Lena Palmberg

Regulation of differentiated properties and proliferation of arterial smooth muscle cells.

D uring recent years, it has become increasingly evident that arterial smooth muscle cells occur in at least two distinct states, usually referred to as a synthetic and a contractile phenotype. Synthetic-state cells have a fibroblast-like appearance, and their main functions are to proliferate and to produce extracellular matrix components. They are found in the embryo and the young growing organism, where they take part in the formation of the vessel wall. Contractile-state cells have a musclelike appearance and contract in response to chemical and mechanical stimuli. They predominate in the vessels of adults and are primarily involved in the control of blood pressure and flow. However, these cells are able to return to a synthetic phenotype, and this appears to be an important early event in atherogenesis. This review briefly summarizes the present knowledge concerning the regulation of differentiated properties and proliferation of arterial smooth muscle cells. Most of the discussion will deal with studies on cultured cells, which so far are the most abundant. Particular attention will be paid to the role of extracellular matrix components like fibronectin and laminin and of polypeptide mitogens like platelet-derived growth factor (PDGF). It is believed that future studies in this area will help to widen our understanding of vasculogenesis and the initial stages in the pathogenesis of atherosclerosis.

Inhalation of swine dust induces cytokine release in the upper and lower airways

In healthy subjects, acute inhalation of swine dust causes an influx of inflammatory cells into the airways and increased bronchial responsiveness. The exposure may also cause fever and generalized symptoms. It seems likely that proinflammatory cytokines are involved in the response to inhaled swine dust. Nasal and bronchoalveolar lavage (BAL) were performed before, and 7 and 24 h after the start of 3 h exposure to swine dust, during a period of work in a swine confinement building, in 22 healthy subjects. Lavage fluids were analysed with regard to the cellular response and concentrations of interleukin (IL)-1 alpha, IL-1 beta, IL-6 and tumour necrosis factor-alpha (TNF-alpha). Each subject carried personal samplers for exposure measurements. Inhalable dust and airborne endotoxin, 3-hydroxylated (2-OH) fatty acid and muramic acid were measured. Bronchial responsiveness to methacholine was investigated 1-2 weeks before and 7 h after the start of the exposure. Exposure caused fever (> 38 degrees C) in three subjects, and approximately 25% of the subjects experienced symptoms. Bronchial responsiveness to methacholine increased by 3.5 (1.6-4.8) doubling doses (median (25th-75th percentile)). Following exposure, granulocytes increased more than 50 fold in BAL fluid and more than 40 fold in nasal lavage fluid. IL-1 alpha and IL-1 beta increased significantly in BAL fluid (p < 0.05) and nasal lavage fluid (p < 0.01). IL-6 increased 25 fold in BAL and 15 fold in nasal lavage fluid (p < 0.001). TNF-alpha was below detection limit (0.25 ng.L-1) in most subjects before exposure and increased following exposure to 3.8 (2.4-5.7) and 1.3 (0.6-2.3) ng.L-1 in BAL and nasal lavage fluid, respectively, (p < 0.001). Total inhalable dust was 20.5 (14.6-30.0) mg.m-3 and the concentrations of airborne endotoxin, 3-OH fatty acid and muramic acid were 1.2 (0.8-1.4), 3.5 (2.2-4.5) and 0.9 (0.3-1.9) microgram.m-3, respectively. There was a significant correlation between the IL-6 response in BAL fluid and exposure to dust endotoxin activity and 3-OH fatty acids (p < 0.05). Otherwise, no significant correlations were found between exposure and the cytokine response. We conclude that exposure to swine dust causes an intense upper and lower airway inflammation, which involves the proinflammatory cytokines interleukin-1, interleukin-6 and tumour necrosis factor-alpha.

Induction of IL-8 production in human alveolar macrophages and human bronchial epithelial cells in vitro by swine dust

BACKGROUND Exposure to swine dust causes an intense airway inflammation with increased levels of interleukin 8 (IL-8) and predominantly neutrophils in the nasal and bronchoalveolar lavage fluids of healthy human subjects. It is not clear which components in the swine house environment are responsible for the airway reaction. The aim of the present study was to evaluate and compare the effect in vitro of swine dust components on human alveolar macrophages and bronchial epithelial cells. METHODS Normal human bronchial epithelial cells (NHBE), human pulmonary epithelial carcinoma cell line (A549), and human alveolar macrophages were stimulated with swine dust, lipopolysaccharides (LPS; present in Gram negative bacteria), grain dust (swine feed components), and glucans (a structural component of fungi) in a dose response manner (1–100 μg/ml). RESULTS Swine dust at a concentration of 100 μg/ml increased IL-8 production 20 fold in NHBE cells, 28 fold in A549 cells, and 15 fold in macrophages. LPS (100 μg/ml) stimulated all three cell types significantly, in macrophages to the same extent as swine dust, but in NHBE and A549 cells swine dust was 5–8 times as potent. Grain dust (100 μg/ml) had no effect in A549 cells but stimulated NHBE cells and macrophages. Glucans (100 μg/ml) stimulated A549 cells and macrophages but not NHBE cells. Both glucans and grain dust were weaker stimuli than swine dust and LPS. The LPS content of swine dust solution was 2.16 (0.2) ng/100 μg and of grain dust was 0.53 (0.04) ng/100 μg. CONCLUSIONS Swine dust is a strong stimulus for IL-8 production in both bronchial epithelial cells and human alveolar macrophages, whereas LPS has different potency in these cells.

Free, soluble interleukin-17 protein during severe inflammation in human airways

Studies in rodents indicate that the cytokine, interleukin (IL)-17, links the activation of T-lymphocytes to neutrophilic inflammation. The aim of the current study was to determine whether free, soluble IL-17 protein can be released during severe inflammation in human airways. Fifteen healthy subjects were exposed to a swine confinement in order to induce severe inflammation characterized by high neutrophil numbers in the airways. Bronchoalveolar lavage (BAL) fluid was harvested 2 weeks prior to and 24 h after this exposure and the concentration of IL-17 protein was measured using an enzyme-linked immunosorbent assay. Total and cell differential counts were also performed in BAL fluid. Prior to exposure to the swine confinement, the concentration of IL-17 in BAL fluid was low (<7.8 pg·mL−1) in 14 out of 15 subjects. However, exposure to the swine confinement caused an increase in IL-17 in 13 out of 15 subjects (median IL-17 concentration of 26.9 pg·mL−1). This exposure also caused a 51-fold increase in the concentration of neutrophils in BAL fluid. To conclude, free, soluble interleukin-17 protein can be released during severe inflammation characterized by high neutrophil numbers in human airways. The significance of interleukin-17 in inflammatory airway diseases therefore deserves further evaluation.

Gram positive bacteria induce IL-6 and IL-8 production in human alveolar macrophages and epithelial cells.

Background and Objective: Inhalation of dust from swine confinement buildings results in an acute inflammatory reaction in the respiratory tract. The dust has a high microbial content, dominated by Gram positive bacteria. The aim of the present study was to evaluate the significance of bacteria in the induction of IL-6 and IL-8 release from respiratory epithelial cells and alveolar macrophages. The results would give an indication to what extent the bacteria contribute to the toxic inflammation following exposure to swine dust. Methods: Epithelial cells from a human lung carcinoma cell line (A549) and human alveolar macrophages obtained from healthy subjects by bronchoalveolar lavage, were stimulated with swine dust, LPS, one Gram negative and four Gram positive bacteria strains. The dose-response release of IL-6 and IL-8 were studied. In addition, a bacteria-free supernatant was prepared from each strain and used for stimulation. Results: With a few exceptions, a dose-dependent IL-6 and IL-8 release was demonstrated from both cell types after stimulation with bacteria. In epithelial cells, Escherichia coli was the most potent bacteria at the highest concentration of 400 bacteria/cell regarding secretion of both IL-6 and IL-8 (P < 0.001), followed by Staphylococcus hominis and Staphylococcus lentus. In alveolar macrophages, S. lentus was the most potent strain (P < 0.001) in inducing cytokine release (P < 0.001), followed by S. hominis and E. coli concerning IL-6 secretion or Micrococcus luteus and E. coli with respect to IL-8 secretion (P < 0.001). Differences in potency between the various bacteria could be demonstrated, both within the two cell types as well as between the epithelial cells and macrophages. Bacteria-free supernatants were also able to induce cytokine release in both cell types. In macrophages the supernatants were even more potent stimuli than whole bacteria. Conclusions: The results indicate that bacteria or bacterial products could be an important contributing factor to the inflammatory reaction following exposure to swine dust.

Adult human arterial smooth muscle cells in primary culture Modulation from contractile to synthetic phenotype

SummarySmooth muscle cells were isolated enzymatically from adult human arteries, grown in primary culture in medium containing 10% whole blood serum, and studied by transmission electron microscopy and [3H]thymidine autoradiography. In the intact arterial wall and directly after isolation, each smooth muscle cell had a nucleus with a wide peripheral zone of condensed chromatin and a cytoplasm dominated by myofilament bundles with associated dense bodies. After 1–2 days of culture, the cells had attached to the substrate and started to spread out. At the same time, a characteristic fine-structural modification took place. It included nuclear enlargement, dispersion of the chromatin and formation of large nucleoli. Moreover, myofilament bundles disappeared and an extensive rough endoplasmic reticulum and a large Golgi complex were organized in the cytoplasm. This morphological transformation of the cells was completed in 3–4 days. It was accompanied by initiation of DNA replication and mitosis.The observations demonstrate that adult human arterial smooth muscle cells, when cultivated in vitro, pass through a phenotypic modulation of the same type as arterial smooth muscle cells from experimental animals. This modulation gives the cells morphological and functional properties resembling those of the modified smooth muscle cells found in fibroproliferative lesions of atherosclerosis. Further studies of the regulation of smooth muscle phenotype and growth may provide important clues for a better understanding of the pathogenesis of atherosclerosis.

Fluticasone and budesonide inhibit cytokine release in human lung epithelial cells and alveolar macrophages

Glucocorticoids are potent anti‐inflammatory agents capable of influencing cytokine release in a number of cell types. The aim of the present study was to investigate whether glucocorticoids, frequently used in the treatment of asthma, interfere with cytokine secretion by lung epithelial cells and alveolar macrophages in vitro. Inhalation of swine dust induces airway inflammation with influx of inflammatory cells and release of proinflammatory cytokines in the lungs. Therefore, human lung epithelial cells (A549) and human alveolar macrophages were stimulated with swine dust or lipopolysaccharide (LPS), and the inhibitory effect of budesonide and fluticasone propionate on cytokine release was studied in a dose‐response (10−13–10−8 M) manner. The time course for the steroid effect was also investigated. Both steroids caused a dose‐dependent, almost total, inhibition of swine dust‐induced IL‐6 and IL‐8 release from epithelial cells and LPS‐induced IL‐6 and TNF‐α from alveolar macrophages. The steroids only partially inhibited IL‐8 release from alveolar macrophages. Budesonide was approximately 10 times less potent than fluticasone propionate. Preincubation with the steroids did not inhibit cytokine release more than simultaneous incubation with stimulus and steroid. In conclusion, budesonide and fluticasone propionate, in concentrations that probably occur in the airway lining fluid during inhalational therapy, inhibited cytokine release from human lung epithelial cells (IL‐6, IL‐8) and alveolar macrophages (TNF‐α, IL‐6, IL‐8). In vitro, the onset of this effect was rapid.

Saliva is one likely source of leukotriene B4 in exhaled breath condensate

Leukotriene (LT)B4 in exhaled breath condensate (EBC) has been reported to be elevated in airway inflammation. The origin of leukotrienes in EBC is, however, not established. The aims of this study are to measure LTB4 levels in EBC collected in two challenges characterised by a strong neutrophilic airway inflammation and to compare LTB4 levels in EBC with levels in sputum and saliva. LTB4 and α-amylase were measured in EBC from 34 healthy subjects exposed in a pig confinement building or to a lipopolysaccharide provocation. These markers were also measured in induced sputum in 11 of the subjects. For comparison, LTB4 and α-amylase were measured in saliva from healthy subjects. Only four out of 102 EBC samples had detectable LTB4 (28–100 pg·mL-1). α-amylase activity was detected in the LTB4-positive samples. In contrast, LTB4 was detected in all examined sputum supernatants in the same study (median 1,190 pg·mL-1). The median LTB4 level in saliva was 469 pg·mL-1. High levels of leukotriene B4 in saliva and the presence of leukotriene B4 in exhaled breath condensate only when α-amylase was detected, indicate that leukotriene B4 found in exhaled breath condensate is the result of saliva contamination. As leukotriene B4 was consistently present in sputum supernatants, exhaled breath condensate may be inappropriate for monitoring airway leukotriene B4.

Release of prostaglandin D2 and leukotriene C4 in response to hyperosmolar stimulation of mast cells

Background:  Mannitol‐induced bronchoconstriction in subjects with exercise‐induced asthma is associated with increased urinary excretion of 9α, 11β‐PGF2, a metabolite of prostaglandin D2 (PGD2) serving as a mast cell marker. It has however been questioned whether or not human mast cells release PGD2 and leukotriene C4 (LTC4) after osmotic challenge with mannitol in vitro.

Exhaled nitric oxide and bronchial responsiveness in healthy subjects exposed to organic dust

Inhalation of organic dust from swine houses causes an intense inflammatory reaction in the respiratory tract, and increased bronchial responsiveness to methacholine in healthy subjects. The aims of the present study were to investigate whether exhaled nitric oxide (NO) is a marker of the inflammation caused by exposure to organic dust (swine dust), whether there is a relationship between an increase in exhaled NO and bronchial responsiveness, and also whether wearing a half-mask influences the airway reaction (assessed by exhaled NO) and the increased bronchial responsiveness. Thirty-three healthy nonatopic, nonsmoking subjects were exposed during 3 h of light work in a swine confinement building. Eleven of the subjects were wearing a half-mask and 22 were unprotected. Lung function, bronchial responsiveness and exhaled NO were measured before and after exposure. The provocative concentration causing a 20% fall in forced expiratory volume in one second fell by 2.7 (2.1–4.1) (median (25th–75th percentiles)) doubling concentration steps in subjects without a half-mask and by 1.5 (0.9–2.9) doubling concentration steps in subject wearing a mask. Exhaled NO increased from 7.5 (5.7–13.7) parts per billion (ppb) before to 13.4 (10.5–17.5) ppb after exposure in the unprotected group and was unaltered (8.3 (6.1–14.1) to 8.6 (6.6–14.6) ppb) in the group wearing a half-mask. There was no correlation between NO increase and provocative dose causing a 20% fall in the forced expiratory volume in one second decrease. In conclusion, bronchial responsiveness and exhaled nitric oxide increased after exposure to a swine confinement facility. Half-mask abolished the increase in exhaled nitric oxide levels, but influenced the increase in bronchial responsiveness to a minor extent. These results indicate that these two outcome measures reflect different aspects of airway inflammation induced by exposure to a farming environment.