1. This work was funded by BBSRC and GSK. In the present abstract, we show that macrolide antibiotics, in particular erythromycin and clarithromycin inhibit cytokine and mucin release in human respiratory epithelial cells. EFFECT OF 14-MEMBER RING MACROLIDE ANTIBIOTICS ON CYTOKINE AND MUCIN RELEASE IN A549 CELLS J.M. Hunt, K. Bunce, P.J. Barnes and D.F. Rogers, Thoracic Medicine, Faculty of Medicine, National Heart and Lung Institute, Imperial College London, Dovehouse Street, London, SW3 6LY, U.K ABSTRACT Erythromycin is widely used in the treatment of upper and lower respiratory tract infections and has been shown to improve chronic inflammatory processes including diffuse panbronchiolitis, bronchial asthma, and chronic sinusitis, suggesting its direct effects on host cells. Erythromycin has also been shown to inhibit secretion of respiratory glycoconjugate from human airways. Clarithromycin is a semi-synthetic macrolide similar in structure to erythromycin and is used in the treatment of infectious diseases. Clarithromycin differs by the methylation of a hydroxyl group at position 6 of the lactone. In this study we have investigated the effect of macrolide antibiotics, including, erythromycin and clarithromycin on cytomix induced mucin and IL-8 release in A549 cells (pulmonary carcinoma-derived). We have established stimulatory and inhibitory concentrations and time courses. Cytomix (TNF- , IL-1  and IFN-  ) significantly increased IL-8 release at 24h, by 4673% above control, which was significantly decreased by 74% at . Mucin release was significantly stimulated by 241% above control at 48h and significantly inhibited by 360% at . An estimation of mucin concentrations in samples was determined using an Enzyme Linked Lectin Assay (ELLA) and an IL-8 ELISA was used to measure Human Interleukin 8 (IL-8) release in cell culture supernates. Eyrthromycin inhibited cytokine release in a time-dependent (maximal at 72 h out of 30 min, 24h and 48h) and concentration-dependent manner which was significant at  and  (P<0.05, n=6). Mucus production was inhibited by erythromycin after 48h (n=6). Clarithromycin inhibited mucus production and IL-8 release in a concentration dependent-manner, which was greatest at 100  g/ml following cytomix stimulation after 24h (n=5). In conclusion, the results of the present study show that macrolide antibiotics in particular erythromycin and clarithromycin inhibit cytokine and mucin release. METHODS A549 cells (human type II alveolar cell line) were cultured in 25ml Dulbecco’s modified Eagle’s media (DMEM) supplemented with 10% (v/v) foetal calf serum (FCS), and 2mM L-glutamine Humidified incubator (95% air and 5% CO 2 at 37 o C) Media replaced every 3 days upon reaching visual confluence, cells were subcultured REFERENCES 1.Nagai H, Shishido H, Yoneda R, Yamaguchi E, Tamura A, Kurashima A. Long-term low- dose administration of erythromycin to patients with diffuse pan-bronchiolitis. Respiration 1991; 58:145-149. 2.Shirai, T., A. Sato, and K. Chida. 1995. Effect of 14-membered ring macrolide therapy on chronic respiraotry tract infections and polmorphonuclear leukocyte activity. Intern. Med. 34469-474. 3.Nakamura, H., K. Yoshimura, H.A. Jaffe, and R.G. Crystal. 1991. Interleukin-8 gene expression in human bronchial epithelial cells. J. Biol. Chem. 266:19611-19617. 4.Goswami SK, Kivity S, and Marom Z. Erythromycin inhibits respiratory conjugate secretion from human airways in vitro. Am. Rev. Respir Di141:72-78. ACKNOWLEDGEMENTS RESULTS joanne.hunt@imperial.ac.uk AIM To investigated the inhibitory effect of macrolide antibiotics, in particular erythromycin, on mucin and cytokine release in human epithelial cell line A549. FIGURES Erythromycin inhibited cytokine release in a time-dependent (maximal at 72h out of 30 min, 24 and 48h) and concentration- dependent manner which was significant at 10  M and 100  M (P< 0.05 and P<0.01, n=6) (Fig. 1). Cytomix significantly increased IL-8 release at 24h, by 4673% above control, which erythromycin significantly decreased by 74% at 10  M (P< 0.01, n=6) (Fig. 1). Mucus production was inhibited by erythromycin after 48 and 72h (lack of stimulation) (Fig. 2). Mucin release was significantly stimulated by cytomix, 241% above control at 48h, which erythromycin significantly inhibited by 360% at 10  M (P< 0.05, n=6) (Fig. 2). Clarithromycin inhibited mucus production and IL-8 release in a concentration- dependent manner, which was greatest at 100  g/ml following cytomix stimulation after 24h (P< 0.05, n=5) (Fig. 3). CONCLUSIONS Cells where changed to serum-free media 24 h before they were treated with test compounds. Erythromycin was aliquoted in ethanol and clarithromycin was aliquoted in methanol and diluted in serum-free media. Serum-free media containing 0.1, 1, 10 and 100  M of erythromycin and 3, 10 and 100  g/ml of clarithromycin was added to the A549 cells. The cells were then pre-incubated at 37 o C for 30 mins, 24 h, 48 h or 72 h. Cells were then stimulated with cytomix (IL-1  IFN-  and TNF-  ) at 0.1ng/ml (sub-maximal) for 4h. Supernatant was removed and stored at -70 0 C. IL-8 ELISA was performed to measure human interleukin 8 (IL-8) in cell culture supernates. Enzyme linked lectin assay (ELLA) was used to estimate the mucin concentration in the samples. Lectin identifies specific terminal sugars on glycoproteins or N- / O- linked oligosaccharides. Lectin: derived from Helix promatia or edible snail and conjugated to horseradish peroxidase. Brief method:  Samples are incubated on a maxipore plate,  Wash and blocked with buffer  Apply lectin and incubate  ‘Develop’ using OPD (o-Phenylenediamine Dihydrochloride) peroxidase substrate and  Terminate with sulphuric acid  Samples are compared with human airway standard INTRODUCTION Clarithromycin and erythromycin are 14-member ring macrolide antibiotics widely used for the treatment of airway infection and inflammation 1- 2. Precise mechanisms underlying their anti- inflammatory activity remain unclear. Anti-inflammatory mechanism of action not explained by anti-microbial properties. Interleukin (IL)-8 is an important mediator of airway inflammation, for example in COPD 3. Mucus hypersecretion is a major consequence of airway inflammation. Erythromycin has been shown to inhibit the secretion of respiratory conjugate from human airways 4. METHODS 30 mins + EM (100  M) +V+V +V+V +V+V +V+V +V+V +V+V +V+V +V+V [EM] (  M) Stim 24 H 42 H 72 H Figure 1. The Effect of Erythromycin on Cytomix induced IL-8 release, p*< 0.05, p**< 0.01, p***< 0.005 24 H 42 H 72 H Figure 2. The Effect of Erythromycin on Cytomix induced Mucin production, p*< 0.05 Figure 3. The Effect of Clarithromycin on Cytomix induced IL-8 release and Mucin production after 30mins and 24h, p*< 0.05 Lectin Assay at 30mins Lectin Assay at 24 H IL-8 ELISA at 30 mins IL-8 ELISA at 24 H + CAM (100  g) +V+V +V+V +V+V +V+V [CAM] (  g) Stim