Effect of Antibiotics on Polymorphonuclear
Study Objective. To evaluate the effects of various antibiotics -- direct and indirect as a result of bacterial killing -- on polymorphonuclear neutrophil (PMN) apoptosis.
Design. In vitro analysis.
Setting. Research laboratory.
Intervention. Whole blood collected from healthy human subjects was incubated with and without Klebsiella pneumoniae (1.0 x 10 colony-forming units [cfu]/ml) plus ceftazidime 50 µg/ml, gentamicin, ciprofloxacin, trovafloxacin, tetracycline, doxycycline, erythromycin, azithromycin (each 5 µg/ml), or lipopolysaccharide 10 µg/ml. After staining with fluorescein-conjugated annexin V, red blood cells were lysed, and the remaining white blood cells were assessed by flow cytometry with gating on PMNs.
Measurements and Main Results. In the absence of K. pneumoniae infection, antibiotic exposure directly decreased PMN apoptosis by 17.8% (range -25.0% to -13.9%, p=0.008) compared with untreated cells. In the presence of K. pneumoniae, all antibiotic treatments, even those with poor in vitro activity for the bacterial isolate, decreased PMN apoptosis by 26.2% (range -38.0% to -17.8%, p<0.001) compared with untreated control cells and by 36.6% compared with untreated (no antibiotic) K. pneumoniae-stimulated cells (range -46.2% to -28.0%, p<0.001).
Conclusions. All tested antibiotics in clinically relevant concentrations resulted in modest yet consistent decreases in PMN apoptosis. The magnitude of this change increased slightly in the presence of K. pneumoniae infection. In vivo studies are needed to determine whether antibiotic-associated prolongation of PMN survival improves host response to infection.
Polymorphonuclear neutrophils (PMNs) are the most important cell type involved in the early nonspecific host response to bacterial and fungal cell pathogens. They function primarily as phagocytic cells to ingest, degrade, and remove microbial pathogens. The activation of PMNs by microbial stimuli is enhanced by numerous inflammatory cytokines, most notably tumor necrosis factor-a (TNF-a), interleukin 1ß (IL-1ß), IL-3, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), and G-CSF.
With a half-life of 8-20 hours, PMNs are short-lived cells. In the absence of inflammatory stimuli, PMNs undergo genetically programmed cell death, or apoptosis, characterized by cytoplasmic shrinkage, nuclear condensation, membrane blebbing, DNA fragmentation, and formation of apoptotic bodies. Early in the apoptotic process, phosphatidylserine is exposed on the cell surface by flipping from the inner to the outer leaflets of the cytoplasmic membrane. This event is thought to be important for macrophage recognition of cells undergoing apoptosis, thus allowing the cells to be removed after their death with minimal inflammation. In addition, apoptosis of human PMNs is thought to be critical for control of the inflammatory process and maintenance of homeostasis, although its regulatory mechanisms are not completely understood. Numerous pathologic conditions (certain cancers, autoimmune and neurodegenerative disorders, sepsis), microbial pathogens (human immunodeficiency virus, herpes viruses, Mycobacterium tuberculosis), chemicals (N-formyl-methionyl-leucyl-phenylalanine), and drug therapies (cyclosporine, macrolides, cancer chemotherapy, glucocorticoids) have been shown to alter apoptosis, in part through fibroblast-associated (Fas, CD95), caspase-dependent, and cytokine-signaling mechanisms.
Lipopolysaccharide (LPS, endotoxin), G-CSF, GM-CSF, IL-1ß, IL-2, IL-6, and IL-10 have been shown to delay PMN apoptosis, whereas TNF-a appears to accelerate the apoptotic process. Antibiotics possess differential effects on endotoxin release from gram-negative bacteria with the subsequent production and release of inflammatory (e.g., TNF-a, IL-1ß, IL-6) and antiinflammatory (e.g., IL-10) cytokines. In addition, we previously demonstrated antibiotic-mediated upregulation of PMN CD11b, an important chemoattractant-induced leukocyte adhesion molecule, as a result of bacterial killing. The CD11b/CD18 complex contributes to the control of activated PMNs by upregulating apoptosis. Therefore, because endotoxin, various cytokines, and CD11b alter or regulate apoptosis, and given that antibiotics can modulate these important factors, we hypothesized that antibiotic exposure can modify PMN apoptosis.
Study Objective. To evaluate the effects of various antibiotics -- direct and indirect as a result of bacterial killing -- on polymorphonuclear neutrophil (PMN) apoptosis.
Design. In vitro analysis.
Setting. Research laboratory.
Intervention. Whole blood collected from healthy human subjects was incubated with and without Klebsiella pneumoniae (1.0 x 10 colony-forming units [cfu]/ml) plus ceftazidime 50 µg/ml, gentamicin, ciprofloxacin, trovafloxacin, tetracycline, doxycycline, erythromycin, azithromycin (each 5 µg/ml), or lipopolysaccharide 10 µg/ml. After staining with fluorescein-conjugated annexin V, red blood cells were lysed, and the remaining white blood cells were assessed by flow cytometry with gating on PMNs.
Measurements and Main Results. In the absence of K. pneumoniae infection, antibiotic exposure directly decreased PMN apoptosis by 17.8% (range -25.0% to -13.9%, p=0.008) compared with untreated cells. In the presence of K. pneumoniae, all antibiotic treatments, even those with poor in vitro activity for the bacterial isolate, decreased PMN apoptosis by 26.2% (range -38.0% to -17.8%, p<0.001) compared with untreated control cells and by 36.6% compared with untreated (no antibiotic) K. pneumoniae-stimulated cells (range -46.2% to -28.0%, p<0.001).
Conclusions. All tested antibiotics in clinically relevant concentrations resulted in modest yet consistent decreases in PMN apoptosis. The magnitude of this change increased slightly in the presence of K. pneumoniae infection. In vivo studies are needed to determine whether antibiotic-associated prolongation of PMN survival improves host response to infection.
Polymorphonuclear neutrophils (PMNs) are the most important cell type involved in the early nonspecific host response to bacterial and fungal cell pathogens. They function primarily as phagocytic cells to ingest, degrade, and remove microbial pathogens. The activation of PMNs by microbial stimuli is enhanced by numerous inflammatory cytokines, most notably tumor necrosis factor-a (TNF-a), interleukin 1ß (IL-1ß), IL-3, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), and G-CSF.
With a half-life of 8-20 hours, PMNs are short-lived cells. In the absence of inflammatory stimuli, PMNs undergo genetically programmed cell death, or apoptosis, characterized by cytoplasmic shrinkage, nuclear condensation, membrane blebbing, DNA fragmentation, and formation of apoptotic bodies. Early in the apoptotic process, phosphatidylserine is exposed on the cell surface by flipping from the inner to the outer leaflets of the cytoplasmic membrane. This event is thought to be important for macrophage recognition of cells undergoing apoptosis, thus allowing the cells to be removed after their death with minimal inflammation. In addition, apoptosis of human PMNs is thought to be critical for control of the inflammatory process and maintenance of homeostasis, although its regulatory mechanisms are not completely understood. Numerous pathologic conditions (certain cancers, autoimmune and neurodegenerative disorders, sepsis), microbial pathogens (human immunodeficiency virus, herpes viruses, Mycobacterium tuberculosis), chemicals (N-formyl-methionyl-leucyl-phenylalanine), and drug therapies (cyclosporine, macrolides, cancer chemotherapy, glucocorticoids) have been shown to alter apoptosis, in part through fibroblast-associated (Fas, CD95), caspase-dependent, and cytokine-signaling mechanisms.
Lipopolysaccharide (LPS, endotoxin), G-CSF, GM-CSF, IL-1ß, IL-2, IL-6, and IL-10 have been shown to delay PMN apoptosis, whereas TNF-a appears to accelerate the apoptotic process. Antibiotics possess differential effects on endotoxin release from gram-negative bacteria with the subsequent production and release of inflammatory (e.g., TNF-a, IL-1ß, IL-6) and antiinflammatory (e.g., IL-10) cytokines. In addition, we previously demonstrated antibiotic-mediated upregulation of PMN CD11b, an important chemoattractant-induced leukocyte adhesion molecule, as a result of bacterial killing. The CD11b/CD18 complex contributes to the control of activated PMNs by upregulating apoptosis. Therefore, because endotoxin, various cytokines, and CD11b alter or regulate apoptosis, and given that antibiotics can modulate these important factors, we hypothesized that antibiotic exposure can modify PMN apoptosis.
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