Spontaneous Secretion Of IL-6 Research Articles

Multiple Myd88-dependent Toll-like receptor signaling pathways mediate inflammation in a mouse model of primary Sjögren’s syndrome

Abstract Primary Sjögren’s syndrome (pSS) is an autoimmune disease characterized by exocrine gland dysfunction and immune hyperactivity. The adaptor Myd88 is critical for immune function, as most TLRs utilize Myd88 for signal transduction. Previous work by our group found Myd88 is required for pSS disease. Our objective was to identify the specific Myd88-dependent TLR pathways that mediate salivary and systemic inflammation. We used the pSS mouse model NOD.B10Sn-H2b/J (NOD.B10), Myd88-deficient NOD.B10 mice (NOD.B10Myd88−/−), and C57BL/10 controls. For all experiments we used female mice that were at least 26 weeks old, the age that NOD.B10 females display clinical disease. We isolated spleens from NOD.B10 mice and C57BL/10 controls and performed RNA-sequencing. We then harvested salivary tissue or spleens from NOD.B10, NOD.B10Myd88−/−, and C57BL/10 mice. We performed qPCR and flow cytometry to assess expression of Myd88-dependent TLRs. We cultured salivary tissue from NOD.B10 and NOD.B10Myd88−/− mice and performed cytokine arrays on the supernatants. Finally, we treated salivary tissue with a TLR4 inhibitor and quantified spontaneous IL-6 secretion. We identified dysregulation of numerous TLR-related networks in pSS splenocytes. We found upregulation of Myd88 dependent-TLRs in both the spleen and salivary tissue from NOD.B10 mice. Salivary tissue from Myd88-sufficient NOD.B10 females exhibited spontaneous secretion of IL-6, MCP-1 and TNFa, and this was diminished in NOD.B10Myd88−/− mice. Finally, TLR4 blockade reduced IL-6 secretion in NOD.B10 salivary tissue. Our preliminary data suggest Myd88-dependent pathways contribute to the inflammatory landscape in pSS, and inhibition of such will likely have therapeutic utility.

Tofacitinib regulates synovial inflammation in psoriatic arthritis, inhibiting STAT activation and induction of negative feedback inhibitors

BackgroundPsoriatic arthritis (PsA) is a chronic inflammatory disease, characterised by synovitis and destruction of articular cartilage/bone. Janus-kinase and signal transducer and activator of transcription (JAK-STAT) signalling pathway is implicated in...

AB0089 Tofacitinib Mediates Synovial Angiogenesis in Psoriatic Arthritis

BackgroundPsoriatic Arthritis (PsA) is a common, chronic immune-mediated inflammatory disease, characterised by synovitis, progressive destruction of articular cartilage/bone, and is associated with psoriasis. Janus Kinase and Signal Transducer and Activator...

Hypoxia and STAT3 signalling interactions regulate pro-inflammatory pathways in rheumatoid arthritis

To examine the effect of hypoxia on Signal Transducer and Activator of Transcription 3 (STAT3)-induced pro-inflammatory pathways in rheumatoid arthritis (RA). Detection of phospho-STAT3 was assessed in RA synovial tissue and fibroblasts (RASFC) by immunohistology/immunofluorescence. Primary RASFCs and a normal synoviocyte cell line (K4IM) were cultured under hypoxic and normoxic conditions±Stat3-siRNA, HIF-siRNA or WP1066 (JAK2-inhibitor). HIF1α, p-STAT3, p-STAT1 and Notch-1IC protein expression were analysed by western blot. Functional mechanisms were quantified by invasion chamber, matrigel and migration assays. IL-6, IL-8, IL-10 and matrixmetalloproteinases (MMP)-3 were quantified by ELISA. Notch-1 receptor, its DLL-4 ligand and downstream target genes (hrt-1, hrt-2) were quantified by real-time PCR. The effect of WP1066 on spontaneous secretion of pro/anti-inflammatory cytokines and Notch signalling was examined in RA synovial explants ex vivo. p-STAT3 was increased in RA synovium compared with control (p<0.05). Hypoxia induced p-STAT3, p-STAT1 and HIF1α expression, an effect blocked by Stat3-siRNA and WP1066. Hypoxia-induced cell invasion, migration and cytokine production were inhibited by Stat3-siRNA (p<0.05) and WP1066 (p<0.05). While HIF1α siRNA inhibited hypoxia-induced p-STAT3 detection, Stat3-siRNA also inhibited hypoxia-induced HIF1α. Furthermore, hypoxia-induced Notch-1IC, DLL4, hrt-1 and -2 expression were significantly inhibited by WP1066 (p<0.05). Finally, in RA synovial explant cultures ex vivo, WP1066 decreased spontaneous secretion of IL-6, IL-8 and MMP3 (p<0.05), Notch-1 mRNA (p<0.05) and induced IL-10 (p<0.05). This is the first study to provide evidence of a functional link between HIF1α, STAT3 and Notch-1 signalling in the regulation of pro-inflammatory mechanisms in RA, and further supports a role for STAT blockade in the treatment of RA.

Adaptive and Innate Immune Responsiveness toBorrelia burgdorferi sensu latoin Exposed Asymptomatic Children and Children with Previous Clinical Lyme Borreliosis

Why some individuals develop clinical manifestations in Lyme borreliosis (LB) while others remain asymptomatic is largely unknown. Therefore, we wanted to investigate adaptive and innate immune responsiveness to Borrelia burgdorferi sensu lato in exposed Borrelia-antibody-positive asymptomatic children (n = 20), children with previous clinical LB (n = 24), and controls (n = 20). Blood samples were analyzed for Borrelia-specific interferon (IFN)-γ, interleukin (IL)-4, and IL-17 secretion by ELISPOT and Borrelia-induced IL-1β, IL-6, IL-10, IL-12(p70), and tumor necrosis factor (TNF) secretion by Luminex. We found no significant differences in cytokine secretion between groups, but a tendency towards an increased spontaneous secretion of IL-6 was found among children with previous clinical LB. In conclusion, the adaptive or innate immune responsiveness to Borrelia burgdorferi sensu lato was similar in Borrelia-exposed asymptomatic children and children with previous clinical LB. Thus, the immunological mechanisms of importance for eradicating the spirochete effectively without developing clinical manifestations of LB remain unknown.

Cryopreserved peripheral blood mononuclear cells are suitable for the assessment of immunological markers in type 1 diabetic children

Cryopreserved peripheral blood mononuclear cells (PBMC) are commonly used when assessing immune responses in clinical trials, both for practical reasons and to minimize interassay variation, as samples are often collected and studied over time. This study investigated the effect of cryopreservation on cytokine and chemokine secretion, and on expression of regulatory T-cell associated markers, in samples from children with type 1 diabetes. PBMC were cultured before and after cryopreservation either with GAD 65 or PHA. Secretion of cytokines (IL-5, -6, -10, -12, -13 -17, IFN-γ and TNF-α) and chemokines (IP-10, MCP-1, MIP-1α, MIP-1β and RANTES) was analysed in cell supernatants using multiplex fluorochrome technique (Luminex). Expression of FOXP3 and TGF-β mRNA was detected by multiplex real-time RT-PCR. Increased spontaneous secretion of IL-6, -10, -12, -13, IFN-γ and MCP-1, and mRNA expression of FOXP3 and TGF-β, was detected after cryopreservation. Stimulation with GAD 65 induced higher levels of IL-6, IFN-γ, TNF-α and MIP-1α, whereas lower secretion was found for IL-10 and IL-13 in cryopreserved PBMC. Stimulation with PHA induced lower secretion of IP-10, MCP-1 and RANTES and FOXP3 mRNA expression after cryopreservation. Thus, cryopreserved PBMC were suitable to assess the immunological markers included in this study, even though their expression could differ from freshly handled cells.

Spontaneous expression of IL-4 mRNA in lymphocytes from children with atopic dermatitis.

Normal lymphocytes do not generally produce or secrete lymphokines in the resting or unstimulated state and only express or release cytokines following activation. Recently, the spontaneous production of intracellular interferon-gamma (IFN-gamma) and spontaneous secretion of IL-6 has been documented in patients with atopic dermatitis. These findings indicated that lymphocytes had been previously activated in vivo. Such in vivo activation may also be associated with spontaneous production of IL-4. As measurement of IL-4 secretion by immunoassay is complicated by poor sensitivity, and only provides information on the net amount of cytokine present after secretion, adsorption, consumption and degradation have occurred, IL-4 mRNA expression in peripheral blood lymphocytes from children with atopic dermatitis and controls was examined by polymerase chain reaction (PCR)-assisted mRNA amplification. Spontaneous expression of IL-4 mRNA was detected in four of eight patients with severe atopic dermatitis. Following stimulation in vitro, seven of eight atopic patients demonstrated detectable IL-4 mRNA. In comparison, no spontaneous expression of IL-4 mRNA was found in controls, and only six of 10 controls expressed IL-4 mRNA in stimulated cultures. The spontaneous expression of IL-4 mRNA in unstimulated cultures from children with atopic dermatitis supports the possibility that previous in vivo activation has occurred, and suggests that IL-4 production is increased in vivo in atopic dermatitis. This in vivo activation together with the constitutive expression of IL-4 mRNA are likely to contribute to the spontaneous in vitro production of IgE in atopic patients.

Increased spontaneous secretion of IL-6 from B cells of patients with B chronic lymphatic leukaemia (B-CLL) and autoimmunity

We studied B cells from 18 patients with B-CLL, six of them with autoimmune haemolytic anaemia, for spontaneous secretion of IL-6. Our aim was to determine whether the increased incidence of autoimmune disease found in B-CLL patients is associated with enhanced spontaneous IL-6 secretion. IL-6 was measured by the effect of B cell supernatants on the proliferation of an IL-6 dependent plasmacytoma cell line T1165. The highest IL-6 values (7.4 +/- 1.8 U/ml) were measured in supernatants derived on day 3 of culture from lymphocytes of the six patients with B-CLL and concomitant autoimmune disease. The maximal IL-6 values for 10 patients with B-CLL only were 2.8 +/- 0.3 U/ml and for 10 age-matched controls, 0.8 +/- 0.3 U/ml (P less than 0.01, each group compared with the other). We conclude that there is an association between B-CLL, autoimmune disease and the spontaneous in vitro secretion of IL-6. Further studies are needed to determine whether the IL-6 secretion plays a role in the pathogenesis of autoimmune disease in patients with B-CLL.

Defective response to Toll‐like receptor 3 and 4 ligands by activated monocytes in chronic hepatitis C virus infection

Toll-like receptors (TLR) have a critical role in innate immunity against pathogens. We investigated the cytokine response to TLR stimulation in peripheral blood cells of subjects infected with hepatitis C virus (HCV) and/or human immunodeficiency virus (HIV) in the Women Interagency HIV Study (WIHS) cohort. Interleukin (IL)-6 in response to TLR3 and TLR4 ligands such as polyinosinic-polycytidylic acid and lipopolysaccharide was significantly compromised in HCV-infected women. High spontaneous secretion of IL-6 suggested pre-existing cell activation as a factor mediating reduced responses to TLR3 and TLR4 stimulation. To a lesser extent, tumour necrosis factor-alpha and IL-1beta responses to TLR stimulation were also compromised. Monocytes, but not B cells or NK cells, were identified as the cell population spontaneously secreting cytokines and also as the cells responding to TLR stimulation. These results highlight a functional defect in antigen-presenting cells of women with HCV infection or co-infection. In women with existing HIV co-infection, decreased cytokine function of antigen-presenting cells suggests another mechanism contributing to immune dysfunction in addition to the HIV-associated CD4 defect.

Decreased production of inflammatory cytokines by circulating monocytes and dendritic cells in type 2 diabetic men with atherosclerotic complications

Antigen-presenting cells (APCs) are involved in the development of atherosclerosis, whose complications represent the main cause of death in diabetic patients. Nevertheless, their role in atherogenesis is poorly understood. We compared the number of circulating monocyte and dendritic cell (DC) subsets as well as their capacity to produce inflammatory cytokines IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) in type 2 diabetic men with ( n=11) and without ( n=44) chronic atherosclerotic complications. Identification and enumeration of peripheral blood (PB) lymphoid subsets, monocytes, myeloid (CD33 strong+), CD16 +, and plasmacytoid (CD33 −/dim+) DCs as well as of their spontaneous and stimulated production of IL-1β, IL-6, and TNF-α were performed at the single-cell level by flow cytometry. Our results show that type 2 diabetic men with atherosclerotic complications display a significantly reduced spontaneous secretion of IL-6 by monocytes and CD16 + DCs and of TNF-α by CD16 + DCs as compared to patients without atherosclerotic complications. Spontaneous secretion of IL-1β by monocytes and CD16 DCs and of IL-6 by CD33 + and plasmacytoid DCs was detected in patients without atherosclerotic complications but not in the other patients with complications. Taken together, these results indicate that type 2 diabetic men with atherosclerotic complications display both quantitatively and functionally impaired immunological responses by circulating APCs. The decreased patterns of inflammatory cytokine production by these cells may influence the inflammatory response mediated by APCs as well as the antigen-specific responses mediated by other cells such as T cells.

Labor Affects Cytokine Production in Newborns

To investigate whether the mode of delivery or the drugs given to the mother during labor may affect the newborns' immune system. Three groups of term newborns were included: A, spontaneously delivered with i.v. analgesia (n = 37); B, spontaneously delivered with epidural analgesia (n = 26); and C, delivered by cesarean section under general anesthesia (n = 29). Natural killer (NK) cell cytotoxicity, mitogenic response, and the capacity of peripheral blood mononuclear cells (PBMCs) to produce interleukin (IL)-1 beta, IL-2, IL-3, IL-6, and tumor necrosis factor (TNF)-alpha were examined. NK cell cytotoxicity increased significantly in all three groups of newborns on the second day of life. Decreased IL-2 production was observed in newborns delivered by cesarean section. Spontaneous IL-1 beta secretion was higher in newborns to mothers treated with epidural analgesia. Spontaneous IL-6 secretion was elevated in infants to mothers undergoing general anesthesia and surgery or epidural analgesia. TNF-alpha production was increased in newborns delivered by cesarean section. The immune response of the newborn is affected by the mode of delivery and/or drugs given to the mother during labor.

Effect of IL-6 on Alveolar Fibroblast Proliferation in Interstitial Lung Diseases

Alveolar macrophage–fibroblast interaction may be involved in the pathogenesis of interstitial lung diseases (ILD). Herein, we compared IL-6 secretion from alveolar macrophages (AM) and alveolar fibroblasts (AFb) recovered from patients with sarcoidosis (SA) and with diffuse interstitial fibrosis (DIF). Moreover, we evaluated the effect of IL-6 on thein vitroAFb proliferation in both diseases. AM and AFb from SA patients showed increased spontaneous secretion of IL-6 compared with cells from DIF subjects. Tumor necrosis factor-α (TNFα) and interleukin-1 (IL-1) enhanced IL-6 secretion and IL-6 mRNA transcription in AFb of SA patients. Addition of anti-IL-6 MoAbs increased AFb proliferation capacity in SA, but suppressed it in DIF. These results show that only SA AM and AFb secrete high levels of IL-6 which have suppressive effect on AFb proliferation. This may indicate a potential role of IL-6 in the fibrogenesis of ILD.

Alveolar type II epithelial cells produce interleukin-6 in vitro and in vivo. Regulation by alveolar macrophage secretory products.

The aims of this study were (a) to determine if rat alveolar type II (ATII) cells and human pulmonary epithelial-derived cells (A549 cell line) could generate IL-6 in vitro, (b) to characterize the cytokine regulation of IL-6 gene and protein expression in these cells, and (c) to detect the in vivo expression of immunoreactive IL-6 by human ATII cells. Rat ATII cells in primary culture secreted bioactive IL-6 and immunostained with an anti-IL-6 antiserum. Spontaneous IL-6 secretion by rat ATII cells amounted to 5,690 +/- 770 pg/ml/10(6) cells (n = 12) and was fivefold higher than spontaneous rat alveolar macrophages IL-6 secretion (1,052 +/- 286 pg/ml/10(6) cells, n = 8, P = 0.001). Rat alveolar macrophage conditioned media (CM) increased IL-6 secretion by rat ATII cells through the effect of IL-1 and TNF. IL-6 gene expression and IL-6 secretion by A549 cells was induced by IL-1 beta, TNF alpha, and by human alveolar macrophages and THP1 cells CM. Induction was abolished when CM were preincubated with anti-IL-1 beta and anti-TNF alpha antibody. The combination of IFN gamma and LPS induced the expression of IL-6 mRNA by A549 cells whereas LPS alone had no effect. Immunohistochemical staining evidenced the expression of immunoreactive IL-6 by hyperplastic ATII cells in fibrotic human lung, a condition in which alveolar macrophages are known to be activated. ATII cells in normal human lung did not express immunoreactive IL-6. Our findings demonstrate that ATII cells may be an important source of IL-6 in the alveolar space thereby participating to the regulation of the intra-alveolar immune response.

High Levels of Interleukin-6 Are Associated With Low Tumor Burden and Low Growth Fraction in Multiple Myeloma

AbstractInterleukin-6 (IL-6) is a multifunctional cytokine postulated to play a central role as a growth factor for multiple myeloma (MM). We evaluated the spontaneous secretion of IL-6 in supernatants of Ficoll-Hypaque-enriched bone marrow (BM) cultures from 35 patients with MM. The levels of IL-6 were correlated with biological and clinical characteristics of the disease. High levels of IL-6 production defined a subgroup of patients with low tumor burden as determined by lower serum β2-microglobulin (B2M) (P = .02) and lower percentage of myeloma cells infiltrating the bone marrow (P = .003), higher synthetic rates of monoclonal protein (P = .006), and low proliferative compartments as measured by the percentage of Ki-67-positive myeloma cells. Patients with high proliferative fractions (Ki-67-positive myeloma cells > 20%) had significantly lower levels of IL-6 when compared with patients with low proliferative fractions (P = .005). Our findings do not support IL-6 as a major growth factor for MM, but demonstrate an association of high levels of IL-6 secretion with low tumor cell burden and low proliferative fraction.

Immunological Characteristics of Umbilical Cord Blood Cells: Phenotypic and Functional Analysis

The purpose of this study was to phenotype and assay immunological function on cord blood from 70 samples. Immunophenotyping indicated similar numbers of CD2, 3, and 8-positive cells as in adult bone marrow. CD4-positive cells were increased and CD19, 20, and CD56-positive cells were decreased in numbers. Proliferative responses to nonspecific mitogens were lower in cord blood cells than in adult cells, as was NK activity and spontaneous secretion of IL-6 and TNF-alpha. This study confirms the relative immaturity of cord blood cells even when some functional assays appear normal.