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Mechanisms of the Macrolide-Induced Inhibition of Superoxide Generation by Neutrophils

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ABSTRACT

The effect of macrolides on the superoxide (O2 −) production by neutrophils was studied. Resting neutrophils become primed by lipopolysaccharide (LPS) or N-formyl-methionyl-leucyl-phenylalanine (fMLP), and primed neutrophils generate O2 − in response to fMLP or adhesion, respectively. Both LPS-primed fMLP-stimulated O2 − generation by macrolide-treated neutrophils and adhesion-stimulated O2 − generation by macrolide-treated fMLP-primed neutrophils were inhibited. Macrolide inhibition of O2 − generation was dependent on serum or pH. Serum could be substituted by NaHCO3. The intensity of inhibition was azithromycin = roxithromycin > clarithromycin > erythromycin, in that order. Non-antimicrobial derivatives of erythromycin, that is, EM703 and EM900, inhibited O2 − generation at pH 7.4. NH4Cl abolished the activity of azithromycin (AZ) only when added to neutrophils with AZ but not after incubation with AZ, suggesting that NH4Cl prevented the influx of AZ. AZ did not affect the expression of alkaline phosphatase, CD11b, and cytochrome b558 in both resting and LPS-primed neutrophils. These results suggested that macrolides did not affect granule mobilization but inhibited O2 − generation selectively.

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Abbreviations

AZ:

Azithromycin

CL:

Clarithromycin

DMSO:

Dimethyl sulfoxide

ER:

Erythromycin

FITC:

Fluorescein isothiocyanate

fMLP:

N-formyl-methionyl-leucyl-phenylalanine

HEPES:

H-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid

LPS:

Lipopolysaccharide

IgG:

Immunoglobulin G

MFI:

Mean channel of fluorescence intensity

mAb:

Monoclonal antibody

PBS:

Phosphate-buffered saline

PMA:

Phorbol myristate acetate, O2 −, superoxide anion

RO:

Roxithromycin

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ACKNOWLEDGMENTS

We thank Pfizer Pharmaceutical Inc., Tokyo, Japan, for providing azithromycin; Toshiaki Sunazuka of The Kitasato Institute for providing the erythromycin derivatives EM703 and EM900; Michio Nakamura of Nagasaki University for providing the anti-cytochrome b558 antibody; Floyd McIntire for providing the Escherichia coli LPS; and Tomofumi Miyamoto of Kyushu University and Sadafumi Omura of Technology Licensing Organization, Kyushu, for their helpful comments and encouragement.

This work was supported by a grant-in-aid (26463137) to T.F. from the Ministry of Health of Japan.

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Authors and Affiliations

  1. Section of Periodontology, Division of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan

    Kohji Nozoe, Yoshitomi Aida, Takao Fukuda, Terukazu Sanui & Fusanori Nishimura

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  1. Kohji Nozoe
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  2. Yoshitomi Aida
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Correspondence to Yoshitomi Aida.

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Nozoe, K., Aida, Y., Fukuda, T. et al. Mechanisms of the Macrolide-Induced Inhibition of Superoxide Generation by Neutrophils. Inflammation 39, 1039–1048 (2016). https://doi.org/10.1007/s10753-016-0333-3

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