Dysferlin Deficiency Enhances Monocyte Phagocytosis
2008; Elsevier BV; Volume: 172; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2008.070327
ISSN1525-2191
AutoresKanneboyina Nagaraju, Rashmi Rawat, Edina Veszelovszky, Rachana Thapliyal, Akanchha Kesari, Susan Sparks, Nina Raben, Paul H. Plötz, Eric P. Hoffman,
Tópico(s)Nuclear Structure and Function
ResumoDysferlin deficiency causes limb-girdle muscular dystrophy type 2B (LGMD2B; proximal weakness) and Miyoshi myopathy (distal weakness). Muscle inflammation is often present in dysferlin deficiency, and patients are frequently misdiagnosed as having polymyositis. Because monocytes normally express dysferlin, we hypothesized that monocyte/macrophage dysfunction in dysferlin-deficient patients might contribute to disease onset and progression. We therefore examined phagocytic activity, in the presence and absence of cytokines, in freshly isolated peripheral blood monocytes from LGMD2B patients and in the SJL dysferlin-deficient mouse model. Dysferlin-deficient monocytes showed increased phagocytic activity compared with control cells. siRNA-mediated inhibition of dysferlin expression in the J774 macrophage cell line resulted in significantly enhanced phagocytosis, both at baseline and in response to tumor necrosis factor-α. Immunohistochemical analysis revealed positive staining for several mononuclear cell activation markers in LGMD2B human muscle and SJL mouse muscle. SJL muscle showed strong up-regulation of endocytic proteins CIMPR, clathrin, and adaptin-α, and LGMD2B muscle exhibited decreased expression of decay accelerating factor, which was not dysferlin-specific. We further showed that expression levels of small Rho family GTPases RhoA, Rac1, and Cdc 42 were increased in dysferlin-deficient murine immune cells compared with control cells. Therefore, we hypothesize that mild myofiber damage in dysferlin-deficient muscle stimulates an inflammatory cascade that may initiate, exacerbate, and possibly perpetuate the underlying myofiber-specific dystrophic process. Dysferlin deficiency causes limb-girdle muscular dystrophy type 2B (LGMD2B; proximal weakness) and Miyoshi myopathy (distal weakness). Muscle inflammation is often present in dysferlin deficiency, and patients are frequently misdiagnosed as having polymyositis. Because monocytes normally express dysferlin, we hypothesized that monocyte/macrophage dysfunction in dysferlin-deficient patients might contribute to disease onset and progression. We therefore examined phagocytic activity, in the presence and absence of cytokines, in freshly isolated peripheral blood monocytes from LGMD2B patients and in the SJL dysferlin-deficient mouse model. Dysferlin-deficient monocytes showed increased phagocytic activity compared with control cells. siRNA-mediated inhibition of dysferlin expression in the J774 macrophage cell line resulted in significantly enhanced phagocytosis, both at baseline and in response to tumor necrosis factor-α. Immunohistochemical analysis revealed positive staining for several mononuclear cell activation markers in LGMD2B human muscle and SJL mouse muscle. SJL muscle showed strong up-regulation of endocytic proteins CIMPR, clathrin, and adaptin-α, and LGMD2B muscle exhibited decreased expression of decay accelerating factor, which was not dysferlin-specific. We further showed that expression levels of small Rho family GTPases RhoA, Rac1, and Cdc 42 were increased in dysferlin-deficient murine immune cells compared with control cells. Therefore, we hypothesize that mild myofiber damage in dysferlin-deficient muscle stimulates an inflammatory cascade that may initiate, exacerbate, and possibly perpetuate the underlying myofiber-specific dystrophic process. Limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy, a distal myopathy, are both caused by recessively inherited mutations in the dysferlin gene.1Liu J Aoki M Illa I Wu C Fardeau M Angelini C Serrano C Urtizberea JA Hentati F Hamida MB Bohlega S Culper EJ Amato AA Bossie K Oeltjen J Bejaoui K McKenna-Yasek D Hosler BA Schurr E Arahata K de Jong PJ Brown Jr, RH Dysferlin, a novel skeletal muscle gene, is mutated in Miyoshi myopathy and limb girdle muscular dystrophy.Nat Genet. 1998; 20: 31-36Crossref PubMed Scopus (758) Google Scholar, 2Bashir R Britton S Strachan T Keers S Vafiadaki E Lako M Richard I Marchand S Bourg N Argov Z Sadeh M Mahjneh I Marconi G Passos-Bueno MR Moreira Ede S Zatz M Beckmann JS Bushby K A gene related to Caenorhabditis elegans spermatogenesis factor fer-1 is mutated in limb-girdle muscular dystrophy type 2B.Nat Genet. 1998; 20: 37-42Crossref PubMed Scopus (557) Google Scholar Both disorders show a loss of dysferlin protein at the plasma membrane in myofibers, which leads to abnormalities in vesicle traffic and membrane repair.3Han R Bansal D Miyake K Muniz VP Weiss RM McNeil PL Campbell KP Dysferlin-mediated membrane repair protects the heart from stress-induced left ventricular injury.J Clin Invest. 2007; 117: 1805-1813Crossref PubMed Scopus (137) Google Scholar, 4Glover L Brown Jr, RH Dysferlin in membrane trafficking and patch repair.Traffic. 2007; 8: 785-794Crossref PubMed Scopus (126) Google Scholar However, dysferlin is also expressed in many other cell types, including CD14+ monocytes.5De Luna N Freixas A Gallano P Caselles L Rojas-Garcia R Paradas C Nogales G Dominguez-Perles R Gonzalez-Quereda L Vilchez JJ Marquez C Bautista J Guerrero A Salazar JA Pou A Illa I Gallardo E Dysferlin expression in monocytes: a source of mRNA for mutation analysis.Neuromuscul Disord. 2007; 17: 69-76Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 6Ho M Gallardo E McKenna-Yasek D De Luna N Illa I Brown Jr, RH A novel, blood-based diagnostic assay for limb girdle muscular dystrophy 2B and Miyoshi myopathy.Ann Neurol. 2002; 51: 129-133Crossref PubMed Scopus (92) Google Scholar Thus, nonmyofiber cells may possibly contribute to the disease process. Dysferlin is a C2 domain-containing 230-kDa transmembrane protein, principally localized to the intracellular face of the plasma membrane.7Matsuda C Aoki M Hayashi YK Ho MF Arahata K Brown Jr, RH Dysferlin is a surface membrane-associated protein that is absent in Miyoshi myopathy.Neurology. 1999; 53: 1119-1122Crossref PubMed Google Scholar, 8Britton S Freeman T Vafiadaki E Keers S Harrison R Bushby K Bashir R The third human FER-1-like protein is highly similar to dysferlin.Genomics. 2000; 68: 313-321Crossref PubMed Scopus (60) Google Scholar The dysferlin gene is a large 55-exon gene localized to 2p13.1Liu J Aoki M Illa I Wu C Fardeau M Angelini C Serrano C Urtizberea JA Hentati F Hamida MB Bohlega S Culper EJ Amato AA Bossie K Oeltjen J Bejaoui K McKenna-Yasek D Hosler BA Schurr E Arahata K de Jong PJ Brown Jr, RH Dysferlin, a novel skeletal muscle gene, is mutated in Miyoshi myopathy and limb girdle muscular dystrophy.Nat Genet. 1998; 20: 31-36Crossref PubMed Scopus (758) Google Scholar, 2Bashir R Britton S Strachan T Keers S Vafiadaki E Lako M Richard I Marchand S Bourg N Argov Z Sadeh M Mahjneh I Marconi G Passos-Bueno MR Moreira Ede S Zatz M Beckmann JS Bushby K A gene related to Caenorhabditis elegans spermatogenesis factor fer-1 is mutated in limb-girdle muscular dystrophy type 2B.Nat Genet. 1998; 20: 37-42Crossref PubMed Scopus (557) Google Scholar Dysferlin was originally thought to play a role in membrane vesicle fusion events through its extensive sequence homology to the Caenorhabditis elegans FER-1 protein, which is known to be important for vesicle fusion during spermatogenesis.9Washington NL Ward S FER-1 regulates Ca2+-mediated membrane fusion during C. elegans spermatogenesis.J Cell Sci. 2006; 119: 2552-2562Crossref PubMed Scopus (109) Google Scholar More recent data regarding isolated myofibers are consistent with a role for dysferlin in membrane vesicle trafficking and membrane repair.10Bansal D Miyake K Vogel SS Groh S Chen CC Williamson R McNeil PL Campbell KP Defective membrane repair in dysferlin-deficient muscular dystrophy.Nature. 2003; 423: 168-172Crossref PubMed Scopus (772) Google Scholar, 11Lennon NJ Kho A Bacskai BJ Perlmutter SL Hyman BT Brown Jr, RH Dysferlin interacts with annexins A1 and A2 and mediates sarcolemmal wound-healing.J Biol Chem. 2003; 278: 50466-50473Crossref PubMed Scopus (314) Google Scholar Muscle samples from patients with a dysferlin deficiency show numerous extrastructural membrane defects when analyzed by electron microscopy, including tears in the plasma membrane and an accumulation of subsarcolemmal vesicles and vacuoles.12Selcen D Stilling G Engel AG The earliest pathologic alterations in dysferlinopathy.Neurology. 2001; 56: 1472-1481Crossref PubMed Scopus (134) Google Scholar Although it is clear that there are cell-autonomous membrane abnormalities in dysferlin-deficient myofibers, the presentation and progression of LGMD2B/Miyoshi patients include enigmatic histological and clinical features that are not fully explained by the myofiber defects.13Argov Z Sadeh M Mazor K Soffer D Kahana E Eisenberg I Mitrani-Rosenbaum S Richard I Beckmann J Keers S Bashir R Bushby K Rosenmann H Muscular dystrophy due to dysferlin deficiency in Libyan Jews. Clinical and genetic features.Brain. 2000; 123: 1229-1237Crossref PubMed Scopus (81) Google Scholar, 14McNally EM Ly CT Rosenmann H Mitrani Rosenbaum S Jiang W Anderson LV Soffer D Argov Z Splicing mutation in dysferlin produces limb-girdle muscular dystrophy with inflammation.Am J Med Genet. 2000; 91: 305-312Crossref PubMed Scopus (90) Google Scholar Patients are typically quite healthy until their late teens, and some patients show athletic prowess at a young age. There are presymptomatic elevations in serum creatine kinase but little evidence of weakness before disease onset. Also, disease onset can be more acute than in other dystrophies, and in some instances it is associated with an environmentally related muscle insult. Finally, muscle biopsies from patients early in the disease process can show striking inflammatory infiltrates in perivascular, perimysial, and endomysial areas of the muscle.15Gallardo E Rojas-Garcia R de Luna N Pou A Brown Jr, RH Illa I Inflammation in dysferlin myopathy: immunohistochemical characterization of 13 patients.Neurology. 2001; 57: 2136-2138Crossref PubMed Scopus (178) Google Scholar In fact, this relatively acute onset and the presence of inflammatory infiltrates often lead to LGMD2B being misinterpreted as polymyositis, an autoimmune disease of muscle.16Nguyen K Bassez G Krahn M Bernard R Laforet P Labelle V Urtizberea JA Figarella-Branger D Romero N Attarian S Leturcq F Pouget J Levy N Eymard B Phenotypic study in 40 patients with dysferlin gene mutations: high frequency of atypical phenotypes.Arch Neurol. 2007; 64: 1176-1182Crossref PubMed Scopus (208) Google Scholar The inflammatory infiltrates in dysferlin-deficient muscle have been described.12Selcen D Stilling G Engel AG The earliest pathologic alterations in dysferlinopathy.Neurology. 2001; 56: 1472-1481Crossref PubMed Scopus (134) Google Scholar, 14McNally EM Ly CT Rosenmann H Mitrani Rosenbaum S Jiang W Anderson LV Soffer D Argov Z Splicing mutation in dysferlin produces limb-girdle muscular dystrophy with inflammation.Am J Med Genet. 2000; 91: 305-312Crossref PubMed Scopus (90) Google Scholar, 15Gallardo E Rojas-Garcia R de Luna N Pou A Brown Jr, RH Illa I Inflammation in dysferlin myopathy: immunohistochemical characterization of 13 patients.Neurology. 2001; 57: 2136-2138Crossref PubMed Scopus (178) Google Scholar, 17Confalonieri P Oliva L Andreetta F Lorenzoni R Dassi P Mariani E Morandi L Mora M Cornelio F Mantegazza R Muscle inflammation and MHC class I up-regulation in muscular dystrophy with lack of dysferlin: an immunopathological study.J Neuroimmunol. 2003; 142: 130-136Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 18Rowin J Meriggioli MN Cochran EJ Sanders DB Prominent inflammatory changes on muscle biopsy in patients with Miyoshi myopathy.Neuromuscul Disord. 1999; 9: 417-420Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 19Cenacchi G Fanin M De Giorgi LB Angelini C Ultrastructural changes in dysferlinopathy support defective membrane repair mechanism.J Clin Pathol. 2005; 58: 190-195Crossref PubMed Scopus (88) Google Scholar Both dysferlin-deficient muscle biopsies and polymyositis biopsies often show inflammation; however, there are several differences between the muscle inflammation in dysferlin deficiency and that in other inflammatory muscle diseases.17Confalonieri P Oliva L Andreetta F Lorenzoni R Dassi P Mariani E Morandi L Mora M Cornelio F Mantegazza R Muscle inflammation and MHC class I up-regulation in muscular dystrophy with lack of dysferlin: an immunopathological study.J Neuroimmunol. 2003; 142: 130-136Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar In general, dysferlin-deficient muscle biopsies show approximately twice as many macrophages but half as many CD8+ T cells as in polymyositis.15Gallardo E Rojas-Garcia R de Luna N Pou A Brown Jr, RH Illa I Inflammation in dysferlin myopathy: immunohistochemical characterization of 13 patients.Neurology. 2001; 57: 2136-2138Crossref PubMed Scopus (178) Google Scholar Specifically, the endomysial and perivascular infiltrates in dysferlin-deficient muscle have been reported to consist of CD4+ T cells (40.6 ± 22.8%), macrophages (36.7 ± 23.7%), and CD8+ T cells (11.1 ± 6.6%).15Gallardo E Rojas-Garcia R de Luna N Pou A Brown Jr, RH Illa I Inflammation in dysferlin myopathy: immunohistochemical characterization of 13 patients.Neurology. 2001; 57: 2136-2138Crossref PubMed Scopus (178) Google Scholar Nonnecrotic dysferlin-deficient fibers appear free of all types of infiltrates, suggesting a relative paucity of cytotoxic T-cell-mediated myofiber death, despite the extensive inflammatory cell infiltrates in the muscle. Inflammation is also prominent in other muscular dystrophies, such as fascioscapulohumeral dystrophy, in which a high percentage of B cells and CD4+ T cells are observed in perivascular sites, and in Duchenne dystrophy, in which macrophages and T cells are present, for the most part in necrotic fibers.20Arahata K Ishihara T Fukunaga H Orimo S Lee JH Goto K Nonaka I Inflammatory response in facioscapulohumeral muscular dystrophy (FSHD): immunocytochemical and genetic analyses.Muscle Nerve. 1995; 2: S56-S66Crossref Scopus (132) Google Scholar Dysferlin is normally expressed in CD14+ monocytes, and CD14+ cells show dysferlin deficiency in LGMD2B and Miyoshi myopathy.6Ho M Gallardo E McKenna-Yasek D De Luna N Illa I Brown Jr, RH A novel, blood-based diagnostic assay for limb girdle muscular dystrophy 2B and Miyoshi myopathy.Ann Neurol. 2002; 51: 129-133Crossref PubMed Scopus (92) Google Scholar Detection of dysferlin in CD14+ cells has become a blood-based test for the diagnosis of dysferlin deficiency, although there have been no published comparisons of the sensitivity and specificity of the muscle biopsy test versus the CD14+ blood test. Efficient membrane regulation is necessary for a number of critical monocyte/macrophage functions, including receptor-mediated phagocytosis, cytokine secretion, and receptor signaling regulation through Rho family small GTPases such as Rac1, RhoA, and Cdc42.21Kwiatkowska K Sobota A Signaling pathways in phagocytosis.Bioessays. 1999; 21: 422-431Crossref PubMed Scopus (155) Google Scholar, 22Caron E Hall A Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPases.Science. 1998; 282: 1717-1721Crossref PubMed Scopus (806) Google Scholar, 23Bokoch GM Knaus UG The role of small GTP-binding proteins in leukocyte function.Curr Opin Immunol. 1994; 6: 98-105Crossref PubMed Scopus (39) Google Scholar, 24Cox D Chang P Zhang Q Reddy PG Bokoch GM Greenberg S Requirements for both Rac1 and Cdc42 in membrane ruffling and phagocytosis in leukocytes.J Exp Med. 1997; 186: 1487-1494Crossref PubMed Scopus (368) Google Scholar Given the role of dysferlin in membrane regulation in muscle cells and the presence of dysferlin in monocytes, we hypothesized that dysferlin deficiency may alter the ability of monocytes to perform such functions via small Rho family GTPases. Because monocytes are the precursors to tissue macrophages, we further hypothesized that abnormal macrophage function could play a role in the inflammatory reaction seen in skeletal muscle of many LGMD2B patients. To test the function of dysferlin-deficient monocytes, we designed a human trial of monocyte function in dysferlin-deficient patients and controls. These findings were then validated in vivo in dysferlin-deficient mice (SJL and AJ mouse strains) and in vitro in a J774 macrophage cell culture model using siRNA to specifically knock down dysferlin. Our findings suggest that overaggressive signaling in muscle macrophages resulting from dysferlin deficiency in these cells may play a key role in disease onset and progression. Samples from 13 dysferlin-deficient patients were used in this study. DNA samples were used for mutation analysis, peripheral blood for phagocytosis assay, and muscle biopsies for immunohistochemistry and mRNA expression profiling. Flash-frozen diagnostic muscle biopsies from dysferlin-deficient patients were received by the Research Center for Genetic Medicine at Children's National Medical Center as part of an ongoing, gratis molecular diagnostics program. Biopsies were tested for dystrophin and dysferlin by Western blotting (duplicate blots); immunostaining for merosin (lamina-α2), dystrophin, and α-sarcoglycan; and histological examination (hematoxylin and eosin staining). Biopsies were assigned a tentative diagnosis of primary dysferlin deficiency (LGMD2B, Miyoshi myopathy) if they showed a loss of dysferlin by Western blotting (0 to 10% of normal control levels) but a normal dystrophin Western blot and normal α-sarcoglycan and merosin immunostaining. Muscle biopsies showing complete dysferlin deficiency (0% relative to controls) were then selected for mRNA profiling (patients 1, 2, 4, 7, 10, 11, 12, and 13) and immunohistochemical staining (patients 1, 3, 4, 5, and 6) as described below (Table 1).Table 1Characteristics of Dysferlin-Deficient PatientsNo.Age at biopsySexDysferlinZygosityMutationsPhagoIHCMicroarray119F0%Compound heterozygotec.610C>T c.4192_4193insCXXX217M0%Compound heterozygotec.1834C>T splice mut (I-50)XndX3.n/aF0%Compound heterozygotec.3113G>A c.1749_1750insTndXnd4.31F0%Compound heterozygotec.2779delG c.4253G>AndXX527M0%Heterozygousc.3992G>TndXnd627F0%ESE detected*Exon splicing enhancer (ESE) was detected in exon 20 (c.2197 T>C).ndXnd769M0%None detectedndndX8n/aM0%Compound heterozygote*Exon splicing enhancer (ESE) was detected in exon 20 (c.2197 T>C).c.1834C>T splice mut(I-50)†Patient 8 is a full brother to patient 2 and is assumed to show dysferlin deficiency and have the same mutations, although this was not directly tested.Xndnd926M10%Not detectedXndnd1026F0%Compound heterozygotec.2643 + 1G>A c.4167 + 1G>CndndX1123M0%Homozygotec.5835_5838DelndndX1237M0%Heterozygotec.2641A>CndndX1319F0%Heterozygotec.3113G>AndndXnd, not done; IHC, immunohistochemistry.* Exon splicing enhancer (ESE) was detected in exon 20 (c.2197 T>C).† Patient 8 is a full brother to patient 2 and is assumed to show dysferlin deficiency and have the same mutations, although this was not directly tested. Open table in a new tab nd, not done; IHC, immunohistochemistry. The dysferlin gene is difficult to assay for gene mutations, given its large size (55 exons) and the lack of common mutations (most patients have private mutations). We screened DNA samples from all 13 patients for gene mutations by denaturing high-performance liquid chromatography of each dysferlin exon, followed by sequencing of exons containing heteroduplexes by denaturing high-performance liquid chromatography. Pathogenic mutations were detected in 10 of the 13 patients, and a possible exon splicing enhancer mutation was detected in 1 additional patient (patient 6) (Table 1). One of the affected patients was a full sibling to a second mutation-positive patient and therefore was assumed to have the same underlying mutations (patients 2 and 8 in Table 1). Under the auspices of an institutional review board-approved protocol conducted through the Children's National Medical Center General Clinical Research Center, we also contacted dysferlin-deficient patients via their referring physicians and invited them to participate in the monocyte study. Four of these patients (patients 1, 2, 8, and 9) volunteered and were brought into the Children's National Medical Center Clinical Research Center for blood draws for monocyte studies using peripheral blood. Four age- and sex-matched healthy adults with normal muscle function were also recruited as controls under the same protocol. All patients gave informed consent. All animal experiments were conducted in accordance with institutional guidelines. Five-month-old male and female SJL/J (dysferlin-deficient, n = 16), AJ/J (dysferlin-deficient, n = 6), Mdx (dystrophin-deficient, n = 6), and C57BL/6J (normal control, n = 16) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were housed in an individually ventilated cage system with a 12-hour light and dark cycle. Blood from tail bleeds of mice was collected in heparinized tubes and stored on ice until assayed. The mice were then euthanized by carbon dioxide gas, and death was ensured by cervical dislocation and verified by checking for a heartbeat. Peritoneal macrophages were collected after euthanasia by injecting 10 ml of ice-cold phosphate-buffered saline (PBS) containing 10% fetal bovine serum into the peritoneal cavity. The recovered peritoneal fluid was washed in chilled PBS. Viability, as determined by trypan blue dye exclusion, was >98%. For some experiments peritoneal macrophages were elicited in C57/BL6 and SJL/J mice and isolated 3 days after the injection of 3% thioglycollate broth. These peritoneal macrophages were washed with Dulbecco's modified Eagle's medium twice and then resuspended in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum at a concentration of 1 × 106/ml and treated with 1 μg/ml of lipopolysaccharide for 4 hours at 37°C in a CO2 incubator and lysates were prepared as described below. Hind limb muscle tissues were dissected and flash-frozen in isopentane-chilled liquid nitrogen and stored at −80°C until processed for immunohistochemistry and Western blotting. Immunohistochemical staining was performed on muscle biopsies that had been obtained from five patients (patients 1, 3, 4, 5, and 6) for routine diagnosis at the Research Center for Genetic Medicine. These analyses were used to determine the activation status of the infiltrated macrophages, as described previously.25Nagaraju K Raben N Villalba ML Danning C Loeffler LA Lee E Tresser N Abati A Fetsch P Plotz PH Costimulatory markers in muscle of patients with idiopathic inflammatory myopathies and in cultured muscle cells.Clin Immunol. 1999; 92: 161-169Crossref PubMed Scopus (76) Google Scholar, 26Nagaraju K Raben N Loeffler L Parker T Rochon PJ Lee E Danning C Wada R Thompson C Bahtiyar G Craft J Hooft Van Huijsduijnen R Plotz P Conditional up-regulation of MHC class I in skeletal muscle leads to self-sustaining autoimmune myositis and myositis-specific autoantibodies.Proc Natl Acad Sci USA. 2000; 97: 9209-9214Crossref PubMed Scopus (246) Google Scholar Frozen sections of human muscle biopsy samples were stained with mouse anti-human HLA-A, -B, -C (W6/32) (Harlan Sera Labs, Leicestershire, UK), HLA-DR (Tu 36) (BD Pharmingen, Franklin Lakes, NJ), CD86 (IT2.2), or DAF/CD55 (55C02) (Calbiochem, San Diego, CA) antibody. Frozen sections of muscle tissues from SJL/J (dysferlin-deficient) and C57BL/6J (control) mice were stained with rat anti-mouse MoMa-2 (Serotec, Oxford, UK) or with biotinylated anti-mouse CD11c (HL3) or ICAM-1 (3E2) (BD Pharmingen). Anti-mouse horseradish peroxidase, anti-rat horseradish peroxidase, streptavidin-horseradish peroxidase (DAKO, Carpinteria, CA), or anti-mouse IgG CyTM3 (Jackson ImmunoResearch, West Grove, PA) was used as the secondary antibody, as appropriate. Isotype-matched mouse Igs were used instead of primary antibodies as negative controls. Phagocytosis was assayed using the Vybrant phagocytosis assay kit from Molecular Probes (Eugene, OR). For human samples, heparinized whole blood was used within 2 hours of collection. Heparinized mouse blood was used within 30 minutes after collection. All materials used for the assay were prechilled on ice for 20 minutes. Heparinized whole blood (25 μl) was placed in 5-ml tubes, and 75 μl of Hanks' balanced salt solution containing 5% fetal bovine serum was added, along with 10 μl of fluorescent Escherichia coli bioparticles (Molecular Probes). The tubes were vortexed and incubated for 10 minutes at 37°C, then transferred to ice, and 100 μl of chilled trypan blue solution was added. The cell suspensions were washed with fluorescence-activated cell sorting buffer (phosphate-buffered saline containing 2% fetal calf serum, 1% bovine serum albumin, and 0.05% sodium azide), and the red blood cells were lysed by the addition of ammonium chloride, potassium chloride lysing solution at room temperature. The remaining cells were washed twice with fluorescence-activated cell sorting buffer, and fluorescence data were collected on 20,000 cells using a FACScan flow cytometer and CellQuest software. Monocyte gating was established using forward and side scatter profiles and further verified by MOMA staining. The phagocytic activity index was calculated by multiplying the percentage of monocytes phagocytosing the fluorescent bioparticles by the mean fluorescent intensity of the monocytes, as determined by CellQuest software. For cytokine incubations, samples were treated with 20 ng/ml of tumor necrosis factor (TNF)-α for 30 minutes at room temperature. All assays were performed using either duplicate or triplicate samples. Western blotting of muscle tissue was performed as described previously.27Nagaraju K Casciola-Rosen L Rosen A Thompson C Loeffler L Parker T Danning C Rochon PJ Gillespie J Plotz P The inhibition of apoptosis in myositis and in normal muscle cells.J Immunol. 2000; 164: 5459-5465PubMed Google Scholar Macrophage, spleen, and muscle homogenates were prepared in lysis buffer (1% Nonidet P-40, 20 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1 mmol/L ethylenediaminetetraacetic acid) with 1 mmol/L dithiothreitol and protease inhibitors. Lysates (20 to 100 μg) were electrophoresed on 4 to 12% Bis-Tris Gels (Invitrogen, Carlsbad, CA) and transferred to a nitrocellulose membrane. Immunoblots were performed with antibodies to dysferlin (230 kDa), CIMPR (275 kDa), clathrin (180 kDa), adaptin-α (112 kDa), Cdc-42 (21 kDa), Rac-1 (21 kDa), and RhoA (21 kDa) (Cell Signaling, Beverly, MA). All blots were stripped and blotted for vinculin (117 kDa) or β tubulin (55 kDa) to assess equal gel loading. The autoradiograms were scanned using an Arcus II scanner (Agfa, Mortsel, Belgium) and volume analysis was performed using Quantity one software (Bio-Rad discovery series; Bio-Rad, Hercules, CA). The ratios of CIMPR, clathrin, adaptin-α, Cdc42, Rac-1, and RhoA to vinculin or β-tubulin were calculated for dysferlin-deficient and control mice. The J774 mouse macrophage cell line (TIB67) was obtained from American Type Culture Collection (Rockville, MD) and grown to 60 to 70% confluence in six-well plates in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, then transfected with various concentrations of siRNA oligonucleotides targeting four different sequences within the dysferlin gene (DYSF1, 5′-CCCGAACTATGCCGCCATGAA-3′; DYSF2, 5′-ATCTGTCATCGGAGAATTTAA-3′; DYSF3, 5′-TACCCTGAGCTTTGGCGTTAA-3′; DYSF4, 5′-CAGGATAAGGACTACACCATT-3′) [4 for silencing; Qiagen, Valencia, CA]) using oligofectamine (Invitrogen). Transfection was performed at 50, 100, and 250 nmol/L of control or DYSF siRNA in the presence of serum. We used a cocktail composed of equal concentrations of four siRNAs: DYSF1r (CGAACUAUGCCGCCAUGAAUU) r(UUCAUGGCGGCAUAGUUCGGG), DYSF2 r(CUGUCAUCGGAGAAUUUAAUU) r(UUAAAUUCUCCGAUGACAGAU), DYSF3 r(CCCUGAGCUUUGGCGUUAAUU) r(UUAACGCCAAAGCUCAGGGUA), DYSF4 r(GGAUAAGGACUACACCAUUUU) r(AAUGGUGUAGUCCUUAUCCUG). Negative control siRNA was used at the same total concentration: control r(UUCUCCGAACGUGUCACGU)d(TT)(ACGUGACACGUUCGGAGAA)d(TT). siRNAs were added to 350 μl of OptiMEM (Invitrogen) and incubated for 30 minutes at room temperature. Separately, 5 μl of oligofectamine was mixed with 20 μl of OptiMEM and incubated for 30 minutes at room temperature. The oligofectamine was then mixed with siRNA and incubated for 30 minutes at room temperature. The mixture was overlaid on cells cultured in antibiotic-free medium in six-well plates. After 18 hours, the medium was replaced, and the ability of the cells to phagocytose fluorescent E. coli bioparticles was assessed by fluorescence-activated cell sorting analysis as described above. The results were expressed in terms of phagocytosis activity index. Dysferlin expression in these experiments was verified by Western blotting. The expression profiles used in these studies were from a 128-muscle biopsy, 256-Affymetrix (Santa Clara, CA) profile data set that we described as part of a previously published study of Emery-Dreifuss muscular dystrophy.28Bakay M Wang Z Melcon G Schiltz L Xuan J Zhao P Sartorelli V Seo J Pegoraro E Angelini C Shneiderman B Escolar D Chen YW Winokur ST Pachman LM Fan C Mandler R Nevo Y Gordon E Zhu Y Dong Y Wang Y Hoffman EP Nuclear envelope dystrophies show a transcriptional fingerprint suggesting disruption of Rb-MyoD pathways in muscle regeneration.Brain. 2006; 129: 996-1013Crossref PubMed Scopus (235) Google Scholar A full description of the data set can be found in our previous article, although the published data focused on patients harboring emerin and lamin A/C mutations. The present analysis is the first analysis of the data related to dysferlin deficiency (LGMD2B), although the
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