Artificial surfactant (Surfactant TA) modulates adherence and superoxide production of neutrophils.

Neutrophils cause lung injuries by releasing proteases and active oxygen radicals in patients with acute respiratory distress syndrome (ARDS). Artificial surfactant is used to replace native surfactant whose functions are deteriorated by serum-derived inhibitors in these patients. We investigated potential interactions between exogenous surfactant (Surfactant TA) and neutrophils in in vivo and in vitro experimental models. Neutrophil alveolitis was induced in hamster lungs by the intratracheal administration of bleomycin (5 mg/kg) on Day 0. Some of the animals were followed by replacement with Surfactant TA (5 and 10 mg/100 g body weight) on Day 1. Alveolar cells were harvested by lung lavage on Day 2. The numbers of the neutrophils obtained from the lungs treated with bleomycin and Surfactant TA were unchanged, but the superoxide production from these cells was significantly decreased when compared with control animals (no Surfactant TA). From the in vitro experiments, Surfactant TA was shown to inhibit adherence and superoxide production of human neutrophils. These effects were derived from the heat-resistant components of Surfactant TA and were mimicked by treatment with liposomes of dipalmitoyl phosphatidylcholine. Surfactant-TA-treated neutrophils were demonstrated to have picnotic nuclei and to express Fas antigens, which were characteristic of apoptotic cells. These results suggest that exogenous Surfactant TA may play an important role not only in improving surfactant functions but in preventing neutrophils from further activation, probably through enhancing apoptosis.

[Effects of surfactant TA on adherence and structure of human peripheral blood neutrophils].

Artificial surfactant is used to treat patients with the adult respiratory distress syndrome; we studied its effects on neutrophil function. Neutrophils were isolated from healthy volunteers and their adherence to plastic surfaces was used as an index of their function. Surfactant TA (S-TA, 0.16-5 mg/mL) dose-dependently inhibited neutrophil adherence stimulated by n-formyl-methionyl-leucyl-phenyl-alanine, phorbol myristate acetate, and tumor necrosis factor. This inhibition was observed whether untreated S-TA or heated S-TA was used. Electron microscopy revealed an increase in the number of apoptotic neutrophils with pyknotic nuclei and smooth cell surfaces after treatment with S-TA which suggests that neutrophils apoptosis was increased. The number of apoptotic neutrophils increased with increasing time of incubation with S-TA, and was also high in the presence of G-CSF, which inhibits neutrophil apoptosis. These results show that S-TA can inhibit neutrophil function, and they suggest that S-TA therapy for the adult respiratory distress syndrome not only corrects the surface-tension abnormality, but can also inhibit infiltration of neutrophils.

[Effects of vitamin E deficiency on bleomycin-induced pulmonary fibrosis in hamsters].

We examined the effect of free radicals on lung defense mechanism in bleomycin (BLM)-induced pulmonary fibrosis in hamsters. The concentration of vitamin E (VE) in the lung tissue increased significantly after intratracheal BLM administration. VE deficient hamsters showed increased lipid peroxide values in the lung tissue at a very early stage after BLM treatment. This was followed by decreased superoxide dismutase activities in the lung tissue. The results indicate that; 1) VE appears to be necessary for the prevention of lipid peroxidation in BLM-induced oxidant lung injury, 2) VE deficiency produces a large number of free radicals at the early stage after BLM treatment, and might induce protease-antiprotease imbalance within the lung, which probably causes an emphysematous change in the BLM-treated lung at a late stage after BLM administration.

[The study of the lung accumulation of I-123 IMP by the broncho-alveolar lavage].

We studied the accumulated portion and the movement of I-123 IMP in the lung. Ten subjects were studied. They were four patients with fibrosing lung disease, two with lung cancer, and four with other lung disease. They underwent the broncho-alveolar lavage (BAL) for the diagnosis of their disease. 1.5 mCi of I-123 IMP was injected into the ante-cubital vein. The BAL examination was carried out about 40 minutes after the injection of I-123 IMP. The subjects' blood was sampled at the same time. The total BAL liquid (BAL-T) was divided into the fluid component (BAL-F) and the cell component (BAL-C) by centrifugation. The radioactivities in BAL-T, BAL-F, BAL-C, and serum (B-S) were measured by the well-counter. The average of BAL-T/B-S, BAL-F/B-S and BAL-C/B-S were 6.86, 4.26 and 2.71 respectively. It was confirmed that I-123 IMP was transported from the pulmonary capillary to the alveolar space and was taken up by the alveolar cells. It was considered that the analysis of the I-123 IMP release from the lung showed not only the endothelial cell uptake function but also the interstitial and material cells' amine transport and uptake function.

[The effect of foamy alveolar macrophages presented in bleomycin-injured rat lungs in pulmonary fibrosis].

Foamy alveolar macrophages (FAM) are observed in lungs injured by Bleomycin (BLM), but their relation to pulmonary fibrosis is not clearly understood. We purified FAM from BLM-instilled rat lungs by density gradient centrifugation on Percoll, and studied the effect of FAM on pulmonary fibrosis. The cells lavaged from the rat lungs 14 days after the administration of BLM (B) or saline (S), were applied on Percoll. After centrifugation, the cells layered on each interface were collected and named as SI, SII, SIII, and BI, BII, BIII in order of gravity. The BI layer included 8.5% of unfractionated cells (U). These BI cells were viable (88%), significantly larger than the others, nonspecific esterase positive cells, and included much ferritin and lysozyme, and were morphologically identified as alveolar macrophages (AM). Therefore, we called the BI cells FAM. We estimated the capacity of FAM (2.5 X 10(5] to synthesize DNA (3H-thymidine uptake) and RNA (3H-uridine uptake), and the activities of silica-stimulated FAM to cause proliferation of mouse thymocytes (IL-1 activity) and rat lung fibroblasts (FP activity), and to produce PGE2. FAM has a lower mitogenic activity but did not have been protein synthetic activity as compared with the others. Silica-stimulated FAM released less IL-1 than BII or BIII, and induced less fibroblast growth than BII, but induced as much as BIII, possibly because of the increased capacity of BIII cells to produce PGE2, which is known to inhibit fibroblast growth. In this way, FAM were considered to be "already activated" rather than "highly activated" cells, but the presence of FAM suggested that smaller or denser AM might receive bleomycin stimulation and release fibrogenic mediators (IL-1 or MDGF) into the alveolar spaces during FAM formation, and that AM might participate in the fibrogenic responses.

A novel neutrophil adherence test effectively reflects the activated state of neutrophils.

We developed a novel and convenient neutrophil adherence test for ascertaining the activated state of neutrophils. Human peripheral blood neutrophils were placed in wells of a 96-well flat-bottom culture plate, and incubated in the presence or absence of neutrophil stimulants for varying periods of time at 37 C in a CO2 incubator (5% CO2, 95% air). After non-adherent cells were completely removed by vibration on a vortex mixer, residual adherent cells were fixed and stained with crystal violet containing 12% formaldehyde and 10% ethanol. After thorough washing, 1% SDS was added to the plate, and the absorbance of each well was measured at 570 nm. Our results correlate well with those of a previously reported method using 51Cr-labeled cells.

Asymmetry of chromatin subunits probed with histone H1 in an H1-DNA complex.

Treatment of nucleosomes with a low concentration of sodium dodecyl sulfate removed all proteins except histone H1 from DNA, thus confirming our previous observation on sheared chromatin. No redistribution of H1 occurred during this procedure for isolation of the H1-DNA complex. The H1-DNA complex was isolated from a nucleosome monomer, doubly labeled in its protein and DNA and fractionated according to the length of DNA, and then the distribution of H1 was analyzed quantitatively. The results indicated that the monomer consisted of two subspecies, one containing 160 base pairs of DNA and one molecule of H1, and the other containing 140 base pairs of DNA and no H1. Since no monomer with two molecules of H1 was found, it is concluded that the nucleosome core has a binding site for H1 on only one side, and thus that the nucleosome is not a dyad.