Guinea pig lung eosinophil: purification and prostaglandin production.

Guinea pig lung eosinophils have been purified from bronchoalveolar lavage (BAL) fluid, and the production of prostaglandin E2 (PGE2), thromboxane B2 (TXB2), and 6-keto-PGF1 alpha was measured after stimulation with phorbol myristate acetate (PMA), platelet-activating factor (PAF), and formyl-methionyl-leucyl-phenylalanine (fMLP). A combination of plating and discontinuous Percoll gradient centrifugation was used to purify eosinophils. The purity of eosinophils in fraction E (interface between Percoll density 1.057-1.068) was around 96%, and the viability was 99%, with a mean yield of 2.7 x 10(6) cells per guinea pig. Similarly, in fraction D (interface between Percoll density 1.047-1.057), the mean purity of eosinophils was 76% and the viability of cells was 99%, with a mean yield of 1.1 x 10(6) cells per guinea pig. Purified eosinophils produced TXB2 predominantly after stimulation with PMA, fMLP, and PAF. The 6-keto-PGF1 alpha was slightly increased in cell supernatants after stimulation with PMA and fMLP, but PGE2 was not elevated with any stimulus. The highly purified eosinophils of fraction E generated amounts of TXB2 and 6-keto-PGF1 alpha similar to the amount generated by eosinophils contaminated with some macrophages (fraction D). These results suggest a role for eosinophils in the production of TXA2 by guinea pig lung.

Guinea pig lung cells. Method of isolation and partial purification, identification, ultrastructure, and cell count.

Guinea pig lung cells (over 700 x 10(6) cells/lung) were obtained following gentle digestion of lung tissues with a solution of protease type VII (50 micrograms/ml). The viability of these cells was over 86% as estimated by the trypan blue exclusion technique. The cell suspensions were elutriated into eight fractions, which were characterized by selected staining techniques (Alcian blue, esterase, and Papanicolaou) and by electron microscopy. Differential cell counts were done to establish the percentages of each type of cells in the overall population. Electron microscopy analyses of the cell populations have allowed the identification of most of the various isolated cell types and showed that the cellular organelles and ultrastructures were well preserved. These cell populations will be used for characterizing lung immunologic, metabolic, and endocrine functions, as well as for studying cell interactions.

Stimulation of release of prostaglandins and thromboxanes from isolated guinea pig lung cells by bradykinin, f-Met-Leu-Phe, phorbol myristate, ionophore A23187, and leukotrienes.

Guinea pig lungs were perfused for 15 min with a solution of protease type VII (0.05 mg/ml) and dispersed to yield a suspension of morphologically intact and metabolically active lung cells (over 250 X 10(6) cells per animal). The viability of these cells assessed with the Trypan blue exclusion technique was more than 80%. Treatment of these mixed cells (1 X 10(6) cells/ml) with chemicals such as phorbol myristate acetate (1 X 10(-9) to 1 X 10(-7) M), f-Met-Leu-Phe (5 X 10(-6) to 1 X 10(-4) M), and the calcium ionophore A23187 (3.1 X 10(-7) to 2.5 X 10(-6) M), and with autacoids such as bradykinin (1 X 10(-5) to 1 X 10(-4) M), leukotriene B4 (1 X 10(-13) to 1 X 10(-7) M), and leukotriene D4 (1 X 10(-10) to 1 X 10(-7) M) stimulated to a variable degree the release of prostaglandin E2 and thromboxane B2 (measured with a novel enzyme immunoassay). It is suggested that icosanoid release from the lungs is the result of direct chemical or hormonal stimulation of the cells and not a consequence of vascular changes. Studies are in progress to purify lung cell populations and characterize the cells responsible for the release of these icosanoids.

A comparative study on the hemolytic action of short asbestos fibers on human, rat, and sheep erythrocytes.

The hemolytic activity of short asbestos fibers isolated by a sedimentation procedure was studied using human (HRBC), rat (RRBC), and sheep (SRBC) red blood cells. The initial velocity (Vi) of hemolysis is proportional to the concentration of fibers with HRBC and RRBC, but not with SRBC. Initial velocity (Vi) is 14.30 for HRBC, 5.75 for RRBC, and 3.60 for SRBC at a concentration of 1,000 micrograms of fibers/ml. Maximum velocity (Vm) is reached at 400 micrograms/ml with HRBC and its value is 16.7. With RRBC and SRBC, Vm is reached at 600 micrograms/ml and its value is 10 and 3.6, respectively. The 50% hemolytic concentration (HC50) at 60 min of incubation is 75 micrograms/ml for HRBC, 240 micrograms/ml for RRBC, and 260 micrograms/ml for SRBC. It appears that the sensitivity against the short asbestos fibers of the three types of RBC used is in the following order: HRBC greater than RRBC greater than SRBC.

Hemolysis by chrysotile asbestos fibers. I. Influence of the sialic acid content in human, rat, and sheep red blood cell membranes.

Complete removal of accessible sialic acids from human, rat, and sheep red cell membranes does not inhibit the hemolytic properties of chrysotile asbestos fibers. After 60 min of contact, the percentages of hemolysis are comparable between the treated and the nontreated groups. Nevertheless, with low concentrations of fibers the initial velocity of hemolysis of rat and sheep red blood cells is lower in the neuraminidase-treated group. Although sialic acids are not essential for the expression of the hemolytic activity of asbestos, we suggest that these acids could play a role during the first seconds or minutes of the hemolytic process by increasing the electrostatic attraction between the red blood cell membranes and the fibers. However, it is clear that asbestos fibers provoke hemolysis by acting on some other cell membrane component(s).

Secretion of thromboxane B2 and prostaglandin E2 by guinea-pig lung cells isolated by centrifugal elutriation and by macrophages obtained by bronchoalveolar lavage.

Guinea-pig lung cells were partially purified by an elutriation technique after enzymatic digestion of the lung. Eight fractions were obtained and the cell content of each fraction was analysed by staining and by electron microscopy. After stimulation with selected agonists, the secretion of eicosanoids has been measured in the supernatants of each elutriation fraction and in the supernatants of alveolar macrophages obtained by bronchoalveolar lavage. The cell response was characterised by a marked secretion of thromboxane B2 (TXB2) and prostaglandin E2 (PGE2) from the alveolar macrophages and a more discrete secretion of TXB2 from the cells of the elutriated fractions. The secretion of TXB2 varied in the cell fractions as a function of the cellular composition and the agonist used. Platelet activating factor (PAF) and ethoxy PAF, phorbol myristate acetate (PMA) and ionophore A-23187 stimulated most cell types although PMA produced a more important stimulation of the fractions containing pneumocytes and eosinophils, and ionophore A-23187 appeared to stimulate Foa-Kurloff cells. The chemotactic peptide fMLP stimulated markedly the fractions containing the inflammatory cells (mast cells, macrophages, neutrophils) and endothelial cells. Pretreatment of the cells with cytochalasin B potentiated the release of TXB2 and PGE2 by macrophages following stimulation with PAF, leukotriene D4 (LTD4) and formyl-methionyl-leucyl-phenylalanine (fMLP) but not by mixed lung cells or by elutriation fractions. In conclusion, the recovery of a large number of partially purified lung cell types was made possible by mild protease digestion and elutriation.(ABSTRACT TRUNCATED AT 250 WORDS)

The hemolytic activity of chrysotile asbestos fibers: a freeze-fracture study.

Short asbestos fibers isolated by a sedimentation procedure have a strong hemolytic activity. In the presence of ferritin particles, hemolysis by chrysotile fibers is inhibited at least during the first 10 min. Freeze-fracture studies show that after 20 sec or 2 min of contact between the fibers and the RBC membrane, the intramembranous particles remain randomly distributed over the whole surface of the P-face. On the E-face of the asbestos-treated red blood cell membranes, the number of intramembranous particles is significantly reduced. With the transmission electron microscopy, it is not possible to resolve the trilaminar structure of the ghost membrane around the deeply buried asbestos fibers. It is postulated that the membrane defects brought about by asbestos are caused by the adsorption of one membrane constituent, possibly phospholipids, on the chrysotile fibers.