Production of tumour necrosis factor alpha by primary cultured rat alveolar epithelial cells.

Tumour necrosis factor alpha(TNF-alpha) is one of the most important pro-inflammatory cytokines, which plays an important role in host defense and acute inflammation related to tissue injury. The major source of TNF-alpha has been shown to be immune cells such as macrophages and neutrophils. In the present study, we demonstrated that LPS-treatment on alveolar epithelial cells isolated from adult rat lungs also induced a dose- and time-dependent release of TNF-alpha. The purity and identity of these cells were examined by immunofluorescent staining and confocal microscopy with antibodies for cytokeratin and pro-surfactant protein C, markers for epithelial cells and type II pneumocytes respectively. Positive staining of TNF-alpha was observed throughout the cell layer and localized intracellularly. LPS-induced TNF-alpha production from alveolar epithelial cells was blocked not only by cycloheximide, an inhibitor of protein translation, but also by actinomycin D, an inhibitor of gene transcription. The mRNA of TNF-alpha rapidly increased within 1 h of LPS stimulation. These data suggest that LPS-induced TNF-alpha production from alveolar epithelial cells is primarily regulated at the transcriptional level, which is different from that of macrophages and neutrophils. TNF-alpha produced by alveolar epithelial cells may function as an alert signal in host defense to induce production of other inflammatory mediators.

Effects of the stress response in septic rats and LPS-stimulated alveolar macrophages: evidence for TNF-alpha posttranslational regulation.

We have previously demonstrated that induction of the stress response, by heat stress or sodium arsenite, administered 18 h before initiation of sepsis in rats, significantly decreased mortality and lung injury. As a possible mechanism underlying this effect, we hypothesized that the induction of the stress response, prior to bacterial endotoxin (lipopolysaccharide, LPS) stimulation, would cause a decrease in synthesis and/or release of tumor necrosis factor-alpha (TNF-alpha), making the animals more resistant to sepsis. Rats exposed to Salmonella typhosa LPS demonstrated a rise in plasma TNF-alpha. In contrast, rats exposed to heat stress or to sodium arsenite 18 h prior to LPS had significantly lower levels of plasma TNF-alpha. To examine the mechanisms by which the stress response mediates this decrease, we studied cultured alveolar macrophages. Similar to in vivo studies, TNF released into supernatants of alveolar macrophages treated with LPS was significantly higher than from cells exposed to the stress response prior to LPS. The decrease in TNF-alpha protein release was not accompanied by a similar decrease in TNF-alpha mRNA levels or by a decrease in cell-associated TNF-alpha, suggesting possible posttranslational regulation of TNF-alpha. To determine whether the decrease in TNF-alpha release was due to binding and sequestration by heat shock proteins (HSP), TNF-alpha was purified by immunoprecipitation. Under these conditions, TNF-alpha and HSP72kDa coprecipitated from cells that had received stress treatment prior to LPS. These data implicate HSP in posttranslational control of TNF-alpha release in LPS-stimulated alveolar macrophages exposed to the stress response.

Epithelial dysfunction in acute lung injury.

More than a quarter century has passed since Ashbaugh and colleagues postulated that abnormalities of surfactant are causally related to the abrupt and severe organ dysfunction that occurs in individuals with acute lung injury (ALI). In this time, much progress has been made in expanding our understanding of the normal functions of the alveolar epithelium and how these functions may be disrupted in the context of ALI. Alveolar epithelial cells are a key structural component of the spatial separation of gas and plasma, essential for normal gas exchange in the lung. In addition, alveolar epithelial cells synthesize, secrete, and take up surfactant, which, by reducing surface tension, is a key determinant of intra-alveolar pressure. Surfactant is qualitatively and quantitatively abnormal in lung injury due to changes in synthesis, secretion, intra-alveolar metabolism, and biophysical inhibition by protein and lipid inhibitors. Alveolar epithelial cells also subserve additional host defense functions, such as modulation of lymphocyte, macrophage, and neutrophil function; production of prostanoids, complement proteins, and nitric oxide; and display of major histocompatibility complex II molecules and intracellular adhesion molecule-1. Although the physiologic and pathogenic significance of some these functions is not absolutely clear, they are of potential relevance in the context of lung injury, wherein they may participate in the process of alveolar injury and repair.

Basic fibroblast growth factor and growth factor receptor gene expression in 85% O2-exposed rat lung.

Lungs exposed to elevated O2 concentrations suffer an initial loss of type I pneumocytes, followed by a reparative type II pneumocyte hyperplasia. We hypothesized that type II pneumocyte hyperplasia after exposure of young adult rats to 85% O2 in vivo would be temporally related to 1) an increased concentration of intrapulmonary basic fibroblast growth factor (bFGF), a potent stimulator of type II pneumocyte DNA synthesis in vitro, and 2) an upregulation of pneumocyte receptors for bFGF (FGF-R). Increased rat lung bFGF mRNA, relative to air-exposed control animals, was observed at 4 days of exposure, with no increase at days 6 and 14 of exposure. Parallel changes were observed with bFGF receptor (flg) mRNA. Nuclear runoff assays confirmed increased transcription of both bFGF and flg genes in response to 85% O2, whereas increased translation at 6 days of exposure was confirmed by protein immunoanalysis. Immunohistochemistry demonstrated a broad distribution of bFGF throughout the lung, including the alveolar epithelium, which increased after 6 and 14 days of exposure to 85% O2. Our findings are compatible with a role for bFGF in O2-mediated pneumocyte hyperplasia.

Bronchogenic carcinoma in patients seropositive for human immunodeficiency virus.

The purpose of this report is to describe an association between bronchogenic carcinoma and HIV. Three HIV-seropositive patients are described who developed bronchogenic cancer (two large cell, one adenocarcinoma) before developing an AIDS-defining illness. A critical review of the literature revealed 22 other patients in which the association of HIV infection and lung cancer is reported. These patients are characterized by a relatively young age at diagnosis (median, 43 years) and prevalence of the adenocarcinoma subtype (13 of 25 patients). Twenty of 21 patients had a history of smoking. Among 21 patients for whom data were available, 6 patients (28 percent) had AIDS at time of diagnosis of lung cancer while 11 patients (55 percent) did not have AIDS or AIDS-related complex at diagnosis.

Sodium arsenite induces heat shock protein-72 kilodalton expression in the lungs and protects rats against sepsis.

OBJECTIVE: To examine the hypothesis that induction of heat shock proteins by a nonthermal mechanism would confer protection against experimental sepsis. DESIGN: Prospective, blind, randomized, laboratory study. SETTING: University research laboratory. SUBJECTS: Sixty-two adult male Sprague-Dawley rats (weight range 250 to 350 g). INTERVENTIONS: Administration of sodium arsenite or saline in an animal model of sepsis by cecal ligation and perforation. MEASUREMENTS AND MAIN RESULTS: Sixty-two rats were randomly divided into two groups: group 1 received sodium arsenite (6 mg/kg iv) and group 2 received saline injection, in a blinded fashion. Eighteen hours after receiving sodium arsenite or saline, cecal ligation and perforation were performed and the animals were monitored for mortality for 96 hrs. Sodium arsenite injection, in the absence of an increase in body temperature, induced heat shock protein of 72-kilodalton molecular weight expression in the lung, which was detected 2 hrs after injection, peaked between 9 and 24 hrs, and returned to baseline by 48 hrs. Prior administration of sodium arsenite conferred significant protection against cecal ligation and perforation-induced mortality at 18 hrs (p = .002) and 24 hrs (p v .026) after cecal ligation and perforation, and correlated with expression of heat shock proteins in the lungs. However, at 48 and 96 hrs, when heat shock protein expression returned to basal values, the mortality rates of both groups were indistinguishable. CONCLUSIONS: We conclude that in vivo injection of sodium arsenite induces expression of HSP-72 in the lungs, and confers transient protection against experimental sepsis during the period that heat shock proteins are also expressed.

Vitamin D stimulates DNA synthesis in alveolar type-II cells.

Alveolar type-II cells are responsible for alveolar epithelial cell proliferation during growth and development and in response to lung injury. Based on the observation of abnormal lung development in rachitic rat pups and the expression of receptors for vitamin D by fetal alveolar epithelial cells, the present study examined the influence of 1,25-dihydroxy vitamin D (DHD) on the proliferation of primary cultures of fetal, neonatal and adult alveolar epithelial cells. The ontogony of vitamin D responsiveness was examined, using fetal (days 18, 19 and 22 = term), neonatal (days 7 and 18) alveolar epithelial cells as well as adult alveolar type-II cells. Maximal stimulation of [3H]thymidine incorporation occurred in neonatal d18 cells: (250 +/- 4.8%, n = 4, P < 0.05). Incubation of adult type-II cells, in the presence of 10(-9) M DHD increased thymidine incorporation into DNA (149.1 +/- 33.2%, mean +/- S.E., n = 3, P < 0.001) compared to control cells maintained in basal medium. Exposure to DHD also increased thymidine incorporation after stimulation with a mixture of conventional progression factors (insulin (10 micrograms/ml) (I), cholera toxin (10 micrograms/ml) (C) and EGF (20 ng/ml) (E)) (349.4 +/- 42.9% vs. 213.5 +/- 23.6%, n = 6, P < 0.005). Autoradiographic labeling indices of adult type-II cells increased from 3.1 +/- 0.6% for cells cultured in basal medium to 7.2 +/- 1.7% in cells exposed to DHD from the time of plating and I, C, E from 20-68 h in culture (n = 4, P < 0.05). Although no increase in the number of adult type-II cells was observed in these experiments, flow cytometric analysis of nuclear DNA content revealed an increased proportion of cells in the S and G2 phases of the cell cycle (basal: S = 2.6%, G2/M = 3.0%, DHD+GF: S = 4.7%, G2/M = 5.6%, P < 0.05 for each comparison). These data demonstrate that vitamin D3 is a growth factor for alveolar type-II cells and suggest the possibility that local elaboration of vitamin D may provide a novel mechanism of modulation of epithelial proliferation in the context of lung development and repair.

Induction of heat stress proteins is associated with decreased mortality in an animal model of acute lung injury.

This study examined the hypothesis that transient, whole-body hyperthermia would reduce lung damage and/or mortality in a previously described animal model of acute lung injury. Normal, adult Sprague-Dawley rats were randomly assigned either to a heated (n = 40) or to a sham-heated (n = 49) group. Heated animals were warmed to 41 to 42 degrees C 18 h before intratracheal instillation of phospholipase A2. Forty-eight hours after phospholipase A2 exposure, the two groups were compared in a blinded fashion for mortality rate, PaO2, AaPO2, lung wet/dry weight ratio, alveolar inflammatory cell number, and lung histopathology. Heated, injured animals exhibited a reduced mortality rate and less lung damage than did unheated animals: mortality (zero versus 27%, p < 0.001); AaPO2 (22 +/- 3 versus 36 +/- 15 mm Hg, p < 0.002); lung lavage cell counts (5.3 +/- 3 versus 16.9 +/- 7 x 10(6)/ml, p < 0.05); lung wet/dry weight ratio (4.1 +/- 0.6 versus 5.1 +/- 0.7, p < 0.025); parenchymal lung injury fraction (0.10 versus 0.51, p < 0.001). Transcription and translation of heat shock proteins (HSP70) were examined by Northern and Western analysis. Pulmonary tissue HSP70 mRNA was elevated 1 h after heating. HSP72 protein levels were increased over baseline levels between 12 and 72 h after whole-body hyperthermia, but they were unchanged in sham-heated animals. These data indicate that thermal pretreatment associated with the induction of HSP72 protein synthesis, attenuates tissue damage and mortality in experimental lung injury.

The effect of low-potassium-dextran versus Euro-Collins solution for preservation of isolated type II pneumocytes.

Limited availability of donor organs is a major factor restricting the clinical application of lung transplantation. Improvements in preservation techniques are essential for prolonging storage time and improving lung function following transplantation. The present investigation used primary cultures of adult rat alveolar type II cells as a model for evaluating lung-preservation solutions. Type II cells were plated onto tissue-culture plastic at a density 5 x 10(5) cells/cm2 and maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (D10) for 40 hr. Cells were then exposed to Euro-Collins solution or a low-potassium-dextran solution (LPD). At designated time points, measurements of lactate-dehydrogenase (LDH) release, protein content, and incorporation of 3H-thymidine into cellular DNA were made. During 12 hr of "storage" at 37 degrees C, cells maintained in LPD released less LDH (14.3 +/- 1.2% of cellular total, mean +/- SEM, n = 5) than their counterparts stored in EC (20.6 +/- 1.6%, P less than 0.05). During the 36 hr following a 6-hr exposure to preservative solutions, LPD-treated cells incorporated more thymidine per mg of protein (2566 +/- 419.8 cpm/micrograms protein, mean +/- SEM, n = 6) compared with cells maintained continuously in D10 (1431 +/- 351, P less than 0.05). By contrast, cells exposed to EC incorporated less thymidine (82.2 +/- 62.8 cpm/micrograms protein) than either cells maintained in LPD or D10 (P less than 0.01 for each comparison). These results suggest that LPD solution is less cytotoxic than EC and that LPD enables higher levels of metabolic activity in recovering epithelial cells. In vitro cultures of type II epithelial cells are a useful model system for the study of lung preservation and posttransplantation lung injury.

Limited division of low-density adult rat type II pneumocytes in serum-free culture.

A previously reported serum-free medium for the culture of adult rat type II pneumocytes has been further defined by the addition of 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer and 700 U/ml catalase. Both additions significantly enhanced [3H]thymidine incorporation into pneumocyte DNA between days 5 and 6 of culture. In the presence of 100 micrograms/ml bovine pituitary extract, both insulin-like growth factor (IGF) I (20 ng/ml) and II (25 ng/ml) further increased [3H]thymidine incorporation into pneumocyte DNA over that observed with the basal medium. When type II pneumocytes were plated at 2.75 X 10(5) cells/cm2 there was a plating efficiency of 50 +/- 5%, and the increased DNA synthesis in response to IGFs was not associated with an increase in cell number. When the plating number was reduced to 1.6 X 10(5) cells/cm2 the plating efficiency was only 23 +/- 2%. At this low cell density a doubling of cell number was observed in response to IGF-I between days 4 and 9 of culture.

Acute lung injury induced by phospholipase A2. Structural and functional changes.

On the basis of the observation that serum levels of phospholipase A2 (PLA2) are elevated in pancreatitis and systemic sepsis, and the association of these conditions with the subsequent development of acute lung injury, the present investigation examined the structural and physiologic consequences of intratracheal administration of PLA2 to adult male rats. Rats received direct intratracheal instillation of either control vehicle or 40,000 units/kg of PLA2 repurified from Naja naja venom. Animals treated with PLA2 showed higher cumulative mortality (33% versus 0%, n = 79; p less than 0.01) than did their control littermates. The PLA2-treated animals showed histologic evidence of acute lung injury characterized by interstitial and alveolar edema, accumulation of inflammatory cells, and alveolar wall thickening, which reached maximal severity 48 h after enzyme instillation. Forty-eight hours after PLA2 administration experimental animals had lower arterial oxygen tensions (73.9 +/- 7.66 mm Hg versus 96.7 +/- 2.52 mm Hg, mean +/- SEM; p less than 0.01), higher alveolar-arterial oxygen gradients (35.3 +/- 6.3 mm Hg versus 18.8 +/- 1.42 mm Hg, p less than 0.01), and higher wet-dry lung weight ratios (5.08 +/- 0.26, mean +/- SEM, n = 7 versus 3.29 +/- 0.08, n = 3; p less than 0.002) than did control animals. Lung lavage from experimental animals 48 h after PLA2 instillation showed increased total cell counts [(26.6 +/- 5.04) x 10(6) cells versus (4.69 +/- 1.48) x 10(6) cells; p less than 0.01], an increased percentage of neutrophils (34.2 +/- 4.6% versus 1.25 +/- 0.25%, mean +/- SEM; p less than 0.01), and increased protein concentrations in lavage fluid (0.38 +/- 0.06 mg/ml, mean +/- SEM, n = 4 versus 0.27 +/- 0.02 mg/ml, n = 5; p less than 0.05). The histologic and physiologic abnormalities had largely resolved by 240 h. These results suggest that PLA2 may be a potent mediator of lung inflammation and that intratracheal administration of PLA2 to adult rats may provide a useful experimental model of acute lung injury.

Effects of two extracellular matrices on morphologic and biochemical properties of human type II cells in vitro.

Previous observations have suggested that differentiated functions of adult rat type II alveolar cells are affected in part by cell-matrix interactions. We examined several aspects of differentiated adult human type II cells cultured on either bovine corneal endothelial cell extracellular matrix (BCECM) or matrix derived from the Englebreth-Holm-Swarm tumor (EHS). Compared to cells cultured on BCECM, adult human type II cells grown on EHS assumed a more cuboidal shape, had a more defined apical-basal polarity, and appeared to contain a greater number of lamellar bodies and neutral lipid inclusions. These cells also incorporated a greater percentage of [14C]acetate into saturated phosphatidylcholine (SPC) than did their counterparts grown on BCECM. In contrast, the relative incorporation of [14C]acetate into phosphatidylglycerol (PG) was lower in cells grown on EHS than cells cultured on BCECM. The histochemical stain for alkaline phosphatase was useful in identification of human type II cells. Alkaline phosphatase expression was elevated in cells cultured on EHS compared to those cultured on BCECM. These results suggest that maintenance of a differentiated morphology, lipid synthesis, and expression of alkaline phosphatase activity by primary cultures of adult human type II cells are also influenced by cell-matrix interactions. All markers of differentiated function of type II cells except synthesis of PG are better maintained on EHS than on BCECM. Under the conditions of these experiments, synthesis of SPC and PG appears to be independently regulated.

Pulmonary complications of AIDS: a clinical strategy.

Infectious and noninfectious forms of pulmonary disease are the most common complications of acquired immune deficiency syndrome (AIDS), and many are amenable to treatment. We describe the clinical and radiologic features of the most common causes of lung disease in AIDS patients and review the drugs available for treatment. In addition, we provide a strategy for the clinical assessment and management of patients with human immunodeficiency virus infection who have lung infiltrates.

Alkaline phosphatase: a marker of alveolar type II cell differentiation.

In an effort to identify type II cells by a method independent of staining phospholipid inclusions, we evaluated a histochemical technique for alkaline phosphatase activity in normal rat lung, in freshly isolated type II cells, and in primary culture of type II cells. In the adult rat alveolus, alkaline phosphatase staining selectively identified type II cells, although nonciliated bronchiolar (Clara) cells and loose perivascular connective tissue also stained for alkaline phosphatase activity. In cell suspensions of type II cells and other dissociated lung cells, alkaline phosphatase staining correlated closely with the modified Papanicolaou technique and was particularly useful in distinguishing type II cells from alveolar macrophages. To determine if alkaline phosphatase was related to the differentiated phenotype of type II cells, we studied conditions known to affect other type II cell functions. When type II cells were cultured on plastic substrata, the intensity of alkaline phosphatase staining decreased with increasing time in culture. To quantitate the apparent decrease in alkaline phosphatase activity, we used a biochemical assay to study the expression of alkaline phosphatase by type II cells. The specific activity of alkaline phosphatase in type II cells declined with increasing time in tissue culture on plastic substrata. Alkaline phosphatase activity was maintained, however, by culturing cells on Englebreth-Holm-Swarm (EHS) tumor matrix. Cells that had reduced levels of alkaline phosphatase activity following 48 h of culture on plastic substrata could be "rescued" by removing them from the plastic substratum and reculturing them for 48 h on EHS matrix. Alkaline phosphatase activity was also increased by culturing type II cells in the presence of cAMP or sodium butyrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Corticosteroids as adjunctive therapy in treatment of Pneumocystis carinii pneumonia in patients with acquired immunodeficiency syndrome.

10 patients with the acquired immunodeficiency syndrome whose respiratory failure due to Pneumocystis carinii pneumonia (PCP) was deteriorating rapidly had 7 days of intravenous methylprednisolone added to their antibiotic regimen. 8 similar patients were treated with antibiotic therapy alone. 9 of the 10 methylprednisolone-treated patients survived their episode of PCP, compared with 2 of the 8 conventionally treated patients. Clinical improvement was evident within 2 days of the start of steroid therapy, and in none of the 10 patients did clinical deterioration or recurrence of PCP occur on cessation of steroid therapy. In 1 steroid-treated patient disseminated herpes zoster developed 2 days after discontinuation of methylprednisolone. Methylprednisolone seems to be a useful adjunctive therapeutic agent for patients with AIDS in whom Pneumocystis carinii is the sole respiratory pathogen.

Amiodarone pulmonary toxicity: functional and ultrastructural evaluation.

Pulmonary function, chest radiographic appearances, and the cellular composition of bronchoalveolar lavage fluid were assessed in 13 patients who were receiving amiodarone treatment. Eight of the patients had developed clinical and radiological evidence of lung disease and five were symptom free. The proportions of lymphocytes (mean 8.6 (SD 6.9)) and neutrophils (mean 3.4 (3.3)) obtained by bronchoalveolar lavage were similar in patients with and without lung complications. Electron microscopic examination of alveolar macrophages showed intralysosomal inclusion bodies in all subjects, regardless of clinical state. There was no significant difference in the mean number of inclusion bodies per macrophage transection between those with and those without lung disease. The differential cell count in bronchoalveolar lavage fluid and the presence of macrophage inclusion bodies were therefore not useful as markers of disease activity. Among those who developed clinical and radiological evidence of lung disease, the cumulative drug dose per kilogram of body weight and the duration of treatment (mean 16.5 (SD 9.0) months) were significantly correlated with the degree of lung restriction as measured by total lung capacity and forced vital capacity. It is concluded that, while the severity of the restrictive pulmonary defect that is induced by amiodarone is largely dose related, the development of lung toxicity is to some extent idiosyncratic.

Lung inflammation in scleroderma: clinical, radiographic, physiologic and cytopathological features.

Twenty-five patients with systemic sclerosis were studied by chest radiography, lung function, esophageal motility, gallium-67 (67Ga) lung scanning and bronchoalveolar lavage (BAL). Alveolar inflammation, as defined by an elevation of proportional BAL lymphocyte or neutrophil counts, or increased thoracic uptake of 67Ga was found in 16 patients. An NIH gallium index greater than 65 index units identified a subgroup of patients with a significantly higher proportional BAL lymphocyte count (13.7 +/- 8.5 vs 5.6 +/- 3.1, p less than 0.0005). The presence of an abnormal chest radiograph correlated with physiologic evidence of lung restriction (p less than 0.01), and an elevation of proportional BAL lymphocyte count (15.5 +/- 8.2 vs 6.6 +/- 5.1, p less than 0.01). Eight patients receiving oral penicillamine therapy had significantly lower BAL lymphocyte counts compared to untreated patients (4.7 +/- 3.6 vs 11.3 +/- 7.7, p less than 0.05). We suggest that alveolar inflammation in scleroderma is characterized by lymphocyte accumulation and increased thoracic uptake of gallium.

Autofluorescence of alveolar macrophages: problems and potential solutions.

Despite recent advances in immunofluorescence technology, the study of alveolar macrophage cell surface proteins is hampered by the presence of autofluorescence. We suggest several approaches that might overcome this problem and enable specific immunofluorescent detection of cellular proteins and automated analysis of alveolar macrophages.