Defect of autologous mixed lymphocyte reaction and interleukin-2 in aged individuals.

The proliferative responsiveness of T cells of aged individuals is known to be depressed in both autologous mixed lymphocyte reaction (AMLR) and in PHA-stimulated cultures. In the present study we confirm previous results and also report decreased IL-2 and normal IFN-gamma production (PHA-induced) in aged subjects as compared to young normals. In addition, similar percentages of T lymphocytes expressing surface IL-2 receptors both in the peripheral blood and after different stimulations, i.e. AMLR and PHA, were detected in young and aged individuals. The addition of exogenous IL-2 induces a sharp increase of spontaneous and AMLR proliferation in young individuals, whereas the increase is only slight in aged subjects. The experiments reported herein suggest that in general the T cell proliferation in AMLR is not completely dependent on the presence of IL-2 in the cultures and that aged subjects are probably defective in the production of other factor(s) presumably involved in AMLR proliferation, since the addition of exogenous IL-2 does not produce T-cell proliferation comparable to normal young subjects. The possible meanings of these experimental evidences in AMLR and in the defective immune responses of aged subjects are discussed.

Alpha 1-acid glycoprotein (alpha 1-AGP) on the membrane of human lymphocytes: possible involvement in cellular activation.

A glycoprotein termed alpha 1-acid glycoprotein (alpha 1-AGP) is a component of normal human serum; its concentration is often increased in several pathological disorders, including acute inflammation and cancer. Inhibitory effects of alpha 1-AGP on some in vitro T and B cell function assays have been reported but our recent data indicated that alpha 1-AGP is indeed a T cell mitogen at physiological concentrations. The present study was designed to investigate: (a) the relationship between this glycoprotein and two other glycoproteins of the T and B cell membrane, i.e. the T3 and Ia antigens; (b) the ability of lymphocytes to take up exogenous alpha 1-AGP; (c) the different expression of alpha 1-AGP on the T cell membrane upon different activation pathways, i.e., autologous non-T-cells (B cells and monocytes) phytohemagglutinin and anti-T3 monoclonal antibody (MAb) stimulations. The data reported herein show no competition at the membrane level between anti-alpha 1-AGP and anti-T3 or anti-Ia MAbs. In addition, (1) the lymphocytes were able to absorb alpha 1-AGP from the culture medium and (2) the expression of this glycoprotein was enhanced upon T cell stimulation (all three stimulants employed induced an increase of alpha 1-AGP positive T cells), thus suggesting a possible role of this glycoprotein in in vitro T cell activation.

Anti-human thyroglobulin autoantibodies assayed by an enzyme-linked immunosorbent assay.

A rapid and simple enzyme-linked immunosorbent assay (ELISA) for anti-thyroglobulin IgG antibodies is described. The method is based on a 'sandwich' using purified human thyroglobulin adsorbed to polystyrene microplates, human serum and anti-human IgG antiserum conjugated to alkaline phosphatase. The sensitivity of the method is about 8 ng/ml, as evaluated with a purified anti-thyroglobulin antibody preparation. High concentrations of antibodies were observed, as expected, in autoimmune thyroid disease; however, the majority of normal subjects have detectable, although very low, antibody levels. We conclude the method is suitable for current clinical use.

Abnormalities of circulating T cell subsets in atopy: influence of specific immunotherapy.

Blood samples of patients with severe respiratory allergic diseases contain increased numbers of T cells bearing surface HLA-DR antigens, indicating the presence of activated T cells. In the same group of patients, MLR3 and MLR4, two monoclonal antibodies (Mab) directed to subsets of activated peripheral T cells, recognize T cell percentages within the normal range. Thus, it seems possible that specialized subsets of activated T cells (HLA-DR+/MLR3-MLR-) are represented in the peripheral blood of atopic patients. Such cells are lacking in patients after specific immunotherapy. Similar results--an increased percentage of 5/9+ T cells in untreated patients and normal counts of 5/9+ T cells in treated ones--were obtained in the two groups of patients by using another Mab, 5/9, which serves as a reliable marker of helper T cells in resting peripheral T lymphocytes. These data further support the concept of a T cell imbalance in allergic patients and suggest a possible role of specific immunotherapy in correcting the modification of peripheral T cell abnormalities.

Atypical thyroid dermopathy: clinical and immunological features of two cases.

Two cases of pretibial and atypical dermopathy in Graves' disease, showing many similarities in clinical pictures and natural histories, are reported. In both patients we observed abnormalities of some immunological parameters, namely reduction of Fc-positive T lymphocytes and positive TBI indexes. These abnormalities were concomitant to the appearance of dermopathy and apparently unrelated to other clinical manifestations of Graves' disease.

Inhibitory effect of an antibody against alpha 1-acid glycoprotein (alpha 1-AGP) on autologous mixed lymphocyte reaction and anti-T3 T-lymphocyte activation.

The action of an anti-alpha 1-AGP antibody on AMLR, anti-T3 and PHA T-lymphocyte proliferative response was evaluated. We observed a strong dose-dependent inhibition on T-lymphocyte proliferative responsiveness to autologous non-T cells and to anti-T3 stimulus, whereas PHA activation was unaffected. A lower degree of inhibition of the proliferative response was also observed on pretreating both T and non-T cells with the antibody; the addition of anti-alpha 1-AGP in the culture containing cells pretreated with the antibody showed a further inhibition of thymidine incorporation. The data suggest a direct influence of the antibody on membrane alpha 1-AGP and support a positive role of this glycoprotein (distinct from Ia and T3 antigens) on both anti-T3 and autologous non-T cell T-lymphocyte responsiveness, thus indicating the involvement of alpha 1-AGP in the T3-Ti antigen-specific pathway of T-cell activation.

Does normal lymphocyte DNA synthesis in response to PHA exclude cell-mediated immunodepression?

PHA stimulation assay was the first in vitro method for evaluating the T-cell function, and this T-cell proliferative response has been routinely used to discriminate between normal subjects and patients with deficiency in cell-mediated immunity. However, [3H]thymidine incorporation into lymphocyte DNA can be studied by using additional in vitro assay methods since they measure different lymphocyte activation pathways. In the present study we selected three different tests to investigate the reliability of this single approach: PHA induced lymphocyte DNA synthesis; T lymphocyte DNA synthesis to anti-T3 monoclonal antibody (OKT3); autologous mixed lymphocyte reaction (AMLR). In addition, IL-2 receptor expression on the membrane of T-cell stimulated in AMLR both with PHA and anti-T3 was evaluated. This study was performed in various groups of subjects: normal young controls, aged healthy individuals, and patients with Alzheimer's disease (AD), Retinitis Pigmentosa (RP), and with cell-mediated immunodeficiency and clinical evidence of recurrent viral infections (ID). The data reported herein show heterogeneity of results in each group studied and demonstrate the necessity of employing more than one laboratory test for the routine evaluation of T-cell-mediated immunity.

T cell activation surface markers and autologous mixed lymphocyte reaction do not differ in true and pseudo food allergy.

Eighteen patients affected by itching, urticaria, eczema, angioedema, and asthma related to food-stuff intake were studied and classified in two groups (true food allergy and pseudoallergy) on the basis of clinical data, skin prick tests, total and specific IgE levels (PRIST and RAST) and double-blind challenge test. Autologous mixed lymphocyte reaction (AMLR) and T cell activation markers were thought to be tests possibly useful to discriminate between 'true' food allergy and 'pseudoallergy'. The present study failed to show either a significant increase in T cell activation markers (MLR4, Ia) or a significant decrease in AMLR proliferation in such subjects as compared to normal controls. In addition, we found no differences between 'true' allergic and 'pseudoallergic' patients on the basis of the parameters evaluated. Although the AMLR defect was reported both in asthma and in dermatitis, and therefore was thought to be related to atopy, the present data do not confirm this hypothesis in 'true' food allergy.

Circulating T cell subsets in euthyroid Graves' disease.

T cell subpopulations recognized by surfaces markers of different functional meaning have been evaluated in 12 female patients with euthyroid Graves' disease and in 2 patients with ophthalmopathy and Hashimoto's thyroiditis. We have used the following markers: i) receptors for Fc fragments of IgG; ii) antigens recognized by the monoclonal antibodies MLR4, 5/9, BT 2/9 (anti-DR). In the 12 patients with euthyroid Graves' disease a marked decrease of TG cells (which proved to exert suppressor function in several in vitro systems) was observed, as previously reported in hyperthyroid Graves' disease. The 2 Hashimoto's patients with eye changes had normal or high TG. 5/9+ T cells (which contain cells with helper activity in vitro), as well as MLR4+ and BT 2/9+ cells (activated T cells) were normal in the majority of patients, but elevated in the 2 Hashimoto's thyroiditis. The observed abnormality of TG cells in euthyroid Graves' disease might be consistent with the hypothesized autoimmune pathogenesis of endocrine ophthalmopathy.

Activation of T lymphocytes and autologous mixed lymphocyte reaction in allergic patients.

In allergic patients the authors previously observed high proportions of circulating T lymphocytes bearing Ia antigens, assumed to be "activated" T cells. In the present investigation they employed other T cell activation markers (4F2, insulin receptor, MLR4) which differ in the kinetics of appearance upon the surface of stimulated T cells. They report high proportions of Ia and 4F2-positive T cells, normal levels of MLR4-positive T lymphocytes and no insulin binding on T cells. However, T cells of allergic subjects are able to express insulin receptors in PHA-induced culture, such as normal subjects do. The authors conclude that these data, supported by similar observations in autoimmune diseases, indicates differences between in vivo and in vitro features of expression of T cell activation markers. In addition the autologous mixed lymphocyte reaction (AMLR) in atopic patients was studied. The results indicate that AMLR responsiveness is defective in allergic patients.

Reaction of T lymphocytes with anti-T3 induces translocation of C-kinase activity to the membrane and specific substrate phosphorylation.

Reaction of the T cell membrane with monoclonal antibodies to T3 can initiate cellular activation, and this is associated with increased intracellular Ca2+ and inositol-trisphosphate (IP3) release. We therefore studied the possible involvement of Ca2+/phospholipid-dependent kinase (C-kinase) in these phenomena. Quantitative assays of exogenous substrate phosphorylation in unstimulated cells showed Ca2+/phospholipid-dependent kinase activity in the cytosol, but no comparable activity in the particulate fractions corresponding to membrane and cytoskeleton material. At concentrations of soluble anti-T3 that partially activate T cells in the absence of macrophages, there was a 50 to 60% decrease in C-kinase activity in the cytosol, with a comparable increase in activity in the membrane fraction. A similar transfer of activity was also induced with the known C-kinase activator, 12-O-tetradecanoyl-phorbol-13-acetate, although redistribution was more rapid in onset, more complete, and more sustained. Redistribution of enzyme activity was additionally confirmed by qualitative assays of endogenous substrate phosphorylation. Labeling of intact cells followed by immunoprecipitation analysis with anti-T3 indicated signal-dependent phosphorylation of two components of the T3 complex and an unidentified 94,000 substrate that was resistant to reduction and alkylation. These findings are consistent with an important role for C-kinase in transduction of membrane events by the T3-Ti complex.

Thyroglobulin-induced T-cell in vitro proliferation in Hashimoto's thyroiditis: identification of the responsive subset and effect of monoclonal antibodies directed to Ia antigens.

Recently it was reported that the peripheral blood and thyroid gland of patients with Hashimoto's thyroiditis contain activated (Ia+ and/or MLR4+) T cells and high levels of 5/9+ ("helper") T lymphocytes. In normal individuals the 5/9 monoclonal antibody recognizes a T-cell fraction that includes all T lymphocytes with inducer activities. Here, circulating 5/9+ and 5/9- T lymphocytes were isolated from patients with Hashimoto's disease, and the proliferative response induced by human thyroglobulin was investigated. The results show that the total thyroglobulin-induced lymphocyte DNA synthesis is confined to the 5/9+ T-cell fraction. Further subfractionation of 5/9+ into MLR4+ and MLR4- cells clearly indicates that no substantial differences exist in their proliferative capacities. Whether 5/9, MLR4, and Ia antigens, all expressed on the thyroglobulin-responsive T-cell subset, are involved in thyroglobulin-induced cell proliferation, was also analyzed. Although both 5/9 and MLR4 monoclonal antibodies had no effect, complete inhibition of antigen-induced blastogenesis was observed upon addition of monoclonal antibodies (D1/12 and BT2/9) directed to common determinants of Ia antigens. This inhibitory effect was also observed when T or non-T fractions were separately incubated with the monoclonal antibodies before culture. These results indicate that in humans, as in animals, the major histocompatibility complex may play a role in autoimmune thyroiditis. The data show that (a) the thyroglobulin-induced proliferative response is confined to a subset (5/9+) of T lymphocytes and (b) Ia antigens are involved in thyroglobulin-induced lymphocyte DNA synthesis in Hashimoto's disease.

Inhibition of T3 mediated T-cell proliferation by Ca2+-channel blockers and inhibitors of Ca2+/phospholipid-dependent kinase.

The potential roles of Ca2+ ions in the response of T lymphocytes to stimulation with monoclonal antisera to the T3 antigen were investigated by means of pharmacological agents that predominantly inhibit the flux of Ca2+ ions into cells (verapamil, nifedipine) or the activity of Ca2+-dependent kinases (trifluoperazine, polymyxin B). As assessed by uptake of [3H]thymidine, proliferation induced with anti-T3 +/- recombinant IL-2 at 72 h was inhibited by greater than 80% in the presence of nifedipine at 50 microM, and almost completely arrested (greater than 95% inhibition) with the other agents at the same concentration. Further quantitative assays of the effects of polymyxin B and trifluoperazine on C-kinase labelling of exogenous substrate showed a major reduction with both agents, but inhibition was substantially greater with polymyxin B that with trifluoperazine (IC50 = 14 and 70 microM respectively). These results were confirmed by qualitative assessment of Ca2+/phospholipid-dependent phosphorylation of endogenous substrates, which demonstrated major phosphoproteins of MW 56,000, 52,000, 43,000, and 20,000, and dose-dependent reduction in labelling in the presence of polymyxin B. Similar results were obtained under more physiological conditions in intact cells labelled with 32P orthophosphate. These findings indicate several possible roles for Ca2+ in T-cell activation, and several possible levels of activity, including modulation of calmodulin-dependent kinases and effects on Ca2+/phospholipid-dependent kinases and Ca2+ channels.