Search

Cigarette smoke is a risk factor for the development of several diseases, but the exact mechanism responsible has not been well-characterized. Because modification, or adducting, of biomolecules is thought to mediate the toxic effects observed from exposure to a wide variety of harmful chemicals, this study investigated the ability of cigarette smoke to produce specific adducts on a peptide to gain insight into the likely effect on cellular proteins. We describe the modification of the ε-amino group of lysine contained in a test peptide with stable fluorescent adducts derived from monofunctional aldehydes occurring in cigarette smoke and malonaldehyde, a product of lipid peroxidation. Utilizing high-performance liquid chromatography, fluorescent measurements, and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy, the 1,4-dihydropyridine-3,5-dicarbaldehyde and 4-methyl-1,4-dihydropyridine-3,5-dicarbaldehyde derivatives of lysine were identified as products of exposure to cigarette smoke extract and malonaldehyde. These data suggest that cigarette smoke may promote the modification of proteins, like those associated with oxidized low-density lipoprotein, and may contribute to smoking-related disease.

Polycyclic aromatic hydrocarbons (PAH) are metabolized to electrophiles that can bind to DNA bases and destabilize the N-glycosyl bond, causing rapid depurination of the adducted bases. Recent studies support depurination of DNA as a mechanism central to the genesis of H-ras mutations in PAH-treated mouse skin. Depurinating adducts account for 71% of all DNA adducts formed in mouse skin treated with benzo[a]pyrene (BP). This study analyzed urine of cigarette smokers, coal smoke-exposed women, and nonexposed controls for the presence and quantities of the depurinated BP-adducted DNA bases, 7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua) and 7-(benzo[a]pyren-6-yl)adenine (BP-6-N7Ade). Since these adducted bases originate from reaction of the BP radical cation with double-stranded DNA and not with RNA or denatured DNA, their presence in urine is indicative of DNA damage. Urine samples were fractionated by a combination of SepPak extraction and reverse-phase HPLC, and then analyzed by tandem mass spectrometry and capillary electrophoresis with laser-induced fluorescence. BP-adducted bases were detected in the urine from three of seven cigarette smokers and three of seven women exposed to coal smoke, but were not detected in urine from the 13 control subjects. Concentrations were estimated to be 60−340 and 0.1−0.6 fmol/mg of creatinine equivalent of urine for coal smoke-exposed women (maximum possible BP intake of ca. 23 000 ng/day) and cigarette smokers (BP intake of ca. 800 ng/day), respectively, exhibiting a sensitive response to BP exposures. BP-6-N7Gua was present at ca. 20−300 times the concentration of BP-6-N7Ade in the urine of coal smoke-exposed women, but was not detected in the urine of cigarette smokers. This difference may be due to the remarkably different BP exposures experienced by the two groups of PAH-exposed individuals. These results justify more extensive studies of depurinated BP-adducted DNA bases as potential biomarkers of PAH-associated cancer risk.

We have isolated a unique, basement membrane proteoglycan from the Engelbreth-Holm-Swarm (EHS) sarcoma. This proteoglycan, estimated to be 0.75 X 10(6) daltons, was found to contain about equal amounts of protein and covalently linked heparan sulfate. Antibody prepared against this proteoglycan reacts with the basement membrane matrix in the tumor and with the basement membranes in skin, kidney, and cornea. These studies indicate that the heparan sulfate proteoglycan is a normal constituent of basement membranes that presumably plays an important role in the organization of basement membrane components and that also may determine the permeability of basement membranes to acidic molecules.

1. Cultured bovine bronchial epithelial cells express a large number of β-adrenoceptors of the β2 subtype which are coupled to activation of adenylate cyclase and synthesis of cyclic AMP. The mechanisms involved in agonist-induced desensitization of β-adrenoceptor function in these cells were investigated. 2. Preincubation of cells with the β-adrenoceptor agonist isoprenaline induced a rapid desensitization of adenylate cyclase activity stimulated by isoprenaline (40% at 30 min) with little or no decrease in forskolin stimulation or in the number of β-adrenoceptors detected by radioligand binding, indicating that uncoupling of β-adrenoceptors from adenylate cyclase had occurred. 3. Uncoupling of β-adrenoceptors from the stimulatory guanine-nucleotide-binding protein Gs was indicated by the loss of GTP effects on agonist competition for radioligand binding. Results from subcellular fractionation by sucrose density gradient centrifugation suggest that rapid internalization or sequestration of β-adrenoceptors occurred along with the desensitization. 4. Preincubation with isoprenaline for longer times led to a marked decrease in total receptor number (85% at 18 h), indicating that receptor down-regulation also occurs. Recovery of receptors after down-regulation was prevented by cyclobeximide and by actinomycin D, suggesting that degradation of both receptor protein and receptor mRNA may be involved in down-regulation.

Because cells of the mononuclear phagocyte system are known to produce fibronectin and because alveolar macrophages are activated in many interstitial lung diseases, the present study was designed to evaluate a role for the alveolar macrophage as a source of the increased levels of fibronectin found in the lower respiratory tract in interstitial lung diseases and to determine if such fibronectin might contribute to the development of the fibrosis found in these disorders by being a chemoattractant for human lung fibroblasts. Production of fibronectin by human alveolar macrophages obtained by bronchoalveolar lavage and maintained in short-term culture in serum-free conditions was demonstrated; de novo synthesis was confirmed by the incorporation of [14C]proline. This fibronectin had a monomer molecular weight of 220,000 and was antigenically similar to plasma fibronectin. Macrophages from patients with idiopathic pulmonary fibrosis produced fibronectin at a rate 20 times higher than did normal macrophages; macrophages from patients with pulmonary sarcoidosis produced fibronectin at 10 times the normal rate. Macrophages from 6 of 10 patients with various other interstitial disorders produced fibronectin at rates greater than the rate of highest normal control. Human alveolar macrophage fibronectin was chemotactic for human lung fibroblasts, suggesting a functional role for this fibronectin in the derangement of the alveolar structures that is characteristic of these disorders.

The effects of the simple bioactive lipid mediator lysophosphatidic acid (LPA) on cAMP accumulation were investigated in cultured human airway smooth muscle cells (ASMC). Pretreatment of cells with LPA induced an increase in subsequent stimulation of cAMP accumulation by forskolin and by isoproterenol. When included during the assay of cAMP accumulation rather than as a pretreatment, LPA inhibited forskolin stimulation but enhanced isoproterenol stimulation. Both effects of LPA on forskolin stimulation were completely blocked by pertussis toxin treatment, whereas the effects on isoproterenol stimulation appeared relatively insensitive to pertussis toxin. The protein kinase C activator phorbol-12-myristate-13-acetate (PMA) sensitized forskolin stimulation to a similar extent as did LPA, and the combination of LPA plus PMA caused markedly more sensitization than either agent alone. In contrast, PMA inhibited isoproterenol stimulation and markedly decreased the sensitization induced by LPA. Serum also induced sensitization, and sensitization by LPA plus serum was no greater than that with LPA alone. LPA-induced sensitization appeared to be independent of protein kinase C activation because it was unchanged in cells treated to down-regulate protein kinase C. LPA also stimulated polyphosphoinositide hydrolysis, and this stimulation was partially inhibited by pertussis toxin treatment. These results suggest that LPA activates receptors coupled to both the pertussis toxin-sensitive G protein Gi and the pertussis toxin-insensitive G protein Gq. The complex effects of LPA, PMA, and pertussis toxin on cAMP accumulation in these cells are consistent with the expression of the type 2 isozyme of adenylyl cyclase in these cells.

We have found that sera from humans with Chagas' disease and Rhesus monkeys infected with Trypanosoma cruzi contain IgM and IgG antibodies, which react with structures in a variety of connective tissues. These antibodies react with laminin but not with various other purified connective tissue components like collagen types I, III, IV, and V, fibronectin, heparan sulfate (BM-1) proteoglycan, or chondronectin. The tissue-reacting antibodies were isolated by absorption to a laminin-Sepharose column. The bound fraction contained all the tissue-reacting antibodies. These antibodies strongly stained trypomastigotes and amastigotes, but weakly stained epimastigotes. These studies show that sera from T. cruzi-infected primates contain antilaminin antibodies, which may be produced by those host in response to a laminin-like molecule present in the parasite.

Fibroblast replication is regulated by exogenous signals provided by growth factors, mediators that interact with the target cell surface and signal the cell to proliferate. A useful model of growth regulation, the "dual control model," suggests that growth factors can be grouped either as competence factors or as progression factors, and that optimal replication of fibroblasts requires the presence of both types of growth factors. Although most growth factors are soluble mediators, recent studies have demonstrated that, for some cell types, the extracellular matrix can replace the requirement for a competence factor. Since fibronectin is an important constituent of the extracellular matrix that interacts with specific domains on the fibroblast surface, we examined the ability of fibronectin to act as a competence factor to promote the growth of human diploid fibroblasts. To accomplish this, fibronectins purified from two sources, human plasma and human alveolar macrophages, were tested for their ability to (a) stimulate fibroblast replication in serum-free medium containing characterized progression factors (insulin or alveolar macrophage-derived growth factor); (b) provide a growth-promoting signal early in G1. Fibronectin stimulated fibroblast replication in a dose-dependent manner in the presence of a fixed dose of a progression factor. Conversely, fibronectin conferred on previously unresponsive fibroblasts the ability to replicate in a dose-dependent manner when cultured with increasing amounts of a progression factor. Moreover, fibronectin signaled growth-arrested fibroblasts to traverse G1 approximately 4 h closer to S phase. No differences were observed in the ability of plasma or macrophage fibronectins to provide a competence signal for fibroblast replication. Since fibronectin is a major component of the extracellular matrix, these observations suggest that it may provide at least one of the signals by which the matrix conveys the "competence" that permits fibroblasts to replicate in the presence of an appropriate progression signal.

Different types of immunocompetent cells, including T lymphocytes and alveolar macrophages, account for pulmonary host defense. Taking advantage of the availability of the monoclonal antibody technique, cell culture facilities, pure recombinant cytokines, and molecular probes for their genes, in the last few years it has been possible to keenly study the different steps that lead to the compartmentalization of immune response in human lung. Furthermore, the immunological analysis of cells retrieved from bronchoalveolar lavage (BAL) allowed recognition of the importance of immune mechanisms in the evolution of immune-mediated pulmonary disorders. The purpose of this review is to summarize recent advances on the immunologic characterization of lung lymphocytes in health and disease. Following a brief description of the pathways through which the pulmonary lymphoid system contributes to removing potentially harmful inhaled antigenic materials, available laboratory techniques to evaluate the lymphoid component of the pulmonary immune system and their byproducts are discussed. These techniques cover methods for preparing lymphocytes from the BAL fluid and for characterizing lung lymphocytes both in cell suspensions and pulmonary tissue biopsies. Other sections of this review describe the techniques for measuring the immunologic effector functions of lung lymphocytes. We also provide the reader with a flavor of the molecular biology methods used to characterize lymphocytes in the pulmonary microenvironment. The final sections of the review article highlight the pathogenetic role envisaged for lymphoid cells in pulmonary disease states and emphasize the importance of the BAL analysis in the clinical management of the most relevant immune-mediated lung diseases.