Myosin in striated muscle exists in at least two immunologically distinct forms. Human autoantibodies specific for either form show selective muscle fiber reactivity. One group of antibodies reacts with type 2b (white) muscle fibers, the other with type 2a (red) and type 1 (intermediate) fibers. All of these antibodies react with the A band of glycerinated myofibrils. Both groups of antibodies react with cardiac muscle. Muscle-fiber-specific antibodies are detected in approximately 1% of sera submitted for diagnostic screening. Sera from eight patients were studied further. The two patients with antibodies against type 2b (white) muscle myosin had rheumatoid arthritis. The six patients with antibodies against type 2a (red) and type 1 (intermediate) myosin had clinical features which included various autoimmune and infectious disorders. MUSCLE 81NERVE 2:37-43 1979
MYOSIN AUTOANTIBODIES REACT1NG WITH SELECTIVE MUSCLE FIBER TYPES BRIAN L. McDONALD, BSc, ROGER L. DAWKINS, MD, and JENNIFER ROBINSON, AAlMT
Autoantibodies reactive with striated muscle can be classified into three categories. First, there is the antistriated (antistriational) muscle antibody associated with a thyrnorna.’” ‘The second group is characterized by reactivity with smooth as well as striated ~ n u s c l eThird, .~ thcrc are the “zebra” antibodies, so called because of their striped, fibertype-specific reaction pattern on longitudinally sectioned muscle of mixed histochemical fiber type.5 The present article describes a subcategory of fiber-selective antibodies characterized by their reactivity with myosin, and distinguished from zebra antibodies by t.heir striationa! pattern on glycerinated myofibrils. One example of this type of antibody has been r e p o r t ~ dWe . ~ present eight fur-
From the Department of Clinical Immunology Royal Perth Hospital. Perih Western Australia Acknowledgments The authors wish to acknowledge Mr H Whyte lor 111s technical assistance with the histochemistry, Mr H Upenicks for his aid in the preparation of photographs and Ms J Hendriks and Mrs A Sharpe for their help in preparing the manuscript This investigation was supported by grants from the Muscular Dystrophy Associabon of America and lrom the National Health and Medical Research Council of Australia Address reprint requests to Dr Dawkins at the Department of Clinical Irn rnunology Royal Perth Hospital Perth Western Australia 6001 Rece ved for pJbliCatiOn June 27, 978 revised mawscript accepted fopublication October 17, 1978 0148-639X/0201/0037 $00 OOiO 0 1979 Houghton Mifflin Professional Publishers
Fiber-Type-Specific Autoantibodies
ther samples and compare these sera with the antibodies produced by immunization of guinea pigs with human myosin. MATERIALS AND METHODS
‘l’he antibodies reported here were found in the sera of eight patients (see table 1). Sera were selected from among approximately 1,000 Submitted for autoantibody screening. Criteria for selection in this study were (1) selective fiber staining of sectioned guinea pig mixed skeletal muscle, (2) staining of‘cardiac muscle, and (3) evidence of striational staining. Three patients had rheumatoid arthritis, and one of these patients was receiving penicillamine therapy. another two patients had liver disease. The remaining three patients had miscellaneous infections of autoimmune diseases.
Human Sera.
Inbred strain- 13 guinea pigs were immunized with human crude myosin (see below). Animals were injected subcutaneously on days 0, 14. and 21. Each immunization mixture included 50% complete Freund‘s adjuvant and 250 p g protein antigen in a total volume of 0.5 mi. Animals were bled by direct cardiac puncture while anesthetized by haloihane and an oxygen/nitrogen dioxide mixture. Early bleeds were on-day 28, and late bleeds were after day 3 5 . Experimental Sera.
MUSCLE & NERVE
JanIFeb 1979
37
Table 1. Summary of patients' clinical features. Muscle fiber ?ype specificity and occurrence of any other autoantibodies detected by immunofluorescence Patients
Clinical features"
Fiber type
Other autoantibodies
1 2 3 4
RA, thyroiditis RA (penicillarnine-treated) RA SBE PMISlviral pericaiditis Liver disease (cholestasis) Liver disease (?viral) MG (no thyrrisma)
2b (white) 2b (white) 2ail (rediintermediate) 2a'l (red: interrnediate) 2ail (redlintermediate) 2 d l (red: intermediate) 2ail (red: inte rmediate) 2 d l (rediinterrnediate)
ANAb
5 6 7 8
-
SMAC ~
-
' R A = rheumatoid arthritis, SBE = subacute bacterial endocarditis (S viridans) PM/S = postrnyocardia/infarcbon syndrome. MG = myasthenia gravis bAntrnuclearantibody CSrnooth-musc/eantbody
Crude myosin, column-purified myosin, arid C proteinHwere prepared from human thigh muscle of undefined fiber type (fig. 1). The method is summarized in figure 2. For in-vitro investigations, protein preparations were used at a concentration of 3-5 rng/rnl. All protein preparations were analyzed by SDS acrylamide-gel electropho-
Antigens.
Figure 1 . Preparation of antigens using 0.1% SDS10% polyacrylamide gels of proteins. a = crude myosin; b = column-purified myosin, c = C protein and F protein. Geis were run in the direction of the arrow. H = myosin heavy chains; C = C protein; F = F protein; L = hght chains of myosin. L a the lowest-rnoiecuiar-weight light chain, is in low concentratfor; and is poorly resolved
38
Fiber-Type-Specific Autoantibodies
resis using 10% acrylamide gels after the method of Mieber and Osborn.'2 Fluid-phase absorptions were perfbriried by incubation of 0.05 rnl serum with 0.05 ml antigen overnight at 4°C. The mixture was made up t o 0.5 ml with 0.15 M phosphate-bul'fered saline, pH 7.2. Precipitated protein was removed by centrifugation at 10,000 x g for 20 min at 4°C before the mixture was reacted with substrate for iinmunofluorexence.
Absorptions.
Human and animal sera were screened for antibodies on 6 p m freshfrozen sections of a composite block of tissues which included rat heart, stomach, liver, kidney, esophagus, niouse stomach, and guinea pig mixed skeletal muscle. Tissues were air-dried on slides before serum was applied for 30 min. After tissues were washed in phosphate-buffered saline solution (PBS) for 10 min, appropriate conjugate was applied for 30 min. After a further wash for 1 hr, tissues were mounted in 90% glycerol (pH 8.6). In addition, sera were tested on guinea pig glycerinated myofibrils'O and on sections of guinea pig muscles (soleus, gastrocnemius, and white vastus Iateralis). Human antibodies were detected by means of Burroughs Wellcome (Beckenham, England) fluorescein isothiocyanate (F1TC)-conjugated sheep antihuman IgG, IgM, and IgA, and by means of Behringwerke (Marburg, West Germany) FITCconjugated, monospecific rabbit antihuman IgG, IgM, and IgA. Guinea pig antibodies were detected by Behringwerke FITC-conjugated rabbit anti-guinea pig Ig. A Leitz Orthoplan UV/visible microscope with epifluorescence and phase-contrast optics was used. Photomicrographs were taken
Indirect Immunofluorescence.
MUSCLE & NERVE
JaniFeb 1979
HUMAN MUSCLE (STORE -70°C)
! Extracted Huxley’s buffer (0.6 M KCI, 10 mM PP,, 1 mM MgCI,)
4
SUPERNATANT
+ 14 vols distilled water PRECiPlTATE
PRECIPITATE DISCARDED
SUPERNATANT DISCARDED
Washed x 2 (Huxley’s buffer diluted 1120)
Dissolved in myosin buffer (0.4 M KCI, 0.03 M PO,, 1 mM EDTA) Ammonium sulfate to 1.40 M 4°C-1
hr
SUPERNATANT
PRECIPITATE DISCARDED
Ammonium sulfate to 1.66 M 4°C-1 hr PREClPlTATE
SUPERNATANT DISCARDED
Either
Figure 2 Preparation of thickfilament antlgeos (After the method described b y Offer G, Moos C, and Starr R A new protein o f vertebrate skeletal rnyohbrUs Extraction, purification and charactensation J Mot Bl01 74 653-676, I973 )
with a Leitz Ortholux camera on Kodak Tri-X film. Blocking Studies. T h e above procedure was modified by preincubation of the substrate with the putativc blocking serum. For example, muscle substrate was incubated with guinea pig antimyosin, after which it was washed and incubated with human serum. It was then further washed and stained with conjugated antihuman immunoglobulin. Controls included addition of anti-guinea pig conjugate to detect antibody binding during preincubation and use of normal guinea pig serum. All conjugates were absorbed exhaustively to remove any cross-species antiimmunoglobulin activity. Antihuman immunoglobulin activity in the FITC-conjugated anti-guinea pig serum was removed by overnight incubation (4°C) with equal
Fiber-Type-Specific Autoantibodies
volumes of normal human serum. Precipitate was removed by centrifugation. Conjugated antiirnmunoglobulin sera were shown not to contain crossspecies reactivity in the imrnunofluorescence system when tested against positive sera from the same species used for absorption. Plastic 50-mm-diameter Petri dishes were used; these cont.ained 4 ml of 0.5% agarose (Calbiochem A-grade 121852) in 0.4 M KC1-0.03 M phosphate buffer, pH 7.2. Center wells were 10 mni, arid the surrounding six wells 6 mm, in diameter. T h e reactants added to each well are described in the figure legends. Diffusions took place at 4°C and could usually be read by the second day.
Ouchterlony Gel Diffusion.
Histochemistry. Serial
sections near those used for immunofluorescence were stained for the oxida-
MUSCLE & NERVE
JaniFeb 1979
39
Figure 3 . indirect immunofluorescence with patients’ sera on send sections of guinea p’g gastrocnemius muscle. (A)Serum of patient 1 , showing reactivity w ~ t hthe majority of fibers. (B) Serum of pattent 5, showing reactivity with those fibers that are negative with the serum of patient 1 . x 200
tive enzyme succinate dehydrogenase, the glycolytic enzyme lactate dehydroger~ase,~ and myosin A7’Pa~e.~ Nomenclature of fiber types is based on combinatioris of enzymes. Type 2a (red) fibers are those with high oxidative, high glycolytic, and high ATPase actiyity (fast-twitch oxidati\re/glycolytic). Type 2b (white) fibers have high glycolytic, high ATPase, and low oxidative activity (fast-twitch glycolytic). ‘Type 1 (intermediate) fibers have intermediate oxidative and low ATPase activity (slowtwitch oxidative).” Enzymes wcre quantitated on the basis o f intensity o f the histochemical staining reaction. RESULTS
Immunofluorescence. All
Figure 4 ImmunoNuorescence (IF) and phase contrast (phase) of guinea p/g glycerinated myof/br//sreacted with serum from patient 1, showing a broad A band and a negative MIH zone x 1,350
eight sera were identified by their zebra (fiber-selective) pattern o f staining on muscle sections of mixed fiber type (fig. 3 ) . On examination of. longitudinally sectioned fibers, broad striational staining o f inyofibrils was apparent. On glycerinated guinea pig myofibrils, the eight sera gave a broad A-band and a negative central A-band region, which was referred to as the M/H zone because it bvas tiif’ficultto accurately assign the negative area to the I41 line or to the H zone by fluorescence (fig. 4). Since A-band staining implied reactivity with myosin 01‘ C protein, absorptions were performed. In all eight sera, activity was removed by myosin but not by C protein. All fiberselective antibodies were shown to be of the 1gG class because of their reactivity with anti-1gC and their lack of reactivity with othcr conjugates. ‘The same crude myosin absorbed all antimyosin sera.
ison between succinic dehydrogenase (SDH) reactivity and the reactivity of a n antibody from each of‘these two groups is shown in figure 5 . Kesults of these studies are summarized in table 1 . Guinea pigs imniunized with human thigh muscle myosin produced antibodies. T h e thigh muscles were not defined histochemically, but were generally of niixed fiber type. Sera from the early bleeds showed reactivity with type ‘La fibers alone. Later bleeds also showed weak reactivity with intermediate fibers (figs. 6 and 7). The sera on glycerinated fibers gave a pattern indistinguishable from the human fiber-selective antibodies. Similarly, reactivity was absorbcd with myosin but not with C protein.
Six of the eight sera reacted with type 2a (red) arid type 1 (intermediate) fibers, and two reacted with type 2b (white) fibers. A compar-
Blocking Experiments. Although the guinea pig sera and the six huinan sera reacted with in yo sir^ from the same fiber types, no blocking could be dem-
Histochemistry.
40
Fiber-Type-Speciflc Autoantibodies
MUSCLE 8 NERVE
JaniFeb 1979
Figure 5 Serial sections of two guinea pig gastrocnemius muscies (A) and (C) in transverse section (A) stained for succinic dehydrogenase (SDH) and (C) stained by /nd/rectimmunofiuorescence w/th serum of patient 1 This shows antibody reactivity with the fibers that have low SDH activity Converseiy, in seriai sections cut in longitudinal section (B) and (D), the fiber showing the highest SDH activity is that fiber with which a guinea pig antimyosin antibody reacts by immunofluorescence (0) x 200
Figure 6 lmmunoiiuorescence on serial sections of guinea pig gastrocnemius mclscie (A) Guinea pig antimyosin (B) Serum of patient 5, showing stainmg of the same fiber types x 200
Figure 7 Indirect immunofhorescence on cross sectionai guinea pig gastrocnemius muscle The serum is from a guinea pig five weeks after immunization with myosin Fiber-speofic activity ranges from 3 + intensity to negative x 250
Fiber-Type-Specific Autoantibodies
MUSCLE & NERVE
Jan/Feb 1979
41
Table 2. Blocking studies irnmunofluorescence results ofexperiments with hu-ian and guinea pig sera on the same sections of guinea pig muscle
First serum Patient 1 Patient 6 Patient 6 GP GP GP
Second serum
Conluqate
GPb GP GP Patient 1 Patient 6 Patient 6
Anti-GPC Anti-GP Antihuman Antihuman Antihuman Anti-GP
IFa result
3+ 3+ 3+il+
3+ 3+il+ 3+
Fiber type 2a (red) 2a (red) 2atl (red/intermediate) 2b (white) 2a 1 (red: inte rmediate) 2a (red)
YF
= immunofluwescence. bGP = guinea pig
"Conjugate ant/-GP = NTC-labeied anb-guinea pig /mmunog/obuhn
onstrated by immunofluorescence. However, both antibodies were shown to be bound to the same fibers (table 2). All sera showing fiberselective striational staining gave a precipitation line with crude myosin, purified myosin, and crude actomyosin, but not with C protein or thin-filament preparation (fig. 8). Activity was removed by absorption with myosin. All myosin preparations used contained light and heavy chains.
Ouchterlony Gel Diffusion.
DISCUSSION
The human and experimental antibodies reported here are characterized by their reactivity with alternate striations of particular muscle fiber types and by their specificity for myosin. The cxperimental antibodies were produced by immunization of guinea pigs with crude myosin. The human antibodies were detected in approximately 1% of sera submitted for diagnostic screening. In most respects, the experimental and human antibodies were identical, but blocking could not be demonstrated. Although these antibodies have been shown to react with myosin, the precise antigenic determinant remains to be established. Since the reaction pattern was very similar to that demonstrated histochemically, it was concluded that the precise specificity was related to the myosin isoenzymes previously characterized by biochemical and histochemical approaches.1-6All of the experimental sera reacted with type 2a and type I fibers. Of the eight human sera, six were similar, but two reacted with type 2b (white) fibers." Fairfax and GroschelStewart4 have previously described a patient with Coxsackie pericarditis and an autoantibody specific for the myosin of red (type 2a) fibers. Experimental antibodies against red (type 2a) and intermediate (type 1) fibers'*6and against white (type 2b) fibers' have been reported.
42
Fiber-Type-Specific Autoantibodies
Figure 8. Ouchterlony gel diffusion of a fiber-selective striafional antibody. Well 1 = crude myosin; 2 = pure myosin; 3 = C protein; 4 = pure myosin, 5 = actomyosin preparation, 6 = buffer. Reactions can b e seen with crude myosin and pure myosin. A very weak precipitation was found with actomyosin, aifhough it is not visible in this photograph. None was found with C protein.
In the past, the antibodies described here may have been confused with the nonstrialional zebra antibodies described by Feltkarnp and FeltkampVr0om.j Use of glycerinated fibers can facilitate differentiation of the two categories of fiber-selecrive antibodies. The antimyosin antibodies gave Aband staining, whereas true Lebra antibodies are negative and will only be detected if sections of mixed muscle are used. The fiber-selective striational antibodies occur approximately four times as frequently as the zebra antibodies. It is of some interest that six of the eight human antimyosins reacted with type 2dtype 1 (redhtermediate) rather than type 2b (white) myosin, and that the corresponding experimental antibodies had similar reactivity. In contrast, zebra antibodies react more frequently with type 2b (white) fibers, as do experimental antibodies produced by immuniza-
MUSCLE & NERVE
JaniFeb 1979
tion with the mitochondria1 fraction of skeletal muscle.' T h e clinical significance of human antimyosin antibodies is quite different from that of antistriational antibodies found in association with a thymoma, a n d recognition o f t h e two groups is essential to maintenance of the high diagnostic specificity that a n antistriational antibody has for a thymoma. Of the present cases, t w o patients with rheumatoid arthritis had antibodies to myosin from type 2b fibers. T h e remaining six patients with type 'Ldtype
1 myosin antibodies were heterogeneous in terms of their clinical features. Interestingly, the one patient with myasthenia gravis had n o evidence of a thymoma o r of the antistriational antibody that one would expect to find with a thymoma.1° l'hree of the six patients may have had viral infections and may have similarities with the patient described p r e v i o ~ s l y .T~h e association between antiactin arid virus infections is well established arid may be a mechanism involved in the production of these antimyosin antibodies. . I
REFERENCES 1. Arndt I, Pepe F 4 : Antigenic specificity of red and whit, muscle myosin. J Hbtochern Cytorlwm 23:159-168, 1975. 2. Dawkins RL, Gihb DGA, Holt PG: Ba,wRnunrch tn ,M~oEogj: Inlerridionol Cungrerc Sene.\ .\.'o. 294. Amsterdam, Excerpla Medica, 197 1, pp 605-609. 3. DubowitL V,Brooke MH: ,Muscle Biopsj: A 'Vludern Approuch, V o l 2 . Ncw York, WB Saunders, 1973, pp 31-32. 4. Fairfax AJ, Groschel-Stewart U: Myosin autoantibodies detected by immunofluorescence. Clin Exp Irnmunol28:27--34, 1977. 5. Feltkarnp T E W , Feltkamp-Vroom T M : Antibodies against, the various types of skeletal muxle fibres. I m m z t n o l u ~9:275279, 1965. 6. Groschel Stewart U, Doniach D: Immunological evidence tor human myosin isoenzymes. Irnrnunologr 17:99 1-994, 1969. 7. McDonald BL, Dawkins RL, Holhorow EJ: Evidence for
Fiber-Type-Specific Autoantibodies
8. 9. 10. 11.
12.
irrimLinologicalcross reactivity hetweeri smooth and skeletal muscle. C h Exp Immicnol 27:269-272. 1977. Ofler G, Moos C, Starr R: A new protein o f vertebrate skcleta1 nryofibrils. Extraction, put-ification and characterisati0n.J .\.lo/ B i d 743653-616, 1973. Pearce AGV: H?storhemzstr~,3rd ed. London, Churchill Livingstone, 1972, pp 1342-1347. Peers J, McDonald BL, Dawkins RL: T h e reactivity of the aritistriational antibodics associated with th\,moma and myasthenia grai-is. Cljn Exp IlnVlMn(Jl 27:66-73. 197'7. Peter JB, Barnard KJ, Edgerton VR: Gillespie CA, Stempel KE: Metabolic profiles of three fibre types of skeletal muscle in guinea pigs and rabbits. Biochpmistry 11:2627-2633, 1972. Weher K , Osborn M : The reliability of molecular weight determinations by dodecyl sulphate polyacrylamide gel e1ectrophoresis.J Bid C h r m 244:4406-4412, 1969.
MUSCLE & NERVE
JaniFeb 1979
43
MYOSIN AUTOANTIBODIES REACT1NG WITH SELECTIVE MUSCLE FIBER TYPES BRIAN L. McDONALD, BSc, ROGER L. DAWKINS, MD, and JENNIFER ROBINSON, AAlMT
Autoantibodies reactive with striated muscle can be classified into three categories. First, there is the antistriated (antistriational) muscle antibody associated with a thyrnorna.’” ‘The second group is characterized by reactivity with smooth as well as striated ~ n u s c l eThird, .~ thcrc are the “zebra” antibodies, so called because of their striped, fibertype-specific reaction pattern on longitudinally sectioned muscle of mixed histochemical fiber type.5 The present article describes a subcategory of fiber-selective antibodies characterized by their reactivity with myosin, and distinguished from zebra antibodies by t.heir striationa! pattern on glycerinated myofibrils. One example of this type of antibody has been r e p o r t ~ dWe . ~ present eight fur-
From the Department of Clinical Immunology Royal Perth Hospital. Perih Western Australia Acknowledgments The authors wish to acknowledge Mr H Whyte lor 111s technical assistance with the histochemistry, Mr H Upenicks for his aid in the preparation of photographs and Ms J Hendriks and Mrs A Sharpe for their help in preparing the manuscript This investigation was supported by grants from the Muscular Dystrophy Associabon of America and lrom the National Health and Medical Research Council of Australia Address reprint requests to Dr Dawkins at the Department of Clinical Irn rnunology Royal Perth Hospital Perth Western Australia 6001 Rece ved for pJbliCatiOn June 27, 978 revised mawscript accepted fopublication October 17, 1978 0148-639X/0201/0037 $00 OOiO 0 1979 Houghton Mifflin Professional Publishers
Fiber-Type-Specific Autoantibodies
ther samples and compare these sera with the antibodies produced by immunization of guinea pigs with human myosin. MATERIALS AND METHODS
‘l’he antibodies reported here were found in the sera of eight patients (see table 1). Sera were selected from among approximately 1,000 Submitted for autoantibody screening. Criteria for selection in this study were (1) selective fiber staining of sectioned guinea pig mixed skeletal muscle, (2) staining of‘cardiac muscle, and (3) evidence of striational staining. Three patients had rheumatoid arthritis, and one of these patients was receiving penicillamine therapy. another two patients had liver disease. The remaining three patients had miscellaneous infections of autoimmune diseases.
Human Sera.
Inbred strain- 13 guinea pigs were immunized with human crude myosin (see below). Animals were injected subcutaneously on days 0, 14. and 21. Each immunization mixture included 50% complete Freund‘s adjuvant and 250 p g protein antigen in a total volume of 0.5 mi. Animals were bled by direct cardiac puncture while anesthetized by haloihane and an oxygen/nitrogen dioxide mixture. Early bleeds were on-day 28, and late bleeds were after day 3 5 . Experimental Sera.
MUSCLE & NERVE
JanIFeb 1979
37
Table 1. Summary of patients' clinical features. Muscle fiber ?ype specificity and occurrence of any other autoantibodies detected by immunofluorescence Patients
Clinical features"
Fiber type
Other autoantibodies
1 2 3 4
RA, thyroiditis RA (penicillarnine-treated) RA SBE PMISlviral pericaiditis Liver disease (cholestasis) Liver disease (?viral) MG (no thyrrisma)
2b (white) 2b (white) 2ail (rediintermediate) 2a'l (red: interrnediate) 2ail (redlintermediate) 2 d l (red: intermediate) 2ail (red: inte rmediate) 2 d l (rediinterrnediate)
ANAb
5 6 7 8
-
SMAC ~
-
' R A = rheumatoid arthritis, SBE = subacute bacterial endocarditis (S viridans) PM/S = postrnyocardia/infarcbon syndrome. MG = myasthenia gravis bAntrnuclearantibody CSrnooth-musc/eantbody
Crude myosin, column-purified myosin, arid C proteinHwere prepared from human thigh muscle of undefined fiber type (fig. 1). The method is summarized in figure 2. For in-vitro investigations, protein preparations were used at a concentration of 3-5 rng/rnl. All protein preparations were analyzed by SDS acrylamide-gel electropho-
Antigens.
Figure 1 . Preparation of antigens using 0.1% SDS10% polyacrylamide gels of proteins. a = crude myosin; b = column-purified myosin, c = C protein and F protein. Geis were run in the direction of the arrow. H = myosin heavy chains; C = C protein; F = F protein; L = hght chains of myosin. L a the lowest-rnoiecuiar-weight light chain, is in low concentratfor; and is poorly resolved
38
Fiber-Type-Specific Autoantibodies
resis using 10% acrylamide gels after the method of Mieber and Osborn.'2 Fluid-phase absorptions were perfbriried by incubation of 0.05 rnl serum with 0.05 ml antigen overnight at 4°C. The mixture was made up t o 0.5 ml with 0.15 M phosphate-bul'fered saline, pH 7.2. Precipitated protein was removed by centrifugation at 10,000 x g for 20 min at 4°C before the mixture was reacted with substrate for iinmunofluorexence.
Absorptions.
Human and animal sera were screened for antibodies on 6 p m freshfrozen sections of a composite block of tissues which included rat heart, stomach, liver, kidney, esophagus, niouse stomach, and guinea pig mixed skeletal muscle. Tissues were air-dried on slides before serum was applied for 30 min. After tissues were washed in phosphate-buffered saline solution (PBS) for 10 min, appropriate conjugate was applied for 30 min. After a further wash for 1 hr, tissues were mounted in 90% glycerol (pH 8.6). In addition, sera were tested on guinea pig glycerinated myofibrils'O and on sections of guinea pig muscles (soleus, gastrocnemius, and white vastus Iateralis). Human antibodies were detected by means of Burroughs Wellcome (Beckenham, England) fluorescein isothiocyanate (F1TC)-conjugated sheep antihuman IgG, IgM, and IgA, and by means of Behringwerke (Marburg, West Germany) FITCconjugated, monospecific rabbit antihuman IgG, IgM, and IgA. Guinea pig antibodies were detected by Behringwerke FITC-conjugated rabbit anti-guinea pig Ig. A Leitz Orthoplan UV/visible microscope with epifluorescence and phase-contrast optics was used. Photomicrographs were taken
Indirect Immunofluorescence.
MUSCLE & NERVE
JaniFeb 1979
HUMAN MUSCLE (STORE -70°C)
! Extracted Huxley’s buffer (0.6 M KCI, 10 mM PP,, 1 mM MgCI,)
4
SUPERNATANT
+ 14 vols distilled water PRECiPlTATE
PRECIPITATE DISCARDED
SUPERNATANT DISCARDED
Washed x 2 (Huxley’s buffer diluted 1120)
Dissolved in myosin buffer (0.4 M KCI, 0.03 M PO,, 1 mM EDTA) Ammonium sulfate to 1.40 M 4°C-1
hr
SUPERNATANT
PRECIPITATE DISCARDED
Ammonium sulfate to 1.66 M 4°C-1 hr PREClPlTATE
SUPERNATANT DISCARDED
Either
Figure 2 Preparation of thickfilament antlgeos (After the method described b y Offer G, Moos C, and Starr R A new protein o f vertebrate skeletal rnyohbrUs Extraction, purification and charactensation J Mot Bl01 74 653-676, I973 )
with a Leitz Ortholux camera on Kodak Tri-X film. Blocking Studies. T h e above procedure was modified by preincubation of the substrate with the putativc blocking serum. For example, muscle substrate was incubated with guinea pig antimyosin, after which it was washed and incubated with human serum. It was then further washed and stained with conjugated antihuman immunoglobulin. Controls included addition of anti-guinea pig conjugate to detect antibody binding during preincubation and use of normal guinea pig serum. All conjugates were absorbed exhaustively to remove any cross-species antiimmunoglobulin activity. Antihuman immunoglobulin activity in the FITC-conjugated anti-guinea pig serum was removed by overnight incubation (4°C) with equal
Fiber-Type-Specific Autoantibodies
volumes of normal human serum. Precipitate was removed by centrifugation. Conjugated antiirnmunoglobulin sera were shown not to contain crossspecies reactivity in the imrnunofluorescence system when tested against positive sera from the same species used for absorption. Plastic 50-mm-diameter Petri dishes were used; these cont.ained 4 ml of 0.5% agarose (Calbiochem A-grade 121852) in 0.4 M KC1-0.03 M phosphate buffer, pH 7.2. Center wells were 10 mni, arid the surrounding six wells 6 mm, in diameter. T h e reactants added to each well are described in the figure legends. Diffusions took place at 4°C and could usually be read by the second day.
Ouchterlony Gel Diffusion.
Histochemistry. Serial
sections near those used for immunofluorescence were stained for the oxida-
MUSCLE & NERVE
JaniFeb 1979
39
Figure 3 . indirect immunofluorescence with patients’ sera on send sections of guinea p’g gastrocnemius muscle. (A)Serum of patient 1 , showing reactivity w ~ t hthe majority of fibers. (B) Serum of pattent 5, showing reactivity with those fibers that are negative with the serum of patient 1 . x 200
tive enzyme succinate dehydrogenase, the glycolytic enzyme lactate dehydroger~ase,~ and myosin A7’Pa~e.~ Nomenclature of fiber types is based on combinatioris of enzymes. Type 2a (red) fibers are those with high oxidative, high glycolytic, and high ATPase actiyity (fast-twitch oxidati\re/glycolytic). Type 2b (white) fibers have high glycolytic, high ATPase, and low oxidative activity (fast-twitch glycolytic). ‘Type 1 (intermediate) fibers have intermediate oxidative and low ATPase activity (slowtwitch oxidative).” Enzymes wcre quantitated on the basis o f intensity o f the histochemical staining reaction. RESULTS
Immunofluorescence. All
Figure 4 ImmunoNuorescence (IF) and phase contrast (phase) of guinea p/g glycerinated myof/br//sreacted with serum from patient 1, showing a broad A band and a negative MIH zone x 1,350
eight sera were identified by their zebra (fiber-selective) pattern o f staining on muscle sections of mixed fiber type (fig. 3 ) . On examination of. longitudinally sectioned fibers, broad striational staining o f inyofibrils was apparent. On glycerinated guinea pig myofibrils, the eight sera gave a broad A-band and a negative central A-band region, which was referred to as the M/H zone because it bvas tiif’ficultto accurately assign the negative area to the I41 line or to the H zone by fluorescence (fig. 4). Since A-band staining implied reactivity with myosin 01‘ C protein, absorptions were performed. In all eight sera, activity was removed by myosin but not by C protein. All fiberselective antibodies were shown to be of the 1gG class because of their reactivity with anti-1gC and their lack of reactivity with othcr conjugates. ‘The same crude myosin absorbed all antimyosin sera.
ison between succinic dehydrogenase (SDH) reactivity and the reactivity of a n antibody from each of‘these two groups is shown in figure 5 . Kesults of these studies are summarized in table 1 . Guinea pigs imniunized with human thigh muscle myosin produced antibodies. T h e thigh muscles were not defined histochemically, but were generally of niixed fiber type. Sera from the early bleeds showed reactivity with type ‘La fibers alone. Later bleeds also showed weak reactivity with intermediate fibers (figs. 6 and 7). The sera on glycerinated fibers gave a pattern indistinguishable from the human fiber-selective antibodies. Similarly, reactivity was absorbcd with myosin but not with C protein.
Six of the eight sera reacted with type 2a (red) arid type 1 (intermediate) fibers, and two reacted with type 2b (white) fibers. A compar-
Blocking Experiments. Although the guinea pig sera and the six huinan sera reacted with in yo sir^ from the same fiber types, no blocking could be dem-
Histochemistry.
40
Fiber-Type-Speciflc Autoantibodies
MUSCLE 8 NERVE
JaniFeb 1979
Figure 5 Serial sections of two guinea pig gastrocnemius muscies (A) and (C) in transverse section (A) stained for succinic dehydrogenase (SDH) and (C) stained by /nd/rectimmunofiuorescence w/th serum of patient 1 This shows antibody reactivity with the fibers that have low SDH activity Converseiy, in seriai sections cut in longitudinal section (B) and (D), the fiber showing the highest SDH activity is that fiber with which a guinea pig antimyosin antibody reacts by immunofluorescence (0) x 200
Figure 6 lmmunoiiuorescence on serial sections of guinea pig gastrocnemius mclscie (A) Guinea pig antimyosin (B) Serum of patient 5, showing stainmg of the same fiber types x 200
Figure 7 Indirect immunofhorescence on cross sectionai guinea pig gastrocnemius muscle The serum is from a guinea pig five weeks after immunization with myosin Fiber-speofic activity ranges from 3 + intensity to negative x 250
Fiber-Type-Specific Autoantibodies
MUSCLE & NERVE
Jan/Feb 1979
41
Table 2. Blocking studies irnmunofluorescence results ofexperiments with hu-ian and guinea pig sera on the same sections of guinea pig muscle
First serum Patient 1 Patient 6 Patient 6 GP GP GP
Second serum
Conluqate
GPb GP GP Patient 1 Patient 6 Patient 6
Anti-GPC Anti-GP Antihuman Antihuman Antihuman Anti-GP
IFa result
3+ 3+ 3+il+
3+ 3+il+ 3+
Fiber type 2a (red) 2a (red) 2atl (red/intermediate) 2b (white) 2a 1 (red: inte rmediate) 2a (red)
YF
= immunofluwescence. bGP = guinea pig
"Conjugate ant/-GP = NTC-labeied anb-guinea pig /mmunog/obuhn
onstrated by immunofluorescence. However, both antibodies were shown to be bound to the same fibers (table 2). All sera showing fiberselective striational staining gave a precipitation line with crude myosin, purified myosin, and crude actomyosin, but not with C protein or thin-filament preparation (fig. 8). Activity was removed by absorption with myosin. All myosin preparations used contained light and heavy chains.
Ouchterlony Gel Diffusion.
DISCUSSION
The human and experimental antibodies reported here are characterized by their reactivity with alternate striations of particular muscle fiber types and by their specificity for myosin. The cxperimental antibodies were produced by immunization of guinea pigs with crude myosin. The human antibodies were detected in approximately 1% of sera submitted for diagnostic screening. In most respects, the experimental and human antibodies were identical, but blocking could not be demonstrated. Although these antibodies have been shown to react with myosin, the precise antigenic determinant remains to be established. Since the reaction pattern was very similar to that demonstrated histochemically, it was concluded that the precise specificity was related to the myosin isoenzymes previously characterized by biochemical and histochemical approaches.1-6All of the experimental sera reacted with type 2a and type I fibers. Of the eight human sera, six were similar, but two reacted with type 2b (white) fibers." Fairfax and GroschelStewart4 have previously described a patient with Coxsackie pericarditis and an autoantibody specific for the myosin of red (type 2a) fibers. Experimental antibodies against red (type 2a) and intermediate (type 1) fibers'*6and against white (type 2b) fibers' have been reported.
42
Fiber-Type-Specific Autoantibodies
Figure 8. Ouchterlony gel diffusion of a fiber-selective striafional antibody. Well 1 = crude myosin; 2 = pure myosin; 3 = C protein; 4 = pure myosin, 5 = actomyosin preparation, 6 = buffer. Reactions can b e seen with crude myosin and pure myosin. A very weak precipitation was found with actomyosin, aifhough it is not visible in this photograph. None was found with C protein.
In the past, the antibodies described here may have been confused with the nonstrialional zebra antibodies described by Feltkarnp and FeltkampVr0om.j Use of glycerinated fibers can facilitate differentiation of the two categories of fiber-selecrive antibodies. The antimyosin antibodies gave Aband staining, whereas true Lebra antibodies are negative and will only be detected if sections of mixed muscle are used. The fiber-selective striational antibodies occur approximately four times as frequently as the zebra antibodies. It is of some interest that six of the eight human antimyosins reacted with type 2dtype 1 (redhtermediate) rather than type 2b (white) myosin, and that the corresponding experimental antibodies had similar reactivity. In contrast, zebra antibodies react more frequently with type 2b (white) fibers, as do experimental antibodies produced by immuniza-
MUSCLE & NERVE
JaniFeb 1979
tion with the mitochondria1 fraction of skeletal muscle.' T h e clinical significance of human antimyosin antibodies is quite different from that of antistriational antibodies found in association with a thymoma, a n d recognition o f t h e two groups is essential to maintenance of the high diagnostic specificity that a n antistriational antibody has for a thymoma. Of the present cases, t w o patients with rheumatoid arthritis had antibodies to myosin from type 2b fibers. T h e remaining six patients with type 'Ldtype
1 myosin antibodies were heterogeneous in terms of their clinical features. Interestingly, the one patient with myasthenia gravis had n o evidence of a thymoma o r of the antistriational antibody that one would expect to find with a thymoma.1° l'hree of the six patients may have had viral infections and may have similarities with the patient described p r e v i o ~ s l y .T~h e association between antiactin arid virus infections is well established arid may be a mechanism involved in the production of these antimyosin antibodies. . I
REFERENCES 1. Arndt I, Pepe F 4 : Antigenic specificity of red and whit, muscle myosin. J Hbtochern Cytorlwm 23:159-168, 1975. 2. Dawkins RL, Gihb DGA, Holt PG: Ba,wRnunrch tn ,M~oEogj: Inlerridionol Cungrerc Sene.\ .\.'o. 294. Amsterdam, Excerpla Medica, 197 1, pp 605-609. 3. DubowitL V,Brooke MH: ,Muscle Biopsj: A 'Vludern Approuch, V o l 2 . Ncw York, WB Saunders, 1973, pp 31-32. 4. Fairfax AJ, Groschel-Stewart U: Myosin autoantibodies detected by immunofluorescence. Clin Exp Irnmunol28:27--34, 1977. 5. Feltkarnp T E W , Feltkamp-Vroom T M : Antibodies against, the various types of skeletal muxle fibres. I m m z t n o l u ~9:275279, 1965. 6. Groschel Stewart U, Doniach D: Immunological evidence tor human myosin isoenzymes. Irnrnunologr 17:99 1-994, 1969. 7. McDonald BL, Dawkins RL, Holhorow EJ: Evidence for
Fiber-Type-Specific Autoantibodies
8. 9. 10. 11.
12.
irrimLinologicalcross reactivity hetweeri smooth and skeletal muscle. C h Exp Immicnol 27:269-272. 1977. Ofler G, Moos C, Starr R: A new protein o f vertebrate skcleta1 nryofibrils. Extraction, put-ification and characterisati0n.J .\.lo/ B i d 743653-616, 1973. Pearce AGV: H?storhemzstr~,3rd ed. London, Churchill Livingstone, 1972, pp 1342-1347. Peers J, McDonald BL, Dawkins RL: T h e reactivity of the aritistriational antibodics associated with th\,moma and myasthenia grai-is. Cljn Exp IlnVlMn(Jl 27:66-73. 197'7. Peter JB, Barnard KJ, Edgerton VR: Gillespie CA, Stempel KE: Metabolic profiles of three fibre types of skeletal muscle in guinea pigs and rabbits. Biochpmistry 11:2627-2633, 1972. Weher K , Osborn M : The reliability of molecular weight determinations by dodecyl sulphate polyacrylamide gel e1ectrophoresis.J Bid C h r m 244:4406-4412, 1969.
MUSCLE & NERVE
JaniFeb 1979
43
