Philip F. Watson

A cytotoxic T lymphocyte antigen‐4 (CTLA‐4) gene polymorphism is associated with autoimmune Addison's disease in English patients

Recent studies have demonstrated an association between a microsatellite polymorphism of the CTLA‐4 gene, specifically a 106 base pair allele, and both Graves disease and autoimmune hypothyroidism. The aim of the present study was to determine whether the same polymorphism of the CTLA‐4 gene was associated with autoimmune Addisons disease.

Intrathyroidal cytokine gene expression in Hashimoto's thyroiditis

Hashimoto’s thyroiditis (HT) is an organ‐specific autoimmune disease in which cytokines are likely to have a role in the initiation and perpetuation of the disease. Using the reverse transcription‐polymerase chain reaction (RT‐PCR) we analysed the cytokine profile in four HT tissue samples. Furthermore, cell fractionation was carried out on two tissue samples and cytokine profile was studied in CD4+ and CD8+ T cells, in addition to the residual cellular infiltrate composed of CD4− and CD8− cells. Our results showed IL‐1β, IL‐4 and IL‐6 mRNA expression in three out of four tissue samples, whereas IL‐1α, IL‐2, IL‐8, IL‐10, interferon‐gamma (IFN‐γ) and tumour necrosis factor‐alpha (TNF‐α) were expressed in all tissue samples studied. Expression of IL‐1α and IL‐1β was absent in both CD4+ and CD8+ subsets. However, IL‐2, IL‐4, IL‐6, IL‐8, IFN‐γ and TNF‐α mRNA were detected in both CD4+ and CD8+ subsets. IL‐10 was expressed in the CD4+ subset in one sample, whereas it was negative in both CD8+ fractions. All the cytokines studied were expressed in the residual infiltrate. These results suggest a mixed Th1 and Th2 response in HT, both of which may have an important role in the pathophysiology of the thyroid destructive process through cell‐mediated cytotoxicity, and/or humoral autoimmunity manifested by autoantibody production.

Cytokines and thyroid function.

Cytokines play a crucial role in autoimmune thyroid disease (ATD) through various mechanisms. They are produced in the thyroid by intrathyroidal inflammatory cells, in particular lymphocytes, as well as by the thyroid follicular cells (TFC) themselves and may thus act in a cascade to enhance the autoimmune process (Fig. 1). Cytokines upregulate the inflammatory reaction through stimulation of both T and B cells, resulting in antibody production and tissue injury. In addition, intrathyroidal cytokines induce immunological changes in TFC including enhancement of both major histocompatibility complex (MHC) class I and class II molecule expression, and upregulation of adhesion and complement regulatory molecule expression. Cytokines can also modulate both growth and function of TFC and have a role in extrathyroidal complications of ATD, most importantly thyroid-associated ophthalmopathy (TAO), where they induce fibroblast proliferation and enhance the production of glycosaminoglycans (GAG), resulting in proptosis and the other clinical features of the disease. In addition to these effects, exogenous administration of cytokines has been associated with impairment of thyroid function ranging from the appearance of autoantibodies alone to the development of frank thyroid dysfunction. Cytokines have also been implicated in subacute thyroiditis (SAT) and amiodarone-induced thyroid dysfunction, as well as in thyroid function abnormalities occurring in patients with non-thyroidal illnesses (NTI). Genetic variations in cytokine genes represent potential risk factors for ATD, and disease associations have been described for polymorphisms in IL-1ra and TNF beta genes. Recent experimental evidence suggests the possibility of novel cytokine-based therapeutic approaches for ATD and its complications, in particular TAO.

Association of HLA‐DQA1*0501 with Graves’ disease in English Caucasian men and women

OBJECTIVEu2003A recent report has demonstrated a stronger association between the HLA‐DQA1*0501 allele and Graves’ disease in Caucasian men than in women. Our aim was to confirm this association in a larger series of male Caucasian patients.

Immunoprecipitation of melanogenic enzyme autoantigens with vitiligo sera: evidence for cross-reactive autoantibodies to tyrosinase and tyrosinase-related protein-2 (TRP-2).

In the present study we describe the detection of TRP‐2 antibodies in vitiligo patients using in vitro35S‐labelled human TRP‐2 in a radioimmunoassay. Of 53 vitiligo sera examined in the assay, three (5.9%) were found to be positive for TRP‐2 antibodies. In contrast, 20 control sera, sera from 10 patients with Hashimotos thyroiditis and sera from 10 patients with Graves disease were all negative. All three patients positive for TRP‐2 antibodies (mean age 54 years, age range 50–63 years) had had vitiligo of the symmetrical type for more than 1 year and all of them also had an associated autoimmune disorder: Graves disease in one and autoimmune hypothyroidism in two. In addition, antibodies to the melanogenic enzyme tyrosinase were present in their serum. To examine any immunological cross‐reactivity between TRP‐2 and tyrosinase, the three vitiligo sera positive for TRP‐2 antibodies were preabsorbed with COS‐7 cell extract containing either expressed TRP‐2 or tyrosinase, and subsequently used in the radioimmunoassay. These absorption studies indicated that preincubation with both proteins inhibited the immunoreactivity of the positive sera in the immunoassay using in vitro translated 35S‐TRP‐2. This antibody cross‐reactivity suggests the humoral response to the two melanogenic enzymes in these patients may not be entirely independent.

Autoantibodies to human melanocyte‐specific protein Pmel17 in the sera of vitiligo patients: a sensitive and quantitative radioimmunoassay (RIA)

In the present study we describe the in vitro transcription‐translation of human melanocyte‐specific protein Pmel17 cDNA and subsequent use of the resulting 35S‐labelled Pmel17 in an RIA to analyse vitiligo sera for the presence of Pmel17 antibodies. Of 53 vitiligo sera examined in the assay, three (5.9%) were found to be positive for Pmel17 antibodies. In contrast, sera from 20 healthy controls, 10 patients with Hashimotos thyroiditis and 10 patients with Graves disease (GD) were all negative for Pmel17 antibodies. All three patients positive for Pmel17 antibodies (aged 50–63 years) had had vitiligo of the symmetrical type for >u20031 year and all of them also had an associated autoimmune disorder: GD in one and autoimmune hypothyroidism in two. In addition, all three patients had antibodies to the melanogenic enzymes tyrosinase, tyrosinase‐related protein‐1 (TRP‐1) and tyrosinase‐related protein‐2 (TRP‐2) in their serum. Absorption studies indicated that preincubation with COS‐7 cell extract containing expressed Pmel17 absorbed out the immunoreactivity of the three sera positive in the RIA, confirming the anti‐Pmel17 reactivity of the sera from these patients. In contrast, COS‐7 cell extracts containing either expressed tyrosinase, TRP‐1 or TRP‐2 did not remove the anti‐Pmel17 reactivity of the three sera in the RIA. This lack of cross‐reactivity suggests that the humoral response to Pmel17 in these patients is specific and independent of the antibody reactivity to tyrosinase, TRP‐1 and TRP‐2.

Somatic hypermutation in antoimmune thyroid disease

Summary: Autoimmune thyroid disease is one of the most common autoimmune diseases. There is typically patient antibody (Ab) reactivity to one or more of the antigens thyroglobulin (Tg), thyroid peroxidase (TPO) and the thyroid stimulating hormone receptor (TSFlr). With the advent of combinatorial library technology, there has been an enormous increase in the number of sequences from Ab to Tg and TPO, The repertoire of both Tg and TPO Ab is restricted and indicates the importance of somatic hypermutation in the development of the high affinity, Ab response. However, there are still too few sequences to determine patterns in which the mutation occurs, which residues are introduced during substitution and how individual substitutions affect the affinity of the Ab, Ab to the TSHr are of far greater pathological significance than those to Tg and TPO, but the current repertoire of Ab to the TSHr has yet to include the high affinity IgG Ab characteristic of patient serum Ab, Instructive analysis of the role of somatic hypermutation in the development of TSHr Ab therefore still awaits the isolation of the pathologically active repertoire. Despite this, the Ab response in thyroid autoimmunity remains one of the best characterised of human autoimmune diseases.

No restriction of intrathyroidal T cell receptor Vα families in the thyroid of Graves’disease

Recently it has been reported that the intrathyroidal T cells in Graves’disease display restriction in Vα T cell receptor (TcR) gene family usage, although this is not found with TcR Vβ gene families in the same individuals. We have performed a qualitative analysis of TcR Vα family usage in 12 patients with Graves’disease by reverse transcription and polymerase chain reaction (PCR) amplification of RNA extracted from isolated, unstimulated intrathyroidal lymphocytes and from snap‐frozen whole thyroid specimens. No restriction was observed, with 10–15 Vα gene families being amplified in all cases. The pattern of usage was similar to that in peripheral blood lymphocytes derived from normal subjects (n = 3) and from patients with Graves’disease (n = 3), as well as that present in the thyroids of patients with non‐autoimmune toxic multinodular goitre (n= 4). These results indicated that there is no marked restriction of the unselected intrathyroidal T cell population in patients with Graves’disease who have been treated with antithyroid drugs.

Cytokines and Graves' disease

Cytokines are an extraordinarily diverse group of molecules, with pleiotropic and often overlapping effects. They are crucial to the autoimmune response, and, in particular, regulation of CD4+ and CD8+ T-cell function depends on the balance of cytokines produced during an immune response. It is also now clear that cytokines are produced by a wide array of cells, including the thyroid follicular cells (TFCs). Intrathyroidal lymphocytes produce a heterogeneous pattern of cytokines and we have summarized the likely effects of these. In Graves disease, TFCs can themselves express immunologically important molecules as the result of cytokine stimulation and these could contribute to the perpetuation of the autoimmune process. In addition, cytokines have a number of generally inhibitory effects on thyroid hormone production which would tend to counter the stimulatory effects of thyroid-stimulating hormone receptor antibodies in Graves disease.

Molecular mapping of epitopes on melanocyte-specific protein Pmel17 which are recognized by autoantibodies in patients with vitiligo

Previously, we reported the identification of Pmel17 autoantibodies in some patients with vitiligo. Here, we have determined the B cell epitopes on Pmel17 which are recognized by these autoantibodies. Deletion derivatives of Pmel17 cDNA were constructed using either subcloning of specific cDNA fragments or polymerase chain reaction amplification. Full‐length Pmel17 cDNA and its truncated derivatives were then translated in vitro to produce [35S]‐labelled proteins. The radiolabelled ligands were used subsequently in radiobinding assays to investigate the reactivity of sera from vitiligo patients. Two epitope regions were identified: one located at the C‐terminal end of Pmel17 between amino acids 634–644 and one in a central region of the protein between amino acids 326–341. Computer analysis of the potential B cell epitopes on Pmel17 revealed that the epitope domain encompassing amino acids 326–341 was located in an area of the protein which was predicted to be highly antigenic. In contrast, the epitope identified at the C‐terminal of Pmel17 (amino acids 634–644) was located in a region of the protein predicted to have low antigenicity. The amino acid sequences of the identified Pmel17 epitopes were compared to the amino acid sequences of the related melanogenic enzymes tyrosinase, tyrosinase‐related protein‐1 and tyrosinase‐related protein‐2. However, no sequence homology was found between either of the Pmel17 epitopes and the aforementioned proteins. This finding is consistent with our previous study in which we were unable to show the presence of Pmel17 antibodies which were cross‐reactive with either tyrosinase, tyrosinase‐related protein‐1 or tyrosinase‐related protein‐2. It also suggests that the IgG response to Pmel17 is distinct from the antibody response to the other melanocyte‐specific antigens.