Ronald W. Walenga

Excessive inflammatory response of cystic fibrosis mice to bronchopulmonary infection with Pseudomonas aeruginosa.

In cystic fibrosis (CF), defective function of the cystic fibrosis transmembrane conductance regulator (CFTR) in airway epithelial cells and submucosal glands results in chronic pulmonary infection with Pseudomonas aeruginosa. The pulmonary infection incites an intense host inflammatory response, causing progressive suppurative pulmonary disease. Mouse models of CF, however, fail to develop pulmonary disease spontaneously. We examined the effects of bronchopulmonary infection on mice homozygous for the S489X mutation of the CFTR gene using an animal model of chronic Pseudomonas endobronchial infection. Slurries of sterile agarose beads or beads containing a clinical isolate of mucoid P. aeruginosa were instilled in the right lung of normal or CF mice. The mortality of CF mice inoculated with Pseudomonas-laden beads was significantly higher than that of normal animals: 82% of infected CF mice, but only 23% of normal mice, died within 10 d of infection (P = 0.023). The concentration of inflammatory mediators, including TNF-alpha, murine macrophage inflammatory protein-2, and KC/N51, in bronchoalveolar lavage fluid in CF mice 3 d after infection and before any mortality, was markedly elevated compared with normal mice. This inflammatory response also correlated with weight loss observed in both CF and normal littermates after inoculation. Thus, this model may permit examination of the relationship of bacterial infections, inflammation, and the cellular and genetic defects in CF.

Infant animal model of pulmonary mycotoxicosis induced by Stachybotrys chartarum

In recent years cases of often fatal pulmonary hemorrhage in infants have been associated with water damaged homes and the toxigenic fungusStachybotrys chartarum. The fungal spores contain mycotoxins which could be injurious to the rapidly developing lung. In order to understand the developmental pathophysiology of this disease we developed an infant rat model of stachybotrytoxicosis describing the effects of fungal spores on survival, growth, histopathology of the lung and respiration. Conidia ofS. chartarum were instilled intratracheally (1.0–8.0 × 105/gm wt.) in 4-dold Sprague-Dawley rat pups. Two control groups received either sterile PBS or a suspension of spores extensively extracted with ethanol to remove toxins. Lethal dose response was determined (LD50 = 2.7 × 105 spores/gm wt.). All dead pups had extensively hemorrhagic lungs. Growth of surviving animals was impaired in a dose-dependent manner. Changes of pulmonary function parameters in rats treated with 1.1 × 105 spores/g were consistent with an increased respiratory resistance. Histology of lungs revealed fresh hemorrhage, sparse hemosiderin-laden macrophages, and evidence of inflammation including thickened alveolar septa infiltrated by lymphocytes and mononuclear cells and intra-alveolar macrophages. Significant increases (p = 0.001) in numbers of macrophages (2-fold), lymphocytes (5-fold) and neutrophils (7-fold) were found in BAL fluid. Hemoglobin was elevated 2-fold (p = 0.004). Proinflammatory mediator IL-1β increased more than 6-fold and TNF-α30-fold (p = 0.001). Extracted spores had a minimal effect on all examined parameters in BAL fluid indicating that mycotoxins are primarily responsible for the hemorrhagic and inflammatory response.

15-HETE-substituted diglycerides selectively regulate PKC isotypes in human tracheal epithelial cells

Human tracheal epithelial (TE) cells selectively incorporate their major lipoxygenase product, 15-hydroxyeicosatetraenoic acid (15-HETE), into the sn-2 position of phosphatidylinositol (PI) (S. E. Alpert and R. W. Walenga. Am. J. Respir. Cell Mol. Biol. 8: 273-281, 1993). Here we investigated whether 15-HETE-PI is a substrate for receptor-mediated generation of 15-HETE-substituted diglycerides (DGs) and whether these 15-HETE-DGs directly activate and/or alter conventional diacylglycerol-induced activation of protein kinase C (PKC) isotypes in these cells. Primary human TE monolayers incubated with 0.5 microM 15-[3H]-HETE or 15-[14C]HETE for 1-2 h were stimulated with 1 nM to 1 microM platelet-activating factor (PAF) for 30 s to 6 min, and the radiolabel in the medium, cellular phospholipids, and neutral lipids was assessed by high-performance liquid and thin-layer chromatography. PAF mobilized radiolabel from PI in a dose-dependent manner (22 +/- 5% decrease after 1 microM PAF) without a concomitant release of free intra- or extracellular 15-HETE. 14C-labeled DGs were present in unstimulated TE monolayers incubated with 15-[14C]HETE, and the major 14C band, identified as sn-1,2-15-[14C]HETE-DG, increased transiently in response to PAF. Western blots of freshly isolated and cultured human TE cells revealed PKC isotypes alpha, betaI, betaII, delta, epsilon, and zeta. In vitro, cell-generated sn-1, 2-15-[14C]HETE-DG selectively activated immunoprecipitated PKC-alpha and inhibited diacylglycerol-induced activation of PKC-alpha, -delta, -betaI, and -betaII. Our observations indicate that 15-HETE-DGs can modulate the activity of PKC isotypes in human TE cells and suggest an intracellular autocrine role for 15-HETE in human airway epithelia.Human tracheal epithelial (TE) cells selectively incorporate their major lipoxygenase product, 15-hydroxyeicosatetraenoic acid (15-HETE), into the sn-2 position of phosphatidylinositol (PI) (S. E. Alpert and R. W. Walenga. Am. J. Respir. Cell Mol. Biol. 8: 273-281, 1993). Here we investigated whether 15-HETE-PI is a substrate for receptor-mediated generation of 15-HETE-substituted diglycerides (DGs) and whether these 15-HETE-DGs directly activate and/or alter conventional diacylglycerol-induced activation of protein kinase C (PKC) isotypes in these cells. Primary human TE monolayers incubated with 0.5 μM 15-[3H]-HETE or 15-[14C]HETE for 1-2 h were stimulated with 1 nM to 1 μM platelet-activating factor (PAF) for 30 s to 6 min, and the radiolabel in the medium, cellular phospholipids, and neutral lipids was assessed by high-performance liquid and thin-layer chromatography. PAF mobilized radiolabel from PI in a dose-dependent manner (22 ± 5% decrease after 1 μM PAF) without a concomitant release of free intra- or extracellular 15-HETE. 14C-labeled DGs were present in unstimulated TE monolayers incubated with 15-[14C]HETE, and the major 14C band, identified as sn-1,2-15-[14C]HETE-DG, increased transiently in response to PAF. Western blots of freshly isolated and cultured human TE cells revealed PKC isotypes α, βI, βII, δ, ε, and ζ. In vitro, cell-generated sn-1,2-15-[14C]HETE-DG selectively activated immunoprecipitated PKC-α and inhibited diacylglycerol-induced activation of PKC-α, -δ, -βI, and -βII. Our observations indicate that 15-HETE-DGs can modulate the activity of PKC isotypes in human TE cells and suggest an intracellular autocrine role for 15-HETE in human airway epithelia.

Prostaglandin E2 Attenuates Hyperoxia-Induced Injury in Cultured Rabbit Tracheal Epithelial Cells

ABSTRACT: We assessed the kinetics of hyperoxia-induced prostaglandin E2 (PGE2) production by cultured rabbit tracheal epithelial (TE) cells with different inherent capacities to generate PGE2 and the role of endogenous PGE2 production in protecting these cells from hyperoxic injury. Rabbit TE cells grown to confluence with or without lipid supplements [0.1% Excyte III (Miles-Pentex) and 1 /μM arachidonic acid] were exposed for 2 h to control (5% CO2/air) or hyperoxic (5% CO2/90% O2) atmospheres at a gas-fluid interface. Serial cell culture effluents collected during exposure were analyzed for PGE2 by enzyme-linked immunoassay. Basal PGE2 production by lipid-supplemented cells was approximately 3-fold greater than that by unsupplemented cultures (p < 0.01). In lipid-supplemented cells, PGE2 production doubled after 15 min of hyperoxic exposure (p < 0.05) and then declined to approximately 50% of initial levels, whereas exposure to 5% CO2/air did not significantly change PGE2 production. In unsupplemented cells, neither control nor hyperoxic exposure altered PGE2 production. Hyperoxia-exposed TE cells had decreased ability to convert 10 μM exogenous arachidonic acid to PGE2, suggesting hyperoxia-induced inhibition of the enzymes involved in PGE2 synthesis. Lipid-supplemented cells were less susceptible to hyperoxic injury than unsupplemented monolayers, as evidenced by increased viability (trypan blue exclusion) and decreased generation of lipid peroxides (thiobarbituric acid reactive substances). Addition of exogenous PGE2 to unsupplemented cultures at concentrations that were produced by lipid-supplemented cells (2 ng/mL every 15 min) during hyperoxic exposure eliminated these differences in hyperoxia-induced lipid peroxidation and cytotoxicity. Our observations suggest that hyperoxic exposure inhibits TE cell cyclooxygenase activity and that endogenously produced airway epithelial cell PGE2 has a cytoprotective role against hyperoxia-induced injury.

Functional Consequences of Abnormal Fatty Acid Profiles in Cultured Airway Epithelial Cells

Maintenance of serum-free conditions for the culture of TE or other airway epithelial cells provides a defined environment in which to explore the regulation of cellular functions. Yet TE cells appear to be dependent on the medium for essential, if not all, polyunsaturated fatty acids. At present, some laboratories routinely use serum to support the growth of airway epithelial cells, presumably in part through recognition that cells of mammalian origin require an exogenous source of lipids. While 5% FBS can increase the linoleic and arachidonic acid content of cultured rabbit and human TE cells, it does not fully restore the fatty acid composition of cultured TE cells to that of freshly isolated cells, particularly in the case of human TE cells. Equally good, if not better, repair of membrane fatty acid composition can be achieved by addition of a defined, commercial non-serum source of lipids (Excyte III) plus exogenous arachidonic acid. Cultured TE cells maintained in serum-free medium have been shown to be deficient in prostaglandin and HETE production, both at baseline and in response to physiological stimuli compared to TE cells with greater endogenous content of arachidonic acid. Differences between lipid supplemented and unsupplemented cultured TE cells in cAMP response to PGE2 and in susceptibility to hyperoxic injury have been observed. Other cellular functions regulated by the fatty acid composition of membrane lipids may also be impaired in lipid unsupplemented cells. It is evident that the maintenance of as normal as possible membrane fatty acid content is essential to the use of cultured TE cells as experimental models of airway epithelium.