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Endocrinology Vol. 144, No. 12 5422-5429
Copyright © 2003 by The Endocrine Society

Complement Activation by Direct C4 Binding to Thyroperoxidase in Hashimoto’s Thyroiditis

Stéphanie Blanchin, Valérie Estienne, Josée-Martine Durand-Gorde, Pierre Carayon and Jean Ruf

Institut National de la Santé et de la Recherche Médicale, Unité 555, Faculté de Médecine Timone, Université de la Méditerranée, F-13385 Marseille, France

Address all correspondence and requests for reprints to: Jean Ruf, D.Sc., Unité 555, Institut National de la Santé et de la Recherche Médicale Faculté de Médecine Timone, 27, Boulevard Jean Moulin, F-13385 Marseille Cedex 5, France. E-mail: Jean.Ruf{at}medecine.univ-mrs.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Biosynthesis of thyroid hormones is an oxidative process that generates reactive oxygen species (ROS) and involves thyroperoxidase (TPO) that is one of the main autoantigens involved in autoimmune thyroid diseases. The ectodomain of TPO consists of a large N-terminal myeloperoxidase-like module followed by a complement control protein (CCP)-like module and an epidermal growth factor-like module. The presence of these two additional gene modules suggests that they may play some crucial, hitherto unsuspected role associated with thyroid function. Because the CCP module is a constituent of the molecules involved in the activation of C4 complement component, we investigated the possibility that C4 may bind to TPO and activate the complement pathway in autoimmune conditions. We showed that TPO via its CCP module directly activated complement without any mediation by Ig. We suggested that this additional complement pathway requires the production of ROS and specially hydroxyl radicals that aggregate TPO and oxidize methionines of C4. Moreover, we found, in patients with Hashimoto’s thyroiditis, that thyrocytes overexpress C4 and all the downstream components of the complement pathway. These results indicate that TPO has some as yet unknown function, which may contribute along with other mechanisms to the massive cell destruction observed in Hashimoto’s thyroiditis. Investigating this complement pathway, therefore, would provide an excellent means of reaching a better understanding of the etiology of other degenerative diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AUTOIMMUNE THYROID DISEASES (AITD) include a broad spectrum of pathological disorders ranging from thyroid hyperfunction in Graves’ disease (GD) to thyroid destruction in Hashimoto’s thyroiditis (HT). AITD can be adopted as models for other organ-specific autoimmune and/or degenerative diseases because they have been extensively investigated in the past, resulting in the identification of diverse T-and B-cell mediated pathological mechanisms (1, 2). The etiology of AITD involves genetic and environmental factors, especially iodine, which triggers the process of thyroid hormone synthesis. During hormone formation, reactive oxygen species (ROS) are produced that can be detrimental to thyroid function (3). However, both the initial step and the sequence of subsequent events resulting in AITD still remain to be elucidated. A relationship certainly exists between the endocrine function of the thyroid gland and thyroid autoimmunity because iodine plays an important role in the physiological and pathological mechanisms, as do the main thyroid autoantigens, thyroglobulin (Tg), and thyroperoxidase (TPO), which are the substrate-enzyme pair involved in hormone synthesis (4). However, the respective roles played by Tg and TPO in AITD are still a matter of debate. Tg has been known for a long time to trigger thyroiditis per se in experimental models (5), whereas TPO has mainly been reported to act via induced autoantibodies that are associated with thyroid damage (6, 7) or enzymatic impairment (8).

TPO is a membrane-bound enzyme acting on the apical side of thyrocytes facing the closed colloidal space. The extracellular part of human TPO (amino acids 1–848) consists of three associated gene modules. The largest of these is the N-terminal gene module (amino acids 1–739), which shows 44% homology with myeloperoxidase. The other two gene modules show homologies with unrelated proteins, i.e. the complement control protein (CCP) gene module of complement receptor type 1 (amino acids 742–794) and the epidermal growth factor (EGF) gene module of human fibrillin 1 (amino acids 795–839), respectively (9). These two additional modules are also present in the C1s subunit of complement component C1 and mannose-binding lectin-associated serine proteases, a family of proteolytic enzymes that activate complement component C4 (10, 11).

Complement is an important mediator of inflammatory tissue damage. The early steps in complement activation can take either the classical, lectin, or alternative pathways, whereas the subsequent steps leading to the formation of a membrane-attack complex are similar in all three cases (12). The various complement components (C1–C9) interact in a highly regulated enzymatic cascade. C4 is an essential component of the complement system, which is involved in the propagation of the complement cascade via the classical (antigen-antibody complex mediated) and lectin (bacteria mediated) pathways. Before being activated, C4 is cleaved into the C4b subunit and, the C3 and C5 convertases are formed (13). Efficient C4 cleavage occurs at CCP module binding sites where the C4b subunit is retained (14). The presence of the CCP module in TPO, therefore, raises the question as to what role it may play in the complement activation during AITD.

Here we report on the overexpression of C4 and all the subsequent components in the complement cascade by HT thyrocytes. In concert with the antibody-dependent classical pathway, the complement activation consequent to direct C4 binding to the CCP-like module of TPO is thus being able to provide to the massive cell destruction observed in the acute phase of HT.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Human sera
Blood was collected from four healthy volunteers and used as source of complement. The sera were removed, pooled and aliquots were stored at -80 C until the time of use to preserve complement activity (15). The quantitative determinations of C3 and C4 were performed by immunonephelometry using an automatic laser nephelometer (BNII, Dade Behring, Marburg, Germany). In the pool of sera, the C3 and C4 contents (1.20 and 0.27 g/liter, respectively) were in the normal ranges. To remove C4 from the pool of human sera, a 10-fold molar excess of C4 antibody (Sigma, St. Louis, MO) was mixed to the sera for 1.5 h at 37 C. Complexes were then precipitated by protein G cell suspension (Omnisorb, Calbiochem-Novabiochem Corp., La Jolla, CA) and removed by centrifugation according to the manufacturer’s instructions. Efficient depletion of C4 (0.07 g/liter) but not C3 (1.19 g/liter) in the pool of sera was monitored as described above. Quantitative determination of TPO autoantibodies was performed using a competitive luminescence immunoassay kit (Lumitest anti-TPOn, BRAHMS, Berlin, Germany) and a luminometer (Berilux, BRAHMS). The TPO antibody titer of the pool of sera was negative.

Thyroid tissues
Normal thyroid tissue specimens were obtained from para-adenomatous tissues from three patients with macrofollicular nontoxic adenomas (diagnosed on the basis of histopathological examination at thyroidectomy). Autoimmune thyroid tissues were from five patients who underwent thyroidectomy for GD and two patients suffering from HT. Each patient’s tissue was used to prepare a single primary thyrocyte cell culture.

Thyroid antigens
Human Tg was purified from colloid goiter by performing slice extraction, sodium phosphate precipitation and gel filtration (16). Human TPO was purified from GD tissue by monoclonal antibody-assisted chromatography (17). TPO recombinant peptides r-pep1 and r-pep2 corresponding to the myeloperoxidase- and CCP + EGF-like modules of the human TPO, respectively, were produced in Chinese hamster ovary cells and purified by continuous elution electrophoresis as previously described (18).

Primary human thyrocyte cultures
Primary cell cultures were prepared as previously described by Estienne et al. (19). Briefly, the tissue was minced into small fragments. The tissue fragments were washed in Coon modified Ham’s F-12 medium (Sigma) containing 2.6 g/liter sodium carbonate (Merck & Co., Darmstadt, Germany), penicillin (100 IU/ml)/streptomycin (100 µg/ml) (Life Technologies, Grand Island, NY), and kanamycin (100 µg/ml), (Life Technologies). To separate the epithelial cells from the connective tissue, the fragments were digested by collagenase I (1 mg/ml) (Life Technologies) and dispase II (2.4 U/ml) (Roche Diagnostics, Meylan, France) in a calcium- and magnesium-free PBS (pH 7.4) at 37 C for 15 min. The enzymatic digestion step then repeated for 60 min with fresh enzyme mixture. The digested tissue was filtered through a sterile gauze. Culture medium was added and the cells were washed by performing several centrifugations at 100 x g for 5 min. After red cell lysis for 5 min at 37 C by 0.16 M NH4Cl, 0.17 M Tris buffer (pH 7.2), the cells were seeded at a density of 2 x 106 cells per 25 cm2 Falcon tissue culture flask (Becton Dickinson, Franklin Lakes, NJ) in 7 ml of culture medium supplemented with 5% fetal calf serum (Valbiotech, Paris, France) in the presence of bovine TSH, 1 U/liter (Sigma) to maintain the thyrocytes in a stimulating environment and the following five nutrients: human insulin, 10 mg/liter (Roche Diagnostics); somatostatin, 10 µg/liter (Novabiochem, Läufelfingen, Switzerland); human transferrin, 6 mg/liter (Roche Diagnostics); hydrocortisone, 10-8 M (Calbiochem, La Jolla, CA); and glycyl-histidyl-lysine acetate, 10 µg/liter (Calbiochem). The culture medium also contained L-glutamine (2 mM) (Life Technologies) and nonessential amino acids (ICN Pharmaceuticals, Costa Mesa, CA). The cell cultures were incubated at 37 C in a humidified atmosphere containing 5% CO2. Twenty-four hours later, the nonadherent cells (lymphocytes) were removed by aspiration of the culture supernatants and fresh medium was added to the bound cells.

Thyrocyte lysis assay
Confluent GD thyrocytes were incubated in the culture medium with and without 1 µM KI (Fluka, Buchs, Switzerland) and gradual doses of pooled normal human sera, heated for 30 min at 56 C to inactivate C1q (20), were added to provide complement. After 96 h, the thyrocyte lysis induced by the C4 binding to membrane TPO was determined by counting the dead cells in the culture supernatant using the Trypan blue exclusion method with a Malassez cytometer. After centrifuging, the preparation to remove the dead cells, the culture supernatants were kept frozen for subsequent Western blotting detection of the thyroid hormones bound to Tg (see below). To block the C4-induced cytolytic effect, similar experiments were performed using gradual doses of either a human C4 rabbit antibody (Sigma) or dimethyl sulfoxide (DMSO) mixed with the ED50 of the pool of sera in the cell culture.

RT-PCR
Total RNA was extracted from freshly prepared thyroid cell cultures using Trizol (Life Technologies) according to the manufacturer’s instructions. The concentration of the RNA was determined by measuring the OD at 260 nm of an aliquot of the final preparation. The integrity of the RNA was confirmed by electrophoresis. RT-PCR was performed using 0.5 µg of total RNA, the primers listed in Table 1Go (19, 21) and the Titan One Tube RT-PCR System (Roche Diagnostics) according to the manufacturer’s instructions. PCR products were analyzed by electrophoresis on 2% agarose gels.


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  		TABLE 1. 5' and 3' primers used for PCR

 
Immunofluorescence
GD cells grown on coverslips were used at 80% confluence. Cells were washed with PBS (pH 7.4) containing 0.9 mM CaCl2 and 0.45 mM MgCl2. For total staining, cells were fixed and permeabilized for 30 sec with cold acetone-methanol (1:1). For surface staining, cells were fixed for 30 min with 2% cold paraformaldehyde and quenched with 50 mM cold NH4Cl for 10 min. The following steps are common to the both procedures. For the C4 staining, cells were first incubated with pooled normal human sera at 1/5 dilution to provide C4, and then incubated with a human C4 rabbit antibody (Sigma) at 1/100 dilution. For the TPO staining, cells were incubated with a murine TPO monoclonal antibody, mAb 47 (22), at 1/500 dilution. Rhodamine-labeled antimouse (Immunotech, Marseille, France) and a fluorescein isothiocyanate-labeled antirabbit (Sigma) secondary antibody were used. Stained cells were mounted in Mowiol (Calbiochem) and viewed in an Olympus fluorescence microscope using a x50 oil immersion lens. Pictures were acquired with the Kodak DC290 Zoom Digital Camera (Eastman Kodak Co., New Haven, CT) equipped with the Kodak MDS290 software program (Eastman Kodak Company). The Photoshop software program (version 6; Adobe System Inc.) was used to edit and superimpose the images for colocalization viewing.

SDS-PAGE and Western blotting
To study the presence of C4 component in normal, GD, and HT thyroid cells, freshly prepared cell suspensions were incubated overnight in culture medium. The adherent cells were then washed with PBS and scraped into 1 ml of 1% Triton X-100 in PBS with a protease inhibitor cocktail (Sigma). The cells were lysed for 1 h on ice with vortex-mixing. The lysate was then centrifuged at 12,000 rpm for 15 min at 4 C. The supernatant was recovered and quantified by performing a microBCA assay (Pierce, Rockford, IL). Fifty micrograms of proteins from normal, GD and HT lysates were dissolved in 10 µl sample buffer [65 mM Tris-HCl buffer (pH 6.8), containing 2% sodium dodecyl sulfate, 10% glycerol, and 0.025% Bromophenol Blue] and electrophoresed under nonreducing conditions onto 10% acrylamide, 80 x 100 mm, 0.5 mm thick minigel using the Tricine SDS-PAGE method (23). Proteins were then directly electrotransferred onto a 0.45 µm Immobilon-P polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membrane was saturated with PBS (pH 7.4), containing 3% BSA. Western blotting experiments were then carried out by incubating the blotted membrane for 2 h with a human C4 rabbit antibody (Sigma) at 1/100 dilution. The membrane was then washed six times for 5 min in PBS. The second, antirabbit antibody labeled with horseradish peroxidase (Sigma) was incubated for 2 h at room temperature under shaking. After several additional washes, the blot was developed with 4-chloro 1-naphtol as the substrate.

To determine whether any thyroid hormones bound to Tg were released by the GD thyrocytes during the thyrocyte lysis assay described above, the culture media were centrifuged, the corresponding supernatants were freeze-dried in a Speed-Vac concentrator (Savant Instruments, Holbrook, NY) and dissolved with sample buffer. The samples were electrophoresed onto 8% acrylamide minigel as above. Proteins were transferred onto a polyvinylidene difluoride membrane. After saturation of the membrane, proteins were incubated for 2 h with T4 antibody labeled with horseradish peroxidase (Ortho-Clinical Diagnostics, Amersham, UK). After a washing step, the blot was developed as above.

ELISA
To study the ability of C4 to recognize TPO, wells of microtiter plates (Nunc, Roskilde, Denmark) were filled with PBS containing 300 ng of either Tg, the whole human TPO molecule, or the TPO recombinant peptides r-pep1 and r-pep2. Uncoated wells served as blanks. After being incubated overnight at 4 C in a humidified atmosphere, the wells were washed and saturated with BSA. They were then filled with 600 ng of either purified C4 (Sigma) or purified C4b (Calbiochem) in PBS, 1% BSA, and left to incubate for 1 h at 37 C. After a washing step, the C4 and C4b binding levels were both detected using a human C4 rabbit antibody (Sigma) at 1/50,000 dilution. C4 antibody binding was revealed using an antirabbit second antibody labeled with alkaline phosphatase (Sigma) and p-nitrophenyl phosphate as the appropriate substrate. The OD was read at 405 nm using a Vmax microtiter plate reader (Molecular Devices, Wokingham, UK).

To study the activation of the complement pathway, wells were coated with human TPO and saturated with BSA as above. They were then filled with serial dilutions of pooled normal human sera in HEPES-buffered saline [HBS, 20 mM HEPES, 150 mM NaCl (pH 7.4)] containing 0.15 mM CaCl2, 1 mM MgCl2 and 1% BSA and incubated for 1 h at 37 C. HBS was used instead of PBS in the subsequent steps. Any unbound material was then removed by extensive washing. Complement activation induced the formation of C3b, which was detected using a rabbit antihuman C3c antibody directed against a subunit of C3b (DAKOCytomation, Glostrup, Denmark) at 1/90,000 dilution. At such a dilution, C3c antibody was found to bind to C3b but not to C4b (not shown) and its binding was revealed as above.

Fenton oxidative reaction
Human TPO and C4 (1 µM) dissolved in 0.01 M Tris-HCl buffer (pH 7.2) were submitted to Fenton chemistry (24) for 2 h at 37 C using freshly prepared cocktails that contained 100 µM CuSO4, 0.5–3.5 mM H2O2 and 0.125–0.75 mM ascorbate. All the reactions were stopped by performing a freeze-drying step. The materials were then dissolved in the sample buffer for SDS-PAGE as above and loaded onto 8% acrylamide minigels. After running, the proteins from the gel were stained with G-250 Coomassie brillant blue.

Amino acid analysis
C4 were analyzed before and after the Fenton reaction for the presence of methionine sulfoxide on a Beckman (Fullerton, CA) 6300 analyzer, using the derivatization by the ninhydrin after amino acid separation by HPLC. The analyses were performed at the Institut de Biologie et Chimie des Protéines, Unité Mixte de Recherche 5086 Centre National de la Recherche Scientifique (Lyon, France).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
C4 is differentially expressed in normal and pathological thyroid tissues
The expression of C4 was investigated in several normal, GD, and HT primary thyrocyte cultures using RT-PCR, and the results were found to be consistently similar. A representative pattern is shown in Fig. 1Go. Total RNA was prepared from each primary culture and RT-PCR was performed with specific primers for C4 (Table 1Go). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was included as an internal standard to ensure that the RNA in the various samples were of comparable levels. Figure 1AGo shows that the levels of the RT-PCR products gradually increased between the RNA of normal, GD and HT thyrocytes, in that order. The expression of C4 in thyrocytes was confirmed at the protein level by Western blotting experiments with a C4 antibody (Fig. 1BGo). A similar band was detected at 200 kDa, corresponding to the whole C4 molecule in each tissue. The intensity of the band gradually increased between normal, GD and HT lysates. In addition, a 190- to 195-kDa band corresponding to the C4b-activated fragment of C4 and a 40- to 45-kDa band corresponding to the C4d subunit of C4b, were detected only in HT thyrocytes.



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  		FIG. 1. Expression of C4 by thyrocytes. A, RT-PCR analysis of C4 and GAPDH in normal (N) and autoimmune (GD and HT) thyroid cells. Markers (M, in bp) are shown on the left and the negative control without RNA (-) is shown on the right. B, Production of the C4 protein in N, GD, and HT thyrocytes. Cellular lysates from thyrocytes were analyzed to determine their C4 content by Western blotting performed with a specific human C4 antibody. Arrows show the intact C4 and its C4b and C4d fragments, respectively. Markers (M, in kDa) are shown on the left.

 
C4 colocalizes with TPO in GD thyrocytes
Immunofluorescence experiments (Fig. 2Go) were carried out in which C4 (green) and TPO (red) were stained in fixed thyrocytes. TSH-stimulated GD thyrocytes that overexpress C4 were used here. The labeling was limited to the cell-surface or extended to the intracellular compartment after permeabilization treatment. In both cases, the combined C4 and TPO staining patterns showed extensive colocalization of the two proteins (yellow). These results show that the secreted C4 bound to the membrane-attached TPO (surface labeling) and that these two proteins were produced by the thyrocytes (intracellular labeling).



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  		FIG. 2. Colocalization of C4 and TPO determined by immunofluorescence. A–D, Thyrocytes were fixed and permeabilized with methanol-acetone buffer and stained with a TPO mAb (in red) and a human C4 antibody (in green). A and C, Negative controls (not incubated with the primary antibody). E, Superimposed B and D panels. F–I, Thyrocytes were fixed with 2% paraformaldehyde (no permeabilization) and stained as above with a TPO mAb (in red) and a human C4 antibody (in green). F and H, Negative controls. J, Superimposed G and I panels.

 
C4 binds to the CCP module of TPO
To determine whether C4 specifically associated with the CCP module of TPO, the whole human TPO molecule and the TPO recombinant peptides r-pep1 and r-pep2, were tested in ELISA to measure the direct binding of purified whole C4 molecule and its C4b activated fragment. Tg is devoid of CCP modules and was used as a negative control. As expected, the results given in Fig. 3Go show that both C4 and, to a lesser extent, C4b bound to the TPO whole molecule and to the r-pep2 that contains the CCP module of TPO. C4 and C4b bound more strongly to r-pep2 than to TPO. Tg and r-pep1, which both are devoid of CCP modules, bound only weakly to C4 and C4b in similar proportions as the result of unspecific interactions.



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  		FIG. 3. Specific binding of C4 and C4b to the CCP module of TPO. Purified Tg, TPO and recombinant TPO peptides r-pep1 and r-pep2 were coated onto ELISA plates. Wells, after saturation with BSA, were incubated with purified C4 and C4b. The binding behavior of C4 and C4b was detected using a specific human C4 antibody and revealed with a second, species-specific, labeled antibody. The results given are those obtained after subtracting the blank value (uncoated wells), expressed as the OD at 405 nm. The values are the means ± SD of triplicates.

 
C4 activates the complement cascade
ELISA was used to detect the production level of C3b, the protein-bound activated fragment of C3, after incubating coated TPO with the pool of normal human sera to provide complement. C3b shows up downstream of C4 and C2 activation in the complement cascade and was taken here to signal complement activation. These results are shown in Fig. 4Go. A dose-response curve was obtained with the full pool of human sera, whereas background level of C3b production was observed with the C4-depleted pool of human sera.



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  		FIG. 4. Activation of complement pathway by TPO. Purified TPO was coated onto ELISA plates. After being saturated with BSA, the wells were incubated with serial dilutions of full and C4-depleted pooled normal human sera. The activation of the complement cascade was monitored by checking C3b, which appears downstream of C4 and C2 in the complement pathway. The results given are those obtained after subtracting the blank value (uncoated wells) and expressed as the OD at 405 nm. The values are the means ± SD of triplicates.

 
C4-mediated lysis of thyrocytes
To test whether C4 binding to TPO may activate the complement cascade in vivo, we used GD thyrocyte cultures expressing functional TPO at the cell-surface in a cytolytic test. Thyrocytes were grown 96 h under culture conditions favoring thyroid hormone synthesis. Tg production and iodine organification by thyrocytes were monitored by testing T4 bound to Tg that was released into the culture supernatant in Western blotting experiments (Fig. 5AGo). The results of the cytolytic test (Fig. 5BGo) show that the number of dead cells increased with the dose of the pool of sera used. The cytolytic effect was reversed in a dose-dependent manner by coincubating as above the ED50 of the pool of sera with gradual doses of C4 antibody (Fig. 5CGo). Control experiments carried out on similar lines, omitting iodine from the culture medium to avoid hormone synthesis resulted in the absence of T4 bound to Tg in the culture supernatant (Fig. 5AGo) and the loss of cytotoxicity (Fig. 5BGo).



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  		FIG. 5. Complement-mediated lysis of human thyrocytes. A, Expression of T4 bound to Tg in the thyrocyte culture. Supernatants from 5 thyrocyte cultures prepared with and without KI were analyzed by performing SDS-PAGE and Western blotting experiments for the presence of T4 bound to Tg. A representative result is shown. B, Thyrocyte lysis as a function of the dose of human serum as source of complement. Thyrocytes were incubated with various doses of pooled normal human sera with and without KI. The dead cells present in the culture supernatants were counted using the Trypan blue-exclusion method. Results are expressed as the percentage of the maximum number of dead cells (100% ranged from 2 to 3 x 104 dead cells) after 96 h of culture. Results from 5 cultures are shown. C, Reversal of the cytolytic effect induced by a human C4 antibody. The ED50 of the pool of human sera was mixed with gradual doses of a C4 antibody in the thyrocyte culture. Results are expressed as above (100% ranged from 1 to 1.5 x 104 dead cells) (mean ± SD, n = 5).

 
Role of ROS in complement activation
Because thyroid hormone synthesis is an oxidative process which generates ROS, we looked at the role of ROS on the TPO and C4 at the molecular level. ROS were generated by Fenton chemistry and were found to aggregate TPO but not C4. Maximum TPO aggregation, as assessed by densitometric measurement (not shown), was reached using 100 µM CuSO4, 1 mM H2O2, and 0.25 mM ascorbate (Fig. 6AGo). Persistence of TPO aggregates in SDS-PAGE under reducing conditions suggests that dityrosine bonds were formed during the oxidative reaction (not shown). On the other hand, methionine residues of proteins are particularly sensitive to oxidation by ROS. Effectively, the amino acid analysis of C4 before and after the oxidative conditions described above for TPO showed that ROS oxidized methionines of C4. The calibration procedure used here for the amino acid analysis showed that oxidized methionine eluted in front position from the HPLC column, and we found that 52.60% of C4 methionines were changed to oxidized methionines after Fenton reaction (Table 2Go). To link the putative role of ROS in activating complement and the lysis of hormone-producing thyrocytes, we used DMSO, a specific hydroxyl radical scavenger. Interestingly, we found that DMSO inhibited in a dose-dependent manner the C4-mediated lysis of thyrocytes (Fig. 6BGo).



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  		FIG. 6. Role of ROS in complement activation. A, SDS-PAGE patterns of TPO and C4 in various oxidative conditions stained with G-250 Coomassie brillant blue. M, Markers of molecular weight. From left to right: 1 µM TPO alone (lane 1), TPO + 100 µM CuSO4 + 0.5 mM H2O2 + 0.125 mM ascorbate (lane 2), TPO + CuSO4 + 1 mM H2O2 + 0.25 mM ascorbate (lane 3), TPO + CuSO4 + 2 mM H2O2 + 0.45 mM ascorbate (lane 4), TPO + CuSO4 + 3.5 mM H2O2 + 0.75 mM ascorbate (lane 5), 1 µM C4 alone (lane 6), in lanes 7–10, are C4 plus CuSO4 and ascorbate as in lanes 2–5. B, Influence of DMSO on ROS-mediated thyrocyte lysis by complement. The ED50 of the pool of human sera was mixed with gradual doses of DMSO in the thyrocyte culture. Results are expressed as in Fig. 5Go (100% ranged from 1 to 1.5 x 104 dead cells) (mean ± SD, n = 5).

 

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  		TABLE 2. Amino acid analysis of native and oxidized C4

 
Differential levels of C2–C9 expression in various thyrocytes
Starting with C4 activation, the final cytolytic events in the complement pathway require the local presence of all the downstream components of the cascade in the thyroid. Primers designed for all the mRNA of the complement components of the classical pathway except C1 (C1q, C1r, and C1s) were used (Table 1Go). Figure 7Go shows the pattern of complement gene expression in normal, GD and HT thyrocyte cell cultures. Normal thyrocytes slightly expressed all but C7, C8 mRNA and GD thyrocytes overexpressed the normal thyrocytic pattern of complement components, whereas HT thyrocytes overexpressed all the mRNA tested. Complement components expression is probably underestimated in HT thyrocytes considering the low expression of the corresponding GAPDH internal control.



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  		FIG. 7. RT-PCR analysis of C2–C9 and GAPDH. RT-PCR procedures were performed with the total RNA extracted from N, GD, and HT thyrocytes. See Fig. 1Go for details.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present data show for the first time that complement can be activated by direct C4 binding to the CCP-like module of TPO. C4 perfectly colocalized with TPO on thyrocytes and specifically bound to the CCP-like module of TPO. Giving a strong evidence for the C4-mediated activation of complement via TPO, the full but not C4-depleted pool of normal human sera led to C3b binding to coated TPO. Furthermore, the lysis of functional thyrocytes induced by the pool of human sera was reversed by complexing C4 with a specific antibody. Because the pool of sera used as source of complement throughout this study was negative for TPO autoantibodies, the results obtained here indicated that TPO specifically activated the classical complement pathway without any mediation by Ig. This finding is reminiscent of other antibody-independent pathways, whereby complement is activated by the direct C1q and/or C3 binding to the ß-amyloid peptides in Alzheimer’s disease, a neurodegenerative disease (25). The common denominator between AITD and neurodegenerative diseases is the inflammatory process that is signaled here by the presence in lysate of tissue from HT patients of membrane-bound C4b and its C4d subunit, a marker of acute inflammation (25, 26) arising from inactivation of C4b by factor I cleavage. In both diseases, the inflammation depends on the local synthesis of various ROS, cytokines and factors including complement components. Immunocompetent cells invading the target tissue after injury secrete inflammatory mediators. Alternatively, pathological tissue itself can release some of these mediators under the influence of genetic, hormonal and environmental factors (27). We established here that whereas C4 is expressed in normal thyrocytes, it is overexpress together with all the downstream complement components in HT. These results convincingly argued for a direct C4-mediated activation of complement through TPO in patients with AITD as reported here ex vivo on human thyrocytes in stimulating culture medium.

ROS are produced during thyroid hormone synthesis, an oxidative process that requires iodine and an H2O2-generating system (28). ROS have been reported to cleave (4) or aggregate (29) Tg in the thyroid. The oxidative reactions were expected to alter TPO that is ROS-exposed to the same extent as Tg at the thyroid cell-surface. Effectively, we found here that TPO aggregated under ROS production probably by forming dityrosine bridges as reported to occur in ß-amyloid under exposure to peroxidase and H2O2 (30). This phenomenon is likely to increase the C4 affinity for TPO by multiplying the CCP modules—as observed here using r-pep2 which is smaller than TPO (20 vs. 110 kDa) for coating in ELISA—because C4 activation via the classical pathway involves substrate recognition sites located in both of the CCP modules of C1s (14). In addition, ROS were previously found to activate C5 by oxidizing methionine residues without requiring any proteolytic cleavage (31, 32). Here, we found that 52.60% of C4 methionine residues were oxidized by ROS. Because C3, C4, and C5 all belong to the {alpha}-2 macroglobulin protein family, C4 may therefore be ROS-activated like C5 in thyroid tissue without requiring any mediation by the C1r, C1s serine proteases linked to the C1q-TPO autoantibody complexes. In agreement with the putative role of ROS and specially hydroxyl radicals in the process of complement activation, we observed firstly that GD thyrocyte cultures prepared in stimulating medium without iodide did not trigger ROS-related hormone synthesis and were therefore not lysed by complement, and secondly that DMSO, one of the most specific hydroxyl radical scavengers (33), inhibited the lysis of GD thyrocytes. Unfortunately, the scarcity of HT tissue precluded performing a comparative study between efficiency of HT and the more available GD thyrocytes for complement cytolysis.

During HT development, complement can be activated both by the classical antibody-dependent pathway (6) and the TPO pathway which proceeds from C4 binding to TPO and C4 oxidative activation mediated by ROS as described here. The liver is the main organ responsible for synthesizing and secreting C4 into the circulation, but many extrahepatic sites producing moderate quantities of C4 have also been described (34, 35). The tissue expression of C4 is inducible or enhanced by interferon-{gamma} (36) that is produced by immunocompetent cells invading thyrocytes at inflammatory sites (37, 38, 39). Normal thyrocytes were found here to produce relatively low levels of C4, whereas higher levels of C4 were detected in autoimmune thyrocytes. This last feature adds to our picture of degenerative diseases involving the production of ROS, cytokines, and complement components. Complement-mediated injury is regulated by many factors; among these CD59 has been identified as a widely distributed glycoprotein that inhibits membrane C5b-9 (terminal complement component) formation. Increased expression of thyrocytes membrane-anchored CD59 was found in patients with AITD (40). Impairment of CD59 expression by some unknown mechanisms may facilitate the activation of complement pathways and increase the risk for destructive thyroiditis. Our present findings support the idea that etiological mechanisms involved in HT could not be so very different from those involved in other degenerative diseases. In the past, inflammation has been involved as both cause and aggravating effect in various diseases and it was observed that complement connects the innate inflammatory response with the adaptive immunity by promoting T-cell responses in viral infection (41). Moreover, in the thyroid gland, the ROS-induced inflammation process is likely to produce interferon-{gamma} that constrains thyrocytes to act as professional antigen-presenting cells and initiate the autoimmune process (42). It is unclear whether thyrocytes act in disease initiation or progression due in part to divergent data reported on the B7 costimulatory molecules expression by thyrocytes (43, 44, 45). Without looking for an argument, thyrocytes by themselves obviously play a major role in AITD interacting with immunologically active molecules (40). Hence, the sequence of events that contribute to cell failure may be similar in both autoimmune and degenerative pathologies. These events may start with an increase in ROS production and the resulting inflammatory and protein modification processes, leading to the occurrence of complement components acting on aggregated molecules. Accordingly, amyloid aggregates within the islet of the pancreas have been observed in type 2 (insulin resistant) diabetic patients that are either of cryptic type 1 (autoimmune diabetes) or evolve with time to type 1 (46). Applied to the thyroid model, it is possible that TPO aggregates may form in the thyroid during overt or silent GD, transforming the pathological process into degenerative HT.

These studies have been performed in vitro or ex vivo on human materials giving us the opportunity to evaluate separately various agents potentially involved in thyrocyte lysis during HT. However, it must be kept in mind that the physiopathological scenario we proposed for HT remains to be demonstrated in vivo conditions where the thyroid gland is under the influence of a multitude of factors. Further investigations on the complement cascade activation via C4 binding to TPO and specially on the role of ROS in such an in vivo activation are now required to prove our hypothesis conclusively and to elucidate the inflammatory process leading to destructive HT, which represents the archetype for other degenerative diseases.


   Acknowledgments
 
We thank Prof. J. F. Henry and Dr. C. De Micco for providing us with the thyroid specimens. We also thank Dr. P. J. Lejeune for providing us with the human sera.


   Footnotes
 
S.B. and V.E. are recipients of grants from the Association pour le Développement des Recherches Biologiques et Médicales and the Fondation pour la Recherche Médicale.

Abbreviations: AITD, Autoimmune thyroid diseases; CCP, complement control protein; DMSO, dimethyl sulfoxide; EGF, epidermal growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GD, Graves’ disease; HT, Hashimoto’s thyroiditis; ROS, reactive oxygen species; Tg, thyroglobulin; TPO, thyroperoxidase.

Received July 22, 2003.

Accepted for publication August 18, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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