Enhanced production of gamma-interferon by thyroid-derived T cell clones from patients with Hashimoto''s thyroiditis.

T lymphocytes from thyroid infiltrate and peripheral blood (PB) of four patients with Hashimoto's thyroiditis (HT) were analysed at clonal level for their ability to secrete interleukin 2 (IL-2) and gamma-interferon (gamma-IFN). As controls, T cell clones from PB of four normal donors and from spleen of two trauma victims were used. While no abnormality was found in the capacity to produce IL-2, the proportion of gamma-IFN-producing (IFN-P) T cell clones derived from HT infiltrates was significantly higher (P less than 0.0005) than that of IFN-P clones derived from normal or patient PB. Most of CD4+ and CD8+ IFN-P clones from thyroid infiltrates, as well as a proportion of CD4+ PB-derived clones of patients with HT, released higher amounts of gamma-IFN than control clones. A relationship could be demonstrated between high gamma-IFN production and natural killer (NK) activity in T cell clones from thyroid and PB of HT patients. In fact, the percentage of IFN-P clones with NK potential (NK+) was remarkably higher (P less than 0.0005) in thyroid infiltrates than in normal spleen or PB. The proportion of IFN-P NK+ clones from patient PB was also significantly increased (P less than 0.02) but, unlike thyroid-derived clones in which the majority of IFN-P NK+ clones were CD8+, most PB-derived IFN-P NK+ clones from the same patients expressed the CD4+ phenotype. Almost all thyroid NK+ clones could be triggered to produce more gamma-IFN, while gamma-IFN synthesis by NK-negative thyroid clones was comparable to that of control clones. In view of the multiple effects ascribed to gamma-IFN in the cascade of events leading to immune responses, the abnormal potential to gamma-IFN secretion shown by intrathyroidal T lymphocytes may be of importance in the pathogenesis of autoimmune thyroiditis.     

Production of interleukin (IL)-5 and IL-10 accompanies T helper cell type 1 (Th1) cytokine responses to a major thyroid self-antigen, thyroglobulin, in health and autoimmune thyroid disease

Tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma exert detrimental effects in organ-specific autoimmune disease, while both destructive and protective roles have been demonstrated for interleukin (IL)-10, IL-4 and IL-5. We examined the production of these cytokines by peripheral blood mononuclear cells (PBMC) from patients with Hashimoto's thyroiditis (HT), Graves' disease (GD) and healthy controls, upon exposure to a thyroid self-antigen, human thyroglobulin (Tg), in the presence of autologous serum. Initially, TNF-alpha and IL-2 were produced in all three groups, accompanied by IL-10. Release of IFN-gamma, IL-4 and, notably, IL-5 ensued. Both patient groups exhibited increased TNF-alpha, IL-2, IFN-gamma and IL-10 responses, and PBMC from HT patients secreted lower amounts of IL-5 than male, but not female, controls. Enhanced TNF-alpha production by HT cells also occurred in the presence of pooled normal sera, indicating a dependency on intrinsic cellular factors. Conversely, higher production of TNF-alpha and IL-5 occurred in the presence of autologous sera than in the presence of pooled normal sera in both patient groups, indicating a dependency on serum constituents. Complement appeared to promote the production of IL-2 and particularly IL-5, the levels of which were reduced by neutralization of complement by heat- or zymosan treatment. The production of IFN-gamma and IL-2 of the three groups together correlated directly with the serum anti-Tg activity. Moreover, TNF-alpha, IFN-gamma, IL-5 and IL-10 responses were markedly inhibited by partial denaturation of Tg by boiling. We hypothesize that autoantibodies and complement may promote mixed Th1/Th2 cell cytokine responses by enhancing the uptake of autoantigens by antigen-presenting cells.     

Peripheral blood and intrathyroidal T cell clones from patients with thyroid autoimmune diseases

For a better understanding of the pathogenesis of thyroid autoimmune diseases, we have studied morphological and functional properties of T clones from peripheral blood lymphocytes (PBL) and from intrathyroidal lymphocytes (ITL) obtained from 3 patients with Graves' disease or 1 Hashimoto's thyroiditis. Investigations were carried out on clones cultured alone or cocultured with autologous thyrocytes. Clonage efficiency ranged from 30% to 33% for PBL and 10% to 36% for ITL. A predominance of CD4-positive clones was observed whatever the origin of the lymphocytes or the autoimmune pathology. Gamma interferon (IFN-gamma) was detected in the majority (17/19) of the clones tested. Intracytoplasmic interleukin (IL-4) was secreted in 7/19 clones and both cytokines were produced in 5/19 clones. In coculture a proliferative response and tumour necrosis factor (TNF-alpha) production were observed with 6 clones (4 from Graves thyrocytes and 2 from thyroiditis). No cytotoxic clone was derived from Graves or thyroiditis tissues. These data demonstrate that the large majority of T clones are principally CD4-T cells; all the clones secreted TNF-alpha and a large majority produced IFN-gamma. Only a few clones produced IL-4 alone or associated with IFN-gamma. Six T clones induced proliferative response and of TNF-alpha secretion in coculture. Further investigations must be performed on these antigen-reactive T clones to analyse their role in the pathogenesis of the human thyroid autoimmune diseases.     

Cytokines, thyroid autoantibody synthesis and thyroid cell survival in culture

In autoimmune thyroid disease lymphoid cells infiltrating the thyroid gland occur in conspicuous aggregates or as a diffusely distributed population invading the thyroid follicles. Consequently cytokines secreted by activated T cells or macrophages could influence neighbouring thyroid cells as well as other lymphocytes. We have investigated this possibility using recombinant cytokines. Thyroid cell survival was assessed in terms of mitochondrial dehydrogenase activity in monolayers exposed to tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-1 (IL-1 alpha and beta) and interleukin-2 (IL-2) in the presence or absence of thyroid-stimulating hormone (TSH). Neither TNF-alpha nor IL-2 affected thyroid cell survival, IFN-gamma was usually inhibitory and IL-1 alpha slightly enhanced cell survival in some experiments. However, the effects were small and variable and were not enhanced by potentially synergistic combinations of cytokines, longer periods of exposure, or different culture conditions. In contrast, IFN-gamma, IL-2 and TNF-alpha inhibited the ability of thyroid lymphocytes from patients with Graves' disease and Hashimoto's thyroiditis to synthesize autoantibodies to thyroid peroxidase (TPO) and thyroglobulin (Tg). Comparison of lymphoid populations isolated by digestion and/or mechanical disaggregation indicated that a population of activated B cells, plasma cells and T cells, intimately associated with thyroid cells since they could only be extracted by digestion, was influenced by cytokines. Our studies suggest that in addition to its well-recognized ability to induce MHC class II antigens on thyroid cells, IFN-gamma may inhibit thyroid cell proliferation and TNF-alpha, IFN-gamma and IL-2 may down-regulate thyroid autoantibody synthesis.     

Single-cell analysis of intrathyroidal lymphocytes shows differential cytokine expression in Hashimoto's and Graves' disease

Most human organ-specific autoimmune diseases such as Hashimoto's thyroiditis (HT) are considered to be Th1 mediated, and a quantitative dominance of Th1 cells in thyroid infiltrates from both Graves' disease (GD) and HT affected glands has been reported. However, Th2 dominance would be expected in GD, where thyroid hyperfunction induced by stimulating antibodies predominates over tissue destruction. We have analyzed the interleukin-4 (IL-4), interferon-γ (IFN-γ) production by T cells at the single-cell level, both in infiltrating lymphocytes isolated from digested GD and HT thyroid glands and in derived T cell lines, by direct intracellular cytokine detection. Results showed a heterogeneous pattern of cytokine production in bulk GD infiltrates and derived T cell lines, and a similar pattern was observed in the much larger HT infiltrates. Both type 1 and type 2 cytokines were simultaneously produced by the infiltrating populations, and T cells with both patterns as well as intermediate patterns similar to ThO cells could be detected ex vivo. However, the larger T lymphocytes, presumably activated and responsible for the autoimmune damage, predominantly produced IL-4 in GD and IFN-γ in HT. The specificity of the Th2 responses in GD was suggested by the enrichment in IL-4 production after antigen-specific expansion of two oligoclonal T cell lines. These data show that both type 1 and type 2 cytokines are produced in the thyroid glands affected by autoimmunity and that the difference between diseases may be the effect of a functionally dominant population at a given time. This in vivo chronically activated antigen-specific population, producing type 1 or type 2 cytokines locally, may be responsible for the effect finally leading to one of the disease states.     

In vivo activated cytotoxic T cells in the thyroid infiltrate of patients with Hashimoto''s thyroiditis.

High proportions of T8+ cells with inverted T4/T8 ratio were found in freshly isolated thyroid lymphocytes from patients with Hashimoto's thyroiditis. In addition, about one third of thyroid infiltrating cells expressed the TAC antigen, whereas in patient peripheral blood (PB) or normal lymphocytes from PB or lymphoid organs the percentage of TAC-positive cells was consistently lower than 10%. Following negative selection with OKT4 or OKT8 monoclonal antibodies and complement, TAC+ T cells were enriched in the T8+ cell population. Thyroid infiltrating T cells from two patients underwent two different cloning procedures. In the first, single T cells were initially activated with phytohaemagglutinin (PHA) and interleukin 2 (IL-2), in the other with recombinant IL-2 (rIL-2) alone. The majority of T cell clones obtained by initial PHA-stimulation (55-65%) had the T8+ phenotype, but the frequency of T8+ clones obtained by stimulating T cells with rIL-2 alone was even higher (78 & 71%, respectively). The majority of T8+ clones elicited by PHA (35/37 & 36/38) and all the T8+ clones (36/36 & 22/22) obtained from thyroid infiltrates with initial stimulation by rIL-2 displayed cytolytic activity. Most of cytolytic T8+ clones obtained from thyroid infiltrates with both cloning procedures, displayed NK activity against human K562 and MOLT-4 target cells, but not against a NK-resistant target, such as Raji cells. These data suggest that in Hashimoto's disease a considerable proportion of thyroid infiltrating T cells are in vivo activated T8+ cytolytic T cells with NK activity, which may be of importance in determining or maintaining the tissue damage of the target gland.     

Tumour necrosis factor-alpha synthesis by cerebrospinal-fluid-derived T cell clones from patients with multiple sclerosis.

T cell clones derived from cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) were analysed for their ability to produce interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2) and interleukin-4 (IL-4). The CSF-T clones were compared for their ability to produce cytokines with autologous peripheral T clones and with liver-infiltrating T cell clones from patients with chronic active hepatitis. IL-4 production was also compared with that by peripheral T clones derived from atopic patients. All the CSF-T clones (both CD4+ and CD8+) produced large amounts of IFN-gamma and particularly of TNF-alpha. These cytokines were synthesized in significantly larger amounts by CSF T clones than by reference clones. Moreover, they were capable of secreting IL-2, but not IL-4. We conclude that the CSF-CD4+ T clones could constitute a subset with functional properties similar to those of T helper 1 (Th1)inflammatory cells of the mouse; and that the large amounts of TNF produced by CSF T cell clones strongly suggest a significant role for this cytokine in MS immunopathogenesis.     

CD4+ T-cell clones from autoimmune thyroid tissue cannot be classified according to their lymphokine production

In order to define whether CD4+ T cells from autoimmune and non-autoimmune thyroid tissue could be classified according to their mediator production, lymphokine production was studied in 63 thyroid-derived CD4+ T-cell clones from four patients with Graves' disease, one with Hashimoto's thyroiditis, and one with non-toxic goitre (9-12 clones per patient). The production of interleukin 2 (IL-2), gamma interferon (IFN-gamma), tumour necrosis factor alpha (TNF-alpha), lymphotoxin (LT), interleukin 6 (IL-6) and transforming growth factor beta (TGF-beta) was assessed at the mRNA level by slot-blot analysis in unstimulated clones as well as after activation with monoclonal anti-CD3 (OKT3) and IL-2. No lymphokine production was found in unstimulated clones, whereas 56% of the clones produced all six lymphokines simultaneously after stimulation. In the remaining 44% usually not more than one lymphokine was missing from the complete panel. Lymphokine mRNA concentrations varied between different clones and different patients, but, in this small sample, not between the diseases from which the clones were originated. There was a significant correlation between IL-6, LT, and IL-2 mRNA levels and T-cell helper function, which was estimated by the stimulation of thyroid microsomal autoantibody production using autologous peripheral B cells. TGF-beta and IFN-gamma mRNA expression was unrelated to T-cell help. The results demonstrate that intrathyroid T cells from autoimmune and non-autoimmune thyroid disorders cannot be classified according to their lymphokine production, unlike some results with in vitro-induced mouse T-cell clones, where two populations, Th1 and Th2, have been described. Single T cells are capable of producing a whole panel of lymphokines and thus are capable of triggering a multitude of different processes.     

自身免疫甲状腺病患者血清中IL-12和IL-18水平的分析

目的:提供自身免疫甲状腺病(AITD)患者体内Th1/Th2平衡紊乱的依据。方法:应用酶联免疫吸附法(ELISA)测定27例Graves病(GD)、24例甲状腺功能正常的桥本甲状腺炎(HT)、25例甲状腺功能低下的HT患者及20例正常对照者血清中IL12和IL18的浓度,并检测GD患者的甲状腺刺激性抗体。结果:GD患者与甲状腺功能正常的HT患者血中IL12、IL18水平无明显差异,但均高于正常对照者的相应水平。甲状腺功能低下的HT患者血中IL12和IL18的水平与正常对照者无差异。在GD和甲功正常的HT,IL18与IL12呈明显正相关。在GD,IL12和IL18均与其甲状腺刺激性抗体(TSAb)活性呈正相关。在甲状腺功能正常的HT还存在IL12和IL18二者与甲状腺球蛋白抗体(TgAb)的显著性正相关。结论:提示Th1型细胞在GD和HT两种AITD的发病中均起重要作用。通过抑制Th2型免疫反应,促进向Th1型的转变来治疗GD时,有可能导致病情恶化。     

CD4(+) T cell clones producing both interferon-gamma and interleukin-10 predominate in bronchoalveolar lavages of active pulmonary tuberculosis patients.

The pattern of cytokine production in T cell clones derived from bronchoalveolar lavages (BAL) of active pulmonary tuberculosis (TB) patients was analyzed in clones obtained by limiting dilution procedures which expand with high efficiency either total T lymphocytes, independently of their antigen-recognition specificity, or Mycobacterium tuberculosis-specific T cells. BAL-derived clones, representative of CD4(+) cells from five patients with active TB, produced significantly higher amounts of IFN-gamma than BAL-derived CD4(+) clones from three inactive TB donors or four controls (with unrelated, noninfectious pathology). Average IL-4 and IL-10 production did not differ significantly in the three groups. Although these data suggest a predominant Th1 response to M. tuberculosis infection in the lungs, the majority of BAL-derived CD4(+) clones produced both IFN-gamma and IL-10 and the percentage of clones with this pattern of cytokine production was significantly higher in clones derived from BAL of active TB patients than from controls. Only rare clones derived from peripheral blood (PB)-derived CD45RO(+) CD4(+) T cells of both patients (nine cases) and controls (four cases) produced both IFN-gamma and IL-10; instead, the IL-10-producing clones derived from PB T cells most often also produced IL-4, displaying a typical Th2 phenotype. Higher average amounts of IFN-gamma and IL-10 were produced by BAL-derived CD8(+) clones of four active TB patients than of four controls, although the frequency of CD8(+) clones producing both IFN-gamma and IL-10 was lower than that of CD4(+) clones. The M. tuberculosis-specific BAL-derived T cell clones from three active TB patients were almost exclusively CD4(+) and produced consistently high levels of IFN-gamma often in association with IL-10, but very rarely with IL-4. Unlike the BAL-derived clones, the M. tuberculosis-specific clones derived from PB CD45RO(+) CD4(+) T cells of three different active TB patients and two healthy donors showed large individual variability in cytokine production as well as in the proportion of CD4(+), CD8(+), or TCR gamma/delta(+) clones. These results indicate the predominance of CD4(+) T cells producing both the proinflammatory cytokine IFN-gamma and the anti-inflammatory cytokine IL-10 in BAL of patients with active TB.     

Cytokine regulation of HLA on thyroid epithelial cells

The regulation of class I and class II HLA expression in human thyroid follicular cells was studied in vitro. Tumour necrosis factor-alpha (TNF-alpha) enhanced the expression of class I antigen on thyrocytes, but these cytokines had little effect on the expression of class II antigen. Interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) did not affect class I and class II antigen expression. The combination of interferon-gamma (IFN-gamma) with TNF-alpha or IL-1 beta enhanced the induction of class I and class II antigens, compared with the effect of IFN-gamma alone. Neither class I nor class II expression was induced by IL-6 alone or in combination with IFN-gamma. These findings suggest that TNF-alpha and IL-1 beta may have an important role in inappropriate expression of HLA antigens on thyrocytes in thyroid gland.     

Binding of peripheral blood and thyroidal T lymphocytes to thyroid cell monolayers: possible role of "homing-like" receptors in the pathogenesis of thyroid autoimmunity.

The specific binding of blood lymphocyte populations to non-antigenic receptors on high endothelial cells in peripheral lymphoid organs is well established. Such "homing" receptors in target tissues may also play a role in the early phase of organ specific autoimmunity. In this study binding of peripheral blood and thyroid-derived T cells to human thyroid (THY) cells was examined. Binding was measured as the percentage 51Cr-labelled T cells bound to THY monolayers. Interferon-gamma (IFN-gamma) enhanced the binding of peripheral blood T cells (PBL-T) from patients with Graves' disease (GD) and normals, but not from patients with Hashimoto's thyroiditis (HT), to allogeneic THY monolayers. The binding of T cells from patients with HT to untreated allogeneic THY monolayers was significantly greater than that for T cells from patients with GD or normals. TSH inhibited the IFN-gamma enhancement of binding of PBL-T from 4 normals and one HT patient to both allogeneic (n = 5) and autologous (n = 4) THY monolayers. Intra-thyroid T cells (ITL-T) bound to autologous THY monolayers significantly more than PBL-T from the same patient, while ITL from patients with HT or Graves' disease bound more than their PBL-T to both allogeneic and autologous THY monolayers. ITL-T, but not PBL-T, bound significantly more to autologous THY than to autologous thyroid-derived fibroblast monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Th1 cytokine pattern in sarcoidosis is expressed by bronchoalveolar CD4+ and CD8+ T cells

The pathogenesis of pulmonary sarcoidosis has been related to an increased production of Th1-like cytokines. However, cytokine expression in sarcoidosis has not been systematically studied at a single-cell level. We therefore investigated the expression of IL-2, IL-4, IL-13, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) intracellularly in bronchoalveolar lavage (BAL) and peripheral blood CD3+ T lymphocytes from patients with pulmonary sarcoidosis (radiologic stage II-III, n = 8) and normal controls (n = 9) by flow cytometry. In contrast to IL-4 and IL-13, the percentage of T lymphocytes expressing intracellular IL-2 (49.3 +/- 21.3% versus 14.5 +/- 15.6%), IFN-gamma (75.5 +/- 14.9% versus 32.6 +/- 18.7%) and TNF-alpha (68.3 +/- 18.7% versus 36.8 +/- 20.8%) was significantly higher in patients with sarcoidosis than in normal controls (each P < 0.005). In contrast to BAL lymphocytes, expression of these cytokines in peripheral blood lymphocytes did not differ between patients with sarcoidosis and normal controls. Close correlations were observed between the percentages of BAL lymphocytes expressing intracellular IL-2, IFN-gamma and TNF-alpha, but not for IL-4 or IL-13. Analysis of the expression of these cytokines in T lymphocyte subsets revealed IL-2, IFN-gamma, and TNF-alpha in CD4+ as well as CD8+ T lymphocytes, suggesting a contribution of TC1 cells to the production of proinflammatory cytokines in sarcoidosis. We conclude that a Th1-like cytokine pattern can be observed in CD4+ as well as in CD8+ BAL T lymphocytes in patients with pulmonary sarcoidosis.     

Increased numbers of Th2-like CD4+ T cells in target organs and in the allergen-specific repertoire of allergic patients. Possible role of IL-4 produced by non-T cells.

Phytohemagglutinin (PHA)-induced human T cell clones (TCC) derived from conjunctival flogistic tissues of 3 patients with vernal conjunctivitis produced unusually high amounts of interleukin-4 (IL-4) and no, or limited amounts of, gamma-interferon (IFN-gamma). Allergen (Dermatophagoides pteronyssinus or Lolium perenne group I)-specific TCC derived from peripheral blood of two atopic donors produced significantly higher amounts of IL-4 and significantly lower amounts of IFN-gamma than TCC specific for bacterial antigens (tetanus toxoid and PPD) contemporarily established from the same donors. These data provide evidence for a compartimentalization of Th2-like helper T cells in target organs and in the allergen-specific T cell repertoire of allergic patients. Non-B, non-T bone marrow cells could produce IL-4, but not IL-2 or IFN-gamma, in response to cross-linkage of Fc epsilon type I receptors. These cells may further contribute to the maintenance and amplification of allergic inflammation.     

Increased T helper 1 cytokine responses by circulating T cells are present in women with recurrent pregnancy losses and in infertile women with multiple implantation failures after IVF

BACKGROUND: We aimed to study T-helper 1 (Th1) and Th2 intracellular cytokine expression in peripheral blood lymphocytes of women with recurrent spontaneous abortions (RSA) or infertility with multiple implantation failures after IVF cycles. METHODS: Twenty-six women with three or more RSA and 23 with two or more IVF failures (14 with no history of spontaneous abortion (SAB) and nine with more than one SAB) comprised the two study groups. Twenty-one non-pregnant healthy multiparous women served as controls. Proportions (%) of lymphocytes containing IFN-gamma, TNF-alpha, IL-4 and IL-10 and the Th1/Th2 ratios of IFN-gamma/IL-4, IFN-gamma/IL-10, TNF-alpha/IL-4 and TNF-alpha/IL-10 in CD3+, CD3+/CD8- (T helper) and CD3+/CD8+ (T suppressor) cells were measured by 4-colour flow cytometry. RESULTS: RSA women demonstrated significantly higher Th1/Th2 ratios of IFN-gamma/IL-4 (P < 0.01), TNF-alpha/IL-4 and TNF-alpha/IL-10 (P < 0.05 each) in CD3+/CD8- T helper cells than those of controls. The proportion of TNF-alpha producing CD3+/CD8- cells (P < 0.05), and the Th1/Th2 ratios of TNF-alpha/IL-4 (P < 0.05) and TNF-alpha/IL-10 (P < 0.005) in CD3+/CD8- cells were significantly higher in women with multiple IVF failures without SAB as compared with those of controls. CONCLUSIONS: The prevalence of dominant Th1 immune responses in peripheral blood lymphocytes may reflect the systemic contribution of Th1 cytokines to RSA or multiple implantation failures in IVF cycles.     

Localized Wegener's granulomatosis: predominance of CD26 and IFN-gamma expression

The immune response in Wegener's granulomatosis (WG) has been characterized as a predominant, potentially pathogenic Th1-like reaction by blood T cells and T-cell clones from diseased tissues. To elucidate further the immunopathogenic mechanisms, this study analysed the phenotypes of inflammatory infiltrates in frozen nasal biopsies with involvement of the upper respiratory tract only (localized or 'initial phase' WG) and with multi-organ involvement, including systemic vasculitis (generalized WG). The expression and production of Th1 and Th2 cytokines were examined in tissue specimens and peripheral blood mononuclear cells (PBMCs) of localized and generalized WG. The number of CD3+ T cells in inflammatory infiltrates ranged from 50 to 70%, together with approximately 30% CD14+ monocytes/macrophages. An average of 40% of T cells expressed CD26 in nasal biopsies of localized WG, compared with about 16% in specimens of generalized WG. In parallel, a higher number of interferon-gamma (IFN-gamma)-positive cells were detected in nasal tissue of localized than in generalized WG. PBMCs from localized WG similarly exhibited higher spontaneous IFN-gamma production in contrast to generalized WG (207 vs. 3 pg/ml, p<0.05). Interleukin-4 (IL-4) mRNA was found in higher amounts in generalized than in localized WG. IL-4 production was negligible in both disease and controls. In addition, both IL-10 mRNA and IL-10 protein levels of activated PBMCs from localized WG were elevated when compared with generalized disease (574 vs. 154 pg/ml, p<0.05) or healthy controls (574 vs. 246 pg/ml, p<0.05). It is conluded that in nasal tissues, mainly CD4+/CD26+ T cells as well as IFN-gamma-positive cells may support a polarized Th1-like immune response. Furthermore, the data suggest that this in situ immune response is already initiated and established in localized WG, accompanied by increased peripheral IFN-gamma and IL-10 production.     

Bee venom phospholipase A2-specific T cell clones from human allergic and non-allergic individuals: cytokine patterns change in response to the antigen concentration.

Protein antigens with both allergenic and immunoprotective properties represent appropriate molecules to study IgE and IgG regulation. We have established a panel of T cell clones specific to bee venom phospholipase A2 (PLA) from human individuals allergic, hyposensitized or immune (protected) to bee sting. All clones obtained were CD3+, CD4+ and expressed alpha, beta T cell receptor. Depending on the T cell clone, maximal stimulation required 1 to 100 micrograms/ml of PLA, and the addition of interleukin (IL)-2 and/or IL-4 increased their antigen-dependent proliferation. Following antigen stimulation, the clones produced IL-4, interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor. Most clones also produced tumor necrosis factor alpha (TNF-alpha) and tumor necrosis factor beta (TNF-beta), and some produced IL-5 and/or IL-2. Both absolute and relative amounts of secreted cytokines depended on the antigen concentration. At low antigen doses, IL-4 was produced but little or not IFN-gamma, whereas at higher PLA concentrations significant amounts of both IL-4 and IFN-gamma were obtained. Thus, these PLA-specific T cell clones could be classified according to the changes in the ratio of IL-4/IFN-gamma production in response to increasing antigen concentrations. Clones derived from allergic and hyposensitized individuals required higher critical amounts of antigen for IFN-gamma induction, and expressed increasing IL-4/IFN-gamma ratios with increasing concentrations of PLA. Modulation of cytokine patterns by the dose of the antigen may be a driving force for IgE or IgG formation resulting in allergy or immunoprotection.     

Colonic epithelial cell lines as a source of interleukin-8: stimulation by inflammatory cytokines and bacterial lipopolysaccharide.

Cytokines produced by intestinal epithelial cells may function as signals to neighbouring immune and inflammatory cells. We investigated production of the neutrophil and T-lymphocyte chemotactic cytokine interleukin-8 (IL-8) by intestinal epithelial cells using four colonic adenocarcinoma cell lines, T84, CaCo-2, HT29 and SW620, as a model system. These cell lines secreted substantial amounts of IL-8 if stimulated with IL-1 beta, tumour necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma), except CaCo-2 cells, which responded only to IL-1 beta. Bacterial lipopolysaccharide (LPS) was also an efficient stimulus of IL-8 release in SW620 and HT29 cells, whereas T84 and CaCo-2 cells were completely unresponsive to LPS, IL-8 secretion was greater at 4 hr after stimulation and was accompanied by induction of IL-8 messenger RNA. In T84 cells IFN-gamma and epidermal growth factor (EGF) stimulated IL-8 secretion synergistically with TNF-alpha, whereas in SW620 cells this synergism occurred only between IFN-gamma and TNF-alpha. IL-4, IL-10 and transforming growth factor-beta (TGF-beta), which can down-regulate IL-8 production in macrophages, had no effect on IL-8 generation by our cell lines. Adenocarcinoma cell culture supernatants also induced rapid transients of intracellular calcium in neutrophils. Depending on cell line and stimulus, supernatant bioactivity was completely or partially abrogated by neutralizing antibodies to IL-8, indicating that the cell lines investigated also generate other neutrophil-activating factors. IL-8 and possibly other chemokines generated by colonic adenocarcinomas may help to attract tumour-infiltrating leucocytes. Possibly, normal intestinal epithelial cells also have the potential to secrete this potent chemoattractant and thus might contribute to inflammatory responses of the intestinal mucosa, for example in inflammatory bowel disease.     

Association of Interleukin-1B and Interleukin-4 Gene Variants with Autoimmune Thyroid Diseases in Tunisian Population

Autoimmune thyroid diseases (AITD) including Graves’ disease (GD) and Hashimoto’s thyroiditis (HT) are complex genetic diseases. Cytokines IL-1B and IL-4 play a role in the pathogenesis of AITD. This study was conducted on Tunisian patients with GD or HT to investigate the association of IL-1B and IL-4 gene polymorphisms with the risk and the prognosis of AITD. A total of 358 healthy controls and 341 patients with AITDs (249 HT and 92 GD) were genotyped for IL-1B+3953C/T and IL-4 intron 3 VNTR polymorphisms. A significant association was found between IL-1B+3953C/T polymorphism and GD or HT, both in the dominant and additive models. The IL-1B+3953T allele was associated with GD (p = 0.0003, OR = 1.93, CI = 1.34-2.78) and HT (p = 0.009, OR = 1.43, CI = 1.09-1.88). The IL-4 VNTR polymorphism was associated only with HT risk both in additive (p = 0.03, OR = 0.31, CI = 0.11-0.86) and recessive (p = 0.03, OR = 3.04, CI = 1.13-8.17) models. No significant association was found between IL-1B+3953C/T polymorphism and change in the serum concentrations of TSH and FT4 in GD and HT patients. In HT patients, the IL-1B+3953T allele (p = 0.009, OR = 0.42, CI = 0.22-0.83) and the IL-1B+3953T/T genotype (p = 0.03, OR = 0.21, CI = 0.04-1.07) were more frequent in the absence than in the presence of an anti-TPO antibody. The proportion of HT patients with the P1P2 genotype of the IL-4 gene was significantly higher in the absence than in the presence of the anti-TPO antibody (p = 0.04, OR = 0.39, CI = 0.17-0.89). These preliminary results suggest that IL-1B and IL-4 gene polymorphisms may be associated with GD and HT susceptibility and may represent prognostic factors for predicting the severity of HT.     

Inflammatory cytokines in small intestinal mucosa of patients with potential coeliac disease

T helper cell type 1 (Th1) response to gluten has been implicated in the pathogenesis of coeliac disease (CD). To characterize immunological activation and mild inflammations leading to overt CD in potential coeliac patients, jejunal biopsies were obtained from family members of patients with CD or dermatitis herpetiformis (DH). Nine family members and one latent CD, eight CD patients and eight normal controls furnished jejunal biopsy specimens. Immunohistochemical staining of sections for interleukin-1alpha (IL-1alpha), IL-2, IL-4, interferon-gamma (IFN-gamma), tumour necrosis factor alpha (TNF-alpha), CD3, gammadelta-T cell receptor (gammadelta-TCR), and alphabeta-TCR was carried out with monoclonal antibodies. Further, expression of IL-4 and IFN-gamma messenger RNA was detected by radioactive in situ hybridization in these same samples. In lamina propria, CD patients and potential CD patients had higher densities of IL-2 (P = 0.028, P = 0.043), IL-4 (P = 0.021, P = 0.034) and IFN-gamma positive cells (P = 0.000, P = 0.009) than did controls. Moreover, CD patients showed a higher density of TNF-alpha positive cells (P = 0.012, P = 0.001) than the other two groups, and expression of IFN-gamma mRNA (P = 0.035) was higher in them than in the other two study groups. Additionally, higher densities of TNF-alpha and IFN-gamma positive cells occurred in potential CD patients with high gammadelta-TCR+ intraepithelial lymphocytes (IELs). Our findings support the hypothesis that lamina propria T cells and macrophages, through their secretion of cytokines, play a central role in the pathogenesis of coeliac disease. The inflammatory cytokines found in potential CD specimens strongly suggest that these inflammatory markers can be identified long before visible villous changes have occurred.