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Technical Briefs |
1 Department of Clinical Chemistry, Medisch Spectrum Twente Hospital Group, 7500 KA Enschede, The Netherlands
aaddress correspondence to this author at: Laboratorium Medisch Spectrum Twente, Postbus 50.000, 7500 KA Enschede, The Netherlands; Fax 31-53-487-3075, e-mail labmst{at}euronet.nl
The enzyme transglutaminase (tTg; EC 2.3.2.13) catalyzes,
among others, the formation of 
-(
-glutamyl)-lysine bonds between
substrate proteins, leading to cross-linked protein polymers
(1). The enzyme is synthesized by a broad spectrum of cell
types and is widely distributed in human organs (2).
Induction and activation of tTG is part of the apoptotic cascade and
plays an effector role in this process (3)(4).
The enzyme is present in preapoptotic cells and enables the production
of a highly cross-linked protein scaffold in apoptotic cells, joining
cytoplasmic and membrane proteins and thus maintaining cellular
integrity during the formation of apoptotic bodies. This cross-linking
of proteins stabilizes the apoptotic bodies and limits the leakage of
intracellular components into the extracellular space
(1)(5).
Recently, tissue tTg has been shown to be the key autoantigen of the so-called anti-endomysium antibodies (IgA type), which are diagnostic for celiac disease (6). IgG antibodies against tTG (IgG anti-tTG) can be found in other autoimmune diseases in humans (7) and animals (1). There is ample evidence of the relationship between autoimmune disease and dysbalanced apoptosis, but the mechanisms remain hypothetical (8)(9). It has been suggested that a deficiency of the apoptotic removal of autoreactive immune cells breaks the self-tolerance (10). A second hypothesis is that the clearance of apoptotic bodies is deficient, with the result that their contents come into contact with the immune system, leading to autoimmunity as well (11)(12)(13). Accordingly, the demonstration of autoantibodies against tTg may be of great interest in the diagnosis and follow-up of autoimmune diseases and may throw a new light on their etiology. We therefore developed a time-resolved fluoroimmunoassay (TRFIA) to measure IgG anti-tTG.
The TRFIA method was as follows. tTg from guinea pig liver (Sigma) was used as antigen. F(ab')2 fragments from antibodies raised in rabbits against human IgG (Dako A/S) labeled with Eu3+, using the reagent and procedure from Wallac Oy, served as the tracer. Samples (diluted 1000-fold) were incubated in antigen-coated polystyrene microtiter plates (Polysorp®; Nunc). The time-resolved fluorescence was measured (1420 Victor 2; Wallac Oy), and the concentration of the anti-tTg was calculated. All measurements were performed in duplicate. To a patient serum containing a high concentration of IgG anti-tTg [20 000 (arbitrary) kilounits/L], was assigned. This was used as the calibrator. The assay has a measuring range of 5–1000 kilounits/L with a between-run imprecision (CV) of <12%. The reference values were calculated as the 95th percentile of concentrations in sera from healthy blood bank donors (n = 40) and found to be <18 kilounits/L. Anti-double-stranded DNA (anti-dsDNA) (14), IgM rheumatoid factor (15), IgA anti-gliadin, and IgA anti-tTG (reference values <20 kilounits/L) were measured by TRFIA. Anti-proteinase 3 was measured by ELISA (DLD Diagnostika). C-Reactive protein was assayed by immunoturbidimetry (Tina-quant®; Roche). Antinuclear antibodies were detected by immunofluorescence on Hep-2000® cells (Biomedical Diagnostics) and identified by immunoblotting using the InnoliaTM ANA reagent set (Innogenetics).
Sera were selected from our serum bank (in which they were stored at
-80 °C for periods of 1 week to 8 years) on the basis of their
antibody pattern and clinical diagnosis: (a) 30 sera from
patients with systemic lupus erythematosus (SLE; n = 15) with
increased anti-dsDNA; (b) serum samples with different
anti-nuclear antibodies [SSA and SSA/SSB (Ro, La; n = 13), Sm/RNP
and RNP (n = 21)], anti-nucleolar antibodies (immunofluorescence;
n = 7), anti-histidyl-tRNA synthetase (Jo-1; n = 5),
anti-centromere protein B (Cenp-B; n = 14), or anti-topoisomerase
I (Scl-70; n = 7); and (c) serum samples containing
other autoantibodies, such as IgM rheumatoid factor (n = 30),
anti-proteinase 3 (n = 14), and IgA anti-gliadin (n = 9). The
samples contained in general not more than one detectable antibody (in
addition to anti-tTG) other than the one mentioned. All patients
described here suffered from chronic inflammation. To exclude the
possibility that IgG anti-tTG is an aspecific marker for this
condition, we tested sera (n = 14) with increased C-reactive
protein concentrations. We found IgG anti-tTG concentrations comparable
to those in control subjects (Fig. 1
). The procedures followed were in accordance with the policies
of the Ethical Review Board of the hospital group.
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High concentrations of IgG anti-tTG (range, 53–3300 kilounits/L) were
found in patients with SLE (increased anti-dsDNA) and in patients with
SSA/SSB antibodies (but with anti-dsDNA within reference values; Fig. 1
). It is remarkable that these are the very nuclear antigens present
in the blebs in the surface of apoptotic cells, which are the
precursors of apoptotic bodies (16). The IgA anti-tTG
concentration was 5–16 kilounits/L in the SLE sera and 18–58
kilounits/L in the SSA/SSB group. From these results one can conclude
that celiac disease in unlikely, at least in the majority of the
patients.
The apoptotic bodies and several components of their contents are autoimmunogenic (17). Tissue tTg is also present in the apoptotic bodies (1)(4), but that does not necessarily explain why it also is immunogenic.
Immunoglobulins against tTG substrate proteins have been demonstrated in various autoimmune diseases. One example is the presence of antibodies against gliadin, the protein that causes the symptoms in celiac disease, which is a preferred tTg substrate (18). It is supposed that dietary gliadin in gliadin-tTg complexes creates antigenic neoepitopes, which can initiate an immune response directed to both gliadin and tTg (19). Autoantibodies often are directed against different components of the same molecular complex (5)(8). A similar mechanism may cause the simultaneous formation of antibodies against tTg and its substrates (of which little is known) during apoptosis. tTG-substrate complexes typically exist only intracellularly, thus avoiding contact with the immune system. However, in situations of an imbalance between the supply and clearance of apoptotic bodies, the immune system can detect them, leading to an autoimmune response. This mechanism was demonstrated in SLE, where nucleosomes, which usually are cleared rapidly, expose the elementary antigens, leading to the formation of anti-DNA antibodies (12).
Our findings seem to fit into the theory that in certain autoimmune diseases, such as SLE, the content of apoptotic bodies comes into contact with the immune system, leading to an autoimmune response.
The difference in IgG anti-tTG concentrations between the SLE or SSA/SSB groups and the other groups is striking: patients with other detectable anti-nuclear antibodies and anti-proteinase 3 antibodies had normal or only slightly increased IgG anti-tTg concentrations (range, 0–55 kilounits/L). Perhaps the pathogenic mechanism in the latter groups of autoimmune diseases is different.
In the SLE patients, we did not find a longitudinal correlation between the concentrations of anti-DNA antibodies, which are useful in the follow-up of these patients, and anti-tTG. This suggests that anti-tTG may provide additional clinical information. In patients with Sjögrens syndrome, the concentrations of SSA/SSB antibodies do not correlate with exacerbations (20). Perhaps IgG anti-tTG has clinical value in monitoring these individuals.
Our results are only the first step in exploring the clinical value of IgG anti-tTG assays in patients with autoimmune diseases. In addition to the more fundamental aspects concerning the link between apoptosis and autoimmunity, its role in diagnosis, including sensitivity and specificity, and in the monitoring of patients still has to be elucidated and is the object of further investigations.
References
The following articles in journals at HighWire Press have cited this article:
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  D. Villalta, N. Bizzaro, E. Tonutti, and R. Tozzoli IgG Anti-Transglutaminase Autoantibodies in Systemic Lupus Erythematosus and Sjogren Syndrome Clin. Chem., July 1, 2002; 48(7): 1133 - 1133. [Full Text] [PDF]
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M. Di Tola, L. Sabbatella, A. Picarelli, G. van der Sluijs Veer, and I. Vermes Presence of AntiTissue Transglutaminase Antibodies as a Sign of Tissue Lesion The authors of the article cited above respond: Clin. Chem., February 1, 2002; 48(2): 393 - 394. [Full Text] [PDF]
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