6031-03b

Journal of Histochemistry & Cytochemistry Production by T-cells in
Intravenous Liposomal Prednisolone Downregulates In Situ TNF-
Experimental Autoimmune Encephalomyelitis
Jens Schmidt, Josbert M. Metselaar and Ralf Gold can be found at:
Journal of Histochemistry & Cytochemistry
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Volume 51(9): 1241–1244, 2003
The Journal of Histochemistry & Cytochemistry
B R I E F R E P O R T
Intravenous Liposomal Prednisolone Downregulates In Situ
TNF-
Production by T-cells in Experimental
Autoimmune Encephalomyelitis

Jens Schmidt,1 Josbert M. Metselaar, and Ralf Gold Department of Neurology, University of Würzburg, Würzburg, Germany (JS,RG); and Department of Pharmaceutics, Utrecht University, Utrecht, The Netherlands (JMM) Multiple sclerosis (MS) relapses are treated with high-dose IV glucocortico- steroids. Here we investigated mechanisms of long-circulating polyethylene glycol-coatedliposomes encapsulating prednisolone (PL) in adoptive transfer experimental autoimmune K E Y W O R D S
encephalomyelitis. Rats received IV 10 mg/kg PL 6, 18, or 42 hr before sacrifice at disease maximum. In formalin-fixed, paraffin-embedded spinal cord we employed a nonfluores- cent immunohistochemical (IHC) double labeling. We stained for tumor necrosis factor-␣ (TNF-␣) in combination with a T-cell antigen. Compared with PBS-containing liposomes, PL at 18 hr, and more at 42 hr, significantly reduced the rate of TNF-␣ double-labeled T-cells.
This correlated with an ameliorated disease score at day 5 after PL 42 hr. Our results help to further understand mechanisms of action of drug targeting by liposomal steroids, with possible implications for treatment of autoimmune disorders such as MS.
(J Histochem Cytochem 51:1241–1244, 2003)
Multiple sclerosis (MS) is a common autoimmune
ing polyethylene glycol (PEG)-coated liposomes en- disorder of the central nervous system, with T-cell in- capsulating prednisolone (prednisolone liposomes, PL) filtration and demyelination as pathological hallmarks for drug delivery of GS. This principle of drug target- (Noseworthy et al. 2000). The main goal of therapeu- ing improved therapeutic efficacy in a rat model of ar- tic strategies is prevention of ongoing tissue destruc- thritis (Metselaar et al. in press). In experimental au- tion with subsequent permanent functional deficits.
toimmune encephalomyelitis (EAE), a dose of IV Relapses are treated by very high-dose IV glucocorti- 10 mg/kg PL achieved much higher and prolonged tis- costeroids (GS) (pulse therapy). The common regimen sue levels of the GS and was superior to a fivefold in MS involves IV therapy with 10 mg/kg methylpred- higher dose of free GS with regard to reduced cellular nisolone or prednisolone for 3–5 days (reviewed in inflammation and clinical benefit (Schmidt et al. in Brusaferri and Candelise 2000). Clinical (Oliveri et al.
1998) and experimental (Schmidt et al. 2000) data At lower concentrations, GS effects are mainly demonstrated that an ultra-high dose may be superior mediated by the classical GS receptor. Only at ultra- to the “standard” high dose of 10 mg/kg GS. Re- high tissue concentrations are alternative nongenomic cently, we used a novel formulation of long-circulat- mechanisms of action “activated,” which explains thesuperior efficacy of high and ultra-high doses in thetreatment of some autoimmune disorders (Gold et al.
2001).
Correspondence to: Jens Schmidt, MD, Neuromuscular Diseases In these experiments we investigated in situ effects Section, National Institute of Neurological Disorders and Stroke,National Institutes of Health, Building 10, Room 4N 252, 10 Cen- of PL treatment in EAE using an immunohistochemi- ter Drive, MSC 1382, Bethesda, MD 20892. E-mail: [email protected] cal (IHC) double labeling method. We show that a single IV injection of 10 mg/kg PL reduces the percent- Received for publication March 3, 2003; accepted May 22, age of TNF-␣-positive T-cells in the lesion as part of 1Present address: National Institutes of Health, Bethesda, MD.
the downregulation of the inflammatory response.
Our results help to understand therapeutic effects of IV, which showed no difference compared to saline in- liposomal GS, and ultimately may have implications jections in previous experiments. For tissue prepara- for treatment of autoimmune disorders such as MS.
tion, anesthetized animals were sacrificed and per- Female Lewis rats (Charles River; Sulzfeld, Ger- fused through the left ventricle with HAES-steril 6% many) were 6–8 weeks old. All culture media and sup- (Fresenius; Bad Homburg, Germany), followed by plements were obtained from Gibco BRL (Eggenstein, paraformaldehyde 4% in 0.1 M phosphate buffer.
Germany). Encephalitogenic T-cells for in vivo experi- The spinal cord was removed, postfixed, dehydrated, ments were generated and maintained as previously described (Schmidt et al. 2000). Briefly, primed T-cells Five-␮m cross-sections of spinal cord were deparaf- (3 ϫ 105/ml) were restimulated with guinea pig myelin finized and rehydrated. After blocking of non-specific basic protein (MBP, 20 ␮g/ml) in RPMI 1640 supple- binding with 10% BSA in 0.05 M Tris-buffered saline mented with 1% normal rat serum, 100 U/ml penicil- (0.15 M sodium, TBS) for 30 min, sections were incu- lin, 100 ␮g/ml streptomycin, and 2 mM glutamine, bated with a polyclonal rabbit anti-TNF␣ antibody using freshly isolated and irradiated (3000 rad) thy- (Serotec, via Biozol; München, Germany) at a dilution mocytes (1.5 ϫ 107/ml) as antigen-presenting cells.
of 1:100 in TBS with 1% BSA, incubated overnight at Adoptive transfer (AT)-EAE was induced by IV injec- 4C. The specificity was proved by preadsorption of tion of 10–12 ϫ 106 freshly activated MBP-specific the primary antibody with rat TNF-␣. Except after T-cells in the tail vein. Animals were inspected daily by BSA blocking, all other steps were followed by wash- an observer masked to the respective treatment, using ing with TBS. The primary antibody was detected a 6 grade score: 0, healthy; 1, weight loss, limp tip of with a biotinylated goat anti-rabbit IgG antibody tail; 2, limp tail, mild paresis; 3, moderate paraparesis, (Vector; Wertheim, Germany), which was pread- ataxia; 4, tetraparesis; 5, moribund; 6, dead (Schmidt sorbed 1:1 with normal rat serum, diluted 1:50 in TBS et al. 2000). Disease onset in all animals started at day with 1% BSA and incubated for 45 min. An alkaline 2 and was maximal at day 5. Bavarian state authori- phosphatase-bound avidin–biotin complex (Dako; Hamburg, Germany) was applied for 30 min, followed Liposomes were prepared by the film-extrusion by Vector red (Vector) as chromogenic substrate for method (Metselaar et al. in press). Briefly, a lipid solu- 7–10 min. After blocking of all excess avidin–biotin tion was prepared in ethanol containing dipalmitoyl binding sites with an AB blocking kit (Vector), the phosphatidylcholine (DPPC; Lipoid GmbH, Ludwig- next primary antibodies were applied. T-cells were de- shafen, Germany), PEG 2000–distearyl phosphatidyl- tected with a mouse monoclonal antibody to a pan- ethanolamine (PEG–DSPE), and cholesterol (Sigma Chemi- T-cell antigen (B 115-1, dilution 1:500; from HyCult cal; Poole, UK) in a molar ratio of 1.85:0.15:1.0. A Biotechnology via Sanbio, Beutelsbach, Germany), in- lipid film was created by rotary evaporation. The film cubated for 1 hr at room temperature. Endogenous was hydrated with a solution of 100 mg/ml predniso- peroxidase activity was blocked with 3% H2O2 and lone phosphate (Bufa; Uitgeest, The Netherlands) in 0.2 M sodium azide in methanol. The primary anti- sterile water. The resulting lipid dispersion was sized body was detected with a biotinylated goat anti- by multiple extrusions through polycarbonate filter mouse IgG antibody preabsorbed 1:1:1 with sera from membranes to a diameter of 90–100 nm. Mean parti- rabbit and rat at 37C for 15 min, diluted 1:200 in TBS cle size was determined by dynamic light scattering with 1% BSA, and incubated for 45 min. A horserad- with a Malvern 4700 system (Malvern; Malvern, UK).
ish-peroxidase-bound avidin–biotin complex (Dako) Phospholipid content was determined with a phos- was applied for 30 min, followed by 3,3Ј-diaminoben- phate assay (Metselaar et al. in press) and pred- zidine-tetrahydrochloride-nickel (DAB-Ni, black; Vec- nisolone phosphate concentration by reversed-phase tor) as chromogenic substrate for 3–4 min. For each HPLC. Each 1 ml of liposomal preparation contained staining we added three control sections by omitting ف4.5 mg prednisolone phosphate and an average of either one or both of the primary antibodies. All sec- tions were dehydrated and mounted in Vitro-clud (R.
For therapeutic studies we used prednisolone PEG Langenbrinck; Emmendingen, Germany). In one lum- liposomes (PL). The treatment regimen for AT-EAE bar (intumescentia lumbalis) spinal cord cross-section, essentially followed the protocol used in previous an observer blinded to the respective treatment ana- studies (Schmidt et al. 2000; Schmidt et al. in press).
lyzed 10 fields of a 10 ϫ 10 square grid at a ϫ400 en- All experiments were performed in groups of five ani- largement. The localization of the 10 fields followed a mals each and were reproduced at least once, some in- standardized graphic pattern that was applicable to all jection time points even three times. Under general an- sections and that yielded equal areas of gray and white esthesia, 10 mg/kg body weight PL was injected into a matter. Data for TNF-␣-positive T-cells were ex- tail vein at 6, 18, or 42 hr before sacrifice at day 5.
pressed as the ratio of double-labeled T-cells and the Negative controls received PBS-containing liposomes total number of T-cells in percent. Statistical analysis Liposomal Prednisolone Reduces TNF- in T-cells Treatment of AT-EAE with a single IV injection of 10 mg/ kg PL at indicated time points before sacrifice at day 5, comparedto PBS–liposomes (same experiment as in Table 1). Quantification IHC double labeling for detection of TNF-␣ (visualized by of IHC double labeling for TNF-␣ and T-cell markers in one lumbar Vector red) in combination with a T-cell marker antigen (visualized spinal cord cross-section, analyzed as detailed in the text. Percent- by DAB-Ni, black) in a 5-␮m formalin-fixed, paraffin-embedded age of TNF-␣-positive T-cells is given as mean Ϯ SD. Each symbol cross-section of spinal cord from a control group AT-EAE rat at day represents data from one rat (nϭ5 per group); data were repro- 5. Solid arrows indicate double labeled TNF-␣-producing T-cells.
duced at least once with similar results. *pϽ0.05 for PL 18 hr vs Open arrow indicates a TNF-␣-negative T-cell. Arrowhead shows a controls; **pϽ0.01 for PL 42 hr vs controls.
TNF-␣-producing cell, which is not a T-cell. Bar ϭ 10 ␮m.
described a novel formulation of liposomal steroids to of the data was performed by Student’s t-test, consid- deliver ultra-high steroid doses by drug targeting with ering pϽ0.05 and pϽ0.01 as significant p-values.
fewer systemic doses of the free GS in treatment of Groups of five female Lewis rats received one IV in- EAE (Schmidt et al. in press). In these experiments we jection of 10 mg/kg PL at 6 hr, 18 hr, or 42 hr before observed high steroid tissue concentrations soon after sacrifice at the peak of the disease course of AT-EAE injection and accumulation of the liposomes in the in- on day 5. TNF-␣ double-labeled T-cells were detected flamed CNS. In contrast to the short-lived actions of in spinal cord by a nonfluorescent IHC double label- free GS, the effects of PL were clearly prolonged, and ing technique (Figure 1). At 18 hr after the injection of induction of T-cell apoptosis and reduction of T-cell 10 mg/kg PL, the rate of TNF-␣-producing T-cells was and macrophage infiltration in the CNS peaked at clearly reduced compared to controls (Figure 2). Fur- 42 hr after treatment. At this time point, a high frac- thermore, PL at 42 hr significantly decreased the rate tion of PL has been degraded after phagocytic uptake of TNF-␣-producing T-cells. PL at 6 hr had no effect, and by extracellular proteases, leading to ultra-high which was in accord with previous findings (Schmidt tissue levels of the active drug prednisolone. We ob- et al. in press). In contrast to all other groups, the served tissue levels greater than 10 moles/liter for up peak of the disease was significantly ameliorated after to 18 hr after PL, which is well within the range of PL at 42 hr (Table 1), which correlated with the re- nongenomic steroid actions and may have been opera- duced percentage of TNF-␣-positive T cells. Experi- tive in the reduction of the percentage of TNF-␣-posi- ments were reproduced at least once with similar re-sults.
Treatment of AT-EAE with a single IV injection of The therapeutic goal in treatment of MS relapses is 10 mg/kg PL at indicated time points before sacrifice at day 5, reduction of cellular inflammation as efficiently as compared to PBS–liposomes (same experiment as in Figure 2)a possible to prevent ongoing tissue destruction and axon loss. The dosing of GS as mainstay of therapy in MS relapses is a continuous matter of debate. With re- gard to our previous findings in EAE (Schmidt et al.
2000), one of the major issues of steroids dosing is to reach ultra-high tissue levels, exerting multiple path- aClinical score is given as mean Ϯ SD (nϭ5 per group). Each individual time ways of steroid action according to a new model of point was reproduced at least once with similar results in five independentexperiments.
steroid mechanisms (Gold et al. 2001). Recently we bpϽ0.05 for PL 42 hr vs all other groups.
tive T-cells. Two injections of PL proved superior to Taken together, our experiments using a nonfluo- two injections of a free GS at a fivefold-higher dose rescent immunohistochemical double labeling tech- with regard to clinical and in situ effects.
nique demonstrate that a single injection of PL down- TNF-␣, mainly secreted by T-cells and macro- regulates the rate of TNF-␣-producing T-cells in phages, is one of the most critical cytokines in the pro- spinal cord of EAE rats. Analyses of the production of cess of demyelination in the course of MS (reviewed in TNF-␣ in situ during the demyelination vs the remy- Steinman et al. 2002) and EAE (overview in Kassiotis elination phase maybe useful for a better understand- and Kollias 2001). Its proinflammatory actions have ing of experimental treatment strategies for MS and long been established, but during the disease course TNF-␣ can also exert antiinflammatory properties(Steinman et al. 2002), which may explain the failure of neutralizing TNF-␣-antibodies in therapeutic stud- Supported by funds from the state of Bavaria, Germany.
ies of the heterogenous disease MS (The Lenercept The invaluable technical assistance of Gabriele Köllner Study Group 1999), and the worsening of EAE in a and Helga Brünner is gratefully acknowledged. We thank TNF-␣-deficient mouse model (Liu et al. 1998). More- Louis van Bloois for his help with preparing the liposomes.
over, TNF-␣ has been shown to have neuroprotectiveproperties by promoting oligodendrocyte progenitorsand remyelination (Arnett et al. 2001). The model of a Literature Cited
dualistic role for TNF-␣ is further supported by a re- Arnett HA, Mason J, Marino M, Suzuki K, Matsushima GK, Ting cent study in EAE in which distinct TNF-␣ signaling JP (2001) TNF alpha promotes proliferation of oligodendrocyteprogenitors and remyelination. Nature Neurosci 4:1116–1122 pathways were operative either in the beneficial reduc- Brusaferri F, Candelise L (2000) Steroids for multiple sclerosis and tion of autoreactive T-cells or in detrimental effects optic neuritis: a meta-analysis of randomized controlled clinical during the acute phase of the disease (Kassiotis and Kollias 2001). Our AT-EAE treatment with PL is only Gold R, Buttgereit F, Toyka KV (2001) Mechanism of action of glu- cocorticosteroid hormones: possible implications for therapy of a model to investigate rather short-lived therapeutic neuroimmunological disorders. J Neuroimmunol 117:1–8 effects during the acute phase of CNS inflammation.
Kassiotis G, Kollias G (2001) Uncoupling the proinflammatory However, we show how liposomal GS may interfere from the immunosuppressive properties of tumor necrosis factor with the complex cytokine network, finally exerting a (TNF) at the p55 TNF receptor level: implications for pathogen-esis and therapy of autoimmune demyelination. J Exp Med beneficial therapeutic effect in our model.
The aim of the present study was to further eluci- Liu J, Marino MW, Wong G, Grail D, Dunn A, Bettadapura J, date the in situ mechanisms of action of a single IV PL Slavin AJ, et al. (1998) TNF is a potent anti-inflammatory cyto-kine in autoimmune-mediated demyelination. Nat Med 4:78–83 injection on the level of specific immune cells. Other Metselaar JM, Wauben MHM, Wagenaar-Hilbers JPA, Boerman techniques such as RT-PCR, Western blotting, and OC, Storm G (in press) Joint targeting of glucocorticoids with ELISA in homogenized spinal cord lack specificity at long-circulating liposomes induces complete remission of experi- single-cell level. Second, these techniques cannot al- Noseworthy JH, Lucchinetti C, Rodriguez M, Weinshenker BG ways be performed in formalin-fixed tissue of per- (2000) Multiple sclerosis. N Engl J Med 343:938–952 fused animals, which is necessary to provide high- Oliveri RL, Valentino P, Russo C, Sibilia G, Aguglia U, Bono F, quality sections to reliably quantify mechanisms of Fera F, et al. (1998) Randomized trial comparing two differenthigh doses of methylprednisolone in MS: a clinical and MRI action on immune cells in situ. The simple but efficient double labeling technique described here enables anal- Schmidt J, Gold R, Schonrock L, Zettl UK, Hartung HP, Toyka KV ysis of cytokine production of specific immune cells (2000) T-cell apoptosis in situ in experimental autoimmune en- and, in contrast to fluorescent double labeling, allows cephalomyelitis following methylprednisolone pulse therapy.
Brain 123:1431–1441 morphological evaluation at the same time. In addi- Schmidt J, Metselaar JM, Wauben MHM, Toyka KV, Storm G, tion, the stable chromogens facilitate analysis for a Gold R (in press) Drug targeting by long-circulating liposomal long time, which is not possible with fading fluores- glucocorticosteroids increases therapeutic efficacy in a model of cent labels. In situ hybridization is a more sensitive Steinman L, Martin R, Bernard C, Conlon P, Oksenberg JR (2002) technique which, however, is clearly more cost- and Multiple sclerosis: deeper understanding of its pathogenesis re- labor-intensive and less feasible for quantification of veals new targets for therapy. Annu Rev Neurosci 25:491–505 experiments with large numbers of tissue specimens.
The Lenercept Multiple Sclerosis Study Group and The University of British Columbia MS/MRI Analysis Group (1999) TNF neu- Second, the latter technique lacks analysis at the pro- tralization in MS: results of a randomized, placebo-controlled multicenter study. Neurology 53:457–465 Volume 51(10): 1391, 2003
The Journal of Histochemistry & Cytochemistry
Intravenous Liposomal Prednisolone Downregulates In Situ TNF-␣ Production by T-cells in Experimental Au-toimmune Encephalomyelitis. Jens Schmidt, Josbert M. Metselaar, Ralf Gold (in J Histochem Cytochem 51(9):1241–1244, 2003).
Figure 2, as it appeared on page 1243, contained an error of omission in the labeling of the x-axis.

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