Showing papers in "American Journal of Respiratory Cell and Molecular Biology in 1990"

Myofibroblasts and Subepithelial Fibrosis in Bronchial Asthma

Abstract: A thickened bronchial epithelial basement membrane has long been regarded as a histopathologic characteristic of bronchial asthma. As we had previously demonstrated that this phenomenon is due to the deposition of interstitial collagens and fibronectin, we have now sought to determine the nature of the cell responsible for this process by studying endobronchial biopsies from eight normal and seven asthmatic volunteers by immunohistochemistry and electron microscopy. Biopsies were stained with PR 2D3, a monoclonal antibody to myofibroblasts of the pericrypt sheath of the colon and a monoclonal antibody to alpha-smooth muscle actin. The thickness of the subepithelial collagen and the organelle content of the cells therein were determined by electron microscopy. The subepithelial collagen thickness in the normal subjects ranged from 2.16 to 6.26 microns, while that in the asthmatic subjects ranged from 3.75 to 11.1 microns (Mann-Whitney test; P = 0.05). Elongated cells in the collagen layer were identified by staining with PR 2D3. As this antibody also stains smooth muscle, consecutive frozen sections were stained for alpha-smooth muscle actin and the number of positive cells per millimeter of basement membrane was subtracted from the count for PR 2D3. This yielded a count of 4.9 to 9.4 cells/mm in the normal subjects and 11.9 to 20.6 cells/mm in the asthmatics (P = 0.001). There was a highly significant correlation between the depth of subepithelial collagen and the number of PR 2D3-positive, alpha-smooth muscle actin-positive cells (Spearman rank correlation; r = 0.764 and P = 0.006). Electron microscopy confirmed the myofibroblastic nature of these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Pulmonary surfactant protein A enhances the host-defense mechanism of rat alveolar macrophages.

Abstract: The effects of surfactant, surfactant lipids, and surfactant protein A (SP-A) on the surface phagocytosis of [3H]thymidine-labeled Staphylococcus aureus (SAE) by rat alveolar macrophages were studied. Alveolar macrophages only ingest SAE when the bacteria are opsonized with rat serum prior to incubation with alveolar macrophages. Preincubation or “opsonization” of the bacteria with surfactant did not result in phagocytosis by the macrophages. However, preincubation of the macrophages with surfactant increased the phagocytosis of rat serum-opsonized bacteria by approximately 70% when compared to the control macrophages. The factor present in surfactant causing the stimulation of the phagocytosis is probably SP-A. Preincubation of macrophages with human SP-A enhanced the phagocytosis to the same extent as whole surfactant, whereas preincubation with surfactant lipids had no effect on the phagocytosis. The SP-A-induced enhancement of the phagocytosis is time, temperature, and concentration dependent. Phagocy...

Pulmonary response to silica or titanium dioxide: inflammatory cells, alveolar macrophage-derived cytokines, and histopathology.

Abstract: We investigated the effects of silica (SiO2) and titanium dioxide (TiO2) on the pulmonary recruitment of inflammatory cells and the ability of alveolar macrophages (AMs) to release the pro-inflammatory cytokines, interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF). Rats were intratracheally instilled with 5 to 100 mg/kg of the materials, and bronchoalveolar lavage cell populations and AM cytokine release were characterized on days 1, 7, 14, and 28. Both dusts elicted dose-related increases in neutrophils, lymphocytes, and AMs; however, this response was more pronounced and persistent with SiO2. SiO2 at ≤ 50 mg/kg increased AM release of IL-1 and TNF at all time points; lower SiO2 doses had either a transient or no effect on AM-derived cytokines. TiO2 did not result in AM IL-1 release and increased TNF release transiently at doses ≤ 50 mg/kg. Both dusts primed AMs to release increased levels of IL-1 and TNF upon in vitro stimulation with lipopolysaccharide. Histopathology (day 28) demonstrated doser...

Is the bronchus-associated lymphoid tissue (BALT) an integral structure of the lung in normal mammals, including humans?

Abstract: In the respiratory tract, lymphoid aggregates with a specialized epithelium have been called bronchus-associated lymphoid tissue (BALT) and compared to the organized lymphoid tissue of the gut (GALT), e.g., Peyer's patches. BALT might play a central role in antigen uptake, initiating immune responses and disseminating primed lymphoid cells in the respiratory tract. In the present study, lungs of mice, rats, guinea pigs, rabbits, pigs, cats, and humans have been studied with respect to the presence and number of BALT and the dependence of BALT on age and microbial stimulation. BALT is not a constitutive structure in all these species. Its frequency varies widely, from 100% in rabbits and rats, 50% in guinea pigs, 33% in pigs, to its absence in cats and all normal human lungs. BALT seems to be a lymphoid structure which is not present in all the species studied but can develop in the lung after stimulation. This is in contrast to lymphoid organs, such as lymph nodes or Peyer's patches, which can always be f...

Degradation of airway neuropeptides by human lung tryptase.

Abstract: Several lines of evidence suggest a possible role for mast cell proteases in modulating the biologic effects of neuropeptides. To explore the potential of such interactions in human airway, we examined the activity of human tryptase, the major secretory protease of human lung mast cells, against several neuropeptides with proposed regulatory functions in human airway. Using highly purified tryptase obtained from extracts of human lung, we determined the sites and rates of hydrolysis of vasoactive intestinal peptide (VIP), peptide histidine-methionine (PHM), calcitonin gene-related peptide (CGRP), and the tachykinins substance P (SP), neurokinin A (NKA), and neurokinin B (NKB). Tryptase hydrolyzes VIP rapidly at several sites (Arg12, Arg14, Lys20, and Lys21) with an overall kcat/Km of 1.5 × 105 M−1 s−1 and hydrolyzes PHM primarily at a single site (Lys20) with a kcat/Km of 1.9 × 104 M−1 s−1. Tryptase also rapidly hydrolyzes CGRP at two sites (Arg18 and Lys24) with a kcat/Km of 2.1 × 105 M−1 s−1. The tachyk...

Human Alveolar Macrophage Gene Expression of Interleukin-8 by Tumor Necrosis Factor-α, Lipopolysaccharide, and Interleukin-1β

Abstract: The alveolar macrophage (AMO) in its pivotal position for pulmonary host defense may play a prominent role in the orchestration of polymorphonuclear leukocyte (PMN) diapedesis. We demonstrate that the hu- man AMO may participate in these inflammatory events through the production of a novel neutrophil chemotactic factor, interleukin-8 (IL-8). The induction of AMO-derived IL-8 by tumor necrosis factor (TNF), lipopolysaccharide (LPS), and interleukin-1 (IL-1β) was shown to be both dose and time depen- dent. Maximal IL-8 gene expression, as assessed by Northern blot analyses, was achieved with 20 ng/ml and 1 µg/ml, respectively, for each of the cytokines and LPS. A kinetic study of TNF-, IL-1β-, and LPS-treated AMOs showed significant steady-state IL-8 mRNA accumulation post-stimulation at 1 h, peaking by 8 h, with a decline over the next 16 h. Immunohistochemical staining using rabbit anti-human IL-8 antibody demonstrated significant immunolocalization of cell-associated IL-8 antigen at 4 h, with persistenc...

Neutrophil attractant/activation protein-1 (NAP-1 [interleukin-8]).

Abstract: Neutrophil attractant/activation protein-1 (NAP-1 [interleukin-8]) is an 8,400 D protein that is a chemoattractant and granule release stimulus for neutrophils. NAP-1 was first purified from culture fluids of lipopolysaccharide-stimulated human blood mononuclear leukocytes. It was subsequently isolated from lipopolysaccharide-stimulated lung macrophages, mitogen-stimulated lymphocytes, and virus-infected fibroblasts. Interleukin-1 or tumor necrosis factor induces NAP-1 mRNA in many cells, including monocytes, fibroblasts, and endothelial cells. NAP-1 belongs in a family of host defense small proteins, which have a degree of sequence and structural similarity. Noteworthy are the four half-cystine residues in each protein, which are in register when the protein sequences are suitably aligned. Based on cloning data and N-terminal sequence analyses, NAP-1 is secreted as a 79 residue protein after cleavage of a 20 residue signal peptide. The commonly isolated 77 and 72 residue forms are probably extracellular cleavage products. NAP-1 has considerable charge heterogeneity. Charge and length variants all have chemotactic activity. In contrast to many chemoattractants, NAP-1 does not attract monocytes. Intradermal injection of NAP-1 causes neutrophil infiltration. The wide spectrum of cell sources and production stimuli suggests that NAP-1 mediates neutrophil recruitment in host defense and disease.

Bombesin Increases Fetal Lung Growth and Maturation In Utero and in Organ Culture

Abstract: Pulmonary neuroendocrine cells (PNECs) in fetuses synthesize gastrin-releasing peptide (GRP, or mammalian bombesin) at high levels, but the role of this hormone in lung development has been obscure. The present study demonstrates that bombesin administered for 2 to 4 d toward the end of gestation in utero led to increased DNA (days 17 and 18) and saturated phosphatidylcholine (SPC) synthesis (day 18) in a dose-dependent fashion in fetal lung. These kinetics coincide with the timing of endogenous GRP gene activation in untreated fetal mouse lung, where GRP mRNA is detectable on day 16 and peaks at day 18. Electron microscopy on in vivo bombesin-treated fetal lung showed an increase in the number of cells containing lamellar bodies on both days 17 and 18, consistent with increased growth and/or maturation of type II cells. In mouse fetal lung organ cultures, the addition of bombesin led to accelerated uptake of [3H]thymidine into DNA, [3H]leucine into protein, and [3H]choline into SPC, indicating that incre...

The macrophage mannose receptor: current status.

Abstract: Sugar-specific recognition of oligosaccharide chains present on macromolecules and particles has emerged as an important mechanism by which macrophages carry out a variety offunctions including host defense, molecular scavenging, and cell-cell recognition. Among the oligosaccharide determinants that are recognized by macrophages, terminal mannose residues are the most thoroughly studied. More than a decade ago, in vivo experiments showed that a variety of glycoproteins bearing high mannose chains, including the lysosomal enzyme {j-glucuronidase, were rapidly and specifically cleared from the bloodstream after intravenous infusion (1). It is now widely appreciated that in vivo clearance of mannose glycoconjugates is mediated by a receptor found in liver Kupffer and endothelial cells and in mononuclear phagocytes (2). With the exception of lysosomal hydrolases, which possess high mannose chains as a remnant of the mannose-phosphate targeting marker, glycoproteins and membrane glycoconjugates of most higher organisms bear processed or complex N-linked oligosaccharide chains (3). As such, mannose is nearly always relegated to a penultimate or more inferior position in the carbohydrate structure. Lower organisms, however, including many infectious agents, have little facility to process N-linked oligosaccharide chains and, consequently, express abundant mannose, glucose, and other sugars on their surfaces. It is not surprising, then, that higher organisms have evolved sugar-specific recognition molecules as part oftheir host-defense system. Macrophages recognize, in addition to mannose and L-fucose, molecules and particles bearing galactose, glucose, and N-acetylneuraminic acid residues (2). The mannose receptor has served as an excellent model for studies of receptor recycling in the macrophage. The availability of several high affinity ligands and anti-receptor antibodies have permitted a detailed analysis of receptor movement in macrophages. {j-Glucuronidase, a lysosomal glycosidase that can be easily purified from rat preputial glands, and mannose-derivitized bovine serum albumin (mannoseBSA) , bind the mannose receptor with high affinity. Many other glycoconjugates have been used as ligands as well, including ricin A-chain, yeast mannan and invertase, ovalbumin, and ribonuclease B. Binding and internalization studies have shown that the mannose receptor is both phagocytic (4)

Clearance of neutrophil-derived myeloperoxidase by the macrophage mannose receptor.

Abstract: Uptake of neutrophil-derived myeloperoxidase by the macrophage mannose receptor was studied. Rat bone marrow-derived macrophages internalized 75% of [125I]myeloperoxidase through a mannose-specific process. Uptake via the mannose receptor is highly sensitive to treatment with oxidants. Treatment of rat macrophages with 1 mM H2O2 for 30 min resulted in a 94% reduction in uptake of myeloperoxidase. By Percoll gradient fractionation studies, 38% of internalized myeloperoxidase was delivered to the lysosomal compartment during a 15-min chase period, similar to findings for delivery of other ligands for this receptor. Once in the lysosome, the myeloperoxidase remained enzymatically active for several hours, with 50% activity remaining at 8 h. Finally, myeloperoxidase-containing macrophages had an increased capacity to down-regulate their own mannose receptors or receptors on neighboring macrophages, possibly through the myeloperoxidase-mediated production of oxidized halogens. Thus, the macrophage mannose rece...

Surfactant Apoprotein A (SP-A) Is Synthesized in Airway Cells

Abstract: The pulmonary surfactant apoproteins A, B, and C (SP-A, SP-B, and SP-C, respectively) function in concert with surfactant phospholipids to reduce surface pressure in the alveolus. Surfactant apoproteins also regulate surfactant synthesis, secretion, adsorption, and recycling. SP-A and B have been localized by immunocytochemistry to alveolar epithelial (type II) cells, alveolar macrophages, and nonciliated bronchiolar epithelial (Clara) cells. In contrast, in situ hybridization to SP-A and B mRNA in human lung has shown SP-A and B transcripts in type II cells, but only SP-B message in Clara cells, implying that synthesis of SP-A occurs exclusively in type II cells. In this report, in situ hybridization to SP-A mRNA was performed on adult and developing rabbit lung and on human lung. SP-A transcripts were found in type II cells and bronchiolar epithelium of both species. The distribution of SP-A message-containing cells in the bronchiolar epithelium of rabbits and humans was similar to the distribution of Clara cells in these two species. These data indicate that SP-A is not only synthesized in type II cells but also in Clara cells.

Specific binding of endothelin on human bronchial smooth muscle cells in culture and secretion of endothelin-like material from bronchial epithelial cells.

Abstract: Endothelin, synthesized by endothelial cells, is the most potent vasoconstrictor and bronchoconstrictor agent known. We investigated endothelin release from human bronchial epithelial cells and the binding of the peptide to autologous bronchial smooth muscle cells in culture. Epithelial and smooth muscle cells were isolated by enzymatic digestion of bronchial tissue obtained on surgery, and cultured to confluency by standard methods. Epithelial cells stained positively for cytokeratin filaments. Smooth muscle cells stained uniformly for alpha-smooth muscle actin. Immunoreactive endothelin contents in the supernatants of epithelial cells extracted on C8 Amprep columns were evaluated by radioimmunoassay. Epithelial cells released appreciable amounts of immunoreactive endothelin into the culture medium (from 0.65 to 2.1 pmol/ml). A single specific binding site for [125I]endothelin 1 was identified on bronchial smooth muscle cells with an apparent Kd of 113 pM and a maximal binding capacity of 22.1 fmol/106 c...

Smooth muscle cell markers in developing rat lung.

Abstract: We employed a panel of antibodies directed against cytoskeletal and contractile proteins in a developmental study to follow the differentiation and distribution of smooth muscle-like cells in the rat lung. We observed that, in the mesenchyme around developing airways and vessels, desmin replaces vimentin as the predominant intermediate filament as specialization toward smooth muscle occurs. Normally, desmin and smooth muscle myosin were expressed together in the cells and their acquisition appeared indicative of terminal differentiation of smooth muscle. In this regard, the maturation of vascular smooth muscle is delayed in the lung relative to that surrounding the developing air passages. alpha-smooth muscle actin-containing cells form a thicker coat around the primitive airway tubes and extend farther down the tree than desmin or smooth muscle myosin-positive cells. This suggests that the alpha-actin is a marker for initial differentiation of smooth muscle cells and that these cells arise from the enveloping mesenchyme. In the pseudoglandular and canalicular lung, alpha-actin-containing cells were also found in regions of epithelial tube cleft formation, suggesting an association with the process of branching morphogenesis. In addition, a large complement of alpha-actin-positive but smooth muscle myosin-negative cells were observed in the saccular interstitium during the period of secondary saccule formation and capillary reorganization that leads to final alveolarization. In summary, we note an association of smooth muscle-like, alpha-actin-containing cells with areas and periods of remodeling during normal pulmonary development. This observation may have relevance to the repair process in the adult lung.

Expression of Mucin Synthesis and Secretion in Human Tracheobronchial Epithelial Cells Grown in Culture

Abstract: The effects of culture conditions on growth and differentiation of human tracheobronchial epithelial (HTBE) cells have been defined. Epithelial cells were dissociated from tissues by protease treatment and were plated on tissue culture dishes in F12 medium supplemented with insulin, transferrin, epidermal growth factor, hydrocortisone, cholera toxin, bovine hypothalamus extract, and retinol. HTBE cells did not express any mucociliary function (ciliogenesis or mucin secretion) on tissue culture plastic, but they could be passaged 3 to 5 times with a total of 10 to 25 population doublings. Cells from early passages re-express both these functions when transplanted to tracheal grafts. When tissue culture plates were coated with collagen film or collagen gel substrata, cell attachment and proliferation were stimulated. However, the expression of mucous cell function in culture occurred only when cells were plated on collagen gel substrata and vitamin A (retinol) was present in the medium. Mucous cell differentiation under optimal conditions was defined by ultrastructural studies, by immunologic studies with mucin-specific monoclonal antibodies, and by carbohydrate and amino acid compositional analyses of mucin-like glycoproteins purified from culture medium. These results demonstrate for the first time that HTBE cells can express mucin synthesis and secretion under appropriate culture conditions.

Hypoxic contraction of cultured pulmonary vascular smooth muscle cells.

Abstract: The cellular events involved in generating the hypoxic pulmonary vasoconstriction response are not clearly understood, in part because of the multitude of factors that alter pulmonary vascular tone. The goal of the present studies was to determine if a cell culture preparation containing vascular smooth muscle (VSM) cells could be made to contract when exposed to a hypoxic atmosphere. Cultures containing only fetal bovine pulmonary artery VSM cells were assessed for contractile responses to hypoxic stimuli by two methods. In the first, tension forces generated by cells grown on a flexible growth surface (polymerized polydimethyl siloxane) were manifested as wrinkles and distortions of the surface under the cells. Wrinkling of the surface was noted to progressively increase with time as the culture medium bathing the cells was made hypoxic (PO2 approximately 25 mmHg). The changes were sometimes reversible upon return to normoxic conditions and appeared to be enhanced in cells already exhibiting evidence of some baseline tone. Repeated passage in culture did not diminish the hypoxic response. Evidence for contractile responses to hypoxia was also obtained from measurements of myosin light chain (MLC) phosphorylation. Conversion of MLC to the phosphorylated species is an early step in the activation of smooth muscle contraction. Lowering the PO2 in the culture medium to 59 mmHg caused a 45% increase in the proportion of MLC in the phosphorylated form as determined by two-dimensional gel electrophoresis. Similarly, cultures preincubated for 4 h with 32P and then exposed to normoxia or hypoxia for a 5-min experimental period showed more than twice as much of the label in MLCs of the hypoxic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of guinea pig tracheal epithelial cells maintained in biphasic organotypic culture: cellular composition and biochemical analysis of released glycoconjugates.

Abstract: An air-liquid interface (biphasic) primary culture system in which guinea pig tracheal epithelial cells maintain morphologic characteristics of differentiated epithelium has been developed in this laboratory. In this report, we compared quantitatively cell populations of 8-day cultures to those of epithelial mucosa in intact trachea. In addition, high molecular weight glycoconjugates released by the cultured cells were isolated and characterized. Quantitative morphometric analysis revealed similar volume densities of ciliated, secretory, basal, and “other” cells in cultures and in intact tracheal surface epithelium, although the cultures tended to have smaller cells and contained fewer basal cells. High molecular weight glycoconjugates released apically by cell cultures and excluded from Sepharose CL-4B columns contained approximately 5% hyaluronic acid but undetectable amounts of other proteoglycans, such as chondroitin sulfate, heparan sulfate, and dermatan sulfate. The hyaluronidase-resistant glycoconj...

The insulin-like growth factors and the lung.

Abstract: The insulin-like growth factors (IGF-I and IGF-II) are peptides of about 7,500 D with structural homology to proinsulin that are capable of stimulating cellular proliferation and inducing differentiation. They are each encoded by single, large, complex genes that direct the transcription of multiple mRNAs. Both genes are expressed in most organs and tissues, predominantly by cells of mesenchymal origin. Developmental factors are important in their regulation, with IGF-II's expression predominating prenatally and IGF-I's postnatally. In the fetus, placental lactogen can stimulate the synthesis of both IGF-I and IGF-II. After birth, however, growth hormone and nutritional status are the major regulators of IGF-I. In addition, a variety of other factors exert tissue-specific stimulation of IGF-I and IGF-II expression. The actions of the IGFs are mediated by interaction with the type 1 IGF cell surface receptor, which, like the IGFs, is expressed in most tissues. The biologic effects of the IGFs are modulated...

Cell Kinetics of Normal Adult Hamster Bronchial Epithelium in the Steady State

Abstract: To establish the progenitor role of bronchial epithelial cells in the steady state, we undertook a quantitative autoradiographic study in normal hamsters. Groups of 7 hamsters were killed 1 h and 1, 2, 3, 4, 7, and 14 d after an intraperitoneal injection of [3H]thymidine (2 microCi/g body wt). Autoradiograms were prepared from 861 Epon sections, 2 microns thick, of left intrapulmonary hilar bronchi. Epithelial cells were classified into 1 of 7 categories: basal-1 (B1) and basal-2 (B2), depending on nuclear height; secretory cells denoted as S1 with zero to 4 granules, S2 with 5 or more granules with intervening cytoplasm, and S3 with abundant granules completely filling the cytoplasm; ciliated (C); and indeterminate (IN). Mean silver grain counts decreased significantly over time only for B1 cells (P less than 0.05), with a cell cycle time of 20.6 d and a DNA synthetic time of 7.5 h. Labeled cells, 1 h after thymidine injection, comprised 30.5% S1, 27.8% B1, 22.8% B2, 6.8% IN, 6.4% S2, 5.7% C, and 0% S3 cells. Labeling indices of individual cell categories (LIc), at 1 h after labeling, were highest for B1 followed by B2 cells, reflecting their proliferative intensity. Labeling index of all epithelial cells combined did not change with time, indicating that there was no major cell death or label dilution. The LIc decreased significantly over time only for B1 and B2 cells (P less than 0.001 and P less than 0.002, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of a reconstituted basement membrane on expression of surfactant apoproteins in cultured adult rat alveolar type II cells.

Abstract: Pulmonary surfactant, which is composed of phospholipids and three lung-specific apoproteins, is synthesized and secreted by alveolar type II cells. Previous work from this laboratory (Biochim. Biophys. Acta 1987; 931:143-156) has shown that cell-extracellular matrix interactions and cuboidal cell shape affect both the ultrastructural appearance and pattern of phospholipids synthesized by cultured rat type II cells. In the present study, we have examined the effects of cell-matrix interactions and cell shape on the ability of adult rat type II cells to express the surfactant apoproteins in culture. Isolated adult rat type II cells were cultured for 2, 4, and 8 days on either tissue culture plastic, on an extract of the Engelbreth-Holm-Swarm (EHS) tumor, or on laminin-coated plastic dishes. Expression of surfactant proteins A, B, and C (SP-A, SP-B, and SP-C) was evaluated by Northern analysis using specific rat cDNA probes for these mRNAs. SP-A content was determined by enzyme-linked immunosorbent assay using a polyclonal antibody raised against rat SP-A purified from lavage. Type II cells cultured on plastic dishes assumed an attenuated morphology soon after being placed in culture. Except for an occasional positive signal on day 2 of culture, these cells were uniformly negative for the presence of mRNA for SP-A, SP-B, or SP-C. Type II cells cultured on plastic did not contain SP-A. In contrast, type II cells cultured on EHS gels formed three-dimensional aggregates on the surface of the substratum; these aggregates were composed of polarized cells that had their apical surfaces directed inward. Type II cells cultured on this substratum showed a positive signal for mRNA for all three surfactant proteins; the abundance of these mRNAs, however, was significantly below that seen in freshly isolated type II cells. While the abundance of mRNA for SP-A and SP-B steadily increased with time in culture under these conditions, the abundance of SP-C mRNA decreased, suggesting that SP-C is regulated independently of SP-A and SP-B. These cultures were also positive for SP-A content, which increased with increasing time in culture. Type II cells cultured on laminin-coated dishes initially spread more slowly across the culture surface than cells on plastic, but were extremely attenuated by day 8 in culture. These cells contained neither SP-A, nor mRNA for any of the three surfactant proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Iloprost inhibits neutrophil-induced lung injury and neutrophil adherence to endothelial monolayers.

Abstract: We hypothesized that Iloprost, a long-acting prostacyclin analog, would inhibit neutrophil (PMN)-induced lung injury and decrease PMN adherence to vascular endothelium. Human PMNs infused into isolated buffer-perfused rat lungs subsequently stimulated with phorbol myristate acetate (PMA) resulted in lung injury as assessed by the accumulation of [125I]bovine serum albumin (125I-BSA) in lung parenchyma and alveolar lavage fluid. Addition of Iloprost to the lung perfusate, prior to activation of the PMNs, reduced lung injury as assessed by a decrease in the accumulation of 125I-BSA in the lung. This protective effect was not due to the vasodilatory effect of Iloprost. Protection by Iloprost was not linked to a reduction in PMA-induced PMN superoxide production since Iloprost did not reduce the amount of superoxide released into lung perfusate. In vitro, Iloprost caused a dose-depedent inhibition of PMA-stimulated PMN adherence to endothelial cells. Iloprost did not affect the number of Mol adhesion molecule...

Human Ciliated Bronchial Epithelial Cells: Expression of the HLA-DR Antigens and of the HLA-DR Alpha Gene, Modulation of the HLA-DR Antigens by Gamma-Interferon and Antigen-presenting Function in the Mixed Leukocyte Reaction

Abstract: HLA-DR class II molecules are expressed by a variety of nonlymphoid cells, including the respiratory epithelium. However, it is not known if ciliated bronchial epithelial cells express the HLA-DR genes, if the expression of class II molecules on their surface can be modulated by immune mediators and, finally, if these cells, like other HLA-DR-positive epithelial cells, have the potential to serve as antigen-presenting cells. To answer these questions, we collected ciliated bronchial epithelial cells by brushing and by suction during fiberoptic bronchoscopy and by scraping surgically resected bronchi. The number of cells recovered by brushing or suction during fiberoptic bronchoscopy was similar (P greater than 0.2), but lower than that obtained by scraping surgically resected bronchi (P less than 0.01); however, compared with brushing, suction of ciliated bronchial epithelial cells resulted in a better viability (P less than 0.05). HLA-DR antigens on ciliated bronchial epithelial cells were detected by immunofluorescence using the PTF 29.12 and the L243 monoclonal antibodies, both recognizing HLA-DR molecules on the vast majority of ciliated bronchial epithelial cells. Cytoplasmic dot blot analysis demonstrated that ciliated bronchial epithelial cells had mRNA HLA-DR transcripts, and Northern blot hybridizations showed that the size of the HLA-DR messages was the same observed in other HLA-DR-positive cells. Interestingly, ciliated bronchial epithelial cells showed a significant decline of HLA-DR expression after 5 days in culture, but the addition of gamma-interferon to the cell cultures was associated with the persistence of the expression of class II antigens on the cell surface (P less than 0.01 with control cultures at 5 days). Finally, while ciliated bronchial epithelial cells were ineffective in stimulating allogeneic T cell proliferation in a 6-day primary mixed leukocyte reaction (MLR), the addition of phorbol myristate acetate to the MLR was able to induce a significant T cell proliferation (P less than 0.001, all comparisons). Thus, human ciliated bronchial epithelial cells express HLA-DR surface antigens and have mRNA molecules for the HLA-DR genes, and the expression of the surface class II antigens can be modulated in vitro by immune mediators.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of activated eosinophils and neutrophils on guinea pig airway epithelium in vitro.

Abstract: Epithelial shedding is a characteristic feature of asthmatic airways and has been attributed to eosinophil products. We have examined the interaction of purified intraperitoneal guinea pig eosinophils with or without platelet-activating factor (PAF, 10(-7) M) or lyso-PAF (10(-7) M) with guinea pig tracheal epithelium in vitro. At 0, 4, 14, and 24 h, the percentage of ciliation of the tracheal circumference (CTC) was measured by light microscopy and the ciliary beat frequency (CBF) by photometry. PAF-activated eosinophils (50 x 10(6) cells/ml) disrupted the epithelium, mean CBF and CTC being reduced by 77.8 +/- 5.8% (mean +/- SEM; P less than 0.001 versus control) and 94.2 +/- 1.4% (P less than 0.001) over 24 h, respectively. PAF (10(-7) M) alone had no significant effect. Lyso-PAF with eosinophils (50 x 10(6) cells/ml) also reduced mean CBF and CTC but to a lesser extent. Eosinophils alone also led to a reduction of 36.2 +/- 11.4% in mean CBF and 53.0 +/- 15.5% in CTC, but these changes were not significant. The PAF antagonist, WEB 2086 (10(-6) M), significantly inhibited the mean CBF and CTC reduction due to PAF-activated eosinophils by 61.5 +/- 17.2% (P less than 0.01) and 20.8 +/- 6.5% (P less than 0.05), respectively. In addition, catalase (1,125 U/ml) partially inhibited the mean CBF and CTC reduction induced by PAF-activated eosinophils. Intraperitoneal neutrophils (PMN) (50 x 10(6) cells/ml) also disrupted epithelium but to a lesser extent (24-h reduction: 34.2 +/- 12.7% for mean CBF and 60.2 +/- 13.2% for CTC, respectively). Stimulation with PAF (10(-7) M) had no further effect. Marked exfoliation of the epithelial layer was observed after 14 h of incubation with activated eosinophils. We concluded the PAF-activated eosinophils are capable of grossly disrupting ciliated epithelium and may contribute to epithelial damage observed in asthma.

Endothelial Heparan Sulfate Proteoglycan. I. Inhibitory Effects on Smooth Muscle Cell Proliferation

Abstract: Proliferation of smooth muscle cells is an important component of pulmonary arterial morphogenesis, both during normal development and pathologic remodeling. However, little is known of the factors that regulate smooth muscle proliferation in these vessels. To investigate the hypothesis that factors produced by endothelial cells may regulate smooth muscle cell growth, we studied the effects of culture medium conditioned by fetal bovine pulmonary arterial endothelium on proliferation of smooth muscle cells in culture. This conditioned medium contains an inhibitor of smooth muscle proliferation that is degraded by nitrous acid, heparinase, and heparitinase, but resists degradation by protease, boiling, and chondroitin ABC lyase, indicating that the inhibitor is structurally similar to heparin. Inhibitor release occurs in both growing and confluent endothelial cell cultures and in the presence and absence of serum. A growth-inhibiting proteoglycan purified to homogeneity from endothelial cell-conditioned medium has physicochemical characteristics similar to those of the prototypic basement membrane heparan sulfate proteoglycan of the Englebreth-Holm-Swarm tumor: an overall size of approximately 10(6) D, heparan sulfate chains of 60,000 D, and a buoyant density of 1.33 g/ml. Antibody raised against the tumor basement proteoglycan recognizes this endothelial heparan sulfate proteoglycan, and Western blotting after SDS-PAGE demonstrates that the core proteins of both proteoglycans migrate as a doublet at apparent molecular weights of 450,000 and 360,000 D. Heparan sulfate glycosaminoglycan prepared from purified medium proteoglycan is a potent inhibitor of smooth muscle cell growth, exhibiting activity approximately 1,000 times greater than that of heparin. These results indicate that endothelial cells cultured from fetal bovine pulmonary arteries produce a basement membrane heparan sulfate proteoglycan that is a potent inhibitor of smooth muscle proliferation. This proteoglycan may mediate endothelial regulation of smooth muscle growth during development or pathologic pulmonary arterial remodeling.

Macrophages: important physiologic and pathologic sources of polypeptide growth factors.

Abstract: The monocyte-macrophage, a bone marrow-derived cell recognized by Metchnikoff more than 100 years ago as important for host defense, has more recently been identified as a mediator of a variety of essential immunologic and nonimmunologic biologic activities (1). Macrophages also have been identified as a central component of the acute inflammatory phase of tissue repair, and have been found essential for normal wound healing (2). Thus, the critical role for macrophages in wound healing provides a useful paradigm for examining their possible roles in other physiologic and pathologic processes. Recently, polypeptide growth factors have been identified as soluble mediators of many biologic activities considered intrinsic for tissue repair, including cellular proliferation, chemotaxis, and differentiation (3). Because wound macrophages synthesize many polypeptide growth factors, they may provide an important endogenous source of factors to the wound. Macrophage-derived growth factors include platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-tJ), basic fibroblast growth factor (bFGF) , and transforming growth factor alpha (TGF-a) (4). Locally applied PDGF enhances and accelerates recruitment of macrophages into experimental wounds, and subsequently activates them to synthesize increased amounts of endogenous growth factors, including TGF-tJ (2,5); PDGFtreated wounds have increased wound strength, increased extracellular matrix (granulation tissue) generation, and enhanced fibroblast recruitment and activation (5, 6). In experimental wounds rendered deficient in repair due to elimination of wound macrophages (i.e., glucocorticoid treatment or total body irradiation), PDGF is ineffective in reversing the deficit, indicating a requirement for wound macrophages to transduce the PDGF signal (2). In contrast, in wounds in which local fibroblast activity is abrogated (i.e., surface irradiation), PDGF enhances macrophage recruitment into the wound and reverses the wound-healing deficit.

Extracellular glutathione suppresses human lung fibroblast proliferation.

Abstract: Alveolar epithelial lining fluid glutathione (GSH) is markedly decreased in patients with idiopathic pulmonary fibrosis (IPF). Because patients with IPF have exaggerated numbers of fibroblasts in their lower respiratory tract, we hypothesized that GSH can suppress lung fibroblast proliferation. To verify this hypothesis, we examined the ability of GSH to suppress human lung fibroblast (ATCC; HFL-1) proliferation in vitro in the presence of either IPF bronchoalveolar lavage fluid (BAL) or calf serum (CS). Both CS at a concentration of 10% and IPF BAL markedly increased fibroblast proliferation when compared to cells grown without CS or IPF BAL (10% CS = 93 +/- 4%, P less than 0.001; IPF BAL = 47 +/- 4%, P less than 0.001). In the presence of physiologic concentrations of GSH (0 to 500 microM), both CS- and IPF BAL-mediated fibroblast proliferation were markedly reduced, with 500 microM GSH inducing complete inhibition. Interestingly, glutathione disulfide (GSSH) and S-methylglutathione did not suppress proliferation, whereas various sulfhydryl-containing molecules (cysteine, N-acetylcysteine, 2-mercaptoethanol, and low concentrations of dithiothreitol) induced an inhibition of fibroblast proliferation similar to that observed with GSH. Most of the suppressive effect of GSH was mediated at the cell level since incubation of fibroblasts with 500 microM GSH for 1 h completely blocked the ability of the cells to subsequently proliferate in the presence of untreated 10% CS. Treatment of CS with 500 microM GSH for 1 h followed by removal of GSH by molecular sieve chromatography had no detectable effect on fibroblast proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)

Entactin: Structure and Function

Abstract: Entactin is an integral and ubiquitous component of the basement membrane. The amino acid sequences of the mouse and human molecules have been determined and exhibit 85% sequence identity. The molecule is organized into three structural domains, an N-terminal globule (I) is linked to a smaller C-terminal globule (III) by a rigid stalk (II) largely consisting of cysteine-rich EGF-like homology repeats and a cysteine-rich thyroglobulin homology repeat. The molecule binds calcium ions and supports cell adhesion. However, its major function may be the assembly of the basement membrane. The carboxyl globule binds tightly to one of the short arms of laminin at the inner rodlike segment. This same region is also believed to be responsible for the attachment of entactin to type IV collagen at approximately 80 nm from its carboxyl noncollagenous end. Entactin therefore could serve as a bridge between the two most abundant molecules in the basement membrane. Supporting evidence for this role has been obtained from ...

Human alveolar macrophage and blood monocyte interleukin-6 production.

Abstract: Interleukin-6 (IL-6) modulates a number of processes relevant to host immunity and inflammation. We investigated the capacity of the human alveolar macrophage to elaborate IL-6 in response to lipopolysaccharide (LPS), recombinant interleukin-1 (rIL-1), and recombinant tumor necrosis factor (rTNF), and compared macrophage IL-6 production to that of blood monocytes and lung fibroblasts. Unstimulated and TNF-stimulated alveolar macrophages and monocytes produced little or no detectable IL-6. In contrast, macrophages and monocytes produced large amounts of IL-6 in response to LPS and monocytes produced lesser but readily detectable amounts in response to rIL-1. Monocytes and alveolar macrophages differed significantly in their capacity to produce IL-6, with macrophages making more IL-6 in response to LPS and less IL-6 in response to rIL-1 than autologous blood monocytes. Monocytes aged in vitro produced little detectable IL-6 in response to LPS or rIL-1, suggesting that differences in cell maturity may account for the diminished capacity of the alveolar macrophage to produce IL-6 in response to IL-1 but not its enhanced capacity to produce IL-6 in response to LPS. Mononuclear phagocytes and lung fibroblasts also differed in their ability to produce IL-6. Lung fibroblasts produced more IL-6 in response to rIL-1 and less IL-6 in response to LPS than monocytes and macrophages. In addition, monocytes and macrophages elaborated electrophoretically identical IL-6 moieties that differed from those produced by lung fibroblasts. These differences could be at least partially attributed to differences in sialylation and/or glycosylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical localization of peptide-containing nerves in human airways: age-related changes.

Abstract: Bronchial reactivity changes during childhood, indicating possible changes in neural control. Nerves supplying the intrapulmonary airways were therefore studied in autopsy tissue from 14 normal infants (0 to 3.5 yr), 3 children (8.3 to 10.75 yr), and 4 adults (17 to 24 yr). An indirect immunofluorescence technique was used to study the distribution and relative number of nerve fibers containing the general neuronal markers protein gene product 9.5 and synaptophysin. Nerve subpopulations were identified using antisera to neuropeptide tyrosine, vasoactive intestine polypeptide, somatostatin, substance P, calcitonin gene-related peptide, and the enzyme tyrosine hydroxylase. Between birth and 3 yr, the distribution and relative number of immunoreactive nerves shown by both the general neuronal markers and specific antisera did not change. Neuropeptide tyrosine-immunoreactive nerves were the most common peptide-containing nerve subpopulation identified in the human lung, supplying bronchial smooth muscle, submucosal glands, cartilage, and submucosa. Other peptide-containing nerves exhibited distinct distribution patterns. Two differences in the airway innervation were identified between cases aged 0 to 3.5 yr and the older age groups. Relatively fewer peptide-containing nerves occurred in the adult bronchioli and respiratory unit, but the relative number of vasoactive intestinal polypeptide-containing nerves supplying the bronchial and bronchiolar smooth muscle was greater in the two older age groups. Given these apparent age-related differences in the number of peptide-containing nerves supplying the human airway, studies on the development of peptide receptors are indicated.

In vitro dissolution of uniform cobalt oxide particles by human and canine alveolar macrophages.

Abstract: Intracellular dissolution of inhaled particles is an important pathway of clearance of potentially toxic materials. To study this process, monolayers of human and canine alveolar macrophages (AM) were maintained alive and functional in vitro for more than 2 wk. Complete phagocytosis of moderately soluble, monodisperse 57Co3O4 test particles of four different sizes was obtained by optimizing the cell density of the monolayer and the particle-to-cell ratio. The fraction of the initial particle mass that was soluble increased over time when the particles were ingested by AM but remained constant when in culture medium alone. Smaller particle sizes had a faster characteristic intracellular dissolution rate constant than did larger particles.The dissolution rates differed between AM obtained from two human volunteers as compared to those obtained from six mongrel dogs. These in vitro dissolution rates were very similar to in vivo translocation rates previously obtained from human and canine lung clearance stud...

Epithelial progenitor cells in the rat trachea

Abstract: Highly pure populations of basal and secretory cells from the rat trachea have been inoculated into denuded tracheal grafts to determine the differentiation pathways of these two cell types. The basal cell inoculum resulted in an epithelium comprised of only basal and ciliated cells, while the secretory cell inoculum gave rise to an epithelium comprised of secretory, basal, and ciliated cells. These results show that, in the rat trachea, the secretory cell is the major progenitorial cell type and the basal cell has only limited progenitorial capacity.