Antibiotic-induced cell wall fragments of Staphylococcus aureus increase endothelial chemokine secretion and adhesiveness for granulocytes

Abstract

Antibiotics release inflammatory fragments, such as lipoteichoic acid (LTA) and peptidoglycan (PG), from the cell wall of Staphylococcus aureus. In this study, we exposed S. aureus cultures to a number of beta-lactam antibiotics (imipenem, flucloxacillin, and cefamandole) and protein synthesis-inhibiting antibiotics (erythromycin, clindamycin, and gentamicin) and investigated whether supernatants of these cultures differ in their capacity to stimulate endothelial cells (EC). After 24 h of incubation, endothelial adhesiveness for leukocytes, surface expression of various adhesion molecules, and secretion of the chemokines interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) were measured. Supernatants of beta-lactam-exposed cultures (designated beta-lactam supernatants) enhanced the adhesiveness of EC for granulocytes, whereas those of protein synthesis-inhibiting antibiotic-exposed cultures (designated protein synthesis-inhibitor supernatants) did not. This hyperadhesiveness coincided with a higher intercellular adhesion molecule-1 expression on the surface of the stimulated EC. In addition, EC stimulated with beta-lactam supernatants secreted significantly higher concentrations of the chemokines IL-8 and MCP-1 than those stimulated with protein synthesis-inhibitor supernatants. The finding that the concentrations of LTA and PG in beta-lactam supernatants were much higher than those in protein synthesis-inhibitor supernatants suggests that the observed differences in stimulatory effect between these supernatants are a result of differences in the release of cell wall fragments, although the presence of other stimulatory factors in the supernatants cannot be excluded. In conclusion, our results argue for a release of LTA and PG from S. aureus after exposure to beta-lactam antibiotics that enhances the development of a systemic inflammatory response by stimulating EC such that adhesiveness for granulocytes is increased and large amounts of IL-8 and MCP-1 are secreted.

FIG. 1

Release of LTA and PG from S. aureus ATCC 25923 after culture for 4 h in the absence (contr) or presence of various antibiotics at concentrations 20 times the MIC. Antibiotics studied were the protein synthesis-inhibitors (PSI) erythromycin (ery), clindamycin (clinda), and gentamicin (genta) and the β-lactam antibiotics imipenem (imi), flucloxacillin (fluclox), and cefamandole (cefa). After 4 h of incubation with the antibiotics, bacterial supernatants were collected by centrifugation and filtration of the cultures, and the amounts of LTA and PG were measured by means of a specific sandwich ELISA and an SLP assay, respectively. Data are the means + standard errors of the means (error bars) of the results of three to four separate experiments.

FIG. 2

Adherence of granulocytes to EC that were incubated for 24 h with various supernatants of S. aureus cultures. The supernatants were collected after 4 h of culture in the absence (contr) or presence of different protein synthesis-inhibiting (PSI) and β-lactam antibiotics at concentrations 20 times the MIC. After stimulation, the EC were washed and incubated with 4 × 10⁵ to 6 × 10⁵ granulocytes, with (▧) or without () preincubation with anti-CD18 MAb, for 30 min. After removal of the nonadherent granulocytes by washing, the remaining cells were stained with Giemsa and counted by light microscopy. The number of adherent granulocytes is presented as a percentage of the total number of granulocytes added. The binding of granulocytes to unstimulated EC amounted to 3.2%. Data were corrected for this background adherence and are the means of the results of three to four separate experiments. ery, erythromycin; clinda, clindamycin; genta, gentamicin; imi, imipenem; fluclox, flucloxacillin, cefa, cefamandole.

FIG. 3

Endothelial surface expression of ICAM-1 after stimulation for 24 h with various supernatants of S. aureus. The bacterial supernatants were collected after 4 h of culture in the absence (contr) or presence of different protein synthesis-inhibiting (PSI) and β-lactam antibiotics at concentrations of 20 times the MIC. Stimulated EC were washed, trypsinized, and incubated with a mouse anti-human ICAM-1 MAb for 30 min. Next, the EC were incubated with peroxidase-conjugated goat anti-mouse Ig MAb for 30 min, and 5,000 cells of each sample were analyzed by flow cytometry. As a measure of ICAM-1 surface expression the MFI, which was 117 ± 9 for unstimulated EC, was used. Data were corrected for this background ICAM-1 expression and are the means + standard errors of the means (error bars) of the results of three to four separate experiments. ery, erythromycin; clinda, clindamycin; genta, gentamicin; imi, imipenem; fluclox, flucloxacillin; cefa, cefamandole.

FIG. 4

Secretion of the chemokines IL-8 () and MCP-1 (▧) by EC stimulated with various supernatants of S. aureus. The bacterial supernatants were collected after 4 h of culture in the absence (contr) or presence of different protein synthesis-inhibiting (PSI) and β-lactam antibiotics at concentrations 20 times the MIC. After 24 h of incubation with these supernatants, the culture medium was collected and chemokine concentrations were measured by means of specific sandwich ELISAs. Data are the means + standard errors of the means (error bars) of the results of three to four separate experiments; no correction for background levels was made. ery, erythromycin; clinda, clindamycin; genta, gentamicin; imi, imipenem; fluclox, flucloxacillin; cefa, cefamandole.