Prednisolone decreases cellular adhesion molecules required for inflammatory cell infiltration in dystrophin-deficient skeletal muscle

Neuromuscular Disorders 14 (2004) 483–490 www.elsevier.com/locate/nmd

Prednisolone decreases cellular adhesion molecules required for inflammatory cell infiltration in dystrophin-deficient skeletal muscle Michelle Wehling-Henricksa, James J. Leec, James G. Tidballa,b,* a

Department of Physiological Science, University of California, 5833 Life Science Building, Los Angeles, CA 90095-1527, USA b Department of Pathology and Laboratory Medicine, University of California, Los Angeles, CA, USA c Department of Biochemistry and Molecular Biology, Mayo Clinic Scottsdale, Scottsdale, AZ, USA Received 2 February 2004; received in revised form 1 April 2004; accepted 28 April 2004

Abstract The mechanism of prednisolone’s efficacy in the dystrophic pathology is unclear. Prednisolone’s anti-inflammatory functions may be particularly important considering the significance of inflammatory cells in dystrophinopathy. In other pathologies, prednisolone’s antiinflammatory effects can be mediated by reducing cellular adhesion molecule (CAM) expression. The goal of this study was to examine the effects of prednisolone on inflammation and CAM expression in dystrophic muscle. Dystrophin-deficient, mdx mice were treated with 0.75 mg/kg prednisolone from 2 to 4 weeks of age. Prednisolone reduced macrophages (2 59%, 257%), CD4þ T-cells (250%, 2 60%), CD8þ T-cells (2 58%, 2 48%), and eosinophils (2 36%, 225%) in quadriceps and soleus muscles, respectively. Prednisolone-treated mice also exhibited decreased vascular P-selectin (282%) and ICAM-1 (252%) expression and fewer L-selectin (279%) and ICAM-1 (257%) expressing mononuclear cells in quadriceps. Prednisolone reduced sarcolemmal damage and degeneration as well. Our data show that prednisolone is an effective anti-inflammatory in dystrophic muscle and may function by modulating CAM expression. q 2004 Elsevier B.V. All rights reserved. Keywords: mdx; Mouse; Muscular dystrophy; Prednisolone; Inflammation

1. Introduction The glucocorticoid prednisone, or its active form prednisolone (both of which will be referred to as prednisolone), is commonly prescribed to Duchenne muscular dystrophy (DMD) patients because of its efficacy in maintaining muscle strength and function and slowing the disease process [1 –3]. Unfortunately, prednisolone treatment is often accompanied by undesirable side effects including excessive weight gain, growth retardation, and cushingoid appearance. In some cases, patients choose to discontinue prednisolone therapy because the consequences of such side effects are perceived to outweigh the benefits of treatment. Despite the routine use of prednisolone in the treatment of DMD, the specific manner in which it functions is not well defined. If the mechanisms of prednisolone * Corresponding author. Address: Department of Physiological Science, University of California, 5833 Life Science Building, Los Angeles, CA 90095-1527, USA. Tel.: þ1-310-206-3395; fax: þ 1-310-825-8489. E-mail address: [email protected] (J.G. Tidball). 0960-8966/$ - see front matter q 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2004.04.008

function were better understood, alternative pharmaceutical treatment, with fewer negative side effects, could feasibly be developed. Previous studies suggest a variety of mechanisms through which prednisolone could mediate improvements in DMD patients. Daily prednisolone treatment induced decreases in 3-methylhistidine excretion and increases in creatinine excretion in DMD patients suggesting that the drug may function by inhibiting muscle proteolysis [4,5]. These data are supported by findings showing that prednisolone can inhibit protease activity in vitro [6]. However, a broad analysis of skeletal muscle protease activity and structural proteins in normal and degenerating conditions indicate that the prednisolone-induced decrease in proteolytic activity is not sufficient to account for the observed benefits in DMD patients [7]. Prednisolone may also function by improving calcium handling and reducing the basal cytosolic calcium concentration as shown in C2C12 muscle cells [8]. Reducing cytosolic calcium levels could prevent the activation of calcium-dependent proteases observed in dystrophin-deficient muscle [9].

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Additionally, prednisolone has been shown to enhance myogenesis [10] and prevent apoptosis [11] in cultures derived from normal and dystrophic human and mouse skeletal muscle. It is suggested that these observations may indicate improved repair and regeneration in dystrophic muscle following prednisolone treatment. These data are in contrast with findings showing that prednisolone actually decreases the cell titer in normal and dystrophic human muscle cell cultures but increases utrophin expression via post-translational mechanisms [12]. Prednisolone may also reduce the pathology of dystrophin-deficient muscle through its anti-inflammatory and immunosuppressive activity. Prednisolone has strong antiinflammatory effects in a broad range of inflammatory pathologies including multiple sclerosis [13], allergic inflammation [14], polymyositis, dermatomyositis [15,16], and even DMD [17]. These anti-inflammatory functions of prednisolone may be important in its therapeutic function because depletion of T-lymphocyte or macrophage populations from dystrophin-deficient mdx mice improves muscle histology and, most importantly, lessens sarcolemmal damage, which could prevent down-stream pathological events [18,19]. Because prednisolone can reduce macrophage and T-cell activation [20,21], its function in reducing dystrophinopathy may involve reducing damage caused by those cells. The anti-inflammatory effects of prednisolone may be mediated by down-regulation of cellular adhesion molecules (CAMs) that are required for inflammatory cell invasion. CAMs are cell surface molecules expressed on vascular endothelium and leukocytes. The interaction of ligand –receptor pairs is necessary for inflammatory cell diapedesis and in pathological conditions; up-regulation of CAMs such as the selectins and ICAM-1 enhances inflammatory cell infiltration. The selectins are a family of heterogeneous proteins involved in leukocyte tethering and rolling, and ICAM-1 is an immunoglobin superfamily protein that is crucial for firm adhesion of leukocytes to vascular endothelium. Pharmacologically decreasing the expression of CAMs provides a potential strategy to modulate an inflammatory response. In vivo studies show that prednisolone therapy reduces expression of CAMs on blood mononuclear cells from MS and allergic asthmatic patients [14,22] and on vascular endothelium in patients with polymyositis and dermatomyositis [15,16]. These findings are supported by in vitro studies, which show that prednisolone directly decreases CAM expression on human umbilical vein endothelial cells and human brain microvessel endothelial cells after stimulation with pro-inflammatory cytokines [23 –26]. Based on these findings and the observation that CAMs are up-regulated in DMD muscle [27], we hypothesize that a beneficial effect of prednisolone in DMD is anti-inflammatory-based and mediated, at least in part, by a reduction in the expression of CAMs. The goal of this study is to test the anti-inflammatory effect of prednisolone and determine if prednisolone

treatment affects the expression of CAMs in dystrophindeficient skeletal muscle. To investigate the hypothesis, we administered prednisolone to mdx mice and immunohistochemically analyzed the effect of treatment upon inflammatory cell populations (CD4þ T-cells, CD8þ T-cells, macrophages, and eosinophils) and adhesion molecules (L-selectin, P-selectin, E-selectin, and ICAM-1) in the skeletal muscle. We also evaluated the effect of prednisolone on membrane integrity and central-nucleation, which provide indices of muscle degeneration and regeneration.

2. Materials and methods 2.1. Animals Mdx mice were obtained from our breeding colony in the UCLA vivarium where they were housed during the experimental treatment. Original breeding pairs were purchased from Jackson Labs, Bar Harbor, ME. All animal procedures were in accordance with guidelines set forth by the UCLA Animal Research Committee. Following treatment, animals were sacrificed at 4 weeks of age. Tissues were immediately frozen in liquid nitrogen-cooled isopentane and subsequently stored in isopentane-filled vials at 2 80 8C. 2.2. Prednisolone treatment Male and female mice were randomly divided into experimental and control groups at 2 weeks of age. Prednisolone-treated animals received 0.75 mg/kg per day of prednisolone 21-hemisuccinate sodium salt (Sigma, St Louis, MO) in sterile phosphate-buffered saline (PBS) via intraperitoneal injection 5 times per week from 2 weeks until 4 weeks of age. Animals were weighed and dosage was adjusted prior to each injection. Control animals received intraperitoneal injections of equal volumes of sterile PBS only. 2.3. Antibodies Monoclonal antibodies that were used for immunohistochemistry were obtained from the supernatants of CD4 hybridoma, CD8 hybridoma or F4/80 hybridoma cultures (hybridomas obtained from American Type Culture Collection, Bethesda, USA). Anti-CD8 and anti-CD4 were concentrated by ammonium sulfate precipitation, and then dialyzed against PBS. Anti-F4/80 was obtained by affinity chromatography using agarose-bound mouse anti-rat IgG (Sigma) to separate rat IgG from the supernatant of F4/80 hybridomas. The concentration of affinity isolated IgG’s was determined by ELISA, and samples were diluted to 0.1– 0.2 mg/ml for use in immunohistochemistry. The rabbit anti-murine eosinophil granule major basic protein (mMBP) is polyclonal [28]. Rat anti-CD62P was used at a working

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concentration of 0.6 mg/ml and rat anti-CD62L was used at a working concentration of 1 mg/ml (both from BD Pharmingen, San Diego, CA). Hamster anti-ICAM-1 was diluted to 5 mg/ml for use and biotinylated rat anti-CD62E was diluted to 20 mg/ml for use (both from Research Diagnostics Inc., Flanders, NJ). 2.4. Inflammatory cell analysis One quadriceps and one soleus muscle from each animal were cut mid-belly into 10 mm-thick cross-sections and transferred to microscope slides coated with 0.2% gelatin and 0.02% chromium potassium sulfate for immunohistochemical analysis. Tissue sections were air-dried at room temperature for at least 30 min, fixed in ice-cold acetone for 10 min, and air-dried again for 10 min. After quenching the sections in 0.3% hydrogen peroxide in PBS for 5 min, they were washed in PBS for 5 min and blocked in a buffer containing 0.05% Tween-20, 0.2% gelatin, and 3% BSA for at least 30 min. For mononuclear cell analysis, sections were incubated with rat anti-CD8, rat anti-CD4, rabbit antimMBP for eosinophils, or rat anti-F4/80 for macrophages for 2 h, then with a host-specific biotinylated secondary antibody (Vector Laboratories, Burlingame, CA) for 30 min, and finally with horseradish peroxidase avidin-D (Vector Laboratories) for 30 min. Sections were washed 3 times for 5 min in PBS between incubations and developed using 3-amino-9-ethyl carbazole (AEC, red) as substrate. Negative control sections, for which incubation with the primary antibody was omitted, were prepared during each staining to ensure antibody specificity. The concentrations of macrophages, eosinophils, CD4þ cells and CD8þ cells were determined by counting the number of positively stained cells in at least two cross-sections per muscle, measuring the area of the crosssectioned muscle, and subsequently calculating the concentration of immunolabeled cells per volume of tissue section. The concentration of cells per section provides a relative measure of cell density in the muscle, but is not identical to cell concentration in the whole muscle, because an individual, labeled cell may be present in more than one section. 2.5. Adhesion molecule analysis Only quadriceps muscles were used for adhesion molecule analysis because the small cross-sectional area of the solei did not contain a sufficient number of vessels for statistical comparison. Sections were incubated with rat anti-CD62L (Pharmingen, San Diego, CA) for L-selectin, hamster anti-ICAM-1 (Research Diagnostics, Flanders, NJ), or rat anti-CD62P (Pharmingen) for P-selectin for 3 h, then with a host-specific biotinylated secondary antibody (Vector Laboratories) for 30 min, followed by horseradish peroxidase avidin-D (Vector Laboratories) for 30 min. For CD62E (E-selectin) staining, a biotin-conjugated rat anti-CD62E

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antibody (Research Diagnostics) was used. The 30-min incubation with a biotinylated secondary was omitted for sections stained with the anti-CD62E. All sections were washed 3 times for 5 min in PBS between incubations and developed using 3-amino-9-ethyl carbazole (AEC, red) as substrate. Negative control sections, for which incubation with the primary antibody was omitted, were prepared during each staining to ensure antibody specificity. A biotinylated isotype control antibody (Research Diagnostics) was used as a negative control in CD62E staining. Adhesion molecule expression was analyzed by counting the number of positively stained mononuclear cells and blood vessels. The concentrations of CD62L and ICAM-1 positive mononuclear cells were determined using the same method as for the inflammatory cell counts. The number of CD62P, CD62E, or ICAM-1 positively stained vessels in each of two sections per animal were counted and expressed per square millimeter. Based on the convention used by Lefer et al. [29], a non-capillary vessel was counted as positively stained if greater than 50% of the circumference of the vessel was labeled. Vasculature was confirmed as such by staining with anti-CD31 (Pharmingen) (not shown). 2.6. Assay for sarcolemmal damage Procion red (Sigma), an extracellular dye, was used to identify muscle cells with damaged membranes. Soleus muscles were carefully dissected, mounted in a cuvette at rest length, and incubated in a 0.2% solution of procion red in Kreb’s Ringer (Sigma) with 1 mM NaHCO3 and 2.5 mM CaCl2 for 1 h. Muscles were then washed twice, for 5 min in Kreb’s Ringer, frozen in liquid nitrogen-cooled isopentane, and stored at 2 80 8C in isopentane-filled vials. Twentymicrometer thick cross-sections were cut from the soleus muscles, transferred to coated microscope slides, and analyzed using fluorescent microscopy. The cytoplasm of fibers with membrane damage fluoresces due to the infiltration of the dye into the fiber. Positive fibers were counted and expressed as a percentage of the total number of fibers in each tissue section. At least two sections per animal were quantified. Only soleus muscles were analyzed for sarcolemmal damage because their anatomy allows for removal of the muscle with sufficient tendinous attachments on both ends for mounting. 2.7. Index of degeneration and regeneration Degeneration and regeneration of mdx skeletal muscle was measured by quantifying the number of fibers with centrally located nuclei and expressing that number as a percentage of the total number of fibers in a tissue section. As a result of degeneration, the myonuclei migrate from the periphery of the muscle fiber to the center of the cell where they remain indefinitely. The percentage of fibers with central nuclei is an indication of the cumulative damage

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the muscle has experienced [30]. At least two sections per muscle, per animal were quantified. 2.8. Statistics Statistical significance for all analyses was determined using the two-tailed Student’s t-test.

3. Results Daily prednisolone treatment significantly reduced the concentration of specific inflammatory cell populations in mdx skeletal muscle (Figs. 1 and 2). Macrophages were reduced by 59% in quadriceps (treated: 4968 mm23, sem ¼ 1166; untreated: 12,111 mm23, sem ¼ 1401) and 57% in solei (treated: 15,657 mm 23 , sem ¼ 2562; untreated: 36,092 mm23, sem ¼ 4739). CD4þ T-cells were reduced by 50% in quadriceps (treated: 274 mm23, sem ¼ 76; untreated: 563 mm23mm3, sem ¼ 99) and 60% in solei (treated: 506 mm 23, sem ¼ 51; untreated: 1282 mm23, sem ¼ 182). CD8þ T-cells were reduced by

58% in quadriceps (treated: 57 mm23, sem ¼ 8; untreated: 136 mm 23, sem ¼ 18) and 48% in solei (treated: 200 mm23, sem ¼ 13; untreated: 387 mm23, sem ¼ 50). Eosinophils were significantly reduced in quadriceps (36% reduction; treated: 2477 mm23, sem ¼ 267; untreated: 3850 mm23, sem ¼ 439) and showed a strong trend of reduction in soleus (25% reduction; treated: 10,714 mm23, sem ¼ 1751; untreated: 14,175 mm23, sem ¼ 3417). These data show that prednisolone is an effective immunosuppressant in dystrophin-deficient skeletal muscle. The expression of cell adhesion molecules was reduced in mdx quadriceps following prednisolone treatment. The concentration of mononuclear cells expressing L-selectin or ICAM-1 was significantly reduced by 79 and 57%, respectively (Figs. 3 and 4). The number of vessels expressing P-selectin, or ICAM-1 was also significantly reduced by 82 and 52%, respectively (Figs. 3 and 5). Though not statistically significant, we did observe a 47% decrease in the number of ICAM-1 positive capillaries after prednisolone treatment. A relatively small proportion of vessels stained positive for E-selectin, however, there was no difference between prednisolone-treated and control

Fig. 1. Inflammatory cells in mdx quadriceps. Representative images of labeled inflammatory cells identified for quantification. (A) Macrophages stained with anti-F4/80. (B) Eosinophils stained with anti-mMBP. (C) CD4þ T-cells stained with anti-CD4. (D) CD8þ T-cells stained with anti-CD8. All images are the same magnification. Bar, 50 mm.

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4. Discussion

Fig. 2. Prednisolone reduces inflammatory cell concentrations in mdx muscle. (A) Concentrations of inflammatory cells in control and prednisolone-treated quadriceps. n ¼ 5 in each group. (B) Concentrations of inflammatory cells in control and prednisolone-treated solei. n ¼ 7 prednisolone-treated and six controls. Control, black bars; prednisolonetreated, striped bars. Asterisk, differs from control at P , 0:05: Error bars, standard error of the mean.

animals. No capillaries stained positively for E-selectin or P-selectin. The decreases in adhesion molecule expression observed after treatment suggest that the anti-inflammatory effect of prednisolone function involves inhibition of inflammatory cell diapedesis. The effect of prednisolone treatment upon sarcolemmal integrity was assessed using the vital dye, procion red. If the membrane of a muscle fiber was damaged, the dye infiltrates the cytoplasm and is visible when viewed with fluorescent optics. We observed a 48% decrease in the number of soleus muscle fibers with damaged membranes in prednisolonetreated animals (Fig. 6). This finding demonstrates that prednisolone treatment decreases sarcolemmal damage, a serious consequence of dystrophin-deficiency. We also measured the number of fibers with centrally located nuclei, a morphological indicator of degeneration and regeneration. Prednisolone treatment reduced the number of fibers with centrally located nuclei in soleus muscles from 45.4 to 31.9% and from 28.6 to 18.2% in quadriceps muscles (Fig. 7). Though these decreases are not statistically significant, there is a definite trend to suggest that prednisolone therapy decreases the degenerative effects of the dystrophic pathology in skeletal muscle.

Our findings support the hypothesis that beneficial effects of prednisolone in treating dystrophinopathy may occur through its broad anti-inflammatory effects, and that these effects may result from down-regulations of CAMs on mdx leukocytes and vasculature. We observed statistically significant decreases in macrophages, CD4þ T-cells, and CD8þ T-cells in both muscles analyzed. Eosinophils were significantly reduced in quadriceps, and showed a strong trend towards reduction in soleus muscles as well. Since each of these inflammatory cell types can contribute to the dystrophic pathology [18,19,31], reducing their concentration in dystrophic muscle would be expected to have beneficial effects. In the same prednisolonetreated tissues, we observed significant decreases in the number of mononuclear cells expressing L-selectin or ICAM-1, and in the number of vessels expressing P-selectin or ICAM-1. Although we cannot differentiate between whether the decrease in mononuclear cells that express L-selectin or ICAM-1 is due to decreased CAM expression or reduced concentration of inflammatory cells, the significant reduction in the expression of P-selectin and ICAM-1 on the vasculature clearly shows that prednisolone can down-regulate CAM expression. These observations suggest that prednisolone may exert its anti-inflammatory effects in DMD via downregulation of specific CAMs, which may provide a mechanism that may be exploited in the development of new therapeutic strategies for DMD. The observed decrease in ICAM-1 expression on vessels, capillaries, and mononuclear cells may be the most functionally important finding because ICAM-1 participates in the facilitation of firm adhesion between the leukocyte and endothelium. Firm adhesion is the final step before extravasation of the inflammatory cell into tissue and represents a process that if obstructed, will inhibit the infiltration of inflammatory cells. Though we do not present direct evidence of a prednisolone-mediated decrease in CAMs here, previous studies have used a variety of in vitro systems to establish that prednisolone can directly decrease CAM expression [23–26]. It is a novel finding that this is a mechanism through which prednisolone functions in dystrophin-deficient skeletal muscle. Another major finding of our study is that prednisolone treatment decreases membrane damage in dystrophindeficient skeletal muscle. Damage to the sarcolemma is a significant component of the dystrophic pathology because it can compromise the functional integrity of the muscle and lead to further pathological events including the activation of proteases. Membrane lesions permit the un-regulated influx of calcium and result in elevated cytosolic calcium levels that can activate calcium dependent proteases, such as calpains, that are increased in concentration and activity in dystrophic tissue [9]. It is possible that the observed reduction in membrane damage following prednisolone treatment is due to a steroidspecific effect of membrane stabilization [32], or it may be due to the anti-inflammatory effect of prednisolone. Previously, we showed that reducing the concentration of macrophages in

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Fig. 3. Cellular adhesion molecule expression in mdx quadriceps. Representative images of CAM staining used for quantification. (A) ICAM-1-positive vessel. (B) ICAM-1-stained mononuclear cells. (C) E-selectin-stained vessel. (D) ICAM-1-stained capillaries. (E) P-selectin-stained vessel. (F) L-selectin-stained mononuclear cells. A and B are at the same magnification; C –F are at the same magnification. Bars, 50 mm.

dystrophin-deficient skeletal muscle reduces membrane damage [19]. Since we observed a significant decrease in the concentration of macrophages with prednisolone treatment in the current study, it is possible that this is the mechanism by which prednisolone reduces sarcolemmal damage.

Our previous studies demonstrating the significant improvements in muscle histology and membrane integrity following the depletion of inflammatory cell populations [18,19] show the extent to which inflammation contributes to dystrophinopathy and suggest that the anti-inflammatory

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Fig. 4. Mononuclear cells expressing cellular adhesion molecules are reduced in quadriceps of prednisolone-treated mdx mice. Concentrations of mononuclear cells expressing either CD62L, or ICAM-1. Control, black bars; prednisolone-treated, striped bars. n ¼ 5 animals per group. Asterisk, differs from control at P , 0:05: Error bars, standard error of the mean.

effects of prednisolone contribute significantly to its efficacy in the treatment of DMD. However, previous investigators have concluded that the benefits of prednisolone treatment in DMD are not primarily attributable to its anti-inflammatory function [33]. Those investigators hypothesized that if the primary beneficial effect of prednisolone were via its antiinflammatory function, an immunosuppressant, such as azathioprine, should yield functional benefits similar to prednisolone. Because azathioprine and prednisolone produced reductions in inflammatory cells, but only prednisolone yielded functional benefits, it was concluded that prednisolone does not primarily function as an immunosuppressive agent [34]. Although the differences in the reduction of inflammatory cells between prednisolone and azathioprine treatment were not statistically significant, biopsies from azathioprine-treated patients contained 25% more total mononuclear cells, 54% more CD8þ T-cells, 27% more CD4þ T-cells, and 150% more B-cells than biopsies

Fig. 5. Prednisolone treatment reduces the number of vessels that express cellular adhesion molecules in mdx quadriceps. Number of vessels expressing either CD62P, CD62E, or ICAM-1. Control, black bars; prednisolone-treated, striped bars. n ¼ 5 animals per group. There was no effect on expression of CD62E. Asterisk, differs from control at P , 0:05: Error bars, standard error of the mean.

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Fig. 6. Sarcolemmal damage is reduced with prednisolone treatment. The number of procion red-positive fibers is expressed as a percentage of the total number of fibers. Control, black bars; prednisolone-treated, striped bars. n ¼ 7 prednisolone-treated and six controls. Asterisk, differs from control at P , 0:05: Error bars, standard error of the mean.

from prednisolone-treated patients. This suggests that perhaps the anti-inflammatory effect of azathioprine as dosed in the study may not have been sufficient to yield functional benefits, or that the anti-inflammatory mechanism of prednisolone function is superior to that of azathioprine in the treatment of dystrophic inflammation. In addition, subsequent studies of the effects of anti-inflammatory drugs on mdx mouse muscle function have shown that not all anti-inflammatory drugs have equivalent effects on dystrophic muscle function [35]. In that investigation, seven non-steroidal anti-inflammatory drugs were tested, but only prednisolone and two others were observed to improve post-exercise muscle strength in mdx mice. Our current findings verify the anti-inflammatory effects of prednisolone in dystrophin-deficient muscle and indicate a mechanism through which prednisolone can reduce

Fig. 7. Prednisolone-treated mdx mice exhibit a trend of reduced degeneration and regeneration in skeletal muscle. The number of centralnucleated fibers is expressed as a percentage of the total number of fibers. Control, black bars; prednisolone-treated, striped bars. Quadriceps, n ¼ 7 prednisolone-treated and six controls; soleus, n ¼ 5 animals in each group. These differences are not statistically significant. Error bars, standard error of the mean.

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inflammation. We also present evidence to show that reducing the concentration of inflammatory cells in mdx muscle leads to improvements in indices of muscle damage and degeneration and therefore, suggest that the immunosuppressive effects of prednisolone are an important component of its efficacy in DMD. Our hope is that elucidation of the mechanisms through which prednisolone functions in DMD will lead to improved therapies for use until a cure is achieved. Ongoing studies in our lab are focused on further investigation of the role of CAMs in the dystrophin-deficient pathology.

[15]

[16]

[17]

[18]

Acknowledgements [19]

This investigation was supported by a grant from the National Institutes of Health (AR47721). [20]

References [21] [1] Mendell JR, Moxley RT, Griggs RC, et al. Randomized, double-blind six-month trial of prednisone in Duchenne’s muscular dystrophy. N Engl J Med 1989;320:1592–6. [2] Griggs RC, Moxley RT, Mendell JR, et al. Prednisone in Duchenne dystrophy. Arch Neurol 1991;48:383–8. [3] Backman E, Henriksson KG. Low-dose prednisolone treatment in Duchenne and Becker muscular dystrophy. Neuromuscul Disord 1995;5:233 –41. [4] Kawai H, Adachi K, Nishida Y, Inui T, Kimura C, Saito S. Decrease in urinary excretion of 3-methylhistidine by patients with Duchenne muscular dystrophy during glucocorticoid treatment. J Neurol 1993; 240:181–6. [5] Rifai Z, Welle S, Moxley RT, Lorenson M, Griggs RC. Effect of prednisone on protein metabolism in Duchenne dystrophy. Am J Physiol 1995;268:E67 –E74. [6] Banik NL, Matzelle D, Terry E, Hogan EL. A new mechanism of methylprednisolone and other corticosteroids action demonstrated in vitro: inhibition of a proteinase (calpain) prevents myelin and cytoskeletal protein degradation. Brain Res 1997;748:205 –10. [7] Haycock JW, Falkous G, Maltin CA, Delday MI, Mantle D. Effect of prednisone on protease activities and structural protein levels in rat muscle in vivo. Clin Chim Acta 1996;249:47–58. [8] Metzinger L, Passaquin A-C, Leijendekker WJ, Poindron P, Ruegg UT. Modulation by prednisolone of calcium handling in skeletal muscle cells. Br J Pharmacol 1995;116:2811–6. [9] Spencer MJ, Croall DE, Tidball JG. Calpains are activated in necrotic fibers from mdx dystrophic mice. J Biol Chem 1995;270:10909–14. [10] Passaquin AC, Metzinger L, Leger JJ, Warter J-M, Poindron P. Prednisolone enhances myogenesis and dystrophin-related protein in skeletal muscle cell cultures from mdx mouse. J Neurosci Res 1993;35: 363–72. [11] Sklar RM, Brown RH. Methylprednisolone increases dystrophin levels by inhibiting myotube death during myogenesis of normal human muscle in vitro. J Neurol Sci 1991;101:73–81. [12] Courdier-Fruh I, Barman L, Briguet A, Meier T. Glucocorticoidmediated regulation of utrophin levels in human muscle fibers. Neuromuscul Disord 2002;12:S95–S104. [13] Leussink VI, Jung S, Merschdorf U, Toyka KV, Gold R. High-dose methylprednisolone therapy in multiple sclerosis induces apoptosis in peripheral blood leukocytes. Arch Neurol 2001;58:91–7. [14] Liu MC, Proud D, Lichtenstein LM, et al. Effects of prednisone on the cellular responses and release of cytokines and mediators after

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32] [33]

[34]

[35]

segmental allergen challenge of asthmatic subjects. J Allergy Clin Immunol 2001;108:29 –38. Matsubara S, Hirai S, Sawa Y. Pulsed interventions methylprednisone therapy for inflammatory myopathies: evaluation of the effect by comparing two consecutive biopsies from the same muscle. J Neuroimmunol 1997;76:75–80. Lundberg I, Kratz AK, Alexanderson H, Patarroyo M. Decreased expression of interleukin-1a, interleukin-1b, and cell adhesion molecules in muscle tissue following corticosteroid treatment in patients with polymyositis and dermatomyositis. Arthritis Rheum 2000;43:336–48. Kissel JT, Burrow KL, Rammohan KW, Mendell JR, CIDD Study Group. Mononuclear cell analysis of muscle biopsies in prednisone-treated and untreated Duchenne muscular dystrophy. Neurology 1991;41:667–72. Spencer MJ, Montecino-Rodriguez E, Dorshkind K, Tidball JG. Helper (CD4þ) and cytotoxic (CD8þ) T-cells promote the pathology of dystrophin-deficient muscle. Clin Immunol 2001;98:235–43. Wehling M, Spencer MJ, Tidball JG. A nitric oxide synthase transgene ameliorates muscular dystrophy in mdx mice. J Cell Biol 2001;155:123 –31. Wong PK, Cuello C, Bertouch JV, et al. Effects of pulse methylprednisolone on macrophage chemotactic protein-1 and macrophage inflammatory protein-1 alpha in rheumatoid synovium. J Rheumatol 2001;28:2634 –6. Ferman RM, Church JA. Immunologic and virologic effects of glucocorticoids on human immunodeficiency virus infection in children: a preliminary study. Pediatr Infect Dis J 1994;13:212–6. Elovaara I, Lalla M, Spare E, Lehtimaki T, Dastidar P. Methylprednisolone reduces adhesion molecules in blood and cerebrospinal fluid in patients with MS. Neurology 1998;51:1703–8. Yoshida N, Yoshikawa T, Nakamua Y, et al. Methylprednisolone inhibits neutrophil–endothelial interactions induced by interleukin1b under flow conditions. Life Sci 1997;60:2341 –7. Dufour A, Corsini E, Gelati M, et al. Modulation of ICAM-1, VCAM1 and HLA-DR by cytokines and steroid in HUVECs and human brain endothelial cells. J Neurol Sci 1998;157:117–21. Gelati M, Corsini E, Dufour A, et al. High-dose methylprednisolone reduces cytokine-induced adhesion molecules on human brain endothelium. Can J Neurol Sci 2000;27:241 –4. Heimburger M, Larfars G, Bratt J. Prednisolone inhibits cytokineinduced adhesive and cytotoxic interactions between endothelia cells and neutrophils in vitro. Clin Exp Immunol 2000;199:441–8. De Bleecker JL, Engel AG. Expression of cell adhesion molecules in inflammatory myopathies and Duchenne dystrophy. J Neuropathol Exp Neurol 1994;53:369– 76. Lee JJ, McGarry MP, Farmer SC, et al. Interleukin-5 expression in the lung epithelium of transgenic mice leads to pulmonary changes pathognomonic of asthma. J Exp Med 1997;185:2143–56. Lefer DJ, Jones SP, Girod WG, et al. Leukocyte–endothelial cell interactions in nitric oxide synthase-deficient mice. Am J Physiol 1999;276:H1943–50. Carnwath JW, Shotton DM. Muscular dystrophy in the mdx mouse: histopathology of the soleus and extensor digitorum longus muscles. J Neurol Sci 1987;80:39–54. Cai B, Spencer MJ, Nakamura G, Tseng-Ong L, Tidball JG. Eosinophilia of dystrophin-deficient muscle is promoted by perforin-mediated cytotoxicity by T cell effectors. Am J Pathol 2000;156:1789–96. Sessa G, Weissmann G. Phospholipid spherules (liposomes) as a model for biological membranes. J Lipid Res 1968;9:310–8. Griggs RC, Moxley RT III, Mendell JR, et al. Duchenne dystrophy: randomized, controlled trial of prednisone (18 months) and azathioprine (12 months). Neurology 1993;43:520–7. Kissel JT, Lynn DJ, Rannohan KW, et al. Mononuclear cell analysis of muscle biopsies in prednisone- and azathioprine-treated Duchenne muscular dystrophy. Neurology 1993;43:532–6. Granchelli JA, Pollina C, Hudecki MS. Pre-clinical screening of drugs using the mdx mouse. Neuromuscul Disord 2000;10:235–9.