Inflammatory response in facioscapulohumeral muscular dystrophy (FSHD): Immunocytochemical and genetic analyses

zyx zy zyxwvuts To investigate the nature of the inflammatory response in facioscapulohumeral muscular dystrophy (FSHD), we analyzed mononuclear cells in muscle sections obtained from 18 FSHD patients and 8 controls. Monoclonal antibodies reactive for T cells, T cell subsets, B cells, and NK cells were used for cell typing. Macrophages were identified by acid phosphatase reaction. The localization of perforin, granzyme A, MHC-I and -11, dystrophin, and a-actinin antigens was also examined. We found that all FSHD patients, both familiar and sporadic cases, had greater amounts of mononuclear cellular infiltrates in muscle than controls, in whose specimens only few extra vascular mononuclear cells were counted. Seventy-two percent (13 of 18) of the patients had more than 50 inflammatory mononuclear cells per 1000 muscle fibers, and 33% (6 of 18) patients had numerous inflammatory cells exceeding 600 per 1000 muscle fibers (1835 & 482 SE). Nonnecrotic fibers invaded by mononuclear cells with either T8+, perforin+, or granzyme A + were not observed in FSHD, while a few degenerating fibers were superficially invaded by T cells and macrophages. Occasional T cells were observed moving through the blood vessel wall. The increased number of necrotic fibers was paralleled by an increased number of inflammatory cells ( r = 0.783, P = 0.0001). Genetic analysis, using the probes p13E11, pFR-1, D4S139, and D4S163, was done in 6 patients (3 familiar, 3 sporadic) who had numerous inflammatory infiltrates. These 6 patients had small (<28 kb) EcoRl fragments associated with the disease, and the disease was linked to 4q35. These results suggest that, in chromosome b l i n k e d FSHD: (1) inflammatory changes in muscle are a common histological feature; (2) mononuclear cellular infiltrates may enhance muscle fiber damage; but (3) T-cell-mediated cytotoxicity directed against muscle fibers is unlikely. We speculate that the immune effector mechanism in FSHD is different from that in previously reported inflammatory myopathies and Duchenne muscular dystrophy. 0 1995 John Wiley & Sons, Inc. Key words: facioscapulohumeral muscular dystrophy inflammatory response lymphocyte subsets genetic diagnosis MUSCLE & NERVE Suppl2:S56-S66 1995 INFLAMMATORY RESPONSE IN FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY (FSHD): IMMUNOCYTOCHEMICAL AND GENETIC ANALYSES KllCHl ARAHATA, MD, TADAYUKI ISHIHARA, MD, HlDETOSHl FUKUNAGA, MD, SATOSHI ORIMO, MD, JE HYEON LEE, PhD, KANAKO GOTO, BS, and IKUYA NONAKA, MD From the National Institute of Neuroscience (NCNP), Tokyo, Japan (Drs Arahata, Orimo, and Lee, Ms Goto, and Dr Nonaka); National HigashiSaitama Hospital, Saitama, Japan (Dr. Ishihara); National Minami-Kyushu Hospital, Kagoshima, Japan (Dr. Fukunaga); and Kanto Chuo Hospital, Tokyo, Japan (Dr. Orimo). Acknowledgments: We thank Dr. Hideo Sugita (President, NCNP, Japan) for his helpful discussion and advice, Drs. ~ u n eR. Frants, ciscawi]. menga, Egben Bakker, and G ~ ~ B. van J Ommen ~ ~ (Department of G ~ . netics Leiden Universitv. Leiden. The Netherlands) and Barbara Weiffenbach (Collaborative Research lnc , Waltham, MA) for their very kind technical help, and providing us the plasmids This study was supported by Research Grants 91A1208 and 5A-2 from the Ministry of Health and S56 FacioscapulohurneralMuscular Dystrophy Welfare, Japan, Grants-in-Aid for Scientific Research (B) and Developmental Scientific Research from the Ministry of Education, Science, and Culture, Japan; a research grant from the Uehara Memorial Foundation; and by a research grant from the Muscular Dystrophy Association (USA) (K.A ) zyxwv Address reprint requests to Dr Kiichi Arahata. MD, Department of NeurOmUSCUlar Research, National Institute of Neuroscience, NCNP, 4-1-1 ogawa-higashi' Kodaira, Tokyo ' 8 7 , Japan CCC 0148-639X/95/S20S56-11 0 1995 John Wiley & Sons Inc MUSCLE & NERVE Supplement 2 1995 of these cellular infiltrates in FSHD muscle reF a c i o s c a p u l o h u m e r a l muscular dystrophy mains unclear. (FSHD) is an inherited disease transmitted in an We have recently examined a total of 158 Japautosomal-dominant f a ~ h i o n . ' ~ , ~T'h~e ' dis~~~~ anese individuals that included 18 unrelated ease locus has been mapped to chromosome FSHD families with 38 affected and 73 healthy 4q,28,394',47,48251 although genetic heterogeneity family members, as well as 35 Japanese controls, exists in rare fa mi lie^.^^,^^ Both males and females for genetic diagnosis, using the probes p13E-11 have a 50% chance of inheriting the gene from an and pFR- 1. I 7 In our patient population, the probes affected parent, but the gene may also be defective DNA rearrangements associated with because of a new m ~ t a t i o n . ' ~A~ recently ~ ~ , ~ ~ , ~detected ~ FSHD; i.e., most of the Japanese FSHD patients identified probe, p13E-11 (D4F104Sl), usually de(97.4%) had specific small (<28 kb) EcoRI fragtects specific smaller (<28 kb) EcoRI fragments ments which cosegregated with the disease, and that cosegregate with FSHD'7,27,4','6,49,'2; delethe disease was linked to chromosome 4q35. In tions of the tandemly repeated 3.2- (or 3.3) -kb contrast, none of the patients with neurogenic KpnI units (D4Z4) in the EcoRI fragment are thought to cause the d i ~ e a s e . ~ ~ ~ ' ~ ~ HOW~ " ~ ' ~ " "scapuloperoneal .~~ muscular atrophy had small ever, the molecular genetic mechanisms underlyEcoRI fragments associated with the disease. ing the disease are still unclear. T h e p13E-11 In the present study, we examined subsets of mononuclear cells in the muscles of 18 patients probe has since been mapped to the 4q35-qter region distal to the D4S139 locus (cen-D4S139with FSHD and 8 controls, using qualitative and quantitative immunocytochemistry to elucidate the D4F35S 1-D4F104S 1-FSHD-tel),4694Y754 and the role of the inflammatory response in FSHD. We probe is considered to have immediate diagnostic value for FSHD, although this probe can be used also accomplished DNA-based genetic analysis of as a tightly linked polymorphic marke~-.'~*~' the 6 FSHD patients who had numerous mononuInflammatory changes in muscle are frequently clear cellular infiltrates in muscle, using the probes (40-80%) observed in FSHD patients and somep13E-11 and pFR-1. times become very marked.8,9,'2 In 1948, Ogryzlo described a small focal accumulation of lymphocytes in muscle in a sporadic patient with facioMATERIALS AND METHODS scapulohumeral atrophy.33 In 1955, Nattrass reported an adult patient with severe polymyositis Analysis of Mononuclear Cell Subsets in Muscle. who was initially thought to have sporadic FSHD Clinical Materials. Limb muscle specimens (obbecause of the presence of symmetrical wasting of tained from biceps bracii) were examined for difacial, scapular, and upper-arm muscles3' More agnostic purposes, after informed consent was obtained from the subjects. The muscle specimens than 15 similar patients have since been reported,7,11,19,23,29,38,44,55 and the presence of FSH were flash frozen in isopentane chilled with liquid nitrogen. Specimens from 18 patients with FSHD syndrome due to an inflammatory myopathy has been ~ o n s i d e r e d .Facial ~ muscle involvement has [9 familiar, 9 sporadic; 6-62 years old; 29 +- 18 been reported in about 7-11% of patients diagyears (SE)] and 8 controls (unrelated, with no nosed with p o l y m y o s i t i ~ . " ~Massive ~~ inflammaknown myopathy and no histologic abnormality by tory cellular infiltration in muscle also occurs in ordinary examinations) were studied. The diagnothose FSHD patients who have a positive family sis of FSHD was based on the clinical, histological, In addition, in the first family reand electromyographic finding^.^','"^"^^ Nine of the 18 FSHD patients had elevated serum creatine ported by Bacq et aL5 both the affected mother kinase (CK) activity; and the overall mean value and her daughter had inflammatory changes in was 378 65 (SE) U/L (normal 12-280). muscle, suggesting the possibility that the inflamImmunoreugents. The antibodies used in this matory response in FSHD is intrinsically involved study were: monoclonal antibodies reactive for T with the disease process. Although some studies have analyzed inflammatory cellular subsets in pacells, T-cell subsets (T4, TS), B cells, NK cells, MHC-I and -11 antigens, and polyclonal antitients with a clinical diagnosis of FSH myopathy of perforin and -granzyme A antigens. The specificFSHD,'5x21genetic analysis has not been accomity of each antibody has been described previousplished. We still do not know the relationship beIy.2-4,13,34Monoclonal antibodies for dystrophin tween inflammatory infiltrates and dystrophic muscle histology (necrosis, phagocytosis, regener(4C5) and a-actinin (AB- 1) were also used in some cases. Affinity-purified fluorescein isothiocyanate ation, variation in fiber size, and proliferation of (F1TC)-labeled goat F(ab')2 anti-mouse and anticonnective tissues), and therefore, the significance zyxwvut zyxw zyxwvutsrq zy * Facioscapulohumeral Muscular Dystrophy zyxwvuts MUSCLE & NERVE Supplement 2 1995 S57 zyx zyxwvu zyxwvutsrq zyxwvuts zyxwv rabbit IgG were obtained from Tago Inc., and used as the second layer antibodies. Immunocytochemistry. Four-micron serial cryostat sections were thawed on gelatinized cover slips, fixed in 100% ice-cold acetone for 5 min, air-dried for 30 min, soaked in 0.1% Nonidet-P40 for 3 min, and then preincubated with PBS containing 2% BSA and 5% heat-inactivated normal goat serum at pH 7.4. The sections were incubated with primary antibodies for 2 h at 37°C and with secondary antibodies (10 pg/mL) for 1 h at room temperature. The sections were mounted in a glycerolbased medium, and observed under a Zeiss Axiophot microscope with epifluorescence. Specificity of the immunostaining was examined by replacement of the primary antibody with control mouse myeloma IgG or preimmune rabbit serum. Quantitative Analysis of Mononuclear Cells in Muscle. For quantitative analysis, a region that contained at least 1000 muscle fibers was selected at random in each specimen, and the same region was analyzed in each serial section. We counted all muscle fibers and all reactive cells for each antibody in the randomly chosen region, and calculated the average cell number per 1000 muscle fi- bers. The analysis was done according to sites of accumulation, as described.23'" Genetic Analysis of FSHD Patients with Numerous In. flammatory Infiltrates in Muscle. Probes. T h e probes used in this study were p13E-11 (0.8-kb insert in pBS isolated by SacIIEcoRI doubledigestion, kindly provided by Dr. Rune R. Frants) and our own pFR-1 l 7 (1.1-kb fragment isolated by HindIII/HincII double digestion from the 4.9-kb KpnI fragment of the plasmid pSMl kindly supplied by Dr. Barbara Weiffenbach). We also used other 4q35 markers (D4S139, D4S163). These probes were labeled with 32P-dCTP for Southern blot analysis, using a Random Primed DNA Labeling Kit (Boehringer). Southern Blot Hybridization. Fifteen micrograms of genomic DNA (isolated from peripheral blood lymphocytes) was digested with the restriction enzyme EcoRI at 37°C for 12 h, as recommended by the manufacturer's protocol. The DNA was separated on 0.3% HGT agarose gel for 72 h at 15 mA (0.5 V/cm), and was then transferred to H ybond N (Amersham). Hybridization was carried out overnight at 65°C. The filter was thoroughly washed in 1 x SSC/O.l% SDS for 1 h at 60"C, fol~ ~~ Table 1. Summary of pathological and immunocytological findings of 18 FSHD cases ~ Case 1 2 3 4 5 6 Total cellst (n) No of nec* Cell Nec Rege Size Nuc Ang Conn 3757 2342 2019 1654 632 607 10 7 18 15 4 2 4 4 4 4 3 3 3 3 4 4 3 2 2 2 3 2 1 1 4 4 4 4 2 4 2 2 2 1 1 2 2 3 1 2 2 1 4 3 4 267 149 101 72 66 65 52 35 33 32 19 16 2 3 1 1 0 0 0 0 1 0 0 0 2 2 2 2 2 1 1 1 2 1 1 1 2 2 1 1 0 0 0 0 1 0 0 2 3 1 1 0 1 0 0 1 0 0 1 2 4 1 2 4 1 1 1 0 1 1 2 1 1 1 2 1 1 0 1 0 3 4 3 2 1 0 0 1 4 4 1 2 4 1 1 1 1 0 0 0 0 Mean 2 SE 662 2 252 3.6 2 1.3 Controls (n = 8) 9.2 2 2.1 SE Age Sex 13 1 4 M M M 6 9 F F 61 38 1 7 8 9 10 11 12 13 14 15 16 17 18 ~~~ 7 6 a 15 15 - + + F F 2 17 58 43 32 45 17 + + - M 10 6 Fa* - M - M + + + + + M - M F - M F F F M Qualitative analysis of muscle pathology§ 4 3 4 EcoRl (kb)Ii 17 13 177 13 24 24 3 1 1 1 1 1 1 zyxwvut zyxwvutsrqp zyxwvutsrqponml - zyxwvutsrqp 0 1 0 0 0 0 0 0 *Family history. j 7 cells + B cells macrophages NK cells per 1000 muscle fibers. #Number of necrotic fibers per 1000 muscle fibers. 50 = Not found; 1: slight; 2 mild; 3: moderate; 4: marked. (Cell.: cellular infiltrations; Nec.. necrotic fibers, Rege.. regenerated fibers; Slze: vanation In fiber size diameter; Nuc . internal nuclei; Ang: small angular fibers; Conn : connective tissue proliferation). "Size of the FSHD-associated genomic EcoRl fragment which is detected by the probes p13E-11 and pFR-1 Father of case 1. + S58 + Facioscapulohumeral Muscular Dystrophy MUSCLE & NERVE Supplement 2 1995 these patients had numerous inflammatory cells, exceeding 600 per 1000 muscle fibers [835 482 (SE), range 607-3757]; the remaining 12 patients had small to moderate amounts of inflammatory cells [76 & 21 (SE), range 16-2671. There was no significant difference in total number of the inflammatory cells between FSHD patients who had a positive family history and those who had no family history (clinically new mutations). Although the average number of necrotic fibers (confirmed by complement C9 and C5b-9 membrane attack complex) per 1000 muscle fibers was low [3.6 ? 1.3 (SE)] in FSHD, this number increased in parallel with the number of the inflammatory cells ( r = 0.783, P = 0.0001) (Table 1, Fig. 1). Chronic dystrophic changes, characterized by the presence of either regenerated fibers, fiber size variation, increased connective tissue elements, or internal nuclei, were found in all FSHD muscles examined (Table 1). In the 6 FSHD patients who had numerous cellular infiltrates, B cells were most abundant at 5.0% of perivascular sites, accounting for 25.4 the total. The T4/T8 ratio at the perivascular site was 1.51 i 0.55, and N K cells were least abundant (Table 2). Macrophages accounted for about one third of all mononuclear cells. At the endomysial site, T cells were most abundant (Table 2), inflammatory cells were diffusely scattered, and there were no nonnecrotic muscle fibers invaded by mononuclear cells that included T 8 + , perforin+, or granzyme A+ cells. Only a few degenerating * zyxwvutsrq zyxwvutsrqpo zyx zyxwvutsr zyxwvutsrqpon zyxwvu -2 0 2 4 6 8 10 12 14 16 18 20 nec. fibers FIGURE 1. Positive correlation is shown between the total number of inflammatory cells and the number of necrotic muscle fibers per 1000 muscle fibers in FSHD. ( r = 0.783, P = 0.0001). lowed by autoradiography for 2 4 h using a BAS 2000 image analyzer, and for 3 days using Konica AX film with an intensifying screen. RESULTS inflammatory Infiltrates. A summary of the pathological and immunocytochemical findings is shown in Table 1. In control muscles, only a few [9.2 k 2.1 (SE) per 1000 muscle fibers] extravascular mononuclear cells were observed. All 18 patients with FSHD (9 familiar, 9 sporadic) had much higher total numbers of inflammatory mononuclear cells (T cells B cells + macrophages + N K cells), with the mean value being 662 k 252 (SE) per 1000 muscle fibers (range 16-3757). Six of + ~~ zyxw * ~ Table 2. Quantitative analysis of mononuclear cells in FSHD Perivascular cells Cell type or marker FSHD with numerous (>600 cells per 1000 fibers) cellular infiltrates ( n 25.4 2 5.0* B cells (B1 + ) (%) Macrophages (%) 23.6 -+ 12.9 5 1 . 0 2 11 9 T cells ( T l l + ) (%) 46.5 2 11.6 T 8 + K cells (%) 61.1 2 14.1 T 4 + n cells (%) 1.51 2 0 55* T4+K8+ ratio B/T ratio 0.52 i 0.14* 8.1 2 3.3 Leu-7+ cells (%) = Perirnysial cells Endomysial cells 8.1 t 5.3t 35.6 2 11.1 56.4 2 12.6 64.5 2 18.7 42.2 2 14.4 0.86 2 0.37 0.16 2 0 . l O t 11.1 2 4.7 63252* 2362101 701 2 1 1 5 6 5 9 2 160 4 2 5 2 168 0 79 2 0 67 010+008* 101 2-46 1.9 2 32.4 5 66.5 2 53 7 2 52.0 -+ 0.96 2 0.03 2 17.6 2 04210 2262197 7 9 0 2 175 5 7 O r 173 4752191 0 84 2 0 77 0 04 2 0 01 2 0 0 2 191 6) zyxwvuts FSHD with sparse to mild (<300 cells per 1000 fibers) cellular infiltrates (n 4.7 2 7.4 B cells (B1+) (%) Macrophages (%) 37.1 2 29.0 58.2 2 28.8 T cells ( T l l + ) (%) 57.1 2 31.6 T 8 + n cells (%) T 4 + n cells (%) 57.2 2 31.4 T 4 + n 8 + ratio 0.85 2 0.73 BIT ratio 0.05 2 0.08 18.6 2 17 6 Leu-7+ cells (%) Values are expressed as mean ? SD = 12) 3.9 17.7 18 1 19.8 20.5 0.67 0.06 14 3 Significantly dffferent from the values in FSHD with sparse to mild cellular infdtrates 'P Facioscapulohumeral Muscular Dystrophy MUSCLE & NERVE < 0 005 and f P < 0 025 Supplement 2 1995 S59 zyxwvutsrqpo zyxwvutsrqp zyx zyx FIGURE 2. Muscle biopsies of FSHD patients often have a relatively normal appearance, although there is some variation in size, in the presence or absence of an internal nucleus, in endomysial fibrosis, and in numbers of mononuclear cells (a: H & E). Changes in fiber size diameter often become prominent with large and very small fibers being exhibited (b: mGT). Consecutive frozen sections obtained from FSHD patients were stained with H & E (c) and rnGt (e), and immunoreacted for dystrophin (d) and a-actinin (f). Note, partially degenerating fibers in the center are focally surrounded by and superficially invaded by mononuclear cells, but no nonnecrotic fibers are invaded by mononuclear cells. (a and b: x125; c-f: x250). fibers were surrounded by and superficially invaded by mononuclear cells (Fig. 2). In contrast, blood vessels were frequently surrounded by a compact collection of cells (Fig. 3) and were often invaded by T4+ and T8+ T cells (Fig. 4). Presumably, these cells were passing through the vessel wall. The overall perivascular (PV), perimysial (PMY), and endomysial (EMY) average cell counts per 1000 fibers in FSHD were, respectively: T cells, 97, 56, 277; B cells, 43, 6, 30; Leu-7+ cells, 14, 5 , 33; L e u - l l + cells, 2, 1, 2; and macrophages, 40, 32, 81. The T4/T8 ratios were 1.1, 0.7, and 1.0, respectively. MHC-I expression was not detected in control muscle fibers, but was found in the surface membranes of a few muscle fibers in FSHD (Fig. 3). Vascular endothelial cells and mononuclear cells expressed MHC-I in both control and FSHD muscles. MHC-I1 expression was limited to vascular endothelial cells and some mononuclear cells (Fig. 3). zyxwvut S60 FacioscapulohumeralMuscular Dystrophy MUSCLE & NERVE Supplement 2 1995 zyxwvutsrqpon zyxwvutsrqp zyxwv zyxwvutsrqpo FIGURE 3. Inflammatory cells in FSHD. Serial sections were stained with H & E and reacted for T11, E l , HLA-I, and HLA-II markers, and acid phosphatase (AP).The perivascular exudates are intermingled with predominantly T and B cells. HLA-I antigen is expressed in all inflammatory cells, vascular endothelial cells, and in the plasma membrane of some adjacent muscle fibers. HLA-II antigen is expressed in vascular endothelial cells and in some inflammatory cells, but not in the plasma membrane of the muscle fiber ( ~ 2 5 0 ) . Genetic analysis was accomplished in the 6 FSHD patients (from 5 unrelated families) who had numerous mononuclear cellular infiltrates. The disease in these patients was linked to 4q35 (Fig. 5) and the patients’ specimens exhibited small (<28 kb) EcoRI fragments (13-24 kb) that cosegregated with the disease (Figs. 5-7, and Table 1). Genetic Analysis. DISCUSSION The presence of inflammatory cellular infiltrates in the skeletal muscle of patients with FSHD is one of the characteristic features of the disease, and can be observed in 40-80% of patient^,^"'^^ while general histological changes in FSHD muscle are rather nonspecific (variations in fiber size, scattered small angulated fibers, etc.). In the present Facioscapulohumeral Muscular Dystrophy study, 6 of 18 (33%) FSHD patients had marked inflammatory cellular exudates in muscle, with a mean value of 1835 ? 482 (SE) cells per 1000 muscle fibers (range 607-3756), these changes being paralleled by increasing numbers of necrotic fibers. Genetic analysis revealed that FSHD was linked to chromosome 4q35 in these 6 patients (3 familiar, 3 sporadic) and that they had FSHDassociated small EcoRl fragments (<28 kb) detected by the probes p13E-ll and pFR-1 (Table 1, Figs. 5-7). The remaining 12 patients had less marked inflammatory infiltrates, but still showed more inflammatory cells [76 21 (SE), range 162671 than the controls [9.2 k 2.1 (SE)]. Thus, our findings suggest that inflammatory cellular response in muscle is a common histological feature of chromosome 4-associated FSHD in both famil- * zyxwvut MUSCLE & NERVE Supplement 2 1995 S61 zyxwvutsrqpon zyxwvutsrqponm zyxwvutsrqponmlkjihgfedc zyxwvutsrq FIGURE 4. Perivascular inflammatory cells in FSHD. The T4+ cells are most prominent at perivascular sites. Both T4+ and T8+ cells are seen occasionally moving through the vessel wall (arrow), but they do not invade the adjacent nonnecrotic muscle fibers (x410). I I Markers Alleles I14S139 P, p, 3,1,5,6 UJS163 A,u,3,4,5,6 p13E-ll/ EcoRI 46 47 48 49 50 D4S139 / TaqI 46 47 48 49 5 0 kb 16 I 47 47.034.623.1- FIGURE 5. Southern blot analysis of a FSHD patient (case 6:49) and his family in whose muscle specimen numerous inflammatory infiltrates were shown (p13E-11 probe). Each affected member had a small (24 kb) fragment (arrow) associated with the disease; this feature was not detected in the healthy family member^.^^,^' Other chromosome 4q35 markers (D4S139, D4S163) revealed that the disease in this family was associated with chromosome 4q35. S62 Facioscapulohumeral Muscular Dystrophy zyxw MUSCLE 8, NERVE Supplement 2 1995 - zyxwv n zyxwvutsr FSHDwith numerous cellular infiltrates u EZ72 OtherFSHD 1 2 3 4 5 6 7 8 zyxwvu zyxwvutsrqponmlk Y v) c kb 0Controls v ?! 4 zy zyxw J + 48.538.433.529.924.822.6- m n 0 b n 5 z I 10 19.4- + 17.115.0- 4 12.1- 10.1- 8.68.3- 2 I I 1 5 20 2'5 k 2 8 k b EcoRl fragment size detected by the probes pl3E-11 and pFR-1 FIGURE 7. Size distribution of the genomic EcoRl fragment associated with FSHD in 23 unrelated Japanese families and 35 controls. Probes p13E-11 and pFR-1 were used. All 6 patients (five families) who had numerous inflammatory infiltrates in muscle are shown (black columns). zyxwvutsrqponm (CTL)-mediated muscle fiber injuries occur, and endomysial mononuclear cells (mostly T cells) focally surround, invade, and finally replace nonnecrotic muscle fiber^."'^'^ Perforin+ and granzyme A + cells appear among these invading cells, and 10-12% of the endomysial T 8 + cells are shown to be double positive for p e r f 0 1 - h .In ~ ~FSHD muscle, in contrast, we found no invasion of T8+, perforin+, or granzyme A + cells into nonnecrotic fibers (Fig. 8), although a few degenerating fibers were focally surrounded by andlor superficially invaded by mononuclear cells (most being T cells and macrophages) (Fig. 2). These findings suggest that there is no CTL-mediated immune effector mechanism directed against muscle fibers in FSHD. There are several similarities in the mononuclear cell subsets at perivascular sites in FSHD and DM,*,14i.e., the high percentage of B cells, the high T4/T8 ratios, and the close proximity of T 4 + cells to B cells. Nonnecrotic muscle fibers invaded by mononuclear cells are also essentially absent in both diseases (CTL-mediated muscle fiber injury is thus unlikely in these diseases). However, w e found no complement deposition on blood vessels in FSHD (data not shown). Perifascicular atrophy and ischemic changes or infarction of muscle are not observed in FSHD. Therefore, the immune effector mechanism of FSHD must be different from that observed in DM. Comparison of the immunocytochemical findings in FSHD and DMD is of interest, since patients with these muscular dystrophies often show zyxwvutsr zyxwvuts FIGURE 6. Genetic analysis of FSHD patients in whose muscle specimens numerous inflammatory infiltrates were shown. Southern blot tests for EcoR1-digested genomic DNA from the families of cases 1 and 2 (pFR-1 probe). Both the father of case 1 (lane 1: case 3) and his son (lane 3: case 1) had small (17 kb) EcoRl fragment (arrow). Case 2 (lane 8) showed a small (13 kb) EcoRl fragment (arrowhead) which was absent in both parents (lanes 5 and 6 ) , suggesting the presence of a de novo DNA rearrangement in the patient. iar and sporadic patients. However, patients with polymyositis who have facial muscle involvement (about 8-1 1%) have been rep~rted,"~" but we do not know whether the disease in these reported patients is linked to chromosome 4q. We speculate that at least some of the familiar patients with polymyositis who have facial and shoulder-girdle ~ e a k n e s s ~ and , ~ , *the ~ sporadic patients who have restricted distribution in the face and shoulder musc~es7,11,19,23,29,38,44,55 represent clinical variants of chromosome 4-linked FSHD. Most of these patients show only limited clinical benefit with corticosteroid treatment, and have progressive muscle WeakneSS.23,29,30,32,38,55 The nature of the inflammatory response in FSHD muscle seems to differ from that observed in inflammatory myopathies (Fig. 8). In polymyositis (PM) and inclusion body myositis (IBM), but not in dermatoinyositis (DM), cytotoxic T-cell FacioscapulohumeralMuscular Dystrophy MUSCLE & NERVE Supplement 2 1995 563 Macrophages Nec fibers \ 5 i zyx zyxwvutsrqponm y 7 cells 100 200 Nonnecrotic fibers invaded by mononuclear cells 300 /SO0 60D, zyxwvutsrq FIGURE 8. Endomysial T cell counts, frequency of necrotic muscle fibers, and frequency of nonnecrotic muscle fibers invaded by mononuclear cells per 1000 muscle fibers in inclusion body myositis (IBM), polymyositis (PM), Duchenne muscular dystrophy (DMD), and FSHD. Values represent means in 8 biopsy specimens each of IBM, PM, DMD,3 and 18 biopsy specimens of FSHD. zy myopathy with FSH distribution numerous inflammatory infiltrates in m ~ s c l e . ~ , ' ~ 'inflammatory ~~ shows rapid progression of weakness, occasional myIn DMD, although the number of nonnecrotic fialgia, and marked to moderately elevated serum CK bers invaded by T cells is low and the invading cells level, which may respond transiently to corticosteremain at a superficial level, it is possible that the roid treatment but not sustained.23,29930,32,38.5j CTL-mediated immune effector mechanism may Finally, the finding that abundant T cells accuexis t2,3,14 (Fig. 8). In contrast, in FSHD, only demulate in the perivascular site of FSHD muscle is generating fibers are focally surrounded and suof note. In the blood vessels, occasional mononuperficially invaded by mononuclear cells (Fig. 2), clear cells were observed moving through vessel nonnecrotic fibers are not invaded by mononuwalls (Fig. 4). The migration of mononuclear cells clear cells (Fig. 8), and the surfaces of the adjacent from the bloodstream is known to be initiated by a muscle fibers appear normal (Fig. 4). Amounts of necrotic fibers also differ in the two diseases. DMD variety of signaling molecules produced locally by cells (mainly in connective tissue) or by complemuscles have 4.1 times more necrotic fibers than ment activation, by which vessel walls become more FSHD (Fig. 8), but the number of endomysial inflammatory T cells is 4.7 times greater in FSHD permeable.' In 50-70% of FSHD patients, exuda(Fig. 8).2,3,14 These findings suggest that the cellutive retinal vasculopathy with capillary telangiectalar infiltrations in FSHD muscle do not simply sis, microaneurysms, and capillary closure have represent nonspecific events triggered by muscle been reported.".l6 Although retinal vasculopathy fiber necrosis of dystrophic origin. Therefore, has not been proven as part of the disease, associit is conceivable that different immune effector ation of the two diseases is extremely interesting. mechanisms may be responsible for these two disLeaky capillaries in the retina imply analogous capillary abnormality in muscle. Our immunocytoeases. We found the increasing number of necrotic chemical observations in FSHD muscle, together fibers in FSHD to parallel the increased number of with our genetic analysis of FSHD patients, suggest inflammatory cells (Fig. 1). Thus, it appears that the presence of a T-cell-mediated effector reinflammatory infiltrates in FSHD may accelerate sponse directed against either connective tissue secondary muscle fiber breakdown, since the celand/or vascular elements and/or altered muscle filular response in FSHD may not be caused simply ber component(s) produced by dystrophic muscle by muscle fiber necrosis, as discussed above. This fiber degeneration or by other means, in FSHD possibility is supported by clinical observations that muscle. zyxwvu '' S64 Facioscapulohurneral Muscular Dystrophy MUSCLE & NERVE Supplement 2 1995 zyxwvutsrqp zyxwvutsrqponm zyxwvutsrqpon zyxwvutsrqpo REFERENCES 1. Albert B, Bray D, Lewis J, Raff M, Roberts K, Watson J D (eds): Molecular Biology of the Cell (3rd ed). New York, Garland Publishing Co., Inc., 1944, pp 1164-1 165. 2. Arahata K, Engel AG: Monoclonal antibody analysis of mononuclear cells in myopathies. 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