Multiple sclerosis: Mitoxantrone promotes differential effects on immunocompetent cells in vitro

Journal of Neuroimmunology 168 (2005) 128 – 137 www.elsevier.com/locate/jneuroim

Multiple sclerosis: Mitoxantrone promotes differential effects on immunocompetent cells in vitro Oliver Neuhaus a,*, Heinz Wiendl b, Bernd C. Kieseier a, Juan J. Archelos c, Bernhard Hemmer a, Olaf Stu¨ve a,d, Hans-Peter Hartung a a Department of Neurology, Heinrich-Heine-Universita¨t, Moorenstr. 5, 40225 Du¨sseldorf, Germany Department of Neurology, Clinical Research Group for Multiple Sclerosis and Neuroimmunology, Universita¨t Wu¨rzburg, Josef-Schneider-Straße 11, 97080 Wu¨rzburg, Germany c Department of Neurology, Multiple Sclerosis Research Group, Medizinische Universita¨t, Auenbruggerplatz 22, 8036 Graz, Austria Department of Neurology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, TX 75201, United States b

d

Received 12 November 2004; accepted 10 January 2005

Abstract Mitoxantrone is an anti-neoplastic anthracenedione derivative that, based on its immunosuppressive properties, is approved for the treatment of severe forms of relapsing – remitting or secondary progressive multiple sclerosis (MS). Whether the beneficial clinical effects of mitoxantrone in MS are due to a broad immunosuppression, or whether there is a specific mechanism of action remains unknown. Peripheral blood mononuclear cells (PBMCs) from untreated or interferon-h-treated patients with MS or from healthy donors were stimulated in the presence or absence of mitoxantrone. Irrespective of the source of the cells and the cellular phenotype, mitoxantrone inhibited proliferation of activated PBMCs, B lymphocytes, or antigen-specific T-cell lines (TCLs) stimulated on antigen-presenting cells (APCs) in a dose-dependent manner. For functional analysis, TCLs or APCs were incubated separately with mitoxantrone. Pre-incubation of APC more effectively impaired TCL proliferation than pre-incubation of TCLs. Production of cytokines, expression of activation markers, matrix metalloproteinases, and chemokine receptors were not influenced substantially by mitoxantrone. In contrast, in dendritic cells (DCs), mitoxantrone interfered with the antigen-presenting capabilities. For evaluation of apoptotic cell death of target cells, annexin-V-conjugates and a DNA fragmentation assay were applied. Mitoxantrone induced apoptosis of PBMCs, monocytes and DCs at low concentrations, whereas higher doses caused cell lysis. Our observations suggest that the beneficial effects of mitoxantrone in MS result (i) from its immunosuppressive action based on nonspecific cytotoxic effects on lymphocytes, (ii) by inducing programmed cell death of professional APCs, such as DCs. D 2005 Elsevier B.V. All rights reserved. Keywords: Multiple sclerosis; Mitoxantrone; Immunosuppression; Immunomodulation

1. Introduction Multiple sclerosis (MS) is the most common inflammatory disorder of the central nervous system (CNS) and the leading cause of neurological disability in younger adults (Noseworthy et al., 2000; Hemmer et al., 2002; Kieseier and Hartung, 2003). Therapeutic options in MS are limited

* Corresponding author. Tel.: +49 211 8117880; fax: +49 211 8118469. E-mail address: [email protected] (O. Neuhaus). 0165-5728/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2005.01.024

(Hohlfeld and Wiendl, 2001; Neuhaus et al., 2003; Wiendl and Kieseier, 2003). For the long-term treatment of MS, two classes of agents are currently available: (i) Immunomodulatory agents with pleiotropic effects, which include the reduction of inflammatory mediators such as pro-inflammatory cytokines and matrix metalloproteinases (MMPs). Interferon-h (IFN-h) and glatiramer acetate (GA) are the two approved agents (Yong, 2002; Neuhaus et al., 2003). (ii) Immunosuppressive agents are global inhibitors of crucial components of the immune system. Their administration leads to general immune dysfunction. Their beneficial

O. Neuhaus et al. / Journal of Neuroimmunology 168 (2005) 128 – 137

effects in MS are limited by systemic adverse effects. Examples are azathioprine and cyclophosphamide. Mitoxantrone is an anti-neoplastic anthracenedione derivative related to the anthracyclins doxorubicin and daunorubicin. It interacts with topoisomerase-2 and causes single and double strand breaks by intercalating the DNA (Smith, 1983). In addition, as a potent immunosuppressive agent targeting proliferating immune cells (Fidler et al., 1986a,b; Mauch et al., 1992; Gbadamosi et al., 2003), it has proven efficacy in the treatment of severe active relapsing – remitting (RR) and secondary progressive (SP) MS (Mauch et al., 1992; Noseworthy et al., 1993; Bastianello et al., 1994; Gonsette, 1996; Edan et al., 1997; Millefiorini et al., 1997; Van de Wyngaert et al., 2001; Hartung et al., 2002). It reduces both attack and progression rates and has been approved for the treatment of patients with secondary progressive and worsening forms of MS (Jain, 2000; Edan et al., 2003; Neuhaus et al., 2005). However, treatment duration is limited to 2 to 3 years or 140 mg/m2 cumulative dose because of possible cardiotoxic side effects (De Castro et al., 1995; Ghalie et al., 2002). Recently, evidence has surfaced that some immunosuppressive agents additionally exhibit immunomodulatory properties. For example, cyclophosphamide shifts immune responses from TH1 towards TH2 by a mechanism yet unknown (Karni et al., 2004). Mitoxantrone has also been claimed to act not only as an immunosuppressive but also as an immunomodulatory drug (Jain, 2000). Already in the 1980s, Fidler and colleagues observed a decreased secretion of pro-inflammatory cytokines and enhanced suppressor Tcell functions induced by mitoxantrone (Fidler et al., 1986a,b). More recently, longitudinal observations revealed short-term inhibitory effects on most subsets of T lymphocytes and a persistent decrease of B cells (Gbadamosi et al., 2003). In this study, we analyzed immunological effects of mitoxantrone in vitro to explore its potential immunomodulatory activity in MS. We observed that apart from its potent immunosuppressive effects on T and B cells, the predominant action site of mitoxantrone in MS (and probably other autoimmune disorders) is the antigenpresenting cell (APC) that is induced to undergo apoptosis and cell lysis by mechanisms that have yet to be elucidated.

2. Materials and methods 2.1. Patients and controls In order to assess the impact of immunomodulatory treatment, peripheral blood mononuclear cells (PBMCs) were obtained with informed consent from (i) patients with MS receiving IFN-h1a or IFN-h1b, n = 17, f : m = 13 : 4, mean age: 37 years, expanded disability status scale (EDSS) (Kurtzke, 1983) range 0.0– 6.0; (ii) untreated MS patients, n = 15, f : m = 11 : 4, mean age: 36 years, EDSS range 0.0 –

129

4.5; or (iii) n = healthy donors (HD), n = 9, f : m = 5 : 4, mean age: 31 years. Proliferative activity was analyzed in n = 15 IFN-h1-treated, n = 12 untreated MS patients and n = 6 HD. Surface flow cytometry was carried out on cells retrieved from n = 4 IFN-h1-treated, n = 5 untreated MS patients and n = 5 HD. Transcription of MMPs and chemokine receptors was assessed in n = 5 untreated MS patients and n = 5 HD. Cell death analysis was performed in n = 2 IFN-h1-treated, n = 3 untreated MS patients and n = 8 HD. 2.2. Cell cultures Unless specified, all cell cultures were established and maintained using RPMI 1640 medium (Gibco, Gaithersburg, USA) supplemented with 5% pooled and heat-inactivated human AB serum (kindly provided by Drs. G. Lanzer and A. Vadon, Blood Transfusion Unit, Medical University, Graz, Austria) containing 2 mM glutamine, 100 U/ml penicillin, 100 Ag/ml streptomycin (all from Gibco), 20 Ag/ml ciprofloxacin (Bayer Vital, Leverkusen, Germany) and incubated at 37 -C in an atmosphere of 5% CO2. PBMCs were isolated by density centrifugation gradient (Lymphoprep, density 1.077 g/ml; Axis-Shield, Oslo, Norway). 2.3. Dendritic cell culture Monocytes were obtained from Ficoll (Biocoll separating solution; Biochrom, Berlin, Germany) separated PBMCs of HD. B cells were removed using magnetic beads directed against CD22 (Miltenyi Biotec, Bergisch Gladbach, Germany) and monocytes were obtained after 1 h adherence in RPMI supplemented with 10% fetal calf serum (FCS; Gibco) at 37 -C. Monocytes, more than 90% pure as assessed by flow cytometry, were cultured in RPMI plus 10% FCS supplemented with granulocyte-macrophagecolony stimulating factor (GM-CSF, 100 ng/ml; Novartis, Basel, Switzerland) and interleukin (IL)-4 (40 ng/ml; PeproTech, Rocky Hill, USA). After 6 days the cells exhibited an immature DC phenotype (CD14 CD1a+ HLA-DRlow CD86low CD80low/ CD83 ). Maturation was induced by incubation of the immature DCs with lipopolysaccharide (LPS; 5 Ag/ml, S. thyphi; Sigma-Aldrich, St. Louis, USA). High levels of surface HLA-DR and costimulatory molecules (CD86, CD80) identified mature DCs. Effects of mitoxantrone on maturation and survival of DCs were induced at the stage of immature DCs. Mitoxantrone was added to the DCs on day 6 and effects quantified 24 and 48 h after application. 2.4. Proliferative activity For proliferation analysis, 1 105 freshly isolated PBMCs per well were stimulated in triplicates with phytohemagglutinin (PHA, 10 Ag/ml; Sigma) for 5 days. Mitoxantrone (Wyeth-Lederle, Newbridge, Ireland) at various concentrations (0.02 – 2000 ng/ml) was added at day 4, since presence

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in vitro of mitoxantrone for the whole 5 days induced cell death at concentrations exceeding 2 ng/ml (data not shown). Sixteen to 18 h before the end of incubation, [methyl-3H]thymidine (0.5 ACi per well; Amersham Biosciences, Buckinghamshire, UK) was added. Cells were harvested and [methyl]-3H-thymidine incorporation was measured using a liquid scintillation counter (Packard BioScience, Meriden, USA). For selective analysis of B-cell proliferation, B cells were enriched in PBMCs using paramagnetic beads directed against non-B cells (Dynal, Oslo, Norway) and activated with pokeweed mitogen (PWM, 2 Ag/ml; Sigma). T-cell lines (TCLs) reactive against tetanus toxoid (TT; Massachusetts Biologic Laboratories, Worcester, USA) or GA (Teva Pharmaceutical Industries, Petah Tiqva, Israel) were selected using the split-well technique and stimulated with TT or GA, respectively, on irradiated (40 Gy) APCs for 48 h as described previously (Neuhaus et al., 2000). For functional analysis, TCLs or APCs were incubated separately with mitoxantrone for 24 h and washed three times before antigen stimulation for additional 48 h. 2.5. Surface markers and intracellular cytokine expression For phenotypical characterization by flow cytometry, cells were stained with FITC- or PE-labeled monoclonal antibodies (mAb) directed against CD3, CD4, CD8, CD14, CD19, CD27, CD45RO, CD45RA, CD69, CD80, VLA-4, ICAM-1 (all from Becton Dickinson, San Jose, USA), CD64 (Medarex, Annandale, USA), CD1a, CD80, CD83, CD86, HLA-DR (BD PharMingen, San Diego, USA) and the corresponding isotype controls (Becton Dickinson). Unless stated above, DCs were stained with anti-CD86 mAb (Diaclone, Besanc¸ on, France) and anti-HLA-DR mAb (Coulter, Miami, USA). Intracellular production of IFN-g and IL-4 by PBMCs was determined as described previously (antibodies from BD PharMingen) (Neuhaus et al., 2000). Using a FACScalibur (Becton Dickinson), data from 5000 cells per sample were accumulated and the results were analyzed as dot plots representing the relative fluorescence intensity.

(MMP-10), and -3 (MMP-11), tissue inhibitor of metalloproteinase (TIMP)-1, -2, and -3, and GAPDH were used as described previously (Kieseier et al., 2001). Briefly, three-fold serial dilutions of competitive standard DNA were combined with a definite amount of sample cDNA, and PCR was performed in 50 Al reactions containing 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris – HCl pH 9.0, and 0.1% Triton X-100 in the presence of 200 AM dNTP (Pharmacia, Freiburg, Germany), 50 pmol sense and antisense MMP primers, 1 U taq DNA polymerase (Perkin Elmer, Branchburg, USA), and 1 ACi 32P-dCTP (Amersham). Amplification was carried out using 35 cycles (95 -C, 30 s; 57 -C, 30 s; 72 -C, 120 s) in a Hybaid Omnigene thermal cycler (MWG Biotech, Ebersberg, Germany). Ten microliters of the reaction products were electrophoresed on a 6% polyacrylamide gel, which were read and quantitated on a Cyclon phosphor imaging system (Packard). MMP RNA levels were determined by plotting the ratio of sample cDNA to standard DNA against the standard dilution using a double-logarithmic scale. Chemokine receptor expression was detected on the RNA level by RNase protection. Multi-probe template sets for human chemokine receptors (hCR-5 and hCR-6) to be used in RNase protection assays were obtained from BD PharMingen. RNase protection assays were performed according to the manufacturer’s instructions using 10 Ag of total RNA from human mononuclear cells to analyze the expression of the RNA of CCR1, CCR2, CCR3, CCR4, CCR5, CCR8, CXCR1, CXCR2, CXCR3, and CXCR4. GAPDH served as house keeping gene. Equal amounts of tRNA were used as control to exclude incomplete digestion of the probes. Each multiprobe RNase assay included probes specific for ribosomal L32 and GAPDH RNA to assure equal amounts of input RNA in each assay and to control for lane to lane variation during PAGE. Protected probes were run on a 5% polyacrylamide gel under temperature control. After drying, gels were exposed on phosphor screens for use with a Cyclon phosphor imaging system. Undigested probes served as markers. 2.7. Detection of cell death

2.6. Expression of matrix metalloproteinases and chemokine receptors Total cellular RNA was extracted from the cultured PBMCs according to the method of Chomczynski and Sacchi (Chomczynski and Sacchi, 1987) and used as a template for cDNA synthesis using AMV reverse transcriptase (Gibco). For quantitation of human MMP RNA expression polymerase chain reaction (PCR) using a synthetic multi-competitor standard DNA containing tandem arrays of 5Vand 3Vpriming sites for cDNA of different MMPs and glycerinaldehyde phosphate dehydrogenase (GAPDH) was performed. Primer pairs for interstitial collagenase (MMP-1), matrilysin (MMP-7), gelatinase A (MMP-2), and B (MMP-9), stromelysin-1 (MMP-3), -2

Apoptotic cells were visualized by flow cytometry using annexin-V-conjugates (Trevigen, Gaithersburg, USA). These conjugates bind to phosphatidylserine which is exposed on the cell surface of early apoptotic cells. DNA fragmentation as another characteristic of apoptosis was measured by detection of incorporated biotinylated nucleotides into the DNA by terminal deoxynucleotidyl transferase (TdT) by flow cytometry (FlowTACS; Trevigen). Necrotic cell death was measured by propidium iodide uptake (2 Ag/ml; Sigma). 2.8. Statistical analysis Significance was assessed by two-sided Student’s t-test (*p < 0.05, **p < 0.01).

O. Neuhaus et al. / Journal of Neuroimmunology 168 (2005) 128 – 137

A

3. Results

**

Mitoxantrone inhibited proliferation of activated PBMCs in a dose-dependent manner irrespective of the source of the PBMCs (IFN-h-treated vs. untreated MS patients vs. HD) used (Fig. 1A). Proliferation of PWM-activated B cells was

[Methyl]-3H-thymidine incorporation (cpm × 10-3)

20

3.1. Antigen-specific and antigen-nonspecific proliferation is reduced by mitoxantrone

*

TT-reactive TCL GA-reactive TCL

15 ** *

10

5

** ** ** **

** **

200

2,000

0

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No Ag

MS IFN-β1 MS no treatment

** ** *

100

0

80

B

** ** **

** ** **

20 0 0

2

20

200

2,000

Mitoxantrone (ng/ml)

[Methyl]-3H-thymidine incorporation (cpm × 10-3)

B

25

B cells+ PWM

*

APC + mitoxantrone TCL + mitoxantrone

* *

10

5 *

* * * *

0 No Ag

20

20

*

15

60 40

2

Ag + mitoxantrone (ng/ml)

HD

[Methyl]-3H-thymidine incorporation (cpm × 10-3)

Proliferation (% control)

131

0

2

20

200

2,000

Ag + mitoxantrone (ng/ml) **

15 10 5

**

**

0 0

2

20

200

2,000

Mitoxantrone (ng/ml) Fig. 1. Inhibition of T- and B-cell-proliferation by mitoxantrone. Peripheral blood mononuclear cells (PBMCs) were isolated and (A) T cells were stimulated with phytohemagglutinin for 5 days and treated with mitoxantrone at day 4 at different concentrations. Sixteen to 18 h before end of incubation, cells were labeled with tritiated thymidine, harvested and thymidine incorporation was measured using a liquid scintillation counter. Proliferation is given as % of the stimulation-induced proliferation without treatment. Each value represents the mean + SD of n = 15 MS patients receiving IFN-h1 (black bars), n = 12 untreated MS patients (hatched bars), and n = 6 HD (white bars). The corresponding absolute values given as the control value (100%) are as follows: MS IFN-h, 43,921 cpm; MS no treatment, 53,619 cpm; HD, 48,565 cpm. (B) For selective analysis of Bcell proliferation, B cells were enriched from PBMCs using paramagnetic beads directed against non-B cells and activated with pokeweed mitogen. Proliferation is given as mean cpm + SD from one representative experiment (HD). In this experiment, B-cell percentage was increased from 13.4% to 44.2% of lymphoid cells as assessed by CD19 surface expression. Significance was assessed by two-sided Student’s t-test comparing untreated cells (0 ng/ml) to mitoxantrone-treated cells at different concentrations (2 – 2000 ng/ml), respectively (*p < 0.05; **p < 0.01).

Fig. 2. Inhibition of antigenic T-cell line stimulation by mitoxantrone. (A) T-cell lines (TCLs) reactive against tetanus toxoid (TT, n = 2) or glatiramer acetate (GA, n = 9) were selected using the split-well technique and stimulated with TT (2 Ag/ml; black bars) or GA (50 Ag/ml; white bars), respectively, on irradiated autologous antigen-presenting cells (APCs). TCL cells were incubated with antigen on APCs for 48 h in the presence or absence of mitoxantrone at different concentrations. Proliferation is given as mean cpm + SD from one representative experiment (HD). Note that with TT, the background proliferation without antigen (Ag) is usually high. (B) For functional analysis, TCLs as effector cells or APCs were preincubated separately with different concentrations of mitoxantrone for 24 h, washed and then co-incubated in the presence of GA for additional 48 h. Proliferation is given as mean cpm T SD from one representative experiment (MS). Similar results were observed with isolated monocytes or B cells as APCs. Note that at lower concentrations (2 – 20 ng/ml), mitoxantrone inhibited proliferation significantly more effectively when pre-incubated with APCs than with TCL cells. Two-sided Student’s t-tests compared (i) mitoxantrone-untreated but Ag-stimulated TCLs to those in the absence of Ag (first and second bars, respectively), and (ii) mitoxantrone-untreated Ag-stimulated TCLs (second bars) with the respective mitoxantrone-treated TCLs (third to sixth bars). *p < 0.05; **p < 0.01.

also impaired dose-dependently (Fig. 1B), at mitoxantrone doses comparable to PBMCs (significantly at 20 ng/ml or higher). Antigen-specific proliferation of TCLs stimulated by their respective antigens presented on autologous APCs was inhibited as well (Fig. 2A). Interestingly, the doses needed to affect TCL proliferation were 10-fold lower than that required for PBMC or B-lymphocyte proliferation (Fig. 1). Functionally, separate pre-incubation of APCs (B cells,

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monocytes or irradiated PBMCs containing the latter) with mitoxantrone yielded a higher diminution of TCL proliferation than separate pre-incubation of effector cells suggesting that APCs are more susceptible to the actions of mitoxantrone than T lymphocytes (Fig. 2B). 3.2. Mitoxantrone has no significant impact on surface markers and intracellular cytokine expression Both in PHA-stimulated PBMCs and in PBMCs left unstimulated for 5 days, as well as in PBMC subsets or

isolated monocytes cultured for 24 h, surface expression of activation markers and adhesion molecules and intracellular production of IL-4 and IFN-g were not influenced substantially by mitoxantrone (Table 1). 3.3. Gene transcription of matrix metalloproteinases and chemokine receptors is not influenced by mitoxantrone Both semiquantitative RT-PCR and RNase protection assay revealed a constitutive expression of various MMPs, TIMPs, and chemokine receptors by human PBMCs in

Table 1 Influence of mitoxantrone on surface marker and cytokine expression Marker Viable cellsa Annexin-V CD3 CD4 CD8 CD19 CD20 CD27 CD45RO CD45RA CD69 CD14 CD64 CD80 CD86 HLA-DR CD11a VLA-4 ICAM-1 IL-4 IFN-g

PHA-stimulated PBMC 5 days [%] < M < M < M < M < M < M < M < M < M < M < M < M < M < M < M < M < M < M < M < M < M

97.8 T 1.6 96.4 T 2.9 1.7 T 0.6 3.4 T 1.7 84.8 T 6.3 83.1 T 6.3 48.7 T 14.9 56.8 T 20.1 31.8 T 13.8 23.6 T 10.8 4.9 T 3.3 4.7 T 2.7 n.d. 44.6 T 22.1 41.2 T 18.6 76.5 T 12.1 70.8 T 10.8 14.1 T 8.4 16.2 T 8.5 5.4 T 9.3 11.3 T 16.0 n.d.

Unstimulated PBMC 5 days [%] ns ns ns ns ns ns

ns ns ns ns

99.4 T 0.9 96.0 T 4.0 1.1 T 0.4 0.8 T 0.0 73.6 T 24.7 77.7 T 25.9 43.1 T17.9 57.6 T 7.9 19.2 T 7.3 18.3 T 7.1 7.5 T 3.5 4.3 T 3.6 n.d. 24.3 T 22.9 30.4 T 23.0 39.1 T 8.4 43.6 T 8.9 41.1 T 4.2 34.5 T 6.5 0.7 T 1.2 1.7 T 2.4 n.d.

n.d.

n.d.

n.d.

n.d.

7.2 5.8 5.4 T 3.7 6.2 T 3.2 n.d.

1.7 3.0 6.3 T 1.3 4.5 T 1.8 n.d.

88.6 T 5.5 87.3 T 6.7 31.0 T 20.3 29.3 T 21.1 6.7 T 3.6 7.1 T 3.3 0.3 T 0.2 0.2 T 0.2

ns

ns ns

68.6 T 8.7 60.5 T 24.8 2.4 T 2.1 3.8 T 0.8 n.d.

ns n.d. ns

Unstimulated PBMC 24 h [%] ns ns ns ns ns ns

ns ns *

ns

ns

ns ns

99.7 T 0.3 97.0 T 0.7 6.0 T 2.0 7.3 T 0.8 67.4 T 8.3 75.8 T 5.3 36.4 T 10.5 32.4 T 5.6 31.6 T 4.7 38.2 T 0.5 7.2 T 2.4 9.3 T 3.6 9.9 T 2.9 12.4 T 4.1 58.0 T 6.1 63.8 T 0.4 38.4 T 7.9 46.4 T 14.8 50.6 T 11.9 34.6 T 11.6 14.6 T 6.3 25.6 T 15.9 1.1 T 0.8 1.7 T 0.6 2.5 T 0.6 4.2 T 0.9 3.2 T 3.9 11.2 T 7.7 7.8 T 3.0 14.0 T 4.5 15.2 T 5.9 19.7 T 6.4 97.9 T 0.4 97.3 T 0.3 85.5 T 9.4 88.9 T 3.5 4.9 T 2.2 12.7 T 1.3 3.8 T 1.0 2.6 T 0.4 9.7 T 4.7 8.4 T 7.2

Unstimulated monocytes 24 h [%] ns ns ns

89.5 T 4.7 12.2 T 9.3 3.2 T 0.6 2.8 T 2.9 0.1 T 0.1 0.3 T 0.2 n.d.

**

ns ns

ns n.d. ns n.d. ns n.d. ns n.d. ns n.d. ns n.d. ns ns ns ns ns ns ns ns ns **

96.4 T 0.9 97.8 T 1.0 89.5 T 8.9 92.2 T 10.6 88.4 T 0.5 87.8 T 6.2 94.4 T 6.5 95.9 T 4.7 95.2 T 2.2 96.8 T 3.4 96.6 T 4.0 99.0 T 0.2 84.5 T 9.0 89.5 T 2.0 29.3 T 9.2 28.3 T 7.3 80.6 T 6.0 77.7 T 5.8 n.d.

ns ns ns ns ns ns ns ns ns

ns n.d. ns

Results are given as mean T SD from up to 11 experiments (5-day culture) and 8 experiments (1-day culture), respectively. Cells from n = 4 IFN-h-treated MS patients, n = 5 untreated MS patients and n = 6 HD were analyzed. a Propidium iodide negative cells. <, no mitoxantrone; M, mitoxantrone 200 ng/ml; n.d., not determined; ns, not significant. * p < 0.05. ** p < 0.01.

O. Neuhaus et al. / Journal of Neuroimmunology 168 (2005) 128 – 137 Table 2 RNA expression in PBMC with and without mitoxantrone treatment in vitro No mitoxantrone

Mitoxantrone

0.25 T 0.04 0.76 T 0.02 0.32 T 0.05 0.56 T 0.03 0.83 T 0.08 n.d. n.d.

0.29 T 0.01 0.71 T 0.04 0.37 T 0.01 0.51 T 0.05 0.81 T 0.09 n.d. n.d.

ns ns ns ns ns – –

Tissue inhibitors of metalloproteinase TIMP-1 0.73 T 0.04 TIMP-2 n.d. TIMP-3 n.d.

0.69 T 0.08 n.d. n.d.

ns – –

a-Chemokine receptors CXCR1 CXCR2 CXCR3 CXCR4

0.34 T 0.01 0.21 T 0.03 0.52 T 0.01 n.d.

0.29 T 0.01 0.27 T 0.09 0.51 T 0.02 n.d.

ns ns ns –

h-Chemokine receptors CCR1 CCR2 CCR3 CCR4 CCR5 CCR8

0.21 T 0.03 0.67 T 0.08 n.d. 0.47 T 0.05 0.51 T 0.03 0.59 T 0.04

0.24 T 0.01 0.68 T 0.02 n.d. 0.50 T 0.02 0.48 T 0.07 0.61 T 0.02

ns ns – ns ns ns

Metalloproteinases MMP-1 (Collagenase-1) MMP-2 (Gelatinase A) MMP-3 (Stromelysin-1) MMP-7 (Matrilysin) MMP-9 (Gelatinase B) MMP-10 (Stromelysin-2) MMP-11 (Stromelysin-3)

MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; n.d., not detectable; ns, not significant. RNA expression levels are given as a fraction of the house keeping gene GAPDH [arbitrary units]. MMPs and TIMPs were measured by RT-PCR, chemokine receptors by RNAse protection. Values are given as mean T SD from n = 5 untreated MS patients and n = 5 HD. Sub-analysis revealed no significant differences between the two groups.

vitro. However, no significant modulation of the expression of any of these RNAs detectable was noted under the treatment with mitoxantrone in vitro (Table 2). 3.4. Mitoxantrone induces apoptotic cell death at low concentrations and necrotic cell death at high concentrations Using labeled annexin-V as an early apoptosis marker, it appeared that in lymphocytes the mechanism of cell death induced by mitoxantrone is bi-directional: apoptosis at in vitro doses below 20 ng/ml and necrosis at doses above 200 ng/ml (Fig. 3A). Direct comparison of lymphoid and monocytic cells obtained from the same individual revealed a higher susceptibility of monocytes to mitoxantroneinduced cell lysis. Whilst at 20 ng/ml, 16% of lymphocytes and 20% of monocytes were in the early apoptotic stage, 0% of lymphocytes and 19% of monocytes were propidium iodide positive, i.e., had undergone late apoptosis or even necrotic cell death (Fig. 3A). At higher doses of mitoxantrone, the percentages of annexin-V/propidium iodide

133

double-positive monocytes still substantially outnumbered those of the respective lymphocytes (Fig. 3A). Consistent with these findings, our phenotypical assessment revealed that the percentage of viable monocytes (propidium iodide negative cells) was reduced from 90% (no mitoxantrone) to 12% (mitoxantrone 200 ng/ml) without influencing their phenotype (Table 1), indicating a higher susceptibility of monocytes than lymphocytes to mitoxantrone-induced cell death. Interestingly, the culture of monocytes for 24 –48 h induced a high percentage of apoptotic cells, even in the absence of mitoxantrone as assessed by annexin-V conjugation (Fig. 3A) or by detection of DNA fragmentation using TdT (Fig. 3B), indicating an increased susceptibility of monocytes to any apoptogenic signals as compared to lymphocytes. The most prominent effects were observed at the highest dose we applied in vitro, 2000 ng/ml. In a 48 h culture in the presence of PHA, virtually all lymphocytes and monocytes had undergone necrotic cell death, although the ratio of annexin-V-negative and -positive cells is inversed (75 : 21 in lymphocytes vs. 25 : 75 in monocytes; Fig. 3A). In a 24 h culture without any mitogen, DNA fragmentation as measured by TdT had occurred in 11% of CD3+ T lymphocytes, and, to a higher extent, in 30% of CD20+ B lymphocytes (Fig. 3B). In monocytes, however, mitoxantrone at this highest dose fully disintegrated the cells, as indicated by complete loss of CD14 expression (Fig. 3B), which was not observed at the 10-fold lower dose of 200 ng/ml (Fig. 3B, Table 1). 3.5. Mitoxantrone inhibits maturation of dendritic cells Mitoxantrone promoted a dose dependent inhibitory effect on the expression of surface molecules characterizing mature DCs (major histocompatibility complex (MHC) class II, CD80, CD86). Although decreased surface expression of MHC class II, CD80, and CD86 was already seen at concentrations of 2 ng/ml, significant inhibition was observed at concentrations of 200 ng/ml and beyond (Fig. 4A). Even at low concentrations of 2 ng/ml mitoxantrone, a high proportion was already rendered apoptotic (5% annexin-V positive cells in untreated or immature DCs vs. 37% annexin-V positive cells at 2 ng/ml mitoxantrone). At the maximum dose of 2000 ng/ml, most of the DCs stained double positive for annexin-V and propidium iodide, reflecting a high proportion of necrotic cells (Fig. 4B).

4. Discussion In our study, mitoxantrone inhibited non-antigen-specific proliferation of activated peripheral blood lymphocytes (both T cells and B cells) as well as antigen-specific T-cell lines in a dose-dependent manner. The fact that no differences were

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observed between the effect of mitoxantrone on cells from IFN-h-treated patients, untreated MS patients or HD was surprising. It has been demonstrated by other groups that IFN-h has antiproliferative effect on numerous cell types,

and one would assume that there would be an additive effect with mitoxantrone. Our proliferation data would suggest two things: (i) add-on therapy of IFN-h to mitoxantrone, at least given in a higher dose as in our study, may not significantly

O. Neuhaus et al. / Journal of Neuroimmunology 168 (2005) 128 – 137

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Mitoxantrone (ng/ml) Fig. 4. Effects of mitoxantrone on dendritic cells (DCs). DCs were cultured as described in the text. (A) Surface expression of MHC class II, CD80 and CD86 was analyzed by flow cytometry. (B) Evaluation of cell death. Left histogram, % annexin-V positive DCs. Right histogram, % propidium iodide positive cells. As positive control, DCs were activated with phytohemagglutinin (PHA) in the presence of CD95L. Results are from one representative experiment (HD).

reduce the proliferation of encephalitogenic T cells and B cells in MS. (ii) In addition, the antiproliferative effects of mitoxantrone are not disease-specific. They may rather explain the beneficial clinical effects of mitoxantrone on a number of neoplastic and autoimmune disorders.

By contrast, analysis of significance revealed that mitoxantrone promoted differential inhibitory effects on the proliferation of leukocyte subgroups. The effect on nonantigen-specific mitogen-activated peripheral blood T cells and B cells was significantly diminished compared to that on

Fig. 3. Effects of mitoxantrone on cell death. (A) Peripheral blood mononuclear cells (PBMCs) were stimulated with phytohemagglutinin for 48 h, the last 18 h in the presence or absence of mitoxantrone or dexamethasone 1 AM as positive control. Apoptosis was measured using annexin-V-conjugates that bind to phosphatidylserine which is exposed on the cell surface of early apoptotic cells. Necrosis was measured by propidium iodide uptake. Results are from one representative experiment (HD). Upper left dot plot, gating of lymphoid and monocytoid cells. Other dot plots, binding of annexin-V vs. uptake of propidium iodide. The numbers represent % lymphoid/monocytoid cells. Note that dexamethasone induces apoptosis (the annexin-V+/propidium iodide+ cells are already in the late apoptotic state). (B) PBMCs, enriched B lymphocytes or monocytes were cultured for 24 h in the presence or absence of mitoxantrone. DNA fragmentation was measured by detection of incorporated biotinylated nucleotides into the DNA by terminal deoxynucleotidyl transferase (TdT), and plotted against CD3, CD20 and CD14, respectively.

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antigen-specific T cells (Fig. 1). This is an interesting observation that may explain why mitoxantrone has significant effects on MS exacerbation rate and disease progression, while no significant complications due to global immunosuppression have been observed in clinical trials (Hartung et al., 2002). Antigen-induced T-cell line proliferation was inhibited significantly at concentrations 10-fold lower than T-cell or B-cell proliferation (Fig. 2A); functional sub-analyses revealed that this effect was apparently assigned to the APCs (Fig. 2B). These findings suggest that mitoxantrone susceptibility is dependent either on the target cell type or the strength of the stimulus. Phenotypical analyses revealed only minor effects of doses of mitoxantrone that are already very effective in curtailing proliferation. In T and B cells, both surface expression of activation markers, intracellular expression of cytokines, as well as chemokine receptor and MMP expression on the RNA level remained virtually uninfluenced by mitoxantrone at concentrations up to 200 ng/ ml (Tables 1 and 2). It appears difficult to interpret how these findings relate to the observation that a most potent effect of mitoxantrone in MS patients is the virtual shut down of new magnetic resonance imaging (MRI) activity (Edan et al., 1997; Van de Wyngaert et al., 2001). It is speculated that effects on activation markers or MMPs other than the ones tested may reflect the impact of mitoxantrone on MRI activity. In contrast to T and B lymphocytes, partially marked effects were observed in DCs (Fig. 4A). These data further strengthen the suggestion of a selective susceptibility of specific leukocyte subgroups to the action of mitoxantrone. In T and B lymphocytes and in DCs, the mechanism of cell death induced by mitoxantrone appears to be apoptosis at lower concentrations and cell lysis at higher concentrations (Fig. 3, Fig. 4B). In contrast, in monocytes, necrosis was observed already at lower concentrations (Fig. 3, Table 1). Furthermore, the presence of high percentages of apoptotic monocytes in cell culture conditions even in the absence of mitoxantrone suggests the particular fragility of this leukocyte subtype. Previous pharmacokinetic studies in oncology revealed maximum serum concentrations of mitoxantrone between 308 and 839 ng/ml and terminal half-lives between 38.4 and 71.5 h at doses of 12 – 15 mg/m2 body surface (Canal et al., 1993; Hu et al., 1992; Repetto et al., 1999). Thus, in the first 3.9 to 5.4 half-lives or 8.6 to 11.8 days after infusion, maximum serum concentrations were shown to exceed 20 ng/ml, whereas the rest of the time post infusion (approximately 80 days at a 3-month dosage regime), concentrations were below 20 ng/ml. It remains unclear whether the major impact of mitoxantrone in MS depends on the short-time immunosuppressive effects by inhibition of immune cell proliferation (Fidler et al., 1986a,b) and induction of cell lysis both leading to leukocyte reduction in the blood post infusion (Fidler et al., 1986a). Our findings suggest that additional immunological effects of mitoxantrone may be executed at lower concentrations. Consistent

with this notion, the clinical effects of mitoxantrone in MS have been reported to last up to 1 year following discontinuation of therapy (Gonsette, 1996). At low concentrations, mitoxantrone presumably achieves its immunological effects by inducing apoptosis (Bhalla et al., 1993). Among the predominant target cells are B lymphocytes and DCs, i.e., cells that are involved in antigen presentation (Fidler et al., 1986a). Given the increasing recognition that DCs invade the inflamed brain parenchyma (Pashenkov et al., 2003), mitoxantrone may therefore interfere with (auto)antigen presentation and hence clonal activation of autoreactive T cells both in the periphery and in the CNS. Why APCs appear to be selectively susceptible to the actions of mitoxantrone remains elusive at present. An overall conclusion that could be drawn from our findings is that mitoxantrone acts as a cytotoxic agent targeting cell types with different susceptibilities and fragilities. In the proliferation assays (Figs. 1 and 2), the dose-dependent decrease in proliferation could be related to fewer cells incorporating [methyl-3H]-thymidine because of cell death. Phenotypical analyses (Fig. 3, Table 1) and evaluation of MMP and chemokine receptor RNA levels (Table 2) revealed only marginal effects of mitoxantrone which could be explained by the fact that these techniques use living cells and gate out dead cells. Thus, our observations may be the result of whether or not target cells die, implicating a mechanism of action of mitoxantrone based on cytotoxicity with selective target cell susceptibilities rather than on specific immunomodulatory properties. As indicated above, our data caution against the concurrent therapeutic use of mitoxantrone and IFN-h. Similarly, a possible combination therapy of mitoxantrone and GA is not supported by our findings: The propagation of anti-inflammatory GA-reactive T cells, which is one of the reported mechanisms of GA (Neuhaus et al., 2000), may be impaired by concurrent administration of mitoxantrone. In contrast, a sequential therapeutic approach with an initial regimen consisting of mitoxantrone followed by IFN-h or GA may present a reasonable option for patients who are forced to discontinue mitoxantrone as they reach the cumulative lifetime dose. Any such combination therapies should be carefully assessed in the setting of controlled clinical trials. In parallel to our in vitro studies of immunological effects of mitoxantrone, analyses of its ex vivo effects in MS patients have recently been communicated that suggest an impact on the disturbed TH1/TH2 cytokine balance (Gbadamosi et al., 2003). We failed to observe significant cytokine shifts in vitro. More recently, Chan and co-workers reported in an ex vivo study a predominant susceptibility of CD19 positive B lymphocytes to mitoxantrone-induced immediate cell death, corroborating our findings that this drug preferentially targets APCs (Chan et al., 2004). Mitoxantrone may target the immunopathological pathways culminating in neural tissue damage at several crucial checkpoints. Further evaluations of the mechanisms of action of mitoxantrone in MS are warranted.

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