Iris F. Ueki

Oxidative Stress Causes Mucin Synthesis Via Transactivation of Epidermal Growth Factor Receptor: Role of Neutrophils

Oxidative stress has been implicated in the pathogenesis of inflammatory diseases of airways. Here we show that oxidative stress causes ligand-independent activation of epidermal growth factor receptors (EGFR) and subsequent activation of mitogen-activated protein kinase kinase (MEK)-p44/42 mitogen-activated protein kinase (p44/42mapk), resulting in mucin synthesis in NCI-H292 cells. Exogenous hydrogen peroxide and neutrophils activated by IL-8, FMLP, or TNF-α increased EGFR tyrosine phosphorylation and subsequent activation of p44/42mapk and up-regulated the expression of MUC5AC at both mRNA and protein levels in NCI-H292 cells. These effects were blocked by selective EGFR tyrosine kinase inhibitors (AG1478, BIBX1522) and by a selective MEK inhibitor (PD98059), whereas a selective platelet-derived growth factor receptor tyrosine kinase inhibitor (AG1295), a selective p38 MAPK inhibitor (SB203580), and a negative compound of tyrosine kinase inhibitors (A1) were without effect. Neutrophil supernatant-induced EGFR tyrosine phosphorylation, activation of p44/42mapk, and MUC5AC synthesis were inhibited by antioxidants (N-acetyl-l-cysteine, DMSO, dimethyl thiourea, or superoxide dismutase); neutralizing Abs to EGFR ligands (EGF and TGF-α) were without effect, and no TGF-α protein was found in the neutrophil supernatant. In contrast, the EGFR ligand, TGF-α, increased EGFR tyrosine phosphorylation, activation of p44/42mapk, and subsequent MUC5AC synthesis, but these effects were not inhibited by antioxidants. These results implicate oxidative stress in stimulating mucin synthesis in airways and provide new therapeutic approaches in airway hypersecretory diseases.

Permselectivity of the glomerular capillary wall to macromolecules. II. Experimental studies in rats using neutral dextran

To determine the permselectivity characteristics of the glomerular capillary wall, known molecular size fractions of [3H]dextran, prepared by gel chromatography, were infused into normally hydrated Wistar rats, thus permitting simultaneous measurement of Bowmans space/plasma water (BS/P) and urine/plasma water (U/P) concentration ratios, along with glomerular pressures and flows. Since (BS/P)inulin = 1.01 +/- 0.01 SE(n = 34, radius = approximately 14 A) and since (BS/P)dextran/(BS/P)inulin equaled (U/P)dextran/(U/P)inulin for dextrans ranging in molecular radius from 21 to 35 A, these findings validate that dextrans are neither secreted nor reabsorbed. For dextran radii of 20, 24, 28, 32, 36, 40, and 44 A, (U/P)dextran/(U/P)inulin averaged 0.99, 0.92, 0.69, 0.42, 0.19, 0.06, and 0.01, respectively. In accord with theoretical predictions that these fractional dextran clearances should vary appreciably with changes in glomerular transcapillary pressures and flows, an increase in glomerular plasma flow rate, induced in these same rats by plasma volume expansion, resulted in a highly significant lowering of fractional clearance of all but the smallest and largest dextrans studied. These findings emphasize that fractional solute clearances alone are inadequate to describe the permselective properties of the glomerular capillary wall unless glomerular pressures and flows are also known. This sensitivity of fractional dextran clearance to changes in plasma flow indicates that dextrans are transported across the capillary not only by bulk flow but also to an important extent by diffusion.

Multiple TLRs activate EGFR via a signaling cascade to produce innate immune responses in airway epithelium.

Toll-like receptors (TLRs) are critical for the recognition of inhaled pathogens that deposit on the airway epithelial surface. The epithelial response to pathogens includes signaling cascades that activate the EGF receptor (EGFR). We hypothesized that TLRs communicate with EGFR via epithelial signaling to produce certain innate immune responses. Airway epithelium expresses the highest levels of TLR2, TLR3, TLR5, and TLR6, and here we found that ligands for these TLRs increased IL-8 and VEGF production in normal human bronchial epithelial cells. These effects were prevented by treatment with a selective inhibitor of EGFR phosphorylation (AG-1478), a metalloprotease (MP) inhibitor, a reactive oxygen species (ROS) scavenger, and an NADPH oxidase inhibitor. In an airway epithelial cell line (NCI-H292), TNF-alpha-converting enzyme (TACE) small interfering RNA (siRNA) was used to confirm that TACE is the MP involved in TLR ligand-induced IL-8 and VEGF production. We show that transforming growth factor (TGF)-alpha is the EGFR ligand in this signaling cascade by using TGF-alpha neutralizing antibody and by showing that epithelial production of TGF-alpha occurs in response to TLR ligands. Dual oxidase 1 (Duox1) siRNA was used to confirm that Duox1 is the NADPH oxidase involved in TLR ligand-induced IL-8 and VEGF production. We conclude that multiple TLR ligands induce airway epithelial cell production of IL-8 and VEGF via a Duox1--> ROS--> TACE--> TGF-alpha--> EGFR phosphorylation pathway. These results show for the first time that multiple TLRs in airway epithelial cells produce innate immune responses by activating EGFR via an epithelial cell signaling cascade.

Tumor necrosis factor α-converting enzyme mediates MUC5AC mucin expression in cultured human airway epithelial cells

Ectodomain shedding of epidermal growth factor receptor (EGFR) ligands [e.g., transforming growth factor type α (TGF-α)] and EGFR phosphorylation are implicated in mucin production in airway epithelial cells. Tumor necrosis factor α-converting enzyme (TACE) is reported to cleave precursor of TGF-α, with release of soluble mature TGF-α in various epithelial tissues. We hypothesized that TACE increases the shedding of TGF-α, resulting in EGFR phosphorylation and inducing mucin production in human airway epithelial (NCI-H292) cells. To examine this hypothesis, we stimulated NCI-H292 cells with phorbol 12-myristate 13-acetate (PMA, an activator of TACE) and pathophysiologic stimuli [lipopolysaccharide (LPS) and supernatant from the Gram-negative bacterium Pseudomonas aeruginosa (PA sup)]. PMA, PA sup, and LPS increased MUC5AC gene expression and mucin protein production, effects that were prevented by pretreatment with AG1478, a selective inhibitor of EGFR phosphorylation and by preincubation with an EGFR-neutralizing Ab or with a TGF-α-neutralizing Ab, implicating ligand (TGF-α)-dependent EGFR phosphorylation in mucin production. These stimuli induced release of soluble TGF-α, EGFR phosphorylation, and MUC5AC expression, which were blocked by the metalloprotease inhibitors tumor necrosis factor-α protease inhibitor-1 and tissue inhibitor of metalloprotease-3. We specifically knocked down the expression of metalloprotease TACE by using small interfering RNA for TACE. Knockdown of TACE inhibited PMA-, PA sup-, and LPS-induced TGF-α shedding, EGFR phosphorylation, and mucin production. From these results, we conclude that TACE plays a critical role in mucin production by airway epithelial cells by means of a TACE ligand–EGFR cascade in response to various stimuli.

Human eosinophils induce mucin production in airway epithelial cells via epidermal growth factor receptor activation.

Eosinophil recruitment and mucus hypersecretion are characteristic of asthmatic airway inflammation, but eosinophils have not been shown to induce mucin production. Because an epidermal growth factor receptor (EGFR) cascade induces MUC5AC mucin in airways, and because EGFR is up-regulated in asthmatic airways, we examined the effect of eosinophils on MUC5AC mucin production in NCI-H292 cells (a human airway epithelial cell line that produces mucins). Eosinophils were isolated from the peripheral blood of allergic patients, and their effects on MUC5AC mucin gene and protein synthesis were assessed using in situ hybridization and ELISAs. When IL-3 plus GM-CSF or IL-3 plus IL-5 were added to eosinophils cultured with NCI-H292 cells, MUC5AC mucin production increased; eosinophils or cytokines alone had no effect. Eosinophil supernatant obtained by culturing eosinophils with IL-3 plus GM-CSF or IL-3 plus IL-5 also increased MUC5AC synthesis in NCI-H292 cells, an effect that was prevented by selective EGFR inhibitors (AG1478, BIBX1522). Supernatant of activated eosinophils induced EGFR phosphorylation in NCI-H292 cells. Supernatant of activated eosinophils contained increased concentrations of TGF-α protein (an EGFR ligand) and induced up-regulation of TGF-α expression and release in NCI-H292 cells. A blocking Ab to TGF-α reduced activated eosinophil-induced MUC5AC synthesis in NCI-H292 cells. These results show that activated eosinophils induce mucin synthesis in human airway epithelial cells via EGFR activation, and they implicate TGF-α produced by eosinophils and epithelial cells in the EGFR activation that results in mucin production in human airway epithelium.

Immunocytochemical localization of arachidonate 15-lipoxygenase in erythrocytes, leukocytes, and airway cells.

In reticulocytes, the enzyme 15-lipoxygenase (15-LO) is believed to contribute to cellular differentiation, and in leukocytes and airway cells 15-LO generates inflammatory mediators. The recent availability of antibodies to 15-LO now allows us to determine which specific cells contain the enzyme, to characterize its subcellular localization, and to determine its expression at the translational level. A polyclonal antibody to recombinant human reticulocyte 15-LO was used with a standard immunofluorescent technique. In rabbit red blood cells, fluorescence appeared during the course of anemia. Early reticulocytes did not fluoresce, but more mature reticulocytes showed increased fluorescent intensity. Late reticulocytes contained little fluorescence. Among human leukocytes, only eosinophils fluoresced. In human trachea, 15-LO immunofluorescence was localized to epithelial cells, and both basal and ciliated cells fluoresced. In all cells studied, fluorescence was localized to the cytoplasm and was variable in degree among cells in each preparation. We conclude that the 15-LO of airway cells and eosinophils is immunologically related to the reticulocyte 15-LO. Furthermore, the variable fluorescence among cells (e.g., in epithelium) and during development (e.g., reticulocytes) suggests a role of 15-LO in cell growth and development.

Pseudomonas lipopolysaccharide accelerates wound repair via activation of a novel epithelial cell signaling cascade.

The surface of the airway epithelium represents a battleground in which the host intercepts signals from pathogens and activates epithelial defenses to combat infection. Wound repair is an essential function of the airway epithelium in response to injury in chronic airway diseases, and inhaled pathogens such as Pseudomonas bacteria are implicated in the pathobiology of several of these diseases. Because epidermal growth factor receptor (EGFR) activation stimulates wound repair and because LPS activates EGFR, we hypothesized that LPS accelerates wound repair via a surface signaling cascade that causes EGFR phosphorylation. In scrape wounds of NCI-H292 human airway epithelial cells, high concentrations of LPS were toxic and decreased wound repair. However, lower concentrations of LPS accelerated wound repair. This effect was inhibited by treatment with a selective inhibitor of EGFR phosphorylation (AG 1478) and by an EGFR neutralizing Ab. Metalloprotease inhibitors and TNF-α-converting enzyme (TACE) small interfering RNA inhibited wound repair, implicating TACE. Additional studies implicated TGF-α as the active EGFR ligand cleaved by TACE during wound repair. Reactive oxygen species scavengers, NADPH oxidase inhibitors, and importantly small interfering RNA of dual oxidase 1 inhibited LPS-induced wound repair. Inhibitors of protein kinase C isoforms αβ and a TLR-4 neutralizing Ab also inhibited LPS-induced wound repair. Normal human bronchial epithelial cells responded similarly. Thus, LPS accelerates wound repair in airway epithelial cells via a novel TLR-4→protein kinase C αβ→dual oxidase 1→reactive oxygen species→TACE→TGF-α→EGFR phosphorylation pathway.

Neutrophil-dependent goblet cell degranulation: role of membrane-bound elastase and adhesion molecules

We examined the effect of the neutrophil chemoattractants interleukin (IL)-8 and N-formyl-methionyl-leucyl-phenylalanine on goblet cell (GC) degranulation in guinea pigs. Chemoattractants caused time-dependent neutrophil recruitment and GC degranulation in vivo. NPC 15669 (an inhibitor of leukocyte infiltration) prevented both responses, implicating neutrophils. ICI 200,355 (an inhibitor of neutrophil elastase and proteinase-3) or secretory leukocyte protease inhibitor (an inhibitor of elastase but not of proteinase-3) abolished IL-8-induced GC degranulation, implicating elastase. Incubating tracheal segments with IL-8 plus neutrophils caused GC degranulation in vitro, an effect due to activation of the neutrophils themselves (and not an effect present in the supernatant). Chemoattractant increased surface staining of elastase and the cleavage of elastase-specific fluorogenic substrate by neutrophils. Pretreatment with anti-intercellular adhesion molecule-1, anti-CD18, or anti-CD11b antibody inhibited the chemoattractant-induced GC degranulation in vitro, implicating adhesion molecules. These studies suggest that chemoattractants cause neutrophil-dependent GC degranulation involving adhesive interactions between cells, with elastase activity occurring at the cell interface, causing GC secretion. The findings, reproduced in human airways, suggest novel methods of therapeutic intervention.We examined the effect of the neutrophil chemoattractants interleukin (IL)-8 and N-formyl-methionyl-leucyl-phenylalanine on goblet cell (GC) degranulation in guinea pigs. Chemoattractants caused time-dependent neutrophil recruitment and GC degranulation in vivo. NPC 15669 (an inhibitor of leukocyte infiltration) prevented both responses, implicating neutrophils. ICI 200,355 (an inhibitor of neutrophil elastase and proteinase-3) or secretory leukocyte protease inhibitor (an inhibitor of elastase but not of proteinase-3) abolished IL-8-induced GC degranulation, implicating elastase. Incubating tracheal segments with IL-8 plus neutrophils caused GC degranulation in vitro, an effect due to activation of the neutrophils themselves (and not an effect present in the supernatant). Chemoattractant increased surface staining of elastase and the cleavage of elastase-specific fluorogenic substrate by neutrophils. Pretreatment with anti-intercellular adhesion molecule-1, anti-CD18, or anti-CD11b antibody inhibited the chemoattractant-induced GC degranulation in vitro, implicating adhesion molecules. These studies suggest that chemoattractants cause neutrophil-dependent GC degranulation involving adhesive interactions between cells, with elastase activity occurring at the cell interface, causing GC secretion. The findings, reproduced in human airways, suggest novel methods of therapeutic intervention.

Permselectivity of of the glomerular capillary wall. Studies of experimental glomerulonephritis in the rat using neutral dextran.

Polydisperse [3h] dextran was infused into eight Munich-Wistar rats in the early autologous phase of nephrotoxic serum nephritis (NSN), thereby permitting direct measurements of pressures and flows in surface glomeruli and fractional clearances for dextrans [(U/P) dextran/(U/P) inulin] ranging in radius from 18 to 42 A. Despite glomerular injury, evidenced morphologically and by a marked reduction in the glomerular capillary ultrafiltration coefficient, the glomerular filtration rate remained normal because of a compensating increase in the mean net ultrafiltration pressure. In NSN rats, as in normal controls, inulin was found to permeate the glomerular capillary wall without measurable restriction, and dextrans were shown to be neither secreted nor reabsorbed. For dextran radii of 18, 22, 26, 30, 34, 38, and 42 A, (U/P) dextran/(U/P) inulin in NSN and control rats, respectively, averaged 0.90 vs. 0.99, 0.81 vs. 0.97, 0.63 vs. 0.83, 0.38 vs 0.55, 0.20 vs. 0.30, 0.08 vs. 0.11, and 0.02 vs. 0.03. Using a theory based on macromolecular transport through pores, the results indicate that in NSN rats, effective pore radius is the same as in controls, approximately 50 A. In NSN, however, the ratio of total pore surface area to pore length, a measure of the number of pores, is reduced to approximately 1/3 that of control, probably due to a reduction in capillary surface area. These results suggest that proteinuria in glomerular disease is not due simply to increases in effective pore radius or number of pores, as previously believed. Using a second theoretical approach, based on the Kedem-Katchalsky flux equations, dextran permeability across glomerular capillaries was found to be slightly lower, and reflection coefficient slightly higher in NSN than in control rats.

Pseudomonas aeruginosa induces MUC5AC production via epidermal growth factor receptor

Hypersecretory disease associated with Pseudomonas aeruginosa (PA) infections is characterised by increased goblet cells and increased mucin production. Recently, an epidermal growth factor receptor (EGFR) signalling cascade was shown to be a common pathway through which many stimuli induce mucin MUC5AC expression in airways by differentiation to a goblet cell phenotype. This study looked at whether PA products induce EGFR expression and activation and thus result in mucin MUC5AC production. Human airway epithelial (NCI–H292) cells were stimulated with PA culture supernatant (Sup). MUC5AC protein production, MUC5AC and EGFR messenger ribonucleic acid (mRNA) expression, and phosphorylated EGFR and phosphorylated p44/42 mitogen-activated protein kinase (MAPK) were all examined using enzyme-linked immunosorbent assay, by in situ hybridisation and by immunoblotting. PA Sup induced MUC5AC mRNA and subsequent protein expression, EGFR and p44/42 MAPK phosphorylation and EGFR mRNA expression. Induction of MUC5AC mRNA and protein expression and EGFR and p44/42 MAPK phosphorylation were inhibited completely by pretreatment with a selective EGFR tyrosine kinase inhibitor. Pretreatment with a selective inhibitor of MAPK kinase prevented MUC5AC production and p44/42 MAPK phosphorylation but not EGFR phosphorylation. The authors conclude that PA products induce mucin MUC5AC production in human airway epithelial cells via the expression and activation of epidermal growth factor receptor.