IL-17A Gene Expression Research Articles

Neither IL-17A mRNA Nor IL-17A Protein Are Detectable in Langerhans Cell Histiocytosis Lesions

Langerhans cell histiocytosis (LCH) is a rare disease characterized by heterogeneous lesions including CD207(+)/CD1a(+) dendritic cells that can result in significant morbidity and mortality. The etiology of LCH remains speculative, and neoplastic and inflammatory origins have been debated for decades. A recent study identified abundant interleukin-17 (IL-17A) protein in dendritic cells in LCH lesions as well as in plasma from patients with active disease. Furthermore, it identified dendritic cells as a novel source of IL-17A expression. However, subsequent studies from our research group failed to identify any IL-17A gene expression from CD207(+) dendritic cells or CD3(+) T cells in LCH lesions. In this study, further investigation once again fails to identify any cells in LCH lesions with IL-17A gene expression. Furthermore, IL-17A antigen is undetectable in LCH lesion lysates with western blotting, immunoprecipitation, spectral analysis, and enzyme-linked immunosorbent assay (ELISA). Western blots, immunoprecipitation, and ELISA experiments also demonstrate that antibodies used in original studies that established the IL-17A hypothesis for pathogenesis of LCH recognize nonspecific proteins. We conclude that evidence for IL-17A as a significant factor in LCH remains inadequate and clinical trials targeting IL-17A remain unjustified.

Peripheral and Islet Interleukin-17 Pathway Activation Characterizes Human Autoimmune Diabetes and Promotes Cytokine-Mediated β-Cell Death

OBJECTIVECD4 T-cells secreting interleukin (IL)-17 are implicated in several human autoimmune diseases, but their role in type 1 diabetes has not been defined. To address the relevance of such cells, we examined IL-17 secretion in response to β-cell autoantigens, IL-17A gene expression in islets, and the potential functional consequences of IL-17 release for β-cells.RESEARCH DESIGN AND METHODSPeripheral blood CD4 T-cell responses to β-cell autoantigens (proinsulin, insulinoma-associated protein, and GAD65 peptides) were measured by IL-17 enzyme-linked immunospot assay in patients with new-onset type 1 diabetes (n = 50). mRNA expression of IL-17A and IFNG pathway genes was studied by qRT-PCR using islets obtained from subjects who died 5 days and 10 years after diagnosis of disease, respectively, and from matched control subjects. IL-17 effects on the function of human islets, rat β-cells, and the rat insulinoma cell line INS-1E were examined.RESULTSA total of 27 patients (54%) showed IL-17 reactivity to one or more β-cell peptides versus 3 of 30 (10%) control subjects (P = 0.0001). In a single case examined close to diagnosis, islet expression of IL17A, RORC, and IL22 was detected. It is noteworthy that we show that IL-17 mediates significant and reproducible enhancement of IL-1β/interferon (IFN)-γ–induced and tumor necrosis factor (TNF)-α/IFN-γ–induced apoptosis in human islets, rat β-cells, and INS-1E cells, in association with significant upregulation of β-cell IL17RA expression via activation of the transcription factors STAT1 and nuclear factor (NF)-κB.CONCLUSIONSCirculating IL-17+ β-cell–specific autoreactive CD4 T-cells are a feature of type 1 diabetes diagnosis. We disclose a novel pathway to β-cell death involving IL-17 and STAT1 and NF-κB, rendering this cytokine a novel disease biomarker and potential therapeutic target.

Mannan oligosaccharide modulates gene expression profile in pigs experimentally infected with porcine reproductive and respiratory syndrome virus12

This study characterized gene expression in peripheral blood mononuclear cells (PBMC) and bronchoalveolar lavage fluid (BALF) cells from control- or mannan oligosaccharide (MOS)-fed pigs with or without porcine reproductive and respiratory syndrome virus (PRRSV) at d 7 postinfection (PI). Weaned pigs (3 wk old) fed 0 or 0.2% MOS (Bio-Mos) diets were intranasally inoculated with PRRSV or a sterile medium at 5 wk of age. Total RNA (3 pigs/treatment) was extracted from cells. Double-stranded cDNA was amplified, labeled, and further hybridized to the Affymetrix GeneChip Porcine Genome Array consisting of 23,937 probe sets representing 20,201 genes. Microarray data were analyzed in R using packages from the Bioconductor project. Differential gene expression was tested by fitting a mixed linear model equivalent to a 2 × 2 factorial ANOVA using the limma package. Dietary MOS and PRRSV changed the expression of thousands of probe sets in PBMC and BALF cells (P < 0.05). The MOS × PRRSV interaction altered the expression of more nonimmune probe sets in PBMC (977 up, 1,128 down) than in BALF cells (117 up, 78 down). The MOS × PRRSV interaction (P < 0.05) for immune probe sets in PBMC affected genes encoding key inflammatory mediators. In uninfected pigs, gene expression of IL-1α, IL-6, myeloid differentiation factor 88, Toll-like receptor (TLR) 4, major histocompatibility complex (MHC) II, and dead box polypeptide 58 increased in PBMC of MOS-fed pigs (P < 0.05). This suggests that MOS enhances disease resistance in pigs and supports the fact that MOS induced a rapid increase in leukocytes at d 3 and 7 PI. Within infected pigs, however, MOS reduced the expression of IL-1β, IL-6, IL-8, macrophage inflammatory protein (MIP)-1α, MIP-1β, monocyte chemotactic protein (MCP)-1, and TLR4 genes in PBMC (P < 0.05). This finding may explain why fever was ameliorated in infected pigs fed MOS by d 7 PI. The expression of IL-1β, IL-6, MIP-1β, MCP-1, and TLR4 genes was confirmed by quantitative real-time reverse-transcription PCR. In BALF cells of infected pigs, MOS reduced the gene expression of TLR4, MHCII, and molecules associated with the complement system, but increased the gene expression of MHCI. In short, MOS regulated the expression of nonimmune and immune genes in pig leukocytes, perhaps providing benefits by enhancing the immune responses of the pigs to an infection, while preventing overstimulation of the immune system.

In vitro inhibition of compression-induced catabolic gene expression in meniscal explants following treatment with IL-1 receptor antagonist

BackgroundDamage to the knee meniscus may result in tears that are difficult or unable to heal, and are often treated by partial removal of the damaged tissue. In vitro, 20% dynamic compressive strains on meniscal tissue explants have resulted in an increase in the release of sulfated glycosaminoglycans (GAG) and nitric oxide (NO) from the tissue explants and increased expression of matrix metalloproteinases (MMP) and interleukin-1α (IL-1α). The objective of this study was to explore the efficacy of IL-1 blockade on the expression of a wide range of genes, as well as NO and GAG release, following dynamic compression of porcine meniscal explants. MethodsExplants were dynamically compressed for 2 h at 1 Hz to 0, 10, or 20% strain with and without a pretreatment of 500 ng/ml interleukin-1 receptor antagonist (IL-1RA). Relative changes in gene expression of IL-1α, MMP-1, -3, -13, A Disintegrin and Metalloproteinase with ThromboSpondin 4 (ADAMTS-4), ADAMTS-5, iNOS, aggrecan, and COX-2, as well as changes in NO and GAG release, were measured with standard biochemical assays. ResultsExpression of IL-1α, MMP-3, MMP-13, and ADAMTS-4 in superficial explants was significantly downregulated at 20% dynamic strain compared to 10% strain following treatment with IL-1RA. GAG and NO release were not significantly influenced by IL-1RA treatment. ConclusionsTreatment of meniscal explants with IL-1RA inhibited the expression of many catabolic genes following a single bout of high dynamic strain. IL-1RA may therefore be a potential therapy option during the acute phase of meniscal tear or meniscectomy treatment.

IL-17A and IL-17F Gene Expression is Strongly Induced in the Mucosa of H. pylori-Infected Subjects From Kenya and Germany

Helicobacter pylori infection is the major cause of gastritis. Immunologically, H. pylori gastritis is associated with an infiltration of immune cells into gastric mucosa and the upregulation of various cytokines. Here, we analysed the gene expression of IL-1- and IL-17-related cytokines in regard to H. pylori infection in 85 German and 51 Kenyan patients with reflux-related or dyspeptic symptoms, respectively. Degree of gastritis and density of colonization were assessed histologically in accordance with the updated Sydney classification. Gene expression levels of cytokines IL-1β, IL-8, IL-18, IL-33, IL-17A, IL-17F and IL-23 as well as IL-23R were analysed by real-time RT-PCR. In both populations, H. pylori-infected individuals had significant higher inflammatory scores for activity and chronicity than H. pylori-negative subjects (P values between 0.006 and <0.0001). IL-8 mRNA was induced up to 6-fold in H. pylori-infected patients (P < 0.05), while the expression levels of IL-1β, IL-18, IL-23, IL-33 and IL-23R did not differ with respect to the H. pylori status in both groups. Most strikingly, a significant induction of both IL-17A and IL-17F was noted in H. pylori-infected individuals of both ethnic groups. Almost all IL-17F-positive samples revealed co-expression of IL-17A (40/42, 95.2%). Analysing IL-17A and IL-17F transcript levels of these 40 'double-positive' samples, a highly significant positive correlation between both genes was identified (P < 0.001). Taken together, H. pylori infection leads to a strong upregulation of both IL-17A and IL-17F in the gastric mucosa suggesting a regulatory link between both genes.

High gene expression of inflammatory markers and IL-17A correlates with severity of injection site reactions of Atlantic salmon vaccinated with oil-adjuvanted vaccines

BackgroundTwo decades after the introduction of oil-based vaccines in the control of bacterial and viral diseases in farmed salmonids, the mechanisms of induced side effects manifested as intra-abdominal granulomas remain unresolved. Side effects have been associated with generation of auto-antibodies and autoimmunity but the underlying profile of inflammatory and immune response has not been characterized. This study was undertaken with the aim to elucidate the inflammatory and immune mechanisms of granuloma formation at gene expression level associated with high and low side effect (granuloma) indices.Groups of Atlantic salmon parr were injected intraperitoneally with oil-adjuvanted vaccines containing either high or low concentrations of Aeromonas salmonicida or Moritella viscosa antigens in order to induce polarized (severe and mild) granulomatous reactions. The established granulomatous reactions were confirmed by gross and histological methods at 3 months post vaccination when responses were known to have matured. The corresponding gene expression patterns in the head kidneys were profiled using salmonid cDNA microarrays followed by validation by real-time quantitative PCR (qPCR). qPCR was also used to examine the expression of additional genes known to be important in the adaptive immune response.ResultsGranulomatous lesions were observed in all vaccinated fish. The presence of severe granulomas was associated with a profile of up-regulation of innate immunity-related genes such as complement factors C1q and C6, mannose binding protein, lysozyme C, C-type lectin receptor, CD209, Cathepsin D, CD63, LECT-2, CC chemokine and metallothionein. In addition, TGF-β (p = 0.001), IL-17A (p = 0.007) and its receptor (IL-17AR) (p = 0.009) representing TH17 were significantly up-regulated in the group with severe granulomas as were arginase and IgM. None of the genes directly reflective of TH1 T cell lineage (IFN-γ, CD4) or TH2 (GATA-3) responses were differentially expressed.ConclusionsGranulomatous reactions following vaccination with oil-based vaccines in Atlantic salmon have the profile of strong expression of genes related to innate immune responses. The expression of TGF-β, IL-17A and its receptor suggests an involvement of TH17 T cell lineage and is in conformity with strong infiltration of neutrophils and macrophages into inflamed areas. Arginase upregulation shows that macrophages in these reactions are alternatively activated, indicating also a TH2-profile. To what extent the expression of IL-17A and its receptor reflects an autoimmune vaccine-based reaction remains elusive but would be in conformity with previous observations of autoimmune reactions in salmon when vaccinated with oil-based vaccines.

Pharmacologic Inhibition of MEK–ERK Signaling Enhances Th17 Differentiation

The cytokines and transcription factors that promote Th17 cell development have been extensively studied. However, the signaling pathways that antagonize Th17 differentiation remain poorly characterized. In this study, we report that pharmacologic inhibition of MEK-ERK signaling enhances the in vitro differentiation of Th17 cells and increases their gene expression of il-17a, il-17f, il-21, il-22, and il-23r. IL-2, which suppresses Th17 differentiation via STAT5 activation, also acts through ERK signaling to inhibit Th17 generation. In turn, ERK signaling is found to potentiate the production of IL-2 and activate STAT5, suggesting the existence of an autoregulatory loop to constrain Th17 development. Finally, compared with the transfer of untreated Th17 cells, the transfer of ERK-inhibited Th17 cells leads to accelerated onset and exacerbated colitis in immunodeficient mice. Our data indicate that MEK-ERK signaling negatively regulates Th17 differentiation in a Th cell-intrinsic manner.

Abstract C58: IL-1 receptor regulates calprotectin-dependent accumulation of pathogenic bacteria in oral carcinoma

Abstract The oral epithelium represents the mucosal immune system's initial line of defense against invasive bacterial and viral pathogens. Invasion of bacterial pathogens into oral epithelial cells increases expression of inflammatory cytokines, including interleukin-1α (IL-1α). Head and neck squamous cell carcinomas (HNSCC) express more IL-1α and have greater numbers of colonizing bacteria than normal oral tissues. Previously, we showed that exogenous IL-1α induces expression of antimicrobial proteins (AMP), including calprotectin, a heterodimer of S100A8 and S100A9 and increases IL-1 receptor-dependent resistance of buccal carcinoma cells to the invasive bacterial pathogen, Listeria monocytogenes. Since S100A9 is down-regulated in HNSCC, we knocked down calprotectin using siRNA against S100A8 and S100A9 in human buccal carcinoma cells (TR146) to further characterize the role of calprotectin in the IL-1α-dependent pathogen response. First, we examined the time course IL-1α expression in TR146 cells in response to coincubation with L. monocytogenes. We then assessed the exogenous IL-1α dose-dependent regulation of intracellular (calprotectin) and extracellular (human β-defensin-2; h-BD2) AMPs using comparative real-time RT-PCR. Lastly, we examined the role of calprotectin, in response to exogenous IL-1α, for modulation of resistance to L. monocytogenes invasion in TR146 cells expressing siRNA against S100A8 and S100A9 (A8A9c10) or non-specific siRNA (Neg3) using an antibiotic protection assay. We found that L. monocytogenes increases IL-1α gene expression in a time-dependent manner in wild type (WT) TR146 cells. Exogenous IL-1α induces a dose-dependent increase in expression of h-BD2 and calprotectin subunits in Neg3, A8A9c10 and WT TR146 cells. Furthermore, exogenous IL-1α provides IL-1 receptor-mediated protection against L. monocytogenes invasion in WT TR146 cells. In calprotectin-silenced A8A9c10 cells, however, exogenous IL-1α increases the proportion of intracellular bacteria when compared to Neg3 cells (123% to 77% of the vehicle control, respectively; p&amp;lt;0.0299 one-tailed Student's t-test with equal variance). Invasion by L. monocytogenes is independent of carcinoma cell size and viability of extracellular bacteria. These data strongly suggest that IL-1α signaling through the IL-1 receptor regulates calprotectin-mediated, cell-autonomous resistance to bacterial invasion as modeled by L.monocytogenes. Exogenous IL-1α increases expression of IL-8 and GM-CSF in HNSCC, which enhances invasion of inflammatory tumor-promoting immune cells and malignant progression. Therefore, for the first time, we show the loss of calprotectin in oral carcinoma cells, combined with increased IL-1α, as seen in HNSCC, can lead to increased bacterial colonization of oral carcinoma cells and expression of proinflammatory signals. These events may contribute to tumor growth and progression. Citation Information: Cancer Res 2009;69(23 Suppl):C58.

Interleukin-17A is not expressed by CD207+ cells in Langerhans cell histiocytosis lesions

Interleukin-17 (IL-17A) is a pro-inflammatory cytokine that has recently been implicated in pathogenesis of Langerhans Cell Histiocytosis (LCH), a potentially fatal disease characterized by lesions including CD207+ (langerin +) histiocytes. However, in this study we were unable to identify IL-17A gene expression in Langerhans cell lesions, and plasma levels of IL-17A did not correlate with disease activity. Therefore, this study does not support a central role for IL-17A in LCH pathogenesis.

The differential roles of Act-1 and NF-κB in the regulation of various IL-17A-induced genes in A549 cells (134.95)

Abstract It has been shown that IL-17A is a potent inducer of human beta-defensin (HBD)-2 in airway epithelia through the Act1-dependent NF-kB transcriptional regulation. In our recent findings, the induction of HBD-2 is very robust in human primary airway epithelia and human bronchial epithelial-1 (HBE-1). In contrast, A549, an alveolar epithelial cell line, has a very weak IL-17A-stimulated HBD-2 gene expression. We found A549 cell has an impaired NF-kB activation specific to IL-17A cytokine, since other pro-inflammatory cytokines could stimulate significant NF-kB and HBD-2 gene expression. In addition, over-expression of Act-1 protein in HBE-1 can significantly induce HBD-2 promoter activity in the absence of IL-17A, while in A549, no HBD-2 induction was observed regardless of the presence of IL-17A. Interestingly, the IL-17A-stimulated genes that have been discovered so far are mostly dependent on Act-1 and NF-kB activation. However, we found the extent of the regulation by Act-1 and NF-kB varies between different genes. By use of siRNA knockdown, we found some IL-17A-induced genes are strictly regulated by Act1 but only partially by NF-kB. Therefore, IL-17A can stimulate certain gene expressions even under a deficient NF-kB activation in A549 cells. These findings suggested there might be some molecules in addition to NF-kB regulated by Act1 to stimulate IL-17A gene expression.

IFN-β1a Inhibits the Secretion of Th17-Polarizing Cytokines in Human Dendritic Cells via TLR7 Up-Regulation

IFN-beta, an effective therapy against relapsing-remitting multiple sclerosis, is naturally secreted during the innate immune response against viral pathogens. The objective of this study was to characterize the immunomodulatory mechanisms of IFN-beta targeting innate immune response and their effects on dendritic cell (DC)-mediated regulation of T cell differentiation. We found that IFN-beta1a in vitro treatment of human monocyte-derived DCs induced the expression of TLR7 and the members of its downstream signaling pathway, including MyD88, IL-1R-associated kinase 4, and TNF receptor-associated factor 6, while it inhibited the expression of IL-1R. Using small interfering RNA TLR7 gene silencing, we confirmed that IFN-beta1a-induced changes in MyD88, IL-1R-associated kinase 4, and IL-1R expression were dependent on TLR7. TLR7 expression was also necessary for the IFN-beta1a-induced inhibition of IL-1beta and IL-23 and the induction of IL-27 secretion by DCs. Supernatant transfer experiments confirmed that IFN-beta1a-induced changes in DC cytokine secretion inhibit Th17 cell differentiation as evidenced by the inhibition of retinoic acid-related orphan nuclear hormone receptor C and IL-17A gene expression and IL-17A secretion. Our study has identified a novel therapeutic mechanism of IFN-beta1a that selectively targets the autoimmune response in multiple sclerosis.

IL-1 alpha gene expression and protein production by fibroblasts from patients with systemic sclerosis.

We examined IL-1 alpha and IL-1 beta gene expression and protein production in human dermal fibroblasts from patients with systemic sclerosis (SSc) to investigate the abnormal function of SSc fibroblasts. Human dermal fibroblasts were biopsied from 13 patients with SSc, three patients with rheumatoid arthritis (RA) and five healthy normal controls (NC). Cells were cultured in serum-free media and total RNA was collected from second or third passage fibroblasts. In cultured SSc fibroblasts, IL-1 alpha and IL-1 beta mRNAs were constitutively expressed and intracellular pro-IL-1 alpha was present. These observations suggest that an autocrine effect of IL-1 alpha contributes to the fibrosis in SSc.

Different T cell subsets in the nodule and synovial membrane: Absence of interleukin‐17A in rheumatoid nodules

To determine gene expression of the interleukin-17 (IL-17) family members (IL-17A-F) in rheumatoid subcutaneous nodules, and to assess the cytokines involved in regulating IL-17A expression. Total RNA was isolated from 19 nodules obtained from 16 different patients with rheumatoid arthritis (RA). Reverse transcription-polymerase chain reaction (PCR) was used to screen for gene expression of the IL-17 subtypes (IL-17A-F) in all nodules. Quantitative real-time PCR was used to measure the expression of interferon-gamma (IFN gamma), IL-6, IL-23, IL-12, and transforming growth factor beta (TGFbeta), relative to GAPDH as control, in a subset of 10 nodules. IL-17A gene expression was present in only 1 of 19 nodules, IL-17B in 17 of 19 nodules, IL-17C in 18 of 19 nodules, IL-17D in 16 of 19 nodules, and IL-17E in 3 of 19 nodules. IL-17F was absent in all samples. Cytokines that stimulate IL-17A production (IL-6, IL-23) as well as those that inhibit IL-17A production (IL-12, IFN gamma, TGFbeta) were present in the majority of nodules. Quantitative real-time PCR showed a similar pattern of gene expression for the individual cytokines between the different nodules. The mean +/- SD expression of IL-6 relative to GAPDH was 2.28 +/- 2.2 ng, and that of TGFbeta was 2.96 +/- 1.14 ng. There was a lower relative expression of IL-23 (0.05 +/- 0.05 ng), while the expression of IFN gamma was 0.67 +/- 0.68 ng and that of IL-12 was 0.48 +/- 0.23 ng. IL-17 family members are varyingly expressed in rheumatoid nodules. The paucity of IL-17A in nodules suggests an important difference from that observed in the synovium. The expression of IL-23 below a critical threshold level seems the most likely explanation for the virtual absence of IL-17A. The presence of tissue destruction within the nodule despite the absence of IL-17A suggests that IL-17A may be an important amplifier rather than an absolute requirement for inflammation in RA.

IL‐22 directly induces keratinocyte hyperplasia, up‐regulates anti‐microbial peptide and pro‐inflammatory cytokine gene expression in mouse skin

IL‐22 is a member of the IL‐10 family of cytokines that signals through the class II cytokine receptor heterodimer IL‐22R/IL‐10R2. It is secreted by activated CD4+ T cells and NK cells and has been shown to regulate keratinocyte hyperplasia and differentiation in vitro. Since the IL‐22 receptor is expressed on a variety of epithelial tissues, the IL‐22/IL‐22R pathway is believed to have effects on epithelial cells. The fact that IL‐23 induced ear swelling is significantly decreased in IL‐22 deficient mice when compared with the control mice further highlights the role of IL‐22 in dermal inflammation and acanthosis.To directly examine the role of IL‐22 in vivo, IL‐22 or saline was injected into the ears of wild type Balb/c mice every other day for two weeks. RNA from the mouse ears was prepared for quantitative RT‐PCR analysis two weeks after the first injection. At the end of the two weeks, IL‐22 induced keratinocyte hyperplasia in the mouse ear, as well as increased defensin β1, S100A8 and S100A9 transcripts. IL‐22 also significantly induced IL‐1α gene expression with a trend towards increased IL‐6 and TNFα transcripts. These results further confirmed that IL‐22 directly induces pro‐inflammatory gene expression and proliferation of keratinocytes that may lead to the recruitment of immune cells and contribute to the development of skin inflammation in vivo.

Interleukin-1 receptor type 1-deficient mice fail to develop social stress-associated glucocorticoid resistance in the spleen

Frequent or chronic stress as a result of repeated or persistent exposure to social challenges has been shown to affect the glucocorticoid (GC) responsiveness of immune cells in mice. Lipopolysaccharide-stimulated splenocytes of mice that were repeatedly subjected to social disruption were less sensitive to the anti-inflammatory actions of GC as evident from an increased production of pro-inflammatory cytokines and enhanced cell survival. The development of functional GC resistance was accompanied by the accumulation of GC-insensitive CD11b(+) cells in the spleen. These cells were shown to exhibit impaired nuclear translocation of the GC receptor and lack of GC-induced suppression of NF-kappaB. Similar impairments in GC receptor function have been reported after in vitro treatment of various cell lines with interleukin (IL)-1. The aim of this study was to elucidate whether IL-1 is a critical factor for the development of GC resistance in socially stressed mice. In the first experiment, we investigated if repeated social stress alters plasma levels and tissue gene expression of IL-1alpha and IL-1beta. It revealed that recurrent stressor exposure significantly increased splenic and hepatic mRNA expression and the plasma protein level of IL-1beta, and hepatic mRNA expression of IL-1alpha. In the second experiment, IL-1 receptor type 1 (IL1R1)-deficient mice were subjected to the stressor and both the tissue distribution of CD11b(+) cells and the GC sensitivity of the splenocytes were compared to wildtype mice. Mice lacking the IL1R1 exhibited adrenal hypertrophy, thymic involution, and elevated serum corticosterone levels in response to the stressor but did not show splenic accumulation of CD11b(+) cells and failed to develop GC resistance. These findings suggest that IL-1 plays a critical role in the development of the social stress-associated GC resistance in the murine spleen.

The interaction with Sp1 and reduction in the activity of histone deacetylase 1 are critical for the constitutive gene expression of IL-1α in human melanoma cells

A375-6 human melanoma cells are sensitive to the antiproliferative effect of IL-1. After a long period of culturing, we have obtained cells resistant to IL-1. The resistant clone A375-R8 constitutively produced IL-1 alpha. In this study, we identified a sequence, CGCC, located at -48 to -45 upstream of the transcription start site, to be essential for the constitutive IL-1 alpha gene activation. Specificity protein 1 (Sp1) and Sp3 bound to the nucleotide containing the sequence. Although the binding level to the nucleotide and expression level of Sp1 and Sp3 are comparable in A375-R8 and A375-6 cells, transactivation activity of Sp1 is higher in A375-R8 cells as compared with A375-6 cells. Sp3 could not transactivate the IL-1 alpha promoter. These results suggest that Sp1 but not Sp3 is important for IL-1 alpha gene activation. Trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC), greatly augmented the IL-1 alpha promoter activity in A375-6 cells to the level comparable with that in A375-R8 cells. TSA also induced IL-1 alpha mRNA expression in A375-6 cells. Sp1 and Sp3 bound to HDAC1 in A375-R8 and A375-6 cells. The chromatin immunoprecipitation assay revealed the binding of Sp1 and HDAC1 to the promoter region of the IL-1 alpha gene. The activities of HDAC bound to Sp1 and Sp3, and that of HDAC1 was lower in A375-R8 cells as compared with A375-6 cells. These results indicate that the reduction in the activity and interaction of HDAC1 with Sp1 are critical for the constitutive IL-1 alpha gene expression.

Differential cytokine gene expression in CD4 + and CD8 + T cell subsets of calves

The distinct patterns of cytokine expression in CD4 + and CD8 + T cells are well understood in mice and humans. However, little information is available about cytokine expression in bovine CD4 + and CD8 + T cells. In this study, mRNA expression of 19 different cytokines was analyzed in CD4 + and CD8 + T cells of calves with or without Concanavalin A (Con A) stimulation. CD4 + and CD8 + T cell populations were enriched to 98% purity by positive selection using magnetic cell sorting (MACS). CD4 + T cells spontaneously expressed the mRNAs of interleukin-1α (IL-1α), IL-1β, IL-2, IL-6, IL-7, IL-8, IL-10, IL-18, IFN-γ, TNF-α, TNF-β and TGF-β, and augmented the mRNA expression of IL-10, IFN-γ and TNF-β after Con A stimulation. The mRNAs of IL-3, IL-4, IL-5, IL-13 and GM-CSF were newly expressed in Con A-stimulated CD4 + T cells. CD8 + T cells displayed spontaneous mRNA expression of IL-6, IL-18, TNF-α, TNF-β and TGF-β, and newly expressed the mRNA of IL-2, IL-7, interferon-gamma (IFN-γ) and GM-CSF after Con A stimulation. It was found that CD4 + T cells expressed the mRNA of 17 cytokines except for IL-12 and IL-15, while CD8 + T cells expressed only the mRNA of 9 cytokines after Con A stimulation. The profile of cytokine mRNA expression was substantially different in the CD4 + and CD8 + T cells of calves, indicating that CD4 + T cells can be distinguished from CD8 + T cells by the cytokine gene expression of IL-1α, IL-1β, IL-3, IL-4, IL-5, IL-8, IL-10 and IL-13. Differential cytokine expression between CD4 + and CD8 + T cells serve to interpret an individual function of T cell subsets in the immune system of calves.

Alveolar type II cells inhibit fibroblast proliferation: role of IL-1α

Alveolar type II (ATII) cells inhibit fibroblast proliferation in coculture by releasing or secreting a factor(s) that stimulates fibroblast production of prostaglandin E2 (PGE2). In the present study, we sought to determine the factors released from ATII cells that stimulate PGE2 production in fibroblasts. Exogenous addition of rat IL-1alpha to cultured lung fibroblasts induced PGE2 secretion in a dose-response manner. When fibroblasts were cocultured with rat ATII cells, IL-1alpha protein was detectable in ATII cells and in the coculture medium between days 8 and 12 of culture, correlating with the highest levels of PGE2. Furthermore, under coculture conditions, IL-1alpha gene expression increased in ATII cells (but not fibroblasts) compared with either cell cultured alone. In both mixed species (human fibroblasts-rat ATII cells) and same species cocultures (rat fibroblasts and ATII cells), PGE2 secretion was inhibited by the presence of IL-1 receptor antagonist (IL-1Ra) or selective neutralizing antibody directed against rat IL-1alpha (but not IL-1beta). Conditioned media from cocultures inhibited fibroblast proliferation, and this effect was abrogated by the addition of IL-1Ra. Addition of keratinocyte growth factor (KGF) resulted in an earlier increase in PGE2 secretion and fibroblast inhibition (day 8 of coculture). This effect was inhibited by indomethacin but was not altered by IL-1Ra. We conclude that in this coculture system, IL-1alpha secretion by ATII cells is one factor that stimulates PGE2 production by lung fibroblasts, thereby inhibiting fibroblast proliferation. In addition, these studies demonstrate that KGF enhances ATII cell PGE2 production through an IL-1alpha-independent pathway.

Effects of PM10 in human peripheral blood monocytes and J774 macrophages.

The effects of PM10, one of the components of particulate air pollution, was investigated using human monocytes and a mouse macrophage cell line (J774). The study aimed to investigate the role of these nanoparticles on the release of the pro-inflammatory cytokine TNF-α and IL-1α gene expression. We also investigated the role of intracellular calcium signalling events and oxidative stress in control of these cytokines and the effect of the particles on the functioning of the cell cytoskeleton. We showed that there was an increase in intracellular calcium concentration in J774 cells on treatment with PM10 particles which could be significantly reduced with concomitant treatment with the calcium antagonists verapamil, the intracellular calcium chelator BAPTA-AM but not with the antioxidant nacystelyn or the calmodulin inhibitor W-7. In human monocytes, PM10 stimulated an increase in intracellular calcium which was reduced by verapamil, BAPTA-AM and nacystelyn. TNF-α release was increased with particle treatment in human monocytes and reduced by only verapamil and BAPTA-AM. IL-1α gene expression was increased with particle treatment and reduced by all of the inhibitors. There was increased F-actin staining in J774 cells after treatment with PM10 particles, which was significantly reduced to control levels with all the antagonists tested. The present study has shown that PM10 particles may exert their pro-inflammatory effects by modulating intracellular calcium signalling in macrophages leading to expression of pro-inflammatory cytokines. Impaired motility and phagocytic ability as shown by changes in the F-actin cytoskeleton is likely to play a key role in particle clearance from the lung.

ST2 protein induced by inflammatory stimuli can modulate acute lung inflammation

We have investigated gene and protein expression of ST2/ST2L in a murine alveolar macrophage (AM) cell line, MH-S, reacting to inflammatory stimuli in vitro and in the lung tissue of an acute lung injury model in vivo. We have also analyzed the effect of soluble ST2 protein on inflammatory response of MH-S cells. Lipopolysaccharide (LPS) and proinflammatory cytokines such as IL-1β, IL-6, and TNF-α induced ST2 mRNA expression in MH-S cells. In an acute lung injury model, protein and mRNA expression levels of ST2 increased to the maximal level at 24–72 h after the LPS challenge. Furthermore, pretreatment with ST2 protein significantly reduced the protein production and gene expression of IL-1α, IL-6, and TNF-α in LPS-stimulated MH-S cells in vitro. These results suggest that increases in endogenous ST2 protein in AM, which is induced by inflammatory stimuli, such as LPS and proinflammatory cytokines, may modulate acute lung inflammation.