HLA-DM and invariant chain are expressed by thyroid follicular cells, enabling the expression of compact DR molecules

Marta Catálfamo1, Laurence Serradell2, Carme Roura-Mir1, Edgardo Kolkowski1, Mireia Sospedra1, Marta Vives-Pi1, Francesca Vargas-Nieto1, Ricardo Pujol-Borrell1 and Dolores Jaraquemada1,2

1 Unitat d'Immunologia, Hospital Universitari Germans Trias i Pujol,
2 Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain

Correspondence to: D. Jaraquemada, Unitat d'Immunologia, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, Carretera de Canyet s/n, 08916 Badalona, Spain


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Thyroid follicular cells (TFC) in Graves' disease (GD) hyperexpress HLA class I and express ectopic HLA class II molecules, probably as a consequence of cytokines produced by infiltratingT cells. This finding led us to postulate that TFC could act as antigen-presenting cells, and in this way be responsible for the induction and/or maintenance of the in situ autoimmune T cell response. Invariant chain (Ii) and HLA-DM molecules are implicated in the antigen processing and presentation by HLA class II molecules. We have investigated the expression of these molecules by TFC from GD glands. The results demonstrate that class II+ TFC from GD patients also express Ii and HLA-DM, and this expression is increased after IFN-{gamma} stimulation. The level of HLA-DM expression by TFC was low but sufficient to catalyze peptide loading into the HLA class II molecules and form stable HLA class II-peptide complexes expressed at the surface of TFC. These results have implications for the understanding of the possible role of HLA class II+ TFC in thyroid autoimmune disease.

Keywords: class II molecules, peptides, thyroid autoimmunity, thyroid follicular cell


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Human autoimmune thyroid diseases (AITD), i.e. Hashimoto's thyroiditis, Graves' disease and primary myxedema, are characterized by an intense immune response to well characterized thyroid autoantigens such as thyroglobulin (Tg), thyroid peroxidase (TPO) and the thyrotropin receptor (TSH-R). This response includes high-affinity antibodies, some of which are produced in situ (1), against all these antigens and is maintained for long periods of time. Th lymphocytes specific for these antigens are therefore expected to direct and possibly modulate this response. The recognition of thyroid follicular cells by the thyroid-specific T lymphocytes is considered central to AITD pathogenesis (2,3).

TFC express class II molecules in glands affected by AITD (2,4). They also overexpress class I molecules as well as associated molecules such TAP (5), LMP-2 and LMP-7 (6). This phenomenon has led us to postulate that presentation of autoantigens may play a key role in either breaking the tolerance to peripheral autoantigens or in the perpetuation of the autoimmune response. Expression of HLA class II by itself is not sufficient for efficient antigen presentation. The nature and functionality of class II molecules expressed by TFC depends on the simultaneous expression of associated molecules such as Ii and HLA-DM.

Class II {alpha} and ß chains assemble in the endoplasmic reticulum (ER) forming large complexes with the invariant chain (Ii-{alpha}-ß)3 which are transported to endo-lysosomal compartments by signals in the Ii cytoplasmic domain. Ii participates in the assembly of {alpha}ß complexes in the ER, occupying the peptide binding site of the {alpha}ß dimer with a region spanning residues 81–104 of Ii, which interacts with MHC in the same way as a peptide, thus preventing the binding of free peptides in the ER (7,8). This region corresponds to class-II-associated invariant chain peptide (CLIP) (9). Class II–Ii trimers are transported to the secretory route and targeted to the endocytic pathway (10) where Ii chain is cleaved, leaving CLIP bound to the class II molecule peptide-binding site. CLIP peptides are removed by immunogenic or autologous peptides generated by endosomal processing, thus forming mature {alpha}ß peptide stable complexes which are transported to the cell surface. In these endo-lysosomal compartments, HLA-DM molecules act as the main catalyzer in the release of CLIP peptides and binding of other peptides to class II molecules (11). Cells lacking DM express class II molecules in their surface, mostly loaded with Ii-derived CLIP peptides (12).

DM molecules are MHC-encoded membrane glycoprotein heterodimers formed by two subunits DM{alpha} (33–35 kDa) and DMß (30–31 kDa), with structural homology with both class I and class II molecules (13). The cellular distribution of DM is similar to class II molecules and their expression is also induced by IFN-{gamma}. DM molecules are expressed at low levels in professional APC, are not expressed at the cell membrane and have a relatively long half-life (14).

The capacity of DM to release CLIP and form stable complexes has been reproduced in vitro (1518). DM kinetics as an enzyme catalyzing CLIP release from class II molecules are of three to 12 class II molecules per minute and per DM molecule using MHC-CLIP complexes as a preferential substrate for DM rather than stable MHC-peptide complexes (19). DM molecules also act as a chaperone for {alpha} and ß complexes preventing their aggregation at low pH. DM binds to empty {alpha}ß molecules from where CLIP has been removed and stabilizes them until they bind peptide (20). Specific signals in the cytoplasmic domain target DM molecules to compartments of the endocytic pathway including early and late endosomes as well as the MHC class II compartment (MIIC) (21,22). Finally, DM also function as a peptide editor, preventing binding of low-affinity peptides and favoring the binding of high-affinity peptides, thus helping the formation of mature complexes (23). Recently, a new molecule, HLA-DO, also codified within the MHC class II region has been described associated to DM, which has the capacity to modulate DM function (24,25).

The presence or absence of Ii and DM in class II-expressing TFC is very relevant since it will determine the access of autologous peptides to the class II molecules. In the absence or suboptimal amounts of Ii, the majority of class II molecules at the surface would be expected to be unstable and bound to unselected peptides. If DM was absent, a majority of peptides would be CLIP variants. On the other hand, if both DM and Ii are expressed in sufficient amounts, the array of peptides presented by class II molecules would be varied and probably from tissue-specific molecules, some of which could be important in the maintenance of the autoimmunity. We have studied the Ii and DM expression by class II-expressing TFC from GD glands.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Patients
Thyroid tissue samples were obtained at surgery from seven patients with GD, i.e. TB212 (73%), TB242 (0%), TB250 (46%), TB255 (42%), TB258 (40%), TB260 (45%) and TB378 (49%), expressing different levels of class II molecules (in brackets) and one with multinodular goitre (MNG) (TB359), class II. Clinical diagnosis was made on the basis of the usual thyroid function tests and were confirmed by histopathology. The protocol had been approved by the ethical committee of the HUGTiP.

Cell lines
M1 (26) is a human fibroblast cell line. TEB158 is an Epstein–Barr virus-transformed lymphoblastoid B cell line (LCL) from one thyroid donor, which was used as control in all experiments. All cell lines were maintained in standard culture conditions in culture medium RPMI + 10% FCS.

TFC suspensions
Samples of thyroid tissue obtained at surgery were digested as described (27). Cell suspensions were cultured overnight in RPMI 1640 culture medium supplemented with 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin and 10% heat-inactivated FCS at 37°C/5% CO2 in tissue culture flasks. Non-adherent mononuclear cells were collected and cryopreserved. The adherent cell population (TFC) was harvested, extensively washed and stored frozen or directly used. For the induction of HLA class II expression in TFC and M1 cells, cultures were exposed to 500 U/ml IFN-{gamma}. After 24 or 48 h, cells were pelleted for total RNA extraction or stained and analyzed by flow cytometry. Recombinant human IFN-{gamma} was a kind gift from G. R. Adolf (Boehringer Institute, Vienna, Austria).

Antibodies
The following mAb were used: MIC-18 (mAb anti-TPO, from P. Carayon, University of Marseille, France), EDU-1 (specific for a monomorphic HLA-class II determinant, from R. Vilella, Hospital Clínic, Barcelona, Spain), DA6.147 (28) (also specific for a monomorphic HLA-class II determinant, a gift from E. O. Long, NIAID, Rockville, MD), VIC-Y1 (29) (anti-human Ii chain cytoplasmic domain, from W. Knapp, Vienna, Austria), R-DMB/C (30) (a rabbit anti-DMß serum, from P. Jensen, Emory University School of Medicine, Atlanta, GA), MaP.DMB/C (31) (specific for a cytoplasmic tail peptide of DMß chain) and CerCLIP.1 (15) (specific for CLIP-associated class II molecules) (both from P. Cresswell, Yale University School of Medicine, New Haven, CT). Human serum with high-titer TPO antibodies was also used for immunofluorescence staining of cryostat sections.

Immunofluorescence staining
Series of 5 µm cryostat sections from a class II+ GD gland (TB212) were stained by double-indirect immunofluorescence (32) combining a human anti-TPO serum (a patient's serum with high-titer TPO antibodies, used at 1:10,000) with either anti-class II (EDU-1), anti-Ii (VIC-Y1) mAb or a rabbit anti-HLA-DMß serum (R-DMB/C), using TRITC-conjugated goat anti-human Ig and FITC-conjugated goat anti-mouse IgG or goat anti-rabbit Ig, as second reagents (all from Southern Biotechnology, Birmingham, AL). For double class II/DM staining, EDU-1 and R-DMB/C were used with TRITC–goat anti-mouse IgG and FITC–goat anti-rabbit Ig respectively as secondary antibodies. To check possible cross-reactions among the antibodies and non-specific binding to the tissue, sections were stained following the above protocols but replacing the primary antibody by normal mouse serum or replacing the rabbit anti-R-DMB/C antiserum by normal rabbit serum and by omitting each of the layers in turn. To block non-specific binding, 1% of BSA was added to the PBS used to dilute antibodies. Between incubations (30 min for each layer), preparations were washed in PBS.

Flow cytometry
LCL and overnight cultured TFC were fixed and permeabilized before staining with anti-TPO, anti-class II, anti-Ii and anti-DM antibodies (33), and the same was done after 48 h culture in the presence of IFN-{gamma}. Live cells were used for CerCLIP.1 staining, and surface class II and TPO controls. Antibody binding was detected with GAM–FITC. Samples of 10,000 cells were analyzed on a FACScan using the Lysys II software (both from Becton Dickinson, San Jose, CA).

Northern blot hybridization
Total RNA was isolated using the guanidine thiocyanate method (34). Samples (10 µg) were separated by electrophoresis on formaldehyde agarose gels and transferred overnight to a Hybond N+ membrane (Amersham, Buckinghamshire, UK) using 20xSSC as transfer buffer. Hybridization was performed with 2x106 c.p.m./ml of an {alpha}-32P-labeled 0.8 kb DMA probe (from P. Cresswell), at high stringency (68°C) in solution containing 7% SDS, 0.25 M Na2HPO4, 1% BSA and 1 mM EDTA. After 20 h hybridization, the membrane was washed in 20 mM phosphate buffer, 1 mM EDTA and 1% SDS at 68°C, and exposed at –70°C. Hybridization with a 1.2 kb Ii probe and with a 1.3 kb HLA-DR{alpha} (both from E. Long, NIAID, Rockville, MD) was performed using the same conditions. Membranes were subjected to autoradiography.

Western blots
Cells at a concentration of 5x107 cells/ml were lysed in lysis buffer (0.01 M Tris, pH 7.4, 0.15 M NaCl, 1% Nonidet P40, 5 mM EDTA, 10 mM iodoacetamide, 1 mM PMSF, 2 µM pepstatine, 5 µM aprotinin and 5 µM leupeptin), and aliquots corresponding to 1.5x106 and 0.75x106 cells were heated for 4 min at 95°C, loaded onto a 12% acrylamide gel and electrophoresed in SDS–PAGE. Proteins were transferred to nitrocellulose. Blots were blocked for 30 min in Blotto (TBS, 0.05% Tween 20 and 5% skimmed milk), incubated for 2 h with a rabbit anti-DMß serum (30), in TBSN/milk. After three (10 min) washes in TBSN, blots were incubated for 1 h with an anti-mouse IgG conjugated to horseradish peroxidase in TBSN and the bands developed using the enhanced chemiluminescence ECL kit (Amersham). The bands were analyzed by densitometry using a Scanjet IIc scanner (Hewlett Packard) connected to Power Macintosh 4400/200 and the Scan Analysis program (Biosoft, Cambridge, UK). Ratios between densitometry results for DM and DR from 1.5x106 and 7x105 cell samples were calculated.

Immunoprecipitation
Cells (107) were washed twice with cold PBS and labeled following the standard protocol with 0.5 mCi Na125I (Amersham) using lactoperoxidase/H2O2 and washed again 3 times in cold PBS. Radiolabeled cells were lysed at 107 cells/ml in lysis buffer containing 2% NP-40, 6 mM CHAPS, 50 mM Tris–HCl, pH 8, 150 mM NaCl, 5 mM EDTA, 1 mM PMSF, 10 mM iodoacetamide, 5 µM aprotinin, 5 µM leupeptin and 2 µM pepstatin. After 30 min incubation at 4°C, cell lysates were cleared of nuclei and debris by centrifugation at 14,000 g for 5 min at 4°C. Lysates were precleared with an irrelevant antibody, normal rabbit serum and Protein A–Sepharose beads (Pharmacia), and mixed with the relevant antibody overnight at 4°C. Antigen–antibody complexes were isolated with 10 µl rabbit anti-mouse Ig bound-Protein A–sepharose beads. After one wash with lysis buffer and four with buffer containing 6 mM CHAPS, 50 mM Tris, pH 8, 150 mM NaCl and 5 mM EDTA, samples were resuspended in 100 µl non-reducing sample buffer and left for 1 h at room temperature in order to establish complex stability. One-half of the sample, boiled at 100°C for 3 min (B), and the other half (NB) were electrophoresed on SDS–12% polyacrylamide gels. Gels were dried and subjected to autoradiography.


    Results
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 References
 
TFC from GD glands express Ii and HLA-DM
Expression of HLA-DR, Ii and HLA-DM in TFC was studied by indirect immunofluorescence in thyroid tissue sections from GD patients, using anti-TPO antibodies in parallel to define TFC in the sections (Fig. 1b, d and fGo, labeled red). As seen in Fig. 1Go, all three molecules were expressed with different intensities by TPO+ cells (Fig. 1a, c and eGo, labeled green) from a class II+ GD thyroid gland, TB212. Class II staining (Fig. 1aGo) was visible on the apical pole of TFC lining the coloid space of the follicles as well as in some infiltrating cells (possibly B lymphocytes, dendritic cells, macrophages and activated T cells). Ii was detected in the cytoplasm of TPO+ TFC around follicles and also in infiltrating cells (Fig. 1cGo). Ii was distributed all over the cytoplasm of TFC, in contrast with HLA-DM (Fig. 1eGo), which was detected in TPO+ TFC showing a distribution around the nuclei and near the basal pole. DM staining was also observed in infiltrating mononuclear cells. DM+ TFC (Fig. 1gGo) were also class II+ (Fig. 1hGo). Three thyroid glands (TB212, TB250 and TB255) expressing class II in the TFC were analyzed for the expression of DR, Ii and DM, and showed very similar patterns in all samples (not shown). Control staining with normal mouse, normal rabbit and normal human sera were negative in all cases. Anti-CD3 mAb only stained interstitial infiltrating cells (data not shown).



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Fig. 1. TFC from GD glands express HLA-DM and Ii. Cryostat sections of double staining of TPO (b, d and f, red), HLA class II (a, green; h, red), Ii (c, green) and HLA-DM (e and g, green) of class II+ gland TB212.

 
HLA-DM and Ii message are detected in unstimulated GD TFC by Northern blots and can be induced by stimulation with IFN-{gamma}
Total RNA was extracted from different TFC samples, separated from the infiltrating population by overnight culture and processed (unstimulated) or cultured for 48 h in the presence of 500 U/ml human rIFN-{gamma} and processed (stimulated). Expression of HLA-DR, Ii and HLA-DM was detected by hybridization with specific probes, and results from one experiment are shown in Fig. 2Go. Total RNA from a LCL (TEB158) was used as a positive control, and RNA extracts from a human fibroblast cell line (M1) were used as a negative control for basal expression and positive for IFN-{gamma} induction. Basal expression of Ii and DR was detected in class II+ GD TFC (TB378, TB250 and TB260) but not in a class II multinodular TFC sample (TB359). Basal expression of HLA-DM was also detectable in all three samples, although the intensity of the signal was much lower (see Fig. 2Go) than for HLA-DR and Ii. In all cases, culture in the presence of IFN-{gamma} greatly increased expression of DM, Ii and DR message in positive samples, and induced their expression in negative samples. The induction of DM and Ii message was as efficient as the induction of DR expression and went parallel to the induction of all three genes in M1 cells. A total of seven other GD samples (TB212, 228, 242, 255, 258, 269 and 270) were tested in separate experiments showing the same pattern: very low basal expression of DM in class II+ samples, no signal in class II samples and a strong increase of message expression upon IFN-{gamma} stimulation (data not shown).



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Fig. 2. HLA-DM message is detected in unstimulated GD TFC by Northern blotting and can be induced by stimulation with IFN-{gamma}. Total RNA samples from HLA class II+ (TB378, TB250 and TB260) GD glands and HLA class II (TB359) MNG gland TFC were hybridized with HLA-DR{alpha}, Ii and DMA probes.

 
The level of HLA-DM expression in the cytoplasm of TFC is very low
Cytoplasmic expression of HLA-DM, Ii and DR molecules was also studied in overnight cultured and IFN-{gamma}-treated TFC by flow cytometry. Figure 3AGo show the cytoplasmic expression of TPO, class II, Ii and HLA-DM by unstimulated (–) and stimulated (st) class II+ TFC (TB255). HLA-DM expression in unstimulated cells was very low and there was hardly any detectable increase after stimulation, whereas cytoplasmic DR and Ii expression were higher without stimulation, and their increase after IFN-{gamma} treatment was clearly detectable. The DM data contrast with the message data shown in Fig. 2Go, since the DM message expression was efficiently induced in all cells tested. The expression of DM in stimulated cultured TFC was indeed visible by immunofluorescence in the positive cells (stimulated TB255, ~10% DM+ cells), within the cytoplasm, in vesicles near the plasma membrane (Fig. 3BGo). Basal expression of HLA-DM by unstimulated TB255 cultured cells was visible but very low (not shown).




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Fig. 3. Expression of HLA-DM in class II+ TFC is low but detectable by immunofluorescence. (A) Cytoplasmic staining of HLA-DR, Ii and HLA-DM in basal (–) and stimulated (st) conditions in GD TFC (TB255) and LCL, detected by flow cytometry. (B) Double-immunofluorescence staining of HLA-DM (right panels) and TPO (left panels) of IFN-{gamma}-stimulated TFC (TB255).

 
DR protein induction by IFN-{gamma} is more efficient than the induction of HLA-DM protein in TFC
Low expression levels of DM in TFC could be related to the relatively low level of class II expression by these cells, compared to LCL. To compare the level of DR and DM proteins before and after IFN-{gamma} treatment, Western blots were performed on TFC, stimulated TFC and LCL samples, the bands analyzed by densitometry, and the relative ratio DM to DR calculated. Figure 4Go show the expression of both proteins by all samples and their calculated relative ratio for the 1:1 dilution. The data show that HLA-DM is expressed by class II+ TFC (45% DR+ TB260) at a lower level relative to DR than in LCL (DM:DR ratio 0.52 and 0.33 respectively). In addition, an increase of DM expression after IFN-{gamma} treatment was clearly visible by Western blot analysis, although the DM:DR ratio in stimulated cells was lower (0.18). This indicates that DR induction by IFN-{gamma} is more efficient than DM induction, explaining the failure to detect a clear increase of DM protein expression in stimulated TFC by flow cytometry.



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Fig. 4. HLA-DM induction by IFN-{gamma} is less efficient than induction of HLA-DR in TFC. Western blot analysis of HLA-DM and HLA-DR{alpha} corresponding to 1.5x106 (1:1) and 0.7x106 (1:2) of unstimulated (TB260) and stimulated (TB260 IFN-{gamma}) GD TFC and LCL as control of expression. The DM:DR ratio was estimated by densitometric analysis.

 
Immunoprecipitation of 125I-labeled HLA-DR molecules expressed by TFC demonstrates the expression of `compact' SDS-resistant surface HLA-DR complexes
Mature, peptide-bound HLA class II complexes are mostly resistant to SDS denaturation at room temperature and migrate as dimers in SDS–PAGE (`compact' forms) (35). Since TFC express Ii and DM, transport of DR complexes to the endo-lysosomal compartments would be secured by Ii and loading of peptides produced in the endo-lysosomes would be facilitated by DM molecules, capable of displacing CLIP peptides. However, levels of DM were relatively low so, only if these levels were not suboptimal, DR molecules expressed by TFC should therefore be mature, SDS-resistant, peptide-bound DR dimers. To characterize the conformational characteristics of class II molecules expressed by TFC, surface (125I-labeled) DR complexes were immunoprecipitated with a DR{alpha}-specific mAb (DA6.147) from a detergent-solubilized total cell extract of IFN-{gamma}-stimulated TFC (TB258). Control samples were LCL extracts. Samples were incubated in non-reducing SDS sample buffer and either boiled or kept at room temperature prior to their analysis by SDS–PAGE. Results shown in Fig. 5Go demonstrate that most DR molecules expressed by TFC are SDS-resistant `compact' forms ({alpha}ß), which dissociate into {alpha} and ß monomers when boiled, releasing an increasing number of low mol. wt peptides (smear at the bottom of each lane). This suggests that despite low levels, DM molecules expressed by TFC are sufficient and fully functional.



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Fig. 5 . Immunoprecipitation of 125I-labeled HLA-DR molecules expressed by TFC demonstrates the expression of `compact' SDS-resistant surface complexes. Peptides bound to these molecules are heterogeneous. Immunoprecipitation of HLA-DR–peptide complexes with mAb (DA6.147) and analysis by SDS–PAGE in non-reducing conditions with (B) or without (NB) treatment at 95°C. The figure shows {alpha}ß complexes (NB) and {alpha} and ß chains dissociation after treatment (B).

 
Functionality of DM molecules is confirmed by the absence of CLIP-associated DR molecules on the surface of TFC
mAb CerCLIP.1 recognizes CLIP peptides associated to DR molecules. Expression of such complexes at the surface of the cells would imply that peptide exchange in the endosomal compartments was incomplete. That is the case of LCL, where the rate of class II expression is extremely high and there are always some molecules associated with CLIP which are transported to the cell surface. We have compared surface staining of class II and CerCLIP.1 in LCL and class II+ and class II TFC, using surface TPO as a positive control for TFC. As seen in Fig. 6Go, CerCLIP.1 was mostly negative in class II+ (TB255 and TB258) and class II (TB242) TFC samples compared to the positivity in LCL, showing CLIP-class II complexes in LCL but no such complexes in class II+ TFC. These data confirm the immunoprecipitation data showing that most class II molecules expressed by TFC are compact, i.e. associated to stabilizing peptides, again suggesting a very high efficiency of DM in the removal of CLIP peptides from class II molecules in TFC.



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Fig. 6. CLIP-associated class II molecules are nearly absent from the surface of TFC, confirming that TFC class II are compact molecules. CerCLIP.1 mAb-specific HLA class II-CLIP complexes was used for surface staining of HLA class II+ (TB255 and TB258) and HLA class II-(TB242) GD TFC and LCL. The figure also shows the surface staining of HLA class II (EDU-1) and TPO (MIC-18). HLA-DM expression by thyroid follicular cells HLA-DM expression by thyroid follicular cells HLA-DM expression by thyroid follicular cells HLA-DM expression by thyroid follicular cells HLA-DM expression by thyroid follicular cells HLA-DM expression by thyroid follicular cells

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
TFC in autoimmunity express class II molecules (4,36), presumably due to the effect of cytokines synthesized in situ by inflammatory cells and T lymphocytes (5,37). The role of these class II molecules is not clear, although it has been demonstrated that they can present exogenous peptides (38) and it has been postulated that they may be involved in the induction and/or maintenance of the in situ autoimmune T cell response (2). However, since TFC do not express co-stimulatory molecules even upon IFN-{gamma} treatment the function of these ectopic class II molecules remains unknown. The data presented in this report demonstrate that they are structurally capable of binding endogenous peptides and therefore to present them. We have been able to demonstrate the expression of Ii and DM molecules by TFC from autoimmune glands and their induction by IFN-{gamma} treatment in vitro. Expression of Ii was high enough to ensure efficient transport of class II molecules to the peptide loading compartment. The expression of HLA-DM by these cells has important implications if we accept that autoantigen presentation by TFC may have a role in the disease process. It is known that DM interact with class II molecules bound to endogenous peptides (CLIP or others) incapable of forming high stability class II–peptide complexes. DM acts as a peptide editor favoring the binding of peptides and the formation of highly stable long-life complexes. DM has therefore a strong influence in the repertoire of peptides ultimately bound to class II molecules expressed at the cell surface (23).

We have also demonstrated that surface HLA class II molecules expressed by TFC are compact SDS-stable complexes. Immunoprecipitations were made on IFN-{gamma} stimulated samples to increase the expression of class II, although the original cells (TB258) were class II+ (40%). That, together with the lack of expression of CLIP-bound complexes at the surface, indicates that class II molecules expressed by TFC are loaded with peptides other than CLIP.

These results are important in relation to the presentation of autoantigens by class II molecules expressed by TFC. Highly specialized endocrine epithelial cells such as TFC which are responsible for the synthesis of thyroid hormones contain highly tissue-specific antigens. TFC secrete thyroglobulin which is transported to the follicular lumen where it is the major component of the coloid. Thyroglobulin is the precursor of and a biological storage system for thyroid hormones. A specialization of the TFC is their unique capacity of endocytosis by which thyroglobulin–hormone complexes enter the cells, and are hydrolysed in the lysosomes to release T3 and T4 hormones. Another thyroid-specific autoantigen is the enzyme TPO, which localizes mainly on the surface at the apical pole and also in the cytoplasm of the follicular cells. Other molecules, such as the TSH receptor, are expressed at much lower density at the opposite basal pole. TSH-R is a major target for autoantibodies in GD and these autoantibodies act mostly as TSH-R agonists, activating TFC function including hormone synthesis, endocytosis and even class II expression (39,40). Whether from the cytoplasm (41) or by internalization (42), these molecules should have access to the endocytic pathway and therefore peptides from their degradation could to bind to class II molecules on their way to the surface. Efficient transport of class II to the peptide-loading compartment and peptide exchange is possible since TFC express Ii and DM. Peptide exchange by DM in these cells was efficient enough to almost completely prevent expression at the surface of CLIP-associated complexes. A possible explanation of the high efficiency of DM in TFC is the putative absence of HLA-DO from these cells (24,25), since expression of DO appears to be limited to some cells (B cells, thymic epithelium) and is not induced by IFN-{gamma}. HLA-DO down-regulates DM function reducing its efficiency as a peptide editor. Absence of DO should facilitate maximum efficiency for DM action.

The peptide pool bound to these TFC class II compact surface molecules should contain at least some tissue-specific peptides and may be capable of interacting with tissue specific T cells. The lack of expression of conventional co-stimulatory molecules has questioned a role for these complexes at least in the induction of the autoimmune response. Only professional APC express B7 in autoimmune thyroid infiltrates (43). Class II+ TFC could be cooperating with these APC in the infiltrates in such a way that APC would stimulate naive cells and TFC maintain their activation. This is assuming that the needs for co-stimulation in the maintenance of a response are less astringent or that the TFC are able to stimulate T cells trans-co-stimulated by other APC. This has been demonstrated in vitro with class II+ TFC, incapable of inducing primary allogeneic responses but capable if co-cultured with a B7.1-expressing transfected cell. Some clones could respond to TFC in a B7-independent manner producing IL-4 in contrast with the production of IL-2 + IL-4 if the presenting cells were LCL (44). Non-professional APC could also induce incomplete responses or induce different functional capacity in the T cells such as changes in the cytokine profiles in response to antigen (45). On the other hand, conventional APC present in the autoimmune tissue are able to uptake cell debris containing the same tissue-specific antigens as those presumably presented by class II on TFC. In addition, differential processing by APC and TFC may generate different epitopes which could lead to differential recruitment and/or signaling to T cells depending on the type of the presenting cells (46). In vitro, TFC can induce proliferation of T cell clones (4749), can be targets of cytotoxic T lymphocytes, induce IL-4 production by T cell clones and be recognized by {gamma}{delta} cells (50 and M. Catálfamo, unpublished data). Their role in AITD pathogenesis is likely to be complex and will be progressively unveiled as knowledge on the regulation of antigen presentation, T cell activation and differentiation expands.


    Acknowledgments
 
We thank Drs G. R. Adolf, P. Cresswell, E. Long, S. Kovats, W. Knapp, P. Jensen and R. Vilella for kindly supplying us with reagents. We would like to thank Dr M. Martí for critically reading the manuscript. This work was supported by grants from the Fondo de Investigaciones Sanitarias of the Spanish Health Ministry (FIS 94/0807), from the DGES of the Spanish Education Ministry (PM95-0191) and in part by a grant from the ISCI Program of the European Commission (CI1*CT92-0071). M. C. is supported by the FIS (94/0807), L. S. by the TMR program of the European Commission (ERBFMBICT961295), C. R.-M. by the Generalitat de Catalunya (GRQ 93-2015) and M. S. by the FPI program of the Spanish Education Ministry.


    Abbreviations
 
AITDautoimmune thyroid diseases
APCantigen-presenting cell
CLIPclass II-associated invariant chain peptide
ERendoplasmic reticulum
GDGraves' disease
Iiinvariant chain
LCLEpstein–Barr virus-transformed lymphoblastoid B cell line
MNGmultinodular goitre
TFCthyroid follicular cells
TPOthyroid peroxidase
TSHthyrotropin
TSH-Rthyrotropin receptor

    Notes
 
Transmitting editor: M. Feldmannn

Received 10 December 1997, accepted 26 October 1998.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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