Journal of Immunological Methods, 130 (1990) 1-8
1
Elsevier JIM05573
Anti-actin antibodies: a n e w test for an old p r o b l e m * M. Fusconi, F. Cassani, D. Zauli, M. Lenzi, G. Ballardini, U. Volta and F.B. Bianchi Cattedra di Semeiotica Medica, Istituto di Clinica Medica Generale e Terapia Medica, University of Bologna, Bolognia, Italy (Received 10 November 1989, revised received 14 December 1989, accepted 29 January 1990)
Smooth muscle antibodies with anti-actin specificity are commonly regarded as markers of autoimmune liver disease. However, there are interpretational problems because different techniques have been used for their identification and therefore the results are difficult to compare. The present paper reports the results of a new method for the identification of anti-actin antibodies (indirect immunofluorescence on cryostat sections of liver from rats chronically injected with phalloidin). The results have been compared with those obtained by four other techniques: demonstration by immunofluorescence of kidney peritubular reactivity (SMAT), of anti-microfilament antibodies (on HEp-2 cells and vinblastine-treated peripheral blood mononuclear cells) and counterimmunoelectrophoresis with purified muscle actin as antigen. The new method proved to be the most sensitive and specific. Furthermore, its reproducibility was found to be high, the interpretation easy and the cost low. The clinical significance of anti-actin antibodies in patients with chronic liver disease is also discussed. Key words: Smooth muscle antibody; Anti-actin antibody; Autoimmune hepatitis; Chronic liver disease; Immunofluorescence
Introduction
Since liver-specific autoantibodies or target antigens have not yet been proved to be relevant in the pathogenesis of autoimmune chronic hepatitis, its diagnosis remains largely inaccurate. In fact, the internationally accepted criteria (Leevy et al., 1976) are both the absence of any other known cause of liver damage and the presence of nonorgan specific autoantibodies, namely antinuclear (ANA) and anti-smooth muscle antibodies (SMA). This kind of approach leads to two different biases. On the one hand, at least theoretically, it is impossible to exclude the possibility that 'primary' Correspondence to: F.B. Bianchi, Cattedra di Semeiotica Medic.a, Istituto di Clinica Medica Generale e Terapia Medica, Policlinico S. Orsola, Via Massarenti 9, 40138 Bologna, Italy. * This work was supported by CNR Grant 88.01942.04.
autoimmune mechanisms can be associated with other causes of liver damage. This can be relevant in populations with a high prevalence of viral infections or alcohol abuse. On the other hand, ANA and SMA are known to be heterogeneous families of autoantibodies, which are directed against different antigens. It is well known that SMA immunofluorescence (IFL) patterns are produced by antibodies to different cytoskeletal proteins and that only the anti-actin (anti-microfilament) specificity is associated with autoimmune liver disease (Kurki et al.. 1983). Other SMA, directed against different cytoskeletal components (namely intermediate filaments), are present, often at high prevalence, in virus-induced liver disease (Bretherton et al., 1983). The availability of a marker able to identify accurately autoimmune cases of chronic liver disease (CLD) is of great interest from a therapeutic point
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
of view. Although data are present in the literature on the prevalence of anti-actin antibodies in CLD (Pedersen et al., 1982; Bretherton et al., 1983; Kurki et al., 1983; Cunningham et al., 1985), the methods employed are different and the results difficult to compare. The absence of a reliable, easy and low cost method for the identification of anti-actin antibodies prompted us to set up a new test and to compare its results with those obtained by four currently used ones.
Materials and methods
Serum samples These were obtained from 223 non-consecutive patients with CLD, defined by internationally accepted criteria (Leevy et al., 1976). 38 were from patients with HBV related (HBV-rel) CLD (24 HBsAg positive and 14 antiHBs positive); 38 with alcoholic CLD (ALD); 49 with autoimmune (AUTO) CLD (defined on the basis of the presence of antinuclear, ANA, a n d / o r anti-smooth muscle, SMA, autoantibodies and the exclusion of other known causes of liver damage); 28 with cryptogenic (CRYPTO) CLD, 50 with primary biliary cirrhosis (PBC) and 20 with anti-liver-kidney microsomai antibodies (LKM) positive CLD. Sera from 83 age- and sex-matched blood donors (BD) were used as controls.
with a Leitz Orthoplan microscope equipped with an epiillumination.
A ctin-containing structures Actin-containing structures in the different substrates were detected by IFL using both a polyclonal anti-actin antibody (Miles, U.S.A.) and rhomdamine-conjugated phalloidin (generously provided by Prof. T. Wieland, Max-Plank-Institut, Heidelberg, F.R.G.), which binds specifically to actin (Wulf et al., 1979). To assess the specificity of the reaction, inhibition experiments were performed by incubating the substrate with a mixture of conjugated and free phalloidin at a ratio of 1 to 2000, as described earlier (Wulf et al., 1979).
Detection of SMA This was performed by indirect IFL. Sera were considered to be SMA positive, according to Bottazzo et al. (1976), when a fluorescent reaction was detectable on kidney vessel wall only (SMAv) or on kidney vessels walls, glomerular and peritubular structures (SMAT). Positive sera were titrated by doubling dilutions to an end point.
Detection of anti-microfilament antibodies (anti-MF) Anti-MF antibodies were assayed by indirect IFL on HEp-2 cells (Kallestadt, F.R.G.) and on vinblastine-treated peripheral blood mononuclear cells (MC), which have been shown to be a good substrate for the detection of anti actin specificity in autoimmune CLD (Zauli et al., 1985).
Immunofluorescence (I FL) The indirect method was performed on different substrates according to a standard procedure. 4 /~m cryostat sections of rat liver, kidney and stomach were used for the identification of ANA, SMA, anti-mitochondrial antibodies (AMA) and LKM antibodies. Sera were tested at a dilution of 1/40 in phosphate-buffered saline (PBS) and incubated for 20 min at room temperature in a moist chamber. After three washes of 10 min each with PBS, the slides were incubated with a fluorescein-conjugated sheep anti-human immunoglobulin (IgG, IgA, IgM, Wellcome, U.K.) at a dilution of 1/60 in PBS for 20 min in a moist chamber. After three more washes with PBS, slides were mounted in PBS/glycerol (1/1, v/v) and observed, under code, by two independent readers
Detection of anti-liver cell actin antibodies (ALCA) The test was performed by indirect IFL using 4 /~m cryostat liver sections from rats chronically intoxicated with phalloidin (a generous gift from Prof. T. Wieland). The drug was administered intraperitoneally for 19 days at a dose of 500 /~g/kg in 0.5 ml. This sublethal dose leads to an enormous deposition of microfilamentous material, consisting mainly of actin, at the biliary and sinusoidal portion of the hepatocytes (Gabbiani et al., 1975).
Detection of anti-muscle actin antibodies (AAA ) Anti-muscle actin antibodies were assayed by counterimmunoelectrophoresis (CIE) (Lenzi et al., 1988) using purified actin extracted from acetone
J
-..
w.
Fig. 1. Counterimmunoelectrophoresisdetection of anti-muscle actin antibodies (AAA). Positive sera give precipitin lines showing reactions of identity.
powder of rabbit skeletal muscle (Pardee and Spudich, 1982) as antigen. The purity of the Gactin preparation was assesed by polyacrylamide gel electrophoresis, which showed a single band at a molecular weight of 43,000. In C I E experiments actin was used at a concentration of 0.3 m g / m l . A reaction of identity was present with positive sera (Fig. 1). As positive control a polyclonal rabbit anti-actin antibody (Miles, U.S.A.) was used.
Statistical analysis Statistical comparisons were performed using the Wilcoxon rank sum test.
Fig. 2. lmmunofluorescence localization of actin in liver of phalloidin-treated rats using rhodamine-labelled phalloiodin. Actin is deposited at the periphery of the hepatocytes. Anti-liver cell actin (ALCA) positive sera display sera display this pattern. Original magnification 25 x.
On rat kidney the reactivity was present in vessels, glomerular mesangium and peritubular fibrils with an interrupted pattern (Fig. 3). Also the brush border was faintly reactive. On rat stomach the positivity was restricted to the muscularis mucosae, intergland fibers and, to a lesser extent, the cytoplasm of parietal ceils with a filamentous pattern. On HEp-2 cells the positivity of the two probes was confined to the microfilaments, with the typical pattern of linear cables distributed on the
Results
Morphological identification of actin-containing structures On liver sections from phalloidin-treated rats staining was present in the vessels and, more strongly, in blocks of material located at the periphery of hepatocytes, mainly around bile canaliculi. The sinusoids were positive and enlarged (Fig. 2). This pattern is identical to that described by Gabbiani et al. (1975) and corresponds to microfilamentous deposits at the periphery of liver ceils. In normal liver strong positivity was detected in the arterial and venous walls, while faint reactivity was present at the periphery of the hepatocytes.
Fig. 3. Immunofluorescence localization of actin on rat kidney by rhodarnine-labelled phalloidin. Vessel walls, glomerular mesangium and peritubular stuctures, with an interrupted pattern, are positive. SMAT positive sera show identical reactivity. Original magnification 25 x.
Fig. 4. Immunofluorescence localization of actin on HEp-2 cells and peripheral blood mononuclear cells by rhodamine-labelled phalloidin. Actin cables are detected as 'classical' stress fibers on HEp-2 cells (A) or microvillar projections on mononuclear cells (B). Anti-MF positive sera display these reactivities. Original magnification 50 ×.
longitudinal axis of the cells (Fig. 4A). On MC the pattern of positivity was that of cytoplasmic staining with peripheral microvilli (Fig. 4B). Sera were considered to be positive for SMAT, anti MF and ALCA when they gave the above IFL patterns on rat kidney, HEp-2 cells, MC and phalloidin-treated liver, respectively.
Prevalence of SMA 73 of the 223 sera from CLD patients (32%) were positive for SMA: 21 (9%) with a SMA v pattern and 52 (23%) with a SMAv pattern. Ten of the 83 BD studied were SMA positive (12%), always with a SMA v pattern and a low titer ( < 1/80). The prevalence of SMA among the different subgroups is reported in Table I. It is
apparent that SMA positive sera are significantly associated with the A U T O CLD group. 18 of the 21 SMA T sera were from patients with A U T O CLD and PBC. Titers of the 21 SMA T positive sera (median > 1/320; range 1 / 8 0 - > 1/320) were significantly higher ( P < 0.01) than those of the SMA v positive sera (median 1/80; range 1 / 4 0 - > 1/320). The detection of the SMAT pattern was difficult in cases where the titer was low and other autoantibodies reacting with kidney tubules (antimitochondrial and anti liver-kidney microsomal antibodies) were present.
Prevalence of anti-MF The anti-MF data is shown in Table II. The two cellular substrates gave different results, with
TABLE I PREVALENCE OF ANTI-SMOOTH MUSCLE ANTIBODIES (SMA) AND THEIR PATTERN IN DIFFERENT SUBGROUPS OF PATIENTS WITH CHRONIC LIVER DISEASE AUTO (49) SMA positive T V
11(22%) 28 (57%)
Total SMA negative
39 (79%) 10 (21%)
CRYPTO (28)
ALD (38)
HBV-rel (38)
(0%) (0%)
0 (0%) 10 (26%)
0 (0%) 28 (100%)
10 (26%) 28 (74%)
0 0
PBC (50)
LKM (20)
BD (83)
2 (5%) 10 (26%)
7(14%) 2 (4%)
1 (5%) 2 (10%)
0 (0%) 10 (12%)
12 (31%) 26 (69%)
9 (18%) 41 (82%)
3 (15%) 17 (85%)
10 (12%) 73 (88%)
Key: AUTO, autoimmune chronic liver disease; CRYPTO, cryptogenic chronic liver disease; ALD, alcoholic chronic liver disease; HBV-rel, hepatitis B virus-induced chronic liver disease; PBC, primary biliary cirrhosis; LKM, Anti-liver-kidney microsomal antibody positive chronic liver disease; BD, blood donors.
TABLE 1I PREVALENCE OF ANTI-MICROFILAMENT ANTIBODIES (ANTI-MF) AS DETERMINED ON HEP-2 CELLS AND VINBLASTINE TREATED MONONUCLEAR CELLS (MC) IN RELATION TO SMA POSlTIVITY AND PA'VTERN IN DIFFERENT SUBGROUPS OF PATIENTS WITH CHRONIC LIVER DISEASE AUTO ( 4 9 ) Anti-MF (HEp-2) SMA T positive SMA v positive SMA negative Total Anti-MF (MC) SMA T positive SMA v positive SMA negative Total
CRYPTO (28)
ALD ( 3 8 )
HBV-rel (38)
10/11 (91%) 0/0 (0%) 0/28 ( 0 % ) 0/0 (0%) 0/10 ( 0 % ) O/28 (0%)
0/0 (0%) 0/10 (0%) 0/28 (O%)
2/2 (100%) 7/7(100%) 0/10 (0%) 0/2 (0%) O/26 (O%) 0/41 (O%)
1/1 (100%) 0/0 (0%) 0/2 ( 0 % ) 0/10 (0%) 0/17 ( 0 % ) 0/73 (0%)
10/49 (20%)
0/28 (0%)
0/38 (0%)
2/38
7/50 (14%)
1/20
11/11 (100%) 9/28 (32%) 0/10 ( 0 % )
0/0 (0%) 0/0 (0%) 0/28 (0%)
0/0 (0%) 3/10 (30%) 0/28 (0%)
1/2 (50%) 6/10 (60%) 0/26 (0%)
7/7 (100%) 2/2 (100%) 0/41 ( 0 % )
1/1 (100%) 0/0 (0%) 0/2 (0%) 0/10 (0%) 0/17 ( 0 % ) 0/73 (0%)
20/49 (40%)
0/28 (0%)
3/38 (8%)
7/38 (18%)
9/50 (18%)
1/20
(5%)
CPB (50)
LKM (20)
(5%)
(5%)
BD (83)
0/83 (0%)
0/83 (0%)
Key: AUTO, autoimmune chronic liver disease; CRYPTO, cryptogenic chronic liver disease; ALD, alcoholic chronic liver disease; HBV-rel, hepatitis B virus-induced chronic liver disease; PBC, primary biliary cirrhosis; LKM, anti-liver-kidneymicrosomal antibody positive chronic liver disease; BD, blood donors.
a higher percentage of positive results when MC were used. In fact, 20 of the 223 (9%) sera were positive on HEp-2 cells while 40 (18%) were reactive on MC. All the sera positive for anti-MF (tested on HEp-2 or MC) were SMA positive. All but one of the anti-MF positive sera on HEp-2 cells were confirmed on MC, the exception being one serum positive for SMA T, A L C A and AAA. Conversely, 21 of the 40 anti M F positive sera on MC were negative on HEp-2 cells. None of the 83 BD sera were anti M F positive.
Prevalence of ALCA A positive reaction was detected in 28 of the 223 sera from C L D patients (12%). All 28 A L C A positive sera were also SMA positive, while none of the SMA negative sera were A L C A positive. N o n e of the 83 control sera were positive. All the 21 SMA r sera were positive for ALCA. In addition, seven SMA v positive sera showed this reactivity. The prevalences of A L C A among the different subgroups is reported in Table III. The detection of A L C A was always easy, even when
TABLE 11I PREVALENCE OF ANTI-LIVER CELL ACTIN ANTIBODIES (ALCA) IN RELATION TO SMA POSITIVITY AND PATTERN IN DIFFERENT SUBGROUPS OF PATIENTS WITH CHRONIC LIVER DISEASE AUTO ( 4 9 )
CRYPTO (28)
ALD (38)
HBV-rel (38)
CBP (50)
LKM (20)
BD (83)
SMATpositive SMA v positive SMA negative
11/11 (100%) 4/28 (14%) 0/10 ( 0 % )
0/0 (0%) 0/0 ( 0 % ) 0/28 (0%)
0/0 (0%) 0/10(0%) 0/28 (0%)
2/2 (100%) 2/10 (20%) 0/26 ( 0 % )
7/7 (100%) 1/2 (50%) 0/41 ( 0 % )
1/1 (100%) 0/2 (0%) 0/17 ( 0 % )
0/0 (0%) 0/10(0%) 0/73 (0%)
Total
15/49 (31%)
0/28 (0%)
0/38 (0%)
4/38 (10%)
8/50 (16%)
1/20
0/83 (0%)
(5%)
Key: AUTO, autoimmune chronic liver disease; CRYPTO, cryptogenic chronic liver disease; ALD, alcoholic chronic liver disease; HBV-rel. hepatitis B virus-induced chronic liver disease; PBC, primary biliary cirrhosis; LKM, anti-liver-kidneyrnicrosomal antibody positive chronic liver disease; BD, blood donors.
6 TABLE IV PREVALENCE OF ANTI-MUSCLE ACTIN ANTIBODIES (AAA) IN RELATION TO SMA POSITIVITY AND PATYERN IN DIFFERENT SUBGROUPS OF PATIENTS WITH CHRONIC LIVER DISEASE AUTO ( 4 9 ) SMA T positive SMAv positive SMA negative Total
CRYPTO (28)
ALD ( 3 8 )
HBV-rel (38)
CBP (50)
LKM (20)
BD (83)
9/11 (89%) 3/28 (11%) 0/10 (0%)
0/0 (0%) 0/0 (0%) 0/28 (0%)
0/0 (0%) 1/10 (10%) 0/28 (0%)
2/2 (100%) 1/10 (10%) 0/26 ( 0 % )
6/7 (86%) 1/2 (50%) 0/41 ( 0 % )
1/1 (100%) 0/2 (0%) 0/17 ( 0 % )
0/0 (0%) 0/10 (0%) 0/73 (0%)
12/49 (24%)
0/28 (0%)
1/38 (3%)
3/38
7/50 (14%)
1/20
0/83 (0%)
(8%)
(5%)
Key: AUTO, autoimmune chronic liver disease; CRYPTO, cryptogenic chronic liver disease; ALD, alcoholic chronic liver disease; HBV-rel, hepatitis B virus-induced chronic liver disease; PBC, primary biliary cirrhosis; LKM, anti-liver-kidney microsomal antibody positive chronic liver disease; BD, blood donors.
the titer was low o r o t h e r c y t o p l a s m i c a u t o a n t i b o d i e s were s i m u l t a n e o u s l y present.
Prevalence of AAA 24 of the 223 sera f r o m C L D p a t i e n t s were positive b y C I E with a reaction of identity. N o n e of the 83 c o n t r o l s were reactive by this m e t h o d . All the 24 positive sera were S M A positive, 18 with the S M A T a n d six with the S M A v p a t t e r n . T h e prevalence of A A A a m o n g the d i f f e r e n t subg r o u p s is s u m m a r i z e d in T a b l e IV.
Comparison of SMA, anti-MF (HEp-2 and MC), ALCA and AAA T h e results o b t a i n e d with the five techniques are shown in T a b l e V. It is a p p a r e n t that within the S M A x s u b g r o u p all the m e t h o d s gave almost the s a m e results, C I E b e i n g slightly less sensitive t h a n the others. T h e results are different within the S M A v s u b g r o u p , b e c a u s e o f the high p r o p o r tion of a n t i - M F positive sera on M C (38%); A L C A , A A A a n d a n t i - M F were d e t e c t e d on H E p - 2 cells in 13%, 12% a n d 0% of these sera, respectively. In
TABLE V RELATIONSHIP OF ANTI-MF ON HEP-2 CELLS, ANTI-MF ON MC, ALCA AND AAA WITH SMA POSITIVITY AND PATTERN IN ALL THE SERA FROM PATIENTS WITH CHRONIC LIVER DISEASE
SMA T positive
Total SMA v positive
16 (76%) 3 (14%)
ALCA
AAA
+ +
+ +
+ +
-
+
1
(5%)
-
+
+
+
(5%)
+
-
+
+
20 (95%)
20 (95%)
21 (100%)
18 (86%) +
21 (100%) 2
(4%)
-
+
+
4
(8%)
-
+
+
-
-
+ +
-
+
(6%) 11 (21%)
-
1
(2%)
-
-
+
-
1
(2%)
-
-
-
+
30 ( 5 7 % )
SMA negative
Anti-MF (MC)
1
3
Total
Anti-MF (HEp-2)
.
52 (100%) 150 ( 1 0 0 % )
. 0 (0%)
.
.
.
20 (38%) .
.
7 (13%) .
6 (12%)
TABLE VI 'FALSE' POSITIVE AND 'FALSE' NEGATIVE RESULTS IN THE DETERMINATION OF ANTI ACTIN ANTIBODIES OBTAINED WITH THE DIFFERENT TECHNIQUES (A serum was arbitrarily assumed to be postive for anti-actin antibodies when it was reactive in at least two of the five tests.)
SMA-r Anti-MF (HEp-2) Anti-MF (MC) ALCA AAA
"False' positive
'False' negative
0/21 0/20 11/40 1/28 1/24
9/52 10/53 1/33 3/45 7/49
(0%) (0%) (27%) (4%) (4%)
(17%) (19%) (3%) (7%) (14%)
order to evaluate the disease sensitivity and specificity of each test, we arbitrarily assumed that a serum was positive for anti-actin antibodies when it was reactive in at least two of the five tests. By this method, 30 out of the 73 SMA positive sera (41%) were positive for anti-actin antibodies. Of these, 21 gave a SMA T and nine a SMAv pattern. Titers of SMA v and anti-actin positive sera were not significantly different from those of SMA v positive and anti-actin negative sera. A 'false' positive reaction was present in 11 out of the 40 actin-MF positive sera on MC (27%), since they were negative by all the other techniques. These sera were mainly found in the HBV and alcohol related CLD. 'False' positive results were conversely extremely rare with ALCA and AAA (one serum each). The proportion of 'false' negative results was high with HEp-2 cells (19%), SMA T (17%) and AAA (14%), but low with MC (3%). These results are summarized in Table VI. The method which gave the lowest incidence of 'false' positive and negative results (4% and 7%, respectively) was the determination of ALCA.
Discussion
These data show that the five different tests used for the identification of anti-actin antibodies (SMAT; anti-MF on HEp-2 cells and MC; ALCA and AAA in counterimmunoelectrophoresis) give different results, mainly among SMA v positive sera. All SMA T positive sera contain anti-actin antibodies, while only a small proportion of SMA v positive sera (18% in our series, the percentage
possibly depending on case selection) show this specificity. The above observations are important since the only internationally accepted 'positive' criterion for the identification of autoimmune chronic hepatitis is the presence of antinuclear antibodies (ANA) a n d / o r anti-smooth muscle antibodies (SMA), regardless of their immunomorphological pattern. It is well known that viral infections can lead to SMA production, mainly with anti intermediate filament (vimentin) specificity (Brown et al., 1986) and always showing a SMA v pattern. In a series of 14 patients with acute hepatitis B 11 (79%) were SMA v positive at the clinical onset of the disease, while only three (21%) were still SMA v positive after 4 months (personal unpublished data). All sera were negative for SMAT and anti actin antibodies. Thus HBV-induced autoimmunity does not seem to be directed against actin. Preliminary data show that a proportion of autoimmune hepatitis cases are positive for anti-HCV antibodies (Esteban et ai., 1989). However, no information is yet available on the specificity of A N A and SMA in such cases. In this context, the availability of a clinically useful method for the identification of anti-actin antibodies is relevant for both diagnostic and therapeutic purposes. The most reliable test among the five studied is definitely indirect immunofluorescence on cryostat liver sections from rats chronically intoxicated by phalloidin, a test which detects antibodies to liver cell actin (ALCA). It is easy, reproducible and of low cost. The presence of other autoantibodies does not interfere with the interpretation of the immunofluorescence pattern. This is in contrast with the SMAT test, when antimitochondrial or anti-liver-kidney microsomal antibodies are simultaneously present or the SMA T titre is low. However, the most important feature of this test is, its reliability in terms of low 'false' positive and 'false' negative results (particularly with SMA v positive sera). Since no gold standard is at present available for the identification of anti-actin antibodies, we are obliged to arbitrarily choose the criterion of positivity in at least two tests. The SMA T test proved to the specific, but rather insensitive. The same applies to anti-MF on HEp-2 cells and to CIE. Interestingly, the concordance between anti-MF antibodies on HEp-2 cells
and peripheral blood mononuclear cells was low. In fact, the latter substrate gave a high percentage of 'false' positive sera (27%), most of them being in the HBV or alcohol related groups. The difference might be due to the fact that microfilaments contain, in addition to actin, several actinassociated proteins and that different cells express different microfilament components. Different fixation procedures, not usually revealed for the commercially available HEp-2 cells, could also play a role. CIE can give rise to interpretational problems, since precipitation bands can sometimes be faint and thus difficult to be unequivocally identified. An ELISA procedure has been described (Bretherton et al., 1983), but its sensitivity, which is similar to that of immunomorphological tests on isolated cells, is counterbalanced by a loss in specificity. Western blotting using both purified actin and crude extracts from different tissues (rabbit skeletal muscle, liver from phalloidintreated rats) as a source of antigen, gave, in our hands, consistently negative results. This has also been confirmed by other authors (Gabbiani G., personal communication), using the same model. The present data confirm that anti-actin antibodies are mainly found in autoimmune hepatitis and occasionally in other subgroups, where SMA differing from anti-actin antibodies are not infrequently found. It is therefore advisable to search specifically for anti actin antibodies in SMA positive sera from patients with chronic liver disease.
References Bottazzo, G.F., Florin-Chriestensen, A., Fairfax, A., Swana, G., Doniach, D. and Groeschel-Stewart, U. (1976) Classification of smooth muscle antibodies detected by immunofluorescence. J. Clin. Pathol. 29, 403. Bretherton, L., Brown, C., Pedersen, J.S., Toh, B.H., Clarke, F.M., Mackay, I.M. and Gust, I.D. (1983) ELISA assay for
IgG autoantibody to G actin: comparison of chronic active hepatitis and acute viral hepatitis. Clin. Exp. Immunol. 51, 611. Brown, C., Pedersen, J.S., Underwood, J.R.0 Gust, I.D. and Toh, B.H. (1986) Autoantibodies to intermediate filaments in acute viral hepatitis A, B, and non-A, non-B are directed against vimentin. J. Clin. Lab. Immunol. 19, 1. Cunningham, A.L., Mackay, I.R., Frazer, I.H., Brown, C., Pedersen, J.S., Toh, B.H., Tait, B.D. and Clarke, F.M. (1985) Antibody to G-actin in different categories of alcoholic liver disease: quantification by an ELISA and significance for alcoholic cirrhosis. Clin. lmmunol. Immunopathol. 34, 158. Esteban, J.l., Esteban, R., Viladomiu, L., Lopez-Talavera, J.C., Gonzales. A., Hernandez, J.M., Roget, M., Vargas, V., Buti, M., Houghton, M., Choo, Q.-L. and Kuo, G. (1989) Hepatitis C virus among risk groups in Spain. Lancet i, 294. Gabbiani, G., Montesano, R., Tuchweber, B., Salas, M. and Orci, L. (1975) Phalloidin-induced hyperplasia of actin filaments in hepatocytes. Lab. Invest. 33, 562. Kurki, P., Miettinen, A., Salaspuro, M., Virtanen, I. and Stenman, S. (1983) Cytoskeleton antibodies in chronic active hepatitis, primary biliary cirrhosis, and alcoholic liver disease. Hepatology 3, 297. Leevy, C.M., Popper, H. and Sherlock, S. (1976) Disease of the liver and biliary tract. Standardization of nomenclature, diagnostic criteria and diagnostic methodology. Fogarty Int. Cent. Proc. 22, 76. Lenzi, M., Fusconi, M., Selleri, L., Caselli, A., Cassani, F., Bianchi, F.B. and Pisi, E. (1988) Counterimmunoelectrophoresis (CIE) for the detection of anti-liver-kidney microsome (LKM) antibodies in the sera of patients with chronic liver disease. J. Immunol. Methods 111,253. Pardee, J.D. and Spudich, J.A. (1982) Purification of muscle actin. Methods Enzymol. 85, 164. Pedersen, J.S., Toh, B.H., Mackay, I.R., Tait, B.D., Gust, I.D., Kastelan, A. and Hadzic, N. (1982) Segregation of autoantibody to cytoskeletal filaments, actin and intermediate filaments with two types of chronic active hepatitis. Clin. Exp. lmmunol. 48, 527. Wulf, E., Deboben, A., Bautz, F.A., Faulstick, M. and Wieland, T. (1979) Fluorescent phallotoxin, a tool for visualization of cellular actin. Proc. Natl. Acad. Sci. U.S.A. 76, 4498. Zauli, D., Crespi, C., Bianchi, F.B. and Pisi, E. (1985) lmmunofluorescent detection of anti-cytoskeleton antibodies using vinblastin-treated mononuclear cells. J. Immunol. Methods. 82, 77.
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Elsevier JIM05573
Anti-actin antibodies: a n e w test for an old p r o b l e m * M. Fusconi, F. Cassani, D. Zauli, M. Lenzi, G. Ballardini, U. Volta and F.B. Bianchi Cattedra di Semeiotica Medica, Istituto di Clinica Medica Generale e Terapia Medica, University of Bologna, Bolognia, Italy (Received 10 November 1989, revised received 14 December 1989, accepted 29 January 1990)
Smooth muscle antibodies with anti-actin specificity are commonly regarded as markers of autoimmune liver disease. However, there are interpretational problems because different techniques have been used for their identification and therefore the results are difficult to compare. The present paper reports the results of a new method for the identification of anti-actin antibodies (indirect immunofluorescence on cryostat sections of liver from rats chronically injected with phalloidin). The results have been compared with those obtained by four other techniques: demonstration by immunofluorescence of kidney peritubular reactivity (SMAT), of anti-microfilament antibodies (on HEp-2 cells and vinblastine-treated peripheral blood mononuclear cells) and counterimmunoelectrophoresis with purified muscle actin as antigen. The new method proved to be the most sensitive and specific. Furthermore, its reproducibility was found to be high, the interpretation easy and the cost low. The clinical significance of anti-actin antibodies in patients with chronic liver disease is also discussed. Key words: Smooth muscle antibody; Anti-actin antibody; Autoimmune hepatitis; Chronic liver disease; Immunofluorescence
Introduction
Since liver-specific autoantibodies or target antigens have not yet been proved to be relevant in the pathogenesis of autoimmune chronic hepatitis, its diagnosis remains largely inaccurate. In fact, the internationally accepted criteria (Leevy et al., 1976) are both the absence of any other known cause of liver damage and the presence of nonorgan specific autoantibodies, namely antinuclear (ANA) and anti-smooth muscle antibodies (SMA). This kind of approach leads to two different biases. On the one hand, at least theoretically, it is impossible to exclude the possibility that 'primary' Correspondence to: F.B. Bianchi, Cattedra di Semeiotica Medic.a, Istituto di Clinica Medica Generale e Terapia Medica, Policlinico S. Orsola, Via Massarenti 9, 40138 Bologna, Italy. * This work was supported by CNR Grant 88.01942.04.
autoimmune mechanisms can be associated with other causes of liver damage. This can be relevant in populations with a high prevalence of viral infections or alcohol abuse. On the other hand, ANA and SMA are known to be heterogeneous families of autoantibodies, which are directed against different antigens. It is well known that SMA immunofluorescence (IFL) patterns are produced by antibodies to different cytoskeletal proteins and that only the anti-actin (anti-microfilament) specificity is associated with autoimmune liver disease (Kurki et al.. 1983). Other SMA, directed against different cytoskeletal components (namely intermediate filaments), are present, often at high prevalence, in virus-induced liver disease (Bretherton et al., 1983). The availability of a marker able to identify accurately autoimmune cases of chronic liver disease (CLD) is of great interest from a therapeutic point
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of view. Although data are present in the literature on the prevalence of anti-actin antibodies in CLD (Pedersen et al., 1982; Bretherton et al., 1983; Kurki et al., 1983; Cunningham et al., 1985), the methods employed are different and the results difficult to compare. The absence of a reliable, easy and low cost method for the identification of anti-actin antibodies prompted us to set up a new test and to compare its results with those obtained by four currently used ones.
Materials and methods
Serum samples These were obtained from 223 non-consecutive patients with CLD, defined by internationally accepted criteria (Leevy et al., 1976). 38 were from patients with HBV related (HBV-rel) CLD (24 HBsAg positive and 14 antiHBs positive); 38 with alcoholic CLD (ALD); 49 with autoimmune (AUTO) CLD (defined on the basis of the presence of antinuclear, ANA, a n d / o r anti-smooth muscle, SMA, autoantibodies and the exclusion of other known causes of liver damage); 28 with cryptogenic (CRYPTO) CLD, 50 with primary biliary cirrhosis (PBC) and 20 with anti-liver-kidney microsomai antibodies (LKM) positive CLD. Sera from 83 age- and sex-matched blood donors (BD) were used as controls.
with a Leitz Orthoplan microscope equipped with an epiillumination.
A ctin-containing structures Actin-containing structures in the different substrates were detected by IFL using both a polyclonal anti-actin antibody (Miles, U.S.A.) and rhomdamine-conjugated phalloidin (generously provided by Prof. T. Wieland, Max-Plank-Institut, Heidelberg, F.R.G.), which binds specifically to actin (Wulf et al., 1979). To assess the specificity of the reaction, inhibition experiments were performed by incubating the substrate with a mixture of conjugated and free phalloidin at a ratio of 1 to 2000, as described earlier (Wulf et al., 1979).
Detection of SMA This was performed by indirect IFL. Sera were considered to be SMA positive, according to Bottazzo et al. (1976), when a fluorescent reaction was detectable on kidney vessel wall only (SMAv) or on kidney vessels walls, glomerular and peritubular structures (SMAT). Positive sera were titrated by doubling dilutions to an end point.
Detection of anti-microfilament antibodies (anti-MF) Anti-MF antibodies were assayed by indirect IFL on HEp-2 cells (Kallestadt, F.R.G.) and on vinblastine-treated peripheral blood mononuclear cells (MC), which have been shown to be a good substrate for the detection of anti actin specificity in autoimmune CLD (Zauli et al., 1985).
Immunofluorescence (I FL) The indirect method was performed on different substrates according to a standard procedure. 4 /~m cryostat sections of rat liver, kidney and stomach were used for the identification of ANA, SMA, anti-mitochondrial antibodies (AMA) and LKM antibodies. Sera were tested at a dilution of 1/40 in phosphate-buffered saline (PBS) and incubated for 20 min at room temperature in a moist chamber. After three washes of 10 min each with PBS, the slides were incubated with a fluorescein-conjugated sheep anti-human immunoglobulin (IgG, IgA, IgM, Wellcome, U.K.) at a dilution of 1/60 in PBS for 20 min in a moist chamber. After three more washes with PBS, slides were mounted in PBS/glycerol (1/1, v/v) and observed, under code, by two independent readers
Detection of anti-liver cell actin antibodies (ALCA) The test was performed by indirect IFL using 4 /~m cryostat liver sections from rats chronically intoxicated with phalloidin (a generous gift from Prof. T. Wieland). The drug was administered intraperitoneally for 19 days at a dose of 500 /~g/kg in 0.5 ml. This sublethal dose leads to an enormous deposition of microfilamentous material, consisting mainly of actin, at the biliary and sinusoidal portion of the hepatocytes (Gabbiani et al., 1975).
Detection of anti-muscle actin antibodies (AAA ) Anti-muscle actin antibodies were assayed by counterimmunoelectrophoresis (CIE) (Lenzi et al., 1988) using purified actin extracted from acetone
J
-..
w.
Fig. 1. Counterimmunoelectrophoresisdetection of anti-muscle actin antibodies (AAA). Positive sera give precipitin lines showing reactions of identity.
powder of rabbit skeletal muscle (Pardee and Spudich, 1982) as antigen. The purity of the Gactin preparation was assesed by polyacrylamide gel electrophoresis, which showed a single band at a molecular weight of 43,000. In C I E experiments actin was used at a concentration of 0.3 m g / m l . A reaction of identity was present with positive sera (Fig. 1). As positive control a polyclonal rabbit anti-actin antibody (Miles, U.S.A.) was used.
Statistical analysis Statistical comparisons were performed using the Wilcoxon rank sum test.
Fig. 2. lmmunofluorescence localization of actin in liver of phalloidin-treated rats using rhodamine-labelled phalloiodin. Actin is deposited at the periphery of the hepatocytes. Anti-liver cell actin (ALCA) positive sera display sera display this pattern. Original magnification 25 x.
On rat kidney the reactivity was present in vessels, glomerular mesangium and peritubular fibrils with an interrupted pattern (Fig. 3). Also the brush border was faintly reactive. On rat stomach the positivity was restricted to the muscularis mucosae, intergland fibers and, to a lesser extent, the cytoplasm of parietal ceils with a filamentous pattern. On HEp-2 cells the positivity of the two probes was confined to the microfilaments, with the typical pattern of linear cables distributed on the
Results
Morphological identification of actin-containing structures On liver sections from phalloidin-treated rats staining was present in the vessels and, more strongly, in blocks of material located at the periphery of hepatocytes, mainly around bile canaliculi. The sinusoids were positive and enlarged (Fig. 2). This pattern is identical to that described by Gabbiani et al. (1975) and corresponds to microfilamentous deposits at the periphery of liver ceils. In normal liver strong positivity was detected in the arterial and venous walls, while faint reactivity was present at the periphery of the hepatocytes.
Fig. 3. Immunofluorescence localization of actin on rat kidney by rhodarnine-labelled phalloidin. Vessel walls, glomerular mesangium and peritubular stuctures, with an interrupted pattern, are positive. SMAT positive sera show identical reactivity. Original magnification 25 x.
Fig. 4. Immunofluorescence localization of actin on HEp-2 cells and peripheral blood mononuclear cells by rhodamine-labelled phalloidin. Actin cables are detected as 'classical' stress fibers on HEp-2 cells (A) or microvillar projections on mononuclear cells (B). Anti-MF positive sera display these reactivities. Original magnification 50 ×.
longitudinal axis of the cells (Fig. 4A). On MC the pattern of positivity was that of cytoplasmic staining with peripheral microvilli (Fig. 4B). Sera were considered to be positive for SMAT, anti MF and ALCA when they gave the above IFL patterns on rat kidney, HEp-2 cells, MC and phalloidin-treated liver, respectively.
Prevalence of SMA 73 of the 223 sera from CLD patients (32%) were positive for SMA: 21 (9%) with a SMA v pattern and 52 (23%) with a SMAv pattern. Ten of the 83 BD studied were SMA positive (12%), always with a SMA v pattern and a low titer ( < 1/80). The prevalence of SMA among the different subgroups is reported in Table I. It is
apparent that SMA positive sera are significantly associated with the A U T O CLD group. 18 of the 21 SMA T sera were from patients with A U T O CLD and PBC. Titers of the 21 SMA T positive sera (median > 1/320; range 1 / 8 0 - > 1/320) were significantly higher ( P < 0.01) than those of the SMA v positive sera (median 1/80; range 1 / 4 0 - > 1/320). The detection of the SMAT pattern was difficult in cases where the titer was low and other autoantibodies reacting with kidney tubules (antimitochondrial and anti liver-kidney microsomal antibodies) were present.
Prevalence of anti-MF The anti-MF data is shown in Table II. The two cellular substrates gave different results, with
TABLE I PREVALENCE OF ANTI-SMOOTH MUSCLE ANTIBODIES (SMA) AND THEIR PATTERN IN DIFFERENT SUBGROUPS OF PATIENTS WITH CHRONIC LIVER DISEASE AUTO (49) SMA positive T V
11(22%) 28 (57%)
Total SMA negative
39 (79%) 10 (21%)
CRYPTO (28)
ALD (38)
HBV-rel (38)
(0%) (0%)
0 (0%) 10 (26%)
0 (0%) 28 (100%)
10 (26%) 28 (74%)
0 0
PBC (50)
LKM (20)
BD (83)
2 (5%) 10 (26%)
7(14%) 2 (4%)
1 (5%) 2 (10%)
0 (0%) 10 (12%)
12 (31%) 26 (69%)
9 (18%) 41 (82%)
3 (15%) 17 (85%)
10 (12%) 73 (88%)
Key: AUTO, autoimmune chronic liver disease; CRYPTO, cryptogenic chronic liver disease; ALD, alcoholic chronic liver disease; HBV-rel, hepatitis B virus-induced chronic liver disease; PBC, primary biliary cirrhosis; LKM, Anti-liver-kidney microsomal antibody positive chronic liver disease; BD, blood donors.
TABLE 1I PREVALENCE OF ANTI-MICROFILAMENT ANTIBODIES (ANTI-MF) AS DETERMINED ON HEP-2 CELLS AND VINBLASTINE TREATED MONONUCLEAR CELLS (MC) IN RELATION TO SMA POSlTIVITY AND PA'VTERN IN DIFFERENT SUBGROUPS OF PATIENTS WITH CHRONIC LIVER DISEASE AUTO ( 4 9 ) Anti-MF (HEp-2) SMA T positive SMA v positive SMA negative Total Anti-MF (MC) SMA T positive SMA v positive SMA negative Total
CRYPTO (28)
ALD ( 3 8 )
HBV-rel (38)
10/11 (91%) 0/0 (0%) 0/28 ( 0 % ) 0/0 (0%) 0/10 ( 0 % ) O/28 (0%)
0/0 (0%) 0/10 (0%) 0/28 (O%)
2/2 (100%) 7/7(100%) 0/10 (0%) 0/2 (0%) O/26 (O%) 0/41 (O%)
1/1 (100%) 0/0 (0%) 0/2 ( 0 % ) 0/10 (0%) 0/17 ( 0 % ) 0/73 (0%)
10/49 (20%)
0/28 (0%)
0/38 (0%)
2/38
7/50 (14%)
1/20
11/11 (100%) 9/28 (32%) 0/10 ( 0 % )
0/0 (0%) 0/0 (0%) 0/28 (0%)
0/0 (0%) 3/10 (30%) 0/28 (0%)
1/2 (50%) 6/10 (60%) 0/26 (0%)
7/7 (100%) 2/2 (100%) 0/41 ( 0 % )
1/1 (100%) 0/0 (0%) 0/2 (0%) 0/10 (0%) 0/17 ( 0 % ) 0/73 (0%)
20/49 (40%)
0/28 (0%)
3/38 (8%)
7/38 (18%)
9/50 (18%)
1/20
(5%)
CPB (50)
LKM (20)
(5%)
(5%)
BD (83)
0/83 (0%)
0/83 (0%)
Key: AUTO, autoimmune chronic liver disease; CRYPTO, cryptogenic chronic liver disease; ALD, alcoholic chronic liver disease; HBV-rel, hepatitis B virus-induced chronic liver disease; PBC, primary biliary cirrhosis; LKM, anti-liver-kidneymicrosomal antibody positive chronic liver disease; BD, blood donors.
a higher percentage of positive results when MC were used. In fact, 20 of the 223 (9%) sera were positive on HEp-2 cells while 40 (18%) were reactive on MC. All the sera positive for anti-MF (tested on HEp-2 or MC) were SMA positive. All but one of the anti-MF positive sera on HEp-2 cells were confirmed on MC, the exception being one serum positive for SMA T, A L C A and AAA. Conversely, 21 of the 40 anti M F positive sera on MC were negative on HEp-2 cells. None of the 83 BD sera were anti M F positive.
Prevalence of ALCA A positive reaction was detected in 28 of the 223 sera from C L D patients (12%). All 28 A L C A positive sera were also SMA positive, while none of the SMA negative sera were A L C A positive. N o n e of the 83 control sera were positive. All the 21 SMA r sera were positive for ALCA. In addition, seven SMA v positive sera showed this reactivity. The prevalences of A L C A among the different subgroups is reported in Table III. The detection of A L C A was always easy, even when
TABLE 11I PREVALENCE OF ANTI-LIVER CELL ACTIN ANTIBODIES (ALCA) IN RELATION TO SMA POSITIVITY AND PATTERN IN DIFFERENT SUBGROUPS OF PATIENTS WITH CHRONIC LIVER DISEASE AUTO ( 4 9 )
CRYPTO (28)
ALD (38)
HBV-rel (38)
CBP (50)
LKM (20)
BD (83)
SMATpositive SMA v positive SMA negative
11/11 (100%) 4/28 (14%) 0/10 ( 0 % )
0/0 (0%) 0/0 ( 0 % ) 0/28 (0%)
0/0 (0%) 0/10(0%) 0/28 (0%)
2/2 (100%) 2/10 (20%) 0/26 ( 0 % )
7/7 (100%) 1/2 (50%) 0/41 ( 0 % )
1/1 (100%) 0/2 (0%) 0/17 ( 0 % )
0/0 (0%) 0/10(0%) 0/73 (0%)
Total
15/49 (31%)
0/28 (0%)
0/38 (0%)
4/38 (10%)
8/50 (16%)
1/20
0/83 (0%)
(5%)
Key: AUTO, autoimmune chronic liver disease; CRYPTO, cryptogenic chronic liver disease; ALD, alcoholic chronic liver disease; HBV-rel. hepatitis B virus-induced chronic liver disease; PBC, primary biliary cirrhosis; LKM, anti-liver-kidneyrnicrosomal antibody positive chronic liver disease; BD, blood donors.
6 TABLE IV PREVALENCE OF ANTI-MUSCLE ACTIN ANTIBODIES (AAA) IN RELATION TO SMA POSITIVITY AND PATYERN IN DIFFERENT SUBGROUPS OF PATIENTS WITH CHRONIC LIVER DISEASE AUTO ( 4 9 ) SMA T positive SMAv positive SMA negative Total
CRYPTO (28)
ALD ( 3 8 )
HBV-rel (38)
CBP (50)
LKM (20)
BD (83)
9/11 (89%) 3/28 (11%) 0/10 (0%)
0/0 (0%) 0/0 (0%) 0/28 (0%)
0/0 (0%) 1/10 (10%) 0/28 (0%)
2/2 (100%) 1/10 (10%) 0/26 ( 0 % )
6/7 (86%) 1/2 (50%) 0/41 ( 0 % )
1/1 (100%) 0/2 (0%) 0/17 ( 0 % )
0/0 (0%) 0/10 (0%) 0/73 (0%)
12/49 (24%)
0/28 (0%)
1/38 (3%)
3/38
7/50 (14%)
1/20
0/83 (0%)
(8%)
(5%)
Key: AUTO, autoimmune chronic liver disease; CRYPTO, cryptogenic chronic liver disease; ALD, alcoholic chronic liver disease; HBV-rel, hepatitis B virus-induced chronic liver disease; PBC, primary biliary cirrhosis; LKM, anti-liver-kidney microsomal antibody positive chronic liver disease; BD, blood donors.
the titer was low o r o t h e r c y t o p l a s m i c a u t o a n t i b o d i e s were s i m u l t a n e o u s l y present.
Prevalence of AAA 24 of the 223 sera f r o m C L D p a t i e n t s were positive b y C I E with a reaction of identity. N o n e of the 83 c o n t r o l s were reactive by this m e t h o d . All the 24 positive sera were S M A positive, 18 with the S M A T a n d six with the S M A v p a t t e r n . T h e prevalence of A A A a m o n g the d i f f e r e n t subg r o u p s is s u m m a r i z e d in T a b l e IV.
Comparison of SMA, anti-MF (HEp-2 and MC), ALCA and AAA T h e results o b t a i n e d with the five techniques are shown in T a b l e V. It is a p p a r e n t that within the S M A x s u b g r o u p all the m e t h o d s gave almost the s a m e results, C I E b e i n g slightly less sensitive t h a n the others. T h e results are different within the S M A v s u b g r o u p , b e c a u s e o f the high p r o p o r tion of a n t i - M F positive sera on M C (38%); A L C A , A A A a n d a n t i - M F were d e t e c t e d on H E p - 2 cells in 13%, 12% a n d 0% of these sera, respectively. In
TABLE V RELATIONSHIP OF ANTI-MF ON HEP-2 CELLS, ANTI-MF ON MC, ALCA AND AAA WITH SMA POSITIVITY AND PATTERN IN ALL THE SERA FROM PATIENTS WITH CHRONIC LIVER DISEASE
SMA T positive
Total SMA v positive
16 (76%) 3 (14%)
ALCA
AAA
+ +
+ +
+ +
-
+
1
(5%)
-
+
+
+
(5%)
+
-
+
+
20 (95%)
20 (95%)
21 (100%)
18 (86%) +
21 (100%) 2
(4%)
-
+
+
4
(8%)
-
+
+
-
-
+ +
-
+
(6%) 11 (21%)
-
1
(2%)
-
-
+
-
1
(2%)
-
-
-
+
30 ( 5 7 % )
SMA negative
Anti-MF (MC)
1
3
Total
Anti-MF (HEp-2)
.
52 (100%) 150 ( 1 0 0 % )
. 0 (0%)
.
.
.
20 (38%) .
.
7 (13%) .
6 (12%)
TABLE VI 'FALSE' POSITIVE AND 'FALSE' NEGATIVE RESULTS IN THE DETERMINATION OF ANTI ACTIN ANTIBODIES OBTAINED WITH THE DIFFERENT TECHNIQUES (A serum was arbitrarily assumed to be postive for anti-actin antibodies when it was reactive in at least two of the five tests.)
SMA-r Anti-MF (HEp-2) Anti-MF (MC) ALCA AAA
"False' positive
'False' negative
0/21 0/20 11/40 1/28 1/24
9/52 10/53 1/33 3/45 7/49
(0%) (0%) (27%) (4%) (4%)
(17%) (19%) (3%) (7%) (14%)
order to evaluate the disease sensitivity and specificity of each test, we arbitrarily assumed that a serum was positive for anti-actin antibodies when it was reactive in at least two of the five tests. By this method, 30 out of the 73 SMA positive sera (41%) were positive for anti-actin antibodies. Of these, 21 gave a SMA T and nine a SMAv pattern. Titers of SMA v and anti-actin positive sera were not significantly different from those of SMA v positive and anti-actin negative sera. A 'false' positive reaction was present in 11 out of the 40 actin-MF positive sera on MC (27%), since they were negative by all the other techniques. These sera were mainly found in the HBV and alcohol related CLD. 'False' positive results were conversely extremely rare with ALCA and AAA (one serum each). The proportion of 'false' negative results was high with HEp-2 cells (19%), SMA T (17%) and AAA (14%), but low with MC (3%). These results are summarized in Table VI. The method which gave the lowest incidence of 'false' positive and negative results (4% and 7%, respectively) was the determination of ALCA.
Discussion
These data show that the five different tests used for the identification of anti-actin antibodies (SMAT; anti-MF on HEp-2 cells and MC; ALCA and AAA in counterimmunoelectrophoresis) give different results, mainly among SMA v positive sera. All SMA T positive sera contain anti-actin antibodies, while only a small proportion of SMA v positive sera (18% in our series, the percentage
possibly depending on case selection) show this specificity. The above observations are important since the only internationally accepted 'positive' criterion for the identification of autoimmune chronic hepatitis is the presence of antinuclear antibodies (ANA) a n d / o r anti-smooth muscle antibodies (SMA), regardless of their immunomorphological pattern. It is well known that viral infections can lead to SMA production, mainly with anti intermediate filament (vimentin) specificity (Brown et al., 1986) and always showing a SMA v pattern. In a series of 14 patients with acute hepatitis B 11 (79%) were SMA v positive at the clinical onset of the disease, while only three (21%) were still SMA v positive after 4 months (personal unpublished data). All sera were negative for SMAT and anti actin antibodies. Thus HBV-induced autoimmunity does not seem to be directed against actin. Preliminary data show that a proportion of autoimmune hepatitis cases are positive for anti-HCV antibodies (Esteban et ai., 1989). However, no information is yet available on the specificity of A N A and SMA in such cases. In this context, the availability of a clinically useful method for the identification of anti-actin antibodies is relevant for both diagnostic and therapeutic purposes. The most reliable test among the five studied is definitely indirect immunofluorescence on cryostat liver sections from rats chronically intoxicated by phalloidin, a test which detects antibodies to liver cell actin (ALCA). It is easy, reproducible and of low cost. The presence of other autoantibodies does not interfere with the interpretation of the immunofluorescence pattern. This is in contrast with the SMAT test, when antimitochondrial or anti-liver-kidney microsomal antibodies are simultaneously present or the SMA T titre is low. However, the most important feature of this test is, its reliability in terms of low 'false' positive and 'false' negative results (particularly with SMA v positive sera). Since no gold standard is at present available for the identification of anti-actin antibodies, we are obliged to arbitrarily choose the criterion of positivity in at least two tests. The SMA T test proved to the specific, but rather insensitive. The same applies to anti-MF on HEp-2 cells and to CIE. Interestingly, the concordance between anti-MF antibodies on HEp-2 cells
and peripheral blood mononuclear cells was low. In fact, the latter substrate gave a high percentage of 'false' positive sera (27%), most of them being in the HBV or alcohol related groups. The difference might be due to the fact that microfilaments contain, in addition to actin, several actinassociated proteins and that different cells express different microfilament components. Different fixation procedures, not usually revealed for the commercially available HEp-2 cells, could also play a role. CIE can give rise to interpretational problems, since precipitation bands can sometimes be faint and thus difficult to be unequivocally identified. An ELISA procedure has been described (Bretherton et al., 1983), but its sensitivity, which is similar to that of immunomorphological tests on isolated cells, is counterbalanced by a loss in specificity. Western blotting using both purified actin and crude extracts from different tissues (rabbit skeletal muscle, liver from phalloidintreated rats) as a source of antigen, gave, in our hands, consistently negative results. This has also been confirmed by other authors (Gabbiani G., personal communication), using the same model. The present data confirm that anti-actin antibodies are mainly found in autoimmune hepatitis and occasionally in other subgroups, where SMA differing from anti-actin antibodies are not infrequently found. It is therefore advisable to search specifically for anti actin antibodies in SMA positive sera from patients with chronic liver disease.
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