IFN-λ1 in CHO cells: its expression and biological activity

Background

Many studies have investigated the characteristics and biological activities of type III interferon (IFN), finding that it has similar features to type I IFN but also unique actions because it is recognized by a different receptor.

Results

A full-length recombinant human IFN-λ1 (rhIFN-λ1) cDNA was cloned into the pDF expression vector and stably expressed in Flp-In-CHO cells. After four purification steps (ammonium sulfate precipitation, SP Sepharose chromatography, Blue Sepharose 6 fast flow affinity chromatography and molecular sieve chromatography), the rhIFN-λ1 had a purity of about 90% and was found to have the predicted biological activities. The anti-viral activity of rhIFN-λ1 was determined as 10⁶ IU/mg using the vesicular stomatitis virus (WISH-VSV) assay system. The anti-proliferation activity of rhIFN-λ1 was measured using the MTS method and the growth inhibition ratio was 57% higher than that for recombinant human IFN-α2b (rhIFN-α2b) when the rhIFN-λ1 concentration was 1000 IU/ml. rhIFN-λ1 had lower natural killer cell cytotoxicity than rhIFN-α2b.

Conclusion

The Flp-In-CHO system is suitable for stably expressing rhIFN-λ1 that possesses the predicted anti-viral, anti-proliferation and natural killer cell cytotoxicity-promoting activities.

     

Pneumocystis cell wall β-glucan stimulates calcium-dependent signaling of IL-8 secretion by human airway epithelial cells

Background

During pregnancy asthma may remain stable, improve or worsen. The factors underlying the deleterious effect of pregnancy on asthma remain unknown. Oxytocin is a neurohypophyseal protein that regulates a number of central and peripheral responses such as uterine contractions and milk ejection. Additional evidence suggests that oxytocin regulates inflammatory processes in other tissues given the ubiquitous expression of the oxytocin receptor. The purpose of this study was to define the role of oxytocin in modulating human airway smooth muscle (HASMCs) function in the presence and absence of IL-13 and TNFα, cytokines known to be important in asthma.

Method

Expression of oxytocin receptor in cultured HASMCs was performed by real time PCR and flow cytomery assays. Responses to oxytocin was assessed by fluorimetry to detect calcium signals while isolated tracheal rings and precision cut lung slices (PCLS) were used to measure contractile responses. Finally, ELISA was used to compare oxytocin levels in the bronchoalveloar lavage (BAL) samples from healthy subjects and those with asthma.

Results

PCR analysis demonstrates that OXTR is expressed in HASMCs under basal conditions and that both interleukin (IL)-13 and tumor necrosis factor (TNFα) stimulate a time-dependent increase in OXTR expression at 6 and 18 hr. Additionally, oxytocin increases cytosolic calcium levels in fura-2-loaded HASMCs that were enhanced in cells treated for 24 hr with IL-13. Interestingly, TNFα had little effect on oxytocin-induced calcium response despite increasing receptor expression. Using isolated murine tracheal rings and PCLS, oxytocin also promoted force generation and airway narrowing. Further, oxytocin levels are detectable in bronchoalveolar lavage (BAL) fluid derived from healthy subjects as well as from those with asthma.

Conclusion

Taken together, we show that cytokines modulate the expression of functional oxytocin receptors in HASMCs suggesting a potential role for inflammation-induced changes in oxytocin receptor signaling in the regulation of airway hyper-responsiveness in asthma.     

Regulation of endogenous ENaC functional expression by CFTR and ΔF508-CFTR in airway epithelial cells

The functional expression of the epithelial sodium channel (ENaC) appears elevated in cystic fibrosis (CF) airway epithelia, but the mechanism by which this occurs is not clear. We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) alters the trafficking of endogenously expressed human ENaC in the CFBE41o? model of CF bronchial epithelia. Functional expression of ENaC, as defined by amiloride-inhibited short-circuit current (I(sc)) in Ussing chambers, was absent under control conditions but present in CFBE41o? parental and ΔF508-CFTR-overexpressing cells after treatment with 1 μM dexamethasone (Dex) for 24 h. The effect of Dex was mimicked by incubation with the glucocorticoid hydrocortisone but not with the mineralocorticoid aldosterone. Application of trypsin to the apical surface to activate uncleaved, \"near-silent\" ENaC caused an additional increase in amiloride-sensitive I(sc) in the Dex-treated cells and was without effect in the control cells, suggesting that Dex increased ENaC cell surface expression. In contrast, Dex treatment did not stimulate amiloride-sensitive I(sc) in CFBE41o? cells that stably express wild-type (wt) CFTR. CFBE41o? wt cells also had reduced expression of α- and γ-ENaC compared with parental and ΔF508-CFTR-overexpressing cells. Furthermore, application of trypsin to the apical surface of Dex-treated CFBE41o? wt cells did not stimulate amiloride-sensitive I(sc), suggesting that ENaC remained absent from the surface of these cells even after Dex treatment. We also tested the effect of trafficking-corrected ΔF508-CFTR on ENaC functional expression. Incubation with 1 mM 4-phenylbutyrate synergistically increased Dex-induced ENaC functional expression in ΔF508-CFTR-overexpressing cells. These data support the hypothesis that wt CFTR can regulate the whole cell, functional, and surface expression of endogenous ENaC in airway epithelial cells and that absence of this regulation may foster ENaC hyperactivity in CF airway epithelia.     

Regulation of glycosyltransferases and Lewis antigens expression by IL-1β and IL-6 in human gastric cancer cells

Inflammation of stomach mucosa has been postulated as initiator of gastric carcinogenesis and the presence of pro-inflammatory cytokines can regulate specific genes involved in this process. The cellular expression pattern of glycosyltransferases and Lewis antigens detected in the normal mucosa changed during the neoplassic transformation. The aim of this work was to determine the regulation of specific fucosyltransferases and sialyltransferases by IL-1β and IL-6 pro-inflammatory cytokines in MKN45 gastric cancer cells. IL-1β induced significant increases in the mRNA levels of FUT1, FUT2 and FUT4, and decreases of FUT3 and FUT5. In IL-6 treatments, enhanced FUT1 and lower FUT3 and FUT5 mRNA expression were detected. No substantial changes were observed in the levels of ST3GalIII and ST3GalIV. The activation of FUT1, FUT2 and FUT4 by IL-1β is through the NF-κB pathway and the down-regulation of FUT3 and FUT5 by IL-6 is through the gp130/STAT-3 pathway, since they are inhibited specifically by panepoxydone and AG490, respectively. The levels of Lewis antigens after IL-1β or IL-6 stimulation decreased for sialyl-Lewis x, and no significant differences were found in the rest of the Lewis antigens analyzed, as it was also observed in subcutaneous mice tumors from MKN45 cells treated with IL-1β or IL-6. In addition, in 61 human intestinal-type gastric tumors, sialyl-Lewis x was highly detected in samples from patients that developed metastasis. These results indicate that the expression of the fucosyltransferases involved in the synthesis of Lewis antigens in gastric cancer cells can be specifically modulated by IL-1β and IL-6 inflammatory cytokines.     

Induction of triggering receptor expressed on myeloid cells (TREM-1) in airway epithelial cells by 1,25(OH)₂ vitamin D₃

The airway epithelium plays a role in host defense through the binding of innate immune receptors, which leads to the activation of inflammatory mediators, including antimicrobial peptides. The active form of vitamin D, 1,25(OH)(2)D(3), induces the expression of the antimicrobial peptide LL-37 in both myeloid cells and airway epithelial cells (AEC). Here, we demonstrate that mRNA encoding triggering receptor expressed on myeloid cells (TREM)-1 was induced up to 12-fold by 1,25(OH)(2)D(3) in normal human bronchial epithelial (NHBE) cells and in well-differentiated cultures of six airway epithelial cell lines from patients with cystic fibrosis and healthy individuals. TREM-2 and DAP12 were also expressed in airway cultures, but not induced by vitamin D. Induction occurs through a vitamin D response element identified in its proximal promoter region, and was regulated by PU.1 expressed in the AEC. Activation of TREM-1 by a cross-linking antibody led to an induction of both human β-defensin-2 and TNF-α mRNA, demonstrating its functionality in these cells. Our results expand on the role played by the airway epithelium in innate immunity and suggest that vitamin D can modulate the innate immune defense of the airway epithelium, and could potentially be developed as an adjunctive therapy for airway infections.     

Regulation of glucocorticoid receptors and Na−K ATPase activity by hydrocortisone in proximal tubular epithelial cells

Summary The effect of hydrocortisone (HC) in modulating glucocorticoid receptors (GR) and sodium-potassium adenosine triphosphatase (Na−K ATPase) activity was studied in primary cultures of immunoisolated murine proximal tubular epithelial cells (PTEC). Utilizing monoclonal antibody against stage-specific embryonic antigen-1, a homogeneous population of PTEC was obtained in high yield. The cells were cultured to confluence and further treated for 48 h in serum-free growth medium containing no HC (control); 50 nM HC; or 50 nM HC plus 20 nM of the antiglucocorticoid, RU 38486. PTEC treated with 50 nM HC had 56% of GR binding and 160% Na−K ATPase activity as compared to controls (P<0.01). GR binding was abolished by incubation in RU 38486 whereas Na−K ATPase fell below control values (P<0.05). Brief incubations of HC-treated PTEC with 0.5 mM ouabain resulted in a fall in GR binding without a change in Na−K ATPase activity. These data indicate that in PTEC, HC regulates GR binding and they suggest that stimulation of Na−K ATPase activity is a direct biological response to this receptor-hormone interaction. Thus, primary cultures of immunoaffinity-isolated PTEC offer a good model system for investigating the molecular basis underlying the regulation of GR binding and postreceptor events influenced by glucocorticoids.     

TLR3 promotes MMP-9 production in primary human airway epithelial cells through Wnt/β-catenin signaling

Background

Airway epithelial cells (AEC) act as the first line of defence in case of lung infections. They constitute a physical barrier against pathogens and they participate in the initiation of the immune response. Yet, the modalities of pathogen recognition by AEC and the consequences on the epithelial barrier remain poorly documented.

Method

We investigated the response of primary human AEC to viral (polyinosinic-polycytidylic acid, poly(I:C)) and bacterial (lipopolysaccharide, LPS) stimulations in combination with the lung remodeling factor Transforming Growth Factor-β (TGF-β).

Results

We showed a strong production of pro-inflammatory cytokines (Interleukin (IL)-6, Tumor Necrosis Factor α, TNFα) or chemokines (CCL2, CCL3, CCL4, CXCL10, CXCL11) by AEC stimulated with poly(I:C). Cytokine and chemokine production, except CXCL10, was Toll Like Receptor (TLR)-3 dependent and although they express TLR4, we found no cytokine production after LPS stimulation. Poly(I:C), but not LPS, synergised with TGF-β for the production of matrix metalloproteinase-9 (MMP-9) and fibronectin. Mechanistic analyses suggest the secretion of Wnt ligands by AEC along with a degradation of the cellular junctions after poly(I:C) exposure, leading to the release of β-catenin from the cell membrane and stimulation of the Wnt/β-catenin pathway.

Conclusion

Our results highlight the cross talk between TGF-β and TLR signaling in bronchial epithelium and its impact on the remodeling process.

     

Transforming growth factor-β regulates the expression of anosmin (KAL-1) in human retinal pigment epithelial cells

In a microarray analysis of human retinal pigment epithelial cells (HRPE) treated with TGF-β, in addition to the alteration of a number of known Extracellular matrix (ECM)-related genes regulated by TGF-β, we found a significant increase in the expression of Kallmann Syndrome (KAL)-1 gene, that codes for the protein anosmin-1. Enhanced expression of KAL-1 by TGF-β was validated by real-time PCR analysis. In in vitro experiments, TGF-β receptor inhibitor abolished TGF-β-induced expression of KAL-1. Immunofluorescence staining showed increased presence of anosmin-1 in TGF-β treated HRPE cells, with distinct localization at the intercellular junctions. Treatment of HRPE cells with TGF-β enhanced secretion of anosmin-1 and the release of anosmin-1 was further augmented by heparin sulfate. Enhanced secretion of anosmin-1 in the presence of TGF-β and heparin was also observed in other ocular cells such as corneal epithelial and corneal fibroblast cultures. The role of anosmin-1, a protein with adhesion functions, in retinal structure, function and pathology has not been known and remains to be investigated.     

TGF-β1 induced epithelial to mesenchymal transition (EMT) in human bronchial epithelial cells is enhanced by IL-1β but not abrogated by corticosteroids

Background

Chronic persistent asthma is characterized by ongoing airway inflammation and airway remodeling. The processes leading to airway remodeling are poorly understood, and there is increasing evidence that even aggressive anti-inflammatory therapy does not completely prevent this process. We sought to investigate whether TGFβ₁ stimulates bronchial epithelial cells to undergo transition to a mesenchymal phenotype, and whether this transition can be abrogated by corticosteroid treatment or enhanced by the pro-inflammatory cytokine IL-1β.

Methods

BEAS-2B and primary normal human bronchial epithelial cells were stimulated with TGFβ₁ and expression of epithelial and mesenchymal markers assessed by quantitative real-time PCR, immunoblotting, immunofluorescence microscopy and zymography. In some cases the epithelial cells were also incubated with corticosteroids or IL-1β. Results were analyzed using non-parametric statistical tests.

Results

Treatment of BEAS-2B or primary human bronchial epithelial cells with TGFβ₁ significantly reduced the expression level of the epithelial adherence junction protein E-cadherin. TGFβ₁ then markedly induced mesenchymal marker proteins such as collagen I, tenascin C, fibronectin and α-smooth muscle actin mRNA in a dose dependant manner. The process of mesenchymal transition was accompanied by a morphological change towards a more spindle shaped fibroblast cell type with a more motile and invasive phenotype. Corticosteroid pre-treatment did not significantly alter the TGFβ₁ induced transition but IL-1β enhanced the transition.

Conclusion

Our results indicate, that TGFβ₁ can induce mesenchymal transition in the bronchial epithelial cell line and primary cells. Since asthma has been strongly associated with increased expression of TGFβ₁ in the airway, epithelial to mesenchymal transition may contribute to the contractile and fibrotic remodeling process that accompanies chronic asthma.     

Methylation of secreted frizzled-related protein 1 (SFRP1) promoter downregulates Wnt/β-catenin activity in keloids

Keloid, a benign skin disorder, forms during wound healing in genetically susceptible individuals. To better control keloid and understand the molecular mechanisms, this study screened gene hypermethylations of GEO database microarray data on keloids and identified the hypermethylation of the secreted frizzled related protein-1 (SFRP1) promoter. Subsequently, hypermethylation and mRNA and protein levels were assessed in 57 cases of keloid vs. normal skin tissues. Fibroblasts from tissues were isolated for the assessment of gene regulation in vitro. The methods used were bioinformatic analysis, lentiviral infection carrying SFRP1 cDNA, qRT-PCR, western blot, immunohistochemistry, luciferase reporter assay, methylation-specific PCR and methylated DNA immunoprecipitation-qPCR, ELISA, and/or 5-Aza-2′-deoxycytidine treatment. The data revealed that the SFRP1 promoter was hypermethylated in keloid tissues, compared with that in normal skin tissues. The SFRP1 promoter methylation contributed to the downregulation of SFRP1 mRNA and protein in keloid tissues and keloid fibroblasts. The 5-Aza treatment significantly upregulated SFRP1 mRNA and protein level in keloid fibroblasts. Furthermore, the knockdown of DNMT1 expression, and not the expression of DNMT3a or DMNT3b, was responsible for the hypermethylation of the SFRP1 promoter and upregulation of SFRP1 mRNA and protein in keloid fibroblasts. In addition, the infection of lentivirus carrying SFRP1 cDNA significantly inhibited the signaling activity of Wnt/β-catenin and the mRNA and protein expression of β-catenin and α-SMA in keloid fibroblasts. In summary, the lost SFRP1 expression-induced Wnt/β-catenin signaling due to the hypermethylation of the SFRP1 promoter could associate with keloid development, suggesting that SFRP1 might be a therapeutic target for keloid treatment.     

Clara cell 10-kDa protein gene transfection inhibits NF-κB activity in airway epithelial cells

Background

Clara cell 10-kDa protein (CC10) is a multifunctional protein with anti-inflammatory and immunomodulatory effects. Induction of CC10 expression by gene transfection may possess potential therapeutic effect. Nuclear factor κB (NF-κB) plays a key role in the inflammatory processes of airway diseases.

Method/Results

To investigate potential therapeutic effect of CC10 gene transfection in controlling airway inflammation and the underlying intracellular mechanisms, in this study, we constructed CC10 plasmid and transfected it into bronchial epithelial cell line BEAS-2B cells and CC10 knockout mice. In BEAS-2B cells, CC10''s effect on interleukin (IL)-1β induced IL-8 expression was explored by means of RT-PCR and ELISA and its effect on NF-κB classical signaling pathway was studied by luciferase reporter, western blot, and immunoprecipitation assay. The effect of endogenous CC10 on IL-1β evoked IL-8 expression was studied by means of nasal explant culture. In mice, CC10''s effect on IL-1β induced IL-8 and nuclear p65 expression was examined by immunohistochemistry. First, we found that the CC10 gene transfer could inhibit IL-1β induced IL-8 expression in BEAS-2B cells. Furthermore, we found that CC10 repressed IL-1β induced NF-κB activation by inhibiting the phosphorylation of IκB-α but not IκB kinase-α/β in BEAS-2B cells. Nevertheless, we did not observe a direct interaction between CC10 and p65 subunit in BEAS-2B cells. In nasal explant culture, we found that IL-1β induced IL-8 expression was inversely correlated with CC10 levels in human sinonasal mucosa. In vivo study revealed that CC10 gene transfer could attenuate the increase of IL-8 and nuclear p65 staining in nasal epithelial cells in CC10 knockout mice evoked by IL-1β administration.

Conclusion

These results indicate that CC10 gene transfer may inhibit airway inflammation through suppressing the activation of NF-κB, which may provide us a new consideration in the therapy of airway inflammation.