055 Rostock, Germany Department of Neurology, Clinical Research Group for Multiple Sclerosis and Neuroimmunology, Julius Maximilians UniÕersity, D-97080 Wurzburg, ¨ Germany
Received 15 October 1998; revised 21 December 1998; accepted 28 December 1998
Abstract Apoptosis of autoreactive T cells has been recognized as an important mechanism of immune homeostasis in autoimmune PNS diseases. To examine whether T cells are induced to undergo apoptosis by macrophages ŽMØ. via reactive oxygen species ŽROS. neuritogenic P2-specific T line cells were exposed to peritoneal MØ of Lewis rats in vitro. ROS production was stimulated by phorbol myristate acetate ŽPMA. and inhibited by catalase. Flow cytometric analysis of apoptosis as measured by TUNEL technique revealed that 5–15% of all P2-cells were apoptotic, if cultured alone. This percentage increased slightly, but did not exceed 20% when P2-cells were cocultivated with PMA-stimulated MØ in a two-chamber culture plate separated by a cell-impermeable membrane. Direct cocultivation caused apoptotic cell death of more than 40% of P2-cells, which was completely abrogated by catalase. These results suggest a requirement for close cell–cell contact for apoptosis induction of T cells via MØ-derived ROS. Thus MØ may contribute to termination of inflammation in vivo through the release of highly reactive oxygen species that mediate apoptotic cell death of autoaggressive T lymphocytes. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Apoptosis; Lewis rats; Macrophage; T cells
1. Introduction Apoptosis is regarded as the major physiologic mechanism of elimination of autoreactive T cells in a number of autoimmune diseases ŽGold et al., 1997.. Mediators of T cell apoptosis in vivo are not yet identified. Possible candidates would be products of the oxidative burst, i.e., reactive oxygen species ŽROS., in the inflammatory infiltrates ŽButtke and Sandstrom, 1994.. Aims to obtain information on the origin of ROS and to determine their possible role for termination of autoimmune processes are, therefore, the focus of current research. Abbreviations: MØ, macrophages; P2-cells, P2-specific T line cells; ROS, reactive oxygen species; TUNEL, terminal transferase ddUTP nick end labeling ) Corresponding author. Department of Neurology, Neurologische Universitatsklinik, Josef-Schneider-Strasse 11, D-97080 Wurzburg, Germany. ¨ Tel.: q 49-931-2015755; Fax q 49-931-2013488; E-m ail: [email protected] 1 Present address: Department of Neurology, University of Graz, Austria.
Among inflammatory cells, macrophages ŽMØ. are good candidates as inducers of autoreactive T cell apoptosis, since ŽI. MØ kill developing peripheral T cells ŽMunn et al., 1996. as well as permanent T line cells ŽWu et al., 1995. with high efficacy in vitro, ŽII. MØ are abundant at the target site in autoimmune inflammatory lesions ŽBruck ¨ et al., 1996., ŽIII. MØ produce ROS in high quantity upon activation ŽVan Furth, 1992., and ŽIV. the mechanism of T cell killing by MØ involves apoptosis in vitro and in vivo ŽAliprantis et al., 1996.. ROS produced by locally accumulated MØ could, therefore, be important mediators of apoptotic cell death for autoreactive T cells. We have tested this hypothesis by in vitro activation of peritoneal MØ of Lewis rats with phorbol myristate acetate ŽPMA. and their cocultivation with neuritogenic P2-specific T line cells ŽP2-cells. derived from lymph nodes of the same rat strain. Cocultures were performed by incubating the P2 cells directly with the peritoneal MØ or in twochamber culture plates separated by a cell-impermeable membrane. Apoptosis was examined in both cell compartments by the TUNEL technique and flow cytometry.
0165-5728r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 Ž 9 9 . 0 0 0 0 6 - 5
E. Mix et al.r Journal of Neuroimmunology 95 (1999) 152–156
153
2. Materials and methods 2.1. Cell preparation and cocultiÕation The T cell line P2 was established from lymph nodes of Lewis rats immunized with the peripheral myelin component P2 protein as described previously ŽZettl et al., 1995.. P2-cells were used on days 3 to 6 after the sixth in vitro restimulation cycle with P2 protein as antigen and irradiated thymocytes as antigen-presenting cells. MØ were freshly prepared from the peritoneal exudates of Lewis rats by adherence. Briefly, peritoneal exudate cells of two to three animals were washed once in PBS without Ca2q and Mg 2q and resuspended in culture medium consisting of RPMI 1640 ŽGibco, Grand Island, NY. supplemented with 1% normal rat serum, 2 mM glutamine and antibiotics. Cells were allowed to adhere to the surface of 200 ml tissue culture flasks precoated with fetal calf serum by incubation of 378C for 45 min. Non-adherent cells were removed by rinsing with warm 5 mM EDTA-PBS and discarded. The remaining adherent cells were removed by 2 min incubation at 48C with culture medium and washed once. The resulting cell suspension contained ) 90% viable macrophages as tested by trypan blue exclusion and labeling with the MØ-specific monoclonal antibody ED1 ŽSerotec, Raleigh, NC.. Cocultivation of P2-cells and MØ was performed in 24-well culture plates ŽCostar, Cambridge, MA. containing inserts with a cell impermeable membrane Žpore size 1.0 mm.. 10 6 MØ were applied in 0.5 ml to the outer well and 10 5 P2-cells in 0.5 ml to the insert Žseparated cocultivation.. Alternatively, cocultivation was done without using the inserts by direct application of the same cell numbers and volumes to the outer wells of culture plates Ždirect cocultivation.. For control MØ and P2-cells were cultivated alone by adding another 0.5 ml medium to the respective cell suspensions. For stimulation of the oxidative burst PMA ŽSigma, St. Louis, MO. was added to the MØ at a final concentration of 10y7 M. For the inhibition of H 2 O 2 catalase ŽSigma. was added at a final concentration of 10y3 unitsrml. All cultures were run for 18 h at 378C. 2.2. TUNEL technique and FACS analysis For quantitation of apoptotic cells, cell cultures were transferred to 1.5-ml conical reaction tubes ŽEppendorf, Hamburg, Germany. followed by fixation, permeabilization and staining by the terminal transferase ddUTP end labeling ŽTUNEL. technique as described previously ŽGold et al., 1994.. Briefly, cells were centrifuged for 5 min at 400 = g and room temperature ŽRT. and fixed for 12 min in 200 ml of 4% buffered paraformaldehyde. After one washing cells were permeabilized in 200 ml of 0.025 Nonidet P-40 ŽSigma. for 15 min at RT. After another washing 20 ml cell suspension were mixed with 35 ml of a tailing reaction mixture containing the following compo-
Fig. 1. Histogram of cocultivated P2-cells and MØ after labeling by TUNEL technique Žgreen Fluorescent ddUTP, y-axis. for quantitation of apoptotic cells and by anti-ED1 monoclonal antibodies Žred Fluorescent ED1, x-axis. for identification of MØ. For details of staining and FACS, see Section 2.
nents: 0.15 nmol FITC-12-ddUTP ŽBoehringer, Mannheim, Germany., 0.85 nmol dTTP and 1 nmol dGTP, dATP and dCTP ŽPharmacia, Uppsala, Sweden., 5 units terminal deoxynucleotidyl-transferase ŽTdT; Promega, Madison, WI. and bi-distilled water. The samples were rotated under light protection Ž400 rpm. at 378C for 90 min and analyzed by FACS. To identify MØ and P2-cells in direct cocultures, cells were stained for the MØ-specific antigen ED1 prior to TUNEL technique. In a first step mouse anti-rat ED1 monoclonal antibodies were added to the permeabilized cells diluted 1:80 in 50 ml staining buffer Ž1% BSA and 0.1% NaN3 in PBS. and incubated on ice for 30 min followed by two washes with the same buffer. In a second step anti-mouse IgG1-Tricolor ŽMedac, Hamburg, Germany. diluted 1:20 in 50 ml staining buffer was added, incubated and washed as in the first staining step followed by TUNEL technique. Labeled cells were analyzed for apoptotic T cells and MØ in a FACScan ŽBecton Dickinson, San Jose, ´ CA. on the basis of 10,000 cells gated by forward vs. side scatter. Fig. 1 illustrates the window settings for quantitation of apoptotic green fluorescent cells among ED1q red fluorescent MØ and ED1y P2-cells in an example of direct cocultivation.
3. Results and discussion T cell elimination from many sites of inflammation, e.g., in autoimmune diseases, occurs mainly via apoptosis ŽGold et al., 1997.. Macrophages are supposed to induce T cell apoptosis in inflammatory lesions either by soluble mediators or by direct cell–cell interaction ŽMunn et al.,
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154
1996; Wu et al., 1996.. Direct cell–cell contact via adhesion molecules has been shown to be a prerequisite for Fas-mediated apoptosis of human peripheral T cells triggered by monocytes ŽWu et al., 1996.. An alternative way of apoptosis induction involves the action of ROS, which are produced by activated MØ ŽVan Furth, 1992. and monocytes ŽWesch et al., 1998.. We have investigated the possibility that T cell apoptosis is induced by MØ-derived ROS in a dual approach of cocultivation with and without addition of the ROSantagonist catalase. Peripheral myelin protein P2-specific T line cells were exposed either directly to rat peritoneal MØ Ždirect coculture. or separated by a semipermeable membrane Žseparated coculture.. The main results of our study were the following ŽTable 1.: ŽI. T cells and MØ showed a baseline frequency of apoptotic cells of less than 10%, if the cells were cultured alone in culture medium. A slight increase of apoptotic cells to about 15% was observed in T cells exposed to the stimulator of the oxidative burst, PMA, whether they were cultivated in the inserts of plates for separated cocultivation or in the ‘normal’ wells of plates for direct cocultivation. Since the slight increase of T cell apoptosis seen after addition of PMA could be suppressed by catalase, it may be the result of ‘endogenous’ ROS production of stimulated T line cells. MØ did not undergo a PMA-stimulated apoptosis. ŽII. In direct cocultures of T cells with MØ ED1y T cells revealed a 2-fold increase of apoptotic cells compared to the T cell cultures alone Žabout 20%., which was further augmented by PMA Žto about 40%.. The PMA-induced augmentation could be suppressed by catalase to values of cocultures without PMA, but not to the ‘basal’ rate of single T cell cultures. The remaining T cell apoptosis may, therefore, be due to the action of apoptosis inducers other than ROS, e.g., tumor necrosis factor-a ŽTNF-a ., nitric oxygen radicals ŽNO., etc. ŽZheng et al., 1995; Zettl et al.,
1997.. Also, Fas-mediated apoptosis cannot be excluded to account for the non-catalase inhibitable rate of apoptotic T cells ŽDeas et al., 1997.. The apoptotic rate of ED1q MØ was not affected neither by PMA nor by cocultivated T cells. ŽIII. In separated cocultures only a minor increase of the rate of apoptotic T cells was seen with PMA, i.e., to about 15% in the single T cell culture and to about 20% in the coculture with MØ. These slight increases were inhibited by catalase. However, since they did not exceed the 20% T cell apoptosis in direct cocultures with PMA q catalase, they may be the result of indirect ROS-stimulation via soluble mediators like TNF-a ŽZheng et al., 1995. The low frequency of apoptosis observed in MØ Ž5–10%. argue against the existence of a relevant apoptosis-inducing effect of T cells on MØ. MØ may be generally resistant to ROS-induced apoptosis. Since apoptosis of MØ-exposed activated T cells was abrogated to a considerable degree by catalase, we conclude that H 2 O 2 is the major mediator of MØ cytotoxicity to T cells in our in vitro system that mimics the conditions of autoimmune demyelinating cells in the peripheral nervous system of the rat. The mechanism of action may not involve the Fas-dependent pathway of apoptosis, since ŽI. Wesch et al. Ž1998. found recently that monocyte-mediated apoptotic cell death of mitogen activated T cells is Fas-independent, and ŽII. Deas et al. Ž1997. reported that Fas-mediated T cell death does not rely on ROS. The situation may be different in other cell types like endothelial cells, in which H 2 O 2 induces Fas expression ŽSuhara et al., 1998.. Several mechanisms may account for the apoptosis inducing effect of H 2 O 2 on activated neuritogenic T cells used in our study: Firstly, peroxidation of plasma membrane lipids may be the first step of the apoptotic process as it has recently been described for polyunsaturated fatty acid-induced apoptosis in leukemia ŽFinstad et al., 1998. and in cancer cells ŽHawkins et al., 1998.. This pathway
Table 1 Percentages of apoptotic cells after cocultivation of rat P2-specific T line cells ŽP2. and peritoneal macrophages ŽMØ. with and without stimulation by PMAa Cells
MØ alone P2 alone MØ q P2 a
Direct cocultivation
Separated cocultivation
Medium alone
qPMA
qPMAq catalase
Medium alone
qPMA
qPMAq catalase
7.1 " 2.3 Ž6.0. b 8.2 " 3.4 Ž6.1. ED1q: 5.1 " 3.2 Ž2.7. c ED1y: 21.9 " 4.0 Ž18.5.
8.6 " 4.1 Ž4.3. 14.8 " 8.0 Ž10.3. 7.0 " 5.7 Ž2.5. 37.8 " 6.4(43.2)
9.9 " 4.3 Ž8.1. 12.2 " 9.3 Ž5.1. 8.2 " 7.2 Ž1.7. 20.2 " 14.3 Ž7.8.
n.d. 10.3 " 0.8 Ž10.1. MØ: 5.2 " 1.0 Ž6.1. P2: 10.3 " 1.2 Ž10.8.
n.d. 14.0 " 1.3 Ž14.2. 10.8 " 1.0 Ž10.0. 20.8 " 10.0 Ž15.5.
n.d. 6.8 " 1.8 Ž6.4. 10.2 " 3.3 Ž8.8. 12.8 " 1.8 Ž11.1.
P2 cells and peritoneal MØ were cultivated either alone or together in microtiter plates separated by a cell impermeable membrane Žseparated cocultivation. or unseparated Ždirect cocultivation.. Three types of cultures were performed: Ž1. Cultivation in medium alone, Ž2. additional stimulation of oxidative burst with PMA, and Ž3. additional stimulation of oxidative burst with PMA and destroying of H 2 O 2 by catalase. Apoptosis was detected by TUNEL technique and flow cytometry. b Data are given as means" S.E.M. Žmedians in parenthesis. of three independent experiments. c In the direct coculture, MØ were identified by fluorescence labeled anti-ED1 monoclonal antibodies. ED1q cells represent the peritoneal MØ, whereas the majority of ED1y cells Ž) 90%. are P2 T cells. The highest values of apoptotic cells were found for T cells directly exposed to PMA-stimulated MØ Žprinted in bold..
E. Mix et al.r Journal of Neuroimmunology 95 (1999) 152–156
can be antagonized endogenously by the anti-apoptotic mitochondrial protein bcl-2 ŽHaldar et al., 1995. and exogenously by the antioxidant aminoguanidine ŽGiardino et al., 1998., which prevent lipid peroxidation. Secondly, there may be primary intracellular targets for the action of membrane diffusible H 2 O 2-like mitochondrial membranes ŽShenker et al., 1998. and transcription factors ŽLos et al., 1998.. Apoptosis, which is induced primarily by intracellular oxidative processes is typically prevented by thiol-type antioxidants. For example, exogenously applied thioredoxin ŽIwata et al., 1997; Los et al., 1998. as well as endogenously generated glutathione ŽIwata et al., 1997. suppress apoptosis in Jurkat T cells with similar efficacy as catalase. Furthermore, differences in surface thiols correlate with differential sensitivities of T cell subsets to oxidative stress ŽLawrence et al., 1996.. In summary, the catalase-sensitive T cell apoptosis in our PMA-stimulated cocultures with macrophages may be mediated by plasma membrane lipid peroxidation andror mitochondrial dysfunction described, e.g., by Slater et al. Ž1995.. Concerning the catalase-resistant T cell apoptosis in our system Fas and TNF-a receptors are suggestive to be involved. Their activation could be controlled by T cell receptor ŽTCR. ligation with partial agonists that do not induce full TCR signaling, but rather lead to peripheral tolerance in vivo ŽCombadiere et al., 1998.. Our in vitro system may replicate the in vivo situation in inflammatory lesions, where the proportion of MØ to T cells is about 10:1 ŽBruck ¨ et al., 1996., the T cells are activated via T cell receptor ligation and proinflammatory cytokines and cell–cell contact between different cell types easily occurs. From our results it is concluded that a selective intervention in autoimmune inflammation, e.g., in autoimmune demyelinating diseases, should not necessarily aim to prevent cell–cell contact between MØ and T cells and apoptosis-mediating ROS, but rather attempt to downregulate apoptosis-protective gene products like bcl-2 in T cells. Acknowledgements We thank Dr. K.V. Toyka for helpful comments on the manuscript, Mrs Alexandra L. Bunz for culture of the P2-cell line and Mrs. Ines Tschertner for preparation of the MØ. The secretarial help of Mrs. Iris Kell and Mrs. Andrea Freund is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft ŽZe 326r5-1 and Go 459r3-1. and Gemeinnutzige Hertie¨ Stiftung. References Aliprantis, A.O., Diez-Roux, G., Mulder, L.C.F., Zychlinsky, A., Lang, R.A., 1996. Do macrophages kill through apoptosis? Immunol. Today 17, 573–576.
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Bruck, W., Sommermeier, N., Bergmann, M., Zettl, U., Gobel, H.H., ¨ ¨ Kretzschmar, H.A., Lassmann, H., 1996. Macrophages in multiple sclerosis. Immunobiology 195, 588–600. Buttke, T.M., Sandstrom, P.A., 1994. Oxidative stress as a mediator of apoptosis. Immunol. Today 15, 7–10. Combadiere, B., Reis e Sousa, C., Trageser, C., Zheng, L.X., Kim, C.R., Lenardo, M.J., 1998. Differential TCR signaling regulates apoptosis and immunopathology during antigen responses in vivo. Immunity 9, 305–313. Deas, O., Dumont, C., Mollereau, B., Metivier, D., Pasquier, C., Bernard-Pomier, G., Hirsch, F., Charpentier, B., Senik, A., 1997. Thiol-mediated inhibition of FAS and CD2 apoptotic signaling in activated human peripheral T cells. Int. Immunol. 9, 117–125. Finstad, H.S., Myhrstad, M.C., Heimli, H., Lomo, J., Blomhoff, H.K., Kolset, S.O., Drevon, C.A., 1998. Multiplication and death-type of leukemia cell lines exposed to very long-chain polyunsaturated fatty acids. Leukemia 12, 921–929. Giardino, I., Fard, A.K., Hatchell, D.L., Brownlee, M., 1998. Aminoguanidine inhibits reactive oxygen species formation, lipid peroxidation, and oxidant-induced apoptosis. Diabetes 47, 1114–1120. Gold, R., Schmied, M., Giegerich, G., Breitschopf, H., Hartung, H.-P., Toyka, K.V., Lassmann, H., 1994. Differentiation between cellular apoptosis and necrosis by the combined use of in situ tailing and nick translation techniques. Lab. Invest. 71, 219225. Gold, R., Hartung, H.-P., Lassmann, H., 1997. T-cell apoptosis in autoimmune diseases: termination of inflammation in the nervous system and other sites with specialized immune-defense mechanisms. Trends Neurosci. 20, 399–404. Haldar, S., Jena, N., Croce, C.M., 1995. Inactivation of Bcl-2 by phosphorylation. Proc. Natl. Acad. Sci. USA 92, 4507–4511. Hawkins, R.A., Sangster, K., Arends, M.J., 1998. Apoptotic death of pancreatic cancer cells induced by polyunsaturated fatty acids varies with double bond number and involves an oxidative mechanism. J. Pathol. 185, 61–70. Iwata, S., Hori, T., Sato, N., Hirota, K., Sasada, T., Mitsui, A., Hirakawa, T., Yodoi, J., 1997. Adult T cell leukemia ŽALT.-derived factorrhuman thioredoxin prevents apoptosis of lymphoid cells induced by L-cystine and glutathione depletion: possible involvement of thiolmediated redox regulation in apoptosis caused by pro-oxidant state. J. Immunol. 158, 3108–3117. Lawrence, D.A., Song, R., Weber, P., 1996. Surface thiols of human lymphocytes and their changes after in vitro and in vivo activation. J. Leukocyte Biol. 60, 611–618. Los, M., Khazaie, K., Schulze-Osthoff, K., Bauerle, P.A., Schirrmacher, ¨ V., Chlichlia, K., 1998. Human T cell leukemia virus-I ŽHTLV-I. tax-mediated apoptosis in activated T cells requires an enhanced intracellular prooxidant state. J. Immunol. 161, 3050–3055. Munn, D.H., Pressey, J., Beall, A.C., Hudes, R., Alderson, M.R., 1996. Selective activation-induced apoptosis of peripheral T cells imposed by macrophages. A potential mechanism of antigen-specific peripheral lymphocyte deletion. J. Immunol. 156, 523–532. Shenker, B.J., Guo, T.L., Shapiro, I.M., 1998. Low-level methylmercury exposure causes human T-cells to undergo apoptosis: evidence of mitochondrial dysfunction. Environ. Res. 77, 149–159. Slater, A.F., Stefan, C., Nobel, I., van den Dobbelsteen, D.J., Orrenius, S., 1995. Signalling mechanisms and oxidative stress in apoptosis. Toxicol. Lett. 82, 149–153. Suhara, T., Fukuok, K., Sugimoto, T., Morimoto, S., Nakahashi, T., Hata, S., Shimizu, M., Ogihara, T., 1998. Hydrogen peroxide induces up-regulation of Fas in human endothelial cells. J. Immunol. 160, 4042–4047. Van Furth, R. ŽEd.., 1992. Mononuclear Phagocyte Biology of Monocytes and Macrophages. Kluver Academic, Dordrecht, The Netherlands. Wesch, D., Marx, S., Kabelitz, D., 1998. Monocyte-dependent death of freshly isolated T lymphocytes: induction by phorbolester and mitogens and differential effects of catalase. J. Immunol. 161, 1248–1256.
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Wu, M.X., Daley, J.F., Rasmussen, R.A., Schlossman, S.F., 1995. Monocytes are required to prime peripheral blood T cells to undergo apoptosis. Proc. Natl. Acad. Sci. U.S.A. 92, 1525–1529. Wu, M.X., Ao, Z., Hegen, M., Morimoto, C., Schlossman, S.F., 1996. Requirement of FasŽCD95., CD45, and CD11arCD18 in monocytedependent apoptosis of human T cells. J. Immunol. 157, 707–713. Zettl, U.K., Gold, R., Toyka, K.V., Hartung, H.-P., 1995. Intravenous glucocorticosteroid treatment augments apoptosis of inflammatory T
cells in experimental autoimmune neuritis ŽEAN. of the Lewis rat. J. Neuropathol. Exp. Neurol. 54, 540–547. Zettl, U.K., Mix, E., Zielasek, J., Stangel, M., Hartung, H.-P., Gold, R., 1997. Apoptosis of myelin-reactive T cells induced by reactive oxygen and nitrogen intermediates in vitro. Cell. Immunol. 178, 1–8. Zheng, L., Fisher, G., Miller, R.E., Peschon, J., Lynch, D.H., Lenardo, M.J., 1995. Induction of apoptosis in mature T cells by tumor necrosis factor. Nature 377, 348–351.
Apoptosis induction by macrophage-derived reactive oxygen species in myelin-specific T cells requires cell–cell contact Eilhard Mix a , Uwe K. Zettl b
a,b
, Jurgen Zielasek b, Hans-Peter Hartung ¨
b,1
, Ralf Gold
b,)
a Department of Neurology, UniÕersity of Rostock, D-18055 Rostock, Germany Department of Neurology, Clinical Research Group for Multiple Sclerosis and Neuroimmunology, Julius Maximilians UniÕersity, D-97080 Wurzburg, ¨ Germany
Received 15 October 1998; revised 21 December 1998; accepted 28 December 1998
Abstract Apoptosis of autoreactive T cells has been recognized as an important mechanism of immune homeostasis in autoimmune PNS diseases. To examine whether T cells are induced to undergo apoptosis by macrophages ŽMØ. via reactive oxygen species ŽROS. neuritogenic P2-specific T line cells were exposed to peritoneal MØ of Lewis rats in vitro. ROS production was stimulated by phorbol myristate acetate ŽPMA. and inhibited by catalase. Flow cytometric analysis of apoptosis as measured by TUNEL technique revealed that 5–15% of all P2-cells were apoptotic, if cultured alone. This percentage increased slightly, but did not exceed 20% when P2-cells were cocultivated with PMA-stimulated MØ in a two-chamber culture plate separated by a cell-impermeable membrane. Direct cocultivation caused apoptotic cell death of more than 40% of P2-cells, which was completely abrogated by catalase. These results suggest a requirement for close cell–cell contact for apoptosis induction of T cells via MØ-derived ROS. Thus MØ may contribute to termination of inflammation in vivo through the release of highly reactive oxygen species that mediate apoptotic cell death of autoaggressive T lymphocytes. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Apoptosis; Lewis rats; Macrophage; T cells
1. Introduction Apoptosis is regarded as the major physiologic mechanism of elimination of autoreactive T cells in a number of autoimmune diseases ŽGold et al., 1997.. Mediators of T cell apoptosis in vivo are not yet identified. Possible candidates would be products of the oxidative burst, i.e., reactive oxygen species ŽROS., in the inflammatory infiltrates ŽButtke and Sandstrom, 1994.. Aims to obtain information on the origin of ROS and to determine their possible role for termination of autoimmune processes are, therefore, the focus of current research. Abbreviations: MØ, macrophages; P2-cells, P2-specific T line cells; ROS, reactive oxygen species; TUNEL, terminal transferase ddUTP nick end labeling ) Corresponding author. Department of Neurology, Neurologische Universitatsklinik, Josef-Schneider-Strasse 11, D-97080 Wurzburg, Germany. ¨ Tel.: q 49-931-2015755; Fax q 49-931-2013488; E-m ail: [email protected] 1 Present address: Department of Neurology, University of Graz, Austria.
Among inflammatory cells, macrophages ŽMØ. are good candidates as inducers of autoreactive T cell apoptosis, since ŽI. MØ kill developing peripheral T cells ŽMunn et al., 1996. as well as permanent T line cells ŽWu et al., 1995. with high efficacy in vitro, ŽII. MØ are abundant at the target site in autoimmune inflammatory lesions ŽBruck ¨ et al., 1996., ŽIII. MØ produce ROS in high quantity upon activation ŽVan Furth, 1992., and ŽIV. the mechanism of T cell killing by MØ involves apoptosis in vitro and in vivo ŽAliprantis et al., 1996.. ROS produced by locally accumulated MØ could, therefore, be important mediators of apoptotic cell death for autoreactive T cells. We have tested this hypothesis by in vitro activation of peritoneal MØ of Lewis rats with phorbol myristate acetate ŽPMA. and their cocultivation with neuritogenic P2-specific T line cells ŽP2-cells. derived from lymph nodes of the same rat strain. Cocultures were performed by incubating the P2 cells directly with the peritoneal MØ or in twochamber culture plates separated by a cell-impermeable membrane. Apoptosis was examined in both cell compartments by the TUNEL technique and flow cytometry.
0165-5728r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 Ž 9 9 . 0 0 0 0 6 - 5
E. Mix et al.r Journal of Neuroimmunology 95 (1999) 152–156
153
2. Materials and methods 2.1. Cell preparation and cocultiÕation The T cell line P2 was established from lymph nodes of Lewis rats immunized with the peripheral myelin component P2 protein as described previously ŽZettl et al., 1995.. P2-cells were used on days 3 to 6 after the sixth in vitro restimulation cycle with P2 protein as antigen and irradiated thymocytes as antigen-presenting cells. MØ were freshly prepared from the peritoneal exudates of Lewis rats by adherence. Briefly, peritoneal exudate cells of two to three animals were washed once in PBS without Ca2q and Mg 2q and resuspended in culture medium consisting of RPMI 1640 ŽGibco, Grand Island, NY. supplemented with 1% normal rat serum, 2 mM glutamine and antibiotics. Cells were allowed to adhere to the surface of 200 ml tissue culture flasks precoated with fetal calf serum by incubation of 378C for 45 min. Non-adherent cells were removed by rinsing with warm 5 mM EDTA-PBS and discarded. The remaining adherent cells were removed by 2 min incubation at 48C with culture medium and washed once. The resulting cell suspension contained ) 90% viable macrophages as tested by trypan blue exclusion and labeling with the MØ-specific monoclonal antibody ED1 ŽSerotec, Raleigh, NC.. Cocultivation of P2-cells and MØ was performed in 24-well culture plates ŽCostar, Cambridge, MA. containing inserts with a cell impermeable membrane Žpore size 1.0 mm.. 10 6 MØ were applied in 0.5 ml to the outer well and 10 5 P2-cells in 0.5 ml to the insert Žseparated cocultivation.. Alternatively, cocultivation was done without using the inserts by direct application of the same cell numbers and volumes to the outer wells of culture plates Ždirect cocultivation.. For control MØ and P2-cells were cultivated alone by adding another 0.5 ml medium to the respective cell suspensions. For stimulation of the oxidative burst PMA ŽSigma, St. Louis, MO. was added to the MØ at a final concentration of 10y7 M. For the inhibition of H 2 O 2 catalase ŽSigma. was added at a final concentration of 10y3 unitsrml. All cultures were run for 18 h at 378C. 2.2. TUNEL technique and FACS analysis For quantitation of apoptotic cells, cell cultures were transferred to 1.5-ml conical reaction tubes ŽEppendorf, Hamburg, Germany. followed by fixation, permeabilization and staining by the terminal transferase ddUTP end labeling ŽTUNEL. technique as described previously ŽGold et al., 1994.. Briefly, cells were centrifuged for 5 min at 400 = g and room temperature ŽRT. and fixed for 12 min in 200 ml of 4% buffered paraformaldehyde. After one washing cells were permeabilized in 200 ml of 0.025 Nonidet P-40 ŽSigma. for 15 min at RT. After another washing 20 ml cell suspension were mixed with 35 ml of a tailing reaction mixture containing the following compo-
Fig. 1. Histogram of cocultivated P2-cells and MØ after labeling by TUNEL technique Žgreen Fluorescent ddUTP, y-axis. for quantitation of apoptotic cells and by anti-ED1 monoclonal antibodies Žred Fluorescent ED1, x-axis. for identification of MØ. For details of staining and FACS, see Section 2.
nents: 0.15 nmol FITC-12-ddUTP ŽBoehringer, Mannheim, Germany., 0.85 nmol dTTP and 1 nmol dGTP, dATP and dCTP ŽPharmacia, Uppsala, Sweden., 5 units terminal deoxynucleotidyl-transferase ŽTdT; Promega, Madison, WI. and bi-distilled water. The samples were rotated under light protection Ž400 rpm. at 378C for 90 min and analyzed by FACS. To identify MØ and P2-cells in direct cocultures, cells were stained for the MØ-specific antigen ED1 prior to TUNEL technique. In a first step mouse anti-rat ED1 monoclonal antibodies were added to the permeabilized cells diluted 1:80 in 50 ml staining buffer Ž1% BSA and 0.1% NaN3 in PBS. and incubated on ice for 30 min followed by two washes with the same buffer. In a second step anti-mouse IgG1-Tricolor ŽMedac, Hamburg, Germany. diluted 1:20 in 50 ml staining buffer was added, incubated and washed as in the first staining step followed by TUNEL technique. Labeled cells were analyzed for apoptotic T cells and MØ in a FACScan ŽBecton Dickinson, San Jose, ´ CA. on the basis of 10,000 cells gated by forward vs. side scatter. Fig. 1 illustrates the window settings for quantitation of apoptotic green fluorescent cells among ED1q red fluorescent MØ and ED1y P2-cells in an example of direct cocultivation.
3. Results and discussion T cell elimination from many sites of inflammation, e.g., in autoimmune diseases, occurs mainly via apoptosis ŽGold et al., 1997.. Macrophages are supposed to induce T cell apoptosis in inflammatory lesions either by soluble mediators or by direct cell–cell interaction ŽMunn et al.,
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1996; Wu et al., 1996.. Direct cell–cell contact via adhesion molecules has been shown to be a prerequisite for Fas-mediated apoptosis of human peripheral T cells triggered by monocytes ŽWu et al., 1996.. An alternative way of apoptosis induction involves the action of ROS, which are produced by activated MØ ŽVan Furth, 1992. and monocytes ŽWesch et al., 1998.. We have investigated the possibility that T cell apoptosis is induced by MØ-derived ROS in a dual approach of cocultivation with and without addition of the ROSantagonist catalase. Peripheral myelin protein P2-specific T line cells were exposed either directly to rat peritoneal MØ Ždirect coculture. or separated by a semipermeable membrane Žseparated coculture.. The main results of our study were the following ŽTable 1.: ŽI. T cells and MØ showed a baseline frequency of apoptotic cells of less than 10%, if the cells were cultured alone in culture medium. A slight increase of apoptotic cells to about 15% was observed in T cells exposed to the stimulator of the oxidative burst, PMA, whether they were cultivated in the inserts of plates for separated cocultivation or in the ‘normal’ wells of plates for direct cocultivation. Since the slight increase of T cell apoptosis seen after addition of PMA could be suppressed by catalase, it may be the result of ‘endogenous’ ROS production of stimulated T line cells. MØ did not undergo a PMA-stimulated apoptosis. ŽII. In direct cocultures of T cells with MØ ED1y T cells revealed a 2-fold increase of apoptotic cells compared to the T cell cultures alone Žabout 20%., which was further augmented by PMA Žto about 40%.. The PMA-induced augmentation could be suppressed by catalase to values of cocultures without PMA, but not to the ‘basal’ rate of single T cell cultures. The remaining T cell apoptosis may, therefore, be due to the action of apoptosis inducers other than ROS, e.g., tumor necrosis factor-a ŽTNF-a ., nitric oxygen radicals ŽNO., etc. ŽZheng et al., 1995; Zettl et al.,
1997.. Also, Fas-mediated apoptosis cannot be excluded to account for the non-catalase inhibitable rate of apoptotic T cells ŽDeas et al., 1997.. The apoptotic rate of ED1q MØ was not affected neither by PMA nor by cocultivated T cells. ŽIII. In separated cocultures only a minor increase of the rate of apoptotic T cells was seen with PMA, i.e., to about 15% in the single T cell culture and to about 20% in the coculture with MØ. These slight increases were inhibited by catalase. However, since they did not exceed the 20% T cell apoptosis in direct cocultures with PMA q catalase, they may be the result of indirect ROS-stimulation via soluble mediators like TNF-a ŽZheng et al., 1995. The low frequency of apoptosis observed in MØ Ž5–10%. argue against the existence of a relevant apoptosis-inducing effect of T cells on MØ. MØ may be generally resistant to ROS-induced apoptosis. Since apoptosis of MØ-exposed activated T cells was abrogated to a considerable degree by catalase, we conclude that H 2 O 2 is the major mediator of MØ cytotoxicity to T cells in our in vitro system that mimics the conditions of autoimmune demyelinating cells in the peripheral nervous system of the rat. The mechanism of action may not involve the Fas-dependent pathway of apoptosis, since ŽI. Wesch et al. Ž1998. found recently that monocyte-mediated apoptotic cell death of mitogen activated T cells is Fas-independent, and ŽII. Deas et al. Ž1997. reported that Fas-mediated T cell death does not rely on ROS. The situation may be different in other cell types like endothelial cells, in which H 2 O 2 induces Fas expression ŽSuhara et al., 1998.. Several mechanisms may account for the apoptosis inducing effect of H 2 O 2 on activated neuritogenic T cells used in our study: Firstly, peroxidation of plasma membrane lipids may be the first step of the apoptotic process as it has recently been described for polyunsaturated fatty acid-induced apoptosis in leukemia ŽFinstad et al., 1998. and in cancer cells ŽHawkins et al., 1998.. This pathway
Table 1 Percentages of apoptotic cells after cocultivation of rat P2-specific T line cells ŽP2. and peritoneal macrophages ŽMØ. with and without stimulation by PMAa Cells
MØ alone P2 alone MØ q P2 a
Direct cocultivation
Separated cocultivation
Medium alone
qPMA
qPMAq catalase
Medium alone
qPMA
qPMAq catalase
7.1 " 2.3 Ž6.0. b 8.2 " 3.4 Ž6.1. ED1q: 5.1 " 3.2 Ž2.7. c ED1y: 21.9 " 4.0 Ž18.5.
8.6 " 4.1 Ž4.3. 14.8 " 8.0 Ž10.3. 7.0 " 5.7 Ž2.5. 37.8 " 6.4(43.2)
9.9 " 4.3 Ž8.1. 12.2 " 9.3 Ž5.1. 8.2 " 7.2 Ž1.7. 20.2 " 14.3 Ž7.8.
n.d. 10.3 " 0.8 Ž10.1. MØ: 5.2 " 1.0 Ž6.1. P2: 10.3 " 1.2 Ž10.8.
n.d. 14.0 " 1.3 Ž14.2. 10.8 " 1.0 Ž10.0. 20.8 " 10.0 Ž15.5.
n.d. 6.8 " 1.8 Ž6.4. 10.2 " 3.3 Ž8.8. 12.8 " 1.8 Ž11.1.
P2 cells and peritoneal MØ were cultivated either alone or together in microtiter plates separated by a cell impermeable membrane Žseparated cocultivation. or unseparated Ždirect cocultivation.. Three types of cultures were performed: Ž1. Cultivation in medium alone, Ž2. additional stimulation of oxidative burst with PMA, and Ž3. additional stimulation of oxidative burst with PMA and destroying of H 2 O 2 by catalase. Apoptosis was detected by TUNEL technique and flow cytometry. b Data are given as means" S.E.M. Žmedians in parenthesis. of three independent experiments. c In the direct coculture, MØ were identified by fluorescence labeled anti-ED1 monoclonal antibodies. ED1q cells represent the peritoneal MØ, whereas the majority of ED1y cells Ž) 90%. are P2 T cells. The highest values of apoptotic cells were found for T cells directly exposed to PMA-stimulated MØ Žprinted in bold..
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can be antagonized endogenously by the anti-apoptotic mitochondrial protein bcl-2 ŽHaldar et al., 1995. and exogenously by the antioxidant aminoguanidine ŽGiardino et al., 1998., which prevent lipid peroxidation. Secondly, there may be primary intracellular targets for the action of membrane diffusible H 2 O 2-like mitochondrial membranes ŽShenker et al., 1998. and transcription factors ŽLos et al., 1998.. Apoptosis, which is induced primarily by intracellular oxidative processes is typically prevented by thiol-type antioxidants. For example, exogenously applied thioredoxin ŽIwata et al., 1997; Los et al., 1998. as well as endogenously generated glutathione ŽIwata et al., 1997. suppress apoptosis in Jurkat T cells with similar efficacy as catalase. Furthermore, differences in surface thiols correlate with differential sensitivities of T cell subsets to oxidative stress ŽLawrence et al., 1996.. In summary, the catalase-sensitive T cell apoptosis in our PMA-stimulated cocultures with macrophages may be mediated by plasma membrane lipid peroxidation andror mitochondrial dysfunction described, e.g., by Slater et al. Ž1995.. Concerning the catalase-resistant T cell apoptosis in our system Fas and TNF-a receptors are suggestive to be involved. Their activation could be controlled by T cell receptor ŽTCR. ligation with partial agonists that do not induce full TCR signaling, but rather lead to peripheral tolerance in vivo ŽCombadiere et al., 1998.. Our in vitro system may replicate the in vivo situation in inflammatory lesions, where the proportion of MØ to T cells is about 10:1 ŽBruck ¨ et al., 1996., the T cells are activated via T cell receptor ligation and proinflammatory cytokines and cell–cell contact between different cell types easily occurs. From our results it is concluded that a selective intervention in autoimmune inflammation, e.g., in autoimmune demyelinating diseases, should not necessarily aim to prevent cell–cell contact between MØ and T cells and apoptosis-mediating ROS, but rather attempt to downregulate apoptosis-protective gene products like bcl-2 in T cells. Acknowledgements We thank Dr. K.V. Toyka for helpful comments on the manuscript, Mrs Alexandra L. Bunz for culture of the P2-cell line and Mrs. Ines Tschertner for preparation of the MØ. The secretarial help of Mrs. Iris Kell and Mrs. Andrea Freund is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft ŽZe 326r5-1 and Go 459r3-1. and Gemeinnutzige Hertie¨ Stiftung. References Aliprantis, A.O., Diez-Roux, G., Mulder, L.C.F., Zychlinsky, A., Lang, R.A., 1996. Do macrophages kill through apoptosis? Immunol. Today 17, 573–576.
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