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Regular Articles |
From the Laboratory of Immunobiology for Research and Application
to Diagnosis,*
Centre for Transfusions and Tissue Bank,
Division of Endocrinology
and
Pathology,
Hospital Universitari
"Germans Trias i Pujol," and the Institute for Fundamental
Biology,
Autonomous University of Barcelona,
Barcelona; and the Immunology Division,¶
Hospital
Clínic i Provincial de Barcelona, Barcelona, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Histologically, HT is characterized by lymphocytic infiltration that may progressively replace thyroid follicles. Lymphoid infiltrates are also present in GD glands, but in this condition most of the thyroid remains intact except for the signs of hyperfunction. Cases that share features of HT and GD occur are occasionally observed.16 Lymphoid infiltrates often organize themselves as follicle-like structures containing germinal centers (GCs) similar to those in secondary lymphoid follicles (LFs) of LNs. Intrathyroidal B lymphocytes can synthesize Tg, TPO, and TSH-R antibodies in vitro, and this suggested that they are an important source of thyroid autoantibodies.17-21
Ectopic or extranodal secondary LFs are found in other autoimmune lesions, such as rheumatoid arthritis synovium,22-24 myasthenia gravis thymus,25 and Sjögren’s disease salivary glands,26 but also in chronically infected tissues such as hepatitis C liver27 and Helicobacter pylori gastritis mucosa.28 The formation of extranodal LFs might be a normal development in the course of a maintained immune response but it is not known if it contributes to response effectiveness. In autoimmune diseases it has been interpreted as a sign of the intensity of the response but not as a significant step in their pathogenesis. The real frequency of lymphoid follicle formation in autoimmune tissue has been difficult to estimate, because these structures are irregularly distributed, scattered all over the affected tissue, and can be easily missed during routine histopathological examination.
Lymphoid follicles with GCs are crucial sites in the development of the anamnestic immune responses because they are the sites where cells undergo somatic hypermutation and affinity maturation. Newly formed GCs are oligoclonal B cell populations derived from one to three B cell clones.29,30 Somatic hypermutation can generate autoreactive B cells and requires the existence of tolerance mechanisms to keep them under control.31,32 The discovery of RAG and Tdt expression in GCs has suggested that secondary VDJ rearrangement may be another process that contributes to GC function.33-35
The presence of well-organized B cell structures in AITD glands may be relevant to pathogenesis, not only for the production of autoantibodies but also for the development and maintenance of autoimmune response. B cells in intrathyroidal LFs are in a privileged location to capture large amounts of self-antigens and to present them to T lymphocytes. It has been suggested that, as they are outside the limits of lymphoid organs, they may bypass normal peripheral tolerance mechanisms more easily.32 The importance of extranodal LF formation for the development of autoimmune disease was established in recent experiments by Ludewig and colleagues36 using the RIP-GP mouse model; these authors reported a positive correlation between neogenesis of lymphoid tissue and development of autoimmune diabetes. The prevention of diabetes in NOD mice incorporating a Igµ null mutation37 constitutes compelling evidence for the role of B cell in endocrine autoimmune disease.
Previous studies by ourselves,38 and by other authors have described the surprisingly frequent occurrence and complex organization of intrathyroidal LFs. This prompted the present study, whose results indicate that intrathyroidal LFs in AITD patients are indeed functional. Our data include first evidence that chemokines capable of organizing and self-perpetuating LFs are generated in AITD glands and that secondary rearrangement of immunoglobulin genes may take place in these structures. In addition, we have found that intrathyroidal secondary LFs are more prevalent than previously estimated, correlate with autoantibody titer, and seem to result from the expansion of a few seeding B cells that are specific for thyroid autoantigens.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thyroid tissue was obtained from 67 patients, 35 with GD, 8 with HT, 22 with multinodular goiter (MNG), and 2 previously healthy multiorgan cadaveric donors. MNG glands were considered nonautoimmune thyroid tissue. Clinical diagnosis was based on usual thyroid function tests, including free thyroxine, tri-iodothyronine, and TSH plasma levels. TPO and Tg antibodies were measured by enzyme-linked immunosorbent assay (Immunowell, San Diego, CA), normal ranges are 42 to 100 and 67 to 115 IU/ml, respectively. Antibodies to TSH-R were measured by radioimmunoassay (Brahms Diagnostica, Berlin, Germany) and all values >1.5 IU/L were considered positive. In the statistical analysis, negative samples were assigned the value to the lower limit of detection of the corresponding assay (ie, 24.0, 36.0, and 1.0 IU, respectively). Diagnoses were confirmed by histopathological examination of the glands. Tissue samples from one thymus, six palatine tonsils (PTs), and five LNs were used as reference lymphoid tissue.
Several blocks from most specimens were treated separately; some were formalin-fixed for standard histopathology tests and other were snap-frozen in isopentane and stored at -70°C. Series of sequential cryostat sections (4 µm) obtained from frozen blocks of thyroid, tonsil, thymus, and LN were used for immunofluorescence staining, terminal dUTP nick-end labeling techniques, and also for RNA extraction under RNase-free conditions.
Dispersed thyroid cells and intrathyroidal lymphocytes from 42 (26 GD, 3 HT, 13 MNG) glands were prepared by enzymatic digestion as described elsewhere.39 The cells were filtered through a 500-µm mesh and cultured in RPMI 1640, 10% fetal calf serum, and antibiotics. Adherent cells, which included thyroid follicular cells, were separated from infiltrating thyroid lymphocytes by adherence to plastic after 18 to 24 hours of culture and separately cryopreserved in fetal calf serum containing 10% dimethyl sulfoxide. The lymphocyte-to-thyroid follicular cell ratio was assessed by fluorescence-activated cell sorting as described elsewhere.39
Identification of Germinal Centers, Immunofluorescence Staining, and Image Analysis
Thyroid blocks, with an average weight of 5 g, were
systematically screened for LFs by examining 1 out of 10 sequential
cryostat sections stained by hematoxylin and eosin (H&E) and confirmed
by either immunofluorescence staining with peanut agglutinin and
anti-IgD (cryostat sections)40
or with anti-CD20
(formalin-fixed sections). Ten to 80 consecutive 4-µm sections were
stained for phenotypic markers, adhesion molecules, and related
functional markers by simple or double-indirect immunofluorescence or
by immunohistochemistry technique following published
protocols41
and using the antibodies listed in Table 1
. Epithelial cells were identified with
high titer (1:105) anti-TPO patient’s serum.
Affinity-purified fluorochrome-labeled conjugated antisera (specific
for some IgG subclasses) were used as secondary antibodies (all from
Southern Biotechnology, Birmingham, AL). In all cases, the controls
included both using nonimmune sera or unrelated mAb as
primary antibodies and testing the effect of omitting each of the
layers. Biotin-labeled TPO and Tg (a kind gift from Pharmacia-Upjohn,
Freiburg, Germany) and fluorescein isothiocyanate-streptavidin were
used to detect B and plasma cells bearing Ig that were specific for
these antigens. The controls included blocking staining with unlabeled
antigens.
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TdT-Mediated dUTP Nick-End Labeling Assay
Apoptosis was detected by using a commercial kit (In situ cell death detection; Boehringer Mannheim, Mannheim, Germany) following the manufacturer’s instructions. Two substrates were used and the protocol was adjusted accordingly; 1) sections from formalin-fixed paraffin-embedded blocks (4 µm) were dewaxed, rehydrated, and digested with proteinase K (20 µg/ml), and 2) cryostat sections (4 µm) were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100/0.1% sodium citrate solution. dNTP-fluorescein isothiocyanate was detected with an anti-fluorescein alkaline phosphatase-conjugated antiserum and Fast red. The sections were counterstained with Mayer’s hematoxylin for 30 seconds. Apoptotic nuclei were counted using the Openlab software and expressed as number of positive nuclei per mm2.
Microdissection, RNA Extraction, and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Amplification
To study the RNA from the LFs, these structures were identified by
H&E staining and microdissected from 4 to 10 consecutive thyroidal
sections under a stereomicroscope (SV8, Zeiss). RNA was extracted from
sections devoid of LFs and from control normal lymphoid organs. To
study chemokine expression, RNA was extracted from frozen tissue
blocks. Genomic DNA was removed by digestion with DNase-I and the RNA
was reprecipitated with 0.4 µmol/L of nuclease-free glycogen
(Boehringer-Mannheim) as a carrier; then, it was quantified in a
spectrophotometer at OD260 nm. cDNA was prepared
in a final volume of 10 µl by mixing 200 ng to 1 µg of total RNA,
20 pmol Oligo-(dT15) (Pharmacia-Biotech), 40
U/µl RNase inhibitor (Clontech), 10 mmol/L dNTP mix
(Pharmacia-Biotech), 0.1 mol/L dithiothrcitol (DTT), 1x SuperScript
buffer and 200 U/µl of SuperScript-II enzyme. For RAG2, the antisense
primer used for the retrotranscription and for PCR amplification was
the same. The reaction was performed at 42°C for 45 minutes and the
product was diluted 5 to 10 times in TE (10mM TRIS/1mM EDTA, (TE) pH 8)
for subsequent experiments. For PCR reactions 1 to 3 µl of cDNA was
added to the mixture containing 0.2 mmol/L dNTP, 0.3 µmol/L each
primer, 1x PCR buffer and 300 mU of DynaZyme II (Finnzymes, Oy,
Finland) to a final volume of 20 µl in hot start. The following
programs and primers were used: 1) GAPDH, 30 cycles at 94°C for 30
seconds; denaturation, 65°C for 45 seconds; annealing/elongation
(sense primer 5'-TCTTCTTTTGC GTCGCCAG-3', antisense primer
5'-AGCCCCAGCCTTCTCCA-3'), amplicon 371 bp. 2) RAG1 35 cycles at
94°C for 30 seconds; denaturation, 56°C for 30 seconds;
annealing at 72°C for 40 seconds; elongation (sense primer
5'-ACTTTCCCTTCATCCTGCTTA-3', antisense primer
5'-TTTTTCTCCTCCTCTTGCTTC-3'), amplicon 653 bp. 3) RAG2, 30 cycles of
denaturation at 94°C for 30 seconds; annealing at 63°C for 30
seconds; extension at 72°C for 40 seconds (sense primer
5'-GCCACAGTCATAGTGGGCAGTCA G-3', antisense primer
5'-CAAAGGGAGTGGAATCCCCTGG-3'), amplimer 534 bp. 4) SDF-1, 30 cycles of
denaturation at 94°C for 30 seconds; annealing at 60°C for 20
seconds; and extension at 72°C for 30 seconds (sense primer
5'-GTCGTGGTCGTGCTGGTC-3', antisense primer 5'-CGGGCTACAATCTGCAGG-3'),
amplicon 155 bp. 5) SLC, 35 cycles at 94°C for 30 seconds;
denaturation, 65°C for 45 seconds; annealing/elongation (sense primer
5'-AAGGCAGTGATGGAGGGG-3', antisense primer 5'-CTGGGCTGGT TTCTGTGG-3'),
amplicon 238 bp. 6) BLC, 35 cycles at 94°C for 30 seconds;
denaturation, 51°C for 20 seconds; annealing at 72°C for 30
seconds; elongation (sense primer 5'-CGACATCTCTGCTTCTC-3', antisense
primer 5'-ACTTCCATCATTCTTTG-3'), amplicon 255 bp. All amplifications
were followed by a final extension at 72°C for 7 minutes.
The PCR products were analyzed by 1 to 3.5% agarose gel
electrophoresis and transferred to nylon membranes (Hybond+, Amersham).
The membranes were hybridized with specific labeled probes
[
-32P]ATP 10µCi/µl) at high stringency
conditions (RAG1 5'-CCCTTACTGTTGAGACTGC-3'; RAG2 5'-GGACAAAA
AGGCTGGCCCAA-3'; SDF-1 5'-TGCCTCAGCGACGGG-3; SLC 5'-CTTGGTT
CCTGCTTCCG-3'; BLC 5'-ACAACCATTCCCACGG-3'). In the case of RAG1, both
primers annealed to the same exon (exon 2) so a sample of
unretrotranscribed RNA was introduced as an additional control. To
estimate the amount of chemokine message in tissue samples, hybridized
membranes were exposed and the autoradiographies were either counted on
a Phosphorimager (BioRad, Richmond, CA) using Quantity One software or
assessed by densitometry (TDI Systems). The results were normalized
according to the amount of GAPDH message as estimated by densitometry
of gels stained with ethidium bromide. Preliminary experiments were
performed to establish the conditions under which the reactions were
within the exponential phase of amplification. To better compare the
levels of the different chemokines and refer them to a physiological
substrate, the values obtained from thyroid samples were divided by the
average value from PT for each chemokine. Therefore, the results are
given as percentages of reference PT values.
Statistical Analysis
Data distribution was first analyzed using the Sigma Stat software (Microsoft Corp., Seattle, WA). Parametric (Student’s t-test) and nonparametric (Mann-Whitney) tests were applied to normal and nonnormally distributed data, as indicated.
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Results |
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Typical secondary LFs, similar to those found in lymphoid organs
were easily detected by screening H&E sections. Eight of 8 (100%)
glands from HT, 14 of 26 (53.8%) from GD, and 1 of 22 (4.5%) from MNG
patients contained LFs (Table 2)
.
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. This cellular
distribution was confirmed by staining for a series of phenotypic
markers. Among them, anti-CD20 (Figure 1b)
and peanut
agglutinin-fluorescein isothiocyanate (Figure 1c)
specifically labeled
the GCs. Peanut agglutinin stained both the GCs and the vascular
endothelium, a feature that was useful to trace a given GC in
consecutive sections. The MZ consisted of typical CD19low IgD+ IgM+
CD38- CD23-/low+ follicular B lymphocytes (data not shown), but some
areas were rich in CD3+ cells, predominantly of the CD5+ CD3+
CD4+ phenotype but also containing CD3+ CD8+
cells; these areas corresponded to the T-cell rich MZ areas observed in
lymphoid node LFs (Figure 1
; d, e, and f). Staining for CD83 (Figure 1g)
revealed a rich network of DCs that included extensive areas of the
MZ and of the surrounding infiltrate, but normally did not reach the
inner GC area. Immunofluorescence staining confirmed the presence
within the GC of the two characteristic B cell populations: IgM-/low
IgD- CD23- CD77+ CD38+ centroblasts and CD77+ CD38+ CD23+
centrocytes. A framework of follicular dendritic cells, polarized
toward the light zone, was identified by mAb 7D6 against the long form
of CD21(CD21L) (Figure 1h)
.
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. GC and LF areas in GD and HT glands
were significantly different (P = 0.0020 and
P = 0.0174, respectively, t-test). As
expected, the presence of LFs was associated by the presence of
extensive lymphoid infiltration (P < 0.000005,
Mann-Whitney). LFs were detected in heavily infiltrated glands (defined
as containing >25% of lymphocytes over total amount of dispersed
cells, as assessed by flow cytometry of 24-hour thyroid primary
cultures (data not shown), but this relationship was not symmetrical;
some glands with extensive infiltration lacked LFs.
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Both in the whole study group and in the AITD group, the presence
of intrathyroidal LFs was associated to significantly higher levels of
TPO (P = 0.00860, Mann-Whitney test) but not of
Tg autoantibodies, although a tendency toward association was observed
(P = 0.053). In the GD group, intrathyroidal LFs
were associated to higher levels of TSH-R antibodies
(P = 0.0146, Mann-Whitney test). The specificity
of the B cells in intrathyroidal LFs was demonstrated by the binding of
Tg and TPO to LFs in a HT gland (16 of 28 LFs) and in four GD glands
(17 of 27; Figure 3b
). Two types of cells
bound thyroid antigens: numerous small cells with a distribution
similar to that of GC B cells and strongly positive cells with a strong
cytoplasmic staining scattered all over the LFs and the diffuse
lymphoid infiltrates. Double immunofluorescence revealed that most
cells that bound to TPO or Tg were IgG+. The distribution of Tg+ or
TPO+ cells was markedly uneven among the LFs, which tended to either be
totally negative or contain numerous positive cells. This suggested
that, as in canonical LFs, intrathyroidal CGs consist of an oligoclonal
B cell population. Blocking experiments with unlabeled TPO and Tg gave
negative results, thus confirming the specificity of the reaction.
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Proliferation in the LFs is linked to somatic
hypermutation and Ig repertoire diversification. AITD GCs contained
many cells positive for the proliferation marker Ki67, which were
oriented toward the dark zone; this was also the case in control tonsil
and LN tissue samples (Figure 4a)
.
Two-color immunofluorescence with anti-IgM antibodies confirmed that
most Ki67-positive cells were lymphoblasts with slight or no IgM
staining. Cell proliferation is high in AITD GCs, especially in HT
glands; the level of proliferation is similar to that detected in
tonsil GCs and well above that of LNs (Figure 4b)
. Collaboration
between B and T lymphocytes through CD40-CD40L interaction is central
to GC functions.42
As in lymphoid LFs,43
intrathyroidal LFs contained in their GCs a small polarized population
of CD40+ B cells and some scattered B lymphocytes bearing the
IgD+CD38-CD40L+ phenotype in the T-cell-rich area. CD40 was expressed
by a population of large-sized dendritic cells located outside the LFs,
in the areas of diffuse lymphocytic infiltration where T lymphocytes
predominate. These cells probably correspond to the dendritic cells
stained by CD83 in other sections and are equivalent to the dendritic
interdigitating cells described in the paracortical area of LNs (data
not shown).
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. Positive nuclei were
located mainly in the light zone, but some labeled nuclei were detected
in the rest of the GC areas as well as outside them, but not in the
thyroid follicular epithelium, as reported.44
The number
of apoptotic cells per square mm in LFs from HT was as high as in the
tonsils (370.2 ± 186 versus 325 ± 99.4;
P = not significant) and significantly higher than in
GD glands (126.4 ± 57.6) (HT versus GD;
P < 10-4, t-test).
B-lymphocyte apoptosis in lymphoid organ LFs is triggered by CD95-CD95L
(Fas/FasL) interaction and is modulated by the expression of Bcl-2 and
other anti-apoptotic factors. As expected, centroblasts were Fas+
Bcl2- (Figure 4, d and f)
whereas most centrocytes were Fas- Bcl-2+.
The IgD+ naïve B cells in the MZ were also bcl-2-positive. In
general, the distribution of these two molecules was similar to that
observed in tonsils and LNs, but in intrathyroidal LFs the level of
Bcl-2 seemed to be higher than in lymphoid organ LFs.
It has been reported that the RAG1 and RAG2 recombinase genes are
expressed in secondary lymphoid organs during active immune response
(see Discussion). We assessed the expression of RAG1 and RAG2 in AITD
glands by RT-PCR and Southern blotting using thymus, tonsil, and LN as
positive controls. RNA was extracted from 4 to 10 consecutive cryostat
sections and, when feasible, intrathyroidal GCs were microdissected.
All sections containing GCs were positive for RAG1 and RAG2 (HT,
n = 3; GD, n = 4) irrespective of
diagnosis, whereas sections that did not contain GCs were negative (HT,
n = 1) (Figure 5)
. Based
on partially normalized densitometric data, the levels of RAG1 and RAG2
mRNA levels in most glands containing LFs were several times higher
than in tonsil and LN samples, and in some cases higher than in samples
from the thymus. Tissue samples obtained from the same glands but
without LFs showed lower RAG1 and RAG2 levels. There were no clear
differences between HT and GD glands.
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These two categories of molecules determine the development,
organization, and self-perpetuation of LFs in the lymphoid organs. The
distribution of ICAM-3 (CD50) and ICAM-1 (CD54) was similar to that
observed in LN LFs,45
ie, ICAM-3 was expressed mainly in
the MZ and the surrounding area and in a few cells of the GC (both
centroblasts and centrocytes), whereas ICAM1 was expressed by GC cells
and especially in the follicular dendritic cell-rich area (Figure 6)
. The distribution of CLA (cutaneous
lymphocyte-associated antigen), a carbohydrate domain that is the
ligand for L-selectin (CD62L) and a marker for high
endothelial venules (HEVs),46
was studied in parallel with
that of factor VIII, an endothelial marker.38
In the rich
capillary network that surrounds the intrathyroidal LFs revealed by
FVIII staining, 
20% of the endothelial cells (Figure 6, c and d)
were positive for CLA (Figure 6e)
. Besides, occasional CD3+ CLA+ were
detected in the areas of lymphoid infiltration. The endothelium of MNG
and GD thyroids lacking LFs was either completely negative or only
showed a very low expression of CLA.
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shows the results from one
representative amplification experiment. mRNA from the three chemokines
was detected in the positive control and their level of expression was
clearly high in the two HT samples. Because the cDNA samples had been
normalized by GADPH expression, the observed fourfold increase of SLC
and SDF1 levels over PT levels indicates a remarkably active synthesis
of chemokine message in the HT glands. BLC message was also higher, but
only when compared to normal or MNG thyroid tissue. In the GD gland
studied, chemokine expression was not different from that in normal or
MNG tissue (Figure 7b)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Histopathological and immunohistochemistry methods have been applied and, when appropriate, observations were confirmed by confocal digital microscopy (deconvolution-based) and computerized morphometry. The antigen specificity of B and plasma cells was studied using a classical approach: binding of labeled antigen and visualization by immunofluorescence.47 For some molecules, such as chemokines and RAG1 and RAG2 gene products, we used RT-PCR-Southern blot, but in these cases the substrates were normalized and the LFs were selected by microdissection. Despite many technical difficulties and limited sample availability, human material was used as substrate to ensure the relevance of the findings to human disease. In thyroid patients, late surgery and prolonged treatment with thionamides that are immunomodulators,48 may be a limitation. However, our results cannot be attributed to this treatment, because intrathyroidal LFs were described well before thionamides were available and HT patients had not been treated with thionamides.
The presence of intrathyroidal LFs in AITD was considered to be a very characteristic feature of HT but not of GD. By their nature, LFs are scattered over the thyroid gland and can be easily missed, especially in GD, where infiltration is much less intense. By using a screening protocol that takes into account the average size of LFs and checking 1 in 10 sections, the possibility of missing LFs was reduced to the minimum. However, because we did not screen >5 g of each gland, our data could still be an underestimate. It is plausible that, if the entire glands had been screened, the frequency of LFs in GD would have been higher, thus implying that LFs are a regular feature. The association between high titer of antibodies against TPO and TSH-R and the presence of intrathyroidal LFs in AITD glands suggests that these structures play a role in antibody production. One possibility is that the formation of intrathyroidal LFs determines transition from self-limited responses to thyroid antigens to AITD, as suggested by the experimental model of Ludewig and colleagues.36 Our results showed binding of Tg and TPO to B and plasma cells, and their distribution among LFs suggests that most intrathyroidal LFs are actually committed to thyroid antigen immune response. The elucidation of the relative contribution of intrathyroidal LFs to the circulating levels of thyroid autoantibodies requires experiments that are difficult to justify in human patients, but the clinical observation of a quick reduction in autoantibody levels after thyroidectomy in GD patients (A Belfiore, personal communication),49 suggests that this contribution is important. If the autoimmune response was driven from the regional LNs, the persistence of antigen trapped in their follicular dendritic cell50 would maintain the response for a much longer period of time.
The similarity of HT and GD LFs to PTs and LN LFs, respectively, similarities that also apply to the levels of proliferation and apoptosis, may indicate a difference in the pathogenesis of each of these two forms of AITD. PTs and intrathyroidal HT LFs show a high degree of activation. It has been reported that PT LFs are activated by lipopolysaccharide and other bacterial products present in the pharynx.51 Current evidence and our data suggest that intrathyroidal LFs are stimulated by high concentrations of thyroid proteins, thus explaining their state of activation. It is relevant that B lymphomas that arise from HT glands have features similar to those of the MALT lymphomas,52 which are caused by monoclonal expansion of B cells chronically stimulated by antigens of the gastrointestinal tract. Similarly, B lymphomas in HT patients, which are histopathologically similar to the MALT lymphomas, could result from chronic stimulation of GC lymphocytes by thyroid autoantigens.53 The intrathyroidal LFs from GD patients are less active, and this may be because of a lower concentration of TSH-R, the most important self-antigen in this entity. Besides, as TSH-R is also expressed outside the thyroid,3 the response to it may be less dependent on intrathyroidal stimulation. In fact, it has been proposed that GD is a systemic disease.54
The complete parallelism between intrathyroidal LFs and secondary LFs in peripheral lymphoid organs suggests similar functional capabilities, probably including those not specifically studied in this work, such as Ig class switch. RAG1 and RAG2 expression, the visualization of HEV and the detection of the main chemokines that determine the organization of LFs have not been reported before and need to be discussed.
The RAG1 and RAG2 recombination activation genes play a central role in the rearrangement of V(D)J gene segments during lymphocyte development in the primary lymphoid organs.55 The finding of RAG1 and RAG2 expression in splenic B cells stimulated in vitro with LPS+IL-4 and in GC B cells of immunized mice suggested that at least some peripheral B cells can be induced to re-express or up-regulate these genes.33,34,56-58 This RAG re-expression was initially interpreted as evidence for the occurrence of peripheral receptor editing (receptor revision) aimed at rescuing autoreactive B cells generated by somatic hypermutation. The demonstration that Ig cross-linking inhibited RAG expression in human tonsil B cells led to the suggestion that receptor revision was rather aimed at rescuing cells whose receptor lost affinity during somatic hypermutation.59 Contrary to the former data, more recent experiments using GFP as a reporter of RAG2 induction have suggested that RAG expression in GCs may be because of the presence of a small percentage of immature RAG-expressing B cells in the spleen and LNs.60 In any case, these immature B cells probably contribute to the immune response and RAG expression should now be considered a feature of active GCs.61 RAG expression in intrathyroidal GCs was remarkably high, in some cases well above the levels detected in thymus, and probably indicates the high activity of these GCs. Our present data do not allow us to establish which of the following two not mutually exclusive alternatives explains the high level of expression of RAG: 1) a very active receptor revision and 2) the presence of a high proportion of immature B cells or, in general, of recent bone marrow emigrants. We believe that the phenotype of B cells in intrathyroidal LFs and the cloning of B cells/plasma cells bearing high-affinity antibodies to Tg and TPO from these cells favor the first alternative. Plausibly, B lymphocytes recruited from blood into intrathyroidal LFs have low-affinity receptors62 and may require extensive receptor revision to generate high-affinity B cells. Interestingly, evidence for over-active receptor revision has been found in human systemic lupus erythematosus (SLE)63 and, more recently, in ectopic LFs from rheumatoid arthritis synovium.64
Selective lymphocyte migration into peripheral lymphoid organs is regulated by adhesion molecules and chemokines. HEVs are specialized structures that allow rapid and selective lymphocyte trafficking from the blood into the secondary lymphoid tissues. Cutaneous lymphocyte-associated antigen (CLA), a HEV addressin, was detected in intrathyroidal small vessels that are morphologically similar to LN HEV. Secondary lymphoid tissue chemokine (SLC), a HEV-expressed chemokine that activates lymphocyte integrins, was also detected in intrathyroidal LFs. The distribution of HEVs around the intrathyroidal GCs indicates that this process takes place in the thyroid and in the LN. In addition to cell recruitment, the formation of LFs requires cytokines and chemokines that provide the signals for tissue organization.65 Among them, SDF-166 and BLC act synergistically: the former directs the migration of naive B cells to the GC and the latter induces the subsequent exit of activated B cells.67 Responsiveness to SDF-1 seems to be regulated during B lymphocyte activation and correlates with the location of B cells within the secondary lymphoid organ. The relatively high level of transcription of these chemokines found by us in intrathyroidal LFs underlines the parallelism between canonical and intrathyroidal LFs, and the high level of activation of the latter. We are currently working to determine the cell source of each of these chemokines, and whether the location of their receptors is in accordance with the suggestion that intrathyroidal GCs can organize and perpetuate themselves (MP Armengol, manuscript in preparation).
The major question that arises from this work is whether intrathyroidal LFs containing GCs are involved in pathogenesis. It is difficult to answer this question, because the role of the physiological ectopic LFs that appear during the response to infection has not yet been elucidated. It is not clear what advantage is obtained by the immune system with the placement of these advanced posts in the target tissue. The LFs in areas very rich in antigen and outside the lymphoid tissue might allow a fast expansion of the response, which thus would reach a high level of intensity. The recruitment of weakly autoreactive B cells in the thyroid and the extensive revision of the receptors in these cells (which thus would acquire high-affinity surface Igs), may be important for the production of high-affinity autoantibodies. In a normal LF, autoreactive B cells would be eliminated by apoptosis as they would encounter soluble antigen31 but this does not happen to the B cells in intrathyroidal GCs despite the availability of very high levels of antigen, thus suggesting that the microenvironment in ectopic LFs is permissive for autoimmune responses. Later, self-antigen presentation by autoreactive B cells and perhaps also by both classical and rogue antigen presenting cells (APCs), such as thyrocytes expressing high levels of histocompatibility leucocyte antigen (HLA) and adhesion molecules68-70 may be crucial for the maintenance and expansion of the autoimmune response. In this scenario it is easy to predict that autoimmune responses would be more difficult to control once LFs with GCs develop in the target tissue.
The event that initiates AITD still has to be identified, but sudden changes in the level of iodine intake are probably important.70 It is not difficult to conceive that an initial nonspecific inflammatory response induced by local iodine toxicity could lead to the secretion of inflammatory cytokines and chemokines by macrophages, dendritic cells, and the thyroid epithelial follicular cells themselves. In certain circumstances, endothelial cells may acquire the features of HEVs and recruit B and T lymphocytes whereas some stromal cells evolve into follicular dendritic cells. In this case, the simultaneous onset of the immune response in the LNs is not so crucial, as the lymphocytes in the thyroid glands could promote the immune response.
The possible involvement of intrathyroidal lymphoid tissue in AITD suggests that the local administration of immunosuppressive therapy could control the disease. This is not so important in the case of AITD, because the currently available treatment is overall satisfactory; however, it may be important for other tissue-specific autoimmune diseases because the administration of a local treatment would result in side effects that are less severe than systemic immunosuppressive treatment. Overall, our results support the relevance of the events in the target organ in the pathogenesis of organ-specific autoimmunity.
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Acknowledgements |
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Footnotes |
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Supported by the "Fondo de Investigaciones Sanitarias" (project 98/1123, to M. P. A.; and grants 98/1123 and 99/1063).
Presented in part at the second International Autoimmunity Congress, Tel Aviv, March, 8 to 11, 1999.
Accepted for publication April 13, 2001.
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