Azithromycin modulates neutrophil function and circulating inflammatory mediators in healthy human subjects

Abstract

Effects on human neutrophils and circulating inflammatory mediators were studied in 12 volunteers who received azithromycin (500 mg/day, p.o.) for 3 days. Blood was taken 1 h before treatment, 2.5, 24 h and 28 days after the last dose. An initial neutrophil degranulating effect of azithromycin was reflected in rapid decreases in azurophilic granule enzyme activities in cells and corresponding increases in serum. The oxidative response to a particulate stimulus was also acutely enhanced. These actions were associated with high plasma and neutrophil drug concentrations. A continuous fall in chemokine and interleukin-6 serum concentrations, within the non-pathological range, accompanied a delayed down-regulation of the oxidative burst and an increase in apoptosis of neutrophils up to 28 days after the last azithromycin dose. Neutrophils isolated from blood at this time point still contained detectable drug concentrations. Acute neutrophil stimulation could facilitate antibacterial effects of azithromycin, while delayed, potentially anti-inflammatory activity may curtail deleterious inflammation.

Introduction

Macrolides are widely used for the therapy of bacterial infections and as immunosuppressive agents Mazzei et al., 1993, Williams and Sefton, 1993, Dumont, 2000. In recent years, a variety of reports have been published demonstrating anti-inflammatory effects of macrolide antibacterial agents. For instance, in carrageenin-induced pleurisy in the rat, roxithromycin, clarithromycin and erythromycin exerted anti-inflammatory activity which was thought to depend on their ability to prevent the production of pro-inflammatory mediators and cytokines (Ianaro et al., 2000). In zymosan-induced peritonitis in rats, roxithromycin was reported to be active through a mechanism different from that of conventional non-steroidal anti-inflammatory agents such as indomethacin (Agen et al., 1993).

Inhibition of leukocyte responses in vitro has also been reported with macrolide antibiotics. While inhibition of the production of several cytokines from mononuclear cells has been reported with some macrolides Takeshita et al., 1989, Morikawa et al., 1996, a majority of the anti-inflammatory effects of this compound class are directed towards neutrophilic granulocytes in vitro Anderson, 1989, Labro, 2000, Čulić et al., 2001. The macrolide that has been most studied in this respect is erythromycin, which has been used in Japan for over a decade in the therapy of diffuse panbronchiolitis, an endemic chronic airway disease characterised by massive infiltration and excessive activation of neutrophils in the lung (Shoji, 1998).

The effects of macrolide antibacterials on inflammatory mediator release and neutrophil functions in vitro have been reviewed recently Labro, 2000, Čulić et al., 2001. As a class, most macrolides inhibit the oxidative burst and chemotaxis of neutrophils, but also stimulate the degranulation of these cells in vitro. The actions of the macrolides on cytokine production in vitro are not so clear-cut. Clarithromycin and azithromycin have been shown to inhibit the production of interleukin-1, granulocyte/monocyte colony stimulating factor and tumour necrosis factor alpha from mononuclear and other cells, but to stimulate interleukin-6 and interleukin-10 production Morikawa et al., 1996, Khan et al., 1999. Erythromycin has also been reported to inhibit the production of the neutrophil chemotactic chemokine, interleukin-8, and of the adhesion molecule CD11b by bronchial epithelial cells and neutrophils Khair et al., 1995, Lin et al., 2000, but also to stimulate apoptosis of neutrophils in vitro (Aoshiba et al., 1995), an action recently observed with azithromycin (Koch et al., 2000).

Azithromycin differs from other macrolide antibacterials in that it exhibits unusual pharmacokinetic properties. It is rapidly accumulated by cells and tissues, particularly by blood leukocytes, and is only slowly release from these sites, giving a plasma half-life of over 40 h (Zuckerman, 2000). A spontaneous observation from clinical practice further stimulated our interest in the effects of azithromycin on neutrophils. During routine treatment of infected patients with antibiotics, it was observed by one of us (VP-B) that after several days of treatment, myeloperoxidase content of white blood cells was frequently reduced. Since azithromycin is widely used in this clinic, the suspicion was raised that the reduction may in some way have been related to the treatment. Together with this finding and the limited and sometimes contradictory data on the effects of azithromycin on neutrophil function and biochemistry in vitro, we decided to study the effects of a standard antibacterial dose regimen of azithromycin, 500 mg/day for 3 days, on various neutrophil functions in human subjects. In addition, we included assays of some serum inflammatory mediators to clarify potential effects of the macrolide on in vivo mediator release. During human inflammatory diseases, neutrophils may be primed or even defective (Viedma Contreras, 1999). The present study was, therefore, performed on healthy human volunteers to obtain information on resting neutrophils, as a basis for future studies on patients with inflammatory disorders. Some of these data have been presented in abstract form Novak Mirčetić, et al., 2001, Čulić et al., 2002.