BRIEF REPORT |
Correspondence to: Jens Schmidt, Neuromuscular Diseases Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Building 10, Room 4N 252, 10 Center Drive, MSC 1382, Bethesda, MD 20892. E-mail: schmidtj@ninds.nih.gov
![]() |
Summary |
---|
![]() ![]() ![]() ![]() |
---|
Multiple sclerosis (MS) relapses are treated with high-dose IV glucocorticosteroids. Here we investigated mechanisms of long-circulating polyethylene glycol-coated liposomes encapsulating prednisolone (PL) in adoptive transfer experimental autoimmune 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 nonfluorescent 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 51:12411244, 2003)
Key Words: glucocorticosteroids, drug targeting, long-circulating liposomes, multiple sclerosis, EAE, neuroinflammation, autoimmunity, immunohistochemical double, labeling
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() |
---|
MULTIPLE SCLEROSIS (MS) is a common autoimmune disorder of the central nervous system, with T-cell infiltration and demyelination as pathological hallmarks (
At lower concentrations, GS effects are mainly mediated by the classical GS receptor. Only at ultra-high tissue concentrations are alternative nongenomic mechanisms of action "activated," which explains the superior efficacy of high and ultra-high doses in the treatment of some autoimmune disorders (
In these experiments we investigated in situ effects of PL treatment in EAE using an immunohistochemical (IHC) double labeling method. We show that a single IV injection of 10 mg/kg PL reduces the percentage of TNF--positive T-cells in the lesion as part of the downregulation of the inflammatory response. Our results help to understand therapeutic effects of liposomal GS, and ultimately may have implications for treatment of autoimmune disorders such as MS.
Female Lewis rats (Charles River; Sulzfeld, Germany) were 68 weeks old. All culture media and supplements were obtained from Gibco BRL (Eggenstein, Germany). Encephalitogenic T-cells for in vivo experiments were generated and maintained as previously described (
Liposomes were prepared by the film-extrusion method (4.5 mg prednisolone phosphate and an average of 60 µmol phospholipid.
For therapeutic studies we used prednisolone PEG liposomes (PL). The treatment regimen for AT-EAE essentially followed the protocol used in previous studies (
Five-µm cross-sections of spinal cord were deparaffinized and rehydrated. After blocking of non-specific binding with 10% BSA in 0.05 M Tris-buffered saline (0.15 M sodium, TBS) for 30 min, sections were incubated with a polyclonal rabbit anti-TNF antibody (Serotec, via Biozol; München, Germany) at a dilution of 1:100 in TBS with 1% BSA, incubated overnight at 4C. The specificity was proved by preadsorption of the primary antibody with rat TNF-
. Except after BSA blocking, all other steps were followed by washing with TBS. The primary antibody was detected with a biotinylated goat anti-rabbit IgG antibody (Vector; Wertheim, Germany), which was preadsorbed 1:1 with normal rat serum, diluted 1:50 in TBS with 1% BSA and incubated for 45 min. An alkaline phosphatase-bound avidinbiotin complex (Dako; Hamburg, Germany) was applied for 30 min, followed by Vector red (Vector) as chromogenic substrate for 710 min. After blocking of all excess avidinbiotin binding sites with an AB blocking kit (Vector), the next primary antibodies were applied. T-cells were detected with a mouse monoclonal antibody to a pan-T-cell antigen (B 115-1, dilution 1:500; from HyCult Biotechnology via Sanbio, Beutelsbach, Germany), incubated for 1 hr at room temperature. Endogenous peroxidase activity was blocked with 3% H2O2 and 0.2 M sodium azide in methanol. The primary antibody was detected with a biotinylated goat anti-mouse IgG antibody preabsorbed 1:1:1 with sera from rabbit and rat at 37C for 15 min, diluted 1:200 in TBS with 1% BSA, and incubated for 45 min. A horseradish-peroxidase-bound avidinbiotin complex (Dako) was applied for 30 min, followed by 3,3'-diaminobenzidine-tetrahydrochloride-nickel (DAB-Ni, black; Vector) as chromogenic substrate for 34 min. For each staining we added three control sections by omitting either one or both of the primary antibodies. All sections were dehydrated and mounted in Vitro-clud (R. Langenbrinck; Emmendingen, Germany). In one lumbar (intumescentia lumbalis) spinal cord cross-section, an observer blinded to the respective treatment analyzed 10 fields of a 10 x 10 square grid at a x400 enlargement. The localization of the 10 fields followed a standardized graphic pattern that was applicable to all sections and that yielded equal areas of gray and white matter. Data for TNF-
-positive T-cells were expressed as the ratio of double-labeled T-cells and the total number of T-cells in percent. Statistical analysis of the data was performed by Student's t-test, considering p<0.05 and p<0.01 as significant p-values.
Groups of five female Lewis rats received one IV injection of 10 mg/kg PL at 6 hr, 18 hr, or 42 hr before sacrifice at the peak of the disease course of AT-EAE on day 5. TNF- double-labeled T-cells were detected in spinal cord by a nonfluorescent IHC double labeling technique (Fig 1). At 18 hr after the injection of 10 mg/kg PL, the rate of TNF-
-producing T-cells was clearly reduced compared to controls (Fig 2). Furthermore, PL at 42 hr significantly decreased the rate of TNF-
-producing T-cells. PL at 6 hr had no effect, which was in accord with previous findings (
-positive T cells. Experiments were reproduced at least once with similar results.
|
|
|
The therapeutic goal in treatment of MS relapses is reduction of cellular inflammation as efficiently as 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 regard to our previous findings in EAE (-positive T-cells. Two injections of PL proved superior to two injections of a free GS at a fivefold-higher dose with regard to clinical and in situ effects.
TNF-, mainly secreted by T-cells and macrophages, is one of the most critical cytokines in the process of demyelination in the course of MS (reviewed in
can also exert antiinflammatory properties (
-antibodies in therapeutic studies of the heterogenous disease MS (
-deficient mouse model (
has been shown to have neuroprotective properties by promoting oligodendrocyte progenitors and remyelination (
is further supported by a recent study in EAE in which distinct TNF-
signaling pathways were operative either in the beneficial reduction of autoreactive T-cells or in detrimental effects during the acute phase of the disease (
The aim of the present study was to further elucidate the in situ mechanisms of action of a single IV PL injection on the level of specific immune cells. Other techniques such as RT-PCR, Western blotting, and ELISA in homogenized spinal cord lack specificity at single-cell level. Second, these techniques cannot always be performed in formalin-fixed tissue of perfused animals, which is necessary to provide high-quality sections to reliably quantify mechanisms of action on immune cells in situ. The simple but efficient double labeling technique described here enables analysis of cytokine production of specific immune cells and, in contrast to fluorescent double labeling, allows morphological evaluation at the same time. In addition, the stable chromogens facilitate analysis for a long time, which is not possible with fading fluorescent labels. In situ hybridization is a more sensitive technique which, however, is clearly more cost- and labor-intensive and less feasible for quantification of experiments with large numbers of tissue specimens. Second, the latter technique lacks analysis at the protein level, which we assessed here.
Taken together, our experiments using a nonfluorescent immunohistochemical double labeling technique demonstrate that a single injection of PL downregulates the rate of TNF--producing T-cells in spinal cord of EAE rats. Analyses of the production of TNF-
in situ during the demyelination vs the remyelination phase maybe useful for a better understanding of experimental treatment strategies for MS and other neuroinflammatory diseases.
![]() |
Footnotes |
---|
1 Present address: National Institutes of Health, Bethesda, MD.
![]() |
Acknowledgments |
---|
Supported by funds from the state of Bavaria, Germany.
The invaluable technical assistance of Gabriele Köllner and Helga Brünner is gratefully acknowledged. We thank Louis van Bloois for his help with preparing the liposomes.
Received for publication March 3, 2003; accepted May 22, 2003.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() |
---|
Arnett HA, Mason J, Marino M, Suzuki K, Matsushima GK, Ting JP (2001) TNF alpha promotes proliferation of oligodendrocyte progenitors and remyelination. Nature Neurosci 4:1116-1122[Medline]
Brusaferri F, Candelise L (2000) Steroids for multiple sclerosis and optic neuritis: a meta-analysis of randomized controlled clinical trials. J Neurol 247:435-442[Medline]
Gold R, Buttgereit F, Toyka KV (2001) Mechanism of action of glucocorticosteroid hormones: possible implications for therapy of neuroimmunological disorders. J Neuroimmunol 117:1-8[Medline]
Kassiotis G, Kollias G (2001) Uncoupling the proinflammatory from the immunosuppressive properties of tumor necrosis factor (TNF) at the p55 TNF receptor level: implications for pathogenesis and therapy of autoimmune demyelination. J Exp Med 193:427-434
Liu J, Marino MW, Wong G, Grail D, Dunn A, Bettadapura J, Slavin AJ et al. (1998) TNF is a potent anti-inflammatory cytokine in autoimmune-mediated demyelination. Nat Med 4:78-83[Medline]
Metselaar JM, Wauben MHM, Wagenaar-Hilbers JPA, Boerman OC, Storm G (in press) Joint targeting of glucocorticoids with long-circulating liposomes induces complete remission of experimental arthritis. Arthritis Rheum
Noseworthy JH, Lucchinetti C, Rodriguez M, Weinshenker BG (2000) Multiple sclerosis. N Engl J Med 343:938-952
Oliveri RL, Valentino P, Russo C, Sibilia G, Aguglia U, Bono F, Fera F et al. (1998) Randomized trial comparing two different high doses of methylprednisolone in MS: a clinical and MRI study. Neurology 50:1833-1836[Abstract]
Schmidt J, Gold R, Schonrock L, Zettl UK, Hartung HP, Toyka KV (2000) T-cell apoptosis in situ in experimental autoimmune encephalomyelitis following methylprednisolone pulse therapy. Brain 123:1431-1441
Schmidt J, Metselaar JM, Wauben MHM, Toyka KV, Storm G, Gold R (in press) Drug targeting by long-circulating liposomal glucocorticosteroids increases therapeutic efficacy in a model of multiple sclerosis. Brain
Steinman L, Martin R, Bernard C, Conlon P, Oksenberg JR (2002) Multiple sclerosis: deeper understanding of its pathogenesis reveals new targets for therapy. Annu Rev Neurosci 25:491-505[Medline]
TNF neutralization in MS: results of a randomized, placebo-controlled multicenter study. (1999) Neurology 53:457-465