Effects of gamma-interferon on DR antigen expression, growth, 3,5,3'-triiodothyronine secretion, iodide uptake, and cyclic adenosine 3',5'-monophosphate accumulation in cultured human thyroid cells.

We have examined, using the same system of human thyroid cells in culture, the effects of the cytokine human gamma-interferon (gamma IFN) on the expression of DR antigen, cell pro-liferation, cAMP accumulation, and the differentiated functions, iodide uptake and T3 secretion. gamma IFN elicited a dose- and time-dependent increase in DR expression, with a maximum effect on day 5 of culture. The cytokine, at the same concentrations and experimental conditions as those found to be effective in inducing DR antigen expression, caused on day 5 of culture a dose-dependent inhibition of [3H]thymidine incorporation, DNA content, cell count, as well as TSH-stimulated (but not basal) iodide uptake and T3 secretion. The gamma IFN suppressive influence on the differentiated functions was not merely due to a reduction in cell number, but was also apparent when results were expressed per micrograms DNA. Since the cytokine did not inhibit TSH- or forskolin-stimulated cAMP accumulation and showed a suppressive influence toward 8-bromo-cAMP- and forskolin-stimulated T3 secretion, its inhibitory effect seems to be exerted at a site located distal to cAMP formation. Although gamma IFN alone was devoid of any effect on cAMP accumulation, it enhanced forskolin-stimulated (as well as TSH-activated) cAMP in the presence of 3-isobutyl-1-methylxanthine, an inhibitor of cAMP degradation. Thus, it would seem that gamma IFN also exerts an influence on cAMP formation (rather than degradation) at a step subsequent to TSH binding to its receptor. The effects we observed seem specific to gamma IFN, since alpha IFN, although capable of inhibiting human thyrocyte multiplication, lacked any influence on DR antigen expression, cAMP accumulation, or T3 secretion by human thyroid cells. In what way, if any, is gamma IFN-induced DR antigen expression on human thyrocytes, an event believed to be critical in the pathophysiology of autoimmune thyroid disease, related to decreased thyroid function and growth is presently unknown.

Treatment of multiple sclerosis with copolymer-1 (Copaxone): implicating mechanisms of Th1 to Th2/Th3 immune-deviation.

The synthetic polypeptide copolymer-1 (Cop-1; Copaxone; Glatiramer Acetate) has been recently approved as an effective treatment in relapsing multiple sclerosis (MS). A large body of evidence demonstrates that Cop-1 induces active suppression of CNS-inflammatory disease in animal models. However, Cop-1-mediated suppressor mechanisms have not yet been elucidated in humans. A 12-month open study following clinical and immunological parameters of ten relapsing MS patients treated with Cop-1 is presented. Relapse rates and disability scores (EDSS) were evaluated prior to and after 12 months of treatment. The immunological parameters assessed prior to and at 3 months' interval during treatment included serum levels of soluble IL-2 receptor (sIL-2R) and IL-10 as well as leukocyte cytokine mRNA expression of TNF alpha, IL-4 and TGF-beta. Copaxone treatment was found to lead to a significant reduction in the mean annual relapse rate (from 1.4 prior to treatment to 0.6 during treatment) and stabilization of disability in 90% of the patients. The treatment was accompanied by an elevation of serum IL-10 levels, suppression of the pro-inflammatory cytokine TNF alpha mRNA, and an elevation of the anti-inflammatory cytokines TGF-beta and IL-4 mRNAs in PBLs. These results suggest that the beneficial clinical effects of Copaxone in MS patients may be attributed to changes in activation of T cell subsets and a shift from Th1 to Th2/Th3 cytokine profile, probably leading to Cop-1-driven mechanisms of bystander suppression.

Immunoregulatory effects of interferon-beta and interacting cytokines on human vascular endothelial cells. Implications for multiple sclerosis autoimmune diseases.

The mechanism(s) of action responsible for the anti-inflammatory effects mediated by interferon (IFN)-beta are still elusive although suggestions include anti-viral effects, the enhancement of natural killer (NK) or suppressor T cell activity and opposition to the effects of inflammatory cytokines. As vascular endothelial cells are active participants in inflammatory and demyelinating processes, we decided to examine the effects of IFN-beta on the expression of major histocompatibility complex (MHC) gene products and intercellular adhesion molecule (ICAM)-1 on human vascular endothelial cells (ECs). Human umbilical ECs demonstrated constitutive expression of ICAM-1 and MHC class I molecules but did not express MHC class II molecules. Basal expression of ICAM-1 molecules was enhanced by TNF alpha and to a lesser extent by IFN-beta, but was not affected by IFN-gamma. MHC class I expression on ECs was enhanced by IFN-beta, IFN-gamma, and tumor necrosis factor (TNF)-alpha. Furthermore, a synergistic effect was observed to combinations of these interacting cytokines. Incubation of ECs with IFN-gamma, but not IFN-beta, induced class II expression in a dose dependent manner. Moreover, co-incubation of ECs with IFN-beta and IFN-gamma resulted in significant down-regulation of class II molecules expression which was directly dependent on IFN-beta concentration. Northern blot analysis of DR alpha and Beta 2-microglobulin mRNA expression suggested that cytokine-mediated regulation of MHC molecules is at the transcriptional level, while modulation of ICAM-1 expression appears to be at the transcriptional as well as post-transcriptional level. Thus, our study demonstrated that IFN-beta and interacting cytokines exert complex immunoregulatory effects on endothelial cells with differential modulatory effects on various cell surface markers. Understanding the biological significance of these immunomodulatory effects mediated by IFN-beta may have important implications for cytokine-based strategies in the treatment of inflammatory and autoimmune diseases.

Does stem cell self renewal and progenitor cell commitment operate through an effector-memory cell mechanism?

We propose a model for stem cell self renewal and transition into commitment towards a variety of cell lineages. In this model the production of both "effector cells" (as represented by the mature cells in the different cell lineages) and of progenitor "memory" lymphocytes, takes place concomitantly. The experimental evidence supporting this model is as follows: Pure lymphocytic suspensions (PLS) are established and persist in culture when nude mouse-spleen and lymph-node cells are maintained on X-irradiated fibroblast monolayers in the presence of the S-phase cytotoxic agent cytosine arabinoside (Ara-C). From these PLS the following colony types can be initiated by the corresponding inducing (stimulating) factors (CSF): histiocytes (tissue macrophages) - CSF-1; granulocytes-macrophages (GM) - CSF-GM; mast cells - MMSF; granular-NK mucus secreting cells - IL-2; and multilineage colonies - IL-3. Mitotically active blast cells (formed by transformation of lymphocytes), condense into motile small cells when the stimulatory factor is removed. These "memory" lymphocytes are committed as they carry the receptors for the specific CSF; they respond by retransformation into blast cells. A dramatic increase in mast-cell colony forming cells is found in bone marrow, spleen and lymph-nodes of mice infected with Schistosoma mansoni. By maintaining PLS with both Ara-C and each of the CSFs and then titrating the incidence of CFC in the residual PLS, we find that each one of the CSFs acts on an independent set of cells in the PLS to produce the corresponding colony type. Finally, the concept suggests that the various blast cells carrying the receptors, undergo condensation into memory lymphocytes when dissociated from the environment prevailed by the corresponding CSF. In this way pluripotential blast-cells condense into motile lymphocytes which are committed to pluripotentiality.

Granular natural-killer cells develop into mucus-secreting cells.

Colonies of granular natural-killer cells selectively develop in lymph-node cell cultures of nude mice after stimulation with rat T-cell growth factor. When these cells are grown on X-irradiated monolayers prepared from 16-18-day-old mouse embryos, they are triggered to synthesize and secrete a sulphated glycoprotein that can be identified as mucus. As a result of an erosive process of the granules, the mucoid material accumulates in pools in the cytoplasm matrix. The secretion is operated through a process of budding of double-membrane-bound vesicles. The successful triggering of mucous synthesis is interpreted by the successful growth of those mesenchymal cells in the embryonic monolayer that function in the induction of epithelial morphogenesis in the developing embryo.

Selection of human TcR V gene families in autologous mixed lymphocyte reactions: relevance to autoimmune immunopathology.

We have postulated that in vivo autologous mixed lymphocyte reactions (AMLRs) are one mechanism in the development of the intrathyroidal lymphocytic infiltration of human autoimmune thyroid disease. Such a mechanism would explain the significant numbers of self-reactive T cells present in thyroid infiltrates as evidenced by cloning studies. However, infiltrating T cells in a variety of autoimmune disease including autoimmune thyroid disease, demonstrate bias in their use of T cell receptor (TcR) V gene families. In order to examine whether such TcR V gene bias may occur secondary to non-antigen specific in vivo AMLRs rather than secondary to specific autoantigen driven mechanisms we have examined the human TcR repertoire after prolonged AMLRs in vitro. Using 5 healthy donors in 1, 2 and 3 weeks AMLRs we showed stimulation indices of 3.1-6.5 after 3 weeks. The hTcR V alpha and V beta gene repertoire was assessed using the PCR technique and revealed an almost complete repertoire of V gene families at the beginning of the studies while at the end of 3 weeks a mean of only 5.2 V alpha genes were transcribed. Less restriction was seen in the hTcR V beta repertoire with a mean of 9 V beta genes used. These data demonstrate that the AMLR is able to mimic the marked bias in hTcR V gene family use seen within the inflammatory infiltrates of autoimmune diseases.

Divergent effects of ischemia/reperfusion and nitric oxide donor on TNFalpha mRNA accumulation in rat organs.

We previously showed that serum TNFalpha bioactivity in rats is proportional to the extent of graded tissue injury caused by laparotomy, intestinal ischemia, and reperfusion and that the spleen is an important source of TNFalpha secretion in this condition. TNFalpha production varies, depending on the type and duration of tissue injury. It is also affected by other mediators, such as nitric oxide (NO). TNFalpha is known to increase NO production, but the effect of NO on the production of TNFalpha has not yet been fully elucidated. In this study we determined the levels of TNFalpha mRNA in rat organs after graded injury caused by anesthesia, laparotomy, intestinal ischemia, and reperfusion and evaluated the effects of the NO donor S-nitroso-N-acetylpenicillamine (SNAP) on it. Samples from different organs were removed, and TNFalpha gene expression was evaluated by semiquantitative RT-PCR. TNFalpha mRNA was not detected in the intestine (the ischemic organ) and in the kidney, brain, heart, or liver after all 4 experimental protocols. In the mesenteric lymph node (draining the ischemic organ) a basal level of expression of TNFalpha mRNA was detected in the control (anesthesia alone) group, which was increased significantly after ischemia. In the spleen (a remote immune organ not directly involved in the ischemia), a significant gradual increase in TNFalpha mRNA, which correlated to the severity of the experimental protocol, was observed. In the lung (a central participant in post-injury multiple organ failure), all interventions increased TNFalpha mRNA. Infusion of SNAP exerted a differential effect on TNFalpha mRNA: diminished its accumulation in the lymph node, enhanced it in the lung, and had no effect in the spleen. The divergent organ pattern of TNFalpha transcription emphasizes the importance of its localized expression, which is critical to the understanding of its autocrine and paracrine actions in ischemia and reperfusion.

Effect of hyperbaric oxygen on tissue distribution of mononuclear cell subsets in the rat.

In a previous study we found a significant temporary decrease in the ratio of CD4/CD8 (helper, inducer/suppressor, cytotoxic) T lymphocytes in the peripheral blood of healthy human volunteers after exposure to a single commonly used profile of hyperbaric oxygen (HBO). The transient nature of the changes suggested redistribution of T-cell subsets. The purpose of the present study was to verify such a redistribution and to locate possible target organs in an animal model. A single exposure of rats to HBO (0.28 MPa) induced a highly significant rapid decrease in the CD4/CD8 ratio in peripheral blood count (P < 0.0001), confirming our previous findings in humans. HBO also induced a significant increase in the CD4/CD8 ratio in the lungs and lymph nodes (P < 0.001) and a significant decrease in the ratio in the spleen (P < 0.01). Furthermore, exposure to HBO induced a significant increase in T cells bearing surface interleukin-2 receptors in the blood, spleen, lungs, and lymph glands (P < 0.001) and a significant decrease in T cells expressing alpha beta-receptors in the lungs (P < 0.001) and lymph glands (P < 0.05). Our findings suggest rapid T-cell activation after a brief exposure to HBO, with shifts of CD4 and CD8 subsets and variations in T-cell receptor type. These rapid changes in the parameters of cell-mediated immunity may represent the activation of protective mechanisms against the toxic effect of oxygen or the early stages of pulmonary oxygen toxicity.

Regulation of HLA-DR and costimulatory B7 molecules in human thyroid carcinoma cells: differential binding of transcription factors to the HLA-DRalpha promoter.

The consequence of autoantigen presentation by thyroid cells is dependent on the magnitude of expression of both HLA class II antigens (mainly HLA-DR) and costimulatory molecules, such as B7 (CD80 and CD86). Autoimmune thyrocytes are induced to express HLA-DR by interferon-gamma (IFN-gamma). The costimulatory signal leading to autoantibody production or cytotoxic T-cell immune response could be provided by antigen presenting cells (APCs) attracted to the thyroid by the primary autoimmune stimulus. Malignant thyrocytes can express HLA-DR antigens either constitutively, as a result of a nonimmunologic stimulus, or on induction with IFN-gamma after triggering of an immune response. However, their ability to express B7 molecules, which may determine enhanced antitumoral immune response mainly in the absence of intrathyroidal macrophages, has not yet been studied. The regulation of HLA-DR gene expression in APCs, such as B cells, is mediated by a series of short DNA consensus sequences located in the promoter, termed the W, X, and Y boxes, which bind several known transcription factors. We have previously characterized the expression of HLA-DR in four human thyroid carcinoma cell lines and found differences between constitutive and high- or moderate-induced expression of the protein and mRNA. Evaluation of B7 expression on the surface of thyroid cancer cells and understanding the mechanisms of HLA-DR gene expression may help in designing efficient immune response to thyroid tumors. Using the electrophoretic mobility shift assay (EMSA), we have demonstrated differences between the four thyroid cell lines in the binding of transcription factors to each of the three boxes. The binding to the promoter in each of the cell lines resulted in a single band, probably representing a complex of proteins formed via protein-protein interactions. Using flow cytometry we have shown that the B7 molecule was absent in the four thyroid cell lines and could not be induced by IFN-gamma. The absence of surface B7 molecules from the malignant thyroid cells may lead to either suppression of antitumoral cytotoxic T cell response or demand the cooperation of infiltrating APCs to favor immune response. Differences previously found in HLA-DR expression in the four human malignant thyroid cell lines may be explained by the variation in the binding of transcription factors to the boxes in the HLA-DRalpha promoter. The binding patterns of nuclear proteins derived from the four thyroid cell lines or from the B lymphocyte cell line--Raji--to each of the boxes or to the whole promoter exhibit similarities, thus suggesting similar DNA-protein interactions.

Coincidence of vitiligo and Paget's bone disease in a patient.

Paget's bone disease developed in a patient with vitiligo. Scrupulous physical examination excluded further systemic or cutaneous involvement. The immunological workup revealed a reversed CD4/CD8 ratio due to a very low CD4 cell percentage and almost negligible responses to PHA as well as Con A, T cell mitogens. The pathogenic significance of these results, which point to phenotypic and functional T cell defects, is discussed.

Sebaceous adenomas, squamous cell carcinoma and skin infections in a patient with carcinoma of the colon, rectum and bladder.

Sebaceous adenomas and squamous cell carcinoma developed in a male patient in addition to viral, mycotic and bacterial infections, several years after the removal of three malignant tumors from his lower gastrointestinal and urinary tract. Skin tests with trichophytin, candidin, and mixed bacteria were negative. Various aspects regarding cutaneous changes associated with colorectal and bladder carcinomas are discussed.

Psoriasis, necrobiosis lipoidica, granuloma annulare, vitiligo and skin infections in the same diabetic patient.

A diabetic patient is described presenting psoriasis, necrobiosis lipoidica diabeticorum, granuloma annulare, and vitiligo and with a history of recurrent erysipelas and mycotic infections. Scrupulous physical examination excluded further systemic or cutaneous involvement. The immunological workup revealed both phenotypic and functional defects in cellular immunity.

Long-standing dermatological manifestations in a patient with chronic heavy metal intoxication.

A patient with chronic metal intoxication is described, presenting during four years after the cessation of her exposure to industrial substances, maculo-papular eruptions with several ulcerated lesions and excoriations on her abdomen and buttocks. She also had pallor of her face, greyish-dark discoloration of the hair, while the fingernails were brittle and sensitive. Scrupulous physical examination excluded further cutaneous involvement. The immunological workup revealed both phenotypic and functional defects in cellular immunity.

Vitiligo, rheumatoid arthritis and pernicious anemia.

A patient with a 46-year history of vitiligo who also presented rheumatoid arthritis and pernicious anemia is described. Meticulous physical examination excluded further systemic or cutaneous involvement. The immunological workup revealed a low CD4 cell percentage with T cells mostly composed of CD8 cells, a discrepancy between the high percentage of cumulative CD4 + CD8 cells and the measured CD3 proportions, very low NK cytotoxicity toward K562 cells, and almost negligible responses to PHA, Con A and PWM mitogens. The results point to severe T and NK cell functional defects. The pathogenetic significance of these data is discussed.

Effect of a single exposure to hyperbaric oxygen on blood mononuclear cells in human subjects.

We studied the effect of a single exposure to a therapeutic profile of hyperbaric oxygen on blood mononuclear cell subset. Twenty healthy volunteers were exposed to 0.28 MPa for 90 min. Thirteen breathed pure oxygen and seven were control subjects exposed to compressed air at the same pressure. Venous blood samples were drawn before HBO exposure, immediately on exit from the chamber, and 24 h later. Immediately after the exposure, a significant increase was observed in the percentage and absolute number of CD8 (suppressor/cytotoxic) T cells, with a concomitant decrease in the CD4 (helper/inducer) T cells. These changes resulted in a decreased CD4:CD8 ratio. A rise was also observed in the number of HLA-DR antigen-bearing cells, with a transient increase in monocytes. There was no change in the total count and percentage of T cells (CD3), B cells, and NK cells. Twenty-four hours after HBO exposure there was a partial reversal of the decrease in the mean CD4:CD8 ratio, but it was still significantly lower than preexposure values. The fast reversibility of the change in the CD4:CD8 ratio suggests specific HBO-induced shifts and sequestration of T-cell subpopulations.

Immature cells identified by the H-1 hematology analyzer predicts thymidine incorporation after mitogenic stimulation.

The incorporation of thymidine by lymphocytes stimulated by phytohemagglutinin (PHA) was correlated with the number of immature cells identified by the Technicon H-1 as blasts. The number of blasts and thymidine incorporation plateaued on day 3. At that time, a high linear correlation between the two variables was found (r = 0.949, p less than 0.01). The H-1 evaluated the samples in 60 seconds as compared to 3 hours of processing required to calculate thymidine incorporation. We conclude that the number of immature cells identified by the Technicon H-1 can predict the degree of thymidine incorporation after stimulation of peripheral blood cells with PHA. The H-1 may replace the more time consuming and dangerous use of radioactive thymidine incorporation in evaluating patients' functional immunologic status.

Decreased natural killer (NK) activity in patients with myeloproliferative disorders.

Natural killer (NK) cell number and activity were measured in 26 patients with myeloproliferative disorders and the results were compared with 16 age-matched control patients. The percent of Leu-11b-positive cells was 11% +/- 3% in the patients, compared with 12% +/- 4% in the control patients. Ten of 26 patients, however, had NK activity lower than all of the control values at three different effector to target cell ratios (E:T) (P less than 0.005). The values of those patients with low unstimulated NK activity remained low despite stimulation with interleukin-2 (IL-2) or alpha-interferon (alpha-IFN), whereas the values of those patients with normal unstimulated activity responded to IL-2 and alpha-IFN like the control patients. Three of the ten patients with low NK activity had a history of malignant neoplasms. None of the 16 patients with normal NK activity had a history of malignant neoplasms (P less than 0.05). We conclude that patients with myeloproliferative disorders frequently have low endogenous NK cell activity in vitro. The dysfunction of the NK system appears to be intrinsic because the relative number of NK cells was similar to control values and the response to stimulation with IL-2 and alpha-IFN was suboptimal. There may be a relationship between low NK activity and the development of malignant disease in such patients.

Ex vivo secretion of tumor necrosis factor and interleukin-2 by rat splenocytes after intestinal ischemia and shock.

We studied the ex vivo secretion of tumor necrosis factor (TNF) and interleukin 2 (IL-2) by splenocytes after circulatory shock induced by intestinal ischemia and reperfusion in rats. Shock was induced by total occlusion of the superior mesenteric artery followed by reperfusion. In a second group, vascular occlusion was maintained throughout the experimental protocol. A third group of sham rats and a fourth group of control rats with a negligible surgical procedure were also studied. "Spontaneous" (untriggered) secretion of TNF by splenocytes was higher in the ischemia-reperfusion group than in all other groups (p < 0.01), but did not increase significantly after stimulation with LPS. Splenocytes from control rats exhibited a marked increase in TNF secretion after stimulation with LPS to values similar to those in the ischemia-reperfusion group. A diminished, though statistically significant increase in LPS-stimulated secretion of TNF was detected in the sham and ischemia only groups of rats (p < 0.05) from untriggered values in each. Untriggered secretion of IL-2 was similar in all groups. However, when compared to control rats, splenocytes from the three surgically manipulated groups exhibited suppressed secretion of IL-2 in response to stimulation with Con A (p < 0.05). These results support the role played by TNF in mediation of shock and point to spleen macrophages as a source of TNF after intestinal ischemia and reperfusion. Our results also demonstrated postinjury alteration in immune function manifested by depressed ability of splenocytes to increase the production of IL-2 after stimulation with Con A.

Altered interleukin-2 secretion in patients with primary fibromyalgia syndrome.

Interleukin-2 (IL-2) production was studied in T lymphocytes and isolated CD4+ T lymphocytes from 12 patients with primary fibromyalgia syndrome and 10 healthy volunteers. The dose and time kinetics of IL-2 production by concanavalin A-stimulated T cells and CD4+ T cells of fibromyalgia patients differed from findings in controls by 1) a need for a higher concentration of mitogen in order to achieve optimal IL-2 secretion, and 2) a delay in the peak time of optimal IL-2 secretion. Unlike normal IL-2 secretion, which was higher after removal of CD8+ T cells, the pattern and degree of IL-2 secretion by cells from fibromyalgia patients were not changed following removal of CD8+ T cells. Addition of calcium ionophore in assays using suboptimal concanavalin A concentrations did not correct the reduction in IL-2 secretion by fibromyalgia patient T cells, but addition of phorbol myristate acetate induced normal secretion of IL-2. These findings suggest that there is a defect in the IL-2 pathway, which is related to protein kinase C activation and does not involve impairment of Ca2+ elevation, in patients with fibromyalgia.