Amoxycillin/clavulanic acid combinations increase transmigration of leucocytes through endothelial cell monolayers: endothelial cells play a key role

R. Hofbauera,*, D. Moserb, B. Gmeinerc, A. D. Kayed, S. Kapiotisa, O. Wagnera and M. Frasse

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Postoperative inflammation is still viewed as an unresolved problem. During inflammation, leucocytes play a tremendous role and migrate from intravascular spaces into the tissue to attack microorganisms. Different agents, e.g. anaesthetic drugs, are able to influence leucocyte recruitment. Previous studies have investigated the influence of amoxycillin on chemotaxis of leucocytes alone. The aim of our study was to examine the effect of amoxycillin/clavulanic acid (co-amoxiclav) on leucocyte migration through endothelial cell monolayers (ECMs). Human umbilical endothelial cells were cultured on microporous membranes, achieving a monolayer. Polymorphonuclear neutrophil leucocytes (PMNLs) were used in a migration assay. The numbers of untreated PMNLs migrating through untreated ECMs were used as control and set as 100%. PMNLs and/or ECMs were pretreated with co-amoxiclav using clinically relevant as well as higher and lower concentrations. Co-amoxiclav was able to increase PMNL migration through ECMs significantly (P < 0.05) when both cell types were treated (291 ± 18.7%). When PMNLs or ECMs were treated alone, it could be shown that ECMs were more affected than PMNLs. The greatest effect was shown when both cell types, PMNLs and ECMs, were treated. In conclusion, co-amoxiclav was identified as a potent drug to increase leucocyte transmigration through ECMs. ECMs were also critically involved. Co-amoxiclav also affects endothelial cells.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
During inflammation, polymorphonuclear neutrophil leucocytes (PMNLs) play a tremendous role. They migrate rapidly from the vascular spaces into the tissue to destroy invading microorganisms. During migration through endothelial cell monolayers (ECMs), PMNLs undergo morphological changes from rounded, relatively smooth cells to elongated, ruffled cells with pseudopodia.1,2,3 The influence of non-steroidal anti-inflammatory drugs, e.g. ibuprofen, or anaesthetics, e.g. ketamine or thiopental, on leucocyte function was recently investigated by our group, using an in-vitro double chamber migration assay including ECMs.4,5,6

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Informed consent was given by the volunteers and by the mothers of newborn babies for umbilical cord collection. The study was approved by the Institutional Review Board of the University of Vienna.

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 Percoll–Ficoll 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, 10–7 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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Figure 1 shows the effect of relevant plasma concentrations of co-amoxiclav (4 mg/L) on leucocyte migration through ECMs. The migration of untreated leucocytes through untreated ECMs, used as control in each single experiment, was set as 100%. The following observation was noted when a clinically relevant concentration of co-amoxiclav was used: if only PMNLs were treated with co-amoxiclav, the migration rate increased to 160 ± 12.3% S.D. (P < 0.05 compared with control). Treatment of ECMs alone increased leucocyte migration significantly to 254 ± 17.3% (P < 0.05 compared with control). When both cell types (PMNLs and ECMs) were pretreated with co-amoxiclav, the amount of leucocyte migration through ECMs increased significantly to 291 ± 18.7% S.D. (P < 0.05 compared with control). Statistical analysis between the treated cell types showed a significant difference (P < 0.05) between the groups in which PMNLs were treated alone versus the group in which PMNLs and ECMs were treated, as well as between the groups in which PMNLs and ECMs were each treated alone. The greatest effect was seen when both PMNLs and ECMs were treated compared with treatment of one of the two cell lines alone.



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Figure 1. Influence of clinically relevant plasma concentrations of co-amoxiclav (4 mg/L) on leucocyte migration through ECMs. This figure shows the migration rates when only polymorphonuclear neutrophil leucocytes were treated (treat PMNL), when only ECMs were pretreated (treat ECM), and when both cell types were treated with co-amoxiclav (both treat). The amount of migration of untreated PMNLs through untreated ECMs was used as control and set as 100%. *P < 0.05 compared with control.

 
Figure 2 shows the influence of different dosages of co-amoxiclav (0.4, 4 and 40 mg/L) on leucocyte migration through ECMs when both cell types were pretreated. The migration rate of treated PMNLs through treated ECMs was measured using a lower concentration (105 ± 9.7%, P was not significant compared with control), and a higher concentration (415 ± 22.8%, P < 0.05 compared with control). Statistical analysis between the different concentrations used in the leucocyte migration experiments showed significant differences (P < 0.05) between all groups (low concentration, medium concentration and high concentration). The results show a dose-dependent effect.



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Figure 2. Influence of clinically relevant plasma concentrations (mean conc; 4 mg/L), lower (low conc; 0.4 mg/L) and higher (high conc; 40 mg/L) concentrations of co-amoxiclav on leucocyte migration through ECMs when PMNLs and ECMs were treated with co-amoxiclav. Clinically relevant and high concentrations lead to a significant increase of leucocyte migration compared with control. The amount of migration of untreated PMNLs through untreated ECMs was used as control and set as 100%. *P < 0.05 compared with control.

 
Figure 3 shows the results of the adhesion assay. Co-amoxiclav (4 mg/L) increased the adhesion of PMNLs to ECMs when using a relevant plasma concentration. Treatment of both cell types increased PMNL adhesion to 275 ± 16.2% S.D. (P < 0.05 compared with control), PMNLs treated alone to 177 ± 12.4% S.D. (P < 0.05 compared with control) and ECMs treated alone to 230 ± 16.9% S.D. (P < 0.05 compared with control).



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Figure 3. Influence of clinically relevant plasma concentrations of co-amoxiclav (4 mg/L) on leucocyte adhesion to ECMs when only PMNLs or ECMs alone, or both cell types were treated with co-amoxiclav. The adhesion of untreated PMNLs to untreated ECMs was used as control and set as 100%. *P < 0.05 compared with control.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Co-amoxiclav is a widely used antimicrobial agent. In this study, we identified co-amoxiclav as a potent elevator of leucocyte transmigration through ECMs using a double-chamber co-culture assay. Higher concentrations lead to a greater increase in leucocyte transmigration. The greatest effect was seen when both cell types, PMNLs and ECMs, were treated, simulating the in-vivo situation.

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.


    Notes
 
* Correspondence address. Department of Medical and Chemical Laboratory Diagnostics, Level 5H, University of Vienna, Waehringer Guertel 18–20, A-1090, Austria. Tel: +43-1-40400-5387; Fax: +43-1-40400-4545; E-mail: roland.hofbauer{at}akh-wien.ac.at Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 11 January 1999; returned 29 March 1999; revised 12 April 1999; accepted 12 May 1999