Airway epithelial cells produce B cell-activating factor of TNF family by an IFN-beta-dependent mechanism

Activation of B cells in the airways is now believed to be of great importance in immunity to pathogens, and it participates in the pathogenesis of airway diseases. However, little is known about the mechanisms of local activation of B cells in airway mucosa. We investigated the expression of members of the B cell-activating TNF superfamily (B cell-activating factor of TNF family (BAFF) and a proliferation-inducing ligand (APRIL)) in resting and TLR ligand-treated BEAS-2B cells and primary human bronchial epithelial cells (PBEC). In unstimulated cells, expression of BAFF and APRIL was minimal. However, BAFF mRNA was significantly up-regulated by TLR3 ligand (dsRNA), but not by other TLR ligands, in both BEAS-2B cells (376-fold) and PBEC (224-fold). APRIL mRNA was up-regulated by dsRNA in PBEC (7-fold), but not in BEAS-2B cells. Membrane-bound BAFF protein was detectable after stimulation with dsRNA. Soluble BAFF protein was also induced by dsRNA (> 200 pg/ml). The biological activity of the epithelial cell-produced BAFF was verified using a B cell survival assay. BAFF was also strongly induced by IFN-beta, a cytokine induced by dsRNA. Induction of BAFF by dsRNA was dependent upon protein synthesis and IFN-alphabeta receptor-JAK-STAT signaling, as indicated by studies with cycloheximide, the JAK inhibitor I, and small interfering RNA against STAT1 and IFN-alphabeta receptor 2. These results suggest that BAFF is induced by dsRNA in airway epithelial cells and that the response results via an autocrine pathway involving IFN-beta. The production of BAFF and APRIL by epithelial cells may contribute to local accumulation, activation, class switch recombination, and Ig synthesis by B cells in the airways.     

The role of zinc in caspase activation and apoptotic cell death

In addition to its diverse role in many physiological systems, zinc (Zn) has now been shown to be an important regulator of apoptosis. The purpose of this review is to integrate previously published knowledge on Zn and apoptosis with current attempts to elucidate the mechanisms of action of this biometal. This paper begins with an introduction to apoptosis and then briefly reviews the evidence relating Zn to apoptosis. The major focus of this review is the mechanistic actions of Zn and its candidate intracellular targets. In particular, we examine the cytoprotective functions of Zn which suppress major pathways leading to apoptosis, as well as the more direct influence of Zn on the apoptotic regulators, especially the caspase family of enzymes. These two mechanisms are closely related since a decline in intracellular Zn below a critical threshold level may not only trigger pathways leading to caspase activation but may also facilitate the process by which the caspases are activated. Studies by our laboratory in airway epithelial cells show that Zn is co-localized with the precursor form of caspase-3, mitochondria and microtubules, suggesting this Zn is critically placed to control apoptosis. Further understanding the different pools of Zn and how they interact with apoptotic pathways should have importance in human disease.     

Regulation of caspase activation and apoptosis by cellular zinc fluxes and zinc deprivation: A review.

Non-toxic agents that target intracellular signalling pathways in apoptosis may have potential therapeutic use in many diseases. One such agent is the transition metal Zn, a dietary cytoprotectant and anti-oxidant, which stimulates cell proliferation and suppresses apoptosis. Zn is maintained in discrete subcellular pools that are critical for the functional and structural integrity of cells. The present review initially describes the current state of knowledge on the cellular biology of Zn, especially the critical free or loosely bound (labile) pools of Zn, which are thought to regulate apoptosis. We then review the evidence relating Zn to apoptosis, including studies from our laboratory showing potent synergy between intracellular Zn deficiency and the short chain fatty acid butyrate in induction of caspase activation and the downstream events of apoptosis. Our studies have also reported the suppressive effects of micromolar concentrations of Zn on caspase-3 activation in cell-free models. Other key issues that will be discussed include the identification of the putative molecular targets of Zn and the evidence that systemic changes in labile Zn levels are sufficient to alter susceptibility to apoptosis and lead to physiopathological changes in the human body. Finally, we propose that labile Zn may serve as a coordinate regulator of mitosis and apoptosis to regulate tissue growth.     

The antiestrogen ICI 182,780 induces early effects on the adult male mouse reproductive tract and long-term decreased fertility without testicular atrophy

Background  

Estrogen receptors (ER) have important physiological roles in both the female and male reproductive systems. Previous studies using the estrogen receptor-α knockout mouse (αERKO) or antiestrogen treatment in adult rodents have shown that ERα is essential for normal function of the male reproductive tract. In the present study, time-response effects of the antiestrogen ICI 182,780 were determined to better understand ERα function in the adult male.     

New insights into the role of zinc in the respiratory epithelium

Over the past 30 years, many researchers have demonstrated the critical role of zinc (Zn), a group IIb metal, in diverse physiological processes, such as growth and development, maintenance and priming of the immune system, and tissue repair. This review will discuss aspects of Zn physiology and its possible beneficial role in the respiratory epithelium. Here we have detailed the mechanisms by which Zn diversely acts as: (i) an anti-oxidant; (ii) an organelle stabilizer; (iii) an anti-apopototic agent; (iv) an important cofactor for DNA synthesis; (v) a vital component for wound healing; and (vi) an anti-inflammatory agent. This paper will also review studies from the authors' laboratory concerning the first attempts to map Zn in the respiratory epithelium and to elucidate its role in regulating caspase-3 activated apoptosis. We propose that Zn, being a major dietary anti-oxidant has a protective role for the airway epithelium against oxyradicals and other noxious agents. Zn may therefore have important implications for asthma and other inflammatory diseases where the physical barrier is vulnerable and compromised.     

Visualization of labile zinc and its role in apoptosis of primary airway epithelial cells and cell lines

The respiratory epithelium is vulnerable to noxious substances, resulting in the shedding of cells and decreased protection. Zinc (Zn), an antioxidant and cytoprotectant, can suppress apoptosis in a variety of cells. Here we used the novel Zn-specific fluorophore Zinquin to visualize and quantify labile intracellular Zn in respiratory epithelial cells. Zinquin fluorescence in isolated ciliated tracheobronchial epithelial cells and intact epithelium from sheep and pigs revealed an intense fluorescence in the apical and mitochondria-rich cytoplasm below the cilia. Zinquin fluorescence was quenched by the Zn chelator N,N,N', N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and increased by the Zn ionophore pyrithione. We also assessed whether changes in intracellular labile Zn would influence susceptibility of these cells to apoptosis by hydrogen peroxide. Our results confirm that Zn deficiency enhanced hydrogen peroxide-induced caspase activation from 1.24 +/- 0.12 to 2.58 +/- 0.53 units. microg protein(-1). h(-1) (P 相似文献   

Use of a zinc fluorophore to measure labile pools of zinc in body fluids and cell-conditioned media

Here we describe a rapid and sensitive zinquin-based fluorometric assay that enables one to monitor levels of labile Zn(II) in body fluids, buffers, and cell-conditioned culture media as well as changes in these pools in disease. Labile pools of Zn(II) are free or loosely bound pools and more tightly bound but zinquin-accessible pools in contrast to the fixed pools of Zn(II) within metalloproteins. In human plasma, mean labile Zn(II) was 8.1 microM (SEM 0.53; n = 81) and constituted about 70% of the total plasma Zn(II) and >90% of human plasma albumin Zn(II). Plasma labile Zn(II) was significantly depleted after 7 days of Zn(II) deprivation in mice, despite only small changes in body weight. Labile Zn(II) concentrations were also measured in the induced sputum plugs, saliva, and urine of normal adults and were 1.30 microM (SEM 0.27; n = 73), 0.11 microM (SEM 0.11; n = 6), and 0.23 microM (SEM 0.08; n = 8), respectively. Urinary labile Zn(II) concentration was significantly increased in some patients with type II diabetes mellitus (overall mean was 0.90 microM, SEM 0.30; n = 12). The technique may be particularly useful in assessing extracellular Zn(II) levels in diseases associated with altered Zn(II) homeostasis, identifying those subjects most in need of Zn(II) supplementation, and defining the optimum concentrations of available Zn(II) in buffers and culture media.     

Involvement of redox events in caspase activation in zinc-depleted airway epithelial cells

Airway epithelial cells (AEC) contain both pro- and anti-apoptotic factors but little is known about mechanisms regulating apoptosis of these cells. In this study we have examined the localization of pro-caspase-3 and Zn(2+), a cellular regulator of pro-caspase-3, in primary sheep and human AEC. Zn(2+) was concentrated in both cytoplasmic vesicles and ciliary basal bodies, in the vicinity of both pro-caspase-3 and the antioxidant Cu/Zn superoxide dismutase (Cu/Zn SOD). Depletion of intracellular Zn(2+) in sheep AEC, using the membrane permeant Zn(2+) chelator TPEN, increased lipid peroxidation in the apical cell membranes (as assessed by immunofluorescence with anti-hydroxynonenal) as well as increasing activated pro-caspase-3 and apoptosis. There were smaller increases in caspase-2 and -6 but not other caspases. Activation of caspase-3 in TPEN-treated AEC was inhibited strongly by N-acetylcysteine and partially by vitamin C and vitamin E. These findings suggest that cytoplasmic pro-caspase-3 is positioned near the lumenal surface of AEC where it is under the influence of Zn(2+) and other anti-oxidants.