Acetylcholine and substance P stimulate bronchial epithelial cells to release eosinophil chemotactic activity

Weinvestigated a role of neuroregulation in the release of eosinophilchemotactic activity (ECA) from bovine bronchial epithelial cells(BBEC). BBEC were stimulated with acetylcholine (ACh) and substance P(SP), and the supernatant fluids were tested for ECA by a blind-wellchemotactic chamber technique. BBEC released ECA in response to ACh andSP in a dose- and time-dependent manner. Checkerboard analysis showedthat ECA in regard to ACh and SP was chemotactic rather thanchemokinetic. Partial characterization revealed that ECA involved bothlipids and peptides. The release of ECA in response to ACh and SP wasinhibited by nonspecific and 5-specific lipoxygenase inhibitors and bycycloheximide (P < 0.01). Molecular-sieve columnchromatography revealed that these mediators induced three molecularmass peaks (near 25 kDa, 9 kDa, and 400 Da, respectively). The lowestpeak, which represented the predominant activity, was blocked byleukotriene B₄-receptor antagonist (P < 0.01) but not by platelet-activating factor-receptor antagonist. The releaseof leukotriene B₄ in the supernatant fluids was increased in response to ACh and SP stimulation (P < 0.01).Platelet-activating factor was not detected. These results raise thepossibility of a role of neuroregulation for the elaboration of ECA inthe airway.

     

Outer membrane protein A from Klebsiella pneumoniae activates bronchial epithelial cells: implication in neutrophil recruitment

Aside from its mechanical barrier function, bronchial epithelium plays an important role both in the host defense and in the pathogenesis of inflammatory airway disorders. To investigate its role in lung defense, the effect of a bacterial cell wall protein, the outer membrane protein A from Klebsiella pneumoniae (kpOmpA) on bronchial epithelial cells (BEC) was evaluated on adhesion molecule expression and cytokine production. Moreover, the potential implication of this mechanism in kpOmpA-induced lung inflammation was also determined. Our in vitro studies demonstrated that kpOmpA strongly bound to BEAS-2B cells, a human BEC line, and to BEC primary cultures, resulting in NF-kappaB signaling pathway activation. Exposure to kpOmpA increased ICAM-1 mRNA and cell surface expression, as well as the secretion of IL-6, CXC chemokine ligand (CXCL)1, CXCL8, C-C chemokine ligand 2, CXCL10 by BEAS-2B cells, and BEC primary cultures (p < 0.005). We analyzed in vivo the consequences of intratracheal injection of kpOmpA to BALB/c mice. In kpOmpA-treated mice, a transient neutrophilia (with a maximum at 24 h) was observed in bronchoalveolar lavage and lung sections. In vivo kpOmpA priming induced bronchial epithelium activation as evaluated by ICAM-1 and CXCL1 expression, associated with the secretion of CXCL1 and CXCL5 in bronchoalveolar lavage fluids. In the lung, an increased level of the IL-6, CXCL1, CXCL5, CXCL10 mRNA was observed with a maximum at 6 h. These data showed that kpOmpA is involved in host defense mechanism by its ability to activate not only APC but also BEC, resulting in a lung neutrophilia.     

Regulation of SLPI and elafin release from bronchial epithelial cells by neutrophil defensins

Secretory leukocyte proteinase inhibitor (SLPI) is a serine proteinase inhibitor that is produced locally in the lung by cells of the submucosal bronchial glands and by nonciliated epithelial cells. Its main function appears to be the inhibition of neutrophil elastase (NE). Recently, NE was found to enhance SLPI mRNA levels while decreasing SLPI protein release in airway epithelial cells. Furthermore, glucocorticoids were shown to increase both constitutive and NE-induced SLPI mRNA levels. In addition to NE, stimulated neutrophils also release alpha-defensins. Defensins are small, antimicrobial polypeptides that are found in high concentrations in purulent secretions of patients with chronic airway inflammation. Like NE, defensins induce interleukin-8 production in airway epithelial cells. This induction is sensitive to inhibition by the glucocorticoid dexamethasone and is prevented in the presence of alpha(1)-proteinase inhibitor. The aim of the present study was to investigate the effect of defensins on the production of SLPI and the related NE inhibitor elafin/SKALP in primary bronchial epithelial cells (PBECs). Defensins significantly increase SLPI protein release by PBECs in a time- and dose-dependent fashion without affecting SLPI mRNA synthesis. In the presence of alpha(1)-proteinase inhibitor, the defensin-induced SLPI protein release is further enhanced, but no effect was observed on SLPI mRNA levels. Dexamethasone did not affect SLPI protein release from control or defensin-treated PBECs. In addition, we observed a constitutive release of elafin/SKALP by PBECs, but this was not affected by defensins. The present results suggest a role for defensins in the dynamic regulation of the antiproteinase screen in the lung at sites of inflammation.     

Neutrophil chemotactic factors promote leukocytosis. A common mechanism for cellular recruitment from bone marrow.

We investigated cellular responses in a rabbit to i.v. administration of five established chemotactic factors (leukotriene B4 (LTB4), platelet-activating factor (PAF), C5a, N-Formyl-Met-Leu-Phe (F-MLF), and IL-8), and each exerted a characteristic effect on circulating white blood cell levels. All five factors induced a rapid and transient leukopenia. The blood was nearly devoid of circulating neutrophils 5 min after administration of each chemotactic factor. Other leukocytes were also variably depleted during the leukopenic phase, including eosinophils, basophils, monocytes, and lymphocytes. The lymphocyte numbers remained significantly depressed (approximately 30%) for as long as 3 h after administration of PAF or f-MLF. Each chemotactic factor produced a marked neutrophilia (i.e., 250-400% of baseline levels) after the initial leukopenia. Eosinophil numbers were elevated along with the neutrophil response in the C5a- and LTB4-treated animals. Basophil levels were significantly elevated only in LTB4-treated animals. The cellular response to PAF, f-MLF, and IL-8 appeared to be specific for the neutrophils. The kinetic profiles of the neutrophilia induced by PAF (10 micrograms/kg) or f-MLF (2.5 micrograms/kg) were similar, with maximal responses occurring 3 to 4 h after administration. In contrast, LTB4 (10 micrograms/kg), IL-8 (2.5 micrograms/kg), and C5a (5 micrograms/kg) induced a more rapid neutrophilia, with peak responses occurring 1 to 1.5 h after injection, and remaining elevated for 3 to 4 h. In all animals the neutrophilia was accompanied by a relative increase in the number of nonsegmented neutrophils (bands), suggesting that a major component of leukocytosis is caused by the release of bone marrow reserves. Phenidone (10 mg/kg), a dual cyclooxygenase/5-lipoxygenase inhibitor, affected neither the neutropenia nor the neutrophilia induced by C5a, f-MLF, or PAF. The protein synthesis inhibitor actinomycin D also failed to suppress neutrophil responses induced by either C5a or PAF. These results suggest that leukocytosis is a common response induced by all neutrophil chemotactic factors. Leukocytosis appears to be a direct result of the dynamic adaptive response of neutrophils to chemotactic factor stimulation without involvement of a secondary mediator system.     

Viral inhibition of IL-1- and neutrophil elastase-induced inflammatory responses in bronchial epithelial cells

Previously, we elucidated the intracellular mechanisms by which neutrophil elastase (NE) up-regulates inflammatory gene expression in bronchial epithelial cells. In this study, we examine the effects of both IL-1 and NE on inflammatory gene expression in 16HBE14o- bronchial epithelial cells and investigate approaches to abrogate these inflammatory responses. IL-1 induced IL-8 protein production in time- and dose-dependent fashions, an important observation given that IL-8 is a potent neutrophil chemoattractant and a key inflammatory mediator. IL-1 and NE were shown to activate the p38 MAPK pathway in 16HBE14o- cells. Western blot analysis demonstrated IL-1R-associated kinase 1 (IRAK-1) degradation in response to stimulation with both IL-1 and NE. In addition, the expression of dominant negative IRAK-1 (IRAK-1delta), IRAK-2delta, or IRAK-4delta inhibited IL-1- and NE-induced NF-kappaB-linked reporter gene expression. Dominant negative versions of the intracellular adaptor proteins MyD88 (MyD88delta) and MyD88 adaptor-like (Mal P/H) abrogated NE-induced NF-kappaB reporter gene expression. In contrast, only MyD88delta was found to inhibit IL-1-induced NF-kappaB reporter activity. We also investigated the vaccinia virus proteins, A46R and A52R, which have been shown to antagonize IL-1 signaling. Transfection with A46R or A52R cDNA inhibited IL-1- and NE-induced NF-kappaB and IL-8R gene expression and IL-8 protein production in primary and transformed bronchial epithelial cells. Furthermore, cytokine array studies demonstrated that IL-1 and NE can up-regulate the expression of IL-6, oncostatin M, epithelial cell-derived neutrophil activating peptide-78, growth-related oncogene family members, vascular endothelial growth factor, and GM-CSF, with induction of these proteins inhibited by the viral proteins. These findings identify vaccinia virus proteins as possible therapeutic agents for the manifestations of several inflammatory lung diseases.     

Bronchial epithelial cells release monocyte chemotactic activity in response to smoke and endotoxin

An increase in mononuclear phagocytes occurs within the airways during airway inflammation. Bronchial epithelial cells could release monocyte chemotactic activity and contribute to this increase. To test this hypothesis, bovine bronchial epithelial cells were isolated and maintained in culture. Bronchial epithelial cell culture supernatant fluids were evaluated for monocyte chemotactic activity. Epithelial cell culture supernatant fluids attracted significantly greater numbers of monocytes compared to media alone and the number of monocytes attracted increased in a time dependent manner. Endotoxin and smoke extract induced a dose and time dependent release of monocyte chemotactic activity compared with cells cultured in media (52.5 +/- 2.6 (endotoxin), 30.5 +/- 2.3 (smoke) vs 20.5 +/- 2.2 cells/high power field (HPF) p less than 0.001). The released activity was chemotactic by checkerboard analysis. Stimulation of the epithelial cells by opsonized zymosan, calcium ionophore (A23187), and PMA also resulted in an increase in monocyte chemotactic activity (p less than 0.01). Because the release of activity was blocked by the lipoxygenase inhibitors, nordihydroguaiaretic acid and diethycarbamazine, epithelial cell monolayers were cultured with 3 microCi [3H]arachidonic acid for 24 h and then exposed to A23187, PMA, or both stimuli, for 4, 8, and 24 h. Analysis of the released 3H activity was performed with reverse-phase HPLC and revealed that the major lipoxygenase product was leukotriene B4. These data suggest that monocytes may be recruited into airways in response to chemotactic factors released by bronchial epithelial cells.     

Hog barn dust extract stimulates IL-8 and IL-6 release in human bronchial epithelial cells via PKC activation.

Hog barn workers have an increased incidence of respiratory tract symptoms and demonstrate an increase in lung inflammatory mediators, including interleukin (IL)-8 and IL-6. Utilizing direct kinase assays for protein kinase C (PKC) activation, we demonstrated that dust from hog confinement facilities, or hog dust extract (HDE), augments PKC activity of human airway epithelial cells in vitro. A 5% dilution of HDE typically stimulates an approximately twofold increase in human bronchial epithelial cell (HBEC) PKC activity compared with control medium-treated cells. This increase in PKC is observed with 15 min of HDE treatment, and kinase activity reaches peak activity by 1-2 h of HDE treatment before returning to baseline PKC levels between 6 and 24 h. The classic PKC inhibitor, calphostin C, blocks HDE-stimulated PKC activity and associated IL-8 and IL-6 release. Desensitization to HDE stimulation of PKC activation does not appear to occur because subsequent exposures to HDE after an initial exposure result in further augmentation of PKC. Detoxification of HDE with polymyxin B to remove endotoxin did not change PKC activation or IL-8 release, suggesting that endotoxin is not solely responsible for HDE augmentation of PKC. These data support the hypothesis that HDE exposure augments HBEC IL-8 and IL-6 release via a PKC-dependent pathway.     

Bradykinin stimulates Ca2+ mobilization in NCB-20 cells leading to direct inhibition of adenylylcyclase. A novel mechanism for inhibition of cAMP production.

The modulation of neuronal adenylylcyclase by Ca2+, acting via calmodulin, is a long-established example of a positive interaction between the Ca2(+)-mobilizing and cAMP-generating systems. In the present study, concentrations of Ca2+ that stimulate brain adenylylcyclase inhibit the adenylylcyclase of NCB-20 plasma membranes. These inhibitory effects of Ca2+ have been characterized and seem to be exerted at the catalytic unit of the enzyme; they are independent of calmodulin, Gi, and phosphodiesterase. To determine whether this inhibition of adenylylcyclase by Ca2+ could occur in the intact cell, cAMP accumulation was measured in response to bradykinin. Bradykinin, which mobilizes Ca2+ in NCB-20 cells, as a consequence of stimulating inositol phosphate production, causes a transient inhibition of prostaglandin E1 stimulation of cAMP accumulation. The inhibitory action of bradykinin is attenuated significantly by treatment of cells with the cell-permeant Ca2+ chelator, 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid. It seems likely that the inhibition of adenylylcyclase by low concentrations of Ca2+ represents a novel means for a negative interaction between Ca2(+)-mobilizing and cAMP-generating systems.     

Effects of bile acids on biliary epithelial cells: proliferation,cytotoxicity, and cytokine secretion

Hydrophobic bile acids, which are known to be cytotoxic for hepatocytes, are retained in high amount in the liver during cholestasis. Thus, we have investigated the effects of bile acids with various hydrophobicities on biliary epithelial cells. Biliary epithelial cells were cultured in the presence of tauroursodeoxycholate (TUDC), taurocholate (TC), taurodeoxycholate (TDC), taurochenodeoxycholate (TCDC), or taurolithocholate (TLC). Cell proliferation, viability, apoptosis and secretion of monocyte chemotactic protein-1 (MCP-1) and of interleukin-6 (IL-6) were studied. Cell proliferation was increased by TDC, and markedly decreased by TLC in a dose dependent manner (50-500 microM). Cell viability was significantly decreased by TLC and TCDC at 500 microM. TLC, TDC and TCDC induced apoptosis at high concentrations. The secretion of MCP-1 and IL-6 was markedly stimulated by TC. TUDC had no significant effect on any parameter. These findings demonstrate that hydrophobic bile acids were cytotoxic and induced apoptosis of biliary epithelial cells. Furthermore, TC, a major biliary acid in human bile, stimulated secretion of cytokines involved in the inflammatory and fibrotic processes occurring during cholestatic liver diseases.     

Peroxynitrite stimulates L-arginine transport system y(+) in glial cells. A potential mechanism for replenishing neuronal L-arginine

We have reported previously that peroxynitrite stimulates L-arginine release from astrocytes, but the mechanism responsible for such an effect remains elusive. To explore this issue, we studied the regulation of L-[(3)H]arginine transport by either exogenous or endogenous peroxynitrite in glial cells. A 2-fold peroxynitrite-mediated stimulation of l-arginine release in C6 cells was found to be Na(+)-independent, was prevented by 5 mm L-arginine and, although only in the presence of Na(+), was blocked by 5 mm L-alanine or L-leucine. Peroxynitrite-mediated stimulation of L-arginine uptake was trans-stimulated by 10 mm L-arginine and was inhibited in a dose-dependent fashion (k(i) of approximately 40 microm) by the system y(+) inhibitor N-ethylmaleimide in C6 cells. Endogenous production of peroxynitrite in lipopolysaccharide-treated astrocytes triggered an increased L-arginine transport activity without affecting Cat1 l-arginine transporter mRNA levels. However, Western blot analyses of peroxynitrite-treated astrocytes and C6 glial cells revealed a 3-nitrotyrosinated anti-Cat1-immunopositive band, strongly suggesting peroxynitrite-mediated Cat1 nitration. Furthermore, peroxynitrite stimulation of L-arginine release was abolished in fibroblast cells homozygous for a targeted inactivation of the Cat1 gene. Finally, peroxynitrite-triggered L-arginine released from astrocytes was efficiently taken up by neurons in an insert-based co-culture system. These results strongly suggest that peroxynitrite-mediated activation of the Cat1 transporter in glial cells may serve as a mechanism focused to replenish L-arginine in the neighboring neurons.     

Kallikrein stimulates arachidonic acid release and production of prostaglandins from TEA3A1 endocrine thymic epithelial cells.

Using TEA3A1 rat endocrine thymic epithelial cells, we demonstrated that kallikrein (EC 3.4.21.35) not only stimulated the release of arachidonic acid (AA) and its metabolites from TEA3A1 cells but also enhanced the intracellular synthesis of prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) by approx. 2-fold. The stimulatory effect of kallikrein was dose- and time-dependent and could be blocked by aprotinin, a kallikrein inhibitor. It was found that the phospholipase A inhibitors ONO RS082 [2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid], and mepacrine (6-chloro-9-[(4-dimethylamino)-1-methyl)]amino-2-methoxyacridine; quinacrine) also inhibited the kallikrein-stimulated release of AA and its metabolites. It is suggested that the kallikrein-induced stimulatory effect might be mediated through a phospholipase A2 pathway. The effect of bradykinin was studied and no significant stimulation was observed, even at a high dose (10 micrograms/ml). This suggested that the formation of kinin does not have a role in the kallikrein-induced stimulation of AA release from TEA3A1 cells. Furthermore, the effect of kallikrein was also totally abolished by adding pepstatin A, a known inhibitor of renin, pepsin and cathepsin D which does not inhibit kallikrein itself. This indicates that kallikrein did not act on the phospholipase-like enzyme directly. There is at least one more enzyme, a pepstatin A-inhibitable proteinase, that acts as a mediator for kallikrein-induced regulation of AA release.     

Relationship of ornithine decarboxylase activity and cAMP metabolism to proliferation of normal human bronchial epithelial cells

The mechanisms of action of extracellular mitogens for normal human bronchial epithelial cells (NHBE) were investigated by observing their effects on selected biochemical pathways when the cells were incubated in serum-free media. We find that (a) epidermal growth factor (EGF) stimulates ornithine decarboxylase (ODC) activity and the rate of cell division without stimulating cAMP; (b) alone, pituitary extract (PEX) does not stimulate ODC activity, cAMP levels, or cell division; (c) when PEX is added to medium containing EGF there is a further increase in both ODC activity and the rate of cell division, again with no increase in cAMP levels; (d) in contrast, alone, L-epinephrine (EPI) stimulates an increase in both ODC and cAMP but does not stimulate cell division; (e) when EPI is added to medium containing both EGF and PEX a further increase in the rate of cell division is noted; (f) the specific inhibitor of ODC, alpha-(difluoromethyl)-ornithine (DMFO), also inhibits NHBE cell proliferation; and (g) the beta-adrenergic receptor antagonist propranolol inhibits the mitogenic action and ODC induction by EPI observed under condition e. We conclude that an increase in ODC activity is necessary but not sufficient for an increase in proliferation of NHBE cells. In contrast, cAMP stimulation is not necessary for an increase in NHBE cell division. However, in the presence of undefined factors in PEX, increases in cAMP levels result in a synergistic increase in the rate of EGF-stimulated clonal growth. By correlating the biochemical pathways invoked by EGF, PEX, EPI, and combinations thereof with their mitogenic actions, we have better defined the role each of these different mitogens plays in stimulating epithelial cell division.     

Crystallization of human interleukin-8. A protein chemotactic for neutrophils and T-lymphocytes

Interleukin-8 is a 72-residue peptide which is chemotactic for neutrophils and T-lymphocytes. It crystallizes in space group P3(1)21 or P3(2)21 with unit cell dimensions of a = b = 40.3 and c = 90.1 A. X-ray diffraction data have been measured to 1.6-A resolution.     

Simvastatin attenuates release of neutrophilic and remodeling factors from primary bronchial epithelial cells derived from stable lung transplant recipients

Obliterative bronchiolitis (OB), the major cause of chronic lung allograft dysfunction, is characterized by airway neutrophilia, inflammation, and remodeling, with progressive fibroproliferation and obliteration of small airways that ultimately leads to patient death. Statins have potential anti-inflammatory effects and have been demonstrated to confer a survival advantage in lung transplant patients. We postulated that the beneficial effects of simvastatin in lung transplantation are in part due to inhibition of the epithelial production of key mediators of neutrophil chemotaxis, inflammation, and airway remodeling. Our objective was to assess the effect of simvastatin on a unique population of primary bronchial epithelial cells (PBECs) derived from stable lung allografts, with specific reference to airway neutrophilia and remodeling. PBEC cultures were stimulated with IL-17 or transforming growth factor (TGF)-beta, with and without simvastatin. Supernatant levels of factors critical to driving airway neutrophilia and remodeling were measured. IL-17 upregulated IL-8, IL-6, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), and VEGF, whereas TGF-beta increased IL-6, GM-CSF, matrix metalloproteinase (MMP)-2, and MMP-9. Simvastatin attenuated effects of both IL-17 and TGF-beta. We have demonstrated the ability of simvastatin to attenuate release of airway neutrophilic and remodeling mediators and to inhibit their upregulation by TGF-beta and IL-17. These data illustrate the potential of simvastatin to alleviate neutrophilic airway inflammation and remodeling in the transplanted lung and may have additional relevance to other neutrophilic airway conditions, such as chronic obstructive pulmonary disease.     

Nerve growth factor stimulates proliferation, adhesion and thymopoietic cytokine expression in mouse thymic epithelial cells in vitro

Thymic epithelial cells, which constitute a major component of the thymic microenvironment, provide a crucial signal for intrathymic T cell development and selection. Neuroimmune networks in the thymic microenvironment are thought to be involved in the regulation of T cell development. NGF is increasingly recognized as a potent immunomodulator, promoting “cross-talk” between various types of immune system cells. The present study clearly shows that NGF stimulates mouse thymic epithelial cell activities in vitro including cell proliferation, thymocyte adhesion to thymic epithelial cells, and the expression of cell adhesion molecules such as ICAM-1 and VCAM-1, and thymopoietic factors including IL-7, GM-CSF, SDF-1, TARC and TECK. Thus, our data are of considerable clinical importance showing that trophic NGF activity could be used to enhance the thymus regeneration and develop methods to improve host immunity when the immune function is depressed due to thymic involution.     

An in vitro test for measuring cytotoxicity of mercuric chloride to human kidney epithelial cells by specific enzyme release

Mercuric compound toxicity is well documented in animals and man for practically all organs. The recent development of cell culture techniques appeared as a novel fruitful tool in toxicology, especially in renal toxicology. Heavy metal induced renal cell alterations can be evaluated by membrane permeability damages.The present study evaluates mercuric chloride nephrotoxic effect in human kidney epithelial cells by measuring the release of two specific nephrotoxicity marker enzymes, Gamma Glutamyl Transferase (GGT) and Alkaline Phosphatase (ALP) in the culture medium. Cultured kidney epithelial cells were exposed to different HgCl₂ concentrations (5, 10, 20, and 50 g). Cultures were examined after 6 and 24 hours exposure. A good correlation between mercury dose and toxic effect, and exposure time and toxic effect was found. Enzymes were significantly released into the culture medium for 5 g and 10 g HgCl₂/ml after 6 hours exposure; and after 24 hours exposure, enzymes were released for 5 g/ml only.It appears that the specific tubular enzyme release in the culture medium is a good in vitro test for quantification of specific tubular damage.