Abstract
Anti-actin IgA antibodies have been found in sera of coeliacs. Our aim was to define the prevalence and clinical significance of anti-actin IgA in coeliacs before and after gluten withdrawal. One hundred and two biopsy-proven coeliacs, 95 disease controls and 50 blood donors were studied. Anti-actin IgA were evaluated by different methods: (a) antimicrofilament positivity on HEp-2 cells and on cultured fibroblasts by immunofluorescence; (b) anti-actin positivity by enzyme-linked immuosorbent assay (ELISA); and (c) presence of the tubular/glomerular pattern of anti-smooth muscle antibodies on rat kidney sections by immunofluorescence. Antimicrofilament IgA were present in 27% of coeliacs and in none of the controls. Antimicrofilament antibodies were found in 25 of 54 (46%) coeliacs with severe villous atrophy and in three of 48 (6%) with mild damage (P < 0·0001). In the 20 patients tested, antimicrofilaments IgA disappeared after gluten withdrawal in accordance with histological recovery. Our study shows a significant correlation between antimicrofilament IgA and the severity of intestinal damage in untreated coeliacs. The disappearance of antimicrofilament IgA after gluten withdrawal predicts the normalization of intestinal mucosa and could be considered a useful tool in the follow-up of severe coeliac disease.
Keywords: anti-actin antibodies, antimicrofilament antibodies, coeliac disease, intestinal villous atrophy, mucosal recovery
INTRODUCTION
Coeliac disease (CD) is an immune-mediated enteropathy triggered by ingestion of gluten in genetically susceptible individuals and histologically characterized by different grades of small intestine damage that consist of villous atrophy, crypt hyperplasia and an increased number of intraepithelial lymphocytes [1]. IgA anti-endomysial antibodies (EmA) are considered the most reliable serological marker of CD [2]. After the recent identification of tissue transglutaminase (tTG) as the main, if not the sole endomysial antigen, a very sensitive enzyme-linked immunosorbent assay (ELISA), using human recombinant tissue transglutaminase (h-tTG) as antigen, has been set up [3]. Despite the high sensitivity and specificity of these serological tests for diagnosis of CD, anti-endomysial antibodies do not correlate with histopathological features [4]. Moreover, anti-tTG antibodies are known to be more correlated with dietetic compliance than with intestinal biopsy morphology [5]. At present, intestinal biopsy with demonstration of gluten-dependent mucosal damage remains the gold standard procedure for a definitive diagnosis of coeliac disease [6].
Recently, Clemente et al. described a very high prevalence of IgA antibodies against actin filaments (AAA) in coeliac patients with severe or moderate intestinal villous atrophy, showing that the finding of these antibodies correlates with the degree of mucosal damage [7]. Actin is known as the major component of microfilaments of cell cytoskeleton and it is regarded as the target of smooth muscle antibodies with the tubular pattern (SMA-T), detected by indirect immunofluorescence (IFL) on kidney sections and closely associated with type 1 autoimmune hepatitis (AIH) [8].
On isolated cells (fibroblasts, HEp-2 cells, peripheral blood mononuclear cells), anti-actin antibodies react with the microfilaments giving the pattern of parallel ‘stress fibres’ [9].
The cytoskeleton network is the basis of the ultrastructural architecture of the enterocytes and it is known that gluten challenge in CD patients causes rapid distortion of microvillus structure with disorganization of actin network on the intestinal mucosa [10,11].
The aim of this study was to define the prevalence of IgA AAA in CD patients before and after gluten withdrawal, using different methods for their detection. Moreover, we have correlated the presence of AAA with small intestinal mucosal morphology before and after gluten withdrawal to evaluate clinical significance of these antibodies.
MATERIALS AND METHODS
Study population
One hundred and two consecutive biopsy-proven CD patients, 69 adults (M/F 19/50, median age 32 years, range 15–74), 33 children (M/F 14/19, median age 6 years, range 2–14), were studied at the time of diagnosis. Intestinal biopsy specimens were graded according to Marsh's classification, modified by Oberhuber [12] as follows: increase in intra-epithelial lymphocytes (IEL) (grade 1), increase in IEL with crypt hyperplasia (grade 2), mild villous flattening (grade 3a), marked villous flattening (grade 3b) and total villous flattening (grade 3c). From grade 1 to grade 3a the histological damage was considered mild, grades 3b and 3c were considered severe.
As a control population we enrolled 20 patients with dyspepsia and normal intestinal biopsy, 15 patients with inflammatory bowel disease (seven ulcerative colitis (UC) and eight Crohn's disease), 11 with post-enteritis syndrome, 29 with type I AIH and 20 with primary biliary cirrhosis (PBC). We also tested 50 age- and sex-matched blood donors as normal controls. All sera were stored at −20°C until use.
This study was approved by the ethical committee of our hospital; in each case, informed consent was obtained before inclusion in the study.
Indirect immunofluorescence
Detection of IGA EmA.
IgA EmA were detected by indirect immunofluorescence using human umbilical cord cryostat sections (4 µm) as substrate. Sera were tested at the initial dilution of 1 : 5 and, when positive, were titred up to the endpoint [2].
Detection of SMA on rat tissue sections.
SMA were detected by IFL as described previously [13]. Briefly, sera diluted 1 : 40 in phosphate buffered saline (PBS) were tested on cryostatic frozen sections of rat liver, kidney and stomach. A fluorescein-conjugated secondary antibody, either polyspecific or IgA-specific (anti-human polyvalent immunoglobulins IgA, IgG, IgM and anti-IgA FITC conjugate, respectively, Sigma ImmunoChemicals, St Louis, MO, USA), was used diluted 1 : 100 in PBS. The positivity for SMA was assessed under a fluorescence microscope (Orthoplan, Leitz, Wetzlar, Germany).
The identification of the SMA patterns was made according to Bottazzo et al. [8]: (1) SMA-V (vessels): staining of small/medium-size vessel walls; (2) SMA-G (glomeruli): staining, in addition to vessels of glomerular mesangial cells also; (3) SMA-T (tubuli): staining of vessels, glomerular and peritubular structures. According to Bottazzo et al. sera were regarded as anti-actin positive only when SMA-T or SMA-G pattern was present.
Detection of SMA-T/G antibodies was performed blindly by two independent investigators (F.C. and A.G.)
Detection of antimicrofilament antibodies (anti-MF) on HEp-2 cells and on human fibroblasts.
All samples were tested by indirect IFL on HEp-2 (Euroimmun, Labordiagnostika, Lubeck, Germany) for detection of IgA anti-MF antibodies; briefly, serum samples were diluted 1 : 5 in PBS and incubated in a humid chamber for 30 min; after three PBS washings, the secondary antibody (anti-human IgA FITC Conjugate, Sigma ImmunoChemicals) at a dilution of 1 : 100 was added.
IgA anti-MF antibodies were also assayed by indirect IFL on human fibroblasts kindly provided by Dr A. Ripalti (Department of Clinical and Experimental Medicine, Section of Microbiology, University of Bologna, Italy). Fibroblasts were grown to semiconfluence on coverslips in Dulbecco's solution with 10% fetal calf serum in 5% carbon dioxide atmosphere, and then fixed with acetone at −20°C for 10 min. Sample sera were diluted 1 : 5 in PBS and the secondary antibody (anti-human IgA FITC conjugate, Sigma ImmunoChemicals) was diluted 1 : 100. The positivity for anti-MF (staining of parallel ‘stress fibres’) was assessed independently and blinded by two investigators (F.C. and A.G.). Positive reactions were titred by the double dilution method.
Enzyme-linked immunosorbent assay (ELISA)
Detection of IgA h-tTG.
IgA h-tTG were detected by ELISA using a commercial kit (Eurospital, Trieste, Italy) as reported previously [14]; the cut-off was fixed at seven arbitrary units (AU). The optical density (OD) was read at 450 nm.
Detection of anti-actin antibodies (AAA) by ELISA.
ELISA was performed using as antigen lyophilized actin commercially available from rabbit muscle (Sigma Chemicals, St Louis, MO, USA).
Briefly, each well of microtitre plates (Dynatech Laboratories Inc., Chantilly, VA, USA) was coated overnight at 4°C with 100 µl (10 µg/ml) of actin diluted in bicarbonate buffer, pH 7·5. After three washings with PBS Tween 0·05%, 100 µl of blocking buffer (0·05 m Tris-HCL, 0·15 m NaCl, 0·01 m EDTA, 0·1% Tween-20, pH 7·4) was added to each well for 60 min at room temperature. One hundred µl of the each serum diluted 1 : 100 in PBS/powdered milk 3% were added to wells and incubated for 60 min at room temperature. After three washing steps with PBS Tween 0·05%, 100 µl of peroxidase-conjugated antibody antihuman IgA (Dako, Denmark) diluted 1 : 500 in PBS/powdered milk 3% was added to each well and incubated for 60 min at room temperature. After the washing steps the reaction was developed with o-phenylenediamine dihydrochloride (Sigma) and H2O2 in citrate buffer for 15 min, and the colourimetric reaction was measured at 492 nm by spectrophotometer. The cut-off was established as the mean optical density at 492 nm ± 5 standard deviations in 100 further healthy blood donors (0·080 + 5 × 0·004 = 0·100).
Absorption studies
Five sera positive for anti-tTG, AAA by ELISA and for anti-MF by IFL were incubated overnight at 4°C, under agitation, with increasing amounts (10 µg, 50 µg, 250 µg, 500 µg) of rabbit muscle actin diluted in bicarbonate buffer (pH 7·5) and guinea-pig liver transglutaminase (Sigma) diluted in 0·05 m Tris-Hcl, 0·15 m Nacl, 5 mm Cacl2 buffer (pH 7·5). Antibody–antigen complexes were separated from sera by centrifugation at 100·000 g for 30 min. ELISA for AAA (sera diluted 1 : 100 were added in duplicate to wells, peroxidase-conjugated antibody antihuman IgA diluted 1 : 500 in PBS/powdered milk 3% was used as secondary antibody, as described previously) and anti-tTG (sera diluted 1 : 26 were added in duplicate to wells, using commercial Eurospital kit as described previously) and IFL on HEp-2 cells and fibroblasts (sera diluted 1 : 5 as described previously) were then performed with an unabsorbed and absorbed serum.
Follow-up study
Coeliac patients positive for IgA AAA at the time of diagnosis were revaluated after 12 months of gluten-free diet (GFD). Follow-up study included detection of AAA, EmA and anti-tTG antibodies together with intestinal biopsy. An accurate dietetic interview was used to evaluate the compliance to gluten free diet.
Statistical analysis
The comparison of categorical variables was performed using χ2 and Fisher's exact test when applicable. Nominal variables were correlated by contingency table. A probability (P) value less than 0·05 was considered significant.
RESULTS
Anti-MF IgA antibodies on fibroblasts (Fig. 1a) were found in 28 (27%) of the 102 CD patients studied (median titre 1 : 160, range 1 : 40–1 : 1280). Of these 28 cases, 26 (25% of total CD patients) were also positive for anti-MF IgA on HEp-2 cells (Fig. 1b) and 15 (14·7% of total CD patients) for IgA AAA by ELISA. EmA and anti-tTG were found positive, respectively, in 101 (99%) and 100 (98%) coeliac patients. All the 145 control sera were negative for EmA, anti-tTG and both IFL-detected anti-MF and ELISA-detected AAA. SMA with the T/G pattern were found in 10·7% (11/102) coeliac patients and in 82% (24/29) AIH patients. By IFL with an IgA-specific secondary conjugate, the SMA T/G was due to IgA antibodies in five (4·9%) CD patients and never in AIH patients. Table 1 summarizes the results obtained with the different methods. Table 2 shows the detailed spectrum of reactivities of the 28 CD sera positive for anti-MF antibodies on fibroblasts.
Fig. 1.
Case no. 4. Anti-microfilaments antibodies (titre 1 : 160) on fibroblasts (a, magnification 40×) and HEp-2 cells (b, magnification 40×) at initial dilution of 1 : 5 with antihuman IgA FITC conjugate as secondary antibodies. Typical positive actin stress fibres (microfilaments) appear as cytoplasmic parallel filaments. Negative effect on microfilaments staining on HEp-2 cells after absorption of serum with 500 µg of rabbit muscle actin. One hundred µl of absorbed serum at dilution of 1 : 5 was used (c, magnification 40×).
Table 1.
Prevalence of different reactivities in CD patients and in pathological controls
| IgA anti-MF on fibroblasts | IgA anti-MF on Hep-2 cells | IgA AAA by ELISA | SMA T/G† | IgA SMA T/G | |
|---|---|---|---|---|---|
| CD total cases (n = 102) | 28 (27%) | 26 (25%) | 15 (14·7%) | 11 (10·7%) | 5 (4·9%) |
| CD grades 1–3a (n = 48) | 3 (6%)* | 3 (6%) | 3 (6%) | 5 (10·4%) | 2 (4%) |
| CD grades 3b–c (n = 54) | 25 (46%)* | 23 (42%) | 12 (22%) | 6 (11%) | 3 (5·5%) |
| Dyspepsia (n = 20) | 0 | 0 | 0 | 0 | 0 |
| IBD (n = 15) | 0 | 0 | 0 | 0 | 0 |
| Post-enteritis syndrome (n = 11) | 0 | 0 | 0 | 0 | 0 |
| AIH type 1 (n = 29) | 0 | 0 | 0 | 24 (82%) | 0 |
| PBC (n = 20) | 0 | 0 | 0 | 0 | 0 |
Grades 3b–c versus 1–3a, P < 0·0001 (Fisher's test).
IFL on rat tissue with antihuman total Ig as secondary antibodies.
Table 2.
Reactivities of the 28 coeliac patients anti-MF positive
| Patient no. | Anti-MF on fibroblasts* | Anti-MF on HEp-2* | IgA AAA ELISA OD | SMA T/G | IgA SMA T/G | Anti-tTg AU† | EmA* |
|---|---|---|---|---|---|---|---|
| 3 | 1 : 40 | 1 : 40 | neg | neg | neg | 14 | 1:160 |
| 4 | 1 : 160 | 1 : 160 | 0·224 | pos | pos | 19 | 1 : 80 |
| 6 | 1 : 640 | 1 : 320 | neg | pos | neg | 32 | 1 : 320 |
| 9 | 1 : 320 | 1 : 320 | neg | neg | pos | 20 | 1 : 80 |
| 11 | 1 : 320 | 1 : 320 | neg | neg | neg | 11 | 1:160 |
| 23 | 1 : 640 | 1 : 320 | 0·254 | pos | neg | 12 | 1 : 160 |
| 25 | 1 : 1280 | 1 : 1280 | 0·542 | neg | neg | 15 | 1 : 320 |
| 30 | 1 : 40 | 1 : 40 | neg | pos | pos | 13 | 1 : 80 |
| 45 | 1 : 320 | 1 : 320 | neg | neg | neg | 16 | 1 : 80 |
| 47 | 1 : 320 | 1 : 320 | neg | pos | neg | neg | 1 : 160 |
| 49 | 1 : 320 | 1 : 160 | 0·264 | neg | neg | 20 | 1 : 320 |
| 50 | 1 : 80 | 1 : 80 | neg | neg | neg | 15 | 1 : 160 |
| 51 | 1 : 40 | 1 : 40 | 0·188 | pos | neg | 26 | 1 : 320 |
| 54 | 1 : 320 | 1 : 320 | neg | neg | neg | 25 | 1 : 320 |
| 58 | 1 : 40 | 1 : 20 | 0·136 | pos | pos | 21 | 1 : 320 |
| 63 | 1 : 320 | 1 : 160 | 0·246 | neg | neg | neg | 1 : 320 |
| 68 | 1 : 640 | 1 : 640 | 0·288 | pos | neg | 9 | 1 : 80 |
| 69 | 1 : 160 | 1 : 160 | 0·214 | neg | neg | 22 | 1 : 40 |
| 74 | 1 : 40 | 1 : 10 | neg | neg | neg | 15 | 1 : 160 |
| 75 | 1 : 40 | 1 : 40 | neg | pos | neg | 14 | 1 : 80 |
| 77 | 1 : 160 | 1 : 160 | 0·256 | neg | neg | 13 | 1 : 80 |
| 78 | 1 : 80 | 1 : 80 | 0·198 | neg | neg | 16 | 1 : 40 |
| 81 | 1 : 80 | 1 : 80 | 0·208 | neg | neg | 11 | 1 : 40 |
| 82 | 1 : 640 | 1 : 640 | 0·274 | neg | neg | 19 | 1 : 320 |
| 87 | 1 : 40 | 1 : 20 | neg | pos | neg | 12 | 1 : 160 |
| 91 | 1 : 160 | 1 : 80 | 0·236 | neg | neg | 16 | 1 : 160 |
| 96 | 1 : 40 | neg | neg | neg | neg | 10 | 1 : 40 |
| 99 | 1 : 40 | neg | 0·148 | pos | pos | 12 | 1 : 40 |
Anti-MF and EmA values are expressed as titres.
AU: arbitrary units.
Taking into account the mucosal histology, anti-MF antibodies were found in 25 (46%) of the 54 CD patients with severe villous atrophy (grades 3b–c) and in only three (6%) of the 48 with mild intestinal damage (grades 1–3a) (P < 0·0001). Among childhood CD patients, anti-MF antibodies were positive in nine (47%) of the 19 patients with severe villous atrophy but in none of those with mild intestinal damage (P = 0·0039). Sixteen (45·7%) of the 35 adult CD patients with severe villous atrophy and only three (8·8%) of the 34 with grades 1–3a of mucosal damage were anti-MF positive (P = 0·0009) (Table 3).
Table 3.
Prevalence of IgA anti-MF in the two groups of CD patients and correlation with histological grade
| CD patients | IgA anti-MF on fibroblasts |
|---|---|
| Adult CD (n = 69) | 19 (27·5%) |
| (a) Grades 1–3a (n = 34) | 3 (8·8%)* |
| (b) Grades 3b–c (n = 35) | 16 (45·7%)* |
| Childhood CD (n = 33) | 9 (27·2%) |
| (c) Grades 1–3a (n = 14) | 0† |
| (b) Grades 3b–c (n = 19) | 9 (47%)† |
Grades 3b–c versus 1–3a. P = 0·0009 (Fisher's test)
grades 3b–c versus 1–3a. P = 0·0039 (Fisher's test).
As shown in Fig. 2, a progressive decrease of anti-actin reactivity by ELISA and MF-pattern positivity by IFL was seen after absorption with actin, but not with tTG.
Fig. 2.
IgA Anti-actin reactivity by ELISA of five anti-tTG/AAA/anti-MF positive sera. Mean of percentage (± standard deviation) of residual absorbance (optical density at 492 nm) is on the y-axis. Absorbance was reduced by preincubation with increasing amounts of actin, whereas it remained virtually unaltered by preincubation with tTG.
After preincubation with 500 µg of rabbit actin, anti-actin reactivity by ELISA decreased > 75% and anti-MF antibodies on fibroblasts and HEp-2 (Fig. 1c) cells disappeared almost completely. On the contrary, absorption with guinea-pig liver transglutaminase did not modify anti-actin reactivity by ELISA and anti-MF antibodies by IFL, which remained unaltered, but abolished anti-tTG reactivity selectively by ELISA (Fig. 3).
Fig. 3.
IgA Anti-tTG reactivity by ELISA of five anti-tTG/AAA/anti-MF positive sera. Mean of percentage (± standard deviation) of residual absorbance (optical density at 450 nm) is on the y-axis. Preincubation with actin did not modify absorbance, which remained unaltered.
After 1 year of gluten-free diet 20 of the 28 patients positive for IgA anti-MF antibodies (12 of these also positive for IgA AAA by ELISA) accepted the follow-up re-evaluation, eight patients declined participation. The anti-actin reactivity disappeared by both ELISA and IFL in all of them, whereas IgA EmA and anti-tTG persisted positive in six of these 20 cases; tTG and EmA were not significantly modified, respectively, in four and five of these six patients. Dietetic interview revealed strict compliance to a gluten-free diet in all 20 patients. In all patients a second intestinal biopsy showed complete regrowth of intestinal mucosa. Table 4 shows details of the serological and histological features of 20 anti-MF positive coeliacs revaluated after 1 year of GFD.
Table 4.
Serological and histological features of the 20 anti-MF positive coeliacs revaluated after 1 year of GFD
| Patient no. | Age (years) | Histology at diagnosis | EmA at diagnosis* | Anti-tTG at diagnosis† | IgA Anti-MF at diagnosis | Histology after GFD | EmA after GFD* | Anti-tTG after GFD† | IgA anti-MF after GFD |
|---|---|---|---|---|---|---|---|---|---|
| 3 | 33 | 2 | 1 : 160 | 14 AU | 1 : 40 | normal‡ | neg | neg | neg |
| 4 | 30 | 3c | 1 : 80 | 19 AU | 1 : 160 | normal | 1 : 80 | 18 AU | neg |
| 6 | 43 | 3c | 1 : 320 | 32 AU | 1 : 640 | normal | 1 : 320 | 9 AU | neg |
| 9 | 32 | 3c | 1 : 80 | 20 AU | 1 : 320 | normal | 1 : 80 | 21 AU | neg |
| 11 | 53 | 3b | 1 : 160 | 11 AU | 1 : 320 | normal | 1 : 160 | 11 AU | neg |
| 23 | 43 | 3b | 1 : 160 | 12 AU | 1 : 640 | normal | neg | neg | neg |
| 25 | 29 | 3b | 1 : 320 | 15 AU | 1 : 1280 | normal | 1 : 40 | 15 AU | neg |
| 30 | 28 | 3c | 1 : 80 | 13 AU | 1 : 40 | normal | 1 : 80 | 9 AU | neg |
| 45 | 30 | 3c | 1 : 80 | 16 AU | 1 : 320 | normal | neg | neg | neg |
| 47 | 29 | 3b | 1 : 160 | neg | 1 : 320 | normal | neg | neg | neg |
| 49 | 47 | 2 | 1 : 320 | 20 AU | 1 : 320 | normal | neg | neg | neg |
| 50 | 37 | 1 | 1 : 160 | 15 AU | 1 : 80 | normal | neg | neg | neg |
| 51 | 40 | 3c | 1 : 320 | 26 AU | 1 : 40 | normal | neg | neg | neg |
| 54 | 36 | 3b | 1 : 320 | 25 AU | 1 : 320 | normal | neg | neg | neg |
| 58 | 21 | 3c | 1 : 320 | 21 AU | 1 : 40 | normal | neg | neg | neg |
| 63 | 25 | 3c | 1 : 320 | neg | 1 : 320 | normal | neg | neg | neg |
| 68 | 39 | 3b | 1 : 80 | 9 AU | 1 : 640 | normal | neg | neg | neg |
| 69 | 41 | 3b | 1 : 40 | 22 AU | 1 : 160 | normal | neg | neg | neg |
| 87 | 48 | 3c | 1 : 160 | 12 AU | 1 : 40 | normal | neg | neg | neg |
| 96 | 14 | 3c | 1 : 40 | 10 AU | 1 : 40 | normal | neg | neg | neg |
Cut-off > 1 : 5.
Cut-off > 7 AU.
Intraepithelial lymphocytes < 40 per 100 epithelial cells, absence of crypthyperplasia and normal villous architecture.
DISCUSSION
Smooth muscle antibodies of the IgG class with anti-actin specificity are regarded commonly as markers of type I autoimmune hepatitis, and different techniques have been used for their identification [9,15]. SMA-T/G and anti-MF antibodies are present in about 80% patients with AIH-1, where the two reactivities are strictly associated, both being regarded as anti-actin antibodies [16].
Similar anti-MF antibodies belonging, however, to the IgA class, have also been reported in CD [7]. This study confirms that in patients with untreated CD IgA anti-MF antibodies are a common finding. In our hands IFL on fibroblasts was the most sensitive method for the detection of anti-actin antibodies in CD. By IFL on kidney sections, the prevalence of SMA T/G was definitely lower, possibly because of the masking effect of the concomitant positivity for antireticulin antibodies, detected in 41% (data not shown) of our CD patients and in 16 (57%) of the 28 coeliacs anti-MF positive; moreover, different sera dilution were used (1 : 40 on kidney sections vs. 1 : 5 on cultured fibroblasts). With regard to AAA IgA, the lower sensitivity of ELISA in comparison with IFL on fibroblasts could be explained by the use of monomeric actin (G-actin) as antigen for ELISA. In CD patients, as already speculated for AIH, anti-actin antibodies probably recognize filamentous or polymerized actin (F-actin), which offers a higher number of potential epitopes than unpolymerized actin (G-actin) [17–19].The polymerization of monomeric actin in high amounts [20] may explain why G-actin successfully removed the MF-pattern in absorption experiments.
It has been shown a co-localization of tissue transglutaminase with stress fibres in vascular smooth muscle cells and immunoprecipitation experiments have demonstrated that transglutaminase co-immunoprecipitated with myosin, suggesting that this pattern of distribution was due to cross-linking of transglutaminase with myosin but not with actin [21]. The results of absorption experiments suggest that actin is the target of the anti-MF pattern; our results also suggest that anti-actin and antitTG are separate reactivities, giving different morphological patterns in IFL experiments.
Antibodies to cytoskeleton components such as anti-intermediate filaments (IMF) and anti-MF antibodies of IgA class have been also found in inflammatory bowel disease. However, their occurrence in these conditions is less frequent than in CD and lack clinical significance, probably being the effect only of antigen leakage in the course of mucosal damage [22]. Instead, in CD setting the appearance of AAA IgA could be enhanced by cross-linking of cytoskeleton components with tTG as actin is a good substrate of tTG [23]. Generation of novel neoepitopes by tTG-catalysed cross-linking reactions or post-translational modifications of cryptic self-antigen, unmasked during cell injury, is supposed as a hypothetical mechanism of CD-related autoimmune disorders [24–26]. Immune reaction towards cytoskeleton components with appearance of specific AAA of the IgA class, resulting from actin-network involvement, could be included with the secondary autoimmune phenomena observed in CD.
In our study, a similar prevalence of anti-MF antibodies was observed between childhood and adult CD patients. A significant difference was found in the prevalence of anti-MF IgA between patients with different degrees of mucosal damage. In fact, about half of our CD patients with marked or total villous flattening resulted positive for anti-MF IgA, in contrast with 6% of those with mild intestinal mucosal damage. The predictive positive value of anti-MF IgA antibodies for severe villous atrophy (grades 3b and c) is estimated to be 89%. None of the pathological and normal controls had anti-MF IgA, indicating a very high specificity for CD.
Compared with the original report by Clemente et al. [7], we found a lower prevalence of anti-MF antibodies in CD patients; this discrepancy could be due both to selection criteria (a higher prevalence of cases with severe mucosal damage in Clemente's study) and to differences in the tests used (different commercial kits of HEp-2 cells and different secondary antibody used). Although Clemente et al. described the disappearance of AAA after GFD, the correlation between AAA and mucosal morphology was investigated before GFD, while no correlation analysis was performed after GFD. Our results indicate that IgA anti-MF antibodies disappeared totally in all 20 cases tested after 1 year of GFD, showing a high correlation with histological recovery, assessed by second intestinal biopsy; on the other hand, both EmA and anti-tTG antibodies persisted in about 30% of the 20 patients. Despite the limited number of cases, almost entirely concerning adult patients, our results, obtained by a complete follow-up study, show for the first time a significant correlation between disappearance of anti-MF IgA and the normalization of intestinal mucosa, suggesting that these antibodies could be helpful for monitoring the response to GFD in severe coeliac disease, especially when anti-tTG or EmA antibodies after GFD are still present.
In conclusion, our study shows that anti-MF IgA antibodies with anti-actin specificity are present in a significant proportion of patients with untreated CD.
Although anti-MF IgA antibodies, due to their global low sensitivity, cannot replace EmA and anti-tTG in the diagnostic algorithm of CD, their finding is associated strictly with flat mucosa and is predictive of a severe gluten-sensitive enteropathy; moreover, they could be considered a further tool for the follow-up of severe coeliac disease in addition to traditional markers.
References
- 1.Trier JS. Celiac sprue. N Engl J Med. 1991;325:1709–19. doi: 10.1056/NEJM199112123252406. [DOI] [PubMed] [Google Scholar]
- 2.Volta U, Molinaro N, de Franceschi L, et al. IgA anti-endomysial antibodies on human umbilical cord tissue for celiac disease screening. Save both money and monkeys. Dig Dis Sci. 1995;40:1902–5. doi: 10.1007/BF02208653. [DOI] [PubMed] [Google Scholar]
- 3.Sblattero D, Berti I, Trevisiol C, et al. Human recombinant tissue transglutaminase ELISA: an innovative diagnostic assay for celiac disease. Am J Gastroenterol. 2000;95:1253–7. doi: 10.1111/j.1572-0241.2000.02018.x. [DOI] [PubMed] [Google Scholar]
- 4.Dickey W, Hughes DF, McMillan SA. Disappearance of endomysial antibodies in treated celiac disease does not indicate histological recovery. Am J Gastroenterol. 2000;95:712–4. doi: 10.1111/j.1572-0241.2000.01838.x. [DOI] [PubMed] [Google Scholar]
- 5.Ciacci C, Cavallaro R, della Valle N, et al. The use of serum tTG-ab assay in patients on gluten-free diet as a measure of dietetic compliance. Gastroenterology. 2002;122:588. doi: 10.1053/gast.2002.31598. [DOI] [PubMed] [Google Scholar]
- 6.Farrell RJ, Kelly CP. Celiac sprue. N Engl J Med. 2002;346:180–8. doi: 10.1056/NEJMra010852. [DOI] [PubMed] [Google Scholar]
- 7.Clemente MG, Musu MP, Frau F, et al. Immune reaction against the cytoskeleton in coeliac disease. Gut. 2000;47:520–6. doi: 10.1136/gut.47.4.520. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Bottazzo GF, Florin-Christensen A, Fairfax A, et al. Classification of smooth muscle autoantibodies detected by immunofluorescence. J Clin Pathol. 1976;29:403–10. doi: 10.1136/jcp.29.5.403. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Fusconi M, Cassani F, Zauli D, et al. Anti-actin antibodies: a new test for an old problem. J Immunol Meth. 1990;130:1–8. doi: 10.1016/0022-1759(90)90291-3. [DOI] [PubMed] [Google Scholar]
- 10.Bailey DS, Freedman AR, Price SC, et al. Early biochemical responses of the small intestine of coeliac patients to wheat gluten. Gut. 1989;30:78–85. doi: 10.1136/gut.30.1.78. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Holmgren Peterson K, Magnusson KE, et al. Confocal laser scanning microscopy of small-intestinal mucosa in celiac disease. Scand J Gastroenterol. 1995;30:228–34. doi: 10.3109/00365529509093269. [DOI] [PubMed] [Google Scholar]
- 12.Oberhuber G, Granditsch G, Vogelsang H. The histopathology of coeliac disease: time for a standardized report scheme for pathologists. Eur J Gastroenterol Hepatol. 1999;11:1185–94. doi: 10.1097/00042737-199910000-00019. [DOI] [PubMed] [Google Scholar]
- 13.Cassani F, Cataleta M, Valentini P, et al. Serum autoantibodies in chronic hepatitis C: comparison with autoimmune hepatitis and impact on the disease profile. Hepatology. 1997;26:561–6. doi: 10.1002/hep.510260305. [DOI] [PubMed] [Google Scholar]
- 14.Volta U, Rodrigo L, Granito A, et al. Celiac disease in autoimmune cholestatic liver disorders. Am J Gastroenterol. 2002;97:2609–13. doi: 10.1111/j.1572-0241.2002.06031.x. [DOI] [PubMed] [Google Scholar]
- 15.Zamanou A, Samiotaki M, Panayotou G, et al. Fine specificity and subclasses of IgG anti-actin autoantibodies differ in health and disease. J Autoimmun. 2003;20:333–44. doi: 10.1016/s0896-8411(03)00036-2. [DOI] [PubMed] [Google Scholar]
- 16.Muratori P, Muratori L, Agostinelli D, et al. Smooth muscle antibodies and type 1 autoimmune hepatitis. Autoimmunity. 2002;35:497–500. doi: 10.1080/0891693021000054066. [DOI] [PubMed] [Google Scholar]
- 17.Kurki P. Determination of anti-actin antibodies by a solid-phase immunoenzymatic assay and by indirect immunofluorescence technique. Clin Immunol Immunopathol. 1978;11:328–38. doi: 10.1016/0090-1229(78)90057-0. [DOI] [PubMed] [Google Scholar]
- 18.Andersen P, Small JV, Andersen HK, et al. Reactivity of smooth-muscle antibodies with F- and G-actin. Immunology. 1979;37:705–9. [PMC free article] [PubMed] [Google Scholar]
- 19.Cancado EL, Abrantes-Lemos CP, Vilas-Boas LS, et al. Thermolabile and calcium-dependent serum factor interferes with polymerized actin, and impairs anti-actin antibody detection. J Autoimmun. 2001;17:223–8. doi: 10.1006/jaut.2001.0540. [DOI] [PubMed] [Google Scholar]
- 20.Cooper JA, Buhle EL, Jr, Walker SB, et al. Kinetic evidence for a monomer activation step in actin polymerization. Biochemistry. 1983;26:2193–202. doi: 10.1021/bi00278a021. [DOI] [PubMed] [Google Scholar]
- 21.Chowdhury ZA, Barsigian C, Chalupowicz GD, et al. Colocalization of tissue transglutaminase and stress fibers in human vascular smooth muscle cells and human umbilical vein endothelial cells. Exp Cell Res. 1997;231:38–49. doi: 10.1006/excr.1996.3448. [DOI] [PubMed] [Google Scholar]
- 22.Zauli D, Crespi C, Dall’Amore P, et al. Antibodies to cytoskeleton components and other autoantibodies in Inflammatory bowel disease. Digestion. 1985;32:140–4. doi: 10.1159/000199232. [DOI] [PubMed] [Google Scholar]
- 23.Huang YP, Seguro K, Motoki M, et al. Cross-linking of contractile proteins from skeletal muscle by treatment with microbial transglutaminase. J Biochem. 1992;112:229–34. doi: 10.1093/oxfordjournals.jbchem.a123882. [DOI] [PubMed] [Google Scholar]
- 24.Molberg O, McAdam SN, Korner R, et al. Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease. Nat Med. 1998;4:713–7. doi: 10.1038/nm0698-713. [DOI] [PubMed] [Google Scholar]
- 25.Schuppan D. Current concepts of celiac disease pathogenesis. Gastroenterology. 2000;119:234–42. doi: 10.1053/gast.2000.8521. [DOI] [PubMed] [Google Scholar]
- 26.Schuppan D, Ciccocioppo R. Coeliac disease and secondary autoimmunity. Dig Liver Dis. 2002;34:13–5. doi: 10.1016/s1590-8658(02)80053-6. [DOI] [PubMed] [Google Scholar]