Nitric oxide is involved in interleukin-1alpha-induced cytotoxicity in polarised human thyrocytes

Abstract

Nitric oxide (NO) is a well-known mediator of autoimmune processes. In the thyroid gland, it is produced in response to interleukin 1 (IL-1) and may mediate cytokine action at an early stage of autoimmune thyroiditis. In this study, we have investigated whether NO is involved in cytokine-induced cytotoxic effects and epithelial barrier alterations in thyrocytes. Human thyroid epithelial cells were cultured as tight polarised monolayers on a permeable support and exposed or not to IL-1 (100 U/ml), alone or in combination with interferon-(IFN-; 100 U/ml) added to the basal compartment. NO production was not detected in control thyrocytes, but was significantly induced by the combination of IL-1 with IFN-, in a time-dependent fashion. Similarly, expression of the inducible isoform of nitric oxide synthase (NOSII), determined by immunoblot and immunofluorescence confocal microscopy, was not detected in control cells, but was markedly induced after 48-h exposure to both cytokines. This treatment significantly increased the release of cytosolic lactate dehydrogenase (LDH) in the apical and basolateral media and decreased transepithelial electrical resistance. Although IFN-was not sufficient to induce NO production, it could by itself decrease transepithelial resistance and synergised the IL-1 effect on LDH release. The NOS inhibitor, -nitro-argininemethyl ester, suppressed the cytokine-induced NO production and decreased the LDH release, but failed to prevent the loss of transepithelial resistance. These results indicated that human thyrocytes express NOSII and produce NO in response to IL-1 +IFN-and suggest that NO acts as a mediator of cytokine-induced cytotoxicity in the thyroid gland and may promote the exposure of autoantigens to the immune system. In contrast, NO does not appear to mediate the cytokine-induced disruption of the thyroid epithelial barrier.

Interferon-gamma down-regulates claudin-1 and impairs the epithelial barrier function in primary cultured human thyrocytes

“…It is likely therefore that loss of barrier function is not secondary to cytokine-mediated cytotoxicity. Similar observations were recently reported by van den Hove et al (14) in studies on nitric oxide as mediator of IL-1a and IFN-g interactions. The maintenance of an epithelial barrier is dependent on the functional interplay of several tight junction proteins (15).…”

N-Acetylcysteine and 15 Deoxy-Δ12,14-Prostaglandin J2 Exert a Protective Effect Against Autoimmune Thyroid Destruction in Vivo but Not Against Interleukin-1α/Interferon γ-Induced Inhibitory Effects in Thyrocytes in Vitro

“…It is, however, clear that the nature of ROS will be determinant in terms of cell survival, some of them being more deleterious than others. 41 Among reactive oxygen species, reactive nitrogen species, especially NO, are known to be induced by IL-1␣/IFN␥ in human thyrocytes 27,30,43 and are partially responsible for the inhibitory effects of Th1 cytokines on thyrocytes. 22 In addition, although NAC partially reduces the production of nitrite, ROS immunoflorescence detected by DCFH-DA fluorescence is completely abolished.…”

“…22,30 When cells were co-incubated with IL-1␣/IFN␥ and NAC or 15dPGJ2, nitrite levels were significantly reduced as compared with Th1-treated cells, but remained higher than in control cells ( Figure 1F). …”

“…25,26 In human, but not in rat thyrocytes, NO has also been identified as mediating the inhibitory actions of Th1 cytokines, but only partially. 22,[27][28][29][30] Intracellular factors other than NO should therefore mediate Th1 cytokine-induced inhibitory effects. ROS may represent, among others, alternative candidates, as suggested by previous studies.…”

Expression of TPO and ThOXs in human thyrocytes is downregulated by IL-1α/IFN-γ, an effect partially mediated by nitric oxide

“…Previous papers (19,25,40) reported that some cytokine-induced effects in the thyroid gland could be mediated by nitric oxide (NO). The three NO synthase (NOS) isoforms are present and functionally active in the thyroid gland (9,10).…”