Showing papers in "American Journal of Respiratory Cell and Molecular Biology in 1999"

A Polymorphism* in the 5 ′ Flanking Region of the CD14 Gene Is Associated with Circulating Soluble CD14 Levels and with Total Serum Immunoglobulin E

Abstract: Total serum immunoglobulin (Ig)E levels are genetically regulated, but the mechanism of inheritance is not well understood. Cytokines produced by T-helper (Th)1 and Th2 lymphocytes control IgE synthesis. Bacterial antigens may favor the development of Th1 cells from naive CD4-positive T cells through a CD14-dependent pathway. CD14 is constitutively expressed on the surface of monocytes and macrophages, and is also present in serum in a soluble form (sCD14). The CD14 gene maps to chromosome 5q31.1, a candidate region for loci regulating total serum IgE. We hypothesized that genetic variants in the CD14 gene could influence Th-cell differentiation and thus total serum IgE. We identified a C-to-T transition at base pair −159 from the major transcription start site (CD14/−159). Among 481 children recruited from a general population sample, frequency of allele C was 51.4%. TT homozygotes had significantly higher sCD14 levels than did carriers of both the CC and CT genotypes (P = 0.01). TT homozygotes also had ...

Inhibition of Myofibroblast Apoptosis by Transforming Growth Factor β1

Abstract: Fibroblast differentiation to the myofibroblast phenotype is associated with α–smooth-muscle actin (α-SMA) expression and regulated by cytokines. Among these, transforming growth factor (TGF)-β1 and interleukin (IL)-1β can stimulate and inhibit myofibroblast differentiation, respectively. IL-1β inhibits α-SMA expression by inducing apoptosis selectively in myofibroblasts via induction of nitric oxide synthase (inducible nitric oxide synthase [iNOS]). Because TGF-β is known to inhibit iNOS expression, this study was undertaken to see if this cytokine can protect against IL-1β–induced myofibroblast apoptosis. Rat lung fibroblasts were treated with IL-1β and/or TGF-β1 and examined for expression of α-SMA, iNOS, and the apoptotic regulatory proteins bax and bcl-2. The results show that TGF-β1 caused a virtually complete suppression of IL-1β–induced iNOS expression while preventing the decline in α-SMA expression or the myofibroblast subpopulation. TGF-β1 treatment also completely suppressed the IL-1β–induced ...

Both Erk and p38 kinases are necessary for cytokine gene transcription.

Abstract: A critical feature of sepsis-induced acute lung injury is the release of cytokines from endotoxin (LPS)- stimulated alveolar macrophages (AM). LPS is also known to activate various members of the mitogen- activated protein kinase (MAPK) family in other types of cells. In this study, we evaluated whether multiple members of the MAPK family regulate cytokine gene expression in LPS-stimulated AM. We found that LPS activates both the extracellular signal–regulated kinase (Erk) and p38 kinases, and that this activation is augmented when the cells are cultured in serum. Inhibition of either the Erk (with PD98059) or p38 (with SB203580) kinase pathway resulted in only a partial reduction in cytokine (interleukin-6 and tumor necrosis factor) messenger RNA accumulation and cytokine release, whereas inhibition of both pathways simultaneously resulted in a decrease in cytokine gene expression to near-control levels. Nuclear run-on assays showed that the effect of these MAPK pathways on LPS-induced expression of the ...

Activity of abundant antimicrobials of the human airway

Abstract: Human airways produce several antimicrobial factors; the most abundant are lysozyme and lactoferrin. Despite their likely importance in preventing infection, and their possible key role in the pathogenesis of cystic fibrosis (CF), we know little about their antibacterial activity in the context of the CF airway. We found that abundant airway antimicrobial factors kill common CF pathogens, although Burkholderia was relatively resistant. To study the antibacterial activity, we developed a rapid, sensitive, and quantitative in vitro luminescence assay. Because NaCl concentrations may be elevated in CF airway surface liquid, we tested the effect of salt on antibacterial activity. Activity of individual factors and of airway lavage fluid was inhibited by high ionic strength, and it was particularly sensitive to divalent cations. However, it was not inhibited by nonionic osmolytes and thus did not require hypotonic liquid. The inhibition by ionic strength could be partially compensated by increased concentrations of antibacterial factors, thus there was no one unique salt concentration for inhibition. CF airway secretions also contain abundant mucin and elastase; however, these had no effect on antibacterial activity of lysozyme, lactoferrin, or airway lavage fluids. When studied at low NaCl concentrations, CF and non-CF airway lavage fluids contained similar levels of antibacterial activity. These results suggest approaches toward developing treatments aimed at preventing or reducing airway infections in individuals with CF.

Human neutrophils secrete gelatinase B in vitro and in vivo in response to endotoxin and proinflammatory mediators.

Abstract: Bacterial sepsis is characterized by a systemic inflammatory state, with activation of numerous cell types. Phagocytes participate in this phenomenon by secreting various proinflammatory cytokines and enzymes. Matrix metalloproteinases (MMPs) such as gelatinases are produced by phagocytes and are thought to play an important role in processes of cell transmigration and tissue remodeling. In this work, we show that endotoxin (lipopolysaccharide [LPS]) and other inflammatory mediators, such as tumor necrosis factor (TNF), interleukin-8, and granulocyte colony-stimulating factor, induce a rapid (within 20 min) release of gelatinase-B (MMP-9) zymogen in whole human blood, as determined by gelatin zymography. The polymorphonuclear neutrophil was identified as the cell responsible for this rapid secretion, as a result of the release of preformed enzymes stored in granules. Normal human subjects given LPS intravenously showed a similar pattern of proMMP-9 secretion, with maximum plasma levels reached 1.5 to 3 h ...

Th1- and Th2-Type Cytokines Regulate the Expression and Production of Eotaxin and RANTES by Human Lung Fibroblasts

Abstract: Eosinophils (Eos) and fibroblasts are known to play a major role in the pathogenesis of bronchial asthma and fibrotic lung disease. Therefore, we investigated whether Th1 and Th2 cytokines stimulate the production of Eo-activating chemokines by lung fibroblasts. Analyses of the culture supernatant using multiple steps of high-performance liquid chromatography demonstrated that interleukin (IL)-4 preferentially stimulates lung fibroblasts to secrete a peak of eosinophil chemotactic activity (ECA) which, upon N-terminal analyses, showed similar sequence to eotaxin, whereas interferon (IFN)-gamma had negligible effect on the release of this chemokine. In contrast, tumor necrosis factor (TNF)-alpha stimulated lung fibroblasts to release two peaks of activity that were found to correspond to eotaxin and regulated on activation, normal T cells expressed and secreted (RANTES), respectively. Interestingly, IL-4 synergized with TNF-alpha to increase greatly the production of three biochemically distinct eotaxin forms. In contrast, IFN-gamma synergized with TNF-alpha to increase RANTES production. Neither IL-2, IL-5, IL-6 nor IL-10 had an effect on lung fibroblasts' capacity to express or release eotaxin and RANTES. Upon appropriate cytokine stimulation, lung fibroblasts were also found to express messenger RNA for monocyte chemotactic protein (MCP)-3 and MCP-4 but not eotaxin-2. However, no ECA like MCP-3 or MCP-4 was detected. These observations suggest that the release of Th1 or Th2 cytokines in the lung tissue polarizes lung fibroblasts to produce either RANTES or eotaxin as major Eo attractants.

Deficient Hydrophilic Lung Surfactant Proteins A and D with Normal Surfactant Phospholipid Molecular Species in Cystic Fibrosis

Abstract: Chronic bacterial colonization of the lungs, with an excessive inflammatory response, is the major cause of morbidity and mortality in cystic fibrosis. Lung surfactant exhibits a spectrum of potential immunomodulatory properties: phospholipid components inhibit cellular inflammatory responses, whereas the hydrophilic surfactant proteins A (SP-A) and D (SP-D) are integral components of the innate host defense response of the lungs against bacterial infection. Consequently, alteration to the relative proportions of lung surfactant components may alter the susceptibility of the lungs to bacterial colonization. In this study, bronchoalveolar lavage (BAL) samples were collected at diagnostic fiberoptic bronchoscopy from 11 control children, 13 children with cystic fibrosis, and 11 children with acute lung infection. Electrospray ionization mass spectrometry analysis demonstrated negligible changes to the molecular species or total BAL concentrations of phosphatidylcholine, phosphatidylglycerol, or phosphatidyl...

Rhinovirus Replication Causes RANTES Production in Primary Bronchial Epithelial Cells

Abstract: The mechanisms by which rhinovirus (RV) infections produce lower airway symptoms in asthmatic individuals are not fully established. To determine effects of RV infection on lung epithelial cells, primary human bronchial epithelial (BE) cells were infected with either RV16 or RV49, and viral replication, cell viability, and cell activation were measured. Both viral serotypes replicated in BE cells at 33°C (ΔTCID50 / ml = 2 to 2.5 log units) and at 37°C (ΔTCID50 /ml = 1.6 log units), but only high doses of RV49 (106 TCID50 /ml) caused cytopathic effects and reduced cell viability. In addition, regulated on activation, normal T cells expressed and secreted (RANTES) secretion was increased in epithelial cells infected with RV16 or RV49 (243 and 398 pg/ml versus 13 pg/ml uninfected control cells), and a similar pattern was seen for RANTES messenger RNA. RV infection also caused increased secretion of interleukin-8 and granulocyte macrophage colony-stimulating factor, but did not alter expression of either inte...

Mucin gene expression during differentiation of human airway epithelia in vitro. Muc4 and muc5b are strongly induced.

Abstract: Mucus hypersecretion is characteristic of chronic airway diseases. However, regulatory mechanisms are poorly understood. Human airway epithelial cells grown on permeable supports at the air‐liquid interface (ALI) develop a mucociliary morphology resembling that found in vivo . Such cultures provide a model for studying secretory cell lineage, differentiation, and function, and may provide insight regarding events leading to mucus hypersecretion. The mucin gene expression profile of well-differentiated human airway epithelial cells in culture has not yet been established. We compared expression of all the currently described mucin genes in poorly differentiated (conventional cultures on plastic) and well-differentiated (ALI) human nasal and bronchial epithelial cells. Differentiation-dependent upregulation of MUC3, MUC5AC, MUC5B, and MUC6 messenger RNA (mRNA) was demonstrated using reverse transcriptase‐polymerase chain reaction (RT‐PCR). Northern blot analysis showed a similar increase for MUC4 and demonstrated that induction of MUC4 and MUC5B expression depended on retinoic acid. MUC1, MUC2, MUC7, and MUC8 mRNAs were also detected by RT‐PCR, but these genes did not appear to be strongly regulated as a function of differentiation. Mucin gene expression was similar in bronchial and nasal cells. Thus, mucociliary differentiation of human airway epithelia in vitro entails upregulation of several mucin genes. Bernacki, S. H., A. L. Nelson, L. Abdullah, J. K. Sheehan, A. Harris, C. W. Davis, and S. H. Randell. 1999. Mucin gene expression during differentiation of human airway epithelia in vitro : MUC4 and MUC5B are strongly induced. Am. J. Respir. Cell Mol. Biol. 20:595‐604.

Surfactant Protein-A Binds Group B Streptococcus Enhancing Phagocytosis and Clearance from Lungs of Surfactant Protein-A–Deficient Mice

Abstract: Surfactant protein-A (SP-A) gene–targeted mice clear group B streptococcus (GBS) from the lungs at a slower rate than wild-type mice. To determine mechanisms by which SP-A enhances pulmonary clearance of GBS, the role of SP-A in binding and phagocytosis of GBS was assessed in SP-A (−/−) mice infected with GBS in the presence and absence of exogenous SP-A. Coadministration of GBS with exogenous SP-A decreased GBS colony counts in lung homogenates of SP-A (−/−) mice. SP-A bound to GBS in a calcium-dependent manner. Although pulmonary infiltration with macrophages was not altered in SP-A (−/−) versus wild-type mice after GBS infection, the number of alveolar macrophages with phagocytosed bacteria was lower in the SP-A (−/−) mice than in the wild-type mice. When SP-A was coadministered with GBS, phagocytosis was significantly increased. Oxygen radical production by alveolar macrophages from SP-A (−/−) mice infected with GBS was decreased compared with wild-type controls and was increased when SP-A (−/−) mice ...

Cooperativity between Oxidants and Tumor Necrosis Factor in the Activation of Nuclear Factor (NF)- κ B

Abstract: The transcription factor nuclear factor (NF)-κB is activated by oxidative stress or cytokines and is critical to the activation of inflammatory genes. Here, we report that hydrogen peroxide or 3-morpholinosydnonimine, which simultaneously releases nitric oxide and superoxide, synergize with the cytokine tumor necrosis factor (TNF)-α to activate NF-κB in rat lung epithelial cells, suggesting that signaling pathways elicited by reactive oxygen species (ROS)/reactive nitrogen species (RNS) are different from TNF-induced signaling. These findings were substantiated by observations that levels of IκB-α did not change after exposure to ROS/RNS, whereas a rapid depletion of IκB-α was observed in cells exposed to TNF. In addition, the proteosome inhibitor MG132 did not affect activation of NF-κB by ROS/RNS, whereas it abolished the TNF response. Transfection of a dominant negative Ras construct prevented the activation of NF-κB by ROS/RNS, demonstrating the requirement for Ras in the activation of NF-κB by oxidan...

Enhanced Insulin-Like Growth Factor Binding Protein–Related Protein 2 (Connective Tissue Growth Factor) Expression in Patients with Idiopathic Pulmonary Fibrosis and Pulmonary Sarcoidosis

Abstract: Connective tissue growth factor is a recently described chemoattractant and fibroblast mitogen which, because of sequence homology and weak binding to insulin-like growth factor (IGF)-1, has been proposed as the eighth member of the IGF binding protein (IGFBP) superfamily, named IGFBP-related protein 2 (IGFBP-rP2). Previous studies have implicated IGFBP-rP2 in a number of heterogeneous fibrotic pathologies, including renal fibrosis, dermal scleroderma, and bleomycin-induced pulmonary fibrosis in mice. Because profibrogenic cytokines may be produced by inflammatory cells, we developed a multiplex competitive reverse transcription/polymerase chain reaction to quantify IGFBP-rP2 transcripts in bronchoalveolar lavage cells from healthy subjects and patients with idiopathic pulmonary fibrosis (IPF) and pulmonary sarcoidosis. IGFBP-rP2 messenger RNA expression was enhanced > 10-fold (P 40-fold (P 90-fold (P < 0.005) in stage III/IV sarcoidosis patients by comparison with healthy nonsmoking control subjects. We suggest these increases are predominantly associated with lymphocyte- and neutrophil-driven IGFBP-rP2 production. These findings, together with previous reports implicating other IGFBPs in the pathogenesis of pulmonary fibrosis, suggest that the complex network of IGFBPs within the human lung is an important determinant of the outcome of the fibroproliferative response to injury.

Relaxation of contracted rabbit tracheal and human bronchial smooth muscle by Y-27632 through inhibition of Ca2+ sensitization.

Abstract: The mechanism of Ca2+ sensitization of contraction has not been elucidated in airway smooth muscle (SM). To determine the role of a small G protein, rhoA p21, and its target protein, rho-associated coiled coil-forming protein kinase (ROCK), in receptor-coupled Ca2+ sensitization of airway SM, we studied the effect of (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexane carboxamide dihydrochloride, monohydrate (Y-27632), a ROCK inhibitor, on isometric contractions in rabbit tracheal and human bronchial SM. Y-27632 completely reversed 1 microM carbachol (CCh)-induced contraction of intact trachea with a concentration producing half-maximum inhibition of effect (IC50) of 1.29 +/- 0.2 microM (n = 5). Although 4beta-phorbol 12,13-dibutyrate (1 microM)-induced Ca2+ sensitization was relatively resistant to Y-27632 in alpha-toxin-permeabilized trachea, CCh (100 microM) plus guanosine triphosphate (GTP) (3 microM)- and guanosine 5'-O-(3'-thiotriphosphate) (10 microM)-induced contractions were relaxed completely by Y-27632 with IC50 of 1.44 +/- 0.3 (n = 6) and 1.15 +/- 0.3 microM (n = 6). Endothelin-1 (1 microM) plus GTP (3 microM)- developed force was also reversed by Y-27632 with IC50 of 4. 10 +/- 1.1 microM (n = 6) in the alpha-toxin-permeabilized bronchus. Both the rabbit and human SM expressed rhoA p21, ROCK I, and its isoform ROCK II. Collectively, rho/ROCK-mediated Ca2+ sensitization plays a central role in the sustained phase of airway SM contraction, and selective inhibition of this pathway may become a new strategy to resolve airflow limitation in asthma.

Developmental Mucin Gene Expression in the Human Respiratory Tract

Abstract: The epithelial surface of the respiratory tract is coated with a protective film of mucus secreted by epithelial goblet and submucosal gland cells. Histology of the airway mucosa and composition of secretions during the second trimester of fetal life are known to differ from the normal adult in that these secretions show similarities with those of hypersecretory disorders. To provide information regarding cell-specific expression of mucin genes and their relation to developmental patterns of epithelial cytodifferentiation, we studied the expression of eight different mucin genes (MUC1–MUC4, MUC5AC, MUC5B, MUC6, MUC7) in human embryonic and fetal respiratory tract using in situ hybridization. These investigations demonstrated that MUC4 is the earliest gene expressed in the foregut at 6.5 wk, followed by MUC1 and MUC2 from 9.5 wk of gestation in trachea, bronchi, epithelial tubules, and terminal sacs before epithelial cytodifferentiation. In contrast, MUC5AC, MUC5B, and MUC7 are expressed at later gestation...

Eotaxin. An essential mediator of eosinophil trafficking into mucosal tissues.

Abstract: Eosinophil accumulation in the peripheral blood and tissues is a hallmark feature of several important medical diseases, including atopic disorders (allergic rhinitis, asthma, and eczema), parasitic infections, and numerous systemic diseases (e.g., Churg Strauss syndrome, eosinophilic pneumonia, eosinophilic gastroenteritis, and the idiopathic hypereosinophilic syndrome) (1). The findings that eosinophils normally account for only a small percent of circulating or tissue-dwelling cells and that their numbers markedly and selectively increase under specific disease states indicate the existence of molecular mechanisms that regulate the selective generation and accumulation of these leukocytes. The pathologic role of eosinophils primarily occurs in tissues; therefore, a major focus of scientific investigation on eosinophils has been to elucidate the processes involved in eosinophil tissue recruitment. Numerous mediators have been identified as eosinophil chemoattractants, including diverse molecules, such as lipid mediators (platelet-activating factor, leukotrienes), bacterial products (formylmethionyl leucylphenylalanine [FMLP]), and recently, chemokines such as RANTES (regulated on activation, normal T cells expressed and secreted) and macrophage inflammatory protein (MIP)-1 a (2). However, none of these mediators selectively promote eosinophil recruitment; they are therefore not considered to be the primary mediators of the tissue eosinophilia observed in numerous hypereosinophilic disorders. In contrast to these molecules, eotaxin is an eosinophilselective chemoattractant and therefore has been the subject of recent intensive research. Eotaxin was initially discovered using a biologic assay in guinea pigs designed to identify the molecules responsible for allergen-induced eosinophil accumulation in the lungs. Using an in vivo chemotaxis assay in guinea-pig skin, the partial amino acid sequence for the protein responsible for eosinophil chemoattraction in the bronchoalveolar fluid in allergen-challenged guinea pigs was determined (3). This facilitated the genetic cloning of the genes and complemenatry DNA (cDNA) for guinea pig, murine, and human eotaxin, and the identification of eotaxin as a member of the CC chemokine family most homologous to the macrophage chemoattractant protein (MCP) subfamily (4–7). This subfamily of eotaxin and MCP chemokines is clustered on human chromosome 17q11, a region clustered with other CC chemokines (such as MIP-1, I-309, RANTES, and HCC-1, -2) (8). Interestingly, this region has been recently linked to asthma susceptibility (9). Recently, another chemokine, which is active primarily on eosinophils, has been identified, and this chemokine has been termed eotaxin-2 (10). Eotaxin-2 is only distantly related to eotaxin because it is only 39% homologous and is located in a different chromosomal position (7q11). The cloning of these genes and the production of recombinant protein enabled the biologic activity of eotaxin to be examined. Eotaxin was distinguished from all other chemokines because it was found to be a potent eosinophil-selective chemoattractant having minimal activity on other leukocytes. Eotaxin was also identified as a potent activator of eosinophils capable of inducing superoxide generation and the release of granule proteins. Eotaxin was subsequently shown to also be active on human peripheral blood basophils, stimulating chemoattraction in vitro and weak histamine release from IL-3–primed basophils (11). Administration of eotaxin in vivo has been performed in guinea pigs, rodents, and primates, and all of these studies have consistently demonstrated a strong preference of eotaxin for eosinophils in vivo because the eosinophil is the only cell that is recruited. Interestingly, the activity of eotaxin in vivo is enhanced by the copresence of interleukin (IL)-5 (12). IL-5, an eosinophil-selective hematopoietin that regulates eosinophil growth, differentiation, and survival, is a potent stimulus for the release of eosinophils and eosinophil precursors from the bone marrow into the peripheral circulation (13). The increased level of circulating eosinophils and the ability of IL-5 to prime eosinophils to have enhanced responsiveness to eotaxin probably accounts for the synergy between these two cytokines (14). The specific activity of eotaxin is mediated by the selective expression of the eotaxin receptor, CC chemokine receptor-3 (CCR-3), a seven-transmembrane–spanning G-protein–linked genetically polymorphic receptor primarily ex( Received in original form July 1, 1999 )

Cellular sources of enhanced brain-derived neurotrophic factor production in a mouse model of allergic inflammation.

Abstract: The aim of this study was to investigate production and cellular sources of brain-derived neurotrophic factor (BDNF) production in allergic asthma. For this purpose a mouse model of chronic and severe ovalbumin (OVA)-induced airway inflammation was developed. Allergen-exposed mice developed elevated immunoglobulin E titers; airway inflammation with influx of lymphocytes, monocytes, and eosinophils; and airway hyperresponsiveness. In addition to an influx of inflammatory cells, interleukin (IL)-4 and IL-5 production were enhanced, macrophages showed morphologic signs of activation, and airway epithelium was thickened and displayed a goblet-cell hyperplasia with a marked mucus production. BDNF was detected using in situ hybridization and enzyme-linked immunosorbent assay. Constitutive expression of BDNF messenger RNA (mRNA) was observed in the respiratory epithelium of sensitized and nonsensitized mouse lungs. In addition, BDNF mRNA was detected in airway inflammatory infiltrations and bronchoalveolar lavage fluid (BALF) cells of OVA-sensitized and aerosol-challenged mice. Highest BDNF protein levels were detected in BALF after long-term allergen aerosol exposure. Analysis of BDNF production by isolated lymphocyte subsets revealed T but not B cells as a cellular source of BDNF. In addition, activated alveolar macrophages were identified as BDNF-positive cells. These data indicate that in allergic airway inflammation BDNF production is upregulated and immune cells serve as a source of BDNF.

Forkhead Transcription Factor HFH-4 Expression Is Temporally Related to Ciliogenesis

Abstract: Members of the forkhead/winged-helix family of transcription factors are expressed in tissue-specific patterns and play critical roles in development and cell differentiation. The expression of forkhead family member hepatocyte nuclear factor-3/forkhead homologue 4 (HFH-4) has been localized by RNA-blot analysis and in situ hybridization to the proximal airway of the lung (trachea, bronchi, and bronchioles) with onset at mouse embryonic day (E) 14.5 and is present in the choroid plexus, ependymal cells, oviduct, and testis. We hypothesized that the restricted expression of HFH-4 messenger RNA suggests a function common to these tissues and therefore a cell-specific role for HFH-4. Accordingly, an anti–HFH-4 antibody was generated and used for cell-specific localization of protein expression to begin to identify the functions of HFH-4. We found HFH-4 expression in proximal airway ciliated epithelial cells, but not Clara cells or alveolar epithelial cells. HFH-4 was also expressed in ciliated epithelial cel...

Development of eosinophilic airway inflammation and airway hyperresponsiveness requires interleukin-5 but not immunoglobulin E or B lymphocytes.

Abstract: We previously defined a role for B cells and allergen-specific immunoglobulins in the development of allergic sensitization, airway inflammation, and airway hyperresponsiveness (AHR), using a 10-d protocol in which allergen exposure occurred exclusively via the airways, without adjuvant. In the present protocol, normal and B-cell–deficient (μMt−/−) mice were sensitized intraperitoneally to ovalbumin (OVA) and challenged with OVA via the airways in order to examine the requirements for AHR with this protocol. T-cell activation (antigen-specific proliferative responses and Th2-type cytokine production) and eosinophil infiltration in the peribronchial regions of the airways, with signs of eosinophil activation and degranulation, occurred in both experimental groups. In contrast to the 10-d protocol, increased in vivo airway responsiveness to methacholine and in vitro tracheal smooth-muscle responses to electrical field stimulation were observed in both normal and B-cell–deficient mice, and these responses we...

Surfactant Protein D Stimulates Phagocytosis of Pseudomonas aeruginosa by Alveolar Macrophages

Abstract: Surfactant protein (SP)-D is an oligomeric glycoprotein belonging to the family of collagen-like lectins known as collectins, which have previously been shown to stimulate phagocytosis and other immune cell functions. The hypothesis investigated in this study was that SP-D would stimulate the phagocytosis of an important pulmonary pathogen, Pseudomonas aeruginosa. SP-D, isolated from the lavage fluid of silica-treated rats, significantly enhanced the uptake of three of six strains of P. aeruginosa by rat alveolar macrophages as analyzed by both fluorescence and electron microscopy. SP-D had only minimal effects on phagocytosis of Haemophilus influenzae. SP-D bound to live P. aeruginosa, and binding was inhibited by chelation of calcium and by a competing saccharide, inositol. In vitro killing assays demonstrated that macrophage-mediated killing of one of the mucoid strains of P. aeruginosa was modestly enhanced by SP-D. P. aeruginosa was not measurably aggregated by SP-D either macroscopically or microsco...

The Attenuated Fibroblast Sheath of the Respiratory Tract Epithelial–Mesenchymal Trophic Unit

Abstract: Interactions between epithelial, mesenchymal, and neural tissue and also the extracellular matrix are necessary to initiate numerous cellular functions of the lung (1). The most common of these functions include differentiation during lung growth, repair of damaged tissue, and regulation of the inflammatory response. Each of these processes requires a localized response to a specific stimulus. Fibroblasts, especially those in close proximity to the airway epithelium, are likely regulators of local responses. In a recent commentary, Smith and colleagues discussed the possibility that resident fibroblasts may act as sentinel cells for these responses (2). In addition to their role as connective tissue cells, fibroblasts also produce cytokines and chemokines in response to various stimuli. Their fixed position in the tissue suggests that they can respond in a local manner to bacterial products, tissue injury, or other environmental factors. The relationship between cytokines and inflammatory cells in asthmatic airways also indicates a similar role for fibroblasts. In the asthmatic lung, the fibroblast plays a key role as a resident mesenchymal cell beneath the epithelium, receiving and sending information to epithelial and inflammatory cells (3, 4). Additionally, these fibroblasts are thought to be responsible for the subepithelial fibrosis associated with asthma. The significance of resident fibroblasts in the airway during inflamation has been described; however, the concept of an anatomically distinct group of fibroblasts associated with airway epithelium has not been explored. In 1990, Brewster and associates described a layer of subepithelial fibroblasts in the bronchi of normal and asthmatic human subjects that were positioned to allow close interaction with the epithelium, neural tissues, and extracellular matrix (5). The population of cells was shown to comprise fibroblasts and myofibroblasts, and individual cells were reported to be as large as 100 m m in diameter. A detailed description of the subepithelial layer of resident fibroblasts in the rat trachea was reported by Evans and coworkers (6). In tissue sections, the cells appear as a layer of attenuated cell processes closely opposed to the lamina reticularis of the basement membrane zone, about 1.9 m m beneath the epithelial basal lamina. The cells are intermeshed with each other, about 40 m m in diameter with the attenuated portions about 0.55 m m in thickness. The cell is thicker near the nucleus and contains abundant rough endoplasmic reticulum near the nucleus. There were no apparent bundles of microfilaments in the thin or thick portions of the cell as there are in myofibroblasts. The cells were determined to be stellate in shape. They exist as a layer of large, flat cells covering about 70% of the interstitial surface of the lamina reticularis, and make numerous contacts with the lamina densa of the basement membrane zone (approximately 7,000 times per mm 2 ). This layer of thin mesenchymal cells was named the attenuated fibroblast sheath (6), with properties of a layer of similar cells in the gut identified as the pericryptal fibroblast sheath (7). On the basis of the data from this previous paper (6), we constructed a three-dimensional model of the attenuated fibroblast sheath (Figure 1). The total attenuated fibroblast sheath, with its large surface area and close proximity to the epithelial/environmental interface, defines an anatomic unit of resident fibroblasts that could respond in a local manner to various stimuli. In this role, the attenuated fibroblast sheath represents the mesenchymal component of interactions with the epithelium, extracellular matrix, neural tissues, and migratory cells of the inflammatory response. The anatomic and functional relationship between the attenuated fibroblast sheath, epithelial and neural tissue, and also the extracellular matrix appears to serve as an epithelial–mesenchymal trophic unit. The epithelial–mesenchymal trophic unit would allow local exchange of information between the different tissue elements in response to various stimuli. This concept is similar to that of other investigators, with the exception that it recognizes that the subepithelial fibroblasts exist as a specific layer of resident fibroblasts beneath the epithelium instead of being randomly distributed in the lamina propria (2–4). Of the tissues in the epithelial–mesenchymal trophic unit, the least is known about the attenuated fibroblast sheath. Examples of the attenuated fibroblast sheath can be seen in most published electron microscope studies of the upper respiratory tract as a thin layer of attenuated cell processes immediately beneath the epithelium. Present in all animal species examined to date, the attenuated fibroblast sheath extends from the proximal to the distal regions of the conducting airways (5, 6, 8). Conceptually, it continues into the gas exchange region as interstitial fibroblasts in ( Received in original form May 14, 1999 and in revised form July 9, 1999 )

The involvement of Fas-Fas ligand pathway in fibrosing lung diseases.

Abstract: Pulmonary fibrosis begins with alveolitis, which progresses to destruction of lung tissue and excess collagen deposition. This process could be the result of DNA damage and a form of apoptosis. Therefore, we hypothesized that Fas ligand (FasL), which induces apoptosis in cells expressing Fas antigen (Fas), is associated with pulmonary fibrosis. We examined frozen lung tissues from seven patients with idiopathic pulmonary fibrosis (IPF), and bronchoalveolar lavage fluid (BALF) cells from 19 patients with IPF and from 17 patients with interstitial pneumonia associated with collagen vascular diseases (CVD-IP). We used five frozen lungs with normal lung parenchyma and BALF cells from 10 patients with solitary pulmonary nodule as controls. Reverse transcription-polymerase chain reaction (RT-PCR) showed that FasL messenger RNA (mRNA) was expressed in BALF cells from all patients with IPF and from 15 of 16 patients with CVD-IP. FasL mRNA was not detected in BALF cells except in one of 10 controls. RT in situ PCR detected FasL mRNA in inflammatory cells in BALF from patients with IPF. Immunohistochemistry detected FasL protein in infiltrating lymphocytes and granulocytes in all of seven frozen lung tissues of IPF, but in none of five control lung tissues. Additionally, the expression of Fas appeared to be upregulated in bronchiolar and alveolar epithelial cells in IPF compared with normal lung parenchyma by immunohistochemistry. We conclude that Fas and FasL were upregulated in fibrosing lung diseases and may associate with DNA damage or apoptosis of bronchiolar and alveolar epithelial cells in this disorder.

Interleukin-10 Gene Transfer to the Airway Regulates Allergic Mucosal Sensitization in Mice

Abstract: The objective of this study was to investigate the effect of airway gene transfer of interleukin (IL)-10, a cytokine with potent anti-inflammatory and immunoregulatory activities, on allergic mucosal sensitization. We used a recently described murine model that involves repeated exposures to aerosolized ovalbumin (OVA), daily for 10 d, in the context of granulocyte macrophage colony-stimulating factor (GM-CSF) expression in the airway environment achieved by intranasal delivery of a replication-deficient adenovirus carrying the GM-CSF transgene. The resulting inflammatory response was characterized by a T-helper 2 cytokine profile and marked airway eosinophilia. After complete resolution of the inflammatory response (Day 28), a single exposure to OVA reconstituted airway eosinophilia and induced airway hyperresponsiveness. We show that concurrent expression of IL-10 inhibited GM-CSF-driven OVA-specific inflammation in a dose-dependent manner. Specifically, IL-10 decreased the number of mononuclear cells, ...

Human Lung Dendritic Cells Have an Immature Phenotype with Efficient Mannose Receptors

Abstract: Dendritic cells (DC) can be present at distinct stages of differentiation within the immune system. Sallusto and colleagues have recently described an in vitro culture system suitable for analyzing the maturation processes of DC (Sallusto and colleagues, J. Exp. Med. 1994;179:1109-1118). Monocytes cultured for 6 d in the presence of granulocyte macrophage colony-stimulating factor and interleukin-4 develop into immature DC with a high endocytic capacity but a low capacity to stimulate T cells. When challenged by lipopolysaccharide, these cells upregulate costimulatory molecules, express CD83, and become mature DC. CCR1 and CCR5 chemokine receptors are highly expressed on immature DC and downregulated on mature DC. This in vitro system was used to characterize human lung DC. Lung DC were shown to express some characteristics of in vitro immature DC. These are: (1) low expression of the costimulatory molecules CD40, CD80, and CD86; (2) poor expression of the differentiation marker CD83 and no CD1a; and (3) good capacity to incorporate dextran. Lung DC express moderate levels of CCR1 and CCR5. However, lung DC, like in vitro mature DC, express high levels of major histocompatibility complex Class II molecules, show low expression of CD14 and CD64, and are characterized by their high capacity to stimulate allogeneic T cells to proliferate during mixed leukocyte reactions (MLRs). Although lung DC express low levels of CD80 and CD86, the important role of these costimulatory molecules in inducing high MLR was demonstrated by using blocking antibodies. Therefore, while lung DC have overall a phenotype and an endocytic capacity close to in vitro immature DC, they share, like in vitro mature DC, a powerful capacity to stimulate T cells.

CD34+/Interleukin-5R α Messenger RNA+ Cells in the Bronchial Mucosa in Asthma: Potential Airway Eosinophil Progenitors

Abstract: Eosinophil differentiation is thought to occur by the action of interleukin (IL)-5 on CD34+ progenitor cells. The allergen-induced increase in eosinophil numbers in isolated airway preparations in vitro, and detection of increased numbers of circulating CD34+ cells in atopic subjects, led us to the hypothesis that the eosinophil infiltration of the airway in asthma may result from local mucosal differentiation, in addition to recruitment from the bone marrow. We examined CD34+ cell numbers by immunohistochemistry and IL-5 receptor α (IL-5Rα) messenger RNA (mRNA) expression by in situ hybridization in bronchial biopsies from atopic asthmatic patients, and from atopic and nonatopic control subjects. CD34+ cell numbers were increased in the airway in atopic asthmatic and atopic nonasthmatic subjects. In contrast, CD34+/ IL-5Rα mRNA+ cells were increased in asthmatic subjects when compared with both atopic and nonatopic control subjects. Airway numbers of CD34+/IL-5Rα mRNA+ cells were correlated to airway cal...

Expression of monocyte chemotactic protein (MCP)-1, MCP-2, and MCP-3 by human airway smooth-muscle cells. Modulation by corticosteroids and T-helper 2 cytokines.

Abstract: We have demonstrated that, in addition to their contractile function, human airway smooth-muscle cells (HASMC) are able to express and to secrete chemokines of the monocyte chemotactic protein (MCP)/ eotaxin subfamily. This group of chemokines is believed to play a fundamental role in the development of allergic airway diseases such as asthma. The expression levels of MCP (MCP-1, -2, and -3) messenger RNA (mRNA) were compared with those of regulated on activation, normal T cells expressed and secreted (RANTES) mRNA in HASMC in culture. HASMC express MCP and RANTES mRNA after stimulation with interleukin (IL)-1β, tumor necrosis factor-α, and interferon-γ. MCP mRNA was maximal at 8 h, whereas RANTES mRNA expression was delayed to 24 h after stimulation. Further, significant differences were observed in the induction patterns of MCP and RANTES mRNA expression after stimulation with the individual cytokines. Dexamethasone (DEX) significantly inhibited cytokine-induced accumulation of MCP and RANTES mRNA, in c...

Characterization of matrix metalloproteinases produced by rat alveolar macrophages.

Abstract: Evidence presented in the accompanying article (Gibbs, D. F., T. P. Shanley, R. L. Warner, H. S. Murphy, J. Varani, and K. J. Johnson. 1999. Role of matrix metalloproteinases in models of macrophage-dependent acute lung injury: evidence for alveolar macrophage as source of proteinases. Am. J. Respir. Cell Mol. Biol. 20:1145–1154) implicates alveolar macrophage matrix metalloproteinases (MMPs) in two models of acute lung inflammation in the rat. As a prerequisite to understanding which specific MMPs might be involved in the injury and how they might function, it was necessary to know the spectrum of enzymes present. To this end, alveolar macrophages were obtained from normal rat lungs by bronchoalveolar lavage, placed in culture with and without various agonists, and assessed by a variety of techniques for MMPs. The identification process involved characterization by gelatin, β-casein, and κ-elastin zymography, with confirmation of identity by Western blot/immunoprecipitation. Message levels of detected MM...

Glucocorticoids Inhibit Proliferation, Cyclin D1 Expression, and Retinoblastoma Protein Phosphorylation, but Not Activity of the Extracellular-Regulated Kinases in Human Cultured Airway Smooth Muscle

Abstract: We have previously shown that glucocorticoids inhibit mitogen-stimulated proliferation of human cultured airway smooth muscle (ASM) cells. The present study analyzed the effect of glucocorticoids on key regulatory pathways leading to passage of cells through the restriction point of the cell cycle, including those mediated by extracellular-regulated kinases (ERK) 1 and 2; the ERK upstream regulator MAPK kinase (MEK1); cyclin D1 levels; and levels and phosphorylation of retinoblastoma protein (pRb). Fluticasone propionate, a new inhaled glucocorticoid, was at least 10-fold more potent than dexamethasone in inhibiting thrombin-stimulated DNA synthesis and increases in cell number. Thrombin-stimulated increases in the levels and hyperphosphorylation of pRb were inhibited by glucocorticoids, which also reduced thrombin-stimulated cyclin D1 protein and messenger RNA (mRNA) levels. PD98059 (10 microM), an inhibitor of MEK1 activation, markedly attenuated thrombin stimulation of ERK activity and phosphorylation, DNA synthesis, and cyclin D1 levels. However, glucocorticoids had no effect on ERK activity or phosphorylation at 5 min, 2 h, or 12 h after addition of thrombin. In conclusion, glucocorticoid-induced reduction of cyclin D1 mRNA and protein levels, and of pRb phosphorylation, is sufficient to account for inhibition of ASM proliferation. Furthermore, these inhibitory effects of glucocorticoids on cyclin D1 and pRb occur on a component of the mitogen signaling cascade that is either downstream of or parallel to the ERK pathway.

Allergen-Induced Increase in Airway Responsiveness, Airway Eosinophilia, and Bone-Marrow Eosinophil Progenitors in Mice

Abstract: Increases in bone-marrow (BM) inflammatory cell progenitors are associated with allergen-induced airway hyperresponsiveness and inflammation in asthmatics and dogs. Here, for the first time, we compare the time course of airway hyperresponsiveness, inflammation, and marrow progenitor responses in a mouse model of airway allergen challenge. Sensitized BALB/c mice were studied at 2, 12, 24, 48, and 72 h after intranasal ovalbumin or saline challenges. Outcome measurements included airway responsiveness, airway inflammation as assessed via bronchoalveolar lavage (BAL) and lung tissue sections, and BM eosinophil colony-forming units (Eo-CFU) as enumerated using a semisolid culture assay with optimal concentrations of interleukin-5. We observed significant increases in BAL fluid eosinophils, neutrophils, lymphocytes, and macrophages by 2 h after the second of two intranasal allergen challenges (P < 0.05). Significant increases in airway responsiveness or BM Eo-CFU were observed at 24 h and persisted until 48 h...

Overexpression of alveolar macrophage gelatinase B (MMP-9) in patients with idiopathic pulmonary fibrosis: effects of steroid and immunosuppressive treatment.

Abstract: Alveolar macrophages (AM) express gelatinase B, a member of the matrix metalloproteinase family involved in the degradation and remodeling of extracellular matrix components. We evaluated the expression of gelatinase B in the course of idiopathic pulmonary fibrosis (IPF) by studying alveolar macrophages in culture AM and bronchoalveolar lavage fluid from 12 untreated patients with IPF, 11 patients with IPF under treatment with steroid and immunosuppressive agents, and 10 control subjects. By using zymography and quantitative image analysis, latent gelatinase B, as well an 88-kD active form, were investigated in culture medium (24 h) of AMs and were found to be significantly increased (P < 0.01) in untreated patients exhibiting severe IPF when compared with control subjects (4.1 +/- 1.7 versus 0.3 +/- 0.2 10(5) arbitrary units [AU]/10(4) AM for the 92-kD form). Concomitant studies of gelatinase B levels associated with cultured AM extracts or freshly harvested AM showed similar results, both at the mRNA and protein levels, respectively. Immunocytochemical studies on freshly harvested AM demonstrated that the enzyme was located mainly at the cell, suggesting some involvement of gelatinase B in AM migration. In contrast, gelatinase B activity secreted by AM tended to be normal in patients with IPF under steroid and immunosuppressive treatment. Simultaneously, level of the gelatinase B activity in epithelial lining fluid was increased in untreated IPF patients, whereas it was normal in treated patients. These results suggest that AM of patients with IPF are primed for gelatinase B expression and that steroid and immunosuppressive treatment induces negative modulation of the gelatinase B overexpression. We conclude that gelatinase B may play a role in lung remodeling in IPF.