High-dose growth hormone does not affect proinflammatory cytokine (tumor necrosis factor-alpha, interleukin-6, and interferon-gamma) release from activated peripheral blood mononuclear cells or after minimal to moderate surgical stress

High-dose GH therapy, with GH doses 10-20 times the normal replacement dose for GH-deficient adults, has been used as an anti-catabolic agent in a number of different patient groups. A recent study, however, has shown an increase in mortality in critically ill patients treated with high-dose GH. The increased mortality was associated with multiorgan failure, septic shock, and uncontrolled infection, suggesting that GH may have altered the immune response. The GH receptor and GH are both expressed in peripheral blood mononuclear cells (PBMCs); thus, GH could act as either an endocrine or an autocrine modulator of the immune response. We have examined the hypothesis that high-dose GH therapy may induce proinflammatory cytokines, which are implicated in septic shock. To do this we measured cytokine production by PBMCs incubated in conditions that simulated high-dose GH therapy, and we measured cytokine levels in patients undergoing laparoscopic cholecystectomy who were randomized to receive either high-dose GH therapy (13 IU/m2 x day) or placebo. To confirm the biological activity of GH in our cell culture system we used a Stat5 functional assay. In this assay GH induced a bell-shaped curve, with a maximal response at GH levels between 100-1,000 ng/mL. PBMCs from healthy volunteers were incubated with GH in doses from 1-1,000 ng/mL for 6-72 h under resting conditions and after activation with endotoxin and the mixed lymphocyte reaction. Studies were repeated with PBMCs from six individuals using a GH dose of 100 ng/mL (the level of GH found after high-dose GH therapy) and an endotoxin dose that gave a submaximal response (0.01 ng/mL). GH had no effect on cell proliferation or the production of tumor necrosis factor-alpha (TNFalpha), interleukin-6 (IL-6), or interferon-gamma (IFNgamma). In patients undergoing laparoscopic cholecystectomy there was a time-related effect of surgery on cytokine levels. There was a rise in IL-6 and a fall in TNFalpha at 24 h after surgery; however, high-dose GH therapy had no effect on the cytokine response. We considered the possibility that endogenous GH production by PBMCs could influence the cytokine response in activated PBMCs; however, incubation of PBMCs in the presence of the GH receptor antagonist, B2036, had no effect on TNFalpha, IL-6, or IFNgamma production by PBMCs in either the mixed lymphocyte reaction or when activated by endotoxin. These results suggest that high-dose GH therapy does not alter the proinflammatory cytokine response to surgery or endotoxin. The results do not exclude an effect of GH on the immune response, but they suggest that the mortality seen in critically ill patients may be due to factors other than immune modulation.     

T cell receptor phenotypes in autoimmune thyroid disease

We have examined T cell receptor expression by peripheral blood and thyroidal lymphocytes in Graves' disease and Hashimoto's thyroiditis, using monoclonal antibodies directed against three beta chain variable region families and against the gamma chain. There was no abnormal distribution of positive staining with the beta chain reagents (compared to normal peripheral blood) in either the thyroid or the blood. However, thyroidal lymphocytes contained an excess of gamma-chain-bearing T cells, compared to peripheral blood, in five of the seven patients in whom simultaneous samples were available. The gamma-chain-positive T cells were not altered in the blood lymphocyte population of untreated Graves' patients. These results suggest that the T cell response in autoimmune thyroid disease is polyclonal and that there may be a role (such as cytotoxicity) for T cells expressing the gamma delta type of T cell receptor in thyroiditis.     

Association analysis of the cytotoxic T lymphocyte antigen-4 (CTLA-4) and autoimmune regulator-1 (AIRE-1) genes in sporadic autoimmune Addison's disease

Although autoimmune Addison's disease (AAD) may occur as a component of the monogenic autoimmune polyendocrinopathy type 1 syndrome (APS1), it is most commonly found as an isolated disorder or associated with the autoimmune polyendocrinopathy type 2 syndrome (APS2). It is likely that sporadic (non-APS1) AAD is inherited as a complex trait; however, apart from the major histocompatibility complex, the susceptibility genes remain unknown. We have examined polymorphisms at two non-major histocompatibility complex candidate susceptibility loci in sporadic (non-APS1) AAD: the cytotoxic T lymphocyte antigen-4 (CTLA-4) gene and the autoimmune regulator (AIRE-1) gene. DNA samples from AAD subjects (n = 90) and local controls (n = 144 for CTLA-4; n = 576 for AIRE-1) were analyzed for the CTLA-4A/G polymorphism in exon 1 of the CTLA-4 gene and for the common mutant AIRE-1 allele (964de113) in United Kingdom subjects with APS1, by using the restriction enzymes Bst7II and BsrBI, respectively. There was an association of the G allele at CTLA-4A/G in AAD subjects (P = 0.008 vs. controls), which was stronger in subjects with AAD as a component of APS2 than in subjects with isolated AAD. In contrast, the mutant AIRE-1 964del13 allele was carried in one each of the 576 (0.2%) control subjects and the 90 (1.1%) AAD subjects as a heterozygote (P = 0.254, not significant), suggesting that this common AIRE-1 gene abnormality does not have a major role in sporadic (non-APS1) AAD.     

细胞因子对体外培养的人球后成纤维细胞增殖及合成透明质酸、胶原蛋白的影响

目的探讨甲状腺相关眼病（ＴＡＯ）的细胞免疫机制，并为临床上开展细胞因子或抗细胞因子疗法提供理论依据。方法采用Ｔｈ１细胞代表细胞因子干扰素（ＩＦＮ）γ和Ｔｈ２细胞代表细胞因子白细胞介素（ＩＬ）４，刺激体外培养的人球后成纤维细胞（ＲＦ），并用液体闪烁测量和放射自显影技术观察ＲＦ的增殖和合成透明质酸（ＨＡ）、Ⅳ型胶原蛋白的能力。结果ＩＦＮγ明显刺激ＲＦ增殖及合成ＨＡ，抑制其合成Ⅳ型胶原蛋白；ＩＬ４刺激ＲＦ增殖和合成Ⅳ型胶原蛋白，却抑制其合成ＨＡ；ＩＦＮγ（１００Ｕ／ｍｌ）和ＩＬ４（１μｇ／Ｌ）共同作用于ＲＦ时，可拮抗其各自对ＲＦ的刺激增殖作用，对ＨＡ和Ⅳ型胶原蛋白合成的刺激或抑制作用也得到中和。结论在ＴＡＯ发生发展中，ＩＦＮγ主要起炎症的致病作用，而ＩＬ４则对ＴＡＯ起修复和调节作用，ＩＬ４可以拮抗ＩＦＮγ对ＲＦ的致病作用     

Thyroid follicular cells produce interleukin-8.

Interleukin-8 (IL-8) is a potent proinflammatory cytokine known to be produced by several cell types. To elucidate whether endocrine cells can also make IL-8, we have tested supernatants from eleven thyroid follicular cell primary cultures. IL-8 was readily detected under basal conditions (range 3.4-32.1 ng/ml from 1 x 10(5) cells in 3 days) and was increased 4-20 fold by stimulation with IL-1. TSH and tumor necrosis factor had an inconsistent effect, while gamma-interferon reduced basal and IL-1-stimulated IL-8 production. Since IL-8 can act as a chemoattractant for lymphocytes, these observations may explain in part the accumulation of lymphocytes within the gland in autoimmune thyroiditis.     

Immunological effects of Graves' disease therapy

Graves' disease is caused by thyroid stimulating antibodies and is accompanied by other autoimmune phenomena predisposing ultimately towards hypothyroidism. Antithyroid drugs directly alter the natural history of the condition, causing a remission in 50% of cases; part of this is likely to be due to their immunomodulatory effect. Surgery and radioiodine treatment are also accompanied by immunological changes which may affect outcome. In particular, some cases of hypothyroidism may be the result of a hastening of the natural progression to destructive thyroiditis.     

Thyroid cells in Graves'' disease and Hashimoto''s thyroiditis stimulate allogeneic T cells when pretreated with phorbol ester

OBJECTIVE: To assess the capacity of thyroid follicular cells to function as antigen presenting cells, we have examined their ability to stimulate allogeneic T cells. DESIGN: Thyroid follicular cells were pretreated with interferon-gamma or phorbol myristate acetate, washed thoroughly, and their capacity to induce allogeneic T cell proliferation was determined. PATIENTS: Thyroid cells were prepared using thyroidectomy specimens from eight patients with Graves' disease, one with Hashimoto's thyroiditis and two with non-toxic multinodular goitre. MEASUREMENTS: T cell 3H-thymidine incorporation was measured after a 16-hour incubation period with the isotope, 3 days after co-culture of T cells and thyroid cells. RESULTS: Four of the eight thyroid cell preparations from thyroid autoimmunity patients failed to stimulate T cells, although there was a significant, weak stimulation for the whole group (P < 0.05): interferon-gamma pretreatment had no effect on this. Phorbol myristate acetate pretreatment significantly increased the ability of thyroid cells (from both autoimmune and multinodular glands) to stimulate T cells (P < 0.05); this was time and concentration dependent. Cell fixation after PMA treatment did not abolish this stimulatory activity, which could be transferred by supernatants from unfixed cells; exogenous interleukin-1 did not mimic the activity. CONCLUSIONS: Thyroid cells expressing major histocompatibility complex class II molecules only weakly and inconsistently stimulate allogeneic T cells, compared to thyroid cells pretreated with phorbol myristate acetate, a difference which may be due to the expression of an unidentified co-stimulatory signal induced by phorbol ester.     

Endometriosis: Serum-soluble CD23 in patients with endometriosis and the effect of treatment with danazol and leuprolide acetate depot injection

Activated B cells have recently been shown to produce solubleCD23 from their membranes. The serumsoluble CD23 concentrationin 21 patients with pelvic pain diagnosed as having endometriosisand confirmed by histology, and in 18 patients without pelvicpain, who had a normal pelvis during laparoscopic sterilization,was studied by chemiluminescent enzyme-linked immunosorbentassay. The endometriosis patients were randomized to 3 monthsof either danazol or leuprolide acetate injection. Serum wastaken before and after 3 months of therapy. The serum-solubleCD23 concentration was significantly elevated in patients withendometriosis when compared with the controls (P < 0.0001).There was no correlation between soluble CD23 concentrationsand the severity of endometriosis (r = 0.48, P > 0.05). Theserum concentration of soluble CD23 decreased significantlyon treatment with danazol but not leuprolide acetate (P <0.05). We conclude that the elevation of soluble CD23 in patientswith endometriosis suggests that there is activation of B cells,which respond to danazol but not leuprolide acetate injection.     

Autoantibodies against tyrosine hydroxylase in patients with non-segmental (generalised) vitiligo

Vitiligo is an acquired idiopathic hypomelanotic skin disorder characterised by depigmented macules because of loss of cutaneous melanocytes. Although the exact cause of vitiligo remains obscure, evidence suggests that autoimmunity plays a role in the pathogenesis of the disease. Previously, tyrosine hydroxylase (TH) was identified as a putative autoantigen in vitiligo using phage-display technology. In this study, the prevalence of TH antibodies in patients with vitiligo was investigated. A radioimmunoassay (RIA) was used to detect TH antibodies in sera from patients with either non-segmental vitiligo (n=79), segmental vitiligo (n=8) or other autoimmune diseases without concomitant vitiligo (n=91). Sera from healthy individuals (n=28) were also tested. Patients with segmental vitiligo, healthy controls and patients with other autoimmune diseases without concomitant vitiligo were all negative for TH antibody reactivity. Of 79 patients with non-segmental vitiligo, 18 (23%) were positive for TH antibodies in the RIA, and a significant increase in the prevalence of TH antibodies in patients with non-segmental vitiligo was evident when compared with controls (P=0.003). TH antibody prevalence was also significantly elevated in patients with active vitiligo compared to those with stable disease (P=0.009). Overall, the results indicate that TH is an antibody target in non-segmental but not in segmental vitiligo and that TH antibodies appear to be more frequent in patients with active vitiligo.     

Function-blocking autoantibodies to the melanin-concentrating hormone receptor in vitiligo patients

Vitiligo is a common depigmenting skin disorder resulting from the loss of melanocytes in the cutaneous epidermis. Although the cause of the disease remains obscure, autoimmune mechanisms are thought to be involved. Recently, melanin-concentrating hormone receptor (MCHR)-binding autoantibodies have been identified in vitiligo patients. In the present study, we aimed to determine if MCHR autoantibodies could also affect receptor function either by direct activation or by blocking its response to melanin-concentrating hormone. The results indicated that 10/18 (56%) vitiligo patient IgG samples inhibited the function of MCHR expressed in a Chinese hamster ovary cell line. In contrast, neither control (n=20) nor SLE patient (n=10) IgG samples blocked receptor function. Compared with healthy controls, MCHR function-blocking autoantibodies were found at a significantly increased frequency in the vitiligo patient group (P=0.0004). No MCHR-activating autoantibodies were detected in any of the vitiligo patient, SLE patient or control IgG samples that were analysed. In addition, vitiligo patient IgGs were tested for MCHR autoantibodies that could mediate antibody-dependent cell-mediated cytotoxicity via the receptor. However, this could only be demonstrated in two vitiligo patient sera. Overall, this work has provided additional evidence that MCHR is a B-cell autoantigen in vitiligo and has demonstrated the existence of MCHR function-blocking autoantibodies further to the receptor-binding autoantibodies previously reported.