Analysis of T-cell clones in systemic lupus erythematosus

BACKGROUND AND OBJECTIVE: It is fairly well established that T-helper (TH)((1)) cells play a role in the pathogenesis of organ-specific autoimmune diseases, while their role and their relationship with TH((2)) cells is far from being defined in systemic lupus erythematosus (SLE). To address this issue, six female patients who fulfilled the American Rheumatism Association criteria for the diagnosis of SLE were studied. DESIGN AND METHODS: We analyzed the intracellular production of cytokines by T-cells from the peripheral blood (PB). Then, we established T-cell clones (TCC) from the peripheral blood (PB) of all cases as well as from the synovial fluid of one patient with an articular flare-up. RESULTS: The percentages of IL-4 positive and IFN-g positive PB T-cells were not different between SLE patients and normal controls. When 93 TCC (67 CD4(+), 23 CD8(+)) from the PB of 5 different SLE patients were compared to 118 TCC (94 CD4(+), 23 CD8(+)) from 5 healthy controls no statistical difference was observed between SLE and controls in terms of TH((1)), TH((2)) or TH((0)) phenotype. However, SLE clones showed a reduced ability to secrete IL-10 (p = 0. 002). In contrast, the analysis of the 30 clones obtained from synovial fluid revealed that 11/23 CD4(+) clones were TH((1)), 12/23 were TH((0)), 2/7 CD8(+ )clones were TH((1)) and 5/7 were TH((0)). No TH((2)) clones were obtained from the synovial fluid. INTERPRETATION AND CONCLUSIONS: The data suggest that the T-cell subsets operating in actively inflamed organs of SLE may belong to the TH((1)) and TH((0)) subsets.     

TSH-R expression and cytokine profile in orbital tissue of active vs. inactive Graves' ophthalmopathy patients

OBJECTIVE: From in vitro studies using cultures of orbital fibroblasts, it has become clear that cytokines play an important role in the orbital inflammation in Graves' ophthalmopathy (GO). Orbital fibroblasts seem to be the key target cells of the autoimmune attack, and they are able to express the TSH receptor (TSH-R). In vivo data on the presence of cytokines in orbital tissues are sparse, and mostly limited to samples obtained from patients with endstage, inactive GO; the same holds true for the presence of the TSH-R. The aim of the present study was to determine whether the cytokine profile and TSH-R expression differ in the active vs. the inactive stage of GO. DESIGN AND MEASUREMENTS: Orbital fat/connective tissue was obtained from six patients with active, untreated GO undergoing emergency orbital decompression, and from 11 patients with inactive GO subjected to rehabilitative decompressive surgery. The mRNA levels of various cytokines and the TSH-R were assessed by real-time polymerase chain reaction (PCR) using the LightCycler. Data are expressed as ratios (unknown mRNA/beta-actin mRNA). RESULTS: Active GO patients had much higher TSH-R expression than inactive patients: 4/0-24 (median value/range) vs. 0/0-9, P = 0.01. TSH-R expression was related to the Clinical Activity Score (r = 0.595, P = 0.015). Patients with active GO compared to those with inactive GO had higher mRNA levels of the proinflammatory cytokines interleukin-1beta (IL-1beta) (445/153-877 vs. 0/0-455, P = 0.001), IL-6 (1583/968-18825 vs. 559/0-7181, P = 0.01), IL-8 (1422/38-7579 vs. 32/0-1081, P = 0.046) and IL-10 (145/58-318 vs. 27/0-189, P = 0.002). In active GO there also existed a trend towards a predominance of T helper 1 (Th1)-derived cytokines as evident from higher IL-2 (37/0-158 vs. 0/0-68, P = 0.043), interferon-gamma (IFN-gamma) (20/0-79 vs. 0/0-16, P = 0.12) and IL-12 (2.3/0-14.8 vs. 0/0-1.6, P = 0.10) mRNAs. IL-1 receptor agonist (IL-1RA), IL-2 receptor (IL-2R), IL-3, IL-4, IL-5, IL-13, IL-18 and tumour necrosis factor-alpha (TNF-alpha) mRNAs were similar in both groups. CONCLUSIONS: These data show that at the mRNA level, TSH-R expression is largely present only during the active stages of GO. The active phase is characterized by the presence of proinflammatory and Th1-derived cytokines, whereas other cytokines, among them Th2-derived cytokines, do not seem to be linked to a specific stage of GO.     

Interleukin-6 stimulates thyrotropin receptor expression in human orbital preadipocyte fibroblasts from patients with Graves' ophthalmopathy.

The thyrotropin receptor (TSHR) is the thyroid autoantigen against which stimulating autoantibodies are directed in Graves' hyperthyroidism. Recent evidence suggests that TSHR may also serve as an orbital autoantigen in Graves' ophthalmopathy (GO) and that expression of this protein is increased in the fatty connective tissues of the orbit in this condition. It has been shown that orbital fibroblasts from patients with GO increase thyrotropin (TSH)-dependent cyclic adenosine monophosphate (cAMP) production and TSHR gene expression when cultured under conditions known to stimulate adipocyte differentiation. In the current study, we wanted to determine whether treatment of these cells with particular cytokines (each 1 ng/mL) during differentiation might further augment TSHR expression. We found that exposure to interleukin (IL)-6 increased TSHR expression above control levels in cells from patients with GO. In contrast, this cytokine did not affect TSHR expression in normal orbital cells. Neither IL-4 nor IL-1alpha had a significant stimulatory effect in either normal or Graves' cultures. These findings suggest that IL-6 may play a role in the pathogenesis of GO by increasing expression of the putative autoantigen within the adipose/connective tissues of the orbit.     

Relative overexpression of macrophage-derived cytokines in orbital adipose tissue from patients with graves' ophthalmopathy

Graves' ophthalmopathy (GO) is an autoimmune disorder involving the adipose and connective tissues of the orbit. The study of cytokines present in these tissues may reveal the nature of the cells and immune responses involved in GO pathogenesis. In the current study, we performed relative quantification of the expression of cytokine genes in orbital adipose tissue from patients with GO (n = 6) and normal individuals (n = 2). Real-time RT-PCR was performed using fluorescent probes and primers for cytokines including IL-1 beta, IL-2, IL-4, IL-5, IL-8, IL-10, IFN-gamma, and TNF-alpha. Results showed IL-1 beta to be the gene having the greatest fold expression increase over normal in four of six patients. TNF-alpha was increased in all six GO patients. In addition, IL-8, IL-10, and IFN-gamma were increased in five of six GO patients. We found no evidence of either IL-4 or IL-5 expression in any of the GO or normal samples. The increased expression of the macrophage-derived cytokines IL-1 beta, TNF-alpha, and IL-10 suggests the presence of macrophage activation and ongoing antigen presentation within the orbit in GO. In addition, the overexpression of IFN-gamma, without evidence of IL-4 or IL-5 expression, supports the concept that cell-mediated, rather than humoral, immunity plays the predominant role in pathogenesis of this disorder.     

An elevation of serum immunoglobulin E provides a new aspect of hyperthyroid Graves' disease

In hyperthyroid Graves' disease, short-term methimazole is sufficient to induce lasting remission in some patients, but even long-term treatment fails to do so in others. We have evaluated the role of autoimmune abnormalities in the helper T cell type 2 (TH2)-interleukin-13 (IL-13)-TSH receptor system in maintaining hyperthyroidism by comparing IgE levels in patients with various thyroid diseases. One hundred and ninety-three patients with hyperthyroid Graves' disease were treated with methimazole, and blood samples were obtained to measure serum levels of T4, T3, TSH, thyroglobulin, antimicrosomal antibody, TSH binding inhibitory Ig (TBII), thyroid-stimulating antibody, thyroid stimulation-blocking antibody, IgE, interferon-gamma, IL-4, and IL-13. Elevation of serum IgE (> or = 170 U/mL) was found in 35.5% of patients with hyperthyroid Graves' disease, and serum levels of T4, T:1, antimicrosomal antibody, and TBII were significantly greater in patients with IgE elevation than in those with normal serum IgE. During methimazole treatment, there was a parallel decrease in the serum T4 concentration in the presence or absence of an IgE elevation. However, there was a significantly smaller decrease in TBII in patients with elevated IgE than in those with normal IgE. As a result, the remission rate was significantly greater in patients with normal IgE than in those with IgE elevation. Serum levels of IL-13 were elevated in 64.7% of patients with IgE elevation in the absence of detectable TH1 marker, interferon-gamma. These findings suggest that in one third of patients with hyperthyroid Graves' disease, TH2 cells are stimulated and secrete excess amounts of IL-13, which subsequently stimulates B cells to synthesize more TSH receptor antibody and IgE, so that during methimazole treatment TBII decreases less in patients with IgE elevation, producing a lower remission rate.     

Ia+ T cells in new onset Graves' disease

The Ia (immune-associated, DR) antigen is a cell surface glycoprotein which is absent on normal circulating T lymphocytes but present on activated T lymphocytes. We studied the expression of this antigen on circulating T lymphocytes from patients with untreated hyperthyroid Graves' disease. All patients (n = 33) with recent onset hyperthyroid Graves' disease studied had an increased percentage and number of circulating Ia+ T cells. Patients with non-Graves's hyperthyroidism or Graves' disease patients more than 1 yr after thyroid ablation had normal values for Ia+ T cells. Other cell surface activation antigens, recognized by monoclonal antibodies 4F2 and 5E9, were not present on circulating T cells in patients with Graves' disease. The 100% positivity for increased numbers of Ia antigen-bearing T cells in hyperthyroid Graves' disease contrasts with our finding of 70% positivity in another HLA-DR-associated disease, recent onset type I diabetes mellitus. The pathogenic significance of these cells is not known, but they seem to represent selective activation of the immune system in patients with untreated hyperthyroid Graves' disease.     

Childhood Graves' ophthalmopathy: results of a European questionnaire study

OBJECTIVE: Evaluation of the frequency of Graves' ophthalmopathy (GO) and its management in children and adolescents up to 18 years old with Graves' hyperthyroidism. STUDY DESIGN: This was a questionnaire study (QS) among members of the European Thyroid Association and the European Society for Paediatric Endocrinology. Approximately 300 QS were sent to members with electronic addresses and 110 QS were returned from 25 countries: 52 respondents said they had no experience with Graves' disease in this age group, but 67 respondents (23 paediatric and 44 adult endocrinologists) completed the QS. RESULTS: Out of 1963 patients with juvenile Graves' hyperthyroidism seen by respondents in the last 10 years, 641 (33%) had GO; about one-third of GO cases were < or =10 years old, and two-thirds were 11-18 years old. The prevalences of GO among juvenile Graves' hyperthyroidism were 36.6, 27.3 and 25.9% in countries in which the smoking prevalence among teenagers was > or =25, 20-25 and <20% respectively (P < 0.0001 by chi(2) test). When confronted with the standard case of a 13-year-old girl with Graves' hyperthyroidism and moderately severe active GO, the diagnostic approach included on average 4.9 biochemical tests (TSH, free thyroxine (FT(4)) and TSH.R-Ab, 100-88% of respondents) and 2.4 specific investigations (thyroid ultrasound by 69%, orthopsy/visual fields/visual acuity by 64% and orbital magnetic resonance imaging or computed tomography by 63%). Antithyroid drugs were the treatment of choice for 94% of respondents; 70% recommended a wait-and-see policy and 28% corticosteroids for the co-existing GO. In variants of the standard case, a younger age did not affect therapeutic approach very much. Recurrent hyperthyroidism would still be treated with antithyroid drugs by 66%, and with (131)I by 25%. Worsening of GO or active GO when euthyroid would convince about two-thirds of respondents to initiate treatment of GO, preferably with steroids. CONCLUSION: GO occurs in 33% of patients with juvenile Graves' hyperthyroidism; its prevalence is higher in countries with a higher prevalence of smoking among teenagers. The diagnostic approach to the standard case of a 13-year-old with Graves' hyperthyroidism and moderately severe active GO involves on average five biochemical tests; thyroid as well as orbital imaging is done in 84% of cases. Antithyroid drugs remain the treatment of choice for 94% of respondents, and even so in case of recurrences (66%). For GO, 70% recommend a wait-and-see policy; intervention, preferably with steroids, is advocated by two-thirds of respondents in cases of worsening or still-active eye disease despite euthyroidism.     

Regulation of Graves' hyperthyroidism with naturally occurring CD4+CD25+ regulatory T cells in a mouse model

Graves' hyperthyroidism can be efficiently induced in susceptible mouse strains by repeated immunization with recombinant adenovirus coding the TSH receptor (TSHR). This study was designed to evaluate the role(s) played by naturally occurring CD4(+)CD25(+) regulatory T cells in the development of Graves' hyperthyroidism in resistant C57BL/6 and susceptible BALB/c mice. Depletion of CD4(+)CD25(+) T cells rendered some C57BL/6 mice susceptible to induction of hyperthyroidism. Thus, hyperthyroidism developed in 30% of the CD4(+)CD25(+) T cell-depleted C57BL/6 mice immunized with adenovirus expressing the TSHR A-subunit (AdTSHR289) vs. 0% of those immunized with AdTSHR289 alone. This immunological manipulation also enhanced disease severity in susceptible BALB/c mice, as reflected by a significant increase in mean T(4) levels by CD4(+)CD25(+) T cell depletion. The immunoenhancing effect of CD4(+)CD25(+) T cell depletion appears to be attributable to an increase in thyroid-stimulating antibody production and/or a decrease in thyroid-blocking antibody synthesis, but not immune deviation to either T helper 1 or 2 cells. Interestingly, unlike BALB/c mice, some hyperthyroid C57BL/6 mice showed some intrathyroidal lymphocytic infiltration with follicular destruction. These results indicate that CD4(+)CD25(+) T cells play a role in disease susceptibility and severity in adenovirus-TSHR-induced Graves' hyperthyroidism. Overall, the imbalance between effector and regulatory T cells appears to be crucial in the pathogenesis of Graves' disease.     

Temporal relationship between onset of Graves' ophthalmopathy and onset of thyroidal Graves' disease

The temporal relationship between the onset of Graves' ophthalmopathy and the onset of thyroidal Graves' disease was evaluated in 125 consecutive patients with Graves' ophthalmopathy. Thyroidal Graves' disease--past or present--was clinically evident in 99 patients (79%): hyperthyroidism in 3 cases. Thyroid disease preceded the eye disease in 37 patients, it occurred simultaneously with the eye disease in 39 patients, and it developed after the eye disease in 23 patients (in 16 cases within one yr after the onset of eye disease). The age at the onset of thyroid disease (38.7 +/- 12.9 yr) was lower than the age at the onset of ophthalmopathy (41.8 +/- 12.5 yr; p less than 0.001). Among the 26 clinically euthyroid patients (21%) laboratory evidence of thyroidal Graves' disease was found in 14 cases (11%): abnormal TRH test, n = 9; normal TRH test but abnormal T3-suppression test, n = 4; normal TRH and T3-suppression tests but positive thyroid stimulating antibodies, n = 1). We conclude that Graves' ophthalmopathy as a rule develops at a time when thyroid autoimmunity also exists. This strongly suggests a common factor in the pathogenesis of thyroidal and ocular expressions of Graves' disease.     

CD30 expression and interleukin-4 and interferon-gamma production of intrathyroidal lymphocytes in Graves' disease.

We reported that serum levels of interleukin-5 (IL-5) and soluble CD30, mainly secreted from T helper 2 (Th2) cells, were increased in Graves' disease. To clarify the immune balance of Th1/Th2 within the Graves' thyroid gland, we have compared the expression of CD30, a preferential marker for T cells producing type 2 cytokines, and the production of interferon-gamma (IFN-gamma) and IL-4 between intrathyroidal lymphocytes (ITL) and peripheral blood lymphocytes (PBL). In PBL, none of these parameters were different between patients and normal subjects. The proportion of CD30+ cells in ITL was markedly higher (5.1%+/-2.8%, p < 0.0001) than that in patients' PBL (0.4%+/-0.3%). Likewise, both the proportions of IFN-gamma+ (14.8%+/-5.5%) and IL-4+ cells (2.4%+/-0.5%) in ITL were higher than those in PBL (9.6%+/-2.5%; p < 0.01, 1.5%+/-0.4%; p < 0.0001, respectively). The proportion of type 0 (both IFN-gamma and IL-4 positive, 1.0%+/-0.4% p < 0.001), type 1 (IFN-gamma positive, 14.0%+/-5.6%, p < 0.01) or type 2 cells (IL-4 positive, 1.4%+/-0.5%, p < 0.05) in ITL was significantly higher as compared with those in PBL (0.4%+/-0.1%, 9.0%+/-2.4%, 1.1%+/-0.3%, respectively). The ratios of ITL/PBL in CD30+ (23.3+/-30.6) and type 0 cells (2.5+/-1.2) were higher than the ratios in other subsets. The proportion of CD30+ cells correlated with the proportion of type 0 cells (r = 0.686, p < 0.01), but not with type 1 or type 2 cells. These findings suggest that there is no obvious deviation of Th2/Th1 profile in the Graves' thyroid gland, although intrathyroidal CD30+ T cells and Th0 cells may play some role in the development of autoimmune abnormalities in Graves' disease.     

Dominant infiltration of T(H)1-type CD4+ T cells at the retrobulbar space of patients with thyroid-associated ophthalmopathy.

Lymphocyte infiltration in the retrobulbar space is a prominent histological feature of thyroid-associated ophthalmopathy (TAO). We have characterized phenotypic and functional features of T cells derived from retrobulbar infiltrates of 3 TAO patients to better understand their roles in the disease. One hundred four T-cell clones (TCC) were directly established from cells of retrobulbar tissues using a highly efficient cloning procedure. Phenotypic analysis of TCC showed approximately 70% to 80% were CD3+ CD4+ CD8- T cells, and approximately 20% to 30% were CD3+ CD8+ CD4- T cells. None of the TCC were CD3+ CD4- CD8- T cells. Analysis of the cytokine profile of TCC, as documented by the ability to express interferon-gamma, interleukin (IL)-2, IL-4, and IL-10 demonstrated that the majority of TCC expressed T helper (T(H))1-like profile in both the mRNA and protein levels. A few TCC showed T(H)0-like profile, but no TCC showed T(H)2-like profile. These results suggest that T(H)1-type CD4+ T cells play important roles in the pathogenesis of TAO.     

Peripheral T lymphocyte cytokine profile (IFNgamma, IL-2, IL-4) and CD30 expression/release during measles infection.

BACKGROUND AND OBJECTIVE: Measles virus infection (MVI) has been reported to be characterized by an imbalanced Th(1/2)-type cytokine profile. CD30 has been proposed as a receptor preferentially associated with the Th(0/2)-type cytokine pattern. The aim of this study was therefore to define the peripheral T lymphocyte cytokine profile and to test which CD30 expression pattern it was associated with in MVI. DESIGN AND METHODS: The design of the study was a prospective evaluation with comparative analysis. The serum levels of the soluble form of CD30 (sCD30) were determined at diagnosis and at weekly intervals up to 4 weeks, using an ELISA, in 23 males (median age 19), who developed MVI while serving in the Italian army and who were admitted to the Infectious Disease Unit of the Military Hospital in Padua. In 10 of the patients at diagnosis we studied the lymphoid immunophenotype and, after non-specific ex vivo stimulation, the expression of IFNgamma, IL-2 and IL-4 by peripheral T cells using flow cytometry single cell analysis. In 3 patients such evaluations were also performed 7 weeks later. RESULTS: At diagnosis, we found (i) reduction of IFNgamma+/CD4+ T cells (p=0.048 vs controls) in the absence of substantial variation of IL-2+ and IL-4+ T cells (p=ns vs controls); (ii) expansion of CD30+/ CD4+ and CD30+/CD8+ T cell subsets (p<0.01 vs controls); (iii) high sCD30 values (median 61 U/mL; p<0.001 vs controls); (iiii) a context of lymphopenia (0. 728+/-0.292 lymph x10(9)/L). sCD30 remained elevated up to 4 weeks from MVI onset [median values 53, 49, 50, 34 U/mL after 1, 2, 3 and 4 weeks, respectively (p=ns between different time points)]. In 3 patients tested 7 weeks after diagnosis, we still observed decreased IFNgamma production by CD4+ and CD8+ T cells (p=0.05 and <0.01, respectively vs controls) and reduction of CD4+ and CD8+/IL-2+ T cells (p<0.01). INTERPRETATION AND CONCLUSIONS: MVI was characterized by featuresof inadequate Th/Tc(1) activation associated with increased circulating CD30+ T cells and elevated sCD30 levels, supporting a correlation between Th/Tc status and CD30 expression/release pattern in vivo.     

Reduction in the suppressor-inducer T cell subset and increase in the helper T cell subset in thyroid tissue from patients with Graves' disease

The expression of surface markers associated with activation and characterization was compared among T cells in thyroid glands and peripheral blood of 10 patients with Graves' hyperthyroidism receiving chronic antithyroid drug therapy, in peripheral blood of 15 patients with untreated hyperthyroid Graves' disease, and in peripheral blood of 21 normal subjects using two-color flow cytometry. In the chronically treated Graves' disease patients, the percentage of activated T cells (HLA-DR+ T cells) among total T cells was significantly higher in thyroid tissue than in peripheral blood, and the increase in percent activated T cells was also significant among both helper/inducer T cell (CD4+ cell) and suppressor/cytotoxic T cell (CD8+ cell) subsets. The percentage of activated T cells in peripheral blood was not significantly different between chronically treated hyperthyroid Graves' patients and normal subjects, whereas the percentage of activated T cells in the peripheral blood from untreated hyperthyroid Graves' disease patients was significantly higher than that in normal subjects or chronically treated hyperthyroid Graves' patients. The percentages of CD4+ cells and CD8+ cells among total T cells were not different between thyroid tissues and peripheral blood in patients with chronically treated hyperthyroid Graves' disease. When CD4+ were further divided into helper T cells (CD4+2H4- cells) and suppressor-inducer T cells (CD4+2H4+ cells) using two-color flow cytometry, the percentage of helper T cells among CD4+ cells was significantly higher in thyroid tissue than in peripheral blood, resulting in an increased ratio of CD4+2H4- cells to CD4+2H4+ cells. The percentage of CD4+2H4+ cells in peripheral blood, however, was not significantly different among untreated and chronically treated Graves' disease patients and normal subjects. From the findings of abnormalities in intrathyroidal T cell subsets, we suggest that the decrease in the function of suppressor T cells within the thyroids of Graves' disease patients may be due to a decrease in CD4+2H4+ cells within thyroid tissue.     

Activated (Ia+) T-lymphocytes and their subsets in autoimmune thyroid diseases: analysis by dual laser flow microfluorocytometry

Peripheral blood lymphocytes of patients with autoimmune thyroid diseases were studied using monoclonal antibodies reacting with cell surface antigens of activated T-cells (Ia+T), as well as their helper-inducer (Ia+TH/I) and suppressor-cytotoxic (Ia+TS/C) subsets, using two-color dye labeling and dual laser activated cell sorter analyses. Compared to normal subjects, hyperthyroid Graves' disease patients had significantly higher percent Ia+T values in association with an increase in percent Ia+TH/I as well as a reduction in percent Ia+TS/C; whereas patients with hypothyroid Hashimoto's thyroiditis as well as those with postpartum thyroiditis studied in the hyperthyroid phase also had a significant but lesser increase in percent Ia+T-cells, but their percent Ia+TH/I subset was significantly decreased, whereas the percent Ia+TS/C subset was increased; and patients with toxic nodular goiter or factitious hyperthyroidism (nonimmunogenic causes of hyperthyroidism) had a significant increase in percent Ia+T-cells without a significant difference in their Ia+T subsets or their ratios in comparison to controls. These studies demonstrated the feasibility of detecting Ia+T-cells and their subset characteristics using two-color dye labeling and dual laser flow microfluorocytometric methodology. In both the active and treated phases of Graves' disease, Hashimoto's thyroiditis, and postpartum thyroiditis, the percent Ia+T-cells was increased compared to normal subjects, with the highest values occurring in hyperthyroid Graves' disease. Furthermore, patients with hyperthyroid Graves' disease had the opposite changes in percent Ia+TH/I and Ia+TS/C subsets as compared to patients with either untreated hypothyroid Hashimoto's disease or the hyperthyroid phase of postpartum thyroiditis, suggesting that the pathogenic mechanisms involved in Hashimoto's disease and the destructive hyperthyroidism of painless thyroiditis are similar, and that they are both distinctly different from that of hyperthyroid Graves' disease.     

Modulation of helper T cell function by prostaglandins

Objective. To determine the influence of prostaglandins on the production of interleukins 2, 4, and 5 (IL-2, IL-4, and IL-5), interferon-γ (IFNγ), granulocytemacrophage colony-stimulating factor, and transforming growth factor β1 by CD4+ T cells. Methods. TH0, TH1, and TH2 T cell clones were stimulated in the presence and absence of the prostaglandin E₁ (PGE₁) analog misoprostol and PGE₂. Lymphokine production was analyzed by using a semiquantitative polymerase chain reaction with lymphokine-specific primer sets and/or by determining lymphokine activity in bioassays. Results. PGE₂ and misoprostol have distinct effects on different functional T helper cells. TH1 cells, which predominantly produce IL-2 and IFNγ, are completely inhibited, while TH2 cells, which preferentially produce IL-4 and IL-5, are largely unaffected. Misoprostol and PGE₂ are equivalent in their ability to modulate T cell function. In the presence of prostaglandins, THO-like helper cells, which are characterized by the coproduction of multiple lymphokines, function as TH2 cells; however, they do not differentiate into TH2 T cells. Conclusion. Prostaglandins that are produced in inflamed tissue can regulate the functional capabilities of infiltrating T cells. In the presence of PGE₂, TH1-like responses are suppressed and TH0-like responses are shifted toward a TH2-like pattern dominated by the production of IL-4 and IL-5. Inhibition of prostaglandin production by antiinflammatory agents might restore TH1 responses with local production of IL-2 and IFNγ.     

Early recovery of aggressive cytotoxic cells and improved immune resurgence with post-transplant immunotherapy for multiple myeloma

A phase I/II trial evaluated early administration and dose escalation of interleukin (IL)-2 with granulocyte macrophage colony stimulating factor (GM-CSF) post-transplant. Following melphalan (200 mg/m(2)) and an autologous transplant, IL-2 was initiated (day 0) and continued for 4 weeks. GM-CSF (250 mcg/m(2)/day) began on day 5. Fifteen of 19 patients completed therapy. No treatment-related deaths occurred. IL-2 (1 x 10(6) IU/m(2)/day) was not tolerated in two of six patients due to > or =grade 3 fatigue/diarrhea (n=1) or supraventricular tachycardia (n=1). The maximum tolerated dose of IL-2 was 6 x 10(5) IU/m(2)/day; this dose was well tolerated by 11 of 13 patients. Neutrophil and platelet engraftment occurred on day 13 (median; range 10-17 days) and day 13 (median; range 0-74 days), respectively. When compared to control patients, there was a marked increase in the number of CD3+ T cells (P=0.005), CD4+ T cells (P=0.01), CD8+ T cells (P=0.001) and CD4+CD25+Treg cells (P=0.015) post-transplant. Cytotoxicity directed against myeloma cells was markedly increased when compared to control patients (P=0.017). This unique trial design using early administration of IL-2 with GM-CSF during the period of lymphodepletion, demonstrated a marked increase in the number and function of early cytotoxic effector T cells, without suppression of engraftment.     

Thyrotropin receptor expression in orbital adipose/connective tissues from patients with thyroid-associated ophthalmopathy.

The TSH receptor (TSHR) is the autoantigen responsible for the hyperthyroidism of Graves' disease. However, whether this receptor plays a role in the development of Graves' ophthalmopathy (GO) is unclear. Expression of TSHr is augmented in orbital tissues from patients with GO, and in newly differentiated adipocytes derived from precursor cells within the orbit. Our recent studies suggest that interleukin-6 (IL-6), a cytokine elevated in the circulation of Graves' patients, stimulates TSHr expression in vitro in orbital preadipocyte fibroblasts. This cytokine might play a role in the pathogenesis of GO by stimulating TSHr expression within the fatty connective tissues of the orbit, allowing the receptor to act there as an autoantigen. Whether IL-6 also stimulates adipogenesis in the orbit is unclear at present, but such an effect could contribute to the increased volume of orbital adipose/connective tissue characteristic of this condition. Other cytokines, including IFN-gamma and TGF-beta, inhibit TSHr expression and adipogenesis by orbital fibroblasts, effects that would seem to favor disease remission. The initiation and subsequent clinical severity of GO may therefore be influenced by competing inhibitory and stimulatory cytokine effects occurring simultaneously within the orbit. Some of these may impact the expression of TSHr, the putative orbital autoantigen in this condition.     

Microsomal antigen-reactive lymphocyte lines and clones derived from thyroid tissue of patients with Graves' disease

Thyroid mononuclear cells (TMC) were maintained in long term cocultures with thyroid fibroblasts and thyroid epithelial cells from patients with Graves' disease, using medium supplemented with thyroid microsomal antigen (McAg) and IL-2. The TMC consisted predominantly of T4+ (CD4+, helper) and, to a lesser extent, T8+ (CD8+, cytotoxic/suppressor) lymphocytes, with a small number of macrophages and natural killer cells. The average T4+ to T8+ ratio was 3.2. From these cultures we obtained thyroid T cell lines and clones reactive to thyroid antigens. T Cell lines were tested in a microproliferation assay using thyroglobulin (Tg), McAg, tetanus toxoid, and IL-2. Of 14 lines from 6 patients, 2 proliferated in response to McAg when TMC plus thyroid fibroblasts were used as antigen-presenting cells. Clones of thyroid lymphocytes were obtained by culturing cells at limiting dilution with IL-2, McAg, and different types of autologous accessory cells. Peripheral blood mononuclear cells plus skin fibroblasts provided the best source of accessory cells, allowing near 100% cloning efficiency. Of 26 clones tested, 6 recognized McAg, 2 were Tg reactive, and 3 were autoreactive. All phenotyped clones were of the T4+ phenotype. Our method results in production of thyroid T cell lines and clones. The fibroblasts probably provided growth factors and/or collaborated with peripheral blood mononuclear cells as antigen-presenting cells. These lines and clones from patients with Graves' disease were predominantly helper T cells, in contrast to the previously demonstrated cytotoxic/suppressor cell predominance in cells from patients with Hashimoto's thyroiditis. This difference in cell function may help explain the differing clinical courses of these two closely related autoimmune thyroid diseases. The availability of long term microsomal antigen-specific T cell clones should allow careful analysis of the role these cells play in thyroid autoimmunity.     

Effects of TH1 and TH2 cytokines on CD8+ cell response against human immunodeficiency virus: implications for long-term survival.

CD8+ cells from long-term survivors [LTS; infected with human immunodeficiency virus (HIV) for 10 or more years and having CD4+ cell counts of > or = 500 cells per microliters] have a 3-fold greater ability to suppress HIV replication than do CD8+ cells from patients who have progressed to disease (progressors) during the same time period. A change in the pattern of cytokines produced in the host from those that typically favor cell-mediated immunity (T helper 1, TH1 or type 1) to those that down-regulate it (T helper 2, TH2 or type 2) was investigated as a cause of this reduced CD8+ cell anti-HIV function. Treatment of CD8+ cells from LTS with the TH1 cytokine interleukin (IL)-2 enhanced their anti-HIV activity, whereas exposure of these cells to TH2 cytokines IL-4 or IL-10 reduced their ability to suppress HIV replication and to produce IL-2. IL-2 could prevent and reverse the inhibitory effects of IL-4 and IL-10. Moreover, prolonged exposure of CD8+ cells from some progressors to IL-2 improved the ability of these cells to suppress HIV replication. These observations support previous findings suggesting that strong CD8+ cell responses play an important role in maintaining an asymptomatic state in HIV infection. The data suggest that the loss of CD8+ cell suppression of HIV replication associated with disease progression results from a shift in cytokine production within the infected host from a TH1 to a TH2 pattern. Modulation of these cytokines could provide benefit to HIV-infected individuals by improving their CD8+ cell anti-HIV activity.     

Remission of Graves' hyperthyroidism and A/G polymorphism at position 49 in exon 1 of cytotoxic T lymphocyte-associated molecule-4 gene

We studied whether a patient with Graves' disease will go into remission during antithyroid drug (ATD) treatment. Remission of Graves' hyperthyroidism is predicted by a smooth decrease in TSH receptor antibody (TRAb) during ATD treatment. Cytotoxic T cell lymphocyte-associated molecule-4 (CTLA-4) may play an important role in the development of Graves' hyperthyroidism and in its remission. We studied A/G polymorphism at position 49 in exon 1 of the CTLA-4 gene in 144 Japanese Graves' patients. We intended to reveal the possible association of CTLA-4 gene polymorphism with the remission of Graves' hyperthyroidism. All patients with Graves' disease were treated with ATD. Thyroid-stimulating antibody and TSH binding inhibitory Ig were measured as TRAb. We analyzed CTLA-4 genotypes and alleles with PCR. We calculated the frequencies of CTLA-4 genotypes and alleles. A significant increase in the frequency of the G allele was seen in Graves' patients compared with controls (P = 0.0095). Graves' patients were divided into three groups (A, B, and C) according to time of TRAb disappearance after the start of ATD treatment. In group A patients TRAb had disappeared within 1 yr after the start of ATD treatment, in group B TRAb had disappeared between the beginning of the second year and the end of the fifth year of treatment, and in group C TRAb continued to be positive after 5 yr of ATD treatment. The frequencies of the GG genotype and the G allele were significantly higher in group C patients with persistently positive TRAb over 5 yr of ATD treatment than in the other groups (P < 0.0001). Group C patients did not have the AA genotype. The periods of time until remission were significantly shorter in the AA genotype. Graves' patients with the G allele need to continue ATD treatment for longer periods.