The inhibition of calcium signaling in T lymphocytes from old mice results from enhanced activation of the mitochondrial permeability transition pore

Mechanisms of Ageing and Development 123 (2002) 707– 724 www.elsevier.com/locate/mechagedev

The inhibition of calcium signaling in T lymphocytes from old mice results from enhanced activation of the mitochondrial permeability transition pore Michael W. Mather *, Hagai Rottenberg Department of Microbiology and Immunology, MCP Hahnemann Uni6ersity School of Medicine, 2900 Queen Lane, Philadelphia, PA 19129, USA Received 31 August 2001; received in revised form 30 November 2001; accepted 30 November 2001

Abstract Aging attenuates calcium signaling in T lymphocytes from old mice. Aging also attenuates the sustained elevation of cell free calcium by ionomycin, which is similar to the T cell receptor signal. In T lymphocytes from young mice, the ionomycin-induced elevation of cell free calcium was inhibited by collapsing the mitochondrial membrane potential by uncouplers and ionophores, and activation of the permeability transition. In T lymphocytes from old mice, the mitochondrial membrane potential was largely collapsed, but cyclosporin and N-methyl-val-4-cyclosporin, inhibitors of the permeability transition, restored the mitochondrial potential, as well as the ionomycin-induced elevation of cell free calcium. In addition, the generation of reactive oxygen species in the presence of mitochondrial electron transport inhibitors was relatively enhanced in T lymphocytes from old mice. The association between low rhodamine 123 fluorescence and attenuated calcium signaling in T lymphocytes from old mice is also shown to be a consequence of the collapsed mitochondrial potential. These results suggest that Ca2 + signaling is attenuated in T lymphocytes from old mice because of an enhanced activation of the permeability transition. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Calcium signaling; Calcium release-activated channel; Mitochondrial permeability transition pore; Immunosenescence; Membrane potential; Reactive oxygen species

Abbre6iations: B6, C57BL/6; [Ca2 + ]in, cell free calcium; CCCP, carbonyl cyanide m-chlorophenyl-hydrazone; CRAC, calcium release-activated calcium; D2, DBA/2; DHE, dihydroethidium; DiOC6(3), 3,3%- dihexyloxacarbocyanine iodide; ER, endoplasmic reticulum; FAD, flavin adenine dinucleotide; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; FSC, forward scattering; HBSS, hanks’ balanced salts solution; MEM, minimum essential medium eagle; MV-4-CS, N-methyl-val-4-cyclosporin; NF-AT, nuclear factor of activated T cells; PBS, phosphate buffered saline; PE, phycoerythrin; P-gp, P-glycoprotein; PPIX, protoporphyrin IX; PTP, permeability transition pore; Rh123, rhodamine 123; ROS, reactive oxygen species; SEM, standard error of the mean; SR, sarcoplasmic reticulum; SSC, side scattering; TCR, T cell receptor; DCm, mitochondrial membrane potential. * Corresponding author. Tel.: + 1-215-9918256; fax: +1-215-8434152. E-mail address: [email protected] (M.W. Mather). 0047-6374/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 0 1 ) 0 0 4 1 6 - X

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1. Introduction Aging is associated with a wide spectrum of degenerative diseases, including immunosenescence (Masoro, 1995). Oxidative stress is the direct cause of aging-associated cell damage in most tissues (Ames et al., 1993), and many of the gene products that are differentially expressed in muscle and brain from old mice are related to stress response and oxygen metabolism (Lee et al., 1999, 2000). Caloric restriction, which reduces oxidative stress and delays aging, reverses many of these changes (Lee et al., 1999, 2000), and ameliorates immunosenescence (Weindruch and Sohal, 1997). Antioxidants were also reported to ameliorate immunosenescence (Meydani et al., 1995). Mitochondrial dysfunction has emerged as the direct cause of aging-associated oxidative stress (Ames et al., 1993; Wallace et al., 1995; Beal, 1995). Reactive oxygen species (ROS) are generated mostly by mitochondria (Chance et al., 1979), and mitochondrial DNA (Wallace et al., 1995), proteins (Yan and Sohal, 1998) and lipids (Kowaltowski and Vercesi, 1999) are the most susceptible targets of aging-associated oxidative damage in the cell. Recently, we observed that in lymphocytes from old mice, electron transport is inhibited, the mitochondrial flavin adenine dinucleotide (FAD) is more oxidized, and the mitochondrial permeability transition pore (PTP) is more activated (Rottenberg and Wu, 1997). The enhanced activation of PTP was also observed in brain and liver mitochondria isolated from old mice (Mather and Rottenberg, 2000). PTP is a large nonspecific channel that is activated by calcium and ROS and is inhibited by cyclosporin (Zoratti and Szabo, 1995). Activation of PTP collapses the mitochondrial membrane potential, DCm, inhibits oxidative phosphorylation and Ca2 + sequestration by mitochondria, and may induce apoptosis or necrosis (Zoratti and Szabo, 1995; Marchetti et al., 1996; Bernardi et al., 1999). Age-induced immune dysfunction has been studied extensively in humans and rodents (Miller, 1996a). Most studies suggest that the decline of the immune response is largely due to T cell dysfunction, which is associated with shifts in the composition of the T cell subset populations

(Pawelec et al., 1997). In both rodents and humans the most significant change in the composition of T cell subsets is a shift from a low memory/naive ratio to a high memory/naive ratio (Miller, 1996a; Pawelec et al., 1997; Miller, 1996b). This change may account for some of the observed immune dysfunctions in old age. One of the most extensively documented dysfunctions in T cells from old rodents is an attenuation of calcium signaling (Miller, 1996b), which was reported to occur also in humans (cf. Sulger et al., 1999). The expansion of the memory T cell subset in old mice was shown to be associated with the attenuation of calcium signaling (Miller, 1996b). Activation of the TCR receptor induces a sustained elevation of [Ca2 + ]in, which activates the nuclear factor of activated T cells (NF-AT) transcription factors, and initiates the transcription of genes of the immune response (Dolmetsch et al., 1998; Crabtree, 1999). It is possible to bypass receptor-dependent activation, and activate T cells artificially by ionomycin, a calcium ionophore that induces a sustained elevation of [Ca2 + ]in and T cell proliferation (Thoman and Weigle, 1988). In most T cells from old mice, ionomycin is much less effective in raising [Ca2 + ]in, suggesting that there is a modulation of the mechanisms of calcium signaling in these cells (Miller, 1996b). 4b-Phorbol 12-myristate 13-acetate, which stimulates protein kinase C, reduces the K0.5 for the [Ca2 + ]in activation of NF-AT (Negulescu et al., 1994) and enhances the ionomycin-induced activation of T cells from old mice (Thoman and Weigle, 1988). The small fraction of ionomycin resistant cells in T cell preparations from young mice, similar to the majority of cells from old mice, are memory cells that do not produce IL-2 in response to con A or enterotoxin B (Miller et al., 1991). More recently, it was reported that the calcium signaling attenuation in T lymphocytes from old mice is associated with high P-glycoprotein (P-gp) activity (Witkowski and Miller, 1999). However, P-gp expression had little effect on immune function (Eisenbraum and Miller, 1999), and the underlying cause of the attenuation of calcium signaling in these cells has not been elucidated.

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Thapsigargin, an inhibitor of the endoplasmic reticulum (ER) Ca2 + -ATPase, releases Ca2 + from internal stores, and activates the calcium release-activated Ca2 + (CRAC) channels in T lymphocytes (Zweifach and Lewis, 1996). The thapsigargin-induced sustained elevation of [Ca2 + ]in in Jurkat T cells was shown to depend critically on mitochondrial calcium uptake, which is driven by the DCm (Hoth et al., 1997; Makowska et al., 2000). The Mitochondria remove Ca2 + from the vicinity of the CRAC channels, thus preventing their inactivation. Inhibition of mitochondrial Ca2 + uptake also inhibits the T cell receptor-induced sustained elevation of [Ca2 + ]in and the nuclear translocation of NF-AT (Hoth et al., 2000). Ionomycin also induces sustained elevation of [Ca2 + ]in by releasing Ca2 + from ER, which activates the CRAC channels, similar to thapsigargin (Mason and Grinstein, 1993; Morgan and Jacob, 1994). Since the sustained elevation of [Ca2 + ]in by ionomycin was shown to be defective in T cells from old mice (Miller, 1996b), and we observed that PTP, which inhibits Ca2 + uptake by mitochondria, is more activated in lymphocytes from old mice (Rottenberg and Wu, 1997), it occurred to us that the enhanced PTP activation could be the underlying cause of the attenuation of calcium signaling in T cells from old mice. We now show that the sustained elevation of [Ca2 + ]in by low concentrations of ionomycin in T lymphocytes also depends on DCm, and is inhibited by uncouplers, ionophores, and the activation of the PTP. In T lymphocytes from old mice, ionomycin is ineffective in sustaining the elevation of [Ca2 + ]in, but treatment with cyclosporin A, or the cyclosporin derivative N-methyl-val-4cyclosporin (MV-4-CS), which inhibits PTP, but not calcineurin (Baumann et al., 1992; Schweizer et al., 1993), restored the ionomycin-sustained elevation of [Ca2 + ]in in most cells. Most T cells isolated from old mice had very low DCm, but cyclosporin or MV-4-CS restored DCm. These results support the conclusion that the attenuation of Ca2 + signaling in T cells from old mice results primarily from the enhanced activation of PTP.

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2. Materials and methods

2.1. Mice Aging (19–20 mo), old (25–26 mo), and young (3–4 mo) male C57BL/6 × DBA/2 F1 mice, and aging (20–21 mo) and young (3 –4 mo) male C57BL/6 × C3H F1 mice, were obtained from NIA-contracted colonies maintained by Charles River Laboratories and Harlan Sprague Dawley. Only healthy mice, without tumors or abnormal spleens, were used in experiments. The protocol was approved by the Institutional Animal Care and Use Committee.

2.2. Preparation of T cells Lymphocytes were prepared from freshly removed mouse spleens essentially as described (Rottenberg and Wu, 1997) in supplemented hanks’ balanced salts solution (HBSS) (containing 0.3% BSA, 1% fetal calf serum (FCS), 15 mM HEPES (pH 7.4)). T cell enriched preparations were isolated by passing the spleen lymphocyte suspension in supplemented HBSS through a mouse T cell recovery column (Accurate/Cedarlane Labs), containing immobilized goat antimouse IgG to remove B cells, at 4 °C. The T cells were concentrated by centrifugation (600 g) and resuspended at  5×106/ml in MEM medium (+1% FCS). Unless otherwise noted, the T cells were incubated at 37 °C in a humidified CO2 incubator for 1–2 h and diluted to 1× 106/ml in MEM + 1% FCS medium before initiation of experimental procedures.

2.3. Assay of cytosolic [Ca 2 + ]in [Ca2 + ]in was determined by flow cytometry of T cell suspensions loaded with the green fluorescent probe fluo-3-AM (Molecular Probes). Cells (1.0× 106/ml) were incubated in medium at 37 °C in a humidified CO2 incubator for 1 h with 5 mM fluo-3-AM. To study the kinetics of the change in [Ca2 + ]in after addition of ionomycin, samples were withdrawn every 2–5 min, followed by immediate collection of flow cytometry data. To determine the effect of various reagents on

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[Ca2 + ]in in the steady state, test reagents (in ethanol, dimethyl sulfoxide, or water) were then added to the cell suspensions and incubation continued for 10 min, followed by immediate collection of flow cytometry data (at least 10 000 events per sample), using a Becton–Dickinson FACScan or FACSort flow cytometer. An appropriate T cell population was chosen for analysis (Lysis II software) by setting bitmap gates on a plot of forward scattering (FSC) vs. side scattering (SSC). Average [Ca2 + ]in was calculated from the average fluorescence as described (Greimers et al., 1996); Fmax was taken as the average maximal fluorescence in a fraction of cells with high Ca2 + after the addition of 1 mg/ml ionomycin; the value of KD of the Ca2 + -fluo-3 complex was taken as 390 nM (Tsien, 1988).

an argon laser, excitation at 488 nm) (Kunz et al., 1997). T cell samples (1.0× 106 cells/ml) were maintained in medium at 37 °C until measurement. The mean fluorescence intensity of the (gated) T cell population was taken as a measure of the relative degree of oxidation.

2.7. Assay for superoxide generation The relative extent of superoxide generation was estimated by incubation of T cells (106 cells/ml) in a medium that contained 2 mM dihydroethidium (DHE, Molecular Probes) for 1 h at 37 °C. The fluorescence of the oxidation product (ethidium) was determined by flow cytometry (F2).

2.8. Immunotyping of T cells 2.4. Assay of Zcm Mitochondrial membrane potential was determined by flow cytometry using the probe DiOC6(3), as described (Rottenberg and Wu, 1998). Briefly, the cells were incubated for 30 min with 0.2 or 0.6 nM DiOC6(3), at 37 °C, in a humidified CO2 incubator, then additions were made and the incubation continued for an additional 30 min at 37°. The average relative magnitude of DCm is obtained from the fluorescence intensity ratio of sample or control cells vs. cells treated with the uncoupler carbonyl cyanide m-chlorophenyl-hydrazone (CCCP).

2.5. Assay of rhodamine 123 efflux The protocol was essentially as described (Eisenbraum and Miller, 1999). Briefly: T cells (2×106/ ml) were incubated with 1 mM Rh123 at 37 °C for 10 min. The cells were cooled on ice and washed three times with cold medium, then resuspended and incubated for 30 min at 37 °C. Samples for FACS analysis were prepared by mixing a sample with four volumes of cold stop buffer, followed by centrifugation and resuspension in cold stop buffer.

The relative size of T cell subsets containing CD4, CD8, and CD44 receptors was determined by immunostaining with anti-receptor mAb conjugated to FITC or (PE) (Pharmingen). Corresponding fluorescent labeled isotype antibodies (Pharmingen) were utilized as controls. Cell samples collected during experiments are washed and suspended in cold wash buffer (PBS containing 2% FCS and 0.1% sodium azide), and stored on ice until immunolabeling (within 8 h). 5× 105 cells (107/ml) are incubated with pre-determined optimal amounts of each antibody (singly and in combinations of two) for 30 min on ice. The stained samples are washed twice and analyzed within 1 h on a FACScan flow cytometer. Crossover fluorescence was compensated by cross-channel subtraction during data collection. An appropriate T cell population was chosen for analysis (Lysis II software) by setting bitmap gates on a plot of FSC vs. SSC.

3. Results

2.6. Assay of mitochondrial FAD oxidation

3.1. Mitochondrial Ca 2 + uptake is critical for the ionomycin-induced sustained ele6ation of [Ca 2 + ]in in mouse spleen T cells

Oxidized FAD fluorescence was detected in the F1 window (530 nm) of the flow cytometer (with

It was shown recently that mitochondrial Ca2 + uptake, which is driven by DCm, is critical for

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store-operated calcium signaling in Jurkat T cells (Hoth et al., 1997; Makowska et al., 2000; Hoth et al., 2000). We found that a low concentration (75 ng/ml) of ionomycin increased the free calcium concentration in mouse spleen T lymphocytes. The free calcium level reached a steady state 4 min after the addition of ionomycin and remained stable for up to 40 min (results not shown). The results summarized in Fig. 1 show that in mouse spleen T lymphocytes the ionomycin-induced sustained, steady state, elevation of [Ca2 + ]in is also critically dependent on mitochondrial Ca2 + uptake. Fig. 1A shows histograms of the fluorescence of the calcium indicator Fluo-3, and the fluorescence of the DCm indicator DiOC6(3), in T cells from a young mouse. Treatment with a low concentration (75 ng/ml) of ionomycin resulted in a 20 fold elevation of [Ca2 + ]in that lasted at least 40 min in the majority of T cells (\75%) (panel b), but had no effect on DCm (panel i). The magnitude of DCm is proportional to the ratio of DiOC6(3) fluorescence in the test system to the fluorescence in the presence of the uncoupler CCCP (shown as a background in all the DiOC6(3) histograms) (Rottenberg and Wu, 1998). The elevation of [Ca2 + ]in by ionomycin was reversed in most of the cells 10– 15 min after the addition of a low concentration of CCCP (panel c), or a low concentration of valinomycin (panel d), which collapsed DCm (panels j and k). The sustained elevation of [Ca2 + ]in was also inhibited by EGTA, which chelates external Ca2 + (panel e), and partially inhibited by Ni, which inhibits the CRAC channels (panel g). These results suggest that in T cells the sustained elevation of [Ca2 + ]in by ionomycin depends both on the maintenance of DCm, and on Ca2 + influx through CRAC channels, as is the case with the sustained elevation of [Ca2 + ]in by thapsigargin (Hoth et al., 1997). However, when the ionomycin concentration was increased to 500 ng/ml, DCm collapsed (panel l), and the elevation of [Ca2 + ]in was reversed (panel g). Fig. 1B shows the relationships between the percentage of cells with high DCm and the percentage of cells with high [Ca2 + ]in as a function of the ionomycin concentration. Increasing the concen-

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tration of ionomycin above 0.4 mg/ml collapses DCm, and inhibits Ca2 + sequestration by the mitochondria (Rottenberg and Scarpa, 1974). Thus, similar to the effect of valinomycin or CCCP, the collapse of DCm by high concentrations of ionomycin resulted in inactivation of the CRAC channel, which gradually reversed the elevation of [Ca2 + ]in. Since PTP activation also inhibits Ca2 + sequestration by the mitochondria, it can be expected to reverse the ionomycin-induced elevation of [Ca2 + ]in. Although there are many reagents that enhance the activation of PTP in isolated mitochondria, few can be considered sufficiently specific when applied to intact cells. We have used the mitochondrial benzodiazepine receptor ligand protoporphyrin IX (PPIX), which was shown to enhance the activation of PTP in lymphocytes (Marchetti et al., 1996). Fig. 1C summarizes the effects of a low concentration of PPIX on the ionomycin-induced elevation of [Ca2 + ]in. In T cells treated with ionomycin, PPIX attenuates DCm (results not shown) and increases the percentage of cells with low [Ca2 + ]in. The PTP inhibitor cyclosporin reverses this effect of PPIX. Moreover, the cyclosporin derivative MV-4-CS, which inhibits PTP but not calcineurin (Baumann et al., 1992; Schweizer et al., 1993), also reverses the effect of PPIX. These results suggest that PTP activation inhibits the sustained elevation of [Ca2 + ]in by the CRAC channels.

3.2. Cyclosporin and MV-4 -CS restore the ionomycin-induced ele6ation of [Ca 2 + ]in in T cells from old mice Fig. 2A shows typical histograms of the effect of low ionomycin concentrations on [Ca2 + ]in distribution, 10 min after the addition of ionomycin. These measurements are obtained in live cells at a particular time point after the addition of ionomycin. Although the [Ca2 + ]in level is relatively stable after 5 min, small differences between preparations occur. The histograms shown here are typical. The average results from several such experiments with different batches of cells are shown below (Fig. 3). In T cells

Fig. 1. Mitochondrial control of the ionomycin-induced elevation of [Ca2 + ]in in T lymphocytes. (A) Histograms of flow cytometric measurements of [Ca2 + ]in with the probe fluo-3,  10 min after the addition of the indicated reagent (first row), and flow cytometric measurements of DCm with the probe DiOC6(3) (second row, see Experimental Procedures for protocols). From left to right: control (no additions); + 75 ng/ml ionomycin; + 5 mM CCCP and 75 ng/ml ionomycin; + 0.1 mM valinomycin and 75 ng/ml ionomycin; + 2.5 mM EGTA and 75 ng/ml ionomycin; +2 mM Ni2 + and 75 ng/ml ionomycin; +0.5 mg/ml ionomycin. In the fluo-3 histograms the percentage of cells and average [Ca2 + ]in in the major fractions marked by horizontal bars are indicated. In the DiOC6(3) histograms a low potential (50 mM CCCP) histogram (unfilled) is overlaid for comparison. (B) The effect of ionomycin concentration on DCm and on [Ca2 + ]in. The fraction of cells with high [Ca2 + ]in (filled circles) and high DCm (filled squares) is plotted vs. the concentration of ionomycin. (C) The effect of PPIX (2 mM) on the percentage of cells with low [Ca2 + ]in in the presence of ionomycin (75 ng/ml): control: ionomycin only; +PPIX: ionomycin + PPIX; + PPIX+ CS: ionomycin + PPIX+cyclosporin (1 mM); + PPIX+ MV4CS: ionomycin + PPIX+MV-4-Cs.

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from 3-month-old B6/D2 F1 mice, without ionomycin, the majority of T cells (75%), had fairly uniform [Ca2 + ]in levels. Ionomycin, at a low concentration (75 ng/ml) increased the average [Ca2 + ]in about 6 fold, and in the fraction of cells with relatively high [Ca2 + ]in (71% of cells) up to 20 fold. When cyclosporin A was added together with ionomycin there was a large increase in mean [Ca2 + ]in, but only a slight increase in the percentage of cells with high [Ca2 + ]in (82% of cells). In T cells from 20-month-old B6/D2 F1 mice, without ionomycin, there was less uniform distribution of [Ca2 + ]in levels. The ionomycin effect was greatly attenuated and less than half the cells showed a high level of [Ca2 + ]in. When cyclosporin was added together with ionomycin, [Ca2 + ]in was increased greatly, and a large fraction of cells (66%) exhibited high [Ca2 + ]in. In T cells from 26-month-old B6/D2 F1 mice, [Ca2 + ]in was similar to that of 20-month-old mice, without ionomycin, but the ionomycin effect was very small, hardly increasing the average [Ca2 + ]in, which was elevated slightly in only 26% of the cells. The fact that cyclosporin, which inhibits PTP, enhances the ionomycin effect suggests that the activation of PTP, which collapses DCm, inhibits the sustained elevation of [Ca2 + ]in. We also tested the effect of MV-4-CS, which inhibits PTP, but does not inhibit calcineurin and has no effect on the immune response (Baumann et al., 1992; Schweizer et al., 1993). Fig. 2B shows histograms from similar experiments with 3-month- and 20month-old B6 ×C3H F1 mice, which show that MV-4-CS, similar to cyclosporin, enhances the ionomycin-induced elevation of [Ca2 + ]in, particularly in old mice. Fig. 3A shows a summary of the results of similar experiments on the ionomycin effect with seven batches (12 mice) of T cells from 3- to

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4-month-old mice, three batches (5 mice) of cells from 20-month-old mice, and four batches (10 mice) of T cells from 26-month-old mice (all mice were from the B6/D2 F1 strain). The ionomycininduced elevation of the average [Ca2 + ]in was significantly higher in 3- to 4-month-old mice (3099 12 nM) than in 20-month-old mice (1499 2 nM, PB 0.0003). The ionomycin-induced elevation of the average [Ca2 + ]in was even smaller in 26-month-old mice (9597, PB 0.00002). In both young and old mice, cyclosporin enhanced the ionomycin effect (5239 18, and 347968 nM, respectively), and there was only a slightly significant difference (PB0.07) in [Ca2 + ]in after treatment with ionomycin+ cyclosporin between 4-month-old and 20-month-old mice. Fig. 3B summarizes the same experiments described in Fig. 3A in terms of the percentage of responding cells. The percentage of T cells that sustained high [Ca2 + ]in after treatment with ionomycin was reduced from 729 5% in 3- to 4month-old to 489 1% in 20-month-old (PB0.0002), and further decreased to 2294% in 26-month-old (PB0.000001). Upon treatment with both cyclosporin and ionomycin, [Ca2 + ]in was elevated in 829 5% of cells of 4-month-old, and in 699 3% of 20-month-old mice. The difference in the response to ionomycin between T cells from 4-month- and 20-month-old mice is best demonstrated by a titration of [Ca2 + ]in with ionomycin9 cyclosporin (Fig. 3C). Without cyclosporin, the increase of [Ca2 + ]in was much larger in T cells from young mice up to 0.4 mg/ml ionomycin; above this concentration, as ionomycin uncoupled DCm, (Fig. 1B), the difference vanished. When cyclosporin was added together with ionomycin, the maximal [Ca2 + ]in was greatly increased in both preparations and reached the same maximal level, although the maximum was obtained at somewhat higher iono-

Fig. 2. Effect of age on the ionomycin-induced elevation of [Ca2 + ]in in T lymphocytes. (A) Histograms from flow cytometric [Ca2 + ]in assays of typical batches of T cells isolated from 3 months (3M), 20 months (20M), and 26 months (26M) old B6 × DBA/2 F1 mice. First row, with no addition (control); second row, with 75 ng/ml ionomycin ( + ionomycin); third row, with 75 ng/ml ionomycin+1 mM cyclosporin A ( +cyclosporin and ionomycin). (B) 3 Months (3M), and 20months (20M) old B6 ×DBA/2 F1 mice. First row, control; second row, with 75 ng/ml ionomycin; third row, with 75 ng/ml ionomycin +1 mM MV-4-CS (+ mVCs and ionomycin). The mean [Ca2 + ]in of the T cell sample is printed on each panel. A horizontal bar indicates a region containing cells with a high [Ca2 + ]in, and the percent of cells in this region is marked near the bar.

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Fig. 2.

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mycin concentration in T cells from old mice. Cyclosporin also enhanced the susceptibility of the cells to the collapse of [Ca2 + ]in by high ionomycin concentrations, most likely because the increased [Ca2 + ]in enhanced Ca2 + transport by mitochondria and thus enhanced the uncoupling of DCm.

3.3. Mitochondrial membrane potential is 6ery low in T cells from old mice and is restored by cyclosporin and MV-4 -CS Fig. 4A shows the effect of cyclosporin on DiOC6(3) fluorescence in T cells from 3-, 20-, and

Fig. 3.

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26-month-old B6/D2 F1 mice, and the effect of MV-4-CS on DiOC6(3) fluorescence in T cells from 3-, and 20-month-old B6/C3H F1 mice. Even in T cells from 3-month-old mice, there was a significant fraction of cells with low DCm. However, in T cells from 20-month-old mice this fraction was much larger, and in T cells from 26-month-old mice only a small fraction of the cells maintained high DCm, in vitro. Nevertheless, as in lymphocytes (Rottenberg and Wu, 1997), DCm in T cells could be restored to high values by treatment with cyclosporin, an inhibitor of PTP. In the presence of cyclosporin there was no difference in DCm between T cells from young and 20-month-old mice. The cyclosporin derivative MV-4-CS had a similar effect on DCm in T cells from young and old mice. Fig. 4B shows the summary of similar experiments with four preparations (10 mice) of T cells from 26-month-old mice, three preparations (6 mice) of T cell from 20-month-old mice, and seven preparations (14 mice) of T cells from 3month-old mice (all mice were from the B6/D2 F1

Fig. 3. The effect of ionomycin and cyclosporin on [Ca2 + ]in in T lymphocytes from young and old mice. (A) Summary of the results of experiments with several batches of T cells from 3, 20, and 26 months old B6 ×DBA/2. The first column shows the average [Ca2 + ]in of T cells without ionomycin (unfilled), the second column shows the average [Ca2 + ]in for T cells incubated with 75 ng/ml ionomycin, and the third column shows the average [Ca2 + ]in of T cells incubated with 1 mM cyclosporin A and 75 ng/ml ionomycin (See text for more detailed description of these results). There were significant differences between [Ca2 + ]in in young and old ionomycin treated cells: 3/20, P B0.0003 (**); 3/26, PB 0.000001 (***). (B) Summary of the same experiments described in Fig. 3A in terms of the percentage cells with high [Ca2 + ]in. The first column shows percentage cells with high [Ca2 + ]in after ionomycin treatment (unfilled), and the second column shows percentage cells with high [Ca2 + ]in after ionomycin + cyclosporin treatment (filled). The differences between T cells from young and old mice after ionomycin treatment were large and highly significant: 3/20, PB0.006 (**); 3/26 PB 0.0002 (***), but the difference between T cells from young and old mice after ionomycin + cyclosporin treatment was small and not significant PB0.07(*). (C) [Ca2 + ]in in T cells as a function of ionomycin concentration: with 1 mM cyclosporin A (filled symbols), or without cyclosporin (empty symbols). Triangles =cells from 3 months old mice; circles =cells from 20 months old mice.

Fig. 4. The effect of age on DCm in mouse splenic T lymphocytes. (A) Histograms from flow cytometric DCm assays of T cells from 3, 20, and 26 months old B6× DBA/2 F1 mice and 3, and 20 months old B6 × C3H F1 mice (last two columns). Cells were loaded with DiOC6(3) and incubated without and with 1 mM cyclosporin A or MV-4-CS (mVCs). A low potential ( + 50 mM CCCP) histogram is overlaid on the panels. (B) Summary of the results of experiments similar to those shown in Fig. 4A with several batches of T cells. The average fluorescence ratio F/FCCCP (a measure of DCm) of T cells from mice of different ages without (open bars) and with (hatched bars) 1 mM cyclosporin A is shown (see text for a more detailed description of these results). The difference in DCm (F/FCCCP) between T cells from young and old mice (without cyclosporin) was highly significant: 3/20, P B0.002 (*); 3/26, PB 0.001 (**).

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strain). The results are summarized as the average ratio (9 SEM) of the total fluorescence in controls (9 cyclosporin) to the fluorescence after treatment with the uncoupler CCCP. In T cells from 26-month-old mice, DCm was much lower than in T cells from 3-month-old mice (F/FCCCP = 1.429 0.04 vs. 3.1390.21, P B 0.0009). In T cells from 20-month-old mice, DCm was slightly higher than in 26-month-old (1.539 0.21), but still significantly lower than that of young mice (PB0.002). Cyclosporin increased DCm in T cells from both young and old mice. After cyclosporin treatment of T cells there was no difference in DCm between 3-month- and 20month-old mice, and only a marginally significant difference (PB 0.06) between 3-month- and 26month-old mice. Comparison of the effect of cyclosporin on DCm and [Ca2 + ]in in old and young mice, as summarized in Fig. 3A,B, and Fig. 4B, suggests that the attenuated response of [Ca2 + ]in to ionomycin of T cells from old mice resulted from the enhanced activation of PTP, since cyclosporin (and MV-4-CS) restored both DCm and normal response of [Ca2 + ]in to ionomycin in the majority of cells in old mice.

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mitochondrial electron transport, which stimulates superoxide generation to its maximal level. Fig. 5B shows a summary of the results of experiments in which the generation of superoxide by mitochondria in T cells from young and old mice was estimated from the oxidative conversion of the non fluorescent molecule DHE to ethidium (Budd and Nicholls, 1996). The cells were treated with the mitochondrial electron transport inhibitor antimycin A or myxothiazole, which enhance the generation of superoxide. The enhancement of ethidium fluorescence was several fold higher in T cells from old mice. The observation that ROS accumulation in the mitochondria of T cells from old mice is enhanced to a much larger extent by inhibition of the electron transport chain is compatible with the conclusion that the redox equilibrium is shifted to a more oxidized state.

3.4. Increased mitochondrial FAD oxidation and mitochondrial generation of superoxide in T cells from old mice We have shown previously that in spleen lymphocytes from old inbred mice the mitochondrial FAD is more oxidized than in lymphocytes from young mice (Rottenberg and Wu, 1997). Fig. 5A shows that the mitochondrial FAD in lymphocytes from 26-month-old C57BL/6 × DBA F1 mice was also more oxidized than in young mice. Moreover, in T cells isolated from these lymphocytes, mitochondrial FAD was even more oxidized. Since the redox systems inside the mitochondria are in redox equilibrium, and oxidation of these systems enhances PTP activation (Chernyak and Bernardi, 1996), it is likely that this oxidation contributes to the enhanced activation of PTP. To test the capacity of mitochondria to generate superoxide, in situ, it is necessary to inhibit

Fig. 5. FAD oxidation and superoxide generation in young and old mice. (A) The mean mitochondrial FAD fluorescence (F1) of four batches of lymphocytes from 10 young (4 mo) mice (filled squares), three batches from 8 old (26 mo) mice (open squares), four batches of T cells from the same young mice (filled circles), and three batches of T cells from the same old mice (open triangles). (B) Enhancement of superoxide generation by inhibitors of mitochondrial electron transport in young and old mice. Samples were incubated for 1 h with 2 mM DHE and either 10 mM antimycin A (anti), or 10 mM myxothiazole (myx). Young =filled bars; old =unfilled bars.

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Fig. 6. Rh123 fluorescence in T cells from young and old mice. (A) Typical kinetics of the increase of Rh123 fluorescence in T cells incubated with 1 mM Rh123, with or without 50 mM verapamil. Young (3M) = empty symbols; old (20M) = filled symbols. Without verapamil =circles; with verapamil = squares. (B) Summary of Rh123 efflux experiments. Rh123 efflux assay as described in Experimental Procedures. The average of four preparations of T cells form 4-month-old young mice (Y) and four preparations of T cells from 21month-old mice (O). The fluorescence was measured immediately after washing the cells (0) and after 30 min incubation of the washed cells (Makowska et al., 2000). The statistical significance of the difference in fluorescence between young and old after washing was PB 0.04 (*); and after 30 min efflux P B 0.005 (**).

3.5. Rh123 fluorescence in T cells is largely determined by the magnitude of Zcm An association between the inability of T cells to retain the fluorescence of the cationic probe Rh123 and the attenuation of calcium signaling was reported recently (Witkowski and Miller, 1999). These observations were interpreted as demonstrating an association between high activation of P-gp and the attenuation of calcium signaling. We therefore wanted to rule out the possibility that our results depend on the reported difference in P-gp activation. Fig. 6A shows the effect of verapamil, a potent inhibitor of P-gp, on the rate of the fluorescence increase in T cells from old and young mice after addition of Rh123. It is observed that the rate of the increase in Rh123 fluorescence is much slower in T cells from old mice, and initially it is not enhanced by verapamil in either old or young mice. This indicates that the initial uptake of Rh123 is not affected by P-gp activity and the difference in the rate of accumulation of Rh123 is most likely due

to the difference in DCm (Rottenberg and Wu, 1998). Even after a longer incubation, where there is some enhancement of uptake by verapamil, the enhancement is similar in magnitude in T cells from old and young mice, and the difference between T cells from young and old mice is the same with or without verapamil. Therefore, the difference between young and old mice in Rh123 fluorescence appears to result largely from the difference in DCm. Since the uptake of DiOC3(6) is much faster than Rh123 (Rottenberg and Wu, 1998), and Rh123 is a much better substrate for P-gp, it is unlikely that the difference we observed in DiOC3(6) fluorescence between T cells from young and old mice resulted from differences in P-gp activity. Moreover, the results presented in Fig. 4A raise doubts regarding the interpretation of the previously published Rh123 efflux experiments (Witkowski and Miller, 1999). If DCm is indeed higher in T cells from young mice, there must also be a higher concentration of Rh123 in the mitochondrial matrix. It is well known that at high dye concentration (\ 1 nM) the fluorescence of cationic dyes that accumulate in the mitochondria is partially quenched (Rottenberg and Wu, 1998). Therefore, in an efflux assay, where the fluorescence that is retained by the cell is measured, cells with a high concentration of quenched dye in the mitochondrial matrix will appear to retain the dye more strongly. Fig. 6B shows a summary of the results of Rh123 efflux experiments from four batches of T cells from 20month-old mice and four batches of T cells from 3-month mice. It is observed that there is significantly higher fluorescence initially in T cells from young mice, indicating higher accumulation of dye because of the higher DCm. Since the higher concentration of dye in the mitochondria is associated with a higher fraction of quenched dye (Rottenberg and Wu, 1998), it is expected that, at equal rate of efflux, the fluorescence decay in cells with high DCm will be slower. This is indeed what we observed: the relative decrease of fluorescence after 30 min incubation is higher in T cells from old mice than in T cells from young mice. While these results are compatible with previous observations (Witkowski and Miller, 1999), we did not

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find a clear distinction between fractions of cells with high and low Rh123 fluorescence during the

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efflux assay. Rather, as the efflux progressed in time, the low fluorescence fraction increased and the high fluorescence fraction decreased. Moreover, if the cells were loaded over a longer time period (and/or higher Rh123 concentration) the distribution between the low and high fluorescence fraction was altered (results not shown). We believe that these results suggest that the magnitude of DCm in each cell and the Rh123 concentration in the cell determine the size of the fraction of quenched Rh123. Since the specific Rh123 fluorescence (i.e. fluorescent Rh123/total Rh123) is higher in T cells from old mice because of their lower DCm, the loss of fluorescence is faster, but it does not necessarily indicate faster efflux.

3.6. Memory cells and the permeability transition It is well known that in T cells from old mice the ratio of memory cells to naive cells is higher (Miller, 1996a). It has also been reported frequently that the ratio of CD8 cells to CD4 cells is

Fig. 7.

Fig. 7. Immunotyping of T lymphocytes from young and old mice. (A) Typical dotplots of T lymphocytes from 3 month old mice (left panels) and from 26 month old mice (right panels) (see Experimental Procedures). Top Panels show dotplots from T cells doubly-labeled with FITC conjugated anti-CD4 (F1) and PE-conjugated anti-CD8 (F2). The percentages of CD4and CD8-labeled cells are indicated. Center panels show dotplots of T cells doubly labeled with FITC conjugated antiCD4 (F1) and PE conjugated anti-CD44 (F2), gated on a pre-determined CD4 positive region. The percentages of antiCD4 labeled cells in the high (memory) and the low (naive) CD44 regions are indicated. Lower panels show dotplots of T cells labeled with FITC conjugated anti-CD8 and PE conjugated anti-CD44, gated on a pre-determined CD8 positive region. The percentages of anti-CD8 labeled cells in the high (memory) and the low (naive) CD44 regions are indicated. (B) Summary of similar results of immunotyping of four batches (10 mice) of 3 – 4 month old (open bars) and three batches (8 mice) of 26 month old (hatched bars) mice. Bars marked ‘CD4’ show average fractions of CD4+ in total CD4+ + CD8+ cells; bars marked ‘CD8’ show average of CD8+ in total CD4+ +CD8+ cells. Bars marked ‘4/44h’ show average fraction of CD44High cells in CD4+ cells. Bars marked ‘8/44h’ show average fraction of CD44h cells in CD8+ cells. Bars marked ‘44h’ show average fraction of CD44High cells in total CD4+ +CD8+ cells. The statistical significance of the difference between young and old is indicated: PB0.003 (*); P B 0.0002 (**); P B0.00003 (***).

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higher in spleen T cells from old mice. CD44 is a cell surface protein that is highly expressed in memory/effector cells and serves as a marker for memory cells (Miller et al., 1997). Fig. 7A shows dot plots of CD4/CD8 distribution in 3-month- and 26-month-old mice and dot plots of CD4/CD44 and CD8/CD44. In T cells from 26-month-old mice the percentage of cells expressing a high level of CD44 was very high, particularly in the CD8 subset. Since there was also a large increase in the fraction of CD8 cells in T cells from old mice, the majority of T cells in the spleen of old mice were memory cells. Fig. 7B shows a summary of similar experiments with four batches (8 mice) of 3-monthold mice and three batches (10 mice) of 26-monthold mice. The reversal of the CD8/CD4 ratio and the large increase in the fraction of memory cells (CD44high), particularly in the CD8 subset is striking. Together, the percentage of memory T cells (CD8 +CD4), increased from 13% in 3-month-old mice to 65% in 26-month-old mice, a difference of 52%. The increase in the percentage of cells resistant to ionomycin, from 28% to 78%, is about the same ( 50%, Fig. 2D), in agreement with the reported association between memory cells and ionomycin resistance (Miller 1996b). Similarly, the increase of the low DCm (i.e. PTP activated) fraction from 40 to 90% was about the same. Thus, in old mice :50% more of the T cell populations were memory cells, had a lower DCm, and were resistant to ionomycin. This is compatible with the notion that the age-induced expansion of the memory cell population is associated with the expansion of the population with enhanced PTP. To prove an association between the memory phenotype and enhanced activation of the mitochondrial permeability transition, it would be necessary to sort the T cells from both young and old mice on the basis of their mitochondrial membrane potential and analyze the distribution of CD44 in each fraction.

cytosolic calcium on mitochondrial metabolism, calcium uptake and release by mitochondria participates, often critically, in cellular calcium signaling (Ichas et al., 1997). The position of mitochondria in close proximity to the internal calcium stores of the sarcoplasmic reticulum (SR) (Rizzuto et al., 1993), and ER (Csordas et al., 1999), and adjacent to calcium channels on the plasma membrane (Hoth et al., 1997; Rutter et al., 1998), facilitates this role. The role of mitochondria in calcium signaling in T lymphocytes was examined in studies of the effects of agents that collapse DCm on the thapsigargin-induced, CRAC channels-mediated elevation of [Ca2 + ]in in Jurkat T cells (Hoth et al., 1997; Makowska et al., 2000), and on T cell receptor-induced activation (Hoth et al., 2000). It was concluded that unless Ca2 + is removed by mitochondria from the vicinity of the CRAC channels, the channels are inactivated, and the Ca2 + signal decays quickly. Because Ca2 + signaling in T lymphocytes serves to activate several transcription factors, and their activation can be differentially regulated by the amplitude and frequency of the signal (Dolmetsch et al., 1998), it can be predicted that mitochondrial dysfunction can not only attenuate the signaling (Hoth et al., 1997), but can also determine the fate of the cell by differentially affecting the activation of different transcription factors. Here we show, that the mitochondrial uncoupler CCCP, and low concentrations of valinomycin, which collapse DCm, reversed the ionomycin-induced elevation of [Ca2 + ]in in mouse spleen T lymphocytes. Interestingly, a high concentration of ionomycin, which also collapses DCm, reduced rather than elevated the sustained level of [Ca2 + ]in. These observations further substantiate the role of mitochondria in calcium signaling in T cells. We also show that PTP activation can regulate Ca2 + signaling in T cells. Similar results were reported recently for oligodendrocyte progenitor cells (Smaili and Russel, 1999).

4. Discussion

4.1. The role of mitochondria in the regulation of Ca 2 + signaling in T lymphocytes In addition to the control that is exerted by

4.2. Membrane potential, redox state and PTP acti6ation in T lymphocytes from old mice We have previously shown that in spleen lymphocytes there is a fraction of cells with low

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DCm, and that this fraction is much larger in lymphocytes from old mice (Rottenberg and Wu, 1997). We show here, that in isolated T cells the low potential fraction is much larger than in lymphocytes, in both young and old mice. When we compared DCm in a batch of lymphocytes and in the T cell fraction isolated from this batch of lymphocytes, the size of the low potential fraction was about 3 fold larger in the T cell subset, both in young and old mice (results not shown). Considering that the T cell subset constitutes about 30% of the total lymphocyte population, these results suggest that most of the low potential spleen lymphocytes are T cells. This conclusion was confirmed by sorting the lymphocytes on the basis of DiOC6(3) fluorescence, which resulted in a T cell enriched, low potential fraction (results not shown). Unless stimulated to proliferate, isolated T cells gradually undergo spontaneous apoptosis that is preceded by loss of DCm. Since we also showed that DCm could be restored by cyclosporin, and MV-4-CS, which are potent inhibitors of PTP, it appears that PTP activation is the principal cause of the low DCm in T cells. The enhanced activation of PTP in T cells from old mice was associated with enhanced oxidation of mitochondrial FAD. Since the redox state of FAD is in equilibrium with the redox state of the NAD(P) pool and glutathione, which control the activation of PTP (Chernyak and Bernardi, 1996), it is likely that the highly oxidized state of the redox systems is a primary cause of the enhanced activation of PTP in old mice. Our finding that aging results in a reduction of DCm and enhanced oxidation of T cell mitochondria, is similar to what was observed in rat liver mitochondria (Hagen et al., 1997). Moreover, we showed recently that aging also enhances the activation of PTP in brain and liver mitochondria (Mather and Rottenberg, 2000). It was also reported recently that caloric restriction, which reduces oxidative stress and ameliorates immunosenescence, protects against the induction of PTP (Kristal and Yu, 1998).

4.3. The mechanism of the attenuation of calcium signaling in T lymphocytes from old mice The attenuation of calcium signaling in T

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lymphocytes from old mice has been studied extensively for more than a decade (reviewed in Miller, 1996b). However, neither the mechanism of this attenuation, nor its etiology, has been elucidated. Miller suggested earlier that this defect is shared by all memory cells, regardless of age, and therefore the etiology of this defect is linked to the expansion of the memory subset in old mice (Miller, 1996b). More recently, Miller focused his attention on the relationships between P-gp activity and the attenuation of calcium signaling in T lymphocytes from old mice (Witkowski and Miller, 1999). His data were interpreted to suggest that the attenuation of calcium signaling is strongly associated with the expansion of the T lymphocyte population that expresses a high level of P-gp. However, our results suggest a different interpretation (see below). Our study suggests a reasonable explanation for both the mechanism and the etiology of the attenuation of calcium signaling in T cells from old mice. The finding of enhanced activation of PTP in T lymphocytes from old mice, together with the evidence for the role of mitochondrial DCm in the ionomycin-induced sustained elevation of [Ca2 + ]in, and the fact that inhibitors of PTP restore both near normal DCm and the sustained elevation of [Ca2 + ]in in T lymphocytes from old mice, provides strong evidence for a role of PTP activation in the attenuation of Ca2 + signaling in T lymphocytes from old mice. Moreover, the etiology of this age-induced defect is consistent with current understanding of the relationship between aging and oxidative stress. The fact that this defect appears to be associated with the large expansion of the memory cell subset in T lymphocytes from old mice, is probably related to the fact that this subset of cells has a much longer life span than the cells of the naive subset. Much of the evidence for a causal relationship between activation of PTP and resistance to elevation of [Ca2 + ]in by ionomycin rests on the effect of cyclosporin, which is a potent inhibitor of PTP. A more potent effect of cyclosporin is inhibition of calcineurin, which is the basis for its clinical use as an immunosuppressive drug (Liu et al.,

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1991). However, MV-4-CS does not inhibit calcineurin and is not immunosuppressive (Baumann et al., 1992); yet the effect of MV-4-CS on both DCm and [Ca2 + ]in was identical to that of cyclosporin, ruling out the involvement of calcineurin in these effects.

4.4. The acti6ity of P-gp in T cells and the effect of cyclosporin on Zcm, and [Ca 2 + ]in It was previously concluded that the reported difference in Rh123 fluorescence between T cells from old and young mice, resulted from enhanced P-gp activity in old mice (Witkowski and Miller, 1993). However, our results on Rh123 uptake challenge this interpretation. Moreover, we used DiOC6(3) for our measurements of DCm, and this probe is not as good a substrate of P-gp as Rh123 (Rottenberg and Wu, 1998). Similarly, it was reported that Fluo-3 (unlike Fluo-3-AM) is not transported significantly by P-gp (Nelson et al., 1998), and therefore it is unlikely that P-gp activity had a significant effect on our measurements of [Ca2 + ]in. It was recently demonstrated that CD4 memory T cells sorted on the basis of their Rh123 fluorescence, exhibited several differences in their response to activation, in addition to calcium signaling (Eisenbraun et al., 2000). Since our results suggest that the difference in Rh123 fluorescence between T lymphocytes from young and old mice results from differences in DCm, which could affect many other cellular processes besides calcium signaling (e.g. ATP level, pHin, ROS production, apoptotic signals), it is likely that the activation of PTP in T cells from old mice attenuates other aspects of the T cells activation, in addition to Ca2 + signaling.

Acknowledgements This work was supported by Grant AG13779 from the National Institutes of Health (to H.R.). We are especially grateful to Dr Akhil Vaidya for his principled support during a difficult time, which, among other things, allowed the completion of this work.

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