Cationic proteins increase the permeability of cultured rabbit tracheal epithelial cells: modification by heparin and extracellular calcium.

Airway inflammation is a consistent finding in asthma, and increased amounts of eosinophil-derived cationic proteins are present in bronchoalveolar lavage fluid from asthmatic subjects. Tracheal instillation of a variety of naturally occurring and synthetic cationic proteins has been shown to induce airway hyperresponsiveness in animal models. Cationic proteins may alter the barrier function of airway epithelium, allowing increased access of agonists to underlying nerves and airway smooth muscle. To examine the effect of cationic proteins on airway epithelial cell function, rabbit tracheal epithelial cells were isolated and cultured on collagen-coated filter membranes. Both apical and basolateral exposure of the cell cultures to poly-L-lysine and poly-L-arginine decreased transepithelial electrical resistance (Rt) over 60 min. There were no discernable light microscopic changes in the morphology of the cultures at 60 min after poly-L-lysine exposure, but permeability to mannitol was increased compared to controls. Evidence for the critical role of cationic charge included the following observations: (1) Poly-L-aspartate, an anionic polyamino acid, had no significant effect on Rt, and (2) the addition of heparin prior to the addition of poly-L-lysine blocked the reduction in Rt. Furthermore, when applied after poly-L-lysine addition, heparin reversed the decrease in Rt in a time-dependent fashion. Increasing the [Ca2+] in the medium from 1 to 10 mM resulted in significant attenuation of the response to polycation addition. These findings suggest that cationic proteins significantly alter the barrier properties of airway epithelium and that cationic charge is a crucial factor. This alteration is not an "all or none" phenomenon, since subsequent addition of heparin resulted in a reversal of the effect. While the precise mechanisms responsible for these observations remain to be elucidated, cationic proteins may be modifying the interaction of extracellular calcium with tight junctions thereby resulting in increased permeability. The barrier function of the epithelium may be perturbed in asthma and a variety of other airway diseases through the presence of cationic proteins derived from inflammatory cells within the airway lumen and/or the subepithelium.

Hydrocortisone promotes the maturation of Na(+)-dependent ion transport across the fetal pulmonary epithelium.

The pulmonary epithelium's change from Cl(-)-dependent fluid secretion to Na(+)-dependent fluid absorption in late gestation appears to be important in the transition of the lung from a fluid-filled organ in utero to an air-filled organ after birth. This maturational process may be regulated in part by hormones. We examined the effects of hydrocortisone on ion transport across monolayer cultures of distal pulmonary epithelial cells isolated from the fetal rat. Hydrocortisone pretreatment enhanced terbutaline (10(-5) M) stimulation of short-circuit current (Isc) but only in monolayers derived from immature fetal cells (day 18 of gestation), stimulating basal Isc by 270% in control monolayers and by 329% in hydrocortisone-pretreated monolayers. Amiloride (10(-4) M) inhibited terbutaline-stimulated Isc by different amounts, depending on gestational age and pretreatment; Isc fell 40% in control monolayers derived from immature cells, 68% in hydrocortisone-pretreated monolayers from immature fetal cells, and approximately 70% in both control and hydrocortisone-pretreated monolayers from mature fetal cells (day 21 of gestation). The basal Isc in monolayers derived from immature cells was also variably inhibited by amiloride with Isc decreasing 26% in control monolayers and 57% in hydrocortisone-pretreated monolayers. The differential responses of terbutaline-stimulated Isc to benzamil, dimethylamiloride, and bumetanide suggested that Isc sensitivity to amiloride was dependent predominantly on Na+ channel activity regardless of gestational age or pretreatment. We conclude that hydrocortisone promotes the maturation of transepithelial Na+ transport in fetal rat lung epithelium by altering Na+ entry into the cell through Na+ channels. Hydrocortisone also enhances beta-adrenergic agonist stimulation of ion transport.

Alterations in eicosanoid production by rat alveolar type II cells isolated after silica-induced lung injury.

Although alveolar type II cells in primary culture have been shown to produce eicosanoids and exposure of type II cells to silica in vitro alters eicosanoid production, the production of eicosanoids by alveolar type II cells isolated after acute lung injury in vivo has not been evaluated. Therefore, we investigated the production of arachidonic acid (AA) metabolites by alveolar type II cells isolated after silica-induced lung injury. Alveolar type II cells were isolated from rats 14 days after intratracheal silica instillation and from untreated animals. Type II cells were separated into normotrophic and hypertrophic populations by centrifugal elutriation, and secreted eicosanoids were determined under basal and stimulated conditions by enzyme immunoassay on the day of isolation and after 1 day in culture. Under basal conditions, freshly isolated type II cells from silica-treated animals produced more prostaglandin (PG) E2 than 6-keto-PGF1 alpha or thromboxane B2 (TxB2). Production of all three prostanoids increased with increasing cell size. The calcium ionophore A23187 stimulated a less than 2-fold increase in PGE2 and 6-keto-PGF1 alpha production in all groups of cells. In contrast, this calcium ionophore greatly enhanced TxB2 and leukotriene C4 (LTC4) production by normotrophic type II cells from both untreated and silica-treated animals. Incubation with exogenous AA suggested that the increased capability of the hypertrophic cells to synthesize PGE2 and TxB2 was due primarily to an increase in arachidonate availability. The hypertrophic type II cells also appear to have increased prostacyclin synthase activity. There were no differences in the catabolism of PGE2 between the normotrophic and the hypertrophic type II cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics of low-dose methotrexate in adult asthmatics.

Several reports have been published on the disposition of methotrexate (MTX) when given in low dosages, but none in asthmatic patients. To address this matter, pharmacokinetic studies were performed in nine patients with severe, steroid-requiring asthma (ages 18-76 yrs) after the sixth weekly intramuscular dose of MTX. Theophylline pharmacokinetic studies were also performed at baseline prior to the start of MTX treatment and at the time of the MTX studies. Total systemic clearance of MTX, given as mean (SD), was 122.6 (25.1) ml/minute, volume of distribution at steady state 0.49 (0.2) L/kg, half-life 3.1 (0.3) hours, mean residence time 5.0 (0.6) hours, and renal clearance 89.1 (36.3) ml/minute. These values are similar to those previously reported for other patient populations receiving low-dose MTX. Eight of these patients were evaluated for changes in theophylline pharmacokinetics. Theophylline clearance at week 6 decreased an average of 19% from baseline and correlated inversely with total MTX clearance. Percentage change in theophylline clearance from baseline was inversely correlated with MTX nonrenal clearance, but was not statistically significant. This finding may indicate competition for clearance pathways between MTX and theophylline.

Effect of low-dose methotrexate on the disposition of glucocorticoids and theophylline.

Low-dose methotrexate (MTX) therapy has been recently proposed as alternative therapy for patients with severe steroid-requiring asthma. Several questions have been raised regarding the mechanism of action, including alteration of pharmacokinetics of two medications used in these patients, specifically, glucocorticoids and theophylline. To address this question, pharmacokinetic studies were performed at baseline and after 6 weeks of treatment with either intramuscular MTX or placebo (folic acid). Plasma concentrations of theophylline were measured by fluorescence polarization immunoassay (TDx, Abbott Diagnostics, Abbott Park, Ill.). Prednisolone, methylprednisolone, and cortisol concentrations were measured by high-performance liquid chromatography. Fifteen adults were enrolled in the double-blind, placebo-controlled trial. No change in prednisolone pharmacokinetic parameters was found. Theophylline clearance decreased an average of 19% in patients randomized to receive MTX, from 48.0 +/- 2.0 ml/hr/kg to 38.9 +/- 3.6 ml/hr/kg (p less than 0.05). This change resembles change observed with theophylline and phenobarbital clearance in which a high degree of interpatient variability is observed. The most likely explanation for the change in theophylline clearance is inhibition of hepatic microsomal enzyme activity. Three patients complained of adverse effects, and dosage was reduced in one patient. The variable effect of MTX on theophylline clearance indicates that theophylline concentration monitoring should be performed in patients receiving both drugs.

Ontogeny of ion transport across fetal pulmonary epithelial cells in monolayer culture.

The transition of the fetal lung from a fluid-secreting to a fluid-absorbing organ is dependent on ion transport across the pulmonary epithelium. This study examined the ion transport characteristics of distal pulmonary epithelial cells isolated from rat fetuses in late gestation and maintained in differentiation-arrested monolayer cultures. The response to inhibitors of active ion transport suggested the presence of apical to basolateral Na+ transport in monolayers derived from each gestational age. However, amiloride inhibition of short-circuit current (Isc) varied with gestational age, decreasing Isc by 30% in monolayers derived from day 18 fetuses and by 55% in monolayers from day 21 fetuses. A portion (10%) of the residual Isc remaining after amiloride addition to monolayers from day 18 fetuses could be inhibited by bumetanide, suggesting the induction of net Cl- transport. Ion-substitution experiments confirmed the presence of Na+ and inducible Cl- transport mechanisms in monolayers from day 18 fetuses and only Na+ transport mechanisms in monolayers from day 21 fetuses. beta-Adrenergic stimulation increased Isc but maintained the age-dependent characteristics of Na(+)- and Cl(-)-dependent ion transport. In summary, monolayer cultures of fetal pulmonary epithelial cells exhibit age-dependent differences in ion transport properties that are consistent with a transition or maturation of the distal pulmonary epithelium from an epithelium capable of Na+ absorption and Cl- secretion preterm to one capable of only Na+ absorption at term.

Lack of benefit of methotrexate in severe, steroid-dependent asthma. A double-blind, placebo-controlled study.

OBJECTIVE: To determine the effect of low-dose methotrexate in asthmatic patients on steroid use, asthma symptom scores, pulmonary function, airway reactivity, blood cellular components, and immunoglobulin E levels. DESIGN: A randomized, double-blind, parallel, placebo-controlled, 13-week clinical trial with follow-up of patients in an open trial of methotrexate at the conclusion of the double-blind study. SETTING: An asthma care outpatient clinic. PATIENTS: From February 1988 to March 1990, 19 patients with severe, steroid-dependent asthma were enrolled in the study. Two of these patients were excluded from analysis. INTERVENTIONS: Patients were administered methotrexate or placebo intramuscularly, to assure complete absorption, once weekly during the 13-week study. RESULTS: Patients on methotrexate and placebo both significantly decreased their steroid dose by 39.6% (95% CI, 25.1% to 54.1%, P = 0.001) and 40.2% (CI, 17.9% to 67.4%, P = 0.003), respectively. Pulmonary function did not differ significantly between the methotrexate and placebo groups. In addition, airway reactivity and symptom scores were unchanged on methotrexate or placebo. No significant toxicities were seen during the course of the 13-week blinded study, but one patient on methotrexate and prednisone in the follow-up period developed Pneumocystis carinii pneumonia and died. Despite continuing methotrexate for up to 1 year, and increasing methotrexate to 30 mg weekly, no significant benefit of methotrexate on asthma control could be shown. CONCLUSION: Our study does not support the use of methotrexate in the treatment of severe asthma.

Prostaglandin and leukotriene production by alveolar type II cells in vitro.

Alveolar type II cells were isolated from adult rats, cultured for 22 h, and individual eicosanoids in the media from unstimulated and stimulated cells were quantified by immunoassay. Stimulation with the calcium ionophore A23187 significantly increased the media levels of prostaglandins (prostaglandin and 6-keto-prostaglandin F1 alpha greater than thromboxane B2). In contrast to previous reports, increased media levels of leukotrienes were also recovered from cells incubated with A23187, but only for cells in culture for less than or equal to 24 h. The production of leukotriene C4 was confirmed by a combination of high-performance liquid chromatography and spectrophotometric analysis. The profile of eicosanoids produced by cultures of alveolar type II cells was distinctly different than that of similarly cultured alveolar macrophages. Finally, stimulation of alveolar type II cell cultures with either a phorbol ester or phospholipase C increased media prostaglandin levels but failed to increase leukotriene levels. We conclude that primary cultures of alveolar type II cells are capable of the de novo metabolism of arachidonic acid to both cyclooxygenase and lipoxygenase products and that the production of leukotrienes is dependent on both time in culture and agonist. Thus alveolar type II cells are a potential source for the production of these eicosanoids in vivo, and the particular lipid mediators produced may vary depending on the pathophysiologic stimulus.

Modulation of bioelectric properties across alveolar type II cells by substratum.

Rat alveolar type II cells were cultured on collagen-coated filters (CCF) and human amnionic basement membrane (ABM) to determine the effect of culture substratum on the development of monolayer bioelectric properties. Monolayers cultured on both substrata rapidly developed bioelectric properties with similar time courses, monolayer capacitance values (approximately 1 muF/cm2), current-voltage relationships, and responses to stimulants and inhibitors of active ion transport. Increasing seeding densities tended to increase monolayer bioelectric properties regardless of culture substratum. Monolayers cultured on ABM had higher resistance values (491 vs. 291 omega.cm2) and lower short-circuit currents (2.85 vs. 4.51 muA/cm2) than monolayers with similar cell densities cultured on CCF. These differences in monolayer bioelectric properties were not due to differences in substratum resistance or capacitance effects. The relationships between monolayer bioelectric properties were also affected by the culture substratum. In additional experiments, cells cultured on contracted gels formed monolayers with high short-circuit currents (9.25 muA/cm2). Cell morphology varied depending on the culture substratum, with cells cultured on contracted gels appearing the most cuboidal, whereas the flattest and most attenuated cells were those cultured on ABM. On the basis of these observations, we conclude that culture substratum significantly affects the development of bioelectric properties across alveolar type II cell monolayers. In vivo the bioelectric properties across the alveolar epithelium may also vary with changes in cellular substratum or cell density (e.g., after acute lung injury) and possibly with cell morphology (e.g., alveolar type I vs. alveolar type II cells).

The Na+/H+ antiporter in rat alveolar type II cells and its role in stimulated surfactant secretion.

We used the pH-sensitive fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to identify Na+/H+ exchange in freshly isolated rat alveolar type II cells and alveolar type II cells in primary culture. The intracellular pH (pHi) of freshly isolated alveolar type II cells was 7.36 +/- 0.05 (n = 3). When freshly isolated alveolar type II cells were acid loaded with nigericin in sodium-free buffer, the pHi dropped to 6.59 +/- 0.04 and remained low in sodium-free buffer. When acid-loaded cells were subsequently incubated with NaCl, pHi increased in a dose-dependent manner. Amiloride (0.1 mM) inhibited the sodium-induced increase in pHi. When the acid-loaded cells were resuspended in an unbuffered choline chloride solution, the cells secreted H+ in a sodium-dependent and amiloride-inhibitable manner. Alveolar type II cell monolayers, which were cultured for 22 h on glass coverslips and then loaded with BCECF, had a resting pHi of 7.48 +/- 0.05 (n = 4). Nigericin acidified these cultured cells in the absence of sodium and NaCl increased the pHi of these acid loaded cells as observed in freshly isolated cells. Secretagogues of pulmonary surfactant, 12-O-tetradecanoylphorbol 13-acetate (TPA) and terbutaline, did not change pHi. Inhibition of the Na+/H+ antiporter by the addition of amiloride to a Na+ containing medium or the substitution of choline for Na+ did not inhibit stimulated phosphatidylcholine secretion. We conclude that pHi regulation in rat alveolar type II cells is in part mediated by an amiloride-sensitive Na+/H+ antiporter, but this system appears not to be involved in TPA- or terbutaline-induced pulmonary surfactant secretion in primary culture.

The effect of neutrophil migration and prolonged neutrophil contact on epithelial permeability.

The effect of neutrophil migration and prolonged neutrophil contact on epithelial permeability was examined. Although neutrophil migration was not associated with a change in epithelial permeability, prolonged neutrophil-epithelial contact following migration resulted in an increase in epithelial permeability. These results were not altered by catalase, a specific neutrophil elastase inhibitor, methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone or cyclohexamide. This suggests that neutrophil migration does not occur via an H2O2-induced reversible mechanism of junctional opening, which we describe herein.

The effect of substratum and serum on the lipid synthesis and morphology of alveolar type II cells in vitro.

To determine the effect of various culture conditions on the maintenance of lipid synthesis and morphology in alveolar type II cells, we cultured isolated adult rat alveolar type II cells on either plastic or denuded human amnionic basement membrane (ABM) in medium supplemented with either fetal bovine, porcine, horse, rat, or human serum. Lipid synthesis was assessed by incubation with [1-14C]acetate and determination of the distribution of radiolabel into individual lipid classes. Cells cultured on ABM incorporated significantly higher percentages of acetate into either phosphatidylcholine (PC) or phosphatidylglycerol (PG), and retained lamellar inclusions and a more characteristic cuboidal shape for longer periods than did cells cultured on plastic. Compared to other sera, cells cultured in the presence of rat serum incorporated the highest percentages of acetate into PC and saturated PC, had the best preservation of lamellar-body ultrastructure, and also appeared to contain more multivesicular bodies. The percent composition of linoleic acid, an essential fatty acid, was found to vary widely among the different sera. Supplementing media with linoleic acid resulted in a marked increase in acetate incorporation into saturated PC and a decreased incorporation into PG. We conclude that for maintenance of differentiated function of adult rat alveolar type II cells in primary culture (1) ABM is preferable to plastic as a culture substratum, (2) rat serum is preferable to fetal bovine serum as a serum supplement, and (3) the regulation of lipid synthesis by linoleic acid causes disparate effects on PG and saturated PC synthesis.

Epithelial permeability produced by phagocytosing neutrophils in vitro.

Neutrophils are thought to increase alveolar permeability in many types of lung injury. To investigate the contribution of neutrophils to the development of permeability pulmonary edema, we have developed an in vitro cell culture system for studying alveolar epithelial permeability. Rat alveolar type II cells, cultured for 6 to 12 days on collagen-coated Millipore filters, form a morphologically and pharmacologically polarized epithelium. The filters are mounted between 2 lucite chambers, and electrical resistance (permeability to ions) and spontaneous potential difference across the monolayer are measured continually or at frequent intervals. When neutrophils and the phagocytosable particle, opsonized zymosan (but not neutrophils or opsonized zymosan alone), were added to the apical side, the potential difference and transepithelial resistance fell dramatically after 20 min, which indicates an increase in epithelial permeability. The increase in epithelial permeability was inhibited by serum alpha-1-protease inhibitor (250 micrograms/ml), methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone (0.02 mM) (an elastase inhibitor), catalase (2,500 units/ml), and superoxide dismutase (330 units/ml). In experiments with a lower concentration of phagocytosing neutrophils, a slower rate of decrease in resistance occurred, and in 3 of 13 studies, there was a definite recovery of the resistance to initial values. This study demonstrated that phagocytosing but not resting neutrophils increase the permeability of the epithelial monolayers to ions and suggests that the increased permeability in this system is mediated in part by both neutral protease(s) and oxygen radicals.

Stimulation of net active ion transport across alveolar type II cell monolayers.

The active transcellular transport of electrolytes across the alveolar epithelium probably plays an important role in alveolar fluid homeostasis by helping to maintain the alveolus relatively free of fluid. To better understand the factors regulating active ion transport across alveolar epithelial cells, we examined the effect of a number of pharmacologically active agents on the bioelectric properties of alveolar type II cells in primary culture. Alveolar type II cells were isolated from adult male rats and cultured on collagen-coated Millipore filters for 6-14 days. The bioelectric properties of these monolayers were determined in Ussing-type chambers. The addition of 10(-3) M 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) increased the short-circuit current (Isc) from 2.9 +/- 0.75 to 6.9 +/- 0.73 microA/cm2 (means +/- SE; n = 8) and decreased the transepithelial resistance. Cholera toxin, 3-isobutyl-1-methylxanthine, and terbutaline sulfate produced similar increases in Isc and decreases in resistance. The Isc stimulated by 8-BrcAMP was Na but not Cl dependent and could be blocked by amiloride but not by furosemide. Thus 8-BrcAMP and agents that increase intracellular cAMP can stimulate a Na-dependent net active ion transport across alveolar type II cell monolayers. Similar regulatory mechanisms may be involved in controlling solute and fluid movement across the alveolar epithelium in vivo.