Biomedical subjects
Publications and source records attributed to Andrew J Ghio.
To determine the impact of acute hypoxia on prohepcidin concentrations in humans, we measured concentrations of this peptide in serum collected during and after a transient period (30 min) of hypoxia and during normoxia. Prohepcidin concentrations were significantly lower 150 min after the end of hypoxia than after normoxia (p=0.028).
We report on a healthy 26-year-old male who had an exercise-induced pulmonary hemorrhage (EIPH) within 24 hours of running a marathon. There were no symptoms, abnormalities on exam, or radiographic infiltrates. He routinely participated in bronchoscopy research and the EIPH was evident on gross inspection of bronchoalveolar lavage fluid, examination of the cytospin, and measurement of hemoglobin, iron, and ferritin concentrations. EIPH in humans may occur without any evidence on clinical presentation; its incidence may be far greater than currently suspected.
Complement, a complex protein system, plays an essential role in host defense through bacterial lysis, stimulation of phagocytosis, recruitment of immune cells to infected tissue, and promotion of the inflammatory response. Although complement is most well-characterized in serum, complement activity is also present in the lung. Here we further characterize the complement system in the normal and inflamed lung. By Western blot, C5, C6, and factor I were detected in bronchoalveolar lavage (BAL) at lower levels than in serum, whereas C2 was detected at similar levels in BAL and serum. C4 binding protein (C4BP) was not detectable in BAL. Exposure to lipopolysaccharide (LPS) elevated levels of C1q, factor B, C2, C4, C5, C6, and C3 in human BAL and C3, C5, and factor B in mouse and rat BAL. Message for C1q-B, C1r, C1s, C2, C4, C3, C5, C6, factor B, and factor H, but not C9 or C4BP, was readily detectable by RT-PCR in normal mouse lung. Exposure to LPS enhanced factor B expression, decreased C5 expression, and did not affect C1q-B expression in mouse and rat lung. BAL from rats exposed to LPS had a greater ability to deposit C3b onto bacteria through complement activation than did BAL from control rats. In summary, these data demonstrate that complement levels, expression, and function are altered in acute lung injury and suggest that complement within the lung is regulated to promote opsonization of pathogens and limit potentially harmful inflammation.
We tested the hypothesis that oxidative stress and biological effect after ozone (O3) exposure are dependent on changes in iron homeostasis. After O3 exposure, healthy volunteers demonstrated increased lavage concentrations of iron, transferrin, lactoferrin, and ferritin. In normal rats, alterations of iron metabolism after O3 exposure were immediate and preceded the inflammatory influx. To test for participation of this disruption in iron homeostasis in lung injury following O3 inhalation, we exposed Belgrade rats, which are functionally deficient in divalent metal transporter 1 (DMT1) as a means of iron uptake, and controls to O3. Iron homeostasis was disrupted to a greater extent and the extent of injury was greater in Belgrade rats than in control rats. Nonheme iron and ferritin concentrations were higher in human bronchial epithelial (HBE) cells exposed to O3 than in HBE cells exposed to filtered air. Aldehyde generation and IL-8 release by the HBE cells was also elevated following O3 exposure. Human embryonic kidney (HEK 293) cells with elevated expression of a DMT1 construct were exposed to filtered air and O3. With exposure to O3, elevated DMT1 expression diminished oxidative stress (i.e., aldehyde generation) and IL-8 release. We conclude that iron participates critically in the oxidative stress and biological effects after O3 exposure.
Alveolar macrophages express many proteins important in iron homeostasis, including the iron importer divalent metal transport 1 (DMT1) and the iron exporter ferroportin 1 (FPN1) that likely participate in lung defense. We found the iron regulatory hormone hepcidin (HAMP) is also produced by alveolar macrophages. In mouse alveolar macrophages, HAMP mRNA was detected at a low level when not stimulated but at a high level when exposed to lipopolysaccharide (LPS). LPS also affected the mRNA levels of the iron transporters, with DMT1 being upregulated and FPN1 downregulated. However, iron had no effect on HAMP expression but was able to upregulate both DMT1 and FPN1 in alveolar macrophages. IL-1 and IL-6, which are important in HAMP augmentation in hepatocytes, also did not affect HAMP expression in alveolar macrophages. In fact, the LPS-induced alterations in the expression of HAMP as well as DMT1 and FPN1 were preserved in the alveolar macrophages isolated from IL-1 receptor or IL-6-deficient mice. When alveolar macrophages were loaded with transferrin-bound (55)Fe, the subsequent release of (55)Fe was inhibited significantly by LPS. In addition, treatment of these cells with either LPS or HAMP caused the diminishment of the surface FPN1. These findings are consistent with the current model that HAMP production leads to a decreased iron efflux. Our studies suggest that iron mobilization by alveolar macrophages can be affected by iron and LPS via several pathways, including HAMP-mediated degradation of FPN1, and that these cells may use unique regulatory mechanisms to cope with iron imbalance in the lung.
Propionibacterium acnes (PA) is a gram-positive anaerobic bacterium implicated as a putative etiologic agent of sarcoidosis. To characterize the pulmonary immune response to PA, C57BL/6 and BALB/c mice were intraperitoneally sensitized and intratracheally challenged with heat-killed bacteria. C57BL/6 mice challenged with PA developed a cellular immune response characterized by elevations in Th1 cytokines/chemokines, increased numbers of lymphocytes and macrophages in lung lavage fluid, and peribronchovascular granulomatous inflammation composed of T- and B-lymphocytes and epithelioid histiocytes. T-lymphocytes in the lung lavage fluid showed a marked CD4+ cell predominance. In contrast, C57BL/6 mice challenged with Staphylococcus epidermidis (SE), another gram-positive commensal of human skin, and BALB/c mice challenged with PA, showed only a modest induction of Th1 cytokines, less pulmonary inflammation, and no granulomatous changes in the lung. Enhancement of Toll-like receptor expression was seen in PA-exposed C57BL/6 mice within 24 h after exposure, suggesting that induction of innate immunity by PA contributes to the robust, polarized Th1 immune response elicited by this bacterium. These findings suggest that PA-induced pulmonary inflammation may be a useful model for testing the contributions of both bacterial and host factors in the development, maintenance, and resolution of granulomatous inflammation in the lung.
Catalytically active iron in the lung causes oxidative stress and promotes microbial growth that can be limited by intracellular sequestration of iron within ferritin. Because cellular iron uptake requires membrane ferrireductase activity that in the gut can be provided by duodenal cytochrome b (Dcytb), we sought Dcytb in the lung to test the hypothesis that it contributes to epithelial iron regulation by reducing Fe(3+) for cellular iron transport. Dcytb expression was found in respiratory epithelium in vitro and in vivo and was responsive to iron concentration. Iron transport was measured in human bronchial epithelial (HBE) cells using inductively coupled plasma atomic emission spectroscopy and was demonstrated to be partially inhibited in the presence of Dcytb-blocking antibody, suggesting that Dcytb reduces Fe(3+) for cellular iron transport. A definite source of reducing equivalents for Dcytb was sought but not identified. We found no evidence that ascorbate was involved but did demonstrate that O(2)(-). production decreased when Dcytb function was blocked. The presence of Dcytb in airway epithelial cells and its regulation by iron therefore may contribute to pulmonary cytoprotection.
Ambient particulate matter is increasingly recognized as a significant contributor to human cardiopulmonary morbidity and mortality in the United States and worldwide. We sought to determine whether exposure to ambient particulate matter would alter alveolar fluid clearance in mice. Mice were exposed to a range of doses of a well-characterized particulate matter collected from the ambient air in Düsseldorf, Germany through a single intratracheal instillation, and alveolar fluid clearance and measurements of lung injury were made. Exposure to even very low doses of particulate matter (10 microg) resulted in a significant reduction in alveolar fluid clearance that was maximal 24 h after the exposure, with complete resolution after 7 d. This was paralleled by a decrease in lung Na,K-ATPase activity. To investigate the mechanism of this effect, we measured plasma membrane Na,K-ATPase abundance in A549 cells and Na,K-ATPase activity in primary rat alveolar type II cells after exposure to particulate matter in the presence or absence of the combined superoxide dismutase and catalase mimetic EUK-134 (5 microM). Membrane but not total protein abundance of the Na,K-ATPase was decreased after exposure to particulate matter, as was Na,K-ATPase activity. This decrease was prevented by the combined superoxide dismutase/catalase mimetic EUK-134. The intratracheal instillation of particulate matter results in alveolar epithelial injury and decreased alveolar fluid clearance, conceivably due to downregulation of the Na,K-ATPase.
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Toxicological investigation suggests that exposures to complex secondary organic aerosol (SOA) products can result in adverse health effects in biological systems. However, the mechanism of adverse health effects is not yet understood. One of the major restrictions in studies of health effects of SOA is a particle exposure technique. In this study, we applied an innovative soft targeting technology using magnetic nanoparticles (MNP) to deliver SOAs onto target biological systems under a magnetic field. The exploratory exposure technology using MNP was demonstrated for the SOAs created from the reaction of ozone with alpha-pinene in an indoor Teflon film chamber. SOA increased the release of the proinflammatory mediator interleukin-8 by respiratory epithelial cells. These results support that MNP can effectively deliver SOAs to epithelial cells in vitro resulting in a significant biological effects.
Exposure to synthetic fibers with employment in textile mills can be associated with an elevated risk of interstitial lung disease (ILD). A mechanism of injury has not been determined. ILD can follow exposures to inorganic fibers (e.g., asbestos) which are associated with a mobilization of iron and catalysis of an oxidative stress. We describe 2 patients with ILD associated with exposure to synthetic textile fibers who demonstrated carbon-based ferruginous bodies suggesting an in vivo accumulation of iron by synthetic fibers after deposition in the lung. These iron-laden bodies varied from perfectly linear fibers to almost particulate matter. Linear structures were irregularly interrupted by deposition of iron-abundant material. The capacity of these synthetic fibers to complex iron and generate an oxidative stress is confirmed in vitro.
STUDY OBJECTIVES: Bronchoscopy with BAL is being used increasingly in the investigation of acute and chronic lung inflammation. The scope of the acute phase response induced by the procedure is not fully evaluated. The purpose of the study is to characterize the acute phase response induced by bronchoscopy with BAL. DESIGN: Observational study. SETTING: A human study research facility. PARTICIPANTS: Normal nonsmoking volunteers. INTERVENTION: A total of 28 subjects were recruited. Under local anesthesia, the subjects underwent bronchoscopy with a videofiberoptic bronchoscope. One subsegment of the lingular segment of the left upper lobe and the right middle lobe were lavaged each with 170 to 270 mL of sterile normal saline solution. MEASUREMENTS AND RESULTS: CBC count, serum levels of indexes of iron homeostasis, fibrinogen, C-reactive protein (CRP), and plasma mediators related to neutrophil migration and endothelial cell activation, including interleukin (IL)-8, angiotensin converting enzyme (ACE), soluble intercellular adhesion molecule (sICAM)-1, and nitrite/nitrate, were measured. Measurements of these plasma markers were done immediately before, immediately after, and 24 h after bronchoscopy. Changes in acute phase response were detected primarily at 24 h after the procedure. WBCs, primarily neutrophils, increased by approximately 50%. Fibrinogen increased by 25% while CRP increased by more than sevenfold. Serum ferritin increased by 25% while serum iron, total iron-binding capacity, and transferrin saturation decreased, indicating dysregulation of iron homeostasis. There were no changes in IL-8, ACE, sICAM-1, or nitrite/nitrate plasma levels. CONCLUSIONS: Bronchoscopy with BAL induces a variety of acute phase responses that includes peripheral neutrophilia, dysregulation of iron homeostasis, and increased levels of fibrinogen and CRP. Human research that employs BAL may need to consider the biological effects induced by the procedure-related acute phase response.
Exposure to ambient pollutant particle (APP) is associated with increased cardiovascular morbidity and mortality. Recent evidence indicates that APP-induced vasoconstriction may be an important mechanism. APP constricts systemic arteries and increases blood pressure in human. APP decreases the diameter of pulmonary arterioles in animals. Intratracheal instillation of APP increases pulmonary artery resistance in isolated buffer-perfused lungs, and APP constricts isolated arterial rings. APP-induced vasoconstriction may be secondary to the release of inflammatory mediators from lung cells, which then activate vascular endothelial and smooth muscle cells. The vasoconstriction may also be caused by alterations in autonomic nervous system balance. Some soluble metals (e.g., vanadium) can produce acute vasoconstriction in in vitro and in vivo systems, and contribute to the systemic health effects of APP since they can more easily permeate the alveolar-capillary membrane than the whole particle. Both APP and its associated metals have been shown to enhance the release of endothelin 1 and reactive oxygen species, activate epithelial growth factor receptor and mitogen-activated protein kinases, and inhibit nitric oxide vasodilator activity. The vasoactive properties of APP and metals raised the possibility that patients with vascular diseases may be more susceptible to APP-induced adverse health effects, and that people who are regularly exposed to high amount of metals, e.g., vanadium contained in certain dietary and muscle-building regimens or in the air of boiler making plants, may have increased risk for vascular diseases. Understanding how metals induce vasoconstriction may lead to the development of novel vasodilator therapies for vascular diseases.
Iron is essential for many aspects of cellular function. However, it also can generate oxygen-based free radicals that result in injury to biological molecules. For this reason, iron acquisition and distribution are tightly regulated. Constant exposure to the atmosphere results in significant exposure of the lungs to catalytically active iron. The lungs have a mechanism for detoxification to prevent associated generation of oxidative stress. Those same proteins that participate in iron uptake in the gut are also employed in the lung, to transport iron intracellularly and sequester it in an inactive form within ferritin. The release of metal is expedited (as transferrin and ferritin) from lung tissue to the respiratory lining fluid for clearance by the mucocilliary pathway or to the reticuloendothelial system for long-term storage. This pathway is likely to be the major method for the control of oxidative stress presented to the respiratory tract.
Several features of the clinical presentation and changes in physiology and pathology following exposure to many diverse ambient air pollution particles are comparable, suggesting a common mechanism for their biological effect. We propose that a mechanism of biological effect common to many ambient air pollution particles is a disruption of iron homeostasis in cells and tissues. Among traits shared by every particle-related lung injury is the introduction of a solid-liquid interface into the respiratory tract. All surfaces of particulate matter have some concentration of oxygen-containing functional groups. As a result of its electropositivity, Fe(3+) has a high affinity for oxygen-donor ligands and will react with these groups at the particle surface. Retained particles accumulate metal from available sources in a cell and tissue, and this complexed iron mediates oxidant generation. In addition to complexation onto the solid-liquid interface provided by the surface of particulate matter (PM), there are several alternative pathways by which metal homeostasis in the lower respiratory tract can be disrupted following exposure to ambient air pollution particles to affect an oxidative stress. Evidence suggests that disruption in iron homeostasis following exposures to ambient air pollution particles is an initial event in their biological effect. An association between metal equilibrium in the lower respiratory tract and biological effect in the lung could explain the observed differential toxicity of ultrafine, fine, and coarse particles and disparities in host susceptibility.
Lung injury after asbestos exposure is associated with an oxidative stress that is catalyzed by iron in the fiber matrix, complexed to the surface, or both. We tested the hypothesis that the cellular response to asbestos includes the transport and sequestration of this iron through (1) generation of superoxide for ferrireduction, (2) up-regulation of divalent metal transporter-1 (DMT1) for intracellular transport of Fe2+, and (3) increased production of cellular ferritin where the metal is stored in a catalytically less reactive state. BEAS-2B cells with normal and elevated Cu,Zn superoxide dismutase (SOD) expression were employed for in vitro investigations. After exposure of these cells to asbestos, we demonstrated by fluorescence methodology a significantly increased generation of SOD with ferrireductive capacity. Fiber exposure also increased DMT1 protein and mRNA expression in the BEAS-2B cells. Incubation with asbestos elevated cellular iron and ferritin concentrations, and these responses were diminished in cells with an enhanced expression of SOD. Finally, fiber exposure increased supernatant concentrations of interleukin 8, but this inflammatory mediator was actually increased in cells with elevated SOD expression. We conclude that the response of respiratory epithelial cells to asbestos includes oxidant-mediated mechanisms to sequester catalytically active iron associated with the fiber.
Tissue factor expression in sepsis activates coagulation in the lung, which potentiates inflammation and leads to fibrin deposition. We hypothesized that blockade of factor X binding to the tissue factor-factor VIIa complex would prevent sepsis-induced damage to the lungs and other organs. Acute lung injury was produced in 15 adult baboons primed with killed Escherichia coli [1 x 10(9) colony-forming units (CFU)/kg], and then 12 h later, they were given 1 x 10(10) CFU/kg live E. coli by infusion. Two hours after live E. coli, animals received antibiotics with or without monoclonal antibody to tissue factor intravenously to block tissue factor-factor X binding. The animals were monitored physiologically for 34 h before being killed and their tissue harvested. The antibody treatment attenuated abnormalities in gas exchange and lung compliance, preserved renal function, and prevented tissue neutrophil influx and bowel edema relative to antibiotics alone (all P < 0.05). It also attenuated fibrinogen depletion (P < 0.01) and decreased proinflammatory cytokines, e.g., IL-6 and -8 (P < 0.01), in systemic and alveolar compartments. Similar protective effects of the antibody on IL-6 and -8 expression and permeability were found in lipopolysaccharide-stimulated endothelial cells. Blockade of factor X binding to the tissue factor-factor VIIa complex attenuates lung and organ injuries in established E. coli sepsis by attenuating the neutrophilic response and inflammatory pathways.
BACKGROUND: Oxidative stress has been proposed as a mechanism of injury underlying obliterative bronchiolitis. Catalytically reactive iron is a potential source of reactive oxygen species in transplanted tissue. Using samples acquired from surveillance bronchoalveolar lavage (BAL), we tested the postulate that there is a disruption of iron equilibrium in transplanted lung, which can worsen with time. METHODS: A control group of 5 healthy, non-smoking volunteers underwent BAL. Five bilateral lung transplant patients underwent surveillance BAL with transbronchial lung biopsies. The BAL fluid concentrations of protein, albumin, total iron, lactoferrin, ferritin, transferrin receptor and total iron binding capacity were measured. RESULTS: The mean ages in the control and transplant groups were 25.0 +/- 2.4 and 34.6 +/- 5.0 years, respectively. Patients were transplanted for cystic fibrosis (n = 3), primary ciliary dyskinesia (n = 1) and bronchiolitis obliterans (n = 1). Surveillance bronchoscopies were performed at 100.6 +/- 63.3, 175.0 +/- 87.7 and 259.2 +/- 82 days post-transplant. No significant differences were noted in BAL protein, albumin and total iron binding capacity (TIBC) levels between the 2 groups. The BAL iron, transferrin, transferrin receptor, lactoferrin and ferritin levels were significantly elevated in transplant patients relative to controls. With time after transplantation, there were increases in lavage iron, transferrin receptor, lactoferrin and ferritin concentrations. CONCLUSIONS: Abnormally high levels of iron and its homeostatic proteins were found in the lung allografts, and levels appeared to increase with time. This supports a disruption in the normal homeostasis of this metal after transplantation and a potential role for a catalyzed oxidative stress in bronchiolitis obliterans. The use of iron-depleting therapy is a possible means for preventing injury in the lung allograft.