PMC:1064873 / 14989-23420 JSON TXT

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LappsTest

2_test

{"project":"2_test","denotations":[{"id":"15535835-10898505-4156888","span":{"begin":316,"end":318},"obj":"10898505"},{"id":"15535835-7306232-4156889","span":{"begin":1252,"end":1254},"obj":"7306232"},{"id":"15535835-11244051-4156890","span":{"begin":2559,"end":2560},"obj":"11244051"},{"id":"15535835-11739506-4156891","span":{"begin":2636,"end":2638},"obj":"11739506"},{"id":"15535835-7543512-4156892","span":{"begin":3393,"end":3394},"obj":"7543512"},{"id":"15535835-10023769-4156893","span":{"begin":3531,"end":3532},"obj":"10023769"},{"id":"15535835-14688368-4156894","span":{"begin":3644,"end":3646},"obj":"14688368"},{"id":"15535835-12039028-4156895","span":{"begin":4971,"end":4972},"obj":"12039028"},{"id":"15535835-7869026-4156896","span":{"begin":5133,"end":5135},"obj":"7869026"},{"id":"15535835-9182920-4156897","span":{"begin":5231,"end":5233},"obj":"9182920"},{"id":"15535835-12039028-4156898","span":{"begin":5591,"end":5592},"obj":"12039028"},{"id":"15535835-11527983-4156899","span":{"begin":7371,"end":7372},"obj":"11527983"},{"id":"15535835-12754507-4156900","span":{"begin":7539,"end":7540},"obj":"12754507"},{"id":"15535835-12626585-4156901","span":{"begin":7541,"end":7542},"obj":"12626585"},{"id":"15535835-12538698-4156902","span":{"begin":7697,"end":7699},"obj":"12538698"}],"text":"Results\n\nResistance to IL-10 inhibition of IFN-γ production in RA CD4+ T cells\nThe CD28 costimulatory pathway is crucial for effective antigen-specific T-cell cytokine production, and IL-10 can directly suppress this response by inhibiting CD28 tyrosine phosphorylation and binding of phosphatidylinositol 3-kinase [24]. To evaluate the responsiveness of RA CD4+ T cells to IL-10, purified PB CD4+ T cells from three patients with active RA and from three healthy controls were stimulated by immobilized anti-CD3 antibody and anti-CD28 antibody with or without diluted concentrations of IL-10 for 36 hours, and IFN-γ production was measured by ELISA. As shown in Fig. 1, IFN-γ production by activated normal CD4+ T cells was mostly inhibited at concentrations as low as 1 ng/ml IL-10. However, RA CD4+ T cells were able to produce significant amounts of IFN-γ in the presence of 1 ng/ml IL-10, and the maximal but not complete inhibition by IL-10 was obtained at 10–100 ng/ml.\nWe thus compared the levels of IFN-γ production by CD4+ T cells after CD3 and CD28 costimulation in the presence of 1 ng/ml IL-10 in RA patients with active disease (multiple inflammatory joints, CRP level ≥ 10 mg/l) and inactive disease (in remission, CRP level \u003c 10 mg/l) [26] and in healthy controls. There were no statistically significant differences in IFN-γ production without IL-10 among these three groups (Fig. 2a), but the inhibitory effect of IL-10 on IFN-γ production was significantly limited in the active RA group as compared with the inactive RA group and healthy controls (percentage decrease: active RA, 2.9 ± 14.4%; inactive RA, 45.6 ± 14.4%; controls, 65.8 ± 7.9%) (Fig. 2b). As a consequence, CD4+ T cells from active RA patients produced higher levels of IFN-γ in the presence of 1 ng/ml IL-10 than did normal CD4+ T cells (Fig. 2a).\nIn addition, we compared IL-2 production by CD4+ T cells after CD3 and CD28 costimulation in the presence of IL-10 in active RA patients and in healthy controls. Similarly, IL-2 production was not affected by 1 ng/ml IL-10 in RA patients (percentage decrease, -2.1 ± 13.8%), while it was significantly reduced in healthy controls (61.1 ± 13.7%; P \u003c 0.05). Taken together, these results indicate that RA CD4+ T cells become less susceptible to the immunoregulatory effect of IL-10 during the active phase.\n\nIncreased expression of cell surface IL-10R1 on RA CD4+ T cells\nThe functional receptor complex of IL-10 consists of two subunits, the primary ligand-binding component IL-10R1 and the accessory component type 2 IL-10R [1]. IL-10R1 expression plays a critical role in cellular responses to IL-10 [29]. To examine whether the resistance to IL-10 inhibition in RA CD4+ T cells was due to limited receptor expression, the cell surface expression of IL-10R1 on PB CD4+ T cells from active RA patients and from healthy controls was determined by flow cytometric analysis. As shown in Fig. 3a,3b, the intensity of IL-10R1 expression on CD4+ T cells was significantly increased in RA patients compared with in healthy controls. These results suggest that the intracellular signal transduction pathway of IL-10 may be impaired in CD4+ T cells of active RA.\n\nDefective IL-10-mediated STAT3 phosphorylation in RA CD4+ T cells\nThe interaction of IL-10R with IL-10 induces tyrosine phosphorylation and activation of the latent transcription factors STAT1 and STAT3 [3]. Macrophage-specific STAT3-deficient mice demonstrated that STAT3 plays a dominant role in IL-10-mediated anti-inflammatory responses [5], which has recently been confirmed in human macrophages by studies of dominant-negative STAT3 overexpression [30]. The induction of STAT1 and STAT3 phosphorylation by IL-10 in PB CD4+ T cells from active RA patients and from healthy controls was examined using western blotting. STAT3 phosphorylation was dose-dependently induced after IL-10 activation for 20 min in normal CD4+ T cells (Fig. 4a,4b). In contrast, STAT3 was phosphorylated in freshly isolated PB CD4+ cells from RA patients and this STAT3 phosphorylation was detectable for up to 6 hours. STAT3 phosphorylation was augmented only when activated by as much as 10 ng/ml IL-10. Both sustained STAT3 phosphorylation and defective IL-10-induced STAT3 phosphorylation were found in RA ST CD4+ T cells (Fig. 4c). On the other hand, IL-10-induced STAT1 phosphorylation was not detected in either RA CD4+ T cells or normal CD4+ T cells (Fig. 4a). These results indicate that STAT3 is the major IL-10-activated STAT in CD4+ T cells, and IL-10-induced STAT3 activation may be diminished in active RA, in association with sustained STAT3 phosphorylation.\n\nIL-6-mediated STAT3 phosphorylation and inhibition of IL-10 effect in normal CD4+ T cells\nSTAT3 is activated by many cytokines and growth factors such as the IL-6 family of cytokines (IL-6, IL-11, leukemia inhibitory factor, and oncostatin M), platelet-derived growth factor, and epidermal growth factor, in addition to IL-10 [4], but previous studies have demonstrated that IL-6 is the major factor in RA synovial fluid that induces constitutive activation of STAT3 in mononuclear cells [31]. Since IL-6 is also abundant in sera of active RA patients, frequently detected at \u003e 1 ng/ml [27], we examined whether persistent exposure of CD4+ T cells to high concentrations of IL-6 in the blood circulation was responsible for their sustained STAT3 activation and resistance to IL-10 inhibition in active RA. Both STAT1 and STAT3 phosphorylation was activated by IL-6 in normal CD4+ T cells (data not shown), in agreement with previous observations [4]. Normal CD4+ T cells were thus incubated for 20 min with culture medium containing 30% serum from active RA patients and neutralizing anti-IL-6 antibody or control antibody, and STAT phosphorylation was examined by western blot analysis. RA serum was able to induce tyrosine phosphorylation of STAT3 but not STAT1, and this STAT3 activation was mostly abolished by neutralization of IL-6 activity (Fig. 5a). These results indicate that IL-6 is the dominant STAT3-activating factor contained in sera of active RA patients. The lack of STAT1 activation by RA serum suggests that much higher concentrations of IL-6 may be required for STAT1 activation as compared with STAT3 activation, or that inhibitors of STAT1 signaling may be present in RA serum.\nWe next examined whether IL-6 could suppress the effect of IL-10 to inhibit IFN-γ production by CD4+ T cells. After preincubation with or without 10 ng/ml IL-6 for 36 hours, normal CD4+ T cells were stimulated by CD3 and CD28 costimulation in the presence or absence of 1 ng/ml IL-10 for 36 hours, and the IFN-γ production was measured by ELISA. IL-6 pretreatment of normal cells reduced IL-10-mediated inhibition of IFN-γ production (Fig. 5b), indicating that high concentrations of IL-6 could modulate T-cell responsiveness to IL-10. Taken together, these findings suggest that persistent exposure to serum IL-6 may have a role in both the induction of STAT3 activation and the resistance to the inhibitory effect of IL-10 in RA CD4+ T cells.\n\nHigh expression of SOCS1 mRNA in RA CD4+ T cells\nIL-6 induces two potent inhibitors of JAKs (SOCS1 and SOCS3 proteins) that not only act as mediators of negative feedback inhibition, but also play a major role in crosstalk inhibition by opposing other cytokine-signaling pathways [7]. SOCS3 has recently been shown to specifically inhibit STAT3 activation induced by IL-6 but not by IL-10, thereby regulating the divergent action of IL-6 and IL-10 [8,9]. On the contrary, SOCS1 is able to partially inhibit IL-10-mediated STAT3 activation and cellular responses, as well as IFN-γ-mediated STAT1 activation [32]. To determine whether SOCSs were involved in the defective IL-10-induced STAT3 activation of RA CD4+ T cells, the levels of SOCS1 and SOCS3 mRNA expression in PB CD4+ T cells from active RA patients and from healthy controls were compared by semiquantitative real-time PCR. The RA CD4+ T cells contained higher levels of SOCS1 but lower levels of SOCS3 transcripts than did control CD4+ T cells (Fig. 6a). Constitutive expression of SOCS1 mRNA in RA CD4+ T cells was comparable with the expression in normal CD4+ T cells stimulated by 10 ng/ml IL-6 (Fig. 6b), supporting its functional significance. Defective IL-10-induced STAT3 activation therefore appears to be due at least in part to an abundance of SOCS1 in RA CD4+ T cells."}

biosemtest

pmc-enju-pas

bionlp-st-ge-2016-coref

bionlp-st-ge-2016-test-proteins

bionlp-st-ge-2016-uniprot

UBERON-AE

{"project":"UBERON-AE","denotations":[{"id":"T17463","span":{"begin":6947,"end":6952},"obj":"http://purl.obolibrary.org/obo/UBERON_0001977"},{"id":"T17462","span":{"begin":6337,"end":6342},"obj":"http://purl.obolibrary.org/obo/UBERON_0001977"},{"id":"T17461","span":{"begin":6151,"end":6156},"obj":"http://purl.obolibrary.org/obo/UBERON_0001977"},{"id":"T17460","span":{"begin":5835,"end":5840},"obj":"http://purl.obolibrary.org/obo/UBERON_0001977"},{"id":"T17459","span":{"begin":5681,"end":5686},"obj":"http://purl.obolibrary.org/obo/UBERON_0001977"},{"id":"T17458","span":{"begin":5330,"end":5335},"obj":"http://purl.obolibrary.org/obo/UBERON_0000178"},{"id":"T17457","span":{"begin":5050,"end":5064},"obj":"http://purl.obolibrary.org/obo/UBERON_0001090"}],"text":"Results\n\nResistance to IL-10 inhibition of IFN-γ production in RA CD4+ T cells\nThe CD28 costimulatory pathway is crucial for effective antigen-specific T-cell cytokine production, and IL-10 can directly suppress this response by inhibiting CD28 tyrosine phosphorylation and binding of phosphatidylinositol 3-kinase [24]. To evaluate the responsiveness of RA CD4+ T cells to IL-10, purified PB CD4+ T cells from three patients with active RA and from three healthy controls were stimulated by immobilized anti-CD3 antibody and anti-CD28 antibody with or without diluted concentrations of IL-10 for 36 hours, and IFN-γ production was measured by ELISA. As shown in Fig. 1, IFN-γ production by activated normal CD4+ T cells was mostly inhibited at concentrations as low as 1 ng/ml IL-10. However, RA CD4+ T cells were able to produce significant amounts of IFN-γ in the presence of 1 ng/ml IL-10, and the maximal but not complete inhibition by IL-10 was obtained at 10–100 ng/ml.\nWe thus compared the levels of IFN-γ production by CD4+ T cells after CD3 and CD28 costimulation in the presence of 1 ng/ml IL-10 in RA patients with active disease (multiple inflammatory joints, CRP level ≥ 10 mg/l) and inactive disease (in remission, CRP level \u003c 10 mg/l) [26] and in healthy controls. There were no statistically significant differences in IFN-γ production without IL-10 among these three groups (Fig. 2a), but the inhibitory effect of IL-10 on IFN-γ production was significantly limited in the active RA group as compared with the inactive RA group and healthy controls (percentage decrease: active RA, 2.9 ± 14.4%; inactive RA, 45.6 ± 14.4%; controls, 65.8 ± 7.9%) (Fig. 2b). As a consequence, CD4+ T cells from active RA patients produced higher levels of IFN-γ in the presence of 1 ng/ml IL-10 than did normal CD4+ T cells (Fig. 2a).\nIn addition, we compared IL-2 production by CD4+ T cells after CD3 and CD28 costimulation in the presence of IL-10 in active RA patients and in healthy controls. Similarly, IL-2 production was not affected by 1 ng/ml IL-10 in RA patients (percentage decrease, -2.1 ± 13.8%), while it was significantly reduced in healthy controls (61.1 ± 13.7%; P \u003c 0.05). Taken together, these results indicate that RA CD4+ T cells become less susceptible to the immunoregulatory effect of IL-10 during the active phase.\n\nIncreased expression of cell surface IL-10R1 on RA CD4+ T cells\nThe functional receptor complex of IL-10 consists of two subunits, the primary ligand-binding component IL-10R1 and the accessory component type 2 IL-10R [1]. IL-10R1 expression plays a critical role in cellular responses to IL-10 [29]. To examine whether the resistance to IL-10 inhibition in RA CD4+ T cells was due to limited receptor expression, the cell surface expression of IL-10R1 on PB CD4+ T cells from active RA patients and from healthy controls was determined by flow cytometric analysis. As shown in Fig. 3a,3b, the intensity of IL-10R1 expression on CD4+ T cells was significantly increased in RA patients compared with in healthy controls. These results suggest that the intracellular signal transduction pathway of IL-10 may be impaired in CD4+ T cells of active RA.\n\nDefective IL-10-mediated STAT3 phosphorylation in RA CD4+ T cells\nThe interaction of IL-10R with IL-10 induces tyrosine phosphorylation and activation of the latent transcription factors STAT1 and STAT3 [3]. Macrophage-specific STAT3-deficient mice demonstrated that STAT3 plays a dominant role in IL-10-mediated anti-inflammatory responses [5], which has recently been confirmed in human macrophages by studies of dominant-negative STAT3 overexpression [30]. The induction of STAT1 and STAT3 phosphorylation by IL-10 in PB CD4+ T cells from active RA patients and from healthy controls was examined using western blotting. STAT3 phosphorylation was dose-dependently induced after IL-10 activation for 20 min in normal CD4+ T cells (Fig. 4a,4b). In contrast, STAT3 was phosphorylated in freshly isolated PB CD4+ cells from RA patients and this STAT3 phosphorylation was detectable for up to 6 hours. STAT3 phosphorylation was augmented only when activated by as much as 10 ng/ml IL-10. Both sustained STAT3 phosphorylation and defective IL-10-induced STAT3 phosphorylation were found in RA ST CD4+ T cells (Fig. 4c). On the other hand, IL-10-induced STAT1 phosphorylation was not detected in either RA CD4+ T cells or normal CD4+ T cells (Fig. 4a). These results indicate that STAT3 is the major IL-10-activated STAT in CD4+ T cells, and IL-10-induced STAT3 activation may be diminished in active RA, in association with sustained STAT3 phosphorylation.\n\nIL-6-mediated STAT3 phosphorylation and inhibition of IL-10 effect in normal CD4+ T cells\nSTAT3 is activated by many cytokines and growth factors such as the IL-6 family of cytokines (IL-6, IL-11, leukemia inhibitory factor, and oncostatin M), platelet-derived growth factor, and epidermal growth factor, in addition to IL-10 [4], but previous studies have demonstrated that IL-6 is the major factor in RA synovial fluid that induces constitutive activation of STAT3 in mononuclear cells [31]. Since IL-6 is also abundant in sera of active RA patients, frequently detected at \u003e 1 ng/ml [27], we examined whether persistent exposure of CD4+ T cells to high concentrations of IL-6 in the blood circulation was responsible for their sustained STAT3 activation and resistance to IL-10 inhibition in active RA. Both STAT1 and STAT3 phosphorylation was activated by IL-6 in normal CD4+ T cells (data not shown), in agreement with previous observations [4]. Normal CD4+ T cells were thus incubated for 20 min with culture medium containing 30% serum from active RA patients and neutralizing anti-IL-6 antibody or control antibody, and STAT phosphorylation was examined by western blot analysis. RA serum was able to induce tyrosine phosphorylation of STAT3 but not STAT1, and this STAT3 activation was mostly abolished by neutralization of IL-6 activity (Fig. 5a). These results indicate that IL-6 is the dominant STAT3-activating factor contained in sera of active RA patients. The lack of STAT1 activation by RA serum suggests that much higher concentrations of IL-6 may be required for STAT1 activation as compared with STAT3 activation, or that inhibitors of STAT1 signaling may be present in RA serum.\nWe next examined whether IL-6 could suppress the effect of IL-10 to inhibit IFN-γ production by CD4+ T cells. After preincubation with or without 10 ng/ml IL-6 for 36 hours, normal CD4+ T cells were stimulated by CD3 and CD28 costimulation in the presence or absence of 1 ng/ml IL-10 for 36 hours, and the IFN-γ production was measured by ELISA. IL-6 pretreatment of normal cells reduced IL-10-mediated inhibition of IFN-γ production (Fig. 5b), indicating that high concentrations of IL-6 could modulate T-cell responsiveness to IL-10. Taken together, these findings suggest that persistent exposure to serum IL-6 may have a role in both the induction of STAT3 activation and the resistance to the inhibitory effect of IL-10 in RA CD4+ T cells.\n\nHigh expression of SOCS1 mRNA in RA CD4+ T cells\nIL-6 induces two potent inhibitors of JAKs (SOCS1 and SOCS3 proteins) that not only act as mediators of negative feedback inhibition, but also play a major role in crosstalk inhibition by opposing other cytokine-signaling pathways [7]. SOCS3 has recently been shown to specifically inhibit STAT3 activation induced by IL-6 but not by IL-10, thereby regulating the divergent action of IL-6 and IL-10 [8,9]. On the contrary, SOCS1 is able to partially inhibit IL-10-mediated STAT3 activation and cellular responses, as well as IFN-γ-mediated STAT1 activation [32]. To determine whether SOCSs were involved in the defective IL-10-induced STAT3 activation of RA CD4+ T cells, the levels of SOCS1 and SOCS3 mRNA expression in PB CD4+ T cells from active RA patients and from healthy controls were compared by semiquantitative real-time PCR. The RA CD4+ T cells contained higher levels of SOCS1 but lower levels of SOCS3 transcripts than did control CD4+ T cells (Fig. 6a). Constitutive expression of SOCS1 mRNA in RA CD4+ T cells was comparable with the expression in normal CD4+ T cells stimulated by 10 ng/ml IL-6 (Fig. 6b), supporting its functional significance. Defective IL-10-induced STAT3 activation therefore appears to be due at least in part to an abundance of SOCS1 in RA CD4+ T cells."}

GO-BP

GO-CC

GO-MF

sentences

simple1

BioNLP16_DUT

BioNLP16_Messiy

DLUT931

bionlp-st-ge-2016-test-ihmc

bionlp-st-ge-2016-spacy-parsed

bionlp-st-ge-2016-test-tees

test3

testone

PMC:1064873 / 14989-23420 JSONTXT

Annnotations TAB JSON ListView MergeView

PMC:1064873 / 14989-23420 JSON TXT