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Interference of cyclosporin with lymphocyte proliferation: Effects on mitochondria and lysosomes of cyclosporin-sensitive or -resistant cell clones
CELLULAR
IMMUNOLOGY
93, 486-496 (1985)
Interference of Cyclosporin with Lymphocyte Proliferation: Effects on Mitochondria and Lysosomes of Cyclosporin-Sensitive or -Resistant Cell Clones’ MARIANNE
KOPONEN,~ ALFONS GRIEDER,
ROLF HAUSER,*
AND FRANCIS LOOR?
Preclinical Research Department, Sandoz Ltd., 4002 Basel, Switzerland; *Toxicology Department, Sandoz Ltd., 4132 Muttenz, Switzerland; and tLaboratoire d’lmmunologie, Universite’ Louis Pasteur, 67048 Strasbourg, France Received September 24, 1984; accepted February 21, 1985
Cyclosporin was previously shown to interfere with-but not to abolish-the increased activities of lysosomes and mitochondria consequent to a mitogenic activation of normal mouse lymphocytes. This was evident from the fluorescence profiles of cell populations after vital staining with euchrysine (giving a lysosomal-specific red fluorescence)and rhodamine- I23 (giving a mitochondrial-specific green fluorescence).Fluorescence profiles of the population of cells not exposed to a mitogen were also altered by cyclosporin, with lower lysosomal and mitochondrial fluorescence of these cell populations. In order to find out more precisely what could be the direct effects of cyclosporin on those cellular organelles, our cyclosporin-sensitive (BE7) and cyclosporin-resistant (LB7) lymphoblastoid cell lines were tested and showed clearcut differences. Only minor effects could be detected for the lysosomal and mitochondrial activities of the resistant cells. On the contrary, cyclosporin caused, in the cells of the sensitive clone BE7, a clear decreaseof mitochondrial activity together with an unexpected increase of the red fluorescence of euchrysine. The latter might not correspond to a real increase of the lysosomal activity of such cells. Indeed electron microscopy studies do not show higher numbers of lysosomes; rather they show that numerous vacuoles appear in the cytoplasm of the cyclosporin-treated BE7 cells (but not in the cells of the resistant clone and not in untreated cells of either types). 0 1985 Academic Press, Inc.
INTRODUCTION The immunomodulatory compound cyclosporin A (CsA) is known to interfere with lymphocyte activation (l), but how this happens is still unclear. The binding of a mitogen to lymphocytes initiates a series of early membranous events. This causesvarious physiological changes in the cytoplasm, and eventually the initiation of nuclear DNA synthesis (2). In this cascadeof biochemical events, the exact site(s) of interference of cyclosporin are not easy to establish. Cyclosporin markedly reduces the mitogen-induced increases of mitochondrial and lysosomal activities displayed by concanavalin A (Con A)-treated lymphocytes. However they remain ’ Supported by grants from the French Science Foundations MRT (82.L.O071),CNRS (ATP1082) and INSERM (PRC 127.036). z Present address: Institute of Clinical Immunology, Inselspital, 30 IO Berne, Switzerland. 486 0008-8749185$3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reproduclion in any form resxved.
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substantially higher than those of untreated cells (3). Such incomplete blocking effects of cyclosporin are difficult to interpret. They may show either that each individual Con A-responding cell is partly blocked in their lysosomal and mitochondrial maturation or that some cells are totally blocked while others would not be blocked at all. Furthermore spleen and lymph node cell suspensions exposed to cyclosporin in absenceof Con A also display various alterations of the mitochondrial and lysosome-specific fluorescence patterns. These effects, too, are difficult to interpret since they are obtained with complex populations of cells in vitro. We have now used our two cloned lymphoblastoid T cells of different susceptibility to cyclosporin (4) in order to look at how their lysosomes and mitochondria might be affected by cyclosporin. This results in clear cut differences: (1) Only the cyclosporin-sensitive (Cs’) cell clone shows a decreaseof uptake of rhodamineinto its mitochondria upon treatment with cyclosporin. (2) Cyclosporin causes an increase of the vital red euchrysine staining (lysosome specificity) of the Css cell clone only, while the resistant (CsR) cell clone is only slightly affected. Preliminary studies by electron microscopy also reveal that cyclosporin causes an extensive vacuolization of the cytoplasm of the Css clone cells. MATERIALS
AND METHODS
Cell cultures and cyclosporin treatment. The cyclosporin-sensitive (BE7) and cyclosporin-resistant (LB7) lymphoblastoid T-cell clones and the culture conditions have been previously described (4, 5). CsA (Sandoz, Basle, Switzerland) was used on the cells at concentrations ranging from 0.1 to 1.0 pg/ml (final ethanol solvent concentration was 0.1%). The cells were assayedat OS- 1.O X 1O5cells/ml in tissue culture trays (24 wells, l-ml cultures; Costar 3524, Cambridge, Mass.). Fluorescence procedures. The staining procedure, the fluorescence microscopy of the stained cells, and the flow cytometry analyses of the stained cell suspensions were performed as described previously (3, 6). The mitochondria were detected with rhodamine(R-123; Eastman Kodak, Rochester, N.Y.) at a final concentration of 10 pg/ml for 10 min at 37°C and lysosomes were vitally stained with euchrysine (acridine orange; Merck, Darmstadt, West Germany) at 5 pg/ml for 5 min at room temperature in the dark. The samples were observed by fluorescence microscopy and analyzed by flow cytometry without delay, using an Ortho Cytofluorograf 50 HH (Ortho Diagnostic Instruments, Westwood, Mass.). Electron microscopy. Following incubation alone or with cyclosporin (1 pg/ml), the cells were sedimented and washed with 0.9% NaCl. They were then repelleted by centrifugation (15OOg,10 min) and prefixed with 2.5% glutaraldehyde in 0.13 M balanced phosphate buffer, pH 7.2. After a thorough rinsing in the same buffer the samples were postfixed in phosphate-buffered 1.3% recycled osmium tetroxide at 4°C (Simec, Birsfelden, Switzerland). The samples were dehydrated through a graded series of ethanol and propylene oxide (Fluka, Buchs, Switzerland) prior to embedding in Beem capsules using Epon 8 12 (7). During the dehydration the samples were stained with 2% uranyl acetate at the 80% ethanol step. Ultrathin sections were cut on a LKB Ultratome III 8000 at a thickness of 60-80 nm (silver interference color) and stained on the grid with 2% uranyl acetate and 0.4% lead citrate (3 min) (8). The sections were examined in a Siemens 101 electron microscope.
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RESULTS Efects of cyclosporin on mitochondria (Fig, 1). The presence of cyclosporin did not change the fluorescence pattern of the CsR cell clone LB7, indicating that the uptake of R-123; thus, the mitochondrial activity was virtually unaffected. The uptake of R-123 into mitochondria of the Css cell clone BE7 was decreased by cyclosporin after 24 hr in culture: there was a shift of the population profile towards lower fluorescence levels. This effect was cyclosporin dose dependent. The dose of 0.1 pg/ml cyclosporin (which has only little effect on the proliferation of the clone, measured at 3-4 days) was still effective. The effects were more pronounced at the doses of 0.3 pg/ml (which is about the LDsOfor this line), and of 1.O pg/ml (which
FIG. 1. Population analysis by vital rhodaminestaining. X axis, green (mitochondria-specific) fluorescence intensity: Y axis, number of cells in each channel. Comparison of 24-hr cultures of Cssensitive clone BE7 (left) and the Cs-resistant clone LB7 (right). Bottom curves, controls without cyclosporin, top curves, with cyclosporin (0.1, 0.3, 1.O &ml as indicated; 0.1% ethanol as solvent). The numbers represent the numbers of cells from channel 55 to channel 5 12 (out of 50,000 cells).
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gives 100% inhibition of proliferation of this line, without detectably affecting cell survival at 24 hr and is still essentially reversible at that time (5)). A large proportion of the Css cells treated for 24 hr with 0.3 or 1.0 Fg/ml cyclosporin showed the same low R-123 uptake as a minority of cells from the control cultures. The effects of cyclosporin on R-123 uptake were not very marked at earlier times (not shown). They also appeared less pronounced after 48-hr incubation. This might be due to unspecific changes in absorption of R-123 by the cells/mitochondria, since the BE7 cells are Cs sensitive and eventually die if cyclosporin is not removed; indeed, after such a time of exposure to cyclosporin, they show a lower capacity to grow upon removal of the drug (5). Eficts of cyclosporin on lysosomes. Cyclosporin treatment of Css cells (BE7) or of CsR cells (LB7) had very different consequences on their lysosome-specific (red) fluorescence when vitally stained by euchrysine. This is shown both by the cell distribution displays and by using two arbitrarily selected windows (windows 1 and 2 for lower and higher red fluorescence levels, respectively). BE7 and LB7 cells were exposed to various concentrations of cyclosporin in the medium (0, 0.1, 0.3, and 1.O pg/ml, with 0.1% ethanol as solvent in each case). After various times they were tested for euchrysine red (lysosome-specific) and green (marker for the available DNA, mostly a cell mass marker) fluorescence. Two independent experiments are shown in Figs. 2-4 and Tables 1 and 2. Within 4 hr of cyclosporin addition to the Css cells, it induced a concentration-dependent increase in the red fluorescence which was more pronounced at 6 hr and very strong after 24 hr (Fig. 2, Table 1). The 0. I-pg/ml dose was only marginally effective except at 24 hr (Table 1). On the contrary, cyclosporin treatment of the CsR cells did not change their vital staining by euchrysine, except in the sample exposed to the highest dose (1 pg/ml) for 24 hr (Table 1, Fig. 3). However this effect was no longer found after 48 hr in the presence of cyclosporin, as though the CsRcells had succeededin “correcting” some action of cyclosporin on the structures stained red by euchrysine (Table 2, Fig. 4). The red fluorescence level of the Css cells continued to increase from 24 to 48 hr for doses of 0.3 and 1.0 yg/ml cyclosporin (Table 2, Fig. 4). The level of red fluorescence per cell is very heterogeneousin the cyclosporintreated Css cells, particularly after 24 and 48 hr of treatment. During the same culture time, the cells showed a decreased green fluorescence and became more homogeneous at a low fluorescence level. This might correspond to a smaller cell size and/or a lower DNA content. Electron microscopy of cyclosporin-treated cells. All studies done so far revealed that CsA at 1 pg/ml induced a vacuolization of the cytoplasm of the Css cells. This was particularly evident after 24 hr in the presence of CsA (Fig. 5a), but it started to be detectable within 3 hr of incubation with CsA (not shown). The CsR cells were not much affected by CsA except for the presence of small vesicles at 24 hr (Fig. 5b). These effects were specific of CsA since the solvent alone (ethanol, 0.1%) did not cause them in either cell clone (Figs. 5c, d). It is relevant here that only a few lysosomes were detected in cells of both clones, even in conditions (Css cells, 1 pg CsA/ml, 24 hr) where the vital staining with euchrysine resulted in high red fluorescence of the cells. Further data on the dose and time dependence of these effects of CsA, as well as on the emergence of lipid bodies and other alterations of the cellular organization, are still preliminary (9).
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FIG. 2. Scatterplots of red (X axis; lysosome-specific)and green (Y axis; DNA content/cell size indic:ator) fluorescence from vital euchrysine-stained clone BE7 (Cs’). In vitro exposure for 6 and 24 hr for va.sious concentrations of cyclosporin (0.1, 0.3, 1.0 &ml in 0.1% ethanol). Each point in scatterplot repre the bivariable measurements of a single cell.
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FIG. 3. CsR ceil clone LB7 after exposure to cyclosporin for 6 and 24 hr. Same analyses as for clone BE7 in Fig. 2 for the 0.3 and 1.0 &ml cyclosporin concentrations.
DISCUSSION Cyclosporin interferes with the concanavalin A-induced activation of mixed lymphocyte populations, at or prior to the steps which involve increased lysosomal and mitochondrial activities (3). The latter were evaluated, as in the present study, by the specific fluorescence emission of euchrysine (lysosomes) and of rhodamine123 (mitochondria) by individual cells of the population. The fluorescence profiles of the cyclosporin-treated population were close but by no means identical to the background fluorescence levels of unstimulated cells (3). The heterogeneity of a cell population and the existence of intercellular interactions remain major difficulties when trying to interpret any modification of a biological or biochemical parameter by a drug or a factor. Were the observed alterations reflecting mainly changes in
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ET AL,
R G. 4. Population analyses of clones BE7 (Cs’) and LB7 (CsR) after in vitro exposure for 48 hr cyclelsporin (0.1, 0.3, and 1.O &ml cyclosporin in 0.1% ethanol). Vital staining by euchrysine.
to
the relative proportions of different cell types of the population? Or were they 43n the contrary the signs of more basic alterations of lysosomal and mitochondn ial acti vities that would actually affect each individual cell?
493
CYCLOSPQRIN EFFECTS ON LYSOSOMES AND MITOCHONDRIA TABLE 1 Percentage of Cells Falling in Window 2” Time in culture (W Cyclosporinb kImI)
4
6
24'
BE7 (Cs’)
0 0.1 0.3 1.0
2.4 3.7 5.5 8.2
0.95 2.0 3.8 10.4
9.7 22.0 38.0 79.2
LB7 (CsR)
0 0.1 0.3 1.0
2.7 2.0 2.0 2.6
0.91 1.0 0.89 0.85
9.5 10.5 13.0 19.2
Cell clone
’ Higher red euchrysine fluorescence b In 0.1% ethanol. ‘Same samples as in Figs. 2 and 3.
The present data were obtained with a Cs-sensitive clone and a Cs-resistant clone and they allow us to better delineate the effects of cyclosporin on single cells. The Css cells are stopped in the early G, phase by cyclosporin (4) (like concanavalin Aactivated spleen cells (9, 10)) whereas the CsRcells are not (4). The difference is not due to different representation or specificity of the membranous cyclosporin-binding sites (I 1). We show here that cyclosporin differently affects both cells with regard to their uptakes of rhodamineand of euchrysine and to the cytoplasm organization. The large increase of the euchrysin red fluorescence emitted by cyclosporintreated Css clone cells could suggest a higher lysosome content. However, electron TABLE 2 Percentage of Cells Falling in Window 2” Time in culture (W Cyclosporin b h/ml)
24
48'
BE7 (Cs’)
0 0.1 0.3 I.0
5.5 5.9 8.8 31.2
2.5 4.1 15.6 61.1
LB7 (CsR)
0 0.1 0.3 1.0
0.8 2.3 2.2 6.6
0.3 0.4 0.6 0.6
Cell clone
’ Higher red euchrysine fluorescence. b In 0.1% ethanol. ‘Same samples as in Fig. 4.
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FIG. 5. Electron microscopic analyses of Css (a, c) and CsR(b, d) clone cells incubated for 24 hr with (a, b) or without (c, d) cyclosporin (1 &ml in 0.1% ethanol in medium at 37’C). Note the numerous cytoplasmic vacuoles in the cyclosporin-treated Cs’ cell (a). (X6 120).
microscopic analyses showed there was no corresponding increase in the number or size of lysosomes. Moreover, direct measurements of activities of several lysosomal enzymes show that cyclosporin doses which affect the euchrysine fluorescence of
CYCLOSPORIN
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the Css cells have little effect on the enzyme content per cell (Renard, P., Fonteneau, P., and Loor, F., manuscript in preparation). Therefore, two possibilities remain: either each lysosome of a cyclosporin-treated Csscell takes up much more euchrysine or becomes more acidic (the red fluorescence of euchrysine requires an acid pH), or the structures detected are not bona jidr lysosomes. The first alternative implies that cyclosporin alters the lysosomal membrane permeability of Css cells, while CsR cells have more stable lysosomal membranes. The second alternative however finds some support in the presence of numerous, possibly autophagic, vacuoles in the cytoplasm of the Css cells at the time they display high red euchrysine fluorescence, and in contrast to their absencein CsRcells. Such vacuoles might pick up euchrysine and fluoresce red if their intracellular pH is acidic. This is the case of the prelysosomal vacuoles in the receptor-mediated endocytosis process ( 12). Weakly basic compounds also inhibit membrane recycling and vacuolise the lysosomes and the Golgi apparatus (13, 14). A variety of autonomic drugs induce cytoplasmic vacuolization in different cells in tissue culture (15). The vacuolization found here seems specific, as it only affects the Css cells, and rather fast, since small vesicles were already detectable within 3 hr of cyclosporin treatment (9). For what concerns other immunosuppressors, large numbers of vacuoles as well as lipid droplets were reported to occur in the cytoplasm of macrophages and reticular cells exposed to steroids (16). Lipid droplets also appear in cyclosporin-treated cells but this does not correlate with their Cs sensitivity (5, 6). A low frequency of a Css clone cell type among normal lymphocytes would explain the contrasting effects of cyclosporin on the red euchrysine fluorescence vital staining of Css clone cell (this study) and of concanavalin A-treated cells (3): in the latter case indeed, the vast majority of the cells showed a decreased red fluorescence when cyclosporin was present during the culture (3). Thus, the particular behavioral response of our Css clone to the presence of cyclosporin may occur in only a minority of a whole spleen or lymph node cell suspension and pass unnoticed. However this type of cell may be common among normal lymphocytes but cyclosporin-induced membranous alterations may allow their recognition by macrophages, their scavenging, and their disappearance from the lymphoid cell cultures. The uptake of rhodamineinto the mitochondria of Css cells was reduced by cyclosporin whereas the CsR clone was not detectably affected. The reduced mitochondrial activity of the Css clone could simply be a consequence of the arrest of the cycling Css cells in early Gr , a cell cycle phase characterized by a low mitochondrial content per cell. However, it seemsthat cyclosporin interferes more directly with the mitochondrial activity or replication, thereby causing the G, arrest. The rhodaminefluorescence distribution profile of cyclosporin-treated Css cells peaks at a rather low fluorescence level which is characteristic of a very small minority of normally growing Css cells. Actually, many of the cyclosporin-treated cells have a very low mitochondrial content/activity which is probably the lowest possible in early G1 phase or even an abnormally reduced one. Interference of cyclosporin with mitochondria function may thus be a major reason for the G, arrest of the Css cells. This does not imply that cyclosporin necessarily works on the mitochondria themselves. Indeed, the recent report that cyclosporin works on the ol-amanitin-sensitive RNA polymerase II (17) suggeststhat effects of cyclosporin
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on mitochondria are indirect; DNA transcription in mitochondria is carried out by a simpler, cu-amanitin-resistant RNA polymerase, but it is noteworthy that mitochondria import a number of nucleus transcribed proteins (18). Inhibition of RNA polymerase II by cyclosporin would block the synthesis and transport of nuclear transcribed proteins into mitochondria. Much mitochondrial maturation is required in order to achieve the high levels of ATP needed for the generation of dNTP up to the levels required for DNA polymerase (Y activation (19). Therefore, deficient mitochondrial function would eventually impair nuclear DNA synthesis and induce the early G, -blocking of cyclosporin-treated cells. The lack of inhibition of mitochondrial activity of CsR cells may have several nonexclusive causes.In the caseof the presently studied LB7 cell clone, transcription is cyclosporin resistant, at least in vitro (Brack, C., Cammisuli, S., Gaveriaux, C., and Loor, F., unpublished experiments). Though this provides a satisfactory explanation for the mechanisms both of cyclosporin action and of cyclosporin-resistance, other mechanisms may operate or be preponderant in other cell types. REFERENCES 1. Wiesinger, D., and Borel, J. F., Immunobiology 156, 454, 1979. 2. Hume, D. A., and Weidemann, M. J., “Mitogenic Lymphocyte Transformation.” Elsevier/Biomedical Press,Amsterdam, 1980. 3. Koponen, M., Grieder, A., and Loor, F., Immunology 53, 55, 1984. 4. Koponen, M., Grieder, A., and Loor, F., Exp. Cell Res. 140, 237, 1982. 5. Koponen, M., and Loor, F., Ann. Immunol. (Paris) 134C, 171, 1983. 6. Koponen, M., and Loor, F., Exp. Cell Res. 149, 499, 1983. 7. Luft, J. H., J. Biophys. Biochem. Cytol. 9,409, 1961. 8. Venable, J. H., and Coggeshall, R., J. Cell Biol. 25, 407, 1965. 9. Koponen, M., “Interference of Cyclosporin with Lymphocyte Activation.” Ph.D. thesis, Biocenter, University of Basel, Switzerland, 1984. 10. Koponen, M., Gerson, D. F., and Loor, F., Agents Actions, 15, 600, 1984. 11. Koponen, M., and Loor, F., Ann. Immunol. (Paris) 134D, 201, 1983. 12. Helenius, A., Mellman, I., Wall, D., and Hubbard, A., Trends Biochem. Sci. 8, 245, 1983. 13. Fedorko, M. E., Hirsch, J. G., and Cohn, 2. A., J. Cell Biol. 38, 392, 1968. 14. Steinman, R. M., Mellman, I. S., Miiller, W. A., and Cohn, Z. A., J. Cell Biol. 96, 1, 1983. 15. Yang, W. C. T., Strasser,F. F., and Pomerat, C. M., Exp. Cell Res. 38, 495, 1965. 16. Miyata, K., and Takaya, K., Cell Tissue Rex 230, 57, 1983. 17. Brack, C., Mattaj, 1. W., Gautschi, J., and Cammisuli, S., Exp. Cell Res. 151, 314, 1984. 18. Wirtz, K. W. A., Biochim. Biophys. Acta 344, 95, 1974. 19. Spadari, S., Sala, F., and Pedrali-Noy, G., Trends Biochem. Sci. I, 29, 1982.
IMMUNOLOGY
93, 486-496 (1985)
Interference of Cyclosporin with Lymphocyte Proliferation: Effects on Mitochondria and Lysosomes of Cyclosporin-Sensitive or -Resistant Cell Clones’ MARIANNE
KOPONEN,~ ALFONS GRIEDER,
ROLF HAUSER,*
AND FRANCIS LOOR?
Preclinical Research Department, Sandoz Ltd., 4002 Basel, Switzerland; *Toxicology Department, Sandoz Ltd., 4132 Muttenz, Switzerland; and tLaboratoire d’lmmunologie, Universite’ Louis Pasteur, 67048 Strasbourg, France Received September 24, 1984; accepted February 21, 1985
Cyclosporin was previously shown to interfere with-but not to abolish-the increased activities of lysosomes and mitochondria consequent to a mitogenic activation of normal mouse lymphocytes. This was evident from the fluorescence profiles of cell populations after vital staining with euchrysine (giving a lysosomal-specific red fluorescence)and rhodamine- I23 (giving a mitochondrial-specific green fluorescence).Fluorescence profiles of the population of cells not exposed to a mitogen were also altered by cyclosporin, with lower lysosomal and mitochondrial fluorescence of these cell populations. In order to find out more precisely what could be the direct effects of cyclosporin on those cellular organelles, our cyclosporin-sensitive (BE7) and cyclosporin-resistant (LB7) lymphoblastoid cell lines were tested and showed clearcut differences. Only minor effects could be detected for the lysosomal and mitochondrial activities of the resistant cells. On the contrary, cyclosporin caused, in the cells of the sensitive clone BE7, a clear decreaseof mitochondrial activity together with an unexpected increase of the red fluorescence of euchrysine. The latter might not correspond to a real increase of the lysosomal activity of such cells. Indeed electron microscopy studies do not show higher numbers of lysosomes; rather they show that numerous vacuoles appear in the cytoplasm of the cyclosporin-treated BE7 cells (but not in the cells of the resistant clone and not in untreated cells of either types). 0 1985 Academic Press, Inc.
INTRODUCTION The immunomodulatory compound cyclosporin A (CsA) is known to interfere with lymphocyte activation (l), but how this happens is still unclear. The binding of a mitogen to lymphocytes initiates a series of early membranous events. This causesvarious physiological changes in the cytoplasm, and eventually the initiation of nuclear DNA synthesis (2). In this cascadeof biochemical events, the exact site(s) of interference of cyclosporin are not easy to establish. Cyclosporin markedly reduces the mitogen-induced increases of mitochondrial and lysosomal activities displayed by concanavalin A (Con A)-treated lymphocytes. However they remain ’ Supported by grants from the French Science Foundations MRT (82.L.O071),CNRS (ATP1082) and INSERM (PRC 127.036). z Present address: Institute of Clinical Immunology, Inselspital, 30 IO Berne, Switzerland. 486 0008-8749185$3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reproduclion in any form resxved.
CYCLOSPORIN
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ON LYSOSOMES
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substantially higher than those of untreated cells (3). Such incomplete blocking effects of cyclosporin are difficult to interpret. They may show either that each individual Con A-responding cell is partly blocked in their lysosomal and mitochondrial maturation or that some cells are totally blocked while others would not be blocked at all. Furthermore spleen and lymph node cell suspensions exposed to cyclosporin in absenceof Con A also display various alterations of the mitochondrial and lysosome-specific fluorescence patterns. These effects, too, are difficult to interpret since they are obtained with complex populations of cells in vitro. We have now used our two cloned lymphoblastoid T cells of different susceptibility to cyclosporin (4) in order to look at how their lysosomes and mitochondria might be affected by cyclosporin. This results in clear cut differences: (1) Only the cyclosporin-sensitive (Cs’) cell clone shows a decreaseof uptake of rhodamineinto its mitochondria upon treatment with cyclosporin. (2) Cyclosporin causes an increase of the vital red euchrysine staining (lysosome specificity) of the Css cell clone only, while the resistant (CsR) cell clone is only slightly affected. Preliminary studies by electron microscopy also reveal that cyclosporin causes an extensive vacuolization of the cytoplasm of the Css clone cells. MATERIALS
AND METHODS
Cell cultures and cyclosporin treatment. The cyclosporin-sensitive (BE7) and cyclosporin-resistant (LB7) lymphoblastoid T-cell clones and the culture conditions have been previously described (4, 5). CsA (Sandoz, Basle, Switzerland) was used on the cells at concentrations ranging from 0.1 to 1.0 pg/ml (final ethanol solvent concentration was 0.1%). The cells were assayedat OS- 1.O X 1O5cells/ml in tissue culture trays (24 wells, l-ml cultures; Costar 3524, Cambridge, Mass.). Fluorescence procedures. The staining procedure, the fluorescence microscopy of the stained cells, and the flow cytometry analyses of the stained cell suspensions were performed as described previously (3, 6). The mitochondria were detected with rhodamine(R-123; Eastman Kodak, Rochester, N.Y.) at a final concentration of 10 pg/ml for 10 min at 37°C and lysosomes were vitally stained with euchrysine (acridine orange; Merck, Darmstadt, West Germany) at 5 pg/ml for 5 min at room temperature in the dark. The samples were observed by fluorescence microscopy and analyzed by flow cytometry without delay, using an Ortho Cytofluorograf 50 HH (Ortho Diagnostic Instruments, Westwood, Mass.). Electron microscopy. Following incubation alone or with cyclosporin (1 pg/ml), the cells were sedimented and washed with 0.9% NaCl. They were then repelleted by centrifugation (15OOg,10 min) and prefixed with 2.5% glutaraldehyde in 0.13 M balanced phosphate buffer, pH 7.2. After a thorough rinsing in the same buffer the samples were postfixed in phosphate-buffered 1.3% recycled osmium tetroxide at 4°C (Simec, Birsfelden, Switzerland). The samples were dehydrated through a graded series of ethanol and propylene oxide (Fluka, Buchs, Switzerland) prior to embedding in Beem capsules using Epon 8 12 (7). During the dehydration the samples were stained with 2% uranyl acetate at the 80% ethanol step. Ultrathin sections were cut on a LKB Ultratome III 8000 at a thickness of 60-80 nm (silver interference color) and stained on the grid with 2% uranyl acetate and 0.4% lead citrate (3 min) (8). The sections were examined in a Siemens 101 electron microscope.
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RESULTS Efects of cyclosporin on mitochondria (Fig, 1). The presence of cyclosporin did not change the fluorescence pattern of the CsR cell clone LB7, indicating that the uptake of R-123; thus, the mitochondrial activity was virtually unaffected. The uptake of R-123 into mitochondria of the Css cell clone BE7 was decreased by cyclosporin after 24 hr in culture: there was a shift of the population profile towards lower fluorescence levels. This effect was cyclosporin dose dependent. The dose of 0.1 pg/ml cyclosporin (which has only little effect on the proliferation of the clone, measured at 3-4 days) was still effective. The effects were more pronounced at the doses of 0.3 pg/ml (which is about the LDsOfor this line), and of 1.O pg/ml (which
FIG. 1. Population analysis by vital rhodaminestaining. X axis, green (mitochondria-specific) fluorescence intensity: Y axis, number of cells in each channel. Comparison of 24-hr cultures of Cssensitive clone BE7 (left) and the Cs-resistant clone LB7 (right). Bottom curves, controls without cyclosporin, top curves, with cyclosporin (0.1, 0.3, 1.O &ml as indicated; 0.1% ethanol as solvent). The numbers represent the numbers of cells from channel 55 to channel 5 12 (out of 50,000 cells).
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gives 100% inhibition of proliferation of this line, without detectably affecting cell survival at 24 hr and is still essentially reversible at that time (5)). A large proportion of the Css cells treated for 24 hr with 0.3 or 1.0 Fg/ml cyclosporin showed the same low R-123 uptake as a minority of cells from the control cultures. The effects of cyclosporin on R-123 uptake were not very marked at earlier times (not shown). They also appeared less pronounced after 48-hr incubation. This might be due to unspecific changes in absorption of R-123 by the cells/mitochondria, since the BE7 cells are Cs sensitive and eventually die if cyclosporin is not removed; indeed, after such a time of exposure to cyclosporin, they show a lower capacity to grow upon removal of the drug (5). Eficts of cyclosporin on lysosomes. Cyclosporin treatment of Css cells (BE7) or of CsR cells (LB7) had very different consequences on their lysosome-specific (red) fluorescence when vitally stained by euchrysine. This is shown both by the cell distribution displays and by using two arbitrarily selected windows (windows 1 and 2 for lower and higher red fluorescence levels, respectively). BE7 and LB7 cells were exposed to various concentrations of cyclosporin in the medium (0, 0.1, 0.3, and 1.O pg/ml, with 0.1% ethanol as solvent in each case). After various times they were tested for euchrysine red (lysosome-specific) and green (marker for the available DNA, mostly a cell mass marker) fluorescence. Two independent experiments are shown in Figs. 2-4 and Tables 1 and 2. Within 4 hr of cyclosporin addition to the Css cells, it induced a concentration-dependent increase in the red fluorescence which was more pronounced at 6 hr and very strong after 24 hr (Fig. 2, Table 1). The 0. I-pg/ml dose was only marginally effective except at 24 hr (Table 1). On the contrary, cyclosporin treatment of the CsR cells did not change their vital staining by euchrysine, except in the sample exposed to the highest dose (1 pg/ml) for 24 hr (Table 1, Fig. 3). However this effect was no longer found after 48 hr in the presence of cyclosporin, as though the CsRcells had succeededin “correcting” some action of cyclosporin on the structures stained red by euchrysine (Table 2, Fig. 4). The red fluorescence level of the Css cells continued to increase from 24 to 48 hr for doses of 0.3 and 1.0 yg/ml cyclosporin (Table 2, Fig. 4). The level of red fluorescence per cell is very heterogeneousin the cyclosporintreated Css cells, particularly after 24 and 48 hr of treatment. During the same culture time, the cells showed a decreased green fluorescence and became more homogeneous at a low fluorescence level. This might correspond to a smaller cell size and/or a lower DNA content. Electron microscopy of cyclosporin-treated cells. All studies done so far revealed that CsA at 1 pg/ml induced a vacuolization of the cytoplasm of the Css cells. This was particularly evident after 24 hr in the presence of CsA (Fig. 5a), but it started to be detectable within 3 hr of incubation with CsA (not shown). The CsR cells were not much affected by CsA except for the presence of small vesicles at 24 hr (Fig. 5b). These effects were specific of CsA since the solvent alone (ethanol, 0.1%) did not cause them in either cell clone (Figs. 5c, d). It is relevant here that only a few lysosomes were detected in cells of both clones, even in conditions (Css cells, 1 pg CsA/ml, 24 hr) where the vital staining with euchrysine resulted in high red fluorescence of the cells. Further data on the dose and time dependence of these effects of CsA, as well as on the emergence of lipid bodies and other alterations of the cellular organization, are still preliminary (9).
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FIG. 2. Scatterplots of red (X axis; lysosome-specific)and green (Y axis; DNA content/cell size indic:ator) fluorescence from vital euchrysine-stained clone BE7 (Cs’). In vitro exposure for 6 and 24 hr for va.sious concentrations of cyclosporin (0.1, 0.3, 1.0 &ml in 0.1% ethanol). Each point in scatterplot repre the bivariable measurements of a single cell.
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FIG. 3. CsR ceil clone LB7 after exposure to cyclosporin for 6 and 24 hr. Same analyses as for clone BE7 in Fig. 2 for the 0.3 and 1.0 &ml cyclosporin concentrations.
DISCUSSION Cyclosporin interferes with the concanavalin A-induced activation of mixed lymphocyte populations, at or prior to the steps which involve increased lysosomal and mitochondrial activities (3). The latter were evaluated, as in the present study, by the specific fluorescence emission of euchrysine (lysosomes) and of rhodamine123 (mitochondria) by individual cells of the population. The fluorescence profiles of the cyclosporin-treated population were close but by no means identical to the background fluorescence levels of unstimulated cells (3). The heterogeneity of a cell population and the existence of intercellular interactions remain major difficulties when trying to interpret any modification of a biological or biochemical parameter by a drug or a factor. Were the observed alterations reflecting mainly changes in
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ET AL,
R G. 4. Population analyses of clones BE7 (Cs’) and LB7 (CsR) after in vitro exposure for 48 hr cyclelsporin (0.1, 0.3, and 1.O &ml cyclosporin in 0.1% ethanol). Vital staining by euchrysine.
to
the relative proportions of different cell types of the population? Or were they 43n the contrary the signs of more basic alterations of lysosomal and mitochondn ial acti vities that would actually affect each individual cell?
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CYCLOSPQRIN EFFECTS ON LYSOSOMES AND MITOCHONDRIA TABLE 1 Percentage of Cells Falling in Window 2” Time in culture (W Cyclosporinb kImI)
4
6
24'
BE7 (Cs’)
0 0.1 0.3 1.0
2.4 3.7 5.5 8.2
0.95 2.0 3.8 10.4
9.7 22.0 38.0 79.2
LB7 (CsR)
0 0.1 0.3 1.0
2.7 2.0 2.0 2.6
0.91 1.0 0.89 0.85
9.5 10.5 13.0 19.2
Cell clone
’ Higher red euchrysine fluorescence b In 0.1% ethanol. ‘Same samples as in Figs. 2 and 3.
The present data were obtained with a Cs-sensitive clone and a Cs-resistant clone and they allow us to better delineate the effects of cyclosporin on single cells. The Css cells are stopped in the early G, phase by cyclosporin (4) (like concanavalin Aactivated spleen cells (9, 10)) whereas the CsRcells are not (4). The difference is not due to different representation or specificity of the membranous cyclosporin-binding sites (I 1). We show here that cyclosporin differently affects both cells with regard to their uptakes of rhodamineand of euchrysine and to the cytoplasm organization. The large increase of the euchrysin red fluorescence emitted by cyclosporintreated Css clone cells could suggest a higher lysosome content. However, electron TABLE 2 Percentage of Cells Falling in Window 2” Time in culture (W Cyclosporin b h/ml)
24
48'
BE7 (Cs’)
0 0.1 0.3 I.0
5.5 5.9 8.8 31.2
2.5 4.1 15.6 61.1
LB7 (CsR)
0 0.1 0.3 1.0
0.8 2.3 2.2 6.6
0.3 0.4 0.6 0.6
Cell clone
’ Higher red euchrysine fluorescence. b In 0.1% ethanol. ‘Same samples as in Fig. 4.
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FIG. 5. Electron microscopic analyses of Css (a, c) and CsR(b, d) clone cells incubated for 24 hr with (a, b) or without (c, d) cyclosporin (1 &ml in 0.1% ethanol in medium at 37’C). Note the numerous cytoplasmic vacuoles in the cyclosporin-treated Cs’ cell (a). (X6 120).
microscopic analyses showed there was no corresponding increase in the number or size of lysosomes. Moreover, direct measurements of activities of several lysosomal enzymes show that cyclosporin doses which affect the euchrysine fluorescence of
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the Css cells have little effect on the enzyme content per cell (Renard, P., Fonteneau, P., and Loor, F., manuscript in preparation). Therefore, two possibilities remain: either each lysosome of a cyclosporin-treated Csscell takes up much more euchrysine or becomes more acidic (the red fluorescence of euchrysine requires an acid pH), or the structures detected are not bona jidr lysosomes. The first alternative implies that cyclosporin alters the lysosomal membrane permeability of Css cells, while CsR cells have more stable lysosomal membranes. The second alternative however finds some support in the presence of numerous, possibly autophagic, vacuoles in the cytoplasm of the Css cells at the time they display high red euchrysine fluorescence, and in contrast to their absencein CsRcells. Such vacuoles might pick up euchrysine and fluoresce red if their intracellular pH is acidic. This is the case of the prelysosomal vacuoles in the receptor-mediated endocytosis process ( 12). Weakly basic compounds also inhibit membrane recycling and vacuolise the lysosomes and the Golgi apparatus (13, 14). A variety of autonomic drugs induce cytoplasmic vacuolization in different cells in tissue culture (15). The vacuolization found here seems specific, as it only affects the Css cells, and rather fast, since small vesicles were already detectable within 3 hr of cyclosporin treatment (9). For what concerns other immunosuppressors, large numbers of vacuoles as well as lipid droplets were reported to occur in the cytoplasm of macrophages and reticular cells exposed to steroids (16). Lipid droplets also appear in cyclosporin-treated cells but this does not correlate with their Cs sensitivity (5, 6). A low frequency of a Css clone cell type among normal lymphocytes would explain the contrasting effects of cyclosporin on the red euchrysine fluorescence vital staining of Css clone cell (this study) and of concanavalin A-treated cells (3): in the latter case indeed, the vast majority of the cells showed a decreased red fluorescence when cyclosporin was present during the culture (3). Thus, the particular behavioral response of our Css clone to the presence of cyclosporin may occur in only a minority of a whole spleen or lymph node cell suspension and pass unnoticed. However this type of cell may be common among normal lymphocytes but cyclosporin-induced membranous alterations may allow their recognition by macrophages, their scavenging, and their disappearance from the lymphoid cell cultures. The uptake of rhodamineinto the mitochondria of Css cells was reduced by cyclosporin whereas the CsR clone was not detectably affected. The reduced mitochondrial activity of the Css clone could simply be a consequence of the arrest of the cycling Css cells in early Gr , a cell cycle phase characterized by a low mitochondrial content per cell. However, it seemsthat cyclosporin interferes more directly with the mitochondrial activity or replication, thereby causing the G, arrest. The rhodaminefluorescence distribution profile of cyclosporin-treated Css cells peaks at a rather low fluorescence level which is characteristic of a very small minority of normally growing Css cells. Actually, many of the cyclosporin-treated cells have a very low mitochondrial content/activity which is probably the lowest possible in early G1 phase or even an abnormally reduced one. Interference of cyclosporin with mitochondria function may thus be a major reason for the G, arrest of the Css cells. This does not imply that cyclosporin necessarily works on the mitochondria themselves. Indeed, the recent report that cyclosporin works on the ol-amanitin-sensitive RNA polymerase II (17) suggeststhat effects of cyclosporin
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on mitochondria are indirect; DNA transcription in mitochondria is carried out by a simpler, cu-amanitin-resistant RNA polymerase, but it is noteworthy that mitochondria import a number of nucleus transcribed proteins (18). Inhibition of RNA polymerase II by cyclosporin would block the synthesis and transport of nuclear transcribed proteins into mitochondria. Much mitochondrial maturation is required in order to achieve the high levels of ATP needed for the generation of dNTP up to the levels required for DNA polymerase (Y activation (19). Therefore, deficient mitochondrial function would eventually impair nuclear DNA synthesis and induce the early G, -blocking of cyclosporin-treated cells. The lack of inhibition of mitochondrial activity of CsR cells may have several nonexclusive causes.In the caseof the presently studied LB7 cell clone, transcription is cyclosporin resistant, at least in vitro (Brack, C., Cammisuli, S., Gaveriaux, C., and Loor, F., unpublished experiments). Though this provides a satisfactory explanation for the mechanisms both of cyclosporin action and of cyclosporin-resistance, other mechanisms may operate or be preponderant in other cell types. REFERENCES 1. Wiesinger, D., and Borel, J. F., Immunobiology 156, 454, 1979. 2. Hume, D. A., and Weidemann, M. J., “Mitogenic Lymphocyte Transformation.” Elsevier/Biomedical Press,Amsterdam, 1980. 3. Koponen, M., Grieder, A., and Loor, F., Immunology 53, 55, 1984. 4. Koponen, M., Grieder, A., and Loor, F., Exp. Cell Res. 140, 237, 1982. 5. Koponen, M., and Loor, F., Ann. Immunol. (Paris) 134C, 171, 1983. 6. Koponen, M., and Loor, F., Exp. Cell Res. 149, 499, 1983. 7. Luft, J. H., J. Biophys. Biochem. Cytol. 9,409, 1961. 8. Venable, J. H., and Coggeshall, R., J. Cell Biol. 25, 407, 1965. 9. Koponen, M., “Interference of Cyclosporin with Lymphocyte Activation.” Ph.D. thesis, Biocenter, University of Basel, Switzerland, 1984. 10. Koponen, M., Gerson, D. F., and Loor, F., Agents Actions, 15, 600, 1984. 11. Koponen, M., and Loor, F., Ann. Immunol. (Paris) 134D, 201, 1983. 12. Helenius, A., Mellman, I., Wall, D., and Hubbard, A., Trends Biochem. Sci. 8, 245, 1983. 13. Fedorko, M. E., Hirsch, J. G., and Cohn, 2. A., J. Cell Biol. 38, 392, 1968. 14. Steinman, R. M., Mellman, I. S., Miiller, W. A., and Cohn, Z. A., J. Cell Biol. 96, 1, 1983. 15. Yang, W. C. T., Strasser,F. F., and Pomerat, C. M., Exp. Cell Res. 38, 495, 1965. 16. Miyata, K., and Takaya, K., Cell Tissue Rex 230, 57, 1983. 17. Brack, C., Mattaj, 1. W., Gautschi, J., and Cammisuli, S., Exp. Cell Res. 151, 314, 1984. 18. Wirtz, K. W. A., Biochim. Biophys. Acta 344, 95, 1974. 19. Spadari, S., Sala, F., and Pedrali-Noy, G., Trends Biochem. Sci. I, 29, 1982.