Bronchoalveolar lavage fluid from asthmatic subjects is mitogenic for human airway smooth muscle.

Airway smooth muscle proliferation may contribute to the airway wall remodeling seen in asthma. In this study we tested for the presence of airway smooth muscle mitogenic activity in bronchoalveolar lavage (BAL) fluid obtained from 12 atopic asthmatics before and serially after segmental allergen challenge, and from four normal subjects who did not undergo allergen challenge. Mitogenic effect was assessed by coincubating BAL fluid with human airway smooth muscle cells, and measuring its effect on (3)[H]thymidine incorporation and cell number. Induction of ERK phosphorylation and cyclin D(1) protein abundance were also assessed. Compared with serum-free medium alone, BAL fluid obtained from normal subjects increased thymidine incorporation, cell number, ERK phosphorylation, and cyclin D(1) abundance. BAL fluid from asthmatic subjects prior to allergen challenge induced even greater increases in all measures, except for cell number, which was similar to that observed with normal subjects' BAL fluid. Incubation with lavage fluid obtained 48 h after segmental allergen challenge in atopic asthmatics caused yet further increases in thymidine incorporation, cell number, and cyclin D(1) protein abundance. Molecular sieving of prechallenge BAL fluid from three asthmatic subjects demonstrated that mitogenic activity was present exclusively in the > 10 kD fraction. These results provide the first direct demonstration that fluid lining the airways of asthmatics contains excess mitogenic activity for human airway smooth muscle, and that this activity increases further after allergen challenge.

Relationship of cellular transmigration and airway response after allergen challenge.

We examined the relationship between eosinophil migration into the bronchoalveolar space and change in FEV(1) after endobronchial allergen challenge (EBAC) in atopic asthmatic (AA) and atopic nonasthmatic (ANA) subjects. The purpose of this study was to obtain continuous, intrasubject controlled assessment of the relationship between cell migration in control and allergen-challenged segments in the same individuals over 96 h. In AA subjects, the eosinophil (Eos) count in the bronchoalveolar lavage fluid (BALF) increased from a baseline of 7,896 +/- 3,865 to 416,476 +/- 231,012 Eos/ml by 72 h (p = 0.001) in the challenged segment post-EBAC. For ANA subjects, the postsegmental challenge count was 29,874 +/- 474 Eos/ml (p = 0.03 versus baseline and p < 0.05 AA peak versus ANA peak). In both groups, there was a comparable decrease in peripheral blood eosinophil count beginning 5 h after challenge, which resolved at 24 h. In AA subjects, 416,476 +/- 231,012 Eos/ml was obtained from the allergen-challenged segment and 23,522 +/- 8,298 Eos/ml was obtained from the sham-challenged segment (p < 0.001) at 72 h. In contrast, there was no difference in the Eos count obtained from the BALF between the antigen- and sham-challenged segments of ANA subjects. We also found that increased airway neutrophils were present in equal numbers in allergen-challenged and sham-challenged segments in both AA and ANA subjects. We conclude that augmented eosinophil migration after EBAC is a characteristic of atopic asthma and is not present in atopic subjects who do not have asthma. We find that BAL eosinophilia in ANA patients as well as neutrophilia in both ANA and AA subjects are nonspecific consequences of bronchoscopy. Finally, we find no relationship between specific airway eosinophil migration into the BALF and FEV(1) < 72 h after challenge; however, at 96 h, there is a substantial decrease in FEV(1) that accompanies BALF eosinophilia.

The Chicago Asthma Consortium: a community coalition targeting reductions in asthma morbidity.

The problem of asthma in Chicago remains a complex one, and it is too early to know whether any programs and efforts have had a discernible effect, but the Chicago Asthma Consortium continues to expand its membership and to define its mission. The successes have come from harnessing the passion of the individual members to move the projects forward. As the focus of the consortium moves to addressing system-wide problems in asthma care and the delivery of that care, the consortium is undertaking the construction of a guide for future efforts. In this way, the consortium will fulfill its vision of creating a comprehensive, community-wide plan for the management of asthma, impacting on the unacceptable current levels of morbidity and mortality of the disease.

Partial characterization of a novel mitogen-activated protein kinase/extracellular signal-regulated kinase activator in airway smooth-muscle cells.

We demonstrated previously that in bovine tracheal myocytes, pretreatment with either forskolin or histamine significantly reduces both platelet-derived growth factor (PDGF)- and epidermal growth factor- induced Raf-1 activation but fails to inhibit extracellular signal-regulated kinase (ERK) activation substantially, evidence of a Raf-1-independent ERK activation pathway. To identify Raf-1-independent upstream signaling intermediates of mitogen-activated protein kinase/ERK kinase-1 (MEK1), the dual-function kinase required and sufficient for ERK activation in these cells, lysates from forskolin and PDGF-treated bovine tracheal myocytes were resolved using ion exchange chromatography. Kinase activity for MEK1 was assessed by in vitro phosphorylation assay. In all experiments, the major peak of MEK1 phosphorylation activity was detected in fractions 18 through 26 (80 to 160 mM NaCl), with the peak fraction eluting at a NaCl concentration of 140 mM. The ability of these fractions to activate MEK1 was confirmed by examining the phosphorylation of myelin basic protein, a known substrate for ERKs, in the presence of functional MEK1 and ERK1. Fractions containing kinase activity were also probed with antibodies against MEK kinase-1, Raf-1, A-Raf, B-Raf, Mos, and Tpl-2. None of these proteins was detected in fractions containing peak kinase activity, suggesting the presence of a novel PDGF-stimulated, forskolin-insensitive MEK1 kinase. Further separation of fractions holding peak MEK phosphorylation activity by gel filtration suggested an apparent molecular mass of 40 to 45 kD. We conclude that PDGF-induced activation of MEK1 in bovine tracheal myocytes is mediated at least in part by a novel kinase.

Mitogen-activated signaling in cultured airway smooth muscle cells.

Airway hyperresponsiveness and excess smooth muscle mass coexist in patients with asthma and bronchopulmonary dysplasia. This increase in airway smooth muscle mass, which in part relates to smooth muscle proliferation, may increase bronchoconstrictor-induced airway narrowing, even in the absence of excessive force generation. Thus, there is need for a precise understanding of the events involved in airway smooth muscle mitogenesis. This review examines the inflammatory substances and growth factors that induce airway smooth muscle proliferation, and the signaling pathways that may be involved in the transduction of these extracellular signals to the cell nucleus. Also discussed are various antimitogenic substances and potential mechanisms underlying the inhibition of cell proliferation. Central to the discussion are the extracellular signal regulated kinases (ERKs), serine/threonine kinases of the mitogen-activated protein kinase (MAP kinase) superfamily, which upon activation, translocate from the cytoplasm to the nucleus after mitogenic stimulation. Insight gained from studies of cultured airway smooth muscle growth and mitogen-activated signaling may shed light on parallel mechanisms that may operate in asthma and in bronchopulmonary dysplasia, and may lead to therapeutic interventions against airway remodeling.

Children's and women's ability to fire handguns. The Pediatric Practice Research Group.

OBJECTIVES: To evaluate whether strength differences between children and women might keep children from firing handguns and to determine how many young children can fire available handguns. DESIGN: One- and two-index finger trigger-pull strength was tested using a standard protocol. Data on trigger-pull settings of 64 commercially available handguns were obtained. SETTING AND PARTICIPANTS: Convenience sample of well children and their mothers at four Chicago (Ill)-area pediatric practices for health supervision visits, and of siblings of emergency department patients, during an 8-week period. INTERVENTION: None. MAIN OUTCOME MEASURE: One- and two-index finger trigger-pull strength of mothers and children. RESULTS: Twenty-five percent of 3- to 4-year-olds, 70% of 5- to 6-year-olds, and 90% of 7- to 8-year-olds have a two-finger trigger-pull strength of at least 10 lb, the fifth percentile one-finger trigger-pull strength of adult women. Forty (62.5%) of 64 handguns require trigger-pull strength of less than 5 lb; 19 (30%) of 64 require 5 to 10 lb. CONCLUSIONS: Significant overlap exists in the trigger-pull strength of young children and women, limiting the potential use of increased trigger-pull settings to discourage firearm discharge by children. Young children are strong enough to fire many handguns now in circulation.

Bronchoalveolar lavage fluid from immature rats with hyperoxia-induced airway remodeling is mitogenic for airway smooth muscle.

We previously demonstrated that hyperoxia-exposed immature rats develop airway smooth muscle layer thickening; this remodeling appears partially attributable to smooth muscle hyperplasia. In this study, we tested the hypothesis that excess mitogenic activity for airway smooth muscle cells is present within the lungs of hyperoxia-exposed immature rats. We assessed the proliferative effect of bronchoalveolar lavage (BAL) fluid from air- and O2-exposed animals on cultured rat tracheal smooth muscle cells. BAL fluids from air- or O2-exposed immature rats increased DNA synthesis ([3H]-thymidine incorporation at 24 h of incubation) and cell number (compared with DMEM-treated control cells, at 2 days of incubation), but BAL fluid from O2-exposed animals had significantly greater mitogenic effects. This excess mitogenic activity was lipid inextractable and was ablated by trypsin digestion, indicating that at least one polypeptide growth factor was responsible; molecular sieve fractionation demonstrated a molecular weight of > 10 kD. Because platelet-derived growth factor (PDGF) has been identified in other models of hyperoxia exposure, we tested the further hypothesis that PDGF contributes to the observed excess mitogenic activity. Addition of neutralizing anti-PDGF antibodies to BAL-stimulated smooth muscle cultures did not reduce BAL fluid-induced mitogenesis. These data indicate that the lungs of O2-exposed rats contain excess mitogenic activity for airway smooth muscle, attributable to non-PDGF polypeptide growth factors. It is conceivable that this abnormal mitogenic activity contributes to O2-induced airway smooth muscle remodeling observed in immature rats in vivo.

In vivo hyperoxic exposure increases cultured lung fibroblast proliferation and c-Ha-ras expression.

Exposure to hyperoxia has been demonstrated to alter the cell number of lung fibroblasts in vivo. The precise mechanism of lung fibroblast proliferation after hyperoxic exposure has not been elucidated, however. We examined the growth characteristics of lung fibroblasts isolated from 21-day-old rats exposed to air or 100% O2 for 8 days. Cell proliferation was assessed by hemocytometry, [3H]thymidine incorporation, and fractional labeling with the thymidine analog bromodeoxyuridine. Under all conditions tested, fibroblasts isolated from O2-exposed rats grew more rapidly than those from air-exposed rats. Conditioned medium from fibroblasts isolated from hyperoxia-exposed rats failed to increase the [3H]thymidine incorporation of control cells to that observed in cells isolated from hyperoxia-exposed animals, suggesting that an autocrine growth factor was not responsible for the excess proliferation. Sensitivity to exogenous growth factors was assessed by measuring the response to increasing concentrations of insulin-like growth factor-1 (IGF-1). Relative to 1% fetal bovine serum (FBS), concentrations of IGF-1 between 3 and 30 ng/ml significantly increased the [3H]thymidine incorporation of fibroblasts derived from hyperoxic animals, whereas control cells were unresponsive to IGF-1 stimulation. The apparent sensitivity to IGF-1 led us to assess the effect of in vivo hyperoxic exposure on the expression of c-Ha-ras, which encodes a membrane-bound, GTP-binding/hydrolyzing protein essential for progression through G1 in the cell cycle. ras mRNA levels in quiescent, control cells were minimal but increased following serum stimulation. The c-Ha-ras expression of lung fibroblasts from hyperoxia-exposed animals, on the other hand, was substantial in quiescent cells and remained high after serum exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Hyperoxia increases airway cell S-phase traversal in immature rats in vivo.

Exposure of 21-day-old Sprague-Dawley rats to hyperoxia (> 95% O2 for 8 days) causes thickening of the airway epithelial and smooth muscle layers. To test the hypothesis that hyperoxic exposure increases airway layer DNA synthesis, we labeled the nuclei of cells undergoing S-phase by administering the thymidine analog bromodeoxyuridine (BrdU). BrdU was administered on days 3 and 4, 5 and 6, or 7 and 8 of air or O2 exposure, and the lungs were harvested immediately thereafter. Histologic sections were stained with an avidin-biotin-immunoperoxidase stain that revealed BrdU incorporation into nuclei, and a hematoxylin counterstain. After 4 days of air or O2 exposure, there was no difference in BrdU fractional labeling between control and hyperoxic animals. Thereafter, fractional BrdU labeling of the small airway (circumference < 1,000 microns) epithelium and smooth muscle layer was significantly increased in O2-exposed animals (P < 0.01, unpaired t test). The fractional labeling of larger, central airway smooth muscle layer cells was also increased after 8 days of O2 exposure (P < 0.01). In another cohort of O2-exposed animals, measurements of airway layer dimensions demonstrated increases in small airway epithelial and smooth muscle layer thickness that paralleled the time course seen for BrdU incorporation. We conclude that O2 exposure of immature rats increases airway epithelial and smooth muscle layer cellular DNA synthesis. These data suggest that hyperplasia of airway epithelial and smooth muscle layer cells may contribute to hyperoxia-induced airway remodeling.

Pentoxifylline does not protect against hyperoxic lung injury in rats.

Hyperoxia has been used extensively as a model of acute lung injury. The drug pentoxifylline has been shown to have a protective effect in other models of lung injury. We sought to determine whether pentoxifylline protects against hyperoxic lung injury in rats by decreasing the accumulation of neutrophils within the lung. A total of 84 rats were studied. Twenty four rats were randomized into four groups. Two groups of rats were pretreated for 48 h with either pentoxifylline (20 mg.kg-1) or saline, and then exposed to > 95% O2 for 60 h while treatments continued. Two groups of control rats received the same treatment regimens as the O2-exposed animals, but breathed room air. Neutrophil accumulation in the lung was quantified both by histology and myeloperoxidase activity. Lung neutrophil accumulation increased in the oxygen-exposed group receiving pentoxifylline as compared to oxygen- or air-exposed rats receiving saline injections. Total glutathione was higher in lung homogenates from the hyperoxic, pentoxifylline-treated group than in homogenates from the other three groups. To study survival, 60 rats were exposed to > 95% O2 for 120 h, 30 rats were pretreated with pentoxifylline, and 30 received saline. Survival after 120 h of exposure to hyperoxia was not altered by pentoxifylline treatment (pentoxifylline treated: 6 out of 30 survived; saline treated: 2 out of 30 survived). We conclude that pentoxifylline does not reduce mortality or lung injury in rats exposed to hyperoxia and is associated with an increase in lung neutrophil accumulation.

Recovery of airway structure and function after hyperoxic exposure in immature rats.

We have previously demonstrated that hyperoxic exposure (> 95% O2 for 8 d) induces airway cholinergic hyperresponsiveness and remodeling in 21-d-old rats. To examine the potential relationship between airway hyperresponsiveness and remodeling in these animals, we exposed rats to air or hyperoxia for 8 d, returned them to air-breathing, and measured airway responsiveness to inhaled acetylcholine (ACh) and layer thicknesses immediately after or 16 or 48 d after cessation of air or O2 exposure. The ACh concentration required to increase resistance by 100% (EC200ACh) was calculated by linear interpolation. Small airway (circumference < 1,000 microns) and medium-sized, conducting airway (1,000 to 3,000 microns) epithelial and smooth muscle layer mean thicknesses and fractional areas (layer area/luminal cross-sectional area) were determined from lung sections by contour tracing using a digitizing pad and computer. As we reported previously, after 8 d of O2 exposure, group mean log EC200ACh was significantly reduced relative to that in control animals (p < 0.001). Similarly, hyperoxic exposure was associated with significant increases in all parameters of airway layer thickness assessed (p < 0.05). However, by 16 d after cessation of O2 exposure, there were no longer statistically significant differences in log EC200ACh, airway layer thickness, or fractional area between control and O2-exposed animals. Further studies, in a second cohort of animals killed 0, 3, 6, 8, or 13 d after cessation of O2 exposure, demonstrated progressive reductions in small airway epithelial and smooth muscle layer thicknesses, confirming that hyperoxia-induced airway remodeling resolves by approximately 2 wk after termination of O2 exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Airway reopening pressure in isolated rat lungs.

In a previous modeling study, we predicted that the yield pressure for airway reopening (Pyield) should depend on airway fluid surface tension (gamma) and airway radius (R), according to the relationship Pyield = 8.3 gamma/R. To test this prediction, we studied tantalum bronchograms of isolated perfused rat lungs from three rats by using microfocal X-ray imaging. Thirty-two airways with diameters ranging from 300 to 2,400 microns were recorded as the airways were collapsed and reinflated. Airway pressure was reduced transiently to -40 cmH2O to produce airway closure. Airway pressure was then slowly increased from 0 to 25 cmH2O. In each airway, the observed diameter remained constant until a Pyield was reached; at this pressure, airways were seen to "pop" open, allowing clear identification of airway reopening pressure. When Pyield was plotted against diameter at maximum inflation, the experimental data were in approximate agreement with predictions of Pyield made assuming a gamma of 35 dyn/cm. The close correspondence of the measured values with these predictions suggests that surfactant is present in these airways and facilitates airway reopening.