Tumour necrosis factor-alpha (TNF-alpha) increases nuclear factor kappaB (NFkappaB) activity in and interleukin-8 (IL-8) release from bovine mammary epithelial cells

Epithelia play important immunological roles at a variety of mucosal sites. We examined NFkappaB activity in control and TNF-alpha treated bovine mammary epithelial monolayers (BME-UV cells). A region of the bovine IL-8 (bIL-8) promoter was sequenced and a putative kappaB consensus sequence was identified bioinformatically. We used this sequence to analyse nuclear extracts for IL-8 specific NFkappaB activity. As a surrogate marker of NFkappaB activation, we investigated IL-8 release in two models. Firstly in BME-UV monolayers, IL-8 release in the presence of pro- and anti-inflammatory agents was determined by enzyme-linked immunosorbent assay (ELISA). Secondly, we measured IL-8 secretion from a novel model of intact mucosal sheets of bovine teat sinus. IL-8 release into bathing solutions was assessed following treatment with pro- and anti-inflammatory agents. TNF-alpha enhanced NFkappaB activity in bovine mammary epithelial monolayers. p65 NFkappaB homodimer was identified in both control and TNF-alpha treated cells. Novel sequencing of the bovine IL-8 promoter identified a putative kappaB consensus sequence, which specifically bound TNF-alpha inducible p50/p65 heterodimer. TNF-alpha induced primarily serosal IL-8 release in the cell culture model. Pre-treatment with anti-TNF or dexamethasone inhibited TNF-alpha induced IL-8 release. High dose interleukin-1beta (IL-1beta) induced IL-8 release, however significantly less potently than TNF-alpha. Bovine mammary mucosal tissue released high basal levels of IL-8 which were unaffected by TNF-alpha or IL-1beta but inhibited by both dexamethasone and anti-TNF. These data support a role for TNF-alpha in activation of NFkappaB and release of IL-8 from bovine mammary epithelial cells.

Interleukin 3 and schizophrenia:the impact of sex and family history

Chromosome 5q21-33 has been implicated in harboring risk genes for schizophrenia. In this paper, we report evidence that multiple single nucleotide polymorphisms in and around interleukin 3 (IL3) are associated with the disease in the Irish Study of High-Density Schizophrenia Families (ISHDSF), the Irish Case-Control Study of Schizophrenia (ICCSS) and the Irish Trio Study of Schizophrenia (ITRIO). The associations are sex-specific and depend on the family history (FH) of schizophrenia. In all three samples, rs31400 shows female-specific and FH-dependent associations (P=0.0062, 0.0647 and 0.0284 for the ISHDSF, ICCSS and ITRIO, respectively). Several markers have similar associations in one or two of the three samples. In haplotype analyses, identical risk and protective haplotypes are identified in the ISHDSF and ITRIO samples in several multimarker combinations. For ICCSS, the same haplotypes are implicated; however, the risk haplotypes observed in the family samples become protective. Several significant markers, rs440970, rs31400 and rs2069803, are located in and around known estrogen response elements, promoter and enhancer of the IL3 gene. They may explain the sex-specific associations and be functional for the expression of IL3 gene.

A role for whey acidic protein four-disulfide-core 12 (WFDC12) in the regulation of the inflammatory response in the lung

Introduction: Secretory leucocyte protease inhibitor and elafin are members of the whey acidic protein (WAP), or WAP four disulfide-core (WFDC), family of proteins and have multiple contributions to innate defence including inhibition of neutrophil serine proteases and inhibition of the inflammatory response to lipopolysaccharide (LPS). This study aimed to explore potential activities of WFDC12, a previously uncharacterised WFDC protein expressed in the lung. Methods: Recombinant expression and purification of WFDC12 were optimised in Escherichia coli. Antiprotease, antibacterial and immunomodulatory activities of recombinant WFDC12 were evaluated and levels of endogenous WFDC12 protein were characterised by immunostaining and ELISA. Results: Recombinant WFDC12 inhibited cathepsin G, but not elastase or proteinase-3 activity. Monocytic cells pretreated with recombinant WFDC12 before LPS stimulation produced significantly lower levels of the pro-inflammatory cytokines interleukin-8 and monocyte chemotactic protein-1 compared with cells stimulated with LPS alone. Recombinant WFDC12 became conjugated to fibronectin in a transglutaminase-mediated reaction and retained antiprotease activity. In vivo WFDC12 expression was confirmed by immunostaining of human lung tissue sections. WFDC12 levels in human bronchoalveolar lavage fluid from healthy and lung-injured patients were quantitatively compared, showing WFDC12 to be elevated in both patients with acute respiratory distress syndrome and healthy subjects treated with LPS, relative to healthy controls. Conclusions: Together, these results suggest a role for this lesser known WFDC protein in the regulation of lung inflammation.

Evidence supporting a role for N-epsilon-(3-formyl-3,4-dehydropiperidino)lysine accumulation in Muller glia dysfunction and death in diabetic retinopathy

Purpose: Recent evidence suggests that neuroglial dysfunction and degeneration contributes to the etiology and progression of diabetic retinopathy. Advanced lipoxidation end products (ALEs) have been implicated in the pathology of various diseases, including diabetes and several neurodegenerative disorders. The purpose of the present study was to investigate the possible link between the accumulation of ALEs and neuroretinal changes in diabetic retinopathy.

Methods: Retinal sections obtained from diabetic rats and age-matched controls were processed for immunohistochemistry using antibodies against several well defined ALEs. In vitro experiments were also performed using a human Muller (Moorfields/Institute of Ophthalmology-Muller 1 [ MIO-M1]) glia cell line. Western blot analysis was used to measure the accumulation of the acrolein-derived ALE adduct N epsilon-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine) in Muller cells preincubated with FDP-lysine-modified human serum albumin (FDP-lysine-HSA). Responses of Muller cells to FDP-lysine accumulation were investigated by analyzing changes in the protein expression of heme oxygenase-1 (HO-1), glial fibrillary acidic protein (GFAP), and the inwardly rectifying potassium channel Kir4.1. In addition, mRNA expression levels of vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF alpha) were determined by reverse transcriptase PCR (RT-PCR). Apoptotic cell death was evaluated by fluorescence-activated cell sorting (FACS) analysis after staining with fluorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide.

Results: No significant differences in the levels of malondialdehyde-, 4-hydroxy-2-nonenal-, and 4-hydroxyhexenal-derived ALEs were evident between control and diabetic retinas after 4 months of diabetes. By contrast, FDP-lysine immunoreactivity was markedly increased in the Muller glia of diabetic rats. Time-course studies revealed that FDP-lysine initially accumulated within Muller glial end feet after only a few months of diabetes and thereafter spread distally throughout their inner radial processes. Exposure of human Muller glia to FDP-lysine-HSA led to a concentration-dependent accumulation of FDP-lysine-modified proteins across a broad molecular mass range. FDP-lysine accumulation was associated with the induction of HO-1, no change in GFAP, a decrease in protein levels of the potassium channel subunit Kir4.1, and upregulation of transcripts for VEGF, IL-6, and TNF-alpha. Incubation of Muller glia with FDP-lysine-HSA also caused apoptosis at high concentrations.

Conclusions: Collectively, these data strongly suggest that FDP-lysine accumulation could be a major factor contributing to the Muller glial abnormalities occurring in the early stages of diabetic retinopathy.

Developmentally regulated IL6-type cytokines signal to germ cells in the human fetal ovary

Fetal ovarian development and primordial follicle formation are imperative for adult fertility in the female. Data suggest the interleukin (IL)6-type cytokines, leukaemia inhibitory factor (LIF), IL6, oncostatin M (OSM) and ciliary neurotrophic factor (CNTF), are able to regulate the survival, proliferation and differentiation of fetal murine germ cells (GCs) in vivo and in vitro. We postulated that these factors may play a similar role during early human GC development and primordial follicle formation. To test this hypothesis, we have investigated the expression and regulation of IL6-type cytokines, using quantitative reverse transcription polymerase chain reaction and immunohistochemistry. Expression of transcripts encoding OSM increased significantly across the gestational range examined (8-20 weeks), while expression of IL6 increased specifically between the first (8-11 weeks) and early second (12-16 weeks) trimesters, co-incident with the initiation of meiosis. LIF and CNTF expression remained unchanged. Expression of the genes encoding the LIF and IL6 receptors, and their common signalling subunit gp130, was also found to be developmentally regulated, with expression increasing significantly with increasing gestation. LIF receptor and gp130 proteins localized exclusively to GCs, including oocytes in primordial follicles, indicating this cell type to be the sole target of IL6-type cytokine signalling in the human fetal ovary. These data establish that IL6-type cytokines and their receptors are expressed in the human fetal ovary and may directly influence GC development at multiple stages of maturation.

The E3 ubiquitin ligase Pellino3 protects against obesity-induced inflammation and insulin resistance

Diet-induced obesity can induce low-level inflammation and insulin resistance. Interleukin-1β (IL-1β) is one of the key proinflammatory cytokines that contributes to the generation of insulin resistance and diabetes, but the mechanisms that regulate obesity-driven inflammation are ill defined. Here we found reduced expression of the E3 ubiquitin ligase Pellino3 in human abdominal adipose tissue from obese subjects and in adipose tissue of mice fed a high-fat diet and showing signs of insulin resistance. Pellino3-deficient mice demonstrated exacerbated high-fat-diet-induced inflammation, IL-1β expression, and insulin resistance. Mechanistically, Pellino3 negatively regulated TNF receptor associated 6 (TRAF6)-mediated ubiquitination and stabilization of hypoxia-inducible factor 1α (HIF1α), resulting in reduced HIF1α-induced expression of IL-1β. Our studies identify a regulatory mechanism controlling diet-induced insulin resistance by highlighting a critical role for Pellino3 in regulating IL-1β expression with implications for diseases like type 2 diabetes.