Interleukin-5 enhances eosinophil adhesion to bronchial epithelial cells

BACKGROUND: Eosinophil-bronchial epithelial cell interactions are thought to be central to the pathogenesis of asthma, both in terms of the epithelium as a source of pro-inflammatory mediators and as a target for eosinophil-mediated damage. We have therefore investigated adhesion interactions between these two cell types. OBJECTIVES: To determine the role of eosinophil and epithelial activation on eosinophil adhesion to bronchial epithelium and to characterize the adhesion receptors mediating eosinophil adhesion. METHODS: Eosinophils were purified from human peripheral blood by immunomagnetic selection and adhesion to confluent cultures of the airway epithelial cell lines A549 and BEAS-2B was studied. RESULTS: Stimulation of A549 cells with TNFalpha, IFNgamma or a combination of 50 ng/mL of TNFalpha, IFNgamma and IL-1 (cytomix) did not effect eosinophil binding despite an increase in ICAM-1 expression. Similarly stimulation of eosinophils with PAF or IL-5 had no effect on eosinophil binding to medium- or cytokine-treated A549 cells. In contrast stimulation of BEAS-2B cells with cytomix caused a significant increase in eosinophil adhesion. This was associated with an increase in expression of ICAM-1 and induced expression of VCAM-1. Treatment of eosinophils with Mn2+ and IL-5 but not eotaxin, RANTES or PAF also significantly enhanced eosinophil adhesion to medium-treated BEAS-2B cells. Using blocking mAbs we were able to demonstrate that the increased adhesion resulting from stimulation of eosinophils or BEAS-2B cells was in both cases mediated by a combination of CD18 and alpha4 integrins. CONCLUSIONS: This study demonstrates a selective role for IL-5 in mediating integrin-dependent eosinophil adhesion to airway epithelium and once again emphasizes the importance of this cytokine in controlling eosinophil activation in diseases such as asthma.     

Diverse effects of eosinophil cationic granule proteins on IMR-32 nerve cell signaling and survival

Activated eosinophils release potentially toxic cationic granular proteins, including the major basic proteins (MBP) and eosinophil-derived neurotoxin (EDN). However, in inflammatory conditions including asthma and inflammatory bowel disease, localization of eosinophils to nerves is associated with nerve plasticity, specifically remodeling. In previous in vitro studies, we have shown that eosinophil adhesion to IMR-32 nerve cells, via nerve cell intercellular adhesion molecule-1, results in an adhesion-dependent release of granule proteins. We hypothesized that released eosinophil granule proteins may affect nerve cell signaling and survival, leading to nerve cell remodeling. Culture in serum-deprived media induced apoptosis in IMR-32 cells that was dose-dependently abolished by inclusion of MBP1 but not by EDN. Both MBP1 and EDN induced phosphorylation of Akt, but with divergent time courses and intensities, and survival was independent of Akt. MBP1 induced activation of neural nuclear factor (NF)-kappaB, from 10 min to 12 h, declining by 24 h, whereas EDN induced a short-lived activation of NF-kappaB. MBP1-induced protection was dependent on phosphorylation of ERK 1/2 and was related to a phospho-ERK-dependent upregulation of the NF-kappaB-activated anti-apoptotic gene, Bfl-1. This signaling pathway was not activated by EDN. Thus, MBP1 released from eosinophils at inflammatory sites may regulate peripheral nerve plasticity by inhibiting apoptosis.     

Regulation of the release of eosinophil cationic protein by eosinophil adhesion

BACKGROUND: Varying release of eosinophil granule proteins depending on the stimulus and environmental factors has previously been reported. OBJECTIVE: To investigate the degranulation from adherent eosinophils by using mixed granulocytes. METHODS: Granulocytes isolated by Percoll gradient centrifugation were incubated on plates coated with plasma and tissue fibronectin, fibrinogen or human serum albumin (HSA) and stimulated with Mn2+, phorbol-myristate-acetate (PMA), formyl-methionyl-leucyl-phenylalanine (f-MLP) and combinations thereof, respectively. The release of eosinophil cationic protein (ECP) was measured by radioimmunoassay. RESULTS: Unstimulated eosinophils incubated in wells coated with plasma and tissue fibronectin, fibrinogen or HSA did not release any ECP. Furthermore, Mn2+ (5 mmol/L) did not induce release of ECP despite the fact that adhesion of eosinophils to these four proteins was induced. PMA stimulated a dose-dependent release of ECP. Contemporaneous stimulation of eosinophils with PMA and Mn2+ induced a dramatically increased release of ECP regardless of which protein the eosinophils were adhering to. A small but significant release of ECP was found when eosinophils incubated on plates coated with fibrinogen and HSA were stimulated by f-MLP. Contemporaneous stimulation of eosinophils with f-MLP and Mn2+ did not induce any synergistic effect on the release of ECP. On the contrary, Mn2+ inhibited the release of ECP induced by f-MLP from eosinophils. Serum-opsonized Sephadex particles stimulated a potent increase of the release of ECP up to 12%-14% in the presence of plasma fibronectin and, in particular, fibrinogen. The kinetics of eosinophil adhesion and degranulation showed that the cellular adhesion preceded the degranulation response and that the degranulation patterns depend on the stimuli and environment. CONCLUSION: The present study indicated that cellular adhesion plays an important role in the regulation of eosinophil degranulation, but that adhesion and degranulation can be induced separately.     

Modest intracellular acidification suppresses death signaling in ouabain-treated cells

The signaling cascade resulting in the death of several types of cells treated with ouabain or other cardiotonic steroids (CTS) remains poorly understood. Recently, we observed that ouabain kills epithelial and endothelial cells via its interaction with Na⁺, K⁺ -ATPase, but independently of inhibition of Na⁺, K⁺ -ATPase-mediated ion fluxes and inversion of the [Na⁺]_i/[K⁺]_i ratio. Here, we report that the death of ouabain-treated epithelial cells from the Madin-Darby canine kidney (C7-MDCK) and endothelial cells from porcine aortae is suppressed by acidification of medium from pH 7.4 to 7.0, i.e. under conditions when pH_i was decreased from 7.2 to 6.9. The rescue of ouabain-treated C7-MDCK cells was also detected under selective intracellular acidification caused by inhibition of Na⁺/H⁺ exchanger. In these cells, neither Na⁺, K⁺ pump activity nor [³H]-ouabain binding was significantly affected by modest acidification. The death of ouabain-treated cells was independent of inhibition of RNA and protein synthesis with actinomycin D and cycloheximide. In contrast, both compounds sharply attenuated the protective action of acidified medium. Thus, our results show that very modest intracellular acidification is sufficient to inhibit the Na⁺ _i/K⁺ _i-independent death signal triggered in epithelial and endothelial cells by CTS. They also suggest that the protective action of acidification is mediated by de novo expression of genes involved in inhibition of the cell death machinery.     

Vascular cell adhesion molecule-1 and eosinophil adhesion to cultured human umbilical vein endothelial cells in vitro.

Cultured human umbilical vein endothelial cell (EC) monolayers stimulated with 10 ng/ml tumor necrosis factor demonstrate a time-dependent increase in the expression of the vascular cell adhesion molecule-1 (VCAM-1) with maintained maximal expression at 24 h following EC activation. A monoclonal antibody (mAb) directed against VCAM-1 (1G11) significantly inhibited the adhesion of eosinophils, but not neutrophils, to EC, which had been activated by tumor necrosis factor-alpha for 24 h, but only when eosinophils had been pretreated with an mAb directed against the common beta chain of the CD11/CD18 complex. In the absence of pretreatment with anti-CD18, mAb 1G11 had no significant effect on eosinophil adhesion. These results suggest that eosinophils bind to VCAM-1. However, the functional capacity in this model of the eosinophil receptor for VCAM-1 is likely to be minor compared with the activity of the CD11/CD18 leukocyte adhesion molecules.     

The promotion of eosinophil degranulation and adhesion to conjunctival epithelial cells by IgE-activated conjunctival mast cells.

BACKGROUND: Allergen-mediated mast cell activation is a key feature of ocular allergic diseases. Evidence of eosinophil-derived mediators in tears and conjunctival biopsy specimens has been associated with chronic ocular allergic inflammation. OBJECTIVE: To examine the role of conjunctival mast cell mediators in eosinophil adhesion to conjunctival epithelial cells and eosinophil degranulation. METHODS: Conjunctival cells were obtained by enzymatic digestion of cadaveric conjunctival tissues. Eosinophils were obtained from peripheral blood samples using negative magnetic bead selection. The effect of IgE-activated mast cell supernates on eosinophil degranulation and adherence to epithelial cells was compared with supernates obtained from mast cells pretreated with a degranulation inhibitor (olopatadine). Eosinophil adhesion was measured by eosinophil peroxidase assay, and eosinophil degranulation was measured by eosinophil-derived neurotoxin radioimmunoassay. RESULTS: IgE-activated conjunctival mast cell supernates stimulated adhesion of eosinophils to epithelial cells (20.4% +/- 6.3% vs 3.1% +/- 1.0%; P = .048). Degranulation was not required for this process (no effect of olopatadine). IgE-activated mast cell supernates stimulated eosinophil-derived neurotoxin release (108.89 +/- 8.27 ng/10(6) cells vs 79.45 +/- 5.21 ng/10(6) cells for controls, P = .02), which was significantly inhibited by pretreatment of mast cells with a degranulation inhibitor (79.22 +/- 4.33 ng/10(6) cells vs 61.09 +/- 5.39 ng/10(6) cells for olopatadine pretreated and untreated, respectively, P = .02). CONCLUSIONS: Mediators released from conjunctival mast cells promote eosinophil adhesion to conjunctival epithelial cells and eosinophil degranulation. Degranulation inhibition studies suggest that different mast cell mediators are involved in regulation of these events.     

Notch信号通路对脑缺血大鼠神经干细胞移植后神经再生的影响

目的 探讨抑制Notch信号通路促进脑缺血大鼠神经干细胞（NSCs）移植后神经再生的机制。方法 采用大脑中动脉阻塞MCAO）方法构建局灶性脑缺血模型,体外培养NSCs,移植入纹状体缺血区。将40只SD大鼠随机分为假手术组、模型组、移植组（移植神经干细胞）、氮-［氮-(3,5-二氟苯乙酰)-L-丙氨酰］-S-苯基甘氨酸丁酯］（DAPT）+移植组。采用HE染色观察各组大鼠神经元损伤程度,利用免疫组织化学、Western blotting检测各组大鼠脑组织中Notch1、Hes1及Hes5的表达情况。结果 与假手术组相比,模型组神经元损伤严重,出现核固缩和核溶解,Notch1、Hes1及Hes5阳性细胞表达明显增多,Notch1、Hes1及Hes5蛋白表达显著上调（P＜0.05）。与模型组相比,移植组和DAPT+移植组神经元损伤均不同程度缓解,Notch1、Hes1及Hes5阳性细胞均有部分表达,各项蛋白表达均有所降低（P＜0.05）;DAPT+移植组神经元损伤明显恢复,较移植组各项蛋白阳性细胞和蛋白表达进一步降低(P＜0.05)。结论 抑制Notch信号通路可促进脑缺血大鼠神经干细胞移植后的神经再生,其机制主要与下调Notch1、Hes1及Hes5的表达有关。     

Urokinase-type plasminogen activator modulates airway eosinophil adhesion in asthma

Eosinophils migrate from the vascular circulation to the inflamed airways during asthma exacerbations. While the mechanism(s) of this process is not known, the expression of urokinase-type plasminogen activator receptor (uPAR) has been found to modulate neutrophil adhesion and migration to inflammatory sites. We hypothesized that increased expression of uPAR and its ligand, uPA, enhance eosinophil adhesion in patients with asthma. Patients with allergic asthma underwent segmental bronchoprovocation with allergen; 48 h later, peripheral blood and airway (from bronchoalveolar lavage fluid) eosinophils were isolated. uPA and uPAR protein expression were measured by flow cytometry and Western blot; mRNA was quantified by real-time PCR. Eosinophil adhesion to intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 was assessed by eosinophil peroxidase activity. Airway eosinophils expressed significantly more uPA and uPAR protein and uPAR mRNA than peripheral blood eosinophils. Removal of cell-bound uPA and/or addition of exogenous uPA had no effect on blood eosinophil adhesion to ICAM-1 or VCAM-1. In contrast, exogenous uPA stimulated ICAM and VCAM adhesion of airway eosinophils. N-formyl-methionyl-leucyl-phenylalanine-activated airway eosinophil adherence to VCAM-1 and ICAM-1 (VCAM-1, 52.8 +/- 4.7%; ICAM-1, 49.2 +/- 5.3%) was increased over blood eosinophil adhesion (VCAM-1, 38.4 +/- 3.6%; ICAM-1, 27.7 +/- 4.9%; P < 0.05). Removal of cell-bound uPA from airway eosinophils decreased adhesion to blood cell levels; reintroduction of exogenous uPA completely restored adhesion levels. These data suggest that constitutive uPA primes, and exogenous uPA can activate, airway eosinophil adhesion following segmental allergen challenge and that increased uPA expression may be a mechanism of increased eosinophil infiltration and function in asthma.     

Heterogeneity of phasic cholinergic signaling in neocortical neurons

Acetylcholine (ACh) is a neurotransmitter critical for normal cognition. Here we demonstrate heterogeneity of cholinergic signaling in neocortical neurons in the rat prefrontal, somatosensory, and visual cortex. Focal ACh application (100 muM) inhibited layer 5 pyramidal neurons in all cortical areas via activation of an apamin-sensitive SK-type calcium-activated potassium conductance. Cholinergic inhibition was most robust in prefrontal layer 5 neurons, where it relies on the same signal transduction mechanism (M1-like receptors, IP(3)-dependent calcium release, and SK-channels) as exists in somatosensory pyramidal neurons. Pyramidal neurons in layer 2/3 were less responsive to ACh, but substantial apamin-sensitive inhibitory responses occurred in deep layer 3 neurons of the visual cortex. ACh was only inhibitory when presented near the somata of layer 5 pyramidal neurons, where repetitive ACh applications generated discrete inhibitory events at frequencies of up to approximately 0.5 Hz. Fast-spiking (FS) nonpyramidal neurons in all cortical areas were unresponsive to ACh. When applied to non-FS interneurons in layers 2/3 and 5, ACh generated mecamylamine-sensitive nicotinic responses (38% of cells), apamin-insensitive hyperpolarizing responses, with or without initial nicotinic depolarization (7% of neurons), or no response at all (55% of cells). Responses in interneurons were similar across cortical layers and regions but were correlated with cellular physiology and the expression of biochemical markers associated with different classes of nonpyramidal neurons. Finally, ACh generated nicotinic responses in all layer 1 neurons tested. These data demonstrate that phasic cholinergic input can directly inhibit projection neurons throughout the cortex while sculpting intracortical processing, especially in superficial layers.     

Apocynin derivatives interrupt intracellular signaling resulting in decreased migration in breast cancer cells

Cancer cells are defined by their ability to divide uncontrollably and metastasize to secondary sites in the body. Consequently, tumor cell migration represents a promising target for anticancer drug development. Using our high-throughput cell migration assay, we have screened several classes of compounds for noncytotoxic tumor cell migration inhibiting activity. One such compound, apocynin (4-acetovanillone), is oxidized by peroxidases to yield a variety of oligophenolic and quinone-type compounds that are recognized inhibitors of NADPH oxidase and may be inhibitors of the small G protein Rac1 that controls cell migration. We report here that while apocynin itself is not effective, apocynin derivatives inhibit migration of the breast cancer cell line MDA-MB-435 at subtoxic concentrations; the migration of nonmalignant MCF10A breast cells is unaffected. These compounds also cause a significant rearrangement of the actin cytoskeleton, cell rounding, and decreased levels of active Rac1 and its related G protein Cdc42. These results may suggest a promising new route to the development of novel anticancer therapeutics.     

The endoplasmic reticulum and mitochondria as elements of the mechanism of intracellular signaling in the nerve cell

Experimental data obtained in our laboratory from studies of intracellular signals arising within nerve cells during excitation are summarized. Measurements of transmembrane ion currents in conditions of fixed membrane potential and intracellular free Ca ion concentrations, using fluorescent probes, yielded the time and spatial characteristics of transient elevations in the Ca concentration (the “calcium signal”) in various types of mouse and rat neurons. These studies showed that intracellular structures—the endoplasmic reticulum and mitochondria—had significant roles in forming these signals; these structures can take up Ca from the cytosol and liberate Ca into the cytosol; the contribution of these processes was extremely variable, depending on the internal organization of different functional types of neurons. Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 84, No. 10, pp. 979–984, October, 1998.     

Effect of rotavirus infection on intracellular calcium homeostasis in cultured cells.

The effect of rotavirus infection on intracellular [Ca2+] was studied in a model system (MA-104 cells). In cells infected at high multiplicity with the OSU strain of rotavirus, production of infectious viruses was maximal at 6 hr postinfection. Cell death, as measured by incorporation of ethidium bromide, started at 6 hr and was complete at 15 hr postinfection. At 4 hr postinfection, intracellular [Ca2+], measured by quin2 fluorescence, was not modified, but Ca2+ permeability was increased. With progression of the infection, intracellular [Ca2+] and Ca2+ pools increased due to the failure of regulatory mechanisms to compensate increased Ca2+ entry. These effects were blocked by cycloheximide added up to 5 hr postinfection, but not by actinomycin D. Reduced extracellular [Ca2+] afforded protection of cell death induced by infection, under conditions at which production of infectious viruses was not affected. The cytopathic effect of rotavirus on host cells appears to be mediated by an increase in intracellular [Ca2+] induced by the synthesis of a viral product. The failure of ionic homeostasis of the enterocyte might be involved in the development of diarrhea.     

钙动员诱导人外周血单核细胞分化为树突状细胞的信号转导

目的初步探讨钙离子载体（CI）在体外迅速诱导人外周血单核细胞（PBMC）分化为树突状细胞（DC）的信号转导途径。方法分离健康献血者的PBMC,在体外用人重组粒／巨噬细胞集落刺激因子（rhGM-CSF）和CI培养40h或rhGM-CSF和TNF-α培养5d,部分PBMC用环胞菌素A（CsA）预处理30min后,再加入CI或TNF-α;相差显微镜下观察细胞的形态;流式细胞仪检测细胞表面CD14、CD80、CD86、CD83、HLS-DR等分子的表达;MTT比色法检测其对同种异体T淋巴细胞的刺激增殖作用;凝胶电泳迁移率变动分析（EMSA）检测不同方法培养的细胞其核转录因子-κB（NF-κB）的活化水平。结果健康献血者的PBMC经rhGM-CSF与CI培养40h或rhGM-CSF与TNF-α培养5d,均可获得DC的典型形态和表面分子的表达,包括CD14表达下调,CD80、CD86及HLA-DR等分子表达的上调,以及较强刺激同种异体T淋巴细胞增殖的作用;其中CI诱导的DC其CD80、CD86、CD83、HLA-DR等分子的上调更明显,刺激T淋巴细胞的增殖能力更强。经TNF-α及CI所诱导分化的DC均具有较好的NF-κB活性。但经CI诱导的DC,其形态、表面标志物、对T淋巴细胞的刺激增殖能力及NF-κB的活性,均受到CsA的抑制;而TNF-α所诱导的DC却不受CsA的影响。结论CI比TNF-α更迅速、更高效地诱导PBMC向DC分化的原因,是信号转导途径的不同,但不论上游信号转导途径有何不同,两者最终都通过激活NF-κB诱导细胞的分化。