Circulating lymphocyte subpopulations in Hashimoto thyroiditis

Peripheral blood and T and B lymphocytes and [¹²⁵I]thyroglobulin-binding lymphocytes were investigated in twenty-two euthyroid Hashimoto thyroiditis patients and in twenty-two age- and sex-matched normal subjects. Although the total lymphocyte count in Hashimoto patients (mean±SEM = 1226±187/mm³) was lower than in normal subjects (1603±156/mm³) this difference was not statistically significant. There was, however, a statistically significant reduction in the proportion of circulating T lymphocytes in the Hashimoto patients (mean±SEM = 57·4±2·5%) as assessed by the sheep red-cell rosette method when compared with the normal controls (mean±SEM = 66·7±1·8%). The proportion of B lymphocytes in the peripheral blood as assessed by indirect immunofluorescence, was not significantly different being 21·6±2·1% in the Hashimoto patients and 20·2±1·1% in normal subjects.

[¹²⁵I]thyroglobulin-binding lymphocytes, as assessed by autoradiography were present in the circulation of nineteen Hashimoto patients with a mean frequency of 8·37±1·15/10⁴ lymphocytes and in thirteen normal subjects with a mean of 8·84±0·93/10⁴ lymphocytes. There was no difference in the degree of [¹²⁵I]thyroglobulin binding between the two groups as determined by grain count analysis. There was no apparent correlation between age or thyroglobulin antibody titres and the frequency of [¹²⁵I]thyroglobulin-binding lymphocytes. Thyroglobulin-binding lymphocytes were increased 100-fold in a Hashimoto thyroid biopsy in comparison to the patient's peripheral blood.

     

In situ characterization of T lymphocyte subpopulations in leprosy in the mangabey monkey.

Leprosy in the mangabey monkey is an experimental model which is similar both clinically and histologically to human lepromatous leprosy. The immunopathology of these diseases was compared using monoclonal antibodies against T lymphocyte subpopulations in frozen tissue sections with an immunoperoxidase technique. In both mangabey and human lepromatous granulomas OKT4 (or Leu 3a) and Leu 2a cells were scattered among macrophages with greater numbers of Leu 2a as compared with OKT4 (or Leu 3a) cells. The results suggest that from an immunopathological standpoint experimental leprosy in mangabeys will provide a suitable model for the investigation of the pathogenesis of human lepromatous leprosy and for the evaluation of new antileprosy vaccines.     

HLA-DR-positive leucocyte subpopulations in human skin include dendritic cells, macrophages, and CD7-negative T cells.

The immunophenotypes of the HLA-DR-positive leucocyte populations in normal human skin were studied using an extensive panel of monoclonal antibodies, which included antibodies from the Third International Leucocyte Differentiation Antigen Workshop (3rd LDAW). Langerhans' cells (LC) in the epidermis stained with antibodies from CD15c, Groups 10, 12a, 12b and 15, of the myeloid panel and from CD39 of the B-cell panel. However, LC did not react with CD14 antibodies or 63D3, which are frequently used to stain tissue macrophages. In addition to epidermal LC (26 cells/linear mm) a significant population of CD1a-positive cells was identified in the papillary dermis (7 cells/linear mm of overlying epidermis). The dermal HLA-DR-positive leucocytes consisted of three cell populations. The most numerous cell type stained with antibodies to monocytes/macrophages. There were fewer, though substantial, numbers of T lymphocytes (mainly CD7-negative) and the least numerous was the population of CD1a-positive cells. The CD1a-positive cells and the population of dermal cells that stain with monocyte/macrophage markers are both potential antigen-presenting cells for the skin-associated immune system.     

Expression of bcl-2, Bax and Fas in oxyphil cells of Hashimoto thyroiditis

Immunoreactivity for bcl-2, Bax and Fas was analysed in 16 cases with Hashimoto thyroiditis. Bcl-2-expression was constantly seen in regular thyrocytes and in the mantle-zone of lymphofollicular infiltrates. However, thyrocytes in the vicinity of lymphoid infiltrates and, especially, mitochondria-rich oxyphil cells exhibited reduced staining or none at all for bcl-2. Bax was found to be weakly reactive or negative in normal thyrocytes and was not up-regulated in bcl-2-deficient epithelial cells. In contrast, expression of Fas was markedly increased both in typical thyrocytes and in oxyphil cells within areas of lymphocytic infiltration. In conclusion, focal lack of bcl-2 expression together with up-regulation of Fas is a constant feature of Hashimoto thyroiditis. The reaction pattern of oxyphil cells is identical to that of affected typical thyrocytes without proliferation of mitochondria. Loss of bcl-2 with up-regulation of Fas is therefore likely to precede oncocytic change. Whether these alterations are involved in the process of oncocytic transformation remains to be clarified, however. Received: 25 June 1999 / Accepted: 14 January 1999     

Class II antigens in Hashimoto thyroiditis. I. Synthesis and expression of HLA-DR and HLA-DQ by thyroid epithelial cells

Aberrant expression of HLA-DR antigens on epithelial cells is seen in various organ-specific autoimmune disorders including Hashimoto thyroiditis (HT). Expression of HLA-DQ has so far not been demonstrated on these cells. We report here that thyroid epithelial cells (TEC) in HT, in addition to the known aberrant expression of HLA-DR, coexpress HLA-DQ antigens. Furthermore we provide evidence that class II antigens are synthesized by TEC themselves by demonstration of intracellular HLA-DR gamma-chain. These findings support the theory that TEC may be able to present (auto)antigens in vivo thus perhaps contributing to the perpetuation of thyroid destruction. As expression of class II antigens on TEC was never observed in non- or weakly infiltrated areas, we propose that infiltration by T cells is necessary to induce this aberrant expression of class II antigens.     

Excess iodine promotes apoptosis of thyroid follicular epithelial cells by inducing autophagy suppression and is associated with Hashimoto thyroiditis disease

The incidence of the autoimmune thyroid disease Hashimoto thyroiditis (HT) has increased in recent years, and increasing evidence supports the contribution of excess iodine intake to thyroid disease. In this study, we examined the status of autophagy and apoptosis in thyroid tissues obtained from patients with HT, and we determined the effects of excessive iodine on the autophagy and apoptosis of thyroid follicular cells (TFCs) in an attempt to elucidate the effects of excess iodine on HT development. Our results showed decreases in the autophagy-related protein LC3B-II, and increases in caspase-3 were observed in thyroid tissues from HT patients. Interestingly, the suppression of autophagy activity in TFCs was induced by excess iodine in vitro, and this process is mediated through transforming growth factor-β1 downregulation and activation of the Akt/mTOR signaling pathway. In addition, excess iodine induced autophagy suppression and enhanced reactive oxygen species (ROS) production and apoptosis of TFCs, which could be rescued by the activation of autophagy. Taken together, our results demonstrated that excess iodine contributed to autophagy suppression and apoptosis of TFCs, which could be important factors predisposing to increased risk of HT development.     

The IL18 gene and Hashimoto thyroiditis in children

Interleukin 18 (IL18) stimulates interferon-γ production in Th1 cells which are prominent in the thyroid of Hashimoto thyroiditis (HT). We investigated the association between the IL18 gene and HT. There were 116 children with HT and 1272 controls. rs187238 and rs1946518 in the promoter region of the IL18 gene were genotyped. Differences in genotype, allele, carrier, and haplotype distributions between patients and controls were compared. A Pc value <0.05 was considered significant. The frequency of the C/G genotype of rs187238 was significantly higher in patients and conferred a risk of HT (OR, 1.96; 95% CI, 1.30–2.95; Pc, 0.0021). So did the frequencies of allele C (OR, 1.73; 95% CI, 1.22–2.44; Pc, 0.0035) and carrier C (OR, 1.96; 95% CI, 1.31–2.92; Pc, 0.0017), however the frequency of the G/G genotype was significantly lower in patients than in controls (OR, 0.51; 95% CI, 0.34–0.76; Pc, 0.0034). There was no association between HT and rs1946518. The CT haplotype was significantly more frequent in patients than in controls and conferred a risk of HT (OR, 1.76; 95% CI, 1.24–2.49; Pc, 0.0049). We concluded that the IL18 gene was associated with HT in children. The rs187238C allele and CT haplotype conferred a risk of HT.     

Interaction of rubella virus with human immune cells. I. Permissiveness of lymphocyte subpopulations

The ability of rubella virus to infect and replicate in various lymphocyte subpopulations was examined. Purified populations of B-cells, CD4+ T-cells and CD8+ T-cells were found to support high levels of viral replication. In addition, when mixed PBMC were infected in vitro, viral antigen was shown to be expressed on the surfaces of CD4+ and CD8+ T-cells by flow cytometry indicating that RV does not have a selective tropism for a specific type of lymphoreticular cell.     

In vivo effects of leptin on lymphocyte subpopulations in mice

Leptin, a hormone-cytokine mainly produced by the adipose tissue, has pleitropic effects on many biological system including metabolic, endocrine, and immune system. Although it is well known that leptin controls food intake on hypothalamic regions of brain, the role of leptin in hematopoietic and immune processes has been mainly investigated with in vitro and transgenic mouse studies. The aim of this study was to investigate the effects of peripheral leptin on lymphocyte subpopulation. Initially forty male Swiss albino mice were divided into five groups. Mice in group I (Control) were given serum physiologic (SP) and group L100, group L250, group L500, and group L1000 were given 100, 250, 500 and 1000 μg/kg/day recombinant mouse leptin, respectively. Leptin or SP was injected subcutaneously for the next 6 days. Daily food/water intake was recorded for each group. At the end of the study, whole blood samples (500 μl) were obtained via intracardiac punction in anesthetized mice. Leptin levels and lymphocyte subpopulations in blood samples were analyzed. We show that no in vivo dose-dependent effect of leptin is existed on lymphocyte subpopulations count in mice. Treatment of mice with high-dose leptin led to increase only CD4+ cells (P<0.05). In addition, high-dose leptin slightly increased CD3+ cells but this was not statistically confirmed (P=0.08). Notably, it was found that leptin caused insignificant changes on body weight and food intake in normal body weight mice. The data support that high-dose leptin has proliferative effect on CD4+ cells in vivo. However, more in vivo study needs to be examined to clarify how leptin affect lymphocyte subpopulations.     

Studies on lymphocyte subpopulations and the effect of age on immune competence in turkeys

We characterized the lymphocyte subpopulations and investigated the effect of age on cellular and humoral immunity, development of lymphoid organs, and the relative proportions of CD4⁺ and CD8⁺ cells in turkeys. The mitogenic responses of peripheral T cells were poorly developed at hatch but developed rapidly after hatch and reached adult levels by 2 weeks-of-age. The average percentage of CD4⁺ cells was 45, 29.8, and 26.3 in the thymi, peripheral blood, and spleens, respectively, in turkeys. The mean percentage of CD8⁺ cells in the thymi, peripheral blood, and spleens of turkeys was 53.8, 13.6, and 15.5, respectively. Age did not influence the relative proportions of CD4⁺ and CD8⁺ T cells in the spleens and peripheral blood of turkeys. The mean percentages of IgM⁺ cells in the bursae and spleens were 78.5 and 26.8, respectively. Day-old turkeys did not develop detectable antibodies to either thymus dependent or independent antigens. However, 2 week or older turkeys showed good humoral responses. Inoculation of BSA at hatch induced tolerance, whereas injection of SRBC did not. Analysis of relative organ weights of turkey lymphoid organs showed that spleens and thymi developed rapidly during the first week-of-age.     

In situ identification of idiotype-positive cells participating in the immune response to phosphorylcholine

The phosphorylcholine idiotype (Id)/anti-Id system has been used to study the role of antigen-specific cells in antigen-induced microenvironmental changes. Anti-Id staining of lymph nodes following PC immunization shows the presence of Id on follicular dendritic cells at 12 h and in plasma cells beginning at day 3. Germinal centers began to form at day 3, peaking in size and number at days 8-10. Scattered Id-positive small lymphocytes are present in germinal centers but with rare exceptions over 98% of germinal center cells are Id-negative. Idiotype-positive small lymphocytes are depleted from primary follicles adjacent to germinal centers but not from distant, unstimulated nodes. These results extend previous studies showing architectural alterations in lymph nodes following antigenic stimulation and demonstrate antigen-specific cells are a prominent component of these antigen-induced microenvironmental changes.     

T lymphocyte subpopulations and Ia-positive T cells in patients with immunodeficiency.

T lymphocyte subpopulations (T gamma and Tmu) were studied in a group of 36 adult patients with immunodeficiency. Proportions and numbers of Ia(+) T cells were also studied in comparison to 46 normal adult controls. Values for per cent and total numbers of T gamma and Tmu cells indicated no uniform abnormality. Mean normal percentage of Ia(+) T cells was 2.4% whereas 16 to 29 immunodeficient patients showed elevated proportions and absolute numbers of Ia(+) T cells. Striking fluctuation in proportions of Ia(+) T cells was noted in serial studies of five immunodeficient subjects in contrast to similar analyses of normal controls. A correlation (P less than 0.01) was recorded between absolute numbers of Ia(+) T cells in immune deficiency patients and numbers of T mu cells. Depletion of T gamma cells by EA rosetting in patients with late-onset primary acquired hypogammaglobulinaemia did not result in significant change in IgG or IgM synthesis with T gamma-depleted T cells were co-cultured with normal B cells. Depletion of Ia(+) T cells likewise did not significantly influence Ig synthesis in co-culture with normal or immune-deficient B cells. These studies emphasize the complexity of defects present among any large group of patients with immune deficiency.     

T lymphocyte subpopulations and B lymphocyte cells in caecum and spleen of chicks infected with Salmonella enteritidis

The effect of low and high doses of Salmonella enteritidis PT4 (SE) on immunocompetent cells in caecum and spleen of one-day-old chicks was investigated. Subsets of T lymphocytes positive for CD3, CD4, CD8 and B lymphocytes (Bu1b-positive cells) were counted in the caecum after immunohistochemical staining and the relative percentage of these cells in the spleen was analysed using a FACScan cytometer on days 7, 10, 14, 21, and 27 post-inoculation (pi). In the low dose group, the number of CD3+ and CD4+ cells in the caecum had significantly increased at day 10 pi. Both CD8+ and Bu1b+ cells were significantly higher on day 14 pi in this group. In the high-dose group, the number of CD4+ cells had significantly increased at day 7 pi. CD3+, CD8+, and Bu1b+ cells showed prolonged proliferation at days 7 up to 21 pi. Splenic lymphocytes demonstrated significant changes only in the high dose group. The percentage of splenic CD4+ cells was decreased at day 7 pi. A decrease in CD3+ and CD8+ cells was found at day 14 pi in this group.     

Rare detection of phenotypically immature lymphocytes in Hashimoto thyroiditis and rheumatoid arthritis

It has been suggested that recombination activating gene (RAG)-dependent revision of the immunoglobulin genes in germinal centres may contribute to local production of autoantibodies in Hashimoto thyroiditis (HT) and rheumatoid arthritis (RA). To test this hypothesis we examined HT and RA tissues for expression of RAG and terminal deoxynucleotidyl transferase (TdT) in situ. Paraffin-embedded tissues from 19 HT patients and from 20 RA patients were subjected to immunohistochemistry using TdT-specific antibodies. Expression of the RAGs was studied by in situ hybridisation. Tonsil sections were used as a control. Expression of TdT and RAGs was detected in extrafollicular lymphocytes in control tonsil sections. By contrast, only rare TdT-expressing cells were identified in 11 of 19 HT and in 2 of 20 RA samples. Germinal centre B-cells were consistently TdT- and RAG-negative. These results suggest that local RAG-dependent receptor revision in germinal centres is unlikely to contribute to production of autoantibodies in HT and RA. The presence of TdT-positive extrafollicular cells may represent an influx of immature cells in the context of chronic immune stimulation.     

In situ injury-induced release of basic-fibroblast growth factor from corneal epithelial cells.

Basic-fibroblast growth factor (b-FGF) binds to heparan sulfate proteoglycan in Bowman's layer of the cornea. The mechanism by which the molecule is deposited in Bowman's layer is the subject of controversy since b-FGF lacks a signal peptide sequence for extracellular secretion. Using immunofluorescence, the authors studied the presence and distribution of b-FGF in the bovine cornea and the conditions under which it could be released and bound to Bowman's layer. The results indicate that corneal epithelium contains b-FGF but that uninjured corneas do not contain detectable levels of b-FGF in Bowman's layer. Injury to the corneal epithelium results in the binding of b-FGF to Bowman's layer. Removal of the intact corneal epithelium without cell injury does not result in the binding of b-FGF to Bowman's layer. These findings suggest that one mechanism for the release of b-FGF from corneal epithelial cells is passive leakage after cell injury with secondary binding to Bowman's layer.     

In situ identification of T lymphocyte subsets and HLA-DR expressing cells in the human skin tuberculin reaction

T lymphocyte subsets and HLA-DR expressing cells were studied with an immunohistochemical double staining technique in frozen sections of human skin 6, 48 and 96 hr after intradermal PPD injections. The number of lymphocytes reacting with monoclonal Leu 1 antibodies (all mature peripheral T cells) increased with time. The majority of the T lymphocytes at 48 and 96 hr reacted with Leu 3a ('helper/inducer' phenotype) antibodies and a few with Leu 2a ('suppressor/cytotoxic' phenotype) antibodies. Apposition of T lymphocytes of both subsets to HLA-DR expressing cells occurred in the dermis as well as in the epidermis. The study gives a morphological picture of the cell-mediated immune reactions in delayed type of hypersensitivity consistent with in vitro experiments on proliferative responses to soluble antigens.     

Human lymphocyte subpopulations: rosette formation with sheep, human and horse red blood cells

Rosette formation between human lymphocytes and horse red blood cells could be promoted by a low pH medium, overnight incubation and a temperature of 4 degrees C. The percent of sheep, horse and human rosette-forming cells in the peripheral blood were 71.7 +/- 1.8, 30.5 +/- 2.8 and 28.3 +/- 3.4 respectively. However, their percentages in thymuses were 97.1 +/- 1.1, 91.4 +/- 2.4 and 89.0 +/- 3.4. Using preparations of isolated subpopulations, it was observed that the horse and human red cell rosette-forming cells were probably also "early" sheep red cell rosette-forming cells. Rosette formation with all three types of red blood cells were inhibited by a preparation of Fetuin-glycopeptide.     

Restricted kappa/lambda light chain ratio by flow cytometry in germinal center B cells in Hashimoto thyroiditis

To determine the diagnostic significance of the kappa/lambda ratio in germinal center (GC) B cells in Hashimoto thyroiditis (HT), we used 4-color flow cytometry to immunophenotype 27 samples (21 patients) of well-characterized HT B-cell clonality was analyzed further by polymerase chain reaction (PCR) of the immunoglobulin heavy chain (IgH) and bcl-2/IgH fusion genes using DNA extracted from aspirate smears and/or paraffin-embedded tissues. By flow cytometric analysis, the CD10+ GC B cells had a higher mean +/- SD kappa/lambda ratio than the CD10- B cells (5.1 +/- 3.3 vs 2.0 +/- 0.8; P < .0001, Student t test). In 18 samples (67%), CD10+ GC B cells had a kappa/lambda ratio greater than 3.07 (the upper limit of kappa/lambda ratio reported in reactive nodes; range, 3.2-14.4 in the 18 cases). Cases tested by PCR showed no evidence of a clonal proliferation. None of 21 cases developed lymphoma during clinical follow-up of up to 3 years. The kappa/lambda ratio of CD10+ GC B cells in HT can be skewed markedly beyond that reported in reactive lymph nodes. This finding frequently is present in HT. Pathologists should be familiar with this phenomenon to prevent misdiagnosis of follicular lymphoma in patients with HT.     

Oxidative stress and antioxidant enzyme activities in patients with Hashimoto’s thyroiditis

We investigated the parameters of oxidative stress in 71 Hashimoto’s thyroiditis patients. They were divided into three sub-groups according to the thyroid function: group I—euthyroid subjects; group II—hypothyroid subjects; and group III—subjects treated with Levothyroxin. Thirty healthy subjects were studied as controls. The level of lipid peroxidation (malondialdehyde, MDA) in the plasma and the antioxidant defences such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) activities in erythrocytes were measured. Concentrations of MDA and SOD activity were not different in sub-groups of patients and controls. CAT activity was significantly lower in group II in comparison with both controls (p = 0.01) and group III (p = 0.02) as well as in group I compared to the controls (p = 0.04). Activity of GPX in erythrocytes in hypothyroidism was significantly higher compared to controls (p = 0.02). GPX activity in both groups I and III tended to be lower in comparison with controls. Our results indicate a deficiency of cellular antioxidative defense in Hashimoto’s thyroiditis patients in all stages of disease. Accordingly, we suppose that the supplementation with antioxidants from an early stage of the disease, in addition to thyroid hormone replacement, may have a positive benefit in the treatment.