Dicloxacillin and erythromycin at high concentrations increase ICAM-1 expression by endothelial cells: a possible factor in the pathogenesis of infusion phlebitis

Peter Lanbeck1,*, Inga Odenholt1 and Kristian Riesbeck2

Departments of 1 Infectious Diseases and 2 Medical Microbiology, Malmö University Hospital, Lund University, SE 205 02 Malmö, Sweden

Received 21 August 2003; returned 20 October 2003; revised 28 October 2003; accepted 5 November 2003


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: Antimicrobial agents are important risk factors for infusion phlebitis, but the risk varies between different antibiotics. Erythromycin and dicloxacillin are known to induce phlebitis frequently, as well as to exert toxic effects on cultured endothelial cells. The pathogenesis of infusion phlebitis is unclear, but chemical toxicity is thought to lead to inflammation and subsequent thrombosis. In the present study, endothelial cells were exposed to antibiotics at the range of concentrations used for intravenous administration, followed by analysis of pro-inflammatory and pro-coagulant surface molecules.

Methods: Primary human umbilical vein endothelial cells (HUVEC) and the endothelial hybrid cell line EaHy926 were exposed to dicloxacillin, erythromycin, benzylpenicillin and cefuroxime (all at 6250 mg/L) for 60 min, followed by washing. After 5 or 24 h additional incubation, cells were analysed for E-selectin (CD62E), tissue factor (TF) or intercellular adhesion molecule 1 (ICAM-1, CD54) density by flow cytometry.

Results: Despite constitutive expression of ICAM-1 (34%) in HUVEC, 6250 mg/L of dicloxacillin or erythromycin significantly increased the number of cells with ICAM-1 expression by 37% and 30%, respectively. In contrast, cefuroxime and benzylpenicillin did not up-regulate ICAM-1 above background levels. A similar pattern was seen with the endothelial cell line EaHy926. The E-selectin and TF density were not affected by the antibiotics examined.

Conclusions: The results of this study support the theory that endothelial cells that are affected by high concentrations of antibiotics may initiate an inflammatory response through expression of ICAM-1. This is a novel finding in the pathogenesis of infusion phlebitis.

Keywords: phlebitis/chemically induced, antibiotics, adhesion molecules, CD54


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Infusion phlebitis is the most common side effect of intravenous therapy1,2 and intravenously administered antibiotics are among the important risk factors for this condition.24 We have shown recently in a clinical prospective observational study that antibiotics differ in their tendency to cause infusion phlebitis.3 We have also demonstrated that different antibiotics, at the concentrations used for intravenous administration, vary in their toxic effects on cultured endothelial cells.5,6

Although infusion phlebitis is a common event, its pathogenesis is not fully understood. The most prevalent opinion is that chemical irritation of the endothelium leads to a sterile inflammation that in some cases is followed by thrombosis.2,7 Histological studies have shown varying results. In the only study on humans, published in India in 1989, vacuolation of the endothelial cells was followed by leucocyte infiltration and destruction of the endothelial lining.8 In dogs, the early event was attachment of granulocytes to the endothelium, followed by migration to subendothelial layers and subsequent induction of thrombosis.9 In contrast, studies in rabbits have shown an alternative process, where shedding of endothelium by toxicity leads to inflammation, with granulocyte migration and subsequent thrombosis.10

The attachment of granulocytes to the endothelium is regulated by several surface molecules on the endothelial cells, such as endothelial leucocyte adhesion molecule 1 (E-selectin, ELAM-1, CD62E) and intercellular adhesion molecule 1 (ICAM-1, CD54).11,12 E-selectin makes the granulocytes roll on the endothelium, facilitating their further attachment to the endothelial cells by binding to ICAM-1. Expression of both E-selectin and ICAM-1 is up-regulated by various cytokines through the nuclear factor-{kappa}B (NF-{kappa}B) signalling pathway.

In addition to adhesion molecules, tissue factor (TF) can be found on endothelial cells stimulated by cytokines or thrombin. TF is the primary cellular initiator of the coagulation cascade.13,14 When endothelial cells are shed as a result of injuries, cells of the subendothelial layers present a high number of TF molecules. Our previous studies show that the antibiotics dicloxacillin and erythromycin, at high concentrations, reduce DNA synthesis in endothelial cells more than benzylpenicillin and cefuroxime, and that these antibiotics also differ in their tendency to cause infusion phlebitis.5,6 If the hypothesis of sterile inflammation with granulocyte adherence were valid, then endothelial cells that are affected by antibiotics should increase their expression of E-selectin, ICAM-1 and TF.

Human umbilical vein endothelial cells (HUVEC) are primary cells that are used widely for endothelial cell research. However, primary cells have to be obtained a short time before experiments, and have a limited life span. In contrast, continuous cell lines grow in a predictable manner, and might divide for many years. Only a few continuous cell lines with an endothelial phenotype are available, one of which is the fusion cell line EaHy926. This cell line has many characteristics in common with HUVEC, but also differs in many properties.12

The present study was designed to examine whether the toxic effects of these different antibiotics were followed by up-regulation of these molecules, and to compare the results obtained with primary cells with those obtained with a continuous cell line.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell cultures

Pooled primary HUVEC were obtained from Cascade Biologics (Portland, OR, USA). Cells were cultured in Medium 200 (Cascade Biologics) supplemented with low serum growth supplement including 2% fetal bovine serum (FBS) (LSGS; Cascade Biologics), and additional FBS. The final concentration of FBS was 10%. HUVEC were maintained in 5% CO2 atmosphere at 37°C. Passage was done at 70–80% confluence at a ratio of 1:2 (cells from one culture flask were trypsinized and split into two flasks), and cells were used for experiments at the third to fourth passage.

The endothelial hybrid cell line EaHy926 (at >50 passages) was cultured in RPMI 1640 medium (Life Technologies, Paisley, UK) supplemented with 10% FBS and 12 mg/L gentamicin in 5% CO2 atmosphere at 37°C, as described previously.15 Passage was carried out once or twice a week at a ratio of 1:3.

Antibiotics

Benzylpenicillin sodium (Bensylpenicillin; AstraZeneca, Södertälje, Sweden), cefuroxime sodium (Zinacef; GlaxoSmithKline, Gothenburg, Sweden), dicloxacillin sodium (Diclocil; Bristol-Myers Squibb, Bromma, Sweden) and erythromycin lactobionate (Abboticin; Abbot, Solna, Sweden) were obtained from the hospital pharmacy as powder in bottles for clinical use. Solutions were prepared freshly for every experiment. Stock solutions were made according to the manufacturers’ instructions. Benzylpenicillin, cefuroxime and dicloxacillin were dissolved in sterile water to a concentration of 100 000 mg/L and erythromycin to 50 000 mg/L. Further dilutions to 12 500, 6250 and 3100 mg/L were done in isotonic saline.

Incubation with antibiotics

The cells, at a density of 5 x 105 cells/mL, were seeded in 12-well plastic culture plates, where each well contained 1 mL of the respective medium with 10% FBS, but without antibiotics. They were grown to subconfluence for 24–36 h, after which they were washed twice with phosphate-buffered saline (PBS), and re-fed with 0.5 mL medium and 0.5 mL antibiotic solution (dicloxacillin, erythromycin, benzylpenicillin, cefuroxime) or isotonic saline. The final concentrations in the wells were 6250, 3100 and 1600 mg/L. Untreated control cells were re-fed with only 1 mL medium. After 30 min (EaHy926) or 60 min (HUVEC and EaHy926) incubation with antibiotics, the cells were washed twice with PBS and re-fed with 1 mL of medium without antibiotic and grown for 5 or 24 h. Experiments with antibiotic concentration of 6250 mg/L and exposure time 60 min were repeated at least three times. To include a positive control stimulus, untreated control cells were incubated with tumour necrosis factor-{alpha} (TNF-{alpha}) (Genzyme, Cambridge, MA, USA) 10 ng/mL, for 1, 5 or 24 h.

Flow cytometry analysis

After 5 h (E-selectin and TF) or 24 h (ICAM-1), cells were washed twice with PBS and trypsinized at 37°C for 3 min. Trypsin was neutralized by the addition of medium with 10% FBS. Cells from two or three wells were then transferred to flow cytometry tubes, and cold PBS with 0.5% BSA was added. EaHy926 cells and HUVEC were centrifuged (4°C) at 253g for 10 min and 176g for 15 min, respectively. Thereafter, cells were incubated with either fluorescein isothiocyanate (FITC)-conjugated mouse IgG1 anti-human CD54 (ICAM-1) monoclonal antibody (mAb) (Dako, Glostrup, Denmark; code F 7143), FITC-labelled mouse IgG1 anti-human tissue factor mAb (American Diagnostica Inc., Stanford, CT, USA; No. 4507 CJ) or R-Phycoerythrin conjugated mouse IgG1 anti-human CD62E (E-selectin) mAb (BD PharMingen, San Diego, CA, USA; No. VI A090). After 30 min on ice, EaHy926 cells and HUVEC were washed with PBS (0.5% BSA) three and two times, respectively. Flow cytometry analyses were done with an XL Coulter EPICS (BD; Hyaleah, FL, USA). In experiments with EaHy926 and HUVEC, 104 cells and 5 x 103 cells were analysed, respectively. Gates were set from cells incubated without specific antibodies (<2%). Results are presented either as the percentage of cells staining positive above the gate or as mean fluorescence intensity (MFI) of all cells. The results of untreated cells were subtracted from the results of cells incubated with antibiotics, saline or TNF-{alpha}. Data from three to four repeated experiments are presented as mean ± S.D.

Statistics

Data were compared with one-way analysis of variance with correction for mass significance (Tukey-B), with significance set at 0.05. Calculations were done using the statistical software SPSS for Mac 6.1.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
HUVEC and EaHy926 constitutively expressed ICAM-1 on 34 ± 4% (mean ± S.D.) and 18.5 ± 7.3% of cells, respectively. The corresponding MFIs were 3.27 ± 0.3 and 3.07 ± 1.0 with HUVEC and EaHy926, respectively.

When HUVEC were incubated for 60 min with 6250 mg/L of the different antibiotics, dicloxacillin and erythromycin significantly increased ICAM-1 expression compared with cells that were incubated with saline, benzylpenicillin or cefuroxime (Figure 1a). Raw data from a typical experiment with this concentration and exposure time are shown in Figure 2. The effect was dose dependent for dicloxacillin with all three concentrations, and for erythromycin between 1600 and 3100 mg/L, as shown in Figure 3. In one additional experiment, the antibiotics were passed through a 0.45 µm filter. Similar results were seen, which excludes the possibility of interference of microparticulate matter.



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Figure 1. The increase of ICAM-1-positive cells in primary (a) HUVEC and (b) the endothelial hybrid cell line EaHy926 after exposure to dicloxacillin, erythromycin, benzylpenicillin or cefuroxime at 6250 mg/L for 60 min. Isotonic saline and TNF-{alpha} were negative and positive controls, respectively. Each value represents the mean of three separate experiments in HUVEC and four experiments with dicloxacillin, and three for the other antibiotics in EaHy926. Error bars represent the S.D. In the right part of the diagram, the increase in the MFI (mean ± S.D.) of all cells are displayed. The asterisks show that dicloxacillin and erythromycin significantly differed from the other antibiotics, saline and each other in HUVEC, and that dicloxacillin alone varied from all others, except erythromycin in EaHy926 (one-way analysis of variance, significance level 0.05). The differences in MFI were significant in a similar way to the other data.

 


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Figure 2. Representative flow cytometry histograms from one of three experiments of ICAM-1 expression on HUVEC. The antibiotics (6250 mg/L) were incubated for 60 min. Gating was set at <2% on untreated cells that were not incubated with any specific antibody. The number of positive cells (%) and MFI are indicated in the different panels.

 


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Figure 3. Flow cytometry histograms from an experiment demonstrating ICAM-1 expression on HUVEC. Cells were exposed to dicloxacillin and erythromycin at 1600, 3100 and 6250 mg/L for 60 min. For untreated cells without the specific antibody, a gate was set at <2% of the cells. The number of positive cells (%) and MFI are indicated in the different panels.

 
EaHy926 is an immortalized hybrid cell line displaying an endothelial phenotype. We included EaHy926 for comparison with primary HUVEC, but the response was considerably weaker (Figure 1b). In EaHy926, only the effect of dicloxacillin was significantly different, compared with benzylpenicillin, cefuroxime and saline, but not with erythromycin. With longer exposure times (30 and 60 min) and increasing concentrations (1600–12 500 mg/L), there was a tendency to increased ICAM-1 expression in EaHy926 (data not shown).

Incubation with TNF-{alpha} for 1, 5 or 24 h raised ICAM-1 expression to >90% in both cell types. The MFI data demonstrate that the level of stimulation with TNF-{alpha} was substantially greater than the effect of antibiotics in both cell systems (Figure 1).

The effects of the antibiotics on TF and E-selectin expression in HUVEC were also studied. Expression of both these molecules peaks at 4–6 h after stimulation, and often returns to baseline by 24 h.11,16 After 5 h of incubation with TNF-{alpha}, E-selectin and TF were expressed in 60% and 28% of HUVEC, respectively. Baseline levels of these two molecules were 3% and 4% of the cells, respectively. Saline or antibiotics did not, in any case, increase the expression by >7%.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study showed that exposure of endothelial cells to a high concentration (6250 mg/L) of erythromycin and dicloxacillin up-regulates the expression of ICAM-1, but not E-selectin or TF. Similar high concentrations can occur locally at the endothelium in veins when antibiotics are administered intravenously.17 Our previous studies demonstrate that erythromycin and dicloxacillin at this high concentration inhibits DNA synthesis, measured as incorporation of [3H]thymidine, in an incubation time-related manner.5,6 In contrast, cefuroxime and benzylpenicillin do not interfere with DNA replication, and in the present study these two antibiotics did not affect ICAM-1 expression. Furthermore, in an observational clinical study,3 the Cox proportional hazards for phlebitis for dicloxacillin and erythromycin were 7.17 and 4.06, respectively, compared with patients not receiving antibiotics. The hazards for benzylpenicillin and cefuroxime were 2.27 and 2.07, respectively. The high risk for infusion phlebitis with erythromycin has been demonstrated in other studies.4,18 In addition, another group has demonstrated that macrolides at 2000 mg/L exert toxic effects on HUVEC.19,20

When an antibiotic is given intravenously, the endothelium is intermittently exposed to high concentrations of the drug in question. A common theory of the underlying cause of infusion phlebitis is that the chemical irritation of the vessel wall causes toxicity and subsequent sterile inflammation. The present study showed that one prerequisite of inflammation leading to attachment of leucocytes, i.e. increased surface expression of ICAM-1, can be caused by the two phlebitogenic antibiotics, erythromycin and dicloxacillin.

In-line filters have been shown to reduce the frequency of infusion phlebitis associated with antibiotics.7,21 The filters cleared the antibiotic solutions from microparticulate matter. In the present study, dicloxacillin and erythromycin induced ICAM-1 expression even after use of a microfilter, indicating that microparticulate matter did not cause the observed effects.

Incubation with TNF-{alpha} caused a higher expression of ICAM-1 than dicloxacillin and erythromycin. Furthermore, TNF-{alpha} at 5 h caused up-regulation of tissue factor, and at both 5 and 24 h up-regulated the expression of E-selectin, an effect that was not seen with the two antibiotics. TNF-{alpha}, as other cytokines, increases the expression of ICAM-1 and E-selectin through the NF-{kappa}B signalling pathway. Since E-selectin was not affected by dicloxacillin and erythromycin, the ICAM-1 induction was probably caused by another mechanism of stimulation. A recent study has shown that ICAM-1 expression can be activated by the cellular repair protein p53 in an NF-{kappa}B-independent manner.22 One effect of the p53 protein is cell cycle arrest,23 and we have previously demonstrated that DNA synthesis was inhibited in endothelial cells exposed to dicloxacillin and erythromycin,6 indicating cell cycle arrest. Irradiation and hypoxia can also activate ICAM-1, and it has been shown that the anti-tumour agents cisplatin and 5-fluorouracil induce ICAM-1 in an NF-{kappa}B-independent manner.24 We propose that the toxicity of antibiotics may act in a similar way as these other cell stress stimuli.

In the present study, pooled HUVEC were used. The basal expression of ICAM-1, E-selectin and TF, as well as the expression after incubation with TNF-{alpha}, is an indication that the cells were in good condition. These expression levels were also in agreement with levels found in single donor cells in other published studies.12,15,2527 It is unlikely, from an immunological point of view, that the present results would be caused by interactions between cells from different donors.

To avoid the use of primary cell lines from umbilical veins, the employment of cell lines such as EaHy926, a hybrid cell line with several characteristics of endothelial cells, would be of great value. However, this cell line does not express E-selectin on stimulation with TNF-{alpha},12,28 and to our knowledge there are no studies that show that this cell line can express TF. In the present study, similar activation of ICAM-1 expression was seen in EaHy926 as in HUVEC, but, as shown in Figure 1, the activation of EaHy926 was less, and, in the case of erythromycin, not significantly increased compared with control cells. Such discrepancies between primary and established cell lines, also demonstrated in one of our former studies,5 limit the use of this cell line.

Infusion phlebitis is a dynamic process, where initial signs of inflammation such as redness, pain and warmth sometimes are followed by thrombosis with a palpable cord. The present study could not confirm that cellular expression of TF was activated by any of the antibiotics. It seems more probable that the thrombosis of the superficial vein that occurs in more severe cases of infusion phlebitis is caused by chemical toxicity and shedding of the endothelial cells. Interestingly, other studies have shown that ICAM-1 can interact with fibrinogen in a pro-coagulant manner,29,30 indicating that the toxicity exerted by antibiotics leading to ICAM-1 expression also can activate coagulation factors.

In conclusion, the present study has demonstrated that erythromycin and dicloxacillin activate ICAM-1 expression on cultured endothelial cells in a dose-dependent manner. This activation could be one important mechanism of the pathogenesis of infusion phlebitis, leading to leucocyte attachment and possibly also a pro-coagulant effect. This ICAM-1 activation might explain the different tendencies of various antibiotics to cause infusion phlebitis.


    Acknowledgements
 
We are indebted to Mirela Karamehmedovic and Emily Eriksson for maintaining the cells. This study was supported by grants from the Swedish Fund for Research without Animal Experiments and County Skåne, Sweden.


    Footnotes
 
* Corresponding author. Tel: +46-40-331000; Fax: +46-40-336279; E-mail: Peter.Lanbeck{at}inf.mas.lu.se Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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