calcimycin and Pulmonary-Eosinophilia

The formation of leukotriene B4 (LTB4) by neutrophils stimulated with the ionophore A23187 or physiological stimuli in heparinized plasma was investigated. In comparison with neutrophils stimulated (A23187) in a protein-free buffered salt solution, neutrophils stimulated in plasma produced only trace amounts of LTB4. The addition of human recombinant LTA4-hydrolase or erythrocytes to plasma prior to A23187 stimulation strongly and selectively stimulated (> 4-fold) the formation of LTB4 supporting that neutrophils activated in plasma with A23187 release in the extracellular milieu most of LTA4 formed by the cells, and indicating that plasma proteins drastically slow down the further metabolism of LTA4 released by neutrophils. The formation of LTB4 was then investigated in GM-CSF-primed neutrophils stimulated with fMLP in plasma; levels of synthesis were very low and the addition of erythrocytes prior to stimulation strongly enhanced LTB4 synthesis, demonstrating that agonist-stimulated neutrophils also release most of LTA4 generated in the extracellular milieu. Investigations on the fate of LTA4 in plasma revealed that LTA4 was slowly degraded through an unknown process, i.e. not through the previously described non-enzymic hydrolysis resulting in the formation of dihydroxy derivatives of LTA4. Using neutrophils labeled with tritiated arachidonate, we also demonstrated that neutrophils stimulated in plasma with fMLP or A23187, almost exclusively use endogenous arachidonate, as opposed to plasma arachidonate, to generate 5-lipoxygenase products. Finally, experiments performed with purified eosinophils indicated that contrary to neutrophils, the eosinophils do not release LTA4, but directly release LTC4.

Topics: Asthma; Calcimycin; Cell Separation; Eosinophils; Epoxide Hydrolases; Erythrocytes; Granulocytes; Humans; In Vitro Techniques; Leukotriene B4; Neutrophils; Plasma; Pulmonary Eosinophilia; Rhinitis

A model of lung inflammation was developed in Brown Norway rats. Intense lung eosinophilia was induced by a single intravenous injection of Sephadex G-200 particles. The eosinophilia observed was preceded by an increase in cysteinyl leukotrienes found in lung lavage fluids. Theophylline and albuterol were tested in the model and found to be inactive, while dexamethasone was effective. Zileuton, a specific leukotriene inhibitor, was found to effectively inhibit leukotriene formation and the influx of eosinophils into the lungs of these Sephadex-treated animals. Studies with specific leukotriene D4 antagonists of the cysLT1 type receptor indicate that this leukotriene receptor is probably not involved directly in the eosinophilic inflammation. This model appears to be useful in characterizing potential anti-inflammatory effects of inhibitors by evaluating their ability to prevent eosinophil influx into the lung.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Bronchoalveolar Lavage Fluid; Calcimycin; Dextrans; Eosinophils; Hydroxyurea; Ionophores; Leukotriene B4; Leukotriene C4; Lipoxygenase Inhibitors; Male; Pulmonary Eosinophilia; Rats; Theophylline

We have developed a method of induction of airway eosinophilia and neutrophilia in guinea pigs by intravenous injection of various types of Sephadex beads. In the first series of experiments, we have shown that G-50 Sephadex beads (Superfine, 24 mg/kg in conscious animals) induced a large accumulation of inflammatory cells in alveolar walls. The bronchoalveolar lavage (BAL) fluid from animals treated with this dose of Sephadex beads contained about 85 x 10(6) cells as compared to 20 x 10(6) cells in control animals. The eosinophils corresponded to 41% of the BAL cell population as assessed with Wright-Giemsa staining. However, in the BAL fluid from these bead-treated animals, a significant increase of monocytes, lymphocytes, and neutrophils was also observed. We have also tested the potency of G-75, G-100, and G-200 Sephadex beads (Superfine) to induce eosinophilia in guinea pig. Nonlethal intravenous doses of G-75 (14.27 mg/kg), G-100 (8.0 mg/kg), and G-200 (10.71 mg/kg) Sephadex beads were selected and induced variable levels of airway eosinophilia and neutrophilia in conscious guinea pigs. The percentage of eosinophil recovered in the BAL fluid corresponded to 35, 61, and 44% of total cells for G-75, G-100, and G-200, respectively. The neutrophils corresponded to 24, 2, and 12% of the total BAL cells for G-75, G-100, and G-200, respectively. Since the size of the beads did not seem to correlate with the intensity of airway eosinophilia and neutrophilia, the effect of lower doses of the G-50 Sephadex beads (9.86-0.43 mg/kg) on the inflammatory cell infiltration was also tested. Results showed that there was a correlation between the neutrophil number and the number of beads (r = 0.996), whereas the number of eosinophils was less directly correlated to the bead number (r = 0.812). The alveolar eosinophils were purified from BAL fluid by centrifugation on a continuous Percoll gradient (65%) to separate eosinophils from neutrophils. Normodense eosinophils (density 1.087-1.100 g/ml) obtained from Sephadex-treated animals were found at the bottom of the continuous Percoll gradient (25 x 10(6); 98% purity). These highly purified eosinophils released thromboxane A2 (TxA2) following stimulation with 2 microM ionophore A23187. The method of accumulation and purification of guinea pig alveolar eosinophils is simple, rapid, and provides a large number of pure normodense cells for further biological studies.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Animals; Bronchoalveolar Lavage Fluid; Calcimycin; Dextrans; Embolism; Guinea Pigs; Injections, Intravenous; Leukocytosis; Microspheres; Neutrophils; Pulmonary Eosinophilia; Thromboxane A2