Transforming growth factor-beta(1) modifies fibroblast growth factor-2 production in type II cells.

Transforming growth factor (TGF)-beta(1) is an inflammatory cytokine that plays multiple roles in pulmonary fibrosis. In vascular epithelium, it has been shown to regulate production and activity of fibroblast growth factor (FGF)-2, a potent type II cell mitogen in the lung. Such a relationship could have important consequences in prefibrotic change in the lung alveolus, where reepithelialization of alveolar surfaces is crucial. The goal of this study was to determine if FGF-2 production by alveolar type II cells is modulated by TGF-beta(1) or FGF-1, another type II cell mitogen. Isolated rat type II cells were exposed to 0 to 40 ng/mL of TGF-beta(1) or 0 to 500 ng/mL of FGF-1 in serum-free medium for 1 to 3 days. Using a specific immunoassay, significant increases in FGF-2 protein in type II cell lysates were achieved after 1 day of exposure to 100 ng/mL of FGF-1 and after 3 days of treatment with 8 ng/mL of TGF-beta(1). Similarly, transcripts for FGF-2 were dramatically increased with TGF-beta(1) or FGF-1, as were those for FGF receptor (FGFR)-1. These interactions were dramatically effected by the addition of heparin, a model sulfated extracellular matrix (ECM). Heparin as low as 0.01 mg/mL significantly downregulated expression of TGF-beta(1) and FGF-1-stimulated FGF-2 and FGFR-1. These results demonstrate important regulatory links between FGF-2, sulfated ECMs, and both TGF-beta(1) and FGF-1, which could contribute to the modulation of normal cell turnover, development, and repair processes attendant to fibrosis in the lung.

Vanadium stimulates human bronchial epithelial cells to produce heparin-binding epidermal growth factor-like growth factor: a mitogen for lung fibroblasts.

The bronchial epithelium is a potential source of growth factors that could mediate airway fibrosis during the progression of diseases such as asthma and chronic bronchitis. We report that conditioned medium (CM) from normal human bronchial epithelial cells (NHBECs) contains mitogenic activity for human lung fibroblasts that is blocked by the epidermal growth factor receptor (EGF-R) tyrosine kinase inhibitor AG1478 and by neutralizing antibodies raised against heparin-binding epidermal growth factor-like growth factor (HB-EGF). Neutralizing antibodies against other EGF-R ligands (EGF and transforming growth factor-alpha) or other antibodies against growth factors (platelet-derived growth factors, insulin-like growth factor-1) had no affect on the mitogenic activity of NHBEC-CM. HB-EGF messenger RNA (mRNA) expression in NHBEC was detected by reverse transcriptase/polymerase chain reaction and Northern blot analysis. HB-EGF protein was detected by enzyme-linked immunosorbent assay. Vanadium pentoxide (V2O5), a fibrogenic metal associated with occupational asthma, caused a several-fold increase in HB-EGF mRNA expression and protein, whereas the inert metal titanium dioxide had no effect on HB-EGF expression. V2O5-induced HB-EGF mRNA expression was inhibited by the EGF-R tyrosine kinase inhibitor AG1478, the p38 mitogen-activated protein (MAP) kinase inhibitor SB203580, and the MAP kinase kinase inhibitor PD98059. Finally, HB-EGF induced the production of fibroblast growth factor (FGF)-2 by human lung fibroblasts and anti-FGF-2 antibody partially blocked the mitogenic activity of NHBEC-CM on fibroblasts. These data suggest that HB-EGF is a fibroblast mitogen produced by NHBECs and that induction of an FGF-2 autocrine loop in fibroblasts by HB-EGF accounts for part of this mitogenic activity.

TGF-beta1 and fibroblast growth factor-1 modify fibroblast growth factor-2 production in type II cells.

Fibroblast growth factor (FGF)-2, which stimulates DNA synthesis by type II cells in the lung, has been shown to be regulated by transforming growth factor (TGF)-beta1, an important inflammatory cytokine, in vascular epithelium. The goal of this study was to determine if FGF-2 production by alveolar type II cells is modulated by TGF-beta1 or FGF-1, which also stimulates DNA synthesis by type II cells. Isolated rat type II cells were exposed to 0-40 ng/ml of TGF-beta1 or 0-500 ng/ml of FGF-1 in serum-free medium for 1-5 days. With a specific immunoassay, significant increases of FGF-2 protein in type II cell lysates to levels above those in control cells were achieved after 1 day of exposure to 100 ng/ml of FGF-1 and after 3 days of treatment with 8 ng/ml of TGF-beta1. Similarly, transcripts for FGF-2 were dramatically increased above those in control cells with TGF-beta1 or FGF-1, as were those for FGF receptor-1. These results demonstrate important regulatory links between FGF-2 and both TGF-beta1 and FGF-1 in the alveolar epithelium that could contribute to the regulation of normal cell turnover, development, and the repair processes after injury in the lung.

Three-dimensional organization of the lamina reticularis in the rat tracheal basement membrane zone.

The airway basement membrane zone is a region specialized for the attachment of the epithelium with the matrix. The epithelium is attached to the lamina densa, which, in turn, is connected to types I and III collagen of the lamina reticularis with anchoring fibrils. The purpose of this study was to define the three-dimensional organization of the structural proteins of the lamina reticularis in the rat trachea. We approached this problem by using whole mounts to look down on the flat surface of the basement-membrane zone rather than a cross section of its thin profile. Fluorescent microscopy with long working distance water immersion objectives and scanning electron microscopy revealed that the structural proteins are arranged as a mat of large fibers oriented along the longitudinal axis of the airway. Smaller fibers are crosslinked with the larger fibers to complete this structure. Other small fibers are oriented around the large fibers and an amorphous material covers individual fibers. The large fibers oriented along the longitudinal axis of the airway are consistent with prior descriptions of fibers composed of collagen III with some collagen I and V; small fibers encircling the large fibers may be collagen VI. The crosslinking fibers are made up of elastin and probably elastin-associated microfibrils. The amorphous proteins covering the fibrous framework may contain proteoglycans and other nonstructural proteins reported to be in the lamina reticularis. The present studies demonstrate that the structural proteins of the lamina reticularis in the rat trachea are arranged as fibers in a highly organized manner.

Sulfation of extracellular matrices modifies growth factor effects on type II cells on laminin substrata.

The alveolar basement membrane contains a variety of extracellular matrix (ECM) molecules, including laminin and sulfated glycosaminoglycans of proteoglycans. These mixtures exist within microdomains of differing levels of sulfate, which may specifically interact to be key determinants of the known capacity of the type II cell to respond to certain growth factors. Isolated type II cells were exposed to either acidic fibroblast growth factor (FGF-1), basic fibroblast growth factor (FGF-2), or keratinocyte growth factor (KGF; FGF-7) on culture wells precoated with laminin alone or in combination with chondroitin sulfate (CS), high-molecular-weight heparin, or their desulfated forms. Desulfated heparin significantly elevated FGF-1- and FGF-2-stimulated DNA synthesis, whereas desulfated CS and N-desulfated heparin elevated FGF-7-stimulated DNA synthesis by type II cells on laminin substrata. When FGF-1 was mixed into the various test matrix substrata, DNA synthesis was significantly increased in all cases. These results demonstrated that decreased levels of sulfate in ECM substrata act to upregulate responses to heparin-binding growth factors by alveolar epithelial cells on laminin substrata.

Biosynthesis of sulfated extracellular matrices by alveolar type II cells increases with time in culture.

The aim of this study was to determine the extent to which sulfate incorporated into biosynthesized basement membrane (BM) components increased as isolated type II cells progress toward a more type I cell-like phenotype from 7 to 21 days in culture. Specific sulfate cytochemistry, using high iron diamine, showed that type I-like cells in 21-day cultures deposited a more highly sulfated extracellular matrix. Biosynthetic labeling experiments using [35S]cysteine or [35S]sulfate as precursors confirmed the increased capacity of 21-day type I-like cells to biosynthesize sulfated BM components compared with type II-like cells in 7-day cultures, including a novel sulfated laminin. These biochemical changes in sulfation of BM components coincide with the established phenotypic transition from type II to type I cells during prolonged culture. More importantly, the data suggest that regulation of sulfation constitutes a potential mechanism by which type I and type II cells alter their environment in such a manner as to stabilize phenotype and modulate responses to growth factors.

Basement membranes and pulmonary development.

Basement membranes (BM) are specialized extracellular matrices (ECM) which serve as complex interfaces between epithelia, peripheral nerves, or muscle cells and their surrounding tissue microenvironments. Their composition is known to include type IV collagen, laminin, entactin, heparan sulfate proteoglycan (HSPG, perlecan), and chondroitin sulfate proteoglycan (CSPG). By immunohistochemistry, collagen IV, laminin, and entactin are detectable from day 14 of gestation on, and become progressively more prominent with time. Perleaan has not been examined in prentatal lungs, but is widely distributed and abundant in all lung MBs from birth throughout development. CSPG has a somewhat discontinuous and lightly reactive appearance in alveolar BMs at birth but the staining becomes continuous and darker in the adult. This contrasts with glycosaminoglycan, chondroitin sulfate, which is prominently expressed in prenatal and early postnatal stages, but progressively diminishes with advancing development. As an interface between cell populations and surrounding ECMs, BMs act as a physical barrier to some cells and molecules, while serving as attachment points and binding sites for others. Basic fibroblast growth factor is an example of the latter, because it localizes with all BM components by immunostaining throughout development and reflects the multifactorial array of potential effectors in the complex processes of proliferation and differentiation.

Phenobarbital-type P-450 inducers protect rats against metaldehyde toxicity.

The mechanism of metaldehyde toxicity is unclear. It may be due to the compound itself or, at least in part, to acetaldehyde resulting from the hydrolysis of metaldehyde in the stomach. In this study, we orally dosed rats with twice the LD50 of metaldehyde following no pretreatment (control) or pretreatment with 1 of 3 different cytochrome P-450 inducers either phenobarbital or o,p'-DDD (inducers of cytochromes P-450 IIB and IIIA) or 3-methylcholanthrene (an inducer of P-450 IA). Our results show strong protection against metaldehyde poisoning afforded by the phenobarbital-DDD P-450 inducers, but only weak protection with 3-methylcholanthrene pretreatment. Acetaldehyde administered at the same molarity failed to produce the clinical signs of metaldehyde toxicity and no clinical differences were observed between any of the pretreated groups.

Sulfation of extracellular matrices modifies responses of alveolar type II cells to fibroblast growth factors.

The pulmonary alveolar basement membrane (BM) associated with alveolar type II cells has been shown to be significantly less sulfated than that of type I cells. To examine the biological significance of this observation, we measured the incorporation of 5-bromodeoxyuridine (BrdU) as an indicator of DNA synthesis in isolated rat type II cells cultured for 72-120 h on substrata that were naturally sulfated, not sulfated, or chemically desulfated in serum-free, hormonally defined media, with and without selected growth factors. The percentage of cells incorporating BrdU was significantly elevated by desulfated chondroitin sulfate in the presence of fibroblast growth factor-2 (FGF-2 or basic FGF) and depressed by heparin in the presence of either FGF-1 or acidic FGF or FGF-2. This depressive effect was lost by removing sulfate from the heparin. Some responses were dependent on the period of time in culture and concentration and molecular weight of the substrata. These observations support the notion that sulfation per se of certain components of BM is a key determinant of type II cell responses to select growth factors that may define patterns of proliferation and differentiation.

Evaluation of an HPTLC method for the determination of strychnine and crimidine in biological samples.

In an attempt to improve the sensitivity and selectivity of thin-layer chromatographic analysis for the detection of strychnine and crimidine in biological samples, a rapid high-performance thin-layer chromatographic (HPTLC) method with densitometry is described. Fortified dog serum and stomach content samples were analyzed after extraction with chloroform. Quantitation was achieved by densitometry in the ultraviolet (UV) range (260 nm) of HPTLC silica gel 60 plates. Detection of trace levels as low as 5 ng proved feasible. Linearity was obtained over a range of 10-250-ng deposits for crimidine and 12.5-250-ng deposits for strychnine with simple or F254 plates. No interferences were observed in the UV spectra (220-380 nm) when peaks obtained with HPTLC of the standard substances and positive biological contents were scanned.

Cell surface-bound elastase and cathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases.

Serine proteinases of human polymorphonuclear neutrophils play an important role in neutrophil-mediated proteolytic events; however, the non-oxidative mechanisms by which the cells can degrade extracellular matrix in the presence of proteinase inhibitors have not been elucidated. Herein, we provide the first report that human neutrophils express persistently active cell surface-bound human leukocyte elastase and cathepsin G on their cell surface. Unstimulated neutrophils have minimal cell surface expression of these enzymes; however, phorbol ester induces a 30-fold increase. While exposure of neutrophils to chemoattractants (fMLP and C5a) stimulates modest (two- to threefold) increases in cell surface expression of serine proteinases, priming with concentrations of lipopolysaccharide as low as 100 fg/ml leads to striking (up to 10-fold) increase in chemoattractant-induced cell surface expression, even in the presence of serum proteins. LPS-primed and fMLP-stimulated neutrophils have approximately 100 ng of cell surface human leukocyte elastase activity per 10(6) cells. Cell surface-bound human leukocyte elastase is catalytically active, yet is remarkably resistant to inhibition by naturally occurring proteinase inhibitors. These data indicate that binding of serine proteinases to the cell surface focuses and preserves their catalytic activity, even in the presence of proteinase inhibitors. Upregulated expression of persistently active cell surface-bound serine proteinases on activated neutrophils provides a novel mechanism to facilitate their egress from the vasculature, penetration of tissue barriers, and recruitment into sites of inflammation. Dysregulation of the cell surface expression of these enzymes has the potential to cause tissue destruction during inflammation.

Heterogeneity of sulfated microdomains within basement membranes of pulmonary airway epithelium.

The purpose of this study was to determine whether the cytochemically defined distribution of sulfated macromolecules is significantly different in microdomains of basement membranes (BMs) associated with different levels of pulmonary airways. The high iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) technique, which is highly specific for sulfate esters of glycosaminoglycans and some glycoproteins, was used as a probe to compare the BM of trachea, bronchi, and three different sizes of bronchioles. When HID-reactive sites were counted and statistically compared, significant differences were found between the three known anatomically distinct layers of the BM--lamina lucida (LL), lamina densa (LD), and lamina reticularis (LR)--relative to the airway level. The highest concentration of HID reactivity in trachea, bronchi, and large bronchiole was found in LR and the lowest in LD. By comparison, HID-reactive sites were found to be more concentrated in the LL in medium and small bronchioles. HID reactivity was consistently low in LD as compared with LL and LR in all five locations. The overall degree of HID reactivity in BMs was clearly highest in large bronchioles and lowest in medium and small bronchioles. This cytochemically detectable heterogeneity in the distribution of HID reactivity in BM microdomains may represent specific compositional differences in pulmonary BMs which are important determinants of epithelial cell function and might be expected to impact key biologic processes in normal and pathologic states.

Immunohistochemical localization of chondroitin sulfate, chondroitin sulfate proteoglycan, heparan sulfate proteoglycan, entactin, and laminin in basement membranes of postnatal developing and adult rat lungs.

Histologic preparations of lungs from 1-, 5-, 10-, 18-, and 25-day-old postnatal and adult rats were examined immunohistochemically with antibodies specific against chondroitin sulfate (CS), basement membrane chondroitin sulfate proteoglycan (BM-CSPG), heparan sulfate proteoglycan (HSPG), entactin, and laminin. A monoclonal antibody specific for the glycosaminoglycan portion (CS) of CSPG and a monoclonal antibody against the core protein of CSPG were used in an immunoperoxidase sequence to stain extracellular matrix (ECM) components of pulmonary basement membranes (BMs). Anti-CS stained airway BM strongly and alveolar BM weakly in the adult rat lung, as well as in vascular and airway adventitia. In developing lungs, immunoreactivity was strong in all ECM sites, including BM, at day 1 postnatal, and progressively diminished thereafter except in vascular and airway adventitia. Anti-CSPG stained alveolar, airway, and vascular BMs, in addition to smooth muscle external laminae (EL), in the adult and developing rat. Immunostaining for CSPG required hyaluronidase digestion, whereas CS staining was lost with the same treatment. A polyclonal antibody to the core protein of HSPG was found to be similarly distributed to CSPG by immunoperoxidase staining in adult and developing rat lungs, with the notable exception that little immunoreactivity for HSPG was found in smooth muscle EL. Commercially obtained polyclonal antibodies to entactin and laminin gave immunostaining comparable to that seen with CSPG, except that entactin showed particular affinity for EL. These results offer a more detailed perspective on previous survey observations of CSPG, HSPG, and entactin in the rat lung, and describe the immunoreactivity of CS for the first time.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical localization of epidermal growth factor and acidic and basic fibroblast growth factors in postnatal developing and adult rat lungs.

Histologic preparations of lungs form 1-, 5-, 10-, 18-, and 25-day-old rats and adult rats were probed immunohistochemically with specific antibodies for the distribution of epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), and basic fibroblast growth factor (bFGF). Immunoperoxidase staining of sections of adult rat lungs with a rabbit polyclonal antibody to bovine EGF was strong in ciliated cells of airways and mast cells, nonciliated cells of bronchioles, smooth muscle, type II cells of alveoli, and interstitial and epithelial cells of alveolar septal regions. Developing postnatal lungs had moderate and somewhat diffuse immunoreactivity in all epithelial cells, and more intense staining in vascular smooth muscle. Immunoperoxidase staining of adult rat lungs with a rabbit polyclonal antibody against bovine aFGF was distributed in identical fashion to EGF, with the exception of mast cells which were not reactive. These same sights were localized using an immunoperoxidase sequence with a rabbit polyclonal antibody to the leu 60-leu 98 fragment of aFGF (aFGFfr) with less background. In postnatal developing lungs, immunoreactivity with aFGF and aFGFfr preparations was diffuse and moderately intense in epithelial cells and vascular smooth muscle. Immunoperoxidase staining of adult rat lung sections with a monoclonal antibody to bovine bFGF was strong in hyaluronidase-digested preparations. Reactivity was principally confined to alveolar and vascular basement membrane regions and external laminae of smooth muscle. In early postnatal development, immunoreactivity for bFGF was found in basement membranes beneath developing epithelium and the endothelium of vessel wall intima and in the adventitia, with reactivity increasing with advancing age.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural and functional relationships between type II pneumocytes and components of extracellular matrices.

Type II pneumocytes of the pulmonary alveolus are dynamic cells with multiple functional capabilities in vivo, including secretion of surface-active lipoproteins and cell renewal of the epithelial lining of the alveolus, involving its differentiation into another cell type (the type I pneumocyte). The factors that influence and control these processes, which are vital to the function of the alveolus, have begun to be more clearly understood in recent years, in large part because of the development of adequate in vitro systems, which permit the manipulation of relevant variables. These appear to be a complex interaction between insoluble components of extracellular matrices, principally of the basement membrane, and soluble factors that include hormones and growth factors. This review focuses particularly on those components of extracellular matrices that specifically and nonspecifically impact on type II cell function, and it attempts to bring together the diverse technical approaches used to define and examine these relationships cytochemically and functionally.