Abstract
Concurrent Session
2:00 PM
Saturday, February 19, 2000
932 Chronic Ethanol Ingestion Alters Surfactant Composition and Increases Acute Lung Injury in Septic Rats
Alcohol abuse increases the incidence and the mortality of the acute respiratory distress syndrome (ARDS) in septic patients. ARDS is characterized by significant disruptions in surfactant composition and function. We previously determined that chronic ethanol ingestion in rats, via depletion of the critical antioxidant glutathione, impaired surfactant secretion in vitro and increased endotoxin-mediated edematous injury in isolated lungs that were perfused ex vivo. Therefore, we hypothesized that ethanol ingestion impairs surfactant composition in vivo and thereby renders the lung susceptible to acute injury during sepsis. We tested this hypothesis in a model of chronic ethanol ingestion followed by surgically induced abdominal sepsis. Rats were fed an isocaloric liquid diet with or without ethanol (36% total calories) for 6 wks. Sepsis was induced via cecal ligation and perforation (CLP) under inhaled anesthesia. Septic rats developed significant changes in appearance and physiologic parameters over the 20 to 24 hour period after surgery, and prior to sacrifice, compared to sham-treated rats in which intravascular catheters were placed but no CLP was performed. Although both septic groups had similar degrees of tachypnea, hypotension, and arterial lactate levels prior to sacrifice, ethanol-fed rats had lower (p<0.05) fractions (54±4.1% vs. 62±3.7%) of their surfactant phospholipid pools in the large aggregate (LA) form, which is the bioactive component of surfactant. Furthermore, the ratio of small aggregates, which are not bioactive, to large aggregates (SA/LA) were higher (p<0.05) in the ethanol-fed rats during sepsis. In parallel, ethanol-fed rats had lower (p<0.05) arterial pO2 levels (64.8 vs. 77.1 mm Hg), and more (p<0.05) neutrophil infiltration, as reflected by tissue levels of myeloperoxidase, compared to control-fed rats. We conclude that chronic ethanol ingestion altered surfactant composition in septic rats, and that these alterations were associated with more severe lung injury. We speculate that alcohol abuse, via similar effects on surfactant composition and function, predisposes patients to ARDS following sepsis and/or other acute insults. We speculate further that glutathione replacement could potentially improve surfactant homeostasis and decrease the severity of acute lung injury in these susceptible patients.
933 Vitamin D Inhibits Pulmonary Surfactant Protein Gene Expression in Human Fetal Lung Explants Cultured In Vitro
Respiratory distress syndrome (RDS), the leading cause of neonatal morbidity and mortality, is due the lack of pulmonary surfactant in premature lungs. Surfactant, a lipoprotein complex produced after 80% of gestation is complete, acts to reduce surface tension at the air/liquid interface of the lung alveolus. Previously, 1α,25-dihydroxycholecalciferol (vitamin D), a steroid hormone, has been reported to increase expression of pulmonary surfactant in fetal rat lung explants and to accelerate the physiological maturation of fetal rat lung. However, the effect of vitamin D on surfactant protein gene expression has not been reported. Our interests lie in defining the molecular mechanisms by which steroids regulate surfactant protein gene expression. We have investigated the effect of various concentrations of vitamin D on expression of three major surfactant proteins (SP-A, SP-B, and SP-C) in human fetal lung explants cultured in vitro in the absence or presence of cyclic AMP (cAMP). Levels of expression of the surfactant genes were determined by Northern analysis of RNA isolated from the explants after incubation for various times in culture using radiolabeled surfactant protein cDNAs as probes. While vitamin D has been reported to accelerate fetal lung maturity and increase expression of surfactant lipids, it appears to decrease surfactant protein gene expression in human fetal lung in vitro. The effect of vitamin D on surfactant protein gene expression is most apparent when the tissue is incubated in the presence of cAMP, indicating that the hormone antagonizes the stimulatory effect of cAMP. The effect of vitamin D is more apparent as the time of culture increases. These unexpected results suggest complex actions of steroid hormones on the surfactant system in fetal lung.
934 the Role of Phosphatidyl Glycerol Hydrolysis in Phospholipase A2-Mediated Surfactant Dysfunction
Patients with ARDS and asthma demonstrate increased secretory A2 phospholipase (sPLA2) activity and surfactant dysfunction. Our lab and others have demonstrated that generation of lysophospholipids by sPLA2's can lead to surfactant dysfunction. However, clues from several studies suggest that other mechanisms including disruption of the interaction between phosphatidylglycerol (PG) and surfactant apoproteins may also be important. In this study, we examine the potential role of sPLA2-mediated hydrolysis of PG on surfactant dysfunction. Group I (porcine pancreatic) and Group II sPLA2 (recombinant human Group IIA-transfected COS cell line) were incubated with surfactant (Survanta®, 1.0 mg phospholipid/ml in buffered saline with 5 mM CaCl2) labeled with either [3H]-PG or [3H]-DPPC for 2 hours at 37°C. Samples were then analyzed for: (1) hydrolysis of PG and PC using lipid extraction, TLC and scintillation counting; (2) surfactant function (γmin) using a pulsating bubble surfactometer. Our results demonstrate significant hydrolysis of [3H]-PG with group I (1.10,000 U/ml, max=94%) and group II (100–10,000, max=83%). At the same dose ranges, group I caused similar hydrolysis of [3H]-DPPC, while group II did not (max=4%). Both enzymes caused significant surfactant dysfunction (increased γmin) at submaximal concentrations (group I = 500 U/ml, group II = 1000 U/ml). Based on previous experiments, the amount of lysophospholipids (% total lipid) generated at those enzyme concentrations were significantly less than required to account for surfactant dysfunction. These data suggest that the hydrolysis of PG is an important independent mechanism in sPLA2-mediated surfactant dysfunction.
935 Acute Lung Injury Edema Fluid Regulates Fibroblast Proliferation and Differential Gene Expression
Pro-collagen peptide levels in pulmonary edema fluid from patients with acute lung injury (ALI) predict a poor outcome and/or fibrosing alveolitis. We hypothesized that ALI edema fluid could regulate fibroblast proliferation through its effects on specific gene expression. Edema fluids were obtained and processed within 4 hrs of mechanical ventilation in patients with either ALI or hydrostatic (HY) edema as controls. Human lung fibroblasts were incubated with dilutions (1/2 to 1/200) of edema fluid and plasma (both in serum free MEM) using samples pooled from 10 patients/group. Cell number was counted daily for 4–8 days and gene expression profiling was performed by 32P-RT-PCR on total RNA from ALI and HY edema fluid-exposed fibroblasts in the presence of 588 gene-specific primers using cDNA arrays. Fibroblasts exhibited a dose-dependent proliferative response to ALI fluid that was significantly greater than the HY edema fluid, whereas the effect of plasma on proliferation did not differ between groups. Gene expression profiling revealed 10 genes that were expressed at >3-fold different levels in the ALI fluid exposed fibroblasts (8 hr@1/2 dilution), as compared with the HY edema fluid. These results indicate that ALI edema fluid is a potent modulator of fibroblast proliferation and gene expression, and demonstrate that soluble pulmonary edema fluid factors may be important determinants of the extent of the fibroproliferative response seen in acute lung injury.
Supported by the NIH - HL58655, HL51856 and VA MERIT Award
936 Reactive Nitrogen Species Correlate with Airway Inflammation in Lung Transplants
Patients who have undergone lung transplantation have high concentrations of reactive nitrogen species (RNS) in bronchoalveolar lavage fluid (BALF) and serum. This study investigated myeloperoxidase (MPO) concentration in BALF and potential use of RNS as markers of airway inflammation in lung transplantation patients (n=40). All patients underwent spirometry, transbronchial biopsies (TBBx) and bronchoalveolar lavage as post transplant surveillance. BALF and serum urea and nitrite (nitrite plus reduced nitrate) were assayed. Most serum nitrite was represented by reduced nitrate (NO3). BALF MPO and differential cell counts also were done. Observed FEV1 was corrected to baseline value. MPO concentration was 0.26±.05 µg/mL and correlated significantly with serum nitrite, BALF percent neutrophil's, “B score” (degree of airway inflammation on TBBx) and change in FEV1. Total nitrite in BALF and serum averaged 6.3±.5 and 49±5 µM, respectively. Based on the ratio of BALF and serum urea, epithelial lining fluid (ELF) nitrite was 320±30 µM. Serum nitrite concentration correlated significantly with percent neutrophil's in BALF, change in FEV1 and “B score”. BALF nitrotyrosine did not correlate with clinical variables. “B score” could be predicted by the change in FEV1 and serum nitrite: Bscore=2.091+(0.00654 serum NO2)-(0.0183 correctedFEV1) (R=0.498, P<0.05) (sensitivity 89%, specificity 38%, positive predictive value 67%, negative predictive value 71%). MPO is found in association with increased RNS, supporting the hypothesis that it has a role in inflammation. Measurement of Serum RNS and BALF RNS and MPO may be useful non-invasive markers of airway inflammation in human lung allografts. A prospective study will test this hypothesis.
937 Inhibition of Isocyanate-Induced Inflammation by Sensory Nerves
Exposure to isocyanates causes airway inflammation and decrements in lung function. Sensory nerves containing tachykinin neuropeptides innervate the lung and may regulate airway inflammation and hyperreactivity. To examine the role of sensory nerves in irritant-induced lung inflammation, we studied genetically manipulated mice with altered innervation of the airways. We previously generated transgenic mice that overexpress nerve growth factor (NGF) from the lung-specific Clara cell secretory protein (CCSP) promoter. CCSP-NGF mice exhibit hyperinnervation of the airways by sympathetic and tachykinin-containing sensory nerve fibers (Hoyle et al., Am. J. Cell Mol. Biol. 18:149-157, 1998). We are also studying mice with a targeted mutation in the gene encoding the low affinity NGF receptor p75, which are deficient in tachykinin-containing sensory nerve fibers. CCSP-NGF transgenic, NGFR knockout, and wild-type C57BL/6 mice were exposed to an aerosol of hexamethylene hexamethylene diisocyanate (HDI), a component of polyurethane spray paints, at 10 mg/m3 for 5 hours. Inflammation was assessed 1 day after exposure. The number of neutrophils recovered in lavage fluid was significantly greater in NGFR knockout mice (median 44,500; n=8; p=0.02 by Mann-Whitney U test) compared to wild-type mice (median 20,600; n=15), whereas the number in CCSP-NGF transgenic mice was significantly decreased (median 8,500; n=8; p<0.02). These results show that the magnitude of HDI-induced neutrophilic inflammation inversely correlates with the sensory innervation of the lung and suggest that airway sensory nerves protect the lung from inflammation induced by HDI.
938 Recurrent Aspiration of Milk Produces Experimental Asthma in a Murine Model: Increased Serum Ige, Eosinophilic Inflammation, Cytokine Imbalance and Goblet Cell Hyperplasia
Aspiration of milk into the respiratory tract is a common respiratory problem in infants and young children. While an association between aspiration of milk and exacerbation and/or development of asthma has been repeatedly suggested, the mechanistic links between these conditions are poorly understood. We have recently developed a novel diagnostic method for pulmonary aspiration (Am J Respir Crit Care Med 1999, In Press). In addition, we have described that recurrent aspiration produces altered airway function in a murine model (Am J Respir Crit Care Med 1999, 159:4871). In this study, we evaluated the inflammatory changes within the airways and the immune response to milk aspiration in our mouse model. Under light anesthesia, BALB/c mice received daily intranasal instillations of cow's milk. Control animals received sterile physiologic saline in a similar manner. Bronchoalveolar lavage (BAL) was performed at various time points for cell differentials and cytokine measurements. The latter were measured in the supernatant fluid by ELISA and expressed in terms of pg/ml as % control (mean±SE). In addition, serum IgE concentrations were measured by ELISA and expressed as pg/ml (mean±SE). Mouse lungs were then fixed in formalin and stained with hematoxylin/eosin and period acid schiff. We found recurrent aspiration of milk produced eosinophilic inflammation as demonstrated by an increased number of eosinophils in the BAL when compared to control animals: 18.0±4.8% (n=8) vs 0.2±0.2% (n=10), respectively. Histologic evaluation of lung sections revealed the presence of significant inflammatory changes characterized by a large number of lymphocytes and eosinophils around the airways. Furthermore, milk aspiration produced thickening of the airway epithelium and marked goblet cell hyperplasia. These changes were associated with increased levels of IL-5 and decreased levels of IFN-γ in the BAL when compared to control animals. Furthermore, total serum IgE concentrations were enhanced by aspiration of milk when compared to control mice: 267.0±23.7 (n=6) and 127.0±14.5 pg/ml (n=6), respectively. Our findings demonstrate recurrent milk aspiration is capable of producing eosinophilic inflammation within the lung associated with a disruption of the normal Th1/Th2 cytokine balance and goblet cell hyperplasia in a murine model. These changes reproduce the inflammatory and immune alterations present in human asthma and could lead and/or contribute to the development of altered airway function.
939 Allergic Inflammation in Mice with Genetically Altered Innervation of the Airways
Sensory nerves containing tachykinin neuropeptides innervate the lung and may regulate airway inflammation and hyperreactivity. To examine the role of sensory nerves in allergic inflammation, we studied genetically manipulated mice with altered innervation of the airways. We previously generated transgenic mice that overexpress nerve growth factor (NGF) from the lung-specific Clara cell secretory protein (CCSP) promoter. CCSP-NGF mice exhibit hyperinnervation of the airways by sympathetic and tachykinin-containing sensory nerve fibers (Hoyle et al., Am. J. Cell Mol. Biol. 18:149-157, 1998). We are also studying mice with a targeted mutation in the gene encoding the low affinity NGF receptor p75, which are deficient in tachykinin-containing sensory nerve fibers. We examined allergic inflammation in CCSP-NGF transgenic, NGFR knockout, and wild-type C57BL/6 mice after sensitization and airway challenge with ovalbumin (OVA). Mice were sensitized on days 1 and 8 by i.p. injection of 50 µg OVA adsorbed to 50 mg aluminum hydroxide. On days 15-21, mice were exposed to an aerosol of 1% OVA. Inflammation was examined by lung lavage 1-3 days following the last challenge. OVA challenge induced a robust eosinophilic inflammatory response in all sensitized mice, and no significant difference in the magnitude of the response was observed between the genetically altered mice and age-matched wild-type controls. Peak BAL eosinophils (x 105) were: 29.8±3.8 for CCSP-NGF transgenic mice (n=6) and 37.1±5.6 for nontransgenic mice (n=6; p=0.16); 28.6±9.0 for NGFR knockout mice (n=5) and 30.8±8.9 (n=5; p=0.17) for wild-type mice. Our results suggest that sensory nerves do not significantly affect allergic inflammation in mice.