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Original Studies |
Allergy and Clinical Immunology Service (M.B., G.P., F.P., N.F., A.M.R., M.B.), Department of Internal Medicine (DI.M.I.), University of Genoa, Genoa, Italy; INSERM U119 (D.O.), Marseilles, France; Laboratory of Immunology (C.G.), Endocrinology Section, Institute of Clinica Medica, University of Palermo, Palermo, Italy; Institute of Clinica Chirurgica R (G.T.), University of Genoa, Italy
Address all correspondence and requests for reprints to: Prof. Marcello Bagnasco, M.D., Allergy and Clinical Immunology Service, Department of Internal Medicine-DI.M.I. University of Genoa, Viale Benedetto XV,6, 16132 Genova, Italy.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD28-CTLA-4/B7 ligand pairs play an important role as a costimulatory system for T-cells (see refs. 3, 4, 5 for review). At least two B7 proteins exist, termed B7.1 (CD80) and B7.2 (CD86), belonging to the immunoglobulin supergene family and sharing about 25% structural homologies. CD86 gene is located in human chromosome 3q13-q23 (6) in proximity to the CD80 gene previously mapped on chromosome 3q13-q21.
Both B7 molecules have been found expressed on professional antigen-presenting cells (APC): dendritic cells, activated macrophages, and activated B cells. On the other hand, B7.1 has not been found on resting T, B, NK cells or monocytes (7).
Although the stimuli able to induce B7.1 and B7.2 expression on B cells and monocytes are similar (8), B7.2 is induced more rapidly than B7.1 and is usually expressed at higher levels (9).
Both B7.1 and B7.2 can interact with their counterpart T-cell surface molecules, CD28 and CTLA-4. CTLA-4 shares significant sequence similarity with CD28; however, its expression is confined to activated T cells (10). Different from CD28, which is able to deliver potent activation signals, CTLA-4 signaling displays an inhibitory effect (11).
There is agreement that several human organ-specific autoimmune
diseases, using Hashimoto’s thyroiditis (HT) as a prototype, are
characterized by the presence at the level of the target organ of
so-called type 1 helper (Th1) and/or cytotoxic (Tc1) T-cells,
displaying CD4 or CD8 phenotype (12, 13, 14, 15) and, at least in some
instances, their autoantigen-specificity has been demonstrated. The
type 1 functional phenotype is characterized by the secretion of
IFN
, IL2, TNFß, and in some instances TNF
(16). Infiltrating
Th1 cells are believed to induce tissue lesions through activation of
cytolytic effector cells and secretion of lymphokines. In addition type
1 cytokines (notably IFN) are potent inducers of MHC antigens and
adhesion molecule expression (such as CD54/ICAM1) on thyroid follicular
cells: these phenomena could contribute to damage in the target cell by
cytolytic effectors (17, 18, 19), as well as to the possible (although
controversial) function of thyroid follicular cells as
autoantigen-presenting cells (see refs. 20, 21 for review).
Data from experimentally induced animal autoimmune diseases, such as experimental allergic enkephalomyelitis (EAE), suggest that autoantigen stimulation under experimental conditions might be able to induce the generation of Th2 cells that could prevent autoimmune diseases mediated by Th1 autoreactive effector cells (22). Moreover, Kuchroo et al. (23) showed, using the EAE model, that the interaction of the costimulatory molecules with their counterreceptors CD28 and CTLA-4 on T-helper precursor cells, during antigenic presentation, leads to the polarization of T-helper responses: B7.1 preferentially acts as a costimulator for the generation of Th1-type cells, while B7.2 costimulation induces the differentiation toward the Th2 functional phenotype.
To investigate the possible role of B7 molecules in the development of human autoimmune thyroid disease, we have analyzed in the present study the possible expression of B7.1 or B7.2 on thyroid tissue surgically removed from patients with HT, Graves’ disease (GD), or nonautoimmune disorders. We specifically addressed the question of whether such molecules might be present on thyroid epithelial cells. In fact, it has been recently reported that epithelial cells in chronic inflammatory disorders (mainly chronic HCV hepatitis) (24) may express B7 antigens. Only data concerning GD are currently available in literature (25–27, see below). Using both in situ analysis and immunofluorescence on isolated cells, we provide evidence for B7.1 antigen expression on follicular cells in HT.
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Materials and Methods |
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We examined surgically-removed thyroid tissue from 14 patients (11 females, 3 males) with HT, 9 with GD, 12 with nontoxic goiter (NTG), and 3 with papillary cancer (PC)(all females). Their ages ranged from 22 to 66 yr. The diagnosis was established on the basis of commonly accepted clinical and laboratory parameters and typical histologic features. The patients with HT underwent surgery because of suspected thyroid lymphoma on the basis of cytology performed by fine needle aspiration biopsy (which was not confirmed by histological examination) or because of the presence of large goiters with tracheal dislocation. After removal, thyroid specimens were snap-frozen in 2-methylbutane, then stored at -80 C until they were sectioned.
In addition, adequately sized specimens from 6 HT, 4 GD, and 5 NTG thyroids were processed as described below for follicular cells isolation and culture.
Monoclonal antibodies, mitogens, and cytokines
The following mouse monoclonal antibodies (mAbs) were used:
Other mAbs, antinatural killer cell subsets, GL183 and EB6 IgG1 (generous gifts of Professor. A Moretta, Genoa) were also tested as negative irrelevant controls for thyroid follicular cell staining in parallel experiments.
A human serum with high concentration of antithyroid peroxidase and antithyroglobulin antibodies (>400 U/mL and > 5000 U/mL respectively, by ELISA) and control autoantibody-negative pool of human sera were also used in other experiments (see below).
The following recombinant human cytokines, purchased from Genzyme General, Boston, MA were used in culture with thyroid follicular
cells, as described below: IFN 500-2000 U/mL, IL1ß 100–400
pg/mL, TNF 200–800 ng/mL, IL10 200 ng/mL, IL4 80 ng/mL,
TGFß1 10 ng/mL, and combination of IFN +TNF (20, 32).
Immunohistochemistry and double staining
Tissue sections 4 µm thick were cut with a Reichert-Jung cryostat (Leica Corp. Instruments GmbH, Nussloca, Germany), placed on alcohol-cleaned glass slides, exposed to absolute acetone for 10 min, and then air-dried. An immunoenzymatic alkaline phosphatase monoclonal anti-alkaline phosphatase complex (APAAP) procedure was used throughout, according to previously described methods (33, 34, 35). Briefly, the sections were submitted to appropriate dilution of each mAb, then washed in TBS pH 7.6, and incubated with rabbit antimouse immunoglobulins, followed by APAAP complex; finally the sections were incubated in substrate solution, containing basic new fucsin, naphthol As-Bi phosphate, and levamisole (all reagents were purchased from Sigma Chemical Co.). Control sections were set up with irrelevant isotype-matched mAbs. The preparations were counterstained with Carazzi’s hematoxylin.
For double staining, streptavidin-peroxidase and streptavidin-AP were used in combination, as previously described (35, 36). To this end, the sections were pretreated with H2O2 and methanol to inhibit endogenous peroxidase activity, then washed by immersion in TBS. Single-step horseradish peroxidase-coupled antihuman thyroglobulin mAb (Dako Corp.) was used. Incubation was carried out for 60 min at room temperature, followed by washing in TBS. Diaminobenzidine (DAB) was used as chromogenic substrate, as described (35, 36). Then APAAP procedure was performed as described above.
The preparations were mounted in buffered glycerol and examined by means of a Leitz Laborlux microscope (Wild Microscopes, Rockleigh, NJ) equipped for microphotography.
Human B7 protein-transfected murine cells
Murine L fibroblasts were stably transfected with human B7.1 and B7.2 complimentary DNAs using Ca++/P method (calcium phosphate coprecipitation technique) and selected by G418 resistance, as previously described (37).
After 10–14 days, G418-resistant colonies were expanded, and the human B7.1/CD80+ and B7.2/CD86+ transfected L cells (hereafter referred as LB7.1 and LB7.2) were screened for B7 expression by immunofluorescence flow cytometry.
The parental L cells, LB7.1 and LB7.2, were maintained in DMEM medium (ICN Flow, CA) supplemented with 10% heat-inactivated fetal calf serum, 100 U/mL penicillin, 100 µg/mL streptomycin sulfate, and 2 mM L-glutamine at 37 C in a humidified atmosphere.
These cells (either in suspension or after cytocentrifugation) were used as positive and negative controls for B7 antigens immunofluorescence or APAAP staining.
Follicular thyroid cell isolation and culture
Thyroid follicular cell suspensions were prepared as previously described, with minor modifications (34, 38). Briefly, fresh surgical thyroid specimens were minced in small pieces and teased apart with a scalpel in HBSS medium with Ca++ and Mg++ (Sigma Chemical Co.) supplemented with 2 mM L-glutamine, penicillin, and streptomycin. The resulting suspension was digested by 3-h incubation at 37 C in a shaker bath with collagenase type II S (Sigma Chemical Co.) at the final concentration of 5 mg/mL supplemented with 1% FCS. At the end of incubation, HBSS without Ca++ and Mg++ supplemented with 2 mM L-glutamine, antibiotics and 10% FCS were added in ice, and the suspension was filtered through a 60-mesh screen and extensively washed. After red cell lysis, the cells were resuspended in RPMI 1640 culture medium (ICN Flow, CA) supplemented with 2 mM glutamine, antibiotics, and 10% FCS, and plated in 25 cm2 tissue culture flasks at 37 C in humidified atmosphere.
After 24 h, the supernatant, containing nonadherent cells, was collected and purified by centrifugation over standard Ficoll-Urovision gradient. The suspension containing lymphocytes was evaluated by indirect immunofluorescence.
The adherent cells were cultured up to 10–14 days, viability (evaluated by trypan blue exclusion) being constantly more than 95%, and immunofluorescence analysis was performed at different times. To this end, the cells were recovered by short incubation with trypsin 0.25% and EDTA 1 mM, followed by extensive washing with culture medium.
At days 2–7 aliquots of cells were transferred to 6-well tissue culture dishes (Costar) and cultured with the above-mentioned stimuli and flow cytometry performed 24 and 48 h later.
Flow cytometry analysis
Indirect immunofluorescence flow cytometry analysis was performed as previously described (34, 38) with the above mentioned mAbs and an FITC-labeled antimouse immunoglobulins antiserum (GAM-FITC, Coulter Corp., Miami, FL) or an FITC-labeled antihuman IgG antiserum (Dako Corp.) when appropriated. Incubations were carried out for 30 min at 4 C. Cytofluorimetric reading was performed by an Epics Elite cytofluorimeter (Coulter Corp., Miami, FL).
For flow cytometry analysis on thyroid follicular cells, TGA+ TPO+ cells (cells stained with a human serum with high concentrations of antithyroid peroxidase and antithyroglobulin antibodies: more than 400 U/mL and more than 5000 U/mL respectively by ELISA) were gated.
Isolation and culture of peripheral and intrathyroid lymphocytes
Peripheral and intrathyroid mononuclear cells isolated by standard density gradient, as previously described (39) from 3 HT and 3 GD patients were cultured in triplicate for 48 h (5 days for cultures containing the CD2 and CD28 mAbs) at the concentrations of 2 x 105/well in 0.2 mL, using round-bottomed microtiter plates (Sterilin), in culture medium in the presence or absence of human rIL2 100 U/mL (Genzyme General). Lymphocytes were stimulated with the following stimuli: PHA (M-form)1%, CD3 mAb 289 0.5 µg/mL, CD2 mAbs (CD2.1+CD2.9) (1:10000 final ascite dilution), CD28 mAb 248 0.5 µg/mL. Cultures were pulsed with [3H]TdR, harvested and counted as above described.
In two HT patients it was possible to obtain adequate numbers of both thyroid follicular cells and intrathyroid lymphocytes for coculture experiments. Intrathyroid lymphocytes were purified from nonadherent cells as described in the previous section. Peripheral blood lymphocytes were prepared by standard density gradient centrifugation followed by plastic adherence.
Peripheral and intrathyroid lymphocytes at the concentration of 105/well in 0.2 mL were cocultured with irradiated (5 x 104/well in 0.2 mL) follicular cells in the presence or absence of CD3 (289 0, 5 µg/mL) and B7.1 (2D10.4 10 µg/mL) mAbs in culture medium (RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, and antibiotics). After 5 days incubation at 37 C in a humidified atmosphere containing 5% CO2 in air, each well was pulsed with thymidine ([3H]TdR) (1 µCi/well) 18 h before being harvested on glass wool filters using an automated cell harvester (Titertek D-001, Flow Lab.), and then [3H]TdR incorporation was measured in a ß-counter (Beckman Coulter, Inc., Fullerton CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We carried out experiments on thin cryostatic sections of thyroid
tissue surgically removed from patients with HT or GD or nonautoimmune
disease (NTG and PC). We first stained the sections by B7.1 mAb using
the APAAP method. Positive and negative control cytospins of human
B7.1-transfected and wild-type murine fibroblasts were simultaneously
stained, which confirmed the specificity of the mAb and the reliability
of the method (Fig. 1e
). The results
obtained showed clear-cut staining for B7.1 of follicular structures
(whole follicles or follicular portions) in 12 out of 14 HT specimens,
although to a variable extent (from isolated follicular portions, Fig. 1f, up to the majority of the follicules identified in the
microscopical fields, Fig. 1a, depending on individual specimens). No
follicular staining was observed in tissue from any of the patients
with GD (Fig. 1d) except one, who exhibited very weak positivity on a
single follicular portion. In addition, in tissues from both patients
with HT and GD, B7.1 staining of infiltrating mononuclear cells,
although variable, was observed. No significant B7.1 positivity was
observed in any of the tissue sample from patients with NTG or PC. The
specificity of B7.1 follicular staining in HT was confirmed by the lack
of staining with other isotype-matched mAb tested as controls, such as
antinatural killer cells subsets GL183 and EB6 (data not shown).
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). Moreover, colocalization
of B7.1 and MHC class II antigens on follicular structures was
demonstrated by double staining (Fig. 2b).
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B7.2 is not expressed on thyroid follicular cells
We then tested B7.2 mAb, using APAAP method, and invariably
demonstrated a positive B7.2 staining of infiltrating mononuclear cells
in both HT and GD (Fig. 3). However, we
failed to demonstrate any significant positive staining for B7.2 on
follicular structures in both HT and GD, as well as in nonautoimmune
specimens.
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In 6 of our HT specimens in which an adequate amount of tissue was available, we isolated follicular cells and evaluated B7.1 and B7.2 antigen expression by immunofluorescence flow cytometry. The expression of other antigens known to be expressed by thyroid cells in culture, such as HLA Class II (DR) and ICAM1, was also evaluated. Follicular cells were also isolated from 4 samples of GD and from 5 samples of NTG thyroid for control.
After isolation, follicular cells were cultured up to 10–14 days, and
immunofluorescence was performed at days 2–7. The purity of follicular
cells was indicated by their immunofluorescence profile when
specifically stained with a human serum containing high concentrations
of thyroid autoantibodies followed by FITC-labeled antihuman IgG (Fig. 4, upper right panel). The
results obtained with B7.1 and B7.2 mAbs substantially confirmed what
had been observed by in situ analysis: follicular positivity
for B7.1 was evidenced, although the variability was high (Fig. 4, left panel), and the proportions of B7.1-positive follicular
cells never exceeded 25% [(10%; MFI 0, 35) (13%; MFI 3, 8) (15,
5%; MFI 5, 6) (23%; MFI 27) (24, 5%; MFI 36) (25%; MFI 34)]. B7.1
positivity remained substantially unchanged in the absence of any
stimulus up to the overall culture period. On the other hand, no B7.1
positivity was observed in cells isolated from GD or NTG thyroid
specimens (Fig 4, left panel). As well, in agreement with
in situ findings, no B7.2 positivity was demonstrated in any
of the cell preparations tested (Fig. 5, upper and middle panels). Control immunofluorescence
analyses performed with DR or ICAM-1 mAbs gave the expected results:
follicular cells in GD and HT strongly expressed DR antigens (Fig. 4, middle right panel), and spontaneous upregulation of ICAM1
(which was not constitutively expressed in GD) during the culture
period was observed confirming previous data (18).
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500-2000 U/mL,
IL1ß 100–400 pg/mL, TNF 200–800 ng/mL, IL10 200 ng/mL, IL4 80
ng/mL, TGFß1 10 ng/mL, LPS 5–25 µg/mL and the combination of IFN
+TNF. Only IL1ß (400 pg/mL) and LPS (25 µg/mL) were able to
induce B7.1 expression in follicular cells that did not express such
molecules at baseline (Fig. 6). Of note,
stimulations with IFN and/or TNF were invariably able to
upregulate follicular ICAM1 expression, tested as control on the basis
of previously reported data (18). No up- or down-regulation of B7.1
occurred on follicular cells of HT patients with anyone of the tested
cytokines (data not shown).
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We also evaluated CD28 expression using both in situ
analysis and immunofluorescence on isolated cells. A large proportion
of infiltrating mononuclear cells was CD28 positive (Fig. 7). This was confirmed by phenotypical
analysis on lymphocytes recovered from tissue digestion in HT
specimens: most of the isolated cells were CD28-positive (up to 60%)
and CD3-positive, similarly to peripheral blood (not shown).
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and 2).
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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been perceived for a long time that the ability of autoimmune thyroid follicular cells to actively interact with lymphocytes implies their capacity to function as autoantigen-presenting cells (see refs. 20, 21 for review). However, as mentioned above, costimulatory signals delivered by professional antigen-presenting cells are strictly required for a productive immune response.
The question of whether thyroid follicular cells could express costimulatory molecules was previously raised in three different studies (25, 26, 27). Using the B7/BB1 mAb, Tandon et al. (25) failed to show any follicular staining in a series of GD patients and in one HT specimens, whereas positive infiltrating cells were observed. Similar results in GD were obtained by Lombardi et al. (26) and Matsuoka et al. (27), using specific anti-B7.1 and B7.2 mAbs. In the latter study, experimental evidence concerning the role of B7/CD28/CTLA4 system in intrathyroid T cell costimulation by professional antigen-presenting cells in GD was provided (27).
In the present study we have addressed the same question, thanks to the availability to us of a relatively large number of frozen HT thyroid tissue specimens obtained in the last year by our thyroid surgery unit.
The results we obtained clearly demonstrate, in our opinion, that B7.1 antigen is frequently expressed on follicular cells in HT thyroids, although to a variable extent. Such evidence derives from both evaluation of isolated follicular cells and from in situ analysis. Isolated cell analysis in a few samples yielded variable percentages of positivity, whereas controls and GD invariably gave negative results. Morphological evidence was supported by colocalization of B7.1 and thyroid autoantigens. On the other hand, no or very poor follicular staining was observed in GD, whereas positive staining of infiltrating cells was present, consistent with the above-mentioned reports (25, 26, 27). No B7.1 positivity was observed in nonautoimmune thyroids.
As far as B7.2 was concerned, no follicular positivity was observed in either HT, GD, or nonautoimmune thyroid specimens, as shown by both in situ and cytofluorimetric evaluation. This is of interest, considering the hypothesized differential role of costimulation via B7.1 or B7.2 in the polarization of T helper response toward the Th1- or Th2-type. The molecular basis of such possible difference between the two ligands is not fully clarified: both molecules display similar effect on T-cell proliferation and IL-2 production, as demonstrated by experiments of coculture with different B7 transfectants (2 and Battifora M; unpublished data), however, B7.2 has been reported to be more effective than B7.1 in inducing IL4 release, under appropriate conditions, by fresh normal peripheral blood mononuclear cells (47).
The differential expression of the two B7 antigens in thyroid tissue of HT is reminiscent of a report concerning human multiple sclerosis, another Th1-cell-mediated autoimmune disease. In specific lesions of the central nervous systems, the expression of B7.1, but not B7.2, was observed on different cell structures, whereas in nonspecific (vascular) lesions, B7.2 predominated (15).
Thus, the expression of B7.1 (and not B7.2) on follicular thyroid cells in HT could provide a local costimulatory signal for T-lymphocyte differentiation toward the Type 1 cytokine secretion pattern and maintenance of a sustained Th1 response. Note that CD28 antigen is expressed in a large proportion of infiltrating lymphocytes, as demonstrated by analysis of isolated cells, and that they are able to proliferate in response to CD28 stimulation even in the absence of IL2. Moreover we obtained preliminary evidence of inhibition of lymphocyte proliferation when cocultured by B7.1 mAb.
Thus, it is conceivable that, at least in part and/or in some phases of the natural history of cronic autoimmune thyroiditis, follicular cells may effectively act as autoantigen-presenting cells capable of initiating or perpetuating the autoimmune injury. The finding of colocalization of MHC class II and B7.1 antigens on the same follicular structures is consistent with such a hypothesis.
On the other hand no relevant B7.1 expression occurs in GD. The major histological differences between HT and GD are the infiltrate amount and the damage to follicular structures.
We observed that B7.1 can be induced on thyroid follicular cells from nontoxic goiter by LPS or high doses of IL1ß. We previously showed (38) that large amounts of IL1ß are present within HT thyroid: possible sources are infiltrating mononuclear cells, or even thyroid follicular cells by themselves. IL1ß proved to be also responsible for induction on thyroid follicular cells of Fas/CD95 molecule, able to mediate apoptosis via interaction with its ligand (FasL) constitutively expressed on the same cells (38). Thus this cytokine appears to be crucial in HT, and its secretion may be promoted by products of Th1 infiltrating cells, far more abundant in HT than in GD.
Moreover B7/CD28/CTLA4 interaction proved to be required for full display of cytotoxic activity (dependent upon Th1-Tc1 cells), in addition to other adhesion/activation systems (48, 49, 50, 51). Thus, follicular B7.1 expression may also be consistent with a role of cellular effectors in thyroid follicular damage of HT.
Finally, a role of B7 antigen interaction with their T-cell ligands in preventing T-cell apoptosis has been demonstrated (52, 53). Thus, follicular B7.1 expression in HT may contribute to maintenance of large lymphocyte infiltrates by increasing their survival, whereas in GD interaction of infiltrating T-cells with MHC Class II-positive, B7-negative follicular cells may result in inactivation or apoptosis.
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Acknowledgments |
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Footnotes |
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Received May 23, 1997.
Revised June 29, 1998.
Accepted July 20, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-interferon by
thyroid-derived T cell clones from patients with Hashimoto’s
thyroiditis. Clin Exp Immunol. 69:323–331.[Medline]
induces HLA-DR expression by thyroid
epithelium. Clin Exp Immunol. 61:265–273.[Medline]
treated thyroid follicular cells inhibits IL2
and support IL4 production by B7-dependent human T cells. Eur J
Immunol. 27:62–71.[Medline]
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