Investigation of the anti-inflammatory and membrane-stabilizing potential of spiramycin in vitro

A. J. Therona,*, C. Feldmanb and R. Andersona

a Medical Research Council Unit for Inflammation and Immunity, Department of Immunology, Institute for Pathology, University of Pretoria, PO Box 2034, Pretoria 0001; b Department of Medicine, University of the Witwatersrand, Johannesburg, South Africa


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The effects of the 16-member macrolide spiramycin (2.5–80 mg/L) and the 14-member agent clarithromycin on the production of superoxide by activated human neutrophils were compared in vitro and related to membrane-stabilizing activity. Superoxide production was measured by lucigenin-enhanced chemiluminescence with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (1 µM) as the stimulus, and membrane-stabilizing activity was measured by a haemolytic procedure. Clarithromycin, but not spiramycin, caused dose-related inhibition of superoxide production by activated neutrophils and also protected erythrocytes against haemolysis, while spiramycin possessed only weak membrane-stabilizing activity. These observations underscore the apparent association between the anti-inflammatory and membrane-stabilizing properties of macrolides.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although they are primarily antimicrobial, macrolide antibiotics have been reported to possess anti-inflammatory properties which may explain their usefulness in the treatment of a variety of acute and chronic inflammatory disorders, including bronchial asthma, sub-acute sclerosing panbronchiolitis and ischaemic heart disease, including unstable angina and acute myocardial infarction.1 Macrolides have been reported to suppress the pro-inflammatory activities of neutrophils,2 eosinophils,3 monocytes,4 Tlymphocytes5 and bronchial epithelial cells.6

The anti-inflammatory activities of macrolides are well documented, but the molecular and biochemical basis of these potentially beneficial, albeit secondary, properties is not completely understood. However, some insights may be provided by the finding that only 14- and 15- member macrolides, and not 16-member agents such as spiramycin and josamycin, possess significant anti-inflammatory activity.1,3 We have reported previously that the anti-inflammatory and cytoprotective properties of 14-member (clarithromycin, erythromycin and roxithromycin) and 15-member (azithromycin) macrolides are closely correlated with their membrane-stabilizing activity.2 However, the membrane-stabilizing and anti-inflammatory potential of 16-member macrolides was not investigated. In the current study we have compared the effects of spiramycin with those of clarithromycin on the generation of superoxide by stimulated human neutrophils in vitro in relation to the membrane-stabilizing activities of these agents.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Macrolides

Clarithromycin and spiramycin were kindly provided by the Johannesburg-based South African affiliates of Abbott Laboratories (Abbott Park, IL, USA) and Rhône–Poulenc Rorer (Vitry-sur-Seine, France), respectively. Unless stated, all other agents used were obtained from Sigma Chemical Co. (St Louis, MO, USA).

Neutrophils

Human neutrophils were obtained from heparinized (5 units of preservative-free heparin/mL) venous blood of healthy adult volunteers and separated from mononuclear leucocytes by centrifugation on Histopaque-1077 (Sigma Diagnostics) cushions at 400g for 25 min at room temperature. The resultant pellet was suspended in phosphate-buffered saline (PBS; 0.15 M, pH 7.4) and sedimented with 3% gelatin for 15 min at 37°C to remove most of the erythrocytes. After centrifugation, erythrocytes were removed by selective lysis with 0.84% ammonium chloride at 4°C for 10 min. The neutrophils, which were routinely of high purity and viability, were resuspended to 1 x 107 per mL in PBS and held on ice until used.

Superoxide generation

Superoxide generation was measured using a lucigenin (bis-N-methylacridinium nitrate)-enhanced chemiluminescence (LECL) method.2 Neutrophils were pre-incubated for 15 min at room temperature in 900 µL Hanks' balanced salt solution (HBSS) containing 0.2 mM lucigenin in the presence or absence of the macrolides (1.25–80 mg/L) followed by 15 min at 37°C. Spontaneous and stimulusactivated LECL responses were then recorded in an LKB Wallac 1251 chemiluminometer (Turku, Finland) after the addition of 100 µL of N-formyl-L-methionyl-L-leucyl-phenylalanine (FMLP; 1 µM) which was used as the stimulus of membrane-associated oxidative metabolism. LECL readings were integrated for 5 s intervals and recorded as mV/s.

Membrane stabilization

The membrane-stabilizing potential of spiramycin and clarithromycin was measured using a haemolytic assay.2 Sheep erythrocytes were washed three times and resuspended to 0.5% in HBSS. The erythrocytes (final concentration 0.05%) were then co-incubated with the macrolides (40 or 80 mg/L) for 30 min at 37°C followed by addition of the membrane-destabilizing lysophospholipid, lysophosphatidylcholine (LPC) at concentrations (0.6–0.75 mg/L) that caused partial haemolysis. After 5 min the erythrocytes were pelleted by centrifugation and the supernatants were assayed spectrophotometrically at 405 nm for haemoglobin content.

Statistical analysis

The results of each series of experiments are expressed as mean ± S.E.M. Where appropriate, levels of statistical significance were calculated by Student's t-test.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Superoxide production

The effects of the test macrolides on superoxide production by FMLP-activated neutrophils are shown in Figure 1Go. Clarithromycin caused dose-related inhibition of superoxide production which was statistically significant (P < 0.05) at all concentrations tested, while spiramycin, at concentrations of 2.5–20 mg/L potentiated the generation of this reactive oxidant by the cells, with very slight inhibition observed at the highest concentration tested (80 mg/L).



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Figure 1. Effects of clarithromycin ({square}) and spiramycin (•) on the FMLP-activated chemiluminescence responses of human neutrophils. The results of 14 experiments are expressed as the mean percentages of the macrolide-free control systems ± S.E.M. The values shown are those for the peak chemiluminescence responses. The absolute values for unstimulated and FMLP activated neutrophils were 306 ± 34 and 1005 ± 61 mV/s, respectively. *Statistically significant (P < 0.05).

 
Membrane stabilization

The effects of the test macrolides on the resistance of sheep erythrocytes to lysis by LPC are shown in Figure 2Go. Clarithromycin, at both concentrations tested (40 and 80 mg/L), caused significant (P < 0.05) protection against LPC-mediated haemolysis, while spiramycin had only modest membrane-stabilizing activity, which was statistically significant (P < 0.05) at the higher concentration tested.



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Figure 2. The effects of clarithromycin () and spiramycin (40 and 80 mg/L) () on lysophosphatidylcholine-mediated haemolysis of sheep erythrocytes, compared with control ({blacksquare}). *Statistically significant (P < 0.05).

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The inhibitory effects of 14- and 15-member macrolides and ketolides on the production of pro-inflammatory, toxic reactive oxidants by neutrophils are well established2,7,8 and have been attributed to the membrane-stabilizing properties of these agents2,8 and to their inhibitory effects on the phospholipase D/phosphatidic acid phosphohydrolase pathway.7 Since this latter pathway is prone to inhibition by membrane-stabilizing agents,7 these two mechanisms are not mutually exclusive. The inhibitory effects of macrolides and ketolides on superoxide production by activated neutrophils, which are related to membrane-stabilizing mechanisms, may be achieved by interference with the protein motions, both lateral and rotational, which are required for optimum functioning of NADPH oxidase, the superoxide-generating system of phagocytes.2 Interference with phospholipase D/phosphatidic acid phosphohydrolase may also contribute to the anti-oxidative interactions of these agents with neutrophils by decreasing the production of second messengers involved in activation of the latent oxidase.7

In the current study we investigated the anti-oxidative and membrane-stabilizing potential of the 16-member macrolide spiramycin. Unlike clarithromycin, which was effective at therapeutically relevant concentrations,2 this agent had no significant inhibitory effects on superoxide production by activated neutrophils. Indeed, production of this reactive oxidant was in fact increased in the presence of spiramycin at concentrations of 2.5–20 mg/L, with only slight inhibition observed at the highest concentration tested. These findings are in agreement with two previous studies which reported either no effects9 or slight stimulation10 of FMLP-activated superoxide production by neutrophils co-incubated with spiramycin at the concentration range used in the present study.

Interestingly, the inability of spiramycin to inhibit the oxidative burst in stimulated neutrophils was associated with weak membrane-stabilizing activity in comparison with that of clarithromycin. The reason for this remains to be established, but may result from structural differences between these agents and/or lower uptake of spiramycin by eukaryotic cells relative to that of clarithromycin. The detection of membrane stabilization, in which cytolysis is the end-point, necessitated the use of high concentrations of clarithromycin and does not exclude more subtle membrane-stabilizing actions such as inhibition of superoxide production at lower concentrations of this macrolide. The physicochemical and biochemical mechanism by which 14- and 15-member macrolides promote membrane stabilization remains to be established, but does not appear to be achieved by direct inhibition of phospholipase A2 or by neutralizing interactions with bioactive phospholipids.2

In conclusion, the 16-member macrolide spiramycin had no meaningful inhibitory action on superoxide production by activated neutrophils in vitro, which contrasts with the effects of clarithromycin. Spiramycin's relative lack of anti-inflammatory properties appears to correlate with the relatively weak membrane-stabilizing activity of this agent.


    Notes
 
* Corresponding author. Tel: +27-12-3192425; Fax: +27-12-3230727. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Wales, D. & Woodhead, M. (1999). The anti-inflammatory effects of macrolides. Thorax 54, Suppl. 2, S58–62.[Free Full Text]

2 . Anderson, R., Theron, A. J. & Feldman, C. (1996). Membrane-stabilising, anti-inflammatory interactions of macrolides with neutrophils. Inflammation 20, 693–705.[ISI][Medline]

3 . Kohyama, T., Takizawa, H., Kawasaki, S., Akiyama, N., Sato, M. & Ito, K. (1999). Fourteen-member macrolides inhibit interleukin-8 release by human eosinophils from atopic donors. Antimicrobial Agents and Chemotherapy 43, 907–11.[Abstract/Free Full Text]

4 . Schultz, M. J., Speelman, P., Zaat, S., van Deventer, S. J. H. & van der Poll, T. (1998). Erythromycin inhibits tumor necrosis factor alpha and interleukin 6 production induced by heat-killed Streptococcus pneumoniae in whole blood. Antimicrobial Agents and Chemotherapy 42, 1605–9.[Abstract/Free Full Text]

5 . Konno, S., Adachi, M., Asano, K., Okamoto, K. & Takahashi, T. (1993). Anti-allergic activity of roxithromycin: inhibition of interleukin-5 production from mouse T-lymphocytes. Life Sciences 52, PL25–30.[ISI][Medline]

6 . Kawasaki, S., Takizawa, H., Ohtoshi, T., Takeuchi, N., Kohyama, T., Nakamura, H. et al. (1998). Roxithromycin inhibits cytokine production by and neutrophil attachment to human bronchial epithelial cells in vitro. Antimicrobial Agents and Chemotherapy 42, 1499–502.[Abstract/Free Full Text]

7 . Perry, D. K., Hand, W. L., Edmondsen, D. E. & Lambeth, J. D. (1992). Role of phospholipase D-derived diradylglycerol in the activation of the human neutrophil respiratory burst oxidase: inhibition by phosphatidic acid phosphohydrolase inhibitors. Journal of Immunology 149, 2749–58.[Abstract/Free Full Text]

8 . Feldman, C., Anderson, R., Theron, A. J., Mokgobu, I., Cole, P. J. & Wilson, R. (1999). The effects of ketolides on bioactive phospholipid-induced injury to human respiratory epithelium in vitro. European Respiratory Journal 13, 1022–8.[Abstract/Free Full Text]

9 . Labro, M. T., el Benna, J. & Babin-Chevaye, C. (1989). Comparison of the in vitro effect of several macrolides on the oxidative burst of human neutrophils. Journal of Antimicrobial Chemotherapy 24, 561–72.[Abstract]

10 . Moutard, I., Gressier, B., Brunet, C., Dine, T., Luyckx, M., Templier, F. et al. (1998). In vitro interaction between spiramycin and polymorphonuclear neutrophils oxidative metabolism. Pharmacological Research 37, 197–201.[ISI][Medline]

Received 21 October 1999; returned 24 January 2000; revised 17 February 2000; accepted 13 March 2000





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