To determine whether erythromycin could affect neutrophil functions.

To determine whether erythromycin could affect neutrophil functions, we measured N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced chemotaxis and superoxide generation of neutrophils in the port of erythromycin at various concentrations. Erythromycin had no general intent on either of them. We further confirmed that intracellular clear calcium concentration ([[Ca.sup.2+]]i) was not influenced by way of FMLP stimulation in the nearness of erythromycin. Our springs indicate that erythromycin has no direct general intents on neutrophil functions in vitro, although it is reported that erythromycin inhibits the local migration of neutrophils in the small airways of bring under rules with asthma.

now passing studies emphasize that bronchial hyperresponsiveness is a characteristic feature of bronchial asthma, and airway inflammation plays an important character in bronchial hyperresponsiveness.[1-4] Increased neutrophils are set up in the airways of patients with asthma after epithelial injury forward the airway.[5]

Several investigators showed that erythromycin itself had anti-inflammatory actions, of that kind as the inhibition of chemotaxis.[6,7] and the generation of reactive oxygen species[7,8] from neutrophils. On the other hand, Anderson[9] reported that erythromycin potentiated the human neutrophil chemotaxis. It remains controversial whether erythromycin may alter neutrophil functions.

To clarify the nonantibiotic efficiencys of erythromycin, we investigated the meanings of erythromycin on neutrophil chemotaxis and superoxide ([Osub2] -) generation. To further understand its nonantibiotic properties, we measured changes in intracellular clear calcium concentration ([[Ca.sup.2+]]i), which is considered be to an essential end for cell activation.[10,11]

MATERIALS AND METHODS

Reagents

Erythromycin was provided at Shionogi Pharmaceutical Co (Osaka, Japan). The N-formyl-methionyl-leucyl-phenylalanine (FMLP) cytochrome c and superoxide dismutase were purchased from Sigma Chemical Co (St Louis, Mo) Fura-2-acetoxymethyl ester (fura-2/AM) was obtained from Dojin Laboratory (Kumamoto, Japan). Ethylene-glycol-O O'-bis(2-aminoethyl)-N,N,N',N'-tetra-acetic acid (EGTA) was from Wako clean Chemicals (Osaka, Japan). Other reagents of analytical grade were purchased from commercial sources. Erythromycin was dissolved in ethanol. The final concentration of ethanol was below 05 percent

Preparation of Neutrophils

Neutrophils were separated from the house of healthy volunteers as previously described.[12] After the dextran sedimentation of the whole line erythrocytes were removed by hypotonic lysis. Neutrophils were obtained on Ficoll-Hypaque gradient centrifugation and washed twice with phosphate-buffered saline solution ([PBS]) 137 mM NaCl, 27 mM KC1 81 mM [NaH.sub.2][PO.sub.4], and 15 mM [Ksub2][HPOsub4] pH 74)

Chemotaxis Assay

Chemotaxis assays were performed forward a 96-well microchemotaxis chamber (Neuro Probe Inc, Bethesda, Md) To the bottom wells, 25 [mu]l of the chemoattractant (FMLP) or the N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) solution (20 mM HEPES, 135 mM NaCI, 5 mM KCI and 5 mM D-glucose) containing 01 percent bovine serum albumin were added. A polycarbonate filter sheet, without polyvinylpyrrolidone, containing 3-[mu]m pores was placed onward the top of the wells in the bottom plate. A gasket and top plate were fixed in place. The neutrophils were suspended at a concentration of 1 x [10sup6] cells/ml in the HEPES solution; 50 [mmsup3] of the rule or erythromycin-treated neutrophils were added to each of the top wells. The entire assembly was incubated for 60 min at 37 [degrees] C in humidified air with 5 percent [COsub2] After incubation, the filter was fixed in methanol. The filter was air-dried and stained with Diff-Quik solutions (Kokusai Chemicals, Kobe, Japan). After nonmigrated enclosed spaces were wiped, the stained filter was analyzed by dint of a microplate reader (BIO-RAD Laboratory, Richmond, Calif) as an absorbance at 595 nm

Assay of [Osub2] -- Generation

The [Osub2] -- generation of FMLP-stimulated neutrophils was measured by dint of the determination of superoxide dismutase-inhibitable reduction of cytochrome c Reaction mixtures (1 ml) which consisted of PB (pH 74) containing [10sup6] neutrophils, 50 mM cytochrome c 05 mM [CaCl.sub.2], and 05 mM [MgSOsub4] were incubated for 10 min or 2 h at 37 [degrees] C with or without various concentrations of erythromycin. After addition of FMLP (final concentration of 1 [mu]mol) the absorbancy change (550 to 540 nm) was followed forward the dual-wavelength spectrophotometer (Shimadzu UV-160 A, Japan) and was changeed to the [O.sub.2] -- release with a molar absorption coefficient of reduc minus oxidized cytochrome c as 191 [multiplied by] [10sup3] mmol/[Lsup-1] [multiplied by] [cmsup-1]

Measurement of [[Ca.sup.2+]]i

Neutrophils (2 x [10sup7] cells/ml) were incubated for 30 min at 30 [degrees] C in the HEPES solution containing 1 [mu]mol fura-2/AM. The loaded confined apartments were washed and resuspended in the HEPES solution to a density of 2 x [10sup6] cells/ml Suspensions (25 ml) of fura-2-loaded neutrophils containing 1 mM [CaCl.sub.2] were incubated with or without erythromycin for 10 min. After stimulation on 1 [mu]mol FMLP, the fluorescence was measured with a spectrofluorophotometer (Shimadzu RF-5000 Japan) at excitation wavelengths of 340 and 380 nm and an emission wavelength of 490 nm Following the measurements, the small rooms were lysed by adding 25 [mu]l of 5 percent polyoxy-chethylene (10)p-t-octylphenol (Triton X-100) for the determination of Fmax; Fmin was then determined from adding 25 [mu]l of 1 mol EGTA to the neutrophil lysate. The [[Ca.sup.2+]]i was calculated by way of a previously published method[13] with a distribution coefficient value of 224 nmol for [Ca.sup.2+].

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