calcimycin and Rhinitis

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

Second generation antihistamines have been reported to have anti-inflammatory properties in addition to their potency as H1 antagonists. In this in-vitro study, we evaluated the effect of terfenadine on platelet activating factor-(PAF)-induced or N-formyl-methionyl-leucyl-phenylamine-(FMLP)-induced human eosinophil and neutrophil chemotactic responses; and on superoxide anion generation from human eosinophils and neutrophils activated by either PAF, calcium ionophore (A23187) or phorbol myristate acetate. Since eosinophil degranulation is also associated with tissue inflammation, we further examined the effect of terfenadine on the PAF-induced release of eosinophil cationic protein. The peak concentration of terfenadine-related materials in serum of adult individuals after 60 mg of oral administration has been reported to be 351 +/- 0.4 ng/mL. We therefore used 100 to 1000 ng/mL concentrations of terfenadine. Purified normodense-eosinophils and neutrophils were obtained by discontinuous gradient from allergic subjects. We observed that terfenadine had greater inhibitory effects on eosinophils than neutrophils in both chemotactic response and superoxide generation. Terfenadine, at concentrations of 500 and 1000 ng/mL, significantly inhibited PAF-induced and FMLP-induced eosinophil chemotaxis, whereas 1000 ng/mL of terfenadine was necessary to suppress neutrophil chemotaxis. Terfenadine, at concentrations achievable at standard dosing regimens, has anti-inflammatory properties in vitro.

Topics: Adult; Asthma; Calcimycin; Cell Degranulation; Cell Separation; Chemotaxis, Leukocyte; Dose-Response Relationship, Drug; Eosinophils; Female; Humans; Male; Middle Aged; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Platelet Activating Factor; Rhinitis; Superoxides; Terfenadine

Peripheral blood neutrophils from patients with allergic rhinitis and from normal subjects were incubated for 5 min at 37 degrees C with 0.15 microM calcium ionophore A23187 in the absence or presence of exogenous arachidonic acid (2.5 to 10 microM). In neutrophils from allergic patients, the leukotriene B4 (LTB4) level was significantly increased by exogenous arachidonic acid in a concentration-dependent manner (16.2 +/- 4.2 and 38.1 +/- 6.8 pmol/5 min per 2 X 10(6) cells in the absence and presence of 10 microM arachidonic acid, respectively; P less than 0.005; n = 8). The LTB4 level in neutrophils from healthy subjects was only 0.97 +/- 0.17 pmol/5 min per 2 x 10(6) cells (n = 5) and was not enhanced by exogenous arachidonate. When cells from allergic patients were challenged in the presence of exogenous [1-14C]arachidonic acid, released LTB4 was radiolabeled and the incorporated radioactivity increased with the labeled arachidonate concentration. Labeled LTB4 was never detectable after incubating neutrophils from normal donors with exogenous labeled arachidonate. When neutrophils were incubated with [1-14C]arachidonate for 1 h, the different lipid pools of the two cell populations were labeled but both types of neutrophils produced unlabeled LTB4 in response to ionophore stimulation. The hydrolysis of choline and ethanolamine phospholipids into diacyl-, alkenylacyl- and alkylacyl-species revealed that solely the alkylacyl-subclass of phosphatidylcholine was unlabeled. We conclude (i) that neutrophils from allergic patients stimulated by low ionophore concentration produce more LTB4 than neutrophils from healthy subjects and incorporate exogenous arachidonate, (ii) that endogenous arachidonate converted to LTB4 by the 5-lipoxygenase pathway may provide only from 1-O-alkyl-2-arachidonoyl-glycero-3-phosphocholine.

Topics: Adult; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cell Division; Chromatography, High Pressure Liquid; Female; Humans; Hypersensitivity, Immediate; Leukotriene B4; Male; Neutrophils; Phospholipids; Rhinitis

The leukotriene generating capacities of ionophore stimulated human eosinophils and neutrophils were compared using specific radioimmunoassays for LTB4 and LTC4. Mixed granulocyte preparations (neutrophils and eosinophils) produced both LTB4 and LTC4 in a time-dependent fashion which was maximal at 10 and 15 min, respectively. Following the separation of eosinophils (greater than 75%) and neutrophils (greater than 90%) by metrizamide gradients, LTC4 production was predominantly from eosinophils, whereas neutrophils were the principal source of LTB4. The concentrations of leukotrienes produced by the eosinophil and neutrophil rich cell preparations were directly proportional to the concentration of ionophore. Following purification of eosinophil derived products by RP-HPLC the LTC4 immunoreactivity corresponded to the elution profile of a synthetic LTC4 marker. Furthermore, in 32 atopic subjects (21 bronchial asthmatics and 11 non-asthmatics) the amounts of LTC4 produced by unseparated leucocytes were directly proportional to the percentage of eosinophils in the total cell suspension. Preferential generation of LTB4 by neutrophils was also demonstrated by immunoreactivity of ionophore stimulated supernatants subjected to RP-HPLC, as well as by its characteristic u.v. absorbance and GC-MS profile and the ability to promote directional neutrophil locomotion (chemotaxis). These experiments support the concept that eosinophils accumulate in tissues partly as a result of the response to neutrophil derived LTB4, and that these cells contribute to the production of sulphidopeptide leukotrienes with subsequent amplification of the acute allergic response.

Topics: Adolescent; Adult; Asthma; Calcimycin; Eosinophils; Granulocytes; Humans; Leukotriene B4; Middle Aged; Neutrophils; Radioimmunoassay; Rhinitis; SRS-A; Urticaria