a Department of Medical and Chemical Laboratory Diagnostics, University of Vienna, Austria b Department of Maxillofacial Surgery, University of Vienna, Austria c Institute of Medical Chemistry, University of Vienna, Austria e Department of Internal Medicine I, University of Vienna, Austria d Department of Anaesthesiology and Intensive Care Medicine, Texas Tech. University, Health Science Center, Lubbock, TX, USA
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Abstract |
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Introduction |
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Under conditions that allow bacterial multiplication, ß-lactam antibiotics such as co-amoxiclav exhibit bacteriostatic activity, affecting extracellular bacteria as reported for Gram-positive bacteria.7,8 Following surgery, wound infections play an important role.9,10,11,12 Co-amoxiclav combinations are used widely for the prophylactic and therapeutic treatment of wound infections.13,14,15,16,17,18 However, little is known about the influence of co-amoxiclav on leucocyte functions in a co-culture system. Different groups have investigated the influence of co-amoxiclav on leucocyte function.19,20 Previous studies investigated leucocyte chemotaxis under agarose gel with no endothelial cells present.21 The aim of this study was to examine the effect of co-amoxiclav on leucocyte transmigration through ECMs in an in-vitro cell co-culture system.
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Materials and methods |
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Isolation of PMNLs
Forty millilitres of heparinized (10 µL/L) whole blood was taken from seven healthy female (n = 4) and male (n = 3) volunteers. PMNLs were isolated using a PercollFicoll method, described by Metcalf.22 Freshly isolated neutrophils were washed twice in phosphate buffer solution (PBS; Sigma, St Louis, MS) and resuspended in medium M199 (Sigma). The cells were counted in a cell counter (Sysmex, Tokyo, Japan). Cell viability was measured using Trypan Blue exclusion staining (Merck, Darmstadt, Germany). A concentration of 5 x 106 PMNLs/mL was used in 200 µL aliquots (106 PMNLs) in the migration assay.
Isolation of human endothelial cells
Human umbilical vein endothelial cells were isolated using a modified method (half concentration of collagenase and double incubation time) described by Jaffe et al.,23 and cultured on fibronectin-coated microporous membranes (polyethylene terephthalate membranes, 3.0 µm pore size, low pore density (8 x 105 pores/cm2); Becton Dickinson, San Jose, CA, USA) producing a monolayer.24 The quality of the monolayer was controlled using an inverted microscope (IMT-100, Olympus, Tokyo, Japan).
Leucocyte transmigration assay
Cultured ECMs and freshly isolated PMNLs were preincubated for 30 min with co-amoxiclav (SmithKline Beecham, King of Prussia, PA, USA) diluted in medium M199 supplemented with fetal calf serum (Gibco-BRL, Gaitherburg, MD, USA), simulating a relevant plasma concentration (4 mg/L).25 In addition, lower (0.4 mg/L) and higher (40 mg/L) concentrations were used.
After preincubation (PMNLs and/or ECMs for 30 min), PMNLs were placed in a two-chamber, 24-well system and incubated (37°C, 5% CO2, 95% humidity) for 3 h in the presence of the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP, 107 M). The assay is a modification of the chemotaxis assay described previously,26,27,28,29 and has been published as migration assay.4,5,6,30 The chemoattractant was placed into the lower chamber of the double-chamber system under the microporous filter. After 3 h, the rate of leucocyte migration (untreated PMNLs through treated ECMs, treated PMNLs through untreated ECMs, treated PMNLs through treated ECMs, and as a control, untreated PMNLs through untreated ECMs) was measured with the help of the fluorescence dye calcein-acetomethylester (calcein-AM, 5 mmol/L; Molecular Probes, Eugene, OR, USA)31,32,33 using a fluorometer (Perseptive Biosystems, Hamburg, Germany), and calculated as percentage of control.
Leucocyte adhesion assay
Leucocyte adhesion (n = 7) was measured using an assay reported by De Clerk et al.34 Briefly, endothelial cells were grown to monolayers on 24-well plates (Costar, Cambridge, MA, USA). PMNLs treated with a clinically relevant concentration of co-amoxiclav (4 mg/L) for 30 min, or untreated PMNLs, were stained with calcein-AM (10 µmol/L) and washed twice in PBS (Sigma). PMNLs were seeded on treated or untreated ECMs and incubated for 3 h at 37°C. Non-adherent PMNLs were removed using a standard procedure similar to ELISA techniques, and PMNLs adherent to endothelial cells were measured using a fluorometer (Perseptive Biosystems).
Statistical analysis
Student's t-test was used for comparisons between the different groups and the control (GraphPAD InStat, Version 1.14; GraphPAD Software Inc., San Diego, CA, USA). ANOVA was used for comparisons between different concentrations of amoxycillin/clavulanate both within and between the groups. The results of seven experiments are expressed as means ± S.D. A probability value of P < 0.05 was considered statistically significant.
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Results |
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Discussion |
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Neutrophils play an important role in the defence against invading microorganisms. Therefore, PMNLs adhere to the vascular endothelium and become firmly attached. PMNLs then roll against the endothelial cell wall, and their shape changes from rounded to elongated cells enabling migration into the tissue to phagocytose bacteria.1,2,3 Therefore, we investigated the influence of co-amoxiclav on migration of PMNLs in the presence of ECMs.
Recently, we investigated the influence of a non-steroidal anti-inflammatory drug (NSAID), ibuprofen,5 on leucocyte migration through ECMs, using the same double-chamber migration assay. In this investigation, the treatment of PMNLs and ECMs with therapeutic plasma concentrations of ibuprofen was able to reduce the amount of PMNL migration to approximately 40%, thereby confirming the expected inhibitory effect of a NSAID. In the current study, co-amoxiclav was identified as a potent elevator of PMNL migration. This assay allows easy and multiple investigation of drug influences.
The possible role of anaesthetic drugs for wound infections is under discussion. Recently, the effect of ketamine4 as well as of thiopental6 on leucocyte migration through ECMs was investigated. A significant reducing effect of these anaesthetic drugs could be found. However, wound infections are still a major problem in surgical intensive care units.9,10,11,12 Co-amoxiclav is well established in the treatment of wound infections as well as a prophylactic treatment.14,18 Therefore, we investigated the effect of co-amoxiclav on the amount of leucocyte transmigration through ECMs.
Previous studies have described a stimulating effect of amoxycillin on leucocyte function. Cuffini et al.19 have demonstrated a significant increase in phagocytosis by human PMNLs using co-amoxiclav or other combinations with clavulanic acid. Roques et al.20 have investigated the effect of amoxycillin in vivoand in vitro. They found increased adhesion and chemotaxis of PMNLs. Grec et al.21 have investigated the effect of amoxycillin on the chemotaxis of human granulocytes alone under agarose in vitro. This group found a stimulating effect of amoxycillin on granulocyte chemotaxis. In concordance with these findings, our study also demonstrated such an effect. Moreover, we used a three-dimensional assay to include a monolayer of endothelial cells for investigation of the effects of co-amoxiclav on leucocyte transmigration. The treatment of both cell types simulated the effect after an iv injection or iv resorption in humans. Using this assay, we could investigate co-amoxiclav simulating in-vivo conditions.
In addition to treating both cell types with co-amoxiclav, the design of the migration assay allows treatment of either PMNLs or ECMs alone. The further aim of the study was to reveal which cell type was more affected by co-amoxiclav. Endothelial cells were significantly (P < 0.05 between treatment of PMNLs and ECMs alone) more affected by a clinically relevant concentration (4 mg/L) of co-amoxiclav. The greatest effect was shown when both cell types were treated, compared with treatment of one cell line alone.
The increasing effect on neutrophil transmigration might be caused by an elevated adhesion of PMNLs to the endothelium. In this study, we found a greater adhesion effect of co-amoxiclav when both cell types were treated with co-amoxiclav. These findings are in concordance with the results of our migration assay.
In conclusion, the results of this study indicate that co-amoxiclav influences human leucocyte and endothelial cell function, and co-amoxiclav was identified as a potent elevator of leucocyte migration. Endothelial cells were more affected than leucocytes. The greatest effect was shown when both cell types, PMNLs and ECMs, were treated. Co-amoxiclav may have a stimulating effect in the recruitment of leucocytes into the tissue.
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Notes |
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References |
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Received 11 January 1999; returned 29 March 1999; revised 12 April 1999; accepted 12 May 1999