- Email: [email protected]
Smooth muscle antibodies of actin and “non-actin” specificity
<:L.,N,<‘,\L
IMIIUN~LOG’~
Smooth
AND
Muscle
P. KURKI,* “Department +Department
9, 443-453 (1978)
IMMUNOPATHOL.OGY
Antibodies of Actin and “Non-actin” Specificity
E. LINDER,*
of Serolo,qy
of Urology.
A. MIETTINEN,*
AND 0. ALFTHAN~
and Bacteriology. Uni\,ersity of He/sin&. Finland. and Second Surgical Clinic, Uni\,ersity Central Hospital o.f Helsinki. Finlund
Received July 13. I977 The specificity and immunoglobulin class of smooth muscle antibodies (SMA) were studied in the sera of patients with urogenital cancer, acute respiratory infection. and chronic hepatitis and in the sera of blood donors by the indirect immunofluorescence technique (IFL). SMA activity could be neutralized with skeletal muscle actin in all 20 sera from patients with chronic hepatitis but in only 21 out of 57 other SMA-positive sera. The IFL staining patterns of anti-actin and anti-“non-actin” SMA were different; antiactin antibodies reacted with the glomerular mesangia and 1 bands of myofibrils, whereas anti- “non-actin” antibodies reacted with the periphery of myofibrils and with intercalated discs. The specificity and diagnostic significance of SMA are discussed.
INTRODUCTION
Smooth muscle antibodies (SMA) are of diagnostic importance in liver diseases (1) but occur also in malignancies (Z), in chronic renal disease (3), in some viral infections (4-lo), and in Mycoplasrna pneunzoniae infections (11). SMA can also be demonstrated in a considerable percentage of normal subjects (2, 3). SMA in chronic active hepatitis and carcinoma are mainly of the IgG class (2. 12), whereas viral infections are often followed by the transient appearance of IgM-SMA (4, 5). The antibody specificities of SMA are incompletely characterized. The antigen in chronic active hepatitis appears to be actin (13, 14). but SMA in other diseases may have additional specificities. This is suggested by the observation that SMA activity in some sera cannot be neutralized with actin (15-19) and by the different IFL staining patterns produced by individual SMApositive sera (15, 19). To evaluate the diagnostic signfkance of different types of SMA, we have investigated the specificity and immunoglobulin class of SMA in sera from patients with chronic hepatitis, urogenital cancer, and acute respiratory infections and the sera of blood donors. This was done by absorption experiments and by observing different staining patterns of SMA-positive sera by indirect immunofluorescence (IFL). MATERIALS
AND METHODS
Serum samples were obtained from 140 patients with urogenital carcinoma, 48 patients with acute respiratory tract infection. and 79 blood donors. The diagnostic criteria for urogenital carcinoma in this material have been described earlier (3). The diagnosis of acute respiratory infection was made by a physician and was 443 0090-1?29/78/0094-0443$01.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproductmn m any form reserved
444
KlRKl
El
41
based on anamnesis and physical examination. In addition. 20 SMA-positive XIX from patients with chronic hepatitis were examined. These sera were sent to the Department of Serology and Bacteriology, University of Helsinki, for routine tissue antibody determinations. Tissues
The tissue specimens were snap-frozen in isopentane cooled with liquid nitrogen. Unfixed, 5-pm cryostat sections of rat liver, kidney, stomach, heart, and skeletal muscle were used as the substrates for the immunofluorescence studies. Cryostat sections fixed in acetone at -20°C for IO min or in 2% formaldehyde at room temperature for IO min were used in some experiments. Skeletal Muscle
Myojihi1.s
Skeletal muscle myofibrils were isolated from glycerinated muscle (16). Rat skeletal muscle was cleaned from the surrounding connective tissue and immersed for 48 hr in a solution containing 50% glycerol in 0.1 M KCI. 1 mM MgCl,, IO n-~&1 imidazole, pH 7.0, cut into 3-mm pieces, and homogenized in a Dual1 30 homogenizer (Kontes Glass Co., Vineland, New Jersey). The suspension containing the isolated myofibrils was smeared onto microscope slides and fixed in acetone at -20°C for 10 min. The sera were allowed to react with cryostat sections for 30 min. Serum dilutions of 1: 10, 1:50, 1: 100, 1:500, 1:5000. and 1:50,000 were used for the determination of the SMA titer. Unless otherwise stated, the SMA-positive sera were used in a 1: 10 dilution. The fluorescein isothiocyanate (FITC) labeled sheep anti-human immunoglobulin (National Bacteriological Laboratory, Stockholm, Sweden) had a molar F/P ratio of 2.9 and contained 6.5 mg of protein/ml. Specific FITC-labeled antisera to human IgG, IgA, and IgM (Meloy Laboratories, Springfield, Virginia) had molar F/P ratios of 2.9, 2.4, and 4.2. respectively, and contained 10, IO. and 9 mg of protein/ml, respectively. A Wild M 20 uv microscope equipped with an iodine quartz lamp (Osram, 100 W). a Wild dark-field condenser, a phase-contrast condenser, and an FITC interference filter combination (Optisk Laboratorium. Lyngby. Denmark) were used. The sera were considered positive for SMA if de&rite staining was observed in the muscularis mucosae and in interglandular smooth muscle fibers of rat stomach (Fig. 3a3 and in the renal arteriolar walls of rat kidney (Fig. 3). The titers are expressed as reciprocals of the endpoint dilutions. The smooth muscle homogenate was prepared from a myomatous human uterus. The uterus was cut into pieces, ground in a meat grinder, and homogenized in phosphate-buffered saline (PBS), pH 7.4. at 4°C in a homogenizer (UltraTurrax, Janke & Kunkel, Staufen, GFR). The homogenate was centrifuged at 5OOOgfor 30 min to remove connective tissue. The remaining solution was used in absorption experiments at a concentration of 12 mg of protein/ml and stored at - 20°C. Actin was extracted from the acetone powder of bovine skeletal muscle 01
SPECIFICITY
OF
SMOOTH
MUSCLE
ANTIBODIES
445
human uterus at 4°C (20). The extraction buffer was 2 mJ4 Tris-HCl, 0.5 mM ATP, 2 m&I CaCl,, pH 7.5. This solution was concentrated and dialyzed against 0.6 M KCl, 0.5 n&f ATP, 0.5 m&I MgCl,, pH 7.0 (dialyzing buffer). F-actin was obtained from the pellet after centrifugation of the solution at 80,OOOg for 60 min. The pellet was washed twice in the dialyzing buffer and suspended in PBS. G-actin was obtained by dialyzing F-actin against the extraction buffer. After centrifugation at 80,000 for 60 mitt, the supernatant contained G-actin. The actin preparations used in absorption experiments contained 1 mg of protein/ml and 0.05% Na-azide. Actin preparations were stored at +4”C for up to 4 weeks. The composition of the actin preparations and of the smooth muscle homogenate was examined using SDS-polyacrylamide gel electrophoresis (21). The actin
a FIG. bovine
b
1. SDS-polyacrylamide gel electrophoresis skeletal muscle and (df 150 pg of human
c
d
of (a) 10 pg. (b) 20 pg. and (c) 60 pg of actin smooth .muscle homogenate. A. actin.
from
446
KlWKI
El
Al
preparation gave a single band. The actin content of the smooth muscle homogenate was approximately 20 pg/12 mg of homogenate protein, as estimated from the SDS-polyacrylamide gels loaded with smooth muscle homogenate and different concentrations of actin (Fig. I). In absorption experiments, sera with low SMA titers (s 100) were diluted 1: 10. and sera with high SMA titers (>lOO) were diluted 1: 100 in the F- or G-actin preparations or in the smooth muscle homogenate. Thus, the concentration of the smooth muscle homogenate was 120-1200 mg of protein/ml of undiluted serum. and the concentration of the actin preparations was IO-100 mg of protein/ml of undiluted serum. Serial twofold dilutions of SMA-positive sera were used when the neutralization capacities of the smooth muscle and F-actin preparations were compared. Controls were diluted in PBS or in the extraction buffer. The sera were incubated overnight at 4°C and centrifuged at SOOOgfor 5 min. The supernatants were then tested for SMA activity. The sera used in G-actin absorptions were dialyzed against the extraction buffer to prevent the polymerization of actin. Fat G-actin absorptions, it was necessary to use acetone-fixed tissue sections to keep the tissue actin insoluble. RESULTS
Of 140 sera from cancer patients, 48 (3455) were SMA positive. and 36 out of 4X sera (75%) from patients with acute respiratory infections and 14 out of 79 sera (18%) from blood donors were SMA positive. SMA titers in the sera from patients with acute respiratory infections and from blood donors did not exceed 50. Two cancer patients. E. H. and V. F., had SMA titers of 500 and 5000, respectively. The sera from patients with chronic hepatitis contained SMA in titers of up to 50,000. The immunoglobulin class of SMA was determined with FITC-labeled heavy chain specific for anti-human IgG, IgA, and IgM antibodies in 57 sera from the patients with cancer or acute infection and from blood donors (Table 1). With the exception of one serum containing IgM-SMA from a patient with acute respirator)
Number positive studied Urogenital carcinoma Acute respiratory infections
and
‘I SMA were assayed by indirect kidney. Ir Indirect immunofluorescence
of SMAsera
Immunoglobulin
class
IgG
IgA
3‘l
34
4
13
12
i
immunofluorescence using
heavy
using chain-specific
unfixed
cryostat
anti-immuooglobulin
of SMA” IgM
I sections
of rat stomach
conjugate>.
SPECIFICITY
OF
SMOOTH
ML-SCLE
TABLE ABSORPTION
OF SMA-POWWE
SERA
WITH
Number of SMApositive sera Urogenital carcinoma Acute respiratory infections Chronic hepatitis Blood donors
447
ANTIBODIES
2
ACTIN
AR‘D
SMOOTH
MUSCLE
HOMOGENATE
Number of sera neutralized”
by
Actin
Smooth muscle homogenate
34
14 (41%)
34 (100%)
13 20 10
4 (319) 20 (100%) 3 (33%;)
13 (lOO%c) Not tested 10 (lOO%c)
” The sera were incubated with smooth muscle homogenate containing 120- 1200 mg of protein/ml of serum or lo- 100 mg of F-actiniml of serum at +4”C for 16 hr and centrifuged. The supernatants were tested for SMA by IFL.
infection, all SMA-positive sera contained IgG-SMA. SMA of the IgA class were detected in 5 out of 13 SMA-positive sera from patients with acute respiratory infections. Ahsorptions The SMA activity could be neutralized in all sera tested with the smooth muscle homogenate. The sera from patients with chronic hepatitis were not tested fol neutralization with smooth muscle homogenate. SMA activity could be neutralized with bovine skeletal muscle actin in all 20 sera from patients with chronic hepatitis but in only 21 out of 57 (37%) sera from patients with other diseases and from blood donors (Table 2). It was shown that the capacity of the F-actin preparation at a concentration of 1 mg/ml to neutralize anti-actin antibodies corresponded to that of the smooth muscle homogenate at a concentration of 60 mg of protein/ml. No differences were seen in the absorbing effect of F-actin of human or bovine origin. The results of absorption experiments with 12 SMA-positive sera showed no differences between the bovine F- and G-actin. Based on the absorption experiments, the SMA were classified as the anti-actin and anti-“nonactin” types. Both types of SMA could be detected using tissue sections fixed in acetone at -20°C for 10 min. After fixation with 2% formaldehyde at room temperature for 10 min only anti-actin could be detected. Distribution of Actin and “Non-actin” Autoantigens by IFL Smooth muscle. By definition, all sera reacted with the muscularis mucosae and interglandular smooth muscle fibers in the rat stomach (Fig. 2a) and with the renal arteriolar walls of the rat kidney (Fig. 3). Some antibodies related to “reticulin’” antibodies reacted with connective tissue elements in the smooth muscle tissue and often mimicked SMA. These connective tissue antibodies were not neutralized by the smooth muscle homogenate. Li\,er. The high titer SMA of the anti-actin type reacted with the periphery of hepatocytes giving the “polygonal“ staining pattern. The high titer SMA of the “non-actin” type tended to react with the sinusoidal walls of hepatocytes. Kidney. Of the 20 SMA-positive sera of the actin type 15 reacted with the glomerular mesangium, and 7 out of the 32 sera of the “non-actin” type reacted with
448
KURKI
E7 Al.
FIG. 2. (a) Rat stomach stained with an SMA-positive serum by indirect immunofluorescence. Note the reaction of SMA with the muscularis mucosae (mm) and interglandular smooth muscle fibers (Ig). x500. (b) Reaction of SMA of the “non-actin” type (patient V.F.) with rat cardiac myofibrils. A &eai\ striational staining and staining of intercalated discs (arrows) is seen. x I 100.
SPECIFICITY
FIG. 3. Indirect non-actin” types.
OF SMOOTH
MCSCLE
ANTIBODIES
immunofluorescent staining of the rat kidney with Staining of the glomeruli (arrows) by the anti-actin
449
SMA of the (a) actin and (b antibodies is seen. x500.
450
KIiKKI
FTC;. 4. Immunofluorescent SMA of the anti-actin type (arrows). x 1200.
.E7- Al
(a, c. e) and phase-contrast (b, d. 0 pairs of glycerinated myofibrilh. were used by indirect IFL. Fluorescence is mostly localized in the I bands
the glomerular mesangium (Fig. 3). Some high titer anti-actin sera reacted with the peri- or intertubular structures and with the brush border of the proximal tubules. One serum (that of patient V.F.) containing SMA of the “non-actin” type reacted with the peritubular structures. Heart and isolated skeletal muscle myofibrils. Five out of the 20 sera of actin type and 22 out of the 32 sera of the “non-actin” type reacted with the intercalated discs (Fig. 2b). The reaction with the intercalated discs could be inhibited with the smooth muscle homogenate but not with actin. Some sera of the non-actin type also reacted with the periphery of the cardiac and skeletal muscle fibers. SMA of the actin type gave a striational staining pattern on the cardiac and skeletal muscle by IFL. The fluorescence was mostly localized in I bands as revealed by observing the isolated myofibrils by IFL and phase-contrast microscopy (Fig. 4). In all sera the antibodies reacting with I bands could be neutralized with actin. The distribution of antigens corresponding to SMA in kidney and heart is summarized in Table 3. DISCUSSION
It was possible to confirm the actin specificity of SMA in chronic hepatitis (13. 14). On the other hand, in the sera of blood donors and patients with urogenital
IFL
staining
of
Kidney Specificity of SMA
_____
Actin “Non-actin” ” Serum
Number sera
of
20 32 dilution.
I: 10. Unfixed
Arterial wall
Heart Glomerular mesangium
Intercalated disc
15 (75’:) 7 (12“;)
5 (‘5’;) 22 (hW)
20 ( loot;) 32 I IOOc”r) cryostat
sections
were
used a\r substrate\
1 hand Cl (4.i’ ; 1 =. 117’cl
SPECIFICITY
OF SMOOTH
MUSCLE
ANTIBODIES
451
cancer or acute respiratory infections, the SMA positivity was due to anti-actin antibodies in only 21 out of 57 sera. Actin preparations used in this and in previous studies have been relatively inefficient in neutralizing SMA activity (13, 14, 16). It has been necessary to use actin in concentrations of 100-320 mg/ml of serum to neutralize the high titer SMA. This might be due to the destruction of antigenic determinants during the extraction procedure. Antigenic differences between actins (22, 23) are unlikely to be responsible for this inefficiency, because we used actin from both human smooth muscle and bovine skeletal muscle. The preparation of unpolymerized actin (G-actin) proved to be just as inefficient in neutralizing anti-actin antibodies as the preparation of polymerized actin (F-actin). Other factors that should be considered are the minor contaminants in the actin preparations which might be antigenically active or block the antigenic site(s) of actin. The IFL staining pattern of anti-actin antibodies in rat kidney and heart was the same as that described by others (14, 15). The antibodies reacted with glomerular mesangium [“anti-glomerulus antibodies” (12)], per-i- or intertubular structures, the brush border of the renal proximal tubules (14), and the I bands of skeletal muscle (24), in addition to smooth muscle. The autoantigens reacting with the “non-actin” type of SMA in our material were not identified. SMA of myosin specificity have occasionally been detected in human sera (16, 18). Two types of SMA of myosin specificity have been described. One cross-reacts with myosins in skeletal and cardiac muscle, liver, and kidney, while the other is restricted to smooth muscle (18). Recently a smooth muscle protein, “janin,” was described (17). It neutralized the SMA activity of 18 out of 104 sera from patients with non-liver diseases. In our material the staining of intercalated discs was typical for anti “non-actin” antibodies. In this respect they differed from anti-myosin antibodies. The localization of the non-actin autoantigens is similar to that of “desmin,” a subunit protein of the intermediate (100 A) filaments, which are abundant in smooth muscle tissue (25). Using cultured cells we have localized a “non-actin“ SMA specificity in intermediate filaments (19). autoantigen with “desmin” and ‘janin” is The possible identity of “non-actin” under investigation. SMA have been determined only by indirect immunofluorescence. Attempts have been made to standardize the IFL test for SMA (2). However, no generally accepted criteria for SMA positivity have been established. This could explain the marked variation in reported incidences of SMA (3). To avoid false positive SMA, we have recorded the staining of smooth muscle in the arteriolar walls of the rat kidney and in the rat gastric mucosa and muscularis mucosae (2). False positive results in SMA determination can be due to heterophile and “reticulin” antibodies reacting with structures located in the smooth muscle tissue of the rat stomach ( 17,27). In our material these antibodies were often seen in sera from patients with urogenital cancer (3). Heterophile and “reticulin” antibodies were not neutralized by the smooth muscle homogenate. The observation that some SMA fail to react with formaldehyde-fixed tissue sections indicates that also false negative results in the SMA assay may occur if fixation is used. High titer SMA have usually been thought to occur only in chronic active hepatitis (28). We found that two cancer patients without liver involvement had IgG-SMA in titers of 500 and 5000, respectively. These results indicate that high
452
KI'RKI
ET AL
titer SMA are not necessarily anti-actin antibodies. Therefore, an assay for antiactin antibodies could be useful in the serological diagnostics of liver diseases. Most SMA of the non-actin type fail to react with the glomerular mesangium and the periphery of hepatocytes. Thus, the demonstration of “anti-glomerulus antibodies” (12) and the pericellular staining of hepatocytes. “bite canalicular antibodies” (291, could be used as markers for anti-actin antibodies in high titer SMA sera. However, it is doubtful if actin or “non-actin” specificity of SMA can be distinguished with certainty by the conventional IFL test. This is due to the difficulty in defining weak staining patterns produced by many SMA sera (15) and the observed presence of several types of SMA in the same sera. Our finding that SMA of actin and “non-actin” specificity react with striated muscle must be taken into consideration in the interpretation of tests for skeletal (31) and heart muscle (30) antibodies. ACKNOWLEDGMENTS We are grateful to Ms. Jaana Gtuschkoff. Ms. Pipsa Kaipainen. and Mr. Raimo Laakia for techmcal assistance. We also thank Ms. Elina Arvitommi. Ms. Marjatta Janhita. and Ms. Aila Lehtonen for collecting the serum specimens. This work was supported by grants from the Finnish Medical Research Council. the Sigrid Jusetiu\ Foundation. the Finnish Culture Foundation. and the Finnish Foundation for Cancer Research.
REFERENCES I. Johnson. G. D.. Hotborow. E. J.. and Glynn, L. E.. I.cr/lc,c,r 2, 27X. 1965. 2. Whitehouse. J. M. A.. and Hotborow. E. J.. Byif. Med. ./. 4. 51 I. 1971. 3. Kurki. P.. Linder. E.. Miettinen. A.. Atfthan. 0.. Heikkinen. A.. and Pasternack. A.. Irrf. .i Cmcer 19, 332. 1977. 4. Farrow, L. J.. Hotborow. E. J., Johnson, G. D.. Lamb. S. G.. Stewart, J. S.. Taylor. P. E.. and Zuckerman. A. J.. &it. Met/. J. 2, 693. 1970. 5, Hotborow. E. J.. Hemsted, E. H.. and Mead. S. V.. Brit. .IJcc/. J. 3. 323. 1973. 6. Ajdukiewicz. A. B.. Dudtey. F. J.. Fox. R. A.. Doniach. D.. and Sherlock. S.. L.ti)~~.<.t 1, X03. 1972. 7. Smith. J. A.. Francis. T. I.. and David-West. T. S., J. Pl/fl~o/. 109, 83. 1973. 8. McMiltan. S. A.. and Haire. M.. C/i/?. Erp. /~r~m~r/~o/. 21, 339. 1975. 9. Andersen. P.. and Andersen. H. K.. C/i,/. Erp. It>~!,~lrrfol. 22, ‘2. 1975. 10. Loza-Tulimowska, M.. Semkow. R.. Michatak. T.. and Nowostawski. A.. A(r
SPECIFICITY
23. GrBschel-Stewart. 50, 271,
OF
SMOOTH
MUSCLE
ANTIBODIES
U.. Ceurremans. S.. Lehr, I., Mahlmeister,
4.53
C.. and Paar. E.. Hisroc,hpnfis/r.!
1977.
Chaponnier, C.. Kohler. L.. and Gabbiani. G.. C/it?. Exp. Ittrmrrnclf. 27, 278, 1977. Lazarides, E.. and Hubbard. B. D., Proc. Nal. Acad. Sri. USA 73, 4344. 1976. Rizzetto. M.. and Doniach. D., J. C/in. Path/. 26, 841. 1973. Hawkins, B. R., McDonald, B. L., and Dawkins. R. L.. J. C/in. Porhol. 30, 299, 1977. Doniach, D., In “Laboratory Diagnosis of Immunologic Disorders” (G. N. Vyas. D. P. Stites. and G. Brecher. Eds.). p. 163. Grune & Stratton, New York. 1975. 29. Diederichsen, H.. Acfa Med. Stand. 186, 299, 1969. 30. Nicholson. G. C.. Dawkins, R. L.. McDonald, B. L., and Wetherall. J. D.. C&r. Intmuito/. Immlrrloprlthol. 7, 349. 1977. 31. Peers, J., McDonald, B. L., and Dawkins. R. L.. Clir~. Exp. Immrrnol. 27, 66. 1977. 24.
25. 26. 27. 28.
IMIIUN~LOG’~
Smooth
AND
Muscle
P. KURKI,* “Department +Department
9, 443-453 (1978)
IMMUNOPATHOL.OGY
Antibodies of Actin and “Non-actin” Specificity
E. LINDER,*
of Serolo,qy
of Urology.
A. MIETTINEN,*
AND 0. ALFTHAN~
and Bacteriology. Uni\,ersity of He/sin&. Finland. and Second Surgical Clinic, Uni\,ersity Central Hospital o.f Helsinki. Finlund
Received July 13. I977 The specificity and immunoglobulin class of smooth muscle antibodies (SMA) were studied in the sera of patients with urogenital cancer, acute respiratory infection. and chronic hepatitis and in the sera of blood donors by the indirect immunofluorescence technique (IFL). SMA activity could be neutralized with skeletal muscle actin in all 20 sera from patients with chronic hepatitis but in only 21 out of 57 other SMA-positive sera. The IFL staining patterns of anti-actin and anti-“non-actin” SMA were different; antiactin antibodies reacted with the glomerular mesangia and 1 bands of myofibrils, whereas anti- “non-actin” antibodies reacted with the periphery of myofibrils and with intercalated discs. The specificity and diagnostic significance of SMA are discussed.
INTRODUCTION
Smooth muscle antibodies (SMA) are of diagnostic importance in liver diseases (1) but occur also in malignancies (Z), in chronic renal disease (3), in some viral infections (4-lo), and in Mycoplasrna pneunzoniae infections (11). SMA can also be demonstrated in a considerable percentage of normal subjects (2, 3). SMA in chronic active hepatitis and carcinoma are mainly of the IgG class (2. 12), whereas viral infections are often followed by the transient appearance of IgM-SMA (4, 5). The antibody specificities of SMA are incompletely characterized. The antigen in chronic active hepatitis appears to be actin (13, 14). but SMA in other diseases may have additional specificities. This is suggested by the observation that SMA activity in some sera cannot be neutralized with actin (15-19) and by the different IFL staining patterns produced by individual SMApositive sera (15, 19). To evaluate the diagnostic signfkance of different types of SMA, we have investigated the specificity and immunoglobulin class of SMA in sera from patients with chronic hepatitis, urogenital cancer, and acute respiratory infections and the sera of blood donors. This was done by absorption experiments and by observing different staining patterns of SMA-positive sera by indirect immunofluorescence (IFL). MATERIALS
AND METHODS
Serum samples were obtained from 140 patients with urogenital carcinoma, 48 patients with acute respiratory tract infection. and 79 blood donors. The diagnostic criteria for urogenital carcinoma in this material have been described earlier (3). The diagnosis of acute respiratory infection was made by a physician and was 443 0090-1?29/78/0094-0443$01.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproductmn m any form reserved
444
KlRKl
El
41
based on anamnesis and physical examination. In addition. 20 SMA-positive XIX from patients with chronic hepatitis were examined. These sera were sent to the Department of Serology and Bacteriology, University of Helsinki, for routine tissue antibody determinations. Tissues
The tissue specimens were snap-frozen in isopentane cooled with liquid nitrogen. Unfixed, 5-pm cryostat sections of rat liver, kidney, stomach, heart, and skeletal muscle were used as the substrates for the immunofluorescence studies. Cryostat sections fixed in acetone at -20°C for IO min or in 2% formaldehyde at room temperature for IO min were used in some experiments. Skeletal Muscle
Myojihi1.s
Skeletal muscle myofibrils were isolated from glycerinated muscle (16). Rat skeletal muscle was cleaned from the surrounding connective tissue and immersed for 48 hr in a solution containing 50% glycerol in 0.1 M KCI. 1 mM MgCl,, IO n-~&1 imidazole, pH 7.0, cut into 3-mm pieces, and homogenized in a Dual1 30 homogenizer (Kontes Glass Co., Vineland, New Jersey). The suspension containing the isolated myofibrils was smeared onto microscope slides and fixed in acetone at -20°C for 10 min. The sera were allowed to react with cryostat sections for 30 min. Serum dilutions of 1: 10, 1:50, 1: 100, 1:500, 1:5000. and 1:50,000 were used for the determination of the SMA titer. Unless otherwise stated, the SMA-positive sera were used in a 1: 10 dilution. The fluorescein isothiocyanate (FITC) labeled sheep anti-human immunoglobulin (National Bacteriological Laboratory, Stockholm, Sweden) had a molar F/P ratio of 2.9 and contained 6.5 mg of protein/ml. Specific FITC-labeled antisera to human IgG, IgA, and IgM (Meloy Laboratories, Springfield, Virginia) had molar F/P ratios of 2.9, 2.4, and 4.2. respectively, and contained 10, IO. and 9 mg of protein/ml, respectively. A Wild M 20 uv microscope equipped with an iodine quartz lamp (Osram, 100 W). a Wild dark-field condenser, a phase-contrast condenser, and an FITC interference filter combination (Optisk Laboratorium. Lyngby. Denmark) were used. The sera were considered positive for SMA if de&rite staining was observed in the muscularis mucosae and in interglandular smooth muscle fibers of rat stomach (Fig. 3a3 and in the renal arteriolar walls of rat kidney (Fig. 3). The titers are expressed as reciprocals of the endpoint dilutions. The smooth muscle homogenate was prepared from a myomatous human uterus. The uterus was cut into pieces, ground in a meat grinder, and homogenized in phosphate-buffered saline (PBS), pH 7.4. at 4°C in a homogenizer (UltraTurrax, Janke & Kunkel, Staufen, GFR). The homogenate was centrifuged at 5OOOgfor 30 min to remove connective tissue. The remaining solution was used in absorption experiments at a concentration of 12 mg of protein/ml and stored at - 20°C. Actin was extracted from the acetone powder of bovine skeletal muscle 01
SPECIFICITY
OF
SMOOTH
MUSCLE
ANTIBODIES
445
human uterus at 4°C (20). The extraction buffer was 2 mJ4 Tris-HCl, 0.5 mM ATP, 2 m&I CaCl,, pH 7.5. This solution was concentrated and dialyzed against 0.6 M KCl, 0.5 n&f ATP, 0.5 m&I MgCl,, pH 7.0 (dialyzing buffer). F-actin was obtained from the pellet after centrifugation of the solution at 80,OOOg for 60 min. The pellet was washed twice in the dialyzing buffer and suspended in PBS. G-actin was obtained by dialyzing F-actin against the extraction buffer. After centrifugation at 80,000 for 60 mitt, the supernatant contained G-actin. The actin preparations used in absorption experiments contained 1 mg of protein/ml and 0.05% Na-azide. Actin preparations were stored at +4”C for up to 4 weeks. The composition of the actin preparations and of the smooth muscle homogenate was examined using SDS-polyacrylamide gel electrophoresis (21). The actin
a FIG. bovine
b
1. SDS-polyacrylamide gel electrophoresis skeletal muscle and (df 150 pg of human
c
d
of (a) 10 pg. (b) 20 pg. and (c) 60 pg of actin smooth .muscle homogenate. A. actin.
from
446
KlWKI
El
Al
preparation gave a single band. The actin content of the smooth muscle homogenate was approximately 20 pg/12 mg of homogenate protein, as estimated from the SDS-polyacrylamide gels loaded with smooth muscle homogenate and different concentrations of actin (Fig. I). In absorption experiments, sera with low SMA titers (s 100) were diluted 1: 10. and sera with high SMA titers (>lOO) were diluted 1: 100 in the F- or G-actin preparations or in the smooth muscle homogenate. Thus, the concentration of the smooth muscle homogenate was 120-1200 mg of protein/ml of undiluted serum. and the concentration of the actin preparations was IO-100 mg of protein/ml of undiluted serum. Serial twofold dilutions of SMA-positive sera were used when the neutralization capacities of the smooth muscle and F-actin preparations were compared. Controls were diluted in PBS or in the extraction buffer. The sera were incubated overnight at 4°C and centrifuged at SOOOgfor 5 min. The supernatants were then tested for SMA activity. The sera used in G-actin absorptions were dialyzed against the extraction buffer to prevent the polymerization of actin. Fat G-actin absorptions, it was necessary to use acetone-fixed tissue sections to keep the tissue actin insoluble. RESULTS
Of 140 sera from cancer patients, 48 (3455) were SMA positive. and 36 out of 4X sera (75%) from patients with acute respiratory infections and 14 out of 79 sera (18%) from blood donors were SMA positive. SMA titers in the sera from patients with acute respiratory infections and from blood donors did not exceed 50. Two cancer patients. E. H. and V. F., had SMA titers of 500 and 5000, respectively. The sera from patients with chronic hepatitis contained SMA in titers of up to 50,000. The immunoglobulin class of SMA was determined with FITC-labeled heavy chain specific for anti-human IgG, IgA, and IgM antibodies in 57 sera from the patients with cancer or acute infection and from blood donors (Table 1). With the exception of one serum containing IgM-SMA from a patient with acute respirator)
Number positive studied Urogenital carcinoma Acute respiratory infections
and
‘I SMA were assayed by indirect kidney. Ir Indirect immunofluorescence
of SMAsera
Immunoglobulin
class
IgG
IgA
3‘l
34
4
13
12
i
immunofluorescence using
heavy
using chain-specific
unfixed
cryostat
anti-immuooglobulin
of SMA” IgM
I sections
of rat stomach
conjugate>.
SPECIFICITY
OF
SMOOTH
ML-SCLE
TABLE ABSORPTION
OF SMA-POWWE
SERA
WITH
Number of SMApositive sera Urogenital carcinoma Acute respiratory infections Chronic hepatitis Blood donors
447
ANTIBODIES
2
ACTIN
AR‘D
SMOOTH
MUSCLE
HOMOGENATE
Number of sera neutralized”
by
Actin
Smooth muscle homogenate
34
14 (41%)
34 (100%)
13 20 10
4 (319) 20 (100%) 3 (33%;)
13 (lOO%c) Not tested 10 (lOO%c)
” The sera were incubated with smooth muscle homogenate containing 120- 1200 mg of protein/ml of serum or lo- 100 mg of F-actiniml of serum at +4”C for 16 hr and centrifuged. The supernatants were tested for SMA by IFL.
infection, all SMA-positive sera contained IgG-SMA. SMA of the IgA class were detected in 5 out of 13 SMA-positive sera from patients with acute respiratory infections. Ahsorptions The SMA activity could be neutralized in all sera tested with the smooth muscle homogenate. The sera from patients with chronic hepatitis were not tested fol neutralization with smooth muscle homogenate. SMA activity could be neutralized with bovine skeletal muscle actin in all 20 sera from patients with chronic hepatitis but in only 21 out of 57 (37%) sera from patients with other diseases and from blood donors (Table 2). It was shown that the capacity of the F-actin preparation at a concentration of 1 mg/ml to neutralize anti-actin antibodies corresponded to that of the smooth muscle homogenate at a concentration of 60 mg of protein/ml. No differences were seen in the absorbing effect of F-actin of human or bovine origin. The results of absorption experiments with 12 SMA-positive sera showed no differences between the bovine F- and G-actin. Based on the absorption experiments, the SMA were classified as the anti-actin and anti-“nonactin” types. Both types of SMA could be detected using tissue sections fixed in acetone at -20°C for 10 min. After fixation with 2% formaldehyde at room temperature for 10 min only anti-actin could be detected. Distribution of Actin and “Non-actin” Autoantigens by IFL Smooth muscle. By definition, all sera reacted with the muscularis mucosae and interglandular smooth muscle fibers in the rat stomach (Fig. 2a) and with the renal arteriolar walls of the rat kidney (Fig. 3). Some antibodies related to “reticulin’” antibodies reacted with connective tissue elements in the smooth muscle tissue and often mimicked SMA. These connective tissue antibodies were not neutralized by the smooth muscle homogenate. Li\,er. The high titer SMA of the anti-actin type reacted with the periphery of hepatocytes giving the “polygonal“ staining pattern. The high titer SMA of the “non-actin” type tended to react with the sinusoidal walls of hepatocytes. Kidney. Of the 20 SMA-positive sera of the actin type 15 reacted with the glomerular mesangium, and 7 out of the 32 sera of the “non-actin” type reacted with
448
KURKI
E7 Al.
FIG. 2. (a) Rat stomach stained with an SMA-positive serum by indirect immunofluorescence. Note the reaction of SMA with the muscularis mucosae (mm) and interglandular smooth muscle fibers (Ig). x500. (b) Reaction of SMA of the “non-actin” type (patient V.F.) with rat cardiac myofibrils. A &eai\ striational staining and staining of intercalated discs (arrows) is seen. x I 100.
SPECIFICITY
FIG. 3. Indirect non-actin” types.
OF SMOOTH
MCSCLE
ANTIBODIES
immunofluorescent staining of the rat kidney with Staining of the glomeruli (arrows) by the anti-actin
449
SMA of the (a) actin and (b antibodies is seen. x500.
450
KIiKKI
FTC;. 4. Immunofluorescent SMA of the anti-actin type (arrows). x 1200.
.E7- Al
(a, c. e) and phase-contrast (b, d. 0 pairs of glycerinated myofibrilh. were used by indirect IFL. Fluorescence is mostly localized in the I bands
the glomerular mesangium (Fig. 3). Some high titer anti-actin sera reacted with the peri- or intertubular structures and with the brush border of the proximal tubules. One serum (that of patient V.F.) containing SMA of the “non-actin” type reacted with the peritubular structures. Heart and isolated skeletal muscle myofibrils. Five out of the 20 sera of actin type and 22 out of the 32 sera of the “non-actin” type reacted with the intercalated discs (Fig. 2b). The reaction with the intercalated discs could be inhibited with the smooth muscle homogenate but not with actin. Some sera of the non-actin type also reacted with the periphery of the cardiac and skeletal muscle fibers. SMA of the actin type gave a striational staining pattern on the cardiac and skeletal muscle by IFL. The fluorescence was mostly localized in I bands as revealed by observing the isolated myofibrils by IFL and phase-contrast microscopy (Fig. 4). In all sera the antibodies reacting with I bands could be neutralized with actin. The distribution of antigens corresponding to SMA in kidney and heart is summarized in Table 3. DISCUSSION
It was possible to confirm the actin specificity of SMA in chronic hepatitis (13. 14). On the other hand, in the sera of blood donors and patients with urogenital
IFL
staining
of
Kidney Specificity of SMA
_____
Actin “Non-actin” ” Serum
Number sera
of
20 32 dilution.
I: 10. Unfixed
Arterial wall
Heart Glomerular mesangium
Intercalated disc
15 (75’:) 7 (12“;)
5 (‘5’;) 22 (hW)
20 ( loot;) 32 I IOOc”r) cryostat
sections
were
used a\r substrate\
1 hand Cl (4.i’ ; 1 =. 117’cl
SPECIFICITY
OF SMOOTH
MUSCLE
ANTIBODIES
451
cancer or acute respiratory infections, the SMA positivity was due to anti-actin antibodies in only 21 out of 57 sera. Actin preparations used in this and in previous studies have been relatively inefficient in neutralizing SMA activity (13, 14, 16). It has been necessary to use actin in concentrations of 100-320 mg/ml of serum to neutralize the high titer SMA. This might be due to the destruction of antigenic determinants during the extraction procedure. Antigenic differences between actins (22, 23) are unlikely to be responsible for this inefficiency, because we used actin from both human smooth muscle and bovine skeletal muscle. The preparation of unpolymerized actin (G-actin) proved to be just as inefficient in neutralizing anti-actin antibodies as the preparation of polymerized actin (F-actin). Other factors that should be considered are the minor contaminants in the actin preparations which might be antigenically active or block the antigenic site(s) of actin. The IFL staining pattern of anti-actin antibodies in rat kidney and heart was the same as that described by others (14, 15). The antibodies reacted with glomerular mesangium [“anti-glomerulus antibodies” (12)], per-i- or intertubular structures, the brush border of the renal proximal tubules (14), and the I bands of skeletal muscle (24), in addition to smooth muscle. The autoantigens reacting with the “non-actin” type of SMA in our material were not identified. SMA of myosin specificity have occasionally been detected in human sera (16, 18). Two types of SMA of myosin specificity have been described. One cross-reacts with myosins in skeletal and cardiac muscle, liver, and kidney, while the other is restricted to smooth muscle (18). Recently a smooth muscle protein, “janin,” was described (17). It neutralized the SMA activity of 18 out of 104 sera from patients with non-liver diseases. In our material the staining of intercalated discs was typical for anti “non-actin” antibodies. In this respect they differed from anti-myosin antibodies. The localization of the non-actin autoantigens is similar to that of “desmin,” a subunit protein of the intermediate (100 A) filaments, which are abundant in smooth muscle tissue (25). Using cultured cells we have localized a “non-actin“ SMA specificity in intermediate filaments (19). autoantigen with “desmin” and ‘janin” is The possible identity of “non-actin” under investigation. SMA have been determined only by indirect immunofluorescence. Attempts have been made to standardize the IFL test for SMA (2). However, no generally accepted criteria for SMA positivity have been established. This could explain the marked variation in reported incidences of SMA (3). To avoid false positive SMA, we have recorded the staining of smooth muscle in the arteriolar walls of the rat kidney and in the rat gastric mucosa and muscularis mucosae (2). False positive results in SMA determination can be due to heterophile and “reticulin” antibodies reacting with structures located in the smooth muscle tissue of the rat stomach ( 17,27). In our material these antibodies were often seen in sera from patients with urogenital cancer (3). Heterophile and “reticulin” antibodies were not neutralized by the smooth muscle homogenate. The observation that some SMA fail to react with formaldehyde-fixed tissue sections indicates that also false negative results in the SMA assay may occur if fixation is used. High titer SMA have usually been thought to occur only in chronic active hepatitis (28). We found that two cancer patients without liver involvement had IgG-SMA in titers of 500 and 5000, respectively. These results indicate that high
452
KI'RKI
ET AL
titer SMA are not necessarily anti-actin antibodies. Therefore, an assay for antiactin antibodies could be useful in the serological diagnostics of liver diseases. Most SMA of the non-actin type fail to react with the glomerular mesangium and the periphery of hepatocytes. Thus, the demonstration of “anti-glomerulus antibodies” (12) and the pericellular staining of hepatocytes. “bite canalicular antibodies” (291, could be used as markers for anti-actin antibodies in high titer SMA sera. However, it is doubtful if actin or “non-actin” specificity of SMA can be distinguished with certainty by the conventional IFL test. This is due to the difficulty in defining weak staining patterns produced by many SMA sera (15) and the observed presence of several types of SMA in the same sera. Our finding that SMA of actin and “non-actin” specificity react with striated muscle must be taken into consideration in the interpretation of tests for skeletal (31) and heart muscle (30) antibodies. ACKNOWLEDGMENTS We are grateful to Ms. Jaana Gtuschkoff. Ms. Pipsa Kaipainen. and Mr. Raimo Laakia for techmcal assistance. We also thank Ms. Elina Arvitommi. Ms. Marjatta Janhita. and Ms. Aila Lehtonen for collecting the serum specimens. This work was supported by grants from the Finnish Medical Research Council. the Sigrid Jusetiu\ Foundation. the Finnish Culture Foundation. and the Finnish Foundation for Cancer Research.
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SPECIFICITY
23. GrBschel-Stewart. 50, 271,
OF
SMOOTH
MUSCLE
ANTIBODIES
U.. Ceurremans. S.. Lehr, I., Mahlmeister,
4.53
C.. and Paar. E.. Hisroc,hpnfis/r.!
1977.
Chaponnier, C.. Kohler. L.. and Gabbiani. G.. C/it?. Exp. Ittrmrrnclf. 27, 278, 1977. Lazarides, E.. and Hubbard. B. D., Proc. Nal. Acad. Sri. USA 73, 4344. 1976. Rizzetto. M.. and Doniach. D., J. C/in. Path/. 26, 841. 1973. Hawkins, B. R., McDonald, B. L., and Dawkins. R. L.. J. C/in. Porhol. 30, 299, 1977. Doniach, D., In “Laboratory Diagnosis of Immunologic Disorders” (G. N. Vyas. D. P. Stites. and G. Brecher. Eds.). p. 163. Grune & Stratton, New York. 1975. 29. Diederichsen, H.. Acfa Med. Stand. 186, 299, 1969. 30. Nicholson. G. C.. Dawkins, R. L.. McDonald, B. L., and Wetherall. J. D.. C&r. Intmuito/. Immlrrloprlthol. 7, 349. 1977. 31. Peers, J., McDonald, B. L., and Dawkins. R. L.. Clir~. Exp. Immrrnol. 27, 66. 1977. 24.
25. 26. 27. 28.