Cliii. exp. Immunol. (1977) 27, 278-284.
Fixation of human anti-actin autoantibodies on skeletal muscle fibres CHRISTINE CHPO\NNIER, IAURE KOHLER* & G. GABBIANI Uinversity of Genieva, Genieva, Siteterlaild
DepartmeutmoJPatholoq),
(Received 2 September 1976)
SUMMXARY
Sera from fixve patients xaith chronic aggressive hepatitis containing smooth muscle autoantibodies xere tested bv means of indirect immunofluorescence for their binding to isolated rabbit skeletal muscle mrofibrils. In all cases, the immunofluorescent staining was sharply localized to I bands. After incubation of these sera with skeletal muscle troponin-tropomyosin complex, purified troponin opururified tropomxosin)no changes in immunofluorescent staining of myofibrils were noted. However, the staining was abolished after incubation of the sera with skeletal muscle actin. In double immunodiffusion experiments, a single precipitation line was obtained after diffusion of the sera against crude or purified actin. It is concluded that, at least for the sera examined, smooth muscle autoantibodies are anti-actin autoantibodies. The high titre of such autoantibodies and their availability in clinical immunology laboratories make them a useful tool to study actin distribution in muscular and non-muscular cells.
INTRODUCTION Smooth muscle autoantibodies have been described in the serum of patients with chronic aggressive hepatitis, infectious mononucleosis and malignant tumours (Johnson, Holboroxv & Gh nn, 1965; Andersen et a!., 1976). The observation that incubation of several smooth muscle autoantibodies containing sera with platelet actin (thrombosthenin-A) abolishes the ability of such sera to bind to smooth muscle or platelets, suggested that these autoantibodies are directed against actin (anti-actin antibodies or AAA) (Gabbiani et all., 1973b). Actin is one of the major components of striated muscle where it is localized in I bands together with regulatory proteins. It is generally accepted that actins from platelets, smooth or striated muscle are xerv similar (Pollard & W~eihing, 1974). The present study deals xith the fixation of AAA on striated muscle. Moreover, immunodiffusion of AAA against skeletal muscle actin, in one hand, and the effect of the previous incubation of AAA-containing sera With proteins present in I bands of striated muscle, on the other hand, have been inxestigated. MATERIALS ANI) METHODS
Mjyofibrils isolation. The technique described by Perry & Grey (1956) and Perry & Zydowo (1959) with minor modifications was used. The minced muscles from the back and legs of a rabbit were homogenized in 5 vol. of a buffer containing 40 mxi tris-HCI pH 7-0 and 25 mxt KCI. After 15 min of centrifugation at 600 g, the supernatant was disregarded. These operations were repeated several times. The pellets were washed eight times in 9 vol. of buffer containing 40 mm tris-HCl pH 7 1 and 0 1 xi KC1. \lyofibrils were then stored at 4 C in the last buffer with 2 mxi 2-mercapto-ethanol. Protein extrachtion. 4ctit. This was prepared from rabbit skeletal muscle following the technique of Spudich & Watt l)epartment of Biochemistry, Faculty of Sciences, Unixersity of Geneva, Geneva, Switzerland. Correspondence: Dr G. Gabbiani, Department of Pathology, University of Geneva, 40 Boulevard de la Cluse, 1211 * From the
Geneva 4, Switzerland.
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(1971). Ten grams of acetonic powder were extracted at 0C by agitation in 200 ml of a buffer composed of 2 mM tris-HCl pH 8-0; 0-2 mm ATP; 0-5 mm 2-mercapto-ethanol and 0-2 mm CaCl2. The solution was filtered through three layers of gauze. The residue was washed with 100 ml of buffer and filtered again through three layers of gauze. The supernatants were clarified by centrifugation at 10,000 g for 3 hr. Actin polimerization was obtained at room temperature in 0 5 M KCl and 2 mM MgCl2 during 20 min. The KC1 concentration was then increased up to 0-6 M under slight agitation during 40 min. The solution was ultracentrifuged for 3 hr at 80,000 g. The pellet was redissolved in the same buffer used for the acetonic powder extraction and dialysed against the same buffer during 72 hr under agitation with frequent changes of buffer. Gactin was clarified by ultracentrifugation at 80,000 g during 3 hr and then polymerized with 50 mm KC1. Troponin (TP) and tropomyosin (TM). These were isolated from native actomyosin (NAM) (Hartshorne & Mueller, 1969). Myofibrils (collected as described above) were washed at 4°C in 1 vol. of a solution containing 1-2 M KCl; 80 mm NaHCO3; 20 mm Na2CO3 and 6 vol. of Weber's solution (0-6 M KCI, 40 mm NaHCO3 and 10 mm Na2CO3) and homogenized rapidly in this solution (for 30 sec at the maximal speed of a Sorvall Omni-mixer). After 30 min, myofibrils were centrifuged at 2500 g during 20 min and the supernatant slowly diluted 1/10 with 15 mm 2-mercapto-ethanol in water. After 60 min, NAM was obtained by centrifugation at 2000 g during 20 min. The pellets were quickly washed first with 20 mm Tris-maleate buffer and then with 100 mm Tris-maleate buffer (both at pH 7-1). The NAM powder was prepared after washing several times in alcohol ether. The NAM powder was left with agitation overnight at 4°C in 1 M KCl and 2 mm DTT. The solution was centrifuged at 40,000 g during 3 hr and the supernatant dialysed against 4 vol. of water and 2 mm DTT to obtain a final dilution of 0 2 M KCl and 2 mm DTT. TP-TM complex was precipitated between 40% and 60% saturation (NH4)2SO4. TM and TP were isolated chromatographically (Eisenberg & Kielley, 1974) on hydroxyapatite (Biorad, in the wet form) column (2-5 x 25 cm). Hydroxyapatite was washed ten times to eliminate the smallest particles and then equilibrated in 1 M KC1, 2 mm DTT, 1 mM P04, pH 7 0. One hundred milligrams of TP-TM complex in 10 ml of 2 mm imidazol HCI, pH 7 0, 1 M KCI, 5 mm 2-mercapto-ethanol were eluted with a linear P04 gradient (from 1 M KC1, 2 5 mm DTT, 1 mM P04 at pH 7 0, to 1 M KCl, 2 5 mm DTT, 0 2 M P04 at pH 7-0). The different peaks were dialysed against 0 5 M KCl, 2 mm DTT, 2 mm EDTA and 2 mm imidazol. AAA sera. AAA containing sera were obtained from five patients with chronic aggressive hepatitis. Their activity in staining frozen sections of smooth muscle was judged as follows: cryostat sections of rat small intestine were fixed for 5 min in cold acetone (Merck Company, Darmstadt, Germany) and were then treated for 15 min with the serum to be tested (diluted when necessary in phosphate-buffered saline or PBS, pH 7.2). The sections were then washed in PBS and stained for 15 min with fluorescein-conjugated IgG fraction of goat antiserum to human IgG (code 64-170 Miles Seravac, Lausanne, Switzerland). After rewashing with PBS and mounting in 90% glycerol in PBS, the level of fluorescence was compared with that found in control sections, treated with normal human serum (NHS) instead of AAA serum. Photographs were taken on a Zeiss UV photomicroscope, using Anscochrome colour slide film 500 day light (Gaf Corp. New York). The pattern of AAA labelling to smooth muscle of muscularis externa or muscularis mucosa was qualitatively similar on the five sera. The dilutions at which immunofluorescent staining was detectable were 1/1280 (two sera), 1/640 and 1/320 (two sera). These dilutions represented the titres of the sera. Tests of these sera for anti-mitochondrial or anti DNA antibodies and for antinuclear factor were negative. Immunodiffusion. Tests were performed according to the Ouchterlony technique. Agarose (Behring-Wercke A.G., Marburg-Lahn, Germany) was dissolved in 2 mm tris-HCI buffer at pH 8-0 containing 0-2 mM ATP, 0 5 mM 2-mercaptoethanol and 0-2 mm CaCl2 (Norberg, Lidman & Fagraeus, 1975). Immunoabsorption. Immunoabsorption tests were performed as follows: 0- 1 ml of serum was added to 0 9 ml of the different solutions of contractile proteins, incubated for 60 min at room temperature (with agitation every 15 min), and left overnight at 4°C. After incubation with actin, a precipitate was present at the bottom of the tube. This precipitate was eliminated by centrifugation of the sera for 10 min at 4600 g. As a control, we incubated 0-1 ml of the sera with 0-9 ml of the buffers in which the various contractile proteins were dissolved. In each case, the AAA titres of these sera were tested on cryostat sections of rat small intestine and compared with that of serum incubated with buffers and with that of original untreated serum.
Immunofluorescent staining. Most of the staining was performed with the serum having a titre of 1/1280. Three hundred microlitres of the standard myofibril suspension were centrifuged at 1000 g and washed twice in PBS. After the last washing they were resuspended in 100 pl of PBS and incubated for 30 min at room temperature with 10 pl of AAA serum. In addition 100 Id of myofibril suspension in PBS were incubated with 10 ,l of NHS or 10 p1 of AAA serum previously incubated with either: (1) actin; (2) the complex TP-TM; (3) TP or (4) TM. After the first incubation, the myofibrils were centrifuged at 1000 g and washed in PBS three times. They were then resuspended in 100,ul of PBS and incubated for 30 min at room temperature with 20,ul of fluorescein-conjugated, y-globulin fraction of goat anti-serum to human IgG. After washing three times in PBS, the myofibrils were suspended in 2 ml of foetal calf serum and cytocentrifuged on glass slides by means of a Shannon Eliott (London, England) cytocentrifuge. The slides were mounted in 90% glycerol in PBS and examined on a Zeiss UV microscope.
RESULTS In double immunodiffusion tests, all AAA sera gave a single strong precipitation line with either crude or purified rabbit skeletal muscle actin (Fig. 1, 2). No precipitation was noted after immunodiffusion
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FIG. 1. Immunodiffusion experiment according to the Ouchterlony technique. AAA serum has been placed in the centre against decreasing concentrations of crude (a) and purified (b) actin. In (a), the concentrations are: 2 4 mg/ml upper right; 1 2 mg/ml middle right; 0 60 mg/ml lower right; 0 30 mg/ml lower left; 0-15 mg/ml middle left and 0 075 mg/ml upper left. In (b) the concentrations are: 3 mg/ml upper right; 1 5 mg/ml middle right; 0 75 mg/ml lower right; 0 375 mg/ml lower left; 0-19 mg/ml middle left and 0 095 mg/ml upper left.
FIG. 2. SDS-Polvacrylamide gel electrophoresis showing crude actin (a) and purified actin (b) from rabbit skeletal muscle used for immunodifffusion and absorption tests.
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FIG. 3. SDS-Polyacrylamide gel electrophoresis showing TP-TM complex (a); the same complex with actin added as a marker (b); purified TP (c); purified TM (d). All these proteins were extracted from rabbit skeletal muscle and used for absorption experiments.
.
f
h
FIG. 4. Isolated myofibrils after immunofluorescent staining with AAA sera alone or previously incubated with contractile proteins present in I-band of striated muscle. (Magnification x 700.) (a) Myofibril treated first with AAA serum followed by IgG fraction of goat antiserum to human IgG: the stain is localized in bands; (b) these bands in the phase constrast micrograph of the same myofibril appear to be I bands; (c) myofibril treated first with AAA serum previously incubated with TM-TP complex followed by IgG fraction of goat antiserum to human IgG: the staining is similar to that of (a); (d) phase contrast micrograph of the myofibril in (c) showing that the positive staining is localized in I bands; (e) myofibril treated first with AAA serum previously incubated with TP followed by IgG fraction of goat antiserum to human IgG: the stain is similar to that of (a) and (c); (f) phase contrast micrograph of the myofibril in (e) showing that the fluorescent staining is localized in I bands; (g) myofibril treated first with AAA serum previously incubated with TM, followed by IgG fraction of goat antiserum to human IgG: the fluorescent staining is similar to that of (a), (c) and (e); (h) phase contrast micrograph ofthe myofibril in (g) showing that the fluorescent staining is localized in I bands; (i) myofibril treated first with AAA previously incubated with actin, followed by IgG fraction of goat antiserum to human IgG: no immunofluorescent staining; (j) phase contrast micrograph of the myofibril in (i).
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Christine Chaponnier, Laure Kohler & G. Gabbiani
of AAA sera with purified TP or TM (Fig. 3). No precipitation also was noted after immunodiffusion of actin against NHS. When myofibrils were incubated with NHS, no fluorescence was present after staining with fluorescein-conjugated anti-human IgG. On the other hand, the staining was strongly positive when myofibrils were treated first with AAA (alone or previously incubated with the complex TP-TM, TP or TM) (Fig. 4). Comparison of immunofluorescence with phase contrast micrographs of the same myofibrils, showed that the immunofluorescent staining with AAA sharply corresponded to I bands. When myofibrils were treated with AAA previously incubated with purified actin, no immunofluorescent staining was present after fluorescein-conjugated goat anti-human IgG (Fig. 4).
DISCUSSION These results show that incubation of isolated myofibrils with AAA results in a sharp staining of Ibands. The contractile proteins composing I bands are actin, TP and TM. The persistence of AAA activity after incubation of AAA sera with the complex TP-TM or with the single purified proteins in one hand and the abolition of AAA activity after incubation of AAA sera with purified striated muscle actin on the other hand, support the previous assumption (Gabbiani et al., 1973b) that the sera tested contain specific anti-actin antibodies. This assumption is confirmed by the immunodiffusion experiments showing a single precipitation line when the sera are diffused against either crude or purified actin. In analogy with the previous experiments using thrombosthenin-A (Gabbiani et al., 1973b), incubation of AAA sera with skeletal muscle actin abolishes the staining of not only I bands in striated muscle but also of the cytoplasm of smooth muscle cells, platelets and cultivated fibroblasts. Although a full comparison of the amino-acid sequences of actins from striated muscle, smooth muscle, platelets and fibroblasts cannot yet be made, it is nevertheless well established that these actins are very similar (Probst & Luischer, 1972; Pollard & Weihing, 1974). Hence, it can be concluded that, the tested sera from patients with chronic aggressive hepatitis contain crossreacting anti-actin autoantibodies. A recent work by Lidman et al. (1976) using thirty anti-smooth muscle antibodies from patients with chronic aggressive hepatitis has also shown that the staining activity of these sera is abolished after incubation with skeletal muscle actin. AAA have been shown to fix on structures containing important amounts of actin such as platelets, megakaryocytes, the peripheral part of intestinal epithelial cells and stress lines of cultivated fibroblasts (Gabbiani etal., 1973b; Gabbiani, Majno & Ryan, 1973a). The demonstration of the selective fixation of AAA to I bands of striated muscle more precisely define their specificity. After the description of anti-actin activity in sera of patients with chronic aggressive hepatitis (Gabbiani et al., 1973b), artificial anti-actin antibodies have been produced experimentally by injecting in rabbit the purified protein (Trenchev, Sneyd & Holborow, 1974; Lazarides & Weber, 1974). Such experimental antibodies have the same specificity as the human ones but relatively low titres when compared to the human ones. This was explained on the basis of the low antigenicity of actin which has a similar structure throughout the philogenetic scale. The reasons why humans undergoing viral hepatitis or other forms of viral diseases produce, at least in a certain proportion of cases, high titre anti-actin antibodies remain mysterious. In any event, the high titre of AAA sera and their availability in clinical immunology laboratories makes them an ideal candidate for the study of the presence and distribution of actin in non-muscular cells. AAA have been previously used to demonstrate for the first time the presence of actin in such cells as interstitial cells of the lung alveoli (Kapanci et al., 1974), beta-cells of Langerhans islets (Gabbiani et al., 1974), adrenocortical cells (Gabbiani, Chaponnier & Liischer, 1975b), fibroblasts of granulation tissue (myofibroblasts) (Gabbiani et al., 1972) and neoplastic cells of the skin, oral cavity, larynx and mammary gland (Gabbiani, Trenchev & Holborow, 1975d). The present study on the specificity of AAA for actin constitutes a further argument supporting the conclusions reached in all these studies. In the last few years, more and more evidences have accumulated showing that contractile proteins are present in many if not all eucaryotic cells (Pollard & Weihing, 1974). Here, they have been correlated to such functions as cell contraction (Gabbiani et al., 1972), cell locomotion (Pollard & Weihing, 1974;
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Durham, 1974), intracellular transport or endo- and exocytosis (Lacy et al., 1968; Orci, Gabbay & Malaisse, 1972). Contractile proteins have been identified in the cytoplasm of several cell types by means of biochemical techniques (Adelstein et al., 1972; Ostlund, Pastan & Adelstein, 1974; Bray & Thomas, 1975) or of electron microscopy, on the basis of the morphology of filaments (Goldman, 1971); actin has also been identified on the basis of the capacity to bind heavy meromyosin, giving a characteristic arrowhead appearance (Ishikawa, Bishoff & Holtzer, 1969). These findings have permitted the study of the distribution of individual contractile protein in different structures under various conditions. Cells in which cytoplasmic filaments increase considerably in vivo following changes in their microenvironment have been described. They include epidermal cells (Gabbiani & Ryan, 1974) or fibroblasts (Gabbiani et al., 1972), during the healing of an open wound, epidermal cells during experimental production of malignant tumours (Malech & Lentz, 1974), hepatocytes during liver regeneration (Gabbiani & Ryan, 1974) or after treatment with small amounts of phalloidin (one of the poisons of the mushroom Amanita Phalloides) (Gabbiani, et. al., 1975c) and aortic endothelial cells during the first stages of hypertension (Gabbiani, Badonnel & Rona, et al., 1975a). In all these cases, one can observe increased staining with antibodies against contractile proteins, particularly AAA. In conclusion, the identification of specific and powerful anti-actin autoantibodies in the serum of patients with chronic aggressive hepatitis constitutes an useful adjunction to the investigations on the presence and function of contractile proteins in non-muscular cells. We thank Dr D. Bray for his advice and help for the extraction of contractile proteins; Mrs F. Gabbiani, Miss M. Flohr and Miss M. Bouland for their technical help and Messrs. J-C. Rumbeli and E. Denckinger for the photographic work. This work was supported in part by The Fonds National Suisse pour la Recherche Scientifique (grant no. 3.0330.73) and by the Foundation Sandoz.
REFERENCES ADELSTEIN, R.S., CONTI, M.A., JOHNSON, G.S., PASTAN, I. & POLLARD, T.D. (1972) Isolation and characterization of myosin from cloned mouse fibroblasts. Proc. nat. Acad. Sci. (Wash.), 69, 3693. ANDERSEN, P., HOLGAARD, J., ANDERSEN, I. & ANDERSEN, H.K. (1976) Smooth-muscle antibodies and antibodies to cytomegalo virus and epstein-barr virus in leukaemia and lymphoma. Acta path. microbial. scand. Sect. C, 84, 86. BRAY, D. & THOMAS, C. (1975) The actin content of fibroblasts. Biochem. j. 147, 221. DURHAM, A.C.H. (1974) A unified theory of the control of actin and myosin in nonmuscle movements. Cell, 2, 123. EISENBEEG, E. & KIELLEY, W.N. (1974) Troponin-Tropomyosin complex. J. biol. Chem. 249, 4742. GABBIANI, G., HiRSCHEL, B.J., RYAN, G.B., STATKOV, P.R. & MAJNO, G. (1972) Granulation tissue as a contractile organ. A study of structure and function. 7. exp. Med. 135, 719. GABBIANI, G., MAJNO, G. & RYAN, G.B. (1973a) The fibroblast as a contractile cell. The myo-fibroblast. Academic Press, London & New York. GABBIANI, G., RYAN, G.B., LAMELIN, J.P., VASSALLI, P., MAJNO, G., BOUVIER, C.A., CRUCHAUD, A. & LUSCHER, E.F. (1973b) Human smooth muscle autoantibody: its identification as anti-actin antibody and a study of its bindings to 'non-muscular' cells. Amer. J. Path. 72, 473. GABBIANI, G. & RYAN, G.B. (1974) Development of contractile apparatus in epithelial cells during epidermal and liver regeneration. J. Submicr. Cytol. 6, 143. GABBIANI, G., MALAISSE-LAGAE, F., BLONDEL, B. & ORCI, L. (1974) Actin in pancreatic islet cells. Endocrinology, 95, 1630. GABBIANI, G., BADONNEL, M.C. & RONA, G. (1975a) Cytoplasmic contractile apparatus in aortic endothelial cells of hypertensive rats. Lab. Invest. 32, 277. GABBIANI, G., CHAPONNIER, C. & LUSCHER, E.F. (1975b)
Actin in the cytoplasm of adrenocortical cells. Proc. Soc. exp. Biol. (N.Y.), 149, 618. GABBIANI, G., MONTESANO, R., TUCHWEBER, B., SALAS, M. & ORCI, L. (1975c) Phalloidin-induced hyperplasia of actin filaments in rats hepatocytes. Lab. Invest. 33, 562. GABBIANI, G., TRENCHEV, P. & HOLBOROW, E.J. (1975d) Increase of contractile proteins in human cancer cells. Lancet, ii, 796. GOLDMAN, R.D. (1971) The role of three cytoplasmic fibers in BHK-21 cell motility. I. Microtubules and the effects of colchicine. J. Cell Biol. 51, 752. HARTSHORNE, P.J. & MUELLER, H. (1969) The preparation of tropomyosin and troponin from natural actomyosin. Biochem. biophys. Acta (Amst.), 175, 301. ISHIKAWA, H., BISHOFF, R. & HOLTZER, H. (1969) Formation of arrow-head complexes with heavy meromyosin in a variety of cell types. ]. Cell Biol. 43, 312. JOHNSON, G.D., HOLBOROW, E.J. & GLYNN, L.E. (1975) Antibody to smooth muscle in patients with liver disease. Lancet, ii, 878. KAPANcI, Y., AssiMAcopouos, A., IRLE, C., ZWAHLEN, A. & GABBIANI, G. (1974) Contractile interstitial cells in pulmonary alveolar septa: a possible regulator of ventilation perfusion ratio ? Ultrastructural, immunofluorescence, and in vitro studies. ]. Cell Biol. 60, 375C. LACY, P.E., HOWELL, S.L., YOUNG, D.A. & FINK, C.J. (1968) New hypothesis of insuline secretion. Nature (Lond.), 219, 1177. LAZARIDES, E. & WEBER, K. (1974) Actin antibody: the specific visualization of actin filaments in non-muscle cells. Proc. nat. Acad. Sci. (Wash.), 71, 2268. LIDMAN, K., BIBERFELD, G., FAGRAEUS, A., NORBERG, R., TORSTENSSON, R. & UTTER, G. (1976) Anti-actin specificity of human smooth muscle antibodies in chronic active hepatitis. Clin. exp. Immunol. 24, 266.
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MALECH, H.L. & LENTZ, T.L. (1974) Microfilaments in epidermal cancer cells. 5. Cell Biol. 60, 473. NORBERG, R., LIDMAN, K. & FAGRAEUS, A. (1975) Effects of cytochalasin-B on fibroblasts, lymphoid cells and platelets revealed by human anti-actin antibodies. Cell, 6, 507. ORCI, L., GABBAY, K.H. & MALAISSE, W.J. (1972) Pancreatic beta-cell web: its possible role in insulin secretion. Science, 175, 1128. OSTLUND, R.E., PASTAN, I. & ADELSTEIN, R.S. (1974) Myosin in cultured fibroblasts. J. biol. Chem. 249, 3903. PERRY, S.V. & GREY, J.C. (1956) A study of the effects of substrate concentration and certain relaxing factors on the magnesium-activated myofibrillar adenosin triphosphatase. Biochem. 5. 64, 184. PERRY, S.V. & ZYDOWO, M. (1959) The nature of the extra-
protein fraction from myofibrils of striated muscle. Biochem. 5. 71, 220. POLLARD, T.D. & WEIHING, R.R. (1974) Actin and myosin and cell movement. CRC Crit. Rev. Biochem. 2, 1. PROBST, E. & LUSCHER, E.F. (1972) Studies on thrombosthenin-A, the actin like moiety of the contractile protein from blood platelets. I. Isolation, characterization and evidence for two forms of thrombosthenin-A. Biochem. biophys. Acta, 278, 577. SpuDicH, J.A. & WATT, S. (1971) The regulation of rabbit skeletal muscle contraction. 5. biol. Chem. 246, 4866. TRENCHEv, P., SNEYD, P. & HOLBOROW, E. (1974) Immunofluorescent tracing of smooth muscle contractile protein antigens in tissues other than smooth muscle. Clin. exp. Immunol. 16, 125.
Fixation of human anti-actin autoantibodies on skeletal muscle fibres CHRISTINE CHPO\NNIER, IAURE KOHLER* & G. GABBIANI Uinversity of Genieva, Genieva, Siteterlaild
DepartmeutmoJPatholoq),
(Received 2 September 1976)
SUMMXARY
Sera from fixve patients xaith chronic aggressive hepatitis containing smooth muscle autoantibodies xere tested bv means of indirect immunofluorescence for their binding to isolated rabbit skeletal muscle mrofibrils. In all cases, the immunofluorescent staining was sharply localized to I bands. After incubation of these sera with skeletal muscle troponin-tropomyosin complex, purified troponin opururified tropomxosin)no changes in immunofluorescent staining of myofibrils were noted. However, the staining was abolished after incubation of the sera with skeletal muscle actin. In double immunodiffusion experiments, a single precipitation line was obtained after diffusion of the sera against crude or purified actin. It is concluded that, at least for the sera examined, smooth muscle autoantibodies are anti-actin autoantibodies. The high titre of such autoantibodies and their availability in clinical immunology laboratories make them a useful tool to study actin distribution in muscular and non-muscular cells.
INTRODUCTION Smooth muscle autoantibodies have been described in the serum of patients with chronic aggressive hepatitis, infectious mononucleosis and malignant tumours (Johnson, Holboroxv & Gh nn, 1965; Andersen et a!., 1976). The observation that incubation of several smooth muscle autoantibodies containing sera with platelet actin (thrombosthenin-A) abolishes the ability of such sera to bind to smooth muscle or platelets, suggested that these autoantibodies are directed against actin (anti-actin antibodies or AAA) (Gabbiani et all., 1973b). Actin is one of the major components of striated muscle where it is localized in I bands together with regulatory proteins. It is generally accepted that actins from platelets, smooth or striated muscle are xerv similar (Pollard & W~eihing, 1974). The present study deals xith the fixation of AAA on striated muscle. Moreover, immunodiffusion of AAA against skeletal muscle actin, in one hand, and the effect of the previous incubation of AAA-containing sera With proteins present in I bands of striated muscle, on the other hand, have been inxestigated. MATERIALS ANI) METHODS
Mjyofibrils isolation. The technique described by Perry & Grey (1956) and Perry & Zydowo (1959) with minor modifications was used. The minced muscles from the back and legs of a rabbit were homogenized in 5 vol. of a buffer containing 40 mxi tris-HCI pH 7-0 and 25 mxt KCI. After 15 min of centrifugation at 600 g, the supernatant was disregarded. These operations were repeated several times. The pellets were washed eight times in 9 vol. of buffer containing 40 mm tris-HCl pH 7 1 and 0 1 xi KC1. \lyofibrils were then stored at 4 C in the last buffer with 2 mxi 2-mercapto-ethanol. Protein extrachtion. 4ctit. This was prepared from rabbit skeletal muscle following the technique of Spudich & Watt l)epartment of Biochemistry, Faculty of Sciences, Unixersity of Geneva, Geneva, Switzerland. Correspondence: Dr G. Gabbiani, Department of Pathology, University of Geneva, 40 Boulevard de la Cluse, 1211 * From the
Geneva 4, Switzerland.
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(1971). Ten grams of acetonic powder were extracted at 0C by agitation in 200 ml of a buffer composed of 2 mM tris-HCl pH 8-0; 0-2 mm ATP; 0-5 mm 2-mercapto-ethanol and 0-2 mm CaCl2. The solution was filtered through three layers of gauze. The residue was washed with 100 ml of buffer and filtered again through three layers of gauze. The supernatants were clarified by centrifugation at 10,000 g for 3 hr. Actin polimerization was obtained at room temperature in 0 5 M KCl and 2 mM MgCl2 during 20 min. The KC1 concentration was then increased up to 0-6 M under slight agitation during 40 min. The solution was ultracentrifuged for 3 hr at 80,000 g. The pellet was redissolved in the same buffer used for the acetonic powder extraction and dialysed against the same buffer during 72 hr under agitation with frequent changes of buffer. Gactin was clarified by ultracentrifugation at 80,000 g during 3 hr and then polymerized with 50 mm KC1. Troponin (TP) and tropomyosin (TM). These were isolated from native actomyosin (NAM) (Hartshorne & Mueller, 1969). Myofibrils (collected as described above) were washed at 4°C in 1 vol. of a solution containing 1-2 M KCl; 80 mm NaHCO3; 20 mm Na2CO3 and 6 vol. of Weber's solution (0-6 M KCI, 40 mm NaHCO3 and 10 mm Na2CO3) and homogenized rapidly in this solution (for 30 sec at the maximal speed of a Sorvall Omni-mixer). After 30 min, myofibrils were centrifuged at 2500 g during 20 min and the supernatant slowly diluted 1/10 with 15 mm 2-mercapto-ethanol in water. After 60 min, NAM was obtained by centrifugation at 2000 g during 20 min. The pellets were quickly washed first with 20 mm Tris-maleate buffer and then with 100 mm Tris-maleate buffer (both at pH 7-1). The NAM powder was prepared after washing several times in alcohol ether. The NAM powder was left with agitation overnight at 4°C in 1 M KCl and 2 mm DTT. The solution was centrifuged at 40,000 g during 3 hr and the supernatant dialysed against 4 vol. of water and 2 mm DTT to obtain a final dilution of 0 2 M KCl and 2 mm DTT. TP-TM complex was precipitated between 40% and 60% saturation (NH4)2SO4. TM and TP were isolated chromatographically (Eisenberg & Kielley, 1974) on hydroxyapatite (Biorad, in the wet form) column (2-5 x 25 cm). Hydroxyapatite was washed ten times to eliminate the smallest particles and then equilibrated in 1 M KC1, 2 mm DTT, 1 mM P04, pH 7 0. One hundred milligrams of TP-TM complex in 10 ml of 2 mm imidazol HCI, pH 7 0, 1 M KCI, 5 mm 2-mercapto-ethanol were eluted with a linear P04 gradient (from 1 M KC1, 2 5 mm DTT, 1 mM P04 at pH 7 0, to 1 M KCl, 2 5 mm DTT, 0 2 M P04 at pH 7-0). The different peaks were dialysed against 0 5 M KCl, 2 mm DTT, 2 mm EDTA and 2 mm imidazol. AAA sera. AAA containing sera were obtained from five patients with chronic aggressive hepatitis. Their activity in staining frozen sections of smooth muscle was judged as follows: cryostat sections of rat small intestine were fixed for 5 min in cold acetone (Merck Company, Darmstadt, Germany) and were then treated for 15 min with the serum to be tested (diluted when necessary in phosphate-buffered saline or PBS, pH 7.2). The sections were then washed in PBS and stained for 15 min with fluorescein-conjugated IgG fraction of goat antiserum to human IgG (code 64-170 Miles Seravac, Lausanne, Switzerland). After rewashing with PBS and mounting in 90% glycerol in PBS, the level of fluorescence was compared with that found in control sections, treated with normal human serum (NHS) instead of AAA serum. Photographs were taken on a Zeiss UV photomicroscope, using Anscochrome colour slide film 500 day light (Gaf Corp. New York). The pattern of AAA labelling to smooth muscle of muscularis externa or muscularis mucosa was qualitatively similar on the five sera. The dilutions at which immunofluorescent staining was detectable were 1/1280 (two sera), 1/640 and 1/320 (two sera). These dilutions represented the titres of the sera. Tests of these sera for anti-mitochondrial or anti DNA antibodies and for antinuclear factor were negative. Immunodiffusion. Tests were performed according to the Ouchterlony technique. Agarose (Behring-Wercke A.G., Marburg-Lahn, Germany) was dissolved in 2 mm tris-HCI buffer at pH 8-0 containing 0-2 mM ATP, 0 5 mM 2-mercaptoethanol and 0-2 mm CaCl2 (Norberg, Lidman & Fagraeus, 1975). Immunoabsorption. Immunoabsorption tests were performed as follows: 0- 1 ml of serum was added to 0 9 ml of the different solutions of contractile proteins, incubated for 60 min at room temperature (with agitation every 15 min), and left overnight at 4°C. After incubation with actin, a precipitate was present at the bottom of the tube. This precipitate was eliminated by centrifugation of the sera for 10 min at 4600 g. As a control, we incubated 0-1 ml of the sera with 0-9 ml of the buffers in which the various contractile proteins were dissolved. In each case, the AAA titres of these sera were tested on cryostat sections of rat small intestine and compared with that of serum incubated with buffers and with that of original untreated serum.
Immunofluorescent staining. Most of the staining was performed with the serum having a titre of 1/1280. Three hundred microlitres of the standard myofibril suspension were centrifuged at 1000 g and washed twice in PBS. After the last washing they were resuspended in 100 pl of PBS and incubated for 30 min at room temperature with 10 pl of AAA serum. In addition 100 Id of myofibril suspension in PBS were incubated with 10 ,l of NHS or 10 p1 of AAA serum previously incubated with either: (1) actin; (2) the complex TP-TM; (3) TP or (4) TM. After the first incubation, the myofibrils were centrifuged at 1000 g and washed in PBS three times. They were then resuspended in 100,ul of PBS and incubated for 30 min at room temperature with 20,ul of fluorescein-conjugated, y-globulin fraction of goat anti-serum to human IgG. After washing three times in PBS, the myofibrils were suspended in 2 ml of foetal calf serum and cytocentrifuged on glass slides by means of a Shannon Eliott (London, England) cytocentrifuge. The slides were mounted in 90% glycerol in PBS and examined on a Zeiss UV microscope.
RESULTS In double immunodiffusion tests, all AAA sera gave a single strong precipitation line with either crude or purified rabbit skeletal muscle actin (Fig. 1, 2). No precipitation was noted after immunodiffusion
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FIG. 1. Immunodiffusion experiment according to the Ouchterlony technique. AAA serum has been placed in the centre against decreasing concentrations of crude (a) and purified (b) actin. In (a), the concentrations are: 2 4 mg/ml upper right; 1 2 mg/ml middle right; 0 60 mg/ml lower right; 0 30 mg/ml lower left; 0-15 mg/ml middle left and 0 075 mg/ml upper left. In (b) the concentrations are: 3 mg/ml upper right; 1 5 mg/ml middle right; 0 75 mg/ml lower right; 0 375 mg/ml lower left; 0-19 mg/ml middle left and 0 095 mg/ml upper left.
FIG. 2. SDS-Polvacrylamide gel electrophoresis showing crude actin (a) and purified actin (b) from rabbit skeletal muscle used for immunodifffusion and absorption tests.
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FIG. 3. SDS-Polyacrylamide gel electrophoresis showing TP-TM complex (a); the same complex with actin added as a marker (b); purified TP (c); purified TM (d). All these proteins were extracted from rabbit skeletal muscle and used for absorption experiments.
.
f
h
FIG. 4. Isolated myofibrils after immunofluorescent staining with AAA sera alone or previously incubated with contractile proteins present in I-band of striated muscle. (Magnification x 700.) (a) Myofibril treated first with AAA serum followed by IgG fraction of goat antiserum to human IgG: the stain is localized in bands; (b) these bands in the phase constrast micrograph of the same myofibril appear to be I bands; (c) myofibril treated first with AAA serum previously incubated with TM-TP complex followed by IgG fraction of goat antiserum to human IgG: the staining is similar to that of (a); (d) phase contrast micrograph of the myofibril in (c) showing that the positive staining is localized in I bands; (e) myofibril treated first with AAA serum previously incubated with TP followed by IgG fraction of goat antiserum to human IgG: the stain is similar to that of (a) and (c); (f) phase contrast micrograph of the myofibril in (e) showing that the fluorescent staining is localized in I bands; (g) myofibril treated first with AAA serum previously incubated with TM, followed by IgG fraction of goat antiserum to human IgG: the fluorescent staining is similar to that of (a), (c) and (e); (h) phase contrast micrograph ofthe myofibril in (g) showing that the fluorescent staining is localized in I bands; (i) myofibril treated first with AAA previously incubated with actin, followed by IgG fraction of goat antiserum to human IgG: no immunofluorescent staining; (j) phase contrast micrograph of the myofibril in (i).
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of AAA sera with purified TP or TM (Fig. 3). No precipitation also was noted after immunodiffusion of actin against NHS. When myofibrils were incubated with NHS, no fluorescence was present after staining with fluorescein-conjugated anti-human IgG. On the other hand, the staining was strongly positive when myofibrils were treated first with AAA (alone or previously incubated with the complex TP-TM, TP or TM) (Fig. 4). Comparison of immunofluorescence with phase contrast micrographs of the same myofibrils, showed that the immunofluorescent staining with AAA sharply corresponded to I bands. When myofibrils were treated with AAA previously incubated with purified actin, no immunofluorescent staining was present after fluorescein-conjugated goat anti-human IgG (Fig. 4).
DISCUSSION These results show that incubation of isolated myofibrils with AAA results in a sharp staining of Ibands. The contractile proteins composing I bands are actin, TP and TM. The persistence of AAA activity after incubation of AAA sera with the complex TP-TM or with the single purified proteins in one hand and the abolition of AAA activity after incubation of AAA sera with purified striated muscle actin on the other hand, support the previous assumption (Gabbiani et al., 1973b) that the sera tested contain specific anti-actin antibodies. This assumption is confirmed by the immunodiffusion experiments showing a single precipitation line when the sera are diffused against either crude or purified actin. In analogy with the previous experiments using thrombosthenin-A (Gabbiani et al., 1973b), incubation of AAA sera with skeletal muscle actin abolishes the staining of not only I bands in striated muscle but also of the cytoplasm of smooth muscle cells, platelets and cultivated fibroblasts. Although a full comparison of the amino-acid sequences of actins from striated muscle, smooth muscle, platelets and fibroblasts cannot yet be made, it is nevertheless well established that these actins are very similar (Probst & Luischer, 1972; Pollard & Weihing, 1974). Hence, it can be concluded that, the tested sera from patients with chronic aggressive hepatitis contain crossreacting anti-actin autoantibodies. A recent work by Lidman et al. (1976) using thirty anti-smooth muscle antibodies from patients with chronic aggressive hepatitis has also shown that the staining activity of these sera is abolished after incubation with skeletal muscle actin. AAA have been shown to fix on structures containing important amounts of actin such as platelets, megakaryocytes, the peripheral part of intestinal epithelial cells and stress lines of cultivated fibroblasts (Gabbiani etal., 1973b; Gabbiani, Majno & Ryan, 1973a). The demonstration of the selective fixation of AAA to I bands of striated muscle more precisely define their specificity. After the description of anti-actin activity in sera of patients with chronic aggressive hepatitis (Gabbiani et al., 1973b), artificial anti-actin antibodies have been produced experimentally by injecting in rabbit the purified protein (Trenchev, Sneyd & Holborow, 1974; Lazarides & Weber, 1974). Such experimental antibodies have the same specificity as the human ones but relatively low titres when compared to the human ones. This was explained on the basis of the low antigenicity of actin which has a similar structure throughout the philogenetic scale. The reasons why humans undergoing viral hepatitis or other forms of viral diseases produce, at least in a certain proportion of cases, high titre anti-actin antibodies remain mysterious. In any event, the high titre of AAA sera and their availability in clinical immunology laboratories makes them an ideal candidate for the study of the presence and distribution of actin in non-muscular cells. AAA have been previously used to demonstrate for the first time the presence of actin in such cells as interstitial cells of the lung alveoli (Kapanci et al., 1974), beta-cells of Langerhans islets (Gabbiani et al., 1974), adrenocortical cells (Gabbiani, Chaponnier & Liischer, 1975b), fibroblasts of granulation tissue (myofibroblasts) (Gabbiani et al., 1972) and neoplastic cells of the skin, oral cavity, larynx and mammary gland (Gabbiani, Trenchev & Holborow, 1975d). The present study on the specificity of AAA for actin constitutes a further argument supporting the conclusions reached in all these studies. In the last few years, more and more evidences have accumulated showing that contractile proteins are present in many if not all eucaryotic cells (Pollard & Weihing, 1974). Here, they have been correlated to such functions as cell contraction (Gabbiani et al., 1972), cell locomotion (Pollard & Weihing, 1974;
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Durham, 1974), intracellular transport or endo- and exocytosis (Lacy et al., 1968; Orci, Gabbay & Malaisse, 1972). Contractile proteins have been identified in the cytoplasm of several cell types by means of biochemical techniques (Adelstein et al., 1972; Ostlund, Pastan & Adelstein, 1974; Bray & Thomas, 1975) or of electron microscopy, on the basis of the morphology of filaments (Goldman, 1971); actin has also been identified on the basis of the capacity to bind heavy meromyosin, giving a characteristic arrowhead appearance (Ishikawa, Bishoff & Holtzer, 1969). These findings have permitted the study of the distribution of individual contractile protein in different structures under various conditions. Cells in which cytoplasmic filaments increase considerably in vivo following changes in their microenvironment have been described. They include epidermal cells (Gabbiani & Ryan, 1974) or fibroblasts (Gabbiani et al., 1972), during the healing of an open wound, epidermal cells during experimental production of malignant tumours (Malech & Lentz, 1974), hepatocytes during liver regeneration (Gabbiani & Ryan, 1974) or after treatment with small amounts of phalloidin (one of the poisons of the mushroom Amanita Phalloides) (Gabbiani, et. al., 1975c) and aortic endothelial cells during the first stages of hypertension (Gabbiani, Badonnel & Rona, et al., 1975a). In all these cases, one can observe increased staining with antibodies against contractile proteins, particularly AAA. In conclusion, the identification of specific and powerful anti-actin autoantibodies in the serum of patients with chronic aggressive hepatitis constitutes an useful adjunction to the investigations on the presence and function of contractile proteins in non-muscular cells. We thank Dr D. Bray for his advice and help for the extraction of contractile proteins; Mrs F. Gabbiani, Miss M. Flohr and Miss M. Bouland for their technical help and Messrs. J-C. Rumbeli and E. Denckinger for the photographic work. This work was supported in part by The Fonds National Suisse pour la Recherche Scientifique (grant no. 3.0330.73) and by the Foundation Sandoz.
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