Tilmicosin-induced bovine neutrophil apoptosis is cell-specific and downregulates spontaneous LTB4 synthesis without increasing Fas expression

Abstract

-The pathology of bacterial pneumonia, such as seen in the bovine lung infected with Mannheimia haemolytica, is due to pathogen virulence factors and to inflammation initiated by the host. Tilmicosin is a macrolide effective in treating bacterial pneumonia and recent findings suggest that this antibiotic may provide anti-inflammatory benefits by inducing polymorphonuclear neutrophilic leukocyte (PMN) apoptosis. Using an in vitro bovine system, we examined the cellspecificity of tilmicosin, characterized the changes in spontaneous leukotriene B 4 (LTB 4 ) synthesis by PMN exposed to the macrolide, and assessed its effects on PMN Fas expression. Previous findings demonstrated that tilmicosin is able to induce PMN apoptosis. These results were confirmed in this study by the Annexin-V staining of externalized phosphatidylserine and the analysis with flow cytometry. The cell-specificity of tilmicosin was assessed by quantification of apoptosis in bovine PMN, mononuclear leukocytes, monocyte-derived macrophages, endothelial cells, epithelial cells, and fibroblasts cultured with the macrolide. The effect of tilmicosin on spontaneous LTB 4 production by PMN was evaluated via an enzyme-linked immunosorbent assay. Finally, the mechanisms of tilmicosin-induced PMN apoptosis were examined by assessing the effects of tilmicosin on surface Fas expression on PMN. Tilmicosin-induced apoptosis was found to be at least partially cell-specific, as PMN were the only cell type tested to die via apoptosis in response to incubation with tilmicosin. PMN incubated with tilmicosin under conditions that induce apoptosis spontaneously produced less LTB 4 , but did not exhibit altered Fas expression. In conclusion, tilmicosin-induced apoptosis is specific to PMN, inhibits spontaneous LTB 4 production, and occurs through a pathway independent of Fas upregulation. macrolide / neutrophil / apoptosis / pasteurellosis / inflammation

Anti-Inflammatory Benefits of Antibiotic-Induced Neutrophil Apoptosis: Tulathromycin Induces Caspase-3-Dependent Neutrophil Programmed Cell Death and Inhibits NF-κB Signaling and CXCL8 Transcription

“…Macrolides are also known to modulate host immunity and inflammation (5,22). Recent observations indicate that the macrolides azithromycin and 16-ring tilmicosin, another veterinary antibiotic, may induce neutrophil apoptosis and promote the resolution of inflammation via mechanisms that remain obscure (5,7,8,25,26). Once terminally differentiated neutrophils reach the site of inflammation, they may alter their proinflammatory signals to an anti-inflammatory response, and induction of neutrophil apoptosis can contribute to this effect (17,42,45).…”

“…Purified neutrophil preparations were obtained from peripheral blood drawn from the jugular veins of healthy Holstein cows into BD Vacutainers containing 1.5 ml anticoagulant acid citrate dextrose (ACD solution A; Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ), as previously described (7,8,26). Briefly, the blood was pooled and spun at 1,200 ϫ g for 20 min at 4°C.…”

“…This class of antibiotics is known to influence the recruitment and infiltration of neutrophils (23,26,31) and to alter their ability to secrete histotoxic compounds (23) and proinflammatory cytokines (31,50,53). Recent findings also indicate that some macrolides may induce cellular death by apoptosis (3,5,7,8,26) and block NF-B signaling in T lymphocytes, macrophages, or epithelial cells (27,36,56). However, the precise mechanisms underlying the anti-inflammatory and proapoptotic activities of macrolides in neutrophils remain unclear.…”

Evaluation of annexin V and Calcein-AM as markers of mononuclear cell apoptosis during human immunodeficiency virus infection

“…Annexin V-FITC is presently employed in evaluation and quantification of cellular apoptosis by flow cytometry due to its specific bind to phosphatidylserine residues [14,15,30]. Thus, viable cells are classified as annexin V negative, while the apoptotic cells are annexin V positive [31].…”

“…Annexin V was shown to be useful for detection of apoptotic cells because of its binding affinity for negatively charged phospholipids like phosphatidylserine, exposed at the beginning of apoptotic process [13][14][15]. The conjugation between fluorescein isothiocyanate (FITC) and annexin V allows identifying and quantifying the apoptotic cells by flow cytometry [13,16].…”