Cell culture and DNA transfection

Madin-Darby canine kidney (MDCK) cells (European Cell Collection, 85011435) were grown in minimum essential medium (Life Technologies, Milan, Italy) supplemented with 10% fetal bovine serum, 10% Earle's balanced salt solution, 50 u/ml penicillin, 50 μg/ml streptomycin, and 1% non-essential amino acids. Cells were maintained in a 5% CO₂ humidified atmosphere. Human tTG cDNA,²¹ cloned in the eukaryotic expression vector pSG5 (Stratagene, La Jolla, California, USA), was used to transfect MDCK cells by calcium phosphate precipitation. A stable cell clone, MDCK-tTG, expressing recombinant tTG was obtained by cotransfecting MDCK cells with pSV2-Neo (Clontech, Palo Alfo, California, USA) in a 10:1 ratio. Cotransfected cells were cultured in a selective medium containing 400 μg/ml G418 (Life Technologies).

MDCK-tTG cells were harvested in 10 mM Tris/HCl pH 7.5, 1 mM ethylenediamine-tetraacetic acid (EDTA), and sonicated for 10 seconds. Protein content was estimated by the method of Bradford with bovine serum albumin (BSA) (Sigma, St Louis, Missouri, USA) as the standard.²²

IgG and IgA purification

IgG and IgA antibodies from normal and CD patients were purified using Sepharose beads conjugated with protein A or rabbit antibodies to human IgA (Sigma). Serum was buffered with 0.1 M Tris/HCl pH 8.0 and applied to the anti-human IgA Sepharose equilibrated in the same buffer. The flow through fraction, containing IgG, was collected and subsequently applied to protein A Sepharose. Bound antibodies were eluted with 0.1 M glycine buffer pH 3.0; fractions were collected into tubes containing 1 M Tris/HCl pH 8.0 to prevent antibody denaturation. Protein content was determined using the method of Bradford.²²

Monoclonal antibodies

Monoclonal antibodies (single chain antibody fragments) to tTG were prepared as described previously.²⁰ Briefly, total RNA was prepared from intestinal biopsies from three previously untreated CD adult patients, cDNA was synthesised using random hexamers, and Ig V regions were amplified using specific V region primers and assembled before cloning into the phagemid vector pDAN5.²³ Antibodies to tTG were isolated by recursive cycles of binding to recombinant human tTG, washing, and elution of phage particles expressing antibody fragments on their surface. In the present work, clones 2.18, 3.7, and 4.2 were isolated from CD libraries whereas clone D51 was selected from a naïve library²³ made from peripheral lymphocytes from healthy donors. The CD antibodies are reported according to the donor patient, and given reference numbers 2, 3, and 4 followed by the individual clone reference number.

Soluble antibodies were obtained by expression in HB2151 (K12, ara Δ(lac-pro), thi/F` proA⁺B⁺, lacI^qZΔM15), a non-suppressor Escherichia coli strain. Phages from individual colonies were infected into HB2151, grown to OD₆₀₀ 0.2, induced with 1 mM isopropyl-β-d-thiogalactopyranoside, and further grown overnight at 28°C. Antibodies were purified from supernatants of induced bacterial cultures by Ni-NTA chromatography (Qiagen, Germany) and dialysed against phosphate buffered saline (PBS) to obtain a concentration of approximately 0.2 mg/ml.

Enzyme linked immunosorbent assay (ELISA) for anti-tTG antibodies

To measure serum IgA and IgG anti-tTG titres, we used an ELISA method. U bottomed microtitre plates (Falcon, Franklin Lakes, New Jersey, USA) were coated with 1 μg of human recombinant tTG per well in 100 μl of coating buffer (50 mM Tris/HCl, 150 mM NaCl, 5 mM CaCl₂, pH 7.5) for two hours at 37°C. Wells were washed three times with washing buffer (50 mM Tris/HCl, 150 mM NaCl, 10 mM EDTA, 0.1% Tween-20, pH 7.4). The plates were incubated in washing buffer overnight at 4°C. Sera, diluted 1:100 in the same buffer, were added to the wells and incubated for one hour at room temperature (RT). After three washes, the wells were incubated with 100 μl of peroxidase conjugated rabbit antihuman IgA or peroxidase conjugated rabbit antihuman IgG (Dako, Denmark) diluted respectively 1:1000 and 1:3000 in the same buffer for one hour at RT. Unbound antibodies were removed with three washes and colour was developed by addition of 100 μl of 1 M sodium citrate, 1 M citric acid, 1 mg/ml o-phenylenediamine-hydrochloride (Sigma), and 0.06% H₂O₂ (Carlo Erba, Italy), pH 5, at RT for 30 minutes in the dark. Absorbances were read on an ELISA reader (SpectraCount, Packard, Milan, Italy) at 450 nm. All samples were tested in duplicate. Values were expressed as percentage of a reference pool of sera obtained from untreated coeliac patients.

In vitro tTG assay

Enzyme activity was assayed by a radiometric method based on Ca²⁺ dependent incorporation of [³H]spermidine (Nen Life Science Products, Boston, Massachusetts, USA) into N,N-dimethylated casein (DMC). Briefly, the assay mixtures (100 μl) containing 125 mM Tris/HCl buffer, pH 8.0, 10 mM dithiotreitol, 5 mM CaCl₂, 0.16 μM [³H]spermidine (15 Ci/mmol), 0.2 mg DMC and, as enzymatic source MDCK-tTG cell homogenate (7 μg of proteins), were incubated at 37°C for one hour. The inhibition studies were performed by preincubation of the enzyme with different amounts of IgG and IgA in 25 mM Tris/HCl pH 8.0 (final volume 20 μl) at 20°C for 10 minutes. Preincubation of tTG with different amounts of monoclonal antibodies was performed in the absence or presence of 5 mM CaCl₂. Blanks were simultaneously run with 5 mM ethylene glycol-bis(β-aminoethyl ether) N,N,N`,N`-tetraacetic acid (EGTA). The reactions were stopped with 10% trichloroacetic acid containing 2 mM unlabelled spermidine. After centrifugation, the precipitates were washed twice with the same solution, dissolved in 0.5 ml of 0.2 N NaOH, and finally counted in 5 ml of Pico-Fluor 40 scintillation mixture (Packard).

In situ tTG activity assay

Umbilical cord cryostat sections (7 μm thick) were mounted on slides with 2% triethoxysilyl-propylamin (Merck, Germany) in acetone, air dried for 60 minutes at RT, and fixed in acetone for 10 minutes. Sections were preincubated with 1% BSA in 100 mM Tris/HCl, pH 7.4, for 30 minutes at RT, and then for two hours at RT with biotinylated monodansyl-cadaverine (bio-MDC) (Molecular Probes, Leiden, the Netherlands). This substrate solution was prepared by adding 10 μl of 10 mM bio-MDC and 25 μl of 200 mM CaCl₂ solution to 965 μl of 100 mM Tris/HCl, pH 7.4. The tTG reaction was stopped with 25 mM EDTA for five minutes. Incorporation of labelled substrate was visualised by incubation in a humid chamber for 30 minutes at RT with streptavidin fluorescein isothiocyanate conjugate (Dako) diluted 1:50 in PBS. Finally, sections were washed twice in PBS and mounted in 10% glycerol in PBS. In control sections, 200 mM CaCl₂ was replaced by 200 mM EDTA. Photographs were taken using an Axiophot (Zeiss, Germany) and colour reversal film (Kodak, UK).

Effects of CD patient IgG and IgA on tTG activity in vitro

Table 1⇓ shows that addition of 10 μg of IgG and IgA from six and five CD patients, respectively, into the assay mixture inhibited the incorporation of [³H]spermidine into DMC catalysed by recombinant tTG expressed in the MDCK-tTG cell line. A different rate of inhibition among patients was also observed. In fact, the extent of inhibition due to IgG and IgA ranged from 8.3 (4.3)% to 24 (1)% and from 11.1 (1.5)% to 39 (1)%, respectively. The data reported in table 1⇓ also showed that there was no correlation between the degree of inhibition and serum IgG or IgA titres measured by ELISA. This behaviour was confirmed when a dose dependent inhibition assay was performed using IgG and IgA from two different patients (Nos 3 and 4 in table 1⇓). In fact, as shown in fig 1⇓, the inhibition exhibited by IgG from patient No 4 (titre 37%) was dose dependent and reached 80% when 60 μg of antibody were used with respect to basal values obtained by preincubating the enzyme in the absence of antibody. In contrast, IgG from patient No 3 (titre 89%) exerted 20% inhibition at the higher concentration used. Finally, IgG purified from healthy controls had no effect on tTG activity.

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Figure 1

Effect of purified serum IgG from coeliac patients (Nos 3 and 4) and from healthy controls on tissue transglutaminase (tTG) activity. Increasing amounts of IgG from patient No 4 (titre 24%), IgG from patient No 3 (titre 89%), and IgG from a pool of control patients (titre 2%) were incubated with 7 μg of proteins from Madin-Darby canine kidney (MDCK)-tTG cells at 37°C for one hour in a 100 μl final volume. The activity of tTG was calculated as a percentage of basal activity (no IgG addition) within MDCK-tTG cells. Data are presented as means (SD) from three separate experiments done in duplicate.

Effects of monoclonal anti-tTG antibodies on tTG activity in vitro

The monoclonal antibody CUB 7402 displayed a dose dependent inhibitory effect towards the catalytic activity of the enzyme expressed in MDCK-tTG cells with respect to basal values, as shown in fig 2⇓. Because calcium ions influence tTG activity by inducing structural changes in the protein, we preincubated the enzyme, before the enzymatic assay, with different amounts of antibodies in 25 mM Tris/HCl pH 8.0 in the presence of 5 mM CaCl₂. Preincubation caused loss of the inhibitory effect due to CUB 7402.

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Figure 2

Effect of the monoclonal antibody anti-tissue transglutaminase (tTG) (CUB 7402) on tTG activity in response to Ca²⁺. Increasing concentrations of CUB 7402 were preincubated at 20°C for 10 minutes in 20 μl of 25 mM Tris/HCl, pH 8.0, in the absence or presence of 5 mM CaCl₂, with 7 μg of proteins from Madin-Darby canine kidney (MDCK)-tTG cells. The samples were further incubated for one hour at 37°C in a 100 μl final volume. The activity of tTG was calculated as a percentage of basal activity (no CUB 7402 added) within MDCK-tTG cells. Data are presented as means (SD) from three separate experiments performed in duplicate.

The same experiments were performed using monoclonal antibodies to tTG derived from both intestinal lymphocytes from three patients with CD and from peripheral blood lymphocytes from healthy subjects. The results reported in fig 3⇓ show that the dose related inhibition caused by 4.2, 2.18, and 3.7 clones was almost the same (ranging from 20% to 40%). However, when preincubation was performed in the presence of CaCl₂, inhibition was still present; in particular, when 2.18 clone was used, enhanced inhibition was observed.

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Figure 3

Effect of single chain monoclonal antibodies on tissue transglutaminase (tTG) activity in response to Ca²⁺. Human monoclonal antibodies (0.5, 1.0, and 1.5 μg) were preincubated at 20° C for 10 minutes in 20 μl of 25 mM Tris/HCl, pH 8.0, in the absence (−) or presence (+) of 5 mM CaCl₂, with 7 μg of proteins from Madin-Darby canine kidney (MDCK)-tTG cells. The samples were further incubated for one hour at 37°C in a 100 μl final volume. The activity of tTG was calculated as a percentage of basal activity (no antibodies added) within MDCK-tTG cells. Data are presented as means (SD) from three separate experiments performed in duplicate.

Finally, only a weak inhibitory effect was evoked by the anti-tTG monoclonal antibody derived from peripheral blood lymphocytes from healthy subjects, suggesting that the anti-tTG antibodies found in CD have special properties which may play a role in the disease.

Effect of antibodies on tTG activity in situ

tTG activity, as detected in umbilical cord sections by incorporation of the labelled substrate bio-MDC, resulted in the presence of fluorescent signals (fig 4A⇓). Such activity was clearly affected by addition of antibodies. Anti-tTG CUB 7402 (fig 4B⇓) and single chain monoclonal antibodies from the intestinal mucosa of coeliac patients (fig 4C⇓) significantly decreased signalling. Both IgG and IgA from coeliac serum, but not from controls, significantly decreased the signalling (data not shown).

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Figure 4

Effect of specific antibodies on tissue transglutaminase (tTG) activity in human umbilical cord. The signal corresponds to incorporation of substrate biotinylated monodansyl-cadaverine (A). Sections treated with the anti-tTG CUB 7402, diluted 1:25 (B), and with monoclonal antibody 4.2, undiluted (C), showed a significantly lower signal (original magnification ×400).