help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ashhab, Y.
Right arrow Articles by Pujol-Borrell, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ashhab, Y.
Right arrow Articles by Pujol-Borrell, R.
The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 8 2873-2882
Copyright © 1999 by The Endocrine Society

Original Studies

A One-Tube Polymerase Chain Reaction Protocol Demonstrates CC Chemokine Overexpression in Graves’ Disease Glands

Yaqoub Ashhab, Orlando Dominguez, Mireia Sospedra, Carme Roura-Mir, Anna Lucas-Martín and Ricardo Pujol-Borrell

Immunology (Y.A., M.S., C.R.-M., A.L.-M., R.P.-B.) and Endocrinology (A.L.-M.) Divisions, University Hospital Germans Trias i Pujol, Badalona, 08916 Barcelona; and Almirall-Prodesfarma Research Center (Y.A., O.D.), 08980 Barcelona, Spain

Address all correspondence and requests for reprints to: Prof. R. Pujol-Borrell, Immunology Unit, University Hospital Germans Trias i Pujol, Carretera. del Canyet s/n, Badalona, 08916 Barcelona, Spain. E-mail: ricardo.pujol{at}uab.es


   Abstract
Top
 Abstract
Introduction
 Experimental Subjects
 Materials and Methods
 Results
 Discussion
 References
 
An adaptation of mixed oligonucleotide primed amplification of complementary DNA to detect the profile of CC chemokines in biological samples is presented. By introducing normalization, two correction coefficients, performing a single amplification reaction, and five parallel hybridizations, intrasample and intersample comparisons can be reliably made. This protocol of single tube PCR CC chemokine profiling was applied to tissue samples from an autoimmune thyroid condition, Graves’ disease, and from a nonautoimmune condition, multinodular goiter. Results demonstrate overexpression of CC chemokines in Graves’ disease, statistically significant for macrophage inflammatory protein-1{alpha} and -1ß, which correlated with the aberrant human leukocyte antigen class II expression by thyrocytes, as assessed by flow cytometry. Overexpression of CC chemokines probably plays a major role in determining the characteristics of the lymphocytes migrating to the thyroid gland and influences the course of the disease. The study of chemokine profile should be more informative than the study of isolated chemokines and cytokines, and as it can be applied to fine needle aspiration biopsies, it may be useful to clinical research.


   Introduction
Top
 Abstract
 Introduction
Experimental Subjects
 Materials and Methods
 Results
 Discussion
 References
 
IT HAS BECOME progressively clear that in vivo, cytokines act in distinct combinations that determine the final effect on the target cells. The patterns of cytokines secreted by Th1 and Th2 cells constitute paradigmatic combinations that specifically drive particular types of immune response, but other such combinations are probably yet to be unveiled. It is therefore of great interest to measure multiple cytokines simultaneously. Current assays that measure cytokine protein or message are costly (enzyme-linked immunosorbent assay and RIA techniques), labor intensive (Northern blotting and ribonuclease protection assays), or not quantitative (RT-PCR). New technologies have been developed that allow the assessment of multiple gene expression in a given sample, e.g. the hybridization of amplified complementary DNA (cDNA) to arrays of oligonucleotides immobilized onto a microchip (1). Due to its cost, such technology is available to few laboratories, and there is a need to develop simpler and cheaper methods to assess the expression of multiple genes of clinical interest in biological samples. While using mixed oligonucleotide primed amplification of cDNA (2) to identify new chemokines, we realized the potential of this method to simultaneously measure multiple messenger ribonucleic acid (mRNA) species sharing short sequence motifs in biological samples. We have developed a protocol that enables the detection of transcripts of five members of the CC chemokine family in a single assay and applied it to study the profiles of CC chemokines in Grave’s disease (GD), the most prevalent clinical form of thyroid autoimmune disease.

Chemoattractant cytokines (chemokines) constitute the largest family of structurally related cytokines described (reviewed in Refs. 3, 4). Four families have been defined based on a cysteine motif, i.e. CXC, CC, C, and CX3C, where the C is a cysteine and X stands for any amino acid residue. Chemokines are known mainly as chemoattractant factors by virtue of their ability to regulate the recruitment of immune cells to inflammation sites (reviewed in Refs. 5, 6). The CXC family is primarily chemotactic for neutrophils, whereas the CC family is primarily chemotactic for monocytes and macrophages, T lymphocytes, basophils, and eosinophils. Chemokines are produced by a wide variety of cell types in response to exogenous factors such as lipopolysaccharides, viruses, and inflammatory cytokines such as interleukin-1{alpha} (IL-1{alpha}), tumor necrosis factor-{alpha} (TNF{alpha}), and interferon-{gamma} (IFN{gamma}) (7, 8). Some chemokines have important effects on regulation of the immune response through actions such as the stimulation of lymphocyte proliferation (8, 9) and activation (reviewed in Ref. 10) and the regulation of adhesion molecules (8, 11).

Autoimmune thyroid diseases (AITD) define a spectrum of disorders in which there is a strong cellular and humoral immune response to the thyroid glands (reviewed in Ref. 12). GD, Hashimoto’s thyroiditis, and primary myxedema are the three main clinical entities grouped under the term AITD. Hashimoto thyroiditis was the first disease suspected to be autoimmune and still today is the paradigm of organ-specific disease (reviewed in Ref. 13). One of the histopathological hallmarks of thyroid glands affected by autoimmune AITD is leukocytic infiltration, mainly by mononuclear cells, including T and B lymphocytes and macrophages (reviewed in Ref. 14). The cellular make-up of the infiltrate varies with the type of AITD, the stage of the disease, and the therapy used, but it is also patient dependent. In GD there is a predominance of T lymphocytes, of which CD4+ are often more abundant than CD8+, but there is also a small, although significant, proportion of {gamma}{delta} T lymphocytes (15, 16, 17) and natural killer T cells (Roura-Mir, C., et al., in preparation). This cellular infiltrate sometimes organizes itself into germinal centers that share many of the features of lymph node germinal centers (14, 18) (Armengol, P., et al., unpublished observation). Several cytokines, such as IL-1{alpha}, TNF{alpha}, IFN{gamma}, IL-6, IL-2, IL-4, and IL-10, have been detected in AITD (19–23; reviewed in Ref. 24), but their specific roles in the pathogenesis of AITD are debated (24), probably because of the limitations of current assays. One of these new assays is simultaneous measurement of intracellular cytokines, which we applied recently to AITD (21).

The presence and role of chemokines in AITD have not yet been studied in detail, even if the degree and organization of the infiltrate strongly suggest that chemokines must be directly involved. Only Kasai et al. (25) showed the production of monocyte chemoattractant protein-1 (MCP-1) by human thyrocytes in primary cultures stimulated by IL-1{alpha}, TNF{alpha}, and IFN{gamma}. To test our newly developed, single tube PCR protocol, it seemed appropriate to use it to investigate the CC chemokine profiles in AITD, an area of study in our laboratory. In this article we present the basis for the CC chemokine profiling protocol and its performance on AITD tissue samples, which enabled quick demonstration of elevated CC chemokine expression and showed that their profile is different from that of multinodular goiter (MNG).


   Experimental Subjects
Top
 Abstract
 Introduction
 Experimental Subjects
Materials and Methods
 Results
 Discussion
 References
 
Samples of thyroid tissue were obtained at surgery from patients with GD and MNG. Essential clinical data are listed in Table 1Go. All GD patients were treated with carbimazole up to the time of surgery, and no patient was treated for less than 2 yr. Diagnosis was confirmed by histopathological examination of the tissue. Tissue was divided into small blocks, snap-frozen, and kept at -70 C until used. Normal thyroid tissue was not available. The procedures to obtain the samples and their use in this work were approved by the ethical committee of the University Hospital Germans Trias i Pujol.


View this table:
[in this window]
[in a new window]
  		Table 1. Summary of patient’s data

 

   Materials and Methods
Top
 Abstract
 Introduction
 Experimental Subjects
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

Thyroid glands primary cultures were prepared from collagenase-trypsin-deoxyribonuclease-dispersed glands as previously described (26). Intrathyoidal lymphocytes were obtained by washing the thyroid cell monolayers with fresh culture medium after overnight culture. The purity of the preparations was assessed by fluorescence-activated cell sorter analysis, using a monoclonal antibody to CD3 (data not shown).

The monocytic cell line U937 (American Type Culture Collection, Manassass, VA) was grown in RPMI 1640 (BioWhittaker, Inc., Walkersville, MD) supplemented with 10% FCS (Life Technologies, Inc., Paisley, Scotland). Cells were stimulated with 10 ng/mL PMA (Sigma Chemical Co., St. Louis, MO) and 100 ng/mL lipopolysaccharides (Sigma Chemical Co.) and harvested 4 h after stimulation. To increase the stability of the chemokine-labile transcripts, anisomycin (Sigma Chemical Co.; 10 µg/mL) was added 1 h before collection (27). This cell line was used as source of positive control cDNA for the different chemokines.

RT-PCR

To isolate total RNA from thyroid tissue and from cells in culture, the single step acid-phenol method (28) was used. Tissue blocks were homogenized at 24,000 rpm (Ultra-Turrax T25, IKA Laborteknik, Staufen, Germany) for 30 s in 1 mL cell lysing solution on ice. To eliminate residual genomic DNA, RNA preparations adjusted at 0.25–50 µg/µL were treated with 5 U deoxyribonuclease I (RQ1, Promega Corp., Madison, WI) for 30 min at 37 C in a solution containing 10 mmol/L bis-Tris-HCl (pH 6.5), 1 mmol/L ethylenediamine tetraacetate, 5 mmol/L MgCl2, 5 mmol/L dithiothreitol, and 30 U RNasin (CLONTECH Laboratories, Inc., Palo Alto, CA). RNA was precipitated with 75% ethanol and 0.3 mol/L NaAc in the presence of 20 µg nuclease-free glycogen (Roche Molecular Biochemicals, Mannheim, Germany) as carrier. Removal of genomic DNA was confirmed by amplifying a fragment of the macrophage inflammatory protein-1{alpha} (MIP-1{alpha}) gene for 40 cycles using MP{alpha}-63- and MP{alpha}-445-specific primers (see Table 2Go). In this experiment, treated RNA samples were run in parallel with gDNA, which served as a positive control.


View this table:
[in this window]
[in a new window]
  		Table 2. Primers and oligonucleotides probes used in this paper

 
Three micrograms of RNA were reverse transcribed with 200 U SuperScript II (Life Technologies, Inc.) following the manufacturer’s instructions. Normalization of cDNA input was achieved by semiquantitative PCR. Briefly, serial dilutions of all samples were amplified (24 cycles) for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with 1 µCi [{alpha}-33P]deoxy-ATP (NEN Life Science Products, Boston, MA) added to the reaction mixture. Bands corresponding to amplified GAPDH were cut out from low melting agarose gel and solubilized, and radioactivity was measured in a ß-scintillation counter (Kontron Instruments Ltd., Milan, Italy) because a phosphorimager was not available at this stage of the project. Sample dilutions were adjusted on the basis of these counts in such a way that they contained approximately the same number of cDNA molecules.

Gene-specific primers are listed in Table 2Go, and degenerate (polyvalent) primers aimed at CC chemokine conserved motifs are given in Fig. 1Go. Oligonucleotide-specific probes (Table 2Go) were aimed at different exons to avoid colinearity between the gene and the mRNA. Analysis of primers and oligonucleotide probes was made using the Oligo program (National Biosciences, Inc., Plymouth, MN).



View larger version (42K):
[in this window]
[in a new window]
  		Figure 1. Top panel, Multiple amino acid alignment of CC chemokines. Conserved CC and WVQ motifs are shaded, and shown below are the approximate locations of gene-specific primers (GSP), degenerate primers (DP), and the sequence-specific oligonucleotide (SSO) in the cDNA. Lower panel, DNA sequence alignment of the five CC chemokines showing the primer regions (boxed) with the conserved bases in boldface. The degenerate, sense, and antisense primers are shown below, where the degenerate bases are: M, C or A; R, G or A; and S, G or C. The calculated annealing temperatures according to the Oligo program are: 2YH1, 64.4 C; 2YH2, 76.1 C; 2YH3, 68.1 C; and 2YH4, 65.3 C. The expected sizes of amplicons produced by degenerate primers are: MIP-1{alpha}, 178 bp; MIP-1ß, 178 bp; MCP-1, 181 bp; MCP-3, 181 bp; and RANTES, 178 bp.

 
CC chemokine-positive controls and optimization of the polyvalent primers

cDNA from stimulated U937 cells was used as substrate to amplify the cDNA of each chemokine included in the profiling protocol using gene-specific primers (Table 2Go) out-flanking the sequences used by the polyvalent primers (Fig. 1Go). Bands of the expected size were excised from low melting agarose gel, and DNA was extracted with a QIAquik gel extraction kit (QIAGEN, Hilden, Germany). These amplified CC chemokine cDNAs were used as positive controls to establish the optimal amplification conditions when using the polyvalent primers and to assess their amplification efficiency for each of the CC chemokines studied. For the polyvalent primers and after exploratory experiments, a 1:1 molar ratio mix of 2YH1:2YH2 and a 4:1 molar ratio of 2YH3:2YH4 were found to be appropriate for sense and antisense priming, respectively. Other conditions of the reactions were adjusted as follows: 2 µmol/L primers, 1.5 mmol/L MgCl2, and 200 µmol/L deoxy-NTPs; 50 C annealing temperature; and 40 mU/µL DNA polymerase (DynaZyme II, DNA polymerase, Finnzymes OY, Espoo, Finland).

Detection of multiple CC chemokines

PCR products from each thyroid gland were immobilized in parallel onto five membranes (Hybond-N+ nylon, Amersham Pharmacia Biotech, Aylesbury, UK) using a slot blotting apparatus (Bio-Dot SF, Bio-Rad Laboratories, Inc., Hercules, CA). Each membrane could accommodate 22 duplicate samples plus the controls. Oligonucleotide probes were labeled with [{alpha}-32P]ATP (Amersham Pharmacia Biotech) as described previously (29). Membranes were prehybridized at 62–65°C for 1 h with hybridization mixture: 2 x PBS, 1% blocking reagent (Roche Molecular Biochemicals), 0.1% N-lauroylsarcosine, and 0.02% SDS. Membranes were hybridized at 50°C for 2 h in the same buffer containing 2–4 x 105 cpm/mL and washed as previously described (30). The stringency of the conditions applied in this step made it possible to detect single mismatches. The five membranes, each with a different probe, were washed simultaneously in the same oven. Specific radioactivity in each slot was measured by radioimaging (Molecular Imager GS-525, Bio-Rad Laboratories, Inc.). To compare readings from different membranes corresponding either to the different chemokines present in the same amplification product or to different samples altogether, we calculated the approximate number of copies of specific chemokine transcript in each amplified sample by extrapolation from the signals produced by two calibrated positive controls blotted in the same membrane; this was the hybridization correction factor. Two negative controls were also included in all membranes: 1) the product of an amplification reaction containing all of the reagents except H2O instead of cDNA, and 2) a mixture of amplified CC chemokine sequences except for the chemokine to be probed in the membrane. These two controls never hybridized with their corresponding CC chemokine-specific probe.

Units and statistical analysis

Results are given as arbitrary units that represent the corrected number of copies of chemokine cDNA present in the amplified sample and reflect the number of copies in the initial sample (see first paragraph of Results). The Mann-Whitney rank sum test was used to analyze the differences in the expression levels of chemokines between GD and MNG groups of patients. The correlation between each two variables was assessed by the Spearman test.


   Results
Top
 Abstract
 Introduction
 Experimental Subjects
 Materials and Methods
 Results
Discussion
 References
 
Single tube PCR assessment of CC chemokine profiles

Simultaneous amplification by RT-PCR of cDNAs from several member of a multigene family with multiple degenerate primers aimed at conserved regions, that we called polyvalent primers, is likely to result in unequal amplification of the different cDNAs. To circumvent this problem, we first prepared template cDNA from each of the five CC chemokines using the sets of specific primers listed in Table 2B. We determined the relative efficiency of amplification for each chemokine by amplifying an equal number of copies of the five CC chemokine template cDNAs using the polyvalent primers (Fig. 1Go) in a radiolabeling PCR (31). The amount of amplified product was measured by radioimaging of the gel. The efficiency of amplification was determined at 20 and 200 molecules/µL, and the results from repeated experiments were consistent. Previous titration experiments had established the ranges of template concentration and cycles within which the reaction maintained exponential kinetics, and those were similar for the five chemokines.

As summarized in Fig. 2Go, the efficiency of amplification followed the hierarchy: MIP-1ß > MCP-1 > MIP-1{alpha} > RANTES (regulated on activation, normal T cell expressed and secreted) >MCP-3, and the amount of amplified product was within the same order of magnitude. An efficiency of amplification coefficient was calculated for each chemokine using the amplification of MIP-1ß as an arbitrary reference point: (cpm chemokine amplification product)/(cpm MIP-1ß amplification). The PCR products were directly immobilized onto nylon membranes to obviate the need for gel electrophoresis and DNA transfer. The specificity of the amplification was confirmed by Southern blotting, using internal oligoprobes (Table 2C). Some of the bands were sequenced as part of another project and were found to contain the corresponding chemokine sequence (data not shown).



View larger version (25K):
[in this window]
[in a new window]
  		Figure 2. Amplification efficiency of the polyvalent primers using cDNA-positive controls as the substrate template. The efficiency of amplification coefficients were calculated for the different chemokines with respect to MIP-1ß. The results from three experiments ± SD are shown.

 
To ensure that we were working within the exponential amplification interval of our protocol in all 20 samples of thyroid tissue, we first analyzed them by RT-PCR using the gene-specific primers (Table 2B). These cDNAs were first normalized according to their GAPDH levels. MIP-1{alpha}, MIP-1ß, and MCP-1 were detected on ethidium bromide-stained gel after 30 cycles. RANTES gave weak bands only after 40 cycles. MCP-3 was not detected. Glands were classified into high and low expressing groups depending on the amount of chemokine transcripts detected (results not shown). Two glands from each group were selected to construct representative amplification curves with the polyvalent primers following the single tube PCR profiling protocol. The radioactive signals of the membranes were quantified by radioimaging. Values were corrected for hybridization (see Materials and Methods) and by dividing them by the efficiency of amplification coefficient and plotted (Fig. 3Go). MIP-1{alpha}, MIP-1ß, and MCP-1 were within the linear phase for the four thyroid glands around 32 cycles. For RANTES, the best interval was around 36 cycles. MCP-3 was not detected. The conditions applied to all samples were based on these results: for MIP-1{alpha}, MIP-1ß, and MCP-1, 32 cycles; and for RANTES and MCP-3, 36 cycles.



View larger version (47K):
[in this window]
[in a new window]
  		Figure 3. Amplification curve of four chemokines using TB255 (GD) and TB287 (GD) as examples of CC chemokines high expressing glands, and TB238 (MNG) and TB280 (MNG) as examples of low expressing glands. {blacktriangleup}, TMB255; {blacktriangledown}, TMB287; {blacksquare}, TMB280; •, TMB238.

 
The profile of CC chemokine expression is different in thyroid glands from GD and MNG patients

The above-modified single tube PCR protocol was applied to a panel of 10 GD and 10 MNG thyroid glands. Normal thyroid samples were not included as controls due to the difficulty in obtaining this type of tissue. We could have used samples from the nonaffected lobe of thyroid glands excised because of nodules, but we (32, 33), as others (34), have found in the past that such samples often contain areas of focal thyroiditis that makes them inadequate controls. Normal thyroid glands from young organ donors (the only appropriate control) are very difficult to obtain. Normalized cDNAs from the 20 glands were tested in 2 independent reactions. Transcripts of MIP-1{alpha}, MIP-1ß, MCP-1, and RANTES were detected in all 20 samples from both GD and MNG glands (Fig. 4Go), whereas MCP-3 was not detected in any sample. GD thyroid glands had a tendency to contain higher levels of CC chemokine transcripts (MIP-1{alpha}, MIP-1ß, MCP-1, and RANTES) than MNG glands. Statistical comparison showed that MIP-1{alpha} and MIP-1ß expression is higher in thyroid glands from GD than in glands from MNG (P < 0.05; Fig. 5Go). A strong correlation was found (r = 0.81; P < 0.05) among the levels of MIP-1ß and MIP-1{alpha} transcripts.




View larger version (103K):
[in this window]
[in a new window]
  		Figure 4. a and b, The expression levels of the different chemokines in the 20 samples. Each bar represents the mean of two independent reactions for the same thyroid tissue. The autoradiographic signals show the raw data, whereas the graph bars show the normalized expression level (in arbitrary units), which reflect the number of copies of the different transcripts. a, MIP-1{alpha} and MIP-1ß results; b, MCP-1 and RANTES results.

 


View larger version (25K):
[in this window]
[in a new window]
  		Figure 5. Summary of the results of CC chemokine expression in GD and MNG glands as assessed by one-tube chemokine profile. Each dot represents the average of two experiments. Arbitrary units reflect the number of copies of the different transcripts (see text and Fig. 4Go for details).

 
Correlation of CC chemokine profiles and clinical and immunopathological features of the cases

The levels of chemokines did not correlate with age, sex, or the titer of thyroid antibodies or with other clinical data. The histopathological data available were nonquantitative, and no obvious correlation was found with them. Expression of human leukocyte antigen (HLA) class I and class II in thyrocytes, a phenomenon probably linked to pathogenesis (35, 36, 37), had been studied by flow cytometry in all but three glands (TB287, TB300, and TB310) as part of a published study (23). We found a significant correlation between the levels of MIP-1{alpha} and MIP-1ß transcripts and the expression of HLA class II (r = 0.676 and r = 0.678 respectively; P < 0.05 for both).

Applicability of the present method to fine needle aspiration biopsy specimen

We knew from another ongoing project of our group that the fine needle biopsy specimen from thyroid autoimmune glands normally contains 104–105 lymphocytes/sample. As these specimens would be the type of sample in which we would like to use the CC chemokine profiling protocol, we assessed its applicability by preparing serial dilutions of mRNA corresponding to 103, 3 x 103, 104, and 3 x 104 intrathyroidal lymphocytes and used them as substrates. Results from three GD glands (TB255, TB257, and TB258) demonstrated that it is perfectly feasible to detect MIP-1{alpha}, MIP-1ß, MCP-1, and RANTES transcripts from as few as 103 intrathyroidal lymphocytes (Fig. 6Go).



View larger version (67K):
[in this window]
[in a new window]
  		Figure 6. Testing the applicability of our single tube PCR protocol for low numbers of cells. Intrathyroidal lymphocytes were extracted from 3 GD glands: TB255, TB257, and TB258. cDNAs corresponding to the indicated number of cells were amplified with polyvalent primers. The amplified products were slot blotted in parallel in 4 membranes, then each hybridized with the specific oligoprobe. 30c, Thirty amplification cycles in the thermocycler; 40c, 40 amplification cycles in the thermocycler.

 

   Discussion
Top
 Abstract
 Introduction
 Experimental Subjects
 Materials and Methods
 Results
 Discussion
References
 
The above results demonstrate that cDNAs of five CC chemokines can be amplified and quantified simultaneously using a single tube PCR protocol that employs mixed/degenerate primers that we call polyvalent. The pros and cons of measuring mRNAs by RT-PCR instead of protein have been debated, but there is some consensus that it can be used as long as the correlation between levels of amplified product and template is maintained, as demonstrated by our titration experiments and correlation with protein levels have been demonstrated in the same or independent studies. This approach is certainly justified when it is the only applicable method, as in the case of very small samples. In this context, the technical interest of our protocol derives from two facts: 1) because each sample undergoes only one retrotranscription and one amplification step, the main sources of RT-PCR variability are avoided, and therefore, chemokine levels can be compared intrasample; and 2) because the cDNAs are normalized and correction coefficients for hybridization and amplification efficiency are used, chemokine profiles from different samples can also be compared.

As presented, this protocol may seem complex because we had to go through a number of normalization steps and introduce several corrections to the results. However, once this initial work was completed, it is was easily applied several times to the 20 samples in a very short time. It is certainly applicable to extremely small samples such as those generated from fine needle biopsies as demonstrated by our intrathyroidal lymphocyte "titration" experiment. On the other hand, this single tube PCR CC chemokine profiling protocol could serve as basis for CXC chemokine or chemokine receptor profiling protocols. We anticipate from our experiments that a more extensive use of mixed primers would not reduce the reproducibility of the reaction, and unrelated cDNAs could be simultaneously be amplified.

Because our group has a long standing interest in thyroid autoimmunity, and to our knowledge there are no reports on the CC chemokine profiles in thyroid autoimmune glands, we applied our one-tube PCR protocol to a panel of Graves’ disease and multinodular goiter thyroid samples. The highly organized lymphomononuclear cell infiltration present in autoimmune thyroid glands suggests the involvement of many chemoattractant factors, among them, and prominently, CC chemokines. It is known that in these glands there is a high proportion of activated T cells of the Th1 type with a memory phenotype, which have been reported to express the CXCR3 and CCR5 chemokine receptors (38). It was not surprising to detect transcripts of the three known ligands of CCR5, i.e. MIP-1ß, MIP-1{alpha}, and RANTES, at higher levels in GD than in MNG glands. As MIP-1ß attracts preferentially CD4+ and MIP-1{alpha} attracts CD8+ T lymphocytes (7), the relative dominance of MIP-1ß over MIP-1{alpha} in GD glands is in keeping with the higher proportion of CD4+ over CD8+ cells found in GD glands (reviewed in Ref. 12 ; 21).

The finding of CC chemokine transcripts in MNG is in agreement with the histopathology of this poorly understood entity, in which there is some degree of focal lymphocytic infiltration even if, in general, it is much less prominent than that in the classical autoimmune thyroid disorders (39). This is the reason why it has been repeatedly proposed that MNG also has an immunological pathogenesis (40, 41). It would be of great interest to expand the study to the ligands of CXCR3 and to investigate the CC chemokine profile in Hashimoto’s thyroiditis glands, in which CD8+ lymphocytes are particularly predominant (21).

The strong correlation between MIP-1{alpha} and MIP-1ß transcript levels is explained because these cytokines share the same cellular source (42) (activated mononuclear cells including T lymphocytes) and regulatory elements in their gene promoters (43), and this confirms the reliability of our protocol.

The most likely sources of the chemokines detected in both GD and MNG are the intrathyroidal inflammatory cells, such as macrophages and lymphocytes. Activated monocytes/macrophages produce MCP-1, MIP-1{alpha}, MIP-1ß, and RANTES, whereas activated T lymphocytes produce MIP-1{alpha}, MIP-1ß, and RANTES (reviewed in Ref. 44). Another possible source of these chemokines could be the thyroid follicular epithelial cells. This latter possibility is particularly interesting because it would indicate that these chemokines are implicated in the initial phase of the disease and not only in the perpetuating stage. In fact, MCP-1 has been detected in primary cultures of human thyrocytes, and its expression is stimulated by the proinflammatory cytokines, IL-1, TNF{alpha}, and IFN{gamma} (25), all of which have been repeatedly detected in thyroid tissues of GD and MNG patients (23, 45, 46). The correlation between the levels of MIP-1ß and MIP-1{alpha} and the degree of HLA class II expression in thyrocytes was expected, as all of these genes are in part regulated by IFN{gamma} (47) and TNF{alpha} (48). MCP-3 was not detected in any of the 20 thyroid tissues by either standard RT-PCR or our protocol. Overexpression of MCP-3 has been reported in inflammatory conditions characterized by the recruitment of basophils, such as allergic diseases, and its absence may only indicate that this chemokine is not involved in GD (49).

One possible clinical application of our single tube PCR profiling protocol in GD patients is in the prediction of recurrence after carbimazol therapy, an unsolved clinical management problem. In principle, assessing cytokine and/or chemokines profiles in a fine needle aspiration biopsy specimen may provide useful information, but the amount of material available is so small that only a technique such as that presented here could be used. We believe that these results are encouraging, and that in untreated patients, in an early stage of the disease and using fine needle biopsy specimens, the differences would be more dramatic and therefore potentially useful for clinicians. Once thermocyclers designed for quantitative PCR become affordable, these or similar tests may become useful to clinicians.

In summary, we present a simple PCR method designed to obtain profiles of transcripts of related genes and we applied it successfully to assess CC chemokine profiles in GD thyroid samples. The finding of CC chemokines at higher levels in GD thyroid gland expands our knowledge on the physiopathology of autoimmune thyroid diseases and points to possible mechanisms that regulate the organization of intrathyroidal lymphocytic infiltrates.

Received January 4, 1999.

Revised April 21, 1999.

Accepted May 5, 1999.


   References
Top
 Abstract
 Introduction
 Experimental Subjects
 Materials and Methods
 Results
 Discussion
 References
 

  1. Heller RA, Schena M, Chai A, et al. 1997 Discovery and analysis of inflammatory disease-related genes using cDNA microarrays. Proc Natl Acad Sci USA. 94:2150–2155.[Abstract/Free Full Text]
  2. Lee C, Wu X, Gibbs RA, Cook R, Munzy D, Caskey CT. 1988 Generation of cDNA probes directed by amino acid sequence: cloning of urateoxidase. Science. 239:1288–1291.[Abstract/Free Full Text]
  3. Baggiolini M, Dewald B, Moser B. 1997 Human chemokines: an update. Annu Rev Immunol. 15:675–705.[CrossRef][Medline]
  4. Vaddi K, Keller M, Newton RC. 1997 The chemokine facts book. London: Academic Press.
  5. Luster A. 1998 Chemokines–chemotactic cytokines that mediate inflammation. N Engl J Med. 338:436–445.[Free Full Text]
  6. Baggiolini M. 1998 Chemokines and leukocyte traffic. Nature. 392:565–568.[CrossRef][Medline]
  7. Taub D, Colon K, Lloyd AR, Oppenheim J, Kelvin D. 1993 Preferential migration of activated CD4+ and CD8+ T cells in response to MIP-1{alpha} and MIP-1ß. Science. 260:355–358.[Abstract/Free Full Text]
  8. Szabo MC, Butcher EC, McIntyre BW, Schall TJ, Bacon KB. 1997 RANTES stimulation of T lymphocyte adhesion and activation: role for LFA-1 and ICAM- 3. Eur J Immunol. 27:1061–1068.[Medline]
  9. Turner L, Ward SG, Westwick J. 1995 RANTES-activated human T lymphocytes. J Immunol. 155:2437–2444.[Abstract]
  10. Taub DD, Ortaldo JR, Turcovski-Corrales SM, Key ML, Longo DL, Murphy WJ. 1996 Chemokines costimulate lymphocyte cytolysis, proliferation, and lymphokine production. J Leukocyte Biol. 59:81–89.[Abstract]
  11. Carr MW, Alon R, Springer TA. 1996 The C-C chemokines MCP-1 differentially modulates the avidity of ß1 and ß2 integrins on T lymphocytes. Immunity. 4:179–187.[CrossRef][Medline]
  12. Weetman AP, McGregor AM. 1994 Autoimmune thyroid disease: further developments in our understanding. Endocr Rev. 15:788–829.[Abstract/Free Full Text]
  13. Doniach D, Roitt IM. 1974 Thyroid auto-allergic diseases. In: Gell PGH, Coombs RRA, Lachman PJ, eds. Clinical aspects of immunology. Oxford: Blackwell; 1355–1386.
  14. Livolsi VA. 1994 The pathology of autoimmune thyroid disease: a review. Thyroid. 4:333–339.[Medline]
  15. Rutenfranz I, Kruse A, Kirchner H. 1991 In situ hybridization of interferon-gamma producing peripheral blood mononuclear cells. Scand J Immunol. 34:169–178.[CrossRef][Medline]
  16. Roura C, Alcalde L, Vargas F, et al. 1993 gamma delta T lymphocytes in endocrine autoimmunity: evidence of expansion in Graves’ disease bot not in type-1 diabetes. Clin Exp Immunol. 92:288–295.[Medline]
  17. Catalfamo M, Roura-Mir C, Sospedra M, et al. 1996 Self-reactive cytotoxic {gamma}-{delta} T lymphocytes in Graves’ disease specifically recognize thyroid epithelial cells. J Immunol. 156:804–811.[Abstract]
  18. McLachlan SM, Prummel MF, Dallow RL, Wiersinga WM, Rapoport B. 1993 Amplification by polymerase chain reaction of immunoglobulin heavy and light chain genes from orbital tissue of patients with Graves’ ophthalmopathy. Autoimmunity. 16:149–152.[Medline]
  19. Watson PF, Pickerill AP, Davis R, Weetman AP. 1994 Analysis of cytokine gene expression in Graves’ disease and multinodular goiter. J Clin Endocrinol Metab. 79:355–360.[Abstract]
  20. Ajjan RA, Watson PF, Weetman AP. 1996 Cytokines and thyroid function. Adv Neuroimmunol. 6:359–386.[Medline]
  21. Roura-Mir C, Catalfamo M, Sospedra M, Alcalde L, Pujol-Borrell R, Jaraquemada D. 1997 Single-cell analysis of the intrathyroidal lymphocytes shows differential cytokine expression in Hashimoto’s and Graves’ disease. Eur J Immunol. 27:3290–3302.[Medline]
  22. Fisfalen ME, Palmer EM, Van Seventer GA, et al. 1997 Thyrotropin-receptor and thyroid peroxidase-specific T cell clones and their cytokine profile in autoimmune thyroid disease. J Clin Endocrinol Metab. 82:3655–3663.[Abstract/Free Full Text]
  23. Sospedra M, Tolosa E, Armengol P, et al. 1997 Hyperexpression of Transporter in antigen processing-1 (TAP-1) in thyroid glands affected by autoimmunity: a contributory factor to the breach of tolerance to thyroid antigens? Clin Exp Immunol. 100:98–106.
  24. Weetman AP, Ajjan RA, Watson PF. 1997 Cytokines and Graves’ disease. Bailliere Clin Endocrinol Metab. 11:481–497.[CrossRef][Medline]
  25. Kasai K, Banba N, Motohashi S, Hattori Y, Manaka K, Shimoda S. 1996 Expression of monocyte chemoattractant protein-1 mRNA and protein in cultured human thyrocytes. FEBS Lett. 394:137–140.[CrossRef][Medline]
  26. Tolosa E, Roura C, Martí M, Belfiore A, Pujol-Borrell R. 1992. Induction of adhesion molecule-1 (ICAM-1) but not of lymphocyte function associated antigen-3 (LFA-3) in thyroid follicular cells. J Autoimmun. 5:119–135.
  27. Edwards D, Mahadevan L. 1992 Protein synthesis inhibitors differentially superinduce c-fos and c-jun by three distinct mechanisms: lack of evidence for labile repressors. EMBO J. 11:2415–2424.[Medline]
  28. Chomczynski P, Sacchi N. 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 162:156–159.[Medline]
  29. Sambrook J. Fritsch E, Maniatis T. 1989 Molecular cloning: a laboratory manual. Cold Spring Harbor: Cold Spring Harbor Laboratory.
  30. Wood W, Gitschier J, Lasky L, Lawn R. 1985 Base composition-independent hybridization in tetrathylammonium chloride: a method for oligonucleotide screening of highly complex gene libraries. Proc Natl Acad Sci USA. 82:1585–1588.[Abstract/Free Full Text]
  31. Gause WC, Adamovicz J. 1995 Use of PCR to quantitate relative differences in gene expression. In: Dieffenbach CW, Dveksler GS, eds. PCR primer: laboratory manual. Cold Spring Harbor: Cold Spring Harbor Laboratory; 293–311.
  32. Lucas-Martin A, Foz-Sala M, Todd I, Bottazzo GF, Pujol-Borrell R. 1988 Occurrence of thyrocite HLA class II expression in a wide variety of thyroid diseases: its correlation with lymphocytic infiltration and thyroid autoantibodies. J Clin Endocrinol Metab. 66:367–375.[Abstract/Free Full Text]
  33. Tolosa E, Roura C, Catalfamo M, et al. 1992 Expression of intracellular adhesion molecule-1 in thyroid follicular cells in autoimmune, non-autoimmune and neoplastic diseases of the thyroid gland: discordance with HLA. J Autoimmun. 5:107–118.[CrossRef][Medline]
  34. Okayasu I, Fujiwara M, Hara Y, Tanaka Y, Rose NR. 1995 Association of chronic lymphocytic thyroiditis and thyroid papillary carcinoma. A study of surgical cases among Japanese, and white and African Americans. Cancer. 76:2312–2318.[CrossRef][Medline]
  35. Bottazzo GF, Pujol-Borrell R, Hanafusa T, Feldmann M. 1983 Hypothesis: role of aberrant HLA-DR expression and antigen presentation in the induction of endocrine autoimmunity. Lancet. 2:1115–1119.[Medline]
  36. Sospedra M, Obiols G, Babi LF, et al. 1995 Hyperinducibility of HLA class II expression of thyroid follicular cells from Graves’ disease. A primary defect?. J Immunol. 154:4213–4222.[Abstract]
  37. Marelli-Berg FM, Weetman A, Frasca L, Deacock SJ, Imami N, Lombardi G, Lechler RI. 1997 Antigen presentation by epithelial cells induces anergic immunoregulatory CD45RO+ T cells and deletion of CD45RA+ T cells. J Immunol. 159:5853–5861.[Abstract]
  38. Qin S, Rottman JB, Myers P, et al. 1998 The chemokine receptors CXCR5 and CCR5 mark subsets of T cells associated with certain inflammatory reactions. J Clin Invest. 101:746–754.[Medline]
  39. Costa A, Ricci C, Benedetto V. 1987 Immunological features of simple endemic goitre. In: Pinchera A, Ingbar SH, McKenzie JM, Fenzi GF, eds. Thyroid autoimmunity. New York, London: Plenum Press; 549–550.
  40. Doniach D, Chiovato L, Hanafusa T, Bottazzo GF. 1982 The implications of "thyroid-growth-immunoglobulins" (TGI) for the understanding of sporadic nontoxic nodular goitre. Springer Semin Immunopathol. 5:433–446.[CrossRef][Medline]
  41. Balsamo A, Botto Micca F, Merlin PA, De Filippis V, Stramignoni A. 1987 Some aspects of cell mediated autoimmunity in endemic nodular goitre. In: Pinchera A, Ingbar SH, McKenzie JM, Fenzi GF, eds. Thyroid autoimmunity. New York, London: Plenum Press; 579–581.
  42. Johnatty RN, Taub DD, Reeder SP, Turcovski-Corrales SM, Cottam DW, Stephenson TJ, Rees RC. 1997 Cytokine and chemokine regulation of proMMP-9 and TIMP-1 production by human peripheral blood lymphocytes. J Immunol. 158:2327–2333.[Abstract]
  43. Irving SG, Zipfel PF, Balke J, et al. 1990 Two inflammatory mediator cytokine genes are closely linked and variably amplified on chromosome 17q. Nucleic Acids Res. 18:3261–3270.[Abstract/Free Full Text]
  44. Ward SG, Backon J, Westwick J. 1998 Chemokines and lymphocytes: more than attraction. Immunity. 9:1–11.[CrossRef][Medline]
  45. Grubeck-Loebenstein B, Buchan G, Chantry D, et al. 1989 Analysis of intrathyroidal cytokine production in thyroid autoimmune disease: thyroid follicular cells produce interleukin-1{alpha} and interleukin-6. Clin Exp Immunol. 77:324–330.[Medline]
  46. Aust G, Scherbaum WA. 1996 Expression of cytokines in the thyroid: thyrocytes as potential cytokine producers. Exp Clin Endocrinol Diabetes. 104(Suppl 4):64–67.
  47. Todd I, Pujol-Borrell R, Hammond LJ, Bottazzo GF, Feldmann M. 1985 Interferon-{gamma} induces HLA-DR expression by thyroid epithelium. Clin Exp Immunol. 61:265–273.[Medline]
  48. Buscema M, Todd I, Deuss U, Hammond L, Mirakian R, Pujol-Borrell R, Bottazzo GF. 1989 Influence of tumor necrosis factor-{alpha} on the modulation by interferon-{gamma} of HLA class II molecules in human thyroid cells and its effect on interferon-{gamma} binding. J Clin Endocrinol Metab. 69:433–439.[Abstract/Free Full Text]
  49. Stafford S, Li H, Forsythe PA, Ryan M, Bravo R, Alam R. 1997 Monocyte chemotactic protein-3 (MCP-3)/fibroblast-induced cytokine (FIC) in eosinophilic inflammation of the airways and the inhibitory effects of an anti-MCP-3/FIC antibody. J. Immunol. 158:4953–4960.



This article has been cited by other articles:


Home page
 Rheumatology (Oxford)Home page
A. Antonelli, C. Ferri, P. Fallahi, S. M. Ferrari, D. Giuggioli, M. Colaci, A. Manfredi, S. Frascerra, F. Franzoni, F. Galetta, et al.
CXCL10 ({alpha}) and CCL2 ( ) chemokines in systemic sclerosis a longitudinal study
Rheumatology, January 1, 2008; 47(1): 45 - 49.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
M. Rotondi, L. Chiovato, S. Romagnani, M. Serio, and P. Romagnani
Role of Chemokines in Endocrine Autoimmune Diseases
Endocr. Rev., August 1, 2007; 28(5): 492 - 520.
[Abstract] [Full Text] [PDF]

Home page
 Eur J EndocrinolHome page
A. Antonelli, P. Fallahi, M. Rotondi, S. M. Ferrari, P. Romagnani, M. Grosso, E. Ferrannini, and M. Serio
Increased serum CXCL10 in Graves' disease or autoimmune thyroiditis is not associated with hyper- or hypothyroidism per se, but is specifically sustained by the autoimmune, inflammatory process.
Eur. J. Endocrinol., May 1, 2006; 154(5): 651 - 658.
[Abstract] [Full Text] [PDF]

Home page
 Eur J EndocrinolHome page
A. Antonelli, M. Rotondi, P. Fallahi, P. Romagnani, S. M. Ferrari, A. Paolicchi, E. Ferrannini, and M. Serio
Increase of interferon-{gamma} inducible {alpha} chemokine CXCL10 but not {beta} chemokine CCL2 serum levels in chronic autoimmune thyroiditis
Eur. J. Endocrinol., February 1, 2005; 152(2): 171 - 177.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. P. Martin, E. C. Coronel, G.-i. Sano, S.-C. Chen, G. Vassileva, C. Canasto-Chibuque, J. D. Sedgwick, P. S. Frenette, M. Lipp, G. C. Furtado, et al.
A Novel Model for Lymphocytic Infiltration of the Thyroid Gland Generated by Transgenic Expression of the CC Chemokine CCL21
J. Immunol., October 15, 2004; 173(8): 4791 - 4798.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M.-P. Armengol, C. B. Cardoso-Schmidt, M. Fernandez, X. Ferrer, R. Pujol-Borrell, and M. Juan
Chemokines Determine Local Lymphoneogenesis and a Reduction of Circulating CXCR4+ T and CCR7 B and T Lymphocytes in Thyroid Autoimmune Diseases
J. Immunol., June 15, 2003; 170(12): 6320 - 6328.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
P. Romagnani, M. Rotondi, E. Lazzeri, L. Lasagni, M. Francalanci, A. Buonamano, S. Milani, P. Vitti, L. Chiovato, M. Tonacchera, et al.
Expression of IP-10/CXCL10 and MIG/CXCL9 in the Thyroid and Increased Levels of IP-10/CXCL10 in the Serum of Patients with Recent-Onset Graves' Disease
Am. J. Pathol., July 1, 2002; 161(1): 195 - 206.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
M. Imaizumi, A. Pritsker, P. Unger, and T. F. Davies
Intrathyroidal Fetal Microchimerism in Pregnancy and Postpartum
Endocrinology, January 1, 2002; 143(1): 247 - 253.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
M. A. GarcIa-Lopez, D. Sancho, F. Sanchez-Madrid, and M. Marazuela
Thyrocytes from Autoimmune Thyroid Disorders Produce the Chemokines IP-10 And Mig and Attract CXCR3+ Lymphocytes
J. Clin. Endocrinol. Metab., October 1, 2001; 86(10): 5008 - 5016.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
C. Simchen, I. Lehmann, D. Sittig, M. Steinert, and G. Aust
Expression and Regulation of Regulated on Activation, Normal T Cells Expressed and Secreted in Thyroid Tissue of Patients with Graves' Disease and Thyroid Autonomy and in Thyroid-Derived Cell Populations
J. Clin. Endocrinol. Metab., December 1, 2000; 85(12): 4758 - 4764.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ashhab, Y.
Right arrow Articles by Pujol-Borrell, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ashhab, Y.
Right arrow Articles by Pujol-Borrell, R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals