In vitro effects of spiramycin and dirithromycin on IL1 beta production by human LPS-stimulated mononuclear cells.

Polymorphonuclear neutrophils are the predominant cells in acute inflammatory lesions and their functions and recruitment are regulated by cytokines, including IL1, TNF and IL8. Antibiotic modulation of inflammatory effects has stimulated investigations of antibiotics for their potential activity as immunomodulators over their primary bactericidal or bacteriostatic activities. This study reports the influence of macrolides, spiramycin and dirithromycin on IL1 beta production. Mononuclear cells, isolated from healthy human volunteers, were preincubated with macrolides (0.1 to 500 micrograms/ml) and stimulated by Escherichia coli lipopolysaccharide. Then, IL1 beta production was detected by western blotting analysis. At therapeutic concentrations, dirithromycin and spiramycin seemed to enhance IL1 beta production by LPS-stimulated cells, with +37 per cent and +28 per cent at 1 microgram/ml respectively. At supratherapeutic concentrations, these drugs seemed to inhibit IL1 beta production through protein kinase C inhibition, with inhibitory concentrations 50 per cent of 378 micrograms/ml for dirithromycin and 234 micrograms/ml for spiramycin. So, macrolides may modulate the host defence system through their influence on cytokine production.

In vitro interaction between spiramycin and polymorphonuclear neutrophils oxidative metabolism.

PMNs are a major component of body defense against microbial invasion, involving reactive oxygen species in great quantity, which could benefit from antibiotic therapy. Recently, possible antibiotic effects on phagocyte functions (impairment or stimulation of reactive oxygen species production) were studied. In our study, an in vitro evaluation was made on macrolide activity on phagocyte respiratory burst functions, using assay of superoxide anion (O2.-) in response to four stimuli systems: N-formyl Met-Leu-Phe (fMLP), an analogue of bacterial chemotactic factors; 4 beta-phorbol 12-myristate 13-acetate (PMA), a direct activator of protein kinase C (PKC); calcium ionophore (A23187), which acts directly on calcium influx; and a bacterial strain, Staphylococcus aureus. We have shown that spiramycin, at therapeutic plasma concentrations, increased O2.- generation by bacteria and fMLP-stimulated PMNs, with rate of 26% for 1 microgram ml-1 and 34% for 5 micrograms ml-1, respectively. This pro-oxidant effect, however, weaker, was observed when PMNs were stimulated by PMA. A weak anti-oxidant effect was observed with A23187. For higher concentrations, spiramycin decreased strongly O2.- production, with IC50 values of 74 micrograms ml-1, 154 micrograms ml-1, 296 micrograms ml-1 and 400 micrograms ml-1 when PMNs were stimulated with bacteria, A23187, fMLP and PMA, respectively. The effect of spiramycin seemed to result from an intracellular mechanism by intervention of PMN oxidative metabolism (NADPH-oxidase activation), rather than a simple chemical interaction, because no effect has been observed in acellular models. For higher spiramycin concentrations, the decrease of O2.- production observed could not be taken into consideration because this concentration was not used in therapy. The enhanced of O2.- production observed could be used in therapy, so as to increase PMNs bactericidal activity.

Azithromycin impact on neutrophil oxidative metabolism depends on exposure time.

Several antimicrobial agents have already been investigated relating to their influence on neutrophil ROS generation. Azithromycin provides, a dose-related anti-oxidant effect, after 15 min incubation, with the stimulating agent FMLP, as well with PMA or S. aureus. This finding was however obtained with concentrations not considered in therapeutics. Since short incubation times are not representative of the physiological situation, and since azithromycin is characterized by prolonged high concentrations within phagocytes, the same experiments were performed over 2 and 4 h exposures. A time-dependent anti-oxidant effect was then reported. The maximum effect was obtained with PMA (IC50 were 856 and 30 micrograms/ml for 15 min and 4 h incubation times respectively). Time-dependent modifications of neutrophil oxidative metabolism seem to be correlated with intracellular concentrations. Depressed oxidative metabolism might be related neither to azithromycin cellular toxicity, nor to superoxide scavenging properties. By increasing exposure periods, therapeutic concentrations could therefore lead to an anti-inflammatory effect, potentially of clinical interest since associated with bacteriostatic activity.

In vitro interaction between dirithromycin or its metabolite, erythromycylamine, and oxidative polymorphonuclear metabolism.

The direct stimulation by neutrophil-infectious bacteria induces an increase in the production of reactive oxygen species which is an important host defense mechanism. Antibiotics that enter rapidly and are concentrated in neutrophils, can stimulate or damage this function. In this study, an in vitro evaluation has been made of the macrolide, dirithromycin, and its active metabolite, erythromycylamine, on the superoxide anion generation by neutrophils in three systems of stimulation: the oligopeptide fMLP, an analogue of bacterial chemotactic factors; the phorbol ester PMA, a direct activator of protein kinase C; and a bacteria strain, Staphylococcus aureus. It has been demonstrated that dirithromycin, at therapeutic plasma concentrations, and its active metabolite have a significant pro-oxidant effect on the two systems: fMLP and bacteria. This effect is greater for dirithromycin than that for erythromycylamine. At higher non-therapeutic concentrations, these substances decrease superoxide generation in the three systems. The effects of these two agents seem to be the result of an intracellular mechanism resulting in the intervention of the oxidative metabolism of neutrophils since no effect was found in the cell-free systems. Therefore, this in vitro study suggests that at therapeutic concentrations, dirithromycin and erythromycylamine could benefit therapy by stimulation of the oxidative metabolism of neutrophils.