Polymorphonuclear leukocyte (PMN) infiltration is a cardinal feature of peritonitis. CXC chemokine ligands 1 and 8 (CXCL1 and CXCL8), and the cytokine granulocyte colony-stimulating factor (G-CSF) are the key mediators of PMN accumulation. Increasing evidence points to an important role of human peritoneal fibroblasts (HPFB) in the response of the peritoneum to infection. We have examined the synthesis of PMN-targeting cytokines by HPFB exposed to intraperitoneal milieu as represented by peritoneal dialysate effluent (PDE) from patients undergoing peritoneal dialysis. PDE obtained during peritonitis, but not during infection-free periods, significantly increased production of CXCL1, CXCL8, and G-CSF by HPFB. The effect was largely blocked by antibodies to interleukin-1beta (IL-1beta), whereas neutralization of tumor necrosis factor-alpha (TNFalpha) had no major effect. Similar pattern of inhibition was observed when HPFB were exposed to conditioned media from endotoxin-stimulated peritoneal macrophages. Significance of IL-1beta stimulation was further shown in experiments with recombinant cytokines. Compared with TNFalpha, exposure of HPFB to recombinant IL-1beta resulted in a much higher release of PMN-targeting cytokines. The assessment of mRNA degradation revealed that the IL-1beta-induced transcripts of CXCL1, CXCL8, and G-CSF were more stable compared with those induced by TNFalpha. These data indicate that HPFB can be a significant source of PMN-targeting cytokines when stimulated with IL-1beta in the inflamed peritoneum.

Epidermal and mucosal epithelial cells are integral to host defense. They not only act as a physical barrier but also utilize pattern recognition receptors, such as the Toll-like receptors (TLRs), to detect and respond to pathogens. Members of the interferon regulatory factor (IRF) family of transcription factors are key components of TLR signaling as they impart specificity to downstream responses. Although IRF6 is a critical regulator of epithelial cell proliferation and differentiation, its role in TLR signaling has not previously been addressed. We show here that IRF6 is activated by IRAK1 as well as by MyD88 but not by TRIF or TBK1. Co-immunoprecipitation experiments further demonstrated that IRF6 can interact with IRAK1. Gene silencing in epithelial cells along with gene promoter reporter assays showed that IRAK1 mediates TLR2-inducible CCL5 gene expression at least in part by promoting IRF6 activation. Conversely, IRAK1 regulated CXCL8 gene expression independently of IRF6, thus identifying a molecular mechanism by which TLR2 signaling differentially regulates the expression of specific chemokines in epithelial cells. Bioinformatics analysis and mutagenesis-based experiments identified Ser-413 and Ser-424 as key regulatory sites in IRF6. Phosphomimetic mutation of these residues resulted in greatly enhanced IRF6 dimerization and trans-activator function. Collectively, our findings suggest that, in addition to its importance for epithelial barrier function, IRF6 also contributes to host defense by providing specificity to the regulation of inflammatory chemokine expression by TLR2 in epithelial cells.

The mechanisms leading to the radiation-induced lung responses of alveolitis and fibrosis are largely unknown. Herein we investigated whether CXC receptor 1 and 2 antagonism with CXCL8((3-72))K11R/G31P (G31P), a protein that reduces neutrophil chemotaxis in acute inflammatory response models, decreases the lung response to radiation. Mice of the AKR/J (alveolitis/pneumonitis responding) and KK/HIJ (fibrosis) strains received 18 Gy whole-thorax irradiation and a subset of these mice were treated with G31P (500 µg/kg) three times per week from the day of irradiation until euthanasia due to respiratory distress symptoms or 20 weeks after radiation treatment. Irradiated mice of both strains receiving G31P survived longer than mice receiving radiation alone. Radiation- and G31P-treated AKR/J mice surviving to the end of the experiment developed significantly less alveolitis, as measured histologically, than mice receiving radiation alone, but this difference was not evident in KK/HIJ mice. Using immunohistochemistry, G31P treatment was shown to increase the numbers of Gr-1-positive cells (neutrophils) in the lungs of unirradiated mice relative to control mice injected with saline, but the antagonist did not alter the numbers of Gr-1-positive cells in the lungs of radiation-treated mice. We conclude that G31P treatment reduces radiation-induced alveolitis but not fibrosis in mice.

The aim of the present study was to investigate the role of chemokine (C-X-C motif) ligand 8 (CXCL8) in the proliferation, invasiveness and metastasis of colon cancer and its role in the induction of epithelial-mesenchymal transition (EMT) via activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/nuclear factor-κB (NF-κB) pathway. The plasmid vector containing CXCL8 cDNA was transfected into LoVo cells using Lipofectamine 2000 reagent. Real-time PCR and western blot analyses were performed to determine expression of CXCL8. MTT growth inhibition, scratch and Transwell invasion assays were conducted to assess cell proliferation, migration and invasiveness of the CXCL8-transfected LoVo cells. Western blot analyses were conducted to measure the levels of phosphorylation of protein in the PI3K/Akt/NF-κB pathway in the CXCL8-transfected LoVo cells. Expression levels of CXCL8 mRNA and protein were significantly increased in the CXCL8-transfected LoVo cells compared with levels in the control and empty-vector cells (P<0.05). Overexpression of CXCL8 increased proliferation of the LoVo cells and significant differences in cell viability were observed 48 h after transfection (P<0.05) and remained significant at 72 and 96 h. CXCL8-transfected LoVo cells had a significantly higher migration rate and doubled invasion. The CXCL8-transfected LoVo cells exhibited an EMT-like phenotype, compared with control and empty-vector cells, with decreased expression of E-cadherin accompanied by increased expression of N-cadherin, vimentin and α-SMA. Overexpression of CXCL8 activated the PI3K/Akt/NF-κB pathway by promoting the phosphorylation of PI3K, Akt and NF-κB. Subcutaneous tumors were generated by subcutaneous injection of LoVo parental cells or CXCL8-transfected LoVo cells in BALB/c nude mice. The tumor growth was more rapid in the CXCL8-transfected group than that noted in the parental cell group. In conclusion, overexpression of CXCL8 induced cell proliferation, migration and invasion of colon cancer LoVo cells. CXCL8 may act through induction of EMT via the PI3K/AKT/NF-κB signaling axis.

The etiology of Autism Spectrum Disorders (ASDs) as severe neurodevelopmental ailments is not known. However, several evidences point to dysregulation of immune system as an underlying cause of ASD. In the present study we evaluated the mRNA expression levels of TNF-α, TGF-β, IFN-γ, CXCL8, IL-1β, IL-2, 1L-4, IL-6, IL-17 in whole blood samples of 30 ASD patients and 41 age and sex-matched healthy subjects with means of real-time PCR. TNF-α, IL-6 and IL-17 have been shown to be significantly up-regulated in ASD patients compared with healthy subjects (P < 0.0001, P = 0.001 and P < 0.0001 respectively). IL-2 has been shown to be significantly down-regulated in total ASD patients (P < 0.0001). No significant difference has been found in expression levels of other cytokines between patients and healthy subjects. The present study provides further evidences for dysregulation of immune response in ASD patients.

Infiltrating immune cells including monocytes/macrophages have been implicated in the pathogenesis of neovascular age-related macular degeneration (nAMD). The aim of this study was to investigate the cytokine and chemokine expression and secretion profile of peripheral blood mononuclear cells (PBMCs) from nAMD patients and the relationship between the cytokine/chemokine expression profile and clinical phenotype of nAMD, including macular fibrosis, macular atrophy or the responsiveness to anti-VEGF therapy.

One hundred sixty-one nAMD patients and 43 controls were enrolled in this study. nAMD patients were divided into subgroups based on the presence/absence of (1) macular atrophy, (2) macular fibrosis and (3) responsiveness to anti-VEGF therapy; 25-30 ml of peripheral blood were obtained from all participants and 5 ml were used for serum collection, and the remaining were used for PBMC isolation using density gradient centrifugation. Intracellular cytokine expressions by PBMCs following phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulation were examined using flow cytometry. Cytokine productions in lipopolysaccharides (LPS)-or 1% oxygen -treated PBMC were measured using cytometric bead array (CBA) assay. In addition, cytokine and chemokine levels in the serum were also measured by CBA assay.

PBMCs from nAMD patients secreted higher levels of IL-8, CCL2 and VEGF, especially following LPS and 1% oxygen stimulation, than those from controls. 60~80% of IL-8 producing cells were CD11b+CD3- monocytes. The percentage of CD11b+CD3- IL-8+ was significantly increased in nAMD patients compared to controls. PBMCs from nAMD patients without macular fibrosis produced the highest levels of IL-8 and CCL2, whilst PBMCs from nAMD patients with macular atrophy produced highest levels of VEGF. In addition, PBMCs from patients who partially responded to anti-VEGF produced higher levels of IL-8 compared to the cells from complete responders. Interestingly, serum level of CCL2 was not increased in nAMD patients although there was a trend of increased IL-8 in nAMD patients.

PBMCs, in particular monocytes, may contribute to CNV development in nAMD through secreting elevated levels of IL-8, CCL2 and VEGF after they are recruited to the macula. Apart from VEGF, IL-8 and CCL2 may be additional targets for nAMD management.

Immunostimulatory CpG-C oligodeoxyribonucleotides (ISS-ODNs) represent a promising strategy to enhance vaccine efficacy. We have shown that the CpG-C ISS-ODN C274 stimulates macaque blood dendritic cells (DCs) and B cells and augments SIV-specific IFN-gamma responses in vitro. To further explore the potential of C274 for future vaccine studies, we assessed the in vivo effects of locally administered C274 (in naive and healthy infected macaques). Costimulatory molecules were marginally increased on DCs and B cells within cells isolated from C274-injected lymph nodes (LNs). However, cells from C274-injected LNs exhibited heightened responsiveness to in vitro culture. This was particularly apparent at the level of CD80 (less so CD86) expression by CD123(+) plasmacytoid DCs and was further boosted in the presence of additional C274 in vitro. Notably, cells from C274-injected LNs secreted significantly elevated levels of several cytokines and chemokines upon in vitro culture. This was more pronounced when cells were exposed to additional stimuli in vitro, producing IFN-alpha, IL-3, IL-6, IL-12, TNF-alpha, CCL2, CCL3, CCL5, and CXCL8. Following C274 administration in the absence of additional SIV Ag, endogenous IFN-gamma secretion was elevated in LN cells of infected animals, but SIV-specific responses were unchanged. Endogenous and SIV-specific responses decreased in blood, before the SIV-specific responses rebounded by 2 wk after C274 treatment. Elevated IFN-alpha, CCL2, and CCL5 were also detected in the plasma after C274 injection. Thus, locally administered C274 has local and systemic activities, supporting the potential for CpG-C ISS-ODNs to boost immune function to enhance anti-HIV vaccine immunogenicity.

The phagocytic clearance of apoptotic cells is critical for tissue homeostasis; a number of non-professional phagocytic cells, including epithelial cells, can both take up and process apoptotic bodies, including the release of anti-inflammatory mediators. These observations are particularly important in the case of human intrahepatic biliary cells (HiBEC), because such cells are themselves a target of destruction in primary biliary cirrhosis, the human autoimmune disease. To address the apoptotic ability of HiBECs, we have focused on their ability to phagocytize apoptotic blebs from autologous HiBECs. In this study we report that HiBEC cells demonstrate phagocytic function from autologous HiBEC peers accompanied by up-regulation of the chemokines CCL2 [monocyte chemotactic protein-1 (MCP-1)] and CXCL8 [interleukin (IL)-8]. In particular, HiBEC cells express the phagocytosis-related receptor phosphatidylserine receptors (PSR), implying that HiBECs function through the 'eat-me' signal phosphatidylserine expressed by apoptotic cells. Indeed, although HiBEC cells acquire antigen-presenting cell (APC) function, they do not change the expression of classic APC function surface markers after engulfment of blebs, both with and without the presence of Toll-like receptor (TLR) stimulation. These results are important not only for understanding of the normal physiological function of HiBECs, but also explain the inflammatory potential and reduced clearance of HiBEC cells following the inflammatory cascade in primary biliary cirrhosis.

We examined the effect of resveratrol (RVT) and its two derivatives (3,3',4,4'-tetrahydroxy-trans-stilbene and 3,3',4,4',5,5'-hexahydroxy-trans-stilbene) on human peritoneal mesothelial cell (HPMC)-dependent angiogenesis in vitro. To this end, angiogenic activity of endothelial cells (HUVEC, HMVEC, and HMEC-1) was monitored upon their exposure to conditioned medium (CM) from young and senescent HPMCs treated with stilbenes or to stilbenes themselves. Results showed that proliferation and migration of endothelial cells were inhibited in response to indirect (HPMC-dependent) or direct RVT activity. This effect was associated with decreased secretion of VEGF and IL-8/CXCL8 by HPMCs treated with RVT, which confirmed the experiments with recombinant forms of these angiogenic agents. Angiogenic activity of endothelial cells treated with CM from HPMCs exposed to RVT analogues was more effective. Improved migration was particularly evident in cells exposed to CM from senescent HPMCs. Upon direct treatment, RVT derivatives stimulated proliferation (but not migration) of HUVECs, and failed to affect the behaviour of HMVEC and HMEC-1 cells. These compounds stimulated production of VEGF and IL-8/CXCL8 by HPMCs. Studies with neutralizing antibodies against angiogenic factors revealed that augmented angiogenic reactions of endothelial cells exposed to CM from HPMC treated with RVT analogues were related to enhanced production of VEGF and IL-8/CXCL8. Collectively, these findings indicate that RVT and its synthetic analogues divergently alter the secretion of the angiogenic factors by HPMCs, and thus modulate HPMC-dependent angiogenic responses in the opposite directions. This may have implications for the attempts of practical employment of the stilbenes for treatment of pathologies proceeding with abnormal vascularisation of the peritoneal tissue.

CXCL8 is secreted by both normal human thyrocytes (NHT) and thyroid cancer cell lines. CXCL8 displays several tumor-promoting effects and recent evidences indicate that its concentrations within the tumor microenvironment can impact the clinical course of the malignancy. Aim of this study was to compare the basal secretion of CXCL8 among NHT and thyroid cancer cell lines (TPC-1 and BCPAP), and to assess the specific cell response to TNF-α in terms of CXCL8 secretion. NHT primary cultures, TPC-1 and BCPAP cell lines were cultured with or without TNF-α (0, 0.1, 1, 10, and 100 ng/ml). CXCL8 levels were measured in the cell supernatants after 24 h. In basal condition, significant differences in the mean levels of CXCL8 were observed among the three cell types: NHT (110.5 ± 56.2 pg/ml), TPC1 (467.4 ± 43.2 pg/ml), and BCPAP (1731.8 ± 493.3 pg/ml), (F = 35.06; p < 0.0001). TNF-α significantly and in a dose-response manner induced CXCL8 secretion in NHT (F = 25.53; p < 0.00001), TPC-1 (F = 13.38; p < 0.0001), and BCPAP (F = 9.88; p < 0.001) cells. The magnitude of the TNF-α effect (fold-increase vs. basal level of CXCL8) differed significantly among the three cell types (F = 10.47; p < 0.0001). BCPAP were identified as the cells showing the highest basal secretion of CXCL8 and the less responsive to TNF-α. NHT, TPC-1, and BCPAP display significant differences in the secretion of both basal and TNF-α-induced CXCL8 secretion. These results indicate that the mechanisms regulating the secretion of CXCL8 differ in tumor cells harboring different genetic alterations suggesting that specific strategies aimed at inhibiting CXCL8 secretion will be required.

BACKGROUND

Posttranslational deimination or citrullination by peptidylarginine deiminases (PAD) regulates the biological function of proteins and may be involved in the development of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. This posttranslational modification of arginine was recently discovered on inflammatory chemokines including CXCL8 and CXCL10, and significantly reduced their biological activity. To evaluate the importance of these modified chemokines in patients, methods for the detection and quantification of citrullinated chemokines are needed. Since citrullination only results in an increase of the protein mass with one mass unit and the loss of one positive charge, selective biochemical detection is difficult. Therefore, we developed an antibody-based method to specifically detect and quantify citrullination on a protein of interest.

RESULTS

First, the citrullinated proteins were chemically modified with antipyrine and 2,3-butanedione at low pH. Such selectively modified citrullines were subsequently detected and quantified by specific antibodies raised against a modified citrulline-containing peptide. The specificity of this two-step procedure was validated for citrullinated CXCL8 ([Cit(5)]CXCL8). Specific detection of [Cit(5)]CXCL8 concentrations between 1 and 50 ng/ml was possible, also in complex samples containing an excess of contaminating proteins. This novel detection method was used to evaluate the effect of lipopolysaccharide (LPS) on the citrullination of inflammatory chemokines induced in peripheral blood mononuclear cells (PBMCs) and granulocytes. LPS had no significant effect on the induction of CXCL8 citrullination in human PBMCs and granulocytes. However, granulocytes, known to contain PAD, were essential for the production of significant amounts of [Cit(5)]CXCL8.

CONCLUSIONS

The newly developed antibody-based method to specifically detect and quantify chemically modified citrullinated proteins is proven to be effective. This study furthermore demonstrates that granulocytes were essential to obtain significant levels of [Cit(5)]CXCL8. For human PBMCs and granulocytes stimulation with LPS did not affect the citrullination of CXCL8.

Citrullination, a posttranslational modification (PTM) recently discovered on inflammatory chemokines such as interleukin-8 (IL-8/CXCL8) and interferon-γ-inducible protein-10 (IP-10/CXCL10), seriously influences their biological activity. Citrullination or the deimination of arginine to citrulline is dependent on peptidylarginine deiminases (PADs) and has been linked to autoimmune diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA). Chemokines are to date the first identified PAD substrates with receptor-mediated biological activity. We investigated whether cytokines that play a crucial role in RA, like interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α), may be citrullinated by PAD and whether such a PTM influences the biological activity of these cytokines. IL-1β and TNF-α were first incubated with PAD in vitro and the occurrence of citrullination was examined by Edman degradation and a recently developed detection method for citrullinated proteins. Both techniques confirmed that human TNF-α, but not IL-1β, was citrullinated by PAD. Citrullination of TNF-α reduced its potency to stimulate chemokine production in vitro on human primary fibroblasts. Concentrations of the inflammatory chemokines CXCL8, CXCL10 and monocyte chemotactic protein-1 (MCP-1/CCL2) were significantly lower in supernatants of fibroblasts induced with citrullinated TNF-α compared to unmodified TNF-α. However, upon citrullination TNF-α retained its capacity to induce apoptosis/necrosis of mononuclear cells, its binding potency to Infliximab and its ability to recruit neutrophils to the peritoneal cavity of mice.

Severe acute respiratory syndrome (SARS), caused by a novel coronavirus, emerged in early 2003 as a major international health crisis. We report on serum cytokine levels, viral load and clinical parameters over the course of the disease in a cohort of nine adult SARS patients treated with steroids and interferon alfacon-1 at North York General Hospital in Toronto, Ontario. Considerable variation among SARS patients with respect to circulating viral load and patterns of SARS-CoV-evoked cytokine responses was recorded. No single cytokine profile was observed in all patients, yet serum concentrations of interferon (IFN)-gamma, interleukin (IL)-10, CXCL10, CCL5 and CXCL8 were found to be elevated above normal levels during the course of the disease in all patients. Expression levels for IL-10, IFN-gamma and CXCL10 consistently peaked within 4 days of peak viral load. IL-12p70, IL-4 and tumour necrosis factor-alpha concentrations were consistently highest within 5 days of peak viral load. These results suggest that elevated levels of inflammatory cytokines are sensitive correlates of disease severity, including lung abnormalities and viral load in serum, and may provide a tool for monitoring disease progression in affected individuals.

BACKGROUND

Immune-mediated polyarthopathy (IMPA) is common in dogs, and is monitored by serial arthrocenteses.

OBJECTIVE

Plasma C-reactive protein (CRP), interleukin-6 (IL-6), and CXCL8 (interleukin-8) would serve as noninvasive markers of joint inflammation in IMPA.

METHODS

Nine client-owned dogs with idiopathic IMPA; 6 healthy controls.

METHODS

Prospective study. Plasma CRP, IL-6, and CXCL8 were measured by ELISA at baseline, 2, and 4 weeks during treatment with prednisone at 50 mg/m(2) /day. Arthrocenteses, the canine brief pain inventory (CBPI), and accelerometry collars were used to assess joint inflammation, lameness, and mobility at all 3 time points.

RESULTS

C-reactive protein concentrations were higher in IMPA dogs (median 91.1 μg/mL, range 76.7-195.0) compared with controls (median <6.3 μg/mL, <6.3-13.7; P = .0035), and were significantly lower at week 2 (10.6 μg/mL, <6.3-48.8) and week 4 (<6.3 μg/mL, <6.3-24.4; P < .001). C-reactive protein was correlated with median CBPI scores (r = 0.68; P = .0004), joint cellularity (r = 0.49, P = .011), and mobility by accelerometry (r = -0.42, P = .048). Plasma IL-6 concentrations were also higher in IMPA dogs (median 45.9 pg/mL), compared with controls (median <15.7 pg/mL; P = .0008). IL-6 was lower in IMPA dogs by week 4 (<15.7 pg/mL; P = .0099), and was modestly correlated with CBPI scores (r = 0.47, P = .023). CXCL8 did not differ significantly between IMPA and healthy dogs.

CONCLUSIONS

Plasma CRP and IL-6 might be useful surrogate markers of synovial inflammation and disease activity in dogs with IMPA.

BACKGROUND

Indoleamine 2,3-dioxygenase (IDO) induces generation of regulatory T cells but suppresses Th17 cells and therefore might attenuate neutrophilic inflammation. The role of IDO in neutrophilic airway diseases such as chronic obstructive pulmonary disease (COPD) remains unknown. We evaluated IDO activity and expression and interleukin (IL)-10 and IL-17A levels in sputum from patients with COPD.

METHODS

IDO activity and cytokine concentrations in sputum supernatants from patients with COPD of varying severity and in smoking and non-smoking control subjects were determined by high-performance liquid chromatography and ELISA, respectively.

RESULTS

Patients with COPD had reduced sputum IDO activity and expression and IL-10 levels, with increased IL-17A, IL-6 and CXCL8 concentrations and sputum neutrophils. These changes were significantly correlated with disease severity. IDO activity was decreased, but to a lesser extent, in normal smokers compared with non-smoking controls.

CONCLUSIONS

Patients with COPD have a progressive reduction in IDO activity with reversal of the balance between IL-10 and IL-17A, resulting in chronic airway neutrophilic inflammation.

Human infection with Paracoccidioides brasiliensis may result in three major outcomes: paracoccidioidomycosis-infection (PI), which is observed in healthy carriers living in endemic areas and the adult form (AF) and juvenile form (JF) of the disease. In this study we proposed to examine the intracellular expression of IFN-gamma, TNF-alpha, IL-2, IL-10, IL-12, CXCL8, CXCL9 and CXCL10 by peripheral blood mononuclear cells (PBMC) of patients with the JF and AF of the disease, as well as of PI individuals stimulated with PMA plus ionomycin, LPS or anti-CD3 plus anti-CD28, by flow cytometry. The results showed that PI individuals present a higher percentage of cells producing IFN-gamma, TNF-alpha, IL-2, CXCL9 and CXCL10 when compared to AF and JF patients. IFN-gamma was predominantly detected in CD3(+)CD8(+) T cells, whereas IL-2 and TNF-alpha were mainly expressed in CD3(+)CD4(+) cells. Monocytes of PI individuals also presented higher expression of CD80 and lower expression of CD86 when compared to JF and AF patients, and higher expression of HLA-DR, only when compared to JF patients. These results indicate that the differential production of cytokines and chemokines, as well as the expression of co-stimulatory molecules involved in antigen presentation, may influence the outcome of PCM infection.

Non-typeable Haemophilus influenzae (NTHi) is one of the most frequently involved pathogens in bacterial exacerbations of chronic obstructive pulmonary disease (COPD). In the airways, the main tissue target of NTHi is bronchial epithelium, where this pathogen can further amplify the inflammatory and structural changes induced by proinflammatory cytokines such as tumour necrosis factor-alpha (TNF-alpha). Therefore, the aim of this study is to investigate, in primary cultures of human bronchial epithelial cells, the effects of NTHi on signal transduction pathways, apoptotic events and chemokine production activated by TNF-alpha. Moreover, we also evaluated the effects exerted on such cellular and molecular phenomena by a corticosteroid drug. p38 mitogen-activated protein kinase (MAPK) phosphorylation was analyzed by Western blotting, using an anti-phospho-p38 MAPK monoclonal antibody. Apoptosis was assayed by active caspase-3 expression. Interleukin-8 (IL-8/CXCL8) was detected in cell-free culture supernatants by ELISA. TNF-alpha induced a significant increase in p38 MAPK phosphorylation. NTHi was able to potentiate the stimulatory actions of TNF-alpha on caspase-3 expression and, to a lesser extent, on IL-8 secretion. These effects were significantly (P less than 0.01) inhibited by a pharmacological pre-treatment with budesonide. These results suggest that TNF-alpha is able to stimulate, via activation of p38 MAPK signalling pathway, IL-8 release and airway epithelial cell apoptosis; the latter effect can be markedly potentiated by NTHi. Furthermore, budesonide can be very effective in preventing, through inhibition of p38 MAPK phosphorylation, both structural and proinflammatory changes elicited in bronchial epithelium by TNF-alpha and NTHi.

Airway smooth muscle (ASM) cells play a critical role in the pathophysiology of asthma due to their hypercontractility and their ability to proliferate and secrete inflammatory mediators. microRNAs (miRNAs) are gene regulators that control many signaling pathways and thus serve as potential therapeutic alternatives for many diseases. We have previously shown that miR-708 and miR-140-3p regulate the MAPK and PI3K signaling pathways in human ASM (HASM) cells following TNF-α exposure. In this study, we investigated the regulatory effect of these miRNAs on other asthma-related genes. Microarray analysis using the Illumina platform was performed with total RNA extracted from miR-708 (or control miR)-transfected HASM cells. Inhibition of candidate inflammation-associated gene expression was further validated by qPCR and ELISA. The most significant biologic functions for the differentially expressed gene set included decreased inflammatory response, cytokine expression and signaling. qPCR revealed inhibition of expression of CCL11, CXCL10, CCL2 and CXCL8, while the release of CCL11 was inhibited in miR-708-transfected cells. Transfection of cells with miR-140-3p resulted in inhibition of expression of CCL11, CXCL12, CXCL10, CCL5 and CXCL8 and of TNF-α-induced CXCL12 release. In addition, expression of RARRES2, CD44 and ADAM33, genes known to contribute to the pathophysiology of asthma, were found to be inhibited in miR-708-transfected cells. These results demonstrate that miR-708 and miR-140-3p exert distinct effects on inflammation-associated gene expression and biological function of ASM cells. Targeting these miRNA networks may provide a novel therapeutic mechanism to down-regulate airway inflammation and ASM proliferation in asthma.

BACKGROUND

We determined previously that hypoxia results in increased 15-lipoxygenase type 2 (15-LOX-2) expression and CXCL8 secretion in macrophages. This study sought to determine whether 15-LOX-2 expression links directly with the secretion of inflammatory molecules in macrophages and also investigated its subsequent effects on T cell migration.

METHODS

Adenovirus-mediated gene delivery caused overexpression of 15-LOX-2 in human macrophages. We used cytometric bead array to measure chemokine secretion, and assessed T cell migration by counting cells in chemotaxis chambers. Expression of chemokine receptors was determined by FACS analysis. Using siRNA, we reduced 15-LOX-2 expression in human macrophages. We used scrambled siRNA as control.

RESULTS

Macrophages that overexpress 15-LOX-2 showed increased secretion of chemokine CXCL10 after 24h incubation. In addition, preconditioned medium from 15-LOX-2-overexpressing cells increased T cell migration and surface expression of CXCR3, the CXCL10 receptor. Knockdown of 15-LOX-2 expression decreased CXCL10 secretion from hypoxic macrophages and also reduced T cell migration.

CONCLUSIONS

In macrophages, overexpression of 15-LOX-2 results in increased secretion of CXCL10 and CCL2. Products released in response to increased 15-LOX-2 activation lead to increased expression of CD69, the T cell activation marker as well as increased T cell migration. Therefore, increased expression of 15-LOX-2 induced by hypoxia may participate in T cell recruitment in diseases such as atherosclerosis.

BACKGROUND

The development of the in vitro cell culture model has greatly facilitated the ability to study gene expression and regulation within human tissues. Within the human uterus, the upper (fundal) segment and the lower segment may provide distinct functions throughout pregnancy and during labour. We have established primary cultured human myometrial cells, isolated from both upper and lower segment regions of the pregnant human uterus, and validated them for the purpose of studying human pregnancy and labour. The specific objectives of this study were to monitor the viability and characterize the expression profile using selected cellular, contractile and pregnancy associated markers in the primary cultured human myometrial cells. Labour has been described as an inflammatory process; therefore, the ability of these cells to respond to an inflammatory stimulus was also investigated.

METHODS

Myometrial cells isolated from paired upper segment (US) and lower segment (LS) biopsies, obtained from women undergoing Caesarean section deliveries at term prior to the onset of labour, were used to identify expression of; α smooth muscle actin, calponin, caldesmon, connexin 43, cyclo-oxygenase-2 (COX-2), oxytocin receptor, tropomyosin and vimentin, by RT-PCR and/or immunocytochemistry. Interleukin (IL)-1β was used to treat cells, subsequently expression of COX-2 mRNA and release of interleukin-8 (CXCL8), were measured. ANOVA followed by Bonferroni's multiple comparisons test was performed.

RESULTS

We demonstrate that US and LS human myometrial cells stably express all markers examined to at least passage ten (p10). Connexin 43, COX-2 and vimentin mRNA expression were significantly higher in LS cells compared to US cells. Both cell populations respond to IL-1β, demonstrated by a robust release of CXCL8 and increased expression of COX-2 mRNA from passage one (p1) through to p10.

CONCLUSIONS

Isolated primary myometrial cells maintain expression of smooth muscle and pregnancy-associated markers and retain their ability to respond to an inflammatory stimulus. These distinct myometrial cell models will provide a useful tool to investigate mechanisms underlying the process of human labour and the concept of functional regionalization of the pregnant uterus.

Previous studies have demonstrated that neutrophils isolated from the blood of healthy donors do not respond to IL-10 in terms of either activation of signal transducer and activator of transcription-3 (STAT3) tyrosine phosphorylation or induction of suppressor of cytokine signalling (SOCS)-3 protein, unlike autologous mononuclear cells. This was explained by the fact that circulating neutrophils of healthy donors express only IL-10R2, but not IL-10R1, the latter IL-10R chain being essential for mediating IL-10 responsiveness. In this study, we report that peripheral blood neutrophils of septic patients constitutively display, besides IL-10R2, also abundant levels of surface IL-10R1. Consequently, septic neutrophils are promptly responsive to IL-10 in vitro, as revealed by a direct IL-10-mediated induction of STAT3 tyrosine phosphorylation and SOCS-3 gene transcription, mRNA and protein expression. Consistent with the presence of a fully functional IL-10R, modulation of LPS-induced CXCL8, CCL4, tumour necrosis factor-alpha and IL-1ra gene expression was also rapidly induced by IL-10 in septic, but not normal, neutrophils. Collectively, these data uncover that neutrophils of septic patients are predisposed to be promptly responsive to IL-10, presumably to help limiting their pro-inflammatory state. They also fully validate our previous observations, herein in the context of a human disease, that responsiveness of human neutrophils to IL-10 is strictly dependent upon the modulation of IL-10R1 expression.

Differentiation therapy is an attractive approach aiming at reversing malignancy and reactivating endogenous differentiation programs in cancer cells. Olive leaf extract, known for its antioxidant activity, has been demonstrated to induce apoptosis in several cancer cells. However, its differentiation inducing properties and the mechanisms involved are still poorly understood. In this study, we investigated the effect of Chemlali Olive Leaf Extract (COLE) for its potential differentiation inducing effect on multipotent leukemia K562 cells. Results showed that COLE inhibits K562 cells proliferation and arrests the cell cycle at G0/G1, and then at G2/M phase over treatment time. Further analysis revealed that COLE induces apoptosis and differentiation of K562 cells toward the monocyte lineage. Microarray analysis was conducted to investigate the underlying mechanism of COLE differentiation inducing effect. The differentially expressed genes such as IFI16, EGR1, NFYA, FOXP1, CXCL2, CXCL3, and CXCL8 confirmed the commitment of K562 cells to the monocyte/macrophage lineage. Thus our results provide evidence that, in addition to apoptosis, induction of differentiation is one of the possible therapeutic effects of olive leaf in cancer cells.

BACKGROUND

There is large variation in the therapeutic response to inhaled corticosteroids (ICS) in COPD patients. We present a pooled analysis of our previous studies investigating the effects of corticosteroids on lung macrophages, in order to robustly determine whether corticosteroid sensitivity in COPD cells is reduced compared to controls, and also to evaluate the degree of between individual variation in drug response.

METHODS

Data from 20 never smokers (NS), 27 smokers (S) and 45 COPD patients was used. Lung macropahges had been stimulated with lipopolysaccharide (LPS), with or without the corticosteroid dexamethasone, and tumour necrosis factor (TNF)-α, interleukin (IL)-6 and chemokine C-X-C motif ligand (CXCL) 8 production was measured.

RESULTS

There was no difference in the anti-inflammatory effects of corticosteroids when comparing group mean data of COPD patients versus controls. The inhibition of TNF-α and IL-6 was greater than CXCL8. The effects of corticosteroids varied considerably between subjects, particularly at lower corticosteroid concentrations.

CONCLUSIONS

We confirm that overall corticosteroid sensitivity in COPD lung macrophages is not reduced compared to controls. The varied effect of corticosteroids between subjects suggests that some individuals have an inherently poor corticosteroid response. The limited suppression of lung macrophage derived CXCL8 may promote neutrophilic inflammation in COPD.

Chronic subdural hematoma (CSDH) is a relatively common disorder in neurosurgery on elderly patients, though the mechanism that causes the disease remains unclear. Studies have suggested that local anticoagulation and inflammatory changes may be important in its pathogenesis. Most studies have used a basic bivariate statistical analysis to assess complex immunological responses in patients with this disorder, hence a more sophisticated multivariate statistical approach might be warranted. Our objective was to assess the association and correlation between the pro- and anti-inflammatory responses in a cohort of patients with chronic subdural hematoma (n=57) using an exploratory and confirmatory factor analysis. Thirteen assigned pro-inflammatory (TNF-α, IL-1β, IL-2, IL-2R, IL-6, IL-7, IL-12, IL-15, IL-17, CCL2, CXCL8, CXCL9 and CXCL10) and five assigned anti-inflammatory (IL-1RA, IL-4, IL-5, IL-10 and IL-13) cytokines from blood and hematoma fluid samples were examined. Exploratory factor analysis indicated two major underlying immunological processes expressed by the cytokines in both blood and hematoma fluid, but with a different pattern and particularly regarding the cytokines IL-13, IL-6, IL-4 and TNF-α. Scores from confirmatory factor analysis models exhibited a higher correlation between pro- and anti-inflammatory activities in blood (r=0.98) than in hematoma fluid samples (r=0.92). However, correlations of inflammatory processes between blood and hematoma fluid samples were lower and non-significant. A structural equation model showed a significant association between increased anti-inflammatory activity in hematoma fluid samples and a lower risk of recurrence, but this relationship was not statistically significant in venous blood samples. Moreover, these findings indicate that anti-inflammatory activities in the hematoma may play a role in the risk of a recurrence of CSDH.